Cloning, sequencing and enhanced expression of the Trichoderma reesei endoxylanase II (pI 9) gene xln2

Saarelainen, Ritva; Paloheimo, Marja; Fagerstrom, Richard M.; Suominen, Pirkko; Nevalainen, Helena

doi:10.1007/bf00279891

Cited by 64 publications

(31 citation statements)

References 35 publications

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“…The probes for Northern analyses were the entire cDNAs of cbh1, cbh2 (15), egl1 (16), and egl2 (previously called egl3) (17) released from vector sequences. The probes for the ␤-xylanases xyn1 and xyn2 (18,19) were 350-bp fragments prepared by polymerase chain reaction (8). The membranes were hybridized with the actin fragment and a glyceraldehyde-3-phosphate dehydrogenase encoding (gpd1) cDNA fragment as internal RNA loading controls.…”

Section: Methodsmentioning

confidence: 99%

ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei

Aro

Saloheimo

Ilmén

et al. 2001

Journal of Biological Chemistry

300

240

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A novel yeast-based method to isolate transcriptional activators was applied to clone regulators binding to the cellulase promoter cbh1 of the filamentous fungus Trichoderma reesei (Hypocrea jecorina). This led to the isolation of the cellulase activator ace2 encoding for a protein belonging to the class of zinc binuclear cluster proteins found exclusively in fungi. The DNA-binding domain of ACEII was expressed as a glutathione S-transferase fusion protein in Escherichia coli, and ACEII was shown to bind in vitro to the 5-GGCTAATAA site present in the cbh1 promoter. This site also contains the proposed binding sequence of the xylanase activator XlnR of Aspergillus niger. Mutation of the GGC triplet abolished ACEII binding. The function of ACEII was studied by analyzing the effects of ace2 deletion in the hypercellulolytic T. reesei strain ALKO2221. Deletion of the ace2 gene led to lowered induction kinetics of mRNAs encoding the major cellulases cellobiohydrolases I and II and endoglucanases I and II and to 30 -70% reduced cellulase activity when the fungus was grown on medium containing Solka floc cellulose. The expression level of the gene encoding xylanase was also affected. ace2 deletion led to lowered xyn2 expression in cellulose-induced cultivation. Cellulase induction by sophorose was not affected by ace2 deletion.

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Section: Methodsmentioning

confidence: 99%

ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei

Aro

Saloheimo

Ilmén

et al. 2001

Journal of Biological Chemistry

300

240

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“…However, despite several attempts at optimization, including modification of codon usage of heterologous genes for Microbiology 158 Microscopy has been used to visually identify bottlenecks along the secretory pathway of heterologous proteins. For example, the expression and secretion of homologous CBHI and a heterologous barley endopeptidase (EPB) in a RUT-C30-based transformant (Saarelainen et al, 1997) was studied using in situ hybridization, indirect immunofluorescence and immunoelectron microscopy (Nykänen et al, 1997). Recombinant EPB appeared trapped in the ER and in spherical vesicles in the apical regions, whereas both cbh1 mRNA and CBHI were distributed throughout the hyphae and particularly localized close to the plasma membrane in elongated vesicles and in the cell walls (Nykänen et al, 1997).…”

Section: Rut-c30 As a Host For Heterologous Protein Productionmentioning

confidence: 99%

Trichoderma reesei RUT-C30 – thirty years of strain improvement

2012

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The hypersecreting mutant Trichoderma reesei RUT-C30 (ATCC 56765) is one of the most widely used strains of filamentous fungi for the production of cellulolytic enzymes and recombinant proteins, and for academic research. The strain was obtained after three rounds of random mutagenesis of the wild-type QM6a in a screening program focused on high cellulase production and catabolite derepression. Whereas RUT-C30 achieves outstanding levels of protein secretion and high cellulolytic activity in comparison to the wild-type QM6a, recombinant protein production has been less successful. Here, we bring together and discuss the results from biochemical-, microscopic-, genomic-, transcriptomic-, glycomic-and proteomic-based research on the RUT-C30 strain published over the last 30 years.

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“…H. jecorina forms two specific endo-␤-1,4-xylanases, Xyn1 and Xyn2 (EC 3.2.1.8), and the respective genes (xyn1 and xyn2) have been cloned (27,34). Expression of xyn1 is induced by D-xylose and is repressed by glucose in a Cre1-dependent manner (18,44), whereas the expression of xyn2 is partially constitutive and further induced by xylobiose, xylan, cellulose, and sophorose (44).…”

mentioning

confidence: 99%

Transcriptional Regulation of xyn2 in Hypocrea jecorina

Würleitner¹,

Pera²,

Wacenovsky³

et al. 2003

Eukaryot Cell

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The xylanase system of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) consists of two specific xylanases, Xyn1 and Xyn2, which are simultaneously expressed during growth on xylan but respond differentially to low-molecular-weight inducers. Using in vivo footprinting analysis of xylan-induced and noninduced mycelia, we detected two adjacent nucleotide sequences (5-AGAA-3 on the noncoding strand and 5-GGGT AAATTGG-3, referred to as the xylanase-activating element [XAE], on the coding strand, respectively) to bind proteins. Among these, binding to the AGAA-box is only observed under noninduced conditions, whereas binding to XAE is constitutive. Electrophoretic mobility shift assay with heterologously expressed components of the H. jecorina Hap2/3/5 protein complex and the cellulase regulator Ace2 suggests that these two transactivators form the protein complex binding to XAE. H. jecorina transformants, containing correspondingly mutated versions of the xyn2 promoter fused to the Aspergillus niger goxA gene as a reporter, revealed that the elimination of protein binding to the AGAA-box resulted in a threefold increase in both basal and induced transcription, whereas elimination of Ace2 binding to its target in XAE completely eliminated transcription under both conditions. Destruction of the CCAAT-box by insertion of a point mutation prevents binding of the Hap2/3/5 complex in vitro and results in a slight increase in both basal and induced transcription. These data support a model of xyn2 regulation based on the interplay of Hap2/3/5, Ace2 and the AGAA-box binding repressor.

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Cloning, sequencing and enhanced expression of the Trichoderma reesei endoxylanase II (pI 9) gene xln2

Cited by 64 publications

References 35 publications

ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei

ACEII, a Novel Transcriptional Activator Involved in Regulation of Cellulase and Xylanase Genes of Trichoderma reesei

Trichoderma reesei RUT-C30 – thirty years of strain improvement

Transcriptional Regulation of xyn2 in Hypocrea jecorina

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