Engineering NADH oxidation for decreasing ethanol yield from glucose in Saccharomyces cerevisiae.
Stephanie Heux, Jean Marie Sablayrolles, Rémy Cachon, Sylvie Dequin
UMR SPO-Microbiologie, INRA, 2 place Pierre Viala, Montpellier, 34060, France
The concentration ratio NADH/NAD + has a significant influence on metabolite formation. Our objective is to analyse how metabolism during growth on glucose might be altered when the intracellular NADH pool is decreased, and to evaluate a strategy based on cofactor engineering to reduce the ethanol content in wine. We expressed in S. cerevisiae the water-forming NADH oxidase from Lactococcus lactis encoded by noxE, which catalyses the oxidation of NADH by reducing molecular O 2 to H 2 O. The noxE gene was cloned under the control of a yeast promoter and integrated in the genome of the wine-derived strain V5. During batch fermentations under controlled microaeration conditions, expression of the NADH oxidase (0,4 U/mg protein) caused a redistribution of metabolic flux as a result of a lower NADH availability. The yields in ethanol, glycerol and hydroxyglutarate were significantly decreased, while acetaldehyde accumulated early in the process. The formation of acetate and acetoin was also markedly increased. The NADH-dependent butanediol dehydrogenase reaction was severely limited, which strongly decreased the efficiency of acetaldehyde elimination. To overcome the undesirable effects on growth and fermentation performance caused by toxic levels of acetaldehyde, O 2 was transferred exclusively during the wine fermentation stationary phase. This strategy allowed to complete the fermentation without affecting growth, while ethanol content was significantly decreased.