Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH

As compared to individual expression of C. boidinii FDH in E. coli BL21 (DE3) that gave an intracellular enzyme activity of 400 units/gCDW, co-expression of the FDH with the ketoreductase (Candida tenuis xylose reductase; XR) resulted in a substantial decline in FDH activity. The remaining FDH activity of only 85 U/gCDW was strongly limiting the overall catalytic activity of the whole cell system. Combined effects from increase in FDH gene copy number, supply of rare tRNAs in a Rosetta strain of E. coli, dampened expression of the ketoreductase, and induction at low temperature (18°C) brought up the FDH activity threefold to a level of 250 U/gCDW while reducing the XR activity by just 19% (1140 U/gCDW). The E. coli whole-cell catalyst optimized for intracellular FDH activity showed improved performance in the synthesis of (S)-1-(2-chlorophenyl)ethanol, reflected in a substantial, up to 5-fold enhancement of productivity (0.37 g/gCDW) and yield (95% based on 100 mM ketone used) as compared to the reference catalyst. For xylitol production, the benefit of enhanced FDH expression was observed on productivity only after elimination of the mass transfer resistance caused by the cell membrane.Expression engineering of C. boidinii FDH is an important strategy to optimize E. coli whole-cell reductase catalysts that employ intracellular formate oxidation for regeneration of NADH. Increased FDH-activity was reflected by higher reduction yields of D-xylose and o-chloroacetophenone conversions provided that mass transfer limitations were overcome.Enzymatic reductions are widely used for the preparation of single-isomer alcohols, amino acids and other fine chemicals. Reductases and dehydrogenases applied in biocatalysis usually utilize NADP(H) or NAD(H) as cofactors. The in situ recycling of the high priced and labile coenzymes remains a dictate of economic considerations. Candida boidinii formate dehydrogenase (CbFDH) has been employed as a workhorse for NADH-regeneration for d