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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
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A High-Throughput Cell-Free Enzyme Screening System Using Redox-Responsive Hydrogel Beads as Artificial Compartments.

Taisei Koga1, Yui Okawa1, Tomoyuki Ito2

  • 1Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan.

ACS Synthetic Biology
|February 13, 2025
PubMed
Summary

Researchers created a novel hydrogel bead system for high-throughput enzyme screening. This method successfully identified new mutations to engineer active microbial transglutaminase (MTGz) for protein cross-linking applications.

Keywords:
cell-free protein synthesisfluorescence-activated droplet sorterhigh-throughput screeninghydrogel beadsmicrobial transglutaminasenext-generation sequencing

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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.