Cloning, functional expression and promoter analysis of xylanase III gene from Trichoderma reesei

Abstract

In this study, the xyn3 gene from the filamentous mesophilic fungus Trichoderma reesei (Hypocrea jecorina) PC-3-7 was cloned and sequenced. Analysis of the deduced amino acid sequence of XYN III revealed considerable homology with xylanases belonging to glycoside hydrolase family 10. These results show that XYN III is distinguishable from XYN I and XYN II, two other T. reesei xylanases that belong to the glycosidase family 11. When xyn3 was expressed in Escherichia coli, significant activity was observed in the cell-free extract, and higher activity (13.2 U/ml medium) was recovered from the inclusion bodies in the cell debris. The sequence of the 5'-upstream region of the gene in the parent strain QM9414 is identical to that of PC-3-7, although the expression level of xyn3 in PC-3-7 has been reported to be at least 1,000 times greater than in QM9414. These results suggest that xyn3 expression in T. reesei QM9414 is silenced. The consensus sequences for ACEI, ACEII, CREI, and the Hap2/3/5 protein complex are all present in the upstream region of xyn3. Deletion analysis of the upstream region revealed that two regions containing consensus sequences for the known regulatory elements play important roles for xyn3 expression.

Application ofTrichoderma reeseiCellulase and Xylanase Promoters through Homologous Recombination for Enhanced Production of Extracellular β-Glucosidase I

“…To place the bgl1 coding region in the context of the xyn3 up-and downstream regions, a blunted 2.4-kb KpnI-SpeI fragment from pCRbgl1 was inserted into the blunted SpeI site of plasmid pBxyn3ag-D0, 7) fusing it to a xyn3 downstream region, which was interrupted by a 3.1-kb DNA fragment of amdS. The resulting plasmid was opened at the SphI site in the 5 0 of the bgl1 coding region, blunted, and …”

“…Functional analyses of the promoter regions of several genes encoding cellulases and xylanases in T. reesei have been reported, [2][3][4][5][6] and we also have tried the analysis of promoters in our previous studies, [7][8][9] but information on how to select a suitable promoter to improve the saccharification of a particular biomass is lacking. Since cellobiohydrolase CBHI (Cel7A) constitutes up to 60% of secreted proteins, 10) the cbh1 promoter is considered the strongest promoter in T. reesei, hence used widely to increase the production of homo-or heterologous protein for specific purposes.…”

Identification of Major Facilitator Transporters Involved in Cellulase Production during Lactose Culture ofTrichoderma reeseiPC-3-7

“…The PC-3-7 strain, which exhibits increased cellulase activity under inducing conditions, has been used as a model of cellulase induction in our laboratory. [20][21][22] This mutant was developed following several rounds of random mutagenesis. 23,24) During screening for hyper-cellulolytic strains, we found that this strain exhibited an absence of carbon catabolite repression, and that it produced high amounts of cellulases during induction by cellulose, sophorose, and the monosaccharide L-sorbose.…”

Xylanase Gene Transcription in Trichoderma reesei Is Triggered by Different Inducers Representing Different Hemicellulosic Pentose Polymers

“…With regard to its xylanases, four have been purified and characterized: two members of glycosyl hydrolase family 11 (GH11) (XYN1 and XYN2) (3,4), the GH10 member XYN3 (5,6), and the GH30 member XYN4 (7). While the first three are all endo-␤-1,4-xylanases, XYN4 is classified as a xylan 1,4-␤-xylosidase, because it produces D-xylose as the main end product from xylan.…”