products of secondary metabolism:wiley encyclopedia

BiotechnologySecond Edition Volume 7

Products of Secondary Metabolism

VCH

A Wiley company

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BiotechnologySecond EditionFundamentalsVolume 1 Biological Fundamentals Volume 2 Genetic Fundamentals and Genetic Engineering Volume 3 Bioprocessing Volume 4 Measuring, Modelling, and Control

Special TopicsVolume 9 Enzymes, Biomass, Food and Feed Volume 10 Special Processes Volumes l l a and b Environmental Processes Volume 12 Legal, Economic and Ethical Dimensions

ProductsVolume 5 Recombinant Proteins, Monoclonal Antibodies, and Therapeutic Genes Volume 6 Products of Primary Metabolism Volume 7 Products of Secondary Metabolism Volume 8 Biotransformations

A Multi-Volume Comprehensive Treatise

Second, Completely Revised Edition Edited by H.-J. Rehm and G. Reed in cooperation with A. Piihler and P. StadlerVolume 7

Biotechnology

Products of Secondary MetabolismEdited by H. Kleinkauf and H. von Dohren

VCH

A Wiley company

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Series Editors: Prof. Dr. H.-J. R eh m Institut f u r Mikrobiologie Universitat Munster CorrensstraBe 3 D-48149 Munster

Dr. G. R e e d 1914 N. Prospect Ave. #61 Milwaukee, WI 53202-1401USA Prof. Dr. P. J. W. Stadler Bayer AG Verfahrensentwicklung Biochemie Leitung Friedrich-Ebert-StraBe 217 D-42096 Wuppertal

FRGProf. Dr. A. Piihler Biologie VI (Genetik) Universitat Bielefeld

Volume Editors: Prof. Dr. H. Kleinkauf Dr. H. von D o h r en Institut f u r Biochemie Technische Universitat Franklin-StraBe 29 A-10587 Berlin Germany

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This book was carefully produced. Nevertheless, authors, editors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate.

Executive Editor: Dr. Hans-Joachim Kraus Editorial Director: Karin Dembowsky Production Manager: Hans-Jochen Schmitt Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data: A catalogue record for this book is available from the British Library

Die Deutsche Bibliothek - CIP-Einheitsaufnahme Biotechnology : a multi volume comprehensive treatise I ed. by H.-J. Rehm and G. Reed. In cooperation with A. Piihler and P. Stadler. 2., completely rev. ed. -VCH. ISBN 3-527-28310-2 (Weinheim ...) NE: Rehm, Hans J. [Hrsg.] Vol. 7. Products of secondary metabolism I ed. by H. Kleinkauf and H. von Dohren - 1997 ISBN 3-S27-28317-X OVCH Verlagsgesellschaft mbH, D-69451 Weinheim (Federal Republic of Germany), 1997 Printed on acid-free and chlorine-free paper.All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form -by photoprinting, microfilm, or any other means-nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. Composition and Printing: Zechnersche Buchdruckerei, D-67330 Speyer. Bookbinding: J. SchSiffer, D-67269 Griinstadt. Printed in the Federal Republic of Germany

Preface

In recognition of the enormous advances in biotechnology in recent years, we are pleased to present this Second Edition of Biotechnology relatively soon after the introduction of the First Edition of this multi-volume comprehensive treatise. Since this series was extremely well accepted by the scientific community, we have maintained the overall goal of creating a number of volumes, each devoted to a certain topic, which provide scientists in academia, industry, and public institutions with a well-balanced and comprehensive overview of this growing field. We have fully revised the Second Edition and expanded it from ten to twelve volumes in order to take all recent developments into account. These twelve volumes are organized into three sections. The first four volumes consider the fundamentals of biotechnology from biological, biochemical, molecular biological, and chemical engineering perspectives. The f next four volumes are devoted to products o industrial relevance. Special attention is given here to products derived from genetically engineered microorganisms and mammalian cells. The last four volumes are dedicated to the description of special topics. The new Biotechnology is a reference work, a comprehensive description of the state-of-the-art, and a guide to the original literature. It is specifically directed to microbiologists, biochemists, molecular biologists, bioengineers, chemical engineers, and food and pharmaceutical chemists working in industry, at universities or at public institutions. A carefully selected and distinguished Scientific Advisory Board stands behind the

series. Its members come from key institutions representing scientific input from about twenty countries. The volume editors and the authors of the individual chapters have been chosen for their recognized expertise and their contributions to the various fields of biotechnology. Their willingness to impart this knowledge to their colleagues forms the basis of Biotechnology and is gratefully acknowledged. Moreover, this work could not have been brought to fruition without the foresight and the constant and diligent support of the publisher. We are grateful to VCH for publishing Biotechnology with their customary excellence. Special thanks are due to Dr. HansJoachim Kraus and Karin Dembowsky, without whose constant efforts the series could not be published. Finally, the editors wish to thank the members of the Scientific Advisory Board for their encouragement, their helpful suggestions, and their constructive criticism. H.-J. Rehm G. Reed A. Puhler P. Stadler

Scientific Advisory Board

Pro$ Dr. M. J. BekerAugust Kirchenstein Institute of Microbiology Latvian Academy of Sciences Riga, Latvia

Pro$ Dr. T. K. GhoseBiochemical Engineering Research Centre Indian Institute of Technology New Delhi, India

Pro$ Dr. J. D. BuLockWeizmann Microbial Chemistry Laboratory Department of Chemistry University of Manchester Manchester, UK

Pro$ Dr. I. GoldbergDepartment of Applied Microbiology The Hebrew University Jerusalem, Israel

Pro$ Dr. C. L. CooneyDepartment of Chemical Engineering Massachusetts Institute of Technology Cambridge, MA, USA

Pro$ Dr. G. GomaDepartement de GCnie Biochimique et Alimentaire Institut National des Sciences AppliquCes Toulouse, France

Pro$ Dr. H. W. DoelleDepartment of Microbiology University of Queensland St. Lucia, Australia

Sir D. A. HopwoodDepartment of Genetics John Innes Institute Norwich, UK

Prof Dr. J. DrewsF. Hoffmann-La Roche AG Basel, Switzerland

Pro$ Dr. E. H. HouwinkOrganon International bv Scientific Development Group Oss. The Netherlands

Pro$ Dr. A. FiechterInstitut fur Biotechnologie Eidgenossische Technische Hochschule Zurich, Switzerland

Pro$ Dr. A. E. HumphreyCenter for Molecular Bioscience and Biotechnology Lehigh University Bethlehem, PA, USA

VIII

Scientific Advisory Board

Prof. Dr. I. KarubeResearch Center for Advanced Science and Technology University of Tokyo Tokyo, Japan

Prof. Dr. K. SchiigerlInstitut fur Technische Chemie Universitat Hannover Hannover, Germany

Prof. Dr. M . A. LachanceDepartment of Plant Sciences University of Western Ontario London, Ontario, Canada

Prof. Dr. P. SensiChair of Fermentation Chemistry and Industrial Microbiology Lepetit Research Center Gerenzano, Italy

Prof. Dr. Y. LiuChina National Center for Biotechnology Development Beijing, China

Prof. Dr. Y. H. TanInstitute of Molecular and Cell Biology National University of Singapore Singapore

Prof. Dr. J. F. MartinDepartment of Microbiology University of Leon Leon, Spain

Prof. Dr. D. ThomasLaboratoire de Technologie Enzymatique UniversitC de Compibgne Compibgne, France

Prof. Dr. B. MattiassonDepartment of Biotechnology Chemical Center University of Lund Lund, Sweden

Prof. Dr. W . VerstraeteLaboratory of Microbial Ecology Rijksuniversiteit Gent Gent, Belgium

Prof. Dr. M . RoehrInstitut fur Biochemische Technologie und Mikrobiologie Technische Universitat Wien Wien, Austria

Prof. Dr. E.-L. WinnackerInstitut fur Biochemie Universitat Munchen Munchen, Germany

Prof. Dr. H. SahmInstitut fur Biotechnologie Forschungszentrum Julich Julich, Germany

Contents

H. von Dohren, H. KleinkaufGeneral Aspects of Secondary Metabolism 1 H. von Dohren, U. Grafe Regulation of Bacterial Antibiotic Production 57 K. Chater, M. Bibb Screening of Novel Receptor-Active Compounds of Microbial Origin 107 H. Tanaka, S. Omura Microbial Lipids 133 C. Ratledge Microbial Siderophores 199 G. Winkelmann, H. Drechsel Advances in the Molecular Genetics of PLactam Antibiotic Biosynthesis 247 P. 15.Skacrud, T. Schwecke, H. v. Liempt, M. B. Tobin Peptide Antibiotics 277 H. Kleinkauj H. von Dohren Lantibiotics 323 R. Jack, F. Gotz, G. Jung

Introduction

9 Glycopeptide Antibiotics (Dalbaheptides) 369 G. Lancini, B. Cavalleri 10 Aminoglycosides and Sugar Components in Other Secondary Metabolites 397 W. Piepersberg, J. Distler 11 Products from Basidiomycetes 489 G. Erkel, T. Anke 12 Cyclosporins: Recent Developments in Biosynthesis, Pharmacology and Biology, and Clinical Applications 535 J. Kallen, V. Mikol, V. F. J. Quesniaux, M. D. Walkinshaw,E. Schneider-Scherzer, K. Schorgendorfer, G. Weber, H. Fliri 13 Secondary Products from Plant Cell Cultures 593 J. Berlin 14 Biotechnical Drugs as Antitumor Agents 641 U. Grafe, K . Dornberger, H.-P. Saluz

Index 707

Contributors

Prof. Dr. Timm AnkeLehrbereich Biotechnologie Universitat Kaiserslautern Postfach 3049 D-67618 Kaiserslautern Germany Chapter I1

Prof. Keith ChaterJohn Innes Centre Norwich Research Park Colney Lane Colney, Norwich NR4 7UH UK Chapter 2

Dr. Jochen BerlinGesellschaft fur Biotechnologische Forschung Mascheroder Weg 1 D-38124 Braunschweig Germany Chapter 13

Jurgen DistlerBergische Universitat G H Mikrobiologie - FB 19 Gauss-StraSe 20 D-42097 Wuppertal Germany Chapter 10

Dr. Mervin BibbJohn Innes Centre Norwich Research Park Colney Lane Colney, Norwich NR4 7UH UK Chapter 2

Dr. Hans von DohrenInstitut fur Biochemie Technische Universitat Franklin-Str. 29 D-10587 Berlin Germany Chapters I, 7

Dr. Bruno CavalleriMMDRI - Lepetit Research Center Via R. Lepetit, 34 1-21040 Gerenzano (Varese) Italy Chapter 9

Dr. Klausjiirgen DornbergerHans-Knoll-Institut fur Naturstoff-Forschung Bereich Naturstoffchemie BeutenbergstraBe 11 D-07745 Jena Germany Chapter 14

XI1

Contributors

Dr. Hartmut DrechselMikrobiologie und Biotechnologie Universitat Tubingen Auf der Morgenstelle 1 D-72076 Tubingen Germany Chapter 5

Prof. Dr. Gunter JungUniversitat Tubingen Institut fur Organische Chemie Auf der Morgenstelle 18 D-72076 Tubingen Germany Chapter 8

Dr. Gerhard ErkelLehrbereich Biotechnologie Universitat Kaiserslautern Postfach 3049 D-67618 Kaiserslautern Germany Chapter I1

Dr. Jorg KallenSandoz Pharma Ltd. Preclinical Research CH-4002 Basel Switzerland Chapter I2

Dr. Hans FliriRhBne Poulonc Rorer S.A. Centre de Recherche de Vitry-Alfortville 13, quai Jules Guesde F-94403 Vitry-sur-Seine Cedex France Chapter 12

Prof. Dr. Horst KleinkaufInstitut fur Biochemie Technische Universitat Franklin-Str. 29 D-10587 Berlin Germany Chapter 7

Dr. Friedrich GotzLehrstuhl fur Mikrobielle Genetik Universitat Tubingen Auf der Morgenstelle 18 D-72076 Tubingen Germany Chapter 8

Dr. Giancarlo LanciniMMDRI - Lepetit Research Center Via R. Lepetit, 34 1-21040 Gerenzano (Varese) Italy Chapter 9

Prof. Dr. Udo GrafeHans-Knoll-Institut fur Naturstoff-Forschung Bereich Naturstoffchemie BeutenbergstraSe 11 D-07745 Jena Germany Chapters I, 14

Dr. Henk van LiemptDRL, BT-FDG SudstraSe 125 D-53175 Bonn Germany Chapter 6

Dr. Ralph JackUniversitat Tubingen Institut fur Organische Chemie Auf der Morgenstelle 18 D-72076 Tubingen Germany Chapter 8

Dr. Vincent MikolSandoz Pharma Ltd. Preclinical Research CH-4002 Basel Switzerland Chapter 12

Contributors

XI11

Prof. Dr. Satoshi OmuraSchool of Pharmaceutical Sciences Kitasato University The Kitasato Institute 9-1, Shirokane 5-chome Minato-ku, Tokyo 108 Japan Chapter 3

Dr. Kurt SchorgendorferBiochemie GmbH A-6330 Kufstein-Schaftenau Austria Chapter 12

Prof. Dr. Wolfgang PiepersbergBergische Universitat G H Mikrobiologie - FB 19 Gauss-Strafie 20 D-42097 Wuppertal Germany Chapter 10

Dr. Torsten SchweckeInstitute of Biochemistry University of Cambridge Tennis Court Road Cambridge, CB2 1QW UK Chapter 6

Dr. Valkrie F.J. QuesniauxSandoz Pharma Ltd. Preclinical Research CH-4002 Basel Switzerland Chapter 12

Dr. Paul L. SkatrudInfectious Diseases Research Eli Lilly and Company Lilly Corporate Center Indianapolis, IN 46285 USA Chapter 6

Prof. Colin RatledgeThe University of Hull Department of Applied Biology Hull HU6 7RX UK Chapter 4

Dr. Haruo TanakaSchool of Pharmaceutical Sciences Kitasato University The Kitasato Institute Minato-ku, Tokyo 108 Japan Chapter 3

Prof. Dr. habil. Hans-Peter SaluzHans-Knoll-Institut fur Naturstoff-Forschung Bereich Naturstoffchemie BeutenbergstraSe 11 D-07745 Jena Germany Chapter 14

Dr. Matthew B. TobinInfectious Diseases Research Eli Lilly and Company Lilly Corporate Center Indianapolis, IN 46285 USA Chapter 6

Dr. Elisabeth Schneider-ScherzerBiochemie GmbH A-6330 Kufstein-Schaftenau Austria Chapter 12

Dr. Malcolm D. WalkinshawSandoz Pharma Ltd. Preclinical Research CH-4002 Basel Switzerland Chapter 12

XIV

Contributors

Dr. Gerhard WeberBiochemie GmbH A4330 Kufstein-Schaftenau Austria Chapter 12

Prof. Dr. Giinther WinkelmannMikrobiologie und Biotechnologie Universitat Tubingen Auf der Morgenstelle 1 D-72076 Tubingen Germany Chapter 5

BiotechnologyEdited by H.-J. Rehm and G. Reed OVCH Verlagsgesellschaft mbH, 1997

This volumes provides an overview of secondary metabolites illustrating most aspects of their discovery, formation, exploitation, and production. Compared to the first edition the focus when has clearly shifted towards the molecular genetic background of the producing organisms. These efforts serve not only our understanding of the production processes to permit improvements by genetic manipulations, but also promote our appreciation of the environmental significance of secondary metabolites. The term secondary metabolite has been discussed widely, and a shift in perception took place in the last years. From a playground of nature leading to mostly disparable products ideas focus now on special purpose products promoting evolutionary advantages. This shift is connected to the impressive elucidation of the genetics of multistep synthetic processes of secondary metabolite formation. Genes encoding biosynthetic reaction sequences have been found clustered together with resistance or export genes and are under the control of specific signals. Biosynthetic functions or unit operations reside on modules, and these modules in their functional protein state interact to assure the fidelity of the multistep processes. The genetic burden for many of these processes seems remarkable, and genes assembled from modules often display sizes of 10 to more than 45 kilobases. Since some of the now established microbial genomes are devoid of such multistep pathways, their unique placement in other genomes indicates important functions for their producers.

Still largely unconnected to the background of their producers secondary metabolites generally are high-value compounds established mainly in pharmacology, veterinary medicine, agriculture, and biochemical and medical research. The introductory chapter points to product fields and to the genetic investigation of biosynthetic unit operations. Regulatory mechanisms are then considered in the most advanced fields of the prokaryotes. As the central field of present drug discovery approaches target-based screenings are discussed. Compound groups considered are lipids siderophores, aminoglycosides, and peptides (p-lactams, dalbaheptides, cyclosporins, lantibiotics). Producer groups presented are basidiomycetes and plant cells. As a target group antitumor drugs are evaluated. An updated chapter on macrolides as secondary metabolites including reprogramming strategies will be included in Volume 10 of the Second Edition of Biofechnofogy(see also Volume 4 of the First Edition). Further chapters to be consulted are especially on biopolymers and surfactants (Volume 6), on the overproduction of metabolites and the treatment of producer organisms like bacilli, streptomycetes and filamentous fungi (Volume 1) as well as on reactor modeling (Volume 3). We thank our colleagues for their valuable contributions, the publisher for their patience and cooperativity, and the series editors for many helpful suggestions. Berlin, March 1997 Hans von Dohren Horst Kleinkauf

BiotechnologyEdited by H.-J. Rehm and G. Reed OVCH Verlagsgesellschaft mbH, 1997

1 General Aspects of Secondary Metabolism

HANS VON DOHRENBerlin, Germany

UDOGRAFEJena, Germany1 Introduction: The Importance of Secondary Metabolites as Drugs 2 2 Secondary Metabolism, an Expression of Cellular and Organismic Individuality 11 2.1 Roles of Secondary Metabolites in Producing Organisms 11 2.2 Regulation of Microbial Secondary Metabolism 17 2.2.1 Genetic Organization of Product Formation 17 2.2.2 Regulatory Mechanisms 23 2.2.3 Genetic Instability 26 2.2.4 Developmental Processes 27 2.2.5 The A-Factor and the Signal Cascade of Cytodifferentiation in Streptomyces 27 2.2.6 Overproduction of Microbial Secondary Metabolites and Precursor Pools 29 2.2.7 Biotechnical Production of Secondary Metabolites 31 3 The Biosynthetic Pathways 31 3.1 Precursors and the Main Biosynthetic Pathways 31 3.2 Secondary Metabolites Formed through Biosynthetic Modifications of a Single Precursor 31 3.3 Polyketides 32 3.4 Terpenes 35 3.5 Sugar-Derived Oligomeric Structures 35 3.6 Oligo- and Polypeptides 36 3.7 Biosynthetic Modifications of Structures and Precursor-Directed Biosyntheses 37 4 Variability of Structures of Secondary Metabolites 38 4.1 Secondary Metabolites as Products of Biological Unit Operations 38 4.2 Structural Classifications of Secondary Metabolites 38 5 Future Perspectives: New Products of the Secondary Metabolism 40 6 References 41

2


crease in resistant nosocomial and opportunistic pathogens (particularly dangerous to immunosuppressed AIDS and tumor patients) requires both improvement of known drugs and search for new drugs (GRAFE, 1992; LANCINI LORENZETTI, and 1993; HUTCHINSON, 1994). Microbial products such as doxorubicin, Today, bioactive secondary metabolites of microorganisms and of plants, and their syn- bleomycin, and mitomycin C are indispensathetic derivatives as well, are among the most ble as cancerostatics (Fox, 1991). The same is frequently used therapeutics in human and true for plant metabolites such as the vinca alveterinary medicine (Scrip, 1993). The inven- kaloids, taxol, and their chemical derivatives tion of antibiotic therapy contributed greatly which exert excellent antitumor activity by into the successful control of most of the epi- teraction with the cellular mitotic system and demic infectious diseases and even promoted (NOBLE, 1990 Fox, 1991; HEINSTEIN 1994 POTIERet al., 1994). their disappearance. Moreover, it contributed CHANG, However, even the non-therapeutic fields to the general increase in the lifespan of man, not only in industrialized countries. New ap- of application, such as in animal husbandry plications for bioactive biotechnical products and plant protection, contributed to a high in medical care like their use as immunosup- degree to the continuing interest in secondary pressants or antiatherosclerotics, and as ani- metabolite production. Last but not least, natmal growth promoters and pesticides in agri- ural products of biotechnical and agricultural culture rendered research on new secondary origin play an important role as biochemical metabolites an apparently endless story (SAN- tools in molecular biology and in the investiGLIER and LARPENT, 1989; ComitC Editorial, gation of cellular functions. 1992; LANCINI LORENZE-ITI, and 1993; VINMore than loo00 antibiotics and similar bioactive secondary metabolites have been ING and STUTTARD, 1995). isolated so far from microbes, and a comparaIn the past, natural products supplied 5 4 % of the annual increase in the worlds total bly higher number of drugs was derived from pharmaceutical market. The list of the 25 plants and even from animals (see, e.g., maworldwide best-selling drugs for application rine tunicates, molluscs, toxic insects, snakes, in humans in 1992 includes a series of drugs and toads) (BERDYet al., 1980; LAATSCH, of microbial origin which are used either in 1994). Approximately 500 new representatheir native structures or as chemical derivatives of low-molecular weight compounds are tives (see, e.g., Mevacor, Cefaclor or other published every year. In addition to this huge and still growing cephalosporins, Augmentin, Sandimmun) number of bioactive molecules, more than (Scrip, 1993). Many plant products, from digitalis glyco- 1OOOOO derivatives as representatives of some sides and neuroactive alkaloids to the pyre- few basic structures (e. g., p-lactams, macrothrines, serve as therapeutics for human dis- lides, aminoglycosides, tetracyclines, anthraeases and as agricultural agents (Comitk Edi- cyclines) were obtained by means of synthetic 1994). Irrespective torial, 1992). Sometimes, the experiences derivatizations (LAATSCH, made in folk medicine initiated the discovery of this plethora of drug molecules a little of new plant-derived antitumor drugs, anti- more than a hundred basic structures gained neuralgic, antihypertonic, antidepressant, in- practical importance. We owe much progress in the detection of secticidal, nematicidal, and other bioactive new drug structures to modern physicocompounds. Antiinfective chemotherapy once was the chemical approaches such as mass-spectromeclassical domain of biotechnical drug produc- try, high-field nuclear magnetic resonance tion due to the discovery of p-lactam antibiot- spectroscopy and X-ray diffractometry. Comics, such as penicillins, cephalosporins, clavu- pilations of the numerous structural data lanates, and carbapenems. Even today, the in- (BERDY et al., 1980; BYCROFT, 1988;

1 Introduction: The Importance of Secondary Metabolites as Drugs

I Introduction: The Importance of Secondary Metabolites as Drugs

3

LAATSCH, 1994) provide indispensable assistance in the identification of new drug molecules. Thus, the enormous number of already known metabolites from microbes and plants increased the detection and isolation of already known structures dramatically. A compilation of about 200 recently described products illustrates the current trends in screening efforts (Tab. 1). These have been published during the last two years. It is evident from these data that highly selective screens prevail and yet the majority of compounds originate from the classical Actinomycete pool. Rare bacteria and fungi, marine microorganisms and plants now have a significant share. It is obvious that well-known organisms again contribute with newly isolated substances to new, e. g., receptor targeted screens. Strategies of such screens are discussed in this volume in Chapter 3 by TANAKA and OMURA. The development of new drugs from natural sources is common practice of the pharmaceutical industry. 6000 to loo00 chemicals have to be tested in a given assay system to obtain one single compound suitable as a therapeutical agent (OMURA,1992; KROHN et al., 1993). No wonder that research and development for a new approved drug may cost up to one billion US$. In most cases, a new natural leading structure is intensively modified by chemical means to improve its activity and to reduce side effects. Chemistry is also extremely helpful if rather rare natural products occurring in low amounts or in organisms from sensitive ecological areas have been proposed as drugs. For example, 40000 yew trees, i. e., the whole population of Northern America, would be required to produce 25 kg of taxol, a new promising cancerostatic drug, and even this amount would not be sufficient to treat every cancer patient. Fortunately, taxol derivatives of similar activity (taxotere) can be obtained by chemical derivatization of taxoid metabolites which are obtainable in large quantities from the dried and leaves of European yews (HEINSTEIN CHANG,1994). Alternatively, cell cultures (ELLISet al., 1996) or endophytic fungi such as Pestalotiopsis microspora (STIERLEet al., 1994, 1995; STROBEL al., 1996) of Taxus et species could be exploited for production.

From the recently completed chemical synthesis of taxol it is evident that, as in bicyclic plactams, classical approaches cannot compete with natural producers. Instead, increasing attention is given to the recruitment of biocatalysts for certain key reactions in metabolite production. In addition, directed biosynthesis in microbial cultures (THIERICKE and ROHR, 1993), production of plant products in cell cultures (BERLIN, Chapter 14, this volume), and cell free in vitro systems of enzymatic synthesis and peptide and protein producing translation systems are considered as complementary methods in structure-function studies (ALAKHOV and VON DOHREN, unpublished data). Only 30% of the total developmental efforts have been spent to the search for a new drug. However, for the estimation of its efficacy and evaluation of safety often more than 50% are needed. Taking into account a quota of approximately 1:15000 for a hit structure, the challenges of modern pharmaceutical development become visible. In general, natural products seem to offer greater chances than synthetically derived agents. Hence, a great research potential is still dedicated to the discovery of new natural drugs and their biotechnical production. Classical strategies of drug development are being more and more supplemented by new biomedical approaches and ideas and by the use of genetically engineered microbes and cells as screening organisms (TOMODA OMURA, and 1990; ELDERet al., 1993). These tools initiated a renaissance in the search for new leading structures. New sources of bioactive material, such as marine organisms, and new microbes from ecological niches promoted the recent advances in the discovery of drugs (WILLIAMS and VICKERS 1986; RINEHART and SHIELD 1988; MONAGHAN TKACZ,1990; JACOB and and ZASLOFF,1994; JENSENand FENICAL, 1994) (Tab. 1). Present research activities were also stimulated by the discovery of block busters (Scrip, 1993) such as cyclosporin A (KAHAN, 1987), avermectins (CAMPBELL,1989), acarbose (MULLER, 1989), and monacolin (ENDO, 1979) in microbial cultures. A series of very promising new screening drugs (zaragozic acid, squalestatins) (HASUMI, 1993), erbstatin

4

I General Aspects of Secondary Metabolism

Tab. 1 Selected Natural Products Detected by Screening Efforts Published in 1995196 .

Compound Reference

Producing Organisms

Structural Type'

Selected Properties

Research Group Involved

Antimicrobial Drugs: Actinomycete Griseusin (unidentified) derivatives Streptomyces BE-24566B violaceus-niger Amicenomycin Streptomyces sp.

PK PK PK-GLYC PK, mod. PEP acyl AA PEP PK PEP PK PEP PK, mod. PK PK PEP + PK PK PK, mod. PK PK PK PEP ALK PEP

antibacterial antibacterial antibacterial antibacterial, MDR strains antibacterial antibacterial antibacterial antibacterial antibacterial antifungal antibacterial antibacterial antilegionella antibacterial antibacterial antibacterial antibacterial antibacterial antibacterial antibacterial antibacterial antibacterial antibacterial antibacterial antibacterial, cytotoxic antibacterial antibacterial antibacterial antibacterial

Institute of Microbial Chemistry Banyu Pharm. Co. Institute of Microbial Chemistry Yamanouchi Pharm. Co. and PT Kalbe Pharma Lepetit Institute of Microbial Chemistry Lepetit Hoechst Hans Knoll Institute and Univ. Tiibingen Bristol Myers Squibb Lepetit Cheil Foods & Chem. Inc and NIH Korea GBF GBF Sankyo Univ. Tiibingen and Hans Knoll Institute Lederle Institute of Microbial Chemistry RIKEN Institute of Microbial Chemistry Institute of Microbial Chemistry Institute of Microbial Chemistry Univ. Alcala Hans Knoll Institute Univ. Tlibingen, Univ. Gattingen, Hans Knoll Institute Univ. Lund and Univ. Kaiserslautern Panlabs Bristol Myers Squibb

Kalimantacins A21459 Epoxyquinomycins GE 37468 Phencomycin Chrysoapermin Bacillaene GE2270 AL072 Ripostatin Sorangiolid Thiomarinol Echinoserine 07F275 Pyralomycins RS-22 Ochracenomycins Azicemycins Amythiamycin APHE31~

Alcaligenes sp. Actinoplanes sp. Amycolatopsis Streptomyces sp. Streptomyces sp. Apiocrea chrysosperma Bacillus subtilis Planobispora rosea Streptomyces sp. Sorangium cellulosum Sorangium cellulosum Alteromonas rava (marine) Streptomyces tendae unidentified fungus Actinomadura spiralis Streptomyces violaceusniger Amycolatopsis sp. Amycolatopsis sulphurea Amycolaotopsis sp. Streptoverticillium griseocarnum Streptomyces aurantiacus Streptomyces griseoviridus Lachnum payraceum Penicillium chrysogenum Penicillium chrysogenum

Aurantimycin Cineromycins Papyracon Cephem derivatives Sorrentanone

TERP PEP, mod. PK

I Introduction: The Importance of Secondary Metabolites as DrugsTab. 1 (Continued) .

5

Compound Reference Benzastatin

Producing Organisms Streptomyces nitrosporeus Sorangium cellulosum Fusarium sp. unidentified mushroom Sphaerellopsis filu Bacillus polymyxa Mycogone rosea Actinomycete Streptomyces sp. Sorangium cellulosum Sorangium cellulosum Apiocrea chrysosperma Pterula sp. Fusarium sambucinum Favolaschia A ureobasidium pullulans Streptomyces sp. Streptomyces aurantiogriseus Bacillus sp. Sporomiella australis Streptomyces sp. Streptomyces sp. Actinomadura spinosa Actinomadura spinosa Ascotricha amphitricha Mucor hiemalis Fusarium sp. Fusarium sp. Tricothecium sp. Streptomyces sp. Stachybotrys sp.

Structural Type ' ALK

Selected Properties antifungal, antiviral free radical scavenger antifungal antifungal antifungal antibacterial, antifungal antifungal, antibacterial antifungal, antibacterial antifungal antifungal antifungal cytotoxic antifungal antifungal antifungal antifungal antifungal antifungal antifungal antifungal antifungal antifungal antibacterial antifungal antifungal antifungal antifungal antifungal antifungal, cytotoxic antifungal antifungal antifungal antiviral, HIV HIV protease inhib., endothelin antag.

Research Group Involved KRIBB

Jerangolides BE29602 Dibefurin Darlucins Fusaricidin Helioferin Azalomycin Liposidolide Chirosazol Ratjadon Chrysospermin Hydroxystrobilurin Fusacandin Favolon Aureobasidins Phosmidosine NP-1OlA YM-47522 Australifungin AKD-2 UK-2AIBICID Prodimicin Pradimicin Ascosteroside Epothilone Fusarielin Saricandin Furanocandin Siamycin L-671,776, derivatives

PK PK-GLYC PK PK-mod. PEP-PK PEP-PK PK PK PK PK PEP PK PK-GLYC TERP PEP NUC A-mod. PK PK PK PEP PK PK TERP-GLYC PK-AA PK PK PK-GLYC PEP PK-AA

GBF Banyu Pharm. Co. Abbott Univ. Kaiserslautern and Univ. Munich Wakunaga Pharm. Co. and PT Kalbe Pharma Hans Knoll Institute Hoechst, AgrEvo RIKEN GBF GBF Hans Knoll Institute Univ. Kaiserslauern and Univ. Munich Abbott Univ. Kaiserslautern and Univ. Munich Takara Shuzo Co. RIKEN and SynPhar Lab. Inc. Hokkaido Univ. Yamanouchi Pharm. Co Merck Sharp & Dohme Univ. Osaka City Osaka Univ. and Suntory Ltd. Meijo U, Toyama Pref. Univ. Toyama Pref. Univ. and Bristol Myers Squibb Bristol Myers Squibb GBF Univ. Tokyo Abbott Meiji Seika Kaishi Ltd and Mitsubishi Chem. Corp. Bristol Myers Squibb Ciba-Geigy

6


Tab. 1 (Continued) . Compound Reference Benzastatins Triterpenesulfates Quinoxapeptides Karalicin AH-758 Eulicin Sattabacin Sattazolin GE20372 Isochromophilones Producing Organisms Streptomyces nitrosporeus Fusarium compactum Betula papyrifera Pseudomonas fluorescens Streptomyces sp. Streptomyces sp. Bacillus sp. Bacillus sp. Streptomyces sp. Penicillium sp. Structural Type' ALK TERP PEP-PK PK PK PEP-mod. A-mod. AA-mod. PEP PK Selected Properties free radical scavenger antifungal, antiviral rhinovirus protease inhib. antiviral: HIV1,2,RT antiviral: HSV antiviral: HSV antiviral: HIVl antiviral: HSV antiviral: HSV antiviral: HIV antiviral: HIV DGAT, ACTAT inhib. Research Group Involved KRIBB Abbott Merck Sharp & Dohme Univ. Cagliari and Univ. Cattolin (Rome) Kumamoto Univ. Jikei Univ., Institute of Microbial Chemistry Univ. Cagliari and Univ. Rome Univ. Cagliari and Univ. Rome Lepetit Kitasato

Antitumor Drugs Sch5290011 Gliocladiurn sp. Micromonospora sp. Rakicidins Actinomadura Esperamicin verrucosospora Ossamycin Streptomyces hygroscopicus Acetophthalidin Penicillium sp. (marine)

PEP PEP-PK PK-GLYC PK PK PEP-TERP NUC-mod. PK PEP PEP PK PK PK PK-GLYC PK PEP-PK PK, mod. PK PK

antitumor cytotoxic antitumor cytotoxic cell cycle inhibitor cell cycle inhib. proliferation mod. antitumor antitumor cytotoxic cytotoxic cytocidal antitumor antitumor cancerostatic aromatase inhib. anticancer oncogen function inhib. cytotoxic

Schering-Plough Bristol Myers Squibb Bristol Myers Squibb Lilly RIKEN RIKEN Toyama Pref. Univ. Kirin Brewery Co. Bristol Myers Squibb GBF Univ. Tokyo Kitasato Kyowa Hakko Kogyo Snow Brand Milk Co. and Kamagawa Univ. Institute of Microbial Chemistry and Showa College Fujisawa Nippon Kayaku Keio Univ. and Institute of Microbial Chemistry Abbott

Tryprostatins Sparoxomycin Cochleamycins Himastatin Chondramide Anguinomycin Clovalicin Clecarmycin Piericidin derivatives Hydroxymycotrienine FR901537 Medelamine Naphthablin Macquarimicin

Aspergillus fumigatus Streptomyces sparsogenus Streptomyces sp. Streptomyces hygroscopicus Chondromyces crocatu'S Streptomyces sp. Sporothrix sp. Streptomyces sp. Streptomyces sp. Bacillus sp. Bacillus sp. Streptomyces sp. Streptomyces sp. Micromonospora sp.

I Introduction: The Importance of Secondary Metabolites as Drugs Tab. 1. (Continued) Compound Reference Thiazinotrienomycin Cremeduycin Tryprostatin Sch50673,6 Terpentecin FD-211 Cytogenin Producing Organisms Streptomyces sp. Streptomyces cremeus Aspergillus fumigatus Nattrassia mangiferae Streptomyces sp. Myceliophthora lutea Streptoverticillium eurocidium Streptomyces sp. Penicillium patulum Streptomyces sp. Streptosporangium amethystogenes unidentified fungus Structural Type' PK-PEP Selected Properties cytostatic (cancer) Research Group Involved Institute of Microbial Chemistry and Showa College Univ. Illinois RIKEN Schering-Plough Kyowa Hakko Kogyo

7

A, mod. PEP, mod. PK PKPK PK

Enaminedonin Dihydroepiepoformin EI-1507-1/2 TAN-15 11 CJ-12,371,2

PEP-PK PK-mod. PK PEP-PK PK

cytotoxic cell cycle inhib. antitumor antitumor: topoisomerase inhib. cytotoxic: MDR Taisho Pharm. Co. antitumor Institute of Chemotherapy (Shizuoka) and Institue of Microbial Chemistry detransforming RIKEN tumor cells IL-1 receptor Upjohn antag. IL-1-converting Kyowa Hakko Kogyo enz. inhib. induces Takeda cytokines DNA gyrase Pfizer inh.

Pharmacological Activities FR901,483 Cladybotryum sp. Streptomyces prunicolor PA-48,153 27-0-demethyl- Steptomyces hygroscopicus Rapamycin Streptomyces sp. NFAT 68,133 Penicillium sp. Stevastatin Streptomyces sp. Trichstatin Cytosporin Leustroducsin Plactins TAN1323CID Monamidocin A-72363 Trachyspic acid Carbazoquinocins Cytospora sp. Streptomyces platensis Agonomycetales Streptomyces purpurescens Streptomyces sp. Streptomyces nobilis Talaromyces trachyspermus Streptomyces violaceus

ALK-P-ester PK PK-AA PK PEP-PK PK PK PK-P-ester PEP PK PEP GLYC PK AA-PK

immunosuppr. immunosuppr. immunosuppr. immnuosuppr. immunosuppr. immunosuppr., histidine decarboxylase inhib. angiotensin bdg. inhib. t hrombocytosis inhib. stimulates fibrinolytic activity angiogenesis inhib. fibrinogen rec. antag. heparanase inhib. hep a r a na se inhib. antioxidant

Fujisawa Pharm. Co. Shionogi Smith Kline Beecham Abbott Nippon Kayaku Kyowa Hakko Kogyo Merck Sharp & Dohme Sankyo Co. Tokyo Noko Univ. Takeda Nippon Roche Sankyo Co. Sankyo and Univ. Tokyo Univ. Tokyo

8


Tab. 1 (Continued) .

Compound Reference Phenopyrazin,

Producing Organisms Penicillium sp. Streptomyces sp.

Structural Type' PK-AA PK ALK PK PK, mod. PK PK PK-AA PK PEP PK-AA PK PK TERP-PK PEP ALK TERP PK PK-S PEP PEP-PK PK PK TERP-mod. TERP-PK TERP-PK PK

Selected Properties radical scavenger protein kinase inhib. protein kinase C inhib. protein tyrosine kinase inhib. myoinositol Pase inhib. myosin light chain kinase inhib. endothelin converting enzyme inhib. endothelin rec. antag. endothelin rec. antag. endothelin rec. antag. endothelin rec. antag. vasodilatory endothelin rec. antag. entothelin rec. bdg. platelet aggr. inhib. platelet aggr. inhib. squalene synth. inhib. cell adhesion inhib. Willebrand factor rec. antag. ACAT inhib. ACAT inhib. ACAT inhib. ACAT inhib. DGAT inhib. ACAT inhib. ACAT inhib. cholesterol esterase inhib. cholesteryl ester transfer protein inhib.

Research Group Involved Kitasato Ciba-Geigy Ciba Geigy Ciba Geigy and Panlabs Lepetit Kyowa Hakko Kogyo Fujisawa Asahi Kyowa Hakko Kogyo Kyowa Hakko Kogyo Ciba-Geigy Mercian Corp. Xenova and Parke Davis Xenova Taisho Pharm. Kitasato Sankyo Kitasato Nippon Roche Tokyo Noko Univ. Tokyo Noko Univ. KRIBB Kitasato and Pfizer Kitasato Kitasato Sankyo Tokyo Tanabe Co. and Univ. Tokyo Cornell Univ. and Schering-Plough

Balmoralmycin

Staurosporine Streptomyces longisporoflavus analogs Paeciloquinones Paecilomyces carneus Factor AIC MS-444 WS79089B Stachybocin RES-1149 RES-701 L-671,776 derivatives Mer-A2026 ET Drirnane-sesquiterpenes Bassiatin Herquline Schizostatin Macrosphelide Sulfobacins Lateritin Isohalobacillin GERI-BP002-A Pyripyropenes Amidepsine Terpendole Epi-cochlioquinone F1839 CETPI unidentified fungus Micromonospora sp. Streptosporangium roseum Stachybotrys sp. Aspergillus sp. Streptomyces sp. Stachybotrys sp. Streptomyces pactum Penicillium sclerotium Aspergillus ustus Beauveria bassina Penicillium herquei Schizophyllum commune Microsphaeropsis sp. Chryseobacter sp. Gibberella lateritium Bacillus sp. Aspergillus fumigatus Aspergillus furnigatus Humicola sp. Albophoma yamanashiensis Stachybotrys bisbyi Stachybotrys Cytospora (insect associated)

I Introduction: The Importance of Secondary Metabolites as DrugsTab. 1 (Continued) .~~

9

Compound Reference Fluvirucin Thermorubin Salfredins Panosialins Xenovulene Arisugacin Nerfilin I Michigazones Aestivophoerin Lavanduquinocin Epolactaene MQ-387 YL-01869P YM 4714112 Poststatin Cathstatins BE-40644 RPR113228 Andrastin Saquayamycins

Producing Organisms Streptomcyces sp. Thermoactinomyces sp. Crucibulum sp. Streptomyces sp. Acremonium strictum Penicillium sp. Streptomyces halstedii Streptomyces halstedii

Structural Type' PK-GLYC PK PK-mod. PK-mod. TERP TERP PEP-PK PEP

Selected Properties phospholipase inhib. aldose reductase inhib. aldose reductase inhib. glycosidase inhib. GABA-benzodiazepine receptor binding AChE-inhib. neurite outgrowth ind. neuronal cell protecting neuronal cell protecting neuronal cell protecting neuritogenic aPase N inhib. matrix metalloproteinase inhib. elastase inhib. Pro-endopeptidase inhib. proteinase inhib.

Research Group Involved Univ. Keio UNITIKA Co. and Univ. Osaka Shionogi Kitasato Xenova Kitasato Somtech and Univ. Tokyo Univ. Tokyo Univ. Tokyo Univ. Tokyo RIKEN and Kaken Pharm. Co. KRIBB Sankyo

PK-mod. Streptomy ces purpeofuscus Streptomyces virdochromogenes Penicillium sp. (marine) PK Streptomyces nayagawaensis Actinomadura ultramentaria Flexibacter sp. Streptomyces virdochromogenes Microascus longirostris Actinoplanes sp. PEP PEP-mod. PEP PEP PEP-mod. PK

Chrysosporium lobatum TERP Penicillium sp. Actinomycetes TERP-PK PK

Yamanouchi Pharm. Co. Institute of Microbial Chemistry SynPhar Lab Inc. and Institute of Marine Bioscience (Halifax) t hioredoxin Tsukuba Res. and inhib. Banyo Pharm. Co. farnesyl protein R h h e Poulenc Rorer transferase inhib. farnesyl protein Kitasato and Keio Univ. transferase inhib. farnesyl protein Keio Univ. and Institute of Microbial Chemistry transferase inhib. plant growth regulator plant growth regulator herbicidal Univ. Tokyo and Ajinimoto Nippon Kayaku Univ. Paul Sabatier (Toulouse)

Agricultural Uses Streptomy ces Rotihibin graminofaciens Streptomyces sp. Pironetin

PEP-PK PK PK

Phthoxazolin

Streptomyces griseoaurantiacus

10


Tab. 1 (Continued) .

Compound Reference Methylstreptimidon-derivatives Fudecalone Arohynapene Xanthoquinodin Hydrantomycin Iturins Trichorzins Azalom ycin Phthoxazolines Phenamide Patulodin Gualamycin NK-374200 Melanoxadin Albocycline CI-4 Oligosperons Isocoumarins Milbemycins Sulfinemycin Musacins Lachnumlactone

Producing Organisms Streptomyces sp. Penicillium sp. Penicillium sp. Humicola sp. Streptomyces sp. Bacillus subtilis Trichoderma harzianum Actinomycete Streptomyces hygroscopicus Streptomyces sp. Streptomyces albospinus Penicillium urticae Streptomyces sp. Taralomyces sp. Trichoderma sp. Streptomyces sp. Pseudomonas sp. (marine) Arthrobotyrys oligospora Lachnum sp. (Ascomycete) Streptomyces sp. Streptomyces albus Streptomyces griseoviridis Lachnum papyraceum

Structural Type' PK-mod. PK PK PK PK PEP-PK PEP PK PK-mod. AA-mod. PK GLYC NUC-PEP PK PEP TERP PK PK PK-mod.

Selected Properties herbicidal anticoccidial anticoccidial anticoccidial herbicidal antibiotic phytopathogens antifungal antifungal antifungal antifungal antifungal acaricidal insecticidal melanine bios. inhib. melanogenesis inhib. chitinase inhib. nematocidal nematocidal antihelminthic antihelminthic antihelminthic nematocidal, cytotoxic

Research Group Involved Hoechst India Kitasato Kitasato Kitasato Kitasato USDA, Univ. Texas and Univ. Purdue CNRS (Paris) Hoechst and AgrEvo Merck Sharp and Dohme Kitasato Monsanto Osaka Univ. Nippon Kayaku Co. Nippon Kayaku Co. Teikyo Univ. and Tokyo Univ. Kitasato Shimizu Labs. Australian National Univ. Univ. Kaiserslautern (FRG) and Univ. Lund (Sweden) Smith Kline Beecham Lederle Univ. Gottingen, Univ. Tubingen, Hans Knoll Institute Univ. Lund and Univ. Kaiserslautern

PK

'*

Structural type: PEP - peptide, PK - polyketide, TERP - terpenoid, GLYC - glycoside, A A - amino acid, NUC - nucleoside, mod. - modified. Property: antag. - antagonist; bios. - biosynthesis; ind. - inducer; inhib. - inhibitor; rec. - receptor. Group identification: Univ. - University of.

(AZUMA, 1987), bestatin (OCHIAI, 1987), topostins (SUZUKI al., 1990), etc., are to be et introduced into future therapy. The large-scale biotechnical production of bioactive compounds has been developed in a highly effective manner. Fermentations of high-producing microorganisms are carried

out up to a volume of more than 300 m3. The yield is sometimes more than 40 g L-' (VANDAMME, 1984), and up to 1OOgL-' in penicillin fermentations. This demonstrates the efficiency of strain selection which supported knowledge of biosynthesis and strain genetics. Optimum bioprocess control and suitable fer-

2 Secondary Metabolism, an Expression of Cellular and Organismic Individuality

11

mentation equipment were developed as further prerequisites of a highly efficienct production of biotechnical drugs. As an introduction to this volume, this chapter summarizes some of the general aspects of secondary metabolism in microorganisms such as:

small, but systematically defined groups of organisms (e.g., special species and genera of microbes, plants, animals) and point to the enormous variability of chemical structures (ComitC Editorial, 1992). In microbes, the capacity to generate secondary metabolites is frequently lost by genomic mutations, but this feature misses any concomitant effect on the - the biological role of bioactive compounds vegetative development of the pertinent 1989; OLESKIN, 1994). An strains (SHAPIRO, in the producer strains, inverse correlation is usually observed be- the biosynthetic pathways and their organitween specific growth rate and the formation zation, - natural and induced variations of second- of secondary metabolites such as antibiotics. ary metabolite structures and problems of Particular features of morphological differentiation in surface or submerged cultures, such their structural classification. as the formation of spores and conidia, seem Finally, future perspectives of drug screen- to be related to the production capacity of secondary metabolism. Moreover, a maxiing from microbial sources are discussed. mum production rate of antibiotics and other secondary metabolites (pigments, alkaloids, mycotoxins, enzyme inhibitors, etc.) has frequently been observed when growth-promoting substrates were depleted from the me1992). This phenomenon was dium (DEMAIN, called catabolite regulation (DEMAIN, 1974). This may be one of the reasons for the phase-dependency of biosynthesis of many microbial drugs. Thus, during the microbial growth phase (trophophase) secondary metabolism is often suppressed, but increased later during the 1986). Sometimes this idiophase (VINING, 2.1 Roles of Secondary feature is not present and depends on the parMetabolites in Producing ticular strains and growth conditions. For inOrganisms stance, the formation of phytotoxins by some phytopathogenic microbes such as Alternaria The majority of bioactive products of mi- and Fusarium strains is not a subject of catabcroorganisms and plants is generated by sec- olite regulation and even occurs in a growthondary metabolism. This part of the meta- associated manner (REUTER,1989). On the bolic machinery of microbes, plants, and ani- other hand, the production of antifungal efmals may play no essential role in the vegeta- fectors including peptaibol trichorzianine may tive development of the producing organisms, be induced, as shown in Trichoderma harbut seems to convey advantages to the perti- zianum by cell walls of the plant pathogen et nent species concerning its long-term survival Botrytis cinerea (SCHIRMBOCK al., 1994). in the biological community and environment Likewise, certain plant metabolites may in(LUCKNER al., 1977; KLEINKAUF VON duce the synthesis of peptide antibiotics in et and DOHREN, 1986; WILLIAMS al., 1989; the respective pathogenic Pseudomonas et and WHITE,1994; MO et LUCKNER, 1989 VINING, 1992; WILLIAMS strains (MAZZOLA et al., 1992; CAVALIER-SMITH, OLESKIN, al., 1995). In general, the phase-dependency 1992; 1994; VINING STUTTARD, and 1995) (Tab. 2). or specific inducibility indicates that the secFurther interpretations imply the formation ondary metabolism is strictly governed by inof certain secondary metabolites by relatively herent regulatory systems (see Sect. 2.2).


12Tab. 2.


Presumed Roles of Secondary Metabolites in Their Producer Organism Endogenous role in the Exogenous role in the producing organism environment endogenous regulatory protection against competing signals triggering morphoorganisms - genesis endogenous signals regulating regulation of commensalism mating processes such as and cohabitation pheromones protection against endogenous detoxification physicochemical noxes (UV light) of metabolites acquisition of trace elements supply of special building material of the cell wall detoxification of trace elements endogenous reserve material not accessible to other microorganisms

Most of the secondary metabolites are biosynthesized in microbes and plants via complex multistep pathways involving many enzymatic and even non-enzymatic events. These appear to be integrated in a coordinated manner into the global microbial processes of cytodifferentiation such as formation of spores, conidia, and aerial mycelia (LUCKNER, 1989), or in the processes of invasion or defense. The same is true for plants in which secondary metabolite formation occurs in different tissues, e. g., roots, leaves, flowers, and seeds. Hence, it seems obvious that secondary metabolism does not reflect an occasional feature but is the result of a very long evolutionary development. As was shown for the tetracycline antibiotics from Sfrepfomyces spp. more than 200 genes may affect the biosynthetic pathway (VANEKand HOSTALEK, 1985). No wonder that speculation about the endogenous function and roles of secondary metabolites in the producing organisms themselves never came to an end (VANEK et al., 1981; VINING, 1992; OLESKIN, 1994; VINING STUTTARD, 1995). and To maintain such a great number of genes, generally linked into clusters, during evolution should be of advantage to the pertinent organism. Obviously, in plants many secondary metabolites are involved in the protection against microorganims and animals (CUND-

1992; JOHNSON and ADAMS, 1992). Others act as chemoattractants or as repellents towards insects fructifying flowers or damaging plant tissues. A series of plant hormones (cytokinins, gibberelic acid, jasmonic acid, etc.) are similar in structure but per definitionem are not secondary metabolites. Another function of secondary metabolites in plants is the detoxification of poisonous metabolites via an endogenous compartmen1989). The role of tized storage (LUCKNER, secondary metabolism in microbes is even more difficult to understand. Cellular efforts needed for secondary pathways are rather low in the wild-type strains (only a small amount of the overall substrate intake is converted to bioactive secondary metabolites). This part of metabolism would possibly have been eliminated during phylogenesis without any selective advantage of secondary metabolite production. It appears to be a generally accepted view that microbial secondary metabolites play an important but not generalizable rote, at least in special situations, e. g., in warranting the survival in particular environmental systems, during limitation of nutrient supply or even in the course of morphological development (LUCKNER al., et 1977; KLEINKAUFand VON DOHREN, 1986; VINING, 1992; KELLet al., 1995; VINING and STUTTARD, 1995). From this point of view,LIFFE,


13

lated by secondary metabolites in heterothe formation of large amounts of antibiotics logous populations. by high-producing strains (substrate conver>0.1) would be considsion rates Yglucose,drug The self-protecting mechanism in antibiotered as a pathophysiological problem (VAic-producing microbes should be mentioned NEK et al., 1981). In order to better understand the general roles of secondary metabol- as a further evidence of an ecological function ites in microbes one could refer to the color of antibiotics, as a weapon against competiet et of hairs and feathers in animals, their odorous tors (ZAHNER al., 1983; BRUCKNER al., 1989,1992; WILLIAMS and pheromones, and other metabolic products 1990; CUNDLIFFE, 1992). By this means the miwhich do not contribute per se to the vegeta- MAPLESTONE, tive life of the pertinent species. But they crobe prevents suicide due to its own secondcould have outstanding importance during ary metabolite either by enzymatic modificathe adaptation to changing media, in the pro- tions of the drug, by alteration of its biologitection against competing organisms, and in cal target, or by an active transport-directed the regulation of sexual and asexual processes export (see, e. g., the tetracycline efflux) of genetic exchange. General discussions of (JOHNSON and ADAMS, 1992; NIKAIDO, secondary metabolite formation in microbes 1994). Usually, resistance mechanisms of the consider four major fields of importance antibiotic-producing microorganisms are the (LUCKNER al., 1977; KLEINKAUF et and VON same as in antibiotic-resistant bacteria. The DOHREN,1986; LUCKNER, 1989; WILLIAMS analysis of the gene sequences encoding reet al., 1989, 1992; VINING, 1992; CAVALIER- sistance determinants support the idea that SMITH,1992; OLESKIN, 1994; VININGand the transfer of resistance occurs from the antibiotic producers to the non-producing miSTU~TARD, 1995) (Tab. 2): crobes (JOHNSONand ADAMS, 1992; SA1995; DAV(1) The formation of secondary metabolites LYERS et al., 1995; HIRAMATSU, facilitates the adaptation to metabolic im- IES, 1994). In addition, the emergence of new balances as a kind of a metabolic valve, types of resistance factors by the formation of which is needed to remove an excess of mosaic genes has been analyzed in P-lactamtoxic, endogenous metabolites that other- resistant pneumococci (SPRATT,1994; COFwise are accumulated during a partial lim- FEY et al., 1995). The great variation of both active and inacitation of substrates. (2) Secondary metabolism could be a source tive secondary metabolites, that were obof individual building blocks of cells or of served in microorganims and plants supplied metabolic reserves which warrant the in- the main arguments against their determined dividuality and particular functionality of function. Obviously, the formation of a bioactive secondary metabolite, such as an antithe given strain. (3) Secondary metabolites could be regarded biotic, rather appears as an exception than as as endogenous signals triggering particu- a rule. Frequently, many inactive shunt-melar stages of morphogenesis and the ex- tabolites and congenors are produced in adchange of genetic material (see Fig. 1). dition to the few active metabolites. It is not This hypothesis was particularly sup- reasonable to believe that all these metaboported by the observation that the major- lites are needed in a single organism. It might ity of the good producers (e.g., actino- be that a function of a secondary metabomycetes, fungi, bacteria) display a life cy- lite could become apparent only in a particucle involving several stages of morpholog- lar, exceptional situation or in special stages of development. Hence, the selection presical differentiation. (4) Secondary metabolite formation is partic- sure on structures and secondary pathways is ularly important in biosystems as a signal necessarily low (ZAHNERet al., 1983). As a of interspecific communication be- consequence, spontaneously evolving variants tween microbes and other microbes, and mutants could survive with the same plants, and animals. Symbiosis, commen- probability as their parents, and modifications salism, and antagonism could be regu- of secondary pathways and structures would

14


A-factor

VB-factorS

0

autoinducer from Vibrio Rscheri

Butalactin

M0, , J ,

factorfrom Str. vlrldochromugenes

differolide

Q0

$ ?Gennicidin

OH

on

Basidifferquinone

ChOH

no

v

v

-

0

antheridiol (for references, see text).

oogonlol

trlsporlc acid C

sirenin

Fig. 1. Structures of some representatives of signaling molecules from bacteria (streptomycetes) and fungi

be preserved (secondary metabolism as a tochromes, chlorophylls, sexual pheromones playground of evolution) (ZAHNER et al., of fungi and bacteria, etc. might have been 1983). This might explain the existence of the evolved similarly. Some of them may be atnumerous similar structures. According to tested to defined functions of microbial secthis hypothesis, the limited substrate specifici- ondary metabolites (Tab. 2, Fig. 1). A role of secondary metabolism in the adty of some enzymes of secondary metabolism has to be mentioned (LUCKNER, 1989). How- aptation to changing nutrient conditions is a ever, it should be noted that in many multi- realistic position since an excessive supply of step processes this limited specificity is re- metabolic intermediates (precursors) usually stricted to certain steps and thus less re- induces or stimulates drug production (DEstricted structural regions of the compounds MAIN 1974,1984,1992). Growth may become (KLEINKAUF and VON DOHREN, press). A imbalanced and precursors are accumulated in few secondary metabolites, out the pool of during the limitation of a given substrate in the many non-functional metabolites, have the medium, while others are still available in apparently acquired an essential role in excess. Excessive precursors could be regrowth and differentiation. The siderophores, leased into the medium or converted to harde.g., are microbial vehicles of iron transport ly metabolizable products which would not formed in variable structures as constitutive support the growth of competitors. Moreover, parts of the iron uptake system (VON DER colored secondary metabolites, such as pigHELMand NEILANDS, 1987; WINKELMANN, ments, could protect cells and spores from 1991; WINKELMANN DRECHSEL, and Chapter damage by ultraviolet radiation or also could 5, this volume). Per definitionern, they should promote the acquisition of rare elements via not be regarded as secondary metabolites. complex formation as, e. g., siderophores. Highly specialized biomolecules such as cy- Complex formation could also protect the


15

cells from high concentrations of toxic heavy metals. The incorporation of secondary metabolites into cellular structures has been suggested to contribute to their individual characteristics. Thus, streptomycin and its building moiety, streptidine, were established as a constitutent of the cell wall of the producing Sfrepfomyces griseus (DEMAIN, 1984; DISTLER et al., 1992). Otherwise, the production of secondary metabolites (so-called idiolites) (DEMAIN, 1992), could serve as a kind of a metabolic reserve which cannot be metabolized by other microbes. Some antibiotics (anthracyclines, tetracyclines, cyclosporins, etc.), e. g., are stored within the mycelium and their complete degradation requires a series of specialized enzymatic steps. Otherwise, bioconversions of antibiotics are a constitutive part of the self-protecting mechanisms of the producer strain. Moreover, concentrations of several antibiotics were shown to decrease in the course of prolonged cultivation, thus indicating the onset of degradative processes. Some fungi are well-known to degradate their own polyketides such as, e.g., citrinin (BARBER al., et 1988) and zearalenon and even to use them for additional syntheses. Active antibiotics were usually not detected in soil samples, although recently sensitive procedures have permitted the detection of phenazines (COOK et al., 1995). Their complete degradation under natural conditions seems very likely. Most likely, a series of signaling molecules is supplied by the secondary metabolism that possess interspecific (ecological) or speciesdependent functions, e. g., as signals triggering morphogenesis and the exchange of genetic material (Fig. l). By growth inhibition of competing microbes a producer strain could attain an advantage (c. f. the production of herbicidal antibiotics by phytopathogenic bacteria which damage plant tissues and facilitate nutrient acquisition from the host) (KOHMOTOand YODER, 1994; MAZZOLA and WHITE,1994; M o et al., 1995). Vice versa, secondary metabolism could confer a particular advantage in symbiotic systems, such as Pseudomonaslplant roots, to both the producing strain and the symbiont. An example is the control of phytopathogenic Fusarium or

Rhizocfonia fungi on plant roots by products of cohabiting streptomycetes and bacteria. Interspecific effects have also been postulated for volatile compounds which are formed, e. g., by streptomycetes and cyanobacteria. Geosmin, isoborneol, and mucidon are the constituents of the typical earthy odor. It has been shown that sclerin and scleroid from the fungus Sclerofinia liberfiana stimulate the biosynthesis of aminoglycosides by streptomycetes, but also the growth of some plants (KUBOTA al., 1966; OXFORDet al., 1986). et The formation of phytotoxins by phytopathogenic microbes is mentioned as another interspecific communication system (KOHMOTO and YODER, 1994). Constituents of the microbial cell wall (elicitors such as p1,3-1,6glucans from Phytophfora megasperma) are recognized by specific plant cell membrane receptors. Subsequently, a series of protective mechanisms is induced in the plant (e.g., hypersensitivity reactions, de novo synthesis of tissues, secretion of enzymes lysing microorganisms, and formation of antimicrobial phytoalexins). On the other hand, some of the phytoalexins are inactivated by enzymes of phytopathogenic microbes. In the natural habitat genetic information can be transferred from one microbe to another interspecifically. Both biosynthetic procedures and resistance mechanisms thus can be spread among various heterologous species and genera. Apparently this is also true for genetic exchanges between plants and microbes. A recent intriguing example is the discovery of a taxol producing fungus living in taxol producing yew trees (STIERLEet al., 1994). Typical plant hormones such as gibberellins and jasmonic acid are also produced by some microorganisms. Aflatoxins formed via complicated biosynthetic pathways in fungi, such as Aspergillus, have been established in actinomycetes. Sequence analyses of the genes encoding penicillin and cephalosporin biosynthetic clusters (ACV synthase, isopenicillin N-synthase, acyltransferase, deacetoxycephalosporin C-synthase, and deacetoxycephalosporin C-hydroxylase) in Penicillium chrysogenum, Acremonium chrysogenum, and Streptomyces spp. strongly suggested that fungi received the pertinent genes from the prokaryotic actinomycetes during evolution

16


(LANDAN al. 1990 MILLERand INGOLIA, et 1993; BUADESand MOYA, 1996). The production of cephabacins, chitinovorins, clavulanates, olivanic acids, carbapenems, and thiopeptides by unicellular bacteria and streptomycetes may indicate that an original biosynthetic pathway was spread horizontally among different microbes, thus giving rise to evolutionary variations of structures and pathways. The evolution of secondary metabolism even appears to create hybrid structures by the combination of genetic material originating from heterologous hosts. Recently, thiomarinol (SHIOZAWA al., 1993) was isolated et from the marine bacterium Alteromonas rava as a composite compound formed by the esterification of pseudomonic acid (found in Pseudomonas fluorescens) and holomycin (a pyrrothine antibiotic, found in Streptomyces Spa). The involvement of secondary metabolism in the regulation of microbial cytodifferentiation seems to be important, at least in some cases. The morphogenesis of antibiotic-producing microorganisms (streptomycetes, fungi, Mycobacteria, etc.) is obviously mediated by a plethora of biochemical steps, which display a high specificity for the given organism. The pathways are regulated by individual signals in a highly coordinated manner (Fig. 1) (LUCKNER, 1989). During morphogenesis, silent genes are activated that have not been expressed during the growth phase. Accordingly, several endogenous non-antibiotic regulators of the cell cycle were discovered in Streptomyces cultures, and their structure was elucidated (see below) (KHOKHLOV, 1982; GRAFE, 1989 HORINOUCHIand BEPPU, l990,1992a,b, 1995; BEPPU,1992,1995). Correlations between the biogenesis of some peptidic antibiotics and morphogenesis were also described for synchronously growing Bacillus cultures (MARAHIEL al., 1979). Tyrocidin, et gramicidin, and bacitracin are produced during the onset of sporulation, suggesting that their function concerns the control of transcription, spore permeability, dormancy of spores, and their temperature stability (MARAHIEL et al., 1979,1993). The y-butyrolactones represent a particularly important group of endogenous regula-

tors of Streptomyces differentiation (Fig. 2) (KHOKHLOV, 1982; GRAFE 1989; HORINOUCHI and BEPPU,l990,1992a, b, 1995; BEPPU, 1995). They are required as microbial hormone-like substances in few species such as streptomycin, virginiamycin or anthracycline producing strains. These effectors permit the formation of antibiotics and aerial mycelium by some blocked, asporogenous, antibioticnegative mutants even in very low concentrations. Several other autoregulators of morphogenesis have been investigated (see, e. g., factor C) (SZESZAK al., 1991). Otherwise, et germicidin B (PETERSEN al., 1993) from et Streptomyces violaceusniger inhibits germination of its own spores by interference with endogenous ATPase. Antibiotics such as hormaomycin (ROSSNER al., 1990) and pamaet mycin (KONDOet al., 1986) were shown to have autoregulatory functions. Moreover, streptomycetes can produce interspecific inducers such as anthranilic acid and basidifferquinone (Fig. 1) which affect basidiomycetes and the formation of fruiting bodies (AZUMA et al., 1980; MURAOet al., 1984). Moreover, regulatory molecules inducing cytodifferentiation were isolated from fungi and molds confirming that morphogenesis can be mediated by the aid of an agency of specialized endogeneous factors (HAYASHI et al., 1985). They can be regarded as secondary metabolites since they do not possess any function in vegetative development. In addition, sexual factors from fungi and yeasts can be considered as functionalized secondary metabolites. They trigger zygospore formation by haploid cells belonging to different mating types (GOODAY, 1974). During the evolution of signal systems, from the simple pro- and eukaryotes up to the hormonal control in mammalians, some structures and activities have been conserved. The alpha-factor of the yeast Saccharornyces cerevisiae as one of its sexual pheromones, e.g., appears to be partially homologous to the human gonadotropin releasing hormone (LouMAYE et al., 1982). Moreover, inducers of differentiation of Friend leukemia cells were isolated from soil organism such as Chaetomium sp. These chlorine containing substituted diphenols (Fig. 1) also induce morphogenesis (stalk cell differentiation) of Dictyostelium


17

Fig. 2. Regulatory events suggested to be involved in morphogenesis and secondary metabolism of Strepand tomyces griseus (P: promotor) (HORINOUCHI BEPPU,1992a).

discoideum, suggesting the similarity of mammalian and fungal control of the cell cycle (KUBOHARA al., 1993). Recently, the ocet currence of sexual pheromones was even established for the prokaryote Streptococcus faecalis. Its pheromones stimulate or inhibit the transfer of conjugative plasmids from donor to recipient strains (WIRTH al., 1990). et Peptides triggering competence in Bacillus subtilis have been characterized and were termed pheromones (DSOUZAet al., 1994; SOLOMONet al., 1995; HAMOEN et al., 1995).

2.2 Regulation of Microbial Secondary Metabolism 2.2.1 Genetic Organization of Product FormationA large number of biosynthetic genes were isolated and characterized and, in general, they have been found assembled in clusters (Tab. 3). Such clusters may contain information for the biosynthesis of the basic structure of the metabolite, its modification, resistance determinants, e. g., promoting modification of products, targets, altered targets, or export systems, as well as regulatory elements; individual gene products which might as well exert regulatory functions.

18


Tab. 3. Biosynthetic Clusters Identified

Compound A54145 Aflatoxins Actinomycin Anguibactin Astaxanthin Avermectin Avilamycin Bacitracin Bialaphos Carbomycin Carotinoids Carotinoids Carotinoids Clavulanic acid Cephalosporin Cephamycin Coronatin Cyclosporin Daptomycin Daunomycin, Daunorubicin, Doxorubicin Destruxin Elloramycin Fatty acids Fatty acids Fengymycin Ferrichrome Frenolicin Geldanamycin Gramicidin S Granaticin Griseusin HC-toxin HET? Immunomycin Jadomycin B

Type acylpeptidolactone polyketide chromopeptidolactone modified peptide carotinoid polyketide polyketide branched cyclopeptide peptide polyketide terpenoids terpenoids terpenoids modified peptide modified peptide modified peptide modified polyketide cyclopeptide acylpeptidolactone polyketide

0rganismStreptomyces fradiae Aspergillus parasiticus, Aspergillus fzavus Streptomyces chrysomallus Vibrio anguillarum Agrobacterium aurantiacum Streptomyces avermitilis Streptomyces viridochromogenes Bacillus licheniformis Streptomyces viridochromogenes Streptomyces thermotolerans Rhodobacter capsulatus Myxococcus xanthus Synecococcus PCC7942 Streptomyces clavuligerus Acremonium chrysogenum Nocardia lactamdurans Pseudomonas syringae Tolypocladium niveum Streptomyces roseosporus Streptomyces C51'peucetius

Selected References' BALTZet al., 1996' BROWN al. 1996 et MAHANTI al., 1996 et KELLER al., 19962 et CHENet al., 1996 MISAWA al., 1995 et MACNEIL, 1995 BECHTHOLD al., 1996' et HERZOG-VELIKONJA et al., 1994 SCHWARTZ al., 1996 et ARISAWA al., 1995 et ARMSTRONG, 1994 ARMSTRONG, 1994 ARMSTRONG, 1994 HODGSON al., 1995 et MART~N GUTIERREZ, and 1995 COQUE al., 1993, et 1995a,b; PETRICH al., et 1994 BENDER al., 1996 et WEBER al., 1994 et BALTZet al., 1996' YE et al., 1994; GRIMM et al., 1994; FILIPPINI al., et 1995; MADDURI HUTand CHINSON, 1995a, b; DICKENS al., 1996 et BAILEY al., 1996 et DECKER al., 1995 et SUMMERS al., 1995 et ROCKand CRONAN, 1996 Liu et al., 1996' LEONGet al., 1996' BIBBet al., 1994 ALLENand RITCHIE, 1994 TURGAY MARAHIEL, and 1995 SHERMAN al. 1989; et BECHTHOLD al., 1995 et Yu et al., 1994 PITKIN al., 1996 et BLACK and WOLK,1994 MOTAMEDI al., 19962 et YANGet al., 1995b, 1996b

peptidolactone polyketide polyketide polyketide peptide cyclopeptide polyketide polyketide cyclopeptide polyketide polyketide cyclopeptide polyketide? modified polyketide polyketide

Metarhizium anisopliae Streptomyces olivaceus Streptomyces glaucescens Escherichia coli Bacillus subtilis Ustilago maydis Streptomyces roseofulvus Streptomyces hygroscopicus Bacillus brevis ATCC9999 Streptomyces violaceoruber Streptomyces griseus Helminthosporium carbonum Anabaena sp. Streptomyces sp. Streptomyces venezuelae

2 Secondary Metabolism, an Expression of Cellular and Organismic IndividualityTab. 3. (Continued)

19

Compound Me1anin Landomycin 6-Methylsalicylic acid Microcystin Mithramycin unknown Nikkornycin Nodusmicin Nogalamycin Oleandomycin Oxytetracyclin Penicillin Phenazin Pristinamycin A Pristinamycin M Purornycin Pyoverdin Rapamycin Saframycin Soraphen A Sterigmatocystin Streptomycin

Type

Organism

Selected References' TAKANO al.. 1995 et BECHTHOLD al., 1996* et BECKet al., 1990 MEISSNER al., 1996 et LOMBd et al., 1996 ARROWSMITH al., 1992 et BORMANN al., 1996 et LE GOUILL al., 1993 et YLIHONKO al., 1996 et Q U I R ~ S SALAS, and 1995 KIM et al., 1994 SMITH al., 1990, et MACCABE al., 1990; et DfEz et al., 1990 PIERSON al., 1995 et DE CRECY-LAGARD, personal Communication BECKet al.. 1990 TERCERO al., 19% et STINTZI al., 1996 et SCHWECKE al., 1995 et POSPIECH al., 1996 et SCHUPP al., 1995 et BROWN al., 1996 et BEYER al., 1996 et FERNANDEZ-MORENO et al., 1996 COSMINA al., 1993 et SHENand HUTCHINSON, 1994 MERSON-DAVIES and CUNDLIFFE, 1994 DECKER al., 1995 et DAVIS and CHATER, 1990 ARMSTRONG, 1994, HUNDLE al., 1994 et

poly ket ide, glycosylated polyketide cyclopeptide polyketide polyketide modified peptide polyketide polyketide polyketide polyketide modified peptide heterocycle acylpeptidolactone polyketide/peptide modified aminoglucoside branched cycloacylpeptide modified polyketide modified peptide modified polyketide polyketide aminoglycoside

Aspergillus nidulans Colletotrichum lagenarium Streptomyces sp. Penicillium patulum Microcystis aeruginosa Streptomyces argillaceus Streptomyces cinnamonensis Streptomyces tendae Saccharopolyspora hirsuta Streptomyces nogalater Streptomyces antibioticus Streptomyces rimosus Aspergillus nidulans, Penicillium chrysogenum Pseudomonas aureofaciens Streptomyces pristinaespiralis Streptomyces sp. Streptomyces alboniger Pseudomonas fluorescens Streptomyces hygroscopicus Myxococcus xanthus Sorangium cellulosum Aspergillus nidulans Streptomyces glaucescens Streptomyces griseus Streptomyces rochei Bacillus subtilis Streptomyces glaucescens Streptomyces fradiae Streptomyces fradiae Streptomyces coelicolor Erwinia herbicola, Erwinia uredovora

Streptothricin modified aminoglucoside Surfactin Tetracenomycin Tylosin Urdamycin Whi, spore pigment Zeaxanthin peptidolactone polyketide polyketide polyketide polyket ide terpenoid (carotinoid)

Presented at the conference Genetics and Molecular Biology of Industrial Microorganisms. Bloomington 1996. Presented at the symposium Enzymology of Biosynthesis of Natural Products. Berlin 1996. Abstracts available from the authors on request.

'

20


The techniques employed include reverse genetics if sequence data of relevant enzymes is available, the use of homologous gene probes or probes constructed from key sequences, the generation by PCR of specific probes flanked by conserved key motifs, complementation of idiotrophic mutants, expression of pathways or single step enzymes in heterologous hosts, cloning of resistance determinants followed by isolation of flanking sequences, identification and cloning of regulatory genes or sequences (promoters, regulatory protein binding sites, pleiotropic genes, master genes, etc.). To improve product levels, the addition of extra copies of positive regulators (CHATER, 1992; HOPWOOD al., 1995; CHATERand et BIBB,Chapter 2, this volume), extra copies of biosynthetic genes possibly representing bottlenecks (SKATRUD al., Chapter 6, this volet ume), or the alteration of promoters of key enzymes are under investigation. The analysis of clusters has revealed a wealth of information including biosynthetic unit operations and their surprisingly complex organization. The majority of large proteins now known are multifunctional enzymes involved in peptide and polyketide formation, with sizes ranging from 165 kDa to 1.7 MDa. Other systems also forming polyketides, peptides, aminoglycosides, etc., are comprised of non-integrated enzyme activities, still performing the synthesis of highly complex structures. The details of various biosynthetic clusters are described in the respective chapters on regulatory mechanisms (CHATER and BIBB,Chapter 2, this volume), peptides (VON DOHREN and KLEINKAUF, Chapter 7, this volume), plactams (SKATRUD al., Chapter et 6, this volume), lantibiotics (JACK et al., Chapter 8, this volume), and aminoglycosides (PIEPERSBERG DISTLER, and Chapter 10, this volume). Recent highlights of the elucidation of such data have been the rapamycin and immunomycin clusters in Streptomyces, the erythromycin cluster in Succharopolysporu, the surfactin and gramicidin S clusters in Bacillus, various plactam clusters, and the sterigmatocystin cluster in Aspergillus nidulans. An overview of examples is presented in Tab. 3. The amplification of biosynthetic clusters in highly selected strains has been a fascinat-

ing key result, as shown for the industrial penicillin producer (FIERRO et al., 1995; MARTfN and GUTIERREZ,1995). The main findings with regard to sequencing of complete genomic fragments are as follows:

- The identification of biosynthetic genes follows by the detection of core sequences. Such sequences permit the recognition of types of biosynthetic unit operations like polyketide condensation reactions, the specificities of the respective transferase sites (HAYDOCKet al., 1995), the number of elongation steps, amino acid activation sites; in the case of repetitive cycles where certain sites are reused, as in type I1 polyketide forming systems or, e. g., cyclodepsipeptide synthetases, where the number of steps remains uncertain. - Additional genes for modification reactions like oxygenases and transferases are readily identifiable by standard structural alignments as well as possible regulatory proteins. At present, however, the unambiguous correlation of product and biosynthetic machinery is not possible without the support of various genetic techniques or, if not available due to the lack of transformation systems, structural details from protein chemistry of isolated enzymes or multienzymes. To illustrate a few concepts, we will point to some recent examples of cluster analysis: PLactam antibiotics as classical examples of modified peptides are still leading antibacterial drugs. Some efforts have been directed to understand at the molecular level the performance of industrial overproducers selected for decades (SKATRUD al., Chapter 6, this et volume). Following the reverse genetics approach in isolation of the isopenicillin N synthase gene (SAMSON al., 1985), which cataet lyzes the formation of the penem bicycle from the tripeptide precursor ACV, the clustering of biosynthetic genes was demonstrated in both pro- and eukaryotic producers (BARTON et al., 1990). The two key enzymes, ACV synthetase and isopenicillin N synthase showed extensive similarities in both bacteria and fungi, and a horizontal intergenic transfer has been suggested (LANDAN al., 1990; MILLet


21

and INGOLIA, 1993; BUADESand MOYA, 1996). The linkage of these adjacent genes illustrates well basic principles of cluster organet ization (Fig. 3) (AHARONOWITZal., 1992). In bacteria both genes are transcribed unidirectionally within an operon linked to sets of other genes the products of which are required for the modifying reactions of the cephem nucleus to cephamycin, and the formation of the plactamase inhibitor clavulanic acid (WARDand HODGSON, 1993). Such extensive linkages have been termed superclusters. In fungi the encoding genes for ACVS and isopenicillin N synthase are bidirectionally transcribed, separated by intergenic regions of about 1 kbp. A variety of environmental conditions are known to affect fungal plactam production at the transcriptional level (ESPESOand PENALVA,1996; SUAREZand PENALVA,1996; BRAKHAGE and TURNER, 1995). The bidirectionally oriented promoters between acvA (pcbAB) and inpA (pcbC) may permit the asymmetrical expression of both genes, and indeed different levels of expression have been obtained in constructs emER

ploying different reporter genes which allowed to measure the expression of both genes simultaneously (BRAKHAGE al., et 1992; BRAKHAGEand TURNER, 1995; BRAGKHAGE VAN DEN BRULLE, and 1995; THENBERGet al., 1996). Such results suggest possible additional functions for the penicillin tripeptide precursor, besides its role in the formation and the still unclear excretion of penicillins. The 872 bp intergenic region between the A . nidulans acvA (pcbAB) and ipnA (pcbC) permits the complex and sensitive regulation involving several protein factors (for P. chrysogenum, see FENG et al., 1995; CHU et al., 1995). The current knowledge of regulatory factors and putative factors implied by the identification and characterization of trans-acting mutations specifically involved in the regulation of the A. nidulans biosynthetic genes is summarized in Fig. 3b. One of these factors, designated PACC, was shown to activate at least the ipnA gene transcription in response to shifts to alkaline pH values (SHAHet al., 1991; ESPESOet al., et 1993;TILLBURN al., 1995; ARST,1996). For

aE F

&lactarnsABC

'

10kbp

'

aflatoxin

raparnycinc

AN

P'

H

acvA

aat

b -

Fig. 3%Organization of the biosynthetic clusters of plactams, rapamycin, and sterigmatocystin, b regulatory sites identified in the penicillin biosynthetic cluster in Aspergillus nidulans.

22


PACC seven binding sites with different affinities have been mapped in this intergenic region (SUAREZand PENALVA, 1996). Another binding site containing a CCAAT motif was detected, bound by a protein complex designated PENRl (THENBERG et al., 1996). PENRl also binds to a CCAAT-containing DNA region in the promoter of the aat gene encoding acyl-CoA:isopenicillin N acyltransferase which is located 3 of the ipnA gene (LITZKA al., 1996). Deletion analysis and et mutagenesis experiments indicated that the binding of PENRl represses the expression of acvA and increases that of both ipnA and aat (THEN BERG et al., 1996; LITZKA et al., 1996). PENRl thus represents the first example of a regulatory protein controlling the regulation of the whole plactam biosynthesis gene cluster in fungi. However, many promoters of eukaryotic genes are known to contain CCAAT motifs which are bound by distinct gene regulatory proteins (JOHNSON and MCKNIGHT, 1989). At the time being, it is unknown what kind of CCAAT binding protein PENRl represents and whether it is a global acting factor specific for the regulation of /3lactam biosynthesis genes. Using a genetic approach which is feasible for the ascomycete A. nidulans, three recessive trans-acting mutations were identified designated prgAllprgB1 for penicillin regulation (BRAKHAGE and VAN DEN BRULLE, et 1995) and npeEl (P~REZ-ESTEBAN al., 1995). These mutations formally correspond to positively acting regulatory genes. Mutants carrying one of the mutations mentioned produced reduced amounts of penicillin. For prgAl and prgBl it was shown that the expression of both genes acvA and ipnA was affected (BRAKHAGE VAN DEN BRULLE, and 1995), whereas npeEl controls at least ipnA expression (P~REz-ESTEBAN al., 1995). et The major nitrogen regulatory protein NRE of Penicillium chrysogenum has also been found to specifically attach to three GATA/ GATT pairs within this intergenic region (HAAS and MARZLUFF, 1995). The pairwise attachment sites indicate a possible dimeric state of this GATA family transcription factor and as well connect this regulatory site with nitrogen assimilation. This example illustrates that similar biosynthetic genes are un-

der the regime of organizationally specific mechanisms of regulation. The respective regulatory mechanisms will be evaluated comparatively in a variety of pro- and eukaryotic hosts. Regulation of the formation of secondary metabolites in eukaryotes, however, does not need to be this complex, as will be discussed below in the case of sterigmacystin/aflatoxin biosynthesis. As a second example for the organization of biosynthetic information the POlyketide immunosuppressant rapamycin has been selected (SCHWECKE al., 1995). This et polyketide with an iminoacyl residue is of interest as an immunosuppressor in autoimmune disease and transplantation. Its biosynthesis proceeds by 16 successive condensation and 21 modification reactions of 7 acetyl and propionyl residues, respectively, followed by pipecolate onto the cyclohexane carboxylic acid starter unit. The respective cluster has been identified in Streptomyces hygroscopicus by LEADLAY al. (SCHWECKE al., 1995) et et using polyketide synthase gene probes of erythromycin synthase from Saccharopolyspora erythrea). The sequence of 107.3 kbp has been determined as well as the boundary sequences, to assure the completeness of the effort. The key part of the cluster is represented by four genes encoding multifunctional enzymes with sizes of 900 (A), 1070 (B), 660 (C), and 154.1 kDa (P) responsible for the formation of the macrolactam ring. These four genes of 25.7, 30.7, 18.8, and 4.6 kb unambiguously correlate with the structural features of the product, however, module 3 and 6 contain catalytic sites for the reduction of the polyketide intermediates, which actually are not found in rapamycin. The solution of this problem remains to be found and plausible explanations are either non-functionality due to, e.g., point mutations, or a possible transient reduction of the intermediates to facilitate folding, which is reversed later. These key genes are flanked by additional 24 open reading frames, most of which have been assigned tentative functions including modification of the macrolactam, export, and regulation. Standard identification procedures are hampered by the non-availability of genetic operations for this strain.


23

The essential data in this case are the presence of large polyfunctional genes in prokaryotic clusters and the surprising lack of strict correlation of expected biosynthetic unit operations within the predicted modules with the actual gene structures found. A similar observation has also been made in the case of the avermectin biosynthetic cluster (MCNEIL et al., 1995). As a recent eukaryotic example the sterigmatocystin biosynthetic cluster in A. niduluns is considered (BROWN al., 1996). Sterigmaet tocystin is the penultimate intermediate in the biosynthesis of aflatoxins. Both polyketides are highly mutagenic and thus carcinogenic. They spoil food upon fungal colonization, especially by A. flavus and A. parasiticus. These losses may be reduced by a detailed understanding of the regulation of the biosynthetic events. So, e. g., the induction of aflatoxin formation has been shown to be strongly suppressed by jasmonate, a phytohormone (GOODRICHTANRIKULU al., 1995). Deet tailed genetic studies have confirmed the linkage and coregulation of sterigmatocystin and aflatoxin biosynthesis (TRAILet al., 1995a, b; KELLER and ADAMS, 1995; BROWNet al., 1996). The recent sequencing of the sterigmatocystin biosynthetic cluster in A. niduluns revealed within a 60 kb region 25 transcripts, the expression of which is coordinated under conditions of toxin production. The cluster is flanked by genes also expressed under nonproduction conditions. The regulatory gene aflR and its A. flavus homolog both specifically induce gene expression within the cluster. Among the identified genes are a fatty acid synthase, five monoxoygenases, four dehydrogenases, an esterase, an O-methyltransferase, a reductase, and an oxidase, all functionally implied in the proposed reaction sequence. Comparative evaluation of the respective cluster in A. parasiticus shows conservation of clustering, but no strict conservation of the gene order (TRAIL al., 1995a, b; et Yu and LEONARD,1995). Conservation of clustering has been suggested to serve both purposes of global regulation and horizontal movement of biosynthetic activities among species. The striking features of the tremendous efforts so far show the integration of a specific fatty acid synthase into a secondary

product cluster. These types of genes have been commonly referred to as primary pathway enzymes. The respective hexanoyl structure serves as a starter and is elongated by a type I1 system forming an aromatic polyketide. So far, such systems have been found only in prokaryotes. Gene characteristics, however, do not suggest a horizontal transfer as in the plactam case (BROWN al., 1996). et Finally, a specific transcription factor is a key element in the expression of the enzyme system, and no evidence has yet been obtained for complex timing and differential gene expression as in the penicillin pathway in A. niduluns. Inspection of other clusters included in Tab. 3 suggests extensive similarities of certain groups which, at first sight, look like structurally unrelated compounds. Certain types of regulatory genes are implied in the formation of various metabolites. There seems to be a non-species-related separation of type I and type I1 systems, e.g., in polyketide formation, but the various degrees of integration of biosynthetic modules catalyzing unit operations may be dictated by the chemistry of their products. Finally, the clustering of pathways also suggests their genetic transfer between various hosts. Within the evolutionary frame, adaptation of pathways to various targets has been proposed, e. g., for Aspergilli adapting to insect colonization and perhaps moving to other target organisms (WICKLOW al., 1994). The structures of et metabolites with key roles in invasive processes would then adapt to new targets by evolutionary processes.

2.2.2 Regulatory MechanismsMechanisms involved in the regulation of secondary metabolite expression have been reviewed recently, focussing on global control in bacterial systems (DOULL and VINING, 1995), bacterial mechanisms in detail (CHATER and BIBB, Chapter 2, this volume), antibiotic formation in Streptomyces coelicolor (HOPWOOD al., 1995), and autoregulators et (HORINOUCHIand BEPPU, 1995; BEPPU, 1995). Euk

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