1-04: Elimination of glycerol production in anaerobic cultures of Saccharomyces cerevisiae engineered for use of acetic acid as electron acceptor

Minimizing by-product formation is crucial to optimize the use of biomass as a substrate for industrial biotechnology. Glycerol is a major byproduct during anaerobic production of ethanol by S. cerevisiae, the single largest fermentation process in industrial biotechnology. In anaerobic cultures of wild-type S. cerevisiae, glycerol production is essential to reoxidize NADH produced in biosynthetic processes. The present study investigates the possibility to eliminate glycerol production by genetically engineering S. cerevisiae, such that it can reoxidize NADH by the reduction of acetic acid to ethanol via NADH-dependent reactions. Acetic acid is available at significant amounts in lignocellulosic hydrolysates of agricultural residues. Deletion of the two genes encoding NAD-dependent glycerol 3-phosphate dehydrogenase (GPD1 and GPD2) led to elimination of glycerol production and an inability to grow anaerobically. However, when the E. coli mhpF gene, encoding the acetylating NAD-dependent acetaldehyde dehydrogenase (EC 1.2.1.10), was expressed in the gpd1D gpd2D strain, anaerobic growth was restored by supplementation with 2.0 g l^-1 acetic acid. The stoichiometry of acetate consumption and growth was consistent with the complete replacement of glycerol formation by acetate reduction to ethanol as the mechanism for NADH reoxidation. This study provides a proof of principle for the potential of this metabolic engineering strategy to improve ethanol yields, eliminate glycerol production and partially convert acetate, which is a well known inhibitor of yeast performance in lignocellulosic hydrolysates, to ethanol. Further research should address kinetic aspects of acetate reduction and the effect of the elimination of glycerol production on cellular robustness (e.g. osmotolerance).