A hierarchical systems metabolic engineering framework for modular construction of Mycolicibacterium neoaurumcell factories for efficient phytosterol bioconversion.

Androst-4-ene-3,17-dione (AD), Androst-1,4-diene-3,17-dione (ADD), and 9-hydroxy-4-androstene-3,17-dione (9-OHAD) are valuable C19 steroid intermediates derived from low-cost phytosterol bioconversionby Mycolicibacterium neoaurum. However, its slow growth rate, limited sterol uptake and complex metabolic networks restrict efficient and selective steroid biosynthesis. Here, we established a hierarchical systems metabolic engineering framework that sequentially integrates chassis evolution, metabolic flux rewiring, pathway optimization, cofactor regeneration, and transporter engineering for modular construction of steroid-producing cell factories. Atmospheric and room-temperature plasma (ARTP)-mediated mutagenesis generated a fast-growing chassis with higher biomass accumulation, while subsequent multi-level engineering significantly enhanced substrate accessibility and carbon-flux redistribution toward target products.Consequently,three product-specific microbial cell factories were constructed, producing 7.90 g/L AD, 8.30 g/L ADD, and 7.40 g/L 9-OHAD in 15 g/L phytosterols as substrate with molar conversion efficiencies of 76.53%, 80.29%, and 67.32%, respectively. The optimized Mn-AD04 strain accumulated 12.7 g/L of total C19 steroid intermediates, including AD, ADD, and 9-OHAD. In addition, the results indicated that both intracellular cofactor availability and sterol transport can constrain phytosterol bioconversion, while sterol transport represents a particularly effective target for further enhancing steroid production in the highly engineered strains. This work provides a generalizable systems metabolic engineering framework for rational construction of high-performance steroid-producing microorganisms and offers a scalable strategy for efficient microbial production of steroid intermediates.