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PubMed2000

Regulation of 36-kDa beta-1,3-glucanase synthesis in Trichoderma harzianum.

The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.

Blotting, Western↗

PubMed2000

Characterization of an amylase produced by a Trichoderma harzianum isolate with antagonistic activity against Crinipellis perniciosa, the causal agent of witches' broom of cocoa.

An isolate of Trichoderma harzianum showing antagonistic activity against Crinipellis perniciosa, the causal agent of the witches' broom disease of cocoa, produces substantial amounts of hydrolytic enzymes. An amylase purified from isolate 1051 had a molecular mass of about 68.7 kDa. Maximal activity against soluble starch was determined at pH 4.0 and 60 degrees C. The K(m) and V(max) values were 3.5 mg ml(-1) and 1.67 mg min(-1) of reducing sugar. The end products were mostly malto-oligosaccharides. The enzyme also hydrolyzed glycogen, amylopectin, maltotriose, and maltotetraose, but not pullulan or cellobiose. Maltose was only barely hydrolyzed. The purified amylase exerted a discrete hydrolytic effect on the C. perniciosa cell wall in vitro as observed by scanning electron microscopic analysis. While Fe(3+), Al(3+), Zn(2+), and Cu(2+) were effective in inhibiting the purified amylase, Mn(2+) considerably enhanced the activity. Ca(2+), Mg(2+), and Co(2+) showed no substantial effect on enzyme activity.

Amylases↗

PubMed2000

Codon optimization of xylanase gene xynB from the thermophilic bacterium Dictyoglomus thermophilum for expression in the filamentous fungus Trichoderma reesei.

The catalytic domain of the xynB (xylanase) gene from the thermophilic bacterium Dictyoglomus thermophilum was reconstructed by PCR to match the codon preference of Trichoderma reesei. The 0.6-kb DNA fragment encoding the enzyme was first amplified by primer extension with a mixture of eight overlapping oligonucleotides, followed by PCR with outside primers containing restriction enzyme sites for directional cloning into Escherichia coli and T. reesei vectors. The synthetic gene was expressed in both organisms, producing a clearing halo around transformant colonies in plate assay utilizing an overlay of oat spelts xylan. Effective transcription of xyn B in T. reesei was obtained after changing 20 codons.

Amino Acid Sequence↗

PubMed2006

Light accelerates the splicing of srh1 homologue gene transcripts in aerial mycelia of Trichoderma viride.

The expression of the Tvsrh1 gene encoding conidial hydrophobin was investigated during the development of surface-cultivated Trichoderma viride mycelia under different illumination regimes. Three transcripts of the whole gene amplified from the total mRNA were found with lengths of 400, 323 and 272 bp. The 400-bp transcript was slowly converted to the shorter forms in the dark. Light-pulse dramatically increased the rate of conversion, and a permanent illumination of mycelia was most efficient in this process. The sequencing of transcripts revealed that the 400 bp transcript contains two introns, whereas the intermediate one contains only one intron located distally from the 5'-end. The shortest transcript was without introns. The sum of all transcripts remained almost unchanged in the dark and increased upon the light pulse but decreased during development under permanent illumination. The appearance of conidia coincided with the complete conversion of the transcripts. The results showed that the splicing of the two introns was not random but sequential, and that it did not follow the cotranscriptional mechanism. Furthermore, they suggested that mRNA processing could represent another regulation level of gene expression by light during the photo-induced conidiation in T. viride.

Base Sequence↗

PubMed2006

Broad-spectrum antimicrobial activity and high stability of Trichokonins from Trichoderma koningii SMF2 against plant pathogens.

Antimicrobial metabolites produced by Trichoderma koningii SMF2 exhibited antimicrobial activity against a range of Gram-positive bacterial and fungal phytopathogens. Purification of these metabolites was achieved using combinations of gel filtration and high-performance liquid chromatography. Identified by liquid chromatography electrospray ionization tandem mass spectrometry, the active metabolites proved to be three known peptaibols: Trichokonin VI, VII and VIII. The Trichokonins were stable and remained biological active over a wide pH range and at every temperature tested, showing no loss of activity even after autoclaving. Trichokonins were insensitive to proteolytic enzymes. Trichokonin VI takes on typical helical structure and the structure changes only slightly at different temperatures and pH values. The present study presented the potential of Trichokonins to be used as biological control agents.

Alamethicin↗

PubMed2005

BGN16.3, a novel acidic beta-1,6-glucanase from mycoparasitic fungus Trichoderma harzianum CECT 2413.

A new component of the beta-1,6-glucanase (EC 3.2.1.75) multienzymatic complex secreted by Trichoderma harzianum has been identified and fully characterized. The protein, namely BGN16.3, is the third isozyme displaying endo-beta-1,6-glucanase activity described up to now in T. harzianum CECT 2413. BGN16.3 is an acidic beta-1,6-glucanase that is specifically induced by the presence of fungal cell walls in T. harzianum growth media. The protein was purified to electrophoretical homogenity using its affinity to beta-1,6-glucan as first purification step, followed by chomatofocusing and gel filtration. BGN16.3 has a molecular mass of 46 kDa in SDS/PAGE and a pI of 4.5. The enzyme only showed activity against substrates with beta-1,6-glycosidic linkages, and it has an endohydrolytic mode of action as shown by HPLC analysis of the products of pustulan hydrolysis. The expression profile analysis of BGN16.3 showed a carbon source control of the accumulation of the enzyme, which is fast and strongly induced by fungal cell walls, a condition often regarded as mycoparasitic simulation. The likely involvement beta-1,6-glucanases in this process is discussed.

Animals↗

PubMed2006

Production and characterization of a secreted, C-terminally processed tyrosinase from the filamentous fungus Trichoderma reesei.

A homology search of the genome database of the filamentous fungus Trichoderma reesei identified a new T. reesei tyrosinase gene tyr2, encoding a protein with a putative signal sequence. The gene was overexpressed in the native host under the strong cbh1 promoter, and the tyrosinase enzyme was secreted into the culture supernatant. This is the first report on a secreted fungal tyrosinase. Expression of TYR2 in T. reesei resulted in good yields, corresponding to approximately 0.3 and 1 g.L(-1) tyrosinase in shake flask cultures and laboratory-scale batch fermentation, respectively. T. reesei TYR2 was purified with a three-step purification procedure, consisting of desalting by gel filtration, cation exchange chromatography and size exclusion chromatography. The purified TYR2 protein had a significantly lower molecular mass (43.2 kDa) than that calculated from the putative amino acid sequence (61.151 kDa). According to N-terminal and C-terminal structural analyses by fragmentation, chromatography, MS and peptide sequencing, the mature protein is processed from the C-terminus by a cleavage of a peptide fragment of about 20 kDa. The T. reesei TYR2 polypeptide chain was found to be glycosylated at its only potential N-glycosylation site, with a glycan consisting of two N-acetylglucosamines and five mannoses. Also, low amounts of shorter glycan forms were detected at this site. T. reesei TYR2 showed the highest activity and stability within a neutral and alkaline pH range, having an optimum at pH 9. T. reesei tyrosinase retained its activity well at 30 degrees C, whereas at higher temperatures the enzyme started to lose its activity relatively quickly. T. reesei TYR2 was active on both l-tyrosine and l-dopa, and it showed broad substrate specificity.

Amino Acid Sequence↗

PubMed1990

Induction of Trichoderma sporulation by nanosecond laser pulses: evidence against cryptochrome cycling.

An important question in the study of photoreceptor action in morphogenesis is whether the chromophore is unidirectionally photobleached, or whether it is recycled, allowing each receptor molecule to be counted more than once. The common soil fungus Trichoderma harzianum grows vegetatively in the dark and sporulates in response to a pulse of blue or UV-A light. Colonies were grown at 26 degrees C, transferred to 3 degrees C, illuminated with non-saturating light, and then put back at 26 degrees C to sporulate. The fluence-response curves for photoinduction in the cold and at 26 degrees C were identical, indicating that there are no enzymatic transduction processes during irradiation. Regions of the perimeter of dark-grown colonies were given single pulses (maximum duration, 30 ns) at 355 nm with a neodymium laser. We obtained a complete fluence-response curve for the laser pulses, which agreed with data for irradiations in the second to minute range. Photoinduction at 3 degrees C, and validity of Bunsen-Roscoe reciprocity from nanoseconds to minutes, support the hypothesis that the inductive event is a simple first-order photobleaching reaction.

Cold Temperature↗

PubMed1990

Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei.

The enzymatic degradation of cellulose is an important process, both ecologically and commercially. The three-dimensional structure of a cellulase, the enzymatic core of CBHII from the fungus Trichoderma reesei reveals an alpha-beta protein with a fold similar to but different from the widely occurring barrel topology first observed in triose phosphate isomerase. The active site of CBHII is located at the carboxyl-terminal end of a parallel beta barrel, in an enclosed tunnel through which the cellulose threads. Two aspartic acid residues, located in the center of the tunnel are the probable catalytic residues.

Amino Acid Sequence↗

PubMed1994

The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei.

Cellulose is the major polysaccharide of plants where it plays a predominantly structural role. A variety of highly specialized microorganisms have evolved to produce enzymes that either synergistically or in complexes can carry out the complete hydrolysis of cellulose. The structure of the major cellobiohydrolase, CBHI, of the potent cellulolytic fungus Trichoderma reesei has been determined and refined to 1.8 angstrom resolution. The molecule contains a 40 angstrom long active site tunnel that may account for many of the previously poorly understood macroscopic properties of the enzyme and its interaction with solid cellulose. The active site residues were identified by solving the structure of the enzyme complexed with an oligosaccharide, o-iodobenzyl-1-thio-beta-cellobioside. The three-dimensional structure is very similar to a family of bacterial beta-glucanases with the main-chain topology of the plant legume lectins.

Binding Sites↗

PubMed2026

Trichoderma reesei Nsd3 transcription factor: pleiotropic roles in development, stress response, secondary metabolism, and cellulase production.

Trichoderma reesei is known for its ability to secrete high amounts of cellulases, enzymes of fundamental importance in generating products from lignocellulosic biomass. Diverse signaling pathways and transcription factors (TFs) control the cellulolytic repertoire in T. reesei to ensure correct adaptation to the environment. Here, we analyzed RNA-Seq data and identified a new potential regulator of cellulase production in T. reesei: a novel TF named Nsd3, a homolog of NsdC from Aspergilli. Deletion of nsd3 reduced vegetative growth and conidiation on solid medium. Phenotypic characterization of the Δnsd3 strain showed that it is more sensitive to osmotic stress, but more resistant to cell wall and oxidative stresses. Our results showed that Nsd3 is a repressor of cellulase expression by directly regulating key genes in the cellulolytic pathway, an unreported role for this TF in fungi. Loss of nsd3 leads to a faster and more robust induction of cellulolytic genes, and higher cellulase and hemicellulase activities. Transcriptional profiling by RNA-Seq, chromatin accessibility profiling by ATAC-Seq, and protein-DNA interaction assays showed that sugar transporters are important targets of Nsd3 during cellulase expression regulation. Combined with microscopy and gene expression analyses, the ATAC-Seq data also highlighted Nsd3 as a central regulator of cell wall remodeling and organization. Furthermore, the transcriptomics also showed that Nsd3 regulates genes involved in secondary metabolism. These results showed that Nsd3 regulates several physiological processes and provide novel insights into the regulatory system of cellulases in T. reesei that can be used in the design of high-performance strains for biorefinery.IMPORTANCETrichoderma reesei is a key player in the production of hydrolytic enzymes for the degradation of lignocellulose biomass, and transcription factors are important targets for genetic engineering to construct cellulase-hyperproducing strains. Here, we identified the transcription factor Nsd3 and characterized its role as a regulator of cellulase production in T. reesei. We applied two powerful genomics methods (transcriptome sequencing and chromatin accessibility sequencing) to unravel the global role of Nsd3 and its regulatory mechanism. Nsd3 participates in various biological processes in T. reesei, including cell wall remodeling, calcium metabolism, and secondary metabolism, in addition to regulating the expression of sugar transporters. Protein-DNA interaction assays demonstrate that Nsd3 acts through important genes to regulate cellulase expression, including ace4, crt1, stp1, and cel1b. Our study provides mechanistic insights about how Nsd3 regulates diverse physiological processes in T. reesei. This work also applied ATAC-Seq for the first time to study chromatin accessibility in T. reesei.

ATAC-Seq↗

PubMed2006

Tvbgn3, a beta-1,6-glucanase from the biocontrol fungus Trichoderma virens, is involved in mycoparasitism and control of Pythium ultimum.

Even though beta-1,6-glucanases have been purified from several filamentous fungi, the physiological function has not been conclusively established for any species. In the present study, the role of Tvbgn3, a beta-1,6-glucanase from Trichoderma virens, was examined by comparison of wild-type (WT) and transformant strains in which Tvbgn3 was disrupted (GKO) or constitutively overexpressed (GOE). Gene expression analysis revealed induction of Tvbgn3 in the presence of host fungal cell walls, indicating regulation during mycoparasitism. Indeed, while deletion or overexpression of Tvbgn3 had no evident effect on growth and development, GOE and GKO strains showed an enhanced or reduced ability, respectively, to inhibit the growth of the plant pathogen Pythium ultimum compared to results with the WT. The relevance of this activity in the biocontrol ability of T. virens was confirmed in plant bioassays. Deletion of the gene resulted in levels of disease protection that were significantly reduced from WT levels, while GOE strains showed a significantly increased biocontrol capability. These results demonstrate the involvement of beta-1,6-glucanase in mycoparasitism and its relevance in the biocontrol activity of T. virens, opening a new avenue for biotechnological applications.

Amino Acid Sequence↗

PubMed2006

Glycoprotein hypersecretion alters the cell wall in Trichoderma reesei strains expressing the Saccharomyces cerevisiae dolichylphosphate mannose synthase gene.

Expression of the Saccharomyces cerevisiae DPM1 gene (coding for dolichylphosphate mannose synthase) in Trichoderma reesei (Hypocrea jecorina) increases the intensity of protein glycosylation and secretion and causes ultrastructural changes in the fungal cell wall. In the present work, we undertook further biochemical and morphological characterization of the DPM1-expressing T. reesei strains. We established that the carbohydrate composition of the fungal cell wall was altered with an increased amount of N-acetylglucosamine, suggesting an increase in chitin content. Calcofluor white staining followed by fluorescence microscopy indicated changes in chitin distribution. Moreover, we also observed a decreased concentration of mannose and alkali-soluble beta-(1,6) glucan. A comparison of protein secretion from protoplasts with that from mycelia showed that the cell wall created a barrier for secretion in the DPM1 transformants. We also discuss the relationships between the observed changes in the cell wall, increased protein glycosylation, and the greater secretory capacity of T. reesei strains expressing the yeast DPM1 gene.

Biotechnology↗

PubMed1976

Beta-glucosidase of Trichoderma: its biosynthesis and role in saccharification of cellulose.

The extracellular beta-glucosidase of Trichoderma viride generally is present in low levels when the organism is cultured on cellulose because it is inactivated under the acid conditions which develop in the medium while the other enzymes of the cellulase complex are more stable. With the appropriate pH control, inactivation of beta-glucosidase is prevented and the activity of this enzyme increases during growth. In the saccharification of crystalline cellulose, or of cellulose at low concentrations, much of the glucose produced is the result of the cleavage of cellobiose by beta-glucosidase. However when high concentrations (10%) of pretreated cellulose are saccharified, significant quantities of glucose are produced by action of enzymes other than beta-glucosidase.

Cell-Free System↗

PubMed1977

Fungal growth on C1 compounds: quantitative aspects of growth of a methanol-utilizing strain of Trichoderma lignorum in batch culture.

A study was made of some salient parameters that influence growth of the methanol-utilizing fungus Trichoderma lignorum growing in batch culture on a minimal medium containing methanol as the sole source of carbon. Maximum cell yield was recorded at the expense of 1.58 g of methanol per liter. Inhibition was observed with methanol concentrations in excess of 4.7 g/liter. The optimum temperature for fungal growth was 23 degrees C. Growth of the fungus was directly proportional to an inorganic nitrogen concentration up to 0.2 g of NH4NO3 per liter. No inhibition of growth occurred at any concentration of NH4NO3 up to 11 g/liter. The pH of the growth medium decreased from 7.0 to 3.5 during growth of the fungus on methanol, which may have been due, in part, to the accumulation of trace amounts of organic acids in the growth medium. An analysis of the commercial potential of the fungus, as a source of edible protein, indicated that the strain of methanol-utilizing T. lignorum used was uneconomical in terms of the yield and the specific growth rate.

Dietary Proteins↗

PubMed1978

Production of extracellular enzymes in mutants isolated from trichoderma viride unable to hydrolyze cellulose.

Mutant strains not producing cellulases were induced and isolated from the cellulolytic fungus Trichoderma viride. Enrichment of mutants was carried out with the aid of nystatin selection. Mutants were shown to lack the ability to hydrolyze both soluble and crystalline cellulose. Mannanase and xylanase activities were also absent, indicating a common regulation for all these enzymes in T. viride. In some strains aryl-beta-glucosidase activity was also missing. Mutants grew normally, but the amount of proteins secreted into the medium was very low, and in most cases these proteins were qualitatively different from the proteins of the parent strain.

Cellulase↗

PubMed1985

Butanediol production from cellulose and hemicellulose by Klebsiella pneumoniae grown in sequential coculture with Trichoderma harzianum.

The bioconversion of cellulose and hemicellulose substrates to 2,3-butanediol by a sequential coculture approach was investigated with the cellulolytic fungus Trichoderma harzianum E58 and the fermentative bacterium Klebsiella pneumoniae. Vogel medium optimal for the production of the cellulolytic and xylanolytic enzymes of the fungus was found to be inhibitory to butanediol fermentation. This inhibition appeared to be due to a synergistic effect of various ingredients, particularly the salts, present in the fungal medium. The removal or replacement of such ingredients from Vogel medium led to the relief of fermentation inhibition, but the treatments also resulted in a significant decrease in fungal enzyme production. Resting cells of K. pneumoniae could be used for butanediol production in the fungal medium, indicating that the inhibitory effect on solvent production under such conditions was due to the indirect result of growth inhibition of the bacterial cells. The resting-cell approach could be combined with a fed-batch system for the direct conversion of 8 to 10% (wt/vol) of Solka-Floc or aspenwood xylan to butanediol at over 30% of the theoretical conversion efficiencies.

Butylene Glycols↗

PubMed1985

Subcellular fractionation of a hypercellulolytic mutant, Trichoderma reesei Rut-C30: localization of endoglucanase in microsomal fraction.

The growing mycelia of Trichoderma reesei Rut-C30 are richly endowed with endoplasmic reticula and a variety of pleomorphic subcellular bodies. Mycelia of the culture growing in presence of avicel pH101 was fractionated in sucrose density gradients, and several morphologically and biochemically distinct fractions were isolated. Mycelia were homogenized in a Bead Beater, and the homogenate was freed of nucleus and wall fragments by low-speed centrifugation before fractionation. Organelle-free cytosol, which did not penetrate the gradient, contained (of the total) 72% of the vanadate-sensitive ATPase, 26% of carboxymethyl cellulase (CMCase), 2% of cytochrome c reductase, and 13% of the protein. Significant fractions separated on a gradient were light vesicles containing heavily stained material inside and ribosomes attached to the outside surface, intact vesicles resembling condensing vacuoles, large vesicles derived from the plasma membrane, and heavy vesicles containing crystalline material. The light-vesicle fraction contained a large portion of the cell-bound CMCase activity. The particle-bound ATPase and cytochrome c reductase activities were concentrated in heavy fractions. The fractionation in the presence of MgCl2 improved the preservation of subcellular bodies derived from the endoplasmic reticula. Although the CMCase activity of the light-vesicle fraction was 4 times higher than the activity in the heavy-vesicle fraction, the CMCase antibody-binding capacities of both fractions were about the same. This discrepancy between the catalytic activity and the antibody-binding capacity suggests that the heavy vesicles might have contained considerable amount of inactive CMCase compared with that present in the light vesicles.

Cell Fractionation↗