Production in Trichoderma reesei of three xylanases from Chaetomium thermophilum: a recombinant thermoxylanase for biobleaching of kraft pulp

Abstract

Three endoxylanase genes were cloned from the thermophilic fungus Chaetomium thermophilum CBS 730.95. All genes contained the typical consensus sequence of family 11 glycoside hydrolases. Genomic copies of Ct xyn11A, Ct xyn11B, and Ct xyn11C were expressed in the filamentous fungus T. reesei under the control of the strong T. reesei cel7A (cellobiohydrolase 1, cbh1) promoter. The molecular masses of the Ct Xyn11A, Ct Xyn11B, and Ct Xyn11C proteins on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were 27, 23, and 22 kDa, respectively. Ct Xyn11A was produced almost as efficiently as the homologous xylanase II from a corresponding single-copy transformant strain. Ct Xyn11B production level was approximately half of that of Ct Xyn11A. The amount of Ct Xyn11C was remarkably lower. Ct Xyn11A had the highest temperature optimum and stability of the recombinant xylanases and the highest activity at acid-neutral pH (pH 5-7). It was the most suitable for industrial bleaching of kraft pulp at high temperature.

Cell-free pipeline for discovery of thermotolerant xylanases and endo -1,4-β-glucanases

“…This lack of activity may be a result of the absence of the catalytic domains' flanking sequences, but also is likely to be a consequence of the structural heterogeneity across the enzyme class that led to erroneous identification and annotation and is indirectly supported by the observation that the structurally uniform xylanase library contained only three inactive candidates. One of these xylanases originates from a thermophilic organism and has been previously characterized to be active at temperatures above 50 °C (Mäntylä et al, 2007). Despite the fact that the volume of the cell-free expressed enzyme was not adjusted to the expression levels, we observed no correlation between the expression levels and the activity.…”

Deciphering the molecular mechanisms behind cellulase production in Trichoderma reesei, the hyper-cellulolytic filamentous fungus

“…Contemporary recombinant DNA technology, on the other hand, provides more precise methods for obtaining cellulase-overproducing strains. [95][96][97][98][99] In T. reesei, maximum extracellular protein production under optimal culture conditions was reported as 30 g/l 21) from 10% carbon sources. Considering the limitations of carbon utilization, it may be impossible to prepare cellulase-overproducing strains with further high productivity.…”

“…100,101) Generally, a strong cbh1 promoter is used for heterologous protein expression in T. reesei. [95][96][97][98][99] However, for the expression of a heterologous gene encoding an effective glycoside hydrolase, the use of cbh1 promoter brings some limitations: (i) the expression construct should be integrated in the genomic location other than the cbh1 locus because CBHI is an essential enzyme for cellulose saccharification, (ii) the titration of the transcription factor may affect the expression of other hydrolase genes, (iii) the importance of the ratio of the expressed heterologous cellulase in the enzyme cocktail should be taken into account. Taking all these into consideration, evaluation of the cbh1, cbh2, egl1, egl3, and xyn3 promoters was carried out using GUS reporter system to select the promoter of T. reesei cellulase and xylanase genes with suitable expression strength for a target gene.…”

Consistent mutational paths predict eukaryotic thermostability

“…Several crystal structures of proteins from these thermophilic fungi have been determined such as those of two beta 1,4-galactanases from T.heterothallica [13], a glycoside hydrolase from T. terrestris [14], and Get3, Get4 and beta 1,4-xylanase from C. thermophilum [15-17]. The paper-industry utilizes members of the beta 1,4-xylanase family for bio-bleaching of kraft-pulp [18,19]. The biotechnological potential of C. thermophilum is also illustrated by the purification and characterization of its thermostable superoxide dismutase (SOD) [20], an enzyme which is utilized in cosmetic products to reduce free radical damage to the skin.…”