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Combinatorial InVitroFlow-assisted mutagenesis (CombIMut) yields a 41-fold improved CelA2 cellulase.

Georgette Körfer1, Volkan Besirlioglu1, Mehdi D Davari2

  • 1Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.

Biotechnology and Bioengineering
|April 22, 2022
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Summary

This study introduces InVitroFlow, a novel ultrahigh-throughput screening method for enzyme engineering. It successfully identified an improved cellulase variant with significantly enhanced specific activity.

Keywords:
cellulasedirected evolutionflow cytometryin vitro compartmentalizationultrahigh-throughput screening

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Area of Science:

  • Biotechnology
  • Enzyme Engineering
  • Molecular Biology

Background:

  • Enzyme discovery relies on combining diversity generation and ultrahigh-throughput screening (uHTS).
  • Simultaneous amino acid substitutions at multiple positions can yield improved enzyme variants.
  • Efficient screening of large variant libraries is crucial for enzyme evolution.

Purpose of the Study:

  • To screen multisite saturation mutagenesis and OmniChange libraries of CelA2 cellulase using InVitroFlow.
  • To identify improved cellulase variants with enhanced enzymatic activity.
  • To demonstrate the efficacy of InVitroFlow for enzyme engineering.

Main Methods:

  • Development and application of InVitroFlow, a cell-free compartmentalization technology using double emulsions for uHTS.
  • Construction of multisite saturation mutagenesis and OmniChange libraries targeting the active site of CelA2 cellulase.
  • Screening of over 36 million enzyme variants using flow cytometry.

Main Results:

  • Identification of a significantly improved cellulase variant, CelA2-M3 (H288F/H524Q).
  • CelA2-M3 exhibited an 8-fold increase in specific activity compared to the parent variant and a 41-fold increase compared to wildtype CelA2.
  • The study demonstrated the capability of InVitroFlow to analyze up to 10^7 events per hour.

Conclusions:

  • InVitroFlow enables efficient screening of large enzyme libraries, overcoming limitations of traditional methods.
  • The identified CelA2-M3 variant represents a significant advancement in cellulase engineering.
  • This approach facilitates the discovery of enzymes with enhanced functions for various applications.