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PubMed1998

Isolation of a Trichoderma reesei cDNA encoding GTP: a-D-mannose-1-phosphate guanyltransferase involved in early steps of protein glycosylation.

A cDNA coding for GTP: alpha-d-mannose-1-phosphate guanyltransferase (MPG1 transferase) (EC 2.7.7.13) was isolated from a cDNA library of the Trichoderma reesei RutC-30 strain by suppression of the yeast Saccharomyces cerevisiae mutation in the DPM1gene encoding mannosylphosphodolichol (MPD) synthase. The nucleotide sequence of the 1.6 kb-long cDNA revealed an ORF which encodes a protein of 364 amino acids. Sequence comparisons demonstrate 70% identity with the S. cerevisiae guanyl transferase gene (MPG1) and 75% identity with the Schizosaccharomyces pombe homologue. No similarity was found with the MPD synthase encoded by the S. cerevisiae DPM1 gene. The possibility that cloned cDNA encodes a product with a MPD synthase activity was also excluded by transforming a heterozygous S. cerevisiae dpm1::LEU2/DPM1 diploid, which did not lead to the restoration of viability of the dpm1 spores. Simultaneously, a significant increase in MPG transferase activity, as compared with the wild-type yeast, was observed in cellular extracts when the mpg1 cDNA from Trichoderma was expressed in the S. cerevisiae dpm1-6 mutant.

Amino Acid Sequence↗

PubMed2003

The effects of drugs inhibiting protein secretion in the filamentous fungus Trichoderma reesei. Evidence for down-regulation of genes that encode secreted proteins in the stressed cells.

To study the mechanisms of protein secretion as well as the cellular responses to impaired protein folding and transport in filamentous fungi, we have analyzed Trichoderma reesei cultures treated with chemical agents that interfere with these processes, dithiothreitol, brefeldin A, and the Ca(2+)-ionophore A23187. The effects of the drugs on the kinetics of protein synthesis and transport were characterized using metabolic labeling of synthesized proteins. Cellobiohydrolase I (CBHI, Cel7A), the major secreted cellulase, was analyzed as a model protein. Northern analysis showed that under conditions where protein transport was inhibited (treatments with dithiothreitol or brefeldin A) the unfolded protein response pathway was activated. The active form of the hac1 mRNA that mediates unfolded protein response signaling was induced, followed by induction of the foldase and chaperone genes pdi1 and bip1. Concomitant with the activation of the unfolded protein response pathway, the transcript levels of genes encoding secreted proteins, like cellulases and xylanases, were drastically decreased, suggesting a novel type of feedback mechanism activated in response to impairment in protein folding or transport (repression under secretion stress (RESS)). By studying expression of the reporter gene lacZ under cbh1 promoters of different length, it was shown that the feedback response was mediated through the cellulase promoter.

Biological Transport↗

PubMed1988

Differential regulation of synthesis of multiple forms of specific endoglucanases by Trichoderma reesei QM9414.

A method consisting of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent detection of endoglucanases by blotting with a polyclonal antibody against endoglucanase I was used to investigate the effect of induction and carbon catabolite derepression on the synthesis of multiple forms of endoglucanase I by Trichoderma reesei. Five forms appeared upon growth on cellulose, whereas four and only two appeared upon growth on lactose (carbon catabolite derepression) and induction by sophorose in a resting cell system, respectively. All endoglucanases detected resembled endoglucanase I in their specificity, since they exhibited no activity toward xylan or paranitrophenyl-beta-D-lactobioside. A small (25-kilodalton) endoglucanase only appeared during growth on cellulose. None of the multiple forms arose by postsecretional modification. The results indicate that sophorose may not be the only compound mediating cellulose induction of the specific endoglucanases in T. reesei.

Cellulase↗

PubMed2004

Purification, crystallization and preliminary X-ray diffraction analysis of the Trichoderma reesei hydrophobin HFBI.

Hydrophobins are fungal proteins that are capable of altering the hydrophobicity of surfaces by self-assembly at hydrophilic-hydrophobic interfaces. Here, the growth of hydrophobin crystals suitable for X-ray crystallography is reported. The hydrophobin HFBI from Trichoderma reesei was crystallized by vapour diffusion in hanging drops in 30% PEG 4000, 0.1 M sodium citrate pH 4.3 buffer containing 0.2 M ammonium acetate and CYMAL-5 detergent (initial concentration of 2.4 mM). HFBI crystals are hexagonal and belong to space group P6(1) (or P6(5)), with unit-cell parameters a = b = 45.9, c = 307.2 A. The HFBI used in the crystallization experiments was purified from fungal cell walls.

Crystallization↗

PubMed1999

Fractionation of cellulase and beta-glucosidase in a Trichoderma reesei culture liquid by use of two-phase partitioning.

An aqueous two-phase system based on the two polymers poly(ethylene glycol) and dextran has been used for the fractionation of cellulase enzymes present in culture liquid obtained by fermentation with Trichoderma reesei. The activities of beta-glucosidase and glucanases were separated to high degree by using the two-phase systems for a counter-current distribution process in nine transfer steps. While the glucanases had high affinity to the poly(ethylene glycol) rich top phase the beta-glucosidase was enriched in the dextran-containing bottom phase. Multiple counter-current distribution performed indicates the heterogeneity of beta-glucosidase activities assuming at least four isoenzyme forms. One step concentration of beta-glucosidase by using system with 46:1 phase volume ratio resulted in 16 times higher enzyme activity.

Cellulase↗

PubMed1993

Triggering of cellulase biosynthesis by cellulose in Trichoderma reesei. Involvement of a constitutive, sophorose-inducible, glucose-inhibited beta-diglucoside permease.

We prepared [U-14C]cellobiose by cultivating Acetobacter pasteurianus in the presence of [U-14C]glucose and hydrolyzing the [U-14C]cellulose formed with beta-glucosidase-free cellulase from Trichoderma reesei. This 14C-labeled cellobiose was used to investigate the presence of an uptake system for cellobiose in T. reesei. Evidence was obtained for the presence of a high affinity (Km for cellobiose 0.3 microM) but low activity (2.5 milliunits/mg fungal dry weight) cellobiose permease. The permease is formed constitutively, but higher levels are formed after addition of sophorose (glucosyl-beta-1,2-diglucoside), a reputed cellulase inducer. The permease appears to be specific for beta-diglucosides, as the uptake of [U-14C]cellobiose is inhibited by sophorose, gentiobiose (glucosyl-beta-1,3-glucoside), and cellobiose. Under these conditions, cellooligodextrines (n, 4-7; final concentration, 1 mM) are not inhibitors. Glucose, but no other monosaccharides, inhibits the permease. The hypersecretory mutant T. reesei RUT C-30 exhibits elevated permease activities, whereas in T. reesei QM 9979, a mutant strain defective in the induction of cellulases by cellulose or sophorose, strongly reduced permease activities were demonstrated. The results stress a hitherto not recognized point of control in the induction of cellulases by T. reesei at the level of uptake of cellulose oligosaccharides.

Cellulase↗

PubMed1997

Hybridization dominance of kinetics in recombinant ATH-1376 obtained via protoplast fusion between Aspergillus niger and Trichoderma reesei.

Comparisons of the kinetics of mycelium growth, cellulase biosynthesis, and the degradation of filter paper to accumulate reducing-sugar by the filtrates of cultures were carried out among the recombinant strain ATH-1376 and its two parents, Aspergillus niger AMS11 and Trichoderma reesei QM9414. The results showed that both the specific mycelium growth rate and the cellulase biosynthesis rate of the recombinant were dramatically dominant over those of the two parents. In addition, the negative correlation between the specific mycelium growth rate and the cellulase biosynthesis rate of the recombinant ATH-1376 was much lower than those of its parents. In terms of the amount of reducing-sugar accumulated from the hydrolysis of filter paper by culture filtrates, there were great differences among the three different treatments, i.e., fermentation filtrate of single parental strain, mixture of the fermentation filtrates from two parental strains with different ratios (v:v), and filtrate from the mixed culture of the two parental strains. Out of these, the second approach, particularly with the ratio of 1:1, was best for the accumulation of reducing sugar. Within various tested periods of enzymic hydrolysis, the amounts of reducing-sugar produced by the recombinant were 1.19 to 2.26 times as much as the maximum amounts produced in parallel by the mixture of filtrates (1:1) from separate fermentations of the two parental strains. These results suggested that constructing the engineered strains with hybridization dominance of these two typical genera of far-heredity could be effective to overcome the great deficiencies of routine mixculture, single-strain fermentation, or double fed-batch fermentations.

Aspergillus niger↗

PubMed1997

Cellobiohydrolase I from Trichoderma reesei: identification of an active-site nucleophile and additional information on sequence including the glycosylation pattern of the core protein.

(R,S)-3,4-Epoxybutyl beta-cellobioside, but not the corresponding propyl and pentyl derivatives, inactivates specifically and irreversibly cellobiohydrolase I from Trichoderma reesei by covalent modification of Glu212, the putative active-site nucleophile. The position and identity of the modified amino acid residue were determined using a combination of comparative liquid chromatography coupled on-line to electrospray ionization mass spectrometry, tandem mass spectrometry and microsequencing. It was found that the core protein corresponds to the N-terminal sequence pyrGlu1-Gly434 (Gly435) of intact cellobiohydrolase I. In the particular enzyme samples investigated, the asparagine residues in positions 45, 270 and 384 are each linked to a single 2-acetamido-2-deoxy-D-glucopyranose residue.

Amino Acid Sequence↗

PubMed2004

Expression in Trichoderma reesei and characterisation of a thermostable family 3 beta-glucosidase from the moderately thermophilic fungus Talaromyces emersonii.

The gene encoding a thermostable beta-glucosidase (cel3a) was isolated from the thermophilic fungus Talalaromyces emersonii by degenerate PCR and expressed in the filamentous fungus Trichoderma reesei. The cel3a gene encodes an 857 amino acid long protein with a calculated molecular weight of 90.59 kDa. Tal. emersonii beta-glucosidase falls into glycosyl hydrolase family 3, showing approximately 56 and 67% identity with Cel3b (GenBank ) from T. reesei, and a beta-glucosidase from Aspergillus Niger (GenBank ), respectively. The heterologously expressed enzyme, Cel3a, was a dimer equal to 130 kDa subunits with 17 potential N-glycosylation sites and a previously unreported beta-glucosidase activity produced extracellularly by Tal. emersonii. Cel3a was thermostable with an optimum temperature of 71.5 degrees C and half life of 62 min at 65 degrees C and was a specific beta-glucosidase with no beta-galactosidase side activity. Cel3a had a high specific activity against p-nitrophenyl-beta-D-glucopyranoside (Vmax, 512 IU/mg) and was competitively inhibited by glucose (k(i), 0.254 mM). Cel3a was also active against natural cellooligosacharides with glucose being the product of hydrolysis. It displayed transferase activity producing mainly cellobiose from glucose and cellotetrose from cellobiose.

Amino Acid Sequence↗

PubMed1999

Mathematical modeling and optimization of cellulase protein production using Trichoderma reesei RL-P37.

The enzyme cellulase, a multienzyme complex made up of several proteins, catalyzes the conversion of cellulose to glucose in an enzymatic hydrolysis-based biomass-to-ethanol process. Production of cellulase enzyme proteins in large quantities using the fungus Trichoderma reesei requires understanding the dynamics of growth and enzyme production. The method of neural network parameter function modeling, which combines the approximation capabilities of neural networks with fundamental process knowledge, is utilized to develop a mathematical model of this dynamic system. In addition, kinetic models are also developed. Laboratory data from bench-scale fermentations involving growth and protein production by T. reesei on lactose and xylose are used to estimate the parameters in these models. The relative performances of the various models and the results of optimizing these models on two different performance measures are presented. An approximately 33% lower root-mean-squared error (RMSE) in protein predictions and about 40% lower total RMSE is obtained with the neural network-based model as opposed to kinetic models. Using the neural network-based model, the RMSE in predicting optimal conditions for two performance indices, is about 67% and 40% lower, respectively, when compared with the kinetic models. Thus, both model predictions and optimization results from the neural network-based model are found to be closer to the experimental data than the kinetic models developed in this work. It is shown that the neural network parameter function modeling method can be useful as a "macromodeling" technique to rapidly develop dynamic models of a process.

Cellulase↗

PubMed1998

High-resolution crystal structures reveal how a cellulose chain is bound in the 50 A long tunnel of cellobiohydrolase I from Trichoderma reesei.

Detailed information has been obtained, by means of protein X-ray crystallography, on how a cellulose chain is bound in the cellulose-binding tunnel of cellobiohydrolase I (CBHI), the major cellulase in the hydrolysis of native, crystalline cellulose by the fungus Trichoderma reesei. Three high-resolution crystal structures of different catalytically deficient mutants of CBHI in complex with cellotetraose, cellopentaose and cellohexaose have been refined at 1.9, 1.7 and 1.9 A resolution, respectively. The observed binding of cellooligomers in the tunnel allowed unambiguous identification of ten well-defined subsites for glucosyl units that span a length of approximately 50 A. All bound oligomers have the same directionality and orientation, and the positions of the glucosyl units in each binding site agree remarkably well between the different complexes. The binding mode observed here corresponds to that expected during productive binding of a cellulose chain. The structures support the hypothesis that hydrolysis by CBHI proceeds from the reducing towards the non-reducing end of a cellulose chain, and they provide a structural explanation for the observed distribution of initial hydrolysis products.

Amino Acid Substitution↗

PubMed2002

Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei.

Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.

Ascomycota↗

PubMed2004

Effect of pH on cellulase production of Trichoderma reesei RUT C30.

Currently, the high market price of cellulases prohibits commercialization of the lignocellulosics-to-fuel ethanol process, which utilizes enzymes for saccharification of cellulose. For this reason research aimed at understanding and improving cellulase production is still a hot topic in cellulase research. Trichoderma reesei RUT C30 is known to be one of the best hyper producing cellulolytic fungi, which makes it an ideal test organism for research. New findings could be adopted for industrial strains in the hope of improving enzyme yields, which in turn may result in lower market price of cellulases, thus making fuel ethanol more cost competitive with fossil fuels. Being one of the factors affecting the growth and cellulase production of T. reesei, the pH of cultivation is of major interest. In the present work, numerous pH-controlling strategies were compared both in shake-flask cultures and in a fermentor. Application of various buffer systems in shake-flask experiments was also tested. Although application of buffers resulted in slightly lower cellulase activity than that obtained in non-buffered medium, beta-glucosidase production was increased greatly.

Biotechnology↗

PubMed1993

Trichoderma reesei has no true exo-cellulase: all intact and truncated cellulases produce new reducing end groups on cellulose.

Adsorption to and formation of insoluble reducing end groups on cellulose was studied for intact enzymes and catalytic domains, 'cores', of the four major cellulases from Trichoderma reesei, CBH I, CBH II, EG I and EG III. Individual enzymes were incubated with NaBH4-reduced, phosphoric acid swollen Avicel (regenerated cellulose) or with filter paper. Adsorption onto regenerated cellulose was rapid (equilibration reached within 2 min), but was slow onto filter paper (not completed after 24 h). On both substrates, less was bound of the core domains than of the intact enzymes. After reaching a maximum in adsorption, all the core domains except CBH I core were released again. In general, the desorption of the core enzymes was much faster than the rate of substrate conversion. All enzymes produced new reducing end groups on both substrates, and thus none of them is a true exo-cellulase. However, both the rate of formation and the amount was considerably higher for the EG enzymes than for the CBH's, which may justify the classification of cellulases into two groups, although the difference is quantitative rather than qualitative. EG III was the most endo-active of the enzymes, and CBH I the least.

Adsorption↗

PubMed2006

Two crystal structures of Trichoderma reesei hydrophobin HFBI--the structure of a protein amphiphile with and without detergent interaction.

Hydrophobins are small fungal proteins that are highly surface active and possess a unique ability to form amphiphilic membranes through spontaneous self-assembly. The first crystal structure of a hydrophobin, Trichoderma reesei HFBII, revealed the structural basis for the function of this amphiphilic protein--a patch consisting of hydrophobic side chains on the protein surface. Here, the crystal structures of a native and a variant T. reesei hydrophobin HFBI are presented, revealing the same overall structure and functional hydrophobic patch as in the HFBII structure. However, some structural flexibility was found in the native HFBI structure: The asymmetric unit contained four molecules, and, in two of these, an area of seven residues was displaced as compared to the two other HFBI molecules and the previously determined HFBII structure. This structural change is most probably induced by multimer formation. Both the native and the N-Cys-variant of HFBI were crystallized in the presence of detergents, but an association between the protein and a detergent was only detected in the variant structure. There, the molecules were arranged into an extraordinary detergent-associated octamer and the solvent content of the crystals was 75%. This study highlights the conservation of the fold of class II hydrophobins in spite of the low sequence identity and supports our previous suggestion that concealment of the hydrophobic surface areas of the protein is the driving force in the formation of multimers and monolayers in the self-assembly process.

Amino Acid Sequence↗

PubMed2006

Factors affecting foaming behavior in cellulase fermentation by Trichoderma reesei Rut C-30.

Coupling fermentation with in situ foam fractionation may be beneficial to cellulase production in optimizing oligomer inducer generation, minimizing catabolite repression and reducing cellulase degradation by proteases. In this study, the potential factors that may affect the foaming behavior of broth from Trichoderma reesei Rut C-30 fermentation were examined. These factors included solid (both cell and cellulose) concentrations, cellulase activity and extracellular protein concentration. The loss of cellulase activity caused by the foaming process was minimal. The foamate generation was lower in the presence of higher solids (cell and/or cellulose) concentrations. Cellulase appeared to promote the broth foaming ability but its enrichment ratio was not high (lower than 1.2). The enrichment ratios for the individual component enzymes (beta-glucosidase, endo- and exo-glucanases) were found to be similarly low. None of the cellulase components were likely the primary foaming factors. The foam also carried out cells and cellulose solids. The hydrophobicity of cell surface, studied at various fermentation stages and in both media with and without cellulose, increased as the fermentation approached the stationary phase and then decreased gradually after entering the stationary phase.

Cellulase↗

PubMed1996

Cloning and characterisation of genes (pkc1 and pkcA) encoding protein kinase C homologues from Trichoderma reesei and Aspergillus niger.

Oligonucleotides, designed on the basis of conserved flanking amino acid sequence segments within the catalytic domain of eukaryotic protein kinase C (PKC) proteins, were used as primers for polymerase chain reactions to amplify a 427-bp chromosomal DNA fragment from the filamentous fungus Trichoderma reesei. This fragment was then used to isolate genes encoding PKC homologues of T. reesei and Aspergillus niger (pkc1 and pkcA, respectively). The genes contain six (T. reesei) and eight (A. niger) introns, which exhibit notable conservation in position with those found in the corresponding Schizosaccharomyces pombe pkc1+ and Drosophila melanogaster dPKC53Ebr genes. A single 4.2-kb transcript was detected in Northern analyses. The deduced PKC1 (T.reesei, 126 kDa) and PKCA (A. niger, 122 kDa) amino acid sequences reveal domains homologous to the C1 and C3/C4 domains of PKC-related proteins, but lack typical Ca(2+)-binding (C2) domains. Both contain a large, extended N-terminus, which shares a high degree of similarity with the corresponding regions of Saccharomyces cerevisiae PKC1 and S. pombe pkc1+ and pkc2+ proteins, but which is not present in PKCs of Dictyostelium or higher eukaryotes. This extended region can be divided into three subdomains; the N-terminal one contains a hydrophobic helix-turn-helix motif, whereas the C-terminal one contains potential targets for proteolytic processing. A polyclonal antiserum raised against the pseudosubstrate-binding domain of PKC1 recognizes in T. reesei a 115-120 kDa protein in Western blots. Expression of pkc1 cDNA in insect cells directs the synthesis of a PKC1 protein of similar size. The T. reesei PKC1 protein was partially purified and some of its properties examined: it is stimulated about twofold by phospholipids or phorbol esters but is not stimulated by Ca2+. We conclude that these PKC proteins from filamentous fungi represent the Ca(2+)-insensitive fungal homologues of the nPKC family.

Amino Acid Sequence↗

PubMed2005

beta-Glucosidase production by Trichoderma reesei.

The hydrolysis of cellulose to the water-soluble products cellobiose and glucose is achieved via synergistic action of cellulolytic proteins. The three types of enzymes involved in this process are endoglucanases, cellobiohydrolases, and beta-glucosidases. One of the best fungal cellulase producers is Trichoderma reesei RUT C30. However, the amount of beta-glucosidases secreted by this fungus is insufficient for effective cellulose conversion. We investigated the production of cellulases and beta-glucosidases in shake-flask cultures by applying three pH-controlling strategies: the pH of the production medium was adjusted to 5.8 after the addition of seed culture with no additional pH adjustment performed, the pH was adjusted to 6.0 daily, and the pH was maintained at 6.0 by the addition of Tris-maleate buffer to the growth medium. Different carbon sources Solka Floc 200, glucose, lactose, and sorbitol were added to standard Mandels nutrients. The lowest beta-glucosidase activities were obtained when no pH adjustment was done regardless of the carbon source employed. Somewhat higher levels of beta- glucosidase were measured in the culture filtrates when daily pH adjustment was carried out. The effect of buffering the culture medium on beta-glucosidase liberation was most prominent when a carbon source inducing the production of other cellulases was applied.

Cell Culture Techniques↗