Fluorescence-Based Screens for Engineering Enzymes Linked to Halogenated Tryptophan

• Published in: ACS synthetic biology 2024-04, Vol.13 (4), p.1373-1381
• Main Authors: Reed, Kevin B., d’Oelsnitz, Simon, Brooks, Sierra M., Wells, Jordan, Zhao, Minye, Trivedi, Adit, Eshraghi, Selina, Alper, Hal S.
• Format: Article
• Language: English
• Subjects: Fluorescence; Tryptophan
• ISSN: 2161-5063; 2161-5063
• DOI: 10.1021/acssynbio.3c00616

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.