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bioRxiv · 10.1101/2021.08.20.457167

Evolving a generalist biosensor for bicyclic monoterpenes

d'Oelsnitz, S.·Nguyen, V.·Alper, H. S.·Ellington, A. D.
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Abstract

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 systems 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.

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d'Oelsnitz, S., Nguyen, V., Alper, H. S., Ellington, A. D.. 2021-08-21. Evolving a generalist biosensor for bicyclic monoterpenes. https://doi.org/10.1101/2021.08.20.457167

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