Production of Hydroxylated Steroid Intermediates at 10-g Scale via the Original Sterol Modification Pathway in Mycolicibacterium neoaurum

The aerobic catabolism of steroids in bacteria is highly conserved, and the mechanism of steroid degradation in mycobacteria has been extensively studied. However, the branching modification pathways of steroids in mycobacteria remain a mystery, including the likely roles of cytochromes P450. In this study, we unraveled the CYP105S17 converting androst-4-ene-3,17-dione (AD) to 17β-hydroxy-4-androstene-3,16-dione (16-oxo-TS), which was subsequently reduced to 16α,17β-dihydroxy-androst-4-ene-3-one (16α-OH-TS) under reductive conditions in Mycolicibacterium neoaurum. By applying this modification pathway, the genetically modified strains overexpressing CYP105S17 were able to produce 16α-OH-TS at titers 13.0 g/L with a conversion rate of 91.9% (supplemented with 20 g/L phytosterols as the substrate) through a two-stage biotransformation process. This is the first instance of utilizing the native P450 of Mycobacterium to produce 16-hydroxylated steroid intermediates at the 10 g scale. This work provides invaluable perspectives and guidance to researchers seeking to understand the complexities of steroid metabolism in bacteria, and also highlights the great potential of Mycobacterium as a production platform for hydroxylated steroid intermediates or pharmaceuticals.