Mechanistic Insight-Guided Protein-Engineering Strategies Lead to the Control of Cytochrome-P450-Mediated Regioselectivity of Midchain Fatty Acid C–H Activation

Regioselective midchain hydroxylation of fatty acids is an attractive chemical transformation, producing high-value hydroxy fatty acids and lactones. However, achieving such regioselective C–H activation without directing groups remains a significant chemical challenge. Herein, we use the midchain selective wild-type cytochrome P450 monooxygenase CYP116B46 as a scaffold to explore and expand regioselectivity, combining active site engineering and computational modeling to investigate the regioselectivity of CYP116B46 and design a range of variants with selectivity shifted from natural C5 to engineered C6–10 hydroxylation of decanoic acid. Our study suggests that this broad range of C–H activation is achieved by switching between two different substrate binding modes controlled by the protonation state of the fatty acid substrates in the active site. Using a variant with C5 selectivity as an example, we demonstrate that this methodology can be used to improve the activity and selectivity. Overall, we generated a panel of CYP116B46 variants capable of producing six distinct hydroxy acids from the same decanoic acid substrate, highlighting the potential of this strategy for chemical diversification.