Elucidating flavour formation and stress tolerance in brewing yeast hybrids

Lager beer is one of the most popular beverages worldwide, and lager yeast, a natural interspecific hybrid between Saccharomyces cerevisiae and Saccharomyces eubayanus, is one of the most important industrial microorganisms. Its success is likely due to a combination of traits not commonly found in pure yeast species: low-temperature tolerance from S. eubayanus, and maltotriose utilization from S. cerevisiae. However, the diversity of lager yeast in industrial use is currently limited. In an attempt to increase the genetic and phenotypic diversity of lager yeast, we explored the possibility of using interspecific hybridization as a non-GM method to produce de novo hybrids with tailored properties, by mating selected strains of S. cerevisiae and S. eubayanus. In addition to establishing strain development methodology, we aimed to elucidate mechanisms responsible for phenotypes observed in the newly created strains. The new hybrids had not only inherited beneficial properties from both parent strains, but in many cases also showed apparent hybrid vigour, fermenting faster and producing beer with higher concentrations of alcohol and flavour-active esters than the parents. Interspecific hybridization can therefore be applied not only for production of novel non-GM lager yeast strains, but also to improve brewing-relevant traits relative to parent strains. In addition, we developed methodology that allowed creation of hybrids with multiple parent strains and removal of undesirable phenolic off-flavour through the use of fertile allotetraploid intermediates. We also showed that the inherent genomic instability of hybrids could be exploited to generate variant strains by adaptive evolution. Adapted variants outperformed non-adapted strains during wort fermentation, and the majority also possessed several desirable brewing-relevant traits. Ploidy levels of the generated hybrids influenced fermentation properties, as higher ploidy hybrids fermented faster and produced higher concentrations of flavour-active esters. Higher gene copy numbers and expression levels were observed for relevant genes. We also observed significant variation in lipid composition of parent and hybrid strains at different fermentation temperatures and ethanol contents. Strains which performed well at low temperatures and/or high ethanol concentrations were associated with increased concentrations of lipids containing unsaturated fatty acids. Results demonstrated the potential of interspecific hybridization as a valuable tool for developing new lager yeast strains, and may also help to elucidate the evolutionary history of industrial lager yeast strains.