Ultrahigh-throughput screening to identify E. coli cells expressing functionally active enzymes on their surface.

Area of Science:

• Biotechnology
• Enzymology
• Microbiology

Background:

• Enzyme engineering and directed evolution rely on high-throughput screening methods.
• Developing efficient methods for labeling and isolating specific bacterial cells is crucial for these processes.

Purpose of the Study:

• To develop a novel method for covalently labeling bacteria displaying specific enzyme activities.
• To enable the isolation of esterase-proficient bacteria using a biotinylation strategy.

Main Methods:

• Engineered E. coli cells co-displaying lipases/esterases and horseradish peroxidase (HRP).
• Enzymatic hydrolysis of biotin tyramide by surface-displayed enzymes.
• Covalent attachment of biotin labels to bacterial cell surfaces via HRP-mediated radical reaction.
• Isolation of labeled cells using magnetic cell sorting.

Main Results:

• Demonstrated successful covalent attachment of up to 120,000 biotinylated tyramide derivatives per E. coli cell.
• Showed a correlation between cellular esterase activity and the extent of biotinylation.
• Achieved selective isolation of lipase-active bacteria from a 1:10^6 mixture using magnetic cell sorting.

Conclusions:

• The developed strategy enables efficient and selective biotinylation of esterase-proficient bacteria.
• This method offers a powerful tool for ultrahigh-throughput screening of enzyme libraries for hydrolytic enzymes.
• Potential applications include identifying enzymes with improved activity or enantioselectivity.