Discovery of a reductase-producing strain recombinant E. coli CCZU-A13 using colorimetric screening and its whole cell-catalyzed biosynthesis of ethyl (R)-4-chloro-3-hydroxybutanoate

Highlights

• One high-throughput screening strategy for COBE-reducing enzymes was built.

• An NADH-dependent reductase (SsCR) was discovered by genome data mining.

• Highly stereoselective bioreduction of COBE was demonstrated.

• Effective biotransformation of COBE was in butyl acetate–water (10:90, v/v) media.

• Broad substrate specificity was shown.

Abstract

An NADH-dependent reductase (SsCR) was discovered by genome data mining. After SsCR was overexpressed in E. coli BL21, recombinant E. coli CCZU-A13 with high reductase activity and excellent stereoselectivity for the reduction of ethyl 4-chloro-3-oxobutanoate (COBE) into ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-CHBE) was screened using one high-throughput colorimetric screening strategy. After the reaction optimization, a highly stereoselective bioreduction of COBE into (R)-CHBE (>99% ee) with the resting cells of E. coli CCZU-A13 was successfully demonstrated in n-butyl acetate–water (10:90, v/v) biphasic system. Biotransformation of 600 mM COBE for 8 h in the biphasic system, (R)-CHBE (>99% ee) could be obtained in the high yield of 100%. Moreover, the broad substrate specificity in the reduction of aliphatic and aromatic carbonyl compounds was also found. Significantly, E. coli CCZU-A13 shows high potential in the industrial production of (R)-CHBE (>99% ee) and its derivatives.

Introduction

Optically active ethyl (R)-4-chloro-3-hydroxybutanoate ester (CHBE) is an important precursor for the production of (R)-carnitine, (R)-4-amino-3-hydroxybutyric acid, (R)-4-hydroxy-2-pyrrolidone, and other fine chemicals (Ema et al., 2006, Kita et al., 1996, Liu et al., 2014, Yamamoto et al., 2002, Yu et al., 2007). Compared with conventional chemical synthesis, asymmetric bioreduction of prochiral ketone has been used as an economical and practical way for synthesizing highly optically active alcohols (Asako et al., 2009, Breuer et al., 2004, Cao et al., 2011, Ema et al., 2008, Ema et al., 2006, Goldberg et al., 2007, Gröger et al., 2006, Ni et al., 2010, Yamamoto et al., 2004, Ye et al., 2011). Although several carbonyl reduction enzymes for synthesizing (R)-CHBE from ethyl 4-chloro-3-oxobutanoate (COBE) have been found that required inexpensive NADPH for biocatalysis, few studies have addressed reductases that require the relative cheap cofactor NADH as an electron donor (Kita et al., 1996, Liu et al., 2014, Yu et al., 2007). Thus, discovery for new carbonyl reduction enzymes and improving their application potential for the synthesis of (R)-CHBE (>99% ee) are of great interest (He et al., 2014a, Kizaki et al., 2001, Ye et al., 2010b).

It is well-known that laborious traditional biocatalyst discovery is based on the screening from soil samples for searching the microbes with desired enzyme activity (He et al., 2011, Ye et al., 2011). However, this kind of screening strategy is always time consuming. Notably, only less than 1% of microbes in the environment can be culturable (He et al., 2014b). In post-genomic era, genome data mining offers an unprecedented opportunity for searching novel and useful biocatalysts with industrial application potential due to the abundant gene resources in the gene database (Hohne et al., 2010, Wang et al., 2011). Recently, genome data mining has been effectively used to search gene data bases for sequences similar to those of known reductases (He et al., 2014a, Yamamoto et al., 2003, Wang et al., 2011). However, there are a limited data about its application in the biotransformations of COBE into (R)-CHBE (>99% ee).

To obtain more COBE-reducing enzymes, it is important to establish a convenient, rapid and accuracy method for screening the desired biocatalysts. Many high-throughput methods have been employed to screen biocatalysts (He et al., 2011). The use of the conventional chromatographic method is usually time-consuming and inefficient (Yasohara et al., 1999, Liu et al., 2014). In this study, it is the first report that a high-throughput assay strategy for the screening of β-carbonyl carboxylic ester reductase based on ferric perchlorate spectrophotometry at room temperature was built. Moreover, an NADH-dependent reductase (SsCR) from Sporidiobolus salmonicolor was discovered by genome data mining. After SsCR was overexpressed in E. coli BL21, a high activity of reductase-producing strain, recombinant E. coli CCZU-A13, was employed for the efficient synthesis of ethyl (R)-CHBE (>99% ee) from the reduction of COBE. To increase the yield of (R)-CHBE, various parameters (cosubstrate, organic solvent, volumetric phase ratio, reaction temperature, reaction pH, metal ion additive, cell dosage, and substrate loading) on the reductase activity were investigated in the aqueous-organic biphasic media. Subsequently, highly efficient synthesis of ethyl (R)-CHBE and its derivatives by recombinant E. coli CCZU-A13 was successfully demonstrated.