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Small-molecule sensing in plants is dominated by chemical-induced dimerization modules. In the abscisic acid (ABA) system, allosteric receptors recruit phosphatase effectors and achieve nM in vivo responses from µM receptor–ligand interactions. This sensitivity amplification could enable ABA receptors to serve as generic scaffolds for designing small-molecule sensors. To test this, we screened collections of mutant ABA-receptors against 2,726 drugs and other ligands and identified 553 sensors for 6.6% of these ligands. The mutational patterns indicate strong selection for ligand-specific binding pockets. We used these data to develop a sensor design pipeline and isolated sensors for multiple plant natural products, 2,4,6-trinitrotoluene (TNT), and “forever” per- and polyfluoroalkyl substances (PFAS). Thus, the ABA sensor system enables design and isolation of small-molecule sensors with broad chemical scope and antibody-like simplicity. 

Abstract Plants sense abscisic acid (ABA) using chemical-induced dimerization (CID) modules, including the receptor PYR1 and HAB1, a phosphatase inhibited by ligand-activated PYR1. This system is unique because of the relative ease with which ligand recognition can be reprogrammed. To expand the PYR1 system, we designed an orthogonal ‘*’ module, which harbors a dimer interface salt bridge; X-ray crystallographic, biochemical and in vivo analyses confirm its orthogonality. We used this module to create PYR1*^MANDI/HAB1* and PYR1*^AZIN/HAB1*, which possess nanomolar sensitivities to their activating ligands mandipropamid and azinphos-ethyl. Experiments inArabidopsis thalianaandSaccharomyces cerevisiaedemonstrate the sensitive detection of banned organophosphate contaminants using living biosensors and the construction of multi-input/output genetic circuits. Our new modules enable ligand-programmable multi-channel CID systems for plant and eukaryotic synthetic biology that can empower new plant-based and microbe-based sensing modalities. 

Abstract A general method to generate biosensors for user-defined molecules could provide detection tools for a wide range of biological applications. Here, we describe an approach for the rapid engineering of biosensors using PYR1 (Pyrabactin Resistance 1), a plant abscisic acid (ABA) receptor with a malleable ligand-binding pocket and a requirement for ligand-induced heterodimerization, which facilitates the construction of sense–response functions. We applied this platform to evolve 21 sensors with nanomolar to micromolar sensitivities for a range of small molecules, including structurally diverse natural and synthetic cannabinoids and several organophosphates. X-ray crystallography analysis revealed the mechanistic basis for new ligand recognition by an evolved cannabinoid receptor. We demonstrate that PYR1-derived receptors are readily ported to various ligand-responsive outputs, including enzyme-linked immunosorbent assay (ELISA)-like assays, luminescence by protein-fragment complementation and transcriptional circuits, all with picomolar to nanomolar sensitivity. PYR1 provides a scaffold for rapidly evolving new biosensors for diverse sense–response applications.