Applied Mycology and BiotechnologyVolume 2. Agriculture and Food Production 1@ 2002 Elsevier Science B.V. Al! rights reserved

Brewer's Yeast: Genetics and Biotechnology

Julio PolainaInstituto de Agroquímica y Tecnología de Alimentos, Consejo Superior deInvestigaciones Científicas, Apartado de Correos 73, E46100-Burjasot (Valencia),

Spain (E-Mail:jpola,ina@iata.csic.es).

The advance of Science in fue 19th century was a decisive force for fue deve10pment andexpansion of fue modero brewing industry. Correspondingly, fue brewing industrycontributed important scientific achievements, such as Hansen's isolation of pure yeastcultures. Early studies on yeast were connected to fue development of different scientificdisciplines such as Microbiology, Biochemistry and Genetics. An example of this connectionis Winge's discovery of Mendelian inheritance in yeast. However, genetic studies with fuespecific type of yeast used in brewing were hampered by fue complex constitution of thisorganismo The emergence of Molecular Biology allowed a precise characterization of fuebrewer's yeast and fue manipulation of its properties, aimed at fue improvement of thebrewing process and fue quality of fue beer.

1. INTRODUCTIONThe progress of chemistry, physiology and microbiology during the 19th.Century, allowed

a scientific approach to brewing that caused a tremendous advancement on fue production ofbeer. The precursor of such approach was the French microbiologist Louis Pasteur. At thistime, fue Danish brewer Jacob Christian Jacobsen, algO founded fue Carlsberg Brewery andfue Carlsberg Laboratory. In Jacobsen's own words, fue purpose ofthe Carlsberg Laboratorywas: "By independent investigation to test the doctrines already furnished by Science and bycontinued studies to develop them into as fully scientij/c a basis as possible for the operationof mailing, brewing and fermentation". Louis Pasteur (1822-1895) demonstrated thatalcoholic fermentation is a process caused by living yeast cells. His conclusion was thatfermentation is a physiological phenomenon by which sugarsare converted in ethanol as aconsequence of yeast metabolismo In 1876, Pasteur published "Etudes sur la Biere", whichfollowed the trend of his previous book "Etudes sur le Vin", published ten years earlier. InEtudes sur la Biere, he dealt with fue diseases ofbeer and described how the fermenting yeastwas often contaminated by bacteria, filamentous fungi, and other yeasts. However, theimportance of Pasteur in relation with brewing is due to his discovery of yeast as fue agent offermentation. His more specific contributions to this field are not to be considered among hisgreatest achievements. Probably, this had something to do with the fact that he did not likebeer. Pasteur's work in connection with yeast and fue brewing industry has been recentlyreviewed by Anderson [1] and Bamett [2]. A crucial achievement for the developmentofthebrewing industry was accomplished by Emil Christian Hansen (1842-1909). Originallytrained as a house painter and a primary school teacher, E. C. Hansen later became a botanistand a mycologist. In 1877, he was employed as a fermentation physiologist at fue CarlsbergBrewery. Familiar with fue work of Pasteur and facing fue problems of microbialcontamination that often caused serious troubles in breweries, Hansen pursued the idea of

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obtaining pille yeast cultures. To this end, he estimated the amount of yeast cells present in abeer sample. He made serial dilutions of fue sample until he reached an estimatedconcentration ofO.5 cells per mI, and used 1 mI aliquots afilie diluted suspension to inoculatemany individual flasks containing wort. After about a week oí incubation, roughIy half of fuecultures contained a single yeast colony, very few contained. two or more colonies, and nogrowth was observed in fue other half of fue flasks. Hansen concluded from this experimentthat it was possible to obtain a single colony consisting of fue uncontaminated descendants of .an individual cell. He performed additional experiments in which, starting with a mixture oftwo or more types of yeast, he was able to recover pille cultures of each different type.Another important contribution of Hansen to fue work with yeast was fue introduction ofcultures on "solid medium". For this purpose he adapted fue procedure devised by RobertKoch for bacteria. Yeast colonies were grown on glass plates, on fue surface of a jellifiedmedium prepared with gelatin. Hansen's new techniques allowed him to obtain pille culturesof different brewing strains and also to characterize contaminant strains that caused differentbeer diseases. In 1883, fue Carlsberg Brewery started industrial production of lager beer withone of Hansen's pille cultures. This event became a milestone of the industrial revolution,since it meant fue transition from small-scale, artisan brewing to large-scale, moderoproduction. The path led by the Carlsberg Brewery was soon followed by other companies,and in fue next few years fue technique of brewing with pille yeast cultures became standardin Europe and North America and caused an exponential growth of beer production all ayerthe world. An exciting account ofthe work ofHansen has been given by van Wettstein [3].

0jvind Winge was born in Arhus (Denmark) in 1886, shortly after the first industrialbrewing with apure yeast culture. Winge was a very capable biologist who mastereddifferent disciplines, including botany, plant and animal genetics, and mycology. In 1921, hebecame Professor of Genetics, firstly at the Veterinary and Agricultural University ofCopenhagen and several years later at University of Copenhagen. Winge took the position ofDirector of fue Department of Physiology at fue Carlsberg Laboratory in 1933. Whenestablished in bis new position, he recovered fue collection of natural and industrial yeaststrains gathered by Hansen and Albert Klocker, who both had preceded him at fueDepartment of Physiology. Winge faced fue problem that brewer's yeast strains were not ableto sporulate, or did so very poorly, which made them unsuitable for genetic analysis.Therefore, he focused bis attention on baker's yeast (S. cerevisiae), which had long been afavorite organism for biochemical studies, and different varieties of Saccharomyces capableof sporulation (S. ludwigii, S. chevalieri, S. ellipsoideus, and others). With the help of amicromanipulation system of bis own design, Winge carried out dissection of fue asci ofsporulated yeast cultures _and followed the germination of individual spores. He concludedthat Saccharomyces has a normal alternance of unicellular haploid and diploid phases, i. e. it ...

should behave genetically according to Mendel's laws. In collaboration with O. Laustsen,Winge reported the first results oftetrad analysis. After a lag period imposed by World Warn, Winge started a very productive period that is marked by his collaboration with Catherine o;

Roberts. Together, they discovered fue gene that controls homothallism and many genes thatcontrol maltose and sucrose fermentation. They also found that haploid yeast strains mighthave several copies afilie genes involved in fue fermentation ofthese sugars. They coined fueexpression polymeric genes to designate a repeated set of genes that perform fue samefunction. The beginning of fission yeast (Schizosaccharomyces pombe) genetics is alsolinked to Winge. Urs Leupold spent a research stay in Winge's Department of Physiologywhere he established fue mating system and described the first cases of Mendelianinheritance for this yeast [4]. The work ofWinge in connection with yeast has been reviewedby R. K. Mortimer [5]. The birth of yeast genetics had a strong Scandinavian clout sincebesides Winge, fue other prominent figure was Carl C. Lindegren, born in 1896 in Wisconsin,

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USA, in a family of Swedish immigrants. The most transcendent achievement of Lindegrenin connection with yeast genetics was fue discovery of fue mating types. This led todevelopment of stable haploid cultures of both mating types and served to start fue cycle ofmutant isolation and genetic crosses that made of Saccharomyces one of the mostconspicuous organisms for genetic research. Other important achievements were fuediscovery of fue phenomenon of gene conversion and the elaboration of fue first genetic mapsof fue yeast. The work and the controversial personality of Lindegren have been the subjectof an inspiring book chapter [6].

In 1847, the brewer J. C. Jacobsen started fue production ofbottom fermented (lager) beerat a brewery that he built in V alby, in fue outskirts of Copenhagen. He named bis breweryCarlsberg after bis five years old son Carl, who later became a maecenas of arts in Denmark,J. C. Jacobsen was one of the pioneers of industrialization in Denmark. He introduced newprocedures in fue brewing process that soon became standard and gave Carlsberg a rapidsuccess. In 1875-76, J. C. Jacobsen established fue Carlsberg Foundation and fue CarlsbergLaboratory. The Carlsberg Laboratory was divided in two Departments, Physiology andChemistry. As a tradition, both Departments have focused their work main1y, albeit notexclusively, on processes and organisms of special significance for brewing, s'uch as yeastand barley. The first director of fue Department of Chemistry was Johan Kjeldah1, whoinvented fue procedure for fue determination of organic nitrogen that carries bis llame.Undoubtedly, fue most popular contribution of the Department of Chemistry was fue conceptof pH, due to S0ren P. L. S0rensen who was head ofthe Department from 1901 to 1938. Ofoutstanding scientific significance was fue work of fue following director, Kaj U.Lindestr0m-Lang, who devised fue terms primary, secondary, and tertiary structure, todescribe fue structural hierarchy in proteins. The contributions of two former directors of theDepartment of Physiology, Hansen and Winge, have been summarized above. More recentwork carried out with yeast will be dealt with in fue following sections. Together with fuework with yeast, fue Department of Physiology has produced important contributions relatedto chlorophyll biosynthesis [7,8].

2. GENE TIC CONSTITUTION OF BREWER'S YEASTSaccharomyces cerevisiae is one of fue best genetically characterized yeast as its genome

is fully sequenced and analyzed exhaustively [9]. Procedures for genetic manipulation of S.cerevisiae are available on tapo Being a eukaryotic, fue key of its success lies in the selectionof a moJel strain with a perfect heterothallic life cycle [10]. In contrast, brewer's yeast isrefractory to the genetic procedures used with laboratory strains. The main reason is its lowsexual fertility. Like most other industrial yeast, brewing strains do not sporulate or do sowith low efficiency. Even in those cases that they show a suitable sporulation frequency,most spores are not viable. The use of appropriate techniques and patient work, carried outmostlyat fue Carlsberg Laboratory during fue last two decades, has lead to fue elucidation offue genetic constitution of a representative strain of brewer's yeast. Ibis work has beenrecently reviewed by Andersen et al. [11].

2.1. Strain TypesThere are basically two kinds of yeast used in brewing that correspond to fue ale and lager

types of beer. Ale beer is produced by a top-fermenting yeast that works at about roomtemperature, ferments quickly, and produces beer with a characteristic fruity aroma. Thebottom-fermenting lager yeast works at lower temperatures, about 10-14°C, ferments moreslowly and produces beer with a distinct taste. The vast majority of beer productionworldwide is lager. It is difficult to make generalizations conceming fue yeast strains used forthe industrial production of beer, since they are generally ill characterized and very few

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comparative studies have been reported. Bottom fermenting, lager strains are usually labeledSaccharomyces carlsbergensis. Although strains from different sources show differencesregarding cell size, morphology and frequency of spore formation, it is unlikely that fuesedifferences reflect a significant genetic divergence. Only o~e strain, Carlsberg productionstrain 244, has been extensively analyzed and most of fue studies described in fue followingsections have been conducted with this strain.

2.2. Genetic CrossesEarly attempts to carry out conventional genetic analysis with brewer's yeast faced fue

problems of poor sporulation and low viability [12]. To overcome this difficulty, severalresearchers hybridized brewing strains with 1aboratory strains of S. cerevisiae [13-16].Notwithstanding fue poor performance of brewing strains, viable spores were recovered fromthem. Some of fue spores had mating capability and could be crossed with S. cerevisiae togenerate hybrids easier to manipulate. The meiotic offspring of fue hybrids was repeatedlybackcrossed with laboratory strains of S. cerevisiae to bring particular traits of fue brewingstrain into an organism amenab1e to analysis. This procedure was followed to studyflocculence, an important character in brewing [13,17]. Gjermansen and Sigsgaard [18]carried out a detailed analysis of the meiotic offspring of S. carlsbergensis strain 244. Theyobtained viable spore clones of both mating types. Celllines with opposite mating type werecrossed pairwise to generate a number of hybrids that were tested for brewing performance.One of them was as good as fue original strain. Additionally, fue clones derived from strain244 with mating capability served as starting material for further genetic analysis which aredescribed in fue following section.

2.3 kar Mutants and Chromosome TransferNuclear fusion (karyogamy), which takes place following gamete fusion (plasmogamy), is

fue event that instates fue diploid phase in all organisms endowed with sexual reproduction. J.Conde and collaborators carried out a genetic analysis of nuclear fusion in S. cerevisiae byisolating mutations in different genes that control fue process (kar mutations) [19,20]. Thekar mutations served as a basis for a comprehensive study of the molecular mechanisms thatcontrol karyogamy, carried out by Rose and collaborators (see review by Rose) [21]. The karmutations have been particularly useful tools to investigate cytoplasmic inheritance [22-24].Additionally, fue kar mutations supplied new genetic techniques. For instance, fuechromosome number of virtually any Saccharomyces strain can be duplicated upon matingwith a kar2 partner [25]. These new tools and techniques opened a new way for fuecharacterization of fue brewer' s yeast. Nilsson- Tillgren et al. [26] and Dutcher [27],described that when a normal Saccharomyces strain mates with a kar 1 mutant, transfer ofgenetic information occurs at a low frequency between nuclei (Fig. 1). Nuclear transferevents also occurs with kar2 and kar3 mutants [20]. Using strains with appropriate genetic ~

markers, one can select fue transfer of specific chromosomes. Ni1sson- Tillgren et al. [28]used kar l-mediated chromosome transfer to obtain a S. cerevisiae strain that carried an extracopy of chromosome III from S. carlsbergensis. Since fue brewing strain does not matenormally, fue strain used in kar crosses was a meiotic derivative of strain 244 with matingcapability [18]. When disomic strains for chromosome III (al so referred to as chromosomeaddition strains) were crossed to haploid S. cerevisiae strains, normal spore viability wasobtained, allowing tetrad analysis. In this process, one of fue two copies of chromosome IIIcan be lost. If fue original S. cerevisiae copy is lost, fue result is a "chromosome substitutionstrain" carrying a complete S. cerevisiae chromosome set, except chromosome III, whichcomes from S. carlsbergensis. Meiotic analysis of crosses between chromosome III additionstrains and laboratory strains of S. cerevisiae revealed two important facts: (i) fue functional

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equivalence of chromosome 1II for fue brewing strain and S. cerevisiae, since ascosporeviability and chromosome segregation were normal, and (ii) in grite of fue functionalequivalence, fue two copies of chromosome 1II were different since fue overall frequency ofrecombination between them was much lower than that expected for perfect homologues.The new procedure allowed fue anaIysis of entire chromosomes from fue brewing strain,placed into a laboratory yeast that could easily be manipulated genetically. The work with S.carlsbergensis chromosome 1II was followed by fue analysis of chromosomes V, VII, X , XII

and XIII [29-32].

2.4. Molecular AnalysisA clear picture of fue genetic composition of S. carlsbergensis emerged from Southem

hybridization experiments and from fue first gene sequences from this yeast. The paper byNilsson- Tillgren et al. [28], where fue transfer of a chromosome 1II from fue brewing strain toS. cerevisiae was reported, included a detailed Southem analysis of fue HIS4 gene containedin this chromosome. Five yeast strains were used in this analysis. Two were S. cerevisiaestrains carrying mutant alleles of fue HIS4 gene, a point mutation and a deletion respectively.The other three strains were S. carlsbergensis 244, a chromosome 1II substitution strain and achromosome addition strain. DNA samples from each one of fue five strains were digestedwith restriction endonucleases, electrophoresed in an agarose gel and hybridized with alabeled probe that contained fue HIS4 gene. The pattem of bands obtained for fue brewingstrain and fue chromosome addition strain were found to be composed by fue bandscharacteristic of S. cerevisiae, plus other, extra bands, which showed weaker hybridization.This result indicated the presence in fue brewing strain (and also in fue addition strain) of twoversions of chromosome 1II, one virtually indistinguishable from that of S. cerevisiae, andanother with a reduced level of sequence homology. Therefore, fue brewer's yeast must be analloploid, or species hybrid, presumably arisen by hybridization between S. cerevisiae andanother species of Saccharomyces. This conclusion was corroborated by similar analysiscarried out for several other genes [29-36]. Determination of the nucleotide sequence of anumber of S. carlsbergensis genes provided a precise characterization of fue differencebetween fue two types of homologous alleles present in fue brewing yeast. This analysis hasbeen carried out for ILVI and ILV2 [37]; URA3 [38]; HIS4 [39]; ACBI [40]; MET2 [41];METIO [42] and ATFI [43]. Pooled data indicate a nucleotide sequence divergence of 10-20% within coding regions and higher outside.

2.5PloidyFinding a sound answer for the long-standing question of how many chromosomes are

contained in brewer's yeast, has taken a long time. The relative DNA content of S.carlsbergensis 244 has been recently determined by flow cytometry. Results obtained showthat the genetic constitution ofthis strain must be close to tetraploidy [38]. Since it is knownthat S. carlsbergensis is an alloploid generated by fue hybridization of two differentSaccharomyces spp., the question arises of what is fue contribution of each parental species tothe hybrid. Pooled data obtained from gene replacement experiments and meiotic analysis ofgenes located in chromosomes VI, XI, XIII and XIV, suggest that S. carlsbergensis containsfour copies of ~ach one of these chromosomes, two from each parental species [11].However, this can not be generalized to all chromosomes. Results of experiments in which

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cloned from S. cerevisiae and characterized [51-54]. S. carlsbergensis-specific alleles oftheILV genes from fue brewer's strain have algo been cloned [32,37,55,56]. Because of fuegenetic complexity of fue brewing strain (a hybrid with about four copies of each gene, twofrom each parent), fue abolition of fue ILV2 function requires the very laborious task ofeliminating each of the four copies of the gene present in fue yeast. This result has not beenreported so faro An altemative could be to boost fue activity of fue enzymes that direct fuefollowing steps in fue conversion of a-acetolactate into valine: fue reductoisomerase, encodedby ILV5 and possibly fue dehydrase, encoded by ILV3 [57-60]. To achieve fue desired effect,it could be sufficient to manipulate onlyone ofthe four copies ofthe ILV genes present in fuebrewer's yeast. A clever procedure to inhibit fue ILV2 function, by using an antisense RNAof fue gene, has been reported [61]. However, a later note from fue same laboratory statedthat fue reported results were incorrect [62]. Another approach makes use of an enzyme, a-acetolactate decarboxylase, which catalyzes fue direct conversion of acetolactate into acetoin,bypassing fue formation of dyacetyl. This enzyme is produced by different microorganisms[63]. Its use for fue accelerated maturation of beer was suggested years ago [64,65], andcurrently is cornmercially available for this use. An obvious altemative is to express a geneencoding a-acetolactate decarboxylase in fue brewing yeast. This has been carried out bydifferent groups [66-68].

3.2. Beer Attenuation and the Production of Light BeerConversion of barley into wort that can be fermented requires two previous processes:

malting and mashing. During malting, fue barley grain is subjected to partial germination,

Pyruvale

ILV2 °1ald

a- Acelolaclate ~ 0ILV5 0n ~

V Dlacetyl

~a-[J- Dlhydroxy AcelolnIsovalerale

ILV3 1a - Kelolsovalerate

1"

Vallne

Fig. 2. Strategies designed to prevent the presence of diacetyl in beer. 1. Elimination of ILV2. This prevents thesynthesis of the enzyme acetohydroxyacid synthase, required for fue formation of the diacetyl precursor,acetolactate. 2. Overexpression of fue ILV5. This increases fue activity of the enzyme, which converts 0-acetolactate into dihydroxy isovaleriate, fue following intermediate of valine biosynthesis. As a consequence,fue amount of O-acetolactate that can be transformed into diacetyl is reduced. 3. Expression in brewer's yeast offue ald gene encoding bacterial acetolactate decarboxylase. This enzyme avoids fue formation of diacetyl, byconverting fue available acetolactate into acetoin. Commercial preparations of the enzyme are available as beeradditive to accelerate maturation.

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achieved by moistening, and subsequent drying. Germination induces fue synthesis ofamylase and other enzymes that allow fue seed to mobilize its reserves. The dried malt ismilled and fue resulting powder is mixed with water and allowed to steep at warmtemperatures. During mashing, amylases digest the seed's starch, liberating simpler sugars,chiefiy maltose. This process is critical, since fue brewing yeast is unable to hydrolyze starch.The enzymatic action of barley's amylases on starch yields fermentable sugars, but algOoligosaccharides (dextrins) which remain unfermented during brewing. Dextrins represent animportant fraction of fue caloric content of beer. In current brewing practice, it is quitecornmon to add exogenous enzymes. Thus glucoamylase can be added to fue mash toimprove the digestion of fue starch. If fue enzymatic treatment is carried out exhaustively, fuedextrins are completely hydrolyzed, and the result is a light beer with substantially lowercaloric content, for which there is a significant market demand in some parts of the world. Aconvenient altemative to fue addition of exogenous glucoamylase is to endow fue brewer'syeast with the genetic capability of synthesizing ibis enzyme. A variety of S. cerevisiae,formerly classified as a separate species (S. diastaticus), produces glucoamylase. Because ofits close phylogenetic relationship with fue brewing yeast, S. diastaticus is an obvious sourceoribe glucoamylase gene.

The percentage of fue sugar in fue wort that is converted into ethanol and CO2 by fue yeastis called attenuation. Microbial contamination of beer is often associated with a pronouncedincrease in fue attenuation value, which is known as superattenuation. This effect is due to fuefermentation of dextrins, which are hydrolyzed by amylases produced by fue contaminantmicroorganisms. S. diastaticus was characterized as a wild yeast that caused superattenuation[69]. Similarly to fue synthesis of invertase or maltase by Saccharomyces, fue synthesis ofglucoamylase is controlled by a set of at least three polymeric genes, designated STAl, STA2and STA3 [70]. This genetic system is complicated by the existence in normal S. cerevisiaestrains of a gene, designated STAlO, which inhibits fue expression of fue other STA genes[71]. Recently, the STAlO gene has been identified with fue absence of Fl08p, atranscriptional regulator of both glucoamylase and fiocculation gene s [72]'. The sequence offue STA 1 gene was first determined by Yamashita et al. [73]. Different species of filamentousfungi, in particular some of fue genus Aspergillus, produce powerful glucoamylases. Thegene that encodes fue enzyme of A. awamori has been expressed in S. cerevisiae [74].A vailable information about the genetic control of glucoamylase production bySaccharomyces and current technology makes fue construction of brewing strains with ibis

capability relatively easy.

3.3. Beer Filterability and the Action of p-glucanasesBrewing with certain types or batches of barley, or using certain malting or brewing

practices, can yield wort and beer with high viscosity, very difficult to filtrate. When ibisproblem arises, fue beer mar algO present hazes and gelatinous precipitates. Scott [75]pointed out that ibis problem was caused by a deficiency in p-glucanase activity. Thesubstrate of ibis enzyme, p-glucan, is a major component of fue endosperm cell walls ofbarley and other cereals. During the germination of fue grain, p-glucanase degrades fueendosperm cell walls, allowing fue access of other hydrolytic enzymes to fue starch andprotein reserves of fue seed. Insufficient p-glucanse activity during malting gives rige to anexcess of p-glucan in the wort, which causes the problems. The addition of bacterial or fungalp-glucanases to fue mash, or directly to the beer during fue fermentation, is a corrunonremedy. The construction of a brewing yeast with appropriate p-glucanase activity wouldmake unnecessary fue treatment with exogenous enzymes. Suitable organisms to be used as

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sources of fue [3-glucanase gene are Bacillus subtilis and Thricoderma reesei, from which fuecommercial enzyme preparations used in brewing are prepared. The genes from both havebeen characterized [76-79] and brewer's yeast expressing [3-glucanase activity have beenconstructed [80]. An altemative is to make use ofthe gene encoding barley [3-glucanase, theenzyme that naturally acts in malting. This gene has been characterized and expressed in S.cerevisiae [81-83]. However, the barley enzyme has lower thermal resistance than. fuemicrobial enzymes, which is a limitation for its use against the [3-glucans present in wort.Consequently, the enzyme has been engineered to increase its thermal stability [84,85].

3.4. Control of Sulfite Production in Brewer's YeastSulfite has an important, dual function in beer. It acts as an antioxidant and a stabilizing

agent of flavor. Sulfite is formed by fue yeast in fue assimilation of inorganic sulfate, as anintermediate of fue biosynthesis of sulfur-containing amino acids, but its physiologicalconcentration is low. Hansen and Kielland-Brandt [86] have engineered a brewing strain toenhance sulfite level to a concentration that increases flavor stability. The formation of sulfitefrom sulfate is carried out in three consecutive enzymatic steps catalyzed by A TP sulfurylase,adenylsulfate kinase and phosphoadenylsulfate reductase. In S. cerevisiae, fuese enzymes areencoded by MET3, MET14 and MET16 [87-89]. In turn, sulfite is converted firstly intosulfide, by sulfite reductase, and then into homocysteine by homocysteine synthetase. Thislast compound leads to fue synthesis of cysteine, methionine and S-adenosylmethionine. Ithas been proposed that S-adenosylmethionine plays a key regulatory role by repressing fuegenes of fue pathway [90-92]. However, more recent evidence assigns this function tocysteine [93]. Anyhow, because of the regulation of fue pathway, yeast growing in fuepresence of methionine contains very little sulfite. To increase its production in the brewingyeast, Hansen and Kielland-Brandt [86] planned to abolish sulfite reductase activity. Thiswould increase sulfite concentration, as it cannot be reduced. At the same time, the disruptionof fue methionine pathway prevents the formation of cysteine and keeps free from repressionthe genes involved in sulfite formation. Sulphite reductase is a tetramer with an a2 [32structure. The a and [3 subunits are encoded by the METIO and MET5 genes, respectively[42,94]. Hansen and Kielland-Brandt undertook fue construction of a brewing strain withoutMETIO gene function. The allotetraploid constitution of S. carlsbergensis made it extremelydifficult to perform the disruption of fue four functional copies of fue yeast. Therefore, theyused allodiploid strains, obtained as meiotic derivatives of the brewer's yeast. Theseallodiploids contains two homeologous alleles ofthe METIO gene, one similar to the versionnormally found in S. cerevisiae and another which is S. carlsbergensis-specific. It is knownthat some allodiploids can be mated to each other to regenerate tetraploid strains with goodbrewing performance[18]. The functional METIO alleles present in fue allodiploids werereplaced by deletion-harboring, non-functional copies, by two successive steps ofhomologous recombination. New allotetraploid strains with reduced or abolished METIOactivity were then generated by crossing the manipulated allodiploids. The brewingperformance of one ofthese strains, in which the METIO function was totally abolished, metthe expectations. Hansen and Kielland-Brandt [95] have used another strategy to increase fueproduction of sulfite which relies in the inactivation of fue MET2 gene function. The MET2gene encodes O-acetyl transferase. This enzyme catalyzes the biosynthesis of O-acetylhomoserine, which binds hydrogen sulfide to forro homocysteine [96]. Similarly to fueinactivation of MET 10, inactivation of MET2 impedes the formation of cysteine, depressingthe genes required for sulfite biosynthesis.

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3.5. Yeast FlocculationAs beer fennentation proceeds, yeast cells start to flocculate. The flocs grow in size, and

when they reach a certain mass start to settle. Eventually, the great majority of the yeastbiomass sediments. This phenomenon is of great importance to the brewing process becauseit allows separation of the yeast biomass from fue beer, once fue primary fennentation isovero The small fraction afilie yeast that is left in the green beer is sufficient to carry out fuesubsequent step, the lagering. Flocculation is a cell adhesion process mediated by fueinteraction between a lectin protein and mannose [97-99]. Stratford and Assinder [100]carried out an analysis of 42 flocculent strains of Saccharomyces and defined two differentphenotypes. One was fue known pattern observed in laboratory strains that carried the FLOlgene. They found, in some ale brewing strains, a new flocculation pattern characterized bybeing inhibited by fue presence in fue medium of a variety of sugars, including mannose,maltose, sucrose and glucose, whereas fue FLOl type was sensitive only to mannose. Thegenetic analysis of flocculation has revealed fue existence of a polyrneric gene familyanalogous to fue SUC, MAL, STA and MEL families [101,102]. The FLOl gene has beenextensively characterized [103-107], which encodes a large, cell wall protein of 1,537 aminoacids. The protein is highly glycosylated. It has a central domain harboring direct repeats richin serine and threonine (putative sites for glycosylation). Kobayashi et al. [108] have isolateda flocculation gene homolog to FLOl that corresponds to fue new pattern described byStratford and Assinder [100]. This result is consistent with fue hybrid nature afilie brewingyeast. In addition to fue structural genes encoding flocculins, other FLO genes playaregulatory roleo For instance, the FLO8 gene (alias STAIO) encodes a transcriptional activatorthat in addition to flocculation regulates glucoamylase production, filamentous growth and

mating [72,109-113].

3.6. Beer Spoilage Caused by MicroorganismsMicrobial contamination of beer, caused by bacteria or wild yeast is a serious problem in

brewing. To overcome fue contamination, commonly sulfur dioxide and other chemicals areadded, but this practice faces restrictive legal regulation and consumer rejection. An attractivealternative is to endow fue brewing yeast with fue capability of producing anti-microbialcompounds. A specific example is fue expression in S. cerevisiae of fue genes required forthe biosynthesis of pediocin, an antibacterial peptide from Pediococcus acidilactici [114].Another example is fue transfer to brewing strains of fue killer character, conferred by fueproduction of a toxin active against other yeasts [115,116].

3.7. Enhanced Synthesis of Organoleptic CompoundsThe yeast metabolism during beer fennentation gives rige to the fonnation of higher

alcohol, esters and other compounds which make an important contribution to the aroma andtaste of beer. A first group of compounds important to beer flavor are isoamyl and isobutylalcohol and their acetate esters. These compounds derive from fue metabolism of valine andleucine [117]. Two genes, ATFl and LEU4, encoding enzymes involved in fue fonnation offuese compounds, have been successfully manipulated to increase theirs synthesis. ATFlencodes alcohol acetyl transferase. It has been shown that its over-expression causesincreased production ofisoamyl acetate [118]. LEU4encodes a-isopropylmalate synthase, anenzyrne that control s a key step in fue fonnation of isoamyl alcohol from leucine. Thisenzyme is inhibited by leucine [119,120]. Mutant strains resistant to a toxic analog of leucineare insensitive to leucine inhibition [119]. Mutants ofthis type, obtained from a lager strain,produce increased amounts ofisoamyl alcohol and its ester [121].

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4. CONCLUSIONSDevelopment of molecular biology in fue 20th century has brought many new opportunities

for technical improvements in fue field of brewing industry. The basic scientific questionsconceming fue genetic nature of fue brewer's yeast and different physiological problemsrelated to brewing (secondary fermentation, flocculation; etc.) have been answered.Instruments to construct a new generation of brewer's yeast strains, designed to circurnventcornrnon problems of brewing, have been developed. Afine example is fue work of Hansen .,

and Kielland-Brandt [86] that led to the construction of a brewing yeast with increased sulfiteproduction. Presentir, fue main obstacle for fue development and industrial implementationof improved brewing yeast is not technical but psychological. Public concem about fue safetyof genetic engineering and pressure, often misguided, from various groups, force fue brewingcompanies to refrain from innovation in fuese directions. Nevertheless, it is easy to forecastthat in fue future, genetic engineering will bring to fue brewing industry, as well as to otherfood industries, a plethora of better and safer products.

Acknowledgment. 1 thank Professor Morten Kielland-Brandt for many useful suggestions and criticalreading of the manuscript.

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R. Trav. Lab. Carlsberg Ser. Physiol. 24:381-480.5. Mortimer, R. K. (1993). 0jvind Winge: Founder ofyeast genetics. In: The Early Days ofYeast Genetics.

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