Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

Abstract

ABSTRACT Streptomyces coelicolor CR1 ( Sc CR1) 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 Sc 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 Sc CR1. First, the crystal structure of Sc CR1 complexed with NADH and cosubstrate 2-propanol was solved, and the specific activity of Sc CR1 was increased from 38.8 U/mg to 168 U/mg ( Sc CR1 I158V/P168S ) by structure-guided engineering. Second, directed evolution was performed to improve the stability using Sc CR1 I158V/P168S as a template, affording a triple mutant, Sc CR1 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 Sc CR1 A60T/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 Sc CR1 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 Sc CR1. The catalytic efficiency, thermostability, and substrate tolerance of Sc CR1 were significantly improved by structure-guided engineering and directed evolution. The engineered Sc CR1 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.