Antibiotics-Free\nSteady Bioproduction of Valuable\nChemicals from Organic Wastes by Engineered Vibrio\nnatriegens through Targeted Gene Integration

Bioproduction of chemicals by using engineered bacteria\nis promising\nfor a circular economy but challenged the instability of the introduced\nplasmid by conventional methods. Here, we developed a two-plasmid\nINTEGRET system to reliably integrate the targeted gene into the Vibrio natriegens genome, making it a powerful strain\nfor efficient and steady bioproduction without requiring antibiotic\naddition. The INTEGRET system allows for gene insertion at over 75%\ninserting efficiency and flexibly controllable gene dosages. Additionally,\nsimultaneous gene insertion at four genomic sites was achieved at\n54.3% success rate while maintaining stable inheritance of exogenous\nsequences across multiple generations. The engineered strain could\nefficiently synthesize PHB from the fermentation of diverse organic\nwastes, with an efficiency comparable to those with overexpressed\nplasmid. When the mixture of seawater and molasses was used as the\nfeedstock, it achieved a high PHB yield of 39.41 wt %. An extended\napplication of the INTEGRET system for imparting the riboflavin production\nability to the bacterium was also demonstrated. Our work presents\na reliable and efficient genomic editing tool to facilitate the development\nof sustainable and environmentally benign biological platforms for\nconverting biomass wastes into valuable chemicals.