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

25-Hydroxyvitamin D ₃ (25-OH-VD ₃ ) is the main active form of Vitamin D ₃ 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 ( Cci UPO) represents a promising biocatalyst for the synthesis of 25-OH-VD ₃ . However, its low catalytic efficiency limits further application in the biosynthesis of 25-OH-VD ₃ . To address this, semi-rational design was employed to modify the substrate-binding pocket and non-conserved residues of Cci UPO. The resulting triple mutant I73M/P108K/G245A increased the 25-OH-VD ₃ concentration to 87.83 mg/L, representing a 41.18% increase over the wild type (62.21 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 VD ₃ concentration of 0.5 g/L, the 25-OH-VD ₃ concentration further increased to 152.50 mg/L. The combination of semi-rational engineering and process optimization of Cci UPO offers a feasible, green and efficient strategy for the biosynthesis of 25-OH-VD ₃ .