Semi-rational engineering of cytochrome CYP153A from Marinobacter aquaeolei for improved omega-hydroxylation activity towards oleic acid
文献类型： 外文期刊
作者： Duan, Yan 1 ; Ba, Lina 1 ; Gao, Jianwei 1 ; Gao, Xianxing 1 ; Zhu, Dunming 3 ; de Jong, Rene M. 4 ; Mink, Daniel 5 ; Kalu 1 ;
作者机构： 1.Tsinghua Univ, Natl Engn Lab Ind Enzymes, Dept Chem Engn, One Tsinghua Garden Rd, Beijing 100084, Peoples R China
2.Shanghai Acad Agr Sci, Inst Edible Fungi, 1000 Jinqi Rd, Shanghai 201403, Peoples R China
3.Chinese Acad Sci, Natl Engn Lab Ind Enzymes, Tianjin Inst Ind Biotechnol, Tianjin Airport Econ Area, 32 Xi Qi Dao, Tianjin 300308, Peoples R China
4.DSM Biotechnol Ctr, Alexander Fleminglaan 1, NL-2613 AX Delft, Netherlands
5.DSM Ahead R&D BV Innovat Synth, Urmonderbaan 22, NL-6167 RD Geleen, Netherlands
6.DSM Ahead R&D BV Innovat Synth, Urmonderbaan 22, NL-6
关键词： Cytochrome CYP153A;Oleic acid;Electron transfer;Site-directed saturation mutagenesis;Iterative saturation mutagenesis
期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.813； 五年影响因子：4.697 ）
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收录情况： SCI
摘要： omega-Hydroxy oleic acid is an important intermediate for the synthesis of certain polyesters and polyamides. In this study, a functional CYP153A/ putidaredoxin (Pdx)/putidaredoxin reductase (Pdr) hybrid system was engineered for improved omega-hydroxylation activity towards oleic acid. By the combination of site-directed saturation mutagenesis (SDSM) and iterative saturation mutagenesis (ISM), a best mutant (Variant II) was obtained with mutations at two sites (S120 and P165) at the Pdx interaction interface with CYP153A, and one site (S453) in the substrate binding pocket. The in vitro-reconstituted activity of Variant II with purified Pdx and Pdr was 2.7-fold that of the template, while the whole cell transformation activity was 2.0-fold that of the template. A 96-well format-based screening scheme for CYP153A was also developed, which should be useful for engineering of other P450s with low activity. Kinetic analyses indicated that the activity improvement for CYP153A variants largely resulted from enhanced electron transfer. This further demonstrates the importance of the electron transfer between P450s and the non-native redox partners for the overall performance of hybrid P450 systems.
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