Biotechnology Letters

, Volume 34, Issue 9, pp 1711–1717

Construction of self-cloning, indigenous wine strains of Saccharomyces cerevisiae with enhanced glycerol and glutathione production

Original Research Paper


To improve wine taste and flavor stability, a novel indigenous strain of Saccharomyces cerevisiae with enhanced glycerol and glutathione (GSH) production for winemaking was constructed. ALD6 encoding an aldehyde dehydrogenases of the indigenous yeast was replaced by a GPD1 and CUP1 gene cassette, which are responsible for NAD-dependent glycerol-3-phosphatase dehydrogenase and copper resistance, respectively. Furthermore, the α-acetohydroxyacid synthase gene ILV2 of the indigenous yeast was disrupted by integration of the GSH1 gene which encodes γ-glutamylcysteine synthetase and the CUP1 gene cassette. The fermentation capacity of the recombinant was similar to that of the wild-type strain, with an increase of 21 and 19 % in glycerol and GSH production. No heterologous DNA was harbored in the recombinant in this study.


Glutathione Glycerol Indigenous yeast Saccharomyces cerevisiae Self-cloning Wine 

Supplementary material

10529_2012_954_MOESM1_ESM.docx (20 kb)
Supplementary material 1 (DOCX 19 kb)


  1. Arroyo-López FN, Pérez-Torrado R, Querol A, Barrio E (2010) Modulation of the glycerol and ethanol syntheses in the yeast Saccharomyces kudriavzevii differs from that exhibited by Saccharomyces cerevisiae and their hybrid. Food Microbiol 27:628–637PubMedCrossRefGoogle Scholar
  2. Blomberg A, Adler L (1998) Roles of glycerol and glycerol-3-phosphate dehydrogenase in acquire osmotolerance of Saccharomyces cerevisiae. J Bacteriol 171:1087–1092Google Scholar
  3. Cambon B, Monteil V, Remize F, Camarasa C, Dequin S (2006) Effects of GPD1 overexpression in Saccharomyces cerevisiae commercial wine yeast strains lacking ALD6 Genes. Appl Environ Microbiol 72:4688–4694PubMedCrossRefGoogle Scholar
  4. Chen YF, Yang X, Zhang SJ, Wang XQ, Guo CH, Guo XW, Xiao DG (2012) Development of Saccharomyces cerevisiae producing higher levels of sulfur dioxide and glutathione to improve beer flavor stability. Appl Biochem Biotechnol 166:402–413PubMedCrossRefGoogle Scholar
  5. Cronwright GR, Rohwer JM, Prior BA (2002) Metabolic control analysis of glycerol synthesis in Saccharomyces cerevisiae. Appl Environ Microbiol 68:4448–4456PubMedCrossRefGoogle Scholar
  6. Eglinton JM, Heinrich AJ, Pollnitz AP, Langridge P, Henschke PA, Lopes MB (2002) Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene. Yeast 19:295–301PubMedCrossRefGoogle Scholar
  7. Fan XY, He XP, Guo XN, Qu N, Wang CL, Zhang BR (2004) Increasing glutathione formation by functional expression of the γ-glutamylcysteine synthetase gene in Saccharomyces cerevisiae. Biotechnol Lett 26:415–417PubMedCrossRefGoogle Scholar
  8. Gao HY, Liao XJ, Hu XS (2004) Simultaneous determination of eleven organic acids in fruit juice by reversed phase high performance liquid chromatography. Chin J Anal Chem 32:1645–1648Google Scholar
  9. Gawel R, Sluyter SV, Waters EJ (2007) The effects of ethanol and glycerol on the body and other sensory characteristics of Riesling wines. Aust J Grape Wine Res 13:38–45CrossRefGoogle Scholar
  10. Hill JE, Meyers AM, Koerner TJ (1993) Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast 2:163–167CrossRefGoogle Scholar
  11. Hosry LE, Auezova L, Sakr A, Hajj-Moussa E (2009) Browning susceptibility of white wine and antioxidant effect of glutathione. Int J Food Sci Technol 44:2459–2463CrossRefGoogle Scholar
  12. Kutyna DR, Varela C, Stanley GA, Borneman AR, Henschke PA, Chambers PJ (2012) Adaptive evolution of Saccharomyces cerevisiae to generate strains with enhanced glycerol production. Appl Microbiol Biotechnol 93(3):1175–1184PubMedCrossRefGoogle Scholar
  13. Lambropoulos I, Roussis IG (2007) Protection of some aroma volatiles in a model wine medium by sulphur dioxide and mixtures of glutathione with caffeic acid or gallic acid. Food Res Int 40:176–181CrossRefGoogle Scholar
  14. Lopes MB, Rehman AU, Gockowiak H, Heinrich AJ, Langridge P, Henschke PA (2000) Fermentation properties of a wine yeast overexpressing the Saccharomyces cerevisiae glycerol-3-phosphate dehydrogenase gene (GPD2). Aust J Grape Wine Res 6:208–215CrossRefGoogle Scholar
  15. Michnick S, Roustan JL, Remize F, Barre P, Dequin S (1997) Modulation of glycerol and ethanol yields during alcoholic fermentation in Saccharomyces cerevisiae strains overexpressed or disrupted for GPD1 encoding glycerol-3-phosphate dehydrogenase. Yeast 13:783–793PubMedCrossRefGoogle Scholar
  16. Noble AC, Bursick GF (1984) The contribution of glycerol to perceived viscosity and sweetness in white wine. Am J Enol Vitic 35:110–112Google Scholar
  17. Okieb N, Amada K, Hirano S, Haruki M, Imanaka T, Morikawa M, Kanaya S (1999) Gene cloning and characterization of aldehyde dehydrogenase from a petroleum-degrading bacterium strain HD-1. J Biosci Bioeng 88:7–11CrossRefGoogle Scholar
  18. Papadopoulou D, Roussis IG (2008) Inhibition of the decrease of volatile esters and terpenes during storage of a white wine and a model wine medium by glutathione and N-acetylcysteine. Int J Food Sci Technol 43:1053–1057CrossRefGoogle Scholar
  19. Remize F, Roustan JL, Sablayrolles JM, Barre P, Dequin S (1998) Glycerol overproduction by engineered Saccharomyces cerevisiae wine yeast strains leads to substantial changes in by-product formation and to a stimulation of fermentation rate in stationary phase. Appl Environ Microbiol 65:143–149Google Scholar
  20. Short JM, Fernandez JM, Sorge JA, Huse WD (1988) λ ZAP: a bacteriophage λ expression vector with in vivo excision properties. Nucl Acids Res 16:7583–7600PubMedCrossRefGoogle Scholar
  21. Soden A, Francis IL, Oakey H, Henschke PA (2000) Effects of co-fermentation with Candida stellata and Saccharomyces cerevisiae on the aroma and composition of Chardonnay wine. Aust J Grape Wine Res 6:21–30CrossRefGoogle Scholar
  22. Spiropoulos A, Tanaka J, Flerianos I, Bisson LF (2000) Characterization of hydrogen sulfide formation in commercial and natural wine isolates of Saccharomyces. Am J Enol Vitic 51:233–248Google Scholar
  23. Toro ME, Vazquez F (2002) Fermentation behaviour of controlled mixed and sequential cultures of Candida cantarellii and Saccharomyces cerevisiae wine yeasts. World J Microb Biotechnol 18:347–354Google Scholar
  24. Ugliano M, Fedrizzi B, Siebert T, Travis B, Magno F, Versini G, Henschke PA (2009) Effect of nitrogen supplementation and Saccharomyces species on hydrogen sulfide and other volatile sulfur compounds in Shiraz fermentation and wine. J Agric Food Chem 57:4948–94955PubMedCrossRefGoogle Scholar
  25. Wang ZY, He XP, Zhang BR (2008) Construction of self-cloning industrial brewing yeast with high-glutathione and low-diacetyl production. Int J Food Sci Technol 43:989–994CrossRefGoogle Scholar
  26. Zhang JN, He XP, Guo XN (2005) Genetically modified industrial brewing yeast with high-glutathione and low-diacetyl production. Chin J Biotechnol 21:942–946Google Scholar
  27. Zhang Y, Wang ZY, He XP, Liu N, Zhang BR (2008) New industrial brewing yeast strains with ILV2 disruption and LSD1 expression. Int J Food Microbiol 123:18–24PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  1. 1.College of EnologyNorthwest A&F UniversityYanglingPeople’s Republic of China
  2. 2.The Laboratory of Molecular Genetics and Breeding of Yeast, Institute of MicrobiologyChinese Academy of SciencesBeijingPeople’s Republic of China

Personalised recommendations