Prokaryotic transcription factors can be repurposed as analytical and synthetic tools for precise chemical measurement and regulation. Monoterpenes encompass a broad chemical family that are commercially valuable as flavors, cosmetics, and fragrances, but have proven difficult to measure, especially in cells. Herein, we develop genetically-encoded, generalist monoterpene biosensors by using directed evolution to expand the effector specificity of the camphor-responsive TetR-family regulator CamR from Pseudomonas putida. Using a novel negative selection coupled with a high-throughput positive screen (Seamless Enrichment of Ligand-Inducible Sensors, SELIS), we evolve CamR biosensors that can recognize four distinct monoterpenes: borneol, fenchol, eucalyptol, and camphene. Different evolutionary trajectories surprisingly yielded common mutations, emphasizing the utility of CamR as a platform for creating generalist biosensors. Systematic promoter optimization driving the reporter increased the system’s signal-to-noise ratio to 150-fold. These sensors can serve as a starting point for the high-throughput screening and dynamic regulation of bicyclic monoterpene production strains.

中文翻译：

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开发双环单萜的通用生物传感器

原核转录因子可以重新用作分析和合成工具，用于精确的化学测量和调节。单萜包括广泛的化学家族，它们作为香料、化妆品和香料具有商业价值，但已证明难以测量，尤其是在细胞中。在此，我们通过使用定向进化来扩展来自恶臭假单胞菌的樟脑反应性 TetR 家族调节剂 CamR 的效应子特异性，从而开发了基因编码的通用单萜生物传感器. 使用新的负选择加上高通量正筛选（配体诱导传感器的无缝富集，SELIS），我们开发了 CamR 生物传感器，可以识别四种不同的单萜：冰片、苯酚、桉叶油和莰烯。不同的进化轨迹出人意料地产生了共同的突变，强调了 CamR 作为创建通用生物传感器平台的实用性。驱动记者的系统启动子优化将系统的信噪比提高到 150 倍。这些传感器可以作为双环单萜生产菌株高通量筛选和动态调控的起点。