Structure-Guided Directed Evolution of a Carbonyl Reductase Enables the Stereoselective Synthesis of (2S,3S)-2,2-Disubstituted-3-hydroxycyclopentanones via Desymmetric Reduction

2020 ◽  
Vol 22 (9) ◽  
pp. 3444-3448 ◽  
Author(s):  
Juan Li ◽  
Jinhui Feng ◽  
Xi Chen ◽  
Jingyao Gong ◽  
Yunfeng Cui ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Stereoselective Synthesis ◽  
Carbonyl Reductase

Stereoselective Synthesis of Chiral δ-Lactones via an Engineered Carbonyl Reductase

2021 ◽  
Author(s):  
Tao Wang ◽  
Xiaoyan Zhang ◽  
Yucong Zheng ◽  
Yunpeng Bai
Keyword(s):  
Serratia Marcescens ◽  
Directed Evolution ◽  
Stereoselective Synthesis ◽  
Specific Activity ◽  
Carbonyl Reductase

A carbonyl reductase variant, SmCRM5, from Serratia marcescens was obtained through structure-guided directed evolution. The variant showed improved specific activity (U mg-1) toward most of 16 tested substrates and gave...

Efficient reductive desymmetrization of bulky 1,3-cyclodiketones enabled by structure-guided directed evolution of a carbonyl reductase

2019 ◽  
Vol 2 (10) ◽  
pp. 931-941 ◽  
Author(s):  
Xi Chen ◽  
Hongliu Zhang ◽  
Miguel A. Maria-Solano ◽  
Weidong Liu ◽  
Juan Li ◽  
...  
Keyword(s):  
Directed Evolution ◽  
Carbonyl Reductase

Efficient Stereoselective Synthesis of Structurally Diverse γ‐ and δ‐Lactones Using an Engineered Carbonyl Reductase

ChemCatChem ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 2600-2606 ◽  
Author(s):  
Meng Chen ◽  
Xiao‐Yan Zhang ◽  
Chen‐Guang Xing ◽  
Chao Zhang ◽  
Yu‐Cong Zheng ◽  
...  
Keyword(s):  
Stereoselective Synthesis ◽  
Carbonyl Reductase

scholarly journals Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

2017 ◽  
Vol 83 (12) ◽  
Author(s):  
Min Li ◽  
Zhi-Jun Zhang ◽  
Xu-Dong Kong ◽  
Hui-Lei Yu ◽  
Jiahai Zhou ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Protein Engineering ◽  
Directed Evolution ◽  
Specific Activity ◽  
Streptomyces Coelicolor ◽  
Catalytic Efficiency ◽  
Carbonyl Reductase ◽  
Triple Mutant ◽  
Engineering Strategy ◽  
Substrate Tolerance

ABSTRACT 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°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.

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