Engineering a terephthalate-responsive PobR biosensor for discovering PET-degrading microbes via cell-to-cell communication

Abstract

Abstract Background Poly(ethylene terephthalate) (PET) is one of the most widely used synthetic plastics, yet its environmental accumulation continues to outpace effective recycling. Although PET-degrading enzymes and microorganisms have been reported, discovery of new PET-degrading biological resources remains slow because conventional screening depends on labor-intensive cultivation and chromatographic analysis. A sensitive whole-cell biosensor that reports terephthalic acid (TPA), a key PET hydrolysis product, could substantially accelerate the identification of PET-degrading microbes from environmental samples. Results Here we engineered a TPA-responsive whole-cell biosensor by evolving the Acinetobacter baylyi PobR transcription factor (TF), which naturally responds to 4-hydroxybenzoate but not TPA. A biosensor composed of PobR, its cognate promoter region, GFP, and the evolved A. baylyi MucK transporter did not respond to TPA in its wild type form. We therefore performed directed evolution of PobR using site-saturation mutagenesis at ten ligand-recognition positions and fluorescence-activated cell sorting. The final variant, designated PobR_v10, contained the substitutions S118T, ΔL141, V143Y, M148A, T159I, S160T, S212A, H216F, L220M, and M241L, and detected TPA over a dynamic range of 50–500 µM with a detection limit of 50 µM. To convert this biosensor into a colony-screening platform, we coupled the TPA-responsive circuit to the Bacillus subtilis racE gene in a D-glutamate auxotrophic Escherichia coli host, enabling inducer-dependent growth and fluorescence through cell-to-cell communication on solid medium. The resulting sensor selectively visualized PET hydrolysis around colonies expressing leaf-branch compost cutinase and enabled rapid screening of environmental samples on BHET-containing agar. Five candidate isolates were recovered and validated by HPLC-based assays. Among them, the isolate identified as Rhodococcus qingshengii showed the strongest activity toward PET, BHET, and MHET and exhibited robust growth with PET as the sole added carbon source. Unlike previously reported TphR-based systems, the engineered PobR platform was directly integrated into a growth-coupled spatial screening architecture compatible with colony-level PET hydrolysis detection. Conclusions This study establishes an engineered PobR-based TPA biosensor and demonstrates its application as a rapid cell-to-cell communication platform for discovering PET-degrading microbes on solid media. The system expands the repertoire of TPA-responsive TF biosensors and provides a practical engineering tool for environmental bioprospecting, enzyme discovery, and future PET upcycling workflows.