Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System.

Area of Science:

• Biotechnology
• Enzyme Engineering
• Metagenomics

Background:

• High-throughput single-cell assay techniques are crucial for discovering novel enzymes from metagenomic libraries.
• A previously proposed genetic enzyme screening system (GESS) utilizes a dimethylphenol regulator activated by phenolic compounds.
• GESS has theoretical applicability for screening over 200 enzymes due to natural enzymatic production of phenolic compounds.

Purpose of the Study:

• To detail an optimized screening process for maximizing flow cytometry signals when using GESS.
• To demonstrate the application of GESS and flow cytometry for identifying novel enzymes from metagenomic libraries.
• To validate the screening of specific enzymes including lipase, cellulase, and alkaline phosphatase.

Main Methods:

• Metagenome preprocessing using the genetic enzyme screening system (GESS).
• Operation of a flow cytometry sorter for high-throughput screening.
• Utilized three distinct phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-β-D-cellobioside, phenyl phosphate) with GESS.

Main Results:

• Successfully screened and identified three distinct enzymes: lipase, cellulase, and alkaline phosphatase.
• The screening process was optimized to achieve maximum flow cytometry signals.
• Confirmed enzyme activities using flow cytometry, with potential for further gene identification via sequencing and in vitro analysis.

Conclusions:

• The developed screening process, combining GESS and flow cytometry, is effective for identifying functional enzymes from metagenomic libraries.
• This optimized method enhances the efficiency and signal detection for enzyme screening.
• The approach provides a robust platform for discovering novel enzymes with specific activities.