Co-expression of glutamate dehydrogenase and alcohol dehydrogenase for preparation of (R)-2-chloro-1-phenylethanol

Abstract:

[Background] Alcohol dehydrogenase AdhS can produce (R)-2-chloro-1-phenylethanol by catalyzing asymmetric reduction. However, due to its insufficient ability to regenerate coenzyme NADH, coenzyme regenerator is needed to assist NADH regeneration. Glutamate dehydrogenase can regenerate coenzyme NAD(P)H with glutamate as substrate, and has the potential of coenzyme regeneration. [Objective] This study aimed to clone and express glutamate dehydrogenase gene gdhA, and construct the co-expression system of glutamate dehydrogenase GdhA and alcohol dehydrogenase AdhS in Escherichia coli, for improving conversion efficiency of (R)-2-chloro-1-phenylethanol preparation by AdhS. [Methods] GdhA was cloned from Bacillus subtilis 168 and expressed in Escherichia coli BL21(DE3) for the analysis of coenzyme regeneration activity. Then it was co-expressed with alcohol dehydrogenase AdhS to optimize the co-expression condition. Finally, the influence of different coenzyme regeneration schemes on conversion efficiency for (R)-2-chloro-1-phenylethanol preparation was studied. [Results] Specific activity of glutamate dehydrogenase GdhA to regenerate NADH was 694 U/g. The conversion efficiency for (R)-2-chloro-1-phenylethanol preparation reached 465 U/L through co-expression of AdhS and GdhA and optimization of expression conditions. By comparison, the conversion efficiency for (R)-2-chloro-1-phenylethanol preparation by AdhS was improved to about 3 times with NADH regeneration assisted by GdhA. [Conclusion] Glutamate dehydrogenase GdhA can be used for highly efficient regeneration of NADH, and through its co-expression with alcohol dehydrogenase AdhS, the conversion efficiency for (R)-2-chloro-1- phenylethanol preparation by AdhS have been enhanced significantly.