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1
Lajoie, Denis. "Lactose hydrolases de Trichoderma reesei MCG-80." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp04/mq26227.pdf.
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Zhang, Qin. "COLLECTION OF TRICHODERMA REESEI CELLULASE BY FOAMING." University of Akron / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=akron1195069754.
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Heikinheimo, Lea. "Trichoderma reesei cellulases in processing of cotton /." Espoo : Technical Research Centre of Finland, 2002. http://www.vtt.fi/inf/pdf/publications/2002/P483.pdf.
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Chahal, Parminder Singh. "Cellulase production from lignocellulosic materials by Trichoderma reesei." Thesis, University of Ottawa (Canada), 1987. http://hdl.handle.net/10393/5536.
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Cochet-Giraud, Nelly. "Les Cellulases de Trichoderma reesei production et application /." Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb37604010n.
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Curach, Natalie Claire. "An investigation into the hex1 gene and gene promoter for the enhancement of protein production in Trichoderma reesei." Phd thesis, Australia : Macquarie University, 2005. http://hdl.handle.net/1959.14/71199.
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Supplementary material to figures contained on DVD only available with manuscript.Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Dept. of Biological Sciences, 2005.
Bibliography: p. 221-244.
Introduction -- Materials and methods -- Isolation of the hex1 gene from Trichoderma reesei and Ophiostoma floccosum -- Expression of DsRed under the cbh1 promoter and the hex1 promoter with random integration -- Modified expression vectors containing a fusion to a portion of hex1 gene sequence -- Expression of DsRed from the hex1 locus and the phenotypic characteristics of a hex1 deletion mutant -- Summary and concluding discussion.
For Trichoderma reesei to be developed as an effiecient producer of a large variety of proteins, the expression system requires diversification. In particular, the choice of promoters available needs to be broadened to include promoters which are active in conditions other than those conducive to induction of cellulase expression. Using proteomics, the HEX1 protein was identified as an abundant protein of the cell envelope of T. reesei when grown on a range of carbon sources, suggesting that a strong constitutive promoter drives the expression of this physiologically important protein. This thesis is an exploration into the hex1 gene promoter and the role of hex1 in the maintenance of mycelium integrity in T. reesei with consideration for the application of this gene in the further development of filamentous fungi as protein expression systems. -- The single copy hex1 gene and flanking regions were isolated from T. reesei and another biotechnologically important fungus, Ophiostoma floccosum. The fluorescent reporter protein DsRed1-E5 was expressed under the T. reesei hex1 promoter and promoter activity was monitored by fluorescence CLSM and RNA analysis. During the rapid growth phase of a culture, the hex1 promoter was active in a range of carbon sources and three transcipt types with alternative tsp and splicing sites were discovered for the hex1 gene. The distribution of fluorescence throughout the mycelium suggested spatial regulation of the hex1 promoter as well as temporal regulation. The promoter was continually active in the absence of a functional hex1 gene product suggesting that the hex1 promoter is regulated in part, by negative feedback from the endogenous gene product. Interruption of the hex1 gene produced hyphae that leaked excessive volumes of cytoplasm when physically damaged which may be advantageous for the externalisation of selected protein products. The results indicate that the regulation of the hex1 hene promoter is complex and that the hex1 gene is integral to the maintenance of the integrity of the fungal mycelium.
Mode of access: World Wide Web.
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Poggi-Parodi, Dante. "Une approche de biologie systémique pour développer des souches industrielles performantes de Trichoderma reesei." Electronic Thesis or Diss., Paris 6, 2014. http://www.theses.fr/2014PA066721.
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La compréhension de la régulation de la synthèse et sécrétion de cellulases dans le champignon Trichoderma reesei a fortement évolué ces dernières années. Cependant, le coût de production de cellulases reste encore un des principaux problèmes pour la production de bioéthanol à partir de lignocellulose. C’est dans ce contexte que mon projet de thèse s’inscrit, dans l’amélioration de la production de cellulases de T. reesei et son adaptation aux conditions industrielles. D’abord, nous avons réalisé une analyse du transcriptome sur une lignée de souches améliorées dans des conditions proches du procède industrielle avec lactose comme inducteur. Cette étude nous a permis de trouver des gènes spécifiquement régulés dans la souche plus performante, probablement impliqués dans sa grande capacité de production de cellulases. Dans un deuxième temps, nous avons identifié parmi les gènes régulés du transcriptome lesquels impliquées dans la production de cellulases. A cet effet, des souches mutées ont été construites et leur phénotype évalué. Trois de ces gènes mutés ont affecté la production de cellulase et leur fonction ont dévoilé des nouvelles mécanismes régules. Finalement, nous avons exploré les différences d’expression lorsque nous utilisons un hydrolysat de lignocellulose comme inducteur de cellulases à la place du lactose. Une étude du transcriptome dans ces conditions, nous a permis de caractériser les différences de régulation des gènes entre ces inducteurs. En outre, nous avons identifié un groupe des gènes probablement impliqué dans la détoxification qui peuvent être utilisés dans le futur pour développer une souche résistante aux inhibiteursA lot of progress had been done in recent years to understand the regulation of synthesis and secretion of cellulases in the fungus Trichoderma reesei. However, the production cost of cellulases still remains one of the most important limiting steps in the production of bioethanol from lignocellulose. It is in this context that my PhD project has been developed: to genetic engineering T. reesei strains to increase its cellulase production and its adaptation to industrial conditions. First, we conducted a transcriptome analysis to an improved lineage of industrial strains during cellulase production following conditions close to the industrial process (lactose as inducer and fed-batch culture in bioreactor). We found specifically regulated genes for the most performant strain, possibly involved in its high cellulase production capacity. Then, we identified which regulated genes from transcriptome were involved in cellulase production. For this purpose mutated strains for highly regulated genes were constructed and their phenotype assessed. Three mutated genes showed to impact cellulase production and their function gave us an insight into new mechanisms being regulated. Finally, we explored the expression differences when we used a lignocellulose hydrolysate as cellulases inducer instead of lactose. A transcriptomic study of cellulases production on lignocellulose hydrolysates and lactose, allowed us to characterize the differences in regulated genes between these inducers. In addition, a group of genes probably related to detoxification was identified and could be used in the future to develop an inhibitor resistant strain
8
Askolin, Sanna. "Characterization of the Trichoderma reesei hydrophobins HFBI and HFBII /." [Espoo, Finland] : VTT Technical Research Centre of Finland, 2006. http://www.vtt.fi/inf/pdf/publications/2006/P601.pdf.
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Belshaw, N. J. "The nuclear matrix and gene expression in Trichoderma reesei." Thesis, University of East Anglia, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.296346.
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Chan, Ho Tong Laetitia. "Amélioration du champignon cellulolytique Trichoderma reesei par reproduction sexuée." Thesis, Paris, Institut agronomique, vétérinaire et forestier de France, 2017. http://www.theses.fr/2017IAVF0017.
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Dans le cadre de la production de bioéthanol de deuxième génération, l’ingénierie génétique de Trichoderma reesei est une des solutions envisagées pour diminuer le coût de l’étape d’hydrolyse enzymatique. Elle permet d’améliorer les performances de sécrétion de ce champignon filamenteux producteur de cellulases et les propriétés de ces enzymes. Comme de nombreux champignons industriels, T. reesei a longtemps été considéré comme possédant exclusivement un cycle asexuel. La mise en évidence récente d’un cycle de reproduction sexuée chez ce champignon filamenteux ouvre de nouvelles perspectives d’amélioration des souches utilisées en biotechnologie. Cependant, comme toutes les souches industrielles de T. reesei dérivent de l’isolat sauvage QM6a, elles sont toutes de type sexuel MAT1-2 et stériles en tant que femelles. L’objectif de ce travail de thèse est de mettre en place la reproduction sexuée comme outil génétique et d’amélioration des performances des souches industrielles. Son utilisation en complément des outils d’ingénierie génétique permet de combiner des caractères intéressants ou d’en générer de nouveaux, de stabiliser les souches industrielles en éliminant les mutations non désirées, de s’affranchir des marqueurs de sélection et d’identifier les gènes et mutations responsables d’un caractère d’intérêt. La première partie de ce travail a été consacré à l’optimisation de la reproduction sexuée puis à l’étude des étapes et des mécanismes de la reproduction sexuée entre souches sauvages et hyperproductrices. Dans un deuxième temps, nous avons mis en place la reproduction sexuée entre des souches hyperproductrices femelles stériles, à l’aide de la stratégie originale de la « souche assistante », qui a abouti au rétablissement partiel de leur reproduction sexuée. Afin de compléter ce rétablissement, une étude de l’étape de fécondation que nous supposons être l’étape problématique dans notre stratégie a été initiée. Parallèlement, l’exploitation de la reproduction sexuée entre souches sauvage et hyperproductrices a permis de générer une souche hyperproductrice de cellulases, de type sexuel MAT1-1 donc compatible avec toutes les souches industrielles, femelle fertile et possédant une activité β-glucosidase améliorée. Enfin, la reproduction sexuée associée à la génétique classique (« Bulk Segregant Analysis ») et aux techniques de séquençage haut débit a été mise en oeuvre pour permettre l’identification de mutations impliquées dans des phénotypes d’intérêtGenetic engineering of Trichoderma reesei is one of the solutions considered to reduce the cost of the enzymatic hydrolysis step in second-generation ethanol production processes. It allows the improvement of the secretory performances of this cellulase producer fungus and the properties of its enzymes. Like many industrial fungi, T. reesei has long been considered to possess exclusively an asexual cycle. The recent discovery of a sexual reproduction cycle in this filamentous fungus opens up new prospects of improvement for strains used in biotechnology. However, all industrial strains of T. reesei are derived from the wild isolate QM6a and therefore possess the MAT1-2 mating type and are female sterile.The aim of this work was to develop sexual reproduction as a genetic tool and to improve the performance of the industrial strains. In combination with the genetic engineering tools, it would enable combination of interesting traits or generation of new ones, improve strains stability by purging deleterious mutations, selection markers elimination and identification of genes and mutations responsible of interesting characteristics.The first part of this work was dedicated to the optimization of sexual reproduction and to the study of the steps and mechanisms of sexual reproduction between wild-type and hyperproducer strains. In a second step, we set up sexual reproduction between female sterile hyperproducer strains, using the original "assistant strain" strategy, which resulted in the partial restoration of their sexual reproduction. A study of the fertilization step was also initiated, as we suspect it to be the blocking point in our strategy. In a second part, we took advantage of sexual reproduction between wild-type and hyperproducer strains for (i) the generation of a MAT1-1 mating-type strain compatible with all industrial strains, female fertile and possessing improved β-glucosidase activity and (ii) the implementation of a bulk segregation analysis associated with high-throughput sequencing techniques to identify mutations involved in phenotypes of interest
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Lo, Chi-Ming. "Cellulase Production by Trichoderma Reesei Rut-C30." University of Akron / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=akron1205776927.
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Johansson, Helene. "Extraction and Characterization of Hydrophobin from Trichoderma reesei." Thesis, Linnéuniversitetet, Institutionen för naturvetenskap, NV, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:lnu:diva-8432.
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Hydrophobins are a class of small proteins (7-15kDa) found in filamentous fungi and are among the most surface active proteins known today. Because of this they have received attention for different applications, e.g. for the food industry as an alternative in emulsions. The goal of this project was to culture and extract hydrophobins from Trichoderma reesei and characterize it. This was done from a freeze-dried culture of Trichoderma reesei, which was cultured on PDA-plates and in liquid medium with glucose as carbon source. Extraction was made by breaking the cells, mechanically and by sonication, and then by shaking a seperating funnel to create foam from the surface-active proteins. The foam was washed and freeze-dried and the total protein concentration of the freeze-dried substance was determined with Bradford assay and the hydrophpbin was characterized with SDS-PAGE. The culturing of the fungi was successful. The amount of foam created was, however, less than expected. The Bradford assay gave a total protein concentration of 7.5% in the freeze-dried substance, but the SDS-PAGE didn't give any results. The reason for this probably depends on the culturing and the extraction of the hydrophobin. T. reesei hydrophobin HFBI, expressed in glucose containing media, is bound to the mycelium of the fungi and the breaking of the mycelium might not have been enough to release all the protein, which also would explain the small amounts of foam. One way to improve this could be to grow the fungi on lactose instead. This will result in that T. reesei produces HFBII instead, which is mainly released to the surrounding. The conclusion of the project is that the method for culturing and extraction needs to be improved to obtain hydrophobin from T. reesei.
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Awafo, Victor Ankang. "Biosynthesis of cellulase-system from Trichoderma reesei and its characteristics." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1997. http://www.collectionscanada.ca/obj/s4/f2/dsk2/tape16/PQDD_0002/NQ29881.pdf.
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Kyriacou, Andreas. "Characterization and adsorption of the cellulase components from Trichoderma reesei." Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75770.
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The cellulase enzyme system of the fungus Trichoderma reesei Rut C-30 was fractionated by DEAE ion exchange chromatography into four groups according to their substrate specificity. By analytical isoelectric focusing and activity stains it was revealed that fraction EGI is comprised of endoglucanases specific to cellulosic substrates, and that fractions EGII and EGIII are non-specific endoglucanases that hydrolyze cellulose as well as xylan substrates. The major protein fraction CBHI was shown to be a cellobiohydrolase. Turbidimetric measurement phase contrast microscopy and analysis of the products resulting from the hydrolysis of swollen cellulose demonstrated differences between endoglucanases and cellobiohydrolases. The enzyme component CBHII, previously described as a cellobiohydrolases was shown to be an endoglucanase.The adsorption behavior of the four enzyme fractions was examined, with respect to pH, temperature and ionic strength. This was accomplished by using ($ sp3$H) radiolabeled cellulase fractions as tracers. The adsorption of the cellulases occurred within 60 minutes, and was described by a Langmuir type correlation. Increasing the adsorption temperature increased the saturation uptake of the endoglucanases but not of the cellobiohydrolases. Changes in pH and ionic strength affected both the degree and strength of adsorption of all the fractions, likely due to protein structure conformational changes.
Direct evidence of exchange between adsorbed and free enzymes was obtained for each component using ($ sp3$H) and ($ sp{14}$C) radiolabeled tracers. In simultaneous adsorption of enzyme pairs, CBHI was shown to predominate adsorption. Endoglucanase EGI was preferentially adsorbed over EGII and EGIII. Sequential adsorption studies have shown that interaction between enzyme components largely determine the degree of their adsorption. Evidence suggested both common and distinct adsorption sites exist, and that their occupation depends on which components are involved.
Light microscopy and monitoring of sugar production during cellulose hydrolysis indicated that conditions which limit predominance in adsorption by any one of the cellulase components, enhance synergism and increase degree of hydrolysis.
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Awafo, Victor Ankang. "Biosynthesis of cellulase-system from Trichoderma reseei [i.e. reesei] characteristics." Thesis, McGill University, 1997. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41972.
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There are generally four factors recognized as delimiting in the study of lignocelluloses for fuel ethanol production, viz., the source of the cellulase-system and its quality characteristics for cellulose hydrolysis, the substrate and pretreatment method, the process for cellulase production and bioreactor design, and the ability of yeast to ferment mixed hexose and pentose sugars. Wheat straw (WS) and T. reesei mutants were used in the study to evaluate the production of cellulase-systems. Hydrolysis of cellulose revealed the superiority of mild NaOH pretreatment over steam explosion for cellulase production with T. reesei MCG 80 and QMY-1. Response surface models were capable of predicting that NaOH could be used for the pretreatment of WS at 4% (w/w) without urea in the fermentation medium to yield optimum filter paper activity (FPA) of 9.9 IU/mL (247 IU/g WS) and beta-glucosidase activity ($ beta$GA) of 6.4 IU/mL (159 IU/g WS) under solid-state fermentation (SSF) conditions. Multiple regression analysis with multiple coefficients of correlation, R, between 0.957 and 0.99 from the experimental data showed close agreement between the cellulase activities (FPA and $ beta$GA) from the experiments and predicted values.The superiority of SSF over liquid-state fermentation (LSF) in the production of cellulase-systems was also established, and a prototype pan-bioreactor showed good potential for upgrading cellulase production under SSF conditions. The economics of fuel ethanol production was considered in the optimization model that sought to establish threshold cellulase loadings needed to achieve maximum cellulose hydrolysis for fermentation. High substrate concentrations of up to 7.5% were hydrolyzed with cellulase loadings of 24-30 IU/g and fermented by Pichia stipitis to achieve 90-100% conversion into ethanol.
Crude unextracted cellulase yielded over 90% hydrolysis of delignified wheat straw and proved to be better than extracted cellulase and commercial cellulases for the hydrolysis of pure cellulose and pretreated wheat straw. Studies were also conducted to demonstrate the importance of the ratio of $ beta$GA- to FPA in cellulose hydrolysis which showed that ratios closer to one (1), produced more sugars and lowered the cellobiose content in the hydrolysates. It was also shown that the source of the cellulase is important in eliminating the accumulation of cellobiose during hydrolysis as was demonstrated with cellulase from mixed cultures of T. reesei and Aspergillus phoenicis. Higher $ beta$GA from the latter were implicated since A. phoenicis is a good $ beta$-glucosidase producer.
Delignified wheat straw at 5% concentration when subjected to separate hydrolysis and fermentation and simultaneous hydrolysis and fermentation resulted in similar volumetric productivities (g/L/h) of ethanol.
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Junior, Euclides Matheucci. "Clonagem e caracterização do gene de actina de trichoderma reesei." Universidade de São Paulo, 1993. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-18092015-165016/.
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Não consta resumo na publicação.The gene encoding actin in the cellulolytic filamentus fungus Trichoderma reesei has been isolated and sequenced. The nucleotide sequence reveals that the gene is composed of 6 exons separated by 5 introns within the coding region. The positions of the introns were predicted by comparison of sequence homology to the genes coding for actin with known amino acid sequence and by identification of splice-site signal sequences. The actin protein of Trichoderma reesei shows extensive homology to the actins of other fungi E. nidulans, 95% , T. lanuginosus, 92% and S. pombae. The T. reesei actin promoter has a CT-rich region, CAAT and GC. There is no obvious TATA sequence in the T. reesei actin promoter. The absence of TATA-like sequence were also observed in anothers genes of T. reesei. An important aspect in molecular biology of filamentous fungi is the analysis, under a specific metabolic events, of the mechanism(s) regulating the expression of constitutive and induced genes. The filamentous fungus Trichoderma reesei is considered to be one of the most efficient producer of cellulase, and it serves as a model system for enzymatic cellulose hydrolysis. Expression of the cellulase genes are stringently regulated by the carbon source. Growth on cellulose results in induction of the cellulase transcripts, whereas glucose strongly represses their expression. The availability of a constitutive expressed genes of T. reesei provides not only important information regarding the molecular biology of the fungi, but also is essential for a better understanding of the mechanism(s) controlling the expression of the cellulase transcripts. Under inductive process of the of the major cellulase transcript (cbh1) and its repression by glucose, actin mRNA is constitutively expressed. The present results should be useful for further structural and functional analysis of the elements involved in inductive and constitutive expression of cellulase and actin transcripts.
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Bueno, Indianara Kawana. "Caracterização das linhagens mutantes do fungo Trichoderma reesei RUT-C30Δzface1." Universidade Estadual do Oeste do Paraná, 2018. http://tede.unioeste.br/handle/tede/3702.
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The research for renewable energy sources became even more essential due the imminent depletion of the fossil fuel sources. In this context Brazil has a prominent position on the world stage, since it has already used ethanol from sugar cane for some decades. The second generation ethanol (2G) is produced from the lignocellulosic biomass of the vegetable, which is composed by cellulose, hemicellulose and lignin. The hydrolysis of these compounds requires a specific and high cost enzymatic cocktail. On this scenario, the Trichoderma reesei fungus gains spotlight, since it is one the microorganisms with the highest potential to produce hydroliytic enzymes. Therefore, the attempt to increase the cellulases production of this fungus is an important for the production of biofuels more attractive to the market. The aim of this work is to confirm the deletion of the sequence which codifies the zinc finger motif of the transcription factor ACE1 for cellulose repression from the T. reesei RUT-C30 strain and to characterize the enzymatic production of these mutant strains named T. reesei RUT-C30Δzface1. The enzymatic quantification was carried using the substrates carboxymethyl cellulose, microcrystalline cellulose and Whatman paper filter. The deletion confirmation occurred by the absence of the amplification gene ace1 on the mutants and the amplification of a 429 pb fragment of the RUT-C30 parental strain when the same primers and PCR conditions where used. These results suggest that the deletion of the zinc finger motif of the from ACE1 transcription factor is a prominent way to achieve an economically viable production of bioethanol.
Com a depleção eminente das fontes de combustíveis fósseis, torna-se cada vez mais imprescindível a busca por fontes renováveis de energia. Neste âmbito, o Brasil tem destaque no cenário mundial, pois já utiliza o etanol a partir da cana-de-açúcar há algumas décadas. O etanol de segunda geração (2G) é produzido a partir da massa lignocelulolítica do vegetal, que é composta de celulose, hemicelulose e lignina. A hidrólise desses compostos necessita de um coquetel enzimático específico e de alto custo. Neste cenário, o fungo Trichoderma reesei ganha destaque, pois é um dos microrganismos com maior potencial para produção de enzimas hidrolíticas. Desta forma, as tentativas de aumentar a produção de celulases desse fungo, torna a produção do bioetanol uma alternativa mais atrativa ao mercado. Este trabalho teve como objetivos confirmar a deleção da sequência que codifica o dedo de zinco do fator de transcrição do repressor de celulase ACE1 da linhagem T. reesei RUT-C30 e caracterizar a produção enzimática dessas linhagens mutantes denominadas T. reesei RUT-C30Δzface1. A confirmação de deleção ocorreu pela ausência de amplificação do gene ace1 nos mutantes e amplificação de um fragmento de 479 pb na linhagem parental RUT-C30, quando utilizados os mesmos primers e condições de reação de PCR. A dosagem enzimática com os substratos carboximetilcelulose (CMC), celulose microcristalina (Avicel®) e papel de filtro Whatman (PF), mostraram que o RUT-C30Δzface1 tem a atividade celulolítica aumentada em até 3,2 vezes em Avicel e 2,1 vezes em CMC e PF em comparação à linhagem parental RUT-C30. Em 24 horas de hidrólise os mutantes apresentaram liberação de açúcar 1,4 vezes maior em relação ao RUT-C30. Estes resultados sugerem que a deleção parcial do fator de transcrição ACE1 é um proeminente caminho para a conquista de uma produção de bioetanol economicamente viável.
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Saqib, Abdul Aala Najmus. "Elucidation of the factors involved in cellulose breakdown by Trichoderma reesei." Thesis, University of Surrey, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.479489.
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Joncour, Karine. "An investigation of the transferase activity of cellulase from Trichoderma reesei." Thesis, University of Huddersfield, 1998. http://eprints.hud.ac.uk/id/eprint/4837/.
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A study of the transglycosylationr eactionsc atalysedb y a multi-enzymec omplex, cellulase from Trichoderma reesei, was undertaken. An activated substrate donor, p-nitrophenyl P-D-cellobioside (PNPQ, and various mono- and disaccharide acceptors were tested in the studies which were performed under kinetically controlled conditions. Surprisingly, three main transfer products were obtained as opposed to the single product cited in the literature for the cellulase catalysed reaction. Two were identified as the N-(p-nitrophenyl)-p-D-ceUobioside (a P-(14) linked disaccharide)a nd the N-(p-nitrophenyl)-p-D-gentiobiosylamine(a P-(1-6) linked disaccharide)A. number of experimentalp arametersw ere varied and their effects on the yield of the transglycosylation reaction were determined. The variables investigated included: increasing of substrate concentration, increasing the acceptor concentration, varying the pH and the temperature of the reaction. The effect of the addition of a co-solvent (ACN, t-butanol, dioxane or acetone) was also studied.T he reactionsw ere found to be stereospecificb ut not regioselective. The latter was found to vary with the substrate concentration: at low concentrations (< 1.5 mM), the P-(1-6) linked disaccharide was the preferred transfer product whereasa t higher concentrationst,h e P-(14) linked disaccharide was favoured. Increasing the acceptor concentration was found to increase the transglycosylationy ield (6% to 19%) whereast he addition of co-solvent resulted in a decrease. Ilese results are discussed in relation to the components of the complexw hich are responsiblef or the production of the various transferp roducts. Interestingly,t he use of p-nitrophenyl 1-thio-p-D-glucopyranosidea s an acceptor proved to give a higher yield of the transfer products (-40 %) showing the importanceo f the acceptors tructure in the transglycosylationr eaction Moreover, the transglycosylation studies were also undertaken in the presence of the P-glucosidasein hibitor, 1,5-glucono-S-lactoneT. his resulted in the formation of a single product, the P-(14) linked disaccharide and therefore P-glucosidase was the only cellulase component responsible for producing the P-(1-6) transfer product. A difference in the degree of orientation of the acceptor between the different enzyme components of the cellulase complex was then suggested.
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Carneiro, Andréia Aparecida Jacomassi [UNESP]. "Produção de β-glucanases por Trichoderma reesei e Trichoderma harzianum e aplicação na hidrólise de β-glucanas." Universidade Estadual Paulista (UNESP), 2012. http://hdl.handle.net/11449/103972.
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000687526.pdf: 2190725 bytes, checksum: 76a291fbe3368473b4a2340b9a8996c5 (MD5)As glucanas fúngicas têm sido muito estudadas por apresentarem respostas biológicas benéficas à saúde. O Agaricus blazei, conhecido popularmente como cogumelo do sol, é um basidiomiceto que apresenta propriedades funcionais devido às β-glucanas. Os fungos Trichoderma harzinaum e Trichoderma reesei foram capazes de se desenvolverem em Agaricus blazei em pó como única fonte de carbono produzindo β-1,3-glucanases, analisadas por superfície de resposta. As enzimas hidrolisaram β-glucanas de A. blazei e produziu glucose e diferentes glucooligossacarídeos. As enzimas brutas do T. harzianum e T. reesei hidrolisaram menos de 15 % de glucana e em torno de 40 % de laminarina (β-1,3 glucana comercial), após 60 minutos, respectivamente. Utilizando a β-1,3-glucanase bruta de T. harzianum foram detectados gentiobiose e laminaritriose nos hidrolisados enzimáticos de β- glucana e laminarina, enquanto a laminaritetraose, celotetraose e celotriose foram identifificados e quantificados apenas nos hidrolisados de β-glucana de A. blazei. A ação da β- 1,3-glucanase bruta de T. reesei sobre a laminarina e glucana de A. blazei resultou em glicose e gentiobiose. O que sugere uma possível diferença na ação catalítica das duas enzimas. As enzimas parcialmente purificadas do T. harzianum e T. reesei hidrolisaram 47 e 85 % de laminarina, respectivamente. A β-1,3-glucanase purificada de T. harzianum não degradou a glucana de A. blazei, e a enzima purificada de T. reesei degradou 2,6 % de glucana, após 60 minutos, no entanto, gentiobiose e laminaritriose foram detectados no hidrolisado de laminarina. Utilizando a enzima parcialmente purificada de T. reesei gentiobiose foi detectado no hidrolisado de glucana e de laminarina, e celotriose e laminaritriose foram detectados apenas no hidrolisado de laminarina. A fim de conhecer melhor...
Fungal glucans have been studied extensively because of their biological responses, which have been shown to possess health benefits. Agaricus blazei, commonly known as mushroom of the sun, is a basidiomycete that has functional properties because of its β-glucans. The fungi Trichoderma harzinaum and Trichoderma reesei were able to develop in a powdered form of A. blazei, which served as the only carbon source. The result was the production of β- 1,3-glucanases, which were analyzed using response surface methodology. The enzymes hydrolyzed the β-glucans of A. blazei, and produced glucose and different glucooligosaccharides. The crude enzymes of T. harzianum and T. reesei hydrolyzed less than 15% of glucan and approximately 40% of laminarin after 60 minutes, respectively. Using crude β-1,3-glucanase from T. harzianum, gentiobiose and laminaritriose were detected in enzymatic hydrolysates of β-glucan and laminarin, while laminaritetraose, cellotriose and celotetraose were identified and quantified only in the hydrolysates of the β-glucan of A. blazei. The action of the crude β-1,3-glucanase of T. reesei on the laminarin and glucan of A. blazei resulted in glucose and gentiobiose. These results suggest a possible difference in the catalytic action of the two enzymes. The partially purified enzymes of T. harzianum and T. reesei hydrolyzed 47% and 85% of laminarin, respectively. The purified β-1,3-glucanase from T. harzianum did not degrade the glucan of A. blazei, and the purified enzyme from T. reesei degraded 2.6% of the glucan after 60 minutes; however, gentiobiose and laminaritriose were detected in the hydrolyzates of laminarin. Using partially purified enzymes from T. reesei, gentiobiose was detected in the hydrolyzates of both glucan and laminarin, and laminaritriose and cellotriose and were detected... (Complete abstract click electronic access below)
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Carneiro, Andréia Aparecida Jacomassi. "Produção de β-glucanases por Trichoderma reesei e Trichoderma harzianum e aplicação na hidrólise de β-glucanas /." Rio Claro : [s.n.], 2012. http://hdl.handle.net/11449/103972.
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Orientador: Roberto da SilvaBanca: Adalberto Pessoa Junior
Banca: Inês Conceição Roberto
Banca: Eleonora Cano Carmona
Banca: Eleni Gomes
Resumo: As glucanas fúngicas têm sido muito estudadas por apresentarem respostas biológicas benéficas à saúde. O Agaricus blazei, conhecido popularmente como cogumelo do sol, é um basidiomiceto que apresenta propriedades funcionais devido às β-glucanas. Os fungos Trichoderma harzinaum e Trichoderma reesei foram capazes de se desenvolverem em Agaricus blazei em pó como única fonte de carbono produzindo β-1,3-glucanases, analisadas por superfície de resposta. As enzimas hidrolisaram β-glucanas de A. blazei e produziu glucose e diferentes glucooligossacarídeos. As enzimas brutas do T. harzianum e T. reesei hidrolisaram menos de 15 % de glucana e em torno de 40 % de laminarina (β-1,3 glucana comercial), após 60 minutos, respectivamente. Utilizando a β-1,3-glucanase bruta de T. harzianum foram detectados gentiobiose e laminaritriose nos hidrolisados enzimáticos de β- glucana e laminarina, enquanto a laminaritetraose, celotetraose e celotriose foram identifificados e quantificados apenas nos hidrolisados de β-glucana de A. blazei. A ação da β- 1,3-glucanase bruta de T. reesei sobre a laminarina e glucana de A. blazei resultou em glicose e gentiobiose. O que sugere uma possível diferença na ação catalítica das duas enzimas. As enzimas parcialmente purificadas do T. harzianum e T. reesei hidrolisaram 47 e 85 % de laminarina, respectivamente. A β-1,3-glucanase purificada de T. harzianum não degradou a glucana de A. blazei, e a enzima purificada de T. reesei degradou 2,6 % de glucana, após 60 minutos, no entanto, gentiobiose e laminaritriose foram detectados no hidrolisado de laminarina. Utilizando a enzima parcialmente purificada de T. reesei gentiobiose foi detectado no hidrolisado de glucana e de laminarina, e celotriose e laminaritriose foram detectados apenas no hidrolisado de laminarina. A fim de conhecer melhor... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Fungal glucans have been studied extensively because of their biological responses, which have been shown to possess health benefits. Agaricus blazei, commonly known as "mushroom of the sun," is a basidiomycete that has functional properties because of its β-glucans. The fungi Trichoderma harzinaum and Trichoderma reesei were able to develop in a powdered form of A. blazei, which served as the only carbon source. The result was the production of β- 1,3-glucanases, which were analyzed using response surface methodology. The enzymes hydrolyzed the β-glucans of A. blazei, and produced glucose and different glucooligosaccharides. The crude enzymes of T. harzianum and T. reesei hydrolyzed less than 15% of glucan and approximately 40% of laminarin after 60 minutes, respectively. Using crude β-1,3-glucanase from T. harzianum, gentiobiose and laminaritriose were detected in enzymatic hydrolysates of β-glucan and laminarin, while laminaritetraose, cellotriose and celotetraose were identified and quantified only in the hydrolysates of the β-glucan of A. blazei. The action of the crude β-1,3-glucanase of T. reesei on the laminarin and glucan of A. blazei resulted in glucose and gentiobiose. These results suggest a possible difference in the catalytic action of the two enzymes. The partially purified enzymes of T. harzianum and T. reesei hydrolyzed 47% and 85% of laminarin, respectively. The purified β-1,3-glucanase from T. harzianum did not degrade the glucan of A. blazei, and the purified enzyme from T. reesei degraded 2.6% of the glucan after 60 minutes; however, gentiobiose and laminaritriose were detected in the hydrolyzates of laminarin. Using partially purified enzymes from T. reesei, gentiobiose was detected in the hydrolyzates of both glucan and laminarin, and laminaritriose and cellotriose and were detected... (Complete abstract click electronic access below)
Doutor
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Vasara, Tuija. "Functional analysis of the RHOIII and 14-3-3 proteins of Trichoderma reesei /." Espoo [Finland] : Technical Research Centre of Finland, 2002. http://www.vtt.fi/inf/pdf/publications/2002/P466.pdf.
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Silva, Márcia Josefa da. "Produção de enzimas celulolíticas e xilanolíticas por Trichoderma ressei RUT C-30 em meios com diferentes capacidades de indução." Universidade Federal de Pernambuco, 2014. https://repositorio.ufpe.br/handle/123456789/12147.
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CAPES
A biomassa lignocelulósica destaca-se como matéria-prima alternativa para a produção de combustíveis e outros produtos. Devido à alta complexidade desse material, é necessária uma hidrólise enzimática eficiente com a utilização de um pool enzimático adequado. O objetivo deste trabalho foi estudar o perfil de produção de enzimas celulolíticas e xilanolítica por Trichoderma reesei RUT C-30 em meios com diferentes capacidades de indução. A produção de enzimas foi realizada em biorreator de bancada (Bioflo 110) com 1,3 ou 3 L de volume de trabalho, nas seguintes condições: 500 rpm, 28° C, 2 vvm e pH 5,0. As fontes de carbono investigadas foram: lactose, xilana, pectina, celulose microcristalina, melaço, biomassa de palma forrageira e hidrolisado hemicelulósico. O hidrolisado foi obtido por tratamento hidrotérmico de bagaço de cana-de-açúcar em reator descontínuo de 20 L (Regmed AU/20), com volume de trabalho de 10 L e carga de sólidos de 5% (m/v), a 185°C, por 16 minutos. Em substratos solúveis, a determinação da concentração celular foi realizada por peso seco e as concentrações dos substratos foram obtidas por cromatografia líquida de alta eficiência. Ao final dos cultivos, foram isoladas proteínas extracelulares, que servirão para futura análise do secretoma de T. reesei RUT C-30. Em meio de lactose, os valores de atividades enzimáticas obtidos com 54 horas de cultivo foram: FPase (1,43 UI mL-1), CMCase (15,67 UI mL-1), xilanase (11,91 UI mL-1) e β-glicosidase (0,24 UI mL-1). A velocidade máxima específica de crescimento, μmax, e o coeficiente de rendimento de biomassa no substrato, Yx/s, foram 0,06 h-1 e 0,38 g g-1, respectivamente. Em meio de melaço, μmax e Yx/s foram 0,26 h-1 e 0,52 g g-1, respectivamente, e as atividades enzimáticas insignificantes. No hidrolisado, o crescimento do micro-organismo foi inibido devido à presença de compostos inibidores produzidos no tratamento hidrotérmico. Os valores de atividades enzimáticas com 54 horas foram: FPase (0,06 UI mL-1), CMCase (0,24 UI mL-1), xilanase (1,40 UI mL-1) e β-glicosidase nula. Em meio com celulose, também foram obtidos baixos valores de atividades enzimáticas, porém, a xilanase apresentou valor de 1,52 UI mL-1 com 50 horas de cultivo. A xilanase foi a enzima mais evidente nos cultivos com xilana, atingindo valor máximo de 11,93 UI mL-1, ao final do cultivo. Em meio com pectina: FPase, CMCase, xilanase e β-glicosidase foram: 0,01 UI mL-1, 1,25 UI mL-1, 2,83 UI mL-1 e 0,10 UI mL-1, respectivamente. Nos cultivos em meio à base de palma, observaram-se os seguintes valores com 50 horas: FPase (0,29 UI mL-1), CMCase (3,30 UI mL-1), xilanase (6,21 UI mL-1) e β-glicosidase (0,09 UI mL-1). Entre as fontes investigadas, a lactose é o melhor substrato para a indução de todas as enzimas estudadas, enquanto o melaço favorece o crescimento rápido do micro-organismo. O hidrolisado hemicelulósico e a palma forrageira são potenciais meios para a produção de enzimas, em particular xilanases. Para a utilização do hidrolisado, no entanto, será necessária a sua detoxificação ou, alternativamente, a obtenção de linhagens resistentes aos inibidores por engenharia metabólica e/ou engenharia evolutiva.
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Portnoy, Thomas. "Analyse du transcriptome de Trichoderma reesei pour l'amélioration de la production de cellulases." Paris 6, 2011. http://www.theses.fr/2011PA066388.
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Le champignon filamenteux Trichoderma reesei constitue la principale source de cellulases, enzymes employées dans les procédés industriels d’hydrolyse de la biomasse lignocellulosique nécessaire à la production de bioéthanol de deuxième génération. Ce projet de thèse se base sur l’utilisation de techniques de génétique moléculaire et d’outils de génomique fonctionnelle pour décrire les mécanismes génétiques impliqués dans la synthèse et la sécrétion de ces enzymes afin d’envisager une amélioration des capacités de production des souches de T. Reesei rationnellement et de manière ciblée. Une première partie de ce travail a consisté à préciser la régulation de l’expression des facteurs de transcription centraux de la production de cellulases lors d’une induction par le lactose. Nous avons notamment démontré que xyr1, ace1 et ace2 étaient spécifiquement induits par le lactose, et que CRE1, médiateur de la répression catabolique, était nécessaire à une induction complète de xyr1 et ace2. Dans un deuxième temps, des études transcriptomiques nous ont permis (i) d’identifier les gènes cibles de la répression catabolique dépendante ou non de CRE1, et (ii) de décrire à l’échelle du génome total les régulations mises en jeu lors de l’induction de la production de cellulases. Nos travaux ont notamment permis d’établir l’importance de l’expression génétique basale dans l’acquisition des capacités d’hyperproduction des souches, et d’identifier les principales fonctions cellulaires soumises à des régulations au cours du processus d’induction. L’ensemble de ces résultats permet d’orienter avec précision les stratégies d’amélioration des souches de T. Reesei par ingénierie génétique
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Belmokhtar, Nassim. "Etude de la saccharification enzymatique du miscanthus par les cocktails cellulolytiques de Trichoderma reesei." Thesis, Reims, 2012. http://www.theses.fr/2012REIMS028/document.
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Parmi les ressources d'origines agricole et forestière utilisables aujourd'hui en tant que biomasse à destination énergétique, le miscanthus apparait comme l'une des espèces de graminées les plus prometteuses pour la production de bioéthanol de seconde génération grâce à son haut potentiel en biomasse. Ce procédé dit "2G" convertit la cellulose contenue dans ces biomasses lignocellulosiques en bioéthanol et ce via un procédé intégrant prétraitement physico-chimique, hydrolyse enzymatique et fermentation. Le principal objectif de ce projet de thèse visait à étudier l'impact de l'hétérogénéité tissulaire et structurale du miscanthus sur sa saccharification et s'est décliné en différents volets liés à l'étude de l'efficacité des prétraitements et à l'analyse des performances de différents cocktails enzymatiques de Trichoderma reesei. L'hydrolyse enzymatique est essentiellement limitée par la structure et la porosité des complexes pariétaux qui réduisent l'accessibilité de la cellulose aux cellulases. En plus des constituants hémicelluloses et lignines qui recouvrent la cellulose, les parois cellulaires du miscanthus sont riches en acides hydroxycinnamiques (pCA et FA) qui jouent un rôle important dans la cohésion du réseau pariétal complexe. L'application de prétraitements acide et alcalin sur le miscanthus a ainsi révélé une différence de réactivité en fonction des types cellulaires. Les parois secondaires du sclérenchyme sont plus facilement dégradées par les cellulases fongiques après prétraitement acide. L'étude de la distribution des composés phénoliques au niveau cellulaire par micro spectrophotométrie UV a rapporté une nette diminution de l'absorbance UV dans tous les tissus après chaque prétraitement. Ceci n'expliquant pas totalement les différences de réactivité observées, d'autres facteurs physicochimiques seraient donc impliqués. Une approche visant à évaluer la progression des cellulases au sein des parois par immunocytochimie a également été initiée mais elle s'est heurtée à des problématiques techniques liées à la nature des tissus et aux anticorps employés. Les performances en terme de conversion de la cellulose ont été évaluées avec des cocktails enzymatiques de T. reesei comprenant des activités (hemi-)cellulolytiques variables. Une meilleure efficacité du prétraitement par explosion à la vapeur a ainsi pu être montrée par réduction de la quantité d'enzymes mises en œuvre. Comme c'est le cas pour d'autres graminées, ces travaux ont permis de confirmer le rôle crucial de l'enzyme β-glucosidase, permettant de limiter l'inhibition par le cellobiose et améliorant la cinétique initiale de saccharification. L'amélioration du rendement d'hydrolyse par l'utilisation d'un sécrétome comprenant une bonne activité hémicellulolytique a pu être ensuite démontrée. L'utilisation de cocktails enzymatiques reconstitués à partir d'enzymes pures a enfin permis de définir un mélange "optimal" composé des quatre principales cellulases de T. reesei (CBH1, CBH2, EG1 et EG2) associées à une hémicellulase (XYN1)Among agricultural and forest resources, the grass specie miscanthus has emerged as one of the most promising feedstock candidates for 2G-biofuel production due to its high biomass yield. The biofuels 2G-production process is based on cellulose conversion into bioethanol via physicochemical pretreatment, enzymatic hydrolysis and fermentation. The main objective of this Ph.D. project was to evaluate the effect of tissue and structure heterogeneity of miscanthus on its saccharification by evaluating pretreatment efficiency and analyzing the performance of different Trichoderma reesei cellulolytic cocktails.Enzymatic hydrolysis is mainly hindered by cell wall structure and porosity which limit cellulose accessibility to cellulase. In addition to hemicelluloses and lignin polymers, miscanthus cell walls, contain high amounts of hydroxycinnamic acids (pCA and FA) that play a significant role in cross-linking polymers into cohesive network. Applying acid and alkali pretreatments on miscanthus revealed a distinctive reactivity depending on cell types. Secondary cell walls of sclerenchyma appeared more digested by fungal cellulases after acid pretreatment. Addressing phenolics distribution (lignin and hydroxycinnamic acids) at cell level by UV micro spectrophotometry highlighted a significant decrease in UV absorbance after both pretreatments irrespective to cell type indicating that other physicochemical and structural features are involved in distinct cell wall reactivity. We have also attempted to evaluate cellulase progression into miscanthus cell walls by immunocytochemistry but we have had many technical problems due to the nature of miscanthus tissues and used antibodies. Cellulose conversion ability was then evaluated using enzymatic cocktails of T. reesei which vary in their (hemi-)cellulolytic activities. Higher efficiency of the steam explosion pretreatment was demonstrated by reducing enzymes loading. As reported previously on other grasses, β-glucosidase plays a crucial role by limiting the inhibiting effect of cellobiose and improving the initial saccharification step. We furthermore showed that the use of hemicellulases-improved cocktails allowed significant increase in saccharification yields. We finally identified an optimal reconstituted enzyme mixture composed of four major cellulases of T. reesei (CBH1, CBH2, EG1 and EG2) and the hemicellulase XYN-1
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Carraro, Cláudia Batista. "Identificação de alvos de fosforilação de MAPK em Trichoderma reesei através de fosfoproteômica durante a produção de celulases." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17131/tde-20112018-114017/.
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O fungo filamentoso Trichoderma reesei é uma espécie de grande importância biotecnológica no que tange a degradação de biomassa lignocelulósica para a produção de bioetanol em larga escala. Seu sistema de enzimas celulolíticas é muito eficiente, apesar de ser possuir poucas celulases, sugerindo que o controle dessas enzimas vai além da regulação transcricional. Assim, neste trabalho nós obtivemos o perfil fosfoproteômico de T. reesei cultivado em glicose e bagaço de cana-de-açúcar a partir de análise fosfoproteômica LC-MS/MS por spectral counting. A comparação entre os perfis de fosfoproteínas obtidos das linhagens parental QM6a de T. reesei e dos mutantes knockout para TMK1 e TMK2 permitiu a demonstração de que essas MAPK agem de maneira interconectada com outras vias de transdução de sinal na célula, especialmente a via de TOR e de AMPc-PKA, para regulação da produção de celulases. Além disso, também demonstramos a regulação da resposta a estresse celular por TMK2, e o papel da fosforilação no controle direto de enzimas CAZy. Nossos resultados mostram que a fosforilação desempenha papel importante na regulação dessas enzimas e de outras funções celulares no fungo após a transcrição de seus respectivos genes. O agrupamento desses dados permite melhor entendimento da via de sinalização mediada pelas MAPK TMK1 e TMK2 de T. reesei, e como as modificações pós-traducionais promovidas por elas afetam no sensing de nutrientes celulares e, por consequência, na produção de enzimas celulolíticas, de forma direta ou indireta.The filamentous fungus Trichoderma reesei has great biotechnological importance in regards to the lignocellulosic biomass degradation for large-scale production of bioethanol. Its cellulolytic system is very efficient, despite being composed by only a few cellulases, which suggests that the control of these enzymes goes beyond their transcriptional regulation. Thus, in this study, we performed a spectral counting LC-MS/MS analysis and achieved the phosphoproteomic profile of T. reesei grown either in glucose or sugarcane bagasse as sole carbon source. The comparison between the phosphoproteins profiles obtained from the parental strain QM6a and the knockout mutants for TMK1 and TMK2 allowed us to demonstrate that these MAPK act in an interconnected manner with other signaling transduction pathways, especially the TOR and cAMP-PKA pathways, in order to regulate the cellulases production. Furthermore, we were also able to determine the regulation of cellular stress response by TMK2, and the role of phosphorylation in the direct control of CAZymes. Our results show that phosphorylation plays an important role on the control of these enzymes and other cellular functions in T. reesei after the transcription of their respective genes. Taken together, this data allows better comprehension of the signaling pathways mediated by TMK1 and TMK2 in T. reesei, and how the post-translational modification promoted by these MAPK might affect the nutrient sensing and, therefore, the production of the cellulolytic enzymes, either directly or indirectly.
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Chen, Chi Fan. "Activity-based proteomics profiling for identification and quantification of Trichoderma reesei cellulases." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/38967.
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Evans, Elaine Trene. "Mechanism of action and inhibition of the cellulase system of Trichoderma reesei." Thesis, University of Salford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386381.
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Cortez, Joao Marques. "Biofinishing of cotton fabrics with genetically modified strains of Trichoderma reesei cellulases." Thesis, De Montfort University, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391644.
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Dalal, Pankaj. "Étude physiologique et cinétique de la production des enzymes cellulolytiques chez Trichoderma reesei en culture continue." Paris 11, 1989. http://www.theses.fr/1989PA112091.
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Cette étude vise à une meilleure connaissance de la production des enzymes cellulolytiques par le champignon cellulolytique Trichoderma reesei en culture submergée et à une meilleure compréhension des mécanismes de synthèse et de régulation de ces enzymes. La cinétique de la croissance et la production des enzymes cellulolytiques chez T. Reesei CL-847, mutant résistant à la répression catabolique et hyper producteur, ont été étudiées en culture continue limitée par le lactose, seule source de carbonne, et inducteur du système cellulolytique, et comparées à celles de la souche parentale T. Reesei QM9414. Le fonctionnement stable obtenu dans la conduite en culture continue a permis d'étudier particulièrement l'influence du glucose et du 2-désoxyglucose sur la production et l'activité des enzymes cellulolytiques. La chromafocalisation analytique est une méthode excellente pour la caractérisation des enzymes cellulolytiques. Nous l'avons utilisée pour caractériser les enzymes cellulolytiques excrétées dans des états physiologiques différents pendant la culture continue. Une meilleure compréhension de ces phénomènes génétiques et biochimiques liés à la production de cellulases pourrait permettre dans un proche avenir des développements importants à l'échelle industrielle.
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Morante, Estela Ynés Valencia. "Análise do promotor bidirecional que controla os genes citrato sintase e isocitrato liase do fungo filamentoso Trichoderma reesei." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-28092006-114353/.
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O gene TrCit do fungo filamentoso Trichoderma reesei codifica a proteína citrato sintase, uma enzima chave do ciclo de Krebs. Análise da região 5´ upstream de TrCit mostra que o gene está adjacente ao gene TrIcl (que codifica a proteína isocitrato liase, uma enzima do ciclo de glioxalato), em uma orientação cabeça-cabeça. A região promotora intergênica de 647 pb rica em G + C, apresenta uma ilha CpG, seqüência INR, caixas GC, caixas CAAT, sítios de ligação para diversos fatores de transcrição e é isenta de caixa TATA. O gene TrCit de 1573 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1422 pb produz uma proteína de 474 aminoácidos, com um peso molecular estimado de 52,3 kD. O gene TrIcl de 1880 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1788 pb produz uma proteína de 596 aminoácidos, com um peso molecular estimado de 65,4 kD. A atividade transcricional da região promotora foi analisada utilizando como repórter o gene de higromicina B fosfotransferase (hph). Uma região funcional necessária à transcrição de ambos os genes foi identificada na região central do promotor e contém uma caixa GC que liga o putativo fator de transcrição Sp1 de T. reesei (TrZnFSp1). O gene do putativo fator de transcrição zinc-finger TrZnFSp1 de 1500 pb contém 3 éxons e 2 íntrons. Sua seqüência codificadora de 1344 pb produz uma proteína de 448 aminoácidos, com um peso molecular estimado de 48,4 kD. Os resultados mostram que ambos os genes são transcritos de forma divergente a partir de um promotor bidirecional que compartilha na região central uma caixa GC, necessária para a transcrição de ambos os genes.The TrCit gene from the filamentous fungus Trichoderma reesei codes for the citrate synthase protein, a key enzyme in the Krebs cycle. Analysis of TrCit 5 upstream region showed that it is adjacent to the TrIcl gene that codes for isocitrate lyase protein, an enzyme involved in the glyoxylate cycle. Both genes, on a head-to-head orientation, are separated by an intergenic GC-rich and TATA-less promoter region of 647 base pairs. This bidirectional promoter has diverse cis regulatory elements: a CpG island, two INR sequences, GC boxes, CAAT boxes and several putative interaction sites for different transcription factors. The TrCit gene, 1,573-base pair-long, has an open reading frame of 1,422 base pairs interrupted by two introns. The gene codes for a protein with an estimated molecular weight of 52.3 kD. The TrIcl gene, 1,880-base pair-long, contains 3 exons and 2 introns and a putative coding sequence of 1,788 base pairs. The estimated molecular weight of TrICL is 65.4 kD. he transcriptional activity of the intergenic promoter region was analyzed using hygromicin B phosphotransferase (hph) as a reporter gene. A functional region required for the transcription of both genes was identified in the centre of this promoter. It has a GC box that interacts with a putative transcription factor Sp1 from T. reesei (TrZnFSp1). The results presented in this work show that both genes are divergently transcribed from a bidirectional promoter that shares an essential central GC box.
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Callow, Nicholas V. "Exploring The Controlled Pellet Formation of Trichoderma reesei RUT-C30 for Improved Fermentation." University of Akron / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=akron1427975356.
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Driver, Diane Patricia. "A gene fusion of Cex from Cellulomonas fimi and CbhI from Trichoderma reesei." Thesis, University of British Columbia, 1991. http://hdl.handle.net/2429/29830.
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The proteins Cex from C. fimi and CbhI from T. reesei are exo-glucanases which are composed of separate catalytic and cellulose binding domains. When separated from one another by treatment with a protease, these domains retain their specific functions. Using polymerase chain reaction, a gene fusion was constructed which encodes a polypeptide containing the catalytic domain of Cex and the cellulose binding domain of Cbhl. During DNA sequencing of the fusion clones, an error was detected in the published Cbhl DNA sequence (Shoemaker et al. 1983b). The corrected sequence codes for two prolines, as Fagerstam (1981) determined during his earlier amino acid sequencing of Cbhl.
This fusion protein, expressed in E. coli, was active on the substrates p-nitrophenyl-β-D-cellobioside, p-nitrophenyl-β-D-lactoside, carboxymethyl cellulose and xylan, as is Cex, and was able to bind to microcrystalline cellulose but not to chitin. It can be eluted from cellulose with 8M guanidine-HC1. The fusion protein was found in the culture supernatant of E. coli cultures and presumably leaks from the periplasm in the same manner as Cex (Guo et al. 1988). Adsorption assays were conducted for the Cex/Cbhl fusion on bacterial microcrystalline cellulose (BMCC).Science, Faculty of
Microbiology and Immunology, Department of
Graduate
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Lopes, Douglas Christian Borges. "Functional characterization of Trichoderma reesei xyloglucanase (CEL74A) in the degradation of sugarcane bagasse." Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17131/tde-01022019-104422/.
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O fungo filamentoso Trichoderma reesei é um dos principais fungos utilizados para a produção em larga escala de enzimas devido a sua grande capacidade de produção e secreção de holocelulases para aplicação em processos de sacarificação da biomassa vegetal lignocelulósica. Embora o T. reesei seja utilizado como um dos principais produtores de celulases a nível industrial, diversos processos e estudos são realizados com o objetivo de aprimorar o entendimento de todo o mecanismo de degradação de biomassa vegetal além de prover o aumento da eficiência tanto da produção quanto da atividade das celulases. No presente trabalho foi realizado a construção de uma linhagem mutante para o gene cel74a (codificando para uma xiloglucanase) e a caracterização funcional de CEL74A na regulação gênica de holocelulases durante o cultivo em bagaço de cana-de-açúcar. Os nossos resultados mostraram que deleção de cel74a possivelmente pode estar envolvida no sinergismo da regulação da expressão de holocelulases durante o cultivo em bagaço de cana-de-açúcar. A partir da análise do perfil de expressão gênica, foi possível observar a redução na expressão de todos os genes de celulases testados (cel7a, cel7b e cel6a) embora não tenha afetado a atividade enzimática, ao passo que as hemicelulases (xyn1 e xyn2) apresentaram aumento tanto na expressão quanto na atividade enzimática. Na linhagem ?cel74a, foi observado redução na liberação de glicose, xilose e galactose após a hidrólise de xiloglucano. Além disso, a atividade de CEL74A foi modulada na presença de cálcio e pode ser necessária para a atuação mais eficiente de outras enzimas envolvidas na degradação de xiloglucano. Desta forma, em T. reesei, CEL74A apresenta um papel importante tanto na regulação de genes holocelulolíticos quanto para a degradação eficiente de bagaço de cana-de-açúcar, contribuindo para a elucidação de mecanismos pelos quais este fungo utiliza para a utilização do bagaço de cana-de-açúcar como fonte de carbonoThe filamentous fungus Trichoderma reesei is one of the main fungi used for the large-scale production of enzymes due to their great capacity of production and secretion of holocellulases for application in saccharification processes of lignocellulosic plant biomass. Although T. reesei is used as one of the main producers of cellulases at industrial level, several processes and studies are carried out with the aim of improving the understanding of the whole plant biomass degradation mechanism, as well as increasing the efficiency of both the production and cellulase activity. In the present work the construction of a mutant lineage for the cel74a gene (coding for a xyloglucanase) and the functional characterization of CEL74A in the gene regulation of holocellulases during the cultivation of sugarcane bagasse were carried out. Our results showed that deletion of cel74a may be involved in the regulation of holocellulase expression during sugarcane bagasse cultivation. From the analysis of the gene expression profile, it was possible to observe the reduction in the expression of all tested cellulase genes (cel7a, cel7b and cel6a), although it did not affect the enzymatic activity, whereas the hemicellulases (xyn1 and xyn2) presented increase in both expression and enzymatic activity. In the ?cel74a strain, a reduction in glucose, xylose and galactose release was observed after xyloglucan hydrolysis. In addition, the activity of CEL74A was modulated in the presence of calcium and may be required for the more efficient performance of other enzymes involved in the degradation of xyloglucan. Thus, in T. reesei, CEL74A plays an important role both in the regulation of holocellulolytic genes and in the efficient degradation of sugarcane bagasse, contributing to the elucidation of mechanisms by which this fungus uses for the use of sugarcane bagasse as source of carbon
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Alharake, Jawad. "Study of genetic factors involved in enzyme secretion in hyperproductive strains of Trichoderma reesei." Electronic Thesis or Diss., université Paris-Saclay, 2023. http://www.theses.fr/2023UPASB061.
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Les combustibles fossiles sont un contributeur majeur au réchauffement climatique, et leur nature non renouvelable est un obstacle à la construction de sociétés durables. Dans ce contexte, les biocarburants de seconde génération se présentent comme une alternative attractive et plus respectueuse de l'environnement. Le processus de production de biocarburants de deuxième génération comprend plusieurs étapes incluant le prétraitement physico-chimique de la biomasse lignocellulosique (non comestible), l'hydrolyse enzymatique de la cellulose en glucose et par la fermentation de sucres simples en biocarburants, comme le bioéthanol. Un des problèmes principaux pour une large application est cependant le coût relativement élevé des enzymes hydrolytiques, les cellulases, utilisées pour déconstruire la biomasse lignocellulosique prétraitée en sucres fermentescibles.Le champignon filamenteux Trichoderma reesei est le choix privilégié pour la production industrielle de cellulases car il possède des capacités d'hyperproduction et d'hypersécrétion. Les souches industrielles de T. reesei peuvent sécréter jusqu'à 100 g/L de cellulases dans des fermenteurs industriels contrôlés. En particulier, la souche mutante Rut-C30 est une souche hyperproductrice de référence, mais notre compréhension incomplète de son système de sécrétion performant complique l'amélioration de sa capacité d'hypersécrétion par génie génétique. C'est pourquoi, ce travail visait à éclaircir les voies de régulation contrôlant la sécrétion et les réponses au stress de sécrétion pour identifier des goulots d'étranglement et pour développer de nouvelles souches dotées d'une capacité de sécrétion supérieure dans le futur.Dans ce but, des données transcriptomiques étaient générées à partir de cultures de T. reesei Rut-C30 dans différentes conditions de stress de sécrétion et ont permis d'identifier de composants potentiels de régulation de la sécrétion qui pouvaient être ciblés pour invalidation. Pour compléter cette approche, un datamining de données transcriptomiques obtenues avec d'autres champignons filamenteux cultivés dans des conditions de stress de sécrétion a révélé d'autres gènes cibles potentiellement impliqués dans la régulation de la voie de sécrétion. Finalement, neuf gènes ont été invalidés dans la souche Rut-C30 et les mutants obtenus ont été caractérisés phénotypiquement. Tous ont montré une croissance ralentie et un comportement de sécrétion altéré. Un séquençage de l'ARN a été réalisé sur les mutants ∆res2, ∆rpn4 and ∆snd1 et comparé à celui de Rut-C30 dans les mêmes conditions de culture. Aucun des trois facteurs de transcription n'impacte la transcription des gènes impliqués dans la sécrétion ou dans la réponse au stress de sécrétion dans nos conditions. En revanche, des gènes codant pour des enzymes du métabolisme des lipides sont différentiellement exprimés dans les trois mutants ce qui pourrait affecter la sécrétion indirectement. Les résultats délivrent des premiers indices pour atténuer les goulots d'étranglement de la sécrétion dans T. reesei Rut-C30 et ouvrent la voie vers le développement de souches possédant une capacité de sécrétion amélioréeFossil fuels are a major contributor to global warming, and their non-renewable nature is an impediment for building sustainable societies. In this context, second generation biofuels represent an attractive and more environmentally friendly alternative. The process of second-generation biofuel production consists of several steps including a physicochemical pretreatment of lignocellulosic (non-edible) biomass, the enzymatic hydrolysis of cellulose into glucose and the fermentation of simple sugars into biofuels, such as bioethanol. One of the main bottlenecks for a large implementation of this process is, however, the relatively high cost of hydrolytic enzymes, namely cellulases, used to deconstruct the pretreated lignocellulosic biomass into fermentable sugars.The filamentous fungus Trichoderma reesei is the preferred choice for industrial production of cellulases since it has hyperproduction and hypersecretion capacities. Industrial strains of T. reesei can secrete up to 100 g/L of cellulases in controlled industrial fermenters. In particular, the mutant strain Rut- C30 is a reference hyperproducer strain, but our incomplete understanding of its enhanced secretion system complicates further improvement of its hypersecretion capacity by genetic engineering. Therefore, this work aimed at unravelling the regulatory pathways controlling secretion and the secretion stress response in order to identify bottlenecks and to develop new strains with enhanced secretion capacity in the future.To this end, transcriptomic data were generated from cultivations of T. reesei Rut-C30 in different secretion stress conditions which allowed to identify potential components of secretion regulation that were targeted for deletion. As a complementary approach, mining of transcriptomic data obtained with other filamentous fungi in secretion stress conditions revealed further target genes potentially involved in the regulation of the secretion pathway. Finally, nine genes were deleted in the Rut-C30 strain, and the resulting strains phenotypically characterized. All of them displayed reduced growth and showed altered protein secretion behavior. RNA sequencing was performed on ∆res2, ∆rpn4 and ∆snd1 mutant strains and compared to that of Rut-C30 in the same culture conditions. Neither of the three transcription factors impacts transcription of genes involved in secretion or the secretion stress response in our conditions. However, in all three mutants, genes encoding enzymes of lipid metabolism are differentially expressed which could affect secretion in an indirect way. The results represent first clues to alleviate bottlenecks in secretion in T. reesei Rut-C30 and pave the way to develop strains with still improved secretion capacity
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Ferreira, Ari José Scattone. "Regulação da resposta transcricional a estresses ambientais em fungos: análise de \"microarrays\" de cDNAs de Trichoderma reesei." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-14122006-114540/.
Full textAbstract:
A grande diversidade de organismos que hoje encontramos em nosso planeta se deve à adaptação às diferentes condições ambientais de cada nicho ecológico existente e à resposta adaptativa originária das mudanças dessas condições. Pode-se considerar que a etapa inicial do processo de adaptação seja a reprogramação da expressão gênica dum organismo como resposta imediata a uma nova condição ambiental. De fato parte do genoma de todos os organismos é dedicada à codificação de proteínas relacionadas ao controle dos efeitos nocivos criados por diferentes tipos de estresse como choque térmico, ou osmótico, estresse oxidativo, ou aqueles resultantes de altas concentrações de íons de metais pesados. De forma semelhante, a ausência, ou exaustão, de fontes de macronutrientes, como carbono, nitrogênio, fósforo ou enxofre, exige uma reorganização do padrão de expressão gênica para adequação às novas condições nutricionais, também sendo considerada um estresse ambiental. Visto que a maioria dos estudos de análise da expressão gênica em resposta a estresses ambientais realizados em fungos se refere às leveduras unicelulares Saccharomyces cerevisiae e Schizossacharomyces pombe, nos propusemos a estudar tal resposta no fungo filamentoso multicelular Trichoderma reesei. Dessa forma analisamos por meio da técnica de \"microarrays\" de cDNAs a expressão gênica de aproximadamente 2.000 transcritos desse organismo em resposta a choque térmico, à alta concentração de íons de cádmio II e à ausência de fonte de carbono, ou nitrogênio, por período de 2 horas. Em geral, as respostas aos estresses se compuseram da regulação negativa da transcrição de genes envolvidos em processos com alta demanda de energia como a síntese protéica, evidenciada pela repressão da expressão de genes de proteínas ribossomais e do anabolismo. Em contrapartida, genes codificando proteínas relacionadas à defesa celular, como chaperonas, tiveram sua expressão induzida. As respostas ao choque térmico e ao tratamento com cádmio II se mostraram bastantes semelhantes, enquanto a ausência de fonte de nitrogênio também induziu a expressão de genes relacionados à degradação de proteínas e nucleotídeos. Genes relacionados à utilização de reservas lipídicas foram induzidos tanto na ausência de fonte de carbono quanto de nitrogênio. Foram identificados reguladores transcricionais e componentes de vias de sinalização celular com expressão diferenciada frente a esses diferentes estresses ambientais. A maior parte dos genes cuja expressão se alterou em função dos diversos estresses ambientais estudados ainda não tem função celular conhecida, sendo essa observação, portanto, uma contribuição importante para sua anotação funcional. Uma vez que o fungo filamentoso Trichoderma reesei vem se tornando um organismo de valor biotecnológico por sua característica de alto poder de síntese e secreção de proteínas, esperamos que os dados apresentados forneçam um maior entendimento dos processos celulares desse organismo e possam subsidiar futuros projetos visando uma melhor adaptação do mesmo a ambientes industriais.The diversity of organisms found today in our planet is due to their adaptation to different environmental conditions present in each ecological niche, and to the adaptative response originated from changes in those conditions. The first step in the adaptation process is considered to be the reprogramming of gene expression as an immediate response to a new environmental condition. A fraction of the genome from all living organisms is dedicated to encoding proteins related to the control of deleterious effects created by different types of stresses like heat or osmotic shock, oxidative stress, or by the presence of high concentrations of heavy metal ions. Similarly, the absence or exhaustion of macronutrients as carbon, nitrogen, phosphorous or sulphur sources demand new patterns of gene expression in order to the organisms survive in a limited nutritional condition, which is also considered an environmental stress. Once the gene expression analyses in fungi as a response to environmental stresses have been widely studied in the yeasts Saccharomyces cerevisiae and Schizossacharomyces pombe, we proposed to study such response in the multicellular filamentous fungus Trichoderma reesei. To this purpose, we have utilized the cDNA microarray technique to analyze the gene expression of approximately 2,000 T. reesei transcripts in response to heat shock, to high concentration of cadmium II ions and to a 2-hour absence of carbon or nitrogen source. As a general response to the four studied stresses, we observed on one hand a negative transcriptional regulation of genes involved in processes that demand great amounts of energy, i.e. a negative regulation of protein synthesis, indicated by strong repression of ribosomal protein genes transcription, as well as a negative regulation of anabolism. On the other hand, genes that encode proteins associated with cellular defense, like chaperones, had their expression induced. The responses to heat shock and to cadmium poisoning were quite similar while nitrogen source absence also induced the expression of genes related to protein and nucleotide degradation. Genes implicated in the consumption of lipid reserves were induced in the absence of both carbon and nitrogen sources. We identified some transcription regulators as well as components of signal transduction pathways that have differential patterns of gene expression caused by these different environmental stresses. Most of the genes that had their expression altered in response to the studied environmental stresses has no known function yet. Their expression patterns towards such stresses are therefore an important contribution to their functional annotation. Since the filamentous fungus Trichoderma reesei has become a microorganism of biotechnological value for its high capacity of synthesis and secretion of proteins, we expect that the data presented on this work can provide a better understanding of its cellular processes and may support future projects for a better adaptation of this organism to industrial conditions.
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Hardy, Nicolas. "Identification des critères d’extrapolation du procédé de production de cellulases par Trichoderma reesei en utilisant l’approche « scale-down »." Thesis, Université Paris-Saclay (ComUE), 2016. http://www.theses.fr/2016SACLA017/document.
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Le procédé de production d’éthanol à partir de biomasse lignocellulosique nécessite l’hydrolyse de cette dernière en sucres simples. Cette hydrolyse est le plus souvent réalisée par voie biologique grâce à des enzymes appelées cellulases. La production de ces enzymes représente cependant un verrou économique majeur au développement du procédé à grande échelle. Les cellulases sont généralement produites industriellement par le champignon filamenteux aérobie Trichoderma reesei, doté d’une forte capacité de sécrétion d’enzymes. Les cultures sont réalisées en bioréacteurs aérés et agités mécaniquement. Elles nécessitent de contrôler la concentration des substrats, ce qui requiert la maitrise de conditions hydrodynamiques et physicochimiques. En effet, le milieu de culture de T. reesei devient une suspension de cellules de champignons associées en filaments, de structure complexe, dont la viscosité augmente avec la concentration microbienne selon un comportement rhéofluidifiant. La viscosité est fonction de la morphologie du microorganisme qui peut, elle-même, varier avec les conditions de cultures. Cet accroissement de viscosité est un critère clef de l’extrapolation du procédé, car il affecte le transfert d’oxygène. Afin de maintenir une concentration en oxygène dissous suffisante, l’agitation et l’aération sont en général augmentées, entraînant un accroissement du cisaillement. Cet accroissement impacte en retour la morphologie du champignon, ralentit sa croissance puis diminue la production de cellulases. Ainsi, les conditions hydrodynamiques et rhéologiques engendrées au sein du bioréacteur sont complexes et variables dans le temps. L’interrelation entre conditions opératoires, morphologie, croissance du champignon et viscosité du moût de fermentation impose l’intégration de tous ces phénomènes pour l’optimisation du procédé, notamment à grande échelle. L’objectif de la thèse est de mettre en place une approche, visant à étudier au laboratoire la croissance de T. reesei et sa production d’enzymes, en reproduisant les contraintes hydrodynamiques associées aux conditions de fonctionnement des fermenteurs industriels. Pour ce faire, deux méthodologies originales ont été développées : une méthode de mesure de la viscosité du milieu, optimisée pour les champignons filamenteux, représentative des conditions rencontrées à grande échelle et qui s’appuie sur l’utilisation d’un rhéomètre rotatif équipé d’un rotor hélicoïdal ; une méthode d’analyse d’images associant un microscope motorisé et des algorithmes d’analyse d’images innovants, qui permet de générer des données sur la morphologie du champignon et d’identifier un critère morphologique pertinent basé sur le nombre de « trous » au sein d’un filament. Parallèlement à ces méthodes, différentes contraintes de cisaillement ont été mises en oeuvre en fermentation, afin de reproduire, à l’échelle du laboratoire, les conditions rencontrées à l’échelle industrielle. Ces outils ont été utilisés conjointement et validés lors de cultures non conventionnelles mimant les conditions industrielles en termes de cisaillement. Ils ont permis d’identifier un critère représentatif du cisaillement (EDCF) et d’établir, à partir de ce critère, des corrélations capables de prédire la viscosité du moût de fermentation, le taux de croissance maximum du microorganisme ainsi que certains paramètres morphologiques de la souche. De façon originale, ces corrélations déterminées à l’échelle du laboratoire ont été validées par des mesures effectuées à l’échelle industrielle. Au final, l’approche développée permet d’identifier au plus tôt les contraintes d’extrapolation à ne pas dépasser, afin d’orienter les choix technologiques des fermenteurs industriels impliquant des champignons filamenteuxEthanol production from lignocellulosic biomass requires its transformation into fermentable sugars before the alcoholic fermentation. This step called hydrolysis is catalyzed by cellulases and is often considered as the major technical and economic challenge for the process development. Cellulases are industrially produced by the filamentous fungus Trichoderma reesei, thanks to its high secretion capacity. This fungus is strictly aerobic and is thus cultivated in aerated and stirred bioreactors. The fermentation optimization requires control of physicochemical conditions. Actually the growth of fungi induces an increase of the broth viscosity with shear thinning behavior because of the formation of three-dimensional mycelial structures (from micrometer to millimeter). This viscosity increase has a negative impact on the oxygen transfer. In order to keep the dissolved oxygen concentration higher than a critical limit, it is necessary to increase the power input thereby increasing the shear stress, which may affect the morphology of the fungus as well as its growth and cellulose production. Actually, physico-chemical conditions generated inside the bioreactor are complex and vary with time. These interrelations, between process conditions, morphology, growth and viscosity, require the integration of all these parameters to optimize the full-scale process. The goal of the thesis work was to develop a scale-down approach at lab-scale to mimic hydrodynamic conditions of industrial bioreactors and to study their impact on T. reesei growth and cellulase production. For that purpose, two new tools were developed. The first one consists in a new rheological measurement set-up using a helical rotor dedicated to filamentous fungi preventing mycelium degradation during the measurements. The second one is an original image analyses method that uses specific algorithms. It was then possible to record various morphological data on fungi and to select the most relevant ones (like the number of holes). Meanwhile, a wide range of shear stress conditions were explored in the laboratory bioreactor to reproduce industrial conditions. The new tools we had developed, coupled to these unconventional cultures lead to identifying a shear stress relevant
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Borin, Gustavo Pagotto 1991. "Estudos genômicos da expressão gênica global do fungo filamentoso Trichoderma reesei crescido em bagaço e colmo de cana-de-açúcar = Genomic studies of global gene expression of filamentous fungus Trichoderma reesei grown in bagasse and culm of sugarcane." [s.n.], 2015. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316807.
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Orientadores: Gustavo Henrique Goldman, Juliana Velasco de Castro OliveiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A parede celular vegetal é uma estrutura recalcitrante, composta por polissacarídeos complexos que podem ser quebrados em açúcares fermentáveis. A desconstrução desse material complexo pode ser feita por diversos tipos de enzimas hidrolíticas, que são produzidas naturalmente por uma variedade de microrganismos. Entre eles, o fungo Trichoderma reesei se destaca pela capacidade de produzir e secretar estas enzimas em grandes quantidades. Embora alguns trabalhos utilizando abordagens de proteômica e transcriptômica já tenham sido realizados com esse fungo, ainda não são conhecidos em detalhes os mecanismos moleculares responsáveis pela degradação da parede e a regulação gênica envolvida nesse sistema lignocelulolítico. O presente trabalho tem como objetivo principal a análise da expressão gênica global de T. reesei, crescido por 6, 12 e 24 horas em bagaço e colmo de cana-de-açúcar como fontes únicas de carbono, pela técnica de sequenciamento high-throughput de RNA (RNA-Seq). No transcriptoma de T. reesei foram identificadas sendo hiper-expressas as principais celulases, hemicelulases e proteínas acessórias relacionadas direta ou indiretamente com a desconstrução da parede vegetal. De modo geral, as celulases e hemicelulases apresentaram uma expressão maior do que outras enzimas, e o nível dos seus transcritos foi crescente ao longo do tempo tanto em colmo quanto no bagaço. A grande maioria dos genes de CAZymes e proteínas acessórias hiper-expressos foram compartilhados pelos dois substratos, o que demonstra que a estratégia usada por T. reesei para degradar a parede celular do colmo e do bagaço é similar. Adicionalmente, vários fatores de transcrição, proteínas de função desconhecida e transportadores supostamente envolvidos na assimilação dos açúcares liberados também foram hiper-expressos nas condições amostradas. Para validação do RNA-Seq, foi realizado PCR em tempo real de diversos genes hiper-expressos que codificam para enzimas hidrolíticas, reguladores transcricionais, proteínas acessórias e genes ainda não caracterizados. Para isso, a análise temporal foi ampliada para 30 minutos, 2, 4, 6, 12 e 24 horas de crescimento após o inóculo, o que permitiu uma análise mais detalhada da expressão desses genes. Como objetivo secundário, foi analisado o secretoma deste fungo e os açúcares concomitantemente liberados no sobrenadante. Estas análises indicaram que a desconstrução da parede celular já se inicia dentro de 6 horas pós inoculo, com a liberação de monômeros (principalmente xilose e glicose) dos polissacarídeos e secreção de diversas CAZymes. Ensaios enzimáticos também foram realizados, mostrando atividades celulo e hemicelulolíticas. Assim, descrevemos pela primeira vez o arsenal de enzimas transcritas e secretadas por T. reesei RUT C30, desde pontos inicias de crescimento, em bagaço explodido e colmo de cana-de-açúcar. Por fim, este trabalho também permitiu a identificação de vários genes, com função predita ou não, que podem abrir caminho para a descoberta de novos atuantes na resposta do fungo ao substrato lignocelulósico
Abstract: Plant cell wall is a recalcitrant structure composed of complex polysaccharides which can be broken down into fermentable sugars. The deconstruction of this complex material can be made by a variety of hydrolytic enzymes which are naturally produced by a variety of microorganisms. Among them, stands out the fungus Trichoderma reesei, able to produce and secrete those enzymes in large quantities. Although some studies using transcriptomics and proteomics approaches have been performed with this fungus, the molecular mechanisms responsible for the degradation of the cell wall and gene regulation involved in this lignocellulosic system are not well known. This work has as main objective the analysis of global gene expression of T. reesei grown at 6, 12 and 24 hours in sugarcane bagasse and culm as sole carbon sources by high-throughput RNA sequencing technology (RNA-Seq). In the T. reesei transcriptome, it was identified the major cellulases, hemicellulases and accessory proteins directly or indirectly related to the deconstruction of plant cell wall. In general, cellulases and hemicellulases exhibited higher expression than other enzymes, and the level of their transcripts was increased over the time in both culm and bagasse cultures. Most of up-regulated CAZymes and accessory proteins were shared between the two substrates, which demonstrates the strategy used by T. reesei to degrade the bagasse and culm cell wall is similar. Furthermore, several transcription factors, proteins of unknown function and transporters supposedly involved in the assimilation of sugars were also up-regulated in the sampled conditions. To validate the RNA-Seq data, real-time PCR of several up-regulated genes encoding hydrolytic enzymes, transcriptional regulators, accessory proteins and proteins not yet characterized was carried out. The time points was extended to 30 min, 2, 4, 6, 12 and 24 hours of growth after inoculation, allowing a more detailed analysis of the expression of these genes. As a secondary objective, T. reesei secretome and the sugars released in the supernatant were analyzed. It was shown that the sugarcane cell wall deconstruction begins within the first 6 hours post inoculation, releasing sugar monomers (mainly xylose and glucose) from polysaccharides due to the secretion of several hydrolytic enzymes. Enzymatic assays were also performed, showing cellulosic and hemicellulosic activities. Finally, this is the first study showing the arsenal of enzymes transcribed and secreted by T. reesei grown on steam exploded sugarcane bagasse and culm, at early time points. It was possible identify several genes, with predicted function or not, that can open new paths to discover novel players on the fungus response to lignocellulosic substrate
Mestrado
Microbiologia
Mestre em Genética e Biologia Molecular
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Tejada, Erik Cendel Saenz. "Caracterização do gene do Fator Transcricional 3 da RNA Polimerase B (btf3) de Trichoderma reesei e o efeito de seu nocaute sobre a expressão gênica no estresse por choque térmico." Universidade de São Paulo, 2007. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-23102007-142152/.
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A transcrição pela RNA polimerase II (RNA polimerase B) e a sínteses de proteínas são os mais importantes processos metabólicos em células eucarióticas, e estão envolvidas no controle da expressão gênica. Trichoderma reesei foi utilizado como modelo de estudo para o desenvolvimento deste estudo. Este fungo filamentoso é um microrganismo que vem sendo utilizado por vários laboratórios no mundo para o estudo dos diversos processos biológicos básicos devido à sua grande importância biotecnológica. Foi estabelecido por nosso grupo de pesquisa um banco de dados ESTs (\"Expressed Sequence Tags\") para este microrganismo e, por meio da técnica de \"microarrays\" de cDNAs, determinou-se a reposta transcricional de T. reesei em função da disponibilidade de oxigênio e glicose, assim como a alguns estresses ambientais. Com base nesses resultados foram escolhidos alguns transcritos afetados pela limitação de oxigênio, tais como o gene btf3. Este gene codifica para a proteína reguladora BTF3 (Fator Transcricional 3 da RNA polimerase B), muito conservada em eucariotos, envolvida na transcrição de vários promotores da classe II e que faz parte do complexo que se liga aos polipeptídios nascentes (NAC). Com o intuito de estudar a funcionalidade do gene btf3 em condições normais e em choque térmico foi realizada uma análise transcricional comparativa em larga escala da cepa selvagem de T. reesei QM9414 e do mutante nocaute do gene btf3. A expressão de aproximadamente 2.000 genes foi analisada, por meio de \"microarrays\", em células submetidas ao estresse por choque térmico produzido pelo incremento da temperatura de cultura a 40°C. O nocaute do gene btf3 produziu o incremento da expressão dos genes das vias metabólicas primárias (ND4 e FBA), da defesa celular (DnaJ, HSP70 e RCI) e da síntese de proteínas (eIF2); enquanto que reprimiu genes da estrutura celular (HFBII) e da síntese de RNA (ATF21). No choque térmico, genes que codificam para proteínas associadas com a defesa celular, como as chaperonas Hsp70 e DnaJ, tiveram sua expressão induzida enquanto que proteínas associadas à divisão celular, como histonas e septina B, e à síntese de proteínas, como as proteínas ribossomais, foram reprimidas em ambas as cepas como resultado do estresse. Os resultados obtidos na análise por \"microarray\" foram validados através de PCR quantitativo em tempo real (qPCR). Estes resultados sugerem que BTF3 possa atuar como repressor de alguns genes transcritos pela RNA polimerase II.Transcription by RNA polymerase II (RNA polymerase B) and protein synthesis are the most important metabolic processes in eukaryotic cells, and they are involved in the control of gene expression. Trichoderma reesei was used as model of study for the development of this study. This filamentous fungus is a microorganism that has been used by some laboratories around the world for the study of diverse basic biological questions due to its great biotechnological importance. We had established a data base of ESTs (Expressed Sequence Tags) for this microorganism and, through the technique of cDNA microarray, we had determined the transcriptional response of T. reesei to oxygen and glucose availability, as well as some environmental stresses. Based on such studies we chose some transcripts affected by oxygen limitation such as the btf3 gene, for more detailed investigations. This gene codes for the BTF3 regulatory protein (RNA Polymerase B Transcription Factor 3), a conserved transcriptional factor among eukaryotes that is involved in the transcription of several class II promoters and is part of the nascent polypeptide-associated complex (NAC). In order to study the functionality of the btf3 gene in normal and heat shock conditions, a large-scale transcriptional comparative analysis between T. reesei wildtype strain QM9414 and the btf3 knockout mutant was executed. The expression of approximately 2,000 genes was analyzed using microarrays in cells submitted to heat stress produced by culture temperature increment to 40°C. The knockout of btf3 produces the increment of the expression of genes involved with the primary metabolism pathways (ND4 and FBA), cellular defense (DnaJ, HSP70 and RCI) and protein synthesis (eIF2); whereas it repressed genes related to the structure cell (HFBII) and RNA synthesis (ATF21). In heat shock, genes that encode Hsp70 and DnaJ proteins, associated to cellular defense, as chaperones, had their expression induced. On the other hand, genes for proteins associated to cellular division, such as histones and septin B, and those related to protein synthesis, such as ribossomal proteins were transcriptionally repressed in both strains as a result of the stress. The results obtained in the microarray analysis were validated through quantitative real-time PCR (qPCR). These results suggest that BTF3 can act as a repressor for some genes transcribed by RNA polymerase B.
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Malouf, Philippe. "Study of the relationship of rheology, morphology and biomass concentration of Trichoderma reesei fermentation." Thesis, University of Ottawa (Canada), 2008. http://hdl.handle.net/10393/27706.
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Bioethanol produced from cellulosic materials, abundantly found as wastes, appears to be a viable alternative to fossil fuels. Cellulose is a glucose polymer and must undergo a hydrolysis step by cellulase enzymes prior to be used for bioethanol production. In order to make bioethanol more cost competitive, all aspects of the production are being examined, including trying to improve the production of these enzymes using a filamentous microorganism, Trichoderma reesei. Filamentous fungi broth is widely known for being highly viscous, which limits transport properties, thus hindering the production of cellulase. Therefore, the overall objective of this work is to understand the intimate relationship that exists between enzyme production, morphology of microorganism, rheology of the broth and operating conditions. In this work, the rheology of the fermentation broth is examined in order to understand its impact on the other process variables.
As a preliminary step, the choice of an appropriate rheological instrument was discussed. Fed-batch fermentation runs were performed in two bioreactors: a stirred tank bioreactor with an agitation speed of 200, 300, 400 and 500 RPM and a reciprocating plate bioreactor agitated at 0.25, 0.50, 0.75 and 1.00 Hz. Herschel-Bulkley equation described relatively well rheological data. The effect of morphology and biomass concentration (X) on the consistency index (K) was studied separately. Biomass reconstitution experiments showed that biomass exponent alpha, in K/Xalpha, remained constant. K/X alpha varied during the course of the fermentation runs, underlying the importance of including a morphological parameter in the prediction of K. Although several morphological parameters were analyzed, K/Xalpha was found to correlate well with the average roundness, when the batch and the fed-batch data were considered separately. Parity plot analysis of the models showed good prediction of the experimental K.
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Ries, Laure Nicolas Annick. "Regulation of genes encoding enzymes involved in plant cell wall deconstruction in Trichoderma reesei." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/13045/.
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This study describes the regulation of genes encoding plant cell wall-degrading enzymes in the presence of different carbon sources from the biotechnologically important fungus Trichoderma reesei. It was shown that different carbon sources influence fungal growth rate, biomass production and subsequent enzyme secretion. Several genes were identified and suggested to play a role in the development of conidia and in maintaining polarised growth. RNA-sequencing studies showed an increase in transcript levels of genes encoding enzymes involved in plant cell wall degradation (CAZy) as well as of genes encoding lipases, expansins, hydrophobins, G-protein coupled receptors and transporters when mycelia were cultivated in the presence of a lignocellulosic substrate (wheat straw). The encoded non-CAZy proteins were proposed to have accessory roles in carbohydrate deconstruction. A model for solid substrate recognition in T. reesei was described, based on the comparison with the one proposed for Aspergillus niger. Post-transcriptional regulation mediated by regulatory RNAs was identified for nearly 2% of all T. reesei genes, including genes encoding cell wall-degrading enzymes. Transcriptional regulation studies confirmed that transcription patterns of genes encoding enzymes involved in polysaccharide degradation differed between different carbon sources and that they are fine-tuned and dependent on factors such as culture conditions, consumption rate, assimilation of glucose and the presence of several transcription factors. The analysis of the structure of chromatin in the promoter and coding regions of one of these genes, cbh1, revealed different nucleosome positioning patterns under repressing (glucose) and inducing (sophorose, cellulose) conditions. CRE1, the carbon catabolite repressor in T. reesei was shown to be involved in the repression of many CAZy and non-CAZy encoding genes. Furthermore, CRE1 was also shown to be important for nucleosome positioning within the cbh1 coding region under repressing conditions and proposed to do so by interaction with (a) yet unidentified protein(s).
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Castro, Lilian dos Santos. "Análise global da expressão gênica durante a formação de celulases pelo fungo Trichoderma reesei." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/17/17131/tde-14012016-095203/.
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O fungo filamentoso Trichoderma reesei (Hypocrea jecorina) é o principal produtor de enzimas celulolíticas e hemicelulolíticas utilizadas para a produção de biocombustíveis a partir de biomassa lignocelulósica. Para que o custo de produção de etanol de segunda geração (2G), em escala industrial, seja competitivo, uma mistura eficiente de enzimas hidrolíticas é necessária para degradar a biomassa vegetal em açúcares fermentescíveis. Dada a crescente demanda pelo desenvolvimento de processos que reduzam os custos de enzimas de interesse biotecnológico, o presente trabalho se propôs a analisar o perfil global do transcriptoma de T. reesei em presença de celulose, soforose e glicose como fontes de carbono, a fim de prover um melhor entendimento de como essas enzimas são produzidas por esse microrganismo. Para isso, foi utilizada a abordagem RNA-seq e duas linhagens de T. reesei: QM9414 (parental) e xyr1 (mutante). No Capítulo I, foram anlisados 22 genes que codificam celulases e xilanases, com o intuito de verificar a regulação da expressão gênica pelo fator de transcrição XYR1 em diferentes fontes de carbono. Foi observada uma dependência da fonte de carbono e redução da expressão destes genes que codificam celulases e xilanases quando se comparou a linhagem mutante com a parental, cultivados na presença de celulose e soforose. Na presença de glicose, a maior parte dos genes analisados apresentou aumento de expressão no mutante xyr1. Por meio de análises in silico foi identificado o elemento regulatório cis para o fator de transcrição XYR1 usando quatro conjuntos de dados de genes dependente-condição envolvendo os experimentos RNA-seq e qPCR-TR. Foram identificados dois motivos com o consenso de ligação proposto para o regulador XYR1 (nomeado PWMXYR1). Ao usar esses motivos identificados, foi analisada a presença e disposição dos putativos elementos regulatórios cis nesses conjuntos de dados, sendo que sítios com intervalos curtos foram mais associados a promotores dependentes de XYR1 do que sítios simples. Além disso, a abordagem utilizada permitiu a identificação de sítios de ligação XYR1, na região promotora dos genes cel7a e xyn1 e mapear a potencial sequência alvo do regulador na região promotora do gene cel6a. Adicionalmente, sete outros promotores (genes cel7b, cel61a, cel61b, cel3c, cel3d, xyn3 e swo) apresentaram sítios de ligação putativos de XYR1. Usando a arquitetura do regulador PWMXYR1, foram identificados potenciais genes alvos de regulação direta por XYR1 no genoma de T. reesei. O mapeamento do referido sítio foi realizado, também, nos genes diferencialmente expressos na linhagem mutante xyr1, destacando que a regulação indireta desempenha um papel fundamental nas vias de sinalização. Estes resultados fornecem novas informações sobre os mecanismos de sinalização mediados por XYR1 na regulação de promotores de celulases. No Capítulo II, foi analisado o transcriptoma global da linhagem parental (QM9414) em diferentes fontes de carbono: celulose; soforose e glicose. Aplicando limites rigorosos de corte, 2060 genes foram identificados como diferencialmente expressos em pelo menos uma das fontes de carbono analisadas. Clusterização hierárquica desses genes diferencialmente expressos identificaram-se três possíveis regulons, representando 123 genes controlados por celulose, 154 genes controlados por soforose e 402 genes controlados por glicose. A análise da rede regulatória demonstrou a inter-relação existente entre as condições, permitindo a identicação de 75 genes específicos do crescimento em soforose e 107 de celulose. Esses resultados revelaram novos genes envolvidos na degradação da celulose, tais como: proteínas acessórias, transportadores, fatores de transcrição e CAZymes que respondem especificamente a presença de celulose ou soforose. No Capítulo III, analisou-se o perfil transcricional da linhagem mutante xyr1 comparando com a linhagem parental (QM9414), na presença das três fontes de carbono. As análises de expressão gênica revelaram que 2185 genes foram diferencialmente expressos na condição celulose, 2124 genes na condição soforose e 46 genes em glicose. Esses genes foram utilizados para analisar a inter-relação existente entre as condições, com destaque para grande número de genes exclusivos nas condições de indução (celulose e soforose). Por meio da clusterização hierárquica foram identificados 6 possíveis regulons, sendo 3 deles compostos por genes up-regulados e 3 regulons por genes down-regulados. Na ausência do regulador XYR1, a expressão de genes CAZys, fatores de transcrição, transportadores, entre outros, foram afetadas de modo dependente da fonte de carbono. Essas análises permitiram verificar que o fator de transcrição XYR1 é fundamental no processo de indução de enzimas de interesse biotecnológico, além de participar de outros processos bioquímicos/moleculares. Desse modo, todos os resultados obtidos fornecem informações sobre os eventos moleculares envolvidos na regulação da expressão gênica durante a adaptação de T. reesei frente a diferentes condições ambientais como: diferentes fontes de carbono e necessidades nutricionais. Contribuindo, assim, para uma melhor caracterização desse fungo filamentoso em relação à produção de enzimas e o desenvolvimento de mutantes metabólicos para utilização industrial.The filamentous fungus Trichoderma reesei (Hypocrea jecorina) is a major producer of cellulolytic enzymes used for biofuels production from lignocellulosic biomass. In order to achieve a low cost second generation ethanol production in industrial scale, an efficient mix of hydrolytic enzymes are required for the degradation of plant biomass into fermentable sugars. Given the growing demand for development of processes in order to reduce the cost of enzymes production, the present work proposes a large-scale transcriptomic analysis of T. reesei in presence of cellulose, sophorose, and glucose as carbon sources, in order to provide insights about the mechanisms of enzymes production by this microorganism. For this purpose, RNA-seq approach was employed, as well, as two T. reesei strains: QM9414 (parental) and xyr1 (mutant). In Chapter I, 22 genes encoding cellulases and xylanases were analyzed regarding the regulation through the transcription factor XYR1. This regulation seems to occur in a carbon source dependence manner, and the expression of cellulases and xylanases was diminished in the mutant strain compared to the parental strain in the presence of cellulose and sophorose. In glucose, most of the analyzed genes showed higher expression in the mutant compared to the parental strain. In silico analysis identified a cis regulatory element for XYR1 using four datasets of condition-dependent genes based on RNA-seq and qPCR experiments. Two binding motifs have been identified with the proposed consensus for the XYR1 (named PWMXYR1). Using these motifs, the presence and arrangement of putative cis regulatory elements was analyzed, and the results indicate that the sites with short intervals were stronger associated with XYR1 dependent promoters than single sites, even with high scores. Moreover, this approach allowed the identification of XYR1 binding sites in the promoter region of cel7a and xyn1, as well as, map the potential target sequence in the promoter of cel6a gene. In addition, seven other promoters from genes cel7b, cel61a, cel61b, cel3c, cel3d, xyn3 and swo presented XYR1 putative binding sites. Using the cis-regulatory architecture of PWMXYR1, potential targets for direct regulation by XYR1were identified in a genome wide manner. The putative binding sites were also mapped in the differentially expressed genes identified in the mutant strain xyr1, suggesting that indirect regulation plays a key role in signaling pathways. Taken together, the data provided here provides important information regarding signaling mechanisms mediated by XYR1 in the regulation of cellulases promoters. In Chapter II, the transcriptome of the parental strain (QM9414) was analyzed in three carbon sources, cellulose, sophorose and glucose. By applying a stringent cut-off threshold, 2,060 genes were identified as differentially expressed in at least one of the carbon sources analyzed. Hierarchical clustering of differentially expressed genes identified three possible regulons, representing 123 genes controlled by cellulose, 154 genes controlled by sophorose and 402 genes controlled by glucose. The analysis of the regulatory network demonstrated the interrelation between the conditions, allowing the identification of 75 genes specifically expressed in soforose and 107 in cellulose. These results revealed new players involved in cellulose degradation, such as accessory proteins, transporters, transcription factors and CAZymes, that specifically respond to the presence of either cellulose or sophorose. In Chapter III, a genome wide comparative transcriptional analysis were performed between the mutant xyr1 strain and the parental strain (QM9414) using the three carbon sources. Gene expression analysis revealed 2,185 genes differentially expressed in cellulose, 2124 genes in sophorose and 46 genes in glucose. These differentially expressed genes were used to analyze the relationship between the conditions, revealing a greater number of exclusive genes in theinducing conditions (cellulose and sophorose). The hierarchical clustering analysis revealed 6 possible regulons, being three composed by up-regulated genes and 3 regulons composed by down-regulated genes. In the absence of the regulator XYR1, the expression of CAZy genes, transcription factors among other genes were affected in a carbon-dependent manner. These analyses showed that the transcription factor XYR1 has an essential role in induction of enzymes of biotechnological interest and participates in other biochemical/molecular processes. Taken together these results provide important information about the molecular events, involved in gene expression regulation during T. reesei adaptation to different environments such as: carbon sources and/or nutritional needs, thus contributing to a better understanding of this filamentous fungus regarding the the production of enzymes and the development of mutants for industrial applications.
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Poidevin, Laetitia. "Caractérisation d'enzymes fongiques dégradant les parois végétales et complémentation du sécrétome de Trichoderma reesei." Aix-Marseille 1, 2010. http://www.theses.fr/2010AIX11058.
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La conversion biologique de la lignocellulose en éthanol est une tache complexe. L'approche consiste à extraire les sucres fermentescibles de la cellulose avant de les convertir en éthanol. Actuellement, l'hydrolyse de la biomasse est réalisée en utilisant les enzymes de Trichoderma reesei. Une voie importante pour la réduction des coûts de production repose sur la complémentation du sécrétome de ce champignon. C'est pourquoi le projet de recherche de cette thèse a pour but d'identifier de nouvelles enzymes provenant d'autres champignons qui peuvent améliorer l'efficacité d'hydrolyse de T. Reesei. Si l'estérase de Piromyces equi semle plus adaptée pour des applications pharmaceutiques que pour la production de bioéthanol, le criblage génomique de Podospora anserina a permis de caractériser trois nouvelles enzymes de famille GH6 dont une s'est révélée très active dans les conditions d'hydrolyse. Les enzymes de famille GH61 clonées n'ont cependant pas montré d'activité lors des différentes analyses. De plus, aucune synergie d'action avec les enzymes GH6 ou les enzymes T. Reesei n'a pu être montrée. La deuxième approche, un criblage fonctionnel des sécrétomes de P. Anserina a en revanche conduit à l'obtention de cocktails de protéines montrant des activités complémentaires à T. Reesei. En effet, un des principaux résultats est une amélioration de 17% du taux d'hydrolyse, ce qui ouvre la voie à des investigations plus approfondies sur la ou les enzyme(s) responsable(s) et le développement de nouveaux mélanges enzymatiques efficacesBiological conversion of lignocellulose to ethanol is a complex task. The approach consists in extracting the fermentable sugars from cellulose before converting them to ethanol. To date, the enzymatic hydrolysis of biomass involves enzymes from the filamentous fungus Trichoderma reesei. A promising way to reduce the bioethanol production cost implies the complementation of the T. Reesei enzyme cocktail. Therefore, the present work aimed to identify new enzymes from other fungi able to improve the hydrolysis efficiency of T. Reesei enzymes. While the esterase EstA from Piromyces equi seems to be better adapted for pharmaceutical applications than for bioethanol production, genomic screening of Podospora anserina allowed the characterization of three new interesting family GH6 enzymes. One of them proved to be very active under enzymatic hydrolysis conditions. However, the cloned family GH61 enzymes were not active under any of the conditions tested. Moreover, no synergistic interaction with family GH6 or T. Reesei enzymes could be shown. In a second approach, a functional screening of P. Anserina secretomes led to the identification of enzymes mixtures able to complement T. Reesei. Indeed, an improvement of lignocellulose hydrolysis by 17% was achieved opening the way for more detailed investigations on the responsible enzyme(s) and the development of efficient cellulolytic enzyme mixtures
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Coffman, Anthony M. "Production of Carbohydrases by Fungus Trichoderma Reesei Grown on Soy-based Media." University of Akron / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=akron1381761363.
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Verbeke, Jonathan. "Vers l'optimisation du cocktail cellulolytique de trichoderma reesei par les protéines apparentées aux expansines." Aix-Marseille 1, 2009. http://theses.univ-amu.fr.lama.univ-amu.fr/2009AIX11064.pdf.
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La production industrielle de bioéthanol à partir de biomasse lignocellulosique nécessite l'amélioration de l'efficacité hydrolytique du cocktail enzymatique de Trichoderma reesei. Ce champignon filamenteux présente l'intérêt de sécréter des enzymes cellulolytiques en grande quantité mais l'analyse de son génome montre que la diversité de gènes codant ces enzymes est restreinte. C'est pourquoi, dans ce travail, une complémentation de ce cocktail par des enzymes auxiliaires a été envisagée. Récemment, la présence de la swollénine, une protéine apparentée aux expansines végétales, a été mise en évidence chez T. Reesei. Sa capacité à écarter les fibres de cellulose et l'induction de son gène parallèle à ceux des cellulases laisse penser que cette protéine peut avoir un rôle auxiliaire pendant la cellulolyse. L'étude in silico de séquences comportant des similarités avec les expansines chez les champignons a montré l'existence de plusieurs familles dont une est absente chez T. Reesei. Un représentant de cette famille, la protéine CELA d'Aspergillus fumigatus ainsi qu'une swollénine de cette espèce, SWOAfu, ont donc été choisies pour une expression hétérologue dans T. Reesei. De plus, des constructions chimériques destinées à rapprocher physiquement SWOAfu de la cellobiohydrolase CBH1 de T. Reesei ont été réalisées. Cette protéine de fusion, qui devrait permettre d'augmenter leur synergie a également été exprimée dans T. Reesei. Enfin, pour étudier une implication éventuelle des protéines Endoglucanase-45/Expansin-Like (EEL) de T. Reesei dans l'hydrolyse de la lignocellulose, des études transcriptionnelles ont été réalisées dans des conditions d’induction de cellulases. Une expression constitutive d'une d'entre elles, semblable à celle de l'endoglucanase Cel5b a pu être montrée. Après analyse de leur structure protéique et de leurs promoteurs, les fonctions potentielles des protéines EEL sont discutéesThe industrial production of bioethanol from lignocellulosic biomass requires the increase of the hydrolytic efficiency of the enzymatic pool produced by Trichoderma reesei. This fungus is able to secrete large amounts of cellulolytic enzymes, but its genome shows a low diversity of genes encoding these enzymes. Therefore, in this work, a complementation of this cocktail with auxiliary proteins was envisaged. Recently, the presence of swollenin, a protein related to plant expansins, was brought to light in T. Reesei. Its capacity to loosen cellulose fibers and the induction of its gene parallel to cellulase genes suggests that this protein could have an auxiliary role in cellulose hydrolysis. A database search of sequences presenting similarities with plant expansins in fungi showed that different families exist, one of which is absent in T. Reesei. CELA from Aspergillus fumigatus, a protein belonging to this family, and a swollenin from this species, SWOAfu, were selected for heterologous expression in T. Reesei. Moreover, chimeric protein constructions were realised to approach the catalytic domains of SWOAfu and the cellobiohydrolase CBH1 from T. Reesei. This chimeric protein which should lead to an increase of their synergy was also expressed in T. Reesei. Finally, in order to investigate a potential implication of Endoglucanase-45/Expansin-Like (EEL) proteins of T. Reesei in the cellulolytic process, transcriptional studies were realised under conditions of cellulase induction. A constitutive expression, similarly to the endoglucanase Cel5b, was shown for one of them. Potential functions of the EEL proteins are discussed with regard to their protein and promoter structure
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Tokunaga, Yuki. "Interaction analysis between lignin and carbohydrate-binding module of cellobiohydrolase I from Trichoderma reesei." Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/263699.
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Bonaccorsi, Eric D\'Alessandro. "Regulação da expressão gênica por oxigênio em microrganismos eucariotos: análises de ESTs (Expressed Sequence Tags) e microrrays de cDNA de Trichoderma reesei." Universidade de São Paulo, 2003. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-04052018-110230/.
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Glicose e oxigênio são moléculas essenciais para a maioria dos organismos vivos. Além de sua importância nos processos de produção de energia - glicose como fonte de carbono e energia e oxigênio como aceptor dos elétrons doados por NADH e FADH2 - estes dois compostos funcionam como efetuadores, modulando vários processos metabólicos e fisiológicos nas células. Visto que a mitocôndria é um dos alvos afetados pelas disponibilidades destas duas moléculas, nós isolamos e seqüenciamos o genoma mitocondrial de Trichoderma reesei, um fungo multicelular empregado neste trabalho como sistema modelo. Foi estudado o efeito da variação de concentração de glicose e oxigênio sobre a expressão de transcritos do genoma mitocondrial, bem como sua implicação no metabolismo de glicose. São apresentadas análises da expressão gênica de aproximadamente 2000 transcritos de T. reesei submetido a concentrações limitantes de oxigênio dissolvido, realizadas com o emprego da técnica de microarrays de cDNA. Pelo menos 330 transcritos foram diferencialmente expressos em função da disponibilidade de oxigênio. Aqueles envolvidos nos processos de síntese protéica e divisão celular foram regulados negativamente, enquanto transcritos relacionados com funções de defesa celular e síntese de RNA foram positivamente regulados. Uma fração substancial de outros genes afetados pela baixa disponibilidade de oxigênio não possui, atualmente, funções celulares conhecidas. Esta observação deve contribuir para a posterior anotação funcional do genoma de T. reesei. Também foram identificados reguladores transcricionais diferencialmente expressos em baixas tensões de oxigênio. O perfil de expressão destes reguladores aponta-os como potenciais candidatos ao envolvimento com a expressão de genes afetados pela disponibilidade de oxigênio.Glucose and oxygen are essential molecules in most of living organisms. In addition to their importance in production of energy - glucose as a carbon and energy source and oxygen as an acceptor of electrons donated by NADH and FADH2 - both molecules function as effectors modulating various metabolic and physiological processes in the cell. Because one of the targets affected by both molecules is the mitochondrion, we isolated and sequenced the mitochondrial genome of Trichoderma reesei, a multicellular fungus that is used in this study as a model system. The effect of varying the concentration of glucose and oxygen on the expression of the transcripts of the mitochondrial genome, and its implication on the metabolism of glucose, was studied. Gene-wide expression analyses of nearly 2000 transcripts of T. reesei under limited concentration of dissolved oxygen, using cDNA microarry technique, are presented. At least 330 transcripts were differentially expressed with respect to oxygen availability. Those involved in protein synthesis and cell division processes were downregulated, while transcripts involved in cell defense and RNA synthesis were upregulated. A substantive fraction of other anaerobically affected genes have currently unknown cellular roles, and these results should therefore contribute to further functional annotation of the genome. ln addition, we have identified transcriptional regulators that are differentially expressed at a low oxygen tensions. The expression profile of these regulators points them out as potential candidates involved in the expression of genes affected by oxygen availability.
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Karjalainen, M. (Marika). "Optimizing reaction conditions for an LPMO-enzyme from Trichoderma reesei with a downscaled TTC-assay." Master's thesis, University of Oulu, 2017. http://urn.fi/URN:NBN:fi:oulu-201711293191.
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Abstract
The increasing awareness of the causes and consequences of climate chance has led to actions to reduce the dependency on oil and other finite energy and raw material sources. Plant biomass is used in increasing amounts as a resource for biofuel, biochemical and fiber production. Carbohydrate enzymology has provided new ways to utilize and modify renewable carbon sources, especially the lignocellulolytic systems of fungi. Cellulolytic enzymes work in a synergistic manner on recalcitrant structure of cellulose, hydrolyzing it into soluble oligosaccharides, and eventually, glucose. Lytic polysaccharide monooxygenases (LPMOs) contribute to this system by oxidizing either C1- or C4-carbon from the carbohydrate chain on a crystalline cellulose with the help of copper-core induced radicals, thus creating available substrates for the other cellulolytic enzymes. Since their discovery in 2010, the research on their activities and specificities have increased rapidly, but the analytical methods to investigate this diverse group of enzymes is mostly limited to short and soluble products, which are only a fraction of the oxidation products. In addition, most of the methods require special equipment, wide range of standards and expertise to interpret the results. In this study, HPLC and HPAEC-PAD were tested, unsuccessfully, to quantify soluble products from LPMO-catalysis. A TTC-method, in which 2,3,5-triphenyl-2H-tetrazolium chloride is reduced into red and spectrophotometrically quantifiable formazan by reducing ends from insoluble LPMO-products, was successfully optimized and downscaled, and used to optimize reaction conditions for a type 3 LPMO from Trichoderma reesei, TrAA9A, with Whatman filter paper 1 as a substrate. Experiments were conducted to investigate the effects of pH, temperature, donor, time and the presence/absence of H₂O₂ to the accumulation of reducing ends. The results did not show any substantial differences in the accumulation of aldehydes in different reaction conditions. This study showed that cellulose degrades in the presence of TrAA9A and an electron donor. The greatest effects were observed with longer reaction times and the addition of H₂O₂, both increasing the amount of measured aldehydes in the insoluble products. The highest yield was recorded from the reactions with gallic acid as a donor at pH 6, and in the presence of 0.7 mM H₂O₂. The results from this study could lead to understanding the rate-limiting factors of the LPMOs and further improve the utilization of this enzyme in the degradation of lignocellulosic biomass.
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Gattinger, Loni D. "The enzymatic saccharification of canola meal and its utilization for xylanase production by Trichoderma reesei." Thesis, University of Ottawa (Canada), 1990. http://hdl.handle.net/10393/5643.
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Tests made utilizing canola meal as a substrate for the production of xylanase indicate that Trichoderma reesei produced this enzyme in similar or better yields from canola meal than from expensive carbon sources such as Solka-floc, cellulose, glucose, lactose, sucrose or purified xylans. The effect of culture conditions on xylanase production when canola meal was used as a carbon source was also investigated. The enzyme system produced using canola meal also contained a higher proportion of acetyl-xylan esterase, cellulase, and xylosidase activities, most of which are required for synergistic action and hydrolysis of complex materials. The enzymatic saccharification of canola meal was also investigated. The results show that saccharification of canola meal is mainly brought about by hemicellulases capable of degrading arabinogalactan, arabinoglucan, galactan and galactomannan, while cellulase and xylanase play a minor role. This autoclaving pretreatment also released water soluble polysaccharides consisting mainly of arabinnose and glucose. T. reesei was unable to produce enzymes capable of hydrolyzing these polysaccharides when cultivated on canola meal as substrate. (Abstract shortened by UMI.)
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Santos, Emerson dos [UNESP]. "Utilização de enzimas produzidas por Trichoderma reesei E Aspergillus niger na extração de óleos essenciais." Universidade Estadual Paulista (UNESP), 2008. http://hdl.handle.net/11449/96249.
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Universidade Estadual Paulista (UNESP)
O uso e produção de enzimas têm se tornado uma das áreas de maior interesse da indústria biotecnológica. As enzimas obtidas de microrganismos por processos fermentativos têm sido amplamente pesquisadas e utilizadas em todo mundo. A aplicação de enzimas na extração de óleos é uma alternativa consistente aos processos convencionais, por se tratar de um processo que consome menor energia, melhora a qualidade de vários produtos como, por exemplo, o óleo de oliva, e causa um mínimo de impacto ambiental. Para realizar a extração do óleo, que se encontra nos vacúolos intracelulares, há a necessidade do rompimento das paredes e membranas celulares. Os tratamentos mecânico e térmico causam a ruptura das estruturas celulares, porém, não são suficientes, já que parte do óleo permanece na célula, sem ser extraído. Para aumentar o rendimento no processo de extração do óleo se faz necessário a utilização de um complexo multienzimático que irá atuar sobre os componentes da parede, facilitando a sua liberação. O objetivo deste trabalho foi utilizar uma mistura de enzimas contendo celulases, xilanases e pectinases produzidas por microrganismos para extração de óleos essenciais de plantas. As enzimas foram produzidas pelos fungos T. reesei Rut C-30 e Aspergillus niger, em processo fermentativo incubado em biorreator de 10 L. A composição do meio de cultura, bem como as condições de cultivo, foram estabelecidas a fim de se obter maior produção de enzimas. A produção de enzimas em biorreator para a linhagem Rut C-30 de T. reesei mostrou os melhores níveis em pH 4,0, temperatura de 28º C, agitação de 400 rpm e taxa de aeração de 1,5 vvm, obtendo uma produção de 2,01 U/mg, 1,79 U/mg e 0,2 U/mg de celulase, xilanase e pectinase, respectivamente. A produção utilizando A. niger apresentou melhores níveis enzimáticos nas mesmas condições usadas para T. reesei e foram: 1,21 U/mg, 2,96 U/mg e 4,2 U/mg de celulase,
The use and production of enzyme have become one of the areas of great interest of the biotechnological industry. The enzymes produced by microorganisms in fermentation processes have been widely searched and used in the world. The enzyme application in the oil extraction is a consistent alternative to the conventional processes, because represents a process that consumes less energy, improves the quality of some products as, for example, the olive oil, and cause a minimum of environmental impact. To perform the extraction of the oil, which is found in the intracellular vacuoles, it is necessary break the cellular and membranes walls. The mechanical and thermal treatments cause the rupture of the cellular structures, however, they are not enough, since part of the oil remains in the cell, without be extracted. To increase the yield in the process of oil extraction is necessary to use a multienzymatic complex that will act on the components of the wall, facilitating its release. The objective of this work was to utilize a enzyme mixture containing cellulases, xylanases and pectinases produced by microorganisms to extract plants essential oils. The enzymes had been produced by the microorganisms Trichoderma reesei QM9414 and T. reesei Rut C-30 and Aspergillus niger, in fermentative process incubated in a 10L bioreactor. The composition of the culture medium, and the culture conditions were established in order to obtain higher level of enzyme production. The enzyme production in bioreactor by T. reesei Rut C-30 showed the best levels in pH 4.0, temperature of 28º C, agitation of 400 rpm and tax of aeration of 1,5 vvm, and obtaining production of 2.01 U/mg, 1.79 U/mg and 0.2 U/mg of cellulase, xylanase and pectinase, respectively. The production using A. niger showed the best leves at the same conditions used to T. reesei and the were: 1.21 U/mg, 2.96 U/mg and 4.2 U/mg of cellulase, xylanase and pectinase, respectively. Melampodium divaricatum (Rich.