Pregnenolone Bioproduction in Engineered Methylobacteria: Design and Elaboration

In this study, for the first time, the genes encoding the mammalian steroidogenesis system-cytochrome P450scc (CYP11A1), and its native redox partners adrenodoxin and adrenodoxin reductase-were successfully expressed in the methylobacterium <i>Methylorubrum extorquens</i>. The advantage of using methylobacteria as an expression chassis is that they grow on inexpensive mineral media, use methanol as a carbon and energy source, and do not possess their own sterol catabolism systems. Using recombinant methylobacteria, the valuable steroid pregnenolone was obtained as a sole metabolite from cholesterol. The effect of media composition, bioconversion conditions such as methanol and N-sources content, modes of substrate addition, detergents, methyl-β-cyclodextrin, biomass, and aeration on pregnenolone accumulation was investigated. Under optimized conditions, its yield exceeded 100 mg/L. The results demonstrate a proof of concept relating to the use of bacteria lacking their own steroid degradation systems as microbial chassis for heterologous steroidogenesis systems, including mammalian cytochrome CYP11A1.