Cloning, expression, and directed evolution of carbonyl reductase from Leifsonia xyli HS0904 with enhanced catalytic efficiency.
(R)-[3,5-bis(trifluoromethyl)phenyl] ethanol ((R)-BTPE) is a valuable chiral intermediate for the synthesis of antiemetic drug Aprepitant and Fosaprepitant. A Leifsonia xyli HS0904-derived carbonyl reductase (LXCAR), an effective biocatalyst for the asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone (BTAP) to (R)-BTPE, was overexpressed in Escherichia coli BL21 (DE3). Bioinformatics analysis indicated that the amino acid sequence of recombinant LXCAR showed 89 % similarity to short-chain dehydrogenase/reductase. E. coli recombinant carbonyl reductase crude extract showed a specific activity of 1.54 U/mg, which was 62 times higher than that of L. xyli HS0904 crude extract. By using error-prone polymerase chain reaction and site-directed mutagenesis, the engineered LXCAR demonstrated superior catalytic activity toward BTAP, and the obtained mutant LXCAR-S154Y exhibited nearly 13-fold, 5.4-fold, and 2.3-fold increase in k cat/K m value, k cat value, and specific activity toward BTAP, respectively, compared to the recombinant LXCAR. Additionally, the reduction of BTAP by whole cells of mutant LXCAR-S154Y afforded a best yield of 99.6 % for (R)-BTPE within 2 h at 200 mM BTAP, which was shortened by 28 and 2 h compared to those catalyzed by L. xyli HS0904 cells and recombinant E. coli cells expressing LXCAR, respectively. Moreover, a yield of 82.5 % for (R)-BTPE was achieved within 12 h at an increased BTAP concentration of up to 1,000 mM (256 g/l), representing a 1.9-fold increase over the recombinant LXCAR. Homology modeling and docking analysis revealed the molecular basis for the high catalytic activity of mutant LXCAR-S154Y toward BTAP. The results present here provide a promising alternative for economical and efficient production of chiral alcohols by engineered LXCAR.
College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310032, People's Republic of China.
Neng-Qiang
NQ
Wang
Jing
J
Sun
Jin
J
Huang
Pu
P
Wang
D039903:Actinobacteria / Q000201:enzymology* / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001711:Biotransformation; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D050505:Mutant Proteins; D000429:Alcohol Oxidoreductases