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 incorp...

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Autores principales:	Parinthon Nearmnala, Manutsawee Thanaburakorn, Watanalai Panbangred, Pimchai Chaiyen, Narupat Hongdilokkul
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/dab1a6356dce467a8b7038d09ed89775
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spelling	oai:doaj.org-article:dab1a6356dce467a8b7038d09ed897752021-12-02T17:50:41ZAn in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes10.1038/s41598-021-91204-42045-2322https://doaj.org/article/dab1a6356dce467a8b7038d09ed897752021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91204-4https://doaj.org/toc/2045-2322Abstract 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.Parinthon NearmnalaManutsawee ThanaburakornWatanalai PanbangredPimchai ChaiyenNarupat HongdilokkulNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-14 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q 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
description	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.
format	article
author	Parinthon Nearmnala Manutsawee Thanaburakorn Watanalai Panbangred Pimchai Chaiyen Narupat Hongdilokkul
author_facet	Parinthon Nearmnala Manutsawee Thanaburakorn Watanalai Panbangred Pimchai Chaiyen Narupat Hongdilokkul
author_sort	Parinthon Nearmnala
title	An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
title_short	An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
title_full	An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
title_fullStr	An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
title_full_unstemmed	An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
title_sort	in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/dab1a6356dce467a8b7038d09ed89775
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An in vivo selection system with tightly regulated gene expression enables directed evolution of highly efficient enzymes

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