In this current grant year, the analysis of the impact of over-expression of two genes involved in consumption of reduced sulfur, CYS4 and MET17, on H2S formation in commercial and natural wine strains of Saccharomyces was completed. Interestingly, increasing the level of expression of the CYS4 gene completely eliminated hydrogen sulfide production in four strains, had no effect in others, and in a few resulted in an increase in H2S. Similar results were obtained for MET17. So far, strains that showed reduced volatile sulfur formation with CYS4 did not show any effect with MET17 and those showing an effect with MET17 showed no or increased H2S formation with over-expression of CYS4. Strains that were high produces of H2S tended to decrease sulfide release when CYS4 was present, while the moderate producers showed a stronger response with MET17. Thus, there are multiple underlying genetic causes for the production of hydrogen sulfide. This analysis does indicate that once the cause of H2S release is known for a given strain, it can be corrected genetically. It will also be possible to screen for strains naturally possessing alleles leading to reduced sulfide production to be used in conventional breeding programs.
This research has further clarified the basis for the two phases of hydrogen sulfide release observed during fermentation. The early phase of hydrogen sulfide production occurs shortly after maximal cell biomass is attained, within the first few days of active fermentation, and is related to the relative activities of the enzymes generating and consuming reduced sulfur. The later stage, which occurs at the end of fermentation, is related to the nitrogen recycling behavior of the culture. Genomic data indicates that at this point in time numerous pathways have been induced that shunt nitrogen between amino acid components. When this occurs, there is a net shift of nitrogen from the sulfur containing amino acids to the non-sulfur containing amino acids. If nitrogen levels are in ample supply, this is prevented from occurring. Interestingly, analysis of the pattern of production of hydrogen sulfide of the 12 strains used in this study revealed that many of the strains produce hydrogen sulfide continuously during fermentation. Over-expression of MET17 and CYS4 has the highest impact on the continual producers versus the transient producers.