Multi-gene Regulation Based on Metabolites Analysis Reduces Nucleus Degradation and By-product Accumulation During Phytosterols Conversion to 9-Hydroxyandrost-4-ene-3,17-Dione.

Mycobacterium neoaurum HK86W is capable of converting phytosterols into the steroidal pharmaceutical intermediate 9-hydroxyandrost-4-ene-3,17-dione (9-OH-AD). In this study, systematic biotransformation product profiling during sterol transformation revealed that degradation of the steroidal nucleus and accumulation of multiple by-products are key factors limiting the industrial application of this process. Knockout of two newly identified 3-ketosteroid-Δ¹-dehydrogenase genes (kstD4 and kstD5) effectively reduced degradation of the steroid nucleus caused by Δ¹-dehydrogenation. Meanwhile, the mechanisms underlying the generation of various major by-products were clearly elucidated. Through genetic modifications involving deletion of salA and overexpression of kshA, hsd4A, and hsd4B, a mutant strain HK86-V was successfully constructed to reduce by-product formation. In a 50 g/L phytosterol transformation system, the engineered strain significantly increased 9-OH-AD production from 21.97 g/L to 29.47 g/L, with a molar yield of 80.34%. Furthermore, by reducing the reaction rate and supplementing glucose as an auxiliary carbon source, the yield of 9-OH-AD was further enhanced to 32.14 g/L and the molar yield increased to 87.62% under optimized transformation conditions.