Dual‐Key Genetic Circuit Enables Stable and Self‐Regulated Engineered Bacteria for the Treatment of Ulcerative Colitis

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.