Engineering Streptomyces coelicolor Carbonyl Reductase for Efficient Atorvastatin Precursor Synthesis

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 (ScCR1 I158V/P168S ) by structure-guided engineering. Second, directed evolution was performed to improve the stability using ScCR1 I158V/P168S as a template, affording a triple mutant, ScCR1 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 ScCR1 A60T/I158V/P168S within 9 h, affording an excellent enantiomeric excess (ee) of Ͼ99% and a space-time yield of 255 g liter Ϫ1 day Ϫ1 . 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.

Efficient Asymmetric Synthesis of Ethyl (S)-4-Chloro-3-hydroxybutyrate Using Alcohol Dehydrogenase SmADH31 with High Tolerance of Substrate and Product in a Monophasic Aqueous System

“…For the substrate COBE, we observed that the K m value of SmADH31 was only 0.42 mM, which was lower than that of other ADHs including ChADH (7.5 mM) and ScADH (0.59 mM). 21,22 The SmADH31 k cat /K m value for COBE was 5.5 × 10 2 mM −1 s −1 , which was higher than that of ChADH (26.8 mM −1 s −1 ) and ScADH (34.7 mM −1 s −1 ). All these results indicate that SmADH31 possesses a strong binding affinity and high catalytic efficiency for COBE.…”

Highly efficient synthesis of rosuvastatin intermediate using a carbonyl reductase–cofactor co‐immobilized biocatalyst in the non‐aqueous biosystem

“…Carbonyl reductase (EC 1.1.1.148) is the most widely used industrial oxidoreductase for catalyzing the biotransformation of the pro‐ketone substrate to the corresponding chiral alcohol with the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) because of its high activity, excellent enantioselectivity and environmentally friendly effect 9‐13 . Cui et al 14 .…”

“…Carbonyl reductase (EC 1.1.1.148) is the most widely used industrial oxidoreductase for catalyzing the biotransformation of the pro-ketone substrate to the corresponding chiral alcohol with the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) because of its high activity, excellent enantioselectivity and environmentally friendly effect. [9][10][11][12][13] Cui et al 14 employed two substrate-tolerant carbonyl reductases from Bacillus subtilis and Gluconobacter oxydans to efficiently reduce 2-hydroxy acetophenone to (S)-and (R)-1-phenyl-1,2-ethylene glycol in the biphasic system with a yield of 99% and an e.e. of 99%, respectively.…”

Efficient production of an ezetimibe intermediate using carbonyl reductase coupled with glucose dehydrogenase

“…8 Recently, biocatalyst has attracted more and more attention due to its wide range of natural sources, mild reaction conditions, high catalytic efficiency, excellent stereoselectivity, and environmental friendship. 9 Carbonyl reductases (CRs, EC 1.1.1.148) are a type of nicotinamide coenzyme-dependent oxidoreductases which catalyze the reduction of a carbonyl group to the corresponding chiral alcohol, in the present of the reduced form of the cofactor, NAD[P]H. 7,10,11 CRs have been employed as a valuable biocatalyst for ezetimibe synthesis. As early as 1997, it was reported that (S)-ET-5 was synthesized by whole cells of Schizosaccharomyces octosporus (ATCC 2479) and S. octosporus (ATCC 4206) containing CRs with the yields of 33.0 and 34.0%, respectively, and the e.e.…”