Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae: Role of the cytosolic Mg2+ and mitochondrial K+ acetaldehyde dehydrogenases Ald6p and Ald4p in acetate formation during alcoholic fermentation

Acetic acid plays a crucial role in the organoleptic balance of many fermen ted products. We have investigated the factors controlling the production o f acetate by Saccharomyces cerevisiae during alcoholic fermentation by meta bolic engineering of the enzymatic steps involved in its formation and its utilization. The impact of reduced pyruvate decarboxylase (PDC), limited ac etaldehyde dehydrogenase (ACDH), or increased acetoacetyl coenzyme A synthe tase (ACS) levels in a strain derived from a wine yeast strain was studied during alcoholic fermentation. In the strain with the PDC1 gene deleted exh ibiting 25% of the PDC activity of the wild type, no significant difference s were observed in the acetate yield or in the amounts of secondary metabol ites formed. A strain overexpressing ACS2 and displaying a four- to sevenfo ld increase in ACS activity did not produce reduced acetate levels, In cont rast, strains with one or two disrupted copies of ALD6, encoding the cytoso lic Mg2+-activated NADP-dependent ACDH and exhibiting 60 and 30% of wild-ty pe ACDH activity, showed a substantial decrease in acetate yield (the aceta te production was 75 and 40% of wild-type production, respectively), This d ecrease was associated with a rerouting of carbon flux towards the formatio n of glycerol, succinate, and butanediol, The deletion of ALD4, encoding th e mitochondrial K+-activated NAD(P)-linked ACDH, had no effect on the amoun t of acetate formed. In contrast, a strain lacking both Ald6p and Ald4p exh ibited a long delay in growth and acetate production, suggesting that Ald4p can partially replace the Ald6p isoform, Moreover, the ald6 ald4 double mu tant was still able to ferment large amounts of sugar and to produce acetat e, suggesting the contribution of another member(s) of the ALD family.