title: Application of T-DNA insertional mutagenesis for improving cellulase production in the filamentous fungus Trichoderma reesei.
abstract: 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.
authors.affiliation: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, No.27, Shanda South Road, Jinan 250100, PR China.
authors.forename: Yaohua
authors.initials: Y
authors.lastname: Zhong
authors.forename: Xiaoli
authors.initials: X
authors.lastname: Wang
authors.forename: Haina
authors.initials: H
authors.lastname: Yu
authors.forename: Shaorong
authors.initials: S
authors.lastname: Liang
authors.forename: Tianhong
authors.initials: T
authors.lastname: Wang
mesh_terms: D002480:Cellulase / Q000096:biosynthesis*; D004269:DNA, Bacterial / Q000235:genetics*; D006868:Hydrolysis; D016254:Mutagenesis, Insertional; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D004269:DNA, Bacterial; C036483:T-DNA; D002480:Cellulase