Search

The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1 . In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1 - 1 , found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T . reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions. There was no phenotypic difference between the Δ cre1 and cre1 - 1 mutant strains in any of the experiments done, indicating that the cre1 - 1 gene is practically a null allele. The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T . reesei .

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH‐7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate‐limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate‐binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4–10°C) and more active (two‐ to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45°C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70°C, however, was the 2‐module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three‐dimensional homology models of these enzymes.

The extracellular bga1-encoded β-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 ± 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl- and alkyl-β-d-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-β-d-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is ∼ 5.0, and the optimum temperature was found to be 60 °C. Bga1 is also capable of releasing d-galactose from β-galactans and is thus actually a galacto-β-d-galactanase. β-Galactosidase is inhibited by its reaction product d-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.

A bacterial xylanase gene, Nonomuraea flexuosa xyn11A , was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides. By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures. The carriers tested were the T. reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T. reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof. The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide. The recombinant Xyn11A produced had properties similar to those of the native xylanase. It constituted 6 to 10% of the total proteins secreted by the transformants. About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed. Even in the best Xyn11A producers, the levels of the fusion mRNAs were only ∼10% of the level of cel7A ( cbh1 ) mRNA in the untransformed host strain.

Mashups that combine already existing data into an integrated experience are becoming increasingly popular. So far most mashups have been built around maps and images. However, as the web is growing increasingly ubiquitous media, also multimedia content - sound, video and even small programs - is becoming a candidate for mashup creation, with potentially superior user experience. Currently available methods to implement mashups do not allow effortless access to personal data across domains or provide means to ensure that the user experience is coherent. This paper describes our mobile mashup ecosystem aimed to solve these problems and work as a ubiquitous platform for mobile multimedia mashups. Furthermore, we provide a brief literature review about existing mashup frameworks and end-user programming. In addition, to constitute a basis for future work, we discuss about challenges expected to emerge when the ecosystem is implemented.

Melanocarpus albomyces laccase crystals were soaked with 2,6-dimethoxyphenol, a common laccase substrate. Three complex structures from different soaking times were solved. Crystal structures revealed the binding of the original substrate and adducts formed by enzymatic oxidation of the substrate. The dimeric oxidation products were identified by mass spectrometry. In the crystals, a 2,6-dimethoxy-p-benzoquinone and a C–O dimer were observed, whereas a C–C dimer was the main product identified by mass spectrometry. Crystal structures demonstrated that the substrate and/or its oxidation products were bound in the pocket formed by residues Ala191, Pro192, Glu235, Leu363, Phe371, Trp373, Phe427, Leu429, Trp507 and His508. Substrate and adducts were hydrogen-bonded to His508, one of the ligands of type 1 copper. Therefore, this surface-exposed histidine most likely has a role in electron transfer by laccases. Based on our mutagenesis studies, the carboxylic acid residue Glu235 at the bottom of the binding site pocket is also crucial in the oxidation of phenolics. Glu235 may be responsible for the abstraction of a proton from the OH group of the substrate and His508 may extract an electron. In addition, crystal structures revealed a secondary binding site formed through weak dimerization in M. albomyces laccase molecules. This binding site most likely exists only in crystals, when the Phe427 residues are packed against each other.

The extracellular bga1-encoded beta-galactosidase of Hypocrea jecorina (Trichoderma reesei) was overexpressed under the pyruvat kinase (pki1) promoter region and purified to apparent homogeneity. The monomeric enzyme is a glycoprotein with a molecular mass of 118.8 +/- 0.5 kDa (MALDI-MS) and an isoelectric point of 6.6. Bga1 is active with several disaccharides, e.g. lactose, lactulose and galactobiose, as well as with aryl- and alkyl-beta-D-galactosides. Based on the catalytic efficiencies, lactitol and lactobionic acid are the poorest substrates and o-nitrophenyl-beta-D-galactoside and lactulose are the best. The pH optimum for the hydrolysis of galactosides is approximately 5.0, and the optimum temperature was found to be 60 degrees C. Bga1 is also capable of releasing D-galactose from beta-galactans and is thus actually a galacto-beta-D-galactanase. beta-Galactosidase is inhibited by its reaction product D-galactose and the enzyme also shows a significant transferase activity which results in the formation of galacto-oligosaccharides.

We have previously shown that the Nonomuraea flexuosa Xyn11A polypeptides devoid of the carbohydrate binding module (CBM) have better thermostability than the full-length xylanase and are effective in bleaching of pulp. To produce an enzyme preparation useful for industrial applications requiring high temperature, the region encoding the CBM was deleted from the N. flexuosa xyn11A gene and the truncated gene was expressed in Trichoderma reesei . The xylanase sequence was fused to the T. reesei mannanase I (Man5A) signal sequence or 3′ to a T. reesei carrier polypeptide, either the Man5A core/hinge or the cellulose binding domain (CBD) of cellobiohydrolase II (Cel6A, CBHII). The gene and fusion genes were expressed using the cellobiohydrolase 1 ( cel7A , cbh1 ) promoter. Single-copy isogenic transformants in which the expression cassette replaced the cel7A gene were cultivated and analyzed. The transformants expressing the truncated N. flexuosa xyn11A produced clearly increased amounts of both the xylanase/fusion mRNA and xylanase activity compared to the corresponding strains expressing the full-length N. flexuosa xyn11A . The transformant expressing the cel6A CBD-truncated N. flexuosa xyn11A produced about 1.9 g liter −1 of the xylanase in laboratory-scale fermentations. The xylanase constituted about 25% of the secreted proteins. The production of the truncated xylanase did not induce the unfolded protein response (UPR) pathway. However, the UPR was induced when the full-length N. flexuosa xyn11A with an exact fusion to the cel7A terminator was expressed. We suggest that the T. reesei folding/secretion machinery is not able to cope properly with the bacterial CBM when the mRNA of the full-length N. flexuosa xyn11A is efficiently translated.

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.

DVB-H (digital video broadcasting for handheld) is one of the key technologies that enables new services, such as mobile phone TV, to be used in handhelds. DVB-H enhances mobile features found in the DVB-T standard, enabling new techniques, e.g., loss-free handover. For services such as mobile TV, loss-free handovers must be supported. Different positioning technologies, such as GPS, also play an important role in enhancing today's mobile technologies. The paper proposes a new feature for DVB-H, a new method for signalling cell coverage area, thus improving performance for loss-free handovers. The proposed method is targeted at DVB-H handhelds that have GPS receivers. The benefits of the proposed method for handover and power consumption are evaluated. In addition, its effect on the consumption of bandwidth and receiver memory is estimated.

The aim of this work was to genetically modify the carbon catabolite repression of Trichoderma reesei to obtain mutant strains derepressed in cellulase production. Several genes coding for regulators of cellulase expression have been isolated and characterized from T. reesei including the cre1 gene mediating the carbon catabolite repression in the presence of glucose. Glucose repression has been shown to occur upon binding of CREI protein to specific sequences in the promoter of the major cellulase gene cbh1. CREI target sequences have also been identified in the promoter regions of other cellulase and hemicellulase genes such as e.g. cbh2 and xyn1. The CREI protein of T. reesei is similar to many other fungal proteins mediating glucose repression. It was recently shown, however, that a truncated form of CREI (cre1-1) present in the hypercellulolytic T. reesei strain Rut-C30 is responsible for derepression of (hemi)cellulase gene expression on glucose-containing media. Therefore, to study the effect of cre1 on cellulase and hemicellulase expression, the wild type cre1 gene present in the T. reesei strain QM6a was either replaced by the "Rut-C30 -type" truncated cre1 -1 gene or completely removed. Bioreactor cultivations on lactose and glucose media were carried out with these cre1-1 and delta-cre1 mutant strains and analysed for cellulase and hemicellulase production. Northern analysis indicated remarkably higher expression levels of several (hemi)cellulase genes in both mutant strains on lactose when compared to the non-modified parent strain. In accordance, 20-fold higher cellobiohydrolase and endoglucanase activities and almost 10-fold higher xylanase activities were detected in the lactose culture medium of mutant strains.

Trichoderma reesei has a long history in industrial enzyme production because of its high protein secretion capability and easy processability. Despite its many positive features, further strain engineering is still being pursued to remove any remaining protein production and secretion bottlenecks. Systems biology methods were applied to study protein production in selected proprietary T. reesei strains. Transcriptional analysis using microarrays was performed to identify differentially expressed genes in various cultivation conditions and high-density Comparative Genomic Hybridization (CGH) was used to characterize genomic alterations in the strains. Combined data from CGH and transcriptional analyses was utilized to identify target genes for further improvement of protein production/secretion. Deletions and/or overexpressions of 16 genes involved in protein transport, post-translational modification, basic energy metabolism or regulatory functions were studied in detail.