Four cellulase genes of Trichoderma reesei, cbh1, cbh2, egl1 and egl2, have been replaced by the amdS marker gene. When linear DNA fragments and flanking regions of the corresponding cellulase locus of more than 1 kb were used, the replacement frequencies were high, ranging from 32 to 52%. Deletion of the major cellobiohydrolase 1 gene led to a 2-fold increase in the production of cellobiohydrolase II; however, replacement of the cbh2 gene did not affect the final cellulase levels and deletion of egl1 or egl2 slightly increased production of both cellobiohydrolases. Based on our results, endoglucanase II accounts for most of the endoglucanase activity produced by the hypercellulolytic host strain. Furthermore, loss of the egl2 gene causes a significant drop in the filter paper-hydrolysing activity, indicating that endoglucanase II has an important role in the total hydrolysis of cellulose.

An electrophoretic karyotype of Trichoderma longibrachiatum (reesei) was obtained using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Seven chromosomal DNA bands were separated in the wild-type T. longibrachiatum strain QM6a. The sizes of the chromosomal DNA bands ranged from 2.8 to 6.9 Mb, giving an estimated total genome size of about 33 Mb. The electrophoretic karyotype of the strain QM6a was compared to three hyper-celluloytic mutant strains, QM9414, RutC30 and VTT-D-79125. The chromosome pattern of the mutant QM9414 was quite similar to that of the wild-type QM6a except that the smallest chromosome differed somewhat in size. The VTT-D-79125 and RutC30 strains, which have undergone several mutagenesis steps, showed striking differences in their karyotype compared to the initial parent. The chromosomal DNA bands were identified using the previously characterized T. longibrachiatum genes (egl1, egl2, cbh1, cbh2, pgk1, rDNA) and random clones isolated from a genomic library. In all strains the cellulase genes cbh1, cbh2 and egl2 were located in the same linkage group (chromosome II in the wild-type), while the main endoglucanase, egl1, hybridized to another chromosomal DNA band (chromosome VI in the wild-type).

Two endoxylanases, Nf Xyn11A and Nf Xyn10A, were cloned from a Nonomuraea flexuosa (previously Actinomadura flexuosa) DSM43186 genomic expression library in Escherichia coli. The coding sequences of xyn11A and xyn10A consist of 344 and 492 amino acids, respectively. The catalytic domains belong to family 11 and family 10 of glycoside hydrolases. The C-termini share strong amino acid sequence similarity to carbohydrate-binding module (CBM) families CBM2 and CBM13, respectively. Native Nf Xyn11A, and recombinant Xyn11A expressed in the filamentous fungus Trichoderma reesei, were purified from cultivation media and characterized. The molecular masses of the full-length enzymes determined by mass spectrometry were 32.9 kDa and 33.4 kDa, the recombinant enzyme having higher molecular mass due to glycosylation. In addition, shorter polypeptides with molecular masses of 23.8 kDa and 22.0 kDa were characterized from the T. reesei culture medium, both lacking the C-terminal CBM and the 22.0 kDa polypeptide also lacking most of the linker region. The recombinant polypeptides were similar to each other in terms of specific activity, pH and temperature dependence. However, the 23.8 kDa and 22.0 kDa polypeptides were more thermostable at 80 degrees C than the full-length enzyme. All polypeptide forms were effective in pretreatment of softwood kraft pulp at 80 degrees C.

The chromosomal cellobiohydrolase 1 locus (cbh1) of the biotechnologically important filamentous fungus Trichoderma reesei was replaced in a single-step procedure by an expression cassette containing an endoglucanase I cDNA (egl1) under control of the cbh1 promoter. CBHI protein was missing from 37-63% of the transformants, showing that targeting of the linear expression cassette to the cbh1 locus was efficient. Studies of expression of the intact cbh1-egl1 cassette at the cbh1 locus revealed that egl1 cDNA is expressed from the cbh1 promoter as efficiently as cbh1 itself. Furthermore, a strain carrying two copies of the cbh1-egl1 expression cassette produced twice as much EG I as the amount of CBHI, the major cellulase protein, produced by the host strain. The level of egl1-specific mRNA in the single-copy transformant was about 10-fold higher than that found in the non transformed host strain, indicating that the cbh1 promoter is about 10 times stronger than the egl1 promoter. The 10-fold increase in the secreted EG I protein, measured with an enzyme-linked immunosorbent assay (ELISA), correlated well with the increase in egl1-specific mRNA.

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