Dual-Enzyme Cofactor Recycling Drives Efficient 9α-OH-AD Biosynthesis

9α-Hydroxyandrost-4-ene-3,17-dione (9α-OH-AD) is a valuable intermediate for the manufacture of steroid drugs such as hydrocortisone. Its bioproduction from 4-androstene-3,17-dione (4-AD), however, is limited by the low efficiency of the two-component steroid 9α-hydroxylase (KSH) system and insufficient cofactor regeneration during whole-cell catalysis. In this work, RvKshA and RvKshB were mined and heterologously expressed, and plasmid screening identified E. coli BL21/pET28a + -RvKshA/pETDuet-1-RvKshB as the best-performing strain. Structural analyses based on molecular docking and molecular dynamics simulations clarified the cooperative catalytic mechanism of KshA and KshB, providing guidance for rational system construction. More importantly, to address the NADH supply bottleneck, we established a dual-enzyme coupling strategy by introducing formate dehydrogenase (FDH) for in situ cofactor regeneration. This design enabled efficient steroid hydroxylation, giving a 98.3% conversion at 20 mM 4-AD. When combined with high-density fermentation and fed-batch feeding, the engineered strain achieved a substrate processing capacity of 80 mM and a spatiotemporal yield of 42.8 mM/(L·day). This study provides an integrated enzymatic and bioprocess solution for improving whole-cell steroid hydroxylation efficiency and offers a practical basis for the scalable biosynthesis of 9α-OH-AD.