Engineering Vibrio natriegens for biopesticide production: from pathway optimization to plant protection.

Phytopathogens pose a persistent threat to global crop production, necessitating the development of sustainable biocontrol strategies. Microbial biopesticides offer a promising alternative to chemical pesticides; however, their industrial application is frequently constrained by low production titers and high substrate costs. This study reprograms the phenazine-1-carboxylic acid (PCA) biosynthetic pathway in Vibrio natriegens, a fast-growing marine bacterium with broad carbon utilization capability. A modular plasmid toolbox is constructed through systematic optimization of promoters, ribosome binding sites, replication origins, and antibiotic resistance markers, enabling high-level PCA production. To address plasmid instability and antibiotic dependence, an I-SceI-based genome editing platform is established, facilitating scarless, multi-copy chromosomal integration of the PCA gene cluster. The final engineered strain, Vna6, achieves PCA titers of 102.0 mg/L in shake flasks and 579.8 mg/L in fed-batch fermentation with glucose as the carbon source. Furthermore, Vna6 exhibits broad-spectrum antagonistic activity against both bacterial (Streptomyces scabies) and fungal (Glomerella cingulata) phytopathogens, as confirmed by in vitro inhibition zone assays and in vivo plant protection experiments on Arabidopsis thaliana and radish seedlings. This work establishes V. natriegens as a potenial chassis for biopesticide production and offers a promising strategy for sustainable crop disease management.