Abstract
BackgroundThe high costs of pyridine nucleotide cofactors have limited the applications of NAD(P)-dependent oxidoreductases on an industrial scale. Although NAD(P)H regeneration systems have been widely studied, NAD(P)+ regeneration, which is required in reactions where the oxidized form of the cofactor is used, has been less well explored, particularly in whole-cell biocatalytic processes.Methodology/Principal FindingsSimultaneous overexpression of an NAD+ dependent enzyme and an NAD+ regenerating enzyme (H2O producing NADH oxidase from Lactobacillus brevis) in a whole-cell biocatalyst was studied for application in the NAD+-dependent oxidation system. The whole-cell biocatalyst with (2R,3R)-2,3-butanediol dehydrogenase as the catalyzing enzyme was used to produce (3R)-acetoin, (3S)-acetoin and (2S,3S)-2,3-butanediol.Conclusions/SignificanceA recombinant strain, in which an NAD+ regeneration enzyme was coexpressed, displayed significantly higher biocatalytic efficiency in terms of the production of chiral acetoin and (2S,3S)-2,3-butanediol. The application of this coexpression system to the production of other chiral chemicals could be extended by using different NAD(P)-dependent dehydrogenases that require NAD(P)+ for catalysis.
Highlights
Many approaches have been developed to produce chiral chemicals for the manufacture of a wide range of intermediates in the pharmaceutical, agrochemical, fine chemical and food industries [1]
We developed a coexpression system in which acetoin reductase/2,3-butanediol dehydrogenase (BDH) encoded by the Bacillus subtilis ydjL gene was the producing enzyme and NADH oxidase (NOX) was the cofactorregenerating enzyme
The biocatalytic activities of the crude extracts prepared from E. coli BL21(DE3) and E. coli BL21(DE3) cells were assayed using meso-2,3-BD and (2R,3R)-2,3-BD as the substrates
Summary
Many approaches have been developed to produce chiral chemicals for the manufacture of a wide range of intermediates in the pharmaceutical, agrochemical, fine chemical and food industries [1]. The crude extract of induced E. coli BL21(DE3) (pETDuet-ydjL) cells showed a high (2R,3R)-2,3-BDH activity of 16.0 U mg21 (in the direction of (2R,3R)-2,3-BD production). The biocatalytic activities of the crude extracts prepared from E. coli BL21(DE3) (pETDuet-nox) and E. coli BL21(DE3) (pETDuet-ydjL) cells were assayed using meso-2,3-BD and (2R,3R)-2,3-BD as the substrates.
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