Purification and Cloning of a Ketoreductase used for the Preparation of Chiral Alcohols

Abstract

Abstract The synthesis of the leading candidate compound in an anticancer program required ( S) ‐2‐chloro‐1‐(3‐chlorophenyl)‐ethanol as an intermediate. Other possible candidate compounds used analogues of the S ‐alcohol. Of 119 microbial cultures screened for reduction of the corresponding ketone to the S ‐alcohol, Hansenula polymorpha ATCC 58401 (73.8% ee) and Rhodococcus globerulus ATCC 21505 (71.8% ee) had the highest enantioselectivity for producing the desired alcohol. A ketoreductase from Hansenula polymorpha , after purification to homogeneity, gave the S ‐alcohol with 100% ee. Amino acid sequences from the purified enzyme were used to design PCR primers for cloning the ketoreductase. The cloned ketoreductase required NADP(H), had a subunit molecular weight of 29,220 and a native molecular weight of 88,000. The cloned ketoreductase was expressed in E. coli together with a cloned glucose 6‐phosphate dehydrogenase from Saccharomyces cerevisiae to allow regeneration of the NADPH required by the ketoreductase. An extract of E. coli containing the two recombinant enzymes was used to reduce 2‐chloro‐1‐(3‐chloro‐4‐fluorophenyl)‐ethanone and two related ketones to the corresponding S ‐alcohols. Intact E. coli cells provided with glucose were used to prepare ( S) ‐2‐chloro‐1‐(3‐chloro‐4‐fluorophenyl)‐ethanol in 89% yield with 100% ee.