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Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System
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A fluorescent hydrogel-based flow cytometry high-throughput screening platform for hydrolytic enzymes.

Christian Pitzler1, Georgette Wirtz1, Ljubica Vojcic1

  • 1Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 3, 52074 Aachen, Germany.

Chemistry & Biology
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Summary

We developed a novel high-throughput screening platform using a fluorescent hydrogel to rapidly identify active enzymes. This method significantly enriches enzyme variants, accelerating enzyme discovery and directed evolution.

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Area of Science:

  • Biotechnology
  • Enzyme Engineering
  • Polymer Chemistry

Background:

  • High-throughput screening is crucial for enzyme discovery and directed evolution.
  • Existing methods can be laborious and time-consuming for large variant libraries.

Purpose of the Study:

  • To develop a rapid, high-throughput screening platform for enzyme discovery.
  • To enable efficient directed evolution of enzymes like phytase.
  • To create polymer-hybrid cells for biobased materials.

Main Methods:

  • A coupled enzymatic reaction generating hydroxyl radicals.
  • Initiation of fluorescent hydrogel polymerization around enzyme-expressing cells.
  • Flow cytometry for cell sorting and enrichment of active variants.
  • Directed evolution of Yersinia mollaretii phytase (YmPh).

Main Results:

  • Formation of a fluorescent polymer shell (fur-shell) around active YmPh-expressing E. coli cells.
  • Achieved five-fold enrichment of active cell populations.
  • Successfully evolved improved phytase variants from a library of 10^7 variants.
  • Demonstrated the fur-shell technology's effectiveness in directed evolution.

Conclusions:

  • The fur-shell technology offers a rapid and non-laborious method for identifying highly active enzyme variants.
  • This platform accelerates enzyme discovery and directed evolution processes.
  • The technology facilitates the generation of polymer-hybrid cells for novel biobased materials.