The introduction in modern breweries of tall cylindroconical fermentors, replacing the traditional open fermentation vats, unexpectedly revealed strong inhibition of flavor production by the high CO₂ pressure in the fermentors. We have screened our collection of Saccharomyces cerevisiae strains for strains displaying elevated tolerance to inhibition of flavor production by +0.65 bar CO₂, using a laboratory scale CO₂ pressurized fermentation system. We focused on the production of isoamyl acetate, a highly desirable flavor compound conferring fruity banana flavor in beer and other alcoholic beverages, from its precursor isoamyl alcohol (IAAc/Alc ratio). We selected the most tolerant Saccharomyces cerevisiae strain, saké yeast Kyokai no. 1, isolated a stable haploid segregant seg63 with the same high IAAc/Alc ratio under CO₂ pressure, crossed seg63 with the unrelated inferior strain ER7A and phenotyped 185 haploid segregants, of which 28 displaying a high IAAc/Alc ratio were pooled. Mapping of Quantitative Trait Loci (QTLs) by whole-genome sequence analysis based on SNP variant frequency revealed two QTLs. In the major QTL, reciprocal hemizygosity analysis identified MDS3 as the causative mutant gene, a putative member of the TOR signaling pathway. The MDS3^Seg.63 allele was dominant and contained a single causative point mutation, T2171C, resulting in the F274S substitution. Introduction of MDS3^Seg.63 in an industrial tetraploid lager yeast with CRISPR/Cas9 enhanced isoamyl acetate production by 145% under CO₂ pressure. This work shows the strong potential of polygenic analysis and targeted genetic modification for creation of cisgenic industrial brewer's yeast strains with specifically improved traits. IMPORTANCE The upscaling of fermentation to very tall cylindroconical tanks is known to negatively impact beer flavor. Most notably, the increased CO₂ pressure in such tanks compromises production by the yeast of the desirable fruity "banana" flavor (isoamyl acetate). The cause of the CO₂ inhibition of yeast flavor production has always remained enigmatic. Our work has brought the first insight into its molecular-genetic basis and provides a specific gene tool for yeast strain improvement. We first identified a yeast strain with superior tolerance to CO₂ inhibition of flavor production, and applied polygenic analysis to identify the responsible gene. We narrowed down the causative element to a single nucleotide difference, MDS3^T2171C, and showed that it can be engineered into brewing yeast to obtain strains with superior flavor production in high CO₂ pressure conditions, apparently without affecting other traits relevant for beer brewing. Alternatively, such a strain could be obtained through marker-assisted breeding.

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Journal Article | Research Support, Non-U.S. Gov't

Authors

Souffriau B, Holt S, Hagman A, De Graeve S, Malcorps P, Foulquié-Moreno MR, Thevelein JM

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