In situ high throughput method for H2S detection during micro-scale wine fermentation

Winter, Gal; Curtin, Chris

doi:10.1016/j.mimet.2012.08.003

Cited by 18 publications

(16 citation statements)

References 20 publications

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“…While developing a novel high-throughput assay for H 2 S detection [22] , we noted that S. cerevisiae laboratory strain BY4742 did not produce detectable concentrations of H 2 S when grown in a chemically defined medium. In the current work, after supplementation of medium with cysteine, we detected high concentrations of H 2 S, suggesting this H 2 S was generated by BY4742 solely from cysteine catabolism ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, yeast impaired in V-ATPase activity are able to grow at a similar rate to the wild type under acidic conditions (pH<5), making studies of this complex in S. cerevisiae particularly important. Serendipitously, the medium used for our experiments was at pH 4.5, based on the optimal pH needed for H 2 S detection using the MBR method [22] . It is possible that the role of V-ATPase in H 2 S formation from cysteine is to encourage conversion of HS to H 2 S, downstream of enzymatic release from cysteine.…”

Section: Discussionmentioning

confidence: 99%

“…Screening was performed using a novel high-throughput method that estimated H 2 S excreted in the growth medium during fermentation and allows generation of H 2 S production profiles [22] . For follow-up experiments, H 2 S released during fermentation was detected in the shake-flask headspace using silver nitrate selective gas detector tubes (Komyo Kitagawa, Japan), as described in [23] .…”

Section: Methodsmentioning

confidence: 99%

“…Cultures of strains in the S. cerevisiae BY4742 EUROSCARF deletion collection were pre-grown to stationary phase, each in 200 µl YPD with agitation (150 rpm) in a microtiter plate. The preculture was inoculated at 20 µl into 180 µl of medium supplemented with cysteine and 10% (v/v) of either H 2 S detection mix [22] including 100 mM citric acid and 5 mg/ml of methylene blue, hereafter referred to as MB samples or water, referred to as control samples. Each plate included a well with the wild-type strain (BY4742) as a control in addition to un-inoculated wells to account for medium contamination and non-enzymatic degradation of cysteine to H 2 S. Cultures were inoculated into a microtiter plate in four equal blocks (wells A1 to D6, A7 to D12, E1 to H6 and E7 to H12), representing four replicates of each strain.…”

Section: Methodsmentioning

confidence: 99%

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Formation of Hydrogen Sulfide from Cysteine in Saccharomyces cerevisiae BY4742: Genome Wide Screen Reveals a Central Role of the Vacuole

2014

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Discoveries on the toxic effects of cysteine accumulation and, particularly, recent findings on the many physiological roles of one of the products of cysteine catabolism, hydrogen sulfide (H2S), are highlighting the importance of this amino acid and sulfur metabolism in a range of cellular activities. It is also highlighting how little we know about this critical part of cellular metabolism. In the work described here, a genome-wide screen using a deletion collection of Saccharomyces cerevisiae revealed a surprising set of genes associated with this process. In addition, the yeast vacuole, not previously associated with cysteine catabolism, emerged as an important compartment for cysteine degradation. Most prominent among the vacuole-related mutants were those involved in vacuole acidification; we identified each of the eight subunits of a vacuole acidification sub-complex (V1 of the yeast V-ATPase) as essential for cysteine degradation. Other functions identified included translation, RNA processing, folate-derived one-carbon metabolism, and mitochondrial iron-sulfur homeostasis. This work identified for the first time cellular factors affecting the fundamental process of cysteine catabolism. Results obtained significantly contribute to the understanding of this process and may provide insight into the underlying cause of cysteine accumulation and H2S generation in eukaryotes.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Formation of Hydrogen Sulfide from Cysteine in Saccharomyces cerevisiae BY4742: Genome Wide Screen Reveals a Central Role of the Vacuole

2014

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“…Another source of GSH is nutrients that are commonly added to active dry yeast rehydration medium to improve yeast fermentation performance, which might act as a source for H 2 S production during fermentation (49). The mechanisms of H 2 S release due to GSH addition are not yet understood, but it has been suggested that this might occur via degradation of GSH to the individual amino acids and by subsequent degradation of cysteine to H 2 S by cysteine desulfhydrase activity (49,50). With this in mind, the relative impact of the wine yeast IRC7 genotype in modulating VSC formation might be most important when the concentrations of cysteine and/or GSH are elevated, in combination with low-nitrogen or low-phenolic grape musts (47,48).…”

Section: Discussionmentioning

confidence: 99%

Inactivating Mutations in Irc7p Are Common in Wine Yeasts, Attenuating Carbon-Sulfur β-Lyase Activity and Volatile Sulfur Compound Production

Cordente

Borneman

Bartel

et al. 2019

Appl Environ Microbiol

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During alcoholic fermentation of grape sugars, wine yeasts produce a range of secondary metabolites that play an important role in the aroma profile of wines. In this study, we have explored the ability of a large number of wine yeast strains to modulate wine aroma composition, focusing on the release of the “fruity” thiols 3-mercaptohexan-1-ol (3-MH) and 4-mercapto-4-methylpentan-2-one (4-MMP) from their respective cysteinylated nonvolatile precursors. The role of the yeast geneIRC7in thiol release has been well established, and it has been shown that a 38-bp deletion found in many wine strains cause them to express a truncated version of Irc7p that does not possess cysteine-S-conjugate β-lyase activity. In our data, we find thatIRC7allele length alone does not fully explain the capacity of a strain to release thiols. Screening of a large number of strains coupled with analysis of genomic sequence data allowed us to identify several previously undescribed single-nucleotide polymorphisms (SNPs) inIRC7that, when coupled with allele length, more robustly explain the ability of a particular yeast strain to release thiols from their cysteinylated precursors. We also demonstrate that allelic variation ofIRC7not only affects the release of thiols but modulates the formation of negative volatile sulfur compounds from the amino acid cysteine. The results of this study provide winemakers with an improved understanding of the genetic determinants that affect wine aroma and flavor, which can be used to guide the choice of yeast strains that are fit for purpose.IMPORTANCEVolatile sulfur compounds contribute to wine aromas that may be considered pleasant, such as “tropical,” “passionfruit,” and “guava,” as well as aromas that are considered undesirable, such as “rotten eggs,” “onions,” and “sewer.” During fermentation, wine yeasts release some of these compounds from odorless precursor molecules, a process that is most efficient when performed by yeasts that express active forms of the protein Irc7p. We show that most wine yeasts carry mutations that reduce activity of this protein, affecting the formation of volatile sulfur compounds that impart both pleasant and unpleasant aromas. The results provide winemakers with guidance on the choice of yeasts that can emphasize or deemphasize this particular contribution to wine quality.

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TheIRC7gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source

Santiago

Gardner

2015

Yeast

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Although cysteine desulphydrase activity has been purified and characterized from Saccharomyces cerevisiae, the gene encoding this activity in vivo has never been defined. We show that the full-length IRC7 gene, encoded by the YFR055W open reading frame, encodes a protein with cysteine desulphydrase activity. Irc7p purified to homogeneity is able to utilize L-cysteine as a substrate, producing pyruvate and hydrogen sulphide as products of the reaction. Purified Irc7p also utilized L-cystine and some other cysteine conjugates, but not L-cystathionine or L-methionine, as substrates. We further show that, in vivo, the IRC7 gene is both necessary and sufficient for yeast to grow on L-cysteine as a nitrogen source, and that overexpression of the gene results in increased H 2 S production. Strains overexpressing IRC7 are also hypersensitive to a toxic analogue, S-ethyl-L-cysteine. While IRC7 has been identified as playing a critical role in converting cysteine conjugates to volatile thiols that are important in wine aroma, its biological role in yeast cells is likely to involve regulation of cysteine and redox homeostasis.

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In situ high throughput method for H2S detection during micro-scale wine fermentation

Cited by 18 publications

References 20 publications

Formation of Hydrogen Sulfide from Cysteine in Saccharomyces cerevisiae BY4742: Genome Wide Screen Reveals a Central Role of the Vacuole

Formation of Hydrogen Sulfide from Cysteine in Saccharomyces cerevisiae BY4742: Genome Wide Screen Reveals a Central Role of the Vacuole

Inactivating Mutations in Irc7p Are Common in Wine Yeasts, Attenuating Carbon-Sulfur β-Lyase Activity and Volatile Sulfur Compound Production

TheIRC7gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source

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