Publication: An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
Issued Date
2021-12-01
Resource Type
ISSN
20452322
Other identifier(s)
2-s2.0-85107176579
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Mahidol University
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SCOPUS
Bibliographic Citation
Scientific Reports. Vol.11, No.1 (2021)
Suggested Citation
Parinthon Nearmnala, Manutsawee Thanaburakorn, Watanalai Panbangred, Pimchai Chaiyen, Narupat Hongdilokkul An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes. Scientific Reports. Vol.11, No.1 (2021). doi:10.1038/s41598-021-91204-4 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/79244
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Title
An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
Abstract
In vivo selection systems are powerful tools for directed evolution of enzymes. The selection pressure of the systems can be tuned by regulating the expression levels of the catalysts. In this work, we engineered a selection system for laboratory evolution of highly active enzymes by incorporating a translationally suppressing cis repressor as well as an inducible promoter to impart stringent and tunable selection pressure. We demonstrated the utility of our selection system by performing directed evolution experiments using TEM β-lactamase as the model enzyme. Five evolutionary rounds afforded a highly active variant exhibiting 440-fold improvement in catalytic efficiency. We also showed that, without the cis repressor, the selection system cannot provide sufficient selection pressure required for evolving highly efficient TEM β-lactamase. The selection system should be applicable for the exploration of catalytic perfection of a wide range of enzymes.