d'Oelsnitz Simon, Nguyen Vylan, Alper Hal S, Ellington Andrew D
Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, United States.
Freshman Research Initiative, University of Texas at Austin, Austin, Texas 78712, United States.
ACS Synth Biol. 2022 Jan 21;11(1):265-272. doi: 10.1021/acssynbio.1c00402. Epub 2022 Jan 5.
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 . 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.
原核转录因子可被重新用作精确化学测量和调控的分析及合成工具。单萜类化合物包含一个广泛的化学家族，作为香料、化妆品和香水具有商业价值，但已证明难以测量，尤其是在细胞中。在此，我们通过定向进化来扩展樟脑响应性TetR家族调节因子CamR的效应物特异性，从而开发出基因编码的通用单萜生物传感器。利用一种新型负筛选与高通量正筛选相结合的方法（配体诱导传感器无缝富集，SELIS），我们进化出了能够识别四种不同单萜类化合物的CamR生物传感器：冰片、葑醇、桉叶油素和莰烯。令人惊讶的是，不同的进化轨迹产生了共同的突变，这强调了CamR作为创建通用生物传感器平台的实用性。驱动报告基因的系统启动子优化将系统的信噪比提高到了150倍。这些传感器可作为双环单萜生产菌株高通量筛选和动态调控的起点。
