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

Authors

Junfeng Li, Meiling Xiao, Guizhen Liu, Pei Xu, Jianzhong Liu

Published in

Bioresource technology. Pages 136003. Oct 06, 2026. Epub Oct 06, 2026.

Abstract

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.

PMID:
42838348
Bibliographic data and abstract were imported from PubMed on 07 Oct 2026.

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