Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris

Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P), and the constitutive GAP promoter (P). Since promoters play a crucial role in an expression system an...

Descripción completa

Detalles Bibliográficos
Autores: Garrigos, Javier|||0000-0003-2271-2220, Vuoristo, Kiira, Nieto-Taype, Miguel Angel|||0000-0003-1055-0658, Tähtiharju, Juha, Uusitalo, Jaana, Tukiainen, Pauliina, Schmid, Christian, Tolstorukov, Ilya, Madden, Knut, Penttilä, Merja, Montesinos Seguí, José Luis|||0000-0001-9941-0832, Valero, Francisco|||0000-0003-0429-9620, Glieder, Anton|||0000-0002-5986-9689, Garcia-Ortega, Xavier|||0000-0001-7833-3655
Tipo de recurso: artículo
Fecha de publicación:2021
País:España
Institución:Universitat Autònoma de Barcelona
Repositorio:Dipòsit Digital de Documents de la UAB
Idioma:inglés
OAI Identifier:oai:ddd.uab.cat:255373
Acceso en línea:https://ddd.uab.cat/record/255373
https://dx.doi.org/urn:doi:10.1186/s12934-021-01564-9
Access Level:acceso abierto
Palabra clave:Komagataella phaffii (Pichia pastoris)
Recombinant protein production
Methanol-free bioprocesses
Expression system characterisation
Promoter regulation
Bioprocess development
id ES_fd515aea07a5f8f4efb384da5d166f33
oai_identifier_str oai:ddd.uab.cat:255373
network_acronym_str ES
network_name_str España
repository_id_str
spelling Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastorisGarrigos, Javier|||0000-0003-2271-2220Vuoristo, KiiraNieto-Taype, Miguel Angel|||0000-0003-1055-0658Tähtiharju, JuhaUusitalo, JaanaTukiainen, PauliinaSchmid, ChristianTolstorukov, IlyaMadden, KnutPenttilä, MerjaMontesinos Seguí, José Luis|||0000-0001-9941-0832Valero, Francisco|||0000-0003-0429-9620Glieder, Anton|||0000-0002-5986-9689Garcia-Ortega, Xavier|||0000-0001-7833-3655Komagataella phaffii (Pichia pastoris)Recombinant protein productionMethanol-free bioprocessesExpression system characterisationPromoter regulationBioprocess developmentPichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P), and the constitutive GAP promoter (P). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (P, P) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter P, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Both the P and P-based expression systems outperformed similar P-based expression in chemostat cultivations, reaching ninefold higher specific production rates (q ). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both P and P expression systems were also successfully implemented in 15 L fed-batch cultivations where q and product to biomass yield (Y *) values were similar than those obtained in chemostat cultivations. As an outcome of the macrokinetic characterization presented, the novel P and P were observed to offer much higher efficiency for CalB production than the widely used P-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris -based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest. The online version contains supplementary material available at 10.1186/s12934-021-01564-9. 22021-01-0120212021-01-01Articlehttp://purl.org/coar/resource_type/c_6501VoRhttp://purl.org/coar/version/c_970fb48d4fbd8a85info:eu-repo/semantics/articleapplication/pdfhttps://ddd.uab.cat/record/255373https://dx.doi.org/urn:doi:10.1186/s12934-021-01564-9reponame:Dipòsit Digital de Documents de la UABinstname:Universitat Autònoma de BarcelonaInglésengEuropean Commission https://doi.org/10.13039/501100000780 730976open accesshttp://purl.org/coar/access_right/c_abf2Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.https://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:ddd.uab.cat:2553732026-06-06T12:50:31Z
dc.title.none.fl_str_mv Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
title Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
spellingShingle Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
Garrigos, Javier|||0000-0003-2271-2220
Komagataella phaffii (Pichia pastoris)
Recombinant protein production
Methanol-free bioprocesses
Expression system characterisation
Promoter regulation
Bioprocess development
title_short Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
title_full Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
title_fullStr Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
title_full_unstemmed Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
title_sort Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris
dc.creator.none.fl_str_mv Garrigos, Javier|||0000-0003-2271-2220
Vuoristo, Kiira
Nieto-Taype, Miguel Angel|||0000-0003-1055-0658
Tähtiharju, Juha
Uusitalo, Jaana
Tukiainen, Pauliina
Schmid, Christian
Tolstorukov, Ilya
Madden, Knut
Penttilä, Merja
Montesinos Seguí, José Luis|||0000-0001-9941-0832
Valero, Francisco|||0000-0003-0429-9620
Glieder, Anton|||0000-0002-5986-9689
Garcia-Ortega, Xavier|||0000-0001-7833-3655
author Garrigos, Javier|||0000-0003-2271-2220
author_facet Garrigos, Javier|||0000-0003-2271-2220
Vuoristo, Kiira
Nieto-Taype, Miguel Angel|||0000-0003-1055-0658
Tähtiharju, Juha
Uusitalo, Jaana
Tukiainen, Pauliina
Schmid, Christian
Tolstorukov, Ilya
Madden, Knut
Penttilä, Merja
Montesinos Seguí, José Luis|||0000-0001-9941-0832
Valero, Francisco|||0000-0003-0429-9620
Glieder, Anton|||0000-0002-5986-9689
Garcia-Ortega, Xavier|||0000-0001-7833-3655
author_role author
author2 Vuoristo, Kiira
Nieto-Taype, Miguel Angel|||0000-0003-1055-0658
Tähtiharju, Juha
Uusitalo, Jaana
Tukiainen, Pauliina
Schmid, Christian
Tolstorukov, Ilya
Madden, Knut
Penttilä, Merja
Montesinos Seguí, José Luis|||0000-0001-9941-0832
Valero, Francisco|||0000-0003-0429-9620
Glieder, Anton|||0000-0002-5986-9689
Garcia-Ortega, Xavier|||0000-0001-7833-3655
author2_role author
author
author
author
author
author
author
author
author
author
author
author
author
dc.subject.none.fl_str_mv Komagataella phaffii (Pichia pastoris)
Recombinant protein production
Methanol-free bioprocesses
Expression system characterisation
Promoter regulation
Bioprocess development
topic Komagataella phaffii (Pichia pastoris)
Recombinant protein production
Methanol-free bioprocesses
Expression system characterisation
Promoter regulation
Bioprocess development
description Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (P), and the constitutive GAP promoter (P). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (P, P) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter P, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. Both the P and P-based expression systems outperformed similar P-based expression in chemostat cultivations, reaching ninefold higher specific production rates (q ). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both P and P expression systems were also successfully implemented in 15 L fed-batch cultivations where q and product to biomass yield (Y *) values were similar than those obtained in chemostat cultivations. As an outcome of the macrokinetic characterization presented, the novel P and P were observed to offer much higher efficiency for CalB production than the widely used P-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris -based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest. The online version contains supplementary material available at 10.1186/s12934-021-01564-9.
publishDate 2021
dc.date.none.fl_str_mv 2
2021-01-01
2021
2021-01-01
dc.type.none.fl_str_mv Article
http://purl.org/coar/resource_type/c_6501
VoR
http://purl.org/coar/version/c_970fb48d4fbd8a85
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://ddd.uab.cat/record/255373
https://dx.doi.org/urn:doi:10.1186/s12934-021-01564-9
url https://ddd.uab.cat/record/255373
https://dx.doi.org/urn:doi:10.1186/s12934-021-01564-9
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.relation.none.fl_str_mv European Commission https://doi.org/10.13039/501100000780 730976
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
dc.rights.openaire.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.source.none.fl_str_mv reponame:Dipòsit Digital de Documents de la UAB
instname:Universitat Autònoma de Barcelona
instname_str Universitat Autònoma de Barcelona
reponame_str Dipòsit Digital de Documents de la UAB
collection Dipòsit Digital de Documents de la UAB
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869425528696471552
score 15,300724

Similares en LA Referencia