title: Reconstitution of the In Vitro Activity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System.
abstract: The cytochrome P450 enzyme CYP-sb21 from Sebekia benihana is capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylation in vivo, the in vitro activity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted the in vitro activity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed of seFdx and seFdR from Synechococcus elongatus PCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst using Escherichia coli was also constructed, and the permeability problem was solved by using N-cetyl-N,N,N-trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application.
authors.affiliation: Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, China.
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.affiliation: Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, China.
authors.forename: Lei
authors.initials: L
authors.lastname: Du
authors.affiliation: Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.affiliation: College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, Shandong, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Sun
authors.affiliation: College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, Shandong, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Huang
authors.affiliation: Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, China.
authors.forename: Xianliang
authors.initials: X
authors.lastname: Zheng
authors.affiliation: Department of Biological Engineering, Inha University, Incheon, South Korea eungsoo@inha.ac.kr lishengying@qibebt.ac.cn.
authors.forename: Eung-Soo
authors.initials: ES
authors.lastname: Kim
authors.affiliation: Key Laboratory of Biofuels, Shandong Provincial Key Laboratory of Synthetic Biology, Qingdao Institute of Bioenergy and Bioprocess Technology, Chinese Academy of Sciences, Qingdao, Shandong, China eungsoo@inha.ac.kr lishengying@qibebt.ac.cn.
authors.forename: Shengying
authors.initials: S
authors.lastname: Li
mesh_terms: D039903:Actinobacteria / Q000201:enzymology* / Q000378:metabolism*; D001711:Biotransformation; D016572:Cyclosporine / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D007166:Immunosuppressive Agents / Q000378:metabolism*; D060847:Metabolic Engineering; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D046940:Synechococcus / Q000201:enzymology / Q000235:genetics
chemical_list: D007166:Immunosuppressive Agents; D016572:Cyclosporine; D006899:Mixed Function Oxygenases