Enhancing the biocatalytic manufacture of the key intermediate of atorvastatin by focused directed evolution of halohydrin dehalogenase.
Halohydrin dehalogenases (HHDHs) are biocatalytically interesting enzymes due to their ability to form C-C, C-N, C-O, and C-S bonds. One of most important application of HHDH was the protein engineering of HheC (halohydrin dehalogenase from Agrobacterium radiobacter AD1) for the industrial manufacturing of ethyl (R)-4-cyano-3-hydroxybutanoate (HN), a key chiral synthon of a cholesterol-lowering drug of atorvastatin. During our development of an alternative, more efficient and economic route for chemo-enzymatic preparation of the intermediate of atorvastatin, we found that the HheC2360 previously reported for HN manufacture, had insufficient activity for the cyanolysis production of tert-butyl (3 R,5 S)-6-cyano-3,5-dihydroxyhexanoate (A7). Herein, we present the focused directed evolution of HheC2360 with higher activity and enhanced biocatalytic performance using active site mutagenesis. Through docking of the product, A7, into the crystal structure of HheC2360, 6 residues was selected for combined active sites testing (CASTing). After library screening, the variant V84G/W86F was identified to have a 15- fold increase in activity. Time course analysis of the cyanolysis reaction catalyzed by this variant, showed 2- fold increase in space time productivity compared with HheC2360. These results demonstrate the applicability of the variant V84G/W86F as a biocatalyst for the efficient and practical production of atorvastatin intermediate.
Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025, China.|Abiochem Co. LTD, Shanghai, China.
Yu
Y
Luo
Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025, China.
Yangzi
Y
Chen
Key Laboratory of Combinational Biosynthesis and Drug Discovery, Ministry of Education, Wuhan University School of Pharmaceutical Sciences, 185 Donghu Road, Wuhan 430071, China.
Hongmin
H
Ma
Abiochem Co. LTD, Shanghai, China.
ZhenHua
Z
Tian
Key Laboratory of Combinational Biosynthesis and Drug Discovery, Ministry of Education, Wuhan University School of Pharmaceutical Sciences, 185 Donghu Road, Wuhan 430071, China.
Yeqi
Y
Zhang
Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of Chinese Ministry of Education, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025, China.
Jian
J
Zhang
D016960:Agrobacterium tumefaciens / Q000201:enzymology; D000069059:Atorvastatin / Q000138:chemical synthesis*; D055162:Biocatalysis; D020134:Catalytic Domain; D004926:Escherichia coli / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism; D016296:Mutagenesis; D015202:Protein Engineering / Q000379:methods
D000069059:Atorvastatin; D006867:Hydrolases; C430848:halohydrin dehalogenase