Identification of Yeast Genes Affecting Production of Hydrogen Sulfide and Volatile Thiols from Cysteine Treatment during Fermentation

Abstract

Hydrogen sulfide (H2S), well-known for its undesirable rotten-egg odour, is often produced during fermentation by the yeast Saccharomyces cerevisiae when nitrogen becomes depleted. However, an early burst of H2S generated by yeast from cysteine could contribute to the formation of the fruity varietal thiols 3-mercaptohexan-1-ol (3MH) and 3-mercaptohexyl acetate (3MHA) through reaction with (E)-2-hexenal, which is otherwise rapidly metabolised. The goal of this project is to identify genes and pathways leading to H2S generation from cysteine and thus enhance the tropical aromas in wine that appeal to many consumers. Using a candidate gene approach, TUM1 was for the first time identified to play a crucial role in the early production of H2S from cysteine. Overexpressing TUM1 elevated production of H2S, while its deletion reduced the H2S by half. Furthermore, deletion of either MET17 or MET2 led to an additional delayed burst of H2S, suggesting that a portion of the H2S generated from cysteine is fed directly into the sulfate assimilation pathway. Triple deletants of STR2, STR3 and individual MET genes, were shown to require both MET17 and TUM1 to bypass the transsulfuration pathway and grow on high concentrations of cysteine as the sole sulfur source. These results illustrate that cysteine is not converted to sulfate or sulfite, but rather to sulfide via a novel pathway requiring the action of Tum1p. The failure to identify a specific QTL associated with H2S formation from cysteine using a set of 96 fully sequenced M2 x F15 progeny, suggests multiple genes affect the trait. To identify additional genes, a modified version of bismuth-containing indicator agar resembling grape juice was developed and used to screen both AWRI1631 wine yeast and BY4741 deletion collections. Both Δlst4 and Δlst7 strains were observed to form lighter coloured colonies and produce significantly less H2S than the wild-type on high concentrations of cysteine. Further investigations revealed that deleting genes involved in cysteine transportation such as AGP1, GNP1, MUP1, STP1 and DAL81 all resulted in reduced production of H2S from cysteine. These findings demonstrated, for the first time, that genes involved in regulating cysteine uptake could affect H2S formation from cysteine and therefore selecting wine yeasts with ability to take up supplemented cysteine efficiently could maximise aromatic thiol production. Preliminary results indicate that the higher levels of 3MH/A could be achieved by modulating TUM1 and cysteine supplementation. In addition, polysulfides, that may affect the sensory quality of wine, were detected for the first time in yeast undergoing fermentation on high concentrations of cysteine by the fluorescent probe SSP4. Finally, an up-to-date review of recent study on sulfur metabolism in S. cerevisiae is presented, which includes suggestions for future research in this field. In conclusion, these findings not only have greatly advanced our current understanding of S. cerevisiae cysteine catabolism, but also could be applied to develop better yeast strains, as well as novel winemaking practices to enhance tropical aromas of wines.