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

Abstract

Reprogramming cellular behaviour is one of the hallmarks of synthetic biology. To this end, prokaryotic allosteric transcription factors (aTF) have been repurposed as versatile tools for processing small molecule signals into cellular responses. Expanding the toolbox of aTFs that recognize new inducer molecules is of considerable interest in many applications. Here, we first establish a resorcinol responsive aTF-based biosensor in Escherichia coli using the TetR-family repressor RolR from Corynebacterium glutamicum. We then perform an iterative walk along the fitness landscape of RolR to identify new inducer specificities, namely catechol, methyl catechol, caffeic acid, protocatechuate, L-DOPA, and the tumour biomarker homovanillic acid. Finally, we demonstrate the versatility of these engineered aTFs by transplanting them into the model eukaryote Saccharomyces cerevisiae. This work provides a framework for efficient aTF engineering to expand ligand specificity towards novel molecules on laboratory timescales, which, more broadly, is invaluable across a wide range of applications such as protein and metabolic engineering, as well as point-of-care diagnostics.

Molecular basis for the transcriptional regulation of an epoxide-based virulence circuit inPseudomonas aeruginosa

“…In addition, such studies will provide important insights into the mechanism by which epoxide binding triggers the release of operator DNA in CifR and will increase our molecular understanding of this therapeutically relevant family of bacterial transcription factors. Furthermore, prokaryotic transcription factors can be engineered to activate established cellular response pathways in response to new small-molecule signals (83, 84). Our characterization of the CifR epoxide-sensing circuit may expand this toolbox to include a new class of chemical scaffolds.…”

Functional and structural characterization of an IclR family transcription factor for the development of dicarboxylic acid biosensors

“…To demonstrate the versatility of PcaR as a biosensor beyond previous prokaryotic systems, we implemented PcaR in the model eukaryote S. cerevisiae. We incorporated the pcaR gene into a previously-developed S. cerevisiae chassis and tested the ability of this strain to react to molecules identified in our in vitro assay through induction of the expression of the green fluorescent protein (GFP) reporter gene [25,28]. Given the described activity of PcaR as a dual activator and repressor, we designed and constructed chromosomally integrated genetic circuits based on its repressor activity [4].…”