Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference

The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents,...

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Autores: Bosch, Sandra, Sanchez-Freire, Esther, Pozo, María Luisa del, Česnik, Morana, Quesada, Jaime, Maté, Diana M., Hernández Sánchez, Karel, Qi, Yuyin, Clapés Saborit, Pere, Vasić-Rački, Đurđa, Findrik Blažević, Zvjezdana, Berenguer, José, Hidalgo, Aurelio
Tipo de recurso: artículo
Estado:Versión aceptada para publicación
Fecha de publicación:2021
País:España
Institución:Consejo Superior de Investigaciones Científicas (CSIC)
Repositorio:DIGITAL.CSIC. Repositorio Institucional del CSIC
OAI Identifier:oai:digital.csic.es:10261/238222
Acceso en línea:http://hdl.handle.net/10261/238222
Access Level:acceso abierto
Palabra clave:Aldolases
Directed evolution
Hygromycin B phosphotransferase
in vivo selection
Thermostability
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spelling Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding InterferenceBosch, SandraSanchez-Freire, EstherPozo, María Luisa delČesnik, MoranaQuesada, JaimeMaté, Diana M.Hernández Sánchez, KarelQi, YuyinClapés Saborit, PereVasić-Rački, ĐurđaFindrik Blažević, ZvjezdanaBerenguer, JoséHidalgo, AurelioAldolasesDirected evolutionHygromycin B phosphotransferasein vivo selectionThermostabilityThe use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better “evolvability” of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (Tm) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in Tm compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a Tm 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.This work has been funded through the European Union’s Research and Innovation program Horizon 2020 through grant agreement no. 635595 (CarbaZymes) and by the Spanish Ministry of Economy and Competitiveness through grant BIO-2013-44963R. Institutional grants from the Fundación Ramón Areces and Banco Santander to the CBMSO are also acknowledged. S.B. is the recipient of a Ph.D. fellowship from UAM. D.M.M. was supported by a Research Talent Attraction contract from the Community of Madrid. A generous allocation of computing time at the Scientific Computation Center of the UAM (CCC-UAM) is also acknowledged.Peer reviewedAmerican Chemical SocietyEuropean CommissionMinisterio de Economía y Competitividad (España)Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]202120212021info:eu-repo/semantics/articlehttp://purl.org/coar/resource_type/c_6501Postprintinfo:eu-repo/semantics/acceptedVersionhttp://hdl.handle.net/10261/238222reponame:DIGITAL.CSIC. Repositorio Institucional del CSICinstname:Consejo Superior de Investigaciones Científicas (CSIC)Inglés#PLACEHOLDER_PARENT_METADATA_VALUE##PLACEHOLDER_PARENT_METADATA_VALUE#info:eu-repo/grantAgreement/EC/H2020/635595info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO-2013-44963https://doi.org/10.1021/acssuschemeng.1c00699Síinfo:eu-repo/semantics/openAccessoai:digital.csic.es:10261/2382222026-05-22T06:33:51Z
dc.title.none.fl_str_mv Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference
title Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference
spellingShingle Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference
Bosch, Sandra
Aldolases
Directed evolution
Hygromycin B phosphotransferase
in vivo selection
Thermostability
title_short Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference
title_full Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference
title_fullStr Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference
title_full_unstemmed Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference
title_sort Thermostability Engineering of a Class II Pyruvate Aldolase from Escherichia coli by in Vivo Folding Interference
dc.creator.none.fl_str_mv Bosch, Sandra
Sanchez-Freire, Esther
Pozo, María Luisa del
Česnik, Morana
Quesada, Jaime
Maté, Diana M.
Hernández Sánchez, Karel
Qi, Yuyin
Clapés Saborit, Pere
Vasić-Rački, Đurđa
Findrik Blažević, Zvjezdana
Berenguer, José
Hidalgo, Aurelio
author Bosch, Sandra
author_facet Bosch, Sandra
Sanchez-Freire, Esther
Pozo, María Luisa del
Česnik, Morana
Quesada, Jaime
Maté, Diana M.
Hernández Sánchez, Karel
Qi, Yuyin
Clapés Saborit, Pere
Vasić-Rački, Đurđa
Findrik Blažević, Zvjezdana
Berenguer, José
Hidalgo, Aurelio
author_role author
author2 Sanchez-Freire, Esther
Pozo, María Luisa del
Česnik, Morana
Quesada, Jaime
Maté, Diana M.
Hernández Sánchez, Karel
Qi, Yuyin
Clapés Saborit, Pere
Vasić-Rački, Đurđa
Findrik Blažević, Zvjezdana
Berenguer, José
Hidalgo, Aurelio
author2_role author
author
author
author
author
author
author
author
author
author
author
author
dc.contributor.none.fl_str_mv European Commission
Ministerio de Economía y Competitividad (España)
Consejo Superior de Investigaciones Científicas [https://ror.org/02gfc7t72]
dc.subject.none.fl_str_mv Aldolases
Directed evolution
Hygromycin B phosphotransferase
in vivo selection
Thermostability
topic Aldolases
Directed evolution
Hygromycin B phosphotransferase
in vivo selection
Thermostability
description The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better “evolvability” of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in Thermus thermophilus through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein. To generate a monomeric folding reporter, we have increased the thermostability of the moderately thermostable Hph5 variant of the hygromycin B phosphotransferase from Escherichia coli to meet the method requirements. The final Hph17 variant showed 1.5 °C higher melting temperature (Tm) and 3-fold longer half-life at 65 °C compared to parental Hph5, with no changes in the steady-state kinetic parameters. Additionally, we demonstrate the validity of the reporter by stabilizing the 2-keto-3-deoxy-l-rhamnonate aldolase from E. coli (YfaU). The most thermostable multiple-mutated variants thus obtained, YfaU99 and YfaU103, showed increases of 2 and 2.9 °C in Tm compared to the wild-type enzyme but severely lower retro-aldol activities (150- and 120-fold, respectively). After segregation of the mutations, the most thermostable single variant, Q107R, showed a Tm 8.9 °C higher, a 16-fold improvement in half-life at 60 °C and higher operational stability than the wild-type, without substantial modification of the kinetic parameters.
publishDate 2021
dc.date.none.fl_str_mv 2021
2021
2021
dc.type.none.fl_str_mv info:eu-repo/semantics/article
http://purl.org/coar/resource_type/c_6501
Postprint
info:eu-repo/semantics/acceptedVersion
format article
status_str acceptedVersion
dc.identifier.none.fl_str_mv http://hdl.handle.net/10261/238222
url http://hdl.handle.net/10261/238222
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#
info:eu-repo/grantAgreement/EC/H2020/635595
info:eu-repo/grantAgreement/MINECO/Plan Estatal de Investigación Científica y Técnica y de Innovación 2013-2016/BIO-2013-44963
https://doi.org/10.1021/acssuschemeng.1c00699
Sí
dc.rights.none.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.publisher.none.fl_str_mv American Chemical Society
publisher.none.fl_str_mv American Chemical Society
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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