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

• DOI: 10.1021/acssynbio.2c00364
• Publication Date: 2022-12-06T19:37:24Z
• Authors: Sang-Min Shin; Ramesh K. Jha; Taraka Dale
• Keywords: Pseudomonas putida; Bioreporter

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