Carving the Active Site of CYP153A7 Monooxygenase for Improving Terminal Hydroxylation of Medium‐Chain Fatty Acids

Abstract The P450‐mediated terminal hydroxylation of non‐activated C−H bonds is a chemically challenging reaction. CYP153A7 monooxygenase, discovered in Sphingomonas sp. HXN200, belongs to the CYP153A subfamily and shows a pronounced terminal selectivity. Herein, we report the significantly improved terminal hydroxylation activity of CYP153A7 by redesign of the substrate binding pocket based on molecular docking of CYP153A7−C 8:0 and sequence alignments. Some of the resultant single mutants were advantageous over the wild‐type enzyme with higher reaction rates, achieving a complete conversion of n‐ octanoic acid (C 8:0, 1 mM) in a shorter time period. Especially, a single‐mutation variant, D258E, showed 3.8‐fold higher catalytic efficiency than the wild type toward the terminal hydroxylation of medium‐chain fatty acid C 8:0 to the high value‐added product 8‐hydroxyoctanoic acid.