Tackling the Catch-22 Situation of Optimizing a Sensor and a Transporter System in a Whole-Cell Microbial Biosensor Design for an Anthropogenic Small Molecule.

Whole-cell biosensors provide a convenient detection tool for the high-throughput screening of genetically engineered biocatalytic activity. But establishing a biosensor for an anthropogenic molecule requires both a custom transporter and a transcription factor. This results in an unavoidable "Catch-22" situation in which transporter activity cannot be easily confirmed without a biosensor and a biosensor cannot be established without a functional transporter in a host organism. We overcame this type of circular problem while developing an adipic acid (ADA) sensor. First, leveraging an established cis,cis-muconic acid (ccMA) sensor, an annotated ccMA transporter MucK, which is expected to be broadly responsive to dicarboxylates, was stably expressed in the genome of Pseudomonas putida to function as a transporter for ADA, and then a PcaR transcription factor (endogenous to the strain and naturally induced by ß-ketoadipic acid, BKA) was diversified and selected to detect the ADA molecule. While MucK expression is otherwise very unstable in P. putida under strong promoter expression, our optimized mucK expression was functional for over 70 generations without loss of function, and we selected an ADA sensor that showed a specificity switch of over 35-fold from BKA at low concentrations (typically <0.1 mM of inducers). Our ADA and BKA biosensors show high sensitivity (low detection concentration <10 µM) and dynamic range (∼50-fold) in an industrially relevant organism and will open new avenues for high throughput discovery and optimization of enzymes and metabolic pathways for the biomanufacture of these molecules. In particular, the novel ADA sensor will aid the discovery and evolution of efficient biocatalysts for the biological recycling of ADA from the degradation of nylon-6,6 waste.