Enhancing production of 9α-hydroxy-androst-4-ene-3,17-dione (9-OHAD) from phytosterols by metabolic pathway engineering of mycobacteria

9α-hydroxy-androst-4-ene-3,17-dione (9-OHAD), as an important precursor for a large number of steroidal drugs, can be produced by the mycobacterial degradation of phytosterols’ feedstock. Phytosterols are wastes separated from the production of vegetable oil. Three catabolic pathways were involved in the microbial conversion of phytosterols to form 9-OHAD in Mycobacterium neoaurum MS136, namely sterol uptake, sterol side-chain degradation, and steroid nucleus cleavage and degradation. To enhance the production of 9-OHAD, we systematically studied the expression pattern of the genes during the conversion of phytosterols to 9-OHAD. The genes with significant up-regulation (e.g., hsd, cyp125, fadD19, hsd4A and ksh) were overexpressed to examine their effects on the 9-OHAD production. Thus, the recombinant Mycobacterium neoaurum MS136-F was constructed with the overexpression of these four key genes (e.g., hsd, hsd4A, kshA1 and kshB) and the knockout of the gene kstd that led to the degradation of 9-OHAD. Upon adding 13.0 g/L of commercial phytosterols feedstock (including 6.2 g/L β-sitosterol, 3.0 g/L campesterol, 3.3 g/L stigmasterol and 0.5 g/L others), 6.8 g/L 9-OHAD was obtained by this recombinant strain MS136-F after 108 h’ fermentation, which was 1.45-fold of that of the parental M. neoaurum MS136 (4.7 g/L, the molar conversion rate is 77.2%). In the phytosterols feedstock, β-sitosterol and campesterol were converted to 9-OHAD with a much high molar conversion ratio of 98.5%. However, stigmasterol (3.3 g/L) was hardly used by mycobacteria, only 0.3 g/L of which was converted 9-OHAD. This study has a direct implication for the industrial production of 9-OHAD.