Applied Microbiology and Biotechnology

, Volume 93, Issue 1, pp 131–141 | Cite as

Effect of alternative NAD+-regenerating pathways on the formation of primary and secondary aroma compounds in a Saccharomyces cerevisiae glycerol-defective mutant

  • Vishist K. Jain
  • Benoit Divol
  • Bernard A. Prior
  • Florian F. Bauer
Biotechnological Products and Process Engineering


Saccharomyces cerevisiae maintains a redox balance under fermentative growth conditions by re-oxidizing NADH formed during glycolysis through ethanol formation. Excess NADH stimulates the synthesis of mainly glycerol, but also of other compounds. Here, we investigated the production of primary and secondary metabolites in S. cerevisiae strains where the glycerol production pathway was inactivated through deletion of the two glycerol-3-phosphate dehydrogenases genes (GPD1/GPD2) and replaced with alternative NAD+-generating pathways. While these modifications decreased fermentative ability compared to the wild-type strain, all improved growth and/or fermentative ability of the gpd1Δgpd2Δ strain in self-generated anaerobic high sugar medium. The partial NAD+ regeneration ability of the mutants resulted in significant amounts of alternative products, but at lower yields than glycerol. Compared to the wild-type strain, pyruvate production increased in most genetically manipulated strains, whereas acetate and succinate production decreased in all strains. Malate production was similar in all strains. Isobutanol production increased substantially in all genetically manipulated strains compared to the wild-type strain, whereas only mutant strains expressing the sorbitol producing SOR1 and srlD genes showed increases in isoamyl alcohol and 2-phenyl alcohol. A marked reduction in ethyl acetate concentration was observed in the genetically manipulated strains, while isobutyric acid increased. The synthesis of some primary and secondary metabolites appears more readily influenced by the NAD+/NADH availability. The data provide an initial assessment of the impact of redox balance on the production of primary and secondary metabolites which play an essential role in the flavour and aroma character of beverages.


Redox Saccharomyces cerevisiae Higher alcohol Organic acids Fermentation 



This work was supported by the National Research Foundation, South Africa. We would like to thank Dr. D. F. Malherbe and Dr. V. T. Gururajan for kindly providing the pDMPM and pSTAH vectors, respectively, for cloning purposes. The authors would also like to thank Dr. A. Tredoux for technical assistance and Prof. Martin Kidd for statistical analysis.


  1. Albertyn J, Hohmann S, Thevelein JM, Prior BA (1994) GPD1, which encodes glycerol-3-phosphate dehydrogenase, is essential for growth under osmotic stress in Saccharomyces cerevisiae, and its expression is regulated by the high-osmolarity glycerol response pathway. Mol Cell Biol 14:4135–4144Google Scholar
  2. Ansell R, Granath K, Hohmann S, Thevelein JM, Adler L (1997) The two isoenzymes for yeast NAD+-dependent glycerol 3-phosphate dehydrogenase encoded by GPD1 and GPD2 have distinct roles in osmoadaptation and redox regulation. EMBO J 16:2179–2187CrossRefGoogle Scholar
  3. Bach B, Meudec E, Lepoutre JP, Rossignol T, Blondin B, Dequin S, Camarasa C (2009) New insights into γ-aminobutyric acid catabolism: evidence for γ-hydroxybutyric acid and polyhydroxybutyrate synthesis in Saccharomyces cerevisiae. Appl Environ Microbiol 75:4231–4239CrossRefGoogle Scholar
  4. Berben G, Dumont J, Gilliquet V, Bolle PA, Hilger F (1991) The YDp plasmids: a uniform set of vectors bearing versatile gene disruption cassettes for Saccharomyces cerevisiae. Yeast 7:475–477CrossRefGoogle Scholar
  5. Boulton RB, Singleton VL, Bisson LF, Kunkee RE (1996) Principle and practices of winemaking. Chapman and Hall, New YorkGoogle Scholar
  6. Bro C, Regenberg B, Förster J, Nielsen J (2006) In silico aided metabolic engineering of Saccharomyces cerevisiae for improved bioethanol production. Metab Eng 8:102–111CrossRefGoogle Scholar
  7. Camarasa C, Grivet JP, Dequin S (2003) Investigation by 13C-NMR and tricarboxylic acid (TCA) deletion mutant analysis of pathways for succinate formation in Saccharomyces cerevisiae during anaerobic fermentation. Microbiol 149:2669–2678CrossRefGoogle Scholar
  8. Eriksson P, Andre L, Ansell R, Blomberg A, Adler L (1995) Cloning and characterization of GPD2, a second gene encoding glycerol 3-phosphate dehydrogenase (NAD+) in Saccharomyces cerevisiae and its comparison with GPD1. Mol Microbiol 17:95–107CrossRefGoogle Scholar
  9. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34CrossRefGoogle Scholar
  10. Gordon JH, Dubos R (1970) The anaerobic bacterial flora of the mouse cecum. J Exp Med 132:251–260CrossRefGoogle Scholar
  11. Gururajan VT, Gorwa-Grauslund MF, Hahn-Hägerdal B, Pretorius IS, Cordero Otero RR (2007) A constitutive catabolite repression mutant of a recombinant Saccharomyces cerevisiae strain improves xylose consumption during fermentation. Ann Microbiol 57:85–92CrossRefGoogle Scholar
  12. Harju S, Fedosyuk H, Peterson KR (2004) Rapid isolation of yeast genomic DNA: Bust n' Grab. BMC Biotech 4:8CrossRefGoogle Scholar
  13. Hazelwood LA, Daran JM, van Maris AJA, Pronk JT, Dickinson JR (2008) The Ehrlich pathway for fusel alcohol production: a century of research on Saccharomyces cerevisiae metabolism. Appl Environ Microbiol 74:2259–2266CrossRefGoogle Scholar
  14. Jain VK, Divol B, Prior BA, Bauer FF (2011) Elimination of glycerol and replacement with alternative products in ethanol fermentation by Saccharomyces cerevisiae. J Ind Microbiol Biotechnol. doi: 10.1007/s10295-010-0928-x
  15. Kalapos MP (1999) Methylglyoxal in living organisms: chemistry, biochemistry, toxicology and biological implications. Toxicol Lett 110:145–175CrossRefGoogle Scholar
  16. Kuhn A, van Zyl C, van Tonder A, Prior BA (1995) Purification and partial characterization of an aldo-keto reductase from Saccharomyces cerevisiae. Appl Environ Microbiol 61:1580–1585Google Scholar
  17. Lambrechts MG, Pretorius IS (2000) Yeast and its importance to wine aroma: a review. S Afr J Enol Vitic 21:97–129Google Scholar
  18. Larsson C, Pahlman IL, Ansell R, Rigoulet M, Adler L, Gustafsson L (1998) The importance of the glycerol 3-phosphate shuttle during aerobic growth of Saccharomyces cerevisiae. Yeast 14:347–357CrossRefGoogle Scholar
  19. Louw L, Roux K, Tredoux A, Tomic O, Naes T, Nieuwoudt HH, van Rensburg P (2009) Characterization of selected South African young cultivar wines using FTMIR spectroscopy, gas chromatography, and multivariate data analysis. J Agric Food Chem 57:2623–2632CrossRefGoogle Scholar
  20. Malherbe DF (2010) Characterization and evaluation of glucose oxidase activity in recombinant Saccharomyces cerevisiae strains. PhD Dissertation, Stellenbosch UniversityGoogle Scholar
  21. Mulligan KJ (1996) A procedure to determine diethylene glycol (2,2'-oxybisethanol) and ethylene glycol (1,2-ethanediol) in glycerin and selected products. Lab Inform Bull 12:1–7Google Scholar
  22. Murata K, Saikusa T, Fukuda Y, Watanabe K, Inoue Y, Shimosaka M, Kimura A (1986) Metabolism of 2-oxoaldehydes in yeasts. Possible role of glycolytic bypath as a detoxification system in L-threonine catabolism by Saccharomyces cerevisiae. Eur J Biochem 157:297–301CrossRefGoogle Scholar
  23. Ng LK (2002) Analysis by gas chromatography/mass spectrometry of fatty acids and esters in alcoholic beverages and tobaccos. Anal Chim Acta 465:309–318CrossRefGoogle Scholar
  24. Novotny MJ, Reizer J, Esch F, Saier MH (1984) Purification and properties of D-mannitol-1-phosphate dehydrogenase and D-glucitol-6-phosphate dehydrogenase from Escherichia coli. J Bacteriol 159:986–990Google Scholar
  25. Pronk JT, Yde Steensma H, Van Dijken JP (1996) Pyruvate metabolism in Saccharomyces cerevisiae. Yeast 12:1607–1633CrossRefGoogle Scholar
  26. Rossouw D, Naes T, Bauer FF (2008) Linking gene regulation and the exo-metabolome: a comparative transcriptomics approach to identify genes that impact on the production of volatile aroma compounds in yeast. BMC Genomics 9:530CrossRefGoogle Scholar
  27. Saint-Prix F, Bonquist L, Dequin S (2004) Functional analysis of the ALD gene family of Saccharomyces cerevisiae during anaerobic growth on glucose: the NADP+-dependent Ald6p and Ald5p isoforms play a major role in acetate formation. Microbiol 150:2209–2220CrossRefGoogle Scholar
  28. Schoondermark-Stolk SA, Tabernero M, Chapman J, Ter Schure EG, Verrips CT, Verkleij AJ, Boonstra J (2005) Bat2p is essential in Saccharomyces cerevisiae for fusel alcohol production on the non-fermentable carbon source ethanol. FEMS Yeast Res 5:757–766CrossRefGoogle Scholar
  29. van Dijken JP, Scheffers WA (1986) Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol Rev 32:199–224Google Scholar
  30. Vanderhaegen B, Neven H, Coghe S, Verstrepen KJ, Verachtert H, Derdelinckx G (2003) Evolution of chemical and sensory properties during aging of top-fermented beer. J Agric Food Chem 51:6782–6790CrossRefGoogle Scholar
  31. Xu Y, Zhao GA, Wang LP (2006) Controlled formation of volatile components in cider making using a combination of Saccharomyces cerevisiae and Hanseniaspora valbyensis yeast species. J Ind Microbiol Biotechnol 33:192–196CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Vishist K. Jain
    • 1
  • Benoit Divol
    • 1
  • Bernard A. Prior
    • 1
  • Florian F. Bauer
    • 1
  1. 1.Institute for Wine BiotechnologyStellenbosch UniversityStellenboschSouth Africa

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