C22 steroidal intermediates, including 22‑hydroxy‑23,24‑bisnorchol‑4‑ene‑3‑one (4‑HBC) and its Δ 1 ‑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-Δ 1 -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-Δ kstD 1), 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-i wecA ) 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-Δ kstD1 -i wecA 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.
Guo et al. (Tue,) studied this question.