Showing papers on "Trichoderma reesei published in 2003"

ACEI of Trichoderma reesei is a repressor of cellulase and xylanase expression

Abstract: We characterized the effect of deletion of the Trichoderma reesei (Hypocrea jecorina) ace1 gene encoding the novel cellulase regulator ACEI that was isolated based on its ability to bind to and activate in vivo in Saccharomyces cerevisiae the promoter of the main cellulase gene, cbh1. Deletion of ace1 resulted in an increase in the expression of all the main cellulase genes and two xylanase genes in sophorose- and cellulose-induced cultures, indicating that ACEI acts as a repressor of cellulase and xylanase expression. Growth of the strain with a deletion of the ace1 gene on different carbon sources was analyzed. On cellulose-based medium, on which cellulases are needed for growth, the Δace1 strain grew better than the host strain due to the increased cellulase production. On culture media containing sorbitol as the sole carbon source, the growth of the strain with a deletion of the ace1 gene was severely impaired, suggesting that ACEI regulates expression of other genes in addition to cellulase and xylanase genes. A strain with a deletion of the ace1 gene and with a deletion of the ace2 gene coding for the cellulase and xylanase activator ACEII expressed cellulases and xylanases similar to the Δace1 strain, indicating that yet another activator regulating cellulase and xylanase promoters was present.

Effects of inactivation and constitutive expression of the unfolded- protein response pathway on protein production in the yeast Saccharomyces cerevisiae.

Abstract: One strategy to obtain better yields of secreted proteins has been overexpression of single endoplasmic reticulum-resident foldases or chaperones. We report here that manipulation of the unfolded-protein response (UPR) pathway regulator, HAC1, affects production of both native and foreign proteins in the yeast Saccharomyces cerevisiae. The effects of HAC1 deletion and overexpression on the production of a native protein, invertase, and two foreign proteins, Bacillus amyloliquefaciens alpha-amylase and Trichoderma reesei endoglucanase EGI, were studied. Disruption of HAC1 caused decreases in the secretion of both alpha-amylase (70 to 75% reduction) and EGI (40 to 50% reduction) compared to the secretion by the parental strain. Constitutive overexpression of HAC1 caused a 70% increase in alpha-amylase secretion but had no effect on EGI secretion. The invertase levels were twofold higher in the strain overexpressing HAC1. Also, the effect of the active form of T. reesei hac1 was tested in S. cerevisiae. hac1 expression caused a 2.4-fold increase in the secretion of alpha-amylase in S. cerevisiae and also slight increases in invertase and total protein production. Overexpression of both S. cerevisiae HAC1 and T. reesei hac1 caused an increase in the expression of the known UPR target gene KAR2 at early time points during cultivation.

Activation mechanisms of the HAC1-mediated unfolded protein response in filamentous fungi.

Abstract: Summary The unfolded protein response (UPR) is a regulatory pathway activating genes involved in multiple functions related to folding, quality control and transport of secreted proteins. Characterization of the hac1/hacA genes encoding the UPR transcription factors from the filamentous fungi Trichoderma reesei and Aspergillus nidulans is described in this article. The corresponding gene in Saccharomyces cerevisiae is activated through a non-spliceosomal intron-splicing reaction. The T. reesei hac1 and A. nidulans hacA mRNAs undergo an analogous splicing reaction of a 20-nt-long intron during UPR induction. This splicing changes the reading frame of the mRNA and thus could bring in an activation domain to the HACI/HACA proteins. In addition to the non-spliceosomal splicing, the hac1/A mRNAs of the filamentous fungi are truncated at the 5′-flanking region upon UPR induction. An upstream open reading frame is omitted from the mRNAs due to the truncation, and evidence is presented showing that the truncated T. reesei hac1 mRNA is translated more efficiently than a full-length mRNA. This paper reports a novel combination of two different regulatory mechanisms of a transcription factor gene, both operational at the mRNA level.

Efficient cellulase production by Trichoderma reesei in continuous cultivation on lactose medium with a computer-controlled feeding strategy

Abstract: A low-foaming hydrophobin II deletant of the Trichoderma reesei strain Rut-C30 was used for production of cellulases by continuous cultivation on lactose medium in a laboratory fermenter. The control paradigm of the addition of new medium to the continuous process was based on the growth dynamics of the fungus. A decrease in the rate of base addition to the cultivation for pH-minimum control was used as an indicator of imminent exhaustion of carbon source for growth and enzyme induction. When the amount of base added per 5 min computation cycle decreased below a given value, new medium was added to the fermenter. When base addition for pH control thereafter increased above the criterion value, due to increased growth, the medium feed was discontinued or decreased. The medium feeding protocol employed was successful in locking the fungus in the stage of imminent, but not actual, exhaustion of carbon source. According to the results of a batch cultivation of the same strain on the same medium, this is the phase of maximal enzyme productivity. The medium addition protocol used in this work resulted in a very stable continuous process, in which cellulase productivity was maintained for several hundred hours at the maximum level observed in a batch cultivation for only about 10 h. Despite a major technical disturbance after about 420 h, the process was restored to stability. When the cultivation was terminated after 650 h, the level of enzyme production was still maximal, with no signs of instability of the process.

Bacillus pumilus BpCRI 6, a promising candidate for cellulase production under conditions of catabolite repression

Abstract: Cellulose degrading organisms have been used for the conversion of cellulolytic materials into soluble sugars or solvents in several biotechnological and industrial applications. In this report, a mutant of Bacillus pumilus was obtained after chemical mutagenesis and screened for cellulase production. This mutant named BpCRI 6 was selected for its ability to produce cellulase under catabolite repression. Cellulase yield by BpCRI 6 was four times higher than that of the wild type under optimum growth conditions (pH 6.5, 25°C and Ca 2+ 1mM). In shaking flask cultures, production of cellulase by the wild type was completely repressed in the presence of 25 mM glucose, while BpCRI 6 strain still exhibited a residual cellulase production of 80 and 40% at 25 mM and 40 mM of glucose concentrations respectively. The mutant strain is stable and grows rapidly in liquid and solid media. Under conditions of catabolite repression (40 mM of glucose), the production of cellulase by this mutant is particularly significant when compared to Trichoderma reesei a well-known cellulase producer, which is under control of end-product inhibition. This is the first report of a successful catabolite repression insensitivity of cellulase production by a mutant of B. pumilus.

Influence of fibrolytic enzymes on the hydrolysis and fermentation of pure cellulose and xylan by mixed ruminal microorganisms in vitro.

Abstract: A series of in vitro studies was conducted to determine the effects of adding a commercial enzyme product on the hydrolysis and fermentation of cellulose, xylan, and a mixture (1:1 wt/wt) of both. The enzyme product (Liquicell 2500, Specialty Enzymes and Biochemicals, Fresno, CA) was derived from Trichoderma reesei and contained mainly xylanase and cellulase activities. Addition of enzyme (0.5, 2.55 and 5.1 microL/g of DM) in the absence of ruminal fluid increased (P < 0.001) the release of reducing sugars from xylan and the mixture after 20 h of incubation at 20 degrees C. Incubations with ruminal fluid showed that enzyme (0.5 and 2.55 microL/g of DM) increased (P < 0.05) the initial (up to 6 h) xylanase, endoglucanase, and beta-D-glucosidase activities in the liquid fraction by an average of 85%. Xylanase and endoglucanase activities in the solid fraction also were increased (P < 0.05) by enzyme addition, indicating an increase in fibrolytic activity due to ruminal microbes. Gas production over 96 h of incubation was determined using a gas pressure measurement technique. Incremental levels of enzyme increased (P < 0.05) the rate of gas production of all substrates, suggesting that fermentation of cellulose and xylan was enzyme-limited. However, adding the enzyme at levels higher than 2.55 microL/g of DM failed to further increase the rate of gas production, indicating that the maximal level of stimulation was already achieved at lower enzyme concentrations. It was concluded that enzymes enhanced the fermentation of cellulose and xylan by a combination of pre- and postincubation effects (i.e., an increase in the release of reducing sugars during the pretreatment phase and an increase in the hydrolytic activity of the liquid and solid fractions of the ruminal fluid), which was reflected in a higher rate of fermentation.

Synergistic cellulose hydrolysis can be described in terms of fractal-like kinetics.

Abstract: A fractal-like kinetics model was used to describe the synergistic hydrolysis of bacterial cellulose by Trichoderma reesei cellulases. The synergistic action of intact cellobiohydrolase Cel7A and endoglucanase Cel5A at low enzyme-to-substrate ratios showed an apparent substrate inhibition consistent with a case where two-dimensional (2-D) surface diffusion of the cellobiohydrolase is rate-limiting. The action of Cel7A core and Cel5A was instead consistent with a three-dimensional (3-D) diffusion-based mode of action. The synergistic action of intact Cel7A was far superior to that of the core at a high enzyme-to-substrate ratio, but this effect was gradually reduced at lower enzyme-to-substrate ratios. The apparent fractal kinetics exponent h obtained by nonlinear fit of hydrolysis data to the fractal-like kinetics analogue of a first-order reaction was a useful empirical parameter for assessing the rate retardation and its dependence on the reaction conditions.

High-Yield Production of a Bacterial Xylanase in the Filamentous Fungus Trichoderma reesei Requires a Carrier Polypeptide with an Intact Domain Structure

Abstract: A bacterial xylanase gene, Nonomuraea flexuosa xyn11A, was expressed in the filamentous fungus Trichoderma reesei from the strong cellobiohydrolase 1 promoter as fusions to a variety of carrier polypeptides By using single-copy isogenic transformants, it was shown that production of this xylanase was clearly increased (up to 820 mg/liter) when it was produced as a fusion protein with a carrier polypeptide having an intact domain structure compared to the production (150 to 300 mg/liter) of fusions to the signal sequence alone or to carriers having incomplete domain structures The carriers tested were the T reesei mannanase I (Man5A, or MANI) core-hinge and a fragment thereof and the cellulose binding domain of T reesei cellobiohydrolase II (Cel6A, or CBHII) with and without the hinge region(s) and a fragment thereof The flexible hinge region was shown to have a positive effect on both the production of Xyn11A and the efficiency of cleavage of the fusion polypeptide The recombinant Xyn11A produced had properties similar to those of the native xylanase It constituted 6 to 10% of the total proteins secreted by the transformants About three times more of the Man5A core-hinge carrier polypeptide than of the recombinant Xyn11A was observed Even in the best Xyn11A producers, the levels of the fusion mRNAs were only approximately 10% of the level of cel7A (cbh1) mRNA in the untransformed host strain

Regulation of Trichoderma cellulase formation: lessons in molecular biology from an industrial fungus. A review.

Abstract: The present article reviews the current understanding of regulation of cellulase gene transcription in Hypocrea jecorina (= Trichoderma reesei). Special emphasis is put on the mechanism of action of low molecular weight inducers of cellulase formation, the presence and role of recently identified transactivating proteins (Ace1, Ace2, Hap2/3/5), and the role of the carbon catabolite repressor Cre1. We also report on some recent genomic approaches towards understanding how cellulase inducers signal their presence to the transcriptional apparatus.

The relationship between thermal stability and pH optimum studied with wild-type and mutant Trichoderma reesei cellobiohydrolase Cel7A.

Abstract: The major cellulase secreted by the filamentous fungus Trichoderma reesei is cellobiohydrolase Cel7A Its three-dimensional structure has been solved and various mutant enzymes produced In order to study the potential use of T reesei Cel7A in the alkaline pH range, the thermal stability of Cel7A was studied as a function of pH with the wild-type and two mutant enzymes using different spectroscopic methods Tryptophan fluorescence and CD measurements of the wild-type enzyme show an optimal thermostability between pH 35-56 (Tm, 62 +/- 2 degrees C), at which the highest enzymatic activity is also observed, and a gradual decrease in the stability at more alkaline pH values A soluble substrate, cellotetraose, was shown to stabilize the protein fold both at optimal and alkaline pH In addition, unfolding of the Cel7A enzyme and the release of the substrate seem to coincide at both acidic and alkaline pH, demonstrated by a change in the fluorescence emission maximum CD measurements were used to show that the five point mutations (E223S/A224H/L225V/T226A/D262G) that together result in a more alkaline pH optimum [Becker, D, Braet, C, Brumer, H, III, Claeyssens, M, Divne, C, Fagerstrom, RB, Harris, M, Jones, TA, Kleywegt, GJ, Koivula, A, et al (2001) Biochem J356, 19-30], destabilize the protein fold both at acidic and alkaline pH when compared with the wild-type enzyme In addition, an interesting time-dependent fluorescence change, which was not observed by CD, was detected for the pH mutant Our data show that in order to engineer more alkaline pH cellulases, a combination of mutations should be found, which both shift the pH optimum and at the same time improve the thermal stability at alkaline pH range

Production of β-glucosidase in mixed culture of Aspergillus niger BKMF 1305 and Trichoderma reesei RUT C30

Abstract: Summary The aim of the present study was to investigate a new approach to -glucosidase production of an Aspergillus strain using cheap lignocellulosic material i.e. waste paper in order to substitute glucose, a generally used carbon source, and thereby reduce the production cost. The enzyme production was performed and optimized for the highest -glucosidase yield in cofermentation with a Trichoderma strain to support the degradation of cellulose and to provide the non-cellulolytic Aspergillus with water soluble carbon source. Batch fermentation experiments of Aspergillus niger BKMF 1305 and Trichoderma reesei RUT C30 were carried out in shake flask cultures. The factors influencing the enzyme production, such as the concentrations of nutrients and carbon source, the inoculum ratio of the two species, and the delay in A. niger inoculation were investigated using a 2 3 full factorial design. The results were analyzed with the response surface methodology using commercially available software, Statistica for Windows. All three examined factors were found significant. The highest -glucosidase activity of 3.07 IU/mL was obtained after 7 days of incubation, if 3.3 % Aspergillus and 6.7 % Trichoderma inoculum were added at the same time to modified Mandels’ medium, in which the concentration of nutrients was doubled compared to normal Mandels’ medium and the carbon source concentration was set to 20 g/L waste paper.

d-Xylose Metabolism in Hypocrea jecorina: Loss of the Xylitol Dehydrogenase Step Can Be Partially Compensated for by lad1-Encoded l-Arabinitol-4-Dehydrogenase

Abstract: With the goal of the genetic characterization of the d-xylose pathway in Hypocrea jecorina (anamorph: Trichoderma reesei), we cloned the xdh1 gene, encoding NAD-xylitol dehydrogenase, which catalyzes the second step of fungal d-xylose catabolism. This gene encodes a 363-amino-acid protein which has a mass of 38 kDa, belongs to the zinc-containing alcohol dehydrogenase family, exhibits high sequence identity to the published sequences of xylitol dehydrogenases from yeast origins, but contains a second, additional binding site for Zn2+. The enzyme catalyzed the NAD-dependent oxidation of xylitol and d-sorbitol and the NADH-dependent reduction of d-xylulose and d-fructose. No activity was observed with NADP, l-arabinose, or l-arabinitol. A single 1.4-kb transcript was formed during growth on xylan, d-xylose, l-arabinose, l-arabinitol and, at a lower abundance, xylitol, d-galactose, galactitol, and lactose but not on d-glucose and glycerol. xdh1 deletion mutants exhibited 50% reduced growth rates on d-xylose, whereas growth rates on xylitol remained unaltered. These mutants contained 30% of the xylitol dehydrogenase activity of the parent strain, indicating the presence of a second xylitol dehydrogenase. This activity was shown to be due to lad1-encoded l-arabinitol-4-dehydrogenase, because H. jecorina xdh1 lad1 double-deletion strains failed to grow on d-xylose or xylitol. In contrast, lad1 deletion strains of H. jecorina grew normally on these carbon sources. These results show that H. jecorina contains a single xylitol dehydrogenase which is encoded by xdh1 and is involved in the metabolism of d-xylose and that lad1-encoded l-arabinitol-4-dehydrogenase can compensate for it partially in mutants with a loss of xdh1 function.

Transcriptional Regulation of xyn2 in Hypocrea jecorina

Abstract: The xylanase system of the filamentous fungus Hypocrea jecorina (Trichoderma reesei) consists of two specific xylanases, Xyn1 and Xyn2, which are simultaneously expressed during growth on xylan but respond differentially to low-molecular-weight inducers. Using in vivo footprinting analysis of xylan-induced and noninduced mycelia, we detected two adjacent nucleotide sequences (5′-AGAA-3′ on the noncoding strand and 5′-GGGTAAATTGG-3′, referred to as the xylanase-activating element [XAE], on the coding strand, respectively) to bind proteins. Among these, binding to the AGAA-box is only observed under noninduced conditions, whereas binding to XAE is constitutive. Electrophoretic mobility shift assay with heterologously expressed components of the H. jecorina Hap2/3/5 protein complex and the cellulase regulator Ace2 suggests that these two transactivators form the protein complex binding to XAE. H. jecorina transformants, containing correspondingly mutated versions of the xyn2 promoter fused to the Aspergillus niger goxA gene as a reporter, revealed that the elimination of protein binding to the AGAA-box resulted in a threefold increase in both basal and induced transcription, whereas elimination of Ace2 binding to its target in XAE completely eliminated transcription under both conditions. Destruction of the CCAAT-box by insertion of a point mutation prevents binding of the Hap2/3/5 complex in vitro and results in a slight increase in both basal and induced transcription. These data support a model of xyn2 regulation based on the interplay of Hap2/3/5, Ace2 and the AGAA-box binding repressor.

Superactive cellulase formulation using cellobiohydrolase-1 from Penicillium funiculosum

Abstract: Purified cellobiohydrolase I (glycosyl hydrolase family 7 (Cel7A) enzymes from Penicillium funiculosum demonstrate a high level of specific performance in comparison to other Cel7 family member enzymes when formulated with purified E1cd endoglucanase from A. cellulolyticus and tested on pretreated corn stover. This result is true of the purified native enzyme, as well as recombinantly expressed enzyme, for example, that enzyme expressed in a non-native Aspergillus host. In a specific example, the specific performance of the formulation using puriified recombinant Cel7A from Penicillium funiculosum expressed in A. awamori is increased by more than 200% when compared to a formulation using purified Cel7A from Trichoderma reesei.

Probing pH-dependent functional elements in proteins : Modification of carboxylic acid pairs in Trichoderma reesei cellobiohydrolase Cel6A

Abstract: Two carboxylic acid side chains can, depending on their geometry and environment, share a proton in a hydrogen bond and form a carboxyl-carboxylate pair. In the Trichoderma reesei cellobiohydrolase ...

The Humicola grisea Cel12A enzyme structure at 1.2 A resolution and the impact of its free cysteine residues on thermal stability.

Abstract: As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have extended our previous work on the structural and biochemical diversity of GH 12 homologs to include the most stable fungal GH 12 found, Humicola grisea Cel12A. The H. grisea enzyme was much more stable to irreversible thermal denaturation than the Trichoderma reesei enzyme. It had an apparent denaturation midpoint (Tm) of 68.7°C, 14.3°C higher than the T. reesei enzyme. There are an additional three cysteines found in the H. grisea Cel12A enzyme. To determine their importance for thermal stability, we constructed three H. grisea Cel12A single mutants in which these cysteines were exchanged with the corresponding residues in the T. reesei enzyme. We also introduced these cysteine residues into the T. reesei enzyme. The thermal stability of these variants was determined. Substitutions at any of the three positions affected stability, with the largest effect seen in H. grisea C206P, which has a Tm 9.1°C lower than that of the wild type. The T. reesei cysteine variant that gave the largest increase in stability, with a Tm 3.9°C higher than wild type, was the P201C mutation, the converse of the destabilizing C206P mutation in H. grisea. To help rationalize the results, we have determined the crystal structure of the H. grisea enzyme and of the most stable T. reesei cysteine variant, P201C. The three cysteines in H. grisea Cel12A play an important role in the thermal stability of this protein, although they are not involved in a disulfide bond.