Graduate Thesis Or Dissertation


Nutritional and Genetic Aspects of Sulfite Excretion During Fermentation by Wine Strains of Saccharomyces cerevisiae Public Deposited

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  • Reductive sulfate assimilation, the biological process by which sulfur-containing amino acids and key derivatives are synthesized from sulfate, is broadly shared among bacteria, fungi, and plants. It is the major, if not sole source of methionine and cysteine for Saccharomyces cerevisiae during wine fermentation. Two obligate intermediates formed in the process, sulfite and hydrogen sulfide, are important in winemaking because both compounds can be excreted during fermentation and influence wine quality. Hydrogen sulfide is highly undesirable if it exceeds threshold concentrations and is not reabsorbed by the yeast or lost by evaporation. Winemakers commonly add and monitor levels of sulfite for use as a mild antioxidant and antimicrobial agent because most wine yeasts do not excrete more than 10-30 mg/L. However, too much sulfite, whether excreted or added, can inhibit the Oenococcus oeni¬-mediated malolactic fermentation (MLF) that typically follows the alcoholic fermentation in the production of red and some white wines. Deliberate exploitation of the natural ability of yeast to excrete high amounts of sulfite could potentially replace the need for additions made by winemakers for the production of white wines that are neither aged nor undergo the MLF. This is relevant to organic winemaking because unlike the sulfite additions that are disallowed by current USDA regulations, sulfite produced by yeast is permissible. While the causes of sulfite excretion by yeast during fermentation are not well understood, cultural conditions and genotype are key factors. This project investigated the nutritional and genetic basis for excretion of high levels of sulfite during lab-scale Pinot gris fermentations. The nutritional study examined the question of how nitrogen and pantothenic acid availability affected sulfite excretion by two high-sulfite-excreting and two low-sulfite-excreting commercial strains of Saccharomyces cerevisiae. While nitrogen supplementation generally stimulated sulfite excretion in the low-sulfite producers, a uniform response by the high-sulfite producers was not observed. The response to the form of added nitrogen--ammonia, alanine or yeast extract--was also non-uniform. The addition of pantothenic acid in the presence of low or high nitrogen levels had no effect on excretion by any strain. The genetic analysis uncovered the basis for sulfite excretion in one of the high-sulfite-excreting wine strains. Mating-competent derivatives of one “high” and one “low” sulfite-excreting strain were crossed to generate a hybrid. Meiotic progeny of the hybrid were scored for their ability to excrete sulfite. DNA from “high” and “low” sulfite-excreting progeny was isolated, pooled, sequenced, and subjected to bulk segregant analysis. Three genes were identified as responsible for the high-sulfite excretion phenotype: MET10, ADH2, and SKP2. A new allele of MET10, encoding sulfite reductase, was identified as the most significant factor. Relative to two low sulfite-producing wine strains, eight single nucleotide polymorphisms were observed, of which five resulted in amino acid changes. Four of these five mutations are not present in MET10-932, an allele previously reported to cause a reduction in hydrogen sulfide excretion. ADH2, which encodes alcohol dehydrogenase, and SKP2, that plays a role in the stability of the enzyme that generates the immediate biosynthetic precursor of sulfite, were also implicated. These findings suggest that the high-sulfite excretion phenotype can be introduced into other wine strains of S. cerevisiae by a traditional breeding program.
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