title: Engineering of a novel carbonyl reductase with coenzyme regeneration in E. coli for efficient biosynthesis of enantiopure chiral alcohols.
abstract: The novel anti-Prelog stereospecific carbonyl reductase from Acetobacter sp. CCTCC M209061 was successfully expressed in E. coli combined with glucose dehydrogenase (GDH) to construct an efficient whole-cell biocatalyst with coenzyme NADH regeneration. The enzymatic activity of GAcCR (AcCR with a GST tag) reached 304.9U/g-dcw, even 9 folds higher than that of wild strain, and the activity of GDH for NADH regeneration recorded 46.0U/mg-protein in the recombinant E. coli. As a whole-cell biocatalyst, the recombinant E. coli BL21(DE3)pLysS (pETDuet-gaccr-gdh) possessed a broad substrate spectrum for kinds of carbonyl compounds with encouraging yield and stereoselectivity. Besides, the asymmetric reduction of ethyl 4-chloroacetoacetate (COBE) to optically pure ethyl 4-chloro-3-hydroxybutyrate (CHBE) catalyzed by the whole-cell biocatalyst was systematically investigated. Under the optimal reaction conditions, the optical purity of CHBE was over 99% e.e. for (S)-enantiomer, and the initial rate and product yield reached 8.04μmol/min and 99.4%, respectively. Moreover, the space-time yield was almost 20 folds higher than that catalyzed by the wild strain. Therefore, a new, high efficiency biocatalyst for asymmetric reductions was constructed successfully, and the enantioselective reduction of prochiral compounds using the biocatalyst was a promising approach for obtaining enantiopure chiral alcohols.
authors.affiliation: Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China; School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Wei
authors.affiliation: Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China.
authors.forename: Jia-Xin
authors.initials: JX
authors.lastname: Gao
authors.affiliation: State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China.
authors.forename: Gao-Wei
authors.initials: GW
authors.lastname: Zheng
authors.affiliation: Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Wu
authors.affiliation: Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China; School of Chemistry and Chemical Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China.
authors.forename: Min-Hua
authors.initials: MH
authors.lastname: Zong
authors.affiliation: Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China. Electronic address: wylou@scut.edu.cn.
authors.forename: Wen-Yong
authors.initials: WY
authors.lastname: Lou
mesh_terms: D000091:Acetobacter / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D000438:Alcohols / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D055162:Biocatalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics; D060847:Metabolic Engineering / Q000379:methods*; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D000438:Alcohols; D001426:Bacterial Proteins; D000429:Alcohol Oxidoreductases
keywords: (S)-CHBE; Acetobacter sp; Asymmetric reduction; Chiral alcohols; GAcCR; Whole-cell biocatalysis