Rettberg, N. | Search bioRxiv

bioRxiv2022

Increased volatile thiol release during beer fermentation using constructed interspecies yeast hybrids

Interspecies hybridization has been shown to be a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. Thanks to the popularity of heavily hopped India Pale Ale-style beers, there is an increased demand from brewers for strains that can boost hop aroma. Here, we explored whether hybridization could be used to construct strains with an enhanced ability to release hop-derived flavours through {beta}-lyase activity, which releases desirable volatile thiols. Wild Saccharomyces strains were shown to possess high {beta}-lyase activity compared to brewing strains, however, they also produced phenolic off-flavours (POF) and showed poor attenuation. To overcome these limitations, interspecies hybrids were constructed by crossing pairs of one of three brewing and one of three wild Saccharomyces strains (S. uvarum and S. eubayanus). Hybrids were screened for fermentation ability and {beta}-lyase activity, and selected hybrids showed improved fermentation and formation of both volatile thiols (4MMP, 3MH and 3MH acetate) and aroma-active esters compared to the parent strains. Undesirable traits (e.g. POF) could be removed from the hybrid by sporulation. To conclude, it was possible to boost the release of desirable hop-derived thiols in brewing yeast by hybridization with wild yeast. This allows production of beer with boosted hop aroma with less hops (thus improving sustainability issues).

microbiology↗

bioRxiv2021

Efficient breeding of industrial brewing yeast strains using CRISPR/Cas9-aided mating-type switching

Yeast breeding is a powerful tool for developing and improving brewing yeast in a number of industry-relevant respects. However, breeding of industrial brewing yeast can be challenging, as strains are typically sterile and have large complex genomes. To facilitate breeding, we used the CRISPR/Cas9 system to generate double-stranded breaks in the MAT locus, generating transformants with a single specified mating type. The single mating type remained stable even after loss of the Cas9 plasmid, despite the strains being homothallic, and these strains could be readily mated with other brewing yeast transformants of opposite mating type. As a proof of concept, we applied this technology to generate yeast hybrids with an aim to increase {beta}-lyase activity for fermentation of beer with enhanced hop flavour. First, a genetic and phenotypic pre-screening of 38 strains was carried out in order to identify potential parent strains with high {beta}-lyase activity. Mating-competent transformants of eight parent strains were generated, and these were used to generate over 60 hybrids that were screened for {beta}-lyase activity. Selected phenolic off-flavour positive (POF+) hybrids were further sporulated to generate meiotic segregants with high {beta}-lyase activity, efficient wort fermentation and lack of POF; all traits that are desirable in strains for the fermentation of modern hop-forward beers. Our study demonstrates the power of combining the CRISPR/Cas9 system with classic yeast breeding to facilitate development and diversification of brewing yeast. Key PointsO_LICRISPR/Cas9-based mating type switching was applied to industrial yeast strains C_LIO_LITransformed strains could be readily mated to form intraspecific hybrids C_LIO_LIHybrids exhibited heterosis for a number of brewing-relevant traits C_LI

microbiology↗