Abstract
Engineered bacteria offer a promising therapeutic platform but often display plasmid instability and antibiotic dependence. A synthetic dual-key genetic circuit is established in Escherichia coli Nissle 1917 (EcN) by deleting both asd and thyA genes, generating an auxotrophic chassis that requires dual-plasmid complementation for survival. The "lysis module" restores asd function and incorporates a quorum-sensing system for self-regulated lysis and controlled protein release ("One Key"). By contrast, the "expression module" complements thyA and co-delivers an interleukin-2 (IL-2) mutant and the membrane protein Amuc_1100 to modulate immune balance and repair the intestinal barrier ("Dual Keys"). This dual-key design enabled antibiotic-free plasmid stability, precise population control, and sustained therapeutic protein secretion. Oral administration of the engineered strain significantly alleviated colitis in mice by enhancing regulatory T-cell expansion, restoring epithelial integrity, and reshaping the gut microbiota. This modular system provides a safe, stable, and programmable strategy for live bacterial therapy against immune and mucosal diseases.