Pregnenolone Bioproduction in Engineered Methylobacteria: Design and Elaboration

Area of Science:

• Biotechnology
• Synthetic Biology
• Microbial Engineering

Background:

• Steroid production is crucial for pharmaceuticals and hormones.
• Mammalian steroidogenesis involves complex enzyme systems like cytochrome P450scc (CYP11A1).
• Developing efficient microbial hosts for heterologous steroidogenesis is an ongoing challenge.

Purpose of the Study:

• To express the mammalian steroidogenesis system, including CYP11A1 and its redox partners, in *Methylorubrum extorquens*.
• To utilize *M. extorquens* as a microbial chassis for producing pregnenolone from cholesterol.
• To optimize bioconversion conditions for enhanced pregnenolone yield.

Main Methods:

• Gene cloning and expression of mammalian steroidogenesis components (CYP11A1, adrenodoxin, adrenodoxin reductase) in *Methylorubrum extorquens*.
• Bioconversion of cholesterol to pregnenolone using recombinant *M. extorquens*.
• Optimization of media composition, substrate feeding, and fermentation parameters.

Main Results:

• Successful expression of functional mammalian steroidogenesis genes in *M. extorquens*.
• Production of pregnenolone as the sole metabolite from cholesterol.
• Achieved pregnenolone yield exceeding 100 mg/L under optimized conditions.

Conclusions:

• *Methylorubrum extorquens* serves as a viable microbial chassis for heterologous steroidogenesis due to its lack of endogenous steroid catabolism.
• This study provides a proof of concept for engineering bacteria for the biosynthesis of valuable steroids.
• The developed system offers a cost-effective approach for producing pregnenolone using inexpensive feedstocks.