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However, limitations of<jats:italic>Sc<\/jats:italic>CR1 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<jats:italic>Sc<\/jats:italic>CR1. First, the crystal structure of<jats:italic>Sc<\/jats:italic>CR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of<jats:italic>Sc<\/jats:italic>CR1 was increased from 38.8 U\/mg to 168 U\/mg (<jats:italic>Sc<\/jats:italic>CR1<jats:sub>I158V\/P168S<\/jats:sub>) by structure-guided engineering. Second, directed evolution was performed to improve the stability using<jats:italic>Sc<\/jats:italic>CR1<jats:sub>I158V\/P168S<\/jats:sub>as a template, affording a triple mutant,<jats:italic>Sc<\/jats:italic>CR1<jats:sub>A60T\/I158V\/P168S<\/jats:sub>, whose thermostability (<jats:italic>T<\/jats:italic><jats:sub>50<\/jats:sub><jats:sup>15<\/jats:sup>, defined as the temperature at which 50% of initial enzyme activity is lost following a heat treatment for 15 min) and substrate tolerance (<jats:italic>C<\/jats:italic><jats:sub>50<\/jats:sub><jats:sup>15<\/jats:sup>, defined as the concentration at which 50% of initial enzyme activity is lost following incubation for 15 min) were 6.2\u00b0C 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<jats:italic>Sc<\/jats:italic>CR1<jats:sub>A60T\/I158V\/P168S<\/jats:sub>within 9 h, affording an excellent enantiomeric excess (<jats:italic>ee<\/jats:italic>) of &gt;99% and a space-time yield of 255 g liter<jats:sup>\u22121<\/jats:sup>day<jats:sup>\u22121<\/jats:sup>. These results suggest high efficiency of the protein engineering strategy and good potential of the resulting variant for efficient synthesis of the atorvastatin precursor.<\/jats:p><jats:p><jats:bold>IMPORTANCE<\/jats:bold>Application of the carbonyl reductase<jats:italic>Sc<\/jats:italic>CR1 in asymmetrically synthesizing (<jats:italic>S<\/jats:italic>)-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<jats:italic>Sc<\/jats:italic>CR1. The catalytic efficiency, thermostability, and substrate tolerance of<jats:italic>Sc<\/jats:italic>CR1 were significantly improved by structure-guided engineering and directed evolution. The engineered<jats:italic>Sc<\/jats:italic>CR1 may serve as a promising biocatalyst for the biosynthesis of (<jats:italic>S<\/jats:italic>)-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.<\/jats:p>","DOI":"10.1128\/aem.00603-17","type":"journal-article","created":{"date-parts":[[2017,4,8]],"date-time":"2017-04-08T00:45:25Z","timestamp":1491612325000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":29,"title":["Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis"],"prefix":"10.1128","volume":"83","author":[{"given":"Min","family":"Li","sequence":"first","affiliation":[{"name":"Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering and Shanghai Collaborative Innovation Center for 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