MXPA02002516A - Expression of proteins in genetically modified fungi. - Google Patents

Abstract

The present invention relates to increasing the production of a protein of interest from a fungal host. The invention discloses nucleotide sequences comprising, a regulatory region in operative association with a xylanase secretion sequence and a gene of interest. The gene of interest encodes a protein selected from a pharmaceutical, nutraceutical, industrial, animal feed, food additive and an enzyme. Preferably, the gene of interest encodes a cellulase, hemicellulase, a lignin degrading enzyme, pectinase, protease, or peroxidase. The present invention also relates to vectors and hosts comprising these nucleic acid sequences, and to methods for the production of a protein of interest.

Description

EXPRESSION OF PROTEINS IN GENETICALLY MODIFIED FUNGI Background of the Invention 1. Field of the Invention This invention relates to the genetic modification of fungi to intensify the production of a protein of interest. Additionally, this invention refers to new genetic constructions that dramatically increase the amount of protein produced by fungi contained in these constructions. 2. BACKGROUND OF RELATED ART The use of fungal expression systems for the production of proteins of interest is well known within the art. For example, heterologous proteins have been produced within fungal expression systems for the conversion of biomass, applications in detergents, cellulase substrates, and other enzymes in industrial enzymes. The production of other heterologous proteins of interest, such as food additives or supplements, pharmaceutical compounds, pharmaceutical compounds, antibodies, protein reagents and the like, and industrial proteins is also feasible within the fungal expression systems. Many microbes make enzymes that hydrolyze cellulose, including the wood decomposition fungus Trichoderma, the compost bacteria Thermomonospora, Bacillus, and Cellulomonas; Streptomyces; and the Humicola, Aspergillus and Fusariu fungi. The enzymes elaborated by these microbes are mixtures of proteins with three types of actions useful in the conversion of cellulose glucose: endoglucanases (EG), cellobiohydrolases (CBH), and β-glucosidase. The enzymes EG and CBH collectively refer to "cellulase". EG enzymes cut the cellulose polymer at random locations, opening it for attack by CBH enzymes. As an example, Trichoderma strains produce at least four different EG enzymes, known as EGI, EGII, EGIII, and EGV. The CBH enzymes sequentially release cellobiose molecules from the ends of the cellulose polymer. The cellobiose is the water soluble glucose-linked β-1, 4-dimer. There are two primary CBH enzymes within Trichoderma, CBHI and CBHII. The enzymes of B-glucosidase hydrolyze the c-l-obi sa to glucose. Trichoderma produces a ß-glucoside enzyme. This final step in the hydrolysis of cellulose that is catalyzed by ß-glucosidase is important, because glucose is easily fermented to ethanol by a variety of yeasts while cellobiose does not. Any cellobiose that remains at the end of the hydrolysis represents a loss of ethanol yield. More importantly, cellobiose is an extremely potent inhibitor of the CBH and EG enzymes. The cellobiose decreases the hydrolysis rate of the CBH and EG enzymes of Trichoderma by 50% at a concentration of only 3.3 g / L. The decrease in the rate of hydrolysis necessitates the addition of higher levels of cellulase enzymes, which have an adverse impact on the overall economy of the process. Therefore, the accumulation of cellobiose during hydrolysis is extremely undesirable for the production of ethanol. The accumulation of cellobiose has been a major problem in enzymatic hydrolysis because Trichoderma and the other cellulase-producing microbes make very little β-glucosidase. Less than 1% of the total protein made by Trichoderma is β-glucosidase. The low amount of β-glucosidase results in a shortage or deficit in the ability to hydrolyze cellobiose to glucose and an accumulation of 10 to 20 g / L of cellobiose during hydrolysis. This high level of cellobiose increases the amount of cellulose required by 10 times over that if an adequate amount of ß-glucosidase is present. Several approaches have been proposed to overcome the shortage of ß-glucosidase in the cellulase enzymes.

One approach has been to produce ß-glucosidase using microbes that produce little cellulose, and to add this ß-glucosidase exogenously to the cellulose enzyme to intensify the hydrolysis. The most successful of these ß-glucosidase producing microbes have been Aspergillus niger and Aspergillus phoenicis. The ß-glucosidase of these microbes is commercially available as Novozym 188 from Novo Nordisk. NeverthelessThe quantities required are too expensive for a commercial operation of biomass to ethanol. A second approach to overcome the shortage of ß-glucosidase is to carry out the hydrolysis of cellulose simultaneously with fermentation of glucose by yeast, the so-called simultaneous process of saccharification and fermentation (SSF). In an SSF system, the glucose fermentation removes it from the solution. Glucose is a potent inhibitor of β-glucosidase, so that SSF is an attempt to increase the efficiency of ß-glucas-id sja. However, SSF systems are not yet commercially viable because the operating temperature for the yeast of 28 ° C is too low for the 50 ° C conditions required by the cellulase; The operation at a compromise temperature of 3 ° C is inefficient and promotes microbial contamination. A third approach to overcome the shortage of ß-glucosidase is to use genetic management to overexpress the enzyme and increase its production by Trichoderma. This approach was taken by Barnett, Berka, and Fowler, in "Cloning and Amplification of the Gene Encoding an Extracellular B-glucosidase from Trichoderma Reesei: Evidence for Improved Rates of Saccharification of Cellulosic Substrates," Bio / Technology, Volume 9, June 1991, p. 562-567, hereinafter referred to as "Barnett, et al."; and Fowler, Barnett, and Shoemaker in WO 92/10581"Improved Saccharification of Celluiose by Cloning and Amplification of the B-glucosidase gene of Trichoderma reesei", referred to herein as "Fowler, et al." Both Barnett, et al. as Fowler, et al., describe the insertion of multiple copies of the ß-glucosidase gene in the P40 strain of Trichoderma reseei. Both groups constructed the plasmid pSAS ~ glu, a transformation vector containing the genomic gene - * 'of'; β-glucosidase from T. reesei and the amdS selectable marker. The amdS gene is from Aspergillus nidulans and codes for the enzyme acetamidase, which allows transformed cells to grow in acetamide as a single source of nitrogen. T. reesei does not contain a functional equivalent to the amdS gene and is therefore unable to use acetamide as a nitrogen source. Transfected cells contained 10 to 15 copies of the β-glucosidase gene and produced 5.5 times more β-glucosidase than non-transformed cells. The improved production of β-giucosidase obtained by Barnett, et al., And Fowler, et al., Is not sufficient to mitigate the shortage of β-glucosidase for the hydrolysis of cellulose. The amount of ß-glucosidase produced by natural strains of Trichoderma, for example, must be increased at least 10-fold to meet the requirements of cellulose hydrolysis. When proteins are overexpressed in Trichoderma, one strategy is to link the gene of interest directly to the cbhl promoter or the cbhl secretion signal. Since CBH1 is the most abundant protein produced by Trichoderma under cellulase induction conditions, the cbhl promoter and the secretion signal are thought to be very effective in the direction of secretion transcription of proteins encoded by a gene placed after a genetic construction. This strategy has been successfully used to overexpress Trichoderma proteins and other microorganisms (Margolles-Clark, Kayer, Karman and Penttila, 1996, "Improved Production of Trichoderma harzianum endochitinase by expression in Trichoderma reesei", Ap l. Environ. Microbiol. 6): 2145-2151; Joutsjouki, Torkkeli and Nevalainen, 1993, "Transíormation of Trichoderma reesei with the Hormoconis resinae glucoamylase P (gamP) gene: producton of a heterologus glucoamylase by Trichoderma reesei", Curr. Genet 24: 223-228 Arhunen, Mantyla, Nevalainen and Suominen, 1993, "High frequency one-step gene replacement in Trichoderma reesei 1. Endoglucanase I overproduction", Mol.Ge.Genet 241: 515-522). Another example of an industrial enzyme produced with fungal expression systems includes xylanase. Some of the most important commercial xylanases are classified as xylanases from Family 11. A xylanase enzyme is classified in Family 11 if it possesses the amino acids common to family 11, which include two glutamic acid residues that serve as the essential catalytic residues. These residues are amino acids 86 and 177 by the numbering of xylanase II of Trichoderma reesei. The amino acids common to xylanases from Family 11 are described in Wakarchuck, et al, Pro ein Science 3: 467-475 (1994). "The expression of pharmaceutically important heparologous proteins, for example, insulin (Goeddel D.V. et al., 1979, Proc. Nat. Acad. Sci. 76 106-110), and blood coagulation factor Xa (Smith D.B. 1988, Gene 67: 31-40), has been reported using bacterial expression systems. Similarly, the U.S. 4,751,180 describes the expression of a heterologous protein in yeast that includes insulin IgF-2. Heterologous production of bovine prochymosin has been reported using the filamentous fungus Trichoderma reesei (Harkki A. et al., 1989, Bio / Technol 7: 596-603), with the genetic construct comprising a regulatory region of cbhl (gene of cellobiohydrolase I) and terminator, either the chymosin or cbhl signal sequence, and optionally an intervening region obtained from cbhl. Margolles-Clark E. et al., (1996, App Environ Microbiol., 62: 2152-2155) describes the expression of endochitinase from T. Haraianum using the cbhl promoter of T. reesei. Similarly, the proteins of interest, for example chymosin, have also been produced in the filamentous fungi Aspergillus nidulans and A. awamori, using genetic constructs comprising the regulatory region and secretion signal of the glaA gene (glucoamylase) a signal sequence of either glaA or chymosin, and a region of glucoaminase intervention (EP 215,594). · In both of these latter cases, the transcription of the protein product within the host was not a limiting factor in the production of heterologous protein. However, secretion of the protein product from the host was low and resulted in poor extracellular recovery of the protein.In an attempt to increase the extracellular accumulation of heterologous protein production within a filamentous fungal expression system, Lav / lis (1997, US 5,679,543) describes the use of a multicomponent fusion polypeptide to enhance secretion and extracellular accumulation of a protein of interest. Genetic constructs were complexes that code for a fusion protein comprising four parts and include a signal peptide, a secreted polypeptide or portion thereof (a carrier protein), a cleavable linker polypeptide, and the desired polypeptide for which you want the expression. Increased levels of protein secretion were attributable to the use of a glaA signal sequence fused to full-length glucoamylase (the carrier protein) or another protein that secretes within the host, which was then fused to the cleavable linker and protein interest (chymosin). This construct was found to increase the secretion of the fusion polypeptide when expressed in the filamentous fungus A. nidulans. Although increased secretion was noted within. of US 5,679,543, using these four component fusion proteins, the production of the expression vectors is complex. This expression system requires the use of a six-part genetic construct that expresses a four-part protein product, complex with a variable and expressed carrier protein. In addition, approximately 50% of the desired, expressed product is not recovered since it comprises the carrier protein and this increases the costs associated with handling and purification after expression of the desired protein. Significant processing after secretion that includes ligand cleavage and acidification of the medium is also required for the recovery of the final, desired protein product. A simplified expression system that results in high levels of expression and secretion of a protein of interest from a host cell is required within the art. Preferably, the genetic constructs used within this expression system comprise few component parts, so that the chimeric construction is easy to prepare. In addition, the levels of expression and secretion using this genetic construct must be high and preferably, little or no downstream manipulation is required to collect the protein of interest. It is an object of the invention to overcome the disadvantages of the prior art. The above object is fulfilled by the feature combinations of the main claims, the subclaims disclosing additional advantageous embodiments of the invention.

Brief Description of the Invention The present invention relates to the genetic modification of fungi to intensify the production of a protein of interest. Furthermore, this invention relates to new genetic constructions that dramatically increase the amount of protein produced and secreted by fungi contained in these constructions. Genetic constructs of expression can be used to enhance the production of a protein of interest within fungal expression systems. Genetic constructs that accomplish this task comprise DNA sequences comprising one or more regulatory elements in operative association with a nucleotide sequence encoding a secretion secretion of xylanase, optionally an intervening region and a protein of interest. The present invention relates to a nucleotide sequence, comprising, a regulatory region in the operative association with a xylanase secretion sequence and a gene of interest, wherein at least one of the regulatory region, or gene of interest is not normally associated with the production of xylanase protein. One embodiment of this invention also relates to the nucleotide sequence defined above, wherein the regulatory region is selected from the group consisting of cbhl, cbh2, egl, eg2, eg3, egS, xlnl, and xln2. The present invention also relates to the nucleotide sequence as defined above, wherein the gene of interest is selected from a gene that codes for a protein selected from the group consisting of a pharmaceutical, nutraceutical, industrial, a food for animals, a food supplement or an enzyme. Preferably, the gene of interest codes for an enzyme selected from the group consisting of a β-glucosidase, cellulase, hemicellulase, a lignin degradation enzyme, protease, pectinase and peroxidase. The nucleotide sequence of the present invention as defined above can also compile an intervening sequence. This invention also relates to a vector comprising the nucleotide sequence as described above, and to a transformed filamentous fungus comprising this vector. This invention also relates to a filamentous fungus comprising the nucleotide sequence as defined above. Preferably, the transformed filamentous fungus is selected from the group consisting of Trichoderma, Humicola, Fusariu, Aspergillus, Botrytis, Mycogone, Verticillium, Streptomyces, Colletotrichum, Neurospora, Pleurotus, Penicillum, Cephalosporium, Myrothecium, Papulospora, Achlya, Podospora, Endothia, Mucor, Cochilobbolus, Tolypocladium, Pyricularia, Penicillium, Myceliophthora, Irpex, Stachybotrys, Scorpulariopsis, Chaetomium, Gilocladium, Cephalosporin and Acremonium. This invention encompasses a method for producing a protein of interest within a filamentous fungus comprising, transforming the filamentous water with a nucleic acid sequence comprising, a regulatory region in operative association with a xylanase secretion sequence and a gene of interest , wherein at least one of the regulatory region, or gene of interest is not normally associated with the production of the xylanase protein, and wherein the secretion sequence of _-xylanase is heterologous or homologous to the filamentous fungus, culturing the fungus filamentous, and cause the filamentous fungus to produce the protein of interest. Optionally, the protein of interest can be purified. This invention relates to a method for producing a protein of interest within a filamentous fungus comprising, transforming the filamentous fungus with a nucleic acid sequence comprising, a regulatory region in operative association with a xylanase secretion sequence, a sequence of intervention, and a gene of interest, wherein at least one of the regulatory region, or gene of interest is not normally associated with the production of xylanase protein, and wherein the sequence of xylanase secretion is heterologous or homologous with respect to To the filamentous fungus, grow the filamentous fungus and make the fungus produce the protein of interest. Optionally, the protein of interest can be purified. Additionally,. the amino acid sequence encoded by the intervening sequence can be removed from the protein of interest. The present invention also relates to a protein produced by the methods as described above. As described herein, the use of the xylanase secretion signal resulted in increased levels of expression and secretion of a protein range of interest than the use of secretory signals of the prior art, e.g. a secretion signal cbhl. Since xylanase comprises a much smaller proportion of the total protein produced by Trichoderma than does CBH1 (5% and 60%, respectively), it would be expected that the cbhl secretion signal will be more effective, however, this It is not the case. Additionally, the xylanase secretion signal enhanced protein production in various host expression systems. This brief description of the invention does not necessarily describe all the necessary features of the invention, but the invention may also reside in a sub-combination of the desired characteristics. Other aspects of the present invention will be better understood and the advantages thereof will be more apparent in view of the following detailed description of the preferred embodiments and the accompanying drawings.

Brief Description of the Drawings Figure 1: Restriction map of the pCBGl-TV vector and the amino acid sequence of the mature CBH1 / -glucosidase secretion signal binding (see Example 5) ^ ,, - | ·, Figure 2: Restriction map of the pXBGl-TV vector and the amino acid sequence of the mature xylanase secretion signal binding / ß-glucosidase (see Example 6). Figure 3: Restriction map of vector pC / XBG / Xbal) -TV and the amino acid sequence of mature xylanase secretion signal binding / ß-glucosidase (see Example 7). Figure 4: Southern blot of genomic DNA isolated from RutC30 and M2C38 strains of T. reesei and probed with a labeled DNA fragment comprising the xylanase promoter of M2C38 plus the secretion signal. Figure 5: Southern blot of genomic DNA isolated from RutC30 and M3C38 strains of T. reesei and probed with a labeled DNA fragment comprising the mature ß-glucosidase coding region of M2C38. Figure 6: Shows schematic maps of expression vectors eg2, pEG2-TV (Figure 6a) and pC / XREG2-TV (Figure 6b), which comprises the secretion signal xln2, as described in Example 23. Figure 7: Shows schematic maps of the expression vectors pCMAN-TV ( Figure 7a) of peanuts as described in Example 27. Figure 7b shows the map of pXMAN-TV, and Figure 7c shows the schematic map of pC / XMAN-TV (Figure 7c), both of which comprise the signal of secretion xln2. These last two vectors' are described in Example 28. Figure 8: Shows schematic maps of expression vectors comprising eg2 obtained from Rumicola insolens. Figure 8a shows pChHE2-TV, and Figure 8b shows pC / XhHE2-TV, which comprises the secretion signal xln2, both are described in Example 30.

Figure 9: Shows schematic maps of laccase I expression vectors (lccl). Figure Sa shows pCLl-TV, and Figure 9b shows pC / XLl-TV, which comprises the secretion signal xln2, as described in Example 32. Figure 10: Shows a schematic map of an expression vector comprising xylanase (pC / XHTX4-TV) as described in Example 34.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS This invention relates to genetic modification of fungi to enhance the production of a protein of interest. Furthermore, this invention relates to new genetic constructs that dramatically increase the amount of protein produced by fungi contained in these constructions. The following description is of a preferred embodiment, by way of example only and without limitation to the combination of features necessary to carry out the invention. The genetic modification of fungi as it is. describes in the present invention arises from the use of new genetic constructs used for the expression of a gene of interest. The gene of interest codes for a protein of interest that is expressed at high levels within the host, and additionally, it is secreted from the host. The preferred expression host is a filamentous fungus. Filamentous fungi are characterized by a vegetative mycelium that exhibits a cell wall comprised of complex polysaccharides that include chitin and cellulose. The vegetative growth typically continues through hypal lengthening. Examples of filamentous fungi that can be used within the present invention include, without limitation, Trichoderma, Humicola, Fusarium, Aspergillus, Botrytis, Mycogone, Verticilliu, Streptoces, Colletotrichum, Neurospora, Pleurotus, Penicillum, Cephalosporium, Myrothecium, Papulospora, Achlya, Podospora, Endothia, Mucor, Cochilobbolus, Tolypocladium, Pyricularia, Penicillium, Myceliophthora, Irpex, Stachybotrys, Scorpulariopsis, Chaetoum, Gilocladium, Cephalosporin and Acremonium. Methods for the transformation of filamentous fungi are known in the art (eg EP 870,835, U: S: 5,863,783, Cajeéis et al., Curr Genet., 1991, 20: 309-314, Lorito et al. "', 1993, Curr. Genet 24: 349-356; Goldman et al., 1990, Curr. Genet. 17: 169-174; Penttila, et al. 1987, Gene 6: 155-164; Yelton et al., 1984, Proc. Nati Acad. Sci. USA 81: 14701474; Bajar et al., 1991, Proc. Nati Acad. Sci. USA 88: 8202-8212; Hopwood et al., 1985, "Genetic Manipulation of Streptomyces: a laboratory manual", The John Innes Foundation, Norwich, UK; all of which are incorporated herein by reference). The genetic constructs of the present invention typically comprise a regulatory region, in operative association with a secretion signal, and a gene of interest, and other elements, for example marker terminator sequences, etc., which may be added as required. An intervening sequence between the secretion signal and a gene of interest can be used, if desired.

The Regulatory Region By "regulatory region" or "regulatory element" is meant a portion of nucleic acid typically but not always, in the 5 'direction of a gene, which is typically comprised of DNA. A regulatory element includes promoter elements, baseline promoter elements (nuclei), elements that are inducible in response to an external stimulus, elements that mediate promoter activity such as negative regulatory elements or transcription enhancers. The regulated element as used herein, also includes elements that are active after transcription, for example, regulatory elements that modulate gene expression such as transcription and transcription enhancers, translational repressors and transcription, activation sequences in the 5 'direction and determinants of mRNA instability. Several of these latter elements can be located next to the coding region. ?? the context of this description, the term "regulatory element" or "regulatory region" typically refers to a DNA sequence, usually but not always, in the direction (5 ') of the coding sequence of a structural gene, which controls the expression of the coding region by providing recognition of the A-polymerase and / or other factors required for transcription to start at a particular site. However, it is to be understood that other nucleotide sequences, for example enunciatively and without limitation those located 3 'of the sequence, may also contribute to the regulation of the expression of a coding region of interest. An example of a regulatory element that provides recognition-for RNA polymerase or other transcription factors to ensure initiation at a particular site is a promoter element. A promoter element comprises a basal promoter element, sensitive for the initiation of transcription, as well as other regulatory elements (as listed above) that modify gene expression. Regulatory elements, as used herein, include elements regulated by development, inducible and constitutive. A regulatory element that is regulated by development, or controls the differential expression of a gene under its control, is activated at specific times during the development of the host. An inducible regulatory element is one that is capable of directly or indirectly activating the transcription of one or more DNA sequences or genes in response to an inducer. In the absence of an inducer, DNA sequences or genes will not be transcribed. Typically, the protein factor, which binds specifically to an inducible regulatory element to activate the transcription, is present in an active form which is then converted directly or indirectly to the active form by the inducer. The inducer can be a chemical people such as a protein, metabolite or other chemical agent. A constitutive regulatory element directs the expression of a gene in a continuous manner throughout the length of the host. «*? >; - · - · | There are many suitable regulatory elements that have been cloned and characterized, and which can be used to drive the expression of a gene of interest within a fungal host as described herein. For example, that is not going to be considered limiting in any way, several genes and their associated regulatory regions are cloned: EGIII of T. reesei (Saloheimo M et al., 1988, Gene 63: 11-21), xylanase, xln2 ( Saarelainen et al., Mol. Gen. Genet 241: 497-503, 1993). The use of other regulatory regions for the expression of heterologous genes of interest is also known within the art. For example, the promoter pgk (phosphoglycerate kinase, Vanhanen et al., Gene 106: 129-133, 1991), the constitutive promoter pki obtained from T. reesei (Carmen LM et al. al., 19S9, Phytopathol., 89; 2554-261), the regulatory region obtained from Mucor Miehei carboxyl protease (US 5,679,543). gla. (amyloglucosidase) Cullen D. et al., 1987, Bio / Echnol. 5: 713-719), gdp (glyceraldehyde-3-phosphate dehydrogenase) obtained from Aspergillus (Deane et al., 1999, Enzyme and icrobial Tech, vol 24, pp. 419-424; Pentilla et al., 1987, Gene vol 61, pp. 155-164), tpiA from A. nidulans (McKinight GL et al., 1986, Cell 46: 143-147), alcA from Aspergillus (Lockington, RA, et al., 1986, Gene 33: 137 -149), emy < α-amylase) from A. oryzae (Christensen T. et al., 1988, Bio / Technol, 6: 1419-1422), Trl from T. reesei (Camels et al. (1991, Curr Genet, vol. 20, pp. 309-314), T. reesei cbhl (Harkki, A. et al., 1989, Bio / Technol 7: 596-603), the glucoamylase regulatory region of Aspergillus niger (Nunberg JH et al., 1984, Mol. Cell. Bio., 4: 2306-2315, Boel E. et al., 1984, EMBO J., 3: 1581-1585), fcrpC of A. de A. nidulans (Yelton M. et al., 1984, Proc. Nat. Acad.

Sci.81: 1470-1474), xlnl or xln2 (Torronen at al., 1992, Bio / Technol 10: 1461-1455) or amdS from A. nidulans (Hynes MJ, et al., 1983 Mol. Cell. Genet, 199: 37-45). The use of heterologous regulatory elements, including xlnA, phytase, ATP synthetase, subunit 9 (oliC), tpi (triose phosphate isomerase), adh (alcohol dehydrogenase) amy, glaA, lactase and gpd have also been described in the US 5,863,783. The practice of the present invention is not limited by the choice of the regulatory element used within the genetic construct. However, the preferred regulatory elements are cbhl, cbh2, egl, eg2, eg3, eg5, xlnl and xln2. The cbhl DNA sequence of Trichoderma reesei is deposited in the GenBank under Accession No. D8623. Those skilled in the art are aware that a natural regulatory element can be modified by replacement, substitution, addition or elimination of one or more nucleotides without changing its function. The practice of the invention encompasses and is not restricted by, these alterations to the promoter.

A Secretion Signal A "secretion signal", which can also be referred to as a "secretory sequence" is used to intensify the extracellular location of the protein of interest from the host. As described herein, the preferred secretion signal is a xylanase secretion signal obtained from xylanase. The xylanase secretion signal is the DNA sequence that codes for a xylanase secretion signal peptide. The xylanase secretion signal peptide (or secretory peptide) is the peptide sequence present in the amino terminus of the encoded xylanase enzyme that is subsequently removed during the export of the mature xylanase enzyme out of the host cell. The secretion signal may comprise a pro-peptide, a pre-peptide or both. In a preferred embodiment, the xylanase secretion signal comprises a xylanase secretion signal from a Family 11 xylanase gene. In a more preferred embodiment, the xylanase gene from Family 11 is a xylanase gene from Trichoderma. . In a still more preferred embodiment, the xylanase secretion signal acquires a xylanase secretion signal from the xylanase I gene. { xlnl) of Trichoderma or the xylanase II gene (xln2). The DNA sequences of the secretion signals xlnl and Xln2 of Trichoderma can be found in Figures 3 and 2, respectively, by Torronen, Mach, Messner, Gonzalez, Kalkkinen, Harkkinen and Kubicek, "The Two Major Xylanases from Trichoderma Reesei: Characterization of Both Entries and Genes, Bio / Technology 10: 1461-1465, 1992. in the legends of the figures, as published, are inverted.) Those skilled in the art are aware that a natural secretion signal can be modified by replacement, substitution, addition or elimination of one or more nucleic acids without changing their function The practice of the invention encompasses, and is not limited to, these alterations to the xylanase secretion signal.

Intervention signal Additional nucleotide sequences can be inserted within the region of intervention between the signal sequence and the gene of interest. These sequences can be introduced for a variety of purposes, for example, to increase the length of the guide polypeptide, to aid in the ease of insertion of the gene of interest within the genetic construct, to increase the protein expression level. of interest, to increase the export of the protein of interest from the host (for example US 5,679,543), or to aid in the purification of the protein of interest, for example, using affinity chromatography or other methods that are well known in the art. technique. The leader sequence may be of variable length, from several amino acids to an amino acid sequence that codes for a protein typically exported by the host. Additionally, the leader sequence may also code for amino acids that aid in the isolation of a protein of interest using a cleavable linker. The use of a cleavable linker may be desired if the region of intervention comprises a guide polypeptide that affects the activity of the protein of interest, comprises an affinity tag used for protein purification, or if the guide polypeptide is of considerable length . These cleavable linkers are well known in the art, for example, without limitation, the amino acid methionine, which is cleaved by cyanogen bromide, or known amino acid sequences that are cleaved by proteases, for example, without limitation. , trypsin, collagenase, clostripine, KEX2-yeast protease, factor Xa, subtilisin (eg Martson FAO 1986, Biol. Chem J. 240: 1-12). However, it will understand that. The gene constructs of the present invention may also lack these intervening sequences. The genetic constructs described in Examples 5-7, 23, 27, 28, 30 and 32 contain nine additional base pairs of the DNA sequence as shown in Figures 1-3, and 6-9; the first three encode for the glutamine residue after the secretion signal of the xylanase II gene of Trichoderma reesei and the remaining six result from the insertion and / or modification of unique restriction sites used to bind the xylanase secretion signal to the nucleotide sequence that codes for the protein of interest. These DNA sequences result in the presence of additional amino acids between the signal peptide of xylanase secretion and the protein of interest. These DNA sequences, which may be natural or synthetic, may encode one or more of the amino acids of the mature xylanase protein corresponding to the xylanase secretion signal encoded by the construct or may result from the addition of enzyme sites of restriction necessary to bind the xylanase secretion signal sequence to the gene of interest. The intervening sequence may also comprise nucleotides encoding amino acids cleaved by proteases, or leader polypeptides, as described above. The practice of the invention encompasses, without limitation, the presence of additional AD sequences between the signal of xylanase secretion and the mature ß-glucosidase coding region.

The Gene of Interest: By "gene of interest" is meant any gene that is to be expressed in a transformed cell. A gene of interest comprises the nucleic acid sequence encoding a protein of interest. A protein of interest may include, without limitation, a pharmaceutically active protein, for example growth factors, growth regulators, antibodies, antigens, its derivatives useful for immunization or vaccination and the like, interleukins, insulin, G-CSF, GM-CSF, hPG-CSF, M-CSF or combinations thereof, interferons, for example, interferon-a, interferon-β, interferon-t, blood coagulation factors, eg, factor VIII, factor IX or tPA or combinations thereof. A gene of interest can also code for an industrial enzyme, for example, for use within the production of pulp and paper, textile modification or ethanol. Eligibility of interest: In addition, you can code for a protein supplement, nutraceutical product, or a product of additional value for animal feed, food, or for use in food and food. Examples of these proteins include, without limitation, enzymes, proteases, oxidases, phytases, chitinases, mannanases, laccases, invertases, lipases, cellulases, hemicellulases, lignin degradation enzymes, pectinansas, xylanases, β-gi cosidases, peroxidases. , etc. Analogs of a protein of interest can also be expressed within the chimeric genetic constructs of the present invention. These analogs are typically characterized as having alterations to the amino acid sequence such as insertions, deletions or other variations such as allelic variations and the like. The present invention is further directed to chimeric gene constructs containing a DNA of interest operably linked to a regulatory element and secretory sequence of the present invention. Any gene of interest can be used and manipulated according to the present invention to result in the expression of the protein of interest.

Other Elements The genetic construct may contain -, a transcriptional terminator immediately in the 3 'direction of the nucleotide sequence coding for the protein of interest. Any suitable transcriptional terminator may be used as will be known to those skilled in the art. The practice of this invention is not limited by the choice of the transcriptional terminator. An example of a transcriptional terminator, which is not to be considered limiting in any way, is the transcriptional terminator in the 3 'direction of the gene of interest. Suitable terminators are readily available to one skilled in the art and can be obtained from at least the genes identified above (see "regulatory region"). A . The terminator, which is not to be considered limiting in any way, is described in Examples 5-7, 23, 27, 28, 30, 32 and 34, which comprises 1.9 kb of DNA 3"to the terminator codon of the cbh2 gene of richoderma The DNA sequence of the first 553 base pairs of the transcriptional terminator cbh.2 of Trichoderma reesei, which are located immediately in the 3 '(or 3') direction of the TAA terminator codon, is described (see Figure 2) Chen Gritzali and Stafford, "Nucleotide Sequence and Deduced Primary Structure of Cellobiohydrolase II from Trichoderma reesei", Bio / Technology 5: 274-278, 1987. However, it is to be understood that other terminator sequences, for example, without limitation and without limitation to those obtained from cbhl, xln2 as described in Examples 23, 27, and 28 (Figures 6a and 7a-c) The genetic construct contains a selectable marker which may be present at the 5 'direction or in the 3' direction of genetic construction. {ie, e n the 5 'or 3' end of the construct) in the same plasmid vector or can be co-transformed with the construct into a separate plasmid vector. Selectable marker choices are well known to those skilled in the art and include genes (synthetic or natural) that confer transformed cells the ability to utilize a metabolite that is not normally metabolized by the microbe (e.g., the amdS gene of A nidulans coding for acetamidase and conferring the ability to grow on acetamide as the sole source of nitrogen) or resistance to antibiotics (for example, the hph gene of Escherichia coli that codes for hygromycin ^ -tropheransferase and that confers resistance to hygromycin. If the host strain lacks a functional gene for the chosen marker, then that gene can be used as a marker Examples of these markers include trp, pyr4, pyrG, argB, leu, and the like The corresponding host strain therefore will have a lack of a functional gene that corresponds to the chosen marker, that is, trp, pyr, arg, leu and the like. e used in the genetic constructs described in Examples 5-7, 27 and 28 is the E. coli hph gene expressed from the phosphoglycerate kinase (pgk) promoter of Trichoderma. The use of pyr4 is described in Examples 23, 30 and 32 (Figures 6b, 8a, 8b, 9a and 9b).

The DNA sequence of the E. coli hph gene can be found in Figure 4 of Gritz and Davies, "Plasmid-encoded hygromycin B Resist: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae", Gene 25: 179-188, 1983; the DNA sequence of the pgk promoter of Trichoderma reesei can be found in Figure 2 of Vanhanen, Saloheimo, limen, Knowles and Penttila, "Promoter structure and expression of the 3-phophoglycerate kinase-encoding gene (pgkl) of Trichoderma reesei", Gene 106: 129-133 1991. Therefore, the genetic constructs of the present invention comprise, a regulatory region in operative association with a xylanase secretion sequence and a gene of interest. Preferably, at least one of the regulatory region, or gene of interest, is not normally associated with the production of xylanase protein. One embodiment of the invention comprises the genetic construct of β-glucosidase described in this way. The practice of the present invention is not limited by the method to elaborate the construction, which may include, without limitation, normal techniques of molecular biology such as isolation of plasmid DNA from E. coli by alkaline lysis, digestion of plasmid DNA with construction endonucleases, isolation of fragments from DNA by agarose gel electrophoresis, ligation of DNA fragments with T4-DNA ligase, insertion of unique restriction sites at the ends of the DNA fragments by the polymerase chain reaction or the addition of oligonucleotide linkers, and the formation of blunt ends of DNA fragments with T4-DNA-polymerase. or Klenow fragment of DNA polymerase I from E. coli. Examples 1-7 describe methods for making these genetic constructs. The present invention also describes the preparation of genetic constructs (expression vectors) comprising endoglucanase II from T. reesei and H. insolens, eg2 (Examples 4 and 30), mannanase, peanut, (Examples 27 and 28), laccase, Jccl , (Example 34) and xylanase (Example 38). The expression of several of these proteins of interest within T. reesei is described in Examples 25, 29, 31 and 33. The expression of a protein of Jjitér.és Humicola insolens is described in Example 38. In another embodiment of the present invention, the genetic construct encoding ß-glucosidase, endolugluconas II, mannanase, laccase or xylanase is introduced and expressed in a fungal host to create a genetically modified microbe. The resulting genetically modified microbe produces an increased level of the protein of interest relative to the untransformed microbial host; or when compared to the transformed, the host comprising a secretion signal that is endogenous to the gene that is expressed. For all the proteins of interest examined, and for all the hosts examined, the genetic or chimeric construct comprising the xylanase secretion signal resulted in amounts. increased of the protein of interest that is isolated from the host. For example, what is not to be considered limiting, the increased level of β-glucosidase preferably at least about 10-fold relative to the untransformed microbial host, more preferably at least about 40-fold relative to the microbial host untransformed and more preferably at least about 120 times relative to the untransformed microbial host were observed. Increased levels of expression were also observed with the. other proteins of interest (see examples 25, 29, 33 and 38 for · the expression of endoglucanase II, mannanase, laccase and xylanase respectively). This invention encompasses any method for introducing the genetic construct comprising the gene of interest in the microbial host familiar to those skilled in the art, including but not limited to, treatment with calcium chloride in bacterial cells or fungal protoplasts to weaken the cell membranes, addition of polyethylene glycol to allow the fusion of cell membranes, depolarization of cell membranes by electroporation, transformation mediated with Agrobacterium, firing of DNA through the cell wall and membranes via bombardment with microprojectiles with a particle bomb, etc. . Methods for the transformation of filamentous fungi have been reported (eg, EP 870,835, U.S. 5,863,783, Camels T. et al., Curr Genet, 1991, 20: 309-314, which is incorporated herein by reference). Examples 9, 20 and 35 describe the procedures for introducing the genetic construction of ß-glucosidase into spores of Trichoderma or Hu icola insolens using a particle gun. A 10-fold or greater improvement in β-glucosidase production relative to the untransformed microbial host reflects a significant improvement that is well above the natural variability of the strain and is commercially significant. -glucosidase by this method has been as high as 136 times and could reach more than 1000. The measurement of the degree of improvement of ß-glucosidase production is by growth of culture and measurement of the activity of β-glucosidase, as described in Example 11. It is understood by those skilled in the art that the specific activity of ß-glucosidase from a mixture of enzymes (in IU / mg protein) can be increased by decreasing the amount of cellulase and other proteins in the mixture of This can be done by physical and mechanical separation of the enzyme mixture or by deletion of the cellulase or other genes by recombinant means.These methods have little or no effect on the actual production of ß-glucosidase by the microorganism. These methods may optionally be included, however, in the practice of the present invention. Examples 25, 29 and 33 describe the overexpression of endoglucanase II, mannase, and laccase using the chimeric constructions of the present invention. In all cases, an increased level of intepés protein production was observed to vary from approximately 3 to 10 times or more, increase in activity with respect to that of the non-transformed host. Examples 30 and 31 describe the endoglucanase II production of Hu icola insolens within T. reesei, which also resulted in increased levels of endoglucanase II production. Examples 34-38 describe the production of a xylanase, obtained from T. reesei and its expression within Humicola insolens. These examples demonstrate that a non-native promoter and secretion signal are active in a heterologous filamentous fungus, and that these elements drive the expression of a gene of interest that is active within a heterologous host. In these examples, the gene of interest was obtained from Trichoderma and encoded for xylanase, fused to a xylanase secretion signal also obtained from Trichoderma. This nucleic acid sequence was placed under the control of the cbhl promoter that was also obtained from Trichoderma. The final construction was used to transform Humicola insolens. The activities of the gene of interest within transformed and non-transformed H. insolens were determined (see Example 38) and demonstrated that the cbhl promoter and the xylanase secretion signal obtained from Trichoderma are active in H. insolens. Additionally, the transformed host produces an increase of 2 to 2.5 times in the activity with respect. to the endogenous activity expressed within the host 'not transformed. The result of this experiment indicates that a non-native promoter and secretion signal obtained from a filamentous fungus is active in another filamentous fungus. The present invention is therefore directed to the expression of a gene of interest, and the production of a protein of interest with any filamentous fungus. For example, that is not going to be considered limiting in any way, the host can be selected from Trichoderma, Humicola, Fusarium, Aspergillus, Streptomyces, Colletotrichum, Neurospora, Pleurotus, Penicillu, Cephalosporium, Achlya, Podospora, Endothia, Mucor, Cochilobbolus, Tolypocladium, Pyricularia. The selection of the appropriate host may depend on which protein of interest will be produced. Methods for the introduction of DNA constructs into Trichoderma have been published (Lorito, Hayes, DiPietro and Harman, 1993, "Biolistic Transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA," Curr. Genet 24: 349-356; Goldman, Van ontagu and Herrera-Estrella 1990, "Transformation of Trichoderma harsianum by high-voltage electric pulse", Curr. Genert. 17: 169-174; Pen tila, Nevalainen, Ratto, Salminen and Knowles, 1987, "A versatiie transformation system for the cellulolytic fungus Trichoderma reesei", Gene 6: 155-164), Aspergillus ^ Y iton, Hamer and Timberlake, 1984, "Transformation of Aspergillus nidulans using a trpC plasmid, "Proc. Nati Acad. Sci. USA 81: 14701474), Fusarium (Bajar, Podila and Kolattukudy, 1991, "Identification of fungal cutinase promoter tha is inducible by a plant signal via phosphorylated transacting factor," Proc. Nati. Acad. Sci. USA 88: 8202-8212 ), Tolypocladium (Camels T. et al. Curr Genet., 1991, 20: 309-314). Additionally, EP 870,835 discloses a method for transforming the range of filamentous fungi, including Aspergillus, Colietotrichum, Fusarium, Ne rospora, Pleurotus and Trichoderma - using gene transfer mediated by Agrohacterium. In a preferred embodiment, the microbial host is Trichoderma. In a more preferred embodiment the microbial host is Trichoderma reesei. The genetic constructs used in these published transformation methods are similar to those described in Examples 5-7, 23, 27, 28, 30, 32 and 34 in that they contain a regulatory region in operative association with a protein coding region ( that can code for a selectable marker) and a transcriptional terminator. In most cases, the genetic constructs are linked to a selectable marker gene. In a preferred embodiment, the xylanase secretion signal is native to the microbial host from which the genetically modified microbe is derived (i.e., the source of the xylanase secretion signal is the same type of microbial host as the microbial host). from which the genetically modified microbe is derived). However, any signal of xylanase secretion can be used as described herein.

The protein of interest produced using the genetic methods and constructs as described herein can be used as a crude extract as obtained from the host, or the protein of interest can be purified either partially or extensively using methods that are well known within of the technique including centrifugation, precipitation with salt and pH, exclusion of size, affinity, ion chromatography, etc. Purification of the protein of interest can be improved by using a guide polypeptide comprising an affinity tag and the separation of the tagged protein of interest using a suitable affinity matrix. These affinity tags and their corresponding affinity matrices are well known in the art. In addition, a leader peptide encoded by an intervening sequence can be excised from the expressed protein of interest before, during or after processing of the protein of interest. The above description is not intended to limit the claimed invention in any way, furthermore, the analyzed combination of characteristics may not be absolutely necessary for the inventive solution. The present invention will be further illustrated in the following examples. However, it is to be understood that these examples are for illustrative purposes only and should not be used to limit the scope of the present invention, in any way.

EXAMPLES Example 1 describes the isolation of genomic DNA from the strains RutC30, 2C38, BTR48 of Trichoderma reesei and the genetically modified derivatives of these strains. Examples 2-7 describe the construction of genomic DNA libraries, the cloning of several genes and various genetic constructs of the M2C38 strain of Trichoderma reesei. Examples 9 and 11-15 describe the transformation and expression of the ß-glucosidase genetic constructs in the strains M2C38, BTR48, and RutC30 of Trichoderma reesei. Strains M2C38 and BTR48 of Trichoderma reesei are patented strains of Iogen Corporation and are derived from RutC30 Trichoderma reesei (ATCC 56765, Montenecourt and Eveleigh, 1979, "Selective isolation of high yielding cellulase mutants of T. reesei", Adv. Chem. Ser . £ 1: 28.9-301), which in turn was derived from Qm6A of Trichoderma reesei (ATCC 13631 Mandéis and Reese, 1957"Induction of cellulose in Trichoderma viride as influenced by carbon sources and · -metáis", J Bacterial 73: 269-278). In Example 1 and the subsequent Examples, the restriction endonucleases, T4-DNA polymerase, T4-DNA ligase and Klenow fragment of E. Coli DNA polymerase I were purchased from Gibco / BRL, New England Biolabs, Boehringer Mannheim or Pharmacia and was used as recommended by the manufacturer. Pwo polymerase with reading-proof activity (Boehringer Mannheim) was used in all polymerase chain reactions (PCR) according to the manufacturer's protocol. Hygromycin B from CalBiochem was purchased.

EXAMPLE 1 Isolation of Genomic DNA from Trichoderma reesei To isolate genomic DNA, 50 ml of Potato Dextrose Broth (Different) was inoculated with T. reesei spores collected from a Potato Dextrose Agar plate with a sterile inoculation loop. The cultures were shaken at 200 rpm for 2-3 days at 28 ° C. The mycelia were filtered on a sterile GFA glass microfiber filter (Whatman) and washed with cold deionized water. The fungal cakes were frozen in liquid nitrogen ^ - and - crushed to a powder with a pre-cooled mortar! and pistil; 0.5 g of powdered biomass was dispersed in 5 ml of 100 M Tris, 50 mM EDTA, pH 7.5 plus 1% sodium dodecyl sulfate (SDS). The lysate was centrifuged - (5000 g for 20 minutes, 4 ° C) to pellet the cellular debris. The supernatant was extracted with a volume of saturated phenol in buffer (10 mM Tris, 1 mM STDA, 8.0) followed by extraction with a volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) saturated in buffer in order to remove soluble proteins. DNA was precipitated from the solution by adding 0.1 volume of 3M sodium acetate, pH 5.2 and 2.5 volumes of 95% cold ethanol. After incubation for at least 1 hour at -20 ° C, the DNA was pelleted by centrifugation (5000 g for 23 minutes, 4 ° C), rinsed with 10 ml of 70% ethanol, dried with air and redispersed in 1 ml of 10 mM Tris, 1 mM EDTA, pH 8.0. The RNA was digested by the addition of Ribonuclease A (Boehringer annheim) added to a final concentration of 0.1 mg / ml and incubation at 37 ° C for 1 hour. Sequential extractions with a volume of saturated phenol in buffer and a volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) saturated in buffer were used to remove the ribonuclease from the DNA solution. The DNA was precipitated again with 0.1 volume of 3M sodium acetate, pH 5.2 and 2.5 volumes · of 95% cold ethanol, sedimented by centrifugation, rinsed with 70% ethanol, dried with air and redispersed in 50 μ? of 10 mM Tris, 1 mM EDTA, pH 8.0. The concentration of DNA was determined by measuring the absorbance of the solution at 260 nm (p.Cl in Sambrook, Fritsch and Maniatis, "Molecular Cloning: A Laboratory manual, Second Edition", Cold Spring Harbor Press 1989, hereinafter referred to herein). as Sambrook et al.).

Example 2 Construction of T. reesei Genomic Libraries Two plasmid libraries and a phage library were constructed using the genomic DNA isolated from the M2C38 strain of r. reesei Plasmid libraries were constructed in the vector pUC119 (Viera and Messing, "Insolation of single-stranded plasmid DNA", Methods Enzymol, 153: 3, 1987) as follows: 10 μg of genomic DNA were digested for 20 hours at 37 ° C in a volume of 100 μ? with 2 units / μg of restriction enzymes HindIII, BamHI or EcoRI. The digested DNA was fractionated on a 0.75% agarose gel run in 0.04 Tris-acetate, 1 mM EDTA, and stained with ethidium bromide. Slices of gel corresponding to the sizes of the gene of interest (based on published information and Southern blotting) were excised and subjected to electroelution to rgc. Acquire DNA fragments (Sambrook et al., Pp. 6.28-6.29). These DNA-enriched fractions were ligated into püC119 in order to create gene libraries in the ligation reactions containing 20-20 μg ml of DNA in a 2: 1 molar ratio of vecto: insert, DNA, ImM ATP and 5 units of T4 -ADN-ligase in a total volume of 10-15 at 4 ° C for 16 hours. The HB101 strain of Escherichia coli was electrophoresed with ligation reactions using the Cell Proator System (Gibco / BRL) following the manufacturer's protocol and the transorinants were selected on LB agar containing 70 μg ml of ampicillin. The phage library was constructed in the vector? -DASH (Stratagene, Inc.) as follows: Genomic DNA (3 μg) was digested with 2, 1, 0.5 and 0.2 units ^ g of Bam HI for 1 hour at 37 ° C to generate fragments of 9-23 kB in size. The DNA of each digestion was purified by extraction with a volume of phenol: chloroform: isoamyl alcohol saturated with Tris (25: 24: 1) followed by precipitation with 10 μ? of 3 M sodium acetate, pH 5.2 and 250 μ? of 95% ethanol (-20 ° C). The digested DNA was pelleted by microcentrifugation, rinsed with 0.5 ml of cold 70% ethanol, dried with air and redispersed in 10 μ? of deionized water, sterile. The enrichment of DNA fragments of 9-23 kB in size was confirmed by agarose gel electrophoresis (0.8% agarose in Tris-acetate ^ O ... Q4 - .. M, EDTA lmM). The digested DNA (0.4 μg) was ligated to 1 μg of β-DASH arms predigested with BamHI ('Stratagene) in a reaction containing 2 units of T4-DNA-ligase and lmM ATP in a total volume of 5 μm. at 4 ° C during the night. The ligation mixture was packed into phage particles using GigaPack ® II Gold packaging extracts following the protocol of. maker . The library was titled using the XLl-Blue strain MRA (P2) host of E. coli and found to contain 3 x 105 independent clones.

Example 3 Isolation of T. reesei M2C38 clones from the cellobiohydrolase I (cbhl), cellobiohydrolase II (cbh2) and B-qlucosidase (bqll) genes from the PUC119 libraries. HB101 transformants of E. coli having clones cbhl, cbh2 or ball of recombinant libraries pUC119-Hind III, -BamHI or EcoRI were identified by colony-raising hybridization: 1-3 x 104 were transferred into HyBond nylon membranes ™ (Amersham) the colony-side membranes were placed upward on the transfer paper (VWR 238) saturated with 0.5 M NaOH, 1 M NaCl for 5 minutes to lyse the bacterial cells and denature the DNA; the membranes were then neutralized by placing the colony side up on the transfer paper - (VWR 238) saturated with 1.5 M Tris, pH 7.5 plus NaCl 1 for 5 minutes. The membranes were allowed to air dry for 30 minutes and the DNA was then fixed to the membranes by baking at 80 ° C for 2 hours. Probes labeled with 3P were prepared by PCR amplification of short fragments (0.7-1.5 kB) of the bgll, cbhl and cbh2 coding regions of the enriched mixture of the? Hind III, BamHI or EcoRI fragments, respectively, in a reaction of labeling containing 10-50 ng of target DNA, 0.2 mM each d (GCT) TP, dATP 0.5 μ ?, 20-40 ?? of alpha-32P-dATP, 10 pmol of oligonucleotide primers and 0.5 units of Taq-polymerase in a total volume of 20 μ? . The reaction was subjected to 6-7 cycles of amplification (95 ° C, 2 minutes 56 ° C, 1.5 minutes 70 ° C, 5 minutes). The amplified 32 P labeled DNA was precipitated by the addition of 0.5 ml of 10% trichloroacetic acid (w / v) and 0.5 mg of yeast tRNA. The DNA was pelleted by microcentrifugation, washed twice with 1 ml of 70% ethanol, dried with air and redispersed in 1 M Tris, pH 7.5, 1 mM EDTA. The nylon membranes in which the recombinant pUC119 plasmids were fixed were prehybridized in heat-sealed pouches for 1 hour at 60-65 ° C in 1 M NaCl, 1% SDS, 50 mM Tris, 1 mM EDTA, pH 7.5 and 100 μg / ml salmon sperm DNA, sheared, denatured. Hybridizations were performed in heat-sealed bags in the same buffer with only 50 μg ml of denatured, sheared salmon sperm DNA and 5 x 10 '- 5 x 10' cpm of the bgll, cbhl or cbh2 denatured probe for 16- 20 hours at 60-65 ° C. The membranes were washed once for 15 minutes with 1 M NaCl, 0.5% SDA at 60 ° C, twice for 15 minutes each with 0.3 M NaCl, 0.5% SDS at 60 ° C and once for 15 minutes with NaCl 0.03M, 0.5% SDS at 55 ° C. The membranes were again placed in heat-sealed bags and exposed to an X-ray film of Kodak RP for 16-48 hours at -70 ° C. The X-ray film was revealed following the manufacturer's protocols. Colonies that give strong or weak signals are harvested and cultured in 2xYT medium supplemented with 70 μg / ml ampicillin. Plasmid DNA was isolated from these cultures using the alkaline lysis method (Sambrook et al., Pp. 1.25-1.28) and analyzed by restriction digestion, Southern hybridization (Sambrook et al., Pp. 9.38-9.44) and PCR analysis (Sambrook et al., pp. 14.18-14.19). Clones having the bgl gene were identified by colony-uptake hybridization from the pUC119-Hind III library (Example 2) with a 1.0 kb bgll probe prepared using oligonucleotide primers designed to amplify pb 462-140 ^ 3., - e -.the published sequence of bgl (Barrett et al.). A cali clone, pJEN200, was isolated containing a 6.0 kb Hind III fragment corresponding to the promoter, structural gene and termination sequence. Clones having the cbhl gene will be identified by colony survey hybridization of the pUC119-BamHI library with a 0.7 kb cbhl probe using oligonucleotide primers designed to amplify bp 597-1361 of the cbhl sequence (Shoemaker, Schweikart, Ladner, Gelfand, Kwok, Myambo and Innis, "Molecular cloning of exo-cellobiohydrolyase I derived from Trichoderma reesei strain L27", Bio / Technology 1: 591-596, 1983, hereinafter referred to as Shoemaker et al). A clone of cbhl, pCOR132, was isolated containing a BamHI fragment of 5.7 kb corresponding to the promoter (4.7 kb) and 1 kb of the structural gene cbhl. From this, an EcoRI fragment of 2.5 kb containing the cbhl promoter (2.1 kb) and the 5 'end of the coding region of cbhl (0.4 3b) was subcloned into pUC119 to generate pCBl52. Clones having the cbh2 gene were identified by colony-collation hybridization from the püC119-EcoRI library with a 1.5 kb cbh2 probe prepared using the oligonucleotide primers designed to amplify pb 580-2114 of the published cbh2 sequence (Chen, Gritzali and Stafford, "Nucleotide sequence and deduced primary structure of cellobiohydrolase II from Trichoderma reesei.", Bio / Technology 5: 274-278, 1987, referred to herein as Chen et al.). A clone cbh2, pZUK600, was isolated containing an EcoRI fragment of 4.8 kb corresponding to the promoter (600 bp), structural gene (2.3 kb) and terminator (1.9 kbp).

Example 4 Cloning of cbhl terminator, M2C38 of T. reesei, xylanase II gene (xln2), phosphoglycerate-kinase promoter (pqJc Probes labeled with Digoxigen-ll-düTP were prepared from PCR-amplified coding regions of the cbhl, xln2 and pgk genes by random primer labeling using the DIG labeling and detection equipment (Boehringer Mannheim) and following the manufacturer's protocols . Genomic clones containing the cbhl, xln2 and pgk genes were identified by plaque-library hybridization? -DASH. For each gene of interest, IxlO1 clones were transferred to Nytran naylon membranes (Schleicher and Schull). The phage particles were lysed and the phage DNA was denatured by placing the side of the membrane plate upward on the transfer paper (VWR238) saturated with 0.5 M NaOH, 1 M NaCl durgide-, 5 minutes; the membranes were neutralized by placing the side of the plate upwards in the transfer paper (VWR238) saturated with 1.5 M Tris, pH 7.5 plus 1 M NaCl for 5 minutes, the membranes were allowed to air dry for 5 minutes and the DNA then it was fixed to the membranes by cooking at 80 ° C for 2 hours. The membranes were prehybridized in heat sealed pouches in a solution of 6X SSPE, Denhard 5X, 1% SDS plus 100 .ug / ml sheared salmon sperm DNA, denatured at 65 ° C for 2 hours. The membranes were then hybridized in sealed bags with heat in the same solution containing 50 μg ml of salmon sperm DNA, sheared, denatured and 0.5 μg of probes labeled with digoxigen-dUTP at 65 ° C overnight. The membranes were washed twice for 15 minutes in 2X SSPE, 0.1% SDS at room temperature, twice for 15 minutes in 0.2X SSPE, 0.1% SDS at 65 ° C and once for 5 minutes in 2X SSPE. Clones that positively hybridized were identified by reaction with an anti-digoxigenin / alkaline phosphatase antibody conjugate, 5-bromo-4-cioro-3-indole-phosphate and 4-nitro blue tetrazolium chloride (Boehringer Mannheim) following the protocol of maker. Clones that positively hybridized were further purified by a second round of stoppage with the probes labeled with digoxigen-dl £ P · - The individual clones were isolated and the phage DNA = purified as described in Sambrook, et al. (1989) pp. 2.118-2.121 with the exception that the CsCl gradient step is replaced by extraction with a volume of phenol: chloroform: isoamyl alcohol (25: 24: 1) and a volume of chloroform: isoamyl alcohol (24: 1). The DNA was precipitated with 0.1 volume of 3M sodium acetate and pH 5.2 and 2.5 volumes of 95% cold ethanol. The precipitated phage DNA was washed with 0.5 ml of 70% cold ethanol, dried with air and redispersed in 50 μ? of tris 10 mM, SDTA 1 mM, pH 8.0. The restriction fragments they contain. genes of interest were identified by restriction digests of purified phage DNA and Southern blot hybridization (Sambrook et al., pp. 9.38-9.44) using the same digoxigen-düTP labeled probes used to detect the? -DASH library. The membranes were hybridized and the fragments that positively hybridized were visualized by the same methods used for plaque surveys. Once the desired restriction fragments of each clone? -DASK were identified, restriction digestions were repeated, the fragments were resolved on a 0.8% agarose gel in TAE and the desired bands were excised. The DNA was eluted from the gel slices using the Sephaglas BandPrep (Pharmacia) following equipment - the manufacturer's protocol. Clones having the cbhl gene were identified by library colony hybridization? -DASH (example 2) with a cbhl probe comprising pb 45-2220 of the published cbhl sequence (Shoemaker et al.). A 1.8 kb Ba HI fragment containing the 3 'end of the cbhl coding region (0.5 kb) and the cbhl terminator (1.3 kb) was isolated by restriction digestion of purified phage DNA from a clone? cbhl. This fragment was subcloned into the BamH1 site of the pUC119 plasmid vector of E. coli to generate plasmid pCBITa. Clones that have the xln2 gene were identified by library colony hybridization? -DASH (example 2) with a xln2 probe comprising pb 100-783 of the published xln2 sequence (Saarelainen, Paloheimo, Fagerstrom, Suominen and Nevalainen, "Cloning, sequencing and enhanced expression of the Trichoder to reesei endoxylanase II (pl9) gene xln2" Mol. Gen. Gene. 241: 497-503, - 1993, hereinafter referred to by Saarelainen et al.) - A 5.7 kb kpbl fragment containing the promoter (2.3 kb), the coding region (0.8 kb) and the terminator (2.6 kb), the xln2 gene was isolated by restriction digestion of purified phage DNA from a? -DASH xln2 clone. This fragment was subcloned into the Kpnl site of pUC119 to generate the plasmid pXY 2K-2. Clones having the pgk 'gene were identified by library colony hybridization? -DASH (Example 2) with a pgkl probe comprising 4-1586 of the published pgk sequence (Vanhanen, Penttila, Lehtovaara and Knowles, " Isolation and characterization of the 3phosohoglycerate kinase gene { Pgk) from the filamentous fungus Trichoderma reesei ", Curr.

Genet 15: 181-186, 1989). A 5.0 kb EcoRI fragment containing the promoter (2.9 kb), the coding region (1.6 kb) and the terminator (0.5 kb) of the pgk gene was isolated by restriction digestion of purified phage phage clone DNA. -DASH. This fragment was subcloned into the EcoRI site of pUC119 to generate plasmid pGK 5.0.

Example 5 Construction of pCBGl-TV vector of ß-crlucosidase overexpression This Example describes the construction of a vector containing the cellobiohydrolase I promoter from Trichoderma and the secretion signal and the mature ß-glucosidase coding region. A DNA fragment containing the bgll coding region minus the B-glucosidase secretion signal (bp 474-2679) was amplified by PCR from the template pJEN200 using homologous primers to the published bgll sequence containing either a 5 'sphl site to Val32 of encoded B-glucosidase or a 3' Kpnl site to the bgl leader terminator using Pwo polymerase. This amplified fragment was digested with Sphl and kpnl and inserted into pCB219N digested with Sphl and Kpnl to generate pBgstrf. To make pC3219N, a cbh2 terminator fragment of template pZUK600 was amplified using a primer homologous to pb 2226-2242 of the published 3 'untranslated region of the cbh2 gene (Chen et al., 19S7) containing a KpnJ site at the end 5 'and the püC forward primer (Cat. No. 1224, New England Biolabs) which are downstream of the EcoRI site at the 3' end of cbh2 at ZUK600. This fragment was digested at the managed Kpnl and EcoRI sites, and inserted into the corresponding sites of püC119 to generate pCB219. An EcoRI-NoCI adapter (Catalog No. 35310-010, Gibco / BRL) was inserted into the unique EcoRI site of pCB219 to generate pCB219N. A fragment containing the cbhl promoter and the secretion signal was amplified from pCB152 using a cbhl-specific primer (pb 249-284 of the published cbhl sequence, Shoemaker et al., 1983) containing a 3 'Sphl site to Serl9 from the CBHl encoded and the forward primer pUC (Catalogo No. 1224, New England Biolabs) which is fixed in the 5 'direction of the EcoRI site at the 5' end of cbhl in pCBl52. The product of __ ^ GR of promoter cbhl + secretion signal was digested with Sphl and EcoRI and inserted into the corresponding sites in PBR322L (a derivative of pBR322 in which the region between the Sphl and Salí sites was replaced with the Sphl linker -Notl-Sall) to generate pBR322LCS. To make the expression cartridge, the bgll coding region and the cbh2 terminator were isolated from pBgstrf as a 4.1 kb Sphl / Notl fragment and inserted into pB 322LCS digested with Sphl and Notl. In order to maintain the correct reading frame at the junction of the cbhl secretion signal and the mature 3-glocosidase, the resulting plasmid, pCBGstrf, was linearized at the unique Sphl site and the Sphl site became blunt ended with T4 DNA polymerase The resulting plasmid, pCBGl, was then further modified by conversion of the unique Notl site at the 3 'end of the cbh2 terminator to a unique Xhol site by the addition of the Xhol linkers (Catalog No. 1073, New England Biolabs). The final plasmid, pCBGI-Xho, is the plasmid of the expression cartridge. The hygromycin-phosphotransferase gene (hph) of E. coli used as a selectable marker for T. reesei was amplified by Pwo polymerase of the plasmid pVUlO05 (Van den Wizen, Townsend, Lee and Bedbrok, "A chimaeric hygromycin resistance gene as a selectable marker in plan cells ", Plant Mol. Biol. 5: 299-302, 1989). The primers -, were designed to introduce Sphl and Kpnl sites at the 5 'and 3' ends of the hph coding region (pb 211-1236 of the hph sequence, published, Gritz and Davies, "Plasmid-encoded hygromicin b resistance: the sequence of hygromycin B phosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae "Gene 25: 179-188, 1983), respectively. The PCR product was digested with Sphl and Kpnl and inserted into the corresponding sites in the polylinker region of püC119. The resulting plasmid, pHPTlOO was used as the starting plasmid for the construction of the selection cartridge. Two linker regions were introduced into this plasmid to facilitate cloning of the promoter and terminator fragments. A HindIII-Xbal-Xhol-Sphl linker was inserted between the HindIII and Sphl sites as well as a Kpnl-Notl-SacI linker that was inserted between the Kpnl and SacI sites of the pUC119 polylinker which remains at pHPTlOO. This construction was designated as pHTPl02. The primers used to amplify the pgk promoter (Vanhanen, Sloheimo, limen, Knowles and Penttila, "Promoter structure and expression of the 3-phosphoglycerate kinase gene (pgkl) of Trichoderma reesei", Gene 106 129-133, 1991.}. were digested to introduce an Xhol site and a Sphl site at positions -970 and +1 of the promoter, respectively.These sites were subsequently used to insert the pgk jen promoter ·· the Xhol and Sphl sites of pHPTl02 to generate pHPTÍ15. cbhl of 1.3 kb was amplified with Pwo polymerase from pCBITa using a primer that binds to the 3 'untranslated region of cbhl (bp 1864-1899 of the published cbhl sequence) containing a Kpnl site at pbl877-1882 and the inverted primer pUC (Cat. No., 18432-013, Gibco / BRL) which are fixed in the 3"direction of the EcoRI site at the 3 'end of the cbhl terminator in pCBITa The PCR product of the cbhl terminator was digested with Kpnl and it was inserted into the unique Kpnl site of pHPTllS pair to generate plasmid pHPTl36 from the selection cartridge. To make the transformation vector, the expression cartridge of pCBGl-Xho was isolated as an Xbal / Xhol fragment of 5.6 Kb and inserted between the unique Xbal and Xhol sites in the 5 'direction of the pHPT136 selection cartridge. The final transformation vector, pCBGl-TV, as depicted in Figure 1, was introduced as a circular plasmid in M2C38 of T. reesei via bombardment of microprojectiles as described later in Example 9.

Example 6 Construction of the β-qlucosidase overexpression pXBGl-TV vector This example describes the · μ? vector containing the Trichoderma 'promoter and selection signal, and the mature ß-glucosidase coding region. The ß-glucosidase coding region (bp 474-2680) was amplified with Pwo polymerase from clone pJE 200 of genomic bgll using primer to insert an Xbal site directly upstream of bp 474 in the published bgll sequence (Barrett, et al. ) and a Kpnl site directly in the 3 'direction of bp 2680. The blunt end PCR product was inserted into the Smal site of pUC118 to generate the plasmid designated pBGm.s. Since the Xbal site was driven to be immediately in the 5 'direction of the onset of mature β-glucosidase in Vai32, the cloned fragment did not include the β-glucosidase secretion signal. The plasmid pBGm.s was digested with Xbal and Kpnl and the 2.2 kb fragment containing the bgll coding region minus the secretion signal was isolated and inserted into the Xbal and Kpnl sites in the 5 'direction of the cbh2 terminator in the plasmid pCB219N (described in Example 5, above) to produce the plasmid pBG2X. A 2.3 kb fragment containing the promoter and secretion signal of the xln2 gene (pb 2150 to +99 where +1 indicates the start codon of ATG) was amplified with Pwo polymerase from subclone pXYN2K-2 of genomic xln2 using a specific primer of xln2 containing a Nhel site directly in the 3 'direction of pbl03 of the published xln2 sequence (Saarelainen et al.) and the inverted pUC primer (Catalog No. 18432-013, Gibco / BRL) which is set at address 5"from the kpnl site at the 5 'end of the xln2 gene This PCR product from xln2 was digested with EcoRI (which was amplified as part of the pUC119 polylinker of pXYN2K-2i and Nhel and inserted into the plasmid pBR322L (described in the example 5 above) to generate pBR322LXN The EcoRI site of pBR322LXN was then blunt-ended with Klenow, and Spel linkers (Catalog No. 1086, New England Biolabs) were added to generate pBR322pXN Plasmid p3G2X was cut with Kbal and Notl and a 4.2 kb fragment was isolated, which it contains the coding region bgll followed by the terminator cbh2. This fragment was inserted into the pBR322SpXN plasmid cut with NheJ and NotJ (AZ eJ and Xbal have compatible overhangs). This cloning resulted in the fusion of the xylanase secretion signal directly to the mature β-glucosidase creating the complete expression cartridge pXBG-2. The cbhl terminator in the plasmid pHPT * 136 of the selection cartridge described in Example 5 above was replaced with a 2.6 kb Kpnl fragment having the transcriptional terminator xln2. The xln2 terminator was amplified with Pwo polymerase from the genomic subclone pXYN2K-2 using a primer to introduce a ^ kpnl site directly in the 3 'direction of pb 780 of the published xln2 sequence (Saarelainen et al.) And the forward primer pUC Catalog No 18431-015, Gibco / BRL) which is fixed in the 3 'direction of the 3' end of the xln2 gene in pXY 2K-2. The PCR product of the xln2 terminator was digested with Kpnl and ligated to a 1.5 kb Kpnl fragment from pHPT136 containing the hph gene promoted with pgk in pUC119 to generate plasmid pHPTl36X from the selection cartridge. The construction of the transformation vector comprised the insertion of the expression cartridge directly in the 5 'direction of the pgk promoter of the selection cartridge. Plasmid pXBG2 of expression cartridge was digested with Notl, and the ends blunted using Klenow and then digested with Spel. The pHPTl36X selection cartridge was prepared in a similar manner by Xhol digestion, followed by the filling in reaction to create the blunt ends and then a digestion with Xbal. A blunt-bar linkage of these two fragments was performed to ligate the 6.5 kb Spel / NocI blunt fragment from pXBG2 to the Xbal / Xhol blunt fragment of pHPT136X (Spel and Xbal have compatible overhangs). The final transformation vector, pX3G-TV, as depicted in Figure 2, was linearized in its unique No before transformation of T. reesei M2C38 via microprojectile bombardment, as described later in Example 9.

Example 7 Construction of pC / XBG vector (Xbal) -TV of ß-glucosidase overexpression This Example describes the construction of a vector containing the cellobiohydrolase I promoter of Trichoderma, the secretion signal of xylanase II and the secretion region of mature ß-glucosidase. This Example was carried out to test the combined effects of the cbhl promoter and the xln2 secretion signal on the expression of bgl. A 1.2 kb HindIII fragment comprising pb -1399 to -204 of the cbhl promoter was amplified by PCR using plasmid pBR322LCS containing cbhl promoter (Example 5) as a template in order to insert a unique Xbal site in bp -1393 to 1388. This modified cbhl promoter fragment was digested with HindIII and used to replace pb-1400 to -121 of the xln2 promoter in pXBGl (Example 6) to generate the new pC / XBG1 plasmid of expression cartridge. The 6.4 kb expression cartridge from pC / XBGl was isolated by digestion with Notl followed by formation of blunt ends of the Notl site with Klenow fragment and subsequent digestion with SpeJ. This fragment was then inserted by blunt / bar linkage in the 5 'direction of the hph selection cartridge in pHPT1 6X which was digested with XhoI, blunted in the Xhol site with Klenow and digested with Kbal. The final transformation vector, pC / XBG (Xbal) -TV (Accession No. 209613, deposit date February 3, 1998, American Type Culture Collection, 12301 Parkiawn Drive, Rockville, MD 20852 USA), as shown in Figure 3 was linearized at the unique Xbal and Notl sites at the 5 'end of the cbhl promoter and the 3' end of the xln2 terminator before the transformation of M2C38 from T. reesei via bombardment of microprojectiles, as described later in FIG. Example 9 Example 8 Southern blot of genomic DNA isolated from RutC30 and M2C38 strains of. reesei Genomic DNA was isolated from each strain as described in Example 1. For Southern blotting, 1 μg of DNA was digested with 3-10 units of restriction endonuclease at 37 ° C for at least 2 hours and the products of digestion were resolved on a 0.8% agarose gel in 0.04 M Tris-acetate, 1 mM EDTA. The DNA was transferred by nylon membranes (3oehringer Mannheim) by capillary transfer (Sambrook et al., Pp. 9.38-9.44). In Figures 4 and 5, lanes 2, 4, 6, 8, 10 and 12 contain digested M2C38 DNA and lanes 3 ^ -5 / 9, 11 and 13 contain digested RutC30 DNA. The restriction endonucleases used were BamHI (lanes 2 and 3), EcoRI (lanes 4 and 5), Xbal (lanes 6 and 7), Hiñó III (lanes 8 and 9), SstI (lanes 10 and 11) and Kpnl (lanes). 12 and 13). In both figures, lane 1 contains molecular size standards of? -HindIII (Gibco / BRL, catalog No. 15612-013) and lane 4 contains 1 ng of labeled fragment used to make the probe. The Southern blots are hybridized with a random barley probe labeled with digoxigen-1-dUTP prepared using the DIG labeling and detection equipment (Boehringer Mannheim). The template for the probe used in Figure 4 was a 2.3 kb fragment comprising the T. reesei xln2 promoter and the secretion signal (Saarelainen et al.). The template for the probe used in Figure 5 was a 2.1 kb fragment comprising pb 574-2679 from the mature bgll coding region of T. reesei (Barret, et al.). After the post-hybridization washes, dig-düTP complexes were visualized by incubation with an anti-digoxigenin alkaline phosphatase conjugate (Boehringer Mannheim) followed by reaction with 5-bromo-4-chloro-3-indoyl-phosphate and chloride of tretrazolium blue 4-nitro (Boehringer Mannheim).

EXAMPLE 9 Transformation of RutC30, M2C38 and BTR48 of T. ree ^ sei vi-a bombardment with microprojectiles The PDS-1000 / He system is biolistic (BioRad, E.I.

DuPont de Nemours and Company) was used to transform spores of the strains RutC30, M2C38 and BTR48, of T. reesei and all the procedures were carried out as recommended by the manufacturer. M-10 tungsten particles (average diameter of 0.7 iim) were used as microcarriers.

The following parameters were used in the optimization of the transformation. A breaking pressure of 77.34 g / circle (1100 pounds / square inch) with a helium pressure of 29 mm Hg, a separation distance of 0.95 cm; a travel distance of the macrocarrier of 16 mm, and a target distance of 9 cm. Plates with 1 x 106 spores were prepared on potato dextrose agar (PDA.) Medium. The bombarded plates were incubated at 28 ° C. 4 hours after the bombardment, the spores were subjected to primary selection by coating the selective PDA medium supplemented with 80 units / ml of HygB. The bombarded plates were incubated at 28 ° C. The transformants were observed after 3.6 days of growth; however, additional incubation is necessary to achieve sporulation. After sporulation has occurred, a secondary selection process was performed to isolate individual transformants. Spores were harvested from the plate with an inoculation loop and redispersed in sterile water. This suspension is then filtered through a sterile syringe capped with glass microfibers. This allows the passage of spores while retaining unwanted mycelia. A determination of spore concentration in this suspension is required and subsequent dilutions are placed in palca on PDA plates supplemented with 0.75% Oxgall (Disk) and HygB (40 units / mL) to obtain 20-50 spores per plate. Oxallactivity acts as a colony restrictor, thus allowing the isolation of individual colonies in these secondary selection plates. Isolated colonies can be observed after 2-3 days.

Example 10 Southern blot analysis of genomic DNA isolated from strains RutC30, RC300, RC302, M2C38, RM4-300, R4-301, RM4-302, BTR48 and RB4-301 from T. reesei Genomic DNA was isolated from each strain as described in Example 1. For Southern blots, 1 μg of DNA was digested with 3-10 units of Kpnl or Xbal at 37 ° C for at least 2 hours and the digestion products were resolved on an agarose gel. 0.8% in 0.04 M Tris-acetate, 1 mM EDTA. DNA was transferred through nylon membranes (Boehringer Mannheim) by stapling (Sambrook et al., P.93-8-9.44). The Southern blots were hybridized with a random primed probe labeled with digoxigen-ll-dUTP prepared using the DIG Marking and Detection (Boehringer Mannheim) equipment. The template was a 1.3 kb EcoRI-Bgl II fragment comprising bp 1215-2464 of the published bgll sequence (Barrett et al.). After the posehybridization washes, were dig-düT complexes visualized? by incubation with an anti-digoxigenin alkaline phosphatase conjugate (Boehringer annheim) followed by reaction with chemiluminescent reagent CSPD (Boehringer Mannheim) and exposure to X-ray film (Kodak). The results are summarized in Table 1.

Table 1 Copy number of bglT in parenteral and recombinant T. reesei strains Strains Host Promoter Vector Signal gene bgll total S Secretion native vector genes bgll bgll RUTC30 Same bgll bgll None Present 0 1 RC-300 RuC30 cb l cbhl pCBGl-T Present 1 2 RC-302 RutC30 cbhl xln2 pC / XBGl- Away 1 X TV M2C38 Same Bgll bgll None Present 0 1 RM4-300 M2C38 cbhl cbhl pCBGl-TV Absent 2 2 RM4-301 M2C38 xin2 xln2 pXBGl-TV Present 2 RM4-302 M2C38 cbhl xln2 pC / XBGl- Present 2 TV BTR48 Same bgll bgll None Present 0 1 RB4-301 BTR48 xln2 xln2 DXBGI-TV Absent 2 2 Example 11 Production of ß-glucosidase in liquid cultures This example describes the methods used to determine the amounts of the ß-glucosidase enzyme produced by a strain of Trierma. Individual Trierma colonies are transferred to PDA plates for the propagation of each culture. Sporulation is required for the uniform inoculation of agitated flasks that are used in the culture capacity test to produce β-glucosidase and cellulase. The culture medium is composed of the following: Component g / L (NH4) 2S04 6.35 KH2P04 4.00 MgS04-7H20 2.02 CaCl2-2H20 0.53 CSL (liquor impregnated with maize 6.25 CaC03 10.00 Sources of carbon ** 5-10 Trace Elements * 1 mL / L * Trace element solution contains 5 / gl of FeSo4-7H20; 1.6 g / 1 MnS04-H20 1.4 g / 1 ZnSo4- 7H20. ** 5 g / 1 glucose plus 10 g / 1 Solka floc (when the cbhl or another cellulase promoter is used), 10 g / 1 xilan (when the kln2 promoter is used) or another source of carbon compatible with the promoter that directs the expression of ß-glucosidase. The carbon source can be sterilized separately with an aqueous solution at pH 2 to 7 and added to the remaining medium. The liquid volume per 1 liter flask is 150 mL, the initial pH is 5.5 and each flask is sterilized by steam autoclave for 30 minutes at 121 ° C before inoculation. For both untransformed (ie, native) and transformed cells, spores were isolated from the PDA plates as described in Example 9 and 1-10 x 10 5 spores were used to inoculate each flask. The flasks are shaken at 200 rpm at a temperature of 28 ° C for a period of 6 days. The filtrate containing the secreted protein was collected by filtration through the microfiber glass filters of GF / A (Whatman). The protein concentration was determined using the Bio-Rad Protein Assay (Cat. No. 500-0001) using Trichoderma cellulase as a standard.The activity of β-glucosidase was determined as described in Example 16. detected transoronants for the ability to produce at least 10-fold more ß-glucosidase (in IU / mg) than the untransformed host strain as determined by the IU / ml of β-glucosidase activity of the culture filtrate divided by the protein concentration (in mg / ml) of the culture filtrate.

Example 12 Production of ß-glucosidase by strains RutC30, RC-300, and RC-302 of T. reesei using carbon source Solka floc Based on the previous successes using the cbhl promoter and secretion signal to overexpress proteins in Trichoderma, the Mature ß-glucosidase coding region was placed in the 3 'direction of the cbhl promoter and secretion signal in the genetic construct shown in Figure 1 described in Example 5 (pCBGl-TV). The vector was introduced into RutC30 of T. reesei by bombardment of particles (Example 9) and the resulting transformant RC-300, produced 7 times more β-glucosidase activity than the parental strain (Table 2). It results from the incorporation of a copy of the vector into the chromosomes of the host (Example 10, Table 1). The greatest increase in β-glucosidase activity obtained from a copy of a -construction in which β-glucosidase is expressed using the cbhl promoter and the secretion signal suggests that this strategy is better than that employed by Barnett et al. , and Fowler et al., which resulted in only a 5-fold increase in β-glucosidase activity of 10-15 copies of a construct in which β-glucosidase is expressed from its own promoter and secretion signal. However, the resulting 7-fold increase in β-glucosidase activity has not yet been sufficient to alleviate the shortage of β-glucosidase for cellulose hydrolysis. The unreacted T. reesei RutC30 strain was transformed by particle pumping (Example 9) with a genetic construct of the pC / XBG (Xbal) -TV vector encoding the mature T. reesei β-glucosidase enzyme bound to the secretion signal of xylanase II from T. reesei. The untransformed RutC30 strain and the resulting transformed strain of this host, RC-302, were cultured using the procedures of Example 11 with 10 g / L of Solka floc and 5 g / L of glucose as carbon sources. The results are shown in Table 2. ^ | -. The untransformed strain produced 0.14 iu of ß-glucosidase per mg of protein. The transformant RC-302 with the C3H1 promoter and xylanase signal produced 19 IU / mg of β-glucosidase. This represents an improvement of approximately 136 times cor-respect to the untransformed strain, which is very significant for a cellulose to ethanol process.

Transformant RC-302 with the CBHI promoter and xylanase II secretion signal produced approximately 19 times more β-glucosidase activity than the best transformant RC300 with the CBH1 promoter and the CBH1 secretion signal.

Table 2 Production of ß-glucosidase in strains RutC30, RC-300, and RC-302 of T. reesei in 150 ml of flask cultures Example 13 Production of ß-glucosidase by strains M2C38 and .gR 4-3.02 using the carbon source Solka floc. The vector pCBGl-TV, in which ß-glucosidase is expressed from the CBH1 promoter and secretion signal (Figure 1 and Example 5), was introduced in 2C38 of T. reesei by bombardment of particles (Example 9). The resulting transformant RMN4-300 produced approximately 7-12 times more β-glucosidase activity than the parental strain (Table 3). Strain M2C38 of untransformed T. reesei was transformed by particle bombardment (Example 9) with a genetic construct of the pC / XBG (Xb l) -TV vector encoding the mature T. reesei β-glucosidase enzyme linked to the secretion signal of T. reesei xylanase II. The untransformed strain M2C38 and the transformed strain of this host, RM4-302, were cultured using the procedures of Example 11 with 10 g / L of Solka floc and 5 g / L of glucose as carbon sources. The results are shown in Table 3. The untransformed strain produced 0.35 l of ß-glucosidase per mg of protein. The NMR4-302 transformant with the CBH1 promoter and xylanase II secretion signal produced 14.1 / / mg of β-glucosidase. This represents approximately a 40-fold improvement over the untransformed strain / -jue -is very significant for a cellulose to ethanol process. The RMN4-302 transformant with the CBKI promoter and xylanase II secretion signal produced approximately 3 times more β-glucosidase activity than the transformant with the CBHI promoter and the CBHI secretion signal. This is a significant difference, since the CBHI promoter and the secretion signal do not lead to sufficient production of β-glucosidase to completely suppress the production of cellobiose in 1 hydrolysis.

Table 3 Production of ß-glucosidase in strains M2C38, RM4-300, and RM4 302 of T. reesei in 150 ml of flask cultures EXAMPLE 14 Production of ß-glucosidase by strains M2C38 and RM4-301 of T. reesei using the source of xylan coal Strain M2C38 of T. reesei untransformed - was transformed by bombardment of particles (Example 9) with a genetic construct of the pXBGl-TV vector coding for mature T. reesei ß-glucosidase linked to the xylanase promoter and secretion signal. Untransformed strain M2C38 and a transformed strain of this host, RM4-301, were cultured using the procedures of Example 11 with 5 g / L glucose and 10 g / L xylan as the carbon source. The results are shown in Table 4. The untransformed strain produced 0.16 IU of ß-glucosidase per mg of protein. Transformant F. 4-301 with the xylanase II promoter and the xylanase II secretion signal produced 20.4 IU / mg of β-glucosidase. This represents approximately a 127-fold improvement over the non-transformed strain, which is very significant for a cellulose-to-ethanol process.

Table 4 Production of ß-glucosidase in strains 2C38 and RM4-301 of T. reesei with xylan in 150 ml flask cultures.

EXAMPLE 15 Production of β-glucosidase by BTR-48 and RB48-301 strains using Solka-floc carbon sources The non-transformed strain T. reesei BTR48 was transformed by particle bombardment with a genetic construct of the pXBGl-TV vector coding for the mature T. reesei ß-glucosidase linked to the xylanase promoter and secretion signal. Untransformed strain BTR-48 and a transformed strain of this host, RB48-301, were cultured using the procedures of Example 11 with 5 g / L of glucose and lOg / L of Solka floc as the carbon sources. The results are shown in Table 5. The untransformed strain produced 0.16 IU of ß-glucosidase per mg of protein. Transformant R348-301 with the xylanase II promoter and xylanase II secretion signal produced approximately 21.9 μg / mg of β-glucosidase. This represents approximately a 136-fold improvement over the non-transformed strain, which is very significant for a cellulose-to-ethanol process.

Table 5 Production of ß-glucosidase in strains BTR48 and RB48-301 in T. reesei with Solka floc in flasks of ^ .50 ml.

Promoter Strain Bg Signal Secretion (IU / mg) BTR48 bgll bgll 0.16 RB48 xln2 xln2 21.9 Example 16 Measurement of β-glucosidase activity of a mixture of enzymes The β-glucosidase activity of an enzyme is measured using the Ghose procedures, "Measurement of Cellulase Activities", Puré and Ap l. Chem., 59: 257-268 (1987), as follows. The enzyme sample is diluted, at various concentrations, in 50 mM sodium citrate buffer, pH 4.8, to a volume of 0.5 ml. A convenient range of dilutions is 3 to 24 times the estimated activity of the sample. For example, a sample of 10 units / ml should be diluted 1:30 to 1:40. In spite of the dilutions used, a 0.5 ml sample of citrate buffer is added to each enzyme tube. The substrate is prepared as a 15 mM cellobiose (5.13 g / L). The stocks of diluted enzyme and substrate are preheated separately at 50 ° C for 5 minutes, then an aliquot of 0.5 ml of the substrate is added to each tube with enzyme. The test tubes are incubated for 30 minutes at 50 ° C. The reaction is completed by immersing each tube in a boiling water bath for 5 minutes. The tubes are then vortexed and the amount of sugar produced by each enzyme sample is measured on a YSI glucose analyzer, taking into account the small background of the enzyme. A unit of β-glucosidase activity is defined as the number of micromoles of glucose produced per minute. The activity is calculated based on equation 1 using the average value of each of the aliquots that produces 0.15 to 1.5 mg / ml of glucose.

A = C * G * D (1) where A = activity, ß-glucosidase units / mi (or micromoles of glucose / ml / min) C = 16.7 micromoles / mg / min G = glucose produced, mg / ml D = enzyme dilution, without dimension Example 17 Cellulose hydrolysis The purpose of this procedure was to demonstrate the effectiveness of the ß-glucosidase produced by the transformed Trichoderma by improving the hydrolysis of cellulose. The enzymes used for this study were Iogen's Cellulase, a commercial cellulase enzyme from Iogen Corporation, and the product of R 4-302 grown in a 30-liter fermentation vessel using the procedures described in Example 11, with twice the levels of media concentration listed in that Example. The concentration of enzymes was implemented by ultrafiltration through an Amicon 10,000 MWCO membrane and normalized to the same cellulase activity as the Iogen cellulase. The activities of these two enzymes are shown in Table 6.

Table 6 Enzyme activities used in the cellulose hydrolysis study The cellulose used for this study were pretreated oat shells, prepared as described in Example 6 in FOODY, et al, Improved Pretreatment Process for Conversion of Cellulose to Fuel Ethanol, U.S. 5,916,780. The pre-treated oat husk cellulose samples of 0.5 grams were added to 25 ml flasks with 49.5 grams of 0.05 molar sodium citrate buffer, pH 4.8.

Enzymes were added to the flask in an amount corresponding to 10 FPU per gram of cellulose. The resulting doses of β-glucosidase are listed in Table 6. In both cases, the flasks are shaken at 250 rprn and kept at 50 ° C for 24 hours. At that time, samples were taken, filtered to remove insoluble cellulose, and analyzed for glucose and cellobiose concentration using normal Dionex amperometric impulse HPLC carbohydrate analysis methods. The results are listed in Table 7. Iogen cellulose, the conventional Trichoder a cellulase, converted only 45% of the cellulose to glucose. This is unacceptably low for an ethanoi process. The accumulation of cellobiose was significant, representing 13% of the cellulose. Cellulase with improved β-glucosidase performed much better. The conversion of cellulose to glucose reached 84%. The reason for this excellent performance was that the accumulation of cellobiose was imperceptible due to the abundance of β-glucosidase. 31 Table 7 Improved cellulose hydrolysis by high concentration of β-glucosidase Example 18 Comparison of the genes xln2 and bgll of Trichoderma reesei in strains RutC30 and M2C38 A Southern blot analysis was performed on DNA of M2C38 and RutC30 digested with six different restriction enzymes that cut both inside and outside the regions coding for the mature ß-glucosidase and the xylanase secretion signal (Example 8) to determine if any polymorphism exists between J «as two strains. As shown in Figures 4 and 5, identical bands were found to hybridize with labeled probes prepared from M2C38 fragments encoding the mature β-glucosidase enzyme and the xylanase II promoter plus secretion signal, not indicating polymorphisms and a high degree of DNA sequence homology in these regions between the two strains.

The probes and primers used to identify and clone the M2C38 DNA sequences necessary to make the genetic constructs described in Examples 5-7 are based on published DNA sequences of several genes from several different strains of Trichoderma reesei including QM9414. { pgk, Vanhanen et al., 1989 and cbh2, Chen et al.), the derivatives of QM9414 VTT-D79125 (xln2, Saarelainen et al.) and L27. { cbhl, Shoemaker et al.), and the P40 strain derived from strain RL-P37 (bgll, 3arnett et al.). All these strains, like M2C38, are derived from the strain QM6a (Carter, Allison, Rey and Dunn-Cole an, "Chrornosorna1 and genetic analysis of the electrophoretic karyotype of Trichoderma reesei: mapping of the cellulase and xylanase genes", Molecular Microbiology 6: 2157-2174, 1992). Because RutC30 is the parent derivative of QM6a of M2C38, the inventors are confident that the method as described in Examples 2-4, gara - the 2 - isolation of the gene sequences used to make the expression vectors of ß-glucosidase described in Examples 5-7, will work equally well for the isolation of the same gene sequences of both M2C38 and RutC30. Based on the lineage of the strain described above and the Southern blot data, the inventors also have a high degree of confidence that genetic constructs prepared from RutC30 RNA will contain the identical DNA segments encoding the β-glucosidase enzyme mature and the secretion signal of xylanase II as those DNA preparations of M2C38. Since the prepared DNA constructs of M2C38 (Example 5-7) result in improved expression of β-glucosidase in both M2C38 and RutC30 (Examples 12-14), the inventors are also confident that the genetic constructs made of DNA of RutC30 will result in minimal levels of ß-glucosidase activity improvement in both RutC30 and M2C38.

Examples 19-33 Examples 19-33 summarize the cloning and expression of several heterologous genes of interest within T. reesei using a xylanase secretion signal. Example 19 describes the isolation of auxotrophs pyr4 from strains M2C38 and BTR213 from Tri? Tiod & reesei. Examples 20 and 21 describe the transformation and expression of target enzymes (both endogenous and heterologous) by genetic constructs in strains M2C38 and BTR213 of Trichoderma reesei. Examples 22, 26 and 32 describe the cloning of the T. reesei eg2 and peanut genes, respectively. Examples 23, 27, 28, 30 and 32 describe the construction of several genetic constructs for the expression of target enzymes in Trichoderma reesei. Examples 25, 29, 31 and 33 describe the transformation and expression of genetic constructs of strains 2C38 and BTR213 of T. reesei.

Example 19 Selection of pyr4 auxotrophs from M2C38 and BTR213 The pyr4 gene encodes orotidine-5'-monophosphate decarboxylase, an enzyme required for uridine biosynthesis. Mutations in this gene make the cell unable to grow in the absence of uridine. Mutations in this gene can be selected in the presence of the toxic inhibitor, 5-fluoroorotic acid (FOA). FOA is an analogue of the pyrimidine precursor orotic acid. It is incorporated into wild-type T. reesei cells, thus amending the cells that are capable of uridine biosynthesis. Cells- <They contain a defective pyrus gene that are resistant to FOA but require uridine for growth. To select these mutants, spores of Trichoderma reesei were plated on minimal solid media containing 1.2 mg / ml FOA, 2 mg / ml uridine. The minimum medium consisted of the following: lOg / L of glucose; 10 g / L of KH2P04; 6 g / L of (H4) 2S04; 1 g / L MgSO4-7H20; 3 g / L of sodium tri-citrate-2H20; 5 mg / L FeS0-7H20; 1.6 mg / L of MnSO4-H20; 1.4 mg / L of ZnS04-7H20; 2 mg / ml CaCl2-2H20; pH 5.5. Colonies resistant to spontaneous FOA appear in the space of 3-4 days. The additional selection identified mutants that required uridine for growth. The identification of mutants that specifically contained a defective pyr4 gene was identified by transformation with the plasmid pNCP4hph containing a hygromycin resistance gene and the pyr4 gene of Neurospora crassa. This vector was constructed by isolating the 3.2 kb BglII fragment from pFB6 (Buxton, FP and Radgord, A., 1983, "Cloning of the structural gene for orotidine-5 '-phosphate carboxilase of Neurospora crassa by expression in Escherichia coli. Gen. Genet 190: 403-405) and by cloning it into the unique BamHI site of pHPT136 (Example 5, above) to generate the vector pNCP4hph.The spores were transformed with pNCP4hph using bombardment with microprojectiles and selected -, between of potato dextrose agar containing hygromycin The subsequent growth of hygromycin-resistant transformants in medium lacking uridine identified those in which the defective pyr gene was complemented by the pyr4 gene of N. crassa.

EXAMPLE 20 Transformation of Trichoderma reesei by particle bombardment The PDS-1000 / He Biolistic system (BioRad; EI DuPont de Nemours and Company) was used to transform spores of the strains M2C38, T. reesei BTR213 or the pyr4 auxotrophs of these strains. and all procedures were performed as recommended by the manufacturer. M-10 tungsten particles (average diameter 0.7 μt?) Were used as microcarriers. The following parameters were used in the optimization of the transformation: a breaking pressure of 1100 pounds / inch2, a helium pressure of 29 mm Hg, a separation distance of 0.95 cm, a macroporter travel distance of 16 mm, and a target distance of 9 cm. When transformed with vectors containing the hygromycin-phosphotransferase gene (hph) of E. coli as the selectable marker, pest-epn lxlO6 spores were prepared in Potato Dextrose Agar (PDA) medium. Plaques bombarded at 28 ° C were incubated. Four hours after the bombardment, the spores were subjected to primary selection by coating the selective PDA medium supplemented with 80 units / ml of HygB. The bombardment plates are incubated at 28 ° C. After 3-6 days of growth, the individual transformants are harvested with a sterile stick, placed on individual PDA plates containing 40 units / ml of HygB and incubated at 28 ° C for 3-6 days. When they are transformed with vectors containing the pyr4 gene of N. crassa. As the selected marker, the plates were prepared with 1x10 ° spores in minimal medium (Example 19). The bombarded plates were incubated at 28 ° C. After 3-6 days of growth, the individual transformants are harvested with a sterile toothpick, placed on individual minimal medium plates and incubated at 28 ° C for 3-6 days.

Example 21 Production of target enzymes in liquid cultures Individual colonies of Trichoderma strains, both native and transformed, were transferred to PDA boxes for the propagation of each culture. Sporulation is required for the uniform inoculation of agitated satraqes that are used in the test of the ability of the culture to produce the target enzyme. The culture medium is composed of the following: Component q / L (NH4) 2S04 6.35 KH2P04 4.00 MgS04-7H20 2.02 CaCl2-2H20 0.53 CSL 6.25 CaC03 Elements 1.ml/L trace * Sources of 5-10 carbon * * * Trace element solution contains 5 g / 1 FeSO. * 7H, 0; 1.6 g / 1 MnS04 * H, 0; 1.4 g / 1 ZnS04 * 7H, 0. ** 5 g / 1 glucose plus 10 g / 1 Solka flower (when using the cbhl or other cellulase promoter), 10 g / 1 xilan (when the xln2 promoter is used) or another compatible carbon source the promoter that directs the expression d ^ -la-β-glucosidase. The carbon source can be sterilized separately as an aqueous solution at pH 2 to 7 and added to the remaining medium. The liquid volume per 1 liter flask is 150 ml, the initial pH is 5.5 and each flask is sterilized by steam autoclave for 30 minutes at 121 ° C before inoculation.

For both native and transformed cells, spores are harvested from the plate with a loop of inoculation and redispersed in sterile water. This suspension is then filtered through a sterile syringe tacked with glass microfibers. This allows the passage of spores while retaining unwanted mycelia. After the completion of the concentration of spores in this suspension, l-2xl06 spores are used to inoculate each flask. The flasks are shaken at 200 rmp at a temperature of 28 ° C for a period of 6 days. The filtrate containing the secreted protein is collected by filtration through glass microfiber filters GF / A (W atman). The protein concentration is determined using the Bio-Rad protein assay (Cat. No. 500-0001) using Trichoder a cellulase as a standard.

EXAMPLE 22 Cloning of T. reesei endoglucanase II [I2) gene from strain M2C28 Genomic clones containing non-translated regions with 5 'structural and non-translated 3' of eg2 of. reesei were isolated from the XOASH DNA library of M2C38 of Trichoderma using the previously described methods (Examples 2 and 4 above). An eg2 gene probe labeled with digoxigenin-11-dUTP was prepared from the M2C38 genomic DNA using Pwo polymerase and the primers designed to amplify pb 262-1692 of the published DNA sequence (Saloheimo, Lehtovaara, Penttila, Teeri, Stahlberg, Johansson, Petterson, Claeyssens, Tomme and Knowles, 1988, "EGIII, a new endoclucanase from Trichoderma ressei: the characterization of both gene and enzyme", Gene 63: 11-21 later referred to as Saloheimo et al) and was subsequently used as a probe to detect the ADASH library. An EcoRI fragment of 6.0 kb from one of the clones that positively hybridizes was isolated and boosted in the EcoRI site of pUC119 to generate the pEG2 subclone.

EXAMPLE 23 Construction of PEG2-TV and PC / XREG2-TV vectors of endogucanase II overexpression This example describes the construction of vectors expressing the mature endoglucanase-II coding region under the control of the T. reesei cbhl promoter and the secretion signal eg2 or xln2. Using the pEG2gen subclone of eg2 as a template, the DNA sequence encoding the endoglucanase II protein with its own secretion signal (bp 262-1692) was amplified with pol wo (Boehringer) polymerase and the primers designed to introduce a Sphl site in bp 260-265 and a Kpnl site directly in the 3 'direction of the terminator codon in bp 1692. The resulting PCR product was inserted as a blunt-ended fragment in the Smal site of pUC119 to make the pEG2 plasmid -6 and the sequence was verified. Before an expression cartridge from which this eg2 gene of the cbhl promoter can be expressed, a 2.2 kb fragment containing the cbhl promoter was amplified from the cbhl genomic pCB152 subclone (Example 3, above) using polymerase Pwo a primer set in the direction of the EcoRI site of the pUC119 polylinker and an inverted primer designed to introduce a Sphl site into the cbhl finalizing codon (bp 209-214 of the published cbhl sequence, Shoemaker, Schweikart, Ladner, Gelfand, Kwok, Myambo and Innis, "Molecular cloning of exo-cellobiohydrolase I derived from Trichoderma reesei strain L27", Bio / Technology 1: 691-696, 1983, hereinafter referred to as Shoemaker et al). This amplified phage was digested with EcoRI and Sphl and pBR322L was inserted (Example 5, above), a derivative of pBR322 in which a synthetic Sphl-Notl-Sall linker was inserted between the existing Sphl and Sali sites of pBR322 to generate the plasmid pBRClpro. The eg2 gene was isolated as a 1.4 kb Sphl / Kpnl fragment from pEG2-6 and inserted between the unique Sphl and Kpnl sites in the 5 'direction of the cbh2 transcriptional terminator in plasmid pCB219N (Example 5, above) . A 3.3 kb fragment comprising the gene eg2 and the transcriptional terminator cbh2 was subsequently isolated by digestion with Sphl / Notl and inserted between the Sphl and Notl sites directly in the 3 'direction of the cbhl promoter in pBRClpro. The resulting plasmid, expression cartridge pCEG2, contains the eg2 gene (which codes for the secreted, mature endoglucanase II enzyme and its own secretion signal) linked to the cbhl promoter 7 the bbh.2 terminator sequences. This plasmid was further modified to insert a unique Xhol site at the 3 'end of the cbhl terminator by digestion at the single Notl site, blunting with Klenow and the addition of the Xhol linkers (Cat. No. 1073, New England Biolabs) for elaborate a new pEG2-Xho plasmid of expression cartridge. To elaborate the final transformation factor, pEG2-TV (Figure 6a), pCEG2-Xho was digested as the unique Xhol site at the 3 'end of the cbh2 terminator and then-digested at the Xbal site at bp -1392 at the promoter cbhl in order to isolate the 5.6 kb expression cartridge containing the eg2 gene under the control of the cbhl promoter. This fragment was then inserted in the 5 'direction of the hph selection cartridge in pHPT136 (Example 5, above) that has been digested at the unique Xhol and Xbal site. Before transformation of the T. reesei M2C38 strain, the pEG2-TV transformation vector was digested with Xbal and Nocí, the fragments were separated by agarose gel electrophoresis and the larger band containing the eg2 construct was purified . To elaborate an expression cartridge from which the mature endoglucanase II enzyme is linked to the Xln2 secretion signal under the control of the cbhl promoter, pb 331-1692 of the published eg2 sequence (Saloheimo et al) were amplified with Pwo polymerase and the primers were designed to introduce a unique Nhel site directly in the 5 'direction of pb 331 and a single Kpnl site directly in the 3' direction of pbl692 using the pEG2gen genomic subclone as a template. The resulting fragment of extremes, blots was inserted into the Smal site of püC119 to generate the plasmid pEl and the sequence of the eg2 gene was verified. The 1.3 kb fragment encoding the endoglucanase II enzyme (without a secretion signal) was isolated from pEl by digestion with Nhel / Kpnl and inserted, in the 5 'direction of the cbh.2 terminator in the plasmid pCB219N-HB to generate pCB219N-El. PCB219N-HB is a derivative of pCB219N (Example 5, above) in which a synthetic HindIII-Sphl-Nhel-BamHI linker was inserted between the existing HindIII and BamHI sites in the 5 'direction of the cbh2 terminator fragment of pCB219. A 3.2 kb fragment comprising the coding region eg2 and the terminator sequences cbh2 were isolated by digestion with Nhel / Notl from pCB219N. { -El and was inserted between the Nhel and Nocí sites directly with the 3 'address of the promoter xln2 and the secretion signal in the plasmid pBR322LX (Example 6, above) to generate pXE2. A 1.3 kb HindIII fragment comprising pb -1400 to -121 of the xln2 promoter in pXE2 was replaced by a modified 1.2 kb HindIII fragment comprising pb -1399 to -204 of the cbhl promoter but with a new Xbal site in pb -1393 to -1388 which was prepared by PCR amplification using the pBR322LCS plasmid comprising the cbhl promoter as a template. From the resulting plasmid pC / XRE2-Xba, an approximately 4.9 kb Xbal / Notl fragment comprising the modified cbhl promoter, xln2 secretion signal, eg2 coding region and cbh2 terminator was isolated from pC / XRE2-Xba and used for replacing the Xbal / Notl fragment in pCE2 comprising the cbhl promoter and secretion signal, eg2 coding region and cbh2 terminator for gengr.ar.-the plasmid pC / XRE2 expression cartridge. It is noted that 'the Xbal site in the cbhl promoter of pCE2 is the endoge site located in bp -1497 to -1492. Therefore in plasmid PC / XRE2, of expression cartridge, pb -1496 to -1393 of the cbhl promoter are lost as a result of the fusion of the endoge Xbal site of the native cbhl promoter of pCE2 with the Xbal site of the modified cbhl promoter in pC / XE2-Xba. To make the final transformation vector pC / XRE2-TV (Figure 6b), the eg2 expression cartouches were isolated from pCE2 and pC / XRE2 by digestion with EcoRI (which cuts with the 5 'end of the promoter of the cbhl promoter and the 3 'end of the cbh2 terminator). These fragments were then inserted into the unique EcoRI site of the pyré selection plasmid pNCBgl from N. crassa. PNCBgl was constructed by inserting the 3.2 kb bglII fragment comprising the promoter, coding region and terminator of the pyré gene of N. crassa from pFB6 at the BamHI site in the pUC19 polylinker. The orientation of the insert was chosen such that the complete genetic construct comprising the expression cartridge of eg2 and the selection cartridge pyr4 of N. crassa can be isolated from the pUC sequence by digestion with Xbal. Before the transformation of T. reesei strain BTR213, the transformation vector pC / XRE2-TV was digested with Xbal, the fragments were separated by agarose gel electrophoresis and the larger band containing the eg2 constructs was purified.

EXAMPLE 24 Southern blot analysis of Trichoderma strains 3TR213, 201-2A, 843-2 and 845-2 Genomic DNA was isolated from the native and transformed Trichoderma strains as previously described (Ehemple 1, above). For Southern blots, 1 μg of DNA was digested with 3-10 units of restriction enzyme at 37 ° C for at least 2 hours and the digestion products were resolved on 0.8% agarose gel in 0.04 M Tris-acetate, ADTA 1 mM. DNA was transferred to nylon membrane (Boehringer Mannheim) by capillary transfer (Sambrook et al., Pp. 9.38-9.44). The Southern Blots were hybridized with a random primed probe labeled with digoxigen-ll-dUTP prepared using the DIG labeling and detection equipment (Boehringer Mannheim). The template was an EcoRI-BglII fragment of 1.3 kb isolated from the pEl plasmid (Example 22, above) and comprising pb 331-1692 of the published eg2 sequence (Saloheimo j ^ fc -ál). After the post-hybridization washes, dig-dUTP complexes were visualized by incubation with an anti-digoxigenin alkaline phosphatase conjugate (Boehringer Mannheim) followed by reaction with 5-bromo-4-chloro-3-indoyl-phosphate and chloride of tetrazolium blue 4-nitro. The results are summarized in Table 8.

Table 8 Copy number of eg2 in parenteral T. reeseí strains and recombine tes EXAMPLE 25 Production of endoglucanase II by native and transformed strains of T. reesei Strain BTR213 of T. reesei or its auxotroph pyr4 was transformed by particle bombardment (Example 20) with genetic constructions of vectors pE2-TV and? ß- ~ / ?? &2- TV that encodes paral to endoglucanase II enzyme linked to the secretion signal eg2 or xln2 under the control of the cbhl promoter. The native BRT213 strain and the resulting transformed strains were cultured using the procedures of Example 21 with 10 g / 1 solka flower and 5 g / 1 glucose as a carbon source.

Measurement of endoglucanase activity of an enzyme sample The activity of endoglucanase is determined by measuring the release of reducing sugars from a carboxymethyl cellulose (CMC) substrate. The enzyme sample is diluted to various concentrations in 50 mM sodium citrate buffer, pH 4.8, at a volume of 0.5 ml. A convenient range of dilutions is 2-20 times the estimated activity of the sample. For example, a sample of 1000 units / ml should be diluted 1: 2000 to 1: 20,000. In spite of the dilutions used, a sample of 0.5 ml of citrate buffer is added to each enzyme tube. The substrate is prepared as 1% CMC in 50 mM citrate, pH 4.8. The diluted enzyme stocks and the substrate are preheated separately at 50 ° C for 5 minutes, then an aliquot of 0.5 ml of the substrate is added to each tube with enzyme. The test tubes are incubated for 30 minutes at 50 ° C. The reaction is terminated by the addition of 3 ml of the DNS reagents (1% dinitrosalicylic acid, 1% sodium hydroxide, 0.2% phenol, 0.05% sodium metabisulfite in H50) and al-immersion. Each tube in a boiling water bath for 10 minutes. After boiling, 1 ml of an aqueous solution of 40% potassium sodium tartrate is added to each tube. The tubes are then vortexed, cooled and the amount of soluble reducing sugars that were released from the substrate and reacted with the DNS reagent were determined by measuring the absorbance at 550 nm against a normal curve generated from the solutions containing 0.18-0.5 mg / ml glucose (a reducing sugar) in 50 m citrate, pH 4.8. The culture activity is then calculated by comparing the mi of the culture filtrate required to produce 0.5 mg / ml of reducing sugar as compared to the mi of an endoglucanase enzyme control solution of known activity required to release 0.5 mg / ml of sugar reducer under the same conditions. The culture filtrates were harvested as described in Example 21 and tested for endoglucanase activity. The results are shown in Table 9.

Table 9 Production of endoqlucanase in strains BTR213, 201-2A, 843-2 and 845-2 of T. reesei in flasks cultures of ^ lSQ mi Strain Vector promoter CMCU Source Signal / mg carbon secretion BTR213 none eg2 eg2 solka floc 9.9 201-2A pE2-TV cbhl eg2 solka floc 20 843-2 PC / XRE2-TV cbhl xln2 solka floc 35 845-2 pC / XRE2 -TV cbhl xln2 solka floc 29 The untransformed strain produced 9.9 μl of endoglucanase per mg of protein . The 210-2A transformant with 2 copies of the cbhl promoter and the eg2 secretion signal produced 2 times more endoglucanase or approximately 22 IU / mg whereas the 843-2 and 845-2 transformants with the cbhl promoter and the xln2 secretion signal produced 29-35 IU / mg which is 3.3 times more endoglucanase than the native strain 1.3-1.6 times more than the transformant with the secretion signal eg2.

Example 26 Cloning of the β-mannanase gene from M2C38 of T. reesei This examples describes the cloning of the T. reesei peanut gene from a genomic library. The total M2C38 RNA from T. reesei was isolated and purified as follows: the mycelia of the cellulose-induced cultures were filtered through the Whatmas filter. -and-were washed with 50 mM Tris buffer, 10 m EDTA, 8.0. The fungal cakes were immediately frozen in dry ice and powdered in a mixer. Each gram of biomass was extracted for 10 minutes with 4 volumes of guanidium-4 M EDTA and 1% β-mercaptoethanol and centrifuged at 5000 x g to pellet the cell debris. The supernatants (3.5 ml) were stratified in a pad of 1.5 ml of CsCl 5.7 M - 0.1 M EDTA, pK 7 0 and ultracentrifuged for 16 hours at 30,000 rmp in a SW 50.1 swinging bucket rotor from Beckman. The RNA pellets were washed with 70% ethanol, dissolved in water treated with diethyl pyrocarbonate (Depc?,?), Precipitated with 2.5 M ammonium acetate and 3 volumes of ethanol; the sediment was dissolved in Depc H, 0. and stored at -80 ° C. For the synthesis of first-strand cDNA, 60 μg of total RNA in 20 μ? of depc -?,? was pre-treated with 2 μ? of methylmercuric hydroxide 0.1 m for 10 minutes at room temperature. Samples were cooled on ice and neutralized with 4 μ? of ß-mercaptoethanol 0.7 M. The RNA was then precipitated with 3 mol of ethanol and the pellet was dissolved in 20 μ? of decp -?,? The first strand synthesis was performed with inverted AMV transcriptase (Pharmacia) and oligo-dT primers following the manufacturer's protocols. A mannanase-specific probe from the first strand cDNA was amplified using specific brows for the 5 'and 3' ends of the mannanase coding region (bp 88-1443 of the published peanut sequence, Stalbrand, Saloheimo, Vehmaanpera, Kenrissat and Penttila , 1995, "Cloning and Expression in Sacharomyces cerevisiae of a Trichoderma ressei ß-mannanase gene containing a cellulose binding domain", Environ. Micro 61: 1090-1097 with Taq-polymerase and digoxigenin-11-dUTP (Boehringer) This probe was then used to detect the prepared XDASH library of the 2C38 genomic DNA digested with BamHI as previously described (Examples 2 and 4, above). Clones that positively hybridize were selected and purified.

Example 27 Construction of pAN vector AN-TV Mannanase overexpression This example describes the construction of a vector containing the Trichoder a chhl promoter and secretion signal and the mature mannanase coding region. Using the AASHASH peanut clone as a template, the DNA sequence encoding the secreted Mannanase protein (bp 88-1443) was amplified with Pwo polymerase (Boehringer) and the primers were designed to introduce a Nhel site at 88-90 bp. 93 and a Kpnl site directly in the 3 'direction of the terminator codon at bp 1443. The resulting PCR product was inserted as a blunt-ended fragment into the SamI site of pUC118 to make plasmid pManGNK and the sequence was verified. The peanut fragment was isolated from pManGNK by digestion with Nhel and Kpnl and inserted into the expression cartridge plasmid pBR322LEC which was digested with Nhel and Kpnl to make the pLECMAN expression cartridge plasmid. PBR322LEC was constructed from pBR322LCS containing the 2.3 kb fragment of the T. reesei cbhl gene comprising the promoter signal and secretion sequences (Example 5, above), as follows: Nhel linkers were ligated to PB322LCS which has been linearized by digestion with Sphl and the ends blunted with T4-DNA polymerase (Gibco / BRL); after digestion by Nhel, the plasmid was recirculated with T4-DNA ligase to make the plasmid pBR322LCN. A 1.9 kb Nhel / Notl fragment containing the transcripciomal terminator of the T. reesei cbh2 gene and unique Kpnl and Notl sites from the 5 'and 3' ends of the terminator was isolated by Nhel / Notl digestion of pCB219N-HB ( Example 22, above) and inserted between the unique Nhel and Notl sites in pBR322LCN to make pBR322LEC. To make the final transformation vector pCMA -TV (Figure 7a), pLECMAN was digested at the unique iVotJ site at the 3 'end of the cbh2 terminator, blunted with ^ Klenow and then digested at the unique Xbal site in bp - 1392 in the cbhl promoter in order to isolate the 5.6 kb expression cartridge containing the peanut gene under the control of the cbhl promoter and the secretion signal. This fragment was then inserted in the 5 'direction of the hph selection cartridge at pHPT136 (Example 5, above) which was digested at the unique Xhol site, blunted with Kleno and then digested at the adjacent, unique Xbal site. Before the transformation of the T. reesei strain M2C38, the pCMAN-TV transformation vector was digested with Xbal and Notl, the fragments were separated by agarose gel electrophoresis and the larger band containing the peanut construct was purified.

Example 28 Construction of pXMA-TX vectors and pC / XMAN-TV Mannanase overexpression This example describes the construction of vectors expressing the mature mannanase coding region under the control of the T. reesei xln2 promoter and the secretion signal or the cbhl promoter of T. reesei and the secretion signal xln2. A 3.3 kb Nhel / Notl fragment comprising the peanut coding region and the cbh2 transcriptional terminator was isolated by digestion with NheIjJotJ-, from pBR322LEC (Example 27, above) and the 3 'direction of the xln2 promoter was inserted. T. reesei and the secretion signal sequences contained in plasmid pBR322SpX (Example-6, above) to generate plasmid pX AN of expression cartridge. A 1.2 kb HindIII fragment comprising pb -1399 to -204 of the T. reesei cbhl promoter was isolated by digestion with HindIII pBR322LCS (Example 5, above) was used to replace a 1.3 b HindIII fragment comprising pb - 1400 to -121 of promoter xln2 in pXMAN to generate plasmid pC / XMA-TV of expression cartridge. To make the final transformation vectors pXMAN-TV (Figure 7b) and pC / XMAN-TV (Figure 7c), the expression cartridge was isolated from digestion with pXMAN and pC / XMAN at the unique Notl site at end 3 'of the cbh2 terminator was blunted with Klenow and then digested at the unique Xbal site in bp -1392 at the cbhl & In order to isolate the 5.6 kb expression cartridges containing the peanut gene under the control of the xln2 promoter and the secretion signal or the cbhl promoter and the xln2 secretion signal. These fragments are then inserted in the 5 'direction of the hph selection cartridge at pHPT136X (Example 6, above) that has been digested at the unique Xhol site, blunted with Klenow and then digested at the unique, adjacent Xbal site. Before the transformation of the T. reesei strain M2C38, the transcription vectors pXMAN-TV and pC / XMAN-TV were analyzed by excision with Notl.

Example 29 Mannanase production by native and transfected strains of T. reesei The T. reesei strain M2C38 was transformed by particle bombardment (Example 20) with genetic constructs of the pCMAN-TV and pC / X AN-TV vectors encoding for the mature mannanase enzyme linked to the secretion signal cbhl or xln2 under the control of the cbhl promoter. Native strain M2C38 and the resulting transformed strains were cultured using the procedures of Example 21 with 10 g / 1 solka flower and 5 g / 1 glucose as a carbon source.

Measurement of mannanase activity of an enzyme sample Mannanase activity is determined by measuring the release of reducing sugars from a mannan substrate. The enzyme sample is diluted to various concentrations in sodium citrate buffer, 50 m, pH 4.8, at a volume of 0.5 ml. A convenient range of dilutions is 5-40 times the estimated activated sample. For example, a sample of 10 units / ml should be diluted 1:50 to 1: 400. In spite of the dilutions used, a sample of 0.5 ml of citrate buffer is added to each enzyme tube. The substrate is prepared as a 1% acacia gum mannan in 50 mM citrate, pK 4.3. The stocks of diluted enzyme and substrate are precancelated separately at 50 ° C for 5 minutes, then an aliquot of 0.5 ml of substrate is added to each tube with enzyme. The test tubes are incubated for 30 minutes at 50 ° C. The reaction is terminated by the addition of 3 ml of the DNS reagents (1% dinitrosalicylic acid, 1% sodium hydroxide, 0.2% phenol, 0.05% sodium metabisulfite in H20) and by dipping each tube into a bath of boiling water for 10 minutes. After boiling, 1 ml of an aqueous solution of 40% potassium sodium tartrate is added to each tube. The tubes are then vortexed, cooled and the amount of soluble reducing sugars that were released from the substrate and reacted with the DNS reagent were determined by measuring the absorbance at 550 nm against a normal curve generated from the solutions containing 0.18-0.5 g / l of glucose (a reducing sugar) in 50 mM citrate, pK 4.8. The activity of. The culture is then calculated by compactara-, the mi of the culture filtrate required to produce 0.5 mg / ml of reducing sugar compared to the mi of a mannanase enzyme control solution of known activity required to release 0.5 mg / ml of reducing sugar under the same conditions. The culture filtrates are harvested as described in Example 21 and titrated for mannanase activity as described above. The results are shown in Table 10.

Table 10 Mannanase production in strains M2C38, 5D and 82D of T. reesei in 150 ml flask cultures The untransformed strain produced 3.58 l of mannanase per mg of protein. The 5D transoronant with the cbhl promoter and the secretion signal produced 1.9 times more than mannanase or 6.72 IU / mg while the β-rmante 82D trainee with the cbhl promoter and the x'ln2 secretion signal produced 16.22 IU / mg which is 4.5 times more mannanase than the native strain or 2.4 times more than the transformant with the cbhl secretion signal. Strain M2C38 of T. reesei was transformed by particle bombardment (Example 20) with a genetic construct of the vector pXMAN-TV coding for the mature mannanase enzyme linked to the secretion signal of xln2 under the control of the xln2 promoter. The native 2C33 strains and the resulting transformed strains were cultured using the procedures of Example 21 with 10 g / 1 xinal and 5 g / 1 glucose as the carbon source. The culture filtrates were harvested as described in Example 21 and evaluated for mannanase activity as described above. The results are shown in Table 11.

Table 11 Mannanase production in M2C38 and 17D strains of T. reasei in 150 ml flask cultures The untransformed strain produced 0.3 l of mannanase per mg of protein when xylan is used as the carbon source. The 17D transformant with the xln2 promoter and the secretion signal produced 9.4 times more mannanase or 2.81 IU / mg.

EXAMPLE 30 Construction of the vectors pChHE2-TV v pC / XhHE2-TV expression of endogualucanase 2 from fíuraicola insolens This example describes the construction of vectors expressing the coding region of endoglucanase II from mature H. isolens under the control of the cbhl promoter T. reesei and secretion signal or the cbhl promoter and xln2 secretion signal from T. reesei. In order to clone the g coding region of mature endoglucanase II (cmc3), it was isolated in genomic DNA of Humicola insolens from the biomass of the ATCC22082 strain of H. insolens grown in medium containing 24 g / 1 of Impregnated corn liquor, 24 g / 1 glucose and 0.5 g / 1 CaC03, pH 5.5 at 37 ° C for 48 hours (as described in Barbesgaard, Jensen and Holm, 1984, "Detergent Cellulase, U: S: Pat 4,435,307) using the previously described methods (Example 8) This genomic DNA from H. insolens was then used as a template to amplify the coding region of the mature endoglucanase II enzyme with Pwo polymerase and primers designed to introduce a N eJ site. only directly in the 5 'direction of pb 64 and a single Kpnl site directly in the 3' direction of pb 1182 of the cmc3 sequence of H. insolens (accession number of GenBank X76046) The amplified fragment was digested with Nhel and Kpnl and it was used to replace the peanut gene in the expression cartridge pCMAN plasmid (Example 26) which has been digested with Nhel and Kpnl. In the resultant c c3 expression cartridge plasmid pChHE2, the expression of the cmc3 sequence will be driven by the cbhl promoter and the secretion signal. In order to make the expression cartridge in which the cmc3 gene binds to the secretion signal xln2, pC / HhHE2, the PCR product of cmc3 that has been digested with Nhel and Kpnl was used to replace the eg2 gene in the pCE2 plasmid of expression cartridge (Example 22, above) that has been digested with Nhel and Kpnl. To make the transformation vectors, pChHE2-TV (Figure 8a) and pC / XhHE2-TV (Figure 8b), the cmc3 expression cartridges were isolated from pChHE2 and pC / XHE2 by EcoRI digestion (which cuts into the 5 'end of the cbhl promoter and the 3' end of the cbh2 terminator). These fragments were then inserted into the unique EcoRI site of the plasmid pNCBgl (Example 23, above-) of the pyré selection cartridge of N. crassa. The orientation of the insert was chosen such that the complete genetic construct comprising the cmc3 expression cartridge and the pyr selection cartridge of N. crassa can be isolated away from the pUC sequences by digestion with Xbal. Before transformation of the T. reesei strain M2C38, the transformation vectors pChHE2-TV and pC / XhHE2-TV were digested with Xbal, the fragments were separated by agarose gel electrophoresis and the larger band containing the constructions of cmc3 was purified.

Example 31 Production of endoglucanase 2 from H. insolens by native and transformed strains of T. reesei. Strain 2C38 of T. reesei was transformed by bombardment of particles (Example 20) with genetic constructs of the vectors pChHE2-TV and pC / XhHE2-TV coding for the endoglucanase II enzyme of H. insolens linked to the secretion signal cbhl or xln2 under the control of the cbhl promoter. The native M2C38 strain and the resulting transformed strains were cultured in 14 liter fermentation vessels using the procedures described in Example 21, with twice the average concentration levels listed in that Example.

Measurement of high pK endoglucanase activity of an enzyme sample High-pH endoglucanase activity is determined by measuring the release of reducing sugars from a hydroxylethyl cellulose (HEC) substrate.The sample of enzymes is diluted at various concentrations in 50 mM phosphate buffer, pH 7.0, at a volume of 0.5 ml A convenient range of dilutions is 2-20 times the estimated activity of the sample For example, a sample of 1000 units / ml should be diluted 1: 2000 to 1: 20,000.In spite of the dilutions used, a sample of 0.5 ml of citrate buffer is added to each enzyme tube.The substrate is prepared as 3% HEC in 50 m phosphate, pH 7.0. stock of diluted enzyme and substrate are preheated separately at 60 ° C for 5 minutes, then an aliquot of 0.5 ml of the substrate is added to each tube with enzyme.The test tubes are incubated for 30 minutes at 60 ° C. The reaction is finished by the adi 3 ml of the DNS reagent (1% dinitrosalicylic acid, 1% sodium hydroxide, 0.2% phenol, 0.05% sodium metabisulfite in?,?) and by submerging each tube in a boiling water bath for 10 minutes. After boiling, 1 ml of an aqueous solution of 40% sodium potassium tartrate is added to each tube. The tubes are then vortexed, cooled, and the amount of soluble reducing sugars that were released from the substrate and reacted with the DNS reagent is determined by measuring the absorbance at 550 mM against a normal curve generated from solutions containing 0.18. - 0.5 mg / ml glucose (a reducing sugar) in 50 mM phosphate, pK 7.0. The culture activity is then calculated by comparing the mi of the culture filtrate required to produce 0.5 mg / ml of reducing sugar in comparison to the mi of an endoglucanase enzyme solution of high pH of known activity control required to release 0.5 mg / ml. ml of reducing sugar under the same conditions. The culture filtrates are harvested as described in Example 21 and evaluated for high pH endoglucanase activity as described above. The results are shown in Table 12.

Table 11 Production of endoglucanase of high pH in strains M2C38, 98 and 998A of T. reesei in fermentations of 14 liters The untransformed strain produced 0 IU of high pH endoglucanase activity per mg of protein. The 984A transformant with the cbhl promoter and the secretion signal produced 0.097 IU / mg whereas the 998A transformant with the cbhl promoter and the xln2 secretion signal produced 0.195 IU / mg which is 2 times more high pH endoglucanase activity than the transformant with the secretion signal cbhl.

EXAMPLE 32 CONSTRUCTION OF pCLl-TV vectors and pC / XLl-TV of expression of laccase I of Trametes Versicolor This example describes the construction of vectors expressing the coding region of mature T. versicolor laccase I under the control of the cbhl promoter and secretion signal T. reesei or the cbhl promoter and the secretion signal xln2 T. reesei. A cDNA clone of the T. versicolor laccase I gene (Iccl) was obtained from Edgar Ong in the pBK-CMV vector of phagemid (Ong, Pollock and Smith, 1997, "Cloning and sequence analysis of two lacease complementary DNAs from the ligninolytic basidiomyeete Trametes versicolor ", Gene 196: 113-119, referred to later in the present ... -like Ong et al. ). The mature laccase coding region lacking its native secretion signal was amplified with Pwo and primers designed to introduce a unique Xbal site directly in the 5 'direction of pb 250 and a unique Xhol site directly in the 3' direction of the codon finalizer in bp 1750 of the published iccl sequence (Ong et al.). The amplified sequence was inserted as a blunt-ended fragment into the unique EcoRV site of pBR322 to generate the plasmid pBRLccl and the sequence of the lccl fragment was verified. In order to make the pCLl and pC / XLl plasmids of the lccl expression cartridge, a unique Xhol site was inserted into the 5 'end of the cbh2 transcriptional terminator in the pChHE2 and pC / XhKE2 vectors of the cmc3 expression cartridge by digestion with Kpnl and blunted with T4-DNA polymerase, followed by ligation of the Xhol linkers (Catalog No. 1073, New England Biolabs) to generate the pChHE2-Xho and pC / XhHE2-Xho plasmids of modified cmc3 expression cassette. The lccl gene was isolated as a 1.5 kg fragment by Xbal / Xhol digestion of pBRLccl and used to replace the cxmc3 gene in the pChHE2-Xho and pC / XhHE2-Xho plasmids of expression cartridge that has been digested with Nhel / Xhol. { Nhel and Xbal have compatible projections). The resulting lccl expression cartridge plasmids contain in the lccl gene linked to the secretion signal cbhl (in pCLl) or xln2 (pC / XLlJ ba or the control of the cbhl promoter.) To make the final transformation vectors, pCLl-TV ( Figure 9a) and pC / XLl-TV (Figure 9b), the lccl expression cartridges were isolated by digestion, with EcoRI of pCLl and pC / XLI and inserted into the unique EcoRI site of the pNCBgl plasmid of the pyr4 selection cartridge (Example 23, above) The orientation of the insert was chosen such that the complete genetic construct comprising the Iccl expression cartridge and the pyr4 selection cartridge of N. crassa can be isolated from the pUC sequences by cleavage by Xbal. of strain BTR213aux28 of T. reesei, the transformation vectors pCLl-TV and pC / XLl-TV were digested with Xbal, the fragments were separated by agarose gel electrophoresis and the larger band containing the Iccl constructs was purified. icó.

EXAMPLE 33 Production of T. versicolor laccase I by native and transformed strains of T. reesei Strain BTR213aux28 of T. Reesei and was transformed by particle bombardment (Example 20) with genetic constructs of vectors pCLl-TV and pC / XLl -TV coding for the mature T. versicolor laccase enzyme linked to the secretion signal cbhl or xln2 under the control of the cbhl promoter. The secondary selection of transformants pyr4 * was analyzed by spotting individual colonies and minimal medium (Example 19) with solka floc at 10% induced to the promoter cbhl, CuS04, 1.0 m, acid 2.2 '-azinobis-. { 3-methyl-benzthiazoline-6-sulfonic acid 1.0 mM) (AB S). The laccase-producing transformants are identified by the formation of a dark green halo indicative of an electronic oxidation of the A3TS substrate.

The native strain BTR213 and the resulting transformed strains were cultured using the procedures of Example 21 with 10 g / 1 solka floc and 5 g / 1 glucose as a carbon source.

Measurement of laccase activity of an enzyme sample Laccase activity is determined by oxidation of ABTS at 30 ° C. The assay mixture contains 0.5 mM ABTS, 0.1 M sodium acetate, pH 5.0 and an adequate amount of enzyme. The oxidation of ABTS is followed by measuring the increase in absorbance at 420 nm (e4.0 = 3.6 x 104 i4"'? Cm1) One unit of laccase activity is the amount of enzyme required to oxidize one micromole of ABTS per minute The culture filtrates are harvested as described in Example 21 and are evaluated for laccase activity as described above The highlights indicate that increased levels of laccase were produced with the transformants comprising the secretion signal of the laccase. xylanase (pC / XLl-TV), compared to the untransformed host (BTR213), or when compared to T. reesei transformed with a vector comprising the cbhl regulatory region and the secretory signal Examples 34-38 describe the expression of a modified thermophilic Trlchoderma xylanase in Humicola insolens using the cbhl promoter and the xln2 secretion signal from Trichoderma.

Example 34 Construction of the pC / XHTX4 vector of xln2 expression of. thermophilic reesei The T. reesei xln2 gene, including the promoter and terminator regions, was cloned as described in Example 4, above. A 2.6 kb fragment encoding the xln2 promoter, secretion signal sequence and the first 8 amino acids of the secreted protein was amplified by polymerase chain reaction from the subclone pXYN2 -2 (Example 4) xln2 using polymerase? or (Boehringer) with a reading-proof activity using a specific primer of xln2 to introduce a single PinAI site in bp 118-123 of the published xln2 sequence (Saarelainen et al.) and the inverted pUC primer (Catalog No. 18432- 013, Gibco / BRL). The resulting blunt-ended fragment was inserted into the Smal site of püC119 to generate the pUC / xynssP plasmid. The amplified sequences were then isolated from pUC / xynssP as an EcoRI / BamHI fragment and inserted between the EcoRI and BamHI sites of pBR322L (Example 5) to generate pBR322LXP. A fragment of HindIII of approximately 1.3 kp comprising pb minus 1400 (approximately) at minus 121 of the xln2 promoter in pBR322LXP was replaced by a 1.2 kb HindIII fragment comprising pb -1399 to -204 of the T. reesei cbhl promoter (No. of access of GenBank D86235). The EcoRI site at the 5"end of the chimeric cbhl / xln2 regulatory region was then destroyed by blunting with Klenow and the addition of Spel linkers. The coding region of xln2 was amplified from pXYN2K-2 using primers to introduce a PinAI site in the 5 'direction of bp +99 and a Kpnl site in the 3 * direction of bp +780 of the published xln2 sequence (Saarelainen et al.) and inserted as a blunt fragment in the S site to that of pUC119 to generate pTrxIIm -Pin A modified synthetic xln2 gene was obtained from W. Sung (containing the NITX11 gene described in US Pat., 759,840 and 5,866,408) and a 75 bp PinAI / Apal fragment encoding amino acids 7-33 of secreted xylanase II but with mutations N10H, V27M, M29L ¾ £, isolated and used to replace the same region in pTrxIIm-Pin , to make pHTX4-Pin. Using pUC119 polylinker sites in pHTX4-Pin, the modified xln2 gene was isolated as a 0.6 kb Sphl / Kpnl fragment and inserted between the Sphl and kpnl sites in the 51st direction of the cbh2 terminator in pCB219N Example 5). The resulting plasmid was then digested with PinAI and NotI to isolate a 2.4 kb fragment comprising the sequences coding for amino acids 9-190 of the modified xylanase II enzyme (0.5 kb) and the cbh2 terminator (1.9 kb). This fragment was inserted in the 3 'direction of the xln2 sequences in pBR322LXP, which was digested with PinAI and No1 to make plasmid pC / XHTX4 of the expression cartridge. The final transformation vector pC / XHTX4-TV was prepared by isolating the expression cartridge from pC / XHTX4 by digestion with Notl, blunting with Klenow followed by digestion with Spel. This fragment was inserted in the direction 51 of the hph selection cartridge in the plasmid PHPT136X (Example 6) which was digested with XhoI, blunted with Klenow, then digested with Xbal (Xbal and Spel have compatible overhangs). The final transformation vector pC / XHTX4-TV was linearized by digestion with Notl before the transformation of strain ATCC22082 of Hicola insolens by particle bombardment as described in Example 35. * «- '·' | EXAMPLE 35 Transformation of ATCC strain 22082 from H. insolens with PHTX4-TV via bombardment of microprojects The biolistic PDS-1000 / He system (BioRad; EI DuPont from Nemours de Nemours and Company) was used to transform spores of the strain ATCC22082 from H. insolens and all procedures were performed as recommended by the manufacturer. M-10 tungsten particles (average diameter 0.7 um) were used as microprocessors. The following parameters were used in the optimization of the transformation: burst pressure of 1100 pounds / square inch, a helium pressure of 29 mm Hg, a separation distance of 0.95 cm, a macroporter travel distance of 16 mm and an objective distance of 9 cm. Plates were prepared with 1x10 ° spores on Emerson YPSS agar (Di ugus). The bombarded plates were incubated at 37 ° C. Four hours after the bombardment, the spores were subjected to primary selection by selective YPSS agar coating supplemented with 240 units / ml of HygB. The bombardment plates were incubated at 37 ° C. The transformants could be observed after 5-6 days of growth. The individual colonies were subjected to secondary selection by isolation with a sterile stick and bathed in plates of ag & ^ Individual -YPSS containing 120 units / mi of HygB. These secondary selection plates were further incubated for 5-6 days at 73 ° C.

EXAMPLE 36 Production of thermophilic T. reesei xylanase, modified in liquid cultures of H. insolens This example describes the methods used to express a modified thermophilic T. reesei xylanase enzyme from a strain of Humicola. Individual colonies of H. insolens were transferred to YPSS agar plates for the propagation of each culture. Sporulation is necessary for the uniform inoculation of agitated flasks that are used in the culture capacity test to produce the β-glucosidase and cellulase. The culture medium is composed of the following: Component q / L (NHJ2S04 6.35 KH2P04 4.0 MgS0.-7H, 0 2.02 CaCl2-2H20 0.53 CSL 6.25 CaCO 10.00 Carbon source ** 5-200 Elements trasa * 1 ml / L * Trace element solution contains 5 g / 1 of FeSo4 * 7K, 0 1.6 g / 1 of MnS04 * H, 0; 1.4 g / 1 of ZnSo, * 7H_70. ** glucose, Solka floc. The carbon source can be sterilized separately as an aqueous solution at pH 2 to 7 and added to the remaining medium initially or through the course of fermentation. The liquid volume per 1 liter flask is 150 ml, the initial pH is 5.5 and each flask is sterilized by steam autoclave for 30 minutes at 121 ° C before inoculation. For both native and transformable cells, spores were isolated from the YPSS agar plates as previously described (Example 9) and 1-2x10 'spores were used to inoculate each flask. The flasks were shaken at 200 rpm at a temperature of 37 ° C for a period of 3 days. The filtrate containing the secreted protein was collected by filtration through glass microfiber (Whatman) GF / A filters. The protein concentration was determined using the Bio-Rad J cx Protein Assay of Catalog 500-0001). ": ' Example 37 Determination of thermophilic xylanase activity The xylanase activity of the culture filtrates was measured at both 50 ° C and 65 ° C using a solid azo-xylan substrate (Megazyme) with some modifications of the manufacturer's protocol as described at the moment. The substrate is prepared as follows: 1 g of azo-xylan is added to 35 ml of water preheated to 80 ° C and stirred for 60 minutes; 12.5 ml of acetate 2.0, pH 4.5 was then added and the volume adjusted to 50 ml; the final pH of the substrate was 4.4-4.7. For the 50 ° C test, the culture filtrate was diluted to various concentrations in 0.5 M acetate buffer, pH 4.5. For the test at 65 ° C, the culture filtrate was diluted to various concentrations in 50 mM acetate buffer, pH 4.8. A convenient range of dilutions is 1-5 times the estimated activity of the sample. For example, a sample of 1000 units / ml should be diluted 1: 1000 to 1: 5000. The diluted samples (0.2 ml each) and the substrate were preheated separately at 50 ° C or 65 ° C for 5 minutes, then an aliquot of 0.25 ml of the azo-xylan substrate was added to each tube with enzyme. The sample tubes were incubated for 10 minutes at 50 ° C or 65 ° C. The reaction was terminated by the addition of 1 ml of 95% ethanoi to each tube and vortexed. The unreacted azo-xylan was removed by centrifugation (6 minutes at 2000 x g, room temperature). The amount of azo dye released from the azo-xylan substrate by the xylanase was determined by measuring the absorbances of the supernatants at 590 nm, taking into account the small release of the dye or substrate dye in the absence of enzyme. The culture activity is then calculated by comparing the mi of the culture filtrate required to give an absorbance of 0.5 to 590 nm compared to the mi of a control xylanase enzyme solution of known activity required to give the same absorbance under the same conditions. terms .

Example 38 Production of a thermophylic T. reesei xylanase modified by strain 22082 and 22082-5A of H. insolens The vector pC / XHTX4-TV, in which a modified thermosophilic T. reesei xylanase II of the cbhl promoter is expressed and secretion signal xln2, was introduced into strain 22082 of H. insolens by bombardment of particles as described in Example 35. Untransformed strain 22082 and the transformed strain of this host, 22082-5A were cultured using the methods C iS. of Example 36 with 10 g / 1 of Solka floc and 5 g / 1 of glycolase as carbon sources. The results are shown in Table 14.

Table 14 Production of a modified thermophilic T. reesei xylanase in strains 22082 and 22082-5A of H. insolens in 150 ml flask cultures The untransformed strain produced 15.9 IU of xylanase activity at 50 ° C and 10.55 IU of xylanase activity at 65 ° C per mg of protein. The transformed 22082-5A produced 29.8 IU of xylanase activity at 50 ° C and 24.99 IU of xylanase activity at 65 ° C per mg of protein. This represents a 2.4-fold improvement in the xylanase fie activity at 65 ° C with respect to the untransformed strain. This result indicates that the T. reesei cbhl promoter and the xln2 secretion signal are effective for the expression of a gene of interest, and the secretion of a protein of interest in a heterologous host. All citations are incorporated herein by reference.

While the present invention has been described with respect to what is currently considered to be the most preferred embodiments, it is to be understood that the invention is not limited to the embodiments described. On the contrary, the invention is proposed to encompass the various modifications and equivalent arrangements included within the spirit and scope of the appended claims. The scope of the following claims will be granted as the broadest interpretation to encompass all these equivalent modifications and formulations and functions.

SEQUENCE LIST < 110 > Iogen Corporation < i20 Enhanced Expression of Proteins in Mushrooms Genetically Mcdilicsc < < · .130 > 08-aai794WOl < 140 > PCT / CAOO / 00997 < 1 1 ^ 2000-09-01 < 150 > US 09 / 392,476 < 151 > 1999-09-09 < 160 > 2 < 170s Patentln Ver. 2.1 < 210 > 1 < 211 > 72 < 212 > DNA < 213 > Artificial Sequence < 220 > < 221 > CDS < 222 > (1) .. (72) < 223 > Secretion signal of Trichodema C3HI / 3-giucosidase fusion < 400; > atg tat cgg aag ttg gcc gtc ate teg gee ttc ttg gee here gcc cgt 48 Met Tyr Arg Lys Leu Ala Val lie Be Ala Phe Leu Ala Tiir Ala Arg 1 5 10 15 gct cag teg gca gtt gta ect ect Wing Gln Ser < 210 > 2 < 211 > 120 < 212 > DNA < 213 Artificial Sequence < 220 > < 221 > CDS < 222 > (1) .. (120) < 22 > Xylanase secretion signal II / fusion of B-glucosidase 400 > 2 atg gct tcc ttc acc tec Ctc Ctc gcc ggc gtc g c g c ate ggc 48 Met Val Be Phe Thr Be Leu Leu Wing Gly Val Wing Ala Be Ser Giy 1 5 10 15 gtc ttg gcc gcc ccc gcc gcc gag gtc gaa tcc gtg gct gag aag Val Leu Wing Wing Phe Wing Wing Glu Val Gl Ser Val Val Wing Glu Lys 20 25 30 cgc cag gct aga gtc gta ccc cct Arg Gln Ala Arg Val Val. Phe Phe 35 40

Claims (25)

1. CLAIMS 1. A nucleotide sequence comprising a regulatory region in operative association with a Trichoderma xylanase secretion sequence and a gene of interest, wherein the gene of interest is not associated with the production of a protein of xylanase to the signal of secretion of xylanase from Trichoderma.
2. 2. The nucleotide sequence according to claim 1, wherein the regulatory region is selected from the group consisting of cbhl, cbh2, egl, eg2, eg3, eg5"xlnl and xln2.
3. The nucleotide sequence according to claim 1, wherein the gene of interest is selected from a gene encoding protein selected from the group consisting of a pharmaceutical product, a nutraceutical, an industrial enzyme, an animal feed, a food additive and an enzyme.
4. 4. The nucleotide sequence is claim 1, further comprising a terminator sequence.
5. 5. The nucleotide sequence according to claim 1, further comprising a selectable marker.
6. 6. The nucleotide sequence according to claim 1, further comprising an intervening sequence.
7. 7. A vector comprising the isolated nucleotide sequence of claim 1.
8. 8. A transformed filamentous fungus comprising the vector of claim 7.
9. 9. A transformed filamentous fungus comprising the nucleotide sequence of claim 1.
10. 10. The transformed filamentous fungus according to claim 9, wherein the filamentous fungus is selected from the group consisting of Trichoderma, Humicola, Fusarium, Aspergillus, Mycogone, Vertidllium, Colletotrichum, Neurospora, Botrytis, Pleurotus, Penicillum, Cephalosporium, Myrothecium, Papulospora, Achlya, Podospora, Endothia, Mucor, Cochilobbolus, Tolypocladium, Pyricularia, Penicillium, Mycelioohthora, Irpex, Stachybotrys, Scorpulariopsis, Chaetomium, Gilocladium, Cephalosporin and Acremonium. , * - * - - "=
11. 11. The filamentous fungus transformed according to claim 10, wherein the filamentous fungus is Trichoderma 12.
12. The transformed filamentous fungus according to claim 10, wherein the filamentous fungus is Humicola.
13. A method for producing a protein of interest within a filamentous fungus comprising the steps of: i) transforming the filamentous fungus with a nucleotide sequence comprising, a regulatory region in operative association with a xylanase secretion sequence and a gene of interest, wherein at least one of the regulatory region, or the gene of interest is not normally associated with the production of xylanase protein; ii) cultivate the filamentous fungus, and iii) cause the fungus to produce the protein of interest.
14. A method for producing a protein of interest within a filamentous fungus comprising the steps of: i) transforming the filamentous fungus with the nucleic acid sequence of claim 6 ii) culturing the filamentous fungus, and ^ * »< · -; iii) cause the fungus to produce the protein of interest.
15. The method according to claim 13, wherein, in the step of transforming, the xylanase secretion sequence is heterologous with respect to the filamentous fungus.
16. 16. The method according to claim 13, wherein, in the step of transforming, the xylanase secretion sequence is homologous with respect to the filamentous fungus.
17. The method according to claim 14, wherein, in the step of transforming, the secretion sequence 5 of xylanase is heterologous with respect to the filamentous fungus.
18. 18. The method according to claim 14, wherein, in the step of transforming, the xylanase secretion sequence is homologous with respect to the filamentous fungus.
19. The method according to claim 13, wherein, in the step of causing the fungus to produce, it further comprises purifying the protein of interest.
20. The method according to claim 14, wherein, in the step of causing the fungus to produce, it comprises 15 further purify the protein of interest.
21. 21. The method according to claim 14, wherein, in the step of causing the fungus to produce the protein of interest, it further comprises removing the 2 - - amino acid sequence encoded by the intervention sequence 20 of the protein of interest.
22. 22. A protein produced by the method of claim 14.
23. 23. A protein produced by the method of claim 14. 25. The nucleotide sequence of claim 3, wherein the protein is selected from a group that It consists of ß-glucosidase, cellulase, hemicellulase, a lignin degradation enzyme, pectinase, protease and peroxidase. 25. A vector comprising the isolated nucleotide sequence of claim 24. 26. A transformed filamentous fungus comprising the vector of claim 25. 27. A transformed filamentous fungus comprising the nucleotide sequence of the claim
24. 24. 28. A method for producing a protein of interest selected from a group consisting of β-glucosidase, cellulase, hemicellulase, a lignin degradation enzyme, pectinase, protease and peroxidase from a filamentous fungus comprising the steps of: i) transforming the filamentous fungus with the vector of claim 25. ^ - · · ·: ii) cultivating the filamentous fungus, and i) causing the fungus to produce the protein. 29. A method for producing a de-interest protein selected from a group consisting of β-glucosidase, cellulase, hemicellulase, a lignin degradation enzyme, pectinase, protease and peroxidase from a filamentous fungus comprising the steps of: ) transforming the filamentous fungus with the nucleotide sequence of claim 24. ii) cultivating the filamentous fungus, and iii) causing the fungus to produce the protein. 30. An expression system for producing a protein of interest comprising, a filamentous fungus containing a nucleotide sequence comprising a regulatory region in operative association with a secretion sequence of Trichoderma xylanase and a gene of interest encoding the protein of interest, wherein at least one of the regulatory region, or the gene of interest is not normally associated with the production of xylanase process. 31. The nucleotide sequence according to claim 2, wherein the gene of interest is selected from the group consisting of modified thermophilic endoglucanase II, β-mannanase, laccase and xylanase. 32. The nucleotide sequence according to claim 1, wherein the xylanase secretion sequence of Trichoderma is a xylanase secretion sequence of Family 11. 33. The nucleotide sequence according to claim 1, wherein the sequence of secretion of xylanase from Trichoderma is a sequence of secretion of xylanase II.