Engineering the Fungal Peroxygenase for Efficient and Regioselective Hydroxylation of Vitamin Ds and Sterols

Abstract

Hydroxylation of C25 C–H bonds (referring to sterols) is of great importance in vivo for metabolizing sterols and vitamin Ds. The biocatalytic hydroxylation of C25 C–H bonds is restricted by the selectivity and activity of the enzymes due to the inertness of these bulky compounds. Herein, we employed fungal unspecific peroxygenase from Agrocybe aegerita (AaeUPO) as the catalyst to develop efficient and selective AaeUPO variants through protein engineering. After three rounds of evolution using semirational design, 2 variants, G195A/G241V/G318V (Stev) and Q72K/G195A/G241V (Veco), were determined to be the ideal catalysts, showing a 25- to 27-fold increase in enzyme activity and an improvement in selectivity from 25% to over 93% in gram-scale conversion of vitamin D3 to 25-hydroxyvitamin D3. These two variants exhibited overall enhanced catalytic performance in hydroxylating the C25 C–H bonds of the other 24 sterol and vitamin D analogues. This work provides an enzymatic toolbox to synthesize the highly important vitamins and sterols into the compounds of interest under mild conditions with remarkable regioselectivity and enzyme activity.