title: Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis.
abstract: Streptomyces coelicolorCR1 (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 ofScCR1 observed for practical application include low activity and poor stability. In this work, protein engineering was employed to improve the catalytic efficiency and stability ofScCR1. First, the crystal structure ofScCR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity ofScCR1 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 usingScCR1I158V/P168Sas a template, affording a triple mutant,ScCR1A60T/I158V/P168S, whose thermostability (T5015, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (C5015, 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 lyophilizedScCR1A60T/I158V/P168Swithin 9 h, affording an excellent enantiomeric excess (ee) of >99% and a space-time yield of 255 g liter-1day-1These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor.IMPORTANCEApplication of the carbonyl reductaseScCR1 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 ofScCR1. The catalytic efficiency, thermostability, and substrate tolerance ofScCR1 were significantly improved by structure-guided engineering and directed evolution. The engineeredScCR1 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.
authors.affiliation: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.affiliation: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Zhi-Jun
authors.initials: ZJ
authors.lastname: Zhang
authors.affiliation: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Xu-Dong
authors.initials: XD
authors.lastname: Kong
authors.affiliation: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, People's Republic of China.
authors.forename: Hui-Lei
authors.initials: HL
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Bio-organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, People's Republic of China jiahai@mail.sioc.ac.cn jianhexu@ecust.edu.cn.
authors.forename: Jiahai
authors.initials: J
authors.lastname: Zhou
authors.affiliation: Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for Biomanufacturing, East China University of Science and Technology, Shanghai, People's Republic of China jiahai@mail.sioc.ac.cn jianhexu@ecust.edu.cn.
authors.forename: Jian-He
authors.initials: JH
authors.lastname: Xu
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000069059:Atorvastatin / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D055162:Biocatalysis; D004795:Enzyme Stability; D006885:Hydroxybutyrates / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D015202:Protein Engineering; D048372:Streptomyces coelicolor / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C511345:4-chloro-3-hydroxybutyric acid; D001426:Bacterial Proteins; D006885:Hydroxybutyrates; D000069059:Atorvastatin; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+)
keywords: carbonyl reductase; catalytic activity; ethyl-(S)-4-chloro-3-hydroxybutyrate; protein engineering; thermostability