Effect of alternative NAD+-regenerating pathways on the formation of primary and secondary aroma compounds in a Saccharomyces cerevisiae glycerol-defective mutant
- 464 Downloads
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.
KeywordsRedox 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.
- 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
- Boulton RB, Singleton VL, Bisson LF, Kunkee RE (1996) Principle and practices of winemaking. Chapman and Hall, New YorkGoogle Scholar
- 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
- 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
- Lambrechts MG, Pretorius IS (2000) Yeast and its importance to wine aroma: a review. S Afr J Enol Vitic 21:97–129Google Scholar
- Malherbe DF (2010) Characterization and evaluation of glucose oxidase activity in recombinant Saccharomyces cerevisiae strains. PhD Dissertation, Stellenbosch UniversityGoogle Scholar
- 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
- 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
- van Dijken JP, Scheffers WA (1986) Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol Rev 32:199–224Google Scholar