Development of molecular biology tools for the wine and beer yeast Dekkera bruxellensis

Abstract: Popular Abstract in English Yeasts are eukaryotic microorganisms classified as fungi and consist of a number of species. The english word ‘yeast’ (as well as its equivalent in many other languages) is based on the words ’foam’ and ’to rise’ – and thus direct references to the fermentation process that produce beer and bread (Kurtzman et al., 2011). Some yeast species are known to be pathogens for humans and animals (like different Candida and Cryptococcus species) causing severe infections first of all in immuocompromised people. However, many species are beneficial and used for millenia from mankind, like the baker´s yeast Saccharomyces cerevisiae, which is used for the production of beverages (wine, beer, soft drinks) and bread. S. cerevisiae was the first eukaryote to be sequenced in 1996 and is a member of the so-called conventional yeasts, which have been studied for many years in the laboratories worldwide and its genetic background is well known. Other species are termed as non-conventional yeasts and they represent a vast majority of known yeast species. They comprise of hundreds of species, of which many of them are not even classified yet or their genetic background is still unknown due to a lack of molecular biology tools. The non-conventional yeasts exhibit unusual features, like an extraordinary metabolism, and therefore present a huge untapped potential for the industrial usage (e.g. as leavening agents, for protein production, for wine and beer production, for ethanol production from inexpensive alternative carbon sources). One of these non-conventional yeasts is Dekkera bruxellensis (anamorph Brettanomyces bruxellensis), which is of great importance for the wine and beer industry due to its ability to produce flavours normally considered as unpleasant and associated with the smell of wet dog, leather, barnyard or burnt plastic. But on the other hand, the taste found in specific beers (Belgian lambic and gueuze) and red wines (Chateau de Beaucastel) as well as the characteristic flavour profile of sourdough and feta cheese would not be possible without the contributions of D. bruxellensis yeasts. More recently, D. bruxellensis came into view as the organsim of choice for the production of biofuel from inexpensive alternative carbon sources. D. bruxellensis and S. cerevisiae separated more than 200 million years ago, but nevertheless they exhibit similar characteristic features. Both can produce and tolerate high ethanol concentrations and are so-called Crabtree-positive yeasts (which means they produce ethanol even in the presence of oxygen and under high glucose concentrations rather than producing biomass). These characteristics probably evolved independently through the occupation of similar niches by both species. During my work as a PhD student I focused on the development of molecular genetic tools for the yeast Dekkera bruxellensis, which are indispensable for its characterization. With them we are now in the position to characterize individual genes and thereby analyze their role in the yeast´s metabolism. One could, for example, link a specific gene (which is known to play a crucial role in the flavour production in other, better analyzed yeast species) with the occurence of a specific flavour. And if this gene is involved in the production of off-flavours, one could influence its expression by using specific techniques (e.g. RNA interference). Furthermore, I analyzed the gene ADH3 in D. bruxellensis through its overexpression. The ADH genes code for alcohol dehydrogenases and are known to be the key enzymes in the alcoholic fermentation. There are seven different isoformes known in S. cerevisiae, which catalyze different enzymatic reactions: the isoformes 1, 3, 4 and 5 are responsible for the reduction of acetaldehyde to ethanol during glucose fermentation, whereas the isoform 2 catalyzes the reverse oxidation of ethanol to acetaldehyde. This knowledge is known from the yeast S. cerevisiae, where the ADH genes have been studied intensively. On the other hand, the ADH genes of D. bruxellensis have been studied only poorly to date, so our studies present one of the first attempts for their analysis. Analysis by Professor Jure Piskur revealed that there are several copies of ADH genes present in D. bruxellensis, similar to S. cerevisiae and that there are lineage-specific duplications in D. bruxellensis as well. This information suggests similar functions of the ADH isoformes in D. bruxellensis and S. cereviaise. Our results showed that it is possible to create D. bruxellensis strains exibiting improved ethanol production abilities (and simultaneously show a faster growth rate) – more ethanol production in shorter time could be a desirable trait of industrial´s interest. Not all strains inside the Dekkera/ Brettanomyces genus are known to be beneficial in industrial production plants. There are several strains known to show high spoilage behaviour, resulting in immense economic losses in the wine and beer industry. On the other hand, some strains can outcompete the production organism S. cerevisiae and become even better production strains. What are the phenotypic and genetic backgrounds for this peculiar behaviour in D. bruxellensis yeasts? We were prompted to learn more about the linkage between the genotype and the phenotype in Dekkera/ Brettanomyces yeasts. I analyzed 170 strains, originating from different niches (wine, beer, soft drinks, ethanol production plants), by determining their behaviour in the presence of different stresses (ethanol, pH, sugars, salts and temperature). Simultaneously we sequenced the whole genome of selected strains, their annotation is remaining. As soon as one has the sequences annotated, one could look for similarities that are unique only for strains isolated in similar niches or (more important) for gene clusters that are common only in spoilage strains. With this knowledge it would be easier to identify strains that could cause spoilage and select strains for better performance in industrial processes. There is much more to discover and a lot of research necessary to better understand the species inside the Dekkera/ Brettanomyces genus. I developed the first auxotrophic strains and thereby the first genetic tools. Now, one needs to use these tools to characterise more genes, to improve strains, to reduce the strain´s spoilage ability, to better understand these yeasts. I wish I would have more time to keep on working with this unique and stunning lab pet. We should now focus on the non-conventional yeasts to uncover their for mankind exceptional potential. Another fascinating aspect is a focus on the non-conventional yeasts to uncover their exceptional potential. It´s not possible to imagine a world without farinaceous products; fermented and baked food products are highly important for societies and have cultural and economic importance. Surprisingly, there is only one yeast species, which has been used all along for the bread making: S. cerevisiae. This species converts sugars (which are in the starch of the flour) into carbon dioxide (which raises the dough and is responsible for the air pockets, making the bread fluffy), biomass and ethanol, which is an attribute of baker´s yeast. Nowadays it is known that these abilities are not exclusively attributed to S. cerevisiae, there are other yeasts behaving similar or even better. These are found within the non-conventional yeasts, of which only a few are studied so far. Together with colleagues I analyzed 14 non-conventional yeast strains in view of their role as being alternative baker´s yeasts. Two strains showed to have at least the same leavening abilities than S. cerevisiae, some behaved even better. On top, the bread made with these two strains had exceptional smell and taste. Our results show that there is a huge potential within the non-conventional yeasts to find new strains for industrial applications. This is probably just the tip of the iceberg. The European Union has one of the most stringent regulations in the world when it comes to the use of genetically modified organisms (GMO) in food (Davison, 2010). Natural selection through selection pressure is another technique to improve strains. For this one could stress yeasts in the presence of bacteria and analyze how their metabolism will change. Or one could mimic scaled-down industrial processes under laboratory conditions and thereby improve the strain´s behaviour towards a selected trait. By doing so one can improve strains for the European market and avoid the regulations for the permission of GMO in the food production.