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Genetically Encoded Biosensor-Based Screening for Directed Bacteriophage T4 Lysozyme Evolution.

Seung-Gyun Woo1,2, Seong Keun Kim1, Baek-Rock Oh3

  • 1Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 34141, Korea.

International Journal of Molecular Sciences
|November 20, 2020
PubMed
Summary

Researchers developed a novel biosensor system for high-throughput screening of engineered lysozymes. This system successfully identified two bacteriophage T4 lysozyme (T4L) variants with 1.4-fold increased lytic activity, advancing lysozyme applications.

Keywords:
bacteriophage T4 lysozymedirected evolutiongenetically encoded biosensor

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzyme Engineering

Background:

  • Lysozyme is a model protein for structure-function studies and shows potential in degrading bacterial secondary sludge.
  • High-throughput screening systems for lysozyme engineering are currently lacking, hindering advancements in its applications.

Purpose of the Study:

  • To develop and validate a genetically encoded biosensor system for high-throughput screening of bacteriophage T4 lysozyme (T4L) evolution.
  • To identify T4L variants with enhanced lytic activity for potential applications in sludge degradation and other fields.

Main Methods:

  • Cloning of bacteriophage T4 lysozyme (T4L) into a plasmid under the control of the araBAD promoter for expression in Escherichia coli.
  • Development of a genetic enzyme screening system where increased soluble T4L expression inversely correlated with fluorescence output.
  • Generation of a T4L random mutation library and screening using the developed biosensor system.

Main Results:

  • Successful expression of T4L in Escherichia coli without significant toxicity.
  • Identification of two T4L variants exhibiting a 1.4-fold increase in lytic activity compared to the native T4L.
  • Demonstration of the first genetically encoded biosensor system for bacteriophage T4 lysozyme evolution.

Conclusions:

  • The developed genetically encoded biosensor system enables high-throughput screening for T4L evolution.
  • The identified T4L variants possess enhanced lytic activity, suggesting potential for improved applications.
  • This approach is adaptable for the evolution and application expansion of other lysozymes.