Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli

Chen et al. BMC Biotechnology (2016) 16:70 DOI 10.1186/s12896-016-0301-x RESEARCH ARTICLE Open Access Efficient biosynthesis of ethyl (R)-4-chloro3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli Xiang Chen1,2, Zhi-Qiang Liu1,2, Chao-Ping Lin1,2 and Yu-Guo Zheng1,2* Abstract Background: Ethyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE) is a versatile chiral precursor for many pharmaceuticals. Although several biosynthesis strategies have been documented to convert ethyl 4-chloro-3-oxobutanoate (COBE) to (R)-CHBE, the catalytic efficiency and stereoselectivity are still too low to be scaled up for industrial applications. Due to the increasing demand of (R)-CHBE, it is essential to explore more robust biocatalyst capable of preparing (R)-CHBE efficiently. Results: A stereoselective carbonyl reductase toolbox was constructed and employed into the asymmetric reduction of COBE to (R)-CHBE. A robust enzyme designed as BgADH3 from Burkholderia gladioli CCTCC M 2012379 exhibited excellent activity and enantioselectivity, and was further characterized and investigated in the asymmetric synthesis of (R)-CHBE. An economical and satisfactory enzyme-coupled cofactor recycling system was created using recombinant Escherichia coli cells co-expressing BgADH3 and glucose dehydrogenase genes to regenerate NADPH in situ. In an aqueous/octanol biphasic system, as much as 1200 mmol COBE was completely converted by using substrate fed-batch strategy to afford (R)-CHBE with 99.9 % ee at a space-time yield per gram of biomass of 4.47 mmol∙L−1∙h−1∙g DCW−1. Conclusions: These data demonstrate the promising of BgADH3 in practical synthesis of (R)-CHBE as a valuable chiral synthon. This study allows for the further application of BgADH3 in the biosynthesis of chiral alcohols, and establishes a preparative scale process for producing (R)-CHBE with excellent enantiopurity. Keywords: Burkholderia gladioli, Carbonyl reductases, Ethyl 4-chloro-3-oxobutanoate, Ethyl (R)-4-chloro-3-hydroxybutyrate, Co-expression Background Stereoselective carbonyl reductases (E.C. 1.1.1.x; SCRs) are nicotinamide cofactor-dependent enzymes capable of catalyzing the reversible redox reaction between alcohols and aldehydes/ketones. During the past decade, SCRs have been considerably applied to the synthesis of chiral pharmaceutical intermediates, including anticholesterol drugs [1, 2], β-lactams antibiotics [3], anticancer drugs * Correspondence: 1 Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou 310014, China 2 Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, China [4], and other important drugs [5]. However, the scaleup of SCR-catalyzed reactions were restricted due to the limited commercially available SCRs, narrow substrate specificity, expensive cofactor dependency, and substrate insolubility. Ethyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE) is a versatile precursor for pharmacologically valuable products, such as L-carnitine [6], (R)-4-amino-3-hydroxybutyric acid (GABOB) [7], and (R)-4-hydroxy-pyrrolidone [8]. Several synthetic strategies for optically active CHBE were developed, wherein the enzymatic asymmetric synthesis is the most promising way. Although various biocatalysts have been found to give (S)-CHBE [1, 2, 9–13], (R)-isomer is in great demand yet less attainable. Since then, several © 2016 The Author(s). Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Chen et al. BMC Biotechnology (2016) 16:70 microorganisms and enzymes capable of converting ethyl 4-chloro-3-oxobutanoate (COBE) to (R)-CHBE have been documented, including gox2036 from Gluconobacter oxydans [14], AKRs from Sporobolomyces salmonicolor and Lodderomyces elongisporus [15–17], and a reductase from Bacillus sp. ECU0013 [18]. However, all of them suffer from impediment such as low substrate concentration, unsatisfactory stereoselectivity, or high substrate/catalyst (S/C) ratio. These shortcomings hindered their applications in the industrial synthesis of (R)-CHBE. Exploring more robust SCRs with the ability to prepare enantioenriched (R)-CHBE efficiently is thus of great interest. Herein, we designed and implemented two strategies for identifying novel SCRs, and constructed an enzyme toolbox to screen a robust SCR that can biotransform COBE to (R)-CHBE. As the promising SCR, BgADH3 from Burkholderia gladioli CCTCC M 2012379 was selected for further study. The substrate spectrum of BgADH3 was evaluated toward varied aryl ketones and ketoesters. Furthermore, the practical applicability of BgADH3 was investigated in the asymmetric synthesis of (R)-CHBE using Escherichia coli cells co-expressing BgADH3 and a glucose dehydrogenase (GDH). Since the substrate was poorly soluble and unstable in aqueous environments, biphasic system was established using substrate fed-batch strategy to solve this issue. To our knowledge, this is the first report of SCR from B. gladioli subjected to the asymmetric synthesis of enantioenriched (R)-CHBE in aqueous/octanol biphasic system. Results Identification and screening of SCRs Strain B. gladioli CCTCC M 2012379 isolated from soil samples exhibited activity for catalyzing COBE to (R)CHBE (Additional file 1: Table S1). Genome hunting and data mining strategies were selected to discovering robust SCRs from CCTCC M 2012379. Based on bioinformatics analysis of sequence-similarity with gox2036 [14], which is a known NADH-dependent SCR giving enantiopure (R)CHBE. 35 candidates were cloned or synthesized, and heterologously overexpressed in E. coli BL21 (DE3), wherein BgADH3 displayed high activity toward COBE and afforded (R)-CHBE (Additional file 2: Table S2). Sequence analysis indicated that the BgADH3 gene contained an open reading frame with 1011 bp encoding a 336 amino-acid protein, in which the conserved NADPbinding motiff T97G98XXXG102XG104 and key catalytic residues N202S228Y241K245 were found (Additional file 3: Figure S1). The recombinant BgADH3 with His6-tag mainly presented in the soluble fraction was purified through nickel chelating affinity chromatography [19]. As shown in Fig. 1, the BgADH3 was not homogeneous, and the estimated molecular mass was around 37 kDa, in accordance with its theoretical value (37.3 kDa). The Page 2 of 12 Fig. 1 (...truncated)