Maturation of barley cysteine endopeptidase expressed in Trichoderma reesei is distorted by incomplete processing

Maturation of barley cysteine endopeptidase B (EPB) in Trichoderma reesei was studied with metabolic in hibitors, Western blotting, and immuno microscopy. The inactive 42-kDa recombinant EPB proprotein, first detected in apical cells, was sequentially processed in a time-dependent manner to a secreted polypeptide of 38.5 kDa, and thereafter, to polypeptides of 37.5, 35.5, and 32 kDa exhibiting enzyme activity both in the hyphae and culture medium. The sizes of the different forms of recombinant EPB were in accordance with molecular masses calculated from the deduced amino acid sequence, assuming cleavage at four putative Kex2p sites present in the 42-kDa proprotein. Both the liquid and the zymogram in-gel activity assays indicated that the 32-kDa enzyme produced in T. reesei in vivo was 2 kDa larger and four times less active than the endogenous EPB. Brefeldin A treatment prevented the last Kex2p processing step of EPB from a 35.5- to a 32-kDa protein. This coincided with a significant increase in the immuno-gold label for EPB and in modified Golgi-like bodies, which suggests that the processing step probably took place in medial Golgi. A 30.5-kDa EPB polypeptide was observed when glycosylation was inhibited by tunicamycin (TM) or when deglycosylation was carried out enzymatically. Deglycosylation increased the enzyme activity twofold, which was also indicated by an increased fluorescence by TM treatment in the zymogram in-gel activity assay. Simultaneous incubation with TM and monensin produced a peptide of 31.5 kDa. Therefore, monensin may inhibit the final processing step of an unglycosylated EPB by an unknown protease in the fungus. In any case, the final recombinant EPB product in Trichoderma differs from the mature endogenous 30-kDa enzyme produced in barley.     

Chitinous Material in Trichoderma reesei

Trichoderma reesei was grown for 180h in batch culture in an 8 liter stirred fermenter using a glucose-rich medium. Concentrations of glucose, ammonia, cell dry weight, debris and lipid are presented for two runs. Cell dry weights reached 26.9g/L and 19.6g/L in these runs. The debris from solvent-extracted cells was chitin which accumulated to greater than 75% of the final cell dry weights.     

Proteins associated with the cell envelope of Trichoderma reesei: a proteomic approach

A total of 220 cell envelope-associated proteins were successfully extracted and separated from Trichoderma reesei mycelia actively synthesizing and secreting proteins and from mycelia in which the secretion of proteins are low. Altogether 56 spots were examined by nanoelectrospray tandem mass spectrometry and amino acid sequence was obtained for 32 spots. From these, 20 spots were identified by Advanced BLAST searches against all databases available to BLAST. The most abundant protein in both types of mycelia was HEX1, the major protein in Woronin body, a structure unique to filamentous fungi. Other proteins identified were vacuolar protease A, enolase, glyceraldehyde-3-phosphate dehydrogenase, transaldolase, protein disulfide isomerase, mitochondrial outer membrane porin, diphosphate kinase and translation elongation factor beta. Partial short amino acid sequence obtained from some proteins did not allow them to be assigned to a specific protein in the database by BLAST search. In some cases, the tandem mass spectrometry spectra were too complicated to be able to assign an amino acid sequence with certainty. The number of spots (12) giving a clear signal but finding no match in the databases suggests that a majority of proteins associated with a filamentous fungal cell wall, are novel. Some technical problems related to protein isolation are also discussed.     

丝状真菌瑞氏木霉生产重组蛋白的分子生物学研究进展

瑞氏木霉是自然界中普遍存在并有重要经济意义的一种丝状真菌,作为工业生产菌株生产多种水解酶类已有多年历史。本文报道了用基因工程手段对瑞氏木霉进行遗传改造,构造具新性状的重组菌株,用以过量产生同源和异源蛋白类物质的分子生物学研究进展。包括利用ＣＢＨＩ基因的强启动子在瑞氏木霉中过量表达瑞氏木霉内切葡聚糖酶、小牛凝乳蛋白酶、人抗体片段、哈茨木霉几丁质酶、Ｈｏｒｍｏｃｏｎｉｓｒｅｓｉｎａｅ葡萄糖淀粉酶等同源和异源蛋白以及利用在葡萄糖上强表达的启动子生产纤维素酶等遗传工程进展情况。     

Electrofusion of Trichoderma reesei protoplasts

Protoplasts of Trichoderma reesei were fused according to the method of Zimmermann. For optimizing the fusion parameters the central composite design was used. Genetic evidence for fusion has been obtained by segregation of the auxotrophic markers in the haploid conidia. The parameters which were optimized were: pulse voltage, pulse duration and number of pulses. The optimal parameters for the fusion of T. reesei protoplasts are 90 V pulse voltage, 37 μS pulse duration and six pulses at intervals of 1-0 s.     

Alternatives to Trichoderma reesei in biofuel production

Mutant strains of Trichoderma reesei are considered indisputable champions in cellulase production among biomass-degrading fungi. So, it is not surprising that most R&D projects on bioethanol production from lignocellulosics have been based on using T. reesei cellulases. The present review focuses on whether any serious alternatives to T. reesei enzymes in cellulose hydrolysis exist. Although not widely accepted, more and more data have been accumulated that demonstrate that fungi belonging to the genera Penicillium, Acremonium and Chrysosporium might represent such alternatives because they are competitive to T. reesei on some important parameters, such as protein production level, cellulase hydrolytic performance per unit of activity or milligram of protein.     

Comparative analysis of optimal endoglucanase production by Trichoderma reesei and intergeneric fusants of Trichoderma reesei Saccharomyces cerevisiae

Intergeneric fusants of Trichoderma reesei QM 9414/Saccharomyces cerevisiae NCIM 3288 developed in the authors' laboratory can convert cellulosic materials directly to ethanol in a single step process. The production of endoglucanase in this case is a key factor. The production profile of this enzyme by the intergeneric fusants is different from Trichoderma reesei QM 9414 (WT). The production of endoglucanase was studied seperately by Trichoderma reesei (WT) using optimal production medium which was designed as per the combined screening approach of Plackett-Burman followed by a central composite experimental plan and the intergeneric fusants using optimal production medium obtained by Box-Behnken optimization procedure. Dried grass was used as the cellulosic substance whose concentration was kept constant during the statistical optimization procedure. The concentration of dried grass was later varied keeping the other optimized medium constituents constant to find the final optimum medium composition for endoglucanase production.     

Expression of Barley Endopeptidase B in Trichoderma reesei

The gene for barley endopeptidase B (EPB) has been expressed in the filamentous fungus Trichoderma reesei from the cbh1 promoter. The EPB signal sequence allowed secretion of over 90% of the recombinant protein. Yields reached about 500 mg of immunoreactive protein per liter and exceeded values for any other protein derived from a higher eukaryotic organism produced in T. reesei.     

Shear inactivation of cellulase of Trichoderma reesei

Inactivation of the cellulase of Trichoderma reesei (EC 3.2.1.4) by shear, is of sufficient magnitude to merit consideration in the design of equipment for the enzymatic hydrolysis of cellulose. The inac inactivation constant, k_d, is a function of the flow rate of the enzyme solution through a fine capillary tube. k_d increased slowly at low shear stress, and much more rapidly when the shear stress was greater than 15 dynes cm^?2.     

alpha,alpha-Trehalase of Trichoderma reesei.

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

Glucose-induced secretion of Trichoderma reesei xylanases.

To produce two xylanases with Trichoderma reesei grown on glucose, recombinant strains which carry either the xyn1 or the xyn2 (xylanase I and II [XYN I and XYN II]-encoding) structural genes under the expression signals of the homologous pki1 (pyruvate kinase-encoding) gene were constructed. The two types of transformants secreted XYN I or II, respectively, during growth on glucose, as demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunostaining. The corresponding specific xylanase activities of the best transformants on glucose were 76 and 145 U/mg of protein for XYN I and XYN II, respectively, as opposed to that obtained by the parent strain (26 U/mg of protein). When related to the amount of biomass formed, however, they produced only about 4 to 5 U/g, in contrast to much higher activities (10 to 12 U/g) during growth on xylan. The ultrastructural location of XYN II in the transformant strain producing the highest constitutive XYN II formation (ATX2-12) was investigated by immunoelectron microscopy and compared with that in the wild-type strain growing on xylan. Cell extracts from both types of transformants grown on glucose exhibited a higher intracellular xylanase activity than did the parent strain grown on xylan. By using electron microscopy and immunogold labelling, XYN II was detected in the endoplasmic reticulum, Golgi-like vesicles, secretory vesicles, vacuoles, and cell walls. The immunolabel in the vacuoles was detected preferentially in subapical cells. When a recombinant strain which expressed xyn2 from the pki1 promoter was compared with the parent strain during growth on xylan, the former exhibited a less proliferated endoplasmic reticulum and a smaller number of secretory vesicles; however, a higher density of labelling was observed. The relationship of these findings to the efficacy of protein secretion during growth on glucose is discussed.     

Glycosylation of acetylxylan esterase from Trichoderma reesei

The nature of the N- and O- linked glycosylation of acetylxylan esterase (AXE) of the Trichoderma reesei strain Rut-C30 has been characterized using different enzymatic, chromatographic, and mass spectrometric techniques. The combined data showed that the AXE N-glycan is phosphorylated and highly mannosylated. The predominant N-glycans on the single glycosylation site on AXE can be represented as GlcNAc(2)Man((1-6))P. The linker-substrate binding domain peptide separated from the core by papain digestion is heavily O-glycosylated and consists of mannose, galactose, and possibly glucose as monosaccharide and disaccharide substituents. In addition to glycosylation, sulfation was observed in the linker region. Both N- and O- linked glycans show remarkable heterogeneity. Three isoforms of AXE, separated by 2D SDS-PAGE, are described with pI values of 5.0, 5.3, and 5.9. The three isoforms can be explained by posttranslational modification of the enzyme by glycans, phosphate, and sulfate. Advancing the knowledge on the nature of the glycans produced by T. reesei is elementary for its use as a host for the expression of heterologous glycoproteins of industrial and pharmaceutical importance.     

Morphology of Trichoderma reesei QM 9414 in submerged cultures

The microscopic morphology of Trichoderma reesei QM 9414, growing in submerged culture, was studied by image analysis. The morphology was characterized by the total hyphal length, the total number of tips, the number of actively growing tips, and the length of the main hypha. To describe the growth of a single mycelium a simple model is set-up. The main features of the model are: (1) saturation type kinetics for the tip extension of the individual branches within the mycelium; and (2) random branching with a frequency function, which is proportional to the total hyphal length. The model is used to simulate a population of mycelia, where spore germination is described with a log-normal distribution. From the simulation of the population, the average properties of the mycelia, e.g., the average total hyphal length, are calculated, and by fitting the model to experimental data the model parameters are estimated. Finally, the distribution function with respect to the mycelia properties, that is, number of tips and total hyphal length, is calculated, and it corresponds well with the experimental determination of the distribution function. (c) 1995 John Wiley & Sons Inc.     

Cellular and extracellular localization of endocellulase in Trichoderma reesei

Abstract An endocellulase (1,4-β- d -glucan 4-glucanohydrolase, EC 3.2.1.4) was purified by preparative isoelectric focusing from culture fluids of Trichoderma reesei QM 9414 grown on cellulose. Its properties were studied by affinity titration curves and immunoelectrophoresis. FITC-labeled protein A-antibody was used to document its occurrence in cellulose and in fungal cell walls. Immunogold electron microscopy served to detect endocellulose sites within the outer exopolysaccharide layer of the fungal cell wall.     

Production of cellulases from alkali-treated bagasse in Trichoderma reesei

Summary Most of the mutants of Trichoderma reesei had good cellulase productivity on Avicel but this was low on alkali-treated bagasse, which could be a most promising cellulosic biomass to use as an inexpensive carbon source for cellulase production. Two T. reesei mutants, PC-3-7 and X-31, in which strong cellulase activity is inducible by l-sorbose, were, however, found to produce cellulase on alkali-treated bagasse. They produced about 100 units of CMCase per ml in 5-1 jar fermentor culture with 4% alkali-treated bagasse as carbon source. They also showed higher cellulase productivity than other mutants on other easily saccharified substrates, such as alkali-treated rice straw and Walseth's cellulose.Production of Ethanol from Biomasses Part IV.Production of Ethanol from Biomasses Part IV.     

Inductive formation of celluases by L-sorbose in Trichoderma reesei

Summary L-Sorbose, which is known as an inhibitor of -1,3-glucan synthesis in fungi, induces the production of cellulases in strains belonging to Trichoderma reesei. Especially, mutant strains PC-3–7 and X-31, which were obtained by several steps of mutation from QM 9414, have the most effective cellulase inducibility by L-sorbose comparing with other mutants of Trichoderma reesei. They synthesized cellulases effectively in liquid culture, whenever the alkaline treated sugarcane bagasse was used as a main carbon source for lowering the cost of cellulase production.