A Versatile Transcription Factor Biosensor System Responsive to Multiple Aromatic and Indole Inducers

Abstract

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.

Keywords: CmeR; aTF; aromatics; biosensor; genetic circuits; indoles; salicylic acid.

Figure 1

Construction and inducer profile of an E. coli salicylate biosensor. (A) One-plasmid genetic circuit structure. The IPTG-inducible tac promoter drives CmeR expression, followed by a second engineered salicylate-inducible promoter to drive GFP expression, which contains one copy of the cmeO operator sequence inserted immediately downstream of the −10 sequence. (B) Left panel: Fluorescence measurements of cells carrying the CmeR genetic circuit in the on and off states without and with 1 mM IPTG added, respectively. Right panel: Fluorescence measurements of cells in the presence of 1 mM IPTG and increasing concentrations of salicylate. (C) Fluorescence measurements of E. coli cultures carrying the CmeR genetic circuit exposed to 1 mM of one of 22 molecules in the presence of 1 mM of IPTG. (D) Dose response curves for 11 candidate inducer molecules in the presence of 1 mM IPTG. All data represents plate reader measurements of GFP fluorescence of E. coli cultures performed in triplicate.

Figure 2

Construction and inducer profile of a S. cerevisiae salicylate biosensor. (A) Chromosomally integrated genetic circuit design in S. cerevisiae. CmeR is expressed from the strong constitutive promoter P_TDH3. P_CCW12 is used for expressing Envy GFP in the control strain, which was subsequently engineered in the sensor strain for salicylate responsiveness by inserting two copies of cmeO downstream of the TATA box. (B) Left panel: Fluorescence measurements of cells carrying the control and sensor circuit. Right panel: Fluorescence measurements of the sensor strain in the presence of increasing concentrations of salicylate. (C) Fluorescence measurements of the sensor strain exposed to 1 mM of one of 22 molecules. (D) Dose response curves for 11 candidate inducer molecules. All data represents flow cytometry measurements of GFP fluorescence of S. cerevisiae cultures performed in triplicate.