Ligand-Specific Biosensing for Aromatic Compounds and Neurochemicals in Engineered Probiotics

Microbial biosensors have diverse applications in metabolic engineering and medicine. Specific and accurate quantification of chemical concentrations allows for adaptive regulation of enzymatic pathways and temporally precise expression of diagnostic reporters. Although ideal biosensors should differentiate structurally similar ligands with distinct biological functions, such specific sensors are rarely found in nature and challenging to create.

Using E. coli Nissle 1917, a ‘generally regarded as safe’ microbe, we developed and characterized several biosensor systems that promiscuously recognize aromatic amino acids or neurochemicals. To improve the sensors’ selectivity and sensitivity, we combined rational protein engineering with directed evolution techniques, applicable to both transcription repressors and activators. The generalizable approach involved searching for optimal evolutionary starting points, identifying critical residues in ligand binding, and screening mutagenesis libraries. Our method also provided insights into the previously uncharacterized structures of transcription regulators and elucidated the corresponding specificity control strategies.

We successfully demonstrated the ligand-specific biosensors for phenylalanine, tyrosine, indole-3-acetic acid, phenylethylamine, tyramine, and tryptamine. Each of these structurally similar compounds serves as a distinct biomarker or regulator that influences intestinal functionality and human health. These results lay the groundwork for developing kinetically adaptive microbes for potential applications, ranging from monitoring food quality and detecting diseases to facilitating therapy administration.