An engineered suicide vector-mediated genome editing of Mycobacterium sp. LY-1 for targeted 9α-hydroxyandrost-4-ene-3,17-dione biosynthesis

Mycolicibacterium is a crucial industrial host for steroid biotransformation, yet its genetic engineering has long been limited by inefficient homologous recombination (HR) and strong non-homologous end joining (NHEJ) activity. Here, we established and optimized a suicide plasmid-based genome editing system for Mycobacterium sp. LY-1. The constructed vector p2NILB, combining kanamycin selection with sacB-mediated counter-selection, enabled precise and markerless allelic exchange. Suppression of NHEJ through deletion of ku or ligD , together with optimization of homology arm length, increased gene knockout efficiency to over 70%. This optimized platform was subsequently applied to metabolic engineering of sterol degradation to enhance 9α-hydroxyandrostenedione (9α-OH-AD) biosynthesis. Sequential deletion of five kstD isoenzymes blocked downstream steroid degradation, with kstD1 identified as the major contributor to product loss. Functional analysis revealed KshA2 as the key 9α-hydroxylase, and its overexpression increased the 9α-OH-AD titer to 3.41 g/L. In addition, deletion of the branched-chain acid transporter gene mmpL3 further enhanced 9α-OH-AD production to 4.19 g/L. To overcome substrate utilization limitations under high phytosterol loading conditions, a soybean oil‑Tween 80 solvent system overcame substrate limitation, enabling 15.71 g/L 9α‑OH‑AD from 30 g/L phytosterols. This study provides a robust genome editing framework and an effective strategy for engineering steroid-producing actinobacteria.