Addition of volatile sulfur compounds to yeast at the early stages of fermentation reveals distinct biological and chemical pathways for aroma formation.

Volatile sulfur compounds (VSCs) greatly influence the sensory properties and quality of wine and arise via both biological and chemical mechanisms. VSCs formed can also act as precursors for further downstream VSCs, thus elucidating the pathways leading to their formation is paramount. Short-term additions of exogenous hydrogen sulfide (H2S), ethanethiol (EtSH), S-ethylthio acetate (ETA), methanethiol (MeSH) and S-methylthio acetate (MTA) were made to exponentially growing fermentations of synthetic grape medium. The VSC profiles produced from live yeast cells were compared with those from dead cells and no cells. Interestingly, this experiment allowed the identification of specific biochemical and/or chemical pathways; e.g. most of the conversion of H2S to EtSH, and the further step from EtSH to ETA, required the presence of live yeast cells, as did the conversion of MeSH to MTA. In contrast, the reaction from MTA to MeSH and ETA to EtSH was due primarily to chemical degradation. Ultimately, this research unravelled some of the complex interactions and interconversions between VSCs, pinpointing the key biochemical and chemical nodes. These pathways are highly interconnected and showcase the complexity of both the sulfur pathways in yeast and the reactive chemistry of sulfur-containing compounds.

The role of yeast ARO8, ARO9 and ARO10 genes in the biosynthesis of 3-(methylthio)-1-propanol from L-methionine during fermentation in synthetic grape medium.

3-(methylthio)-1-propanol (methionol), produced by yeast as an end-product of L-methionine (L-Met) catabolism, imparts off-odours reminiscent of cauliflower and potato to wine. Saccharomyces cerevisiae ARO genes, including transaminases Aro8p and Aro9p, and decarboxylase Aro10p, catalyse two key steps forming methionol via the Ehrlich pathway. We compared methionol concentrations in wines fermented by single Δaro8, Δaro9 and Δaro10 deletants in lab strain BY4743 versus wine strain Zymaflore F15, and F15 double- and triple-aro deletants versus single-aro deletants, using headspace-solid phase microextraction coupled with gas chromatography-mass spectrometry.Deletion of two or more aro genes increased growth lag phase, with the greatest delay exhibited by F15 Δaro8 Δaro9. The single Δaro8 deletion decreased methionol by 44% in BY4743 and 92% in F15, while the Δaro9 deletion increased methionol by 46% in F15 but not BY4743. Single deletion of Δaro10 had no effect on methionol.Unexpectedly, F15 Δaro8 Δaro9 and F15 Δaro8 Δaro9 Δaro10 produced more methionol than F15 Δaro8. In the absence of Aro8p and Aro9p, other transaminases may compensate or an alternative pathway may convert methanethiol to methionol. Our results confirm that Ehrlich pathway genes differ greatly between lab and wine yeast strains, impacting downstream products such as methionol.

Yeast genes involved in regulating cysteine uptake affect production of hydrogen sulfide from cysteine during fermentation.

An early burst of hydrogen sulfide (H2S) produced by Saccharomyces cerevisiae during fermentation could increase varietal thiols and therefore enhance desirable tropical aromas in varieties such as Sauvignon Blanc. Here we attempted to identify genes affecting H2S formation from cysteine by screening yeast deletion libraries via a colony colour assay on media resembling grape juice. Both Δlst4 and Δlst7 formed lighter coloured colonies and produced significantly less H2S than the wild type on high concentrations of cysteine, likely because they are unable to take up cysteine efficiently. We then examined the nine known cysteine permeases and found that deletion of AGP1, GNP1 and MUP1 led to reduced production of H2S from cysteine. We further showed that deleting genes involved in the SPS-sensing pathway such as STP1 and DAL81 also reduced H2S from cysteine. Together, this study indirectly confirms that Agp1p, Gnp1p and Mup1p are the major cysteine permeases and that they are regulated by the SPS-sensing and target of rapamycin pathways under the grape juice-like, cysteine-supplemented, fermentation conditions. The findings highlight that cysteine transportation could be a limiting factor for yeast to generate H2S from cysteine, and therefore selecting wine yeasts without defects in cysteine uptake could maximise thiol production potential.

Hydrogen sulfide and its roles in Saccharomyces cerevisiae in a winemaking context.

The rotten-egg odour of hydrogen sulfide (H2S) produced by the yeast Saccharomyces cerevisiae has attracted considerable research interest due to its huge impact on the sensory quality of fermented foods and beverages. To date, the yeast genetic mechanisms of H2S liberation during wine fermentation are well understood and yeast strains producing low levels of H2S have been developed. Studies have also revealed that H2S is not just a by-product in the biosynthesis of the sulfur-containing amino acids, but indeed a vital molecule involved in detoxification, population signalling and extending cellular life span. Moreover, polysulfides have recently emerged as key players in signalling and the sensory quality of wine because their degradation leads to the release of H2S. This review will focus on the recent findings on the production of H2S and polysulfides in S. cerevisiae and summarise their potential roles in yeast survival and winemaking. Recent advances in techniques for the detection of H2S and polysulfides offer an exciting opportunity to uncover the novel genes and pathways involved in their formation from different sulfur sources. This knowledge will not only provide further insights into yeast sulfur metabolism, but could potentially improve the sensory quality of wine.

The yeast TUM1 affects production of hydrogen sulfide from cysteine treatment during fermentation.

The undesirable rotten-egg odour of hydrogen sulfide (H2S) produced by yeast shortly after yeast inoculation of grape musts might be an important source of desirable varietal thiols, which contribute to tropical aromas in varieties such as Sauvign-on Blanc. In this study, we observed that Saccharomyces cerevisiae strains produce an early burst of H2S from cysteine. Both Δmet2 and Δmet17 strains produce a larger burst, likely because they are unable to utilise the H2S in the sulfate assimilation pathway. For the first time, we show that TUM1 is partly responsible for the early production of H2S from cysteine. Overex-pressing TUM1 elevated production of H2S, whilst its deletion yields only half of the H2S. We further confirmed that yeast convert cysteine to H2S by analysing growth of mutants lacking components of the transsulfuration pathway. High concent-rations of cysteine overcame this growth block, but required TUM1 Collectively, the data indicate that S. cerevisiae does not convert cysteine to sulfate or sulfite, but rather to sulfide via a novel pathway that requires the action of Tum1p. The findi-ngs of this study may allow the improvement of commercial yeasts through the manipulation of sulfur metabolism that are better suited towards production of fruit-driven styles.

The IRC7 gene encodes cysteine desulphydrase activity and confers on yeast the ability to grow on cysteine as a nitrogen source.

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 H2 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.

Yeast genes required for conversion of grape precursors to varietal thiols in wine.

Three varietal thiols are important for the tropical fruit aromas of Sauvignon blanc: 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexanol (3MH) and its acetylated derivative 3-mercaptohexyl acetate (3MHA). These thiols are produced by yeast during fermentation from precursors in grape juice. Here we identify genes in Saccharomyces cerevisiae that are required for the transport and cleavage of two thiol precursors: cysteine-4MMP and glutathione-3MH. A full-length copy of IRC7 is absolutely required for the cleavage of both precursors in the tested strains; the deleted form of the enzyme found in most yeast strains is incapable of converting these compounds into detectable thiols. By using strains that overexpress full-length IRC7, we further show that the glutathione transporter OPT1 and the transpeptidase CIS2 are also required for conversion of glut-3MH to its varietal thiol. No transporter for cys-4MMP was identified: a strain deleted for all nine known cysteine transport genes was still capable of converting cys-4MMP to its varietal thiol, and was also able to take up cysteine at high concentrations. Based on these results, we conclude that cysteine and glutathione precursors make a relatively minor contribution to 3MH production from most grape juices.

Transcriptional response of Saccharomyces cerevisiae to low temperature during wine fermentation.

Although the yeast response to low temperature has industrial significance for baking, lager brewing and white wine fermentation, the molecular response of yeast cells to low temperature remains poorly characterised. Transcriptional changes were quantified in a commercial wine yeast, Enoferm M2, fermented at optimal (25 °C) and low temperature (12.5 °C), at two time points during fermentation of Sauvignon blanc grape juice. The transition from early to mid-late fermentation was notably less severe in the cold than at 25 °C, and the Rim15p-Gis1p pathway was involved in effecting this transition. Genes for three key nutrients were strongly influenced by low temperature fermentation: nitrogen, sulfur and iron/copper, along with changes in the cell wall and stress response. Transcriptional analyses during wine fermentation at 12.5 °C in four F1 hybrids of M2 also highlighted the importance of genes involved in nutrient utilisation and the stress response. We identified transcription factors that may be important for these differences between genetic backgrounds. Since low fermentation temperatures cause fundamental changes in membrane kinetics and cellular metabolism, an understanding of the physiological and genetic limitations on cellular performance will assist breeding of improved industrial strains.

Genetic characterization of strains of Saccharomyces uvarum from New Zealand wineries.

We present a genetic characterization of 65 isolates of Saccharomyces uvarum isolated from wineries in New Zealand, along with the complete nucleotide sequence of a single sulfite-tolerant isolate. The genome of the New Zealand isolate averaged 99.85% nucleotide identity to CBS7001, the previously sequenced strain of S. uvarum. However, three genomic segments (37-87 kb) showed 10% nucleotide divergence from CBS7001 but 99% identity to Saccharomyces eubayanus. We conclude that these three segments appear to have been introgressed from that species. The nucleotide sequence of the internal transcribed spacer (ITS) region from other New Zealand isolates were also very similar to that of CBS7001, and hybrids showed complete genetic compatibility for some strains, with tetrads giving four viable progeny that showed 2:2 segregations of marker genes. Some strains showed high tolerance to sulfite, with genetic analysis indicating linkage of this trait to the transcription factor FZF1, but not to SSU1, the sulfite efflux pump that it regulates in order to confer sulfite tolerance in Saccharomyces cerevisiae. The fermentation characteristics of selected strains of S. uvarum showed exceptionally good cold fermentation characteristics, superior to the best commercially available strains of S. cerevisiae.

Nitrogen and carbon assimilation by Saccharomyces cerevisiae during Sauvignon blanc juice fermentation.

To investigate the assimilation and production of juice metabolites by Saccharomyces cerevisiae during winemaking, we compared the metabolite profiles of 63 Sauvignon blanc (SB) grape juices collected over five harvesting seasons from different locations of New Zealand before and after fermentation by the commercial wine yeast strain EC1118 at 15 °C. Metabolite profiles were obtained using gas chromatography-mass spectrometry and nuclear magnetic resonance and the oenological parameters were determined by Fourier transform infrared spectroscopy. Our results revealed that the amino acids threonine and serine were the most consumed organic nitrogen sources, while proline and gamma-aminobutyric acid were the least consumed amino acids during SB juice fermentation. Saccharomyces cerevisiae metabolised some uncommon nitrogen sources (e.g. norleucine, norvaline and pyroglutamic acid) and several organic acids, including some fatty acids, most likely after fermenting the main juice sugars (glucose, fructose and mannose). However, consumption showed large variation between juices and in some cases between seasons. Our study clearly shows that preferred nitrogen and carbon sources were consumed by S. cerevisiae EC1118 independent of the juice fine composition, whilst the consumption of other nutrient sources mainly depended on the concentration of other juice metabolites, which explains the uniqueness of each barrel of wine.

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.

Yeast genes involved in sulfur and nitrogen metabolism affect the production of volatile thiols from Sauvignon Blanc musts.

Two volatile thiols, 3-mercaptohexan-1-ol (3MH), and 3-mercaptohexyl-acetate (3MHA), reminiscent of grapefruit and passion fruit respectively, are critical varietal aroma compounds in Sauvignon Blanc (SB) wines. These aromatic thiols are not present in the grape juice but are synthesized and released by the yeast during alcoholic fermentation. Single deletion mutants of 67 candidate genes in a laboratory strain of Saccharomyces cerevisiae were screened using gas chromatography mass spectrometry for their thiol production after fermentation of SB grape juice. None of the deletions abolished production of the two volatile thiols. However, deletion of 17 genes caused increases or decreases in production by as much as twofold. These 17 genes, mostly related to sulfur and nitrogen metabolism in yeast, may act by altering the regulation of the pathway(s) of thiol production or altering substrate supply. Deleting subsets of these genes in a wine yeast strain gave similar results to the laboratory strain for sulfur pathway genes but showed strain differences for genes involved in nitrogen metabolism. The addition of two nitrogen sources, urea and di-ammonium phosphate, as well as two sulfur compounds, cysteine and S-ethyl-L-cysteine, increased 3MH and 3MHA concentrations in the final wines. Collectively these results suggest that sulfur and nitrogen metabolism are important in regulating the synthesis of 3MH and 3MHA during yeast fermentation of grape juice.

A new yeast genetic resource for analysis and breeding.

We made a library of Saccharomyces cerevisiae F(1) hybrids from all possible crosses of 16 wild-type strains, including two common laboratory strains and two commercial winemaking varieties. Fourteen of the starting strains have been sequenced. Thus, the sequences of both genomes are known in 182 novel hybrids, and the sequence of one genome is known in 56. All tested strains sporulated. Fertilities were in the range 0-100%. Hybrids showed no more variation than parental strains for ethanol production, ethanol tolerance or growth at temperature extremes, but some F(1) s appeared to display hybrid vigour (heterosis). We tested four tetrads from one hybrid for their ability to grow at low temperature or in the presence of an inhibitory concentration of ethanol. Only one F(2) was as tolerant as the most tolerant F(0) parent. A few showed intermediate tolerance, but most were less tolerant than either parent or the F(1) hybrid, consistent with uncoupling of genes contributing to an optimized quantitative trait. The diversity and structure of the library should make it useful for analysis of genetic interactions among diverse strains, quantitative inheritance and heterosis, and for breeding.

Effects of nitrogen catabolite repression and di-ammonium phosphate addition during wine fermentation by a commercial strain of S. cerevisiae.

Two deletion mutants expected to be defective in nitrogen catabolite repression (NCR) were constructed in a commercial wine yeast background M2: a ure2 mutant and a dal80 gzf3 double mutant. Wild-type and both mutant strains were fermented in Sauvignon Blanc grape juice with and without addition of di-ammonium phosphate (DAP). The dal80 gzf3 double mutant exhibited a long fermentative lag phase, which was offset by DAP addition (corresponding to 300 mg/L of N). Neither the NCR mutations nor DAP addition affected the content of volatile thiols in the final wine. Microarray analyses of transcripts in the wild-type and dal80 gzf3 double-mutant strains were performed after 2% and 70% sugars were fermented. Of 80 genes previously identified as NCR-regulated, only 13 were upregulated during fermentation of the dal80 gzf3 double-mutant strain in grape juice. Following DAP addition, 34 of the known NCR genes were downregulated, including 17 that were downregulated even in the NCR mutant strain. The results demonstrate an unexpected complexity of the NCR response that may reflect differences between strains of yeast or differences in gene regulation during alcoholic fermentation compared with standard aerobic growth.

The yeast IRC7 gene encodes a ß-lyase responsible for production of the varietal thiol 4-mercapto-4-methylpentan-2-one in wine.

Three varietal thiols are key aroma compounds in Sauvignon Blanc wines: 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexanol (3MH) and its acetylated derivative 3-mercaptohexyl acetate (3MHA). Screening of Saccharomyces cerevisiae strains identified a clinical isolate with elevated 4MMP production after fermentation. Bulked Segregant Analysis of a cross between this isolate and the laboratory strain revealed a single major locus for 4MMP production near the telomere of chromosome 6. Deletion of the IRC7 gene from this region in YJM450 reduced 4MMP production below detectable levels, but did not affect yields of 3MH, in Sauvignon Blanc wine. Sequencing revealed that the IRC7 gene in YJM450 had been introgressed from a strain of Saccharomyces paradoxus. Most strains of S. cerevisiae, including the laboratory strain S288C, have a 38-bp deletion that inactivates IRC7. Overexpression of a full-length S. cerevisiae allele of IRC7 in a wine yeast, Zymaflore F15, increased 4MMP production in Sauvignon Blanc wine from undetectable levels (<10 ng L(-1)) to concentrations of 1000 ng L(-1), and also increased 3MH and 3MHA. Biochemical analysis of soluble protein extracts showed that both the cerevisiae and paradoxus IRC7 proteins show ß-lyase activity, with a substrate preference for cys-4MMP over cys-3MH.

A distinct population of Saccharomyces cerevisiae in New Zealand: evidence for local dispersal by insects and human-aided global dispersal in oak barrels.

Humans have used S. cerevisiae to make alcoholic beverages for at least 5000 years and now this super-model research organism is central to advances in our biological understanding. Current models for S. cerevisiae suggest that its population comprises distinct domesticated and natural groups as well as mosaic strains, but we generally know little of the forces which shape its population structure. In order to test the roles that ecology and geography play in shaping the S. cerevisiae species we examined nine variable microsatellite loci in 172 strains of S. cerevisiae isolated from two spontaneous grape juice ferments, soil, flowers, apiaries and bark in New Zealand. Bayesian analysis shows that the S. cerevisiae in NZ comprise a subdivided but interbreeding population that out-crosses approximately 20% of the time. Some strains contributing to spontaneous ferments cluster with NZ soil/bark isolates, but others cluster with isolates from French oak barrels. It seems some strains have been globally dispersed by humans in oak barrels while some are locally vectored by insects. These data suggest geography is more important than ecology in shaping S. cerevisiae's population structure.

Saccharomyces paradoxus and Saccharomyces cerevisiae reside on oak trees in New Zealand: evidence for migration from Europe and interspecies hybrids.

Saccharomyces cerevisiae and Saccharomyces paradoxus are used as model systems for molecular, cell and evolutionary biology; yet we know comparatively little of their ecology. One niche from which these species have been isolated is oak bark. There are no reports of these species from oak in the Southern Hemisphere. We describe the recovery of both S. cerevisiae and S. paradoxus from oak in New Zealand (NZ), and provide evidence for introgression between the species. Genetic inference shows that the oak S. cerevisiae are closely related to strains isolated from NZ and Australian vineyards, but that the S. paradoxus strains are very closely related to European isolates. This discovery is surprising as the current model of S. paradoxus biogeography suggests that global dispersal is rare. We test one idea to explain how members of the European S. paradoxus population might come to be in NZ: they were transported here along with acorns brought by migrants approximately 200 years ago. We show that S. paradoxus is associated with acorns and thus provide a potential mechanism for the unwitting global dispersal of S. paradoxus by humans.

Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae.

Laboratory strains of yeast (Saccharomyces cerevisiae) based on S288C ferment grape juice relatively poorly. We show that slow fermentation appears to be inherent to this strain, because the original S288C isolate shows fermentation similar to current laboratory isolates. We demonstrate further that some auxotrophic mutations in the laboratory strain show reduced rates of fermentation in grape juice, with lysine auxotrophs particularly impaired compared with isogenic Lys(+) strains. Supplementing lysine at a 10-fold higher concentration than recommended allowed yeast cultures to reach higher final cell densities and restored the fermentation rate of auxotrophic strains to those of the corresponding wild-type strains. However, even with the additional supplementation, the fermentation rates of S288C strains were still slower than those of a commercial wine yeast strain. Conditions were developed that enable auxotrophic laboratory strains derived from S288C to ferment grape juice to completion with high efficiency on a laboratory scale. Fermentation in media based on grape juice will allow the suite of molecular genetic tools developed for these laboratory strains to be used in investigations of complex ferment characteristics and products.

A database of microsatellite genotypes for Saccharomyces cerevisiae.

A system for genotyping Saccharomyces cerevisiae is described based on a multiplex of ten microsatellite loci and the MAT locus. A database of genotypes has been developed for 246 yeast strains, including a large set of commercial wine yeasts, as well as 35 sequenced natural isolates currently being sequenced. The latter allow us, for the first time, to make direct comparisons of the relationship between DNA sequence data and microsatellite-based genotypes. The genotyping system provides a rapid and valuable system for strain identification as well as studying population genetics of S. cerevisiae.