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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↗

PubMed2006

H+ -mediated coupling of transmembrane Ca2+ fluxes in vegetative Trichoderma viride mycelia suggested by the study of ageing and adaptation to extreme Ca2+ concentrations.

The adaptation to extreme concentrations of Ca(2+) and its consequence on the properties of the (45)Ca(2+) transport were studied in submerged mycelia of Trichoderma viride. The adaptation to low [Ca(2+)](o) did not cause changes in kinetic parameters of the (45)Ca(2+) influx but the adaptation to high [Ca(2+)](o) increased the K(M(Ca2+)). The V(max) of the (45)Ca(2+) influx decreased with the age of (non-adapted) mycelia with concomitant decrease of the K(M(Ca2+)) these changes were prevented in mycelia adapted to high Ca(2+). High [Ca(2+)](o) decreased the stimulation by the uncoupler, 3, 3', 4', 5-tetrachloro salicylanilide (TCS) (30 muM), as compared to the control, whereas the Ca(2+) chelator, EGTA, stimulated it. In the aged mycelia, the stimulation by TCS of the (45)Ca(2+) influx faded away, in parallel with the activity of the H(+)-ATPase. The (45)Ca(2+) efflux from mycelia was affected by TCS in a similar way as the (45)Ca(2+) influx. The results demonstrate the adaptive responses of transport processes participating in the mycelial Ca(2+) homeostasis and ageing are in agreement with a notion that both Ca(2+)-influx and-efflux are coupled by the H(+)-homeostasis at the plasma membrane.

Calcium↗

PubMed2005

Overexpression and characterization of a novel chitinase from Trichoderma atroviride strain P1.

We describe the overexpression and characterization of a new 30 kDa family 18 chitinase (Ech30) from Trichoderma atroviride strain P1. Sequence alignments indicate that the active site architecture of Ech30 resembles that of endochitinases such as hevamine from the rubber tree (Hevea brasiliensis). The ech30 gene was overexpressed in Escherichia coli without its signal peptide and with an N-terminal His-tag. The enzyme was produced as inclusion bodies, from which active chitinase could be recovered using a simple refolding procedure. The enzyme displayed an acidic pH-optimum (pH 4.5-5.0), probably due to the presence of a conserved Asn residue near the catalytic glutamate, which is characteristic for acidic family 18 chitinases. Studies with oligomers of N-acetylglucosamine [(GlcNAc)(n)], 4-methylumbelliferyl (4-MU) labelled GlcNAc oligomers and beta-chitin reveal enzymatic properties typical of an endochitinase: 1) low activity towards short substrates (kinetic parameters for the hydrolysis of 4-MU-(GlcNAc)2 were K(m), 149+/-29 microM and k(cat), 0.0048+/-0.0005 s(-1)), and 2) production of relatively large amounts of trimers and tetramers during degradation of beta-chitin. Detailed studies with GlcNAc oligomers indicated that Ech30 has as many as seven subsites for sugar binding. As expected for a family 18 chitinase, catalysis proceeded with retention of the beta-anomeric configuration.

Acetylglucosamine↗

PubMed2006

MRSP1, encoding a novel Trichoderma secreted protein, is negatively regulated by MAPK.

A novel gene, MRSP1 (MAP kinase repressed secreted protein 1) is strongly overexpressed in the tmkA MAPK mutant of the biocontrol fungus Trichoderma virens. Transcriptional regulation of MRSP1 is determined by presence or absence of TmkA, rather than by light or sporulation, making it a molecular marker for the unusual, negative, regulation by TmkA. The predicted protein is 15.9 kDa, has a secretory signal, and the four-cysteine pattern, C-X29-CP(G)C-X31-C, may define a new cysteine-rich motif. This is a novel protein with functions not known from any other organism. Conservation in ascomycete, basidiomycete, and Dictyostelium homologs, as well as tight MAPK regulation, might indicate important cellular functions.

Cells, Cultured↗

PubMed2005

Directed evolution for engineering pH profile of endoglucanase III from Trichoderma reesei.

The potential of cellulase has been revealed not only in biomass conversion but also in various industrial processes, including food, textiles, laundry, pulp, and paper. Due to the need for alkali-tolerant cellulase with high specific activity at alkaline pH, for example, for application in detergent industry an error-prone PCR approach was employed for enhancing the alkali-tolerant ability of endoglucanase III (EG III) from Trichoderma reesei by error-prone PCR. One mutant (N321T) which exhibited an optimal activity at pH 5.4, corresponded to a basic shift of 0.6 pH unit compared to the wild-type enzyme, was selected and characterized. In addition, two site-directed mutations, N321D and N321H, were designed to study the role of residue at position 321. As expected, the N321D mutation changed enzyme's optimal activity to pH 4.0, resulting in a large decrease in the specific activity. However, the N321H mutated enzyme was active over a broader pH range compared to the wild type, with no much change in the specific activity. These properties suggest that the residue at position 321 is important amino acid residue in determining the pH activity profile of the EG III from T. reesei.

Amino Acid Sequence↗

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↗

PubMed2005

Xylanase production by Trichoderma reesei Rut C-30 grown on L-arabinose-rich plant hydrolysates.

The suitability of L-arabinose-rich plant hydrolysates as carbon sources and inducers of xylanase production in Trichoderma reesei Rut C-30 was tested. Significantly higher xylanase activities were obtained in cultures on oat husk and sugar beet pulp hydrolysates than on lactose. In batch culture with oat husk hydrolysate and lactose, the xylanase activity was about 9 times higher ( approximately 510 IU/ml) than in lactose ( approximately 60 IU/ml). Even higher xylanase activity ( approximately 630 IU/ml) was obtained when the batch cultivations were done on sugar beet pulp hydrolysate and lactose. In a fed-batch culture using oat husk hydrolysate-lactose the xylanase activity was as high as 1350 IU/ml in 4 days. The cellulase production clearly decreased when T. reesei was cultured on both hydrolysates compared to the cultivation on lactose. Moreover, the relative amounts of the xylanases I-III were similar regardless the used carbon source.

Arabinose↗