BIOTECHNOLOGY OF VITAMINS, PIGMENTS AND GROWTH FACTORS

ELSEVIER APPLIED BIOTECHNOLOGY SERIES

Other Titles in this Series: Y. Chisti: Airlift Bioreactors

BIOTECHNOLOGY OF

VITAMINS, PIGMENTS AND

GROWTH FACTORS

Edited by

ERICK J. VANDAMME Laboratory of General and Industrial Microbiology,

State University of Ghent, Belgium

ELSEVIER APPLIED SCIENCE LONDON and NEW YORK

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WITH 51 TABLES AND 113 ILLUSTRATIONS

© 1989 ELSEVIER SCIENCE PUBLISHERS LTD Sof tcover reprint of the hardcover 1st edition 1989

British Library Cataloguing in Publication Data

Biotechnology of vitamins, pigments and growth factors 1. Industrial chemicals: organic compounds I. Vandamme, Erick J. 661'.8

ISBN·13:978-94-010-6991-5

Library of Congress Cataloging-in-Publication Data

Biotechnology of vitamins, pigments, and growth factors/edited by Erick J. Vandamme.

p. cm.-(Elsevier applied biotechnology series) Includes bibliographies and index. ISBN-13: 978-94-01 0-6991-5 e-ISBN -13: 978-94-009-1111-6 001: 10.1007/978-94-009-1111-6

1. Vitamins-Biotechnology. 2. Growth promoting substances--Biotechnology. 3. Pigments (Biologyl--Biotechnology. I. Vandamme, Erick J., 1943- II. Series.

[DNLM: 1. Biotechnology. 2. Growth Substances. 3. Pigments. 4. Vitamins. QT 34 B61545] TP248.65.V57B56 1989 DNLM/DLC for Library of Congress

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To Mireille

PREFACE

Vitamins and related growth factors belong to the few chemicals with a positive appeal to most people; the name evokes health, vitality, fitness, strength .... each one of us indeed needs his daily intake of vitamins, which should normally be provided via a balanced and varied diet. However, current food habits or preferences, or food processing and preservation methods do not always assure a sufficient natural daily vitamin supply, even for a healthy human being; this is all the more true for stressed or sick individuals. Although modern society is seldom confronted with the notorious avitaminoses of the past, they do still occur frequently in overpopulated and poverty- and famine-struck regions in many parts of the world. Apart from their in-vivo nutritional-physiological roles as growth factors for man, animals, plants and micro-organisms, vitamin compounds are now being introduced increasingly as food/feed additives, as medical-therapeutical agents, as health-aids, and also as technical aids. Indeed, today an impressive number of processed foods, feeds, cosmetics, pharmaceuticals and chemicals contain extra added vitamins or vitamin-related compounds, and single or multivitamin preparations are commonly taken or prescribed.

These reflections do indicate that there is an extra need for vitamin supply, other than that provided from plant and animal food resources. Most added vitamins are indeed now prepared chemically and/or biotechnologically via fermentation/bioconversion processes.

Similarly, other related growth factors, provitamins, vitamin-like com­pounds, i.e. special unsaturated fatty acids, gibberellins and certain pigments­some of which are increasingly used in agriculture, food/feed production or processing and as health aids-are equally important biotechnological pro­ducts, where production via microbial fermentation or micro-algal bioconver­sion is now applied industrially. Indeed, biotechnology-based on bacteria, yeasts, fungi and micro-algae-here again has been very instrumental in procuring sufficient amounts of several of these valuable complex molecules via

vii

viii Preface

natural processes, although for certain products there is fierce competition with chemical synthesis. Abundant and excellent literature is available on the chemical properties, biochemistry, nutritional aspects and clinical aspects of vitamins and related products, while that on microbial synthesis and produc­tion methods is rather scarce or difficult to find.

In this respect, this book intends to assemble useful information on the (potential) industrial synthesis of economically important vitamins, growth factors and pigments, with emphasis on biotechnological aspects including microbiology, genetics, biochemistry and bioprocess technology; so far, such information is scattered widely in the scientific literature: for some products, only secrecy and sparse data are available, other excellent volumes deal with only one specific vitamin compound, while others then stress the chemical synthesis processes. Therefore, I felt that there was a scientific need for a biotechnological survey of the world of vitamins and related compounds synthesis.

The help of several colleagues and friends in suggesting potential authors for difficult-to-get chapters has been invaluable in constructing a rather com­prehensive volume; so was the positive interaction with all my contributors. In this respect, I am particularly indebted to: Dr S. Anderson, Genentech, USA; Dr G. C. Barrere, Rhone-Poulenc, France; Dr E. Cerda-Olmedo, University of Seville, Spain; Dr D. De Buyser, N. V. Vandemoortele, Belgium; Dr. D. Defterdarovic, Pliva, Yugoslavia; Professor Dr A. L. Demain, MIT, USA; Dr J. Florent, Rhone-Poulenc, France; Dr A. Furuya, Kyowa Hakko Kogyo Co., Ltd, Japan; Dr H. Nelis, University of Ghent, Belgium; Dr L. Segers, Orffa, Belgium; Dr S. Shimizu, Kyoto University, Japan; Dr G. Smits, Tiense Suikerraffinaderij, Belgium; Professor Dr Y. Tani, Kyoto University, Japan.

The editor also thanks the staff of Elsevier Science Publishers Ltd. I suspect that my wife, Mireille, could only have withstood my 'mental

absence', strengthened with multivitamin preparations, although my sole vitamin-shot was her encouraging and moral support during this biotechnologi­cal enterprise.

E. J . VANDAMME

CONTENTS

Preface . . . . . . vii

List of Contributors . xi

1. Vitamins and Related Compounds via Micro-organisms: a Biotechnological View (E. J. Vandamme) . . . . . . . 1

Fat-Soluble Vitamins and Pigments

2. p-Carotene (Provitamin A) Production with Algae (L. J. Borowitzka & M. A. Borowitzka) . . . . . . . . . . 15

3. Production of Carotenoids with Fungi (E. Cerda-Olmedo 27 4. Microbial Production of Carotenoids other than p-Carotene (H.

J. Nelis & A. P. De Leenheer) ............ 43 5. Vitamin D: The Biotechnology of Ergosterol (P. Margalith) .. 81 6. Algal and Microbial Production of Vitamin E (Y. Tani) . . .. 95 7. Microbial Production of Polyunsaturated Fatty Acids (Vitamin-

F Group) (S. Shimizu & H. Yamada) . . . . . . . . . 105 8. Microbial Production of Vitamin K2 (Menaquinone) and

Vitamin Kl (Phylloquinone) (Y. Tani) . . . . . . . . 123

Water-Soluble Vitamins

9. Microbial Synthesis of Vitamin Bl (Thiamine) (A. Iwashima) 137 10. Microbial Production of Vitamin B2 (Riboflavin) (T. Kutsal &

M. T. Ozbas) . . . . . . . . . . . . . . . . . . . .. 149 11. Microbial Production of D-Ribose (K. Sasajima & M. Yoneda) 167 12. Pantothenic Acid (Vitamin Bs), Coenzyme A and Related

Compounds (S. Shimizu & H. Yamada). . . . . . . . .. 199 13. Microbial Production of Vitamin B6 and Derivatives (Y. Tani) 221

ix

x Contents

14. Microbial Production of Biotin (Y. Izumi & H. Yamada) 231 15. Microbial Production of Vitamin B12 (C. Spalla, A. Grein, L.

Garofano & G. Feroi) . . . . . . . . . . . . . . 257 16. Microbial Production of Orotic Acid (Vitamin B13) (K.

Takayama & A. Furuya) . . . . . . . . . . . . . 285 17. Microbial Reactions for the Synthesis of Vitamin C (L-Ascorbic

Acid) (V. Delic, D. Sunic & D. Vlasic) . . . . . . . . . . . 299

Other Growth Factors

18. 19.

20. 21.

Index.

Microbial Production of ATP (Y. Tani) ........ . Adenosylmethionine, Adenosylhomocysteine and Related Nucleosides (S. Shimizu & H. Yamada) ........ . Other Vitamin-Related Coenzymes (S. Shimizu & H. Yamada) Fungal Gibberellin Production (B. Brueckner, D. Blechschmidt, G. Sembdner & G. Schneider)

337

351 373

383

431

LIST OF CONTRIBUTORS

D. BLECHSCHMIDT, Friedrich Schiller University Jena, Department of General Microbiology, DDR-6900 Jena, Neugasse 24, GDR

L. J. BOROWITZKA, Western Biotechnology Ltd, 2-6 Railway Parade, Bayswater, WA 6063, Australia

M. A. BOROWITZKA, Algal Biotechnology Laboratory, School of Biological and Environmental Sciences, Murdoch University, Murdoch, WA 6150, Australia

B. BRUECKNER, Friedrich Schiller University lena, Department of General Microbiology, DDR-6900 lena, Neugasse 24, GDR

E. CERDA-OLMEDO, Departmento de Genetica y Biotecnia, Universidad de Sevilla, Apartado 1095, Sevilla, Spain

A. P. DE LEENHEER, Laboratoria voor Medische Biochemie en voor Klinische Analyse, Rijksuniversiteit Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium

V. DELle, PLIVA Pharmaceutical, Chemical, Food and Cosmetic Industry, Research Institute, Zagreb, Yugoslavia

G. FERNI, Farmitalia Carlo Erba, Via dei Gracchi 35, 20146, Milano, Italy

A. FURUYA, Kyowa Hakko Kogyo Co. Ltd, Tokyo Research Laboratories, 3-6-6 Asahi-machi, Machida-shi, Tokyo 194, Japan

L. GAROFANO, Farmitalia Carlo Erba, Via dei Gracchi 35, 20146, Milano, Italy

A. GREIN, Farmitalia Carlo Erba, Via dei Gracchi 35, 20146, Milano, Italy

A. IWASHIMA, Department of Biochemistry, Kyoto Prefectural University of Medicine, Kawaramachi-Kirokoji, Kamigyo-ku, Kyoto 602, Japan

Y. IZUMI, Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan

xi

xii List of Contributors

T. KUTSAL, Hacettepe University, Chemical Engineering Department, 06532 Beytepe, Ankara, Turkey

P. MARGALITH, Department of Food Engineering & Biotechnology, Technion-Israel Institute of Technology, Haifa, 32000, Israel

H. J. NELIS, Laboratoria voor Medische Biochemie en voor Klinische Analyse, Rijksuniversiteit Ghent, Harelbekestraat 72, B-9000 Ghent, Belgium

M. T. (hBAS, Hacettepe University, Chemical Engineering Department, 06532 Beytepe, Ankara, Turkey

K. SASAJIMA, Central Research Division, Takeda Chemical Industries Ltd, 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532, Japan

G. SCHNEIDER, Institute of Plant Biochemistry, Academy of Sciences of the GDR, DDR-4050 Halle, Weinberg 3, GDR

G. SEMBDNER, Institute of Plant Biochemistry, Academy of Sciences of the GDR, DDR-4050 Halle, Weinberg 3, GDR

S. SHIMIZU, Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan

C. SPALLA, Farmitalia Carlo Erba, Via dei Gracchi 35, 20146, Milano, Italy

D. SUNIC, PLIVA Pharmaceutical, Chemical, Food and Cosmetic Industry, Research Institute, Zagreb, Yugoslavia

K. TAKAYAMA, Kyowa Hakko Kogyo Co. Ltd, Tokyo Research Laboratories, 3-6-6 Asahi-machi, Machida-shi, Tokyo 194, Japan

Y. TANI, Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

E. J . VANDAMME, Laboratory of General and Industrial Microbiology, Faculty of Agricultural Services, State University of Ghent, Coupure Links 653, B-9000 Ghent, Belgium

D. VLASIC, PLIV A Pharmaceutical, Chemical, Food and Cosmetic Industry, Research Institute, Zagreb, Yugoslavia

H. YAMADA, Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan

M. YONEDA, Corporate Strategy, Takeda Chemical Industries Ltd, 2-17-85 Jusohonmachi, Yodogawa-ku, Osaka 532, Japan

Chapter 1

VITAMINS AND RELATED COMPOUNDS VIA MICRO·ORGANISMS: A BIOTECHNOLOGICAL VIEW

E. J. VANDAMME

Laboratory of General and Industrial Microbiology, Faculty of Agricultural Sciences, State University of Ghent, Coupure Links, 653, B-9000 Ghent,

Belgium

1 HISTORICAL

The start of the history of vitamins can be traced back to 400 Be, when Hippocrates reported that eating liver could cure night-blindness. Much later, in the 16th century, the therapeutical effects of lemon juice against scurvy or scorbut became known; scorbut had inter alia caused the loss of 100 crew members on Vasco da Gama's journey around Cape Hope. The English ship doctor James Lind studied this disease further and described in 1757 in his book A Treatise of Scurvy the beneficial effect of eating fresh vegetables and fruits in preventing it. For another nutritional deficiency disease (already mentioned in 1762 by Oviedo), the Italian doctor Francesco Frapoli used the name pellagra (pella = skin; agra = rough). In the 19th century in Japan, the Hikan child disease (keratomalacia and xerophthalmia) was successfully treated by including ale-fat, cod liver oil or chicken liver in the diet. Trousseau discovered that cod liver oil and also, direct sunlight, had a curing effect on rickets, a disease already well described by Whistler in 1645. In the Far-East, when hulled rice was replaced by de hulled or polished rice, a sharp increase in the occurrence of beriberi was observed. In 1897 Eijkman observed that poultry fed with polished rice developed polyneuritis, a disease very similar to human beriberi. This disease could also be prevented and cured by feeding rice and the silver fleece of the rice kernel. Grijns in 1901 hypothesised that beriberi was caused by a protecting factor, which was obviously lacking in dehulled rice. We now know that all these diseases are a result of nutritional vitamin deficiencies (Machlin, 1984).

Around 1910, Hopkins in the UK and Osborne & Mendel in the USA initiated modern vitamin research and developed a theory, stating that diseases such as scurvy, pellagra, rickets, beriberi, etc., are the result of a lack of certain essential food components. The Polish chemist Casimur Funk isolated in 1912 from rice bran a beriberi-preventing compound, displaying chemical

1

2 E. J. Vandamme

properties of an amine; this led him in 1912 to coin the name vitamin for this type of compounds.

In 1913, McCollum & Davis demonstrated a liposoluble factor A in butter fat and egg yolk, and in 1915, a water-soluble factor B was found in wheat-germ. It was Drummond in 1920 who named the fat-soluble factor, vitamin A; the water-soluble anti-beriberi factor was named vitamin B; the water-soluble anti-scorbut factor vitamin C. In 1925, the fat-soluble anti-rickets factor was named vitamin D. After 1930, discovery and isolation of several other vitamins followed quickly and their structure, nutritional and chemical properties and synthesis were studied in great detail in the following decades.

These aspects of vitamins and growth factors are compiled in several excellent standard references (Goodwin, 1963; Sebrell & Harris, 1971; De Luca, 1978; Machlin, 1984; De Leenheer et al., 1985; Diplock, 1985; Chytil & McCormick, 1986; Adrian, 1988; Friedrich, 1988).

2 VITAMINS: WHAT'S IN A NAME?

Vitamin nomenclature was initially based on the use of letter symbols, alphabetically arranged to time of discovery. Soon, it appeared that one-letter named vitamins were multiple complexes, and this led to the addition of an index to the original letters (Bb B2 , .•• ). Often, when the function of the vitamin became known, an appropriate letter symbol was chosen, i.e. vitamin K, with K as the first letter of the German word Koagulation; other names reflected deficiencies, i.e. aneurin (B1) for anti-polyneuritis vitamin; vitamin PP = "pellagra-preventing" vitamin. Letter names or trivial names are gener­ally more in use rather than the IUPAC names. The division into fat- and water-soluble vitamins as introduced by McCollum & Davis, is still universally applied.

A listing of well-recognised vitamin compounds is presented in Table 1. From a chemical point of view, vitamins are a very heterogeneous mixture of

compounds, yet they can be considered as a single group, since they are all organic compounds which are essential for a healthy development of humans and animals and need to be present in their food, since their body is not able at all--or not sufficiently able-to synthesize them. Indeed, certain vitamins can be formed partially or indirectly in the body:

(a) compounds--often called provitamins--with no apparent vitamin ac­tivity can be converted within the body into a vitamin, i.e. Jj-carotene (in vegetables, fruits) -- vitamin A tryptophan (in protein-rich food) -- nicotinic acid (niacin) 7-dehydrocholesterol (in skin) uv) vitamin D3 (cholecalcifenol)

(b) other vitamin compounds are formed by the intestinal bacterial flora i.e. vitamin K, some B-vitamins, i.e. B12, etc.

Most vitamin compounds thus have to be provided via daily food/feed intake.

Vitamins and Related Compounds via Micro-organisms 3

Table 1 Survey of Vitamins and Growth Factors

Vitamin group Important examples Important natural International Recommended adult sources Units (IU) daily intake

Provitamin A group ,B-carotene Vegetables, I IV = 0,6 pg 2·4 mg,B-carotene y-carotene, fruits carotene a-carotene, I pg = 3·34 IV kryptoxanthine

Vitamin A group Retinol (vitamin A.), Fish oil, liver, I IV = 0·30 pg 0·75 mg retinol 3-dehydroretinol milk and daily retinol

(vitamin A2), products, eggs retinal (vitamin A.-

aldehyde) Provitamin D 7-Dehydrocholesterol,

ergosterol Yeast Vitamin D group Ergocalciferol (D2 ), Fish oil, liver, I IV = 0·025 pg

egg yolk, milk, vitamin D, cholecalciferol (D,) dairy products Illg = 40 IV

Vitamin E group a-Tocoferol, Plant oils, I IV= I·Omg 2·5 pg vitamin D3 ,B-tocoferol, vegetables dl-tocoferol- 30mg y-tocoferol, liver, eggs, acetate 6-tocoferol dairy products

Vitamin F group Linolic acid, Fish oil, algae, (polyunsaturated y-linolenic acid, fungi fatty acids, PVFAs), eicosa-pentaenoic w-3-fatty acids) acid (EPA),

DHA Vitamin K group Phylloquinone (K.) Liver, vegetables O·lmg

or phytomenadion, bacterial flora, phamoquinone (K2) or plant oils

menaquinone, menadion (K,), menadiol (K.)

Vitamin B. Thiamine Cereals, liver, 1 IV = 31lg 0·8-1·6mg meat, vegetables, thiamine dairy products, lllg = 0·333 IV yeast

Vitamin B2 Riboflavin Liver, eggs, 1·2-1·3mg (vitamin G) dairy products,

vegetables, meat, bacterial flora

Vitamin PP Nicotinamide, Liver, cereals, 10-15 mg (vitamin B3 ) nicotinic acid meat, potatoes,

(niacin) vegetables, yeast, trypto-phane-protein

Pantothenic acid Pantothenic acid, Liver, eggs, molasses 10-20mg (vitamin B5 ) coenzyme A vegetables,

cereals Vitamin B6 group Pyridoxine Liver, potato, 2mg

(pyridoxol), vegetables, pyridoxal meat, yeast pyridoxamine

Biotin Biotin Liver, egg yolk, 0·3mg (vitamin H, B8) vegetables, cane

molasses bacterial flora

(continued)

4

Vitamin group

Folic acid (B.-group, Bc ' M)

Vitamin B12 group

Vitamin B13 Vitamin C group

Lipoic acid Inositol

Choline (B7' J) Para-aminobenzoic acid

(PABA) (Bx' H', H2 )

Gibberellins

E. J. Vandamme

Table l---contd.

Important examples Important natural sources

Pteroyl-glutamic acid, Cereals, liver, pteroyl-diglutamic meat, milk,

acid, vegetables pteroyl-trigIutamic

acid Cyanobalamin (B12), Meat, liver, milk, hydroxycobalamin bacterial flora

(B I2b) ,

nitrosocobalamin, (B12c)

Orotic acid Ascorbic acid

(vitamin q, dehydro-ascorbic acid

Citrus fruit, fruit, vegetables, potato

Yeast, meat Yeast, meat,

vegetables Egg yolk, meat,

hops Yeast Plants, fungi

International Units (IU)

1 IU = 50 I'g L-ascorbic acid

Recommended adult daily intake

400 I'g

,(not for animals except, primates, cavia, etc.)

50-600mg

The vitamin content was originally and still often is expressed in Interna­tional units (IU), which relate to the biological activity of a certain amount of pure vitamin towards a specific test animal.

Presently, it is common practice to express the vitamin content as mg or f-tg per 100 g of material; conversion factors for IU values into weight units are also given in Table 1. Recommended daily intake per person per day (FAO/WHO data) is also given.

Quantitative assays for vitamin content, in foodstuffs, concentrated mix­tures, synthetic formulations, tissues, blood, fermentation broths, etc., are very important and ever more sophisticated techniques (e.g. HPLC) are introduced; for several vitamins, i.e. B12, biotin, etc., a microbiological bioassay is still the method of choice (Freed, 1966; Berg & Behagel, 1972; De Leenheer et al., 1985).

3 PHYSIOLOGICAL FUNCTIONS

Vitamins have a catalytic role in the body in enabling optimal synthesis, conversion and degradation of macromolecules such as nucleic acids, proteins, lipids and carbohydrates.

The biochemical (physiological) function of most water-soluble vitamins is well known: they are part of co-enzymes and thus co-responsible for specific biochemical reactions (Machlin, 1984; Diplock, 1985). A survey is given in Table 2.

Vitamins and Related Compounds via Micro-organisms

Table 2 Water-Soluble Vitamins and their Corresponding Coenzymes

Vitamin

Nicotinamide (B3 or PP)

Riboflavin (B2)

Pyridoxine (B6)

Folic acid (Bg)

Biotin (H)

Pantothenic acid (Bs)

Thiamine (B1)

Cyanocobalamin (B12)

Coenzyme

Nicotinamide-adenine dinucleotide (NAD+)

Nicotinamide-adenine dinucleotide phosphate (NADP+)

Flavine adenine mono­nucleotide (FMN)

Flavine adenine di­nucleotide (FAD)

Pyridoxalphosphate (PLP)

Tetrahydrofolic acid

Biotin (biocytin, E-N-biotinyllysine)

Coenzyme A

Thiaminepyrophos­phate (TPP)

B12-coenzyme

Group Transfer

Hydrogen

Hydrogen

Hydrogen

Hydrogen

Amino-group, decar­boxylation

Formyl

Carboxyl

Acyl

Cz-aldehyde, decar­boxylation

Carboxyl

5

As to the function of fat-soluble vitamins and vitamin C, much controversy still exists; their involvement can often be traced down to specific biochemical processes (Table 3), although their exact function is not yet known in all cases (De Luca, 1978; Diplock, 1985).

Much debate also exists about the positive effects of high (mega) doses of certain vitamins, (e.g. vitamin C) on human and animal physiology; on the other hand, several hypervitaminoses, (e.g. A, D, K) are well known (Machlin, 1984).

Some vitamins display different activity toward man or animal, i.e. ergocal­ciferol (D2) is poorly active in poultry, while in other animals and man it is equally active as cholecalciferol (D3)' This observation has great practical repercussions on feed formulations. Others are essential solely for man, (i.e. vitamin C), while most animals (except primates, cavias) can synthesise them.

Compounds which specifically counteract the functioning of vitamins are also known and are named antivitamins or vitamin antagonists (Machlin, 1984). Their negative action can be based on degradation of the vitamin (thiaminase, ascorbase, etc.), or on the complexation of the vitamin into a non-resorbable complex, (i.e. avidin plus biotin). N5-HydroxY-L-arginine is a vitamin B12

antagonist (Perlman et al., 1974). Dicoumarin excludes vitamin K from the prothrombin synthesis system and amethopterin is an antagonist of folic acid; however, both antivitamins are medically important for the treatment of

6 E. I. Vandamme

Table 3 Important Biochemical Actions of Fat-Soluble Vitamins and of Vitamin C

Vitamin

Vitamin A active form: ll-cis-retinol

Vitamin D3 (cholecalciferol) active form: 1,25-diOH-cholecal­ciferol

Vitamin E: ( a-tocoferol)

Vitamin K j :

(phylloquinone)

Vitamin C

Physiological functions

Retinol is part of rhodopsin, the light sensitive molecule in the eye

Biosynthesis of mucopolysaccharides; synthesis and maintenance of epithelial cells

Regulation in Ca2 + and phosphorous metabolism

Antioxidant action towards unsaturated com­pounds, i.e. fatty acids, etc.; membrane integrity

Essential for formation of prothrombine, a blood coagulation factor

Cofactor for carboxylation of protein bound glutamate residues to form y-carboxyglutamates

Cosubstrate of monooxygenases; Role in redox reactions and in

hydroxylation reactions of amino acids and amines (i.e. proline conversion into hydroxyproline in collagene);

Role in hormone-synthesis, iron absorption, etc.

thrombosis and leukemia, respectively. Antivitamins present in our daily food are usually destroyed during processing or cooking.

Excellent information on detailed nutritional, clinical and physiological aspects of vitamins has been assembled by Machlin (1984), Diplock (1985) and Friedrich (1988).

4 TECHNICAL FUNCTION OF VITAMINS, PIGMENTS AND GROWTH FACTORS

In addition to their nutritional, physiological and medical importance, vitamins and vitamin-like compounds have also found large-scale technical applications as antioxidants (vitamin C, E), as acidulant (vitamin C), and as pigments (f3-carotene and analogues) in the food, feed, cosmetic, chemical and pharmaceutical industry (Machlin, 1984; Diplock, 1985; Florent, 1986).

Apart from the synthetic pigments, most of the natural colorants are now being extracted from plants and animals, e.g. annatto, grapes, red beets, paprika, female insects (Coccus cacti). Micro-organisms could be an excellent source of non-toxic food colorants, but except for the fungal Monascus pigments (monascin), used in the Orient, and for astaxanthin from yeast few attempts have been made to produce and introduce them (Lin, 1973;

Vitamins and Related Compounds via Micro-organisms 7

Palleroni et a/., 1978; Institute of Food Technologists, 1980; Wong & Koehler, 1981). Indeed, in addition to the well-known provitamin-carotenoids, a range of anthraquinone pigments, chlorophylls and several others have been demon­strated in bacteria, yeasts, fungi and algae and are attractive as natural colorant.

In this respect, more emphasis should be given to screening and research on other natural pigment synthesis via safe micro-organisms; this would open up a wider application area in agriculture, food, feed, chemicals, cosmetics and pharmaceuticals.

Fungal gibberellins are already widely used as plant growth-regulators in agriculture and horticulture and in the brewing industry (Jefferys, 1970; Palmer, 1974; Brueckner et a/., this volume).

Details about technical applications of certain vitamins, pigments and gibberellins are given in the corresponding chapters in this volume.

5 PRODUCTION AND APPLICATION

The staple food of man, including cereals, rice, potato, vegetables, fruits, milk, fish, meat, eggs, forms his basic source of vitamins and growth factors. Adequate nutrition should thus supply this daily vitamin need, which however increases with physical exercise, pregnancy, lactation, active growth, reconvalescence, drug abuse, stress, air pollution, etc. Pathological situations (intestinal malresorption, stressed intestinal flora, liver/gall diseases, drug, antibiotic or hormone treatment, enzyme deficiencies), can also lead to vitamin shortages despite a sufficient intake. Malnourishment in many countries of the world also asks for direct medical remediations, combined with diet adjustment. Vitamin­enriched and medicated feed is used worldwide to procure healthy livestock.

However, derived concentrates or extracts from these vitamin-rich natural food products find relatively little use in the food, feed, pharmaceutical or cosmetic industry (except for w-3-fatty acids, vitamin E, etc.). Some of the reasons are:

(a) the average daily intake of vitamins does not always seem to be supplied solely via these natural products;

(b) the level of the natural plant/ animal source vitamins is usually relatively low and fluctuates drastically;

(c) their organoleptic presentation and shelf-life is often far from optimal; (d) vitamins are labile molecules during the process of preservation, storage

or preparation of foodstuffs and are generally very sensitive to pH, heat, light, oxygen, etc.; water-soluble vitamins are easily lost by aqueous extraction or manipulation of foods.

These factors have led to the industrial manufacturing of most vitamins and growth factors. Current world production of important vitamins is given in Table 4.

At the moment, several vitamins are produced only or mainly chemically

8 E. I. Vandamme

Table 4 Survey of Vitamins and Growth Factors-Production and Application

Vitamin Industrial Application World production group production (F = food/feed; (see also (E = extraction; M=medical; tons/year Average Producer Table 1) C=chemical T = technical) price company

synthesis; (S/kg) or country a

B = biotechnol- (1988) ogy)

Provitamin A E F 100 450 2, 5, 6, 6a, 15a, group C M 18, 20, 22, 25

8 (fermenta- T tion, algal)

Vitamin A C F 2500 70 33,34,44,46 group M

Provitamin D C F 8, 14a, 18, 34 8 (fermenta- M

tion) Vitamin D C F 25 350

group 8 (fermenta- M tion)

Vitamin E E F 6800 17 9,11,17,18, group C M 33,34

8 (algal) T Vitamin F C F 200,2000 800,50 3, 6, 6a, 10, 18,

group E M (GLA) (EPA, (GLA) (EPA, 26,32, 8 (fermenta- DHA) DHA) 35,37,38

tion, algal) Vitamin K C M 1·3 1500 9,11,17, 18,

group 34,45 Vitamin 8 1 C F 2000 23 2,18,31,39,

M 40,41 Vitamin 8 2 C F 2000 27 2,5,13,18,

8 (fermenta- M 24,30,41 tion)

Vitamin PP C F 8500 4 2,7,18 (vitamin 8 3) M 47

Pantothenic C F 4000 4 6a,18,39,41 acid 8 (fermenta- M (vitamin 8 s) tion)

Vitamin 8 6 C F 1600 25 18,31, 39, 41 group 8 (fermenta- M

tion) 8iotin C F 3 5000 2, 18, 24, 27,

(vitamin H, (8) M 39,43 8 8 )

Folic acid C F 300 115 18,39,41,42,48 (89 ) M

Vitamin 8 12 8 (fermenta- F 5-10 5000 14, 15, 16,21,23, group tion) M 24,26,33,36

(continued)

Vitamin group (see also Table 1)

Vitamin B13

Vitamin C

Lipoic acid Inositol

Choline

PABA (B., H', Hz)

Gibberellins

Vitamins and Related Compounds via Micro-organisms

Industrial production

(E = extraction; C=chemical

synthesis; B = biotechnol­

ogy)

B (fermenta­tion)

C plus

E C E

C

C

B (fermenta­tion)

B (fermenta­tion)

Table 4--contd.

Application (F = food/feed;

M = medical; T = technical)

M

F M T

M F M T F M T M

T

tons/year

100

65000

12-15

World production

Average price

(S/kg) (1988)

8

30

2000

9

Producer company

or country a

21

2,4,11, 18,24,28, 30,31,41,

44

18,33, 41,42,

48 1,4,12, 19,21, 24,29, 41,44,

a Listing of vitamin and growth factor producing companies or countries: 1. Abbott (USA); 2. BASF (FRG); 3. Biocrops Ltd (UK); 4. China; 5. Coors Biotech Inc. (USA); 6. Cyanotech Inc (USA); 6a. Dainippon Ink & Chemicals (Japan); 7. Degussa (FRG); 8. Duphar (The Netherlands); 9. Eastman­Kodak (USA); 10. Efamol Holdings Ltd (UK); 11. Eisa Co. (Japan); 12. Eli Lilly (USA); 13. E. Merck (FRG); 14. Farmitalia-Carlo Erba (Italy); 14a. Fujisawa (Japan); 15. Genex Corp. (USA); 15a. Gist Brocades (The Netherlands); 16. Glaxo (Sefton Chern) (UK); 17. Henkel (FRG); 18. Hoffmann­Laroche (Switzerland); 19. ICI (UK); 20. Koor Foods (Israel); 21. Kyowa Hakko Kogyo (Japan); 22. Martek Inc. (USA); 23. Medimpex (Yugoslavia); 24. Merck S & D (USA); 25. Microbial Products Inc. (USA); 26. Nippon Oil & Fats Co. Ltd (Japan), 27. Nippon Zeon (Japan); 28. Pao Yeh (Taiwan); 29. Pierrel (Italy); 30. Pfizer Inc. (USA); 31. Pliva (Yugoslavia); 32. Q. P. Corporation (Japan); 33. Rhone-Poulenc (France); 34. Riken Vitamin (Japan); 35. RMC (Evening Primrose Oil Company Ltd) (UK); 36. Roussel-UcLaF (France); 37. Shiseido Co. Ltd (Japan); 38. Sturge Biochemicals (UK); 39. Sumitomo (Japan); 40. Synergen Inc. (USA); 41. Takeda (Japan); 42. Tanabe-Kongo (Japan); 43. Tanabe (Japan); 44. USSR; 45. Vanetta (Italy); 46. Western Biotechnology Ltd (Australia); 47. Yuki Gosei (Japan); 48. Yodo Gowa-Duphar (Japan).

(vitamins A, D3 , E, K, Bh H, etc.), although microbiological methods exist or emerge; others are produced (exclusively) via fermentation (D2' B6 , B12 , F) or micro-algal culture (fJ-carotene, F) and for others both chemical and microbial processes are run industrially (vitamin B2), while vitamin C is produced via a

10 E. J. Vandamme

combination of chemical and microbiological reactions (Sakaguchi et al., 1971; Yamada et al., 1971; Ogata et al., 1976; Periman, 1978; Florent, 1986; Borowitzka & Borowitzka, 1988). Details about these bioprocesses form the backbone of this volume!

A broad range of applications now exists for these vitamins and related compounds in food, feed, cosmetics, technical and pharmaceutical preparations:

revitamination: restoring the original vitamin level of a foodstuff; standardisation: addition of vitamin(s) to compensate for natural fluctuations; vitamin-enrichment: extra addition of vitamin(s) to a level higher than the natural one (health food, diet food industry); vitamination: addition of vitamins to products lacking them (feed, cosmetics) ; technical additive (in food, feed, cosmetics), e.g. p-carotene as pigment, vitamin C and E as antioxidants, vitamin C as acidulant, gibberellins as growth hormones for plants; medical applications: to alleviate hypo- or even avitaminoses.

Apart from obtaining these vitamins via a natural way-what microbial and algal biotechnology is all about-fermentation or bioconversion reactions yield the desired enantiomeric vitamin compound, while products from organic synthesis are often racemic mixtures, each displaying a differing biological activity. Furthermore, vitamin yields in fermentation broths are often very high-especially when using mutants or genetically engineered strains-as compared with their natural levels in plants or animals.

In this respect, continued efforts should be made to further explore the potential and power of microbial and algal biotechnology in the field of economic natural vitamin, pigment and growth factor production.

REFERENCES

Adrian, J. (1988). Parat Dictionary of Food and Nutrition, VII. Ellis Horwood Series in Food Science and Technology. Ellis Horwood, Chichester, UK.

Berg, T. M. & Behagel, H. A. (1972). Semi-automated method for microbiological vitamin-assays. Appl. Microbiol., 23,531-42.

Borowitzka, M. A. & Borowitzka, L. J. (eds) (1988). Micro-algal Biotechnology. Cambridge University Press, Cambridge, UK.

Brueckner et al. (1989). Fungal gibberellin production. In Biotechnology of Vitamins, Pigments and Growth Factors, ed. E. J. Vandamme. Elsevier Science Publishers, London, pp. 383-429

Chytil, P. & McCormick, D. B. (eds) (1986). Vitamins and Coenzymes. Methods in Enzymology, Vols 122, 123. Academic Press, New York, London.

De Leenheer, A. P., Lambert, W. E. & Deruyter, M. G. M. (eds) (1985). Modern Chromatographic Analysis of the Vitamins. Marcel Dekker, New York, Basel.

De Luca, H. F. (ed.) (1978). The fat-soluble vitamins. In Handbook of Lipid Research,

Vitamins and Related Compounds via Micro-organisms 11

Vol. 2. Plenum Press, New York, London. Diplock, A. T. (ed.) (1985). Fat-Soluble Vitamins. Their Biochemistry and Applica­

tions. Heineman, London. Florent, J. (1986). Vitamins. In Biotechnology, Vol. 4, Microbial Products II; ed. H.

Pape & H. J. Rehm. VCH, Weinheim, pp. 115-58. Freed, M. (ed.) (1966). Methods of Vitamin Assay, 3rd edn. John Wiley & Sons, New

York. Friedrich, W. (1988). Vitamins. Walter de Gruyter, Berlin, New York. Goodwin, T. W. (1963). The Biosynthesis of Vitamins and Related Compounds.

Academic Press, London. . Institute of Food Technologists (1980). Food colors. Food Technology, 34,77. Jefferys, E. G. (1970). The Gibberellin Fermentation. Adv. App/. Microbio/., 13,

283-316. Lin, C. F. (1973). Isolation and cultural conditions of Monascus sp. for the production

of a pigment in a submerged culture. 1. Ferment. Techno/., 51,407-9. Machlin, L. J. (ed.) (1984). Handbook of Vitamins: Nutritional, Biochemical and

Clinical Aspects. Marcel Dekker, New York, Basel. Ogata, K., Kinoshita, S., Tsunoda, T. & Aida, K. (1976). Microbial Production of

Nucleic Acid Related Substances. Kodansha, Tokyo. Palleroni, N. S., Reichelt, K. E., Mueller, D., Epps, R., Tabenkin, B., Bull, D. N.,

Schnep, W. & Berger, J. (1978). Production of a novel red pigment, rubrolone, by Streptomyces echinoruber sp. 1. Antibiotics, 31, 1218-25.

Palmer, G. J. (1974). The industrial use of gibberellic acid and its scientific basis-a Review. 1. Inst. Brewing, 80, 13-30.

Perlman, D. (1978). Vitamins. Economic Microbiology, 2, 303-26. Perlman, D., Vlietinck, A. J., Matthews, H. W. & Lo, F. F. (1974). Microbial

production of vitamin B12 anti metabolites. I. N5-hydroxY-L-arginine from Bacillus cereus 439. 1. Antibiotics, 27, 826-32.

Sakaguchi, K., Uemara, T. & Kinoshita, S. (1971). Biochemical and Industrial Aspects of Fermentation. Kodansha, Tokyo.

Sebrell, W. H. & Harris, R. S. (eds) (1971). The Vitamins, 2nd edn. Academic Press, New York, London.

Wong, M. C. & Koehler, P. E. (1981). Production and isolation of an antibiotic from Monascus purpureus and its relationship to pigment production. 1. Food Sc., 46, 589.

Yamada, K., Nakahara, T. & Fukui, S. (1971). Petroleum microbiology and vitamin production. In Biochemical and Industrial Aspects of Fermentation, ed. Sakaguchi, T. Uemura & S. Kinoshita. Kodansha, Tokyo, pp. 61-90.

FAT-SOLUBLE VITAMINS AND PIGMENTS

Chapter 2

II-CAROTENE (PROVITAMIN A) PRODUCTION WITH ALGAE

L. J. BOROWITZKA

Western Biotechnology Ltd, 2-6 Railway Parade, Bayswater, WA 6063, Australia

&

M. A. BOROWITZKA

Algal Biotechnology Laboratory, School of Biological and Environmental Sciences, Murdoch University, Murdoch, WA 6150, Australia

1 HISTORICAL

Dunaliella is a unicellular, biflagellate, naked green alga (Chlorophyceae, Dunaliellales), and the type species of this genus, Dunaliella salina (Dunal) Teodoresco is often found in natural hypersaline waters where it colours the brines red (Teodoresco, 1905). This algal species was first recognised as containing high intracellular concentrations of p-carotene by Mil'ko (1963) and Aasen et al. (1969). Initial research on the potential of using this alga as a commercial source of p-carotene began in the Ukraine in the 1960s (cf. Massyuk, 1966; Massyuk & Abdula, 1969) and it was later also proposed as a commercial source of glycerol (Ben-Amotz; 1980; Chen & Chi, 1981; Ben-Amotz & Avron, 1982).

A number of commercial-scale developments on the production of p­carotene from D. salina have commenced in Australia (Curtain et al., 1987; Borowitzka & Borowitzka, 1988a), Israel (Rich, 1978) and the USA (Klaus­ner, 1986), and a number of open-pond and closed reactor experimental units are in development stages. Pilot projects are also under way in China and Chile. Commercial quantities of extracted algal p-carotene and dried D. salina powder rich in p-carotene have been marketed by companies from the US and Australia since 1985. All are targeting the markets for 'natural' food and animal feed colourings, and 'natural' p-carotene (provitamin A) nutritional supplements.

2 CHEMICAL AND PHYSICAL PROPERTIES

p-Carotene is accumulated as droplets in the chloroplast stroma of Dunaliella salina cells, particularly when culture conditions include high light intensities,

15

16

v U c:: 111

205·071

f 105·931 o III .c <{

c:: ~ o c:: ~ c:: :>

c:: :c 1:: 111 )( 111 V N

T. Kutsal & M. T. Ozbas

c:: c:: c:: ~ ~ ~ 0 0 0 c:: c:: c:: ~ ~ ~

c:: c:: c:: :> :> :>

v c:: v -0 L 111 U I

d

v c:: V .... o L 111 U I

C!l. I

III c:: 111 L .... I

iii vv c::C:: Vv 0 0 LL 111111 UU I I

C!l.C!l. I I

U\.!!! .- U U M Ol~

j j

5 10 6790~==~==~=====;~~====~~==~==~~~~====t=========

o 15 Time (min)

Fig. 1. Typical HPLC analysis of D. salina J3-carotene extract.

high temperature, high salinity and deficiency of nitrogen source (Lerche, 1937; Mil'ko, 1963; Mironyuk & Einor, 1968; Semenko & Abdullayev, 1980; Ben-Amotz & Avron; 1983; Borowitzka et at., 1984). p-carotene contents of up to 14% of dry weight have been reported for D. salina (Ben-Amotz et at., 1982; Borowitzka et at., 1984). The p-carotene does not appear to act as a light-harvesting photo-accessory pigment, but rather as a photo-protective 'sunscreen' to the chlorophyll and cell DNA in the high light intensities which characterise the normal salt-lake environments where this alga grows (MacKinney & Chichester, 1960; Ben-Amotz, 1980). Borowitzka & Borowit­zka (1988a) have also proposed that p-carotene acts as a 'carbon sink' in D. salina for excess combined carbon produced when photosynthesis continues under conditions where growth is limited.

The p-carotene in the chloroplast droplets is a mixture of the cis- and trans-isomers. A typical analysis from D. salina (also called D. bardawil) gives the following composition as percentages of total p-carotene: 15-cis-p­carotene, 10%; 9-cis-p-carotene, 41%; all-trans-p-carotene, 42%; other iso­mers, 6% (Ben-Amotz et at., 1982).

For analytical purposes, total p-carotene may be calculated from its extinction in acetone extracts (m~::. at 452 nm is 2592; Bauernfeind, 1981). HPLC is required to separate the isomers, and to characterise the small quantities of other carotenoids also present in typical D. salina extracts (Nelis & de Leenheer, 1983). Figure 1 shows an HPLC analysis of a typical commercial batch of 20% p-carotene suspension in peanut oil produced by Western Biotechnology Limited from D. salina.

3 STRAIN IMPROVEMENT, SELECTION AND MAINTENANCE Dunaliella salina production systems, like those used by Western Biotechnol­ogy Ltd (Fig. 2), have large open ponds situated in, or near, salt lakes or solar

{3-Carotene Production with Algae 17

Fig. 2. Aerial photographs of Western Biotechnology's Dunaliella production facility at Hutt Lagoon, WA, Australia.

salt works. These ecosystems and the ponds are inhabited by wild-type D. salina, non-carotenogenic Dunaliella species, predatory protozoa, halophilic bacteria and brine shrimp (Borowitzka et al., 1986; Moulton et al., 1987b). In the presence of this mixture of organisms, maintenance of cultures dominated by the carotenogenic D. salina is the first priority of pond management. In an environment dominated by the wild-type D. salina, introduction of an

18 L. J. Borowitzka & M. A. Borowitzka

'improved' D. salina strain is extremely difficult, and has not been reported on a commercial scale. In the large, open-air ponds used for the commercial culture of D. salina the major control of the culture available to the operator is salinity (Borowitzka et al., 1986), although some additional control may be exercised by manipulating nutrient concentrations.

In more isolated systems, as for example, concrete or plastic-lined raceway ponds and tubular photobioreactors, wild-type D. salina and other competing organisms may be excluded and the introduction of improved strains can be achieved, at the cost of higher investment in plant and equipment.

Strain improvement, both in terms of growth characteristics and product yield, can be achieved using mutagenesis and selection programmes. Improved strain breeding using mating strains as in Chlamydomonas, a closely related alga, is also possible (Craig et al., 1988). Further into the future, and awaiting additional research, is the use of cell fusion techniques or the genetic engineering manipulations developed for yeast cells (e.g. Spencer & Spencer, 1983).

In the laboratory, D. salina may be isolated into unialgal and axenic culture either by repeated streaking on agar plates or by isolating clonal cultures from single cells. The most commonly used medium for D. salina and related algae is modified Johnson's medium (Borowitzka, 1988), although a range of other media can also be used, as long as the salinity is appropriately adjusted with NaCI.

Laboratory cultures are usually maintained on agar slants or in liquid cultures, and sub-cultured every 1-2 months. Care must be taken to avoid excessive drying out of the high salinity media. We have also successfully maintained Dunaliella species, frozen in liquid nitrogen in our laboratory for periods in excess of 12 months. In the field, stock cultures are best maintained at saturating salinities where the likelihood of contamination by other organisms is minimised.

4 BIOSYNTHESIS AND REGULATION

There is an inverse relationship between growth rate and carotenogensis in D. salina; conditions which enhance accumulation of p-carotene include high light intensity, high temperature, high salinity, nitrogen deficiency, and these same conditions tend to decrease growth rate. This is a typical pattern for secondary metabolite production in stationary phase cultures of algae. Table 1 sum­marises the environmental factors which influence growth and carotenogenesis in D. salina.

The effect of salinity on carotenogenesis is best shown in experiments which investigated the induction of carotenogenesis. In these experiments the NaCI concentration was increased from 15 to 25% (w/v) and the p-carotene content of the D. salina cells increased linearly from <10 to 260 mg (g cell protein)-t, over 5 days, while growth ceased (Borowitzka et al., 1984).

fJ-Carotene Production with Algae

Table 1 Summary of the Influence of Various Environmental Factors on the Biomass and Jj-Carotene Content of Dunaliella salina. + =

Stimulating Effect; - = Inhibitory Effect; 0 = No Effect

Factor Biomass Jj-carotene

Increase in salinity +++ Decrease in salinity + (-t N deficiency + P deficiency + Increase in [C02] supply + 0 Increase in light 0 ++++ Decrease in light 0 Increase in temperature 0 + Decrease in temperature Increase in [02] 0

a Rate of decrease is very slow.

19

Fortuitously, if salinity is reduced by a similar amount, as for example when rain enters open ponds, the decline in p-carotene content of the cells is very slow (Borowitzka et al., 1984) as the p-carotene is not broken down, but rather the cell content is reduced as the cells divide.

5 BIOMASS PRODUCTION

Open-pond D. salina production is perhaps more closely related to farming than to fermentation techniques. Open cultures are influenced by weather (temperature, light, rainfall, wind), seasons, and input of contaminants from the environment. Enclosed ponds, tank or tubular photobioreactors and fermentor systems can reduce the influence of these external factors on the culture; however, these have, as yet, not been applied to D. salina culture on a commercial scale.

Dunaliella salina cultures may be operated either in batch or continuous mode. A two-stage process, using lower salinity conditions for optimum growth rate and biomass production and then changing to high salinity stress conditions favouring carotenogenesis has also been piloted and the disadvan­tages of this two-stage process are discussed in Borowitzka et al. (1984). A single-stage, semi-continuous process at an intermediate salinity appears to be the best at this stage, and this is the process used by Western Biotechnology Ltd (Fig. 3).

In all D. salina culture systems the following are necessary for good growth and carotenogenesis:

(a) Light. Light is the only energy source for metabolism as D. salina is an obligate photoautotroph. The light source may be sunlight or high intensity artificial white light. Growth and carotenogenesis respond

20

H SALINITY BRINE HIG

EV APORATIVE MAKE-UP

CULUM INO

NU TRIENTS

I HOLDING PONDS

l

L. 1. Borowitzka & M. A. Borowitzka

• •

GROWTH PONDS • (50 Ha)

•

1 I I HARVESTERS I

1 1

CULTURE MEDIUM BIOMASS (BRINE)

I 1

DRY EXTRACT fJ-CAROTENE

! CONCENTRATE

1 PURIFY

1 MILL

1 Dunaliella salina FORMULATION

powder containing 3% fJ-Carotene Suspensions in

Vegetable oil (2%, 10%,20%,30%)

Water Dispersibles Water Solubles

Final --- -- ..... Quality control

PACKAGING J PACKAGING J Fig. 3. Flow chart of the Dunaliella salina fJ-carotene production process used by

Western Biotechnology Ltd.

fJ-Carotene Production with Algae 21

differently to particular wavelengths of light (Mil'ko, 1963), and high photon-flux densities favour fj-carotene accumulation.

(b) Temperature. Dunaliella salina is tolerant of a wide range of tempera­tures for growth, from below O°C (Siegel et al., 1984) to above 40°C (Wegmann et al., 1980). The optimum growth temperature lies between 21 and 40°C, and is dependent on the strain and light intensity (Gibor, 1956; Federov et al., 1968; Borowitzka, 1981). Temperatures close to 40°C and higher stimulate carotenogenesis at the expense of growth (Mil'ko, 1963). In practice, the combination of light intensities, day length and temperatures between 30 and 40°C give optimum growth and fj-carotene production at Western Biotechnology's Australian plant in the summer months, although growth and fj-carotene production are sustained throughout the year.

(c) Carbon source. Dunaliella salina is a photoautotroph; it can use only CO2 and possibly bicarbonate (HC03) as carbon source. Growth of D. salina is stimulated by addition of NaHC03 or bubbling with COz, provided there is no precipitation of carbonates (Massyuk, 1965; Drokova & Dovhorouka, 1966). Limited availability of inorganic carbon (C02 and HC03) is the most common growth limiting factor in the high salinity, high pH and temperature conditions used for D. salina culture. Inorganic carbon equilibria and uptake by D. salina under typical growth conditions are discussed by Borowitzka & Borowitzka (1988a).

(d) Nitrogen and phosphorus sources. The best nitrogen source for D. salina is NaN03 (Mil'ko, 1962; Grant, 1968); ammonium salts, urea and nitrites are less effective (Gibor, 1956; Mil'ko, 1962; Borowitzka & Borowitzka, 1988b). Care must be taken to maintain culture pH; despite natural buffering conferred by the inorganic carbon equilibrium (CO~-~HC03~C02)' nitrate uptake can cause alkalinisation, and ammonium uptake acidification of the growth medium, leading to cell death (Borowitzka & Borowitzka, 1988b). The optimum phosphate concentration for growth is approximately 0·02 j.lg litre- 1 K2HP04

(Gibor, 1956); higher concentrations can inhibit growth. (e) NaCI. All known marine and halophilic species of Dunaliella have a

specific requirement for sodium; D. salina has the broadest reported NaCI tolerance range, from 1 to 35% (w/v) NaCI (saturation) (Loeblich, 1972). Selection of the appropriate NaCI concentration for the commer­cial culture depends on growth or carotenogenesis rate required, and the need (if any) to control predators, competitors or bacterial numbers (Borowitzka et al., 1986). Normal salinities used for growth range from 20 to 30% (w/v) NaCI, i.e. about 9-10 times the salinity of sea-water.

(f) Other requirements. Dunaliella salina requires Mg2+, Ca2+, Na+, CI-, SO~-, chelated iron and trace elements for growth. Optimum pH is between 7 and 9. A discussion of the literature reports on concentration and ratios of these components is given in Borowitzka and Borowitzka (1988a). No requirement for vitamins has been demonstrated.

22 L. 1. BOTowitzka & M. A. BOTowitzka

6 PRODUCT RECOVERY AND PURIFICATION

Commercial D. salina production is a young industry, only 3 years old, and companies in development and production phases guard the confidentiality of their processes. In particular, because harvesting and extraction represent major cost areas, technical advantages in these areas are eagerly sought and strongly guarded. Harvesting D. salina is more difficult and costly compared to most commercial algae. Dunaliella salina is a single cell, with no protective cell wall, approximately 20 x 10 /lm in size, and neutrally buoyant in a high specific gravity, high viscosity brine. Cell densities in large cultures tend to be about 1 g litre- 1 and very large volumes therefore have to be processed. Efforts to centrifuge or filter the algae from the brine generally shear-damage the cells leading to tJ-carotene loss by oxidisation. The cells also distort and pass through filters with pore sizes less than 10 /lm. Corrosion of all metal equipment by the brine is also a major problem.

These problems have led to development and patenting of several biological and chemical methods for harvesting the cells, or for pre-concentration of cells prior to use of more conventional harvest methods. Patents issued for Dunaliella harvesting include high pressure filtration using diatomaceous earth (Ruane, 1974a), exploitation of salinity-dependent buoyancy properties in stationary and moving gradients (Bloch et al., 1982), exploitation of the phototactic and gyrotactic responses of the algae (Kessler, 1982, 1985) salinity-dependent hydrophobic adhesion properties of Dunaliella cells (Cur­tain & Snook, 1983) and flocculation (Sammy, 1987). The harvesting of micro algae is reviewed by Mohn (1988).

The process for extraction of tJ-carotene from the biomass depends in part on the harvesting procedure used, and on the market requirements. Extraction using conventional organic solvents is efficient (Ruane, 1974b), but may not be acceptable to customers seeking a 'natural' product. More acceptable alterna­tive extraction methods use hot vegetable oil (Nonomura, 1987; Potts, 1987) or liquid or supercritical gas solvents.

Harvested biomass may be dried, (i.e. spray-drying) rather than extracted, and is marketed as a tJ-carotene-rich supplement for human health or animal feed (Ben-Amotz et al., 1986; Avron et al., 1987; Curtain et al., 1987).

By-products of tJ-carotene production could include glycerol, which can make up 30% of biomass dry weight (see Chen & Chi, 1981, for process proposal and cost analysis) and high quality protein meal remaining after extraction. The amino acid analysis of several Dunaliella spp is summarised in Table 2.2. in Borowitzka and Borowitzka (1988a).

7 BIOLOGICAL PROPERTIES

tJ-Carotene is marketed mainly as an orange colouring, for foods such as margarine, baked goods, and beverages (KUiui, 1981). A smaller market exists

fJ-Carotene Production with Algae 23

for use of {:1-carotene as a pigment supplement in the commercial feeds for crustaceans, such as prawns, which convert it to astaxanthin, the natural red colouring of the shell and flesh.

{:1-Carotene is provitamin A, and provides the main source of vitamin A in human diets. More recently, interest has increased in the role played by {:1-carotene as an anti-oxidant in human nutrition and correlations have been reported between high dietary {:1-carotene intake and low incidences of some cancers (Mathews-Roth, 1982; Schwartz et aZ., 1986). Large-scale trials are underway in the US (National Cancer Institute) and in Europe to confirm the 'anti-cancer' activity of {:1-carotene.

Trials with cattle have also indicated an increase in fertility attributable to supplements of {:1-carotene.

8 CHEMICAL SYNTHESIS

The first commercial synthesis of f3-carotene was established by Hoffman-La Roche in 1956. Since then, the process has been improved by Roche and other companies; patents describe the key steps in the synthesis. Syntheses of {:1-carotene are reviewed by Mayer & Isler (1971).

9 FORMULA nONS AND APPLICA nONS

{:1-Carotene is a lipid- and oil-soluble product. As suspensions or solutions in vegetable oil (Fig. 2) it finds applications in colouring margarine, baked goods and some prepared foods. Conversion to water-soluble or water-dispersible formulations, by forming emulsions or microencapsulated beadlets extends the food applications to beverages including orange drinks, and to confectionery and further prepared foods.

Nutritional supplements can be prepared by encapsulation of oil suspensions or solutions, or by tableting using beadlet forms. Current recommended daily doses for {:1-carotene are around 6 mg; 20-25 mg doses are in use in the 'anti-cancer' trials in the US and Finland. Dried Dunaliella powder is also prepared for use in animal feed supplements.

10 ECONOMICS

Just as the companies in the new industry guard the confidentiality of their processes, costs of commercial production are not available. Moulton et aZ. (1987a), however, present a bioeconomic model for the {:1-carotene production process using D. salina.

Synthesised {:1-carotene is sold for a minimum of US$300 per kg {:1-carotene, and at higher prices depending on the formulation. 'Natural' {:1-carotene

24 L. I. Borowitzka & M. A. Borowitzka

commands higher prices, with the highest price attainable for the nutritional supplement application. Market price for 'natural' IJ-carotene is volatile and in 1987-88 world market demand exceeds supply.

If a price range for formulations of natural IJ-carotene of US$500-1000 is assumed, and IJ-carotene represents 10% of D. salina biomass, production costs for D. salina should not exceed US$50-100 per kg. In fact, costs must be significantly lower, to account for losses at each processing step, capital expenses, marketing, packaging and distribution costs.

REFERENCES

Aasen, A. J., Eimhjellen, K. E. & Liaaen-Jensen, S. (1969). An extreme source of p-carotene, Acta Chim. Scand., 23,2544-5.

Avron, M., Ben-Amotz, A. & Edelstein, S. (1987). Feed supplement. UK patent application 2 189 675A.

Bauernfeind, J. C. (ed.) (1981). Carotenoids as Colourants and Vitamin A Precursors. Academic Press, New York, pp. 938.

Ben-Amotz, A. (1980). Glycerol production in the alga Dunaliella. In Biochemical and Photosynthetic Aspects of Energy Production, ed. A. San Pietro. Academic Press, New York, pp. 191-208.

Ben-Amotz, A. & Avron, M. (1982). The potential use of Dunaliella for the production of glycerol, p-carotene and high protein feed. In Biosaline Research: A Look to the Future, ed. A. San Pietro. Plenum Publishing Corporation, New York, pp. 207-14.

Ben-Amotz, A. & Avron, M. (1983). On the factors which determine the massive p-carotene accumulation in the halotolerant alga Dunaliella salina. Plant Physiol., 72,593-7.

Ben-Amotz, A., Katz, A. & Avron, M. (1982). Accumulation of p-carotene in halotolerant algae: purification and characterisation of p-carotene-rich globules from Dunaliella bardawil (Chlorophyceae). J. Phycol., 18,529-37.

Ben-Amotz, A., Edelstein, S. & Avron, M. (1986). Use of the p-carotene rich alga Dunaliella bardawil as a source of retinol. Br. Poult. Sci., 27,613-9.

Bloch, M. R., Sasson, J., Ginzburg, M. E., Goldman, Z., Ginzburg, B. Z., Garti, N. & Perath, A. (1982). Oil products from algae. US patent no. 4.341 038.

Borowitzka, L. J. (1981). The microflora. Adaptions to life in extremely saline lakes. Hydrobiologia, 81, 33-46.

Borowitzka, M. A. (1988). Algal growth media and sources of algal cultures. In Micro-algal Biotechnology, ed. M. A. Borowitzka & L. J. Borowitzka, Cambridge University Press, Cambridge pp. 456-65.

Borowitzka, L. J., Borowitzka, M. A. & Moulton, T. P. (1984). The mass culture of Dunaliella salina for fine chemicals: from laboratory to pilot plant. Hydrobiologia, 116/117, 115-2l.

Borowitzka, L. J., Moulton, T. P. & Borowitzka, M. A. (1986). Salinity and the commercial production of beta-carotene from Dunaliella salina. Nova Hedwigia, Beih., 83, 224-9.

Borowitzka, M. A. & Borowitzka, L. J. (1988a) Dunaliella. In Microalgal Biotechnology, ed. M. A. Borowitzka & L. J. Borowitzka. Cambridge University Press, Cambridge, pp. 27-58.

Borowitzka, M. A. & Borowitzka, L. J. (1988b) Limits to growth and carotenogenesis in laboratory and large-scale cultures of Dunaliella salina. In Algal Biotechnology, ed. T. Stadler, J. Mollion, M-C. Verdus, Y. Karamanos, H. Morvan & D. Christiaen. Elsevier Applied Science Publishers, Barking, pp. 371-82.

fJ-Carotene Production with Algae 25

Chen, B. J. & Chi, C. H. (1981). Process development and evaluation for algal glycerol production. Biotech. Bioeng., 23, 1267-87.

Craig, R., Reichelt, B. Y. & Reichelt, J. L. (1988). Genetic engineering of micro-algae. In Micro-algal Biotechnology, ed. M. A. Borowitzka & L. J. Borowitzka. Cambridge University Press, Cambridge, pp. 415-55.

Curtain, C. C. & Snook, H. (1983). Method for harvesting algae. US patent no. 511 135.

Curtain, C. c., West, S. M. & Schlipalius, L. (1987). Manufacture of p-carotene from the salt lake alga Dunaliella salina; the scientific and technical background. Aust. J. Biotechnol., 1, 51-7.

Drokova, I. G. & Dovhorouka, S. I. (1966). Carotene-formation in Dunaliella salina Teod. under the effect of some carbon sources. Ukransk. Bot. Zhour., 21, 59-62.

Federov, V. D., Maksimov, V. N. & Kromov, V. M. (1968). Effect of light and temperature on primary production of certain unicellular green algae and diatoms. Fiziol. Rast., 15, 640-5l.

Gibor, A. (1956). The culture of brine algae. Bioi. bull., Woods Hole, 3,223-9. Grant, B. R. (1968). The effect of carbon dioxide concentration and buffer system on

nitrate and nitrite and assimilation by Dunaliella tertiolecta, J. Gen. Micro., 54, 327-36.

Kessler, J. O. (1982). Algal cell harvesting. US patent no. 4324067. Kessler, J. O. (1985). Hydrodynamic focusing of motile algal cells. Nature, 313,

208-10. KI1tui, H. (1981). Industrial and commercial uses of carotenoids. In Carotenoid

chemistry and Biochemistry, ed. G. Britton & T. W. Goodwin. Pergamon Press, Oxford, pp. 309-28.

Klausner, A. (1986). Algaculture: Food for thought. Biotechnology, 4,947-53. Lerche, W. (1937). Untersuchungen iiber die Entwicklung und Fortpflanzung in der

Gattung Dunaliella. Arch. fur Protistenk., 88, 236-9. Loeblich, L. A. (1972). Studies on the brine flagellate Dunaliella salina. PhD thesis,

University of California, San Diego. MacKinney, G. & Chichester, C. O. (1960). Biosynthesis of carotenoids. In

Comparative Biochemistry of Photoreactive Systems, ed. M. B. Allen. Academic Press, New York, pp. 205-15.

Massyuk, N. P. (1965). Carbonate and bicarbonate as stimulators of growth and carotene accumulation in Dunaliella salina Teod. Ukransk. Bot. Zhour., 22, 18-22.

Massyuk, N. P. (1966). Mass culture of the carotene-bearing alga Dunaliella salina Teod. Ukransk. Bot. Zhour., 23, 12-19.

Massyuk, N. P. & Abdula, E. G. (1969). First experiment of growing carotene­containing algae under semi-industrial conditions. Ukransk. Bot. Zhour., 26,21-7.

Mathews-Roth, M. M. (1982). Medical applications and uses of carotenoids. In Carotenoid Chemistry and Biochemistry, ed. G. Britton & T. W. Goodwin. Pergamon Press, Oxford, pp. 297-307.

Mayer, H. & Isler, O. (1971). Total syntheses. In Carotenoids, ed. O. Isler. Birkhauser, Basle, pp. 328-575.

Mil'ko, E. S. (1962). Study the requirement of two Dunaliella spp. in mineral and organic components if the medium, Moscow University Vestnik. Biologya, 6, 21-3.

Mil'ko, E. S. (1963). Effect of various environmental factors on pigment production in the alga Dunaliella salina. Mikrobiologiya, 32,299-307.

Mironyuk, V. I. & Einor, L. O. (1968). Oxygen exchange and pigment content in various forms of Dunaliella salina Teod. under conditions of increasing NaCl content. Gidrobiol Zhournal, 4,23-9.

Mohn, F. H. (1988). Harvesting of micro-algal biomass. In Micro-algal Biotechnology, ed. M. A. Borowitzka & L. J. Borowitzka. Cambridge University Press, pp. 395-414.

26 L. J. Borowitzka & M. A. Borowitzka

Moulton, T. P., Borowitzka, L. J. & Vincent, D. J. (1987a) The mass culture of Dunaliella salina for p-carotene: from pilot plant to production plant, Hydrobiologia, 151/152,99-105.

Moulton, T. P., Sommer, T. R., Burford, M. A. & Borowitzka, L. J. (1987b). Competition between Dunaliella species at high salinity. Hydrobiologia, 151/152, 107-16.

Nelis, H. J. C. F. & De Leenheer, A. P. (1983). Isocratic nonaqueous reversed-phase liquid chromatography of carotenoids. Analyt. Chem., 55,270-75.

Nonomura, A. M. (1987). Process for producing a naturally derived carotene-oil composition by direct extraction from algae. US patent no. 4,680,314.

Potts, W. T. (1987). Extraction of carotenoid pigments from algae. Australian patent application no. 69260/87.

Rich, V. (1978). Israel's place in the sun. Nature, 275, 581-2. Ruane, M. (1974a). Recovery of algae from brine suspensions. Australian patent no.

486999. Ruane, M. (1974b). Extraction of caroteiniferous materials from algae. Australian

patent no. 487 018. Sammy, N. (1987). Method for harvesting algae. Australian patent application no.

70924/87. Schwartz, J., Suda, D. & Light, G. (1986). Beta-carotene is associated with the

regression of hamster buccal puch carcinoma and induction of tumor necrosis factor in macrophages. Biochem. biophys. Res. Commun., 136, 1130-5.

Semenko, V. E. & Abdullayev, A. A. (1980). Parametric control of p~carotene biosynthesis in Dunaliella salina cells under conditions of intensive cultivation. Fiziol. Rast., 27,31-41.

Siegel, B. Z., Siegel, S. M., Speitel, T., Waber, J. & Stoeker, R. (1984). Brine organisms and the question of habitat-specific adaption. Origins of Life, 14, 757-70.

Spencer, J. F. T. & Spencer, D. M. (1983). Genetic improvements of industrial yeasts. Ann. Rev. Microbiol., 37, 121-42.

Teodoresco, E. C. (1905). Organisation et developpement du Dunaliella nouveau genre de Volvocacee-Polyblepharidee. Bot. Zentralblatt, Beih., 18,215-32.

Wegmann, K., Ben-Amotz, A. & Avron, M. (1980). Effect of temperature on glycerol retention in the halotolerant algae Dunaliella and Asteromonas. Plant. Physiol., 66, 1196-7.

Chapter 3

PRODUCTION OF CAROTENOIDS WITH FUNGI

E. CERDA-OLMEDO

Departamento de Genetica y Biotecnia, Universidad de Sevilla, Apartado 1095, E-41080 Sevilla, Spain

1 INTRODUCTION

Humans, like most other animals, need carotenoids but cannot synthesize them. Our main suppliers are the fruits and vegetables, with lesser, mostly indirect contributions from fungi, algae, and bacteria. Carotenoid production is rarely the main objective of a culture, but colourful products rich in carotenoids are obtained from various plants and algae. The oldest example is saffron (Crocus sativus) , still planted in La Mancha, Spain, and elsewhere for the stigmas of its flowers. Many fields of central Mexico become bright orange in the summer with the flowers of cempasuchil (Tagetes ten uifolia , a kind of marigold), whose dried petals are fed to chicken for meat and egg yolk colour. Annatto is extracted from the seeds of a tropical tree, Bixa orellana. The most familiar carotenoid-rich product is probably paprika, a powder made by grinding red peppers (Capsicum annuum).

The carotenoids may be scarce in the diets of humans and animals for various reasons. Many staple foods and feeds, including most of the cheap sources of carbohydrates and fat, are poor in carotenoids, as witnessed by their lack of colour. The long winters of many countries halt the natural production of carotenoids, and these are relatively unstable and may not survive in stored foods and feeds.

Organic chemists have devised several complete syntheses, which now supply considerable amounts of f3-carotene and other purified carotenoids. The current trend towards natural food colours and the discovery of salutary effects of the carotenoids, beyond their important role as provitamin A, stimulate the demand and press towards a biological production.

The fungi can hardly be considered as traditional food colourants, but the relative ease of cultivation and the many possibilities for physiological, genetical, and industrial manipulations make them attractive to biotech­nologists. Several species are potential sources of carotenoids, and a large­scale process has been based on Blakeslea trispora.

27

28 E. Cerda-Olmedo

It seems that the Soviet Union is the only country that uses Blakeslea trispora for the industrial production of carotenoids; the yearly production is estimated at about 200 kg purified fJ-carotene and another 4 Mg fJ-carotene in the form of mycelia used as an additive to animal feed (A. A. Dmitrovskii, pers. comm., 1988).

The abundant literature on carotenoids has been the subject of many books and reviews. The monumental and indispensable treatise edited by Isler (1971) is complemented by newer reviews (Feofilova, 1974; Goodwin, 1976, 1980, 1988). The practical aspects are explained in great detail by Bauernfeind (1981). Updates are provided by the International Symposia on Carotenoids, published every 3 years as a special issue of Pure and Applied Chemistry. There are specialized and recent reviews on the influence of external factors on carotenogenesis in the Mucorales (Lampila et al., 1985a), on Phycomyces (Cerda-Olmedo & Lipson, 1987), on the regulation of carotenogenesis (Bramley & Mackenzie, 1988), on the metabolism and metabolic effects of the carotenoids (Goodwin, 1986), and many other subjects.

2 FUNGAL CAROTENOIDS

Most fungi produce no carotenoids at all and many of the others contain a single major carotenoid. Although the pathway intermediates may be present in amounts widely variable with the strain and the culture conditions, the carotenoid composition of the fungi is usually much simpler than that of the photosynthetic organisms. Here are a few selected examples of fungal carotenoids. fJ-Carotene predominates in the Mucorales Blakeslea trispora, Phycomyces blakesleeanus and Choanephora cucurbitarum, in the yeast Rhodotorula aurea, in Aspergillus giganteus (EI-Jack et al., 1988) and various species of Penicillium; neurosporaxanthin is the end-product of Fusarium aquaeductuum (Bindl et al., 1970); neurosporaxanthin and fJ-carotene, those of Gibberella fujikuroi (Avalos & Cerda-Olmedo, 1987), Neurospora crassa and Verticillium agaricinum (Valadon & Mummery, 1973); torularhodin and fJ-carotene, those of the yeasts Rhodotorula rubra, R. minuta, and Rhodosporidium diobovatum; fJ-carotene, lycopene, and fJ-zeacarotene are found in different strains of the smut Ustilago violacea (Will et al., 1984); canthaxantin in the mushroom Cantharellus cinnabarinus; astaxanthin, in the yeast Phaffia rhodozyma. See Goodwin (1980) for other fungi and additional references.

Figure 1 shows the structure of the carotenoids that have just been mentioned, with their likely biosynthetic pathways. The carotenoids, like all terpenoids, are synthesized from hydroxymethylglutaryl-coenzyme A, which is first converted to mevalonic acid. The specific part of the pathway begins with the condensation of two molecules of geranylgeranyl pyrophosphate to form phytoene, a colourless carotene. Four dehydrogenations transform phytoene into lycopene, the pigment of red tomatoes. Two cyclisations convert lycopene

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30 E. Cerda-Olmedo

into p-carotene. In Phycomyces these reactions are carried out by an enzyme aggregate that contains four copies of a dehydrogenase and two copies of a cyclase (review, Cerda-Olmedo, 1987). In Giberellafujikuroi the production of torulene requires a fifth dehydrogenation by the same enzyme; the final break and oxidation to neurosporaxanthin appear to be carried out in a single step (Avalos & Cerda-Olmedo, 1987). On the other hand, several dehydrogenases have been postulated for Ustilago violacea (Will et al., 1984). The detection of some minor intermediates indicates that the order of the reactions is sometimes altered in these organisms, and may be different in others. Canthaxantin, a likely intermediate in the biosynthesis of astaxanthin in the crustaceans (Castillo, 1980), was not detected in Phaffia rhodozyma (Andrewes et al., 1976).

Many carotenoids found in plants and algae are unknown in the fungi, but most of the fungal carotenoids are also found in plants or algae, and this is fortunate for fungal biotechnology, because the traditional ingredients of our diet are more acceptable to the health authorities and to the consumers than new products. A few carotenoids are exclusive of certain fungi: torulene, neurosporaxanthin, torularhodin, plectaniaxanthin, phillipsiaxanthin, p-y­carotene. The astaxanthin from Phaffia rhodozyma has the opposite chirality (3R, 3' R) to that of the astaxanthin responsible for the admired colours of salmon and lobster (Andrewes & Starr, 1976).

Research towards industrial production has concentrated on the mucorales Blakeslea trispora and Phycomyces blakesleeanus, whose main and nearly only carotenoid is all-trans p-carotene. Commercial advantages of p-carotene are its colour, its high provitamin A activity, its wide distribution in nature, and its many current applications. A disadvantage is its inability to pigment some avian and fish products.

3 NATURAL VARIATIONS IN CAROTENOID CONTENT

Many fungi attracted the attention of carotenoid chemists because of their bright colours, but the strains used in the laboratories may not be optimal for carotenoid production.

Thus, an impressive polymorphism was found in natural isolates of Ustilago violacea (Garber et al., 1978). Fourfold differences in carotenogenesis were found in a few natural isolates of Phycomyces blakesleeanus (Goodwin & Griffiths, 1952). A large systematic search for better starting strains should be rewarding, as it was for many other biological products.

The carotenoid concentrations are usually too low for commercial applica­tion. The accumulation of carotenoids in the fungi is influenced by many environmental variables; their study is a first step in yield improvement. While so doing, one should bear in mind that the fungi are very distantly related organisms, that the regulations of carotenogenesis are not universal, and therefore, that observations on a species need not apply to others.

Production of Carotenoids with Fungi 31

3.1 Light

Blue illumination stimulates the production of carotenoids in many fungi, as it does in various bacteria, algae, and plants (reviews, Harding & Shropshire, 1980; Rau, 1983; Rau & Schrott, 1987). This phenomenon (photo­carotenogenesis) does not occur in many fungi, for example, in Blakeslea trispora (Sutter, 1970).

Photocarotenogenesis presumably protects the cells against the deletereous effects of very strong illuminations and avoids waste in the dark or in dim light, but is of limited interest to biotechnologists, because bright illumination is not practical in large cultures and the final concentrations of carotenoids are not very high. The examples given in Table 1 prove this point, but should not be compared with each other, because they were obtained under very different conditions, most under non-saturating light exposures.

3.2 Sexual Interaction

In many species of the Mucorales the contact zone of mycelia of opposite sex becomes bright yellow (Blakeslee, 1904). The sexual stimulation is easily observed in mated cultures (mixed cultures of strains of opposite sex) (Barnett et al., 1956; Anderson et al., 1958). The beta-carotene content increases 5-15 times over the level found in separate cultures, according to various reports.

Sexual stimulation in the Mucorales occurs in the absence of physical contact when the interacting hyphae are separated by a membrane (Burgeff, 1924). An enhanced carotenogenesis occurs in strains of Blakeslea trispora grown under such conditions (Barnett et al., 1956). A race between chemists of several major biotechnological companies led to the isolation of chemicals, the trisporic acids, present in mated cultures and able to stimulate carotenogenesis in single cultures (Caglioti et al., 1966). The trisporic acids are industrially useless because the increases in p-carotene content do not compensate their cost.

Table 1 Examples of Photocarotenogenesis in some Fungi

Name of fungus

Aspergillus giganteus Fusarium aquaeductuum Gibberella fujikuroi Neurospora crassa Phycomyces blakesleeanus Rhodosporidium diobovatum Rhodotorula minuta Verticillium agaricinum

Coloured carotenoids in illuminated cultures

(Ilg/gt Main component

170 tJ-Carotene 213 ~eurosporaxanthin 159 ~eurosporaxanthin 152 ~eurosporaxanthin 550 tJ-Carotene 700 tJ-Carotene 150 Torulene 445 Torulene

Reference

EI-Jack et al., 1988 Bindl et al., 1970 Avalos & Cerda-Olmedo, 1987 Harding et al., 1969 Bergman et al., 1973 Vaskivnyuk & Getman, 1984 Tada & Shiroishi, 1982 Valadon & Mummery, 1973

a The numerical values are the total concentrations of the carotenoids detected in illuminated cultures, in Ilg/g dry mycelial weight.

32 E. Cerd4-01medo

Since trisporic acids are made from p-carotene (Austin et a/., 1970), the activation of carotenogenesis by trisporic acids in the Mucorales provides a regulatory loop for increased sexual stimulation. Another likely role of the newly synthesized p-carotene is to make sporopollenins, tough polymers found in the cell walls of the zygospores (Gooday et a/., 1973) and of other structures. Lack of trisporic acids and sporopollenins explains the sexual impotence of mutant Mucorales devoid of p-carotene (Sutter, 1975; Khabrova & Zhdanov, 1979), but not that of the carS mutants, which overproduce it. The trisporic acids have no effect on carotenogenesis or morphogenesis in fungi other than the Mucorales.

3.3 Culture Conditions

The accumulation of carotenoids is influenced by all sorts of variations in the culture conditions, but usually only to a minor extent. The best temperatures for carotenogenesis in B/akeslea trispora and Phycomyces b/akes/eeanus are about 26°C. Acetate, leucine, and other amino acids increase the p-carotene content of Phycomyces, presumably because they can be converted directly to hydroxymethylglutaryl-coenzyme A (Lilly et al., 1960; Cerda-Olmedo, 1987). Many of the conditions tried by Lilly and co-workers allowed Phycomyces to exceed 2 mg p-carotene per g dry weight.

The optimization of the media for the industrial production of p-carotene with B/akes/ea trispora was the central concern of a series of successful investigations (Anderson et a/., 1958; Ciegler et al., 1959a, b, 1962). The best media are rich in various grain products, vegetable oils, and hydrocarbons (kerosene, preferably deodorized).

Other fungi have received much less attention. A medium with tomato pressings increased the astaxanthin content of Phaffia rhodozyma to 0·8 mg/g (Johnson & Lewis, 1979). An appropriate carbon-to-nitrogen ratio and a surface-active agent allowed Neurospora crassa to make over 2·5 mg caroten­oids per g dry weight (Krzeminiski & Quackenbush, 1960).

4 CHEMICAL STIMULATION

Mycelial colour changes facilitate the detection of chemicals that influence carotenogenesis in the fungi. The many inhibitors and the weak activators do not interest us now. The accumulation of p-carotene in the Mucorales is stimulated by p-ionone (Mackinney et a/., 1952, 1953; Reyes et a/., 1964) and retinol (Eslava et a/., 1974; Feofilova & Bekhtereva, 1976). These molecules are identical to p-carotene in the p-ring and the adjacent atoms, but they are not incorporated into it (Engel et a/., 1953; Eslava et a/., 1974). Some variations of these structures are active, and others are not (Mackinney et a/., 1952; Engel et a/., 1953; De la Concha & Murillo, 1984; Bejarano et a/., 1988).

p-Ionone and retinol are kept away from industrial fermentors by their high

Production of Carotenoids with Fungi 33

prices. In the production of fJ-carotene by Blakeslea trispora, fJ-ionone may be replaced by citrus pulps (alkali-heated peels, seeds, and rags) (Ciegler et al., 1963b) and by limonene and citrus oils that contain limonene (Ciegler et al., 1963c).

Many aromatic compounds activate carotenogenesis in Phycomyces. These compounds stimulate the pathway and inhibit phytoene dehydrogenase to different extents. Predominance of the stimulation (observed with dimethyl phthalate, 1,2-dimethoxy-4-propenylbenzene, 4-bromoveratrol, veratrol, and others) results in vast accumulations of fJ-carotene. Mixtures of fJ-carotene and partially desaturated acyclic intermediates result when both activities are prominent (cinnamic alcohol, eugenol, safrol, 1,3-dimethoxybencene, 4-methoxyphenol, etc.) (Cerda-Olmedo & Hllttermann, 1986). These intermedi­ates are undesirable in the industrial production of fJ-carotene, but may be of interest for research and other uses.

The simple comparison of the structural formulae is insufficient to establish functional analogy between chemical activators. A genetic test for functional analogy has been made possible by the availability of many mutants of Phycomyces blakesleeanus with various defects in carotene synthesis and its regulation. If mutants of a certain gene are activated by a chemical but not by another, the mechanisms of action of the two chemicals differ in their need for the product of that gene. The activators were thus classified into four groups: light, trisporic acids, fJ-ring containing chemicals (retinol, fJ-ionone), and phenols (dimethyl phthalate, veratrol) (Bejarano et aI., 1988; Bejarano & Cerda-Olmedo, 1989). It is interesting to note that fJ-ionone, trisporic acids, and phenols are not analogues of each other.

A more classical way to establish functional groups is to determine synergisms. Thus, the joint application of activators belonging to different groups leads to higher carotene contents than may be attained with either alone (Govind & Cerda-Olmedo, 1986; Bejarano et al., 1988).

2,6,6-Trimethyl-1-acetylcyclohexene and some related compounds satisfac­torily replace fJ-ionone in industrial media for Blakeslea. Other chemicals (isoniazid, iproniazid) are synergistic with them (Ninet et aI., 1969). Many herbicides are inhibitors of carotenogenesis, but propanil (N-(3,4-dichlorophenyl)propanamide) is an activator (Feofilova et al., 1982).

The addition of microbial biomass to mated Blakeslea cultures stimulates carotenogenesis (Ciegler et al., 1964). About equally effective are the spent Blakeslea mycelia, after the extraction of fJ-carotene in previous fermenta­tions, and cells of various bacteria, yeasts, and fungi.

Although over 100 chemical activators of carotenogenesis are known, most are relatively ineffective, or too expensive, or too toxic for practical applica­tion. The search for activators is far from complete, and the spot test (Cerda-Olmedo & Hllttermann, 1986) should permit the rapid screening of new ones. The results for one fungus are often invalid for another: fJ-ionone stimulates carotenogenesis in Blakeslea and Phycomyces, but inhibits it in Gibberella (Avalos & Cerda-Olmedo, 1986); isoniazid is active for Blakeslea but not for Phycomyces.

34 E. Cerda-Olmedo

5 GENETICAL STIMULATION

5.1 Phycomyces

The genetic analysis of carotenogenesis in Phycomyces blakesleeanus (review, Cerda-Olmedo, 1987) was made possible by the study of its sexual cycle and its genetics (review, Eslava, 1987), the isolation of numerous car mutants with alterations in the biosynthesis of carotene and its regulation (review, Cerda­Olmedo, 1985) and the design of procedures to bypass their sexual distur­bances (Roncero & Cerda-Olmedo, 1982).

Sexual stimulation can be obtained in a single mycelium with a simple genetic trick. Mycelia containing a mixture of nuclei of opposite sex (inter­sexual heterokaryons) accumulate about 10 times more p-carotene than mycelia containing one or the other kind of nuclei (homokaryons) (Murillo & Cerda-Olmedo, 1976). Intersexual heterokaryons express full sexual stimula­tion of carotenogenesis: their p-carotene cannot be further increased by the addition of trisporic acids (Govind & Cerda-Olmedo, 1986). When the constituent nuclei have different genetic backgrounds, the intersexual hetero­karyons tend to sector out, i.e. to produce mycelia of various nuclear proportions and even pure homokaryons. Consaguineous strains, such as the ones developed by Alvarez & Eslava (1983), reduce this danger, but the best solution is a system of balanced lethals, that is, the induction of a recessive lethal mutation in each of the nuclei. This is much easier than it sounds (Murillo et al., 1978).

Attempts to block carotenogenesis in Phycomyces result in a general activation of the pathway that tends to maintain the concentration of the final product, p-carotene. Thus, the addition of an enzyme inhibitor (such as diphenylamine, which blocks phytoene dehydrogenation) causes an accumula­tion of intermediates (phytoene and other partially desaturated acyclic carotenes); the p-carotene content is affected only at high inhibitor concentra­tions. The same is true for inhibitors of lycopene cyclization (nicotine, 2-(4-chlorophenylthio)triethylamine), with accumulation of lycopene and y-carotene, and for mutations that reduce either enzyme activity. Full chemical or mutational blocks cause a 50-fold increase in carotene content; the product, of course, is no longer fJ-carotene, but the untransformed intermediate, phytoene or lycopene (Eslava & Cerda-Olmedo, 1974; Murillo & Cerda­Olmedo, 1976; Murillo et al., 1981).

This end-product inhibition of the pathway is mediated by p-carotene and two gene products (Cerda-Olmedo, 1987; Bejarano et al., 1988), as sum­marized in Fig. 2. Retinol and beta-ionone, which compete with beta-carotene, and recessive carS mutations, which destroy a dispensable gene product, abolish the inhibition and lead to high p-carotene contents. The best carS mutants contain 100 times more fJ-carotene than the wild type.

Other superproducing strains, containing up to 20 times more p-carotene than the wild type, carry recessive mutations in another gene, carD. Mutations

Production of Carotenoids with Fungi

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carS

35

Fig. 2. The genetics of carotenogenesis in Phycomyces blakesleeanus (Cerda-Olmedo, 1987). Four copies of phytoene dehydrogenase (the product of gene carB) and two copies of lycopene cyclase (one of the products of gene carRA, closely linked to carB) convert phytoene into beta-carotene (above). The product of gene carS and a product of gene carRA combine with beta-carotene to repress the synthesis (center). Two other

genes, care and carD, regulate in opposite ways the action of carS (below).

in gene carS are epistatic over those in gene carD: the double mutants do not surpass single carS mutants and are, therefore, of no practical value. There are superproducing strains that considerably exceed the J3-carotene content of the carS mutants, but they have not yet been published.

The highest published J3-carotene level in Phycomyces blakesleeanus corresponds to intersexual heterokaryons with carS mutations and balanced lethals (Murillo et aI., 1978). They contain 25 mg J3-carotene per g dry weight, some 500 times more than the wild type. Their genetic make-up provides constitutive sexual activation and removal of end-product inhibition, and therefore, they do not respond to retinol or J3-ionone. They do not respond to light or dimethyl phthalate either, although no provision is made for endogenous activation of the mechanisms normally triggered by these agents.

36 E. Cerda-Olmedo

When certain Phycomyces strains are grown in the dark, most of the fJ-carotene appears in a red complex, similar to the main carotenoprotein of many plants, which stabilizes fJ-carotene and changes its colour in an attractive way. These strains contain a carS mutation and a cytoplasmic carE mutation (De la Concha & Murillo, 1984). They accumulate about 100 times more fJ-carotene than the wild-type and no attempt has been made to improve their yield.

Some genetic constructs contain considerable amounts of pathway inter­mediates. Lycopene is accumulated by carR mutants, defective in lycopene cyclase. Constitutive sexual activation and partial removal of end-product inhibition led to strains with about 15 mg lycopene per g dry weight (Murillo et al., 1978). A high phytoene content (9 mg/g dry weight) was obtained by growing a carB mutant, defective in phytoene dehydrogenase, with dimethyl phthalate in the light (Bejarano & Cerda-Olmedo, 1989). Other intermedi­ates can only be produced as part of carotene mixtures: phytofluene and ~-carotene in some leaky carB mutants (Eslava & Cerda-Olmedo, 1974; Bejarano et al., 1987); y-carotene in certain heterokaryons (De la Guardia et al., 1971).

5.2 Other fungi

The genetic analysis of carotenogenesis is feasible in many fungi, but has been carried out in few, and only to limited extents (see, for example, Khabrova & Zhdanov, 1979, for Blakeslea trispora; Harding & Turner, 1981, for Neurospora crassa; Will et al., 1984, for Ustilago violacea; Avalos & Cerda-Olmedo, 1987, for Gibberella fujikuroi). Certain mutants of Gibberella are deep orange even when grown in the dark and contain up to 1.7 mg neurosporaxanthin per g dry weight, but they are not analogous to Phycomyces mutants because there is no end-product regulation in Gibberella.

6 INDUSTRIAL PRODUCTION

6.1 Blakeslea

The only fungal production of carotenoids that has been developed to the industrial stage is that of fJ-carotene with mated cultures of Blakeslea trispora. After detailed studies of media and additives, Ciegler and his co-workers at the US Department of Agriculture scaled up their cultures to a volume of 11 litres (Ciegler et al., 1963a). The process has been reviewed in detail (Ciegler, 1965; Hanson, 1967). The production phase (Ciegler et al., 1963a) consists in the joint culture at 28°C for 3 days of mycelia of two wild types of opposite sex in liquid medium (20 g ground maize, 40 g cotton-seed meal, 30 g white grease or vegetable oil, 30 g deodorized kerosene, 50 g citrus molasses, and 0·2 mg thiamine·HCI per litre). The fermented product contains about 17 mg fJ-

Production of Carotenoids with Fungi 37

carotene per g solids or about 1 g/litre medium. An antioxidant, ethoxyquin, is added at the end to stabilize the carotene. The two strains are cultivated separately before inoculation into the production vessels.

Several industry-affiliated research groups have improved the process (review, Ninet & Renaut, 1979, and the patents referred to therein). The procedure presented by a Rhone-Poulenc team in France (Ninet & Renaut, 1979) has two successive mated cultures. First, 0·4litres of separate cultures of each sex are inoculated into 120 litres of medium (70 g corn steep, 50 g corn starch, 0·5 g KH2P04, 0·1 g MnS04, 10 mg thiamin· HCI per litre tap-water) and incubated for 40 h at 26°C with agitation and aeration. Then, 32 litres of the first culture are inoculated into 320 litres of another medium (70 g distillers solubles, 60 g cornstarch, 30 ml soya oil, 30 ml cotton-seed oil, 20 ml kerosene, 0·35 g ethoxyquin, 0·6 g isoniazid, 0·2 g MnS04, 0·5 mg thiamine·HCI, per litre at pH 6·3) and incubated at 26°C for 185 h with agitation and aeration. After 48 h, 1 g p-ionone, 5 ml kerosene, and 42 g glucose are added per litre (the glucose solution is continuously injected to the end of the culture). The production should be about 30 mg p-carotene per g dry weight or about 3 g/litre.

Other sources of carbon and nitrogen have received consideration. One of them is whey, a subproduct of cheese manufacture (Lampila et al., 1985b,c).

Blakeslea allows the production of lycopene and other intermediate products in the presence of appropriate enzyme inhibitors (Ninet et al., 1969; Hsu et al., 1972; Ninet & Renaut, 1979).

6.2 Phycomyces

Production has been studied only under laboratory conditions. The genetic work was carried out on minimal agar; the wild type contains about 0·040 mg p-carotene per g dry weight and the superproducing strains described above, about 25 mg (Murillo et al., 1978). The mycelial dry weight is 10 g/litre in both cases. We have observed that various simple media, some composed of cheap subproducts of biological industries, increase the biomass to about 40g dry mycelia per litre and maintain its p-carotene concentration. Carotenogenesis suffers if the cultures are agitated, whether they are wild type (Lilly et al., 1960) or superproducers, and therefore surface cultures are the most productive.

6.3 Application

Industrial purification of p-carotene (Ninet et al., 1979) is complicated and expensive, and may not be necessary in many practical applications. For use in animal feeds, the carotene-rich mycelium may be dried, blended with various polymers, extruded into filaments, and powdered; the p-carotene in such preparations survives storage at 41°C for 1 year (Gogek, 1968).

38 E. Cerda-Olmedo

7 THE PROSPECTS

The p-carotene content of Blakeslea and Phycomyces mycelia has been enormously increased through optimization of culture conditions and genetic breeding, respectively. Both lines of research are far from exhausted and should lead to further production gains.

The production of xanthophylls (oxidated carotenoids) would be feasible with certain fungi, particularly after yield improvements brought about by new research. Phaffia rhodozyma offers a good start (Johnson et al., 1978; Johnson & Lewis, 1979) and effectively gives the desired pigmentation to pen-reared salmonids (Johnson et al., 1977).

Genes for carotenoid biosynthesis may eventually be inserted and expressed in colourless, but genetically malleable organisms, such as Saccharomyces cerevisiae. Carotenoids would then become important subproducts of well­established industries. More likely is the application of molecular genetics to strain improvement in organisms that already produce carotenoids. Neurospora crassa offers, of course, magnificent prospects for all sorts of genetic manipulations. Efficient transformation of Phycomyces blakesleeanus with exogenous DNA (Arnau et al., 1988; Suarez & Eslava, 1988) may now be combined with the more traditional approaches.

REFERENCES

Alvarez, M. I. & Eslava, A. P. (1983). Isogenic strains of Phycomyces blakesleeanw suitable for genetic analysis. Genetics, 105, 873-9.

Anderson, R. F., Arnold, M., Nelson, G. E. N. & Ciegler, A. (1958). Microbiological production of beta-carotene in shaken flasks. 1. Agric. Food Chem., 6,543-5.

Andrewes, A. G. & Starr, M. P. (1976). (3R, 3'R)-Astaxanthin from the yeast Phaffia rhodozyma. Phytochem., 15,1009-11.

Andrewes, A. G., Phaff, H. J. & Starr, M. P. (1976). Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast. Phytochem., 15, 1003-7.

Arnau, J., Murillo, F. J. & Torres-Martinez, S. (1988). Expression of Tn5-derived kanamycin resistance in the fungus Phycomyces blakesleeanw. Molec. Gen. Genet., 212,375-7.

Austin, D. J., Bu'Lock, J. D. & Drake, D. (1970). The biosynthesis of trisporic acids from beta-carotene via retinal and trisporol. Experientia, 26,348-9.

Avalos, J. & Cerda-Olmedo, E. (1986). Chemical modification of carotenogenesis in Gibberella fujikuroi. Phytochem., 25, 1837-41.

Avalos, J. & Cerda-Olmedo, E. (1987). Carotenoid mutants of Gibberella fujikuroi. Curro Genet., 11, 505-11.

Barnett, H. L., Lilly, V. G. & Krause, R F. (1956). Increased production of carotene by mixed + and - cultures of Choanephora cucurbitarum. Science, 123,141.

Bauernfeind, J. C. (Ed.) (1981). Carotenoids as Colorants and Vitamin A Precursors. Technological and Nutritional Applications. Academic Press, New York.

Bejarano, E. R. & Cerda-Olmedo, E. (1989). Inhibition of phytoene dehydrogena­tion and activation of carotenogenesis in Phycomyces. Phytochem., 28, 1623-6.

Bejarano, E. R, Govind, N. S. & Cerda-Olmedo, E. (1987). Zeta-carotene and other carotenes in a Phycomyces mutant. Phytochem., 26,2251-4.

Production of Carotenoids with Fungi 39

Bejarano, E. R., Parra, F., Murillo, F. J. & Cerda-Olmedo, E. (1988). End-product regulation of carotenogenesis in Phycomyces. Archs Microbiol., 150, 209-14.

Bergman, K., Eslava, A. P. & Cerda-Olmedo, E. (1973). Mutants of Phycomyces with abnormal phototropism. Molec. Gen. Genet., 123, 1-16.

Bindl, E., Lang, W. & Rau, W. (1970). Untersuchungen tiber die lichtabhangige Carotinoidsynthese. VI. Zeitlicher Verlauf der Synthese der einzelnen Carotenoide bei Fusarium aquaeductuum under verschiedenen Induktionsbedingungen. Planta, 94, 156-74.

Blakeslee, A. F. (1904). Sexual reproduction in the Mucorineae. Proc. Am. Acad. Arts Sci., 40,205-319.

Bramley, P. M. & Mackenzie, A. (1988). The regulation of carotenoid biosynthesis. Curro Topics Cell Regul., 29, 291-343.

Burgeff, H. (1924). Untersuchungen tiber Sexualitat und Parasitismus bei Mucorineen. Botan. Abhandl., 4, 1-135.

Caglioti, L., Cainelli, G., Camerino, B., Mondelli, R., Prieto, A., Quilico, A., Salvatori, T. & Selva, A. (1966). The structure of trisporic-C acid. Tetrahedron Suppl., 7, 175-87.

Castillo, R. (1980). On the transformation of beta-carotene 15,15'-3H2 into astaxanthin by the hermit crab Clinabarius erythropus Latreille (1818), Crustacea, Decapoda, Anomoura. Compo Biochem. Physiol., 66A, 695-7.

Cerda-Olmedo, E. (1985). Carotene mutants of Phycomyces. Meth. Enzymol., 110, 220-43.

Cerda-Olmedo, E. (1987). Carotene. In Phycomyces, ed. E. Cerda-Olmedo & E. D. Lipson. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 199-222.

Cerda-Olmedo, E. & Htittermann, A. (1986). Forderung und Hemmung der Carotin­synthese bei Phycomyces durch Aromaten. Angew. Botanik, 60, 59-70.

Cerda-Olmedo, E. & Lipson, E. D. (Eds) (1987). Phycomyces. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Ciegler, A. (1965). Microbial carotenogenesis. Adv. Appl. Microbiol., 7, 1-34. Ciegler, A., Arnold, M. & Anderson, R. F. (1959a). Microbiological production of

carotenoids. IV. Effect of various grains on production of beta-carotene by mated strains of Blakeslea trispora. Appl. Microbiol., 7,94-8.

Ciegler, A., Arnold, M. & Anderson, R. F. (1959b). Microbiological production of carotenoids. V. Effect of lipids and related substances on production of beta­carotene. Appl. Microbiol., 7, 98-1Ol.

Ciegler, A., Nelson, G. E. N. & Hall, H. H. (1962). Microbiological production of carotenoids. VIII. Influence of hydrocarbon on carotenogenesis by mated cultures of Blakeslea trispora. Appl. Microbiol., 10, 132-6.

Ciegler, A., Lagoda, A. A., Sohns, V. E., Hall, H. H. & Jackson, R. W. (1963a). Beta-carotene production in 20-liter fermentors. Biotechnol. Bioengin., 5, 109-2l.

Ciegler, A., Nelson, G. E. N. & Hall, H. H. (1963b). Enhancement of beta-carotene synthesis by citrus products. Appl. Microbiol., 11, 128-3l.

Ciegler, A., Nelson, G. E. N. & Hall, H. H. (1963c). Enhancement of carotenogenesis in Blakeslea trispora by essential citrus oils. Nature, Lond., 198, 1305-6.

Ciegler, A., Pazola, Z. & Hall, H. H. (1964). Stimulation of carotenogenesis by microbial cells. Appl. Microbiol., 12, 150-54.

De la Concha, A. & Murillo, F. J. (1984). Accumulation of a complex form of beta-carotene by Phycomyces blakesleeanus cytoplasmic mutants. Planta, 161, 233-9.

De la Guardia, M. D., Arag6n, C. M. G., Murillo, F. J. & Cerda-Olmedo, E. (1971). A carotenogenic enzyme aggregate in Phycomyces: Evidence from quantitative complementation. Proc. natn. Acad. Sci. U.S.A., 68,2012-15.

El-Jack, M., Mackenzie, A. & Bramley, P. M. (1988). The photoregulation of carotenoid biosynthesis in Aspergillus giganteus mut. alba. Planta, 174, 59-66.

40 E. Cerda-Olmedo

Engel, B. G., Wiirsch, J. & Zimmermann, M. (1953). fIber den Einfluss von beta-Jonon auf die Bildung von beta-Carotin durch Phycomyces blakesleeanus. Helv. Chim. Acta, 36, 1771-6.

Eslava, A. P. (1987). Genetics. In Phycomyces, ed. E. Cerda-Olmedo & E. D. Lipson. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 27-48.

Eslava, A. P. & Cerda-Olmedo, E. (1974). Genetic control of phytoene dehydrogena­tion in Phycomyces. Plant Sci. Lett., 2,9-14.

Eslava, A. P., Alvarez, M. I. & Cerda-Olmedo, E. (1974). Regulation of carotene biosynthesis in Phycomyces by vitamin A and beta-ionone. Eur. J. Biochem., 48, 617-23.

Feofilova, E. P. (1974). Pigmenty mikroorganizmov. Nauka, Moscow. Feofilova, E. P. & Bekhtereva, M. N. (1976). Effect of vitamin A on carotene

biosynthesis by Blakeslea trispora (in Russian). Mikrobiologia, 45, 557-8. Feofilova, E. P., Ushanova, A. E. & Ivanova, G. B. (1982). Effect of dimilin on

carotene production by Blakeslea trispora (in Russian). Mikrobiologia, 51, 267-71.

Garber, E. D., Baird, M. L. & Weiss, L. M. (1978). Genetics of Ustilago violacea. II. Polymorphism of color and nutritional requirements of sporidia from natural populations. Bot. Gaz., 139,261-5.

Gogek, C. J. (1968). Stabilization of crude carotene-containing mycelium. J. Agric. Food Chem., 16, 730-34.

Gooday, G. W., Fawcett, P., Green, D. & Shaw, G. (1973). The formation of fungal sporopollenin in the zygospore wall of Mucor mucedo: a role for the sexual carotenogenesis in the Mucorales. J. Gen. Microbiol., 74, 233-9.

Goodwin, T. W. (Ed.) (1976). Chemistry and Biochemistry of Plant Pigments, 2nd edn, two vols. Academic Press, London.

Goodwin, T. W. (1980). The Biochemistry of the Carotenoids. 2nd edn., Vol. 1, Plants. Chapman and Hall, London/Academic Press, London.

Goodwin, T. W. (1986). Metabolism, nutrition, and function of carotenoids. Ann. Rev. Nutr., 6,273-397.

Goodwin, T. W. (Ed.) (1988). Plant Pigments. Academic Press, London. Goodwin, T. W. & Griffiths, L. A. (1952). Studies in carotenogenesis. 5. Carotene

production by various mutants of Phycomyces blakesleeanus and Phycomyces nitens. Biochem. J., 52, 499-501.

Govind, N. S. & Cerda-Olmedo, E. (1986). Sexual activation of carotenogenesis in Phycomyces blakesleeanus. J. Gen. Microbiol., 132,2775-80.

Hanson, A. M. (1967). Production of pigments and vitamins. In Microbial Technology, ed. H. J. Peppler. Van Nostrand-Rheinhold, Princeton, pp. 222-50.

Harding, R. W. & Shropshire, W. (1980). Photocontrol of carotenoid biosynthesis. Ann. Rev. Plant Physiol., 31,217-38.

Harding, R. W. & Turner, R. V. (1981). Photoregulation of the carotenoid biosynthe­tic pathway in albino and white collar mutants of Neurospora crassa. Plant Physiol., 68,745-9.

Harding, R. W., Huang, P. C. & Mitchell, H. K. (1969). Photochemical studies of the carotenoid biosynthetic pathway in Neurospora crassa. Archs Biochem. Biophys., 129,696-707.

Hsu, W. J., Yokoyama, H. & Coggins, C. W. (1972). Carotenoid biosynthesis in Blakeslea trispora. Phytochem., 11,2985-90.

Isler, O. (Ed.) (1971). Carotenoids. Birkhauser Verlag, Basel. Johnson, E. A. & Lewis, M. J. (1979). Astaxanthin formation by the yeast Phaffia

rhodozyma. J. Gen. Microbiol., 115,173-83. Johnson, E. A., Conklin, D. E. & Lewis, M. J. (1977). The yeast Phaffia rhodozyma as

a dietary pigment source for salmonids and crustaceans. J. Fish. Res. Board Can., 34,2417-21.

Production of Carotenoids with Fungi 41

Johnson, E. A., Villa, T. G., Lewis, M. J. & Phaff, H. J. (1978). Simple method for isolation of astaxanthin from the basidiomycetous yeast Phaffia rhodozyma. Appl. Environ. Microbiol., 35, 1155-9.

Khabrova, A. M. & Zhdanov, V. G. (1979). Study of sex defective mutants of Blakeslea trispora. Mikrobiologia, 48, 1055-9.

Krzeminiski, L. F. & Quackenbush, F. W. (1960). Stimulation of carotene synthesis in submerged cultures of Neurospora crassa by surface-active agents and ammonium nitrate. Archs Biochem. Biophys., 88,64-7.

Lampila, L. E., Wallen, S. E. & Bullerman, L. B. (1985a). A review of factors affecting biosynthesis of carotenoids by the order Mucorales. Mycopathologia, 90, 65-80.

Lampila, L. E., Wallen, S. E., Bullerman, L. B. & Lowry, S. R. (1985b). The effect of strain and type of whey on the production of beta-carotene and other parameters. Lebensmittel-Wissenschaft-Technologie, 18, 366-9.

Lampila, L. E., Wallen, S. E., Bullerman, L. B. & Lowry, S. R. (1985c). The effect of illumination on growth and beta-carotene content of Blakeslea trispora grown in whey. Lebensmittel-Wissenschaft-Technologie, 18,370-73.

Lilly, V. G., Barnett, H. L. & Krause, R. F. (1960). The production of carotene by Phycomyces blakesleeanus. West Virginia Univ. Agr. Exper. Sta., Bulletin 441T, Morgantown.

Mackinney, G., Nakayama, T., Buss, C. D. & Chichester, C. O. (1952). Carotenoid production in Phycomyces. 1. Am. Chem. Soc., 74,3456-7.

Mackinney, G., Nakayama, T., Chichester, C. O. & Buss, C. D. (1953). Biosynthesis of carotene in Phycomyces. 1. Am. Chem. Soc., 75, 236-8.

Murillo, F. J. & Cerda-Olmedo, E. (1976). Regulation of carotene synthesis in Phycomyces. Molec. Gen. Genet., 148, 19-24.

Murillo, F. J., Calder6n, I. L., L6pez-Diaz, I. & Cerda-Olmedo, E. (1978). Carotene-superproducing strains of Phycomyces. Appl. Environ. M icrobiol., 36, 639-42.

Murillo, F. J., Torres-Martinez, S., Arag6n, C. M. G. & Cerda-Olmedo, E. (1981). Substrate transfer in carotene biosynthesis in Phycomyces. Eur. 1. Biochem., 119, 511-16.

Ninet, L. & Renaut, J. (1979). Carotenoids. In Microbial Technology, 2nd edn, Vol. 1, ed. H. J. Peppler & D. Perlman. Academic Press, New York, pp. 529-44.

Ninet, L., Renaut, J. & Tissier, R. (1969). Activation of the biosynthesis of carotenoids by Blakeslea trispora. Biotechnol. Bioeng., 11, 1195-210.

Rau, W. (1983). Photoregulation of carotenoid biosynthesis. In Biosynthesis of isoprenoid compounds, ed. J. W. Porter & S. L. Spurgeon. John Wiley, New York, pp. 123-57.

Rau, W. & Schrott, E. L. (1987). Blue light control of pigment biosynthesis­Carotenoid biosynthesis. In Blue Light Responses, Vol. I, ed. H. Senger. CRC Press, Boca Rat6n, Florida, pp. 43-63.

Reyes, P., Chichester, C. O. & Nakayama, T. O. M. (1964). The mechanism of beta-ionone stimulation of carotenoid and ergosterol biosynthesis in Phycomyces blakesleeanus. Biochim. Biophys. Acta, 90, 578-92.

Roncero, M. I. G. & Cerda-Olmedo, E. (1982). Genetics of carotene biosynthesis in Phycomyces. Curro Genet., 5, 5-8.

Suarez, T. & Eslava, A. P. (1988). Transformation of Phycomyces with a bacterial gene for kanamycin resistance. Molec. Gen. Genet., 212, 120-23.

Sutter, R. P. (1970). Effect of light on beta-carotene accumulation in Blakeslea trispora. 1. Gen. Microbiol., 64, 215-21.

Sutter, R. P. (1975). Mutations affecting sexual development in Phycomyces blakes­leeanus. Proc. natn. Acad. Sci. USA, 72, 127-30.

42 E. Cerda-Olmedo

Tada, M. & Shiroishi, M. (1982). Mechanism of photoregulated carotenogenesis in Rhodotorula minuta. I. Photocontrol of carotenoid production. Plant Cell Physiol., 23,541-7.

Valadon, L. R. G. & Mummery, R. S. (1973). Effect of certain inhibitors of carotenogenesis in Verticillium agaricinum. Microbios, 7, 173-80.

Vaskivnyuk, V. Y. & Getman, E. I. (1984). Biosynthesis of carotenoids in light in continuous cultures of the yeast Rhodosporidium diobovatum (in Russian). Priklad. Biokhim. Mikrobiol., 20, 480-83.

Will, O. H., Ruddat, M., Garber, E. D. & Kezdy, F. J. (1984). Characterization of carotene accumulation in Ustilago violacea using high-performance liquid chroma­tography. Curro Microbiol., 10,57-64.

Chapter 4

MICROBIAL PRODUCTION OF CAROTENOIDS OTHER THAN Ii-CAROTENE

H. J. NELIS & A. P. DE LEENHEER

Laboratories of Medical Biochemistry and Clinical Analysis, Faculty of Pharmaceutical Sciences, State University of Ghent, B-9OOO Ghent, Belgium

I GENERAL ASPECTS

1 HISTORY

1.1 Discovery of Carotenoids in Nature

The early discovery of carotenoids in living organisms is obviously attributable to the conspicuous appearance of these yellow, orange or red pigments. In 1831 Wackenroder prepared 'carotene' in crystalline form (Isler, 1971), whereas xanthophylls were first isolated from autumn leaves by Berzelius in 1837 (Isler, 1971). Following the main-classical chromatographic experiments of Tswett at the beginning of this century (Isler, 1971), it was soon realized that there existed a complex group of carotenoids in nature. The next decades indeed saw the isolation and structural elucidation of a large variety of new derivatives. More recently, the advent of modern spectrometric techniques and the refinement of separation methods have resulted in the ongoing discovery of more new structures, the determination of their absolute configurations and the establishment of routes for their partial or total synthesis. Concurrently, the biochemistry of carotenoids in plants and animals has gradually been unravelled (Goodwin, 1980, 1984).

1.2 Early Observations on the Occurrence of Carotenoids in Micro-organisms

The occurrence of carotenoids in micro-organisms was first reported about 100 years ago, when a number of pigmented non-photosynthetic bacteria (quoted in Ingraham & Baumann, 1934) and fungi (quoted in Valadon, 1976) were isolated. A more systematic survey of carotenoid-producing non­photosynthetic bacteria was conducted in the 1930s (Ingraham & Baumann,

43

44 H. J. Nelis & A. P. De Leenheer

1934). Pioneering work on photosynthetic bacteria was carried out by Van Niel & Smith (1935).

2 CHEMICAL AND PHYSICAL PROPERTIES (Davies, 1976; Moss & Weedon, 1976)

Chemically, carotenoids are polyenes consisting of a number of isoprenoid units (usually 8) arranged in such a way that the two central methyl groups (20 and 20', Fig. 1) are in the 1,6 position, while all remaining substituents are positioned 1,5 relative to each other. A series of conjugated double bonds constitutes a chromophore of variable length, resulting in the characteristic yellow to red colors (absorption maxima 400-500 nm). Figure 1 presents the five basic (no substituents) structures of which all compounds pertinent to this chapter are derived. Full structures of the latter are depicted in Fig. 2, together with their trivial (common), semi-systematic and systematic names (Table 1, legend to Fig. 2).

Unsubstituted carotenoid hydrocarbons, e.g. lycopene, {:I-carotene, and torulene, are commonly designated as 'carotenes'. The collective term xanth­ophylls covers oxygenated derivatives, including alcohols (e.g. lutein and

... 1

20

r~ 2

.' 20 3'

~ ,.' 2'

B 3

20'

~. 2' 4 f

5

Fig. 1. Basic structures of carotenoids of which the compounds of interest (Fig. 2, Table 1) are chemically derived. Systematic IUPAC names in parentheses. 1, lycopene ('IjI,'IjI-carotene); 2,y-carotene (p,'IjI-carotene); 3,p-carotene (p,p-carotene); 4,a­carotene (P, e-carotene); 5, retrodehydro-p-carotene (4'5' -didehydro-4,5'-retro-p, p-

carotene).

Microbial Production of Carotenoids 45

1 "" •• OH

2 HO

3 -",

HO

4

0 0

5

0

OH

6 HO

0

7

8

9

OCH3

10

11 ..., 0

Fig. 2. Structural formulae of carotenoids of interest. See Table 1 for explanation.

zeaxanthin), epoxides (e.g. violaxanthin), ethers (e.g. spheroidene), ketones (e.g. canthaxanthin, astaxanthin, spheroidenone) and acids such as torular­hodin. Rhodoxanthin is an example of a retrocarotenoid, in which all single and double bonds of the conjugated polyene system have shifted by one position. The modern IUPAC systematic nomenclature (IUPAC, 1971) is based on a designation of the end groups of the molecules, using such prefixes as 1jJ (acyclic end-group), f3 or E (cyclohexene end groups) (indicated in Fig. 1). Most natural carotenoids possess the all-trans configuration, although the genuine presence of cis-isomers has been documented, e.g. in photosynthetic bacteria (Koyama et al., 1983). If a compound contains chiral centers (as in zeaxanthin and astaxanthin) the absolute configuration of the optical isomer(s) found in nature is also mentioned in Table 1.

Stru

ctur

e T

rivi

al n

ame

Tab

le 1

S

truc

ture

s an

d N

omen

clat

ure

of C

arot

enoi

ds o

f In

tere

st.

Bas

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Microbial Production of Carotenoids 47

3 BIOLOGICAL PROPERTIES

The extended chromophore in the carotenoid structure points to a biological significance for these pigments in micro-organisms, by suggesting photosensi­tive and antioxidant properties. In algae and photosynthetic bacteria, caroten­oids participate as secondary light-harvesting compounds in the photosynthetic process (Mathis & Schenck, 1982) and exert photoprotective effects as well (Burnett, 1976). As antioxidants they protect vulnerable tissues against the deleterious effects of singlet oxygen and free radicals (Krinsky, 1979). Similar (photo )-protective effects have been invoked in non-photosynthetic bacteria (Mathews & Krinsky, 1965; Mathews-Roth & Krinsky, 1970; Mathews-Roth et al., 1974), although conflicting evidence exists (Schwartzel & Cooney, 19740; Dieringer et al., 1977).

4 INDUSTRIAL APPLICATIONS

The economic significance of carotenoids has been extensively reviewed (KHiui & Bauernfeind, 1981; Marusich & Bauernfeind, 1981; Simpson et al., 1981; KHiui, 1982). There is a continuously growing market for carotenoids as food colorants (KHiui & Bauernfeind, 1981) and pigmenters in animal feeds (Marusich & Bauernfeind, 1981; Simpson et al., 1981). Poultry feeds based on agricultural waste products have to be supplemented with pigments in order to impart a desirable yellow-orange color to egg yolks and/or the skin of broiler chickens (Marusich & Bauernfeind, 1981). Optimal yolk color ensues from the combination of a yellow base pigment (e.g. lutein, p-apo-8'-carotenoic acid ethyl ester) with a small quantity of an orange-red one, preferably a ketocarotenoid (canthaxanthin) or zeaxanthin.

The addition of ketocarotenoids (astaxanthin or canthaxanthin) to fish feeds also intensifies the reddish colour of the flesh of the salmon and the trout (Simpson et al., 1981). So far, the application of natural sources of carotenoids remains confined to animal feeds (Marusich & Bauernfeind, 1981; Simpson et al., 1981). Industrially produced natural carotenoids are mainly derived from vegetable sources, including marigold flowers, alfalfa, red peppers and annatto (Marusich & Bauernfeind, 1981).

5 PRODUCING MICRO-ORGANISMS

There is a wealth of literature on the occurrence of carotenoids in algae, fungi and bacteria, the emphasis being on structural studies and the elucidation of the pertinent biosynthetic pathways. This information has been exhaustively reviewed in an authoritative book by Goodwin (1980).

Our survey of carotenoid-producing organisms is limited to those species which show biotechnological potential, i.e. which could be considered for the

48 H. J. Nelis & A. P. De Leenheer

development of a commercially exploitable, natural source of pigments. The selection of useful organisms (Table 2) was made on the basis of three criteria:

(a) The presence in the organism of carotenoids of interest (cf. the limitative list in Table 1, p-carotene excluded).

(b) The already established industrial use of a given strain. So far this has only been the case for green algae. However, most organisms included in Table 2 have not (yet) found such application, but do show promise to be developed for this purpose. Specifically, this could mean that attempts have already been made, are currently being made or are likely to be made in the future to optimize the pigment levels in these organisms. In order to justify the efforts, the carotenoids involved should be particularly valuable, difficult to synthesize and be absent or rare in cheaper and more accessible natural sources. The recent commercial exploitation of micro-algae as a source of p-carotene proves that there is a potential market for natural carotenoids, even if the (cheaper) synthetic equivalents exist (cf. Chapter 2).

( c) The current interest in a number of carotenoid containing organisms for reasons other than their pigment composition. Alternative applications include the production of biomass, single cell oil or lipid, (other) fine chemicals, or the purification of waste waters. However, the presence of often high quantities of carotenoids in those particular organisms could make them attractive for commercial production of these useful 'by­products' as well.

6 BIOSYNTHESIS (for a review, see Spurgeon & Porter, 1983)

A first series of steps in the formation of carotenoids belongs to the well-known mevalonate pathway, the general biosynthetic scheme of all isoprenoid compounds. Starting from acetate (acetyl CoA), activated isoprene units undergo a sequence of head-to-tail condensations, leading to geranylger­anyl pyrophosphate (Cw). From this point on, all subsequent steps are unique to the biosynthesis of carotenoids. Head-to-head condensation of 2 C20 units yields the key intermediate phytoene, the first colorless carotenoid. Through a series of stepwise dehydrogenations, phytoene is converted to lycopene, from which all other carotenoids can eventually be derived. The biosynthetic interrelationship of the derivatives pertinent to this chapter is illustrated in Fig. 3.

7 REGULATION OF BIOSYNTHESIS

The biosynthesis of carotenoids by micro-organisms is under genetic control, but is also influenced by various environmental factors (Spurgeon & Porter, 1983).

Microbial Production of Carotenoids

Table 2 Micro-organisms of Potential Practical Interest for the Production of

Carotenoids

Species Carotenoid(s) produced

1. Micro-organisms specifically designed as pigment sources 1. Sources of carotenes

A. Fungi Blakeslea trispora

B. Non-photosynthetic bacteria Streptomyces chrestomyceticus, subsp. rubescens

2. Sources of xanthophyllsa A. Green algae

Spongiococcum excentricum Chlorella pyrenoidosa

B. Fungi Dacrymyces deliquescens

C. Non-photosynthetic bacteria Flavobacterium sp. Streptomyces chrestomyceticus, var. aurantioideus

Lycopene

Lycopene

Lutein Lutein

Lutein

Zeaxanthin Unidentified xanthophylls

Mycobacterium phlei Unidentified xanthophylls 3. Sources of monocyclic ketocarotenoids

A. Non-photosynthetic bacteria Deinococcus radiophilus-radiodurans- Derivatives of 4-keto-radiopugnans y-carotene Mycobacterium smegmatis Derivatives of 4-keto­

y-carotene 4. Sources of bicyclic ketocarotenoids

A. Cyanobacteria Anabaena variabilis Aphanizomenon flos-aquae Nostoc commune

B. Green algae (N-deficiency) Dictyococcus cinnabarinus Haematococcus pluvialis

C. Fungi/yeast PhajJia rhodozyma

D. Non-photosynthetic bacteria Brevibacterium KY -4313 Rhodococcus maris / Mycobacterium brevicale 32-MCT Mycobacterium lacticola Brevibacterium 103

5. Sources of retrocarotenoids Pseudomonas extorquens

Canthaxanthin Canthaxanthin Canthaxanthin

Canthaxanthin Astaxanthin

Astaxanthin

Canthaxanthin Canthaxanthin

Astaxanthin Astaxanthin

Rhodoxanthin

II. Micro-organisms designed for applications other than pigment production

A. Fungi/yeast Rhodotorula, Rhodosporidium sp. (red yeasts)

B. Non-photosynthetic bacteria Methylotrophs

C. Photosynthetic bacteria Rhodobacter capsulatus

a Ketocarotenoids excluded.

Torulene, torularhodin

Miscellaneous

Spheroidene, Spheroidenone

49

50 H. J. Nelis & A. P. De Leenheer

~ OCH, 1 IS'

~ JA lc .--e-~

3

Fig. 3. Biosynthetic relationship between the carotenoids of interest. 1, All-trans­phytoene (actual precursor of lycopene may be 15,15'-cis-phytoene); 2, lycopene; 3, spheroidene; 4, spheroidenone; 5, y-carotene; 6, 4-keto-y-carotene; 7, torulene; 8, torularhodin; 9, /J-carotene; 10, zeaxanthin (3R,3'R); 11, rhodoxanthin; 12, canth­axanthin; 13, astaxanthin (3S, 3'S); 14, a-carotene; 15, lutein (3R, 3'R, 6'R). Reactions involved: A, stepwise desaturation (dehydrogenations). B, water addition at 1,2 double bond + dehydrogenation (C-3 and C-4) + methylation of hydroxyl group at C-l. C, keto group addition at C-2. Mechanism not established. 0, cyclization of 1 or 2 terminal end(s) of lycopene, yielding 1/J-end group (/J, tp: y-carotene, 5), 2/J-end groups (/J,/J: /J-carotene, 9) or 1/J- and Ie-end group (/J,e: a-carotene, 14), respectively. E, dehydrogenation at C-3' and C-4'. F, terminal oxidation (-CH3--CH20H-­CHO--COOH). G, keto group addition at C-4. Probably hydroxylation followed by dehydrogenation. H, hydroxylation at C-3 and C-3'. Requires molecular oxygen. Mechanisms unknown. I, Keto group addition at C-4 and C-4'. Probably hydroxylation followed by dehydrogenation. J, Mechanism uncertain. Probably attack of OH+ on zeaxanthin at C-5, loss of H+ at C-4', loss of water and dehydrogenation of hydroxyl

groups at C-3 and C-3'.

7.1 Environmental Factors

Both the pigment patterns and the amount of carotenoids in the cells may be altered by changes in the composition of the growth medium and by the influence of physical factors such as light, temperature and oxygen. Variation of the growth conditions of an organism with the aim to optimize its carote­noid content sometimes has a rational basis (e.g. generation of biosynthetic intermediates), but very often is rather empirical. From the numerous effects reported in the literature (for a compilation, see Goodwin, 1980) only a few

Microbial Production of Carotenoids 51

examples have been selected to illustrate how the carotenoid biosynthesis in certain algae, fungi and bacteria responds to changes in the environment.

7.1.1 Algae

(A) COMPOSITION OF THE GROWTH MEDIUM

Under optimal conditions for proliferation, green algae produce the typical chloroplastidic carotenoids ~-carotene, lutein, zeaxanthin and various epox­ides. However, nutritional imbalance, particularly the lack of nitrogen, often induces the synthesis of secondary extraplastidic (keto )carotenoids, so that the cultures turn orange or red.

(8) PHYSICAL FACTORS

Under autotrophic conditions, light has in general little or no effect on the carotenoid production by algae. An interesting effect of oxygen is noted in some organisms. Anaerobic conditions exclusively lead to carotene formation, whereas in the presence of oxygen synthesis is primarily directed towards xanthophylls.

7.1. 2 Fungi and yeast

(A) COMPOSITION OF THE GROWTH MEDIUM

The nature of the carbon and nitrogen source affects carotenoid formation in fungi in an unpredictable manner. In general, a high carbon to nitrogen ratio promotes carotenogenesis. The stimulatory effect of leucine and valine can be explained in that these amino acids are precursors of ~-hydroxy-~-methyl­glutaryl-CoA, a key intermediate in isoprenoid synthesis. Fluoride is thought to exert its positive influence at the enzymatic level: this toxic chemical preferentially inhibits certain phosphatases, which catalyze the hydrolysis and, hence, cause the depletion of essential phosphorylated intermediates from the isoprenoid pathway.

(8) PHYSICAL FACTORS

A strange effect of temperature on the qualitative carotenoid composition has been noted in certain red yeasts. At low temperatures, ~- and y-carotene predominate, while a temperature increase reportedly favors the formation of torulene and torularhodin. Strict photoregulation of carotenogenesis occurs in a few, thoroughly investigated species (Rau, 1983). The latter only contain negligible amounts of carotenoids when grown in total darkness. A brief exposure to light, in the presence of oxygen, induces de novo synthesis of carotenogenic enzymes and, accordingly, carotenoid production. The exact nature of the photoreceptor is still uncertain and the whole process is under genetic control (see below).

52 H. J. Nelis & A. P. De Leenheer

7.1. 3 Non-photosynthetic bacteria

(A) COMPOSITION OF THE GROWTH MEDIUM

Carotenoid formation is common among hydrocarbon assimilating bacteria. However, the carotenoid pattern may be different according to whether they are grown on hydrocarbon or non-hydrocarbon media. Like in fungi, high carbon to nitrogen ratios commonly increase the pigment yield. Ammonium salts are preferred nitrogen sources. The concentration of thiamine in the medium may both affect the nature of the carotenoids formed (cyclic vs acyclic) and their cellular concentration.

(8) PHYSICAL FACTORS

Photoinduction, as outlined for fungi (see Section 7.1.2(B» has been reported in a number of strains, particularly belonging to the genus Mycobacterium (Rau, 1983). Illumination of cultures may also alter the qualitative carotenoid composition, by converting carotenes, normally produced in the dark, into xanthophylls.

Z 1. 4 Photosynthetic bacteria

(A) COMPOSITION OF THE GROWTH MEDIUM

Few data are available regarding the influence of nutrients in the medium on the carotenoid formation by photosynthetic bacteria. A specific example of the effect of iron will be discussed below (see Section 111,4).

(8) PHYSICAL FACTORS

Oxygen and light are key factors controlling carotenogenesis in photosynthetic bacteria. In general, anaerobic conditions favor the formation of less-oxidized derivatives (e.g. spheroidene), as opposed to ketocarotenoids (spheroide­none), which are produced in the presence of oxygen.

7.2 Inhibitors (Ninet et aI., 1969; Spurgeon & Porter, 1983)

Compounds inhibiting the normal biosynthesis of carotenoids can be classified in two groups. The first group consists of inhibitors that cause the accumula­tion of early intermediates in the pathway, particularly the colorless phytoene. These compounds, e.g. diphenylamine, are very useful academically to elucidate the biosynthetic schemes, but obviously of no interest for practical application. Other inhibitors preferentially block later reactions in the path­way, notably the cyclization of lycopene. Examples of these selective inhibitors include nicotine and CPT A (2-( 4-chlorophenylthio )-triethylamine) and other substituted amines, as well as nitrogenous heterocyclic bases (e.g. imidazole).

Microbial Production of Carotenoids 53

7.3 Genetic Control

The biosynthesis of carotenoids is under the control of different genes that code for the individual enzymes in the pathway. The basis of the photoregula­tion in fungi and non-photosynthetic bacteria is also genetic; the actual control probably takes place at the transcription level in the protein synthesis (Rau, 1983). Illumination may either cause the inactivation of a repressor (derepres­sion) or, alternatively the production of an inducer. Similarly, repression and derepression may also be involved in the environmental control of carotenogenesis.

8 STRAIN IMPROVEMENT

Various bacterial mutants have been prepared that have a defect in the structural gene for one of the carotenogenic enzymes (Spurgeon & Porter, 1983). Three types of mutants with altered carotenoid biosynthesis are of potential practical interest.

8.1 Blocked Mutants-Mutation to Non-production of Undesirable Carotenoids

Mutants blocked at the level of cyclization of lycopene to fJ-carotene could be useful when overproduction of the former is aimed at. The undesired cyclic compounds will not be formed and consequently lycopene will accumulate.

8.2 Mutational Biosynthesis-New Carotenoid Formation

Mutation may lead to an altered and, from a practical standpoint more interesting pigment pattern (qualitative strain improvement). Saperstein et al. isolated various colored mutants following treatment of a Corynebacterium michiganense strain with uranium salts (Saperstein et al., 1954). While the predominating pigments in the yellow parent strain were reportedly fJ­cryptoxanthin and lycopene, an orange mutant and a red back-mutant were found to contain mostly canthaxanthin and lycopene, respectively. In both cases the 'uninteresting' fJ-cryptoxanthin was replaced by more valuable compounds.

8.3 Mutants with Enhanced Pigment Levels

Few examples are known of systematic mutation programs directed towards increasing the carotenoid content of a given species. This may be in part attributed to the lack of objective criteria for the selection of mutants with enhanced pigmentation. It may seem obvious to carry out such selection on the basis of visual inspection. However, quantitative differences in carotenoid

54 H. 1. Nelis & A. P. De Leenheer

content may not always be so easy to recognize. The subjectivity of the interpretation of color differences is exemplified by the results of Russian workers who prepared carotenoid overproducing mutants of Mycobacterium phlei (Voznyakovskaya & Daraseliya, 1972). The parent strain was described as 'yellow-orange' (carotenoid content of 1·7 mg/ g), whereas two more pigmented mutants were judged as 'orange' (4·4 mg/ g) and 'clear orange' (7·9 mg/g), respectively.

Alternatively, it could be assumed that mutants with enhanced carotenoid content are more resistant to photodynamic killing. If a correlation indeed exists between pigmentation and the extent of photoprotection, these mutants would supposedly show a higher survival rate upon illumination, e.g. in the presence of a photosensitizer. However, as mentioned before (see Section 3), a correlation does not necessarily exist between pigment content and the degree of photoprotection supposedly conferred upon the organism by the carotenoid.

9 FERMENTATION OF CAROTENOID-PRODUCING MICRO-ORGANISMS-GENERAL

The production of 'interesting' carotenoids (cf. Fig. 2 and Table 1) through fermentation of selected organisms (Table 2) will be surveyed in the light of the criteria listed in Section 5: a basic distinction will be made between organisms which have been or could be specifically designed for pigment production, and those which are applied otherwise, but may become attractive as pigment sources as well. How important fermentative production of a given carotenoid really is may be judged from the availability of alternative natural sources (briefly discussed in General). For each organism details will be provided on fermentation conditions, optimization strategies to increase the pigment content and the overall yields (cell mass, pigment level, fermentation time). Most of the data are taken from the scientific or the patent literature, except for part of our own unpublished work with hydrocarbon-utilizing bacteria. The study of the carotenoids of Rhodobacter capsulatus is part of a collaborative project coordinated at the State University of Ghent (Professor Dr W. Verstraete, Faculty of Agricultural Sciences), in which the authors of this chapter were responsible for the analytical monitoring of the carotenoid levels in the course of the optimization work.

II FERMENTATION-MICRO-ORGANISMS SPECIFICALLY DESIGNED FOR PIGMENT PRODUCTION

1 CAROTENES: LYCOPENE

1.1 General

Lycopene, the major carotenoid in tomatoes is mainly used as a food colorant (KHiui & Bauernfeind, 1981). However, there is evidence indicating that its

Microbial Production of Carotenoids 55

presence in poultry feeds enhances the color of the egg yolk (Marusich & Bauernfeind, 1981).

1.2 Fungi

As discussed in this book (Chapter 3), the fungus Blakeslea trispora (Muco­rales) synthesizes massive amounts of p-carotene when the two sexual forms of this species are mixed in the same culture. However, there are several approaches to block the p-carotene biosynthesis at the lycopene level.

1. 2.1 Composition of the Growth Medium A medium with a neutral to alkaline pH favors lycopene production by Blakeslea, whereas at more acidic pH's p-carotene is formed as the pre­dominating pigment (Ciegler, 1965; Ninet & Renaut, 1979). In a 1963 patent, a yield of 0·15 g lycopene per liter was claimed, using a broth containing glucose, oleic acid, fish stick liquor and sodium carbonate (Ciegler, 1965; Ninet & Renaut, 1979). The latter was added to maintain the pH above 6·6.

1. 2. 2 Use of Inhibitors As mentioned above, substituted amines and various nitrogenous heterocyclic compounds block the cyclization of lycopene to p-carotene (Ninet et al., 1969; Spurgeon & Porter, 1983). The addition of CPTA to the growth medium indeed results in the accumulation of lycopene in Blakeslea trispora (Hsu et al., 1972). The maximum yield achieved so far, in a patented 5-day fermentation process, was 0·7 g lycopene/liter (Ninet & Renaut, 1979). In a less efficient process using the more readily available triethylamine as an inhibitor, 0·4 g lycopene/liter was produced in a medium based on cotton-seed, cornflour and soybean oil (Ninet & Renaut, 1979). Ninet et al. obtained about 1 g lycopene per liter by adding 0·1 % piperidine to a medium containing, amongst others, soybean oil, cotton-seed oil, kerosene and p-ionone (Ninet et aI., 1969).

1. 2. 3 Mutants Mutants could be prepared which lack the ability to convert lycopene to p-carotene. However, Blakeslea trispora lends itself reluctantly to mutation (Ninet & Renaut, 1979).

1.3 Non.Photosynthetic Bacteria

Lycopene production (0·5 g/liter in 6 days) by a mutant of Streptomyces chrestomyceticus, subsp. rubescens has been reported in the patent literature (Ninet & Renaut, 1979). The medium used contained starch, soybean flour and ammonium sulfate.

2 XANTHOPHYLLS

2.1 General

Among the xanthophylls, the hydroxylated derivatives lutein and zeaxanthin in particular are of high interest as key components in natural pigmenters for

56 H. J. Nelis & A. P. De Leenheer

poultry (Marusich & Bauernfeind, 1981). Important vegetable sources of this pigment include alfalfa meal, corn gluten meal and marigold flowers (Marus­ich & Bauernfeind, 1981). Zeaxanthin acts synergistically with lutein in that it intensifies the egg yolk color obtained with the yellow base pigment. Some micro-organisms containing xanthophylls of unspecified nature have also been tested as pigmenters in poultry feed. However, legal considerations may restrict their practical application.

2.2 Green Algae

In the early 1960s, alga meals were extensively studied for broiler and yolk pigmentation, as alternatives to other xanthophyll sources like com and alfalfa meal. So far the green alga Spongiococcum excentricum represents the only example of a micro-organism industrially exploited for the production of natural xanthophyll (lutein). For some time, the dried fermentation meal of this species, containing 1·6-2·4 g of xanthophylls per kg dry mass was marketed in the US under the name of A-Zanth (Grain Processing Co., Muscatine, Iowa) (Anonymous, 1962). The alga was grown under hetero­trophic conditions, at 28°C, in a medium containing glucose, com steep liquor, urea and mineral salts (Ciegler, 1965; Hanson, 1967). Incremental feeding of nutrients was required because Spongiococcum does not tolerate high sugar concentrations. Another relative disadvantage is that this alga grows slowly in liquid medium so that ample time must be allowed for inoculum development (Hanson, 1967). Lutein was produced at a rate of 294 mg/liter in 9 days.

Of all other green algae tested Chlorella sorokiniana (pyrenoidosa, 7-11-05) in particular showed great promise for further development. Cell yield and xanthophyll content of this species, grown under heterotrophic conditions in 30-liter fermenters, were optimized by Theriault (Ciegler, 1965; Theriault, 1965; Hanson, 1967). Dry cell weights in excess of 100 g/liter and a total xanthophyll content of 467-512 mg/liter were obtained from 230 to 260 g glucose/liter, in 168 h illuminated batch-type fermentations (25°C). Apart from glucose, the medium constituents were urea, phosphate, MgS04·7H20 and trace elements. The yield was further increased in continuous feed runs to 302 g cells/liter and 650 mg xanthophyll/liter, obtained from 520 g/liter of glucose. Again, maximum concentrations of xanthophylls resulted from using low initial levels of nutrients, followed by incremental feeding of additional medium ingredients.

In a patent assigned to F. Hoffmann-La Roche, another, thermophilic strain of Chlorella pyrenoidosa (A 119 Lagos, ATCC 14860) grown at 35°C was used. The culture medium consisted of cerelose, crude invert molasses, ethanol, potassium nitrate, amino acids, guanidine nitrate, MgS04 ·7H20, phosphates, acetate, EDTA salts and thiamine (Wendall et ai., 1964). Although the total carotenoid content amounted to 7·4 mgt g dry weight, the cell yield (28 g/liter in 4 days) was lower than in the process of Theriault.

Microbial Production of Carotenoids 57

2.3 Fungi

Compared to algae, certain fungi belonging to the Dacrymycetaceae family are inferior sources of xanthophylls (Ciegler, 1965; Ninet & Renaut, 1979). In a patented process, the yield did not exceed 40 mg/liter broth (4 mg/g dry biomass, fermentation time 5 days). A typical medium contained glucose, glycerol and corn steep liquor as main ingredients. External illumination was found obligatory for optimal pigment formation, the wavelength determining the ratio of xanthophylls to total carotenoids and the yield of xanthophylls.

2.4 Non-photosynthetic Bacteria

In view of the relative shortage of zeaxanthin in higher plants, microbial production of this valuable carotenoid is of high interest. Only few bacterial species are known to produce free (non-glycosidic) zeaxanthin, among them certain methylotrophs (Urakami & Komagata, 1986) and marine Flavobacteria (McDermott et al., 1973). The latter in particular could be candidates for industrial application. When no special measures are taken, fermentation of a Flavobacterium sp. in a medium containing glucose and corn steep liquor generates approximately 10-40 mg zeaxanthin per liter (Ninet & Renaut, 1979). The yield was increased to 335 mg/liter by supplementation with palmitic esters, methionine, pyridoxine, ferrous salts, by continuous addition of the F nutrients and reduction of the temperature (Ninet & Renaut, 1979).

Some actinomycetes produce high amounts of mostly unidentified xanth­ophylls. Streptomyces chrestomyceticus var. aurantioideus reportedly yields 0·5 g pigment/liter (Ninet & Renaut, 1979). Addition of the dry mycelium to poultry feed was found to 'improve pigmentation' (Anonymous, 1969-1970a). Even higher yields (up to 0·9 g/liter) were obtained with Streptomyces mediolani, grown in a medium based on dextrin, casein and corn steep liquor (fermentation time 5 days) (Ninet & Renaut, 1979). The predominating carotenoids produced were allegedly isorenieratene (an aromatic carotene) and its oxygenated derivatives. However, the efficiency of these compounds as pigmenters in animal feeds has not been established.

Mycobacterium phlei deserves a special mention in that this species produces carotenoids in quantities far exceeding those normally found in non­photosynthetic bacteria. Unfortunately, there is some confusion about its exact pigment composition, possibly because different strains have been investigated. Schlegel reported the presence of mainly glycosides of oxycarotenoids in amounts exceeding 10 mg/g dry cell weight (Schlegel, 1959). The media used consisted of peptone, glucose, glycerol, sodium citrate, magnesium sulfate and phosphates. Ingraham, in a 1935 study (Ingraham & Steenbock, 1935), found that the addition of glycerol and various alcohols and glycols to a similar basic medium markedly stimulated carotenogenesis, although the pigments isolated were different. Other studies reported isorenieratene (leprotene), various carotenes and some xanthophylls as the major pigments (Goodwin &

58 H. J. Nelis & A. P. De Leenheer

Jamikorn, 1956; Hochmannova et al., 1968). More recent patents describe the production of 0·3 g 'oxycarotenoids' /liter in a medium containing beet molasses and urea (Anonymous, 1966-1967, 1969-1970b; Ninet & Renaut, 1979).

3 MONOCYCLIC KETOCAROTENOIDS

3.1 General

Monocyclic, mono-ketocarotenoids have not been systematically studied so far for application as pigmenters in animal feeds. One of the reasons may be that they are not widespread in nature. However, the color (Amax 470-480 nm) and chemical properties (keto group in conjugation with the polyene chain) of these compounds resemble those of their bicyclic counterparts, e.g. canthaxan­thin. Hence they could theoretically become useful if a sufficient supply could be secured.

3.2 Non-photosynthetic Bacteria

Keto-derivatives of y-carotene and dehydro-y-carotene have been demon­strated in radio-resistant pink tetracocci, e.g. Deinococcus radiophilus and radiodurans (formerly designated as Micrococcus) (Bamji & Krinsky, 1966; Lewis & Kumta, 1973). Our own (unpublished) investigation of Deinococcus radiopugnans ATCC 19172 (formerly Micrococcus roseus) also suggested the presence of monocyclic ketocarotenoids in this related species. A systematic study has been devoted to optimizing the growth during fermentation of tetracocci (Shapiro et al., 1977). The iron content of the medium proved to be a crucial factor in this regard. Various compounds increasing the availability of this element, e.g. hydroxamic acids and hemin, acted as growth promoters. Although no quantitative data on the pigments are available, the mere presence of these unique ketocarotenoids and the exotic nature of the bacteria appear to warrant further study.

Similar monocyclic ketocarotenoids have been reported in a Mycobacterium smegmatis strain, grown on hydrocarbons (Tanaka et al., 1968a,b». The effect of various nutrients and added compounds (amino acids, detergents) on growth and pigmentation was examined. The optimal medium contained 2% of an alkane (preferably n-hexadecane), a mixture of ammonium sulfate and urea, or, alternatively, ammonium phosphate, as a nitrogen source, a number of mineral salts and trace elements as well as histidine. However, the maximum carotenoid yield was disappointingly low (0·8 mg/liter). A non­specific part of this consisted of 4-keto-y-carotene and its mono-hydroxy- and mono-methoxy derivatives.

4 BICYCLIC KETOCAROTENOIDS

4.1 General

Canthaxanthin and astaxanthin are widely used as pigmenters in poultry and fish feeds (see above). Although both compounds are manufactured syntheti-

Microbial Production of Carotenoids 59

cally, their high market value makes any suitable natural source potentially attractive. Canthaxanthin is found in substantial quantities in Crustacea (Castillo et al., 1982) and the feathers of birds (Brush, 1981), but not in higher plants. The fungus Cantharellus cinnabarinus, the first discovered natural source of canthaxanthin (Haxo, 1950) can be dismissed from a practical point of view. Astaxanthin occurs in the feathers of birds (Brush, 1981) and flower petals of Adonis annua (Seybold & Goodwin, 1959) and is abundant in Crustacea (Castillo et aI., 1982) and fish (Simpson et al., 1981). Apart from crustacean products (shrimp meal, crayfish waste), no producing organism has been used commercially (Marusich & Bauernfeind, 1981; Simpson et al., 1981).

Although strictly speaking the retrocarotenoid rho do xanthin also belongs to the ketocarotenoids, this compound is discussed in a separate paragraph (see Section 5).

4.2 Cyanobacteria

In view of the revival of interest in the biotechnological potential of Cyanobacteria (formally named blue-green algae) (Lem & Glick, 1985) the large-scale ketocarotenoid production from certain species belonging to the Nostocaceae (Table 1) could theoretically be envisaged. No systematic study of the usefulness of Cyanobacteria as industrial sources of canthaxanthin has been conducted so far. However, the secretion of deadly toxins by certain Nostocaceae (Lem & Glick, 1985) could obviously be prohibitive for any practical application.

4.3 Green Algae

As mentioned in Section I, 7.1.1(A), many green algae, which normally contain the xanthophylls typical of photosynthetic tissues, synthesize secondary (keto)carotenoids at the end of their growth phase, when certain essential nutrients, particularly nitrogen, become depleted (Czygan, 1968). The nature of the ketocarotenoid( s) varies with the type of algae and not· all species synthesize them (Czygan, 1968). Astaxanthin is the most common secondary pigment, mostly accompanied by canthaxanthin and echinenone. The obvious practical potential of secondary carotenoid formation is probably outweighed by the slow nature of the process and the low levels obtained. The two-phase culturing on laboratory scale first involves proliferation of the algae in a mineral medium containing an ample amount of a nitrogen source, e.g. potassium nitrate. Production of ketocarotenoids starts after replacement of the growth medium by a 'production medium' low in nitrate. Heterotrophic conditions (addition of carbohydrates) accelerate the process indirectly by stimulating growth (and primary carotenoid formation), thus causing the nitrogen supply to be exhausted sooner.

60 H. 1. Nelis & A. P. De Leenheer

4.4 Fungi and Yeast

4.4.1 Importance of Phaffia rhodozyma The red yeast Phaffia rhodozyma is unusual among the Deuteromycetes in many respects. Unlike other genera like Rhodotorula, Rhodosporidium and Sporobolomyces, it has the property of fermenting various carbohydrates (Andrewes et al., 1976). In addition, it contains none of the pigments typical of red yeasts (p-carotene, y-carotene, torulene, torularhodin), but rather as­taxanthin (Andrewes et al., 1976). Apart from the genus Peniophora (Hymenomycetes) (Arpin et al., 1966), this carotenoid has never been demonstrated in fungi. The occurrence of astaxanthin is even more unique in that its absolute configuration (3R, 3' R) is opposite to that of astaxanthin from other natural sources (3S,3'S) (Andrewes & Starr, 1976). In general, the chirality of a carotenoid is indeed considered not to be source dependent (Andrewes & Starr, 1976).

Several observations indicating the efficiency of Phaffia rhodozyma as a pigmenter in salmonid and crustacean diets (Johnson et al., 1977, 1980a) and poultry feeds (Johnson et al., 1980b) created much excitement about this organism. It proved superior to crustacean waste products as a feed additive because of its higher nutritional value and pigment content. These promising results appeared to warrant a thorough feasibility study of Phaffia rhodozyma as a potential industrial source of natural astaxanthin.

4.4.2 Fermentation of Phaffia rhodozyma The goal of such a feasibility study was 3-fold: optimization of pigment production, a search for cheaper media and the improvement of the extrac­tability of astaxanthin from the yeast cells.

(A) OPTIMIZATION OF THE GROWTH MEDIUM (Johnson & Lewis, 1979) A 'standard medium' for the propagation of Phaffia rhodozyma contained cerelose, ammonium sulfate, KH2P04 , MgS04 ·7H20, CaClz·2H20 and yeast extract. Essentially, astaxanthin formation is growth-associated, but its pro­duction still continues when growth has stopped upon exhaustion of glucose. An optimum pH of 4·5 yielded maximum cell weight, growth rate and astaxanthin levels. Of all carbon sources tested cellobiose afforded maximum astaxanthin yield but less biomass and a lower growth rate than glucose. However, increasing concentrations of the latter as well as reduced aeration, both suggestive of fermentative conditions, were inhibitory to astaxanthin production but rather caused less oxygenated precursors like echinenone and p-carotene to accumulate. This is consistent with the fact that cellobiose, which can only be utilized aerobically, is stimulatory. Unlike the carbon source, the nature of the nitrogen source affected growth and carotenoid formation only to a very limited extent. Light was totally without effect. The addition of yeast extract to a vitamin-free medium increased pigmentation by a factor of about 4. Carotenogenesis was strongly promoted (astaxanthin level of

Microbial Production of Carotenoids 61

8141lg/g dry weight) in the presence of tomato waste. This can be rationalized in terms of the uptake by the yeast cells of carotenoid precursors. Typical astaxanthin yields are of the order of 2 mg/liter, obtained in a 6O-h fermentation.

(B) USE OF CHEAP MEDIA

Attempts to grow Phaffia rhodozyma on cheap food-processing waste products have been restricted so far to the use of alfalfa residual juice as a substrate for yeast propagation (Okagbue & Lewis, 1984a). This material supports appreci­able growth but totally inhibits astaxanthin formation, which has been attributed to the presence of saponins (Okagbue & Lewis, 1984b). Saponin presumably disturbs the integrity of the plasma membrane of the cells. The effect is indeed partially reversed by unsaturated acids and sterols.

(C) EXTRACfABILITY OF ASTAXANTIUN

A factor seriously limiting the efficient utilization of the astaxanthin from Phaffia rhodozyma by fish and poultry is the high resistance of its cell wall. No pigment is absorbed from intact cells (Johnson et al., 1978). Mechanical rupture is not feasible for large-scale extraction and chemical hydrolysis of the cell wall destroys the astaxanthin. Autolysis of the yeast cells by incubation in distilled water or a citrate buffer has been proposed as a method to facilitate extraction (Okagbue & Lewis, 1984c). Enzymatic digestion using the ex­tracellular enzymes produced by Bacillus circulans is another useful alternative (Johnson et al., 1978; Okagbue & Lewis, 1981, 1985). This can be accom­plished in a 2-step method, involving the cultivation of Phaffia rhodozyma, followed by heat-inactivation of the yeast cells, pH-adjustment and inoculation of Bacillus circulans (Johnson et al., 1978). However, during heat treatment part of the astaxanthin is lost and readjustment of the medium pH is laborious and impractical. These drawbacks are overcome by growing the two organisms in mixed culture (Okagbue & Lewis, 1981, 1985). Carbohydrates cheaper than glucose, e.g. molasses can be used as carbon sources for this purpose (Okagbue & Lewis, 1981). Strict pH control remains however critical to survival and lytic activity of the bacillus (Okagbue & Lewis, 1985). After growth, the biomass is directly amenable to extraction with acetone, resulting in astaxanthin yields of over 90%. Unfortunately, the overall production of astaxanthin is partly suppressed in mixed culture (maximum yield 1·2-1·5 mg/liter) (Okagbue & Lewis, 1981, 1985). A practical advantage of the mixed culture approach is the possibility for re-use of the cell-free culture liquid (Okagbue & Lewis, 1985). After fortification with nutrients, this medium supports growth of Phaffia rhodozyma and at the same time retains the lytic activity required to modify its cell walls. Alternatively, this culture fluid can also be used to digest the walls of yeast cells previously grown in pure culture. A scheme both including mixed culture and recycling of culture filtrate was developed for the enzymatic large-scale processing of Phaffia rhodozyma for inclusion in animal diets (Okagbue & Lewis, 1981, 1985).

62 H. J. Nelis & A. P. De Leenheer

4.5 Non-photosynthetic Bacteria

The phenomenon of carotenoid production by members of the CMN complex (Corynebacterium-Mycobacterium-Nocardia) grown on hydrocarbons has been recognized for a long time (Haas et al., 1941; Haas & Bushnell, 1944; Davis, 1967). The ketocarotenoids astaxanthin and canthaxanthin occur in a limited number of those hydrocarbon utilizing bacteria.

4.5.1 Brevibacterium KY-4313: Canthaxanthin

(A) BASIC WORK

Brevibacterium KY-4313 contains canthaxanthin and its biosynthetic precur­sors echinenone and p-carotene as the predominating carotenoids (Sakurai et al., 1971). This organism has been used as a model to study the biosynthesis of canthaxanthin (Hsieh et al., 1974). However, Japanese investigators, who apparently had designed the bacterium mainly for petrochemical applications, became aware of its industrial potential as a natural source of canthaxanthin. Thus, a systematic optimization of the pigment level through variation of the composition of the growth medium was undertaken (Tanaka et al., 1971). Growth and pigment production were found to be intimately related. Optimal growth (maximum cell yield about 3·5 g/liter) and maximum canthaxanthin levels (0·6 mg/g, 1-2 mg/liter) resulted from using a basic medium containing 2-4% octadecane, 0·25% NH4H 2P04 , phosphates, magnesium sulfate and minerals (pH 7), supplemented with vitamin B12 , a natural nutrient, e.g. malt extract (0·1 %) and a small amount of a non-ionic detergent (0·01 % Tween 40). High carbon to nitrogen ratios are known to favor the production of cellular lipid in hydrocarbon fermentation (Ratledge, 1970). Accordingly, formation of canthaxanthin was also promoted under these conditions. Light did not affect carotenogenesis, while strong aeration was slightly inhibitory.

(B) OPTIMIZATION STRATEGY (Nelis & De Leenheer, unpublished) Hydrocarbon fermentations suffer from a number of drawbacks (Einsele, 1983), including poor yields (low cell weights and slow growth rate), high costs when pure alkanes are used and complex processing of the product (see Section IV, 1). Pigment production by bacteria grown on paraffins is said to be 'very low' (Ninet & Renaut, 1979). Yet the figures cited above for canthaxan­thin are not far below the astaxanthin levels obtained in Phaffia rhodozyma, an organism which continues to be advocated as commercially attractive (Ok­agbue & Lewis, 1985). In our hands Brevibacterium KY-4313 tended to yield more pigment in hydrocarbon than in non-hydrocarbon media. Therefore, a systematic study was devoted to optimizing the canthaxanthin levels and promoting the bacterial growth during hydrocarbon fermentation.

(a) Stimulation of carotenogenesis:

1. Optimization of the growth medium: Variation of the basic medium constituents only marginally increased the canthaxanthin yield. Minor positive

Microbial Production of Carotenoids 63

effects were noted from combining the optimal carbon source, octadecane or (cheaper) paraffin mixtures enriched (50-90%) in this compound, with certain branched alkanes (pristane) or hydrocarbon mixtures rich in cycloalkanes. Ammonium phosphate, ammonium acetate and sodium nitrate were nearly equally effective as nitrogen sources. Yeast extract was slightly superior to malt extract as a growth factor. The incorporation in the medium of chemicals supposedly acting as precursors of carotenoid biosynthetic intermediates had little or no effect. Examples of such compounds include acetate, pyruvate, farnesol, geraniol, leucine and various sources of acetyl CoA. In addition, Brevibacterium KY-4313 proved refractory to the action of a variety of chemicals, which are reportedly carotenogenic in other micro-organisms, particularly Blakeslea trispora: nitrogenous heterocyclic compounds, vegetable oils, /3-ionone, retinol, aromatics. However, in non-hydrocarbon medium (Brain Heart Infusion Broth) a number of substances were found to promote substantially carotenoid biosynthesis, particularly alcohols (propanol and isopropanol), glycerol and retinol. The latter caused accumulation of /3-carotene rather than canthaxanthin. Vegetable oils greatly stimulated growth but suppressed carotenoid formation.

2. Preparation of mutants: Following treatment of Brevibacterium KY-4313 with the mutagen NMU (Voznyakovskaya & Daraseliya, 1972), we isolated a colony that distinguished itself from the bulk of the orange ones by its reddish hue. This 'new', presumably mutant strain, contained about twice as much canthaxanthin as the parent strain, but also more echinenone. Retreatment of the 'new' strain with the same mutagen again permitted the isolation of a slightly more pigmented colony. The latter was subsequently used for all optimization work.

(b) Growth promotion: In view of the existing positive relationship between growth and pigmentation, growth promotion was crucial to increase the overall productivity of canthaxanthin. Two major factors are growth-limiting in hydrocarbon fermentation, i.e. the availability (dispersion) of the insoluble alkane (Einsele & Fiechter, 1971) and the secretion of toxic metabolites (Einsele & Fiechter, 1971; Yamada et al., 1971). The effective dispersion of octadecane, a liquid at the working temperature (30°C) was facilitated by thorough mechanical stirring and the addition of small amounts of a non-ionic detergent.

A major breakthrough came from removing the toxic metabolites, sup­posedly carboxylic acids, produced in the course of the bacterial growth. In one fermentation on gaseous hydrocarbons, detoxification was accomplished using continuous dialysis culture (Coty, 1968). As a result, the cell yield in this particular example dramatically increased from 4 to 47 g/liter. However, dialysis represents technical problems when liquid alkanes are used. We mimicked its effect by regularly (each time when the pH of the medium dropped to a constant minimum value) renewing the aqueous component of the medium. The separation of the aqueous broth from the hydrocarbon layer,

64 H. 1. Nelis & A. P. De Leenheer

containing the cells, is very convenient because, after discontinuing the stirring, the latter is floating on top of the medium. This semi-continuous approach yielded dry cell weights of the order of 8-14 g/liter, as opposed to 3-4 g in the absence of the aqueous medium renewal. Concurrently, the ketocarotenoid yield increased to 13 mg/liter (1-1·5 mg/g dry weight), of which 70% was canthaxanthin and the remainder echinenone, obtained from 50 ml of hydrocarbon (working volume of the fermenter, 1·25 liters) in a 7 -day run. This figure compares favorably with the astaxanthin yield obtained from Phaffia rhodozyma (1·3-1· 5 mg/liter in 60 h using the mixed culture conditions described above).

4.5.2 Rhodococcus maris/Mycobacterium brevicale As part of an investigation of hydrocarbon-oxidizing marine bacteria (Koro­nelli et al., 1981), Russian workers isolated a pigmented Mycobacterium brevicale strain (32-MCT) (Koronelli et al., 1982), later found to be synony­mous with Rhodococcus maris (Koronelli et al., 1987). This organism contains a dark orange peptidolipid with canthaxanthin as the main carotenoid (Koronelli et al., 1987; Pachlavuni, 1987). Details on culturing conditions were not available at the time of writing, except that the organism was grown in a mineral medium containing hexadecane as a carbon source (Pachlavuni et al., 1987). Rhodococcus maris / Mycobacterium brevicale 32-MCT is apparently not equivalent with the Rhodococcus maris type strain described by Nesterenko (Nesterenko et al., 1982). The latter also grows on various hydrocarbons and produces canthaxanthin as well (personal observation, unpublished). However, pigment levels were lower than in Brevibacterium KY-4313 and reproducibility of carotenoid formation and growth on hydrocarbons was poorer (personal observations, unpublished).

4.5.3 Miscellaneous Hydrocarbon-Utilizing Bacteria Mycobacterium lacticola was the first bacterium reported to form astaxanthin in hydrocarbon medium (Haas & Bushnell, 1944). Remarkably, the pigment was absent when the organism was grown on nutrient agar. Brevibacterium 103 is the second known example of a hydrocarbon utilizing bacterium that produces astaxanthin (Iizuka & Nishimura, 1969). A mineral medium containing 8% kerosene yielded approximately 3 g cells/liter, with a pigment content of 30 ",g/g dry cell weight. In view of this poor yield and the availability of Phaffia rhodozyma, there appears to be no future for either of the above bacteria as a useful source of astaxanthin.

4.5.4 Micrococcus roseus: a controversy The presence of canthaxanthin and other ketocarotenoids has also been reported, by one particular research group, in Micrococcus roseus (A TCC 516) grown in a variety of (non-hydrocarbon) media (Cooney et al., 1966; Ungers & Cooney, 1968; Schwartzel & Cooney, 1970, 1972, 1974a,b; Ascenzi & Cooney, 1975; Dieringer et al., 1977; Cooney & Berry, 1981). However, despite this

Microbial Production of Carotenoids 65

extensive evidence, we as well as others (unpublished results) have repeatedly failed to confirm this finding. We re-investigated the pigment composition of Micrococcus roseus ATCC 516 and of the original strain, donated by Cooney, using HPLC and photodiode array detection (Nelis & De Leenheer, 1987). Although a chromatographic peak corresponding in retention to canthaxanthin was observed, its absorption spectrum, recorded with the aid of a photodiode array detector (see Section IV, 2) was in no way consistent with this structure. None of the pigments separated displayed an absorption spectrum indicative of any ketocarotenoid. The reason for this extreme discrepancy of results has remained obscure so far.

5 RETROCAROTENOIDS: RHODOXANTHIN

5.1 General

By virtue of its deep red color, rhodoxanthin, which has been approved as a food additive in certain countries, might be envisaged as an alternative to astaxanthin. The compound occurs in autumn leaves (Ida, 1981), fish (Kat­suyama & Matsuno, 1979) and the feathers of birds (Brush, 1981).

5.2 Non-Photosynthetic Bacteria

Rhodoxanthin was tentatively identified as the main carotenoid in the methylotroph Pseudomonas extorquens (Downs & Harrison, 1974) (obviously equivalent with Protomonas extorquens (Urakami & Komagata, 1986». The pigment was formed during growth in a mineral medium consisting of ammonium sulfate as a nitrogen source and methanol or glycerol as a carbon source. Oxygen and magnesium limitation caused a marked increase in cell pigmentation, suggesting that conditions of growth restriction in general tend to divert the available carbon to pigment synthesis. However, no attempts have been made to quantitate or to optimize the actual carotenoid content.

III FERMENTATION-MICRO-ORGANISMS DESIGNED FOR APPLICATIONS OTHER THAN PIGMENT PRODUCTION

1 INTRODUCTION

Three selected examples will be briefly discussed of carotenoid-containing micro-organisms whose primary biotechnological potential lies in a field other than pigment production. The carotenoids present in those bacteria and yeasts have in general not been approved yet as additives for feeds or foods. However, this situation could obviously change in the future if these organisms gain acceptance for large-scale biomass or single lipid production and, hence, addition to animal feeds.

66 H. 1. Nelis & A. P. De Leenheer

2 FUNGI: RED YEASTS

Various red yeasts belonging to the genera Rhodotorula and Rhodosporidium (Basidiomycetes) have been considered as industrial sources of single cell protein (Lichtfield, 1979; Costa et al., 1984) and microbial fat (Ratledge, 1982). These organisms can be grown on cheap substrates, e_g. domestic seawage, potato hydrolysate, whey, molasses, etc_ (Lichtfield, 1979; Ratledge, 1982).

Concurrently with fat accumulation, enhanced carotenoid synthesis can be reasonably expected_ The major carotenoids in Rhodotorula and Rhodosporidium are p-carotene, j'-carotene, torulene and torularhodin (Simp­son et al., 1971; Goodwin, 1972). Supplementation of poultry feeds with preparations of Rhodotorula mucilaginosa were found to promote egg yolk pigmentation (Schwarz & Margalith, 1965). This yeast was propagated in glucose-peptone-yeast extract media or, alternatively, in chemically defined media containing a carbohydrate (5-10% glucose, beet molasses), ammonium sulfate, urea and/or ammonium lactate as nitrogen sources, as well as minerals and growth factors (yeast extract, thiamin). The total carotenoid yield on a dry weight basis was as high as I-Smg/g (corresponding to 49mg/liter). Unlike in Blakeslea trispora, the addition of p-ionone or kerosene inhibited carotenoge­nesis, whereas non-ionic detergents were only slightly stimulatory. However, a disadvantage of using Rhodotorula mucilaginosa biomass as an additive for poultry feed was the appearance of a violet hue in the egg yolk, caused by the presence of torularhodin (Margalith & Meydav, 1968). Therefore, subsequent efforts were directed towards reducing the torularhodin/torulene ratio. This could be accomplished by adding diphenylamine, ethanol or isopropanol to a glucose-ammonium lactate medium (Margalith & Meydav, 1968). Unlike diphenylamine, the latter two additives also promoted the total carotenoid yield, which however remained below 0-4 mg/g dry weight. The minimal torularhodin/torulene ratio was 0-33, vs 1-00 in the absence of additives. Other workers obtained a value of 0·48 using a glucose-asparagine medium (Peterson et al., 1958). Both figures contrast with the unacceptably high ratio of 6-12, resulting from the use of sucrose-ammonium sulfate (Villoutreix, 1960)_

3 NON-PHOTOSYNTHETIC BACTERIA: METHYLOTROPHS

A number of methylotrophic bacteria are known vitamin B12 producers (Toraya et al., 1975; Sato et al., 1977; Shimizu et al., 1982; Dumenil et al., 1983) and potentially useful sources of single cell protein (Lichtfield, 1979, 1980). The commonly encountered pink color of these bacteria is due to the presence of, mostly unidentified, carotenoids (Shimizu et al., 1982; Dumenil et al., 1983; Urakami & Komagata, 1986), sometimes of a rhodoxanthin-like nature (Downs & Harrison, 1974; Urakami & Komagata, 1986) (see also

Microbial Production of Carotenoids 67

Section 11,5). Attempts have been made to optimize the carotenoid yield from selected methylotrophs by altering the culturing conditions. A Corynebacterium sp. XG was found to form most pigment on methanol, dimethyl amine and methylamine as carbon sources (Dumenil et al., 1983). Other medium ingredients included ammonium sulfate, minerals and yeast extract. Light, pH variation and the nature of the growth factor did not appreciably affect pigmentation. Protaminobacter ruber, another facultative methylotroph, produced 2 mg/liter of an unidentified pink carotenoid in a medium containing 1,2-propanediol as a carbon substrate and methionine and riboflavine as moderately effective carotenogenic agents (Shimizu et al., 1982).

Both examples demonstrate the interest in methylotrophic bacteria for their carotenoid composition. Although their primary biotechnological potential clearly lies in the production of vitamin B12 and biomass, the presence of useful pigments again adds extra potential value to these organisms.

4 PHOTOSYNTHETIC BACTERIA

4.1 General

Purple photosynthetic bacteria, particularly Rhodobacter capsulatus (formerly Rhodopseudomonas capsulatus, Imhoff et al., 1984) are useful sources of single cell protein (Kobayashi & Tchan, 1973; Shipman et aI., 1977; Kobayashi & Kurata, 1978; Lichtfield, 1979, 1980, 1983; Driessens et aI., 1987). Their ability to grow on waste solutions makes them attractive for the purification of polluted industrial effluents, giving highly nutritious cells as a valuable by-product (Kobayashi & Tchan, 1973). Apart from its general nutritional value, as evidenced from its amino acid profile (Kobayashi & Kurata, 1978; Driessens et al., 1987) Rhodobacter capsulatus reportedly also promotes egg production in chickens and increases the survival of fish (Kobayashi, 1972; Kobayashi & Tehan, 1973).

Although the nature of carotenoids in Rhodobacter capsulatus has been established for a long time (for reviews see Liaaen-Jensen, 1978; Schmidt, 1978; Goodwin, 1980), Driessens & Verstraete were the first to recognize the potential of this organism as a useful natural source of pigments (Driessens et al., 1987 and unpublished results). Spheroidene is the predominating caroten­oid in Rhodobacter capsulatus when grown anaerobically, whereas under aerobic conditions this compound is further oxidized to the ketocarotenoid spheroidenone (Goodwin 1980; Spurgeon & Porter, 1983). Because of its favorable color characteristics, the latter in particular is interesting from a practical! economical standpoint. In the course of a thorough study of optimal biomass production by Rhodobacter capsulatus, the co-production of caroten­oids was also examined (Driessens et al., 1987).

68 H. J. Nelis & A. P. De Leenheer

4.2 Production of Carotenoids by Rhodobacter capsulatus

4.2.1 Batch culture Initially, when Rhodobacter capsulatus was grown photo heterotrophically in batch culture (lactic acid as carbon source, ammonium sulfate as nitrogen source, anaerobic conditions and illumination) high pigment contents (5-10 mg/g) were obtained. Spheroidene represented, as predicted, the major carotenoid (>85%), whereas spheroidenone was only present in amounts ranging from 1 to 5%. Subsequent attempts were, for unknown reasons, less successful. Under anaerobic conditions, the maximum levels of spheroidene and sphero­idenone were 1 and 0·3 mg/g, respectively. Corresponding values obtained in aerobic culture were 0·05 and 0·4 mg/g, respectively. Unexpectedly, in one anaerobic fermentation cells containing 0·6 mg spheroidenone and virtually no spheroidene were produced.

However, batch fermentations of Rhodobacter capsulatus are associated with low cell yields (1-2 g/liter) (Kobayashi & Kurata, 1978; Lichtfield, 1983).

4.2.2 Culture in a continuous flow reactor (Driessens et aI., 1987) Culture of Rhodobacter capsulatus in a continuous flow-through reactor, under external illumination and microaerophilic conditions, yielded 5-10 times higher biomass production rates. The photobioreactor used consisted essen­tially of a glass tube having a PVC decanter on top and two ports at the bottom, one for the removal of excess biomass and one for the entry of (recirculated) medium. The PVC decanter contained ports for effluent removal, gas outlet, medium recirculation and nitrogen flushing (when required). Two non-axenic feed solutions were supplied separately, by means of a peristaltic pump. The first one contained the buffered carbon source (preferably calcium lactate) and the second one the nitrogen source (am­monium sulfate) plus minerals, growth factors as well as selective inhibitors of sulfate reducing bacteria (sodium molybdate) and algae (chloroxuron). The bacteria readily flocculated in the reactor, which permits easy harvesting.

The quality of the resulting biomass did not differ from the one obtained in batch culture. Under conditions of iron excess (4 mg Fe2 + /liter) about 1 mg pigment per g dry weight was produced, of which 0·7 mg was spheroidene and 0·3 mg spheroidenone. However, because of their high toxicity, the com­pounds added to suppress competing micro-organisms had to be eliminated, in order to make the end-product nutritionally acceptable. To maintain the non-axenic conditions and yet to avoid contamination the iron content of the medium had to be drastically lowered (down to 251lg Fe2 + /liter). This did not appreciably affect the growth, but dramatically altered the carotenoid profile. A number of unidentified, red lycopene-like compounds were now formed in amounts ranging from 0·4 to 0·8 mgt g, but neither spheroidenone or sphero­idene were present. A compromise in the iron concentration (with incremental addition) still resulted in a similar carotenoid pattern. Although the cells, when added to pOUltry feed, improved the color of the egg yolk without any signs of

Microbial Production of Carotenoids 69

toxicity (provided the sodium molybdate had been eliminated), the uncertainty about the exact pigment composition precludes, for the time being, the use of Rhodobacter capsulatus as a pigmenter for chickens.

IV PROCESSING-ANALYSIS-APPLICATION AND ECONOMICS

1 PRODUCT RECOVERY AND PURIFICATION

Because carotenoids in micro-organisms are localized intracellularly, their recovery essentially comes down to the isolation of the biomass. Well­established approaches for the harvesting of cells include filtration, centrifuga­tion and coagulation/flocculation (Belter, 1979). However, hydrocarbon fer­mentations pose a number of additional recovery problems due to the presence of residual alkanes (Levi et al., 1979; Einsele, 1983). After separation of the aqueous and the organic phases by centrifugation or flotation, often in conjunction with specialized techniques (Various patents, 1973), the residual hydrocarbon, adsorbed on the cells, has to be removed. Solvent extraction or treatment with surfactants have been suggested for this purpose (Einsele & Fiechter, 1971). However, this purification will cause a very substantial part of the carotenoids to leach from the cells and to dissolve in the hydrocarbon (paraffin) or the extraction solvent (personal observations). The resulting 'clean' cells will not only be defatted, but also be devoid of most of the carotenoids. The separation of the carotenoids from the hydrocarbon itself at a later stage is unfeasible. Therefore, in hydrocarbon fermentation a basic incompatibility appears to exist between the recovery of pure biomass and the quantitative isolation of the intracellular carotenoids.

If the dry biomass does contain the pigments (as in non-hydrocarbon fermentation) it could probably be directly added to animal feed, provided the carotenoid concentration is sufficiently high and no toxic compounds are present. In view of the instability of carotenoids, conditions for drying (spray­or flash-drying) should be as mild as possible, to avoid oxidation and cis /trans isomerization. The addition of antioxidants (BHT, ethoxyquin) might be desirable (Wood et aI., 1983). Alternatively, if the pigment quantity in the biomass is too dilute, solvent extraction is required for its isolation (useful organic solvents are mentioned in Section IV, 2).

2 ASSAY METHODS

Traditionally, the determination of carotenoids in biological materials, includ­ing micro-organisms, has always relied on spectrophotometry (Davies, 1976; Britton, 1985). A typical sample pretreatment consists of a total lipid extraction using such polar organic solvents as acetone or methanol, saponifi­cation to remove triglycerides, partition between hexane/methanol (separation

70 H. J. Nelis & A. P. De Leenheer

of non-polar epiphasic from more polar hypophasic derivatives) and open column chromatography (Davies, 1976; Britton, 1985). Each colored band eluting from the column is subjected off-line to spectrophotometric measure­ment at its Amax, with calculation based on the molar absorption coefficient of the (tentatively) identified compound of interest. Often, this complex purifica­tion of total lipid extracts is omitted when only one pigment is presumably present. That this may lead to errors is exemplified in the spectrophotometric assay of astaxanthin in Phaffia rhodozyma, where the interference of colored substances adsorbed from the medium onto the cells led to an overestimation of the astaxanthin content (Okagbue & Lewis, 1984a). This shows that a chromatographic fractionation is an essential step in carotenoid assays.

As a separation method, modern liquid chromatography on microparticulate materials (HPLC) has now superseded open column chromatography because of its superior resolving power, higher sensitivity and improved speed. HPLC has been used for the profiling and quantitation of carotenoids in algae (Braumann & Grimme, 1981; Mantoura & Llewelynn, 1983; Bowles et ai., 1985) and bacteria (Gillan & Johns, 1983; Kester & Thompson, 1984; Korthals & Steenbergen, 1985; Nelis & De Leenheer, 1987).

A recent development is the use of photodiode array absorbance detection (Miller et ai., 1982) in connection with the HPLC of carotenoids (Gillan & Johns, 1983; Mantoura & Llewelynn, 1984; Ruedi, 1985; Nelis & De Leenheer, 1986). This versatile technique permits a (tentative) identification of each chromatographic peak, based on its on-line recorded absorption spectrum. The need for such a second identity criterion (aside from com­parison with a standard compound) was clearly evidenced from the work with Micrococcus roseus (see Section III, 4.5.4).

One issue that is poorly addressed in the literature is the problem of the complete carotenoid extraction from micro-organisms. We observed that even repeated treatment of several bacteria, e.g. Brevibacterium KY-4313 and Micrococcus roseus with acetone or methanol failed to release all the pigment (Nelis & De Leenheer, 1986, 1987). Also, from streptococci, carotenoids were reportedly poorly extractable using a variety of organic solvents and even alcoholic potassium hydroxide (Merritt & Jacobs, 1978). In the latter case, efficient extraction was accomplished using a rather unusual mixture of glycerol-fenol. Other investigators added trichloroacetic acid (Downs & Harrison, 1974) or potassium hydroxide (Taylor, 1980) to promote the liberation of carotenoids from bacteria. In our hands a brief pretreatment of the cells with pure liquefied fenol was found to make them spectacularly more amenable to extraction with methanol: this approach resulted in a total bleaching of the residue, even for the most refractory species (Micrococcus roseus). Fenol can be removed from the extract by partitioning between diethyl ether and an alkaline water phase or, alternatively, by column chromatography on microparticulate C1S mini-columns (Bond Elut, Sep-Pak). The ether or the column eluate is subsequently evaporated to dryness and the residue is redissolved in the chromatographic solvent. An aliquot (50-100 Ill)

Microbial Production of Carotenoids 71

of the final mixture is injected on a reversed phase column, eluted with a non-aqueous mobile phase (Nelis & De Leenheer, 1983, 1986, 1987). When, as in the case of Brevibacterium KY-4313, hydrocarbons are co-extracted, a chromatographic step on a Bond Elut Alumina mini-column is included to separate the carotenoids (canthaxanthin) from the hydrocarbon.

3 CHEMICAL SYNTHESIS--BIOCATALYSIS

The profitability of the biotechnological production of carotenoids has to be judged in the light of the availability of alternative chemical routes for their preparation. Virtually all derivatives can be prepared by total synthesis in economically feasible processes (for an extensive review, see Mayer & Isler, 1971). This applies in particular to the carotenoids of high commercial interest, i.e. f3-carotene, canthaxanthin, astaxanthin, f3-apo-8' -carotenal and f3-apo-8'­carotenoic acid ethyl ester. Leading European manufacturers are F. Hoffmann­La Roche (Basle, Switzerland) and BASF (Ludwigshafen, FRG).

Basically, the C40 carotenoid skeleton is synthesized by combining two or three building units in symmetrical (e.g. C20 + C20, C 19 + C2 + C19 , CiS + C lO + CiS) or asymmetrical schemes (e.g. ~i + C19, C2S + CiS). The two most frequently used methods are the Wittig reaction (condensation of a carbonyl compound with a trimethylphosphonium halide) and the Grignard reaction (condensation of a carbonyl compound with a metal acetylide). New develop­ments continue to appear, particularly with respect to the total synthesis of optically active carotenoids (Mayer, 1979; Muller et aI., 1982). This is a new

A

2

1 2

Fig. 4. Reduction reactions involved in the preparation of an optically active key intermediate in the synthesis of (3R,3/R)-zeaxanthin. A, enantioselective hydrogena­tion of double bond, catalyzed by baker's yeast; B, chemical reduction; C, reduction of keto group, catalyzed by baker's yeast (unwanted side reaction). 1, desired reaction

product (4R, 6R); 2, unwanted reaction product (4S, 6R).

72 H. J. Nelis & A. P. De Leenheer

___ • (>Yo + ?ir' OH

HO~ HO~ OH 0

Fig. 5. Enantioselective reduction catalyzed by baker's yeast as a first step in the synthesis of (3S, 3'S}-astaxanthin.

area in which micro-organisms have found large-scale application. Synthetic procedures for (3R,3'R)-zeaxanthin and (3S,3'S)-astaxanthin indeed involve the use of micro-organisms as biocatalysts for the introduction of chiral centers, by taking advantage of the stereoselective course of microbially catalyzed reactions (Leuenberger, 1985). Thus the optically active synthon for the synthesis of (3R,3'R)-zeaxanthin has been prepared by a combined process consisting of enantioselective reduction of oxo-isophorone (hydroge­nation of double bond) with baker's yeast (Fig. 4A) and chemical hydrogen­ation (Fig. 4B). However, special precautions have to be taken in the microbial process to avoid reduction of the 4-keto group of oxo-isophorone (Fig. 4C). This secondary reaction leads to the unwanted S configuration at C-4. A synthetic route for the preparation of (3S,3'S)-astaxanthin also involves the use of baker's yeast to carry out an enantioselective reduction. In this particular reaction the 3-keto group of 4-hydroxy-6-oxo-isophorone is specifically attacked to yield a tautomeric mixture of optically active ketodiols (Fig. 5). Both reaction products can be converted chemically (acetonization) to a ketal, a key intermediate in the further synthesis of (3S, 3'S)-astaxanthin.

Another type of useful reaction catalyzed by micro-organisms is microbial resolution of racemates. In an improved synthetic process for (3S,3'S)­astaxanthin a Bacillus sp. selectively hydrolyzes the 3R enantiomer of racemic 3-acetoxy-4-oxo-fl-ionone, but leaves the desired S-enantiomer untouched (Fig. 6). The latter is subsequently isolated in high yield by physico-chemical methods.

1

2

Fig. 6. Microbial resolution by a Bacillus sp. of racemic 3-acetoxy-4-oxo-tJ-ionone, a key step in the synthesis of (3S,3'S}-astaxanthin. 1, unwanted reaction product (3S)

(hydrolyzed); 2, desired reaction product (3R) (untouched).

Microbial Production of Carotenoids 73

The efficiency of microbially catalyzed reactions can be enhanced by immobilization of the cells, the use of two-phase biocatalysis (for water insoluble substrates such as carotenoids) or via recombinant DNA technology.

4 APPLICATIONS AND ECONOMICS-FUTURE PROSPECTS

So far there are no examples of microbiological fermentations of carotenoids that are competitive with their total synthesis or their isolation from plants. A limited number of processes, e.g. the production of lutein from green algae have a mere historical value, as their development and use have been discontinued since the sixties. The production of lycopene from fungi is, to the best of our knowledge, not further developed at present. The most suitable candidates for fermentation are carotenoids with chiral centers, which are more difficult to synthesize than others. A process for the production of (3R,3'R)-zeaxanthin from Flavobacterium sp. appears to approach the stage of commercialization. The same presumably applies to Phaffia rhodozyma as a source of astaxanthin, the absolute configuration of which is, however, opposite to the desirable one (3R, 3'R instead of 3S, 3'S). To a lesser extent there may be a future for a fermentative process for canthaxanthin prepara­tion, although no really useful organisms have been found yet. Still ketocarot­enoids remain valuable compounds because of their efficiency as pigmenters in poultry and fish feeds. The future will tell whether a market for 'natural' canthaxanthin and astaxanthin will develop as an alternative to their synthetic counterparts.

REFERENCES

Andrewes, A. G. & Starr, M. P. (1976). (3R, 3'R)-Astaxanthin from the yeast Phaffia rhodozyma. Phytochem., 15,1009-11.

Andrewes, A. G., Phaff, H. J. & Starr, M. P. (1976). Carotenoids of Phaffia rhodozyma, a red-pigmented fermenting yeast. Phytochem., 15, 1003-7.

Anonymous (1962). Algae-derived xanthophylls for pigmentation control. Feeds Illustrated, November issue, 24-25.

Anonymous (1966-1967). Oxycarotenoid preparations. Microbiology Abstracts, Section A, 2, A1056.

Anonymous (1969-1970a). Microbiological production of carotenoid-containing addi­tives for animal feeds. Microbiology Abstracts, Section A, 5, A200l.

Anonymous (1969-1970b). Production of bacterial preparations with a high content of oxy-carotenoids. Microbiology Abstracts, Section A, 5, A2292.

Arpin, N., Lebreton, J. & Fiasson, J.-L. (1966). Recherches chimiotaxinomiques sur les champignons. II. Les caroteno"ides de Peniophora aurantiaca (Bres.) (Basidiomycete). Bull. Soc. Mycol. France, 82,450-59.

Ascenzi, J. M. & Cooney, J. J. (1975). Action of visible light on enzymes in cell envelopes of Micrococcus roseus. Photochem. Photobiol., 21, 307-1l.

Bamji, M. S. & Krinsky, N. I. (1966). The carotenoid pigments of a radiation-resistant Micrococcus species. Biochim. Biophys. Acta, 115, 276-84.

74 H. I. Nelis & A. P. De Leenheer

Belter, P. A. (1979). General procedures for isolation of fermentation products. In Microbial Technology, Vol. II, 2nd edn, ed. H. J. Peppler & D. Perlman. Academic Press, New York, pp. 403-32.

Bowles, N. D., Paerl, H. W. & Tucker, J. (1985). Effective solvents and extraction periods employed in phytoplankton carotenoid and chlorophyll determinations. Can. J. Fish. Aquat. Sci., 42, 1127-31.

Braumann, T. & Grimme, L. H. (1981). Reversed-phase high-performance liquid chromatography of chlorophylls and carotenoids. Biochim. Biophys. Acta, 637, 8-17.

Britton, G. (1985). General carotenoid methods. Meth. Enzym., 111, 113-49. Brush, A. H. (1981). Carotenoids in wild and captive birds. In Carotenoids as Colorants

and Vitamin A Precursors, ed. J. C. Bauernfeind. Academic Press, New York, pp. 539-62.

Burnett, J. H. (1976). Functions of carotenoids other than in photosynthesis. In Chemistry and Biochemistry of Plant Pigments, Vol. 1, 2nd edn, ed. T. W. Goodwin. Academic Press, New York, pp. 655-79.

Castillo, R., Negre-Sadargues, G. & Lenel, R. (1982). General survey of the carotenoids Crustacea. In Carotenoid Chemistry and Biochemistry, ed. G. Britton & T. W. Goodwin. Pergamon Press, Oxford, pp. 211-24.

Ciegler, A. (1965). Microbial carotenogenesis. Adv. Appl. Microbiol., 7, 1-34. Cooney, J. J. & Berry, R. A. (1981). Inhibition of carotenoid synthesis in Micrococcus

roseus. Can. J. Microbiol., 27,421-5. Cooney, J. J., Marks, H. W. & Smith, A. M. (1966). Isolation and identification of

canthaxanthin from Micrococcus roseus. J. Bacteriol., 92, 342-5. Costa, I., Martelli, H. L., da Silva, I. M. & Pomeroy, D. (1984). Produl;llo de

carotenoides por fermental;llo. I. Produc;ao de biomassa de Rhodotorula glutinis var. glutinis. Rev. Microbiol., Sao Paulo, 15, 109-13.

Coty, V. F. (1968). Dialysis to improve cell growth. US patent 3,418,208. Cited in Proteins From Hydrocarbons, Food Technol. Rev., no. 4, ed. S. Gutcho. Noyes Data Corporation, Park Ridge, NJ, pp. 91-3, 1973.

Czygan, F.-C. (1968). Sekundar-Carotinoide in Griinalgen. I. Chemie, Vorkommen und Faktoren welche die Bildung dieser Polyene beeinflussen. Arch. Mikrobiol., 61, 81-102.

Davies, B. H. (1976). Carotenoids. In Chemistry and Biochemistry of Plant Pigments, Vol. 2, 2nd edn., ed. T. W. Goodwin. Academic Press, New York, pp. 38-165.

Davis, J. B. (1967). Petroleum Microbiology. Elsevier, Amsterdam, pp. 311-15. Dieringer, S. M., Singer, J. T. & Cooney, J. J. (1977). Photokilling of Micrococcus

roseus. Photochem. Photobiol., 26, 393-6. Downs, J. & Harrison, D. E. F. (1974). Studies on the production of pink pigment in

Pseudomonas extorquens NCIB 9399 growing in continuous culture. 1. Appl. Bacteriol., 37,65-74.

Driessens, K., Liessens, J., Masduki, S., Verstraete, W., Nelis, H. & De Leenheer, A. (1987). Production of Rhodobacter capsulatus ATCC 23782 with short residence time in a continuous flow photobioreactor. Process. Biochem., 22, 160-4.

Dumenil, G., Laget, M., Cremieux, A., Millon, M. & Brousse, M. (1983). Etude des conditions de pigmentation d'une souche bacterienne methylotrophe facultative. Ann. Pharm. Franc., 41,427-35.

Einse1e, A. (1983), Biomass from higher n-alkanes. In Biotechnology, Vol. 3, ed. H. Dellweg. VCH, Weinheim, pp. 44-81.

Einsele, A. & Fiechter, A. (1971). Liquid and solid hydrocarbons. Adv. Biochem. Eng., 1, 169-94.

Gillan, F. T. & Johns, R. B. (1983). Normal-phase HPLC analysis of microbial carotenoids and neutral lipids. 1. Chromatogr. Sci., 21, 34-8.

Microbial Production of Carotenoids 75

Goodwin, T. W. (1972). Carotenoids in fungi and non-photosynthetic bacteria. Progr. Industr. Microbiol., 11, 29-88.

Goodwin, T. W. (1980). The Biochemistry of the Carotenoids, Vol. I, Plants. Chapman & Hall, London.

Goodwin, T. W. (1984). The Biochemistry of the Carotenoids, Vol. II, Animals. Chapman & Hall, London.

Goodwin, T. W. & Jamikorn, M. (1956). Studies in carotenogenesis. 17. The carotenoids produced by different strains of Mycobacterium phlei. Biochem. J., 62, 269-8l.

Haas, H. F. & Bushnell, L. D. (1944). The production of carotenoid pigments from mineral oil by bacteria. J. Bacteriol., 48, 219-3l.

Haas, H. F., Yantzi, M. F. & Bushnell, L. D. (1941). Microbial utilization of hydrocarbons. Trans. Kansas Acad. Sci., 44,39-45.

Hanson, A. M. (1967). Microbial production of pigments and vitamins. In Microbial Technology, ed. H. J. Peppler. Reinhold, New York, pp. 222-50.

Haxo, F. (1950). Carotenoids of the mushroom Cantharellus cinnabarinus. Bot. Gaz., 111, 228-32.

Hochmannova, J., Kolmanova, A. & Malek, I. (1968). Comparison of pigments produced by several strains of Mycobacterium phlei. Fol. Microbiol., 13, 505-9.

Hsieh, L. K., Lee, T.-C., Chichester, C. O. & Simpson, K. L. (1974). Biosynthesis of carotenoids in Brevibacterium sp. KY-4313. J. Bacteriol., 118,385-93.

Hsu, W. J., Yokoyama, H. & Coggins, C. W. (1972). Carotenoid biosynthesis in Blakeslea trispora. Phytochem., 11, 2985-90.

Ida, K. (1981). Eco-physiological studies on the response of taxodiaceous conifers to shading, with special reference to the behaviour of leaf pigments. I. Distribution of carotenoids in green and autumnal reddish brown leaves of gymnosperms. Bot. Mag. (Tokyo), 94, 41-54.

Iizuka, H. & Nishimura, Y. (1969). Microbiological studies on petroleum and natural gas. X. Carotenoid pigments of hydrocarbon utilizing bacteria. J. Gen. Appl. Microbiol., 15, 127-34.

Imhoff, J. F., Truper, H. G. & Pfennig, N. (1984). Rearrangement of the species and genera of the phototrophic "purple nonsulfur bacteria". Int. J. Syst. Bacteriol.. 34, 340-43.

Ingraham, M. A. & Baumann, C. A. (1934). The relation of microorganisms to carotenoids and vitamin A. J. Bacteriol., 28, 31-40.

Ingraham, M. A. & Steenbock, H. (1935). The relation of micro-organisms to carotenoids and vitamin A. II. The production of carotenoids by Mycobacterium phlei. Biochem. J., 29, 2553-62.

Isler, O. (1971). Introduction. In Carotenoids, ed. O. Isler. Birkhauser, Basle. Switzerland, p. 13.

IUPAC Commission on the Nomenclature of Organic Chemistry and IUPAC-IUB Commission on Biochemical Nomenclature (1971). Tentative rules for the nomen­clature of carotenoids. In Carotenoids, ed. O. Isler. Birkhauser, Basle, Switzerland pp. 851-64.

Johnson, E. A. & Lewis, M. J. (1979). Astaxanthin formation by the yeast Phaffia rhodozyma. J. Gen. Microbiol., 115, 173-83.

Johnson, E. A., Conklin, D. E. & Lewis, M. J. (1977). The yeast Phaffia rhodozyma as a dietary pigment source for salmonids and crustaceans. J. Fish. Res. Board Can., 34,2417-2l.

Johnson, E. A., Villa, T. G., Lewis, M. J. & Phaff, H. J. (1978). Simple method for the isolation of astaxanthin from the Basidiomycetous yeast Phaffia rhodozyma. Appl. Environ. Microbiol., 35, 1155-59.

Johnson, E. A., Villa, T. G. & Lewis, M. J. (1980a). Phaffia rhodozyma as an astaxanthin source in salmonid diets. Aquaculture, 20, 123-34.

76 H. J. Nelis & A. P. De Leenheer

Johnson, E. A., Lewis, M. J. & Grau, C. R. (1980b). Pigmentation of egg yolks with astaxanthin from the yeast Phaffia rhodozyma. Poultry Sci., 59, 1777-82.

Katsuyama, T. & Matsuno, T. (1979). Isolation and identification ofrhodoxanthin from the fish Tilapia nilotica. Bull. lap. Soc. Scient. Fish., 45, 1045.

Kester, A. S. & Thompson, R. E. (1984). Computer-optimized normal-phase high­performance liquid chromatographic separation of Corynebacterium poinsettiae carotenoids. I. Chromatogr., 310, 372-8.

Klaui, H. (1982). Industrial and commercial uses of carotenoids. In Carotenoid Chemistry and Biochemistry, ed. G. Britton & T. W. Goodwin, Pergamon Press, Oxford, pp. 308-17.

Klaui, H. & Bauernfeind, J. C. (1981). Carotenoids as food colors. In Carotenoids as Food Colorants and Vitamin A Precursors, ed. J. C. Bauernfeind. Academic Press, New York, pp. 48-317.

Kobayashi, M. (1972). Utilization of photosynthetic bacteria. In Fermentation Technol­ogy Today (Proceedings of the Wth International Fermentation Symposium), ed. G. Terui, pp. 527-31.

Kobayashi, M. & Kurata, S. (1978). The mass culture and cell utilization of photosynthetic bacteria. Process Biochem., 13,27-30.

Kobayashi, M. & Tchan, Y. T. (1973). Treatment of industrial waste solutions and production of useful by-products using a photosynthetic bacterial method. Water Res., 7, 1219-24.

Koronelli, T. V., Kalyuzhnaya, T. V. & Rozynov, B. V. (1981). Pigmented arctic mycobacteria and their role in the oxidation of hydrocarbons. Mikrobiologiya, 50, 167-70; Microbiology (English translation), SO, 123-5.

Koronelli, T. V., Pahlavuni, I. K. & Rozynov, B. V. (1982). A pigmented amphiphilic biopolymer from marine mycobacteria. Mikrobiologiya, 51,873-5.

Koronelli, T. V., Pakhlavuni, I. K., Voroybyeva, I. A. & Balashov, S. P. (1987). The lipopeptidocarotenoid complex and cells of Rhodococcus maris studied by spectros­copy. Mikrobiologiya, 56,890-91.

Korthals, H. J. & Steenbergen, C. L. M. (1985). Separation and quantification of pigments from natural phototrophic microbial populations. FEMS Microbiol. Ecol., 31,177-85.

Koyama, Y., Takii, T., Saiki, K. & Tsukida, K. (1983). Configuration of the carotenoid in the reaction centers of photosynthetic bacteria (2). Comparison of the resonance Raman lines of the reaction centers with those of 14 cis-trans isomers of f3-carotene. Photobiochem. Photobiophys. 3, 139-50.

Krinsky, N. I. (1979). Carotenoid protection against oxidation. Pure Appl. Chem., 51, 649-60.

Lem, N. W. & Glick, B. R. (1985). Biotechnological uses of Cyanobacteria. Biotechnol. Adv., 3, 195-208.

Leuenberger, H. G. W. (1985). Microbiologically catalyzed reaction steps in the field of vitamin and carotenoid syntheses. In Biocatalysts in Organic Syntheses, ed. J. Tramper, H. C. van der Plas & P. Linko, Studies in Organic Chemistry, Vol. 22. Elsevier, Amsterdam, pp. 99-118.

Levi, J. D., Shennan, J. L. & Ebbon, G. P. (1979). Biomass from liquid n-alkanes. In Economic Microbiology, Vol. 4, Microbial Biomass, ed. A. H. Rose, Academic Press, New York, pp. 361-419.

Lewis, N. F. & Kumta, U. S. (1973). Lycopene accumulation in pigmented radio­resistant Micrococci grown in presence of nicotine. FEBS Lett., 30, 144-6.

Liaaen-Jensen, S. (1978). Chemistry of carotenoid pigments. In The Photosynthetic Bacteria, ed. R. K. Clayton & W. R. Sistrom. Plenum Press, New York, pp. 233-47.

Lichtfield, J. H. (1979). Production of single-cell protein for use in food and feed. In Microbial Technology, Vol. I, 2nd edn, ed. H. J. Peppler & D. Perlman. Academic Press, New York, pp. 93-155.

Microbial Production of Carotenoids

Lichtfield, J. H. (1980). Microbial protein production. BioScience, 30, 387-96. Lichtfield, J. H. (1983). Single-cell proteins. Science, 219,740-6.

77

McDermott, J. C. B., Ben-Aziz, A., Singh, R. K., Britton, G. & Goodwin, T. W. (1973). Recent studies of carotenoid biosynthesis in bacteria. Pure Appl. Chem., 35, 29-45.

Mantoura, R. F. C. & Llewellyn, C. A. (1983). The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase high-performance liquid chromatography. Anal. Chim. Acta, 151, 297-314.

Mantoura, R. F. C. & Llewellyn, C. A. (1984). Trace enrichment of marine algal pigments for use with HPLC-diode array spectroscopy. J. High Resol. Chromatogr. & Chromatogr. Commun., 7,632-5.

Margalith, P. & Meydav, S. (1968). Some observations on the carotenogenesis in the yeast Rhodotorula mucilaginosa. Phytochem., 7, 765-8.

Marusich, W. L. & Bauernfeind, J. C. (1981). Oxycarotenoids in poultry feed. In Carotenoids as Food Colorants and Vitamin A Precursors, ed. J. C. Bauernfeind. Academic Press, New York, pp. 319-462.

Mathews, M. M. & Krinsky, N. I. (1965). The relationship between carotenoid pigments and resistance to radiation in non-photosynthetic bacteria. Photochem. Photobiol., 4, 813-17.

Mathews-Roth, M. M. & Krinsky, N. I. (1970). Studies on the protective function of the carotenoid pigments of Sarcina lutea. Photochem. Photobiol., 11,419-28.

Mathews-Roth, M. M., Wilson, T., Fujimori, E. & Krinsky, N. I. (1974). Carotenoid chromophore length and protection against photosensitization. Photochem. Photobiol., 19,217-22.

Mathis, P. & Schenck, C. C. (1982). The functions of carotenoids in photosynthesis. In Carotenoid Chemistry and Biochemistry, ed. G. Britton & T. W. Goodwin. Pergamon Press, Oxford, pp. 339-47.

Mayer, H. (1979). Synthesis of optically active carotenoids and related compounds. Pure Appl. Chem., 51, 535-64.

Mayer, H. & Isler, O. (1971). Total syntheses. In Carotenoids, ed. O. Isler. Birkhauser, Basel, pp. 325-575.

Merritt, K. & Jacobs, N. J. (1978). Characterization and incidence of pigment production by human clinical group B-streptococci. J. Clin. Microbiol., 8, 105-7.

Miller, J. c., George, S. A. & Willis, B. G. (1982). Multichannel detection in high-performance liquid chromatography. Science, 218, 241-6.

Moss, G. P. & Weedon, B. C. L. (1976). Chemistry of the carotenoids. In Chemistry and Biochemistry of Plant Pigments, Vol. 1, 2nd edn, ed. T. W. Goodwin. Academic Press, London, pp. 149-224.

Muller, R. K., Bernhard, K. & Vecchi, M. (1982). Recent advances in the synthesis and analysis of 3,4-oxygenated xanthophylls. In Carotenoid Chemistry and Biochemistry, ed. G. Britton & T. W. Goodwin, Pergamon Press, Oxford, pp. 27-54.

Nelis, H. J. C. F. & De Leenheer, A. P. (1983). Isocratic nonaqueous reversed-phase liquid chromatography of carotenoids. Anal. Chem., 55,270-75.

Nelis, H. J. & De Leenheer, A. P. (1986). Carotenoid profiling and quantitation in biological materials using non-aqueous reversed phase chromatography and photo­diode array detection. Abstracts of the 10th International Symposium on Micro­chemical Techniques, Antwerp, 25-29 August, 1986, p. 133.

Nelis, H. J. C. F. & De Leenheer, A. P. (1987). Exhaustive extraction, profiling and quantitation of bacterial carotenoids. Abstracts of the 8th International Symposium on Carotenoids, Boston, 27-31 July 1987, p. 9.

Nesterenko, O. A., Nogina, T. M., Kasumova, S. A., Kvasnikov, E. I. & Batrakov, S. G. (1982). Rhodococcus luteus nom. nov. and Rhodococcus maris nom. nov. Int. J. Syst. Bacteriol., 32, 1-14.

78 H. J. Nelis & A. P. De Leenheer

Ninet, L. & Renaut, J. (1979). Carotenoids. In Microbial Technology, 2nd edn, Vol. I, ed. H. J. Peppler & D. Perlman. Academic Press, New York, pp. 529-44.

Ninet, L., Renaut, J. & Tissier, R. (1969). Activation of the biosynthesis of carotenoids by Blakeslea trispora. Biotechnol. Bioeng., 11, 1195-210.

Okagbue, R. N. & Lewis, M. J. (1981). Mixed culture of Bacillus circulans WL-12 and Phaffia rhodozyma on different carbon sources: yeast-wall lytic enzyme production and extractability of astaxanthin. Biotechnol. Lett., 5,731-6.

Okagbue, R. N. & Lewis, M. J. (1984a). Vse of alfalfa residual juice as a substrate for propagation of the red yeast Phaffia rhodozyma. Appl. Microbiol. Biotechnol., 20, 33-39.

Okagbue, R. N. & Lewis, M. J. (1984b). Inhibition of the red yeast Phaffia rhodozyma by saponin. Appl. Microbiol. Biotechnol., 20,278-80.

Okagbue, R. N. & Lewis, M. J. (1984c). Autolysis of the red yeast Phaffia rhodozyma: a potential tool to facilitate extraction of astaxanthin. Biotechnol. Lett., 6,247-50.

Okagbue, R. N. & Lewis, M. J. (1985). Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-12 and on extractability of astaxanthin from the yeast Phajfia rhodozyma. l. Appl. Bacteriol., 59,243-55.

Pachlavuni, I. K. (1987). A carotenoid-containing complex from Rhodococcus maris: some properties and structure. Abstracts of the 8th International Symposium on Carotenoids, Boston, 27-31 July, 1987, p. 12.

Peterson, W. J., Evans, W. R., Leece, E., Bell, T. A. & Etchells, J. L. (1958). Quantitative determination of the carotenoids in yeasts of the genus Rhodotorula. l. Bacteriol., 75, 586-9l.

Ratledge, C. (1970). Microbial conversions of n-alkanes to fatty acids: a new attempt to obtain economical microbial fats and fatty acids. Chem. Ind., 843-54.

Ratledge, C. (1982). Microbial oils and fats: an assessment of their commercial potential. In Progress in Industrial Microbiology, Vol. 16, ed. M. J. Bull. Elsevier, Amsterdam, pp. 119-206.

Rau, W. (1983). Photoregulation of carotenoid biosynthesis. In Biosynthesis of Isoprenoid Compounds, Vol. 2, ed. J. W. Porter & S. L. Spurgeon. Wiley, New York, pp. 123-57.

Riiedi, P. (1985). HPLC-a powerful tool in carotenoid research. Pure Appl. Chern., 57,793-800.

Sakurai, H., Kato, K., Sakai, T., Masuda, Y. & Kuriyama, T. (1971). Studies on microbial utilization of petroleum. I. Separation and characterization of carotenoids produced by a species of Brevibacterium in hydrocarbon media. Bull. Chem. Soc. lapan, 44,481-84.

Saperstein, S., Starr, M. P. & Filfus, J. A. (1954). Alterations in carotenoid synthesis accompanying mutation in Corynebacterium michiganense. l. Gen. Microbiol., 10, 85-92.

Sato, K., Veda, S. & Shimizu, S. (1977). Form of vitamin B12 and its role in a methanol-utilizing bacterium, Protaminobacter ruber. Appl. Environ. Microbiol., 33,515-2l.

Schlegel, H. G. (1959). Conversion of carotenoids to oxycarotenoids by Mycobacterium phlei. l. Bacteriol., 77, 310-16.

Schmidt, K. (1978). Biosynthesis of carotenoids. In The Photosynthetic Bacteria, ed. R. K. Clayton & W. R. Sistrom. Plenum Press, New York, pp. 729-50.

Schwartzel, E. H. & Cooney, J. J. (1970). Isolation and identification of echinenone from Micrococcus roseus. l. Bacteriol., 104,272-74.

Schwartzel, E. H. & Cooney, J. J. (1972). Isolation of 4'-hydroxyechinenone from Micrococcus roseus. l. Bacteriol., 112, 1422-24.

Schwartzel, E. H. & Cooney, J. J. (1974a). Action of light on Micrococcus roseus. Can. l. Microbiol., 20, W15-2l.

Schwartzel, E. H. & Coon~y, J. J. (1974b). Isolation and characterization of pigmentation mutants of Micrococcus roseus. Can. l. Microbiol., 20, 1007-13.

Microbial Production of Carotenoids 79

Schwarz, Y. & Margalith, P. (1965). Production of egg yolk coloring material by a fermentation process. Appl. Microbial., 13, 876-8l.

Seybold, A. & Goodwin, T. W. (1959). Occurrence of astaxanthin in the flower petals of Adonis annua L. Nature, Lond., 184, 1714-15.

Shapiro, A., DiLello, D., Loudis, M. c., Keller, D. E. & Hutner, S. H. (1977). Minimal requirements in defined media for improved growth of some radio-resistant pink tetracocci. Appl. Environ. Microbiol., 33, 1129-33.

Shimizu, S., Sato, K., Hiraoka, M., Yamashita, F. & Kobayashi, T. (1982). Carotenoids formation by a facultative methylotroph, Protaminobacter ruber. J. Ferment. Technol., 60, 163-6.

Shipman, R. H., Fan, L. T. & Kao, I. C. (1977). Single-cell protein production by photosynthetic bacteria. Adv. Appl. Microbial., 21, 161-83.

Simpson, K. L., Chichester, C. O. & Phaff, H. J. (1971). Carotenoid pigments of yeasts. In The Yeasts, Vol. 2, ed. A. H. Rose & J. S. Harrison. Academic Press, New York, pp. 493-515.

Simpson, K. L., Katayama, T. & Chichester, C. O. (1981). Carotenoids in fish feeds. In Carotenoids as Food Colorants and Vitamin A Precursors. Academic Press, New York, pp. 463-538.

Spurgeon, S. L. & Porter, J. W. (1983). Biosynthesis of carotenoids. In Biosynthesis of Isoprenoid Compounds, Vol. 2, ed. J. W. Porter & S. L. Spurgeon. Wiley, New York, pp. 1-122.

Tanaka, A., Nagasaki, T., Inagawa, M. & Fukui, S. (1968a). Studies on the formation of vitamins and their functions in hydrocarbon fermentation. (V) Production of carotenoids by Mycobacterium smegmatis in hydrocarbon media (Part I). Studies on the cultural conditions. J. Ferm. Technol., 46, 468-76.

Tanaka, A., Nagasaki, T. & Fukui, S. (1968b). Studies on the formation of vitamins and their functions in hydrocarbon fermentation. (VI) Production of carotenoids by Mycobacterium smegmatis in hydrocarbon media. (Part II) Isolation and charac­terization of several carotenoids produced by Mycobacterium smegmatis IFO-3080. J. Ferm. Technol., 46,477-87.

Tanaka, A., Kato, K. & Fukui, S. (1971). Studies on the formation of vitamins and their function in hydrocarbon fermentation. (IX) Production of carotenoids from hydrocarbons by Brevibacterium. 1. Ferm. Technol., 49, 778-91.

Taylor, R. F. (1980). Chromatography of carotenoids and retinoids. Adv. Chromatogr., 22, 157-213.

Theriault, R. J. (1965). Heterotrophic growth and production of xanthophylls by Chlorella pyrenoidosa. Appl. Microbiol., 13, 402-16.

Toraya, T., Yongsmith, B., Tanaka, A. & Fukui, S. (1975). Vitamin B\2 production by a methanol utilizing bacterium. Appl. Microbial., 30, 477-9.

Ungers, G. E. & Cooney, J. J. (1968). Isolation and characterization of carotenoid pigments of Micrococcus roseus. 1. Bacteriol., 96,234-4l.

Urakami, T., & Komagata, K. (1986). Occurrence of isoprenoid compounds in Gram-negative methanol-, methane- and methylamine-utilizing bacteria. J. Gen. Appl. Microbiol., 32, 317-4l.

Valadon, L. R. G. (1976). Carotenoids as additional taxonomic characters in fungi: a review. Trans Br. Mycol. Soc., 67, 1-15.

Van Niel, C. B. & Smith, J. H. C. (1935). Studies on the pigments of the purple bacteria. Arch. Mikrobiol., 6,219-29.

Various patents (1973). Recovery techniques. In Proteins From Hydrocarbons, Food Technology Review no. 4, ed. S. Gutcho. Noyes Data Corporation, Park Ridge, NJ, pp. 144-99.

Villoutreix, J. (1960). Influence de divers agents chimiques sur la carotenogenese de Rhodotorula mucilaginosa. Biochim. Biophys. Acta, 40, 434-4l.

Voznyakovskaya, Y. M. & Daraseliya, G. Y. (1972). Mutants of Mycobacterium phlei with increased synthesis of carotenoids. Mikrobiologiya, 41, 886-90.

80 H. 1. Nelis & A. P. De Leenheer

Wendall, M., Farrow, F. & Tabenkin, B. (1964). Preparation of a lutein product. French patent 1,367,027. CA 62 (1965) 9742.

Wood, J. F., Carter, P. M. & Savory, R. (1983). Investigation into the effect of processing on the retention of the carotenoid fractions of Leucaena Leucocephala during storage, and the effects of processing on mimosine concentration. Anim. Feed Sci. Technol., 9, 307-17.

Yamada, K., Nakahara, T. & Fukui, S. (1971). Petroleum microbiology and vitamin production. In Biochemical and Industrial Aspects of Fermentation, ed. K. Saka­guchi, T. Uemura & S. Kinoshita. Kodansha, Tokyo, pp. 61-90.

5 NOTE ADDED IN PROOF

Several biotechnology companies are presently (May 1989) conducting exten­sive research to improve the production of astaxanthin from Phaffia rhod­ozyma. One of them (Gist Brocades) has now marketed the organism as a pigmenter for salmonid diets. Recent papers on Phaffia rhodozyma are by N. F. Haard (1988, Biotechnol, Lett., 10, 609-14) and G.-H. An (1989, Appl. Environ. Microbiol., 55, 116-24). Maximum astaxanthin levels obtained by these investigators range from 1.2 mg/g (Phaffia rhodozyma parent strain) to 2.5 mg/g (mutant strain). Corresponding yields on a volume basis were 15 mg/liter and not specified, respectively.

Chapter 5

VITAMIN 0: THE BIOTECHNOLOGY OF ERGOSTEROL

P. MARGALITH

Department of Food Engineering & Biotechnology, Technion-Israel Institute of Technology, Haifa, 32000, Israel

1 INTRODUCTION

Among the vitamins, vitamin D is outstanding because of its chemical nature, it being the only vitamin with a steroid structure. Vitamin D is necessary for the proper metabolism of minerals and bone formation, in mammals and birds. Biotechnologically speaking, however, one usually refers to the provitamin, ergosterol (1) the main sterol in yeasts and mycelial fungi. The provitamin may be converted to the proper vitamin (D2) which undergoes further metabolism before exerting its hormone like activity in controlling the processes involved in mineral metabolism.

24

HO HO 1 2

Ergosterol is not the only provitamin suitable for antirachitic treatment. 7-dehydrocholesterol (2) as produced in the mammalian body, may also be transformed into vitamin D (D3) with somewhat different chemical and physiological characteristics. Since the latter vitamin has certain advantages in use, ergosterol as a source for vitamin D no longer occupies the same position in the manufacturing of this vitamin.

Under natural circumstances, the human body produces its vitamin D from 7-dehydrocholesterol upon exposure to sunlight. In many geographical areas

81

82 P. Margalith

where clouded skies prevail throughout most of the year not enough vitamin D is biosynthesized. The migration into cities and the increase in indoor professions led to a decline in the synthesis of the natural vitamin and created a demand for a dietary supplement that would protect the human body from rickets.

In rickets, the organic matrix of new bone is not mineralized. This is due to the body's inability to calcify the collagene matrix of the growing bone and results in large areas of uncalcified bone (osteoid). The resultant lack in rigidity of bones leads to the ends becoming entwisted and bent. Also, tooth enamel formation may be affected. Children are specially prone to this disease. Even with an optimal intake of calcium and phosphorus in vitamin D deficient children, the concentration in the blood serum of these minerals is very low. Vitamin D increases the calcium and phosphate absorption and restores the mineral balance which results in the appropriate calcium phosphate deposition in the bone.

The use of liver oil as a prophylactic against rickets, as well as the importance of light as an antirachitic factor, have been recognized since before

HO 7-Dehydrocholesterol

1

HO Cholecalciferol (D3)

Fig. 1.

HO

./" HO

CH3

Ergosterol

1

Ergocalciferol (D2 )

The Biotechnology of Ergosterol 83

the end of the 19th century. Steenbock & Black (1924) showed that irradiation of foods led to the generation of the vitamin. We now know that irradiation may lead to two types of vitamin 0: (1) By irradiation of ergosterol, ergocalciferol (or O 2 ) is formed while (2) irradiation of 7-dehydrocholesterol leads to the production of cholecalciferol (or D 3), see Fig. 1. This chapter will deal chiefly with the production of ergosterol, the provitamin O 2 •

2 CHEMICAL AND PHYSICAL PROPERTIES

Ergosterol is a fat-soluble vitamin with a typical steroid structure. It consists of 4 rings (the cyclopentanoperhydrophenanthrene system, comprising 3 fused 6-membered rings and 1 5-membered ring) and a side chain, in all 28 carbon atoms. Ergosterol differs from cholesterol, the best known sterol of animal origin, in additional carbon (methyl group) at C-24 in the J3-position (referring to the position on the axis of the side chain), as well as additional double bonds between carbons 7: 8 and 22: 23. Like most steroids, ergosterol also has 2 angular methyl groups at carbon 10 and 13 in the J3-position (perpendicular to the plane of the ring). Both sterols have a hydroxyl group at carbon 3 in the J3-position. Many of the chemical and physiological characteristics of these sterols can be attributed to this group.

Contrary to the saponifiable lipids, hydrolysis of steroids with alkali will not make them water-soluble. They are therefore classified as unsaponifiable lipids. Like most 3J3-hydroxysteroids, ergosterol can also be precipitated with digitonin, a steroidal saponin. Although digitonin may be employed for the precipitation of such sterols, the quantitative determination by gravitational methods is usually not sensitive enough. Colorimetric methods are now widely employed, most of which are based on the Liebermann-Buchard reaction. A sterol in chloroform solution is treated with acetanhydrid and sulfuric acid at a suitable temperature. With time a color develops with characteristic absorp­tion. Ergosterol, on the other hand, may be determined spectrophotometri­cally by absorption in UV. The characteristic spectrum of the 5,7 steroid in absolute alcohol has peaks at 271, 282 and 293 nm. For the quantitative determination of ergosterol it is advisable to employ a suitable separation procedure (TLC or GLC) to avoid the interference of related compounds. The molar extinction of ergosterol at 282 nm is 11 900.

3 OCCURRENCE AND PRODUCING ORGANISMS

Although yeasts are known as producers of ergosterol, the sterol was first isolated from rye ergot (sclerotia of C/aviceps purpurea). It is from this plant disease that the name ergosterol was derived (Tanret, 1889). Many organisms were later screened and examined for their sterol content. While procaryotes were found to be practically devoid of true sterols, eucaryotic micro-organisms

84 P. Margalith

could be divided into a number of categories with regard to the role sterols play in their cell biology:

(1) Organisms in which sterols are an important constituent of the cell material:

(a) Organisms that synthesize their own sterols. (b) Organisms unable to synthesize their sterols, their requirements

being met by environmental sources.

(2) Organisms which do not require sterols for vegetative growth, but need such compounds for the differentiation of certain sexual or asexual reproductive organs.

(3) Organisms which have no known requirements for sterols but which may incorporate such compounds if suitably exposed to them (Mar­galith, 1986).

Screening for organisms that are good producers of ergosterol was obviously directed to the first category of organisms that synthesize their own sterols. Although sterols are widespread among photosynthesizing plants including the fast-growing microalgae, phytosterols of green plants usually do not comprise ergosterol. The search was therefore limited to eucaryotic heterotrophs, amongst these the Mucorales (Zygomycotina), the Ascomycotina (including the yeasts and yeastlike fungi), the Basidiomycotina (in the mushrooms) and many of the Fungi Imperfecti.

In most cases ergosterol is not the only sterol to be found, it is usually accompanied by other related sterols such as zymosterol (3) fungisterol (4) or ergosta-5,7,22,24(28)-tetraenol (5). We shall return to these sterols when discussing the biosynthesis of ergosterol. The quantitative aspects of ergosterol formation and distribution have been also studied. There seems to be no great variation in the sterol content of fungi. According to Appleton et al. (1955), molds have an average of c. 0·6% sterols on a dry weight basis. Exceptional higher values were recorded for Penicillium westlinghi (2·2%).

Not surprisingly, yeasts have been the object of many workers interested in the biotechnology of ergosterol and its biosynthesis. The ease with which yeasts can be grown and the familiarity of many microbiologists with these organisms in various food industries, in addition to their being an excellent source for most vitamin Bs, have made yeasts the organism of choice for the production of provitamin D.

The first to study the ergosterol content of yeasts were Bills et al. (1930). They examined 29 strains from 13 species of 5 genera: Endomyces, Nadsonia, Mycoderma, Saccharomyces, and Zygosaccharomyces. Values of 0·2-0·3% (dry weight) have been recorded. Saccharomyces carlsbergensis (strain ATCC 2345) had the highest ergosterol level (2·4%). Usdin & Burell (1952) found 2·7% in Rhodoturula gracilis. A systematic study for ergosterol in yeasts was carried out by Dulaney et al. (1954) who examined 146 cultures of the following genera: Candida, Diploascus, Hansenula, Nematospora,

The Biotechnology of Ergosterol 85

HO 3 HO

4

HO 5

Saccharomyces, Schwanniomyces, Torulopsis, Cryptococcus, Endomyces, Kloeckera, Pichia, Saccharomycodes, Sporobolomyces, Debaryomyces, Endomycopsis, Nadsonia, Rhodotorula, Schizosaccharomyces, Taphrina. With the exception of Saccharomyces, levels of ergosterol never exceeded 0·4%. The best organism was Saccharomyces cerevisiae for which 3·9% was reported. Additional studies were made by Appleton et al. (1955) who reached similar conclusions. They confirmed the advantage of the genus Saccharomyces for the formation of ergosterol. There seems to be considerable variation between species or even strains. However, generally speaking, brewer's yeast had the highest level of sterols, followed by baker's yeast. A method for the production of high sterol yeasts (10% dry weight) was patented by Dulaney (1957).

Not all reports deal with the nature of the sterols found in yeast. In most cases where this was done the ratio of ergosterol to other sterols was 8: 2.

4 MEDIA AND ENVIRONMENTAL FACTORS

Sterol production by yeasts is greatly affected by environmental conditions. High concentrations of assimilable carbohydrates promote the production of ergosterol, while nitrogen-rich media usually lead to decreased levels of sterol biosynthesis (Dulaney et al., 1954; Gal'tsova et al., 1959).

Non-sugar carbon sources for the production of sterols have also been examined. Acetate seems to have a promoting effect when added to sucrose (Starr & Parks, 1962). Ethanol can also serve as a carbon source. An old method for increased sterol production consisted of exposing yeast cells to vapors of ethanol. It was claimed that by this method a sterol level of c. 10%

86 P. Margalith

could be obtained. The most important environmental factor in yeast growth and sterol productivity is, however, the availability of oxygen.

Although fermenting yeasts have a mechanism for the attainment of workable energy (ATP) under anaerobic conditions, it has been known for some time that Saccharomyces yeasts would not proliferate under strictly anaerobic conditions (Nordheim, 1966). An explanation for this incongruity was first suggested by Andreasen & Stier (1953) in the classical papers on the anaerobic nutrition of S. cerevisiae. It was established that sterols (as well as unsaturated fatty acids) cannot be synthesized under anaerobic conditions and only if added to the media may they replace in part oxygen for growth under these conditions. Since growth cannot take place under anaerobic conditions, these are totally unsuitable for sterol production. Growth under semi­anaerobic conditions, i.e. without aeration, yielded only 1/10 of sterols in comparison to fully aerated yeasts (Klein, 1955). There seems to be a linear relationship between oxygen availability and sterol biosynthesis. However, there appear to be no detailed studies describing such a relationship and whether there are any detrimental effects on sterol production due to overaeration.

5 BIOSYNTHESIS AND REGULATION

Earlier workers (Maguigan & Walker, 1940) suggested a relationship between sterols and the total lipid composition, the former comprising 18-19% of the total lipids. This was later found to be inaccurate since yeast strains with high sterol content and low lipid composition were also found (Maugenet & Dupuy, 1964).

Since sterols belong to the lipid fraction of an organism, it is not surprising that both sterols and fatty acids employ acetate as precursors. Indeed, label studies showed that acetate, both the methyl and carboxyl carbons, serves as a source for all the carbons of ergosterol, with the exception of C-28.

From acetate to lanosterol: the pathway by which the 2 carbon acetate molecule is converted to the 5 carbon isoprene molecule, involves the condensation of 3 acetate residues with the elimination of 1 carbon atom as CO2 • From 3-hydroxy-3-methylglutaric acid (HMG) , isopentenyl pyrophos­phate (IPP) and dimethylallylpyrophosphate (DPP) are formed. A stepwise addition of Cs units leads eventually to the formation of farnesylpyrophosphate (FPP)-C1s ; 2 FPPs forming by tail-to-tail condensation squalene, the open chain C30 of sterols. The cyclization of squalene is the second major step in sterol biosynthesis. This is a complex reaction involving an oxygenated intermediate:2,3-oxidosqualene which is formed in presence of oxygen only. Obviously, anaerobically grown yeasts cannot form this intermediate (Fig. 2). Cyclization of the epoxide is initiated by a specific enzyme (squalene cyclase) leading to a series of hydrogen and methyl migrations giving rise to a cyclic triterpenoid having the sterol nucleus (Schechter et aZ., 1970) known as

The Biotechnology of Ergosterol 87

ACETATE

1 MEVALONATE

1 FARNESYLPYROPHOSPHATE

1 SQUALENE

1 SQUALENE EPOXIDE

1 LANOSTEROL

1 ZYMOSTEROL

I FECOSTEROL

1 EPISTEROL

1 ERGOST A-7 ,22,24(28)TRIENOL

1 ERGOST A-5,7 ,22,24(28)TETRAENOL

! ERGOSTEROL

Fig. 2.

lanosterol (6). Interestingly, this compound is also formed during sterol synthesis in animal tissues and is not found in green plants which synthesize the related cycloartenol (7) as the first cyclization product.

HO HO 6 7

88 P. Margalith

Demethylations and alkylations: The tetracyclic lanosterol differs from a true sterol in its 3 'extra' methyl groups at positions 4,4' and 14. In addition, lanosterol shows 2 double bonds at C-24 (25) and C-8 (9). In order to become ergosterol, the ultimate sterol of most fungi and yeasts, de methylation reactions, reduction and rearrangements of double bonds in addition to the alkylation of C-24 have to take place. Lanosterol as such cannot take the place of ergosterol in anaerobic growth of yeast (Nes et al., 1978). This can be explained by the bulky nature of lanosterol. The demethylation reactions similar to squalene oxygenation require the presence of oxygen (for the elimination in the form of CO2) and cannot proceed under anaerobic conditions. The de methylation reactions lead to the formation of the first true sterol compound:zymosterol (3).

Zymosterol differs from ergosterol in a number of features. The double bond is still at 8: 9 and 24: 25, and C-24 must be alkylated with the C-28 methyl group. We have already pointed out that the C-28 carbon is not derived from acetate. The methyl group is derived from S-adenosylmethionine by a transmethylation reaction (24-methyltransferase) yielding fecosterol (8) to be followed by double bond migration from 8: 9 to 7: 8 giving episterol (9); the introduction of an additional double bond (ergosta-7 ,22,24(28)-trienol); the formation of the tetraenol (with the 5: 6 double bond) and the final reduction of C-24(28) to ergosterol (1).

HO HO H 9

The pattern described above is not the only possible pathway for the production of yeast ergosterol. Sterol synthesis, in general, does not follow a rigid pattern but rather a more flexible framework where many alternative pathways may take place. (Fryberg et al., 1973). Because of the complexity of sterol biosynthesis and the diversity in the timing of certain key reactions (in some strains (A~ B~ C, while in others A~ C~ B), the regulation of sterol synthesis has not been sufficiently studied. Of the many enzymic reactions potentially capable of a regulatory function, hydroxy-methyl-glutaryl-CoA reductase is probably the rate-controlling enzyme of sterol biosynthesis (Qureshi et al., 1980).

6 FERMENTATION

Very few publications deal with the ergosterol fermentation. In the 1950s a number of papers were published dealing with the production of ergosterol by

The Biotechnology of Ergosterol 89

various yeasts and fungi. Since yeasts with strong fermentative metabolism were found to be the best producers of ergosterol, most of the studies concentrated on the fermentation of S. cerevisiae strains. Using a molasses­cornsteep medium, cells with an ergosterol content of 7-10% could be obtained. Cell yields of 30-40 g/liter medium and a sterol content of 3-4 g/liter broth were found in shake flask experiments after 4 days of growth at 28°C (Dulaney et al., 1954). Good yields were also obtained with S. fermentati (EI Refai & EI Kady, 1969). Little has been published about fermentation in larger volumes. Nor has the patent literature revealed important new information on the industrial fermentation of the provitamin. Since the early patent of Dulaney (1957) a number of patents were granted to several companies in various countries. Most of these deal with extraction procedures for the isolation and crystallization of ergosterol from the biomass (e.g. Nelson, 1959). Few patents describe a modification of the fermentation procedure by the incorporation of precursors to the medium. The addition of isopentenol (0·92%) and Na-polyphosphate (0·3%) to the growth of S. uvarum UFO 1264 at 26°C for 96 h, yielded 2·1 % ergosterol (dry weight) (Japanese Patent 8158496, 1981). Organisms described in the patent literature comprise: S. cerevisiae, S. carlsbergensis, Candida tropicalis, Fusarium spp and Trichoderma spp. Yields (ergosterol on basis of dry weight or volume of medium) described in the patent literature are generally far lower than those reported in scientific publications. A number of patents have been granted for the growth of yeasts in medium containing hydrocarbons as sole carbon source, e.g. Candida petrophilum ATCC 20226 (Horiguchi et al., Japanese Patent 7392586, 1973). Also here ergosterol yields were rather low (see Table 1 for list of patents).

Isolation of ergosterol from biological sources usually involves the extraction of the fatty component, saponification and re-extraction with an ether. Direct extraction of yeast is not sufficient, usually a preliminary digestion with hot alkali is included prior to the extraction.

Provitamin D3

The equivalent of ergosterol in animal tissues is 7-dehydrocholesterol (2) which can be produced commercially by the bromination of an esterified cholesterol followed by dehalogenation to give the 7 -dehydrocholesteryl ester.

7 THE FORMATION OF VITAMIN D

Both ergosterol and 7-dehydrocholesterol can be transformed into previtamin D by UV irradiation (280-300 nm). Sufficiently low temperatures are main­tained to prevent the formation of side products. This results in the opening of the B ring. The 5,7 conjugated diene is activated yielding the previtamin (10), which undergoes a thermal equilibration to vitamin D. Fission of the 9: 10

8

Tab

le 1

So

me

Pat

ents

for

the

Pro

duct

ion

of E

rgos

tero

l

Nam

e C

ount

ry

Pat

ent

No.

Ye

ar

Org

anis

m

SJle

cial

feat

ures

Erg

oste

rol

cont

aini

ng y

east

U

SA

28

1762

4 19

57

S. c

erev

isia

e N

RR

L s

trai

ns

Erg

oste

rol

from

yea

st

Bri

tain

80

1390

19

58

Rec

over

y E

xtra

ctio

n of

ste

rols

U

SA

28

3754

0 19

58

Rec

over

y ~

Pro

duct

ion

of e

rgos

tero

l Ja

pan

4979

2802

19

71

C.

troJ

lical

is

N-a

lkan

e as

sol

e ~

by y

east

s fr

om h

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The Biotechnology of Ergosterol 91

bond leads to the formation of the secosteroid. Ergosterol thus becomes Vitamin D2 (ergocalciferol) and 7-dehydrocholesterol becomes vitamin D3 (cholecalciferol). The biological activity of 0·025 f.lg of pure cholecalciferol is considered the international unit (IU) of vitamin D.

R

HO 10

8 METABOLIC TRANSFORMATIONS

Vitamin D formed in the skin or absorbed by ingestion through the gut wall, is transported by the bloodstream into the liver where it is hydroxylated at C-25 to yield 25-hydroxycalciferol which is the main circulating form of Vitamin D. It is transported on a specific a-globulin to the kidney where further hydroxylation takes place at C-1 or C-24, apparently in response to the calcium levels in the blood. Low phosphate levels also stimulate 1,25-dihydroxycholecalciferol (11) production which, in turn, enhances calcium absorption. The C-1 hydroxylation is the critical step which enables the vitamin to assume its hormonal activity in regulating the calcium transport as well as bone calcium mobilization. This important reaction is carried out in kidney mitochondria by a mixed function mono-oxygenase involving cytochrom P-450.

OH /;k

HO 11

Regulation of vitamin D metabolism appears to be controlled by the levels of calcium, phosphate and the parathyroid hormone in blood serum. Below 9·5 mg/ml of Ca2 +, 25-dihydroxy vitamin D is formed, above that level 24,25-dihydroxycalciferol is produced with the discontinuation of the la­hydroxylase activity. The enzyme is also stimulated by the homeostatic parathyroid hormone which causes phosphate diuresis in the kidney and enhances 1,25-dihydroxycholecalciferol production. The parathyroid hormone and 1,25-dihydroxycholecalciferol act at the bone site cooperatively in the stimulation of calcium mobilization from bone. Other metabolites of vitamin D

92 P. Margalith

are also formed. However, they are less potent and their metabolic significance is not yet fully understood.

9 APPLICATION AND ECONOMICS

While vitamin D is used for the fortification of many foodstuffs (margarine, milk), large amounts are added to the animal feeds, mostly for swine, chicken and dairy calves. Vitamin D2 and D3 have the same biological activity in rats. However, in chicken, vitamin D2 is only 1/10 or less active in comparison to vitamin D3. In man cholecalciferol is apparently also more active than ergocalciferol. The difference is probably due to the C-24 methyl group and not the C-22 double bond since 22-dihydroergosterol is as effective as provitamin D2. Considering these facts led to the obvious preference of cholecalciferol produced synthetically. Vitamin D3 is sold at about 1 $/1(t IU; or 10 $/kg of a product of 400 000 IU/g(Hirsch, 1984). Considering the worldwide demand for vitamin D at about 1750 X 1012 IU (1985), neither the quantities required nor the price obtained on the market have given great impetus to the improvement in the biotechnological production of provitamin D 2 •

Nevertheless, some efforts have been made to produce biotechnologically a precursor that could be employed for the production of vitamin D3. Avruch et al. (1976) suggested to inhibit the 24-methyltransferase, the enzyme responsible for the C-24 methylation, characteristic to ergosterol. By growing S. cerevisiae in the presence of 25, aza-24,25 dihydrozymosterol at 1·3 J.lM concentration, it was possible to inhibit the transmethylation reaction without affecting the growth of the yeast. Under these conditions little ergosterol was formed but the accumulation of zymosterol (3) and cholesta 5,7,22,24(25)­tetraenol (12) took place in considerable quantities. The cholestatetraenol was suggested as a substitute for the more expensive 7-dehydrocholesterol provita­min. The conversion of this compound to the corresponding cholecalciferol by irradiation would be expected. However, no additional information has become available about this modified process.

HO 12

The Biotechnology of Ergosterol 93

REFERENCES

Andreasen, A. A. & Stier, T. J. B. (1953). Anaerobic nutrition of Saccharomyces cerevisiae. I. Ergosterol requirement for growth in deficient medium. J. Cell. Physiol., 41, 23-36.

Appleton, G. S., Kieber, R. J. & Payne, W. J. (1955). The sterol content of fungi II. Screening of representative yeasts and molds for sterol content. Appl. Microbiol., 3, 249-51.

Avruch, L., Fischer, S., Pierce, H. D. & Oehlschlager, A. C. (1976). The induced biosynthesis of 7-dehydrocholesterols in yeast: potential sources of new provitamin D3 analogs. Can. J. Biochem., 54,657-65.

Bills, C. E., Massengale, O. N. & Prickett, P. S. (1930). Factors determining the ergosterol content of yeast. I. Species. J. BioI. Chem., 87, 259-64.

Dulaney, E. L. (1957). Preparation of ergosterol containing yeast. US Patent 2817624. Dulaney, E. L., Stapley, E. O. & Simpf, K. (1954). Studies on ergosterol production

by yeasts. Appl. Microbiol., 2,371-9. EI-Refai, A. E. M. & EI Kady, I. A. (1969). Utilization of some industrial by-products

for the microbiological production of sterols from Saccharomyces fermentati. J. Bot. UAR, 12, 55-66.

Fryberg, M., Oehlshiager, A. C. & Uorau, A. M. (1973). Biosynthesis of ergosterol in yeast. Evidence for multiple pathways. J. Am. Chem. Soc., 95,5747-57.

Gal'tsova, R. D., Novichkova, A. T. & Vakina, I. P. (1959). Effect of glucose on ergosterol synthesis by yeasts. Mikrobiologiya, 28, 502-6.

Hirsch, A. (1984). Vitamin D. In Kirk Othmer's Encyclopedia of Chemical Technol­ogy, 3rd edn, Vol. 20. Wiley-Interscience, New York, pp. 186-213.

Klein, H. P. (1955). Synthesis of lipids in resting cells of Saccharomyces cerevisiae. J. Bacteriol., 69, 620-7.

Klein, H. P., Eaton, N. R. & Murphy, S. C. (1954). Net synthesis of sterols in resting cells of Saccharomyces cerevisiae. Biochim. biophys. Acta, 13, 591.

Maguigan, W. H. & Walker, E. (1940). Sterol metabolism of microorganisms. I. Yeast. Biochem. J., 34,804-13.

Margalith, P. (1986). Steroid Microbiology. C. C. Thomas, Illinois. Maugenet, J. & Dupuy, P. (1964). Synthese des sterols par la levure. Ann. Technol.

Agric., 13,329-54. Nelson, H. A. (1959). Recovery of ergosterol. US Patent 2 874 171. Nes, W. R., Sekula, B. C., Nes, W. D. & Adler, J. H. (1978). The functional

importance of structural features of ergosterol in yeast. J. Bioi. Chem., 253, 6218-25.

Nordheim, W. (1966). Metabolic behaviour of fermenting yeasts under cytostatic conditions. Brauwiss., 19, 67-9.

Qureshi, A. A., Burger, W. C., Prentice, N., Bird, H. R. & Sunde, M. L. (1980). Suppression of cholesterol and stimulation of fatty acid biosynthesis in chicken livers by dietary cereals supplemented with culture filtrate of Trichoderma viride. J. Nutr., 110, 1014-22.

Schechter, I., Sweat, F. W. & Bloch, K. (1970). Comparative properties of 2,3 oxidosqualene-Ianosterol cyclase from yeast and liver. Biochim. biophys. Acta, 220, 463-8.

Starr, P. R. & Parks, L. W. (1962). Some factors affecting sterol formation in S. cerevisiae. J. Bacteriol., 83, 1042-6.

Steenbock, H. & Black, A. (1924). Fat soluble vitamins XVII. The induction of growth promoting and calcifying properties in a ration by exposure to ultra-violet light. J. BioI. Chem., 61,405-22.

Taoret, C. (1889). Sur un nouveau principe immediat de L'ergot de seigle, l'ergosterine. C. R. Seances Acad. Sci., 108,98.

Usdin, V. R. & Burrell, R. C. (1952). Preliminary study of the lipids of Rhodotorula gracilis. Archs. Biochem. Biophys., 36, 172-7.

Chapter 6

ALGAL AND MICROBIAL PRODUCTION OF VITAMIN E

Y. TANI

Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

1 INTRODUCTION

The fat-soluble vitamin, vitamin E, has received great attention for its usefulness as an antioxidant in clinical and nutritional applications. The demand for the vitamin has increased year after year. The supply of tocopherols has been limited to the chemically synthesized racemate of a-tocopherol or a mixture of a-, f3(y)- and 6-tocopherols extracted from vegetable oils.

Tocopherols are indispensable components of the lipid bilayer of biological membranes; decrease in their content brings about structural and functional damage to the membranes. It is generally accepted that the stabilizing effect of tocopherols on the membranes is mainly related to three functions: (1) reaction with lipid peroxide radicals, (2) quenching of reactive molecular oxygen, and (3) ordering (i.e. restriction of molecular mobility) of the membrane lipid bilayer by formation of tocopherol complexes with fatty acids. The highest physiologically active form among the various tocopherols and tocotrienols so far identified is D-a-tocopherol.

Since a preliminary survey on the occurrence of tocopherols in micro­organisms (Green et al., 1959), its presence has been demonstrated especially in chlorophyll-containing organisms, though not in all of them (Taketomi et al., 1983). Early, a tocopherol-like antioxidant was found in yeasts (Forbes et al., 1958) and a small amount of a-tocopherol was detected in cells of baker's yeast (Diplock et al., 1961). a-Tocopherolquinol and a-tocopherolquinone were found in the algae, Euglena gracilis, in bacteria and in yeasts (Hughes & Tove, 1982). Ruggeri et al. (1985) reported enhancement of a-tocopherol production in temperature-stressed cells of E. gracilis Z grown photo­heterotrophically. However, a study on industrial production of these com­pounds has not been performed so far.

Recently, Tani & Tsumura (1989) have initiated a study aiming towards

95

96 Y. Tani

the development of new microbial processes to produce tocopherols, especially a--tocopherol.

2 CHEMISTRY AND ASSAY OF VITAMIN E

Vitamin E was found as a dietary factor in animal nutrition, considered in the 1920s to be especially important for normal reproduction. a-- and f3-Tocopherols were isolated from wheat germ oil and characterized in 1936. Soon afterwards the third active factor, y-tocopherol, was found in cotton-seed oil. In 1947, the fourth tocopherol, named ~-tocopherol, was isolated from soybean oil. In the ensuing years, the investigation of several vegetable oils disclosed the existence of four tocotrienols. Thus, four tocopherols and four tocotrienols are now known to occur in nature (Fig. 1).

Tocopherols and tocotrienols are white to pale yellow oils. These are soluble in organic solvents such as ethanol and ether, and miscible with other oils. They are easily oxidized to a dark color. The esters are stable. The naturally occurring form is the D-form, and D-a--tocopherol has the 2R, 4'R and 8'R configuration. The absorption maxima are 290-298 nm.

Determination of tocopherols in cells has been described by Tani & Tsumura (1989). Cells were homogenized in ethanol and extracted at 50-60°C for 20 min. After an equal volume of n-hexane and one and a half volumes of water were added to the ethanolic extract and mixed, the n-hexane layer was obtained by centrifugation. The n-hexane extract was employed for high performance liquid chromatography. Column systems used were an M&S packed column (CIS 4·6 x 150 mm) with methanol-dichloromethane (9: 1, v Iv) as the mobile phase or a YMC packed column (Sil, 6 x 150 mm) with n-hexane-dichloromethane-isopropanol (70: 30 : O· 2, by volume). The flow rate was 1 ml/min. Tocopherols were detected at 292 nm.

Rl R2 CHJ CHJ a-Tocopherol

CHJ H B-Tocopherol

H CHJ y-Tocophero 1

H H 0-Tocophero 1

Rl R2 CHJ CH3 a-Tocotrienol

CHJ H B-Tocotrienol

H CHJ y-Tocotri eno 1

H H 6-Tocotrienol

Fig. 1. Chemical structure of vitamin E group.

Vitamin E Production 97

3 BIOSYNTHESIS OF VITAMIN E

It has been accepted that tocopherols are synthesized through two routes, the tocopherol route and tocotrienol route. Homogentisic acid, a precursor of tocopherols, which was derived from the shikimate pathway (the biosynthetic pathway of aromatic amino acids), incorporates phytyl pyrophosphate or geranylgeranyl pyrophosphate. When phytyl pyrophosphate is incorporated, tocopherols are formed and the following additional methylation using S-adenosyl-L-methionine as the methyl donor occurs to form various types of tocopherols (tocopherol route). When geranylgeranyl pyrophosphate is in­corporated, tocotrienols are formed with a similar methylation. Subsequently, tocotrienols can be transformed to tocopherols. NADPH is the reducing coenzyme for the reduction of the side chain (tocotrienol route). In Spinach chloroplasts, the enzymes involved in the a-tocopherol synthesis were found in the chloroplast envelope fraction. The prenyltransferase and methyltransferase activities were found to be tightly bound to the chloroplast envelope membrane. Thus, it is strongly suggested that the tocopherol biosynthesis is related to the occurrence of chloroplasts or plastids.

4 FERMENTATIVE PRODUCTION OF TOCOPHEROL

4.1 Screening of Tocopherol-Producing Micro-organisms

The distribution of tocopherol-producing ability was extensively studied with 162 strains from 26 genera of ascosporogenous and asporogenous yeasts, 74 strains from 15 genera representing molds of the Phycomycetes, Ascomycetes and Fungi Imperfecti, 41 strains from 12 genera of Basidiomycetes, 4 strains of algae belonging to genera of Euglena, Chlorella and Chlamydomonas, and 5 strains of isolated but unidentified algae. Though occurrences of tocopherol in yeast (Diplock et al., 1961) and a-tocopherolquinone and its quinol in eucaryotic and procaryotic micro-organisms (Hughes & Tove, 1982) were reported, no tocopherol was detected in cells and culture filtrates of yeasts, molds and Basidiomycetes under the cultured and detection conditions employed. On the other hand, all algae tested were found to contain tocopherols, mainly the a-type.

Euglena gracilis Z was the best producer of a-tocopherol among all algae tested. It appeared on high performance liquid chromatograms that the tocopherol detected corresponded to the retention time of a-tocopherol and that it was the dominant component, with only a small amount of fJ(y)- and 6-tocopherols. Shigeoka et al. (1986) demonstrated during a study on the intracellular localization of tocopherols in E. gracilis Z, that a-tocopherol constituted more than 97% of total tocopherols.

98 Y. Tani

4.2 Isolation of a-Tocopherol from EuglelUl gracilis Z CeUs

Cells of E. gracilis Z weighing 2·1 g as dry matter were obtained from 80 ml of culture broth. The cell paste was suspended in 100 ml of ethanol and kept at 50°C for 30 min. The colorless cell debris were filtered and re-extracted once in the same manner. The combined extract was partitioned between equal volume of sodium chloride-saturated water and 150 ml of n-hexane. The n-hexane phase was dried by anhydrous sodium sulfate and then concentrated at 50°C under reduced pressure. Resultant lipid residues dissolved in 20 ml of isooctane were passed through Sep-Pak (silica cartridge, Waters Associates) with a flow rate below 0·5 ml/ml and then 5 ml of isooctane was passed to eliminate non-polar carotenoids. After the elution with 15 ml of isooctane-1,4-dioxane (98:2, v/v), the eluate was concentrated, dissolved in n-hexane and then applied on a thin layer chromatography (Kieselgel60F254). The thin layer chromatography was developed with benzene. The band appeared as dark blue against yellow background under UV light (Rf = 0·62) was scraped off and extracted with n-hexane-ethanol (5: 1, v/v). Pale yellow oil, 3·6 mg, was obtained in the overall recovery of about 34% by evaporation.

The isolate dissolved in ethanol showed the typical absorption spectrum of Amax at 292 nm and corresponded to the authentic a-tocopherol. The peak of the retention time of the isolate on a high performance liquid chromatography, 4·5 min, corresponded with that of a-tocopherol. Therefore, the oily substance isolated from E. gracilis Z cells was identified as a-tocopherol.

4.3 Production of a-Tocopherol by Euglena gracilis Z

When E. gracilis Z was grown on a conventional algal medium, Koren-Hutner (KH) medium, the maximum amount of a-tocopherol, 1·98 mg/liter culture broth and 0·26mg/g dry cells, was obtained at 9th day of cultivation. To improve the productivity and simplify the medium composition, components of culture medium and their concentrations were investigated using KH medium as the basal medium. As a result, a medium was derived as an a-tocopherol production medium consisting of glucose (2%), peptone (1·2%), inorganic salts, thiamin and cyanocobalamin, pH 5·0. The amount of a-tocopherol in the culture broth increased with increase of the growth and chlorophyll levels in the cells, and reached a maximum after 10 days of cultivation. The amount of a-tocopherol was 9·9 mg/liter culture broth and 1·1 mg/ g dry cells.

The production of a-tocopherol, however, was much higher under light conditions than in the dark, indicating that chloroplasts whose differentiation is induced by light could be related to a-tocopherol productivity.

The effect of additions of a-tocopherol biosynthesis-related compounds on a-tocopherol production was investigated. L-Tyrosine, L-phenylalanine, iso­pentenyl alcohol, AMP, 4-hydroxyphenylpyruvate and homogentisate ap­parently increased the a-tocopherol productivity. Since L-tyrosine and L­phenylalanine share the biosynthetic pathway with tocopherols, their addition

Vitamin E Production 99

might repress their own biosynthesis and thus increase the biosynthetic flow to a-tocopherol. Homogentisate is a direct precursor of the tocopherol biosynth­esis. Among prenyl alcohols, which might be involved in the tocopherol biosynthesis in the form of pyrophosphate to be incorporated to the phytyl side chain, isopentenyl alcohol was slightly effective on a-tocopherol production.

When these compounds were added simultaneously to the a-tocopherol production medium, a combination of 0·1 % homogentisate and 0·01 % L-tyrosine was the best for a-tocopherol production. Moreover, it was found that higher productivity can be obtained when this mixture was added to the culture medium only at 5th day of cultivation rather than at the beginning. Feeding of ethanol to the medium together with peptone, when glucose was almost consumed, gave higher production of a-tocopherol than that of glucose-peptone in spite of the same cell yield.

Supplementation with ethanol and peptone, and homogentisate and L­tyrosine during cultivation was performed finally. As shown in Fig. 2, the specific production of a-tocopherol increased markedly when the mixture was fed at 5th, 7th and 9th days of the cultivation. On the other hand, when only ethanol and peptone were fed, the amount of a-tocopherol in the culture broth increased according to increase in cell mass but the intracellular content of a-tocopherol was almost the same as that of the non-supplemented culture.

100

+ a;-... .... ...... '" ~ a 50 ... ..

.&: a. 0 u 0 .... I

" 0

0 5 10

(8) (C)

, , ,

15 0 S 10 lS 0 5

Cultivation Time (day)

10 15

.. ... 20 .~

...... '" "0

10 .~ >.

Fig. 2. a-Tocopherol production in a batch culture. During the cultivation on the a-tocopherol production medium (A), 1% ethanol and 0·6% peptone (B), and 1% ethanol, 0·6% peptone, 0·1% homogentisate and 0·01 % L-tyrosine (C) were added at periods indicated by arrow. Concentrations indicated are the final concentration (w/v)

in the medium.

Y. Tani

Table 1 Progress of a-Tocopherol Production by E. gracilis Z

Medium Cell yield a-Tocopherol produced (g /liter)

(mg/liter) (mg/g dry cells)

KH medium 7·0 2·0 0·3 a-Tocopherol production medium 9·0 9·9 1·1

Added L-tyrosine 10·7 14·8 1·3 Added homogentisate 11·9 17·0 1·4 Added L-tyrosine, homogentisate

and ethanol 11·4 49·8 4·3 Fed L-tyrosine, homogentisate,

ethanol and peptone 28·4 143·6 5·1

The amount of a-tocopherol produced in the culture reached 143·6 mg/liter culture broth and 5·1 mg/g dry cells at 20th day of cultivation.

The increase in intracellular production of a-tocopherol by E. gracilis Z during the optimization study on culture conditions is summarized in Table 1. The final productivity in this medium was 71·8-fold that of the original KH medium and the intracellular content was increased 19·6-fold.

5 BIOCONVERSION REACTIONS FOR VITAMIN E SYNTHESIS

As represented in Fig. 1, a-tocopherol is composed of a chroman ring and an aliphatic side chain. The naturally occurring molecule has three chiral centres

"'co~ ~CHO +

Ho4c

Phytol

Wittig reaction

hydrogenation

hydrolys"

Fig. 3. Synthesis of (2R, 4'R, 8'R)-a-tocopherol by coupling optically active chroman aldehyde with a CI5 phosphonium salt derived from natural phytol.

Vitamin E Production 101

R I I •

(4* :C 1 : (4- (5

Fig. 4. Construction of an optically active C14 side chain from S-fJ-hydroxyisobutyric acid.

with the (2R, 4'R, 8'R)-configuration. Chemical synthesis of vitamin E-which is now industrially applied-yields a racemic product. In this respect, several attempts are made to synthesize the natural optically active compound (Leuenberger, 1985).

One example of such synthesis involves the coupling of optically active chroman aldehyde with a CIs-phosphonium salt derived from natural phytol (Wittig reaction), followed by double bond hydrogenation and hydrolysis of the protecting group. The natural side chain precursor, phytol, is the source of the two chiral centres (Fig. 3).

Recently, the total synthesis of the optically active side chain has been reported. The formation of such side chains starts from small optically active building blocks (C: or Cn, which are produced via microbial reactions. In this respect, microbiologically formed S-( + )-fJ-hydroxyisobutyric acid (a C:­synthon) has been used to produce a Cl4-vitamin E side chain according to the

HO I ~COOR

CD/ HO~OH

j ~COOH

Fig. S. Microbial reactions affording optically active S- or R-fJ-hydroxyisobutyric acid (fJ-HIBA). 1. S-fJ-HIBA; Pseudomonas putida or other micro-organisms. 2. S- or R-fJ-HIBA; depending on micro-organisms used. 3. R-fJ-HIBA; Gluconobacter roseus.

4. R-fJ-HIBA; Candida humicola.

102 Y. Tani

I B' A'~ --

I B A~

hyctroly." cyclzatlon -

Fig. 6. Construction of an optically active CI5 side chain.

scheme: C5 + C; + C i + C; (Fig. 4). Several examples of microbial bioconver­sion leading to this optically active S- or R-tJ-hydroxyisobutyric acid are given in Fig. 5.

Another possibility for the synthesis of an optically active C15 side chain is a C; + C; + C; condensation (Fig. 6). The optically active C5-synthon can be produced from a suitable unsaturated precursor via enantioselective microbial reduction (Fig. 7). Saccharomyces cerevisiae and Geotrichum candidum are useful in this respect and formed the C5-synthon, (S)-3-methyl-y-butyro­lactone, in high yield from suitable precursors.

The optically active chroman ring has also been synthesized using (S)-( + )­citramalic acid to introduce chirality; asymmetric hydration of mesaconic acid into the desired (S)-( + )-citramalic acid has been performed with Clostridium tetanomorphum (Fig. 8). This synthon has also been used to form the vitamin D3-metabolite, (25S, 26)-dihydroxycholecalciferol.

A more detailed description of the use of microbially obtained optically active synthons in vitamin E synthesis is given by Leuenberger (1985).

R

i/ i c~

Fig. 7. Bioconversion leading to optically active C5-synthons.

HaOC)

leaoH

I ----HO* ~ OH

Vitamin E Production

Clostridium tetanomorphum

~

Haoc~

HO COOH

v

I I I , I I I I I t'

H~ ¥O~OH

103

Fig. 8. Synthesis of the optically active chroman moiety from trimethylhydroquinone and (S)-citramalic acid.

REFERENCES

Diplock, A. T., Green, J., Edwin, E. E. & Bunyan, J. (1961). Tocopherol, ubiquinones and ubichromenols in yeasts and mushrooms. Nature, Lond., 189, 749-50.

Forbes, M., Zilliken, F., Roberts, G. & Gyorgy, P. (1958). A new antioxidant from yeast. Isolation and chemical studies, J. Am. Chern. Soc., SO, 385-9.

Green, J., Price, S. A. & Gare, L. (1959). Tocopherols in microorganisms, Nature, Lond., 184, 1339.

Hughes, P. E. & Tove, S. B. (1982). Occurrence of a-tocopherolquinone and a-tocopherolquinol in microorganisms. J. Bacteriol., 151, 1397-402.

Leuenberger, H. G. W. (1985). Microbiologically catalyzed reaction steps in the field of vitamin and carotenoid synthesis. In Biacatalysts in Organic Synthesis, eds. J. Tramper, M. C. Van der Plas & P. Linko. Elsevier, Amsterdam, pp. 99-118.

Ruggeri, B. A., Gray, R. J. H., Watkins, T. R. & Tomlins, R. I. (1985). Effects of low-temperature acclimation and oxygen stress on tocopherol production in Euglena gracilis Z. Appl. Env. Microbial., 50, 1404-8.

104 Y. Tani

Shigeoka, S., Onishi, T., Nakano, Y. & Kitaoka, S. (1986). The contents and subcellular distribution of tocopherols in Euglena gracilis. Agric. BioI. Chern., 50, 1063-5.

Taketomi, H., Soda, K. & Katsui, G. (1983). Results of screening test in tocopherols in microbial realm. Vitamins (Japan), 57, 133-8.

Tani, Y. & Tsumura, H. (1989). Screening of tocopherol-producing microorganisms and a-tocopherol production by Euglena gracilis Z. Agric. Bioi. Chern., 53, 305-12.

Chapter 7

MICROBIAL PRODUCTION OF POLYUNSATURATED FATTY ACIDS (VITAMIN-F GROUP)

S. SHIMIZU & H. YAMADA

Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan

1 INTRODUCTION

The classical investigations performed by Burr & Burr in 1929-1930 clearly demonstrated that animals cannot survive without an exogenous source of fat. Subsequent studies have shown that supplementing a fat-free diet with certain essential fatty acids prevents the occurrence of symptoms of fat deficiency. It is now well established that both the n-6 and n-3 polyunsaturated fatty acids (PUF As) are essential in human nutrition. Current information indicates that they have important roles in the structure and function of biological mem­branes. They have recently attracted great interest due to their unique biological activities, such as lowering of plasma cholesterol level, prevention of thrombosis, and so on. In addition, they are precursors for the biosynthesis of a variety of a large family of structurally related C-20 compounds, such as prostaglandins, prostacyclins, thromboxanes and leukotrienes. Such current status of PUF As suggests that they are highly important substances in the pharmaceutical, medical and nutritional fields. These substances are sometimes referred to as the vitamin-F-group.

In this chapter, we summarize current progress in the biotechnology of PUFA production. Emphasis will be placed on possible advantages of micro-organisms as practical sources of PUFAs. Several reviews summarize other various aspects of PUF As and provide excellent background information (Wagner & Folkers, 1964; Rahm & Holman, 1971; Brenner, 1974; Fluco, 1974; Holloway, 1983; Numa, 1984; Dyerberg, 1986; Needleman et al., 1986; Horrobin & Huang, 1987).

2 NATURALLY OCCURRING POLYUNSATURATED FArry ACIDS

The n-6 and n-3 families of PUFAs are distinguished by differing positions of the double bond closest to the methyl terminal group of the fatty acid chain

105

106 S. Shimizu & H. Yamada

n-7

c:::::::.~ --~ ~~~~ c::::::~~ ~~ 16:1 16:2 18:2 18:3 20:3

n-9 c:::::::;:. --~~ ~~- c:::::::;: 18:1 18:2 20:2 20:3

n-6

~ -c::;::::; -- c::;:;:;~- c:::;::;::: 18:2 18:3 GLA 20:3DHGLA 20:4 ARA

n-3

C:::::::::=H ~C::::::::H -~~ .. ~~. C:::::::OOOH

18:3 18:4 20:4 20:5 EPA 22:6 DHA

Fig. 1. Pathways for the biosynthesis of PUFAs of the n-7 (palmitoleic), n-9 (oleic), n-6 (linoleic) and n-3 (a-linolenic) families. PUFAs include the n-7, n-9, n-6 and n-3 families, which are defined by the position of the double bond closest to the methyl end of the fatty acid molecule. Desaturation occurs toward the carboxyl end of the molecule and chain elongation at the carboxyl end, leaving the methyl end unaltered. Usually, PUFAs of each family are not inconvertible. GLA, r-linolenic acid; DHGLA, dihomo-r-linolenic acid; ARA, arachidonic acid; EPA, eicosapentaenoic acid; DHA,

docosahexaenoic acid.

(see Fig. 1). Linoleic acid (9,12-cis-octadecadienoic acid, 18: 2 n-6) and a-linolenic acid (9,12,15-cis-octadecatrienoic acid, 18: 3 n-3) are found abun­dantly in plants. They are synthesized de novo in higher plants and together represent nearly all the polyenoic fatty acids present. Linoleic acid comprises about 50% of the total fatty acids in corn, soybean, sunflower-seed and cotton-seed oils and over 70% of the fatty acids of safflower oil. a-Linolenic acid represents over 50% of the total fatty acids of linseed and perilla oils. In Oenothera biennis (evening primrose) and O. lamarckiana, however, linoleic acid is further desaturated to form y-linolenic acid (6,9,12-cis-octadecatrienoic acid, 18: 3 n-6), which comprises about 10% of the total fatty acids in their seeds. Mosses (Hartmann et al., 1986), algae (Seto et al., 1984) and protozoa (Erwin & Bloch, 1964), some fungi (Gellerman & Schlenk, 1979; Yamada et al., 1987, Shimizu et al., 1988a,1989a) and some marine bacteria (Yazawa et al., 1988) can further add a 2-carbon unit to these C-18 PUFAs to form C-20 PUFAs.

Animals cannot synthesize fatty acids with double bonds at either the n-3 or n-6 positions, and therefore are dependent upon dietary sources for these fatty acids. Most animals, however, possess enzyme systems for further desaturation and chain elongation at the carboxyl end of these fatty acid molecules. The resulting further desaturated or longer-chain derivatives such as y-linolenic acid, dihomo-y-linolenic acid (8,1l,14-cis-eicosatrienoic acid, 20: 3 n-6), arachidonic acid (5,8,1l,14-cis-eicosatetraenoic acid, 20: 4 n-6), eicosa­pentaenoic acid (5,8,1l,14,17-cis-eicosapentaenoic acid, 20:5 n-3, or EPA) and docosahexaenoic acid (4,7,10,13,16,19-cis-docosahexaenoic acid, 22: 6

Microbial Production of PUFAs 107

n-3, or DHA), are important as essential fatty acids in human nutrition or as structural components of cellular membrane phospholipids.

Most aquatic plants can synthesize the longer chain n-3 fatty acids including EPA and DHA from common metabolites. Fish, shellfish and marine mammals at the apex of the aquatic food chains contain relatively large quantities of these n-3 fatty acids, and therefore provide the human diet with a direct source of preformed EPA or DHA. Terrestrial food chains also contain some n-3 PUFAs, but they are dominated by the n-6 PUFA family because most land plants synthesize linoleic acid rather than a-linolenic acid.

A variety of PUFAs other than those of the n-6 and n-3 families have been detected in living organisms. Some of them are also listed together with the PUFAs of the n-6 and n-3 families in Table 1.

Table 1 Several Naturally Occurring PUFAs and their Dietary Sources and Biological Functions

Fatty acid families and their major members

n-6 Linoleic acid 9,12-cis-18: 2 y-Linolenic acid (GLA) 6,9,12-cis-18: 3

Dihomo-y-linolenic acid 8, 11,14-cis-20 : 3 Arachidonic acid 5,8,11 , 14-cis-20 : 4

Docosapentaenoic acid 4,7, 10,13, 16-cis-22 : 5

n-3 a-Linolenic acid 9,12,15-cis-18: 3

Eicosapentaenoic acid (EPA) 5,8,11,14,17-cis-20:5

Docosahexaenoic acid (DHA) 4,7,10,13,16,19-cis-22: 6

n-9 Oleic acid 9-cis-18: 1 Eicosatrienoic acid 5,8,11-cis-20: 3

n-7 Palmitoleic acid 7-cis-16: 1

Major dietary sources

Most vegetable oils

None

None

Meat, liver, brain

None

Some vegetable oils (soy, linseed), leafy vegetables

Fish, shellfish, algae

Fish, shellfish, algae

Animal and vegetable fats

None

None

Biological function, tissue distribution and other remarks

Essential fatty acid, minor component of most tissues

Major fatty acid of evening primrose seed; used as an addi­tive of feed, cosmetics, etc.

Precursor of 1-group of prosta­glandins

Major component of most mem­brane phospholipids; precursor of 2-group of prostaglandins

Frequently found in tissues deficient in n-3 PUF As

Minor component of tissues

Precursor of 3-group of prosta­glandins; prevent thrombosis; used as pharmaceuticals and a feed additive

Minor component of membrane phospholipids in retinal photo­receptors, sperm, etc.

Major component of many tissues Accumulated in total essential

fatty acid deficiency

Prevent apoplexy

108 S. Shimizu & H. Yamada

3 BIOSYNTHESIS

The biosynthesis of linoleic acid and a-linolenic acid in higher plants, lower plants and some micro-organisms takes place via common metabolic C-18 fatty acids, i.e. stearic acid and oleic acid, from acetate fragments produced during the catabolism of carbohydrates (Fluco, 1974). Linoleic acid and a-linolenic acid are usually the terminal products of fatty acid synthesis in higher plants. These two fatty acids are converted to two distinct families of long-chain and more unsaturated fatty acids through the n-6 and n-3 routes, respectively, as shown in Fig. 1. In these routes, the successive chain elongation and desaturation take place toward the carboxyl end of the fatty acid molecule. Thus, the members of the n-6 family have six carbons after the last double bond in the molecule, and the members of the n-3 family have three carbons in the terminal structure. The first step of the biosynthesis of PUF As is the desaturation at the ~6-position of linoleic acid or a-linolenic acid to yield y-linolenic acid or 6,9,12,15-cis-octadecatetraenoic acid (18: 4 n-3), respectively (~6-desaturation). The ~6-desaturation is followed by the chain elongation to the respective C-20 PUFAs, i.e. dihomo-y-linolenic acid and 8,1l,14,17-cis-eicosatetraenoic acid (20:4 n-3). The ~5-position of dihomo-y­linolenic acid in the n-6 route is then desaturated to yield arachidonic acid (~5-desaturation). Usually, arachidonic acid is the terminal product of the n-6 route. The ~5-desaturation on 20: 4 n-3 fatty acid in the n-3 route results in the formation of EPA, which is followed by chain elongation and desaturation to yield DHA. It is usually the end product of the n-3 route.

The n-6 and n-3 routes have been suggested to share the same enzymes or enzyme systems for the transformations of C-18 PUFAs to C-20 PUFAs (Brenner, 1974). Therefore, linoleic acid and a-linolenic acid compete with each other for the same enzymes. In animals, the n-6 family is usually favored in this competition, forming arachidonic acid. Neither isolation nor detailed characterization of enzymes responsible for the desaturation and chain elongation reactions involved in both routes has been performed yet except for ~6 desaturase from rat liver (Okayasu et al., 1981) . Desaturation reactions require CoA, A TP, Mi+ and NAD(P)H as cofactors, suggesting that desaturation enzymes are a kind of oxygenase and that only fatty acyl-CoA derivatives are substrates for the enzymes. Elongation enzymes have also been suggested to utilize only acyl-CoA derivatives as substrates.

The members of these two families of fatty acids constitute nearly all the PUF As in most living organisms. However, small amounts of different families of fatty acids have been detected. 5,8, ll-cis-eicosatrienoic acid (20: 3 n-9) is derived from oleic acid (9-cis-octadecenoic acid, 18: 1 n-9) through the n-9 route which involves the following successive reactions: desaturation at the ~6 position of oleic acid to form 6,9-cis-octadecadienoic acid (18: 2 n-9), followed by its carbon chain elongation to 8,1l-cis-eicosadienoic acid (20:2 n-9) and desaturation at the ~5-position of the latter to 20: 3 n-9 fatty acid (Fig. 1). 7,10,13-cis-eicosatrienoic acid (20: 3 n-7) is derived from palmitoleic acid

Microbial Production of PUFAs 109

(9-cis-hexadecenoic acid, 16: 1 n-7) through the n-7 route, as shown in Fig. 1. These data indicate that there are at least four families of PUF As in organisms.

4 PRODUCTION OF POLYUNSATURATED FATTY ACIDS

The available lipid sources relatively rich in C-18 (y-linolenic acid) and C-20 PUFAs are seeds of evening primrose, and animal tissues, algal cells and fish oils, respectively. For practical purposes, however, these conventional sources are not satisfactory, both in their lipid contents and in the PUF A contents of the resultant lipid products (see Table 2).

Micro-organisms are thought to be very promising lipid sources because of their extremely high growth rates in simple media and the simplicity of their manipulation. Erwin & Bloch (1964) suggested that lower classes of organisms including micro-organisms can be classified into several groups based on their ability to produce PUF As, as shown in Fig. 2. Show (1965) pointed out that some fungi belonging to the Mucorales can accumulate relatively large

Table 2 Sources of PUF As

Source

Fungi Mortierella isabellina Penicillium cyaneum M. elongata lS-5 M. alpina lS-4 M. alpina lS-4b M. alpina lS-4 M. alpina 20-17c M. alpina 20-17d

Others Chlorella minutissima Porphyridium cruentum Euglena gracilis Seed oile

Porcine liver Fish oil'

a FA, fatty acid.

PUFA type

18:3 20:4 20:4 20:3 20:3 20:4 20:5 20:5

20:5 20:4 20:4 18:3 20:4 20:5

b grown with sesame oil extract. C grown at 12°e. d grown with linseed oil at 28°e. e Oenothera biennis. f Scomber scrombrus. g not reported.

FAa content

Total (mg/g)

553 90 88

456 463 474 243 585

30-220

65 50-150

PUFA (% in

total FA)

10 3-11

25 6·6

23·1 58

10·9 7·1

35-40 5-36 5-15 6-10 2-8

10-15

Potential PUFA

productivity (mg/g) (g/Iiter)

55 5·0 1 -g

22 1·0 28 0·6

107 2·17 275 4·3

27 0·5 42 1·35

20-80 0·07 0·1-0·5

0·5 20

110 S. Shimizu & H. Yamada

Metazoa; Des; y-lin Metaphyta;X;a-lin

Green Algae;X;a-lin Ciliates;Des;y-lin Ameba;Desjy-lin I ----- "I Phytomonads;X;a- and y-lin

Yeasts and Fungi; ----- . Des;a-lin .... ~somonads;Des;a- and y-lin Euglenids;Des;X;

_____ a-and r-lin

Cryptomonads;Des(?);a-lin

Red Algae;Des;a-lin

Actinomycetes;Des;O ..-----Eubacteria; Photosynthetic Bocteria;An;O

Blue-green Algae;Des;a-lin

An;Des;O

Hypothetical Primitive Organisms

Fig. 2. Presumed phylogenetic relationships between groups of existing protists, and patterns of fatty acid biosynthesis observed in these groups. An, Anaerobic mechanism for the synthesis of monoenoic acids; Des, oxidative de saturation for the synthesis of monoenoic acids; X, unknown ('plant') mechanism for the synthesis of monoenoic acids; 0, absence of di- and polyunsaturated fatty acids; a-lin, the n-3 route for the synthesis of PUFAs; y-lin, the n-6 route for the synthesis of PUFAs. For details, see

Erwin & Bloch (1964).

amounts of y-linolenic acid in their mycelia. Based on these early observa­tions, several workers have only since early 1980 started to screen for micro-organisms capable of accumulating lipids containing these PUF As in order to obtain more suitable sources for large-scale preparation of these PUFAs.

Recently, Suzuki and co-workers (Suzuki, 1985, 1988; Suzuki & Yokochi, 1986) found several potent producers of y-linolenic acid through their extensive screening for filamentous fungi. They reported that a strain of Mortierella isabellina accumulates about 5 g/liter of y-linolenic acid when grown in a medium containing glucose as a major carbon source. Based on this finding, commercial production of y-linolenic acid with a Mortierella fungus started recently in Japan. Another process is in progress in England.

Yamada and co-workers (Yamada et al., 1987, 1988) found that several fungal strains produce large amounts of lipids rich in arachidonic acid, dihomo-y-linolenic acid or EPA, or all of these C-20 PUFAs. They are new and promising sources for C-20 PUF As.

4.1. "I-Linolenic acid

Suzuki (1985, 1988) reported that several Mortierella strains, belonging to the subgenus Micromucor produce lipids rich in y-linolenic acid in their

Microbial Production of PUFAs 111

mycelia when grown in a liquid medium containing glucose or sugar cane molasses as a major carbon source. They can grow rapidly in a medium containing a high concentration glucose (200 g/liter) under the conditions of D.O., 2 ppm; pH, 4·0; and cultivation temperature, 30°C, yielding a mycelial mass of about 80 g/liter. In the case of M. vinacea, the lipid content of the resultant mycelia was found to be 48%, by weight, which corresponded to 38 g/liter of the lipids. The purified oil obtained from the mycelia contained myristic acid (0·7%, by weight), palmitic acid (27·2), palmitoleic acid (0,9), stearic acid (S'7), oleic acid (43,9), linoleic acid (12·0), ')I-linolenic acid (8·3), arachidic acid (0,6), 11-cis-eicosenoic acid (0·4), behenic acid (0·1) and erucic acid (0·2). The ')I-linolenic acid content in the oil is comparable to that of Oenothera biennis seed oil. The low content of linoleic acid in the oil may be advantageous in obtaining highly pure ')I-linolenic acid preparation in high recovery.

Recently, M. ramanniana (Hansson & Dostcilek, 1988) and Mucor ambiguus (Fukuda & Morikawa, 1987) were evaluated for their productivities of ')I-linolenic acid from a viewpoint of biochemical engineering. The latter fungus immobilized in porous support particles has been shown to excrete lipids containing ')I-linolenic acid into the culture broth and/or the surface of the cell wall in the presence of a nonionic surfactant.

4.2 Arachidonic acid

Yamada et al. (1987, 1988) assayed productivities of C-20 PUFAs in about 300 fungal isolates from natural sources which are capable of growing on an agar plate containing stearic acid or elaidic acid as the main carbon source for growth. They also tested more than 600 stock strains of a wide variety of micro-organisms, i.e. bacteria (23 genera), actinomycetes (4 genera), yeasts (20 genera), filamentous fungi (45 genera) and basidiomycetes (11 genera). Bacteria, actinomycetes, yeasts and basidiomycetes, in general, did not produce detectable amounts of C-20 PUFAs intracellularly. Most C-20 PUFA producers were found to be filamentous fungi belonging to the orders of Mucorales and Entomophthorales. Through this screening, they found that 60 strains of Mortierella and 4S isolates from natural sources produce large amounts of C-20 PUFAs of the n-6 family (i.e. arachidonic acid and dihomo-')I-linolenic acid) together with C-18 PUFAs of the same family (mainly ')I-linolenic acid). Most of the PUFA-producing isolates were found to belong to the genus Mortierella. It should be noted that all of these Mortierella strains found as C-20 PUFA producers belong to the subgenus Mortierella. Neither the stock cultures nor isolates belonging to the subgenus Micromucor showed any detectable accumulation of C-20 PUF As, although they produced n-6 C-18 PUFAs such as ')I-linolenic acid. The arachidonic acid contents of most of these strains accounted for more than 1S% of the total extractable fatty acids. These values represent more than SO% of the PUF As and are particularly high when compared with those in the case of C-18 PUF As (Table

Tab

le 3

C

ompa

riso

n of

Ara

chid

onic

Aci

d P

rodu

ctiv

itie

s in

Mor

tiere

lla F

ungi

and

the

ir M

ycel

ial

Fat

ty A

cid

Com

posi

tion

G

Stra

in

Pro

duct

ivit

yb

Fat

ty a

cid

com

posi

tion

(%

)C

Myc

elia

l T

otal

2

0:4

2

0:4

1

6:0

1

8:0

18

:1

18

:2

18

:3

20

:3

20

:4

20

:5

othe

rs

mas

s F

A

cont

ent

yiel

d (m

g/m

l)

(mg

/g)

(mg

/g)

(mg

/ml)

M.

hygr

ophi

la I

FO

594

1 7·

0 18

3 32

-8

0·23

24

·7

2·9

3704

9·

5 5·

5 1-

6 17

·9

0 0·

5 M

. zy

chae

CB

S 6

52·6

8 7·

7 12

0 19

·6

0·15

23

·2

12-6

29

·3

8·7

6·0

3·3

16·3

0

0·6

M.

elon

gata

CB

S 1

21·7

1 8·

1 61

13

·2

0·11

15

-4

14·0

30

·3

6·7

6·0

3·8

21-7

0

z.t

M.

elon

gata

IS

-5 A

KU

399

9 6·

9 16

6 39

·2

0·27

14

·5

8·0

34·0

7-

6 6·

0 2·

6 23

·6

0 3·

7 M

. pa

rvis

pora

2S-

13 A

KU

399

4 8·

0 20

1 19

·7

0·16

7-

8 9·

0 56

·3

5·3

6·9

1-9

9·8

0 0

M.

schm

ucke

ri N

RR

L 2

761

8·0

205

25-4

0·

20

19·9

12

-4

37-1

70

4 4-

9 4·

9 12

-4

0 1·

0 M

. al

pina

IS

-4 A

KU

399

8 9·

5 31

8 15

1·7

1-44

17

-9

5-9

11·3

9·

8 4-

1 3-

3 47

·7

0 0

M.

alpi

na 2

0-17

AK

U 3

996

9·4

277

109·

7 1-

03

15-8

5·

3 12

·0

18·2

4·

8 2·

3 39

·6

0 0·

7 M

. al

pina

CB

S 2

50-5

3 6·

2 12

4 33

-6

0·21

14

·5

5·9

27-8

11

-4

704

4·0

27-1

0

1-9

M.

alpi

na 1

-83

AK

U 3

995

9·4

300

143·

7 1·

35

18·6

4-

8 12

·3

8·9

4-1

3-4

47-9

0

0 M

. al

pina

CB

S 2

19·3

5 5·

5 13

9 31

-1

0·17

11

·2

4·0

30·5

14

-4

10·9

4-

1 22

-4

0 1-

7

G F

or

deta

ils,

see

Shi

miz

u et

al.

(198

8b).

b V

alue

s ar

e gi

ven

in m

g/m

l cu

ltur

e b

roth

or

mg/

g dr

y m

ycel

ia.

cVal

ues

are

give

n in

wei

ght

%.1

6:1

, pa

lmit

ic a

cid;

18

:0,

stea

ric

acid

; 1

8:1

, ol

eic

acid

; 1

8:2

, li

nole

ic a

cid;

18

:3,

y-li

nole

nic

acid

; 2

0:3

, di

hom

o-y-

lino

leni

c ac

id;

20

:4,

arac

hido

nic

acid

; 2

0:5

, E

PA

. E

PA

and

oth

er P

UF

As

of

the

n-3

fam

ily

wer

e no

t de

tect

ed.

d F

A =

fat

ty a

cid.

Tab

le 4

P

rodu

ctio

n o

f A

rach

idon

ic A

cid

by S

elec

ted

Mor

tier

ella

Str

ains

und

er O

ptim

al C

ultu

re C

ondi

tion

s

Stra

in

Tot

al

Tem

pera

ture

p

H

Cul

ture

M

ycel

ial

Tot

al

20

:4a

gluc

ose

(0C

) pe

riod

yi

eld

FA

a co

nten

t us

ed

(day

s)

(g /l

iter

) (m

g/g

) (%

in

(mg

/g)

(g /l

iter

) to

tal

FA

)

M.

elon

gata

IS

-5

100

24

5·0

4 22

18

8 25

47

·0

M.

alpi

na I

S-4

40

28

6·

0 4

16

474

58

275·

0 M

. al

pina

IS

-4

104

28

6·0

10

22

440

31

136·

4 M

. al

pina

20

-17

20

20

5·

5 5

10

485

52

252·

5

a F

A =

fatt

y ac

id;

20

:4,

arac

hido

nic

acid

.

20

:4

yiel

d (g/l

iter

)

1·0

4·3

3·0

2·5

~ ~ "" s· - ~ $:I..

I::

<"I g. ;3

~

~ ~ ).: '" ~

~

W

114 S. Shimizu & H. Yamada

3). No detectable amount of free fatty acids was found in lipid fractions extracted with chloroform-methanol, suggesting that the PUF As produced are present as triglyceride and/or phospholipid forms . Through this screening, they obtained three isolates, which were taxonomically identified to be Mortierella alpina IS-4 , M. alpina 20-17 and M. elongata IS-5, as potent producers of arachidonic acid.

Subsequently, they investigated the cultural conditions for arachidonic acid production with the three Mortierella strains (Yamada et al., 1987,1988). These three fungi utilize not only glucose but also glycerol, maltose, n-hexadecane and n-octadecane as carbon source for the arachidonic acid production. As shown in Table 4, all the strains produced more than 1 g/liter of arachidonic acid under 5-liter bench scale fermentor conditions. Based on the results of studies on individual and combined factors affecting arachidonic acid produc­tion, they selected M. alpina IS-4 as the most promising producer. Using a 2000-liter fermentor, 22·5 g/liter mycelia (dry weight) containing 44·0%, by weight, of the lipids, in which arachidonic acid comprised 31 ·0% of the total fatty acids, was produced under the conditions of intermittent feeding of glucose and 10 days cultivation at 28°C (Fig. 3(a» . They also reported that the aradchionic acid in the harvested mycelia can be specifically enriched when the mycelia are allowed to stand for a further few days at room temperature. The arachidonic acid content of the resultant mycelia reached nearly 70% of the total fatty acids (Fig. 3(b». Fractionation of the lipids in the mycelia demonstrated that triglycerides (70·1 %) and phospholipids (23,4%) were their

(s)

30 3r-------------A

o 2 , 6 8 to Cult lVOtlon time (cloys)

(b) 20:3 '8'218~ 20:~ \'S'3G '6~

l' ::: ~tlMm

10 I i } 9 § }{

1 ill ,8:1

o 50 100 FA composition (%)

Fig. 3. Production of arachidonic acid by M. alpina IS-4 under the optimal culture conditions. Mortierella alpina IS-4 was cultivated in a 2000-liter fermentor containing 1400 liters of a medium containing 2% glucose and 1 % yeast extract, pH 6·0 at 28°C. Glucose was added to the medium as shown in (a) . Changes in the mycelial fatty acid composition during growth are shown in (b). 16 : 0, Palmitic acid; 18 : 0, stearic acid; 18 :2, linoleic acid; 18 :3G, y-linolenic acid; 20:3, dihomo-y-linolenic acid; 20:4 or

Ara , arachidonic acid.

Microbial Production of PUFAs 115

.Iant meyer SOL lar tormontor

2,OOOL lar termentor

n·he,ana n.tocophero' ~

~ Na.,SO. I_L~ o L1 ,I ~';;:I.~I;;·:~§":"

~entr"ug.. f"ter evaporator

mil' 1 mil ' 2

Fig. 4. Flow sheet for the production of fungal oil of high arachidonic acid content.

major components, and about 50% of the arachidonic acid was present in the phospholipid fraction.

The lipids containing arachidonic acid can be obtained as an oil from the mycelia of M. afpina 1S-4 in a good recovery (80-90%) through the following successive steps: separation of mycelia by filtration , drying, crushing by ball mill, extraction of the lipids with n-hexane , removal of insoluble materials by centrifugation, decolorization and deodorization with active charcoal , and concentration. The resultant purified oil contains myristic acid (0·2%, by weight), palmitic acid (7·0) , palmitoleic acid (0·1), stearic acid (2·8), oleic acid (6·6), linoleic acid (5·9), y-linolenic acid (3 ·9), 11-cis-eicosenoic acid (0·9), 11 ,14-cis-eicosadienoic acid (1 ·3), dihomo-y-linolenic acid (3·9) and arachido­nic acid (67·4) . The overall process is outlined in Fig. 4. The arachidonic acid in the purified oil can be isolated as the methyl or ethyl ester in a good recovery after successive transesterification, liquid-liquid partition chromatog­raphy and high-performance liquid chromatography.

4.3 Dihomo-y-Linolenic Acid

Most arachidonic acid-producing fungi can accumulate small amounts of dihomo-y-linolenic acid, when they are cultivated under the conditions for arachidonic acid production (Yamada et af. , 1988; Shimizu et af. , 1988a). However, the ratio of dihomo-y-linolenic acid to arachidonic acid in mycelia is usually only about 0·2. Yamada et af. (1988) reported that M. afpina IS-4 accumulated 0·6 g/Iiter of dihomo-y-linolenic acid. Because dihomo-y-lino­lenic acid is a precursor of arachidonic acid in the n-6 route, the arachidonic acid accumulated in the mycelia must be produced via dihomo-y-linolenic

116

~2.0

• 1 "';;-1.0

<" c ...J

" l: o

S. Shimizu & H. Yamada

I

I

I I

, I

Time (days)

I

J2.0~

1.0 CII II)

o o ~

" .c!J

20 :

Fig. 5. Time course of the production of dihomo-y-linolenic acid by M. alpina 1S-4. Mortierella alpina 1S-4 was cultivated in a 50-liter bench scale fermentor containing 30 liters of a medium containing 2% glucose, 1 % yeast extract and 0·022% of sesame oil extract, pH 6·0. Glucose was added to the medium at the time indicated by the arrow.

DHGLnA, dihomo-y-linolenic acid; Ara, arachidonic acid.

acid, suggesting that all the arachidonic acid-producing fungi potentially have the ability to produce large amounts of this fatty acid.

Shimizu et al. (1989a) reported that the mycelial dihomo-y-linolenic acid content of M. alpina 1S-4 increases, with an accompanying marked decrease in its arachidonic acid content, on cultivation with sesame oil. They suggested that this unique phenomenon is due to specific repression of the conversion of dihomo-y-linolenic acid to arachidonic acid by the oil. The effective factor(s) causing this phenomenon was found to be present in the non-oil fraction of the oil, after fractionation of the oil with acetone. In a study on optimization of the culture conditions for the production of dihomo-y-linolenic acid by M; alpina 1S-4, a medium containing glucose, yeast extract and the non-oil fraction was found to be suitable for the production. Under the optimal conditions in a SO-liter bench scale fermentor, the fungus produced 2·17 g/liter of dihomo-y­linolenic acid (107 mg/ g dry mycelia) (Fig. S). This value accounted for 23·1 % of the total mycelial fatty acids. The mycelia were also rich in arachidonic acid (S3·S mg/g dry mycelia, 11·2%). Other major fatty acids in the lipids were palmitic acid (24·1%, by weight), stearic acid (7·0), oleic acid (20·1), linoleic acid (6·6) and y-linolenic acid (4·1).

4.4 Eicosapentaenoic Acid (EPA)

4.4.1 EPA -Production under Low Temperature Growth conditions Yamada and co-workers (Shimizu et al., 1988a; Yamada et aI., 1988) investi­gated the growth conditions causing compositional changes in mycelial fatty

Microbial Production of PUFAs 117

acids using the arachidonic acid producers obtained through their screening studies, because they could not find any strains capable of accumulating a detectable amount of EPA under their screening conditions (see Table 3). They found that lowering the cultivation temperature caused the additional accumulation of a PUFA with five double bonds, EPA, by all the arachidonic acid producers tested, as shown in Table 5. In all cases, cultivation at low temperature significantly increased the mycelial phospholipid content. For example, the mycelial lipids extracted from M. alpina 1S-4 grown at 12°C contained 47·6% (by dry weight) of phospholipids and 35·7% of triglycerides. More than 60% of the EPA accumulated in the mycelia was found in the phospholipid fraction. On the other hand, the lipid from the mycelia grown at 28°C contained only 21·6% of phospholipids, the triglyceride fraction compris­ing 73·5% of the total extractable lipids. It should be noted that most of these arachidonic acid producers grow well at low temperature (6-16°C), and yield enough mycelia in simple growth media.

Subsequently, they studied the mechanism involved in this unique phenome­non. The results of experiments with cell-free extracts of M. alpina 1S-4 demonstrated that the enzyme(s) that catalyzes the formation of EPA is produced even when the fungus is grown at high temperature, but that the reaction(s) yielding EPA does not take place at high temperature, as shown in Table 6. Since all the EPA-producing Mortierella strains accumulate PUFAs of the n-6 family (i.e. y-linolenic acid, dihomo-y-linolenic acid and arachidonic acid) in their mycelia and do not accumulate PUFAs of the n-3 family other than EPA, they suggested that an n-6 PUFA, probably arachidonic acid, may be a precursor of EPA. If this is the case, an enzyme(s) or enzyme system catalyzing the methyl-end directed desaturation of arachidonic acid (a17-desaturation) may be activated on cold adaptation. The resultant EPA may be necessary for maintaining a proper membrane fluidity in a low temperature environment (Shimizu et al., 1988a).

Among various arachidonic acid-producing strains, they selected M. alpina 20-17 as the most promising EPA producer. The fungus was found to accumulate about O· 5 g/liter (26·6 mgt g dry mycelia) of EPA on cultivation for 7 days at 12°C. This value accounted for 11 % of the total fatty acids in the extracted lipids. Other major fatty acids in the lipids were palmitic acid (6·4%, by weight), stearic acid (4·8), oleic acid (3·2), linoleic acid (3·1), y-linolenic acid (4·5) and arachidonic acid (63·8) (Shimizu et al., 1988b).

4.4.2 Conversion of Linseed Oil to an Oil Containing EPA Shimizu et al. (1989b) demonstrated that several arachidonic acid-producing Mortierella strains accumulate detectable amounts of EPA in their mycelia when grown in media containing a-linolenic acid. This observation suggests that the n-3 route occurs in the Mortierella fungi as well as animals, although they are lacking the ability to synthesize a-linolenic acid. This route seems to be independent of growth temperature, because EPA production takes place even when they are grown at 28°C. The ability of the Mortierella fungi to

Tab

le 5

F

orm

atio

n of

EP

A a

nd C

hang

es i

n M

ycel

ial

Fat

ty A

cid

Com

posi

tion

in

Mor

tiere

lla F

ungi

at

Var

ious

Gro

wth

tem

pera

ture

a

Stra

in

Gro

wth

EP

A F

atty

aci

d co

mpo

siti

on (

% t

tem

pera

ture

co

nten

t CC

) (m

g/g)

b 16

:0

18:0

18

:1

18:2

18

:3

20:3

20

:4

20:5

ot

hers

M.

alpi

na I

S-4

6 6·

43

8·0

2·0

14·0

15

·0

10·0

3·

3 30

·3

14·8

2·

6 A

KU

399

8 12

8·

09

8·7

2·2

16·8

14

·4

10·1

3·

0 28

·4

14·9

1·

5 16

1·

35

9·6

6·7

14·6

3·

9 4·

4 4·

2 50

·2

2·4

4·0

20

0 12

·0

4·5

12·1

5·

1 3·

4 3·

2 56

·2

0 3·

5 28

0

15·9

5·

9 11

·3

9·8

4·1

3·3

47·7

0

2·0

M.

hygr

ophi

la

12

9·76

16

·0

3·8

30·7

8·

9 11

·3

2·5

13·6

10

·4

2·8

IFO

594

1 28

0

24·7

2·

9 37

·4

9·5

5·5

1·6

17·9

0

0·5

M.

elon

gata

12

15

·5

13·9

2·

5 32

·0

7-4

13·1

4·

9 12

·3

13-9

0

IFO

857

0 28

0

25·2

4·

3 49

·1

3·5

2·5

1·0

13·9

0

0·5

M.

exig

ua

12

5·45

9·

8 1·

5 22

·5

9·9

6·0

3·2

37·5

7·

0 2·

6 IF

O 8

571

28

0 22

·3

7·2

33·1

10

·2

4·7

3·6

18·0

0

0·9

M.

parv

ispo

ra

12

6·97

10

·1

4·0

43·3

7·

0 8·

7 2·

7 15

·3

5·2

3·7

20-2

4 A

KU

399

7 28

0

13·2

3·

7 54

·6

5·5

5·4

1·7

15·5

0

0·4

a F

or d

etai

ls,

See

Shim

izu

et a

l. (1

988a

).

b V

alue

s ar

e gi

ven

in m

g/g

dry

myc

elia

. C

V

alue

s ar

e gi

ven

in w

eigh

t %

. A

ny f

atty

aci

d of

the

n-3

fam

ily o

ther

tha

n E

PA

was

not

det

ecte

d. F

or a

bbre

viat

ions

of

fatty

ac

ids,

see

Tab

le 3

.

.... .... 00

:-->

~

§.

N'

I:: R- ~

~

~ I:> ~

Microbial Production of PUFAs

Table 6 Enzymatic Formation of EPA by a Cell-Free Extract of M. alpina 1S-4

Grown at 28°C'

Omission from reaction mixture

None

Lipid CofactorsC

Lipid, cofactors Enzyme

Reaction time (h)

0 5

10 22 10 10 10 10

EPA found (nmollml) on incubation at

12°e 28°e

o (2)b o (2) 18 (23) o (4) 38 (49) o (3) 48 (67) o (4)

5 (8) o (3) 8 0 1 0 0 0

a For details, see Shimizu et al. (1988a). b Values obtained with the cell-free extract prepared from the mycelia grown at 12°C are given in parentheses. C ATP, CoA, NADPH and MgCI2 •

119

convert added a-linolenic acid to EPA is very promising from a biotechnologi­cal viewpoint because there are various kinds of easily available natural oils containing a-linolenic acid, and it is expected that they can be converted to oils rich in EPA on incubation with these fungi. They examined the potential of such natural oils as precursors of EPA and found that linseed oil, in which a-linolenic acid amounts to about 60% of the total fatty acids, is the most suitable for EPA production. Under optimal cultural conditions, M. alpina 20-17 converted 5·1 % of the a-linolenic acid in the added linseed oil into EPA, the EPA production reaching 1·35 g/liter (41·5 mg/g dry mycelia). This value is 2·8-fold higher than that obtained under low temperature growth conditions. The resultant lipid is rich in either arachidonic acid or EPA, Another advantage of this conversion is that it can be carried out under normal growth temperature conditions (20-30°C). Under such conditions, the fungal growth is rapid and dense, and the energy costs for temperature control may be less than those for cooling.

4.4.3 EPA from Algae Micro-algae belonging to the genera Skeletonema, Phaeodactylium, Dunaliella, ... were also found to be high EPA-producers. Algal ponds for EPA-synthesis and other fine chemicals are already operational in Israel and in the USA (California, Hawaii) (Borowitzka & Borowitzka, 1988).

5 CONCLUSION

Considerable progress has been made in biotechnological production of PUFAs during the past years. Areas of progress include: (a) the finding of

120 S. Shimizu & H. Yamada

potent fungal strains rich in y-linolenic acid; (b) determination of optimal conditions for large-scale submerged culture, which made it possible to operate industrial-scale production of this fatty acid; (c) the finding that C-20 PUFAs can be produced by filamentous fungi and algae efficiently; (d) selection of potent producers of C-20 PUFAs; and (e) determination of culture conditions for selective accumulation of arachidonic acid, dihomo-y-linolenic acid and EPA by the selected fungi or algae. These PUF As may be of considerable importance in the utilization of these algal cells or fungal mycelia or their oils as a feed supplement. In addition, they may be used as pharmaceutical products. Further progress in this field depends on additional technological development in downstream processes for the large-scale isolation of oils containing PUFAs and/or pure PUFAs.

In conclusion, it seems to us that the nutritional, pharmacological and economical potential of micro-organisms as a source of PUFAs are very high.

REFERENCES

Borowitzka, M. A. & Borowitzka, L. J. (1988). Micro-algal Biotechnology. Cambridge University Press, Cambridge, UK.

Brenner, R. R. (1974). The oxidative desaturation of unsaturated fatty acid. Molec. Cell. Biochem. 3,41-52.

Dyerberg, J. (1986). Linolenate-derived polyunsaturated fatty acids and prevention of atherosclerosis. Nutr. Rev. 44, 125-34.

Erwin, J. & Bloch, K. (1964). Biosynthesis of unsaturated fatty acids in micro­organisms. Science, 143, 1006-12.

FIuco, A. J. (1974). Metabolic alterations of fatty acids. Ann. Rev. Biochem., 43, 215-41.

Fukuda, H. & Morikawa, H. (1987). Enhancement of ')I-linolenic acid production by Mucor ambiguus with nonionic surfactant. Appl. Microbiol. Biotechnol. 27, 15-20.

Gellerman, J. L. & Schlenk, H. (1979). Methyl-directed desaturation of arachidonic acid to eicosapentaenoic acid in the fungus, Saprolegnia parasitica. Biochim. Biophys. Acta, 573, 23-30.

Hansson, L. & Dostalek, M. (1988). Effect of culture conditions on mycelial growth and production of ')I-linolenic acid by the fungus Mortierella ramanniana. Appl. Microbiol. Biotechnol., 28, 240-46.

Hartmann, E., Beutelmann, P., Vandekerkhove, 0., Euler, R. & Kohn, G. (1986). Moss cell cultures as sources of arachidonic acid and eicosapentaenoic acids. FEBS Lett., 198,51-5.

Holloway, P. W. (1983). Fatty acid desaturation. In Enzymes, 3rd edn., Vol. XVI, ed. P. D. Boyer, pp. 63-83. Academic Press, New York.

Horrobin, D. F. & Huang, Y.-S. (1987). The role of linoleic acid and its metabolites in the lowering of plasma chloesterol and the prevention of cardiovascular disease. Int. J. Cardiol., 17, 241-55.

Needleman, P., Turk, J., Jakschiik, B. A., Morrison, A. R. & Lefkowith, J. B. (1986). Arachidonic acid metabolism. Ann. Rev. Biochem., 55,69-102.

Numa, S. (Ed.) (1984). Fatty Acid Metabolism and its Regulation. Elsevier, Amsterdam.

Okayasu, T., Nagao, M., Ishibashi, T. & Imai, Y. (1981). Purification and partial characterization of linoleoyl-CoA desaturates from rat liver microsomes. Arch. Biochem. Biophys. 206,21-8.

Microbial Production of PUFAs 121

Rahm, J. J. & Holman, R. T. (1971). Essential fatty acids. In The Vitamins, Chemistry, Physiology, Pathology, Methods, 2nd edn, Vol. III, ed. W. H. Sebrell Jr & R. T. Harris, pp. 303-39. Academic Press, New York.

Seto, A., Wang, H. L. & Hesseltine, C. W. (1984). Culture conditions affect eicosapentaenoic acid content of Chlorella minutissima. J. Am. Oil Chem. Soc., 61, 892-4.

Shimizu, S., Shinmen, Y., Kawashima, H., Akimoto, K. & Yamada, H. (1988a). Fungal mycelia as a novel source of eicosapentaenoic acid, Activation of enzyme(s) involved in eicosapentaenoic acid production at low temperature. Biochem. Biophys. Res. Commun., 150, 335-41.

Shimizu, S., Kawashima, H., Shinmen, Y., Akimoto, K. & Yamada, H. (1988b). Production of eicosapentaenoic acid by Mortierella fungi. J. Am. Oil Chem. Soc., 65,1455-9.

Shimizu, S., Akimoto, K., Kawashima, H., Shinmen, Y. & Yamada, H. (1989a). Production of dihomo-y-linolenic acid by Mortierella alpina IS-4, J. Am. Oil Chem. Soc. 66, 237-41.

Shimizu, S., Kawashima, H., Akimoto, K., Shinmen, Y. & Yamada, H. (1989b). Microbial conversion of an oil containing a-linolenic acid to an oil containing eicosapentaenoic acid. J. Am. Oil Chem. Soc. 66, 342-7.

Show, R. (1965). The occurrence of y-linolenic acid in fungi. Biochim. Biophys Acta, 98,230-37.

Suzuki, O. (1985). y-Rinorensan no biseibutsu seisan (Production of y-linolenic acid by micro-organisms). Hakko to Kogyo (Fermentation and Industry), 43, 1024-31.

Suzuki, O. (1988). Production of y-linolenic acid by fungi and its industrialization. In Proceedings of the World Conference on Biotechnology for the Fats and Oils Industry, Hamburg. American Oil Chemists' Society, Champaign, Illinois, pp. 110-16.

Suzuki, O. & Yokochi, T. (1986). Production of y-linolenic acid by fungi. J. Am. Oil Chem. Soc., 63, 434.

Wagner, A. F. & Folkers, K. (1964). The essential fatty acid group. In Vitamins and coenzymes. Interscience Publishers, New York, pp. 389-406.

Yamada, H., Shimizu, S. & Shinmen, Y. (1987). Production of arachidonic acid by Mortierella elongata IS-5. Agric. BioI. Chem. 51, 785-90.

Yamada, H., Shimizu, S., Shinmen, Y., Kawashima, H. & Akimoto, K. (1988). Production of arachidonic acid and eicosapentaenoic acid by micro-organisms, Proceedings of the World Conference on Biotechnology for the Fats and Oils Industry, Hamburg. American Oil Chemists' Society, Champaign, Illinois, pp. 173-7.

Yazawa, K., Araki, K., Okazaki, N., Watanabe, K., Ishikawa, C., Inoue, A., Numao, N., & Kondo, K. (1988). Production of eicosapentaenoic acid by marine bacteria. J. Biochem., 103, 5-7.

Chapter 8

MICROBIAL PRODUCTION OF VITAMIN ~ (MENAQUINONE) AND VITAMIN K, (PHYLLOQUINONE)

Y. TANI

Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

1 INTRODUCTION

Vitamin K, one of the fat-soluble vitamins, is known as an anti-hemorrhagic factor. As Bentley & Meganathan (1982) pointed out in their review, vitamin K was the first discovered growth factor for micro-organisms, though it was named following the classical approach of animal nutrition in 1935. Dam discovered a new fat-soluble organic compound which was found to possess blood coagulation capacity, while establishing a bleeding condition in chicks fed on a diet formulated for the study of sterol metabolism, and he proposed the name of vitamin K (K for koagulation).

A major function of vitamin K is to act as a cofactor for the carboxylation of protein-bound glutamate residues to form y-carboxyglutamates. A simplified scheme of the blood clotting mechanism is shown in Fig. 1. Of these reactions, only the synthesis of prothrombin appears to be dependent on vitamin K.

Vitamin K is now widely used for various types of bleeding symptoms: hemorrhagic disease of new-born infants and hemorrhagic symptoms caused by administration of antibiotics, salicylic acid or warpharin dosages. Mena­quinone (MK, vitamin K2) has much higher therapeutic activity than phyllo­quinone (vitamin Kl). However, phylloquinone has been used for a long time as a medicine, because the chemical synthesis of MK is rather difficult and the natural sources contain only a small amount of MK.

MK was prepared from bacterially putrefied fish meal as the first crystalline antihemorrhagic vitamin that was clearly different from that of alfalfa, phylloquinone. Its function in bacteria centers around its major role as an electron carrier. Although more information is needed on its distribution in micro-organisms, a correlation has been found between the taxonomical classification of bacteria based on other criteria and the type of isoprenoid quinones found in those cells (Collins & Jones, 1981). However, economic production of MK by a microbiological process has not been demonstrated.

123

124 Y. Tani

(1) Precursor of Prothrombin ---.r~----~ Prothrombin CO2 , O 2

-NH-CH-CO-I Vitamin K (red.)

-NH-CH-CO­I

CH2

I CH2

I COOH

CH2

I HC-COOH

I COOH

(2) Prothrombin + Thromboplastin + Ca2 + ---~~ Thrombin

(3) Thrombin + Fibrinogen ---~. Fibrin (clot) Fig. 1. Blood clotting mechanism.

2 CHEMISTRY OF MENAQUINONES

The chemistry of vitamin K was established by the schools of Dam, Doisy and Karrer, who succeeded in 1939 in isolating two compounds with vitamin K activity, namely MK-7 (vitamin K2(35» from putrefied fish meal and phyllo­quinone from alfalfa meal (Fig. 2). The structure of phylloquinone was proven to be 2-methyl-3-phytyl-l,4-naphthoquinone, and that of MK to be 2-methyl-3-multiprenyl-l,4-naphthoquinone. The multiprenyl side chain of MK is of variable length from 5 to 70 carbon atoms. A number of derivatives of the basic structure also occur in nature; this is reflected in the modification of ring substitutents at the C-2 or C-3 position or both.

Phylloquinone is a yellow viscous oil and MKs are light yellow microcrys­talline plates. Melting points of MKs vary from 35 to 62°C on the length of the multiprenyl side chain. These are soluble in ether, petroleum ether, benzene, n-hexane and acetone, slightly soluble in methanol and alcohol, and insoluble in water. These are stable to air and heat, but very unstable for alkali and UV irradiation. The absorption maxima are 243,248,261,270 and 325 nm with the molecular extinctions coefficient of 18000-19000 at 248 nm.

o

(a) (b)

Fig. 2. Chemical structure of vitamin K. (a) Menaquinone-n (MK-n); (b) phyllo­quinone.

Vitamin K2 and Vitamin Kl Production 125

3 BIOSYNTHESIS OF MENAQUINONES

Intense research based on microorganisms during the last two decades has clearly proven the MK biosynthetic pathway as summarized by Bentley & Meganathan (1982).

The biosynthetic pathway of MK in bacteria is now believed to consist of the biosynthesis of demethyl MK by polyprenylation of 1,4-dihydroxy-2-naphthoate, which is formed as the first naphthalenoid intermediate from shikimate through chorismate(isochorismate) and o-succinylbenzoate, by ca­talysis of a membrane-associated transferase and its subsequent methylation. The methyl group is derived from S-adenosylmethionine. Biosyntheses of MK and aromatic amino acids share a common route until chorismate. The immediate precursor, shikimate (chorismate), is incorporated to non-carboxyl carbon atoms of 2-ketoglutarate to form the naphthoquinone nucleus. The biosynthetic pathway of MK can now be summarized as shown in Fig. 3, to which recent experimental results are added.

4 FERMENTATIVE PRODUCTION OF MENAQUINONES

In spite of the increasing need of vitamin K supply, no microbiological process for MK production has been developed so far. Only recently, work on the fermentative production of this water-insoluble vitamin by micro-organisms was initiated by Tani et al. (1984) and is described in this section.

4.1 Screening of Menaquinone-producing Micro-organisms

A number of micro-organisms were aerobically grown on media consisting of glycerol and peptone as carbon and nitrogen sources, respectively. Washed cells obtained from culture broth were suspended in acetone, and homogen­ized with a homogenizer. Lipophilic materials in the extract were absorbed on SEP-PAK C18 (Waters Associated, Inc.) and eluted with n-hexane. MK in the n-hexane eluate was detected with a spectrophotometer by its typical UV absorption spectrum. The amount of MK was calculated from the absorbance at 248 nm by using the molar extinction coefficient.

For the determination by high performance liquid chromatography, cells were homogenized in methanol and then extracted at 50-6O"C for 20 min. Solid materials were removed by centrifugation. The resultant supernatant was applied on a liquid chromatograph with 4·6 x 1oo-nm column of Cosmosil 5C18

(Nacalai Tesque Co.). The solvent used was methanol-dichloromethane. MK was detected with a UV monitor at 248 nm.

Thin-layer and reversed-phase chromatographies were also applied for the identification of MK. The thin-layer chromatography was performed with development using a solvent system of benzene-cyclohexane. For the

126

HO~COOH HOY 8hlklmata ,

HOO'--oOCOOH HZ~ HO Chorlemeta

~OOH

, ~g

Hoar.. 6- OOH H.<f°O COOH COZt

Z Ieochorleo TPP meta

Y. Tani

HOOC~H Mavaloneta ,

Olmethylallyl-PPI l.opentenyl-PPI , , ,.CHZCHZCOOH ~P

..... HO-", TPP

6

,

COOH 2-8ucclnyH-h clroxr-2,4-cyctohexa - -

COCil CH eOOH c~rtele 2 Z

;

Oerenyt-PPI

,_ I.opentenyt-PPI

Feme.y"""" , ,_ (n-3)xl.opentenyI-PPI ,

Polypr ... yI-PPI

Oemethylmenaqulnone-n

Manaqulnone-n

Fig. 3. Biosynthesis of menaquinone.

reversed-phase chromatography, the thin-layer plate was immersed gently in 5% liquid n-paraffin in n-hexane and stood horizontally in the air to allow evaporation of n-hexane. The reversed-phase chromatography was performed with development using a solvent system of acetone-water to which a few drops of liquid n-paraffin had been added. The MK band was detected under irradiation.

Vitamin K2 and Vitamin Kl Production 127

The distribution of isoprenoid quinones was surveyed in about 900 microbial strains (Tani et al., 1984). The quinone system of molds was tested in 56 strains of 17 genera representing phycomycetes, ascomycetes and fungi imperfecti. All of them lacked MK; their quinone system was coenzyme Q (CoQ). The same result was obtained with 88 strains of yeasts of 32 genera representing ascosporogenous, asporogenous and ballistosporogenous yeasts, and with 28 strains of Basidiomycetes of 14 genera.

MK was found to be widely distributed in procaryotes. About half of the 112 type strains of bacteria of 27 genera contained MK. The quinone types of the bacteria tested could be divided into three groups: MK alone, MK and CoQ, and CoQ alone. MK was found in Gram-positive bacteria, MK and CoQ in Gram-negative facultative anaerobes, and CoQ in Gram-negative aerobes. The only exception to this rule were strains of the genus Flavobacterium, which were Gram-negative facultative anaerobes but had MK mostly or CoQ. Flavobacterium species showed a higher content of MK than other bacteria. Therefore, 550 strains of yellow-pigmented bacteria were further isolated. About 90% of the isolates were MK producers. All actinomycetes tested (40 strains of 6 genera), contained only MK. This coincides well with the classification of actinomycetes as Gram-positive bacteria.

Actinomycetes, though accounting for a high proportion of potent MK producers, yield MK with eight or more isoprene units in the C-3 polyprenyl side chain. In the blood coagulation tests, MK-4, 5 and 6 were shown to have high biological activity, which decreased with the increase in the number of isoprenoid units. Consequently, Flavobacterium species was selected to further study for the improvement of the productivity.

4.2 Menaquinone-6 Production by Wild Type and Mutant Strains of Flavobacterium meningosepticum

Culture conditions for MK-6 production by the wild type strain of F. meningosepticum were investigated by Tani et al. (1984). The principal cultural conditions that can be considered as influencing MK production are carbon source for growth and the oxygen supply to the medium. Among carbohy­drates, ribose is closely related to the biosynthesis of MK, sharing the intermediate of the biosynthesis of aromatic amino acids. In fact, the cellular content of MK increased when ribose was used in place of glycerol as the carbon source; but the amount of MK in the culture broth decreased due to the low cell concentration. When glycerol, glucose or fructose was used as the carbon source, the growth and the productivity of MK were relatively high. The cellular content of MK increased and the cell yield decreased with decrease of aeration.

Prenyl alcohols are involved in MK biosynthesis in the form of pyrophos­phates, which are incorporated into the polyprenyl side chain. Isopentenol was the most effective in increasing the cellular content of MK, followed by dimethylallyl alcohol.

128 Y. Tani

Precursors of the naphthoquinone nucleus, shikimate and 0-

succinylbenzoate, increased the cellular content of MK to some extent. 1-Hydroxy-2-naphthoate (HNA) inhibited the biosynthesis of MK but had little effect on growth. This suggests that the compound is a specific inhibitor of the biosynthesis as an analog of 1,4-dihydroxy-2-naphthoate.

The biosynthetic pathways of MK and aromatic amino acids branch at chorismate after shikimate. The addition of L-tyrosine or its precursor, p-hydroxyphenylpyruvate, increased the cellular content of MK; however, L-tryptophan, L-phenylalanine and their precursors, anthranilate and phenylpyruvate, inhibited the MK production.

Compounds which were found to increase MK production in the experi­ments described above were examined in various combinations for a possible synergistic effect. Isopentenol and L-tyrosine formed the best couple. When isopentenol was fed intermittently to the medium, the amount of MK reached 21·3 mg per liter of culture broth and 3·52 mg per g of dry cells after 3 days of cultivation.

The MK productivity of F. meningosepticum was improved by mutageniza­tion with N-methyl-N'-nitro-N-nitrosoguanidine. A mutant strain resistant to HNA, which was found to be an inhibitor of MK biosynthesis but not of growth, produced MK more abundantly: 34 mg per liter of culture broth and 5·5 mg per g of dry cells (Tani et al., 1986). The mutant strain was less sensitive to inhibition by HNA on MK biosynthesis than the wild-type strain. Mutant strains, which displayed KCN resistance, aromatic amino acid auxotrophy and no-carotenoid productivity, did not show further increase of MK productivity.

Isolation of MK-6 from cells of the mutant strain was performed as follows: to a cell paste weighing about 100 g, 1 liter of acetone-ethyl ether was added and agitated in a mini-jar fermentor for 2 h. Solid materials were filtered and extracted again in a similar manner. Combined extracts were evaporated under reduced pressure to less than 10% in volume. Each 10 ml of the concentrate was partitioned between 500 ml of diethyl ether and 500 ml of sodium chloride-saturated water. 'the non-polar lipids fraction obtained was dried with anhydrous sodium sulfate and then evaporated. Resultant oily materials were dissolved in chloroform and applied on a thin-layer plate which was prepared by spreading a silica gel suspension (40 g/80 ml H20) on glass plates at a thickness of 0·75 mm, followed by activation at 110°C for 2 h after drying overnight at room temperature. Thin-layer chromatography was performed with development with a solvent system of cyclohexane-benzene. The MK band was scraped. MK was eluted with chloroform and crystallized twice from ethanol. Crystalline MK (220·4 mg) was obtained from cells of the mutant strain at a yield of 75%. MK loss occurred in the recrystallization step.

4.3 Menaquinone-5 Production by a Mutant Strain of Flavobacterium meningosepticum

The diversity of the types of isoprenoid quinones present in micro-organisms has been extensively studied as a result of the recent improvement of analytical

Vitamin K2 and Vitamin Kl Production 129

tools. Different types of MK homo logs were dectected in archaebacteria, mycoplasmas, and in Gram-negative and Gram-positive bacteria with or without minor component(s) in relation to taxonomic groupings. However, a change of the type of MK in the same organism has never been noticed.

It was found that an HNA-resistant mutant, strain HNA 12-D, which was derived from an MK-6 producer, produced an increased amount of MK, due to the production of another type of MK in addition to MK-6 (Tani et al., 1985). The newly formed MK was isolated and identified as MK-5. By combined feeding of isopentenol and L-tyrosine, the total amount of MK produced by strain HNA 12-D was 55·6 mg per liter of culture broth and 9·19 mg per g of dry cells in the ratio of MK-6 and MK-5 of 1: 1· 7 after 72 h cultivation, whereas the parent strain produced 25·3 mg per liter of culture broth and 4·14 mg per g dry cells of MK-6.

The MK system of the wild type strain of the bacterium was composed only of MK-6 without any minor components. The quantity of MK-5 produced by the mutant strain was one to two times as much as that of the MK-6. It was the first observation of a change in the MK pattern within the same micro­organism. HNA is a structural analog of 1,4-dihydroxy-2-naphthoate, an intermediate of MK biosynthesis. Whether the change in the specificity of the enzyme, 1,4-dihydroxy-naphthoate: polyprenyl pyrophosphate transferase, which catalyzes the prenylation of the naphthalenic intermediate using both solanesyl pyrophosphate and octaprenyl pyrophosphate as substrates, or that in the supply of polyprenyl pyrophosphates in strain HNA 12-D extended the MK spectrum by the acquisition of HNA resistance remains to be determined.

4.4 Menaquinone-4 Production by a Mutant Strain of Flavobacterium sp.238-7

During further study to develop a new fermentation process for MK production, a HNA-resistant mutant, strain HNA 250-15, of Flavobacterium sp. 238-7, which produced MK-6 as the major component of respiratory quinone with some minor components, showed an increase in MK productivity due to increased production of a minor component of MK (Tani & Sakurai, 1987). The newly-formed MK was identified as MK-4, which now finds a clinical use and which is made by a chemical process.

The MK-4 productivity of strain HNA 250-15 was further improved by making the mutant resistant to usnic acid and menadione. Flavobacterium sp. 238-7 contains MK as the sole quinone in the respiratory chain. Among the uncouplers and respiratory inhibitors tested, an uncoupler, usnic acid, strongly inhibited the growth of strain HNA 250-15 at a low concentration. On subsequent mutagenization of the strain, a mutant, USNr-2, which was resistant to 4 mg of usnic acid per liter, was found to produce a much larger amount of MK-4 than strain HNA 250-15, whereas the MK-6 content was reduced. The productivity of MK-4 was increased more than three times as compared with that of strain HNA 250-15.

130 Y. Tani

Growth of Flavobacterium was strictly inhibited by addition of menadione, an artificial derivative of vitamin K, to the culture medium, and the content of MK was reduced. Derivation of menadione-resistant mutant strains was then carried out for enhancement of the MK productivity using strain USNr-2 as the parent strain. Among the mutant strains obtained, strain K3-15, which was resistant to 20 mg of menadione per liter, showed the highest content of MK-4 and MK-6. The total MK productivity was increased about three times as compared with that of the wild type strain.

The cultural conditions for MK production were investigated with strain Kr15. Glycerol and peptone were the most effective carbon and nitrogen sources, respectively, both for growth and MK production. The amount of MK-4 increased linearly in proportion with the glycerol concentration, although the amount of MK-6 hardly changed with increasing glycerol concentration.

A variety of natural oils were supplemented to the culture medium at the final concentration of 0·05% directly before autoclaving. Among them, cedar wood oil decreased the growth but significantly increased the MK production.

120 12i () 0

EJ MK-4 I a>

100 0 MK-6 100,

::::: E

a> C ! 80 8 01)

loC C :f .. 0

()

60 I 6 loC

:v I :f III - ..

I

340 I 4 :u !D

... l 1: a> a:

20

r1l r .. ,.

I 2

Fig. 4. Improvement of menaquinone production by Flavobacterium sp. 238-7. The left and right bars indicate the amount of MK in mg/liter of culture broth and mg/g of dry celis, respectively, the open and shaded bars denoting MK-6 and MK-4, respectively. The amounts of MK produced by each strain indicated are the values when grown on the basal medium, except that K3-1sa was grown in the optimized medium and K3-1Sb in

the optimized medium supplemented with cedar wood oil.

Vitamin K2 and Vitamin K) Production 131

MK-4 production increased with increasing cedar wood oil concentration, whereas the amount of MK-6 decreased. None of the prenyl alcohols increased the MK production of strain K3-15 at concentrations of 0·5 and 1 mM, in fact the amount of MK-4 decreased. Among L-tryptophan, L-phenylalanine, L-tyrosine, anthranilate and phenylpyruvate, only phenylpyruvate somewhat increased the MK-4 production in contrast with the case of F. menin­gosepticum.

The amount of MK produced by strain KT 15 was 125·4 mg per liter of culture broth and 12·8 mg per g of dry cells, in the ratio of MK-4 and MK-6 of 6: 1, under the optimal culture conditions in the presence of cedar wood oil.

The improvement of MK production by Flavobacterium sp. 238-7 on mutagenization and optimization of the cultural conditions is summarized in Fig. 4. The MK productivity of strain KT 15 was almost 10 times that of the wild type strain. It is noteworthy that the increase of MK was due to an increase in MK-4 but not MK-6. MK-4 was a minor component of the wild type strain. The physiological role of MK-4 in the mutant strain is still uncertain. In the course of cultivation, the cellular content of MK-4 still increased after the growth had reached the stationary phase.

4.5 Extracellular Production of Menaquinone-4 by a Mutant Strain of Flavobacterium sp. 238-7

It could be considered that the MK productivity of strain K3-15 described in the previous section, was restricted by the intracellular level causing metabolic repression of MK biosynthesis. During attempts to further improve the MK production of the mutant strain, cultivation in a detergent-supplemented medium resulted in the extracellular production of MK, especially MK-4, without inhibition of the MK productivity and growth (Tani & Taguchi, 1988).

It was found that a mutant strain, K3-15, of Flavobacterium sp. 238-7 excreted MK into the culture medium supplemented with a detergent. Figure 5 shows the production of MK-4 and MK-6 during the cultivation of strain K3-15 in the basal medium. MK-6, which was the major MK component of the wild type strain, was produced only in the cells during the cultivation. On the other hand, MK-4 was also detected in the culture fluid.

A number of detergents (174 in total, i.e. the 110 nonionics, 30 anionics, 21 cationics and 13 amphoterics) were tested as to their induction of MK excretion into the culture medium. Most of the non-ionic and amphoteric detergents did not affect the cell growth. On the contrary, many anionic and cationic detergents inhibited cell growth. A non-ionic detergent, Rikanon VA 5012 (polyoxyethylene oleyl ether), showed highest ability as to induction of MK excretion into the medium without inhibiting the cell growth. MK-4, which was newly formed in cells of the mutant strain, was selectively excreted into the medium, but MK-6, which was the original MK of the wild type strain, was hardly excreted. In the presence of 0·05% Rikanon VA 5012, the MK-4

132 Y. Tani

10

~ 20

eft ~ .§ ~

0 'a 0 .. .g u ::a 'a

i 0 .. a. laC e ::Ii! 10 C)

CultIvatIon tIme (h)

Fig. 5. Menaquinone production by strain K3-15. 0, growth, .. , intracellular MK-6; V, extracellular MK-6; e, intracellular MK-4; 0, extracellular MK-4; ., total MK-4.

~ 20

£ ~ ~

'a 0 • 0 u :::a S 'a 0

:5 a ~

laC 0 ::E 10 5 ..

C)

CultlvaUon time (h)

Fig. 6. Menaquinone production by strain K3-15 in the presence of Rikanon UA 5012. The symbols are the same as in Fig. 5.

Vitamin K2 and Vitamin Kl Production 133

productivity increased about 20% compared to that without the detergent, and the total amount of MK was 39 mg per liter of culture broth.

Figure 6 shows the fermentation course of MK production on cultivation in the presence of 0·05% Rikanon VA 5012. The cell mass reached a maximum level, about 10 g by cell weight per liter of culture broth, after 30 h cultivation, and then gradually decreased. MK-6 was mainly produced intracellularly and reached about 12 mg per liter of culture broth after 30 h cultivation. A small amount of MK-6 was detected in the culture filtrate at the late stage of the cultivation. On the other hand, the excretion of MK-4 occurred from the beginning of the cultivation and increased in the later logarithmic phase. The extracellular production of MK-4 reached its maximum after 48 h cultivation. The level of intracellular MK-4 was low but increased with increasing cultivation time. The total amount of MK-4 reached about 25 mg per liter of culture broth at 48 h cultivation, and the amount of total MK was 39 mg liter.

MK-4 excretion occurred from the onset of the cultivation and the excreted MK was only MK-4. These facts showed that the excretion of MK-4 was not due simply to cell lysis. The MK-4leakage might be due to a change in the cell membrane structure, as a result of exposure to the detergent during growth.

The role of MK-4 in the mutant strain remains unknown, it might be that MK-4 is not related to the respiratory function, since the growth and growth rate of strain K3-15 did not decrease when most of the MK-4 had been excreted.

5 BIOCONVERSIONS FOR VITAMIN Kl (PHYLLOQUINONE) SYNTHESIS

Natural vitamin Kh [(7'R,1l'R)-phylloquinone] consists of 2-methyl-1,4-naphthoquinone and the optically active phytyl side chain which is attached to position 3. (3R,7R)-Hexahydrofarnesol, constructed from either of the micro­biologically generated optically active lactones [(S)-2-methyl-y-butyrolactone or (S)-3-methyl-y-butyrolactone] (see chapter 6, p. 102) can be lengthened by condensation with a trans-Cs-olefin to (7R, llR)-trans-phytol and yields natural vitamin Kl (Fig. 7).

Fig. 7. Absolute configurations of (7R,1l'R)-phylloquinone.

134 Y. Tani

REFERENCES

Bentley, R. & Meganathan, R. (1982). Biosynthesis of vitamin K (menaquinone) in bacteria. Microbiol. Rev., 46,241-80.

Collins, M. D. & Jones, D. (1981). Distribution of isoprenoid quinone structural types in bacteria and their taxonomical implications. Microbiol. Rev., 45, 316-50.

Tani, Y. & Sakurai, N. (1987). Menaquinone-4 production by a mutant of Flavobacterium sp. 238-7. Agric. BioI. Chem., 51,2409-15.

Tani, Y. & Taguchi, H. (1988). Excretion of menaquinone-4 by a mutant of Flavobacterium sp. 238-7 in a detergent-supplemented culture. Agric. Bioi. Chem., 52,449-54.

Tani, Y., Asahi, S. & Yamada, H. (1984). Vitamin K2 (menaquinone):screening of producing microorganisms and production by Flavobacterium meningosepticum. J. Ferment. Technol., 62,321-7.

Tani, Y., Asahi, S. & Yamada, H. (1985). Production of manaquinone (vitamin K2)-5 by a hydroxynaphthoate-resistant mutant derived from Flavobacterium meningosepticum, a menaquinone-6 producer. Agric. Bioi. Chem., 49, 111-15.

Tani, Y., Asahi, S. & Yamada, H. (1986). Menaquinone (vitamin K2)-6 production by mutants of Flavobacterium meningosepticum, J. Nutr. Sci. Vitaminol., 32, 137-45.

WATER-SOLUBLE VITAMINS

Chapter 9

MICROBIAL SYNTHESIS OF VITAMIN B, (THIAMINE)

A.IwASHIMA

Department of Biochemistry, Kyoto Prefectural University of Medicine, Kyoto, Japan

1 HISTORICAL

Thiamine was discovered in the course of a search for an agent that would cure beriberi. The conquest of beriberi began in 1885 when Takaki (1885) practically eradicated the disease among the Japanese navy by introducing fish, vegetables, meat and barley into the diet. In 1897 Eijkman (1897) showed that an experimental polyneuritis in fowl, which closely resembled the polyneuritic symptoms of beriberi, could be produced by feeding the birds on a diet of polished rice. When they were fed on unpolished rice they did not develop the disease. In 1926 Jansen & Donath (1926) isolated a crystalline hydrochloride of the antineuritic factor from rice bran.

The structure of thiamine, elucidated in the mid-1930s, was established by Williams (1936) as 3-( 4-amino-2-methyl-5-pyridimidinylmethyl)-5-(tJ­hydroxyethyl}-4-methylthiazolium chloride hydrochloride (Fig. 1). The name thiamine derives from the chemical nature of the vitamin, in that it has the thiazole (sulfur-containing) ring attached to a pyrimidine ring with an amine group. Thiamine was first synthesized in 1936 by Williams & Cline (1936) from the pyrimidine and thiazole moieties of thiamine. Within a year, Lohmann & Schuster (1937) isolated thiamine pyrophosphate (TPP) from yeast and showed that it was the cofactor for the decarboxylation of pyruvic acid. In addition to thiamine and TPP, thiamine monophosphate (TMP) and thiamine triphosphate (TIP) have been found in living organisms. These phosphate esters of thiamine are also shown in Fig. 1.

2 CHEMICAL AND PHYSICAL PROPERTIES

The double-salt from thiamine with hydrochloric acid (C12H 17N40SCI HCI; molecular weight, 337·28) consists of monoclinic plates in rosette-like clusters

137

138 A. Iwashima

H1C"",,:xN NH2 1!: I I U-N CH2~~ 4' CH,CH,OR

H,

R= -H Thiamine

Q R= -P-OH

I TMP

OH

0 0 R=

II II TPP -P-O-P-OH (JH I

OH

0 0 0 R= -P-O-P-O-P-OH

ClH elH OH TTP

Thiochrome

CH, W:-Ofl

HJCyNyNH'lr--s 0 0

NJ. Nt:: "" - CHi' ~ C,H,O-P-O-P-OH HJ 6H 6H

Hydroxyethylthlamlne pyrophosphate

Fig. 1. Structures of thiamine and its related compounds.

with slight thiazole odor (Merck Index, 1983). It is soluble in water and methanol, slightly soluble in ethanol, and practically insoluble in ether, benzene and chloroform. In aqueous solution thiamine is most stable between pH 2 and 4, but unstable at alkaline pH (Yurugi et aI., 1979). It is destroyed by heat if the pH of the solution is above 5·5: the rate of destruction increases with the pH. Under dry conditions thiamine is stable to heating at 100°C for 24 h. The absorption maximum of thiamine in the UV light is affected by pH, and it is 243 nm at pH 2·3 (the molar absorption coefficient: 10·6 X 103), and 235 and 265 nm at pH 7·4 (Yurugi et al., 1979).

TPP chloride (commercially available in the dried form) is stable when stored cold in the dark (Yurugi, 1975). In aqueous solution it is stable at pH 2-6 and O°C for 6 months, but it partially decomposes to TMP and thiamine when allowed to stand for several months at pH 5 and 38°C. TIP is stable under dry conditions, whereas it is unstable in aqueous solution, especially to heat, and decomposes to TMP and TPP. In alkaline solution TIP decomposes to TMP and inorganic phosphate. TMP, which is obtained by hydrolysis of TIP and TPP, is quite stable at acidic and neutral pH. Thiamine and thiamine phosphate esters are quantitatively converted to thiochrome (Fig. 1) and its phosphate esters which are compounds exhibiting intense blue fluorescence. This property observed in early studies of thiamine oxidation serves as the basis for the chemical assay for thiamine (Barger et al; 1935). Hydroxyethylthiamine pyrophosphate (Fig. 1), a TPP-activated aldehyde intermediate of the enzyme reactions, it is also known to be present in micro-organisms and mammalian tissues, especially in muscles (Morita et al., 1968).

3 PRODUCING MICRO-ORGANISMS

Thiamine, as well as other vitamins, is produced by micro-organisms in extremely small amounts. Usually it is not formed in great excess over the

Microbial Synthesis of Vitamin B 1 139

organism's own requirement. The production of thiamine by growing cultures of Escherichia coli ATCC 9637 is 1·6 and 1·4 higher than the amount of thiamine in cells grown in the basal medium (21-23 ng/mg dry weight), when cysteine and methionine are added at 1 mg per ml of the minimal medium, respectively (Akagi & Kumaoka, 1963). The addition of some aromatic amino acids is also stimulatory on thiamine production by growing cultures of the same organisms: phenylalanine and histidine cause 1·5- and 1·2-fold stimula­tions of thiamine synthesis at 10 and 1 mM, respectively (Iwashima et al., 1968). Methionine at 1 mM shows an additional effect of 2·6-fold stimulation of the thiamine production in the presence of 10 mM phenylalanine. On the other hand, the addition of purine and pyrimidine bases of nucleic acids to the growing cultures of E. coli is not effective on thiamine production in the cells. Thus, the supplement in the growth medium is not very effective to produce excess thiamine in bacteria and the de novo synthesis of thiamine cannot be demonstrated in non-growing cells under normal circumstances, because the control mechanism of thiamine biosynthesis in micro-organisms severely limits thiamine overproduction in the cells. However, this difficulty can be overcome by the use of a system that removes the normal control of thiamine over its biosynthesis, so that the organisms form excess thiamine. Using Salmonella typhimurium, Newell & Tucker (1966a,b) observed a burst of thiamine synthesis ensued, increasing the cellular level of thiamine four- to fivefold (about 200 ng/mg dry weight) when cells were preincubated with adenosine, washed, and reincubated in minimal medium. Adenosine inhibits the biosynth­esis of the pyrimidine moiety of thiamine, and the growth of organisms in the presence of adenosine therefore lowers the cellular concentration of thiamine. The normal repressible control by thiamine is thereby lost, and the thiamine­synthesizing enzymes become de repressed to overproduce thiamine. The thiamine formed occurs almost completely in the form of TPP and is located intracellularly.

Mutants affecting regulation of thiamine biosynthesis from the pyrimidine and thiazole moieties were isolated from E. coli K12 as resistant strains to growth inhibition by pyrithiamine, a thiamine antagonist (Kawasaki and Nose, 1969). Two mutants, PT-R1 and PT-R3, have approximately a threefold higher cellular thiamine content (90-100 ng/mg dry weight) than the parent strain. Yeast as well as bacteria do not produce large amounts of thiamine under normal circumstances (Stieglitz et al., 1974). Recently, however, thiamine-excreting mutants of yeast have been isolated (Silhankova, 1985a). Two mutants of S. cerevisiae and four mutants of S. carlsbergensis, which excreted up to 920/-tg thiamine per liter of the culture medium, were obtained by employing several rounds of UV mutagenesis and selection. The genetic characterization of these mutants suggested that thiamine excretion phenotype is recessive and under the control of nuclear genes (Silhankova, 1985b).

The most effective method for the production of thiamine by micro­organisms is an enzymatic conjugation of 2-methyl-4-amino-5-hydroxymethyl­pyrimidine (hydroxymethylpyrimidine) and 4-methyl-5-P-hydroxyethylthiazole (hydroxyethylthiazole), the preformed pyrimidine and thiazole moieties of

140 A.lwashima

thiamine. Washed cell suspensions of E. coli ATCC 9637 (15-20mg dry cells) synthesizes 210 ng of thiamine per mg dry weight from 10 IlM each of the two moieties in the presence of 0·2% glucose at pH 7 for 1 h at 37°C (Iwashima et al., 1968). With the cells derepressed thiamine biosynthesis by adenine or thiamine auxotrophs grown in the presence of limited amount of thiamine the production of thiamine is much more enhanced (Kawasaki et al., 1969). The cells suspension of baker's yeast also has the capability of joining the pyrimidine and thiazole moieties of thiamine to form thiamine in the presence of glucose (Ashida, 1942). In S. cerevisiae, thiamine synthesis from both moieties added leads to 20-40 times higher contents of intracellular thiamine in comparison with normal growth conditions (Silhankova, 1985b).

4 BIOSYNTHESIS AND REGULATION

4.1 Biosynthesis

The biosynthesis of thiamine involves the independent formation of the two ring structures and their subsequent condensation (Fig. 2). The enzymatic steps involved in the conversion of the pyrimidine and thiazole moieties to thiamine and TPP were elucidated some years ago (Leder, 1975). Although the biosynthetic pathway for the formation of hydroxymethylpyrimidine has not yet been completely elucidated, enough is known to eliminate the possibility that this pyrimidine and the pyrimidines of nucleic acids do not share a common biosynthetic pathway (Goldstein & Brown, 1963). An important contribution to the understanding of the biosynthesis of the pyrimidine moiety of thiamine in bacteria was the discovery by Newell & Tucker (1968a,b) of mutants of S. typhimurium with a dual growth require­ment for purines as well as for hydroxymethylpyrimidine. The dual growth requirement was satisfied by 5-aminoimidazole ribonucleotide (AIR) alone and

HlC-g:~' NH, H,c:.......,.,jcNH, H,c.......N:rNH, ~ IZ.~ll ----.. r I Q ~ r- I )! 9

N CH,OH N CH,O-P-OH N CH,O-P-O-P-OH 6H 6H 6H

f1~.' _7, 9 Nl. CH,CH.OH N CH.CH.O-P-OH ~ H. H. 6H

PPt

Hlcy:xN NH, 13: N 9 N CHz'~ CH,CH.O-f-OH

H. OH

Fig. 2. The biosynthesis of thiamine from its pyrimidine and thiazole moieties.

Microbial Synthesis of Vitamin B 1 141

by compounds that precede AIR on the purine pathway, indicating that AIR is the last common intermediate on the route to the purines and to the pyrimidine moiety of thiamine. The biosynthetic pathways of hydroxy­methylpyrimidine in micro-organisms differ between bacteria and yeast. In bacteria, hydroxymethylpyrimidine is synthesized from AIR, and N-1, C-4 and C-6 of the pyrimidine are derived from the nitrogen, C-1 and C-2 of glycine, respectively (Estramareix & Lesieu, 1969; Estramareix, 1970; White & Rudolph, 1979), and the C-2 from formate (Kumaoka & Brown, 1967; Estramareix & Lesieu, 1969). These results suggest that the imidazole ring is opened between C-4 and C-S. The remaining three carbon atoms of hydroxy­methylpyrimidine (C-S, C-7 and C-8) originate from the ribose part of AIR which is thus the precursor of all the carbon atoms of this pyrimidine (Estramareix & Therisod, 1984). Since N-3 and the amino group at C-4 have been recently shown to be derived from the amide-N atom from glutamine in E. coli and S. cerevisiae (Tazuya et ai., 1987a), the origin of almost all of the pyrimidine moiety of thiamine is now known for bacteria, although the reactions whereby AIR is converted to the pyrimidine remain unknown. In yeast, on the other hand, C-4 of the pyrimidine originates from formate (David et ai., 1966) and no significant incorporation of the nitrogen atom and C-2 of glycine into the pyrimidine is found (Linnett & Walker, 1968; White & Spenser, 1979). Furthermore, the incorporation of C-3, C-4 and C-S of pentulose into C-6, C-S and C-7 of the pyrimidine has been recently reported in S. cerevisiae (Grue-S0rensen et ai., 1986). The origin of C-2, C-8 and N-1 of hydroxymethylpyrimidine remains to be clarified in the biogenesis of the pyrimidine moiety in yeast.

Relatively little has been discovered regarding the biosynthetic origins of the thiazole moiety of thiamine, but some progress has been made in recent years toward the identification of its precursors. In E. coli and S. typhimurium the direct utilization of C-2 and the nitrogen atom of tyrosine for the formation of the thiazole ring was demonstrated (Estramareix & Therisod, 1972; Bellion et ai., 1976; White & Rudolph, 1978). On the other hand, no incorporation of C-2 of tyrosine into thiamine is observed in yeast, whereas significant incorporations of C-2 and the nitrogen atom of glycine into C-2 and N of the thiazole are shown (Linnett & Walker, 1968, 1969). These observations seem to indicate that bacteria and yeast use different amino acids as precursors of the C-2 and nitrogen portion of the thiazole ring, implying that the pathways in these two micro-organisms are different. Further incorporation studies with deuterated carbohydrates and related compounds showed that the precursor of the S-carbon chain (C-4, C-S, C-6, C-7 and C-8) of the thiazole moiety of thiamine in E. coli is a S-carbon sugar derived from pyruvate and triose phosphate (White, 1978), which might then react with tyrosine and a sulfur compound in an undefined series of steps to yield hydroxyethylthiazole. This has been supported by the incorporation of 1-·deoxY-D-threo-pentulose into the S-carbon chain of the thiazole without carbon-carbon bond cleavage in E. coli (David et ai., 1981). In yeast, the incorporation pattern leads to the inference

142 A.lwashima

that the 5-carbon chain may not be derived from pyruvate but from 2-pentulose, which is generated from the hexose precursors by oxidative as well as non-oxidative pentose phosphate pathway (White and Spenser, 1982). The precursor of the sulfur atom of the thiazole ring is still not known with certainty, but several experimental observations suggest that either cysteine or H2S which could be produced from cysteine, is the most likely precursor of the sulfur atom of hydroxyethylthiazole in bacteria and yeast (Bellion & Kirkley, 1977; Tazuya et al.; 1987b).

4.2 Regulation

As described above, the derepression of thiamine biosynthesis by adenosine was demonstrated for the first time in S. typhimurium (Newell & Tucker, 1966a,b). Adenine and adenosine, in the form of AMP, exert feedback control over the synthesis of AIR by inhibiting the formation of 5-phosphoribosylamine, the first step exclusive to the synthesis of purines and hydroxymethylpyrimidine. The derepression is unaffected by hydroxy­ethylthiazole, but does not occur if thiamine or hydroxymethylpyrimidine is present, or if protein synthesis is prevented during preincubation. Since derepression by adenosine in a hydroxyethylthiazole auxotroph is prevented by thiamine, but not by hydroxymethylpyrimidine, it was suggested that hydroxy­methylpyrimidine influences the state of repression only by being converted to thiamine. Derepression in the absence of adenosine also occurs when a hydroxyethylthiazole auxotroph is grown on a limited supply of thiamine. These studies were then extended to the regulations of the four enzymes in E. coli that catalyze the synthesis of TMP from hydroxymethylpyrimidine and hydroxyethylthiazole: hydroxymethylpyrimidine kinase (EC 2.7.1.49), phos­phomethylpyrimidine kinase (EC 2.7.4.7), hydroxyethylthiazole kinase (EC 2.7.1.50) and thiamine-phosphate pyrophosphorylase (EC 2.5.1.3) (Kawasaki et al., 1969). These enzymes are derepressed by adenine and repressed by thiamine. The derepression is also brought about by preincubating the growing culture with phenylalanine (Kawasaki et al., 1969). This derepression is reversed by thiamine or hydroxyethylthiazole, but not by hydroxymethylpyr­imidine. Since thiamine synthesis is inhibited by phenylalanine in a thiamine regulatory mutant (PT-R1) and the inhibition is reversed by hydroxy­ethylthiazole (Iwashima & Nose, 1970), this inhibition by phenylalanine appears to account for the derepression in the wild type. However, two-fold increase in cellular thiamine levels accompanied by the inhibition of the thiazole synthesis caused by phenylalanine remains unexplained. In a thiamine regulatory mutant (PT-R1) hydroxyethylthiazole kinase and thiamine­phosphate pyrophosphorylase are not subjected to repression by thiamine, suggesting that it is a constitutive mutant of an operator gene controlling the synthesis of two enzymes (Kawasaki & Nose, 1969). Several thiamine regulatory mutants of E. coli affecting other gene loci have been reported (Kawasaki et al., 1976).

Microbial Synthesis of Vitamin B\ 143

5 ASSAY METHODS

5.1 Microbiological Assays

Assay methods based on use of the thiamine requiring fungus Phycomyces blakesleeanus and thiamine requiring strains of several bacteria such as Staphylococcus aureus, E. coli and Lactobacillus species, and S. cerevisiae have been used (Thomas, 1966).

5.2 Chemical Assays

Chemical assay methods are usually preferred to microbiological methods since such analyses can be performed rapidly and they are usually more reliable for routine determination. Although a number of chemical assay methods for thiamine are known, only the coupling reaction with diazotized p­aminoacetophenone (Melnick & Field, 1937) and thiochrome reaction have been developed as precise methods of assay. In recent years, however, the thiochrome method has been used almost exclusively. As described above, thiamine in alkaline solution is oxidised quantitatively to thiochrome. As oxidizing reagents potassium ferricyanide (Hennessy & Cerecedo, 1939) and cyanogen bromide (Fujiwara & Matsui, 1953) have been routinely used. Thiochrome formed from non-phosphorylated thiamine is extracted with isobutanol. Since thiochrome fluoresces intensely under UV illumination, it can be measured readily in a fluorimeter (excitation 365 nm; emission 430 nm). Phosphorylated thiamines can be determined after their hydrolysis to thiamine by phosphatase. Hydroxyethylthiamine gives thiochrome by oxidation with alkaline ferricyanide, but not with cyanogen bromide. Thiamine gives thio­chrome by either oxidizing agent, so that this difference in the oxidation property is used for the simultaneous determination of thiamine and hydroxy­ethylthiamine (Morita et al., 1968). For the differential determination of thiamine and its phosphate esters in biological materials electrophoresis, paper chromatography, thin-layer chromatography and ion-exchange chromatog­raphy can be used to separate thiamine compounds from each other (Yurugi et al., 1979). In recent years, high-performance liquid chromatography (HPLC) has been developed for simultaneous determination of thiamine and its phosphate esters and assay procedures have been established (Kawasaki & Sanemori, 1985). The oxidation of thiamine compounds in samples can be carried out either before the chromatography (precolumn derivatization procedure) or after the chromatography (postcolumn derivatization proce­dure). Therefore, the precolumn derivatization procedure is essentially the HPLC of thiochrome and its phosphate esters. A mixing coil with proportion­ing pump as an additional equipment for thiochrome reaction is required for the postcolumn derivatization procedure. The intensity of the fluorescent products is measured with a spectrophotofluorometer. The minimum detection by these methods has been reported to be 0·1 pmol or 33·7 pg as thiamine hydro­chloride (Sanemori et al., 1980).

144 A.lwashima

5.3 Enzyme Assays

TPP has been estimated manometrically by measuring the evolution of CO2 from pyruvate in the presence of the apoenzyme of yeast pyruvate decar­boxylase (Green et al., 1941), and spectrophotometrically in the same reaction system by measuring acetaldehyde-dependent reduction of NADH2 using alcohol dehydrogenase (Kajiro, 1957).

6 BIOLOGICAL PROPERTIES

6.1 Metabolism

It has been generally accepted that thiamine transport in micro-organisms occurs via active transport (Iwashima, 1980). In animal cells, evidence has been accumulated which shows that thiamine is taken up by Na+-dependent active process (Iwashima, 1980). In both animals and humans thiamine transport across the small intestine is biphasic: mainly by active transport at low or physiological concentrations «2 IlM) and predominantly passive at higher concentrations (Hoyumpa et al., 1975).

Thiamine must be phosphorylated to TPP, a coenzyme form of thiamine, to exert its physiological functions. The formation of TPP from thiamine is catalyzed by thiamine pyrophosphokinase (EC 2.7.6.2) in yeast and animal tissues. In E. coli thiamine is converted to TPP by two successive phosphoryla­tions catalyzed by thiamine kinase (EC 2.7.1.89) and thiamine-monophosphate kinase (EC 2.7.4.16). TPP is further metabolized to TIP by thiamine­diphosphate kinase (EC 2.7.4.15).

Two thiamine cleavage enzymes have been isolated from various natural sources (Murata, 1982). Thiaminase I (EC 2.5.1.2) is a transferase which catalyzes an exchange reaction between a base and a thiazole moiety of thiamine, and thiaminase II (EC 3.5.99.2) mediates in the hydrolysis of thiamine.

6.2 Physiological Functions

The physiological functions of thiamine is exerted in the form of TPP in cells. TPP serves as the coenzyme for a large numbers of enzyme systems in the metabolism of carbohydrates and amino acids, including pyruvate dehydrogenase(cytochrome) (EC 1.2.2.2), pyruvate oxidase (EC 1.2.3.3), pyruvate dehydrogenase(lipoamide) (EC 1.2.4.1), oxoglutarate dehydro­genase(lipoamide) (EC 1.2.4.2), 2-oxoisovalerate dehydrogenase(Iipoamide) (EC 1.2.4.4), transketolase (EC 2.2.1.1), pyruvate decarboxylase (EC 4.1.1.1), benzoyiformate decarboxylase (EC 4.1.1.7), oxalylCoA decarboxylase (EC 4.1.1.8), tartronate-semialdehyde synthase (EC 4.1.1.47), phosphoketolase (EC 4.1.2.9), 2-hydroxy-3-oxoadipate synthase (EC 4.1.3.15), acetolactate synthase (EC 4.1.3.18) and sulfoacetaldehyde lyase (EC 4.4.1.12).

Microbial Synthesis of Vitamin B) 145

The physiological function of TIP remains enigmatic, although evidence is emerging that this form of thiamine is involved in nerve membrane function (Leder, 1975). It is certain that the neuropathy of beriberi is due to thiamine deficiency and it is probable that the anorexia and cardiac manifestation of beriberi are caused by decreased activity of the enzymes for which TPP is coenzyme. However, much remains unclear about the cellular and molecular mechanism of thiamine action correlated to thiamine deficiency symptoms.

7 CHEMICAL SYNTHESIS

Thiamine has been synthesized in two basic ways (Matsukawa et al., 1970). The condensation method, first adapted to the synthesis of thiamine by Williams & Cline (1936), consists of the condensation of the pyrimidine with thiazole derivatives. In another method, which was devised by Todd & Bergel (1937), the pyrimidine moiety is synthesized with an appropriate side chain conductive to the formation of the remaining structure. Matsukawa (1953) synthesized thiothiamine as an intermediate for thiamine synthesis. This is formed by the condensation of 4-amino-5-aminomethyl-2-methylpyrimidine, CS2 and 3-acetyl-3-chloro-1-propanol. Thiothiamine is a water-insoluble crys­talline compound and is readily converted by oxidation into thiamine in good yield.

REFERENCES

Akagi, M. & Kumaoka, H. (1963). Effect of sulfur-containing amino acids on the production of thiamine by Escherichia coli. J. Vitaminol., 9, 183-7.

Ashida, K. (1942). Synthesis of vitamin B) by microbes. Bulletin of the Agricultural Chemical Society of Japan., 18, 723-6.

Barger, G., Bergel, F. & Todd, A. R. (1935). Uber das Thiochrom aus Vitamin B) (Antineurin). Chern. Ber., 68, 2257-62.

Bellion, E. & Kirkley, D. (1977). The origin of the sulfur atom in thiamine. Biochim. biophys. Acta, 497,323-8.

Bellion, E., Kirkley, D. H. & Faust, J. R. (1976). The biosynthesis of the thiazole moiety of thiamine in Salmonella typhimurium. Biochim. biophys. Acta, 437, 229-37.

David, S., Estramareix, B. & Hirshfield, H. (1966). Le formate, precurseur du carbone 4 de la pyrimidine de la thiamine. Bichim. biophys. Acta, 127, 264-5.

David, S., Estramareix, B., Fischer, J-C. & Therisod, M. (1981). 1-Deoxy-o-threo-2-pentulose, the precursor of the five-carbon chain of the thiazole of thiamine. J. Am. Chern. Soc., 103,7341-2.

Eijkman, C. (1897). Eine Beriberiahnliche Krankheit der Huehner. Virchows Arch. Pathol. Anat., 148, 523.

Estramareix, B. (1970). Biosynthese de la pyrimidine de la thiamine. Origine du carbone 6 chez Salmonella typhimurium. Biochim. biophys. Acta, 208, 170-7l.

Estramareix, B. & Lesieu, M. (1969). Biosynthese de la pyrimidine de la thiamine. Origine des carbones 2 et 4 chez Salmonella typhimurium. Biochim. biophys. Acta, 192,375-7.

146 A.lwashima

Estramareix, B. & Therisod, M. (1972). La tyrosine, facteur de la biosynthese du thiazole de la thiamine chez Escherichia coli. Biochim. biophys. Acta, 273,275-82.

Estramareix, B. & Therisod, M. (1984). Biosynthesis of thiamine. 5-Aminoimidazole ribotide as the precursor of all the carbon atoms of the pyrimidine moiety. J. Am. Chem. Soc., 106,3857-60.

Fujiwara, M. & Matsui, K. (1953). Determination of thiamine by the thiochrome reaction. Analyt. Chem., 25, 810-12.

Goldstein, G. A. & Brown, G. M. (1963). The biosynthesis of thiamine. V. Studies concerning precursors of the pyrimidine moiety. Archs. Biochem. Biophys., 103, 449-52.

Green, D. E., Herbert, D. & Subrahmanyan, V. (1941). Carboxylase. J. bioI. Chem., 138, 327-39.

Grue-S(lJrensen, G., White, R. L. & Spenser, I. D. (1986). Thiamin biosynthesis in Saccharomyces cerevisiae. Origin of the pyrimidine unit. J. Am. Chem. Soc., 108, 146-58.

Hennessy, D. J. & Cerecedo, I. R. (1939). The determination of free and phosphory­lated thiamine by a modified thiochrome assay. J. Am. Chem. Soc., 61, 179-83.

Hoyumpa, A. M., Middleton, H. M. III., Wilson, F. A. & Schenker, S. (1975). Thiamine transport across the rat intestine. I. Normal characteristics. Gastroenterology, 68, 1218-227.

Iwashima, A. (1980). Absorption and membrane transport of thiamine. In Vitaminology, Vol. II, ed. Vitamin Society of Japan, Tokyokagakudojin, Tokyo, pp.30-39.

Iwashima, A. & Nose, Y. (1970). Inhibition by phenylalanine of thiazole biosynthesis in Escherichia coli. J. Bacteriol., 104, 1014-16.

Iwashima, A., Kawasaki, T. Nakamura, M. & Nose, Y. (1968). Effect of amino acids and purine bases on thiamine synthesis by Escherichia coli. J. Vitaminol., 14, 203-10.

Jansen, B. C. P. & Donath, W. F. (1926). On the isolation of antiberiberi vitamin. Proc. Kon. Ned. Akad. Wet., 29, 1390.

Kajiro, Y. (1957). Quantitative determination of thiamine pyrophosphate using apocarboxylase and alcohol dehydrogenase. J. Biochem., 44, 827-38.

Kawasaki, T. & Nose, Y. (1969). Thiamine regulatory mutants in Escherichia coli. J. Biochem., 65,417-25.

Kawasaki, T. & Sanemori, H. (1985). Vitamin B1: Thiamines. In Modern Chromato­graphic Analysis of the Vitamins, ed. M. G. M. De Ruyter. Marcel Dekker, New York, pp. 385-411.

Kawasaki, T., Iwashima, A. & Nose, Y. (1969). Regulation of thiamine biosynthesis in Escherichia coli. J. Biochem., 65,407-16.

Kawasaki, T., Sanemori, H., Egi, Y., Yoshida, S. & Yamada, K. (1976). Biochemical studies on pyrithiamine-resistant mutants of Escherichia coli K12. J. Biochem., 79, 1035-42.

Kumaoka, H. & Brown, G. M. (1967). Biosynthesis of thiamine. VI. Incorporation of formate into carbon atom two of the pyrimidine moiety of thiamine. Archs. Biochem. Biophys., 122,378-84.

Leder, I. G. (1975). Thiamine, biosynthesis and function. In Metabolic Pathways, Vol. 7, ed. D. M. Greenberg, Academic Press, New York, pp. 57-85.

Linnett, P. E. & Walker, J. (1968). Biosynthesis of thiamine. Incorporation experi­ments with 14C-Iabelled substrates and with [lSN]glycine in Saccharomyces cerevisiae. Biochem. J., 109, 16l-8.

Linnett, P. E. & Walker, J. (1969). Biosynthesis of thiamine. IV. C-2 of glycine as the precursor of C-2 of the thiazole moiety in yeast. Biochim. biophys. Acta, 184, 381-5.

Microbial Synthesis of Vitamin B \ 147

Lohmann, K. & Schuster, Ph. (1937). Untersuchungen tiber die Cocarboxylase. Biochem. Z., 294, 188-214.

Matsukawa, T. (1953). Chemistry of the thio-thiamine and its related compounds. Vitamins (Kyoto), 6, 299-310.

Matsukawa, T., Hirano, H. & Yurugi, S. (1970). Preparation of thiamine derivatives and analogs. Meth. Enzymol., 18A, 141-62.

Melnick, D. & Field, H. (1937). Chemical determination of vitamin B\. 1. Reactions between thiamine in pure aqueous solution and diazotized p-aminoacetophenone. J. bioi. Chem., 127,505-14.

Merck Index (1983), 10th edn., ed. M. Windholz. Merck & Co. Rahhway, p. 9134. Morita, M., Nishibe, Y. & Mineshita, T. (1968). Natural occurrence of 2-(1-

hydroxyethyl)thiamine in muscular tissues of animals. J. Vitaminol., 14, 230-38. Murata, K. (1982). Actions of two types of thiaminase on thiamine and its analogues.

Ann. N. Y. Acad. Sci., 378, 146-56. Newell, P. C. & Tucker, R. G. (1966a). The de-repression of thiamine biosynthesis by

adenosine. A tool for investigating this biosynthetic pathway. Biochem. J., 100, 512-16.

Newell, P. C. & Tucker, R. G. (1966b). The control mechanism of thiamine biosynthesis. A model for the study of control of converging pathways. Biochem. J., 100, 517-24.

Newell, P. C. & Tucker, R. G. (1968a). Precursors of the pyrimidine moiety of thiamine. Biochem. J., 106,271-77.

Newell, P. C. & Tucker, R. G. (1968b). Biosynthesis of the pyrimidine moiety of thiamine. A new route of pyrimidine biosynthesis involving purine intermediates. Biochem. J., 106, 279-87.

Sanemori, H., Ueki, H. & Kawasaki, T. (1980). Reversed-phase high-performance liquid chromatographic analysis of thiamine phosphate esters at subpicomole levels. Analyt. Biochem., 102,451-5.

Silhankova, L. (1985a). Yeast mutants excreting vitamin B. and their use in the production of thiamine rich beers. J. Inst. Brew., 91, 78-81.

Silhankova, L. (1985b). Genetic control of thiamine excretion and of its suppression in Saccharomyces cerevisiae. J. Inst. Brew., 91,238-41.

Stieglitz, B., Levy, R. & Mateles, R. I. (1974). Thiamine accumulation in yeast. J. Appl. Chem. Biotechnol., 24, 277-82.

Takaki, K. (1885). On the cause and prevention of Kakke. Transactions of Sei-I-Kawi, 4, Suppl., 29-37.

Tazuya, K., Tanaka, M., Morisaki, M., Yamada, K. & Kumaoka, H. (1987a). Origin of nitrogen atoms of the pyrimidine moiety of thiamin. Biochem. Int., 14, 769-77.

Tazuya, K., Yamada, K., Nakamura, K. & Kumaoka, H. (1987b). The origin of the sulfur atom of thiamin. Biochim. biophys. Acta, 924,210-15.

Thomas, M. H. (1966). Microbiological Assay Technics. In Methods of Vitamin Assay, ed. Association of Vitamin Chemists. Interscience Publishers, New York, pp. 37-62.

Todd, A. R. & Bergel. F. (1937). Aneurin. Part VII. A synthesis of aneurin·. J. Chem. Soc., 364-7.

White, R. H. (1978). Stable isotope studies on the biosynthesis of the thiazole moiety of thiamin in Escherichia coli. Biochemistry, 17, 3833-40.

White, R. H. & Rudolph, F. (1978). The origin of the nitrogen atom in the thiazole ring of thiamine in Escherichia coli. Biochim. biophys. Acta, 542,340-7.

White, R. H. & Rudolph, F. B. (1979). Biosynthesis of the pyrimidine moiety of thiamine in Escherichia coli. Incorporations of stable isotope-labeled glycines. Biochemistry, 18, 2632-6.

148 A.lwashima

White, R. L. & Spenser, I. D. (1979). Thiamine biosynthesis in Saccharomyces cerevisiae. Origin of carbon-2 of the thiazole moiety. Biochem. J., 179, 315-25.

White, R. L. & Spenser, I. D. (1982). Thiamin biosynthesis in yeast. Origin of the five-carbon unit of the thiazole moiety. J. Am. Chem. Soc., 104,4934-43.

Williams, R. R. (1936). Structure of vitamin B1 • J. Am. Chem. Soc., 58, 1063-4. Williams, R. R. & Cline, J. K. (1936). Synthesis of vitamin B1 • J. Am. Chem. Soc., 58,

1504-5. Yurugi, S., Iwashima, A. & Nose, Y. (1979). Thiamine. In Data Book for

Biochemistry, Vol. 1., ed. Japanese Biochemical Society. Tokyokagakudojin, Tokyo, pp. 1164-76.

Yurugi, S. (1975). Thiamine (Vitamin Bt). In Experimental Procedures in Biochemistry, Vol. 13, Vitamins & Coenzymes, ed. Japanese Biochemical Society, Tokyo­kagakudojin, Tokyo, pp. 55-73.

Chapter 10

MICROBIAL PRODUCTION OF VITAMIN B2 (RIBOFLAVIN)

T. KUTSAL & M. T. OZBAS

Hacettepe University, Chemical Engineering Department, 06532 Beytepe, Ankara, Turkey

1 INTRODUCTION

Riboflavin is a water-soluble vitamin which can be produced on a commercial scale by various fermentation processes using various micro-organisms. Ribo­flavin is used pure for human nutrition and therapy and in the crude concentrated form for animal feed supplements. Although it is produced by both synthetic and fermentation processes, it is believed that the microbial synthesis should be able to compete quite successfully with the synthetic chemical processes.

2 HISTORICAL

Riboflavin, also called vitamin B2 (lactoflavine, ovoflavine, uroflavine, hepat­oflavine, verdoflavine, vitamin G), is a water-soluble vitamin which exhibits a strong yellowish-green fluorescence.

As early as 1879, A. W. Blyth isolated from whey an impure preparation of flavine (lactochrome) of a red-orange color. In 1933, P. Gyorgy, R. Kuhn, and T. Wagner-Jauregg described the isolation of pure, crystallized lactoflavine and found the vitamin nature of this yellow pigment. The original name of riboflavin was lactoflavine. Ovoflavine, isolated from eggs; hepatoflavine, from liver; lactoflavine, from milk; and verdoflavine, from plants, were all identified as riboflavin. The name riboflavin, indicating that the naturally occurring flavin is a derivative of o-ribose, was adopted in 1952 by the International Commission for the Reform of Biochemical Nomenclature (Kirk & Othmer, 1968; Wagner-Jauregg, 1972).

The structure of riboflavin was confirmed in 1934 by synthesis by R. Kuhn and F. Weygand and P. Karrer and his co-workers. The chemical structure of vitamin B2 is [6,7-dimethyl-9-(o-1'-ribityl)-isoalloxazine], as shown in Fig. l.

149

150 T. Kutsal & M. T. Ozbas

l' 2' 3' 4' 5'

CHz-CHOH-CHOH-CHOH-CHzOH I

H3C N·N ~O ~91il~ ~lOh 43NH

HC N 3 0

Fig. 1. Riboflavin.

3 CHEMICAL AND PHYSICAL PROPERTIES

Riboflavin (C17HzoN40 6), crystallizes from 2 N acetic acid, alcohol, water or pyridine in orange-yellow needles. Its molecular weight is 376·36. The decomposition point is 278-282°C, and it darkens at about 240°C. The vitamin is odorless and has a bitter taste. Riboflavin is soluble in water only to the extent of 10-13 mg in 100 ml at 25-27·5°C, 19 mg in 100 ml at 40°C, and 230 mg in 100 ml at 100°C. The vitamin dissolves in ethanol and is slightly soluble in amyl alcohol, cyclohexanol, benzyl alcohol, phenol and amyl acetate, but is insoluble in ether, chloroform, acetone and benzene. Although alkali dissolves the vitamin well, these solutions are unstable.

Neutral aqueous solutions of riboflavin have a greenish-yellow color. The absorption spectrum shows characteristic absorption maxima at 475, 446, 359-375, 268 and 223 nm. The absorption in the visible part of the spectrum is used for quantitative determination of riboflavin (Wagner-Jauregg, 1972). Neutral aqueous solutions of riboflavin display intense yellowish-green fluores­cence, with a maximum at 565 nm which can be used for quantitative determination of the vitamin. The fluorescence vanishes on the addition of acid or alkali, optimum fluorescence occurs at pH 3-8.

Riboflavin is an amphoteric compound. Its dissociation constants are Ka = 6·3 X 1O-1z and KfJ = 0·5 X 10-5 , the isoelectric point corresponds to a pH of 6·0.

The basic factors affecting the stability of riboflavin in food are heat, light and the reactions which occur in cells during the storage. Neutral solutions of riboflavin can be sterilized by autoclaving for a short time: only slight destruction occurs by heating to 120°C for 6 h. No appreciable destruction of the vitamin can be observed during the cooking of food since it is relatively heat stable, but alkali decomposes riboflavin rapidly (Wagner-Jauregg, 1972; Heimann, 1980). When milk in bottles is exposed to sunlight, 85% of its riboflavin content is destroyed within 2 h.

Riboflavin is stable against air and most common oxidizing agents, but when reduced by reducing agents such as sodium hydrosulfite or hydrogen, riboflavin readily takes up two hydrogen atoms to form the colorless and non-fluorescent 1,1O-dihydro compound, leucoriboflavin, which can be reconstituted by shak­ing an aqueous solution with air. This reduction and reoxidation is also useful

Microbial Production of Vitamin B2 151

in increasing the specificity of riboflavin assay in food and feeds containing interfering fluorescent substances (Kirk & Othmer, 1968).

Irradiation of riboflavin in solution in the visible light or ultraviolet range degrades the side chain, yielding a number of compounds. These compounds are Lumiflavine (6,7 ,9-trimethylisoalloxazine) , C13H 12N40 2, and Lumichrome (6,7-dimethylisoalloxazine), C12H lON40 2. Irradiation with y rays causes loss of riboflavin in solution but not in dry mixes. Sterilization of foods by irradiation or treatment with ethylene oxide may also cause destruction of riboflavin (Kirk & Othmer, 1968).

4 PRODUCING MICRO-ORGANISMS AND SCREENING

Riboflavin can be produced in significant quantity by various fermentation processes using a number of micro-organisms and appropriate media. Vitamin B2 concentrates for the enrichment of poultry and livestock feeds can be prepared more cheaply by fermentation processes (Smith & Berry, 1975).

Riboflavin can be synthesized by many various types of micro-organisms. Bacteria, yeasts, moulds, algae and protozoa are the micro-organisms which can potentially produce riboflavin.

Different natural sources have been used for the production of riboflavin by fermenting micro-organisms. Lactose fermenting yeasts are especially Saccharomyces fragilis, Clostridium butylicum, several species of Lactobacillus, or moulds, e.g., Aspergillus niger, Aspergillus flavus, Peni­cillium chrysogenum, and species of Fusarium. Whey and other milk by­products can be employed as lacteal raw materials by these micro-organisms. Riboflavin was formed in the butyl alcohol-acetone fermentation of cereal grain mashes or molasses with various strains of Clostridium. Among these bacteria Clostridium acetobutylicum especially is one of the best producers of riboflavin (Kirk & Othmer, 1968). A suitable carbohydrate containing mash cereals, rice, corn, whey and potato starch have been used in these commercial anaerobic fermentation processes of Clostridium acetobutylicum (Wagner­Jauregg, 1972).

Most varieties of the yeast species Candida are also able to produce riboflavin (Goodwin, 1959; Goodwin, 1963a,b). Candida guilliermondia, C. tropicalis varrhaggi, C. flareri, C. arborea, C. lactis, C. krusei, C. ghoshi, C. chalmersi, C. lypolytica, C. olea, C. pulcherrima, C. solani and C. melibiosi were found to synthesize significant amount of riboflavin (Goodwin, 1963a; Robinson, 1966).

Among these riboflavin producing micro-organisms, two closely related ascomycetes, Eremothecium ashbyii and Ashbya gossypii (Ainsworth & Sussman, 1965) are capable of synthesizing very large amounts of this vitamin under appropriate conditions (Perlman et al., 1952; Hickey, 1954).

A great number of cheap natural materials and industrial wastes have been used for riboflavin fermentation. Fermentation residues such as grain stillages

152 T. Kutsal & M. T. Ozbas

from the ethyl alcohol and butyl alcohol-acetone fermentations; sugar containing substances such as molasses and corn syrup; fish meal, dried yeast, skim milk, cotton-seed meal, soybean meal, peas, tankage, wheat bran, animal proteins and malt extract are some of the raw materials employed in industrial processes for riboflavin production by Eremothecium ashbyii (Hickey, 1954; Gutcho, 1973).

Ashbya gossypii is also an important ascomycete very similar morphologi­cally to Eremothecium ashbyii. Corn steep liquor, still ages , proteinaceous agents from animal sources such as tankage, animal stick liquor and meat scraps, vegetable proteinaceous agents, such as soybean meals, glutens, linseed and cotton-seed meals have been used as raw materials for commercial production of riboflavin by Ashbya gossypii (Goodwin, 1959; Robinson, 1966).

Lipids may be of importance for high vitamin B2 production by these two ascomycetes (Ozbas & Kutsal, 1986a,b, 1987). The lipids studied included corn oil, soybean oil, cocoanut oil, lard oil, lecithin, menhaden oil, sunflower oil and others. (Hickey, 1954; Lago & Kaplan, 1981).

It is known that algae synthesize riboflavin but detailed work has not been reported in literature (Goodwin, 1963b).

Among the protozoa, Chilomonas paramoecium synthesizes riboflavin when cultured on a medium containing only acetate and inorganic salts (Goodwin, 1963b).

5 BIOSYNTHESIS AND REGULATION

There are many reports on general factors controlling riboflavin biosynthesis. Purines stimulate riboflavin over-production in all of the organisms studied. This holds true not only for complete fermentations, but for cell washed suspensions as well. Glycine, a known purine precursor, stimulates over­production in Candida species and in A. gossypii. The stimulatory effects of purines and glycine in A. gossypii is not due to an effect on growth but is specific for riboflavin oversynthesis (Demain, 1972). One of the proposed mechanisms is described as follows.

The biosynthetic pathways for purines, riboflavin, and pteridines are closely linked. Thus the carbon atoms of glycine are incorporated into these three classes of compounds in structurally analogous positions. A preformed purine can be converted directly into riboflavin with loss of only carbon 8 of the purine ring: other purine carbons and all the ring nitrogens appear finally in the pyrimidine portion for the riboflavin molecule (Wagner-Jauregg, 1972).

According to some workers who studied the biosynthesis of vitamin, riboflavin (IV) is formed in E. ashbyii from the purine (I) (9-ribitylxanthin) t,hrough the unstable 4-ribityl-amino-5-aminouracil (II) and the dioxopteridine (III) (6,7-dimethyl-8-ribityl-lumazine or DMRL) (Fig. 2). It has been synthes­ized by condensation of II with diacetyl and the same pathway has been suggested for the biological synthesis.

Microbial Production of Vitamin B2 153

R = D-Ribityl

III IV

Fig. 2. The biosynthesis of riboflavin.

That lumazine III alone is the precursor of riboflavin (IV) in organisms such as E. ashbyii and A. ashbyii has been established by biochemical studies. All the carbon atoms of the o-xylene moiety of riboflavin are derived from the lumazine III, two molecules of which are converted by riboflavin synthesis into one molecule of riboflavin (IV) and 4-ribitylamino-5-amino-2,6-dihydroxy pyrimidine. In this biochemical reaction the lumazine (III) simultaneously functions as a donor and acceptor of a C4 unit which consists of the two methyl groups and the C atoms 6 and 7. There is also a similar mechanism for riboflavin formation in plants (Wagner-Jauregg, 1972).

Goodwin et al. showed independently that amino acids provide carbon units and not nitrogen in the biosynthesis of riboflavin. These workers used E. ashbyii and found that L-threonine, L-serine or L-tyrosine stimulated riboflavin synthesis whereas L-glutamate, L-aspartate and L-asparagine stimulated both growth and riboflavin synthesis; L-cystein on the other hand inhibited both (Goodwin, 1963a,b; Robinson, 1966).

6 STRAIN IMPROVEMENT

The changes in the culture media and the selection of high riboflavin-producing mutant cultures cause the most important increases in riboflavin productivity.

In 1950, Pfeifer et al. suggested that some improvement in fermentation productivity was possible as a result of culture selection. Paralleling the pilot plant fermentations, a special study was carried out in the laboratory by Pridham of the NRRL fermentation division to obtain and test natural and induced variants of Ashbya gossypii NRRL Y -1056 for riboflavin production. One of the natural variants was chosen as superior to the original strain on the basis of shaker-flask experiments and was tested in a series of pilot plant fermentations. This particular strain gave consistently higher yields of

154 T. Kutsal & M. T. Ozbas

riboflavin than did the standard NRRL Y -1056. The results indicate the desirability of isolating high-producing strains at regular intervals in order to maintain riboflavin yields at the highest possible values (Pfeifer et al., 1950).

7 FERMENTATION/BIOCONVERSION PROCESS

Commercial fermentation processes for production of riboflavin or riboflavin concentrates are relatively recent, having been developed since about 1940. The Ascomycete processes using Eremothecium ashbyii and Ashbya gossypii are now assuming the most important position in the riboflavin manufacturing field. Both organisms are capable of synthesizing very large amounts of riboflavin under appropriate conditions.

7.1 Inoculum

As reported in Section 4, a great number of cheap natural materials and industrial wastes have been used for riboflavin fermentation by these Ascomy­cetes (Hickey, 1954; Goodwin, 1959; Robinson, 1966; Gutcho, 1973; Ozbas, 1985). In order to produce very large amounts of riboflavin, an appropriate combination of these substrates can be chosen and if necessary, several minerals can be added to the medium.

The generally accepted inoculating procedure is to use a 0·25-1·0% inoculum from an actively growing 24-28 h culture (Goodwin, 1959).

7.2 Medium

In laboratories, cultures of E. ashbyii and A. gossypii are grown on solid agar media in slant tubes and Petri dishes. The composition of the culture medium is as follows (g/liter): glucose, 20·0; peptone, 5·0; yeast extract, 5·0; malt extract, 5·0; MgS04 ·7H20, 0·2; K2HP04 , 0·2. The -pH of the medium is adjusted to the desired value with 0·5M H2S04 (Ozbas et al., 1984, 1986a,b). Both liquid and solid media are sterilized at 121°C in an autoclave before inoculation. The initial substrate concentrations can be changed by making the amount of initial carbon in the medium equivalent to 20·0 g/liter glucose while the amounts of other constituents are kept constant.

7.3 Fermentation Course and Parameters

General fermentation conditions that affect micro-organism growth and productivity are pH, temperature, aeration level, initial substrate concentra­tion such as glucose, and growth factors such as DL-methionine, inositol, D( + )-biotin and thiamine.

7. 3.1 Effect of initial pH An initial pH of 6·0-8·0 for Eremothecium ashbyii and 5·5-7·0 for Ashbya gossypii were found to be preferable (Hickey, 1954; Goodwin, 1959). The

Microbial Production of Vitamin B2 155

//:--..-0.15 . , \ 3.00

L: , 0

-~0.10 0 - E

~o 2.00~

It: ::L. QI

~

.~'~. 0

0.05 1.00 ::

, '~i 0 0

6.0 6.5 7.0 7.5 pH

Fig. 3. Effect of initial pH on the specific growth (I-') and riboflavin production (v) rates for E. ashbyii and A. gossypi (30°; stirring rate 100 rpm; SGO = 20·0 g/liter; ., E.

Ashbyii; 0, A. gossypii.

effects of initial pH on specific growth and riboflavin production rates with E. ashbyii and A. gossypii are shown in Fig. 3. The optimum initial pH value is 6·5 for both micro-organisms (Ozbas & Kutsal, 1986a,b).

Riboflavin production by these two ascomycetes fits the third group of the Gaden classification. In these riboflavin fermentations the pH of the medium decreases with time and drops from its initial value of 6·5 to approximately 4·5 .

• ,.,.__e--

::. /:#:~_o_o 0.16 9.0

0.14 8.0

0.12 7.0

0.10 _ 6.0 ....

0.08 ~ 5.0 ::r: It: Co

U 0.06 4.0

0.04 3.0

~Jo\I// "\ 2.01-t -1.0 / \ o _ 0.02 2.0

o 3 /~_'--_--'-_--'-__ ",--_.....L..._--' 0 1.0 o 40 80 120 160 200

II hI

Fig. 4. Changes in medium during fermentation (30°; initial pH = 6·5; stirring rate 750 rpm; aeration rate, 220 cc/min; SGO = 20·0 g/liter; 0, riboflavin (CR); ., dry weight

(X); 0, pH).

156 T. Kutsal & M. T. Ozbas

0

0.15 3.00_ .J::.

b E

";".c 0.10 t7l

'~~\~o-r-o 2.00-;;::

01

1- ",-

e .,.

0.05 I~I .'\.~ 1.00

--. I 0 0 0

20 25 30 35 40 T (oC)

Fig. 5. Effect of temperature on the specific growth and riboflavin production rates for E. ashbyii (stirring rate, 100 rpm; initial pH = 6·5; 0, SGo = 20·0 g/liter; ., SSo =

20·0 g/liter).

At this point the micro-organisms grow rapidly, but no significant amounts of riboflavin are synthesized. Then both riboflavin production and pH increase and riboflavin formation terminates at approximately pH 9·5. Figure 4 shows characteristic changes in the medium during the fermentation process (Ozbas et aI., 1984; Ozbas & Kutsal, 1986a, b).

7.3.2. Effect of temperature The optimum temperature ranges for riboflavin fermentation by Eremothecium ashbyii and Ashbya gossypii were obtained between 25 and 30°C and 26 and 28°C respectively (Hickey, 1954; Gutcho, 1973).

Ozbas & Kutsal investigated the effects of temperature by utilizing glucose and sunflower oil as substrate in the range from 20 to 40°C. For E. ashbyii and A. gossypii maximum specific growth and riboflavin production rates were obtained at 30°C in all medium compositions. At lower temperatures both growth and riboflavin yield were reduced to values similar to those observed at higher temperatures (Figs 5 and 6).

The Ascomycete processes for riboflavin production are aerobic and it is necessary either to aerate or shake the cultures.

Z 3. 3 Effect of initial substrate For the growth of both micro-organisms the main carbon source is glucose. Maximum yields are obtained with an initial glucose concentration of 20·0 g/liter. Glycerol, sunflower oil, whey and several combinations of these substrates can also be used for riboflavin production. The effects of different substrates and their initial concentrations on the specific growth and riboflavin

Microbial Production of Vitamin B2

0.30 lei 4.00

/;or" '\ 300~ :/,/" \\ 2DO! ,./ ~:~ i; ......... 0" ? /0 '0 ~ 1.00

0.20

0.10

1----° 'r'-0 e -----0 0~2~0----~2~5~--~3~0----~35~----4~0~0 T (DC)

157

Fig. 6. Effect of temperature on the specific growth and riboflavin production rates for A. gossypii (stirring rate, 100 rpm; initial pH = 6·5; 0, Sao = 10·0 g/liter, Sso =

1O·0g/liter; e, Sao = 20·0 g/liter).

production rates and riboflavin yields are given in Table 1 (Ozbas & Kutsal, 1986a,b).

7. 3. 4 Fermentor experiments The effects of agitation and aeration rates on the formation of riboflavin were observed at the range of 300-1200 rpm and 70-330 cc/min, respectively for Eremothecium ashbyii. Optimum values were reached as Il = 0·176 h- 1 and v = 4·45 X 10-3 g/liter per hat 750-1000 rpm and Il = 0·181 h- 1 and v = 3·69 X

10-3 g/liter per h at 220 cc/min. At these optimum conditions, adding the sunflower oil to the medium resulting in 5·0 g/liter glucose and 15·0 g/liter sunflower oil showed Il to be 0·281 h- 1 and v = 5·34 X 10-3 g/liter per h. These results suggested that in addition to pH, temperature and initial substrate concentrations, agitation and aeration are also important control parameters on the riboflavin production (Ozbas et al., 1984).

7. 3. 5 Effect of growth factors The effect of some growth factors such as oL-methionine, inositol, 0(+ )-biotin and thiamine for riboflavin production by E. ashbyii and A. gossypii were investigated in various media.

In order to determine initial concentration effects of these growth factors, glucose was used as substrate at the optimum growth conditions. While, o( + )-biotin and thiamine are ineffective in stimulating either growth or flavinogenesis, maximum specific growth and riboflavin production rates and riboflavin yields are obtained in the media containing 0·4 g/liter oL-methionine and 0·2 g/liter inositol for E. ashbyii. It is also observed that all of these four compounds are effective in supporting growth and product formation

Tab

le 1

E

ffec

ts o

f V

ario

us S

ubst

rate

s on

Spe

cifi

c G

row

th (

It)

and

Rib

ofla

vin

Pro

duct

ion

(v)

Rat

es a

nd R

ibof

lavi

n Y

ield

s (Y

P/SC

a)

for

E.

ashb

yii

and

A.

goss

ypii

(30

"C,

Sti

rrin

g R

ate:

100

rpm

, In

itia

l p

H =

6·5

)

Subs

trat

es

Init

ial

Ita

E.

ashb

yii

YP

lSco

Ita

A

. go

ssyp

ii

YP

lSco

subs

trat

e (h

-1)

V

X 1

03

(h -

1)

vX

10

3

conc

entr

atio

n (g

R/g

mo

per

h)

(gR

/gm

o p

er h

) (g

/lit

er)

Glu

cose

20

·0

0·18

54

3·57

3·

6 0·

1538

1·

25

3·1

Gly

cero

l 20

·0

0·17

33

2·27

3·

1 0·

1315

0·

89

1·3

Sunf

low

er o

il 20

·0

0·16

69

1·45

3·

0 0·

2245

1·

00

2·9

Glu

cose

+ g

lyce

rol

5·0

+ 1

5·0

0·20

44

4·16

4·

5 0·

1979

1·

32

1·7

Glu

cose

+ g

lyce

rol

7·5

+ 1

2·5

0·20

06

4·10

3·

7 0·

2153

1·

5 1·

9 G

luco

se +

sun

flow

er o

il 5

·0+

15·

0 0·

2438

5·

56

9·2

0·23

23

1·49

5·

2 G

luco

se +

sun

flow

er o

il 1

0·0

+ 1

0·0

0·23

58

4·75

6·

8 0·

2635

4·

05

8·8

Whe

y 12

·5

0·13

79

1·48

6·

5 W

hey

30·0

0·

1529

0·

24

2·2

a C

alcu

late

d in

the

reg

ion

of e

xpon

enti

al g

row

th (

Mon

od k

inet

ic m

odel

).

.... VI

00

:-'l ~ l Ro

o ~

:-'l

Co

... 0- ~

Tab

le 2

E

ffec

ts o

f V

ario

us G

row

th F

acto

rs o

n Sp

ecifi

c G

row

th (

fJ)

and

Rib

ofla

vin

Prod

ucti

on (

v) R

ates

and

R

ibof

lavi

n Y

ield

s (Y

PlSc

J fo

r E

. as

hbyi

i an

d A

. go

ssyp

ii (3

0°C

, St

irri

ng R

ate:

100

rpm

, In

itial

pH

= 6·

5)

Subs

trat

es

E.

ashb

yii

Yp/sc

o A

. go

ssyp

ii Yp

/sco

fJ vX

10

3 fJ

v X

10

3

(h -

1)

(gR

/g r

no p

er h

) (h

-1)

(g

R/g

rno

per

h)

G+

Ma

0·29

00

4·46

4·

5 G

+I

0·19

57

4·00

3·

8 G

+S

+I

0·15

64

4·22

6·

3 W

+I

0·13

86

1·78

7·

6 G

+M

b

0·17

79

3·84

6·

4 G

+I+

B+

T

0·20

68

3·92

7·

5 G

+S

+M

0·

1221

0·

49

4·9

G+

S+

I+B

+T

0·

2299

2·

64

7·8

W+

I+B

+T

0·

3015

0·

48

4·0

Initi

al s

ubst

rate

con

cent

rati

ons

wer

e (g

/lit

er):

a

Glu

cose

(G

), 2

0·0;

G

+ s

unfl

ower

oil

(S),

5·0

+ 1

5·0;

whe

y (W

),

12·5

; D

L-m

ethi

onin

e (M

), 0

·4;

inos

itol

(I),

O· 2

. b

G,

20·0

; G

+ S

, 10

·0 +

10·

0; W

, 30

·0;

M,

0·08

; I,

0·5

; D

( +

)-b

iotin

(B

), 4

x 1

0-7

; th

iam

ine

(T),

0·0

4.

~ " ~ "" §.: '"tl ~ ~

;: " ::t. c :: ~

$ is ;: s· t:x:I

N .... VI

\C

160 T. Katsal & M. T. Ozbas

with A. gossypii. Optimum initial growth factor concentrations are found as 0·08 g/liter oL-methionine, 0·5 g/liter isositol, 0·4 Jlg/liter 0(+ )-biotin and 0·04 g/liter thiamine for this ascomycete.

It was observed that the addition of these growth factors to the medium containing only whey or glucose as substrate caused riboflavin production by E. asbhyii and A. gossypii to increase (Table 2). Although higher yields were obtained in the media containing sunflower oil compared to that of glucose lower yields were attained with these growth factors in glucose and sunflower oil medium for both micro-organisms (Ozbas & Kutsal, 1987).

7.4 Flow-Sheet

Descriptions of commercial methods for riboflavin production are primarily limited to the patent literature.

Commercial methods for cultivation of E. ashbyii may be surface methods exposed to air, or submerged methods in which air is dispersed throughout the mash, with or without supplementary agitation (Goodwin, 1959; Wagner­Jauregg, 1972).

The procedures generally adopted in industry by using A. gossypii utilize aerated and agitated submerged cultures in liquid media. The air flow rate in liters/min should be at least 0·25 times the volume of the medium in liters. Excessive agitation which interferes with mycelial development reduces the riboflavin production. Analysis of the experimental results indicates that satisfactory yields of riboflavin can be obtained if fermentation variables are closely controlled. In one of these pilot plant experiments, 2·0% glucose, 1·8-2·1 % suitable corn steep liquor, 1·0% animal stick liquor, and a small amount of some antifoam agent had been used. After fermentation for 96-120 h at 30°C, the riboflavin content of the liquor was 500-600 y / cc. The fermented liquor had been evaporated to syrup in conventional evaporators and dried to a concentrate containing 2·5% riboflavin on steam-heated drum dryers. In these experiments, inoculum quantity and age, corn steep liquor from various sources, and use of other nutrients and experimental techniques are also introduced (Pfeifer et al., 1950; Goodwin, 1959).

The commercial production of riboflavin by A. gossypii is shown in Fig. 7 (Pfeifer et al., 1950).

8 PRODUCT RECOVERY AND PURIFICATION

Fermentation solids containing riboflavin such as residues from butyl alcohol­acetone fermentation may be recovered for use as animal feed supplements by drying and powdering to a crude product. Drying is generally accomplished by drum or spray drying procedures. These food grade products usually contain other members of the B-complex, along with minerals and proteinaceous substances.

2 3

4 5

Mix

ing

ta

nk W

ate

r o

ut

10

La

bo

rato

ry c

ult

ure

A

shby

a go

ssyp

ii

Fe

rme

nto

r

-._

- .. w

ate

r in

8

Su

lph

uri

c a

cid

Air

co

mp

ress

or

Eva

po

rato

r

Fig.

7.

Flow

she

et f

or r

ibof

lavi

n pr

oduc

tion

by

ferm

enta

tion

(Pf

eife

r et

al.,

19

50).

1, G

luco

se;

2, c

om s

teep

liq

uor;

3,

ani

mal

stic

k liq

uor;

4,

sulp

huri

c ac

id;

5, s

oybe

an o

il; 6

, ca

ustic

sod

a; 7

, in

ocul

um t

ank;

8,

air

filte

r; 9

, st

erili

zer;

10

, co

oler

.

~

~.

~ \)' s· - "tI ~ ~ g . :s ~

~ I· b:I

N ... 0'1 ...

162 T. Kutsal & M. T. Ozbas

Pure, crystalline riboflavin is obtained from some liquors containing sufficiently high concentrations of riboflavin, such as the fermentation broths of E. ashbyii and A. gossypii.

For the purification of riboflavin from the fermentation broths, it is useful to remove lipids by extraction with ether, in which the vitamin is insoluble. In some cases, salts and glycogen can be eliminated from riboflavin concentrates by fractionate precipitation with alcohol or acetone.

Various methods were developed for recovery of riboflavin from fermented media.

Two methods were described in 1945 by Hines who found first that by bacteriological reduction, riboflavin in fermentation liquors of E. ashbyii could be precipitated as a reddish-brown amorphous product by the action of Streptococci strains (Hickey, 1954; Wagner-Jauregg, 1972; Periman, 1979). Streptococcus faecaiis, S. liquefaciens and S. zymoge may be used in these microbiological methods (Brit. Pat. 621,468).

Some chemical methods can be used also for recovery of riboflavin from fermented mash. Chemical reducing agents, such as sodium dithionite (Na2S20 4), stannous and chromous chlorides, and certain other reducing compounds were employed in these chemical precipitation methods. Precipita­tion could be obtained more rapidly and almost immediately by the use of chemical reducing agents. Of these, sodium dithionite has economic and low toxicity advantages over some of the other useable reducing agents, such as stannous or chromous chlorides (Hickey, 1954; Wagner-Jauregg, 1972).

The chemical precipitation methods operate primarily on solutions contain­ing at least 20/Jog/ml. In these methods the insoluble matter of mashes is removed by centrifugation and riboflavin-containing parts are adjusted to pH 5·0-5·5. A reducing agent such as Na2S204 is added at a temperature of 20-30DC and in a ratio of 5 moles of reducing agent to 1 mole of riboflavin (Brit. Pat. 621,468; US Pat. 2,367,644; US Pat. 2,367,646).

The resulting precipitate is permitted to settle, the supernatant is centrifu­gated. The residue is filtered. The crude precipitate which is obtained by such means can be readily converted to crystalline riboflavin. In these processes, the reddish-brown precipitate is dissolved in a hot polar solvent, such as 75% aqueous isopropyl alcohol. This mixture is heated to dissolve all the precipit­ate, except the inert material present, filtered, and the clear filtrate on cooling gives yellow needlelike crystals of riboflavin which are separated by filtration or centrifugation. Proportion of solvent to precipitate, and the temperature employed in obtaining riboflavin are not critical (Brit. Pat. 621 468; US Pat. 2421142; US Pat 2367646).

Riboflavin can be also purified by adsorption methods. Good adsorbents for riboflavin are Fuller's earth in acid solution, Florisil, Floridin XXF, and Frankonit in neutral solutions. The vitamin is adsorbed very strongly by charcoal, but elution is difficult from this adsorbate. A combination of precipitation and adsorption methods can sometimes be necessary to isolate pure riboflavin. A general method for the preparation of pure D-riboflavin

Microbial Production of Vitamin B2 163

from natural sources has been described which is based on adsorption on Fuller's earth, fractionation with immiscible solvents and acetone, and crystallization from an aqueous acetone-petroleum ether mixture; aqueous alcohol solutions have been used for elution of the adsorbates (Wagner­Jauregg, 1972).

9 (BIO)-ASSA Y METHODS

Riboflavin can be assayed by physico-chemical, microbiological, or biological methods. The most commonly used physico-chemical method involves meas­urement of the characteristic, yellow-green fluorescence of riboflavin which has a maximum at 565 nm at pH 6. For complex mixtures such as foods or feeds, chemical pretreatment of extracts by column chromatography, oxida­tion, reduction, or extraction is necessary to remove interfering fluorescent impurities. Riboflavin can be measured by polarography, and also by photo­metric or fluorometric measurement of lumiflavine formed by the irradiation of riboflavin in alkaline solutions.

As with most vitamins, the first assays developed for riboflavin depended upon the measuring of the biological response of animals. Early studies were carried out in the rat and in the chicken. Animal assays are time-consuming, expensive, and least accurate, but have the advantage of being based on a biological animal response, which is important from the nutritional standpoint. They are particularly useful for assaying riboflavin derivatives since substituent groups on the riboflavin molecule frequently depress or eliminate the biological response.

In animal methods the reference standard is crystalline synthetic riboflavin. For administration in rat growth assays it is useful to prepare a stock solution, which must be kept in a dark bottle in the refrigerator.

Since riboflavin is widely distributed in plant and animal tissues, test substances may occur in a variety of forms. Solutions containing high concentrations of riboflavin can be administered directly in suitable dilutions. Samples of relatively low potency are mixed with a small portion of the basal diet. Test materials with a high fat content may increase the riboflavin requirement of the rat (Pearson, 1967). A human bioassay based on comparative urinary excretions of riboflavin following test doses of a standard vs tablets has been used widely for checking the physiological availability of riboflavin in tablets (Kirk & Othmer, 1968).

One of the microbiological assays of riboflavin is based on the growth of some micro-organisms. Not many bacteria have a requirement for riboflavin, and virtually all of those known are lactic acid bacteria. The classic microbiological assay for riboflavin with Lactobacillus casei which is measured by titrating the lactic acid formed is in use in the original or modified form. A more sensitive assay is afforded by the use of Leuconostoc mesenteroides. This organism is 50 times more sensitive than L. casei. A method for total riboflavin

164 T. Kutsal & M. T. Ozbas

in body fluids and tissues suitable for clinical and nutritional surveys is based on the ciliate protozoan Tetrahymena pyriformis (Pearson, 1967; Kirk & Othmer, 1968).

10 BIOLOGICAL PROPERTIES

The principal forms of riboflavin which exist in living cells are riboflavin 5'phosphate (FMN) and flavin adenine dinucleotide (FAD). Both these phosphates are protein bound and 60-90% of the total riboflavin in natural products is present in the form of the dinucleotide. Riboflavin performs its biological functions in a number of different enzyme systems. Two derivatives of riboflavin, FMN and FAD, serve as prosthetic groups and combine with specific protein enzymes which catalyze oxidation-reduction reactions in cells (Pearson, 1967). The good sources of riboflavin are milk, egg, liver, heart, kidney, green, leafy vegetables, apricot, tomato, beef and poultry meats (Wagner-Jauregg, 1972).

11 CHEMICAL SYNTHESIS

Riboflavin can thus be produced by chemical synthesis or by fermentation processes. Although pure crystallized riboflavin for therapeutic purposes is also made by chemical synthesis, various commercially competitive fermentation processes have recently been developed for riboflavin production (Ozbas & Kutsal, 1986a). Other processes start from D-ribose, which is produced by fermentation (Chapter 11).

In 1891, O. Kiihling synthesized alloxazines by condensation of o-phenylene­diamine hydrochloride with alloxan. Using the same principle, in 1934, R. Kuhn and P. Karrer independently worked out methods for the synthesis of flavins, based on o-xylene, D-ribose and alloxan as starting materials. Later, processes of technical importance were developed by employing many refine­ments in the general synthetic methods of R. Kuhn and P. Karrer (Wagner­Jauregg, 1972).

Processes employing several chemical procedures have also been patented.

12 FORMULATIONS, APPLICATIONS AND ECONOMICS

Riboflavin is essential for the growth and normal health of animals as well as of man. A lack of riboflavin in human or animal diets causes the formation of certain oral, cutaneous and corneal lesions. Vitamin B2 is widely used in the food enrichment, pharmaceutical and feed supplement industries (Kirk & Othmer,·1968).

USP riboflavin for therapeutic purposes is administered orally in tablet form or by injection as a sterile aqueous solution, with niacinamide or other

Microbial Production of Vitamin 8 2 165

solubilizing agent added. All of the vitamin B complex and most of the multivitamin preparations also contain riboflavin (Kirk & Othmer, 1968).

Sterile, supersaturated solutions of riboflavin in normal saline have been employed for intravenous administration (Wagner-Jauregg, 1972).

Some of the cereal products such as flour and bread are enriched with iron, thiamine and niacin as well as riboflavin to compensate for the loss of these nutrients that occurs in the processing of wheat (Kirk & Othmer, 1968).

Riboflavin can be synthesized by most higher plants, yeasts, and lower fungi, and bacteria. The tissues of higher animals are unable to synthesize this vitamin. For the enrichment of poultry and livestock feeds, riboflavin is usually added at concentrations of 2-8 g/ton, depending on the species, age and purpose. The supplement of riboflavin improves health, growth, tissue repair, and reproduction of the animals and also has an economic importance in poultry raising and egg production (Kirk & Othmer, 1968; Wagner-Jauregg, 1972).

The present world consumption of riboflavin is approximately 1·25 million kg for human and animal use combined (Lago & Kaplan, 1981).

Commercial fermentation processes for production of riboflavin or riboflavin concentrates are relatively recent, having been developed in the past 40 years. In 1946 processes using the ascomycete A. gossypii were started. Manufac­turers using the microbiological process are Merck and Co., Inc. (USA) and BASF (FRG). Companies manufacturing riboflavin by chemical synthesis include Hoffmann-LaRoche Inc. (USA and Switzerland), Takeda Chemical Industries Ltd (Japan), Pfizer, Inc. (USA), and E. Merck (FRG) (Perlman, 1979).

REFERENCES

Ainsworth, G. C. & Sussman, A. S. (1965). The Fungi, Academic Press, New York, pp. 37,492.

Brit. Pat. 621,468 (April 11 1949) (to Commercial Solvents Corp.) Demain, A. L. (1972). Riboflavin oversynthesis, In Annual Reviews of Microbiology

Vol. 26, ed. C. E. Clyton, S. Raffel & M. P. Starr. George Benta Inc., USA, pp. 369-88.

Goodwin, T. W. (1959). Production and biosynthesis of riboflavin in micro-organisms. In Progress In Industrial Microbiology, ed. D. J. D. Hockenhull. Heywood & Company, London, pp. 139-177.

Goodwin, T. W. (1963a). Vitamins. In Biochemistry of Industrial Micro-organisms, ed. C. Rainbow & A. H. Rose. Academic Press, London, pp. 151-8.

Goodwin, T. W. (1963b). Riboflavin and related compounds. In The Biosynthesis of Vitamins and Related Compounds, ed. T. W. Goodwin. Academic Press, London, pp.24-35.

Gutcho, S. J. (1973). Chemicals by Fermentation, Noyes Data Corporation, Chemical Technology Review No. 19, New Jersey, pp. 323-25.

Heimann, W. (1980). Fundamentals of Food Chemistry, Ellis Horwood, Chichester, pp. 212-14.

166 T. Kutsal & M. T. Ozbas

Hickey, R. J. (1954). Production of riboflavin by fermentation. In Industrial Fermentations, ed. L. A. Underkofler & R. J. Hickey. Chemical Publishing Co., New York, pp. 157-90.

Kirk, R. E. & Othmer, D. F. (1968). Encyclopedia of Chemical Technology, Vol. 17, John Wiley & Sons, New York, pp. 445-58.

Lago, B. D. & Kaplan, L. (1981). Vitamin fermentations: Bz and B 12• Adv. in Biotech., 3,241-6.

Ozbas, T., Kutsal, T. & CagIar, A. (1984). The production of riboflavin by Eremothecium ashbyii at various media. In Proceedings of the 3rd European Congress on Biotechnology, ed. D. Behrens, Verlag Chemie GmbH, Weinheim, pp. 389-94.

Ozbas, T. (1~~5). Eremotheciu11J ashbyii Ashbya gossypii Mikroorganizmalari lIe Riboflavin Uretimi. MSc thesis, Hacettepe University, Ankara, Turkey.

Ozbas, T. & Kutsal, T. (1986a). Riboflavin production by Eremothecium ashbyii in a batch stirred tank fermenter. Biotechnology Letters, 8,441-44.

Ozbas, T. & Kutsal, T. (1986b). Comparative study of riboflavin production from two micro-organisms: Eremothecium ashbyii and Ashbya gossypii. Enzyme Microb. Techno!., 8, 593-96.

Ozbas, T. & Kutsal, T. (1987). Effects of inositol, D( + )-biotin and thiamine on riboflavin production by Ashbya gossypii. In Proceedings of the 4th European Congress on Biotechnology, ed. O. M. Neijssel, R. R. van der Meer & K. Ch. A. M. Luyben. Elsevier Science Publishers B.V., Amsterdam, pp. 273-76.

Pearson, W. N. (1967). Riboflavin. In The Vitamins, ed. P. Gyorgy & W. N. Pearson. Academic Press, New York, pp. 99-101.

Perlman, D. (1979). Microbial process for riboflavin production. In Microbial Technology, ed. H. J. Peppler & D. Perlman. Academic Press, New York, pp. 521-7.

Perlman, D., Brown, W. E. & Sylvan, B. L. (1952). Fermentation. Ind. Eng. Chem., 44, 1996-2001.

Pfeifer, V. F., Tanner, F. W., Vojnovich, C. Jr & Traufler, D. H. (1950). Riboflavin by fermentation with Ashbya gossypii. Ind. Eng. Chem., 42, 1776-81.

Robinson, F. A. (1966). The Vitamin Co-factors of Enzyme Systems, Pergamon Press, Braunschweig, pp. 160-63.

Smith, J. E. & Berry, D. R. (1975). The Filamentous Fungi, Vol. 1, Edward Arnold, London, pp. 67-8, 115-116.

US Pat. 2367644 (January 16 1945), G. E. Hines, Jr (to Commercial Solvents Corp.). US Pat. 2367646 (January 161945), M. G. W. Millian (to Commercial Solvents Corp.). US Pat. 2421142 (27 May 1947) J. K. Dale (to Commercial Solvents Corp.). Wagner-Jauregg, T. (1972). Chemistry. In The Vitamins, ed. W. H. Sebrell Jr & R. S.

Harris. Academic Press, New York, pp. 3-5.

Chapter 11

MICROBIAL PRODUCTION OF D-RIBOSE

K. SASAJIMAa & M. YONEOAb

a Central Research Division, b Corporate Strategy, Takeda Chemical Industries Ltd, Yodogawa-ku, Osaka 532, Japan

1 HISTORY

o-Ribose and its derivative, 2-deoxy-o-ribose, are components of RNA and DNA, respectively. It is of interest that these pentoses are components of such important biopolymers in the hereditary material of organisms whereas other ubiquitous pentoses, L-arabinose and o-xylose, are usually present in the plant cell walls. Why the pentose components of nucleic acids are o-ribose and 2-deoxy-o-ribose is an unresolved problem; o-ribose and its derivatives have been also found widely in nature.

From the viewpoint of commercial application, o-ribose has long served as the focus for a starting material for the chemical synthesis of riboflavin (Fig. 1), which has been used in large amounts in pharmaceuticals and livestock feed additives (see also Chapter 10). Various methods have been investigated and established for the large-scale production of o-ribose.

Investigations on o-ribose started in 1909 when Levene and Jacobs identified o-ribose as a component of yeast RNA (Levene & Jacobs, 1909). The subsequent endeavors of many investigators were devoted to efficiently preparing o-ribose from yeast RNA, e.g. chemical hydrolysis of yeast RNA (Levene & Clark, 1921; Levene, 1935) and the enzymatic hydrolysis of yeast RNA (Bredereck & Rothe, 1938; Bredereck et aI., 1940). Along this line, the production of o-ribose from 5-amino-4-imidazole-carboxamide riboside by enzymatic hydrolysis with a bacterial nucleosidase has recently been developed (Sano et al., 1977a,b).

On the other hand, many investigators tried to establish a method for the chemical synthesis of o-ribose from such a cheap material as o-glucose. These studies were stimulated by identification of ribitol, the reduced product of o-ribose, as a component of vitamin B2 , riboflavin, and by chemical synthesis of riboflavin in the middle of the 1930s (Karrer et al., 1935a,b; Kuhn et al., 1935a,b; von Euler et al., 1935). The chemical process has been used as a

167

168 K. Sasajima & M. Yoneda

Fig. 1. Formula of riboflavin.

commercial procedure to produce o-ribose for the synthesis of riboflavin. Investigations from 1910 to the 1950s on preparing and chemically synthesizing o-ribose were reviewed succinctly by Jeanlotz & Fletcher (1951) and Overend & Stacey (1955).

In the early 1950s, the pentose phosphate pathway was established as an important metabolic pathway other than glycolysis in carbohydrate metabolism (Wood, 1985). Thus, the biosynthesis of o-ribose was elucidated, which was very important information for developing a commercial procedure for o-ribose production using the transketolase (EC 2.2.1.1) mutants (tkt mutants) of Bacillus species as will be described later.

At the same time, the production of o-ribose by micro-organisms was first reported by Simonart & Godin (1951). About 10 years later, an unidentified bacterium was found to excrete both o-ribose and o-ribose 5-phosphate into a culture medium (Suzuki et al., 1963). A smaller degree of o-ribose accumula­tion was also described by Saito & Sugiyama (1966).

In the late 1950s, active investigations on the development of nucleotide flavour enhancers, inosine monophosphate and guanosine monophosphate, started in Japan. A manufacturing process was established and has been long used to commercially produce these substances. During the course of studies on the development of the process, it was found that the mutants of Bacillus lacking transketolase, a key enzyme of the pentose phosphate path­way, accumulated a large amount of o-ribose (Sasajima & Yoneda, 1971). Subsequent development research established an economical manufacturing process for o-ribose production using these mutants. The commercial produc­tion of o-ribose for riboflavin synthesis started in the mid-1970s. This process has now been widely used for 15 years. The characteristic feature of this process is that mutants of carbohydrate metabolism of Bacillus species are used. In contrast, wild-type strains of Acetobacter suboxidans, A. xylinum, etc., are used in L-sorbose fermentation, which is another remarkable microbial process of sugar production for vitamin C synthesis (Perlman, 1979).

This review focuses on transitions of developments in o-ribose production

Microbial Production of D-Ribose 169

methods and the related basic investigations. In particular, recent microbial methods will be described in detail.

2 OCCURRENCE OF o-RIBOSE AND ITS DERIVATIVES IN NATURE

The occurrence of o-ribose and its derivatives in nature is summarized in Table 1.

o-Ribose was first isolated from yeast RNA and identified by Levene & Jacobs (1909). Thereafter, it was found to be a component of uric acid in the blood (Davis et al., 1922); of crotonoside in the croton bean (Croton tiglium L.) (Cherbuliez & Bernhard, 1932a,b); of coenzymes such as NAD and NADP from yeasts (von Euler & Vestin, 1935; von El,ller & Schlenk, 1937; von Euler et al., 1942) and from blood cells (Warburg et al., 1935), FAD (Ochoa & Rossiter, 1939; Klein & Kohn, 1940), UDP-glucose (Caputto et aI., 1950), and coenzyme A (Baddiley et al., 1953); of vitamin B12 (Beaven et al., 1949; Brink & Folkers, 1949; Holiday & Petrow, 1949; Brink & Folkers, 1950; Brink et al., 1950; Buchanan et aI., 1950a,b); and of poly(ADP-ribose) (Doly & Mandel, 1967). Other coenzymes such as ADP, CDP, and GDP or many nucleoside antibiotics (Suhadolnik, 1970) also contain o-ribose. It is also present in the cell walls of Salmonella typhimurium as a component of lipopolysaccharide (Kauffman et al., 1962) or of Eubacterium saburreum as a component of polysaccharide (Hoffman et al., 1977; Hofstad & Lygre, 1977); in the flavonoids of Phlebodium dictyocallis (Mett.) Gomez as a component of o-glycosidic moieties (Gomez & Wallace, 1986); and in the coccoliths of Emiliania huxleyi (Lohmann) Hay and Mohler as a component of an acidic Ca2 + -binding polysaccharide (Borman et al., 1987).

o-Ribose derivatives such as 2-deoxy-o-ribose, 3-deoxy-o-ribose, 3-amino-3-deoxy-o-ribose, ribitol, ribonic acid, o-riburonic acid, 5-methylthioribose and glut amyl o-ribose have also been found as a component of organisms or as a product of micro-organisms. 2-Deoxy-o-ribose is a component of DNA (Levene et al., 1930; Chargaff et al., 1949; Chargaff & Lipschitz, 1953) which is the carrier of genetic information in all organisms except RNA viruses (Avery et al., 1944; Hershey & Chase, 1952; Watson & Crick, 1953). 3-Deoxy-o-ribose is a component of an antibiotic, cordycepin, produced by Cordyceps militaris (Bentley et al., 1951). 3-Amino-3-deoxy-o-ribose is a component of puromycin produced by Streptomyces alboniger (Baker & Schaub, 1953; Waller et al., 1953) and an antibiotic, 3'-amino-3'-deoxy-adenosine, produced by Helminthosporium species (Gerber & Lechevalier, 1962; Guarino & Kredich, 1963). Ribitol (adonitol), the reduced derivative of o-ribose, was first isolated from Adonis vernalis (Jeanlotz & Fletcher, 1951) and subsequently was found in the roots of Bupleurum falcatum (Wessely & Wang, 1938), in honeydews excreted by the genus Ceroplastes (Hackman & Trikojus, 1952), in pneumo­cocci as a component polysaccharide (Rebers & Heidelberger, 1959, 1961;

170

o-Ribose or derivative

o-Ribose

2-Deoxy-o-ribose

3-Deoxy-o-ribose 3-Amino-3-deoxy-o­

ribose

K. Sasajima & M. Yoneda

Table 1 Occurrence of o-Ribose in Nature

Source

Ribonucleic acid Uric acid Crotonoside

NAD, NADP

FAD

UDP-glucose Coenzyme A Vitamin B12

Poly (ADP-ribose) Lipopolysaccharide of

Salmonella typhimurium Polysaccharide of

Eubacterium saburreum Flavonoids of Phlebodium

dictyocallis (Mett) Coccolith-associated poly­

saccharide of Emiliania huxleyi (Lohmann) Hay & Mohler

Deoxyribonucleic acid

Cordycepin Puromycin

3'-Amino-3'-deoxy-adenosine

Adonis vernalis Roots of Bupreum falcatum Capsular of pneumococci

Ribitol teichoic acid of Gram-positive bacteria

Lipopolysaccharide of Proteus mirabilis

Lipopolysaccharide of Vibrio parahaemolyticus

Reference

Levene & Jacobs, 1909 Davis et aI., 1922 Cherbuliez & Bernhard,

1932a,b von Euler & Vestin,

1935; Warburg et al., 1935; von Euler & Schlenk, 1937; von Euler et al., 1942 Ochoa & Kossiter, 1939; Klein & Kohn, 1940 Caputto et al., 1950 Baddiley et al., 1953 Beaven et aI., 1949; Brink & Folkers, 1949; Holiday & Petrow, 1949; Brink & Folkers, 1950; Brink et aI., 1950; Buchanan et af., 1950a,b Doly & Mandel 1967 Kauffmann et al., 1962

Hofstad & Lygre, 1977; Hoffman et af., 1977 Gomez & Wallace, 1986

Borman et af., 1987

Levene et al., 1930; Chargaff et af., 1949; Chargaff & Lipschitz, 1953 Bentley et aI., 1951 Baker & Schaub, 1953;

Waller et al., 1953 Gerber & Lechevalier, 1962; Guarino & Kredich, 1963 Jeanlotz & Fletcher, 1951 Wessely & Wang, 1938 Rebers & Heidelberger, 1959, 1961; Roberts et ai., 1963 Ward, 1981

Gmeiner, 1977; Gmeiner et af., 1977 Miyano et aI., 1983

D-Ribose or derivative

D-Ribonic acid

D-Riburonic acid

Methylthioribose

Glutamyl D-ribose 5-phosphate

D-Ribose 5-phosphate D-Ribose I-phosphate 5-Phosphoribosyl

I-pyrophosphate D-Ribose 1,5-

bisphosphate

Microbial Production of D-Ribose

Table l--contd.

Source

Oxidation product of D-ribose by pseudomonads

Oxidation product of D-ribose by Rhizobium meliloti

5' -Methylthioadenosine

Brain and kidney of a patient

Pentose phosphate pathway Precursor of nucleotides Precursor of nucleotides

Human erythrocyte

171

Reference

Foster, 1944; Lockwood & Nelson, 1946; Weimberg, 1961 Amemura et at., 1981

Mandel & Dunham, 1912; Suzuki et aI., 1914, 1924 Williams et at., 1986

Vanderheiden, 1970

Roberts et al., 1963), and in the cell walls of Proteus mirabilis (Gmeiner, 1977; Gmeiner et al., 1977) and Vibrio parahaemolyticus (Miyano et al., 1983) as a component of lipopolysaccharides and of various Gram-positive bacteria as a component of ribitol teichoic acid (Ward, 1981). It is also a component of riboflavin (Karrer et al., 1935a; Kuhn et al., 1935a; von Euler et aI., 1935). D-Ribonic acid was the oxidation derivative of D-ribose produced by pseudo­monads (Foster, 1944; Lockwood & Nelson, 1946; Weimberg, 1961). Another oxidation product, D-riburonic acid, was found in the cell wall polysaccharides of Rhizobium meliloti (Amemura et al., 1981). 5-Methylthioribose is a com­ponent of 5'-methylthioadenosine (Mandel & Dunham 1912; Suzuki et at., 1914, 1924) which has an important role in the metabolism of adenosylthi­omethionine (Schlenk & Smith, 1953). Glutamyl D-ribose 5-phosphate was found as an abnormal storage substance in the brain and kidney of a patient suffering from renal failure and neurologic deterioration (Williams et al., 1986). D-Ribose 5-phosphate is an intermediate in the pentose phosphate pathway (Wood, 1985) which plays an important role in the production of D-ribose by the tkt mutants of Bacillus species as will be described later. D-Ribose-l-phosphate and 5-phosphoribosyl I-pyrophosphate take part in purine and pyrimidine nucleotide biosynthesis (Hartmann, 1970). o-Ribose 1,5-bisphosphate was found in human erythrocytes (Vanderheiden, 1970).

3 CHEMICAL AND PHYSICAL PROPERTIES OF o-RIBOSE

The chemical and physical properties of o-ribose and its derivatives have already been described in detail by Jeanlotz & Fletcher (1951), Overend &

172 K. Sasajima & M. Yoneda

"O~C" " " " 0" " "

Fig. 2. Formula of o-ribose.

Stacey (1955) and Windholz et al. (1983). The formulae of o-ribose is shown in Fig. 2. The chemical and physical properties of o-ribose and its derivatives are as follows.

o-Ribose C5H lO0 5 ; mol. wt 150·13, C 40·00%, H 6·71; 053·29%. Melting point of crystalline o-ribose is 87°C (Levene & Jacobs, 1909). Crystals are plates or needles. o-Ribose shows mutarotation, final [«]b - 23'7° (4,5% in water) (Phelps et al., 1934). Soluble in water, slightly insoluble in alcohol. Anhydroribose C5Hs0 4; m.p. 229-230°C (Bredereck et al., 1940). «-Aniline-N-o-ribopyranoside; m.p. 125-127°C, [«m + 63·4°~ + 48·6° (C = 1·0% in pyridine) (Berger & Lee, 1944; Berger et al., 1944). Benzylphenylhydrazone ClsH22N204; chrysanthemum-like crystals, m.p. 128°C (Sasajima & Yoneda, 1971). p-Bromophenylhydrazone; m.p. 166-167°C (van Ekenstein & Blanksma, 1913; Steiger, 1936) Methyl-o-riboside; crystals from ethyl acetate, m.p. 83-84° C, [«]~ - 113·6° (p = 3) (Windholz et al., 1983). Phenylosazone C17H2oN'403; yellow needles from pyridine + water, m.p. 163-164°C.

The infrared absorption of o-ribose from 8 to 15 microns was measured by Kuhn (1950). A polarographic study of o-ribose was described by Cantor & Peniston (1940); and crystallographic properties were reported by Keenan (1926).

4 BIOSYNTHESIS AND METABOLISM OF o-RIBOSE

As already mentioned above, o-ribose is a component of biologically impor­tant substances such as RNA and coenzymes. After the classical elucidation of the glycolytic pathway, the pentose phosphate pathway was established as another important carbohydrate pathway in the early 1950s. The pentose phosphate pathway and related metabolic pathways are shown in Fig. 3. It has the following roles; (1) supplement of the o-ribose frame for nucleic acid biosynthesis (Hartman, 1970); (2) the biosynthesis of o-erythrose 4-phosphate, one of the essential precursors of aromatic amino acids and vitamins (Greenberg, 1969); (3) the biosynthesis of C-7 and C-8 carbohydrate com­ponents of lipopolysaccharide in the cell walls of Gram-negative bacteria (Eidels & Osborn, 1971; Woisetschlager & Hogenauer, 1986), and (4) the

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174 K. Sasajima & M. Yoneda

biosynthesis of ribitol, a component of ribitol teichoic acid in the cell walls of Gram-positive bacteria (Ward, 1981).

The active investigations of two American research groups headed by Bernard Horecker and Ebraim Racker in the early 1950s, following the observation of the oxidation of D-glucose 6-phosphate to 6-phospho-D­gluconate by Warburg & Christian (1936, 1937) and the identification of D-ribose 5-phosphate as one of the oxidation products by Dickens (1938a,b), established the pathway scheme. Thus, the biosynthetic pathway from D­glucose to D-ribose has been elucidated. The roles of the pathway mentioned above were established through subsequent active investigations. Recently, all aspects of the pathway were reviewed by Wood (1985). The tkt mutants of Bacillus species used for commercially producing D-ribose could be isolated on the basis of the knowledge accumulated about the pentose phosphate pathway.

The catabolism of D-ribose in Escherichia coli has also been studied extensively. It was elucidated that D-ribose is used as a carbon and energy source through the pentose phosphate pathway (David & Wiesmeyer, 1970). D-Ribose catabolism is initiated by transportation into the cells through two distinct D-ribose transport systems (David & Wiesmeyer, 1970). Intracellular D-ribose is phosphorylated by ribokinase (EC 2.7.1.15) (David & Wiesmeyer, 1970) and catabolized further through the pentose phosphate pathway. The high-affinity D-ribose transport system consists of a binding protein (Curtis, 1974; Willis & Furlong, 1974; Galloway & Furlong, 1977) and membrane­bound components (Iida et aI., 1984; Lopilato et ai., 1984). The D-ribose binding protein serves not only as a component of the transport system but also as the attractant receptor for the chemotactic response to D-ribose (Adler, 1969; Hazelbauer & Adler, 1971; Galloway & Furlong, 1977). The gene encoding the D-ribose binding protein was recently cloned and sequenced (Groarke et a/., 1983). The D-ribose binding protein of Salmonella typhimu­rium has also been described (Aksamit & Koshland, 1972, 1974).

The existence of the D-ribose operon (rbs) encoding for the genes of the D-ribose binding protein, membrane-bound components, and the ribokinase of E. coli K-12 was first elucidated by Anderson & Cooper (1970). However, the rbs operon in E. coli B/r was determined to be located at 2 min and expressed constitutively (Abou-Sabe & Richman, 1973) while, in E. coli K-12, it is located at 73·5 min (now 83 min) and expressed inducibly (Anderson & Cooper, 1970). The duplicate rbs operon at 2 min represents a transposition of the 83-min region to 2 min in strain E. coli B/r (Abou-Sabe et al., 1982). A silent copy of the 83-min rbs operon is also present in E. coli B/r (Abou-Sabe et ai., 1982). Recently, the rbs operon was investigated by Lopilato et al. (1984).

The catabolism of D-ribose in some other bacteria has also been studied. D-Ribose might be catabolized into the pentose phosphate pathway after being transported and cleaved into C2 and C3 compounds by phosphoketolase (EC 4.1.2.9) in Lactobacillus species, though the initial reaction was not elucidated (Bernstein, 1953; Heath et ai., 1958). In Rhizobium meliloti (Duncan, 1981)

Microbial Production of D-Ribose 175

and R. leguminosarum (Dilworth et al., 1986), o-ribose is metabolized into the pentose phosphate pathway after phosphorylation by ribokinase. Pseudomon­ads oxidized o-ribose into o-ribonic acid (Foster, 1944; Lockwood & Nelson, 1946; Weimberg, 1961). o-Ribose is converted to acetate and propionate in Bacteroides ruminicola (Caldwell & Newman, 1986) and is converted to o-ribulose by o-ribose isomerase (EC 5.3.1.20) of Mycobacterium smegmatis (Izumori et aI., 1975). Recently, the ribokinase gene of Staphylococcus hyicus subsp. hyicus was cloned (Keller et al., 1984).

In yeasts, there are two metabolic pathways; one initiated by phosphoryla­tion of o-ribose through ribokinase (Sable, 1950, 1952) and another by reduction into ribitol (Barnett, 1976), which is oxidized into o-ribulose and, after phosphorylation, metabolized into the pentose phosphate pathway. o-Ribose is reduced by aldose reductase (EC 1.1.1.21) from Pachysolen tannophilus (Morimoto et al., 1987).

50-RIBOSE-PRODUCING MICRO-ORGANISMS

5.1 Wild-Type Micro-organisms

The first o-ribose accumulation was found in the culture medium of Penicillium brevi-compactum (Simonart & Godin, 1951) during studies on the biosynthesis of the components of benzene derivatives (Godin, 1953a,b). Since o-ribose was detected by paper chromatography, the amount of accumulated o-ribose was unknown. About 10 years later, an unidentified bacterium was reported to accumulate both o-ribose and D-ribose 5-phosphate (Suzuki et al., 1963). Pseudomonas reptilivola was also reported to accumulate o-ribose in a culture medium at the rate of 0·12 mg of o-ribose per ml (Saito & Sugiyama, 1966). All the micro-organisms used in these studies were wild-type and not much o-ribose was accumulated. However, the amount of o-ribose produced by the tkt mutants of Bacillus species was remarkable (Sasajima & Yoneda, 1971) and the procedure, with subsequent improvement, has been used to commercially produce o-ribose for riboflavin synthesis for 15 years. A detailed description of the isolation and improvement of o-ribose-producing tkt mutants of Bacillus species is given in the following section.

5.2 Transketolase Mutants (tkt Mutants) of Bacillus Species

5. 2.1 Isolation of tkt Mutants of Bacillus Species Investigations on the production of the nucleotide flavor enhancers, inosine monophosphate and guanosine monophosphate, by enzymatic degradation of yeast RNA started in Japan in the late 1950s (Ogata, 1976). The process was established and has been used for their commercial production ever since. During this time and subsequently, the production of inosine monophosphate and guanosine monophosphate or their precursors inosine and guanosine by

176 K. Sasajima & M. Yoneda

fermentation became a research subject in many academic and company laboratories in Japan. Various types of mutants of purine nucleotide biosynth­esis were isolated (Ogata et al., 1976) and have been used on the industrial scale to produce these nucleotides. During the course of studies on improving inosine-producing mutants of Bacillus species (Nogami et al., 1968), mutants defective in the pentose phosphate pathway were isolated because it was assumed that more o-ribose frame would be supplied for purine nucleotide biosynthesis in such mutants (Sasajima et al., 1970). Contrary to expectation, inosine productivity was not improved in such mutants. However, it was found that a large amount of o-ribose was accumulated in the culture medium by the tkt and o-ribulose 5-phosphate 3-epimerase (EC 5.1.3.1) mutants of Bacillus species (Sasajima & Yoneda, 1971, 1974b).

Although the reactions catalyzed by transketolase had been elucidated, it was not simple to isolate tkt mutants. They had never been isolated at that time because the pentose phosphate pathway is very complicated and, moreover, plays a role in supplying the precursor of aromatic amino acids such as L-tryptophan, L-tyrosine and L-phenylalanine and of aromatic vitamins such as coenzyme Q, vitamin K, and folic acid (Fig. 3). In the beginning, mutants which could not utilize L-arabinose or o-gluconate that are metabolized through the pentose phosphate pathway were isolated. No tkt mutant could be found among them, though aromatic amino acids were complemented in the culture medium used for mutant isolation. However, a o-ribulose 5-phosphate 3-epimerase mutant was isolated during these studies (Sasajima et al., 1970; Sasajima & Yoneda, 1974b).

Next, attempts were made to first isolate mutants requiring shikimic acid, which is an intermediate of aromatic amino acids and vitamins (Fig. 3), and to secondly select non-utilizers of o-gluconate as a carbon source among them. Ten shikimic acid-requiring mutants were thus isolated and two of them were found not to utilize o-gluconate as a carbon source (Sasajima et al., 1970). These mutant strains were proved to be transketolase-deficient mutants by enzymatic determinations (Sasajima & Yoneda, 1974a,b). The other shikimic acid-requiring mutants were assumed to be those deficient in enzymes of aromatic biosynthesis before shikimic acid. The ratio of 2 to 10 coincided exactly with that anticipated. These tkt mutants accumulated about 40 mg of o-ribose per ml in the culture medium (Sasajima & Yoneda, 1971). The o-ribulose 5-phosphate 3-epimerase mutant produced a large amount of both o-ribose and o-ribulose at the same time in a two to one ratio.

Josephson & Fraenkel (1969) independently isolated tkt mutants of Escherichia coli, which did not accumulate any o-ribose (Fraenkel, D.G., 1973, pers. comm.). Eidels & Osborn (1971) also isolated tkt mutants of Salmonella typhimurium to elucidate the synthetic pathway of L-glycero-o­manno-heptose, a component of the lipopolysaccharide in the outer mem­brane. However, it was not described whether o-ribose was produced by the tkt mutants or not.

The tkt and o-ribulose 5-phosphate 3-epimerase mutants could not utilize

Microbial Production of D-Ribose 177

o-gluconate, o-xylose, and L-arabinose which are catabolized through the pentose phosphate pathway (Sasajima & Yoneda, 1974b; Sasajima et al., 1977). The tkt mutants among them, moreover, required aromatic amino acids such as L-tryptophan, L-tyrosine and L-phenylalanine but, unexpectedly, no aromatic vitamins for their growth (Sasajima & Yoneda, 1974b; Sasajima et al., 1977). Aromatic vitamins might be synthesized from these complemented aromatic amino acids.

As the revertants derived from the tkt mutants did not accumulate o-ribose, it was concluded that their o-ribose accumulation depended on transketolase mutations (Sasajima & Yoneda, 1971). The phenomenon was the first example of the production of carbohydrates by carbohydrate metabolism mutant strains.

The investigation on the isolation of the tkt mutants of other bacterial strains such as Bacillus subtilis, B. pumilus, Brevibacterium thiogenitalis, B. ammoniagenes, Arthrobacter globiformis, Aerobacter aerogenes and Micrococcus denitrificans revealed that the tkt mutants of only Bacillus species among them could accumulate o-ribose (Sasajima et aI., 1985b).

A difference in the productivity of pentoses was observed between strains of Bacillus species. For example, the tkt mutants of a strain of Bacillus subtilis IFO 3026 accumulated both o-ribose and o-ribulose at the same time (Sasajima et aI., 1972; Sasajima, 1976; Sasajima & Yoneda, 1984; Sasajima et aI., 1985b). The tkt mutants of another strain of B. subtilis IFO 12114 accumulated only o-ribose (Sasajima & Yoneda, 1984). The difference in the kind of pentoses accumulated might be due to the strain difference in kind or substrate specificity of the phosphatases which would hydrolize intracellulariy accumu­lated o-ribose 5-phosphate and o-ribulose 5-phosphate to o-ribose and o-ribulose during excretion.

Transketolase activity is functional when thiaminepyrophosphate and Mg2+ ion are coexistent (Wood, 1985). It was, therefore, considered that thiamine­requiring mutants would also accumulate o-ribose. This was shown to be so by isolating the thiamine-requiring mutants of Bacillus subtilis IFO 12114 but the amount of o-ribose accumulated was much less than that in the case of the tkt mutants (Sasajima & Yoneda, 1984; Sasajima et al., 1985b). This is probably because it is essential to add a small amount of thiamine to the culture medium for enzymes other than trans keto lase which require it to function. Thus, it is impossible to make transketolase activity complete!y zero by this mutation, and consequently they produce a smaller amount of o-ribose than do the tkt mutants.

5.2.2 Improvement of rrribose-producing tkt mutants The original tkt mutants accumulated o-ribose at the rate of about 40 mg/ml (Sasajima & Yoneda, 1971). Improvement of these mutants by subsequent mutations resulted in isolating high-level o-ribose-producing mutants which produced about 70 mg of o-ribose per ml in the culture medium (Sasajima et al., 1972, 1985b; Sasajima, 1976; Sasajima & Yoneda, 1984). Two mutation

178 K. Sasajima & M. Yoneda

steps at which the yield of o-ribose production was remarkably improved were the following; (1) a mutation of the regulation of D-glucose dehydrogenase (EC 1.1.1.47) synthesis (Yokota et al., 1979; Yokota & Sasajima, 1981) and (2) an asporogenous mutation (Sasajima et al., 1972, 1985b; Sasajima, 1976; Sasajima & Yoneda, 1984)

D-Glucose dehydrogenase in Bacillus species, is known to be synthesized at stage III of sporulation in parallel with morphological changes during spore formation (Warren, 1968), exists only in spores (Fujita et al., 1977) and has the role of supplying energy during germination (Otani et al., 1986). The initiation of sporulation is also known to occur only after the carbon source is depleted. In the improved mutant strain, the enzyme was found in vegetative cells while no morphological changes concerning sporulation occurred (Yokota et al., 1979; Yokota & Sasajima, 1981). It was presumed that o-glucose was converted to o-ribose in the mutant strain through two metabolic pathways, the non-phosphorylated pathway (from o-glucose to the pentose phosphates via D-gluconate) and the phosphorylated pathway (the oxidative branch of the pentose phosphate pathway) (Fig. 4). The gluconate pathway, probably, contributed to the high D-ribose productivity of the improved strain. However, there was also the possibility of o-gluconate as well as o-ribose accumulation. The ratio of o-ribose and o-gluconate production depended on the partial pressure of oxygen during fermentations, i.e. oxygen deficiency led to D-gluconate rather than o-ribose accumulation (Sasajima & Yoneda, 1984). D-Ribose accumulation by the mutant strain was about 60 mg/ml (Sasajima et al., 1972, 1985b; Sasajima, 1976; Sasajima & Yoneda, 1984).

The second-step asporogenous mutation improved the capability of o-ribose production up to 70 mg/ml (Sasajima et al., 1972, 1985b; Sasajima, 1976; Sasajima & Yoneda, 1984). In this case, the mechanism for the improvement was not clear. It might be a mutation by which carbohydrate metabolism is changed in the direction of higher-level o-ribose accumulation. The high-yield

D-Glucose ------- o-Gluconate

1 1 o-Glucose-6-phosphate - 6-Phospho-o-gluconate

1 0-Ribulose-5-phosphate

1 0-Ribose-5-phosphate

1 D-Ribose

Fig. 4. Pathways of o-ribose formation from o-glucose in high-yield mutant strains.

Microbial Production of D-Ribose 179

mutant strain has been used for 15 years to produce o-ribose for riboflavin synthesis on an industrial scale.

5.2.3 Pleiotropic properties of D-ribose-producing tkt mutants As already described, the tkt mutants not only could never utilize carbon sources which are catabolized through the pentose phosphate pathway but also required complementation of aromatic amino acids in the culture medium for their growth. Moreover, it was revealed that the tkt mutants also had another interesting property, i. e. functions concerning the cell surface changed pleiotropically. The pleiotropic change is described below.

The first unexpected phenomenon was that the tkt mutants could not utilize o-glucose as a sole carbon source (Sasajima & Yoneda, 1974b; Sasajima et at., 1977) though o-glucose is the best carbon source for o-ribose production (Sasajima & Yoneda, 1984; Sasajima et at., 1985b). The mutant strain seemed preferentially to utilize other substances in the medium as a carbon and energy source for growth, and later to become able to metabolize o-glucose for o-ribose production. Investigation on the phenomenon revealed that the phosphoenolpyruvate-dependent phosphotransferase system was defective (Sasajima & Kumada, 1979); especially, the transport function of the Enz nglc

(phosphohistidinoprotein-hexose phosphotransferase; EC 2.7.1.69) of the PEP-dependent o-glucose phosphotransferase system (Roseman, 1969; Postma & Roseman, 1976; Saier, 1977; Dills et at., 1980; Postma & Lengeler, 1985), was defective in the tkt mutant.

Another biochemical change was found in the catabolite repression (Maga­sanik, 1961). The utilization of o-mannitol, o-fructose and glycerol was inhibited by o-glucose while utilization of sorbitol was not (Sasajima & Kumada, 1981a). The synthesis of the enzymes of o-mannitol catabolism, such as o-mannitol-1-phosphate dehydrogenase (EC 1.1.1.17), and of the 0-

mannitol transport system was found to be hypersensitive to o-glucose repression. Meanwhile, the synthesis of enzymes of sorbitol catabolism, such as sorbitol dehydrogenase, and of the sorbitol transport system was resistant to o-glucose repression. D-Gluconate, o-xylose, and L-arabinose were also found to repress the synthesis of the enzymes of D-mannitol catabolism. These changes in the regulation of enzyme synthesis might be caused by a defect in the membrane.

Subsequent investigations on various functions concerning the cell surface revealed; (1) the tkt mutant formed chains of cells during the exponential growth phase; (2) the sensitivity to bacteriophages SPlO and SP01 was altered; (3) the cell walls of the mutant strain autolyzed during incubation more slowly than those of the parental strain, and (4) the sporulation frequency of the mutant strain remarkably decreased (Sasajima & Kumada, 1981b, 1983a). The mutant strain was non-motile because of a deficiency in flagellation (Sasajima & Kumada, 1983b). The isolation and characterization of a true revertant (transketolase reversion) confirmed that these pleiotropic properties were

180 K. Sasajima & M. Yoneda

generated by a single mutation in transketolase (Sasajima & Kumada, 1979, 1983a,b).

The investigation on chemical changes in the membrane composition revealed that the SDS-polyacrylamide gel electrophoresis pattern of the membrane proteins and the thin layer chromatography pattern of the membrane lipids were different in the parental and mutant strains (Sasajima et al., 1985a).

The tkt mutants of Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium could not synthesize L-glycero-o-manno-heptose (Eidels & Osborn, 1971), which is a component of the outer membrane lipopolysaccharides and essential for a functional cell surface (Ames et al., 1974; Koplow & Goldfine, 1974; Bayer et al., 1975; Irvin et al., 1975; Smit et al., 1975; van Alphen et al., 1976). It is of interest that the tkt mutants of both Gram-negative and Gram-positive bacteria are defective in cell surface functions, though the mechanisms were probably different. It can be said that transketolase plays an important role not only in carbohydrate metabolism and aromatic biosynthesis but also in the cell surface functions of bacteria.

5. 2.4 Production of l-deoxy-ketoses by tkt mutants of Bacillus pumilus During studies on the relation between o-ribose productivity and transketolase mutation in various bacteria, it was found that the tkt mutants of Bacillus pumilus produced a new monosaccharide, 1-deoxy-o-altro-heptulose (Yokota et al., 1978). Further investigations revealed that 1-deoxy-o-altro-heptulose 7-phosphate was synthesized from o-ribose-5-phosphate and oL-acetoin (Yokota & Sasajima, 1983). The accumulation of o-ribose 5-phosphate in the cells of the tkt mutant strains presumably stimulated formation of 1-deoxy-o­altro-heptulose, which might accumulate in the culture medium after dephos­phorylation during excretion (Yokota & Sasajima, 1983). Subsequent inves­tigations revealed that cell-free extracts of various micro-organisms catalyzed the formation of 1-deoxy-o-fructose, 1-deoxy-o-sorbose, 1-deoxy-o-tagatose, 1-deoxy-o-threo-pentulose, 1-deoxY-L-threo-pentulose and 1-deoxyerythrulose (Yokota & Sasajima, 1984a, b). It is of much interest that, among them, 1-deoxy-o-threo-pentulose was described as a precursor of the thiazole ring of thiamine (Therisod et al., 1981). This substance was also described as being produced by Streptomyces hygroscopicus (Hoeksema & Baczynskyj, 1976; Slechta & Johnson, 1976). It was elucidated that the formation of 1-deoxy­ketoses was catalyzed by pyruvate dehydrogenase (lipoamide) (EC 1.2.4.1) and acetoin dehydrogenase (Yokota & Sasajima, 1986). It was also found that another similar enzyme, 2-ketoglutarate dehydrogenase (EC 1.2.4.2) produced 2,3-dideoxy-hex-4-ulosonic acid (Yokota & Sasajima, 1985).

6 FERMENTATION 6.1 Inoculation

In the case of Penicillium brevi-com pactum (Simonart & Godin, 1951), an unidentified bacterium (Suzuki et al., 1963), and Pseudomonas reptilivora

Microbial Production of D-Ribose

Table 2 Inoculum Medium of Bacillus Species

Constituent

Soluble starch Corn steep liquor K2HP04

KH2P04

Adenosine Guanosine

Concentration (%)

2·0 2·0 0·3 0·1 0·01 0·02

181

(Saito & Sugiyama, 1966), the cells grown on bouillon agar slants were directly inoculated into the fermentation medium. In the case of the tkt mutants of Bacillus species (Sasajima & Yoneda, 1971), the inoculum culture incubated at 37°C was used. The inoculum medium composition is shown in Table 2. Adenosine and guanosine can be excluded from the medium in the case of high-producing mutant strains whose requirements were compensated by further reversion mutations.

6.2 Fermentation Media

The composition of the fermentation media used for o-ribose production are shown in Tables 3, 4, 5 and 6 (Simonart & Godin, 1951; Suzuki et ai., 1963; Saito & Sugiyama, 1966; Sasajima and Yoneda, 1971).

Suzuki et ai. (1963) described that the Mn2+ ion stimulated o-ribose accumulation while o-ribose 5-phosphate was accumulated more in the absence of Mn2+ ion in the case of an unidentified bacterium. Fe2+ and Zn2+ ions are also effective in stimulating o-ribose accumulation.

In the case of Pseudomonas reptilivora, o-ribose accumulation from various carbon sources other than o-glucose was observed (Saito & Sugiyama, 1966). o-Mannose, o-fructose and o-gluconic acid were found to be as good substrates as o-glucose.

Table 3 Fermentation Medium of Penicillium brevi­

compactum

Constituent

o-Glucose NaN03

KCI KH2P04

MgS047H20 FeS047H20

Concentration (%)

5·0 0·2 0·05 0·1 0·05 0·002

182 K. Sasajima & M. Yoneda

Table 4 Fermentation Medium of an Unidentified

Bacterium

Constituent

D-Glucose KHZP04

K zHP04 MgS047HzO CaClz2H2 0 Urea Yeast extract

Concentration (%)

10·0 1·0 1·0 1·0 0·01 0·6 1·0

In the case of Bacillus species, it was also found that various carbon sources were useful for D-ribose accumulation as shown in Table 7 (Sasajima & Yoneda, 1971). D-Glucose, D-mannose, sorbitol, D-mannitol, maltose, and lactose were good substrates. Various kinds of natural nitrogen sources as well as dried yeast are useful (Table 8) (Sasajima & Yoneda, 1971). Corn steep liquor-based medium was used for a large-scale production (Asai et ai., 1978). It was necessary to complement aromatic amino acids to the medium in the case where corn steep liquor was used as nitrogen source (Asai et ai., 1978).

Table 5 Fermentation Medium of Pseudomonas

reptilivora

Constituent

D-Glucose Urea MgS04 KH2P04

Sodium citrate Corn steep liquor Peptone Yeast extract

Concentration (%)

Table 6

5·0 1·0 0·05 0·1 0·3 0·2 0·1 0·05

Fermentation Medium of Bacillus Species

Constituent

D-Glucose (NH4)2S04

CaHP04

Ca3(P04)z CaC03

Dried yeast

Concentration (%)

12·5; 15·0 1·5 0·5 0·5 1·0 2·5

(pH 7·0)

Microbial Production of D-Ribose

Table 7 D-Ribose Formation from Various Carbon Sources

Carbon source

D-Glucose D-Mannose Sorbitol D-Mannitol Maltose Lactose Glycerol Dextrin Soluble starch

D-Ribose formed (mg/ml)

Strain Shi5"

23·7 17·3 28·0 25·8 22·4 12·3 9·6 9·7

11·4

Strain Shi7"

20·0 18·1 7·7 6·1

18·6 14·2 3·4 9·7 7·0

"Strains SHi5 and Shi7 are tkt mutants of a Bacillus species (Sasajima & Yoneda, 1971).

Table 8 Efficiency of Various Nitrogen Sources for D-Ribose

Formation

Nitrogen source

Dried yeast Pharmamedia Meat extract Com steep liquor Yeast extract Peptone Cotton seed meal Cotton seed flour Com gluten meal Soy bean meal

D-Ribose formed (mg/ml)

1·5%

24·4 31·1 19·4 23·2 30·1 17·6 20·8 12·1 22·6 25·1

2·5%

23·7 23·3 30·2 9·3

25·3 15·6 21·6 26·5 15·4 21·4

Strain S27, a transformant from strain Shi7 which com­pensated for both adenine and xanthine requirements (Sasajima & Yoneda, 1971). D-Glucose was used as the carbon source.

6.3 Fennentation Course

183

In the case of Bacillus species tkt mutants, D-ribose accumulation started at 1 or 2 days in proportion to D-glucose assimilation and ceased at 4 days (Sasajima & Yoneda, 1971). The cell population reached about 1010 cells/ml in 2 days. Thus, D-ribose was accumulated during the stationary phase. This implied that only the aged cells excreted D-ribose into the medium.

A large-scale fermentation course using a high-level producing strain is

184

7 pH 6

5

"'C C ..,~

"'C ;f. 10 QI ~

~ QI - '" ::> 0 E v ::>2 VC) :;: I QlO

.8"iii 5

.- ::> a::"'C I .;;; 0::'

K. Sasajima & M. Yoneda

pH

D-Ribose

L-____ L...'-_ .. _........--.----1r-o 50 100

Time (hr)

Fig. 5. Time course of n-ribose fermentation.

shown in Fig. 5 (Sasajima et al., 1972, 1985b; Sasajima, 1976; Sasajima & Yoneda, 1984).

As already described in the section on 'Producing micro-organisms', the improved strains have the ability to produce n-gluconate as well as n-ribose. n-Ribose is made from n-glucose through two pathways: one is a non­phosphorylated pathway via n-gluconate and the other is a phosphorylated pathway via n-glucose 6-phosphate (Fig. 4). Both n-gluconate and n-glucose 6-phosphate are converted to 6-phospho-n-gluconate, the common intermedi­ate of both pathways, and finally converted to n-ribose.

Appropriate culture conditions are required for the smooth conversion of n-gluconate to 6-phospho-n-gluconate. Otherwise, n-gluconate accumulates in the culture medium resulting in a reduced production of n-ribose (Asai et al., 1978; Sasajima & Yoneda, 1984). Effective factors are the pH of media, an oxygen concentration in the medium enough for three oxidative reactions in the pathway from n-glucose to n-ribose (Fig. 4) (Sasajima & Yoneda, 1984), and the temperature of the culture (Asai et al., 1978). The maximum yield of n-ribose was noted at 36·6°C when exponential growth cells were used as the inoculum (Asai et al., 1978).

7 n-RIBOSE RECOVERY AND PURIFICATION In the course of preparing n-ribose by chemical or enzymatic hydrolysis of RNA, n-ribose was first converted to the p-bromophenylhydrazone (van

Microbial Production of D-Ribose 185

Ekenstein & Blanksma, 1913; Steiger, 1936) or the arylamide riboside (Berger & Lee, 1944; Berger et al., 1944). After subsequent purification, o-ribose was recovered by hydrolysis (Jeanlotz & Fletcher, 1951; Overend & Stacey, 1955).

In the case of preparing o-ribose by enzymatic hydrolysis of 5-amino-4-imidazole-carboxamide-riboside with a nucleosidase, o-ribose was separated and purified from the other product, 5-amino-4-imidazole-carboxamide, by column chromatography with ion-exchange resin Dowex 50 (H+) (Sano et al., 1977b). Finally, o-ribose was obtained as the lyophilized form.

Saito & Sugiyama (1966) purified o-ribose from the culture medium of Pseudomonas reptilivora by first separating microbial cells by centrifugation and subsequent column chromatographies with chromatopile and strong-base anion resin (borate form). Finally, they obtained o-ribose in crystalline form from the syrup.

Sasajima & Yoneda (1971) purified and isolated o-ribose from the culture medium of Bacillus species tkt mutant by removing microbial cells, column chromatography with carbon, yeast treatment to assimilate the residual substrate o-glucose and excluding metal and anionic ions with ion exchange resins, amberlite IR120 and amberlite IRA400. o-Ribose was crystallized by adding ethanol to the syrupy solution.

Hough et al. (1948, 1949) described the separation of o-ribose from coexisting sugars by partition chromatography with a powdered cellulose column.

8 ASSAY METHODS OF o-RIBOSE

o-Ribose can be determined by reagents for pentoses, e.g. orcinol reagent (Bial, 1902, 1903; Mejbaum, 1939; Miller et al., 1951; Dische, 1962b), carbazol reagent (Dische, 1962a) and phloroglucinol reagent (Dische, 1962b ). By orcinol reagent o-ribose in the culture medium is overestimated because of coexisting substances in the culture medium (Sasajima & Yoneda, 1971). o-Ribose can be also assayed by densitometry after paper partition chromatog­raphy (Sasajima & Yoneda, 1971), and can be determined by gas-liquid chromatography (Laine & Sweeley, 1973; Petersson, 1974).

9 CHEMICAL SYNTHESIS

The preparative method of o-ribose by chemical synthesis was first described by van Ekenstein & Blanksma (1913). In 1936, Steiger repeated this process (Fig. 6). The starting material was prepared from calcium o-gluconate according to the method of Hockett & Hudson (1934). The process was later modified by Berezovskii and Rodionova (1953).

Another method of o-ribose synthesis was described by Gehrke & Aichner (1927). This process was improved by Austin & Humoller (1934) in L-ribose

186 K. Sasajima & M. Yoneda

CHO COOH COOH

HOtH HOtH H60H I I . boiling aqueous I HCOH electrolyticaloxidation; HCOH _..<:.pyn<.:;"=di::.:.:ne,--+) HCO H __

HtOH HtOH HtOH

tH20H ~H20H 6H20H o-arabinose

sodium amalgam ..

o-arabonic acid

CHO I

HCOH I

HCOH I

HCOH I

CH20H o-ribono-y-Iactone o-ribose

o-ribonic acid

Fig. 6. Chemical synthesis of D-ribose according to the method of van Ekenstein & B1anksma (1913).

o-arabinose triacetyl-J3-o­arabino-pyranosyl

bromide

CHO I

HCOH

Hr[l CH

Zn dust I acetic aCid) HCOAc 0 --

H10AC I CH2

diacetyl-o-arabinal

Hr[l CH I HCO H 0 ...:.pe_r_be_nz_oi_c _ac_id+)

I HCOR

H10H I CH2

o-arabinal

I HCOH

I CH20H

o-ribose

Fig. 7. Chemical synthesis of D-ribose according to the method of Gehrke & Aichner (1927).

reduction

Microbial Production of D-Ribose

CH20H

HtOH

HOtH I HC-O mel"period"le)

I "" HCOH/CHC6H s I . H 2C-O

187

4,6-benzylidene-o-glucose 4,6-benzylidene-o-glucitol 2,4-benzylidene-o-erythrose

3,5-benzylidene-l-deoxy-l- I-nitro-l-deoxy- D-ribose nitro-o-ribitol o-ribitol

Fig. 8. Chemical synthesis of D-ribose according to the method of Sowden (1950).

H 2C-O" /CH3 I CH

HC-O/' "---CH3

I HOCH

I HCOH

I HC-O ....... y,,/CH3

I /CH H2C-O "---CH3

H 2C-O, /,CH3 I "CH .

HC-O/' "CH3 I . - CH3S020CH

I -HCOH

I HC-O, £H3

I "Cli . H 2C-O/ "CH3

1,2: 5,6-diisopropylidene-o-glucose 3-0-mesyl-l,2: 5,6-diisopropylidene-o-glucose

CHO I

HCOH I

CH3S020CH . I HCOH

I HCOH

I CH20H

-CHO CHO I I

CH3S020CH HCOH . I I

HCOH - HCOH I J

HCOCHO HCOH I I

CH20H CH20H

3-0-mesyl-o-glucose 2-0-mesyl-4-0-formyl-D-arabinose o-ribose

Fig. 9. Chemical synthesis of D-ribose according to the method of Smith (1955).

188 K. Sasajima & M. Yoneda

preparation and was used to prepare o-ribose for the chemical synthesis of riboflavin (Karrer et al., 1935b; Kuhn et al., 1935b). The process includes arabinal as an intermediate as shown in Fig. 7.

The formation of o-ribose from o-glucose was described by Sowden (1950) and Smith (1955) as shown in Figs 8 and 9.

Stroh et al. (1964) compared the above four methods and concluded that the best yield was obtained by the method of Smith (1955). The overall yield was 24%.

10 APPLICATION AND ECONOMY

o-Ribose has long been prepared for the chemical synthesis of riboflavin from o-glucose by one of the chemical processes described above. However, a microbial production process was developed in the early 1970s (Sasajima et al., 1972, 1985b; Sasajima, 1976; Sasajima & Yoneda, 1984). The procedure, using the tkt mutant of Bacillus species is economical and prevents environmental pollution by avoiding the use of mercury, which is essential for the electrolytic reduction required in the chemical process (Harris et al., 1972). The industrial microbial production of o-ribose for the chemical synthesis of riboflavin started in 1973; it has given substantial results for about 15 years. Some companies in the world have been using this process to produce o-ribose for the synthesis of riboflavin, which is used not only for pharmaceuticals but also for animal feed additives worldwide in large quantities.

o-Ribose itself has been also used to treat some cases of heart diseases. It was suggested that administered o-ribose bypasses the pentose phosphate pathway and increases the size of the phosphoribosylpyrophosphate pool resulting in stimulation of nucleotide synthesis, maintenance of adenine nucleotide levels, and protection of the myocardium in various heart diseases (Zimmer et al., 1984). Exercise-induced muscle pain and stiffness due to myoadenylate deaminase deficiency was also successfully treated with o-ribose (ZOllner et al., 1986).

D-Ribose is a natural component of biologically important substances such as nucleic acids and coenzymes. It has been also found in many nucleoside antibiotics. Nucleoside analogs have been developed as antiviral agents. It is hoped that o-ribose can be used in the chemical synthesis of drugs to treat viral and bacterial infectious diseases in the future.

REFERENCES

Abou-Sabe, M. & Richman, J. (1973). On the regulation of o-ribose metabolism in E. coli B/r. II. Chromosomal location and fine structure analysis of the o-ribose permease and o-ribokinase structural genes by PI transduction. Molec. Gen. Genet., 122, 303-12.

Microbial Production of D-Ribose 189

Abou-Sabe, M., Pilla, J., Hazuda, D. & Ninfa, A. (1982). Evolution of the D-ribose operon of Escherichia coli B/r. J. Bacteriol., 150, 762-9.

Adler, J. (1969). Chemoreceptors in bacteria. Science, 166, 1588-97. Aksamit, R. & Koshland, D. Jr (1972). A ribose binding protein of Salmonella

typhimurium. Biochem. biophys. Res. Commun., 48, 1348-53. Aksamit, R. R. & Koshland, D. E. Jr (1974). Identification of the ribose binding

protein as the receptor for ribose chemotaxis in Salmonella typhimurium. Biochemistry, 13, 4473-8.

Alphen, W. van, Lugtenberg, B. & Berendsen, W. (1976). Heptose-deficient mutants of Escherichia coli K12 deficient in up to three major outer membrane proteins. Molec. Gen. Genet., 147,263-9.

Amemura, A., Hisamatsu, M., Ghai, S. K. & Harada, T. (1981). Structural studies on a new polysaccharide, containing D-riburonic acid, from Rhizobium me/iloti IFO 13336. Carbohydr. Res., 91,59-65.

Ames, G. F.-L., Spudich, E. N. & Nikaido, H. (1974). Protein composition of the outer membrane of Salmonella typhimurium: effect of lipopolysaccharide mutations. J. Bacteriol., 117,406-16.

Anderson, A. & Cooper, R. A. (1970). Biochemical and genetical studies on ribose catabolism in Escherichia coli. J. Gen. Microbiol., 62,335-9.

Asai, T., Doi, M., Kono, T. & Fukuda, H. (1978). Kinetic study on the production of D-ribose by Bacillus sp. J. Ferment. Technol., 56,91-5.

Austin, W. C. & Humoller, F. L. (1934). The preparation of I-ribose. J. Am. Chem. Soc., 56, 1152-3.

Avery, O. T., MacLeod, C. M. & McCarty, M. (1944). Studies on the chemical nature of the substance inducing transformation of pneumococcal types. J. expo Med., 79, 137-58.

Baddiley, J., Thain, E. M., Novelli, G. D. & Lipmann, F. (1953). Structure of coenzyme A. Nature, Lond., 171, 76.

Baker, B. R. & Schaub, R. E. (1953). Achromycin. Synthetic studies. III. Synthesis of 3-amino-D-ribose, a hydrolytic product. J. Am. Chem. Soc., 75, 3864-5.

Barnett, J. A. (1976). The utilization of sugars by yeasts. Adv. Carbohydr. Chem. Biochem., 32, 125-234.

Bayer, M. E., Koplow, J. & Goldfine, H. (1975). Alterations in envelope structure of heptose-deficient mutants of Escherichia coli as revealed by freeze-etching. Proc. natn. Acad. Sci. U.S.A., 72,5145-9.

Beaven, G. R., Holiday, E. R., Johnson, E. A., Ellis, B., Mamalis, P., Petrow, V. & Sturgeon, B. (1949). The chemistry of anti-pernicious anaemia factors. Part III. 5: 6-Disubstituted benziminazoles as products of acid hydrolysis of vitamin B12 • J. Pharm. Pharmac., 1,957-70.

Bentley, H. R., Cunningham, K. G. & Spring, F. S. (1951). Cordycepin, a metabolic product from cultures of Cordyceps militaris (Linn.) Link. Part II. The structure of cordycepin. J. Chem. Soc., 1951,2301-5.

Berezovskii, V. M. & Rodionova, E. P. (1953). Transformations and synthesis of carbohydrates. VII. Synthesis of D-ribose. Sbornik Statel Obshchel Khim., 2, 939-43; Chem. Abstr., 49,6838 (1955).

Berger, L. & Lee, J. (1944). Arylamine-N-glycosides. Part II. Arylarnine-N-pentosides and complex salt formation studies. J. Org. Chem., 11, 84-90.

Berger, L., Solmssen, U. V., Leonard, F., Wenis, E. & Lee, J. (1944). Arylamine-N­glycosides. Part III. Hydrolysis of arylamine-N-pentosides and the preparation of crystalline D-ribose. J. Org. Chern., 11,91-4.

Bernstein, I. A. (1953). Fermentation of ribose-C4 by Lactobacillus pentosus. J. BioI. Chem., 205, 309-16.

Bial, M. (1902). Die Diagnose der Pentosurie. Deut. med. Wochschr., 28, 253-4.

190 K. Sasajimo & M. Yoneda

Bial, M. (1903). Ueber die Diagnose der Pentosurie mit dem von mir angegebenen Reagens. Deut. med. Wochschr., 29,477-8.

Borman, A. H., Jong, E. W. de, Thierry, R., Westbroek, P., Bosch, L., Gruter, M. & Kamerling, J. P. (1987). Coccolith-associated polysaccharides from cells of Emiliania huxleyi (Haptophyceae). J. Phycol., 23, 118-23.

Bredereck, H. & Rothe, G. (1938). Nucleinsauren, VI. Mitteil: Darstellung der Nucleoside durch enzymatische Hydrolyse der Hefenucleinsaure; zugleich ein Beitrag zur Darstellung der d-Ribose. Ber., 71,408-11.

Bredereck, H., Kothnig, M. & Berger, E. (1940). Uber die d-Ribose (Darstellung einer krystallisierten Anhydroribose). Ber., 73,956-62.

Brink, N. G. & Folkers, K. (1949). Vitamin B12. VI. 5 ,6-Dimethylbenzimidazole , a degradation product of vitamin B12. J. Am. Chem. Soc., 71, 295l.

Brink, N. G. & Folkers, K. (1950). Vitamin B12. X. 5 ,6-Dimethylbenzimidazole , a degradation product of vitamin B12. J. Am. Chem. Soc., 72, 4442-3.

Brink, N. G., Holly, F. W., Shunk, C. H., Peel, E. W., Cahill, J. J. & Folkers, K. (1950). Vitamin B12. IX. 1-a-Ribofuranosido-5,6-dimethylbenzimidazole, a de­gradation product of vitamin B12. J. Am. Chem. Soc., 72, 1866.

Buchanan, J. G., Johnson, A. W., Mills, J. A. & Todd, A. R (1950a). The isolation of a phosphorus-containing degradation product from vitamin B12c• Chem. Ind., 1950, 426.

Buchanan, J. G., Johnson, A. W., Mills, J. A. & Todd, A. R. (1950b). Chemistry of the vitamin BI2 group. Part I. Acid hydrolysis studies. Isolation of a phosphorus­containing degradation product. J. Chem. Soc., 1950, 2845-55.

Caldwell, D. R & Newman, K. (1986). Pentose metabolism by Bacteroides ruminicola subsp. brevis strain BA. Curro Microbio/., 14, 149-55.

Cantor, S. M. & Peniston, Q. P. (1940). The reduction of aldoses at the dropping mercury cathode: estimation of the aldehyde structure in aqueous solutions. J. Am. Chem. Soc. 62, 2113-2l.

Caputto, R, Leloir, L. F., Cardini, C. E. & Paladini, A. C. (1950). Isolation of the coenzyme of the galactose phosphate-glucose phosphate transformation. J. Bioi. Chem., 184, 333-50.

Chargaff, E. & Lipshitz, R (1953). Composition of mammalian desoxyribonucleic acids. J. Am. Chem. Soc., 75, 3658-6l.

Chargaff, E., Vischer, E., Doniger, R, Green, C. & Misani, F. (1949). The composition of the desoxypentose nucleic acids of thymus and spleen. J. Bioi. Chem., 177,405-16.

Cherbuliez, E. & Bernhard, K. (1932a). Recherches sur la graine de croton. I. Sur Ie crotonoside (2-oxy-6-amino-purine-D-riboside). Helv. Chim. Acta, 1S,464-7l.

Cherbuliez, E. & Bernhard, K. (1932b). Remarques sur Ie crotonoside. Helv. Chim. Acta, IS, 978-80.

Curtis, S. J. (1974). Mechanism of energy coupling for transport of D-ribose in Escherichia coli. J. Bacterio/., 120,295-303.

David, J. & Wiesmeyer, H. (1970). Regulation of ribose metabolism in Escherichia coli. I. The ribose catabolic pathway. Biochim. biophys. Acta, 208,45-55.

Davis, A. R, Newton, E. B. & Benedict, S. R (1922). The combined uric acid in beef blood. J. Bioi. Chem., 54,595-9.

Dickens, F. (1938a). Oxidation of phosphohexonate and pentose phosphoric acids by yeast enzymes. I. Oxidation of phosphohexonate. II. Oxidation of pentose phosphoric acids. Biochem. J., 32, 1626-44.

Dickens, F. (1938b). Yeast fermentation of pentose phosphoric acids. Biochem. J., 32, 1645-53.

Dills, S. S., Apperson, A., Schmidt, M. R & Saier, M. H. Jr (1980). Carbohydrate transport in bacteria. Microbiol. Rev., 44,385-418.

Microbial Production of D-Ribose 191

Dilworth, M. J., Arwas, R., McKay, I. A., Saroso, S. & Glenn, A. R. (1986). Pentose metabolism in Rhizobium leguninosarum MNF300 and in cowpea Rhizobium NGR234. J. Gen. Microbiol., 132,2733-42.

Dische, Z. (1962a). Color reaction of ketoses with carbazole and sulfuric acid. In Methods in Carbohydrate Chemistry, Vol. I, ed. R. L. Whistler, M. L. Wolfrom, J. N. BeMiller & F. Shafizadeh. Academic Press, New York, London, pp. 481-2.

Dische, Z. (1962b). Color reactions of pentoses. In Methods in Carbohydrate Chemistry, Vol. I, ed. R. L. Whistler, M. L. Wolfrom, J. N. BeMiller & F. Shafizadeh. Academic Press, New York, London, pp. 484-8.

Doly, J. & Mandel, P. (1967). Mise en evidence de la biosyntbese in vivo d'un polymere compose, Ie polyadenosine diphosphoribose dans les noyaux de foie de poulet. C. R. hebd. Slanc. Acad. Sci., Paris, Ser. D., 264,2687-90.

Duncan, M. J. (1981). Properties of tn5-induced carbohydrate mutants in Rhizobium meliloti. J. Gen. Microbiol., 122,62-7.

Eidels, L. & Osborn, M. J. (1971). Lipopolysaccharide and aldoheptose biosynthesis in transketolase mutants of Salmonella typhimurium. Proc. natn. Acad. Sci. U.S.A., 68,1673-7.

Ekenstein, W. A. van & Blanksma, J. J. (1913). Over o-ribose. Chem. Weekblad., 10, 664.

Euler, H. von & Vestin, R. (1935). Zur Kenntnis der Wirkungen der Co-Zymase. Z. physiol. Chem., 237, 1-5.

Euler, H. von & Schlenk, F. (1937). Co-Zymase. Z. physiol. Chem., 246,64-82. Euler, H. von, Karrer, R., Malmberg, M., Schopp, K., Benz, F., Becker, B. & Frei, P.

(1935). Synthese des Lactoflavins (Vitamin B2 ) und anderer Flavine. Helv. Chim. Acta, 18,522-35.

Euler, H. von, Karrer, P. & Usteri, E. (1942). Der Zucker der Cozymase. Helv. Chim. Acta, 25, 323-5.

Foster, J. W. (1944). Microbiological aspects of riboflavin. III. Oxidation studies with Pseudomonas riboflavina. J. Bacteriol., 48,97-111.

Fujita, Y., Ramaley, R. & Freese, E. (1977). Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis. J. Bacteriol., 132, 282-93.

Galloway, D. R. & Furlong, C. E. (1977). The role of ribose-binding protein in transport and chemotaxis in Escherichia coli K12. Archs. Biochem. Biophys., 184, 496-504.

Gehrke, M. & Aichner, F. X. (1927). Uber das Arabinal. Ber., 60,918-22. Gerber, N. N. & Lechevalier, H. A. (1962). 3'-Amino-3'-deoxyadenosine, an

antitumor agent from Helminthosporium sp. J. Org. Chem., 27, 1731-2. Gmeiner, J. (1977). The ribitol-phosphate-containing lipopolysaccharide from Proteus

mirabilis, strain D52. Investigations on the structure of O-specific chains. Eur. J. Biochem., 74, 171-BO.

Gmeiner, J., Mayer, H., Fromme, I., Kotelko, K. & Zych, K. (1977). Ribitol­containing lipopolysaccharides from Proteus mirabilis and their serological relation­ship. Eur. J. Bioc;hem., 72, 35-40.

Godin, P. (1953a). Etude du metabolisme ternaire de Penicillium brevi-com pactum. I. Analyse chromatographique du milieu de culture. Biochim. biophys. Acta, 11, 114-18.

Godin, P. (1953b). Etude du metabolisme ternaire de Penicillium brevi-com pactum. II. Analyse chromatographique de la solution glucosee substitutee au milieu de culture initial. Biochim. biophys. Acta, 11, 119-21.

Gomez, L. D. & Wallace, J. W. (1986). Flavonoids of Phlebodium. Biochem. Syst. Ecol., 14,407-8.

Greenberg, D. M. (1969). Biosynthesis of amino acids and related compounds. In

192 K. Sasajima & M. Yoneda

Metabolic Pathways, Vol. III, ed. D. M. Greenberg. Academic Press, New York, London, pp. 237-315.

Groarke, J. M., Mahoney, W. C., Hope, J. N., Furlong, C. E., Robb, F. T., Zalkin, H. & Hermodson, M. A. (1983). The amino acid sequence of o-ribose-binding protein from Escherichia coli K12. I. Bioi. Chem., 258, 12952-6.

Guarino, A. J. & Kredich, N. M. (1963). Isolation and identification of 3'-amino-3'­deoxyadenosine from Cordyceps militaris. Biochim. biophys. Acta, 68,317-19.

Hackman, R. H. & Trikojus, V. M. (1952). The composition of the honeydew excreted by Australian coccids of the genus Ceroplastes. Biochem. I., 51, 653-6.

Harris, R. S., Wagner-Jauregg, T., Horwitt, M. K. & Witting, L. A. (1972). Riboflavin. In The Vitamins, Vol. V (2nd edn), ed. W. H. Sebrell & R. S. Harris. Academic Press, New York, London, pp. 1-96.

Hartman, S. C. (1970). Purines and pyrimidines. In Metabolic Pathways, Vol. IV, ed. D. M. Greenberg. Academic Press, New York, London, pp. 1-68.

Hazelbauer, G. L. & Adler, J.' (1971). Role of the galactose binding protein in chemotaxis of Escherichia coli toward galactose. Nature New Bioi., 230, 101-4.

Heath, E. C., Hurwitz, J., Horecker, B. L. & Ginsburg, A. (1958). Pentose fermentation by Lactobacillus plantarum. I. The cleavage of xylulose 5-phosphate by phosphoketolase. I. Bioi. Chem., 231, 1009-29.

Hershey, A. D. & Chase, M. (1952). Independent functions of viral protein and nucleic acid in growth of bacteriophage. I. Gen. Physiol., 36,39-56.

Hockett, R. C. & Hudson, C. S. (1934). Improvements in the preparation of o-arabinose from calcium gluconate. I. Am. Chem. Soc., 56, 1632-3.

Hoeksema, H. & Baczynskyj, L. (1976). A new metabolite from Streptomyces hygroscopicus, II. Identification as 1-deoxy-o-threo-pentulose. I. Antibiot., XXIX, 688-91.

Hoffman, J., Lindberg, B., Hofstad, T. & Lygre, H. (1977). Structural studies of the polysaccharide antigen of Eubacterium saburreum, strain L.452. Carbohydr. Res., 58,439-42.

Hofstad, T. & Lygre, H. (1977). Composition and antigenic properties of a surface polysaccharide isolated from Eubacterium saburreum, strain L452. Acta Pathol. Microbiol. Immunol. Scand. Section B, 85, 14-17.

Holiday, E. R. & Petrow, V. (1949). Vitamin B12 as a 5:6-dimethylbenziminazole derivative. I. Pharm. Pharmac., 1,734-5.

Hough, L., Jones, J. K. N. & Wadman, W. H. (1948). Application of paper partition chromatography to the separation of the sugars and their methylated derivatives on a column of powdered cellulose. Nature, Lond., 162, 448.

Hough, L., Jones, J. K. N. & Wadman, W. H. (1949). Quantitative analysis of mixtures of sugars by the method of partition chromatography. Part IV. The separation of the sugars and their methylated derivatives on columns of cellulose. I. Chem. Soc., 1949,2511-16.

!ida, A., Harayama, S., lino, T. & Hazelbauer, G. L. (1984). Molecular cloning and characterization of genes required for ribose transport and utilization in Escherichia coli K-12. I. Bacteriol., 158,674-82.

Irvin, R. T., Chatterjee, A. K., Sanderson, K. E. & Costerton, J. W. (1975). Composition of the cell envelope structure of a lipopolysaccharide-defective (heptose-deficient) strain and a smooth strain of Salmonella typhimurium. I. Bacteriol., 124,930-41.

Izumori, K., Rees, A. W. & Elbein, A. D. (1975). Purification, crystallization, and properties of o-ribose isomerase from Mycobacterium smegmatis. I. Bioi. Chem., 250, 8085-7.

Jeanlotz, R. W. & Fletcher, H. G. Jr (1951). The chemistry of ribose. Adv. Carbohydr. Chem. Biochem., 6, 135-74.

Microbial Production of D-Ribose 193

Josephson, B. L. & Fraenkel, D. G. (1969). Transketolase mutants of Escherichia coli. 1. Bacteriol., 100, 1289-95.

Karrer, P., Schopp, K. & Benz, F. (1935a). Synthesen von Flavin IV. Helv. Chim. Acta, 18, 426-9.

Karrer, P., Becker, B., Benz, F., Frei, P., Salomon, H. & Schopp, K. (1935b). Zur Synthese des Lactoflavins. Helv. Chim. Acta. 18,1435-48.

Kauffmann, F., Jann, B., KrUger, L., Liideritz, D. & Westphal, O. (1962). Zur Immunochemie der O-Antigen von Enterobacteriaceae. VIII. Analyse der Zucker­bausteine von Polysacchariden weiterer Salmonella- und Arizona-O-Gruppen. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Abt. 1: Orig., 186, 509-16.

Keenan, G. T. (1926). The optical properties of sugars. 1. Wash. Acad. Sci., 16, 433-40.

Keller, G., Schleifer, K. H. & Gotz, F. (1984). Cloning of the ribokinase gene of Staphylococcus hyicus subsp. hyicus in Staphylococcus carnosus. Archs Microbiol., 140, 218-24.

Klein, J. R. & Kohn, H. I. (1940). The synthesis of flavin-adenine dinucleotide from riboflavin by human blood cells in vitro and in vivo. 1. Bioi. Chem., 136, 177-89.

Koplow, J. & Goldfine, H. (1974). Alterations in the outer membrane of the cell envelope of heptose-deficient mutants of Escherichia coli. 1. Bacteriol., 117,527-43.

Kuhn, L. P. (1950). Infrared spectra of carbohydrates. Analyt. Chem., 22,276-83. Kuhn, R., Reinemund, K., Kaltschmitt, H., Strobele, R. & Trischmann, H. (1935a).

Synthetisches 6,7-Dimethyl-9-d-ribo-flavin. Naturwissenschaften, ~, 260. Kuhn, R., Reinemund, K., Weygand, F. & Strobele, R. (1935b). Uber die Synthese

des Lactoflavins (Vitamin B2). Ber., 68, 1765-74. Laine, R. A. & Sweeley, C. C. (1973). O-Methyl oximes of sugars. Analysis as

O-trimethylsilyl derivatives by gas-liquid chromatography and mass spectrometry. Carbohydr. Res., 27, 199-213.

Levene, P. A. (1935). Note on the preparation of crystalline o-mannose and of crystalline o-ribose. 1. Bioi. Chem., 108,419-20.

Levene, P. A. & Clark, E. P. (1921). d-Ribohexosaminic acids. 1. BioI. Chem., 46, 19-33.

Levene, P. A. & Jacobs, W. A. (1909). Uber Inosinsiiure. Ber., 42, 1198-203. Levene, P. A., Mikeska, L. A. & Mori, T. (1930). On the carbohydrate of

thymonucleic acid. 1. Bioi. Chem., 85, 785-7. Lockwood, L. B. & Nelson, G. E. (1946). The oxidation of pentoses by Pseudomonas.

1. Bacteriol., 52, 581-6. Lopilato, J. E., Garwin, J. L., Emr, S. D., Silhavy, T. J. & Beckwith, J. R. (1984).

o-Ribose metabolism in Escherichia coli K-12: genetics, regulation and transport. 1. Bacteriol., 158, 665-73.

Magasanik, B. (1961). Catabolite repression. Cold Spring Harbor Symp. Quant. Bioi., 26,249-62.

Mandel, J. A. & Dunham, E. K. (1912). Preliminary note on a purine-hexose compound. 1. Bioi. 9hem., 11, 85-6.

Mejbaum, W. (1939). Uber die Bestimmung kleiner Pentosemengen, insbesondere in Derivaten der Adenylsiiure. Z. Physiol. Chem., 258, 117-20.

Miller, G. L., Golder, R. H. & Miller, E. E. (1951). Determination of pentoses. Analyt. Chem., 23,903-5.

Miyano, K., Ishibashi, M., Kunita, N., Takeda, Y. & Miwatani, T. (1983). Demon­stration of the presence of alditols and galacturonic acid in Vibrio parahaemolyticus 010 lipopolysaccharide. FEMS Microbiol. Lett., 20,225-8.

Morimoto, S., Tawaratani, T., Azuma, K., Oshima, T. & Sinskey, A. J. (1987). Purification and properties of aldose reductase from Pachysolen tannophilus. 1. Ferment. Technol., 65, 17-21.

194 K. Sasajima & M. Yoneda

Nogami, I., Kida, M., Iijima, T. & Yoneda, M. (1968). Studies on the fermentative production of purine derivatives. Part I. Derivation of guanosine and inosine­producing mutants of a Bacillus strain. Agric. Bioi. Chem., 32, 144-52.

Ochoa, S. & Rossiter, R. J. (1939). Flavin-adenine-dinucleotide in rat tissues. Biochem. J., 33,2008-16.

Ogata, K. (1976). History and prospects. In Microbial Production of Nucleic Acid­Related Substances, ed. K. Ogata, S. Kinoshita, T. Tsunoda & K. Aida. Kodansha Ltd, Tokyo and John Wiley & Sons, New York, London, Sydney, Toronto, pp. xiii-xviii.

Ogata, K., Kinoshita, S., Tsunoda, T. & Aida, K. (1976). Microbial Production of Nucleic Acid-Related Substances. Kodansha Ltd., Tokyo and John Wiley & Sons, New York, London, Sydney, Toronto.

Otani, M., Ihara, N., Umezawa, C. & Sano, K. (1986). Predominance of gluconate formation from glucose during germination of Bacillus megaterium OM B1551 spores. J. Bacteriol., 167, 148-52.

Overend, W. G. & Stacey, M. (1955). Chemistry of ribose and deoxyribose. In The Nucleic Acids, Vol. I, ed. E. Chargaff & J. N. Davidson. Academic Press, New York, pp. 9-80.

Perlman, D. (1979). Ketogenic fermentation processes. In Microbial Technology, Vol. II, ed. H. J. Peppler & D. Perlman. Academic Press, New York, San Francisco, London, pp. 173-7.

Petersson, G. (1974). Gas-chromatographic analysis of sugars and related hydroxy acids as acyclic oxime and ester trimethylsilyl derivatives. Carbohydr. Res., 33, 47-61.

Phelps, F. P., Isbel, H. S. & Pigman, W. (1934). Mutarotation of beta-d-ribose and beta-I-ribose. J. Am. Chern. Soc., 56, 747-8.

Postma, P. W. & Lengeler, J. W. (1985). Phosphoenolpyruvate: carbohydrate phosphotransferase system of bacteria. Microbiol. Rev., 49,232-69.

Postma, P. W. & Roseman, S. (1976). The bacterial phosphoenolpyruvate: sugar phosphotransferase system. Biochim. biophys. Acta, 457,213-57.

Rebers, P. A. & Heidelberger, M. (1959). The specific polysaccharide of type VI Pneumococcus. I. Preparation, properties and reactions. J. Am. Chem. Soc., 81, 2415-19.

Rebers, P. A. & Heidelberger, M. (1961). The specific polysaccharide of type VI Pneumococcus, II. The repeating unit. J. Am. Chem. Soc., 83,3056-9.

Roberts, W. K., Buchanan, J. G. & Baddiley, J. (1963). The specific substance from Pneumococcus type 34(41). The structure of a phosphorus-free repeating unit. Biochem. J., 88, 1-7.

Roseman, S. (1969). The transport of carbohydrates by a bacterial phosphotransferase system. J. Gen. Physiol., 54, 138s-180s.

Sable, H. Z. (1950). Phosphorylation of ribose and adenosine in yeast extracts. Proc. Soc. exptl. Bioi. Med., 75,215-19.

Sable, H. Z. (1952). Pentose metabolism in extracts of yeast and mammalian tissues. Biochim. biophys. Acta, 8,687-97.

Saier, M. H. Jr (1977). Bacterial phosphoenolpyruvate:sugar phosphotransferase systems: structural, functional, and evolutionary interrelationships. Bacteriol. Rev., 41,856-71.

Saito, N. & Sugiyama, S. (1966). D-Ribose formation by Pseudomonas reptilivora. Agric. BioI. Chem., 30,841-6.

Sano, K., Yokozeki, K. & Mitsugi, K. (1977a). Enzymatic production of 5-amino-4-imidazole-carboxamide from 5-amino-4-imidazole-carboxamide-riboside by Bacillus thiaminolyticus. Optimal conditions for the enzyme formation and the enzymatic reaction. Agric. Bioi. Chern., 41,2331-4.

Sano, K., Yokozeki, K. & Mitsugi, K. (1977b). Screening of micro-organisms

Microbial Production of D-Ribose 195

producing 5-amino-4-imidazole-carboxamide and o-ribose from 5-amino-4-imidazole-carboxamide-riboside. Agric. Bioi. Chem., 41,2463-4.

Sasajima, K. (1976). o-Ribose. In Microbial Production of Nucleic Acid-Related Substances, ed. K. Ogata, S. Kinoshita, T. Tsunoda & K. Aida. Kodansha Ltd, Tokyo and John Wiley & Sons, New York, London, Sydney, Toronto, pp. 199-204.

Sasajima, K. & Kumada, T. (1979). Deficiency of o-glucose transport in transketolase mutant of Bacillus subtilis. Inst. Ferment. Res. Commun., 9, 17-26.

Sasajima, K. & Kumada, T. (1981a). Change in the regulation of enzyme synthesis under catabolite repression in Bacillus subtilis pleiotropic mutant lacking transketo­lase. Agric. Bioi. Chem., 45,2005-12.

Sasajima, K. & Kumada, T. (1981b). Cell surface change of Bacillus subtilis pleiotropic mutant lacking transketolase. Inst. Ferment. Res. Commun., 10,3-9.

Sasajima, K. & Kumada, T. (1983a). Cell surface change of Bacillus subtilis pleiotropic mutant lacking transketolase: properties of various revertant strains. Inst. Ferment. Res. Commun., 11,3-9.

Sasajima, K. & Kumada, T. (1983b). Deficiency of flagellation in a Bacillus subtilis pleiotropic mutant lacking transketolase. Agric. Bioi. Chem., 47, 1375-6.

Sasajima, K. & Yoneda, M. (1971). Carbohydrate metabolism mutants of a Bacillus species. Part II. o-Ribose accumulation by pentose phosphate pathway mutants. Agric. Bioi. Chem., 35, 509-17.

Sasajima, K. & Yoneda, M. (1974a). Simple procedures for the preparation of o-ribose 5-phosphate ketol-isomerase, o-ribulose 5-phosphate 3-epimerase and 0-

sedoheptulose 7-phosphate: o-glyceraldehyde 3-phosphate glycolaldehydetrans­ferase. Agric. Bioi. Chem., 38, 1297-1303.

Sasajima, K. & Yoneda, M. (1974b). o-Sedoheptulose 7-phosphate: o-glyceraldehyde 3-phosphate glycolaldehydetransferase and o-ribulose 5-phosphate 3-epimerase mutants of a Bacillus species. Agric. Bioi. Chem., 38, 1305-10.

Sasajima, K. & Yoneda, M. (1984). Production of pentoses by micro-organisms. Biotechnol. Genet. Engineer. Rev., 2, 175-213.

Sasajima, K., Nogami, I. & Yoneda, M. (1970). Carbohydrate metabolism mutants of a Bacillus species. Part I. Isolation of mutants and their inosine formation. Agric. Bioi. Chem., 34,381-9.

Sasajima, K., Fukuhara, T., Matsukura, A., Nakanishi, I. & Yoneda, M. (1972). o-Ribose production by pentose phosphate pathway mutants of Bacillus. Paper presented at the Fourth International Fermentation Symposium, Kyoto, 19-25 March.

Sasajima, K., Kumada, T. & Yokota, A. (1977). A pleiotropy in carbohydrate metabolism of Bacillus subtilis mutant lacking transketolase. Inst. Ferment. Res. Commun., 8, 69-77.

Sasajima, K., Yokota, A. & Kumada, T. (1985a). Alteration of the membrane composition of Bacillus subtilis pleiotropic mutant lacking transketolase. Inst. Ferment. Res. Commun., 12,5-18.

Sasajima, K., Yokota, A. & Yoneda, M. (1985b). o-Ribose production by bacteria. Kagaku to Seibutsu, 23,240-8 (in Japanese).

Schlenk, F. & Smith, R. L. (1953). The mechanism of adenine thiomethylriboside formation. J. Bioi. Chem., 204, 27-34.

Simonart, P. & Godin, P. (1951). Production de pentoses, d'acide 2-ceto gluconique et d'acide glucuronique par Penicillium brevi-compactum. Bull. Soc. Chim. Belg., 60, 446-8.

Slechta, L. & Johnson, L. E. (1976). A new metabolite from Streptomyces hygro­scopicus. I. Fermentation and isolation. J. Antibiot., XXIX, 685-7.

Smit, J., Kamio, Y. & Nikaido, H. (1975). Outer membranes of Salmonella typhimurium: chemical analysis and freeze-fracture studies with lipopolysaccharide mutants. J. Bacteriol., 124, 942-58.

196 K. Sasajima & M. Yoneda

Smith, D. C. C. (1955). The preparation of o-ribose and 2-deoxy-o-ribose from glucose. Chem. Ind., 1955,92-3.

Sowden, J. C. (1950). The condensation of nitromethane with o-erythrose and 2,4-benzylidene-o-erythrose. I. Am. Chem. Soc., 72,808-11.

Steiger, M. (1936). Zur Herstellung von o-Ribose. Helv. Chim. Acta, 19, 189-95. Stroh, H.-H. von, Dargel, D. & Haussler, R. (1964). Vergleichende Untersuchungen

zur Synthese von o-Ribose aus o-Glucose. I. Prakt. Chem., 23,309-17. Suhadolnik, R. J. (1970). Nucleoside Antibiotics. Wiley-Interscience, New York,

London, Sydney, Toronto. Suzuki, T., Tanaka, N., Tomita, F., Mizuhara, K. & Kinoshita, S. (1963). Bacterial

accumulation of ribose and ribose 5-phosphate. I. Gen. Appl. Microbiol., 9, 457-8. Suzuki, U., Shimamura, T. & Matsunaga, S. (1914). Studies on oryzanin VI. I. Tokyo

Chem. Soc., 34, 1123-75 (in Japanese). Suzuki, U., Odaka, S. & Mori, T. (1924). Uber einen neuen schwefelhaltigen

Bestandteil der Hefe. Biochem. Z., 154,278-89. Therisod, M., Fischer, J. C. & Estramareix, B. (1981). The origin of the carbon chain

in the thiazole moiety of thiamine in Escherichia coli: incorporation of deuterated 1-deoxy-o-threo-2-pentulose. Biochem. biophys. Res. Commun., 98,374-9.

Vanderheiden, B. S. (1970). Phosphate esters in human erythrocytes. VII. Further evidence for ribose 1,5-diphosphate as a natural metabolite. Biochim. Biophys. Acta, 215, 242-8.

Waller, C. W., Eryth, P. W., Hutchings, B. L. & Williams, J. H. (1953). Achromycin. The structure of the antibiotic puromycin I. I. Am. Chem. Soc., 75,2025.

Warburg, O. & Christian, W. (1936). Verbrennung von Robison-Ester durch Triphospho-Pyridin-Nucleotid. Biochem. Z., m, 44O-l.

Warburg, O. & Christian, W. (1937). Abbau von Robisonester durch Triphospho­Pyridine-Nucleotid. Biochem. Z., 292,287-95.

Warburg, 0., Christian, W. & Griese, A. (1935). Wasserstoffiibertragendes Co­Ferment, seine Zusammensetzung und Wirkungsweise. Biochem. Z., 282, 157-205.

Ward, J. B. (1981). Teichoic and teichuronic acids: biosynthesis, assembly, and location. Microbiol. Rev., 45, 211-43.

Warren, S. C. (1968). Sporulation in Bacillus subtilis. Biochemical changes. Biochem. I., 109, 811-18.

Watson, J. D. & Crick, F. H. C. (1953). Molecular structure of nucleic acids. A structure for deoxyribose nucleic acid. Nature, Lond., 171, 737-8.

Weimberg, R. (1961). Pentose oxidation by Pseudomonas fragi. I. Bioi. Chem., 236, 629-35.

Wessely, F. & Wang, S. (1938). Uber ein Vorkommen von Adonit. Monatsh. Chem., 72, 168.

Williams, J. C., Verani, R., Alcala, H., Butler, I. J. & Rosenberg, H. S. (1986). Glutamyl ribose-5-phosphate storage disease: nephrotic syndrome and cerebral atrophy. Pediatr. Pathol., 5, 277-94.

Willis, R. C. & Furlong, C. E. (1974). Purification and properties of a ribose binding protein from Escherichia coli. I. Bioi. Chem., 249,6926-9.

Windholz, M., Budavari, S., Blumetti, R. F. & Otterbein, E. S. (1983). The Merck Index. Merck & Co., Rahway, p. 1185.

Woisetschlager, M. & Hogenauer, G. (1986). Cloning and characterization of the gene encoding 3-deoxy-o-manno-octulosonate 8-phosphate synthetase from Escherichia coli. I. Bacteriol., 168,437-9.

Wood, T. (1985). The Pentose Phosphate Pathway. Academic Press, Orlando, San Diego, New York, Austin, London, Montreal, Sydney, Tokyo, Toronto.

Yokota, A. & Sasajima, K. (1981). Derepressed syntheses of sporulation marker enzymes in a Bacillus species mutant. Agric. Bioi. Chem., 45,2417-23.

Yokota, A. & Sasajima, K. (1983). Enzymatic formation of a new monosaccharide,

Microbial Production of D-Ribose 197

I-deoxy-o-altro-heptulose phosphate, from oL-acetoin and o-ribose 5-phosphate by a transketolase mutant of Bacillus purnilus. Agric. Bioi. Chern., 47, 1545-53.

Yokota, A. & Sasajima, K. (1984a). Formation of I-deoxy-o-threo-pentulose and I-deoxY-L-threo-pentulose by cell-free extracts of micro-organisms. Agric. Bioi. Chern., 48, 149-58.

Yokota, A. & Sasajima, K. (1984b). Formation of I-deoxy-o-fructose, I-deoxy-o­sorbose, I-deoxy-o-tagatose and I-deoxy-erythrulose by cell-free extracts of bacteria and Actinomycetes. Agric. BioI. Chern., 48, 1643-5.

Yokota, A. & Sasajima, K. (1985). Formation of 2,3-dideoxy-hex-4-ulosonic acid by carboligase activity of 2-ketoglutarate dehydrogenase in micro-organisms. [nst. Ferrnent. Res. Cornrnun., 12, 19-33.

Yokota, A. & Sasajima, K. (1986). Formation of I-deoxy-ketoses by pyruvate dehydrogenase and acetoin dehydrogenase. Agric. BioI. Chern., 50,2517-24.

Yokota, A., Sasajima, K. & Horii, S. (1978). Accumulation of a new monosaccharide, I-deoxy-o-altro-heptulose (l-deoxy-sedoheptulose) by transketolase mutants of Bacillus purnilus. Agric. Bioi. Chern., 42,2245-52.

Yokota, A., Sasajima, K. & Yoneda, M. (1979). Reactivation of inactivated o-glucose dehydrogenase of a Bacillus species by pyridine and adenine nucleotides. Agric. Bioi. Chern., 43, 271-8.

Zimmer, H.-G., Ibel, H., Suchner, U. & Schad, H. (1984). Ribose intervention in the cardiac pentose phosphate pathway is not species-specific. Science, 223, 712-4.

Zollner, N., Reiter, S., Gross, M., Pongratz, D., Reimers, C. D. Gerbitz, K., Paetzke, I., Deufel, T. & Hiibner, G. (1986). Myoadenylate deaminase deficiency: successful symptomatic therapy by high dose oral administration of ribose. Klin. Wochenschr., 64, 1281-90.

Chapter 12

PANTOTHENIC ACID (VITAMIN 85), COENZYME A AND RELATED COMPOUNDS

S. SHIMIZU & H. YAMADA

Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan

1 INTRODUCTION

Pantothenic acid (R- or D-( + )-N-(2,4-dihydroxy-3,3-dimethyl-1-oxobutyl)-~­alanine; for chemical formula, see Table 1) was first isolated in the 1930s from liver and found to be an essential growth factor for yeasts (Williams and co-workers, 1943). During this same period, it was also identified independ­ently with the chick anti-dermatitis factor, the filtrate factor, the chick anti-pellagra factor and an essential growth factor for lactic acid bacteria. Later these activities were shown to be identical with pantothenic acid. Since the factor could be obtained from a variety of plants and animal tissues, it was designated pantothenic acid, meaning 'from everywhere' by Williams. The compound is also referred to as vitamin B5 • It was independently synthesized by two groups in 1940.

Pantothenic acid occurs in all types of living organisms in a free form and in conjugated forms such as coenzyme A, pantetheine (Lactobacillus bulgaricus factor) and 4'-phosphopantetheine (Acetobacter suboxydans factor) (see Table 1). The coenzyme form of the vitamin is coenzyme A. It was discovered as an essential cofactor for the acetylation of sulfonamide in the liver and the acetylation of choline in the brain by Lipmann and co-workers (1953). Since that time, it has been identified with 'active acetate' and has been found to be essential for a variety of biochemical transacylation reactions. 4'­Phosphopantetheine is also a coenzyme form of pantothenic acid. It functions as a prosthetic group of the acyl carrier protein of fatty acid synthetase, citrate cleaving enzyme and enzymes involved in the synthesis of peptide antibiotics.

These early studies have been reviewed by several authors (Williams, 1943; Lipmann, 1953; Wagner & Folkers, 1964; Vagelos 1973; Vandamme, 1981; Kleinkauf & von Dohren, 1983).

2 CHEMISTRY

Pantothenic acid can be obtained as a colorless, viscous oil by drying under high vacuum in P20 5 . It is an acid and has a marked tendency to absorb water

199

Tab

le 1

P

anto

then

ic A

cid

and

its N

atur

ally

-Occ

urri

ng D

eriv

ativ

es

o-P

anto

then

ic a

cid

CH

lOH

I

I H

OC

H2C

-CH

CO

NH

(CH

2)2C

OO

H

I CH

l

4H17

NO

s M

W:

219·

23

Cal

cium

o-p

anto

then

ate

( C

HlO

H

),

HO

CH

2{-

lHC

ON

H(C

H2

hC

OO

C

a

CH

l 2

CI6

H32

CaN

20to

M

W:

476·

53

Sodi

um o

-pan

toth

enat

e I

CH

lOH

I

I H

OC

H2C

-CH

CO

NH

(CH

2)2C

OO

Na

I CH

3

4H

16N

aN0 5

M

W:

241·

21

4' -P

hosp

hopa

ntot

heni

c ac

id (

Ba

salt

)

o C

H30

H

/I I

I H

O-P

-OC

H2

C-C

HC

ON

H(C

H2

)2C

OO

H

I I

OH

C

H3

4H

16N

OHP

MW

: 31

3·27

Uns

tabl

e, v

isco

us o

il. E

xtre

mel

y hy

dros

copi

c, e

asily

dec

ompo

sed

by

acid

s, b

ases

, he

at.

Solu

ble

in w

ater

, et

hyl a

ceta

te, d

ioxa

ne,

glac

ial

acet

ic a

cid;

mod

erat

ely

solu

ble

in e

ther

, am

yl a

lcoh

ol;

inso

lubl

e in

be

nzen

e, c

hlor

ofor

m.

Sol

utio

ns a

re s

tabl

e be

twee

n pH

5 a

nd 7

. [a

Hi' +

37·5

°. S

till

er e

t al.

(194

0).

Whi

te n

eedl

es.

Mod

erat

ely

hygr

osco

pic.

Sol

uble

in w

ater

, gl

ycer

ol;

slig

htly

sol

uble

in a

lcoh

ol,

acet

one;

inso

lubl

e in

eth

er, b

enze

ne,

chlo

rofo

rm.

Dec

ompo

sed

by b

ases

. S

olut

ions

are

sta

ble

betw

een

pH 5

an

d 7.

mp

195-

196°

C (

dec)

; [a

J~ +

28·2

° (c

= 5

).

Whi

te,

hygr

osco

pic

crys

tals

. D

ecom

pose

d by

aci

ds a

nd b

ases

. S

olut

ions

ar

e st

able

bet

wee

n pH

5 a

nd 7

. m

p 12

2-12

4°C

; [a

J~ +

27·1

° (c

= 2)

. F

or s

olub

ility

, see

cal

cium

pan

toth

enat

e

Solu

ble

in w

ater

; in

solu

ble

in e

than

ol.

Uns

tabl

e to

bas

es.

Fre

e ac

id is

un

stab

le.

[a J~

+ 9·

0° (

c =

3· 3)

. K

ing

& S

tron

g (1

951)

; O

kada

et a

l. (1

967)

.

Pant

othe

noyl

-L-c

yste

ine

(Sa

salt

) C

H,O

H

CO

OH

II

I H

OC

H2C

-CH

CO

NH

(CH

2hC

ON

HC

HC

H2S

H

I CH

,

C12

H22

N20

6S

MW

: 32

2·38

4' -P

hosp

hopa

ntot

heno

yl-L

-cys

tein

e (S

a sa

lt)

o C

H,O

H

CO

OH

II

I .

I I

HO

-r-O

CH

2T

-CH

CO

NH

(CH

2h

CO

NH

CH

CH

2S

H

OH

C

H,

C12

H2,

N20

9PS

MW

: 41

6·42

Pan

teth

eine

C

H30

H

HOCHi-~HCONH(CH2hCONH(CH2hSH

I CH

3

C1H

22N

204S

M

W:

278·

37

Pan

teth

ine

( C

H3

0H

)

HO

CH

2?-

JHC

ON

H(C

H2

hC

ON

H(C

H2

hS

CH

3 2

C22

H42

N40

5S2

MW

: 55

4·72

4' -P

hosp

hopa

ntet

hein

e (S

a sa

lt)

o C

H30

H

II I

. I

HO

-P-O

CH

2C-C

HC

ON

H(C

H2h

CO

NH

(CH

2hS

H

I I

OH

C

H3

CII

H23

N20

7PS

MW

: 35

8·35

Solu

ble

in w

ater

, m

etha

nol,

mod

erat

ely

solu

ble

in e

than

ol;

inso

lubl

e in

et

her.

Uns

tabl

e to

aci

ds a

nd b

ases

. [ll

']D -

14°

(c =

2).

Oht

a et

al.

(196

7).

Solu

ble

in w

ater

; sl

ight

ly s

olub

le in

alc

ohol

. U

nsta

ble

to a

cids

and

bas

es.

Eas

ily o

xidi

zed

in a

ir.

[ll'

]~ 0

° (c

= 2

). B

addi

ley

& M

athi

as (

1954

);

Nag

ase

(196

7).

Syru

p or

gla

ss.

Solu

ble

in w

ater

; sl

ight

ly s

olub

le in

alc

ohol

, in

solu

ble

in

ethe

r, b

enze

ne, c

hlor

ofor

m,

ethy

l ac

etat

e. U

nsta

ble

to a

cids

and

ba

ses.

Eas

ily o

xidi

zed

in a

ir.

[ll'

]~ +

12·2

° (c

= 3

·45)

. La

ctob

acill

us

bulg

aric

us f

acto

r. S

him

izu

et a

l. (1

965)

.

Dis

ulfi

de f

orm

of

pant

ethe

ine.

Gla

ssy,

col

orle

ss t

o lig

ht y

ello

w

subs

tanc

e. U

nsta

ble

to a

cids

. [l

l']~

+ 17

·1°

(c =

3·2

). F

or s

olub

ilit

y an

d re

f.,

see

pant

ethe

ine.

Solu

ble

in w

ater

; sl

ight

ly s

olub

le in

eth

anol

; in

solu

ble

in e

ther

. U

nsta

ble

to a

cids

and

bas

es.

Eas

ily

oxid

ized

in a

ir.

[ll'

]~ +

13·3

° (c

= 2

·25)

; O

xidi

zed

form

, [l

l']~

+ 12

·2°.

Ace

toba

cter

sub

oxid

ans

fact

or.

Bad

dile

y &

Tha

in (

1953

); M

offa

tt &

Kho

rana

(19

61);

Nag

ase

(196

7).

(con

tinu

ed)

Dep

hosp

ho-c

oenz

yme

A

(Li

salt

)

o C

H,O

H

" I

. I

HO

-P-O

CH

2C-C

HC

ON

H(C

H2h

CO

NH

(CH

2hS

H

I I

9 C

H,

i:N

H2

HO

-f=

0

<N

I

N

~tJ N

J

OH

O

H

C21

H'5

N70

12P2

S M

W:

687·

56

Ta

ble

.l-(

con

td.)

Sol

uble

in w

ater

, m

etha

nol;

ins

olub

le i

n ac

eton

e. U

nsta

ble

to a

cids

and

ba

ses.

For

ref

., se

e co

enzy

me

A.

Coe

nzym

e A

o C

H,O

H

II I

. I

HO

-i-O

CH

'r-C

HC

ON

H(C

H2

hC

ON

H(C

H2

hS

H

o C

H1

NH

I

. .

2

HO

-P=

O

I O-C

H2 o

OH

I

HO-P~

HO/~

~IH3

6N70

16P,

S M

W:

767·

55

4' -P

hosp

hopa

ntet

hein

e-S

-sul

fona

te (

Ca

salt

)

o C

H,O

H

II I· I

H

OrO

CH

2r-

CH

CO

NH

CH

2C

H2

CO

NH

CH

2C

H2

SS

0,H

OH

C

H,

CII

HnO

lON

2S2P

M

W:

438·

41

Sol

uble

in

wat

er;

inso

lubl

e in

eth

anol

, et

her,

ace

tone

. D

ecom

pose

d to

pa

nthe

thei

ne-2

' ,4'

-cyc

lic

phos

phat

e an

d 3

' ,5

'-A

DP

in 1

N N

aOH

(1

00°C

, 2

min

). D

ecom

pose

d to

pan

teth

eine

-4'-

phos

phat

e an

d ad

enin

e in

1 N

HC

I (1

()()O

C, 5

min

). E

asil

y ox

idiz

ed in

air

. M

offa

tt &

Kho

rana

(1

961)

; S

him

izu

(197

0);

Shi

miz

u et

al.

(197

9a).

Sol

uble

in

wat

er. [a

]~ +

7·2°

(c =

1·9

5).

Bifi

dus

fact

or.

Yos

hiok

a &

T

amur

a (1

971)

.

(con

tinu

ed)

4' -O

-(!3

-glu

copy

rano

syl)

-D-p

anto

then

ic a

cid

f{CH

2 0H

lH

3?H

o O

CH

2-C

-CH

CO

NH

CH

2CH

2CO

OH

O

H

I H

O

CH

2

OH

C44

H2S

014N

M

W:

791·

68

OA

-612

9A (

Na

salt

)

Tab

le l

-co

ntd

.

Sol

uble

in w

ater

. [c

:r]ii

-18

· 20

(c =

1·0)

. G

row

th f

acto

r of

Leu

cono

stoc

. A

mac

hi e

t at.

(197

1).

Uns

tabl

e to

aci

ds a

nd b

ases

. [c

:r]ii

+ 1

1.60

(c

= 1·

0).

Ant

ibio

tic

prod

uced

by

Str

epto

myc

es s

p. O

A-6

129.

Yos

hiok

a et

al.

(198

3).

I I'

}x£

OH

C

H1

;/

SC

H2

CH

2N

HC

OC

H2

CH

2N

HC

OC

H-r

-CH

20

H

CH

3 C

OO

H

.

C2o

H31

N10

7S

MW

: 45

7·54

Vitamin Bs, Coenzyme A and Related Compounds 205

! • 4 '-~tetheine • , I : ' pantetheine • ' : :--pantothenic aci.d---\ I : :-pantoic acid ~ I ! :: : : i

~H <j>H CH.C;>H 0=6-o-~-oCH'-{:-CHCo-NHCH2CH2CO-NHCH2CH2SH

C~HZ~ --~-~~--f- i-S-al.anine-J,..cysteamineJ, :---S-aletheine I

c;> H .~

Ho-~ ~~~ i NHz_L

Fig. 1. The structure of coenzyme A.

from the air. It breaks down into fJ-alanine and pantoic acid by alkaline hydrolysis. The latter forms a lactone, i.e. D-( - )-pantoyl lactone, readily in acid solution or upon heating. Acid hydrolysis of pantothenic acid gives fJ-alanine and pantoyl lactone. Pantothenic acid is soluble in water, ethyl acetate, dioxane, acetic acid, ether and amylalcohol, but is insoluble in benzene and chloroform.

The structure of pantothenic acid contains a single asymmetric center, so that it is optically active; only the natural D-( + )-isomer has vitamin activity. The absolute configuration of the vitamin has been defined as R (Hill & Chan, 1970). The conformation of the vitamin has also been reported (Fritz & LOwe, 1962).

The calcium salt of pantothenic acid can be obtained as needle crystals from methanol. It is moderately hygroscopic and is rather more stable to heat, air and light than the free acid. It is soluble in water and glycerol and slightly soluble in alcohol and acetone. Reviews by Baddiley (1955) and Wagner & Folkers (1964) summarize early studies on the chemistry of pantothenic acid.

Table 1 lists several naturally-occurring derivatives of pantothenic acid. They can be grouped into three types based on their chemical structures, i.e. simple pantothenate derivatives, pantetheine derivatives in which cysteamine (or its analogs) attaches by an amide linkage and coenzyme A derivatives in which the pantetheine is adenosylylated (see Fig. 1).

Pantothenyl alcohol, an alcohol analog of pantothenic acid, is also a pharmaceutically important unnatural derivative. Unnatural analogs of pan­tothenic acid and coenzyme A were reviewed by Shimizu (1970).

3 BIOSYNTHESIS

Although pantothenic acid cannot be synthesized by animals, micro-organisms and plants are generally able to produce it from the precursors pantoic acid

206 S. Shimizu & H. Yamada

CH,COCOOH 7 ~jH ~ CH"'r-COOH

CH,COCOOH CH,-c=O pyruvic acid a-aoetolactic acid

malony lsemialdehyde

yH,OH HOCH,c-CHCONHCH,cH,aJOH

tlU D-pantothenic acid

\? H,NCNHCH,CH.a>OH N-carl:>am:>yl-Il-alanine

- 13

dihydrouracil ..L uracil

Fig. 2. The pathway for the biosynthesis of pantothenic acid.

and p-alanine, through catalysis of the enzyme pantothenate synthetase (EC 6.3.2.1). Animals, plants and micro-organisms can convert pantothenic acid to 4' -phosphopantetheine and coenzyme A, the metabolically active forms of the vitamin. The pathway for the biosynthesis of pantothenic acid and coenzyme A has been elucidated by several workers since the early 1950s and has been reviewed (Brown & Reynolds, 1963; Plaut et ai., 1974; Abiko, 1975; Brown & Williamson, 1982). The pathway leading to the vitamin and the coenzymes from common precursors can be summarized as shown in Figs 2 and 3.

3.1 /I-Alanine

Three routes to p-alanine have been reported. Several micro-organisms have been reported to form p-alanine by a--decarboxylation of L-aspartic acid (Fig. 2, reaction 1). Confirmatory evidence for this conversion was provided by Williamson & Brown (1979), who purified (to apparent homogeneity) from extracts of Escherichia coli an enzyme that catalyzes the a--decarboxylation of L-aspartic acid to yield p-alanine and CO2 • They also reported that the enzyme is missing in a mutant of E. coli that requires either p-alanine or pantothenate as a nutritional factor, but is present in the wild-type strain and in a revertant strain of the mutant. It has also been suggested that p-alanine is produced by decarboxylation of N-carbamoyl-p-alanine formed from uracil on the basis of the observation that mutants of Salmonella typhimurium lacking the ability to degrade uracil require N-carbamoyl-p-alanine, p-alanine or pantothenate as a nutritional factor (Fig. 2, reactions 2, 3 and 4) (West et al., 1985). p-Alanine may also be produced by transamination of malonylsemialdehyde produced from propionic acid (Fig. 2, reaction 5 or 6), because enzyme activity catalyzing this conversion was detected in several micro-organisms. However, there have been no further studies concerning this reaction.

Vitamin B5 , Coenzyme A and Related Compounds 207

3.2 Pantoic Acid

The route to pantoic acid from pyruvate as shown in Fig. 2 has been elucidated mainly in E. coli and Neurospora crassa. Two enzymes catalyzing the conversion of pyruvate to a-ketoisovalerate (Fig. 2, reactions 7 and 8) in this route are shared by the route for the biosynthesis of the branched chain amino acids. In E. coli, two enzyme activities have been detected for the conversion of a-ketoisovalerate to ketopantoic acid (Fig. 2, reaction 9); one is dependent on tetrahydrofolate and the other is not. The physiological significance of the tetrahydrofolate-independent activity seemed to be ques­tionable because of its high Km values for formaldehyde (10 mM) and a-ketoisovalerate (100 mM). The fact that a mutant mlssmg the tetrahydrofolate-dependent activity requires pantothenate for growth, whereas the tetrahydrofolate-independent activity is found in the same amounts in the same mutant also give concrete evidence supporting the theory that the tetrahydrofolate-dependent enzyme is the one responsible for the ketopantoate needed for the biosynthesis of pantothenate. The tetrahydrofolate-dependent enzyme, i.e. ketopantoate hydroxymethyltransferase (EC 2.1.2.11), has been purified and characterized in some detail. The observation that pantoate, pantothenate and coenzyme A are all allosteric inhibitors of this enzyme also supports this conclusion (Powers & Snell, 1976; Teller et al., 1976).

The reduction of ketopantoic acid to D-pantoic acid (Fig. 2, reaction 10) is catalyzed by an NADPH-dependent enzyme, ketopanoic acid reductase (EC 1.1.1.169). The enzyme activity has been detected in Saccharomyces cerevisiae and E. coli. The same reduction is also catalyzed by a-acetohydroxyacid isomeroreductase (EC 1.1.1.86) which is the responsible enzyme for the conversion of a-acetolactate to a-ketoisovalerate (Fig. 2, reaction 8) (Primer­ano & Burns, 1983). Recently, Shimizu et al. (1988a) isolated ketopantoic acid reductase in a crystalline form from Pseudomonas maltophilia and charac­terized it in some detail. They also demonstrated that this enzyme is due the enzyme for D-pantoic acid formation, necessary for the biosynthesis of pantothenic acid, due to the observation that mutants lacking this enzyme require either D-pantoic acid or pantothenate for growth and the revertants regain this activity.

3.3 Coenzyme A

The pathway for the biosynthesis of coenzyme A from pantothenic acid, L-cysteine and A TP as shown in Fig. 3 was first demonstrated by Brown (1959a,b) based on his studies with Proteus morganii and the early observa­tions in the 1950s with pig liver and other organisms. Later, the validity of this pathway was confirmed by Abiko and co-workers who separated and charac­terized the enzymes involved in this pathway in rat liver (Abiko, 1975).

The first step in this pathway is the phosphorylation of pantothenic acid (Fig. 3, reaction 1) by pantothenate kinase (EC 2.7.1.33). The enzyme has been purified and characterized from rat liver (Abiko et al., 1972) and

208 S. Shimizu & H. Yamada

AlP ~ Pantothenic acid r---" X I : P-Pontothenic acid Pantothenoylcysteine

: AlP_J2 +Cysteine .~: :lSi P-Pantothenoylcysteine ~,

.!;! ~ -g: P-Pantetheine ,QI L 1!: AlP» " If'

: Dephospho-CoA I

6l Pantetheine

~lP +-

: AlP_ L5 I I !... __________________ CoA _________ 1

Fig. 3. The pathway for the biosynthesis of coenzyme A from pantothenic acid, L-cysteine and ATP. For chemical structures of each compound, see Table l.

Abbreviation used: CoA, coenzyme A.

Brevibacterium ammoniagenes (Shimizu et al., 1973). Both enzymes receive allosteric inhibition by coenzyme A, the end product of the pathway. Since such feedback inhibition by coenzyme A is observed only at this step and no other regulation mechanism has been known, this inhibition seems to be the most important mechanism for controlling the cellular level of coenzyme A. Pantetheine is also phosphorylated by the same enzyme to yield 4'­phosphopantetheine (Fig. 3, reaction 7), which can be converted to coenzyme A.

The condensation of 4' -phosphopantothenic acid with L-cysteine to yield 4' -phosphopantothenoyl-L-cysteine (Fig. 3, reaction 2) is catalyzed by 4'­phosphopantothenoyl-L-cysteine synthetase (EC 6.3.2.5). The mammalian enzyme requires A TP as energy for the condensation whereas the bacterial enzyme preferably utilizes CTP (Brown, 1959a). 4'-Phosphopantothenoyl-L­cysteine is then decarboxylated to yield 4' -phosphopantetheine (Fig. 3, reaction 3) by 4' -phosphopantothenoyl-L-cysteine decarboxylase (EC 4.1.1.36). The enzyme has been shown to be independent of pyridoxal 5'-phosphate. This step is the only one which does not require A TP among the five steps in the biosynthesis of this coenzyme.

The pyrophosphate linkage formation between 4' -phosphopantetheine and ATP to yield 3' -dephospho-coenzyme A (Fig. 3, reaction 4) and the phosphorylation of it (Fig. 3, reaction 5) are respectively catalyzed by dephospho-coenzyme A pyrophosphorylase (EC 2.7.7.3) and dephospho­coenzyme A kinase (EC 2.7.1.24). In rat liver, both enzymes are present as a complex or a bifunctional enzyme (Suzuki et al., 1967). The reversibility of the former reaction may be important for controlling cellular levels of coenzyme A and 4' -phosphopantetheine.

The presence of an alternate route to yield 4' -phosphopantetheine via pantetheine (Fig. 3, reactions 6 and 7) has been suggested in Acetobacter

Vitamin B5 • Coenzyme A and Related Compounds 209

suboxydans, Lactobacillus helveticus and others (Brown, 1959b), but confir­matory evidence for this is not available.

3.4 Control Mechanisms for the Biosynthesis

The allosteric inhibition of ketopantoic acid hydroxymethyltransferase of E. coli by o-pantoic acid, pantothenic acid or coenzyme A may be involved as a control mechanism in pantothenate biosynthesis (Powers & Snell, 1976). On the other hand, such inhibition was not observed in the case of ketopantoic acid reductase of P. maltophilia (Shimizu et al., 1988a).

In the pathway to coenzyme A from pantothenic acid, the feedback inhibition of pantothenate kinase by coenzyme A and 4'-phosphopantetheine has been demonstrated to be involved as a control mechanism in the biosynthesis (Abiko et al., 1972; Shimizu et al., 1973; Vallari et at., 1987). Since this inhibition was generally observed regardless of species and the other four steps following this reaction are not inhibited by coenzyme A or 4' -phosphopantetheine significantly, this may be one of the most important mechanisms to control cellular levels of coenzyme A. No other mechanism such as repression has been observed in either pantothenate or coenzyme A biosynthesis.

Pantetheinase, which specifically degrades pantetheine to pantothenic acid and cysteamine, may also be an important enzyme, because coenzyme A can be degraded to pantetheine enzymatically and pantetheine can be reused as a precursor of coenzyme A after phosphorylation by pantothenate kinase. Cellular coenzyme A levels may be influenced by competition between pantetheinase and pantothenate kinase towards their substrate, pantetheine (Wittwer et al., 1983).

4 CHEMICAL AND MICROBIAL PRODUCTION METHODS

4.1 Pantothenic Add

At present, commercial production of pantothenate depends exclusively on chemical synthesis. As outlined in Fig. 4, the conventional chemical process involves reactions yielding racemic pantoyl lactone from isobutyraldehyde, formaldehyde and cyanide, optical resolution of the racemic pantoyl lactone to 0-( - )-pantoyl lactone with quinine, quinidine, cinchonidine, brucine and so on and condensation of 0-( - )-pantoyllactone with p-alanine. This is followed by isolation as the calcium salt and drying to obtain the final product. A problem of this chemical process apart from the use of poisonous cyanide is the troublesome resolution of the racemic pantoyl lactone and the reracemization of the remaining L-( + )-isomer. Therefore, most of the recent studies in this area have been concentrated on development of an efficient method to obtain 0-( - )-pantoyllactone.

210 S. Shimizu & H. Yamada

Optical ________

resolution

~

Fig. 4. Outline of the chemical synthesis of D-pantothenic acid.

To skip this resolution-reracemization step, several microbial or enzymatic methods have been proposed. They roughly fall into two types based on the starting substrate used (Yamada & Shimizu, 1988).

Recently, an efficient combined chemi-enzymatic method, which involves an efficient one-pot synthesis of ketopantoyl lactone as a starting substrate, followed by stereospecific reduction of it to 0-( - )-pantoyl lactone using microbial cells as a catalyst was reported (Hata et al., 1987; Shimizu et al., 1984a, 1987a). As shown in Fig. 5, ketopantoyl lactone is synthesized from isobutyraldehyde, sodium methoxide, diethyl oxalate and formalin. The reaction is performed in one step at room temperature with a yield of 81·0%. Stereospecific reduction of ketopantoyl lactone to 0-( - )-pantoyl lactone is carried out with washed cells of Rhodotorula minuta or Candia parapsilosis as a catalyst and glucose as energy for the reduction. About 50 or 90 g/liter of optically pure 0-( - )-pantoyl lactone can be produced with a molar yield of nearly 100% by R. minuta or C. parapsilosis, respectively. The enzyme catalyzing this conversion has been isolated as a crystalline form from C. parapsilosis cells and characterized. It is a novel carbonyl reductase which specifically catalyzes the reduction of conjugated polyketone compounds (Hata et al.,1989a,b; Shimizu et al., 1988b).

Racemic pantoyllactone can also be used as a starting substrate. Shimizu et ale (1987b) reported that Nocardia asteroides cells specifically oxidize the L-( + )-isomer in a racemic mixture of pantoyllactone to ketopantoyllactone, which is then converted to 0-( + )-pantoyl lactone by the reduction with C. parapsilosis cells as described above. In these two enzymatic steps, the coexisting 0-( - )-isomer remains without any modification (Fig. 6, reactions 1 and 2). Under suitable conditions, 72 g/liter of 0-( - )-pantoyl lactone was obtained from 80 g/liter of oL-pantoyl lactone. Similar specific oxidation and reduction reactions can also be carried out with a single micro-organism as catalyst. On incubation with washed cells of Rhodococcus erythropolis,

Vitamin Bs, Coenzyme A and Related Compounds

Me Me

MeONa ~ ~CHg~-~~CHO ~TOOMe 4MeOH C;::OOEt I COOMe MeONa COOEt Me MeONii

2 x )CHCHO

T'K". [:~CHCH-~~CHO 1 Me )CHCOCOOH +

Me

Me ~COCOOMe

~CLHLCHO Meuet5Me 0 ~ j 'v, HCOOMe + MeOH HCHO

'CH 0 ,.fe 0

Me Me OH

211

Me -t---\°o ___________ • Me7---\-H ~ -alanine ~~/ Microorganism • ~_?O • (R)-(+)-pantothenic acid

o 0

Fig. S. The reaction pathway for the chemicoenzymatic synthesis of 0-( - )-pantoyl lactone.

D-( - )-pantoyl lactone in the reaction mixture reached 18·2 g/liter with a molar yield of 90·5% (optical purity, 94·4% e.e.). This unique conversion proceeds through the successive reactions as follows: (1) the enzymatic oxidation of L-( + )-pantoyl lactone to ketopantoyl lactone (the same enzyme as that in N. asteroides has been suggested to be the responsible enzyme for this oxidation); (2) the rapid and spontaneous hydrolysis of ketopantoyl lactone to ketopantoic acid, and (3) the enzymatic reduction of the ketopantoic acid to D-pantoic acid. The enzyme catalyzing this reduction seemed to be ketopantoic acid reductase, because R. erythropolis cells could not utilize

OH carbonyl OH u: (1) 00 reductase eoH

L-PL DH (D-PL forming) I

carbonyl (2)

° reductase 0 ° L-PL (L-PL forming} J(PL D-PL I f

Spontaneous Chemical (3)t<PH 7) (HC1)1 (5)

OH Ho J(PA reductase MOH ,

OH OH (4) OHOH

KPA D-PA

Fig. 6. Reactions involved in the enzymatic transformation to 0-( - )-pantoyl lactone. Abbreviations used: L-PL, L-( + )-pantoyllactone; D-PL, 0-( - )-pantoyllactone; KPL,

ketopantoyllactone; KPA, ketopantoic acid; D-PA, o-pantoic acid.

212 S. Shimizu & H. Yamada

ketopantoyl lactone as substrate, different from C. parapsilosis (see Fig. 6, reactions 1, 3 and 4) (Shimizu et at., 1987c).

These three enzymatic methods are simple and require no reracemization step, which is necessary for the conventional chemical resolution. A chemical reduction of ketopantoyl lactone which uses rhodium complex as a catalyst has also been reported to yield 0-( - )-pantoyllactone stereospecifically (Ojima et at., 1978).

4.2 Coenzyme A

The production methods for coenzyme A roughly fall into chemical and microbial ones. The chemical methods have been reviewed by Shimizu (1970) and Mautner (1970). They are not practical due to their complexity. Therefore, commercial production is carried out by microbiological methods. Extraction of coenzyme A from yeast cells has been performed since the early 1950s. Usually, cells of baker's or brewer's yeasts, which are relatively rich in coenzyme A, are used as the source. Later, an efficient enzymatic method using Brevibacterium ammoniagenes cells as the catalyst was developed. These microbial methods were reviewed by Shimizu & Yamada (1986).

A successful enzymatic method using the biosynthetic route of coenzyme A from pantothenic acid, L-cysteine and ATP was first reported by Ogata et al. (1970), who found that B. ammoniagenes has all five enzymes necessary for the biosynthesis of coenzyme A in high activities. The above three substrates, when added to a reaction mixture containing the bacterial cells, were converted to coenzyme A with a satisfactory yield (2-3 g/liter). They also found that the same organism can accumulate coenzyme A directly in the culture medium on addition of pantothenic acid, L-cysteine and AMP, adenosine or adenine in the presence of a surfactant, cetylpyridinium chloride, and high levels of glucose (usually 10%), K2HP04 and MgS04 ·7H20. Under optimal conditions, the amount obtained was 5·5 g/liter. Most coenzyme A in the medium was present in the disulfide form due to the vigorous shaking during the reaction. After treatment of the culture filtrate with Duolite S-30, charcoal and Dowex 1 (Cl-), and the reduction of the disulfide, the very pure thiol form was obtained in a high yield. The mechanism of this coenzyme A production has been suggested to be that shown in Fig. 7 (for details, see Shimizu et at., 1979a,b).

In order to improve the product yield further, the regulation mechanism of the biosynthesis was investigated. As described in the previous section, the biosynthesis of coenzyme A in B. ammoniagenes is mainly controlled by the feedback inhibition of pantothenate kinase by coenzyme A. This was the main problem in the practical production, because the over-produced coenzyme A itself stopped the biosynthesis. Two methods to abolish this feedback inhibition have been reported.

A synthetic scheme in which the reaction is initiated by the condensation of 4' -phosphopantothenic acid and L-cysteine or the transadenosylylation of

Villlmin Bs, Coenzyme A and Related Compounds 213

G~( "1TP >kids.~

~Ar4dhenicacid

PRPP AMP Cysteine

~ne Aderiine CoA

Fig. 7. Reaction sequences of coenzyme A production with Brevibacterium am­moniagenes under A TP-generating conditions. Abbreviations used: PRPP, 5-

phosphoribosyl-l-pyrophosphate; CoA, coenzyme A.

4' -phosphopantetheine was investigated, because these routes do not involve phosphorylation of pantothenic acid or pantetheine by pantothenate kinase. Replacement of the enzymatic phosphorylation of pantothenate or pantetheine with chemical phosphorylation followed by the enzymatic reaction increased the yield of coenzyme A 10-20 times. Yields from 4' -phosphopantothenic acid and 4'-phosphopantetheine were 33 g/liter (molar yield based on ATP, 64·5%) and 115 g/liter (100%), respectively (Shimizu et a/., 1983). This method is applicable to coenzyme A production under the A TP-generating conditions (see Fig. 7). 4'-Phosphopantothenic acid (25 g/liter), L-cysteine (15 g/liter), and AMP (33 g/liter) added to the culture broth of B. ammoniagenes were converted to coenzyme A with a yield of 23 g/liter (Shimizu & Yamada, 1984).

Another way to improve the yield is to use mutants derepressed for the feedback inhibition or those showing elevated pantothenate kinase activity. A mutant of B. ammoniagenes that is resistant to oxypantetheine (the cor-

Fig. 8. Time course of coenzyme A production by an oxypantetheine-resistant mutant of Brevibacterium ammoniagenes under ATP-generating conditions. (A) Production from pantothenic acid, L-cysteine and AMP. (B) Production from pantetheine and AMP. For details, see Shimizu et al. (1984b). 0, Coenzyme A (CoA); ., pantothenic

acid (PaA); D, ATP; .; ADP; D., AMP; .6, pH; x, cell growth.

214 S. Shimizu & H. Yamada

responding oxygen analog of pantetheine) was found to have an elevated activity of pantothenate kinase. Under the ATP-generating conditions, the yields of coenzyme A from pantothenic acid (3-6 g/liter), L-cysteine (1-8 g/liter) and AMP (6 g/liter) or from pantetheine (5 g/liter) and AMP (6g/liter) were 9·3 or U·5g/liter, respectively. These values were about three-fold higher than those obtained with the parent strain, and 70-100% of the added AMP was converted to coenzyme A (Shimizu etal., 1984b) (Fig. 8).

Continuous production of coenzyme A through five enzymatic steps using gel-entrapped cells of B. ammoniagenes has also been reported (Shimizu et al., 1979b; Yamada et al., 1980).

Continuous production of coenzyme A through five enzymatic steps using gel-entrapped cells of B. ammoniagenes has also been reported (Shimizu et al., 1979b; Yamada et al., 1980).

4.3 4'-Phosphopantetheine and Other Intermediates in Coenzyme A Biosynthesis

4' -Phosphopantetheine together with other intermediates in coenzyme A biosynthesis can be effectively synthesized by using B. ammoniagenes cells as the catalyst and by modifying the reaction conditions (Shimizu et al., 1979a): 4' -phosphopantothenic acid on omission of L-cysteine from the reaction mixture, 4'-phosphopantetheine on addition of CTP or CTP and GTP in place of A TP, because pantothenic acid is phosphorylated in the presence of CTP or GTP as well as A TP and the condensation of 4' -phosphopantetheine with nucleoside triphosphate is specific for A TP, and 3' -dephospho-coenzyme A on treatment of the reaction mixture containing coenzyme A with 3'-nucleotidase. The amounts of these intermediates obtained by this method are summarized in Table 2.

5 ASSAY METHODS

Test micro-organisms normally used for the microbiological assay of pan­tothenic acid are Lactobacillus plantarum ATCC 8014 (L. arabinosus 17-5), L. casei ATCC 7469 and Saccharomyces uvarum ATCC 9080 (S. carlsbergensis). Lactobacillus plantarum is suitable for determining unconjugated pantothenate in samples. It should be noted that pantetheine, when simultaneously present in a molar ratio to pantothenate of more than O· 5, gives positive errors in the determination. Saccharomyces uvarum also shows almost specific growth response to free pantothenate, but fJ-alanine stimulates its growth. Hence, an assay procedure employing this organism is also the one chosen for determin­ing the pantothenic acid that occurs in natural products together with other pantothenate forms. Lactobacillus casei responds not only to pantothenate but also several conjugated forms of pantothenate. Lactobacillus helveticus ATCC

Tab

le 2

P

rodu

ctio

n o

f th

e In

term

edia

tes

in C

oenz

yme

A B

iosy

nthe

sis

by B

revi

bact

eriu

m a

mm

onill

gene

s·

Pro

duct

Su

bstr

ates

N

ucle

otid

es

Rea

ctio

ns

Dri

ed

Pro

duct

ivity

(m

g/m

l)

invo

lved

b ce

lls

enzy

me

sour

ce

Cul

ture

Im

mob

ilize

d br

othC

ce

lls

4' -P

hosp

hopa

ntot

heni

c ac

id

Pan

toth

enic

aci

d A

TP

1

3-4

1·5-

2·5

4' -P

hosp

hopa

ntot

heni

c ac

id

Pan

toth

enic

aci

d A

MP

1

4-5

4' -P

hosp

hopa

ntet

hein

e 4'

-Pho

spho

pant

o-C

fP

2,3

3-4

1·8

then

ic a

cid

and

L-c

yste

ine

4' -P

hosp

hopa

ntet

hein

e P

anto

then

ic a

cid

ITP

and

CfP

1,

2,3

2-3

0·3

and

L-c

yste

ine

4' -P

hosp

hopa

ntet

hein

e P

anto

then

ic a

cid

GM

Pan

dC

MP

1,

2,3

3-4

and

L-c

yste

ine

4' -P

hosp

hopa

ntet

hein

e P

ante

thei

ne

ITP

7

2-3

0·9

4' -P

hosp

hopa

ntet

hein

e P

ante

thei

ne

GM

P

7 4-

5 3'

-Dep

hosp

hoco

enzy

me

A

Pan

toth

enic

aci

d A

TP

1,

2,3,

4,5

1-2

and

L-c

yste

ine

a F

or

deta

ils,

see

Shi

miz

u et

al.,

197

9a,b

. b

Num

bers

cor

resp

ond

to t

hose

giv

en i

n F

ig.

3.

C In

this

cas

e, t

he n

ucle

osid

e m

onop

hosp

hate

s ad

ded

are

pres

umed

to

be

conv

erte

d to

the

cor

resp

ondi

ng n

ucle

osid

e tr

ipho

spha

tes

and

used

for

th

e re

acti

ons

in a

sim

ilar

man

ner

as s

how

n in

Fig

. 7.

216 S. Shimizu & H. Yamada

12046 and L. bulgaricus Bl are recommended for determination of pan­tetheine (or pantethine), because both these organisms require more than 100 times as much pantothenic acid as pantethine to give the same response. Treatment of samples and details of assay procedures have been described (Bird & Thompson, 1967).

A sensitive enzymatic assay method using pantothenase has been reported (Airas, 1986), but the enzyme is not commercially available. Chemical and physical methods have also been reported. They are often used in determining pantothenic acid in pharmaceutical products. They are not suitable for determination of natural samples because of their low sensitivity.

6 USE AND ECONOMIC ASPECTS

The current world capacity of calcium pantothenate production and its demand are presumed to be about 4000 and 3600-4000 tons/year, respectively. It is mainly used as an additive of animal feed (about 3000 tons/year) and as a pharmaceutical product (about 600 tons/year). Pantothenyl alcohol is used as a source of panthothenate activity for pharmaceutical vitamin products. Pan­tothenyl alcohol itself has no pantothenate activity; in fact, it is a competitive growth inhibitor of several pantothenate-requiring lactic acid bacteria. How­ever, it has been demonstrated to be quantitatively converted to pantothenic acid in the animal body, and to be equivalent to pantothenic acid in man.

Pantethine, the disulfide of pantetheine, and coenzyme A are also used as pharmaceutical products in several countries. They have been suggested to be effective in reducing cholesterol level, curing fatty liver, and related diseases.

Some sulfonate derivatives of pantetheine or coenzyme A (Bifidus factors), such as 4'-phosphopantetheine-S-sulfonate, which were originally isolated from carrot roots have been shown to be growth factors of Bifidobacterium (Yoshioka & Tamura, 1971). Addition of the bifidus factors to dried milk for infants has been suggested to be useful in improving the quality of the milk. A carbapenem antibiotic, OA-6129A, (Table 1) produced by Streptomyces sp. OA-6129, may be an interesting example suggesting a new use of the vitamin as building block for its synthesis (Yoshioka et al., 1983).

ACKNOWLEDGEMENT

Research from the authors laboratory was supported, in part, by a Grant in aid of Scietific Research from the Ministry of Education, Science and Culture of Japan.

Vitamin Bs, Coenzyme A and Related Compounds 217

REFERENCES

Abiko, Y. (1975). Metabolism of coenzyme A. In Metabolic Pathways, 3rd edn, Vol. 7, Metabolism of Sulfur Compounds, ed. D. M. Greenberg. Academic Press, New York, pp. 1-25.

Abiko, Y., Ashida, S. & Shimizu, M. (1972). Purification and properties of D­pantothenate kinase from rat liver. Biochim. biophys. Acta, 268,364-72.

Airas, R. K. (1986). Pantothenase-based assay of pantothenic acid. Meth. Enzymol., 122,33-5.

Amachi, T., Iwamoto, S. & Yoshizumi, H. (1971). A growth factor for malo-lactic fermentation bacteria. Part II. Structure and synthesis of a novel pantothenic acid derivative isolated from tomato juice. Agric. Bioi. Chem., 35, 1222-30.

Baddiley, J. (1955). The structure of coenzyme A. Adv. Enzymol. 16, 1-22. Baddiley, J. & Mathias, A. P. (1954). Coenzyme A. Part IX. The synthesis of

pantothenoylcysteine, its 4'-phosphate, and related compounds as possible precur­sors of the coenzyme. J. Chem. Soc., 2803-12.

Baddiley, J. & Thain, E. M. (1953). Coenzyme A. Part VIII. The synthesis of pantetheine 4'-phosphate (Acetobactor stimulatory factor), a degradation product of the coenzyme. J. Chem. Soc., 1610-15.

Bird, O. D. & Thompson, R. Q. (1967). Pantothenic acid. In The Vitamins, Chemistry, Physiology, Pathology, Methods, 2nd edn, Vol. VII, ed. P. Gyorgy & W. N. Pearson. Academic Press, New York, pp. 209-41.

Brown, G. M. (1959a). The metabolism of pantothenic acid. J. Bioi. Chem., 234, 370-78.

Brown, G. M. (1959b). Assay and distribution of bound form of pantothenic acid. J. Bioi. Chem., 234, 379-82.

Brown, G. M. & Reynolds, J. J. (1963). Biogenesis of the water-soluble vitamins. Ann. Rev. Biochem., 32,419-62.

Brown, G. M. & Williamson, J. M. (1982). Biosynthesis of riboflavin, folic acid, thiamine, and pantothenic acid. Adv. Enzymol., 53,345-81.

Fritz, H. & LOwe, W. (1962). Konformationsbestimmung der Pantothensaure und des Pantoylamides durch Protonenresonanz-spektroskopie. Angew. Chem., 74,751-3.

Hata, H., Shimizu, S. & Yamada, H. (1987). Enzymatic production of D-( - )-pantoyl lactone from ketopantoyl lactone. Agric. Bioi. Chem., 51, 3011-16.

Hata, H., Shimizu, S., Hattori, S. & Yamada, H. (1989a). Ketopantoyl lactone reductase from Candida parapsilosis, Purification and characterization as a conjug­ated polyketone reductase. Biochim. Biophys. Acta, 990, 175-81.

Hata, H., Shimizu, S., Hattori, S., & Yamada, H. (1989b). Ketopantoyl lactone reductase is a conjugated polyketone reductase. FEMS Microbiol. Lett., 58, 87-90.

Hill, R. K. & Chan, T. H. (1970). The absolute configuration of pantothenic acid. Biochem. Biophys. Res. Commun., 38, 181-3.

King, T. E. & Strong, F. M. (1951). Synthesis and properties of pantothenic acid monophosphates. J. Bioi. Chem. 191, 515-21.

Kleinkauf, H. & Dohren, H. von (1983). Non-ribosomal peptide formation on multifunctional proteins. Trends. Biochem. Sci. 8,281-3.

Lipmann, F. (1953). Symposium on chemistry and function of coenzyme A. Fedn. Proc., n,673-715.

Mautner, H. G. (1970). Synthesis of coenzyme A analogs, Meth. Enzymol., 18A, 338-50.

Moffatt, J. G. & Khorana, H. G. (1961). Nucleoside polyphosphates. XII. The total synthesis of coenzyme A. J. Am. Chem. Soc., 83, 663-75.

Nagase, O. (1967). Investigations on pantothenic acid and its related compounds. IV.

218 S. Shimizu & H. Yamada

Chemical studies. (3). Syntheses of D-pantetheine 4'-phosphate and N-D­pantothenoyl-L-cysteine 4'-phosphate. Chem. Pharm. Bull., 15,648-54.

Ogata, K., Shimizu, S. & Tani, Y. (1970). A new preparation method of coenzyme A with micro-organisms. Agric. Bioi. Chem., 34, 1757-9.

Ohta, G., Nagase, 0., Hosokawa, Y., Tagawa, H. & Shimizu, M. (1967). Investiga­tions on pantothenic acid and its related compounds. III. Chemical studies. (2). Synthesis of di-D-pantothenoyl-L-cystine. Chem. Pharm. Bull., 15,644-7.

Ojima, I., Kogure, T., Terasaki, T. & Achiwa, K. (1978). Effective biomimetic route to D-( + )-pantothenic acid using asymmetric hydrogenation catalyzed by a chiral rhodium complex in the key step. I. Org. Chem., 43,3444-6.

Okada, S., Nagase, O. & Shimizu, M. (1967). Investigations on pantothenic acid and its related compounds. VII. Chemical studies. (5). Synthesis of D-pantothenic acid 4'-phosphate. Chem. Pharm. Bull., 15,713-5.

Plaut, G. W., Smith, C. M. & Alworth, W. L. (1974). Biosynthesis of water-soluble vitamins. Ann. Rev. Biochem., 43, 899-922.

Powers, S. G . & Snell, E. E. (1976). Ketopantoate hydroxymethyltransferase, II. Physical, catalytic, and regulatory properties. I. Bioi. Chem. 251, 3786-93.

Primerano, D. A. & Burns, R. O. (1983). Role of acetohydroxy acid isomeroreductase in biosynthesis of pantothenic acid in Salmonella typhimurium. I. Bacteriol., 153, 259-69.

Shimizu, M. (1970). Pantothenic acid and coenzyme A: Preparation of CoA analogs. Meth. Enzymol., 18A, 322-8.

Shimizu, S. & Yamada, H. (1984). Enzymatic process for the high yield production of coenzyme A. In Third European Congress on Biotechnology, Vol. I. Verlag Chemie, Weinheim, pp. 401-5.

Shimizu, S. & Yamada, H. (1986). Coenzymes. In "Biotechnology", Vol. 4, ed. H.-J. Rehm & G. Reed. VCH Verlagsgesellschaft, Weinheim, pp. 159-84.

Shimizu, M., Ohta, G., Nagase, 0., Okada, S. & Hosokawa, Y. (1965). Investigations on pantothenic acid and its related compounds. I. Chemical studies. (1). A novel synthesis of pantethine. Chem. Pharm. Bull., 13, 180-8.

Shimizu, S., Kubo, K., Tani, Y. & Ogata, K. (1973). Purification and properties of pantothenate kinase from Brevibacterium ammoniagenes IFO 12071. Agric. Bioi. Chem., 37,2863-70.

Shimizu, S., Tani, Y. & Ogata, K. (1979a). Synthesis of coenzyme A and its biosynthetic intermediates by microbial processes. Meth. Enzymol., 62, 236-45.

Shimizu, S., Tani, Y. & Yamada, H. (1979b). Synthesis of coenzyme A by immobilized bacterial cells. In Immobilized Microbial Cells, ed. K. Venkatsubramanian. American Chemical Society, Washington, DC, pp. 87-100.

Shimizu, S., Komaki, R., Tani, Y. & Yamada, H. (1983). A high yield method for the preparative synthesis of coenzyme A by combination of chemical and enzymic reactions. FEBS Lett., 151, 303-6.

Shimizu, S., Hata, H. & Yamada, H. (1984a). Reduction of ketopantoyl lactone to D-( - )-pantoyl lactone by micro-organisms. Agric. Bioi. Chem., 48, 2285-9l.

Shimizu, S., Esumi, A., Komaki, R. & Yamada, H. (1984b). Production of coenzyme A by a mutant of Brevibacterium ammoniagenes resistant to oxypantetheine. Appl. Environ. Microbiol., 48, 1118-22.

Shimizu, S., Yamada, H., Hata, H., Morishita, T., Akutsu, S. & Kawamura, M. (1987a). Novel chemoenzymatic synthesis of 0-( - )-pantoyl lactone. Agric. Bioi. Chem., 51, 289-90.

Shimizu, S., Hattori, S., Hata, H. & Yamada, H. (1987b). Stereoselective enzymatic oxidation and reduction system for the production of D-( - )-pantoyl lactone from a racemic mixture of pantoyllactone. Enzyme Microb. Technol., 9,411-16.

Shimizu, S., Hattori, S., Hata, H. & Yamada, H. (1987c). One-step microbial

Vitamin B5 , Coenzyme A and Reloted Compounds 219

conversion of a racemic mixture of pantoyllactone to optically active D-( - )-pantoyl lactone. Appl. Environ. Microbiol., 53, 519-22.

Shimizu, S., Kataoka, M., Chung, M. C. M. & Yamada, H. (1988a). Ketopantoic acid reductase of Pseudomonas maltophilia 845, Purification, characterization and role in pantothenate biosynthesis. J. BioI. Chem., 263, 12077-84.

Shimizu, S., Hattori, S., Bata, H. & Yamada, H. (1988b). A novel fungal enzyme, NADPH-dependent carbonyl reductase, showing high specificity to conjugated polyketones, purification and characterization. Eur. J. Biochem. 174, 37-44.

Stiller, E. T., Harris, S. T., Finkelstein, J., Keresztesy, J. C. & Folkers, K. (1940). Pantothenic acid. VIII. The total synthesis of pure pantothenic acid. J. Am. Chem. Soc., 62, 1785-90.

Suzuki, T., Abiko, Y. & Shimizu, M. (1967). Investigation of pantothenic acid and its related compounds, XII. Biochemical studies (7). Dephospho-CoA pyrophos­phorylase and dephospho-CoA kinase as a possible bifunctional enzyme complex. J. Biochem., 62, 642-9.

Teller, J. H., Powers, S. G. & Snell, E. E. (1976). Ketopantoate hydroxymethyltrans­ferase, I. Purification and role in pantothenate biosynthesis. J. BioI. Chem., 251, 3780-85.

Vagelos, P. R. (1973). Acyl group transfer (Acyl carrier protein). In The Enzymes, 3rd edn, Vol. 8, ed. P. D. Boyer. Academic Press, New York, pp. 155-99.

Vallari, D. S., Jackowski, S. & Rock, C. O. (1987). Regulation of pantothenate kinase by coenzyme A and its thioesters. J. BioI. Chem., 262, 2468-71.

Vandamme, E. J. (1981). Properties, biogenesis and fermentation of the cyclic decapeptide antibiotic, Gramicidin S. In Topics in Enzyme and Fermentation Biotechnology, Vol. 5, ed. A. Wiseman. Horwood-Wiley. New York, London, pp. 185-20l.

Wagner, A. F. & Folkers, K. (1964). Pantothenic acid and coenzyme A. In Vitamins and Coenzymes. Interscience Publishers, New York, pp. 93-137.

West, T. P., Traut, T. W., Shanley, M. S. & O'Donovan, G. A. (1985). A Salmonella typhimurium strain defective in uracil catabolism and fJ-alanine synthesis. J. Gen. Microbiol., 131, 1083-90.

Williams, R. J. (1943). The chemistry and biochemistry of pantothenic acid. Adv. Enzymol., 3, 253-87.

Williamson, J. M. & Brown, J. M. (1979). Purification and properties of L-aspartate-Il'­decarboxylase, an enzyme that catalyzes the formation of fJ-alanine in Escherichia coli. J. Bioi. Chem., 254, 8074-82.

Wittwer, C. T., Burkhard, D., Ririe, K., Rasmussent, R., Brown, J., Wyse, B. W. & Hansen, R. G. (1983). Purification and properties of a pantetheine-hydrolyzing enzyme from pig kidney. J. Bioi. Chem., 258, 9733-8.

Yamada, H. & Shimizu, S. (1988). Microbial and enzymatic processes for the production of biologically and chemically useful compounds. Angew. Chem. Intern. Eng. Ed., 27,622-42.

Yamada, H., Shimizu, S. & Tani, Y. (1980). Synthesis of coenzymes by immobilized cell systems. In Enzyme Engineering, Vol. 5, ed. H. H. Weetall and G. Royer. Plenum Publishing, New York, pp. 405-1l.

Yoshioka, M. & Tamura, Z. (1971). Bifidus factors in carrot. II. The structure of the factor in fraction IV. Chem. Pharm. Bull., 19, 178-85.

Yoshioka, T., Kojima, I., Isshiki, K., Watanabe, A., Shimauchi, Y., Okabe, M. & Fukagawa, Y. (1983). Structure of OA-6129A, Bh B2 and C, new carbapenem antibiotics produced by Streptomyces sp. OA-6129. J. Antibiot., 36, 1473-82.

Chapter 13

MICROBIAL PRODUCTION OF VITAMIN Ba AND DERIVATIVES

Y. TANI

Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

1 INTRODUCTION

In 1934, vitamin B6 (or pyridoxine) was discovered by Gyorgy as a rat pellagra preventive factor. Soon after the determination of its chemical nature the total synthesis of the vitamin was accomplished. Moreover, clinical investigations have clarified that the vitamin has therapeutic and prophylactic uses for many diseases. Since microbiological assays pointed towards several forms of the vitamin B6-active compound the enzymatic conversion of various forms of vitamin B6 into pyridoxal 5 '-phosphate (pyridoxal-P), the active form of the vitamin in vivo, has been intensively investigated. Pyridoxine, pyridoxamine, pyridoxal and their 5 I -phosphate esters are now recognized as principal vitamin B6 compounds (Fig. 1).

Since Gunsalus & Bellamy first reported in 1944 that pyridoxal-P is the coenzyme of L-tyrosine decarboxylase, it has become evident that pyridoxal-P or pyridoxamine 5' -phosphate (pyridoxamine-P) is a coenzyme for various enzyme systems associated with the metabolism of amino acids, amines, saccharides, fatty acids, etc. A large number of vitamin B6 enzymes has now been found, and the mechanisms of vitamin B6 dependent reactions have been extensively investigated.

Several biotechnological methods for synthesis of vitamin B6 compounds have been investigated and are described in this chapter. However, the industrial production is still limited to chemical synthesis processes for pyridoxine and pyridoxal-Po

2 CHEMISTRY AND ASSAY OF VITAMIN B6

Pyridoxine (3-hydroxy-4,5-dihydroxymethyl-2-methylpyridine) exhibits the properties of a stable hydroxylated weak nitrogen base. Hydrolytic agents such

221

222 Y. Tani

R J

HO CH20R2 ,9'4 5

~ 1 61 /""

CH3 N

Pyridoxine: R\ = CH20H, R2 = H Pyridoxal: R\ = CHO, R2 = H Pyridoxamine: R\ = CH2NH2, R2 = H Pyridoxine 5'-phosphate: R\ = CH20H, R2 = P03H2 Pyridoxal 5'-phosphate: Rl = CHO, Rz = P03HZ Pyridoxamine 5'-phosphate: Rl = CHzNHz, Rz = P03H2

Fig. 1. Chemical structure of vitamin B6 •

as mineral acids or aqueous alkali, hot or cold, do not affect the vitamin. With ferric chloride, pyridoxine reacts as a phenolic substance giving a reddish brown coloration. In alkaline solution, pyridoxine on treatment with 2,6-dichloroquinone chlorimide gives an immediate blue color fading to reddish­brown.

Pyridoxine hydrochloride occurs as white platelets, melting point 204-206°C with decomposition. The free base melts at 160°C. The compound is optically inactive. Both base and hydrochloride readily sublime without decomposition. The hydrochloride is freely soluble in water but sparingly in alcohol and acetone. The base is soluble in methanol. Rapid destruction of pyridoxine by light occurs in neutral and alkaline solutions. In 0·1 M hydrochloric acid there is very little destruction.

The tautomeric properties of pyridoxine are well illustrated by the changes in its UV absorption produced by varying the hydrogen ion concentration. The single maximum at 292·5 nm at pH 2 diminishes in intensity at pH 4·5, and concomitantly a new maximum appears at 327·5 nm. This latter band increases in intensity when the pH is changed to 6·75, and the 292·5 nm maximum disappears but a new band appears at 256·0 nm. When the pH is further raised to 10·2, both bands increase in intensity and shift to shorter wavelengths.

The existence of other forms of pyridoxine was recognized as a result of the comparison of microbiological assays on extracts of natural materials with the values based on chemical and animal assays.

When pyridoxine was treated with mild-oxidizing agents, a marked increase in l?io-activity towards the micro-organism, Lactobacillus casei, was observed. Autoclaving of pyridoxine in the presence of the assay medium or amino acids, greatly increased the activity of pyridoxine towards the test organism, Streptococcus faecalis R. The products formed by treating pyridoxine with animating agents and mild-oxidizing agents were, respectively, the amino and aldehyde derivatives of pyridoxine. The compounds are pyridoxamine (2-methyl-3-hydroxy-4-aminomethyl-5-hydroxymethylpyridine) and pyridoxal (2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine) .

The availability of pyridoxal in synthetic form permitted identification of

Microbial Production of Vitamin B6 223

pyridoxal-P, the coenzyme discovered as an essential cofactor for the en­zymatic decarboxylation of amino acids. Pyridoxamine-P was subsequently discovered as a naturally occurring compound by virtue of its differential activity in promoting growth of certain lactic acid bacteria. Finally, pyridoxine 5'-phosphate (pyridoxine-P) (although it plays no known essential role in metabolism) can be formed from pyridoxine by tissue enzymes and further transformed to pyridoxal-P by other tissue enzymes. It appears therefore to also be a naturally occurring compound. Direct analysis of many tissue extracts also indicates its natural occurrence.

A number of microbiological procedures are established for the estimation of vitamin B6. The method with Saccharomyces cerevisiae (S. carlsbergensis, S. uvarum) is widely used for the assay of total vitamin B6 in non-esterified form. The phosphates of vitamin B6 should be converted to the free form by acid or enzymatic hydrolysis before applying the bioassay. A differential assay employs Lactobacillus casei which responds to pyridoxal and Streptococcus faecalis R which responds to pyridoxal and pyridoxamine. Chemical reactions with 2,6-dichloroquinone chlorimide and diazotized p-aminoacetophenone are less sensitive and non-specific but are useful for the detection on paper chromatograms and paper electrophoregrams. Recent progress in high perfor­mance liquid chromatography is applied for quantitative and simultaneous detection of all vitamin B6 compounds by determining UV absorption and fluorescence.

3 BIOSYNTHESIS AND METABOLISM OF VITAMIN B6

The current knowledge of the biosynthesis and metabolism of vitamin B6 is summarized in Fig. 2.

Although the de novo biosynthesis of vitamin B6 has been investigated since the 1940s using micro-organisms, the complete biosynthetic pathway still remains obscure. Clear evidence of a direct precursor in the vitamin B6 biosynthesis was demonstrated for the first time by Tani & Dempsey (1973). In a vitamin B6 requiring WG3 strain mutant derived from Escherichia coli B, the vitamin B6 requirement could be satisfied by glycolaldehyde, and 14C_ glycolaldehyde was incorporated into vitamin B6 without significant dilution. Subsequently, the biosynthetic route of glycolaldehyde in E. coli (Tani et al., 1977) and the incorporated position of glycolaldehyde in vitamin B6 (Hill et al., 1975) were elucidated. Based on these results, a hypothetical sequence of vitamin B6 biosynthesis, is presented in Fig. 3 in which the condensation of glycolaldehyde with a 'C4 compound' forming a 'C6 compound' such as a,p-dihydroxy-p-hydroxymethyl valerate, as well as the following introduction of a 'C3 compound' are involved. Another route, in which all carbon atoms of pyridoxine are derived from trioses, is also proposed (Vella et aI., 1980).

As to the biodegradation of vitamin B6, research was performed with

224 Y. Tani

Glycolate

1 D-Glutamate 2-Ketoglutarate

. • \. .J. P 'd . P Pyridoxamine-P • Pyndoxal-P • yn oxme-Glycolaldehyde

'-. '-. A ~~i~hOOPhat" ATPt~o'Pha". A ~~i~PhO'Pha", """ Pyridoxine • ~ Pyridoxal ~ • Pyridoxamine

Pyridoxine glUCOSi1 II I L-Alani[)yrUvate

Pyridoxine glucuronide ~ Isopyridoxal 4-Pyridoxic acid lactone

1 1 5-Pyridoxic acid lactone

1 5-Pyridoxic acid

1 a-Hydroxymethyl-a' -(N-acetylamino­

methylene )succinate

1 a-Hydroxymethylsuccinate semi al­

dehyde + acetate + NH3 + cO2

4-Pyridoxic acid

1 2-Methyl-3-hydroxy-5-formyl­pyridine-4-carboxylate

1 2-Methyl-3-hydroxypyridine-4,5-dicarboxylate

1 2-Methyl-3-hydroxypyridine-5-carboxylate

1 a-(N -acetylaminomethylene)-succinate

1 Succinate semialdehyde + acetate +NH3 +C02

Fig. 2. Metabolism of vitamin B6 •

pseudomonads, to which pyridoxine and pyridoxamine were fed as carbon and/or nitrogen source (Nyns et al., 1969).

The biosynthesis of pyridoxal-P from pyridoxine is considered to occur through pyridoxine-+ pyridoxine-P-+ pyridoxal-P rather than pyridoxine-+ pyridoxal-+ pyridoxal-P, based on experimental results so far obtained.

Pyridoxine is transformed into pyridoxal by an oxidase or a dehydrogenase. Pyridoxine oxidase activity was found in an enzyme preparation extracted from rabbit liver, and the oxidase reaction of pyridoxine to pyridoxal proceeded only in the presence of a coupling reaction with an aldehyde oxidase which oxidized pyridoxal to 4-pyridoxic acid. Pyridoxine dehydrogenase requires NADP as a coenzyme. Both oxidase and dehydrogenase reactions are

CH3 I

Microbial Production of Vitamin B6

CH2 I C=O CH3

I I

~~~;;~~'::ed) H}r-'-CH'OH C, COrU~d /C"""

HO COOH *CHO

I **CH20H

.. a, f;I-dihydroxy f;I-hydroxymethyl valerate"

Glycolaldehyde

Fig. 3. Hypothetical biosynthetic sequence of pyridoxine.

225

reversible, though the reaction equilibria favour the formation of pyridoxine from pyridoxal.

The phosphorylation of free vitamin B6 is catalyzed by pyridoxal kinase, which phosphorylates pyridoxine and pyridoxamine as well as pyridoxal in the presence of A TP to form the corresponding phosphate esters. The enzyme activity occurs widely in mammalian tissues, bacteria, molds, and yeasts.

Another phosphotransferring reaction using various organic phosphorus compounds was found in many micro-organisms such as molds, yeasts, and bacteria, and found to be catalyzed by the phosphotransferring ability of phosphatase (Tani & Ogata, 1972).

Pyridoxine-P oxidase catalyzes the oxidative deamination of pyridoxamine-P as well as the oxidation of pyridoxine-Po The coenzyme of the enzyme is FMN. The enzyme activity is widely present in bacteria and it is strongly inhibited by compounds phosphorylated at the 5-position of the pyridine ring, but weakly inhibited by compounds having an aldehyde residue at the 4-position of the pyridine ring (Yamamoto et al., 1965). The fact that the enzyme is strongly inhibited by pyridoxal-P (the reaction product) might be attributed to metabolic regulation of the in vivo biosynthesis of pyridoxal-P. It seems likely that pyridoxine-P or pyridoxamine-P stored in cells is oxidized to pyridoxal-P when necessary.

226 Y. Tani

Anaerobic bacteria, clostridia, lack the pyridoxine-P oxidase activity but have the pyridoxamine-P-2-ketoglutarate transaminase activity which is absent in aerobic bacteria. The transaminase purified from cell-free extract of Clostridium kainantoi uses 2-ketoglutarate as the amino group acceptor and D-glutamic acid as the amino group donor (Tani et al., 1972b).

4 PRODUCTION OF VITAMIN B6 BY DE NOVO SYNTHESIS AND BIOCONVERSION

4.1 Production of Vitamin 8 6 by Fermentation

Extensive screening for vitamin B6 producers among micro-organisms has been pedormed. However, the productivity was quite insufficient to establish an industrial microbiological process of vitamin B6 production. Almost all micro-organisms synthesize vitamin B6 intracellularly 0·05-0·3 mg per g of dry cells, and excrete it in amounts of 0·05-0·5 mg per liter of culture medium. Table 1 is a list of relatively high producers. The extracellular vitamin B6 is the free form.

The biosynthesis of vitamin B6 in E. coli is known to be repressed by a feedback control system. However, in a high producer, Flavobacterium sp. 238-7, the amount of vitamin B6 excreted was increased even when cultivated with the addition of each form of vitamin B6 (Tani et al., 1972a). An enzyme which reduces glycolate to glycolaldehyde as the first step of the biosynthesis of vitamin B6 is present in the particulate fraction of Flavobacterium sp. 238~7. The activity and synthesis of the enzyme was not affected by added vitamin B6 (Tani et al., 1984). From these results, Flavobacterium sp. 238-7 can be considered to lack this feedback control system for the vitamin B6 biosynthesis, resulting in the formation of large amounts of vitamin B6 •

A typical time course of vitamin B6 production by Flavobacterium sp. 238-7 in a glycerol-peptone medium is shown in Fig. 4.

Table 1 Extracellular Production of Vitamin B6 by Micro-organisms

Micro-organisms

Bacteria: Klebsiella sp. Achromobacter cycloclastes Flavobacterium sp. 238-7 Bacillus subtilis

Yeasts: Candida albicans (n-paraffin-grown) Saccharomyces marxianus Pichia guilliermondii (n-paraffin-grown)

Vitamin B6 produced (mg/liter)

2·0-4·0 3·0-4·0

20·0 2·0-5·0

0·6 2·0

25·0

Microbial Production of Vitamin B6 227

! 0 -4' ""-.... H

9 3

12 0.6 '" ~ ... 01

~ ,., .... iii ...

""- .... 00 II)

~ <I .. 8 0.4

"tI "tI .... ~ 01

u 0 .... .... ... ... '" ~ I ,q

<I 4 0.2 .c .... ... ~ :-

0 ... " .... '-' I>

0

Cultivation time (h)

Fig. 4. Production of vitamin B6 by Flavobacterium sp. 238-7.

4.2 Production of Pyridoxal-P from Pyridoxine-P and Pyridoxine

As pyridoxine and pyridoxine-P can be chemically synthesized with ease, the industrial production of pyridoxal-P from pyridoxine and pyridoxine-P by pyridoxine-P oxidase catalysis with or without enzymatic phosphorylation is considered to be worthy of study.

Pyridoxine-P is oxidized by a flavoprotein, pyridoxine-P oxidase. It was found that amino acids accelerate the enzyme reaction due to relief of the aldehyde inhibition by pyridoxal-P of the enzyme activity, in which these amino acids combined non-enzymatically with an aldehyde residue of pyridoxal-P to form a Schiff base. In view of applying this enzyme reaction for industrial production of pyridoxal-P, a suitable industrial medium for Alcaligenes faecalis was constructed with corn steep liquor, ammonium sulfate and glycerol. The cells obtained were autolyzed by addition of toluene or ethyl acetate and used as the enzyme source. The reaction proceeded adequately on addition of aspartic acid or glutamic acid, which are relatively cheap, and resulted in a complete conversion of pyridoxine-P to pyridoxal-P.

An acid phosphatase of Escherichia freundii Kl can phosphorylate pyri­doxine as the most effective phosphoryl acceptor among free vitamin B6 using various kinds of organic phosphates as the phosphoryl donor. As E. freundii had pyridoxine-P oxidase and pyridoxine dehydrogenase activities together with the phosphotransferase activity, a considerable amount of pyridoxal-P was formed as well as pyridoxine-P in the phosphotransferring reaction of

228 Y. Tan;

pyridoxine using cells or cell-free extract of the organism (Tani et al., 1969). In particular, an appreciable amount of pyridoxal-P was formed by use of phenylphosphate as the phosphoryl donor under alkaline conditions. Under the optimal reaction conditions, 0·4 g of pyridoxal-P was formed from 2 g of pyridoxine incubated with phenylphosphate and cells for 24 h.

4.3 Production of Pyridoxal from Pyridoxine

Oxidation of pyridoxine to form pyridoxal is catalyzed by pyridoxine de­hydrogenase. A biochemical process for production of pyridoxal from pyrido­xine was studied with the pyridoxine dehydrogenase of dried cells of baker's yeast (Ogata et al., 1968). The reaction equilibrium of the enzyme lies to the reduction of pyridoxal to pyridoxine. However, a large amount of pyridoxal (pyridoxal-carbonyl complex) was accumulated on addition of various car­bonyl reagents to the reaction mixture. For example, the formed pyridoxal was non-enzymatically converted into pyridoxal-semicarbazone on addition of semicarbazide, and the maximum yield of pyridoxal reached 80% based on the amount of pyridoxine added. Pyridoxal-semicarbazone is highly insoluble in water and can easily be isolated by crystallization from the reaction mixture.

4.4 Production of Vitamin B6 Derivatives

A number of vitamin B6 derivatives such as vitamin B6 fatty acid esters and vitamin B6 amino acid compounds are synthesized chemically. On the other hand, few naturally occurring derivatives have been found: glucuronide of pyridoxine is found in human urine, sulfates (3-position) of pyridoxine or pyridoxal are formed by a homogenate of rat liver, and an indole-pyridoxal complex is found in milk.

It was also found that a new derivative of vitamin B6 was formed in the culture filtrates of some strains belonging to the genera Sarcina and Micrococcus, cultivated in medium containing pyridoxine and sucrose, maltose of phenyl-a-glucoside as carbon source. the derivative isolated from the culture broth consisted of two components. These were separated by Dowex 1 x 2 (borate form) column chromatography and identified as pyridoxine 4'-a-D-glucoside and pyridoxine 5'-a-D-glucoside (Fig. 5) by comparison with chemically synthesized samples (Ogata et al., 1969). Under optimal conditions using Micrococcus sp. No. 431, pyridoxine glucoside, 3·5-3·7 g/liter, was produced in the culture broth of the organism from 2 g of pyridoxine per liter.

The pyridoxine glucoside-synthesizing enzyme was purified and charac­terized to be as a-glucosidase having the ability of transglucosidation. The bioactivity of pyridoxine glucoside using S. cerevisiae as the test organism was about 20% of that of its molar equivalent of pyridoxine, and it increased with incubation time, suggesting the gradual hydrolysis of pyridoxine glucoside into pyridoxine.

Microbial Production of Vitamin B6 229

CH,-O _jp~'OH H°fr~OH I OH

H3C ::"'N

Pyridoxine 4' -a-D-glucoside

Fig. 5. Chemical structure of pyridoxine glucoside.

Pyridoxine glucoside is relatively more stable than pyridoxine toward heat and UV irradiation. Pyridoxine glucoside could be transported into rabbit erythrocytes. The transport of pyridoxine glucoside was about 10-20% of that of its molar equivalent of pyridoxine (Kawai et al., 1972).

REFERENCES

Gunsalus, I. C. & Bellamy, W. D. (1944). A function of pyridoxal. 1. BioI. Chern., 155,357-8.

Hill, R. E., Horsewood, P., Spenser, I. D. & Tani, Y. (1975). Biosynthesis of vitamin B6 • Incorporation of glycolaldehyde into pyridoxal. 1. Chern. Soc. Perkin, I, 1622-7.

Kawai, F., Yamada, H. & Ogata, K. (1972). Studies on transglycosidation to vitamin B6 by micro-organisms. VII. Transport of pyridoxine glucoside into rabbit erythro­cytes. 1. Vitarninol., 18, 183-8.

Nyns, E. J., Zach, D. & Snell, E. E. (1969). The bacterial oxidation of vitamin B6 •

VIII. Enzymatic breakdown of a-(N-acetylaminomethylene) succinic acid. 1. Bio!. Chern., 244,2601-5.

Ogata, K., Tochikura, T., Kawata, S., Yamamoto, S. & Kawai, H. (1968). Pyridoxal derivative formation from pyridoxine by baker's yeast, Agric. BioI. Chern., 32, 1479-81.

Ogata, K., Uchida, Y., Kurihara, N., Tani, Y. & Tochikura, T. (1969). Studies on transglycosidation to vitamin B6 by micro-organisms. II. Chemical structure of pyridoxine glucoside. 1. Vitarninol., 15, 160-66.

Gyorgy, P. (1934). Vitamin B6 and pellegra-like dermatitis in rats. Nature, 133, 498-9.

Tani, Y. & Dempsey, W. B. (1973). Glycolaldehyde is a precursor of pyridoxal phosphate in Escherichia coli B. 1. Bacterio!., 116,341-5.

230 y. Tani

Tani, Y. & Ogata, K. (1972). Studies on vitamin B6 metabolism in micro-organisms. Part IX. Microbial phosphorylation of vitamin B6 through a new phosphotransfer­ring reaction (5). Phosphorylation of pyridoxine by several phosphatases. Agric. Bioi. Chem., 36, 173-80.

Tani, Y., Kawata, S., Tochikura, T. & Ogata, K. (1969). Studies on vitamin B6 metabolism in micro-organisms. Part VIII. Microbial phosphorylation of vitamin B6 through a new phosphotransferring reaction (4). Formation of pyridoxal 5'­phosphatae from pyridoxine. Agric. BioI. Chem., 33,314-20.

Tani, Y., Nakamatsu, T., Izumi, Y. & Ogata, K. (1972a). Studies on vitamin B6 metabolism in micro-organisms. Part XI. Extracellular formation of vitamin B6 by marine and terrestrial micro-organisms and its control. Agric. Bioi. Chem., 36, 189-97.

Tani, Y., Ukita, M. & Ogata, K. (1972b). Studies on vitamin B6 metabolism in micro-organisms. Part X. Further purification and characterization of pyridoxamine 5'-phosphate-a-ketoglutarate transaminase from Clostridium kainantoi. Agric. BioI. Chem., 36, 181-8.

Tani, Y., Morita, H. & Ogata, K. (1977). Glycolaldehyde synthesizing pathway involved in vitamin B6 biosynthesis in Escherichia coli B. Agric. BioI. Chem., 41, 1749-54.

Tani, Y., Nishise, H., Morita, H. & Yamada, H. (1984). Vitamin B6 biosynthesis and glycolate reductase in Flavobacterium sp. 238-7. J. Nutr. Sci. Vitaminol., 30, 415-20.

Vella, G. J., Hill, R. E., Mootoo, B. S. & Spenser, I. D. (1980). The status of glycolaldehyde in the biosynthesis of vitamin B6 • J. BioI. Chem., 255, 3042-8.

Yamamoto, S., Tochikura, T. & Ogata, K. (1965). Studies on vitamin B6 metabolism in micro-organisms. Part III. Pyridoxine phosphate and pyridoxamine phosphate oxidation (3). Product inhibition and reactivation with amino compounds. Agric. Bioi. Chem., 29,597-604.

Chapter 14

MICROBIAL PRODUCTION OF BIOTIN

Y. IZUMI & H. YAMADA

Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan

1 HISTORICAL

In 1927, Boas described a skin injury produced in rats by feeding raw egg white, as well as the occurrence in various foodstuffs of a 'protective factor X' that prevented and cured this injury (Boas, 1927). Gyorgy et al. (1939) tracked down this protective factor and called it vitamin H after the German Haut for skin. Kogi & Tonnis (1936) isolated a yeast growth factor, biotin vitamin B8 from egg yolk in the form of its crystalline methyl ester and Kogi (1937) determined its empirical formula. Biotin was later shown to be identical to vitamin H, the protective factor X, and to coenzyme R. Melville et al. (1942) determined the correct structure of biotin, which was later confirmed by total chemical synthesis in the Merck laboratories (Harris et al., 1943, 1944a,b, 1945) and verified by X-ray crystallography (Traub, 1956; Trotter & Hamilton, 1966).

Studies on the biosynthesis of biotin started in 1937 with studies on the nutritional requirements of micro-organisms (Mueller, 1937 a, b; du Vigneaud et al., 1942; Eakin & Eakin, 1942; Dittmer & du Vigneaud, 1944; Dittmer & du Vigneaud, 1944; Lilly & Leonian, 1944; Stokes & Gunness, 1945). Subsequently, through radiochemical and genetic-biochemical studies (Tatum, 1945; Pontecorvo, 1953; Ryan, 1956), part of the biosynthetic pathway was proposed. Thereafter, a hypothetical pathway was established by Okumura et al. (1962a,b) based on their investigations of the microbial production of glutamic acid using biotin-requiring bacteria. Ogata et al. (1965a,b) and Iwahara et al. (1966a,b,c) made extensive investigations of biotin biosynthesis and verified the main pathway described by Okumura et al. (1962a,b) using growing and resting cells: pimelic acid - 7 -keto-8-aminopelargonic acid (KAPA)-7,8-diaminopelargonic acid (DAPA)-dethiobiotin (DTB)­biotin (Izumi & Ogata, 1977). Through the extensive studies of three groups, Eisenberg (1973), Pai (1971) and Izumi (Izumi & Ogata, 1977), the enzymic

231

232 Y. Izumi & H. Yamada

system involved in the biosynthetic pathway from pimelic acid to DTB had been established by 1975.

In the 1950s it became clear that biotin was involved as a cofactor in many biochemical processes, especially a number of carboxyl transfer reactions. Investigations on the function of biotin in the reaction mechanisms were obstructed by the fact that biotin is active only when covalently attached to an e-amino group of a lysyl residue of the enzyme (Kosow et al., 1962), as was suggested earlier by the isolation of biocytin (e-N-biotinyllysine) from yeast extract (Wolf et al., 1952).

Wakil et al. (1958) and Wakil & Gibson (1960) showed that highly purified acetyl-CoA carboxylase, isolated from avian liver extracts, was enriched with respect to biotin and sensitive to inhibition by avidin, suggesting the tight binding of the vitamin to the carboxylase and its probable role in acetyl-CoA carboxylation. Lynen et al. (1959) made the interesting observation that the A TP- and Mi+ -dependent carboxylation of free biotin was catalyzed by a bacterial p-methylcrotonyl-CoA carboxylase. This model reaction led to the formation of an unstable carboxybiotin derivative which was identified as 1'-N-carboxybiotin (Knappe et al., 1961; Lynen et al., 1961). Subsequently, it was demonstrated with several biotin enzymes that the site of carboxylation of the enzyme-bound prosthetic group was the same as that of free biotin (Moss & Lane, 1971).

The existence of separate subsites for the catalysis of each partial reaction has been unequivocally demonstrated with acetyl-CoA carboxylase of Escherichia coli (Fall & Vagelos, 1972; Polakis et al., 1974). These investiga­tions have shown that there are three dissimilar subunits involved in catalysis: (a) the biotin carboxyl carrier protein (CCP), (b) the biotin carboxylase (BC), which catalyzes the carboxylation of the CCP biotin, and (c) the carboxyl transferase (Cf), which catalyzes the carboxyl transfer from the CCP biotin to acetyl-CoA. Wood's group (Wood & Barden, 1977) has unequivocally demonstrated with transcarboxylase that each partial reaction is catalyzed by a separate subunit and that a distinct CCP subunit is present.

Major portions of the amino acid sequence of the CCP from transcar­boxylase of Propionibacterium shermanii and acetyl-CoA carboxylase from Escherichia coli have been determined (Wood & Barden, 1977). Up to now, the sequences of the portions around biotin of the CCP or the corresponding domains have been elucidated with almost all the biotin enzymes including chicken liver acetyl-CoA carboxylase (Takai et al., 1987), human pyruvate carboxylase (Lamhonwah et al., 1987) and human propionyl-CoA carboxylase (Lamhonwah et al., 1987). Recently, Takai et al. (1988) have deduced for the first time the complete amino acid sequence of a biotin enzyme, chicken liver acetyl-CoA carboxylase.

2 CHEMICAL AND PHYSICAL PROPERTIES

The structure of biotin is shown in Fig. 1. The naturally occurring biotin, (+ )-cis-hexahydro-2-oxo-1H-thieno-(3,4-d)-imidazole-4-n-valeric acid, is ex-

Microbial Production of Biotin 233

* asymmetric carbon

(a)

Fig. 1. Chemical structure (a) and absolute configuration (b) of ( + )-biotin.

pressed in many ways: d-biotin, d-( + )-biotin, D-biotin and D-( + )-biotin. Here, we describe it as (+ )-biotin, and unless otherwise stated, the term 'biotin' indicates ( + )-biotin. The structure of crystalline biotin has also been investigated by X-ray diffraction techniques and is now known precisely (Traub, 1956; Trotter & Hamilton, 1966; Stallings & de Titta, 1985). X-ray crystallographic analysis of (+ )-biotin and of the carboxybiotin derivative described previously revealed that the bicyclic ring system has a boat-like configuration (Fig. 1(B». The planar ureido ring projects upward at an angle of 62·0° with respect to the plane (plane A) formed by the four carbon atoms of the tetrahydrothiophene ring. Another plane comprising S-1, C-2, and C-5 tilts upward at an angle of 37 ·6° with respect to plane A (Bonnemere et al., 1965). Because of the cis orientation of the ureido ring with respect to the aliphatic side chain, C6 resides approximately 2·8 A from the 3' -N of the ureido ring (Traub, 1956). Repulsion between the 3'-N and C-6 positions is thought to cause the greater-than-anticipated C3-Cz-C6 angle of 119° (Traub, 1956).

There are seven other forms which can be chemically synthesized: (-)­biotin, (+)- and (- )-epibiotin (cis-form); (+)- and (- )-allobiotin, and ( + )- and ( - )-epiallobiotin (trans-form).

The crystals of ( + )-biotin are colorless, fine long needles: m.p. 232-3°C, [{1']22=91° (c=1, 0·1NNaOH). The isoelectric point is pH3·5. The pH of 0·01 % aqueous solution is 4·5. Solubility in water is about 22 mg/100 ml at 25°C and it is more soluble in hot water or in dilute alkali. Solubility in 95% ethanol is about 80 mg/100 ml at 25°C. It is insoluble in other common organic solvents. It is stable in air, in a wide temperature range, and up to about pH 9. It is also stable after autoclaving in 6 N H2S04 at 120°C for 1 h. Accordingly, most of the naturally occurring biotin bound to protein can be hydrolyzed to free forms by such autoclave treatments without any problem.

Biotin combines with avidin, a protein in raw egg white, and streptavidin, a protein produced by an actinomycete to become inactive.

Various chemical reactions with biotin and their products are shown in Fig. 2.

234 Y. Izumi & H. Yamada

HNiNH HNiNH HNjlNH

V(CH,>.CONH(CH2,.b:~00H V(01,l.COOH V(CH2'.COOH

biocytin r b 0'" "0 L,,'-Cu Qlotln sulfoxide biotin sulfone ctIHIle

? ? f "MoO, Q(CH , COOH Jl.. NH2 )l...N (HOI 24 HN~NH ~-<::HCOOOCH, HNt\H , , thienyl·"aleric acid

Q t(CH3J2S04

s CH,l.CO'l'H 5 CH,l.COCI A "MoO.

CHCOOH biotin acid '-- HN NH ~ H2N NH2 (HNO,)

N-flIotlnyi ft chloride sOO--- I--l -- H - HOOC(CH2'.COOH ammo acld/ l.A). COC(, l.A).

CH,N,/ 5 (CH2'.COOH 5 (CH2,.COOH adipic acid Q NH, JL / biotin diaminoblotln

HN)l...NH HN NH ~ H, (Rane, Ni)

Os (t'U \ CONH V(CH \ COOCH jl -"2'. 2 2" 3 HN NH BalOH), H N NH HIO

biotin amide bIotln~ methyl ester H COCI, 2 H 2 ~HOOC(CH2'5COOH N,H, CH3(CH2'5COOH CH3(CH2'5COOH

Q dethiobiotln ~. pime/Ic acid HN)l...NH JL gonic acid H HNO, HN NH

l...S~CH,l.CO::-- V(CH,>.CONHNH2 biotin azide biotin hydrazide

Fig. 2. General chemical reactions of biotin and their products.

3 PRODUCING MICRO-ORGANISMS AND SCREENING

Using about 1000 strains of molds, bacteria and actinomycetes, Ogata et al. (1965a,b) and Iwahara et al. (1966a,b) investigated the accumulation of biotin-vitamers when pimelic acid was added to media as a precursor. They found a promoting effect of pimelic acid in a large number of strains. On addition of pimelic acid to the medium of Bacillus sphaericus IFO 3525, 20-200 Ilg/ml of 'total biotin', which is the amount of biotin-vitamer given by the bioassay using Saccharomyces cerevisiae and includes DTB, KAP A and DAPA as well as biotin, was accumulated. This was several hundred times more than has hitherto been reported in micro-organisms. The main com­ponent of the biotin-vitamers formed from pimelic acid was (+ )-DTB. Afterwards, Yamada et al. (1983) found this strain also produced relatively large amounts (0·8-1·1Ilg/ml) of biotin from pimelic acid, as well as from DTB, under optimized conditions.

Ogino et al. (1974a,b) demonstrated a production method using an n-paraffin-utilizing bacterium from a new compound, oL-cis-tetrahydro-2-oxo-4-n-pentylthieno-(3,4-d)-imidazoline (oL-TOPTI), which is a biotin analog having a methyl group instead of a carboxyl group of the biotin molecule. TOPTI was chemically synthesized from Nt ,N3-dibenzyl-oL-cis-tetra­hydrothieno-(3,4-d)-imidazoline-2,4-dione (compound VII in Fig. 6) via a Grignard reaction with n-pentyl magnesium bromide, dehydration in the

Microbial Production of Biotin 235

presence of an acidic catalyst, catalytic hydrogenation, and debenzylation with concentrated HBr. In this production method, n-paraffin was used as a carbon and energy source for cell growth with concurrent transformation (co-oxidation of TOPTI to biotin). First, n-paraffin-utilizing micro-organisms that co­oxidized TOPTI were selected from natural sources. Three strains, identified as Corynebacterium sp., were the most excellent producers of biotinol and biotin from TOPTI. Therefore the conversion of TOPTI to biotin was assumed to occur via p-oxidation. In a medium containing 2% n-paraffin and 0·2% urea, with the addition of 50 mg DL-TOPTI/100 ml after 24 h of cultivation, a maximum conversion of about 60% (0·32 mg (± )-biotin/ml) was obtained 96 h after the addition of TOPTI. However, selective degradation of (+)­biotin occurred after prolonged incubation, leaving the ( - )-isomer. Thus, to avoid such degradation, mutants that were incapable of assimilating n-paraffin and of degrading biotin, but capable of utilizing acetate, were derived. One of the mutants produced 0·65 mg/ml of ( ± )-biotin from 0·8 mg/ml of DL-TOPTI (80·5% conversion).

4 BIOSYNTHESIS AND BIODEGRADATION

4.1 Biosynthesis

Biotin can be synthesized by a great number of micro-organisms, while some organisms which cannot synthesize biotin require it for growth. Biotin is also known to be synthesized by plants (Watanabe et al., 1982). Almost all the studies on the biosynthesis of biotin have used micro-organisms. The pathway of biotin biosynthesis has been established, as shown in Fig. 3. All the enzymes which are involved in the reaction from pimelic acid to DTB have been elucidated and were found to be novel types. Table 1 summarizes the properties of pimelyl-CoA synthetase, KAPA synthase, DAPA aminotrans­ferase and DTB synthetase (Izumi et al., 1980; Izumi, 1984).

The final step from DTB to biotin through a sulfur introduction has not been enzymically resolved yet. However, there have been some reports on the biosynthesis using growing cells and resting cells. Yamada et al. (1983) showed that resting cells of B. sphaericus IFO 3525 exhibited high activity of biotin synthesis from DTB. Izumi et at. (1973) also showed that resting cells of Rhodotorula glutinis which form appreciable amounts of biotin from DTB, formed biotin from DTB only in the presence of methionine, particularly the L-form. The isotopic experiment revealed that sulfur contained in one molecule of L-methionine was incorporated into one molecule of biotin.

Parry & Kunitani (1976), Guillerm et al. (1977), Parry & Naidu (1980) and Trainor et al. (1980) have developed a stereospecific synthesis of DTB and examined the mechanism of the conversion of DTB to biotin by using specifically labelled 3H-DTB and growing cells of Aspergillus niger and E. coli, respectively. They concluded that the introduction of sulfur at C-1 and C-4

236 Y. Izumi & H. Yamada

Pimelic acid

Pimelyl CoA

7-Keto-B'amino­pelargonic acid

(KAPA)

78-0iamino­pelargonic acid

(OAPA)

Oethiobiotin (OTB)

Biotin

Fig. 3. Biosynthetic pathway of biotin. CD Pimelyl-CoA synthetase, (6) KAPA synthase, ® OAP A aminotransferase, @) OTB synthetase.

positions of DTB takes place with the loss of two hydrogen atoms at

C-1 and C-4 and without the loss of hydrogen atoms at C-2 or C-3. These results suggest that unsaturation does not occur at C-2 or C-3.

Table 2 (Izumi et al., 1981) shows that among the strains of bacteria and yeasts tested for activities of the four biotin biosynthetic enzymes, only B. sphaericus IFO 3525, a DTB producer as described previously, showed significant activities for all four enzymes.

Microbial Production of Biotin

Table 1 Properties of Biotin Biosynthetic Enzymes

(a) Pimelyl-CoA synthetase of Bacillus megaterium

Optimum temperature 32°C Optimum pH 8·0 Km value: pimelic acid 2·7 x 10-4 M

CoA 5·5 x 10-4 M

ATP 1·5 x to-3 M

Mi+ 1·5 X to-3 M Substrate specificity Pimelic acid

Nucleotide requirement Metal ion requirement Inhibitors

(b) KAP A synthase of Bacillus sphaericus

(Other dicarboxylic acids were inert)

ATP, ADP Mi+, Mn2+ EDT A, a, a' -dipyridyl, o-phenanthroline

Optimum temperature 6O"C Optimum pH 7·0 Thermostability (10 min) 0-6O"C (100%) Amino acid specificity L-Alanine Coenzyme requirement PyridoxaI5'-phosphate Inhibitors Phenylhydrazine, semicarbazide,

hydroxylamine,o-cycloserine, oL-penicillamine, isoniazid, o-phenanthroline, citrate, L-cysteine, glycine, o-alanine L-serine, o-,L-histidine

(c) DAP A Aminotransferase of Brevibacterium divaricatum

Molecular weight 24,000 A.max 280, 320, 410 nm Optimum temperature 37°C Optimum pH 8·5 Thermostability (10 min) O-60°C (100%) pH Stability (30 min) 7·0-10·0 (100%) Amino donor specificity S-Adenosyl-L-methionine Amino acceptor specificity KAPA (100)

Coenzyme requirement Km values: KAPA

Inhibitors

S-adenosyl-L-methionine pyridoxal 5' -phosphate pyridoxamine 5' -phosphate

7-Amino-8-ketopelargonate (1) Pyridoxal 5' -phosphate Pyridoxamine 5' -phosphate 0·69 x to-4 M

0·55 x 10-3 M

0·83 X to-6 M 1·2 X to-6 M

Phenylhydrazine, semicarbazide hydroxylamine, o-cycloserine, oL-penicillamine, isoniazid, p-chloromercuribenzoate, HgCI iodoacetate; Co2+, Ass+, KCN

237

(cOlllinued)

238 Y. Izumi & H. Yamada

Table l---contd.

(d) DTB Synthetase of Pseudomonas graveolens Optimum temperature 7·0 Optimum pH 7 ·0-8·0 Thermostability 0-45°C (100%) Substrate specificity DAPA (100)

Nucleotide requirement

Metal ion requirement Km values: DAP A

HC03 -

ATP Mi+

Inhibitors

4.2 Regulation of Biosynthesis

NH2 NH2 ti Biotin diamino-S COOH carboxylic acid (10)

ATP (100), CTP (20), UTP (10) GTP (20), ITP (10) Mg2+ Mn2+ Fe2+ 1 X l(j-4 M '

1 X 1O-2 M

5 X 10-5 M 3 X 10-3 M

EDT A, a, a' -dipyridyl, o-phenan­throline

The biosynthetic regulation mechanism has also been elucidated at the enzyme level. Eisenberg & Star (1968) have reported that KAPA synthase of E. coli is almost completely repressed by the addition of 5 ng/ml of biotin to the medium. Eisenberg & Krell (1969) and Pai (1969) observed similar repression by biotin of DTB synthetase. Izumi & Ogata (1977) found that KAPA synthase of B. sphaericus and DAPA aminotransferase of Brevibacterium divaricatum were repressed by the addition of O·lllg/ml of biotin to the medium. Moreover, they found that, in contrast to the complete repression of DTB synthetase in B. megaterium by O·25Ilg/ml of biotin, pimelyl-CoA synthetase was not repressed by even 1llg/ml of biotin. The biotin synthesiz­ing reaction by resting cells of B. sphaericus was repressed by 1llg/ml of biotin. In this way, a strong repressive action of biotin has been demonstrated on all the enzymes between pimelyl-CoA and DTB and in the biosynthetic step(s) between DTB and biotin. The corepressor has been found to be biotinyl-AMP in E. coli (Prakash & Eisenberg, 1979). The repressor protein (Eisenberg et al., 1982) and biotin operon (Szybalski & Szybalski, 1982) have also been elucidated.

4.3 Biodegradation

Biotin is known to be degraded by molds, yeasts and bacteria via f3-oxidation of the side chain of the molecule (Izumi & Ogata, 1977; Tanaka et aI., 1988). The biotin-degrading bacterium, Mycoplana sp. No. 166, formed bisnorbiotin, a compound having two less carbon atoms in its side chain than biotin, from

Tab

le 2

B

ioti

n-vi

tam

er P

rodu

cing

Abi

liti

esa

and

Bio

tin

Bio

synt

heti

c E

nzym

e A

ctiv

ities

of

Var

ious

Yea

sts

and

Bac

teri

a

Stra

in

True

bio

tinb

Tota

l bi

otin

Sp

ecifi

c ac

tivity

c (u

nits

/mg

prot

ein)

(p.g

/ml)

(p

.g/m

l) 1

2 3

4

Yea

sts

Sacc

haro

myc

es k

loec

keri

anus

IF

O 0

016

trt

0·71

tr

tr

0·

06

0·21

Li

pom

yces

sta

rkey

i IF

O 0

678

0·01

5 0·

02

tr

2·90

tr

0·

07

~

Spor

obol

omyc

es s

alm

onic

olar

IF

O 0

374

0·08

5 0·

49

0·23

1·

30

tr

0·06

~.

C3 Sp

orob

olom

yces

sal

mon

icol

or I

FO

103

8 0·

068

0·30

0·

57

1·80

tr

0·

03

0- s·

Spor

obol

omyc

es c

opro

philu

s IF

O 1

442

0·10

0 0·

16

0·14

0·

31

tr

0·01

-

Rho

doto

rula

glu

tinis

IF

O 0

415

0·06

6 0·

12

tr

0·10

tr

0·

03

~ a

Bac

teri

a t

Esc

heri

chia

col

i AK

U 0

07

0·01

8 0·

40

1-13

2·

45

tr

0·03

'" S·

Kle

bsie

lla p

neum

onia

e IF

O 1

2059

tr

0·

30

tr

0·18

0·

08

0·03

:lI

Ent

erob

acte

r ae

roge

nes

IFO

120

10

0·02

1 tr

0·

18

0·08

tr

0·

02

.l;.

Alc

alig

enes

faec

alis

IF

O 3

160

tr

1·58

0·

21

0·16

tr

0·

06

~

5·

Bac

illus

meg

ater

ium

NI

8100

tr

1·

90

4·55

tr

tr

0·

10

S· B

acill

us r

oseu

s lA

M 1

257

tr

0·87

0·

52

tr

0·15

0·

01

Bac

illus

sph

aeri

cus

IFO

352

5 0·

067

20·0

1·

05

4·42

0·

03

1·24

B

revi

bact

eriu

m d

ivar

icat

um N

RR

L 2

311

tr

tr

tr

tr

1·25

0·

03

Pse

udom

onas

gra

veol

ens

IFO

346

0 tr

4·

30

tr

tr

tr

1·44

a T

he a

mou

nts

of b

ioti

n-vi

tam

ers

prod

uced

by

orga

nism

s gr

own

with

pim

elic

aci

d.

b T

he a

mou

nt o

f bi

otin

-vit

amer

giv

en b

y th

e bi

oass

ay u

sing

Lac

toba

cillu

s pl

anta

rum

, w

hich

inc

lude

s bi

otin

and

bio

tin

sulf

oxid

e.

C 1,

pim

elyl

-CoA

syn

thet

ase;

2,

KA

PA

syn

thet

ase;

3,

DA

PA

am

inot

rans

fera

se;

4, D

TB

syn

thet

ase.

dT

race

.

~

240 Y. Izumi & H. Yamada

biotin, as well as j3-hydroxybisnordethiobotin and tetranordethiobiotin from DTB in a resting cell system (Osakai et ai., 1986a). Biotinyl-CoA synthetase, the first enzyme involved in biotin degradation, was purified to homogeneity from Mycopiana sp. No. 166 (Yamada et ai., 1984; Tanaka et aI., 1986, 1988). The enzyme was a monomer with a molecular weight of 55 000. The enzyme catalyzes the stoichiometric conversion of biotin, ATP and CoA into biotinyl­CoA, AMP and inorganic pyrophosphate. DTB is also effective as a substrate.

5 STRAIN IMPROVEMENT AND GENETICS

As described previously, a strong feedback repression by biotin seems to be one of the main problems that makes microbial production difficult. This control must be overcome in order to microbially produce large quantities of biotin and its vitamers. Some potent biotin antimetabolites have already been found (Fig. 4) (Izumi & Ogata, 1977). Therefore, it has become possible to use these antimetabolites for the selection of regulatory mutants producing biotin and its vitamers.

Actithiazic acid a-Dehydrobiotin

o ~

N N

H CH3 H3C (CH2)3CHCOOH

a-Methyldethiobiotin

5-(2-Thienylhl­valerie acid

o ~

N N ~ CH3 lS).(CH2)3 CHCOOH

a-Methylbiotin

Amic/enomycin

H2NO(CH2hCHCOOH CH3 NHCOCHCHCH2 CH3

NHCH3

Stravidin

Fig. 4. Various biotin antimetabolites.

Microbial Production of Biotin 241

Pai (1975) and Eisenberg et al. (1975) isolated a-dehydrobiotin-resistant mutants of E. coli, which showed enhanced excretion levels of biotin, derepressed levels of the biotin biosynthetic enzymes, and resistance to repression by biotin. However, the level of their accumulation was still quite low (less than 100 ng/mI). Since actithiazic acid, or acidomycin (ACM) (Izumi et al., 1981) and 5-(2-thienyl)-n-valeric acid (TVA) (Izumi et aI., 1978) were found to be biotin antagonists which inhibited the biosynthetic step of biotin from DTB and the DAPA aminotransferase reaction, respectively, Yamada et al. (1983) and Tanaka et al. (1988) induced ACM- and/or TV A-resistant mutants from B. sphaericus IFO 3525. ACM- and TV A-resistant mutant, AB12, excreted 7·1 times more biotin (5·35Ilg/ml) than the wild type strain. The TVA-resistant mutant, A16, excreted 11·6 times more total biotin (400 Ilg/ml). Strain AB12 accumulated 9'5Ilg/ml of biotin under the optimum reaction conditions using 1 mg/ml of pimelic acid. Strain AB12 was considered to have enhanced biotin synthesizing enzyme activity, resulting in survival even in the presence of ACM, whereas the repression of the enzymes by biotin was still not diminished.

Recently, the production of biotin by bacteria which were transformed by recombinant DNA has been reported. Hirono et al. (1986) and Ifuku et al. (1986, 1987) have derived an a-dehydrobiotin-resistant mutant from E. coli. They transformed E. coli with the biotin operon coding for the biotin biosynthetic enzymes. The maximum production of biotin by the transformant was 121lg/ml under the optimized conditions (Hirono et al., 1986). Osawa et al. (1987, 1989) obtained an ACM- and TVA-resistant mutant of B. sphaericus IFO 3525, followed by transforming of E. coli strain with the plasmid carrying under appropriate culture conditions, the transformant excreted 161lg/ml of biotin from DL-DTB in a 48-h cultivated medium (Osawa et al., 1989). They have recently determined the nucleotide sequence of the bio B gene of B. sphaericus and compared the amino acid sequence of its gene product, biotin synthetase, with that of E. coli, suggesting conservation in the catalytic mechanism for both enzymes (Osawa et aI., 1989).

Since the acetate produced in the culture medium was found to inhibit the growth, Ifuku & Yanagi (1988) derived a tluoroacetate-resistant mutant from the E. coli transformant, which had a level of acetate production 10 times lower than the parent strain. When the mutant was cultivated in a glucose-fed system with a controlled dissolved oxygen level in a jar fermentor, the growth and the accumulation of biotin reached 55 mg (dry cell weight)/ml and 105 Ilg/ml, respectively.

6 FERMENTATION

6.1 Dethiobiotin Production by B. sphaericus IFO 3525 (Ogata, 1970)

Medium: 10 g peptone, 5 g Casamino acids (Difco), 100 g soybean meal, 20 g glycerol, 1 g K2HP04, 0·5 g KCI, 0·5 g MgS04·7H20, 10 mg FeS04·7H20,

242 Y. Izumi & H. Yamada

10 mg MnSOc6HzO, 200 Ilg thiamine· HCI and 1 g pimelic acid in 1 liter of tap-water, pH 7·2.

Inoculum and fermentation course: The cells of the bacterium, B. sphaericus, from a slant culture are incubated in 30 ml of the medium in a 300-ml shaking flask for 4-5 days at 28°C on a reciprocal shaker (140 strokes/min).

6.2 Biotin Production by an ACM- and TVA-resistant Mutant ABU of B. sphaericus IFO 3525 (Yamada et al., 1983)

Medium: 20g glycerol, 50g Proteose peptone (Difco), 5g Casamino acids (Difco, vitamin-free), 1 g KHzP04, 0·5 g KCI, 0·5 g MgS04·7HzO, 10 mg FeS04'7HzO, 10 mg MnS04·4-6HzO and 20llg thiamine·HCI in 1 liter of tap-water, pH 7·0.

Inoculum and fermentation course: Cells of bacterium AB12 were inocu­lated into 3 ml of the medium in a test tube and incubated at 28°C with shaking. After 1 day of cultivation, pimelic acid was added (1 mg/ml) to the medium and cultivation was continued at 37°C (optimum temperature for biotin biosynthesis from DTB) for 3 more days. The addition of the precursor after 1 day of cultivation is important for biotin production in order to avoid repression by the produced biotin itself of the biotin biosynthetic enzymes (see Section 4.2).

7 PRODUcr RECOVERY AND PURIFICATION

Biotin and biotin-vitamers accumulated in the culture media are purified by active carbon treatment and ion-exchange column chromatography (Ogata, 1970). After centrifugation of the culture medium, the pH of the supernatant is adjusted to about 2-3 with concentrated HCI-15 g of active carbon is added to 1 liter of the culture medium and stirred mechanically for 4 h at room temperature. The active carbon is collected on a Buchner funnel and washed with about 200 ml of water. The active carbon cake is suspended in about 200 ml of 50% ethanol-28% ammonia water (18: 1, v/v) mixture and stirred for 4 h at room temperature. The active carbon is filtered off onto a Buchner funnel, and the active carbon cake is washed with ethanol-ammonia mixture three to four times. The filtrates are concentrated to dryness in vacuo at room temperature. The concentrate obtained is dissolved in about 40 ml of 90% ethanol, and insoluble materials are removed by centrifugation; the clear supernatant solution is again concentrated to a volume of about 5 ml in vacuo at room temperature. This concentrated solution is subjected to ion-exchange chromatography. A few milliliters of the solution of biotin-vitamers (contain­ing about 20-500 Ilg of total biotin, pH 7·0) is quantitatively taken up onto a Dowex 1 X2-formate column (0·6 cmz x 19 cm). The column is washed with 100 ml of deionized water, which removes the unadsorbed biotin-vitamers such

Microbial Production of Biotin 243

as DAPA, then the biotin-vitamers (e.g., biotin, biotin sulfoxide and DTB) are eluted with 0·012 M formic acid, and lO-ml fractions are collected. Biotin concentrations are quantitatively determined by microbiological assays with Saccharomyces cerevisiae and Lactobacillus plantarum.

8 BIOASSAY METHODS

There are three methods for the determination of biotin and its vitamers: microbiological, chemical and enzymatic assay methods. Table 3 summarizes the characteristics of the three methods. The microbiological assay has been most commonly used among the three methods. This assay uses the micro­organisms Lactobacillus plantarum ATCC 8014 (Scheiner, 1985), Saccharomyces cerevisiae ATCC 7754 (Gyorgy, 1967) and Bacillus subtilis AKU 236 (Iwahara et al., 1966c). In addition, a biotin-requiring mutant of E. coli, C162, (bio B-, His-) has also been used as a test of the growth­promoting activities of biotin-vitamers (Salib et al., 1979; Osakai et al., 1986b). These micro-organisms have biotin-vitamer specifi.cities for growth, which enables us to assay them differentially. Table 4 shows the microbiological activities of various biotin-vitamers when assayed with L. plantarum, S. cerevisiae and B. subtilis. The microbiological assay also has the advantage of high sensitivity. However, one of the main disadvantages of the method is that it requires the time-consuming cultivation of assay organisms and the laborious preparation of washed cell suspensions. Tanaka et al. (1987a) have developed improved assay methods for biotin using lyophilized cells of L. plantarum and E. coli C162 and glycerol-suspended cells of S. cerevisiae. Figure 5 shows the standard curves by the paper disk plate method and turbidimetric method using such cells. Those lyophilized or glycerol-suspended cells, which are preserved at -20°C, can be used for the assay for more than 1 year or half a year, respectively.

9 BIOLOGICAL PROPERTIES

9.1 Biotin Deficiencies in Animals

Feeding a diet either low in biotin or, more frequently, using a diet amended with with raw egg white, produced biotin deficiencies in the rat, chicken, poultry, pig, monkey, man and other species (Gyorgy & Larger, 1968). Chicken and poultry, however, require an outside source of biotin, even when egg white is replaced by other proteins. The effectiveness of egg white in producing biotin deficiency is due to the presence of the glycoprotein avidin, which forms a complex with biotin that renders the biotin unavailable to the host. Avidin also forms tight complexes when biotin is the prosthetic group of an enzyme, resulting in the loss of enzyme activity. The biotin-binding capacity is present not only in the

Met

hod

Mic

robi

olog

ical

Che

mic

al

DA

CA

m

etho

d

Avi

din­

HA

BA

m

etho

d

Tab

le 3

A

ssay

Met

hods

of

Bio

tin

and

Its

Rel

ated

Com

poun

ds

Pri

ncip

le

Gro

wth

of

L.

plan

taru

m,

S. c

erev

isia

e, B

. su

btili

s an

d E.

col

i

Col

orin

g re

acti

on o

f the

ure

ido

ring

w

ith

p-di

met

hylc

inna

mal

dehy

de

(DA

CA

) un

der

anhy

drou

s co

ndit

ions

(re

ddis

h or

ange

to

pink

A. m

ax =

533

nm

)

Avi

din

is r

eact

ed w

ith

the

dye,

4-

hydr

oxya

zobe

nzen

e-2'

-car

boxy

lic

acid

(H

AB

A)

to f

orm

the

avi

din­

HA

BA

com

plex

(A. m

ax =

500

om),

fo

llow

ed b

y th

e ad

diti

on o

f bio

tin.

Si

nce

the

HA

BA

in t

he c

ompl

ex is

su

bsti

tute

d by

bio

tin,

the

add

ed

biot

in is

ass

ayed

fro

m t

he

colo

rim

etri

cal d

eter

min

atio

n o

f the

re

mai

ning

avi

din-

HA

BA

com

plex

.

Cha

ract

eris

tics

1. H

igh

sens

itivi

ty

2.

Hig

h sp

ecif

icity

to

var

ious

bio

tin­

vita

mer

s 3.

Eve

n cr

ude

sam

ples

can

be

ass

ayed

. 4.

Tim

e-co

nsum

ing

(6-2

7 h)

1. U

reid

o ri

ng-c

onta

inin

g bi

otin

-rel

ated

com

poun

ds

with

no

mic

robi

olog

ical

ac

tivity

can

be

assa

yed.

2.

F

ast a

ssay

3.

S

ampl

e sh

ould

be

drie

d be

fore

ass

ay

1. A

vidi

n-co

mbi

nabl

e co

mpo

unds

(u

reid

o ri

ng c

ompo

unds

) ca

n be

ass

ayed

. 2.

F

ast a

ssay

Ass

ay r

ange

Pap

er d

isc

(aga

r di

ftU

sion

) m

etho

d:

0·1

-50

llg/

ml

(as

(+ )-

biot

in)

Tur

bidi

met

ric

met

hod:

0

·2-8

ng

/ml

10-1

00ll

g/m

l

0·4

-4Il

g

(0·5

Ilg

biot

in/m

l gi

ves

M

= 0

·069

)

Ref

eren

ce

Tan

aka

et a

l. (1

987a

)

McC

orm

ick

&

Rot

h (1

970)

Gre

en (

1970

)

Avi

din-

Giv

en a

mou

nt o

f av

idin

, of

whi

ch

1.

Avi

din-

com

bina

ble

>0·

1 ng

Bio

tin

Hoo

d (1

979)

3H

C4C

)-bi

otin

com

bina

bili

ty (

a) i

s kn

own,

is

com

poun

ds

(ure

ido

ring

(V

aria

ble

acco

rdin

g D

aksh

inam

urti

&

biot

in

reac

ted

wit

h a

sam

ple

cont

aini

ng

com

poun

ds)

can

to t

he u

sed

amou

nts

All

an (

1979

) bi

ndin

g bi

otin

(x),

fol

low

ed b

y th

e fu

rthe

r be

ass

ayed

. of

avi

din:

m

etho

d ad

diti

on o

f ex

cess

3H

-bio

tin.

x is

2.

F

ast

assa

y co

mm

erci

ally

ob

tain

ed f

rom

the

equ

atio

n x

= a

-av

aila

ble

avid

in b

inds

b,

whe

re b

is t

he c

ombi

ned

3H_

1-1

3ll

g b

ioti

n)

biot

in.

Enz

ymat

ic

BC

S-

The

H20

2 fo

rmed

thr

ough

the

cou

pled

1.

H

igh

sens

itivi

ty

10-2

00 n

mol

/ml

Tan

aka

et a

l. A

OD

-re

acti

ons

of b

ioti

nyl-

CoA

syn

thet

ase

2.

Fas

t as

say

(2 h

) (1

987b

) P

OD

(B

CS

) an

d ac

yl-C

oA o

xida

se (

AO

D)

3.

BC

S is

sta

ble

met

hod

is a

ssay

ed b

y th

e pe

roxi

dase

(P

OD

) re

acti

on,

whi

ch g

ives

a f

luor

esce

nt

com

poun

d.

PC

met

hod

The

enz

ymat

ic b

indi

ng o

f bi

otin

in s

itu

1.

Hig

h se

nsiti

vity

5-

2000

pg/

ml

Haa

rasi

lta

to t

he p

yruv

ate

carb

oxyl

ase

(PC

) 2.

F

ast

Ass

ay (

1 h)

(1

978)

ap

opro

tein

of

biot

in-d

efic

ient

bak

ers'

3.

T

he y

east

cel

ls s

houl

d ye

ast

and

the

subs

eque

nt e

stim

atio

n be

pre

pare

d ju

st b

efor

e of

the

PC a

ctiv

ity b

y a

14C

0 2-f

ixat

ion

assa

y.

met

hod

246 Y. Izumi & H. Yamada

Table 4 Microbiological Activities of Various Biotin-Vitamers

Biotin-vitamer Activitya toward

L. plantarum s. cerevisiae

Intermediates of biotin biosynthesis (+ )-biotin 100 100 ( + )-dethiobiotin 0 100 ( ± )-dethiobiotin 0 50 7,8-diaminopelargonic acid 0 10 pimelic acid 0 0

Intermediates of biotin biodegradation ( + )-bisnorbiotin 0 0 ( + )-bisnorbiotin sulfoxide 0 0 ( + )-bisnordethiobotin 0 0

Naturally occurring biotin-related compounds biocytin 0 30 ( + )-biotin amide 0 100 dethiobiotin amide + ( + )-biotin D-sulfoxide 100 100 ( + )-biotin L-sulfoxide 5 0·001-0·1

Chemically synthesized biotin-related compounds ( ± )-oxibiotin 50 10-25 ( + )-selenobiotin 100 ( ± )-carbobiotin 12 15 diaminobiotin 0 10 ( + )-biotin methyl ester 100

a Relative activity of equimolar concentration of each compound.

B. subtilis

100 100 50

Weak o

100 100 100

o o

+ 100

100

egg whites, but also in the egg yolks of avian species and the turtle (Moss & Lane, 1971). Since avidin is heat-labile, prolonged heating of egg white denatures the avidin and destroys its biotin-binding capacity. Another biotin­binding protein similar to avidin, streptavidin, is produced by a Streptomyces strain (Chaiet & Wolf, 1964).

Biotin deficiency produces dermatitis and perosis in chicken and poultry (Gyorgy, 1968); alopecia, seborrheic skin changes, spasticity of the hind legs and cracks in the feet of pigs. The activities of the biotin-dependent enzymes are also decreased (Whitehead, 1981). These enzymes are involved in carboxylation, transcarboxylation, and decarboxylation reactions, and function in the vitally important metabolic processes of glucose and fat synthesis (Moss & Lane, 1971; Wood & Barden, 1977; Whitehead, 1981). Among the most important enzymes are acetyl-CoA carboxylase, pyruvate carboxylase and propionyl-CoA carboxylase.

It was generally believed that the combination of biotin in the feed

Microbial Production of Biotin

5

(A)

E ~4 III c 0 N

.c i 0 bI 03 ~ j is

2

10 50 100 250 1X10 3 5X10 3 20X10 3

Biotin (ng/ml X 71l1/disk)

(B) 5 (e)

2 4

'" ci3 £ .c i &2

02468 Biotin (ng/ml x 100 III/2m!)

0.4 1.2 2.0 Biotin (ng/ml X 100 III/2m!)

247

2.8

Fig. 5. Standard curves of microbiological assays of biotin. (A) Paper disc plate (agar diffusion) method using lyophilized cells of L. plantarum (x) and E. coli (e) and glycerol-suspended cell of S. cerevisiae (0). (B) Turbidimetric method using lyophilized cells of L. plantarum. (C) Turbidimetric method using glycerol-suspended cells of S.

cerevisiae (0) and lyophilized cells of E. coli (e). From Tanaka et al. (1987a).

ingredients plus the biotin produced in the intestine by bacteria supplied sufficient biotin to meet the poultry's requirement. However, since 1966, a number of reports on biotin deficiency in commercial flocks have appeared (Scheiner & DeRitter, 1975). Apparent biotin deficiencies in swine under commercial conditions were also reported (Scheiner & DeRitter, 1975).

In humans, infant seborrheic dermatitis and the related Leiner's disease are

248 Y. Izumi & H. Yamada

biotin-responsive (Svejcar & Homolka, 1950). Biotin deficiency has been reported in individuals during prolonged total parenteral nutrition (Bozian et al., 1981; Mock et al., 1981). Biotin administration has successfully controlled multiple carboxylase deficiency even in unborn infants (Baumgartener et al., 1981; Munnich et al., 1981; Thoene et al., 1981; Roth et al., 1982). It has been suggested that diseases related to biotin metabolism may be more common than previously thought (Tanaka, 1981).

9.2 Growth·Promoting Activity

Biotin deficiency also causes a marked decrease in the activities of several glycolytic enzymes in the liver, e.g. glucokinase, phosphofructokinase and pyruvate kinase. The activities of these enzymes increased rapidly after the administration of biotin (Dakshinamurti & Cheah-Tan, 1968, 1970; Dak­shinamurti & Hong, 1970; Dakshinamurti et al., 1970), while other glycolytic enzymes such as hexose phosphate isomerase were not affected by biotin administration. Boeckx and Dakshinamurti (1974) showed that biotin ad­ministration to biotin-deficient rats resulted in increased stimulation, by more than twofold, of amino acid incorporation into protein, both in vivo and in vitro in rat liver, pancreas, intestinal mucosa and skin. They also found that the synthesis of some proteins such as serum albumin, a major product of the liver protein-synthetic machinery, was stimulated more than twofold, but others were not stimulated at all. The effect of biotin on protein synthesis was preceded by stimulation in the incorporation of orotic acid into nuclear and ribosomal RNA.

Vesely (1982) and Vesely et al. (1984) found that biotin and its analogs at 0·1-1 IJM enhanced soluble guanylate cyclase activity two- to threefold in rat liver, kidney, colon, cerebellum, and heart. Since cyclic GMP, a product of guanylate cyclase reaction, is known to increase the growth of fibroblasts and thymocytes and also to increase RNA and protein synthesis, these results suggest that the growth-promoting effect of biotin might be mediated by cyclic GMP. Spence & Koudelka (1984) also found that addition of biotin in the presence of insulin elicited an increase in the intracellular content of cyclic GMP, followed by an increase in glucokinase in cultured rat hepatocytes.

10 CHEMICAL SYNTHESIS

10.1 Method of Goldberg et 01.

The industrial synthesis of biotin which is presently carried out is based on a method developed by Hoffmann-La Roche, Inc. (Goldberg & Sternbach, 1949; Gyorgy & Larger, 1968). As illustrated in Fig. 6, the synthesis is characterized by the use of a meso-diaminosuccinic acid derivative as a starting material, which contains two groups in the same spatial arrangement as the two amino

Microbial Production of Biotin

° ° 9 /t" /t" /c"

ToaH ~r ~r (a) ~HR ~HR (b) R~ 7R (e) RN ~R (d) R~ ~R (e) CH=CH _ CH-CH -- CH-CH -- HC--CH __ Ht-CH - HC-CH-

tOOH taaH taoH taaH tOOH taoH taaH ot to AcatH)a

R=benzyl "0/ "0

• o

)< RN NR

Ht-tH

Hzt Co "S/

x

O-Camphorsulfonate of (+)-IX

XIU

• IV V Ac=CH3Ca

VI

(j)

Resolution

XIV

249

Fig. 6. Flow sheet of one chemical process for the industrial preparation of ( + )-biotin. (a) Benzylamine; (b) phosgene; (c) acetic anhydride; (d) Zn, a mixture of acetic acid and acetic anhydride; (e) H 2S, HCI; (f) C2H50(CH2hMgBr; (g) H2, Raney nickel; (h) HBr, acetic acid; (i) silver o-camphorsulfonate; (j) isopropanol; (k) sodium di-

ethylmalonate; (I) HBr. From Goldberg & Sternbach (1949).

groups present (in substituted form) in the biotin molecule, i.e. the meso­configuration in diaminosuccinic acid derivatives corresponding to the cis­structure of the two amino groups in a ring compound such as biotin. Moreover, since resolution is carried out at the intermediate stage (XII-XIII) it permits the direct production of the optically and biologically active biotin, ( + )-biotin.

10.2 Synthesis from Sugars

Efficient stereospecific total syntheses of (+ )-biotin from the sugars, 0-

mannose, o-glucose and o-glucosamine, were achieved by Ohrui & Emoto (1975), Ogawa et al. (1977) and Ohrui et al. (1978), respectively. The synthesis from o-mannose is shown in Fig. 7. o-Mannose is first converted to the aldehyde (II) through isopropyridenylation, benzoylation, selective isopropy­denylation, and periodate oxidation. The Wittig reaction and hydrogenation of

250

<" s~. -H COOCH3 H~H" ---.. o 0

'><

VII

Y. Izumi & H. Yamada

~s COOCH3 -H

H H_ OR bR

IX: R=H

X: R=CHlSOZ

~OOCH3 ~COOCH "H H 3

H-· -H -H- -tI -

N3 N3 AcNH HNAc

XI

(t)-Biotin

Fig. 7. Stereospecific synthesis of ( + )-biotin from D-mannose. From Ohrui & Emoto (1975).

the aldehyde yield compound (IV) which has a side chain-like biotin. The treatment of (IV) with NaOCH3 in methanol, followed by the reduction of the resulting aldehyde (V) with NaBH4 produces (VII). Treatment of (VII) with NaS affords a tetrahydrothiophene derivative (VIII) which is converted via (XI) to (X). Treatment of (X) with NaN3 gives a diazido compound (XI). Catalytic reduction of the azido groups of (XI) in a mixture of methanol and acetic anhydride gives a diacetoamido derivative (XII). Treatment of (XII) with Ba(OHb followed by the treatment with phosgene affords ( + )-biotin. Thus, (+ )-biotin is synthesized from D-mannose in a good yield, because a five-membered ring consisting of isopropyridene, which protects the hydroxyl groups of the sugar, is used to fix the molecular conformation during the intramolecular substitution reactions.

11 APPLICATION AND ECONOMICS

The most practical uses of biotin are as a pharmaceutical and as a supplement of culture media for amino acid production, where the biotin-requiring bacteria such as Corynebacterium glutamicum and Brevibacterium ftavum for glutamic acid and lysine production are used (Aida, 1986). Recently, the vitamin has attracted increasing interest as a food and feedstuff supplement (Pearson et al., 1976; Whitehead, 1981; Kornegay, 1985) as can also be seen from the studies of its growth-promoting effect as described in Section 9.2. There may be a marked increase in the demand and supply of the vitamin as a feedstuff supplement if its price becomes lower. At present, the industrial preparation of biotin is carried out through a chemical process. The present price of biotin as a biochemical reagent is US $170·00/10 g (price list of Sigma, 1988). Therefore, the price of bulk supplies can be supposed to be much lower than the reagent price. In order to economically compete with the present chemical process, the present production levels via fermentation processes should be greatly improved (the maximum level so far reported is 105 mg/liter

Microbial Production of Biotin 251

as described in Section 5). If a fermentation process uses a biotin precursor such as pimelic acid or azelaic acid, the cost of such compounds should also be taken into consideration.

REFERENCES

Aida, K. (1986). An overview of the microbial production of amino acids. In Progress in Industrial Microbiology, Vol. 24 (Biotechnology of Amino Acid Production), ed. K. Aida, I. Chibata, K. Nakayama, K. Takinami & H. Yamada. Kodansha­Elsevier, Tokyo, pp. xxi-xxv.

Baumgartener, R., Suormala, T., Wick, H., Bachmann, C. & Jaggi, K. H. (1981). Biotin dependency causing multiple carboxylase deficiency in vivo. Pediatr. Res., 15, 1189.

Boas, M. A. (1927). The effect of desiccation upon the nutritive properties of egg-white. Biochem. J., 21, 712-24.

Boeckx, R. L. & Dakshinamurti, K. (1974). Biotin mediated protein biosynthesis. Biochem. J., 140, 549-56.

Bonnemere, c., Hamilton, J. A., Steinrauf, L. K. & Knappe, J. (1965). Structure of the bis-p-bromoanilide of carbon dioxide biotin. Biochemistry, 4, 240-45.

Bozian, R. c., Moussavian, N. & Piepmeyer, J. L. (1981). Biotin deficiency during prolonged home total parenteral nutrition (TPN). Clin. Res., 29, 622A

Chaiet, L. & Wolf, F. J. (1964). The properties of streptavidin, a biotin-binding protein produced by Streptomycetes. Archs Biochem. Biophys., 106, 1-5.

Dakshinamurti, K. & Allan, L. (1979). Isotopic dilution assay for biotin: Use of [3H]biotin. In Methods in Enzymology, Vol. 62, ed. D. B. McCormick & L. D. Wright. Academic Press, New York, pp. 284-9.

Dakshinamurti, K. & Cheah-Tan, C. (1968). Liver glucokinase of the biotin deficient rat. Can. J. Biochem., 46, 75-80.

Dakshinamurti, K. & Cheah-Tan, C. (1970). Biotin-mediated synthesis of hepatic glucokinase in the rat. ArdIS Biochem. Biophys., 127, 17-21.

Dakshinamurti, K. & Hong, H. C. (1970). Regulation of key hepatic glycolytic enzymes. Enzymol. BioI. Clin., 11, 423-8.

Dakshinamurti, K., Tarrago-Litvak, L. & Hong, H. C. (1970). Biotin and glucose metabolism. Can. J. Biochem., 48, 493-500.

Dittmer, K. & du Vigneaud, V. (1944). Antibiotins. Science, 100, 129-31. Eakin, R. E. & Eakin, E. E. (1942). A biosynthesis of biotin. Science, 96, 187-8. Eisenberg, M. A. (1973). Biotin: biogenesis, transport and their regulation. Adv.

Enzymol., 38,317-72. Eisenberg, M. A & Krell, K. (1969). Dethiobiotin synthesis from 7,8-

diaminopelargonic acid in cell-free extracts of a biotin auxotroph of Escherichia coli K-12. J. Bioi. Chem., 244,5503-9.

Eisenberg, M. A & Star, C. (1968). Synthesis of 7-keto-8-aminopelargonic acid, a biotin vitamer, in cell-free extracts of Escherichia coli biotin auxotrophs. J. Bacteriol., 96, 1291-7.

Eisenberg, M. A, Mea, B., Prakash, O. & Eisenberg, M. R. (1975). Properties of a-dehydrobiotin-resistant mutants of Escherichia coli K-12. J. Bacteriol., 122, 66-72.

Eisenberg, M. A., Prakash, o. & Hsiung, S. C. (1982). Purification and properties of the biotin repressor-a bifunctional protein. J. Bioi. Chem., 257, 15167-73.

Fall, R. R. & Vagelos, P. R. (1972). Acetyl coenzyme A carboxylase. Molecular forms and subunit composition of biotin carboxyl carrier protein. J. Bioi. Chem., 247, 8005-15.

252 Y. Izumi & H. Yamada

Goldberg, M. W. & Sternbach, L. H. (1949). US Patents 2,489,232-2,489,238. Green, N. M. (1970). Spectrophometric determination of avidin and biotin. In Methods

in Enzymology, Vol. 18A, ed. D. B. McCormick & L. D. Wright. Academic Press, New York, pp. 418-24.

Guillerm, G., Frappier, F., Gaudry, M. & Marquet, A. (1977). On the mechanism of conversion of dethiobiotin to biotin in Escherichia coli. Biochimie, 59, 119-26.

Gyorgy, P. (1967). Biotin. In The Vitamins, 2nd edn, Vol. 7, ed. W. H. Sebrell & R. S. Harris. Academic Press, New York, pp. 303-10.

Gyorgy, P. & Larger, B. W. (1968). Biotin. In The Vitamins, 2nd edn, Vol. 2, ed. W. H. Sebrell & R. S. Harris. Academic Press, New York, pp. 262-357.

Gyorgy, P., Kuhn, R. & Lederer, E. (1939). Attempts to isolate the factor (vitamin H) curative of egg white injury. J. Bioi. Chem., 131,745-59.

Harris, S. A., Wolf, D. E., Mozingo, R. & Folkers, K. (1943). Synthetic biotin. Science, fYI, 447-8.

Harris, S. A., Wolf, D. E., Mozingo, R., Anderson, R. C., Arth, G. E., Easton, N. R., Heyl, D., Wilson, A. N. & Folkers, K. (1944a). Biotin. II. Synthesis of biotin. J. Am. Chem. Soc., 66, 1756-7.

Harris, S. A., Easton, N. R., Heyl, D., Wilson, A. N. & Folkers, K. (1944b). Biotin. IV. Synthesis of 4-benzamido-3-ketotetrahydrothiophene. J. Am. Chem. Soc., 66, 1757-9.

Harris, S. A., Wolf, D. E., Mozingo, R., Arth, G. E., Anderson, R. C., Easton, N. R. & Folkers, K. (1945). Biotin. V. Synthesis of dl-biotin, dl-allobiotin and dl-epi­allobiotin. J. Am. Chem. Soc., 67,2096-100.

Hirono, Y., Kojima, T. & Kimura, H. (1986). Japan Patent 14,901. Hood, R. L. (1979). Isotopic dilution assay for biotin: Use of [14C]biotin. In Methods in

Enzymology, Vol. 62, ed. D. B. McCormick & L. D. Wright. Academic Press, New York, pp. 279-83.

Ifuku, O. & Yanagi, M. (1988). Cloning of biotin operon of Escherichia coli and breeding of biotin producers. Hakka to Kogyo (in Japanese), 46, 102-11.

Ifuku, 0., Haze, S., Kishimoto, J., Sakamoto, T. & Yanagi, M. (1986). Japan Patent 202,686.

Ifuku, 0., Haze, S., Sakamoto, T. & Kishimoto, J. (1987). Japan Patent 155,018. Iwahara, S., Emoto, Y., Tochikura, T. & Ogata, K. (19600). Studies on biosynthesis of

biotin by micro-organisms. Part III. The isolation of crystalline desthiobiotin from culture filtrate of Bacillus sphaericus. Agric. Bioi. Chem., 30,64-7.

Iwahara, S., Takasawa, S., Tochikura, T. & Ogata, K. (1966b). Studies on biosynthesis of biotin by micro-organisms. Part IV. Conversion of desthiobiotin to biotin by various kinds of micro-organisms. Agric. Bioi. Chem., 30, 385-92.

Iwahara, S., Takasawa, S., Tochikura, T. & Ogata, K. (1966c). Studies on biosynthesis of biotin by micro-organisms. Part V. degradation of desthiobiotin by molds. Agric. Bioi. Chem., 30, 1069-75.

Izumi, Y. (1984). Studies on the metabolism of biotin and its regulation in micro­organisms. Nippon Nogei Kagaku Kaishi, 58,891-900.

Izumi, Y. & Ogata, K. (1977). Some aspects of the microbial production of biotin. Adv. Appl. Microbio!., 22, 145-76.

Izumi, Y., Sugisaki, K. & Ogata, K. (1973). Incorporation of the sulfur of L­

p5SJmethionine into the biotin molecule by intact cells of Rhodoturula glutinis. Biochim. biophys. Acta, 304,887-90.

Izumi, Y., Fukuda, H., Tani, Y. & Ogata, K. (1978). The mode of action of 5-(2-thienyl)-valeric acid on biotin biosynthesis. Agric. Bioi. Chem., 42, 579-84.

Izumi, Y., Tani, Y. & Yanada, H. (1980). Biotin biosynthesis in micro-organisms. Bull. Inst. Chem. Res. Kyoto Univ., 58,434-47.

Izumi, Y., Kano, Y., Inagaki, K., Kawase, N., Tani, Y. & Yamada, H. (1981). Characterization of biotin biosynthetic enzymes of Bacillus sphaericus, a de­thiobiotin producing bacterium. Agric. Bioi. Chem., 45, 1983-9.

Microbial Production of Biotin 253

Kogl, F. (1937). Wirkstoffprinzip und Pflanzenwachstum. Naturwissenschaften, 25, 465-70.

Kogl, F. & Tonnis, B. (1936). Uber des Bios-Problem. Darstellung von krystallisierten Biotin aus Eigelb. Z. Physiol. Chem., 242, 43-73.

Knappe, J., Ringelmann, E. & Lynen, F. (1961). Zur biochemischen Funktion des Biotins. III. Die dchemische Konstitution kdes enzymatisch gebildeten Carboxy­biotins. Biochem. Z., 335, 168-76.

Kornegay, E. T. (1985). Biotin in swine nutrition. Ann. N. Y. A cad. Sci., 447, 112-21. Kosow, D. P., Huang, S. C. & Lane, M. D. (1962). Propionyl coenzyme A

holocarboxylase synthesis. I. Preparation and properties of the enzyme system. J. BioI. Chem., 237, 3633-9.

Lamhonwah, A. M., Quan, F. & Gravel, R. A. (1987). Sequence homology around in the biotin-binding site of human propionyl-CoA carboxylase and pyruvate car­boxylase. Archs Biochem. Biophys., 254,631-6.

Lilly, V. G. & Leonian, L. H. (1944). Antibiotin effect of desthiobiotin. Science, 99, 205-6.

Lynen, F., Knappe, J., Lorch, E., Juttig, G. & Ringelmann, E. (1959). Die biochemischen Funktion des Biotin. Angew. Chem., 71,481-91.

Lynen, F., Knappe, J., Lorch, E., Juttig, G., Ringelmann, E. & Lachance, J. P. (1961). Zur biochemischen Funktion des Biotins. II. Reinigung und Wirkungsweise der ,8-Methyl-crotonyl-Carboxylase. Biochem. Z., 335, 123-67.

McCormick, D. B. & Roth, J. A. (1970). Colorimetric determination of biotin and analogs. In Methods in Enzymology, Vol. 18A, ed. D. B. McCormick & L. D. Wright. Academic Press, New York, pp. 383-5.

Melville, D. B., Moyer, A. W., Hofmann, K. & du Vigneaud, V. (1942). The structure of biotin: The formation of thiophenevaleric acid from biotin. J. BioI. Chem., 146, 487-92.

Mock, D. M., de Lorimer, A. A., Liebman, W. M., Sweetman, L. & Baker, H. (1981). Biotin deficiency: An unusual complication of parenteral alimentation. New Engl. J. Med., 304,820-23.

Moss, J. & Lane, D. (1971). The biotin-dependent enzymes. Adv. Enzymol., 35, 321-442.

Mueller, J. H. (1937a). Pimelic acid as a growth accessory for a strain of the diphtheria bacillus. Science, 85, 502-3.

Mueller, J. H. (1937b). Pimelic acid as a growth accessory for the diphtheria bacillus. J. Bioi. Chem., 119,121-9.

Munnich, A., Saudubray, J. M., Carre, G., Coude, F. X., Ogier, H., Charpentier, C. & Frezal, J. (1981). Defective biotin absorption in multiple carboxylase deficiency. Lancet, 2, 263.

Ogata, K. (1970). Microbial synthesis of dethiobiotin and biotin. In Methods in Enzymology, Vol. 17A, ed. D. B. McCormick & L. D. Wright. Academic Press, New York, pp. 390-94.

Ogata, K., Tochikura, T., Iwahara, S., Takasawa, S., Ikushima, K., Nishimura, A. & Kikuchi, M. (1965a). Studies on biosynthesis of biotin by micro-organisms. Part I. Accumulation of biotin-vitamers by various micro-organisms. Agric. Bioi. Chem., 29,889-94.

Ogata, K., Tochikura, T., Iwahara, S., Ikushima, K., Takasawa, S., Kikuchi, M. & Nishimura, A. (1965b). Studies on biosynthesis of biotin by micro-organisms. Part II. Identification of biotin-vitamers accumulated by various micro-organisms. Agric. Bioi. Chem., 29, 895-901.

Ogawa, T., Kawano, T. & Matsui, M. (1977). A biomimetic synthesis of (+ )-biotin from D-glucose. Carbohydrate Res., 57, c31-5.

Ogino, S., Fujimoto, S. & Aoki, Y. (1974a). Cooxidation of dl-cis-tetrahydro-2-oxo-4-n-pentyl-thieno-(3,4-d)-imidazoline (dl-TOPTI) by soil isolates of the genus Corynebacterium. Agric. Bioi. Chem., 38, 275-8.

254 Y. Izumi & H. Yamada

Ogino, S., Fujimoto, S. & Aoki, Y. (1974b). Production of biotin by microbial transformation of dl-cis-tetrahydro-2-oxo-4-n-pentyl-thieno-(3 ,4-d)-imidazoline (dl­TOPTI). Agric. Bioi. Chern., 38,707-12.

Ohrui, H. & Emoto, S. (1975). Stereospecific synthesis of ( + )-biotin. Tetrahedr. Lett., 32,2765-6.

Ohrui, H., Sueda, N. & Emoto, S. (1978). An alternative synthesis of (+ )-biotin from carbohydrate, a formal synthesis of ( + )-biotin from D-glucosamine. Agric. Bioi. Chem., 42,865-8.

Okumura, S., Tsugawa, R., Tsunoda T. & Motozaki, S. (1962a). Studies on the L-glutamic acid fermentation. Part III. On the biotin synthetic path in bacteria producing L-glutamic acid. Nippon Nogei Kagaku Kaishi, 36,599-605.

Okumura, S., Tsugawa, R., Tsunoda, T. & Motozaki, S. (1%2b). Studies on the glutamic acid fermentation. Part IV. The universality of biotin biosynthetic pathway. Nippon Nogei Kagaku Kaishi, 36,605-13.

Osakai, M., Izumi, Y., Nakamura, K. & Yamada, H. (1986a). Bacterial degradation of biotin and dethiobiotin: Isolation and identification of {:J-hydroxy and keto metabolites. Agric. Bioi. Chem., SO,311-6.

Osakai, M., Tamura, K., Izumi, Y. & Yamada, H. (1986b). Reinvestigation of compound X, a suspected biotin intermediate: Identification of N-formyl deriva­tives of biotin and dethiobiotin. J. Nutr. Sci. Vitaminol., 32, 279-86.

Osawa, I., Fuji, N. & Kamogawa, K. (1987). Japan Patent 275,684. Osawa, I., Speck, D., Kisou, T., Hayakawa, K., Zinsius, M., Gloeckler, R., Lemoine,

Y. & Kamogawa, K. (1989). Cloning and expression in Escherichia coli and Bacillus subtilis of the biotin synthetase gene from Bacillus sphaericus. Gene, in press.

Pai, C. H. (1969). Biosynthesis of desthiobiotin in cell-free extracts of Escherichia coli. J. Bacteriol., 99, 696-701.

Pai, C. H. (1971). Biosynthesis of biotin: Synthesis of 7,8-diaminopelargonic acid in cell-free extracts of Escherichia coli. J. Bacteriol., 105, 793-800.

Pai, C. H. (1975). Biochemical and genetic characterization of dehydrobiotin resistant mutants of Escherichia coli. Molec. Gen. Genet., 134,345-57.

Parry, R. J. & Kunitani, M. G. (1976). Biotin biosynthesis. 1. The incorporation of specifically tritiated dethiobiotin into biotin. J. Am. Chem. Soc., 98, 4024-26.

Parry, R. J. & Naidu, M. V. (1980). Biotin biosynthesis. Incorporation of 5(RS)-3H­dethiobiotin into biotin. Tetrahedr. Lett., 21, 4783-6.

Pearson, J. A., Johnson, A. R., Hood, R. L. & Fogerty, A. C. (1976). Fatty liver and Kidney syndrome in chicks. I. Effect of biotin in diet. Aust. J. BioI. Sci., 29, 419-28.

Polakis, S. E., Guchhait, R. B., Zwergel, E. E. & Lane, M. D. (1974). Acetyl coenzyme A carboxylase system of Escherichia coli: Studies on the mechanism of the biotin carboxylase- and carboxyl transferase-catalyzed reactions. J. Bioi. Chem., 249,6657-67.

Pontecorvo, G. (1953). The genetics of Aspergillus nidulans. Adv. Genet., 5,208-38. Prakash, O. & Eisenberg, M. A. (1979). BiotinyI5'-adenylate: Corepressor role in the

regulation of the biotin genes of Escherichia coli K-12. Proc. Natn. Acad. Sci. U.S.A., 76, 5593-602.

Roth, K. S., Yang, W., Allan, L., Saunders, M., Gravel, R. A. & Dakshinamurti, K. (1982). Prenatal administration of biotin in biotin responsive multiple carboxylase deficiency. Pediatr. Res., 16, 126.

Ryan, F. J. (1956). Contributions from microbiology to the concept of the gene and allelism. Japan J. Genet., 31,265-73.

Salib, A. G., Frappier, F., Guillerm, G. & Marquet, A. (1979). On the mechanism of conversion of dethiobiotin to biotin in Escherichia coli. III. Isolation of an intermediate in the biosynthesis of biotin from dethiobiotin, Biochem. Biophys. Res. Commun., 88,312-9.

Microbial Production of Biotin 255

Scheiner, J. (1985). Biotin. In Methods of Vitamin Assay, 4th edn, ed. J. Augustin, B. P. Klein, D. A. Becker & P. B. Venugopal. John Wiley & Sons, New York, pp. 535-53.

Scheiner, J. & DeRitter, E. (1975). Biotin content of feedstuffs. J. Agric. Food Chem., 23, 1157-62.

Spence, J. T. & Koudelka, A. P. (1984). Effects of biotin upon the intracellular level of cGMP and the activity of glucokinase in cultured rat hepatocytes. J. Bioi. Chem., 259,6393-6.

Stallings, W. & Titta, G. T. de (1985). Crystallographic investigations of biotin and carboxybiotin derivatives. Ann. N. Y. Acad. Sci., 447, 152-68.

Stokes, J. L. & Gunness, J. (1945). Microbial activity of synthetic biotin, its optical isomers, and related compounds. J. BioI. Chem., 157, 121-6.

Svejcar, J. & Homolka, J. (1950). Experimental experiences with biotin in babies. Ann. Paediat., 174, 175-93.

Szybalski, E. H. & Szybalski, W. (1982). A physical map of the Escherichia coli bio operon. Gene, 19,93-103.

Takai, T., Wada, K. & Tanabe, T. (1987). Primary structure of the biotin-binding site of chicken liver acetyl-CoA carboxylase. FEBS Lett., 2U, 98-102.

Takai, T., Yokoyama, C., Wada, K. & Tanabe, T. (1988). Primary structure of chicken liver acetyl-CoA carboxylase deduced from cDNA sequence. J. Bioi. Chem., 263, 2651-7.

Tanaka, K. (1981). New light on biotin deficiency. N. Engl. J. Med., 304,839-40. Tanaka, M., Yamamoto, H., Izumi, Y. & Yamada, H. (1986). Purification and

properties of biotinyl-CoA synthetase from Mycoplana sp. No. 166. Archs Biochem. Biophys., 251,479-86.

Tanaka, M., Izumi, Y. & Yamada, H. (1987a). Biotin assay using lyophilized and glycerol-suspended cultures. J. Microbiol. Meth., 6,237-46.

Tanaka, M., Izumi, Y. & Yamada, H. (1987b). Enzymatic assay for biotin using biotinyl-CoA synthetase. Agric. BioI. Chem., 51, 2585-6.

Tanaka, M., Izumi, Y. & Yamada, H. (1988). Microbial metabolism and production of biotin. Vitamin (Japan), 62,305-15.

Tatum, E. L. (1945). Desthiobiotin in the biosynthesis of biotin. J. Bioi. Chem., 160, 455-59.

Thoene, J., Baker, H., Yoshino, M. & Sweetmen, L. (1981). Biotin-responsive carboxylase deficiency associated with subnormal plasma and urinary biotin. N. Engl. J. Med., 304,817-20.

Trainor, D., Parry, R. J. & Gitterman, A. (1980). Biotin biosynthesis. 2. Stereochem­istry of sulfur introduction at C-4 of dethiobiotin. J. Am. Chem. Soc., 102, 1467-8.

Traub, W. (1956). Crystal structure of biotin. Nature, Lond., 178,649-50. Trotter, J. & Hamilton, J. A. (1966). The absolute configuration of biotin.

Biochemistry, 5,713-4. Vesely, D. L. (1982). Biotin enhances guanylate cyclase activity. Science, 216,1329-30. Vesely, D. L., Wormser, H. C. & Abramson, H. N. (1984). Biotin analogs activate

guanylate cyclase. Molec. Cell. Biochem., 60, 109-14. Vigneaud, V. du, Dittmer, K., Hauge, E. & Long, B. (1942). The growth-stimulating

effect of biotin for the diphtheria bacillus in the absence of pimelic acid. Science, 96, 186-7.

Wakil, S. J. & Gibson, D. M. (1960). Studies on the mechanism of fatty acid synthesis. VIII. The participation of protein-bound biotin in the biosynthesis of fatty acids. Biochim. Biophys. Acta, 41, 122-9.

Wakil, S. J., Titchener, E. B. & Gibson, D. M. (1958). Evidence for the participation of biotin in the enzymic synthesis of fatty acids. Biochim. Biophys. Acta, 29, 225-6.

Watanabe, K., Yano, S. & Yamada, Y. (1982). The selection of cultured plant cell lines producing high levels of biotin. Phytochemistry, 21,513-6.

256 Y. Izumi & H. Yamada

Whitehead, C. C. (1981). The assessment of biotin status in man and animals. Proc. Nutr. Soc., 40, 165-72.

Wolf, D. E., Valiant, J., Peck, R. L. & Folkers, K. (1952). Synthesis of biocytin. J. Arn. Chern. Soc., 74, 2002-3.

Wood, H. G. & Barden, R. E. (1977). Biotin enzymes. Ann. Rev. Biochern., 46, 385-413.

Yamada, H., Osakai, M., Tani, Y. & Izumi, Y. (1983). Biotin overproduction by biotin analog-resistant mutants of Bacillus sphaericus. Agric. Bioi. Chern., 47, 1011-6.

Yamada, H., Osakai, M. & Izumi, Y. (1984). Formation of biotinyl-CoA synthetase, the first enzyme involved in microbial biotin degradation. Agric. BioI. Chern., 48, 2039-45.

Chapter 15

MICROBIAL PRODUCTION OF VITAMIN B12

C. SPALLA, A. GREIN, L. GAROFANO & G. FERNI

Farmitalia Carlo Erba, Via dei Gracchi 35, 20146, Milano, Italy

1 INTRODUCTION

From the chemical point of view, the term vitamin B12 is synonymous with cyanocobalamin, which is by far the most important component of the large family of the cobalt corrinoids. In this review, however, as in most publica­tions, the word vitamin B12 is given a very broad meaning so as to include all the cobalt corrinoids of the cobalamin group. Other cobalt corrinoids which can be considered analogous or precursors of cobalamins are also included.

Discovered in 1948 and identified as the anti-pernicious anemia factor, vitamin B12 has been thoroughly investigated from several points of view, including biochemical significance, biosynthesis in micro-organisms, production in commercial amounts and applications. The B12 biosynthetic pathway in micro-organisms is now largely known and good production processes are available and will be described in this review.

Due to its implications in extremely important biochemical reactions, vitamin B12 originated wide interest in large and useful applications in human and animal health care. Many analogues and derivatives were obtained and studied to find new activities with particular emphasis towards B12 molecules endowed with anti-cancer activity. Unfortunately, none of these substances were of particular interest and the application of vitamin B12 remained limited to anti-pernicious anemia activity, polyvitaminic specialties and as a feed supplement in husbandry. The interest in the development of vitamin B12 reached a peak in the 1960s and then decreased slowly.

At present, a well consolidated but steady market and good production processes are in the hands of a very limited number of companies. Like other vitamins, B12 found its niche and from the economic point of view it can now be considered as a 'mature product'.

2 HISTORICAL

Vitamin B12, a natural product endowed with important biological properties, was isolated in 1948, almost contemporaneously but independently and from

257

258 C. Spalla et aI.

different sources, by three industrial research groups, i.e. by Glaxo Labora­tories (Smith, 1948a,b) in the UK and by Merck & Co. Laboratories (Rickes et al., 1948a, b) and Lederle Laboratories (Stokstad et al., 1948) in the USA.

The two first groups found this compound in liver extracts and identified it as the anti-pernicious anemia factor responsible for the effective control of the disease. These experimental programs were related to a continuous study over a 20-year period of the therapeutic effect of whole beef liver in pernicious anemia.

In their communication Rickes et al. also reported the presence of significant amounts of vitamin B12 in media fermented by a grisein-producing strain of Streptomyces griseus as well as in culture broths from fermentations by Mycobacterium smegmatis, Lactobacillus arabinosus, Bacillus subtilis, Strep­tomyces roseochromogenes and Streptomyces antibioticus. The vitamin B12 produced was measured by the microbiological assay based on the growth response of Lactobacillus lactis (Domer strain) devised by Shorb (1947, 1948).

It was also at about the same time that Stokstad et al. (1948) reported the production by Flavobacterium solare of a growth promotor, active in the so-called 'animal protein factor' assay in chicks and effective in the treatment of pernicious anemia in the human body which was identified as vitamin B12. Microbial synthesis was also implicated in the early studies on the presence of 'animal protein factor' in manures and feces (Cary et al., 1946; Hartman and Cary, 1946; Rubin & Bird, 1946; Lillie et al., 1948). Subsequent investigations, which showed that the potency of incubated feces and manures was higher than the freshly voided material, confirmed the earlier hypothesis of the importance of micro-organisms in this synthesis (McGinnis et al., 1947; Sahashi et al., 1953). With this background of information, the finding of vitamin B12 in sewage was not unexpected (Hoover et al., 1951), and some efforts have been made to exploit this source (Bernhauer & Friedrich, 1954; Friedrich & Bernhauer 1958).

Although vitamin B12 is present in small amounts in almost every animal tissue, it originates from micro-organisms. Depending on the nature of their nutritional habits and digestive physiology, animals obtain the vitamin from their own intestinal flora or from other animals through their meat diet. An exogenous supply is mandatory for man. The importance of microbial synthesis of this group of vitamins has been summarized as follows by Smith (1950-1951):

It seems probable that the only primary source of vitamin B12 in nature is the metabolic activity of micro-organisms; there is no convincing evidence for its elaboration in tissues of higher plants or animals. It is synthesized by a wide range of bacteria and actinomycetes, though apparently not to any extent by yeasts or fungi.

Robbins et al. (1950) have concurred and stated:

It appears probable that the synthetic activity of micro-organisms, especially bacteria and actinomycetes, is the original source of vitamin B12 in nature.

Microbial Production of Vitamin B12 259

The elucidation of the chemical structure of vitamin B12 is one of the most outstanding examples of the successful collaboration of the chemist, the X-ray crystallographer, and the biologist. The chemistry of this complex molecule has been reviewed by those actively connected with the research and their interpretation of the X-ray crystallography (Brink et al., 1954; Hodgkin et al., 1955, 1957) and the degradative studies leading to the confirmation of the structural formula, proposed by Dr Hodgkin and associates, should be consulted for a guide to the literature (Folkers & Wolf, 1954; Wolf & Folkers, 1954; Bonnett et al., 1955; Folkers et al., 1957).

Ten years were spent from the first efforts of Woodward and Eschenmoser until a full chemical synthesis was achieved (Krieger, 1973; Maugh, 1973). It turned out to be a very difficult and it involved a group in Zurich, and a group in Cambridge (UK) with more than 100 people. The synthesis requires about 70 steps and is of no value for industrial purposes. Today, vitamin B12 is exclusively obtained by fermentation processes utilizing high-producing micro­organisms or, less frequently, sewage.

3 CHEMICAL AND PHYSICAL PROPERTIES

Vitamin B12 (or cyanocobalamin) belongs to the large family of the cobalt corrinoids exhibiting the general formula presented in Fig. 1. The molecule of cobalamins, which are the most interesting cobalt corrinoids, is formed by the following entities: a macrocycle in planar position, constituted by 4 reduced pyrrol rings linked through their N -atoms to a cobalt atom in central position (corrin) and showing 6 side chains (3 acetamide and 3 propionamide residues). Almost perpendicular to the macrocycle a nucleotide, formed by phosphate, ribose and 5,6-dimethylbenzimidazole is linked, through a coordination bond, to the cobalt atom as shown in Fig. 2. The group of the cobalamins includes cyanocobalamin or true vitamin B12 , hydroxocobalmin or vitamin B 12a, methylcobalamin or mecobalamin and 5'-desoxyadenosilcobalamin or cob am­mide or coenzyme B12 of Barker (Barker et al., 1958), characterized by a cyano, hydrozyl, methyl or 5'-desoxyadenosyl radical respectively (Fig. 2).

Other cobaltcorrinoids with different heterocyclic bases (like substituted benzimidazoles or purines), have been found in various micro-organisms, either spontaneously or after the supply of the corresponding base to the fermentation medium. Pseudovitamin B12 and factor III, in which the base is adenosine and 5-hydroxybenzimidazole respectively, are the most frequently encountered. They can be considered analogues of vitamin B12. A list of these compounds is reported by Perlman (1959) and by Marvyn & Smith (1964). Natural analogues designated as 'incomplete' are also known, for instance factor B which is devoid of the ribose-phosphate moiety. All the known analogues showed low activity as growth factors for vertebrates and no therapeutic effect but they are very potent growth factors for the micro­organisms. Cyanocobalamin, hydroxycobalamin, methylcobalamin and vitamin

260 C. Spalla et al.

NH,COH,C

NH,COH,C

~HCOCH,H,C

H,~ '~ ____ ,_

/CH ",,"'" I H,C '0><' ............... 0- I

-----~~P'o OH :

I

--------------------~

Cobalamins

x y

-CN

-OH

-CH,

CH,CH,CONH,

CH,

CH,

x

Trivial name

Vitamin Bl2

Vitamin Bua

Mecobalamin

C obamam ide or coenzyme Bu:

Fig. 1. General structure of cobaltocorrinoids with references to cobalamins (Florent & Ninet, 1979).

B12 coenzyme (5,6-dimethylbenzimidazol-5'-deoxyadenosylcobalamin) are the only compounds of industrial interest.

About the nomenclature, as shown in Fig. 1, the corrinoid molecule without the nucleotide is named cobinamide or factor B. When only the base is absent, the compound is called cobamide and finally the complete form, i.e. with the 5,6 dimethylbenzimidazole attached, is named cobalamin.

The cobalamins are water-soluble compounds which crystallize in red needles and are present in nature in their coenzyme form. Vitamin B12 coenzyme is very unstable to light and only stable at low temperature (-lOOC) in aqueous solution. At room temperature it is easily transformed to hydroxycobalamin which has been firstly isolated from Streptomyces aureofaci-

Microbial Production of Vitamin B12 261

Vitamin B12 V1tamin B12 Coenzyme

Fig. 2. Structures of vitamin BI2 and BI2 coenzyme (Perlman, 1967).

ens. (Pierce et al., 1949, 1950). This compound is also unstable to light and temperature and in the presence of cyanide originates the more stable, well known cyanocobalamin. Methylcobalamin is the active form by which vitamin B12 is involved in methionine biosynthesis in Escherichia coli from which it was first isolated (Guest et al., 1962). As the other native forms mentioned above, it is also unstable to light.

The B12 corrinoids show the best stability at pH ranging from 4 to 6; they are sensitive to ascorbic acid, to thiols, to gamma rays. They are endocellular products and are extracted from the cells by treatment with water, neutral buffer solution, lower alcohols or aqueous acetone. When natural corrinoids like vitamin B12 coenzyme, hydroxycobalamin or methylcobalamin are to be obtained, the extraction must be performed in the dark. Such a procedure is not needed when cyanocorrinoids are wanted. In fact, the addition of cyanide to the cell material transforms the unstable coenzyme forms to the stable cyano-form. This procedure is usually applied in industry. A comprehensive review of the chemical and physical properties of vitamin B12 corrinoids has been written by Friedrich (1975).

4 PRODUCING MICRO-ORGANISMS AND SCREENING

As the research on vitamin B12 progressed, it soon became clear that its best source in nature is represented by micro-organisms. Actually, it is synthesized

262 C. Spalla et al.

by a wide variety of bacteria and actinomycetes, while its production by yeasts, fungi and tissues of higher plants or animals has not been reported. At first, vitamin B12 used for human therapy and as a supplement for animal feeds was obtained as a by-product of antibiotic-producing fermentations. These included processes for the production of streptomycin by S. griseus (Schindler & Reichstein, 1952; Smith & Ball, 1953), neomycin with S. fradiae (Jackson et al., 1951), chlorotetracyclin by S. aureofaciens (Pierce et al., 1950), and others. As the demand for the vitamin grew, it became profitable to employ specific production processes in which vitamin B12 was the only product. A number of processes were tested and some of them operated on a commercial scale (Periman, 1959). The products of some of these fermentations were used mainly for the enrichment of feeds poor in animal protein factor, a use promoted by Ansbacher et al. (1949), while the pure material was isolated to be used in human therapy.

As already mentioned, fermented feces and manures showed higher vitamin B12 content than the unfermented material. Hence, sources for isolation of vitamin B12-producing micro-organisms became, among others, hen feces (Stokstad et al., 1948), poultry litter, intestinal contents of animals, like bovine rumen (Di Marco & Spalla, 1957) as well as sewage sludge. Among the organisms shown to produce vitamin B12 there are bacteria from the following genera; Aerobacter, Agrobacterium, Alcaligenes, Azotobacter, Bacillus, Clostridium, Corynebacterium, Escherichia, Flavobacterium, Methanobacillus, Mycobacterium, Propionibacterium, Proteus, Pseudomonas, Rhizobium, Serratia, Streptococcus, Xanthomonas (Periman, 1959). Significant quantities of vitamin B12 have shown to be produced by the following species of actinomycetes: Nocardia rugosa, N. gardneri, Streptomyces albidoftavus, S. antibioticus, S. aureofaciens, S. aureus,S. farinosus, S. fradiae, S. griseus, S. olivaceus, S. roseochromogenes and S. vinaceus (Periman, 1959).

The isolation of vitamin B12 formed by the above mentioned micro­organisms was carried out, and, in many instances, examination of the isolated materials showed that several types of vitamin B12 and of its analogues were present. It is not unlikely that there are still some undiscovered vitamin B12-related compounds present in some cultures. So, for instance, Friedrich & Bernhauer (1958) reported to have found in a sample of sewage sludge, besides the identified c5-benzimidazolecobamidecyanide, some 17 other coba­mides in their extracts.

Numerous micro-organisms have been considered as a potential source of vitamin B12 for industrial production (Table 1). Because of their rapid growth and high productivity, Propionibacterium shermanii and Pseudomonas de­nitrificans were finally selected for industrial purposes.

The usefulness of an industrial process with methane-utilizing bacteria has still to be proven, while the one based on the use of hydrocarbons does not seem attractive (Florent & Ninet, 1979).

Improvement of productivity is achieved by screening for spontaneous or induced mutants obtained with mutagenic treatments. Whatever the strains

Microbial Production of Vitamin B12

Table 1 Vitamin B12 Production by Different Strains

Strain

Micromonospora sp. Nocardia rugosa

Yield (mg/liter)

11·5 14 25 23

References

Wagman et al. (1969) Farmaceutici Italia (1971a) Uelaf (1960) Speedie & Hull (1960) Pantskhava & Bykhovsky (1966) Kojima et al. (1976) Dumenil et al. (1979; 1981) Nakao et al. (1974)

263

Propionibacterium freudenreichii Propionibacterium shermanii Methanobacillus omelianskii Protaminobacter ruber Coryneformbacterium strain XF Corynebacterium and Rhodopseudomonas Nocardia gardneri

8·8 2·5 0·2 2·3 4·5 Kyowa Hakko Kogyo Co. Ltd

(1970)

and the culture conditions may be, it is necessary to add to the medium some elements essential for vitamin biosynthesis, like cobalt ions and frequently 5,6-dimethylbenzimidazole. The addition of potential precursors such as glycine, threonine, c>-aminolevulinic acid and aminopropanol proved some­times to be beneficial.

5 BIOSYNTHESIS AND REGULATION

Investigations on the biosynthesis of the vitamin B12 molecule had mainly the objective of elucidating the biosynthetic pathway leading to the tetrapyrrole macrocycle and that leading to the incorporation of the nucleotide to form vitamin B12.

With few exceptions all authors agree with the general pathway shown in Fig. 3. Two molecules of c>-aminolevulinic acid condense to form por­phobilinogen which is the basic unit of the macrocycle. Latest studies identify in uroporphyrinogen III the point where the biosynthesis of vitamin B12 and that of porphyrins diverge. One of the most striking structural differences between the corrin and the porphyrin ring system is the lack in the former of a methene bridge between one of the pairs of pyrroline-type rings.

Even if the sequence of methylations in the course of the biosynthesis of the corrin ring remains unknown, early work from Shemin's laboratory showed that all of the methyl groups are derived from methionine (Bray & Shemin, 1963; Shemin & Bray, 1964).

Substances endowed with a strong stimulatory effect on the production of vitamin B12 are betaine and choline. Ansbacher et al. (1949) reported that out of all the microbial processes available in 1949, the best ones were charac­terized by the disappearance of choline from the medium.

Miller & Putter, quoted by Demain et al. (1968), discovered that betaine

264 C. Spalla et al.

Glycine

.--Su-C-C�n-y-l--C-oA----,~lle~~%In:id I-I_X_2 __ '-I-----,~_'

5,6-0imethyl­benzimidazole

r Riboflavin

x4

----------1 CH, units • C02+

Cobyrinic acid

amlnopropanol

NH,

5' -deaxyadenosine

Fig. 3. General pathway for the biosynthesis of cobalamins (Florent & Ninet, 1979).

and choline stimulate vitamin B12 production by Pseudomonas denitrificans; this has been confirmed by Miller & Rosenblum (1960) and by McDaniel (1961). Stern & Friedman (1960) found these two compounds to stimulate by as much as five-to-sixfold the formation of vitamin B12 by Rhizobium trifolii, R. meliloti, Agrobacterium sp. and Bacillus megaterium.

The stimulatory effect of betaine and choline on vitamin B12 production by Pseudomonas denitrificans grown in a chemical defined medium is shown in Fig. 4 (Demain et al., 1968). The effect of betaine and choline on vitamin B12 production by P. denitrificans in an industrial medium was studied by Garofano & Merli of these laboratories (unpublished data). The complex medium currently utilized for industrial production contains high concentration of sugar beet molasses which, being particularly rich in betaine, is a very good

Microbial Production of Vitamin B12 265

20

18

16

14

- 12 e ~10 <.

N 8 eli c: 6 °e ItJ +-:;: 4

2

00 1 2 3 4 5 Additive (mg Iml)

Fig. 4. EtIect of betaine and choline on vitamin BJ2 production by Pseudomonas denitrificans (Demain et aI., 1968).

raw material for vitamin Bt2 production. Even in this case, however, addition to the medium of an extra amount of betaine exerts a stimulatory effect on the produced vitamin Bt2 which increases from 90 to 140 Ilg/ml. Under the same conditions betaine can be substituted by choline with similar results provided the medium be suitably buffered with CaC03 •

As far as the biochemical mechanism· of the stimulation of vitamin B12

production by betaine and choline is concerned, it has been demonstrated that it is not due to the methyl groups' donation to the corrin ring of vitamin Bt2, as methionine only is the precursor of these 'extra' methyl groups (Demain & White, 1971). According to Demain et al. (1968) and White & Demain (1971), betaine is an absolute requirement also for porphyrin over-production and it is needed by P. denitrijicans for over-production of both corrins and porphyrins. Although the site of betaine action in the biosynthetic pathway is unknown, these data point out that betaine may be a positive effector for some early common step of corrin and porphyrin synthesis.

6 IMPROVEMENT OF VITAMIN B12-PRODUCING STRAINS

The industrial production of vitamin Bt2 , likewise that of many other microbial metabolites, is carried out utilizing strains resulting from long lasting and

266 c. Spalla et al.

intense programs of genetic modification directed to improve their characteris­tics, mainly, but not only, by increasing their productivity (Kyowa Hakko Kogyo Co. Ltd, 1970; Nakao et at., 1974; Pliva Co., 1964; Pierrel S.p.A., 1965).

These programs consist essentially of the treatment of the producing micro-organisms with a mutagenic agent and of the selection of the strains bringing mutations leading to some practical advantage (higher productivity, good genetic stability, resistance to higher concentrations of substances present in the medium, higher rate of growth, etc.). As this kind of mutant is extremely rare (of the order of 10-5), their selection obtained by direct examination of all the strains deriving from the mutagenic treatments is a very tedious and long-lasting task. As soon as knowledge on the biosynthetic pathway progresses, this essentially random technique is implemented and more rational and productive techniques are adopted.

Since an inverse correlation had been found between vitamin B12 and porphyrin productions (Di Marco et at., 1961), porphyrin-less mutants of vitamin Bl2-producing strains were searched. This was done in a very simple way with Nocardia rugosa, taking advantage of the fact that porphyrins are excreted in the medium surrounding the colonies on the Petri dishes forming a reddish halo. Mutants without halo were easily detected by direct examination of the isolation plates. Out of 180 haloless isolated colonies, about 30 proved to be really unable to produce porphyrins and two of them showed a significant increase in vitamin B12 production (Marnati & Spalla, unpublished data).

A more sophisticated technique leading to the same results in Propionibacterium shermanii has been described by Barrere et at. (1981). The catalase is an enzyme with a porphyrin moiety. Hence, mutants unable to synthesize porphyrins cannot produce catalase and, on the other hand, among mutants lacking catalase activity there are good chances of finding strains unable to synthesize porphyrins. Catalase-less mutants were easily identified on Petri dishes because they were unable to develop oxygen bubbles from a drop of oxygen peroxide put onto the colony surface.

Other techniques consist of looking for mutants resistant to high concentra­tions of the vitamin BI2 precursor 5,6-dimethylbenzimidazole or to the antimetabolite ethionine and others. The increase of vitamin B12 productivity, like that of other metabolites, has been a stepwise process. A significant example of strain improvement referred to P. denitrificans is shown in Fig. 5.

7 PRODUCTION OF VITAMIN BI2

During the last 30 years, several reviews dealing with research on vitamin B 12 production have been published (Perlman, 1959, 1967; Prescott & Dunn, 1959; Marvyn & Smith, 1964; Friedmann & Cagen, 1970).

Microbial Production of Vitamin B12 267

®

I@ I® ~nloO,<Jg/ml)~(l50,<Jg/ml)

Fig. 5. Mutation and selection program for the improvement of vitamin B12 production by Pseudomonas denitrificans. The mutagens employed are the following ones: 1, ultraviolet; 2, ethyleneimine; 3, nitrosomethyluretane; 4-7, N-methyl-N-nitro-N­nitrosoguanidine; 8,10, ultraviolet-bromouracil; 9,11, psoralene near UV light (Garo-

fano & Merli, unpublished data).

The main objectives of the research work according to Florent & Ninet (1979) have been:

to acquire a better knowledge of the biosynthetic pathway in order to support biochemical and genetic studies; to improve strains which turned out to be the most useful ones (propioni­bacteria and Pseudomonas); to replace traditional sugars by more economical nutrients in the media; to improve extraction and purification techniques.

7.1 Production by Propionibacteria

Several microaerophilic propionibacteria produce cobaltocorrinoids in conven­tional carbohydrate media supplemented with cobalt and without aeration. For this process Propionibacterium freudenreichii A Tee 6207, P. shermanii A Tee 13673 and their mutants which can synthesize their own dimethyl benzimidazole

268 c. Spalla et aI.

STOCK CULTURE Lyophilized with skim milk

1 MAINTENANCE CULTURE

Test tube with medium (a) incubated 4 days at 30°C

1 SEED CULTURE - FIRST STAGE

2-liter Erlenmeyer flask with 0·4 liter of medium (b) incubated 2 days at 30°C without agitation

1 SEED CULTURE - SECOND STAGE

30-liter stainless steel fermentor with 10 liters medium (c), incubated 24 h at 30°C without aeration and with frequent adjustment of pH to 6·5 with aqueous NH40H solution

1 PRODUCfION CULTURE

SOO-liter stainless steel fermentor with 340 liters of medium (d) sterilized 40 min at 120°C, inoculated with 7 liters of second-stage seed culture and incubated at 30°C, during the first 80 h under slight nitrogen pressure (no aeration) and with slow agitation, then during the next 88 h with agitation and aeration (2 m3 h): pH adjusted to 7·0 by aqueous NH40H addition along the whole fermentation

Fig. 6. Vitamin B12 from Propionibacterium shermanii. Semi-pilot plant scale fe~IlI:en­tation process. Medium (a): tryptone, 10 g; yeast extract, 10 g; filtered tomato JUice, 200 ml; agar, 15 g; tap-water to 1 liter; pH adjusted to 7·2. Medium (b): medium (a) without agar. Medium (c): corn-steep liquor, 20 g; dextrose, 90 g; tap-water to 1 liter; pH adjusted to 6·5. Medium (d): corn-steep liquor, 40 g; dextrose (sterilized separ­ately), 100 g; cobalt chloride, 20 mg; tap-water to 1 liter; pH adjusted to 7·0. (Adapted

from Florent & Ninet, 1979).

Microbial Production of Vitamin B 12 269

(DBI) are the most useful ones. Since aeration favours DBI formation, the use of a two-stage culture is advisable in order to obtain the highest yields. In the first stage, anaerobic culture is run to almost total depletion of sugar which promotes the growth of the bacteria and cob amide biosynthesis. Then, in the second stage, an aeration shift leads to DBI biosynthesis and conversion of cobamide to cobalamin. The two stages can be carried out batchwise in the same tank or continuously in two connected fermentors (Speedie & Hull, 1960). Almost no information is available on strain preservation and culture maintenance conditions.

The fermentation media consist mainly of glucose or inverted molasses (50-100 g/liter) with small amounts of ferrous, manganous and magnesium salts in addition to cobalt salts (60-100 mg/liter), of buffering or neutralizing agents and nitrogenous compounds. Among these, yeast preparations, casein hydrolysates and other traditional sources are currently used. Corn steep liquor (50-70 g/liter) is frequently preferred since it supplies some lactic and pantothenic acids which enhance the growth of the bacteria. Generally, the temperature of the culture is 30°C and the pH is controlled at around 6·5-7·0. Reports indicate yields of 25-40 mg/liter of vitamin B12 by propionibacteria. Figure 6 gives an example of a current process using P. shermanii.

7.2 Production by Pseudomonas

Several Pseudomonas species are vitamin B12 producers but currently the most used species is P. denitrificans (Miller & Rosenblum, 1960). In contrast to the propionibacteria fermentation, that of Pseudomonas is characterized by the fact that its growth parallels cobalamin biosynthesis under aerobic conditions throughout the whole fermentation process.

Accordingly, fermentation is conducted with aeration and agitation in a single tank, batchwise or continuously. The strain requires traditional nutri­ents, such as yeast extract, sucrose and several mineral salts in the growth medium. In order to enhance vitamin B12 production, the medium has to be supplemented at the beginning of the culture with 10-25 mg/liter of DBI and 40-200 mg/liter of cobaltous nitrate (McDaniel, 1961; Daniels, 1970).

Likewise, betaine and to some extent choline, have favorable effects in activating some biosynthetic steps as mentioned before. Glutamic acid stimulates the growth of bacteria (Daniels, 1966; Koike & Hattori, 1975). Owing to its low costs and high betaine and glutamic acid content, beet molasses are preferentially used in industrial fermentations at a 60-120 g/liter concentration in conjunction with 2-5 g/liter of ammonium phosphate and some oligoelements. The optimum temperature is 28°C, with a pH around 7·0.

By mutation and selection of proper strains, vitamin B12 production rose from 5 to 120-140 mg/liter within the last 20 years (Long, 1962; Lago & Demain, 1969; Ferni & Pennella, unpublished data). The process is outlined in Fig. 7. In Table 2 the composition of typical media for vitamin B12 production with selected mutants of P. denitrificans is reported.

270 C. Spalla et al.

STOCK CULTURE Lyophilized with skim milk

1 MAINTENANCE CULTURE

Test tube with medium A incubated 4 days at 28°C

1 SEED CULTURE-FIRST STAGE

2-liter round-bottomed flask with 0·6 liter of medium E inoculated with a working culture slants and incubated 48 hr at 28°C on rotating shaker at 120 r.p.m. with 90 mm excursion.

1 SEED CULTURE-SECOND STAGE

40-80 liter stainless steel fermentor with 25-50 liter medium E inoculated with 1-1·2% seed culture first stage, incubated 25-30h at 32°C, 200 r.p.m. agitation 0·2 bar pressure, 0·5 v/vm' aeration.

1 PRODUCTION CULTURE

500-liter stainless steel fermentor with 300-liter medium P inoculated with 5% seed culture second stage, incubated 140-160 hr at 32°C, 0·2 bar pressure. Impeller speed regulated in order to assure sufficient oxygen during the first phase of growth (20-30 hr) and then to maintain constant the oxygen consumption at the level reached during the first phase. The fermentation lasts 140-150 h and gives a production of about 120-130,ug/ml

Fig. 7. Vitamin B12 from Pseudomonas denitrificans. Pilot plant scale production. For media composition see Table 2. (Femi & Pennella, unpublished data.).

Microbial Production of Vitamin B12

Table 2 Media for the Production of Vitamin B12 with Pseudomonas denitrificans

Sugar beet molasses Sucrose Betaine Choline Ammonium phosphate Ammonium sulfate Magnesium sulfate Zinc sulfate 5-6 dimethylbenzimidazole Cobalt chloride Difeo agar Tap water pH

A

100

2 0·25 0·2 0·02

20 1000

7·2

Media (g/liter)

E P

70 105 15 3

0·8 2 2·5 0·2 0·2 0·02 0·08 0·005 0·025

toOO 1000 7·2 7·2

271

An important parameter to follow during the production phase is the level of dissolved . oxygen in the culture. At the beginning of the process the concentration of the oxygen must be sufficiently high in order to avoid limitation in the ratio of growth (p02 > 0). During the production phase, on the contrary, oxygen becomes the growth limiting factor and this somehow induces the onset of vitamin B12 production. It is therefore necessary to supply oxygen (air) in limited amounts so as to maintain the p02levei at a value near to zero.

8 PRODUCTION OF VITAMIN B12 DERIVATIVES

8.1 Hydroxocobalamin

This compound is produced by cultures of numerous bacteria and streptomy­cetes and by P. shermanii (Ilieva, 1971). During the extraction, successive transformations of native cobalamins in their sulfate, nitrate and chloro derivatives, are required before a final hydrolytic treatment by Amberlite IRA 400 (OH-), to generate hydroxocobalamin (Kaczka et al., 1956; Pierrel S.p.A., 1963a). The chemical synthesis from cyanocobalamin can also be performed through sulfite and nitrite derivatives (Pierrel S.p.A., 1963b; Smith, 1965).

8.2 Deoxyadenosylcobalamin (Coenzyme Bu)

Coenzyme B12 is directly extracted from P. shermanii (Chinoin-Gyogyszer, 1971; Ilieva & Popova, 1974), from P. freudenreichii (Sifa, 1964) and from

272 C. Spalla et al.

Nocardia rugosa (Farmaceutici Italia, 1971a), where it can represent up to 80% of the total native cobalamins.

Coenzyme B12 is always endocellular; after harvest of the cells by centrifu­gation, extraction is performed in the cold by an acetone-water mixture, or at 80-100DC during a short time by a 2% phenol-aqueous solution or an ethanol-water mixture. All operations have to be conducted in the cold, under subdued light and avoiding any drastic pH condition, in the presence of cyanide in order to reach a global yield of 80-85% in the extraction process (Kaken Kagaku, 1965; Chinoin-Gyogyszer, 1971).

9 ISOLATION OF VITAMIN B12

During the last 30 years the extraction processes have been improved many times; they usually include the following main steps: solubilization of cobala­mins and conversion to cyanocobalamin and isolation of a crude product, 80% pure (utilizable directly for animal feeding), followed by purification to a 95-98% level (for medical use). Usually the whole broth or an aqueous suspension of harvested cells is heated at 80-120DC for 10-30 min at pH 6·5-8·5 in order to extract the vitamin B12. The conversion to cyanocobal­amin is obtained by treating the heated broth or cell suspension with cyanide or thiocyanate.

The extraction operation units are the classical ones and they are combined to achieve an efficient and inexpensive process. An extraction process presently used is briefly outlined in Fig. 8. For further details see Florent & Ninet (1979).

10 BIOASSAY METHODS

The usefulness and value of microbiological assays in studying biosynthesis of vitamin B12 and in its detection in natural materials cannot be overemphasized. Indeed, considering it in retrospect, the vitamin B12 isolation by the American group of researchers at Merck in comparison to the English group at Glaxo was due to the possibility of the former one to make use, instead of clinical tests, of the microbiological method. This was based on the growth response of Lactobacillus lactis (Dorner strain) to the vitamin as first described by Shorb (1947, 1948).

Although the sensitivity of this micro-organism to vitamin B12 is high, the fact that it responds to ribosides and desoxyribosides and that its growth response is influenced by changes in aeration of the culture as well as by genetic variability of the culture (Shorb & Briggs, 1948), led to its substitution with Lactobacillus leichmanii strains ATCC 4797 and A TCC 7830 which gave more reproducible results (Skeggs et al., 1948). This turbidimetric assay found general acceptance for the analysis of fermentation samples and other

P. denitrificans broth

Filtrate

Organic extract I

Aqueous extract

Organic extract II

Crude vitamin B12

Microbial Production of Vitamin B 12 273

Heating 30 min. at 120°C Cooling, adjust­ment of pH to 8·5 Addition of KCN, agitation 16 h at 25°C Addition of zinc chloride (200 g) Adjustment of pH to 8·0 Addition of filter-aid (200 g), agitation, filtration

! Extraction 3 times with 350 ml of a cresol and carbon tetrachloride mixture (1: 2 ratio)

! Addition of 500 ml n-butanol Extraction 3 times with 100 ml water

! Extraction 3 times with 30 ml of a cresol and carbon tetrachloride mixture (1: 2 ratio)

!

Addition of 200 ml acetone and 100 ml ether

Dissolution in 10 ml methanol Chromatography on activated aluminia (4 g). Elution with 2% acetic acid in meth­anol. Precipitation with ether

! Pure vitamin B12

Fig. 8. Crystalline vitamin B12 isolation process (adapted from Florent & Ninet, 1979).

274 C. Spalla et aI.

materials, although a number of problems were encountered. Further inves­tigations showed that all of the vitamin BI2-requiring lactobacilli respond, at varying degrees, to all of the cobamides but not to cobinamide. These differences in response have been used as the basis for differential-type assays but unsatisfactory results have been obtained when samples contained more than two types of cobamides.

Davis & Mingioli (1950) reported that certain mutants derived by treatment of E. coli (ATCC 9637) with UV light required either vitamin B12 or methionine for growth. One of these (strain 113-3) has been, and still is, widely used as a test organism in bioassay for the determination of the cobamide content of fermentation materials and other natural products. As this strain responds to all of the cobamides as well as to cobinamide (Ford & Hunter, 1955) it is very useful for the detection of all of these factors in bioautographs of paper chromatograms of such solutions (Ford & Holdsworth, 1952). This strain is cultivated in a very simple medium and can be used in the agar-diffusion assay as well as in the turbidimetric assay method which is more sensitive.

The usefulness of the growth response of certain protozoa as a method of measuring the vitamin BI2 content of samples of natural products was first reported by Hutner et al. (1949). They found that Euglena gracilis is vitamin BIZ- and thiamin-requiring. In this case chlorophyll production under light serves as a measure of the vitamin BI2 content in the samples. All of the cobamides stimulate the growth of this organism, but some problems in assaying fermentation samples are encountered as a result of the strain sensitivity to antibiotics which may be present in them (Robbins et al., 1953). This test is recommended for vitamin BI2 determination in serum.

Another protozoal species useful in bioassay for cobamides is Ochromonas malhamensis (Ford, 1953; Hutner et al., 1953) which has been, and still is, widely used since the finding that only 5,6-dimethylbenzimidazolecobamide stimulates its growth. Because of this benzimidazole-cob amide specificity, O. malhamensis is used in substitution of animal tests like chicks and others where, as in man, only these compounds are active.

The possibility of using chemical or physical methods for the estimation of the cobamides content in fermentation samples or other crude biological materials has been attempted. In these cases it is mandatory to perform preliminary separations of the cobamides from other substances contained in the samples by thin-layer or column chromatography (Tortolani et al., 1970a,b; Vogelmann & Wagner, 1973; Tortolani & Mantovani, 1974), or electrophoresis (Tortolani & Ferri, 1974). Afterwards, several classical meth­ods are available to assay isolated fractions or pure products; spectrophot­ometry in the visible spectrum (Farmaceutici Italia, 1971b; Celletti et al., 1976) or in the IR spectrum (Goldstein et al., 1974), atomic absorption (Whitlock et al., 1976), potentiometry (Goldstein & Duca, 1976) and enzymatic analysis (Hermann & Mueller, 1976; Schneider, 1979).

The radioisotope dilution method is widely used (Cooper et al., 1979; Begley

Microbial Production of Vitamin B12 275

& Hall, 1979; Beck, 1979). Also the application of HPLC for rapid corrinoid determination has been reported (Jacobsen et al., 1979).

11 BIOLOGICAL PROPERTIES

The biological properties of vitamin B12 are many and this has never been found for any other natural product; interesting biochemical, haematological, neurological and oncological aspects in man have been reported. This physiological versatility can be explained by the fact that vitamin B12 is involved in the functioning of numerous enzymes, which have vital roles in assuring a normal, undisturbed evolution of the metabolism.

11.1 Biochemical mechanisms

Historically, among vitamin B12-dependent enzyme reactions, the isomeriza­tion of L-glutamic acid to /3-methylaspartic acid is the most important one because its study led to the isolation of vitamin B12 coenzyme (Barker et al., 1958). This enzyme, found in Clostridium tetanomorphum, in Acetobacter suboxydans (Kato et aI., 1968), in Rhodopseudomonas sphaeroides and Rhodospirillum rubrum (Ohmori et al., 1971) was also investigated in the animal system (plasma of the sheep) where it was found to be vitamin B12-independent (Marston et al., 1961). On the other hand, among biochemi­cal disorders caused by vitamin Bl2-deficient diet in man, a decreased polyglutamate biosynthesis has been observed (Reed et al., 1979).

The degradation of propionic acid in higher organisms and its formation in bacteria is performed by methyl-malonyl-CoA which is present in a catalytic equilibrium with succinyl-CoA and regulated by a specific isomerase. Smith & Monty (1959) showed that in liver homogenates of vitamin B1rdeficient rats the methyl-malonyl-CoA-mutase activity was strongly reduced. Successive experiments performed on mitochondria from rat liver (Gurnani et al., 1960), beef liver (Stern & Friedman, 1960), sheep kidney (Lengyel et al., 1960) and extracts from Propionibacterium shermanii demonstrated the necessary pre­sence of adenosylcobalamin for the functioning of methyl-malonyl-CoA­mutase which has been isolated from Propionibacterium shermanii (Kellermeyer et al., 1964), from liver (Cannata et al., 1965) and more recently also reported for Rhodospirillum rub rum and Rhodopseudomonas sphaeroides (Friedrich, 1975).

The presence of methyl-malonyl-CoA-mutase in animal tissues has been reported by Cardinale et al. (1969) while its determination in leucocytes has been described by Whitaker & Giorgio (1973). High levels of this enzyme were observed in leucocytes of vitamin B12-deficient patients as in rat tissues affected by the same disorder. Degradation of methyl-malonyl-CoA through succinyl­CoA is of vital importance because of the toxicity of methylmalonic acid

276 C. Spa//o et aI.

(methylmalonylacidemia). Similarly a defective function of methylmalonyl­CoA racemase due to vitamin Bt2 deficiency causes accumulation of propionic acid which is also toxic (propionicacidemia). The ribonucleotide reductase, an enzyme catalyzing the reduction of ribonucleotides to desoxyribonucleotides has been demonstrated as vitamin Bt2-dependent in Lactobacillus leichmannii and in Euglena gracilis (Beck, 1968). Its vitamin B12 dependency in animals is still under discussion (Hopper, 1979).

In many micro-organisms and animals, the methionine synthetase which necessitates as methyl donor the 5-methylhydrofolic acid, is vitamin Bt2-dependent. The methioninsynthetases (methyltetrahydrofolic acid­homocysteinmethyltransferase) from Escherichia coli and from liver, are very similar and have identical coenzyme requirements. Some micro-organisms can perform this synthesis even through a vitamin Bt2-independent pathway. Similarly, animals may have an alternative way of methionine synthesis: rats deficient in methionine, folic acid and vitamin Bt2 respond to betaine and homocystein utilizing the enzyme betain-homocysteine methyltransferase which catalyses the CH3 transfer from betain to homocysteine under formation of methionine (Weissbach & Tylor, 1970).

Today, the mechanism generally accepted which explains the anti-anemic activity of vitamin Bt2 is based on the interrelationship between the vitamin, folic acid and methionine (Weissbach & Tylor, 1970). DNA synthesis necessitates thymidilic acid which is produced by thymidilatesynthetase and catalyzed by tetrahydrofolic acid derived from CHr-FH4 which in turn is originated by the vitamin Bt2-dependent methioninesynthetase.

In the absence of vitamin Bt2, folic acid is accumulated as CH3-FH4 and the FH4 amount falls under the permissible level needed for the normal function­ing of the entire cycle. In this case, vitamin Bt2 plays, in contrast to folic acid, a secondary role as anti-pernicious anemia factor. The mechanism here described is known as the 'folate trap'.

Vitamin Bt2 is also involved in the nucleic acid synthesis. In fact, experimental data from systems where this synthesis is particularly active like neoplastic mouse cells (Rotherham et al., 1971) and lymphocytes of vitamin Bt2-deficient patients (Haurani, 1973), showed the existence of the inter­relationship thymidine-vitamin Bt2-folic acid. Vitamin Bt2 participation in the functioning of methanesynthetase has been reviewed by Stadtman (1967) and is important insofar as there are production processes of vitamin Bt2 employing methanobacteria, currently under investigation (Florent & Ninet, 1979; Dumenil et al., 1979, 1981).

11.2 Absorption, transport, metabolism and elimination

The physiological pathway of vitamin Bt2 transport is rather well known: intrinsic factor (IF) secreted by the gastric mucosa picks up the minute amounts of vitamin Bt2 out of the huge bulk of food ingested and mixed thoroughly in the stomach. The firmly-bound vitamin Bt2 is well protected

Microbilll Production of Vitamin B12 277

against both the greediness of intestinal micro-organisms and loss in the stools and is safely transported down to the gastrointestinal tract until it reaches a section of the distal ileum, where it is reabsorbed by the intestinal mucosa. This process is mediated by specific receptors of the mucosa cells for the intrinsic-extrinsic-factor complex. Within the intestinal mucosa cells the intrinsic-extrinsic-factor complex is split, and vitamin Bt2 is now transported across the cell for reabsorption from its vascular contact side.

The IF (intrinsic factor of Castle; Castle et al. 1930) is a glycoprotein of 55 000-60 000 mol. wt showing variable sugar moieties according to the animal species bearing it. The IF of pigs and man are very similar and this fact helped much the elucidation of the absorption mechanism it plays. It is assumed that IF possesses two receptor sites, one for the vitamin and the other for the microvilli of the epithelial cells of the ileum. The absorption takes place at a neutral pH and in presence of Ca or Mg ions (Shinton, 1972). As for the formation of the IF-vitamin B12 complex, the presence of 5,6-benzimidazole seems essential (Friedrich, 1975). There are other vitamin Bt2 binder proteins isolated from the blood serum, named transcobalamins (TC). Among them TC I and TC II are the most important ones. The biological turnover of vitamin Bt2 in human liver is much lower than for other soluble vitamins, being its half-life over 12 months.

Vitamin Bt2 deficiency causes pernicious anaemia, a disease which in the past usually led to death within 1 month after detection and inevitably within 3 years.

As mentioned before, classical observations and experiments revealed the cause of the disease in the lack of intrinsic factor produced in the stomach. This factor was necessary for the reabsorption of vitamin B12. The anaemia is hyperchromic, i.e. there is more haemoglobin available than red cells. Maturation and division of erythroblasts are disturbed and the resulting cells are too few and too big, i.e. macrocytic and megalocytic. Similar deficiencies are seen in the neutrophyls which are hypersegmented and diminished in number. These anomalies can be reversed by parenteral injection of extrinsic factor (vitamin B12) which immediately releases the inhibition of cell matura­tion and corrects the anaemia in a few days. Patients affected by pernicious anaemia show a series of disorders like bone marrow depression, transverse myelitis (myelopathy), peripheral neuropathy and subacute combined de­generation (Reynolds, 1979). A report on clinical diseases related to de­ficiencies of vitamin B12 transport proteins is given by Hitzig et al. (1979).

12 FORMULATIONS, APPLICATIONS, ECONOMICS

Vitamin B12 deficiency is revealed best by its serum level, since experiments on the presence and determination of this compound showed that plasma and serum are primarily indicative of this disorder, other tissues or organs being affected only later on (Sullivan, 1970). The normal Bt2 serum level is 200-900 pg/ml and its total binding capacity is about 500-1100 pg/ml so that

278 C. Spalla et al.

the serum is saturated by only about 60% with vitamin B12. A serum B12 level of about 200 pg/ml is considered critical. Clinical deficiency shows serum concentrations of 150-80 pg/ml. Patients affected by leukemia show significant higher serum levels due to a TC I increase. The presence of several cobalamins in the plasma and the prevalence among them of methylcobalamin was demonstrated first by Lindstrand & Stahlberg (1963) and Lindstrand (1964). The optimal requirement of a healthy person for vitamin B12 is 0·5-1/-lg daily according to Herbert (1968). The normal daily turnover of vitamin B12 is 2·5/-lg. In order to have a reabsorbtion of l/-lg of vitamin from an oral dose of 20/-lg, an amount of IF factor is required capable of binding 5-10 /-lg.

A vitamin B12 nutritional-dependent deficiency is rare and observable only after about 10-20 years; it usually occurs in persons who live on a pure vegetable diet. Oral treatment of vitamin B12 deficiency caused by the absence or a defective function of IF allows a low level of reabsorbtion varying from 1 to 3% of a dosage of 100·000 /-lg. Therefore, a maintenance dose of about 300 /-lg daily of cyanocobalamin at intervals of 3-4 days is suggested. According to Berlin et al. (1968) even higher doses (5OO-1OO0/-lg daily) are suggested. Tablets of 10 and 100 mg are available. In case of parenteral treatments in presence of minor vitamin B12 deficiencies a dosage of 500-1000 /-lg of hydroxycobalamin injected 3-4 times at regular intervals within a year is suggested (Heinrich, 1970). Neuropathies caused by vitamin B12 deficiency need massive dosages of the order of 1000/-lg daily for the first week followed by 1000 /-lg twice a week for several months administered by injection (Friedrich, 1975). The assumption that vitamin B12 lacks side-effects even at high doses has to be revised.

Vitamin B12 is available in numerous forms and grades. As the US market is still using almost exclusively cyanocobalamin, the major portion of B12 material in the world keeps being distributed in that form. However, in Europe hydroxycobalamin is preferred since it shows a better uptake in the liver, less urinary excretion and a more sustained serum level than cyanocobal­amin (Heinrich, 1970) and permits a less frequent dosage regimen than needed for the cyano form. Hydroxycobalamin is often combined with cob amide (vitamin B12 coenzymes).

The pharmaceutical use of vitamin B12 varies considerably. In the United States injections of vitamin B12 used as a general boost against tiredness as well as a remedy for miscellaneous aches and pains are virtually unknown; in many other countries, particularly in France, Italy and Spain the above-mentioned indications are the predominant ones. On the other hand, the US is by far the single largest market on a per capita basis, for vitamin B12 used in dietary supplement formulations; straight vitamin B12 tablets are available in US stores in strengths of 100 /-lg. Vitamin B12 finds a fairly large use as feed supplement. Generally it is dosed into all animal feeds in Europe and the USA with the exception of ruminants. The dosage levels are of 10-30 mg/ton of feed for poultry, pigs and for calves as milk replacer.

The overall world market of vitamin B12, including both pharmaceutical and

Microbial Production of Vitamin B12 279

animal feed use, is around 3 tons/year which is not expected to increase in the near future. The bulk price varies from 3 to 4 US $/g. The main producers of this vitamin are Rhone-Poulenc and Roussel-Uelaf (France), Glaxo (UK), Merck (USA) and Medimpex (Yugoslavia).

ACKNOWLEDGEMENT

The help of Anna Tomassoni in the translation and typing of the text is acknowledged.

REFERENCES

Ansbacher, S., Hill, W. H., Tieman, J. W., Downing, J. F. & Caldwell, J. H., (1949). Microbially synthesized APF (animal protein factor). Fedn Proc., 8, 180.

Barker, H. A., Weisbach, H. & Smith, R. D. (1958). A coenzyme containing pseudovitamin B12. Proc. natn. Acad. Sci. U.S.A., 44, 1093-7.

Barrere, G., Geneste, B. & Satatier, A. (1981). Fabrication de la vitamine B12, l'amelioration d'un procede. Pour La Science, Novembre 1981, 56-60.

Beck, R. A., (1979). Essential prerequisites for the analysis of cyanocobalamin in biochemical complex samples using radiometric competitive binding assays. In Vitamin B 12 , ed. B. Zagalak & W. Friedrich. W. de Gruyter, Berlin, New York, pp.675-80.

Beck, W. S. (1968). Desoxyribonucleotide synthesis and the role of B12 in erythropoi­esis. In Vitamins and Hormones, Vol. 26, ed. R. S. Harris, G. G Wool, J. A. Loraine & K. V. Thimann. Academic Press, New York, London, pp. 413-42.

Begley, J. A. & Hall, C. A. (1979). Effect of residual extract products and the type of binders (R or IF) on serum vitamin B12 levels by radioisotope dilution assay. In Vitamin B 12 , ed. B. Zagalak & W. Friedrich. W. de Gruyter, Berlin, New York, pp.655-6.

Berlin, H., Berlin, R. & Brante, G. (1968). Oral treatment of pernicious anaemia with high doses of vitamin B12 without intrinsic factor. Acta Med. Scand., 184,247.

Bemhauer, K. & Friedrich, W. (1954). Uber die vitamine der B13 gruppe. Angew. Chem., 66,776-80.

Bonnett, R., Cannon, J. R., Johnson, A. W., Sutherland, L., Todd, A. R. & Smith, E. L. (1955). Structure of vitamin B12. Nature, Lond., 176,325-8.

Bray, R. C. & Shemin, D. (1963). On the biosynthesis of vitamin B12. J. Bioi. Chem., 238, 1501-8.

Brink, c., Hodgkin, D. C., Lindsey, J., Pickworth, J., Robertson, J. H. & White, J. G. (1954). Structure of vitamin B12. Nature, Lond. 174, 1169-7l.

Cannata, J. J. B., Focesi, A., Mazumder, R. & Ochoa, S. (1965). Metabolism of propionic acid in animal tissues. XII properties of mammalian methylmalonyl­coenzyme A mutase. J. BioI. Chem., 240, 3249-57.

Cardinale, G. J., Dreyfus, P. M., Auld, P. & Abeles, R. H. (1969). Experimental vitamin B12 deficiency: its effect on tissue vitamin B12-coenzyme levels and on the metabolism of Methylmalonyl-CoA. Archs Biochem. Biophys., 131,92-9.

Cary, C. A., Hartman, A. M., Dryden, L. P. & Likely, G. D. (1946). An unidentified factor essential for rat growth. Fedn Proc., 5, 128.

280 C. Spalla et aI.

Castle, W. B., Townsend, W. C. & Heath, C. W. (1930). Observations on etiologic relationship of achylia gastrica to pernicious anemia; nature of reaction between normal human gastric juice and beef muscle leading to clinical improvement and increased blood formation similar to etIect of liver feeding. Am. J. Med. Sci., 180, 305-35.

Celletti, P., Moretti, G. P. & Petrangeli, B. (1976). Determinazione spettrofotometrica ditIerenziale nel visibile di alcune cobalamine in presenza di sostanze interferenti. Farmaco, ed. prat., 31, 413-19.

Chinoin-Gyogyszer (1971). Crystalline coenzyme B12 by fermentation. French patent 2062367.

Cooper, J. P., Jonas, E. & Whithead, V. M. (1979). Vitamin B12 assay: an evolution of radiodilution assay using cobinamide to increase specificity. In Vitamin B 12 , ed. B. Zagalak & W. Friedrich, W. de Gruyter, Berlin, New York, pp. 655-6.

Daniels, J. H. (1966). Inhibition of growth of Pseudomonas denitrificans by aminoacids. Can. J. Microbiol., 12, 1095-8.

Daniels, J. H. (1970). Some factors influencing vitamin B12 production by Pseudomonas denitrificans. Can. J. Microbiol., 16,809-15.

Davis, B. D. & Mingioli, E. S. (1950). Mutants of Escherichia coli requiring methionine or vitamin B12. J. Bacteriol., 60, 17-28.

Demain, A. L. & White, R. F. (1971). Porphyrin overproduction by Pseudomonas denitrificans: essentiality of betaine and stimulation of ethionine. J. Bacteriol., 107, 456-60.

Demain, A. L., Daniels, H. J., Schnable, L. & White, R. F. (1968). Specificity of the stimulatory etIect of betaine on the vitamin B12 fermentation. Nature, Lond. 220, 1324-5.

Di Marco, A. & Spalla, C. (1957). La produzione di cobalamine da fermentazioni con una nuova specie di Nocardia: Nocardia rugosa. Giorn. Microbiol., 4,24-30.

Di Marco, A., Boretti, G. & Spalla, C. (1961). Biosynthesis of cyanocobalamin in Nocardia rugosa. Sci. Repts. 1st. Sup. Sanita (Rome), 1, 355-67.

Dumenil, G., Cremieux, A., Couderc, R., Chevalier, J., Guiraud, H. & Ballarini, D. (1979). Production of Vitamin B12 by gram-variable methanol-utilizing bacteria. Biotechnol. Lett., 1, 371-6.

Dumenil, G., Cremieux, A., Chevalier, J. & Guiraud, H. (1981). Vitamin B12 formation by a gram-variable methanol-utilizing bacterium. Biotechnol. Lett., 3, 285-90.

Farmaceutici Italia (1971a). Cobamide preparation by fermentation of Nocardia rugosa 7892 Fi. French Patent 2,078,641.

Farmaceutici Italia (1971b). Cobalamin analysis of fermentation brews by automatic solvent extraction and photometric assay. French Patent, 2,083,992.

Florent, J. & Ninet, L. (1979). Vitamin B12. Microbial Technology, ed. H. J. Peppler & D. Perlman. Academic Press, New York, pp. 497-519.

Folkers, K. A. & Wolf, D. E. (1954). Chemistry of vitamin B12. In Vitamins and Hormones, Vol. 12, ed. R. S. Harris, G. F. Marriam & K. V. Thimann. Academic Press, New York, pp. 1-51.

Folkers, K. A., Robinson, F. M. & Shunk, C. H. (1957). Vitamin B12 und Intrinsic Factor, ed. H. C. Heinrich. Enke, Stuttgart, pp. 3-31.

Ford, J. E. (1953). Microbiological tests. Nature, Lond., 171, 149-50. Ford, J. E. & Holdsworth, E. S. (1952). An improved bioautographic technique.

Biochem. J., 53, XXII-XXIII. Ford, J. E. & Hunter, S. H. (1955). Role of vitamin B12 in the metabolism of

micro-organisms. In Vitamins and Hormones, Vol. 13, ed. R. S. Harris, G. F. Marriam & K. V. Thimann. Academic Press, New York, pp. 101-36.

Friedman, H. C. & Cagen, L. M. (1970). Microbial biosynthesis of Vitamin B12-like compounds. Ann. Rev. Microbiol., 24, 160-208.

Microbial Production of Vitamin B12 281

Friedrich, W. (1975). Vitamin B12 und verwandte Corrinoide. In Fermente, Hormone, Vitamine, Vol. 111/2, ed. R. Amann & W. Dirscherl. Georg Thieme, Stuttgart, pp. 1-360.

Friedrich, W. & Bernhauer, K. (1958). Die isolierung der Benzimidazol-und 5-Methyl­benzimidazol-cobalamin-Analoga aus Faulschlamm. Chem. Ber., 91,2061-5.

Goldstein, S. & Duca, A. (1976). Determination of cyanobalamin by thermal decomposition of the cyano group using an ion-selective electrode. J. Pharm. Sci., 65, 1831-3.

Goldstein, S., Ciupitoiu, A., Vasilescu, V. & Duca, A. (1974). Determination of Vitamin B12 by I.R. absorption spectrometry of the cyano-group in benzyl alcohol. Can. J. Pharm. Sci., 9, 96-8.

Guest, J. R., Friedman, S., Woods, D. D. & Smith, E. L. (1962). A methyl analogue of cobamide coenzyme in relation to methionine synthesis by bacteria. Nature, Lond., 195,340-2.

Gurnani, S., Mistry, S. P. & Johnson, B. C. (1960). Function of vitamin B12 in methylmalonyl metabolism I. Effect of a cofactor form of B12 on the activity of metbylmalonyl-CoA isomerase. Biochim. biophys. Acta, 38, 187-8.

Hartman, A. M. & Cary, C. A. (1946). Occurrence in foods, of an unidentified factor essential for rat growth. Fedn Proc., 5, 137.

Haurani, F. I. (1973). Vitamin B12 and the megaloblastic development. Science, IS2, 78.

Heinrich, H. C. (1970). Renale Vitamin B12-Ausscheidung. Dtsch. Med. Wschr., 95, 1134.

Herbert, V. (1968). Nutritional requirements for vitamin B12 and folic acid. Am. J. din. Nutr., 21,743-52.

Hermann, R. & Mueller, O. (1976). Enzymatische Vitamin B I2-Bestimmung. Hoppe Seyler's, Z. physiol. Chem., 357,1695-8.

Hitzig, W. H., Frater-Schroeder, M. & Seger, R. (1979). Clinical disease related to deficiencies of vitamin B12 transport proteins. In Vitamin B 12 , ed. B. Zagalak & W. Freidrich. W. De Gruyter, Berlin, New York, pp. 1009-16.

Hodgkin, D. C., Pickworth, J. & Robertson, J. (1955). Structure of vitamin B12. Nature, Lond., 176, 325-8.

Hodgkin, D. C., Kamper, J. Lindsey, J., Mackay, M., Pickworth, J., Robertson, J. H., Shoemaker, C. B., White, J. G., Prosen, R. J. & Trubelood, K. N. (1957). Structure of vitamin B12. Proc. R. Soc., 242, 228-63.

Hoover, S. R., Jasewicz, L. B. & Porges, N. (1951). Vitamin B12 in activated sewage sludge. Science, 114,213.

Hopper, S. (1979). An investigation of a possible role for coenzyme B12 in ribon­ucleotide reduction in rabbit bone marrow. In Vitamin B 12 , ed. B. Zagalak & W. Friedrich. W. de Gruyter, Berlin, New York, pp. 1025-8.

Hutner, S. H., Provasoli, L., Stokstad, E. L. R., Hoffmann, C. E., Belt, M., Franklin, A. L. & Juckes, T. H. (1949). Assay of anti-pernicious anemia factor with Englena. Proc. Soc. exptl. Bioi. Med., 70, 118-20.

Hutner, S. R., Provasoli, L. & Filfus, J. (1953). Nutrition of some phagotrophic fresh-water chrysomonads. Ann. N. Y. Acad. Sci., 56, 852-62.

Ilieva, M. (1971). Nauch. Tr., Vissh Inst. Khranit Vkusova Prom-st., Plovdiv., 18, 371-7.

Ilieva, M. & Popova, Y. (1974). Nauch Tr., Vissh. Inst. Khranit Vkusova Prom-st., Plovdiv., 21, 105-12.

Jackson, W. G., Whitefield, G. B. & De Vries, W. H. (1951). The isolation of Vitamin B12 from neomycin fermentations. J. Am. Chem. Soc., 73,337-41.

Jacobsen, D., Green, R. & Savage, M. (1979). Rapid determination of corrinoids by high performance liquid chromatography. In Vitamin B 12 , ed. B. Zagalak & W. Friedrich. W. de Gruyter, Berlin, New York, pp. 663-4.

282 C. Spalla et al.

Kaczka, E. A., Wolf, D. E. & Folkers, K. (1956). Vitamin B12 analogs and processes for preparing the same. US Patent 2,738,302, Merck and Co., Inc.

Kaken Kagaku, K. K. (1965). Japanese Patent 65/4,440. Kato, R, Hayashi, M. & Kamikubo, T. (1968). Isolation of 5,6-

Dimethylbenzimidazolylcobamide coenzyme as a cofactor for glutamate formation from Acetobacter suboxydans. Biochim. biophys. Acta, 165,233-7.

Kellermeyer, R W., Allen, S. H. G., Stjernholm, R & Wood, H. G. (1964). Methylmalonyl isomerase. IV. Purification and properties of the enzyme from propionibacteria. J. Bioi. Chem., 239,2562-9.

Koike, 1. & Hattori, A. (1975). Growth yield of a denitrifying bacterium Pseudomonas denitrificans under aerobic and denitrifying conditions. J. Gen. Microbiol., 88, 1-10.

Kojima, 1., Salo, H., Maruhashi, K. and Fujiwara, Y. (1976). Abstr. Int. Ferment. Symp. 5th, 1976, No. 7-04, p. 134.

Krieger, J. H. (1973). Vitamin B12: the struggle toward synthesis. Chem & Eng. News, 51, 16-19, 25, 27, 28.

Kyowa Hakko Kogyo Co. Ltd (1970). Vitamin B12 by fermentation. Japanese Patent 70/36159.

Lago, B. D. & Demain, A. L. (1969). Alternate requirement for vitamin B12 or methionine in mutants of Pseudomonas denitrificans, a vitamin B12 producing bacterium. J. Bacteriol., 99,347-9.

Lengyel, Rajarshi & Ochoa, S. (1960). Metabolism of propionic acid in animal tissues. VI Mammalian methylmalonyl isomerase and vitamin B12 coenzymes. Proc. natn. Acad. Sci. U.S.A., 46, 1312-19.

Lillie, R J., Denton, C. A. & Bird, H. R. (1948). Relation of vitamin B12 to the growth factor present in cow manure. J. BioI. Chem., 176, 1477-8.

Lindstrand, K. (1964). Isolation of Methylcobalamin from natural source material. Nature, Lond., 204, 188-9.

Lindstrand, K. & Stahlberg, K. G. (1963). On vitamin B12 forms in Human Plasma. Acta Med. Scand., 174,665.

Long, R A. (1962). Production of vitamin B12. U.S. Patent 3,018,225, Merck and Co., Inc.

McDaniel, L. E. (1961). Production of cobalamins. US Patent 3,000,793: Merck and Co., Inc.

McGinnis, J., Stevens, J. M. & Groves, K. (1947). The in vitro synthesis of a chick growth promoting factor in hen feces. Poultry Sci., 26,432-3.

Marston, H. R, Allen, S. H. & Smith, R M. (1961). Primary metabolic defect supervening on Vitamin B\2 deficiency in sheep. Nature, Lond., 190, 1085-91.

Marvyn, L. & Smith, E. L. (1964). The biochemistry of vitamin BI2 fermentation. Progr. Industr. Microbiol., 5, 152-201.

Maugh, T. H. (1973). Vitamin B12: after 25 years, the first synthesis. Science, 170, 266-7.

Miller, 1. M. & Rosenblum, C. (1960). Production of vitamin B\2 using delta­aminolevulinic acid. US Patent 2,939,822: Merck and Co., Inc.

Nakao, Y., Hisano, K., Kanemaru, T. & Yamano, T. (1974). Vitamin B12 production by cultivation of Rhodopseudo monas and Corynebacterium bacteria in hydrocarbon medium. Japanese Patent 74/15796: Takeda Chemical Ind. Ltd.

Ohmori, H., Ishitani, H., Sato, K., Shimizuki, S. & Fukui, S. (1971). Vitamin BI2 dependent glutamate mutase activity in photosynthetic bacteria. Biochem. biophys. Res. Commun., 43, 156-62.

Pantskhava, E. S., & Bykhovsky, V. Y. (1966). Prod. Mikrobnogo Sin., Akad. Nauk Latv. SSR Inst. Mikrobiol. 81-94: Chem. Abstr., 67 (1967) 10335.

Perlman, D. (1959). Microbial synthesis of cobamides. Adv. Appl. Microbiol., 1, 87-122.

Perlman, D. (1967). Microbial production of therapeutic compounds. In Microbial

Microbial Production of Vitamin B12 283

Technology, ed. H. J. Peppler. Van Nostrand-Reinhold, New Jersey, pp.283-7.

Pierce, J. V., Page, A. C. Jr, Stokstad, E. L. R. & Jukes, T. H. (1949). Crystallization of vitamin B 12b. J. Am. Chem. Soc., 71, 2952.

Pierce, J. V., Page, A. C. Jr, Stokstad, E. L. R & Jukes, T. H. (1950). Studies of some characteristics of vitamin B 12b. J. Am. Chem. Soc., 72, 2615-16.

Pierrel, S. p. A. (1963a). Hydroxocobalamine from cyanocobalamine by ion exchange. French patent 1,325,304.

Pierrel, S. p. A. (1963b). Pure hydroxocobalamine from natural materials by ion exchange. French Patent 1,325,308.

Pierrel, S. p. A. (1965). Preparation of hydroxycobalamin and cyanocobalamin (II) by cultivating Propionibacterium Freundenreichii or Shermanii in a nutrient medium to produce (II) and its analogues. British Patent 1,007,972.

Pliva, T. F. K. P. (1964). Production of vitamin B12 by a bacterial culture of Propionibacterium vanielli petko vic grown on a nitrogen containing medium. Belgian Patent 651,153.

Prescott, S. C. & Dunn, C. G. (1959). The production of Vitamin B12. In Industrial Microbiology, 3rd edn. McGraw-Hill, New York, pp. 482-96.

Reed, B., Dinn, J., McCannon, S., Wilson, P., O'Sullivan, H., Wein, D. G. & Scott, J. M. (1979). Alterations in mammalian cells induced by inactivation of vitamin B12 with nitrous oxide. In Vitamin B 12 , ed. B. Zagalak & W. Friedrich, W. de Gruyter, Berlin, New York, pp. 1061-4.

Reynolds, E. H. (1979). The neurology of vitamin B12 deficiency. In Vitamin B 12 , ed. B. Zagalak & W. Friedrich. W. de Gruyter, Berlin, New York, pp. 1001-8.

Rickes, E. L., Brink, N. G., Koniuszy, E. R., Wood, T. R & Folkers, K. (1948a). Crystallin Vitamin B 12. Science, 107, 396.

Rickes, E. L., Brink, N. G., Koniuszy, F. R, Wood, T. R. & Folkers, K. (1948b). Vitamin B12 cobalt complex. Science, 108, 134.

Robbins, W. J., Hervey, A. & Stebbins, M. E. (1950). Studies on Euglena and vitamin B12. Science, 112, 455.

Robbins, W. J., Hervey, A. & Stebbins, M. E. (1953). Euglena and vitamin B 12. Ann. N. y. Acad. Sci., 56, 818-30.

Rotherham, J., Price, F. M., Otani, T. T. & Evans, V. J. (1971). Some aspects of the role of vitamin B12 and folic acid in DNA-thymidine synthesis in a neoplastic C3H mouse cell strain. J. natn. Cancer Inst., 47, 277.

Rubin, M. & Bird, H. R. (1946). A chick growth factor in cow manure-I. Its non-identity with chick growth factors previously described. J. BioI. Chem., 24, 1155-6.

Sahashi, Y., Iwamoto, K. & Hayashi, J. (1953). Biosynthesis of vitamin B12 in various organisms. J. Biochem., 40, 227-44.

Schindler, O. and Reichstein, T. (1952). Isolierung von Vitamin B12 aus Leberextrakt sowie aus Ruckstanden der Streptomycinifabrikation. He/v. Chim. Acta, 35, 307-16.

Schneider, Z. (1979). Enzymatic estimation of Vitamin B 12. In Vitamin B 12 , ed. B. Zagalak & W. Friedrich. W. de Gruyter, Berlin, New York, pp. 673-4.

Shemin, D. & Bray, R. C. (1964). The biosynthesis of corrin structure of vitamin B 12. Ann. N. Y. Acad. Sci., 112, 615-21.

Shinton, N. Y. (1972). Vitamin B12 and folate metabolism. Br. Med. J., 1,556-9. Shorb, M. S. (1947). Unidentified essential growth factors for Lactobacillus Lactis

found in refined liver extracts and in certain natural materials. J. Bacteriol., 53, 669. Shorb, M. S. (1948). Activity of Vitamin B12 for the growth of Lactobacillus lactis.

Science, 107,397-8. Shorb, R S. & Briggs, G. M. (1948). The effect of dissociation in Lactobacillus lactis

cultures on the requirement for vitamin B 12 • J. Bioi. Chem., 176, 1463-4.

284 C. Spalla et aI.

Sifa (1964). 5,6-dimethylbenzimidazole-cobamide-coenzme. French Patent 1,368,892. Skeggs, H. R., Huff, J. W., Wright, L. D. & Bosshardt, D. K. (1948). The use of

Lactobacillus leichmanii in the microbiological assay of the animal protein factor. J. Bioi. Chem., 176,1459-60. .

Smith, E. L. (1948a). Purification of anti-pernicious anaemia factors from liver. Nature, Lond., 161,638-9.

Smith, E. L. (1948b). Presence of cobalt in the anti-pernicious anaemia factor. Nature, Lond., 162, 144-5.

Smith, E. L. (1950-51). Nutrition. Abstract and review, 20, 795-809. Smith, E. L. (1965). Hydroxocobalamin. US Patent 3,167,539: Glaxo Lab., Ltd. Smith, E. L. & Ball, S. (1953). Nitrous acid treatment for separating impurities from

antipernicious anaemia active material. US Patent 2,630,401. Smith, R. M. & Monty, K. (1959). Vitamin B12 and propionate metabolism. Biochem.

biophys. Res. Commun., 1,105-9. Speedie, J. D. & Hull, G. W. (1960). Cobalamin producing fermentation process. US

Patent 2,951,017. Stadtman, T. C. (1967). Methane fermentation. Ann. Rev. Microbiol., 21, 122-40. Stern, J. R. & Friedman, D. C. (1960). Vitamin B12 and methylmalonyl CoA

isomerase. Biochim. biophys. Res. Commun., 2,82-7. Stockstad, E. L. R., Page, A. C. Jr, Pierce, J., Franklin, A. L., Jukes, T. H., Heinle,

R. W., Epstein, M. & Welch, A. D. (1948). Activity of microbial animal protein factor concentrates in pernicious anaemia. J. Lab. Clin. Med., 33, 860-64.

Sullivan, L. W. (1970). Differential diagnosis and management of the patient with megaloblastic anemia. Am. J. Med., 48,209.

Tortolani, G. & Ferri, P. G. (1974). Electrophoretic separation of vitamin B12 derivatives. J. Chromatogr., 88,430-33.

Tortolani, G. & Mantovani, V. (1974). SP-Sephadex column chromatography of cobalamins contained in liver extracts. J. Chromatogr., 92,201-6.

Tortolani, G., Bianchini, P. & Mantovani, V. (1970a). Separation and determination of cobalamins on an SP-Sephadex column. J. Chromatogr., 53,577-9.

Tortolani, G., Bianchini, P. & Mantovani, V. (1970b). Separazione mediante cromat­ografia su colonna di varie cobalamine. Farmaco, ed. prat., 25, 772-5.

Uelaf (1960). French Patent 1,264,016. Vogelmann, H. & Wagner, F. (1973). Elutionschromatographische Trennungen von

Corrinoiden an dem Unpolaren Adsorbenz Amberlite XAD-2. J. Chromatogr., 76, 359-79.

Wagman, G. H., Gannon, R. D. & Weinstein, M. J. (1969). Production of vitamin B12 by Micromonospora. Appl. Microbiol., 17,648-9.

Weissbach, H. & Tylor, R. T. (1970). Roles of vitamin B12 and folic acid in Methionine synthesis. In Vitamins and Hormones, Vol. 28, ed. R. S. Harris, P. L. Munson & E. Diczfalusy. Academic Press, New York, London, pp. 415-40.

Whitaker, T. R. & Giorgio, A. J. (1973). A direct radioassay of methylmalonyl­coenzymeA mutase using enzymatically synthesized DL-314C methylmalonyl-CoA. Analyt. Biochem., 53, 522-32.

White, R. F. & Demain, A. L. (1971). Catabolism of betaine and its relationship to cobalamin overproduction. Biochim. biophys. Acta, 237, 112-19.

Whitlock, L. L., Melton, J. R. & Billings, T. J. (1976). Determination of vitamin B12 in dry feeds by atomic absorption spectrophotometry. J. Assoc. Analyt. Chem., 59, 580-81.

Wolf, D. E. & Folkers, K. A. (1954). Vitamin B12-II. Chemistry. In The Vitamins, Vol. 1, ed. W. H. Serbell & R. S. Harris. Academic Press, New York, pp. 397-417.

Chapter 16

MICROBIAL PRODUCTION OF OROTIC ACID (VITAMIN B,3)

K. TAKAYAMA* & A. FURUYA

Kyowa Hakko Kogyo Co, Ltd, Tokyo Research Laboratories 3-6-6, Asahi­machi, Machida-shi, Tokyo, 194, Japan

ABBREVIATIONS

ATC, aspartate transcarbamylase; AU, 6-azauracil; CPS, carbamyl phosphate synthetase; CTP; cytidine-trisphosphate. DHO, dihydroorotase; DHOdeh, dihydrooroatate dehydrogenase; OA, orotic acid; OMP, oritidine 5'-mono­phosphate; OMPdec, OMP decarboxylase; OPRT, orotate phosphoribosyl­transferase; OR, orotidine; PRPP, phosphoribosylpyrophosphate; pyr, pyrimi­dine; UMP, uridine-monophosphate; ura, uracil

1 INTRODUCTION

Orotic acid was first discovered in cow's milk in 1905 and was later found to accumulate as an intermediate in the biosynthetic pathway of pyrimidine nucleotides in a variety of mutants of micro-organisms. On the other hand, a similar substance-which was a growth factor for rats, chickens and lactic acid bacteria-had been isolated from distillers' dried solubles and had been known as vitamin B13. It was proved in 1953 that orotic acid and vitamin B13 are identical. Orotic acid is generally formed in mammals, so it is not a vitamin in a strict sense.

Several reviews have been published on the production of orotic acid (Furuya, 1976; Enei, 1984; Kuninaka, 1986) and the biosynthesis of pyrimidine compounds (O'Donovan & Neuhard, 1970; Shiio, 1972).

In this chapter, recent aspects of the microbial production of orotic acid by the de novo pathway will be described.

2 HISTORICAL

Orotic acid has a complex history. It was isolated from the whey of cow's milk (Biscaro & Belloni, 1905) and subsequently found to be present in the milk of

* Present address: Central Research Laboratory, Shikishima Baking Co. Ltd, 3-5, Shirakabe, Higashi-ku, Nagoya, 461 Japan.

285

286 K. Takayama & A. Furuya

various mammals. It was not until 1930 that the chemical structure of orotic acid was perfectly known (Bachstez, 1930). Further investigations on the compound were carried out late in the 1940s. Mitchell and co-workers (1947, 1948) observed that one of the pyrimidine-requiring mutants of Neurospora utilized orotic acid instead of the pyrimidines, while the others, which did not utilize orotic acid, accumulated large quantities of orotic acid in a culture medium. Afterwards, isotopic experiments led to the conclusion that orotic acid was the important intermediate in the biosynthetic pathway of pyrimidine nucleotides.

On the other hand, a growth factor for rats and chickens, was discovered from distillers' dried solubles (DDS) and named vitamin B13 (Novak & Hauge, 1948). Moreover, Wright et al. (1950) observed that DDS was also effective in promoting the growth of Lactobacillus bulgaricus and orotic acid could substitute for DDS. Subsequently, evidence has been given that vitamin B13 was identical to orotic acid (Manna & Hauge, 1953).

The accumulation of orotic acid by mutants of several species of bacteria was reported, but the amounts accumulated were very low. In 1961, the accumula­tion of orotic acid by a uracil-requiring mutant of Micrococcus glutamicus (synon. Corynebacterium glutamicum) (a glutamic acid-producing micro­organism), was reported (Tanaka et al., 1961; Kinoshita & Tanaka, 1963; Konishita et al., 1963). This was the first report aimed at the industrial production of orotic acid by fermentation procedure. Studies on the microbial production of orotic acid have mainly been reported by Japanese researchers.

The industrial production of orotic acid by fermentation started in the middle of the 1970s in Japan.

3 CHEMISTRY AND ASSAY

3.1 Chemistry

Chemical structure and properties of orotic acid (1,2,3,6-tetrahydro-2,6-dioxo-4-pyrimidinecarboxylic acid; uracil-6-carboxylic acid) and oritidine (3-{J-o­ribofuranosylorotic acid, 6-carboxyuridine) are shown in Fig. 1 and Table 1. Data are quoted from the Merck Index (10th edn, 1983). Potassium, sodium,

H~ H

H~~ ~COOH

HOH'd HO OH

(a) (b)

Fig. 1. Chemical structure of orotic acid (a) and orotidine (b).

Microbial Production of Vitamin B13

Table 1 Properties of Orotic Acid and Oritidine

Property Orotic acid

Molecular weight 156·10 Melting point 340-45°C

Absorption max. 282 nm E 7680 (pH 4·4-7·2) Solubility Soluble in water

about 1·7 mg/ml

Orotidine

288·22 Turns brown near 200°C

but failed to melt at 400°C 268nm 9570 (0·1 N HCl) Soluble in hot water,

lower aliphatic alcohols

287

magnesium, calcium and ammonium salts of orotic acid are scarcely soluble in water.

3.2 Assay

Aqueous solutions of orotic acid and orotidine can be easily assayed spectrophotometrically using the UV absorption property (282 and 268 nm, respectively). In the case of a culture broth or a mixture solution, UV detection combined with paper chromatography. TLC or HPLC is a very useful method for determination. With regard to HPLC, ODS column (reverse phase) is preferably applicable. Orotic acid can also be assayed selectively by a microbiological method using Lactobacillus buigaricus 09 (Wright et ai., 1950) and via a colorimetric method using dimethylaminobenzaldehyde (Adachi, 1963).

4 PRODUCING MICRO-ORGANISMS

Micro-organisms which accumulate orotic acid and/or orotidine through the de novo pathway are summarized in Table 2. Accumulation of orotic acid was first reported using a pyrimidine auxotrophic mutant of Neurospora and a maximal accumulation of 1· 3 mg of orotic acid per ml was obtained (Mitchell et ai., 1948). Thereafter, accumulation of orotic acid by Aerobacter aerogenes (Brooke et ai., 1954), Escherichia coli (Yates & Pardee, 1956) and Serratia marinorubra (Belser, 1961) were reported, but these studies focussed on the biosynthetic pathways of pyrimidine derivatives and the accumulated amounts were very low.

Later on, in 1961, it was reported that a uracil-requiring mutant of Corynebacterium giutamicum accumulated 14 mg/ml in a culture medium containing 10% glucose (Tanaka et ai., 1961). This strain was subsequently found to be deleted in orotate phosphoribosyltransferase. Nakayama et ai. (1965) examined the accumulation of orotic acid and orotidine by uracil­requiring mutants derived from the genera Corynebacterium, Brevibacterium,

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Microbial Production of Vitamin B13 289

Bacillus and Escherichia. Consequently, they found that C. glutamicum KY 9824 (ura- + ade-) and B. ammoniagenes KY 7349 (ura-) accumulated large amounts of orotic acid; the maximal amount was 6·0 mg/ml by the latter.

Skodova & Skoda (1969) and Skodova et al. (1969) also reported the accumulation of orotic acid using a uracil-requiring mutant of B. am­moniagenes CCEB 394, obtaining 5·8 mg/ml in a medium containing 5% glucose, and also found a deletion of orotate phosphoribosyltransferase in the mutant. Watanabe et al. (1968) observed the accumulation of orotic acid by an adenine or hypoxanthine-requiring mutant of Candida tropicalis and obtained a maximal accumulation of 7 mg/ml in a medium. Aspartic acid was recognized to be effective as a precursor. Ozaki et al. (1972) found that a uracil-leaky mutant of Streptomyces showdoensis (a showdomycin producer) accumulated orotic acid and orotidine, obtaining 1·2 and 0·8 mg/ml respectively, and confirmed the considerably decreased activity of OMP decarboxylase.

Research on the accumulation of orotic acid from n-paraffin has also been carried out. Kawamoto et al. (1970) observed the accumulation of orotic acid and orotidine, obtaining 6·7 mg/ml and 8·1 mg/ml, respectively, using a uracil-requiring mutant induced from Arthrobacter paraffineus, a hydrocarbon­utilizing bacterium. Takayama et al. (1971) also observed the accumulation of orotic acid and orotidine, obtaining 7·6 mg/ml and 1·0 mg/ml, respectively, using a uracil-requiring mutant induced from Candida lipolytica, a hydrocarbon-utilizing yeast.

Machida & Kuninaka (1969) and Machida et al. (1970) reported that wild-type strains of E. coli K-12 produced orotic acid, but other strains of E. coli did not. Later, Womack & O'Donovan (1978) investigated this observa­tion and concluded that this was caused by a mutation in the pyrF gene encoding OMP decarboxylase. Shimosaka et al. (1984) reported the excretion of orotic acid by an adenosine-sensitive (20 mM) strain induced from E. coli C-600, derived from the K-12 strain. They also found that this growth inhibitory effect was reversed by co-addition of pyrimidines to the medium; the mutant displayed a lower level (7%) of activity for orotate phos­phoribosyltransferase than a parent strain.

Recently, Doi et al. (1988) reported the accumulation of orotic acid and orotidine by a uracil-requiring mutant F-100 induced from Bacillus subtilis IFO 14386; the accumulation was 55 and 27 mM, respectively. Successively, they derived a uridine-producing mutant from the parent strain IFO 14386 which was assumed to display a potent pyrimidine de novo pathway.

5 BIOSYNTHESIS AND REGULATION

5.1 Biosynthesis of pyrimidine nucleotides

The de novo biosynthesis of pyrimidine nucleotides has been studied in detail in bacteria, especially Escherichia coli and Salmonella typhimurium, in fungi and

CO2

[CPS

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Microbial Production of Vitamin B13 291

in mammals. Several reviews have been previously presented (O'Donovan & Neuhard, 1970; Shiio, 1972; Enei, 1984). The pathway appears to be universal in all organisms. The de novo pathway of pyrimidine biosynthesis is shown in Fig. 2.

The atoms of the pyrimidine nucleus are derived from three simple precursors, CO2 , NH3 (from ammonia or glutamine) and aspartic acid. Carbamyl phosphate is an intermediate of pyrimidine and arginine biosynthe­sis. Orotic acid is formed via N-carbamyl aspartic acid and dihydroorotic acid. Orotic acid reacts with PRPP to OMP and successively decarboxylated to yield UMP, which is the starting substance for synthesis of cytidine and thymidine nucleotide.

Orotic aciduria is a genetic disorder of pyrimidine biosynthesis in man in which orotic acid accumulates in the blood and is excreted in the urine. The disorder is alleviated by the feeding of uridine or cytidine.

5.2 Regulation of the biosynthetic pathway of UMP

In Salmonella typhimurium and Escherichia coli, biosynthesis de novo of UMP is catalyzed by six enzymes encoded by six unlinked genes and operons. The first enzyme, carbamyl phosphate synthetase (CPS), is essential for both pyrimidine and arginine biosynthesis. Synthesis of this enzyme is regulated by cumulative repression exerted by a pyrimidine nucleotide and arginine. Synthesis of aspartate transcarbamylase (ATC) , orotate phos­phoribosyltransferase (OPRT) and OMP decarboxylase (OMPdec) are found to be repressed by uri dine nucleotides, whereas synthesis of dihydroorotase (OHO) and dihydroorotate dehydrogenase (OHOdeh) are found to be repressed primarily by cytidine nucleotides.

Carbamyl phosphate synthetase (CPS) is subjected to feedback inhibition by uridine nucleotides, especially UMP, and this inhibition is reversed by ornithine. Aspartate transcarbamylase (ATC) is sensitive to feedback inhibi­tion by CTP and to activation by A TP.

5-Fluorouracil is a powerful antimetabolite of pyrimidine nucleotides. A mutant of S. typhimurium resistant to a combination of 5-tluorouracil and 5-tluorouridine isolated by Jensen et al. (1982) possessed fourfold elevated pools of the pyrimidine nucleotide triphosphate. Furthermore, the specific activities of ATC and OPRT in the mutant were 40-fold and 7-fold higher than in the parent strain when grown in minimal media and the synthesis of these two enzymes was not repressed by pyrimidine compounds.

In Bacillus subtilis, the regulatory mechanism appeared to be considerably different from that of Salmonella (Doi et al., 1989). Synthesis of all six enzymes, catalyzing UMP biosynthesis, was strongly repressed by uracil compounds and CPS was inhibited by UMP. In a mutant strain resistant to uracil analogues such as 2-thiouracil or 6-azauracil, activities of the six enzymes increased to 16-30-fold higher than in the parent strain. Moreover, the

292 K. Takayama & A. Furuya

repression by uracil compounds to the six enzymes and the feedback inhibition by UMP to CPS were remarkably lost.

It has been observed that great differences exist for the regulation of the pathway as a whole, especially for ATC, even though a common pathway is found in all organisms (O'Donovan & Neuhard, 1970).

6 FERMENTATIVE PRODUCTION

6.1 Fermentation

Most orotic acid-accumulating strains require uracil for growth, hence the concentration of uracil in media severely affects the accumulation of orotic acid as well as cell growth. Figure 3(a) and (b) shows the remarkable effects of uracil on the accumulation of orotic acid by C. glutamicum and B. ammoniagenes, respectively (Nakayama et al., 1965). Maximal accumulations were obtained at concentrations of uracil which allowed the micro-organisms to grow to about half-maximal levels. Similar results were obtained with uracil-requiring mutants of other micro-organisms (Brooke et aI., 1954; Tanaka et al., 1961; Konishi et al., 1963; Kawamoto et aI., 1970; Ozaki et al., 1972; Doi et al., 1988).

For the accumulation of orotic acid by a mutant of Candida tropicalis, addition of aspartic acid to the medium is effective. The amino acid has been shown to be a metabolic precursor of orotic acid, and the accumulation of

3.0

_ 2.5

e ...... If

- 2.0 ., " ~

~ 1.5 o ."

"

( a)

~ 1.0 \ &... __ -cr--- ___ 00

~ \ jP', tJ '0, '~ E 0.5 )1 '_, o

o~~~~~ __ ~ __ ~_ o 50 100 150 Uracil (pg/ml)

2.8

_ 2.4

e ...... .. ~2.0 ., " ~ 1.6 o .. o

(b)

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Uracil(pg/ml)

1.0

0.8

0.6 :l 0.4 ~ ..

Q

0·.6

0.2

Fig. 3. Effect of uracil on orotic acid accumulation. (a) C. glutamicum KY 9824, (b) B. ammoniagenes KY 7349 . • , Orotic acid, /':,; orotidine; 0, Growth.

Microbial Production of Vitamin B13 293

orotic acid proceeds in parallel with aspartic acid consumption (Watanabe et al., 1968). However, such effects of aspartic acid were not observed in the case of C. glutamicum (Tanaka et al., 1961).

Recently, a mutant resistant to 5-ftuorouracil was derived from C. glutami­cum ATCC 14275 (a uracil-requiring strain) by Takayama & Matsunaga (1987). The activities of both carbamyl phosphate synthetase (CPS) and aspartate transcarbamylase (ATC) in the mutant were recognized to increase, comparing to the parent strain ATCC 14275 to some extent. By using the improved strain, about 40-50 mg orotic acid per ml was accumulated in a culture medium containing 17% glucose or other carbohydrates employing the technique of submersed culture with stirring and aeration for 3-4 days.

Accumulation of orotic acid and/or orotidine by uracil-requiring mutants can be ascribed to two genetic blocks, that is, orotate phos­phoribosyltransferase (OPRT) and OMP decarboxylase. Since nucleotides permeate poorly through cell membranes, it is assumed that OMP is excreted only after dephosphorylation to orotidine or further degradation to orotic acid. Therefore, when accumulation of orotidine in addition to orotic acid is observed, it is reasonable to assume a deletion of OMP decarboxylase. However, when accumulation of orotic acid alone is observed, it should be confirmed which one of the two enzymes is deleted.

6-Azauracil, a pyrimidine analogue, is a powerful inhibitor of OMP decarboxylase in micro-organisms in vivo. It exerts its effect only after being

Fig. 4. Bacterial cells and crystals of orotate in the orotic acid fermentation broth. (scanning electron micrograph).

294

(a)

(b)

CO,Et H,C" NH,

I + I EtO,CCO CSMe

HN-?

K. Takayama & A. Furuya

CO,Et 0

/ NH =f. NH CO,H NH, + I '--+ EtO,CHC . --+ I 1--+

EtO CH CO CO I HO,CHC=C~ _co , , --- N--":::-O 'N-

H,N H H

Fig. 5. Chemical synthesis of orotic acid.

o r NH Ho,clN)"o

H

converted to 6-aza-UMP, inhibits OMP decarboxylase, and causes the ac­cumulation of OMP, which subsequently inhibits OPRT. As a result, orotic acid is excreted into the medium.

6.2 Isolation and purification

Orotic acid in the fermentation broth usually exists as crystals of ammonium, potassium and/or sodium salt due to its limited solubility (Fig. 4). Crude orotate can be easily separated from the fermentation broth with repeated decantation of supernatant liquid. After this operation, the resulting precipi­tates of crude orotate are, for example, dissolved in a solution of hydrochloric acid or sulfuric acid at an elevated temperature and then the solution is filtered to remove impurities and an excess of acid. For further purification, treatments based on active carbon and on ion exchange resin are generally used.

7 CHEMICAL SYNTHESIS

Synthesis of orotic acid involving the condensation of diethyl oxalacetate with S-methylthiourea is believed to be the most practical method in the laboratory (Fig. 5(a». Hydantoin sometimes arises in reactions designed to make pyrimidines. Diethyl oxaloacetate and urea can be converted into orotic acid under suitable conditions (Fig. 5(b» (Brown, 1984).

8 APPLICATION

Orotic acid has been used as a hepatic drug. However, it may be contrarily said that large doses of orotic acid result in liver disturbance, because of the imbalance between purine and pyrimidine compounds.

Microbial Production of Vitamin B 13 295

Considerable quantities of orotic acid are applied in the field of medicine and in health food in the form of free orotic acid, or its calcium, magnesium, potassium or iron salt. The demand for orotate is estimated worldwide to be about 100 tons/year.

Orotic acid is an intermediate in pyrimidine biosynthesis, hence, expansion of the utilization of orotic acid for the synthesis of various types of pyrimidine compounds is expected in the near future.

REFERENCES

Adachi, S. (1963). A colorimetric determination of orotic acid. (I) Improvement of p-dimethylaminobenzaldehyde method. Vitamin (Japanese), 27, 433-7.

Bachstez, M. (1930). Constitution of orotic acid. Ber. Dtsch. Chem. Ges., 863, 1000-1007.

Belser, W. L. (1961). Uracil biosynthesis in Serratia marinorubra. Biochem. Biophys. Res. Commun., 4,56-60.

Biscaro, G. & Belloni, E. (1905). Sur un nouveau constituant du lait. Monit. Sci., 19, 384.

Brooke, M. S., Ushiba, D. & Magasanik, B. (1954). Some factors affecting the excretion of orotic acid by mutants of Aerobacter aerogenes. J. Bacteriol., 68, 534-40.

Brown, D. J. (1984). Pyrimidines and their benzo derivatives. In Comprehensive Heterocyclic Chemistry, Vol. 3, ed. A. R. Katritzky & C. W. Rees. Pergamon Press, Oxford, pp. 57-155.

Doi, M., Tsunemi, Y., Asahi, S., Akiyama, S. & Nakao, Y. (1988). Bacillus subtilis mutants producing uridine in high yields. Agric. Bioi. Chem., 52, 1479-84.

Doi, M., Asahi, S., Tsunemi, Y. & Akiyama, S. (1989). Mechanism of uridine production by Bacillus subtilis mutants. Appl. Microbiol. Biotechnol. (in press).

Enei, H. (1984). Fermentative production of pyrimidines, pyrimidine nucleosides and pyrimidine nucleotides. Hakko to Kogyo (Japanese), 42, 562-9.

Furuya, A. (1976). Production of other nucleic acid-related substances. In Microbial Production of Nucleic Acid-related Substances, ed. K. Ogata, S. Kinoshita, K. Aida & T. Tsunoda, Kodansha, Tokyo, pp. 184-91.

Handschumacher, R. E. (1958). Bacterial preparation of orotidine-5'-phosphate and uridine-5'-phosphate. Nature, Lond., 18'1., 1090-91.

Jensen, K. F., Neuhard, J. & Schack, L. (1982). RNA polymerase involvement in the regulation of expression of Salmonella typhimurium pyr genes. Isolation and characterization of fluorouracil-resistant mutant with high, constitutive expression of the pyrB and pyrE genes due to mutation in rpoBe. The EMBO Journal, 1, 69-74.

Kawamoto, I., Nara, T., Misawa, M. & Kinoshita, S. (1970). Fermentative production of orotic acid and orotidine from hydrocarbon. Agric. Bioi. Chem., 34, 1142-9.

Kinoshita, S., Tanaka, K. (1963). Production of orotic acid by fermentation method. US Patent, 3 086 917.

Kinoshita, S., Tanaka, K. & Kimura, K. (1963). Production of orotic acid by fermentation method. Japanese Published Examined Patent Application, S38-9950.

Konishi, S., Shiro, T. & Takahashi, M. (1963). Accumulation of orotidine by auxotrophic mutant of Bacillus subtilis. Ann. Mtg. Agr. Chem. Soc. Japan, Abstract (Japanese), p. SO.

Kuninaka, A. (1986). Nucleic acids, nucleotides and related compounds. In Biotechnology, Vol. 4, ed. H. J. Rehm & G. Reed. VCH, Weinhein, pp. 108-9.

296 K. Takayama & A. Furuya

Machida, H. & Kuninaka, A. (1969). Studies on the accumulation of orotic acid by Escherichia coli K12. Agric. Bioi. Chem., 33, 868-75.

Machida, H., Kuninaka, A. & Yoshino, H. (1970). Studies on the accumulation of orotic acid by Escherichia coli K12. Part 2. Mechanism of the accumulation. Agric. Bioi. Chem., 34, 1129-35.

Manna, L. & Hauge, S. M. (1953). A possible relationship of Vitamin B13 to orotic acid. J. Bioi. Chem., 202,91-6.

Merck Index, 10th edn (1983). Merck & Co., Rahway, NJ, USA. Mitchell, H. K. & Houlahan, M. B. (1947). Investigation on the biosynthesis of

pyrimidine nucleotides in Neurospora. Fedn Proc., 6,506-9. Mitchell, H. K., Houlahan, M. B. & Nyc, J. F. (1948). The accumulation of orotic acid

by a pyrimidineless mutant of Neurospora. J. Bioi. Chem., 172,525-9. Nakayama, S., Sato, Z., Tanaka, H. & Kinoshita, S. (1965). Accumulation of orotidine

and orotic acid by pyrimidine-auxotrophic mutant of micro-organisms. J. Agric. Chem. Soc., Japan (Japanese), 39,118-22.

Novak, A. F. & Hauge, S. M. (1948). Isolation of the unidentified growth factor (Vitamin, B13) in distillers' dried solubles. J. BioI. Chem., 174,647-51.

O'Donovan, G. A. & Neuhard, J. (1970). Pyrimidine metabolism in micro-organisms. Bacteriol. Rev., 34,278-343.

Ozaki, M. & Kimura, T. (1972). Mechanism of the accumulation of orotic acid and orotidine by a mutant of Streptomyces showdoensis. Amino Acid· Nucleic Acid, 26, 31-3.

Ozaki, M., Tagawa, S. & Kimura, T. (1972). The accumulation of orotic acid and orotidine by a mutant of Streptomyces showdoensis. Amino Acid. Nucleic Acid, 26, 24-30.

Shiio, I. (1972). Regulation of nucleotide biosynthesis. Seikagaku (Japanese), 44, 7-21. Shimosaka, M., Fukuda, Y., Murata, K. & Kimura, A. (1984). Growth inhibition by

purine derivatives and its reversal by pyrimidine derivatives in a mutant of Escherichia coli K12. Agric. Bioi. Chem., 48, 1303-10.

Skoda, J. & Sorm, F. (1958). Accumulation of nucleic acid metabolites in Escherichia coli exposed to the action of 6-azauracil. Biochim. biophys. Acta, 28, 659-60.

Skodov3, H. & Skoda, J. (1969). Mechanism of overproduction of orotic acid by a mutant of Brevibacterium ammoniagenes. Appl. Microbiol., 17, 188-9.

Skodov3, H., Solinov3, H., Skoda, J. & Dyr, J. (1969). Orotic acid formation by pyrimidine-deficient mutants of Brevibacterium ammoniagenes. Folia Microbiol., 14,145-54.

Sugimoto, H., Iwata, T. & Ishiyama, J. (1962). Studies on phosphodiesterases produced by micro-organisms. (2) The influence of fluoride on the excretion of nucleic acid derivatives by fungi. J. Agric. Chem. Soc., Japan (Japanese), 36, 690-5.

Takayama, K. & Matsunaga, T. (1987). Production of orotic acid by fermentation method. Japanese Patent Application, S62-261715.

Takayama, K., Kawabata, T.& Abe, S. (1971). Production of orotic acid by fermentation method. Japanese Published Examined Patent Application, S51-6756.

Tanaka, K., Nakajima, Y. & Kinoshita, S. (1961). Accumulation of orotic acid by a mutant strain of Micrococcus glutamicus. Ann. Mtg. Agr. Chem. Soc. Japan, Abstract (Japanese), p. 14.

Watanabe, A., Tani, K. & Sasaki, Y. (1968). Accumulation of orotic acid by auxotrophic mutant of Candida tropicalis. Amino Acid. Nucleic Acid (Japanese), 18,9-14.

Womack, J. E. & O'Donovan, G. A. (1978). Orotic acid excretion in some wild-type strains of Escherichia coli K-12. J. Bacteriol., 136,825-7.

Microbial Production of Vitamin B13 297

Wright, L. G., Huff, J. W., Skeggs, H. R., Valentik, K. A. & Bosshardt, D. K. (1950). Orotic acid, a growth factor for Lactobacillus bulgaricus. 1. Am. Chern. Soc., 72,2312-13.

Yates, R. A. & Pardee, A. B. (1956). Pyrimidine biosynthesis in Escherichia coli. 1. BioI. Chern., 221, 743-55.

Chapter 17

MICROBIAL REACTIONS FOR THE SYNTHESIS OF VITAMIN C (L-ASCORBIC ACID)

V. DELlC, D. SUNIC & D. VLASIC

PLIVA Pharmaceutical, Chemical, Food And Cosmetic Industry, Research Institute, Zagreb, Yugoslavia

1 INTRODUCTION

Microbial conversion of several chemical substances deserves special attention, mainly due to its economical and ecological advantages as compared to conventional chemical routes. Industrial production of vitamin C is an example of sophisticated chemical manufacturing involving highly complex and expen­sive chemical and microbial technology. In this process only one step is mediated by micro-organisms. This review is to provide a complete discussion of results, methods and current ideas of L-ascorbic acid (vitamin C) biosynthe­sis by micro-organisms, particularly since this field has only rudimentally been covered in the literature (Razumovskaya, 1962; Kulhanek, 1970; Crawford & Crawford, 1980).

Many excellent articles have been published on L-ascorbic acid chemical synthesis, biosynthesis in plants and animals and its biological role in living organisms (Sebrell & Harris, 1967; Jaffe, 1984). All these interesting issues will only briefly be covered in this review, the main objective being to focus on micro-organisms involved in the biosynthetic steps and conditions to L-ascorbic acid as a final product.

2 HISTORY

L-Ascorbic acid was first isolated by the famous Hungarian biochemist A. Szent-Gyorgyi in 1928, under the name of 'crystalline hexuronic acid'. He used natural material such as ox adrenal cortex, orange juice and cabbage juice for the isolation of a new compound. In the process of conducting this work, Szent-Gyorgyi and his co-workers established the identity of 'hexuronic acid', e.g. the presence of an acidic group and the easy reversible nature of its oxidation. In addition they found that green paprika was a particularly rich

299

300 V. Delic, D. Sunic & D. VlaSic

Table 1 Some Historical, Production and Economical Data on L-Ascorbic Acid Development

Time period Events Production Price References (years) (t/yr) (US$jkg)

1928 First isolation of 'hexuronic Szent-Gyorgyi, 1928 acid'

1930-1934 Description of the structure. 0·05 7000 Reichstein & Grtissner, 1934 Reichstein's first and second chemical synthesis. First pro-ductions of vitamins by chemical synthesis; microbial oxidation as a step in chemical synthesis.

1937-1940 L-Ascorbic acid synthesis on 100-127 Jaffe, 1984 industrial scale.

1945 L-Ascorbic acid from Aspergil- Geiger-Hiiber & Galli, 1945 Ius niger; never reproduced.

Increasing of production c. 17 25·0 Jaffe, 1984. capacity in USA.

1953 Preparation of intermediates Yamazaki & Miki, 1953 by biosynthesis Yamazaki, 1953a, b, c

1953-1960 Many chemical and technical Crawford & Crawford, 1980 modifications; e.g. electro-chemical oxidation

Production in USA c. 3000 6·0 Jaffe, 1984 196(J....1966 Increasing production c.5000 4,0-4·5 Jaffe, 1984

c. 14% year. Total Ullmanns, 1983 production in the world.

1966-1973 Biosynthesis of L-ascorbic 3·25-4·25 Isono et al.. 1968 acid intermediates by Okazaki et al .. 1968 micro-organisms. Okazaki et al.. 1969

Kanzaki & Okazaki, 1970 1974 Inflation; increasing in the 7,15-8·30 Jaffe, 1984

raw material and energy Ullmanns, 1983 cost

1981 Total world production. 34 000-36 000 10·40 Ullmanns, 1983 1982 Biosynthesis of 2-keto-L- 12·0 Sonoyama et al .. 1982

gulonic acid by two-stage fermentation

1984 Biosynthesis of 2-KLG on a Kieslich, 1984 commercial scale in People's Republic of China.

1985 One step biosynthesis of 2-KLG Anderson et al.. 1985 by genetically modified Erwinia herbicola.

1987 Total world production. c.60000 11·00 1987-2000 Predictions: (a) growth of nominal

capacity of main world producers;" (b) intensive efforts on rDNA techniques; (c) use of bio-catalysts from modified enzymes and/or cells and (d) research on various bioreactors, e.g. membrane reactors.

" See listing of world producers in Table 4.

Microbial Production of Vitamin C 301

source of vitamin C (Svirbely & Szent-Gyorgyi, 1933). Detailed descriptions of 'hexuronic acid' led to the discovery of the correct structure of vitamin C, later on named ascorbic acid by Szent-Gyorgyi and Haworth (Crawford & Craw­ford, 1980).

These data, together with those derived from· the study of L-ascorbic acid oxidation products, led Hirst et al. (1933) to propose that this compound was 3-keto-L-sorbosone. The early results of L-ascorbic acid structure elucidation are summarized in several reviews (Haworth & Hirst, 1933; Hirst, 1939; Crawford & Crawford, 1980).

The importance of L-ascorbic acid in human health care was established soon after its discovery and caused tremendous interest in all aspects of its scientific and industrial development. Hence, it is not surprising that up to now L-ascorbic acid has been and still is one of the major products of interest to the pharmaceutical, chemical and food industries. The main stages of the history of L-ascorbic acid over the past 60 years are listed in Table 1.

Its medical importance and the role of L-ascorbic acid in human health have been very well documented in over 22 000 publications that have appeared in the literature from 1966 to 1984 (Jaffe, 1984). Since humans and some animals lack L-gulono-y-lactone oxidase, the key oxidizing enzyme in liver for L-ascorbic acid biosynthesis, they have to consume vitamin C from exogenous sources. The main symptoms of L-ascorbic acid deficiency, mainly due to a dietary lack of fresh fruit and vegetables, are general weakness, fatigue and listlessness followed by a shortage of breath and aching in the bones. As the illness-known from ancient times as scurvy-progresses, the skin becomes dry and rough followed by a swelling of the gums, easy bleeding and a loss of teeth. Obviously the lack of L-ascorbic acid affects many different metabolic processes in living cells such as collagen synthesis, amino acid synthesis, immune response, etc., although the precise mechanisms of its actions are not clear. L-Ascorbic acid, an important reducing agent functioning as an elec­tron carrier, plays an important role in the respiration and oxidative reactions in the cells.

3 CHEMICAL AND PHYSICAL PROPERTIES

3.1 Nomenclature

Names previously used for L-ascorbic acid were: cevitamic acid, hexuronic acid, redoxon, scorbutamin, vitamin C and L-xylo-ascorbic acid. Throughout this review, the name L-ascorbic acid will be used.

In 1965 the trivial name ascorbic acid or L-ascorbic acid was recommended by the IUPAC-IUB Commission on Biochemical Nomenclature as a name for vitamin C. The systematic name for L-ascorbic acid is L-threo-hex-2-enonic acid y-lactone or L-threo-hex-2-enomo l,4-lactone (IUPAC-IUB, 1965).

302

3.2 Properties

V. Delic, D. Sunic & D. VlaJic

6

H0'qCH20H 5 0 4 , =0 or __

HO OH

H~" OH 0 ,~ -0

HO H H --

HO OH (b)

H~O'" H 0 H '--H"'" H"'" __ =0

HO HO OH

(a)

Fig. 1. Chemical constitution of L-ascorbic acid.

L-Ascorbic acid is white, crystalline solid melting at 192°C. Specific rotation in water at the sodium D line is +21·5°C. In solution, L-ascorbic acid has: pKl of 4·17, pK2 of 11·57. The most acidic proton is that of the 3C-hydroxyl group. Crystals of sodium and calcium L-ascorbate have the metals associated with 0-3. Reversible oxidation takes place at + 127 m V.

Infrared, ultraviolet, IH-nuclear magnetic resonance (nmr) and 13C-nmr spectra have all been reported (see: Schauenstein et al., 1948; Billman et al., 1972; The Merck Index, 1983). Ogawa et al. (1977), studied the conformation of L-ascorbic acid in deuterium oxide by 13C-nmr spectroscopy. They con­cluded that in aqueous solution the most favoured rotamer of L-ascorbic acid is in the form (b) (Fig. 1). The rotamer is not present in the crystalline state of L-ascorbic acid. The approximate conformation in the crystal is shown by structure form (a) (Fig. 1).

Elucidation of L-ascorbic acid stereochemistry played an important role in the determination of its stereomolecular structure and in the development of chemical synthesis for industrial production. The configuration of L-ascorbic acid was assignment to the L-series and confirmed by synthesis from o-glucose and L-xylose. Chiral centers at C-2 and C-3 of these sugars were in the correct position to become C-5 and C-4 of L-ascorbic acid, respectively (Reichstein & Griissner, 1934). Other carbohydrates, e.g. L-gulose, L-idose, L-galactose and L-talose have the correct chirality at C-4 and C-5 to be potential raw materials for the synthesis, but their high cost makes them impractical.

4 BIOSYNTHESIS IN PLANTS AND ANIMALS

Since fruit and vegetables are the main natural sources of L-ascorbic acid, biosynthetic pathways in plants were subjected to extensive studies. It has been shown that L-ascorbic acid is a product of hexose phosphate metabolism including conversion of D-glucose or o-galactose to L-ascorbic acid (Fig. 2).

Microbial Production of Vitamin C

o-Glucose o-Galactose

UDP-o-Glucose

1 UDP-Glucuronic acid

D-Glucuronic acid o-Galacturonic acid

1 1 L-Gulonic acid L-Galactonic acid

1 j L-Gulono-y-Iactone

~ L-Gulono-y-Iactone

1 L-Galactono-y-Iactone

1 L-2-Keto-galactono-y-lactone

1 L-Ascorbic acid

(a)

o-Glucose

I

I

I 1

o-Glucuronic acid

1 L-Gulonic acid

j

303

L-Gulono-y-lactone

1 L-2-Keto-gulono- y-lactone

j L-Ascorbic acid

(b)

Fig. 2. Proposed routes of L-ascorbic acid biosynthesis in plants (a) and animals (b), involving inversion of configuration; UDP-uridine diphosphate.

Studies by using radioactive o-glucose C4C-1 of 14C_6, respectively) have shown that the main six-carbon chain is conserved and radioactivity found at C-1 or C-6 atoms of L-ascorbic acid. In this retention of configuration, oxidation occurs at C-1, followed by lactonization, then again oxidation at C-2 and C-3, and finally epimerization at C-5 to give L-ascorbic acid (Loewus, 1980). Alternative biosynthetic pathways involve configurational inversion in which the main precursor is turned to L-galactono-y-Iactone, more active than L-gulono-y-Iactone, which is oxidized at C-2 to give L-ascorbic acid (in o-glucose series epimerization at C-3 occurred prior to the last oxidation).

Biosynthesis of L-ascorbic acid in animals is somewhat simpler (Fig. 2). In mammals, L-ascorbic acid is synthesized in the liver via the glucuronic acid

304 V. Delif, D. Sunif & D. Vlalic

pathway, likewise in plant biosynthesis. Studies with radioactive labelled D-glucose have shown that 14C_1 (or 14C_6) became the 14C_6 (or 14C_1) atom of L-ascorbic acid until the D-glucose chain remained intact (Bums, 1967).

5 BIOSYNTHESIS WITH MICRO-ORGANISMS

After 1896, when Bertrand for the first time described microbial oxidation of sorbitol to sorbose by the micro-organism Bacterium xylinum (subsequently classified as Acetobacter xylinum) (Bertrand, 1896), more than 30 years passed until this microbial reaction was used as a biooxidation step in L-ascorbic acid synthesis. Afterwards, research in the field of microbial biosynthesis of L-ascorbic acid intermediates became more intensive and further knowledge accumulated quickly.

During the past 50 years there have been a lot of attempts to find ways of microbial conversion of intermediates in L-ascorbic acid synthesis which could compete with the Reichstein-Griissner synthesis from an economical point of view. Different micro-organisms were screened for their ability to carry out reactions on carbohydrates as well as methods for new biosynthetic steps in those routes. These reactions, micro-organisms and media are listed in Table 2. L-Ascorbic acid intermediates biosynthetic routes, including micro-organisms, are shown in Fig. 3.

In this review we present names of micro-organisms as reported in the original articles. Since their first presentation in the literature, the taxonomy of these micro-organisms has undergone many changes due to their morphologi­cal, cultural and biochemical properties, e.g. Acetobacter suboxydans has been renamed Gluconobacter oxydans subsp. suboxydans. t For actual names of micro-organisms see Bergey's Manual of Systematic Bacteriology (1984). Most of the micro-organisms mentioned here are mutants with blocked side reactions in 2-keto-L-gulonic acid biosynthetic routes from carbohydrates, which could utilize alternative carbon sources.

5.1 Biooxidation of L-sorbose to 2-keto-L-gulonic acid

In the classical Reichstein-Griissner synthesis (see p.323), L-sorbose is con­verted to 2-keto-L-gulonic acid (2-KLG) by chemical reactions. In this part we are describing the possibilities of L-sorbose conversion to 2-KLG by micro­organisms (Fig. 3, routes 2, 3).

The first biooxidation of L-sorbose to 2-KLG by micro-organisms was described by Tengerdy (1961a, b) who used UV-irradiated mutant strains of Pseudomonas. After 4 days of cultivation in the medium containing 20 g/liter

t 'Names included in the Approved List of Bacterial Names are the only names which are nomenclaturally valid as at the 1st January, 1980. All other names which have appeared in the literature prior to 1st January, 1980 are nomenclaturally invalid' (Skerman et af., 1980).

Tab

le 2

M

icro

-org

anis

ms

and

Med

ia U

sed

in B

iosy

nthe

sis

of

L-A

scor

bic

Aci

d In

term

edia

tes

Mic

ro-o

rgan

ism

s C

onve

rsio

n M

edia

, co

nditi

ons

Ref

eren

ces

(yie

ld)

Ace

toba

cter

D

-Sor

bito

l ~ L

-sor

bose

su

boxy

dans

(>

95

%)

Ace

toba

cter

S

orbi

tol ~ 2

-KL

G

30 l

iter

s o

f th

e m

ediu

m c

onta

inin

g so

rbit

ol 5

; gl

ucos

e 0·

5;

Oba

ta e

t aI.,

197

5 IF

O 3

243

(4%

) ye

ast

ext.

0·5

and

CaC

03

2%

. 28

-29°

C,

150

h,

aera

tion

15-

24 li

ters

/min

P

seud

omon

as s

p.

L-Sorbose~ 2

-KL

G

Gly

cero

l 1;

L-s

orbo

se 2

0; c

om

ste

ep l

iquo

r 10

; N

Z a

min

e T

enge

rdy,

196

1a,

b ~

(15%

) B

5;

K2H

P0

4 X

3H

20

0·7;

KH

2P0

4 0·

3; N

aCl

0·5;

1';

' a M

gS04

x 7

H20

0·

1 an

d F

eS04

x 7

H20

0·

1 g/

lite

r. 1

0 0

-

lite

rs o

f m

ediu

m i

n 14

-lit

er g

lass

fer

men

tors

was

a

inoc

ulat

ed w

ith

2% c

ell

susp

ensi

on f

rom

ino

culu

m

~ m

ediu

m.

28°C

; p

H 7

·0-7

·2;

dow

sil

icon

e oi

l s::.

.. I::

(0

· 25

g/li

ter)

was

use

d as

ant

ifoa

m;

96 h

; ae

rati

on 1

v /v

<"

I g. pe

r m

in;

stir

red

350

rpm

::s

Pse

udom

onas

L-Sorbose~ 2

-KL

G

KH

2P0

4 0·

04;

MgS

04 x

7H

20

0·02

; N

Z-a

min

e B

0·2

5;

Hua

ng,

1962

~

viri

difla

va

yeas

t ex

t. 0·

5 an

d so

rbos

e 2

%,

28°C

, ae

rati

on 1

v/v

per

~

NR

RL

B-9

4

min

; p

H 8

,1-4

day

s iii ::I

Glu

cono

bact

er

L-S

orbo

se ~ 2

-KL

G

L-S

orbo

se 7

; gl

ycer

ol 0

·5;

yeas

t ex

t. 0·

5; M

gS04

x 7

H2O

T

suka

da &

s·

mel

anog

enus

(2

%)

0·5

and

CaC

03

1%

. 25

°C,

pH

6·5

, 22

0 rp

m,

96

h

Per

lman

, ("

)

IFO

329

3 19

720

Glu

cono

bact

er

L-Sorbose~ 2

-KL

G

L-S

orbo

se 7

; ur

ea 0

·5-0

·8;

K2H

P0

4 0·

007;

KH

2P0

4 Y

in e

t al.,

198

0 ox

ydan

s (4

0%)

0·00

3; g

lyce

rol 0

·2;

MgS

04 x

7H

20

0·01

; C

aC0

3 0·

5 N

1197

A

and

com

ste

ep l

iquo

r 0·

5%.

pH

6·0

-6·5

. A

ddit

ion

of

Yan

et a

l., 1

981

urea

or

(NH

4)zH

P0 4

dur

ing

ferm

enta

tion

G

luco

noba

cter

L-Sorbose~ 2

-KL

G

L-S

orbo

se 1

00;

glyc

erol

0·5

; ye

ast

ext.

15·

0 g/

lite

r; s

alts

. F

ujiw

ara

et a

i.,

oxyd

ans

U-1

3 (6

4%)

Cul

tiva

tion

on

rota

ry s

hake

r, 3

0°C

, 4 d

ays

1987

P

seud

oglu

cono

-L-Sorbose~ 2

-KL

G

1000

lit

ers

of

the

ferm

. m

ediu

m c

onta

inin

g L

-sor

bose

15;

N

ogam

i et

ai.,

198

7 ba

cter

sac

char

o-(7

6%)

CaC

03

5; c

om

ste

ep l

iquo

r 2;

dri

ed y

east

0·3

; A

ctco

l ke

toge

nes

0·03

; (N

H4)

2S04

0·0

3; N

a 2S 2

03 x

5H20

0·

5 an

d V

l IF

O 1

4464

F

eS0 4

x 7

H20

0·

1 %

. 0

(con

tinu

ed)

U\

Tab

le 2

-con

td.

w ~

Mic

ro-o

rgan

ism

s C

onve

rsio

n M

edia

, co

nditi

ons

Ref

eren

ces

(yie

ld)

Bac

illus

30

°C, 1

10 r

pm,

aera

tion

900

lite

rs/m

in,

pres

sure

m

egat

eriu

m

0·5

kg/c

m2 ,

4 d

ays,

123

·1 m

g/m

I2-K

LG

for

med

IF

O 1

210S

P

seud

omon

as

Sorbose~ L

-ido

nate

S

orbo

se 5

; com

ste

ep li

quor

2·5

; M

gS04

x 7

H20

0·0

1;

Moc

hizu

ki e

t al.

, ae

rugi

nosa

K

H2P

04

0·03

; K

2HP

04

0·07

; N

aCl 0

·05

and

glyc

erol

19

69a

0·05

%.

Aer

atio

n, 2

SoC

, 12

0h

Glu

cono

bact

er

L-S

orbo

se ~ L

-sor

boso

ne

L-S

orbo

se 7

; gl

ycer

ol 0

·5;

yeas

t ext

. 0·

5; M

gS04

x 7

H2O

K

itam

ura

&

mel

anog

enus

(6

-8%

) 0·

5; C

aC0

3 1 %

. p

H 6

·8, 3

0°C

, 250

rpm

, 7

days

P

erlm

an,

1975

:0::

:: IF

O 3

293

.t::;)

Pse

udom

onas

L

-Sor

boso

ne ~ 2

-KL

G

Gly

cero

l 2·5

; N

a-ci

trat

e 5·

0 g/

lite

r; m

iner

al s

alts

; 2S

oC,

Mak

over

& P

rues

s,

~

pulid

a (2

0%)

over

nigh

t; f

resh

med

ium

(9·

5 m

l) c

onta

inin

g. L

-19

76

.:>'

sorb

oson

e 10

g/l

iter

was

ino

cula

ted

with

0·5

ml

of th

e ~

old

med

ium

, 28

°C

'-l.

I::

P

seud

omon

as

Ca-L-idonate~ C

a-2-

KL

G

Ca-

L-i

dona

te;

mal

tose

(or

glu

cose

) 3;

com

ste

ep li

quor

3;

Gra

y, 1

947a

;:

~.

,""

mild

enbe

rgii

KH

2P0

4 0·

3 an

d M

gS04

x 7

H20

0·1

par

t; 5

0 pa

rts

of

R-P.

mild

enbe

rgii

(gro

wn

on 5

% g

luco

se a

nd 0

·5%

~

yeas

t).

pH

5·5

-6·0

;S

P

seud

omon

as

Ca-

L-i

dona

te

2-ke

to-

Idon

ic a

cid

250;

glu

coni

c ac

id 2

50;

NH

40

Ac

12;

K3P

04

Yam

azak

i, 19

55

~ flu

ores

cens

C

a-n-

gluc

onat

e ~ h

exon

ic

5; M

gS04

1 g

; 10

lite

rs H

20.

pH

6·5

; 2

-3 d

ays;

AcO

H

,""

(1: 1

) ac

ids

adde

d to

pH

5·5

, kep

t 7-S

day

s (4

0%)

Pse

udom

onas

Ca-idonate~ 2

-KL

G

Ca-

idon

ate

and

Ca-

gluc

onat

e (7

: 3)

Fuj

isaw

a et

al.,

flu

ores

cens

(7

7%)

50; ~OAc 1

·2;

KH

2P0

4 0·

5; M

gS04

x 7

H20

0·3

; 19

63a,

b

FeS

0 4 x

7H

20 O

·S g

/lit

er.

Aer

atio

n, 6

day

s P

seud

omon

as

Ca-L-idonate~ 2

-KL

G

Ca-

L-i

dona

te 7

; C

a-n-

gluc

onat

e 3;

yea

st e

xt.

2·5;

T

aked

a et

al.

, ae

rugi

nosa

T-S

1 (S

O%

) (N

H4)

2HP0

4 0·

2; K

H2P

04

0·1

and

MgS

04 0

·025

%.

1964

28

°C,

225

rpm

, 3

days

A

ceto

bact

er

Ca-L-idonate~ 2

-KL

G

Glu

cose

0·5

; co

rn s

teep

liqu

or 1

·0;

KH

ZP0

4 0·

1;

Moc

hizu

ki e

t al.,

m

elan

ogen

us

(27%

) M

gS04

x 7

H20

0·0

5; C

a-5-

keto

-glu

cona

te

1969

b hy

drog

enat

ion

prod

uct 3

4% (

cont

aini

ng id

onic

aci

d 14

·7 a

nd g

luco

nic

acid

7·2

%);

1 li

ter.

2So

C,

shak

e cu

ltur

ed, 7

6 h,

40

g 2-

KL

G f

orm

ed

Ace

toba

cter

C

a-L

-gu

lon

ate-

Ca-

2-K

LG

C

a-L

-gul

onat

e 12

0; c

om s

teep

liqu

or 5

; oc

tade

cyl a

le.

Gra

y, 1

947b

su

boxy

dans

(7

5%)

0·3;

mal

tose

or

sorb

itol

5 a

nd H

20

1000

par

ts;

pH

6·

0-6·

1. 5

0 pa

rts

of in

ocul

um;

25°C

; ae

rate

d; 8

day

s;

90 p

arts

Ca-

2-K

LG

for

med

X

anth

omon

as

L-g

ulo

nat

e-2-

keto

-L-

4 L

iter

sti

rred

fer

men

ter

cont

aini

ng 2

lite

rs o

f pr

oduc

tion

K

ita,

1979

tr

ansl

ucen

s gu

lona

te

med

ium

con

tain

ing

corn

ste

ep li

quor

8;

mea

t di

gest

6;

AT

CC

107

68

(85%

) (N

H4)

zHP

0 41;

glu

cose

10;

CaC

03

3 an

d C

a-L

-gu

lona

te 8

0 g/

lite

r. A

fter

fer

m.

at 2

8-30

°C f

or 4

8 h

with

sti

rrin

g an

d ae

rati

on,

40 g

/lit

er N

a-L

-gul

onat

e fe

rm.

brot

h w

as a

dded

fol

low

ed b

y an

add

nl.

30 g

/lit

er

at -

72

h

Bre

viba

cter

ium

5

-KD

G-

L-i

doni

c ac

id

Cel

ls o

f lo

g ph

ase

wer

e su

spen

ded

in 4

00 m

l of

0·1

M

Sono

yam

a et

at.

, ~

keto

sore

duct

um

phos

phat

e bu

ffer

(pH

6·8

6) m

ixed

with

200

ml

of 6

%

1974

1=

;' a F

ER

M-P

190

5 K

-sal

t of

5-K

DG

and

hel

d at

30°

C f

or 4

8 h

(J-

(AT

CC

219

14)

~

Ace

toba

cter

G

luco

se-

5-ke

togl

ucon

ic

Glu

cose

10%

, 33

h S

tubb

s et

al.

, ~

subo

xyda

ns

acid

19

40

s::..

I:

(90%

) g.

Ace

toba

cter

sp.

G

luco

se-

5-K

DG

G

luco

se 1

0-15

; ye

ast

0·75

-1·0

%;

Ter

amot

o et

al.,

;:s

(89%

) 26

°C, 7

day

s 19

46

~

Ace

toba

cter

o

-Glu

cose

-2,5

-DK

G

Glu

cose

10;

yea

st e

xt.

0·5;

gly

cero

l 0·5

; M

gS04

x 7

H2O

S

tros

hane

&

~ m

eian

ogen

us

(93%

) 0·

5; C

aC0

31%

; pH

7·0.

25-

26°C

, 300

rpm

, 3

days

P

erlm

an,

1977

::!

AT

CC

9937

s·

Ace

toba

cter

o

-Glu

cose

-2,5

-DK

G

2 L

iter

s of

med

ium

(pH

6·0

) co

ntai

ning

glu

cose

110

; co

rn

Kita

& H

all,

(j

ceri

nus

(95%

) st

eep

liqu

or 0

·5;

(NH

4)zH

P0 4

0·5

8; K

H2P

04

1·5;

19

81b

IFO

326

3 M

gS04

x 7

H20

0·05

; ur

ea 0

·5 g

/lit

er;

Cu

S0

4 x

5H2O

1;

nic

otin

ic a

cid

0·3

mg/

lite

r. 1

; ni

cotin

ic a

cid

o· 3 m

g/li

ter.

28°

C,

36 h

, 17

00 r

pm,

aera

tion

o· 75

v /v

x

min

. A

n ad

diti

onal

55

g gl

ucos

e/li

ter

was

add

ed a

t 20

h

and

the

pH

was

mai

ntai

ned

at 5

·5

Erw

inia

sp.

o

-glu

cose

-C

a-2,

5-D

KG

o-

Glu

cose

5·8

; co

m s

teep

liqu

or 1

-13;

(N

H4)

zHP

0 4O

'56;

S

onoy

ama

et a

l.,

SHS

2629

001

(94·

5%)

CaC

03

18·4

; p-2

000

anti

foam

0·0

2%;

pH

6·8

. 19

82

der.

fro

m

1·86

m3/1

0 m

3;

160

rpm

; 3·

6 m

3 ai

r/m

in;

28°C

; 26

h.

SH

S20

06

Dur

ing

ferm

enta

tion

, 23

04 k

g 50

% (

wt/

wt)

o-g

luco

se

(AT

CC

316

26)

..., w

as a

dded

0 -..

,J

(con

tinu

ed)

Tab

le 2

-con

td.

~

Mic

ro-o

rgan

ism

s C

onve

rsio

n M

edia

, co

nditi

ons

Ref

eren

ces

(yie

ld)

Cor

yneb

acte

rium

C

a-5

-DK

G-C

a-2-

KL

G

o-G

luco

se 2

; com

ste

ep li

quor

3;

NaN

03

0·34

5; K

H2P

04

Son

oyam

a et

ai,

sp.

(92·

5)

0·06

7; p

-200

0 an

tifo

am 0

·001

67%

; Z

nS04

X 7

H20

4·9

; 19

82

SHS

7520

01

MnC

l 2 x

4H

20 0

·8;

thia

min

e hy

droc

hlor

ide

0·22

; C

a-de

r. f

rom

o-

pant

othe

nate

0·1

7 m

g/li

ter;

pH

6·9

; 28

°C;

160

rpm

; SH

S 00

07

1·18

air

/min

. N

aN0 3

, o-

gluc

ose

and

Ca-

2,5-

DK

G

(AT

CC

310

90)

adde

d du

ring

fer

men

tati

on

Citr

obac

ter

2,5

-DK

G-2

-KL

G

100

ml m

ediu

m c

ontg

. ce

relo

se 2

; (~)2HP04 1

; K

ita

& H

all,

:"'

fr

eund

ii (3

0%)

KH

2P0

4 1;

MgS

04 X

7H

20 0

·5;

beet

mol

asse

s 2

and

1981

a t::::

I ~

AT

CC

6750

gl

ycin

e 0·

2 g/

lite

r; p

H 6

·7.

Aft

er 2

2 h

15 m

l of a

n ~

Ace

toba

cter

cer

inus

fer

m.

brot

h co

ntg.

15-

20%

2,5

-!='

DK

G w

as a

dded

28°

C;

addn

l. 52

h;

pH

6·5

§

Erw

inia

o

-glu

cose

-2-K

LG

G

luco

se 3

; gl

ycer

ol 2

0; y

east

ext

. 5;

pep

tone

5;

CaC

0 3

And

erso

n et

al.

, he

rbic

ola

(33%

) 7·

5 g/

lite

r; p

H 7

·0.

1 g/

lite

r 2-

KL

G f

orm

ed

1985

~

Est

ell e

t al.,

198

5 Ro

Erw

inia

o

-glu

cose

-2-K

LG

50

0 m

l of m

ediu

m c

ontg

. gl

ucos

e 10

; o-

man

nito

l 20;

com

H

ardy

et a

l. ,

!=' ci

treu

s (5

0%)

stee

p li

quor

10;

yea

st e

xt.

1·5;

cas

amin

o ac

ids

10;

1987

~

ER

l11

6

K2H

P0

4 4;

KH

2P0

4 1;

~Cl1; C

aCI 2

0·01

and

~

K2S

04

2·6

g/li

ter

with

thr

ee f

urth

er a

ddit

ions

of

<"I,

Can

dida

G

alac

ton

ic-

L-a

scor

bic

10 g

/lit

er o

-glu

cose

. 28

°C;

800

rpm

; ov

er 6

0 h

Gal

acto

nic

acid

or

gala

cton

o-y-

Iact

one

0·5;

com

ste

ep

Rol

and

et a

l. ,

norv

egen

sis

acid

ac

id

liqu

or 0

·25;

~CI 0

·1;

glyc

ine

0·7;

MgS

04 x

7H

2O

1985

K

CC

MF

42

(8-9

%)

0·05

; N

a-gl

utam

ate

0·2;

EtO

H 1

·5%

; tra

ce m

etal

s;

pH

4.2

. 30

0C, s

tirr

ing,

aer

atio

n, 4

8 h.

0·4

3 g/

lite

r L-

asco

rbic

aci

d fo

rmed

Microbial Production of Vitamin C

PseudogluconOb~cter Si!cch.ilroketog~

~y~

CHO G. m.,.nog'''' fO

r~p'Ut;d. HO OH

HO ~ CH,OH

L - Sorbosone

HO}COOH

1~P.ut;d' ~X. tran"u"ns :: H

CHZOH

l-Gulonic aCid

CH20H CH20H

HOf

HOf ~g.nus mHO _HO ~~ OH OH

HO HO

CHO CHZOH

O-Glucose D-Sorbltol

p.dlerug~

~g~

L-Sorbose L-Sorbose diacetone

2-Keto- L­gulonlc .. cid diace-tone

® l HHOO~C:O~H COOH ~~HO~OH OH

HO CH20H CH20H

L - Idose L - Idonic acid

~glnosa

Po mildenber9li p tluorescens G mel.ilnogenus

A SUbOXy~ HOfC:~H B '.tosocedu'tum

OH o

1

5-Keto-D-

Erwmia Gluconobact~r

glueo,;, ILao_'d_-=:=:'n='~=~'=S~=-~==;'='~=: __

Acetobacter cerlnus AcetomonCiis albosesamae

Erwima cltreus

Erwinla herbrcola

{ CHZOH

<;Qrynebacterlum Brevibacterium Arthrobact~r

Micrococcus ~P...b):'lococcus Bacillus

~ Crtrobact~r freundir

2,5-0rKeto-O­gluconrc acid

HO~C~H_ HOfC~O=- ~~~C01 OH OH 0

HO HO HO

CH20H CH20H CH,DH 2-Keto-L- Methyl- L-Ascorbic gulonlc acid 2-Keto-L- Clcid

gulonate

309

Fig. 3. Biosynthesis of intermediates in L-ascorbic acid routes by different micro­organisms. 1, Reichstein-Griissner route; 2, L-idose route; 3, L-sorbosone route; 4,

5-keto-o-gluconic and L-idonic acid route; 5, 2,5-diketo-o-gluconic acid route.

L-sorbose, 13 g/liter of substrate was utilized and 3 g/liter of 2-KLG formed. Tengerdy (1961a, b) proposed the one-step reaction involving oxidation of the C-1 of L-sorbose to 2-KLG. Another Pseudomonas mutant (P. viridiflava NRRL B-94), which oxidized L-sorbose to 2-KLG with 50% yield, was isolated by Huang (1962).

Different genera of eubacteria were screened for their ability to oxidize sorbitol and L-sorbose. Isono et al. (1968) found that micro-organisms producing keto-sugar acid from sorbitol were restricted to the genera Acetobacter, Gluconobacter and Pseudomonas whereas those producing acids

310 V. Delic, D. Sunic & D. VlaIic

from L-sorbose were distri1?uted widely in the 12 genera: Acetobacter, Alcaligenes, Aerobacter, Azotobacter, Bacillus, Escherichia, Gluconobacter, Klebsiella, Micrococcus, Pseudomonas, Serratia and Xanthomonas. As shown by Mochizuki et al. (1969a), P. aeruginosa was able to oxidize sorbose only to L-idonic acid. Kanzaki & Okazaki (1970) suggested a multi-step conversion of L-sorbose to 2-KLG by Pseudomonas aeruginosa in which L-idose and L-idonic acid were intermediates. Pseudomonas aeruginosa IFO 3898 converted L­sorbose via L-idose and L-idonic acid to 2-KLG efficiently (Fig. 3, route 2).

There are only a few genera of micro-organisms capable of oxidizing sorbitol to 2-KLG. Some unidentified Acetobacter and Pseudomonas strains were able to produce 2-KLG from sorbitol (Takeda Chemical Industries, 1964). It was reported that these strains, after 150 h of fermentation in the medium containing 5% sorbitol, produced 2-KLG with an overall yield of 8-9%. Acetobacter sp. IFO 3243 cultivated in 30 liters of fermentation medium converted 4% sorbitol to 2-KLG (Obata et al., 1975).

Okazaki et al. (1968, 1969) studied the conversion of sorbitol to 2-KLG by means of G. melanogenus IFO 3292. After 7 days of cultivation in a medium containing 10% sorbitol, the fermentation broth was found to contain neutral and acidic compounds which were identified' as: L-sorbose, 5-keto-o-mannonic acid, 2-keto-o-gluconic acid, 2-KLG, L-idonic acid, o-mannonic acid, 0-

fructose and 5-keto-o-fructose. As a result of the accumulation of these compounds the authors proposed the following metabolic pathway in G. melanogenus: sorbitol--+ L-sorbose--+ L-idose--+ L-idonic acid--+ 2-KLG (Fig. 3, route 2).

In the early 1970s, a number of publications appeared describing the biooxidation of L-sorbose to 2-KLG by Gluconobacter meianogenus IFO 3293. Tsukada & Perlman (1972a, b, c) found conditions for the conversion of 2% of metabolized L-sorbose to 2-KLG and proposed the direct conversion of L-sorbose to 2-KLG. Later on, the 'sorbosone pathway' was found to be involved. Employing this route, L-sorbose was converted to 2-KLG via L-sorbosone as an intermediate (Fig. 3, route 3). The first step of this reaction was reversible and mediated, in both directions, by an enzyme(s) fraction of Pseudomonas putida ATCC 21812 (Makover et al., 1975).

Growing cultures or cell-free extracts of G. melanogenus IFO 3293 were also able to carry out this reaction (Kitamura & Perlman, 1975). The limiting factor of this route was the reversibility of the first step as well as the poor absorption of L-sorbose into the cells (Makover et al., 1975). Oxidation of L-sorbose to L-sorbosone was efficiently improved 2-3 times by the addition of organic solvents or detergents, due to the increased cell membrane permeability (Martin & Perlman, 1975).

Yin et al. (1980) and Yan et al. (1981) established the optimal fermentation medium for producing 2-KLG from L-sorbose by the bacterial strain Gluconobacter oxydans N 1197 A. In the medium containing 7 or 10% of L-sorbose and 0·5-0·7% of urea concentration, the yield of 2-KLG was about 30 or 37 g/liter, respectively.

Microbial Production of Vitamin C 311

It seems likely that researchers in the People's Republic of China are able to produce 2-KLG from L-sorbose (10 g/liter) over L-sorbosone on a commercial scale with a 90% yield using bacteria of the genera Gluconobacter and Bacillus (Kieslich, 1984; Lu et al., 1985; Ning et al., 1988).

Fujiwara et al. (1987) have used the G. oxydans strain, which has a high activity of L-sorbose dehydrogenase, to produce 2-KLG. After a 4-day cultivation of G. oxydans U-13 in the L-sorbose medium 2-KLG was formed with a 64% yield. Also, G. oxydans U-13 was able to produce 2-KLG during cultivation in o-sorbitol medium.

Recently, Nogami et al. (1987) reported the capability of Pseudo­gluconobacter saccharoketogenes IFO 14464 (novel genus, isolated from soil origin) and a concomitant bacterium Bacillus megaterium IFO 12108 to produce 2-KLG in a high yield (76%) during cultivation in a 2 m3 fermenter containing 1000 liters of L-sorbose medium. Bacillus megaterium, which was not directly involved in the oxidation process, promoted the growth of the oxidative strain (P. saccharoketogenes) and increased the yield of 2-KLG. (Fig. 3). Compared with a pure culture of the oxidative strain the mixed culture was able to oxidize higher concentrations of L-sorbose to 2-KLG in a shorter period of time. The bacteria of some other genera could be successfully used instead of B. megaterium, e.g. Pseudomonas, Proteus, Citrobacter, Enterobacter, Erwinia, Xanthomonas and Flavobacterium.

5.2 Biosynthesis of 2-keto-L-gulonic acid from 5-keto-D-gluconic and L­idonic acid

Chemical reduction of 5-keto-o-gluconic acid (5-KDG) resulted in a mixture of L-idonic and o-gluconic acid that could be converted to 2-KLG by various micro-organisms. The first microbial biooxidation of L-idonic acid to 2-KLG was reported by Gray (1947a), who found that Pseudomonas mildenbergii was capable of performing this reaction (Fig. 3, route 4). In this process L-idonic acid or its salts were prepared along with o-gluconic acid by a chemical reduction of the corresponding salts of 5-keto-gluconic acid. L-Idonic acid was subsequently biooxidized by P. mildenbergii to 2-KLG, while o-gluconic acid was oxidized back to 5-keto-gluconic acid by A. suboxydans. 5-Keto-gluconic acid was reused in the process.

Pseudomonas fluorescens oxidized well both calcium-L-idonate and calcium­o-gluconate (Yamazaki & Miki, 1953; Yamazaki, 1953a, b, c). After 7-8 days of cultivation in the medium containing the mixture of L-idonic and o-gluconic acid (ratio 1: 1) the producing strains of genus Pseudomonas were able to convert both of the added compounds to 2-keto-hexonic acids with a 40-50% yield. The main problem in this process was the isolation of 2-KLG from the mixture of 2-keto-hexonic acids obtained at the end of fermentation. 2-Keto­guion ate was isolated in a 50% yield (Yamazaki, 1953c, 1955). 5-Keto-o­gluconate, used in this procedure, was obtained by the biooxidation of o-glucose with A. suboxydans (Yamazaki, 1953a).

312 V. Delic, D. Sunic & D. VlaJic

With a different starting mixture of Ca-idonate and Ca-gluconate (7: 3, respectively) and after a 6-day cultivation of P. f/uorescens, Fujisawa et al. (1963a, b) obtained a 77% yield of 2-KLG based on L-idonic acid. Similar results were obtained by Alieva (1966) using the same micro-organism and by Takeda et al. (1964) using P. aeruginosa T-81 strain.

It seems likely that the ability for oxidation of L-idonic acid to 2-KLG is widely spread in genus Pseudomonas, e.g. P. aeruginosa, P. mildenbergii, P. chlororaphis and P. pseudomallei (Nakanishi et al., 1964a, b). Apart from genus Pseudomonas, some other micro-organisms have also been reported to oxidize L-idonic acid to 2-KLG (Shoemaker, 1956; Chas. Pfizer & Co., 1958: unidentified bacteria ATCC 11867 and ATCC 11868; Mochizuki et al., 1969b: Acetobacter melanogenus and A. suboxydans).

A new approach to L-ascorbic acid intermediate synthesis was applied by Sonoyama et al. (1974b). Instead of chemical agents, they used the cells or a crude enzyme preparation of Brevibacterium ketosoreductum sp. nov. FERM­P 1905 derived from ATCC 21914 to reduce 5-KDG to L-idonic acid.

5.3 Biooxidation of L-gulonic to 2-keto-L-gulonic acid

A combination of chemical and biochemical reactions for the preparation of 2-KLG from L-idonic acid was described by Gray (1947b). In this combination the first step was the chemical conversion of L-idonic to L-gulonic acid, while the second step was the biooxidation of L-gulonic acid to 2-KLG by A. suboxydans. After 8 days of cultivation 75% of L-gulonic acid was oxidized to 2-KLG. As shown by Perlman (1959), P. aeruginosa was able to oxidize even L-gulono-Iactone to 2-KLG in a 44% yield. Some strains of genus Xanthomonas were also able to oxidize L-gulonic acid to 2-KLG. For example, Xanthomonas translucens A TCC 10768 was cultivated in the medium contain­ing 80 g/liter calcium-L-gulonate (added twice during cultivation). In this instance the overall yield of 2-KLG was 85% (Kita, 1979).

5.4 Biosynthesis of 2-keto-L-gulonic acid from o-glucose

Studies on o-glucose biooxidation to keto-gluconic acids began 50 years ago. Bernhauer & Knobloch (1938, 1940) reported biosynthesis of 2-keto-gluconic and 5-keto-gluconic acids from glucose or gluconate by micro-organisms of genus Acetobacter (Fig. 3, route 4, 5). Stubbs et al. (1940, 1943) obtained a 90% yield of 5-keto-gluconic acid from 10% glucose in the medium with A. suboxydans and 82% with an unidentified strain. Teramoto et al. (1946) obtained identical yield of 5-keto-gluconic acid using an unidentified Acetobacter strain. As shown by Razumovskaya & Vasil'eva (1956), A. suboxydans was able to oxidize o-glucose not only to 5-keto-gluconic but also to diketo-gluconic acid.

Advancement along this route was achieved when Katznelson et al. (1953) established oxidation of o-glucose to 2,5-diketo-o-gluconic acid (2,5-DKG) by

Microbial Production of Vitamin C 313

Acetobacter melanogenus MA 6.2 without prior splitting of the o-glucose molecule. The route they proposed was glucose- gluconic acid- 2-keto­gluconic acid-2,5-DKG. The same route was studied by Wakisaka (1964a) who reported the microbial oxidation of glucose to 2,5-DKG by Pseudomonas sesami. Soon afterwards a new species of Pseudomonas genus (P. albosesamae) capable of a high production of 2,5-DKG from o-glucose was isolated (Wakisaka, 1964b, c). Acetobacter fragum (Oga et al., 1972) and A. cerinus (Kita & Hall, 1981b) were also able to produce 2,5-DKG under aerobic conditions in the medium containing o-glucose. As much as 95% of the starting o-glucose was converted to 2,5-DKG by A. cerinus. Stroshane & Perlman (1977) obtained 2,5-DKG in a 93% yield from o-glucose by A. melanogenus ATCC 9937.

The authors proposed a metabolic pathway which included o-gluconic acid and 5-KDG as intermediates. In this biosynthesis the main problem was the instability of 2,5-DKG and decomposition of up to 80% during the isolation process.

The procedure for 2-KLG biosynthesis from o-glucose was improved by Sonoyama et al. (1982, 1983b) who used a two-stage fermentation system (Fig. 4). They reported a process which included biosynthesis in 10 m3 conventional fermenters for the production of calcium-2-KLG from o-glucose over calcium-2,5-DKG by two different micro-organisms. The first step in this process was o-glucose oxidation to Ca-2,5-DKG by the mutant strain SHS 2629001 of Erwinia sp. derived from ATCC 31626 and, subsequently, the stereospecific reduction (to eliminate 2-keto-o-gluconic acid formation) of Ca-2,5-DKG to Ca-2-KLG by the mutant strain SHS 752001 of Corynebacterium sp. derived from ATCC 31090. At the end of cultivation, Erwinia sp. was treated with sodium dodecyl sulfate to decrease the number of viable cells so the broth with accumulated Ca-2,5-DKG could be used directly for the next reduction, mediated by Corynebacterium sp. Thus, in this process, the isolation of Ca-2,5-DKG was avoided. The total fermentation time for this two-stage process was about 100 hand Ca-2-KLG accumulated up to 106·3 mg/ml (84·6% yield from o-glucose). Media used in this process are listed in Table 3.

Compared to some other processes previously described in this review, a two-stage fermentation procedure for 2-KLG production has some advantages that make it a possible candidate for industrial application. These are: (a) high producing strains; (b) elimination of 2,5-DKG isolation from the broth; (c) commercially feasible media for both biosyntheses; (d) reliability and re­producibility and (e) a simplified process for 2-KLG isolation (absence of compounds like o-glucose, Ca-L-idonate, Ca-o-gluconate, Ca-5-KDG, Ca-2-KDG).

Results obtained by Sonoyama and his co-workers on this subject have been very well documented in a number of publications on the mixed cultures of Acetomonas albosesamae ATCC 21998 and Brevibacterium sp. ATCC 31083, as well as on the screening, isolation and description of micro-organisms capable of the oxidation of glucose to 2,5-DKG (Erwinia species) and

314 V. Delic, D. Sunic & D. VlaJic

~ 28'C. lOh. 275 rpm

~ .20 , •• <> of the seed tncdium in

:.:.:: :::.:::: l ~ml rermentor ..... : .. :

l 28"C. 10 h. z.w rpm; 0,(2 ml .ir/min.

1860 tilfa of 1M ferment. medium in 100mJ {ermen.or inoculated with the

cnlirc conterU of the seed fcrmc:nlc:r

3.6 m3 Iit/m.!n. (&Ilk prcuure 1.5 .... /an2; tdd.ition of D1lucosc .. 128"C' 26 h. ,60 rpm;

4200 Ijll'd ollhc: fermen ted broth: 328 m, Ca·2.5-DKGlmL Add.itJon of SOS. after 6 I'll the IQlal viabk; cell populldon docrc.&$Cd

!Torn 5 • 109 t. 9. ,03 c:cUsiml.

D·""""""dd«I as a hydrogen

do"".

11 ~~';7ri."IP' ~ (ATCC 31(90)

l28"'C . .ah

[j 10.600 ml 0/ pn:-

seed medium in a 2·ntrc=fluk

! 28"C. 2' h. 215 rpm

4DO I:llrCl oIlhc: fennent. medium in IO-m3 fennenlor inoculated with the entire content of tl'M: JCCd fennenu~:t

28"C. 18h . '60 rpm; 1-18 m1 lir/min: NINO) odQ"j ..

7430 litra of tbe fermented broth; U16 m& Ca-2-KLGlmi

Fig. 4. Flow-sheet for the production of 2-keto-L-gulonic acid by two-stage fermenta­tion; SDS, sodium dodecylsulfate; Ca-2,5-DKG, calcium-2,5-diketo-o-gluconate; Ca-2-

KLG, calcium-2-keto-L-gulonate.

Microbial Production of Vitamin C

Table 3 Media Used in the Biosynthesis of Calcium 2,S-Diketo-o-Gluconate and Calcium 2-Keto-L-Gulonate by Erwinia sp. SHS 2629001 and

Corynebacterium sp. SHS 725001

Media Concentration (g/liter)

Erwinia sp. Corynebacterium sp.

Agar slant o-glucose 5·0 glycerol 5·0 yeast extract 5·0 5·0 peptone 3·0 5·0 KH2P04 1·0 1·0 MgS04 x 7H2O 0·2 0·2 agar 20·0 20·0

Preseed medium o-glucose 10·0 glycerol 5·0 yeast extract 5·0 peptone 5·0 com steep liquor 30·0 NaN03 1·0 KH2P04 1·0 1·0 MgS04x7H2O 0·2 p-200 antifoam 0·1

Seed medium o-glucose 10·0 glycerol 5·0 com steep liquor 30·0 20·0 NaN03 2·0 KH2P04 1·0 1·0 MgS04 x7H2O 0·2 p-2000 antifoam 0·1 0·05

Fermentation medium o-glucose 58·0 20·0 com steep liquor 11·3 30·0 NaN03 3·45 KH2P04 0·67 (NH4)2HP04 5·6 CaC03 184·0 p-2000 antifoam 0·2 0·0167 ZnS04x 7H2O 4·9 mg/liter MnCl2 x 4H2O 0·8 mg/liter thiamine hydrochloride 0·22 mg/liter Ca-0-Pantothenate 0·17 mg/liter

315

316 V. De/if, D. Sunil & D. Vlalif

reduction of 2,5-DKG to 2-KLG, like Brevibacterium ketosoreductum, Bacillus megaterium, Arthrobacter simplex, Staphylococcus aureus, Micrococcus, Pseudomonas, etc. (Sonoyama et al., 1974a, 1976, 1983a, 1986).

Mutants of Corynebacterium sp. ATCC 31088, 31089 and 31090 with improved bioconversion abilities have been selected after N-methyl-N'-nitro­N-nitrosoguanidine mutagenic treatment. These mutants were blocked in degradative metabolic pathways of 2,5-DKG, reducing 2,5-DKG only to 2-KLG in a high molar yield with the addition of glucose as a hydrogen donor (Sonoyama et al., 1987a, b).

2-KLG is the end product of all described biosynthetic routes starting from D-glucose, L-sorbose or L-gulonic acid, respectively. This is the ultimate intermediate in L-ascorbic acid biosynthesis by micro-organisms (or chemicals). Subsequently, 2-KLG could easily be converted by chemical methylation and cyclization to L-ascorbic acid.

5.5 Direct Biosynthesis of L-Ascorbic Add

Direct biosynthesis by micro-organisms is the most challenging means of L-ascorbic acid synthesis. Until now, there have been only few articles describing micro-organisms and their ability for direct biosynthesis of L­ascorbic acid from different carbohydrates. Various groups of organisms were found to be able to produce L-ascorbic acid from different sugar substrates but concentrations of substrates and yields of L-ascorbic acid are still very low.

Geiger-Huber & Galli (1945) found that Aspergillus niger was able to synthesize L-ascorbic acid (1·5 mg/lOO ml) after 6-8 days of cultivation in the medium containing 15% sucrose. Sucrose was also a suitable substrate for L-ascorbic acid biosynthesis when Aspergillus flavus was used (Ramakrishnan & Desai, 1956). Sastry & Sarma (1957) cultivated A. niger in glucose medium. They proposed the following metabolic pathway as follows: D-glucose­D-glucuronic acid- L-gulono-Iactone- 2-keto-L-gulonolactone- L-ascorbic acid.

Recently, Roland et al. (1985, 1986) reported that strains of the yeast Candida (e.g. C. norvegensis KCC MF 42) were able to produce L-ascorbic acid during cultivation in the medium containing L-galactonic substrates, such as L-galactonic acid, L-galactonic acid esters or L-galactono-y-Iactone. Accord­ing to these results it seems likely that biosynthetic pathways in some yeasts and moulds are similar to those proposed for plants (see Fig. 2).

Some green algae also displayed the ability to produce L-ascorbic acid. Chlorella pyrenoidosa cultivated on a glucose medium for 101 h produced 1·5 g/liter of L-ascorbic acid (Skatrud & Huss, 1987).

5.6 Biocatalysts Involved in Biosynthesis of L-Ascorbic Add Intennediates

Recently, new methods applying biological catalysts-biocatalysts have been developed for conversion of different raw materials to desired end-products.

Microbial Production of Vitamin C 317

These new technologies and processes significantly reduce the cost of bio­synthetic production (Aiba et al., 1973; Rehm & Reed, 1981; Karube et al., 1984; Kulbe & Knopki, 1986).

These methods rely on preparation of biocatalysts such as immobilized enzyme(s), parts of cells or whole cells (Klein & Wagner, 1978; Scott, 1987; Brodelius and Vandamme, 1987). The biocatalysts can be used for the production of various substances in a single-step enzyme reaction (6-aminopenicillanic acid from penicillin G; fructose from glucose, etc.). The application of biocatalysts for multi-step enzyme biosynthesis (for instance, antibiotics) is rudimentally described in the literature (Morikawa et al., 1979, 1980; Vandamme, 1984). This interesting area represents a new type of biosynthesis, and as such this approach to L-ascorbic acid synthesis should not be neglected in the future.

Martin & Perlman (1976) described conversion of L-sorbose to L-sorbosone by biocatalysts. They used immobilized cells of native Gluconobacter melano­genus IFO 3293 entrapped in polyacrylamide gel. Authors concluded that conversion depended on the concentration of monomer used. The higher the monomer concentration, the more L-sorbosone accumulated. Different results were found with immobilized lyophilized cells. In this case a very active preparation of biocatalysts was obtained with low monomer concentration due to the impaired permeability of lyophilized cells, allowing diffusion of the toxic monomers into the cytoplasm. Fujiwara et al. (1987a, b) described isolation, purification and characterization of two enzymes (L-sorbose dehydrogenase and L-sorbosone dehydrogenase) from Gluconobacter oxydans UV -10 involved in conversion of L-sorbose to 2-KLG via L-sorbosone.

The cells of Gluconobacter oxydans strain No.6 VNIVI capable of oxidizing o-sorbitol to L-sorbose were entrapped in polyacrylamide gel. The highest oxidative activity of such a biocatalyst, reused five times, was 3·3 g of L-sorbose per liter per h (Pomortseva & Krasil'nikova, 1983).

The production of L-sorbose by cells of Gluconobacter suboxydans (ATCC 621) immobilized in polyacrylamide gel was carried out in a continuous process. The entrapped cells of this strain were capable of almost completely converting o-sorbitol into L-sorbose at a rate of about 7 kg/m3 per h during a long period of time (Stefanova et al., 1987).

Bailey et al. (1985) developed a mathematical model on K-carrageenan­immobilized cells of Acetobacter suboxydans oxidizing ethyl alcohol to acetic acid. This model, which predicted the cells, substrate and product concentra­tion in reaction with the biocatalyst, could possibly be used for biooxidative studies in the L-ascorbic acid pathway.

Studies on enzyme participation in the pathways of o-sorbitol oxidation were done by Shinaga-,a et al. (1982). They described the solubilization, purification and characteristics of o-sorbitol dehydrogenase from the membrane fraction of Gluconobacter suboxydans var. a IFO 3254.

Recently, Sonoyama & Kobayashi (1987) reported two 2,5-DKG reductases (reductase I and II) isolated from Corynebacterium sp. SHS 752001 mutant

318 V. De/ie, D. Sunie & D. V/aIiC

derived from ATCC 31090. Reductase I differed from reductase II in its molecular weight, its higher specific activity and its affinity for substrate 2,5-DKG. Reductase II is essentially the same enzyme described by Anderson et aZ. (1985) and Miller et aZ. (1987). Both reductases had the ability for the stereospecific reduction of the keto group of 2,5-DKG at the C-5 position and were NADPH-dependent.

6 CLONING OF GENES RELATED TO L-ASCORBIC ACID BIOSYNTHESIS

Molecular biology techniques developed in the last 15 years and the possibility of gene manipulation in vitro (recombinant DNA technologies-rDNA), particularly in microbial cells, resulted in the creation of hybrid features and the non-conventional production of several substances in bioreactors. Gene cloning was extended to many different micro-organisms, showing diversity of application in a wide range of products like hormones, vaccines, enzymes, amino acids, vitamins, antibiotics, etc. (Old & Primrose, 1985; Primrose, 1987). The classical example is cloning of genes for human insulin in the bacterium Escherichia coli (Goeddel et aZ., 1979) and the production of this hormone by fermentation (Frank & Chance, 1983). These techniques were also usefully applied in the construction of improved microbial strains for producing penicillin G-acylase, an enzyme involved in the hydrolysis of penicillin G to 6-aminopenicillanic acid (Mayer et al., 1979; Vandamme, 1984; Garcia & Buesa, 1986).

6.1 Cloning of 2,S-Diketo-D-Gluconic Acid Reductase Gene in Genus Erw;n;a

As described in previous parts of this review, many micro-organisms have the ability to catalyse a reaction on a sugar molecule and to carry out a conversion on the L-ascorbic acid pathway. The shortest route for o-glucose conversion to 2-keto-L-gulonic acid (2-KLG) is the two-step reaction via 2,5-diketo-o­gluconic acid (2,5-DKG) (Fig. 3, route 5). In this route, o-glucose is oxidized by microbial dehydrogenases to give 2,5-DKG. On the other hand, a group of-for example-coryneforming bacteria was able to reduce enzymatically (2,5-DKG reductase) 2,5-DKG to 2-KLG, the last intermediate in L-ascorbic acid synthesis that could be easily chemically converted into L-ascorbic acid. Sonoyama et al. (1982) demonstrated a two-stage fermentation using mutants of bacteria Erwinia sp. and Corynebacterium sp. to convert o-glucose to 2-KLG.

Based on this data, the idea of Anderson et al. (1985) was to combine the traits of these two micro-organisms in a single cell by gene manipulation techniques and to simplify the conversion of o-glucose to 2-KLG. The strategy was to clone the 2,5-DKG reductase gene from Corynebacterium sp. ATCC

Microbial Production of Vitamin C 319

31090 in Erwinia herbicola ATCC 21988 (also known as Acetomonas albosesamae) which was able to oxidize D-glucose into 2,S-DKG. Anderson et al. (198S) isolated and characterized 2,S-DKG reductase from Corynebacterium sp. and through Edman degradation determined a sequence of 40 amino acids from the NH2-terminus of the enzyme. Two synthetic DNA probes, 43 nucleotides long and corresponding to the NH2-terminal amino acid sequence were used to detect by hybridization the 2,S-DKG reductase gene between fragments obtained by Bam HI digestion of total Corynebacterium sp. DNA. Fragments ranging in size from 2·0 to 2·S kb were isolated and cloned in a Bam HI site of plasmid pBR 322. A partial genomic library was obtained in E. coli and the transformed colonies were checked for the presence of 2,S-DKG reductase gene by hybridization. A fragment was subcloned in bacteriophage M13mp9 cut by a combination of two restriction enzymes Pst I and Sma I for further vector construction. A simplified route for suitable vector construction is given in Fig. S. For good 2,S-DKG reductase gene expression in bacterium Erwinia herbicola, deletion was constructed upstream of the ATG start codon. A 'strong' tryptophan (trp) promoter linked to the Shine-Dalgarno sequence (Shine & Dalgarno, 1974) from plasmid pHGH207-1ptrpL\RIS' (De Boer et al., 1983) was attached to the 2,S-DKG reductase coding region instead of the deleted seque~ce. This was accomplished by using deletion primer and Alu I fragments of M13mp9 treated with Klenow fragment of DNA polymerase I, T4 DNA ligase and deoxynucleotide triphosphates (dNTPs) to give heteroduplex mit12 RF (replicative form) molecule used to transform E. coli JM101. Deletion of the upstream region between Nco I restriction site and A TG starting codon was selected by plaque hybridization (A). A replicative form of one isolate mit12L\ having desired deletion was used to excise 2,S-DKG reductase gene by Eco RI and Hind III digestion. A new hybrid vector was constructed from three different parts (B). 2,S-DKG reductase gene (l·S kb) from mit12L\ was mixed with a small fragment (1·0 kb) of plasmid pHGH207-1ptrpL\R1S' digested with Pst I and Eco RI. This small fragment contained tryptophan promoter region essential for 2,S-DKG reduc­tase gene expression. The third part of the hybrid vector was a larger fragment (3·6 kb) of plasmid pBR322 obtained after digestion with Hind III and Pst I. Three parts were mixed and ligated and this mixture was used to transform E. coli MM294. Transformants were selected for ampicillin resistance. Hybrid plasmid pmit12L\ trp1 AmpR could not be used for the selection of transformed cells of Erwinia herbicola since this micro-organism is naturally resistant to ampicillin. Thus, a functional gene for tetracycline resistance was re­constructed, since after pBR322 digestion with Hind III-Pst I loss of tetracy­cline resistance occurred. For this purpose plasmid pBR322 was digested with Eco RI and Sph I to generate the fragment of 0·S6 kb containing the tetracycline promoter region. This fragment was ligated with a S·4 kb fragment from plasmid pmit 12L\/trp1 AmpR isolated from E. coli MM294 and digested with Hind III and Sph I (C). The resulting 2,S-DKG reductase expression vector ptrpl-3S was used to transform Erwinia herbicola and transformants

320 V. Delic, D. Sunic & D. VlaIic

2.5- DKG red. gene

(A)

(B)

(C)

-HindW

S ECOR!

mil 12 6 f Screening for phage

containing deletion

I Hind m

Annealing of deletion primer and Alul fra9m~nt$, DNA pOlymerase I (Klenow), dNTPs. T4 DNA ligase

Transfectron ot E.colt JMIOI w ith heterodupr;;-;;~ ' 2RF

e) E. CORl ePS~ H;nd m pBR 322

Handm

-- 15kb 1·0kb 3 ·6kb

----- I ------Ligation

Transformation J, ~ MM294.

select ion for AmpR (olonies I .

8SR322 lE~:tR! SphI

5 .6kb~ /o.56kb

L1gation

I Transfo,.mat ion of "",,!!,!Ill>1 !....!lS=~. selection for TetR cOlOnies

Erwini. herblcola w ith 2,5- DKG reductase gene on pl.smid ptrpl- 35

Fig. 5. Construction of 2,5-diketo-D-gluconic acid reductase cloning vector and transformation of Erwinia herbicola. Only relevant restrictipn sites are given; AmpR, ampicillin resistance; Tet, R tetracycline resistance; trp, tryptophan promoter; kb, kilo

base; dNTPs, deoxynucleotide tryphosphates.

were selected on tetracycline. Isolated transformants, checked for enzyme activity, showed 54 times higher 2,5-DKG reductase activity in crude extracts in comparison with E. herbicola containing plasmid pBR322. Activity was 50-100 times higher in E. herbicola than in E. coli MM294 when transformed with the same ptrpl-35 plasmid.

Microbial Production of Vitamin C 321

For the production of 2-KLG, E. herbicola with plasmid ptrp1-35 was cultivated in a o-glucose-containing medium and subsequently on the medium containing 2% glycerol. After additional incubation, 1 g/liter 2-KLG was found and analysed by HPLC. Identity of 2-KLG was confirmed by the usual analytical methods (Lazarus & Seymour, 1986).

A similar approach has recently been applied by Hardy et al. (1987) and Grindley et al. (1988) for the construction of 2,5-DKG reductase expression vector. Fragments containing 2,5-DKG reductase sequence were taken from Corynebacterium sp. SHS 7520001 after Sau 3A digestion and inserted into plasmid pUCS digested by Bam HI restriction enzyme. Recombinant plasmid was used for the transformation of Escherichia coli JM83 and colonies of the resulting library were screened by hybridization with the 17-mer DNA synthetic probe to locate the 2,5-DKG reductase gene. Two recombinant plasmids, pCBR10 and pCBR13, were found to contain 2,5-DKG reductase gene approximately 3·0 kb long. These plasmids were used for further vector construction using different promoters (lac, trp, tac, the operator and promoter regions of phage A, the control region of fd coat protein, the promoters of yeast, etc.). Plasmid pPLred332 containing APL promoter region upstream of 2,5-DKG reductase region from pCBR13 was used to transform the mutant strain Erwinia citreus ER 1026 obtained from E. citreus SHS 2003 (ATCC 31623). The newly-formed E. citreus ER 1116 strain was able to convert o-glucose in a single biosynthetic step to 2-KLG after 65 h of cultivation. o-Glucose was added three times during biosynthesis and at the end 19·8 g/liter of 2-KLG was found (overall yield was 49·4%).

6.2 Molecular Biology of Other Micro-organisms Involved in 2-Keto-L-Gulonic Add Biosynthesis

Members of the genus Acetobacter and Gluconobacter are industrially impor­tant because of their ability for high yield conversion of carbohydrates to various oxidation products. In recent years more data on the molecular biology and genetics of these micro-organisms have been accumulated.

Fukaya et al. (1985a) showed that as much as 81·8% of Acetobacter strains harboured mainly multicopy plasmids of low molecular weight while 63·8% of Gluconobacter strains harboured plasmids ranging from 1·0 to 17 Md.

The transformation method for Acetobacter strains was improved by polyethylene glycol and treatment of cells by divalent cations (Ca2+, S~+, Ba2+, Mn2+). Efficiency of transformation was HP transformants per I-'g DNA (Fukaya et al., 1985b).

Construction of shuttle vectors for Gluconobacter suboxydans and Escherichia coli was a further step in the application of recombinant DNA technology for this group of micro-organisms, although efficiency of transfor­mation was low (1ij2 per I-'g DNA) (Fukaya et al., 1985c).

Two phages isolated from 54 different strains of Gluconobacter (Robakis et al., 1985a) were characterized and discovered to be unusually large, containing

322 V. Delit, D. Sunil & D. Vlaiil

double stranded DNA. Construction of a restriction map of bacteriophage A-I genome revealed circular DNA containing cohesive ends (Robakis et al., 1985b). Palleroni (1986) showed that Gluconobacter oxydans IFO 3293 could accept plasmid RSF 1010 both by transformation and conjugation but the yields of transformants were low, and obviously could be improved.

A new approach was used by Kahn and Manning (1988) to create Gluconobacter oxydans strains capable of higher production of 2-KLG from L-sorbose. They carried out additional mutation in G. oxydans UV-lO mutant strain by using insertional transposon mutagenesis (PI:: Tn5). Mutant strain M23-15 produced more 2-KLG (33·3 g/liter) than the parent strain (19·6 g/liter) due to reduced 2-KLG-reductase activity. As a consequence of this reduction, decreased flux of 2-KLG to L-idonic acid occurred. Besides this, new recombinant strains of G. oxydans IFO 3293 were obtained by using plasmid vectors constructed of broad host range plasmid RSF 1010 and a part of chromosomal DNA of the wild type strain containing L-sorbose de­hydrogenase DNA sequence. One of these recombinant strains, obtained by conjugative transfer with constructed plasmid pURU010 into the parent strain (1·8 glliter of 2-KLG) showed much higher conversion of L-sorbose to 2-KLG (28 g/liter).

Some species of the genus Pseudomonas, particularly P. aeruginosa and P. putida, are well characterized genetically and biochemically. This includes the development of host-vector systems for gene cloning by using restriction­negative host strains of P. aeruginosa and P. putida, and the construction of suitable plasmids or bacteriophage DNA as vectors (Bagdasarian & Timmis, 1982; Holloway, 1986).

Broad host range plasmid RSF 1010 is one of the possible candidates for a cloning vector in Pseudomonas cells. Some derivatives of this plasmid (pKT 230; pKT 231) with stable maintenance and propagation in host strains, have several unique cleavage sites, and insertional inactivation inside antibiotic resistance determinants is possible. Recently, new cloning vectors were constructed by inserting the entire sequence of plasmid pUC 13 into derivatives of the RSF 1010 plasmid. These new vectors, named pWS, were stable both in E. coli and Pseudomonas putida (Werneke et al., 1985). With regard to the cloning genes of Pseudomonas, P. putida possesses a potent gene engineering system, even more promising than E. coli.

These findings in those industrially important micro-organisms, which have the capacity for oxidation products from carbohydrates and polyalcohols, have opened up further possibilities for the creation of new routes for L-ascorbic acid biosynthesis by recombinant DNA technology.

7 INDUSTRIAL PROCESS

L-Ascorbic acid was the first vitamin to be produced in commercial quantities (Table 1). It is produced in a very large, integrated and automated process

Microbial Production of Vitamin C 323

involving both batch and continuous operations. Modern industrial production of L-ascorbic acid is based on Reichstein-Griissner synthesis established more than 50 years ago. According to available information, it seems that most L-ascorbic acid producers use this procedure for industrial production.

7.1 The Reichstein-Griissner synthesis

The first chemical synthesis was reported by Reichstein et al. (1933; 1934). L-Ascorbic acid was synthetized from L-xylose over L-xylosone but this route was never commercialized. It is interesting that when Reichstein and co­workers published the first synthesis of L-ascorbic acid, the correct structure was not yet known.

The most important synthesis of L-ascorbic acid for the manufacturing processes is that reported by Reichstein & Griissner (1934), well known in literature as Reichstein's second synthesis (Fig. 3, route 1).

This synthesis from o-glucose included the inversion of the glucose chain, which means the rearrangement of the molecule where C-1 of o-glucose became C-6 of L-ascorbic acid. The main steps of the Reichstein-Griissner synthesis were: reduction at C-1 (o-glucose- o-sorbitol) followed by microbial biooxidation at C-5 (o-sorbitol- L-sorbose), acetone treatment (L-sorbose­L-sorbose diacetone), oxidation at C-6 (L-sorbose diacetone- 2-keto-L-gulonic acid (2-KLG) diacetone), hydrolysis (2-KLG diacetone- 2-KLG), esterifica­tion (2-KLG- 2-KLG methyl ester) and lactonization (2-KLG methyl ester- L-ascorbic acid). o-Sorbitol was oxidized to L-sorbose with Acetobacter xylinum in a 60% yield. Later on A. suboxydans became the micro-organism of choice due to its high effective oxidative ability. The modified Reichstein­Griissner synthesis is a very efficient process for L-ascorbic acid synthesis and served as the basis for modern industrial production.

From an industrial point of view, the Reichstein-Griissner synthesis has two crucial features. The first is low cost of raw material, e.g. o-glucose which is a starting compound, and second, fully protected intermediate L-sorbose diace­tone for the second oxidation which precludes other side reactions.

Many chemical and technological modifications have improved the efficiencies of each step. This multi-step chemical synthesis, which includes micro-organisms for oxidation in one step, remains till now the principal and the most economical process for industrial production of L-ascorbic acid. The process steps including microbial oxydation are outlined in Fig. 6.

o-Sorbitol obtained by the hydrogenation of o-glucose was oxidized by Acetobacter suboxydans into the key intermediate L-sorbose. L-Sorbose is then condensed with acetone to form sorbose diacetone which is oxidized to a diacetone derivative of 2-keto-L-gulonic acid (2-KLG). After hydrolysis and esterification 2-KLG diacetone gives 2-KLG methyl ester which is converted to L-ascorbic acid by cyclization. The procedure requires about 1·7 kg of L-sorbose per kg of L-ascorbic acid with an overall yield of 66% (Jaffe, 1984).

324 V. Delil, D. Sunil & D. V[alil

D-GLUCOSE

! D- SORBITOL

~ FERMENTATION r-------------------------,

Acetob~cter Suboxy'd~ns

Filtered air

L-SORBOSE

~ L - SORBOSE DIACETONE

~ 2 - KETO - L- GULONIC ACID

l L- ASCORBIC ACID

Fig. 6. Synthesis of L-ascorbic acid including biooxidation of D-sorbitol to L-sorbose by Acetobacter suboxydans.

7.2 Product recovery and purification

Some steps of industrial processing include generally sophisticated product purification and recovery.

L-Sorbose isolation from cultivation broth is related with demands for the separation of A. suboxydans biomass accumulated in the course of n-sorbitol fermentation and elimination of other accompanying impurities. The product purification and recovery of L-sorbose as a solid is a continuous process which includes not only filtration, evaporation and crystallization steps but also purification in anion and cation ion exchange columns.

The acetone treatment of L-sorbose results in a mixture of mono- and diacetone-L-sorbose. Because benzene readily extracts diacetone-L-sorbose, diacetone-L-sorbose can be separated in a benzene/water system, while the monoacetone-L-sorbose in the aqueous layer is recovered and recycled.

2-Keto-L-gulonic methyl ester is recovered as a crystalline product. After the

Microbial Production of Vitamin C 325

formation of 2-KLG methyl ester crystals, mother liquors and methanol washes are accumulated for a second recovery of ester crystals.

At the end of the Reichstein-Griissner process crude L-ascorbic acid is filtered and purified by recrystallization. The pure product is isolated and dried. Mother liquors are accumulated and recycled for L-ascorbic acid recovery (Jaffe, 1984).

8 ASSAY METHODS FOR L-ASCORBIC ACID

Since L-ascorbic acid is widely distributed in different plant and animal tissues, as well as in crystalline form added to a variety of products (e.g. in food, drinks, etc.), the determination and quantification of its content deserve a special analytical approach.

Apart from these difficulties L-ascorbic acid is easily subjected to oxidation by moderate oxidants, thus yielding dehydroascorbic acid which frequently interferes with L-ascorbic acid determination.

As analytical details on L-ascorbic acid determination are not within the scope of this review, readers interested in this field of L-ascorbic acid are recommended to a very pertinent review by Pelletier (1985). The main analytical methods will be mentioned here only briefly.

Bioassay as a method was not frequently used for L-ascorbic acid determina­tion. This method based on guinea-pig protection against scurvy, was further applied to determine if certain L-ascorbic acid derivatives or other compounds exhibited anti-scurvy activity.

Most determinations are based on colorimetric methods using blue dye 2,6-dichloroindophenol (Hughes, 1983). In the presence of L-ascorbic acid, dye is reduced, resulting in colourless or pink colour solution. Direct titration of L-ascorbic acid with 2,6-dichloroindophenol is at present the most frequently used oxido-reduction method applicable to diverse types of samples yielding reproducible results.

Murty & Rao (1979) used iodine salts for the determination of L-ascorbic acid. In this case different dye indicators could be used (naphthol blue black, Amaranth, Brilliant Ponceau 5R) in titration mixture, liberating iodine which in the presence of starch yielded a blue colour. Reduction of ferric, cupric and mercuric ions by L-ascorbic acid has been the basis of simple and sensitive methods for measuring L-ascorbic acid. Corresponding ferrous, cuprous or mercurous coloured complexes with different substances were used as a measure of L-ascorbic acid.

Pelletier & Brassard (1977) developed a method for the determination of the total content of L-ascorbic acid, dehydroascorbic acid and diketogluconic acid in the samples on the basis of coupling with 2,4-dinitrophenyl hydrazine.

Gas-liquid chromatography of trimethylsilyl L-ascorbic acid derivatives was also adapted for quantitative determination of L-ascorbic acid in different samples (Schlack, 1974).

326 V. De/ie, D. Sunie & D. V/aIie

High performance liquid chromatography (HPLC) seems to be the most precise method for the determination of L-ascorbic acid in samples of different origins (Geigert et al., 1981; Rose & Nahrwold, 1981) although it remains to be established which method is the most accurate and precise for general application.

Recently developed peroxidase-based colorimetric determination of L­ascorbic acid has certain advantages over previously mentioned analytical methods (Thompson, 1987). Determination is based on the interference of L-ascorbic acid with the horseradish enzyme peroxidase which causes a decrease in the rate of oxidative coupling of chromogenic reagents like 4-aminoantipyrine or 3,5-dichloro-2-hydroxy-phenylsulphonate. The resulting change in absorbance at 510 nm is related to the concentration of L-ascorbic acid.

9 APPLICATION AND ECONOMICS

Pharmaceutical applications represent the largest segment of the L-ascorbic acid market. L-Ascorbic acid is not used only for the prevention and treatment of scurvy, which is of course its primary application, but also for the prevention and treatment of other pathological conditions and maintenance of good health in general. It can be used for the prevention and treatment of certain types of anaemia, some cardiovascular diseases, wound repair and normal healing processes. Due to its stimulative role toward the immune system, L-ascorbic acid is used in the prevention and treatment of various infections such as common colds and others.

L-Ascorbic acid has gained extensive application in the food industry due to

Table 4 Main World Producers of L-Ascorbic Acida

Producers

Hoffmann-La Roche

Takeda Chemicals Ltd Merck BASF Pfizer' PLIVA

City, country

Grenzach, FRG Belvedere, New Jersey, USA DaIry, UK Osaka, Japan Darmstadt, FRG Grenaa, Denmark Croton, Connecticut, USA Zagreb, Yugoslavia

Nominal capacity (t/year)

11000 13500 13500

7000-8000 6000 3000 2000 1200

a Total world production (including Eastern countries, China, India and Brasil) is assumed to be 65 000-70 000 t/year. b According to available information Pfizer's plant in Croton has been closed.

Microbial Production of Vitamin C 327

its antioxidant properties. It is used as an antioxidant in the commercial preparation of oils, fats, meats, beer, soft .drinks, milk products and canned and frozen food. It is also used as a dough and flour conditioner in the flour industry. L-Ascorbic acid is applicable in photography as a developing agent, and in metallurgy as a reducing agent (Jaffe, 1984).

At the present time, L-ascorbic acid is one of the most used and the most widely produced vitamins of importance in the pharmaceutical, food, cosmetic and other industries. The largest world producers are listed in Table 4 (Ullmans, 1983; Kieslich, 1984).

10 CONCLUSION

Although no alternative chemical route has reached the economical impor­tance of Reichstein's second synthesis, current developments of chemical synthesis and biosynthesis by micro-organisms may provide the important basis in the future of L-ascorbic acid production. Many future views are focused on the direct biosynthesis of 2-KLG from o-sorbitol or o-glucose by genetically­manipulated organisms.

Nevertheless, the main effort in the future could be designated as the substitution of the chemical pathway for L-ascorbic acid synthesis with a stereospecific bioconversion in one single step and continuous operation.

ACKNOWLEDGEMENTS

The authors gratefully acknowledge the expert secretarial assistance of Miss Jasenka Korajlija during the preparation of the manuscript, and thank Dr Ivanka Pavusek for suggestions and reading of the manuscript.

REFERENCES

Aiba, S., Humprey, A. E. & Milles, N. F. (1973). Biochemical Engineering. Academic Press, New York and London.

Alieva, R. M. (1966). Formation of 2-keto-L-gulonic acid by resting cells of Pseudomonas fluorescens. Tr. Petergofsk. Bioi. Inst., Leningr. Gos. Univ., 19, 100-103.

Anderson, S., Marks, C. B., Lazarus, R., Miller, J., Stafford, K., Seymour, J., Light, D., Rastetter, W. & Estell, D. (1985). Production of 2-keto-L-gulonate an intermediate in L-ascorbate synthesis, by a genetically modified Erwinia herbicola. Science, 230, 144-9.

Bagdasarian, M. & Timmis, K. N. (1982). Host: vector systems for gene cloning in Pseudomonas. In Current Topics in Microbiology and Immunology, Vol. 96, ed. P. H. Hofschneider & W. Goebel. Springer, Berlin, Heidelberg, New York, pp. 47-67.

328 V. Delit, D. Sunit & D. VlaJit

Bailey, K. M., Venkatasubramanian, K. & Karkare, S. B. (1985). Immobilized live cell reactor dynamics following dilution rate shift to growth conditions: cell synchrony effects. Biotechnol. Bioeng., 27, 1208-13.

Bergey's ManuaL of Systematic BacterioLogy (1984). Vol. 1, ed. N. R. Krieg & J. G. Holt. The Williams & Wilkins Co., Baltimore.

Bernhauer, K. & Knobloch, H. (1938). Decomposition of glucose by Acetobacter suboxydans. Naturwissenschaften, 26,819.

Bernhauer, K. & Knobloch, H. (1940). Oxidations with acetic acid bacteria. VI Comparative study on the formation of reducing-sugar carboxylic acids and the preparation of 2-keto-gluconic acid. Biochem. z., 303, 308-15.

Bertrand, G. (1896). Preparation biochimique du sorbose. C.R. Hebd. Stanco Acad. Sci., Paris, 122, 900-3.

Billman, J. H., Sojka, S. A. & Taylor, P. R. (1972). Investigations of keto-enol tautomerism by carbon-13 nuclear magnetic resonance spectroscopy. 1. Chem. Soc., Perkin Trans., 2, pp. 2034-5.

Brodelius, P. & Vandamme, E. J. (1987). Immobilized cell systems. In BiotechnoLogy, Vol. 7A (Enzyme TechnoLogy), 8, ed. J. F. Kennedy. VCH, Weinheim, pp. 405-64.

Burns, J. J. (1967). Ascorbic acid. In Metabolic Pathways, Vol. 1, ed. D. M. Greenberg. Academic Press, New York, pp. 394-411.

Chas. Pfizer & Co. (1958). 2-0xo-L-gulonic acid. British Patent No. 800 634. Crawford, T. C. & Crawford, S. A (1980). Synthesis of L-ascorbic acid. Adv.

Carbohydr. Chem. Biochem., 37,79-155. De Boer, H. A., Comstock, L. J. & Vasser, M. (1983). The tac promoter: a functional

hybrid derived from the trp and lac promoters. Proc. Natl. Acad. Sci. U.S.A., 80, 21-5.

Estell, D. A, Light, D. R., Rastetter, W. H., Lazarus, R. A. & Miller, J. V. (1985). Biosynthetic 2,5-diketogluconic acid reductase recombinant cells and expression vectors for its production, and its use in preparing 2-keto-L-gulonic acid. European Patent No. 132308.

Frank, B. H. & Chance, R. E. (1983). Two routes for producing human insulin utilizing recombinant DNA technology. Much. med. Wschr., 125 (Suppl. 1), 14-20.

Fujisawa, T., Yamazaki, M., Nishiyama, K., Shimizu, K. & Sawai, H. (1963a). 2-Ketogulonic acid. Japan Patent No. 637725.

Fujisawa, T., Yamazaki, M., Nishiyama, K., Shimizu, K. & Sawai, H. (1963b). Purification of 2-ketogulonic acid. Japan Patent No. 638714.

Fujiwara, A., Hoshino, T. & Sugisawa, T. (1987a). Enzym und Verfahren zu seiner Herstellung. European Patent No. 248 400.

Fujiwara, A, Hoshino, T. & Sugisawa, T. (1987b). Purification and characterization of L-sorbosone dehydrogenase of GLuconobacter. European Patent No. 248 401.

Fujiwara, A, Sugisawa, T., Shinjoh, M., Setoguchi, Y. & Hoshino, T. (1987c). Process for the manufacture of ketogulonic acid. European Patent No. 213 591.

Fukaya, M., Iwata, T., Entani, E., Masai, H., Uozumi, T. & Beppu, T. (1985a). Distribution and characterization of plasmids in acetic acid bacteria. Agric. BioI. Chem., 49, 1349-55.

Fukaya, M., Takayama, K., Okumura, H., Masai, H., Uozumi, T. & Beppu, T. (1985b). Improved transformation method for Acetobacter with plasmid DNA. Agric. BioL. Chem., 49, 2091-7.

Fukaya, M., Okumura, H., Masai, H., Uozumi, T. & Beppu, T. (1985c). Development of a host-vector system for GLuconobacter suboxydans. Agric. BioL. Chem., 49, 2407-11.

Garcia, J. L. & Buesa, J. M. (1986). An improved method to clone penicillin acylase genes: Cloning and expression in Escherichia coLi of penicillin G acylase from KLuyvera citrophila. 1. Biotechnol., 3, 187-95.

Microbial Production of Vitamin C 329

Geiger-Huber, M. & Galli, H. (1945). Uber den Nachweis der L-Ascorbinsaure ais Stoffwechsel-produkt von Aspergillus niger. Helv. Chim. Acta, 28, 248-50.

Geigert, J., Hirano, D. S. & Neidleman, S. L. (1981). High performance liquid chromatographic method for the determination of L-ascorbic acid and D-isoascorbic acid. J. Chromatogr., 206, 396.

Goeddel, D. V., Kleid, D. G., Bolivar, F., Heyneker, H. L., Yansura, D. G., Crea, R., Hirose, T., Kraszewski, A., Itakura, K. & Riggs, A. D. (1979). Expression in Escherichia coli of chemically synthesized genes for human insulin. Proc. Natl. A cad. Sci. U.S.A., 76, 106-10.

Gray, E. B. (1947a). 2-Keto-gulonic acid and its salts. US Patent No.2 421611. Gray, E. B. (1947b). 2-Keto-gulonic acid and its salts. US Patent No.2 421612. Grindley, J. F., Payton, M. A., Van de Pol, H. & Hardy, K. G. (1988). Conversion of

glucose to 2-keto-L-gulonate, an intermediate in L-ascorbate synthesis, by a recombinant strain of Erwinia citreus. Appl. Environ. Microbiol., 54, 1770-5.

Hardy, K., Van de Pol, H., Grindley, J. & Payton, M. A. (1987). Production of a vitamin C precursor using genetically modified organisms. WO 87/00863.

Haworth, W. N. & Hirst, E. L. (1933). Ascorbic acid. Chem. Ind. (London), 52, 645-6.

Hirst, E. L. (1939). The structure and synthesis of vitamin C and its analogs. Fortschr. Chem. Org. Naturst., 2, 132-59.

Hirst, E. L., Percival, E. G. V. & Smith, F. (1933). Ascorbic acid. Nature, Lond., 131, 617.

Holloway, B. W. (1986). Organization of the Pseudomonas genome. In Proceedings of the 5th International Symposium on the Genetics of Industrial Micro-organisms, ed. M. Alacevic, D. Hranueli & Z. Toman. Pliva, Zagreb, pp. 393-401.

Huang, H. T. (1962). 2-Keto-L-gulonic acid. US Patent No.3 043 749. Hughes, D. E. (1983). Titrimetric determination of ascorbic acid in commercial liquid

diets with 2,6-dichloroindophenol. J. Pharmac. Sci., 72, 126-9. Isono, M., Nakanishi, K., Sasajama, K., Motizuki, K., Okazaki, H. & Yoshino, H.

(1968). 2-Keto-L-gulonic acid fermentation. I Paper chromatographic characteriza­tion of metabolic products from sorbitol and L-sorbose by various bacteria. Agric. Bioi. Chem., 32,424-31.

IUPAC-IUB (1965). Commission on Biochemical Nomenclature. Biochim. Biophys. Acta, 107,4.

Jaffe, G. M. (1984). Ascorbic acid. In Encyclopedia of Chemical Technology, Vol. 24, ed. R. E. Kirk & D. F. Othmer. John Wiley & Sons, New York, pp. 8-40.

Kahn, M. S. & Manning, R. F. (1988). Process for the preparation of keto-gulonic acid. European Patent No. 276 832.

Kanzaki, T. & Okazaki, H. (1970). 2-Keto-L-gulonic acid fermentation. IV L-Sorbose metabolism in Pseudomonas aeruginosa. Agric. BioI. Chem., 34, 432-6.

Karube, I., Suzuki, S. & Vandamme, E. J. (1984). Antibiotic production with immobilized living cells. In Biotechnology of Industrial Antibiotics, ed. E. J. Vandamme. Marcel Dekker, New York, Basel, pp. 762-80.

Katznelson, H., Tanebaum, S. W. & Tatum, E. L. (1953). Glucose, gluconate and 2-ketogluconate oxidation by Acetobacter melanogenus. J. Bioi. Chem., 204,43-59.

Kieslich, K. (1984). Present state of biotechnological productions of pharmaceuticals. In Proceedings of the 3rd European Congress on Biotechnology, Vol. IV. VCH, Weinheim, pp. 39-72.

Kita, D. A. (1979). Fermentation process for converting L-gulonic acid to 2-keto-L­gulonic acid. US patent No.4 115 812.

Kita, D. A. & Hall, K. E. (1981a). Preparation of 2-keto-L-gulonic acid. US Patent No. 4245049.

Kita, D. A. & Hall, K. E. (1981b). Process for producing 2,5-diketo-gluconic acid. US Patent No.4 263 402.

330 V. Delii, D. Sunii & D. VlaIiC

Kitamura, I. & Perlman, D. (1975). Conversion of L-sorbose to L-sorbosone by Gluconobacter melanogenus. Biotechnol. Bioeng., 17,349-59.

Klein, J. & Wagner, F. (1978). Immobilized whole cells. Biotechnology, Dechema, 82. Verlage Chemie, Weinheim, New York, pp. 142-64.

Kulbe, K. D. & Knopki, G. (1986). Process for the intrasequential cofactor regeneration in enzymatic synthesis, particularly when producing vitamine C. WO 86/04353.

Kulhanek, M. (1970). Fermentation processes employed in vitamin C synthesis. In Advances in Applied Microbiology, Vol. 12, ed. D. Perlman. Academic Press, New York, London, pp. 11-33.

Lazarus, R. A. & Seymour, J. L. (1986). Determination of 2-keto-L-gulonic and other ketoaldonic and aldonic acids produced by ketogenic bacterial fermentation. Analyt. Biochem., 157,360-6.

Loewus, F. A. (1980). L-Ascorbic acid: metabolism, biosynthesis, function. In Biochemistry of Plants, Vol. 3, ed. J. Preiss. Academic Press, New York, pp. 77-99.

Lu, S., Huang, S. & Yu, J. (1985). Rules of the supply and demand of oxygen and scale up for vitamin C fermentation. Huaxue Fanying Gongcheng Yu Gongyi, 1, 72-82.

Makover, S. & Pruess, D. L. (1976). 2-Keto-L-gulonic acid. Swiss Patent No. 58016l. Makover, S., Ramsey, G. B., Vane, F. M. & Witt, C. G. (1975). New mechanisms for

the biosynthesis and metabolism of 2-keto-L-gulonic acid in bacteria. Biotechnol. Bioeng., 17, 1485-514.

Martin, C. K. A. & Perlman, D. (1975). Stimulation by organic solvents and detergents of conversion of L-sorbose to L-sorbosone by Gluconobacter melanogenus IFO 3293. Biotechnol. Bioeng., 17, 1473-83.

Martin, C. K. A. & Perlman, D. (1976). Conversion of L-sorbose to L-sorbosone by immobilized cells of Gluconobacter melanogenus IFO 3293. Biotechnol. Bioeng., 18, 217-37.

Mayer, H., Collins, J. & Wagner, F. (1979). Cloning of the penicillin G-acylase gene of Escherichia coli ATCC 11105 on multicopy plasmids. In Plasmids of Medical, Environmental and Commercial Importance, ed. K. M. Timmis & A. Puhler. Elsevier/North-Holland Biomedical Press, Amsterdam, pp. 187-94.

Merck Index, The, 10th edn. (1983). Merck & Co., Rahway, New Jersey, p. 844. Miller, J. V., Estell, D. A. & Lazarus, R. A. (1987). Purification and characterization

of 2,5-diketo-o-gluconate reductase from Corynebacterium sp. J. Bioi. Chem., 262, 9016-20.

Mochizuki, K., Kanzaki, T., Kusunoki, T. & Okazaki, H. (1969a). Idonic acid by oxidation of sorbose with Pseudomonas. Japan Patent No. 6908073.

Mochizuki, K., Kanzaki, T., Okazaki, H. & Doi, M. (1969b). 2-Keto-L-gulonic acid by Acetobacter fermentation of L-idonic acid. Japan Patent No. 69 08074.

Morikawa, Y., Karube, I. & Suzuki, S. (1979). Penicillin G production by immobilized whole cells of Penicillium chrysogenum. Biotechnol. Bioeng., 21,261-70.

Morikawa, Y., Karube, I. & Suzuki, S. (1980). Continuous production of bacitracin by immobilized living whole cells of Bacillus sp. Biotechnol. Bioeng., 22, 1015-23.

Murty, N. K. & Rao, K. R. (1979). Vitamine C (ascorbic acid). In Methods in Enzymology, Vol. 62, ed. D. B. McCormick & L. D. Wright. Academic Press, New York, San Francisco, London, p. 12.

Nakanishi, I., Sasajima, K., !sono, M. & Takeda, R. (1964a). Keto acid fermentation. I Oxidation of o-gluconic and L-idonic acids by genus Pseudomonas. Takeda Kenkyusho Nempo, 23,38-47.

Nakanishi, I., Sasajima, K., !sono, M. & Takeda, R. (1964b). Keto acid fermentation. II Differential determination of 2-oxo-o-gluconic and 2-oxo-L-idonic acids. Takeda Kenkyusho Nempo, 23, 48-53.

Ning, W., Tao, Z., Wang, C., Wang, S., Yan, Z. & Yin, G. (1988). Fermentation

Microbial Production of Vitamin C 331

process for producing 2-keto-L-gulonic acid from sorbose. European Patent No. 278 447.

Nogami, I., Shirafuji, H., Oka, M. & Yamaguchi, T. (1987). A method for producing 2-keto-L-gulonic acid. European Patent No. 221707.

Obata, Y., Nara, K., Tarni, K., Mochizuki, K. & Isono, M. (1975). L-Ascorbic acid. Japan Patent No. 75 22113.

Oga, S., Sato, K., Imada, K. & Asano, K. (1972). Fermentative manufacture of 2,5-diketogluconic acid. Japan Patent No. 72 38193.

Ogawa, T., Uzawa, J. & Matsui, M. (1977). A carbon-13 NMR study on the conformation of L-ascorbic acid in deuterium oxide. Carbohydr. Res., 59, 32-5.

Okazaki, H., Kanzaki, T., Doi, M., Nara, K. & Motizuki, K. (1968). 2-Keto-L-gulonic acid. II. Identification of metabolic products from sorbitol. Agric. BioI. Chem., 32, 1250-5.

Okazaki, H., Kanzaki, T., Sasayama, K. & Taketa, Y. (1969). 2-Keto-L-gulonic acid. III Evaluation of the pathway of sorbitol metabolism in Gluconobacter melanoge­nus. Agric. BioI. Chem., 33,207-11.

Old, R. W. & Primrose, S. B. (1985). Principles of Gene Manipulation, 3rd edn. Blackwell Scientific Publications, Oxford.

Palleroni, N. J. (1986). Isolation and characterization of R' plasmids in Gluconobacter oxydans. In Proceedings of the 5th International Symposium on Genetics of Industrial Micro-organisms, ed. M. Alacevic, D. Hranueli & Z. Toman. Pliva, Zagreb, pp. 415-24.

Pelletier, O. (1985). Vitamin C (L-Ascorbic and dehydro-L-ascorbic acids). In Methods of Vitamin Assay, 4th edn, ed. J. Augustin, B. P. Klein, D. A. Becker & P. B. Venugopal. John Wiley & Sons, New York, Chichester, Brisbane, Toronto, Singapore, pp. 303-47.

Pelletier, O. & . Brassard, R. (1977). Determination of vitamin C (L-ascorbic acid and dehydroascorbic acid) in food by manual and automated photometric methods. J. Food Sci., 42, 1471.

Perlman, D. (1959). Preparation of 2-keto-L-gulonic acid by Pseudomonas aeruginosa. US Patent No.2 917 435.

Pomortseva, N. V. & Krasil'nikova, T. N. (1983). Okislenie D-sorbita v L-sorbozu immobilizovanimi kletkami Gluconobacter oxydans. Khim.-Farm. Zh., 17,721-5.

Primrose, S. B. (1987). Modern Biotechnology. Blackwell Scientific Publications, Oxford.

Ramakrishnan, C. V. & Desai, P. J. (1956). Effect of addition of iron, cobalt and ascorbic acid to the medium on the synthesis of ascorbic acid in molds. Current Sci. (India), 25, 189-90.

Razumovskaya, Z. G. (1962). Obzori puti izpolzovanija mikroorganizmov v sinteze vitamina C. Mikrobiologija, 31, 172-8.

Razumovskaya, Z. G. & Vasil'eva, O. A. (1956). Oxidation of glucose by acetic acid bacteria. Ser. Bioi. Nauk, 41, 57-66.

Rehm, H. J. & Reed, G. (1981). Biotechnology, Microbial Fundamentals. Verlag Chemie, Weinheim, Deerfield, Florida, Basel.

Reichstein, T. & Grussner, A. (1934). Eine ergiebige Synthese der 1-Ascorbinsaure. (C-Vitamin). Helv. Chim. Acta, 17,311-28.

Reichstein, T., Grussner, A. & Oppenauer, R. (1933). Synthese der d- und I-Ascorbinsaure (C-Vitamin). Helv. Chim. Acta, 16, 1019-33.

Reichstein, T., Grussner, A. & Oppenauer, R. (1934). Synthese der Ascorbinsaure und verwandter Verbindungen nach der Oson-Blausaure-Methode. Helv. Chim. Acta, 17,510-20.

Robakis, N. K., Palleroni, N. J., Despreaux, C. W., Boublik, M., Baker, C. A., Churn, P. J. & Claus, G. W. (1985a). Isolation and characterization of two phages for Gluconobacter oxydans. J. Gen. Microbiol., 131,2467-73.

332 V. Delic, D. Sunic & D. VlaJic

Robakis, N. K., Palleroni, N. J., Boublik, M. & Despreaux, C. W. (1985b). Construction of a restriction map for the Gluconobacter bacteriophage A-1 genome. J. Gen. Microbiol., 131,2475-7.

Roland, J. F., Cayle, T., Dinwoodie, R. C. & Mehnert, D. W. (1985). Bioconversion production of ascorbic acid. WO 85/01745.

Roland, J. F., Cayle, T., Dinwoodie, R. C. & Mehnert, D. W. (1986). Fermentation production of ascorbic acid from L-galactonic substrate. US Patent No.4 565659.

Rose, R. C. & Nahrwold, D. L. (1981). Quantitative analysis of ascorbic acid and dehydroascorbic acid by high-performance liquid chromatography. Analyt. Biochem., 114,140.

Sastry, K. S. & Sarma, P. S. (1957). Glucuronic acid, a precursor of ascorbic acid in Aspergillus niger. Nature, Lond., 179, 44-5.

Schauenstein, E., Ochsenfeld-Lohr, I., Puxkandl, H. & Stampfer, M. (1948). Spec­trographic absorption studies on L-ascorbic acid. Monatsch. Chem., 79, 487-98.

Schlack, J. E. (1974). Quantitative determination of L-ascorbic acid by gas-liquid chromatography. J. Assoc. Off. Anal. Chem., 37, 1346.

Scott, C. D. (1987). Immobilized cells: a review of recent literature. Enzy. Micr. Tech. Biotech. Res. Rev., 9, 66-73.

Sebrell, W. H. Jr & Harris, R. S. (1967). The Vitamins. Chemistry, Physiology, Pathology Methods, Vol. 1, ed. W. H. Sebrell, Jr & R. S. Harris. Academic Press, New York, pp. 306-50l.

Shinagawa, E., Mutsushita, K., Adachi, O. & Ameyama, M. (1982). Purification and characterization of o-sorbitol dehydrogenase from membrane of Gluconobacter suboxydans vaT. ll'. Agric. BioI. Chem., 46, 135-41.

Shine, J. & Dalgarno, L. (1974). 3'-Terminal sequence of Escherichia coli 16 S ribosomal RNA: Complementary to nonsense triplets and ribosome binding sites. Proc. Natl. Acad. Sci. U.S.A., 71, 1342-6.

Shoemaker, R. N. (1956). Microbiological oxidation of idonic acid to 2-keto-L-gulonic acid. US Patent No.2 741577.

Skatrud, T. J. & Huss, R. J. (1987). L-Ascorbic acid production in Chlorella. European Patent No. 207 763.

Skerman, V. B. D., McGowan, V. & Sneath, P. H. A. (1980). Approved lists of bacterial names. Int. J. Syst. Bacteriol., 30, 225-420.

Sonoyama, T. & Kobayashi, K. (1987). Purification and properties of two 2,5-diketo-o­gluconate reductases from a mutant derived from Corynebacterium sp. J. Ferment. Technol., 65,311-17.

Sonoyama, T., Kageyama, B. & Honjo, T. (1974a). 2-Keto-L-gulonic acid by fermentation. German Patent No.2 413 963.

Sonoyama, T., Kageyama, B. & Honjo, T. (1974b). L-Idonic acid or its salt production by Brevibacterium. Japan Kokai 74 125589.

Sonoyama, T., Tani, H., Kageyama, B., Kobayashi, K., Honjo, T. & Yagi, S. (1976). 2-Keto-L-gulonic acid. German Patent No.2 530 86l.

Sonoyama, T., Tani, H., Matsuda, K., Kageyama, B., Tanimoto, M., Kobayashi, K., Yagi, S., Kyotani, H. & Mitsushima, K. (1982). Production of 2-keto-L-gulonic acid from o-glucose by two-stage fermentation. Appl. Environ. Microbiol., 43, 1064-9.

Sonoyama, T., Yagi, S. & Kageyama, B. (1983a). 2-Keto-L-gulonic acid and a mutant producing it. European Patent No. 88408.

Sonoyama, T., Yagi, S. & Kageyama, B. (1983b). 2-Keto-L-gulonic acid. European Patent No. 88409.

Sonoyama, T., Yagi, S., Kageyama, B. & Tanimoto, M. (1986). 2,5-Diketo-o-gluconic acid-producing Erwinia species. Japan Patent No. 8663278.

Sonoyama, T., Kageyama, B. & Yagi, S. (1987a). Distribution of micro-organisms capable of reducing 2,5-diketo-o-gluconate to 2-keto-L-gulonate. Agric. Bioi. Chem., 51, 2003-4.

Microbial Production of Vitamin C 333

Sonoyama, T., Kageyama, B., Yagi, S. & Mitsushima, K. (1987b). Biochemical aspects of 2-keto-L-gulonate accumulation from 2,5-diketo-D-gluconate by Corynebacterium sp. and its mutants. Agric. BioI. Chem., 51, 3039-47.

Stefanova, S., Koseva, M., Tepavicharova, I. & Beshkov, V. (1987). L-Sorbose production by cells of the strain Gluconobacter suboxydans entrapped in a polyacrylamide gel. Biotechnol. Lett., 9,475-7.

Stroshane, R. M. & Perlman, D. (1977). Fermentation of glucose by Acetobacter melanogen us. Biotechnol. Bioeng., 19, 459-65.

Stubbs, J. J., Lockwood, L. B., Roe, E. T., Taubenkin, B. & Ward, G. E. (1940). Ketogluconic acids from glucose. Bacterial production. Ind. Eng. Chem., 32, 1626-31.

Stubbs, J. J., Lockwood, L. B., Roe, E. T. & Ward, G. E. (1943). 5-keto-gluconic acid by fermentation of glucose. US Patent No.2 318 641.

Svirbely, J. L. & Szent-Gyorgyi, J. (1933). Vitamin C. Biochem. J., 27,279-85. Szent-Gyorgyi, A. (1928). Function of peroxidase systems and chemistry of the adrenal

cortex-carbohydrate derivat. Biochem. J., 22, 1387-409. Takeda Chemical Industries Ltd (1964). Manufacture of 2-keto-L-gulonic acid. French

Patent No. 1376741. Takeda, R., Isono, M., Nakanishi, T. & Sasajima, K. (1964). 2-Keto-L-gulonic acid.

Japan Patent No. 64 2463. Tengerdy, P. R. (1961a). Redox potential changes in the 2-keto-L-gulonic acid

fermentation. I Correlation between redox potential and dissolved oxygen con­centration. J. Biochem. Microbiol. Tech. Eng., 3,241-53.

Tengerdy, P. R. (1961b). Redox potential changes in the 2-keto-L-gulonic acid fermentation. II Relationship betwen redox potential and product formation. J. Biochem. Microbiol. Tech. Eng., 3,255-60.

Teramoto, S., Yagi, R. & Hori, I. (1946). Utilization of oxidative fermentation. IV Basal studies of manufacturing 5-keto-gluconic acid. J. Ferment. Technol., 24, 22-6.

Thompson, R. Q. (1987). Peroxidase-based colorimetric determination of L-ascorbic acid. Analyt. Chem., 59, 1119-21.

Tsukada, Y. & Perlman, D. (1972a). The fermentation of L-sorbose by Gluconobacter melanogen us. I General characteristics of the fermentation. Biotechnol. Bioeng., 14, 799-810.

Tsukada, Y. & Perlman, D. (1972b). The fermentation of L-sorbose by Gluconobacter melanogen us. II. Inducible formation of enzyme catalyzing conversion of L-sorbose to 2-keto-L-gulonic acid. Biotechnol. Bioeng., 14,811-8.

Tsukada, Y. & Perlman, D. (1972c). The fermentation of L-sorbose by Gluconobacter melanogen us. III Investigation of the metabolic pathway from sorbose to 2-keto-L­gulonic acid. Biotechnol. Bioeng., 14, 1035-8.

Ullmanns, (1983). Encyklopiidie der technischen Chemie, Vol. 23. Verlag Chemie, Weinheim, Basel, pp. 685-92.

Vandamme, E. J. (1984). Biotechnology of Industrial Antibiotics. Marcel Dekker, New York, Basel.

Wakisaka, Y. (1964a). 2,5-dioxogluconic acid by fermentation process. Japan Patent No. 64 14493.

Wakisaka, Y. (1964b). Carbohydrates oxidation by Pseudomonas albosesamae. nov. sp. I Taxonomic studies on a newly isolated bacterium P. albosesamae. Agric. Bioi. Chem., 28,369-74.

Wakisaka, Y. (1964c). Carbohydrate oxidation by Pseudomonas albosesamae. nov. sp. II Chemical structure of the product on glucose and gluconate oxidation. Agric. BioI. Chem., 28,819-27.

Werneke, J. M., Sligar, S. G. & Schuler, M. A. (1985). Development of broad­host-range vectors for expression of cloned genes in Pseudomonas. Gene, 38, 73-84.

334 v. Delil, D. Sunil & D. VlaIil

Yamazaki, M. (1953a). Preparation of vitamine C by fermentation. II Isolation of 2-oxo acids. J. Ferment. Technol., 31,86-90.

Yamazaki, M. (1953b). Preparation of vitamine C by fermentation. III Oxidation of Ca-L-idonate and Ca-o-gluconate mixture by Pseudomonas. J. Ferment. Technol., 31,126-30.

Yamazaki, M. (1953c). Preparation of vitamine C by fermentation. IV Oxidation of the mixture of Ca-L-idonate and Ca-o-gluconate by Pseudomonas in shake culture. J. Ferment. Technol., 31, 230-4.

Yamazaki, M. (1955). 2-0xogulonic acid. Japan Patent No. 555331. Yamazaki, M. & Miki, T. (1953). Preparation of vitamine C by fermentation. I

Preparation of Ca-L-idonate and Ca-o-gluconate from Ca-5-keto-o-gluconate. J. Ferment. Techno/., 31, 39-42.

Yan, Z. Z. et al. (1981). Studies on production of vitamin C precursor 2-keto-L-gulonic acid from L-sorbose by fermentation. II Study on fermentation conditions. Wei Sheng Wu Hsueh Pao, 21, 185-91.

Yin, G. L. et al. (1980). Studies on the production of vitamin C precursor 2-keto-L-gulonic acid from L-sorbose by fermentation. I Isolation, screening and identification of 2-keto-L-gulonic acid producing bacteria. Wei Sheng Wu Hsueh Pao, 20, 246-51.

OTHER GROWTH FACTORS

Chapter 18

MICROBIAL PRODUCTION OF ATP

Y. TANI

Research Center for Cell and Tissue Culture, Faculty of Agriculture, Kyoto University, Kyoto 606, Japan

1 INTRODUCnON

A TP plays a central role in energy metabolism as a carrier of chemical energy from catabolic reactions to anabolic reactions. It has two high-energy phosphate bonds which give a free energy of 14-16 kcal/mol on hydrolysis. Since ATP was first isolated in 1929 from a tissue (muscle) having high glycolysis activity, by Lohmann, and Fiske and Subbarow, the mechanism of its formation has been studied through elucidation of the mechanisms of the glycolytic pathway and the respiratory chain. Thus, A TP is an important chemical in biochemical research and medicine. Recently, ATP has also attracted much attention as the energy donor in bioreactors for the produc­tion of useful metabolites.

There are various methods for the preparation of A TP, which include extraction from rabbit muscle, as originated by Lohmann, inhibiting the activity of ATPase with magnesium ion, chemical synthesis using AMP as a starting material, and fermentation. Regarding ATP formation by fermen­tation methods, Lutwak-Mann & Mann in 1935 and Ostern et al. in 1938 found that added AMP or adenosine was phosphorylated to A TP in the alcohol fermentation of yeast.

During the past two decades, several biochemical processes of A TP production were developed in Japan; (1) phosphorylation of adenosine and AMP through glycolysis by yeasts, using glucose as the energy donor; (2) phosphorylation of AMP by Escherichia coli with a hybrid-plasmid containing genes for phosphofructokinase and triose phosphate isomerase, (3) direct ribotidation of adenine through salvage synthesis and phosphorylation by Brevibacterium ammoniagenes, and (4) phosphorylation with or without ribotidation of AMP, adenosine and adenine using the oxidative phosphoryla­tion system of methylotrophic yeast and methanol as the energy donor. These methods will be described in this chapter.

337

338 Y. Tani

2 CHEMISTRY AND ASSAY OF ATP

ATP has the chemical structure shown in Fig. 1, and a molecular weight of 507·2. The absorption maxima are 257nm at pH2 and 259nm at pH7, with the molecular extinction coefficients of 14·7 and 15·4, respeCtively. It is rather stable in neutral solution but unstable in acidic solution; 60% of phosphorus is liberated by heating at 100°C for 10 min in 1 M HCl. The breakage of P-O bonds at y and fJ positions produce free energy of 7-8 kcal (30-40 kJ).

ATP and its related compounds can be determined quantitatively by several methods.

A convenient paper chromatographic method is as follows. The sample on filter paper is developed in a solvent system containing 5 vol. of iso-butyric acid and 3 vol. of 0·5 N ammonium hydroxide. Each compound on paper is detected under UV irradiation. Absorbing parts are cut off and eluted with 5 ml of 0·1 M hydrochloric acid for about 15 h at room temperature. Absor­bancy at 260 nm is measured by a spectrophotometer and the amounts of compounds are calculated from standard curves. Since inosine and AMP are developed on the same position with this solvent system, these compounds were calculated by measuring absorbancy at 250 and 260 nm.

High voltage paper electrophoresis using two kinds of buffers, 0·1 M borate, pH 9·4, and 0·2 M acetate, pH 3·5, is carried out at 3 kV for 30-60 min. Detection and determination of nucleic acid-related compounds are the same as the paper chromatography.

High performance liquid chromatography is carried out with a Hitachi liquid chromatograph system 655 under the following conditions: column, M&S PACK C18 (4·6 mm i.d. x 150mm); mobile phase, 0·2M potassium phosphate buffer, pH 6·0 (solvent A), and methanol (solvent B); gradient, 2% of solvent B (0-6 min) and 10% of solvent B (6-20 min); flow rate, 1·0 ml/min; injection volume, 5 Ill; detection, absorbancy at 260 nm. Figure 2 demonstrates the elution pattern of a reaction mixture to phosphorylate adenosine by a methylotrophic yeast, which will be described below.

ATP and ADP are determined by enzymatic methods using

o 0 0 II II II

HO-PY-O-Pf3-O-pa-O-H2CS' 0 I I I

OH OH OH

OH OH

Fig. 1. Chemical structure of A TP .

Microbial Production of A TP 339

::

o II 10 min

111 20

Fig. 2. High performance liquid chromatography of A TP and its related compounds in the reaction mixture.

hexokinase/glucose 6-phosphate dehydrogenase and pyruvate kinase/lactate dehydrogenase systems, respectively, in measuring the absorbance of NADH at 340nm.

Isolation of A TP and its related compounds can be carried out by a purification process with column chromatography employing Dowex 1 x 2 as ion exchanger.

3 ATP PRODUCTION THROUGH GLYCOLYTIC PATHWAY (SUBSTRATE-LEVEL PHOSPHORYLATION)

3.1 Yeast Cell Reaction Method

It was found by Tochikura et al. (1967) that AMP or adenosine, added to a fermentation system with ground or acetone-dried baker's yeast cells, was effectively phosphorylated to A TP and ADP. In this system, 72% of AMP added as substrate was converted to ATP. The phosphorylation of AMP scarcely occurred with air-dried or fresh yeast cells, while the phosphorylation of adenosine to A TP proceeded only with acetone-dried yeast. The concentra­tion of inorganic phosphate as buffer in the reaction mixture is a special requirement of this method: the phosphorylation of AMP with acetone-dried yeast proceeds at a concentration of 1/3 M but not at 1/9 M or 2/3 M, and that with ground yeast proceeds at 1/4M. The mechanism of ATP formation is thought to be the substrate-level phosphorylation of AMP or adenosine via ADP, using the energy of glycolysis of glucose. The essential features of this method are presumably the inhibition of phosphatase by a high concentration

340 Y. Tani

of inorganic phosphorus and the release of the inhibited glycolysis in high salt concentrations by AMP.

This method was improved to permit easier cell preparation, and by adding higher concentrations of the substrate (Tanaka & Hironaka, 1972). They obtained 15·7 g of ATP·Na2 from 10 g of adenosine in 1·2 liters of the reaction mixture after treatment of the yeast cells with a surfactant, Cation S, and the addition of manganese chloride.

Further improvement of the productivity was studied in detail by Tochikura's group. High amounts of ATP (202 mM, 102·5 g/liter) and ADP (37 mM, 15·8 g/liter) were obtained at 35-h reaction at 28°C in the reaction mixture consisting of 300 g dried yeast cells per liter, 0·4 M adenosine, 1 M inorganic phosphate and 1·5 M glucose initially and with feeding of 0·6 M inorganic phosphate and 0·5 M glucose at 3-h incubation periods.

The mechanism of the A TP production by this method is as follows:

adenosine + ATP adenosine kinase) AMP + ADP (1)

AMP + ATP adenylate kinase) 2ADP (2)

glucose + 2Pi + 2ADP glycolysis) 2 ethanol + 2C02 + 2ATP (3)

By adding one-and-a-half times equation (3) to the sum of equations (1) and (2), equation (4) is derived:

adenosine + 1· 5 glucose + 3Pi ~ A TP + 3ethanol + 3C02 (4)

Screening to find an enzyme source other than baker's yeast has also been carried out. The AMP-phosphorylating activity was searched for among 59 strains of 17 genera of yeasts, and Debaryomyces nilssonii and Hansenula jadinii were found to be good ATP producers. These yeasts could phosphory­late 80-100% of AMP (lOmM) to ATP. It was also found that a hydrocarbon­assimilating yeast, Candida sp., possessed the ability to form A TP from AMP in high yield; the activity was markedly affected by the culture conditions for cell production.

The mechanism of phosphorylation was further studied with respect to the hexokinase isozyme (Kimura et al., 1980). Dried cells of Hansenula jadinii, that had been cultured aerobically with acriflavine, contained three hexokinase isozymes and metabolized glucose to produce A TP in the presence of a high concentration of inorganic phosphorus. The cells cultured aerobically without acriflavine contained two hexokinase isozymes and could not metabolize glucose under the same conditions. Two of the isozymes of the yeast cultured with acriflavine were similar to isozymes of the yeast cultured without acriflavine. The third isozyme was resistant to a high phosphorous concentra­tion. Therefore, the isozyme was considered to be responsible for regeneration of ATP through glycolysis and phosphorylation of nucleotides in the A TP-

Microbial Production of A TP 341

producing system. Recently, genes of hexokinase and glucokinase of Saccharomyces cerevisiae

were cloned and the yeast cells harbouring the hybrid plasmid could phosphorylate glucose about three times as effectively as the original strain (Fukuda et al., 1984). These cells completely converted adenosine (130 mM) into A TP, consuming glucose rapidly by the enhanced glycolytic pathway.

The yeast cell method could also be applied to the production of CfP, UTP and GTP from the corresponding mononucleotides in high yield.

3.2 Escherichia coli-CeU Reaction Method

The production of A TP from AMP by the glycolytic pathway in dried cells of E. coli was achieved by applying recombinant DNA techniques.

A plasmid pLC16-4 which contained an E. coli phosphofructokinase structural gene was introduced into E. coli C600 (Shimosaka et al., 1981). It was designated as C600 (pLC16-4). By gene dosage effect the C600 (pLC16-4) showed a sevenfold higher level of phosphofructokinase than the original strain E. coli C-600. By the use of this plasmid-carrying E. coli C600 (pLC16-4), a high level of A TP was produced in the A TP formation system consisting of AMP and glucose 6-phosphate as the substrate: about 75% of 20 mM AMP was phosphorylated to ATP in a 3-b reaction, while only 25% of AMP was converted to ATP by the original strain C600. However, this method was difficult to operate practically since glucose 6-phosphate is very expensive.

It could also be prepared from sucrose and inorganic phosphate with Leuconestoc mesenteroides sucrose phosphorylase (Vandamme et al., 1987) which yields fructose and glucose-1-phosphate; subsequent action of a phos­phoglucose mutase then results in glucose-6-phosphate formation.

Methods were therefore sought to produce ATP more economically from AMP and glucose, and E. coli B was found to be a suitable strain for this purpose. An ATP production method from AMP and glucose by dried E. coli B-cells dosed with the glucokinase gene was then developed (Yamaguchi et al. , 1984). The glucokinase activity of E. coli B cells was increased about 2·2-fold by culturing the cells in a medium containing D-fructose and ammonium phosphate. After drying, these cells converted AMP to ATP, consuming glucose by the glycolytic pathway. Under the optimal conditions, AMP (309 mM) was almost completely converted into ATP within 3 h. The dried E. coli B cells harbouring a hybrid plasmid (pGK100) carrying the glucokinase gene showed higher A TP-producing activity than cells without hybrid plasmid.

3.3 Continuous ATP Regeneration by Yeast Cells

There has been extensive work on bioreactors which use immobilized enzymes, resting cells and living cells as catalysts (Kennedy, 1987).

A TP regeneration systems so far studied are classified into four categories; chemical synthesis, whole cells (mainly yeasts), organelles, and cell-free enzymes. Chemical synthesis was eliminated because it uses an excess of

342 Y. Tani

expensive reactants and non-aqueous media which must be separated and recovered before and after ATP regeneration. Although organelles such as mitochondria, chloroplasts and chromatophores are efficient in A TP regenera­tion, with very cheap energy sources, they are too unstable. It was considered that cell-free enzyme systems were the most feasible routes for large-scale A TP regeneration processes, in which acetyl phosphate as phosphoryl donor and acetate kinase were employed, considering the stability and cost of the phosphoryl donor, and the stability of the enzyme. On the other hand, the yeast cell system for A TP production was also considered to be suitable for the continuous A TP regeneration in the following respects: (1) the energy source for ATP formation, glucose, is easily supplied; (2) enzyme preparation is easy, and (3) the most abundant reaction intermediate formed as a by-product is energy-rich fructose 1,6-bisphosphate, which can be used as an energy source for further A TP regeneration.

Continuous A TP regeneration from adenosine using enzymes of the glycolytic pathway was demonstrated using a reactor equipped with a semi-permeable membrane by Asada et al. (1981). Firstly, ATP regeneration from adenosine in batch systems with acetone-dried yeast cells, acetone-dried yeast cells immobilized in photo-crosslinked resin gel, and an extracted crude enzyme preparation was carried out. The substrate solution contained 600 mM glucose, 600 mM potassium phosphate buffer, pH 7·3, 20 mM magnesium sulfate and 20 mM adenosine. A crude enzyme preparation was extracted from 150 mg of acetone dried yeast cells by soaking the cells in 1 ml of a medium containing 600 mM potassium phosphate buffer, pH 7· 3, 1 M glucose and 25 mM magnesium sulfate for 3·5 h at 28°C with stirring.

The phosphorylation of adenosine proceeded similarly in all three cases, in which A TP is accumulated abruptly several hours after the beginning of incubation, concomitant with fructose 1,6-bisphosphate accumulation, and reaches more than 75% yield. However, accumulated ATP and fructose 1,6-bisphosphate decreased rapidly with further incubation in the case of whole cells and immobilized cells, while they remained at a high level for a fairly long time in the case of the extracted enzymes, suggesting a decrease in ATP­degrading activity. From this result, the extracted crude enzyme preparation was chosen for use in a continuous ATP regeneration system.

Since the immobilization of the 13 enzymes participating in A TP regenera­tion without loss of activity was difficult, and carbon dioxide gas generated during the reaction must be removed from the system, a membrane-type reactor was chosen. A plug-flow type continuous reaction apparatus consisting of eight chambers, each separated into two compartments by a collodion membrane of 40-50 m thick, was successfully constructed.

The addition of dithiothreitol, a protector of the enzyme-thiol group, increased the duration of the period of ATP formation from 42 to 100 h. With the addition of a yeast extract containing intermediates of the glycolysis, a stable steady state was attained, in which a yield of more than 75% ATP continued for 2 weeks. It was considered that the long-term continuous ATP formation obtained might be due to the protection and stabilization of enzymes

Microbial Production of ATP 343

by the yeast extract which was added to prevent inactivation. Another role of the yeast extract was to act as an energy-rich substance which is necessary to initiate the reaction.

4 ATP PRODUCTION THROUGH SALVAGE SYNTHESIS BY BREVIBACTERIUM AMMONIA GENES

4.1 Culture Method

A culture method to ribosylate and phosphorylate adenine, a cheaper starting material than AMP and adenosine, by B. ammoniagenes for the production of A TP was shown to be useful for the industrial production of A TP by Tanaka et al. (1968).

Brevibacterium ammoniagenes was cultivated for 4 days at 30°C in the medium consisting of 100 g glucose, 6 g urea, 10 g monopotassium phosphate, 10 g dipotassium phosphate, 10 g magnesium sulfate, 10 g yeast extract, 0·1 g calcium chloride and 30 g biotin in 1 liter , pH 7 ·4, with the addition of adenine (2 g/liter) after 3 days. It was found that A TP, AD P and AMP accumulated in amounts of 1·57, 1·59 and 2·16 g/liter of the culture filtrate, respectively. GTP, GDP and GMP similarly accumulated on cultivation with the addition of guanine. The mechanism of phosphorylation is considered to be the further phosphorylation of AMP from adenine, since the accumulation of AMP has been demonstrated with adenine-supplemented cultures of the same organism.

Although adenine can be used as the precursor with this method, a drawback appeared for practical production. Brevibacterium ammoniagenes cells grown with a strictly limited supply of manganese ion showed a high ability to convert adenine to A TP. However, excessive addition of the metal ion (above 30 g/liter) to the culture media severely inhibited ATP production. It became clear that limitation of manganese ions caused large morphological changes in the cells which result in the removal from the cell membrane of a permeability barrier to the excretion of A TP. The optimal concentration of the metal ion was very low, around 10 g/liter, and the optimal range was very narrow.

4.2 CeU Reaction Method

A cell reaction system for the production of A TP from adenine with manganese ion-unlimited cells of B. ammoniagenes was developed by Fujio & Furuya (1983). They first screened agents which allow A TP to permeate through the cell membrane and accumulate in the reaction mixture. Polyoxy­ethylene stearylamine, a cationic detergent, was obtained as the most effective additive. With resting cells of B. ammoniagenes grown in manganese-ion-rich medium, the reaction conditions for A TP production from adenine were optimized at 33°C and pH 7·4. Magnesium ions inhibited the ATP production. For efficient ATP production, it was necessary to maintain the concentration of soluble phosphate between 35 and 150 mM. A practical method fulfilling this

344 Y. Tani

requirement was developed, in which disodium phosphate was added to the reaction mixture intermittently every hour, starting at the third hour of the reaction. Under these optimum conditions, 13 g of ATP·Na2·3H20 per liter was produced from 3·5 g of adenine per liter. The molar ratio of the transformation of adenine to A TP was approximately 83%.

Further improvement of this method was performed by Fujio & Furuya, (1985). The ATP formation in this system ceased within 6-8 h. Simultaneous addition of magnesium ion and phytic acid, a chelator of divalent cations, was found to allow ATP formation to continue longer, and 24·2 g of ATP·Na2·3H20 per liter was accumulated in lOh. However, ATP formation ceased thereafter.

This second cessation was proved to be caused by the lack of magnesium ion active as a co-factor. To provide the active magnesium ion, sufficient phytic acid was added at the beginning of the reaction and magnesium ion was also added intermittently. Under these conditions ATP production continued further, and the rate of ATP production was increased; 37·0 g of ATP·Na2·3H20 per liter was accumulated in a 13-h reaction.

5 ATP PRODUCTION THROUGH OXIDATIVE PHOSPHORYLATION BY METHYLOTROPHIC YEASTS

Industrially applicable methods for biochemical production of A TP have been developed and established based on substrate-level phosphorylation of yeasts and on salvage synthesis in B. ammoniagenes as described above. However, there has been no report on A TP production from AMP or adenosine by use of the oxidative phosphorylation system. Moreover, the energy source substrate was exclusively glucose.

Methylotrophic yeasts reduce NAD+ in the oxidation pathway to supply energy for the assimilation of C1 compounds through glyceraldehyde 3-phosphate by the xylulose monophosphate pathway. The NADH formed is oxidized in the respiratory chain, and ADP is phosphorylated by the proton-driving force in the oxidative phosphorylation system.

C1 compounds are currently attracting much attention as convenient raw material for the chemical industry and for biotechnology. Methanol could be expected to be one of the most useful of these compounds in the very near future because it can be easily produced from natural gas. Methanol is advantageous as a carbon and energy source of microbial growth because of its high purity and miscibility with water in all proportions. Therefore, attempts have been made to establish new processes for fermentative production in which both unique and conventional aspects of metabolism of methylotrophs are utilized.

Recently, an A TP production system from AMP, adenosine and adenine with Zymolyase® and sorbitol-treated cells of a methylotrophic yeast, Candida boidinii (Kloeckera sp.) No. 2201 was developed from the viewpoint described

Microbial Production of A TP 345

above. The system was based on conversion of free energy of methanol to ATP through oxidative phosphorylation in respiration.

5.1 Construction of the ATP-producing System

A reaction system producing A TP from AMP and reduced C1 compounds was constructed with cells of C. boidinii No. 2201 (Tani et al., 1982). Yeast cells were treated with Zymolyase, a cell wall-lytic bacterial enzyme. The treated cells release protoplasts on transfer to a hypotonic reaction mixture from a hypertonic suspension, and then burst to yield intact mitochondria and cytoplasmic enzymes. A reaction mixture containing AMP, methanol (or formate), NAD+, inorganic phosphate and the treated cells was used for ATP production. Production of A TP could be inhibited by replacement of air in the reaction tube by nitrogen gas, and by addition of an uncoupler, an electron­transport inhibitor or an ATPase inhibitor. Involvement of substrate-level phosphorylation in the presence of xylulose 5-phosphate, an initiator of the xylulose monophosphate pathway, was negligible.

Thus, Zymolyase-treated cells of C. boidinii No. 2201 produce ATP from AMP by adenyl ate kinase, oxidation of methanol by formaldehyde and formate dehydrogenases to reduce NAD+ and oxidative phosphorylation of ADP to ATP in the respiratory chain as shown Fig. 3.

The phosphorylation of AMP proceeds by the following scheme:

AMP + ATP .Q4 2ADP ~ 2ATP

ATP for reaction (1), adenylate kinase, is initially supplied from the endogenous pool in the protoplast and then by reaction (2), oxidative

CH30H T'""'\, HCHO ~ CHo-SG-HCOO~ cO2

o H;o2 If. \ ( . \ 2 ~ NAD+ NADH NAD+ NADH

Microbody

H20

ATP

Fig. 3. ATP-producing system from AMP by methylotrophic yeast.

346 Y. Tani

phosphorylation. Thus, oxidative phosphorylation is required twice for the formation of one molecule of A TP in this system.

Reaction conditions for A TP production were optimized in respect of substrate and coenzyme concentrations, pH and temperature, osmotic pres­sure, and oxygen supply. Under optimal conditions, 13 and 4 g/liter of ATP were produced with methanol and formate as C1 substrate, respectively (Tani et a/., 1984a).

Subsequently, cell treatment was simplified to obtain yeast cells with stable ATP production ability, by incubation with added sorbitol (Tani et a/., 1984b). The sorbitol-treated cells did not lose this activity even in the presence of 3 M

methanol or after incubation for 36 h. Under optimal reaction conditions, the amount of ATP in the reaction mixture reached 30 g/liter (Tani et a/., 1984c). The effect of sorbitol treatment on the cell structure of C. boidinii No. 2201 was demonstrated by analysis with transmission electron microscopy (Yone­hara & Tani, 1988). The high ATP-producing activity of the cells was proved to be due to the plasmolysis without destruction of essential organelles for the A TP-producing system.

To prepare stable active cells, the essential elements of the yeast extract in the culture medium were identified: biotin was an essential growth factor; ferric ion and thiamin stimulated growth more or less and zinc ions strongly stimulated growth. Limitation of zinc ions in the culture medium (0·5 mg/liter of zinc chloride) improved the A TP yield from AMP, the conversion rate being 92%, possibly due to repression of AMP deaminase, for which the zinc ion was a cofactor and which affected the A TP-producing activity. A zinc ion-supplemented culture provided fully-active cells as to ATP production. Transmission electron microscopy revealed that the zinc ion was an essential factor for the formation of peroxisomes in which alcohol oxidase and catalase are localized.

S.2 Energy Efficiency of the ATP·produdng System

The AMP phosphorylation system can be characterized as energy conver­sion from methanol to high-energy phosphate bonds in nucleotides as well as the biotransformation of adenine nucleotide. As described below, AMP can be replaced with adenosine or adenine as a substrate for A TP formation.

The sequential conversion system can be illustrated as shown in Fig. 4. NADH produced by the methanol-dissimilation system is located in the cytosol of this yeast cell and cannot permeate through the inner mitochondrial membrane. The electron transport system coupled to the oxidative phos­phorylation is located in the inner mitochondrial membrane. Subsequently, electrons from cytoplasmic NADH may enter mitochondria by the glycerol phosphate shuttle mechanism and reduce FAD bound to the inner membranes. The reduced flavin inside the mitochondria transfers its electrons to the respiratory chain at the level of coenzyme Q. Consequently, the P / 0 ratio in this system is not 3·0 but 2·0.

Microbial Production of A TP

Adenine

Adenosine

1 AMP

\ ADP

ATP ADP

H'"

CH 3 0H

1 HCHO

J\ NATyto'~H

H' cO 2

O2

347

Fig. 4. ATP-producing system from adenosine or adenine by methylotrophic yeast.

The material balance of A TP production from adenosine can be summarized in the equation below:

adenosine + 3Pi + 3/4 methanol + 9/8 O2 -- ATP + 3/4C02 + 9/2 H 20

Table 1 shows the free energy changes of respective reactions involved in the system. The theoretical value of the total energy efficiency, Ke(t), from methanol to A TP is,

Ke(t) = 18·0/(167·91 x 3/4) x 100 = 14·3%

The practical energy efficiency, Ke (p ), was calculated using a result of

Table 1 Free Energy Changes of Reactions Involved in A TP

Production from Adenosine

Reaction

CH30H + 3/202~ CO2 + 2H20 NADH + H+ + 1/202~ NAD+ + H20 Adenosine + 3Pi ~ ATP + 3H20 3ADP + 3Pi ~ 3ATP + 3H20 AMP+ATP~2ADP Adenosine + ATP~ AMP + ADP

- Ll.G(kcallmol)

167·91 52·6

-18·0 -7·3 x 3 o 3·9

348 Y. Tani

adenosine phosphorylation and methanol consumption in a typical reaction (Yonehara & Tani, 1986). The amount of not only adenine nucleotides but also that of IMP, a by-product, as energy stored compounds in the reaction mixture was also estimated. Furthermore, the evaporation of methanol from the reaction mixture was prevented by an air-sealed system.

The practical energy efficiency against the methanol consumption is ex­pressed as follows,

Ke(p) = (2181/51302) X 100 = 4·25%

The ratio of the practical efficiency against the theoretical efficiency is expressed as follows,

Ke = (Ke(p)/Ke(t» X 100 = (4·25/14·3) X 100 = 30%

Thus, the energy efficiency of the ATP-producing system is quite high as in a biochemical energy conversion system.

5.3 ATP Production from Adenosine or Adenine

The use of adenosine or adenine instead of AMP, which is chemically rather unstable and commercially expensive as a starting material for A TP produc­tion, was investigated to construct a more profitable process with sorbitol­treated cells of C. boidinii No. 2201 (Tani & Yonehara, 1985).

The reaction conditions for A TP production from adenosine or adenine were optimized as to initial concentrations of substrates and coenzymes, pH of potassium phosphate buffer, temperature, osmotic pressure and other addi­tives. Efficient A TP production from adenosine was achieved by the addition of pyrophosphate together with magnesium ions. On the other hand, significant A TP production from adenine was obtained but not in high yield under the conditions tested.

Orthophosphate, pyrophosphate and divalent metal ions inhibited the deamination of AMP to IMP, a major by-product. No hypoxanthine nucleo­tide compound was accumulated by addition of an adenosine deaminase inhibitor, coformycin. By successive feeding of potassium phosphate to maintain the phosphorous concentration at over 100 mM, the conversion rate from adenosine to A TP was improved to 70%. Simultaneous feeding of potassium phosphate and adenosine resulted in the accumulation of 100 mM A TP (50·7 g/liter) after 28 h of incubation and the increase of IMP without decrease of A TP for 48 h.

A TP production was further prolonged up to 60 h of incubation and a high amount, 198mM ATP (100 g/liter) , was accumulated (conversion rate of 77-4%) from adenosine by control of pH in the range of 6·5-6-8 during the reaction (Yonehara & Tani, 1987).

Biochemical systems for A TP production so far studied are summarized in Table 2, in which raw material, ATP yield, efficiency in grams of ATP

Microbial Production of A TP 349

Table 2 ATP Production by Biochemical System

System Raw material A TP(g/liter) Efficiency * Molar yield (%)

Culture of Adenine, 1·57 20 B. ammoniagenes glucose Cell of Adenine, 10·9 0·10 83 B. ammoniagenes glucose Cell of yeasts Adenine, 102·5 0·34 50

glucose Cell of E. coli AMP, 14·0 0·14 92

glucose Chromatophore of ADP, 0·4 2·0 80 R. rubrum light Chromatophore of ADP, 200·0 R. capsulata light Cell of blue- ADP, 0·17 56·0 17 green alga light Acetate kinase ADP, 10·0 100

acetyl-P Adenylate kinase ADP 0·24 1·3 60 Cell of AMP, 30·0 1·5 60 C. boidinii methanol

Adenosine, 100·0 4·0 77 methanol Adenine, 4·1 0·20 40 methanol

* ATP produced (g) by 1 g of dry cell weight, protein or chlorophyl.

produced by 1 g of dry cell weight, protein or chlorophyll, and molar yield are compared. The highest amount is obtained by yeast cell methods using glycolysis (substrate level phosphorylation) and oxidative phosphorylation. The efficiency of A TP productivity for cells is more than 10 times higher in the latter than that in the former. The efficiency is quite high in the system using photophosphorylation, but the amount of A TP produced by these methods is quite low and the substrate needed is ADP.

REFERENCES

Asada, M., Yanamoto, K., Nakanishi, K., Matsuno, R., Kimura, A. & Kamikubo, T. (1981). Long term continuous ATP regeneration by enzymes of the alcohol fermentation pathway and kinases of yeast. Eur. J. App!. Microbiol. Biotechnol., 12, 198-204.

Fujio, T. & Furuya, A. (1983). Production of ATP from adenine by Brevibacterium ammoniagenes. J. Ferment. Techno!., 61,261-7.

Fujio, T. & Furuya, A. (1985). Effects of magnesium ion and chelating agents on enzymatic production of ATP from adenine. Appl. Microbiol. Biotechno!., 21, 143-7.

350 Y. Tani

Fukuda, Y., Yamaguchi, S., Hashimoto, H., Shimosaka, M. & Kimura, A. (1984). Cloning of glucose phosphorylation genes in S. cerevisiae by the KU-method and application to ATP production. Agric. BioI. Chem., 48,2877-81.

Kennedy, J. F. (Ed.) (1987). Enzyme Technology, Vol. 7A, Biotechnology, eds. H. J. Rehm & G. Reed. VCH, Weinheim, FRG.

Kimura, A., Tatsutomi, Y., Fukuda, H. & Morioka, H. (1980). Effect of acriflavine on the hexokinase isozyme pattern of a yeast, Hansenula jadinii. Biochim. Biophys. Acta, 629,217-24.

Lutwak-Mann, C. & Mann, T. (1935). Uber die Verkettung der chemischen Umsetz­ungen in der alkoholichen Garung. I. Mitteilung: Bildung uns Spaltung der Adenosintriphosphorsaure und deren Zusammenhang mit den Vorgangen der Zuckerspaltung. Biochem. Z., 281, 140-56.

Ostern, P., Baranowski, T. & Terszakowec, J. (1938). Uber die Phosphorylierung des Adenosins durch Hefe und die Bedeutung dieses Borgangs flir die alkoholische Garung. II. Metteilung, Hoppe-Seyler's Z. Physiol. Chem., 251,258-84.

Shimosaka, N., Fukuda, Y. & Kimura, A. (1981). Application of plasmid to ATP production by E. coli. Agric. Bioi. Chem., 45, 1025-7.

Tanaka, A. & Hironaka, J. (1972). Studies on enzymatic production of ATP. Agric. Bioi. Chem., 36,867-9.

Tanaka, H., Sato, Z., Nakayama, K. & Kinoshita, S. (1968). Production of nucleic acid-related substances by fermentative processes. Part XV. Formation of ATP, GTP and their related substances by Brevibacterium ammoniagenes. Agric. BioI. Chem., 32,721-6.

Tani, Y. & Yonehara, T. (1985). ATP production from adenosine or adenine by a methanol-utilizing yeast. Candida boidinii (Kloeckera sp.) No. 2201. Agric. BioI. Chem., 49, 637-42.

Tani, Y., Mitani, Y. & Yamada, H. (1982). Utilization of Cccompounds: phosphoryla­tion of adenylate by oxidative phosphorylation in Candida boidinii (Kloeckera sp.) No. 2201. Agric. Bioi. Chem., 46, 1097-9.

Tani, Y., Mitani, Y. & Yamada, H. (1984a). ATP production by protoplasts of a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201. Agric. BioI. Chern., 48, 431-7.

Tani, Y., Mitani, Y. & Yamada, H. (1984b). Preparation of ATP-producing cells of a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201. J. Ferment. Technol., 62,99-101.

Tani, Y., Yonehara, T., Mitani, Y. & Yamada, H. (1984c). ATP production by sorbitol-treated cells of a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201, J. Biotechnol., 1, 119-27.

Tochikura, T., Kuwahara, M., Yagi, S., Okamoto, H., Tominaga, Y., Kano, T. & Ogata, K. (1967). Fermentation and metabolism of nucleic acid-related compounds in yeasts. J. Ferment. Technol., 45, 511-29.

Vandamme, E. J., Vanloo, J., Machtelinckx, L. & De Laporte, E. (1987). Microbial sucrose phosphorylase: fermentation process, properties and biotechnical applica­tions. Adv. Appl. Microbiol., 32.

Yamaguchi, S., Fukuda, Y., Shimosaka, M. & Kimura, A. (1984). Phosphorylation of AMP to ATP by dried Escherichia coli B cells, J. Ferment. Technol., 62,29-33.

Yonehara, T. & Tani, Y. (1986). Comparative studies on ATP production from adenosine by a methanol yeast. Agric. BioI. Chem., 50,899-905.

Yonehara, T. & Tani, Y. (1987). Highly efficient production of ATP by a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201. J. Ferment. Technol., 65,255-60.

Yonehara, T. & Tani, Y. (1988). ATP production by a methanol yeast, Candida boidinii (Kloeckera sp.) No. 2201: effects of sorbitol treatment and zinc on cell structure as to ATP production. Agric. BioI. Chem., 52,909-14.

Chapter 19

ADENOSYLMETHIONINE, ADENOSYLHOMOCYSTEINE AND RELATED NUCLEOSIDES

S. SHIMIZU & H. YAMADA

Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan

1 INTRODUCTION

The function of L-methionine as a methyl group donor in biological trans­methylation reactions requires ATP to form so-called active methionine. This compound was identified as S-adenosyl-L-methionine (AdoMet) by Can toni (1952, 1953), who showed, with a liver preparation, that it was synthesized by the enzyme, methionine adenosyltransferase (EC 2.5.1.6) through the follow­ing reaction:

L-methionine + ATP ~ AdoMet + PPi + Pi

Ado Met is the most versatile methyl group donor for many biological transmethylation systems in both prokaryotic and eukaryotic organisms, whereas 5-methyltetrahydrofolic acid, methylcobalamine and betaine are involved in relatively few methylation reactions. Many low molecular weight metabolic compounds receive the methyl group from AdoMet through the reactions catalyzed by AdoMet-dependent transmethylases. These include catechols, norepinephrine, histamine, serotonine, tryptamine, and so on. Much attention has recently been paid to the methylation of membrane phospholipids in relation to regulation of membrane structure and function. The methylation of proteins, RNAs and DNAs has also been suggested to play important roles in regulation of cell differentiation, protein synthesis, car­cinogenesis and so on. Such versatility of AdoMet-dependent transmethylation reactions in biological systems suggests that they play very important roles in cellular regulations. In this respect, transmethylation reactions can be com­parable to phosphorylation reactions.

S-Adenosyl-L-homocysteine (AdoHcy), the product of AdoMet-dependent transmethylation reactions, is known to be a potent inhibitor of a number of AdoMet-dependent transmethylases. The inhibition of AdoMet-dependent reactions by AdoHcy is usually relieved by the enzymatic hydrolysis of

351

352 S. Shimizu & H. Yamada

AdoHcy to adenosine and L-homocysteine. The reaction is catalyzed by AdoHcy hydrolase (EC 3.3.1.1), which was first demonstrated in rat liver by de la Haba & Cantoni (1959). The activity of this enzyme is thought to playa critical role in the control of cellular levels of AdoHcy and thereby influence a variety of methyltransfer reactions.

Since the discovery that AdoHcy hydrolase is an adenosine binding protein (Hershfield & Kredich, 1978), this area of research has expanded very rapidly. One of the most important findings in the last 10 years was that an antiviral agent, arabinosyladenine, attacks this enzyme as a suicide inactivator (Hersh­field, 1979). This observation soon led to further studies which showed that AdoHcy hydrolase is a target for dozens of naturally occurring and artificial adenosine analogs exhibiting antibiotic, antiviral and/or antitumor activities. In connection with these findings, studies on the methylation of DNAs and RNAs in relation to cell growth, differentiation and carcinogenesis also indicated the potential of these nucleosides as pharmacologically active agents.

Another attractive and widely expanding line of investigation on trans­methylation reactions is their relation to the central nervous system. The metabolic relationships between AdoMet, AdoHcy and related nucleosides, and biogenic amines suggests their importance in this field. For example, AdoMet has been suggested as an effective treatment for depression, sleep­lessness and a variety of neuroses. AdoHcy is expected to have unique sedative and anticonvulsive properties.

In addition, AdoMet, after decarboxylation by AdoMet decarboxylase (EC 4.1.1.50), provides the aminopropyl group for the synthesis of polyamines which also play important roles in control of cell growth, differentiation and protein synthesis.

The involvement of methyl and aminopropyl group transfer reactions in such a wide variety of biological phenomena suggests the potential of these nucleosides in the pharmaceutical and chemotherapeutic fields. However, no process for the practical production of these nucleosides had been established before the current work performed by Yamada and co-workers (Shimizu & Yamada, 1984; Yamada et aI., 1984; Yamada & Shimizu, 1985, 1988). In this chapter, we summarize current progress in the biotechnological production of these nucleosides. Several reviews summarize other various aspects of these nucleosides and provide excellent background information (Cantoni, 1975; Salvatore et ai., 1977; Usdin et ai., 1979, 1982; Zappia et ai., 1979; Ueland, 1982; Schlenck, 1983, 1984; Shimizu & Yamada, 1984; Tabor & Tabor, 1984; Borchardt et ai., 1986).

2 CHEMISTRY

The methyl group of the methionine unit in the molecule of Ado Met is activated by the positive charge on the adjacent sulfur atom, which makes it very reactive. Thus, AdoMet is used as a methyl donor in versatile biological

Adenosylmethionine, Adenosylhomocysteine and Related Nucleosides 353

transmethylation reactions. Four stereoisomers are possible for AdoMet due to two asymmetric centers at the sulfonium center and at the a-amino carbon atom in the methionine moiety. Only the natural form, i.e. ( - )-S-adenosyl-L­methionine, is active as the methyl donor (de la Haba et al., 1959). The conformation of AdoMet was studied by Stolowitz & Minch (1981). AdoMet is unstable at room temperature, both as a dry solid and in aqueous solution, being easily decomposed to mainly 5'-deoxymethylthioadenosine (MeSAdo). Several AdoMet analogs, i.e. S-adenosyl-D-methionine, S-adenosyl-2-methylmethionine and S-adenosylethionine have been reported to be synthe­sized enzymatically (see Table 1).

AdoHcy is the demethylated product of Ado Met. The structure contains a single asymmetric center at the a-amino carbon atom in the homocysteine moiety, so that it is optically active. In most cases, only the L-isomer shows potent inhibitor activity toward biological transmethylation reactions which utilize Ado Met as the methyl donor. The conformation of AdoHcy has been reported (Ishida et al., 1982). Many analogs of AdoHcy, modified in the sugar moiety, amino acid portion or purine ring, have been synthesized to obtain information on the structural requirements for binding of AdoHcy to various transmethylases and compounds that resist metabolic degradation. Some of these compounds have been reported to show antiviral and growth inhibitory activity. Recent reviews (Ueland, 1982; Borchardt et al., 1986) provide further information.

Table 1 lists several important derivatives of Ado Met and AdoHcy.

3 BIOSYNTHESIS

3.1 Activated Methyl Cycle

Ado Met is synthesized by the transfer of an adenosyl group from A TP to the sulfur atom of L-methionine. The reaction is unusual in that there is a complete dephosphorylation of A TP, and the energy of ATP is used to activate the methyl group of the methionine unit. AdoHcy is formed when the methyl group of AdoMet is transferred to an acceptor compound such as catechol. In eukaryotic and most prokaryotic organisms, the thioether of AdoHcy is then hydrolyzed by AdoHcy hydrolase to form adenosine and L-homocysteine. This reaction had long been thought to be characteristic in eukaryotic organisms (Duerre, 1962; Miller & Duerre, 1968; Walker & Duerre, 1975). An alternative reaction that yields adenine and S-ribosyl-L-homocysteine for the metabolism of AdoHcy had been suggested to be involved in prokaryotic organisms. However, it was recently demonstrated that most prokaryotic organisms also metabolize AdoHcy through the thioether hydrolysis as well as eukaryotic organisms (Shimizu et al., 1984a, 1988). Although the reaction favors synthesis of AdoHcy in vitro, the metabolic flow is in the hydrolytic direction because adenosine and L-homocysteine are rapidly metabolized in the

Am

ino

acid

mod

ifica

tions

NH

2

~JyN~

':~

HO

O

H

S-A

deno

syl-

D-m

ethi

onin

e CH

3

• I

HO

OC

-CH

-(C

H2)

2-S

- CH

2-

I +

N

H2

S-A

deno

syl-

L-e

thio

nine

CH

2-C

H3

I H

OO

C-C

H-(

CH

2)2

-S- C

H2

-I

+

NH

2

S-A

deno

syl-

2-m

ethy

l-D

L-m

ethi

onin

e

CH

] C

H3

I .

I H

OO

C-C

-(C

H2)

2-S

-CH

2-I

+

NH

2

Tab

le 1

So

me

Der

ivat

ives

of

Ado

Met

and

Ado

Hcy

Che

mic

al s

truc

ture

s of

eac

h am

ino

acid

moi

ety

(R)

are

as s

how

n be

low

lef

t

Acc

umul

ated

in C

andi

da u

tilis

whe

n gr

own

wit

h D

-met

hion

ine

(Sch

lenk

et a

l., 1

978)

; sh

ows

met

hyl

dono

r ac

tivity

in s

ome

tran

smet

hyla

tion

sys

tem

s (N

akam

ura

& S

chle

nk,

1976

)

Acc

umul

ated

in b

aker

's y

east

(S

chle

nk e

t al.,

196

5) a

nd m

olds

(K

usak

abe

et a

l., 1

974)

w

hen

grow

n w

ith L

-eth

ioni

ne

Acc

umul

ated

in C

andi

da u

tilis

whe

n gr

own

with

2-m

ethy

l-D

L-m

ethi

onin

e (S

chle

nk e

t al

., 19

78);

sho

ws

met

hyl d

onor

act

ivity

in

som

e tr

ansm

ethy

lati

on s

yste

ms

(Nak

amur

a &

Sch

lenk

, 19

76)

<.H

~

~

~

§.

I:j.

I:

Roo ;t:

~ ~ ~

S-A

deno

syl-

o-ho

moc

yste

ine

• H

OO

C-C

H-(

CH

2h

-S--

CH

2-

I NH

2

S-A

deno

syl-

L-c

yste

ine

HO

OC

-CH

-CH

2-S

--C

H2-

I NH

2

Sine

fung

in

HO

OC

-CH

-(C

H2

h-C

H-C

H,-

I 1

-N

H2

NH

2

A91

45C

HO

OC

-CH

-(C

H2

h-C

H-C

H=

I

I N

H2

NH

2

5' -D

eoxy

-5' -

S-is

obut

yl-t

hioa

deno

sine

CH

,,,,

CH

-CH

2-S

--C

Hz-

CH

/ , N

ucle

osid

e m

odif

icat

ions

H2N~ C

H-C

H2-

CH

2-S

--C

Hz

base

HO

OC

/ ~

HO

O

H

Synt

hesi

zed

chem

ical

ly a

nd s

how

s in

hibi

tor

activ

ity in

som

e tr

ansm

ethy

lati

on r

eact

ions

(B

orch

ardt

& W

u, 1

974)

Synt

hesi

zed

by l

upin

see

d an

d be

ef li

ver

Ado

Hcy

hyd

rola

ses

(Gur

anow

ski

&

Jaku

bow

ski,

1983

)

Isol

ated

fro

m S

trep

tom

yces

gri

seoi

us a

s an

ant

ifun

gal

anti

biot

ic (

Ham

ill &

Hoe

hn,

1973

); a

pote

nt in

hibi

tor

of s

ome

tran

smet

hyla

tion

rea

ctio

ns (

see

Uel

and,

198

2)

A m

etab

olit

e of

Sin

efun

gin

(Ham

ill

& H

oehn

, 19

73);

a po

tent

inh

ibit

or o

f som

e tr

ansm

ethy

lati

on r

eact

ions

(se

e U

elan

d, 1

982)

Che

mic

ally

syn

thes

ized

as

an a

ntiv

iral

age

nt (

Hil

desh

eim

et a

i., 1

971)

; an

inh

ibit

or o

f po

lyam

ine

synt

hesi

s an

d tr

ansm

ethy

lati

on r

eact

ions

(se

e U

elan

d, 1

982)

Che

mic

al s

truc

ture

s of

eac

h ba

se a

re a

s sh

own.

In

the

case

of S

-ari

ster

omyc

inyl

-L­

hom

ocys

tein

e, t

he r

ibos

e m

oiet

y is

mod

ifie

d as

sho

wn.

(con

tinu

ed)

;... ~

::r ~ I §. s· .'" ;... ~ ~ s:: ~ ~ '" ~ s· '" § >:>

.. ~ S" [ , ~ "" U\ U

\

S-F

orm

ycin

yl-L

-hom

ocys

tein

e

NH

z

N~N\

~NJ-

-C~

S-N

ebra

liny

l-L

-hom

ocys

tein

e

H

N~N

~NJ-

) S

-Tub

erci

diny

l-L

-hom

ocys

tein

e

NH

z

(j)

N

N

S-2

-Chl

oroa

deno

syl-

L-h

omoc

yste

ine

NJ:-N

CI

AN~~

S -I

t' -M

ethy

lade

nosy

l-L

-hom

ocys

tein

e

H-N

-CH

3

ex)

Tab

le l

-co

ntd

.

Syn

thes

ized

by

beef

live

r, l

upin

see

ds a

nd b

acte

rial

Ado

Hcy

hyd

ro la

ses

(Gur

anow

ski

et

at.,

1981

; S

him

izu

eta

l.,

1984

a, b

; S

him

izu

& Y

amad

a, 1

988)

Syn

thes

ized

by

beef

live

r, l

upin

see

ds a

nd b

acte

rial

Ado

Hcy

hyd

ro la

ses

(Gur

anow

ski

et

at.,

1981

; S

him

izu

et a

t., 1

984a

, b;

Shi

miz

u &

Yam

ada,

198

8)

Syn

thes

ized

by

beef

live

r, l

upin

see

ds a

nd b

acte

rial

Ado

Hcy

hyd

rola

ses

(Gur

anow

ski

et

aI.,

1981

; S

him

izu

et a

I.,

1984

a).

A p

oten

t inh

ibit

or o

f va

riou

s tr

ansm

ethy

lati

on

reac

tion

s (C

owar

d et

at.,

197

4; a

lso

see

Uel

and,

198

2)

Syn

thes

ized

by

bact

eria

l A

doH

cy h

ydro

lase

(S

him

izu

et a

I.,

1984

a, b

; Shi

miz

u &

Y

amad

a, 1

988)

; 2-

Chl

oroa

deno

sine

sho

ws

a po

tent

inac

tiva

tor

acti

vity

tow

ard

Ado

Hcy

hyd

rola

se (

Shi

miz

u et

aI.

, 19

84a;

see

als

o U

elan

d, 1

982)

A p

oten

t in

hibi

tor

of

som

e tr

ansm

ethy

lati

on r

eact

ions

(H

offm

an,

1978

); S

ynth

esiz

ed b

y li

ver,

lup

in s

eeds

and

bac

teri

al A

doH

cy h

ydro

lase

s (H

offm

an,

1978

; G

uran

owsk

i et

at

., 19

81;

Shi

miz

u et

at.

, 19

84a)

.... VI

0"1

~

~

§.

N·

I:

R- ~

~ I

S-l

-Met

hyla

deno

syl-

L-h

omoc

yste

ine

HC

N

Hz

, "~

~N~

"""NJ-N

S

-Ino

syl-

L-h

omoc

yste

ine

OH

7JyN

~ """

NJ-N

S

-3-D

eaza

aden

osyl

-L-h

omoc

yste

ine

~~

N~C~~

Syn

thes

ized

by

bact

eria

l A

doH

cy h

ydro

lase

(S

him

izu

et a

l., 1

984a

)

Syn

thes

ized

by

bee

f liv

er,

lupi

n se

eds

and

bact

eria

l A

doH

cy h

ydro

lase

s (G

uran

owsk

i et

ai

., 19

81;

Shi

miz

u et

al.

, 19

84a)

Syn

thes

ized

by

bee

f li

ver

and

lupi

n se

eds

Ado

Hcy

hyd

rola

ses

(Gur

anow

ski

et a

l., 1

981)

; 3-

Dea

za-a

deno

sine

is a

pot

ent c

ompe

titi

ve i

nhib

itor

of

Ado

Hcy

hyd

rola

se

(Gur

anow

ski e

t al.,

198

1)

S-2

-Aza

-3-d

eaza

aden

osyl

-L-h

omoc

yste

ine

Syn

thes

ized

by

bee

f li

ver

and

lupi

n se

eds

Ado

Hcy

hyd

rola

ses

(Gur

anow

ski

et a

i., 1

981)

NH

z

~:X)

N..,

C

S-8

-Aza

aden

osyl

-L-h

omoc

yste

ine

NH

z

N~\

~NJ...N

Syn

thes

ized

by

beef

live

r an

d lu

pin

seed

s A

doH

cy h

ydro

lase

s (G

uran

owsk

i et a

l., 1

981)

(con

tinue

d)

~

f} 5 ~ i " ;;. 5·

;:s s· -" ~ ~ ~ s: ~ ~ .. ~ ~. ~ ~ Ei"" ~

1:1. ~ .., ~

~ ~ .. w ~

S-8

-Am

inoa

deno

syl-

L-h

omoc

yste

ine

NH

2

N~N

~~TJ

-)-

NH

2

N

N

S -(

5' -D

eoxy

aden

osin

e N

'-ox

ide-

5')

-L-h

omoc

yste

ine

NH

2

O'-N

J)-N

) ~N~

S-Py

razo

myc

ynyl

-L-h

omoc

yste

ine

o

H2N~N~

T~~'

/;

HO

C

S-A

rist

erom

ycin

yl-L

-hom

ocys

tein

e

NH

2

0:)

H

OC

H2 ~

HO

O

H

Tab

le l

---c

ontd

.

Syn

thes

ized

by

beef

live

r an

d lu

pin

seed

s A

doH

cy h

ydro

lase

s (G

uran

owsk

i et a

l., 1

981)

Syn

thes

ized

by

beef

live

r, l

upin

see

ds a

nd b

acte

rial

Ado

Hcy

hyd

rola

ses

(Gur

anow

ski

et

aI.,

1981

; Sh

imiz

u et

al.

, 19

84a,

b;

Shim

izu

& Y

amad

a, 1

988)

Syn

thes

ized

by

beef

live

r an

d lu

pin

seed

s A

doH

cy h

ydro

lase

s (G

uran

owsk

i et

al.,

198

1);

pyra

zom

ycin

is a

pot

ent i

nact

ivat

or o

f A

doH

cy h

ydro

lase

(se

e U

elan

d, 1

982)

Syn

thes

ized

by

beef

live

r an

d lu

pin

seed

s A

doH

cy h

ydro

lase

s (G

uran

owsk

i et a

l., 1

981)

; ar

iste

rom

ycin

is a

pot

ent

com

peti

tive

inh

ibit

or o

f A

doH

cy h

ydro

lase

(G

uran

owsk

i et

al.,1

981)

~

00

~

~

§.

N·

I: R­ ~

;;,< l

Adenosylmethionine, Adenosylhomocysteine and Related Nucleosides 359

normal cells. Deamination of adenosine by adenosine deaminase (EC 3.5.4.4) and phosphorylation of adenosine to AMP through the action of adenosine kinase (EC 2.7.1.20) are the responsible reactions for the further metabolism of adenosine. The other product, L-homocysteine, is used to regenerate L-methionine through the transfer of a methyl group from N 5_

methyltetrahydrofolate. This reaction is catalyzed by 5-methyltetrahydrofolate: L-homocysteine methyltransferase (EC 2.1.1.13). Alternatively, it can be methylated to L-methionine by utilizing betaine as a methyl donor with the enzyme, betaine: L-homocysteine S-methyltransferase (EC 2.1.1.5).

These reactions constitute the activated methyl cycle (Fig. 1). The methyl groups enter the cycle in the conversion of L-homocysteine into L-methionine and are then converted to a highly reactive form as Ado Met. The high transfer potential of the methyl group in AdoMet enables it to be transferred to a wide variety of acceptor compounds.

When cellular levels of adenosine or L-homocysteine or both are high, the reaction catalyzed by AdoHcy hydrolase is directed toward synthesis of

ATP

':Hz

lY) ¥l>+r%-~-CH~

.... 2 Hj:. 0 N~N,; C~2./ S-Adenosylmethylthlopropylamlne

~_J--N ~ I«l OH l j--J~ 1<H3 ~- -~-CHz-CHr~

HO OH ~, N:.JLN

S-Adenosylmethionine ~N~I ) HJC-!i-Di2

~

l?=~ HO OH ¥, ~ Methylthloadenoslne

HOOC'CH-CHz-CHrS-CH6 I Adenine ~ lIethylthlor!bO$e-1P

HOOH

S-Adenosylhomocysteine L s-Ribosylhomocysteine ,._~ Methionine

L 5-AdenOSYUJomlysreine hydrolase

CH3- Homocysteine ~ Adenosine

~

\ Ribose I , ' . , ...... _----- -- ------ -- -------------- ----- --- --- -----'

Fig. 1. Major routes for the metabolism of AdoMet and AdoHcy and the activated methyl cycle.

360 S. Shimizu & H. Yammla

AdoHcy and the enzymatic degradation of AdoHcy is inhibited (de la Haba & Cantoni, 1959). The accumulated AdoHcy blocks some metabolically impor­tant AdoMet-dependent transmethylation reactions, because AdoHcy is a potent inhibitor of many AdoMet-dependent transmethylases (Usdin et al., 1979; Borchardt et al., 1986). Furthermore, AdoHcy hydrolase is an adenosine binding protein (Hershfield & Kredich, 1978)\ Various adenosine analogs including adenosine have been shown to inactivate this enzyme irreversibly. As the result of this inactivation, the cellular level of AdoHcy increases, which also results in inhibition of transmethylation reactions. Thus, the hydrolysis of AdoHcy by AdoHcy hydrolase is a key step for the regulation of the activated methyl cycle and AdoHcy itself is thought to be a key regulator compound for transmethylation reactions (Cantoni & Chiang, 1980; Ueland, 1982; Borchardt et al., 1986).

The route which yields adenine and S-ribosyl-L-homocysteine for the degradation of AdoHcy is present in only few bacteria, especially in Entero­bacteriaceae (Duerre, 1962; Miller & Duerre, 1968; Shimizu et al., 1984a, 1988). This route involves the following two reactions: cleavage of glycosyl linkage of AdoHcy by AdoHcy nucleosidase (EC 3.2.2.9), yielding adenine and S-ribosyl-L-homocysteine, and hydrolysis of S-ribosyl-L-homocysteine to L-homocysteine and ribose by S-ribosylhomocysteine hydrolase (EC 3.3.1.3) (see Fig. 1).

3.2. Decarboxylation of AdoMet

Decarboxylation of AdoMet is a key step in the biosynthetic pathway for spermidine and spermine. This reaction is catalyzed by AdoMet decarboxylase (EC 4.1.1.50) (Tabor & Tabor, 1984). The decarboxylated AdoMet, S­adenosylmethylthiopropylamine, donates its aminopropyl group to putrescine to form spermidine, or spermidine to spermine in the presence of aminopropyltransferases. On transfer of the aminopropyl group, the decar­boxylated AdoMet itself is converted to MeSAdo, which has been demonstr­ated to be a potent inhibitor of the aminopropyltransferases and AdoHcy hydrolase (Ueland, 1982; Schlenk 1983). The inhibition by MeSAdo is relieved by the enzymatic cleavage of MeSAdo to adenine and 5-deoxy­methylthioribose-l~phosphate by MeSAdo phosphorylase (EC 2.4.2.28). Thus, this enzyme, as well as AdoHcy hydrolase, plays an important role in regulation of the activated methyl cycle. This enzyme activity has been detected in the organisms having AdoHcy hydrolase, but not in the bacteria having AdoHcy nucleosidase instead of AdoHcy hydrolase. In such bacteria, MeSAdo is hydrolyzed to adenine and ribose by AdoHcy nucleosidase (Shimizu et al., 1988) (see Fig. 1).

3.3 AdoHcy Hydrolase

As mentioned above, AdoHcy hydrolase is the most important enzyme for the regulation of the activated methyl cycle. Hydrolysis of AdoHcy by this enzyme

Adenosylmethionine, Adenosylhomocysteine and Related Nucleosides 361

is reversible, the reaction equilibrium in vitro greatly favors the synthetic direction (de la Haba & Cantoni, 1959), and the specificity of the enzyme for adenosine analogs is broad (Guranowski et al., 1981; Shimizu et aI., 1984a), suggesting that it is also important as a practical catalyst for the preparation of AdoHcy and related nucleosides (Shimizu & Yamada, 1984).

The enzyme activity has been detected in a variety of organisms ranging from mammalians to micro-organisms. The enzyme has been isolated from mammalian tissues (Richards et aI., 1978; Palmer & Abeles, 1979; Fujioka & Takata, 1981), lupin seeds (Guranowski & Pawelkiewicz, 1977) and a bacterium Alcaligenes faecalis (Shimizu et aI., 1984a) and characterized in some detail.

Bacterial AdoHcy hydrolase can be easily purified and crystallized from cells of A. faecalis, in which the cellular content of this enzyme is about 2·5% of the total extractable protein. This value is about 100 times higher than the enzyme content of eukaryotic sources. The purified enzyme has six identical subunits, each of which (mol wt = 48000-50000) binds 1 mol of NAD+, whereas most mammalian enzymes are tetramer whose subunit molecular mass is about 48000 and the plant enzyme is composed of two subunits identical in molecular

Table 2 Properties of AdoHcy Hydrolase of A. faecalis·

Mol. wt. Sedimentation equilibrium Gel filtration HPLC SDS-PAGE

Sedimentation velocity (S20.w) Number of subunits pI Km (mM)

Vm (Ilmol/min per mg)

Optimum temperature (0C) Optimum pH Heat stability CC) pH stability COOH-teiminal amino acid Other substrates

Inhibitor

Inactivator

AdoHcy adenosine DL-homocysteine Hydrolysis Synthesis

Synthesis

Formycin A, neburalin, adenosine N' -oxide, tubercidin, inosine, etc. Adenosine 5' -carboxylate, 5'-deoxyadenosine, etc. Arabinosyladenine, 2'­deoxyadenosine,5'-deoxy­adenosine, adenosine, etc .

• See Shimizu et al. (1984a) for details.

280000 290000 290000 48000 9·79 6 4·7 0·014 0·050 1·0

546·5 7·47

50 8·0-10·5

45 6·0-10·0

Tyr

362 S. Shimizu & H. Yamada

mass (55 (00). The function of NAD+ in the reversible reaction was estab­lished by Palmer & Abeles (1979). The prokaryotic enzyme efficiently catalyzes thioether formation between L-homocysteine and several novel nucleosides, most of which are known as antibiotic, antiviral or antitumor agents (for chemical structures, see Table 1), yielding the corresponding S-nucleosidyl-L-homocysteines. This fact suggests that the enzyme can be used as the catalyst for the production of not only AdoHcy but also these novel nucleoside derivatives. The enzyme is a characteristic adenosine binding protein as well as mammalian enzymes. Properties of the bacterial enzyme are summarized in Table 2.

4 PRODUCTION OF ADOMET

4.1 Microbial or enzymatic production

Since AdoMet was first prepared from L-methionine and A TP with a liver preparation containing methionine adenosyltransferase (Cantoni, 1953), there have been several reports for the preparation of Ado Met. Schlenk and co-workers (Schlenk & DePalma, 1957; Schlenk et ai., 1965) pointed out that several common yeasts, such as Saccharomyces cerevisiae and Candida utiiis, might be suitable sources of AdoMet. They reported that AdoMet is accumulated in growing or washed cells in the presence of relatively large amounts of L-methionine. Several molds were also reported to be suitable sources of AdoMet (Kusakabe et ai., 1974). However, the cellular contents of AdoMet were not satisfactory in these micro-organisms.

i • o U100 ~ "a

0 0

QI "-

§ c

0 0

0 • :8 ~ c • g 50

a • a 0 u • -; ~ 0 "a C

/j.

/j. A

~ i 16 0 0.05 0.10

Methlon"'e adenosyltransferase activity (pmo1/30 m ... /mg)

Fig. 2. Relationship between methionine adenosyltransferase activity and cellular AdoMet content of various yeasts. See Shiozaki et ai. (1984) for details. 0, Saccharomyces sake strains; e, other Saccharomyces strains; 6., yeasts of other genera.

Adenosylmethionine, Adenosylhomocysteine and Related Nucleosides 363

Recently, Yamada and co-workers (Shimizu & Yamada, 1984; Shiozaki et al., 1984; Yamada et al., 1984) assayed a variety of micro-organisms for their ability to accumulate Ado Met intracellularly using several media containing L-methionine. The ability to accumulate significant amounts of AdoMet was found to be widely distributed in yeasts belonging to the genera Saccharomyces and Candida. A group of Japanese traditional sake yeasts, i.e. Saccharomyces sake strains, accumulated especially high levels of Ado Met. The AdoMet contents of most of these strains reached more than 100 mg/g dry cells or 1 mg/ml of culture broth (Fig. 2). They selected S. sake K-6, as the most promising strain.

In a study on the optimization of the culture conditions for the accumulation of AdoMet by S. sake K-6, they found that sucrose and urea are very suitable carbon and nitrogen sources, respectively, for obtaining high density growth

'l 12 i 5 ._ .. \'-// .. _ ... _., ............. _., ... - ... , ............ _._.-

3

i 0"", 0'1 / E E 10 50 0\ / r --Y- 0'1

Qj ~ /i\ E ::.:

I 10 '0 0 ;,<~\ '0 I c « 0

~ 8 I 40 / 0 ..c

/ W a a /

~ ::2 £/ / OJ ~30 I u 6 l: /

E / x / Vl QI / 300 ~ L.. ..c

I u 0

Q 3 / A-·A, C 4 ~ 20 A"'/ "'A U

L.. I 0'1 0 0

I '. 200 .f ::2 I /

, OJ '\ u I

I ~ ~ I

C 2 10 I i A C

A' 100 0

0 I u

OJ ;§ 9 L )e _e_e", 0

0 0 ~ 0 2 3 4 5 6 7

Time (days)

Fig. 3. Typical time course of AdoMet production by S. sake K-6. S. sake K-6 was cultivated in a lO-liter bench scale fermentor with 5 liters of a medium containing 10% sucrose, 1% yeast extract, 1·8% urea, 1% L-methionine, 0·2% glycylglycine, 0·4% KH2P04, 0·01 % MgS04·7H20, biotin (2,Ltg/ml) and 10 ml of a mixture of metal salts and trace elements (20 g of CaCI2·2H20, 0·5 g of MnS04'5H20, 0·5 g of FeS04·7H20, 1·0 g of ZnS04'7H20, 0·01 g of CuS04·5H20, 0·01 g of CoCI2·6H20, 0·01 g of H 3B03 ,

0·01 g of Na2Mo04 and 0·01 g of KI in 1 liter H20), pH 6·3. Cultivation was carried out at 28°C with aeration at 1 vvm and agitation at 300 rpm. Ethanol (50 ml) was added to the medium at the time indicated by arrows in the figure. MgS04·7H20 (0·25 g) was further added to the medium on the 3rd day of the cultivation. See Shiozaki et al.

(1986) for details.

364 S. Shimizu & H. Yamada

Fig. 4. Ultraviolet photomicrograph showing accumulation of Ado Met in vacuoles of S. sake K-6. (0) indicates control cells grown for 3 days without L-methionine. (2) and (5) indicate cells grown with L-methionine for 2 and 5 days, respectively, under the

conditions given in Fig. 3.

without any decrease in intracellular level of AdoMet. They also pointed out that addition of glycylglycine, biotin and CaCl2 to the culture medium increases cellular AdoMet content (Shiozaki et al. , 1986). Under the optimal culture conditions in a lO-liter bench scale fermentor , S. sake K-6 produced lO·8 g/liter of AdoMet (Fig. 3). Almost all the AdoMet was accumulated in vacuoles in the cells (Fig. 4), the extracellular concentration being very low. The maximum AdoMet content of cells reached 260 mgt g dry cells; this compares favorably with the commercially available yeast cells rich in AdoMet which contain only about 20 mg/g solids.

Leakage of AdoMet from vacuoles leads to its rapid decomposition to MeSAdo and it is important to retain vacuolar integrity and high concentra­tions of Ado Met in order to produce satisfactorily large amounts of the nucleosides. Although it is not yet clear why sake yeasts show notably high ability to accumulate AdoMet, one possible explanation is suggested by the data shown in Fig. 2, where methionine adenosyltransferase activities are compared among various kinds of yeasts. The results reveal a positive correlation between accumulation of Ado Met and the enzyme activity. Another interesting characteristic of this yeast is that it is able to grow well in the presence of high concentrations of L-methionine. L-Methionine has been reported to markedly inhibit the growth of common Saccharomyces yeasts (Takahashi & Takahashi, 1968). This may also be an important characteristic for the high accumulation of Ado Met.

Another approach to produce AdoMet was reported by Gross et al. (1983) , who synthesized 7·8 mmol of AdoMet on incubation of a reaction mixture (850 ml) containing 20 mmol of AMP, 0·5 mmol of A TP, 40 mmol of phos­phoenol pyruvate, lO mmol of MgCI2 , 150 mmol of KCl, 20 mmol of L-

Adenosylmethionine, Adenosylhomocysteine and Related Nucleosides 365

L-Methlonine

AMP ;; P\ ATP -"'-_...,..-------"

PEP Pyruvate PPi ~ Pi

Fig. 5. Reaction sequences of AdoMet synthesis using immobilized cell-free enzymes. See Gross et al. (1983) for details. PEP, phosphoenol pyruvate; Met adenosyltransferase, methionine adenosyltransferase; AK, adenylate kinase; PK,

pyruvate kinase; PPase, inorganic pyrophosphatase.

methionine and 1 mmol of dithiothreitol with methionine adenosyltransferase purified from baker's yeast, inorganic pyrophosphatase, adenylate kinase and pyruvate kinase for 6 days at room temperature. In this case, the enzymes immobilized on gels were used as catalysts and the A TP used for the synthesis was supplied in situ from AMP using phosphoenol pyruvate as the source of phosphate, as shown in Fig. 5.

4.2 Methods for Isolation of AdoMet and its Stabilization

Some important problems to be improved for the practical production of AdoMet are its separation from other low molecular weight cell constituents and the stabilization of the finally isolated AdoMet (Shimizu & Yamada, 1984, 1986). The conventional large-scale preparation method involves the following successive steps: extraction of AdoMet from yeast cells with perchloric acid, decolorization of the extract, separation of AdoMet and salt formation. Decolorization can be achieved with synthetic adsorbents, and is indispensable for obtaining a final product of high purity. Separation of Ado Met from other low molecular weight compounds may be carried out on a cationic ion­exchange resin due to its high positive electric charge at the sulfonium center. Adenosine, adenine, MeSAdo and similar compounds having positive electric charges sometimes interfere with the separation of AdoMet. Shiozaki et al. (1986) reported a new and efficient isolation procedure involving freezing/thawing of cells for the extraction of AdoMet and chromatographies on Amberlite IRA-45, XAD-2 and IRC-50. They obtained AdoMet of 96·1% purity in a recovery yield of 69·7% from cells of S. sake K-6 (28 g as dry weight) containing 5·01 g of AdoMet through this procedure.

A variety of salt forms have been proposed for stabilization of AdoMet. They can be grouped into the following types: salts with haloacids (i.e. AdoMet+Cl-, AdoMet+Cl-HCI, AdoMet+r, AdoMet+Br-, etc.), salts with sulfate compounds (i.e. AdoMet+HSO; and AdoMet+HSO;·(H2S04)1_3, and double salts with H2S04 and inosine-5'-monosulfate, adenosine-5'­monosulfate, uridine-5'-monosulfate, etc.), salts with sulfonate compounds (i.e. salts with p-toluenesulfonic acid, p-chlorobenzenesulfonic acid, methane­sulfonic acid, etc., and a double salt with H2S04 and p-toluenesulfonic acid), salts with phosphoric acid (i.e. AdoMet+H3PO;, etc.), salts with dicarboxylic

366 S. Shimizu & H. Yamada

acids (i.e. salts with citric acid, tartaric acid, malic acid, succinic acid, etc.), salt with ascorbic acid, and cyclodextrin complexes of AdoMet+HSOi, AdoMet+HSOi . (H2S04)n , etc. Among these, the p-toluenesulfonate salts and cyclodextrin complexes seem to be considerably stable. The double salt with p-toluenesulfonic acid and H2S04 has been used to treat various liver diseases in Italy and some other countries.

Another problem recently revealed is that most commercially available AdoMet products contain 10-20% of the inactive ( + )-enantiomer, the source of which is unknown (Stolowitz & Minch, 1981). Matos et al. (1987) reported that the ( - )-enantiomer is the sole product of the enzymatic reaction with E. coli methionine adenosyltransferase. This suggests that conversion of the ( - )-enantiomer to the ( + )-enantiomer might take place during isolation or storage.

4.3 Chemical Synthesis of AdoMet

Chemical approaches to prepare AdoMet generally involve the methylation of AdoHcy with appropriate methylating agents such as methyl iodide. However, the chemical methylation is not stereoselective. Molar ratios of the (- )- to ( + )-enantiomer are 55: 45 and 65: 35 for methyl iodide and trimethylsul­fonium iodide, respectively (Matos et al., 1987). In addition, AdoHcy is required for the preparation. It is considerably expensive when compared with L-methionine.

5 PRODUCTION OF AdoHcy

5.1 Enzymatic Methods

The in vitro hydrolysis of AdoHcy by AdoHcy hydrolase greatly favors the synthetic direction and the enzyme is considered to be a useful catalyst for the synthesis of AdoHcy and S-nucleosidylhomocysteines.

Recently, a simple and efficient method for the production of AdoHcy using the reverse reaction of AdoHcy hydrolase has been reported (Shimizu et al., 1984b, 1986a, b; Yamada et al., 1984) based on the finding that bacterial cells are rich sources of AdoHcy hydrolase (Shimizu et aI., 1984a). Shimizu et al. (1986a) found that washed cells of several bacteria belonging to the genera Alcaligenes and Pseudomonas, and some strains of actinomycetes, efficiently converted the added adenosine and L-homocysteine to AdoHcy. They selected A. faecalis AKU 101 as the most promising catalyst. With the washed cells of this bacterium as the catalyst, and adenosine (200 mM) and L-homocysteine (200 mM) as the substrates, they obtained nearly 100% molar conversion with a yield of about 80 g/liter after 40 h at 37°C (see Table 3).

A problem with this synthesis was that the D-isomer of homocysteine could

Adenosylmethionine, Adenosylhomocysteine and Related Nucleosides 367

not be utilized as the substrate. To overcome this, they screened for microbial strains capable of synthesizing AdoHcy from D-homocysteine. Several Pseudomonas strains were found to have this ability. When washed cells of P. putida were used as the catalyst, AdoHcy synthesis from adenosine, and each of the three forms of homocysteine, i.e. L-, D- or DL-homocysteine, proceeded at the same reaction rate. In each case, the product was the L-form of AdoHcy. They demonstrated that, although the P. putida AdoHcy hydrolase as well as the A. faecalis enzyme catalyzes only the condensation of adenosine and L-homocysteine, the bacterium also produces a racemase which catalyzes the interconversion of the D- and L-homocysteine isomers but not that of the AdoHcy produced. For practical purposes, A. faecalis cells are still superior to P. putida cells in that they show high AdoHcy hydrolase activity and low adenosine deaminase activity. In the mixed cell system containing the two strains, the reaction rate greatly decreased when the content of P. putida cells was increased. Therefore, they used P. putida cells just as a source of the racemase. With 200 mM adenosine and 200 mM DL-homocysteine, the molar conversion to AdoHcy was 86%, when a mixture of A. faecalis cells (36 mg/ml) and P. putida cells (4 mg/ml) was used as the catalyst (Table 3). The reaction mechanism for this system is considered to be as shown in Fig. 6 (Shimizu & Yamada, 1984; Shimizu et al., 1986b).

Table 3 AdoHcy Synthesis with Varying Concentrations of Adenosine and L- or DL­

Homocysteine with Cells of A. faecalis or Mixed Cells of A. faecalis and P. putida

Cells used as catalysts

A. faecalis

A. faecalis

A. faecalis: P. putida (9: 1)

Adenosine

100 200 300 400

50 100 100 200 300 100 200 300 400

Concentrations (I1mol/ml) of

Homocysteine L- DL-

100 200 300 400

100 100 200 400 600 100 200 300 400

AdoHcy produced (I1mol/ml)

100 178 199 153 50 53 76

103 71

100 173 173 155

Reaction mixture (1·0 ml) containing 40 mg of air-dried cells, as indicated, 50 Ilmol of potassium phosphate buffer, pH 8·0, and the indicated amounts of adenosine and homocysteine were incubated for 36 h at 37°C. See Shimizu et al. (1986b) for details.

368 S. Shimizu & H. Yamada

AdoHcy hydTo/ase

(A.faecalls) L -Homocysteine + Adenosine ~ " AdoHcy

1: :R.cemase l(P.putlda)

D-Homocysteine Fig. 6. Reaction mechanism for the synthesis of AdoHcy from adenosine and

DL-homocysteine.

5.2 Chemical Methods

Two routes have been used generally for the chemical synthesis of AdoHcy. One involves (1) protection of the 2'- and 3'-hydroxyl groups of adenosine using an isopropylidene protecting group; (2) activation of the nucleoside 5'-position by formation of the 5'-tosylate; (3) condensation of the intermedi­ate 5'-tosylate with S-benzyl-L-homocysteine; and (4) removal of the isopropylidene protecting group using dilute acid (Baddiley & Jamieson, 1955; Borchardt & Wu, 1974). The other route involves replacement of the 5'-hydroxyl group of the adenosine by chlorine followed by the condensation of the resulting 5'-chloro-S'-deoxyadenosine with L-homocysteine (Borchardt et al., 1976). Condensation of unprotected adenosine with a protected homocy­steine in the presence of trialkyl phosphine was also reported (Serafinowski, 1985). These standard procedures have been used for the synthesis of a broad spectrum of AdoHcy analogs.

6 PRODUCTION OF NOVEL S-NUCLEOSIDYLHOMOCYSTEINES

Because A. faecalis AdoHcy hydrolase utilizes a variety of adenosine analogs as substrates (Shimizu et al., 1984a), the cells containing this enzyme can be used as a catalyst for the synthesis of a variety of S-nucleosidylhomocysteines. Shimizu et al. (1984b) reported that neburalin and formycin A, as well as adenosine, were converted to the corresponding S-nucleosidylhomocysteines in high yields on incubation with L-homocysteine as the cosubstrate and dried cells of A. faecalis as the catalyst. They also reported that K-carrageenan­entrapped cells of the same organism efficiently catalyze the conversion of adenosine Nl-oxide and 2-chloroadenosine, in addition to neburalin and formycin A, to the corresponding S-nucleosidylhomocysteine congeners. The immobilized cells showed high stability on repeated reactions, whereas free cells rapidly inactivated (Fig. 7) (Shimizu & Yamada, 1988).

These novel S-nucleosidylhomocysteines may also be promising in the pharmaceutical and chemotherapeutic fields (Ueland, 1982; Borchardt et al., 1986).

Adenosylmethionine, Adenosylhomocysteine and Related Nucleosides 369

o 1 2 3 4 5 6 7 8 9 10 11 Number of repeat

Fig. 7. Synthesis of S-nucleosidylhomocysteines with K-carrageenan entrapped cells of A. faecalis. See Shimizu & Yamada (1988) for details. Adenosine (Ado), formycin A (For), adenosine N1-oxide, (Ado-N-oxide) and 2-chloroadenosine (Cl-Ado) were used

as nucleoside substrates.

7 CONCLUSION

The biotechnological area of research on AdoMet and related nucleosides has advanced considerably in the past few years. Important progress in this area can be summarized as follows: (1) screening of S. sake K-6 as a potent producer of AdoMet; (2) determination of the optimal conditions for large-scale culture, by which Ado Met content of the yeast reached 200-300 mg/g dry cells; (3) finding of A. faecalis as a potent AdoHcy hydrolase producer, which made possible to synthesize AdoHcy and related nucleosides in high yields; and (4) development of a new synthetic scheme for the production of AdoHcy, involving AdoHcy hydrolase and homocysteine racemase as the catalysts. This made possible to use cheap racemic mixture of homocysteine as the amino acid substrate. Rapid expansion of research and accumulation of biochemical and pharmacological information on these nucleosides suggest that their pharmacological values are very high.

ACKNOWLEDGEMENT

Research from the author's laboratory was supported, in part, by a Grant in Aid of Scientific Research from the Ministry of Education, Science and Culture of Japan and a grant from the Uehara Memorial Foundation.

REFERENCES

Baddiley, J. & Jamieson, G. A. (1955). Synthesis of S-(5'-deoxyadenosine-5')­homocysteine, a product from enzymic methylations involving 'active methionine'. J. Chern. Soc., 1085-9.

370 S. Shimizu & H. Yamada

Borchardt, R. T. & Wu, Y. S. (1974). Potential in activator of S-adenosylmethionine­dependent methyltransferases. 1. Modification of the amino acid portion of S-adenosylhomocysteine.l. Med. Chem., 17,862-7.

Borchardt, R. T., Huber, J. A. & Wu, Y. S. (1976). A convenient preparation of S-adenosylhomocysteine and related compounds. J. Org. Chem., 41, 565-7.

Borchardt, R. T., Creveling, C. R. & Ueland, P. M. (Eds) (1986). Biological Methylation and Drug Design, Experimental and Clinical Roles of s­Adenosylmethionine. Humana Press, Clifton, NJ.

Cantoni, G. L. (1952). The role of the active methyl donor formed enzymatically from L-methionine and adenosinetriphosphate. J. Am. Chem. Soc., 74,2942-3.

Cantoni, G. L. (1953). S-Adenosylmethionine; a new intermediate formed enzy­matically from L-methionine and adenosinetriphosphate. J. BioI. Chem., 204, 403-16.

Cantoni, G. L. (1975). Biological methylation: Selected aspects. Ann. Rev. Biochem., 44,435-51.

Cantoni, G. L. & Chiang, P. K. (1980). The role of S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase in the control of biological methylations. In Natural Sulfur Compounds: Novel Biochemical and Structural Aspects, ed. D. Cavallini, G. E. Gaull & V. Zappia. Plenum Press, New York, pp. 67-80.

Coward, J. K., Bussolotti, D. L. & Chang, C.-D. (1974). Analogs of S­adenosylhomocysteine as potential inhibitors of biological transmethylation. Inhibi­tion of several methylases by S-tubercidinylhomocysteine. J. Med. Chem., 17, 1286-9.

Duerre, J. A. (1962). A hydrolytic nucleosidase acting on S-adenosylhomocysteine and on 5'-methylthioadenosine. 1. Bioi. Chern., 237, 3737-41.

Fujioka, M. & Takata, Y. (1981). S-Adenosylhomocysteine hydrolase from rat liver: Purification and properties. J. Bioi. Chem., 256, 1631-5.

Gross, A., Geresh, S. & Whitesides, G. E. (1983). Enzymatic synthesis of S-adenosyl­L-methionine from L-methionine and ATP. Appl. Biochem. Biotechnol., 8,415-22.

Guranowski, A. & Jakubowski, H. (1983). Substrate specificity of S­adenosylhomocysteinas'e: Cysteine is a substrate of the plant and mammalian enzymes. Biochim. Biophys. Acta, 742, 250-6.

Guranowski, A. & Pawelkiewicz, J. (1977). Adenosylhomocysteinase from yellow lupin seeds: Purification and characterization of the enzyme from Lupinus luteus seeds. Eur. J. Biochem., 114,293-9.

Guranowski, A., Montogomery, J. A., Cantoni, G. L. & Chiang, P. K. (1981). Adenosine analogues as substrates and inhibitors of S-adenosylhomocysteine hydrolase. Biochemistry, 20. 110-5.

Haba, G. de la & Cantoni, G. L. (1959). The enzymatic synthesis of S-adenosyl-L­homocysteine from adenosine and homocysteine. J. Bioi. Chem., 234,603-8.

Haba, G. de la, Jamieson, G. A., Mudd, S. H. & Richards, H. H. (1959). S-Adenosylmethione: The relation of configuration at the sulfonium center to enzyme reactivity. J. Am. Chem. Soc., 81,3975-80.

Hamill, R. L. & Hoehn, M. M. (1973). A9145, a new adenine-containing antifungal antibiotic. 1. Discovery and isolation. J. Antibiot., 26, 463-5.

Hershfield, M. S. (1979). Apparent suicide inactivation of human lymphoblast S-adenosylhomocysteine hydrolase by 2'-deoxyadenosine and adenine arabinoside: A basis for direct toxic etIects of analogs of adenosine. J. Bioi. Chem., 254, 22-5.

Hershfield, M. S. & Kredich, N. M. (1978). S-Adenosylhomocysteine hydrolase is an adenosine-binding protein: A target for adenosine toxity. Science, 202. 757-60.

Hildesheim, J., Hildesheim, R. & Lederer, E. (1971). Syntheses d'inhiabiteurs des methyl-transferases: Analogues de la S-adenosyl homocysteine. Biochimie, 53, 1067-71.

Adenosylmethionine, Adenosylhomocysteine and Related Nucleosides 371

Hoffman, J. L. (1978). Biosynthesis of S-N>-methyladenosylhomocysteine, an inhibitor of RNA methyltransferases. J. Bioi. Chem., 253,2905-7.

Ishida, T., Tanaka, A., Inoue, M., Fujiwara, T. & Tomita, K. (1982). Conformational studies of S-adenosyl-L-homocysteine, a potential inhibitor of S-adenosyl-L­methionine-dependent methyltransferases. J. Am. Chem. Soc., 104, 7239-48.

Kusakabe, H., Kuninaka, A. & Yoshino, H. (1974). Accumulation of S­adenosylmethionine by molds. Agric. Bioi. Chem., 38, 1669-72.

Matos, J. R., Raushel, F. M. & Wong, C.-H. (1987). S-Adenosylmethionine: Studies on chemical and enzymatic synthesis. Biotechnol. Appl. Biochem. 9, 39-52.

Miller, C. H. & Duerre, J. A. (1968). S-Ribosylhomocysteine cleavage enzyme from Escherichia coli. J. Bioi. Chem., 243, 92-7.

Nakamura, K. D. & Schlenk, F. (1976). The activity of adenosyl-D-methionine and adenosyl-2-methylmethionine in transmethylations. Archs. Biochem. Biophys., 177, 170-5.

Palmer, J. L. & Abeles, R. H. (1979). The mechanism of action of S­adenosylhomocysteinase. J. BioI. Chem., 254, 1217-26.

Richards, H. H., Chiang, P. K. & Cantoni, G. L. (1978). Adenosylhomocysteine hydrolase. Crystallization of the purified enzyme and its properties. J. Bioi. Chem., 253, 4476-80.

Salvatore, F., Borek, E., Zappia, V., Williams-Ashman, H. G. & Schlenk, F. (eds) (1977). The Biochemistry of Adenosylmethionine. Columbia University Press, New York.

Schlenk, F. (1983). Methylthioadenosine. Adv. Enzymol., 54, 195-265. Schlenk, F. (1984). The discovery of enzymatic transmethylation. Trends Biochem. Sci.

9,34-5. Schlenk, F. & DePalma, R. E. (1957). The formation of S-adenosylmethionine in

yeast. J. Bioi. Chem., 229, 1027-50. Schlenk, F., Zydek, C. R., Ehninger, D. J. & Dainko, J. L. (1965). The production of

S-adenosyl-L-methionine and S-adenosyl-L-ethionine by yeast. Enzymologia, 29, 283-98.

Schlenk, F., Hannum, C. H. & Ferro, A. J. (1978). Biosynthesis of adenosyl-D­methionine and adenosyl-2-methylmethionine by Candida utilis. Archs. Biochem. Biophys., 187, 191-6.

Serafinowski, P. (1985). A convenient preparation of S-adenosylhomocysteine and its analogs. Synthesis, 926-9.

Shimizu, S. & Yamada, H. (1984). Microbial and enzymatic processes for the produc­tion of pharmacologically important nucleosides. Trends Biotechnol. 2, 137-41.

Shimizu, S. & Yamada, H. (1986). Coenzymes. In Biotechnology, Vol. 4, ed. H.-J. Rehm & G. Reed. VCH Vedagsgesellschaft, Weinheim, pp. 159-84.

Shimizu, S. & Yamada, H. (1988). Enzymatic process for the production of pharmacologically useful nucleosides. Ann. N. Y. Acad. Sci., 542, 423-7.

Shimizu, S., Shiozaki, S., Ohshiro, T. & Yamada, H. (1984a). Occurrence of S-adenosylhomocysteine hydrolase in prokaryote cells: Characterization of the enzyme from Alcaligenes faecalis and role of the enzyme in the activated methyl cycle. Eur. J. Biochem., 141,385-92.

Shimizu, S., Shiozaki, S., Ohshiro, T. & Yamada, H. (1984b). High yield synthesis of S-adenosylhomocysteine and related nucleosides by bacterial S-adenosylhomocysteine hydrolase. Agric. BioI. Chem., 48, 1383-5.

Shimizu, S., Shiozaki, S. & Yamada, H. (1986a). High yield production of S-adenosyl­L-homocysteine with microbial cells as the catalyst. J. Biotechnol., 4,81-90.

Shimizu, S., Ohshiro, T., Shiozaki, S. & Yamada, H. (1986b). Production of S-adenosyl-L-homocysteine by bacterial cells with a high content of S­adenosylhomocysteine hydrolase: utilization of a racemic mixture of homocysteine as the substrate. J. Biotechnol., 4,91-100.

372 S. Shimizu & H. Yamada

Shimizu, S., Abe, T. & Yamada, H. (1988). Distribution of methylthioadenosine phosphorylase in eubacteria. FEMS Microbiol. Lett., 51, 177-80.

Shiozaki, S., Shimizu, S. & Yamada, H. (1984). Unusual intracellular accumulation of S-adenosyl-L-methionine by micro-organisms. Agric. Bioi. Chem., 48, 2293-2300.

Shiozaki, S., Shimizu, S. & Yamada, H. (1986). Production of S-adenosyl-L-methionine by Saccharomyces sake. J. Biotechnol., 4,345-54.

Stolowitz, M. L. & Minch, M. J. (1981). S-Adenosyl-L-methionine and S-adenosyl-L­homocysteine, an NMR study. J. Am. Chem. Soc., 103,6015-19.

Tabor, C. W. & Tabor, H. (1984). Methionine adenosyltransferase (S­adenosylmethionine synthetase) and S-adenosylmethionine decarboxylase. Adv. Enzymol., 56, 251-82.

Takahashi, T. & Takahashi, H. (1968). Inhibition of yeast growth by methionine: Part III. Methionine as an inhibitor of conversion from fermentative to respiratory processes. Agric. BioI. Chem., 32, 1448-52.

Ueland, P. M. (1982). Pharmacological and biochemical aspects of S­adenosylhomocysteine and S-adenosylhomocysteine hydrolase. Pharmacol. Rev., 34,223-53.

Usdin, E., Borchardt, R. T. & Creveling, C. R. (Eds) (1979). Transmethylation. Elsevier, Amsterdam.

Usdin, E., Borchardt, R. T. & Creveling, C. R. (Eds) (1982). Biochemistry of S-Adenosylmethionine and Related Compounds. Macmillan Press, London.

Walker, R. D. & Duerre, J. A. (1975). S-Adenosylhomocysteine metabolism in various species. Can. J. Biochem., 53,312-19.

Yamada, H. & Shimizu, S. (1985). Microbial enzymes as catalysts for synthesis of biologically useful compounds. In Biocatalysts in Organic Synthesis, ed. J. Tramper, H. C. van der Plas & P. Linko. Elsevier, Amsterdam, pp. 19-37.

Yamada, H. & Shimizu, S. (1988). Microbial and enzymatic processes for the production of biologically and chemically useful compounds. Angew. Chem. Int. Ed. Engl., 27,622-42.

Yamada, H., Shimizu, S., Shiozaki, S. & Ohshiro, T. (1984). Microbial and enzymatic processes for the production of S-adenosylmethionine, S-adenosylhomocysteine and related nucleosides. In Third European Congress on Biotechnology, Vol. I. Verlag Chemie, Weinheim, pp. 249-53.

Zappia, V., Usdin, E. & Salvatore, F. (Eds) (1979). Biochemical and Pharmacological Roles of Adenosylmethionine and the Central Nervous System. Pergamon Press, Oxford.

Chapter 20

OTHER VITAMIN-RELATED COENZYMES

S. SHIMIZU & H. YAMADA

Department of Agricultural Chemistry, Kyoto University, Kyoto 606, Japan

1 INTRODUCTION

The name 'coenzymes' has been given to a group of dissociable, low molecular weight active components of enzymes which catalyze transfer of chemical groups, hydrogen or electrons. Coenzymes, defined in this way, usually function as cosubstrates for enzymes to couple two otherwise independent reactions. Thus, they can be regarded as transmitters of metabolites. In a wider sense, any catalytically active, low molecular weight, non-protein components of enzymes are also included in the coenzyme group. This definition includes coenzymes which are covalently bound to enzymes as prosthetic groups. In this case, the coenzyme and the apoenzyme together make up the active holoenzyme.

Many coenzymes are biosynthesized from vitamins and also contain a nucleoside (or nucleotide) moiety in their molecules. The relationships of some coenzymes to vitamins and metabolic functions are listed in Table 1. Some coenzymes are produced on an industrial scale and are used as pharmaceuti­cals. For example, ATP, COP-choline, FAD, NAO, coenzyme A, S­adenosylmethionine, coenzyme Q1O, etc., are used for muscular dystrophy, metabolic diseases, clouding of consciousness, cerebral surgery, hepatic or renal dysfunction and so on. They are also used for analysis and research in clinical chemistry, biochemistry, metabolism and pharmacology. Because several coenzymes are already mentioned in the preceding individual chapters describing the corresponding parent vitamins, this chapter outlines a few of the remaining ones. A previous review on coenzymes (Shimizu & Yamada, 1986) seen from a biotechnological point of view may be useful for further understanding.

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376 S. Shimizu & H. Yamada

2 NICOTINAMIDE COFAcrORS

Various aspects of the studies on these cofactors were reviewed in a recent book by Dolphin et al. (1987). A review by Chenault & Whitesides (1987) focussed on the biotechnological aspect of the cofactors.

2.1 Preparation of NAD and NADP

Both extraction from yeast cells and enzymatic synthesis are performed for the commercial production of NAD. Baker's yeast (Saccharomyces cerevisiae) and brewer's yeast (S. carlsbergensis or S. uvarum) have been shown to be favorable sources of NAD. On cultivation in a medium containing adenine and nicotinamide, values of 12 mg/g dry cells and 42 mg/g dry cells for S. cerevisiae and S. carlbsergensis, respectively, were reported (Sakai et al., 1973a, b). The enzymatic process utilizes the salvage route for the biosynthesis of NAD from nicotinamide (or nicotinic acid) and adenine. Brevibacterium ammoniagenes was found to convert the added precursors to NAD efficiently. When the precursors were added to the medium after culturing for 48 h and incubated for a further 120 h, the amount of NAD in the medium reached 2·3 mg/ml. The mechanism involved in the NAD production has been suggested to be that adenine and nicotinamide are first ribosylated to yield A TP and nicotinamide mononucleotide, respectively, which are then condensed by pyrophosphoryla­tion to form NAD (Nakayama et al., 1968). Several combined cell-free enzymatic and chemical syntheses of NAD starting from AMP or ATP have also been reported. For example, on incubation of 40 mmol of nicotinamide mononucleotide (synthesized from AMP in three steps) and AMP for 10 days in a 2000 ml of reaction mixture containing immobilized NAD pyrophos­phorylase, inorganic pyrophosphatase, acetate kinase and adenylate kinase, 39 mmol of NAD was produced (Walt et al., 1980). These procedures seem to be inferior to the extraction from yeast cells and to the enzymatic synthesis with cells of B. ammoniagenes because of their complexity.

NADP is prepared by the phosphorylation of NAD using NAD kinase and ATP. The conditions for the continuous phosphorylation of NAD have been studied with immobilized cells of Achromobacter aceris (Uchida et al., 1978) and B. ammoniagenes (Tanaka et al., 1982). With a column packed with the immobilized A. aceris, 6-8 Ilmol/ml of NADP was obtained on a continuous passage of the substrate solution (25·1 mM) at space velocity of 0·1 h- 1 .

Recently, a thermostable NAD kinase of Corynebacterium flaccumfaciens was shown to be useful for practical preparation of NADP (Matsushita et al., 1986). Brevibacterium ammoniagenes has been reported to produce a novel enzyme, polyphosphate kinase, which catalyzes the phosphorylation of NAD with methaphosphate as a phosphate donor. Continuous phosphorylation of NAD with immobilized cells of this bacterium was also reported (Murata et al., 1979).

NADH and NADPH can be prepared from the corresponding oxidized coenzymes by chemical, enzymatic, or microbial reduction.

Other Vitamin-related Coenzymes 3n

2.2 Regeneration of NADD and NADPD

Many systems for regenerating nicotinamide cofactors are involved in the complex and regulated metabolism of living cells. For example, the pathway for glucose oxidation via glucose-6-phosphate involves two important reactions yielding NADPH, i.e. the oxidation of glucose-6-phosphate by glucose-6-phosphate dehydrogenase and the oxidation of 6-phosphogluconate by 6-phosphogluconate dehydrogenase. Endogeneous NADPH or a catalytic amount of added NADPH can be efficiently regenerated by the simple mixing of microbial cells with glucose in an appropriate reaction mixture. The reductions of various carbonyl compounds by yeast cells occasionally per­formed in synthetic organic chemistry are mainly based on this system. Similarly, oxidation of ethanol to acetic acid by yeasts and oxidation of methanol to CO2 by methylotrophic micro-organisms are considered to be useful for NADH regeneration.

When cell-free enzymes requiring NAD(P)H are to be used, the construc­tion of a suitable regenerating system is an additional requirement. Many systems have been tested for this purpose (for a review, see Chenault & Whitesides, 1987). Among them, the formate/formate dehydrogenase, glucose/ glucose dehydrogenase and glucose-6-phosphate/ glucose-6-phosphate dehydrogenase systems are most widely used, because of the availability of these hydrogen donor substrates and enzymes (Table 2). The formate/formate dehydrogenase system has been used successfully to regenerate a polymer­bound NADH in a membrane-reactor synthesis of several optically active amino acids from the corresponding keto acids (Wandrey et ai., 1984; Hummel et aI., 1986). Successful application of the same system has also been reported for the production of L-phenylalanine with phenylalanine dehydrogenase (Asano & Nakazawa, 1987) and R-( - )-mandelic acid with 2-hydroxyiso­caproate dehydrogenase (Yamazaki & Maeda, 1986).

3 PYRROLOQUINOLINE QUINONE

Pyrroloquinoline quinone (POO), 2,7 ,9-tricarboxy-H -pyrrolo[2,3-f)quinoline-4,5-dione, was found as the prosthetic group of a primary alcohol de­hydrogenase (EC 1.1.99.8) in methylotrophic bacteria. Subsequent work has demonstrated that in addition to the NAD(P)-dependent and flavoprotein dehydrogenases, there is a third group of enzymes, 'quinoprotein oxidoreduc­tases', in which PQQ is involved as the coenzyme. Alcohol, aldehyde and glucose dehydrogenases of acetic acid bacteria and several other bacteria have been found to be POQ-dependent dehydrogenases. Evidence has been provided for the quinoprotein nature of several other microbial and mam­malian enzymes such as amine dehydrogenase, amine oxidase, nitrile hydra­tase, and several more (for review, see Duine et ai., 1986, 1987). These results suggest that PQQ is widely distributed among living organisms, although no

Tab

le 2

Sy

stem

s fo

r R

egen

erat

ion

of N

AD

(P)H

fro

m N

AD

(P)"

Syst

em a

nd r

eact

ion

For

mat

e/fo

rmat

e de

hydr

ogen

ase

HC

OO

-+

NA

D-

CO

2 +

NA

DH

Glu

cose

/ glu

cose

deh

ydro

gena

se

Glu

cose

+ N

AD

(P)-

gluc

onat

e +

NA

DP

(H)

Glu

cose

-6-p

hosp

hate

l glu

cose

-6-p

hosp

hate

de

hydr

ogen

ase

G-6

-P +

NA

D(P

)-6-

PG

+ N

AD

(P)H

Eth

anol

/ alc

ohol

deh

ydro

gena

se-a

ldeh

yde

dehy

drog

enas

e ~H50H +

NA

D-

CH

3CO

O-+

NA

DH

Adv

anta

ge

Hig

h re

duci

ng p

oten

tial

In

expe

nsiv

e re

duct

ant

Eas

y re

mov

al o

f by

-pro

duct

(C

02

)

Hig

h re

duci

ng p

oten

tial

In

expe

nsiv

e re

duct

ant

Hig

h st

abili

ty o

f en

zym

e H

igh

spec

ific

act

ivity

of

enzy

me

Abi

lity

to r

educ

e N

AD

or

NA

DP

H

igh

redu

cing

pot

enti

al

Inex

pens

ive

enzy

me

Hig

h sp

ecif

ic a

ctiv

ity o

f en

zym

e A

bilit

y to

red

uce

NA

D o

r N

AD

P

Inex

pens

ive

enzy

me

(yea

st a

lcoh

ol

dehy

drog

enas

e)

Inex

pens

ive

redu

ctan

t H

igh

spec

ific

act

ivity

of

enzy

mes

V

olat

ility

of

etha

nol

and

acet

alde

hyde

Dis

adva

ntag

e

Hig

h ex

pens

e of

enz

yme

Low

spe

cifi

c ac

tivity

of

enzy

me

Inab

ility

to

redu

ce N

AD

P

Hig

h ex

pens

e of

enz

yme

Dif

ficu

lty i

n re

mov

al o

f by

-pro

duct

(gl

ucon

ate)

Exp

ensi

ve r

educ

tant

D

iffi

culty

in

rem

oval

of

by-p

rodu

ct (

6-PG

)

Low

red

ucin

g po

tent

ial

Pro

duct

inh

ibit

ion

by a

ceta

ldeh

yde

Inab

ility

to

redu

ce N

AD

P

a M

odif

ied

from

Che

naul

t &

Whi

tesi

des

(198

7).

G-6

-P,

gluc

ose-

6-ph

osph

ate;

6-P

G,

6-ph

osph

oglu

cona

te.

~

00

:-> ~

§. ... ;::

R­ ;t:

;;.< l

Other Vitamin-related Coenzymes 379

systematic search has been reported. POO has been shown to have a growth-promoting effect on several bacteria (Ameyama et al., 1984a; Shimao et al., 1984).

Several bacteria have been reported to excrete PQO into their culture medium. Especially, methylotrophic bacteria seem to be favorable sources for PQQ production. Reported fermentation yields range from 0·01 to 10 ,ug/ml (Ameyama et al., 1984b). From a culture filtrate (1100 liters) of Hyphomicrobium X grown with 0·5% methanol, a yield of 1385 mg POO was obtained through the procedures involving absorption with Amberlyst A21 anion exchanger and elution from it (Duine et al., 1987).

4 COENZYME 0

Coenzyme Q (CoQ, ubiquinone) is a 2,3-dimethyl-5-methyl benzoquinone with an isoprenoid side chain composed of dihydroisoprene units. There are various types which are designated according to the number of isoprene units in the side chain: Co06 , COQ7, CoOs, Co09. COQIO, etc. CoQ6-COQIO are widely distributed (the name 'ubiquinone' is from its ubiquitous occurrence). Co01-CoQs were found in several bacteria and yeasts, such as Rhodospirillum rubrum, Escherichia coli and Saccharomyces cerevisiae (Friis et aI., 1966; Daves et aI., 1967; Bentley & Meganathan, 1987). The occurrence of CoOu , C0012 and Co013 was also reported (Natori & Nagasaki, 1979, 1980). Among these homologs, CoOIO has been extensively studied as to practical production, because it is the natural coenzyme in humans and it is used in therapeutic treatment for diseases of such tissues as heart and muscle.

An ethionine-resistant mutant derived from Agrobacterium sp. has been reported to be a potent producer of CoOlO. This mutant produced two types of morphologically distinguishable colonies, rough and white, and smooth and yellow, on an agar plate. Only the former showed excellent production (4·1 mg/ g dry cells), but changed easily to the latter with lower productivity (2·8 mg/g dry cells) during cultivation or on transfer. The concentration of ammonium ions in the production medium and aeration or agitation during the cultivation were found to be important factors affecting cellular COQlO content. Under optimum cultivation conditions, a value of 5·1 mg/g dry cells or 211 ,ug/ml of culture broth was attained with this mutant (Kuratsu et al., 1984a, b, c). A facultative methylotroph, Protaminobacter ruber, produced 1· 52 mg/ g dry cells on cultivation with methanol as sole carbon source for growth. In this case, no other CoO homologs than COQlO were detected in the cells (Natori et aI., 1978). Paracoccus denitrificans (Matsumura et al., 1983) and tobacco cells (Ikeda et al., 1981) have also been shown to be potent producers of CoOlO.

Microbial production of several CoO homo logs other than COQlO has also been studied. An n-alkane-utilizing yeast, Candida tropicalis, produced 4·7 mg COQ9/g dry cells (52,ug/ml of culture broth) on cultivation in a medium containing n-alkanes with successive addition of p-hydroxybenzoate as

380 S. Shimizu & H. Yamada

a precursor of the quinone nucleus. n-Alkanes have been suggested to be superior to other usual growth substrates for the production of CoQ, because the isoprenoid moiety of the CoQ molecule is derived from acetyl-CoA which is thought to be sufficiently available through the degradation of n-alkanes (Shimizu et al., 1970a, b).

REFERENCES

Ameyama, M., Shinagawa, E., Matsushita, K. & Adachi, O. (1984a). Growth stimulation of micro-organisms by pyrroloquinoline quinone. Agric. BioI. Chem., 48, 2909-11.

Ameyama, M., Hayashi, M., Matsushita, K., Shinagawa, E. & Adachi, O. (1984b). Microbial production of pyrroloquinoline quinone. Agric. Bioi. Chem., 48,561-5.

Asano, Y. & Nakazawa, A. (1987). High yield synthesis of L-amino acids by phenylalanine dehydrogenase from Sporosarcina ureae. Agric. Bioi. Chem., 51, 2035-6.

Bentley, R. & Meganathan, R. (1987). Biosynthesis of the isoprenoid quinones, ubiquinone and menaquinone. In Escherichia coli and Salmonella typhimurium, ed. F. C. Neidhardt. American Society for Microbiology, Washington, DC, pp. 512-20.

Chenault, H. K. & Whitesides, G. M. (1987). Regeneration of nicotinamide cofactors for use in organic synthesis. Appl. Biochem. Biotechnol., 14, 147-97.

Daves, G. D. Jr, Muraca, R. F., Whittick, J. S., Friis, P. & Folkers, K. (1967). Discovery of ubiquinones-l, -2, -3, and -4 and the nature of biosynthetic isoprenylation. Biochemistry, 6,2861-6.

Dolphin, D., Avranovic, O. & Poulson, R. (Eds) (1987). Coenzymes and Co/actors, Vol. 2: Pyridine Nucleotide Coenzymes: Chemical, Biochemical, and Medical Aspects, Part B. John Wiley and Sons, New York.

Duine, J. A., Frank, Jzn; J. & Jongejan, J. A. (1986). PQQ and quinoprotein enzymes in microbial oxidations. FEMS Microbiol. Rev., 32, 165-78.

Duine, J. A., Frank, Jzn, J. & Jongejan, J. A. (1987). Enzymology of quinoproteins. Adv. Enzymol., 59, 169-212.

Friis, P., Daves, G. D. Jr & Folkers, K. (1966). Isolation of ubiquinone-5, new member of ubiquinone group. Biochem. Biophys. Res. Commun., 24,252-6.

Hummel, W., Schmidt, E., Wandrey, C. & Kula, M. R. (1986). L-Phenylalanine dehydrogenase from Brevibacterium sp. for production of L-phenylalanine by reductive amination of phenylpyruvate. Appl. Microbiol. Biotechnol., 25, 175-85.

Ikeda, T., Matsumoto, T., Obi, Y., Kisaki, T. & Noguchi, M. (1981). Characteristics of cultured tobacco cell strains producing high levels of ubiquinone-lO selected by a cell cloning technique. Agric. Bioi. Chem., 45, 2259-63.

Kuratsu, Y., Hagino, H. & Inuzuka, K. (1984a). Effect of ammonium ion on coenzyme QlO fermentation by Agrobacterium species. Agric. BioI. Chem., 48, 1347-8.

Kuratsu, Y., Sakurai, M., Hagino, H. & Inuzuka, K. (1984b). Productivity and colony morphology associated with coenzyme QlO production by Agrobacterium species. Agric. Bioi. Chem., 48, 1997-2002.

Kuratsu, Y., Sakurai, M., Hagino, H. & Inuzuka, K. (1984c). Aeration-agitation effect on coenzyme QlO production by Agrobacterium species. J. Ferment. Technol., 62, 305-8.

Matsumura, M., Kobayashi, T. & Aiba, S. (1983). Anaerobic production of ubiquinone-lO by Parococcus denitrificans. Eur. J. Appl. Microbiol. Biotechnol., 17, 85-9.

Other Vitamin-related Coenzymes 381

Matsushita, H., Yokoyama, S. & Obayashi, A. (1986). NADP+ production using thermostable NAD+ kinase of Corynebacterium flaccumfaciens AHU-1622. Can. J. Microbio!., 32,585-90.

Murata, K., Kato, J. & Chibata, I. (1979). Continuous production of NADP by immobilized Brevibacterium ammoniagenes cells. Biotechnol. Bioeng., 21,887-95.

Nakayama, K., Sato, Z., Tanaka, H. & Kinoshita, S. (1968). Production of nucleic acid-related substances by fermentation processes. Part XVII. Production of NAD and nicotinic acid mononucleotide with Brevibacterium ammoniagenes. Agric. Bio!. Chem., 32, 1331-6.

Natori, Y. & Nagasaki, T. (1979). Formation of coenzyme Q n by Pseudomonas M16, a mutant. Agric. Bioi. Chem., 43,797-802.

Natori, Y. & Nagasaki, T. (1980). Occurrence of coenzyme Q 12 and coenzyme Q13 in facultative methanol-oxidizing bacteria. Agric. Bio!. Chem., 44, 2105-10.

Natori, Y., Nagasaki, T., Kobayashi, A. & Fukawa, H. (1978). Production of coenzyme Q10 by Pseudomonas N842. Agric. Bioi. Chem., 42, 1799-1800.

Sakai, T., Uchida, T. & Chibata, I. (1973a). Production of nicotinamide adenine dinucleotide by Saccharomyces carlsbergensis. Agric. Bioi. Chem., 37, 1041-8.

Sakai, T., Uchida, T. & Chibata, I. (1973b). Accumulation of nicotinamide adenine dinucleotide in baker's yeast by secondary culture. Agric. Bioi. Chem., 37, 1049-56.

Shimao, M., Yamamoto, H., Ninomiya, K., Kato, N., Adachi, 0., Ameyama, M. & Sakazawa, C. (1984). Agric. Bioi. Chem., 48,2873-76.

Shimizu, S. & Yamada, H. (1986). Coenzymes. In "Biotechnology", Vol. 4, ed. H.-J. Rehm & G. Reed. VCH Verlagsgesellschaft, Weinheim, pp. 159-84.

Shimizu, S., Tanaka, A. & Fukui, S. (1970a). Studies on the utilization of hydrocar­bons by micro-organisms (X). Production of coenzyme Q by Candida tropicalis pK 233 in hydrocarbon fermentation. (Part III) Effect of compounds related to the respiratory system on coenzyme Q production. J. Ferment. Techno!., 48, 542-8.

Shimizu, S., Tanaka, A. & Fukui, S. (1970b). Studies on the utilization of hydrocar­bons by micro-organisms (XI). Production of coenzyme Q by Candida tropicalis pK 233 in hydrocarbon fermentation. (Part IV) Effect of various medium components on coenzyme Q production. J. Ferment. Technol., 48,549-55.

Tanaka, Y., Hayashi, T., Kawashima, K., Yokoyama, T. & Watanabe, T. (1982). Production of NADP by immobilized cells with NAD kinase. Biotechnol. Bioeng., 24,857-69.

Uchida, T., Watanabe, T., Kato, J. & Chibata, I. (1978). Continuous production of NADP by immobilized Achromobacter aceris cells. Biotechnol. Bioeng., 20, 255-66.

Walt, D. R., Rios-Mercadillo, V. M., Auge, J. & Whitesides, G. M. (1980). Synthesis of nicotinamide adenine dinucleotide (NAD) from adenosine monophosphate (AMP). J. Am. Chem. Soc., 102,7805-6.

Wandrey, c., Wichmann, R., Berke, W., Morr, M. & Kula, M. R. (1984). Continuous cofactor regeneration in membrane reactors. In Third European Congress on Biotechnology, Vol. I. Verlag Chemie, Weinheim, pp. 239-44.

Yamazaki, Y. & Maeda, H. (1986). Enzymatic synthesis of optically pure (R)-( -)­mandelic acid and other 2-hydroxycarboxylic acids: Screening for the enzyme, and its purification, characterization and use. Agric. Bioi. Chem., SO,2621-31.

Chapter 21

FUNGAL GIBBERELLIN PRODUCTION

B. BRUECKNER, D. BLECHSCHMIDT

Friedrich-Schiller-University lena, Dept. of General Microbiology, DDR-6900 lena, Neugasse 24, GDR

&

G. SEMBDNER, G. SCHNEIDER

Institute of Plant Biochemistry, Academy of Sciences of the GDR, DDR-4050 Halle, Weinberg 3, GDR

1 HISTORICAL SURVEY

Micro-organisms are capable of producing a series of plant growth regulators such as gibberellins, auxines, abscisic acid, ethylene, fusicoccin, jasmonic acid, etc., but only the microbiological production of gibberellins is of industrial importance. The story of the identification of gibberellins as a plant growth regulator is a classic example of the interactions between soil micro-organisms and plants. It is well known that gibberellins were first obtained from culture filtrates of the soil fungus Gibberella fujikuroi (Saw.) Wr. (Fusarium monili­forme Sheld). In Japan and other Far East countries this plant-pathogenic fungus is one of the most important pathogens of rice. When it attacks rice seedlings an early disease symptom is a superelongation of the shoots, which are more pale in colour than normal shoots. Farmers have called them bakanae or 'foolish seedlings'. After growing very rapidly, infected plants die. The disease was a serious cause of rice destruction in Japan during the last century.

The first scientific description of the bakanae disease was published in 1898 by Hori, who detected the causative agent and identified it as an imperfect fungus. The Japanese scientist Kurosawa (1926), after several failures, showed that the sterilized filtrate of the culture medium in which Giberella fujikuroi had grown is capable of causing the same marked growth stimulation in rice seedlings without infection of the fungus. He reported that the phytopathoge­nic fungus secretes a toxin which promotes the rice growth. Kurosawa concluded that the activity of the sterile culture filtrate 'was not due to enzyme action but rather to some kind of chemical' (Kurosawa, 1926, cit. Phinney, 1983). This observation was confirmed and extended by other Japanese scientists. The main results established by this work are these:

(a) cultural filtrates of Giberella fujikuroi produced overgrowth of shoots, growth of roots was unaffected;

383

384 B. Brueckner et al.

(b) other plants, including dicotyledons as well as monocotyledons, re­sponded by increased shoot growth;

(c) the rice plant does not respond by forming an antibody against the toxin.

After Kurosawa's report a number of plant pathologists started to isolate the active principle and to study its biological properties. More than 50 publica­tions appeared between 1927 and 1940, mainly in Japan. In 1934 Yabuta's group succeeded in obtaining an active component from the fungus filtrate. However, they showed rather inhibitory effects than stimulatory activity. The substance was given the name fusaric acid (Yabuta et ai., 1934). After changing the composition of the culture medium Yabuta (1935) announced the isolation of the growth stimulant as a fairly pure non-crystalline solid which was called 'gibberellin' according to the bakanae fungus. This was the first appearance of the term in scientific literature. In 1938 Yabuta & Sumiki isolated two crystalline substances, 'gibberellin A' and 'gibberellin B'. Later the terms were reversed because the former was shown to be inactive (Tamura, 1971). After the war the Japanese experiments initiated the studies in other countries too. In the USA, Stodola and his colleagues (1955) developed the fermentation techniques for mass production of gibberellins and showed that the substance they had isolated was composed of two gibberellins, which they named 'gibberellin A' and 'gibberellin X'. Independently, workers at the Imperial Chemical Industries Ltd (ICI) in Great Britain in 1954 obtained a new active substance with similar biological properties but different in chemical and physical properties called 'gibberellic acid' (Curtis & Cross, 1954).

In the 1950s the Japanese workers at the Tokyo University reinvestigated their original gibberellin A preparation and could separate it into four gibberellins and named them gibberellins AI> A 2 , A3 and A4 (Tamura, 1971). Furthermore, they demonstrated that gibberellin A3 was identical with gibberellin X and gibberellic acid, and that gibberellin Al was the same as Stodola's gibberellin A. The successful work with the fungus induced further studies on higher plants. At once the number of publications on gibberellin responses in plants increased exponentially. In 1954 Brian reported the elongation of a pea dwarf cultivar in response to exogenous gibberellin. The spectacular growth response of Brian's dwarf pea to gibberellic acid led the workers to speculate that dwarfism might be due to the absence of endogenous gibberellins and that endogenous gibberellins would be present in non-dwarf cultivars. The next step in the story of gibberellins was the discovery in 1956 that extracts from higher plants contained gibberellin-like substances that induced biological responses identical to those elicited by the fungal gib­berellins (Radley, 1956). The detection of gibberellin-like substances in several species of plants led to studies that resulted in the isolation and identification of gibberellins in higher plants. In 1958 MacMillan and his colleagues were successful in the identification of gibberellin Al from runner bean seeds, and

Fungal Gibberellin Production 385

later on in the identification of gibberellin As from the same material (Phinney, 1983). Up to now, a total of 72 gibberellins have been isolated and structurally elucidated, of which 62 have been isolated from plants and 26 from the fungus.

2 CHEMICAL AND PHYSICAL PROPERTIES

Gibberellins (GAs) exhibit a bifacial nature, on one hand they are products of the secondary metabolism in fungi, especially Gibberella fujikuroi, on the other hand they are biologically active, endogenous hormones in the higher plants. From the chemical point of view, gibberellins represent a group of diterpenoids having a typical tetracyclic ring system that, according to the nomenclature (Rowe, 1968) is called ent-gibberellane (Fig. 1).

The trivial nomenclature attributes gibberellins to a numbered A series (GAl ... GAx). This system was originally dedicated to the plant hormone status of gibberellins, because in addition to having the typical structure and the natural occurrence, the gibberellins have to be biologically active in certain bioassays (MacMillan & Takahashi, 1968). Currently, the group of gibberellins (A-series) comprises altogether 72 different GAs; 26 of them were isolated from fungal sources (Bearder, 1980; Muromtsev & Agnistova, 1984; Takahashi et al., 1986). Also 3-0-acetyl-GA3 was found to be a genuine product of the fungus (Schreiber et al., 1966). The structures and some physical data of fungal gibberellins are compiled in Table 1.

Gibberellins fall into two groups: ~o-gibberellins, which possess the complete diterpenoid skeleton, and C19-gibberellins, which have biogenetically lost the C-20 resulting in a 19,1O-y-lactone structure that is typically for GAs with high bioactivity. Other inevitable features of physiologically important GAs are the 6-carboxy function and the 16(17) methylene group.

The most prominent representative GA is GA3 (=gibberellic acid, ent-3£t', 10, 13-trihydroxy-20-nor-gibberella-1(2), 16(17)dien-7 ,19-dicarboxy-19,1O­lactone) because of its high abundance in microbial fermentation but also because of its high bioactivity.

Due to the structural diversity and the manifoldness of functional groups GAs cover a broad range of polarity, e.g. hydrophilic and lipophilic properties.

Fig. 1. The ent-gibberellane skeleton.

Table 1 Fungal Gibberellins

Structure Formula M mp caC) (a)D

GA, o H C'9H2.0 0 348 255-258 +380

HOJ11y[},~~ (dec.)

CH, COOH 2

GA2 o H C'9H 260 0 350 255 +120

HO~OH (dec.)

CH, COOH CH,

GA, @ C'9H 220 0 346 234-236 +920

~o nn OH (dec.)

HO - H CH 2

CH, COOH

GA.

@ C'OH24O, 332 214-216 _30

co (dec.)

HO 2 (H H ,

CH3 COOH

GA7

@~ C'9H 220 , 330 169-172 +200

202 (dec.)

HO· -(II H 2

CH, (DOH

GAo W C,oH2.04 316 208-211 -220

,. H CH,

CH, COOH

GAIO

~O" CH 3 (DOH (H 3

C,oH20O, 334 245-246 +30

GAil

W C'OH22O, 330 242-244 + 11 0

~O H CH z

CH, COOH

GA'2 ~H, H C2oH 2XO. 332 245-248

HOOC""\~ H CH

CH, COOH z

GA"

"@ C2"H260 7 334 194-196 -480

HOOC\"" H CHz (H, COOH

Table l-contd.

Structure Formula M mp (OC) (a)D

GAl. ~fi:J H CZOH 2,O, 348 242-243 -730

HO~ HOO(~ H (H,

(H3 (DOH

GA"

~ytL, C ZOH 26O. 330 274-276 +50

(H3 (DOH

GAI6

# C I9H z.06 348 157-165

=0 H =

(H3H DOH (H,

GA2• CHO H C2oH 26O, 346 198-203 -880

®CH, HOOC CH3 COOH

GAz5 (DOH H CZOHZ606 362 248-252 -690

~CH' HO DC CH3 COOH

GA36 ~HO H CZOHZ606 362 205-208

HO~CH' HOOC CH (OOH

3

GA37

@ C2oH z6O, 346 228-232

CO

HO ~ H CH, CH3 COOH

GA40 Q H C I9H 2.O, 332 212-213 HO/////%tl

(H3H COOH (H,

GA' l Cz"HzgOg 396 am.

366 174-182

(continued)

GAs.

GA"

3-0-acetyl­GA,

Table l-contd.

Structure

Ho.@,.H ~o

HO :; H (H2

(H3 (OOH

"4P", 4htt" H H (H

(H, COOH 2

H

HO

(O()-j

~ H

~IIIOH H~(H (H, COOH 2

~~H

~O AcO" ,,,,OH

H (H_ (H, COOH '

Formula

""""'''''''"~ )''"'"'' "d'

@~" HO H (H2

(H, (OOH

~

,~~ ~ ~H H(k~H

"~O II ... OH ~ lu .. OH HO - H (H 2 ~gO("· H (H2

rn, (OOH ~ (OO~

iso-GA, d icarboxyllc acid

M mp t'C)

348

348 243-246

364 260-263

364 am.

364 147-150

380 226

glbbe ri c ac Id

Fig. 2. Typical reactions of ring A of GA3 type gibberellins.

(a)D

Fungal Gibberellin Production 389

H H

0) OOH

H; Lewis Ac id

nCH3 100D C CH2 = - 0

fLCH' H

b) H+

n'CH; H H

c) U CH, H+

14CH, OH

Fig. 3. Typical reactions of ring C/D site of gibberellins.

Accordingly GAs differ considerably in solubility and in their partitioning coefficients (Durley & Pharis, 1972; Takahashi et ai., 1986). The pK-values of of monobasic GAs range between 3·8 and 4·3.

The structural elucidation of GAs is based on comprehensive data of mass spectrometry and of IH_ and 13C-NMR investigations (Takahashi et ai., 1986; Hedden, 1987). The stereochemistry of GA3 was finally confirmed by X-ray analysis (Hartsuck & Lipscomb, 1963), where other GAs can be related to.

As far as the chemical reactivity of GAs is concerned the multifunctional substitution pattern and tensions of the ring system induce a series of typical degradation processes, which may also play some role in fermentation and/or processing steps. Thus, already slight alterations of the pH value can lead to irreversible rearrangements and transformations, especially with GA3-type gibberellins as shown in Fig. 2. Allogibberic acid, gibberellenic acid and iso-GA3 are almost concomitants of GA3.

Some general routes of reactions taking place in the C/D-ring site of GAs are outlined in Fig. 3. These reactions depend on the specific surrounding of the 16(17) methylene group, which is accessible to both nucleophilic and electrophilic attack. Quite common is the Wagner-Meerwein rearrangement of the 13-hydroxy-16(17) methylene structure, which for GA3 leads to the formation of gibberic acid (Graebe & Ropers, 1978; Takahashi et ai., 1986).

3 PRODUCING MICRO-ORGANISMS AND SCREENING

As described above, gibberellins were first isolated from culture filtrates of Gibberella fujikuroi. Although several micro-organisms have been tested for their potency in gibberellin formation, the most productive system continues to

390 B. Brueckner et al.

be Gibberella fujikuroi (Saw.) Wr. (Fusarium moniliforme Sheld). In the first publications after the discovery the taxonomic position of the phytopathogenic fungus responsible for the bakanae disease was poorly defined. This led to considerable confusion in the literature in the naming of the gibberellin producer and in relation to the symptoms of the rice disease to a specific species of the fungus. The reason for the controversy was the lack of a systematic nomenclature for the genus. Jefferys (1970) listed all names which have been attributed to producing organisms:

Fusarium heterosporum Fusarium moniliforme Fusarium oxysporum Gibberella fujikuroi Gibberella moniliforme

Nees Sheldon Schlechtendahl (Sawada) Wollenweber (Sheldon) Wineland

In 1931 Wollenweber in his publication on the taxonomy of the genus Fusarium resolved the problem and named the imperfect stage of the bakanae fungus Fusarium moniliforme (Sheldon) and the perfect stage Gibberella fujikuroi (Saw.) Wr. (Phinney, 1983). Borrow et al. (1955) had tested a lot of strains isolated from several host plants for their capacity to synthesize gibberellins. The bakanae effect was produced by most of the rice strains but only by one of the strains from other hosts. He concluded a connection between the ability of isolates to produce gibberellins and the origin of the strains from infected rice.

In contrast to these results Gordon (1960) showed that gibberellins were produced by isolates from rice and other host plants such as maize, sugar cane and cotton. He had acquired a large collection of strains from widely differing hosts and geographical locations. All high-yielding strains of Gibberella fujikuroi have their origin from the best wild-types, which were isolated from infected rice seedlings by an extended screening programme. So in 1966 and 1967 Tamura isolated 1500 strains of Fusarium moniliforme in Japan. The infected rice seedlings were collected from fields on the islands of Hokkaido, Honshu, Shikoku and Kyushu (Phinney, 1983). One of the most cited high producing wild-strains, isolated from infected rice seedlings in Japan is Gibberella fujikuroi (Saw.) Wr., BRL (ACC) 917. This is the C.B.S., Baarn, 'Sawada' strain, and it is also culture No. NRRL 2633 and Commonwealth Mycological Institute culture No. 58290 (Borrow et al., 1955, 1961). The BRL (ACC) 917 was the subject of many physiological, biochemical and genetic studies in connection with biosynthesis of gibberellins. The highest yields reported were approximately 1 g GA per liter of culture filtrate obtained in improved medium with this strain (Rehm, 1980).

Beside Gibberella fujikuroi other fungi were also found to produce GA., but generally in lower amounts. In 1972 J. C. Lozano at the Centro Internacionale de Agricultura Tropical (CIAT) in Cali (Columbia) reported a 'superelonga­tion disease' of cassava plants. The independent work of two research groups has established the occurrence of GA4 in the Deuteromycete Sphaceloma manihoticola causing growth promotion in cassava (Rademacher & Graebe,

Fungal Gibberellin Production 391

1979; Zeigler et al., 1980). G~ appears to be an end-product of gibberellin biosynthesis in Sphaceloma manihoticola, while it is an intermediate in the biosynthesis of GA7, GA3 and GAl in Gibberella fujikuroi.

In the culture filtrate of Sphaceloma manihoticola some other gibberellins could be detected: GA13 (5 mg/liter), GAl4 (1 mg/liter), GA24 (100 Ilg/liter), GA9 (50 Ilg/liter) and GAI5 , GA25, GA36, and GA37 in trace amounts. GA4 is obtained in a very pure state in high amounts of maximum 7 mg/liter (EP 0,024,951). By improving culture conditions and selecting high-producing strains it might be possible to produce G~ in a very pure form.

From mycelia, but not from culture filtrate, of Neurospora crassa, low amounts of gibberellic acid could be isolated (Kawanabe et al., 1983). The authors concluded that gibberellic acid has a regulatory function in the sexual cycle of this fungus. Coolbaugh et al. (1985) reported the detection of several gibberellins from the culture filtrate of the producer of abscisic acid, Cercospora rosicola. Gibberellin-like substances were found to be produced in trace amounts by several groups of micro-organisms, especially soil bacteria, actinomycetes, algae, farns and fungi (Muromtsev & Agnistova, 1984; Brueck­ner & Blechschmidt, 1986). But the structure of these active compounds was not identified unequivocally.

4 BIOSYNTHESIS AND REGULATION

As diterpenes the gibberellins are formed through the isoprenoid biosynthetic pathway, starting from mevalonic acid which is first converted via the isopentenyl, dimethylallyl, geranyl and farnesyl pyrophosphates to geranylger­anyl pyrophosphate (GGPP), being an important pivotal intermediate. As shown in Fig. 4, several points of divergence occur in this pathway which lead to other important groups of compounds.

GGPP is converted further to ent-kaurene, which is the first committed intermediate in gibberellin biosynthesis. The formation of ent-kaurene is catalyzed by ent-kaurene synthetase in a two-step sequence. In the first step (A-activity of the enzyme) GGPP is partially cyclized to form the bicyclic copalyl pyrophosphate which is cyclized again in the second step (B-activity) to give ent-kaurene. Both the A- and B-activity of the enzyme appear in the supernatant after high speed centrifugation. The molecular weight of the purified ent-kaurene synthetase, which exhibited both activities, was estimated at 4·3-4·9 x 105 dalton and the pH optimum of the A- and B-activities were 7·5 and 6·9, respectively. The presence of a divalent cation, preferably Mg2+, is required for both activities. Knowledge about these early stages of gibberellin biosynthesis in Gibberella fujikuroi has been comprehensively reviewed by Sembdner et al. (1980) and Coolbaugh (1983); and a more recent review (Graebe, 1987) provides some supplementary information. It is apparent from the extensive literature that the early stages of GA biosynthesis are identical in the fungus and in higher plants. However, the extraordinary rate of GA

392

~H3 Ho-~-CH2COOH

CH2CH20H

B. Brueckner et al.

mevalonic acid

ATP---1

ATP-i

ATP-i

~OPP J

63-isopentenyl pyrophosphate ----, cytokinin side chains (IPP)

(opp ~ 3.3'- dimethylallyl pyrophosphate

1PP-i (Copp geranyl pyrophosphate -- monoterpenes

IPP-1

A

B

abscisic acid

farnesyl pyrophosphate ~ -- squalene __ sterols

~I OPP phytoe ne - carotenolds ~ ,ChlorOPhyuesters I geranylgeranyl pyrophosphate 0(. tocopherol

~ phylloqUInone plas toquin one I fatty acid esters

~ noncyclic diterpenes

;; ,# I OPP ______ macrocyclic dlterpenes

H copalyl pyrophosphate :::::::::

,H -- ent-isokaurene

! 17

ent- kaurene gibberellins

18

Fig. 4. Gibberellin biosynthetic pathway from mevalonic acid to ent-kaurene.

Fungal Gibberellin Production 393

production in Gibberella fujikuroi indicates that the pathway in the fungus lacks some of the controls working in higher plants.

ent-Kaurene synthesis is a branch point in the pathway which commits the cell (organelle) to the production of either GA or alternative products, the variety of which is much wider in the higher plants than in the fungus (cf. Fig. 4). ent-Kaurene is a direct precursor of GAs, and the key position of ent-kaurene synthetase suggests that its activities A and B might be metabo­lically controlled. However, regulation of ent-kaurene synthetase in Gibberella fujikuroi is much less studied than in higher plants. A number of plant growth inhibitors, like Phosphon D and quarternary ammonium compounds (e.g. AMO-1618, CCC), are known to affect ent-kaurene synthetase, especially its activity A.

Further conversion of ent-kaurene is characterized by stepwise oxidation to form ent-kaurenol, ent-kaurenal, ent-kaurenoic acid and ent-7a­hydroxykaurenoic acid (Fig. 5). These reactions are catalyzed by microsomal monooxygenases, requiring NADPH as the only cofactor and probably containing cytochrome P-450 (Graebe, 1987). The pathway branches, ap­parently at two points, and leads to either ent-7 a, 18-dihydroxykaurenolide or ent-6a, 7 a-dihydroxkaurenoic acid. The intermediates and end products of these branches are not converted to GAs and lost to GA biosynthesis. The first gibberellin compound formed from ent-7 a-hydroxykaurenoic acid by contrac­tion of the B ring is GA12-aldehyde.

The enzymes catalyzing the conversion of ent-kaurenolids into gibberellins possess a low substrate specificity, as was shown by supplying ent-kaurenes other than the true intermediates to Gibberella cultures. The various results obtained by feeding kaurenoids to Gibberella fujikuroi have been comprehen­sively reviewed (Sembdner et aI., 1980; Bearder, 1983).

The three steps of oxidative metabolism of ent-kaurene to ent-kaurenoic acid are the sites of action of highly efficient plant growth retardants acting as inhibitors of GA biosynthesis such as substituted pyrimidines (ancymidol), norbornenodiazetine derivatives (tetcyclacis) and triazole derivatives (paclo­butrazol) (see Sembdner et al., 1987).

GA12-aldehyde being the first compound with ent-gibberellane skeleton is the starting point for two parallel pathways through which all gibberellins are formed in Gibberella fujikuroi (Fig. 6). One metabolic route leads from GA12

via two further non-hydroxylated Czo-stages (GAlS and GA24) to GA9 and subsequent products like GAlO, GAll and GA40• A second pathway starts from GAwaldehyde which is derived from GA12-aldehyde by 3p-hydroxylation. This route leads via GA14 and two further C2o-intermediates (GA37 and GA36) to a number of Cwgibberellins, the origin of which is GA4. Metabolic transformation of C2o-gibberellins is characterized by stepwise oxidation of carbon 20, starting from -CH3, via -CH20H and -CHO, to the -COOH stage. These transformation steps are catalyzed by soluble 2-oxoglutarate­dependent dioxygenases. The conversion of Czo-gibberellins to Cwgibberellins by elimination of C-20 and formation of the characteristic y-Iactone bridge in

394 B. Brueckner et al.

~c~ HJw~' H C"' ,"Ho,~".

~3 H

e nt- kaurenol .. H

Hf "'-(H20H

! ~(H3 -c."'~

H ent- kaurenal H -

Hf ~(HO

~l c."'~ ~~

H ent-kaurenoic ',~H -

Hf ~OOH

1

___ .. ~ ~§H H ::t~7oc. 18-dih'Jdroxy-ac id OH - .

~ H ~ kaurenolide HOH2( --C 0-0

~H ~(:'0, ~_ CHz

a H OH ent-1x..hydroxy ---... ~ H OH ent- 60(,70( -

H3( ">~~OOH kaurenoic acid H3C %.~OOH OH dihydroxy-! kaurenoic acid

~"" GA" - old.hyd.

Hi "(OOH

Fig. 5. Gibberellin biosynthetic pathway from ent-kaurene to GA12-aldehyde.

ring A occurs when the aldehyde oxidation step is reached; tricarboxylic acids like GA13, GA25, and GA41 are not converted to Cwgibberellins. Feeding experiments using 14C_ and 13C-Iabelled precursors demonstrated that C20 is lost as CO2 (see Graebe, 1987).

The main product of GA biosynthesis in Gibberella fujikuroi is GA3, which is formed from G~ via GA7 by 1,2-dehydrogenation (G~-+ GA7) and 13-hydroxylation (GA7-+ GA3). The alternative pathway leading through GAl to GA3 is a minor one and may be due to non-specificity of the dehydrogenat­ing enzyme. However, in Sphaceloma manihoticola GA4 is the main product

'"

.D

'" u

erl,

R

1 0\,

R

Fungal Gibberellin Production

(\-\,

Po thlJay lJithout h yd ro x y la ti 0 n

d uri ng (20 - s toges

R = H

GA 12

GAlS (hydroxy acid)

1

GA25

395

PathlJay lJith early 3r-hydroxylation

at (20 -level

R = OH

GA'4~GA2

GA 3-( hydroxy ac id)

1

GA36

I \A~GA I 13 41

----------------11--- --------- ----GA~

GA-~GA~GA ,:l'/~~ ~ __ ----~ 3

0/ :«\ ~ GAl ---k -0/.

GAS4 GA'6 GA4" 130£-OH~ GAs I ~ 6

GA,s GAS7

Fig. 6. Gibberellin biosynthetic routes after GA12-aldehyde (derived from review articles by Sembdner et aI., 1980; Bearder, 1983; Dathe, 1986; Graebe, 1987).

396 B. Brueckner et al.

and GA7, GA3 and GAl are not formed. In Gibberella fujikuroi 13-hydroxylation is a major reaction in conversion of Cwgibberellins such as GA4 ( -+ GAl) and some subsequent products derived from GA4 by hydroxylation at position 10 (GAI6), 1 (GAS4) and 20 (G~7)' respectively. It is interesting to note that 13-hydroxylation occurs only at the end of the pathway and is catalyzed by soluble enzymes, whereas 3P-hydroxylation is microsomal and occurs at the beginning of the pathway (GA12-aldehyde-+ GAI4-aldehyde). The oxidation of GAwaldehyde to GAl4 also proceeds in fungal microsomes (Hedden, 1983). Opposite to the fungus in higher plants two further pathways starting with early 13-hydroxylation (GAS3) or 3p,13-dihydroxylation (GAlS) at the ~o-level are operating (see Graebe, 1987).

Both the amount and the type of gibberellins formed by the fungus are dependent on the genetic constitution of the strain used (see Section 5) and the fermentation conditions applied (see Section 6). Though rapid progress has been made in the biosynthetic routes and the enzymes catalyzing conversion steps, only little is known about the metabolic regulation of GA biosynthesis. This holds true especially for fungal systems, whereas some more insights have been gained during the last few years into the physiological and biochemical control of GA biosynthesis in higher plants (see Graebe, 1987; Sembdner et ai., 1987).

5 STRAIN IMPROVEMENT AND GENETICS

For the first time the genetics of the fungus Gibberella fujikuroi was investigated by Gordon, who was occasionally successful in crossing some of the strains of his large collection. He could show that the fungus was heterothallic (Gordon, 1960). Physiological-genetic studies on gibberellin production presented evidence for the presence of two genes that control different steps in the gibberellin biosynthetic pathway. Genetic studies were difficult at first because production of perithecia was erratic and a rare process. A number of natural and synthetic media were tested which would support the development of the sexual stage of the fungus. Spector & Phinney (1966, 1968) found a medium containing stems of Citrus medica stimulated perithecial production. When fungal strains of opposite mating type were grown on stems of this tree, perithecia were regularly produced within 3-6 weeks. The number of ascospores present in an ascus varied, a maximum number of eight spores was observed. Mating between a high and low gibberellin-producing strain gave tetrads that segregated 2: 2 in terms of the total amount of produced gibberellins. One of the analyzed tetrads was of special interest, since one of the four strains produced high amounts of G~, GA7 and GA9 but no GAl and GA3 (Spector & Phinney, 1968). Genetic studies with this strain resulted in the identification of two genes, gl and g2. Gene gl is responsible for the con­trol of the overall gibberellin production, and gl-mutants neither produced

Fungal Gibberellin Production 397

gibberellins nor metabolized those added. A second pair of alleles, gene g2, controls 13-hydroxylation since g2+ strains produced the whole spectrum of gibberellins, whereas g2 strains did not synthesize GAl or GA3 but accumu­lated GA4 and GA7.

As indicated above, the best wild strains such as Gibberella fujikuroi ACC 917 produce about 1 g GA3 per liter of culture filtrate in an optimized medium, and this was the yield in the early years of GA manufacturing. Current yields are reported to be many times higher (Martin, 1983). Increased yields and production rates are the result of improvement in strains and in fermentation processes. Strain development of gibberellin-producing organisms had received a great deal of attention. Since most of this research has been done at the ICI Pharmaceutical Division and other industrial laboratories, it is mostly un­published because of its proprietary nature. Like other natural products the techniques included a combination of direct selection for high-producing strains and mutagenesis by several different agents. Conventional mutation processes have been published by Soviet workers (Imshenetsky & Ulyanova, 1962; Erokhina & Sokolova, 1966; Erokhina & Efremov, 1970). Mutants with increased GA-titers of up to 60% were found after UV irradiation, fast neutrons, and gamma treatments from spores or mycelia in the case of non-sporing organisms (Erokhina & Sokolova, 1966).

A mutant strain resulting from UV and ethylenimine treatments gave yields of approximately 2 g/liter in a medium with plant oil as the sole carbon source (USSR Patent 440408). Another way of increasing yields is the screening of mutants with blocked carotenoid biosynthesis as a competing biosynthetic pathway with the same precursor mevalonate. Avalos & Cerda-Olmedo (1987) found that strains with particularly low basal levels of neurosporaxanthin, which usually represents about 75% of total carotenoids, are good gibberellin producers.

In general, Gibberella fujikuroi is a highly suitable organism for the induction and isolation of mutants. The uninucleate microconidia commonly allow the expression of recessive mutations (Avalos et al., 1985). Besides gibberellic acid, the last few years' commercial interest has been concentrated on the production of GA4 and GA7. Gibberellin A3-producing strains can be switched over to increased production of the precursors of GAl and GA3 by increasing the pH value to the range of pH 6-7·5 (Jefferys, 1970). In g2-mutants the hydroxylation at C-13 is blocked and consequently, relatively large amounts of GA4 and GA7 could be isolated without separation from GA3 and GAl' Bearder (1983) described such a g2-mutant R-9, which did not produce GAl and GA3. Mutation and selection for increased product formation are probably the most important factors in improving the yield of gibberellins. There is only a small number of publications concerning methods of parasexual recombination to obtain diploid strains in Gibberella fujikuroi. Crossing both wild strains and mutants as parents Calam et al. (1973) found different recombinants, some of which gave increased yields of gibberellic acid, compared with the already high-yielding parents.

398 B. Brueckner et al.

6 FERMENTATION PROCESS AND PHYSIOLOGY

Beside strain improvement of wild-type strains, medium development ano appropriate cultivation techniques are very important prerequisites for suc­cessful economy of gibberellin production. Jefferys (1970) and Brueckner & Blechschmidt (1986) made suggestions for maximizing production of GA. The following section concentrates primarily on the effects of inoculum­preparation, medium composition and culture conditions on the course of fermentation.

6.1 Inoculum

Inoculum quality and quantity strikingly affect the production of gibberellins. Based on experiments with a mutant strain of Fusarium moniliforme Gancheva & Dimova (1984) established that at the submerged culture fermentation the growth and biosynthesis of gibberellins depended on the age and quantity of vegetative inoculum. The highest yield was obtained when a 48-h vegetative culture at the phase of slowed-up mycelium growth was used as the inoculum. The productive phase continues for a longer time (250-264 h) than with 72-h mycelium (216 h). With increasing age of vegetative inoculum the hyphae broke and autolysis began. The yield of fermentations inoculated with such a mycelium was very small (Gancheva & Dimova, 1984). The necessary number of conidia for inoculation of 1 ml inoculum medium is from 4 x 104 to 6 X 104 •

The best results were found when the inoculation of the fermentation medium was carried out with 10 vol% vegetative mycelium.

Mycelium used as inoculum for large-scale fermentations is prepared in progressively larger submerged-culture stages until sufficient of it is available. For still vigorously growing vegetative culture little adaptation is needed and growth in production fermentors commences quickly (Vass & Jefferys, 1979).

6.2 Nutrient Media and Environmental Factors for GibbereUin Production

The synthesis of secondary metabolites depends principally on biomass. Therefore, the selection of medium components is based on both the aspects of growth and product formation. A criterion for medium composition and other ingredients is a fast enrichment of gibberellins at high concentrations. As carbon sources, glucose and sucrose have frequently been used. However, if the initial concentration of glucose was higher than 30% the specific growth rate and the rate of gibberellin production were decreased (Borrow et al., 1964). In view of the inhibitory effect of high glucose amounts on productivity, feed processes were introduced (Brit. Patent 783 611; US Patent 2906 670; US Patent 2906671). Glucose was added at intervals during the production phase, and the concentration maintained below 4%. Another way to avoid the inhibitory effect of glucose is to use carbohydrate polymers, such as starch and plant meals (Brit. Patent 839652; Fuska et al., 1961; CS Patent 104329) or

Fungal Gibberellin Production 399

combinations of fast and slowly utilized carbon sources (Brit. Patent 919 186; Darken et al., 1959). Darken et al. (1959) obtained yields of 880 mg gibberellic acid per liter in a 7-day period using a basal fermentation medium with com steep liquor, ammonium sulfate, potassium dihydrogen phosphate and a mixture of glycerol (20 g/liter), glucose (10 g/liter), and lactose (20 g/liter).

Moderate, but economically reasonable yields of gibberellin were produced on molasses, sulfite liquors, and skimmed milk (FRG Patent 1081402; Maddox, 1977). The Soviet workers have successfully used plant oils, e.g. sunflower oil (Muromtsev et al., 1968; Muromtsev & Agnistova, 1984; USSR Patent 440 408). They compared growth and gibberellin formation on mediums with adequate amounts of oil and sucrose. The fat was introduced into the initial nutrient medium as a single additive while sugar was supplemented to the liquid in fractional doses. In the oil-containing medium significantly more biomass was formed, and the production phase continued for a long time by high mycelium productivity (Muromtsev & Agnistova, 1984). Sucrose con­sumption for the gibberellic acid synthesis (as calculated per carbon) was 2·3-3·6 times higher than oil consumption. The results in Fig. 7 display the increased yields with plant oil in comparison with sucrose. Besides plant oils, hydrocarbons (Rehm, 1980) and fatty acids (Muromtsev & Dubovaya, 1964) have also been tested for their ability to support growth and gibberellin production.

Very important for the gibberellin fermentation are the quality and quantity of nitrogen. Favorable nitrogen sources are ammonium sulfate, ammonium chloride and slowly assimilable sources such as glycine, ammonium tartrate and natural sources of nitrogen (Jefferys, 1970). The productivity on am­monium acetate was considerably lower than on the other N-sources. It seems possible that the stimulation in productivity by natural sources of carbon and nitrogen such as plant meals, plant oils and com steep liquor could be attributed to the content of precursors.

In submerged culture significant production of gibberellins starts only at the time of nitrogen exhaustion. Production was greater the higher the value of initial nitrogen. Further increases in nitrogen concentrations lead to a decrease of the mycelium productivity because of the decreasing efficiency of oxygen transfer (Borrow et al., 1964). Therefore, the selection of both the initial concentration of nitrogen and of an optimal C/N-ratio is very important. Besides carbon and nitrogen sources magnesium, potassium, phosphate and sulphate were all needed. Trace element requirements would be met by impurities in commercial media (Vass & Jefferys, 1979). For a maximum production of GA Jefferys (1970) recommended a temperature of 31-32°C for growth and for product formation 29°C. The airflow-agitation regime should be as vigorous as possible.

Borrow et al. (1964) showed that the specific growth rate and the gibberellin yield are fairly constant over the range of pH 3·5-6·5. However, the composition of the gibberellin mixture produced depends on the pH value. In a wild-type culture of Gibberella Jujikuroi the normal end-product of the

400 B. Brueckner et aJ.

a) , I , 0 <l

0 , ~

9 0 I

/6 ____ 6 I

2000 <l- 5 5,5 " 55 ~ .... , /~ en ,

" E E

'-... .C-Ol \ 6 c::;;

01

A/ E .- 4 5,0 \

OJ 45 c::;; OJ I \

M \ "" <{ 3 I ,,/~, ~ /

0 0 a. -'"

l:J 1 000 ;::-3 4,5 V............... \ /,/ -", ./0 35 ~ u /t:i. .--\-~~ c::;;

/ \. / 0----,,0 2 4,0 i Y '---'- 30 25

. -----0

.y /0 20

200 3,5 -t!; 10 15 '0 0

3 5 7 9 11 13 15 17 19 days

b) 0 .q

" q , I

I 0 , <l-5

'-... '-... 9 2 ODD- S,S "" 55 Ol E E ->-

.C- "-'.... ./' - ..... x::-:6~::s;:::~:::::::,A_6

c::;; '-... Vl OJ en - 4 5,0 c::;; E I '--- / ~ 0 0 45 OJ ). ./A /o~ "- ~

en 0 M 3 I "- 0

-'" a. '6 Y' ..... ..-<{ / X / , l:J 1 000 ;::-3 4,5 ./ li '_ ................... 0 '--- 35 CI

'0 // / '\ c::;;

2 4,0 A 0 ,--- 3D 25

o 0/ 20 200 3,5 D~o 10 15

3 5 7 9 11 13 15 17 19 day s

Fig. 7. Growth and GA3 production by Fusarium moniliforme (strain F6) with different carbon sources: (a) medium with glucose (fed batch); (b) medium with plant oil (after

Muromtsev & Agnistova, 1984).

gibberellin biosynthesis is GA3. At higher pH values, e.g. at pH 7, smaller quantities of GA3, but increased production of the intermediates GA4, GA7, GA9 , GA12, GAl4 and GA16 could be detected. This pH effect may be ex­plained in terms of increased dissociation of intermediates expelled into the brew of the end-product GA3 at high pH. Therefore, these substances cannot be transported back into the mycelium to be further metabolized (Bearder, 1983). When the fungus was grown on a complex nitrogen source, relatively larger amounts of GAl, GAl6 and GA47 were produced compared with the growth on a synthetic medium (McInnes et aI., 1977). It was suggested by Bearder (1983) that the dehydrogenation of GA4 to GA7 was readily inhibited by inorganic nitrogen. The complex medium slowly releases nitrogen, which inhibits the

Fungal Gibberellin Production 401

GA4 1,2-dehydrogenase preferentially and allows the accumulation of GAb GA47, GA16 and G~. Japanese workers (JP 58,152,499) found that alumi­nium, zinc and copper stimulated the preferable production of gibberellin A4.

6.3 Fermentation Course and Fermentation Process Development

Production of GA is a classical fermentation in which significant production of gibberellins and other secondary metabolites starts only after growth has ceased because of exhaustion of assimilable nitrogenous compounds. Borrow et al. (1961, 1964) defined producing and non-producing phases of the fermenta­tion process. Growth during the balanced phase is exponential, and the uptakes of glucose, nitrogen and the other nutrients remain constant. In the following storage phase, when nitrogen was exhausted, the dry weight continued to increase due to accumulation of lipid (maximum 45%) and storage carbohydrates (maximum 32%). But under production conditions these maxima are not reached because of controlled feeds of carbon source. This phase is characterized by the onset of rapid production of gibberellins. The storage phase is the main producing phase which is associated with the production of gibberellins used for commercial purposes. With optimum carbohydrate feeds this phase can be continued for a long time. Other phases of growth which were described by Borrow et al. (1961) do not occur in production conditions (Vass & Jefferys, 1979).

Bu'Lock et al. (1974) studied the synthesis of two different secondary metabolites of Gibberella fujikuroi, namely the polyketide pigment bikaverin and the diterpenoid gibberellic acid. In batch culture under conditions of increasing nitrogen limitation the fungus first accumulates bikaverin and then, under very strong nitrogen limitation, gibberellins are produced. It is very interesting that this series of events can be reproduced in chemostat cultures at different growth rates. Bu'Lock et al. (1974) concluded that the gibberellin pathway is under the same type of overall regulation as bikaverin synthesis, but that the GA synthesis begins at a level of limited nutrient and a corresponding lower growth rate.

The conventional lag phase is by-passed in large-scale fermentations if high initial glucose concentrations (more than 20%) or the use of ammonium acetate as a nitrogen source is avoided, and if vigorously growing inoculum is used to commence a fast mycelium development (Vass & Jefferys, 1979).

A critical factor in the gibberellin production is the supply of oxygen. Since the demand for oxygen of growing mycelium increases with increasing dry weight, oxygen limitation can be initiated which results in subsequent decrease of GA production in the thickening broth. The dry weight at the end of the exponential growth phase was shown to be dependent on the initial nitrogen concentration (No) and on the rate of agitation of the fermentor (Borrow et

402 B. Brueckner et al.

at., 1964). It is well known that the Q~ of the fungus decreases when the cells switch to storage phase metabolism and the oxygen transfer capacity is no longer saturated. That is why the initial nitrogen concentration, optimum medium composition and nutrient feeds must be selected in such a way that some further proliferation and, therefore, a mixed linear growth and storage phase occurs after the balanced phase (Jefferys, 1970).

The gibberellin fermentation has a long history of development. Early trials of the Japanese workers were based on surface cultivation, and low yields (40-60 mg/liter) after prolonged periods of incubation were reported (Rehm, 1980).

In shake-flask experiments with the same media 200 mg/liter GA3 was obtained. Optimization of medium composition, especially of the initial nitrogen and carbon concentrations, led to a further increase of GA yield up to 1 g/liter. The early gibberellin production, prior to 1961, used a purely batch technique. Later fermentations are batch-fed processes (Vass & Jefferys, 1979). From this time onward the feed regimes have been developed using natural and synthetic carbohydrates and nitrogenous nutrients in the feedstock. The carbohydrate feed rate also must be carefully restricted to limit the storage activity of the fungus without lowering the rate of GA production. In the patent literature it was said that some precursor feeds, e.g. mevalonic acid, kaurene and kauranol, increased the yield of gibberellin A3 (Brit. Patent 957634). However, it seems unlikely that the obtained increases have any economic interest because of the high cost of these compounds.

Holme & Zacharias (1965) and Bu'Lock et at. (1974) described the production of gibberellin A3 in glycine-limited continuous culture conditions and found maximum gibberellic acid synthesis at a growth rate of 0·005 h- 1•

But because of the little specific rates of synthesis and the long period of fermentation the chemostate culture has a theoretical importance only.

Besides submerged fermentation techniques employed throughout the world, solid state fermentation is known to produce the metabolites in most of the cases at high yield, especially if molds are involved (Lindenfelser & Ciegler, 1975). First information on the cultivation of the gibberellin-producing fungus on com grains was published by Focke et at. (1967). Indian workers compared the production of GA3 by solid state fermentation and submerged fermentation. Based on adequate carbohydrate contents they found that in the case of solid medium (wheat bran) the accumulation of GA3 was 1·6 times higher (Kumar & Lonsane, 1987). The degree of biomass formation varied between 9·7 and 11·9 g/kg commercial wheat bran, the maximum quantity of GA3 accumulated at the end of a incubation period of 7-8 days was 1·2 g/kg solid substrate.

Up to now, there is no information on scaling up the extension of the solid state fermentation technique for technical purposes in GA3 production on an industrial scale. Probably this is due to the problems with the process control, especially with oxygen transfer and moisture control on an enlarged scale.

Fungal Gibberellin Production 403

6.4 Fermentation Process Economics

The total cost of a fermentation process comprises many factors, namely the cost of raw materials, fixed costs, costs for separation and purification of the product and so on. Process improvements, especially increase of the yield, include strain improvement, optimization of the medium composition and extension of the production phase. Conditions which lead to increases in the overall rate of production, for example, by decreasing the percentage of by-products, are also very important.

Vass & Jefferys (1979) calculated the changes in production data over a period of 18 years. The process development obtained resulted in the introduction of new strains, transition from a purely batch technique to batch-fed process and modifications in medium compositions (Fig. 6.2). Only those process changes which have shown a cost reduction or yield improve­ment in laboratory experiments were implemented.

As can be seen in Fig. 8 the producing strains of Gibberella fujikuroi (A-D) were developed during this period. For example, the introduction of strain D shows an improvement in volumetric production rate of 35% in comparison with strain C. The authors defined the term volumetric production rate as the

Period- Upto1961 11961

150

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~ § E

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I E

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Fig. 8. Changes in productivity of gibberellic acid up to 1976. Relative values have been calculated from process data of Imperial Chemicals Industries. Horizontally striped histograms indicate yield (mol), open histograms production rate (mol liter-3 ) ,

and vertically striped histograms volume-specific production rate (molliter3(t + tr )-I), where tr denotes the time for turn-round (after Vass & Jefferys, 1979).

404 B. Brueckner et al.

average rate of change of concentration of product with time, where the time includes the period for vessel cleaning and preparations for new fermentation. On the other hand, in the period from the introduction of strain D 1970 to 1976 an improvement in the volumetric production rate of more than 60% was achieved by variations of the medium components in both the initial batching and in the feed-liquor and by control of the supply of assimilable nitrogenous nutrients for the growth period. The feed regime is also controlled during the course of fermentation to nearly maintain a state of oxygen depletion (Vass & Jefferys, 1979).

It is interesting that although the total fermentation costs per batch have increased by 50% during these 18 years, the unit production costs decreased with the increase of gross yield per batch up to 1700%.

Vass & Jefferys (1979) have mentioned that at the end of fermentation a greater percentage of available carbohydrates from the feed was metabolized to cell storage components rather than gibberellins. The decrease of these losses of expensive components seems to be a target for further improvements.

7 PRODUCT RECOVERY AND PURIFICATION

During the fermentation the gibberellins are secreted into the culture medium. Therefore, the fermentation of Gibberella fujikuroi normally results in the production of a mixture of gibberellins among which GA3, GA4 and GA7 predominate. GA3 is the most abundant gibberellin.

Prior to isolation of gibberellins, the mycelium should be separated from the broth by means of filter press, centrifuge, or drum filter with a filter aid. Afterwards, the gibberellins can be prepared from the broth by adsorbing on activated charcoal, by various types of ion exchange processes or by liquid­liquid extraction. But it is very difficult to describe the processes in use on a large commercial basis because these processes are kept as company secrets and are not published. This is why the product recovery and purification of gibberellins can only be described by a general discussion of the kinds of technology available and published in literature and in patent specifications.

The adsorption on active carbon (e.g. charcoal) was the first method for the extraction of GA3 developed by Japanese researchers. Probably it is also used in commercial processes. After the adsorption the elution of the gibberellins is carried out by percolation with water-miscible solvents, e.g. methanol or acetone. The solvents may be used pure or mixed with ammonia (Jefferys, 1970).

However, this method has some disadvantages:

(a) it requires large amounts of solvents; (b) the complete recovery of the gibberellins from the carbon is not

possible; (c) the carbon can be used just once; (d) the purification is poor.

Fungal Gibberellin Production 405

Therefore, subsequent purification processes with buffer-ethyl acetate systems proved to be necessary.

Another possibility for fermentation recovery is the application of ion­exchange resins. A patent filed by the Societe d'Etudes et d' Applications Biochimiques (Brit. Patent 936 548) describes the isolation of the gibberellins and their purification by means of ion-exchange resins. The pre-treatment of the culture filtrate is carried out by additive of alkaline-earth metal hydroxides (e.g. Ba(OHh). This treatment effects the precipitation of impurities such as organic acids, proteins, pigments and others. Moreover, the volume of the resins needed for the ion-exchange processes is reduced to a half or to a fifth of the original amount without pre-treatment (FRG Patent 1288044). After a further purification on a weak cation-exchange resin (e.g. Amberlite IRC 50, H. form) the culture filtrate passes a column of a weak anion-exchange resin (e.g. Amberlite IR 4 B, acetate or formate form). Also the use of strongly alkaline resins (e.g. Amberlite IRA 401, Dowex 1 x 2, chloride form) is described.

The gibberellins are eluted from the resins by slow percolation with 1 N

ammonia or with buffered alkaline solutions of ammonia salts (e.g. ammonium diphosphate). Also the elution is possible with methanol acidified with H2S04

or HCl (Jefferys, 1970). The first fractions of the ammoniacal eluate obtained are sufficiently pure to

make possible the direct production of raw crystals by mere extraction of the gibberellins from those fractions of the eluate with ethyl acetate and concentration of the extract, without recourse to other purifying steps (US Patent 3118909). The obtaining of such fractions represents a characteristic feature and an important advantage of the extraction of gibberellins from a culture broth by means of weak anion-exchange resins. Therefore, in commer­cial processes employing this method it suffices to segregate these fractions to produce crystallized gibberellins. The raw crystals prepared in such a manner contained about 77% gibberellins. The extraction efficiency achieved with this method was near to 100%.

The further purification of these raw products is possible by a second purification step. It is carried out by the adsorption of the products obtained in the first stage onto active carbon and the desorption of the gibberellins by means of a solvent such as ethyl acetate. Evaporation, crystallization of the gibberellins in pure ethyl acetate, and subsequent washing and drying resulted in crystals with a purity of the order of 95% (US Patent 3118909).

However, the application of ion-exchange resins for the fermentation recovery comprises some problems: the procedure requires large amounts of resins, and numerous impurities and organic acids are adsorbed onto the ion-exchange resins together with the gibberellins. Furthermore, the lactone ring of the gibberellin molecule may be destroyed on the anion-exchange resin or during the desorption under alkaline conditions (see Chapter 10). In this way the gibberellin would lose its biological activity.

The Polish patent 0 152 578 avoids these disadvantages of the ion-exchange

406 B. Brueckner et al.

resins by the use of synthetic sorption resins with non-ionic character and with unpolar or middle-polar surfaces of the type Amberlite XAD-4, XAD-2 or XAD-7. Gibberellins are caught quantitatively by resins of this type, whereas the impurities and by-products are bound only to a negligible extent. The gibberellins may be eluted by aqueous solutions of acetone, methanol or ethanol. A further removal of impurities is possible by the choice of selective elution media. The advantage of this procedure is based on the fact that the solvent mixtures for the desorption of the gibberellins and of the impurities are identical. They only differ in their concentrations. For example, the desorption of the gibberellins is carried out with 35-80% methanol and with acetone >70% or <45%, respectively; by comparison the desorption of the impurities is possible by elution with 80-95% methanol and 45-70% acetone.

The third way of gibberellin recovery is the liquid-liquid extraction of the culture filtrate with water-immiscible solvents. These methods were developed as the yields in the fermentation broths were increased. The solvents mostly used are esters such as ethyl acetate or butyl acetate and ketones such as ethyl methyl ketone or methyl isobutyl ketone. However, n butanol or esters can be used either alone or in mixtures. Ethyl acetate appears to be used as solvent on plant-scale processes. Acid conditions (pH 2-4) are needed for the extraction. The subsequent recovery of the gibberellins from the solvent is effected by adsorption on solid sodium or potassium bicarbonate or by the buffer-solvent processes being based on the relative solubilities of the free acid in solvent and of the sodium or potassium salt in an aqueous phase. The extraction efficiencies described in the literature varied between 40 and 100% (Jefferys, 1970). An important disadvantage of the liquid-liquid extraction is the large demand for solvents and their recovery.

Definitely, it can be judged that the cost for each of the three procedures of the recovery described is much higher than the fermentation cost itself because the yields of the recovery processes on the large commercial scale are still relatively low. The recovery cost is the main reason for the expensive price of the product. The procedures mentioned up to now relate to the separation of the gibberellins from the culture medium. But the culture broth contains a mixture of gibberellins amongst which GA3, G~ and GA7 predominate. It is desirable to separate the gibberellins not only from the culture broth but also from each other because the gibberellins GA3 and G~/GA7 have different plant gJ,:owth-regulatory effects, and, thus, the compounds have distinct and specific practical applications.

The separation of GA3 and a mixture of G~/GA7 is possible by a liquid-liquid extraction which is based on the different partition coefficients of GA3 and G~/GA7 between certain organic solvents and water within a particular pH range. For example, the culture filtrate is extracted with an organic water-immiscible solvent (preferably ethyl acetate) at a pH value between 4 and 8·5. The result is an extract rich in G~/GA7 and an aqueous broth rich in GA3. The precipitation of G~/GA7 from the solvent is difficult because other substances inhibit this process. The leI patent EP 83 306 682

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408 B. Brueckner et aJ.

describes the additive of a N-hydrocarbyl-N-arylmethyl amine to an organic extract of a fermentation broth containing GA4/GA7. The reaction caused a selective precipitation of the salts of GA4/GA7 with that amine, leaving the residues of GA3 in solution. The recovery of the precipitated salt is carried out by filtration. The isolation of G~/GA7 is also practicable by a reaction of the amine salt with an organic acid (e.g. citric acid) and subsequent filtration. Preparations obtained by these processes contained the GA4/GA7 mixture in concentrations of about 95%. Consequently, the GA4/GA7 preparation is sufficiently pure so that it may be used directly after isolation and no recrystallization is necessary. Furthermore, the GA4/GA7 mixtures are free from GA3 and other gibberellins. The application of this method on a large commercial scale is described in the ICI patent cited above.

The production and the recovery of the gibberellins are summarized in Fig. 9.

8 ASSAY METHODS

8.1 General

The identification and quantification of endogenous GAs in higher plants is one of the most important aims for analysts (Crozier & Durley, 1983; Crozier, 1987; Sembdner et al., 1988). In microbial fermentations, where GAs represent an abundant group of constituents, the analysis does not require such high sensitivity as in plant hormone analysis, but it has almost to cope with routine work for a large number of samples. This has to be considered especially for sample preparation and prepurification steps. Commonly, the first step of working up of fermentation broths consists of partitioning of the acidified aqueous phase against ethyl acetate. More recently, solid phase extraction using various cartridges became more favoured. This easy-to-handle method, which is mostly based on partitioning at reversed phase material, ensures both high efficiencies and recoveries (Hedden, 1987; Sembdner et al., 1988).

8.2 Physical Methods

For some moderate analytical purposes, purified GA samples can be subjected straight away to spectroscopical analysis. Thus, for the estimation of GA3 its reaction with Folin-Ciocalteu reagent has been used for colorimetry at 730 nm (Muromtsev & Agnistova, 1973).

Another possibility consists of inducing GAs to fluoresce by treating them with sulphuric acid. The obtained fluorescence spectra are characteristic for structurally related GAs (Table 2) and allow the quantification at the ng-level (Schneider, 1988a). However, these spectroscopical methods fail if complex GA mixtures or troublesome admixtures are to be considered.

Fungal Gibberellin Production

Table 1 Experimental Conditions for Fluorimetric Analysis of some Fungal Gibberellins (AE, Exciting Wavelength, AF,

Fluorescence Wavelength)

AE AF Incub. temp. Incub. time (nm) (nm) ("C) (min)

GAl 410 455 80 120 GA3 410 455 20 30 G~ 390 410 80 90 GA7 455 468 80 60 GA9 385 410 80 180

409

More reliable analyses, therefore, usually include some chromatographic steps in order to separate single GAs before quantifying them. In this respect the combination of TLC and fluorimetric assay in situ turned out to be quite versatile (Winkler, 1978). Based on the automated measurement on HPTLC plates this method was reported to be both accurate and effective, especially if large sample series have to be processed. This way GAs can be estimated at concentrations of 1-200 Ilg/ml with SDs as low as 3-4% (Sackett, 1984).

Better resolution of complex GA samples can be achieved by HPLC separation. Especially for the distinction of GA3 in the presence of the biologically inactive concomitant iso-GA3 (see Section 2) HPLC is the method of choice. If non-selective detection at 204-210 nm is used, some simple pre-purification steps, e.g. by cartridge, is mandatory prior to HPLC. Analyses are then performed on RP 8 or RP 18 materials with methanol/water (acidified with, e.g. 0·1% H3P04) (Jensen et al., 1986; Schneider, 1988b). Standard deviations are as good as for HPTLC (1-4%). Automated HPLC-systems guarantee high reproducibility and effectiveness in routine work. Considerable increase in detection sensitivity for GAs can be achieved by the introduction of a suitable chromophor (precolumn derivatization). Thus, GA-p­bromophenacyl esters have been used for the detection at 254 nm (Morriss & Zaerr, 1978). By reaction with 7-methoxy-4-bromomethyl coumarin GAs also become accessible to fluorometric detection (pg-range) at AE = 320 nm and AF = 400 nm (Crozier et al., 1982).

The highest level of information, accuracy and reproducibility in analysing GAs is offered by GC-MS. Free acidic GAs have to be derivatized before. For this, methyl esters and their trimethylsilyl derivatives are commonly used (Crozier & Durley, 1983; Takahashi et al., 1986; Schneider, 1988c). The extraordinary discriminating power of capillary GC sometimes becomes necessary also for the analysis of fungal GAs, if they are isomers or closely related otherwise. Subsequent on-line MS detection including isotope-labelled internal standards, provides highest precision in quantifying GAs at pg­amounts (Hedden, 1986, 1987).

410 B. Brueckner et al.

8.3 Bioassays

Bioassays are based on typical physiological effects of gibberellins (cf. Section 9). Bioassays have been used successfully in gibberellin research since the original purification and isolation of GAs from fungal cultures, and they are still significant tools in GA analysis. Bioassays are most helpful in detection and preliminary quantification of GA-containing fractions and in physiological characterization of the GAs as well as in studies on structure-activity relationships. Limitations in bioassaying are given by the fact that the single GAs possess different physiological potencies. Thus, GAs of low activity like Czo-GAs and GA9 may give only 1 % or less of the response produced by GA3. On the other hand the activity of a given GA differs in different bioassays. Therefore, the biological activity measured by bioassay has always to be related to the identity of the GA and the specific sensitivity of the assay. For practical use only those bioassays the responsiveness of which is well known, should be used. Furthermore, it has to be considered that bioassays normally result in relative GA quantities only, e.g. measured as {tg GA3 equivalents. The sensitivity of a bioassay is characterized by the minimal GA concentration (or GA amount) necessary to give a significant response. Sensitivity of good GA bioassays is of the same order as the most sensitive physical methods. Another characteristic is the concentration range within which the increase is proportional to the dose (Table 3).

Quantification of bioassay results is possible by use of a log dose-response curve (Fig. 10) obtained by measuring different concentrations of a standard GA (normally GA3)' Furthermore, Fig. 10 demonstrates that the deviation ranges are differing in dependence upon the logarithmic dose-responsiveness.

Many physiological effects of gibberellins (d. Section 9) have been put to

Table 3 Sensitivity of GA Bioassays

Assay

Dwarf rice 'Tan-ginbozu' microdrop variant Root application

Dwarf maize--d1

Dwarf pea--cv. 'Meteor' Lettuce bypocotyl-

cv. 'Arctic' a-amylase; barley

endosperm

Minimum GA3 dose concentration

giving significant response

0·1 ng/plant

10-8_10-7 mol/liter 5 ng/plant O· 5-1·0 ng/ plant 5 x 10-7 mol/liter

10-10_10-9 mol/liter

Range of linear log dose-response

0·1-100 ng/plant

10-7_10-5 mol/liter 5-10,Ltg/plant 1 ng-lO ,Ltg/plant 5 x 10-7_10-4 mol/liter

10-9_10-7 mol/liter

Fungal Gibberellin Production 411

Response

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use as a bioassay. However, only three types of bioassays have become widely used due to their ease of performance, reliability, sensitivity and range of response. These are the dwarf mutants, the hypocotyl and the a-amylase bioassays. The very extensive literature dealing with GA bioassays including complete descriptions of their performance is covered by a number of reviews and monographs (see Bailiss & Hill, 1971; Reeve & Crozier, 1975, 1980; Graebe & Ropers, 1978; Hoad, 1983; Bergner, 1988; Bernhardt, 1988).

Some dwarf mutants of various plant species are known to be deficient in endogenous gibberellins and, therefore, can be normalized by exogenous GA. Such GA-sensitive mutants are favoured bioassay objects. Most important are special mutants of Zea mays L., Oryza sativa L. and Pisum sativum L., the dwarfism of which is controlled by a single recessive gene. The GA sensitivity of those mutants is due to a block in GA biosynthesis (see Section 3) preventing the formation of GAl necessary for normal stem growth. GA biosynthesis can be blocked either at a very early stage (d5 mutant of maize, dx

mutant of rice and na mutant of pea) or immediately before GAl formation (d l

mutant of maize, dy mutant of rice and Ie mutant of pea). Mutants belonging to the first type respond to a broad spectrum of different gibberellins, and the second type is sensitive only to GAs having a 3f3-0H group, like GAb GA3 ,

G~, GA7 , etc. Dwarf rice bioassays are widely used either with a microdrop or root application technique. Both dx-mutants (cv. 'Tan-ginbozu') and dy-mutants (cv. 'Katake-Tamamishikai' and 'Waito-C') are applied. Rice seedlings are pre-germinated and-after application of the test solution-

412 B. Brueckner et al.

further cultivated under artificial light and controlled temperature conditions. Measurement of seedling length is performed after 4 days (microdrop assay) and 7 days (root application), respectively. The root application assay is most easy in performance, rather sensitive and well suited for screening of GA activities in extract fractions or even non-purified culture liquids of the fungus. Quantification can be done more precisely by the microdrop variant.

Dwarf maize bioassays can be performed under varying conditions. Favoured are the mutants d 1 and ds which, however, are not available commercially. Normally caryopses are pregerminated and seedlings grown under controlled light and temperature conditions either in soil or nutrient solution. Test solutions are applied as 50 Jll drops into the unfolding first leaf and after about 6 days the length of the second leaf is measured.

Dwarf pea bioassays are in most cases performed with varieties the genotypes of which are not known, like cv. 'Progress Nr. 9' and cv. 'Meteor'. Seeds are germinated in wet sawdust and selected seedlings grown in nutrient solution under controlled light and temperature conditions. About 5 days after application of the test solution (50 Jll to the apex) the length of three internodes above the first complete leaf is measured. By use of suited genotypes and appropriate conditions, quantitation of GA amounts in extract fractions is possible.

Hypocotyl growth in various plant species can be stimulated by low amounts of GA. Among the different bioassays based on this response the lettuce hypocotyl assay is rather widely used. Seeds of Lactuca sativa L. cv. 'Arctic' are germinated and selected seedlings thereafter grown on filter paper wetted with test solution. The assay is easy in performance, but not very sensitive and often disturbed because of non-specific inhibition, e.g. by traces of solvent, acetic acid, etc.

a-Amylase bioassays are based on the GA controlled induction of the enzyme in the aleurone layer of germinating cereal caryopses. Several variants of the assay have been described in the test object, measurement and some other parameters; however, all are rather complicated in performance and reliability. In any variant embryoless parts of cereal caryopses (wheat, barley, rice) are used and incubated with the GA test solution. Sensitivity varies highly depending on the variety, age and provenance of the seed, etc. Enzyme activity is determined either by iodometric measurement of starch degradation or by measuring the formation of reducing sugars. Because of the complicated procedure the assay is only seldom used routinely in screening research, but it may be helpful in physiological studies.

8.4 Immunoassays

Immunoassays are the most recent tools in quantitative GA analysis. During the last years both the radioimmunoassay (RIA) and enzyme immunoassay technique, especially the enzyme-linked immunosorbent assay (ELISA) have

Fungal Gibberellin Production 413

been introduced to GA analysis and have gained considerable importance. The first group of immunoassays that have been developed comprises radioim­munoassays which make use of polyclonal antibodies raised in rabbits. Such assays are available for the fungal gibberellins At. A3, ~, and A24 (Weiler, 1984; Weiler et al., 1986a, b; Knoefel, 1988). They employ tritium or, less frequently, iodine-125-labelled tracers which allow the detection of minimal GA amounts of 0·05-0·1 pmol. Even higher sensitivity could be reached by solid-phase enzyme immunoassays detecting 1 fmol of GA3 methyl ester, 0·5 fmol of G~ methyl ester and 1 fmol of GA7 methyl ester (Atzorn & Weiler, 1983). With the introduction of the monoclonal antibody technology the use of immunoassays in plant hormone analysis has entered a new dimension (Weiler, 1986). Thus, monoclonal antibodies against GAlJ-imide recognizing GA4 and related gibberellins have been applied to analyse the GA spectrum produced by the fungus Sphaceloma manihoticola (Eberle et al., 1986). A series of monoclonal antibodies allowing recognition of, and the discrimination between GAl> GA4 and GA9 , have been derived from immunisations with immunogens in which the proteins were linked to GAl at carbon-3 and to GA4 and G~ at carbon-17 (Knox et al., 1987). Formerly immunogen synthesis had been carried out only via carbon-7. Antisera raised against apolar gibberellins, like G~, proved to be most selective, whereas antisera raised against more polar gibberellins, like GAl and GA3, showed cross-reactivity with few related GAs and, thus, gave 'group-selective' assays. To date the immunoassays available cover only part of the fungal gibberellins; however, the major ones are among them.

There is no doubt that immunoassays will be used to an increasing extent to quantify the levels of gibberellins. They complete the traditional methodical spectrum and can profitably be applied in combination with chromatographic techniques, especially HPLC (see Section 8.2). Nevertheless, we must be aware of their limitations resulting mainly from the fact that each material to be analysed may contain substances that will interfere in the immunoreaction. Therefore, in any case appropriate purification procedures and validation of the assay have to be checked (Pengelly, 1986; Wang et al., 1986; Knoefel, 1988).

9 BIOLOGICAL PROPERTIES

The bakanae disease syndrome in rice plants, including the typical overgrowth symptoms, led to the discovery of gibberellins as the active metabolites of the pathogenic fungus Gibberella fujikuroi (see Section 1). Apparently, the gibberellins are part of the substantial interactions between the parasite and the host and, therefore, might have some functional role in disease develop­ment. However, there is only one piece of evidence that gibberellins have some physiological significance in Gibberella fujikuroi. Nakamura et al. (1985) found a promotive effect of GA3 on conidial germination and elongation of

414 B. Brueckner et al.

young hyphae in Gibberella fujikuroi, but also in Penicillium notatum and Neurospora crassa.

The work with the fungus led to the discovery of gibberellins in higher plants. By use of bioassays (see Section 8.3), originally developed on the base of the action of fungal GAs on higher plants, both the existence of gibberellins in higher plants and their various physiological potencies could be demon­strated. As a result of intensive GA research concerning the natural occur­rence, structural elucidation and quantification, the biosynthesis, metabolism and translocation as well as the physiological actions, the gibberellins were found to represent a group of endogenous plant hormones. Within the hormonal system the gibberellins interact with auxins, cytokinins, abscisic acid, ethylene, and possibly other plant growth regulators, like brassinosteroids and jasmonic acid, in the coordination and regulation of growth and development of plants. The most obvious function of gibberellins is to control the rate of growth and final length of stem internodes. This is best seen in certain dwarf mutants which due to disturbed GA biosynthesis (see Section 8.3 and the review of Graebe, 1987) have very short internodes but, upon treatment with GA, grow to normal height. Gibberellins are also active in the reactions of cold-requiring and long-day plants in response to induction. Furthermore, gibberellins affect sex expression, the formation of flowers-at least in some gymnosperms-and the extension of floral organs, the pollination and fertiliza­tion, the development of seeds and fruits, especially pod growth, the germination of seeds and vegetative propagation organs as well as the breaking of dormancy and the abscission processes. Gibberellin functions are mediated by external factors such as light conditions, temperature, water supply and nutrition; and gibberellins might also be involved in the response of plants to different stress factors.

The biological properties of gibberellins, including the multiple structure­activity relationships and their physiological functions as plant hormones, have been studied most intensively and the published literature contains numerous original papers, many reviews and a number of monographs. Within the Encyclopedia of Plant Physiology, New Series, three volumes deal with the hormonal regulation of plant development and, thus, contain remarkable knowledge on gibberellin physiology (MacMillan, 1980; Scott, 1984; Pharis & Reid, 1985). Besides several early reviews (see Graebe & Ropers, 1978) two special gibberellin books were published by Krishnamoorthy (1975) and Muromtsev & Agnistova (1984). Important aspects of gibberellin physiology were comprehensively summarized into a monograph edited by Crozier (1983). It contains reviews on gibberellin-induced growth in two extensively studied objects; these are excised lettuce hypocotyls (Jones & Moll, 1983) and Avena internodes (Kaufman & Dayanandan, 1983). Many effects of gibberellins on flowering have been described, although their physiological role in flower induction is not completely understood (Zeevaart, 1983). Also, the interac­tions between gibberellins and phytochrome need further investigation (Reid, 1983). However, photoperiodic control of stem growth is clearly mediated by

Fungal Gibberellin Production 415

gibberellins (a recent review is given by Graebe, 1987). Some information on the physiological role of gibberellins in fertilization and reproductive develop­ment has been derived from either GA effects obtained by exogenous application or studies on the endogenous hormonal situation. Present knowl­edge in these fields has been summarized by Tsao & Linskens (1986) concerning the fertilization processes and by Pharis & King (1985) with respect to seed and fruit development. The physiological significance of gibberellins in seed germination is well established; some more recent aspects have been reviewed and discussed in view of gibberellin-deficient mutants (Karssen & Lacka, 1986). A general survey on plant hormones in seed dormancy and germination is given by Lewak (1985).

Gibberellin effects on conifers, especially with respect to flowering, have gained increasing interest and it could be shown that there may be profound differences between angiosperms and gymnosperms as far as GA physiology is concerned (Dunberg & Oden, 1983). On the other hand, gibberellins are known to have widespread effects on cryptogams, when exogenously applied. They promote growth in many species of algae, liverworts, mosses, and ferns, induce spore germination in ferns and regulate differentiation of reproductive organs in fern gametophytes. As analogues processes in the life cycle of higher plants are often similarly influenced by GA treatment, it might be assumed that the mechanism of GA action in green cryptogams is essentially the same as in higher plants (Furuya & Takeno, 1983).

The mechanism of gibberellin action is most intensively studied in cereal aleurone cells known to respond to exogenous gibberellins with the de novo synthesis and secretion of hydrolytic enzymes, predominantly a-amylase isoenzymes (see Section 8.3). During germination of caryopses the embryo is assumed to provide aleurone cells with gibberellins (reviewed by Jacobsen, 1983). The GA action on a-amylase synthesis by affecting gene expression is intensively studied at present. Considering the results published it can be concluded that GA controls the synthesis of certain aleurone cell enzymes by enhancing mRNA transcription. Mechanism of GA action on growth processes is also studied on the level of gene expression but less intensively than in aleurone system (see Sembdner et af., 1987). a-Amylase and some other hydrolytic enzymes are influenced by gibberellins also in systems other than aleurone cells. Furthermore, it is well known that gibberellins can affect either the biosynthesis or the activity of various enzymes covering nearly all aspects of plant metabolism, though the modes of action and physiological roles are understood in a few cases only (see Barendse, 1986).

With respect to gibberellin primary action, a number of investigations deal with the binding properties of radioactively labelled gibberellins to high molecular weight compounds and the characterization of proteinaceous recep­tor sites. However, only little progress is available in gibberellin receptor research up to now. The evident knowledge in this field has been summarized with competency by Stoddart (1986).

According to the data published gibberellins apparently display only low

416 B. Brueckner et al.

activities or they are inactive in animal systems. The acute toxicity on animals is very low. The tolerance limit to GA3 is much higher than to antibiotics; for mice it is given as 6300 and 25 ()()() mg/kg after intravenous and oral application, respectively (Schwartz et al., 1983). The acute toxicity for G~/GA7 on mice is also>500mg/kg (Abdel-Rahman et al., 1977). Neither in animals nor in tissue cultures were teratogenic or carcinogenic effects observed over a period of years. Pharmacological studies have been done with GA3 using predominantly mice, rats and guinea-pigs. Among the results reviewed by Schwartz et al. (1983), there are stimulating effects on the body weight in various animals, influences on endocrinologic functions and on the functions of the macrophaga and changes in mitotic activities as well as protective effects against pathogenic infections, toxicants and X-rays.

10 CHEMICAL SYNTHESIS

10.1 Total Synthesis

The total synthesis of any natural product is a challenge to chemists even if commercial interests might be far beyond. In first attempts for total synthesis of complex GA structures, other totally synthesized compounds, e.g. enmein, were tried to transform to GAs, e.g. GAlS (Somei & Okamoto, 1970). The group of 13-dehydroxy-GAs became linked to total synthesis by converting

Fig. 11. Main routes in conversion of gibberellins.

Fungal Gibberellin Production 417

synthetic epigibberic acid via gibberelline C-methyl ester to GA4 or G~ (Mori et al., 1968). The highly complicated structure of GA3 with eight chiral C-atoms could finally be synthesized in the course of a 34-step sequence by Corey et al. (1978). Using another approach, Lombardo et al. (1980) succeeded in synthesizing GA3 and GAl> too. As many other GAs can be derived from or related to either GA3 and GAl or GA4 (see Fig. 11) all these GAs are formally linked to total synthesis.

10.2 Conversion

Most of the endogenous plant GAs occur in traces only. Thus the microbiolog­ical source of GAs is of high importance in order to get access to substantial amounts of these substances. Commonly the main fungal GAs like GA3, G~ and GA7 act as starting material for preparing CwGAs, and GAl3 for Cw-GAs. Figure 11 compiles the main routes in GA conversion, for references see Takahashi & Yamaguchi (1983). Sometimes a single step like hydrogena­tion leads easily to another GA (GA3~ GAl> GA7~ G~). Other conver­sions need a complex sequence of reactions, e.g. GA3~ GA29•

10.3 Labelling

In principle, many of the reactions used in conversion of GAs (see above) are suitable for introducing stable or radioactive labels into GAs (see Takahashi & Yamaguchi, 1983). For example, hydrogenations with deuterium or tritium lead efficiently to labelled analogues. A widely used method for double labelling of GAs consists of oxidatively removing the exocyclic methylene group followed by refixing it with 13C, 3H2-Wittig reagent.

Sometimes biotransformation by Gibberella fujikuroi or plant enzymes are included in modifying initial products. Other possibilities for introduction of 3H are the catalytic hydrogen exchange with GA3-type gibberellins (Nadeau & Rappaport, 1974) or the 6-H exchange on the 6-aldehyde level (Lischewski, 1985).

10.4 Structural Modification

With respect to structure-activity relationships of GAs in plant systems, structural modifications of natural GAs are of high scientific but also commercial interest. Thus, a series of papers is dealing with the introduction of halogen atoms, e.g. fluorine, chlorine, bromine, and iodine, especially at positions C-l, C-3, C-13 (Banks & Cross, 1977; Cross & Simpson, 1981; Keith et al., 1979).

Quite promising were experiments toward the blockage of metabolic oxidation by the alkylation of C-2, e.g. 2,2-dimethyl GAl or GA4 (Beale et al., 1984). Other facets in modifying GAs are alterations or removal of the 6-carboxy group (Lischewski & Adam, 1980) or the introduction of hetero

418 B. Brueckner et aI.

atoms like N, e.g. as l-azido-, l-amino-GAs (Voigt & Adam, 1982) or amino acid conjugates (Adam et ai., 1977). Physiologically relevant glucosyl conjug­ates of GAs have also been prepared. Thus a series of GA-O-glucosides and GA-glucosyl esters were synthesized (see Schneider, 1983).

11 FORMULATIONS, APPLICATIONS AND ECONOMICS

From the 72 gibberellins only gibberellic acid (GA3) and, to a lesser extent, mixtures of GA,. and GA7 have found practical use. Sometimes it is difficult to calculate the financial return to the grower for the application of gibberellins. For example, climatic effects, environmental factors and differences in varietal response can effect or veil the efficiency of the hormones applied so that a reliable calculation of the economic value is connected with many elements of uncertainty. Moreover, it has been difficult to gather accurate information separating actual commercial use of gibberellins from wishful thinking or potential uses.

The gibberellins are the most widely used group of native plant hormones. In 1980 the world production of gibberellins for commercial use was estimated to be 12-15 tons (Martin, 1983). Countries with a good tradition in the production of gibberellins are the USA (Abbott Laboratories, Eli Lilly), the UK (Imperial Chemical Industries) and Japan (Takeda Chemical Industries). Further countries producing more or less large amounts of gibberellins are the USSR, Hungary, Poland and the People's Republic of China. Well-known trade names are Pro-Gibb, Promalin, Gib-Sol, Gib-Tabs, Activol, Berelex, Auxilin, Gibersib and Gibreskol. The preparations often contain GA3 and small quantities of other gibberellins (At, A4 , A7 and the artefact iso-GA3) together with spreader activators, effervescent mixtures or desiccants. At present, practical uses of gibberellins are most extended in the brewing industry, in increasing both yields and quality of grapes and in stimulating the growth of sugar cane.

In the routine commerical malting of barley the use of GA3 is a widespread method because the development of malt is a costly, time-consuming step in brewing beer. Normally, malt is developed by weighing a certain quantity of barley seed into a tank where germination occurs. During this process en­dogenous gibberellins passing from the germinating embryo to the aleurone layer of the seed induce the synthesis of hydrolytic enzymes, especially a-amylase and diastase. These enzymes degrade endosperm food reserves such as cell wall carbohydrates, storage protein and a limited quantity of starch. The resulting liquor is then drained from the steep for further processing. Small additions of GA3 accelerate the production and release of the enzymes which degrade the hemicellulosic-protein-starch complex into essential brewing ma­terials such as sugar, peptides and amino acids. Today the average steeping and germinating time amounts to 7-10 days. It can be shortened to 1-3 days by the addition of GA3. In commerical use GA3 is applied as an aqueous

Fungal Gibberellin Production 419

spray, or in the final steeping water in concentrations ranging from 0·25 to 1·0 ppm (Palmer, 1974) resp. 0·02S-0·5ppm (Martin, 1983) with respect to the weight of the barley. The economic effect of using GA3 during malting is difficult to evaluate, nevertheless a net gain of 2% production can be estimated (Martin, 1983). At present, the US brewing industry does not use GA3 in the malting process (Martin, 1983), and in the FRG the use of GA3 is also prohibited (Sommer, 1987).

Besides the brewing industry the gibberellins have found a wide propagation in horticulture and agriculture caused by their ability to increase the vegetative growth, to enhance flowering, to increase fruit set, size, development and quality and to delay senescence in numerous plant species.

One of the most important and traditional fields of application of the gibberellins is wine-growing. Most of the GA3 used in the world is applied to grapes. Seedless grape varieties contain only small quantities of endogenous gibberellins. Therefore, for more than 20 years the growers of seedless grapes have been using gibberellins as a safe and practical method of reducing the cost of cultivation by regulating the time of harvesting or by increasing yields and improving the quality of the crop. In Japan, the seeded grape cultivar 'Delaware' is treated with GA3 prior to bloom and 2 weeks later to induce parthenocarpic fruit set and to accelerate apparent fruit maturity. The berries attain their full size, irrespective of seed number. At least 12000 ha are treated in this way (Rappaport, 1980).

GA3 is also being routinely used to increase the crop yields in sugar cane. Treatment of sugar cane with about 62 g GA3 per acre can increase the yield to more than 5 tons and raise the output of sugar from 0·2 to 0·5 tons per acre (Nickell, 1978). These results were achieved with several types of gibberellins including broths, semi-purified and crystalline gibberellins· under Hawaiian plantation conditions (Nickell, 1979).

Besides these main commercial uses the gibberellins have reached a certain importance by a limited, sometimes only local application in selected fields of agriculture, horticulture and fruit growing. For example, in the USA and in the Mediterranean countries the application of GA3 is widespread by reason of the beneficial effects on citrus species. GA3 is widely used by Californian navel orange growers to improve fruit quality by delaying rind senescence and thus reducing surface defects such as 'rind staining', 'puffy rind', 'sticky rind', and 'water spot'. In mandarins GA3 treatment with 100 mg/liter applied in the period between anthesis and 2 weeks post-anthesis increases fruit set, total fruit number and the number of seedless fruits. Ten milligrams of GA3 per liter applied to lemons prior to degreening results in delayed degreening and prolonged storage life of the fruit up to 3 months (Rappoport, 1980). In the USA, sweet cherries are treated with 25-50 ppm GA3 up to 4 weeks before harvest (Martin, 1983). The fruits are larger and firmer, and the GA3 treatment suppresses the development of post-harvest surface pitting and bruising defects during harvest or transport. GA3 is also being used routinely in Europe to increase the crop yield in pears, to induce parthenocarpic fruit

420 B. Brueckner et al.

set, and to treat frost damage of pear (Martin, 1983). In the UK a mixture of GA3 and naphthalene acetic acid is registered for use on Cox's Orange Pippin applies to improve fruit set and yield (Looney, 1979). In the UK, the Netherlands and Canada GA3 is recommended for use on rhubarb. The treatment with the hormone breaks the dormancy in some early cultivars and increases stick yield from plants emerging from dormancy in all cultivars.

GA3 is also effective in breaking dormancy of potatoes. The treatment with GA3 is particularly useful in climates where two potato crops are grown in one season (e.g. in India), by breaking the dormancy of seed potatoes saved from the first crop. Moreover, GA3 hastens shoot emergence and increases the number of stems produced and daughter tubers formed (Rappaport, 1980).

The ability of GA3 to stimulate flowering and to increase the flower number in a wide range of vegetable species has been utilized in the production of seed from poor-bolting lettuce cultivars. GA3 (10 ppm) applied at 4- and 8-leaf stage increased the seed yield about 4-7-fold compared with the unsprayed and not beheaded control (Martin, 1983)_

Yields of certain cultivars of hops are improved and the number of cones are increased up to 35% after spraying with GA3 (Palmer, 1974).

In Mediterranean countries and in South Africa GA3 is used commercially for the production of globe artichokes. By spraying the hormone onto young plants it is possible to increase the head number per plant and to advance the harvest by several weeks, especially at low temperatures which limit bud growth. In such a way total crop yields can be increased by up to 40% without affecting earliness (Thomas, 1985).

Besides these uses, on a large commercial scale numerous possibilities of GA3 application are known in crops with a limited market size: GA3 can be used for the early harvest of chicory, cabbage, mint, parsley and spinach, for the increase of the yields of beans, celery, endives, peppers, watercress and tomatoes under glasshouse conditions, for the seed production of carrots, celery and onions, for the improvement of the germination of celery and onions, for a better fruit set of blueberries and plums, for the increase of the fruit size of lemons and melons and for the induction of parthenocarpic development of many fruits such as black and white currants, figs, straw­berries, apples, pears and tomatoes (Martin, 1983).

In addition to GA3, the gibberellins GA4 and GA7 are going to reach a greater utilization. It is supposed by numerous experts that their importance will rise further in the near future, because the physiological potencies of GA4, GA7 and GA3 differ from each other in some important respects. For example, GA4/GA7 have the remarkable ability to modify the sex expression of cucurbits. Treatment of gynoecious cucumbers with GA4/GA7 (e.g. ProGibb) can enhance male flower formation, and this phenomenon can be used commercially for producing pollen parents in gynoecious cucumber fields (Rappaport, 1980).

In celery seeds, thermodormancy can be broken by GA4/GA7 treatment

Fungal Gibberellin Production 421

(Biddington & Thomas, 1978). This procedure has some importance for the UK with a celery production of about 41000 tons (Thomas, 1985).

In the USA the russet incidence of the apple cultivar 'Golden Delicious' is reduced by spraying with GA4/GA7 (Meador & Taylor, 1987). In Italy five to six treatments of GA4 applied 8-15 days after full bloom reduced russeting of the 'Golden Delicious' apple by 70%.

A recent addition for the growth-regulator treatment of apples is a combination product of a mixture of GA4+ 7 and the cytokinines N6_

benzyl adenine (Promalin). This treatment increases the ratio of length to diameter of 'Golden Delicious' apples, resulting in an apple which better meets market expectation with respect to shape (Looney et al., 1979). In forestry, promoting influences of GA4/GA7 on the flowering and the seed production of conifers were observed in field experiments (Rappaport, 1980). For example, the strobili production in western hemlock (Tsuga heterophylla) and the flowering in white spruce (Picea glauca) were promoted (Pharis et al., 1986; Rottink, 1986). These important economic forest trees have a poor natural regeneration because of the infrequency of good seed years. Thus there is a need for a reliable method to increase and stabilize seed production. However, a true commercial scale application will not be possible before sufficient amounts of GA4/GA7 are available.

Finally, it seems to be worthwhile to mention some outlooks for new and unconventional fields of gibberellin application. Japanese patents describe the addition of gibberellins to a cosmetic cream for the decolorizing of freckles (Japan Patent 58103307 and Japan Patent 58077 808) and to a hair tonic for the promotion of the growth of hair (Japan Patent 8059906).

By adding GA3 to the food of fattened bulls, the daily increase in weight of these animals was increased by 18%. The GA3 stimulated the protein, carbohydrate and fat metabolism (Shamberev et al., 1985). Furthermore, GA3 stimulated the growth of lactic acid bacteria resulting in an increase in biomass yield of 4-8-fold. This effect of GA3 will possibly gain importance for the silage of green fodder (Erzinkyan, 1981). It is to be expected that the commercial interest in the gibberellins will grow further, especially as this group of compounds has very low mammalian toxicity and occurs naturally in many vegetable foodstuffs.

In spite of the applications described above, the scale of gibberellins is relatively low compared with the market of the herbicides, defoliants and desiccants. Some important factors limiting use of gibberellins are:

(a) the relatively high cost and the low availability, especially for GA4+7; (b) the action over a long period of time in contrast to the herbicides which

are expected to produce a rapid effect; (c) potential side-effects, such as inhibition of flowering in fruit trees for a

long period of time after treatment;

422 B. Brueckner et al.

(d) the insufficient reproducibility of the results obtained under controlled conditions in the field, and

(e) the inaccurate knowledge of the ideal time of application and of the concentration required.

12 CONCLUSIONS

In this chapter we have shown that the production of gibberellins is an almost classical fermentation, in which typical phases of growth can be discerned. Gibberella fujikuroi (Saw.) is up to now the only gibberellin-synthesizing fungus of economic importance. High producing strains of Gibberella fujikuroi were developed from wild strains. Obviously, process improvement involve a complex of conditions, which:

lead to increases in the overall rate of production; extend the production phase; hasten the onset of production; and yield fewer by-products (Vass & Jefferys, 1979).

A detailed knowledge of the gibberellin biosynthetic pathway and well­characterized mutants are required for maximizing production. Further impor­tant phenomena in the gibberellin production, which for economic reasons is a fed-batch process, include medium composition, the feed regime and airflow­agitation regime. The separation and purification process constitutes a famous part of gibberellin production cost, and from recovery process improvements there may be a decrease of gibberellin losses. Continuing reductions in the costs make gibberellins more attractive for existing applications and open possibilities for further applications.

In the future, the introduction of molecular genetics in order to increase rate-limiting enzyme levels by means of gene amplification may contribute to strain selection programs. Fermentation process computer control develop­ment is also important for the near future.

REFERENCES

Abdel-Rahman, M., Schneider, B. A. & Frank, J. R. (1977). Plant Growth Regulator Handbook. Plant Growth Regulator Working Group, USA.

Adam, G., Lischewski, M., Sych, F. J. & Ulrich, A. (1977). Synthese von Gibberellin­Aminosaeure-Konjugaten. Tetrahedron, 33,95-100.

Atzorn, R. & Weiler, E. W. (1983). The immunoassay of gibberellins. II. Quantitation of GA3 , GA4 , and GA7 by ultrasensitive solid-phase enzyme innunoassay. Planta, 159, 7-11.

Avalos, J., Casadesus, J. & Cerda-Olmedo, E. (1985). Gibberella fujikuroi mutants obtained with UV radiation and N-methyl-N'-nitro-N-nitrosoguanidine. Appl. Environ. Microbiol., 49, 187-91.

Fungal Gibberellin Production 423

Avalos, J. & Cerda-Olmedo, E. (1987). Carotenoid mutants of Gibberella fujikuroi. Curro Genet., 11, 505-11.

Bailiss, K. W. & Hill, T. A. (1971). Biological assays for gibberellins. Bot. Rev., 37, 437-79.

Banks, R. E. & Cross, B. E. (1977). Reactions of 2-chloro-NN-diethyl-1,1,2-trifluoroethylamine with alcohols. Part 2. Bridgehead fluorination of gibberellins. 1. Chem. Soc. Perkin I; (1977),512-15.

Barendse, G. W. M. (1986). Hormone action on enzyme synthesis and activity. In Hormonal Regulation of Plant Growth and Development, Vol. 3, ed. S. S. Purohit. Agro Botanical Publishers, India, pp. 119-202.

Bateson, J. H. & Cross, B. E. (1974). Reaction of 2-chloro-NN-diethyl-1,1,2-trifluoroethylamine with alcohols. Part 1. Preparation of 2- and 4-fluorogibberellins. 1. Chem. Soc. Perkin I, (1974),2409-13.

Beale, M. H., MacMillan, J., Spray, C. R. & Taylor, D. A. (1984). The synthesis of 2,2-dialkylgibberellins.l. Chem. Soc. Perkin I, (1984), 541-5.

Bearder, J. R. (1980). Plant hormones and other growth substances, their background, structures and occurrence. In Hormonal Regulation of Development, ed. I. J. MacMillan. Encyclopedia of Plant Physiology, New Series, Vol. 9. Springer, Berlin, Heidelberg, New York, pp. 9-112.

Bearder, J. R. (1983). In vivo diterpenoid biosynthesis in Gibberella fujikuroi: The pathway after Ent-kaurene. In The Biochemistry and Physiology of Gibberellins, Vol. 1, ed. A. Crozier. Praeger, New York, pp. 251-388.

Bergner, C. (1988). Gibberellin-Biotests. In Methoden zur Pflanzenhormonanalyse, ed. G. Sembdner, G. Schneider & K. Schreiber. Gustav Fischer, Jena, pp. 167-71.

Bernhardt, D. (1988). a-Amylase-Tests. In Methoden zur Pflanzenhormonanalyse, ed. G. Sembdner, G. Schneider & K. Schreiber. Gustav Fischer, Jena, pp. 171-4.

Biddington, N. L. & Thomas, T. H. (1978). Thermodormancy in celery seeds and its removal by cytokinins and gibberellins. Physiol. Plantarum, 42,401-5.

Borrow, A., Brian, P. W., Chester, V. E., Curtis, P. J., Hemming, H. G., Henehan, C., Jefferys, E. G., Lloyd, P. B., Nixon, I. S., Norris, G. L. F. & Radley, M. (1955). Gibberellic acid, a metabolic product of the fungus Gibberella fujikuroi: Some observations on its production and isolation. 1. Sci. Food Agr., 6, 340-48.

Borrow, A., Brown, S., Jefferys, E. G., Kessel, R. H. J., Lloyd, P. B., Rothwell, A., Rothwell, B. & Swait, J. C. (1964). Metabolism of Gibberella fujikuroi in stirred culture. Can. 1. Microbiol., 10, 407-44.

Borrow, A., Jefferys, E. G., Kessell, R. H. J., Lloyd, E. C., Lloyd, P. B. & Nixon, I. S. (1961). The metabolism of Gibberella fujikuroi in stirred culture. Can. 1. Microbiol., 7, 227-76.

Brian, P. W., Elson, G. W., Hemming, H. G. & Radley, M. (1954). The plant growth promoting properties of gibberellic acid, a metabolic product of the fungus Gibberella fujikuroi. 1. Sci. Food Agr., 5, 602-12.

Brueckner, B. & Blechschmidt, D. (1986). Die mikrobiologische Synthese von Gibberellinen.l. Basic Microbiol., 26,483-97.

Bu'Lock, J. D., Detroy, R. W., Hostalek, Z. & Monin-AI-Shakarchi, A. (1974). Regulation of secondary biosynthesis in Gibberella fujikuroi. Trans. Br. Mycol. Soc., 62, 377-89.

Calam, C. T., Daglish, L. B. & Gaitskell, W. S. (1973). Hybridisation experiments with Penicillium patulum and Fusarium moniliforme. In Genetics of Industrial Micro-organisms, ed. Z. Vanek, Z. Hostalek & J. Cudlin. Academia, Prague, pp. 265-82.

Coolbaugh, R. C. (1983). Early stages of gibberellin biosynthesis. In The Biochemistry and Physiology of Gibberellins, Vol. 1, ed. A. Crozier. Praeger, New York, pp. 53-98.

424 B. Brueckner et al.

Coolbaugh, R. C., AI-Nimri, L. F. & Nester, J. E. (1985). Evidence for gibberellins in culture filtrates of Cercospora rosicola. In Abstracts of the 12th International Conference of Plant Growth Substances, Heidelberg 1985, pp. 12,24.

Corey, E. J., Danheiser, R. L., Chandrasekaran, S., Keck, G. E., Gopalan, B., Larsen, S. D., Sivet, P. and Gras, J. L. (1978). Stereospecific total synthesis of gibberellic acid. I. Am. Chem. Soc., 100, 8034-6.

Cross, B. E. & Simpson, I. C. (1981). Reactions of 2-chloro-N,N-diethyl-1,1,2-trifluoroethylamine with alcohols. Part 3. In the presence of lithium chloride, preparation of chlorogibberellins. I. Chem. Soc. Perkin I, (1981), 98-104.

Crozier, A. (ed.) (1983). The Biochemistry and Physiology of Gibberellins, Vols 1 and 2. Praeger, New York.

Crozier, A. (1987). The principles of plant hormone analysis. In Principles and Practice of Plant Hormone Analysis, Vol. 1, ed. L. Rivier & A. Crozier. Academic Press, London.

Crozier, A. & Durley, R. C. (1983). Modern methods of analysis of gibberellins. In The ·Biochemistry and Physiology of Gibberellins, Vol. I, ed. A. Crozier. Praeger, New York, pp. 485-560.

Crozier, A., Zaerr, J. B. & Morris, R. O. (1982). Reversed- and normal-phase high performance liquid chromatography of gibberellin methoxycoumaryl esters. I. Chromatogr., 238, 157-66.

Curtis, P. J. & Cross, B. E. (1954). Gibberellic acid. A new metabolite from the culture filtrates of Gibberella fujikuroi. Chem. Ind., 1066.

Dathe, W. (1986). Occurrence of gibberellins in fungi and higher plants in relation to their taxonomical connections. In Hormonal Regulation of Plant Growth and Development, Vol. 3, ed. S. S. Purohit. Agro Botanical Publishers, India, pp. 203-44.

Darken, M. A., Jensen, A. L. & Shu, P. (1959). Production of gibberellic acid by fermentation. Appl. Microbiol., 7,301-3.

De Angelis, J. G. (1970). Effect of gibberellic acid treatments in globe artichoke (Cynara scolymus L.). Israel I. Agr. Res., 20, 149-57.

Dunberg, A. & Oden, P. C. (1983). Gibberellins and conifers. In The Biochemistry and Physiology of Gibberellins, Vol. 2, ed. A. Crozier. Praeger, New York, pp. 221-95.

Durley, R. C. & Pharis, R. P. (1972). Partition coefficients of 27 gibberellins. Phytochemistry, 11,317-26.

Eberle, J., Yamaguchi, I., Nakagawa, R., Takahashi, N. & Weiler, E. W. (1986). Monoclonal antibodies against GAB-imide recognize the endogenous plant growth regulator, GA4 and related gibberellins. FEBS Lett., 202,27-31.

Eccher, T. & Boffelli, G. (1981). Effects of dose and time of application of GA4+ 7 on russeting, fruit set and shape of 'Golden Delicious' apples. Sci. Hort., 14,307-14.

Erokhina, L. I. & Efremov, B. D. (1970). Biochemical mutants of Fusarium moniliforme (Sheld). Genetika, 6, 170-72.

Erokhina, L. I. & Sokolova, E. V. (1966). Selection of Fusarium moniliforme Sheld (producer of gibberellins) with application of mutagenic factors. Genetika, 1, 109-15.

Erzinkyan, L. A. (1981). Gibberellin-growth stimulator for lactic acid bacteria. Bioi. Zh. Arm., 34, 1188-90.

Focke, I., Sembdner, G. & Schreiber, K. (1967). Der Einfluss komplexer Naehrsub­strate auf die Gibbellinbildung von Fusarium moniliforme Sheld. Bioi. Zbl., 86, 509-19.

Furuya, M. & Takeno, K. (1983). Gibberellins in algae and higher cryptogams. In The Biochemistry and Physiology of Gibberellins, Vol. 2, ed. A. Crozier. Praeger, New York, pp. 189-220.

Fuska, J., Kuhr, I., Podojil, M. & Sevcik, V. (1961). The influence of the nitrogen source on the production of gibberellic acid in submerse cultivation of Gibberella fujikuroi. Folia Microbiol., 6, 18-21.

Fungal Gibberellin Production 425

Gancheva, V. & Dimova, Ts. (1984). Biosynthesis of gibberellins. II. Influence of the quantity and age of inoculum on the biosynthesis of gibberellins from the strain Fusarium moniliforme IM-11. Acta Microbiol. Bulgarica, 14,74-9.

Gordon, W. L. (1960). Distribution and prevalence of Fusarium moniliforme Sheld (Gibberella fujikuroi (Saw.) Wr. producing substances with gibberellin-like biologi­cal properties. Nature, Lond., 18(J, 698-700.

Graebe, J. E. (1987). Gibberellin biosynthesis and control. Ann. Rev. Plant Physiol., 38,419-65.

Graebe, J. E. & Ropers, H. J. (1978). Gibberellins. In Phytohormones and Related Compounds--a Comprehensive Treatise, Vol. 1, ed. D. S. Letham, P. B. Goodwin & T. J. V. Higgins. Elsevier, Amsterdam, Oxford, New York, pp. 107-204.

Hartsuck, J. A. & Lipscomb, W. N. (1963). Molecular and crystal structure of the di-p-bromobenzoate of the methyl ester of gibberellic acid. J. Am. Chem. Soc., 85, 3414-19.

Hedden, P. (1983). In vitro metabolism of gibberellins. In The Biochemistry and Physiology of Gibberellins, Vol. 1, ed. A. Crozier. Praeger, New York, pp. 99-149.

Hedden, P. (1986). The use of combined gas chromatography-mass spectrometry in the analysis of plant growth substances. In Modern Methods of Plant Analysis, Vol. 3, Gas Chromatography/Mass Spectrometry, ed. H. F. Linskens & J. E. Jackson. Springer, Berlin, Heidelberg, New York, Tokyo, pp. 1-12.

Hedden, P. (1987). Gibberellins. In Principles and Practise of Plant Hormone Analysis, Vol. 1, ed. L. Rivier & A. Crozier. Academic Press, London.

Hoad, G. V. (1983). Gibberellin bioassays and structure-activity relationships. In The Biochemistry and Physiology of Gibberellins, Vol. 2, ed. A. Crozier. Praeger, New York, pp. 57-94.

Holme, T. & Zacharias, B. (1965). Gibberellic acid formation in continuous culture. Biotechnol. Bioeng., 7,405-15.

Hori, S. (1898). Some observations on 'bakanae' disease of the rice plant. Mem. Agr. Res. Sta. (Tokyo), 12, 110-19.

Imshenetsky, A. A. & Ulyanova, O. M. (1962). Experimental variability in Fusarium moniliforme Sheld leading to the formation of gibberellins. Nature, Lond., 195, 62-3.

Jacobsen, J. V. (1983). Regulation of protein biosynthesis in aleurone cells by gibberellin and abscisic acid. In The Biochemistry and Physiology of Gibberellins, Vol. 2, ed. A. Crozier. Praeger, New York, pp. 159-87.

Jefferys, E. G. (1970). The gibberellin fermentation. Adv. Appl. BioI., 13,283-316. Jensen, E., Crozier, A. & Monteiro, A. M. (1986). Analysis of gibberellins and

gibberellin conjugates by ion-suppression reversed-phase high-performance liquid chromatography. J. Chromatogr., 367,377-84.

Jones, R. L. & Moll, C. (1983). Gibberellin-induced growth in excised lettuce hypocotyls. In The Biochemistry and Physiology of Gibberellins, Vol. 2, ed. A. Crozier. Praeger, New York, pp. 95-128.

Karssen, C. M. & Lacka, E. (1986). A revision of the hormone balance theory of seed dormancy: Studies on gibberellin and/or abscisic acid-deficient mutants of Arabidopsis thaliana. In Plant Growth Substances, ed. M. Bopp. Springer, Berlin, Heidelberg, New York, Tokyo, pp. 315-23.

Kaufman, P. B. & Dayanandan, P. (1983). Gibberellin-induced growth in Avena internodes. In The Biochemistry and Physiology of Gibberellins, Vol. 2, ed. A. Crozier. Praeger, New York, pp. 129-57.

Kawanabe, Y., Yamane, H., Murayama, T., Takahashi, N. & Nakamura, T. (1983). Identification of gibberellin A3 in mycelia of Neurospora crassa. Agr. BioI. Chern., 47, 1693-4.

Keith, B., Srivastava, L. M. & Murofushi, N. (1979). Agric. BioI. Chem. (Tokyo), 43, 141-3.

Knoefel, H. D. (1988). Immunologische Methoden. In Methoden zur

426 B. Brueckner et aI.

Pjlanzenhormonanalyse, ed. G. Sembdner, G. Schneider & K. Schreiber. Gustav Fischer, Jena, pp. 195-219.

Knox, J. P., Beale, M. H., Butcher, G. W. & MacMillan, J. (1987). Preparation and characterization of monoclonal antibodies which recognise different gibberellin epitopes. Planta, 170, 86-91.

Krishnamoorthy, H. N. (ed.) .(1975). Gibberellins and Plant Growth. Wiley Eastern Limited, New Delhi.

Kumar, P. K. R. & Lonsane, B. K. (1987). Gibberellic acid by solid state fermentation: Consistent and improved yields. Biotechnol. Bioeng., 30,267-71.

Kurosawa, E. (1926). Experimental studies on the nature of the substance excreted by the 'bakanae' fungus. Trans. Nat. Hist. Soc. Formosa, 16,213-27.

Lewak, S. (1985). Hormones in seed dormancy and germination. Adv. Agric. Biotechnol., 16,95-114.

Lindenfelser, L. A. & Ciegler, A. (1975). Solid-substrate fermentor for ochratoxin A production. Appl. Microbiol., 29, 323-7.

Lischewski, M. (1985). Tritiummarkierungen von Gibberellin At. Z. Chemie, 25, 141-2.

Lischewski, M. & Adam, G. (1980). Gibberelline LXII. Synthese von 7-desoxygenierten Gibberellin-A3-Verbindungen. Tetrahedron, 36, 1237-44.

Lombardo, L., Mander, L. N. & Turner, J. V. (1980). General strategy for gibberellin synthesis: Total synthesis of ( + )-gibberellin At and gibberellic acid. J. Am. Chem. Soc., 102, 6626-8.

Looney, N. E., Pharis, R. P. & Noma, m. (1985). Promotion of flowering in apple (Malus domestica cv. Golden Delicious) trees with gibberellin ~ and C-3 epi-gibberellin ~. Planta (Ber!.), 165(2), 292-4.

MacMillan, J. (ed.) (1980). Hormonal Regulation of Development. l. Molecular Aspects of Plant Hormones. Encyclopedia of Plant Physiology, New Series, Vol. 9. Springer, Berlin, Heidelberg, New York.

MacMillan, J. & Takahashi, N. (1968). Proposed procedure for the allocation of trivial names to gibberellins. Nature, Lond., 217, 170-1.

Maddox, R. (1977). Use of response surface methodology for the rapid optimization of microbiological media. J. Appl. Bact., 43, 197-204.

Martin, G. C. (1983). Commercial uses of gibberellins. In The Biochemistry and Physiology of Gibberellins, Vol. 2, ed. A. Crozier. Praeger, New York, pp. 395-444.

McInnes, A. G., Smith, D. G., Durley, R. C., Pharis, R. P., Arsenault, G. P., MacMillan, J., Gaskin, P. & Vining, L. C. (1977). Biosynthesis of gibberellins in Gibberella fujikuroi. Biosynthesis of gibberellins in Gibberella fujikuroi. Gibberellin ~7' Can. J. Biochem., 55, 728-35.

Meador, D. B. & Taylor, B. H. (1987). Effect of early season foliar sprays of G~+7 on russeting and return bloom of 'Golden Delicious' apple. Hort. Sci., 22,412-15.

Mori, K., Shiozaki, M., Itaya, N., Ogawa, T., Matsui, M. & Sumiki, Z. (1968). Synthesis of substances related to gibberellins XVIII. Total synthesis of (-)­gibberellin A 2 , ~, A9 and A lO• Total synthesis of ( - )-gibberellin A 2 , ~, A7 and A lO • Tetrahedron Lett., 2183-8.

Morris, R. O. & Zaerr, J. B. (1978). 4-Bromophenyacyl esters of gibberellins, useful derivatives for high performance liquid chromatography. Analyt. Lett., 11,73-83.

Morofushi, N., Yamaguchi, I., Ishiogooka, H. & Takahashi, N. (1976). Chemical conversion of gibberellins An to gibberellin ~. Agric. Bioi. Chem., 40, 2471-4.

Muromtsev, G. S. & Agnistova, V. N. (1973). Plant Hormones-Gibberellins. Nauka, Moscow (in Russian).

Muromtsev, G. S. & Agnistova, V. N. (1984). Gibberellins, ed. S. V. Letunova. Nauka, Moscow (in Russian).

Muromtsev, G. S. & Dubovaya, L. P. (1964). The use of plant oils and fatty acids for biosynthesis of gibberellins by Fusarium moniliforme. Mikrobiologia, 33, 1048-54.

Fungal Gibberellin Production 427

Muromtsev, G. S., Rakovsky, Yu. S., Dubovaya, L. P., Temnikova, T. V. & Fedchenko, A. N. (1968). Study of sucrose as a carbon source for the biosynthesis of gibberellins. Prikladnaya Biochimia i Mikrobiologia, 4, 398-407.

Nadeau, R. & Rappaport, L. (1974). The synthesis of CH) GA3 and CH) GAt by palladium catalysed action of carrier-free tritium of gibberellin A3. Phytochemistry, 13, 1337-45.

Nakamura, T., Mitsuoka, K., Sugano, M., Tomita, K. & Murayama, T. (1985). Effects of auxin and gibberellin on conidial germination and elongation of young hyphae in Gibberellafujikuroi and Penicillium notatum. Plant Cell Physiol., 26, 1433-7.

Nickell, L. G. (1978). Plant growth regulators. Controlling biological behavior with chemicals. Chem. Eng. News, 56, 18-34.

Palmer, G. H. (1974). The industrial use of gibberellic acid and its scientific basis-a review. J. Inst. Brew., 80, 13-30.

Pengelly, W. L. (1986). Validation of immunoassays. In Plant Growth Substances, ed. M. Bopp. Springer, Berlin, Heidelberg, New York, Tokyo, pp. 35-43.

Pharis, R. P. & King, R. W. (1985). Gibberellins and reproductive development in seed plants. Ann. Rev. Plant Physiol., 36,517-68.

Pharis, R. P. & Reid, D. M. (ed) (1985). Hormonal Regulation of Development. III. Role of Environmental Factors. Encyclopedia of Plant Physiol., New Series, Vol. 11. Springer, Berlin, Heidelberg, New York, Tokyo.

Pharis, R. P., Tomchuk, D., Beall, F. D., Rauter, M. & Kiss, G. (1986). Promotion of flowering in white spruce (Picea glauca) by gibberellin A 4n , auxin (naphthalene acetic acid), and the adjunct cultural treatments of girdling and calcium nitrate fertilization. Can. J. For. Res., 16,340-5.

Phinney, B. O. (1983). The history of gibberellins. In The Biochemistry and Physiology of Gibberellins, Vol. 1, ed. A. Crozier. Praeger, New York, pp. 19-52.

Rademacher, W. & Graebe, J. E. (1979). Gibberellin A4 produced by Sphaceloma manihoticola, the cause of the superelongation disease of cassava (manihot esculenta). Biochem. Biophys. Res. Commun., 91,35-40.

Radley, M. (1956). Occurrences of substances similar to gibberellic acid in higher plants. Nature, Lond., 178, 1070-1.

Rappaport, L. (1980). Applications of gibberellins in agriculture. In Plant Growth Substances, ed. F. Skoog. Springer, Berlin, Heidelberg, New York, pp. 377-91.

Reeve, D. R. & Crozier, A. (1975). Gibberellin bioassays. In Gibberellins and Plant Growth, ed. H. N. Krishnamoorthy. Wiley Eastern, New Delhi, pp. 35-64.

Reeve, D. R. & Crozier, A. (1980). Quantitative analysis of plant hormones. In Hormonal Regulation of Development. I. Molecular Aspects of Plant Hormones, ed. J. MacMillan. Encyclopedia of Plant Physiol, New Series, Vol. 9. Springer, Berlin, Heidelberg, New York, pp. 203-80.

Rehm, H. J. (1980). Industrielle Mikrobiologie. Springer, Berlin, Heidelberg, New York.

Rehm, H. J. (1981). Growth of Fusarium moniliforme on n-alkanes: Comparison of an immobilization method with conventional process. Eur. J. Appl. Microbiol. Biotech., -11, 139-45.

Reid, D. M. (1983). Gibberellins and phytochrome. In The Biochemistry and Physiology of Gibberellins, Vol. 2, ed. A. Crozier. Praeger, New York, pp. 297-332.

Reid, J. B. (1987). Gibberellin mutants. In A Genetic Approach to Plant Biochemistry, ed. A. D. Blonstein & P. J. King. Springer, Wien, New York, pp. 1-34.

Rottink, B. A. (1986). Comparison of gibberellins A3 and A4n for promoting strobili in western hemlock (Tsuga heterophylla). Can. J. For. Res., 16,389-90.

Rowe, J. W. (1968). The Common and Systematic Nomenclature of Cyclic Diterpenes, 3rd revision. US Forest Product Laboratory, Madison, Wisconsin.

Sackett, P. H. (1984). High performance thin-layer chromatography of gibberellins in fermentation broths. Analyt. Chem., 56, 1600-3.

428 B. Brueckner et al.

Schneider, G. (1983). Gibberellin conjugates. In The Biochemistry and Physiology of Gibberellins, Vol. 1, ed. A. Crozier. Praeger, New York, pp. 389-456.

Schneider, G. (1988a). HPLC von Gibberellinen. In Methoden zur PJlanzenhormonanalyse, ed. G. Sembdner, G. Schneider & K. Schreiber. G. Fischer, Jena, pp. 102-8.

Schneider, G. (1988b). Gaschromatographie von Gibberellinen. In Methoden zur PJlanzenhormonanalyse, ed. G. Sembdner, G. Schreiber & K. Schreiber. G. Fischer, Jena, pp. 127-32.

Schneider, G. (1988c). Spektrofiuorometrie. In Methoden zur PJlanzenhormonanalyse, ed. G. Sembdner, G. Schneider & K. Schreiber. G. Fischer, Jena, pp. 233-40.

Schneider, G., Sembdner, G. & Schreiber, K. (1966). Gibberelline, ihre Derivate und Abbauprodukte. Akademie, Berlin.

Schreiber, K., Schneider, G., Sembdner, G. & Focke, F. (1966). Isolierung von O(2)-Acetyl-gibberellinsaeure als Stoffwechselprodukt von Fusarium moniliforme Sheld. Phytochemistry, 5, 1221-5.

Schwartz, E., Miklussak, S., Vincurova, M., Laczko, V., Krcova, M. & Zemkova, O. (1983). Pharmakologische Wirkungen der Gibberellinsaeure. Pharmazie, 38,716-8.

Scott, T. K. (ed.) (1984). Honnonal Regulation of Development. II. The Function of Hormones from the Level of the Cell to the Whole Plant. Encyclopedia of Plant Physiol., New Series, Vol. 10. Springer, Berlin, Heidelberg, New York, Tokyo.

Sembdner, G., Gross, D., Liebisch, H. W. & Schneider, G. (1980). Biosynthesis and metabolism of plant hormones. In Hormonal Regulation of Development, ed. I. J. MacMillan. Encyclopedia of Plant Physiol., New Series, Vol. 9. Springer, Berlin, Heidelberg, New York, pp. 281-444.

Sembdner, G., Schlie mann , W. & Herrmann, G. (1987). Growth. Progr. Bot., 49, 137-70.

Sembdner, G., Schneider, G. & Schreiber, K. (1988). Methoden zur PJlanzenhor­monanalyse. G. Fischer, Jena.

Shamberev, Y. N., Gavrishuk, V. I., Ivanov, I. S. & Netesa, Y. I. (1985). Effect of gibberellin on the endocrine system and metabolism in bulls. Izv. Timiryasev s-kh. Akad., 2, 138-43.

Somei, M. & Okamoto, T. (1970). A novel method for attacking nonactivated C-H bond and its application to the synthesis of gibberellin A 15 from enmein. Chern. Pharm. Bull., 18, 2135-8.

Sommer, G. (1987). Beer. In Fundamentals of Biotechnology, ed. P. Praeve, U. Faust, W. Sittig & D. A. Sukatsch. Verlagsgesellschaft, Weinheim.

Spector, C. & Phinney, B. O. (1966). Gibberellin production: Genetic control in the fungus Gibberella fujikuroi. Science, 153, 1397-8.

Spector, C. & Phinney, B. O. (1968). Gibberellin biosynthesis: Genetic studies in Gibberella fujikuroi. Physiol. Plant, 21, 127-36.

Stoddart, J. L. (1986). Gibberellin receptors. In Hormones, Receptors and Cellular Interactions in Plants, ed. C. M. Chadwick & D. R. Garrod. Cambridge University Press, Cambridge, London, New York, New Rochelle, Melbourne, Sydney, pp. 91-114.

Stodola, F. H., Raper, K. B., Fennel, D. I., Conway, H. F., Johns, V. E., Langford, C. T. & Jackson, R. W. (1955). The microbial production of gibberellins A and X. Archs Biochem. Biophys., 54,240-5.

Takahashi, N. & Yamaguchi, I. (1983). Preparation and isotope labeling of gib­berellins. In Biochemistry and Physiology of Gibberellins, Vol. 1, ed. A. Crozier. Praeger, New York, pp. 457-84.

Takahashi, N., Yamaguchi, I. & Yamane, H. (1986). Gibberellins. In Chemistry of Plant Hormones, ed. N. Takahashi. CRC Press, Boca Raton, Florida, pp. 57-15l.

Tamura, S. (1971). Gibberellins and other biologically active substances produced by micro-organisms. In Biochemical and Industrial Aspects of Fermentation, ed. K.

Fungal Gibberellin Production 429

Sakaguchi, T. Uemura & S. Kinoshita. Kodansha Scientific Books, Tokyo, pp. 155-73.

Thomas, T. H. (1985). Plant growth regulators in the production and storage of outdoor and glasshouse vegetables. In Growth Regulators in Horticulture, ed. R. Menhenett & M. B. Jackson. Proc. Conf. Univ. Reading, pp. 29-42.

Tsao, T. H. & Linskens, H. F. (1986). Role of growth substances in fertilization. In Hormonal Regulation of Plant Growth and Development, Vol. 3, ed. S. S. Purohit. Agro Botanical Publishers, India, pp. 91-118.

Vass, R. C. & Jefferys, E. G. (1979). Gibberellic acid. In Economic Microbiology, Vol. 3. Secondary Products of Metabolism, ed. A. H. Rose. Academic Press, London, New York, San Francisco, pp. 421-33.

Voigt, B. & Adam, G. (1982). Synthese von l-Amino-gibberellinen. Z. Chemie, 23, 177-8.

Wang, T. L., Griggs, P. & Cook, S. (1986). Immunoassays for plant growth regulators--A help or a hindrance? In Plant Growth Substances, ed. M. Bopp. Springer, Berlin, Heidelberg, New York, Tokyo, pp. 26-34.

Weiler, E. W. (1984). Immunoassays of plant growth regulators. Ann. Rev. Plant Physiol., 35, 85-95.

Weiler, E. W. (1986). Plant hormone immunoassay based on monoclonal and polyclonal antibodies. In Immunology in Plant Science. Modern Methods of Plant Analysis, New Series, Vol. 4, ed. H. F. Linskens & J. E. Jackson. Springer, Berlin, Heidelberg, New York, pp. 1-17.

Weiler, E. W., Eberle, J. & Mertens, R. (1986a). Immunoassays for the quantification of plant growth. In Plant Growth Substances, ed. M. Bopp. Springer, Berlin, Heidelberg, New York, Tokyo, pp. 22-5.

Weiler, E. W., Eberle, J., Mertens, R., Atzorn, R., Feyerabend, M., Jourdan, P. S., Arnscheidt, A. & Wieczorek, U. (1986b). Monoclonal and polyclonal antibody based immunoassay of plant hormones. In Immunology in Plant Science, ed. T. L. Wang. Cambridge University Press, Cambridge, pp. 27-58.

Winkler, V. M. (1978). Gibberellins ~A7 and 6-benzyladenine. In Analytical Methods for Pesticides and Plant Growth Regulators, Vol. X. New and Updated Methods, ed. G. Zweig & J. Sherma. Academic Press, New York, San Francisco, London, pp. 454-9.

Yabuta, T. (1935). Biochemistry of the 'bakanae' fungus of rice. Agr. Hort., 10, 17-22. Yabuta, T., Kambe, K. & Hayashi, T. (1934). Biochemistry of the 'bakanae' fungus of

rice. Part 1. Fusaric acid, a new product of the bakanae fungus. J. Agr. Chem. Soc. Japan, 10, 1059-68.

Yokota, N., Murofushi, N. & Takahashi, N. (1980). Extraction, purification and identification. In Hormonal Regulation of Development I, ed. J. MacMillan. Encyclopedia of Plant Physiology, New Series, Vol. 9. Springer, Berlin, Heidelberg, New York, pp. 113-201.

Zeevaart, J. A. D. (1983). Gibberellins and flowering. In The Biochemistry and Physiology of Gibberellins, Vol. 2, ed. A. Crozier. Praeger, New York, pp. 333-74.

Zeigler, R. S., Powell, L. E. & Thurston, H. D. (1980). Gibberellin A4 production by Sphaceloma manihoticola, causal agent of cassava superlongation disease. Phytopathol., 70,589-93.

INDEX

Acetobacter, 309, 310, 321 Acetobacter cerinus, 313 Acetobacter fraqum, 313 Acetobacter melanogenus, 312, 313 Acetobacter suboxydans, 168, 199, 209,

275, 304, 311, 312, 317, 323, 324 Acetobacter xylinum, 168, 304, 323 Acetomonas albosesamae, 313, 319 Acetyl-CoA carboxylase, 232 Achromobacter aceris, 376 Adenine, 348 Adenosine, 348, 367 Adenosylhomocysteine (AdoHcy), 351-

72 activated methyl cycle, 353-60 biosynthesis, 353-62 chemical synthesis, 368 chemistry, 352-3 derivatives of, 353 enzymatic methods, 366-7 hydrolase, 360-2 production of, 366-8

Adenosylmethionine (AdoMet), 351-72 activated methyl cycle, 353-60 biosynthesis, 353-62 chemical synthesis, 366 chemistry, 352-3 decarboxylation, 360 derivatives of, 353 enzymatic production, 362-5 isolation of, 365-6 microbial production, 362-5 production of, 362-6 stabilization of, 365-6

Adonis vernalis, 169

431

ADP, 338, 339 Aerobacter, 262, 310 Aerobacter aerogenes, 177, 287 Agrobacterium, 262, 264, 379 tJ-Alanine, 206 Alcaligenes, 262, 310 Alcaligenes faecalis, 361, 366, 367, 368,

369 Algae

composition of growth medium, 51 physical factors, 51

5-Aminoimidazaole ribonucleotide (AIR), 140-1

AMP, 337-40, 343,344, 346,376 Antivitamins, 5, 6 Arachidonic acid, 111-15 Arthrobacter globiformis, 177 Arthrobacter parafJineus, 289 Arthrobacter simplex, 316 L-Ascorbic acid, 299-334

application, 326-7 assay methods, 325-6 background history, 299-301 biocatalysts involved in biosynthesis of

intermediates, 316-18 biosynthesis

cloning of genes related to, 318-22 in plants and animals, 302-4 micro-organisms and media used in,

305-8 with micro-organisms, 304-18

chemical properties, 302 development data, 300 direct synthesis of, 316 economics, 326-7

432

L-Ascorbic acid-contd. industrial process, 322-5 nomenclature, 302 physical properties, 302 product recovery and purification,

324-5 Reichstein-Griissner synthesis, 323

Ashbya gossypii, 151-60, 162, 165 Aspergillus flavus, 151, 316 Aspergillus giganteus, 28 Aspergillus niger, 151, 235, 316 Astaxanthin, 58-9

extractability of, 61 ATP, 337-50, 376

assay methods, 338-9 chemical structure, 338 chemistry, 338-9 construction of reaction system

producing, 345-6 energy efficiency of phosphorylation

system, 346-8 production

adenosine or adenine, from, 348-9 biochemical system, by, 349 glycolytic pathway, through, 339-43 oxidative phosphorylation by

methylotrophic yeast, through, 344-9

salvage synthesis, through, 343-4 role in energy metabolism, 337

Azotobacter, 262,310

Bacillus, 72, 168, 171, 174, 175-83, 185, 262, 289, 310

Bacillus circulans, 61 Bacillus megaterium, 237, 238, 264, 311,

316 Bacillus pumilus, 177, 180 Bacillus sphaericus, 234-8, 241, 242 Bacillus subtilis, 177, 243, 258, 289, 291 Bacterium xylinum, 304 Bacteroides rumincola, 175 Bicyclic ketocarotenoids, 58-65 Bifidobacterium, 216 Biotin, 231-56

absolute configuration, 233 antimetabolites, 240 application, 250 assay methods, 244-5 bioassay methods, 243 biodegradation, 238-40 biological properties, 243-8 biosynthesis, 231,235-6

Index

Biotin-contd. chemical properties, 232-3 chemical reactions, 233 chemical structure, 233 chemical synthesis, 248-50 deficiencies in animals, 243-8 economics, 250 fermentation, 241-2 genetics, 240-1 growth-promoting activity, 248 historical background, 231-2 physical properties, 232-3 producing micro-organisms, 234-5 product recovery and purification,

242-3 regulation of biosynthesis, 238 screening, 234-5 strain improvement, 240-1 structure of, 232

Biotin carboxylase (BC), 232 Bixa orellana, 27 Blakeslea trispora, 27, 28, 30-3, 36, 55 Blood clotting mechanism, 124 Brevibacterium, 287, 313 Brevibacterium ammoniagenes, 212-14,

177,208,215,289,292,337, 343-4,376

Brevibacterium divaricatum, 237, 238 Brevibacterium flavum, 250 Brevibacterium ketosoreductum, 312,

316 Brevibacterium KY-4313, 62-4, 71 Brevibacterium thiogenitalis, 177 Bupleurum falcatum, 169

Calcium 2,5-diketo-o-gluconate, 315 Calcium 2-keto-L-gulonate, 315 Calcium pantothenate, 216 Candida, 84 Candida arborea, 151 Candida boidinii, 344-6, 348 Candida chalmersi, 151 Candida flareri, 151 Candida ghoshi, 151 Candida quilliermondia, 151 Candida humicola, 101 Candida krusei, 151 Candida lactis, 151 Candida lipolytica, 151, 289 Candida meliblosi, 151 Candida norvegensis, 316 Candida olea, 151 Candida parapsilosis, 210

Candida puicherrima, 151 Candida solani, 151 Candida tropicalis, 89, 289, 292, 379 Candida tropicalis varrhaggi, 151 Candida utilis, 362 Cantharellus cinnabarinus, 28 Canthaxanthin, 58-9, 62 Capsicum annuum, 27 Carboxyl carrier protein (CCP), 232 Carboxyl transferase (Cf), 232 p-Carotene,15-26

applications, 23 biological properties, 22-3 biosynthesis, 18-19 chemical properties, 15-16 chemical synthesis, 23 economics, 23-4 formulations, 23 HPLC analysis, 16 industrial purification, 37 physical properties, 15-16 product recovery and purification, 22 regulation, 18-19 syntheses, 27

Carotenes, 54-5 Carotenoids

applications, 73 assay methods, 68-71 basic structures, 44 biological properties, 47 biosynthesis and regulation, 48-53 biosynthetic relationships, 50 chemical properties, 44-5 chemical synthesis, 71-3 compounds inhibiting normal

biosynthesis of, 52 content variations, 30-2 culture conditions, 32 discovery in nature, 43 eady observations in micro-organisms,

43-4 economics, 73 environmental factors, 50-2 fungal,28 fungi production, 27-42 future prospects, 73 genetic control, 53 industrial applications, 47 industrial production, of, 28 microbial production, 43-79 micro-organisms of interest in

production of, 47-8 nomenclature, 46 physical properties, 44-5

Index 433

Carotenoids---contd. product recovery and purification, 69 scarcity in diet, 27 strain improvement, 53-4 structural formulae, 45 structure of, 28

Cell reaction method, 341, 343-4 Cempasuchil, 27 Cercospora rosicola, 391 Ceroplastes, 169 Chilomonas paramoecium, 152 Chlamydomonas, 97 Chlorella, 97 Chlorella pyrenoidosa, 56, 316 Chlorella sorokiniana, 56 Choanephora cucurbitarum, 28 Cholecalciferol, 82 Citrobacter, 311 Citrus medica, 396 Claviceps purpurea, 83 Clostridium, 262 Clostridium acetobutylicum, 151 Clostridium butylicum, 151 Clostridium kainantoi, 226 Clostridium tetanomorphum, 102, 275 CMN complex, 62 Cobalamins, 259, 260

biosynthesis of, 264 Cobaltcorrinoids, 259, 260 Coccus cacti, 6 Coenzyme A, 199,207-9,212-14

application, 216 biosynthesis, 214 economic aspects, 216 structure of, 205

Coenzyme Q, 379-80 Coenzymes, 373-81

classification, metabolic functions and sources of, 374-5

Cordyceps militaris, 169 Corynebacterium, 235, 262, 287, 313,

315-19 Corynebacterium glutamicum, 250, 286,

287, 289, 292, 293 Corynebacterium sp. XG, 67 Crocus sativus, 27 Croton bean, 169 Croton tiglium L., 169 Culture method, 343 Cyanobacteria, 59 Cyanocobalamin, 257 Cyclic GMP, 248

Debaryomyces nilssonii, 340

434

Deinococcus radiodurans, 58 Deinococcus radiophiLus, 58 Deinococcus radiopugnans, 58 Deoxyadenosylcobalamin,271-2 I-Deoxy-ketoses, 180 2-DeoxY-D-ribose, 167 Dethiobiotin, 241-2 Dihomo-y-linolenic acid, 115-16 2,5-Diketo-D-gluconic acid reductase,

318-21 Dimethylallypyrophosphate (DPP), 86 DipLoascus, 84 DunalieLLa, 15, 119 DunalieLLa salina, 15

production process requirements, 19-21

strain improvement, selection and maintenance, 16-18

Eicosapentaenoic acid (EPA), 116-19 EmiLiania huxLeyi, 169 Endomyces, 84 Enterobacter, 311 EntomophthoraLes, 111 Enzyme immunoassay, 412 Enzyme-linked immunosorbent assay

(ELISA), 412 Eremothecium ashbyii, 151-62 Ergocalciferol, 82 Ergosterol, 81-93

chemical properties, 83 fermentation, 88-9 occurrence and producing organisms,

83 patents for production of, 90 physical properties, 83 production, 87

Erwinia, 311, 313, 315, 318-21 Erwinia citreus, 321 Erwinia herbicoLa, 319, 320, 321 Escherichia, 262, 289, 310 Escherichia coLi, 139-41, 143, 174, 176,

180, 207, 209, 226, 232, 235, 238, 241, 243, 261, 274, 276, 287, 289, 291, 318, 320-2, 337, 341, 379

Escherichia freundii, 227 Eubacterium saburreum, 169 EugLena, 97 EugLena graciLis, 95, 276 EugLena graciLis Z, 95, 97-100, 98

Famesylpyrophosphate (FPP)-C15 , 86 Fermentation, 54

carotenoid-producing micro-organisms specifically designed for pigment

Index

Fermentation--contd. production, 54-65

micro-organisms designed for applications other than pigment production, 65-9

Flavin adenine dinuleotide (FAD), 164 FLavobacterium, 57, 73, 127, 129-33,

226,262, 311 Flavobacterium meningosepticum, 127-9 Flavobacterium soLare, 258 Fungal gibberellins. See Gibberellins Fungi, 60, 66

blue illumination, 31 carotenoid composition, 28-30 carotenoid production, 27-42 chemical stimulation, 32-3 composition of growth medium, 51 culture conditions, 32 genetical stimulation, 34-6 growth medium, 55 industrial production, 36-8 inhibitors, 55 photocarotenogenesis,31 physical factors, 51 sexual interaction, 31 sources of xanthophylls, 57

Fusarium, 89, 151 Fusarium aquaeductuum, 28 Fusarium heterosporum, 390 Fusarium moniLiforme, 383, 390, 398,

400 Fusarium oxysporum, 390

Geotrichum candidum, 102 GibbereLLa fujikuroi, 28, 30, 36, 383,

385, 390, 391, 393, 394, 396, 397, 399,401,403,404,413,414,417, 422

GibbereLLa moniLiforme, 390 Gibberellins, 7, 383-429

applications, 418-22 assay methods, 408-13 bioassays, 410-12 biological properties, 384, 413-16 biosynthesis and regulation, 391-6 biosynthetic pathway, 394 chemical properties, 385-9 chemical synthesis, 416-18 economics, 418-22 fermentation and recovery flow chart,

407 fermentation process, 398-404

economics of, 403-4

Gibberellins--contd. ftuorimetric analysis, 409 formulations, 418-22 genetics, 396-7 historical survey, 383-5 immunoassays,412-13 labelled analogues, 417 main routes in conversion of, 416, 417 physical properties, 385-9 plant growth regulator, 383 producing micro-organisms, 389-91 product recovery and purification,

404-8 screening, 389-91 strain improvement, 396-7 structural modification, 417-18 structure of, 385 total synthesis, 416-17

Gluconobacter, 309, 310, 321 Gluconobacter melanogen us , 310 Gluconobacter oxydans, 310, 311, 317 Gluconobacter oxydans subsp.

suboxydans, 304 Gluconobacter roseus, 101 Gluconobacter suboxydans, 317, 321,

322 D-Glucose, 312-16 Green algae, 56-7, 59 Growth factors, 2, 3

basic source of, 7 production and application, 7-10 technical function, 6-7

L-Gulonic acid, 312

Hansenula, 84 Hansenula jadinii, 340 Helminthosporium, 169 Hexuronic acid, 299 High performance liquid

chromatography (HPLC), 326, 338

High voltage paper electrophoresis, 338 Hydrocarbon assimilating bacteria, 52 Hydrocarbon-utilizing bacteria, 64 Hydroxocobalamin, 271 3-hydroxy-3-methylglutaric acid (HMG),

86 R-tJ-hydroxyisobutyric acid (tJ-HIBA),

101 Hyphomicrobium X, 379

L-Idonic acid, 311-12

Index

International units (IV), 4 Isopentenyl pyrophosphate (IPP), 86 5-Keto-D-gluconic acid, 311-12 2-Keto-L-gulonic acid, 304-16, 321-2 Klebsiella, 310

435

Lactobacillus, 143, 151, 174 Lactobacillus arabinosus. 258 Lactobacillus bulgaricus, 199, 216, 286,

287 Lactobacillus casei, 214, 163, 222, 223 Lactobacillus helveticus, 209, 214 Lactobacillus lactis, 258,272 Lactobacillus leichmanii, 272, 276 Lactobacillus plantarum, 214, 243, 247 Lactuca sativa L. cv. Arctic, 412 Leuconostoc mesenteroides, 163 y-Linolenic acid, 109-11 Linseed oil, 117-19 Lycopene, 54-5

Menaquinones, 123-33 biosynthesis of, 125 chemistry of, 124 fermentative production of, 125 menaquinone-4, 131-3 menaquinone-5,128-9 menaquinone-6,127-8 screening of micro-organism, 125-8

Methanobacillus, 262 Methionine, 351 Methylotrophic yeast, 344-9 Methyltrophs, 66-7 Micrococcus, 228, 310, 316 Micrococcus denitrificans, 177 Micrococcus glutamicus, 286 Micrococcus roseus, 64-5, 70 Micromucor, 110, 111 Micromucor ramanniana, 111 Micromucor vinacea, 111 Micro-organisms, 1-11 Monascus, 6 Monocyclic ketocarotenoids, 58 Mortierella, 110-13, 117, 118 Mortierella alpina, 114-117, 119 Mortierella elongata, 114 Mortierella isabellina, 110 Mucor ambigus, 111 Mucorales, 109, 111 Mutants

blocked,53 enhanced pigment levels, with, 53-4

436

Mutation to non-production of undesirable carotenoids, 53

Mutational biosynthesis, 53 Mycobacterium, 52, 262 Mycobacterium brevicale, 64 Mycobacterium lactiola, 64 Mycobacterium phlei, 54, 57 Mycobacterium smegmatis, 58, 175, 258 Mycoderma, 84 Mycoplana, 238, 240

NAD,376 NADH,377 NADP, 376 NADPH,377 Nadsonia, 84 Nematospora, 84 Neurospora, 286, 287 Neurospora crassa, 28, 38, 207, 391, 414 Nicotinamide cofactors, 376-7 Nocardia asteroides, 210 Nocardia gardneri, 262 Nocardia rugosa, 262, 266, 272 S-Nucleosidylhomocysteines, 368

Ochromonas malhamensis, 274 Oenothera biennis, 106 Oenothera lamarckiana, 106 Orotic acid, 285-97

application, 294-5 assay, 287 biosynthesis and regulation, 289-92 chemical synthesis, 294 chemistry, 286-7 fermentation, 292-3 historical background, 285-6 isolation, 294 producing micro-organisms, 287-9,

288 properties of, 287 purification, 294 structure of, 286

Orotidine properties of, 287 structure of, 286

Oryza sativa L., 411

Pachysolen tannophilus, 175 Pantetheine (Pantethine), 199,216 Pantoic acid, 207

Index

Pantothenic acid, 199-219 assay methods, 214-16 biosynthesis, 205-9 chemical production, 209-12 chemistry of, 199-205 control mechanisms for biosynthesis,

209 microbial production, 209-12

Pantothenyl alcohol, 205, 216 Paper chromatography, 338 Paprika, 27 Paracoccus denitrificans, 379 Penicillium, 28 Penicillium brevi-compactum, 175, 180,

181 Penicillium chrysogenum, 151 Penicillium notatum, 414 Penicillium westlinghi, 84 Phaeolactylium, 119 Phaffia rhodozyma, 28, 30, 38, 60, 61,

70,73 growth medium, 60 use of cheap media, 61

Phlebodium dictyocallis, 169 5'Phosphate (FMN), 164 4' -Phosphopantetheine, 214 Photocarotenogenesis, 31 Photoinduction, 52 Photosynthetic bacteria, 52, 67-9 Phycomyces, 28, 30 Phycomyces blakesleanus, 28, 30, 32-6,

38, 143 Phylloquinone, 133 Pigments

production, 54-65 technical function, 6-7

Pisum sativum L., 411 Plant growth-regulators, 7 Polyunsaturated fatty acids (PUFAs),

105-21 biosynthesis, 108-9 naturally occurring, 105-7 production, 109-19 sources of, 109

Propionibacterium, 262 Propionibacterium freudenreichii, 267-

9,271-2 Propionibacterium sherman ii, 232, 262,

266-9, 271-2, 275 Protaminobacter ruber, 67, 379 Protective factor X, 231 Proteus, 262, 311 Proteus mirabilis, 171 Proteus morgan ii, 207

Protomonas extorguens, 65 Provitamin A. See p-Carotene Provitamin D 3 , 89 Pseudo-gluconobacter

saccharoketogenes, 311 Pseudomonas, 262, 269, 309-11, 316,

366 Pseudomonas aeruginosa, 310, 312, 322 Pseudomonas albosesamae, 313 Pseudomonas chlororaphis, 312 Pseudomonas denitrificans, 262, 264,

265,267, 269-71 Pseudomonas extorquens, 65 Pseudomonas jluorescens, 311, 312 Pseudomonas graveolens, 238 Pseudomonas maltophilia, 207, 209 Pseudomonas mildenbergii, 311, 312 Pseudomonas pseudomallei, 312 Pseudomonas putida, 310, 322, 367, 101 Pseudomonas reptilivora, 175, 180, 181,

182, 185 Pseudomonas sesami, 313 Pseudomonas viridiflava, 309 Pyridoxal,228 Pyridoxal-P, 221, 223, 227-8 Pyridoxamine 5' -phosphate, 221 Pyridoxamine-P, 221, 223, 225 Pyridoxine, 221, 225, 227-8 Pyridoxine glucoside, 229 Pyridoxine-P, 223, 225, 227-8 Pyrimidine nucleotides, 289-91 Pyrroloquinoline quinone (PQQ), 377-9

Radioimmunoassay (RIA), 412 Red peppers, 27 Red yeasts, 66 Reichstein-Grussner synthesis, 323 Retrocarotenoids, 65 Rhizobium, 262 Rhizobium lequminosarum, 175 Rhizobium melioti, 171, 174,264 Rhizobium tritolii, 264 Rhodobacter capsulatus, 54, 67-9 Rhodococcus erythropolis, 210, 211 Rhodococcus maris, 64 Rhodopseudomonas capsulatus, 67 Rhodopseudomonas sphaeroides, 275 Rhodospirillum rubrum, 275, 379 Rhodosporidium, 66 Rhodosporidium diobovatum, ·28 Rhodotorula, 66 Rhodotorula aurea, 28 Rhodotorula glutinis, 235

Index

Rhodoturula minuta, 28, 210 Rhodoturula mucilaginosa, 66 Rhodotorula rubra, 28 Rhodoturula gracilis, 84 Rhodoxanthin, 65 Riboflavin, 149-66

applications, 164 bioassay methods, 163-4 biological properties, 164 biosynthesis and regulation, 152-3 chemical properties, 150 chemical synthesis, 164

437

economics, 164 fermentation/bioconversion process,

154-61 formulae of, 168 formulations, 164 history, 149 physical properties, 150 producing micro-organisms, 151-2 product recovery and purification,

160-3 screening, 151-2 strain improvement, 153-4 structure of, 149

D-Ribose, 167-97 application, 188 assay methods, 185 biosynthesis, 172-5 chemical properties, 171-2 chemical synthesis, 185-8 economy, 188 fermentation, 180-4 fermentation time course, 184 formation from carbon sources, 183 formation from D-glucose, 178 formula of, 172 metabolism, 172-5 nitrogen sources for, 183 occurrence in nature, 169-71 physical properties, 171-2 producing micro-organisms, 175-80 producing tkt mutants

improvement of, 177-9 pleiotropic properties, 179-80

recovery and purification, 184-5 Rye ergot, 83

Saccharomyces, 84-6 Saccharomyces carlsbergensis, 84, 89,

139,376 Saccharomyces cerevisiae, 38, 85, 86,

89, 92, 102, 140, 141, 143, 207,

438

Saccharomyces cerevisiae--contd. 223,228,234,243,247,341,362, 376,379

Saccharomyces fermentati, 89 Saccharomyces fragilis, 151 Saccharomyces sake, 363-5, 369, 376 Saccharomyces uvarum, 89, 214, 376 Saffron, 27 Salmonella typhimurium, 139, 141, 142,

169, 174, 176, 206, 289, 291 Sarcina, 228 Serratia, 262, 310 Serratia marinorubra, 287 Skeletonema, 119 Sphaceloma manihoticola, 390-1, 394,

413 Spongiococcum excentricum, 56 Staphylococcus aureus, 143,316 Staphylococcus hyicus subsp. hyicus, 175 Sterols

biosynthesis and regulation, 86-8 production, media and environmental

factors, 85-6 Streptococcus, 262 Streptococcus faecalis, 162 Streptococcus faecalis R, 222, 223 Streptococcus liquefaciens, 162 Streptococcus zymoge, 162 Streptomyces, 216, 246 Streptomyces albidoflavus, 262 Streptomyces alboniger, 169 Streptomyces antibioticus, 258, 262 Streptomyces aureofaciens, 260-1, 262 Streptomyces aureus, 262 Streptomyces chrestomyceticus subsp.

rubescens, 55 Streptomyces chrestomyceticus var.

aurantioideus, 57 Streptomyces farinosus, 262 Streptomyces fradiae, 262 Streptomyces griseus, 258, 262 Streptomyces hygroscopicous, 180 Streptomyces mediolani, 57 Streptomyces olivaceus, 262 Streptomyces roseochromogenes, 258,

262 Streptomyces showdoensis, 289 Streptomyces vinaceus, 262

Tagetes tenuifolia, 27 Tetrahymena pyriformis, 164 Thiamine, 137-48

assay methods, 143-4

Index

Thiamine--contd. biological properties, 144 biosynthesis, 140-2 chemical assays, 143-4 chemical properties, 137-8 chemical synthesis, 145 enzyme assays, 144 metabolism, 144 microbiological assays, 143 physical properties, 137-8 physiological functions, 144-5 producing micro-organisms, 138-40 regulation, 142 structure of, 137

Thiamine monophosphate (TMP), 137-8 Thiamine pyrophosphate (TPP), 137-8 Thiamine triphosphate (TIP), 137-8 Tocopherol, fermentative production of,

97-100 a-Tocopherol

isolation of, 98 production of, 98-100

Tocopherols, 95 producing micro-organisms, 97

Transketolase mutants, 175-80 Trichoderma spp., 89 Trimethylhydroquinone, 103

UMP, 291-2 Ustilago violacea, 28, 30, 36

Verticillium agaricinum, 28 Vibrio parahaemolyticus, 171 Vitamin A, 2 Vitamin antagonists, 5 Vitamin B, 2, 137-48 Vitamin B), 2 Vitamin B2 , 149-66 Vitamin Bs , 199-219 Vitamin B6 , 221-9

assayof,221-3 biodegradation of, 223 biosynthesis, 223-6 chemistry of, 221-3 de novo synthesis and bioconversion,

226-9 derivatives, 228 fermentation, 226 metabolism, 223-6 structure of, 222

Vitamin B12, 66, 67, 257-84 absorption, 276-7

Vitamin B12-contd. analogues of, 259 applications, 277-9 bioassay methods, 272-5 biochemical mechanisms, 275-6 biological properties, 275-7 biosynthesis, 263-5 chemical properties, 259-61 chemical structure, 259 deficiency, 277-8 derivatives, 271-2 economics, 277-9 elimination, 276-7 forms and grades of, 278 formulations, 277-9 historical background, 257-9 improvement of producing strains,

265-6 isolation of, 272 metabolism, 276-7 nutritional-dependent deficiency, 278 pharmaceutical use of, 278 physical properties, 259-61 producing micro-organisms, 261-3 production of, 266-71 regulation, 263-5 screening, 261-3 transport, 276-7

Vitamin B12 coenzyme, 260-1, 271-2, 275

Vitamin B12a, 259 Vitamin B13 , 285-97 Vitamin C, 2, 5, 299-334 Vitamin compounds, 2 Vitamin content, 4 Vitamin 0, 2, 81-93

application and economics, 92 chemical nature, 81 formation of, 89-91 metabolic transformations, 91-2

Index

Vitamin O2 , 5, 81, 92 Vitamin 0 3 ,5,81,92 Vitamin deficiencies, 1 Vitamin E, 95-104

assayof,96

439

bioconversion reactions for synthesis of,100-2

biosynthesis of, 97 chemistry of, 96

Vitamin H, 231 Vitamin K, 2,123,133

chemical structure of, 124 Vitamin Kb 133 Vitamin K 2 , 123-33 Vitamin nomenclature, 2-4 Vitamin PP, 2 Vitamins

basic source of, 7 fat-soluble, 5 history of, 1 nutritional, clinical and physiological

aspects of, 6 physiological functions, 4-6 production and application, 7-10 research history, 1-2 technical function, 6-7

Xanthomonas, 262, 310, 311 Xanthomonas translucens, 312 Xanthophylls, 55-8 X-ray crystallography, 259

Yeast cell reaction method, 339-41 Yeast cell regeneration, 341-3

Zea mays L., 411