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PubMed1988

EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme.

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.

Amino Acid Sequence↗

PubMed1989

A lignin peroxidase-encoding cDNA from the white-rot fungus Phlebia radiata: characterization and expression in Trichoderma reesei.

The nucleotide sequence of a cDNA coding for a lignin peroxidase (Lgp) of the white-rot fungus, Phlebia radiata, has been determined. By amino acid (aa) sequencing, it has been shown that the protein product of this gene is the LIII Lgp of Pb. radiata. The isolated gene and the putative aa sequence are about 60% homologous to published Lgp sequences from the fungus, Phanerochaete chrysosporium. The aa thought to be involved in the catalysis of LIII are revealed by comparison with the yeast cytochrome c peroxidase. The P. radiata Lgp-encoding gene (lgp3) was expressed in the fungus, Trichoderma reesei, under the cellobiohydrolase-encoding cbh1 gene promoter. Lgp3 mRNA was produced by the T. reesei transformants. No Lgp protein, however, could be detected.

Agaricales↗

PubMed1991

Promoter structure and expression of the 3-phosphoglycerate kinase-encoding gene (pgk1) of Trichoderma reesei.

Transcription of the 3-phosphoglycerate kinase (PGK)-encoding gene (pgk1) of Trichoderma reesei results in two transcripts due to two main transcription start points (tsp) which are differentially regulated during the growth cycle. The nucleotide sequence of the promoter reveals a number of putative regulatory elements present also in the PGK promoter of Saccharomyces cerevisiae: a 20-nt long sequence similar to the CTTCC-repeat region of the upstream activating sequence UAS, the eukaryotic heat-shock consensus sequence, HSE, and a putative eukaryotic cAMP regulatory sequence. The functionality of the putative HSE sequence was examined, but no clear effect could be seen on the total amount of pgk1 mRNA at elevated temperatures nor on transcription initiation from the upstream tsp, preceded by the HSE sequence.

Amino Acid Sequence↗

PubMed1993

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei.

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.

Amino Acid Sequence↗

PubMed1993

Isolation of Trichoderma reesei genes highly expressed on glucose-containing media: characterization of the tef1 gene encoding translation elongation factor 1 alpha.

Genes that are highly expressed on glucose-containing media were isolated from the filamentous fungus, Trichoderma reesei. A cDNA bank was prepared from glucose-grown fungus, the bank was screened with the same cDNA as a probe, and clones giving the strongest signal were isolated. This resulted in the isolation of previously uncharacterized genes. Five of the genes, representing the most abundant transcripts, corresponded to 1-3% of the total mRNA population and were clearly more highly expressed than the phosphoglycerate kinase-encoding gene (pgk1) of T. reesei. Based on sequence homology, one of the genes was identified as tef1, encoding translation elongation factor 1 alpha (TEF). The T. reesei TEF is most related to the Mucor racemosus TEF3, showing an overall amino acid similarity of 85%. Interestingly, an exon of only 2 bp seems to be present in T. reesei tef1, comprising the first 2 bp of the Gly15 codon.

Amino Acid Sequence↗

PubMed1994

Sequence of the pyr4 gene encoding orotidine-5'-phosphate decarboxylase from the biocontrol fungus Trichoderma harzianum.

The pyr4 gene, encoding orotidine-5'-phosphate decarboxylase (OMP decarboxylase) from the biocontrol fungus Trichoderma harzianum, has been isolated by hybridization, using a polymerase chain reaction (PCR)-derived fragment as a probe. The PCR primers corresponded to conserved regions of OMP decarboxylase-encoding genes from other filamentous fungi. A 2490-bp genomic fragment, which complemented a pyr4-auxotrophic T. reesei mutant, was sequenced. The gene showed high homology to pyr4 genes from other pyrenomycetes.

Amino Acid Sequence↗

PubMed1994

Sequence analysis and expression studies of a gene encoding a novel serine + alanine-rich protein in Trichoderma harzianum.

The promoters of constitutive genes have proven themselves to be useful parts of expression vectors for genetic engineering in different organisms. Based on a differential screening of an induced cDNA library, we have used a new approach to isolate constitutively expressed genes which can be used for this purpose. Following this strategy, we have isolated constitutively expressed cDNA clones of the mycoparasitic fungus Trichoderma harzianum. One of these cDNA clones corresponds to a gene (cob4) that encodes a novel serine + alanine-rich protein. The predicted amino acid (aa) sequence of this protein (171 aa, deduced M(r) 15,564, pI 12.49) displays a motif that is also found in structural proteins of different species and in a yeast thermoinducible protein. Northern (RNA) blot analysis has demonstrated that cob4 is expressed during growth when glucose or cell walls of a phytopathogenic fungus are provided as carbon source. Hybridizations of T. harzianum genomic DNA indicate that this gene most probably belongs to a multigene family.

Alanine↗

PubMed1994

A protein kinase-encoding gene, pkt1, from Trichoderma reesei, homologous to the yeast YPK1 and YPK2 (YKR2) genes.

A gene (pkt1) was isolated from the filamentous fungus Trichoderma reesei, which exhibits high homology with the yeast YPK1 and YKR2 (YPK2) genes. It contains a 2123-bp ORF that is interrupted by two introns, and it encodes a 662-amino-acid protein with a calculated M(r) of 72,820. During active growth, pkt1 is expressed as two mRNAs of 3.1 and 2.8 kb which differ in the 3' untranslated region due to the use of two different polyadenylation sites.

Amino Acid Sequence↗

PubMed1994

Isolation and sequence of an endochitinase-encoding gene from a cDNA library of Trichoderma harzianum.

There are no reports of gene sequences coding for extracellular chitinolytic enzymes from filamentous fungi, even though these enzymes are considered critical to the biological control of plant pathogenic fungi. The purpose of this paper was to report the isolation of a gene (ThEn-42) encoding endochitinase (Ech) from Trichoderma harzianum strain P1, describe its sequence, and to determine whether it was related to genes coding for enzymes with similar functions from prokaryotic or other eukaryotic sources. A clone containing a 1096-bp foreign cDNA fragment was isolated from thalli grown under induced conditions. This cDNA molecule was sequenced and found to lack a portion of the 5' terminus. Polymerase chain reaction (PCR) was used to isolate a fragment from the lambda gt11 library which contained the 5' terminus plus an overlap region with the 1096-bp cDNA clone. The full-length cDNA sequence, consisting of 1554 bp, contained an open reading frame (ORF) expressing a protein of 424 amino acids (aa). Southern analysis of genomic DNA indicated that there is only a single gene in strain P1 with sequence identity to the sequence described in this report. One region within the protein, thought to be required for catalytic activity of the enzyme, was highly conserved between genes coding for Ech from Th, Serratia marcescens, Bacillus circulans, Streptomyces plicatus, Vibrio parahemolyticus and Kluyveromyces lactis.

Amino Acid Sequence↗

PubMed1995

Structure, organization and promoter expression of the actin-encoding gene in Trichoderma reesei.

The single gene encoding actin (Act) in the cellulolytic filamentous fungus Trichoderma reesei (Tr) has been isolated and characterized. The gene contains five introns located in identical positions when compared to the putative ancestral actin genes (act) present in Thermomyces lanuginosus and Aspergillus nidulans. The 5' untranslated region (UTR) of the gene contains a TATA-like sequence (TAATA), a C + T-rich region and a potential CCAAT motif. This region was used as a homologous promoter to direct expression of hygromycin-B-resistance-encoding gene as a dominant-selectable Tr marker.

Actins↗

PubMed1996

Isolation and characterization of a cDNA from Trichoderma harzianum P1 encoding a 14-3-3 protein homolog.

A full-length cDNA close, Th1433, (GenBank accession No. U24158), was isolated and characterized from the filamentous fungus, Trichoderma harzianum. The deduced amino acid (aa) sequence showed an acidic 30-kDa protein homologous to the 14-3-3 proteins, a family of putative kinase regulators originally characterized in mammalian brain tissue. The greatest homology, 71% identical aa, was found to BMH1, the corresponding protein from Saccharomyces cerevisiae and to the epsilon isoform from sheep brain. Southern analysis of genomic DNA indicated that Th1433 is a member of a small genomic family. At least two genes encoding 14-3-3-like proteins exist in T. harzianum. Northern analysis showed the highest level of expression during the first day after inoculation of the culture with conidial spores.

14-3-3 Proteins↗

PubMed1996

The alpha-glucuronidase-encoding gene of Trichoderma reesei.

The Trichoderma reesei cDNA coding for alpha-glucuronidase (GLRI), which releases glucuronic acid attached to xylose units of xylan, was cloned and sequenced. The deduced N-terminal amino acid (aa) sequence of the protein was verified by sequencing of the purified GLRI. The aa sequence of the GLRI displayed no similarity with any aa sequence available in the data bases.

Amino Acid Sequence↗

PubMed1996

Mutants of Trichoderma reesei are defective in cellulose induction, but not basal expression of cellulase-encoding genes.

Four mutants of Trichoderma reesei defective in cellulose utilization were characterized at the molecular level. Genomic analysis of the cellulase-encoding genes (cel) and transcript induction using two well-established inducers of the cel system--the insoluble polymer, cellulose and the soluble inducer, sophorose,--revealed that these mutants are defective in the transcription of cel genes. The results also indicate that the cel genes are coordinately expressed and most probably are regulated by the same mechanism. Using a heterologous gene construct, in which the hygromycin-B-resistance-encoding gene was placed under the control of the promoter of the major cel gene, cbh1, we showed that the mutants synthesize basic levels of cellulase, but are defective in the cel induction.

Cellulase↗

PubMed1995

Production of recombinant proteins in the filamentous fungus Trichoderma reesei.

The potential of the filamentous fungus Trichoderma reesei for producing heterologous proteins has recently been demonstrated with a number of secreted proteins. Rate-limiting steps and ways of improving the production have been studied, especially using antibody Fab fragments. Major improvements have been achieved by producing the foreign protein fused to the fungal cellulase cellobiohydrolase I. In addition to the strong inducible cbh1 promoter, other promoters, which are regulated in a different manner, have been developed.

Genetic Engineering↗

PubMed2005

Production of cellulase by Trichoderma reesei from dairy manure.

Cellulase production by the fungi Trichoderma reesei was studied using dairy manure as a substrate. Data showed that T. reesei RUT-C30 had higher cellulase production than T. reesei QM 9414 and that a homogenized manure, treated by a blender to reduce fiber size, led to higher cellulase production. The cellulase production was further optimized by growing T. reesei RUT-C30 on homogenized manure. The effects of manure concentration, pH, and temperature on cellulase production were investigated with optimal parameter values determined to be 10 g/l manure (dry basis), 25.5 degrees C, and pH 5.7, respectively. Elimination of CaCl2, MgSO4, nitrogen sources (NH4+ and urea) and trace elements (Fe2+, Zn2+, Co2+ and Mn2+) from the original salt solution had no negative influence on the cellulase production, while phosphate elimination did reduce cellulase production. Based on above results, the final medium composition was simplified with manure additives being KH2PO4, tween-80 and CoCl2 only. Using this medium composition and a reaction time of 6-8 days, a maximum cellulase production activity of 1.74 IU/ml of filter paper activity, 12.22 IU/ml of CMCase activity, and 0.0978 IU/ml of beta-glucosidase was obtained. This filter paper activity is the highest ever reported in cellulase production from agricultural wastes.

Animals↗

PubMed2004

Functional analysis of tvsp1, a serine protease-encoding gene in the biocontrol agent Trichoderma virens.

Serine proteases are highly conserved among fungi and considered to play a key role in different aspects of fungal biology. These proteases can be involved in development and have been related to pathogenesis or biocontrol processes. A gene (tvsp1) encoding an extracellular serine protease was cloned from Trichoderma virens, a biocontrol agent effective against soilborne fungal pathogens. The gene was expressed in Escherichia coli and a polyclonal antibody was raised against the recombinant protein. The expression pattern of tvsp1 was determined and its physiological role was addressed by mutational analysis. Strains of T. virens in which tvsp1 was deleted (PKO) or constitutively overexpressed (POE) were not affected in growth rate, conidiation, extracellular protein accumulation, antibiotic profiles nor in their ability to induce phytoalexins in cotton seedlings. Tvsp1 overexpression, however, significantly increased the ability of some strains to protect cotton seedlings against Rhizoctonia solani. Our data show that Tvsp1 is not necessary for the normal growth or development of T. virens, but plays a role in the biocontrol process.

Base Sequence↗

PubMed2004

Insight into Trichoderma reesei's genome content, organization and evolution revealed through BAC library characterization.

Trichoderma reesei is an important industrial fungus known for its ability to efficiently secrete large quantities of protein as well as its wide variety of biomass degrading enzymes. Past research on this fungus has primarily focused on extending its protein production capabilities, leaving the structure of its 33 Mb genome essentially a mystery. To begin to address these deficiencies and further our knowledge of T. reesei's secretion and cellulolytic potential, we have created a genomic framework for this fungus. We constructed a BAC library containing 9216 clones with an average insert size of 125 kb which provides a coverage of 28 genome equivalents. BAC ends were sequenced and annotated using publicly available software which identified a number of genes not seen in previously sequenced EST datasets. Little evidence was found for repetitive sequence in T. reesei with the exception of several copies of an element with similarity to the Podospora anserina transposon, PAT. Hybridization of 34 genes involved in biomass degradation revealed five groups of co-located genes in the genome. BAC clones were fingerprinted and analyzed using fingerprinted contigs (FPC) software resulting in 334 contigs covering 28 megabases of the genome. The assembly of these FPC contigs was verified by congruence with hybridization results.

Chromosome Mapping↗

PubMed2004

The fungal STRE-element-binding protein Seb1 is involved but not essential for glycerol dehydrogenase (gld1) gene expression and glycerol accumulation in Trichoderma atroviride during osmotic stress.

Fungi counteract extracellular osmotic pressure by producing intracellular polyols to prevent loss of water. In yeast osmotic signaling involves a MAP-kinase pathway culminating at the STRE-binding transcription factors Msn2/4. We investigated the role of a putative STRE-binding orthologue of Trichoderma atroviride, Seb1, in osmotic stress signaling. T. atroviride, subjected to osmotic stress (10% glucose or glycerol, 1M KCl or NaCl), responds by raising its intracellular glycerol level. In contrast to Aspergillus nidulans, no erythritol is accumulated. Accumulation of glycerol levels under osmotic stress is strongly reduced in a seb1 deletion strain. To investigate glycerol biosynthesis in T. atroviride, the genes encoding glycerol dehydrogenase (gld1) and glycerol-3-phosphate dehydrogenase (gfd1) were cloned and characterized. Although both genes contain STRE-elements in their 5'-non-coding regions, only gld1 mRNA accumulates in response to osmotic stress, whereas expression of gfd1 remains at a constitutive level. In comparison to A. nidulans gld1 transcript levels in T. atroviride rise very slowly under conditions of salt stress. Deletion of seb1 results in a delayed accumulation of the gld1 transcript, but final levels match those in the wild-type whereas gfd1 transcript accumulation remains unaffected. Assays for glycerol dehydrogenase and glycerol-3-phosphate dehydrogenase enzymatic activities reveal an increase of the former--whereas the latter remains mainly unaffected--in the wild-type and the Deltaseb1 strain under different kinds of osmotic stress. The data suggest that Seb1 is only involved in, but not essential for osmotic stress response which is in contrast to the yeast orthologues Msn2/4.

5' Untranslated Regions↗