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.
