Combinatorial metabolic engineering of Mycobacterium neoaurum LY-2 for efficient androstenedione production from phytosterols.

Androstenedione (AD) is an important precursor for the synthesis of corticosteroids, sex hormones, and other high-value steroid pharmaceuticals. Microbial conversion of phytosterols provides a sustainable route for AD production, but current production levels remain insufficient for practical industrial application. In this study, a modular metabolic engineering strategy was developed to improve AD biosynthesis from phytosterols in Mycobacterium neoaurum LY-2. Competing C19 and C22 steroid pathways were first blocked to attenuate AD degradation, generating the AD-accumulating strain Mbn12 with a titer of 5.14 g/L. Sterol catabolic flux was then enhanced by relieving transcriptional repression and improving acetyl-CoA and propionyl-CoA utilization, which increased AD production to 6.08 g/L. To support high-flux sterol oxidation, cofactor regeneration and oxidative stress resistance were further strengthened, raising AD production to 6.67 g/L. Finally, moderate cell envelope remodeling improved substrate transport and increased AD production to 6.98 g/L. The final engineered strain Mbn30 produced 20.55 g/L AD from 30 g/L phytosterols in a 5 L fermenter, representing a 48.6% improvement over Mbn12. This work provides a scalable engineering framework for AD production and offers guidance for the microbial manufacture of other steroid intermediates.