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Controlled Continuous Evolution of Enzymatic Activity Screened at Ultrahigh Throughput Using Drop-Based

R G Rosenthal1,2, X Diana Zhang1, K Ilić Đurđić1,3,4

  • 1School of Engineering and Applied Sciences, Harvard University, Cambridge, MA 02138, USA.

Angewandte Chemie (International Ed. in English)
|April 5, 2023
PubMed
Summary
This summary is machine-generated.

This study introduces a continuous evolution platform for enzyme optimization, significantly reducing labor and capital costs. The new method enhances enzyme catalytic efficiency, turning waste products into valuable feedstocks.

Keywords:
CRISPR-Directed EvolutionContinuous EvolutionDroplet MicrofluidicsProtein EngineeringSustainable Chemistry

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Area of Science:

  • Biotechnology
  • Enzyme Engineering
  • Synthetic Biology

Background:

  • Enzymes are crucial catalysts for drug development and industrial processes, but often require optimization.
  • Directed evolution is a common method for enzyme optimization but is labor- and capital-intensive due to multiple molecular biology steps and limited screening throughput.

Purpose of the Study:

  • To develop an effective and broadly applicable continuous evolution platform for ultrahigh-throughput enzyme evolution.
  • To enable controlled exploration of enzyme fitness landscapes by directly measuring enzymatic activity.

Main Methods:

  • A drop-based microfluidics platform was utilized for continuous evolution.
  • The platform integrates cell growth, mutagenesis (using nCas9 chimera and mutagenesis polymerase), and screening with minimal human intervention.
  • In vivo gene diversification was achieved using sgRNAs tiled along the target gene.

Main Results:

  • The platform was successfully used to evolve alditol oxidase, altering its substrate specificity towards glycerol.
  • A variant with a 10.5-fold increase in catalytic efficiency was identified.
  • This demonstrates the platform's ability to convert waste products into valuable feedstocks.

Conclusions:

  • The developed continuous evolution platform offers an effective and broadly applicable solution for enzyme engineering at ultrahigh throughput.
  • This technology significantly reduces the labor and capital investment required for enzyme optimization.
  • The platform facilitates the development of novel biocatalysts with tailored substrate specificities, with potential applications in waste valorization and green chemistry.