Abstract
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.
Similar content being viewed by others
References
Allen T, Herbst-Johnstone M, Girault M, Butler P, Logan G, Jouanneau S, Nicolau L, Kilmartin PA (2011) Influence of grape harvesting steps on varietal thiol aromas in Sauvignon Blanc wines. J Agric Food Chem 59:10641–10650
Anfang N, Brajkovich M, Goddard MR (2008) Co-fermentation with Pichia kluyveri increases varietal thiol concentrations in Sauvignon Blanc. Aust J Grape Wine Res 15:1–8
Benkwitz F, Tominaga T, Kilmartin PA, Lund C, Wohlers M, Nicolau L (2012) Identifying the chemical composition related to the distinct flavor characteristics of New Zealand Sauvignon Blanc wines. Am J Enol Vitic 63:62–72
Borneman AR, Desany BA, Riches D, Affourtit JP, Forgan AH, Pretorius IS, Egholm M, Chambers PJ (2011) Whole-genome comparison reveals novel genetic elements that characterize the genome of industrial strains of Saccharomyces cerevisiae. PLoS Genet 7:1–10
Bradbury JE, Richards KD, Niederer HA, Lee SA, Dunbar PR, Gardner RC (2006) A homozygous diploid subset of commercial wine yeast strains. Anton Leeuw 89:27–37
Capone DL, Jeffery DW (2011) Effects of transporting and processing Sauvignon Blanc grapes on 3-mercaptohexan-1-ol precursor concentrations. J Agric Food Chem 59:4659–4667
Cardenas ME, Cutler NS, Lorenz MC, Di Como CJ, Heitman J (1999) The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev 13:3271–3279
Chen EJ, Kaiser CA (2002) Amino acids regulate the intracellular trafficking of the general amino acid permease of Saccharomyces cerevisiae. PNAS 99:14837–14842
Coetzee C, du Toit WJ (2012) A comprehensive review on Sauvignon Blanc aroma with a focus on certain positive volatile thiols. Food Res Intern 45:287–298
Cohen J (1988) Statistical power analysis for the behavioral sciences, 2nd edn. Lawrence Erlbaum Associates, Hillsdale
Cooper TG (2002) Transmitting the signal of excess nitrogen in Saccharomyces cerevisiae from the Tor proteins to the GATA factors: connecting the dots. FEMS Microbiol Rev 26:223–238
Courchesne WE, Magasanik B (1988) Regulation of nitrogen assimilation in Saccharomyces cerevisiae: roles of the URE2 and GLN3 genes. J Bacteriol 170:708–713
Crespo JL, Helliwell SB, Wiederkehr C, Demougin P, Fowler B, Primig M, Hall MN (2004) NPR1 kinase and RSP5-BUL1/2 ubiquitin ligase control GLN3-dependent transcription in Saccharomyces cerevisiae. J Biol Chem 279:37512–37517
Deed N, van Vuuren H, Gardner R (2011) Effects of nitrogen catabolite repression and di-ammonium phosphate addition during wine fermentation by a commercial strain of S. cerevisiae. Appl Microbiol Biotechnol 89:1537–1549
Edskes HK, Hanover JA, Wickner RB (1999) Mks1p is a regulator of nitrogen catabolism upstream of Ure2p in Saccharomyces cerevisiae. Genet 153:585–594
Fedrizzi B, Pardon KH, Sefton MA, Elsey GM, Jeffery DW (2009) First identification of 4-S-glutathionyl-4-methylpentan-2-one, a potential precursor of 4-mercapto-4-methylpentan-2-one, in Sauvignon Blanc juice. J Agric Food Chem 57:991–995
Feller A, Boeckstaens M, Marini AM, Dubois E (2006) Transduction of the nitrogen signal activating Gln3-mediated transcription is independent of Npr1 kinase and Rsp5-Bul1/2 ubiquitin ligase in Saccharomyces cerevisiae. J Biol Chem 281:28546–28554
Field D, Wills C (1998) Abundant microsatellite polymorphism in Saccharomyces cerevisiae, and the different distributions of microsatellites in eight prokaryotes and S. cerevisiae, result from strong mutation pressures and a variety of selective forces. PNAS 95:1647–1652
Field D, Eggert L, Metzgar D, Rose R, Wills C (1996) Use of polymorphic short and clustered coding-region microsatellites to distinguish strains of Candida albicans. FEMS Immunol Med Microbiol 15:73–79
Ganguli D, Kumar C, Bachhawat AK (2007) The alternative pathway of glutathione degradation is mediated by a novel protein complex involving three new genes in Saccharomyces cerevisiae. Genet 175:1137–1151
Gietz RD, Schiestl RH, Willems AR, Woods RA (1995) Studies on the transformation of intact yeast cells by the LiAc/SS-DNA/PEG procedure. Yeast 11:355–360
Grant-Preece PA, Pardon KH, Capone DL, Cordente AG, Sefton MA, Jeffery DW, Elsey GM (2010) Synthesis of wine thiol conjugates and labeled analogues: fermentation of the glutathione conjugate of 3-mercaptohexan-1-ol yields the corresponding cysteine conjugate and free thiol. J Agric Food Chem 58:1383–1389
Hanna M, Xiao W (2006) Isolation of nucleic acids. Methods Mol Biol 313:15–20
Hansen J, Johannesen PF (2000) Cysteine is essential for transcriptional regulation of the sulfur assimilation genes in Saccharomyces cerevisiae. Mol Gen Genet 263:535–542
Harsch MJ, Lee SA, Goddard MR, Gardner RC (2010) Optimized fermentation of grape juice by laboratory strains of Saccharomyces cerevisiae. FEMS Yeast Res 10:72–82
Hiraishi H, Miyake T, Ono B-I (2008) Transcriptional regulation of Saccharomyces cerevisiae CYS3 encoding cystathionine γ-lyase. Curr Genet 53:225–234
Holt S, Cordente AG, Williams SJ, Capone DL, Jitjaroen W, Menz IR, Curtin C, Anderson PA (2011) Engineering Saccharomyces cerevisiae to release 3-mercaptohexan-1-ol during fermentation through overexpression of an S. cerevisiae gene, STR3, for improvement of wine aroma. Appl Environ Microbiol 77:3626–3632
Howell KS, Klein M, Swiegers JH, Hayasaka Y, Elsey GM, Fleet GH, Hoj PB, Pretorius IS, de Barros Lopes MA (2005) Genetic determinants of volatile-thiol release by Saccharomyces cerevisiae during wine fermentation. Appl Environ Microbiol 71:5420–5426
Kumar C, Sharma R, Bachhawat AK (2003) Utilization of glutathione as an exogenous sulfur source is independent of γ-glutamyl transpeptidase in the yeast Saccharomyces cerevisiae: evidence for an alternative glutathione degradation pathway. FEMS Microbiol Ecol 219:187–194
Lee SA, Rick FE, Dobson J, Reeves M, Clark H, Thomson M, Gardner RC (2008) Grape juice is the major influence on volatile thiol aromas in Sauvignon Blanc. Aust NZ Grapegr Winemaker 1002:78–86
Linderholm AL, Findleton CL, Kumar G, Hong Y, Bisson LF (2008) Identification of genes affecting hydrogen sulfide formation in Saccharomyces cerevisiae. Appl Environ Microbiol 74:1418–1427
Liti G, Carter DM, Moses AM, Warringer J, Parts L, James SA, Davey RP, Roberts IN, Burt A, Koufopanou V, Tsai IJ, Bergman CM, Bensasson D, O’Kelly MJT, van Oudenaarden A, Barton DBH, Bailes E, Nguyen Ba AN, Jones M, Quail MA, Goodhead I, Sims S, Smith F, Blomberg A, Durbin R, Louis EJ (2009) Population genomics of domestic and wild yeasts. Nat 458:337–341
Lund CM, Thompson MK, Benkwitz F, Wohler MW, Triggs CM, Gardner R, Heymann H, Nicolau L (2009) New Zealand Sauvignon Blanc distinct flavor characteristics: sensory, chemical, and consumer aspects. Am J Enol Vitic 60:1–12
Magasanik B, Kaiser CA (2002) Nitrogen regulation in Saccharomyces cerevisiae. Gene 290:1–18
Maw GA (1961) Ability of S-methyl-l-cysteine to annul the inhibition of yeast growth by l-ethionine and by S-ethyl-l-cysteine. J Gen Microbiol 25:441–449
Maw GA (1963) The uptake of some sulphur-containing amino acids by a brewer's yeast. J Gen Microbiol 31:247–259
Melcher K, Entian K-D (1992) Genetic analysis of serine biosynthesis and glucose repression in yeast. Curr Genet 21:295–300
Mestres M, Busto O, Guasch J (2000) Analysis of organic sulfur compounds in wine aroma. J Chromatogr A 881:569–581
Momose Y, Iwahashi H (2001) Bioassay of cadmium using a DNA microarray: genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium. Environ Toxicol Chem 20:2353–2360
Nakagawa S, Cuthill IC (2007) Effect size, confidence interval and statistical significance: a practical guide for biologists. Biol Rev 82:591–605
Ono B-I, Hazu T, Yoshida S, Kawato T, Shinoda S, Brzvwczy J, Paszewski A (1999) Cysteine biosynthesis in Saccharomyces cerevisiae: a new outlook on pathway and regulation. Yeast 15:1365–1375
Patel P, Herbst-Johnstone M, Lee SA, Gardner RC, Weaver R, Nicolau L, Kilmartin PA (2010) Influence of juice pressing conditions on polyphenols, antioxidants, and varietal aroma of Sauvignon Blanc microferments. J Agric Food Chem 58:7280–7288
Peña-Gallego A, Hernández-Orte P, Cacho J, Ferreira V (2012) S-cysteinylated and S-glutathionylated thiol precursors in grapes. A review. Food Chem 131:1–13
Penninckx MJ (2000) A short review on the role of glutathione in the response of yeasts to nutritional, environmental and oxidative stresses. Enzyme Microb Technol 26:737–742
Peyrot des Gachons C, Tominaga T, Dubourdieu D (2000) Measuring the aromatic potential of Vitis vinifera L. Cv. Sauvignon Blanc grapes by assaying S-cysteine conjugates, precursors of the volatile thiols responsible for their varietal aroma. J Agric Food Chem 48:3387–3391
Peyrot des Gachons C, Tominaga T, Dubourdieu D (2002) Sulfur aroma precursor present in S-glutathione conjugate form: identification of S-3-(hexan-1-ol)-glutathione in must from Vitis vinifera L. cv. Sauvignon Blanc. J Agric Food Chem 50:4076–4079
Roland A, Schneider R, Guernevé CL, Razungles A, Cavelier F (2010a) Identification and quantification by LC-MS/MS of a new precursor of 3-mercaptohexan-1-ol (3MH) using stable isotope dilution assay: elements for understanding the 3MH production in wine. Food Chem 121:847–855
Roland A, Vialaret J, Razungles A, Rigou P, Schneider R (2010b) Evolution of S-cysteinylated and S-glutathionylated thiol precursors during oxidation of Melon B. and Sauvignon Blanc musts. J Agric Food Chem 58:4406–4413
Roland A, Schneider R, Razungles A, Cavelier F (2011) Varietal thiols in wine: discovery, analysis and applications. Chem Rev 111:7355–7376
Roncoroni M, Santiago M, Hooks DO, Moroney S, Harsch MJ, Lee SA, Richards KD, Nicolau L, Gardner RC (2011) The yeast IRC7 gene encodes a β-lyase responsible for production of the varietal thiol 4-mercapto-4-methylpentan-2-one in wine. Food Microbiol 28:926–935
Schacherer J, Shapiro JA, Ruderfer DM, Kruglyak L (2009) Comprehensive polymorphism survey elucidates population structure of Saccharomyces cerevisiae. Nat 458:342–345
Schneider R, Charrier F, Razungles A, Baumes R (2006) Evidence for an alternative biogenetic pathway leading to 3-mercaptohexanol and 4-mercapto-4-methylpentan-2-one in wines. Anal Chim Acta 563:58–64
Sosa E, Aranda C, Riego L, Valenzuela L, DeLuna A, Cantú JM, González A (2003) Gcn4 negatively regulates expression of genes subjected to nitrogen catabolite repression. Biochem Biophys Res Commun 310:1175–1180
Spiropoulos A, Bisson LF (2000) MET17 and hydrogen sulfide formation in Saccharomyces cerevisiae. Appl Environ Microbiol 66:4421–4426
Strudwick N, Brown M, Parmar VM, Schröder M (2010) Ime1 and Ime2 are required for pseudohyphal growth of Saccharomyces cerevisiae on nonfermentable carbon sources. Mol Cell Biol 30:5514–5530
Student (1908) The probable error of a mean. Biometrika 6:1–25
Subileau M, Schneider R, Salmon J-M, Degryse E (2008a) New insights on 3-mercaptohexanol (3MH) biogenesis in Sauvignon Blanc wines: cys-3MH and (E)-hexen-2-al are not the major precursors. J Agric Food Chem 56:9230–9235
Subileau M, Schneider R, Salmon J-M, Degryse E (2008b) Nitrogen catabolite repression modulates the production of aromatic thiols characteristic of Sauvignon Blanc at the level of precursor transport. FEMS Yeast Res 8:771–780
Swiegers JH, Pretorius I (2007) Modulation of volatile sulfur compounds by wine yeast. Appl Microbiol Biotechnol 74:954–960
Swiegers JH, Willmott R, Hill-Ling A, Capone DL, Pardon KH, Elsey GM, Howell KS, de Barros Lopes MA, Sefton MA, Lilly M, Pretorius IS (2006) Modulation of volatile thiol and ester aromas by modified wine yeast. In: Bredie WLP, Petersen MA (eds) Developments in food science, vol 43. Elsevier, Amsterdam, pp 113–116
Swiegers JH, Capone DL, Pardon KH, Elsey GM, Sefton MA, Francis IL, Pretorius IS (2007) Engineering volatile thiol release in Saccharomyces cerevisiae for improved wine aroma. Yeast 24:561–574
Taillandier P, Ramon-Portugal F, Fuster A, Strehaiano P (2007) Effect of ammonium concentration on alcoholic fermentation kinetics by wine yeasts for high sugar content. Food Microbiol 24:95–100
Tehlivets O, Hasslacher M, Kohlwein SD (2004) S-Adenosyl-l-homocysteine hydrolase in yeast: key enzyme of methylation metabolism and coordinated regulation with phospholipid synthesis. FEBS Lett 577:501–506
Thibon C, Marullo P, Claisse O, Cullin C, Dubourdieu D, Tominaga T (2008) Nitrogen catabolic repression controls the release of volatile thiols by Saccharomyces cerevisiae during wine fermentation. FEMS Yeast Res 8:1076–1086
Tominaga T, Dubourdieu D (2006) A novel method for quantification of 2-methyl-3-furanthiol and 2-furanmethanethiol in wines made from Vitis vinifera grape varieties. J Agric Food Chem 54:29–33
Tominaga T, Masneuf I, Dubourdieu D (1995) A S-cysteine conjugate, precursor of aroma of white Sauvignon. J Int Sci Vigne Vin 29:227–232
Tominaga T, Darriet P, Dubourdieu D (1996) Identification of 3-mercaptohexyl acetate in Sauvignon wine a powerful aromatic compound exhibiting box tree odour. Vitis 35:207–210
Tominaga T, Furrer A, Henry R, Dubourdieu D (1998a) Identification of new volatile thiols in the aroma of Vitis vinifera L. var. Sauvignon Blanc wines. Flavour Fragr J 13:159–162
Tominaga T, des Peyrot GC, Dubourdieu D (1998b) A new type of flavor precursors in Vitis vinifera L. cv. Sauvignon Blanc: S-cysteine conjugates. J Agric Food Chem 46:5215–5219
Wach A (1996) PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae. Yeast 12:259–265
Wach A, Brachat A, Pöhlmann R, Philippsen P (1994) New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808
Wang XD, Bohlscheid JC, Edwards CG (2003) Fermentative activity and production of volatile compounds by Saccharomyces grown in synthetic grape juice media deficient in assimilable nitrogen and/or pantothenic acid. J Appl Microbiol 94:349–359
Wiles AM, Cai H, Naider F, Becker JM (2006) Nutrient regulation of oligopeptide transport in Saccharomyces cerevisiae. Microbiol 152:3133–3145
Winter G, Henschke PA, Higgins VJ, Ugliano M, Curtin C (2011) Effects of rehydration nutrients on H2S metabolism and formation of volatile sulfur compounds by the wine yeast VL3. AMB Express 1:36
Acknowledgments
This work was funded by a grant from the Foundation of Research Science and Technology in New Zealand (contract UOAX0404) with the support of New Zealand Winegrowers. We are grateful to Andy Frost (Pernod Ricard NZ, Blenheim) for supplying grape juice and Laura Nicolau (Wine Science, University of Auckland) for access to and assistance with GC-MS analysis.
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
(PDF 370 kb)
Rights and permissions
About this article
Cite this article
Harsch, M.J., Gardner, R.C. Yeast genes involved in sulfur and nitrogen metabolism affect the production of volatile thiols from Sauvignon Blanc musts. Appl Microbiol Biotechnol 97, 223–235 (2013). https://doi.org/10.1007/s00253-012-4198-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-012-4198-6