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

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.