Structure-Guided Co-Evolution of Fungal Unspecific Peroxygenase for Improved VD ₃ C25-Hydroxylation by Substrate-Binding Pocket Engineering

Fungal unspecific peroxygenases (UPOs) can directly utilize H₂O₂ as an oxygen and electron donor for selective C-H bond oxyfunctionalization. However, long-time duration of genetic modification and expression in <i>Pichia pastoris</i> limited its quick evolution. Herein, we propose a novel structure-guided combinatorial evolution strategy(SBPCS) to accelerate the iterative cycle of multipoint mutagenesis for C25 hydroxylation of VD₃ by <i>Aae</i>UPO. Through mutational screening of key residues within the substrate-binding pocket, combined with molecular dynamics (MD) simulations, we observed shortened distance and a stabilized dihedral angle between the substrate's C25 atom and the heme Fe═O center. After two rounds of coevolution, a triple mutant (G195A/V244I/S272A, M3) was successfully obtained, demonstrating a 5.16-fold increase in C25-hydroxylation efficiency, with a increased the yield of 25(OH)VD₃ from 45.8 to 354.40 mg/L. Our study elucidate how the geometry of the heme-VD₃ complex effect catalytic efficiency during selective hydroxylation and guided a rational design for accelerating <i>Aae</i>UPO coevolution toward VD₃ C25-hydroxylation.