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Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii

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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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Acknowledgements

The financial support of Fundação para a Ciência e a Tecnologia (FCT), Portugal, is acknowledged: project AshByofactory PTDC/EBB-EBI/101985/2008 and grant SFRH/BD/30229/2006 to Orquidea Ribeiro.

The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. IBB–Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, Universidade do Minho, Campus de Gualtar, 4710-057, Braga, Portugal

    Orquídea Ribeiro & Lucília Domingues

  2. VTT Technical Research Centre of Finland, P.O. Box 1000, 02044 VTT, Espoo, Finland

    Marilyn Wiebe, Marja Ilmén & Merja Penttilä

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  1. Orquídea Ribeiro
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  2. Marilyn Wiebe
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  3. Marja Ilmén
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  4. Lucília Domingues
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Correspondence to Lucília Domingues.

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Ribeiro, O., Wiebe, M., Ilmén, M. et al. Expression of Trichoderma reesei cellulases CBHI and EGI in Ashbya gossypii . Appl Microbiol Biotechnol 87, 1437–1446 (2010). https://doi.org/10.1007/s00253-010-2610-7

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