Uncovering shared enzymes and secondary routes across β-oxidation cycles in Mycolicibacterial phytosterol side-chain degradation
Mycolicibacterium strains are among the most effective biofactories for converting phytosterols into active pharmaceutical intermediates. Despite the main route resembling fatty acid β-oxidation, the specific enzymes and their precise roles are poorly defined. In this study, we performed a comprehensive gene knock-out analysis in the industrial 4-AD-producing strain, Mycolicibacterium neoaurum HGMS9 strain, a variant of M. neoaurum B-3805. Our results demonstrate that the three β-oxidation cycles responsible for phytosterol side-chain degradation share a suite of core enzymes. Specifically, seven enzymes, including the acyl-CoA dehydrogenase ChsE1/E2, ChsE4/E5, the hydratase, ChsH1/H2 and the aldolase Ltp2, function in both the second and third β-oxidation cycles to generate the final products, 4-AD. Importantly, we identified five potential secondary routes that divert metabolism to produce eight off-route C22, C23 and C24 intermediates. These compounds represent valuable precursors for the synthesizing advanced steroids like corticosteroids and bile acids. This study not only clarifies the enzymatic steps of phytosterol side-chain degradation but also establishes a metabolic blueprint for engineering high-yielding Mycolicibacterium strains.