Zhai, Xinghui; Yuan, Chenyang; Sun, Xiaoxuan; Sun, Wen; He, Beiru; Zhang, Ruijie; Zhang, Baoguo

PROCESS BIOCHEMISTRY

2025, ,

Zhai, Xinghui; Yuan, Chenyang; Sun, Xiaoxuan; Sun, Wen; He, Beiru; Zhang, Ruijie; Zhang, Baoguo

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 (3a alpha-H-4 alpha-(3'-Propionic acid)-5 alpha-hydroxy-7a(3-methylhexahydro-1-indanone-O-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 alpha-methyl group instead of a (3-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.