Rapid construction of metabolite biosensors using domain-insertion profiling

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Single-fluorescent protein biosensors (SFPBs) offer single-cell analyte quantification in vivo.
• Challenges in rational design, particularly green fluorescent protein (GFP) insertion for allosteric coupling, limit SFPB development.
• Existing methods for SFPB construction are often slow and labor-intensive.

Purpose of the Study:

• To introduce an unbiased, high-throughput method for constructing SFPBs.
• To overcome the rate-limiting step of rational design in SFPB development.
• To demonstrate the utility of the new method for creating metabolite biosensors.

Main Methods:

• Domain-insertion profiling with DNA sequencing (DIP-seq) was developed.
• DIP-seq combines rapid library creation of potential SFPBs with high-throughput activity assays.
• A proof-of-concept SFPB for the regulatory sugar trehalose was constructed using DIP-seq.

Main Results:

• DIP-seq identified allosteric hotspots for GFP insertion in a trehalose-binding protein.
• High-dynamic range SFPBs for trehalose were successfully generated.
• The developed biosensors demonstrated robust functionality in vivo.

Conclusions:

• DIP-seq significantly accelerates the construction of metabolite biosensors.
• This method provides a novel tool for investigating protein allostery.
• DIP-seq enables efficient discovery of functional SFPBs for various analytes.