MET2 affects production of hydrogen sulfide during wine fermentation

The production of hydrogen sulfide (H₂S) during yeast fermentation contributes negatively to wine aroma. We have mapped naturally occurring mutations in commercial wine strains that affect production of H₂S. A dominant R₃₁₀G mutant allele of MET2, which encodes homoserine O-acetyltransferase, is present in several wine yeast strains as well as in the main lab strain S288c. Reciprocal hemizygosity and allele swap experiments demonstrated that the MET2 _R310G allele confers reduced H₂S production. Mutations were also identified in genes encoding the two subunits of sulfite reductase, MET5 and MET10, which were associated with reduced H₂S production. The most severe of these, an allele of MET10, showed five additional phenotypes: reduced growth rate on sulfate, elevated secretion of sulfite, and reduced production in wine of three volatile sulfur compounds: methionol, carbon disulfide and methylthioacetate. Alleles of MET5 and MET10, but not MET2, affected H₂S production measured by colour assays on BiGGY indicator agar, but MET2 effects were seen when bismuth was added to agar plates made with Sauvignon blanc grape juice. Collectively, the data are consistent with the hypothesis that H₂S production during wine fermentation results predominantly from enzyme activity in the sulfur assimilation pathway. Lower H₂S production results from mutations that reduce the activity of sulfite reductase, the enzyme that produces H₂S, or that increase the activity of l-homoserine-O-acetyltransferase, which produces substrate for the next step in the sulfur assimilation pathway.     

Development of a method to measure hydrogen sulfide in wine fermentation

A hydrogen sulfide (H2S) detecting tube was developed for the quantitative determination of H2S produced by yeast during laboratory scale wine fermentations. The detecting tube consisted of a small transparent plastic tube packed with an H2S-sensitive color-indicating medium. The packed medium changed color, with the color change progressing upward from the bottom of the tube, upon exposure to H2S produced by yeast during fermentation. A calibration study using a standard H2S gas showed that the length of the portion that darkened was directly related to the quantity of H2S (microg) with a high correlation coefficient (r2=0.9997). The reproducibility of the H2S detecting tubes was determined with five repetitive measurements using a standard H2S solution [5.6 microg/200 ml (28 ppb)], which resulted in a coefficient of variation of 3.6% at this level of H2S. With the sulfide detecting tubes, the production of H2S was continuously monitored and quantified from laboratory scale wine fermentations with different yeast strains and with the addition of different levels of elemental sulfur to the grape juice. This sulfide detecting tube technology may allow winemakers to quantitatively measure H2S produced under different fermentation conditions, which will eventually lead winemakers to better understand the specific factors and conditions for the excessive production of H2S during wine fermentation in a large production scale.     

Impact of fermentation rate changes on potential hydrogen sulfide concentrations in wine

The correlation between alcoholic fermentation rate, measured as carbon dioxide (CO2) evolution, and the rate of hydrogen sulfide (H2S) formation during wine production was investigated. Both rates and the resulting concentration peaks in fermentor headspace H2S were directly impacted by yeast assimilable nitrogenous compounds in the grape juice. A series of model fermentations was conducted in temperature-controlled and stirred fermentors using a complex model juice with defined concentrations of ammonium ions and/or amino acids. The fermentation rate was measured indirectly by noting the weight loss of the fermentor; H2S was quantitatively trapped in realtime using a pre-calibrated H2S detection tube which was inserted into a fermentor gas relief port. Evolution rates for CO2 and H2S as well as the relative ratios between them were calculated. These fermentations confirmed that total sulfide formation was strongly yeast strain-dependent, and high concentrations of yeast assimilable nitrogen did not necessarily protect against elevated H2S formation. High initial concentrations of ammonium ions via addition of diammonium phosphate (DAP) caused a higher evolution of H2S when compared with a non-supplemented but nondeficient juice. It was observed that the excess availability of a certain yeast assimilable amino acid, arginine, could result in a more sustained CO2 production rate throughout the wine fermentation. The contribution of yeast assimilable amino acids from conventional commercial yeast foods to lowering of the H2S formation was marginal.     

Interactions between Torulaspora delbrueckii and Saccharomyces cerevisiae in wine fermentation: influence of inoculation and nitrogen content

Alcoholic fermentation by an oenological strain of Torulaspora delbrueckii in association with an oenological strain of Saccharomyces cerevisiae was studied in mixed and sequential cultures. Experiments were performed in a synthetic grape must medium in a membrane bioreactor, a special tool designed to study indirect interactions between microorganisms. Results showed that the S. cerevisiae strain had a negative impact on the T. delbrueckii strain, leading to a viability decrease as soon as S. cerevisiae was inoculated. Even for high inoculation of T. delbrueckii (more than 20× S. cerevisiae) in mixed cultures, T. delbrueckii growth was inhibited. Substrate competition and cell-to-cell contact mechanism could be eliminated as explanations of the observed interaction, which was probably an inhibition by a metabolite produced by S. cerevisiae. S. cerevisiae should be inoculated 48 h after T. delbrueckii in order to ensure the growth of T. delbrueckii and consequently a decrease of volatile acidity and a higher isoamyl acetate production. In this case, in a medium with a high concentration of assimilable nitrogen (324 mg L^?1), S. cerevisiae growth was not affected by T. delbrueckii. But in a sequential fermentation in a medium containing 176 mg L^?1 initial assimilable nitrogen, S. cerevisiae was not able to develop because of nitrogen exhaustion by T. delbrueckii growth during the first 48 h, leading to sluggish fermentation.     

The wine yeast strain-dependent expression of genes implicated in sulfide production in response to nitrogen availability

Sulfur metabolism in S. cerevisiae is well established, but the mechanisms underlying the formation of sulfide remain obscure. Here we investigated by real time RT-PCR the dependence of expression levels of MET3, MET5/ECM17, MET10, MET16 and MET17 along with SSU1 on nitrogen availability in two wine yeast strains that produce divergent sulfide profiles. MET3 was the most highly expressed of the genes studied in strain PYCC4072, and SSU1 in strain UCD522. Strains behaved differently according to the sampling times, with UCD522 and PYCC4072 showing the highest expression levels at 120h and 72h, respectively. In the presence of 267mg assimilable N/l, the genes were more highly expressed in strain UCD522 than in PYCC4072. MET5/ECM17 and MET17 were only weakly expressed in both strains under any condition tested. MET10 and SSU1 in both strains, but MET16 only in PYCC4072, were consistently up-regulated when sulfide production was inhibited. This study illustrates that strain genotype could be important in determining enzyme activities and therefore the rate of sulfide liberation. This linkage, for some yeast strains, of sulfide production to expression levels of genes associated to sulfate assimilation and sulfur amino acid biosynthesis could be relevant for defining new strategies for genetic improvement of wine yeasts.     

Re-assessment of the influence of yeast strain and environmental factors on glycerol production in wine

Increasing glycerol production is of concern for wine-makers in improving the quality of certain wines. We have compared the impact of strain and relevant environmental factors influencing glycerol production under the same conditions, i.e. standardized conditions simulating enological fermentation. The glycerol production of 19 industrial wine strains ranged from 6.4 to 8.9 g l-1 and varied significantly between strains. The production of acetate and succinate was also found to differ substantially depending on the strain but no significant strain-dependent variation was observed for acetaldehyde. Interestingly, high glycerol production was not correlated to high production of acetate or acetaldehyde, which are undesirable in wine. A detailed study with two low or two high glycerol-producing strains showed that temperature and the initial concentration of nitrogen had little effect on the amount of glycerol formed, although agitation or a nitrogen source composed mainly of ammoniacal nitrogen slightly enhanced glycerol production. The influence of environmental factors remained minor while the predominant factor for glycerol variability in wine was attributed to the strain. Taking into account wine-making constraints, the results indicate that achieving a high glycerol content in wine requires the selection or improvement of yeast strains rather than the control of growth and cultivation conditions.     

Isolation of sulfite reductase variants of a commercial wine yeast with significantly reduced hydrogen sulfide production

The production of hydrogen sulfide (H₂S) during fermentation is a common and significant problem in the global wine industry as it imparts undesirable off-flavors at low concentrations. The yeast Saccharomyces cerevisiae plays a crucial role in the production of volatile sulfur compounds in wine. In this respect, H₂S is a necessary intermediate in the assimilation of sulfur by yeast through the sulfate reduction sequence with the key enzyme being sulfite reductase. In this study, we used a classical mutagenesis method to develop and isolate a series of strains, derived from a commercial diploid wine yeast (PDM), which showed a drastic reduction in H₂S production in both synthetic and grape juice fermentations. Specific mutations in the MET10 and MET5 genes, which encode the catalytic α- and β-subunits of the sulfite reductase enzyme, respectively, were identified in six of the isolated strains. Fermentations with these strains indicated that, in comparison with the parent strain, H₂S production was reduced by 50–99%, depending on the strain. Further analysis of the wines made with the selected strains indicated that basic chemical parameters were similar to the parent strain except for total sulfite production, which was much higher in some of the mutant strains.     

A genetic approach of wine yeast fermentation capacity in nitrogen-starvation reveals the key role of nitrogen signaling

Background

In conditions of nitrogen limitation, Saccharomyces cerevisiae strains differ in their fermentation capacities, due to differences in their nitrogen requirements. The mechanisms ensuring the maintenance of glycolytic flux in these conditions are unknown. We investigated the genetic basis of these differences, by studying quantitative trait loci (QTL) in a population of 133 individuals from the F2 segregant population generated from a cross between two strains with different nitrogen requirements for efficient fermentation.

Results

By comparing two bulks of segregants with low and high nitrogen requirements, we detected four regions making a quantitative contribution to these traits. We identified four polymorphic genes, in three of these four regions, for which involvement in the phenotype was validated by hemizygote comparison. The functions of the four validated genes, GCN1, MDS3, ARG81 and BIO3, relate to key roles in nitrogen metabolism and signaling, helping to maintain fermentation performance.

Conclusions

This study reveals that differences in nitrogen requirement between yeast strains results from a complex allelic combination. The identification of three genes involved in sensing and signaling nitrogen and specially one from the TOR pathway as affecting nitrogen requirements suggests a role for this pathway in regulating the fermentation rate in starvation through unknown mechanisms linking nitrogen signaling to glycolytic flux.

Electronic supplementary material

The online version of this article (doi: 10.1186/1471-2164-15-495) contains supplementary material, which is available to authorized users.     

Production of fermentation aroma compounds by Saccharomyces cerevisiae wine yeasts: effects of yeast assimilable nitrogen on two model strains

The contribution of yeast fermentation metabolites to the aromatic profile of wine is well documented; however, the biotechnological application of this knowledge, apart from strain selection, is still rather limited and often contradictory. Understanding and modeling the relationship between nutrient availability and the production of desirable aroma compounds by different strains must be one of the main objectives in the selection of industrial yeasts for the beverage and food industry. In order to overcome the variability in the composition of grape juices, we have used a chemically defined model medium for studying yeast physiological behavior and metabolite production in response to nitrogen supplementation so as to identify an appropriate yeast assimilable nitrogen level for strain differentiation. At low initial nitrogen concentrations, strain KU1 produced higher quantities of esters and fatty acids whereas M522 produced higher concentrations of isoacids, gamma-butyrolactone, higher alcohols and 3-methylthio-1-propanol. We propose that although strains KU1 and M522 have a similar nitrogen consumption profile, they represent useful models for the chemical characterization of wine strains in relation to wine quality. The differential production of aroma compounds by the two strains is discussed in relation to their capacity for nitrogen usage and their impact on winemaking. The results obtained here will help to develop targeted metabolic footprinting methods for the discrimination of industrial yeasts.     

Occurrence of acid-labile sulfide in cadmium-binding peptide 1 from fission yeast

Two kinds of Cd-binding peptides (Cd-BP1 and Cd-BP2) are induced in fission yeast upon addition of CdCl2 to the culture medium (l). It was also reported that Cd-BP1 and Cd-BP2 consisted of the same components, unit peptides (Cys3, Glu3, Gly1) and Cd atoms, though the respective amounts of components in each molecule were different (1, 2). Now, we have found that Cd-BP1 contains about 1 mol of acid-labile sulfide per mol, and Cd-BP2 contains no labile sulfide. The existence of the labile sulfide explains the unique physicochemical characteristics of Cd-BP1. Since acid-labile sulfide has not been found in metallothioneins or other metallothionein-like metal-binding proteins, the occurrence of labile sulfide in Cd-BP1 is the first instance in this field.     

Isolation and characterization of low hydrogen sulphide producing wine yeast

Variants of the wine yeast Saccharomyces cerevisiae var. montrachet no. 522 which retain more and excrete less hydrogen sulphide (H₂S) into the growth medium have been isolated and characterized. Wine produced using these low excretors has been found to contain less H₂S.     

Comparative genomics: a revolutionary tool for wine yeast strain development

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Highlights► Phylogeny of Saccharomyces cerevisiae reveals industry-relevant groupings. ► Comparative genomics reveals genomic variation across industrial yeast strains. ► Next-gen sequencing in next-gen wine yeast strain development. ► Precise mapping of QTLs for marker-assisted breeding of industrial yeast. ► Discovery of putative mobile gene cluster that uses novel type of transposition.