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

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.