Engineering E. coli Nissle 1917 for production and outer membrane vesicle-mediated delivery of a computationally designed GLP-1R/GIPR/GCGR tri-agonist

Microbial cell factories provide a genetically programmable route for the production of therapeutic biomolecules, while bacterial outer membrane vesicles (OMVs) offer a biologically derived vehicle for biomolecular delivery. Integrating recombinant therapeutic peptide production with OMV biogenesis could therefore provide a unified strategy for peptide production and delivery. Here, we engineered Escherichia coli Nissle 1917 (EcN) to produce OMVs associated with GGG, a computationally designed peptide agonist targeting the glucagon-like peptide-1 receptor (GLP-1R), glucose-dependent insulinotropic polypeptide receptor (GIPR), and glucagon receptor (GCGR). The tri-agonist peptide (GGG) was identified from a constrained library of 983,040 candidate sequences through computational screening and structural evaluation against GLP-1R, GIPR, and GCGR. The selected peptide was subsequently integrated into an EcN ΔnlpI-based OMV production system using an EcN-compatible T7 expression architecture. The resulting vesicles exhibited characteristic nanoscale properties and were enriched with the GGG-containing fusion cargo. The cargo showed limited passive release in buffer, whereas GGG-containing species were progressively detected in the soluble fraction following incubation of intact OMV preparations in fresh whole blood. Importantly, the TEV-processed GGG–6×His product recovered from the OMV-associated preparation retained functional activity at GLP-1R, GIPR, and GCGR, with apparent EC50 values of 1.84, 1.50, and 5.08 nM, respectively. In high-fat-diet-fed mice, repeated OMV-GGG administration produced sustained reductions in body mass and adiposity together with improved glucose regulation, while maintaining lean and fat-free body compartments under the tested conditions. No overt histopathological abnormalities were observed during the 5-week treatment period. These findings establish a proof-of-concept microbial production and OMV-associated formulation framework that integrates genetically encoded production of a computationally designed multi-receptor peptide with vesicle-associated delivery and functional validation. The study demonstrates the feasibility of coupling genetically encoded peptide production with OMV-associated formulation and provides a basis for further development of OMV-based peptide delivery systems.