Metabolic engineering of Mycobacterium fortuitum for high-level production of sitolactone from biorenewable phytosterols

Recent advances in steroid drug synthesis have increasingly focused on leveraging biosynthetic intermediates derived from phytosterols, an economical and abundant raw material for microbial metabolism. Sitolactone (3aα-H-4α-(3’-Propionic acid)-5α-hydroxy-7aβ-methylhexahydro-1-indanone-Δ-lactone, or HIL) serves as a key precursor for the synthesis of steroid drugs, particularly those either devoid of a methyl group or featuring an α-methyl group instead of a β-methyl group at the C-10 position. In preliminary investigations, we achieved HIL production with a purity of only 63.9 % through the inactivation of the CAR1, CAR2, and FadE30 genes in Mycobacterium fortuitum ATCC 6842. To address the accumulation of by-products, we subsequently identified and inactivated three isoenzymes of FadE30 (FadE31, FadE32, FadE33), leading to an HIL purity exceeded 99 %. The optimized strain demonstrated the ability to convert 10 g/L of phytosterols into 4.84 g/L of HIL within 72 hours, achieving a molar yield of 90.2 %—the highest reported yield to date. Bioconversion experiments using phytosterol concentrations at 50 g/L yielded a maximum space-time productivity of 3.66 g/L/d with an 81.87 % molar yield, marks the highest yield recorded. These findings provide a cost-effective and efficient method for producing 19-norsteroid intermediates in pharmaceutical manufacturing while contributing valuable insights into steroid nucleus metabolism.