Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter

The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris (syn. Komagataella spp. ) is one of the strongest promoters for heterologous gene expression. Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harbori...

Descripción completa

Detalles Bibliográficos
Autores: Cámara, Elena|||0000-0003-4271-7555, Monforte, Sergi|||0000-0001-9752-5273, Albiol i Sala, Joan|||0000-0001-5626-429X, Ferrer, Pau|||0000-0002-5287-4127
Tipo de recurso: artículo
Fecha de publicación:2019
País:España
Institución:Universitat Autònoma de Barcelona
Repositorio:Dipòsit Digital de Documents de la UAB
Idioma:inglés
OAI Identifier:oai:ddd.uab.cat:266676
Acceso en línea:https://ddd.uab.cat/record/266676
https://dx.doi.org/urn:doi:10.1002/bit.26947
Access Level:acceso abierto
Palabra clave:Pichia pastoris (Komagataella spp.)
Mxr1
Mit1
AOX1 promoter
Methanol metabolism
Heterologous gene dosage
Recombinant protein production
id ES_24e01fd9debaa6850ff2d670ccdea004
oai_identifier_str oai:ddd.uab.cat:266676
network_acronym_str ES
network_name_str España
repository_id_str
spelling Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoterCámara, Elena|||0000-0003-4271-7555Monforte, Sergi|||0000-0001-9752-5273Albiol i Sala, Joan|||0000-0001-5626-429XFerrer, Pau|||0000-0002-5287-4127Pichia pastoris (Komagataella spp.)Mxr1Mit1AOX1 promoterMethanol metabolismHeterologous gene dosageRecombinant protein productionThe methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris (syn. Komagataella spp. ) is one of the strongest promoters for heterologous gene expression. Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harboring more than two copies of a Rhizopus oryzae lipase gene (ROL) have previously shown a decrease in cell growth, lipase production, and substrate consumption, as well as a significant transcriptional downregulation of methanol metabolism. This pointed to a potential titration effect of key transcriptional factors methanol expression regulator 1 (Mxr1) and methanol-induced transcription factor (Mit1) regulating methanol metabolism caused by the insertion of multiple expression vectors. To prove this hypothesis, a set of strains carrying one and four copies of ROL (1C and 4C, respectively) were engineered to coexpress one or two copies of MXR1*, coding for an Mxr1 variant insensitive to repression by 14-3-3 regulatory proteins, or one copy of MIT1. Small-scale cultures revealed that growth, Rol productivity, and methanol consumption were improved in the 4C-MXR1* and 4C-MIT1, strains growing on methanol as a sole carbon source, whereas only a slight increase in productivity was observed for re-engineered 1C strains. We further verified the improved performance of these strains in glycerol-/methanol-limited chemostat cultures. 22019-01-0120192019-01-01Articlehttp://purl.org/coar/resource_type/c_6501SMURhttp://purl.org/coar/version/c_71e4c1898caa6e32info:eu-repo/semantics/articleapplication/pdfhttps://ddd.uab.cat/record/266676https://dx.doi.org/urn:doi:10.1002/bit.26947reponame:Dipòsit Digital de Documents de la UABinstname:Universitat Autònoma de BarcelonaInglésengAgencia Estatal de Investigación https://doi.org/10.13039/501100011033 CTQ2013-4239-RAgencia Estatal de Investigación https://doi.org/10.13039/501100011033 CTQ2016-74959-RAgència de Gestió d'Ajuts Universitaris i de Recerca https://doi.org/10.13039/501100003030 2014/SGR-452open accesshttp://purl.org/coar/access_right/c_abf2Aquest material està protegit per drets d'autor i/o drets afins. Podeu utilitzar aquest material en funció del que permet la legislació de drets d'autor i drets afins d'aplicació al vostre cas. Per a d'altres usos heu d'obtenir permís del(s) titular(s) de drets.https://rightsstatements.org/vocab/InC/1.0/info:eu-repo/semantics/openAccessoai:ddd.uab.cat:2666762026-06-06T12:50:31Z
dc.title.none.fl_str_mv Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
title Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
spellingShingle Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
Cámara, Elena|||0000-0003-4271-7555
Pichia pastoris (Komagataella spp.)
Mxr1
Mit1
AOX1 promoter
Methanol metabolism
Heterologous gene dosage
Recombinant protein production
title_short Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
title_full Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
title_fullStr Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
title_full_unstemmed Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
title_sort Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter
dc.creator.none.fl_str_mv Cámara, Elena|||0000-0003-4271-7555
Monforte, Sergi|||0000-0001-9752-5273
Albiol i Sala, Joan|||0000-0001-5626-429X
Ferrer, Pau|||0000-0002-5287-4127
author Cámara, Elena|||0000-0003-4271-7555
author_facet Cámara, Elena|||0000-0003-4271-7555
Monforte, Sergi|||0000-0001-9752-5273
Albiol i Sala, Joan|||0000-0001-5626-429X
Ferrer, Pau|||0000-0002-5287-4127
author_role author
author2 Monforte, Sergi|||0000-0001-9752-5273
Albiol i Sala, Joan|||0000-0001-5626-429X
Ferrer, Pau|||0000-0002-5287-4127
author2_role author
author
author
dc.subject.none.fl_str_mv Pichia pastoris (Komagataella spp.)
Mxr1
Mit1
AOX1 promoter
Methanol metabolism
Heterologous gene dosage
Recombinant protein production
topic Pichia pastoris (Komagataella spp.)
Mxr1
Mit1
AOX1 promoter
Methanol metabolism
Heterologous gene dosage
Recombinant protein production
description The methanol-regulated alcohol oxidase promoter (PAOX1) of Pichia pastoris (syn. Komagataella spp. ) is one of the strongest promoters for heterologous gene expression. Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harboring more than two copies of a Rhizopus oryzae lipase gene (ROL) have previously shown a decrease in cell growth, lipase production, and substrate consumption, as well as a significant transcriptional downregulation of methanol metabolism. This pointed to a potential titration effect of key transcriptional factors methanol expression regulator 1 (Mxr1) and methanol-induced transcription factor (Mit1) regulating methanol metabolism caused by the insertion of multiple expression vectors. To prove this hypothesis, a set of strains carrying one and four copies of ROL (1C and 4C, respectively) were engineered to coexpress one or two copies of MXR1*, coding for an Mxr1 variant insensitive to repression by 14-3-3 regulatory proteins, or one copy of MIT1. Small-scale cultures revealed that growth, Rol productivity, and methanol consumption were improved in the 4C-MXR1* and 4C-MIT1, strains growing on methanol as a sole carbon source, whereas only a slight increase in productivity was observed for re-engineered 1C strains. We further verified the improved performance of these strains in glycerol-/methanol-limited chemostat cultures.
publishDate 2019
dc.date.none.fl_str_mv 2
2019-01-01
2019
2019-01-01
dc.type.none.fl_str_mv Article
http://purl.org/coar/resource_type/c_6501
SMUR
http://purl.org/coar/version/c_71e4c1898caa6e32
dc.type.openaire.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv https://ddd.uab.cat/record/266676
https://dx.doi.org/urn:doi:10.1002/bit.26947
url https://ddd.uab.cat/record/266676
https://dx.doi.org/urn:doi:10.1002/bit.26947
dc.language.none.fl_str_mv Inglés
eng
language_invalid_str_mv Inglés
language eng
dc.relation.none.fl_str_mv Agencia Estatal de Investigación https://doi.org/10.13039/501100011033 CTQ2013-4239-R
Agencia Estatal de Investigación https://doi.org/10.13039/501100011033 CTQ2016-74959-R
Agència de Gestió d'Ajuts Universitaris i de Recerca https://doi.org/10.13039/501100003030 2014/SGR-452
dc.rights.none.fl_str_mv open access
http://purl.org/coar/access_right/c_abf2
https://rightsstatements.org/vocab/InC/1.0/
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://rightsstatements.org/vocab/InC/1.0/
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_ 1869404725088092160
score 15.300724

Similares en LA Referencia