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At least 19 recordsLinked to original sources

PubMed1991

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains.

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and beta-octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but - unlike in extracellular culture fluids from Trichoderma - CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.

Antibodies, Monoclonal↗

PubMed1990

Stereochemical course of hydrolysis and hydration reactions catalysed by cellobiohydrolases I and II from Trichoderma reesei.

Cellobiohydrolase I from Trichoderma reesei catalyzes the hydrolysis of methyl beta-D-cellotrioside (Km = 48 microM, kcat = 0.7 min-1) with release of the beta-cellobiose (retention of configuration). The same enzyme catalyzes the trans-hydration of cellobial (Km = 116 microM, kcat = 1.16 min-1) and lactal (Km = 135 microM, kcat = 1.35 min-1), presumably with glycosyl oxo-carbonium ion mediation. Protonation of the double bond is from the direction opposite that assumed for methyl beta-cellotrioside, but products formed from these prochiral substrates are again of beta configuration. Cellobiohydrolase II from the same microorganism hydrolyzes methyl beta-D-cellotetraoside (Km = 4 microM, kcat = 112 min-1) with inversion of configuration to produce alpha-cellobiose. The other reaction product, methyl beta-cellobioside, is in turn partly hydrolysed by cellobiohydrolase II to form methyl beta-D-glucoside and D-glucose, presumably the alpha-anomer. Reaction with cellobial is too slow to permit unequivocal determination of product configuration, but clear evidence is obtained that protonation occurs from the si-direction, again opposite that assumed for protonating glycosidic substrates. These results add substantially to the growing evidence that individual glycosidases create the anomeric configuration of their reaction products by means that are independent of substrate configuration.

Cellulose 1,4-beta-Cellobiosidase↗

PubMed2026

Multi-omics analysis reveals Protein Kinase A-associated regulatory remodeling during adaptation of Trichoderma reesei to lignocellulosic substrate.

The filamentous fungus Trichoderma reesei is a major industrial source of holocellulolytic enzymes, and its response to complex carbon sources is regulated by nutrient-sensing mechanisms, including the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. Here, we integrated transcriptomics, quantitative proteomics, and phosphoproteomics to analyze PKAc1-associated responses in the parental strain QM9414 and a Δpkac1 strain cultivated under glucose or sugarcane bagasse conditions. Deletion of pkac1 was associated with altered growth-related phenotypes and reduced extracellular activities of selected biomass-depolymerizing enzymes. Multi-omics profiling revealed condition-dependent changes affecting subsets of carbohydrate-active enzymes (CAZymes) genes and proteins, nutrient transporters, stress-associated proteins, and regulatory factors. Phosphoproteomics identified phosphorylation-state changes associated with pkac1 deletion, including reduced phosphorylation at sites enriched for the PKA consensus motif. In silico peptide docking was used to prioritize candidate PKAc1-associated substrates for future validation, including a Sec 7-derived peptide with favorable docking behavior relative to the control peptide. Together, these data support a working model in which PKAc1 contributes to regulatory and phosphorylation-state remodeling during adaptation to sugarcane bagasse, with effects on the magnitude and/or timing of selected CAZyme-related outputs in T. reesei.

Trichoderma↗

PubMed1989

Isolation and characterization of the 3-phosphoglycerate kinase gene (pgk) from the filamentous fungus Trichoderma reesei.

The 3-phosphoglycerate kinase gene (pgk) from Trichoderma reesei was isolated by hybridization with the corresponding Saccharomyces cerevisiae PGK gene. The 1,545 nt long nucleotide sequence of the cloned gene codes for a 416 amino acid protein. The coding sequence contains two introns of 219 and 75 nt, respectively, at positions identical to those corresponding genes from the other filamentous fungi Aspergillus nidulans and Penicillum chrysogenum. This gene codes for two mRNAs of about 1.65 kb and 1.85 kb. The PGK protein of Trichoderma shows extensive homology to the PGKs of other fungi A. nidulans (77%), P. chrysogenum (73%) and Saccharomyces cerevisiae (69%). However, the PGKs of the two other filamentous fungi, A. nidulans and P. chrysogenum, seem to be more closely related to each other than to the T. reesei enzyme.

Amino Acid Sequence↗

PubMed1990

Pilot scale production of a heterologous Trichoderma reesei cellulase by Saccharomyces cerevisiae.

Cellobiohydrolase II of Trichoderma reesei was produced in laboratory and pilot scale using a transformant strain of Saccharomyces cerevisiae harbouring a multicopy expression plasmid. Different strategies were compared for concentration and partial purification of the enzyme produced in a 200 1 pilot cultivation. After efficient separation of biomass and sub-cellular particulate matter, a combination of ultrafiltration and adsorbent treatment for removal of protein impurities was used to provide a concentrate for chromatographic purification. Effective purification of the CBH II protein was obtained by passing the concentrate through a column of DEAE Sepharose, on which almost all the yeast proteins were adsorbed. The purified enzyme reacted with antibodies prepared against T. reesei CBH II and catalyzed partial solubilization of crystalline cellulose to soluble sugars.

Biotechnology↗

PubMed1989

Mechanism by which cellulose triggers cellobiohydrolase I gene expression in Trichoderma reesei.

The expression of cellobiohydrolase I mRNA from Trichoderma reesei, measured by Northern blot hybridization, is controlled by the nature of carbon sources used in the culture medium. Cellulose and the soluble disaccharide sophorose, but not glycerol or glucose, act as inducers. Cellobiohydrolase I mRNA was undetectable when antibodies to the major members of the cellulolytic system were present in the culture medium prior to the addition of cellulose. These antibodies had no repressive effect if sophorose was used as an inducer. The results strongly suggest that a low constitutive cellulolytic system catalyzes the formation of a soluble inducer from cellulose and that this inducer triggers the expression of the cellobiohydrolase I gene transcript, most probably at the transcription level.

Cellulose↗

PubMed1991

Monoclonal antibodies against core and cellulose-binding domains of Trichoderma reesei cellobiohydrolases I and II and endoglucanase I.

Cellulases from Trichoderma reesei form an enzyme group with a common structural organization. Each cellulase enzyme is composed of two functional domains, the core region containing the active site and the cellulose-binding domain (CBD). To facilitate the specific detection of each domain, monoclonal antibodies (mAb) against cellobiohydrolase I (CBHI), cellobiohydrolase II (CBHII) and endoglucanase I (EGI) were produced. Five mAb were obtained against CBHI, ten against CBHII and eight against EGI. The location of the antigenic epitope for each antibody was mapped by allowing the antibodies to react with truncated cellulases, synthesized from deleted cDNA in Saccharomyces cerevisiae. Proteolytic fragments of Trichoderma cellulases, obtained by papain digestion, were used to confirm the results. Specific antibodies were detected against the core and the CBD epitopes for all three cellulases. Using the truncated enzymes, it was possible to locate the epitopes to a reasonably short region within the protein. To obtain a quantitative assay for each enzyme, a specific mAb against each antigen was chosen, based on the affinity to the corresponding antigen on Western-blot staining and on filter blots of the cellulolytic yeasts. The mAb were used to quantitative the corresponding enzymes in T. reesei culture medium. Specific quantitation of each cellulase enzyme has not been possible by biochemical assays or using polyclonal antibodies, due to their cross-reactions. Now, these mAb can be specifically used to recognize and quantitate different domains of these three important cellulolytic enzymes.

Antibodies, Monoclonal↗

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↗

PubMed1978

alpha,alpha-Trehalase of Trichoderma reesei.

A simple adsorption and elution of the trehalase of Trichoderma reesei on bentonite increased the specific activity 70-80 times, with a recovery of 90%. This alpha,alpha-trehalase has an optimum pH of 4.4, a pl of 5.7, a Km of 3.1 X 10(-3) M, and a specific activity of 50 mumol/mg. min-1.

Adsorption↗

PubMed1992

Mechanisms of thermoinactivation of endoglucanase I from Trichoderma reesei QM 9414.

The mechanism of irreversible thermoinactivation of endoglucanase I from Trichoderma reesei has been determined at 70 degrees C at the pH of maximum enzyme activity. The time-course of thermoinactivation did not follow first-order kinetics and kinetic constants of the process were dependent on enzyme concentration, suggesting that aggregation was the main process leading to irreversible inactivation. The enzyme was extremely resistant to urea, which in fact seemed to stabilize it against temperature. Disulphide exchange, deamidation and hydrolysis of peptide bonds were also responsible for the loss of enzyme activity at 70 degrees C.

Ammonium Sulfate↗

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↗

PubMed1986

Homology between cellulase genes of Trichoderma reesei: complete nucleotide sequence of the endoglucanase I gene.

The filamentous fungus Trichoderma reesei produces several endoglucanases (EG) and cellobiohydrolases (CBH) which are involved in cellulose hydrolysis in a complex synergistic manner. We have cloned and sequenced the gene and the full-length cDNA coding for the major endoglucanase EG-I, and compared this to the cbh1 gene sequence to clarify the relationship between the EG and CBH classes of cellulases. The deduced 437-amino acids (aa) long EG-I protein with a 22-aa long signal peptide is 45% identical in aa sequence with CBH-I. The best conserved region is found at the C terminus and shows about 70% homology. The data suggest that the two enzymes have arisen from a common ancestor by gene duplication. Despite this, the intron positions have not been conserved in these genes which both contain two short introns. The deduced EG-I sequence contains six putative N-glycosylation sites, and a putative O-glycosylated region is found near the C terminus, closely resembling a similar region at the C terminus of CBH-I. Comparison of the aa sequences suggests that the evolutionary divergence of EG-I from CBH-I has involved four separate 10-20 aa "deletions" from the ancestral protein.

Amino Acid Sequence↗

PubMed1991

Structural and functional analysis of Trichoderma reesei endoglucanase I expressed in yeast Saccharomyces cerevisiae.

The function of the domains of Trichoderma reesei endoglucanase I (EGI) has been studied. Truncated EGI proteins were expressed from the 3'-end deleted cDNAs in the yeast Saccharomyces cerevisiae under the control of the ADC1 expression cassette. EGI protein was detected by monoclonal antibody EI-2 and EGI activity as cleared zones around growing colonies on agar plates containing hydroxyethylcellulose (HEC) covalently stained with Ostazin brilliant red (OBR). The results showed that the The-Ser-rich hinge region and the conserved 'tail' are not necessary for the efficient synthesis and secretion of EGI in yeast, but the intact core region is necessary for the enzymatic activity.

Blotting, Western↗

PubMed1990

The development of a heterologous transformation system for the cellulolytic fungus Trichoderma reesei based on a pyrG-negative mutant strain.

Six uridine auxotroph mutants of Trichoderma reesei QM 9414 were isolated by resistance to 5-fluoroorotic acid and one strain was identified as OMP-decarboxylase negative (pyr-) by a radiometric enzyme assay. Transformation to uridine prototrophy was achieved with the pyr4 gene of Neurospora crassa (up to 1500 transformants/micrograms) and with pyrA of Aspergillus niger (700-800 transformants/micrograms). In many transformants the PYR+ function seems to be present as extrachromosomal DNA. There is evidence for a correlation between the stability of transformants and integration of the vector in the genome whereas unstable transformants are obtained when autonomous replication of the plasmid occurs.

Blotting, Southern↗

PubMed1990

The competitive inhibition of Trichoderma reesei C30 cellobiohydrolase I by guanidine hydrochloride.

The p-nitrophenylcellobiosidase (PNPCase) activity of Trichoderma reesei cellobiohydrolase I (CBH I) was competitively inhibited by concentrations of guanidine hydrochloride (Gdn HCl) that did not affect the tryptophan fluorescence of this enzyme. The Km of CBH I, 3.6 mM, was increased to 45.4 mM in the presence of 0.14 M Gdn HCl, the concentration that was required to inhibit the enzyme by 50%. A similar concentration of lithium chloride and urea had little effect on the PNPCase activity of CBH I. Maximal inhibition was pH dependent, occurring in the range of pH 4.0 to 5.0, which is in the range for maximal activity. Analysis of the inhibition data indicated that 1.2 molecules of Gdn HCl combined reversibly with 1 molecule of CBH I. Other hydrolases and proteases were also inhibited by Gdn HCl. It is suggested that the inhibition of CBH I by Gdn HCl occurs as a result of the interaction between the positively charged guanidinium group of Gdn HCl and the carboxylate group of glutamic acid 126, postulated to be in the catalytic center of this enzyme.

Binding Sites↗

PubMed1992

Chromosomal and genetic analysis of the electrophoretic karyotype of Trichoderma reesei: mapping of the cellulase and xylanase genes.

An electrophoretic karyotype has been established for Trichoderma reesei strain QM6a, and several of its derivatives, by pulsed-field gel electrophoresis. All strains examined appear to have seven chromosomes with a total genome size of approximately 33 megabases (Mb). The sizes of the chromosomal bands in strain QM6a are approximately 6.2, 6.0, 5.1, 4.2 (doublet), 3.6 and 3.2 Mb. Genes encoding the cellulase complex and xylanases of T. reesei have been mapped, as have several other genes.

Cellulase↗

PubMed1991

Enzyme production by recombinant Trichoderma reesei strains.

The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail.

Animals↗

PubMed1989

Monoclonal antibodies against different domains of cellobiohydrolase I and II from Trichoderma reesei.

Monoclonal antibodies have been produced against two functionally different domains present in two cellobiohydrolases from Trichoderma reesei (CBH I and CBH II). Four groups of antibodies were obtained, which specifically recognized (Western blotting, ELISA) (a) the core protein within CBH I, (b) the core protein within CBH II, (c) the BA region of CBH I, and (d) the ABB' region of CBH II. No cross-reactivities within these four groups were observed. The antibodies reacted also specifically with proteins of similar size to CBH I and CBH II (SDS-PAGE) from other Trichoderma strains (Western blotting), whereas no reaction was observed with cellulases from other fungal sources. Analysis of culture filtrates of T. reesei QM 9414 harvested at various times of growth on cellulose under buffered conditions (pH 5-6) indicated the presence of only single bands of CBH I and CBH II, even after prolonged cultivation (160 h). Cultivation on cellulose in unbuffered media, however, showed the appearance (Western blotting) of additional lower molecular weight proteins, which reacted with the monoclonal antibodies directed against the cores of CBH I and II, but not with those recognizing the respective BA and ABB' regions. The appearance of these lower molecular weight bands was most pronounced in unbuffered media, supplemented with a 3-fold (w/w) amount of organic nitrogen (peptone). Analysis of some commercial cellulase preparations from T. harzianum revealed the same pattern of lower molecular weight proteins, in contrast to samples from other fungal cellulases. Those samples or preparations, showing a multiple pattern of CBH I and CBH II, exhibited higher activities of an acid proteinase. These results imply that the use of unbuffered, high nitrogen-supplemented culture conditions for production of cellulases may lead to considerable proteolytic modification of the secreted cellobiohydrolases.

Antibodies, Monoclonal↗