Engineering a\nNovel Probiotic Toolkit in Escherichia coli <i>Nissle 1917</i> for\nSensing and Mitigating Gut Inflammatory Diseases

Inflammatory bowel\ndisease (IBD) is characterized by chronic intestinal\ninflammation with no cure and limited treatment options that often\nhave systemic side effects. In this study, we developed a target-specific\nsystem to potentially treat IBD by engineering the probiotic bacterium Escherichia coli <i>Nissle 1917</i> (EcN).\nOur modular system comprises three components: a transcription factor-based\nsensor (NorR) capable of detecting the inflammation biomarker nitric\noxide (NO), a type 1 hemolysin secretion system, and a therapeutic\ncargo consisting of a library of humanized anti-TNFα nanobodies.\nDespite a reduction in sensitivity, our system demonstrated a concentration-dependent\nresponse to NO, successfully secreting functional nanobodies with\nbinding affinities comparable to the commonly used drug Adalimumab,\nas confirmed by enzyme-linked immunosorbent assay and in vitro assays.\nThis newly validated nanobody library expands EcN therapeutic capabilities.\nThe adopted secretion system, also characterized for the first time\nin EcN, can be further adapted as a platform for screening and purifying\nproteins of interest. Additionally, we provided a mathematical framework\nto assess critical parameters in engineering probiotic systems, including\nthe production and diffusion of relevant molecules, bacterial colonization\nrates, and particle interactions. This integrated approach expands\nthe synthetic biology toolbox for EcN-based therapies, providing novel\nparts, circuits, and a model for tunable responses at inflammatory\nhotspots.