Engineered 3-Ketosteroid 9α-Hydroxylases in Mycobacterium neoaurum: an Efficient Platform for Production of Steroid Drugs

3-Ketosteroid 9α-hydroxylase (Ksh) consists of a terminal oxygenase (KshA) and a ferredoxin reductase and is indispensable in the cleavage of steroid nucleus in microorganisms. The activities of Kshs are crucial factors in determining the yield and distribution of products in the biotechnological transformation of sterols in industrial applications. In this study, two KshA homologues, KshA1_N and KshA2_N, were characterized and further engineered in a sterol-digesting strain, Mycobacterium neoaurum ATCC 25795, to construct androstenone-producing strains. kshA1_N is a member of the gene cluster encoding sterol catabolism enzymes, and its transcription exhibited a 4.7-fold increase under cholesterol induction. Furthermore, null mutation of kshA1_N led to the stable accumulation of androst-4-ene-3,17-dione (AD) and androst-1,4-diene-3,17-dione (ADD). We determined kshA2_N to be a redundant form of kshA1_N Through a combined modification of kshA1_N, kshA2_N, and other key genes involved in the metabolism of sterols, we constructed a high-yield ADD-producing strain that could produce 9.36 g liter^-1 ADD from the transformation of 20 g liter^-1 phytosterols in 168 h. Moreover, we improved a previously established 9α-hydroxy-AD-producing strain via the overexpression of a mutant KshA1_N that had enhanced Ksh activity. Genetic engineering allowed the new strain to produce 11.7 g liter^-1 9α-hydroxy-4-androstene-3,17-dione (9-OHAD) from the transformation of 20.0 g liter^-1 phytosterol in 120 h.IMPORTANCE Steroidal drugs are widely used for anti-inflammation, anti-tumor action, endocrine regulation, and fertility management, among other uses. The two main starting materials for the industrial synthesis of steroid drugs are phytosterol and diosgenin. The phytosterol processing is carried out by microbial transformation, which is thought to be superior to the diosgenin processing by chemical conversions, given its simple and environmentally friendly process. However, diosgenin has long been used as the primary starting material instead of phytosterol. This is in response to challenges in developing efficient microbial strains for industrial phytosterol transformation, which stem from complex metabolic processes that feature many currently unclear details. In this study, we identified two oxygenase homologues of 3-ketosteroid-9α-hydroxylase, KshA1_N and KshA2_N, in M. neoaurum and demonstrated their crucial role in determining the yield and variety of products from phytosterol transformation. This work has practical value in developing industrial strains for phytosterol biotransformation.