title: Rapid asymmetric reduction of ethyl 4-chloro-3-oxobutanoate using a thermostabilized mutant of ketoreductase ChKRED20.
abstract: Ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE) is an important chiral intermediate for the synthesis of "blockbuster" drug statins. The carbonyl reductase ChKRED20 from Chryseobacterium sp. CA49 was found to catalyze the bio-reductive production of (S)-CHBE with excellent stereoselectivity (>99.5 % ee). Perceiving a capacity for improvement, we sought to increase the thermostability of ChKRED20 to allow a higher reaction temperature. After one round of error-prone PCR (epPCR) library screening followed by the combination of beneficial mutations, a triple-mutant MC135 was successfully achieved with substantially enhanced thermostablity. The activity of MC135 at 50 °C was similar to the wild type. However, at its temperature optima of 65 °C, the mutant displayed 63 % increase of activity compared to the wild type and remained >95 % activity after being incubated for 15 days, while the wild type had a half-life of 11.9 min at 65 °C. At a substrate/catalyst ratio of 100 (w/w), the mutant catalyzed the complete conversion of 300 g/l substrate within 1 h to yield enantiopure (S)-CHBE with an isolated yield of 95 %, corresponding to a space-time yield of 1824 mM/h.
authors.affiliation: Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, China.|Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu, 610041, China.|Graduate University of the Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Feng-Jiao
authors.initials: FJ
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, China.|Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu, 610041, China.|Graduate University of the Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Xiao-Qiong
authors.initials: XQ
authors.lastname: Pei
authors.affiliation: Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, China.|Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu, 610041, China.|Graduate University of the Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Ren
authors.affiliation: Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 610041, China. wuzhl@cib.ac.cn.|Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu, 610041, China. wuzhl@cib.ac.cn.
authors.forename: Zhong-Liu
authors.initials: ZL
authors.lastname: Wu
mesh_terms: D000090:Acetoacetates / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D045247:Chryseobacterium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D006358:Hot Temperature; D007536:Isomerism; D007700:Kinetics; D009154:Mutation; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000090:Acetoacetates; D001426:Bacterial Proteins; C111982:ethyl 4-chloro-3-oxobutanoate; D010088:Oxidoreductases
keywords: (S)-4-Chloro-3-hydroxybutanoate; Carbonyl reductase; Directed evolution; Ketoreductase; Thermostability