Toward a generalized and high-throughput enzyme screening system based on artificial genetic circuits

Area of Science:

• Biotechnology
• Synthetic Biology
• Enzyme Engineering

Background:

• Large-scale enzyme screening is crucial for developing cost-effective biological processes for sustainable biobased chemical production.
• Existing methods for enzyme discovery from metagenomic libraries can be limited in throughput and sensitivity.

Purpose of the Study:

• To introduce a novel genetic circuit, the Genetic Enzyme Screening System (GESS), for high-throughput enzyme screening.
• To enable sensitive and quantitative discovery of novel enzymes from diverse microbial metagenomes.

Main Methods:

• Designed a genetic circuit with two AND logic gates to link enzyme activity to reporter gene expression.
• The first AND logic detects the target enzyme and substrate, producing a phenol compound.
• The second AND logic detects the phenol compound and an inducible transcription factor to activate reporter gene expression, quantifiable by flow cytometry.

Main Results:

• The developed genetic circuit demonstrated approximately 100-fold higher cellular fluorescence compared to negative controls.
• The system allows for sensitive quantification of phenolic derivatives using flow cytometry.
• Successfully screened various enzymes including cellulase, lipase, and alkaline phosphatase, demonstrating broad applicability.

Conclusions:

• The Genetic Enzyme Screening System is a highly sensitive and quantitative tool for discovering and engineering novel enzymes at the single-cell level.
• This approach facilitates rapid identification of enzymes with marginal activities from metagenomic libraries.
• The system offers a widely applicable toolkit for advancing sustainable biobased chemical production.