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Background   Candida parapsilosis  CCTCC M203011 catalyzes the stereoinversion of ( R )-1-phenyl-1,2-ethanediol (PED) through oxidation and reduction. Its NAD + -linked ( R )-carbonyl reductase (RCR) catalyzes the oxidization of ( R )-PED to 2-hydroxyacetophenone (HAP), and its NADPH-dependent ( S )-carbonyl reductase (SCR) catalyzes the reduction of HAP to ( S )-PED. The reactions require NAD +  and NADPH as cofactors. However, even if NAD +  and NADPH are added, the biotransformation of ( S )-PED from the ( R )-enantiomer by an  Escherichia coli  strain co-expressing RCR and SCR is slow and gives low yields, probably as a result of insufficient or imbalanced redox cofactors. To prepare ( S )-PED from the ( R )-enantiomer in one-step efficiently, plus redox cofactor regeneration, we introduced pyridine nucleotide transhydrogenases (PNTs) from  E .  coli  to the metabolic pathway of ( S )-PED.    Results  The PNTs were successfully introduced into the  E .  coli  strain RSAB. Most of the PNT activities occurred in the cell membrane of  E .  coli . The introduction of PNTs increased intracellular NAD +  and NADH concentrations and decreased the NADPH pool without affecting the total nucleotide concentration and cell growth properties. The presence of PNTs increased the NADH/NAD +  ratio slightly and reduced the NADPH/NADP +  ratio about two-fold; the ratio of NADPH/NADP +  to NADH/NAD +  was reduced from 36 to 17. So, the PNTs rebalanced the cofactor pathways: the rate of RCR was increased, while the rate of SCR was decreased. When the ratio of NAD + /NADPH was 3.0 or higher, the RSAB strain produced ( S )-PED with the highest optical purity, 97.4%, and a yield of 95.2% at 6 h. The introduction of PNTs stimulated increases of 51.5% and 80.6%, respectively, in optical purity and yield, and simultaneously reduced the reaction time seven-fold.    Conclusions  In this work, PNTs were introduced into  E .  coli  to rebalance the cofactor pools within the engineered ( S )-PED pathways. The efficient one-step production of ( S )-PED plus NAD + –NADPH  in - situ  regeneration was realized. This work provided new insights into cofactor rebalancing pathways, using metabolic engineering methods, for efficient chiral alcohol production.

microbial cell factories

Efficient one-step production of (S)-1-phenyl-1,2-ethanediol from (R)-enantiomer plus NAD+–NADPH in-situ regeneration using engineered Escherichia coli