Functional Link Between IRC7 Allelic Variation and Carbon-Sulfur-β-Lyase Activity in Brewing Saccharomyces cerevisiae Strains

Abstract

The IRC7 gene encodes a β-lyase enzyme critical for the release of volatile thiols during beer fermentation, contributing to aroma development in fermented beverages. In this study, 15 Saccharomyces cerevisiae ale brewing strains were analyzed to investigate IRC7 allelic diversity and its functional implications. Using a nested PCR approach combined with sequencing, three genotypes were identified: IRC7^L/IRC7^L, IRC7^L / IRC7^S, and IRC7^S / IRC7^S. Allelic segregation in two sporulation-competent heterozygous strains was achieved through spore dissection and haplo-selfing, enabling the generation of homozygous monosporic derivatives. In total, 18 IRC7 sequences were characterized, including 15 IRC7^L alleles carrying different combinations of 9 amino acid substitutions and 3 truncated IRC7^S variants. Two substitutions previously associated with reduced enzymatic activity, G101D and T185A, were frequently detected in IRC7^L alleles. Protein stability predictions indicated a destabilizing effect of these mutations, and sequence similarity analysis highlighted substantial variability among closely related IRC7^L variants. In vitro β-lyase activity assays performed using three model substrates (L-cysteine, S-ethyl-L-cysteine, and S-methyl-L-cysteine) showed that strains carrying IRC7^L generally exhibited higher enzymatic activity, while the presence of the IRC7^S allele or the T185A substitution in IRC7^L allele was associated with reduced activity in a substrate-dependent manner. Monosporic derivatives confirmed that the IRC7^S allele markedly impairs β-lyase activity and that T185A negatively affects activity toward S-methyl-L-cysteine. Overall, these findings expand the current knowledge of IRC7 genetic and functional diversity in brewing strains and its implications for flavor development. The results provided a basis for implementing genotype-informed yeast selection, whereas the mating-competent monosporic derivatives carrying IRC7^L allele generated in this study represent valuable resources for future breeding strategies aimed at optimizing thiol release and aroma complexity in beer.