Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides

[EN] Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthet...

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Autores: Viña-González, Javier, González-Pérez, David, Ferreira, Patricia, Martínez, Ángel T., Alcalde Galeote, Miguel
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2015
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/173884
Acceso en línea:http://hdl.handle.net/10261/173884
Access Level:acceso abierto
Palabra clave:Aryl-alcohol oxidase
Focused Directed Evolution
Saccharomyces cerevisiae
Chimeric Signal Peptides
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spelling Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal PeptidesViña-González, JavierGonzález-Pérez, DavidFerreira, PatriciaMartínez, Ángel T.Alcalde Galeote, MiguelAryl-alcohol oxidaseFocused Directed EvolutionSaccharomyces cerevisiaeChimeric Signal Peptides[EN] Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, and yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO from Pleurotus eryngii was replaced by those of the mating -factor and the K1 killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae. The secretion of these AAO constructs increased in the following order: prepro-AAO > preproK-AAO > preKpro-AAO > preproK-AAO. The chimeric preproK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Met[1]-Val109 and Phe392-Gln566), and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the preproK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature, whereas the pH activity profiles and the Kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except for P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability.This study was supported by the European Commission projects Indox FP7-KBBE-2013-7-613549 and Cost-Action CM1303-Systems Biocatalysis and by the National Projects Dewry (BIO201343407-R) and Cambios (RTC-2014-1777-3).Peer reviewedAmerican Society for MicrobiologyEuropean CommissionMinisterio de Economía y Competitividad (España)Martínez, Angel T. [0000-0002-1584-2863]Alcalde, Miguel [0000-0001-6780-7616]Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]201920192015info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Publisher's versioninfo:eu-repo/semantics/publishedVersionhttp://hdl.handle.net/10261/173884reponame: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#info:eu-repo/grantAgreement/EC/FP7/613549info: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/RTC2014-1777-3http://dx.doi.org/10.1128/AEM.01966-15Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/1738842026-05-22T06:33:51Z
dc.title.none.fl_str_mv Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides
title Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides
spellingShingle Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides
Viña-González, Javier
Aryl-alcohol oxidase
Focused Directed Evolution
Saccharomyces cerevisiae
Chimeric Signal Peptides
title_short Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides
title_full Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides
title_fullStr Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides
title_full_unstemmed Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides
title_sort Focused Directed Evolution of Aryl-Alcohol Oxidase in Saccharomyces cerevisiae by Using Chimeric Signal Peptides
dc.creator.none.fl_str_mv Viña-González, Javier
González-Pérez, David
Ferreira, Patricia
Martínez, Ángel T.
Alcalde Galeote, Miguel
author Viña-González, Javier
author_facet Viña-González, Javier
González-Pérez, David
Ferreira, Patricia
Martínez, Ángel T.
Alcalde Galeote, Miguel
author_role author
author2 González-Pérez, David
Ferreira, Patricia
Martínez, Ángel T.
Alcalde Galeote, Miguel
author2_role author
author
author
author
dc.contributor.none.fl_str_mv European Commission
Ministerio de Economía y Competitividad (España)
Martínez, Angel T. [0000-0002-1584-2863]
Alcalde, Miguel [0000-0001-6780-7616]
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Aryl-alcohol oxidase
Focused Directed Evolution
Saccharomyces cerevisiae
Chimeric Signal Peptides
topic Aryl-alcohol oxidase
Focused Directed Evolution
Saccharomyces cerevisiae
Chimeric Signal Peptides
description [EN] Aryl-alcohol oxidase (AAO) is an extracellular flavoprotein that supplies ligninolytic peroxidases with H2O2 during natural wood decay. With a broad substrate specificity and highly stereoselective reaction mechanism, AAO is an attractive candidate for studies into organic synthesis and synthetic biology, and yet the lack of suitable heterologous expression systems has precluded its engineering by directed evolution. In this study, the native signal sequence of AAO from Pleurotus eryngii was replaced by those of the mating -factor and the K1 killer toxin, as well as different chimeras of both prepro-leaders in order to drive secretion in Saccharomyces cerevisiae. The secretion of these AAO constructs increased in the following order: prepro-AAO > preproK-AAO > preKpro-AAO > preproK-AAO. The chimeric preproK-AAO was subjected to focused-directed evolution with the aid of a dual screening assay based on the Fenton reaction. Random mutagenesis and DNA recombination was concentrated on two protein segments (Met[1]-Val109 and Phe392-Gln566), and an array of improved variants was identified, among which the FX7 mutant (harboring the H91N mutation) showed a dramatic 96-fold improvement in total activity with secretion levels of 2 mg/liter. Analysis of the N-terminal sequence of the FX7 variant confirmed the correct processing of the preproK hybrid peptide by the KEX2 protease. FX7 showed higher stability in terms of pH and temperature, whereas the pH activity profiles and the Kinetic parameters were maintained. The Asn91 lies in the flavin attachment loop motif, and it is a highly conserved residue in all members of the GMC superfamily, except for P. eryngii and P. pulmonarius AAO. The in vitro involution of the enzyme by restoring the consensus ancestor Asn91 promoted AAO expression and stability.
publishDate 2015
dc.date.none.fl_str_mv 2015
2019
2019
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/10261/173884
url http://hdl.handle.net/10261/173884
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#
info:eu-repo/grantAgreement/EC/FP7/613549
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/RTC2014-1777-3
http://dx.doi.org/10.1128/AEM.01966-15
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
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