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Heterologous production of cellobiose dehydrogenases from the basidiomycete Coprinopsis cinerea and the ascomycete Podospora anserina and their effect on saccharification of wheat straw

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Abstract

Cellobiose dehydrogenases (CDHs) are extracellular glycosylated haemoflavoenzymes produced by many different wood-degrading and phytopathogenic fungi. Putative cellobiose dehydrogenase genes are recurrently discovered by genome sequencing projects in various phylogenetically distinct fungi. The genomes from the basidiomycete Coprinopsis cinerea and the ascomycete Podospora anserina were screened for candidate cdh genes, and one and three putative gene models were evidenced, respectively. Two putative cdh genes were selected and successfully expressed for the first time in Aspergillus niger. CDH activity was measured for both constructions (CDHcc and CDHpa), and both recombinant CDHs were purified to homogeneity and subsequently characterised. Kinetic constants were determined for several carbohydrates including β-1,4-linked di- and oligosaccharides. Optimal temperature and pH were 60 °C and 5 for CDHcc and 65–70 °C and 6 for CDHpa. Both CDHs showed a broad range of pH stability between 4 and 8. The effect of both CDHs on saccharification of micronized wheat straw by an industrial Trichoderma reesei secretome was determined. The addition of each CDH systematically decreased the release of total reducing sugars, but to different extents and according to the CDH concentration. Analytical methods were carried out to quantify the release of glucose, xylose and gluconic acid. An increase of glucose and xylose was measured at a low CDHcc concentration. At moderated and high CDHcc and CDHpa concentrations, glucose was severely reduced with a concomitant increase of gluconic acid. In conclusion, these results give new insights into the physical and chemical parameters and diversity of basidiomycetous and ascomycetous CDHs. These findings also demonstrated that CDH drastically influenced the saccharification on a natural substrate, and thus, CDH origin, concentration and potential enzymatic partners should be carefully considered in future artificial secretomes for biofuel applications.

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Acknowledgments

We thank Régine Lebrun and Sabrina Lignon, IBSM-CNRS, Plate-forme Protéomique, Marseille, France for in-gel tryptic digestion and protein identification by mass spectrometry. This study was funded by the French National Research Agency (ANR, program E-TRICEL 344 ANR-07-BIOE-006).

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Authors and Affiliations

  1. INRA, UMR1163 Biotechnologie des Champignons Filamenteux, Aix-Marseille Université, ESIL Polytech Marseille, 163 avenue de Luminy, CP 925, 13288, Marseille Cedex 09, France

    Annick Turbe-Doan, Yonathan Arfi, Eric Record & Anthony Levasseur

  2. Aix-Marseille Univ, UMR1163 BCF, ESIL Polytech Marseille, 13288, Marseille, France

    Annick Turbe-Doan, Yonathan Arfi, Eric Record & Anthony Levasseur

  3. Departamento de Biotecnologıa y Ciencias Alimentarias, Instituto Tecnologico de Sonora, Cd. Obregon, 85000, Sonora, Mexico

    Isabel Estrada-Alvarado

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  1. Annick Turbe-Doan
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  2. Yonathan Arfi
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  4. Isabel Estrada-Alvarado
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Correspondence to Anthony Levasseur.

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Turbe-Doan, A., Arfi, Y., Record, E. et al. Heterologous production of cellobiose dehydrogenases from the basidiomycete Coprinopsis cinerea and the ascomycete Podospora anserina and their effect on saccharification of wheat straw. Appl Microbiol Biotechnol 97, 4873–4885 (2013). https://doi.org/10.1007/s00253-012-4355-y

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  • DOI: https://doi.org/10.1007/s00253-012-4355-y

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