Genetic Code Expansion in Probiotics Enables the Secretion of Covalent Protein Drugs in Mice

Oral delivery of therapeutic proteins remains a formidable challenge. Although engineered microbes have emerged as promising platforms for localized drug synthesis in the gut, their functional capacity has been restricted to the 20 canonical amino acids, limiting the chemical diversity of biologic payloads. Here, we demonstrate that integrating genetic code expansion (GCE) into bacterial therapy overcomes this fundamental constraint. We engineered the probiotic <i>Escherichia coli</i> Nissle 1917 (EcN) to incorporate the noncanonical amino acid fluorosulfate-l-tyrosine (FSY), enabling <i>in situ</i> secretion of a site-specifically modified covalent anti-IL-23 nanobody exhibiting picomolar binding potency (5.9 pM). Oral administration of this engineered EcN strain, followed by FSY supplementation, significantly ameliorated colitis in a murine model. This approach thereby establishes a versatile and generalizable platform that substantially expands the functional scope and therapeutic potential of live biotherapeutics.