Yeast genes involved in sulfur and nitrogen metabolism affect the production of volatile thiols from Sauvignon Blanc musts.

Two volatile thiols, 3-mercaptohexan-1-ol (3MH), and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical varietal aroma compounds in Sauvignon Blanc (SB) wines. These aromatic thiols are not present in the grape juice but are synthesized and released by the yeast during alcoholic fermentation. Single deletion mutants of 67 candidate genes in a laboratory strain of Saccharomyces cerevisiae were screened using gas chromatography mass spectrometry for their thiol production after fermentation of SB grape juice. None of the deletions abolished production of the two volatile thiols. However, deletion of 17 genes caused increases or decreases in production by as much as twofold. These 17 genes, mostly related to sulfur and nitrogen metabolism in yeast, may act by altering the regulation of the pathway(s) of thiol production or altering substrate supply. Deleting subsets of these genes in a wine yeast strain gave similar results to the laboratory strain for sulfur pathway genes but showed strain differences for genes involved in nitrogen metabolism. The addition of two nitrogen sources, urea and di-ammonium phosphate, as well as two sulfur compounds, cysteine and S-ethyl-L-cysteine, increased 3MH and 3MHA concentrations in the final wines. Collectively these results suggest that sulfur and nitrogen metabolism are important in regulating the synthesis of 3MH and 3MHA during yeast fermentation of grape juice.

The yeast IRC7 gene encodes a ß-lyase responsible for production of the varietal thiol 4-mercapto-4-methylpentan-2-one in wine.

Three varietal thiols are key aroma compounds in Sauvignon Blanc wines: 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexanol (3MH) and its acetylated derivative 3-mercaptohexyl acetate (3MHA). Screening of Saccharomyces cerevisiae strains identified a clinical isolate with elevated 4MMP production after fermentation. Bulked Segregant Analysis of a cross between this isolate and the laboratory strain revealed a single major locus for 4MMP production near the telomere of chromosome 6. Deletion of the IRC7 gene from this region in YJM450 reduced 4MMP production below detectable levels, but did not affect yields of 3MH, in Sauvignon Blanc wine. Sequencing revealed that the IRC7 gene in YJM450 had been introgressed from a strain of Saccharomyces paradoxus. Most strains of S. cerevisiae, including the laboratory strain S288C, have a 38-bp deletion that inactivates IRC7. Overexpression of a full-length S. cerevisiae allele of IRC7 in a wine yeast, Zymaflore F15, increased 4MMP production in Sauvignon Blanc wine from undetectable levels (<10 ng L(-1)) to concentrations of 1000 ng L(-1), and also increased 3MH and 3MHA. Biochemical analysis of soluble protein extracts showed that both the cerevisiae and paradoxus IRC7 proteins show ß-lyase activity, with a substrate preference for cys-4MMP over cys-3MH.

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae.

Laboratory strains of yeast (Saccharomyces cerevisiae) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys(+) strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.

New precursor of 3-mercaptohexan-1-ol in grape juice: thiol-forming potential and kinetics during early stages of must fermentation.

Two volatile thiols, 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA), are key aroma impact compounds in many young white wines, especially of the variety Sauvignon blanc (SB). Although great effort has been invested to identify their precursors in recent years, the origin of the majority of 3MH and 3MHA generated during wine fermentation still cannot be explained. Here we demonstrate that supplying an external source of hydrogen sulfide to grape juice hugely increases its thiol-forming potential. We further describe the discovery of (E)-2-hexen-1-ol as an additional new thiol precursor and demonstrate that it possesses, together with (E)-2-hexenal, an immense thiol-forming potential during fermentation. Both C6-compounds are extremely rapidly metabolized by yeast during the first hours after inoculation, even under commercial conditions, and can be interconverted during this phase depending on their initial concentration in the grape juice. Spiking grape juice with additional acetaldehyde greatly enhanced the (E)-2-hexen-1-ol to (E)-2-hexenal conversion rate. Delaying the metabolization of the two unsaturated C6-thiol precursors by yeast, at the same time as increasing hydrogen sulfide production early in fermentation, opens up a great opportunity to tap into this enormous potential 3MH and 3MHA source in grape juice and extends the possibility of thiol production to other non-grape-based alcoholic beverages as well.