Engineering of the Pyruvate Dehydrogenase Bypass inSaccharomyces cerevisiae: Role of the Cytosolic Mg²⁺and Mitochondrial K⁺Acetaldehyde Dehydrogenases Ald6p and Ald4p in Acetate Formation during Alcoholic Fermentation

Abstract

Acetic acid plays a crucial role in the organoleptic balance of many fermented products. We have investigated the factors controlling the production of acetate by Saccharomyces cerevisiae during alcoholic fermentation by metabolic engineering of the enzymatic steps involved in its formation and its utilization. The impact of reduced pyruvate decarboxylase (PDC), limited acetaldehyde dehydrogenase (ACDH), or increased acetoacetyl coenzyme A synthetase (ACS) levels in a strain derived from a wine yeast strain was studied during alcoholic fermentation. In the strain with the PDC1 gene deleted exhibiting 25% of the PDC activity of the wild type, no significant differences were observed in the acetate yield or in the amounts of secondary metabolites formed. A strain overexpressing ACS2 and displaying a four-to sevenfold increase in ACS activity did not produce reduced acetate levels. In contrast, strains with one or two disrupted copies of ALD6, encoding the cytosolic Mg 2؉ -activated NADP-dependent ACDH and exhibiting 60 and 30% of wild-type ACDH activity, showed a substantial decrease in acetate yield (the acetate production was 75 and 40% of wild-type production, respectively). This decrease was associated with a rerouting of carbon flux towards the formation of glycerol, succinate, and butanediol. The deletion of ALD4, encoding the mitochondrial K ؉ -activated NAD(P)-linked ACDH, had no effect on the amount of acetate formed. In contrast, a strain lacking both Ald6p and Ald4p exhibited a long delay in growth and acetate production, suggesting that Ald4p can partially replace the Ald6p isoform. Moreover, the ald6 ald4 double mutant was still able to ferment large amounts of sugar and to produce acetate, suggesting the contribution of another member(s) of the ALD family.

Decreasing acetic acid accumulation by a glycerol overproducing strain of Saccharomyces cerevisiae by deleting the ALD6 aldehyde dehydrogenase gene

“…This finding contrasts with studies on ALD6, which have shown that the growth rate of ald6D strains was significantly less than that of the parental strain on glucose (Meaden et al, 1997;Navarro-Aviñ o et al, 1999). The phenotype might be strain-dependent, however, as other reports concur with our findings (Wang et al, 1998;Remize et al, 2000).…”

“…As the ALD4 and ALD6 genes have been shown to have a role in the oxidation of acetaldehyde to acetic acid (Meaden et al, 1997;Wang et al, 1998;Remize et al, 2000), these were chosen as primary targets for modification. Deletion of the ALD4 gene had no obvious effect on the biosynthesis of acetic acid in GPD2-OP cells when grown anaerobically (unpublished results).…”

“…The ALD6 gene product, which is activated by Mg 2+ and preferentially uses NADP + , is functional in both ethanoland glucose-grown cells (Meaden et al, 1997;Wang et al, 1998). This gene, together with ALD4, appears to contribute to the oxidation of acetaldehyde during fermentation (Remize et al, 2000).…”

Response to acetaldehyde stress in the yeast Saccharomyces cerevisiae involves a strain‐dependent regulation of several ALD genes and is mediatedby the general stress response pathway

“…On the other hand, in cells growing in ethanol, further addition of ethanol or acetaldehyde induces an increase in the mRNA steady-state levels only for ALD6, while the mitochondrial counterpart (ALD4 ) does not show a significant modification in its expression. This is consistent with the ability of cytosolic ALDH to partially compensate mitochondrial ALDH absence when its activity is sufficient (Boubekeur et al, 1999(Boubekeur et al, , 2001Remize et al, 2000).…”

“…Traditionally, cytosolic enzymes have been considered responsible for the formation of acetate from glucose while the main role of mitochondrial enzymes would take place during growth on ethanol (Wang et al, 1998;Saigal et al, 1991). However, growth behaviour analysis of mutants in genes encoding aldehyde dehydrogenases have indicated that mitochondrial and cytosolic enzymes may -at least partially and under several conditions -compensate each other (Boubekeur et al, 1999(Boubekeur et al, , 2001Remize et al, 2000).…”