Engineered Probiotics for Closed-Loop Control of Disease-Associated Gut Metabolites in Gut-On-Chip Models

PROJECT SUMMARY Engineered commensal microbes represent a promising platform for controlling microbial metabolism in the gut microbiota for therapeutic outcomes. While strains have been successfully engineered to either reduce the concentration of a toxic metabolite or produce a therapeutic one, strains capable of controlling the level of a

metabolite within a narrow window have not been developed. Such ‘smart probiotics’, able to dynamically respond to the environment and either produce or consume a compound based on the local concentration, would be particularly useful for stabilizing metabolites which play a concentration-dependent role in host health and

disease. For example, ulcerative colitis and Crohn’s disease have been linked to microbially produced hydrogen sulfide (H2S), with a growing consensus that low levels of this molecule have anti-inflammatory properties and support a healthy epithelium, whereas high concentrations of H2S are genotoxic, inhibit mitochondrial function

and butyrate oxidation, and potentially weaken the mucosal barrier. Given that H2S concentration varies spatially and temporally throughout the mucosa, controlling H2S within a tight range is not possible with current small- molecule sulfide donors, which release sulfide regardless of local concentration. We propose a new synthetic

biology-based approach to controlling microbial metabolites in situ, in which the engineered microbe uses a transcription factor responsive to the metabolite of interest to dynamically balance the expression of metabolic pathways for production and consumption of the metabolite. This will produce a stable, titratable concentration

in a manner analogous to a thermostat. In this proposal, we will demonstrate this technology by developing engineered strains of E. coli Nissle to dynamically control the level of H2S in situ, incorporating mathematical modeling and a human organ-chip platform into the design-built-test cycle to achieve robust and stable operation

in the complex gut environment. If successful, the proposed research will establish the design rules for a novel synthetic biology control strategy applicable to many gut metabolites with concentration-dependent roles in disease, identify and mitigate host factors that impact engineered strain performance, and facilitate greater

translatability of synthetic probiotics.