Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes

Abstract

BackgroundTranscription factor-based biosensors are useful tools for the detection of metabolites and industrially valuable molecules, and present many potential applications in biotechnology and biomedicine. However, the most common approach to develop biosensors relies on employing a limited set of naturally occurring allosteric transcription factors (aTFs). Therefore, altering the ligand specificity of aTFs towards the detection of new effectors is an important goal.ResultsHere, the PcaV repressor, a member of the MarR aTF family, was used to develop a biosensor for the detection of hydroxyl-substituted benzoic acids, including protocatechuic acid (PCA). The PCA biosensor was further subjected to directed evolution to alter its ligand specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Ligand recognition of Van2 was explored in vitro using a range of biochemical and biophysical analyses, and extensive in vivo genetic-phenotypic analysis was performed to determine the role of each amino acid change upon biosensor performance.ConclusionsThis is the first study to report directed evolution of a member of the MarR aTF family, and demonstrates the plasticity of the PCA biosensor by altering its ligand specificity to generate a biosensor for aromatic aldehydes.

Transcription Factor Based Small‐Molecule Sensing with a Rapid Cell Phone Enabled Fluorescent Bead Assay

“…Even if only a small portion of these are unique allosteric TFs, this study indicates that there are numerous potential sensing proteins to explore. Moreover, for targeted identification of TFs for specific analytes, TFs can be identified through genomic and experimental screens, [22, 43] mutated or evolved to change ligand specificity or sensitivity, [41, 44, 45] or engineered from known binding domains [46–48] . This combination of native and engineered specificity yields countless TF‐based sensing opportunities.…”

Transcription Factor Based Small‐Molecule Sensing with a Rapid Cell Phone Enabled Fluorescent Bead Assay

“…[42] Even if only asmall portion of these are unique allosteric TFs,this study indicates that there are numerous potential sensing proteins to explore. Moreover,f or targeted identification of TFs for specific analytes,T Fs can be identified through genomic and experimental screens, [22,43] mutated or evolved to change ligand specificity or sensitivity, [41,44,45] or engineered from known binding domains. [46][47][48] This combination of native and engineered specificity yields countless TF-based sensing opportunities.…”

Optimizing Calcium Detection Methods in Animal Systems: A Sandbox for Synthetic Biology

“…Just as GECIs and synthetic dyes have undergone their own optimization, cell-based biosensor optimization via synthetic biology needs to similarly apply rational methods. More strategies that can expand the range of de novo allosteric transcription factors for Ca 2+ detection will be vital to biosensor optimization [208]. In particular, the use of directed evolution to change specificity patterns can enhance Ca 2+ detection by introducing intracellular synthetic circuits not limited to natural sources of Ca 2+ -sensing machinery.…”