Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii

Orquídea Ribeiro, Marilyn Wiebe, Marja Ilmén, Lucília Domingues, Merja Penttilä

Research output: Contribution to journalArticleScientificpeer-review

27 Citations (Scopus)

Abstract

To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-β-d-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.

Original languageEnglish
Pages (from-to)1437-1446
JournalApplied Microbiology and Biotechnology
Volume87
Issue number4
DOIs
Publication statusPublished - 1 Jul 2010
MoE publication typeA1 Journal article-refereed

Fingerprint

Cellulose 1,4-beta-Cellobiosidase
Cellulases
Trichoderma
Saccharomyces cerevisiae
Fungi
Proteins
Protein Sorting Signals
Glycosylation
Recombinant Proteins
Culture Media
Plasmids
Western Blotting
Monoclonal Antibodies

Keywords

  • Ashbya gossypii
  • Cellulases heterologous expression
  • Recombinant protein production
  • Trichoderma reesei cellobiohydrolase I
  • Trichoderma reesei endoglucanase I

Cite this

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title = "Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii",
abstract = "To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-β-d-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.",
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Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii. / Ribeiro, Orquídea; Wiebe, Marilyn; Ilmén, Marja; Domingues, Lucília; Penttilä, Merja.

In: Applied Microbiology and Biotechnology, Vol. 87, No. 4, 01.07.2010, p. 1437-1446.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii

AU - Ribeiro, Orquídea

AU - Wiebe, Marilyn

AU - Ilmén, Marja

AU - Domingues, Lucília

AU - Penttilä, Merja

N1 - CA2: TK402 CA2: TK400 ISI: BIOTECHNOLOGY & APPLIED MICROBIOLOGY

PY - 2010/7/1

Y1 - 2010/7/1

N2 - To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-β-d-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.

AB - To explore the potential of Ashbya gossypii as a host for the expression of recombinant proteins and to assess whether protein secretion would be more similar to the closely related Saccharomyces cerevisiae or to other filamentous fungi, endoglucanase I (EGI) and cellobiohydrolase I (CBHI) from the fungus Trichoderma reesei were successfully expressed in A. gossypii from plasmids containing the two micron sequences from S. cerevisiae, under the S. cerevisiae PGK1 promoter. The native signal sequences of EGI and CBHI were able to direct the secretion of EGI and CBHI into the culture medium in A. gossypii. Although CBHI activity was not detected using 4-methylumbelliferyl-β-d-lactoside as substrate, the protein was detected by Western blot using monoclonal antibodies. EGI activity was detectable, the specific activity being comparable to that produced by a similar EGI producing S. cerevisiae construct. More EGI was secreted than CBHI, or more active protein was produced. Partial characterization of CBHI and EGI expressed in A. gossypii revealed overglycosylation when compared with the native T. reesei proteins, but the glycosylation was less extensive than on cellulases expressed in S. cerevisiae.

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KW - Recombinant protein production

KW - Trichoderma reesei cellobiohydrolase I

KW - Trichoderma reesei endoglucanase I

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EP - 1446

JO - Applied Microbiology and Biotechnology

JF - Applied Microbiology and Biotechnology

SN - 0175-7598

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ER -