Investigating the b-lyase activity of the brewer’s yeast Saccharomyces cerevisiae

<p><strong>This study investigates how thiol release is affected by the genetics of the brewer’s yeast Saccharomyces cerevisiae. Polyfunctional thiols are sulphur-containing compounds that contribute desirable tropical fruit aromas to beer and wine. The release of thiols like 4-methyl-4-sulfanyl-pentan- 2-one (4MSP), 3-sulfanyl-hexan-1-ol (3SH), and 3-sulfanyl-hexyl acetate (3SHA) has been linked to the activity of the β-lyase enzyme Irc7p in S. cerevisiae during fermentation. This enzyme cleaves the carbon-sulphur bond of amino acid-bound thiol precursors, releasing free volatile thiols and assimilable nitrogen. However, within yeast, there exists a common 38 bp deletion in the IRC7 gene, as well as other deleterious SNPs, which negatively impact enzyme function and thiol release. Another factor that limits Irc7p activity is transcriptional repression via the nitrogen catabolite repression (NCR) pathway. In the presence of freely assimilable nitrogen, Ure2p inactivates the transcription factors Gln3p and Gat1p, which subsequently reduces IRC7 transcription. Due to the nitrogen-rich environment during beer and wine fermentation, NCR is activated and IRC7 expression is repressed.</strong></p><p>To develop an informed approach for developing strains with increased varietal thiol release, the genetic variation of URE2 and IRC7 was investigated in a diverse S. cerevisiae population. Several variant alleles were identified in IRC7 and URE2, which distinguished a full-length, functional IRC7 allele and a number of URE2 variants. To compare β-lyase (Irc7p) activity between different strains’ genetics, IRC7 transcript abundance was measured and a β-lyase-dependent growth assay, using S-methyl-L-cysteine (SMC) as a sole nitrogen source, was developed. Methylamine was used in these growth assays to trigger the NCR response present during industrial fermentations without providing utilisable nitrogen.</p><p>Furthermore, several URE2 variants were selected for their potential to disrupt NCR. The URE2 variants were then introduced, using CRISPR-Cas9, into a lab strain (BY4741) expressing a functional IRC7 allele. These variants were characterised by measuring IRC7 expression (regulated by URE2 and NCR), and through the novel β-lyase-dependent growth experiments. Of the three URE2 variants introduced, there was one variant (E74G) that increased IRC7 expression by 5-fold. Overall, my results show that the methods established can be used to identify genetic variants, in non-GMO yeast strains, that contribute to increased volatile thiol release, and can enable the development of strains with enhanced thiol aroma profiles for winemaking and beer fermentation.</p>