Cofactor engineering in Saccharomyces cerevisiae: Expression of a H2O-forming NADH oxidase and impact on redox metabolism.
The pyridine nucleotides NAD(H) and NADP(H) play major roles in the formation of by-products. To analyse how Saccharomyces cerevisiae (S. cerevisiae) metabolism during growth on glucose might be altered when intracellular NADH pool is decreased, we expressed noxE encoding a water-forming NADH oxidase from Lactococcus lactis (L. lactis) in the S. cerevisiae strain V5. During batch fermentation under controlled microaeration conditions, expression of the NADH oxidase under the control of a yeast promoter lead to large decreases in the intracellular NADH concentration (five-fold) and NADH/NAD+ ratio (six-fold). This increased NADH consumption caused a large redistribution of metabolic fluxes. The ethanol, glycerol, succinate and hydroxyglutarate yields were significantly reduced as a result of the lower NADH availability, whereas the formation of more oxidized metabolites, acetaldehyde, acetate and acetoin was favoured. The biomass yield was low and consumption of glucose, at concentration above 10%, was impaired. The metabolic redistribution in cells expressing the NADH oxidase was a consequence of the maintenance of a redox balance and of the management of acetaldehyde formation, which accumulated at toxic levels early in the process.
UMR Sciences pour l'Oenologie, Microbiologie, INRA, 2 place Viala, F-34060 Montpellier Cedex 1, France.
Stéphanie
S
Heux
Rémy
R
Cachon
Sylvie
S
Dequin
D049109:Cell Proliferation; D003067:Coenzymes / Q000235:genetics / Q000378:metabolism*; D004734:Energy Metabolism / Q000502:physiology; D005947:Glucose / Q000378:metabolism*; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism*; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014867:Water / Q000378:metabolism*
D003067:Coenzymes; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014867:Water; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D005947:Glucose; D010100:Oxygen