Fluorescence-Based Screens for Engineering Enzymes Linked to Halogenated Tryptophan.

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.

Citation

Kevin B Reed, Simon d'Oelsnitz, Sierra M Brooks, Jordan Wells, Minye Zhao, Adit Trivedi, Selina Eshraghi, Hal S Alper. Fluorescence-Based Screens for Engineering Enzymes Linked to Halogenated Tryptophan. ACS synthetic biology. 2024 Apr 19;13(4):1373-1381

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PMID: 38533851

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