Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production

[EN] Aspergillopepsin B was identified in culture broths of Aspergillus awamori by in situ detection of its proteolytic activity and by immunodetection with anti-aspergillopepsin B antibodies. Severe thaumatin degradation was observed after in vitro treatment of thaumatin with purified aspergillopep...

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Authors: Moralejo, Francisco J., Cardoza, Rosa E., Gutiérrez , Santiago 1965-, Lombraña, Marta, Fierro, Francisco, Martín Martín, Juan Francisco
Format: article
Status:Published version
Publication Date:2002
Country:España
Institution:Universidad Rey Juan Carlos
Repository:BULERIA. Repositorio Institucional de la Universidad de León
OAI Identifier:oai:buleria.unileon.es:10612/17799
Online Access:https://journals.asm.org/doi/full/10.1128/aem.68.7.3550-3559.2002
https://hdl.handle.net/10612/17799
Access Level:Open access
Keyword:Biología
Filamentous fungi
aspergillopepsin B
antisense RNA expression
Thaumatin
2414 Microbiología
2414.01 Antibióticos
2414.06 Hongos
2414.07 Metabolismo Microbiano
2415.01 Biología Molecular de Microorganismos
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oai_identifier_str oai:buleria.unileon.es:10612/17799
network_acronym_str ES
network_name_str España
repository_id_str
spelling Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin ProductionMoralejo, Francisco J.Cardoza, Rosa E.Gutiérrez , Santiago 1965-Lombraña, MartaFierro, FranciscoMartín Martín, Juan FranciscoBiologíaFilamentous fungiaspergillopepsin Bantisense RNA expressionThaumatin2414 Microbiología2414.01 Antibióticos2414.06 Hongos2414.07 Metabolismo Microbiano2415.01 Biología Molecular de Microorganismos[EN] Aspergillopepsin B was identified in culture broths of Aspergillus awamori by in situ detection of its proteolytic activity and by immunodetection with anti-aspergillopepsin B antibodies. Severe thaumatin degradation was observed after in vitro treatment of thaumatin with purified aspergillopepsin B. The pepB gene encoding aspergillopepsin B of A. awamori was cloned and characterized. It is located in chromosome IV of A. awamori, as shown by pulsed-field gel electrophoresis, and encodes a protein of 282 amino acids with high similarity to the aspergillopepsin B of Aspergillus niger var. macrosporus. The pepB gene is expressed at high rates as a monocistronic 1.0-kb transcript in media with casein at acidic pH values. An antisense cassette constructed by inserting the pepB gene in the antisense orientation downstream from the gpdA promoter resulted in a good level of antisense mRNA, as shown by reverse transcription-PCR. Partial silencing of the pepB gene by the antisense mRNA resulted in a 31% increase in thaumatin yield. However, significant residual degradation of thaumatin still occurred. To completely remove aspergillopepsin B, the pepB gene was deleted by double crossover. Two of the selected transformants lacked the endogenous pepB gene and did not form aspergillopepsin B. Thaumatin yields increased by between 45% in transformant APB 7/25 and 125% in transformant 7/36 with respect to the parental strain. Reduction of proteolytic degradation by gene silencing with antisense mRNA or total removal of the aspergillopepsin B by directed gene deletion was a very useful method for improving thaumatin production in A. awamori.SIAmerican Society for Microbiology ASM JournalsMicrobiologiaFacultad de Ciencias de la Salud2002info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionhttps://journals.asm.org/doi/full/10.1128/aem.68.7.3550-3559.2002https://hdl.handle.net/10612/17799reponame:BULERIA. Repositorio Institucional de la Universidad de Leóninstname:Universidad Rey Juan CarlosInglésinfo:eu-repo/semantics/openAccessoai:buleria.unileon.es:10612/177992026-06-24T12:43:27Z
dc.title.none.fl_str_mv Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production
title Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production
spellingShingle Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production
Moralejo, Francisco J.
Biología
Filamentous fungi
aspergillopepsin B
antisense RNA expression
Thaumatin
2414 Microbiología
2414.01 Antibióticos
2414.06 Hongos
2414.07 Metabolismo Microbiano
2415.01 Biología Molecular de Microorganismos
title_short Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production
title_full Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production
title_fullStr Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production
title_full_unstemmed Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production
title_sort Silencing of the Aspergillopepsin B (pepB) Gene of Aspergillus awamori by Antisense RNA Expression or Protease Removal by Gene Disruption Results in a Large Increase in Thaumatin Production
dc.creator.none.fl_str_mv Moralejo, Francisco J.
Cardoza, Rosa E.
Gutiérrez , Santiago 1965-
Lombraña, Marta
Fierro, Francisco
Martín Martín, Juan Francisco
author Moralejo, Francisco J.
author_facet Moralejo, Francisco J.
Cardoza, Rosa E.
Gutiérrez , Santiago 1965-
Lombraña, Marta
Fierro, Francisco
Martín Martín, Juan Francisco
author_role author
author2 Cardoza, Rosa E.
Gutiérrez , Santiago 1965-
Lombraña, Marta
Fierro, Francisco
Martín Martín, Juan Francisco
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Microbiologia
Facultad de Ciencias de la Salud
dc.subject.none.fl_str_mv Biología
Filamentous fungi
aspergillopepsin B
antisense RNA expression
Thaumatin
2414 Microbiología
2414.01 Antibióticos
2414.06 Hongos
2414.07 Metabolismo Microbiano
2415.01 Biología Molecular de Microorganismos
topic Biología
Filamentous fungi
aspergillopepsin B
antisense RNA expression
Thaumatin
2414 Microbiología
2414.01 Antibióticos
2414.06 Hongos
2414.07 Metabolismo Microbiano
2415.01 Biología Molecular de Microorganismos
description [EN] Aspergillopepsin B was identified in culture broths of Aspergillus awamori by in situ detection of its proteolytic activity and by immunodetection with anti-aspergillopepsin B antibodies. Severe thaumatin degradation was observed after in vitro treatment of thaumatin with purified aspergillopepsin B. The pepB gene encoding aspergillopepsin B of A. awamori was cloned and characterized. It is located in chromosome IV of A. awamori, as shown by pulsed-field gel electrophoresis, and encodes a protein of 282 amino acids with high similarity to the aspergillopepsin B of Aspergillus niger var. macrosporus. The pepB gene is expressed at high rates as a monocistronic 1.0-kb transcript in media with casein at acidic pH values. An antisense cassette constructed by inserting the pepB gene in the antisense orientation downstream from the gpdA promoter resulted in a good level of antisense mRNA, as shown by reverse transcription-PCR. Partial silencing of the pepB gene by the antisense mRNA resulted in a 31% increase in thaumatin yield. However, significant residual degradation of thaumatin still occurred. To completely remove aspergillopepsin B, the pepB gene was deleted by double crossover. Two of the selected transformants lacked the endogenous pepB gene and did not form aspergillopepsin B. Thaumatin yields increased by between 45% in transformant APB 7/25 and 125% in transformant 7/36 with respect to the parental strain. Reduction of proteolytic degradation by gene silencing with antisense mRNA or total removal of the aspergillopepsin B by directed gene deletion was a very useful method for improving thaumatin production in A. awamori.
publishDate 2002
dc.date.none.fl_str_mv 2002
dc.type.none.fl_str_mv info:eu-repo/semantics/article
info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.none.fl_str_mv https://journals.asm.org/doi/full/10.1128/aem.68.7.3550-3559.2002
https://hdl.handle.net/10612/17799
url https://journals.asm.org/doi/full/10.1128/aem.68.7.3550-3559.2002
https://hdl.handle.net/10612/17799
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
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 ASM Journals
publisher.none.fl_str_mv American Society for Microbiology ASM Journals
dc.source.none.fl_str_mv reponame:BULERIA. Repositorio Institucional de la Universidad de León
instname:Universidad Rey Juan Carlos
instname_str Universidad Rey Juan Carlos
reponame_str BULERIA. Repositorio Institucional de la Universidad de León
collection BULERIA. Repositorio Institucional de la Universidad de León
repository.name.fl_str_mv
repository.mail.fl_str_mv
_version_ 1869402680024104960
score 15,300724

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