Biosynthesis of (R)-1,2-phenylethanediol and (R)-4-chloro-1,2-phenylethanediol by using two recombinant cells expressing enantiocomplementary epoxide hydrolases

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Abstract

Enantioconvergent biosynthesis of (R)-phenyl-1,2-ethanediol and (R)-4-chlorophenyl-1,2-ethanediol using two types of recombinant Escherichia coli cells expressing epoxide hydrolase of Caulobacter crescentus (CcEH) and a triple-point mutated EH of Mugil cephalus was investigated. The CcEH gene in pET28b(+) was expressed at 15 °C, and the reaction conditions such as biocatalyst ratio, reaction temperature and detergent addition were optimized to give best enantiopurity and yield. When two recombinant E. coli cells were used in sequential mode, (R)-phenyl-1,2-ethandiol with 90% enantiopurity and 95% yield was obtained in batch reaction. From 100 mM racemic 4-chlorostyrene oxide, (R)-4-chlorophenyl-1,2-ethanediol with 92% enantiopurity and 71% yield could be obtained.

Introduction

Many valuable pharmaceuticals consist of optically active compounds. These chiral compounds are synthesized from various chiral synthetic intermediates [1]. Chiral epoxides and diols can be used as chiral synthetic intermediates because they can undergo various reactions with nucleophiles, electrophiles, acids, and bases. Chiral epoxides can be produced by epoxide hydrolase (EH)-catalyzed kinetic resolution of racemic epoxides substrate [2]. EH is the enzyme that catalyzes the enantioselective biohydrolysis reaction. EHs are useful biocatalysts because they are cofactor-independent hydrolase with a high enantioselectivity and broad substrate specificities [3].

In a kinetic resolution, chiral epoxide can be obtained in 50% theoretical yield, since the preferred enantiomer of racemic mixture is hydrolyzed into diol by EH and then removed. Chiral diols are also useful synthetic starting materials for the synthesis of chiral pharmaceuticals. Chiral diols can be prepared from racemic epoxides by using EH-catalyzed enantioconvergent process with yields greater than 50% [4]. Enantioconvergent process is a deracemization procedure where both enantiomers of racemic epoxides must proceed in enantio- and regio-complementary fashions to give single enantiomeric diol product [5], [6]. Herein, one enantiomer of racemic epoxides should be converted to the diol with different charity via an inversion of chiral configuration (Fig. 1). Chemocatalysts for the enantioconvergent biohydrolysis of racemic epoxides have never been devised so far, to the best knowledge of authors, because the stereochemical requirements of the reaction are too complex to be achieved by conventional chemocatalysts.

Some EHs have enantio- and regio-complementary specificities for racemic epoxides. In EH-mediated enantioconvergent biohydrolysis of racemic epoxides, the attack of EH at the hindered α carbon of (S)-enantiomer caused the inversion of configuration to form (R)-diol, whereas (R)-enantiomer is transformed to (R)-diol with retention of chiral configuration by a predominant attack of EH at the less hindered β carbon atom. Various microbial EHs from Aspergillus niger, Agrobacterium radiobacter AD1, Beauveria sulfurescens, and Nocardia EH1 in combination with the plant EHs from potato and mung bean were used for enantioconvergent process [7], [8], [9], [10].

Recently, we have developed recombinant EH of Caulobacter crescentus (CcEH) to prepare (R)-4-chlorophenyl-1,2-ethandiol with yield higher than 50% [11]. We have also developed a marine fish EH from Mugil cephalus (McEH) [12], and conducted the enantioconvergent biohydrolysis of racemic styrene oxide in combination with CcEH. In the recombinant CcEH and McEH-catalyzed enantioconvergent biohydrolysis of racemic styrene oxide, CcEH was shown to attack predominantly (S)-enantiomer at α carbon atom and to produce (R)-diol with the inversion of chiral configuration [4]. On the contrary, recombinant McEH preferentially hydrolyzed (R)-enantiomer into (R)-diol with the retention of configuration. Hence, the combined use of two EHs leads to the preparation of chiral diols from racemic epoxides. Chiral diols were prepared with high enantiopurity more than 90% ee. Although the chiral diol was successfully prepared with yield more than 50%, the enantiopurity was high only at low concentration such as 5 mM [11]. Herein, we report on enantioconvergent biohydrolysis of racemic styrene oxide and 4-chlorostyrene oxide to prepare the corresponding chiral diols at 100 mM. The EH gene of C. crescentus was functionally expressed at low temperature to enhance the efficiency of recombinant whole-cell based enantioconvergent biohydrolysis reaction. The EH from M. cephalus with three point mutations (McEH mutant) was employed as the counter partner of CcEH. Two recombinant E. coli cells expressing CcEH and the mutated McEH were sequentially added to batch enantioconvergent biohydrolysis reaction.

Section snippets

Media and culture conditions

In order to enhance the functional expression of CcEH gene, the recombinant plasmid containing CcEH gene was expressed in various expression vector and in the presence of molecular chaperone. The resulting E. coli BL21 (DE3) was grown in LB broth containing 50 μg kanamycin/ml at 37 °C for 2 h at 180 rpm. When the OD600 reached 0.4-0.6, 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) was added in the culture. After induction, the culture was incubated at 15 °C overnight with a shaking speed of 180 rpm.

High-level expression and characterization of CcEH

In order to enhance the active protein portion of CcEH, we investigated the effects of expression temperature and chaperones on the functional expression of CcEH gene. The recombinant CcEH gene was inserted in pET and pColdI expression vectors and then expressed at different culture temperature. The CcEH gene was successfully expressed in pColdI, pET 21b(+) and pET 28b(+) at low temperature, 15 °C. When the hydrolytic activities were compared, the recombinant E. coli possessing pET 28b(+) vector

Conclusion

An enantioconvergent biohydrolysis reaction was performed by using two recombinant CcEH and McEH possessing enantiocomplementary biohydrolysis activities. The CcEH gene in pET28b(+) was expressed at 15 °C, and the triple-point mutated McEH gene was expressed in the presence of pGro7 containing the genes for groES-groEL. The effects of recombinant whole-cell biocatalyst ratio, reaction temperature and detergent addition on the enantiopurity and yield of the (R)-diol were analyzed. By the combined

Acknowledgment

This work was supported by the Marine and Extreme Genome Research Center Program, Ministry of Land, Transportation and Maritime Affairs.

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