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.

Divergent Directed Evolution of a TetR-type Repressor Towards Aromatic Molecules

“…Conversely, sensors for homovanillic acid and caffeic acid showed no activity in S. cerevisiae compared to E. coli. In particular, we have previously seen caffeic acid-responsive biosensors in E. coli that did not function in S. cerevisiae, further emphasizing these differences 24 .…”

“…aTFs have often evolved to respond to a limited set of inducers, which restricts their use in many applications 22 . The existing repertoire of aTFs could be expanded by continuous bioprospecting 23,24 , creating synthetic chimeras 25,26 , or by directed evolution to alter their ligand speci city 8 . Directed evolution is widely used to introduce bene cial properties to proteins, where rounds of genetic sequence diversi cation (either random or structure-guided) are followed by variant library screening or selection 27,28 .…”

Divergent Directed Evolution of a TetR-type Repressor Towards Aromatic Molecules

“…Most aTFs have evolved towards specific ligands, yet some exhibit promiscuity towards multiple small molecules, and are sometimes called "generalists" 24,[49][50][51] . Thus, we sought to determine if expression from P 2 could be induced by other molecules of interest.…”

“…aTFs have often evolved to respond to a limited set of inducers, which restricts their use in many applications 22 . The existing repertoire of aTFs could be expanded by continuous bioprospecting 23,24 , creating synthetic chimeras 25,26 , or by directed evolution to alter their ligand specificity 8 . Directed evolution is widely used to introduce beneficial properties to proteins, where rounds of genetic sequence diversification (either random or structure-guided) are followed by variant library screening or selection 27,28 .…”

An intelligent synthetic bacterium with sound-integrated ability for chronological toxicant detection, degradation, and lethality

“…The wild MarR-P marO sensor in E. coli required 24 h as a response time, which was six times longer than ours ( 27 ). Using CmeR in E. coli could only detect 0.1–1 mM salicylate in 20–24 h ( 28 ). There were many other ways to optimize the biosensor, such as promoter and RBS engineering, replication origin engineering, regulator protein engineering and cascaded amplifiers ( 25, 29–31 ).…”