RT Journal Article
SR Electronic
T1 Evolving a generalist biosensor for bicyclic monoterpenes
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2021.08.20.457167
DO 10.1101/2021.08.20.457167
A1 Simon d’Oelsnitz
A1 Vylan Nguyen
A1 Hal S. Alper
A1 Andrew D. Ellington
YR 2021
UL http://biorxiv.org/content/early/2021/08/21/2021.08.20.457167.abstract
AB 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.Competing Interest StatementA.D.E has equity in GRO Biosciences, a company developing protein therapeutics. The other authors declare no conflict of interest.