Improvement of carbonyl reductase activity for the bioproduction of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate |
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| Institution: | 1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China;2. Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China;1. Zhejiang Provincial Engineering Technology Research Center of Marine Biomedical Products, School of Food and Pharmacy, Zhejiang Ocean University, Zhoushan 316022, China;2. Zhejiang Changxing Pharmaceutical CO., LTD, Huzhou 313108, China;1. Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China;2. Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu 610041, China;3. University of Chinese Academy of Sciences, Beijing 10049, China;1. Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China;2. Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China |
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| Abstract: | tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key chiral intermediate for the side chain synthesis of rosuvastatin. In this study, random mutagenesis, site-saturation mutagenesis and combinatorial mutagenesis methods were applied to improve the activity of a synthesized stereoselective short chain carbonyl reductase (SCR) to prepare (3R,5S)-CDHH. After screened by high-throughput screening method and high-performance liquid chromatography, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser, were obtained, and the enzyme activities of mutants were improved by 1.60- and 1.91-fold compared with parent enzyme, respectively. The catalytically efficiencies (kcat/Km) of mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser exhibited 5.11- and 8.07-fold improvements in initial activity toward (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH), respectively. In the asymmetric reduction, mut-Phe145Tyr/Thr152Ser catalyzed 500 g L?1 of (S)-CHOH to produce (3R,5S)-CDHH with >99% yield and >99% e.e., and the highest space-time yield achieved at 752.76 mmol L?1 h?1 g?1 wet cell weight within 8 h bioconversion. This study provides a foundation for the preparation of (3R,5S)-CDHH by carbonyl reductase. |
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| Keywords: | Carbonyl reductase Directed evolution Single-batch reaction |
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