Semi-rational design and modification of CciUPO for the efficient biosynthesis of 25-hydroxyvitamin D₃

25-Hydroxyvitamin D₃ (25-OH-VD₃) is the main active form of Vitamin D₃ and has broad applications in clinical treatment and agriculture. Although current industrial production predominantly relies on chemical synthesis, the growing demand for green and efficient manufacturing has accelerated the development of microbial enzymatic conversion methods. Due to its high reaction specificity, the unspecific peroxygenase from Coprinopsis cinerea (CciUPO) represents a promising biocatalyst for the synthesis of 25-OH-VD₃. However, its low catalytic efficiency limits further application in the biosynthesis of 25-OH-VD₃. To address this, semi-rational design was employed to modify the substrate-binding pocket and non-conserved residues of CciUPO. The resulting triple mutant I73M/P108K/G245A increased the 25-OH-VD₃ concentration to (87.83 ± 3.47) mg/L, representing a 41.18% increase over the wild type [(62.21 ± 3.02) mg/L]. The mechanism for enhanced catalytic efficiency was elucidated through analysis of the substrate-binding pocket, enzyme-substrate interactions, and molecular dynamics simulations. Subsequently, the fermentation conditions and multi-enzyme cascade reaction were optimized. Under optimized conditions with a substrate VD₃ concentration of 0.5 g/L, the 25-OH-VD₃ concentration further increased to (152.50 ± 1.95) mg/L. The combination of semi-rational engineering and process optimization of CciUPO offers a feasible, green and efficient strategy for the biosynthesis of 25-OH-VD₃.