title: Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli.
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.
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China.|Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China.|Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, China.
authors.forename: Zhi-Qiang
authors.initials: ZQ
authors.lastname: Liu
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China.|Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, China.
authors.forename: Chao-Ping
authors.initials: CP
authors.lastname: Lin
authors.affiliation: Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, Hangzhou, 310014, China. zhengyg@zjut.edu.cn.|Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, 310014, China. zhengyg@zjut.edu.cn.
authors.forename: Yu-Guo
authors.initials: YG
authors.lastname: Zheng
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D042725:Burkholderia gladioli / Q000201:enzymology* / Q000235:genetics; D002087:Butyrates / Q000302:isolation & purification / Q000378:metabolism*; D002384:Catalysis; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D024861:Genetic Enhancement / Q000379:methods*; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D013237:Stereoisomerism
chemical_list: D002087:Butyrates; C121244:ethyl 4-chloro-3-hydroxybutanoate; D000429:Alcohol Oxidoreductases; C019642:alcohol dehydrogenase (NADP+)
keywords: Burkholderia gladioli; Carbonyl reductases; Co-expression; Ethyl (R)-4-chloro-3-hydroxybutyrate; Ethyl 4-chloro-3-oxobutanoate