Liu, Xiangcen; He, Beiru; Zhang, Jingxian; Yuan, Chenyang; Han, Suwan; Du, Guilin; Shi, Jiping; Sun, Junsong; Zhang, Baoguo

APPLIED MICROBIOLOGY AND BIOTECHNOLOGY

2023, 107, 1432-0614

Liu, Xiangcen; He, Beiru; Zhang, Jingxian; Yuan, Chenyang; Han, Suwan; Du, Guilin; Shi, Jiping; Sun, Junsong; Zhang, Baoguo

Androst-4-ene-3,17-dione (AD) and 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) are important drug intermediates that can be biosynthesized from phytosterols. However, the C9 hydroxylation of steroids via 3-ketosteroid 9 alpha-hydroxylase (KSH) limits AD and 4-HBC accumulation. Five active KshAs, the oxidation component of KSH, were identified in Mycobacterium fortuitum ATCC 35855 for the first time. The deletion of kshAs indicated that the five KshA genes were jointly responsible for C9 hydroxylation during phytosterol biotransformation. MFKD Delta kshA, the five KshAs deficient strain, blocked C9 hydroxylation and produced 5.37 g/L AD and 0.55 g/L 4-HBC. The dual function reductase Opccr knockout and 17 beta-hydroxysteroid dehydrogenase Hsd4A enhancement reduced 4-HBC content from 8.75 to 1.72% and increased AD content from 84.13 to 91.34%, with 8.24 g/L AD being accumulated from 15 g/L phytosterol. In contrast, hsd4A and thioesterase fadA5 knockout resulted in the accumulation of 5.36 g/L 4-HBC from 10 g/L phytosterol. We constructed efficient AD (MFKD Delta kshA Delta opccr_hsd4A) and 4-HBC (MFKD Delta kshA Delta hsd4A Delta fadA5) producers and provided insights for further metabolic engineering of the M. fortuitum ATCC 35855 strain for steroid productions.