Enhancing the bioconversion of phytosterols to 22-hydroxy-23,24- bisnorchol-4-ene-3-one in Mycobacterium neoaurum ZS-15 through genetic modification of kstD1 and wecA

C22 steroidal intermediates, including 22‑hydroxy‑23,24‑bisnorchol‑4‑ene‑3‑one (4‑HBC) and its Δ1‑dehydrogenated derivative 1,4‑HBC serve as key precursors for synthesizing progestational and adrenocortical hormones. However, the accumulation of multiple products coupled with the inherently poor cell permeability remains a critical challenge for producing high-purity and high-yield 4-HBC in Mycobacterium neoaurum, the most valuable producer for C22 steroidal intermediates. M. neoaurum ZS-15 is a promising cell factory accumulating high yield HBCs (90.67% 1,4-HBC, 8.11% 4-HBC) and minimal C19 by-products through transforming phytosterols (20 g/L). In order to enhance the industrial potential of ZS-15, we first identified the key 3-ketosteroid-Δ1-dehydrogenase (KstD) which is responsible for catalyzing 4-HBC to produce 1,4-HBC. Then, it was knocked out by a modified gene editing system constructed in this study. Subsequently, we achieved the aim of improving cell permeability and promoting the product titer through the strategy of inhibiting a cell wall synthesis-related gene. In this work, KstD1 was predicted (by molecular docking) and confirmed (via CRISPR/dCas9-mediated knockdown) as the key isoenzyme for converting 4-HBC to 1,4-HBC in ZS-15. A modified CRISPR/Cas12a gene editing system (pRH2502-FnCpf1cg and pCR-Hyg-tetO) tailored for ZS-15 was developed through promoter substitution and plasmid backbone fusion, and a kstD1 knockout strain (M-ΔkstD1), which increased 4-HBC proportion in the product to 96.00% (at 20 g/L phytosterols), was constructed. The knockdown of three cell wall synthesis-related genes revealed that inhibiting the expression of N-acetylglucosamine-1-phosphate transferase (M-iwecA) led to the largest increase in 1,4-HBC titer relative to ZS-15, despite causing slight reductions in growth rate and biomass. Consequently, the highest 4-HBC titer at flask scale (28.80 g/L from 40 g/L phytosterols) was achieved by strain M-ΔkstD1-iwecA via combinatorial engineering of kstD1 knockout and wecA knockdown, attaining a 95% molar yield and 93.12% purity. Our study demonstrates that inhibition of wecA significantly enhances cell permeability, thereby improving phytosterol utilization efficiency in ZS-15, and knockout of kstD1 results in the main product converting to 4-HBC from 1,4-HBC. These findings provide a robust foundation for developing high-yield 4-HBC production systems and offer novel metabolic engineering strategies for steroid intermediate biosynthesis.