Cloning of a Gene Suppressing Hydrogen Sulfide Production by <i>Saccharomyces Cerevisiae</i> and its Expression in a Brewing Yeast

Tezuka, Hidetoshi; Mori, Toshio; Okumura, Yasushi; Kitabatake, Katsuaki; Tsumura, Yoshihiro

doi:10.1094/asbcj-50-0130

Cited by 42 publications

(25 citation statements)

References 6 publications

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“…Previous research has identified MET17 (also known as MET25 and MET15) and CYS4, both located downstream of sulfite reductase, as controlling genes in the pathway affecting sulfide leakage (13,28,42,43). The MET17 gene encodes the enzyme O-acetyl homoserine-O-acetyl serine sulfhydrylase and is responsible for incorporating the sulfide, along with O-acetylhomoserine, into homocysteine.…”

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confidence: 99%

“…The strain in which overexpression had an effect was later shown to carry a disrupted allele of MET17 (24). Likewise, overexpression of CYS4 in four native isolate strains did not lead to any significant reduction in H 2 S production (24), although overexpression of CYS4 did reduce H 2 S in a brewing strain (43). These findings suggest that simple increases in enzyme activity of reactions that consume reduced sulfide are insufficient to reduce sulfide formation and therefore not a viable strategy for the construction of genetically modified strains with a reduced capacity to produce sulfide.…”

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Identification of Genes Affecting Hydrogen Sulfide Formation inSaccharomyces cerevisiae

Linderholm

Findleton

Kumar

et al. 2008

Appl Environ Microbiol

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A screen of the Saccharomyces cerevisiae deletion strain set was performed to identify genes affecting hydrogen sulfide (H 2 S) production. Mutants were screened using two assays: colony color on BiGGY agar, which detects the basal level of sulfite reductase activity, and production of H 2 S in a synthetic juice medium using lead acetate detection of free sulfide in the headspace. A total of 88 mutants produced darker colony colors than the parental strain, and 4 produced colonies significantly lighter in color. There was no correlation between the appearance of a dark colony color on BiGGY agar and H 2 S production in synthetic juice media. Sixteen null mutations were identified as leading to the production of increased levels of H 2 S in synthetic juice using the headspace analysis assay. All 16 mutants also produced H 2 S in actual juices. Five of these genes encode proteins involved in sulfur containing amino acid or precursor biosynthesis and are directly associated with the sulfate assimilation pathway. The remaining genes encode proteins involved in a variety of cellular activities, including cell membrane integrity, cell energy regulation and balance, or other metabolic functions. The levels of hydrogen sulfide production of each of the 16 strains varied in response to nutritional conditions. In most cases, creation of multiple deletions of the 16 mutations in the same strain did not lead to a further increase in H 2 S production, instead often resulting in decreased levels.

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Identification of Genes Affecting Hydrogen Sulfide Formation inSaccharomyces cerevisiae

Linderholm

Findleton

Kumar

et al. 2008

Appl Environ Microbiol

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“…Recently, capillary electrophoresis-electrospray ionization (CE-ESI)-MS has emerged as a powerful analytical tool, and a number of CE-ESI-MS methods have been developed for the analysis of charged species such as carboxylic acids, phenolic compounds, amino acids, metal species, tetramines, and herbicides (25). While metabolite-profiling analysis of the glutathione synthesis pathway in baker's yeast was recently reported (15), to our knowledge, the metabolomic analysis of bottom-fermenting yeast has not yet been reported.The physiology of sulfur metabolism in Saccharomyces yeasts, particularly in regard to SO 2 and H 2 S production, has received significant attention (11,17,28). Although the biosynthesis of these two compounds is interconnected, it would be desirable to increase SO 2 and to decrease H 2 S in beer.…”

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“…The physiology of sulfur metabolism in Saccharomyces yeasts, particularly in regard to SO 2 and H 2 S production, has received significant attention (11,17,28). Although the biosynthesis of these two compounds is interconnected, it would be desirable to increase SO 2 and to decrease H 2 S in beer.…”

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Development of Bottom-Fermenting Saccharomyces Strains That Produce High SO ₂ Levels, Using Integrated Metabolome and Transcriptome Analysis

Yoshida

Imoto

Minato

et al. 2008

Appl Environ Microbiol

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Sulfite plays an important role in beer flavor stability. Although breeding of bottom-fermenting Saccharomyces strains that produce high levels of SO 2 is desirable, it is complicated by the fact that undesirable H 2 S is produced as an intermediate in the same pathway. Here, we report the development of a high-level SO 2 -producing bottom-fermenting yeast strain by integrated metabolome and transcriptome analysis. This analysis revealed that O-acetylhomoserine (OAH) is the rate-limiting factor for the production of SO 2 and H 2 S. Appropriate genetic modifications were then introduced into a prototype strain to increase metabolic fluxes from aspartate to OAH and from sulfate to SO 2 , resulting in high SO 2 and low H 2 S production. Spontaneous mutants of an industrial strain that were resistant to both methionine and threonine analogs were then analyzed for similar metabolic fluxes. One promising mutant produced much higher levels of SO 2 than the parent but produced parental levels of H 2 S.The bottom-fermenting yeast Saccharomyces pastorianus is used to produce beer and has been proposed to be a natural hybrid between Saccharomyces cerevisiae and Saccharomyces bayanus (30). Bottom-fermenting yeasts have two types of genes, one set highly homologous (more than 90% identity) to those of S. cerevisiae and the other less so but highly homologous to S. bayanus (i.e., non-S. cerevisiae [Lg type]) (8,14,27,33). One way in which S. pastorianus differs from baker's yeast (S. cerevisiae) is its tendency to produce higher levels of both sulfite (SO 2 ) and hydrogen sulfide (H 2 S).It is well known that sulfur compounds in beer make significant contributions to flavor and aroma. SO 2 , for example, acts as an antioxidant, which slows the development of oxidation haze and staling of flavors in beer. In contrast, H 2 S has an aroma of rotten eggs and is also a precursor of other compounds with undesirable sensory characteristics. SO 2 and H 2 S are produced by yeast during reductive sulfate assimilation (Fig. 1). Inorganic sulfate is taken up through a sulfate permease and reduced to SO 2 by enzymes encoded by MET3, MET14, and MET16. SO 2 is then reduced to H 2 S by SO 2 reductase encoded by MET5 and MET10 (29). The next intermediate, homocysteine, which is synthesized from H 2 S and O-acetylhomoserine (OAH) by OAH sulfhydrylase encoded by MET17, leads to the formation of cysteine, methionine, and S-adenosylmethionine (SAM). SAM transcriptionally represses all of the genes involved in sulfate assimilation. Park and Bakalinsky previously reported that SSU1 encodes an SO 2 efflux pump that exports intracellular SO 2 through the plasma membrane (18).In the postgenomic era, systematic and high-throughput analyses of mRNA and proteins have become central to recent functional genomics initiatives. Metabolomics entails the analysis of all cellular metabolites and has become a powerful new tool for gaining insight into functional biology. Measurement of numerous metabolites within a cell and tracking concentration changes as a fu...

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“…The physiology of sulfur metabolism in Saccharomyces species of yeast, particularly in regard to SO 2 and H 2 S production, has received considerable attention (Hansen and Kielland-Brandt, 1996;Omura and Shibano, 1995;Takahashi et al, 1980;Tezuka et al, 1992). SO 2 and H 2 S are produced by yeast during the assimilatory reduction of sulfate to H 2 S, which is essential for biosynthesis of the sulfur-containing amino acids methionine and cysteine ( Fig.…”

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confidence: 99%

A novel mechanism regulates H₂S and SO₂ production in Saccharomyces cerevisiae

Yoshida

Imoto

Minato

et al. 2010

Yeast

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Sulfite (SO 2 ) plays an important role in flavour stability in alcoholic beverages, whereas hydrogen sulfide (H 2 S) has an undesirable aroma. To discover the cellular processes that control SO 2 and H 2 S production, we screened a library of Saccharomyces cerevisiae deletion mutants. Deletion of 12 genes led to increased H 2 S productivity. Ten of these genes are known to be involved in sulfur-containing amino acid metabolism, whereas UBI4 functions in the ubiquitin-proteasome system and SKP2 encodes an F-box-containing protein whose function is unknown. We found that the skp2 mutant accumulated H 2 S and SO 2 , because the adenosylphophosulfate kinase Met14p is a substrate of SCF Skp2 and more stable in the skp2 mutant than in the wild-type strain. Furthermore, the skp2 mutant grew more slowly than the wild-type strain under nutrient-limited conditions. Metabolome analysis showed that the concentration of intracellular cysteine is lower in the skp2 mutant than in the wild-type strain. The slow growth of the skp2 mutant was due to a lower concentration of intracellular cysteine, because the addition of cysteine suppressed the slow growth. In the skp2 mutant, the cysteine biosynthesis proteins Str2p, Str3p and Str4p are more stable than in the wild-type strain. Moreover, supplementation with methionine, S -adenosylmethionine, S -adenosylhomocysteine and homocysteine also suppressed the slow growth. Overexpression of STR1 or STR4 caused a more severe defect in the skp2 mutant. These results suggest that the balance of methionine and cysteine biosynthesis is important for yeast cell growth. Thus, Skp2p is one of the key components regulating this balance and H 2 S/SO 2 production.

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Cloning of a Gene Suppressing Hydrogen Sulfide Production by Saccharomyces Cerevisiae and its Expression in a Brewing Yeast

Cited by 42 publications

References 6 publications

Identification of Genes Affecting Hydrogen Sulfide Formation inSaccharomyces cerevisiae

Identification of Genes Affecting Hydrogen Sulfide Formation inSaccharomyces cerevisiae

Development of Bottom-Fermenting Saccharomyces Strains That Produce High SO ₂ Levels, Using Integrated Metabolome and Transcriptome Analysis

A novel mechanism regulates H₂S and SO₂ production in Saccharomyces cerevisiae

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Cloning of a Gene Suppressing Hydrogen Sulfide Production by Saccharomyces Cerevisiae and its Expression in a Brewing Yeast

Cited by 42 publications

References 6 publications

Identification of Genes Affecting Hydrogen Sulfide Formation inSaccharomyces cerevisiae

Identification of Genes Affecting Hydrogen Sulfide Formation inSaccharomyces cerevisiae

Development of Bottom-Fermenting Saccharomyces Strains That Produce High SO 2 Levels, Using Integrated Metabolome and Transcriptome Analysis

A novel mechanism regulates H2S and SO2 production in Saccharomyces cerevisiae

Contact Info

Product

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Development of Bottom-Fermenting Saccharomyces Strains That Produce High SO ₂ Levels, Using Integrated Metabolome and Transcriptome Analysis

A novel mechanism regulates H₂S and SO₂ production in Saccharomyces cerevisiae