Glutarate metabolism in Pseudomonas putida is regulated by two distinct glutarate sensing transcription factors

ABSTRACT Transcription factor based biosensors can be leveraged to screen thousands of genetics designs for optimal production in engineered microbes. In this study we characterize two glutarate sensing transcription factors (CsiR and GcdR) from Pseudomonas putida. The genomic contexts of CsiR homologs were analyzed and DNA binding sites were bioinformatically predicted. Both CsiR and GcdR were purified and shown to bind upstream of their coding sequencing in vitro. CsiR was shown to dissociate from DNA in vitro when exogenous glutarate was added confirming it acts as a genetic repressor. Both transcription factors were then engineered into plasmid based biosensors and their respective sensing performance features calculated. Both sensor plasmids were then reintroduced into P. putida and were evaluated for their ability to sense flux through glutarate when grown on various lysine metabolites as sole carbon sources. These findings provide information describing glutarate flux in P. putida, and potentially useful tools for future metabolic engineering and synthetic biology efforts.