Abstract
In the filamentous fungus Trichoderma reesei, endoglucanase III (EGIII) is coordinately expressed with other cellulases during growth on cellulose, its derivatives, and L-sorbose. To elucidate EGIII induction mechanism, we cloned and sequenced the upstream region of egl3 encoding EGIII. Two GGCTAA motifs, a putative binding site for ACEII and xylanase regulator Xyr1, were found on the template strand of the egl3 upstream region. Deletion analysis of the egl3 upstream region using the beta-glucuronidase (GUS) reporter system revealed that removal of regions containing the GGCTAA motifs and the region between −1,045 and −1,002 bp containing GGCTAT motif severely affected GUS inducibility. Furthermore, mutation of the two GGCTAA motifs and the GGCTAT motif of this region led to a significant decrease in GUS activity. These data indicate that both GGCTAA and GGCTAT are key motifs for egl3 expression, and that egl3 induction may also be controlled by Xyr1. This hypothesis was supported by in vitro electrophoretic mobility shift assay, in which heterologously expressed Xyr1 specifically bound not only GGCTAA but also GGCTAT motif.
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Aro N, Saloheimo A, Ilmén M, Penttilä M (2001) ACEII, a novel transcriptional activator involved in regulation of cellulase and xylanase genes of Trichoderma reesei. J Biol Chem 276:24309–24314
Aro N, Ilmén M, Saloheimo A, Penttilä M (2003) ACEI of Trichoderma reesei is a repressor of cellulase and xylanase expression. Appl Environ Microbiol 69:56–65
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254
Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol Biol Rep 5(4):387–405
Kawamori M, Morikawa Y, Takasawa S (1985) Inductive formation of cellulases by L-sorbose in Trichoderma reesei. Appl Microbiol Biothechnol 22:235–236
Kawamori M, Morikawa Y, Takasawa S (1986) Induction and production of cellulases by L-sorbose in Trichoderma reesei. Appl Microbiol Biotechnol 24:449–453
Marui J, Tanaka A, Mimura S, de Graaff LH, Visser J, Kitamoto N, Kato M, Kobayashi T, Tsukagoshi N (2002) A transcriptional activator, AoXlnR, controls the expression of genes encoding xylanolytic enzymes in Aspergillus oryzae. Fungal Genet Biol 35:157–169
Nogawa M, Goto M, Okada H, Morikawa Y (2001) L-Sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei. Curr Genet 38:329–334
Ogasawara W, Yosuke S, Furukawa T, Shimada R, Nakagawa S, Kawamura M, Yagyu T, Kosuge A, Xu J, Nogawa M, Okada H, Morikawa Y (2006) Cloning, functional expression and promoter analysis of xylanase III gene from Trichoderma reesei. Appl Microbiol Biotechnol 72:995–1003
Okada H, Tada K, Sekiya T, Yokoyama K, Takahashi A, Tohda H, Kumagai H, Morikawa Y (1998) Molecular characterization and heterologous expression of the gene encoding a low-molecular-mass endoglucanase from Trichoderma reesei QM9414. Appl Environ Microbiol 64:555–563
Penttilä M, Nevalainen H, Rättö M, Salminen E, Knowels JKC (1987) A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei. Gene 61:155–164
Rauscher R, Würleitner E, Wacenovsky C, Aro N, Stricker AR, Zeilinger S, Kubicek CP, Penttilä M, Mach RL (2006) Transcriptional regulation of xyn1, encoding xylanase I in Hypocrea jecorina. Eukaryotic Cell 5(3):447–456
Saloheimo A, Aro N, Ilmén M, Penttilä M (2000) Isolation of the ace1 gene encoding a Cys(2)-His(2) transcription factor involved in regulation of activity of the cellulase promoter cbh1 of Trichoderma reesei. J Biol Chem 275:5817–5825
Sandgren M, Shaw A, Ropp TH, Wu S, Bott R, Cameron AD, Ståhlberg J, Mitchinson C, Jones TA (2001) The X-ray crystal structure of the Trichoderma reesei family 12 endoglucanase 3, Cel12A, at 1.9 A resolution. J Mol Biol 308(2):295–310
Schmoll M, Kubicek CP (2003) Regulation of Trichoderma cellulase formation: lessons in molecular biology from an industrial fungus. A review. Acta Microbiol Immunol Hung 50:125–145
Seiboth B, Hartl L, Pail M, Fekete E, Karaffa L, Kubicek CP (2004) The galactokinase of Hypocrea jecorina is essential for cellulase induction by lactose but dispensable for growth on D-galactose. Mol Microbiol 51(4):1015–1025
Stricker AR, Grosstessner-Hain K, Würleitner E, Mach RL (2006) Xyr1 (xylanase regulator 1) regulates both the hydrolytic enzyme system and D-xylose metabolism in Hypocrea jecorina. Eukaryot Cell 5(12):2128–2137
Takashima S, Iikura H, Nakamura A, Masaki H, Uozumi T (1996) Analysis of Cre1 binding sites in the Trichoderma reesei cbh1 upstream region. FEMS Microbiol Lett 145(3):361–366
van Paij NN, Visser J, de Graaff LH (1998) Isolation and analysis of xlnR, encoding a transcriptional activator coordinating xylanolytic expression in Aspergillus niger. Mol Microbiol 27(1):131–142
Würleitner E, Pera L, Wacenovsky C, Cziferszky A, Zeilinger S, Kubicek CP, Mach RL (2003) Transcriptional regulation of xyn2 in Hypocrea jecorina. Eukaryot Cell 2(1):150–158
Xu J, Nogawa M, Okada H, Morikawa Y (1998) Xylanase induction by L-sorbose in Trichoderma reesei PC-3–7. Biosci Biotechnol Biochem 62:1555–1559
Xu J, Nogawa M, Okada H, Morikawa Y (2000) Regulation of xyn3 gene expression in Trichoderma reesei PC-3–7. Appl Microbiol Biotechnol 54:370–375
Zeilinger S, Mach RL, Kubicek CP (1998) Two adjacent protein binding motifs in the cbh2 (cellobiohydrolase II-encoding) promoter of the fungus Hypocrea jecorina (Trichoderma reesei) cooperate in the induction by cellulose. J Biol Chem 273:34463–34471
Zeilinger S, Ebner A, Marosits T, Mach RL, Kubicek CP (2001) The Hypocrea jecorina HAP 2/3/5 protein complex binds to the inverted CCAAT-box (ATTGG) within the cbh2 (cellobiohydrolase II-gene) activating element. Mol Genet Genomics 266:56–63
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This work was supported by a grant (Project ID: 06A43502c) from the New Energy and Industrial Technology Development Organization project of the Ministry of Industry and Technology of Japan.
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Shida, Y., Furukawa, T., Ogasawara, W. et al. Functional analysis of the egl3 upstream region in filamentous fungus Trichoderma reesei . Appl Microbiol Biotechnol 78, 515–524 (2008). https://doi.org/10.1007/s00253-007-1338-5
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DOI: https://doi.org/10.1007/s00253-007-1338-5