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Evolving a Generalist Biosensor for Bicyclic Monoterpenes

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    Research Article

    Evolving a Generalist Biosensor for Bicyclic Monoterpenes
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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2022, 11, 1, 265–272
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    https://doi.org/10.1021/acssynbio.1c00402
    Published January 5, 2022
    Copyright © 2022 American Chemical Society
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    Abstract

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    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.

    Copyright © 2022 American Chemical Society

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acssynbio.1c00402.

    • Response measurements of transcription factor homologues and evolved variants to several monoterpenes; Circuit designs, key sensor performance metrics, and the sequences of promoters, sensor mutants, and whole plasmids used in this study, provided in tabular format; Analyses of the ligand structures and the CamR homology model as well as metrics on its quality (PDF)

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    ACS Synthetic Biology

    Cite this: ACS Synth. Biol. 2022, 11, 1, 265–272
    Click to copy citationCitation copied!
    https://doi.org/10.1021/acssynbio.1c00402
    Published January 5, 2022
    Copyright © 2022 American Chemical Society
    Request reuse permissions

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