Antibiotics-Free Steady Bioproduction of Valuable Chemicals from Organic Wastes by Engineered <i>Vibrio natriegens</i> through Targeted Gene Integration

Bioproduction of chemicals by using engineered bacteria is promising for a circular economy but challenged the instability of the introduced plasmid by conventional methods. Here, we developed a two-plasmid INTEGRET system to reliably integrate the targeted gene into the <i>Vibrio natriegens</i> genome, making it a powerful strain for efficient and steady bioproduction without requiring antibiotic addition. The INTEGRET system allows for gene insertion at over 75% inserting efficiency and flexibly controllable gene dosages. Additionally, simultaneous gene insertion at four genomic sites was achieved at 54.3% success rate while maintaining stable inheritance of exogenous sequences across multiple generations. The engineered strain could efficiently synthesize PHB from the fermentation of diverse organic wastes, with an efficiency comparable to those with overexpressed plasmid. When the mixture of seawater and molasses was used as the feedstock, it achieved a high PHB yield of 39.41 wt %. An extended application of the INTEGRET system for imparting the riboflavin production ability to the bacterium was also demonstrated. Our work presents a reliable and efficient genomic editing tool to facilitate the development of sustainable and environmentally benign biological platforms for converting biomass wastes into valuable chemicals.