Fluorescence-Based Screens for Engineering Enzymes Linked to Halogenated Tryptophan

Area of Science:

• Biotechnology and Synthetic Biology
• Enzyme Engineering and Directed Evolution
• Metabolic Engineering and Bioproduction

Background:

• Directed evolution is crucial for enzyme discovery but often hindered by low-throughput screening methods.
• Transcription factor (TF) biosensors offer a potential solution for high-throughput screening by linking enzyme activity to reporter gene expression.
• Developing versatile TF biosensors that can function as both activators and repressors is key to expanding their applicability.

Purpose of the Study:

• To demonstrate the feasibility of a refactorable transcription factor (TF) biosensor system for directed evolution.
• To validate the utility of specific TF biosensors for high-throughput screening of halogenase enzymes.
• To establish a biosensor-directed evolution scheme for identifying enzymes involved in halogenated chemical production.

Main Methods:

• Engineered 5-halo- or 6-halo-tryptophan-specific TF biosensors for *in vivo* detection of halogenated tryptophan.
• Validation of biosensors in two distinct halogenase-specific halo-tryptophan accumulation screens.
• Refactoring the TF circuit to respond to substrate depletion for screening downstream enzyme variants.

Main Results:

• Successfully isolated the 5-tryptophan-halogenase, XsHal, from a mixed pool with 100% efficiency using the TF biosensor.
• Generated a targeted library of the 6-tryptophan halogenase, Th-Hal, and isolated functional variants with 100% efficiency.
• Prototyped a high-throughput biosensor-directed evolution scheme for screening promiscuous halogenated tryptophan-converting enzymes.

Conclusions:

• The developed TF biosensor system significantly enhances the throughput and accuracy of screening halogenases and related enzymes.
• This work represents a substantial advancement towards the rapid bioproduction of valuable halogenated chemicals.
• The refactorable TF system provides a powerful platform for future enzyme discovery and engineering efforts in biocatalysis.