Asymmetric synthesis of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase from Candida albicans with two co-existing recombinant Escherichia coli strains

Cai, Ping; An, Mingdong; Xu, Sheng; Yan, Ming; Hao, Ning; Li, Yan; Lin, Xu

doi:10.1080/09168451.2015.1012145

Bioscience, Biotechnology, and Biochemistry

2015

DOI: 10.1080/09168451.2015.1012145

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Asymmetric synthesis of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase from Candida albicans with two co-existing recombinant Escherichia coli strains

Ping Cai

Mingdong An

Sheng Xu

et al.

Abstract: (2015) Asymmetric synthesis of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase from Candidaalbicans with two co-existing recombinant Escherichiacoli strains, Bioscience, Biotechnology, and Biochemistry, 79:7, 1090-1093,

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“…Compared with chemical synthesis, biocatalytic asymmetric synthesis of (S)-CHBE from ethyl-4-chloro-3-oxobutyrate (COBE) has attracted more and more attention because it provides eco-friendly reaction conditions, satisfactory selectivity, 100% theoretical yield, and fewer by-products, with simpler and easier operations (3)(4)(5)(6). Therefore, several biocatalytic approaches for the synthesis of (S)-CHBE from COBE employing different carbonyl reductases have been developed (3,(7)(8)(9)(10)(11). However, these methods usually require expensive NADH or NADPH as a cofactor, which represents a great challenge for practical application.…”

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Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

Zhang

Kong

et al. 2017

Appl Environ Microbiol

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Streptomyces coelicolor CR1 (ScCR1) has been shown to be a promising biocatalyst for the synthesis of an atorvastatin precursor, ethyl-(S)-4-chloro-3-hydroxybutyrate [(S)-CHBE]. However, limitations of ScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. First, the crystal structure of ScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of ScCR1 was increased from 38.8 U/mg to 168 U/mg (ScCR1 I158V/P168S ) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1 I158V/P168S as a template, affording a triple mutant, ScCR1 A60T/I158V/P168S , whose thermostability (T 50 15 , defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C 50 15 , defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2°C and 4.7-fold higher than those of the wild-type enzyme. Interestingly, the specific activity of the triple mutant was further increased to 260 U/mg. Protein modeling and docking analysis shed light on the origin of the improved activity and stability. In the asymmetric reduction of ethyl-4-chloro-3-oxobutyrate (COBE) on a 300-ml scale, 100 g/liter COBE could be completely converted by only 2 g/liter of lyophilized ScCR1 A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of Ͼ99% and a space-time yield of 255 g liter Ϫ1 day Ϫ1 . These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor.IMPORTANCE Application of the carbonyl reductase ScCR1 in asymmetrically synthesizing (S)-CHBE, a key precursor for the blockbuster drug Lipitor, from COBE has been hindered by its low catalytic activity and poor thermostability and substrate tolerance. In this work, protein engineering was employed to improve the catalytic efficiency and stability of ScCR1. The catalytic efficiency, thermostability, and substrate tolerance of ScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineered ScCR1 may serve as a promising biocatalyst for the biosynthesis of (S)-CHBE, and the protein engineering strategy adopted in this work would serve as a useful approach for future engineering of other reductases toward potential application in organic synthesis.

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Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

Zhang

Kong

et al. 2017

Appl Environ Microbiol

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Asymmetric synthesis of lipitor chiral intermediate using a robust carbonyl reductase at high substrate to catalyst ratio

Tang

2016

Journal of Molecular Catalysis B: Enzymatic

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Asymmetric synthesis of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase from Candida albicans with two co-existing recombinant Escherichia coli strains

Abstract: (2015) Asymmetric synthesis of (S)-4-chloro-3-hydroxybutanoate by sorbose reductase from Candidaalbicans with two co-existing recombinant Escherichiacoli strains, Bioscience, Biotechnology, and Biochemistry, 79:7, 1090-1093,

Cited by 2 publications

References 18 publications

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

Asymmetric synthesis of lipitor chiral intermediate using a robust carbonyl reductase at high substrate to catalyst ratio

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