Production Of Chiral Pharmaceutical Intermediate Ethyl R-4-chloro-3-hydroxybutyrate In Recombinant E.coli Systems

Oxido-reductases have many applications in chemical synthesis, especially in asymmetric synthesis of chiral compounds. A big burden in the industrialization of oxido-reductases catalyzed reaction is the requirement of expensive coenzymes NAD(P)+/NAD(P)H. Various approaches to the regeneration of NAD(P)+/NAD(P)H have been studied, such as chemical, enzymatic, electrochemical and photochemical methods etc. In this paper, whole cells were used for biocatalysis and a novel method of NADPH regeneration was explored.Ethyl (i?)-4-chloro-3-hydroxybutanoate (i?-CHBE) is a chiral building block for the synthesis of pharmaceutical target compounds. Aldehyde reductase (ALR, E.C.I. 1.1.2) from Sporobolomyces salmonicolor can reduce ethyl 4-chloro-3-oxobutanoate (COBE) to (R)-CHBE. When glucose is oxidized to glucolactone by glucose dehydrogenase (GDH, E.C. 1.1.1.47), NADP+ is reduced to NADPH, therefore, this reaction can be coupled to the reduction of COBE to (R)-CHBE.In this work, two expression vectors have been constructed: pQE30-alr0307 harboring an NADPH-dependent aldehyde reductase gene from Sporobolomyces salmonicolor ZJU0307, and pQE30-gdh0310 harboring a glucose dehydrogenase gene from Bacillus megaterium ZJU0310. Two recombinant strains, E. coli M15 (pQE30-alr0307) and E. coli Ml 5 (pQE30-gdh0310), are mixed after being cultivated separately and applied in the asymmetric reduction from ethyl 4-chloro-3-oxobutanoate to (R)-4-chloro-3-hydroxybutanoate. The former strain acted as catalyst and the later functioned in NADPH regeneration. In this two-strain system, the biotransformation reaction was completed effectively without any glucose dehydrogenase or NADPH/NADP+ added. The new strategy has the potential to be employed in other biotransformation systems with coenzyme regeneration.