1 Department of Biology, Centre for Applied Synthetic Biology, and Centre for Structural and Functional Genomics, Concordia University, Montréal, Quebec H4B 1R6, Canada.
2 PROTEO, Quebec Network for Research on Protein Function, Structure, and Engineering, Québec City, Quebec G1 V 0A6, Canada.
3 Department of Chemistry and Biochemistry, Concordia University, Montréal, Quebec H4B 1R6, Canada.

Allosteric transcription factor (aTF) biosensors are valuable tools for engineering microbes toward a multitude of applications in metabolic engineering, biotechnology, and synthetic biology. One of the challenges toward constructing functional and diverse biosensors in engineered microbes is the limited toolbox of identified and characterized aTFs. To overcome this, extensive bioprospecting of aTFs from sequencing databases, as well as aTF ligand-specificity engineering are essential in order to realize their full potential as biosensors for novel applications. In this work, using the TetR-family repressor CmeR from Campylobacter jejuni, we construct aTF genetic circuits that function as salicylate biosensors in the model organisms Escherichia coli and Saccharomyces cerevisiae. In addition to salicylate, we demonstrate the responsiveness of CmeR-regulated promoters to multiple aromatic and indole inducers. This relaxed ligand specificity of CmeR makes it a useful tool for detecting molecules in many metabolic engineering applications, as well as a good target for directed evolution to engineer proteins that are able to detect new and diverse chemistries.