Engineering the Steroid Hydroxylating System from Cochliobolus lunatus in Mycolicibacterium smegmatis

14 alpha-hydroxylated steroids are starting materials for the synthesis of contraceptive and anti-inflammatory compounds in the steroid industry. A synthetic bacterial operon containing the cytochrome P450 CYP103168 and the reductase CPR64795 of the fungus Cochliobolus lunatus able to hydroxylate steroids has been engineered into a shuttle plasmid named pMVFAN. This plasmid was used to transform two mutants of Mycolicibacterium smegmatis named MS6039-5941 and MS6039 that accumulate 4-androstene-3,17-dione (AD), and 1,4-androstadiene-3,17-dione (ADD), respectively. The recombinant mutants MS6039-5941 (pMVFAN) and MS6039 (pMVFAN) were able to efficiently express the hydroxylating CYP system of C. lunatus and produced in high yields 14 alpha OH-AD and 14 alpha OH-ADD, respectively, directly from cholesterol and phytosterols in a single fermentation step. These results open a new avenue for producing at industrial scale these and other hydroxylated steroidal synthons by transforming with this synthetic operon other Mycolicibacterium strains currently used for the commercial production of steroidal synthons from phytosterols as feedstock.

Automated summary

A synthetic bacterial operon containing the cytochrome P450 CYP103168 and the reductase CPR64795 of the fungus Cochliobolus lunatus has been engineered into a shuttle plasmid, which was then used to transform Mycolicibacterium smegmatis mutants for efficient expression of the hydroxylating CYP system and production of hydroxylated steroids. This new approach allows for direct synthesis of hydroxylated steroidal products from cholesterol and phytosterols in a single fermentation step, offering a potential avenue for industrial-scale production of these compounds.