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

• Main Authors: Zhang Rongzhen, Xu Yan, Xiao Rong, Zhang Botao, Wang Lei
• Format: Article
• Language: English
• Published: BMC 2012-12-01
• Series: Microbial Cell Factories
• Subjects: Redox cofactor regeneration; Chiral alcohol; Alcohol dehydrogenases/reductases; Metabolic pathway; One-step stereoinversion
• Online Access: http://www.microbialcellfactories.com/content/11/1/167

<p>Abstract</p> <p>Background</p> <p><it>Candida parapsilosis</it> CCTCC M203011 catalyzes the stereoinversion of (<it>R</it>)-1-phenyl-1,2-ethanediol (PED) through oxidation and reduction. Its NAD⁺-linked (<it>R</it>)-carbonyl reductase (RCR) catalyzes the oxidization of (<it>R</it>)-PED to 2-hydroxyacetophenone (HAP), and its NADPH-dependent (<it>S</it>)-carbonyl reductase (SCR) catalyzes the reduction of HAP to (<it>S</it>)-PED. The reactions require NAD⁺ and NADPH as cofactors. However, even if NAD⁺ and NADPH are added, the biotransformation of (<it>S</it>)-PED from the (<it>R</it>)-enantiomer by an <it>Escherichia coli</it> strain co-expressing RCR and SCR is slow and gives low yields, probably as a result of insufficient or imbalanced redox cofactors. To prepare (<it>S</it>)-PED from the (<it>R</it>)-enantiomer in one-step efficiently, plus redox cofactor regeneration, we introduced pyridine nucleotide transhydrogenases (PNTs) from <it>E</it>. <it>coli</it> to the metabolic pathway of (<it>S</it>)-PED.</p> <p>Results</p> <p>The PNTs were successfully introduced into the <it>E</it>. <it>coli</it> strain RSAB. Most of the PNT activities occurred in the cell membrane of <it>E</it>. <it>coli</it>. 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 (<it>S</it>)-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.</p> <p>Conclusions</p> <p>In this work, PNTs were introduced into <it>E</it>. <it>coli</it> to rebalance the cofactor pools within the engineered (<it>S</it>)-PED pathways. The efficient one-step production of (<it>S</it>)-PED plus NAD⁺–NADPH <it>in</it>-<it>situ</it> regeneration was realized. This work provided new insights into cofactor rebalancing pathways, using metabolic engineering methods, for efficient chiral alcohol production.</p>