Graduation Semester and Year
Fall 2025
Language
English
Document Type
Thesis
Degree Name
Master of Science in Biomedical Engineering
Department
Bioengineering
First Advisor
Justyn Jaworski
Second Advisor
Kytai Nguyen
Third Advisor
Georgios Alexandrakis
Fourth Advisor
Mark Pellegrino
Abstract
Tryptophan (Trp) is the essential amino acid precursor to the vital neurotransmitter serotonin. Live bacterial therapeutics engineered to produce the serotonin precursor 5-hydroxytryptophan (5-HTP) in the gut represent a promising strategy to modulate serotonin levels and may be generalized to enhance or modify related biochemical pathways of interest. A key metabolic hurdle is that native Escherichia coli possess an enzyme, tryptophanase A (TnaA), which diverts Trp away from 5-HTP synthesis to produce indole, which is a signaling molecule implicated in bacterial biofilm formation. Th following research examines the impact of inhibiting TnaA as a dual-action strategy to simultaneously enhance 5-HTP metabolic flux and control biofilm development. First, the role of indole in E. coli was characterized. A genetic knockout of the tnaA gene resulted in a nearly 60% reduction in biofilm biomass. Pharmacological inhibition of TnaA using N-acetyl-tryptophan (NAT) similarly reduced both indole production and biofilm formation. Furthermore, NAT was found to have a novel secondary function, which was attenuating biofilm enhancement caused by reactive oxygen species (ROS). This strategy was applied to an engineered probiotic E. coli Nissle 1917 strain harboring the enzymatic machinery for 5-HTP synthesis. Through additional recombineering, a safer probiotic form of E. coli Nissle was produced that is able to make 5-HTP as the precursor of serotonin. In the course of this work, a new fluorescence-based strategy for 5-HTP detection and quantification was identified, which enabled us to easily and sensitively measure 5-HTP. In conclusion, targeting the TnaA enzyme, either genetically or with an inhibitor like NAT, is a highly effective strategy for modulating activity and biofilms formation for live bacterial therapeutics. This approach successfully "plugs" a metabolic leak within the bacteria to improve therapeutic yield. Because this simultaneously reduces biofilm formation, it can enhance the safety of engineered probiotics.
Keywords
Keywords: Biofilm; Escherichia coli; indole signaling; N-acetyl tryptophan; reactive oxygen species; tryptophanase
Disciplines
Biological Engineering | Molecular, Cellular, and Tissue Engineering
License
This work is licensed under a Creative Commons Attribution 4.0 International License.
Recommended Citation
Monsef, Mohammad Amin, "INDOLE PATHWAY MODULATION FOR IMPROVED 5-HTP PRODUCTION AND BIOFILM CONTROL" (2025). Bioengineering Theses. 228.
https://mavmatrix.uta.edu/bioengineering_theses/228
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