Reversal of coenzyme specificity of 2,3‐butanediol dehydrogenase from Saccharomyces cerevisae and in vivo functional analysis

Abstract

Saccharomyces cerevisiae NAD(H)-dependent 2,3-butanediol dehydrogenase (Bdh1), a medium chain dehydrogenase/reductase is the main enzyme catalyzing the reduction of acetoin to 2,3-butanediol. In this work we focused on altering the coenzyme specificity of Bdh1 from NAD(H) to NADP(H). Based on homology studies and the crystal structure of the NADP(H)-dependent yeast alcohol dehydrogenase Adh6, three adjacent residues (Glu(221), Ile(222), and Ala(223)) were predicted to be involved in the coenzyme specificity of Bdh1 and were altered by site-directed mutagenesis. Coenzyme reversal of Bdh1 was obtained with double Glu221Ser/Ile222Arg and triple Glu221Ser/Ile222Arg/Ala223Ser mutants. The performance of the triple mutant for NADPH was close to that of native Bdh1 for NADH. The three engineered mutants were able to restore the growth of a phosphoglucose isomerase deficient strain (pgi), which cannot grow on glucose unless an alternative NADPH oxidizing system is provided, thus demonstrating their in vivo functionality. These mutants are interesting tools to reduce the excess of acetoin produced by engineered brewing or wine yeasts overproducing glycerol. In addition, they represent promising tools for the manipulation of the NADP(H) metabolism and for the development of a powerful catalyst in biotransformations requiring NADPH regeneration.

Metabolic Impact of Redox Cofactor Perturbations on the Formation of Aroma Compounds in Saccharomyces cerevisiae

“…Overall, any modification of the balance between the oxidized and reduced forms of redox cofactors is expected to have far-reaching effects on the functional metabolic network. In agreement with this finding, altered metabolite patterns were identified in previous studies that focused on the impact of redox perturbations on fermentation kinetics and the formation of products from central carbon metabolism, such as glycerol, acetate, and succinate (19)(20)(21)(22)(23)(24).…”

“…The strains used in this study were constructed from S. cerevisiae strain 59A, a haploid derivative of the wine yeast strain Lalvin EC1118 (29). The 59A-NADH-Bdh and 59A-NADPH-Bdh strains were obtained previously by overexpressing the BDH1 gene encoding the NADH-dependent 2,3-butanediol dehydrogenase (Bdh1p) or a mutated version of this gene encoding a NADPH-dependent Bdh1p (21,30). The S. cerevisiae strains were grown in yeast extract-peptone-dextrose (YPD) medium (1% Bacto yeast extract, 2% Bacto peptone, and 2% glucose).…”

“…To this end, fermentations on chemically defined fermentation medium, as found in grape must, were performed using strains overexpressing native NADH-dependent or engineered NADPHdependent butanediol dehydrogenase (Bdh). On the basis of the amount of added acetoin, this biological tool enables specific modulation of the NAD(P) ϩ /NAD(P)H balance (21,22,23). This experimental design allowed the study of the metabolic changes involved in the maintenance of redox balance in response to increased NAD(P)H demand.…”

Engineering of 2,3-Butanediol Dehydrogenase To Reduce Acetoin Formation by Glycerol-Overproducing, Low-AlcoholSaccharomyces cerevisiae

“…In addition to the decreased acetoin accumulation, the most obvious effect of BDH1 or mutated BDH1 overexpression was decreased diacetyl production. Five enzymes having in vitro diacetyl reductase activity in S. cerevisiae, including Bdh1, which reduces diacetyl as the second best substrate after acetoin, have been identified previously (9,12,24,38,43). We have shown that the overexpression or deletion of BDH1 results in a twofold decrease or increase in diacetyl levels, respectively, suggesting that Bdh1 is a rate-limiting enzyme in diacetyl reduction.…”

Reducing alcohol levels in wines through rational and evolutionary engineering of Saccharomyces cerevisiae

“…An engineered NADPH-dependent Bdh1 enzyme was obtained by substituting 3 amino acids of the cofactor binding site (Ehsani et al, 2009b). Overproduction of the native and mutated enzymes in glycerol-overproducing strains showed that the availabilities of Bdh1 and NADH limited the BDH reaction, with the major rate-controlling factor being the amount of Bdh1 enzyme (Ehsani et al, 2009a). These strategies made it possible to redirect 85-90% of the accumulated acetoin to 2,3-butanediol.…”