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 Escherichia coli, 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 Ideonella sakaiensis-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.