A High-Sensitivity Genetically Encoded Biosensor for Terephthalic Acid Detection in PET Degradation

The accumulation of polyethylene terephthalate (PET) waste poses a serious environmental challenge due to its durability and resistance to degradation. Enzymatic PET hydrolysis offers a sustainable solution, but efficient high-throughput screening tools for PET-degrading enzymes remain limited. Here, we report a genetically encoded biosensor (GEB) for terephthalic acid (TPA)─the primary monomer released during PET degradation─that enables rapid and sensitive detection of enzymatic activity. We engineered a TphR-based biosensor in <i>Escherichia coli</i>, combining an optimized transcriptional system with diverse TPA uptake transporters to enhance intracellular TPA accumulation. This dual strategy improved the signal intensity and broadened the detection range. The best-performing configuration, integrating a high-affinity transporter with fine-tuned genetic components, achieved a detection limit of 1 μM TPA─a 1,000-fold sensitivity improvement over the initial design. We validated the system using PETases, including <i>Ideonella sakaiensis</i>-derived FAST-PETase, and benchmarked it against HPLC assays. The biosensor reliably distinguished PETase variants based on hydrolytic activity, demonstrating its utility for directed evolution, metagenomic screening, and enzyme engineering. This work establishes a rapid, scalable, and ultrasensitive biosensor platform for monitoring PET hydrolysis. The engineered GEB offers a robust, low-cost alternative to conventional analytics, accelerating the discovery and optimization of PET-degrading enzymes for plastic upcycling and circular bioeconomy applications.