Construction of Low H2S Producing Strains of Wine Yeast

The aim of this one year project was to evaluate the impact of altered alleles derived from the low-sulfide producing strain UCD932 for their potential to reduce sulfide formation in high-sulfide producers. UCD932 is a native wine isolate previously shown to not produce sulfide under any enological circumstances evaluated. However, it is not as strong of a fermentor as other strains and has limited commercial applicability. The sequences of the genes of the sulfate reduction pathway were analyzed in UCD932 and genetic crosses were used to define those genes associated with the low sulfide production trait. In most crosses only one gene, subsequently identified as MET10UCD932 seemed to be required to reduce sulfide production while in other crosses it seemed that more than one gene was required. UCD932 contains mutations in CYS4HOM6MET5and MET6 in addition to the mutation in MET10. Allele swap technology, the replacement of one form of a gene by a natural variant from another strain, was used to evaluate the role of these genes in sulfide production in UCD522. UCD522 is a well known high sulfide producer. Replacement of MET10UCD522 with MET10UCD932eliminated H2S production by UCD522. Replacement of other alleles appeared to have no effect. Similarly, replacement of the MET10 genes of two native high sulfide producers, UCD940 and UCD950, with MET10UCD932also eliminated sulfide production in those two genetic backgrounds in both synthetic and actual grape juice. Thus, allele swap of the existing MET10 gene of a commercial or native strain with the MET10 gene from UCD932 is an effective strategy to reduce sulfide production. Further tests demonstrated that this allele did not affect other enological properties of the strains such as sulfite tolerance, fermentation rate or progression, or aroma profile with the exception of the loss of sulfides. A patent application has been submitted for the use of the MET10UCD932 allele to reduce sulfide formation. In one other high sulfide producing strain, UCD939, replacement of the existing MET10 gene with MET10UCD932 appeared to be a lethal event. Thus there are likely combinations of genes of this pathway that do not permit cell growth or that are toxic. An important goal for this grant was the analysis of the potential impact on strain competitiveness due to the change in allele at MET10. To test this, mixtures of equal concentrations of modified and unmodified UCD522 were inoculated into synthetic and actual juices. The two strains can be easily distinguished on BiGGY agar as one, MET10UCD932 gives white colonies on this medium and the other, MET10UCD522, gives tan colonies. At the end of fermentation, the relative percentage of the strain carrying MET10UCD932 had decreased from 50%to 20-25%indicating that there is an advantage in the wild to hydrogen sulfide formation. This is not surprising and explains why so many strains have evolved to produce sulfide. It also suggests that while the percentage of the allele swap strain decreased, it is still competitive and would likely dominate a fermentation when used following normal inoculation practices.