Repurposing conformational changes in ANL superfamily enzymes to rapidly generate biosensors for organic and amino acids.
Repurposing conformational changes in ANL superfamily enzymes to rapidly generate biosensors for organic and amino acids.
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DOI：
10.1038/s41467-023-42431-y
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发表时间：
2023-10-21
影响因子：
16.6
通讯作者：
Wang, Meng
中科院分区：
文献类型：
--
作者：
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
生物传感器是检测、实时成像和定量分子的强大工具，但快速构建不同的遗传编码生物传感器仍然具有挑战性。在这里，我们报告了一种方法，快速转换酶为基因编码的循环排列的荧光蛋白为基础的指标，以检测有机酸（GECFINDER）。ANL超家族酶在催化过程中经历铰链介导的配体偶联结构域运动。我们将一种循环排列的荧光蛋白引入酶铰链中，将配体诱导的构象变化转化为显着的荧光信号变化。我们获得了11个用于检测苯丙氨酸、谷氨酸等氨基酸的GECFINDER。GECFINDER-Phe 3和GECFINDER-Glu可以在体外高效准确地定量生物样品中的靶分子。这种方法简化了氨基酸定量，而不需要复杂的设备，有可能在低资源环境中作为临床应用的即时检测工具。我们还开发了一种基于GECFINDER的液滴微流控高通量筛选方法，用于获得高产工业菌株。我们的方法为利用酶作为生物传感器设计、创建和应用的未开发蓝图资源提供了基础。生物传感器具有广泛的潜在应用，但快速构建基因编码的生物传感器仍然具有挑战性。在这里，作者报告了一种方法，用于快速转换ANL超家族酶为有机酸的生物传感器，基于其结合后的构象变化。
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. Biosensors have a wide number of potential applications, but rapidly constructing genetically encoded biosensors remains challenging. Here, authors report a method for rapidly converting ANL superfamily enzymes into biosensors for organic acids, based on their conformational changes upon binding.