High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypii
Autor(a) principal: | |
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Data de Publicação: | 2014 |
Outros Autores: | , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/1822/31895 |
Resumo: | Ashbya gossypii has been recently considered as a host for the expression of recombinant proteins. The production levels achieved thus far were similar to those obtained with Saccharomyces cerevisiae for the same proteins. Here, the β-galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2-micron plasmids carrying the native signal sequence at higher levels than those secreted by S. cerevisiae laboratorial strains. Four different constitutive promoters were used to regulate the expression of β-galactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant β-galactosidase in A. gossypii, leading to 2- and 8-fold higher extracellular activity than the AgGPD promoter and the heterologous promoters, respectively. In similar production conditions, the levels of active β-galactosidase secreted by A. gossypii were up to 37 times higher than those secreted by recombinant S. cerevisiae and approximately 2.5 times higher than those previously reported for the β-galactosidase-high producing S. cerevisiae NCYC869-A3/pVK1.1. The substitution of glucose by glycerol in the production medium led to a 1.5-fold increase in the secretion of active β-galactosidase by A. gossypii. Recombinant β-galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger β-galactosidase and recombinant β-galactosidase produced by yeast. These results highlight the potential of A. gossypii as a recombinant protein producer and open new perspectives to further optimize recombinant protein secretion in this fungus. |
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High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypiiAshbya gossypiiAspergillus niger β-galactosidaseRecombinant β-galactosidase secretionA. gossypii GPD and TEF promotersSaccharomyces cerevisiae PGK1 and ADH1 promotersrecombinant beta-galactosidase secretionAspergillus niger beta-galactosidaseScience & TechnologyAshbya gossypii has been recently considered as a host for the expression of recombinant proteins. The production levels achieved thus far were similar to those obtained with Saccharomyces cerevisiae for the same proteins. Here, the β-galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2-micron plasmids carrying the native signal sequence at higher levels than those secreted by S. cerevisiae laboratorial strains. Four different constitutive promoters were used to regulate the expression of β-galactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant β-galactosidase in A. gossypii, leading to 2- and 8-fold higher extracellular activity than the AgGPD promoter and the heterologous promoters, respectively. In similar production conditions, the levels of active β-galactosidase secreted by A. gossypii were up to 37 times higher than those secreted by recombinant S. cerevisiae and approximately 2.5 times higher than those previously reported for the β-galactosidase-high producing S. cerevisiae NCYC869-A3/pVK1.1. The substitution of glucose by glycerol in the production medium led to a 1.5-fold increase in the secretion of active β-galactosidase by A. gossypii. Recombinant β-galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger β-galactosidase and recombinant β-galactosidase produced by yeast. These results highlight the potential of A. gossypii as a recombinant protein producer and open new perspectives to further optimize recombinant protein secretion in this fungus.Project AshByofactory (grant PTDC/EBB-EBI/101985/2008 - FCOMP-01-0124-FEDER-009701), MIT-Portugal Program (PhD grant SFRH/BD/39112/2007 to Tatiana Q. Aguiar) and grant SFRH/BDP/63831/2009 to Carla OliveiraAmerican Chemical SocietyUniversidade do MinhoMagalhães, FredericoAguiar, Tatiana QuintaOliveira, Carla Cristina Marques deDomingues, Lucília20142014-01-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/31895eng8756-79381520-603310.1002/btpr.184424851247http://onlinelibrary.wiley.com/journal/10.1021/(ISSN)1520-6033info:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:48:40Zoai:repositorium.sdum.uminho.pt:1822/31895Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:46:58.008859Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypii |
title |
High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypii |
spellingShingle |
High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypii Magalhães, Frederico Ashbya gossypii Aspergillus niger β-galactosidase Recombinant β-galactosidase secretion A. gossypii GPD and TEF promoters Saccharomyces cerevisiae PGK1 and ADH1 promoters recombinant beta-galactosidase secretion Aspergillus niger beta-galactosidase Science & Technology |
title_short |
High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypii |
title_full |
High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypii |
title_fullStr |
High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypii |
title_full_unstemmed |
High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypii |
title_sort |
High-level expression of Aspergillus niger β-galactosidase in Ashbya gossypii |
author |
Magalhães, Frederico |
author_facet |
Magalhães, Frederico Aguiar, Tatiana Quinta Oliveira, Carla Cristina Marques de Domingues, Lucília |
author_role |
author |
author2 |
Aguiar, Tatiana Quinta Oliveira, Carla Cristina Marques de Domingues, Lucília |
author2_role |
author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Magalhães, Frederico Aguiar, Tatiana Quinta Oliveira, Carla Cristina Marques de Domingues, Lucília |
dc.subject.por.fl_str_mv |
Ashbya gossypii Aspergillus niger β-galactosidase Recombinant β-galactosidase secretion A. gossypii GPD and TEF promoters Saccharomyces cerevisiae PGK1 and ADH1 promoters recombinant beta-galactosidase secretion Aspergillus niger beta-galactosidase Science & Technology |
topic |
Ashbya gossypii Aspergillus niger β-galactosidase Recombinant β-galactosidase secretion A. gossypii GPD and TEF promoters Saccharomyces cerevisiae PGK1 and ADH1 promoters recombinant beta-galactosidase secretion Aspergillus niger beta-galactosidase Science & Technology |
description |
Ashbya gossypii has been recently considered as a host for the expression of recombinant proteins. The production levels achieved thus far were similar to those obtained with Saccharomyces cerevisiae for the same proteins. Here, the β-galactosidase from Aspergillus niger was successfully expressed and secreted by A. gossypii from 2-micron plasmids carrying the native signal sequence at higher levels than those secreted by S. cerevisiae laboratorial strains. Four different constitutive promoters were used to regulate the expression of β-galactosidase: A. gossypii AgTEF and AgGPD promoters, and S. cerevisiae ScADH1 and ScPGK1 promoters. The native AgTEF promoter drove the highest expression levels of recombinant β-galactosidase in A. gossypii, leading to 2- and 8-fold higher extracellular activity than the AgGPD promoter and the heterologous promoters, respectively. In similar production conditions, the levels of active β-galactosidase secreted by A. gossypii were up to 37 times higher than those secreted by recombinant S. cerevisiae and approximately 2.5 times higher than those previously reported for the β-galactosidase-high producing S. cerevisiae NCYC869-A3/pVK1.1. The substitution of glucose by glycerol in the production medium led to a 1.5-fold increase in the secretion of active β-galactosidase by A. gossypii. Recombinant β-galactosidase secreted by A. gossypii was extensively glycosylated, as are the native A. niger β-galactosidase and recombinant β-galactosidase produced by yeast. These results highlight the potential of A. gossypii as a recombinant protein producer and open new perspectives to further optimize recombinant protein secretion in this fungus. |
publishDate |
2014 |
dc.date.none.fl_str_mv |
2014 2014-01-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/1822/31895 |
url |
http://hdl.handle.net/1822/31895 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
8756-7938 1520-6033 10.1002/btpr.1844 24851247 http://onlinelibrary.wiley.com/journal/10.1021/(ISSN)1520-6033 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
American Chemical Society |
publisher.none.fl_str_mv |
American Chemical Society |
dc.source.none.fl_str_mv |
reponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação instacron:RCAAP |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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RCAAP |
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RCAAP |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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