An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
| Autor: | Narupat Hongdilokkul, Pimchai Chaiyen, Parinthon Nearmnala, Watanalai Panbangred, Manutsawee Thanaburakorn |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Models Molecular Molecular biology Protein Conformation Science Repressor Computational biology Protein Engineering Article Gene Expression Regulation Enzymologic 03 medical and health sciences Structure-Activity Relationship In vivo Gene expression Escherichia coli Selection Genetic Promoter Regions Genetic Selection (genetic algorithm) Selection system chemistry.chemical_classification Multidisciplinary 030102 biochemistry & molecular biology Base Sequence Chemistry Molecular engineering Escherichia coli Proteins Gene Expression Regulation Bacterial Directed evolution Enzymes RNA Bacterial 030104 developmental biology Enzyme Experimental evolution Mutation Molecular evolution Nucleic Acid Conformation Medicine Diffusion limited enzyme Directed Molecular Evolution |
| Zdroj: | Scientific Reports, Vol 11, Iss 1, Pp 1-14 (2021) Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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