Disentangling Metabolite Detection from Pathway Stress Using Biosensors

Genetically encoded biosensors are powerful tools for linking metabolite production to fluorescent outputs, but stresses imposed by engineered pathways can confound their signals. In this study, we developed four fluorescence-based biosensors for inhibitors of protein tyrosine phosphatase 1B (PTP1B), a therapeutic target for cancer and diabetes, and compared their performance in the presence of heterologous terpenoid pathways, a promising source of new inhibitors. When expressed in <i>Escherichia coli</i>, all four could reliably detect an exogenously supplied PTP1B inhibitor, but only one remained functional alongside terpenoid pathways, where pathway-derived stress reduced overall fluorescence. This sensor, which links PTP1B inhibition to the assembly of a split T7 RNA polymerase, exhibited a high dynamic range with minimal toxicity, a common liability of T7-based circuits. Variants of this sensor with different sensitivities to inhibition and stress allowed us to separate the terpenoid pathways that made PTP1B inhibitors from those that did not. Our findings show how pathway-specific stresses (e.g., terpene synthase expression) can alter biosensor signals as strongly as their target analytes and provide a framework for disentangling these confounding effects in high-throughput screens.