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

• Published in: Nature communications Vol. 14; no. 1; p. 6680
• Main Authors: Wang, Jin, Xue, Ning, Pan, Wenjia, Tu, Ran, Li, Shixin, Zhang, Yue, Mao, Yufeng, Liu, Ye, Cheng, Haijiao, Guo, Yanmei, Yuan, Wei, Ni, Xiaomeng, Wang, Meng
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group 21.10.2023; Nature Publishing Group UK; Nature Portfolio
• Subjects: Amino acids; Biological properties; Biological samples; Biosensors; Catalysis; Coding; Conversion; Coupling (molecular); Enzymes; Fluorescence; Genetic code; Glutamic acid; High-throughput screening; In vitro methods and tests; Industrial strains; Ligands; Microfluidics; Organic acids; Phenylalanine; Proteins
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-023-42431-y

Abstract Biosensors 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.