A novel whole-cell biocatalyst with NAD+ regeneration for production of chiral chemicals

Abstract

Background: The 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 findings: Simultaneous overexpression of an NAD(+) dependent enzyme and an NAD(+) regenerating enzyme (H(2)O 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/significance: A 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.

Figure 1. SDS-PAGE analysis of recombinant E. coli.

Lane M, marker proteins; lane 1, E. coli BL21(DE3); lane 2, E. coli BL21(DE3) (pETDuet); lane 3, E. coli BL21(DE3) (pETDuet-ydjL); lane 4, E. coli BL21(DE3) (pETDuet-nox); and lane 5, E. coli BL21(DE3) (pETDuet-ydjLnox).

Figure 2. A recombinant E. coli whole-cell biocatalyst with BDH and the enzymatic NAD regeneration system for the production of chiral AC and (2S,3S)-2,3-BD.

Figure 3. Effects of biocatalysis conditions on the reaction rate.

A. pH; B. substrate concentration. E. coli BL21(DE3) (pETDuet-ydjLnox) was incubated with meso-2,3-BD in the reaction.

Figure 4. Time course of the recombinant E. coli whole-cell biocatalyst-mediated (3S)-AC production from meso-2,3-BD by the recombined E. coli.

The concentration of (3S)-AC (□) and meso-2,3-BD (▪) in E. coli BL21(DE3) (pETDuet-ydjLnox) ; concentrations of (3S)-AC (○) and meso-2,3-BD (•) in E. coli BL21(DE3) (pETDuet-ydjL); and concentrations of (3S)-AC (▵) and meso-2,3-BD (▴) in E. coli BL21(DE3).

Figure 5. GC analyses of substrates and products of the catalytic reaction (* Isoamyl alcohol was used as the internal standard).

A. Conversion of meso-2,3-BD to (3S)-AC: A1-before the reaction; A2-after the reaction; B. Conversion of (2R,3R)-2,3-BD to (3R)-AC: B1-before the reaction; B2-after the reaction; C. The starting material and products of kinetic resolution from a mixture of 2,3-BD: C1-before the reaction; C2-after the reaction.