Engineered S. cerevisiae strains overexpressing GPD1 coding for glycerol-3-phosphate dehydrogenase (GPDH) and lacking the acetaldehyde dehydrogenase Ald6 display a large diversion of the carbon flux from ethanol towards glycerol, without accumulating acetate. Although GPD1 ald6 strains have a great potential for reducing the ethanol content in wines, one major side effect is the accumulation of acetoin, having a negative sensory impact on wine. Acetoin is reduced to 2,3-butanediol by the NADH-dependent 2,3-butanediol dehydrogenase Bdh1. In order to investigate the influence of potential factors limiting this reaction, we overexpressed BDH1 and BDH1221,222,223, coding for the native NADH-dependent Bdh1 and an engineered NADPH-dependent Bdh1 enzyme, respectively, in a glycerol overproducing wine yeast. We show that both the amount of Bdh1 and the NADH availability limit the BDH reaction. During wine fermentation, however, the major limiting factor was the synthesis level of Bdh1. Consistent with this, overproduction of native or engineered Bdh1 made it possible to redirect 85-90% of the accumulated acetoin into 2,3-butanediol, a sensory neutral compound. In addition, the production of diacetyl, a compound causing off-flavour in alcoholic beverages, whose production was increased in glycerol overproducing yeast cells, was decreased by half. The production of higher alcohols and esters, which was slightly decreased or unchanged in GPD1 ald6 cells compared to the control, was not further modified in BDH1 cells. Overall, rerouting carbons towards glycerol and 2,3-butanediol represents a new milestone in the engineering of a low alcohol yeast with desirable organoleptic features, permitting to decrease the ethanol content in wines by up to 3 degrees.
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