# Metabolic reprogramming of abscisic acid-producing strain Botrytis cinerea TB-31 toward terpenoid biosynthesis using a CRISPR/Cas9 ribonucleoprotein system

## TL;DR

Researchers developed a CRISPR-based system in a fungus to enhance the production of valuable terpenoids by redirecting its metabolism.

## Contribution

A CRISPR/Cas9 system was established in Botrytis cinerea TB-31 for efficient gene editing and metabolic reprogramming.

## Key findings

- The △bcaba1234 strain showed significant metabolic flux rewiring toward terpenoid precursor biosynthesis.
- The CRISPR system enabled efficient gene knockout and heterologous protein expression in the fungal strain.
- The study provides a foundation for using B. cinerea TB-31 as a terpenoid-producing chassis.

## Abstract

Compared with conventional microbial hosts, filamentous fungi have distinct advantages for the industrial-scale biosynthesis of high-value chemical compounds. However, current research on strain engineering and fermentation optimization strategies for synthetic biology applications is limited in filamentous fungi, especially in industrial production strains. In this study, we established a CRISPR/Cas9-based gene editing system in Botrytis cinerea strain TB-31, an important filamentous fungal platform for the study of the biosynthesis and regulation of the sesquiterpenoid abscisic acid (ABA). This system enables efficient single- and multigene knockout, large-fragment deletion, and heterologous protein expression. Among the engineered mutant strains, the △bcaba1234 strain with complete ablation of the ABA biosynthetic gene cluster (BGC) demonstrated significant metabolic flux rewiring, redirecting cellular resources toward terpenoid precursor biosynthesis; this metabolic reprogramming proves pivotal for high-value terpenoid biosynthesis. This study not only establishes an efficient genome editing tool for the ABA-producing strain B. cinerea TB-31 but also provides a foundation for its development as a new potential terpenoid-producing chassis strain.