文献类型：期刊文献

英文题名：Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

作者：Li, Min[1,2];Zhang, Zhi-Jun[1,2];Kong, Xu-Dong[1,2];Yu, Hui-Lei[1,2];Zhou, Jiahai[3];Xu, Jian-He[1,2]

机构：[1]East China Univ Sci & Technol, Lab Biocatalysis & Synthet Biotechnol, State Key Lab Bioreactor Engn, Shanghai, Peoples R China;[2]East China Univ Sci & Technol, Shanghai Collaborat Innovat Ctr Biomfg, Shanghai, Peoples R China;[3]Chinese Acad Sci, Shanghai Inst Organ Chem, State Key Lab Bioorgan & Nat Prod Chem, Shanghai, Peoples R China

年份：2017

卷号：83

期号：12

外文期刊名：APPLIED AND ENVIRONMENTAL MICROBIOLOGY

收录：;EI(收录号：20172203724045);WOS:【SCI-EXPANDED(收录号:WOS:000402386500024)】；

基金：This work was financially supported by the National Natural Science Foundation of China (no. 21406067 and 21536004), the Ministry of Science and Technology, People's Republic of China (no. 2011CB710800, 2011AA02A210, and 2012AA022201), and the Shanghai Commission of Science and Technology (no. 11431921600).

语种：英文

外文关键词：carbonyl reductase; protein engineering; catalytic activity; thermostability; ethyl-(S)-4-chloro-3-hydroxybutyrate

摘要：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 (ScCR1I158V/P168S) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1I158V/P168S as a template, affording a triple mutant, ScCR1A60T/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 degrees 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 ScCR1A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of > 99% and a space-time yield of 255 g liter(-1) day(-1). 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.