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Cerevisia

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Selecting and generating superior yeasts for the brewing industry

Jan Steenselsa,b , Tim Snoeka,b , Esther Meersmana,b , Martina Picca Nicolinoa,b , Elham Aslankoohia,b ,Joaquin F. Christiaensa,b, Rita Gemayela,b, Wim Meerta,b, Aaron M. Newa,b, Ksenia Pougacha,b,Veerle Saelsa,b, Elisa van der Zandea,b, Karin Voordeckersa,b, Kevin J. Verstrepena,b,∗

a VIB Laboratory for Systems Biology, Gaston Geenslaan 1, B-3001 Leuven, Belgiumb Laboratory for Genetics and Genomics, Centre of Microbial and Plant Genetics (CMPG), KU Leuven, Gaston Geenslaan 1, B-3001 Leuven, Belgium

Introduction

Fermented foods and beverages have been consumed byhumans for over 8000 years. These fermentations were at firsta spontaneous process, without any control or knowledge of themicrobial driving force. This led to irregular and often inferior endproducts. It was not until the late 19th century that it was proposedto use a well-defined microbial starter culture for wine production,consisting of one pure yeast strain. Although this greatly increasedthe reproducibility of the fermentations, the main difficulty wasto select a strain with all beneficial characteristics necessary foran efficient and high-quality fermentation. Until recently, theappropriate tools and knowledge where lacking to make a well-considered and scientifically-founded choice about which strain topick. This explains why, even today, most beer yeasts are used basedon historical rather than scientific reasons. However, the currentknowledge and technologies now allow selection of superior yeastsstrains that are optimally suited to perform their industrial tasks. Inour research team, for example, we are using state-of-the-art tech-nologies to select and generate yeast strains with improved ethanoltolerance, fermentation speed, attenuation and flavour production.

From selective breeding to genetic modification

Selective breeding of crops and livestock started at the verybeginning of agriculture, about 10,000 years ago. Farmers intu-itively chose the best plants to harvest seeds from to use for nextyear’s planting, and superior animals were used for breedingprograms (Sleper and Poehlman, 2006). Although this suitedthe human agenda, breeding programs bypass one of the mostimportant laws of nature: that of natural selection. Humansdetermine artificially which characteristics should be maintainedand enhanced, and which have to go. The power of such breedingcan hardly be underestimated. In many cases, yields of today’sagricultural crops and livestock are an order of magnitude higherthan that of their feral progenitors. Moreover, many of today’s

∗ Corresponding author at: VIB Laboratory for Systems Biology, Bio-Incubator,Gaston Geenslaan 1, B-3001 Leuven, Belgium.

E-mail address: Kevin.Verstrepen@biw.vib-kuleuven.be (K.J. Verstrepen).

crops and livestock are clearly the product of human selection,and do not stand a chance outside the protected farms and fields.One of the most striking examples is the Belgian Blue cattle breed.These bovine body-builders are so muscular that the birth channelhas become too narrow for the broad-shouldered neonatal calves,so Caesarian sections are necessary for each birth.

Compared to the breeding and improvement of crops and live-stock, microbes are lagging far behind. There are several importantreasons for this lag. First of all, it was not until the pioneer-ing microscopy work of Antoni van Leeuwenhoek in 1680, manythousand years after farmers started breeding livestock and crops,that humankind started to realize that microbes exist. Two cen-turies later, the pioneering work of Louis Pasteur demonstrated theimportance of microbes in pathogenesis and food production. Buteven after these discoveries, it has not been easy to breed microbesfor human use. It is relatively easy to cross two desired animals fromyour herd, but the same cannot be said for mating two yeast cells(each 5 �m) having the desired phenotype, present in a culture ofbillions of cells that look exactly the same. The latter requires spe-cialized tools and a scientific approach which only became availablein the last years and is still not accessible to most end users (bakers,brewers, winemakers, etc.).

Genetic modification is a much more recent tactic for theimprovement of our microbes, crops and livestock. The basic prin-ciple of selective breeding and genetic modification is exactly thesame: try to get as many “beneficial” fragments of DNA in oneorganism. The difference between these two approaches lies in twoimportant factors. In contrast to selective breeding, where piecesof DNA of the parents are randomly intermingled, genetic modi-fication allows the insertion of pieces of DNA in the host genomewith striking accuracy and precision. In addition, genetic modifica-tion is not restricted to combining pieces of DNA from organismsthat can sexually reproduce. Using genetic modification, it is pos-sible to transfer a gene from a bacterium to the genome of, e.g. acotton plant. This is not as far-fetched as it sounds: about 50% ofcotton plants possess a gene of the bacterium Bacillus thuringiensis,because of which they produce a compound which is lethal for cer-tain parasites, but harmless for humans (Aroian, 2011). The resultis two-fold: these plants do not require large amounts of harmfulpesticides and also have a higher yield (Thomson, 2006). The cot-ton industry is certainly no exception in using this technique: a

1373-7163/$ – see front matter © 2012 the Associations of the Former Students of the Belgian Brewing Schools. Published by Elsevier B.V. All rights reserved.http://dx.doi.org/10.1016/j.cervis.2012.08.001

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significant part of the world production of, e.g. soy beans, corn andcanola consists of genetically modified crops (77%, 26% and 21%,resp.) (GMO Compass, 2010).

The possibilities of GMOs are limited only by our knowl-edge. Genetic modification requires a profound understanding andknowledge of the genes responsible for certain characteristics. Thebig advantage is that it is possible to work much faster and moreaccurately than selective breeding. For example: it is in principlepossible to generate muscled cattle, such as the Belgian Blue, with asingle genetic modification, instead of spending centuries on breed-ing programs (Grobet et al., 1997). Moreover, we are not limitedanymore to pieces of DNA present in nature: it has become possi-ble to generate large stretches of DNA by chemical synthesis, andthus create “tailor-made” genes. This emerging field of “syntheticbiology” has already managed to create artificial life in a test tubeby chemically synthesizing fully functional genomes from scratch(Gibson et al., 2010).

In the nineties, there was a short but fierce race to design indus-trially interesting microbes through genetic modification. In themedical sector, this race already started a bit earlier, and nowadaysmany GMOs are used for the production of a variety of medicines(Marris, 2001). In the battle against diabetes, for example, expres-sion of an oleosin-human pro-insulin protein (the precursor ofinsulin) in the seeds of transgenic safflower accounts for the biggestpart of the industrial (pro-)insulin production (Institute of Sciencein Society, 2007). In the more conservative food industry, somegenetically modified microbes did try to make their claim to fame.In fact, many GMO food products were quite successful, untilenvironmental organizations started fierce (and not always scien-tifically fair) campaigns. Helped by unrelated food scandals, such asthe Belgian dioxine crisis and bovine spongiform encephalopathy(mad cow disease), genetically modified food products disappearedfrom the shelves in supermarkets. By contrast, genetically modifiedplants used for the production of cattle feed and other derived prod-ucts, are becoming increasingly important, and more than 10% ofthe crops produced worldwide are now genetically modified. How-ever, the brewing industry is not (yet) following this trend, andmany breweries are (still) refusing the use of genetically modifiedingredients and yeasts. Whereas the discussion about whether ornot to use GMOs in the food industry is a very complex and inter-esting matter, we will not further address the pros and cons of thetechnology here (for further reading, see Marris, 2001; Naranjo,2005; Schuller and Casal, 2005; Singh et al., 2006; Saerens et al.,2010).

Genetic modification of microbes

Over the past years, incredible progress has been made withthe generation of superior yeast strains through genetic modifi-cation. Several research teams, including our research group, havedeveloped yeast strains that could be very interesting for the brew-ing industry should they consider the use of genetically modifiedyeasts (e.g. yeasts that produce very specific flavours, yeasts thatproduce less diacetyl, yeasts that can ferment starch and/or dex-trins) (Hammond, 1995; Verstrepen et al., 2001, 2004a,b; Cebolleroet al., 2007; Fleet, 2008; Nevoigt, 2008; Saerens et al., 2010).

When the field of genetic modification of microbes was emerg-ing, the food industry also became interested in this exciting newfield. The possibilities of the GMO-technique are almost unlimited.It is possible to create microbes that produce low or high levelsof a specific enzyme, produce beneficial antioxidants, produce bet-ter and totally natural aroma compounds, grow more efficiently oninferior nutrients or have a higher tolerance to different stresses.Yeasts with novel and very interesting industrial properties werecreated to meet the needs of the consumers. One of the most

surprising examples was probably the use of a brewer’s yeast thatexpressed a gene that allowed cells to degrade complex sugarsand even starch. This allowed the production of beer with lessresidual sugar (and thus fewer calories) (Hammond, 1995). Despitethe great demand for low calorie beers, the beer was never com-mercialized. Partly because of major (but not always scientificallycorrect) protests of pressure groups, and partly because of com-pletely unrelated food safety scandals, such as the British mad-cowdisease and the Belgian dioxin affair, consumers became cautiousand even mistrustful regarding modern, industrial food production(Marris, 2001). This suspicion also spread to everything related togenetic modification, even though there was no connection at all.As a result, the food industry stopped investing in and producingGMO food in the late nineties.

Slowly but steadily, the food industry is starting to appreci-ate the beneficial applications of genetically modified microbes. Inrecent years, GMOs have begun to reappear in the food industry.For example, a yeast strain that can break down certain undesir-able aroma compounds, of particular concern to the wine industry,was recently patented in the USA (Chambers and Pretorius, 2010)and is now used by a few winemakers in the USA and Canada.Inside our faculty of Bioscience Engineering, a lot of work is doneto genetically modify microbes to meet industrial needs. Our ownresearch group is working on creating yeast strains with specificbeneficial characteristics to produce beer, wine, chocolate and bio-fuel. We have yeasts that produce various levels of fruity aromacompounds, that can ferment faster or more completely, producehigher or lower levels of alcohol, stick together (flocculate) moreor less, are more resistant to high levels of alcohol and even yeaststhat carry a “genetic barcode” to be consequently distinguishedfrom other yeasts (Verstrepen et al., 2001, 2003a,b,c, 2004a,b, 2005;Verstrepen and Klis, 2006; Saerens et al., 2006, 2008; Brown et al.,2010) (Fig. 1).This is mainly for strategic reasons: even though noGMOs can be used at this point, brewers assume that these superiormicrobes might make their way to the market within the next fewyears.

Improving industrial yeast strains using non-GMOtechniques

Because consumers and producers are reluctant to use genet-ically modified microbes, much attention is now dedicated toselecting and breeding superior yeasts that are not genetically mod-ified (Table 1). We use the same basic principles that farmers havebeen using since the start of agriculture: we test many differentnatural yeasts to find the very best strains, and then we cross thesestrains to generate even better strains. Even though yeast plays suchan important role in beer production, brewers are lagging behind inyeast selection. For some microbes, e.g. baker’s yeast, the progressis more significant. Bakers can nowadays choose from a plethoraof yeast strains, where each yeast strain has specific characteristicsregarding fermentation speed, capacity to make the dough rise androbustness in freezing or heating conditions. All these strains werecreated by selective crossing of different yeast varieties, and henceare not GMOs.

The reason why the brewing industry is lagging behind con-cerning the use of new, superior yeasts is, paradoxically, preciselybecause yeast plays such a significant role in beer quality: brew-ers are reluctant to swap “their” yeast for another because it mightchange the unique characteristics of their beer. The irony is that thechoices made by brewers are mostly based on trial and error – goingwith whatever works – and not on the systematic screening of theyeast natural diversity that could yield even better quality beers.Fortunately, breweries are beginning to see the value in makingscientifically informed decisions about the yeasts they use for their

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Fig. 1. Examples of experiments to improve industrially relevant phenotypes in yeast strains. (A) Genetically modified yeasts in which one of the most important genes foraroma production is fused to a gene which codes for a green fluorescent protein (GFP). This way, the enzyme that accounts for the production of aroma compounds lights upgreen, so we are able to study its activity and localization in the cell. (B) Genetic modification of a yeast strain to fine-tune the level of flocculation. 1: control, 2–9: gradualincrease of flocculation.

beer production. In fact, in cooperation with some Belgian brew-ers, our research group is already engaging in large-scale studiesfocusing on the selection or creation of better yeasts for beer pro-duction in a non-GMO way. With success, we might add, some ofthese yeasts already found their way to the market.

The basic strategy of our research is simple. We are charac-terizing hundreds of existing, natural yeasts to find yeasts withspecial, industrially relevant properties. Our extensive screening ofhundreds of different yeast strains (including Saccharomyces cere-visiae, but also other yeasts suitable for food production, such as S.bayanus, S. pastorianus, Schizosaccharomyces pombe, Kluyveromyceslactis, Debaryomyces hansenii and Hanseniaspora uvarum) in a broadvariety of conditions allows us to map the (industrially relevant)

Table 1Yeast properties that our lab is investigating and improving in brewer’s yeast (non-GMO unless stated otherwise).

Increased production of specific flavours (fruity, flowery, banana, apple, roses,etc.)

Specific flocculation behaviour (strong or weak flocculation, early or late onset)Increased or decreased attenuationMetabolism of dextrins and starch (through GMO)Decreased production of diacetyl (reduced need for lagering)Special, novel fermentation characteristics (use of non-conventional yeasts)Release of extra natural flavours (bioflavouring through the use of special

yeasts or enzymes)

characteristics of these different yeast strains. After processingthese data with specialized software, we can select precisely whichyeast strains can be used for industrial fermentations, or whichyeasts are interesting for breeding programs (Fig. 2). For example,we have exploited the “heterosis” effect to create superior offspringby crossing strains that possess important trait(s) for industrial fer-mentations (e.g. high ethanol tolerance), leading to new strainswith an even better phenotype.

Mating is especially powerful to improve many industrially rel-evant phenotypes, such as ethanol tolerance, which are not easy tomodify with the techniques usually used for GMOs. These traitsare often poorly characterized and involve a very large numberof genes. Instead of classic mating, we now also utilize anothertechnique, called “genome shuffling”, which is in principle a mul-tiplexed mating involving many different strains (instead of justtwo parents in classic breeding). By mating several strains, wegenerate millions of new strains that carry mosaic genomes thatconsists of a “shuffled” mixture of the genomes of the parentalstrains. Repeated rounds of genome shuffling make it possible torapidly obtain strains that show improvement in complex traits (forreview, see Gong et al., 2009). Genome shuffling is basically crossingat a large scale: usually the first step of the approach is to irradi-ate a population of cells to increase genetic diversity. These cellsare mixed and allowed to mate, after which only the best progeny(for instance the most ethanol-tolerant ones) are selected. This

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Fig. 2. Graphical representation (heat map) of different characteristics of industrial yeast strains. Every row consists of data from a different yeast strain, every column is adifferent characteristic. “Yellow” is a low score, and “red” is a high score for this certain characteristic. The dendrogram on the left represents the genetic relatedness of theyeasts, based on an AFLP fingerprint exploiting transposon TY1 insertion site polymorphisms. The colour code on the top right indicates the origin of the yeast strains. Thiskind of analysis allows us to select yeasts with specific beneficial traits, for example to use in industry, or for breeding.

procedure is repeated several times, until cells that show sub-stantial improvement are obtained. In our laboratory, we are nowexploring the possibility to scale-up this approach and instead ofusing one or few strains, exploit the natural variation of hundredsof industrial yeast strains at once.

Apart from improving existing, well-established fermentationprocesses (such as beer fermentations), our lab is now pioneeringresearch aimed at the development of starter cultures that accu-rately mimic the best spontaneous fermentations. Whereas thisis of course interesting for the beer industry, we are also focus-ing on cocoa fermentation. Fermentation of cocoa beans is oneof the last large-scale truly spontaneous industrial fermentationprocesses. Whereas spontaneous fermentation brings the advan-tage of a complex, intriguing and characteristic flavour palate, an

inevitable downside is the variable quality and efficiency. We arenow selecting and improving mixtures of specific microbes withoptimal fermentation characteristics to optimize fermentation effi-ciency, flavour formation and reducing downstream processingcosts.

Conclusion

The choice of the yeast strains used in industrial fermenta-tions is often still based on historical rather than scientific reasons.However, this is rapidly changing and the beer, wine, biofuel andcocoa producers are starting to realize the enormous potential ofselection and creation of superior microbes. With today’s state-of-the-art-technology and knowledge, it becomes possible to rapidly

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enhance industrially relevant phenotypes. By thoroughly screen-ing the natural diversity of yeast and using this knowledge inhigh-throughput improvement methods such as genome shuffling,new, superior non-GMO yeast strains can be created. By contrast,superior microbes obtained with recombinant DNA technique (theGMOs) are not yet widely applicable in industry at this moment. Themain hurdle for the use of GMOs in food production remains publicperception and the average consumer remains suspicious towardsthe so-called “frankenfood”. In the medical sector, the taboo ofGMOs has largely disappeared, but in the food industry this trendis much slower. This is not necessarily a bad thing, because it hasgiven us a pause to develop better technologies, to consider thepossible dangers and to improve the guiding principles. Meanwhilehowever, a few hundred GMO yeast strains with interesting indus-trially relevant characteristics are patiently but eagerly waiting inour lab’s freezer. Moreover, some companies have released genet-ically modified yeast strains that reduce the need for a malolacticfermentation in wine production. Although the yeast is only usedby a few specific winemakers, it might be the advent of a new gen-eration of genetically modified yeasts used in industrial beverageproduction. In the meantime, our research team is mostly concen-trating on using non-GMO technologies to create superior yeaststhat can be readily used in food production.

Acknowledgments

The authors thank all lab members for their help and sugges-tions. J.S. acknowledges financial support from the IWT BaekelandProgram and Barry Callebaut. Research in the lab of KJV is sup-ported by the Human Frontier Science Program, ERC, VIB, EMBO YIPProgram, KU Leuven, FWO, and IWT, as well as several breweries.

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