Development of Engineered Ferredoxin Reductase Systems for the Efficient Hydroxylation of Steroidal Substrates

9α-Hydroxy-4-androstene-3,17-dione (9OHAD), formed by the 9α-hydroxylation reaction catalyzed by 3-ketosteroid-9-hydroxylase (KSH), is an important precursor for the synthesis of adrenocortical hormones. KSH, a key enzyme complex in microbial steroid catabolism, contains a Rieske oxygenase (KshA) and a ferredoxin reductase (KshB). In this study, first we determined that the activity of KshB was rate-limiting in 9OHAD production. Thus, several potential alternative reductases were screened; a toluene 2,3-dioxygenase (TDO) reductase showed the highest activity toward NADH. In pathway optimization, the addition of a Rieske [2Fe–2S] cluster to KshB or TDO resulted in improved 9OHAD yields, which implies an improved efficiency of electron transfer. A sufficient supply system of NADH was ensured by introducing formate dehydrogenase (FDH) to construct an NADH regeneration system. The biosynthesis of 9OHAD was then optimized in a whole Escherichia coli cell catalysis system expressing FDH, KshA, and a variant of TDO reductase containing five point mutations and an added Rieske [2Fe–2S] cluster, which resulted in the final production of 5.24 g/L 9OHAD from 4-androstene-3,17-dione with 99.3% yield. This research provides detailed insight into the electron-transfer system for steroid hydroxylation reactions.