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Comparison of the Heterologous Expression of Trichoderma reesei Endoglucanase II and Cellobiohydrolase II in the Yeasts Pichia pastoris and Yarrowia lipolytica

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Abstract

The sequences encoding the genes for endoglucanase II and cellobiohydrolase II from the fungus Trichoderma reesei QM9414 were successfully cloned and expressed in Yarrowia lipolytica under the control of the POX2 or TEF promoters, and using either the native or preproLip2 secretion signals. The expression level of both recombinant enzymes was compared with that obtained using Pichia pastoris, under the control of the AOX1 promoter to evaluate the utility of Y. lipolytica as a host strain for recombinant EGII and CBHII production. Extracellular endoglucanase activity was similar between TEF-preoproLip2-eglII expressed in Y. lipolytica and P. pastoris induced by 0.5 % (v/v) methanol, but when recombinant protein expression in P. pastoris was induced with 3 % (v/v) methanol, the activity was increased by about sevenfold. In contrast, the expression level of cellobiohydrolase from the TEF-preproLip2-cbhII cassette was higher in Y. lipolytica than in P. pastoris. Transformed Y. lipolytica produced up to 15 mg/l endoglucanase and 50 mg/l cellobiohydrolase, with the specific activity of both proteins being greater than their homologs produced by P. pastoris. Partial characterization of recombinant endoglucanase II and cellobiohydrolase II expressed in both yeasts revealed their optimum pH and temperature, and their pH and temperature stabilities were identical and hyperglycosylation had little effect on their enzymatic activity and properties.

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Acknowledgments

This work was financially supported by grants from Chulalongkorn University Dutsadi Phiphat Scholarship, Chulalongkorn University Graduate Scholarship to Commemorate the 72nd Anniversary of His Majesty King Bhumibol Adulyadej, and Mrs. Ratanavalee In-ochanon from the PTT Public Company Limited. The authors thank Florence Bordes and Alain Marty for precious help with using the Yarrowia expression system and Jean-Marc Nicaud for readily supplying plasmids and Yarrowia strains.

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Authors and Affiliations

  1. Biological Science Program, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok, 10330, Thailand

    Nassapat Boonvitthya

  2. Department of Botany, Faculty of Science, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok, 10330, Thailand

    Nassapat Boonvitthya & Warawut Chulalaksananukul

  3. Biofuels by Biocatalysts Research Unit, Chulalongkorn University, 254 Phayathai Road, Bangkok, 10330, Thailand

    Nassapat Boonvitthya & Warawut Chulalaksananukul

  4. PTT Research & Technology Institute, PTT Public Co. Ltd, Ayutthaya, 13170, Thailand

    Vorakan Burapatana

  5. Université de Toulouse; INSA, UPS, INP; LISBP, 135 Avenue de Rangueil, 31077, Toulouse, France

    Sophie Bozonnet & Michael J. O’Donohue

  6. INRA, UMR792 Ingénierie des Systèmes Biologiques et des Procédés, 31400, Toulouse, France

    Sophie Bozonnet & Michael J. O’Donohue

  7. CNRS, UMR5504, 31400, Toulouse, France

    Sophie Bozonnet & Michael J. O’Donohue

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  1. Nassapat Boonvitthya
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Correspondence to Warawut Chulalaksananukul.

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Boonvitthya, N., Bozonnet, S., Burapatana, V. et al. Comparison of the Heterologous Expression of Trichoderma reesei Endoglucanase II and Cellobiohydrolase II in the Yeasts Pichia pastoris and Yarrowia lipolytica . Mol Biotechnol 54, 158–169 (2013). https://doi.org/10.1007/s12033-012-9557-0

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