Single mutations of ketoreductase ChKRED20 enhance the bioreductive production of (1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol
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(1S)-2-chloro-1-(3, 4-difluorophenyl) ethanol ((S)-CFPL) is an intermediate for the drug ticagrelor, and is manufactured via chemical approaches. To develop a biocatalytic solution to (S)-CFPL, an inventory of ketoreductases from Chryseobacterium sp. CA49 were rescreened, and ChKRED20 was found to catalyze the reduction of the ketone precursor with excellent stereoselectivity (>99 % ee). After screening an error-prone PCR library of the wild-type ChKRED20, two mutants, each bearing a single amino acid substitution of H145L or L205M, were identified with significantly increased activity. Then, the two critical positions were each randomized by constructing saturation mutagenesis libraries, which delivered several mutants with further enhanced activity. Among them, the mutant L205A was the best performer with a specific activity of 178 μmol/min/mg, ten times of that of the wild-type. Its k cat/K m increased by 15 times and half-life at 50 °C increased by 70 %. The mutant catalyzed the complete conversion of 150 and 200 g/l substrate within 6 and 20 h, respectively, to yield enantiopure (S)-CFPL with an isolated yield of 95 %.
KeywordsTicagrelor Bioreduction Ketoreductase Directed evolution Carbonyl reductase
We thank Professor Ganggang Wang and Dr. Zhongchuan Liu of the Chengdu Institute of Biology for the assistance with structural analysis.
Compliance with ethical standards
This study was funded by the National Natural Science Foundation of China (21372216 and 21572220), the Open Fund of Key Laboratory of Environmental and Applied Microbiology (KLCAS-2016-08 and KLCAS-2015-01), and the Key Research Program (KGZD-EW-606-14) of the Chinese Academy of Sciences.
Conflict of interest
The authors declare that they have no competing interests.
This article does not contain any studies with human participants or animals performed by any of the authors.
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