A High-Throughput Cell-Free Enzyme Screening System Using Redox-Responsive Hydrogel Beads as Artificial Compartments

Area of Science:

• Biotechnology
• Biochemistry
• Materials Science

Background:

• Developing efficient screening systems for enzyme discovery is crucial for biotechnology.
• Hydrogel beads offer potential as micro-compartments for high-throughput screening.
• Cell-free protein synthesis and fluorescence-activated droplet sorting (FADS) are powerful tools for enzyme engineering.

Purpose of the Study:

• To develop a rapid, simple, and high-throughput screening system for recombinant enzymes using disulfide-bonded hydrogel beads (HBs).
• To validate the use of HBs for cell-free protein synthesis, enzymatic staining, and genetic recovery via FADS.
• To identify novel mutations for engineering active enzymes, specifically microbial transglutaminase (MTGz).

Main Methods:

• Production of redox-responsive hydrogel beads (HBs) using a microfluidic method.
• Cell-free protein synthesis of enzyme mutants within HBs.
• Enzymatic reaction-based fluorescent staining of HBs.
• Fluorescence-activated droplet sorting (FADS) for genetic information recovery.
• Next-generation sequencing (NGS) to identify mutation sites.

Main Results:

• Validated the expression of microbial transglutaminase zymogen (MTGz) and cross-linking-based fluorescent staining within HBs.
• Identified novel mutation sites (N25 and N27) in the propeptide domain of MTGz through NGS analysis of sorted HBs.
• Engineered an active MTGz by introducing the identified mutations.

Conclusions:

• Hydrogel beads serve as effective artificial compartments for FADS-based enzyme selection.
• The developed system enables the discovery and engineering of enzymes for peptide and protein cross-linking.
• This approach holds significant potential for advancing enzyme engineering and biocatalysis.