Application of T-DNA insertional mutagenesis for improving cellulase production in the filamentous fungus Trichoderma reesei

A T-DNA-tagged mutant library created by Agrobacterium-mediated transformation (AMT) was assayed for improvement of cellulase production. After 96-well plate screening for rapid growth on cellulose substrates followed by plate-clearing zone assay, three putative mutants, TA-32, TB-87 and TE-6, with enhanced cellulolytic ability were isolated, exhibiting 38%, 51% and 31% increase in total cellulase activity than the parental strain QM9414, respectively. Endoglucanase, cellobiohydrolase and β-glucosidase activities as well as the hydrolysis efficiencies of the mutants were also improved. Moreover, T-DNA was shown to be integrated at a single site in the genomes of TA-32 and TE-6 while inserted at two copies into the genome of TB-87. Further, the sequences flanking the T-DNA insertion sites were successfully rescued, demonstrating the increased utility of T-DNA insertional mutagenesis for improvement of cellulase production as well as subsequent identification of the tagged genes relevant to cellulolytic ability.