Structure-Guided Engineering of KshA Yields a High-Performance 9α-Hydroxylase for Synthesis of 9α–OH-AD

9α-hydroxy-4-androstene-3,17-dione (9α–OH-AD) is a crucial steroid pharmaceutical intermediate synthesized from androst-4-ene-3,17-dione (4-AD) via catalysis by 9α-hydroxylase (KSH), which comprises KshA and KshB subunits. KshB supplies electrons to activate the [2Fe–2S] center in KshA, enabling 4-AD hydroxylation. However, KSH’s stability and activity limit industrial 9α–OH-AD production. This study identified KshA as a key bottleneck and revealed four mutation hotspots through structure-guided mutagenesis. Ultimately, a triple mutant KshA L263A/G321N/D325 K with superior performance was obtained. This variant KSH exhibited a 10.3-fold increase in activity and a 5.04-fold improvement in k cat / K m in comparison to the wild-type. Molecular simulations indicated enhanced structural stability and substrate accessibility. The engineered strain Escherichia coli BL21-pET28a + -KshA L263A/G321N/D325 K /pETDuet-1-KshB-FDH achieved 119.8 mM 9α–OH-AD from 4-AD in fed-batch transformation, demonstrating a high-performance KSH variant for efficient industrial production.