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

Woo, Seung-Gyun; Kim, Seong Keun; Oh, Baek-Rock; Lee, Seung-Goo; Lee, Dae-Hee

doi:10.3390/ijms21228668

Open AccessArticle

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

by

Seung-Gyun Woo

^1,2,

Seong Keun Kim

¹,

Baek-Rock Oh

³,

Seung-Goo Lee

^1,2,*

and

Dae-Hee Lee

^1,2,*

¹

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

²

Department of Biosystems and Bioengineering, KRIBB School of Biotechnology, University of Science and Technology (UST), Daejeon 34113, Korea

³

Microbial Biotechnology Research Center, Jeonbuk Branch Institute, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeongeup 56212, Korea

^*

Authors to whom correspondence should be addressed.

Int. J. Mol. Sci. 2020, 21(22), 8668; https://doi.org/10.3390/ijms21228668

Submission received: 14 October 2020 / Revised: 5 November 2020 / Accepted: 16 November 2020 / Published: 17 November 2020

(This article belongs to the Special Issue Microbial Systems and Synthetic Biology)

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Abstract

Lysozyme is widely used as a model protein in studies of structure–function relationships. Recently, lysozyme has gained attention for use in accelerating the degradation of secondary sludge, which mainly consists of bacteria. However, a high-throughput screening system for lysozyme engineering has not been reported. Here, we present a lysozyme screening system using a genetically encoded biosensor. We first cloned bacteriophage T4 lysozyme (T4L) into a plasmid under control of the araBAD promoter. The plasmid was expressed in Escherichia coli with no toxic effects on growth. Next, we observed that increased soluble T4L expression decreased the fluorescence produced by the genetic enzyme screening system. To investigate T4L evolution based on this finding, we generated a T4L random mutation library, which was screened using the genetic enzyme screening system. Finally, we identified two T4L variants showing 1.4-fold enhanced lytic activity compared to native T4L. To our knowledge, this is the first report describing the use of a genetically encoded biosensor to investigate bacteriophage T4L evolution. Our approach can be used to investigate the evolution of other lysozymes, which will expand the applications of lysozyme.

Keywords: bacteriophage T4 lysozyme; genetically encoded biosensor; directed evolution

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MDPI and ACS Style

Woo, S.-G.; Kim, S.K.; Oh, B.-R.; Lee, S.-G.; Lee, D.-H. Genetically Encoded Biosensor-Based Screening for Directed Bacteriophage T4 Lysozyme Evolution. Int. J. Mol. Sci. 2020, 21, 8668. https://doi.org/10.3390/ijms21228668

AMA Style

Woo S-G, Kim SK, Oh B-R, Lee S-G, Lee D-H. Genetically Encoded Biosensor-Based Screening for Directed Bacteriophage T4 Lysozyme Evolution. International Journal of Molecular Sciences. 2020; 21(22):8668. https://doi.org/10.3390/ijms21228668

Chicago/Turabian Style

Woo, Seung-Gyun, Seong Keun Kim, Baek-Rock Oh, Seung-Goo Lee, and Dae-Hee Lee. 2020. "Genetically Encoded Biosensor-Based Screening for Directed Bacteriophage T4 Lysozyme Evolution" International Journal of Molecular Sciences 21, no. 22: 8668. https://doi.org/10.3390/ijms21228668

APA Style

Woo, S.-G., Kim, S. K., Oh, B.-R., Lee, S.-G., & Lee, D.-H. (2020). Genetically Encoded Biosensor-Based Screening for Directed Bacteriophage T4 Lysozyme Evolution. International Journal of Molecular Sciences, 21(22), 8668. https://doi.org/10.3390/ijms21228668

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

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