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Cloning, expression, and directed evolution of carbonyl reductase from Leifsonia xyli HS0904 with enhanced catalytic efficiency.
Appl Microbiol Biotechnol 2014; 98(20):8591-601AM

Abstract

(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.

Authors+Show Affiliations

College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, 310032, People's Republic of China.No affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

24788330

Citation

Wang, Neng-Qiang, et al. "Cloning, Expression, and Directed Evolution of Carbonyl Reductase From Leifsonia Xyli HS0904 With Enhanced Catalytic Efficiency." Applied Microbiology and Biotechnology, vol. 98, no. 20, 2014, pp. 8591-601.
Wang NQ, Sun J, Huang J, et al. Cloning, expression, and directed evolution of carbonyl reductase from Leifsonia xyli HS0904 with enhanced catalytic efficiency. Appl Microbiol Biotechnol. 2014;98(20):8591-601.
Wang, N. Q., Sun, J., Huang, J., & Wang, P. (2014). Cloning, expression, and directed evolution of carbonyl reductase from Leifsonia xyli HS0904 with enhanced catalytic efficiency. Applied Microbiology and Biotechnology, 98(20), pp. 8591-601. doi:10.1007/s00253-014-5770-z.
Wang NQ, et al. Cloning, Expression, and Directed Evolution of Carbonyl Reductase From Leifsonia Xyli HS0904 With Enhanced Catalytic Efficiency. Appl Microbiol Biotechnol. 2014;98(20):8591-601. PubMed PMID: 24788330.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Cloning, expression, and directed evolution of carbonyl reductase from Leifsonia xyli HS0904 with enhanced catalytic efficiency. AU - Wang,Neng-Qiang, AU - Sun,Jing, AU - Huang,Jin, AU - Wang,Pu, Y1 - 2014/05/01/ PY - 2014/01/27/received PY - 2014/04/10/accepted PY - 2014/04/09/revised PY - 2014/5/3/entrez PY - 2014/5/3/pubmed PY - 2015/6/17/medline SP - 8591 EP - 601 JF - Applied microbiology and biotechnology JO - Appl. Microbiol. Biotechnol. VL - 98 IS - 20 N2 - (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. SN - 1432-0614 UR - https://www.unboundmedicine.com/medline/citation/24788330/Cloning_expression_and_directed_evolution_of_carbonyl_reductase_from_Leifsonia_xyli_HS0904_with_enhanced_catalytic_efficiency_ L2 - https://dx.doi.org/10.1007/s00253-014-5770-z DB - PRIME DP - Unbound Medicine ER -