# Pregnenolone Bioproduction in Engineered Methylobacteria: Design and Elaboration

## TL;DR

Researchers engineered a type of bacteria to produce the steroid pregnenolone using a mammalian enzyme system, achieving high yields in a cost-effective way.

## Contribution

This is the first demonstration of using methylobacteria to express mammalian steroidogenesis enzymes for pregnenolone production.

## Key findings

- Pregnenolone was successfully produced as the sole metabolite from cholesterol using recombinant methylobacteria.
- Optimized conditions achieved pregnenolone yields exceeding 100 mg/L.
- Methylobacteria lacking sterol catabolism systems can serve as a chassis for heterologous steroidogenesis.

## Abstract

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 Methylorubrum extorquens. 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.