title: The impact of a single-nucleotide mutation of bgl2 on cellulase induction in a Trichoderma reesei mutant.
abstract: BACKGROUND
  The filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.
  
  
  RESULTS
  To analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.
  
  
  CONCLUSIONS
  We conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188 Japan.
authors.forename: Yosuke
authors.initials: Y
authors.lastname: Shida
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188 Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Yamaguchi
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188 Japan ; Japan Science and Technology Agency (JST), 4-1-8 Honcho, Kawaguchi, Saitama 332-0012 Japan.
authors.forename: Mikiko
authors.initials: M
authors.lastname: Nitta
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188 Japan.
authors.forename: Ayana
authors.initials: A
authors.lastname: Nakamura
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188 Japan.
authors.forename: Machiko
authors.initials: M
authors.lastname: Takahashi
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188 Japan.
authors.forename: Shun-Ichi
authors.initials: S
authors.lastname: Kidokoro
authors.affiliation: Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Mori
authors.affiliation: Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Tashiro
authors.affiliation: Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka, 812-8581 Japan.
authors.forename: Satoru
authors.initials: S
authors.lastname: Kuhara
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 Japan.
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Matsuzawa
authors.affiliation: Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba Central 6, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8566 Japan.
authors.forename: Katsuro
authors.initials: K
authors.lastname: Yaoi
authors.affiliation: School of Pharmacy, Iwate Medical University, 2-1-1 Nishitokuta, Yahaba, Iwate 028-3694 Japan.
authors.forename: Yasumitsu
authors.initials: Y
authors.lastname: Sakamoto
authors.affiliation: School of Pharmacy, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142-8555 Japan.
authors.forename: Nobutada
authors.initials: N
authors.lastname: Tanaka
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188 Japan.
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Morikawa
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188 Japan.
authors.forename: Wataru
authors.initials: W
authors.lastname: Ogasawara
keywords: Cellobiose; Cellulase induction; Gene regulation; Hypocrea jecorina; Trichoderma reesei; β-Glucosidase