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.

2-Oxobutyrate is an important intermediate in the chemical, drug, and food industries. Whole cells ofPseudomonas stutzeri SDM, containing NAD-independent lactate dehydrogenases, effectively converted 2-hydroxybutyrate into 2-oxobutyrate. Under optimal conditions, the biocatalytic process produced 2-oxobutyrate at a high concentration (44.4 g liter ؊1 ) and a high yield (91.5%).

Sulfane sulfur, such as inorganic and organic polysulfide (HSn− and RSn−, n > 2), is a common cellular component, produced either from hydrogen sulfide oxidation or cysteine metabolism. In Pseudomonas aeruginosa PAO1, LasR is a quorum sensing master regulator. After binding its autoinducer, LasR binds to its target DNA to activate the transcription of a suite of genes, including virulence factors. Herein, we report that the production of hydrogen sulfide and sulfane sulfur were positively correlated in P. aeruginosa PAO1, and sulfane sulfur was able to modify LasR, which generated Cys188 persulfide and trisulfide and produced a pentasulfur link between Cys201 and Cys203. The modifications did not affect LasR binding to its target DNA site, but made it several-fold more effective than unmodified LasR in activating transcription in both in vitro and in vivo assays. On the contrary, H2O2 inactivates LasR via producing a disulfide bond between Cys201 and Cys203. P. aeruginosa PAO1 had a high cellular sulfane sulfur and high LasR activity in the mid log phase and early stationary phase, but a low sulfane sulfur and low LasR activity in the declination phase. Thus, sulfane sulfur is a new signaling factor in the bacterium, adding another level of control over LasR-mediated quorum sensing and turning down the activity in old cells.