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

Abstract: The non-conventional wine and beer yeast Dekkera bruxellensis is of great interest for the food industry and academic research. This Crabtree-positive yeast is a good ethanol producer and can compete the baker´s yeast Saccharomyces cerevisiae, in ethanol and pH tolerance and could be an even better organism for the biofuel production from alternative carbon sources. D. bruxellensis is considered to be one of the main spoilage organisms in the wine and biofuel industry; on the other hand, it is ‘indispensable’ as a contributor to the flavor profile of Belgium lambic and gueuze beers. It also adds characteristic aromatic properties to some red wines and sourdough. There are not many molecular tools available for this yeast species. During my PhD project I characterized some D. bruxellensis promoters and analyzed their expression activity in response to different carbon sources. This led me to the construction of a D. bruxellensis expression vector. Furthermore I analyzed the role of alcohol dehydrogenase gene isoform III (ADH3) in the glucose fermentation and ethanol production. I have also analyzed a collection of 170 strains of the Dekkera/ Brettanomyces genus isolated from different niches from different parts of the world. These strains have been phenotypically characterized regarding the traits as ethanol, pH and temperature resistance, utilization of different carbon and nitrogen sources and osmotolerance. Some of the strains were studied with further genetic analyses. This gives us a better knowledge of the relationship of the phenotype and the genotype in this genus. Non-conventional yeasts represent a vast potential for industrial application, but most of them have not been characterized yet. I have analyzed 14 non-conventional yeasts for their ability to be alternatives to baker´s yeast. Seven of these strains have been used for baking bread. Two strains (Kazachstania gamospora, Wickerhamomyces subpelliculosus) exhibited the same or even better leavening abilities (production of carbon dioxide, resistance towards high sodium chloride and sugar concentrations) comparing to commercial baker´s yeast. On the other hand, the selected strains exhibited an extraordinary taste. This could be further used in the industry for the production of bread with new flavours.  In conclusion, the research presented in this thesis broadens the knowledge about Dekkera/ Brettanomyces yeasts (particularly D. bruxellensis) by delivering molecular tools to manipulate this species. These tools are necessary to further identify and characterize genes of interest. Additionally, the identification of two alternative baker´s yeasts picked out of the huge bulk of non-conventional yeasts illustrates how fruitful research into yet uncharacterized species can be to discover their useful qualities and abilities.