Article ID Journal Published Year Pages File Type
23191 Journal of Biotechnology 2014 10 Pages PDF
Abstract

•Directed evolution of P450 BM3 for terminal fatty acid hydroxylation.•High throughput screening based on LC-MS for simultaneous detection of fatty acid metabolites.•Strong impact of residues on regioselectivity distant of the catalytic site.

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, I263, 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.

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Physical Sciences and Engineering Chemical Engineering Bioengineering
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