Repurposing conformational changes in ANL superfamily enzymes to rapidly generate biosensors for organic and amino acids

Abstract

AbstractBiosensors are powerful tools for detecting, real-time imaging, and quantifying molecules, but rapidly constructing diverse genetically encoded biosensors remains challenging. Here, we report a method to rapidly convert enzymes into genetically encoded circularly permuted fluorescent protein-based indicators to detect organic acids (GECFINDER). ANL superfamily enzymes undergo hinge-mediated ligand-coupling domain movement during catalysis. We introduce a circularly permuted fluorescent protein into enzymes hinges, converting ligand-induced conformational changes into significant fluorescence signal changes. We obtain 11 GECFINDERs for detecting phenylalanine, glutamic acid and other acids. GECFINDER-Phe3 and GECFINDER-Glu can efficiently and accurately quantify target molecules in biological samples in vitro. This method simplifies amino acid quantification without requiring complex equipment, potentially serving as point-of-care testing tools for clinical applications in low-resource environments. We also develop a GECFINDER-enabled droplet-based microfluidic high-throughput screening method for obtaining high-yield industrial strains. Our method provides a foundation for using enzymes as untapped blueprint resources for biosensor design, creation, and application.