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A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei 总被引:15,自引:0,他引:15
An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per microgram of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli beta-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter. 相似文献
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Summary Production of cellulases by three different genetically improved strains of Trichoderma reesei: MCG 77, RUT-C30 and CL-847, has been assessed on various fermentation media. The three strains produce high levels of enzymes when grown on purified cellulose as the main carbon energy source; when grown on lactose, decrease in enzyme yield and productivity, differs significantly from strain to strain. 相似文献
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Sophorose (2-O-beta-glucopyranosyl-D-glucose) induces carboxymethyl cellulase in Trichoderma reesei QM6a mycelium with 1.5 to 2 h. The induction response to sophorose concentration, although complicated by the metabolism of sophorose, shows saturation kinetics. Most of the cellulase appears after most of the sophorose has been taken up, but the presence of an inducer is required to maintain cellulase synthesis because enzyme production ceases after separation of the mycelium from the induction medium. Cellulase appears simultaneously in the medium and in the mycelium, and no appreciable levels accumulate in the mycelium. Response to pH suggest either that synthesis and secretion of the enzyme are closely associated or concurrent events affected by surface interactions with the medium. Effects of temperature and pH on cellulase induction by sophorose are similar to those reported for induction by cellulose. The kinetics of absorption by mycelium differs from that of other beta-linked saccharides and glucose, the uptake of sophorose being much slower. Under our cultural conditions, sophorose appears to induce an incomplete array of cellulase enzymes, as indicated by enzymatic and electrophoretic studies. 相似文献
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Transcriptional regulation of biomass-degrading enzymes in the filamentous fungus Trichoderma reesei 总被引:4,自引:0,他引:4
Foreman PK Brown D Dankmeyer L Dean R Diener S Dunn-Coleman NS Goedegebuur F Houfek TD England GJ Kelley AS Meerman HJ Mitchell T Mitchinson C Olivares HA Teunissen PJ Yao J Ward M 《The Journal of biological chemistry》2003,278(34):31988-31997
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Studies of the cellulolytic system of the filamentous fungus Trichoderma reesei QM 9414. Substrate specificity and transfer activity of endoglucanase I. 总被引:1,自引:0,他引:1 下载免费PDF全文
M Claeyssens H van Tilbeurgh J P Kamerling J Berg M Vrsanska P Biely 《The Biochemical journal》1990,270(1):251-256
Endoglucanase I from the filamentous fungus Trichoderma reesei catalyses hydrolysis and glycosyl-transfer reactions of cello-oligosaccharides. Initial bond-cleaving frequencies determined with 1-3H-labelled cello-oligosaccharides proved to be substrate-concentration-dependent. Using chromophoric glycosides and analysing the reaction products by h.p.l.c., kinetic data are obtained and, as typical for an endo-type depolymerase, apparent hydrolytic parameters (kcat., kcat./Km) increase steadily as a function of the number of glucose residues. At high substrate concentrations, and for both free cellodextrins and their aromatic glycosides, complex patterns (transfer reactions) are, however, evident. In contrast with the corresponding lactosides and 1-thiocellobiosides, and in conflict with the expected specificity, aromatic 1-O-beta-cellobiosides are apparently hydrolysed at both scissile bonds, yielding the glucoside as one of the main reaction products. Its formation rate is clearly non-hyperbolically related to the substrate concentration and, since the rate of D-glucose formation is substantially lower, strong indications for dismutation reactions (self-transfer) are again obtained. Evidence for transfer reactions catalysed by endoglucanase I further results from experiments using different acceptor and donor substrates. A main transfer product accumulating in a digest containing a chromophoric 1-thioxyloside was isolated and its structure elucidated by proton n.m.r. spectrometry (500 MHz). The beta 1-4 configuration of the newly formed bond was proved. 相似文献
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The properties and enzymic activity of endoglucanases (EC 3.2.1.4) of the fungus Trichoderma reesei were studied by means of immunological methods and by using polyglycosidic substrates. Endoglucanases exist in the culture liquid as a series of immunologically related components. The most active endoglucanase component has an Mr of 43 000 and pI value of 4.0. The most abundant components have a value of pI about 5.0, an Mr of 56 000-67 000 and specific activity only one-fifth of that of the pI-4.0 component. During purification and storage the endoglucanases are spontaneously modified; the relative proportion of components having greater Mr values, more alkaline pI values and lower specific activities is increased. The hexose content of the endoglucanase components is 2-7%. Endoglucanases hydrolyse soluble beta-1,4 glycans. The enzymes described here differ from endoglucanase preparations described previously in not showing activity towards insoluble substrates. The role of endoglucanases in wood hydrolysis is consequently limited to the stage where wood constituents are already in soluble form. 相似文献
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Marja P. Vaheri Matti E. O. Vaheri Veli S. Kauppinen 《Applied microbiology and biotechnology》1979,8(1-2):73-80
Summary Production and release of cellulolytic enzymes by Trichoderma reesei QM 9414 were studied under induced and non-induced conditions. For that purpose, a method was developmed to produce cellulases by Trichoderma reesei QM 9414 using the soluble inducer, cellobiose, as the only carbon source. The production was based on continuous feeding of cellobiose to a batch culture. For optimum production, the cellobiose supply had to be adjusted according to the consumption so that cellobiose was not accumulated in the culture. With a proper feeding program the repression and/or inactivation by cellobiose could be avoided and the cellulase production by Trichoderma reesei QM 9414 was at least equally as high as with cellulose as the carbon source.During the cultivation, specific activities against filter paper, carboxymethyl cellulose (CMC) and p-nitrophenyl glucoside were analyzed from the culture medium as well as from the cytosol and the cell debris fractions. There was a base level of cell debris bound hydrolytic activity against filter paper and p-nitrophenyl glucoside even in T. reesei grown non-induced on glycerol. T. reesei grown on cellobiose was induced to produce large amounts of extracellular filter paper and CMC hydrolyzing enzymes, which were actively released into the medium even in the early stages of cultivation. -Glucosidase was mainly detected in the cell debris and was not released unless the cells were autolyzing. 相似文献
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Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei. 总被引:4,自引:0,他引:4 下载免费PDF全文
Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression. 相似文献
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《Enzyme and microbial technology》2011,48(7):335-341
Metabolic measurements and screening of Trichoderma reesei have conventionally been performed during the hyphal stage of fungal development. To determine if flow cytometric measurements of protein expression could be made on germinating spores we created a gene construct, placing the Renilla reniformis green fluorescent protein gene under control of the cellobiohydrolase I (cbh1) promoter and terminator of T. reesei. This vector was transformed into T. reesei and GFP expression was measured in germlings by flow cytometry. Fluorescence associated with GFP expression was observed in germlings grown under conditions known to induce cellulases in Trichoderma. Spores were mutated using UV light and germinating spores were screened for increased GFP expression using high-speed cell sorting, to select for strains with genetic changes associated with increased protein expression. Secondary screens for cellulase production were conducted in microtitre plates. Flow cytometric screening of germinating spores expressing GFP yielded a mutant with improved ability to hydrolyse biomass. 相似文献
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Culture filtrates of CL-847 strain of Trichoderma reesei grown on different carbon sources have been compared. The highest enzyme production is obtained with Whatman C 41 cellulose: 17.9 mg/mL of soluble proteins and 13.7 units of filter paper (FP) activity. Wood pulps gave lower production values and more viscous culture media. About one-third of maximal enzyme production is obtained on lactose as the sole carbon source. Addition of 0.5% cellulosic inducer to 6% lactose media enhances enzyme production up to the following levels: 14.1 mg/mL of soluble proteins and 8.4 units of FP activity. 相似文献
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He Ronglin Ding Ruihua Heyman John A. Zhang Dongyuan Tu Ran 《Journal of industrial microbiology & biotechnology》2019,46(11):1603-1610
Journal of Industrial Microbiology & Biotechnology - The selection of improved producers among the huge number of variants in mutant libraries is a key issue in filamentous fungi of industrial... 相似文献
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Chambergo FS Valencia EY Ferreira-Júnior JR Camilo CM Campana PT 《International journal of biological macromolecules》2012,50(1):19-24
Superoxide dismutases (SODs; EC 1.15.1.1) are part of the antioxidant system of aerobic organisms and are used as a defense against oxidative injury caused by reactive oxygen species (ROS). The cloning and sequencing of the 788-bp genomic DNA from Trichoderma reesei strain QM9414 (anamorph of Hypocrea jecorina) revealed an open reading frame encoding a protein of 212 amino acid residues, with 65-90% similarity to manganese superoxide dismutase from other filamentous fungi. The TrMnSOD was purified and shown to be stable from 20 to 90 °C for 1 h at pH from 8 to 11.5, while maintaining its biological activity. 相似文献