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 kshA and kshB genes from Mycobacterium smegmatis mc2155 and optimized their heterologous expression in E. coli, 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.