Multilevel Optimization of 3-Ketosteroid-9α-Hydroxylase for Enhanced 9α-Hydroxy-4-androstene-3,17-dione Production

9α-Hydroxylation represents a critical modification step in steroid pharmaceutical synthesis, where 9α-hydroxy-4-androstene-3,17-dione (9α-OH-AD) serves as an important intermediate for producing high-potency steroids, such as dexamethasone. The biosynthesis of 9α-OH-AD is catalyzed by a 3-ketosteroid-9α-hydroxylase (KSH) system comprising Rieske oxygenase (KshA) and ferredoxin reductase (KshB). In this study, we cloned the <i>kshA</i> and <i>kshB</i> genes from <i>Mycobacterium smegmatis</i> mc²155 and optimized their heterologous expression in <i>E. coli</i>, enhancing 9α-OH-AD yield by 4.9-fold. We implemented a 17β-carbonyl reductase (17β-CR)-mediated NADH regeneration system to ensure sufficient cofactor supply, which increased 9α-OH-AD production by 5.8-fold, while computer-aided design yielded the optimal KshA mutant A83V-G186L-A249W with a remarkable 13.8-fold improvement in catalytic efficiency compared to the wild-type. Through biotransformation optimization, the engineered microbial cell factory demonstrated a 17.2 times enhancement over the starting strain at the flask level, with a fed-batch strategy. This enzymatic conversion strategy establishes an eco-efficient platform for the sustainable synthesis of steroid therapeutics.