Engineering cytochrome P450 BM3 of Bacillus megaterium for terminal oxidation of palmitic acid

被引:21
|
作者
Bruehlmann, Fredi [1 ]
Fourage, Laurent [2 ,3 ]
Ullmann, Christophe [2 ]
Haefliger, Olivier P. [1 ]
Jeckelmann, Nicolas [1 ]
Dubois, Cedric [1 ]
Wahler, Denis [2 ,4 ]
机构
[1] Firmenich Co, Corp R&D, CH-1211 Geneva 8, Switzerland
[2] Proteus, F-30035 Nimes 1, France
[3] Total New Energies R&D Biotechnol, F-92069 Paris, France
[4] SEPPIC, F-81105 Castres, France
关键词
Cytochrome P450; Bacillus megaterium; Directed evolution; Palmitic acid; Terminal oxidation; FATTY-ACIDS; ELECTRON-TRANSFER; LABORATORY EVOLUTION; SUBSTRATE; HYDROXYLATION; MECHANISM; P450BM-3; REGIOSELECTIVITY; CYP102A1; BINDING;
D O I
10.1016/j.jbiotec.2014.05.002
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Directed evolution via iterative cycles of random and targeted mutagenesis was applied to the P450 domain of the subterminal fatty acid hydroxylase CYP102A1 of Bacillus megaterium to shift its regioselectivity towards the terminal position of palmitic acid. A powerful and versatile high throughput assay based on LC-MS allowed the simultaneous detection of primary and secondary oxidation products, which was instrumental for identifying variants with a strong preference for the terminal oxidation of palmitic acid. The best variants identified acquired up to 11 amino acid alterations. Substitutions at F87,1263, and A328, relatively close to the bound substrate based on available crystallographic information contributed significantly to the altered regioselectivity. However, non-obvious residues much more distant from the bound substrate showed surprising strong contributions to the increased selectivity for the terminal position of palmitic acid. (C) 2014 Elsevier B.V. All rights reserved.
引用
收藏
页码:17 / 26
页数:10
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