An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
Copyright policy )An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
AbstractIn 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 suppressingcisrepressor 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 thecisrepressor, 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.
- Mahidol University Thailand
- Vidyasirimedhi Institute of Science and Technology Thailand
Models, Molecular, Artificial intelligence, Protein Conformation, Science, Protein Engineering, Gene, Biochemistry, Article, Gene Expression Regulation, Enzymologic, Computational biology, Structure-Activity Relationship, Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins, Selection (genetic algorithm), Biochemistry, Genetics and Molecular Biology, In vivo, Escherichia coli, Genetics, Selection, Genetic, Promoter Regions, Genetic, Molecular Biology, Experimental Evolution, Biology, Base Sequence, Escherichia coli Proteins, Evolutionary Dynamics of Genetic Adaptation and Mutation, Q, Metabolic Engineering and Synthetic Biology, R, Mutant, Life Sciences, Gene Expression Regulation, Bacterial, Computer science, RNA, Bacterial, Enzyme, FOS: Biological sciences, Mutation, Directed evolution, Repressor, Medicine, Nucleic Acid Conformation, Gene expression, Directed Molecular Evolution
Models, Molecular, Artificial intelligence, Protein Conformation, Science, Protein Engineering, Gene, Biochemistry, Article, Gene Expression Regulation, Enzymologic, Computational biology, Structure-Activity Relationship, Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins, Selection (genetic algorithm), Biochemistry, Genetics and Molecular Biology, In vivo, Escherichia coli, Genetics, Selection, Genetic, Promoter Regions, Genetic, Molecular Biology, Experimental Evolution, Biology, Base Sequence, Escherichia coli Proteins, Evolutionary Dynamics of Genetic Adaptation and Mutation, Q, Metabolic Engineering and Synthetic Biology, R, Mutant, Life Sciences, Gene Expression Regulation, Bacterial, Computer science, RNA, Bacterial, Enzyme, FOS: Biological sciences, Mutation, Directed evolution, Repressor, Medicine, Nucleic Acid Conformation, Gene expression, Directed Molecular Evolution
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