The endophytic fungus named FSN006 was isolated from the inner bark of Juglans mandshurica. It grew quickly and formed circular colony on PDA plate. The upper side of the colony was white, while the lower side of the colony and the conditioned medium were light yellow as a result of significant yellow pigment substances were produced and secreted by the fungi. Green elliptic conidia appeared when cultured on CMX plate. Based on the morphology identification and ITS sequence, it was clear that this fungus belonged to the Deuteromycotina, HyPhomycetes, Moniliales, Trichoderma longibrachiatum. The conditioned medium of FSN006 showed a high anti-tumor ability against liver cancer cell-HepG2, and reached its IC50 concentration after being diluted 20 times, while the IC50 concentration of curcumine was(11.49 +/- 0.12) mg x L(-1). In addition, there was preeminent selective inhibiting effect against the normal liver cell strain HL-7702 and its caner counter strain HepG2. The inhibiting effect against strain HL-7702 was only one quarter of that against HepG2 at the concentration of IC50. Therefore, the fermentation of FSN006 may provide a possible way to produce anticancer drug with higher efficiency and lower toxicity.

A novel endoglucanase from Trichoderma reesei, EGIII, has been purified and its catalytic properties have been studied. The gene for that enzyme (egl3) and cDNA have been cloned and sequenced. The deduced EGIII protein shows clear sequence homology to a Schizophyllum commune enzyme (M. Yaguchi, personal communication), but is very different from the three other T. reesei cellulases with known structure. Nevertheless, all the four T. reesei cellulases share two common, adjacent sequence domains, which apparently can be removed by proteolysis. These homologous sequences reside at the N termini of EGIII and the cellobiohydrolase CBHII, but at the C termini of EGI and CBHI. Comparison of the fungal cellulase structures has led to re-evaluation of hypotheses concerning the localization of the active sites.

The cre1 genes of the filamentous fungi Trichoderma reesei and T. harzianum were isolated and characterized. The deduced CREI proteins are 46% identical to the product of the glucose repressor gene creA of Aspergillus nidulans, encoding a DNA-binding protein with zinc fingers of the C2H2 type. The cre1 promoters contain several sequence elements that are identical to the previously identified binding sites for A. nidulans CREA. Steady-state mRNA levels for cre1 of the T. reesei strain QM9414 varied depending on the carbon source, being low on glucose-containing media. These observations suggest that cre1 expression may be autoregulated. The T. reesei strain Rut-C30, a hyper-producer of cellulolytic enzymes, was found to express a truncated form of the cre1 gene (cre1-1) with an ORF corresponding to a protein of 95 amino acids with only one zinc finger. Unlike QM9414 the strain Rut-C30 produced cellulase mRNAs on glucose-containing medium and transformation of the full-length cre1 gene into this strain caused glucose repression of cbh1 expression, demonstrating that cre1 regulates cellulase expression.

A method is presented for the isolation of genes encoding hydrolytic enzymes without any knowledge of the corresponding proteins. cDNA made from the organism of interest is cloned into a yeast vector to construct an expression library in the yeast Saccharomyces cerevisiae. Colonies producing hydrolytic enzymes are screened by activity plate assays. In this work, we constructed a yeast expression library from the filamentous fungus Trichoderma reesei and isolated a new beta-1,4-endoglucanase gene on plates containing beta-glucan. This gene, egl5, codes for a previously unknown small protein of 242 amino acids. Despite its small size, the protein contains two conservative domains found in Trichoderma cellulases, namely the cellulose-binding domain (CBD) and the linker region that connects the CBD to the catalytic core domain. Molecular modelling of the EGV CBD revealed some interesting structural differences compared to the CBD of the major cellulase CBHI from T. reesei. The catalytic core of EGV is unusually small for a cellulase and represents a new family of cellulases (Family K) and of glycosyl hydrolases (Family 45) together with the endoglucanase B of Pseudomonas fluorescens and the endoglucanase V of Humicola insolens on the basis of hydrophobic cluster analysis.

Two different cellobiohydrolases, CBHI and CBHII, of the filamentous fungus Trichoderma reesei both hydrolyse highly crystalline cellulose. Cellulolytic strains of the yeast Saccharomyces cerevisiae were constructed by transferring cDNAs coding for these enzymes into yeast on an expression plasmid. These cellulolytic yeasts were able to secrete efficiently the large, heterologous proteins to the culture medium. The recombinant cellulases were observed to be heterogeneous in Mr due, at least partly, to variable N-glycosylation. Recombinant CBHII was able to bind to crystalline cellulose, although slightly less efficiently than the native enzyme. Both of the two recombinant cellulases were able to degrade amorphous cellulose. In a fermenter cultivation, around 100 micrograms/ml of CBHII was secreted into the yeast growth medium.

The relationship of the important cellulase producing asexual fungus Trichoderma reesei to its putative teleomorphic (sexual) ancestor Hypocrea jecorina and other species of the Trichoderma sect. Longibrachiatum was studied by PCR-fingerprinting and sequence analyses of the nuclear ribosomal DNA region containing the internal transcribed spacers (ITS-1 and ITS-2) and the 5.8S rRNA gene. The differences in the corresponding ITS sequences allowed a grouping of anamorphic (asexual) species of Trichoderma sect. Longibrachiatum into Trichoderma longibrachiatum, Trichoderma pseudokoningii, and Trichoderma reesei. The sexual species Hypocrea schweinitzii and H. jecorina were also clearly separated from each other. H. jecorina and T. reesei exhibited identical sequences, suggesting close relatedness or even species identity. Intraspecific and interspecific variation in the PCR-fingerprinting patterns supported the differentiation of species based on ITS sequences, the grouping of the strains, and the assignment of these strains to individual species. The variations between T. reesei and H. jecorina were at the same order of magnitude as found between all strains of H. jecorina, but much lower than the observed interspecific variations. Identical ITS sequences and the high similarity of PCR-fingerprinting patterns indicate a very close relationship between T. reesei and H. jecorina, whereas differences of the ITS sequences and the PCR-fingerprinting patterns show a clear phylogenetic distance between T. reesei/H. jecorina and T. longibrachiatum. T. reesei is considered to be an asexual, clonal line derived from a population of the tropical ascomycete H. jecorina.

Transformation systems developed for Trichoderma spp. were utilized to improve the biocontrol efficiency of the mycoparasitic fungus Trichoderma harzianum by increasing the copy number of the basic proteinase gene prb1. The transformants were stable and carried from two to ten copies of prb1. High levels of expression of prb1 during fungus-fungus interaction were detected when T. harzianum and Rhizoctonia solani were confronted in vitro. In liquid cultures the proteinase was induced by cell walls of R. solani. Under greenhouse conditions, incorporation of T. harzianum transformants into pathogen-infested soil significantly reduced the disease caused by R. solani in cotton plants.

A 72-kDa N-acetyl-beta-D-glucosaminidase was purified from the mycoparasitic fungus Trichoderma harzianum P1; antibodies were raised against it, and aa-sequences were obtained. The antibody reacted with a single 72-kDa protein band in culture filtrates of T. harzianum grown on chitin, and was subsequently used to clone the corresponding nag1 gene from a lambdagt11 cDNA expression library. It was interrupted by two short introns and encoded a protein of 580 amino acids. The deduced protein sequence contained aa-sequence areas of high similarity to N-acetyl-glucosaminidases from other eukaryotes such as Candida albicans, and invertebrate and vertebrate animal tissues. The highest similarity was observed with the corresponding gene from the silkworm. The aa-sequence of a tryptic fragment of purified N-acetyl-beta-D-glucosaminidase from T. harzianum corresponded to a deduced aa sequence from a portion of the cloned gene, thus verifying that the protein is encoded by nag 1. Southern analysis showed that nag 1 is present as a single-copy gene in T. harzianum. Expression of nag1-mRNA was strongly induced upon growth on chitin, N-acetyl-glucosamine and the cell walls of Botrytis cinerea used as a carbon source. The appearance of the corresponding N-acetyl-beta-D-glucosaminidase protein, as determined by Western analysis, paralleled the pattern of nag 1 expression, thereby suggesting that its formation is regulated at the level of transcription.

Regulation of formation of the extracellular xylanase system of Trichoderma reesei QM 9414 during growth on xylan, cellulose, and replacement onto a number of soluble inducers was investigated by Northern analysis of xyn1 and xyn2 transcripts and by the use of the Escherichia coli hph (hygromycin B-phosphotransferase-encoding) gene as a reporter. Whereas the xyn1 promoter is active in the presence of xylan and xylose, and virtually silenced in the presence of glucose, the xyn2 promoter enables basal transcription at a low level, but is enhanced in the presence of xylan and xylobiose and also of sophorose or cellobiose. The respective regulatory nucleotide regions were localized on a 221-base pair fragment and a 55-base pair fragment of the xyn1 and xyn2 5'-upstream noncoding sequences, respectively. Electrophoretic mobility shift assays, using cell-free extracts, identified induction-specific protein-DNA complexes: one complex of high mobility was observed under basal, noninduced conditions (glucose) with xyn2, which was in part replaced by a slow-migrating complex upon induction by xylan or sophorose. Both complexes bound to a CCAAT box. With xyn1, the induced complex also binds to a CCAAT box, but this binding is not observed in the presence of the carbon catabolite repressor Cre1, which binds to a nearby located consensus motif.

The cDNA copies of the two endo-beta-1,4-glucanase genes, egl1 and egl3, from the filamentous fungus Trichoderma reesei were expressed in yeast Saccharomyces cerevisiae under the control of the yeast phosphoglycerate kinase gene promoter. Active EGI and EGIII enzyme was produced and secreted by yeast into the growth medium. The recombinant EGI enzyme was larger and more heterogeneous in size than the native enzyme secreted by Trichoderma, due to differences in the extent of N-glycosylation between these two organisms. The morphology of the yeast cells producing EGI or EGIII was clearly different from control strain.