Abstract
14α-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 14αOH-AD and 14αOH-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.
Highlights
Steroids represent one of the most widely used compounds as pharmaceuticals such as anti-inflammatory, immunosuppressive, anti-allergic, and anti-cancer drugs [1].The oxidation state of the steroid nucleus rings and the presence of different attached functional groups determine the biological functions of steroid molecule [2]
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
Some fungi have been reported to carry out hydroxylations at almost all stereogenic centers of the steroid molecule [5] and they have been widely applied for the industrial production of steroids [6]
Summary
Steroids represent one of the most widely used compounds as pharmaceuticals such as anti-inflammatory, immunosuppressive, anti-allergic, and anti-cancer drugs [1]. The heterologous co-expression of hydroxylating CYPs and their natural redox partners as putative industrial biocatalyzers has been addressed in different host such as yeasts (e.g., Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe), and bacteria (e.g., Escherichia coli, Bacillus subtilis, Bacillus megaterium, Corynebacterium glutamicum) [10,14,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31] All these bioconversion processes are based in whole-cell biocatalysts and require previously purified steroid synthons as substrates. We show that a synthetic operon containing the CYP103168 and its natural partner CPR64795 of C. lunatus can be engineered in M. smegmatis resulting in the production of 14α-hydroxylated synthons directly from sterols in a single fermentation step
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