Asymmetric synthesis of lipitor chiral intermediate using a robust carbonyl reductase at high substrate to catalyst ratio

doi:10.1016/j.molcatb.2015.11.001

Journal of Molecular Catalysis B: Enzymatic

Volume 123, January 2016, Pages 67-72

https://doi.org/10.1016/j.molcatb.2015.11.001 Get rights and content

Highlights

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RpCR shares low sequence identity with known COBE carbonyl reductases.
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RpCR exhibits a high k_cat/K_m of 1747 s⁻¹ mM⁻¹.
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The substrate to catalyst ratio and space-time yield of RpCR reached 70 and 1480 g L⁻¹ d⁻¹.

Abstract

An NADPH-dependent carbonyl reductase (RpCR) from Rhodococcus pyridinivorans was discovered by genome mining for the asymmetric reduction of ethyl 4-chloro-3-oxo-butanoate (COBE). RpCR has been soluble expressed in Escherichia coli BL21(DE3). The highest activity is determined at pH 5.0 and 50 °C toward COBE. The apparent K_m and k_cat/K_m are 0.39 mM and 1747 s⁻¹ mM⁻¹, endowing RpCR with high catalytic efficiency in reduction of COBE. Employing merely 0.1 g recombinant RpCR-GDH in a toluene-aqueous biphasic system, as much as 7.0 g COBE could be asymmetrically reduced into ethyl (S)-4-chloro-3-hydroxybutanoate [(S)-CHBE] (>99% ee) without addition of external cofactor, achieving molar isolation yield of 91%, substrate to biocatalyst ratio of 70 and space-time yield of 1480 g L⁻¹ d⁻¹. Our results indicate the robust RpCR could be potentially applied in the preparation of optically pure (S)-CHBE.

Graphical abstract

Introduction

Asymmetric reduction of ketones is one of the most important and practical approaches for the production of chiral secondary alcohols, which is essential for the synthesis of industrially important chemicals such as pharmaceuticals, agrochemicals and natural products [1], [2], [3]. As an alternative to chemical methodologies, bioreductive preparation of (R) or (S)-enantiomers of alcohols using isolated enzymes or whole-cell systems has been extensively investigated due to its high enantio-, regio- and chemoselectivities, mild conditions, reproducibility and easy operation [4], [5]. However, two major challenges for the scale-up application of bioreduction systems are the lack of efficient biocatalysts and the necessity of expensive cofactors such as NAD(P)H/NAD(P)⁺ [6], [7]. The employment of robust biocatalysts with high cofactor utilization efficiency could reduce or even avoid the addition of external cofactors. Consequently, exploration of robust biocatalysts is of special interest to solve above issues [8], [9].

Ethyl (S)-3-hydroxyl-4-chlorobutanoate [(S)-CHBE] is a versatile and important chiral intermediate for the production of chiral drugs, including the cholesterol lowering 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) reductase inhibitors (namely statins) [10], [11], which rank the class I best-selling drugs due to their excellent therapeutic effect and low side effect. The asymmetric reduction of ethyl 3-oxo-4-chlorobutanoate (COBE) into (S)-CHBE is the most promising approach. In recent years, various microorganisms have been identified for the efficient synthesis of (S)-CHBE and well-reviewed by Ye et al. [11]. However the application of wild-type strains is often hindered by their complicated dehydrogenases/reductases systems with variable stereospecificity and low expression level of key reductases [11], [12]. An effective solution is discovering novel reductases by genome mining and their heterogeneous overexpression [11], [13]. A number of enantioselective carbonyl reductases have been identified with S-selectivity in the asymmetric reduction of COBE, including S1 from Candida magnolia [14], ARII from Sporobolomyces salmonicolor [15], CPE from Candida parapsilosis [16], PsCRI and PsCRII from Pichia stipitis [17], [18], ScCR from Streptomyces coelicolor [19], SOU1 from Candida albicans [20] and DhCR from Debaryomyces hansenii [21]. The highest catalytic efficiency (V_max = 349 μmol min⁻¹ mg⁻¹) was reported for ARII, however at a high substrate concentration (K_m = 1.49), and the ee was relatively lower for application [15]. A carbonyl reductase CPE identified by Wang et al. displayed 99% ee and a low K_m (0.19 mM), while its V_max (200 μmol min⁻¹ mg⁻¹) was not as high as ARII [19].

Besides high stability and enantioslelectivity, the lower K_m and higher V_max (or higher k_cat/K_m) is one of the crucial parameters of biocatalysts in large-scale application. Biocatalysts with high k_cat/K_m value could often reach the maximum velocity and maintain the activity for a longer time even at a low substrate concentration [22]. In current study, a novel carbonyl reductase from Rhodococcus pyridinivorans (RpCR) was identified by genome mining, and soluble expressed in Escherichia coli BL21(DE3). RpCR exhibited a high enantioselectivity, lower K_m than CPE and higher catalytic efficiency in the asymmetric reduction of COBE. Furthermore, the synthesis of (S)-CHBE employing RpCR at high substrate loading was achieved in an organic solvent-aqueous biphasic system to evaluate its potential for industrial applications.

Section snippets

Cloning and expression of RpCR coding gene in E. coli BL21(DE3)

Genomic DNA was extracted from R. pyridinivorans using a TIANamp Bacteria DNA Kit from Tiangen (Shanghai). Primers with HindIII and XhoI restriction sites were designed according to the RpCR coding gene (rpcr) sequence (GenBank accession No.: EHK80525.1). The PCR product of rpcr was double digested with HindIII and XhoI and then inserted into the expression vector pET28a. The resultant plasmid, pET28-rpcr, was transformed into E. coli BL21(DE3). The cells were cultivated at 37 °C in LB medium

Screening of recombinant reductases

Genome mining strategy was adopted to explore novel enzymes capable of reducing COBE. Twenty oxidoreductases sharing 40–60% amino acid sequence identities with known COBE reductases were selected from NCBI databases. All the potential COBE reductases were heterogeneous expressed in E. coli. The carbonyl reductase (GenBank accession No.: EHK80525.1) from R. pyridinivorans displayed the highest activity and enantioselectivity toward COBE, which was designated as RpCR and chosen for further

Conclusions

The COBE reductase RpCR from R. pyridinivorans was discovered by genome mining. RpCR displays high substrate affinity and catalytic efficiency toward COBE. RpCR is a promising biocatalyst for the synthesis of (S)-CHBE considering its high stereoselectivity, yield and final product concentration. The substrate to catalyst ratio and the space-time yield of RpCR in asymmetric reduction of COBE are about 70 and 1480 g L⁻¹ d⁻¹, respectively. To our best knowledge, this is the first report on newly

Acknowledgements

We are grateful to National Natural Science Foundation of China (21276112, 21506073), Natural Science Foundation of Jiangsu Province (BK20150003), the Fundamental Research Funds for the Central Universities (JUSRP51409B), the Program of Introducing Talents of Discipline to Universities (111-2-06), and a project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions for the financial support of this research.

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Asymmetric synthesis of lipitor chiral intermediate using a robust carbonyl reductase at high substrate to catalyst ratio

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Cloning and expression of RpCR coding gene in E. coli BL21(DE3)

Screening of recombinant reductases

Conclusions

Acknowledgements

Curr. Opin. Chem. Biol.

J. Mol. Catal. B: Enzym.

Bioresour. Bioprocess.

Biotechnol. Adv.

Tetrahedron: Asymmetry

Tetrahedron: Asymmetry

J. Mol. Catal. B: Enzym.

Bioresour. Technol.

Bioresour. Technol.

Bioresour. Technol.

Bioresour. Technol.

J. Biotechnol.

Chem. Biol. Interact.

J. Biol. Chem.

Bioresour. Technol.

Bioresour. Technol.

Nature