PT  - JOURNAL ARTICLE
AU  - Leopoldo F. M. Machado
AU  - Andrew Currin
AU  - Neil Dixon
TI  - Directed evolution of the PcaV allosteric transcription factor to generate a biosensor for aromatic aldehydes
AID  - 10.1101/689232
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 689232
4099  - http://biorxiv.org/content/early/2019/07/09/689232.short
4100  - http://biorxiv.org/content/early/2019/07/09/689232.full
AB  - Background Genetically encoded 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 transcript factors (aTFs). Therefore, altering the substrate specificity of aTFs towards the detection of new effectors is an important goal.Results Here, 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 substrate specificity towards vanillin and other closely related aromatic aldehydes, to generate the Van2 biosensor. Substrate 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.Conclusions This 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 substrate specificity to generate a biosensor for aromatic aldehydes.aTFallosteric transcription factorPCAprotocatechuic acidEBDeffector binding domainDBDDNA binding domainRNAPRNA polymeraseDMSOdimethyl sulfoxideRFUrelative fluorescence unitOE-PCRoverlap-extension polymerase chain reactionDEdirected evolutionFACSfluorescence activated cell sortingMTBEmethyl-tert-butyl etherEMSAelectrophoretic mobility shift assaySEC-MALSsize-exclusion chromatography coupled to multi-angle light scatteringSPRSurface Plasmon Resonance