# Multi-layered engineering of Aspergillus terreus enhances biosynthesis of the plant-derived fungicide physcion

## TL;DR

Researchers improved the production of the plant-derived fungicide physcion in a fungus by engineering its biosynthesis pathway and reducing byproduct formation.

## Contribution

Identification of a detoxification bottleneck and development of two improved fungal platforms for physcion and emodin production.

## Key findings

- Deleting CYP-H6231 increased physcion titer by 1.8-fold and improved product purity.
- Engineering the SAM pathway and enzyme fusion provided only modest improvements due to reduced strain robustness.
- Structural modeling identified key residues in CYP-H6231 for substrate recognition and catalysis.

## Abstract

Emodin and its derivatives are important bioactive anthraquinones from rhubarb, with diverse pharmacological activities. Physcion, an O-methylated derivative of emodin, is a promising plant-derived fungicide and pharmaceutical lead. However, plant extraction yields are low and land-intensive, while microbial production is hampered by inefficient conversion and byproduct accumulation.

Here, we identify a cytochrome P450 enzyme (CYP-H6231) that, with its dedicated redox partner cytochrome P450 reductase (CPR-H10273), converts emodin to ω-hydroxyemodin in Aspergillus terreus. Deletion of CYP-H6231 increased physcion titer by 1.8-fold and significantly improved product purity. Further engineering, via 3-O-methyltransferase overexpression, SAM pathway enhancement, and enzyme fusion, yielded only modest improvement (up to 37%), likely due to compromised strain robustness from the loss of CYP-H6231 mediated detoxification. Structural modeling and mutagenesis of CYP-H6231 revealed key residues for substrate recognition and catalysis.

This study reveals a detoxification bottleneck in anthraquinone biosynthesis and establishes two improved A. terreus platforms for scalable production of physcion and emodin, respectively, highlighting trade-offs between pathway efficiency and cellular fitness.

The online version contains supplementary material available at 10.1186/s12934-025-02857-z.