designer yeasts for the fermentation industry

UDC 663.12:663.1 reviewISSN 1330-9862

(FTB-1195)

Designer Yeasts for the Fermentation Industry

of the 21st

Century

Isak S. Pretorius*, Maret du Toit and Pierre van Rensburg

Institute for Wine Biotechnology, Department of Viticulture and Oenology,Stellenbosch University, Stellenbosch 7600, South Africa

Received: October 25, 2002Accepted: January 24, 2003

Summary

The budding yeast, Saccharomyces cerevisiae, has enjoyed a long and distinguished hi-story in the fermention industry. Owing to its efficiency in producing alcohol, S. cerevisiaeis, without doubt, the most important commercial microorganism with GRAS (GenerallyRegarded As Safe) status. By brewing beer and sparkling wine, mankind’s oldest domesti-cated organism made possible the world’s first biotechnological processes. With the emer-gence of modern molecular genetics, S. cerevisiae has again been harnessed to shift thefrontiers of mankind’s newest revolution, genetic engineering. S. cerevisiae is at the fore-front of many of these developments in modern biotechnology. Consequently, the indu-strial importance of S. cerevisiae has extended beyond traditional fermentation. Today, theproducts of yeast biotechnologies impinge on many commercially important sectors, inclu-ding food, beverages, biofuels, chemicals, industrial enzymes, pharmaceuticals, agricultureand the environment. Nevertheless, since ethyl alcohol produced by yeast fermentation islikely to remain the foremost worldwide biotechnological commodity for the foreseeablefuture, this review focuses on advances made with respect to the development of tai-lor-made yeast strains for the fermented beverage and biofuel industries.

Key words: Saccharomyces cerevisiae, tailor-made yeast strains, fermented beverages, biofuelindustry

Introduction

The power with which microorganisms have causedirreversible revolutions in the existence and activities ofhumankind during the course of history and with whichthese mostly invisible forms of life still influence theweal and woe of this planet can best be illustrated onthe basis of the phenomenal list of achievements of thewell known yeast, Saccharomyces cerevisiae. This simple,unicellular fungus boasts an unequalled track record,making it a true pioneer among microorganisms. As amatter of fact, since the beginning of recorded civilisa-tion, this versatile trailblazer has been instrumental inplanting the most important milestones on the road of

the development of modern humankind. S. cerevisiaetherefore can be regarded as the key to the most hiddensecrets of life, because, of all the microorganisms, it wasthe first to be (i) domesticated by people for the produc-tion of food (e.g. bread in ancient Rome in 100 BC) andbeverages (e.g. beer and wine in Assyria, Caucasia, Mes-opotamia and Sumer in 7000 BC), (ii) observed micro-scopically (by Antonie van Leeuwenhoek), (iii) de-scribed as a living biochemical agent of transformation(by Louis Pasteur), (iv) used as a host for the productionof the first recombinant vaccine (against hepatitis B) andthe first food enzyme (the milk coagulation enzyme,

3I. S. PRETORIUS et al.: Designer Yeasts for the Fermentation Industry, Food Technol. Biotechnol. 41 (1) 3–10 (2003)

* Corresponding author

chymosin, for cheese making), and (v) used to reveal theentire nucleotide sequence of a eukaryotic genome. Onthe basis of the last-mentioned frontier-shifting mile-stone of modern science, there already are strong indica-tions that S. cerevisiae will probably also become the firsteukaryotic cell of which the complexities of the transcrip-tomes, proteomes and metabolomes will be unlocked. Inthis way, it will once again be the forerunner when thetwo strongest forces in modern day science, namelybiotechnology and information technology, are com-bined in a breathtaking new discipline, bioinformatics.This will lead to the third millennium finally biddingfarewell to the industrial economy and entering the ex-citing era of the information economy and the bio-econ-omy.

There is no doubt that the pace of scientific andtechnological innovation will be much faster in the Bio--economy and that S. cerevisiae, as one of the most popu-lar scientific models and commercial microorganisms,will continue to play a leading role in the future. It isexpected that the traditional production of ethyl alcohol(for the beer, wine, distilled beverage and fuel indus-tries) and yeast biomass (for the food and animal feedindustries) will continue to provide the largest quantityof the world’s fermentation products in the foreseeablefuture. This assumption is based on the fact that S. cere-visiae currently remains responsible for the productionof all four of the leading commodity products of fer-mentation (in terms of worldwide tonnage per year), na-mely 60 million tons of beer, 30 million tons of wine,800 000 tons of single cell protein and 600 000 tons ofbaker’s yeast. However, it is an accomplished fact that,in the future, S. cerevisiae will become increasingly im-portant as a host organism for the production of low vol-ume, high value biotechnological products (e.g. commerci-ally important enzymes, chemicals, therapeutic proteinsand other pharmaceutical products).

In the light of these 21st century developments andforecasts, the demand for suitable designer yeasts for awide variety of existing and new products becomes in-creasingly greater and more urgent. Since S. cerevisiae andethanol are likely to remain, respectively, the world’spremier commercial microorganism and biotechnologi-cal commodity for many years to come, this overview isaimed at highlighting the most important targets for thedevelopment of tailored S. cerevisiae strains for the fer-mented beverage and biofuel industries and providing avision for the use of such yeasts.

Tailoring of Yeast Strains for the FermentedBeverage Industry

The healthful benefits of fermented alcoholic bever-ages, now largely attributed to the antimicrobial activityof yeast-derived ethanol, predate recorded history. Beer,wine and distilled spirits were some of the first com-modities to be bartered by early civilisations engaged ininternational trade. Then, as now, the most successfulproducers of beer, wine, brandy and whisky were thosewho grasped the market forces of supply and demand,and whose products met the prevailing definition ofquality (1). Today’s fierce competition for market sharehas led to increased diversity and innovation within the

fermented beverage industry, much to the benefit of theconsumer. This, in turn, has created an urgent quest forcustomised brewing, wine, brandy and whisky yeaststrains. The knowledge of the physiology of genetic ref-erence strains of S. cerevisiae that is now available is agreat help in directing the modification of industrialyeasts toward practical goals, even though the industrialstrains and species are less characterised and their ge-netic make-up is more complex (2).

Strain fingerprinting

In the fermented beverage industry it is very impor-tant that the microorganisms associated with the variousindustrial processes have food grade status. Therefore,with the prominence of S. cerevisiae as starter culture inalcoholic beverages, it is important to ensure the iden-tity and safety of each of the newly developed or se-lected strains. This quest for novel yeast strains that aredesigned to perform specific tasks set by the beverageindustries has led to the development of rapid and reli-able methods for yeast strain differentiation. Moleculargenetic techniques can be used to discriminate betweenyeast strains that have similar physiological characteris-tics. The methods most frequently used today are elec-trophoretic karyotyping, restriction fragment lengthpolymorphisms (RFLP), random amplified polymorphicDNA (RAPD), polymerase chain reaction (PCR), PCRwith specific primers, temperature gradient gel electro-phoresis (TGGE) or denaturing gradient gel electropho-resis (DGGE) of PCR-amplified products, amplifiedfragment length polymorphisms (AFLP) and Fou-rier-transform infrared microspectroscopy (3–6).

Improvement of fermentation performance

The primary selection criteria applied to most straindevelopment programmes in the fermented beverage in-dustry relate to the overall objective of achieving abetter than 98 % conversion of sugar to alcohol and car-bon dioxide, at a controlled rate and without the develop-ment of off-flavours. The growth and fermentation prop-erties of yeast starter cultures have yet, however, to begenetically defined. What makes the genetic definitionof these attributes even more complex is the fact that lagphase, rate and efficiency of sugar conversion, resistanceto inhibitory substances and total time of fermentationare affected strongly by the physiological condition ofthe yeast, as well as by the physicochemical and nutri-ent properties of the grape must.

This complexity explains why it is so difficult to de-fine all the key genetic determinants of a yeast's fermen-tation performance that may be candidates for geneticengineering. However, general targets include increasedstress tolerance and resilience; improved polysaccharidedegradation (e.g. starch, pectin, cellulose and hemicellu-lose), sugar uptake and assimilation; increased ethanoltolerance; improved nitrogen assimilation; enhanced re-sistance to microbial metabolites and toxins; resistanceto heavy metals and agrochemical residues; tolerance tochemical preservatives; and reduced foam formation(2,4,7,8).

As sterols, trehalose, glycogen and aquaporins playmultiple roles in increasing the survival of S. cerevisiae

4 I. S. PRETORIUS et al.: Designer Yeasts for the Fermentation Industry, Food Technol. Biotechnol. 41 (1) 3–10 (2003)

cells exposed to several physical and chemical stresses,they have important implications for the general stresstolerance, resilience, fitness and vigour of active driedyeast starter cultures upon reactivation. As a result,there is a strong incentive to develop yeast strains witha superior ability to accumulate these compounds. How-ever, due to the complex stress response mechanisms inyeast, it is not yet clear whether the deletion of theATH1 trehalase gene and the modification of the expres-sion levels of the genes involved in the metabolism oftrehalose (TPS1, TPS2, ATH1), glycogen (GSY1, GSY2)and sterols (SUT1 and/or SUT2), and in the synthesis ofaquaporins (AQY1, AQY2) will result in an improve-ment in yeast viability and vitality (2,4).

An imbalance in the high levels of carbon and lowlevels of nitrogen in some of the raw materials used inthe fermented beverage industry (e.g. grape must) is an-other cause of poor fermentative performance. Sluggishor stuck fermentations occur because nitrogen depletionirreversibly arrests hexose transport. The main focus toensure the efficient utilisation of sugars under condi-tions of nitrogen limitation is to increase the rate of gly-colytic flux by replacing any non-functional mutant al-leles of genes encoding the key glycolytic enzymes, toenhance the efficiency of hexose (especially fructose) up-take, and to alleviate the assimilation of proline and ar-ginine (accounting for 30 to 65 % of the total amino acidcontent of grape juice) from nitrogen catabolite repres-sion. In the case of incomplete fermentations, the prefer-ence of, for example, wine yeasts for glucose over fruc-tose can lead to excessive residual fructose levels thatcompromise the quality of the wine. It is hypothesisedthat the rate of alcohol production by yeast is limitedprimarily by the rate of sugar uptake, especially the up-take of fructose, in the presence of high sugar levels du-ring the early phase of fermentation and during the fi-nal stages of nitrogen depletion coupled with nutrientlimitation. Therefore, several laboratories focus on pho-sphorylation by the HXK1- and HXK2-encoded hexoki-nases and the GLK1-encoded glucokinase, as well as onhexose transporters encoded by HXT1-HXT18 andSNF3. There is anecdotal evidence that the overexpres-sion of the S. pastorianus FSY1-encoded fructose/H+

symporter, together with some of the other HXT1-HXT18and SNF3-encoded hexose transporters and the HXK1--encoded hexokinase (with the highest affinity for fruc-tose, but still a significantly lower affinity than for glu-cose), result in improved glucose and fructose uptakeduring wine fermentations. Furthermore, the deletion ofthe URE2-encoded repressor of the PUT1-encoded pro-line oxidase and PUT2-encoded pyrroline-5-carboxylatedehydrogenase represents the first step towards the de-velopment of wine yeasts that can efficiently assimilatethe abundant supply of proline and arginine in grapejuice under fermentative conditions (2,4,7,8).

Since S. cerevisiae is only able to metabolise mono-,di- and tri-hexoses, while some raw materials used forthe production of potable ethanol (e.g. beer wort andwhisky grain mash) contain substantial amounts ofpolysaccharides such as starch, there is considerable in-terest in improving the fermentation performance by de-veloping yeast strains that are able to use a wider rangeof carbohydrates. For example, in the whisky and brew-

ing industries, modified S. cerevisiae strains that are ableto hydrolyse and ferment polysaccharides would con-vert these raw materials to ethanol much more effecti-vely than the current starter culture strains. In an attemptto avoid the use of expensive enzyme preparations (oftencontaining unwanted, contaminating or side activities)in the production of grain whisky, low carbohydrate di-abetic beers and low calorie »light« beers, several hete-rologous amylase-encoding genes have been expressedin S. cerevisiae (9). In some instances, these amylolyticstrains have also been improved with respect to their ef-fectiveness to ferment hexoses other than glucose (e.g.maltose and melibiose).

Another thrust to improve the fermentation perfor-mance of S. cerevisiae starter culture strains is to increasetheir resistance to toxic microbial metabolites (e.g. etha-nol, acetic acid, medium chain fatty acids, etc.), zymo-cins (yeast-derived killer toxins or zymocins), chemicalpreservatives (e.g. sulphite) and agrochemicals contain-ing heavy metals (e.g. copper). For example, the modifi-cation of the expression of the SUT1, SUT2, PMA1 andPMA2 genes results in increased sterol accumulationand cell membrane ATPase activity, thereby increasingthe resistance to ethanol (2). In addition, the genetic,mycoviral determinants and other genes encoding killertoxins (zymocidal peptides) and immunity factors canbe incorporated into S. cerevisiae starter culture strains tomake them insensitive to the zymocins of contaminatingwild yeasts. With respect to resistance to agrochemicals,an increase in the copy number of the CUP1 copper che-latin gene enables yeast to tolerate higher levels of cop-per residues in the fermentation medium (4).

Improvement of processing efficiency

The main objectives of product processing, fining(e.g. addition of adsorptive compounds followed by set-tling or precipitation) and clarification (e.g. sedimenta-tion, racking, centrifugation, filtration, etc.) include theremoval of excess amounts of certain components andmicrobial cells to achieve clarity and to ensure the physi-cochemical stability of the end product. The fining andclarification of fermented beverages often include ex-pensive and laborious practices that generate large vol-umes of lees for disposal, thereby causing a loss of pro-duct and the removal of important aroma and flavourcompounds from the remaining product. In order to mi-nimise the disadvantages of these harsh fining and clari-fication practices, an increasing spectrum of relativelyexpensive commercial enzyme preparations (e.g. prote-ases, pectinases, glucanases, xylanases, arabinofuranosi-dases, etc.) are often added to some fermentation media(e.g. grape must and wine). As an alternative strategy tothe addition of costly enzyme preparations that oftencontain unwanted contaminating or side activities, star-ter culture yeasts are being developed to secrete proteo-lytic and polysaccharolytic enzymes that would removehaze-forming proteins and filter-clogging polysaccha-rides, respectively. To this end, the overexpression of se-veral bacterial, fungal and yeast genes resulted in thedevelopment of proteolytic, pectinolytic, glucanolyticand xylanolytic yeasts (9,10).

A second target for the improvement of clarificationand filtration is to efficiently remove all yeast cells from


the liquid phase of the fermentor, tank or barrel (11).The regulated expression of the flocculation genes is im-portant to guarantee a high suspended yeast count for arapid fermentation rate during the fermentation process,while efficient settling is needed to minimise problemswith wine clarification at the end of sugar conversion.Yeast flocculation is particularly important for the pro-duction of bottle-fermented sparkling wine, and the con-trolled onset of yeast flocculation at the appropriatetime during sparkling wine production can simplify thiscostly process. The expression of the FLO1 flocculingene, linked to the late-fermentation HSP30 promoter,can be induced by a heat-shock treatment, confirmingthat controlled flocculation is indeed possible duringfermentation (12). Cell aggregation also plays a key rolein the production of flor sherry, during which a relatedcellular process results in the flotation of the yeast cells,thereby forming a velum (biofilm) on the surface of thewine. By placing the MUC1 (also known as FLO11) mu-cin gene under the control of the HSP30 promoter, theformation of the biofilm can be promoted at the end offermentation, thereby simplifying the development ofthe flor.

Improvement of sensory quality

The single most important factor in the fermentedbeverage industry is the organoleptic quality (appear-ance, aroma and flavour) of the final product. The end-less variety of flavours stems from a complex, comple-tely non-linear system of interactions among manyhundreds of compounds. The bouquet of a beer, wine,brandy or whisky is determined by the presence of awell-balanced ratio of desirable flavour compounds andmetabolites, and the absence of undesirable ones. Theperceived flavour of these products is the result of abso-lute amounts and specific ratios of many of these inter-active compounds, rather than being attributable to asingle »impact« compound. Subtle combinations of tracecomponents derived from the raw material usually elicitthe characteristic flavour and aroma notes, whereas theproducts of yeast fermentation (e.g. esters, alcohols, etc.)contribute to the generic background flavour and aro-ma, as well as to the complexity and intensity of thearoma and taste of the final product (13). However, theflavour of the product immediately after fermentation ordistillation only approximates that of the finished prod-uct. After the sudden and dramatic changes in composi-tion during fermentation and distillation, chemical con-stituents generally react slowly during ageing to moveto their equilibria, resulting in gradual changes in fla-vour. The harmonious complexity of some products,such as wine, brandy and whisky, subsequently can beincreased further by volatile extraction during oak bar-rel ageing.

There is an obvious need for the development ofS. cerevisiae starter culture strains that could impart spe-cific desirable characteristics to beer, wine, brandy andwhisky. To this end, significant progress has been madein the construction of yeasts producing colour- and aro-ma-liberating enzymes (e.g. pectinases, glycosidases,glucanases, arabinofuranosidases, etc.) and ester-modify-ing enzymes (e.g. alcohol acetyl transferases, esterases,isoamyl acetate hydrolysing enzyme, etc.) (11). Further-

more, yeasts have been developed that produce optimallevels of glycerol (the overexpression of GPD1, GPD2and FPS1, together with the deletion of the ALD6 acetal-dehyde dehydrogenase gene), fusel oils (e.g. isobutyl al-cohol, isoamyl alcohol, etc.), and phenolic acids (modi-fied expression of the yeast PAD1 phenyl acrylic aciddecarboxylase gene, as well as the expression of bacte-rial pdc �-coumaric acid decarboxylase and padc phenolicacid decarboxylase genes).

The bioadjustment of acidity in wine can be achie-ved by recombinant wine yeasts containing combinati-ons of genes cloned from Schizosaccharomyces pombe andlactic acid bacteria (14,15). A wine yeast that containsthe S. pombe mae1 malate permease gene and the mae2malic enzyme gene converts malic acid to ethanol (ma-loethanolic fermentation), whereas a transformant carry-ing the mae1 gene, together with the Oenococcus oeni(mleA), Lactococcus lactis (mleS) or Lactobacillus delbrueckii(mleS) malolactic enzyme gene, converts malic acid intolactic acid (malolactic fermentation). The maloethanolicwine yeast would be preferred for low pH wines fromthe cooler wine-producing regions, while the malolacticwine yeast would provide the best solution for high pHwines from the warmer regions. In the case of high pHwines, the production of additional lactic acid duringfermentation can be achieved by incorporating the Lacto-bacillus casei LDH1 lacticodehydrogenase gene into themalolactic wine yeast strain (2,4). These yeasts also pre-clude the requirement for the use of bioamine-formingmalolactic bacteria in red wine and certain styles ofwhite wine that need to undergo malolactic fermenta-tion.

Since yeast can also be responsible for the produc-tion of unwanted byproducts, such as hydrogen sulphide,dimethyl sulphide, sulphite, diacetyl and phenolic off--flavours, there is an incentive to delete some endoge-nous genes from commercial strains of S. cerevisiae. Forexample, yeasts carrying disrupted alleles of the MET14adenosylphosphosulphate kinase, MET10 sulphite re-ductase, MRX1 methionine sulphoxide reductase and/or MET2 homoserine O-acetyl transferase have beenconstructed so that the production of sulphite and sul-phide can be controlled during fermentation. Some ofthese modified yeasts have been shown to produce re-duced levels of sulphide and optimal levels of sulphite,which facilitate increased flavour stability (2,4,16). Fur-thermore, two different strategies have been used in at-tempts to reduce the levels of diacetyl in beer. In thefirst, diacetyl formation was reduced in ILV2 mutantsand in strains in which the expression of ILV5 was in-creased. The second strategy consisted of expressing theALDC acetolactate decarboxylase gene from Enterobacteraerogenes in S. cerevisiae. This transformant producedconsiderably less diacetyl during fermentation than theparent yeast (16).

Biopreservation of spoilage microorganisms

Uncontrolled microbial growth before, during or af-ter fermentation can alter the chemical composition ofthe end product, thereby detracting from its sensoryproperties of appearance, aroma and flavour. Unspoiledraw material and sound, hygienic fermentation practicesare the cornerstones of the industry’s strategy to prevent


the uncontrolled proliferation of spoilage microbes.Added safety is provided by the addition of chemicalpreservatives, such as sulphur dioxide, dimethyl dicar-bonate, benzoic acid, fumaric acid and sorbic acid,which control the growth of unwanted microbial con-taminants. However, the excessive use of these chemicalpreservatives is harmful to the quality of the end prod-uct and is confronted by mounting consumer resistance.Consumer preferences have shifted to products that areless heavily preserved with chemicals, less processed, ofhigher quality, more natural and healthier. Therefore,biopreservation with yeast-derived metabolites (e.g. for-mation of SO2 or hydrogen peroxide during wine fer-mentations), antimicrobial enzymes (e.g. lysozyme, chiti-nases, endoglucanases, etc.) and peptides (zymocins andbacteriocins) is currently being considered as an alterna-tive strategy to chemical preservation. However, the useof purified antimicrobial enzymes and bacteriocins is ex-pensive, resulting in an increase in retail costs. Thisproblem might be circumvented by expressing effectiveantimicrobial enzymes and peptides in wine yeaststarter culture strains, thereby addressing the wine in-dustry’s call for wines of higher quality and purity. Tothis end, the hen egg white lysozyme gene (HEL1), thePediococcus acidilactici pediocin gene (PED1) and theLeuconostoc carnosum leucocin gene (LCA1) have beenused to engineer bactericidal yeasts (4,16–18). The anti-fungal yeast CTS1-encoded chitinase and EXG1-encodedexoglucanase have also been expressed in S. cerevisiae(2,19). The main approach in the construction of zymoci-dal strains entails the inclusion of a combination of my-coviral killer toxin determinants of S. cerevisiae (e.g. aK1/K2 double killer) and zymocin-encoding genes fromother yeasts (e.g. Hanseniaspora, Kluyveromyces, Pichia,etc.) into S. cerevisiae starter culture strains. The idealwould be to incorporate all of these antimicrobial activi-ties into a single starter culture strain, thereby counter-acting all contaminating spoilage bacteria, yeasts andmoulds (2,16,20,21).

Improvement of wholesomeness

It is generally accepted that moderate drinking ofalcoholic beverages (especially wine) can be socially be-neficial and effective in the management of stress andthe reduction of coronary heart disease. The principalprotective compounds found in products such as wineinclude the phenolic compounds, resveratrol, salicylicacid and alcohol (4,22). However, prudent consumers ofthese products are increasingly fastidious about the pre-sence of undesirable compounds in fermented bever-ages. These unwanted compounds include suspectedcarcinogens, such as ethyl carbamate, neurotoxins, suchas biogenic amines, and asthmatic chemical preserva-tives, such as sulphites. The most finicky among thesefussy consumers are even concerned about high levelsof alcohol in fermented beverages. When yeast starterstrains are developed, it therefore is of the utmost im-portance to focus on these health aspects and to developyeasts that may enhance the benefits (e.g. production ofresveratrol, carnitine, etc.) and reduce the risks (e.g. eli-minating ethyl carbamate and biogenic amines, and re-ducing the levels of alcohol) associated with moderatealcohol consumption (2,16).

With regard to the production of resveratrol duringfermentation, progress has already been made by con-structing a wine yeast that expresses the 4CL9/216 co-en-zyme A ligase, VST1 stilbene synthase and BGL1 �-glu-cosidase genes (23). The development of a bactericidalyeast that is deleted for the CAR1 arginase gene (block-ing the secretion of urea, the precursor for the formationof ethyl carbamate) or that is transformed with heterolo-gous urease genes (enabling the degradation of urease)would reduce the levels of added sulphite, yeast-deri-ved ethyl carbamate and bioamines formed by bacterialcontaminants. The bioreduction of the levels of alcoholin fermented beverages can be achieved by redirectingthe carbon flux away from ethanol formation and to-wards the production of glycerol and gluconic acid. Asignificant increase in the level of extracellularly accu-mulated glycerol and concomitant decreases in ethanolconcentrations have been achieved by the overexpressi-on of the endogenous GPD1 and GPD2-encoded glyc-erol-3-phosphate dehydrogenase isozymes of S. cerevisiae,together with the constitutive expression of its FPS1-en-coded glycerol transport facilitator (2,4,24). Similar de-creases in ethanol levels have been achieved by the ex-pression of the Aspergllus niger GOX1 glucose oxidasegene in S. cerevisiae (25).

Tailoring of Yeast Strainsfor the Biofuel Industry

Fuel ethanol, which has a higher octane rating thangasoline, accounts for approximately two-thirds of theworld’s total annual ethanol production of more than 31billion litres (26). The primary reasons for consideringthe expanded use of biofuel ethanol concern sustainableresource supply, enhanced security and the realisationof macroeconomic benefits for rural communities andthe economy at large (27,28). All indications are thatdramatic changes in energy supply will occur in the 21st

century, particularly relating to oil. It is estimated thatworld oil production will peak within the next 25 yearsand then follow a permanent decline. The time requiredto develop and deploy new technologies that allow asmooth transition from oil to plant biomass as the onlyforeseeable sustainable source of biofuels is running out.Furthermore, more than two-thirds of the world’s re-maining oil reserves lie in the Middle East (includingthe Caspian Sea), which is an increasingly prominentconcern in the context of post-Cold War considerationsrelating to security of nations (27,28). If a transition fromfossil fuels to biofuels becomes affordable, this unwel-come dependence on the Middle Eastern cartels control-ling the production, manufacturing and marketing of oilcould diminish and the world’s security could be im-proved in many ways. A transition to biofuel ethanolwould increasingly limit the ability of oil-exportingcountries and regimes to shape world events and woulddemocratise the world’s energy markets. Since biomassfeedstocks are available as both residues and dedicatedcrops at a lower price on a per energy basis than oil, itis foreseen that the agricultural sector and the economichealth of rural communities could be promoted, withhuge benefits for the economy at large. In addition tothese political and socioeconomic considerations, a tran-


sition from fossil fuel to biofuel ethanol would also con-tribute to a cleaner environment (26). The incompletecombustion of fossil fuels emits smog-forming gases,while their extraction, processing and combustion resultin the pollution of the air, water and soil, and are thushazardous to the environment and to public health. Byusing biomass-derived ethanol, the net reduction in thelevels of carbon dioxide (the main polluting greenhousegas) could range between 60 and 90 % relative to gaso-line-consuming vehicles (26).

In the light of the above-mentioned aspects relatingto oil exhaustion, a possible interruption in oil supplycaused by political meddling, a superior net perfor-mance of biofuel ethanol in comparison with gasoline,and a significant reduction in pollution levels when fos-sil fuel is replaced by bioethanol as a sustainable energysource for fuel transportation, it can be expected that thedemand for cheap, renewable substrates and cost-effec-tive ethanol production processes will become evermore urgent. In this regard, plant biomass is the onlyforeseeable sustainable source of fuel bioethanol becauseof its relatively low cost and plentiful supply (26–28).The central technological impediment to more wide-spread utilisation of this important resource is the gen-eral absence of low-cost technology for overcoming therecalcitrance of plant biomass (28). A promising strategyto overcome this obstruction involves the metabolic en-gineering of S. cerevisiae strains for use in an integratedprocess, known as direct microbial conversion (DMC) orconsolidated bioprocessing (CBP) (26,28). Such an inte-grated process differs from the earlier SHF (separate hy-drolysis and fermentation) and SSF (simultaneous sac-charification and fermentation in which enzymes fromexternal sources are used) strategies in that the produc-tion of polysaccharide-degrading enzymes, the hydroly-sis of biomass and the fermentation of the resulting sugarsto ethanol takes place in a single process via a polysac-charide-fermenting yeast strain (9). The CBP strategy of-fers a very large cost reduction if S. cerevisiae strains thatpossess the required combination of substrate utilisationand product formation properties can be developed. Thedesired substrate utilisation properties include the pro-duction of a hydrolytic enzyme system allowing highrates of hydrolysis and the utilisation of the resultinghydrolysis products under anaerobic conditions. The de-sired product formation properties include high productselectivity and high concentrations. An efficient polysac-charide-degrading yeast with this combination of prop-erties has not been described to date. The often-underes-timated diversity of yeast species encompasses organismswith a broad range of properties that differ from S. cere-visiae and could be useful for CBP. However, S. cerevisiaehas received the most attention with regard to the he-terologous expression of amylases, pectinases, cellulasesand hemicellulases, as well as the production of ethanol.

While industrial strains of S. cerevisiae are unable toferment polysaccharides, certain strains produce saccha-rolytic enzymes with limited activity on polysacchari-des. These endogenous genes include the glucoamylasegenes (STA1, STA2, STA3, and SGA1), the pectinase ge-nes (PGU1 and PGL1) and the glucanase genes (EXG1,EXG2, BGL2 and SSG1) (9). These genes are of interestbecause their encoded enzymes may augment the effec-

tiveness of heterologous polysaccharolytic componentsand because the expression of natural S. cerevisiaesaccharases may provide clues to the effective expres-sion of recombinant enzymes.

A large number and wide variety of genes encodingamylases, pectinases, cellulases and hemicellulases havebeen cloned from various bacteria, yeasts, filamentousfungi, plants and animals and have successfully been ex-pressed in S. cerevisiae (9,28). However, limited growthon non-native polysaccharide substrates has been repor-ted for only a small number of these recombinant stra-ins of S. cerevisiae. Furthermore, this research is at pres-ent more advanced with respect to �-linked substrates(starch) than �-linked substrates (cellulose and its deriv-atives). The growth of S. cerevisiae on starch, under aero-bic conditions in most, if not all, cases, has been demon-strated in several studies. A significant breakthroughstudy has achieved the utilisation of 100 g/L starch withthe production of 44 g/L ethanol and 8 g/L cells. Al-though the ethanol and cell yields on starch were simi-lar to those on glucose, the specific growth rate was ne-arly 10-fold lower on starch (28).

With respect to �-linked substrates, the growth ofrecombinant S. cerevisiae has been demonstrated on cel-lobiose and cellodextrins (28). Anaerobic SSF with Avi-cel as the substrate has also been investigated, using anS. cerevisiae strain expressing endo/exoglucanase and�-glucosidase originating from a species of Bacillus. Thisstrain produced filter paper activity under both aerobicand anaerobic conditions, but was not shown to grow orto produce ethanol in the absence of added cellulase(28).

Despite only a relatively small number of studies,significant progress has been made in the area of utilisa-tion of non-native substrates by virtue of the heterolo-gous expression of saccharolytic enzymes. In the case ofstarch, some results are encouraging, not only with re-gard to the feasibility of starch utilisation, but also in re-spect of the overall feasibility of enabling the utilisationof non-native substrates. Increased rates of growth andhydrolysis, as well as the use of insoluble substrates,would appear to be logical objectives for starch utilisa-tion (28). Work aimed at the microbial utilisation of�-linked substrates was undertaken much later than thework aimed at starch, with the first such studies appear-ing in 1998, and is less advanced. Growth on cellobioseand cellodextrins provides a point of departure that canbe built upon in future work. A logical next step wouldappear to be anaerobic growth in the absence of addedcellulase enzymes, perhaps first on non-crystalline cellu-lose and then on crystalline cellulose (28).

Concluding Remarks and Future Prospects

Genetic engineering has rapidly revolutionised sev-eral areas of basic and applied yeast biology. The pastdecade has seen marked advances in the depth andbreadth of scientific understanding of the fields of mo-lecular yeast genetics, physiology and biotechnology.However, no genetically engineered yeast is currentlyused in the commercial production of fermented bever-ages and fuel bioethanol. And, in the case of fermentedbeverages, this will continue to be the case unless both


the consumers and the industry are satisfied that theproducts derived from genetically engineered yeasts aresafe, of high quality and beneficial. The successful com-mercialisation of transgenic yeasts for the fermentationindustry will depend on a multitude of scientific, techni-cal, economic, marketing, safety, regulatory, legal andethical issues. However, we are optimistic that, in thelong run, these hurdles will be overcome and that ge-netically tailored yeast strains will be of immense im-portance to the fermentation industry of the 21st cen-tury.

Acknowledgements

Ms. Marisa Honey for critical reading of this manu-script. The research conducted in the Institute for WineBiotechnology is supported by grants from the NationalResearch Foundation (NRF) and the South African WineIndustry (Winetech).

References

This article represents a summary of several com-prehensive review articles and book chapters that haverecently been published. Due to the limited space allo-cated to this paper, all references are not listed here. Thereader therefore is referred to the original scientific arti-cles cited in the following listed papers.

1. L. F. Bisson, A. L. Waterhouse, S. E. Ebeler, M. A. Walker,J. T. Lapsley, Nature, 418 (2002) 696–699.

2. I. S. Pretorius, F. F. Bauer, Trends Biotechnol. 20 (2002) 426–432.

3. I. S. Pretorius, T. J. van der Westhuizen, O. P. H. Aug-sustyn, S. Afr. J. Enol. Vitic. 20 (1999) 61–74.

4. I. S. Pretorius, Yeast, 16 (2000) 675–729.

5. P. Raspor, F. Cus, K. P. Jemec, T. Zagorc, N. Cadez, J. Ne-manic, Food Technol. Biotechnol. 40 (2002) 95–102.

6. M. Wenning, H. Seiler, S. Scherer, Appl. Environ. Microbiol.68 (2002) 4717–4721.

7. R. Snow: Genetic improvement of wine yeast. In: Yeast Ge-netics-Fundamental and Applied Aspects, J. F. T. Spencer, D.M. Spencer, A. R. W. Smith (Eds.), Springer-Verlag, NewYork (1983) pp. 439–459.

8. I. S. Pretorius, Aust. N.Z. Wine Indust. J. 14 (1999) 42–47.

9. I. S. Pretorius: Utilization of polysaccharides by Saccharom-yces cerevisiae. In: Yeast Sugar Metabolism, F. K. Zimmer-

mann, K.-D. Entian (Eds.), Technomic Publishing, Pennsy-lvania (1997) pp. 459–501.

10. P. van Rensburg, I. S. Pretorius, S. Afr. J. Enol. Vitic. 21(2000) 52–73.

11. I. S. Pretorius: The improvement of wine yeasts. In: FungalBiotechnology, D. Arora (Ed.), Marcel Decker (2002) (inpress).

12. K. J. Verstrepen, F. F. Bauer, G. Michiels, F. Derdelinckx,F. Delvaux, I. S. Pretorius, Eur. Brew. Conv. Monogr. 28(2000) 30–42.

13. M. G. Lambrechts, I. S. Pretorius, S. Afr. J. Enol. Vitic. 21(2000) 97–129.

14. H. Volschenk, M. Viljoen, J. Grobler, F. Bauer, A. Lonva-ud-Funel, M. Denayrolles, R. E. Subden, H. J. J. van Vuu-ren, Am. J. Enol. Vitic. 48 (1997) 193–196.

15. H. Volschenk, M. Viljoen, J. Grobler, B. Petzold, F. F. Bau-er, R. Subden, R. A. Young, A. Lonvaud, M. Denayrolles,H. J. J. van Vuuren, Nature Biotechnol. 15 (1997) 253–257.

16. S. Dequin, Appl. Microbiol. Biotechnol. 56 (2001) 577–588.

17. M. du Toit, I. S. Pretorius, S. Afr. J. Enol. Vitic. 21 (2000)74–96.

18. H. Schoeman, M. A. Vivier, M. Du Toit, L. M. T. Dicks, I.S. Pretorius, Yeast, 15 (1999) 647–656.

19. M. Carstens, M. A. Vivier, P. van Rensburg, I. S. Pretorius,Ann. Microbiol. (2002) (in press).

20. G. M. Walker: Yeast Physiology and Biotechnology, John Wi-ley & Sons, New York (1998).

21. J. R. M. Hammond: Brewer’s yeast. In: The Yeasts, Vol. 5.Yeast Technology, A. H. Rose, J. S. Harrison (Eds.), Acade-mic Press, London (1993) pp. 7–67.

22. G. O. Armstrong, M. G. Lambrechts, E. P. G. Mansvelt, D.P. van Velden, I. S. Pretorius, S. Afr. J. Sci. 97 (2001)279–282.

23. J. W. Becker: Plant defence genes expressed in tobacco andyeast, MSc Thesis, Stellenbosch University (2002).

24. E. Nevoigt, R. Pilger, E. Mast-Gerlach, U. Schmidt, S. Frei-hammer, M. Eschenbrenner, L. Garbe, U. Stahl, FEMS Ye-ast Res. 2 (2002) 225–232.

25. D. F. Malherbe, M. Du Toit, R. R. Cordero Otero, P. vanRensburg, I. S. Pretorius, Appl. Microbiol. Biotechnol. (2002)(submitted).

26. J. Zaldivar, J. Nielsen, L. Olsson, Appl. Microbiol. Biotec-hnol. 56 (2002) 17–34.

27. L. R. Lynd, C. E. Wyman, T. U. Gerngross, Biotechnol.Progr. 15 (1999) 777–793.

28. L. R. Lynd, P. J. Weimar, W. H. van Zyl, I. S. Pretorius,Microbiol. Mol. Biol. Rev. 66 (2002) 506–577.

Konstruirani tipovi kvasaca

za fermentativnu industriju u 21. stolje}u

Sa`etak

Klijaju}i kvasac, Saccharomyces cerevisiae, ima dugu i bitnu ulogu u fermentacijskoj in-dustriji. Zbog svoje sposobnosti da proizvede alkohol, Saccharomyces cerevisiae je nedvojbe-no najva`niji komercijalni mikroorganizam sa statusom GRAS (generally regarded assafe). Budu}i da je najstariji udoma}eni mikroorganizam ~ovje~anstva omogu}io proizvod-nju piva i pjenu{avog vina, pridonio je razvoju i prvih svjetskih biotehnolo{kih procesa.Pojavom moderne molekularne genetike, Saccharomyces cerevisiae je ponovno pridonio po-micanju granica najnovije ljudske revolucije, geneti~kog in`enjerstva. Saccharomyces cerevi-


siae je najistaknutiji u razvoju novih biotehnolo{kih grana pa se njegova industrijska va-`nost prote`e izvan granica tradicionalne fermentacije. Danas proizvodi biotehnologijekvasca zadiru u mnoga komercijalno va`na podru~ja, uklju~uju}i hranu, pi}a, biolo{ka go-riva, kemikalije, industrijske enzime, farmaceutske proizvode, agrikulturu i okoli{. Ned-vojbeno, budu}i da se etilni alkohol proizvodi fermentacijom kvasca, vjerojatno }e ostativode}i svjetski biotehnolo{ki proizvod u predvidivoj budu}nosti. Ovaj je rad usmjeren nanapredak postignut razvojem sojeva kvasca posebno konstruiranih za fermentirana pi}a iindustriju biolo{kih goriva.


designer yeasts for the fermentation industry

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