Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae

Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be...

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Autores: Martín-Díaz, Javier, Paret, Carmen, García-Ruiz, Eva, Molina-Espeja, Patricia, Alcalde Galeote, Miguel
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2018
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/165826
Acceso en línea:http://hdl.handle.net/10261/165826
Access Level:acceso abierto
Palabra clave:Peroxygenases
Neutral genetic drift
In vivo DNA shuffling
Saccharomyces cerevisiae
Directed evolution
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spelling Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiaeMartín-Díaz, JavierParet, CarmenGarcía-Ruiz, EvaMolina-Espeja, PatriciaAlcalde Galeote, MiguelPeroxygenasesNeutral genetic driftIn vivo DNA shufflingSaccharomyces cerevisiaeDirected evolutionUnspecific peroxygenase (UPO) is a highly promiscuous biocatalyst and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae so that neutral mutations were simply added and recombined in vivo. With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded and active UPO variants were generated. After only 8 rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with modifications in peroxidative and peroxygenative activities, kinetic thermostability and enhanced tolerance to organic solvents. With an average 4.6 substitutions introduced per clone, neutral mutations covered roughly 10% of the protein sequence. As such, this study opens new avenues of UPO design by bringing together neutral genetic drift and DNA recombination in vivo.This work was supported by the European Union [FP7-KBBE-2013-7-613549-INDOX; H2020-BBI-PPP-2015-2-720297-ENZOX2], the CSIC [project PIE-201580E042], and the Spanish Ministry of Economy, Industry and Competitiveness [projects BIO2013-43407-R.DEWRY and BIO2016-79106-R. LIGNOLUTION].Peer reviewedAmerican Society for MicrobiologyEuropean CommissionMinisterio de Economía, Industria y Competitividad (España)Consejo Superior de Investigaciones Científicas (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]201820182018info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/165826reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/EC/FP7/613549info:eu-repo/grantAgreement/EC/H2020/720297info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2013-43407-Rinfo:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2016-79106-Rhttp://dx.doi.org/10.1128/AEM.00808-18Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1658262026-05-22T06:33:51Z
dc.title.none.fl_str_mv Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae
title Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae
spellingShingle Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae
Martín-Díaz, Javier
Peroxygenases
Neutral genetic drift
In vivo DNA shuffling
Saccharomyces cerevisiae
Directed evolution
title_short Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae
title_full Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae
title_fullStr Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae
title_full_unstemmed Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae
title_sort Shuffling the neutral drift of unspecific peroxygenase in Saccharomyces cerevisiae
dc.creator.none.fl_str_mv Martín-Díaz, Javier
Paret, Carmen
García-Ruiz, Eva
Molina-Espeja, Patricia
Alcalde Galeote, Miguel
author Martín-Díaz, Javier
author_facet Martín-Díaz, Javier
Paret, Carmen
García-Ruiz, Eva
Molina-Espeja, Patricia
Alcalde Galeote, Miguel
author_role author
author2 Paret, Carmen
García-Ruiz, Eva
Molina-Espeja, Patricia
Alcalde Galeote, Miguel
author2_role author
author
author
author
dc.contributor.none.fl_str_mv European Commission
Ministerio de Economía, Industria y Competitividad (España)
Consejo Superior de Investigaciones Científicas (España)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Peroxygenases
Neutral genetic drift
In vivo DNA shuffling
Saccharomyces cerevisiae
Directed evolution
topic Peroxygenases
Neutral genetic drift
In vivo DNA shuffling
Saccharomyces cerevisiae
Directed evolution
description Unspecific peroxygenase (UPO) is a highly promiscuous biocatalyst and its selective mono(per)oxygenase activity makes it useful for many synthetic chemistry applications. Among the broad repertory of library creation methods for directed enzyme evolution, genetic drift allows neutral mutations to be accumulated gradually within a polymorphic network of variants. In this study, we conducted a campaign of genetic drift with UPO in Saccharomyces cerevisiae so that neutral mutations were simply added and recombined in vivo. With low mutational loading and an activity threshold of 45% of the parent's native function, mutant libraries enriched in folded and active UPO variants were generated. After only 8 rounds of genetic drift and DNA shuffling, we identified an ensemble of 25 neutrally evolved variants with modifications in peroxidative and peroxygenative activities, kinetic thermostability and enhanced tolerance to organic solvents. With an average 4.6 substitutions introduced per clone, neutral mutations covered roughly 10% of the protein sequence. As such, this study opens new avenues of UPO design by bringing together neutral genetic drift and DNA recombination in vivo.
publishDate 2018
dc.date.none.fl_str_mv 2018
2018
2018
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Publisher's version
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/165826
url http://hdl.handle.net/10261/165826
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv #PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
#PLACEHOLDER_PARENT_METADATA_VALUE#
info:eu-repo/grantAgreement/EC/FP7/613549
info:eu-repo/grantAgreement/EC/H2020/720297
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2013-43407-R
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO2016-79106-R
http://dx.doi.org/10.1128/AEM.00808-18
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv American Society for Microbiology
publisher.none.fl_str_mv American Society for Microbiology
dc.source.none.fl_str_mv reponame:DIGITAL.CSIC. Repositorio Institucional del CSIC
instname:Consejo Superior de Investigaciones Científicas (CSIC)
instname_str Consejo Superior de Investigaciones Científicas (CSIC)
reponame_str DIGITAL.CSIC. Repositorio Institucional del CSIC
collection DIGITAL.CSIC. Repositorio Institucional del CSIC
repository.name.fl_str_mv
repository.mail.fl_str_mv
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