Overexpression of XYR1 to increase cellulases production in Trichoderma harzianum

The filamentous fungus Trichoderma harzianum is a potential candidate for different cellulases and hemicellulases production and is also well adapted to bioreactor cultivations. The different cellulase genes are generally regulated in a coordinated way and by at least three transcriptional activators including XYR1, ACE2 and HAP2/3/5 complex. Among them, XYR1 is clearly the major cellulase activator and indispensable for cellulase expression. The objective of the present work was to develop a mutant of the wild-type T. harzianum which was isolated from the Amazon rainforest that presents a xyr1 overexpression. For the identification of the T. harzianum xyr1 it was used the respective orthologues from Trichoderma reesei by BLASTP and it was cloned in a vector containing the aminoglycoside 3'-phosphotransferase gene which enables resistance against geneticin. This vector contains pyruvate kinase (pki1) of T. reesei for constitutive expression the All the transformants were cultivated using steam explosion pretreated sugar cane bagasse followed by delignification with NaOH as the carbon source in shake flask in order to select the clones that presented cellulases production at least 2-folds higher than the wild-type. The strains selected were purified twice for mitotic stability, and the integration of the expression cassettes was verified by PCR analysis. The xyr1 over expression was further analyzed using CMC, beech wood xylan and sugar cane bagasse by RT-PCR.

 

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