title: Generation of a glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis.
abstract: We obtained a novel glucose de-repressed mutant of Trichoderma reesei using disparity mutagenesis. A plasmid containing DNA polymerase δ lacking proofreading activity, and AMAI, an autonomously replicating sequence was introduced into T. reesei ATCC66589. The rate of mutation evaluated with 5-fluoroorotic acid resistance was approximately 30-fold higher than that obtained by UV irradiation. The transformants harboring incompetent DNA polymerase δ were then selected on 2-deoxyglucose agar plates with hygromycin B. The pNP-lactoside hydrolyzing activities of mutants were 2 to 5-fold higher than the parent in liquid medium containing glucose. Notably, the amino acid sequence of cre1, a key gene involved in glucose repression, was identical in the mutant and parent strains, and further, the cre1 expression levels was not abolished in the mutant. Taken together, these results demonstrate that the strains of T. reesei generated by disparity mutagenesis are glucose de-repressed variants that contain mutations in yet-unidentified factors other than cre1.
authors.affiliation: a Applied Microbiology and Biotechnology Laboratory , College of Bioresource Sciences, Nihon University , Fujisawa , Japan.
authors.forename: Hidekazu
authors.initials: H
authors.lastname: Iwakuma
authors.affiliation: a Applied Microbiology and Biotechnology Laboratory , College of Bioresource Sciences, Nihon University , Fujisawa , Japan.
authors.forename: Yoshiyuki
authors.initials: Y
authors.lastname: Koyama
authors.affiliation: a Applied Microbiology and Biotechnology Laboratory , College of Bioresource Sciences, Nihon University , Fujisawa , Japan.
authors.forename: Ayako
authors.initials: A
authors.lastname: Miyachi
authors.affiliation: a Applied Microbiology and Biotechnology Laboratory , College of Bioresource Sciences, Nihon University , Fujisawa , Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Nasukawa
authors.affiliation: a Applied Microbiology and Biotechnology Laboratory , College of Bioresource Sciences, Nihon University , Fujisawa , Japan.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Matsumoto
authors.affiliation: b Neo-Morgan Laboratory Incorporated , Kawasaki , Japan.
authors.forename: Shuntaro
authors.initials: S
authors.lastname: Yano
authors.affiliation: a Applied Microbiology and Biotechnology Laboratory , College of Bioresource Sciences, Nihon University , Fujisawa , Japan.
authors.forename: Jun
authors.initials: J
authors.lastname: Ogihara
authors.affiliation: a Applied Microbiology and Biotechnology Laboratory , College of Bioresource Sciences, Nihon University , Fujisawa , Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Kasumi
mesh_terms: D004258:DNA Polymerase III / Q000235:genetics; D003847:Deoxyglucose / Q000378:metabolism*; D008855:Microscopy, Electron, Scanning; D016296:Mutagenesis; D014242:Trichoderma / Q000235:genetics* / Q000378:metabolism / Q000648:ultrastructure
chemical_list: D003847:Deoxyglucose; D004258:DNA Polymerase III
keywords: Trichoderma reesei; cellulase; cre1; disparity mutagenesis; glucose de-repression