# Engineering of plant-derived P450scc for de novo biosynthesis of pregnenolone

## TL;DR

This study engineered plant enzymes to efficiently produce pregnenolone, a steroid precursor, in yeast, achieving a major breakthrough in scalable biosynthesis.

## Contribution

The study introduces a novel enzyme engineering strategy for plant-derived P450scc to enable high-efficiency pregnenolone biosynthesis in yeast.

## Key findings

- Structure-guided engineering of DlCYP87A improved catalytic efficiency in microbial systems.
- A 1.46 g/L pregnenolone titer was achieved in a 5-liter fermentation system.
- This is the first gram-scale de novo biosynthesis of pregnenolone using engineered yeast.

## Abstract

In contrast to the extensively researched animal CYP11A1 system, the catalytic mechanism of sterol side-chain cleavage by plant-derived cytochrome P450scc enzymes remains poorly understood. Through the integration of computational structural biology and enzyme channel engineering, this study successfully elucidated the key intermediates in the stepwise hydroxylation-cleavage catalytic process of Digitalis purpurea-derived DlCYP87A enzyme. Building on this foundation, we implemented structure-guided rational design to precisely engineer the substrate channel and catalytic pocket, systematically delineating their structure-activity relationships, which ultimately overcame the critical catalytic bottleneck of low conversion efficiency in heterologous microbial systems expressing plant-derived P450scc. This study established an efficient steroid synthesis system in Saccharomyces cerevisiae through integrated systematic enzyme engineering and transcriptome-guided organelle optimization. In a 5-liter fermentation system, engineered strain P4 achieved a pregnenolone titer of 1.46 g/L. This achievement represents the first gram-scale breakthrough in de novo pregnenolone biosynthesis, laying a crucial technological foundation for scalable bio-manufacturing of steroid precursors and pioneering a new industrial production pathway.