MET17 and Hydrogen Sulfide Formation in Saccharomyces cerevisiae

Hydrogen sulfide (H2S) is an undesirable by-product of alcoholic fermentation by yeast. Very low levels of H2S can be detected due to its low aroma threshold (11), so trace amounts can have a profound effect on final product quality. Subtle changes in yeast metabolic behavior appear to have a major impact on the presence or absence of this spoilage character. It is highly desirable, therefore, to have yeast strains available for wine production that will not produce and release H2S. Lowering the amount of H2S produced could be achieved by supplementation of grape juice with one or more organic sources of sulfur: methionine, cysteine, glutathione, or the intermediate homocysteine, as transsulfuration pathways exist, thereby negating the need for sulfate reduction (7, 34). However, these sulfur-containing compounds can be metabolized to other volatile sulfur compounds by Saccharomyces and other yeasts and bacteria present in fermenting juice (8), producing characters (feces or rotten seafood) as objectionable as or more objectionable than that of H2S (rotten egg). Several environmental factors have been implicated in the appearance of hydrogen sulfide: high residual levels of elemental sulfur (26, 27), presence of sulfur dioxide (13, 17, 31), presence of organic compounds containing sulfur (1, 19), pantothenate deficiency (35, 36), high threonine content (37), high methionine or cysteine content relative to other amino acids (1, 19, 37), and nitrogen limitation (12, 13, 37). Volatile-sulfur-compound production varies widely among commercial strains and natural isolates in response to these environmental conditions (1, 9, 12, 15, 37). This variation suggests that differences in internal levels of enzymatic activities or their regulation have a profound effect on the appearance of hydrogen sulfide. If the basis of this genetic variation can be identified, then it may be possible to construct commercial strains with reduced sulfur production. The MET17 gene (also called MET15 or MET25) encodes a sulfhydrylase (SHLase) capable of using either O-acetylserine (OAS) or O-acetylhomoserine (OAH) as the substrate in vitro and is the last step of the sulfate reduction pathway. met17 mutants are methionine auxotrophs, and no OAS/OAH SHLase activity is detectable (22). Only O-acetylhomoserine appears to be the substrate in vivo, with O-acetylserine serving instead as an inducer of the sulfate reduction pathway (22, 23, 25), in contrast to the situation in bacteria (18). A mutational analysis indicated that the transsulfuration pathway is the only pathway by which cysteine is synthesized in Saccharomyces (7) and that the direct conversion of O-acetylserine to cysteine does not occur. The MET17 gene, therefore, encodes the only enzymatic activity responsible for incorporation of reduced sulfide in this yeast. The levels of OAS/OAH SHLase can vary 10-fold in different strains of Saccharomyces cerevisiae (22). One hypothesis is that low levels of reduced sulfur incorporation result in “leakiness” of reduced sulfur and subsequent release of H2S (16, 19). Thus, strains that are high producers of H2S should have relatively low levels of OAS/OAH SHLase activity. In support of this hypothesis, Omura et al. (21) reported that H2S formation was reduced in a brewing yeast strain overexpressing the MET17 gene, which encodes OAS/OAH SHLase. Our objective in this study was to determine if low levels of OAS/OAH SHLase activity accounted for increased H2S production in two commercial yeast strains: one, UCD522, which produces high levels of H2S under commercial conditions, and another, UCD713, which is a low-to-moderate-level producer of this compound. Additional objectives of this study were (i) to overexpress the MET17 gene in two commercial strains of S. cerevisiae to further test the hypothesis that increased levels of expression would increase efficiency of incorporation of sulfur and reduce H2S formation and (ii) to study the consequences of the overproduction of OAS/OAH SHLase during alcoholic fermentation simulating wine production conditions.