| Autor: |
Reed KB; McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E. Dean Keeton Street Stop C0400, Austin, Texas 78712, United States., d'Oelsnitz S; Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, Texas 78712, United States., Brooks SM, Wells J; McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E. Dean Keeton Street Stop C0400, Austin, Texas 78712, United States., Zhao M; McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E. Dean Keeton Street Stop C0400, Austin, Texas 78712, United States., Trivedi A; McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E. Dean Keeton Street Stop C0400, Austin, Texas 78712, United States., Eshraghi S; McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E. Dean Keeton Street Stop C0400, Austin, Texas 78712, United States., Alper HS; McKetta Department of Chemical Engineering, The University of Texas at Austin, 200 E. Dean Keeton Street Stop C0400, Austin, Texas 78712, United States.; Institute for Cellular and Molecular Biology, The University of Texas at Austin, 2500 Speedway Avenue, Austin, Texas 78712, United States. |
| Abstrakt: |
Directed evolution is often limited by the throughput of accurate screening methods. Here we demonstrate the feasibility of utilizing a singular transcription factor (TF)-system that can be refactored in two ways (both as an activator and repressor). Specifically, we showcase the use of previously evolved 5-halo- or 6-halo-tryptophan-specific TF biosensors suitable for the detection of a halogenated tryptophan molecule in vivo . We subsequently validate the biosensor's utility for two halogenase-specific halo-tryptophan accumulation screens. First, we isolated 5-tryptophan-halogenase, XsHal, from a mixed pool of halogenases with 100% efficiency. Thereafter, we generated a targeted library of the catalytic residue of 6-tryptophan halogenase, Th-Hal, and isolated functioning halogenases with 100% efficiency. Lastly, we refactor the TF circuit to respond to the depletion of halogenated tryptophan and prototype a high-throughput biosensor-directed evolution scheme to screen for downstream enzyme variants capable of promiscuously converting halogenated tryptophan. Altogether, this work takes a significant step toward the rapid and higher throughput screening of halogenases and halo-tryptophan converting enzymes to further reinforce efforts to enable high-level bioproduction of halogenated chemicals. |
