Compositions for degrading cellulosic material

Abstract

The present invention relates to cellulolytic compositions for degrading or converting cellulose-containing material and methods of producing and using the compositions.

Description

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a divisional application of U.S. application Ser. No. 12/130,838, filed May 30, 2008, which claims the benefit of U.S. Provisional Application No. 60/941,251, filed May 31, 2007, the contents of which are fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

[0003] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

REFERENCE TO A DEPOSIT OF BIOLOGICAL MATERIAL

[0004] This application contains a reference to deposits of biological material, which deposits are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Field of the Invention

[0005] The present invention relates to cellulolytic protein compositions for degrading or converting cellulose-containing material and methods of producing and using the compositions.

Description of the Related Art

[0006] Cellulose is a polymer of the simple sugar glucose covalently bonded by beta-1,4-linkages. Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases. Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble beta-1,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobiose to glucose.

[0007] The conversion of cellulosic feedstocks into ethanol has the advantages of the ready availability of large amounts of feedstock, the desirability of avoiding burning or land filling the materials, and the cleanliness of the ethanol fuel. Wood, agricultural residues, herbaceous crops, and municipal solid wastes have been considered as feedstocks for ethanol production. These materials primarily consist of cellulose, hemicellulose, and lignin. Once the cellulose is converted to glucose, the glucose is easily fermented by yeast into ethanol.

[0008] WO 2005/074647 discloses isolated polypeptides having cellulolytic enhancing activity and polynucleotides thereof from Thielavia terrestris. WO 2005/074656 discloses an isolated polypeptide having cellulolytic enhancing activity and a polynucleotide thereof from Thermoascus aurantiacus. U.S. Published Application Serial No. 2007/0077630 discloses an isolated polypeptide having cellulolytic enhancing activity and a polynucleotide thereof from Trichoderma reesei.

[0009] It would be an advantage in the art to improve the ability of cellulolytic protein compositions to degrade or convert cellulosic material.

[0010] The present invention relates to cellulolytic protein compositions improved in their ability to degrade or convert cellulosic material.

SUMMARY OF THE INVENTION

[0011] The present invention relates to filamentous fungal host cells, comprising: (a) a first polynucleotide encoding a native or heterologous polypeptide having cellulolytic enhancing activity; (b) a second polynucleotide encoding a native or heterologous beta-glucosidase; and (c) one or more (several) third polynucleotides encoding native or heterologous cellulolytic enzymes selected from the group consisting of a Trichoderma reesei cellobiohydrolase I (CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and a Trichoderma reesei endoglucanase I (CEL7B), and orthologs or variants thereof.

[0012] The present invention also relates to methods of producing a cellulolytic protein composition, comprising: (a) cultivating such filamentous fungal host cells under conditions conducive for production of the cellulolytic protein composition; and (b) recovering the cellulolytic protein composition. The present invention also relates to cellulolytic protein compositions obtained by such a methods.

[0013] The present invention also relates to cellulolytic protein compositions, comprising: (a) a polypeptide having cellulolytic enhancing activity; (b) a beta-glucosidase; and (c) one or more (several) cellulolytic enzymes selected from the group consisting of a Trichoderma reesei cellobiohydrolase I (CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and a Trichoderma reesei endoglucanase I (CEL7B), and orthologs or variants thereof.

[0014] The present invention also relates to methods for degrading or converting a cellulosic material, comprising: treating the cellulosic material with an effective amount of such a cellulolytic protein composition.

[0015] The present invention further relates to methods for producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an effective amount of such a cellulolytic protein composition; (b) fermenting the saccharified cellulosic material of step (a) with one or more (several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

BRIEF DESCRIPTION OF THE FIGURES

[0016] FIG. 1 shows a restriction map of pMJ04.

[0017] FIG. 2 shows a restriction map of pCaHj527.

[0018] FIG. 3 shows a restriction map of pMT2188.

[0019] FIG. 4 shows a restriction map of pCaHj568.

[0020] FIG. 5 shows a restriction map of pMJ05.

[0021] FIG. 6 shows a restriction map of pSMai130.

[0022] FIG. 7 shows the DNA sequence and amino acid sequence of an Aspergillus oryzae beta-glucosidase native signal sequence (SEQ ID NOs: 91 and 92).

[0023] FIG. 8 shows the DNA sequence and amino acid sequence of a Humicola insolens endoglucanase V signal sequence (SEQ ID NOs: 95 and 96).

[0024] FIG. 9 shows a restriction map of pSMai135.

[0025] FIG. 10 shows a restriction map of pSMai140.

[0026] FIG. 11 shows a restriction map of pSaMe-F1.

[0027] FIG. 12 shows a restriction map of pSaMe-FX.

[0028] FIG. 13 shows a restriction map of pAlLo47.

[0029] FIG. 14 shows a restriction map of pSaMe-FH.

DEFINITIONS

[0030] Cellulolytic enhancing activity: The term "cellulolytic enhancing activity" is defined herein as a biological activity that enhances the hydrolysis of a cellulose-containing material by proteins having cellulolytic activity. For purposes of the present invention, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulose-containing material by cellulolytic protein under the following conditions: 1-50 mg of total protein containing 80-99.5% w/w cellulolytic protein/g of cellulose in PCS and 0.5-20% w/w protein of cellulolytic enhancing activity for 1-7 day at 50.degree. C. compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g of cellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST.RTM. 1.5 L (Novozymes A/S, Bagsvrd, Denmark) in the presence of 3% Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 3% Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae according to Example 22 of WO 02/095014) of cellulase protein loading is used as a standard of the cellulolytic activity.

[0031] The polypeptides having cellulolytic enhancing activity have at least 20%, preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 100% of the cellulolytic enhancing activity of the mature polypeptide of SEQ ID NO: 2, 4, 6, 8, 10, 12, or 14.

[0032] The polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a cellulose-containing material catalyzed by proteins having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 0.1-fold, more at least 0.2-fold, more preferably at least 0.3-fold, more preferably at least 0.4-fold, more preferably at least 0.5-fold, more preferably at least 1-fold, more preferably at least 3-fold, more preferably at least 4-fold, more preferably at least 5-fold, more preferably at least 10-fold, more preferably at least 20-fold, even more preferably at least 30-fold, most preferably at least 50-fold, and even most preferably at least 100-fold.

[0033] Cellulolytic activity: The term "cellulolytic activity" is defined herein as cellulase activity (e.g., endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof) that hydrolyzes a cellulose-containing material. Cellulolytic protein may hydrolyze or hydrolyzes carboxymethyl cellulose (CMC), thereby decreasing the viscosity of the incubation mixture. The resulting reduction in viscosity may be determined by a vibration viscosimeter (e.g., MIVI 3000 from Sofraser, France). Determination of cellulase activity, measured in terms of Cellulase Viscosity Unit (CEVU), quantifies the amount of catalytic activity present in a sample by measuring the ability of the sample to reduce the viscosity of a solution of carboxymethyl cellulose (CMC). The assay is performed at the temperature and pH suitable for the cellulolytic protein and substrate.

[0034] For purposes of the present invention, cellulolytic activity is determined by measuring the increase in hydrolysis of a cellulose-containing material by a cellulolytic composition under the following conditions: 1-50 mg of cellulolytic protein/g of cellulose in PCS for 1-7 day at 50.degree. C. compared to a control hydrolysis without addition of cellulolytic protein.

[0035] Endoglucanase: The term "endoglucanase" is defined herein as an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. No. 3.2.1.4), which catalyses endohydrolysis of 1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. For purposes of the present invention, endoglucanase activity is determined using carboxymethyl cellulose (CMC) hydrolysis according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268.

[0036] Cellobiohydrolase: The term "cellobiohydrolase" is defined herein as a 1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91), which catalyzes the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1,4-linked glucose containing polymer, releasing cellobiose from the reducing or non-reducing ends of the chain. For purposes of the present invention, cellobiohydrolase activity is determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279 and by van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288. In the present invention, the Lever et al. method was employed to assess hydrolysis of cellulose in corn stover, while the method of van Tilbeurgh et al. was used to determine the cellobiohydrolase activity on a fluorescent disaccharide derivative.

[0037] Beta-glucosidase: The term "beta-glucosidase" is defined herein as a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues with the release of beta-D-glucose. For purposes of the present invention, beta-glucosidase activity is determined according to the basic procedure described by Venturi et al., 2002, J. Basic Microbiol. 42: 55-66, except different conditions were employed as described herein. One unit of beta-glucosidase activity is defined as 1.0 .mu.mole of p-nitrophenol produced per minute at 50.degree. C., pH 5 from 4 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium citrate, 0.01% TWEEN.RTM. 20.

[0038] Family 1, Family 3, Family 5, Family 6, Family 7, Family 9, Family 12, Family 45, Family 61, or Family 74 glycoside hydrolase: The term "Family 1, Family 3, Family 5, Family 6, Family 7, Family 9, Family 12, Family 45, Family 61, or Family 74 glycoside hydrolase" or "Family GH1, Family GH3, Family GH5, Family GH6, Family GH7, Family GH9, Family GH12, Family GH45, Family GH61, or Family GH74" is defined herein as a polypeptide falling into the glycoside hydrolase Family 1, Family 3, Family 5, Family 6, Family 7, Family 9, Family 12, Family 45, Family 61, or Family 74, respectively, according to Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696. Presently, Henrissat lists the GH61 Family as unclassified indicating that properties such as mechanism, catalytic nucleophile/base, catalytic proton donors, and 3-D structure are not known for polypeptides belonging to this family.

[0039] Cellulose-containing material: The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemi-cellulose, and the third is pectin. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1-4)-D-glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.

[0040] The cellulose-containing material can be any material containing cellulose. Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. The cellulose-containing material can be, but is not limited to, herbaceous material, agricultural residues, forestry residues, municipal solid wastes, waste paper, and pulp and paper mill residues. The cellulose-containing material can be any type of biomass including, but not limited to, wood resources, municipal solid waste, wastepaper, crops, and crop residues (see, for example, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp. 23-40, Springer-Verlag, New York). It is understood herein that the cellulose-containing material is preferably in the form of lignocellulose, e.g., a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix.

[0041] In a preferred aspect, the cellulose-containing material is corn stover. In another preferred aspect, the cellulose-containing material is corn fiber. In another preferred aspect, the cellulose-containing material is corn cobs. In another preferred aspect, the cellulose-containing material is switch grass. In another preferred aspect, the cellulose-containing material is rice straw. In another preferred aspect, the cellulose-containing material is paper and pulp processing waste. In another preferred aspect, the cellulose-containing material is woody or herbaceous plants. In another preferred aspect, the cellulose-containing material is bagasse.

[0042] The cellulose-containing material may be used as is or may be subjected to pretreatment, using conventional methods known in the art. For example, physical pretreatment techniques can include various types of milling, irradiation, steaming/steam explosion, and hydrothermolysis; chemical pretreatment techniques can include dilute acid, alkaline, organic solvent, ammonia, sulfur dioxide, carbon dioxide, and pH-controlled hydrothermolysis; and biological pretreatment techniques can involve applying lignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212; Ghosh, P., and Singh, A., 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566, American Chemical Society, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson, L., and Hahn-Hagerdal, B., 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander, L., and Eriksson, K.-E. L., 1990, Production of ethanol from lignocellulosic materials: State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

[0043] Pre-treated corn stover: The term "PCS" or "Pre-treated Corn Stover" is defined herein as a cellulose-containing material derived from corn stover by treatment with heat and dilute acid. For purposes of the present invention, PCS is made by the method described herein.

[0044] Full-length polypeptide: The term "full-length polypeptide" is defined herein as a precursor form of a polypeptide having biological activity, wherein the precursor contains a signal peptide region and alternatively also a propeptide region, wherein upon secretion from a cell, the signal peptide is cleaved and alternatively also the propeptide is cleaved yielding a polypeptide with biological activity.

[0045] Signal peptide: The term "signal peptide" is defined herein as a peptide linked in frame to the amino terminus of a polypeptide and directs the encoded polypeptide into a cell's secretory pathway.

[0046] Signal peptide coding sequence: The term "signal peptide coding sequence" is defined herein as a peptide coding region that codes for an amino acid sequence linked in frame to the amino terminus of a polypeptide and directs the encoded polypeptide into a cell's secretory pathway.

[0047] Propeptide: The term "propeptide" is defined herein as a peptide linked in frame to the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is linked in frame to the amino terminus of a polypeptide and the signal peptide region is linked in frame to the amino terminus of the propeptide region.

[0048] Propeptide coding sequence: The term "propeptide coding sequence" is defined herein as a peptide coding region that codes for an amino acid sequence linked in frame to the amino terminus of a polypeptide forming a proenzyme or propolypeptide (or a zymogen in some cases).

[0049] Catalytic domain: The term "catalytic domain" is defined herein as a structural portion or region of the amino acid sequence of a beta-glucosidase or an endoglucanase that possesses the catalytic activity of the beta-glucosidase or the endoglucanase.

[0050] Beta-glucosidase fusion protein: The term "beta-glucosidase fusion protein" is defined herein as a polypeptide that exhibits beta-glucosidase activity and comprises at least both a beta-glucosidase catalytic domain and an endoglucanase catalytic domain.

[0051] Components of a beta-glucosidase fusion protein: The term "components of a beta-glucosidase fusion protein" is defined herein as individual (cleaved) fragments of the beta-glucosidase fusion protein, wherein each fragment has beta-glucosidase activity and includes either the endoglucanase and the beta-glucosidase catalytic domain or the beta-glucosidase catalytic of the fusion protein. For example, the presence of a cleavage site, e.g., Kex2 site, between the endoglucacase and beta-glucosidase components of the fusion protein can result in the production of a polypeptide having endoglucanase activity and another polypeptide having beta-glucosidase activity.

[0052] Cellulose binding domain: The term "cellulose binding domain (CBD)" is defined herein as a portion of the amino acid sequence of an endoglucanase (cellulase) that is involved in the cellulose binding activity of the endoglucanase. Cellulose binding domains generally function by non-covalently binding the endoglucanase to cellulose, a cellulose derivative, or a polysaccharide equivalent thereof. CBDs typically function independent of the catalytic domain.

[0053] Beta-glucosidase fusion construct: The term "beta-glucosidase fusion construct" refers to a nucleic acid construct that is composed of different genes or portions thereof in operable linkage. The components include from the 5' end a DNA molecule comprising at least an endoglucanase catalytic domain and a DNA molecule comprising at least a beta-glucosidase catalytic domain.

[0054] Isolated polypeptide: The term "isolated polypeptide" as used herein refers to a polypeptide that is isolated from a source. In a preferred aspect, the polypeptide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by SDS-PAGE.

[0055] Substantially pure polypeptide: The term "substantially pure polypeptide" denotes herein a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which it is natively or recombinantly associated. It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99%, most preferably at least 99.5% pure, and even most preferably 100% pure by weight of the total polypeptide material present in the preparation. The polypeptides of the present invention are preferably in a substantially pure form, i.e., that the polypeptide preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated. This can be accomplished, for example, by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

[0056] Mature polypeptide: The term "mature polypeptide" is defined herein as a polypeptide having biological activity, e.g., enzyme activity, which is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.

[0057] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" is defined herein as a nucleotide sequence that encodes a mature polypeptide having biological activity.

[0058] Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

[0059] For purposes of the present invention, the degree of identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues.times.100)/(Length of Alignment-Total Number of Gaps in Alignment)

[0060] For purposes of the present invention, the degree of identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 3.0.0 or later. The optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides.times.100)/(Length of Alignment-Total Number of Gaps in Alignment)

[0061] Homologous sequence: The term "homologous sequence" is defined herein as sequences with an E value (or expectancy score) of less than 0.001 using the blastp (for protein databases) or tblastn (for nucleic acid databases) algorithms with the BLOSUM62 matrix, wordsize 3, gap existence cost 11, gap extension cost 1, no low complexity filtration, and a mature protein sequence as query. See Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402.

[0062] Polypeptide Fragment: The term "polypeptide fragment" is defined herein as a polypeptide having one or more (several) amino acids deleted from the amino and/or carboxyl terminus of the mature polypeptide or a homologous sequence thereof, wherein the fragment has activity as the mature polypeptide thereof.

[0063] Subsequence: The term "subsequence" is defined herein as a nucleotide sequence having one or more (several) nucleotides deleted from the 5' and/or 3' end of the mature polypeptide coding sequence or a homologous sequence thereof, wherein the subsequence encodes a polypeptide fragment having activity as the mature polypeptide thereof.

[0064] Allelic variant: The term "allelic variant" denotes herein any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0065] Isolated polynucleotide: The term "isolated polynucleotide" as used herein refers to a polynucleotide that is isolated from a source. In a preferred aspect, the polynucleotide is at least 1% pure, preferably at least 5% pure, more preferably at least 10% pure, more preferably at least 20% pure, more preferably at least 40% pure, more preferably at least 60% pure, even more preferably at least 80% pure, and most preferably at least 90% pure, as determined by agarose electrophoresis.

[0066] Substantially pure polynucleotide: The term "substantially pure polynucleotide" as used herein refers to a polynucleotide preparation free of other extraneous or unwanted nucleotides and in a form suitable for use within genetically engineered protein production systems. Thus, a substantially pure polynucleotide contains at most 10%, preferably at most 8%, more preferably at most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polynucleotide material with which it is natively or recombinantly associated. A substantially pure polynucleotide may, however, include naturally occurring 5' and 3' untranslated regions, such as promoters and terminators. It is preferred that the substantially pure polynucleotide is at least 90% pure, preferably at least 92% pure, more preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, even more preferably at least 98% pure, most preferably at least 99%, and even most preferably at least 99.5% pure by weight. The polynucleotides of the present invention are preferably in a substantially pure form, i.e., that the polynucleotide preparation is essentially free of other polynucleotide material with which it is natively or recombinantly associated. The polynucleotides may be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or any combinations thereof.

[0067] cDNA: The term "cDNA" is defined herein as a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell.

[0068] cDNA lacks intron sequences that are usually present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps before appearing as mature spliced mRNA. These steps include the removal of intron sequences by a process called splicing. cDNA derived from mRNA lacks, therefore, any intron sequences.

[0069] Nucleic acid construct: The term "nucleic acid construct" as used herein refers to a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or which is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic. The term nucleic acid construct is synonymous with the term "expression cassette" when the nucleic acid construct contains the control sequences required for expression of a coding sequence of the present invention.

[0070] Control sequence: The term "control sequences" is defined herein to include all components necessary for the expression of a polynucleotide encoding a polypeptide of the present invention. Each control sequence may be native or foreign to the nucleotide sequence encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleotide sequence encoding a polypeptide.

[0071] Operably linked: The term "operably linked" denotes herein a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence such that the control sequence directs the expression of the coding sequence of a polypeptide.

[0072] Coding sequence: When used herein the term "coding sequence" means a nucleotide sequence, which directly specifies the amino acid sequence of its protein product. The boundaries of the coding sequence are generally determined by an open reading frame, which usually begins with the ATG start codon or alternative start codons such as GTG and TTG and ends with a stop codon such as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA, synthetic, or recombinant nucleotide sequence.

[0073] Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0074] Expression vector: The term "expression vector" is defined herein as a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide of the present invention and is operably linked to additional nucleotides that provide for its expression.

[0075] Host cell: The term "host cell", as used herein, includes any cell type that is susceptible to transformation, transfection, transduction, and the like with a nucleic acid construct or expression vector comprising a polynucleotide.

[0076] Modification: The term "modification" means herein any chemical modification of a mature polypeptide or a homologous sequence thereof; as well as genetic manipulation of the DNA encoding such a polypeptide. The modification can be a substitution, a deletion and/or an insertion of one or more (several) amino acids as well as replacements of one or more (several) amino acid side chains.

[0077] Artificial variant: When used herein, the term "artificial variant" means a polypeptide produced by an organism expressing a modified nucleotide sequence of a mature polypeptide coding sequence or a homologous sequence thereof. The modified nucleotide sequence is obtained through human intervention by modification of the polynucleotide sequence or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

[0078] The present invention relates to filamentous fungal host cells, comprising: (a) a first polynucleotide encoding a native or heterologous polypeptide having cellulolytic enhancing activity; (b) a second polynucleotide encoding a native or heterologous beta-glucosidase; and (c) one or more (several) third polynucleotides encoding native or heterologous one or more (several) cellulolytic enzymes selected from the group consisting of a Trichoderma reesei cellobiohydrolase I (CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and a Trichoderma reesei endoglucanase I (CEL7B), and orthologs or variants thereof.

[0079] The present invention also relates to methods of producing a cellulolytic protein composition, comprising: (a) cultivating such filamentous fungal host cells under conditions conducive for production of the cellulolytic protein composition; and (b) recovering the cellulolytic protein composition. The present invention also relates to cellulolytic protein compositions obtained by such a methods.

[0080] The present invention also relates to cellulolytic protein compositions, comprising: (a) a polypeptide having cellulolytic enhancing activity; (b) a beta-glucosidase; and (c) one or more (several) cellulolytic enzymes selected from the group consisting of a Trichoderma reesei cellobiohydrolase I (CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and a Trichoderma reesei endoglucanase I (CEL7B), and orthologs or variants thereof.

[0081] In a preferred aspect, the filamentous fungal host cell produces a cellulolytic protein composition comprising a polypeptide having cellulolytic enhancing activity of the mature polypeptide of SEQ ID NO: 8; a beta-glucosidase fusion protein of SEQ ID NO: 106; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 52, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 54, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 56. In a preferred aspect, the filamentous fungal host cell is Trichoderma reesei, in particular Trichoderma reesei RutC30.

[0082] In another preferred aspect, the filamentous fungal host cell produces a cellulolytic protein composition comprising a polypeptide having cellulolytic enhancing activity of the mature polypeptide of SEQ ID NO: 8; a beta-glucosidase fusion protein of SEQ ID NO: 106; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 52, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 54, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 56, and further produces one or more (several) enzymes selected from the group consisting of a Trichoderma reesei endoglucanase II (CEL5A) of the mature polypeptide of SEQ ID NO: 58, a Trichoderma reesei endoglucanase V (CEL45A) of the mature polypeptide of SEQ ID NO: 62, and a Trichoderma reesei endoglucanase III (CEL12A) of the mature polypeptide of SEQ ID NO: 60. In another preferred aspect, the filamentous fungal host cell is Trichoderma reesei, in particular Trichoderma reesei RutC30.

[0083] In another preferred aspect, the filamentous fungal host cell produces a cellulolytic protein composition comprising a polypeptide having cellulolytic enhancing activity of the mature polypeptide of SEQ ID NO: 8; a beta-glucosidase fusion protein of SEQ ID NO: 106; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 52, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 54, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 56, and further produces a Thielavia terrestris cellobiohydrolase of the mature polypeptide of SEQ ID NO: 64. In another preferred aspect, the filamentous fungal host cell is Trichoderma reesei, in particular Trichoderma reesei RutC30.

[0084] In another preferred aspect, the filamentous fungal host cell produces a cellulolytic protein composition comprising a polypeptide having cellulolytic enhancing activity of the mature polypeptide of SEQ ID NO: 8; a beta-glucosidase fusion protein of SEQ ID NO: 106; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 52, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 54, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 56, and further produces (1) one or more (several) enzymes selected from the group consisting of a Trichoderma reesei endoglucanase II (CEL5A) of the mature polypeptide of SEQ ID NO: 58, a Trichoderma reesei endoglucanase V (CEL45A) of the mature polypeptide of SEQ ID NO: 62, and a Trichoderma reesei endoglucanase III (CEL12A) of the mature polypeptide of SEQ ID NO: 60, and/or (2) further produces a Thielavia terrestris cellobiohydrolase of the mature polypeptide of SEQ ID NO: 64. In another preferred aspect, the filamentous fungal host cell is Trichoderma reesei, in particular Trichoderma reesei RutC30.

[0085] In another preferred aspect, the cellulolytic protein composition comprises a polypeptide having cellulolytic enhancing activity of the mature polypeptide of SEQ ID NO: 8; a beta-glucosidase fusion protein of SEQ ID NO: 106; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 52, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 54, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 56.

[0086] In another preferred aspect, the cellulolytic protein composition comprises a polypeptide having cellulolytic enhancing activity of the mature polypeptide of SEQ ID NO: 8; a beta-glucosidase fusion protein of SEQ ID NO: 106; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 52, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 54, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 56 and further comprises one or more (several) enzymes selected from the group consisting of a Trichoderma reesei endoglucanase II (CEL5A) of the mature polypeptide of SEQ ID NO: 58, a Trichoderma reesei endoglucanase V (CEL45A) of the mature polypeptide of SEQ ID NO: 62, and a Trichoderma reesei endoglucanase III (CEL12A) of the mature polypeptide of SEQ ID NO: 60.

[0087] In another preferred aspect, the cellulolytic protein composition comprises a polypeptide having cellulolytic enhancing activity of the mature polypeptide of SEQ ID NO: 8; a beta-glucosidase fusion protein of SEQ ID NO: 106; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 52, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 54, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 56 and further comprises a Thielavia terrestris cellobiohydrolase of the mature polypeptide of SEQ ID NO: 64.

[0088] In another preferred aspect, the cellulolytic protein composition comprises a polypeptide having cellulolytic enhancing activity of the mature polypeptide of SEQ ID NO: 8; a beta-glucosidase fusion protein of SEQ ID NO: 106; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 52, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 54, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 56 and further comprises (1) one or more (several) enzymes selected from the group consisting of a Trichoderma reesei endoglucanase II (CEL5A) of the mature polypeptide of SEQ ID NO: 58, a Trichoderma reesei endoglucanase V (CEL45A) of the mature polypeptide of SEQ ID NO: 62, and a Trichoderma reesei endoglucanase III (CEL12A) of the mature polypeptide of SEQ ID NO: 60, and/or (2) further comprises a Thielavia terrestris cellobiohydrolase of the mature polypeptide of SEQ ID NO: 64.

Polypeptides Having Cellulolytic Enhancing Activity and Polynucleotides Thereof

[0089] Any polypeptide having cellulolytic enhancing activity that is useful in processing cellulose-containing material can be used in the compositions of the present invention. A polynucleotide encoding such a polypeptide having cellulolytic enhancing activity can be used to produce the compositions.

[0090] In a first aspect, isolated polypeptides having cellulolytic enhancing activity, comprise the following motifs:

TABLE-US-00001 [ILMV]-P-X(4,5)-G-X-Y-[ILMV]-X-R-X-[EQ]-X(4)-[HNQ] and [FW]-[TF]-K-[AIV],

wherein X is any amino acid, X(4,5) is any amino acid at 4 or 5 contiguous positions, and X(4) is any amino acid at 4 contiguous positions.

[0091] The isolated polypeptide comprising the above-noted motifs may further comprise:

TABLE-US-00002 H-X(1,2)-G-P-X(3)-[YW]-[AILMV], [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV], or H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and [EQ]-X-Y-X(2)- C-X-[EHQN]-[FILV]-X-[ILV],

wherein X is any amino acid, X(1,2) is any amino acid at 1 position or 2 contiguous positions, X(3) is any amino acid at 3 contiguous positions, and X(2) is any amino acid at 2 contiguous positions. In the above motifs, the accepted IUPAC single letter amino acid abbreviation is employed.

[0092] In a preferred embodiment, the isolated polypeptide having cellulolytic enhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV]. In another preferred embodiment, the isolated polypeptide having cellulolytic enhancing activity further comprises [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV]. In another preferred embodiment, the isolated polypeptide having cellulolytic enhancing activity further comprises H-X(1,2)-G-P-X(3)-[YW]-[AILMV] and [EQ]-X-Y-X(2)-C-X-[EHQN]-[FILV]-X-[ILV].

[0093] In a second aspect, isolated polypeptides having cellulolytic enhancing activity, comprise the following motif:

TABLE-US-00003 [ILMV]-P-x(4,5)-G-x-Y-[ILMV]-x-R-x-[EQ]-x(3)-A- [HNQ],

wherein x is any amino acid, x(4,5) is any amino acid at 4 or 5 contiguous positions, and x(3) is any amino acid at 3 contiguous positions. In the above motif, the accepted IUPAC single letter amino acid abbreviation is employed.

[0094] In a third aspect, isolated polypeptides having cellulolytic enhancing activity comprise or consist of amino acid sequences that have a degree of identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have cellulolytic enhancing activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides comprise or consist of an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

[0095] A polypeptide having cellulolytic enhancing activity preferably comprises the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide comprises amino acids 20 to 326 of SEQ ID NO: 2, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises amino acids 20 to 326 of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 2. In another preferred aspect, the polypeptide consists of amino acids 20 to 326 of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of amino acids 20 to 326 of SEQ ID NO: 2.

[0096] A polypeptide having cellulolytic enhancing activity preferably comprises the amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 4. In another preferred aspect, the polypeptide comprises amino acids 18 to 240 of SEQ ID NO: 4, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises amino acids 18 to 240 of SEQ ID NO: 4. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 4. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 4. In another preferred aspect, the polypeptide consists of amino acids 18 to 240 of SEQ ID NO: 4 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of amino acids 18 to 240 of SEQ ID NO: 4.

[0097] A polypeptide having cellulolytic enhancing activity preferably comprises the amino acid sequence of SEQ ID NO: 6 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 6. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 6. In another preferred aspect, the polypeptide comprises amino acids 20 to 258 of SEQ ID NO: 6, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises amino acids 20 to 258 of SEQ ID NO: 6. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 6 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 6. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 6. In another preferred aspect, the polypeptide consists of amino acids 20 to 258 of SEQ ID NO: 6 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of amino acids 20 to 258 of SEQ ID NO: 6.

[0098] A polypeptide having cellulolytic enhancing activity preferably comprises the amino acid sequence of SEQ ID NO: 8 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 8. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 8. In another preferred aspect, the polypeptide comprises amino acids 19 to 226 of SEQ ID NO: 8, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises amino acids 19 to 226 of SEQ ID NO: 8. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 8 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 8. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 8. In another preferred aspect, the polypeptide consists of amino acids 19 to 226 of SEQ ID NO: 8 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of amino acids 19 to 226 of SEQ ID NO: 8.

[0099] A polypeptide having cellulolytic enhancing activity preferably comprises the amino acid sequence of SEQ ID NO: 10 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 10. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 10. In another preferred aspect, the polypeptide comprises amino acids 20 to 304 of SEQ ID NO: 10, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises amino acids 20 to 304 of SEQ ID NO: 10. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 10 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 10. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 10. In another preferred aspect, the polypeptide consists of amino acids 20 to 304 of SEQ ID NO: 10 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of amino acids 20 to 304 of SEQ ID NO: 10.

[0100] A polypeptide having cellulolytic enhancing activity preferably comprises the amino acid sequence of SEQ ID NO: 12 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 12. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 12. In another preferred aspect, the polypeptide comprises amino acids 23 to 250 of SEQ ID NO: 12, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises amino acids 23 to 250 of SEQ ID NO: 12. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 12 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 12. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 12. In another preferred aspect, the polypeptide consists of amino acids 23 to 250 of SEQ ID NO: 12 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of amino acids 23 to 250 of SEQ ID NO: 12.

[0101] A polypeptide having cellulolytic enhancing activity preferably comprises the amino acid sequence of SEQ ID NO: 14 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 14. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 14. In another preferred aspect, the polypeptide comprises amino acids 20 to 249 of SEQ ID NO: 14, or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises amino acids 20 to 249 of SEQ ID NO: 14. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 14 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 14. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 14. In another preferred aspect, the polypeptide consists of amino acids 20 to 249 of SEQ ID NO: 14 or an allelic variant thereof; or a fragment thereof that has cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of amino acids 20 to 249 of SEQ ID NO: 14.

[0102] Preferably, a fragment of the mature polypeptide of SEQ ID NO: 2 contains at least 277 amino acid residues, more preferably at least 287 amino acid residues, and most preferably at least 297 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 4 contains at least 185 amino acid residues, more preferably at least 195 amino acid residues, and most preferably at least 205 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 6 contains at least 200 amino acid residues, more preferably at least 212 amino acid residues, and most preferably at least 224 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 8 contains at least 175 amino acid residues, more preferably at least 185 amino acid residues, and most preferably at least 195 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 10 contains at least 240 amino acid residues, more preferably at least 255 amino acid residues, and most preferably at least 270 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 12 contains at least 175 amino acid residues, more preferably at least 190 amino acid residues, and most preferably at least 205 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 14 contains at least 200 amino acid residues, more preferably at least 210 amino acid residues, and most preferably at least 220 amino acid residues.

[0103] Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 1 contains at least 831 nucleotides, more preferably at least 861 nucleotides, and most preferably at least 891 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 3 contains at least 555 nucleotides, more preferably at least 585 nucleotides, and most preferably at least 615 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 5 contains at least 600 nucleotides, more preferably at least 636 nucleotides, and most preferably at least 672 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 7 contains at least 525 nucleotides, more preferably at least 555 nucleotides, and most preferably at least 585 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 9 contains at least 720 nucleotides, more preferably at least 765 nucleotides, and most preferably at least 810 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 11 contains at least 525 nucleotides, more preferably at least 570 nucleotides, and most preferably at least 615 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 13 contains at least 600 nucleotides, more preferably at least 630 nucleotides, and most preferably at least 660 nucleotides.

[0104] In a fourth aspect, isolated polypeptides having cellulolytic enhancing activity are encoded by polynucleotides comprising nucleotide sequences that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11, or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has cellulolytic enhancing activity. In a preferred aspect, the mature polypeptide coding sequence is nucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ ID NO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 of SEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 67 to 796 of SEQ ID NO: 11, or nucleotides 77 to 766 of SEQ ID NO: 13.

[0105] The nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or a subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having cellulolytic enhancing activity from strains of different genera or species. In particular, such probes can be used for hybridization with the genomic or cDNA of the genus or species of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 14, preferably at least 25, more preferably at least 35, and most preferably at least 70 nucleotides in length. It is, however, preferred that the nucleic acid probe is at least 100 nucleotides in length. For example, the nucleic acid probe may be at least 200 nucleotides, preferably at least 300 nucleotides, more preferably at least 400 nucleotides, or most preferably at least 500 nucleotides in length. Even longer probes may be used, e.g., nucleic acid probes that are at least 600 nucleotides, at least preferably at least 700 nucleotides, more preferably at least 800 nucleotides, or most preferably at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin). Such probes are encompassed by the present invention.

[0106] A genomic DNA or cDNA library prepared from such other strains may, therefore, be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having cellulolytic enhancing activity. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that is homologous with SEQ ID NO: 1; or a subsequence thereof; the carrier material is preferably used in a Southern blot.

[0107] For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 11, or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, its full-length complementary strand, or a subsequence thereof, under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film.

[0108] In a preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 1. In another preferred aspect, the nucleic acid probe is nucleotides 388 to 1332 of SEQ ID NO: 1. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 2, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 1. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pEJG120 which is contained in E. coli NRRL B-30699, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pEJG120 which is contained in E. coli NRRL B-30699.

[0109] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 3. In another preferred aspect, the nucleic acid probe is nucleotides 98 to 821 of SEQ ID NO: 3. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 4, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 3. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTter61C which is contained in E. coli NRRL B-30813, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pTter61C which is contained in E. coli NRRL B-30813.

[0110] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 5. In another preferred aspect, the nucleic acid probe is nucleotides 126 to 978 of SEQ ID NO: 5. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 6, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 5. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTter61D which is contained in E. coli NRRL B-30812, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pTter61D which is contained in E. coli NRRL B-30812.

[0111] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 7. In another preferred aspect, the nucleic acid probe is nucleotides 55 to 678 of SEQ ID NO: 7. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 8, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 7. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTter61E which is contained in E. coli NRRL B-30814, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pTter61E which is contained in E. coli NRRL B-30814.

[0112] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 9. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 912 of SEQ ID NO: 9 In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 10, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 9. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTter61G which is contained in E. coli NRRL B-30811, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pTter61G which is contained in E. coli NRRL B-30811.

[0113] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 11. In another preferred aspect, the nucleic acid probe is nucleotides 67 to 796 of SEQ ID NO: 11. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 12, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 11. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pDZA2-7 which is contained in E. coli NRRL B-30704, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pDZA2-7 which is contained in E. coli NRRL B-30704.

[0114] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 13. In another preferred aspect, the nucleic acid probe is nucleotides 77 to 766 of SEQ ID NO: 13. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 14, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 13. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTr333 which is contained in E. coli NRRL B-30878, wherein the polynucleotide sequence thereof encodes a polypeptide having cellulolytic enhancing activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pTr333 which is contained in E. coli NRRL B-30878.

[0115] For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon sperm DNA, and either 25% formamide for very low and low stringencies, 35% formamide for medium and medium-high stringencies, or 50% formamide for high and very high stringencies, following standard Southern blotting procedures for 12 to 24 hours optimally.

[0116] For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2.times.SSC, 0.2% SDS preferably at least at 45.degree. C. (very low stringency), more preferably at least at 50.degree. C. (low stringency), more preferably at least at 55.degree. C. (medium stringency), more preferably at least at 60.degree. C. (medium-high stringency), even more preferably at least at 65.degree. C. (high stringency), and most preferably at least at 70.degree. C. (very high stringency).

[0117] For short probes of about 15 nucleotides to about 70 nucleotides in length, stringency conditions are defined as prehybridization, hybridization, and washing post-hybridization at about 5.degree. C. to about 10.degree. C. below the calculated T.sub.m using the calculation according to Bolton and McCarthy (1962, Proceedings of the National Academy of Sciences USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6 mM EDTA, 0.5% NP-40, 1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per ml following standard Southern blotting procedures for 12 to 24 hours optimally.

[0118] For short probes of about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below the calculated T.sub.m.

[0119] In a fifth aspect, isolated polypeptides having cellulolytic enhancing activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which encode an active polypeptide having cellulolytic enhancing activity.

[0120] In a preferred aspect, the mature polypeptide coding sequence is nucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ ID NO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 of SEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 67 to 796 of SEQ ID NO: 11, or nucleotides 77 to 766 of SEQ ID NO: 13. See polynucleotide section herein.

[0121] In a sixth aspect, isolated polypeptides having cellulolytic enhancing activity are artificial variants comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; or a homologous sequence thereof. Preferably, amino acid changes are of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

[0122] Examples of conservative substitutions are within the group of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, In, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

[0123] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline, and alpha-methyl serine) may be substituted for amino acid residues of a wild-type polypeptide. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for amino acid residues. "Unnatural amino acids" have been modified after protein synthesis, and/or have a chemical structure in their side chain(s) different from that of the standard amino acids. Unnatural amino acids can be chemically synthesized, and preferably, are commercially available, and include pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

[0124] Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

[0125] Essential amino acids in the parent polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities of essential amino acids can also be inferred from analysis of identities with polypeptides that are related to a polypeptide according to the invention.

[0126] Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display (e.g., Lowman et al., 1991, Biochem. 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0127] Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.

[0128] The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14, is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1.

[0129] A polypeptide having cellulolytic enhancing activity may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a nucleotide sequence is produced by the source or by a strain in which the nucleotide sequence from the source has been inserted. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0130] A polypeptide having cellulolytic enhancing activity may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus polypeptide having cellulolytic enhancing activity, or a Gram negative bacterial polypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide having cellulolytic enhancing activity.

[0131] In a preferred aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide having cellulolytic enhancing activity.

[0132] In another preferred aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having cellulolytic enhancing activity.

[0133] In another preferred aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having cellulolytic enhancing activity.

[0134] The polypeptide having cellulolytic enhancing activity may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide having cellulolytic enhancing activity; or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide having cellulolytic enhancing activity.

[0135] In a preferred aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having cellulolytic enhancing activity.

[0136] In another preferred aspect, the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaea saccata polypeptide having cellulolytic enhancing activity.

[0137] In a more preferred aspect, the polypeptide is a Thielavia terrestris polypeptide having cellulolytic enhancing activity. In a most preferred embodiment, the polypeptide is a Thielavia terrestris NRRL 8126 polypeptide having cellulolytic enhancing activity, e.g., the mature polypeptide of SEQ ID NO: 2, 4, 6, 8, or 10, or fragments thereof that have cellulolytic enhancing activity.

[0138] In another more preferred aspect, the polypeptide is a Thermoascus aurantiacus polypeptide having cellulolytic enhancing activity, e.g., the mature polypeptide of SEQ ID NO: 12.

[0139] In another more preferred aspect, the polypeptide is a Trichoderma reesei polypeptide having cellulolytic enhancing activity. In another most preferred aspect, the polypeptide is a Trichoderma reesei RutC30 (ATCC 56765) polypeptide having cellulolytic enhancing activity e.g., the mature polypeptide of SEQ ID NO: 14, or fragments thereof that have cellulolytic enhancing activity.

[0140] It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0141] Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0142] Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms from natural habitats are well known in the art. The polynucleotide may then be obtained by similarly screening a genomic or cDNA library of such a microorganism. Once a polynucleotide sequence encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are well known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

[0143] Polypeptides having cellulolytic enhancing activity also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof having cellulolytic enhancing activity. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) encoding a polypeptide h having cellulolytic enhancing activity. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.

[0144] For further details on polypeptides having cellulolytic enhancing activity and polynucleotides thereof, see WO 2005/074647, WO 2005/074656, and U.S. Published Application Serial No. 2007/0077630, which are incorporated herein by reference.

[0145] Polynucleotides comprising nucleotide sequences that encode polypeptides having cellulolytic enhancing activity can be isolated and utilized to practice the methods of the present invention, as described herein.

[0146] The polynucleotides comprise or consist of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, that encode a polypeptide having cellulolytic enhancing activity.

[0147] In a preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 1. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pEJG120 that is contained in Escherichia coli NRRL B-30699. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 1. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in plasmid pEJG120 that is contained in Escherichia coli NRRL B-30699. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or the mature polypeptide thereof, which differ from SEQ ID NO: 1 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 1 that encode fragments of SEQ ID NO: 2 that have cellulolytic enhancing activity.

[0148] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 3. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pTter61C that is contained in Escherichia coli NRRL B-30813. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 3. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in plasmid pTter61C that is contained in Escherichia coli NRRL B-30813. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 4 or the mature polypeptide thereof, which differ from SEQ ID NO: 3 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 3 that encode fragments of SEQ ID NO: 4 that have cellulolytic enhancing activity.

[0149] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 5. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pTter61D that is contained in Escherichia coli NRRL B-30812. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 5. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in plasmid pTter61D that is contained in Escherichia coli NRRL B-30812. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 6 or the mature polypeptide thereof, which differ from SEQ ID NO: 5 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 5 that encode fragments of SEQ ID NO: 6 that have cellulolytic enhancing activity.

[0150] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 7. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pTter61E that is contained in Escherichia coli NRRL B-30814. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 7. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in plasmid pTter61E that is contained in Escherichia coli NRRL B-30814. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 8 or the mature polypeptide thereof, which differ from SEQ ID NO: 7 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 7 that encode fragments of SEQ ID NO: 8 that have cellulolytic enhancing activity.

[0151] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 9. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pTter61G that is contained in Escherichia coli NRRL B-30811. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 9. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in plasmid pTter61G that is contained in Escherichia coli NRRL B-30811. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 10 or the mature polypeptide thereof, which differ from SEQ ID NO: 9 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 9 that encode fragments of SEQ ID NO: 10 that have cellulolytic enhancing activity.

[0152] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 11. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pDZA2-7 that is contained in Escherichia coli NRRL B-30704. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 11. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in plasmid pDZA2-7 that is contained in Escherichia coli NRRL B-30704. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 12 or the mature polypeptide thereof, which differ from SEQ ID NO: 11 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 11 that encode fragments of SEQ ID NO: 12 that have cellulolytic enhancing activity.

[0153] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 13. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pTr3337 which is contained in Escherichia coli NRRL B-30878. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 13. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in plasmid pTr3337 which is contained in Escherichia coli NRRL B-30878. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 14 or the mature polypeptide thereof, which differ from SEQ ID NO: 13 or the mature polypeptide coding sequence thereof by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 13 that encode fragments of SEQ ID NO: 14 that have cellulolytic enhancing activity.

[0154] The present invention also relates to mutant polynucleotides comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, in which the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. In a preferred aspect, the mature polypeptide is amino acids 20 to 326 of SEQ ID NO: 2, amino acids 18 to 240 of SEQ ID NO: 4, amino acids 20 to 258 of SEQ ID NO: 6, amino acids 19 to 226 of SEQ ID NO: 8, or amino acids 20 to 304 of SEQ ID NO: 10, amino acids 23 to 250 of SEQ ID NO: 12, or amino acids 20 to 249 of SEQ ID NO: 14. In another preferred aspect, the mature polypeptide coding sequence is nucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ ID NO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 of SEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 67 to 796 of SEQ ID NO: 11, or nucleotides 77 to 766 of SEQ ID NO: 13.

[0155] As described earlier, the techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof.

[0156] The polynucleotide may also be a polynucleotide comprising or consisting of a nucelotide sequence encoding a polypeptide having cellulolytic enhancing activity that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 1, SEQ ID NO: 5, or SEQ ID NO: 11, or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 13, or (iii) a full-length complementary strand of (i) or (ii); or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein. In a preferred aspect, the mature polypeptide coding sequence is nucleotides 388 to 1332 of SEQ ID NO: 1, nucleotides 98 to 821 of SEQ ID NO: 3, nucleotides 126 to 978 of SEQ ID NO: 5, nucleotides 55 to 678 of SEQ ID NO: 7, nucleotides 58 to 912 of SEQ ID NO: 9, nucleotides 67 to 796 of SEQ ID NO: 11, or nucleotides 77 to 766 of SEQ ID NO: 13.

Beta-Glucosidases and Polynucleotides Thereof

[0157] Any polypeptide having beta-glucosidase activity useful in processing cellulose-containing material can be a component of the compositions of the present invention. A polynucleotide encoding such a polypeptide having beta-glucosidase activity can be used to produce the compositions.

[0158] In a first aspect, isolated polypeptides having beta-glucosidase activity comprise or consist of amino acid sequences that have a degree of identity to the mature polypeptide of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have beta-glucosidase activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides comprise or consist of an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28.

[0159] A polypeptide having beta-glucosidase activity preferably comprises the amino acid sequence of SEQ ID NO: 16 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 16. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 16. In another preferred aspect, the polypeptide comprises amino acids 20 to 861 of SEQ ID NO: 16, or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide comprises amino acids 20 to 861 of SEQ ID NO: 16. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 16 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 16. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 16. In another preferred aspect, the polypeptide consists of amino acids 20 to 861 of SEQ ID NO: 16 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of amino acids 20 to 861 of SEQ ID NO: 16.

[0160] A polypeptide having beta-glucosidase activity preferably comprises the amino acid sequence of SEQ ID NO: 18 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 18. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 18. In another preferred aspect, the polypeptide comprises amino acids 20 to 861 of SEQ ID NO: 18, or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide comprises amino acids 20 to 861 of SEQ ID NO: 18. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 18 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 18. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 18. In another preferred aspect, the polypeptide consists of amino acids 20 to 861 of SEQ ID NO: 18 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of amino acids 20 to 861 of SEQ ID NO: 18.

[0161] A polypeptide having beta-glucosidase activity preferably comprises the amino acid sequence of SEQ ID NO: 20 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 20. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 20. In another preferred aspect, the polypeptide comprises amino acids 20 to 863 of SEQ ID NO: 20, or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide comprises amino acids 20 to 863 of SEQ ID NO: 20. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 20 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 20. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 20. In another preferred aspect, the polypeptide consists of amino acids 20 to 863 of SEQ ID NO: 20 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of amino acids 20 to 863 of SEQ ID NO: 20.

[0162] A polypeptide having beta-glucosidase activity preferably comprises the amino acid sequence of SEQ ID NO: 22 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 22. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 22. In another preferred aspect, the polypeptide comprises amino acids 37 to 878 of SEQ ID NO: 22, or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide comprises amino acids 37 to 878 of SEQ ID NO: 22. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 22 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 22. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 22. In another preferred aspect, the polypeptide consists of amino acids 37 to 878 of SEQ ID NO: 22 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of amino acids 37 to 878 of SEQ ID NO: 22.

[0163] A polypeptide having beta-glucosidase activity preferably comprises the amino acid sequence of SEQ ID NO: 24 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 24. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 24. In another preferred aspect, the polypeptide comprises amino acids 32 to 744 of SEQ ID NO: 24, or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide comprises amino acids 32 to 744 of SEQ ID NO: 24. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 24 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 24. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 24. In another preferred aspect, the polypeptide consists of amino acids 32 to 744 of SEQ ID NO: 24 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of amino acids 32 to 744 of SEQ ID NO: 24.

[0164] A polypeptide having beta-glucosidase activity preferably comprises the amino acid sequence of SEQ ID NO: 26 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 26. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 26. In another preferred aspect, the polypeptide comprises amino acids 20 to 860 of SEQ ID NO: 26, or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide comprises amino acids 20 to 860 of SEQ ID NO: 26. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 26 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 26. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 26. In another preferred aspect, the polypeptide consists of amino acids 20 to 860 of SEQ ID NO: 26 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of amino acids 20 to 860 of SEQ ID NO: 26.

[0165] A polypeptide having beta-glucosidase activity preferably comprises the amino acid sequence of SEQ ID NO: 28 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 28. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 28. In another preferred aspect, the polypeptide comprises amino acids 20 to 860 of SEQ ID NO: 28, or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide comprises amino acids 20 to 860 of SEQ ID NO: 28. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 28 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 28. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 28. In another preferred aspect, the polypeptide consists of amino acids 20 to 860 of SEQ ID NO: 28 or an allelic variant thereof; or a fragment thereof that has beta-glucosidase activity. In another preferred aspect, the polypeptide consists of amino acids 20 to 860 of SEQ ID NO: 28.

[0166] Preferably, a fragment of the mature polypeptide of SEQ ID NO: 16 contains at least 720 amino acid residues, more preferably at least 760 amino acid residues, and most preferably at least 800 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 18 contains at least 720 amino acid residues, more preferably at least 760 amino acid residues, and most preferably at least 800 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 20 contains at least 770 amino acid residues, more preferably at least 800 amino acid residues, and most preferably at least 830 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 22 contains at least 720 amino acid residues, more preferably at least 760 amino acid residues, and most preferably at least 800 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 24 contains at least 620 amino acid residues, more preferably at least 650 amino acid residues, and most preferably at least 680 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 26 contains at least 720 amino acid residues, more preferably at least 760 amino acid residues, and most preferably at least 800 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 28 contains at least 720 amino acid residues, more preferably at least 760 amino acid residues, and most preferably at least 800 amino acid residues.

[0167] Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 15 contains at least 2160 nucleotides, more preferably at least 2280 nucleotides, and most preferably at least 2400 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 17 contains at least 2160 nucleotides, more preferably at least 2280 nucleotides, and most preferably at least 2400 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 19 contains at least 2310 nucleotides, more preferably at least 2400 nucleotides, and most preferably at least 2490 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 21 contains at least 2160 nucleotides, more preferably at least 2280 nucleotides, and most preferably at least 2400 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 23 contains at least 1860 nucleotides, more preferably at least 1950 nucleotides, and most preferably at least 2040 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 25 contains at least 2160 nucleotides, more preferably at least 2280 nucleotides, and most preferably at least 2400 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 27 contains at least 2160 nucleotides, more preferably at least 2280 nucleotides, and most preferably at least 2400 nucleotides.

[0168] In a preferred aspect, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from an Aspergillus oryzae beta-glucosidase gene. In a most preferred embodiment, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from an Aspergillus oryzae beta-glucosidase gene comprising the mature polypeptide coding sequence of SEQ ID NO: 15 that encodes the mature polypeptide of SEQ ID NO: 16.

[0169] In another preferred aspect, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from an Aspergillus oryzae beta-glucosidase mutant gene. In a most preferred embodiment, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from an Aspergillus oryzae beta-glucosidase gene comprising the mature polypeptide coding sequence of SEQ ID NO: 17 that encodes the mature polypeptide of SEQ ID NO: 18.

[0170] In another preferred aspect, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from an Aspergillus fumigatus beta-glucosidase gene. In a most preferred embodiment, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from an Aspergillus fumigatus beta-glucosidase gene comprising the mature polypeptide coding sequence of SEQ ID NO: 19 that encodes the mature polypeptide of SEQ ID NO: 20.

[0171] In another preferred aspect, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from a Penicillium brasilianum strain IBT 20888 beta-glucosidase gene. In a most preferred embodiment, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from a Penicillium brasilianum strain IBT 20888 beta-glucosidase gene comprising the mature polypeptide coding sequence of SEQ ID NO: 21 that encodes the mature polypeptide of SEQ ID NO: 22.

[0172] In another preferred aspect, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from a Trichoderma reesei strain No. QM9414 beta-glucosidase gene. In another most preferred embodiment, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from a Trichoderma reesei strain No. QM9414 beta-glucosidase gene comprising the mature polypeptide coding sequence of SEQ ID NO: 23 that encodes the mature polypeptide of SEQ ID NO: 24 (GenBank.TM. accession no. U09580). In another preferred aspect, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from an Aspergillus niger beta-glucosidase gene. In a most preferred embodiment, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from an Aspergillus niger beta-glucosidase gene comprising the mature polypeptide coding sequence of SEQ ID NO: 25 that encodes the mature polypeptide of SEQ ID NO: 26 (GenBank.TM. accession no. AJ132386).

[0173] In another preferred aspect, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from an Aspergillus aculeatus beta-glucosidase gene. In a most preferred embodiment, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from an Aspergillus aculeatus beta-glucosidase gene comprising the mature polypeptide coding sequence of SEQ ID NO: 27 that encodes the mature polypeptide of SEQ ID NO: 28 (EMBL accession no. D64088).

[0174] The Aspergillus oryzae polypeptide having beta-glucosidase activity can be obtained according to WO 2002/095014. The Aspergillus oryzae polypeptide variant having beta-glucosidase activity can be obtained according to WO 2004/099228. The Aspergillus fumigatus polypeptide having beta-glucosidase activity can be obtained according to WO 2005/047499. The Penicillium brasilianum polypeptide having beta-glucosidase activity can be obtained according to WO 2007/019442. The Trichoderma reesei strain No. QM9414 polypeptide having beta-glucosidase activity can be obtained according to U.S. Pat. No. 6,022,725. The Aspergillus niger polypeptide having beta-glucosidase activity can be obtained according to Dan et al., 2000, J. Biol. Chem. 275: 4973-4980. The Aspergillus aculeatus polypeptide having beta-glucosidase activity can be obtained according to Kawaguchi et al., 1996, Gene 173: 287-288.

[0175] In a preferred aspect, the mature polypeptide of SEQ ID NO: 16 is encoded by a polynucleotide contained in the plasmid which is contained in E. coli DSM 14240. In another preferred aspect, the mature polypeptide of SEQ ID NO: 20 is encoded by the polynucleotide contained in plasmid pEJG113 which is contained in E. coli NRRL B-30695. In another preferred aspect, the mature polypeptide of SEQ ID NO: 22 is encoded by a polynucleotide contained in plasmid pKKAB which is contained in E. coli NRRL B-30860.

[0176] In a second aspect, isolated polypeptides having beta-glucosidase activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has beta-glucosidase activity.

[0177] The nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, or a subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having beta-glucosidase activity from strains of different genera or species, as described supra.

[0178] For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the mature polypeptide coding sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, its full-length complementary strand, or a subsequence thereof, under very low to very high stringency conditions, as described supra.

[0179] In a preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 15. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 2584 of SEQ ID NO: 15. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 16, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 15. In another preferred aspect, the nucleic acid probe is the polynucleotide contained in E. coli DSM 14240, wherein the polynucleotide sequence thereof encodes a polypeptide having beta-glucosidase activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in E. coli DSM 14240.

[0180] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 17. In another preferred aspect, the nucleic acid probe is nucleotides 58 to 2584 of SEQ ID NO: 17. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 18, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 17.

[0181] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 19. In another preferred aspect, the nucleic acid probe is nucleotides 73 to 1413 of SEQ ID NO: 19. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 20, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 19. In another preferred aspect, the nucleic acid probe is the polynucleotide contained in plasmid pEJG113 which is contained in E. coli NRRL B-30695, wherein the polynucleotide sequence thereof encodes a polypeptide having beta-glucosidase activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pEJG113 which is contained in E. coli NRRL B-30695.

[0182] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 21. In another preferred aspect, the nucleic acid probe is nucleotides 67 to 1377 of SEQ ID NO: 21. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 22, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 21. In another preferred aspect, the nucleic acid probe is the polynucleotide contained in plasmid pKKAB which is contained in E. coli NRRL B-30860, wherein the polynucleotide sequence thereof encodes a polypeptide having beta-glucosidase activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in E. coli NRRL B-30860.

[0183] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 23. In another preferred aspect, the nucleic acid probe is nucleotides 88 to 2232 of SEQ ID NO: 23. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 24, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 23.

[0184] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 25. In another preferred aspect, the nucleic acid probe is nucleotides nucleotides 59 to 2580 of SEQ ID NO: 25. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 26, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 25.

[0185] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 27. In another preferred aspect, the nucleic acid probe is nucleotides 59 to 2580 of SEQ ID NO: 27. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 28, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 27.

[0186] For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are as defined herein.

[0187] For long probes of at least 100 nucleotides in length, the carrier material is finally washed as defined herein.

[0188] For short probes of about 15 nucleotides to about 70 nucleotides in length, stringency conditions are as defined herein.

[0189] For short probes of about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed as defined herein.

[0190] In a third aspect, the polypeptides having beta-glucosidase activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which encode an active polypeptide.

[0191] In a sixth aspect, the polypeptides having beta-glucosidase activity are artificial variants comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28; or a homologous sequence thereof. Methods for preparing such artificial variants are described supra.

[0192] The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28, is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1.

[0193] In another preferred aspect, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from a Family 1 beta-glucosidase gene.

[0194] In another preferred aspect, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from a Family 3 beta-glucosidase gene.

[0195] In another preferred aspect, the polypeptide having beta-glucosidase activity is encoded by a polynucleotide obtained from a Family 5 beta-glucosidase gene.

[0196] Examples of other beta-glucosidases that can be used as sources for the polynucleotides in the present invention include, but are not limited to, an Aspergillus oryzae beta-glucosidase (WO 02/095014; WO 04/099228); Aspergillus aculeatus beta-glucosidase (Kawaguchi et al., 1996, Gene 173: 287-288); Aspergillus avenaceus beta-glucosidase (GenBank.TM. accession no. AY943971); Aspergillus fumigatus beta-glucosidase (GenBank.TM. accession no. XM745234); Aspergillus kawachii beta-glucosidase (GenBank.TM. accession no. AB003470); Aspergillus niger beta-glucosidase (GenBank.TM. AJ132386); Magnaporthe grisea beta-glucosidase (GenBank.TM. accession no. AY849670); Phanerochaete chrysosporium beta-glucosidase (GenBank.TM. accession no. AB253327); Talaromyces emersonii beta-glucosidase (GenBank.TM. accession no. AY072918), and Trichoderma reesei beta-glucosidase (GenBank.TM. accession nos. U09580, AB003110, AY281374, AY281375, AY281377, AY281378, and AY281379). Variants of beta-glucosidases may also be used as sources for the polynucleotides such as those described in WO 04/099228.

[0197] Other beta-glucosidases are disclosed in more than 13 of the Glycosyl Hydrolase families using the classification according to Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696.

[0198] A polypeptide having beta-glucosidase activity may also be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" is used as defined herein. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0199] A polypeptide having beta-glucosidase activity may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus polypeptide having beta-glucosidase activity, or a Gram negative bacterial polypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide having beta-glucosidase activity.

[0200] In a preferred aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide having beta-glucosidase activity.

[0201] In another preferred aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having beta-glucosidase activity.

[0202] In another preferred aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having beta-glucosidase activity.

[0203] The polypeptide having beta-glucosidase activity may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide having beta-glucosidase activity; or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide having beta-glucosidase activity.

[0204] In a preferred aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having beta-glucosidase activity.

[0205] In another preferred aspect, the polypeptide is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaea saccata polypeptide having beta-glucosidase activity.

[0206] It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0207] Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0208] Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes, as described herein.

[0209] Polypeptides having beta-glucosidase activity also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof having beta-glucosidase activity. A fused polypeptide is produced as described herein.

[0210] Polynucleotides comprising or consisting of nucleotide sequences that encode polypeptides having beta-glucosidase activity can be isolated and utilized to practice the methods of the present invention, as described herein.

[0211] The polynucleotides comprise or consist of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which encode a polypeptide having beta-glucosidase activity.

[0212] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 15. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in E. coli DSM 14240. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 15. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 58 to 2584 of SEQ ID NO: 15. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in E. coli DSM 14240. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 16 or the mature polypeptide thereof, which differ from SEQ ID NO: 15 or the mature polypeptide coding sequence thereof by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 15 that encode fragments of SEQ ID NO: 16 that have beta-glucosidase activity.

[0213] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 17. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 17. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 58 to 2584 of SEQ ID NO: 17. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 18 or the mature polypeptide thereof, which differ from SEQ ID NO: 17 or the mature polypeptide coding sequence thereof by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 17 that encode fragments of SEQ ID NO: 18 that have beta-glucosidase activity.

[0214] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 19. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pEJG113 which is contained in E. coli NRRL B-30695. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 19. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 58 to 2580 of SEQ ID NO: 19. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in plasmid pEJG113 which is contained in E. coli NRRL B-30695. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 20 or the mature polypeptide thereof, which differ from SEQ ID NO: 19 or the mature polypeptide coding sequence thereof by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 19 that encode fragments of SEQ ID NO: 20 that have beta-glucosidase activity.

[0215] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 21. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pKKAB which is contained in E. coli NRRL B-30860. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 21. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 109 to 2751 of SEQ ID NO: 21. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in plasmid pKKAB which is contained in E. coli NRRL B-30860. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 22 or the mature polypeptide thereof, which differ from SEQ ID NO: 21 or the mature polypeptide coding sequence thereof by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 21 that encode fragments of SEQ ID NO: 22 that have beta-glucosidase activity.

[0216] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 23. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 23. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 88 to 2232 of SEQ ID NO: 23. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 24 or the mature polypeptide thereof, which differ from SEQ ID NO: 23 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 23 that encode fragments of SEQ ID NO: 24 that have beta-glucosidase activity.

[0217] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 25. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 25. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 88 to 2232 of SEQ ID NO: 23. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 26 or the mature polypeptide thereof, which differ from SEQ ID NO: 25 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 25 that encode fragments of SEQ ID NO: 26 that have beta-glucosidase activity.

[0218] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 27. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 27. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 88 to 2232 of SEQ ID NO: 23. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 28 or the mature polypeptide thereof, which differ from SEQ ID NO: 27 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 27 that encode fragments of SEQ ID NO: 28 that have beta-glucosidase activity.

[0219] The present invention also relates to mutant polynucleotides comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, in which the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28.

[0220] As described earlier, the techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof.

[0221] The polynucleotide may also be a polynucleotide comprising or consisting of a nucleotide sequence encoding a polypeptide having beta-glucosidase activity that hybridizes under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, (iii) a full-length complementary strand of (i) or (ii); or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein.

[0222] In each of the preferred aspects above, the mature polypeptide is amino acids 20 to 861 of SEQ ID NO: 16, amino acids 20 to 861 of SEQ ID NO: 18, amino acids 20 to 863 of SEQ ID NO: 22, amino acids 37 to 878 of SEQ ID NO: 22, amino acids 32 to 744 of SEQ ID NO: 24, amino acids 20 to 860 of SEQ ID NO: 26, or amino acids 20 to 860 of SEQ ID NO: 28, and the mature polypeptide coding sequence is nucleotides 58 to 2584 of SEQ ID NO: 15, nucleotides 58 to 2584 of SEQ ID NO: 17, nucleotides 58 to 2580 of SEQ ID NO: 19, nucleotides 109 to 2751 of SEQ ID NO: 21, nucleotides 88 to 2232 of SEQ ID NO: 23, nucleotides 59 to 2580 of SEQ ID NO: 25, or nucleotides 59 to 2580 of SEQ ID NO: 27.

Beta-Glucosidase Fusion Polypeptides and Polynucleotides Thereof

[0223] The beta-glucosidase can also be in the form of a beta-glucosidase fusion protein. A beta-glucosidase fusion polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding a polypeptide having beta-glucosidase activity to a nucleotide sequence (or a portion thereof) encoding a polypeptide having endoglucanase activity and a nucleotide sequence encoding a signal peptide operably linked to the nucleotide sequence (or a portion thereof) encoding the polypeptide having endoglucanase activity. Techniques for producing fusion polypeptides are known in the art, and include, for example, ligating the coding sequences encoding the polypeptides so that they are in frame and expression of the fused polypeptide is under control of the same promoter(s) and terminator. Fusion proteins may also be constructed using intein technology in which fusions are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

[0224] The fusion protein having beta-glucosidase activity comprising at least the catalytic domain of an endoglucanase linked in frame to a signal peptide increases secretion of the fusion protein compared to the absence of at least the catalytic domain of the endoglucanase. The increase in secretion of the fusion protein having beta-glucosidase activity is at least 25%, preferably at least 50%, more preferably at least 100%, even more preferably at least 150%, even more preferably at least 200%, most preferably at least 500%, and even most preferably at least 1000% compared to the absence of at least the catalytic domain of the endoglucanase.

[0225] In each of the preferred aspects below, the components of the beta-glucosidase fusion protein construct are operably linked from the 5' end to the 3' end of the construct.

[0226] In a preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a catalytic domain of an endoglucanase; and a polynucleotide comprising a sequence encoding a catalytic domain of a beta-glucosidase.

[0227] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a mature polypeptide of an endoglucanase; and a polynucleotide comprising a sequence encoding a catalytic domain of a beta-glucosidase.

[0228] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a catalytic domain of an endoglucanase; and a polynucleotide comprising a sequence encoding a mature polypeptide of a beta-glucosidase.

[0229] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a mature polypeptide of an endoglucanase; and a polynucleotide comprising a sequence encoding a mature polypeptide of a beta-glucosidase.

[0230] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a catalytic domain of an endoglucanase; and a polynucleotide comprising a sequence encoding a full-length polypeptide of a beta-glucosidase.

[0231] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a mature polypeptide of an endoglucanase; and a polynucleotide comprising a sequence encoding a full-length polypeptide of a beta-glucosidase.

[0232] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a full-length polypeptide of an endoglucanase (signal peptide and mature polypeptide); and a polynucleotide comprising a sequence encoding a full-length polypeptide of a beta-glucosidase.

[0233] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a catalytic domain of an endoglucanase, and optionally a linker and/or a cellulose binding domain; and a polynucleotide comprising a sequence encoding a catalytic domain of a beta-glucosidase.

[0234] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a mature polypeptide of an endoglucanase, and optionally a linker and/or a cellulose binding domain; and a polynucleotide comprising a sequence encoding a catalytic domain of a beta-glucosidase.

[0235] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a catalytic domain of an endoglucanase, and optionally a linker and/or a cellulose binding domain; and a polynucleotide comprising a sequence encoding a mature polypeptide of a beta-glucosidase.

[0236] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a mature polypeptide of an endoglucanase, and optionally a linker and/or a cellulose binding domain; and a polynucleotide comprising a sequence encoding a mature polypeptide of a beta-glucosidase.

[0237] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a catalytic domain of an endoglucanase, and optionally a linker and/or a cellulose binding domain; and a polynucleotide comprising a sequence encoding a full-length polypeptide of a beta-glucosidase.

[0238] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a mature polypeptide of an endoglucanase, and optionally a linker and/or a cellulose binding domain; and a polynucleotide comprising a sequence encoding a full-length polypeptide of a beta-glucosidase.

[0239] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a catalytic domain of an endoglucanase; a polynucleotide comprising a sequence encoding another signal peptide; and a polynucleotide comprising a sequence encoding a catalytic domain of a beta-glucosidase.

[0240] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a mature polypeptide of an endoglucanase; a polynucleotide comprising a sequence encoding another signal peptide; and a polynucleotide comprising a sequence encoding a catalytic domain of a beta-glucosidase.

[0241] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a catalytic domain of an endoglucanase; a polynucleotide comprising a sequence encoding another signal peptide; and a polynucleotide comprising a sequence encoding a mature polypeptide of a beta-glucosidase.

[0242] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a mature polypeptide of an endoglucanase; a polynucleotide comprising a sequence encoding another signal peptide; and a polynucleotide comprising a sequence encoding a mature polypeptide of a beta-glucosidase.

[0243] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a catalytic domain of an endoglucanase, and optionally a linker and/or a cellulose binding domain; a polynucleotide comprising a sequence encoding another signal peptide; and a polynucleotide comprising a sequence encoding a catalytic domain of a beta-glucosidase.

[0244] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a mature polypeptide of an endoglucanase, and optionally a linker and/or a cellulose binding domain; a polynucleotide comprising a sequence encoding another signal peptide; and a polynucleotide comprising a sequence encoding a catalytic domain of a beta-glucosidase.

[0245] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a catalytic domain of an endoglucanase, and optionally a linker and/or a cellulose binding domain; a polynucleotide comprising a sequence encoding another signal peptide; and a polynucleotide comprising a sequence encoding a mature polypeptide of a beta-glucosidase.

[0246] In another preferred aspect, the beta-glucosidase fusion protein construct comprises a polynucleotide comprising a sequence encoding a signal peptide; a polynucleotide comprising a sequence encoding a mature polypeptide of an endoglucanase, and optionally a linker and/or a cellulose binding domain; a polynucleotide comprising a sequence encoding another signal peptide; and a polynucleotide comprising a sequence encoding a mature polypeptide of a beta-glucosidase.

[0247] In each of the preferred aspects above, the components of the beta-glucosidase fusion protein constructs further comprise a promoter region.

[0248] A polynucleotide encoding a catalytic domain, mature polypeptide, or full-length polypeptide having beta-glucosidase activity or endoglucanase activity may be obtained from any organism. For purposes of the present invention, the term "polypeptide" will be understood to include a full-length polypeptide, mature polypeptide, or catalytic domain; or portions or fragments thereof that have beta-glucosidase or endoglucanase activity. The term "obtained from" is used as defined herein.

[0249] Many endoglucanases have a multidomain structure consisting of a catalytic domain separated from a cellulose binding domain (CBD) by a linker peptide (Suurnakki et al., 2000, Cellulose 7: 189-209). The catalytic domain contains the active site whereas the CBD interacts with cellulose by binding the enzyme to it (van Tilbeurgh et al., 1986, FEBS Letters 204: 223-227; Tomme et al., 1988, European Journal of Biochemistry 170: 575-581).

[0250] Polynucleotides encoding polypeptides having beta-glucosidase activity are described earlier.

[0251] A polynucleotide encoding a polypeptide having endoglucanase activity may be obtained from a gene encoding a bacterial polypeptide. For example, the polypeptide may be a Gram positive bacterial polypeptide including, but not limited to, a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus polypeptide, e.g., a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus, Streptomyces lividans, or Streptomyces murinus polypeptide; or a Gram negative bacterial polypeptide including, but not limited to, an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide.

[0252] Examples of bacterial endoglucanases that can be used as sources for the polynucleotides in the methods of the present invention include, but are not limited to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); and Thermobifida fusca endoglucanase V (WO 05/093050).

[0253] A polynucleotide encoding a polypeptide having endoglucanase activity may also be obtained from a gene encoding a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide; or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide.

[0254] In a preferred aspect, a polynucleotide encoding a polypeptide having endoglucanase activity may be obtained from a gene encoding a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide.

[0255] In another preferred aspect, a polynucleotide encoding a polypeptide having endoglucanase activity may be obtained from a gene encoding an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium funiculosum, Penicillium purpurogenum, Phanerochaete chrysosporium, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaea saccata polypeptide.

[0256] Examples of fungal endoglucanases that can be used as sources for the polynucleotides in the methods of the present invention include, but are not limited to, a Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263; GenBank.TM. accession no. M15665); Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene 63:11-22; GenBank.TM. accession no. M19373); Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563; GenBank.TM. accession no. AB003694); Trichoderma reesei endoglucanase IV (Saloheimo et al., 1997, Eur. J. Biochem. 249: 584-591; GenBank.TM. accession no. Y11113); and Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GenBank.TM. accession no. Z33381); Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884); Aspergillis kawachii endoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439); Chrysosporium sp. C1 (U.S. Pat. No. 6,573,086; GenPept accession no. AAQ38150); Corynascus heterothallicus (U.S. Pat. No. 6,855,531; GenPept accession no. AAY00844); Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GenBank.TM. accession no. L29381); Humicola grisea var. thermoidea endoglucanase (GenBank.TM. accession no. AB003107); Melanocarpus albomyces endoglucanase (GenBank.TM. accession no. MAL515703); Neurospora crassa endoglucanase (GenBank.TM. accession no. XM_324477); Piromyces equi (Eberhardt et al., 2000, Microbiology 146: 1999-2008; GenPept accession no. CAB92325); Rhizopus oryzae (Moriya et al., 2003, J. Bacteriology 185: 1749-1756; GenBank.TM. accession nos. AB047927, AB056667, and AB056668); and Thielavia terrestris (WO 2004/053039; EMBL accession no. CQ827970).

[0257] Other endoglucanases are disclosed in more than 13 of the Glycosyl Hydrolase families using the classification according to Henrissat B., 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat B., and Bairoch A., 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696.

[0258] The techniques used to isolate or clone a polynucleotide encoding a polypeptide having endoglucanase activity are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides from such genomic DNA can be effected, e.g., by using the well known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shared structural features. See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used.

[0259] It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0260] Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0261] In a preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from an endoglucanase I gene.

[0262] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from an endoglucanase II gene.

[0263] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from an endoglucanase III gene.

[0264] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from an endoglucanase IV gene.

[0265] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from an endoglucanase V gene. In a more preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene. In a most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene comprising SEQ ID NO: 29 that encodes the polypeptide of SEQ ID NO: 30.

[0266] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from an endoglucanase VI gene.

[0267] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Family 5 endoglucanase gene. In a more preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Myceliophthora thermophila CBS 117.65 endoglucanase gene. In a most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Myceliophthora thermophila CBS 117.65 endoglucanase gene comprising SEQ ID NO: 31 that encodes the polypeptide of SEQ ID NO: 32. In another more preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a basidiomycete CBS 495.95 endoglucanase gene. In another most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a basidiomycete CBS 495.95 endoglucanase gene comprising SEQ ID NO: 33 that encodes the polypeptide of SEQ ID NO: 34. In another more preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a basidiomycete CBS 494.95 endoglucanase gene. In another most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a basidiomycete CBS 494.95 endoglucanase gene comprising SEQ ID NO: 35 that encodes the polypeptide of SEQ ID NO: 36.

[0268] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Family 6 endoglucanase gene. In another more preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL6B endoglucanase gene. In another most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL6B endoglucanase gene comprising SEQ ID NO: 37 that encodes the polypeptide of SEQ ID NO: 38. In another more preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL6C endoglucanase gene. In another most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL6C endoglucanase gene comprising SEQ ID NO: 39 that encodes the polypeptide of SEQ ID NO: 40.

[0269] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Family 7 endoglucanase gene. In another more preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL7C endoglucanase gene. In another most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL7C endoglucanase gene comprising SEQ ID NO: 41 that encodes the polypeptide of SEQ ID NO: 42. In another more preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL7E endoglucanase gene. In another most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL7E endoglucanase gene comprising SEQ ID NO: 43 that encodes the polypeptide of SEQ ID NO: 44. In another more preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL7F endoglucanase gene. In another most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Thielavia terrestris NRRL 8126 CEL7F endoglucanase gene comprising SEQ ID NO: 45 that encodes the polypeptide of SEQ ID NO: 46. In another more preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Cladorrhinum foecundissimum ATCC 62373 CEL7A endoglucanase gene. In another most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Cladorrhinum foecundissimum ATCC 62373 CEL7A endoglucanase gene comprising SEQ ID NO: 47 that encodes the polypeptide of SEQ ID NO: 48.

[0270] In another most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Trichoderma reesei strain No. VTT-D-80133 endoglucanase gene comprising SEQ ID NO: 49 that encodes the polypeptide of SEQ ID NO: 50 (GenBank.TM. accession no. M15665).

[0271] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Family 9 endoglucanase gene.

[0272] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Family 12 endoglucanase gene.

[0273] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Family 45 endoglucanase gene. In a more preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Family 45 endoglucanase gene. In a most preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the Family 45 endoglucanase is encoded by a polynucleotide obtained from a Humicola insolens endoglucanase V gene comprising SEQ ID NO: 29 that encodes the polypeptide of SEQ ID NO: 30, or othologs and variants thereof. Other preferred Family 45 endoglucanases and the polynucleotides thereof are obtained from Chrysosporium sp. C1 (U.S. Pat. No. 6,573,086; GenPept accession no. AAQ38150); Corynascus heterothallicus (U.S. Pat. No. 6,855,531; GenPept accession no. AAY00844); Piromyces equi (Eberhardt et al., 2000, Microbiology 146: 1999-2008; GenPept accession no. CAB92325); and Rhizopus oryzae (Moriya et al., 2003, J. Bacteriology 185: 1749-1756; GenBank.TM. accession nos. AB047927, AB056667, and AB056668).

[0274] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from a Family 74 endoglucanase gene.

[0275] In a preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from a Family 1 beta-glucosidase gene.

[0276] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from a Family 3 beta-glucosidase gene.

[0277] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from a Family 5 beta-glucosidase gene.

[0278] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from an Aspergillus oryzae beta-glucosidase gene. In a most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from an Aspergillus oryzae beta-glucosidase gene comprising SEQ ID NO: 15 that encodes the polypeptide of SEQ ID NO: 16 or an Aspergillus oryzae beta-glucosidase mutant gene comprising SEQ ID NO: 17 that encodes the polypeptide of SEQ ID NO: 18.

[0279] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from an Aspergillus fumigatus beta-glucosidase gene. In a most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from an Aspergillus fumigatus beta-glucosidase gene comprising SEQ ID NO: 19 that encodes the polypeptide of SEQ ID NO: 20.

[0280] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from a Penicillium brasilianum strain IBT 20888 beta-glucosidase gene. In a most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from a Penicillium brasilianum strain IBT 20888 beta-glucosidase gene comprising SEQ ID NO: 21 that encodes the polypeptide of SEQ ID NO: 22.

[0281] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from a Trichoderma reesei strain No. QM9414 beta-glucosidase gene. In another most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from a Trichoderma reesei strain No. QM9414 beta-glucosidase gene comprising SEQ ID NO: 23 that encodes the polypeptide of SEQ ID NO: 24 (GenBank.TM. accession no. U09580).

[0282] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from an Aspergillus niger beta-glucosidase gene. In a most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from an Aspergillus niger beta-glucosidase gene comprising SEQ ID NO: 25 that encodes the polypeptide of SEQ ID NO: 26.

[0283] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from an Aspergillus aculeatus beta-glucosidase gene. In a most preferred embodiment, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from an Aspergillus aculeatus beta-glucosidase gene comprising SEQ ID NO: 27 that encodes the polypeptide of SEQ ID NO: 28.

[0284] In another preferred aspect, the beta-glucosidase is naturally secreted. In another preferred aspect, the beta-glucosidase is not naturally secreted.

[0285] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide obtained from genes encoding a homologous polypeptide comprising or consisting of an amino acid sequence that has a degree of identity to the amino acid sequences of the full-length polypeptide, mature polypeptide, or catalytic domain of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, or SEQ ID NO: 50 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have endoglucanase activity. In a preferred aspect, the homologous polypeptide has an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the full-length polypeptide, mature polypeptide, or catalytic domain of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, or SEQ ID NO: 50.

[0286] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide obtained from a gene encoding a homologous polypeptide comprising or consisting of an amino acid sequence that has a degree of identity to the amino acid sequences of the full-length polypeptide, mature polypeptide, or catalytic domain of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have endoglucanase activity. In a preferred aspect, the homologous polypeptide comprises or consists of an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the full-length polypeptide, mature polypeptide, or catalytic domain of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28.

[0287] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase is encoded by a polynucleotide comprising or consisting of a nucleotide sequence that hybridizes under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A subsequence of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides.

[0288] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the beta-glucosidase is encoded by a polynucleotide comprising or consisting of a nucleotide sequence that hybridizes under very low stringency conditions, preferably low stringency conditions, more preferably medium stringency conditions, more preferably medium-high stringency conditions, even more preferably high stringency conditions, and most preferably very high stringency conditions with (i) SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.). A subsequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides.

[0289] The nucleotide sequence of SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, or SEQ ID NO: 49, or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, or SEQ ID NO: 50, or a fragment thereof for endoglucanase, and the nucleotide sequence of SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, or SEQ ID NO: 27, or a subsequence thereof, as well as the amino acid sequence of SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, or SEQ ID NO: 28, or a fragment thereof for beta-glucosidase, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having endoglucanase or beta-glucosidase activity from strains of different genera or species, as described supra.

[0290] For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to one of the nucleotide sequences described above under very low to very high stringency conditions, as described supra.

[0291] For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are as defined herein.

[0292] For long probes of at least 100 nucleotides in length, the carrier material is finally washed as defined herein.

[0293] For short probes that are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are as defined herein.

[0294] For short probes that are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed as defined herein.

[0295] In another preferred aspect, the full-length polypeptide, mature polypeptide, or catalytic domain of the endoglucanase described in the section "Polypeptides Having Cellobiohydrolase or Endoglucanase Activity and Polynucleotides Thereof" can also be used to construct beta-glucosidase fusion proteins.

[0296] In a preferred aspect, the beta-glucosidase fusion protein comprises or consists of SEQ ID NO: 104. In another preferred aspect, the beta-glucosidase fusion protein is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 103. In another preferred aspect, the beta-glucosidase fusion protein comprises or consists of SEQ ID NO: 106. In another preferred aspect, the beta-glucosidase fusion protein is encoded by a polynucleotide comprising or consisting of SEQ ID NO: 105.

[0297] As mentioned supra, many endoglucanases have a multidomain structure consisting of a catalytic domain separated from a cellulose binding domain by a linker peptide. In the methods of the present invention, the beta-glucosidase fusion constructs can further comprise a linker located 3' to the sequence comprising the endoglucanase catalytic domain and 5' to the sequence comprising the beta-glucosidase catalytic domain.

[0298] The linker can be obtained from the same gene as the catalytic domain of the endoglucanase or from a different endoglucanase gene. On the other hand, the linker can be synthetic in origin.

[0299] Examples of linkers that can be used in the methods of the present invention include, but are not limited to, linkers obtained from the genes for the Trichoderma reesei cellobiohydrolase I (Srisodsuk et al., 1993, Journal of Biological Chemistry 268: 20765-20761); Hypocrea jecorina (formerly Trichoderma reesei) Cel7A cellobiohydrolase (Mulakala et al., 2005, Proteins 60: 598-605); Humicola insolens endoglucanase V; and Thielavia terrestris NRRL 8126 CEL7C endoglucanase.

[0300] In a preferred aspect, the linker is obtained from a Humicola insolens endoglucanase gene. In another preferred aspect, the linker is obtained from a Trichoderma reesei endoglucanase gene. In a more preferred aspect, the linker is obtained from a Humicola insolens endoglucanase V (eg5) gene.

[0301] In another preferred aspect, the linker is obtained from a Thielavia terrestris endoglucanase gene. In another more preferred aspect, the linker is obtained from a Thielavia terrestris NRRL 8126 CEL7C endoglucanase gene.

[0302] In a preferred aspect, the linker is at least 5 amino acid residues. In a more preferred aspect, the linker is at least 15 amino acid residues. In a most preferred aspect, the linker is at least 25 amino acid residues.

[0303] In a preferred aspect, the linker is between about 5 to 60 amino acid residues. In a more preferred aspect, the linker is between about 15 to 50 amino acid residues. In a most preferred aspect, the linker is between about 25 to 45 amino acid residues.

[0304] Although a number of types of carbohydrate binding domains have been described, the majority thereof are commonly referred to as cellulose-binding domains (CBDs). A typical cellulose-binding domain occurs in a cellulase. Likewise, other sub-classes of CBDs would emcompass, for example, chitin-binding domains (CBDs which typically occur in chitinases), xylan-binding domains (CBDs which typically occur in xylanases), mannan-binding domains (CBDs which typically occur in mannanases), and starch-binding domains.

[0305] CBDs are found as integral parts of large polypeptides or proteins consisting of two or more polypeptide amino acid sequence regions, especially in hydrolytic enzymes (hydrolases) which typically comprise a catalytic domain containing the active site for substrate hydrolysis and a carbohydrate-binding domain (CBD) for binding to the carbohydrate substrate in question. Such enzymes can comprise more than one catalytic domain and one, two or three CBDs, and optionally further comprise one or more (several) polypeptide amino acid sequence regions linking the CBD(s) with the catalytic domain(s), a region of the latter type usually being denoted a "linker". Examples of hydrolytic enzymes comprising a CBD are cellulases, xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases (See P. Tomme et al., Cellulose-Binding Domains--Classification and Properties in Enzymatic Degradation of Insoluble Carbohydrates, John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618, 1996). Most of the known CBDs are derived from cellulases and xylanases.

[0306] A CBD may be located at the N or C terminus or at an internal position of a protein or polypeptide. The region of a polypeptide or protein that constitutes a CBD typically consists of more than about 30 and less than about 250 amino acid residues. For example: those CBDs listed and classified in Family I in accordance with Tomme et al., 1996, supra, consist of 33-37 amino acid residues, those listed and classified in Family IIa consist of 95-108 amino acid residues, those listed and classified in Family VI consist of 85-92 amino acid residues, while one CBD (derived from a cellulase from Clostridium thermocellum) listed and classified in Family VII consists of 240 amino acid residues. Accordingly, the molecular weight of an amino acid sequence constituting a CBD will typically be in the range of from about 4 kDa to about 40 kDa, and usually below about 35 kDa.

[0307] In the methods of the present invention, any CBD may be used. The CBD may be naturally associated with the endoglucanase or may be foreign to the endoglucanase.

[0308] In a preferred aspect, a CBD is obtained from a Trichoderma reesei endoglucanase (EG) gene. In a more preferred aspect, a CBD is obtained from a Trichoderma reesei endoglucanase EGI gene. In another more preferred aspect, a CBD is obtained from a Trichoderma reesei endoglucanase EGII gene. In another more preferred aspect, a CBD is obtained from a Trichoderma reesei endoglucanase EGV.

[0309] In another preferred aspect, a CBD is obtained from a Trichoderma reesei cellobiohydrolase (CBH) gene. In another preferred aspect, a CBD is obtained from a Trichoderma reesei CBHI gene (Terri et al., 1987, Gene 51: 42-52; Linder and Teeri, 1996, Biochemistry 93: 12251-12255). In another preferred aspect, a CBD is obtained from a Trichoderma reesei CBHII gene.

[0310] In another preferred aspect, a CBD is obtained from a Thielavia terrestris endoglucanase gene. In another more preferred aspect, a CBD is obtained from a Thielavia terrestris NRRL 8126 CEL7C endoglucanase gene.

[0311] The beta-glucosidase fusion constructs can further comprise a cleavage site. The cleavage site is preferably located after the sequence comprising at least the endoglucanase catalytic domain and before the sequence comprising at least the beta-glucosidase catalytic domain. Upon secretion of the beta-glucosidase fusion protein, the site is cleaved releasing the polypeptide having beta-glucosidase activity from the fusion protein.

[0312] Examples of cleavage sites include, but are not limited to, a Kex2 site which encodes the dipeptide Lys-Arg (Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-76; Svetina et al., 2000, J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991, Biotechnology 9: 378-381), an Ile-(Glu or Asp)-Gly-Arg (SEQ ID NO: 127) site, which is cleaved by a Factor Xa protease after the arginine residue (Eaton et al., 1986, Biochem. 25: 505-512); a Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 128) site, which is cleaved by an enterokinase after the lysine (Collins-Racie et al., 1995, Biotechnology 13: 982-987); a His-Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I (Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248); a Leu-Val-Pro-Arg-Gly-Ser (SEQ ID NO: 129) site, which is cleaved by thrombin after the Arg (Stevens, 2003, Drug Discovery World 4: 35-48); a Glu-Asn-Leu-Tyr-Phe-Gln-Gly (SEQ ID NO: 130) site, which is cleaved by TEV protease after the Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro (SEQ ID NO: 131) site, which is cleaved by a genetically engineered form of human rhinovirus 3C protease after the Gln (Stevens, 2003, supra).

Polypeptides Having Cellobiohydrolase or Endoglucanase Activity and Polynucleotides Thereof

[0313] In the present invention, polypeptides having cellobiohydrolase or endoglucanase activity and their polynucleotides are preferably selected from the group consisting of a Trichoderma reesei cellobiohydrolase I (CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and a Trichoderma reesei endoglucanase I (CEL7B), and orthologs or variants thereof, and further selected from the group consisting of a Trichoderma reesei endoglucanase II (CEL5A), a Trichoderma reesei endoglucanase III (CEL12A), and a Trichoderma reesei endoglucanase V (CEL45A), and orthologs or variants thereof.

[0314] In a first aspect, isolated polypeptides having cellobiohydrolase I activity comprise or consist of amino acid sequences that have a degree of identity to the mature polypeptide of SEQ ID NO: 52 (Trichoderma reesei cellobiohydrolase I; CEL7A) of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have cellobiohydrolase I activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides comprise or consist of an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 52.

[0315] A polypeptide having cellobiohydrolase I activity preferably comprises the amino acid sequence of SEQ ID NO: 52 or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase I activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 52. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 52. In another preferred aspect, the polypeptide comprises amino acids 18 to 514 of SEQ ID NO: 52, or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase I activity. In another preferred aspect, the polypeptide comprises amino acids 18 to 514 of SEQ ID NO: 52. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 52 or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase I activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 52. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 52. In another preferred aspect, the polypeptide consists of amino acids 18 to 514 of SEQ ID NO: 52 or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase I activity. In another preferred aspect, the polypeptide consists of amino acids 18 to 514 of SEQ ID NO: 52.

[0316] In another first aspect, isolated polypeptides having cellobiohydrolase II activity comprise or consist of amino acid sequences that have a degree of identity to the mature polypeptide of SEQ ID NO: 54 (Trichoderma reesei cellobiohydrolase II; CEL6A) of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, even more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have cellobiohydrolase II activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides comprise or consist of an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 54.

[0317] A polypeptide having cellobiohydrolase II activity preferably comprises the amino acid sequence of SEQ ID NO: 54 or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase II activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 54. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 54. In another preferred aspect, the polypeptide comprises amino acids 25 to 471 of SEQ ID NO: 54, or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase II activity. In another preferred aspect, the polypeptide comprises amino acids 25 to 471 of SEQ ID NO: 54. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 54 or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase II activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 54. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 54. In another preferred aspect, the polypeptide consists of amino acids 25 to 471 of SEQ ID NO: 54 or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase II activity. In another preferred aspect, the polypeptide consists of amino acids 25 to 471 of SEQ ID NO: 54.

[0318] In another first aspect, isolated polypeptides having endoglucanase I activity comprise or consist of amino acid sequences that have a degree of identity to the mature polypeptide of SEQ ID NO: 56 (Trichoderma reesei endoglucanase I; CEL7B) of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have endoglucanase I activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides comprise or consist of an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 56.

[0319] A polypeptide having endoglucanase I activity preferably comprises the amino acid sequence of SEQ ID NO: 56 or an allelic variant thereof; or a fragment thereof that has endoglucanase I activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 56. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 56. In another preferred aspect, the polypeptide comprises amino acids 23 to 459 of SEQ ID NO: 56, or an allelic variant thereof; or a fragment thereof that has endoglucanase I activity. In another preferred aspect, the polypeptide comprises amino acids 23 to 459 of SEQ ID NO: 56. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 56 or an allelic variant thereof; or a fragment thereof that has endoglucanase I activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 56. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 56. In another preferred aspect, the polypeptide consists of amino acids 23 to 459 of SEQ ID NO: 56 or an allelic variant thereof; or a fragment thereof that has endoglucanase I activity. In another preferred aspect, the polypeptide consists of amino acids 23 to 459 of SEQ ID NO: 56.

[0320] In another first aspect, isolated polypeptides having endoglucanase II activity comprise or consist of amino acid sequences that have a degree of identity to the mature polypeptide of SEQ ID NO: 58 (Trichoderma reesei endoglucanase II; CEL5A) of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have endoglucanase II activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides comprise or consist of an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 58.

[0321] A polypeptide having endoglucanase II activity preferably comprises the amino acid sequence of SEQ ID NO: 58 or an allelic variant thereof; or a fragment thereof that has endoglucanase II activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 58. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 58. In another preferred aspect, the polypeptide comprises amino acids 22 to 418 of SEQ ID NO: 58, or an allelic variant thereof; or a fragment thereof that has endoglucanase II activity. In another preferred aspect, the polypeptide comprises amino acids 22 to 418 of SEQ ID NO: 58. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 58 or an allelic variant thereof; or a fragment thereof that has endoglucanase II activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 58. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 58. In another preferred aspect, the polypeptide consists of amino acids 22 to 418 of SEQ ID NO: 58 or an allelic variant thereof; or a fragment thereof that has endoglucanase II activity. In another preferred aspect, the polypeptide consists of amino acids 22 to 418 of SEQ ID NO: 58.

[0322] In another first aspect, isolated polypeptides having endoglucanase III activity comprise or consist of amino acid sequences that have a degree of identity to the mature polypeptide of SEQ ID NO: 60 (Trichoderma reesei endoglucanase III; CEL12A) of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have endoglucanase III activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides comprise or consist of an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 60.

[0323] A polypeptide having endoglucanase III activity preferably comprises the amino acid sequence of SEQ ID NO: 60 or an allelic variant thereof; or a fragment thereof that has endoglucanase III activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 60. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 60. In another preferred aspect, the polypeptide comprises amino acids 17 to 234 of SEQ ID NO: 60, or an allelic variant thereof; or a fragment thereof that has endoglucanase III activity. In another preferred aspect, the polypeptide comprises amino acids 17 to 234 of SEQ ID NO: 60. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 60 or an allelic variant thereof; or a fragment thereof that has endoglucanase III activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 60. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 60. In another preferred aspect, the polypeptide consists of amino acids 17 to 234 of SEQ ID NO: 60 or an allelic variant thereof; or a fragment thereof that has endoglucanase III activity. In another preferred aspect, the polypeptide consists of amino acids 17 to 234 of SEQ ID NO: 60.

[0324] In another first aspect, isolated polypeptides having endoglucanase V activity comprise or consist of amino acid sequences that have a degree of identity to the mature polypeptide of SEQ ID NO: 62 (Trichoderma reesei endoglucanase V; CEL45A) of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have endoglucanase V activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides comprise or consist of an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 62.

[0325] A polypeptide having endoglucanase V activity preferably comprises the amino acid sequence of SEQ ID NO: 62 or an allelic variant thereof; or a fragment thereof that has endoglucanase V activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 62. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 62. In another preferred aspect, the polypeptide comprises amino acids 18 to 242 of SEQ ID NO: 62, or an allelic variant thereof; or a fragment thereof that has endoglucanase V activity. In another preferred aspect, the polypeptide comprises amino acids 18 to 242 of SEQ ID NO: 62. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 62 or an allelic variant thereof; or a fragment thereof that has endoglucanase V activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 62. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 62. In another preferred aspect, the polypeptide consists of amino acids 18 to 242 of SEQ ID NO: 62 or an allelic variant thereof; or a fragment thereof that has endoglucanase V activity. In another preferred aspect, the polypeptide consists of amino acids 18 to 242 of SEQ ID NO: 62.

[0326] In another first aspect, isolated polypeptides having cellobiohydrolase II activity comprise or consist of amino acid sequences that have a degree of identity to the mature polypeptide of SEQ ID NO: 64 (Thielavia terrestris cellobiohydrolase II; CEL6A) of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which have cellobiohydrolase II activity (hereinafter "homologous polypeptides"). In a preferred aspect, the homologous polypeptides comprise or consist of an amino acid sequence that differs by ten amino acids, preferably by five amino acids, more preferably by four amino acids, even more preferably by three amino acids, most preferably by two amino acids, and even most preferably by one amino acid from the mature polypeptide of SEQ ID NO: 64.

[0327] A polypeptide having cellobiohydrolase II activity preferably comprises the amino acid sequence of SEQ ID NO: 64 or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase II activity. In a preferred aspect, the polypeptide comprises the amino acid sequence of SEQ ID NO: 64. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 64. In another preferred aspect, the polypeptide comprises amino acids 18 to 481 of SEQ ID NO: 64, or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase II activity. In another preferred aspect, the polypeptide comprises amino acids 18 to 481 of SEQ ID NO: 64. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 64 or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase II activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 64. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 64. In another preferred aspect, the polypeptide consists of amino acids 18 to 481 of SEQ ID NO: 64 or an allelic variant thereof; or a fragment thereof that has cellobiohydrolase II activity. In another preferred aspect, the polypeptide consists of amino acids 18 to 481 of SEQ ID NO: 64.

[0328] Preferably, a fragment of the mature polypeptide of SEQ ID NO: 52 contains at least 425 amino acid residues, more preferably at least 450 amino acid residues, and most preferably at least 475 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 54 contains at least 390 amino acid residues, more preferably at least 410 amino acid residues, and most preferably at least 430 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 56 contains at least 370 amino acid residues, more preferably at least 390 amino acid residues, and most preferably at least 410 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 58 contains at least 340 amino acid residues, more preferably at least 360 amino acid residues, and most preferably at least 380 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 60 contains at least 190 amino acid residues, more preferably at least 200 amino acid residues, and most preferably at least 210 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 62 contains at least 190 amino acid residues, more preferably at least 200 amino acid residues, and most preferably at least 210 amino acid residues. Preferably, a fragment of the mature polypeptide of SEQ ID NO: 64 contains at least 400 amino acid residues, more preferably at least 420 amino acid residues, and most preferably at least 440 amino acid residues.

[0329] Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 51 contains at least 1275 nucleotides, more preferably at least 1350 nucleotides, and most preferably at least 1425 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 53 contains at least 1170 nucleotides, more preferably at least 1230 nucleotides, and most preferably at least 1290 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 55 contains at least 1110 nucleotides, more preferably at least 1170 nucleotides, and most preferably at least 1230 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 57 contains at least 1020 nucleotides, more preferably at least 1080 nucleotides, and most preferably at least 1140 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 59 contains at least 570 nucleotides, more preferably at least 600 nucleotides, and most preferably at least 630 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 61 contains at least 570 nucleotides, more preferably at least 600 nucleotides, and most preferably at least 630 nucleotides. Preferably, a subsequence of the mature polypeptide coding sequence of SEQ ID NO: 63 contains at least 1200 nucleotides, more preferably at least 1260 nucleotides, and most preferably at least 1320 nucleotides.

[0330] In a second aspect, isolated polypeptides having cellobiohydrolase I activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 51, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 51, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 51 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has cellobiohydrolase I activity.

[0331] In another second aspect, isolated polypeptides having cellobiohydrolase II activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 53, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 53, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 53 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has cellobiohydrolase II activity.

[0332] In another second aspect, isolated polypeptides having endoglucanase I activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 55, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 55, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 55 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has cellobiohydrolase II activity.

[0333] In another second aspect, isolated polypeptides having endoglucanase II activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 57, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 57, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 57 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has endoglucanase II activity.

[0334] In another second aspect, isolated polypeptides having endoglucanase III activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 59, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 59, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 59 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has endoglucanase III activity.

[0335] In another second aspect, isolated polypeptides having endoglucanase V activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 61, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 61, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 61 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has endoglucanase V activity.

[0336] In another second aspect, isolated polypeptides having cellobiohydrolase II activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 63, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 63, (iii) a subsequence of (i) or (ii), or (iv) a full-length complementary strand of (i), (ii), or (iii) (J. Sambrook, E. F. Fritsch, and T. Maniatus, 1989, supra). A subsequence of the mature polypeptide coding sequence of SEQ ID NO: 63 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment that has cellobiohydrolase II activity.

[0337] In a preferred aspect, the mature polypeptide coding sequence is nucleotides 52 to 1542 of SEQ ID NO: 51, nucleotides 73 to 1413 of SEQ ID NO: 53, nucleotides 67 to 1377 of SEQ ID NO: 55, nucleotides 64 to 1254 of SEQ ID NO: 57, nucleotides 49 to 702 of SEQ ID NO: 59, nucleotides 52 to 726 of SEQ ID NO: 61, or nucleotides 52 to 1443 of SEQ ID NO: 63. The nucleotide sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, or a subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64, or a fragment thereof, may be used to design a nucleic acid probe to identify and clone DNA encoding polypeptides having cellobiohydrolase or endoglucanase activity from strains of different genera or species, as described supra.

[0338] For purposes of the present invention, hybridization indicates that the nucleotide sequence hybridizes to a labeled nucleic acid probe corresponding to the mature polypeptide coding sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, its full-length complementary strand, or a subsequence thereof, under very low to very high stringency conditions, as described supra.

[0339] In a preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 51. In another preferred aspect, the nucleic acid probe is nucleotides 52 to 1542 of SEQ ID NO: 51. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 52, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 51.

[0340] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 53. In another preferred aspect, the nucleic acid probe is nucleotides 73 to 1413 of SEQ ID NO: 53. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 54, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 53.

[0341] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 55. In another preferred aspect, the nucleic acid probe is nucleotides 67 to 1377 of SEQ ID NO: 55. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 56, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 55.

[0342] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 57. In another preferred aspect, the nucleic acid probe is nucleotides 64 to 1254 of SEQ ID NO: 57. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 58, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 57.

[0343] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 59. In another preferred aspect, the nucleic acid probe is nucleotides 49 to 702 of SEQ ID NO: 59. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 60, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 59.

[0344] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 61. In another preferred aspect, the nucleic acid probe is nucleotides 52 to 726 of SEQ ID NO: 61. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 62, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 61.

[0345] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 63. In another preferred aspect, the nucleic acid probe is nucleotides 52 to 1443 of SEQ ID NO: 63. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 64, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 63. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pTter6A which is contained in E. coli NRRL B-30802, wherein the polynucleotide sequence thereof encodes a polypeptide having cellobiohydrolase activity. In another preferred aspect, the nucleic acid probe is the mature polypeptide coding region contained in plasmid pTter6A which is contained in E. coli NRRL B-30802.

[0346] For long probes of at least 100 nucleotides in length, very low to very high stringency conditions are as defined herein.

[0347] For long probes of at least 100 nucleotides in length, the carrier material is finally washed as defined herein.

[0348] For short probes that are about 15 nucleotides to about 70 nucleotides in length, stringency conditions are as defined herein.

[0349] For short probes that are about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed as defined herein.

[0350] In a third aspect, isolated polypeptides having cellobiohydrolase or endoglucanase activity are encoded by polynucleotides comprising or consisting of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which encode an active polypeptide.

[0351] In a preferred aspect, the mature polypeptide coding sequence is nucleotides 52 to 1542 of SEQ ID NO: 51, nucleotides 73 to 1413 of SEQ ID NO: 53, nucleotides 67 to 1377 of SEQ ID NO: 55, nucleotides 64 to 1254 of SEQ ID NO: 57, nucleotides 49 to 702 of SEQ ID NO: 59, nucleotides 52 to 726 of SEQ ID NO: 61, or nucleotides 52 to 1443 of SEQ ID NO: 63. See polynucleotide section herein.

[0352] In a sixth aspect, isolated polypeptides having cellobiohydrolase or endoglucanase activity are artificial variants comprising a substitution, deletion, and/or insertion of one or more (or several) amino acids of the mature polypeptide of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64; or a homologous sequence thereof. Methods for preparing such artificial variants are described supra.

[0353] The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64, is 10, preferably 9, more preferably 8, more preferably 7, more preferably at most 6, more preferably 5, more preferably 4, even more preferably 3, most preferably 2, and even most preferably 1.

[0354] A polypeptide having cellobiohydrolase or endoglucanase activity may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" is used as defined herein. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0355] A polypeptide having cellobiohydrolase or endoglucanase activity may be a bacterial polypeptide. For example, the polypeptide may be a gram positive bacterial polypeptide such as a Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, or Oceanobacillus polypeptide having cellobiohydrolase or endoglucanase activity, or a Gram negative bacterial polypeptide such as an E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, or Ureaplasma polypeptide having cellobiohydrolase or endoglucanase activity.

[0356] In a preferred aspect, the polypeptide is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, or Bacillus thuringiensis polypeptide having cellobiohydrolase or endoglucanase activity.

[0357] In another preferred aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having cellobiohydrolase or endoglucanase activity.

[0358] In another preferred aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having cellobiohydrolase or endoglucanase activity.

[0359] The polypeptide having cellobiohydrolase or endoglucanase activity may also be a fungal polypeptide, and more preferably a yeast polypeptide such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia polypeptide having cellobiohydrolase or endoglucanase activity; or more preferably a filamentous fungal polypeptide such as an Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, or Trichoderma polypeptide having cellobiohydrolase or endoglucanase activity.

[0360] In a preferred aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having cellobiohydrolase or endoglucanase activity.

[0361] In another preferred aspect, the polypeptide is an Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Coprinus cinereus, Diplodia gossyppina, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium suiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Magnaporthe grisea, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Pseudoplectania nigrella, Thermoascus aurantiacus, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, or Trichophaea saccata polypeptide having cellobiohydrolase or endoglucanase activity.

[0362] In a more preferred aspect, the polypeptide is a Trichoderma reesei polypeptide having cellobiohydrolase or endoglucanase activity. In another most preferred aspect, the polypeptide is a Trichoderma reesei RutC30 (ATCC 56765) polypeptide, having cellobiohydrolase or endoglucanase activity e.g., the mature polypeptide of SEQ ID NO: 52, or 54, or fragments thereof that have cellobiohydrolase activity, and the mature polypeptide of SEQ ID NO: 56, 58, 60, or 62, or fragments thereof that have endoglucanase activity.

[0363] In another more preferred aspect, the polypeptide is a Thielavia terrestris polypeptide having cellobiohydrolase activity. In another most preferred aspect, the polypeptide is a Thielavia terrestris (NRRL 8126) polypeptide, having cellobiohydrolase II activity e.g., the mature polypeptide of SEQ ID NO: 64, or fragments thereof that have cellobiohydrolase activity.

[0364] It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0365] Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0366] Furthermore, such polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) using the above-mentioned probes, as described herein.

[0367] Polypeptides having cellobiohydrolase or endoglucanase activity also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide or fragment thereof having cellobiohydrolase or endoglucanase activity. A fused polypeptide is produced as described herein.

[0368] Polynucleotides comprising or consisting of nucleotide sequences that encode polypeptides having cellobiohydrolase or endoglucanase activity, can be isolated and utilized to practice the methods of the present invention, as described herein.

[0369] The polynucleotides comprise or consist of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63 of preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, most preferably at least 95%, and even most preferably at least 96%, 97%, 98%, or 99%, which encode a polypeptide having cellobiohydrolase or endoglucanase activity.

[0370] In a preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 51. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 51. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 52 or the mature polypeptide thereof, which differ from SEQ ID NO: 51 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 51 that encode fragments of SEQ ID NO: 52 that have cellobiohydrolase I activity.

[0371] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 53. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 53. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 54 or the mature polypeptide thereof, which differ from SEQ ID NO: 53 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 53 that encode fragments of SEQ ID NO: 54 that have cellobiohydrolase II activity.

[0372] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 55. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 55. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 56 or the mature polypeptide thereof, which differ from SEQ ID NO: 51 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 55 that encode fragments of SEQ ID NO: 56 that have endoglucanase I activity.

[0373] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 57. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 57. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 58 or the mature polypeptide thereof, which differ from SEQ ID NO: 57 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 57 that encode fragments of SEQ ID NO: 58 that have endoglucanase II activity.

[0374] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 59. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 59. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 60 or the mature polypeptide thereof, which differ from SEQ ID NO: 59 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 59 that encode fragments of SEQ ID NO: 60 that have endoglucanase III activity.

[0375] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 61. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 61. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 62 or the mature polypeptide thereof, which differ from SEQ ID NO: 61 by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 61 that encode fragments of SEQ ID NO: 62 that have endoglucanase V activity.

[0376] In another preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 63. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pTter6A which is contained in E. coli NRRL B-30802. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region of SEQ ID NO: 63. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 52 to 1443 of SEQ ID NO: 63. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding region contained in plasmid pTter6A which is contained in E. coli NRRL B-30802. The present invention also encompasses nucleotide sequences that encode a polypeptide comprising or consisting of the amino acid sequence of SEQ ID NO: 64 or the mature polypeptide thereof, which differ from SEQ ID NO: 63 or the mature polypeptide coding sequence thereof by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 63 that encode fragments of SEQ ID NO: 64 that have cellobiohydrolase II activity.

[0377] The present invention also relates to mutant polynucleotides comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, in which the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, or SEQ ID NO: 64. In a preferred aspect, the mature polypeptide is amino acids 18 to 541 of SEQ ID NO: 52, amino acids 25 to 471 of SEQ ID NO: 54, amino acids 23 to 459 of SEQ ID NO: 56, amino acids 22 to 418 of SEQ ID NO: 58, amino acids 17 to 234 of SEQ ID NO: 60, amino acids 18 to 242 of SEQ ID NO: 62, or amino acids 18 to 481 of SEQ ID NO: 64. In another preferred aspect, the mature polypeptide coding sequence is nucleotides 52 to 1542 of SEQ ID NO: 51, nucleotides 73 to 1413 of SEQ ID NO: 53, nucleotides 67 to 1377 of SEQ ID NO: 55, nucleotides 64 to 1254 of SEQ ID NO: 57, nucleotides 49 to 702 of SEQ ID NO: 59, nucleotides 52 to 726 of SEQ ID NO: 61, or nucleotides 52 to 1443 of SEQ ID NO: 63.

[0378] As described earlier, the techniques used to isolate or clone a polynucleotide encoding a polypeptide are known in the art and include isolation from genomic DNA, preparation from cDNA, or a combination thereof.

[0379] The polynucleotide may also be a polynucleotide comprising or consisting of a nucleotide sequence encoding a polypeptide having cellobiohydrolase or endoglucanase activity that hybridize under at least very low stringency conditions, preferably at least low stringency conditions, more preferably at least medium stringency conditions, more preferably at least medium-high stringency conditions, even more preferably at least high stringency conditions, and most preferably at least very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59, SEQ ID NO: 61, or SEQ ID NO: 63, (ii) the cDNA sequence contained in or the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, or SEQ ID NO: 59, SEQ ID NO: 61, or

[0380] SEQ ID NO: 63, (iii) a full-length complementary strand of (i) or (ii); or allelic variants and subsequences thereof (Sambrook et al., 1989, supra), as defined herein. In a preferred aspect, the mature polypeptide coding sequence is nucleotides 52 to 1542 of SEQ ID NO: 51, nucleotides 73 to 1413 of SEQ ID NO: 53, nucleotides 67 to 1377 of SEQ ID NO: 55, nucleotides 64 to 1254 of SEQ ID NO: 57, nucleotides 49 to 702 of SEQ ID NO: 59, nucleotides 52 to 726 of SEQ ID NO: 61, or nucleotides 52 to 1443 of SEQ ID NO: 63.

Nucleic Acid Constructs

[0381] An isolated polynucleotide encoding a polypeptide having cellulolytic enhancing activity, a polypeptide having beta-glucosidase activity, a beta-glucosidase fusion polypeptide, a polypeptide having endoglucanase activity, a polypeptide having cellobiohydrolase activity, or a polypeptide having other cellulolytic enzyme activity may be manipulated in a variety of ways to provide for expression of the polypeptide by constructing a nucleic acid construct comprising an isolated polynucleotide encoding the polypeptide operably linked to one or more (several) control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences. Manipulation of the polynucleotide's sequence prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotide sequences utilizing recombinant DNA methods are well known in the art.

[0382] The control sequence may be an appropriate promoter sequence, a nucleotide sequence that is recognized by a host cell for expression of a polynucleotide encoding such a polypeptide. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any nucleotide sequence that shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.

[0383] Examples of suitable promoters for directing the transcription of the nucleic acid constructs in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Dania (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Fusarium oxysporum trypsin-like protease (WO 96/00787), Trichoderma reesei beta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase); and mutant, truncated, and hybrid promoters thereof.

[0384] The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice may be used in the present invention.

[0385] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease.

[0386] The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice may be used in the present invention.

[0387] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0388] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3' terminus of the nucleotide sequence and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used in the present invention.

[0389] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase.

[0390] The control sequence may also be a signal peptide coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway. The 5' end of the coding sequence of the nucleotide sequence may inherently contain a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding region that is foreign to the coding sequence. The foreign signal peptide coding region may be required where the coding sequence does not naturally contain a signal peptide coding region. Alternatively, the foreign signal peptide coding region may simply replace the natural signal peptide coding region in order to enhance secretion of the polypeptide. However, any signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice, i.e., secreted into a culture medium, may be used in the present invention.

[0391] Effective signal peptide coding regions for filamentous fungal host cells are the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, Humicola insolens endoglucanase V, and Humicola lanuginosa lipase.

[0392] In a preferred aspect, the signal peptide comprises or consists of amino acids 1 to 19 of SEQ ID NO: 2. In another preferred aspect, the signal peptide coding region comprises or consists of nucleotides 330 to 387 of SEQ ID NO: 1.

[0393] In another preferred aspect, the signal peptide comprises or consists of amino acids 1 to 17 of SEQ ID NO: 4. In another preferred aspect, the signal peptide coding region comprises or consists of nucleotides 47 to 97 of SEQ ID NO: 3.

[0394] In another preferred aspect, the signal peptide comprises or consists of amino acids coding region is amino acids 1 to 19 of SEQ ID NO: 6. In another preferred aspect, the signal peptide coding region comprises or consists of nucleotides 69 to 125 of SEQ ID NO: 5.

[0395] In another preferred aspect, the signal peptide comprises or consists of amino acids 1 to 18 of SEQ ID NO: 8. In another preferred aspect, the signal peptide coding region comprises or consists of nucleotides 1 to 54 of SEQ ID NO: 7.

[0396] In another preferred aspect, the signal peptide comprises or consists of amino acids 1 to 19 of SEQ ID NO: 10. In another preferred aspect, the signal peptide coding region comprises or consists of nucleotides 1 to 57 of SEQ ID NO: 9.

[0397] In another preferred aspect, the signal peptide comprises or consists of amino acids 1 to 22 of SEQ ID NO: 12. In another preferred aspect, the signal peptide coding region comprises or consists of nucleotides 1 to 66 of SEQ ID NO: 11.

[0398] In another preferred aspect, the signal peptide comprises or consists of amino acids 1 to 19 of SEQ ID NO: 14. In another preferred aspect, the signal peptide coding region comprises or consists of nucleotides 20 to 76 of SEQ ID NO: 13.

[0399] The control sequence may also be a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region may be obtained from the genes for Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836).

[0400] Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.

[0401] It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in filamentous fungi include the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter may be used as regulatory sequences. Other examples of regulatory sequences are those that allow for gene amplification. In eukaryotic systems, these regulatory sequences include the dihydrofolate reductase gene that is amplified in the presence of methotrexate, and the metallothionein genes that are amplified with heavy metals. In these cases, the nucleotide sequence encoding the polypeptide would be operably linked with the regulatory sequence.

Expression Vectors

[0402] The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector comprising a polynucleotide encoding a polypeptide having cellulolytic enhancing activity, a polypeptide having beta-glucosidase activity, a beta-glucosidase fusion polypeptide, a polypeptide having endoglucanase activity, a polypeptide having cellobiohydrolase activity, or a polypeptide having other cellulolytic enzyme activity, a promoter, and transcriptional and translational stop signals. The expression vectors may include one or more (several) convenient restriction sites to allow for insertion or substitution of the nucleotide sequence encoding the polypeptide at such sites. Alternatively, a polynucleotide encoding such a polypeptide may be expressed by inserting the nucleotide sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0403] The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the nucleotide sequence. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vectors may be linear or closed circular plasmids.

[0404] The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon may be used.

[0405] The vectors preferably contain one or more (several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0406] Selectable markers for use in a filamentous fungal host cell include, but are not limited to, amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof. Preferred for use in an Aspergillus cell are the amdS and pyrG genes of Aspergillus nidulans or Aspergillus oryzae and the bar gene of Streptomyces hygroscopicus.

[0407] The vectors preferably contain an element(s) that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0408] For integration into the host cell genome, the vector may rely on the polynucleotide's sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which have a high degree of identity to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0409] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term "origin of replication" or "plasmid replicator" is defined herein as a nucleotide sequence that enables a plasmid or vector to replicate in vivo.

[0410] Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Research 15: 9163-9175; WO 00/24883). Isolation of the AMA1 gene and construction of plasmids or vectors comprising the gene can be accomplished according to the methods disclosed in WO 00/24883.

[0411] More than one copy of a polynucleotide encoding such a polypeptide may be inserted into the host cell to increase production of the polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0412] The procedures used to ligate the elements described above to construct the recombinant expression vectors are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cells

[0413] Recombinant filamentous fungal host cells, comprising a polynucleotide encoding a polypeptide having cellulolytic enhancing activity, a polypeptide having beta-glucosidase activity, a beta-glucosidase fusion polypeptide, a polypeptide having endoglucanase activity, a polypeptide having cellobiohydrolase activity, or a polypeptide having other cellulolytic enzyme activity can be advantageously used in the recombinant production of the polypeptide. A vector comprising such a polynucleotide is introduced into a host cell so that the vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier. The term "host cell" encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. The choice of a host cell will to a large extent depend upon the gene encoding the polypeptide and its source.

[0414] "Filamentous fungi" include all filamentous forms of the subdivision Eumycota and Oomycota (as defined by Hawksworth et al., 1995, supra). The filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. In contrast, vegetative growth by yeasts such as Saccharomyces cerevisiae is by budding of a unicellular thallus and carbon catabolism may be fermentative.

[0415] In a preferred aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosproium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trametes, or Trichoderma cell.

[0416] In a more preferred aspect, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium lucknowense, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0417] In a most preferred aspect, the filamentous fungal host cell is an Aspergillus oryzae. In another most preferred aspect, the filamentous fungal host cell is a Trichoderma reesei cell. In another most preferred aspect, the filamentous fungal host cell is a Fusarium venenatum cell. In another most preferred aspect, the filamentous fungal host cell is a Chrysosporium lucknowense cell.

[0418] Fungal cells may be transformed by a process involving protoplast formation, transformation of the protoplasts, and regeneration of the cell wall in a manner known per se. Suitable procedures for transformation of Aspergillus and Trichoderma host cells are described in EP 238 023 and Yelton et al., 1984, Proceedings of the National Academy of Sciences USA 81: 1470-1474. Suitable methods for transforming Fusarium species are described by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787. Yeast may be transformed using the procedures described by Becker and Guarente, In Abelson, J. N. and Simon, M. I., editors, Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Volume 194, pp 182-187, Academic Press, Inc., New York; Ito et al., 1983, Journal of Bacteriology 153: 163; and Hinnen et al., 1978, Proceedings of the National Academy of Sciences USA 75: 1920.

Methods of Production

[0419] Methods for producing a cellulolytic protein composition of the present invention, comprise (a) cultivating a filamentous fungal cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0420] Alternatively, methods for producing a cellulolytic protein composition of the present invention, comprise (a) cultivating a recombinant filamentous fungal host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0421] In the production methods, the filamentous fungal cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the protein components are secreted into the nutrient medium, they can be recovered directly from the medium. If not, they can be recovered from cell lysates.

[0422] The protein components of the composition can be detected using the methods described herein or any method known in the art.

[0423] The resulting cellulolytic protein composition may be recovered using methods known in the art. For example, the cellulolytic protein composition may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0424] The protein components of the composition may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.

Methods for Processing Cellulose-Containing Material

[0425] The present invention also relates to methods for degrading or converting a cellulose-containing material, comprising: treating the cellulose-containing material with an effective amount of a cellulolytic protein composition of the present invention.

[0426] The present invention further relates to methods for producing a fermentation product, comprising: (a) saccharifying a cellulose-containing material with an effective amount of a cellulolytic protein composition of the present invention; (b) fermenting the saccharified cellulose-containing material of step (a) with one or more (several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[0427] The methods of the present invention can be used to hydrolyze (saccharify) a cellulose-containing material, e.g., lignocellulose, to fermentable sugars and convert the fermentable sugars to many useful substances, e.g., chemicals and fuels. The production of a desired fermentation product from cellulose-containing material typically involves pretreatment, enzymatic hydrolysis (saccharification), and fermentation.

[0428] The processing of cellulose-containing material according to the present invention can be accomplished using processes known in the art. Moreover, the methods of the present invention can be implemented using any biomass processing apparatus configured to operate in accordance with the invention.

[0429] Hydrolysis (saccharification) and fermentation, separate or simultaneous, include, but are not limited to, separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and cofermentation (SSCF), hybrid hydrolysis and fermentation (HHF), SHCF (separate hydrolysis and co-fermentation), HHCF (hybrid hydrolysis and fermentation), and direct microbial conversion (DMC). It is understood herein that any method known in the art comprising pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination thereof, can be used in the practicing the methods of the present invention.

[0430] A conventional apparatus can include a fed-batch stirred reactor, a batch stirred reactor, a continuous flow stirred reactor with ultrafiltration, and/or a continuous plug-flow column reactor (Fernanda de Castilhos Corazza, Flavio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov, A. V., and Sinitsyn, A. P., 1985, Kinetics of the enzymatic hydrolysis of cellulose: 1. A mathematical model for a batch reactor process, Enz. Microb. Technol. 7: 346-352), an attrition reactor (Ryu, S. K., and Lee, J. M., 1983, Bioconversion of waste cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25: 53-65), or a reactor with intensive stirring induced by an electromagnetic field (Gusakov, A. V., Sinitsyn, A. P., Davydkin, I. Y., Davydkin, V. Y., Protas, 0. V., 1996, Enhancement of enzymatic cellulose hydrolysis using a novel type of bioreactor with intensive stirring induced by electromagnetic field, Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor types include, for example, fluidized bed, upflow blanket, immobilized, and extruder type reactors for hydrolysis and/or fermentation.

[0431] Pretreatment.

[0432] In practicing the methods of the present invention, any pretreatment process known in the art can be used to disrupt the plant cell wall components of the cellulose-containing material. The cellulose-containing material can also be subjected to pre-soaking, wetting, or conditioning prior to pretreatment using methods known in the art. Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment, and biological pretreatment. Additional pretreatments include ultrasound, electroporation, microwave, supercritical CO.sub.2, supercritical H.sub.2O, and ammonia percolation.

[0433] The cellulose-containing material can be pretreated before hydrolysis and/or fermentation. Pretreatment is preferably performed prior to the hydrolysis. Alternatively, the pretreatment can be carried out simultaneously with hydrolysis, such as simultaneously with treatment of the cellulose-containing material with one or more cellulolytic enzymes, or other enzyme activities, to release fermentable sugars, such as glucose and/or maltose. In most cases the pretreatment step itself results in some conversion of biomass to fermentable sugars (even in absence of enzymes).

[0434] Steam Pretreatment. In steam pretreatment, the cellulose-containing material is heated to disrupt the plant cell wall components, including lignin, hemicellulose, and cellulose to make the cellulose and other fractions, e.g., hemicellulase, accessible to enzymes. The cellulose material is passed to or through a reaction vessel where steam is injected to increase the temperature to the required temperature and pressure and is retained therein for the desired reaction time. Steam pretreatment is preferably done at 140-230.degree. C., more preferably at 160-200.degree. C., and most preferably at 170-190.degree. C., where the optimal temperature range depends on addition of a chemical catalyst. Residence time for the steam pretreatment is preferably 1-15 minutes, more preferably 3-12 minutes, and most preferably 4-10 minutes, where the optimal residence time depends on the temperature range and addition of a chemical catalyst. Steam pretreatment allows for relatively high solids loadings, so that the cellulose-containing material is generally only moist during the pretreatment. The steam pretreatment is often combined with an explosive discharge of the material after the pretreatment, which is known as steam explosion, that is, rapid flashing to atmospheric pressure and turbulent flow of the material to increase the accessible surface area by fragmentation (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No. 20020164730).

[0435] A catalyst such as H.sub.2SO.sub.4 or SO.sub.2 (typically 0.3 to 3% w/w) is often added prior to steam pretreatment, which decreases the time and temperature, increases recovery, and improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006, Enzyme Microb. Technol. 39: 756-762).

[0436] Chemical Pretreatment: The term "chemical treatment" refers to any chemical pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin. Examples of suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation (APR), and organosolv pretreatments.

[0437] In dilute acid pretreatment, the cellulose-containing material is mixed with dilute acid, typically H.sub.2SO.sub.4, and water to form a slurry, heated by steam to the desired temperature, and after a residence time flashed to atmospheric pressure. The dilute acid pretreatment can be performed with a number of reactor designs, e.g., plug-flow reactors, counter-current reactors, or continuous counter-current shrinking bed reactors (Duff and Murray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

[0438] Several methods of pretreatment under alkaline conditions can also be used. These alkaline pretreatments include, but are not limited to, lime pretreatment, wet oxidation, ammonia percolation (APR), and ammonia fiber/freeze explosion (AFEX).

[0439] Lime pretreatment is performed with calcium carbonate, sodium hydroxide, or ammonia at low temperatures of 85-150.degree. C. and residence times from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO 2006/11899, WO 2006/11900, and WO 2006/110901 disclose pretreatment methods using ammonia.

[0440] Wet oxidation is a thermal pretreatment performed typically at 180-200.degree. C. for 5-15 minutes with addition of an oxidative agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). The pretreatment is performed at preferably 1-40% dry matter, more preferably 2-30% dry matter, and most preferably 5-20% dry matter, and often the initial pH is increased by the addition of alkali such as sodium carbonate.

[0441] A modification of the wet oxidation pretreatment method, known as wet explosion (combination of wet oxidation and steam explosion), can handle dry matter up to 30%. In wet explosion, the oxidizing agent is introduced during pretreatment after a certain residence time. The pretreatment is then ended by flashing to atmospheric pressure (WO 2006/032282).

[0442] Ammonia fiber explosion (AFEX) involves treating cellulose-containing material with liquid or gaseous ammonia at moderate temperatures such as 90-100.degree. C. and high pressure such as 17-20 bar for 5-10 minutes, where the dry matter content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121:1133-1141; Teymouri et al., 2005, Bioresource Technol. 96: 2014-2018).

[0443] Organosolv pretreatment delignifies cellulose-containing material by extraction using aqueous ethanol (40-60% ethanol) at 160-200.degree. C. for 30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121:219-230). Sulphuric acid is usually added as a catalyst. In organosolv pretreatment, the majority of the hemicellulose is removed.

[0444] Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. and Biotechnol. Vol. 105-108, p. 69-85, and Mosier et al., 2005, Bioresource Technology 96: 673-686, and U.S. Published Application 2002/0164730.

[0445] In one aspect, the chemical pretreatment is preferably carried out as an acid treatment, and more preferably as a continuous dilute and/or mild acid treatment. The acid is typically sulfuric acid, but other acids can also be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride or mixtures thereof. Mild acid treatment is conducted in the pH range of preferably 1-5, more preferably 1-4, and most preferably 1-3. In one aspect, the acid concentration is in the range from preferably 0.01 to 20 wt % acid, more preferably 0.05 to 10 wt % acid, even more preferably 0.1 to 5 wt % acid, and most preferably 0.2 to 2.0 wt % acid. The acid is contacted with the cellulose-containing material and held at a temperature, for example, in the range of 160-220.degree. C., preferably 165-195.degree. C., for periods ranging from seconds to minutes to, e.g., 1 second to 60 minutes.

[0446] In another aspect, pretreatment is carried out as an ammonia fiber explosion step (AFEX pretreatment step).

[0447] In another aspect, pretreatment takes place in an aqueous slurry. In preferred aspects, the cellulose-containing material is present during pretreatment in amounts preferably between 10-80 wt %, more preferably between 20-70 wt %, and most preferably between 30-60 wt %, such as around 50 wt %. The pretreated cellulose-containing material can be unwashed or washed using any method known in the art, e.g., washed with water.

[0448] Mechanical Pretreatment: The term "mechanical pretreatment" refers to various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).

[0449] Physical Pretreatment: The term "physical pretreatment" refers to any pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from cellulose-containing material. For example, physical pretreatment can involve irradiation (e.g., microwave irradiation), steaming/steam explosion, hydrothermolysis, and combinations thereof.

[0450] Physical pretreatment can involve high pressure and/or high temperature (steam explosion). In one aspect, high pressure means pressure in the range of preferably about 300 to about 600 psi, more preferably about 350 to about 550 psi, and most preferably about 400 to about 500 psi, such as around 450 psi. In another aspect, high temperature means temperatures in the range of about 100 to about 300.degree. C., preferably about 140 to about 235.degree. C. In a preferred aspect, mechanical pretreatment is performed in a batch-process, steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden.

[0451] Combined Physical and Chemical Pretreatment: The cellulose-containing material can be pretreated both physically and chemically. For instance, the pretreatment step can involve dilute or mild acid treatment and high temperature and/or pressure treatment. The physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired. A mechanical pretreatment can also be included.

[0452] Accordingly, in a preferred aspect, the cellulose-containing material is subjected to mechanical, chemical, or physical pretreatment, or any combination thereof, to promote the separation and/or release of cellulose, hemicellulose and/or lignin.

[0453] Biological Pretreatment: The term "biological pretreatment" refers to any biological pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from the cellulose-containing material. Biological pretreatment techniques can involve applying lignin-solubilizing microorganisms (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of lignocellulosic biomass, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566, American Chemical Society, Washington, D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson and Hahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates for ethanol production, Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Production of ethanol from lignocellulosic materials: State of the art, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

[0454] Saccharification.

[0455] In the hydrolysis step, also known as saccharification, the pretreated cellulose-containing material is hydrolyzed to break down cellulose and alternatively also hemicellulose to fermentable sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or soluble oligosaccharides. The hydrolysis is performed enzymatically using a cellulolytic protein composition of the present invention. The enzymes components of the composition can also be added sequentially.

[0456] Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions that can be readily determined by one skilled in the art. In a preferred aspect, hydrolysis is performed under conditions suitable for the activity of the enzyme(s), i.e., optimal for the enzyme(s). The hydrolysis can be carried out as a fed batch or continuous process where the pretreated cellulose-containing material (substrate) is fed gradually to, for example, an enzyme containing hydrolysis solution.

[0457] The saccharification is generally performed in stirred-tank reactors or fermentors under controlled pH, temperature, and mixing conditions. Suitable process time, temperature, and pH conditions can readily be determined by one skilled in the art. For example, the saccharification can last up to 200 hours, but is typically performed for preferably about 12 to about 96 hours, more preferably about 16 to about 72 hours, and most preferably about 24 to about 48 hours. The temperature is in the range of preferably about 25.degree. C. to about 70.degree. C., more preferably about 30.degree. C. to about 65.degree. C., and more preferably about 40.degree. C. to 60.degree. C., in particular about 50.degree. C. The pH is in the range of preferably about 3 to about 8, more preferably about 3.5 to about 7, and most preferably about 4 to about 6, in particular about pH 5. The dry solids content is in the range of preferably about 5 to about 50 wt %, more preferably about 10 to about 40 wt %, and most preferably about 20 to about 30 wt %.

[0458] The optimum amounts of the enzymes and polypeptides having cellulolytic enhancing activity depend on several factors including, but not limited to, the mixture of component cellulolytic proteins, the cellulosic substrate, the concentration of cellulosic substrate, the pretreatment(s) of the cellulosic substrate, temperature, time, pH, and inclusion of fermenting organism (e.g., yeast for Simultaneous Saccharification and Fermentation).

[0459] In a preferred aspect, an effective amount of cellulolytic protein(s) to cellulose-containing material is about 0.5 to about 50 mg, preferably at about 0.5 to about 40 mg, more preferably at about 0.5 to about 25 mg, more preferably at about 0.75 to about 20 mg, more preferably at about 0.75 to about 15 mg, even more preferably at about 0.5 to about 10 mg, and most preferably at about 2.5 to about 10 mg per g of cellulose-containing material.

[0460] In another preferred aspect, an effective amount of a polypeptide having cellulolytic enhancing activity to cellulose-containing material is about 0.5 to about 50 mg, preferably at about 0.5 to about 40 mg, more preferably at about 0.5 to about 25 mg, more preferably at about 0.75 to about 20 mg, more preferably at about 0.75 to about 15 mg, even more preferably at about 0.5 to about 10 mg, and most preferably at about 2.5 to about 10 mg per g of cellulose-containing material.

[0461] In another preferred aspect, an effective amount of polypeptide(s) having cellulolytic enhancing activity to cellulose-containing material is about 0.01 to about 50.0 mg, preferably about 0.01 to about 40 mg, more preferably about 0.01 to about 30 mg, more preferably about 0.01 to about 20 mg, more preferably about 0.01 to about 10 mg, more preferably about 0.01 to about 5 mg, more preferably at about 0.025 to about 1.5 mg, more preferably at about 0.05 to about 1.25 mg, more preferably at about 0.075 to about 1.25 mg, more preferably at about 0.1 to about 1.25 mg, even more preferably at about 0.15 to about 1.25 mg, and most preferably at about 0.25 to about 1.0 mg per g of cellulose-containing material. In another preferred aspect, an effective amount of polypeptide(s) having cellulolytic enhancing activity to cellulolytic protein(s) is about 0.005 to about 1.0 g, preferably at about 0.01 to about 1.0 g, more preferably at about 0.15 to about 0.75 g, more preferably at about 0.15 to about 0.5 g, more preferably at about 0.1 to about 0.5 g, even more preferably at about 0.1 to about 0.5 g, and most preferably at about 0.05 to about 0.2 g per g of cellulolytic protein(s).

[0462] Fermentation.

[0463] The fermentable sugars obtained from the pretreated and hydrolyzed cellulose-containing material can be fermented by one or more fermenting microorganisms capable of fermenting the sugars directly or indirectly into a desired fermentation product. "Fermentation" or "fermentation process" refers to any fermentation process or any process comprising a fermentation step. Fermentation processes also include fermentation processes used in the biofuel industry, consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. The fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one skilled in the art.

[0464] In the fermentation step, sugars, released from the cellulose-containing material as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g., ethanol, by a fermenting organism, such as yeast. Hydrolysis (saccharification) and fermentation can be separate or simultaneous. Such methods include, but are not limited to, separate hydrolysis and fermentation (SHF), simultaneous saccharification and fermentation (SSF), simultaneous saccharification and cofermentation (SSCF), hybrid hydrolysis and fermentation (HHF), SHCF (separate hydrolysis and co-fermentation), HHCF (hybrid hydrolysis and fermentation), and direct microbial conversion (DMC).

[0465] Any suitable hydrolyzed cellulose-containing material can be used in the fermentation step in practicing the present invention. The material is generally selected based on the desired fermentation product, i.e., the substance to be obtained from the fermentation, and the process employed, as is well known in the art.

[0466] The term "fermentation medium" is understood herein to refer to a medium before the fermenting microorganism(s) is(are) added, such as, a medium resulting from a saccharification process, as well as a medium, for example, used in a simultaneous saccharification and fermentation process (SSF). "Fermenting microorganism" refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be C.sub.6 and/or C.sub.5 fermenting organisms, or a combination thereof. Both C.sub.6 and C.sub.5 fermenting organisms are well known in the art. Suitable fermenting microorganisms are able to ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, or oligosaccharides, directly or indirectly into the desired fermentation product.

[0467] Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

[0468] Examples of fermenting microorganisms that can ferment C6 sugars include bacterial and fungal organisms, such as yeast. Preferred yeast includes strains of Saccharomyces spp., preferably Saccharomyces cerevisiae.

[0469] Examples of fermenting organisms that can ferment C5 sugars include bacterial and fungal organisms, such as yeast. Preferred C5 fermenting yeast include strains of Pichia, preferably Pichia stipitis, such as Pichia stipitis CBS 5773; strains of Candida, preferably Candida boidinii, Candida brassicae, Candida sheatae, Candida diddensii, Candida pseudotropicalis, or Candida utilis.

[0470] Other fermenting organisms include strains of Zymomonas, such as Zymomonas mobilis; Hansenula, such as Hansenula anomala; Klyveromyces, such as K. fragilis; Schizosaccharomyces, such as S. pombe; and E. coli, especially E. coli strains that have been genetically modified to improve the yield of ethanol.

[0471] In a preferred aspect, the yeast is a Saccharomyces spp. In a more preferred aspect, the yeast is Saccharomyces cerevisiae. In another more preferred aspect, the yeast is Saccharomyces distaticus. In another more preferred aspect, the yeast is Saccharomyces uvarum. In another preferred aspect, the yeast is a Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces marxianus. In another more preferred aspect, the yeast is Kluyveromyces fragilis. In another preferred aspect, the yeast is a Candida. In another more preferred aspect, the yeast is Candida boidinii. In another more preferred aspect, the yeast is Candida brassicae. In another more preferred aspect, the yeast is Candida diddensii. In another more preferred aspect, the yeast is Candida pseudotropicalis. In another more preferred aspect, the yeast is Candida utilis. In another preferred aspect, the yeast is a Clavispora. In another more preferred aspect, the yeast is Clavispora lusitaniae. In another more preferred aspect, the yeast is Clavispora opuntiae. In another preferred aspect, the yeast is a Pachysolen. In another more preferred aspect, the yeast is Pachysolen tannophilus. In another preferred aspect, the yeast is a Pichia. In another more preferred aspect, the yeast is a Pichia stipitis. In another preferred aspect, the yeast is a Bretannomyces. In another more preferred aspect, the yeast is Bretannomyces clausenii (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212).

[0472] Bacteria that can efficiently ferment hexose and pentose to ethanol include, for example, Zymomonas mobilis and Clostridium thermocellum (Philippidis, 1996, supra).

[0473] In a preferred aspect, the bacterium is a Zymomonas. In a more preferred aspect, the bacterium is Zymomonas mobilis. In another preferred aspect, the bacterium is a Clostridium. In another more preferred aspect, the bacterium is Clostridium thermocellum.

[0474] Commercially available yeast suitable for ethanol production includes, e.g., ETHANOL RED.TM. yeast (available from Fermentis/Lesaffre, USA), FALI.TM. (available from Fleischmann's Yeast, USA), SUPERSTART.TM. and THERMOSACC.TM. fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM.TM. AFT and XR (available from NABC--North American Bioproducts Corporation, GA, USA), GERT STRAND.TM. (available from Gert Strand AB, Sweden), and FERMIOL.TM. (available from DSM Specialties).

[0475] In a preferred aspect, the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose utilizing, arabinose utilizing, and xylose and arabinose co-utilizing microorganisms.

[0476] The cloning of heterologous genes into various fermenting microorganisms has led to the construction of organisms capable of converting hexoses and pentoses to ethanol (cofermentation) (Chen and Ho, 1993, Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TALI genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase, Appl. Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle, FEMS Yeast Research 4: 655-664; Beall et al., 1991, Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli, Biotech. Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al., 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470).

[0477] In a preferred aspect, the genetically modified fermenting microorganism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified fermenting microorganism is Zymomonas mobilis. In another preferred aspect, the genetically modified fermenting microorganism is Escherichia coli. In another preferred aspect, the genetically modified fermenting microorganism is Klebsiella oxytoca.

[0478] The fermenting microorganism(s) is typically added to the degraded cellulose or hydrolysate and the fermentation is performed for about 8 to about 96 hours, such as about 24 to about 60 hours. The temperature is typically between about 26.degree. C. to about 60.degree. C., in particular about 32.degree. C. or 50.degree. C., and at about pH 3 to about pH 8, such as around pH 4-5, 6, or 7.

[0479] In a preferred aspect, the fermenting microorganism(s) is applied to the degraded cellulose or hydrolysate and the fermentation is performed for about 12 to about 96 hours, such as typically 24-60 hours. In a preferred aspect, the temperature is preferably between about 20.degree. C. to about 60.degree. C., more preferably about 25.degree. C. to about 50.degree. C., and most preferably about 32.degree. C. to about 50.degree. C., in particular about 32.degree. C. or 50.degree. C., and the pH is generally from about pH 3 to about pH 7, preferably around pH 4-7. However, some, e.g., bacterial fermenting organisms have higher fermentation temperature optima. The fermenting microorganism(s) is preferably applied in amounts of approximately 10.sup.5 to 10.sup.12, preferably from approximately 10.sup.7 to 10.sup.10, especially approximately 2.times.10.sup.8 viable cell count per ml of fermentation broth. Further guidance in respect of using yeast for fermentation can be found in, e.g., "The Alcohol Textbook" (Editors K. Jacques, T. P. Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference.

[0480] For ethanol production, following the fermentation the fermented slurry is distilled to extract the ethanol. The ethanol obtained according to the methods of the invention can be used as, e.g., fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol. A fermentation stimulator can be used in combination with any of the enzymatic processes described herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield. A "fermentation stimulator" refers to stimulators for growth of the fermenting microorganisms, in particular, yeast. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E. See, for example, Alfenore et al., Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process, Springer-Verlag (2002), which is hereby incorporated by reference. Examples of minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

[0481] Fermentation Products:

[0482] A fermentation product can be any substance derived from the fermentation. The fermentation product can be, without limitation, an alcohol (e.g., arabinitol, butanol, ethanol, glycerol, methanol, 1,3-propanediol, sorbitol, and xylitol); an organic acid (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, and xylonic acid); a ketone (e.g., acetone); an aldehyde (e.g., formaldehyde); an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); and a gas (e.g., methane, hydrogen (H.sub.2), carbon dioxide (CO.sub.2), and carbon monoxide (CO)). The fermentation product can also be protein as a high value product.

[0483] In a preferred aspect, the fermentation product is an alcohol. It will be understood that the term "alcohol" encompasses a substance that contains one or more hydroxyl moieties. In a more preferred aspect, the alcohol is arabinitol. In another more preferred aspect, the alcohol is butanol. In another more preferred aspect, the alcohol is ethanol. In another more preferred aspect, the alcohol is glycerol. In another more preferred aspect, the alcohol is methanol. In another more preferred aspect, the alcohol is 1,3-propanediol. In another more preferred aspect, the alcohol is sorbitol. In another more preferred aspect, the alcohol is xylitol. See, for example, Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silveira, M. M., and Jonas, R., 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam, P., and Singh, D., 1995, Processes for fermentative production of xylitol--a sugar substitute, Process Biochemistry 30 (2): 117-124; Ezeji, T. C., Qureshi, N. and Blaschek, H. P., 2003, Production of acetone, butanol and ethanol by Clostridium beijerinckii BA101 and in situ recovery by gas stripping, World Journal of Microbiology and Biotechnology 19 (6): 595-603.

[0484] In another preferred aspect, the fermentation product is an organic acid. In another more preferred aspect, the organic acid is acetic acid. In another more preferred aspect, the organic acid is acetonic acid. In another more preferred aspect, the organic acid is adipic acid. In another more preferred aspect, the organic acid is ascorbic acid. In another more preferred aspect, the organic acid is citric acid. In another more preferred aspect, the organic acid is 2,5-diketo-D-gluconic acid. In another more preferred aspect, the organic acid is formic acid. In another more preferred aspect, the organic acid is fumaric acid. In another more preferred aspect, the organic acid is glucaric acid. In another more preferred aspect, the organic acid is gluconic acid. In another more preferred aspect, the organic acid is glucuronic acid. In another more preferred aspect, the organic acid is glutaric acid. In another preferred aspect, the organic acid is 3-hydroxypropionic acid. In another more preferred aspect, the organic acid is itaconic acid. In another more preferred aspect, the organic acid is lactic acid. In another more preferred aspect, the organic acid is malic acid. In another more preferred aspect, the organic acid is malonic acid. In another more preferred aspect, the organic acid is oxalic acid. In another more preferred aspect, the organic acid is propionic acid. In another more preferred aspect, the organic acid is succinic acid. In another more preferred aspect, the organic acid is xylonic acid. See, for example, Chen, R., and Lee, Y. Y., 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl. Biochem. Biotechnol. 63-65: 435-448.

[0485] In another preferred aspect, the fermentation product is a ketone. It will be understood that the term "ketone" encompasses a substance that contains one or more ketone moieties. In another more preferred aspect, the ketone is acetone. See, for example, Qureshi and Blaschek, 2003, supra.

[0486] In another preferred aspect, the fermentation product is an aldehyde. In another more preferred aspect, the aldehyde is formaldehyde.

[0487] In another preferred aspect, the fermentation product is an amino acid. In another more preferred aspect, the organic acid is aspartic acid. In another more preferred aspect, the amino acid is glutamic acid. In another more preferred aspect, the amino acid is glycine. In another more preferred aspect, the amino acid is lysine. In another more preferred aspect, the amino acid is serine. In another more preferred aspect, the amino acid is threonine. See, for example, Richard, A., and Margaritis, A., 2004, Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers, Biotechnology and Bioengineering 87 (4): 501-515.

[0488] In another preferred aspect, the fermentation product is a gas. In another more preferred aspect, the gas is methane. In another more preferred aspect, the gas is H.sub.2. In another more preferred aspect, the gas is CO.sub.2. In another more preferred aspect, the gas is CO. See, for example, Kataoka, N., A. Miya, and K. Kiriyama, 1997, Studies on hydrogen production by continuous culture system of hydrogen-producing anaerobic bacteria, Water Science and Technology 36 (6-7): 41-47; and Gunaseelan V. N. in Biomass and Bioenergy, Vol. 13 (1-2), pp. 83-114, 1997, Anaerobic digestion of biomass for methane production: A review.

[0489] Recovery.

[0490] The fermentation product(s) can be optionally recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), distillation, or extraction. For example, ethanol is separated from the fermented cellulose-containing material and purified by conventional methods of distillation. Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

[0491] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES

[0492] Chemicals used as buffers and substrates were commercial products of at least reagent grade.

DNA Sequencing

[0493] DNA sequencing was performed using an Applied Biosystems Model 3130X Genetic Analyzer (Applied Biosystems, Foster City, Calif., USA) using dye terminator chemistry (Giesecke et al., 1992, Journal of Virol. Methods 38: 47-60). Sequences were assembled using phred/phrap/consed (University of Washington, Seattle, Wash., USA) with sequence specific primers.

Media and Solutions

[0494] YP medium was composed per liter of 10 g of yeast extract and 20 g of bacto tryptone.

[0495] Cellulase-inducing medium was composed per liter of 20 g of cellulose, 10 g of corn steep solids, 1.45 g of (NH.sub.4).sub.2SO.sub.4, 2.08 g of KH.sub.2PO.sub.4, 0.28 g of CaCl.sub.2, 0.42 g of MgSO.sub.4.7H.sub.2O, and 0.42 ml of trace metals solution.

[0496] Trace metals solution was composed per liter of 216 g of FeCl.sub.3.6H.sub.2O, 58 g of ZnSO.sub.4.7H.sub.2O, 27 g of MnSO.sub.4.H.sub.2O, 10 g of CuSO.sub.4.5H.sub.2O, 2.4 g of H.sub.3BO.sub.3, and 336 g of citric acid.

[0497] STC was composed of 1 M sorbitol, 10 mM CaCl.sub.2, and 10 mM Tris-HCl, pH 7.5.

[0498] COVE plates were composed per liter of 342 g of sucrose, 10 ml of COVE salts solution, 10 ml of 1 M acetamide, 10 ml of 1.5 M CsCl, and 25 g of Noble agar.

[0499] COVE salts solution was composed per liter of 26 g of KCl, 26 g of MgSO.sub.4, 76 g of KH.sub.2PO.sub.4, and 50 ml of COVE trace metals solution.

[0500] COVE trace metals solution was composed per liter of 0.04 g of Na.sub.2B.sub.4O.sub.7.10H.sub.2O, 0.4 g of CuSO.sub.4.5H.sub.2O, 1.2 g of FeSO.sub.4.7H.sub.2O, 0.7 g of MnSO.sub.4.H.sub.2O, 0.8 g of Na.sub.2MoO.sub.2.H.sub.2O, and 10 g of ZnSO.sub.4.7H.sub.2O.

[0501] COVE2 plates were composed per liter of 30 g of sucrose, 20 ml of COVE salts solution, 25 g of Noble agar, and 10 ml of 1 M acetamide.

[0502] PDA plates were composed per liter of 39 grams of potato dextrose agar.

[0503] LB medium was composed per liter of 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride.

[0504] 2.times.YT-Amp plates were composed per liter of 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride, and 15 g of Bacto Agar, followed by 2 ml of a filter-sterilized solution of 50 mg/ml ampicillin after autoclaving.

[0505] MDU2BP medium was composed per liter of 45 g of maltose, 1 g of MgSO.sub.4.7H.sub.2O, 1 g of NaCl, 2 g of K.sub.2HSO.sub.4, 12 g of KH.sub.2PO.sub.4, 2 g of urea, and 500 .mu.l of AMG trace metals solution, the pH was adjusted to 5.0 and then filter sterilized with a 0.22 .mu.m filtering unit.

[0506] AMG trace metals solution was composed per liter of 14.3 g of ZnSO.sub.4.7H.sub.2O, 2.5 g of CuSO.sub.4.5H.sub.2O, 0.5 g of NiCl.sub.2.6H.sub.2O, 13.8 g of FeSO.sub.4.H.sub.2O, 8.5 g of MnSO.sub.4.7H.sub.2O, and 3 g of citric acid.

[0507] Minimal medium plates were composed per liter of 6 g of NaNO.sub.3, 0.52 of KCl, 1.52 g of KH.sub.2PO.sub.4, 1 ml of COVE trace metals solution, 20 g of Noble agar, 20 ml of 50% glucose, 2.5 ml of 20% MgSO.sub.4.7H.sub.2O, and 20 ml of biotin stock solution.

[0508] Biotin stock solution was composed per liter of 0.2 g of biotin.

[0509] SOC medium was composed of 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl.sub.2, and 10 mM MgSO.sub.4, followed by filter-sterilized glucose to 20 mM after autoclaving.

[0510] Mandel's medium was composed per liter of 1.4 g of (NH.sub.4).sub.250.sub.4, 2.0 g of KH.sub.2PO.sub.4, 0.3 g of urea, 0.3 g of CaCl.sub.2, 0.3 g of MgSO.sub.4.7H.sub.2O, 5 mg of FeSO.sub.4.7H.sub.2O, 1.6 mg of MnSO.sub.4.H.sub.2O, 1.4 mg of ZnSO.sub.4.H.sub.2O, and 2 mg of CoCl.sub.2.

Example 1: Construction of pMJ04 Expression Vector

[0511] Expression vector pMJ04 was constructed by PCR amplifying the Trichoderma reesei cellobiohydrolase 1 gene (cbh1, CEL7A) terminator from Trichoderma reesei RutC30 genomic DNA using primers 993429 (antisense) and 993428 (sense) shown below. The antisense primer was engineered to have a Pac I site at the 5'-end and a Spe I site at the 3'-end of the sense primer.

TABLE-US-00004 Primer 993429 (antisense): (SEQ ID NO: 65) 5'-AACGTTAATTAAGGAATCGTTTTGTGTTT-3' Primer 993428 (sense): (SEQ ID NO: 66) 5'-AGTACTAGTAGCTCCGTGGCGAAAGCCTG-3'

[0512] Trichoderma reesei RutC30 genomic DNA was isolated using a DNEASY.RTM. Plant Maxi Kit (QIAGEN Inc., Valencia, Calif., USA).

[0513] The amplification reactions (50 .mu.l) were composed of 1.times. ThermoPol Reaction Buffer (New England Biolabs, Beverly, Mass., USA), 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic DNA, 0.3 .mu.M primer 993429, 0.3 .mu.M primer 993428, and 2 units of Vent DNA polymerase (New England Biolabs, Beverly, Mass., USA). The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA) programmed for 5 cycles each for 30 seconds at 94.degree. C., 30 seconds at 50.degree. C., and 60 seconds at 72.degree. C., followed by 25 cycles each for 30 seconds at 94.degree. C., 30 seconds at 65.degree. C., and 120 seconds at 72.degree. C. (5 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer where a 229 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer's instructions.

[0514] The resulting PCR fragment was digested with Pac I and Spe I and ligated into pAlLo1 (WO 05/067531) digested with the same restriction enzymes using a Rapid Ligation Kit (Roche, Indianapolis, Ind., USA), to generate pMJ04 (FIG. 1).

Example 2: Construction of pCaHj568

[0515] Plasmid pCaHj568 was constructed from pCaHj170 (U.S. Pat. No. 5,763,254) and pMT2188. Plasmid pCaHj170 comprises the Humicola insolens endoglucanase V (CEL45A) full-length coding region (SEQ ID NO: 15, which encodes the amino acid sequence of SEQ ID NO: 16). Construction of pMT2188 was initiated by PCR amplifying the pUC19 origin of replication from pCaHj483 (WO 98/00529) using primers 142779 and 142780 shown below. Primer 142780 introduces a Bbu I site in the PCR fragment.

TABLE-US-00005 Primer 142779: (SEQ ID NO: 67) 5'-TTGAATTGAAAATAGATTGATTTAAAACTTC-3' Primer 142780: (SEQ ID NO: 68) 5'-TTGCATGCGTAATCATGGTCATAGC-3'

[0516] An EXPAND.RTM. PCR System (Roche Molecular Biochemicals, Basel, Switzerland) was used following the manufacturer's instructions for this amplification. PCR products were separated on an agarose gel and an 1160 bp fragment was isolated and purified using a Jetquick Gel Extraction Spin Kit (Genomed, Wielandstr, Germany).

[0517] The URA3 gene was amplified from the general Saccharomyces cerevisiae cloning vector pYES2 (Invitrogen, Carlsbad, Calif., USA) using primers 140288 and 142778 shown below using an EXPAND.RTM. PCR System. Primer 140288 introduced an Eco RI site into the PCR fragment.

TABLE-US-00006 Primer 140288: (SEQ ID NO: 69) 5'-TTGAATTCATGGGTAATAACTGATAT-3' Primer 142778: (SEQ ID NO: 70) 5'-AAATCAATCTATTTTCAATTCAATTCATCATT-3'

[0518] PCR products were separated on an agarose gel and an 1126 bp fragment was isolated and purified using a Jetquick Gel Extraction Spin Kit.

[0519] The two PCR fragments were fused by mixing and amplifed using primers 142780 and 140288 shown above by the overlap splicing method (Horton et al., 1989, Gene 77: 61-68). PCR products were separated on an agarose gel and a 2263 bp fragment was isolated and purified using a Jetquick Gel Extraction Spin Kit.

[0520] The resulting fragment was digested with Eco RI and Bbu I and ligated using standard protocols to the largest fragment of pCaHj483 digested with the same restriction enzymes. The ligation mixture was transformed into pyrF-negative E. coli strain DB6507 (ATCC 35673) made competent by the method of Mandel and Higa, 1970, J. Mol. Biol. 45: 154. Transformants were selected on solid M9 medium (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press) supplemented per liter with 1 g of casamino acids, 500 .mu.g of thiamine, and 10 mg of kanamycin. A plasmid from one transformant was isolated and designated pCaHj527 (FIG. 2).

[0521] The NA2-tpi promoter present on pCaHj527 was subjected to site-directed mutagenesis by PCR using an EXPAND.RTM. PCR System according to the manufacturer's instructions. Nucleotides 134-144 were converted from GTACTAAAACC (SEQ ID NO: 71) to CCGTTAAATTT (SEQ ID NO: 72) using mutagenic primer 141223 shown below.

TABLE-US-00007 Primer 141223: (SEQ ID NO: 73) 5'-GGATGCTGTTGACTCCGGAAATTTAACGGTTTGGTCTTGCATCCC- 3'

Nucleotides 423-436 were converted from ATGCAATTTAAACT (SEQ ID NO: 74) to CGGCAATTTAACGG (SEQ ID NO: 75) using mutagenic primer 141222 shown below.

TABLE-US-00008 Primer 141222: (SEQ ID NO: 76) 5'-GGTATTGTCCTGCAGACGGCAATTTAACGGCTTCTGCGAATCGC-3'

The resulting plasmid was designated pMT2188 (FIG. 3).

[0522] The Humicola insolens endoglucanase V coding region was transferred from pCaHj170 as a Bam HI-Sal I fragment into pMT2188 digested with Bam HI and Xho I to generate pCaHj568 (FIG. 4). Plasmid pCaHj568 comprises a mutated NA2-tpi promoter operably linked to the Humicola insolens endoglucanase V full-length coding sequence.

Example 3: Construction of pMJ05

[0523] Plasmid pMJ05 was constructed by PCR amplifying the 915 bp Humicola insolens endoglucanase V full-length coding region from pCaHj568 using primers HiEGV-F and HiEGV-R shown below.

TABLE-US-00009 Primer HiEGV-F (sense): (SEQ ID NO: 77) 5'-AAGCTTAAGCATGCGTTCCTCCCCCCTCC-3' Primer HiEGV-R (antisense): (SEQ ID NO: 78) 5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3'

[0524] The amplification reactions (50 .mu.l) were composed of 1.times. ThermoPol Reaction Buffer (New England Biolabs, Beverly, Mass., USA), 0.3 mM dNTPs, 10 ng/.mu.l of pCaHj568, 0.3 .mu.M HiEGV-F primer, 0.3 .mu.M HiEGV-R primer, and 2 units of Vent DNA polymerase (New England Biolabs, Beverly, Mass., USA). The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 5 cycles each for 30 seconds at 94.degree. C., 30 seconds at 50.degree. C., and 60 seconds at 72.degree. C., followed by 25 cycles each for 30 seconds at 94.degree. C., 30 seconds at 65.degree. C., and 120 seconds at 72.degree. C. (5 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 937 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0525] The 937 bp purified fragment was used as template DNA for subsequent amplifications with the following primers:

TABLE-US-00010 Primer HiEGV-R (antisense): (SEQ ID NO: 79) 5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3' Primer HiEGV-F-overlap (sense): (SEQ ID NO: 80) 5'-ACCGCGGACTGCGCATCATGCGTTCCTCCCCCCTCC-3'

Primer sequences in italics are homologous to 17 bp of the Trichoderma reesei cellobiohydrolase I gene (cbh1) promoter and underlined primer sequences are homologous to 29 bp of the Humicola insolens endoglucanase V coding region. A 36 bp overlap between the promoter and the coding sequence allowed precise fusion of a 994 bp fragment comprising the Trichoderma reesei cbh1 promoter to the 918 bp fragment comprising the Humicola insolens endoglucanase V coding region.

[0526] The amplification reactions (50 .mu.l) were composed of 1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 1 .mu.l of the purified 937 bp PCR fragment, 0.3 .mu.M HiEGV-F-overlap primer, 0.3 .mu.M HiEGV-R primer, and 2 units of Vent DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 5 cycles each for 30 seconds at 94.degree. C., 30 seconds at 50.degree. C., and 60 seconds at 72.degree. C., followed by 25 cycles each for 30 seconds at 94.degree. C., 30 seconds at 65.degree. C., and 120 seconds at 72.degree. C. (5 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 945 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0527] A separate PCR was performed to amplify the Trichoderma reesei cbh1 promoter sequence extending from 994 bp upstream of the ATG start codon of the gene from Trichoderma reesei RutC30 genomic DNA using the primers shown below (the sense primer was engineered to have a Sal I restriction site at the 5'-end). Trichoderma reesei RutC30 genomic DNA was isolated using a DNEASY.RTM. Plant Maxi Kit.

TABLE-US-00011 Primer TrCBHIpro-F (sense): (SEQ ID NO: 81) 5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' Primer TrCBHIpro-R (antisense): (SEQ ID NO: 82) 5'-GATGCGCAGTCCGCGGT-3'

[0528] The amplification reactions (50 .mu.l) were composed of 1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 100 ng/.mu.l Trichoderma reesei RutC30 genomic DNA, 0.3 .mu.M TrCBHIpro-F primer, 0.3 .mu.M TrCBHIpro-R primer, and 2 units of Vent DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 30 cycles each for 30 seconds at 94.degree. C., 30 seconds at 55.degree. C., and 120 seconds at 72.degree. C. (5 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 998 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0529] The purified 998 bp PCR fragment was used as template DNA for subsequent amplifications using the primers shown below.

TABLE-US-00012 Primer TrCBHIpro-F: (SEQ ID NO: 83) 5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' Primer TrCBHIpro-R-overlap: (SEQ ID NO: 84) 5'-GGAGGGGGGAGGAACGCATGATGCGCAGTCCGCGGT-3'

[0530] Sequences in italics are homologous to 17 bp of the Trichoderma reesei cbh1 promoter and underlined sequences are homologous to 29 bp of the Humicola insolens endoglucanase V coding region. A 36 bp overlap between the promoter and the coding sequence allowed precise fusion of the 994 bp fragment comprising the Trichoderma reesei cbh1 promoter to the 918 bp fragment comprising the Humicola insolens endoglucanase V full-length coding region.

[0531] The amplification reactions (50 .mu.l) were composed of 1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 1 .mu.l of the purified 998 bp PCR fragment, 0.3 .mu.M TrCBH1pro-F primer, 0.3 .mu.M TrCBH1pro-R-overlap primer, and 2 units of Vent DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 5 cycles each for 30 seconds at 94.degree. C., 30 seconds at 50.degree. C., and 60 seconds at 72.degree. C., followed by 25 cycles each for 30 seconds at 94.degree. C., 30 seconds at 65.degree. C., and 120 seconds at 72.degree. C. (5 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 1017 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0532] The 1017 bp Trichoderma reesei cbh1 promoter PCR fragment and the 945 bp Humicola insolens endoglucanase V PCR fragment were used as template DNA for subsequent amplification using the following primers to precisely fuse the 994 bp cbh1 promoter to the 918 bp endoglucanase V full-length coding region using overlapping PCR.

TABLE-US-00013 Primer TrCBHIpro-F: (SEQ ID NO: 85) 5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' Primer HiEGV-R: (SEQ ID NO: 86) 5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3'

[0533] The amplification reactions (50 .mu.l) were composed of 1.times. ThermoPol Reaction Buffer, 0.3 mM dNTPs, 0.3 .mu.M TrCBH1pro-F primer, 0.3 .mu.M HiEGV-R primer, and 2 units of Vent DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 5 cycles each for 30 seconds at 94.degree. C., 30 seconds at 50.degree. C., and 60 seconds at 72.degree. C., followed by 25 cycles each for 30 seconds at 94.degree. C., 30 seconds at 65.degree. C., and 120 seconds at 72.degree. C. (5 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 1926 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0534] The resulting 1926 bp fragment was cloned into a pCR.RTM.-Blunt-II-TOPO.RTM. vector (Invitrogen, Carlsbad, Calif., USA) using a ZEROBLUNT.RTM. TOPO.RTM. PCR Cloning Kit (Invitrogen, Carlsbad, Calif., USA) following the manufacturer's protocol. The resulting plasmid was digested with Not I and Sal I and the 1926 bp fragment was gel purified using a QIAQUICK.RTM. Gel Extraction Kit and ligated using T4 DNA ligase (Roche, Indianapolis, Ind., USA) into pMJ04, which was also digested with the same two restriction enzymes, to generate pMJ05 (FIG. 5). Plasmid pMJ05 comprises the Trichoderma reesei cellobiohydrolase I promoter and terminator operably linked to the Humicola insolens endoglucanase V full-length coding sequence.

Example 4: Construction of pSMai130 Expression Vector

[0535] A 2586 bp DNA fragment spanning from the ATG start codon to the TAA stop codon of an Aspergillus oryzae beta-glucosidase full-length coding sequence (SEQ ID NO: 15 for cDNA sequence and SEQ ID NO: 16 for the deduced amino acid sequence; E. coli DSM 14240) was amplified by PCR from pJaL660 (WO 2002/095014) as template with primers 993467 (sense) and 993456 (antisense) shown below. A Spe I site was engineered at the 5' end of the antisense primer to facilitate ligation. Primer sequences in italics are homologous to 24 bp of the Trichoderma reesei cbh1 promoter and underlined sequences are homologous to 22 bp of the Aspergillus oryzae beta-glucosidase coding region.

TABLE-US-00014 Primer 993467: (SEQ ID NO: 87) 5'-ATAGTCAACCGCGGACTGCGCATCATGAAGCTTGGTTGGATCGAGG- 3' Primer 993456: (SEQ ID NO: 88) 5'-ACTAGTTTACTGGGCCTTAGGCAGCG-3'

[0536] The amplification reactions (50 .mu.l) were composed of Pfx Amplification Buffer (Invitrogen, Carlsbad, Calif., USA), 0.25 mM dNTPs, 10 ng of pJaL660, 6.4 .mu.M primer 993467, 3.2 .mu.M primer 993456, 1 mM MgCl.sub.2, and 2.5 units of Pfx DNA polymerase (Invitrogen, Carlsbad, Calif., USA). The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 30 cycles each for 1 minute at 94.degree. C., 1 minute at 55.degree. C., and 3 minutes at 72.degree. C. (15 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 2586 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0537] A separate PCR was performed to amplify the Trichoderma reesei cbh1 promoter sequence extending from 1000 bp upstream of the ATG start codon of the gene, using primer 993453 (sense) and primer 993463 (antisense) shown below to generate a 1000 bp PCR fragment.

TABLE-US-00015 Primer 993453: (SEQ ID NO: 89) 5'-GTCGACTCGAAGCCCGAATGTAGGAT-3' Primer 993463: (SEQ ID NO: 90) 5'-CCTCGATCCAACCAAGCTTCATGATGCGCAGTCCGCGGTTGACTA- 3'

Primer sequences in italics are homologous to 24 bp of the Trichoderma reesei cbh1 promoter and underlined primer sequences are homologous to 22 bp of the Aspergillus oryzae beta-glucosidase full-length coding region. The 46 bp overlap between the promoter and the coding sequence allowed precise fusion of the 1000 bp fragment comprising the Trichoderma reesei cbh1 promoter to the 2586 bp fragment comprising the Aspergillus oryzae beta-glucosidase coding region.

[0538] The amplification reactions (50 .mu.l) were composed of Pfx Amplification Buffer, 0.25 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic DNA, 6.4 .mu.M primer 993453, 3.2 .mu.M primer 993463, 1 mM MgCl.sub.2, and 2.5 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 30 cycles each for 1 minute at 94.degree. C., 1 minute at 55.degree. C., and 3 minutes at 72.degree. C. (15 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 1000 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0539] The purified fragments were used as template DNA for subsequent amplification by overlapping PCR using primer 993453 (sense) and primer 993456 (antisense) shown above to precisely fuse the 1000 bp fragment comprising the Trichoderma reesei cbh1 promoter to the 2586 bp fragment comprising the Aspergillus oryzae beta-glucosidase full-length coding region.

[0540] The amplification reactions (50 .mu.l) were composed of Pfx Amplification Buffer, 0.25 mM dNTPs, 6.4 .mu.M primer 99353, 3.2 .mu.M primer 993456, 1 mM MgCl.sub.2, and 2.5 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 30 cycles each for 1 minute at 94.degree. C., 1 minute at 60.degree. C., and 4 minutes at 72.degree. C. (15 minute final extension).

[0541] The resulting 3586 bp fragment was digested with Sal I and Spe I and ligated into pMJ04, digested with the same two restriction enzymes, to generate pSMai130 (FIG. 6). Plasmid pSMai130 comprises the Trichoderma reesei cellobiohydrolase I gene promoter and terminator operably linked to the Aspergillus oryzae native beta-glucosidase signal sequence and coding sequence (i.e., full-length Aspergillus oryzae beta-glucosidase coding sequence).

Example 5: Construction of pSMai135

[0542] The Aspergillus oryzae beta-glucosidase mature coding region (minus the native signal sequence, see FIG. 7; SEQ ID NOs: 91 and 92 for signal peptide and coding sequence thereof) from Lys-20 to the TAA stop codon was PCR amplified from pJaL660 as template with primer 993728 (sense) and primer 993727 (antisense) shown below.

TABLE-US-00016 Primer 993728: (SEQ ID NO: 93) 5'-TGCCGGTGTTGGCCCTTGCCAAGGATGATCTCGCGTACTCCC-3' Primer 993727: (SEQ ID NO: 94) 5'-GACTAGTCTTACTGGGCCTTAGGCAGCG-3'

Sequences in italics are homologous to 20 bp of the Humicola insolens endoglucanase V signal sequence and sequences underlined are homologous to 22 bp of the Aspergillus oryzae beta-glucosidase coding region. A Spe I site was engineered into the 5' end of the antisense primer.

[0543] The amplification reactions (50 .mu.l) were composed of Pfx Amplification Buffer, 0.25 mM dNTPs, 10 ng/.mu.1 of pJaL660, 6.4 .mu.M primer 993728, 3.2 .mu.M primer 993727, 1 mM MgCl.sub.2, and 2.5 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 30 cycles each for 1 minute at 94.degree. C., 1 minute at 55.degree. C., and 3 minutes at 72.degree. C. (15 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 2523 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0544] A separate PCR amplification was performed to amplify 1000 bp of the Trichoderma reesei cbh1 promoter and 63 bp of the Humicola insolens endoglucanase V signal sequence (ATG start codon to Ala-21, FIG. 8, SEQ ID NOs: 95 and 96), using primer 993724 (sense) and primer 993729 (antisense) shown below.

TABLE-US-00017 Primer 993724: (SEQ ID NO: 97) 5'-ACGCGTCGACCGAATGTAGGATTGTTATCC-3' Primer 993729: (SEQ ID NO: 98) 5'-GGGAGTACGCGAGATCATCCTTGGCAAGGGCCAACACCGGCA-3'

[0545] Primer sequences in italics are homologous to 20 bp of the Humicola insolens endoglucanase V signal sequence and underlined primer sequences are homologous to the 22 bp of the Aspergillus oryzae beta-glucosidase coding region.

[0546] Plasmid pMJ05, which comprises the Humicola insolens endoglucanase V coding region under the control of the cbh1 promoter, was used as template to generate a 1063 bp fragment comprising the Trichoderma reesei cbh1 promoter and Humicola insolens endoglucanase V signal sequence fragment. A 42 bp of overlap was shared between the Trichoderma reesei cbh1 promoter and Humicola insolens endoglucanase V signal sequence and the Aspergillus oryzae beta-glucosidase mature coding sequence to provide a perfect linkage between the promoter and the ATG start codon of the 2523 bp Aspergillus oryzae beta-glucosidase coding region.

[0547] The amplification reactions (50 .mu.l) were composed of Pfx Amplification Buffer, 0.25 mM dNTPs, 10 ng/.mu.1 of pMJ05, 6.4 .mu.M primer 993728, 3.2 .mu.M primer 993727, 1 mM MgCl.sub.2, and 2.5 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 30 cycles each for 1 minute at 94.degree. C., 1 minute at 60.degree. C., and 4 minutes at 72.degree. C. (15 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 1063 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0548] The purified overlapping fragments were used as templates for amplification using primer 993724 (sense) and primer 993727 (antisense) described above to precisely fuse the 1063 bp fragment comprising the Trichoderma reesei cbh1 promoter and Humicola insolens endoglucanase V signal sequence to the 2523 bp fragment comprising the Aspergillus oryzae beta-glucosidase mature coding region frame by overlapping PCR.

[0549] The amplification reactions (50 .mu.l) were composed of Pfx Amplification Buffer, 0.25 mM dNTPs, 6.4 .mu.M primer 993724, 3.2 .mu.M primer 993727, 1 mM MgCl.sub.2, and 2.5 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 30 cycles each for 1 minute at 94.degree. C., 1 minute at 60.degree. C., and 4 minutes at 72.degree. C. (15 minute final extension). The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 3591 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0550] The resulting 3591 bp fragment was digested with Sal I and Spe I and ligated into pMJ04 digested with the same restriction enzymes to generate pSMai135 (FIG. 9). Plasmid pSMai135 comprises the Trichoderma reesei cellobiohydrolase I gene promoter and terminator operably linked to the Humicola insolens endoglucanase V signal sequence and the Aspergillus oryzae beta-glucosidase mature coding sequence.

Example 6: Expression of Aspergillus oryzae Beta-Glucosidase with the Humicola insolens Endoglucanase V Secretion Signal

[0551] Plasmid pSMai135 encoding the mature Aspergillus oryzae beta-glucosidase linked to the Humicola insolens endoglucanase V secretion signal (FIG. 8) was introduced into Trichoderma reesei RutC30 by PEG-mediated transformation (Penttila et al., 1987, Gene 61 155-164). The plasmid contained the Aspergillus nidulans amdS gene to enable transformants to grow on acetamide as the sole nitrogen source.

[0552] Trichoderma reesei RutC30 was cultivated at 27.degree. C. and 90 rpm in 25 ml of YP medium supplemented with 2% (w/v) glucose and 10 mM uridine for 17 hours. Mycelia were collected by filtration using a Vacuum Driven Disposable Filtration System (Millipore, Bedford, Mass., USA) and washed twice with deionized water and twice with 1.2 M sorbitol. Protoplasts were generated by suspending the washed mycelia in 20 ml of 1.2 M sorbitol containing 15 mg of GLUCANEX.RTM. (Novozymes A/S, Bagsvrd, Denmark) per ml and 0.36 units of chitinase (Sigma Chemical Co., St. Louis, Mo., USA) per ml and incubating for 15-25 minutes at 34.degree. C. with gentle shaking at 90 rpm. Protoplasts were collected by centrifuging for 7 minutes at 400.times.g and washed twice with cold 1.2 M sorbitol. The protoplasts were counted using a haemacytometer and re-suspended in STC to a final concentration of 1.times.10.sup.8 protoplasts per ml. Excess protoplasts were stored in a Cryo 1.degree. C. Freezing Container (Nalgene, Rochester, N.Y., USA) at -80.degree. C.

[0553] Approximately 7 .mu.g of pSMai135 digested with Pme I was added to 100 .mu.l of protoplast solution and mixed gently, followed by 260 .mu.l of PEG buffer, mixed, and incubated at room temperature for 30 minutes. STC (3 ml) was then added and mixed and the transformation solution was plated onto COVE plates using Aspergillus nidulans amdS selection. The plates were incubated at 28.degree. C. for 5-7 days. Transformants were sub-cultured onto COVE2 plates and grown at 28.degree. C.

[0554] Sixty-seven transformants designated SMA135 obtained with pSMai135 were subcultured onto fresh plates containing acetamide and allowed to sporulate for 7 days at 28.degree. C.

[0555] The 67 SMA135 Trichoderma reesei transformants were cultivated in 125 ml baffled shake flasks containing 25 ml of cellulase-inducing medium at pH 6.0 inoculated with spores of the transformants and incubated at 28.degree. C. and 200 rpm for 7 days. Trichoderma reesei RutC30 was run as a control. Culture broth samples were removed at day 7. One ml of each culture broth was centrifuged at 15,700.times.g for 5 minutes in a micro-centrifuge and the supernatants transferred to new tubes. Samples were stored at 4.degree. C. until enzyme assay. The supernatants were assayed for beta-glucosidase activity using p-nitrophenyl-beta-D-glucopyranoside as substrate, as described below.

[0556] Beta-glucosidase activity was determined at ambient temperature using 25 .mu.l aliquots of culture supernatants, diluted 1:10 in 50 mM succinate pH 5.0, in 200 .mu.l of 0.5 mg/ml p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM succinate pH 5.0. After 15 minutes incubation the reaction was stopped by adding 100 .mu.l of 1 M Tris-HCl pH 8.0 and the absorbance was read spectrophotometrically at 405 nm. One unit of beta-glucosidase activity corresponded to production of 1 .mu.mol of p-nitrophenyl per minute per liter at pH 5.0, ambient temperature. Aspergillus niger beta-glucosidase (NOVOZYM.TM. 188, Novozymes A/S, Bagsvrd, Denmark) was used as an enzyme standard.

[0557] A number of the SMA135 transformants produced beta-glucosidase activities several-fold higher than that secreted by Trichoderma reesei RutC30. Of the SMA135 transformants screened, transformant SMA135-04 produced the highest beta-glucosidase activity.

[0558] SDS-PAGE was carried out using CRITERION.RTM. Tris-HCl (5% resolving) gels (Bio-Rad, Hercules, Calif., USA) with the CRITERION.RTM. System (Bio-Rad, Hercules, Calif., USA). Five .mu.l of day 7 supernatants (see above) were suspended in 2.times. concentration of Laemmli Sample Buffer (Bio-Rad, Hercules, Calif., USA) and boiled in the presence of 5% beta-mercaptoethanol for 3 minutes. The supernatant samples were loaded onto a polyacrylamide gel and subjected to electrophoresis with 1.times. Tris/Glycine/SDS as running buffer (Bio-Rad, Hercules, Calif., USA). The resulting gel was stained with BIO-SAFE.RTM. Coomassie Stain (Bio-Rad, Hercules, Calif., USA).

[0559] Of the 38 Trichoderma reesei SMA135 transformants analyzed by SDS-PAGE, 26 produced a protein of approximately 110 kDa that was not visible in Trichoderma reesei RutC30 as control. Transformant Trichoderma reesei SMA135-04 produced the highest level of beta-glucosidase as evidenced by abundance of the 110 kDa band seen by SDS-PAGE.

Example 7: Construction of Expression Vector pSMai140

[0560] Expression vector pSMai140 was constructed by digesting plasmid pSATe111BG41 (WO 04/099228), which carries the Aspergillus oryzae beta-glucosidase variant BG41 full-length coding region (SEQ ID NO: 17 which encodes the amino acid sequence of SEQ ID NO: 18), with Nco I. The resulting 1243 bp fragment was isolated by 1.0% agarose gel electrphoresis using TAE buffer and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0561] Expression vector pSMai135 was digested with Nco I and a 8286 bp fragment was isolated by 1.0% agarose gel electrphoresis using TAE buffer and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions. The 1243 bp Nco I digested Aspergillus oryzae beta-glucosidase variant BG41 fragment was then ligated to the 8286 bp vector fragment, using T4 DNA ligase (Roche, Indianapolis, Ind., USA) according to manufacturer's protocol, to create the expression vector pSMai140 (FIG. 10). Plasmid pSMai140 comprises the Trichoderma reesei cellobiohydrolase I (CEL7A) gene promoter and terminator operably linked to the Humicola insolens endoglucanase V signal sequence and the Aspergillus oryzae beta-glucosidase variant mature coding sequence.

Example 8: Transformation of Trichoderma reesei RutC30 with pSMai140

[0562] Plasmid pSMai140 was linearized with Pme I and transformed into the Trichoderma reesei RutC30 strain as described in Example 6. A total of 100 transformants were obtained from four independent transformation experiments, all of which were cultivated in shake flasks on cellulase-inducing medium, and the beta-glucosidase activity was measured from the culture medium of the transformants as described in Example 6. A number of Trichoderma reesei SMA140 transformants showed beta-glucosidase activities several fold higher than that of Trichoderma reesei RutC30.

[0563] The presence of the Aspergillus oryzae beta-glucosidase variant BG41 protein in the culture medium was detected by SDS-polyacrylamide gel electrophoresis as described in Example 6 and Coomassie staining from the same 13 culture supernatants from which enzyme activity were analyzed. All thirteen transformants that had high .beta.-glucosidase activity, also expressed the approximately 110 KDa Aspergillus oryzae beta-glucosidase variant BG41, at varying yields.

[0564] The highest beta-glucosidase variant expressing transformant, as evaluated by beta-glucosidase activity assay and SDS-polyacrylamide gel electrophoresis, was designated Trichoderma reesei SMA140-43.

Example 9: Construction of Expression Vector pSaMe-F1

[0565] A DNA fragment containing 209 bp of the Trichoderma reesei cellobiohydrolase I gene promoter and the core region (nucleotides 1 to 702 of SEQ ID NO: 15, which encodes amino acids 1 to 234 of SEQ ID NO: 16; WO 91/17243) of the Humicola insolens endoglucanase V gene was PCR amplified using pMJ05 as template and the primers shown below.

TABLE-US-00018 Primer 995103: (SEQ ID NO: 99) 5'-cccaagcttagccaagaaca-3' Primer 995137: (SEQ ID NO: 100) 5'-gggggaggaacgcatgggatctggacggc-3'

[0566] The amplification reactions (50 .mu.l) were composed of 1.times. Pfx Amplification Buffer, 10 mM dNTPs, 50 mM MgSO.sub.4, 10 ng/.mu.l of pMJ05, 50 picomoles of 995103 primer, 50 picomoles of 995137 primer, and 2 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 30 cycles each for 30 seconds at 94.degree. C., 30 seconds at 55.degree. C., and 60 seconds at 72.degree. C. (3 minute final extension).

[0567] The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 911 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0568] A DNA fragment containing 806 bp of the Aspergillus oryzae beta-glucosidase variant BG41 gene was PCR amplified using pSMai140 as template and the primers shown below.

TABLE-US-00019 Primer 995133: (SEQ ID NO: 101) 5'-gccgtccagatccccatgcgttcctccccc-3' Primer 995111: (SEQ ID NO: 102) 5'-ccaagcttgttcagagtttc-3'

[0569] The amplification reactions (50 .mu.l) were composed of 1.times. Pfx Amplification Buffer, 10 mM dNTPs, 50 mM MgSO.sub.4, 100 ng of pSMai140, 50 picomoles of 995133 primer, 50 picomoles of 995111 primer, and 2 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for 30 cycles each for 30 seconds at 94.degree. C., 30 seconds at 55.degree. C., and 120 seconds at 72.degree. C. (3 minute final extension).

[0570] The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 806 bp product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0571] The two PCR fragments above were then subjected to overlapping PCR. The purified overlapping fragments were used as templates for amplification using primer 995103 (sense) and primer 995111 (antisense) described above to precisely fuse the 702 bp fragment comprising 209 bp of the Trichoderma reesei cellobiohydrolase I gene promoter and the Humicola insolens endoglucanase V core sequence to the 806 bp fragment comprising a portion of the Aspergillus oryzae beta-glucosidase variant BG41 coding region by overlapping PCR.

[0572] The amplification reactions (50 .mu.l) were composed of 1.times. Pfx Amplification Buffer, 10 mM dNTPs, 50 mM MgSO.sub.4, 2.5 .mu.l of each fragment (20 ng/.mu.l), 50 picomoles of 995103 primer, 50 picomoles of 995111 primer, and 2 units of high fidelity Pfx DNA polymerase. The reactions were incubated in an EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 programmed for an initial denaturation of 3 minutes at 95.degree. C. followed by 30 cycles each for 1 minute of denaturation, 1 minute annealing at 60.degree. C., and a 3 minute extension at 72.degree. C.

[0573] The reaction products were isolated by 1.0% agarose gel electrphoresis using TAE buffer where a 1.7 kb product band was excised from the gel and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions.

[0574] The 1.7 kb fragment was ligated into a pCR.RTM.4 Blunt Vector (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's instructions. The construct was then transformed into ONE SHOT.RTM. TOP10 Chemically Competent E. coli cells (Invitrogen, Carlsbad, Calif., USA) according to the manufacturer's rapid chemical transformation procedure. Colonies were selected and analyzed by plasmid isolation and digestion with Hind III to release the 1.7 kb overlapping PCR fragment.

[0575] Plasmid pSMai140 was also digested with Hind III to linearize the plasmid. Both digested fragments were combined in a ligation reaction using a Rapid DNA Ligation Kit following the manufacturer's instructions to produce pSaMe-F1 (FIG. 11).

[0576] E. coli XL1-Blue Subcloning-Grade Competent Cells (Stratagene, La Jolla, Calif., USA) were transformed with the ligation product. Identity of the construct was confirmed by DNA sequencing of the Trichoderma reesei cellobiohydrolase I gene promoter, Humicola insolens endoglucanase V signal sequence, Humicola insolens endoglucanase V core, Humicola insolens endoglucanase V signal sequence, Aspergillus oryzae beta-glucosidase variant BG41, and the Trichoderma reesei cellobiohydrolase I gene terminator sequence from plasmids purified from transformed E. coli. One clone containing the recombinant plasmid was designated pSaMe-F1. Plasmid pSaMe-F1 comprises the Trichoderma reesei cellobiohydrolase I gene promoter and terminator and the Humicola insolens endoglucanase V signal peptide sequence linked directly to the Humicola insolens endoglucanase V core polypeptide which are fused directly to the Humicola insolens endoglucanase V signal peptide which is linked directly to the Aspergillus oryzae beta-glucosidase variant BG41 mature coding sequence. The DNA sequence and deduced amino acid sequence of the Aspergillus oryzae beta-glucosidase variant BG fusion protein is shown in SEQ ID NOs: 103 and 104, respectively.

Example 10: Transformation of Trichoderma reesei RutC30 with pSaMe-F1

[0577] Shake flasks containing 25 ml of YP medium supplemented with 2% glucose and 10 mM uridine were inoculated with 5.times.10.sup.7 spores of Trichoderma reesei RutC30. Following incubation overnight for approximately 16 hours at 27.degree. C., 90 rpm, the mycelia were collected using a Vacuum Driven Disposable Filtration System. The mycelia were washed twice in 100 ml of deionized water and twice in 1.2 M sorbitol. Protoplasts were generated as described in Example 6.

[0578] Two micrograms of pSaMe-F1 DNA linearized with Pme I, 100 .mu.l of Trichoderma reesei RutC30 protoplasts, and 50% PEG (4000) were mixed and incubated for 30 minutes at room temperature. Then 3 ml of STC were added and the contents were poured onto a COVE plate supplemented with 10 mM uridine. The plate was then incubated at 28.degree. C. Transformants began to appear by day 6 and were picked to COVE2 plates for growth at 28.degree. C. and 6 days. Twenty-two Trichoderma reesei transformants were recovered.

[0579] Transformants were cultivated in shake flasks on cellulase-inducing medium, and beta-glucosidase activity was measured as described in Example 6. A number of pSaMe-F1 transformants produced beta-glucosidase activity. One transformant, designated Trichoderma reesei SaMeF1-9, produced the highest amount of beta-glucosidase, and had twice the activity of a strain expressing the Aspergillus oryzae beta-glucosidase variant (Example 9).

[0580] Endoglucanase activity was assayed using a carboxymethyl cellulose (CMC) overlay assay according to Beguin, 1983, Analytical Biochem. 131(2): 333-336. Five .mu.g of total protein from five of the broth samples (those having the highest beta-glucosidase activity) were diluted in Native Sample Buffer (Bio-Rad, Hercules, Calif., USA) and run on a CRITERION.RTM. 8-16% Tris-HCl gel (Bio-Rad, Hercules, Calif., USA) using 10.times.Tris/glycine running buffer (Bio-Rad, Hercules, Calif., USA) and then the gel was laid on top of a plate containing 1% carboxymethylcellulose (CMC). After 1 hour incubation at 37.degree. C., the gel was stained with 0.1% Congo Red for 20 minutes. The plate was then destained using 1 M NaCl in order to identify regions of clearing indicative of endoglucanase activity. Two clearing zones were visible, one upper zone around 110 kDa and a lower zone around 25 kDa. The predicted protein size of the Humicola insolens endoglucanase V and Aspergillus oryzae beta-glucosidase variant BG41 fusion is 118 kDa if the two proteins are not cleaved and remain as a single polypeptide; glycosylation of the individual endoglucanase V core domain and of the beta-glucosidase leads to migration of the individual proteins at higher mw than predicted from the primary sequence. If the two proteins are cleaved then the predicted sizes for the Humicola insolens endoglucanase V core domain is 24 kDa and 94 kDa for Aspergillus oryzae beta-glucosidase variant BG41. Since there was a clearing zone at 110 kDa this result indicated that minimally a population of the endoglucanase and beta-glucosidase fusion protein remains intact as a single large protein. The lower clearing zone most likely represents the endogenous endoglucanase activity, and possibly additionally results from partial cleavage of the Humicola insolens endoglucanase V core domain from the Aspergillus oryzae beta-glucosidase.

[0581] The results demonstrated the Humicola insolens endoglucanase V core was active even though it was linked to the Aspergillus oryzae beta-glucosidase. In addition, the increase in beta-glucosidase activity appeared to result from increased secretion of protein relative to the secretion efficiency of the non-fusion beta-glucosidase. By linking the Aspergillus oryzae beta-glucosidase variant BG41 sequence to the efficiently secreted Humicola insolens endoglucanase V core, more beta-glucosidase was secreted.

Example 11: Construction of Vector pSaMe-FX

[0582] Plasmid pSaMe-FX was constructed by modifying pSaMe-F1. Plasmid pSaMe-F1 was digested with Bst Z17 and Eco RI to generate a 1 kb fragment that contained the beta-glucosidase variant BG41 coding sequence and a 9.2 kb fragment containing the remainder of the plasmid. The fragments were separated by 1.0% agarose gel electrphoresis using TAE buffer and the 9.2 kb fragment was excised and purified using a QIAQUICK.RTM. Gel Extraction Kit according to the manufacturer's instructions. Plasmid pSMai135 was also digested with Bst Z17 and Eco RI to generate a 1 kb fragment containing bases homologous to the Aspergillus oryzae beta-glucosidase variant BG41 coding sequence and a 8.5 kb fragment containing the remainder of the plasmid. The 1 kb fragment was isolated and purified as above.

[0583] The 9.2 kb and 1 kb fragments were combined in a ligation reaction using a Rapid DNA Ligation Kit following the manufacturer's instructions to produce pSaMe-FX, which is identical to pSaMe-F1 except that it contained the wild-type beta-glucosidase mature coding sequence rather than the variant mature coding sequence.

[0584] E. coli SURE.RTM. Competent Cells (Stratagene, La Jolla, Calif., USA) were transformed with the ligation product. Identity of the construct was confirmed by DNA sequencing of the Trichoderma reesei cellobiohydrolase I gene promoter, Humicola insolens endoglucanase V signal sequence, Humicola insolens endoglucanase V core sequence, Humicola insolens endoglucanase V signal sequence, Aspergillus oryzae beta-glucosidase mature coding sequence, and the Trichoderma reesei cellobiohydrolase I gene terminator sequence from plasmids purified from transformed E. coli. One clone containing the recombinant plasmid was designated pSaMe-FX (FIG. 12). The DNA sequence and deduced amino acid sequence of the Aspergillus oryzae beta-glucosidase fusion protein is shown in SEQ ID NOs: 105 and 106, respectively.

Example 12: Transformation and Expression of Trichoderma Transformants

[0585] The pSaMe-FX construct was linearized with Pme I and transformed into the Trichoderma reesei RutC30 strain as described in Example 10. A total of 63 transformants were obtained from a single transformation. Transformants were cultivated in shake flasks on cellulase-inducing medium, and beta-glucosidase activity was measured as described in Example 6. A number of pSaMe-FX transformants produced beta-glucosidase activity. One transformant designated SaMe-FX16 produced twice the amount of beta-glucosidase activity compared to Trichoderma reesei SaMeF1-9 (Example 10).

Example 13: Analysis of Trichoderma reesei Transformants

[0586] A fusion protein was constructed as described in Example 9 by fusing the Humicola insolens endoglucanase V core (containing its own native signal sequence) with the Aspergillus oryzae beta-glucosidase variant BG41 mature coding sequence linked to the Humicola insolens endoglucanase V signal sequence. This fusion construct resulted in a two-fold increase in secreted beta-glucosidase activity compared to the Aspergillus oryzae beta-glucosidase variant BG41 mature coding sequence linked to the Humicola insolens endoglucanase V signal sequence. A second fusion construct was made as described in Example 11 consisting of the Humicola insolens endoglucanase V core (containing its own signal sequence) fused with the Aspergillus oryzae wild-type beta-glucosidase coding sequence linked to the Humicola insolens endoglucanase V signal sequence, and this led to an even further improvement in beta-glucosidase activity. The strain transformed with the wild-type fusion had twice the secreted beta-glucosidase activity relative to the strain transformed with the beta-glucosidase variant BG41 fusion.

Example 14: Cloning of the Beta-Glucosidase Fusion Protein Encoding Sequence into an Aspergillus oryzae Expression Vector

[0587] Two synthetic oligonucleotide primers, shown below, were designed to PCR amplify the full-length open reading frame from pSaMeFX encoding the beta-glucosidase fusion protein.

TABLE-US-00020 PCR Forward primer: (SEQ ID NO: 107) 5'-GGACTGCGCAGCATGCGTTC-3' PCR Reverse primer: (SEQ ID NO: 108) 5'-AGTTAATTAATTACTGGGCCTTAGGCAGCG-3'

Bold letters represent coding sequence. The underlined "G" in the forward primer represents a base change introduced to create an Sph I restriction site. The remaining sequence contains sequence identity compared with the insertion sites of pSaMeFX. The underlined sequence in the reverse primer represents a Pac I restriction site added to facilitate cloning into the expression vector pAlLo2 (WO 04/099228).

[0588] Fifty picomoles of each of the primers above were used in a PCR reaction containing 50 ng of pSaMeFX DNA, 1.times. Pfx Amplification Buffer, 6 .mu.l of 10 mM blend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM.RTM. Pfx DNA Polymerase, and 1 .mu.l of 50 mM MgSO.sub.4 in a final volume of 50 .mu.I. An EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 was used to amplify the fragment programmed for 1 cycle at 98.degree. C. for 2 minutes; and 35 cycles each at 96.degree. C. for 30 seconds, 61.degree. C. for 30 seconds, and 68.degree. C. for 3 minutes. After the 35 cycles, the reaction was incubated at 68.degree. C. for 10 minutes and then cooled at 10.degree. C. until further processed. A 3.3 kb PCR reaction product was isolated on a 0.8% GTG-agarose gel (Cambrex Bioproducts One Meadowlands Plaza East Rutherford, N.J., USA) using TAE buffer and 0.1 .mu.g of ethidium bromide per ml. The DNA was visualized with the aid of a DARK READER.TM. (Clare Chemical Research, Dolores, Colo., USA) to avoid UV-induced mutations. A 3.3 kb DNA band was excised with a disposable razor blade and purified with an ULTRAFREE.RTM.-DA spin cup (Millipore, Billerica, Mass., USA) according to the manufacturer's instructions.

[0589] The purified 3.3 kb PCR product was cloned into a pCR.RTM.4Blunt-TOPO.RTM. vector (Invitrogen, Carlsbad, Calif., USA). Four microliters of the purified PCR product were mixed with 1 .mu.l of a 2 M sodium chloride solution and 1 .mu.l of the TOPO.RTM. vector. The reaction was incubated at room temperature for 15 minutes and then 2 .mu.l of the reaction were used to transform One Shot.RTM. TOP10 Chemically Competent E. coli cells according to the manufacturer's instructions. Three aliquots of 83 .mu.l each of the transformation reaction were spread onto three 150 mm 2.times.YT plates supplemented with 100 .mu.g of ampicillin per ml and incubated overnight at 37.degree. C.

[0590] Eight recombinant colonies were used to inoculate liquid cultures containing 3 ml of LB medium supplemented with 100 .mu.g of ampicillin per ml. Plasmid DNA was prepared from these cultures using a BIOROBOT.RTM. 9600 (QIAGEN Inc., Valencia, Calif., USA). Clones were analyzed by restriction enzyme digestion with Pac I. Plasmid DNA from each clone was digested with Pac I and analyzed by 1.0% agarose gel electrophoresis using TAE buffer. All eight clones had the expected restriction digest pattern and clones 5, 6, 7, and 8 were selected to be sequenced to confirm that there were no mutations in the cloned insert. Sequence analysis of their 5' and 3' ends indicated that all 4 clones had the correct sequence. Clones 5 and 7 were selected for further sequencing. Both clones were sequenced to Phred Q values of greater than 40 to ensure that there were no PCR induced errors. Clones 5 and 7 were shown to have the expected sequence and clone 5 was selected for re-cloning into pAlLo2.

[0591] Plasmid DNA from clone 5 was linearized by digestion with Sph I. The linearized clone was then blunt-ended by adding 1.2 .mu.l of a 10 mM blend of dATP, dTTP, dGTP, and dCTP and 6 units of T4 DNA polymerase (New England Bioloabs, Inc., Ipswich, Mass., USA). The mixture was incubated at 12.degree. C. for 20 minutes and then the reaction was stopped by adding 1 .mu.l of 0.5 M EDTA and heating at 75.degree. C. for 20 minutes to inactivate the enzyme. A 3.3 kb fragment encoding the beta-glucosidase fusion protein was purified by gel electrophoresis and ultrafiltration as described above.

[0592] The vector pAlLo2 was linearized by digestion with Nco I. The linearized vector was then blunt-ended by adding 0.5 .mu.l of a 10 mM blend of dATP, dTTP, dGTP, and dCTP and one unit of DNA polymerase I. The mixture was incubated at 25.degree. C. for 15 minutes and then the reaction was stopped by adding 1 .mu.l of 0.5 M EDTA and heating at 75.degree. C. for 15 minutes to inactivate the enzyme. Then the vector was digested with Pac I. The blunt-ended vector was purified by gel electrophoresis and ultrafiltration as described above.

[0593] Cloning of the 3.3 kb fragment encoding the beta-glucosidase fusion protein into the linearized and purified pAlLo2 vector was performed with a Rapid Ligation Kit. A 1 .mu.l sample of the reaction was used to transform E. coli XL10 SOLOPACK.RTM. Gold cells (Stratagene, La Jolla, Calif., USA) according to the manufacturer's instructions. After the recovery period, two 100 .mu.l aliquots from the transformation reaction were plated onto two 150 mm 2.times.YT plates supplemented with 100 .mu.g of ampicillin per ml and incubated overnight at 37.degree. C. A set of eight putative recombinant clones was selected at random from the selection plates and plasmid DNA was prepared from each one using a BIOROBOT.RTM. 9600. Clones 1-4 were selected for sequencing with pAlLo2-specific primers to confirm that the junction vector/insert had the correct sequence. Clone 3 had a perfect vector/insert junction and was designated pAlLo47 (FIG. 13).

[0594] In order to create a marker-free expression strain, a restriction endonuclease digestion was performed to separate the blaA gene that confers resistance to the antibiotic ampicillin from the rest of the expression construct. Thirty micrograms of pAlLo47 were digested with Pme I. The digested DNA was then purified by agarose gel electrophoresis as described above. A 6.4 kb DNA band containing the expression construct but lacking the blaA gene was excised with a razor blade and purified with a QIAQUICK.RTM. Gel Extraction Kit.

Example 15: Expression of the Humicola insolens/Aspergillus Oryzae cel45Acore-cel3A Fusion Gene in Aspergillus oryzae JaL355

[0595] Aspergillus oryzae JaL355 (WO 00/240694) protoplasts were prepared according to the method of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Ten microliters of the purified expression construct of Example 14 were used to transform Aspergillus oryzae JaL355 protoplasts. The transformation of Aspergillus oryzae JaL355 yielded approximately 90 transformants. Fifty transformants were isolated to individual PDA plates and incubated for five days at 34.degree. C.

[0596] Forty-eight confluent spore plates were washed with 3 ml of 0.01% TWEEN.RTM. 80 and the spore suspension was used to inoculate 25 ml of MDU2BP medium in 125 ml glass shake flasks. Transformant cultures were incubated at 34.degree. C. with constant shaking at 200 rpm. After 5 days, 1 ml aliquots of each culture was centrifuged at 12,000.times.g and their supernatants collected. Five .mu.l of each supernatant were mixed with an equal volume of 2.times. loading buffer (10% beta-mercaptoethanol) and loaded onto a 1.5 mm 8%-16% Tris-Glycine SDS-PAGE gel and stained with BIO-SAFE.RTM. Coomassie Blue G250 protein stain (Bio-Rad, Hercules, Calif., USA). SDS-PAGE profiles of the culture broths showed that 33 out of 48 transformants were capable of expressing a new protein with an apparent molecular weight very close to the expected 118 kDa. Transformant 21 produced the best yield and was selected for further studies.

Example 16: Single Spore Isolation of Aspergillus oryzae JaL355 Transformant 21

[0597] Aspergillus oryzae JaL355 transformant 21 spores were spread onto a PDA plate and incubated for five days at 34.degree. C. A small area of the confluent spore plate was washed with 0.5 ml of 0.01% TWEEN.RTM. 80 to resuspend the spores. A 100 .mu.l aliquot of the spore suspension was diluted to a final volume of 5 ml with 0.01% TWEEN.RTM. 80. With the aid of a hemocytometer the spore concentration was determined and diluted to a final concentration of 0.1 spore per microliter. A 200 .mu.l aliquot of the spore dilution was spread onto 150 mm Minimal medium plates and incubated for 2-3 days at 34.degree. C. Emerging colonies were excised from the plates and transferred to PDA plates and incubated for 3 days at 34.degree. C. Then the spores were spread across the plates and incubated again for 5 days at 34.degree. C.

[0598] The confluent spore plates were washed with 3 ml of 0.01% TWEEN.RTM. 80 and the spore suspension was used to inoculate 25 ml of MDU2BP medium in 125 ml glass shake flasks. Single-spore cultures were incubated at 34.degree. C. with constant shaking at 200 rpm. After 5 days, a 1 ml aliquot of each culture was centrifuged at 12,000.times.g and their supernatants collected. Five .mu.l of each supernatant were mixed with an equal volume of 2.times. loading buffer (10% beta-mercaptoethanol) and loaded onto a 1.5 mm 8%-16% Tris-Glycine SDS-PAGE gel and stained with BIO-SAFE.RTM. Commassie Blue G250 protein stain. SDS-PAGE profiles of the culture broths showed that all eight transformants were capable of expressing the beta-glucosidase fusion protein at very high levels and one of cultures designated Aspergillus oryzae JaL355AlLo47 produced the best yield.

Example 17: Construction of pCW087

[0599] Two synthetic oligonucleotide primers shown below were designed to PCR amplify a Thermoascus aurantiacus GH61A polypeptide gene from plasmid pDZA2-7 (WO 2005/074656). The forward primer results in a blunt 5' end and the reverse primer incorporates a Pac I site at the 3' end.

TABLE-US-00021 Forward Primer: (SEQ ID NO: 109) 5'-ATGTCCTTTTCCAAGATAATTGCTACTG-3' Reverse Primer: (SEQ ID NO: 110) 5'-GCTTAATTAACCAGTATACAGAGGAG-3'

[0600] Fifty picomoles of each of the primers above were used in a PCR reaction consisting of 50 ng of pDZA2-7, 1 .mu.l of 10 mM blend of dATP, dTTP, dGTP, and dCTP, 5 .mu.l of 10.times. ACCUTAQ.TM. DNA Polymerase Buffer (Sigma-Aldrich, St. Louis, Mo., USA), and 5 units of ACCUTAQ.TM. DNA Polymerase (Sigma-Aldrich, St. Louis, Mo., USA), in a final volume of 50 .mu.I. An EPPENDORF.RTM. MASTERCYCLER.RTM. 5333 was used to amplify the DNA fragment programmed for 1 cycle at 95.degree. C. for 3 minutes; 30 cycles each at 94.degree. C. for 45 seconds, 55.degree. C. for 60 seconds, and 72.degree. C. for 1 minute 30 seconds. After the 25 cycles, the reaction was incubated at 72.degree. C. for 10 minutes and then cooled at 4.degree. C. until further processing. The 3' end of the Thermoascus aurantiacus GH61A PCR fragment was digested using Pac I. The digestion product was purified using a MINELUTE.TM. Reaction Cleanup Kit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer's instructions.

[0601] The GH61A fragment was directly cloned into pSMai155 (WO 2005/074647) utilizing a blunted Nco I site at the 5' end and a Pac I site at the 3' end. Plasmid pSMai155 was digested with Nco I and Pac I. The Nco I site was then rendered blunt using Klenow enzymes to fill in the 5' recessed Nco I site. The Klenow reaction consisted of 20 .mu.l of the pSma155 digestion reaction mix plus 1 mM dNTPs and 1 .mu.l of Klenow enzyme which was incubated briefly at room temperature. The newly linearized pSMai155 plasmid was purified using a MINELUTE.TM. Reaction Cleanup Kit according to the manufacturer's instructions. These reactions resulted in the creation a 5' blunt end and 3' Pac I site compatible to the newly generated GH61A fragment. The GH61A fragment was then cloned into pSMai155 expression vector using a Rapid DNA Ligation Kit (Roche, Indianapolis, Ind., USA) following the manufacturer's instructions. E. coli XL1-Blue Subcloning-Grade Competent Cells (Stratagene, La Jolla, Calif., USA) were transformed with the ligation product. Identity of the construct was confirmed by DNA sequencing of the GH61A coding sequence from plasmids purified from transformed E. coli. One E. coli clone containing the recombinant plasmid was designated pCW087-8.

Example 18: Construction of pSaMe-Ta61A

[0602] Expression vector pSaMe-Ta61 was constructed by digesting plasmid pMJ09 (WO 2005/056772), which harbors the amdS selectable marker, with Nsi I, which liberated a 2.7 kb amdS fragment. The 2.7 kb amdS fragment was then isolated by 1.0% agarose gel electrphoresis using TAE buffer and purified using a QIAQUICK.RTM. Gel Extraction Kit.

[0603] Expression vector pCW087 was digested with Nsi I and a 4.7 kb fragment was isolated by 1.0% agarose gel electrphoresis using TAE buffer and purified using a QIAQUICK.RTM. Gel Extraction Kit. The 2.7 kb amdS fragment was then ligated to the 4.7 kb vector fragment, using T4 DNA ligase (Roche, Indianapolis, Ind., USA) according to manufacturer's protocol, to create the expression vector pSaMe-Ta61A. Plasmid pSaMe-Ta61A comprises the Trichoderma reesei cellobiohydrolase I (CEL7A) gene promoter and terminator operably linked to the Thermoascus aurantiacus GH61A mature coding sequence.

Example 19: Construction of Trichoderma reesei Strain SaMe-MF268

[0604] A co-transformation was utilized to introduce plasmids pSaMe-FX and pSaMe-Ta61A into Trichoderma reesei RutC30. Plasmids pSaMe-FX and pSaMe-Ta61A were introduced into Trichoderma reesei RutC30 by PEG-mediated transformation (Penttila et al., 1987, supra). Each plasmid contained the Aspergillus nidulans amdS gene to enable transformants to grow on acetamide as the sole nitrogen source.

[0605] Trichoderma reesei RutC30 was cultivated at 27.degree. C. and 90 rpm in 25 ml of YP medium supplemented with 2% (w/v) glucose and 10 mM uridine for 17 hours. Mycelia were collected by filtration using a Vacuum Driven Disposable Filtration System and washed twice with deionized water and twice with 1.2 M sorbitol. Protoplasts were generated by suspending the washed mycelia in 20 ml of 1.2 M sorbitol containing 15 mg of GLUCANEX.RTM. (Novozymes A/S, Bagsvrd, Denmark) per ml and 0.36 units of chitinase (Sigma Chemical Co., St. Louis, Mo., USA) per ml and incubating for 15-25 minutes at 34.degree. C. with gentle shaking at 90 rpm.

[0606] Protoplasts were collected by centrifuging for 7 minutes at 400.times.g and washed twice with cold 1.2 M sorbitol. The protoplasts were counted using a haemacytometer and re-suspended in STC to a final concentration of 1.times.10.sup.8 protoplasts per ml. Excess protoplasts were stored in a Cryo 1.degree. C. Freezing Container (Nalgene, Rochester, N.Y., USA) at -80.degree. C.

[0607] Approximately 4 .mu.g each of plasmids pSaMe-FX and pSaMe-Ta61A were digested with Pme I to facilitate removal of the antibiotic resistance marker, ampR. Following digestion with Pme I the linear fragments were run on a 1% agarose gel using TAE buffer to separate the various fragments. A 7.5 kb fragment from pSaMe-FX and a 4.7 kb fragment from pSaMe-Ta61A were cut out of the gel and purified using a QIAquick Gel Extraction Kit according to the manufacturer's instructions. These purified fragments contain the amdS selectable marker cassette, the Trichoderma reesei cbh1 gene promoter and terminator; additionally, the fragment includes the Humicola insolens EGV core/Aspergillus oryzae BG fusion coding sequence or the T. aurantiacus GH61A coding sequence. The fragments used in transformation did not contain antibiotic resistance markers, as the ampR fragment was removed by this gel purification step. The purified fragments were then added to 100 .mu.l of protoplast solution and mixed gently, followed by 260 .mu.l of PEG buffer, mixed, and incubated at room temperature for 30 minutes. STC (3 ml) was then added and mixed and the transformation solution was plated onto COVE plates using amdS selection. The plates were incubated at 28.degree. C. for 5-7 days. Transformants were sub-cultured onto COVE2 plates and grown at 28.degree. C.

[0608] Over 400 transformants were subcultured onto fresh plates containing acetamide and allowed to sporulate for 7 days at 28.degree. C.

[0609] The Trichoderma reesei transformants were cultivated in 125 ml baffled shake flasks containing 25 ml of cellulase-inducing medium at pH 6.0 inoculated with spores of the transformants and incubated at 28.degree. C. and 200 rpm for 5 days. Trichoderma reesei RutC30 was run as a control. Culture broth samples were removed at day 5. One ml of each culture broth was centrifuged at 15,700.times.g for 5 minutes in a micro-centrifuge and the supernatants transferred to new tubes.

[0610] SDS-PAGE was carried out using CRITERION.RTM. Tris-HCl (5% resolving) gels with the CRITERION.RTM. System. Five .mu.l of day 5 supernatants (see above) were suspended in 2.times. concentration of Laemmli Sample Buffer (Bio-Rad, Hercules, Calif., USA) and boiled in the presence of 5% beta-mercaptoethanol for 3 minutes. The supernatant samples were loaded onto a polyacrylamide gel and subjected to electrophoresis with 1.times. Tris/Glycine/SDS as running buffer (Bio-Rad, Hercules, Calif., USA). The resulting gel was stained with BIO-SAFE.RTM. Coomassie Stain. Transformants showing expression of both the Thermoascus aurantiacus GH61A polypeptide and the fusion protein consisting of the Humicola insolens endoglucanase V core (Ce145A) fused with the Aspergillus oryzae beta-glucosidase as seen by visualization of bands on SDS-PAGE gels were then tested in PCS hydrolysis reactions to identify the strains producing the best hydrolytic broths.

Example 20: Identification of Trichoderma reesei Strain SaMe-MF268

[0611] The transformants showing expression of both the Thermoascus aurantiacus GH61A polypeptide and the Aspergillus oryzae beta-glucosidase fusion protein were cultivated in 125 ml baffled shake flasks containing 25 ml of cellulase-inducing medium at pH 6.0 inoculated with spores of the transformants and incubated at 28.degree. C. and 200 rpm for 5 days.

[0612] The shake flask culture broths were centrifuged 6000.times.g and filtered using STERICUP.TM. EXPRESS.TM. (Millipore, Bedford, Mass., USA) to 0.22 .mu.m prior to hydrolysis. The activities of the culture broths were measured by their ability to hydrolyze the PCS and produce sugars detectable by a chemical assay of their reducing ends.

[0613] Corn stover was pretreated at the U.S. Department of Energy National Renewable Energy Laboratory (NREL), Boulder, Colo., USA, using dilute sulfuric acid. The following conditions were used for the pretreatment: 0.048 g sulfuric acid/g dry biomass at 190.degree. C. and 25% w/w dry solids for around 1 minute. The water-insoluble solids in the pretreated corn stover (PCS) contained 59.2% cellulose as determined by a limit digest of PCS to release glucose and cellobiose. Prior to enzymatic hydrolysis, the PCS was washed with a large volume of double deionized water; the dry weight of the water-washed PCS was found to be 17.73%.

[0614] PCS in the amount of 1 kg was suspended in approximately 20 liters of double deionized water and, after the PCS settled, the water was decanted. This was repeated until the wash water was above pH 4.0, at which time the reducing sugars were lower than 0.06 g per liter. For small volume assays (e.g., 1 ml) the settled slurry was sieved through 100 Mesh screens to ensure ability to pipette. Percent dry weight content of the washed PCS was determined by drying the sample at a 105.degree. C. oven for at least 24 hours (until constant weight) and comparing to the wet weight.

[0615] PCS hydrolysis was performed in a 1 ml volume in 96-deep-well plates (Axygen Scientific) heat sealed by an ALPS 300.TM. automated lab plate sealer (ABgene Inc., Rochester, N.Y., USA). PCS concentration was 10 g per liter in 50 mM sodium acetate pH 5.0. PCS hydrolysis was performed at 50.degree. C. without additional stirring except as during sampling as described. Each reaction was performed in triplicate. Released reducing sugars were analyzed by p-hydroxy benzoic acid hydrazide (PHBAH) reagent as described below.

[0616] A volume of 0.8 ml of PCS (12.5 g per liter in water) was pipetted into each well of 96-deep-well plates, followed by 0.10 ml of 0.5 M sodium acetate pH 5.0, and then 0.10 ml of diluted enzyme solution to start the reaction with a final reaction volume of 1.0 ml and PCS concentration of 10 g per liter. Plates were sealed. The reaction mixture was mixed by inverting the deep-well plate at the beginning of hydrolysis and before taking each sample time point. At each sample time point the plate was mixed and then the deep-well plate was centrifuged (Sorvall RT7 with RTH-250 rotor) at 2000 rpm for 10 minutes before 20 .mu.l of hydrolysate (supernatant) was removed and added to 180 .mu.l of 0.4% NaOH in a 96-well microplate. This stopped solution was further diluted into the proper range of reducing sugars, when necessary. The reducing sugars released were assayed by para-hydroxy benzoic acid hydrazide reagent (PHBAH, Sigma, 4-hydroxy benzyhydrazide): 50 .mu.l of PHBAH reagent (1.5%) was mixed with 100 .mu.l of sample in a V-bottom 96-well THERMOWELL.TM. plate (Costar 6511), incubated on a plate heating block at 95.degree. C. for 10 minutes, then 50 .mu.l of double deionized water was added to each well, mixed and 100 .mu.l was transferred to another flat-bottom 96-well plate (Costar 9017) and absorbance read at 410 nm. Reducing sugar was calculated using a glucose calibration curve under the same conditions. Percent conversion of cellulose to reducing sugars was calculated as:

% conversion=reducing sugars(mg/ml)/(cellulose added(mg/ml).times.1.11)

The factor 1.11 corrects for the weight gain in hydrolyzing cellulose to glucose.

[0617] Following the 1 ml PCS hydrolysis testing, the top candidates were grown in duplicate in 2 liter fermentors.

[0618] Shake flask medium was composed per liter of 20 g of dextrose, 10 g of corn steep solids, 1.45 g of (NH.sub.4).sub.2SO.sub.4, 2.08 g of KH.sub.2PO.sub.4, 0.36 g of CaCl.sub.2, 0.42 g of MgSO.sub.4.7H.sub.2O, and 0.42 ml of trace metals solution. Trace metals solution was composed per liter of 216 g of FeCl.sub.3.6H.sub.2O, 58 g of ZnSO.sub.4.7H.sub.2O, 27 g of MnSO.sub.4.H.sub.2O, 10 g of CuSO.sub.4.5H.sub.2O, 2.4 g of H.sub.3BO.sub.3, and 336 g of citric acid.

[0619] Ten ml of shake flask medium was added to a 500 ml shake flask. The shake flask was inoculated with two plugs from a solid plate culture and incubated at 28.degree. C. on an orbital shaker at 200 rpm for 48 hours. Fifty ml of the shake flask broth was used to inoculate a 3 liter fermentation vessel.

[0620] Fermentation batch medium was composed per liter of 30 g of cellulose, 4 g of dextrose, 10 g of corn steep solids, 3.8 g of (NH.sub.4).sub.2SO.sub.4, 2.8 g of KH.sub.2PO.sub.4, 2.64 g of CaCl.sub.2, 1.63 g of MgSO.sub.4.7H.sub.2O, 1.8 ml of anti-foam, and 0.66 ml of trace metals solution. Trace metals solution was composed per liter of 216 g of FeCl.sub.3.6H.sub.2O, 58 g of ZnSO.sub.4.7H.sub.2O, 27 g of MnSO.sub.4.H.sub.2O, 10 g of CuSO.sub.4.5H.sub.2O, 2.4 g of H.sub.3BO.sub.3, and 336 g of citric acid. Fermentation feed medium was composed of dextrose and cellulose.

[0621] A total of 1.8 liters of the fermentation batch medium was added to a 3 liter fermentor. Fermentation feed medium was dosed at a rate of 0 to 4 g/l/hr for a period of 165 hours. The fermentation vessel was maintained at a temperature of 28.degree. C. and pH was controlled to a set-point of 4.75+/-0.1. Air was added to the vessel at a rate of 1 vvm and the broth was agitated by Rushton impeller rotating at 1100 to 1300 rpm. At the end of the fermentation, whole broth was harvested from the vessel and centrifuged at 3000 rpm x g to remove the biomass. The supernatant was sterile filtered and stored at 35 to 40.degree. C.

[0622] Total protein concentration was determined and broths were re-tested in 50 g PCS hydrolysis reactions as described below. Enzyme dilutions were prepared fresh before each experiment from stock enzyme solutions, which were stored at 4.degree. C.

[0623] Hydrolysis of PCS was conducted using 125 ml screw-top Erlenmeyer flasks (VWR, West Chester, Pa., USA) using a total reaction mass of 50 g according to NREL Laboratory Analytical Protocol #008. In this protocol hydrolysis of PCS (approximately 11.4% in PCS and 6.8% cellulose in aqueous 50 mM sodium acetate pH 5.0 buffer) was performed using different protein loadings (expressed as mg of protein per gram of cellulose) of a 2 liter fermentation broth sample. Testing of PCS hydrolyzing capability was performed at 50.degree. C. with orbital shaking at 150 rpm using an INNOVA.RTM. 4080 Incubator (New Brunswick Scientific, Edison, N.J., USA). Aliquots were taken during the course of hydrolysis at 72, 120, and 168 hours and centrifuged, and the supernatant liquid was filtered using a MULTISCREEN.RTM. HV 0.45 .mu.m membrane (Millipore, Billerica, Mass., USA) by centrifugation at 2000 rpm for 10 minutes using a SORVALL.RTM. RT7 plate centrifuge (Thermo Fisher Scientific, Waltham, Mass., USA). When not used immediately, filtered sugary aliquots were frozen at -20.degree. C. Sugar concentrations of samples diluted in 0.005 M H.sub.2SO.sub.4 were measured after elution by 0.005 M H.sub.2SO.sub.4 at a flow rate of 0.4 ml per minute from a 4.6.times.250 mm AMINEX.RTM. HPX-87H column (Bio-Rad, Hercules, Calif., USA) at 65.degree. C. with quantitation by integration of glucose and cellobiose signal from refractive index detection using a CHEMSTATION.RTM. AGILENT.RTM. 1100 HPLC (Agilent Technologies, Santa Clara, Calif., USA) calibrated by pure sugar samples. The resultant equivalents were used to calculate the percentage of cellulose conversion for each reaction.

[0624] The degree of cellulose conversion to glucose plus cellobiose sugars (conversion, %) was calculated using the following equation:

Conversion.sub.(%)=(glucose+cellobiose.times.1.053).sub.(mg/ml).times.10- 0.times.162/(cellulose.sub.(mg/ml).times.180)=(glucose+cellobiose.times.1.- 053).sub.(mg/ml).times.100/(cellulose.sub.(mg/ml).times.1.111)

In this equation the factor 1.111 reflects the weight gain in converting cellulose to glucose, and the factor 1.053 reflects the weight gain in converting cellobiose to glucose.

[0625] The results of the PCS hydrolysis reactions in the 50 g flask assay described above are shown in Table 1. One strain that produced the highest performing broth was designated Trichoderma reesei SaMe-MF268.

TABLE-US-00022 TABLE 1 Percent conversion to sugars at 168 hour timepoint Percent conversion (glucose plus cellobiose) for protein loading 2.5 mg/g 4.0 mg/g Broth ID-Strain Name cellulose cellulose XCL-461-SaMe-MF268 66.29 80.08 XCL-465-SaMe-MF268 69.13 82.80 XCL-462-SaMe-MF330 62.98 77.99 XCL-466-SaMe-MF330 63.34 77.90 XCL-463-SaMe-MF377 64.03 78.45 XCL-467-SaMe-MF377 64.19 79.06

Example 21: Construction of Vector pSaMe-FH

[0626] Expression vector pSaMe-FH (FIG. 14) was constructed by digesting plasmid pSMai155 (WO 2005/074647) and plasmid pSaMe-FX (Example 11) with Bsp 1201 and Pac 1. The 5.5 kb fragment from pSMai155 and the 3.9 kb fragment from pSaMeFX were isolated by 1.0% agarose gel electrphoresis using TAE buffer and purified using a QIAQUICK.RTM. Gel Extraction Kit. The two fragments were then ligated using T4 DNA ligase according to manufacturer's protocol. E. coli SURE.RTM. Competent Cells were transformed with the ligation product. Identity of the construct was confirmed by DNA sequencing of the Trichoderma reesei cellobiohydrolase I gene promoter, Humicola insolens endoglucanase V signal sequence, Humicola insolens endoglucanase V core sequence, Humicola insolens endoglucanase V signal sequence, Aspergillus oryzae beta-glucosidase mature coding sequence, and the Trichoderma reesei cellobiohydrolase I gene terminator sequence from plasmids purified from transformed E. coli. One clone containing the recombinant plasmid was designated pSaMe-FH. Plasmid pSaMe-FH comprises the Trichoderma reesei cellobiohydrolase I (CEL7A) gene promoter and terminator operably linked to the gene fusion of Humicola insolens Ce145A core/Aspergillus oryzae .beta.-glucosidase. Plasmid pSaMe-FH is identical to that of pSaMe-FX except the amdS selectable marker has been removed and replaced with the hygromycin resistance selectable marker.

Example 22: Isolation of Mutant of Trichoderma reesei SMA135-04 with Increased Cellulase Production and Enhanced Pre-Treated Corn Stover (PCS) Degrading Ability

[0627] PCS (Example 20) was used as a cellulose substrate for cellulolytic enzyme assays and for selection plates. Prior to assay, PCS was washed with a large volume of distilled deionized water until the filtrate pH was greater than pH 4.0. Also, PCS was sieved using 100MF metal filter to remove particles. The washed and filtered PCS was re-suspended in distilled water to a concentration of 60 mg/ml suspension, and stored at 4.degree. C.

[0628] Trichoderma reesei strain SMA135-04 (Example 6) was subjected to mutagenic treatment with N-methyl-N-nitro-N-nitrosoguanidine (NTG) (Sigma Chemical Co., St. Louis, Mo., USA), a chemical mutagen that induces primarily base substitutions and some deletions (Rowlands, 1984, Enzyme Microb. Technol. 6: 3-10). Survival curves were done with a constant time of exposure and varying doses of NTG, and with a constant concentration of NTG and different times of exposure to get a survival level of 10%. To obtain this survival rate, a conidia suspension was treated with 0.2 mg/ml of NTG for 20 minutes at 37.degree. C. with gentle rotation. Each experiment was conducted with a control where the conidia were not treated with NTG and this served as a control for competitive growth of the mutants over the non-mutagenesis culture.

[0629] Primary selection of mutants was performed after the NTG treatment. A total of 8.times.10.sup.6 conidia that survived the mutagenesis were mixed in 30 ml of Mandel's medium containing 0.5% Peptone, 0.1% TRITON.RTM. X-100 (A nonionic surfactant which has a hydrophilic polyethylene oxide group and a hydrocarbon lipophilic or hydrophobic group. The hydrocarbon group is a 4-(1,1,3,3-tetramethylbutyl)-phenyl group.) and 1.5 g of agar. This suspension was then added to a deep plate (150 mm in diameter and 25 mm deep; Corning Inc, NY, USA) and the agar was allowed to harden at room temperature. After hardening the agar, 200 ml of Mandels medium containing 0.5% Peptone, 0.1% TRITON.RTM. X-100, 1.5% agar, and 1.0% PCS was added. The plates were incubated at 28.degree. C. after hardening of the agar. After 3-5 days of incubation, 700 colonies that penetrated through the PCS selection layer before the non-treated control strain were used for secondary selection.

[0630] For secondary selection, three loopfuls of conidia from each isolate were added to 125 ml shake flasks containing 25 ml of cellulase-inducing medium and incubated at 28.degree. C. and 200 rpm for 5 days to induce expression and secretion of cellulases. One ml of each culture broth was centrifuged at 400.times.g for 5 minutes in a micro-centrifuge and the supernatants assayed for hydrolyzing activity of PCS and for total protein yield.

[0631] "Robotic" PCS hydrolysis assay was performed by diluting shake flask broth samples 1:20 in 50 mM sodium acetate pH 5.0. The diluted samples were added to assay plates (96-well flat-bottom plates) at 400 .mu.l sample per g of PCS before dilution. Using a BIOMEK.RTM. FX (Beckman Coulter, Fullerton, Calif.), PCS was added at 10 g of PCS per liter followed by 50 mM sodium acetate pH 5.0 to a total volume of 180 .mu.l. The assay plates were incubated for 5 days at 30.degree. C. in humidified boxes, which were shaken at 250 rpm. In order to increase the statistical precision of the assays, 6 replicates were performed for each sample. However, 2 replicates were performed for the 1:20 sample dilution. After 5 days incubation, the concentrations of reducing sugars (RS) in the hydrolyzed PCS samples were measured using a PHBAH assay, which was modified and adapted to a 96-well microplate format. Using an ORCA.TM. robot (Beckman Coulter, Fullerton, Calif., USA), the growth plates were transported to a BIOMEK.RTM. FX and 9 .mu.l of broth samples were removed from the assay plates and aliquoted into 96-well V-bottom plates (MJ Research, Waltham, Mass., USA). The reactions were initiated by the addition of 135 .mu.l of 0.533% PHBAH in 2% sodium hydroxide. Each assay plate was heated on a TETRAD.RTM. Thermal Cycler (MJ Research, Waltham, Mass., USA) for 10 minutes at 95.degree. C., and cooled to room temperature. After the incubation, 40 .mu.l of the reaction samples were diluted in 160 .mu.l of deionized water and transferred into 96-well flat-bottom plates. Then, the samples were measured for absorbance at 405 nm using SPECTRAMAX.RTM. 250 (Molecular Devices, Sunnyvale, Calif.). The A.sub.405 values were translated into glucose equivalents using a standard curve generated with six glucose standards (0.000, 0.040, 0.800, 0.120, 0.165, and 0.200 mg/ml of deionized water), which were treated similarly to the samples. The average correlation coefficient for the standard curves was greater than 0.98. The degree of cellulose conversion to reducing sugar (RS yield, %) was calculated using the equation described in Example 20.

[0632] Total protein yield was determined using a bicinchoninic acid (BCA) assay. Samples were diluted 1:8 in water to bring the concentration within the appropriate range. Albumin standard (BSA) was diluted at various levels starting with a 2.0 mg/ml concentration and ending with a 0.25 mg/ml concentration in water. Using a BIOMEK.RTM. FX, a total of 20 .mu.l of each dilution including standard was transferred to a 96-well flat bottom plate. Two hundred micro-liters of a BCA substrate solution (BCA Protein Assay Kit, Pierce, Rockford, Ill., USA) was added to each well and then incubated at 37.degree. C. for 45 minutes. Upon completion of the incubation, the optical density of 562 nm was obtained for the 96-well plate using SPECTRAMAX.RTM. 250. Sample concentrations were determined by extrapolation from the generated standard curve by Microsoft Excel (Microsoft Corporation, Redmond, Wash.).

[0633] Of the primary isolates picked, twenty produced broth that showed improved hydrolyzing activity of PCS when compared to broth from strain SMA135-04. These isolates produced cellulolytic broth that was capable of producing 5-15% higher levels of reducing sugar relative to the parental strain. Some isolates, for example, SMai-M104 showed increased performance in hydrolysis of cellulose PCS per volume broth, and additionally secreted higher levels of total protein

[0634] Selection of the best performing Trichoderma reesei mutant strain, SMai-M104, was determined by assessing cellulase performance of broth produced by fermentation. The fermentation was run for 7 days as described in Example 20. The fermentation samples were tested in a 50 g PCS hydrolysis in 125-ml Erlenmeyer flasks with screw caps (VWR, West Chester, Pa., USA). Reaction conditions include: cellulose loading of 6.7%; enzyme loadings of 6 and 12 mg/g cellulose; total reactants of 50 g; 50.degree. C. and pH 5.0. To begin this experiment, each shake flask and cap was weighed and the desired amount of PCS was added to the shake flask and the total weight was recorded. Ten ml of distilled water was added to each shake flask and then all the shake flasks were autoclaved for 30 minutes at 121.degree. C. After autoclaving, the flasks were allowed to cool to room temperature. In order to get the total weight of each flask to 50 grams, 5 ml of 0.5 M sodium acetate pH 5.0 was added followed by cellulase broth to achieve the desired loading, then the appropriate amount of distilled water was added to reach the desired final 50 g weight. The flasks were then placed in an incubator shaker (New Brunswick Scientific, Edison, N.J., USA) at 50.degree. C. and 130 rpm. At days 3, 5 and 7, 1 ml samples were taken from each flask and added to a 96-deep-well plate (2.0-ml total volume). The 96 well-plate was then centrifuged at 3000 rpm for 15 minutes using a SORVALL.RTM. RT7 plate centrifuge (Thermo Fisher Scientific, Waltham, Mass., USA). Following centrifugation, 200 .mu.l of supernatant was transferred to a 96-well 0.45 .mu.m pore size filtration plate (Millipore, Bedford, Mass., USA) and vacuum applied in order to collect the filtrate. The filtrate was then diluted to a proper range of reducing sugars with 0.4% NaOH and measured using a PHBAH reagent (1.5%) as follows: 50 ul of the PHBAH reagent and 100 .mu.l sample were added to a V-bottom 96-well plate and incubated at 95.degree. C. for 10 minutes. To complete the reaction, 50 .mu.l distilled water was added to each well and after mixing the samples, 100 .mu.l of the mix was transferred to another flat-bottom 96-well plate in order to obtain a spectrophotometric reading at A410. The reducing sugar amount was calculated using a glucose calibration curve and percent digestion was calculated as:

% digestion=reducing sugars(mg/ml)/(cellulose added(mg/ml).times.1.11), where the factor 1.11reflects the weight gain in converting cellulose to glucose.

[0635] The PCS hydrolysis assay results showed that one mutant, designated SMai-M104, slightly (approximately 5% increase in glucose) outperformed parental strain SMA135-04, especially at high loading (12 mg/g cellulose).

Example 23: Construction of Trichoderma reesei Strain SMai26-30

[0636] A co-transformation was utilized to introduce plasmids pCW085 (WO 2006/074435), pSaMe-FH, and pCW087 (Example 17) into Trichoderma reesei SMai-M104. Plasmid pCW085 is an expression vector for a Thielavia terrestris NRRL 8126 cellobiohydrolase (CEL6A). All three plasmids were introduced into Trichoderma reesei SMai-M104 by PEG-mediated transformation (Penttila et al., 1987, supra). Each plasmid contained the Escherichia coli hygromycin B phosphotransferase (hph) gene to enable transformants to grow on hygromycin B.

[0637] Trichoderma reesei SMai-M104 was cultivated at 27.degree. C. and 90 rpm in 25 ml of YP medium supplemented with 2% (w/v) glucose and 10 mM uridine for 17 hours. Mycelia were collected by filtration using a Vacuum Driven Disposable Filtration System and washed twice with deionized water and twice with 1.2 M sorbitol. Protoplasts were generated by suspending the washed mycelia in 20 ml of 1.2 M sorbitol containing 15 mg of GLUCANEX.RTM. per ml and 0.36 units of chitinase per ml and incubating for 15-25 minutes at 34.degree. C. with gentle shaking at 90 rpm. Protoplasts were collected by centrifuging for 7 minutes at 400.times.g and washed twice with cold 1.2 M sorbitol. The protoplasts were counted using a haemacytometer and re-suspended in STC to a final concentration of 1.times.10.sup.8 protoplasts per ml. Excess protoplasts were stored in a Cryo 1.degree. C. Freezing Container at -80.degree. C.

[0638] Approximately 10 .mu.g each of plasmids pCW085, pSaMe-FH and pCW087 were digested with Pme I and added to 100 .mu.l of protoplast solution and mixed gently, followed by 260 .mu.l of PEG buffer, mixed, and incubated at room temperature for 30 minutes. STC (3 ml) was then added and mixed and the transformation solution was plated onto PDA containing 1M sucose and 10 mM uridine. The plates were incubated at 28.degree. C. for 16 hours, and then an agar overlay containing hygromycin B (30 .mu.g/ml) final concentration) was then added and incubation continued for 4-6 days. Eighty transformants were sub-cultured onto PDA plates and grown at 28.degree. C.

[0639] The Trichoderma reesei transformants were cultivated in 125 ml baffled shake flasks containing 25 ml of cellulase inducing medium at pH 6.0 inoculated with spores of the transformants and incubated at 28.degree. C. and 200 rpm for 5 days. Trichoderma reesei SMai-M104 was run as a control. Culture broth samples were removed at day 5. One ml of each culture broth was centrifuged at 15,700.times.g for 5 minutes in a micro-centrifuge and the supernatants transferred to new tubes.

[0640] SDS-PAGE was carried out using CRITERION.RTM. Tris-HCl (5% resolving) gels with the CRITERION.RTM. System. Five .mu.l of day 5 supernatants (see above) were suspended in 2.times. concentration of Laemmli Sample Buffer and boiled in the presence of 5% beta-mercaptoethanol for 3 minutes. The supernatant samples were loaded onto a polyacrylamide gel and subjected to electrophoresis with 1.times. Tris/Glycine/SDS as running buffer. The resulting gel was stained with BIO-SAFE.RTM. Coomassie Stain. Transformants showing expression of the Thermoascus aurantiacus GH61A polypeptide and the fusion protein consisting of the Humicola insolens endoglucanase V core (Ce145A) fused with the Aspergillus oryzae beta-glucosidase and Thielavia terrestris cellobiohydrolase 11 as seen by visualization of bands on SDS-PAGE gels were then tested in PCS hydrolysis reactions as described in Example 20 to identify the strains producing the best hydrolytic broths. One transformant that produced the highest performing broth was designated Trichoderma reesei SMai26-30.

[0641] Hydrolysis of PCS by Trichoderma reesei strain SMai26-30 broth was conducted as described in Example 20 with the following modifications. The lot of PCS was different than that used in Example 20, but prepared under similar conditions. In this protocol hydrolysis of PCS (approximately 11.3% in PCS and 6.7% cellulose in aqueous 50 mM sodium citrate pH 5.0 buffer) was performed using different protein loadings (expressed as mg of protein per gram of cellulose) of the Trichoderma reesei strain SMai26-30 fermentation broth. Aliquots were taken during the course of hydrolysis at 48, 120 and 168 hours. The results of the PCS hydrolysis reactions in the 50 g flask assay described above are shown in Table 2.

TABLE-US-00023 TABLE 2 Percent conversion to sugars at 48, 72 and 168 hour timepoint Hours of hydrolysis 48 120 168 mg/ml Percent conversion 2.52 47.2 60.4 64.1 2.52 48.2 61.1 64.8 5.01 67.2 85.0 87.7 5.01 67.9 85.8 88.8 9.98 85.2 95.4 96.0 9.98 85.3 93.6 94.7

DEPOSITS OF BIOLOGICAL MATERIAL

[0642] The following biological materials have been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, 1815 University Street, Peoria, Ill., 61604, and given the following accession numbers:

TABLE-US-00024 Deposit Accession Number Date of Deposit E. coli strain pEJG120 NRRL B-30699 Dec. 19, 2003 E. coli strain pTter61C NRRL B-30823 Jan. 21, 2005 E. coli strain pTter61D NRRL B-30812 Jan. 21, 2005 E. coli strain pTter61E NRRL B-30814 Jan. 21, 2005 E. coli strain pTter61G NRRL B-30811 Jan. 21, 2005 E. coli strain pDZA2-7 NRRL B-30704 Jan. 30, 2004 E. coli strain pTr3337 NRRL B-30878 Sep. 20, 2005 E. coliTOP10 (pEJG113) NRRL B-30695 Oct. 17, 2003 E. coli TOP10 pKKAB NRRL B-30860 Jul. 8, 2005 NN049573 DSM 14240 Apr. 19, 2001

[0643] The strains have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 C.F.R. .sctn. 1.14 and 35 U.S.C. .sctn. 122. The deposits represent substantially pure cultures of the deposited strains. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

[0644] The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

[0645] Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.

Sequence CWU 1

1

13111846DNAThielavia terrestris 1aattgaagga gggagtggcg gagtggccac caagtcaggc ggctgtcaac taaccaagga 60tgggaacagt tcggctcgcc ttgcccgagg gcagcgttcc ctgatgggga cgaaccatgg 120gactggggtc agctgctgta taaaagttca aatcgatgat ctctcagatg gcgctgctgg 180ggtgttctgc gcttttccat cctcgcaacc tggtatccca ctagtccagc gttcggcacc 240atgaagtcgt tcaccattgc cgccttggca gccctatggg cccaggaggc cgccgcccac 300gcgaccttcc aggacctctg gattgatgga gtcgactacg gctcgcaatg tgtccgcctc 360ccggcgtcca actcccccgt caccaatgtt gcgtccgacg atatccgatg caatgtcggc 420acctcgaggc ccaccgtcaa gtgcccggtc aaggccggct ccacggtcac gatcgagatg 480caccaggttc gcacgcctct ctgcgtaggc cccccagcta ctatatggca ctaacacgac 540ctccagcaac ctggcgaccg gtcttgcgcc aacgaggcta tcggcggcga ccactacggc 600cccgtaatgg tgtacatgtc caaggtcgat gacgcggtga cagccgacgg ttcatcgggc 660tggttcaagg tgttccagga cagctgggcc aagaacccgt cgggttcgac gggcgacgac 720gactactggg gcaccaagga cctcaactcg tgctgcggca agatgaacgt caagatcccc 780gaagacatcg agccgggcga ctacctgctc cgcgccgagg ttatcgcgct gcacgtggcc 840gccagctcgg gcggcgcgca gttctacatg tcctgctacc agctgaccgt gacgggctcc 900ggcagcgcca ccccctcgac cgtgaatttc ccgggcgcct actcggccag cgacccgggc 960atcctgatca acatccacgc gcccatgtcg acctacgtcg tcccgggccc gaccgtgtac 1020gcgggcggct cgaccaagtc ggctggcagc tcctgctccg gctgcgaggc gacctgcacg 1080gttggttccg gccccagcgc gacactgacg cagcccacct ccaccgcgac cgcgacctcc 1140gcccctggcg gcggcggctc cggctgcacg gcggccaagt accagcagtg cggcggcacc 1200ggctacactg ggtgcaccac ctgcgctgta agttccctcg tgatatgcag cggaacaccg 1260tctggactgt tttgctaact cgcgtcgtag tccgggtcta cctgcagcgc cgtctcgcct 1320ccgtactact cgcagtgcct ctaagccggg agcgcttgct cagcgggctg ctgtgaagga 1380gctccatgtc cccatgccgc catggccgga gtaccgggct gagcgcccaa ttcttgtata 1440tagttgagtt ttcccaatca tgaatacata tgcatctgca tggactgttg cgtcgtcagt 1500ctacatcctt tgctccactg aactgtgaga ccccatgtca tccggaccat tcgatcggtg 1560ctcgctctac catctcggtt gatgggtctg ggcttgagag tcactggcac gtcctcggcg 1620gtaatgaaat gtggaggaaa gtgtgagctg tctgacgcac tcggcgctga tgagacgttg 1680agcgcggccc acactggtgt tctgtaagcc agcacacaaa agaatactcc aggatggccc 1740atagcggcaa atatacagta tcagggatgc aaaaagtgca aaagtaaggg gctcaatcgg 1800ggatcgaacc cgagacctcg cacatgactt atttcaagtc aggggt 18462326PRTThielavia terrestris 2Met Lys Ser Phe Thr Ile Ala Ala Leu Ala Ala Leu Trp Ala Gln Glu 1 5 10 15 Ala Ala Ala His Ala Thr Phe Gln Asp Leu Trp Ile Asp Gly Val Asp 20 25 30 Tyr Gly Ser Gln Cys Val Arg Leu Pro Ala Ser Asn Ser Pro Val Thr 35 40 45 Asn Val Ala Ser Asp Asp Ile Arg Cys Asn Val Gly Thr Ser Arg Pro 50 55 60 Thr Val Lys Cys Pro Val Lys Ala Gly Ser Thr Val Thr Ile Glu Met 65 70 75 80 His Gln Gln Pro Gly Asp Arg Ser Cys Ala Asn Glu Ala Ile Gly Gly 85 90 95 Asp His Tyr Gly Pro Val Met Val Tyr Met Ser Lys Val Asp Asp Ala 100 105 110 Val Thr Ala Asp Gly Ser Ser Gly Trp Phe Lys Val Phe Gln Asp Ser 115 120 125 Trp Ala Lys Asn Pro Ser Gly Ser Thr Gly Asp Asp Asp Tyr Trp Gly 130 135 140 Thr Lys Asp Leu Asn Ser Cys Cys Gly Lys Met Asn Val Lys Ile Pro 145 150 155 160 Glu Asp Ile Glu Pro Gly Asp Tyr Leu Leu Arg Ala Glu Val Ile Ala 165 170 175 Leu His Val Ala Ala Ser Ser Gly Gly Ala Gln Phe Tyr Met Ser Cys 180 185 190 Tyr Gln Leu Thr Val Thr Gly Ser Gly Ser Ala Thr Pro Ser Thr Val 195 200 205 Asn Phe Pro Gly Ala Tyr Ser Ala Ser Asp Pro Gly Ile Leu Ile Asn 210 215 220 Ile His Ala Pro Met Ser Thr Tyr Val Val Pro Gly Pro Thr Val Tyr 225 230 235 240 Ala Gly Gly Ser Thr Lys Ser Ala Gly Ser Ser Cys Ser Gly Cys Glu 245 250 255 Ala Thr Cys Thr Val Gly Ser Gly Pro Ser Ala Thr Leu Thr Gln Pro 260 265 270 Thr Ser Thr Ala Thr Ala Thr Ser Ala Pro Gly Gly Gly Gly Ser Gly 275 280 285 Cys Thr Ala Ala Lys Tyr Gln Gln Cys Gly Gly Thr Gly Tyr Thr Gly 290 295 300 Cys Thr Thr Cys Ala Ser Gly Ser Thr Cys Ser Ala Val Ser Pro Pro 305 310 315 320 Tyr Tyr Ser Gln Cys Leu 325 3880DNAThielavia terrestris 3accccgggat cactgcccct aggaaccagc acacctcggt ccaatcatgc ggttcgacgc 60cctctccgcc ctcgctcttg cgccgcttgt ggctggccac ggcgccgtga ccagctacat 120catcggcggc aaaacctatc ccggctacga gggcttctcg cctgcctcga gcccgccgac 180gatccagtac cagtggcccg actacaaccc gaccctgagc gtgaccgacc cgaagatgcg 240ctgcaacggc ggcacctcgg cagagctcag cgcgcccgtc caggccggcg agaacgtgac 300ggccgtctgg aagcagtgga cccaccagca aggccccgtc atggtctgga tgttcaagtg 360ccccggcgac ttctcgtcgt gccacggcga cggcaagggc tggttcaaga tcgaccagct 420gggcctgtgg ggcaacaacc tcaactcgaa caactggggc accgcgatcg tctacaagac 480cctccagtgg agcaacccga tccccaagaa cctcgcgccg ggcaactacc tcatccgcca 540cgagctgctc gccctgcacc aggccaacac gccgcagttc tacgccgagt gcgcccagct 600ggtcgtctcc ggcagcggct ccgccctgcc cccgtccgac tacctctaca gcatccccgt 660ctacgcgccc cagaacgacc ccggcatcac cgtgagtggg cttccgttcc gcggcgagct 720ctgtggaaat cttgctgacg atgggctagg ttgacatcta caacggcggg cttacctcct 780acaccccgcc cggcggcccc gtctggtctg gcttcgagtt ttaggcgcat tgagtcgggg 840gctacgaggg gaaggcatct gttcgcatga gcgtgggtac 8804478PRTThielavia terrestris 4Met Arg Phe Asp Ala Leu Ser Ala Leu Ala Leu Ala Pro Leu Val Ala 1 5 10 15 Gly His Gly Ala Val Thr Ser Tyr Ile Ile Gly Gly Lys Thr Tyr Pro 20 25 30 Gly Tyr Glu Gly Phe Ser Pro Ala Ser Ser Pro Pro Thr Ile Gln Tyr 35 40 45 Gln Trp Pro Asp Tyr Asn Pro Thr Leu Ser Val Thr Asp Pro Lys Met 50 55 60 Arg Cys Asn Gly Gly Thr Ser Ala Glu Leu Ser Ala Pro Val Gln Ala 65 70 75 80 Gly Glu Asn Val Thr Ala Val Trp Lys Gln Trp Thr His Gln Gln Gly 85 90 95 Pro Val Met Val Trp Met Phe Lys Cys Pro Gly Asp Phe Ser Ser Ser 100 105 110 His Gly Asp Gly Lys Gly Trp Phe Lys Ile Asp Gln Leu Gly Leu Trp 115 120 125 Gly Asn Asn Leu Asn Ser Asn Asn Trp Gly Thr Ala Ile Val Tyr Lys 130 135 140 Thr Leu Gln Trp Ser Asn Pro Ile Pro Lys Asn Leu Ala Pro Gly Asn 145 150 155 160 Tyr Leu Ile Arg His Glu Leu Leu Ala Leu His Gln Ala Asn Thr Pro 165 170 175 Gln Phe Tyr Ala Glu Cys Ala Gln Leu Val Val Ser Gly Ser Gly Ser 180 185 190 Ala Leu Pro Pro Ser Asp Tyr Leu Tyr Ser Ile Pro Val Tyr Ala Pro 195 200 205 Gln Asn Asp Pro Gly Ile Thr Val Asp Ile Tyr Asn Gly Gly Leu Thr 210 215 220 Ser Tyr Thr Pro Pro Gly Gly Pro Val Trp Ser Gly Phe Glu Phe Met 225 230 235 240 Arg Phe Asp Ala Leu Ser Ala Leu Ala Leu Ala Pro Leu Val Ala Gly 245 250 255 His Gly Ala Val Thr Ser Tyr Ile Ile Gly Gly Lys Thr Tyr Pro Gly 260 265 270 Tyr Glu Gly Phe Ser Pro Ala Ser Ser Pro Pro Thr Ile Gln Tyr Gln 275 280 285 Trp Pro Asp Tyr Asn Pro Thr Leu Ser Val Thr Asp Pro Lys Met Arg 290 295 300 Cys Asn Gly Gly Thr Ser Ala Glu Leu Ser Ala Pro Val Gln Ala Gly 305 310 315 320 Glu Asn Val Thr Ala Val Trp Lys Gln Trp Thr His Gln Gln Gly Pro 325 330 335 Val Met Val Trp Met Phe Lys Cys Pro Gly Asp Phe Ser Ser Ser His 340 345 350 Gly Asp Gly Lys Gly Trp Phe Lys Ile Asp Gln Leu Gly Leu Trp Gly 355 360 365 Asn Asn Leu Asn Ser Asn Asn Trp Gly Thr Ala Ile Val Tyr Lys Thr 370 375 380 Leu Gln Trp Ser Asn Pro Ile Pro Lys Asn Leu Ala Pro Gly Asn Tyr 385 390 395 400 Leu Ile Arg His Glu Leu Leu Ala Leu His Gln Ala Asn Thr Pro Gln 405 410 415 Phe Tyr Ala Glu Cys Ala Gln Leu Val Val Ser Gly Ser Gly Ser Ala 420 425 430 Leu Pro Pro Ser Asp Tyr Leu Tyr Ser Ile Pro Val Tyr Ala Pro Gln 435 440 445 Asn Asp Pro Gly Ile Thr Val Asp Ile Tyr Asn Gly Gly Leu Thr Ser 450 455 460 Tyr Thr Pro Pro Gly Gly Pro Val Trp Ser Gly Phe Glu Phe 465 470 475 51000DNAThielavia terrestris 5ctcctgttcc tgggccaccg cttgttgcct gcactattgg tagagttggt ctattgctag 60agttggccat gcttctcaca tcagtcctcg gctcggctgc cctgcttgct agcggcgctg 120cggcacacgg cgccgtgacc agctacatca tcgccggcaa gaattacccg gggtgggtag 180ctgattattg agggcgcatt caaggttcat accggtgtgc atggctgaca accggctggc 240agataccaag gcttttctcc tgcgaactcg ccgaacgtca tccaatggca atggcatgac 300tacaaccccg tcttgtcgtg cagcgactcg aagcttcgct gcaacggcgg cacgtcggcc 360accctgaacg ccacggccgc accgggcgac accatcaccg ccatctgggc gcagtggacg 420cacagccagg gccccatcct ggtgtggatg tacaagtgcc cgggctcctt cagctcctgt 480gacggctccg gcgctggctg gttcaagatc gacgaggccg gcttccacgg cgacggcgtc 540aaggtcttcc tcgacaccga gaacccgtcc ggctgggaca tcgccaagct cgtcggcggc 600aacaagcagt ggagcagcaa ggtccccgag ggcctcgccc ccggcaacta cctcgtccgc 660cacgagttga tcgccctgca ccaggccaac aacccgcagt tctacccgga gtgcgcccag 720gtcgtcatca ccggctccgg caccgcgcag ccggatgcct catacaaggc ggctatcccc 780ggctactgca accagaatga cccgaacatc aaggtgagat ccaggcgtaa tgcagtctac 840tgctggaaag aaagtggtcc aagctaaacc gcgctccagg tgcccatcaa cgaccactcc 900atccctcaga cctacaagat tcccggccct cccgtcttca agggcaccgc cagcaagaag 960gcccgggact tcaccgcctg aagttgttga atcgatggag 10006516PRTThielavia terrestris 6Met Leu Leu Thr Ser Val Leu Gly Ser Ala Ala Leu Leu Ala Ser Gly 1 5 10 15 Ala Ala Ala His Gly Ala Val Thr Ser Tyr Ile Ile Ala Gly Lys Asn 20 25 30 Tyr Pro Gly Tyr Gln Gly Phe Ser Pro Ala Asn Ser Pro Asn Val Ile 35 40 45 Gln Trp Gln Trp His Asp Tyr Asn Pro Val Leu Ser Cys Ser Asp Ser 50 55 60 Lys Leu Arg Cys Asn Gly Gly Thr Ser Ala Thr Leu Asn Ala Thr Ala 65 70 75 80 Ala Pro Gly Asp Thr Ile Thr Ala Ile Trp Ala Gln Trp Thr His Ser 85 90 95 Gln Gly Pro Ile Leu Val Trp Met Tyr Lys Cys Pro Gly Ser Phe Ser 100 105 110 Ser Cys Asp Gly Ser Gly Ala Gly Trp Phe Lys Ile Asp Glu Ala Gly 115 120 125 Phe His Gly Asp Gly Val Lys Val Phe Leu Asp Thr Glu Asn Pro Ser 130 135 140 Gly Trp Asp Ile Ala Lys Leu Val Gly Gly Asn Lys Gln Trp Ser Ser 145 150 155 160 Lys Val Pro Glu Gly Leu Ala Pro Gly Asn Tyr Leu Val Arg His Glu 165 170 175 Leu Ile Ala Leu His Gln Ala Asn Asn Pro Gln Phe Tyr Pro Glu Cys 180 185 190 Ala Gln Val Val Ile Thr Gly Ser Gly Thr Ala Gln Pro Asp Ala Ser 195 200 205 Tyr Lys Ala Ala Ile Pro Gly Tyr Cys Asn Gln Asn Asp Pro Asn Ile 210 215 220 Lys Val Pro Ile Asn Asp His Ser Ile Pro Gln Thr Tyr Lys Ile Pro 225 230 235 240 Gly Pro Pro Val Phe Lys Gly Thr Ala Ser Lys Lys Ala Arg Asp Phe 245 250 255 Thr Ala Met Leu Leu Thr Ser Val Leu Gly Ser Ala Ala Leu Leu Ala 260 265 270 Ser Gly Ala Ala Ala His Gly Ala Val Thr Ser Tyr Ile Ile Ala Gly 275 280 285 Lys Asn Tyr Pro Gly Tyr Gln Gly Phe Ser Pro Ala Asn Ser Pro Asn 290 295 300 Val Ile Gln Trp Gln Trp His Asp Tyr Asn Pro Val Leu Ser Cys Ser 305 310 315 320 Asp Ser Lys Leu Arg Cys Asn Gly Gly Thr Ser Ala Thr Leu Asn Ala 325 330 335 Thr Ala Ala Pro Gly Asp Thr Ile Thr Ala Ile Trp Ala Gln Trp Thr 340 345 350 His Ser Gln Gly Pro Ile Leu Val Trp Met Tyr Lys Cys Pro Gly Ser 355 360 365 Phe Ser Ser Cys Asp Gly Ser Gly Ala Gly Trp Phe Lys Ile Asp Glu 370 375 380 Ala Gly Phe His Gly Asp Gly Val Lys Val Phe Leu Asp Thr Glu Asn 385 390 395 400 Pro Ser Gly Trp Asp Ile Ala Lys Leu Val Gly Gly Asn Lys Gln Trp 405 410 415 Ser Ser Lys Val Pro Glu Gly Leu Ala Pro Gly Asn Tyr Leu Val Arg 420 425 430 His Glu Leu Ile Ala Leu His Gln Ala Asn Asn Pro Gln Phe Tyr Pro 435 440 445 Glu Cys Ala Gln Val Val Ile Thr Gly Ser Gly Thr Ala Gln Pro Asp 450 455 460 Ala Ser Tyr Lys Ala Ala Ile Pro Gly Tyr Cys Asn Gln Asn Asp Pro 465 470 475 480 Asn Ile Lys Val Pro Ile Asn Asp His Ser Ile Pro Gln Thr Tyr Lys 485 490 495 Ile Pro Gly Pro Pro Val Phe Lys Gly Thr Ala Ser Lys Lys Ala Arg 500 505 510 Asp Phe Thr Ala 515 7681DNAThielavia terrestris 7atgctcgcaa acggtgccat cgtcttcctg gccgccgccc tcggcgtcag tggccactac 60acctggccac gggttaacga cggcgccgac tggcaacagg tccgtaaggc ggacaactgg 120caggacaacg gctacgtcgg ggatgtcacg tcgccacaga tccgctgttt ccaggcgacc 180ccgtccccgg ccccatccgt cctcaacacc acggccggct cgaccgtgac ctactgggcc 240aaccccgacg tctaccaccc cgggcctgtg cagttttaca tggcccgcgt gcccgatggc 300gaggacatca actcgtggaa cggcgacggc gccgtgtggt tcaaggtgta cgaggaccat 360cctacctttg gcgctcagct cacatggccc agcacgggca agagctcgtt cgcggttccc 420atccccccgt gcatcaagtc cggctactac ctcctccggg cggagcaaat cggcctgcac 480gtcgcccaga gcgtaggcgg agcgcagttc tacatctcat gcgcccagct cagcgtcacc 540ggcggcggca gcaccgagcc gccgaacaag gtggccttcc ccggcgctta cagtgcgacg 600gacccgggca ttctgatcaa catctactac cctgttccca cgtcctacca gaaccccggc 660ccggccgtct tcagctgctg a 6818452PRTThielavia terrestris 8Met Leu Ala Asn Gly Ala Ile Val Phe Leu Ala Ala Ala Leu Gly Val 1 5 10 15 Ser Gly His Tyr Thr Trp Pro Arg Val Asn Asp Gly Ala Asp Trp Gln 20 25 30 Gln Val Arg Lys Ala Asp Asn Trp Gln Asp Asn Gly Tyr Val Gly Asp 35 40 45 Val Thr Ser Pro Gln Ile Arg Cys Phe Gln Ala Thr Pro Ser Pro Ala 50 55 60 Pro Ser Val Leu Asn Thr Thr Ala Gly Ser Thr Val Thr Tyr Trp Ala 65 70 75 80 Asn Pro Asp Val Tyr His Pro Gly Pro Val Gln Phe Tyr Met Ala Arg 85 90 95 Val Pro Asp Gly Glu Asp Ile Asn Ser Trp Asn Gly Asp Gly Ala Val 100 105 110 Trp Phe Lys Val Tyr Glu Asp His Pro Thr Phe Gly Ala Gln Leu Thr 115 120 125 Trp Pro Ser Thr Gly Lys Ser Ser Phe Ala Val Pro Ile Pro Pro Cys 130 135 140 Ile Lys Ser Gly Tyr Tyr Leu Leu Arg Ala Glu Gln Ile Gly Leu His 145 150 155 160 Val Ala Gln Ser Val Gly Gly Ala Gln Phe Tyr Ile Ser Cys Ala Gln 165 170 175 Leu Ser Val Thr Gly Gly Gly Ser Thr Glu Pro Pro Asn Lys Val Ala 180 185 190 Phe Pro Gly Ala Tyr Ser Ala Thr Asp Pro Gly Ile Leu Ile Asn Ile 195 200 205 Tyr Tyr Pro Val Pro Thr Ser Tyr Gln Asn Pro Gly Pro Ala Val Phe 210 215 220 Ser Cys Met Leu Ala Asn Gly Ala Ile Val Phe Leu Ala Ala Ala Leu 225 230 235 240 Gly Val Ser Gly His Tyr Thr Trp Pro Arg Val Asn Asp Gly Ala Asp 245 250 255 Trp Gln Gln Val Arg Lys Ala Asp Asn Trp Gln Asp Asn Gly

Tyr Val 260 265 270 Gly Asp Val Thr Ser Pro Gln Ile Arg Cys Phe Gln Ala Thr Pro Ser 275 280 285 Pro Ala Pro Ser Val Leu Asn Thr Thr Ala Gly Ser Thr Val Thr Tyr 290 295 300 Trp Ala Asn Pro Asp Val Tyr His Pro Gly Pro Val Gln Phe Tyr Met 305 310 315 320 Ala Arg Val Pro Asp Gly Glu Asp Ile Asn Ser Trp Asn Gly Asp Gly 325 330 335 Ala Val Trp Phe Lys Val Tyr Glu Asp His Pro Thr Phe Gly Ala Gln 340 345 350 Leu Thr Trp Pro Ser Thr Gly Lys Ser Ser Phe Ala Val Pro Ile Pro 355 360 365 Pro Cys Ile Lys Ser Gly Tyr Tyr Leu Leu Arg Ala Glu Gln Ile Gly 370 375 380 Leu His Val Ala Gln Ser Val Gly Gly Ala Gln Phe Tyr Ile Ser Cys 385 390 395 400 Ala Gln Leu Ser Val Thr Gly Gly Gly Ser Thr Glu Pro Pro Asn Lys 405 410 415 Val Ala Phe Pro Gly Ala Tyr Ser Ala Thr Asp Pro Gly Ile Leu Ile 420 425 430 Asn Ile Tyr Tyr Pro Val Pro Thr Ser Tyr Gln Asn Pro Gly Pro Ala 435 440 445 Val Phe Ser Cys 450 9960DNAThielavia terrestris 9atgaagggac ttttcagtgc cgccgccctc tccctggccg tcggccaggc ttcggcccat 60tacatcttcc agcaactctc catcaacggg aaccagtttc cggtgtacca atatattcgc 120aagaacacca attataacag tcccgttacc gatctcacgt ccgacgatct tcggtgcaat 180gtcggcgccc agggtgctgg gacagacacc gtcacggtga aggccggcga ccagttcacc 240ttcacccttg acacccctgt ttaccaccag gggcccatct ccatctacat gtccaaggcc 300ccgggcgcgg cgtcagacta cgatggcagc ggcggctggt tcaagatcaa ggactggggc 360ccgactttca acgccgacgg cacggccacc tgggacatgg ccggctcata cacctacaac 420atcccgacct gcattcccga cggcgactat ctgctccgca tccagtcgct ggccatccac 480aacccctggc cggcgggcat cccgcagttc tacatctcct gcgcccagat caccgtgacc 540ggcggcggca acggcaaccc tggcccgacg gccctcatcc ccggcgcctt caaggacacc 600gacccgggct acacggtgaa catctacacg aacttccaca actacacggt tcccggcccg 660gaggtcttca gctgcaacgg cggcggctcg aacccgcccc cgccggtgag tagcagcacg 720cccgcgacca cgacgctggt cacgtcgacg cgcaccacgt cctccacgtc ctccgcctcg 780acgccggcct cgaccggcgg ctgcaccgtc gccaagtggg gccagtgcgg cggcaacggg 840tacaccggct gcacgacctg cgcggccggg tccacctgca gcaagcagaa cgactactac 900tcgcagtgct tgtaagggag gccgcaaagc atgaggtgtt tgaagaggag gagaggggtc 96010608PRTThielavia terrestris 10Met Lys Gly Leu Phe Ser Ala Ala Ala Leu Ser Leu Ala Val Gly Gln 1 5 10 15 Ala Ser Ala His Tyr Ile Phe Gln Gln Leu Ser Ile Asn Gly Asn Gln 20 25 30 Phe Pro Val Tyr Gln Tyr Ile Arg Lys Asn Thr Asn Tyr Asn Ser Pro 35 40 45 Val Thr Asp Leu Thr Ser Asp Asp Leu Arg Cys Asn Val Gly Ala Gln 50 55 60 Gly Ala Gly Thr Asp Thr Val Thr Val Lys Ala Gly Asp Gln Phe Thr 65 70 75 80 Phe Thr Leu Asp Thr Pro Val Tyr His Gln Gly Pro Ile Ser Ile Tyr 85 90 95 Met Ser Lys Ala Pro Gly Ala Ala Ser Asp Tyr Asp Gly Ser Gly Gly 100 105 110 Trp Phe Lys Ile Lys Asp Trp Gly Pro Thr Phe Asn Ala Asp Gly Thr 115 120 125 Ala Thr Trp Asp Met Ala Gly Ser Tyr Thr Tyr Asn Ile Pro Thr Cys 130 135 140 Ile Pro Asp Gly Asp Tyr Leu Leu Arg Ile Gln Ser Leu Ala Ile His 145 150 155 160 Asn Pro Trp Pro Ala Gly Ile Pro Gln Phe Tyr Ile Ser Cys Ala Gln 165 170 175 Ile Thr Val Thr Gly Gly Gly Asn Gly Asn Pro Gly Pro Thr Ala Leu 180 185 190 Ile Pro Gly Ala Phe Lys Asp Thr Asp Pro Gly Tyr Thr Val Asn Ile 195 200 205 Tyr Thr Asn Phe His Asn Tyr Thr Val Pro Gly Pro Glu Val Phe Ser 210 215 220 Cys Asn Gly Gly Gly Ser Asn Pro Pro Pro Pro Val Ser Ser Ser Thr 225 230 235 240 Pro Ala Thr Thr Thr Leu Val Thr Ser Thr Arg Thr Thr Ser Ser Thr 245 250 255 Ser Ser Ala Ser Thr Pro Ala Ser Thr Gly Gly Cys Thr Val Ala Lys 260 265 270 Trp Gly Gln Cys Gly Gly Asn Gly Tyr Thr Gly Cys Thr Thr Cys Ala 275 280 285 Ala Gly Ser Thr Cys Ser Lys Gln Asn Asp Tyr Tyr Ser Gln Cys Leu 290 295 300 Met Lys Gly Leu Phe Ser Ala Ala Ala Leu Ser Leu Ala Val Gly Gln 305 310 315 320 Ala Ser Ala His Tyr Ile Phe Gln Gln Leu Ser Ile Asn Gly Asn Gln 325 330 335 Phe Pro Val Tyr Gln Tyr Ile Arg Lys Asn Thr Asn Tyr Asn Ser Pro 340 345 350 Val Thr Asp Leu Thr Ser Asp Asp Leu Arg Cys Asn Val Gly Ala Gln 355 360 365 Gly Ala Gly Thr Asp Thr Val Thr Val Lys Ala Gly Asp Gln Phe Thr 370 375 380 Phe Thr Leu Asp Thr Pro Val Tyr His Gln Gly Pro Ile Ser Ile Tyr 385 390 395 400 Met Ser Lys Ala Pro Gly Ala Ala Ser Asp Tyr Asp Gly Ser Gly Gly 405 410 415 Trp Phe Lys Ile Lys Asp Trp Gly Pro Thr Phe Asn Ala Asp Gly Thr 420 425 430 Ala Thr Trp Asp Met Ala Gly Ser Tyr Thr Tyr Asn Ile Pro Thr Cys 435 440 445 Ile Pro Asp Gly Asp Tyr Leu Leu Arg Ile Gln Ser Leu Ala Ile His 450 455 460 Asn Pro Trp Pro Ala Gly Ile Pro Gln Phe Tyr Ile Ser Cys Ala Gln 465 470 475 480 Ile Thr Val Thr Gly Gly Gly Asn Gly Asn Pro Gly Pro Thr Ala Leu 485 490 495 Ile Pro Gly Ala Phe Lys Asp Thr Asp Pro Gly Tyr Thr Val Asn Ile 500 505 510 Tyr Thr Asn Phe His Asn Tyr Thr Val Pro Gly Pro Glu Val Phe Ser 515 520 525 Cys Asn Gly Gly Gly Ser Asn Pro Pro Pro Pro Val Ser Ser Ser Thr 530 535 540 Pro Ala Thr Thr Thr Leu Val Thr Ser Thr Arg Thr Thr Ser Ser Thr 545 550 555 560 Ser Ser Ala Ser Thr Pro Ala Ser Thr Gly Gly Cys Thr Val Ala Lys 565 570 575 Trp Gly Gln Cys Gly Gly Asn Gly Tyr Thr Gly Cys Thr Thr Cys Ala 580 585 590 Ala Gly Ser Thr Cys Ser Lys Gln Asn Asp Tyr Tyr Ser Gln Cys Leu 595 600 605 11799DNAThermoascus aurantiacus 11atgtcctttt ccaagataat tgctactgcc ggcgttcttg cctctgcttc tctagtggct 60ggccatggct tcgttcagaa catcgtgatt gatggtaaaa agtatgtcat tgcaagacgc 120acataagcgg caacagctga caatcgacag ttatggcggg tatctagtga accagtatcc 180atacatgtcc aatcctccag aggtcatcgc ctggtctact acggcaactg atcttggatt 240tgtggacggt actggatacc aaaccccaga tatcatctgc cataggggcg ccaagcctgg 300agccctgact gctccagtct ctccaggagg aactgttgag cttcaatgga ctccatggcc 360tgattctcac catggcccag ttatcaacta ccttgctccg tgcaatggtg attgttccac 420tgtggataag acccaattag aattcttcaa aattgccgag agcggtctca tcaatgatga 480caatcctcct gggatctggg cttcagacaa tctgatagca gccaacaaca gctggactgt 540caccattcca accacaattg cacctggaaa ctatgttctg aggcatgaga ttattgctct 600tcactcagct cagaaccagg atggtgccca gaactatccc cagtgcatca atctgcaggt 660cactggaggt ggttctgata accctgctgg aactcttgga acggcactct accacgatac 720cgatcctgga attctgatca acatctatca gaaactttcc agctatatca tccctggtcc 780tcctctgtat actggttaa 79912250PRTThermoascus aurantiacus 12Met Ser Phe Ser Lys Ile Ile Ala Thr Ala Gly Val Leu Ala Ser Ala 1 5 10 15 Ser Leu Val Ala Gly His Gly Phe Val Gln Asn Ile Val Ile Asp Gly 20 25 30 Lys Lys Tyr Tyr Gly Gly Tyr Leu Val Asn Gln Tyr Pro Tyr Met Ser 35 40 45 Asn Pro Pro Glu Val Ile Ala Trp Ser Thr Thr Ala Thr Asp Leu Gly 50 55 60 Phe Val Asp Gly Thr Gly Tyr Gln Thr Pro Asp Ile Ile Cys His Arg 65 70 75 80 Gly Ala Lys Pro Gly Ala Leu Thr Ala Pro Val Ser Pro Gly Gly Thr 85 90 95 Val Glu Leu Gln Trp Thr Pro Trp Pro Asp Ser His His Gly Pro Val 100 105 110 Ile Asn Tyr Leu Ala Pro Cys Asn Gly Asp Cys Ser Thr Val Asp Lys 115 120 125 Thr Gln Leu Glu Phe Phe Lys Ile Ala Glu Ser Gly Leu Ile Asn Asp 130 135 140 Asp Asn Pro Pro Gly Ile Trp Ala Ser Asp Asn Leu Ile Ala Ala Asn 145 150 155 160 Asn Ser Trp Thr Val Thr Ile Pro Thr Thr Ile Ala Pro Gly Asn Tyr 165 170 175 Val Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Gln Asn Gln Asp 180 185 190 Gly Ala Gln Asn Tyr Pro Gln Cys Ile Asn Leu Gln Val Thr Gly Gly 195 200 205 Gly Ser Asp Asn Pro Ala Gly Thr Leu Gly Thr Ala Leu Tyr His Asp 210 215 220 Thr Asp Pro Gly Ile Leu Ile Asn Ile Tyr Gln Lys Leu Ser Ser Tyr 225 230 235 240 Ile Ile Pro Gly Pro Pro Leu Tyr Thr Gly 245 250 131172DNATrichoderma reesei 13 ggatctaagc cccatcgata tgaagtcctg cgccattctt gcagcccttg gctgtcttgc 60cgggagcgtt ctcggccatg gacaagtcca aaacttcacg atcaatggac aatacaatca 120gggtttcatt ctcgattact actatcagaa gcagaatact ggtcacttcc ccaacgttgc 180tggctggtac gccgaggacc tagacctggg cttcatctcc cctgaccaat acaccacgcc 240cgacattgtc tgtcacaaga acgcggcccc aggtgccatt tctgccactg cagcggccgg 300cagcaacatc gtcttccaat ggggccctgg cgtctggcct cacccctacg gtcccatcgt 360tacctacgtg gctgagtgca gcggatcgtg cacgaccgtg aacaagaaca acctgcgctg 420ggtcaagatt caggaggccg gcatcaacta taacacccaa gtctgggcgc agcaggatct 480gatcaaccag ggcaacaagt ggactgtgaa gatcccgtcg agcctcaggc ccggaaacta 540tgtcttccgc catgaacttc ttgctgccca tggtgcctct agtgcgaacg gcatgcagaa 600ctatcctcag tgcgtgaaca tcgccgtcac aggctcgggc acgaaagcgc tccctgccgg 660aactcctgca actcagctct acaagcccac tgaccctggc atcttgttca acccttacac 720aacaatcacg agctacacca tccctggccc agccctgtgg caaggctaga tccaggggta 780cggtgttggc gttcgtgaag tcggagctgt tgacaaggat atctgatgat gaacggagag 840gactgatggg cgtgactgag tgtatatatt tttgatgacc aaattgtata cgaaatccga 900acgcatggtg atcattgttt atccctgtag tatattgtct ccaggctgct aagagcccac 960cgggtgtatt acggcaacaa agtcaggaat ttgggtggca atgaacgcag gtctccatga 1020atgtatatgt gaagaggcat cggctggcat gggcattacc agatataggc cctgtgaaac 1080atatagtact tgaacgtgct actggaacgg atcataagca agtcatcaac atgtgaaaaa 1140acactacatg taaaaaaaaa aaaaaaaaaa aa 117214249PRTTrichoderma reesei 14Met Lys Ser Cys Ala Ile Leu Ala Ala Leu Gly Cys Leu Ala Gly Ser 1 5 10 15 Val Leu Gly His Gly Gln Val Gln Asn Phe Thr Ile Asn Gly Gln Tyr 20 25 30 Asn Gln Gly Phe Ile Leu Asp Tyr Tyr Tyr Gln Lys Gln Asn Thr Gly 35 40 45 His Phe Pro Asn Val Ala Gly Trp Tyr Ala Glu Asp Leu Asp Leu Gly 50 55 60 Phe Ile Ser Pro Asp Gln Tyr Thr Thr Pro Asp Ile Val Cys His Lys 65 70 75 80 Asn Ala Ala Pro Gly Ala Ile Ser Ala Thr Ala Ala Ala Gly Ser Asn 85 90 95 Ile Val Phe Gln Trp Gly Pro Gly Val Trp Pro His Pro Tyr Gly Pro 100 105 110 Ile Val Thr Tyr Val Val Glu Cys Ser Gly Ser Cys Thr Thr Val Asn 115 120 125 Lys Asn Asn Leu Arg Trp Val Lys Ile Gln Glu Ala Gly Ile Asn Tyr 130 135 140 Asn Thr Gln Val Trp Ala Gln Gln Asp Leu Ile Asn Gln Gly Asn Lys 145 150 155 160 Trp Thr Val Lys Ile Pro Ser Ser Leu Arg Pro Gly Asn Tyr Val Phe 165 170 175 Arg His Glu Leu Leu Ala Ala His Gly Ala Ser Ser Ala Asn Gly Met 180 185 190 Gln Asn Tyr Pro Gln Cys Val Asn Ile Ala Val Thr Gly Ser Gly Thr 195 200 205 Lys Ala Leu Pro Ala Gly Thr Pro Ala Thr Gln Leu Tyr Lys Pro Thr 210 215 220 Asp Pro Gly Ile Leu Phe Asn Pro Tyr Thr Thr Ile Thr Ser Tyr Thr 225 230 235 240 Ile Pro Gly Pro Ala Leu Trp Gln Gly 245 152771DNAAspergillus oryzae 15ctgttctgct ggttacctgc cacgttatca tgaagcttgg ttggatcgag gtggccgcat 60tggcggctgc ctcagtagtc agtgccaagg atgatctcgc gtactcccct cctttctacc 120cttccccatg ggcagatggt cagggtgaat gggcggaagt atacaaacgc gctgtagaca 180tagtttccca gatgacgttg acagagaaag tcaacttaac gactggaaca ggatggcaac 240tagagaggtg tgttggacaa actggcagtg ttcccagact caacatcccc agcttgtgtt 300tgcaggatag tcctcttggt attcgtttct cggactacaa ttcagctttc cctgcgggtg 360ttaatgtcgc tgccacctgg gacaagacgc tcgcctacct tcgtggtcag gcaatgggtg 420aggagttcag tgataagggt attgacgttc agctgggtcc tgctgctggc cctctcggtg 480ctcatccgga tggcggtaga aactgggaag gtttctcacc agatccagcc ctcaccggtg 540tactttttgc ggagacgatt aagggtattc aagatgctgg tgtcattgcg acagctaagc 600attatatcat gaacgaacaa gagcatttcc gccaacaacc cgaggctgcg ggttacggat 660tcaacgtaag cgacagtttg agttccaacg ttgatgacaa gactatgcat gaattgtacc 720tctggccctt cgcggatgca gtacgcgctg gagtcggtgc tgtcatgtgc tcttacaacc 780aaatcaacaa cagctacggt tgcgagaata gcgaaactct gaacaagctt ttgaaggcgg 840agcttggttt ccaaggcttc gtcatgagtg attggaccgc tcatcacagc ggcgtaggcg 900ctgctttagc aggtctggat atgtcgatgc ccggtgatgt taccttcgat agtggtacgt 960ctttctgggg tgcaaacttg acggtcggtg tccttaacgg tacaatcccc caatggcgtg 1020ttgatgacat ggctgtccgt atcatggccg cttattacaa ggttggccgc gacaccaaat 1080acacccctcc caacttcagc tcgtggacca gggacgaata tggtttcgcg cataaccatg 1140tttcggaagg tgcttacgag agggtcaacg aattcgtgga cgtgcaacgc gatcatgccg 1200acctaatccg tcgcatcggc gcgcagagca ctgttctgct gaagaacaag ggtgccttgc 1260ccttgagccg caaggaaaag ctggtcgccc ttctgggaga ggatgcgggt tccaactcgt 1320ggggcgctaa cggctgtgat gaccgtggtt gcgataacgg tacccttgcc atggcctggg 1380gtagcggtac tgcgaatttc ccatacctcg tgacaccaga gcaggcgatt cagaacgaag 1440ttcttcaggg ccgtggtaat gtcttcgccg tgaccgacag ttgggcgctc gacaagatcg 1500ctgcggctgc ccgccaggcc agcgtatctc tcgtgttcgt caactccgac tcaggagaaa 1560gctatcttag tgtggatgga aatgagggcg atcgtaacaa catcactctg tggaagaacg 1620gcgacaatgt ggtcaagacc gcagcgaata actgtaacaa caccgtggtc atcatccact 1680ccgtcggacc agttttgatc gatgaatggt atgaccaccc caatgtcact ggtattctct 1740gggctggtct gccaggccag gagtctggta actccatcgc cgatgtgctg tacggtcgtg 1800tcaaccctgg cgccaagtct cctttcactt ggggcaagac ccgggagtcg tatggttctc 1860ccttggtcaa ggatgccaac aatggcaacg gagcgcccca gtctgatttc acccagggtg 1920ttttcatcga ttaccgccat ttcgataagt tcaatgagac ccctatctac gagtttggct 1980acggcttgag ctacaccacc ttcgagctct ccgacctcca tgttcagccc ctgaacgcgt 2040cccgatacac tcccaccagt ggcatgactg aagctgcaaa gaactttggt gaaattggcg 2100atgcgtcgga gtacgtgtat ccggaggggc tggaaaggat ccatgagttt atctatccct 2160ggatcaactc taccgacctg aaggcatcgt ctgacgattc taactacggc tgggaagact 2220ccaagtatat tcccgaaggc gccacggatg ggtctgccca gccccgtttg cccgctagtg 2280gtggtgccgg aggaaacccc ggtctgtacg aggatctttt ccgcgtctct gtgaaggtca 2340agaacacggg caatgtcgcc ggtgatgaag ttcctcagct gtacgtttcc ctaggcggcc 2400cgaatgagcc caaggtggta ctgcgcaagt ttgagcgtat tcacttggcc ccttcgcagg 2460aggccgtgtg gacaacgacc cttacccgtc gtgaccttgc aaactgggac gtttcggctc 2520aggactggac cgtcactcct taccccaaga cgatctacgt tggaaactcc tcacggaaac 2580tgccgctcca ggcctcgctg cctaaggccc agtaaggggc aagtcctgat tgtacagagc 2640atttcgagat ttatgatgta catgtttatg aatgacctag ggtagggtaa tacttagtag 2700ggttagttct aattcttgga gtcaagtatt gactcactgg gccgataaaa aaaaaaaaaa 2760aaaaaaaaaa a 277116861PRTAspergillus oryzae 16Met Lys Leu Gly Trp Ile Glu Val Ala Ala Leu Ala Ala Ala Ser Val 1 5 10 15 Val Ser Ala Lys Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser 20 25 30 Pro Trp Ala Asp Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala 35 40 45 Val Asp Ile Val Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr 50 55 60 Thr Gly Thr Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser 65 70 75 80 Val Pro Arg

Leu Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu 85 90 95 Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn 100 105 110 Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala 115 120 125 Met Gly Glu Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro 130 135 140 Ala Ala Gly Pro Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu 145 150 155 160 Gly Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175 Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185 190 Ile Met Asn Glu Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly 195 200 205 Tyr Gly Phe Asn Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys 210 215 220 Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala 225 230 235 240 Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245 250 255 Gly Cys Glu Asn Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265 270 Gly Phe Gln Gly Phe Val Met Ser Asp Trp Thr Ala His His Ser Gly 275 280 285 Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val 290 295 300 Thr Phe Asp Ser Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly 305 310 315 320 Val Leu Asn Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val 325 330 335 Arg Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr 340 345 350 Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His 355 360 365 Asn His Val Ser Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp 370 375 380 Val Gln Arg Asp His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser 385 390 395 400 Thr Val Leu Leu Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu 405 410 415 Lys Leu Val Ala Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly 420 425 430 Ala Asn Gly Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435 440 445 Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu 450 455 460 Gln Ala Ile Gln Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala 465 470 475 480 Val Thr Asp Ser Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln 485 490 495 Ala Ser Val Ser Leu Val Phe Val Asn Ser Asp Ser Gly Glu Ser Tyr 500 505 510 Leu Ser Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp 515 520 525 Lys Asn Gly Asp Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn 530 535 540 Thr Val Val Ile Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp 545 550 555 560 Tyr Asp His Pro Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly 565 570 575 Gln Glu Ser Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn 580 585 590 Pro Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595 600 605 Gly Ser Pro Leu Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln 610 615 620 Ser Asp Phe Thr Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys 625 630 635 640 Phe Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr 645 650 655 Thr Phe Glu Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg 660 665 670 Tyr Thr Pro Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu 675 680 685 Ile Gly Asp Ala Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile 690 695 700 His Glu Phe Ile Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser 705 710 715 720 Ser Asp Asp Ser Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu 725 730 735 Gly Ala Thr Asp Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly 740 745 750 Ala Gly Gly Asn Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val 755 760 765 Lys Val Lys Asn Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu 770 775 780 Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys 785 790 795 800 Phe Glu Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr 805 810 815 Thr Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 820 825 830 Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser Ser 835 840 845 Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 850 855 860 175172DNAAspergillus oryzae 17atgaagcttg gttggatcga ggtggccgca ttggcggctg cctcagtagt cagtgccaag 60gatgatctcg cgtactcccc tcctttctac ccttccccat tacttcgaac caacctagct 120ccaccggcgt aaccgccgac ggagtcatca gtcacggttc ctactagagc gcatgagggg 180aggaaagatg ggaaggggta gggcagatgg tcagggtgaa tgggcggaag tatacaaacg 240cgctgtagac atagtttccc agatgacgtt gacagagaaa gtcaacttaa cgactggaac 300cccgtctacc agtcccactt acccgccttc atatgtttgc gcgacatctg tatcaaaggg 360tctactgcaa ctgtctcttt cagttgaatt gctgaccttg aggatggcaa ctagagaggt 420gtgttggaca aactggcagt gttcccagac tcaacatccc cagcttgtgt ttgcaggata 480gtcctcttgg tattcgtttc tcctaccgtt gatctctcca cacaacctgt ttgaccgtca 540caagggtctg agttgtaggg gtcgaacaca aacgtcctat caggagaacc ataagcaaag 600tcggactaca attcagcttt ccctgcgggt gttaatgtcg ctgccacctg ggacaagacg 660ctcgcctacc ttcgtggtca ggcaatgggt gaggagttca agcctgatgt taagtcgaaa 720gggacgccca caattacagc gacggtggac cctgttctgc gagcggatgg aagcaccagt 780ccgttaccca ctcctcaagt gtgataaggg tattgacgtt cagctgggtc ctgctgctgg 840ccctctcggt gctcatccgg atggcggtag aaactgggaa ggtttctcac cagatccagc 900cactattccc ataactgcaa gtcgacccag gacgacgacc gggagagcca cgagtaggcc 960taccgccatc tttgaccctt ccaaagagtg gtctaggtcg cctcaccggt gtactttttg 1020cggagacgat taagggtatt caagatgctg gtgtcattgc gacagctaag cattatatca 1080tgaacgaaca agagcatttc ggagtggcca catgaaaaac gcctctgcta attcccataa 1140gttctacgac cacagtaacg ctgtcgattc gtaatatagt acttgcttgt tctcgtaaag 1200cgccaacaac ccgaggctgc gggttacgga ttcaacgtaa gcgacagttt gagttccaac 1260gttgatgaca agactatgca tgaattgtac ctctggccct gcggttgttg ggctccgacg 1320cccaatgcct aagttgcatt cgctgtcaaa ctcaaggttg caactactgt tctgatacgt 1380acttaacatg gagaccggga tcgcggatgc agtacgcgct ggagtcggtg ctgtcatgtg 1440ctcttacaac caaatcaaca acagctacgg ttgcgagaat agcgaaactc tgaacaagct 1500agcgcctacg tcatgcgcga cctcagccac gacagtacac gagaatgttg gtttagttgt 1560tgtcgatgcc aacgctctta tcgctttgag acttgttcga tttgaaggcg gagcttggtt 1620tccaaggctt cgtcatgagt gattggaccg ctcatcacag cggcgtaggc gctgctttag 1680caggtctgga tatgtcgatg aaacttccgc ctcgaaccaa aggttccgaa gcagtactca 1740ctaacctggc gagtagtgtc gccgcatccg cgacgaaatc gtccagacct atacagctac 1800cccggtgatg ttaccttcga tagtggtacg tctttctggg gtgcaaactt gacggtcggt 1860gtccttaacg gtacaatccc ccaatggcgt gttgatgaca gggccactac aatggaagct 1920atcaccatgc agaaagaccc cacgtttgaa ctgccagcca caggaattgc catgttaggg 1980ggttaccgca caactactgt tggctgtccg tatcatggcc gcttattaca aggttggccg 2040cgacaccaaa tacacccctc ccaacttcag ctcgtggacc agggacgaat atggtttcgc 2100accgacaggc atagtaccgg cgaataatgt tccaaccggc gctgtggttt atgtggggag 2160ggttgaagtc gagcacctgg tccctgctta taccaaagcg gcataaccat gtttcggaag 2220gtgcttacga gagggtcaac gaattcgtgg acgtgcaacg cgatcatgcc gacctaatcc 2280gtcgcatcgg cgcgcagagc cgtattggta caaagccttc cacgaatgct ctcccagttg 2340cttaagcacc tgcacgttgc gctagtacgg ctggattagg cagcgtagcc gcgcgtctcg 2400actgttctgc tgaagaacaa gggtgccttg cccttgagcc gcaaggaaaa gctggtcgcc 2460cttctgggag aggatgcggg ttccaactcg tggggcgcta tgacaagacg acttcttgtt 2520cccacggaac gggaactcgg cgttcctttt cgaccagcgg gaagaccctc tcctacgccc 2580aaggttgagc accccgcgat acggctgtga tgaccgtggt tgcgataacg gtacccttgc 2640catggcctgg ggtagcggta ctgcgaattt cccatacctc gtgacaccag agcaggcgat 2700tgccgacact actggcacca acgctattgc catgggaacg gtaccggacc ccatcgccat 2760gacgcttaaa gggtatggag cactgtggtc tcgtccgcta tcagaacgaa gttcttcagg 2820gccgtggtaa tgtcttcgcc gtgaccgaca gttgggcgct cgacaagatc gctgcggctg 2880cccgccaggc cagcgtatct agtcttgctt caagaagtcc cggcaccatt acagaagcgg 2940cactggctgt caacccgcga gctgttctag cgacgccgac gggcggtccg gtcgcataga 3000ctcgtgttcg tcaactccga ctcaggagaa ggctatctta gtgtggatgg aaatgagggc 3060gatcgtaaca acatcactct gtggaagaac ggcgacaatg gagcacaagc agttgaggct 3120gagtcctctt ccgatagaat cacacctacc tttactcccg ctagcattgt tgtagtgaga 3180caccttcttg ccgctgttac tggtcaagac cgcagcgaat aactgtaaca acaccgttgt 3240catcatccac tccgtcggac cagttttgat cgatgaatgg tatgaccacc ccaatgtcac 3300accagttctg gcgtcgctta ttgacattgt tgtggcaaca gtagtaggtg aggcagcctg 3360gtcaaaacta gctacttacc atactggtgg ggttacagtg tggtattctc tgggctggtc 3420tgccaggcca ggagtctggt aactccattg ccgatgtgct gtacggtcgt gtcaaccctg 3480gcgccaagtc tcctttcact accataagag acccgaccag acggtccggt cctcagacca 3540ttgaggtaac ggctacacga catgccagca cagttgggac cgcggttcag aggaaagtga 3600tggggcaaga cccgggagtc gtatggttct cccttggtca aggatgccaa caatggcaac 3660ggagcgcccc agtctgattt cacccagggt gttttcatcg accccgttct gggccctcag 3720cataccaaga gggaaccagt tcctacggtt gttaccgttg cctcgcgggg tcagactaaa 3780gtgggtccca caaaagtagc attaccgcca tttcgataag ttcaatgaga cccctatcta 3840cgagtttggc tacggcttga gctacaccac cttcgagctc tccgacctcc atgttcagcc 3900taatggcggt aaagctattc aagttactct ggggatagat gctcaaaccg atgccgaact 3960cgatgtggtg gaagctcgag aggctggagg tacaagtcgg cctgaacgcg tcccgataca 4020ctcccaccag tggcatgact gaagctgcaa agaactttgg tgaaattggc gatgcgtcgg 4080agtacgtgta tccggagggg ggacttgcgc agggctatgt gagggtggtc accgtactga 4140cttcgacgtt tcttgaaacc actttaaccg ctacgcagcc tcatgcacat aggcctcccc 4200ctggaaagga tccatgagtt tatctatccc tggatcaact ctaccgacct gaaggcatcg 4260tctgacgatt ctaactacgg ctgggaagac tccaagtata gacctttcct aggtactcaa 4320atagataggg acctagttga gatggctgga cttccgtagc agactgctaa gattgatgcc 4380gacccttctg aggttcatat ttcccgaagg cgccacggat gggtctgccc agccccgttt 4440gcccgctagt ggtggtgccg gaggaaaccc cggtctgtac gaggatcttt tccgcgtctc 4500aagggcttcc gcggtgccta cccagacggg tcggggcaaa cgggcgatca ccaccacggc 4560ctcctttggg gccagacatg ctcctagaaa aggcgcagag tgtgaaggtc aagaacacgg 4620gcaatgtcgc cggtgatgaa gttcctcagc tgtacgtttc cctaggcggc ccgaatgagc 4680ccaaggtggt actgcgcaag acacttccag ttcttgtgcc cgttacagcg gccactactt 4740caaggagtcg acatgcaaag ggatccgccg ggcttactcg ggttccacca tgacgcgttc 4800tttgagcgta ttcacttggc cccttcgcag gaggccgtgt ggacaacgac ccttacccgt 4860cgtgaccttg caaactggga cgtttcggct caggactgga aaactcgcat aagtgaaccg 4920gggaagcgtc ctccggcaca cctgttgctg ggaatgggca gcactggaac gtttgaccct 4980gcaaagccga gtcctgacct ccgtcactcc ttaccccaag acgatctacg ttggaaactc 5040ctcacggaaa ctgccgctcc aggcctcgct gcctaaggcc cagtaaggca gtgaggaatg 5100gggttctgct agatgcaacc tttgaggagt gcctttgacg gcgaggtccg gagcgacgga 5160ttccgggtca tt 517218861PRTAspergillus oryzae 18Met Lys Leu Gly Trp Ile Glu Val Ala Ala Leu Ala Ala Ala Ser Val 1 5 10 15 Val Ser Ala Lys Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser 20 25 30 Pro Trp Ala Asp Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala 35 40 45 Val Asp Ile Val Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr 50 55 60 Thr Gly Thr Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser 65 70 75 80 Val Pro Arg Leu Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu 85 90 95 Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn 100 105 110 Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala 115 120 125 Met Gly Glu Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro 130 135 140 Ala Ala Gly Pro Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu 145 150 155 160 Gly Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175 Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185 190 Ile Met Asn Glu Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly 195 200 205 Tyr Gly Phe Asn Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys 210 215 220 Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala 225 230 235 240 Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245 250 255 Gly Cys Glu Asn Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265 270 Gly Phe Gln Gly Phe Val Met Ser Asp Trp Thr Ala His His Ser Gly 275 280 285 Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val 290 295 300 Thr Phe Asp Ser Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly 305 310 315 320 Val Leu Asn Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val 325 330 335 Arg Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr 340 345 350 Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His 355 360 365 Asn His Val Ser Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp 370 375 380 Val Gln Arg Asp His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser 385 390 395 400 Thr Val Leu Leu Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu 405 410 415 Lys Leu Val Ala Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly 420 425 430 Ala Asn Gly Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435 440 445 Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu 450 455 460 Gln Ala Ile Gln Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala 465 470 475 480 Val Thr Asp Ser Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln 485 490 495 Ala Ser Val Ser Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr 500 505 510 Leu Ser Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp 515 520 525 Lys Asn Gly Asp Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn 530 535 540 Thr Val Val Ile Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp 545 550 555 560 Tyr Asp His Pro Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly 565 570 575 Gln Glu Ser Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn 580 585 590 Pro Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595 600 605 Gly Ser Pro Leu Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln 610 615 620 Ser Asp Phe Thr Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys 625 630 635 640 Phe Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr 645 650 655 Thr Phe Glu Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg 660 665 670 Tyr Thr Pro Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu 675 680 685 Ile Gly Asp Ala Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile 690 695 700 His Glu Phe Ile Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser 705 710 715

720 Ser Asp Asp Ser Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu 725 730 735 Gly Ala Thr Asp Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly 740 745 750 Ala Gly Gly Asn Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val 755 760 765 Lys Val Lys Asn Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu 770 775 780 Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys 785 790 795 800 Phe Glu Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr 805 810 815 Thr Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 820 825 830 Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser Ser 835 840 845 Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 850 855 860 193060DNAAspergillus fumigatus 19atgagattcg gttggctcga ggtggccgct ctgacggccg cttctgtagc caatgcccag 60gtttgtgatg ctttcccgtc attgtttcgg atatagttga caatagtcat ggaaataatc 120aggaattggc tttctctcca ccattctacc cttcgccttg ggctgatggc cagggagagt 180gggcagatgc ccatcgacgc gccgtcgaga tcgtttctca gatgacactg gcggagaagg 240ttaaccttac aacgggtact gggtgggttg cgactttttt gttgacagtg agctttcttc 300actgaccatc tacacagatg ggaaatggac cgatgcgtcg gtcaaaccgg cagcgttccc 360aggtaagctt gcaattctgc aacaacgtgc aagtgtagtt gctaaaacgc ggtggtgcag 420acttggtatc aactggggtc tttgtggcca ggattcccct ttgggtatcc gtttctgtga 480gctatacccg cggagtcttt cagtccttgt attatgtgct gatgattgtc tctgtatagc 540tgacctcaac tccgccttcc ctgctggtac taatgtcgcc gcgacatggg acaagacact 600cgcctacctt cgtggcaagg ccatgggtga ggaattcaac gacaagggcg tggacatttt 660gctggggcct gctgctggtc ctctcggcaa atacccggac ggcggcagaa tctgggaagg 720cttctctcct gatccggttc tcactggtgt acttttcgcc gaaactatca agggtatcca 780agacgcgggt gtgattgcta ctgccaagca ttacattctg aatgaacagg agcatttccg 840acaggttggc gaggcccagg gatatggtta caacatcacg gagacgatca gctccaacgt 900ggatgacaag accatgcacg agttgtacct ttggtgagta gttgacactg caaatgagga 960ccttgattga tttgactgac ctggaatgca ggccctttgc agatgctgtg cgcggtaaga 1020ttttccgtag acttgacctc gcgacgaaga aatcgctgac gaaccatcgt agctggcgtt 1080ggcgctgtca tgtgttccta caatcaaatc aacaacagct acggttgtca aaacagtcaa 1140actctcaaca agctcctcaa ggctgagctg ggcttccaag gcttcgtcat gagtgactgg 1200agcgctcacc acagcggtgt cggcgctgcc ctcgctgggt tggatatgtc gatgcctgga 1260gacatttcct tcgacgacgg actctccttc tggggcacga acctaactgt cagtgttctt 1320aacggcaccg ttccagcctg gcgtgtcgat gacatggctg ttcgtatcat gaccgcgtac 1380tacaaggttg gtcgtgaccg tcttcgtatt ccccctaact tcagctcctg gacccgggat 1440gagtacggct gggagcattc tgctgtctcc gagggagcct ggaccaaggt gaacgacttc 1500gtcaatgtgc agcgcagtca ctctcagatc atccgtgaga ttggtgccgc tagtacagtg 1560ctcttgaaga acacgggtgc tcttcctttg accggcaagg aggttaaagt gggtgttctc 1620ggtgaagacg ctggttccaa cccgtggggt gctaacggct gccccgaccg cggctgtgat 1680aacggcactc ttgctatggc ctggggtagt ggtactgcca acttccctta ccttgtcacc 1740cccgagcagg ctatccagcg agaggtcatc agcaacggcg gcaatgtctt tgctgtgact 1800gataacgggg ctctcagcca gatggcagat gttgcatctc aatccaggtg agtgcgggct 1860cttagaaaaa gaacgttctc tgaatgaagt tttttaacca ttgcgaacag cgtgtctttg 1920gtgtttgtca acgccgactc tggagagggt ttcatcagtg tcgacggcaa cgagggtgac 1980cgcaaaaatc tcactctgtg gaagaacggc gaggccgtca ttgacactgt tgtcagccac 2040tgcaacaaca cgattgtggt tattcacagt gttgggcccg tcttgatcga ccggtggtat 2100gataacccca acgtcactgc catcatctgg gccggcttgc ccggtcagga gagtggcaac 2160tccctggtcg acgtgctcta tggccgcgtc aaccccagcg ccaagacccc gttcacctgg 2220ggcaagactc gggagtctta cggggctccc ttgctcaccg agcctaacaa tggcaatggt 2280gctccccagg atgatttcaa cgagggcgtc ttcattgact accgtcactt tgacaagcgc 2340aatgagaccc ccatttatga gtttggccat ggcttgagct acaccacctt tggttactct 2400caccttcggg ttcaggccct caatagttcg agttcggcat atgtcccgac tagcggagag 2460accaagcctg cgccaaccta tggtgagatc ggtagtgccg ccgactacct gtatcccgag 2520ggtctcaaaa gaattaccaa gtttatttac ccttggctca actcgaccga cctcgaggat 2580tcttctgacg acccgaacta cggctgggag gactcggagt acattcccga aggcgctagg 2640gatgggtctc ctcaacccct cctgaaggct ggcggcgctc ctggtggtaa ccctaccctt 2700tatcaggatc ttgttagggt gtcggccacc ataaccaaca ctggtaacgt cgccggttat 2760gaagtccctc aattggtgag tgacccgcat gttccttgcg ttgcaatttg gctaactcgc 2820ttctagtatg tttcactggg cggaccgaac gagcctcggg tcgttctgcg caagttcgac 2880cgaatcttcc tggctcctgg ggagcaaaag gtttggacca cgactcttaa ccgtcgtgat 2940ctcgccaatt gggatgtgga ggctcaggac tgggtcatca caaagtaccc caagaaagtg 3000cacgtcggca gctcctcgcg taagctgcct ctgagagcgc ctctgccccg tgtctactag 306020863PRTAspergillus fumigatus 20Met Arg Phe Gly Trp Leu Glu Val Ala Ala Leu Thr Ala Ala Ser Val 1 5 10 15 Ala Asn Ala Gln Glu Leu Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro 20 25 30 Trp Ala Asp Gly Gln Gly Glu Trp Ala Asp Ala His Arg Arg Ala Val 35 40 45 Glu Ile Val Ser Gln Met Thr Leu Ala Glu Lys Val Asn Leu Thr Thr 50 55 60 Gly Thr Gly Trp Glu Met Asp Arg Cys Val Gly Gln Thr Gly Ser Val 65 70 75 80 Pro Arg Leu Gly Ile Asn Trp Gly Leu Cys Gly Gln Asp Ser Pro Leu 85 90 95 Gly Ile Arg Phe Ser Asp Leu Asn Ser Ala Phe Pro Ala Gly Thr Asn 100 105 110 Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Lys Ala 115 120 125 Met Gly Glu Glu Phe Asn Asp Lys Gly Val Asp Ile Leu Leu Gly Pro 130 135 140 Ala Ala Gly Pro Leu Gly Lys Tyr Pro Asp Gly Gly Arg Ile Trp Glu 145 150 155 160 Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175 Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185 190 Ile Leu Asn Glu Gln Glu His Phe Arg Gln Val Gly Glu Ala Gln Gly 195 200 205 Tyr Gly Tyr Asn Ile Thr Glu Thr Ile Ser Ser Asn Val Asp Asp Lys 210 215 220 Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala 225 230 235 240 Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245 250 255 Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265 270 Gly Phe Gln Gly Phe Val Met Ser Asp Trp Ser Ala His His Ser Gly 275 280 285 Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile 290 295 300 Ser Phe Asp Asp Gly Leu Ser Phe Trp Gly Thr Asn Leu Thr Val Ser 305 310 315 320 Val Leu Asn Gly Thr Val Pro Ala Trp Arg Val Asp Asp Met Ala Val 325 330 335 Arg Ile Met Thr Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Arg Ile 340 345 350 Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Trp Glu His 355 360 365 Ser Ala Val Ser Glu Gly Ala Trp Thr Lys Val Asn Asp Phe Val Asn 370 375 380 Val Gln Arg Ser His Ser Gln Ile Ile Arg Glu Ile Gly Ala Ala Ser 385 390 395 400 Thr Val Leu Leu Lys Asn Thr Gly Ala Leu Pro Leu Thr Gly Lys Glu 405 410 415 Val Lys Val Gly Val Leu Gly Glu Asp Ala Gly Ser Asn Pro Trp Gly 420 425 430 Ala Asn Gly Cys Pro Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435 440 445 Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu 450 455 460 Gln Ala Ile Gln Arg Glu Val Ile Ser Asn Gly Gly Asn Val Phe Ala 465 470 475 480 Val Thr Asp Asn Gly Ala Leu Ser Gln Met Ala Asp Val Ala Ser Gln 485 490 495 Ser Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Phe 500 505 510 Ile Ser Val Asp Gly Asn Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp 515 520 525 Lys Asn Gly Glu Ala Val Ile Asp Thr Val Val Ser His Cys Asn Asn 530 535 540 Thr Ile Val Val Ile His Ser Val Gly Pro Val Leu Ile Asp Arg Trp 545 550 555 560 Tyr Asp Asn Pro Asn Val Thr Ala Ile Ile Trp Ala Gly Leu Pro Gly 565 570 575 Gln Glu Ser Gly Asn Ser Leu Val Asp Val Leu Tyr Gly Arg Val Asn 580 585 590 Pro Ser Ala Lys Thr Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595 600 605 Gly Ala Pro Leu Leu Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln 610 615 620 Asp Asp Phe Asn Glu Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys 625 630 635 640 Arg Asn Glu Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr 645 650 655 Thr Phe Gly Tyr Ser His Leu Arg Val Gln Ala Leu Asn Ser Ser Ser 660 665 670 Ser Ala Tyr Val Pro Thr Ser Gly Glu Thr Lys Pro Ala Pro Thr Tyr 675 680 685 Gly Glu Ile Gly Ser Ala Ala Asp Tyr Leu Tyr Pro Glu Gly Leu Lys 690 695 700 Arg Ile Thr Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Glu 705 710 715 720 Asp Ser Ser Asp Asp Pro Asn Tyr Gly Trp Glu Asp Ser Glu Tyr Ile 725 730 735 Pro Glu Gly Ala Arg Asp Gly Ser Pro Gln Pro Leu Leu Lys Ala Gly 740 745 750 Gly Ala Pro Gly Gly Asn Pro Thr Leu Tyr Gln Asp Leu Val Arg Val 755 760 765 Ser Ala Thr Ile Thr Asn Thr Gly Asn Val Ala Gly Tyr Glu Val Pro 770 775 780 Gln Leu Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Arg Val Val Leu 785 790 795 800 Arg Lys Phe Asp Arg Ile Phe Leu Ala Pro Gly Glu Gln Lys Val Trp 805 810 815 Thr Thr Thr Leu Asn Arg Arg Asp Leu Ala Asn Trp Asp Val Glu Ala 820 825 830 Gln Asp Trp Val Ile Thr Lys Tyr Pro Lys Lys Val His Val Gly Ser 835 840 845 Ser Ser Arg Lys Leu Pro Leu Arg Ala Pro Leu Pro Arg Val Tyr 850 855 860 212800DNAPenicillium brasilianum 21tgaaaatgca gggttctaca atctttctgg ctttcgcctc atgggcgagc caggttgctg 60ccattgcgca gcccatacag aagcacgagg tttgttttat cttgctcatg gacgtgcttt 120gacttgacta attgttttac atacagcccg gatttctgca cgggccccaa gccatagaat 180cgttctcaga accgttctac ccgtcgccct ggatgaatcc tcacgccgag ggctgggagg 240ccgcatatca gaaagctcaa gattttgtct cgcaactcac tatcttggag aaaataaatc 300tgaccaccgg tgttgggtaa gtctctccga ctgcttctgg gtcacggtgc gacgagccac 360tgactttttg aagctgggaa aatgggccgt gtgtaggaaa cactggatca attcctcgtc 420tcggattcaa aggattttgt acccaggatt caccacaggg tgttcggttc gcagattatt 480cctccgcttt cacatctagc caaatggccg ccgcaacatt tgaccgctca attctttatc 540aacgaggcca agccatggca caggaacaca aggctaaggg tatcacaatt caattgggcc 600ctgttgccgg ccctctcggt cgcatccccg agggcggccg caactgggaa ggattctccc 660ctgatcctgt cttgactggt atagccatgg ctgagacaat taagggcatg caggatactg 720gagtgattgc ttgcgctaaa cattatattg gaaacgagca ggagcacttc cgtcaagtgg 780gtgaagctgc gggtcacgga tacactattt ccgatactat ttcatctaat attgacgacc 840gtgctatgca tgagctatac ttgtggccat ttgctgatgc cgttcgcgct ggtgtgggtt 900ctttcatgtg ctcatactct cagatcaaca actcctacgg atgccaaaac agtcagaccc 960tcaacaagct cctcaagagc gaattgggct tccaaggctt tgtcatgagc gattggggtg 1020cccatcactc tggagtgtca tcggcgctag ctggacttga tatgagcatg ccgggtgata 1080ccgaatttga ttctggcttg agcttctggg gctctaacct caccattgca attctgaacg 1140gcacggttcc cgaatggcgc ctggatgaca tggcgatgcg aattatggct gcatacttca 1200aagttggcct tactattgag gatcaaccag atgtcaactt caatgcctgg acccatgaca 1260cctacggata taaatacgct tatagcaagg aagattacga gcaggtcaac tggcatgtcg 1320atgttcgcag cgaccacaat aagctcattc gcgagactgc cgcgaagggt acagttctgc 1380tgaagaacaa ctttcatgct ctccctctga agcagcccag gttcgtggcc gtcgttggtc 1440aggatgccgg gccaaacccc aagggcccta acggctgcgc agaccgagga tgcgaccaag 1500gcactctcgc aatgggatgg ggctcagggt ctaccgaatt cccttacctg gtcactcctg 1560acactgctat tcagtcaaag gtcctcgaat acgggggtcg atacgagagt atttttgata 1620actatgacga caatgctatc ttgtcgcttg tctcacagcc tgatgcaacc tgtatcgttt 1680ttgcaaatgc cgattccggt gaaggctaca tcactgtcga caacaactgg ggtgaccgca 1740acaatctgac cctctggcaa aatgccgatc aagtgattag cactgtcagc tcgcgatgca 1800acaacacaat cgttgttctc cactctgtcg gaccagtgtt gctaaatggt atatatgagc 1860acccgaacat cacagctatt gtctgggcag ggatgccagg cgaagaatct ggcaatgctc 1920tcgtggatat tctttggggc aatgttaacc ctgccggtcg cactccgttc acctgggcca 1980aaagtcgaga ggactatggc actgatataa tgtacgagcc caacaacggc cagcgtgcgc 2040ctcagcagga tttcaccgag agcatctacc tcgactaccg ccatttcgac aaagctggta 2100tcgagccaat ttacgagttt ggattcggcc tctcctatac caccttcgaa tactctgacc 2160tccgtgttgt gaagaagtat gttcaaccat acagtcccac gaccggcacc ggtgctcaag 2220caccttccat cggacagcca cctagccaga acctggatac ctacaagttc cctgctacat 2280acaagtacat caaaaccttc atttatccct acctgaacag cactgtctcc ctccgcgctg 2340cttccaagga tcccgaatac ggtcgtacag actttatccc accccacgcg cgtgatggct 2400cccctcaacc tctcaacccc gctggagacc cagtggccag tggtggaaac aacatgctct 2460acgacgaact ttacgaggtc actgcacaga tcaaaaacac tggcgacgtg gccggcgacg 2520aagtcgtcca gctttacgta gatctcgggg gtgacaaccc gcctcgtcag ttgagaaact 2580ttgacaggtt ttatctgctg cccggtcaga gctcaacatt ccgggctaca ttgacgcgcc 2640gtgatttgag caactgggat attgaggcgc agaactggcg agttacggaa tcgcctaaga 2700gagtgtatgt tggacggtcg agtcgggatt tgccgctgag ctcacaattg gagtaatgat 2760catgtctacc aatagatgtt gaatgtctgg tgtggatatt 280022878PRTPenicillium brasilianum 22Met Gln Gly Ser Thr Ile Phe Leu Ala Phe Ala Ser Trp Ala Ser Gln 1 5 10 15 Val Ala Ala Ile Ala Gln Pro Ile Gln Lys His Glu Pro Gly Phe Leu 20 25 30 His Gly Pro Gln Ala Ile Glu Ser Phe Ser Glu Pro Phe Tyr Pro Ser 35 40 45 Pro Trp Met Asn Pro His Ala Glu Gly Trp Glu Ala Ala Tyr Gln Lys 50 55 60 Ala Gln Asp Phe Val Ser Gln Leu Thr Ile Leu Glu Lys Ile Asn Leu 65 70 75 80 Thr Thr Gly Val Gly Trp Glu Asn Gly Pro Cys Val Gly Asn Thr Gly 85 90 95 Ser Ile Pro Arg Leu Gly Phe Lys Gly Phe Cys Thr Gln Asp Ser Pro 100 105 110 Gln Gly Val Arg Phe Ala Asp Tyr Ser Ser Ala Phe Thr Ser Ser Gln 115 120 125 Met Ala Ala Ala Thr Phe Asp Arg Ser Ile Leu Tyr Gln Arg Gly Gln 130 135 140 Ala Met Ala Gln Glu His Lys Ala Lys Gly Ile Thr Ile Gln Leu Gly 145 150 155 160 Pro Val Ala Gly Pro Leu Gly Arg Ile Pro Glu Gly Gly Arg Asn Trp 165 170 175 Glu Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Ile Ala Met Ala Glu 180 185 190 Thr Ile Lys Gly Met Gln Asp Thr Gly Val Ile Ala Cys Ala Lys His 195 200 205 Tyr Ile Gly Asn Glu Gln Glu His Phe Arg Gln Val Gly Glu Ala Ala 210 215 220 Gly His Gly Tyr Thr Ile Ser Asp Thr Ile Ser Ser Asn Ile Asp Asp 225 230 235 240 Arg Ala Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg 245 250 255 Ala Gly Val Gly Ser Phe Met Cys Ser Tyr Ser Gln Ile Asn Asn Ser 260 265 270 Tyr Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ser Glu 275 280 285 Leu Gly Phe Gln Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser 290 295 300 Gly Val Ser Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp 305 310 315 320 Thr Glu Phe Asp Ser Gly Leu Ser Phe Trp Gly Ser Asn Leu Thr Ile 325 330 335 Ala Ile Leu Asn Gly Thr Val Pro Glu Trp Arg Leu Asp Asp Met Ala 340 345 350 Met Arg Ile Met Ala Ala Tyr Phe Lys Val Gly Leu Thr Ile Glu Asp 355 360 365 Gln Pro Asp Val Asn Phe Asn Ala Trp Thr His Asp Thr Tyr Gly Tyr 370 375

380 Lys Tyr Ala Tyr Ser Lys Glu Asp Tyr Glu Gln Val Asn Trp His Val 385 390 395 400 Asp Val Arg Ser Asp His Asn Lys Leu Ile Arg Glu Thr Ala Ala Lys 405 410 415 Gly Thr Val Leu Leu Lys Asn Asn Phe His Ala Leu Pro Leu Lys Gln 420 425 430 Pro Arg Phe Val Ala Val Val Gly Gln Asp Ala Gly Pro Asn Pro Lys 435 440 445 Gly Pro Asn Gly Cys Ala Asp Arg Gly Cys Asp Gln Gly Thr Leu Ala 450 455 460 Met Gly Trp Gly Ser Gly Ser Thr Glu Phe Pro Tyr Leu Val Thr Pro 465 470 475 480 Asp Thr Ala Ile Gln Ser Lys Val Leu Glu Tyr Gly Gly Arg Tyr Glu 485 490 495 Ser Ile Phe Asp Asn Tyr Asp Asp Asn Ala Ile Leu Ser Leu Val Ser 500 505 510 Gln Pro Asp Ala Thr Cys Ile Val Phe Ala Asn Ala Asp Ser Gly Glu 515 520 525 Gly Tyr Ile Thr Val Asp Asn Asn Trp Gly Asp Arg Asn Asn Leu Thr 530 535 540 Leu Trp Gln Asn Ala Asp Gln Val Ile Ser Thr Val Ser Ser Arg Cys 545 550 555 560 Asn Asn Thr Ile Val Val Leu His Ser Val Gly Pro Val Leu Leu Asn 565 570 575 Gly Ile Tyr Glu His Pro Asn Ile Thr Ala Ile Val Trp Ala Gly Met 580 585 590 Pro Gly Glu Glu Ser Gly Asn Ala Leu Val Asp Ile Leu Trp Gly Asn 595 600 605 Val Asn Pro Ala Gly Arg Thr Pro Phe Thr Trp Ala Lys Ser Arg Glu 610 615 620 Asp Tyr Gly Thr Asp Ile Met Tyr Glu Pro Asn Asn Gly Gln Arg Ala 625 630 635 640 Pro Gln Gln Asp Phe Thr Glu Ser Ile Tyr Leu Asp Tyr Arg His Phe 645 650 655 Asp Lys Ala Gly Ile Glu Pro Ile Tyr Glu Phe Gly Phe Gly Leu Ser 660 665 670 Tyr Thr Thr Phe Glu Tyr Ser Asp Leu Arg Val Val Lys Lys Tyr Val 675 680 685 Gln Pro Tyr Ser Pro Thr Thr Gly Thr Gly Ala Gln Ala Pro Ser Ile 690 695 700 Gly Gln Pro Pro Ser Gln Asn Leu Asp Thr Tyr Lys Phe Pro Ala Thr 705 710 715 720 Tyr Lys Tyr Ile Lys Thr Phe Ile Tyr Pro Tyr Leu Asn Ser Thr Val 725 730 735 Ser Leu Arg Ala Ala Ser Lys Asp Pro Glu Tyr Gly Arg Thr Asp Phe 740 745 750 Ile Pro Pro His Ala Arg Asp Gly Ser Pro Gln Pro Leu Asn Pro Ala 755 760 765 Gly Asp Pro Val Ala Ser Gly Gly Asn Asn Met Leu Tyr Asp Glu Leu 770 775 780 Tyr Glu Val Thr Ala Gln Ile Lys Asn Thr Gly Asp Val Ala Gly Asp 785 790 795 800 Glu Val Val Gln Leu Tyr Val Asp Leu Gly Gly Asp Asn Pro Pro Arg 805 810 815 Gln Leu Arg Asn Phe Asp Arg Phe Tyr Leu Leu Pro Gly Gln Ser Ser 820 825 830 Thr Phe Arg Ala Thr Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Ile 835 840 845 Glu Ala Gln Asn Trp Arg Val Thr Glu Ser Pro Lys Arg Val Tyr Val 850 855 860 Gly Arg Ser Ser Arg Asp Leu Pro Leu Ser Ser Gln Leu Glu 865 870 875 232235DNATrichoderma reesei 23atgcgttacc gaacagcagc tgcgctggca cttgccactg ggccctttgc tagggcagac 60agtcactcaa catcgggggc ctcggctgag gcagttgtac ctcctgcagg gactccatgg 120ggaaccgcgt acgacaaggc gaaggccgca ttggcaaagc tcaatctcca agataaggtc 180ggcatcgtga gcggtgtcgg ctggaacggc ggtccttgcg ttggaaacac atctccggcc 240tccaagatca gctatccatc gctatgcctt caagacggac ccctcggtgt tcgatactcg 300acaggcagca cagcctttac gccgggcgtt caagcggcct cgacgtggga tgtcaatttg 360atccgcgaac gtggacagtt catcggtgag gaggtgaagg cctcggggat tcatgtcata 420cttggtcctg tggctgggcc gctgggaaag actccgcagg gcggtcgcaa ctgggagggc 480ttcggtgtcg atccatatct cacgggcatt gccatgggtc aaaccatcaa cggcatccag 540tcggtaggcg tgcaggcgac agcgaagcac tatatcctca acgagcagga gctcaatcga 600gaaaccattt cgagcaaccc agatgaccga actctccatg agctgtatac ttggccattt 660gccgacgcgg ttcaggccaa tgtcgcttct gtcatgtgct cgtacaacaa ggtcaatacc 720acctgggcct gcgaggatca gtacacgctg cagactgtgc tgaaagacca gctggggttc 780ccaggctatg tcatgacgga ctggaacgca cagcacacga ctgtccaaag cgcgaattct 840gggcttgaca tgtcaatgcc tggcacagac ttcaacggta acaatcggct ctggggtcca 900gctctcacca atgcggtaaa tagcaatcag gtccccacga gcagagtcga cgatatggtg 960actcgtatcc tcgccgcatg gtacttgaca ggccaggacc aggcaggcta tccgtcgttc 1020aacatcagca gaaatgttca aggaaaccac aagaccaatg tcagggcaat tgccagggac 1080ggcatcgttc tgctcaagaa tgacgccaac atcctgccgc tcaagaagcc cgctagcatt 1140gccgtcgttg gatctgccgc aatcattggt aaccacgcca gaaactcgcc ctcgtgcaac 1200gacaaaggct gcgacgacgg ggccttgggc atgggttggg gttccggcgc cgtcaactat 1260ccgtacttcg tcgcgcccta cgatgccatc aataccagag cgtcttcgca gggcacccag 1320gttaccttga gcaacaccga caacacgtcc tcaggcgcat ctgcagcaag aggaaaggac 1380gtcgccatcg tcttcatcac cgccgactcg ggtgaaggct acatcaccgt ggagggcaac 1440gcgggcgatc gcaacaacct ggatccgtgg cacaacggca atgccctggt ccaggcggtg 1500gccggtgcca acagcaacgt cattgttgtt gtccactccg ttggcgccat cattctggag 1560cagattcttg ctcttccgca ggtcaaggcc gttgtctggg cgggtcttcc ttctcaggag 1620agcggcaatg cgctcgtcga cgtgctgtgg ggagatgtca gcccttctgg caagctggtg 1680tacaccattg cgaagagccc caatgactat aacactcgca tcgtttccgg cggcagtgac 1740agcttcagcg agggactgtt catcgactat aagcacttcg acgacgccaa tatcacgccg 1800cggtacgagt tcggctatgg actgtcttac accaagttca actactcacg cctctccgtc 1860ttgtcgaccg ccaagtctgg tcctgcgact ggggccgttg tgccgggagg cccgagtgat 1920ctgttccaga atgtcgcgac agtcaccgtt gacatcgcaa actctggcca agtgactggt 1980gccgaggtag cccagctgta catcacctac ccatcttcag cacccaggac ccctccgaag 2040cagctgcgag gctttgccaa gctgaacctc acgcctggtc agagcggaac agcaacgttc 2100aacatccgac gacgagatct cagctactgg gacacggctt cgcagaaatg ggtggtgccg 2160tcggggtcgt ttggcatcag cgtgggagcg agcagccggg atatcaggct gacgagcact 2220ctgtcggtag cgtag 223524744PRTTrichoderma reesei 24Met Arg Tyr Arg Thr Ala Ala Ala Leu Ala Leu Ala Thr Gly Pro Phe 1 5 10 15 Ala Arg Ala Asp Ser His Ser Thr Ser Gly Ala Ser Ala Glu Ala Val 20 25 30 Val Pro Pro Ala Gly Thr Pro Trp Gly Thr Ala Tyr Asp Lys Ala Lys 35 40 45 Ala Ala Leu Ala Lys Leu Asn Leu Gln Asp Lys Val Gly Ile Val Ser 50 55 60 Gly Val Gly Trp Asn Gly Gly Pro Cys Val Gly Asn Thr Ser Pro Ala 65 70 75 80 Ser Lys Ile Ser Tyr Pro Ser Leu Cys Leu Gln Asp Gly Pro Leu Gly 85 90 95 Val Arg Tyr Ser Thr Gly Ser Thr Ala Phe Thr Pro Gly Val Gln Ala 100 105 110 Ala Ser Thr Trp Asp Val Asn Leu Ile Arg Glu Arg Gly Gln Phe Ile 115 120 125 Gly Glu Glu Val Lys Ala Ser Gly Ile His Val Ile Leu Gly Pro Val 130 135 140 Ala Gly Pro Leu Gly Lys Thr Pro Gln Gly Gly Arg Asn Trp Glu Gly 145 150 155 160 Phe Gly Val Asp Pro Tyr Leu Thr Gly Ile Ala Met Gly Gln Thr Ile 165 170 175 Asn Gly Ile Gln Ser Val Gly Val Gln Ala Thr Ala Lys His Tyr Ile 180 185 190 Leu Asn Glu Gln Glu Leu Asn Arg Glu Thr Ile Ser Ser Asn Pro Asp 195 200 205 Asp Arg Thr Leu His Glu Leu Tyr Thr Trp Pro Phe Ala Asp Ala Val 210 215 220 Gln Ala Asn Val Ala Ser Val Met Cys Ser Tyr Asn Lys Val Asn Thr 225 230 235 240 Thr Trp Ala Cys Glu Asp Gln Tyr Thr Leu Gln Thr Val Leu Lys Asp 245 250 255 Gln Leu Gly Phe Pro Gly Tyr Val Met Thr Asp Trp Asn Ala Gln His 260 265 270 Thr Thr Val Gln Ser Ala Asn Ser Gly Leu Asp Met Ser Met Pro Gly 275 280 285 Thr Asp Phe Asn Gly Asn Asn Arg Leu Trp Gly Pro Ala Leu Thr Asn 290 295 300 Ala Val Asn Ser Asn Gln Val Pro Thr Ser Arg Val Asp Asp Met Val 305 310 315 320 Thr Arg Ile Leu Ala Ala Trp Tyr Leu Thr Gly Gln Asp Gln Ala Gly 325 330 335 Tyr Pro Ser Phe Asn Ile Ser Arg Asn Val Gln Gly Asn His Lys Thr 340 345 350 Asn Val Arg Ala Ile Ala Arg Asp Gly Ile Val Leu Leu Lys Asn Asp 355 360 365 Ala Asn Ile Leu Pro Leu Lys Lys Pro Ala Ser Ile Ala Val Val Gly 370 375 380 Ser Ala Ala Ile Ile Gly Asn His Ala Arg Asn Ser Pro Ser Cys Asn 385 390 395 400 Asp Lys Gly Cys Asp Asp Gly Ala Leu Gly Met Gly Trp Gly Ser Gly 405 410 415 Ala Val Asn Tyr Pro Tyr Phe Val Ala Pro Tyr Asp Ala Ile Asn Thr 420 425 430 Arg Ala Ser Ser Gln Gly Thr Gln Val Thr Leu Ser Asn Thr Asp Asn 435 440 445 Thr Ser Ser Gly Ala Ser Ala Ala Arg Gly Lys Asp Val Ala Ile Val 450 455 460 Phe Ile Thr Ala Asp Ser Gly Glu Gly Tyr Ile Thr Val Glu Gly Asn 465 470 475 480 Ala Gly Asp Arg Asn Asn Leu Asp Pro Trp His Asn Gly Asn Ala Leu 485 490 495 Val Gln Ala Val Ala Gly Ala Asn Ser Asn Val Ile Val Val Val His 500 505 510 Ser Val Gly Ala Ile Ile Leu Glu Gln Ile Leu Ala Leu Pro Gln Val 515 520 525 Lys Ala Val Val Trp Ala Gly Leu Pro Ser Gln Glu Ser Gly Asn Ala 530 535 540 Leu Val Asp Val Leu Trp Gly Asp Val Ser Pro Ser Gly Lys Leu Val 545 550 555 560 Tyr Thr Ile Ala Lys Ser Pro Asn Asp Tyr Asn Thr Arg Ile Val Ser 565 570 575 Gly Gly Ser Asp Ser Phe Ser Glu Gly Leu Phe Ile Asp Tyr Lys His 580 585 590 Phe Asp Asp Ala Asn Ile Thr Pro Arg Tyr Glu Phe Gly Tyr Gly Leu 595 600 605 Ser Tyr Thr Lys Phe Asn Tyr Ser Arg Leu Ser Val Leu Ser Thr Ala 610 615 620 Lys Ser Gly Pro Ala Thr Gly Ala Val Val Pro Gly Gly Pro Ser Asp 625 630 635 640 Leu Phe Gln Asn Val Ala Thr Val Thr Val Asp Ile Ala Asn Ser Gly 645 650 655 Gln Val Thr Gly Ala Glu Val Ala Gln Leu Tyr Ile Thr Tyr Pro Ser 660 665 670 Ser Ala Pro Arg Thr Pro Pro Lys Gln Leu Arg Gly Phe Ala Lys Leu 675 680 685 Asn Leu Thr Pro Gly Gln Ser Gly Thr Ala Thr Phe Asn Ile Arg Arg 690 695 700 Arg Asp Leu Ser Tyr Trp Asp Thr Ala Ser Gln Lys Trp Val Val Pro 705 710 715 720 Ser Gly Ser Phe Gly Ile Ser Val Gly Ala Ser Ser Arg Asp Ile Arg 725 730 735 Leu Thr Ser Thr Leu Ser Val Ala 740 252583DNAAspergillus niger 25atgaggttca ctttgatcga ggcggtggct ctgactgccg tctcgctggc cagcgctgat 60gaattggcct actccccacc gtattaccca tccccttggg ccaatggcca gggcgactgg 120gcgcaggcat accagcgcgc tgttgatatt gtctcgcaaa tgacattgga tgagaaggtc 180aatctgacca caggaactgg atgggaattg gaactatgtg ttggtcagac tggcggtgtt 240ccccgattgg gagttccggg aatgtgttta caggatagcc ctctgggcgt tcgcgactcc 300gactacaact ctgctttccc tgccggcatg aacgtggctg caacctggga caagaatctg 360gcataccttc gcggcaaggc tatgggtcag gaatttagtg acaagggtgc cgatatccaa 420ttgggtccag ctgccggccc tctcggtaga agtcccgacg gtggtcgtaa ctgggagggc 480ttctccccag accctgccct aagtggtgtg ctctttgccg agaccatcaa gggtatccaa 540gatgctggtg tggttgcgac ggctaagcac tacattgctt acgagcaaga gcatttccgt 600caggcgcctg aagcccaagg ttttggattt aatatttccg agagtggaag tgcgaacctc 660gatgataaga ctatgcacga gctgtacctc tggcccttcg cggatgccat ccgtgcaggt 720gctggcgctg tgatgtgctc ctacaaccag atcaacaaca gttatggctg ccagaacagc 780tacactctga acaagctgct caaggccgag ctgggcttcc agggctttgt catgagtgat 840tgggctgctc accatgctgg tgtgagtggt gctttggcag gattggatat gtctatgcca 900ggagacgtcg actacgacag tggtacgtct tactggggta caaacttgac cattagcgtg 960ctcaacggaa cggtgcccca atggcgtgtt gatgacatgg ctgtccgcat catggccgcc 1020tactacaagg tcggccgtga ccgtctgtgg actcctccca acttcagctc atggaccaga 1080gatgaatacg gctacaagta ctactacgtg tcggagggac cgtacgagaa ggtcaaccag 1140tacgtgaatg tgcaacgcaa ccacagcgaa ctgattcgcc gcattggagc ggacagcacg 1200gtgctcctca agaacgacgg cgctctgcct ttgactggta aggagcgcct ggtcgcgctt 1260atcggagaag atgcgggctc caacccttat ggtgccaacg gctgcagtga ccgtggatgc 1320gacaatggaa cattggcgat gggctgggga agtggtactg ccaacttccc atacctggtg 1380acccccgagc aggccatctc aaacgaggtg cttaagcaca agaatggtgt attcaccgcc 1440accgataact gggctatcga tcagattgag gcgcttgcta agaccgccag tgtctctctt 1500gtctttgtca acgccgactc tggtgagggt tacatcaatg tggacggaaa cctgggtgac 1560cgcaggaacc tgaccctgtg gaggaacggc gataatgtga tcaaggctgc tgctagcaac 1620tgcaacaaca caatcgttgt cattcactct gtcggaccag tcttggttaa cgagtggtac 1680gacaacccca atgttaccgc tatcctctgg ggtggtttgc ccggtcagga gtctggcaac 1740tctcttgccg acgtcctcta tggccgtgtc aaccccggtg ccaagtcgcc ctttacctgg 1800ggcaagactc gtgaggccta ccaagactac ttggtcaccg agcccaacaa cggcaacgga 1860gcccctcagg aagactttgt cgagggcgtc ttcattgact accgtggatt tgacaagcgc 1920aacgagaccc cgatctacga gttcggctat ggtctgagct acaccacttt caactactcg 1980aaccttgagg tgcaggtgct gagcgcccct gcatacgagc ctgcttcggg tgagaccgag 2040gcagcgccaa ccttcggaga ggttggaaat gcgtcggatt acctctaccc cagcggattg 2100cagagaatta ccaagttcat ctacccctgg ctcaacggta ccgatctcga ggcatcttcc 2160ggggatgcta gctacgggca ggactcctcc gactatcttc ccgagggagc caccgatggc 2220tctgcgcaac cgatcctgcc tgccggtggc ggtcctggcg gcaaccctcg cctgtacgac 2280gagctcatcc gcgtgtcagt gaccatcaag aacaccggca aggttgctgg tgatgaagtt 2340ccccaactgt atgtttccct tggcggtccc aatgagccca agatcgtgct gcgtcaattc 2400gagcgcatca cgctgcagcc gtcggaggag acgaagtgga gcacgactct gacgcgccgt 2460gaccttgcaa actggaatgt tgagaagcag gactgggaga ttacgtcgta tcccaagatg 2520gtgtttgtcg gaagctcctc gcggaagctg ccgctccggg cgtctctgcc tactgttcac 2580taa 258326860PRTAspergillus niger 26Met Arg Phe Thr Leu Ile Glu Ala Val Ala Leu Thr Ala Val Ser Leu 1 5 10 15 Ala Ser Ala Asp Glu Leu Ala Tyr Ser Pro Pro Tyr Tyr Pro Ser Pro 20 25 30 Trp Ala Asn Gly Gln Gly Asp Trp Ala Gln Ala Tyr Gln Arg Ala Val 35 40 45 Asp Ile Val Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr 50 55 60 Gly Thr Gly Trp Glu Leu Glu Leu Cys Val Gly Gln Thr Gly Gly Val 65 70 75 80 Pro Arg Leu Gly Val Pro Gly Met Cys Leu Gln Asp Ser Pro Leu Gly 85 90 95 Val Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Met Asn Val 100 105 110 Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Lys Ala Met 115 120 125 Gly Gln Glu Phe Ser Asp Lys Gly Ala Asp Ile Gln Leu Gly Pro Ala 130 135 140 Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly 145 150 155 160 Phe Ser Pro Asp Pro Ala Leu Ser Gly Val Leu Phe Ala Glu Thr Ile 165 170 175 Lys Gly Ile Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile 180 185 190 Ala Tyr Glu Gln Glu His Phe Arg Gln Ala Pro Glu Ala Gln Gly Phe 195 200 205 Gly Phe Asn Ile Ser Glu Ser Gly Ser Ala Asn Leu Asp Asp Lys Thr 210 215 220 Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Ile Arg Ala Gly 225 230 235 240 Ala Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly 245 250 255 Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly 260 265 270 Phe Gln Gly Phe Val Met Ser Asp Trp Ala Ala His His Ala Gly Val 275 280 285 Ser Gly Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Asp 290

295 300 Tyr Asp Ser Gly Thr Ser Tyr Trp Gly Thr Asn Leu Thr Ile Ser Val 305 310 315 320 Leu Asn Gly Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg 325 330 335 Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Trp Thr Pro 340 345 350 Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Tyr Lys Tyr Tyr 355 360 365 Tyr Val Ser Glu Gly Pro Tyr Glu Lys Val Asn Gln Tyr Val Asn Val 370 375 380 Gln Arg Asn His Ser Glu Leu Ile Arg Arg Ile Gly Ala Asp Ser Thr 385 390 395 400 Val Leu Leu Lys Asn Asp Gly Ala Leu Pro Leu Thr Gly Lys Glu Arg 405 410 415 Leu Val Ala Leu Ile Gly Glu Asp Ala Gly Ser Asn Pro Tyr Gly Ala 420 425 430 Asn Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly 435 440 445 Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln 450 455 460 Ala Ile Ser Asn Glu Val Leu Lys His Lys Asn Gly Val Phe Thr Ala 465 470 475 480 Thr Asp Asn Trp Ala Ile Asp Gln Ile Glu Ala Leu Ala Lys Thr Ala 485 490 495 Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile 500 505 510 Asn Val Asp Gly Asn Leu Gly Asp Arg Arg Asn Leu Thr Leu Trp Arg 515 520 525 Asn Gly Asp Asn Val Ile Lys Ala Ala Ala Ser Asn Cys Asn Asn Thr 530 535 540 Ile Val Val Ile His Ser Val Gly Pro Val Leu Val Asn Glu Trp Tyr 545 550 555 560 Asp Asn Pro Asn Val Thr Ala Ile Leu Trp Gly Gly Leu Pro Gly Gln 565 570 575 Glu Ser Gly Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro 580 585 590 Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gln 595 600 605 Asp Tyr Leu Val Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln Glu 610 615 620 Asp Phe Val Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg 625 630 635 640 Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr 645 650 655 Phe Asn Tyr Ser Asn Leu Glu Val Gln Val Leu Ser Ala Pro Ala Tyr 660 665 670 Glu Pro Ala Ser Gly Glu Thr Glu Ala Ala Pro Thr Phe Gly Glu Val 675 680 685 Gly Asn Ala Ser Asp Tyr Leu Tyr Pro Ser Gly Leu Gln Arg Ile Thr 690 695 700 Lys Phe Ile Tyr Pro Trp Leu Asn Gly Thr Asp Leu Glu Ala Ser Ser 705 710 715 720 Gly Asp Ala Ser Tyr Gly Gln Asp Ser Ser Asp Tyr Leu Pro Glu Gly 725 730 735 Ala Thr Asp Gly Ser Ala Gln Pro Ile Leu Pro Ala Gly Gly Gly Pro 740 745 750 Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr 755 760 765 Ile Lys Asn Thr Gly Lys Val Ala Gly Asp Glu Val Pro Gln Leu Tyr 770 775 780 Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Ile Val Leu Arg Gln Phe 785 790 795 800 Glu Arg Ile Thr Leu Gln Pro Ser Glu Glu Thr Lys Trp Ser Thr Thr 805 810 815 Leu Thr Arg Arg Asp Leu Ala Asn Trp Asn Val Glu Lys Gln Asp Trp 820 825 830 Glu Ile Thr Ser Tyr Pro Lys Met Val Phe Val Gly Ser Ser Ser Arg 835 840 845 Lys Leu Pro Leu Arg Ala Ser Leu Pro Thr Val His 850 855 860 272583DNAAspergillus aculeatus 27atgaagctca gttggcttga ggcggctgcc ttgacggctg cttcagtcgt cagcgctgat 60gaactggcgt tctctcctcc tttctacccc tctccgtggg ccaatggcca gggagagtgg 120gcggaagcct accagcgtgc agtggccatt gtatcccaga tgactctgga tgagaaggtc 180aacctgacca ccggaactgg atgggagctg gagaagtgcg tcggtcagac tggtggtgtc 240ccaagactga acatcggtgg catgtgtctt caggacagtc ccttgggaat tcgtgatagt 300gactacaatt cggctttccc tgctggtgtc aacgttgctg cgacatggga caagaacctt 360gcttatctac gtggtcaggc tatgggtcaa gagttcagtg acaaaggaat tgatgttcaa 420ttgggaccgg ccgcgggtcc cctcggcagg agccctgatg gaggtcgcaa ctgggaaggt 480ttctctccag acccggctct tactggtgtg ctctttgcgg agacgattaa gggtattcaa 540gacgctggtg tcgtggcgac agccaagcat tacattctca atgagcaaga gcatttccgc 600caggtcgcag aggctgcggg ctacggattc aatatctccg acacgatcag ctctaacgtt 660gatgacaaga ccattcatga aatgtacctc tggcccttcg cggatgccgt tcgcgccggc 720gttggcgcca tcatgtgttc ctacaaccag atcaacaaca gctacggttg ccagaacagt 780tacactctga acaagcttct gaaggccgag ctcggcttcc agggctttgt gatgtctgac 840tggggtgctc accacagtgg tgttggctct gctttggccg gcttggatat gtcaatgcct 900ggcgatatca ccttcgattc tgccactagt ttctggggta ccaacctgac cattgctgtg 960ctcaacggta ccgtcccgca gtggcgcgtt gacgacatgg ctgtccgtat catggctgcc 1020tactacaagg ttggccgcga ccgcctgtac cagccgccta acttcagctc ctggactcgc 1080gatgaatacg gcttcaagta tttctacccc caggaagggc cctatgagaa ggtcaatcac 1140tttgtcaatg tgcagcgcaa ccacagcgag gttattcgca agttgggagc agacagtact 1200gttctactga agaacaacaa tgccctgccg ctgaccggaa aggagcgcaa agttgcgatc 1260ctgggtgaag atgctggatc caactcgtac ggtgccaatg gctgctctga ccgtggctgt 1320gacaacggta ctcttgctat ggcttggggt agcggcactg ccgaattccc atatctcgtg 1380acccctgagc aggctattca agccgaggtg ctcaagcata agggcagcgt ctacgccatc 1440acggacaact gggcgctgag ccaggtggag accctcgcta aacaagccag tgtctctctt 1500gtatttgtca actcggacgc gggagagggc tatatctccg tggacggaaa cgagggcgac 1560cgcaacaacc tcaccctctg gaagaacggc gacaacctca tcaaggctgc tgcaaacaac 1620tgcaacaaca ccatcgttgt catccactcc gttggacctg ttttggttga cgagtggtat 1680gaccacccca acgttactgc catcctctgg gcgggcttgc ctggccagga gtctggcaac 1740tccttggctg acgtgctcta cggccgcgtc aacccgggcg ccaaatctcc attcacctgg 1800ggcaagacga gggaggcgta cggggattac cttgtccgtg agctcaacaa cggcaacgga 1860gctccccaag atgatttctc ggaaggtgtt ttcattgact accgcggatt cgacaagcgc 1920aatgagaccc cgatctacga gttcggacat ggtctgagct acaccacttt caactactct 1980ggccttcaca tccaggttct caacgcttcc tccaacgctc aagtagccac tgagactggc 2040gccgctccca ccttcggaca agtcggcaat gcctctgact acgtgtaccc tgagggattg 2100accagaatca gcaagttcat ctatccctgg cttaattcca cagacctgaa ggcctcatct 2160ggcgacccgt actatggagt cgacaccgcg gagcacgtgc ccgagggtgc tactgatggc 2220tctccgcagc ccgttctgcc tgccggtggt ggctctggtg gtaacccgcg cctctacgat 2280gagttgatcc gtgtttcggt gacagtcaag aacactggtc gtgttgccgg tgatgctgtg 2340cctcaattgt atgtttccct tggtggaccc aatgagccca aggttgtgtt gcgcaaattc 2400gaccgcctca ccctcaagcc ctccgaggag acggtgtgga cgactaccct gacccgccgc 2460gatctgtcta actgggacgt tgcggctcag gactgggtca tcacttctta cccgaagaag 2520gtccatgttg gtagctcttc gcgtcagctg ccccttcacg cggcgctccc gaaggtgcaa 2580tga 258328860PRTAspergillus aculeatus 28Met Lys Leu Ser Trp Leu Glu Ala Ala Ala Leu Thr Ala Ala Ser Val 1 5 10 15 Val Ser Ala Asp Glu Leu Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro 20 25 30 Trp Ala Asn Gly Gln Gly Glu Trp Ala Glu Ala Tyr Gln Arg Ala Val 35 40 45 Ala Ile Val Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr 50 55 60 Gly Thr Gly Trp Glu Leu Glu Lys Cys Val Gly Gln Thr Gly Gly Val 65 70 75 80 Pro Arg Leu Asn Ile Gly Gly Met Cys Leu Gln Asp Ser Pro Leu Gly 85 90 95 Ile Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val 100 105 110 Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Gln Ala Met 115 120 125 Gly Gln Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala 130 135 140 Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly 145 150 155 160 Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile 165 170 175 Lys Gly Ile Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile 180 185 190 Leu Asn Glu Gln Glu His Phe Arg Gln Val Ala Glu Ala Ala Gly Tyr 195 200 205 Gly Phe Asn Ile Ser Asp Thr Ile Ser Ser Asn Val Asp Asp Lys Thr 210 215 220 Ile His Glu Met Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly 225 230 235 240 Val Gly Ala Ile Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly 245 250 255 Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly 260 265 270 Phe Gln Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser Gly Val 275 280 285 Gly Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile Thr 290 295 300 Phe Asp Ser Ala Thr Ser Phe Trp Gly Thr Asn Leu Thr Ile Ala Val 305 310 315 320 Leu Asn Gly Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg 325 330 335 Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Tyr Gln Pro 340 345 350 Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Lys Tyr Phe 355 360 365 Tyr Pro Gln Glu Gly Pro Tyr Glu Lys Val Asn His Phe Val Asn Val 370 375 380 Gln Arg Asn His Ser Glu Val Ile Arg Lys Leu Gly Ala Asp Ser Thr 385 390 395 400 Val Leu Leu Lys Asn Asn Asn Ala Leu Pro Leu Thr Gly Lys Glu Arg 405 410 415 Lys Val Ala Ile Leu Gly Glu Asp Ala Gly Ser Asn Ser Tyr Gly Ala 420 425 430 Asn Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala 435 440 445 Trp Gly Ser Gly Thr Ala Glu Phe Pro Tyr Leu Val Thr Pro Glu Gln 450 455 460 Ala Ile Gln Ala Glu Val Leu Lys His Lys Gly Ser Val Tyr Ala Ile 465 470 475 480 Thr Asp Asn Trp Ala Leu Ser Gln Val Glu Thr Leu Ala Lys Gln Ala 485 490 495 Ser Val Ser Leu Val Phe Val Asn Ser Asp Ala Gly Glu Gly Tyr Ile 500 505 510 Ser Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Leu Thr Leu Trp Lys 515 520 525 Asn Gly Asp Asn Leu Ile Lys Ala Ala Ala Asn Asn Cys Asn Asn Thr 530 535 540 Ile Val Val Ile His Ser Val Gly Pro Val Leu Val Asp Glu Trp Tyr 545 550 555 560 Asp His Pro Asn Val Thr Ala Ile Leu Trp Ala Gly Leu Pro Gly Gln 565 570 575 Glu Ser Gly Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro 580 585 590 Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gly 595 600 605 Asp Tyr Leu Val Arg Glu Leu Asn Asn Gly Asn Gly Ala Pro Gln Asp 610 615 620 Asp Phe Ser Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg 625 630 635 640 Asn Glu Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr Thr 645 650 655 Phe Asn Tyr Ser Gly Leu His Ile Gln Val Leu Asn Ala Ser Ser Asn 660 665 670 Ala Gln Val Ala Thr Glu Thr Gly Ala Ala Pro Thr Phe Gly Gln Val 675 680 685 Gly Asn Ala Ser Asp Tyr Val Tyr Pro Glu Gly Leu Thr Arg Ile Ser 690 695 700 Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Lys Ala Ser Ser 705 710 715 720 Gly Asp Pro Tyr Tyr Gly Val Asp Thr Ala Glu His Val Pro Glu Gly 725 730 735 Ala Thr Asp Gly Ser Pro Gln Pro Val Leu Pro Ala Gly Gly Gly Ser 740 745 750 Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr 755 760 765 Val Lys Asn Thr Gly Arg Val Ala Gly Asp Ala Val Pro Gln Leu Tyr 770 775 780 Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe 785 790 795 800 Asp Arg Leu Thr Leu Lys Pro Ser Glu Glu Thr Val Trp Thr Thr Thr 805 810 815 Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Val Ala Ala Gln Asp Trp 820 825 830 Val Ile Thr Ser Tyr Pro Lys Lys Val His Val Gly Ser Ser Ser Arg 835 840 845 Gln Leu Pro Leu His Ala Ala Leu Pro Lys Val Gln 850 855 860 29918DNAHumicola insolens 29atgcgttcct cccccctcct cccgtccgcc gttgtggccg ccctgccggt gttggccctt 60gccgctgatg gcaggtccac ccgctactgg gactgctgca agccttcgtg cggctgggcc 120aagaaggctc ccgtgaacca gcctgtcttt tcctgcaacg ccaacttcca gcgtatcacg 180gacttcgacg ccaagtccgg ctgcgagccg ggcggtgtcg cctactcgtg cgccgaccag 240accccatggg ctgtgaacga cgacttcgcg ctcggttttg ctgccacctc tattgccggc 300agcaatgagg cgggctggtg ctgcgcctgc tacgagctca ccttcacatc cggtcctgtt 360gctggcaaga agatggtcgt ccagtccacc agcactggcg gtgatcttgg cagcaaccac 420ttcgatctca acatccccgg cggcggcgtc ggcatcttcg acggatgcac tccccagttc 480ggcggtctgc ccggccagcg ctacggcggc atctcgtccc gcaacgagtg cgatcggttc 540cccgacgccc tcaagcccgg ctgctactgg cgcttcgact ggttcaagaa cgccgacaat 600ccgagcttca gcttccgtca ggtccagtgc ccagccgagc tcgtcgctcg caccggatgc 660cgccgcaacg acgacggcaa cttccctgcc gtccagatcc cctccagcag caccagctct 720ccggtcaacc agcctaccag caccagcacc acgtccacct ccaccacctc gagcccgcca 780gtccagccta cgactcccag cggctgcact gctgagaggt gggctcagtg cggcggcaat 840ggctggagcg gctgcaccac ctgcgtcgct ggcagcactt gcacgaagat taatgactgg 900taccatcagt gcctgtag 91830305PRTHumicola insolens 30Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro 1 5 10 15 Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20 25 30 Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro 35 40 45 Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50 55 60 Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln 65 70 75 80 Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr 85 90 95 Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 100 105 110 Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln 115 120 125 Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130 135 140 Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe 145 150 155 160 Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu 165 170 175 Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180 185 190 Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val 195 200 205 Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp 210 215 220 Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser 225 230 235 240 Pro Val Asn Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr 245 250 255 Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu 260 265 270 Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys 275 280 285 Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys 290 295 300 Leu 305 311188DNAMyceliophthora thermophila 31cgacttgaaa cgccccaaat gaagtcctcc

atcctcgcca gcgtcttcgc cacgggcgcc 60gtggctcaaa gtggtccgtg gcagcaatgt ggtggcatcg gatggcaagg atcgaccgac 120tgtgtgtcgg gctaccactg cgtctaccag aacgattggt acagccagtg cgtgcctggc 180gcggcgtcga caacgctgca gacatcgacc acgtccaggc ccaccgccac cagcaccgcc 240cctccgtcgt ccaccacctc gcctagcaag ggcaagctga agtggctcgg cagcaacgag 300tcgggcgccg agttcgggga gggcaattac cccggcctct ggggcaagca cttcatcttc 360ccgtcgactt cggcgattca gacgctcatc aatgatggat acaacatctt ccggatcgac 420ttctcgatgg agcgtctggt gcccaaccag ttgacgtcgt ccttcgacca gggttacctc 480cgcaacctga ccgaggtggt caacttcgtg acgaacgcgg gcaagtacgc cgtcctggac 540ccgcacaact acggccggta ctacggcaac atcatcacgg acacgaacgc gttccggacc 600ttctggacca acctggccaa gcagttcgcc tccaactcgc tcgtcatctt cgacaccaac 660aacgagtaca acacgatgga ccagaccctg gtgctcaacc tcaaccaggc cgccatcgac 720ggcatccggg ccgccggcgc gacctcgcag tacatcttcg tcgagggcaa cgcgtggagc 780ggggcctgga gctggaacac gaccaacacc aacatggccg ccctgacgga cccgcagaac 840aagatcgtgt acgagatgca ccagtacctc gactcggaca gctcgggcac ccacgccgag 900tgcgtcagca gcaccatcgg cgcccagcgc gtcgtcggag ccacccagtg gctccgcgcc 960aacggcaagc tcggcgtcct cggcgagttc gccggcggcg ccaacgccgt ctgccagcag 1020gccgtcaccg gcctcctcga ccacctccag gacaacagcg acgtctggct gggtgccctc 1080tggtgggccg ccggtccctg gtggggcgac tacatgtact cgttcgagcc tccttcgggc 1140accggctatg tcaactacaa ctcgatcttg aagaagtact tgccgtaa 118832389PRTMyceliophthora thermophila 32Met Lys Ser Ser Ile Leu Ala Ser Val Phe Ala Thr Gly Ala Val Ala 1 5 10 15 Gln Ser Gly Pro Trp Gln Gln Cys Gly Gly Ile Gly Trp Gln Gly Ser 20 25 30 Thr Asp Cys Val Ser Gly Tyr His Cys Val Tyr Gln Asn Asp Trp Tyr 35 40 45 Ser Gln Cys Val Pro Gly Ala Ala Ser Thr Thr Leu Gln Thr Ser Thr 50 55 60 Thr Ser Arg Pro Thr Ala Thr Ser Thr Ala Pro Pro Ser Ser Thr Thr 65 70 75 80 Ser Pro Ser Lys Gly Lys Leu Lys Trp Leu Gly Ser Asn Glu Ser Gly 85 90 95 Ala Glu Phe Gly Glu Gly Asn Tyr Pro Gly Leu Trp Gly Lys His Phe 100 105 110 Ile Phe Pro Ser Thr Ser Ala Ile Gln Thr Leu Ile Asn Asp Gly Tyr 115 120 125 Asn Ile Phe Arg Ile Asp Phe Ser Met Glu Arg Leu Val Pro Asn Gln 130 135 140 Leu Thr Ser Ser Phe Asp Gln Gly Tyr Leu Arg Asn Leu Thr Glu Val 145 150 155 160 Val Asn Phe Val Thr Asn Ala Gly Lys Tyr Ala Val Leu Asp Pro His 165 170 175 Asn Tyr Gly Arg Tyr Tyr Gly Asn Ile Ile Thr Asp Thr Asn Ala Phe 180 185 190 Arg Thr Phe Trp Thr Asn Leu Ala Lys Gln Phe Ala Ser Asn Ser Leu 195 200 205 Val Ile Phe Asp Thr Asn Asn Glu Tyr Asn Thr Met Asp Gln Thr Leu 210 215 220 Val Leu Asn Leu Asn Gln Ala Ala Ile Asp Gly Ile Arg Ala Ala Gly 225 230 235 240 Ala Thr Ser Gln Tyr Ile Phe Val Glu Gly Asn Ala Trp Ser Gly Ala 245 250 255 Trp Ser Trp Asn Thr Thr Asn Thr Asn Met Ala Ala Leu Thr Asp Pro 260 265 270 Gln Asn Lys Ile Val Tyr Glu Met His Gln Tyr Leu Asp Ser Asp Ser 275 280 285 Ser Gly Thr His Ala Glu Cys Val Ser Ser Thr Ile Gly Ala Gln Arg 290 295 300 Val Val Gly Ala Thr Gln Trp Leu Arg Ala Asn Gly Lys Leu Gly Val 305 310 315 320 Leu Gly Glu Phe Ala Gly Gly Ala Asn Ala Val Cys Gln Gln Ala Val 325 330 335 Thr Gly Leu Leu Asp His Leu Gln Asp Asn Ser Asp Val Trp Leu Gly 340 345 350 Ala Leu Trp Trp Ala Ala Gly Pro Trp Trp Gly Asp Tyr Met Tyr Ser 355 360 365 Phe Glu Pro Pro Ser Gly Thr Gly Tyr Val Asn Tyr Asn Ser Ile Leu 370 375 380 Lys Lys Tyr Leu Pro 385 331232DNABASIDIOMYCETE CBS 495.95 33ggatccactt agtaacggcc gccagtgtgc tggaaagcat gaagtctctc ttcctgtcac 60ttgtagcgac cgtcgcgctc agctcgccag tattctctgt cgcagtctgg gggcaatgcg 120gcggcattgg cttcagcgga agcaccgtct gtgatgcagg cgccggctgt gtgaagctca 180acgactatta ctctcaatgc caacccggcg ctcccactgc tacatccgcg gcgccaagta 240gcaacgcacc gtccggcact tcgacggcct cggccccctc ctccagcctt tgctctggca 300gccgcacgcc gttccagttc ttcggtgtca acgaatccgg cgcggagttc ggcaacctga 360acatccccgg tgttctgggc accgactaca cctggccgtc gccatccagc attgacttct 420tcatgggcaa gggaatgaat accttccgta ttccgttcct catggagcgt cttgtccccc 480ctgccactgg catcacagga cctctcgacc agacgtactt gggcggcctg cagacgattg 540tcaactacat caccggcaaa ggcggctttg ctctcattga cccgcacaac tttatgatct 600acaatggcca gacgatctcc agtaccagcg acttccagaa gttctggcag aacctcgcag 660gagtgtttaa atcgaacagt cacgtcatct tcgatgttat gaacgagcct cacgatattc 720ccgcccagac cgtgttccaa ctgaaccaag ccgctgtcaa tggcatccgt gcgagcggtg 780cgacgtcgca gctcattctg gtcgagggca caagctggac tggagcctgg acctggacga 840cctctggcaa cagcgatgca ttcggtgcca ttaaggatcc caacaacaac gtcgcgatcc 900agatgcatca gtacctggat agcgatggct ctggcacttc gcagacctgc gtgtctccca 960ccatcggtgc cgagcggttg caggctgcga ctcaatggtt gaagcagaac aacctcaagg 1020gcttcctggg cgagatcggc gccggctcta actccgcttg catcagcgct gtgcagggtg 1080cgttgtgttc gatgcagcaa tctggtgtgt ggctcggcgc tctctggtgg gctgcgggcc 1140cgtggtgggg cgactactac cagtccatcg agccgccctc tggcccggcg gtgtccgcga 1200tcctcccgca ggccctgctg ccgttcgcgt aa 123234397PRTBASIDIOMYCETE CBS 495.95 34Met Lys Ser Leu Phe Leu Ser Leu Val Ala Thr Val Ala Leu Ser Ser 1 5 10 15 Pro Val Phe Ser Val Ala Val Trp Gly Gln Cys Gly Gly Ile Gly Phe 20 25 30 Ser Gly Ser Thr Val Cys Asp Ala Gly Ala Gly Cys Val Lys Leu Asn 35 40 45 Asp Tyr Tyr Ser Gln Cys Gln Pro Gly Ala Pro Thr Ala Thr Ser Ala 50 55 60 Ala Pro Ser Ser Asn Ala Pro Ser Gly Thr Ser Thr Ala Ser Ala Pro 65 70 75 80 Ser Ser Ser Leu Cys Ser Gly Ser Arg Thr Pro Phe Gln Phe Phe Gly 85 90 95 Val Asn Glu Ser Gly Ala Glu Phe Gly Asn Leu Asn Ile Pro Gly Val 100 105 110 Leu Gly Thr Asp Tyr Thr Trp Pro Ser Pro Ser Ser Ile Asp Phe Phe 115 120 125 Met Gly Lys Gly Met Asn Thr Phe Arg Ile Pro Phe Leu Met Glu Arg 130 135 140 Leu Val Pro Pro Ala Thr Gly Ile Thr Gly Pro Leu Asp Gln Thr Tyr 145 150 155 160 Leu Gly Gly Leu Gln Thr Ile Val Asn Tyr Ile Thr Gly Lys Gly Gly 165 170 175 Phe Ala Leu Ile Asp Pro His Asn Phe Met Ile Tyr Asn Gly Gln Thr 180 185 190 Ile Ser Ser Thr Ser Asp Phe Gln Lys Phe Trp Gln Asn Leu Ala Gly 195 200 205 Val Phe Lys Ser Asn Ser His Val Ile Phe Asp Val Met Asn Glu Pro 210 215 220 His Asp Ile Pro Ala Gln Thr Val Phe Gln Leu Asn Gln Ala Ala Val 225 230 235 240 Asn Gly Ile Arg Ala Ser Gly Ala Thr Ser Gln Leu Ile Leu Val Glu 245 250 255 Gly Thr Ser Trp Thr Gly Ala Trp Thr Trp Thr Thr Ser Gly Asn Ser 260 265 270 Asp Ala Phe Gly Ala Ile Lys Asp Pro Asn Asn Asn Val Ala Ile Gln 275 280 285 Met His Gln Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Gln Thr Cys 290 295 300 Val Ser Pro Thr Ile Gly Ala Glu Arg Leu Gln Ala Ala Thr Gln Trp 305 310 315 320 Leu Lys Gln Asn Asn Leu Lys Gly Phe Leu Gly Glu Ile Gly Ala Gly 325 330 335 Ser Asn Ser Ala Cys Ile Ser Ala Val Gln Gly Ala Leu Cys Ser Met 340 345 350 Gln Gln Ser Gly Val Trp Leu Gly Ala Leu Trp Trp Ala Ala Gly Pro 355 360 365 Trp Trp Gly Asp Tyr Tyr Gln Ser Ile Glu Pro Pro Ser Gly Pro Ala 370 375 380 Val Ser Ala Ile Leu Pro Gln Ala Leu Leu Pro Phe Ala 385 390 395 351303DNABASIDIOMYCETE CBS 495.95 35ggaaagcgtc agtatggtga aatttgcgct tgtggcaact gtcggcgcaa tcttgagcgc 60ttctgcggcc aatgcggctt ctatctacca gcaatgtgga ggcattggat ggtctgggtc 120cactgtttgc gacgccggtc tcgcttgcgt tatcctcaat gcgtactact ttcagtgctt 180gacgcccgcc gcgggccaga caacgacggg ctcgggcgca ccggcgtcaa catcaacctc 240tcactcaacg gtcactacgg ggagctcaca ctcaacaacc gggacgacgg cgacgaaaac 300aactaccact ccgtcgacca ccacgaccct acccgccatc tctgtgtctg gtcgcgtctg 360ctctggctcc aggacgaagt tcaagttctt cggtgtgaat gaaagcggcg ccgaattcgg 420gaacactgct tggccagggc agctcgggaa agactataca tggccttcgc ctagcagcgt 480ggactacttc atgggggctg gattcaatac attccgtatc accttcttga tggagcgtat 540gagccctccg gctaccggac tcactggccc attcaaccag acgtacctgt cgggcctcac 600caccattgtc gactacatca cgaacaaagg aggatacgct cttattgacc cccacaactt 660catgcgttac aacaacggca taatcagcag cacatctgac ttcgcgactt ggtggagcaa 720tttggccact gtattcaaat ccacgaagaa cgccatcttc gacatccaga acgagccgta 780cggaatcgat gcgcagaccg tatacgaact gaatcaagct gccatcaatt cgatccgcgc 840cgctggcgct acgtcacagt tgattctggt tgaaggaacg tcatacactg gagcttggac 900gtgggtctcg tccggaaacg gagctgcttt cgcggccgtt acggatcctt acaacaacac 960ggcaattgaa atgcaccaat acctcgacag cgacggttct gggacaaacg aagactgtgt 1020ctcctccacc attgggtcgc aacgtctcca agctgccact gcgtggctgc aacaaacagg 1080actcaaggga ttcctcggag agacgggtgc tgggtcgaat tcccagtgca tcgacgccgt 1140gttcgatgaa ctttgctata tgcaacagca aggcggctcc tggatcggtg cactctggtg 1200ggctgcgggt ccctggtggg gcacgtacat ttactcgatt gaacctccga gcggtgccgc 1260tatcccagaa gtccttcctc agggtctcgc tccattcctc tag 130336429PRTBASIDIOMYCETE CBS 495.95 36Met Val Lys Phe Ala Leu Val Ala Thr Val Gly Ala Ile Leu Ser Ala 1 5 10 15 Ser Ala Ala Asn Ala Ala Ser Ile Tyr Gln Gln Cys Gly Gly Ile Gly 20 25 30 Trp Ser Gly Ser Thr Val Cys Asp Ala Gly Leu Ala Cys Val Ile Leu 35 40 45 Asn Ala Tyr Tyr Phe Gln Cys Leu Thr Pro Ala Ala Gly Gln Thr Thr 50 55 60 Thr Gly Ser Gly Ala Pro Ala Ser Thr Ser Thr Ser His Ser Thr Val 65 70 75 80 Thr Thr Gly Ser Ser His Ser Thr Thr Gly Thr Thr Ala Thr Lys Thr 85 90 95 Thr Thr Thr Pro Ser Thr Thr Thr Thr Leu Pro Ala Ile Ser Val Ser 100 105 110 Gly Arg Val Cys Ser Gly Ser Arg Thr Lys Phe Lys Phe Phe Gly Val 115 120 125 Asn Glu Ser Gly Ala Glu Phe Gly Asn Thr Ala Trp Pro Gly Gln Leu 130 135 140 Gly Lys Asp Tyr Thr Trp Pro Ser Pro Ser Ser Val Asp Tyr Phe Met 145 150 155 160 Gly Ala Gly Phe Asn Thr Phe Arg Ile Thr Phe Leu Met Glu Arg Met 165 170 175 Ser Pro Pro Ala Thr Gly Leu Thr Gly Pro Phe Asn Gln Thr Tyr Leu 180 185 190 Ser Gly Leu Thr Thr Ile Val Asp Tyr Ile Thr Asn Lys Gly Gly Tyr 195 200 205 Ala Leu Ile Asp Pro His Asn Phe Met Arg Tyr Asn Asn Gly Ile Ile 210 215 220 Ser Ser Thr Ser Asp Phe Ala Thr Trp Trp Ser Asn Leu Ala Thr Val 225 230 235 240 Phe Lys Ser Thr Lys Asn Ala Ile Phe Asp Ile Gln Asn Glu Pro Tyr 245 250 255 Gly Ile Asp Ala Gln Thr Val Tyr Glu Leu Asn Gln Ala Ala Ile Asn 260 265 270 Ser Ile Arg Ala Ala Gly Ala Thr Ser Gln Leu Ile Leu Val Glu Gly 275 280 285 Thr Ser Tyr Thr Gly Ala Trp Thr Trp Val Ser Ser Gly Asn Gly Ala 290 295 300 Ala Phe Ala Ala Val Thr Asp Pro Tyr Asn Asn Thr Ala Ile Glu Met 305 310 315 320 His Gln Tyr Leu Asp Ser Asp Gly Ser Gly Thr Asn Glu Asp Cys Val 325 330 335 Ser Ser Thr Ile Gly Ser Gln Arg Leu Gln Ala Ala Thr Ala Trp Leu 340 345 350 Gln Gln Thr Gly Leu Lys Gly Phe Leu Gly Glu Thr Gly Ala Gly Ser 355 360 365 Asn Ser Gln Cys Ile Asp Ala Val Phe Asp Glu Leu Cys Tyr Met Gln 370 375 380 Gln Gln Gly Gly Ser Trp Ile Gly Ala Leu Trp Trp Ala Ala Gly Pro 385 390 395 400 Trp Trp Gly Thr Tyr Ile Tyr Ser Ile Glu Pro Pro Ser Gly Ala Ala 405 410 415 Ile Pro Glu Val Leu Pro Gln Gly Leu Ala Pro Phe Leu 420 425 371580DNAThielavia terrestris 37agccccccgt tcaggcacac ttggcatcag atcagcttag cagcgcctgc acagcatgaa 60gctctcgcag tcggccgcgc tggcggcact caccgcgacg gcgctcgccg ccccctcgcc 120cacgacgccg caggcgccga ggcaggcttc agccggctgc tcgtctgcgg tcacgctcga 180cgccagcacc aacgtttgga agaagtacac gctgcacccc aacagctact accgcaagga 240ggttgaggcc gcggtggcgc agatctcgga cccggacctc gccgccaagg ccaagaaggt 300ggccgacgtc ggcaccttcc tgtggctcga ctcgatcgag aacatcggca agctggagcc 360ggcgatccag gacgtgccct gcgagaacat cctgggcctg gtcatctacg acctgccggg 420ccgcgactgc gcggccaagg cgtccaacgg cgagctcaag gtcggcgaga tcgaccgcta 480caagaccgag tacatcgaca gtgagtgctg ccccccgggt tcgagaagag cgtgggggaa 540agggaaaggg ttgactgact gacacggcgc actgcagaga tcgtgtcgat cctcaaggca 600caccccaaca cggcgttcgc gctggtcatc gagccggact cgctgcccaa cctggtgacc 660aacagcaact tggacacgtg ctcgagcagc gcgtcgggct accgcgaagg cgtggcttac 720gccctcaaga acctcaacct gcccaacgtg atcatgtacc tcgacgccgg ccacggcggc 780tggctcggct gggacgccaa cctgcagccc ggcgcgcagg agctagccaa ggcgtacaag 840aacgccggct cgcccaagca gctccgcggc ttctcgacca acgtggccgg ctggaactcc 900tggtgagctt ttttccattc catttcttct tcctcttctc tcttcgctcc cactctgcag 960ccccccctcc cccaagcacc cactggcgtt ccggcttgct gactcggcct ccctttcccc 1020gggcaccagg gatcaatcgc ccggcgaatt ctcccaggcg tccgacgcca agtacaacaa 1080gtgccagaac gagaagatct acgtcagcac cttcggctcc gcgctccagt cggccggcat 1140gcccaaccac gccatcgtcg acacgggccg caacggcgtc accggcctgc gcaaggagtg 1200gggtgactgg tgcaacgtca acggtgcagg ttcgttgtct tctttttctc ctcttttgtt 1260tgcacgtcgt ggtccttttc aagcagccgt gtttggttgg gggagatgga ctccggctga 1320tgttctgctt cctctctagg cttcggcgtg cgcccgacga gcaacacggg cctcgagctg 1380gccgacgcgt tcgtgtgggt caagcccggc ggcgagtcgg acggcaccag cgacagctcg 1440tcgccgcgct acgacagctt ctgcggcaag gacgacgcct tcaagccctc gcccgaggcc 1500ggcacctgga acgaggccta cttcgagatg ctgctcaaga acgccgtgcc gtcgttctaa 1560gacggtccag catcatccgg 158038396PRTThielavia terrestris 38Met Lys Leu Ser Gln Ser Ala Ala Leu Ala Ala Leu Thr Ala Thr Ala 1 5 10 15 Leu Ala Ala Pro Ser Pro Thr Thr Pro Gln Ala Pro Arg Gln Ala Ser 20 25 30 Ala Gly Cys Ser Ser Ala Val Thr Leu Asp Ala Ser Thr Asn Val Trp 35 40 45 Lys Lys Tyr Thr Leu His Pro Asn Ser Tyr Tyr Arg Lys Glu Val Glu 50 55 60 Ala Ala Val Ala Gln Ile Ser Asp Pro Asp Leu Ala Ala Lys Ala Lys 65 70 75 80 Lys Val Ala Asp Val Gly Thr Phe Leu Trp Leu Asp Ser Ile Glu Asn 85 90 95 Ile Gly Lys Leu Glu Pro Ala Ile Gln Asp Val Pro Cys Glu Asn Ile 100 105 110 Leu Gly Leu Val Ile Tyr Asp Leu Pro Gly Arg Asp Cys Ala Ala Lys 115 120 125 Ala Ser Asn Gly Glu Leu Lys Val Gly Glu Ile Asp Arg Tyr Lys Thr 130 135 140 Glu Tyr Ile Asp Lys Ile Val Ser Ile Leu Lys Ala His Pro Asn Thr 145 150 155 160 Ala Phe Ala Leu Val Ile Glu Pro Asp Ser Leu Pro Asn Leu Val Thr 165 170 175 Asn Ser Asn Leu Asp Thr Cys Ser Ser Ser Ala Ser Gly Tyr Arg Glu 180 185 190 Gly Val Ala Tyr Ala Leu Lys Asn Leu Asn Leu Pro Asn Val Ile Met 195 200 205 Tyr Leu Asp Ala Gly His Gly Gly Trp Leu Gly Trp Asp Ala Asn Leu 210 215 220 Gln Pro Gly Ala Gln Glu Leu Ala Lys Ala Tyr Lys Asn Ala Gly Ser 225

230 235 240 Pro Lys Gln Leu Arg Gly Phe Ser Thr Asn Val Ala Gly Trp Asn Ser 245 250 255 Trp Asp Gln Ser Pro Gly Glu Phe Ser Gln Ala Ser Asp Ala Lys Tyr 260 265 270 Asn Lys Cys Gln Asn Glu Lys Ile Tyr Val Ser Thr Phe Gly Ser Ala 275 280 285 Leu Gln Ser Ala Gly Met Pro Asn His Ala Ile Val Asp Thr Gly Arg 290 295 300 Asn Gly Val Thr Gly Leu Arg Lys Glu Trp Gly Asp Trp Cys Asn Val 305 310 315 320 Asn Gly Ala Gly Phe Gly Val Arg Pro Thr Ser Asn Thr Gly Leu Glu 325 330 335 Leu Ala Asp Ala Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly 340 345 350 Thr Ser Asp Ser Ser Ser Pro Arg Tyr Asp Ser Phe Cys Gly Lys Asp 355 360 365 Asp Ala Phe Lys Pro Ser Pro Glu Ala Gly Thr Trp Asn Glu Ala Tyr 370 375 380 Phe Glu Met Leu Leu Lys Asn Ala Val Pro Ser Phe 385 390 395 391203DNAThielavia terrestris 39atgaagtacc tcaacctcct cgcagctctc ctcgccgtcg ctcctctctc cctcgctgca 60cccagcatcg aggccagaca gtcgaacgtc aacccataca tcggcaagag cccgctcgtt 120attaggtcgt acgcccaaaa gcttgaggag accgtcagga ccttccagca acgtggcgac 180cagctcaacg ctgcgaggac acggacggtg cagaacgttg cgactttcgc ctggatctcg 240gataccaatg gtattggagc cattcgacct ctcatccaag atgctctcgc ccagcaggct 300cgcactggac agaaggtcat cgtccaaatc gtcgtctaca acctcccaga tcgcgactgc 360tctgccaacg cctcgactgg agagttcacc gtaggaaacg acggtctcaa ccgatacaag 420aactttgtca acaccatcgc ccgcgagctc tcgactgctg acgctgacaa gctccacttt 480gccctcctcc tcgaacccga cgcacttgcc aacctcgtca ccaacgcgaa tgcccccagg 540tgccgaatcg ccgctcccgc ttacaaggag ggtatcgcct acaccctcgc caccttgtcc 600aagcccaacg tcgacgtcta catcgacgcc gccaacggtg gctggctcgg ctggaacgac 660aacctccgcc ccttcgccga actcttcaag gaagtctacg acctcgcccg ccgcatcaac 720cccaacgcca aggtccgcgg cgtccccgtc aacgtctcca actacaacca gtaccgcgct 780gaagtccgcg agcccttcac cgagtggaag gacgcctggg acgagagccg ctacgtcaac 840gtcctcaccc cgcacctcaa cgccgtcggc ttctccgcgc acttcatcgt tgaccaggga 900cgcggtggca agggcggtat caggacggag tggggccagt ggtgcaacgt taggaacgct 960gggttcggta tcaggcctac tgcggatcag ggcgtgctcc agaacccgaa tgtggatgcg 1020attgtgtggg ttaagccggg tggagagtcg gatggcacga gtgatttgaa ctcgaacagg 1080tatgatccta cgtgcaggag tccggtggcg catgttcccg ctcctgaggc tggccagtgg 1140ttcaacgagt atgttgttaa cctcgttttg aacgctaacc cccctcttga gcctacctgg 1200taa 120340400PRTThielavia terrestris 40Met Lys Tyr Leu Asn Leu Leu Ala Ala Leu Leu Ala Val Ala Pro Leu 1 5 10 15 Ser Leu Ala Ala Pro Ser Ile Glu Ala Arg Gln Ser Asn Val Asn Pro 20 25 30 Tyr Ile Gly Lys Ser Pro Leu Val Ile Arg Ser Tyr Ala Gln Lys Leu 35 40 45 Glu Glu Thr Val Arg Thr Phe Gln Gln Arg Gly Asp Gln Leu Asn Ala 50 55 60 Ala Arg Thr Arg Thr Val Gln Asn Val Ala Thr Phe Ala Trp Ile Ser 65 70 75 80 Asp Thr Asn Gly Ile Gly Ala Ile Arg Pro Leu Ile Gln Asp Ala Leu 85 90 95 Ala Gln Gln Ala Arg Thr Gly Gln Lys Val Ile Val Gln Ile Val Val 100 105 110 Tyr Asn Leu Pro Asp Arg Asp Cys Ser Ala Asn Ala Ser Thr Gly Glu 115 120 125 Phe Thr Val Gly Asn Asp Gly Leu Asn Arg Tyr Lys Asn Phe Val Asn 130 135 140 Thr Ile Ala Arg Glu Leu Ser Thr Ala Asp Ala Asp Lys Leu His Phe 145 150 155 160 Ala Leu Leu Leu Glu Pro Asp Ala Leu Ala Asn Leu Val Thr Asn Ala 165 170 175 Asn Ala Pro Arg Cys Arg Ile Ala Ala Pro Ala Tyr Lys Glu Gly Ile 180 185 190 Ala Tyr Thr Leu Ala Thr Leu Ser Lys Pro Asn Val Asp Val Tyr Ile 195 200 205 Asp Ala Ala Asn Gly Gly Trp Leu Gly Trp Asn Asp Asn Leu Arg Pro 210 215 220 Phe Ala Glu Leu Phe Lys Glu Val Tyr Asp Leu Ala Arg Arg Ile Asn 225 230 235 240 Pro Asn Ala Lys Val Arg Gly Val Pro Val Asn Val Ser Asn Tyr Asn 245 250 255 Gln Tyr Arg Ala Glu Val Arg Glu Pro Phe Thr Glu Trp Lys Asp Ala 260 265 270 Trp Asp Glu Ser Arg Tyr Val Asn Val Leu Thr Pro His Leu Asn Ala 275 280 285 Val Gly Phe Ser Ala His Phe Ile Val Asp Gln Gly Arg Gly Gly Lys 290 295 300 Gly Gly Ile Arg Thr Glu Trp Gly Gln Trp Cys Asn Val Arg Asn Ala 305 310 315 320 Gly Phe Gly Ile Arg Pro Thr Ala Asp Gln Gly Val Leu Gln Asn Pro 325 330 335 Asn Val Asp Ala Ile Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly 340 345 350 Thr Ser Asp Leu Asn Ser Asn Arg Tyr Asp Pro Thr Cys Arg Ser Pro 355 360 365 Val Ala His Val Pro Ala Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr 370 375 380 Val Val Asn Leu Val Leu Asn Ala Asn Pro Pro Leu Glu Pro Thr Trp 385 390 395 400 411501DNAThielavia terrestris 41gccgttgtca agatgggcca gaagacgctg cacggattcg ccgccacggc tttggccgtt 60ctcccctttg tgaaggctca gcagcccggc aacttcacgc cggaggtgca cccgcaactg 120ccaacgtgga agtgcacgac cgccggcggc tgcgttcagc aggacacttc ggtggtgctc 180gactggaact accgttggat ccacaatgcc gacggcaccg cctcgtgcac gacgtccagc 240ggggtcgacc acacgctgtg tccagatgag gcgacctgcg cgaagaactg cttcgtggaa 300ggcgtcaact acacgagcag cggtgtcacc acatccggca gttcgctgac gatgaggcag 360tatttcaagg ggagcaacgg gcagaccaac agcgtttcgc ctcgtctcta cctgctcggc 420tcggatggaa actacgtaat gctcaagctg ctcggccagg agctgagctt cgatgtcgat 480ctctccacgc tcccctgcgg cgagaacggc gcgctgtacc tgtccgagat ggacgcgacc 540ggtggcagga accagtacaa caccggcggt gccaactacg gctcgggcta ctgtgacgcc 600cagtgtcccg tgcagacgtg gatgaacggc acgctgaaca ccaacgggca gggctactgc 660tgcaacgaga tggacatcct cgaggccaac tcccgcgcca acgcgatgac acctcacccc 720tgcgccaacg gcagctgcga caagagcggg tgcggactca acccctacgc cgagggctac 780aagagctact acggaccggg cctcacggtt gacacgtcga agcccttcac catcattacc 840cgcttcatca ccgacgacgg cacgaccagc ggcaccctca accagatcca gcggatctat 900gtgcagaatg gcaagacggt cgcgtcggct gcgtccggag gcgacatcat cacggcatcc 960ggctgcacct cggcccaggc gttcggcggg ctggccaaca tgggcgcggc gcttggacgg 1020ggcatggtgc tgaccttcag catctggaac gacgctgggg gctacatgaa ctggctcgac 1080agcggcaaca acggcccgtg cagcagcacc gagggcaacc cgtccaacat cctggccaac 1140tacccggaca cccacgtggt cttctccaac atccgctggg gagacatcgg ctcgacggtc 1200caggtctcgg gaggcggcaa cggcggctcg accaccacca cgtcgaccac cacgctgagg 1260acctcgacca cgaccaccac caccgccccg acggccactg ccacgcactg gggacaatgc 1320ggcggaatcg gggtacgtca accgcctcct gcattctgtt gaggaagtta actaacgtgg 1380cctacgcagt ggactggacc gaccgtctgc gaatcgccgt acgcatgcaa ggagctgaac 1440ccctggtact accagtgcct ctaaagtatt gcagtgaagc catactccgt gctcggcatg 1500g 150142464PRTThielavia terrestris 42Met Gly Gln Lys Thr Leu His Gly Phe Ala Ala Thr Ala Leu Ala Val 1 5 10 15 Leu Pro Phe Val Lys Ala Gln Gln Pro Gly Asn Phe Thr Pro Glu Val 20 25 30 His Pro Gln Leu Pro Thr Trp Lys Cys Thr Thr Ala Gly Gly Cys Val 35 40 45 Gln Gln Asp Thr Ser Val Val Leu Asp Trp Asn Tyr Arg Trp Ile His 50 55 60 Asn Ala Asp Gly Thr Ala Ser Cys Thr Thr Ser Ser Gly Val Asp His 65 70 75 80 Thr Leu Cys Pro Asp Glu Ala Thr Cys Ala Lys Asn Cys Phe Val Glu 85 90 95 Gly Val Asn Tyr Thr Ser Ser Gly Val Thr Thr Ser Gly Ser Ser Leu 100 105 110 Thr Met Arg Gln Tyr Phe Lys Gly Ser Asn Gly Gln Thr Asn Ser Val 115 120 125 Ser Pro Arg Leu Tyr Leu Leu Gly Ser Asp Gly Asn Tyr Val Met Leu 130 135 140 Lys Leu Leu Gly Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Thr Leu 145 150 155 160 Pro Cys Gly Glu Asn Gly Ala Leu Tyr Leu Ser Glu Met Asp Ala Thr 165 170 175 Gly Gly Arg Asn Gln Tyr Asn Thr Gly Gly Ala Asn Tyr Gly Ser Gly 180 185 190 Tyr Cys Asp Ala Gln Cys Pro Val Gln Thr Trp Met Asn Gly Thr Leu 195 200 205 Asn Thr Asn Gly Gln Gly Tyr Cys Cys Asn Glu Met Asp Ile Leu Glu 210 215 220 Ala Asn Ser Arg Ala Asn Ala Met Thr Pro His Pro Cys Ala Asn Gly 225 230 235 240 Ser Cys Asp Lys Ser Gly Cys Gly Leu Asn Pro Tyr Ala Glu Gly Tyr 245 250 255 Lys Ser Tyr Tyr Gly Pro Gly Leu Thr Val Asp Thr Ser Lys Pro Phe 260 265 270 Thr Ile Ile Thr Arg Phe Ile Thr Asp Asp Gly Thr Thr Ser Gly Thr 275 280 285 Leu Asn Gln Ile Gln Arg Ile Tyr Val Gln Asn Gly Lys Thr Val Ala 290 295 300 Ser Ala Ala Ser Gly Gly Asp Ile Ile Thr Ala Ser Gly Cys Thr Ser 305 310 315 320 Ala Gln Ala Phe Gly Gly Leu Ala Asn Met Gly Ala Ala Leu Gly Arg 325 330 335 Gly Met Val Leu Thr Phe Ser Ile Trp Asn Asp Ala Gly Gly Tyr Met 340 345 350 Asn Trp Leu Asp Ser Gly Asn Asn Gly Pro Cys Ser Ser Thr Glu Gly 355 360 365 Asn Pro Ser Asn Ile Leu Ala Asn Tyr Pro Asp Thr His Val Val Phe 370 375 380 Ser Asn Ile Arg Trp Gly Asp Ile Gly Ser Thr Val Gln Val Ser Gly 385 390 395 400 Gly Gly Asn Gly Gly Ser Thr Thr Thr Thr Ser Thr Thr Thr Leu Arg 405 410 415 Thr Ser Thr Thr Thr Thr Thr Thr Ala Pro Thr Ala Thr Ala Thr His 420 425 430 Trp Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro Thr Val Cys Glu 435 440 445 Ser Pro Tyr Ala Cys Lys Glu Leu Asn Pro Trp Tyr Tyr Gln Cys Leu 450 455 460 431368DNAThielavia terrestris 43accgatccgc tcgaagatgg cgcccaagtc tacagttctg gccgcctggc tgctctcctc 60gctggccgcg gcccagcaga tcggcaaagc cgtgcccgag gtccacccca aactgacaac 120gcagaagtgc actctccgcg gcgggtgcaa gcctgtccgc acctcggtcg tgctcgactc 180gtccgcgcgc tcgctgcaca aggtcgggga ccccaacacc agctgcagcg tcggcggcga 240cctgtgctcg gacgcgaagt cgtgcggcaa gaactgcgcg ctcgagggcg tcgactacgc 300ggcccacggc gtggcgacca agggcgacgc cctcacgctg caccagtggc tcaagggggc 360cgacggcacc tacaggaccg tctcgccgcg cgtatacctc ctgggcgagg acgggaagaa 420ctacgaggac ttcaagctgc tcaacgccga gctcagcttc gacgtcgacg tgtcccagct 480cgtctgcggc atgaacggcg ccctgtactt ctccgagatg gagatggacg gcggccgcag 540cccgctgaac ccggcgggcg ccacgtacgg cacgggctac tgcgacgcgc agtgccccaa 600gttggacttt atcaacggcg aggtatttct tctctcttct gtttttcttt tccatcgctt 660tttctgaccg gaatccgccc tcttagctca acaccaacca cacgtacggg gcgtgctgca 720acgagatgga catctgggag gccaacgcgc tggcgcaggc gctcacgccg cacccgtgca 780acgcgacgcg ggtgtacaag tgcgacacgg cggacgagtg cgggcagccg gtgggcgtgt 840gcgacgaatg ggggtgctcg tacaacccgt ccaacttcgg ggtcaaggac tactacgggc 900gcaacctgac ggtggacacg aaccgcaagt tcacggtgac gacgcagttc gtgacgtcca 960acgggcgggc ggacggcgag ctgaccgaga tccggcggct gtacgtgcag gacggcgtgg 1020tgatccagaa ccacgcggtc acggcgggcg gggcgacgta cgacagcatc acggacggct 1080tctgcaacgc gacggccacc tggacgcagc agcggggcgg gctcgcgcgc atgggcgagg 1140ccatcggccg cggcatggtg ctcatcttca gcctgtgggt tgacaacggc ggcttcatga 1200actggctcga cagcggcaac gccgggccct gcaacgccac cgagggcgac ccggccctga 1260tcctgcagca gcacccggac gccagcgtca ccttctccaa catccgatgg ggcgagatcg 1320gcagcacgta caagagcgag tgcagccact agagtagagc ttgtaatt 136844423PRTThielavia terrestris 44Met Ala Pro Lys Ser Thr Val Leu Ala Ala Trp Leu Leu Ser Ser Leu 1 5 10 15 Ala Ala Ala Gln Gln Ile Gly Lys Ala Val Pro Glu Val His Pro Lys 20 25 30 Leu Thr Thr Gln Lys Cys Thr Leu Arg Gly Gly Cys Lys Pro Val Arg 35 40 45 Thr Ser Val Val Leu Asp Ser Ser Ala Arg Ser Leu His Lys Val Gly 50 55 60 Asp Pro Asn Thr Ser Cys Ser Val Gly Gly Asp Leu Cys Ser Asp Ala 65 70 75 80 Lys Ser Cys Gly Lys Asn Cys Ala Leu Glu Gly Val Asp Tyr Ala Ala 85 90 95 His Gly Val Ala Thr Lys Gly Asp Ala Leu Thr Leu His Gln Trp Leu 100 105 110 Lys Gly Ala Asp Gly Thr Tyr Arg Thr Val Ser Pro Arg Val Tyr Leu 115 120 125 Leu Gly Glu Asp Gly Lys Asn Tyr Glu Asp Phe Lys Leu Leu Asn Ala 130 135 140 Glu Leu Ser Phe Asp Val Asp Val Ser Gln Leu Val Cys Gly Met Asn 145 150 155 160 Gly Ala Leu Tyr Phe Ser Glu Met Glu Met Asp Gly Gly Arg Ser Pro 165 170 175 Leu Asn Pro Ala Gly Ala Thr Tyr Gly Thr Gly Tyr Cys Asp Ala Gln 180 185 190 Cys Pro Lys Leu Asp Phe Ile Asn Gly Glu Leu Asn Thr Asn His Thr 195 200 205 Tyr Gly Ala Cys Cys Asn Glu Met Asp Ile Trp Glu Ala Asn Ala Leu 210 215 220 Ala Gln Ala Leu Thr Pro His Pro Cys Asn Ala Thr Arg Val Tyr Lys 225 230 235 240 Cys Asp Thr Ala Asp Glu Cys Gly Gln Pro Val Gly Val Cys Asp Glu 245 250 255 Trp Gly Cys Ser Tyr Asn Pro Ser Asn Phe Gly Val Lys Asp Tyr Tyr 260 265 270 Gly Arg Asn Leu Thr Val Asp Thr Asn Arg Lys Phe Thr Val Thr Thr 275 280 285 Gln Phe Val Thr Ser Asn Gly Arg Ala Asp Gly Glu Leu Thr Glu Ile 290 295 300 Arg Arg Leu Tyr Val Gln Asp Gly Val Val Ile Gln Asn His Ala Val 305 310 315 320 Thr Ala Gly Gly Ala Thr Tyr Asp Ser Ile Thr Asp Gly Phe Cys Asn 325 330 335 Ala Thr Ala Thr Trp Thr Gln Gln Arg Gly Gly Leu Ala Arg Met Gly 340 345 350 Glu Ala Ile Gly Arg Gly Met Val Leu Ile Phe Ser Leu Trp Val Asp 355 360 365 Asn Gly Gly Phe Met Asn Trp Leu Asp Ser Gly Asn Ala Gly Pro Cys 370 375 380 Asn Ala Thr Glu Gly Asp Pro Ala Leu Ile Leu Gln Gln His Pro Asp 385 390 395 400 Ala Ser Val Thr Phe Ser Asn Ile Arg Trp Gly Glu Ile Gly Ser Thr 405 410 415 Tyr Lys Ser Glu Cys Ser His 420 451011DNAThielavia terrestris 45atgaccctac ggctccctgt catcagcctg ctggcctcgc tggcagcagg cgccgtcgtc 60gtcccacggg cggagtttca cccccctctc ccgacttgga aatgcacgac ctccgggggc 120tgcgtgcagc agaacaccag cgtcgtcctg gaccgtgact cgaagtacgc cgcacacagc 180gccggctcgc ggacggaatc ggattacgcg gcaatgggag tgtccacttc gggcaatgcc 240gtgacgctgt accactacgt caagaccaac ggcaccctcg tccccgcttc gccgcgcatc 300tacctcctgg gcgcggacgg caagtacgtg cttatggacc tcctcaacca ggagctgtcg 360gtggacgtcg acttctcggc gctgccgtgc ggcgagaacg gggccttcta cctgtccgag 420atggcggcgg acgggcgggg cgacgcgggg gcgggcgacg ggtactgcga cgcgcagtgc 480cagggctact gctgcaacga gatggacatc ctcgaggcca actcgatggc gacggccatg 540acgccgcacc cgtgcaaggg caacaactgc gaccgcagcg gctgcggcta caacccgtac 600gccagcggcc agcgcggctt ctacgggccc ggcaagacgg tcgacacgag caagcccttc 660accgtcgtca cgcagttcgc cgccagcggc ggcaagctga cccagatcac ccgcaagtac 720atccagaacg gccgggagat cggcggcggc ggcaccatct ccagctgcgg ctccgagtct 780tcgacgggcg gcctgaccgg catgggcgag gcgctggggc gcggaatggt gctggccatg 840agcatctgga acgacgcggc ccaggagatg gcatggctcg atgccggcaa caacggccct 900tgcgccagtg gccagggcag cccgtccgtc attcagtcgc agcatcccga cacccacgtc 960gtcttctcca acatcaggtg gggcgacatc gggtctacca cgaagaacta g 101146336PRTThielavia terrestris 46Met Thr Leu Arg Leu Pro Val Ile Ser Leu Leu Ala Ser Leu Ala Ala 1 5 10 15 Gly Ala Val Val Val Pro Arg Ala Glu Phe His

Pro Pro Leu Pro Thr 20 25 30 Trp Lys Cys Thr Thr Ser Gly Gly Cys Val Gln Gln Asn Thr Ser Val 35 40 45 Val Leu Asp Arg Asp Ser Lys Tyr Ala Ala His Ser Ala Gly Ser Arg 50 55 60 Thr Glu Ser Asp Tyr Ala Ala Met Gly Val Ser Thr Ser Gly Asn Ala 65 70 75 80 Val Thr Leu Tyr His Tyr Val Lys Thr Asn Gly Thr Leu Val Pro Ala 85 90 95 Ser Pro Arg Ile Tyr Leu Leu Gly Ala Asp Gly Lys Tyr Val Leu Met 100 105 110 Asp Leu Leu Asn Gln Glu Leu Ser Val Asp Val Asp Phe Ser Ala Leu 115 120 125 Pro Cys Gly Glu Asn Gly Ala Phe Tyr Leu Ser Glu Met Ala Ala Asp 130 135 140 Gly Arg Gly Asp Ala Gly Ala Gly Asp Gly Tyr Cys Asp Ala Gln Cys 145 150 155 160 Gln Gly Tyr Cys Cys Asn Glu Met Asp Ile Leu Glu Ala Asn Ser Met 165 170 175 Ala Thr Ala Met Thr Pro His Pro Cys Lys Gly Asn Asn Cys Asp Arg 180 185 190 Ser Gly Cys Gly Tyr Asn Pro Tyr Ala Ser Gly Gln Arg Gly Phe Tyr 195 200 205 Gly Pro Gly Lys Thr Val Asp Thr Ser Lys Pro Phe Thr Val Val Thr 210 215 220 Gln Phe Ala Ala Ser Gly Gly Lys Leu Thr Gln Ile Thr Arg Lys Tyr 225 230 235 240 Ile Gln Asn Gly Arg Glu Ile Gly Gly Gly Gly Thr Ile Ser Ser Cys 245 250 255 Gly Ser Glu Ser Ser Thr Gly Gly Leu Thr Gly Met Gly Glu Ala Leu 260 265 270 Gly Arg Gly Met Val Leu Ala Met Ser Ile Trp Asn Asp Ala Ala Gln 275 280 285 Glu Met Ala Trp Leu Asp Ala Gly Asn Asn Gly Pro Cys Ala Ser Gly 290 295 300 Gln Gly Ser Pro Ser Val Ile Gln Ser Gln His Pro Asp Thr His Val 305 310 315 320 Val Phe Ser Asn Ile Arg Trp Gly Asp Ile Gly Ser Thr Thr Lys Asn 325 330 335 471480DNACladorrhinum foecundissimum 47gatccgaatt cctcctctcg ttctttagtc acagaccaga catctgccca cgatggttca 60caagttcgcc ctcctcaccg gcctcgccgc ctccctcgca tctgcccagc agatcggcac 120cgtcgtcccc gagtctcacc ccaagcttcc caccaagcgc tgcactctcg ccggtggctg 180ccagaccgtc gacacctcca tcgtcatcga cgccttccag cgtcccctcc acaagatcgg 240cgacccttcc actccttgcg tcgtcggcgg ccctctctgc cccgacgcca agtcctgcgc 300tgagaactgc gcgctcgagg gtgtcgacta tgcctcctgg ggcatcaaga ccgagggcga 360cgccctaact ctcaaccagt ggatgcccga cccggcgaac cctggccagt acaagacgac 420tactccccgt acttaccttg ttgctgagga cggcaagaac tacgaggatg tgaagctcct 480ggctaaggag atctcgtttg atgccgatgt cagcaacctt ccctgcggca tgaacggtgc 540tttctacttg tctgagatgt tgatggatgg tggacgtggc gacctcaacc ctgctggtgc 600cgagtatggt accggttact gtgatgcgca gtgcttcaag ttggatttca tcaacggcga 660ggccaacatc gaccaaaagc acggcgcctg ctgcaacgaa atggacattt tcgaatccaa 720ctcgcgcgcc aagaccttcg tcccccaccc ctgcaacatc acgcaggtct acaagtgcga 780aggcgaagac gagtgcggcc agcccgtcgg cgtgtgcgac aagtgggggt gcggcttcaa 840cgagtacaaa tggggcgtcg agtccttcta cggccggggc tcgcagttcg ccatcgactc 900ctccaagaag ttcaccgtca ccacgcagtt cctgaccgac aacggcaagg aggacggcgt 960cctcgtcgag atccgccgct tgtggcacca ggatggcaag ctgatcaaga acaccgctat 1020ccaggttgag gagaactaca gcacggactc ggtgagcacc gagttctgcg agaagactgc 1080ttctttcacc atgcagcgcg gtggtctcaa ggcgatgggc gaggctatcg gtcgtggtat 1140ggtgctggtt ttcagcatct gggcggatga ttcgggtttt atgaactggt tggatgcgga 1200gggtaatggc ccttgcagcg cgactgaggg cgatccgaag gagattgtca agaataagcc 1260ggatgctagg gttacgttct caaacattag gattggtgag gttggtagca cgtatgctcc 1320gggtgggaag tgcggtgtta agagcagggt tgctaggggg cttactgctt cttaaggggg 1380gtgtgaagag aggaggaggt gttgttgggg gttggagatg ataattgggc gagatggtgt 1440agagcgggtt ggttggatat gaatacgttg aattggatgt 148048440PRTCladorrhinum foecundissimum 48Met Val His Lys Phe Ala Leu Leu Thr Gly Leu Ala Ala Ser Leu Ala 1 5 10 15 Ser Ala Gln Gln Ile Gly Thr Val Val Pro Glu Ser His Pro Lys Leu 20 25 30 Pro Thr Lys Arg Cys Thr Leu Ala Gly Gly Cys Gln Thr Val Asp Thr 35 40 45 Ser Ile Val Ile Asp Ala Phe Gln Arg Pro Leu His Lys Ile Gly Asp 50 55 60 Pro Ser Thr Pro Cys Val Val Gly Gly Pro Leu Cys Pro Asp Ala Lys 65 70 75 80 Ser Cys Ala Glu Asn Cys Ala Leu Glu Gly Val Asp Tyr Ala Ser Trp 85 90 95 Gly Ile Lys Thr Glu Gly Asp Ala Leu Thr Leu Asn Gln Trp Met Pro 100 105 110 Asp Pro Ala Asn Pro Gly Gln Tyr Lys Thr Thr Thr Pro Arg Thr Tyr 115 120 125 Leu Val Ala Glu Asp Gly Lys Asn Tyr Glu Asp Val Lys Leu Leu Ala 130 135 140 Lys Glu Ile Ser Phe Asp Ala Asp Val Ser Asn Leu Pro Cys Gly Met 145 150 155 160 Asn Gly Ala Phe Tyr Leu Ser Glu Met Leu Met Asp Gly Gly Arg Gly 165 170 175 Asp Leu Asn Pro Ala Gly Ala Glu Tyr Gly Thr Gly Tyr Cys Asp Ala 180 185 190 Gln Cys Phe Lys Leu Asp Phe Ile Asn Gly Glu Ala Asn Ile Asp Gln 195 200 205 Lys His Gly Ala Cys Cys Asn Glu Met Asp Ile Phe Glu Ser Asn Ser 210 215 220 Arg Ala Lys Thr Phe Val Pro His Pro Cys Asn Ile Thr Gln Val Tyr 225 230 235 240 Lys Cys Glu Gly Glu Asp Glu Cys Gly Gln Pro Val Gly Val Cys Asp 245 250 255 Lys Trp Gly Cys Gly Phe Asn Glu Tyr Lys Trp Gly Val Glu Ser Phe 260 265 270 Tyr Gly Arg Gly Ser Gln Phe Ala Ile Asp Ser Ser Lys Lys Phe Thr 275 280 285 Val Thr Thr Gln Phe Leu Thr Asp Asn Gly Lys Glu Asp Gly Val Leu 290 295 300 Val Glu Ile Arg Arg Leu Trp His Gln Asp Gly Lys Leu Ile Lys Asn 305 310 315 320 Thr Ala Ile Gln Val Glu Glu Asn Tyr Ser Thr Asp Ser Val Ser Thr 325 330 335 Glu Phe Cys Glu Lys Thr Ala Ser Phe Thr Met Gln Arg Gly Gly Leu 340 345 350 Lys Ala Met Gly Glu Ala Ile Gly Arg Gly Met Val Leu Val Phe Ser 355 360 365 Ile Trp Ala Asp Asp Ser Gly Phe Met Asn Trp Leu Asp Ala Glu Gly 370 375 380 Asn Gly Pro Cys Ser Ala Thr Glu Gly Asp Pro Lys Glu Ile Val Lys 385 390 395 400 Asn Lys Pro Asp Ala Arg Val Thr Phe Ser Asn Ile Arg Ile Gly Glu 405 410 415 Val Gly Ser Thr Tyr Ala Pro Gly Gly Lys Cys Gly Val Lys Ser Arg 420 425 430 Val Ala Arg Gly Leu Thr Ala Ser 435 440 491380DNATrichoderma reesei 49atggcgccct cagttacact gccgttgacc acggccatcc tggccattgc ccggctcgtc 60gccgcccagc aaccgggtac cagcaccccc gaggtccatc ccaagttgac aacctacaag 120tgtacaaagt ccggggggtg cgtggcccag gacacctcgg tggtccttga ctggaactac 180cgctggatgc acgacgcaaa ctacaactcg tgcaccgtca acggcggcgt caacaccacg 240ctctgccctg acgaggcgac ctgtggcaag aactgcttca tcgagggcgt cgactacgcc 300gcctcgggcg tcacgacctc gggcagcagc ctcaccatga accagtacat gcccagcagc 360tctggcggct acagcagcgt ctctcctcgg ctgtatctcc tggactctga cggtgagtac 420gtgatgctga agctcaacgg ccaggagctg agcttcgacg tcgacctctc tgctctgccg 480tgtggagaga acggctcgct ctacctgtct cagatggacg agaacggggg cgccaaccag 540tataacacgg ccggtgccaa ctacgggagc ggctactgcg atgctcagtg ccccgtccag 600acatggagga acggcaccct caacactagc caccagggct tctgctgcaa cgagatggat 660atcctggagg gcaactcgag ggcgaatgcc ttgacccctc actcttgcac ggccacggcc 720tgcgactctg ccggttgcgg cttcaacccc tatggcagcg gctacaaaag ctactacggc 780cccggagata ccgttgacac ctccaagacc ttcaccatca tcacccagtt caacacggac 840aacggctcgc cctcgggcaa ccttgtgagc atcacccgca agtaccagca aaacggcgtc 900gacatcccca gcgcccagcc cggcggcgac accatctcgt cctgcccgtc cgcctcagcc 960tacggcggcc tcgccaccat gggcaaggcc ctgagcagcg gcatggtgct cgtgttcagc 1020atttggaacg acaacagcca gtacatgaac tggctcgaca gcggcaacgc cggcccctgc 1080agcagcaccg agggcaaccc atccaacatc ctggccaaca accccaacac gcacgtcgtc 1140ttctccaaca tccgctgggg agacattggg tctactacga actcgactgc gcccccgccc 1200ccgcctgcgt ccagcacgac gttttcgact acacggagga gctcgacgac ttcgagcagc 1260ccgagctgca cgcagactca ctgggggcag tgcggtggca ttgggtacag cgggtgcaag 1320acgtgcacgt cgggcactac gtgccagtat agcaacgact actactcgca atgcctttag 138050459PRTTrichoderma reesei 50Met Ala Pro Ser Val Thr Leu Pro Leu Thr Thr Ala Ile Leu Ala Ile 1 5 10 15 Ala Arg Leu Val Ala Ala Gln Gln Pro Gly Thr Ser Thr Pro Glu Val 20 25 30 His Pro Lys Leu Thr Thr Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val 35 40 45 Ala Gln Asp Thr Ser Val Val Leu Asp Trp Asn Tyr Arg Trp Met His 50 55 60 Asp Ala Asn Tyr Asn Ser Cys Thr Val Asn Gly Gly Val Asn Thr Thr 65 70 75 80 Leu Cys Pro Asp Glu Ala Thr Cys Gly Lys Asn Cys Phe Ile Glu Gly 85 90 95 Val Asp Tyr Ala Ala Ser Gly Val Thr Thr Ser Gly Ser Ser Leu Thr 100 105 110 Met Asn Gln Tyr Met Pro Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser 115 120 125 Pro Arg Leu Tyr Leu Leu Asp Ser Asp Gly Glu Tyr Val Met Leu Lys 130 135 140 Leu Asn Gly Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro 145 150 155 160 Cys Gly Glu Asn Gly Ser Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly 165 170 175 Gly Ala Asn Gln Tyr Asn Thr Ala Gly Ala Asn Tyr Gly Ser Gly Tyr 180 185 190 Cys Asp Ala Gln Cys Pro Val Gln Thr Trp Arg Asn Gly Thr Leu Asn 195 200 205 Thr Ser His Gln Gly Phe Cys Cys Asn Glu Met Asp Ile Leu Glu Gly 210 215 220 Asn Ser Arg Ala Asn Ala Leu Thr Pro His Ser Cys Thr Ala Thr Ala 225 230 235 240 Cys Asp Ser Ala Gly Cys Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Lys 245 250 255 Ser Tyr Tyr Gly Pro Gly Asp Thr Val Asp Thr Ser Lys Thr Phe Thr 260 265 270 Ile Ile Thr Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu 275 280 285 Val Ser Ile Thr Arg Lys Tyr Gln Gln Asn Gly Val Asp Ile Pro Ser 290 295 300 Ala Gln Pro Gly Gly Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser Ala 305 310 315 320 Tyr Gly Gly Leu Ala Thr Met Gly Lys Ala Leu Ser Ser Gly Met Val 325 330 335 Leu Val Phe Ser Ile Trp Asn Asp Asn Ser Gln Tyr Met Asn Trp Leu 340 345 350 Asp Ser Gly Asn Ala Gly Pro Cys Ser Ser Thr Glu Gly Asn Pro Ser 355 360 365 Asn Ile Leu Ala Asn Asn Pro Asn Thr His Val Val Phe Ser Asn Ile 370 375 380 Arg Trp Gly Asp Ile Gly Ser Thr Thr Asn Ser Thr Ala Pro Pro Pro 385 390 395 400 Pro Pro Ala Ser Ser Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser Thr 405 410 415 Thr Ser Ser Ser Pro Ser Cys Thr Gln Thr His Trp Gly Gln Cys Gly 420 425 430 Gly Ile Gly Tyr Ser Gly Cys Lys Thr Cys Thr Ser Gly Thr Thr Cys 435 440 445 Gln Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu 450 455 511542DNATrichoderma reesei 51atgtatcgga agttggccgt catctcggcc ttcttggcca cagctcgtgc tcagtcggcc 60tgcactctcc aatcggagac tcacccgcct ctgacatggc agaaatgctc gtctggtggc 120acgtgcactc aacagacagg ctccgtggtc atcgacgcca actggcgctg gactcacgct 180acgaacagca gcacgaactg ctacgatggc aacacttgga gctcgaccct atgtcctgac 240aacgagacct gcgcgaagaa ctgctgtctg gacggtgccg cctacgcgtc cacgtacgga 300gttaccacga gcggtaacag cctctccatt ggctttgtca cccagtctgc gcagaagaac 360gttggcgctc gcctttacct tatggcgagc gacacgacct accaggaatt caccctgctt 420ggcaacgagt tctctttcga tgttgatgtt tcgcagctgc cgtgcggctt gaacggagct 480ctctacttcg tgtccatgga cgcggatggt ggcgtgagca agtatcccac caacaccgct 540ggcgccaagt acggcacggg gtactgtgac agccagtgtc cccgcgatct gaagttcatc 600aatggccagg ccaacgttga gggctgggag ccgtcatcca acaacgcgaa cacgggcatt 660ggaggacacg gaagctgctg ctctgagatg gatatctggg aggccaactc catctccgag 720gctcttaccc cccacccttg cacgactgtc ggccaggaga tctgcgaggg tgatgggtgc 780ggcggaactt actccgataa cagatatggc ggcacttgcg atcccgatgg ctgcgactgg 840aacccatacc gcctgggcaa caccagcttc tacggccctg gctcaagctt taccctcgat 900accaccaaga aattgaccgt tgtcacccag ttcgagacgt cgggtgccat caaccgatac 960tatgtccaga atggcgtcac tttccagcag cccaacgccg agcttggtag ttactctggc 1020aacgagctca acgatgatta ctgcacagct gaggaggcag aattcggcgg atcctctttc 1080tcagacaagg gcggcctgac tcagttcaag aaggctacct ctggcggcat ggttctggtc 1140atgagtctgt gggatgatta ctacgccaac atgctgtggc tggactccac ctacccgaca 1200aacgagacct cctccacacc cggtgccgtg cgcggaagct gctccaccag ctccggtgtc 1260cctgctcagg tcgaatctca gtctcccaac gccaaggtca ccttctccaa catcaagttc 1320ggacccattg gcagcaccgg caaccctagc ggcggcaacc ctcccggcgg aaacccgcct 1380ggcaccacca ccacccgccg cccagccact accactggaa gctctcccgg acctacccag 1440tctcactacg gccagtgcgg cggtattggc tacagcggcc ccacggtctg cgccagcggc 1500acaacttgcc aggtcctgaa cccttactac tctcagtgcc tg 154252514PRTTrichoderma reesei 52Met Tyr Arg Lys Leu Ala Val Ile Ser Ala Phe Leu Ala Thr Ala Arg 1 5 10 15 Ala Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr 20 25 30 Trp Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser 35 40 45 Val Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser 50 55 60 Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp 65 70 75 80 Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala 85 90 95 Ser Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe 100 105 110 Val Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met 115 120 125 Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe 130 135 140 Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala 145 150 155 160 Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro 165 170 175 Thr Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 180 185 190 Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly 195 200 205 Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly 210 215 220 Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu 225 230 235 240 Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu 245 250 255 Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr 260 265 270 Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr 275 280 285 Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys 290 295 300 Leu Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr 305 310 315 320 Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly 325 330 335 Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu 340 345 350 Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln 355 360 365 Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp 370 375

380 Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr 385 390 395 400 Asn Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr 405 410 415 Ser Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys 420 425 430 Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn 435 440 445 Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr 450 455 460 Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln 465 470 475 480 Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val 485 490 495 Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln 500 505 510 Cys Leu 531413DNATrichoderma reesei 53atgattgtcg gcattctcac cacgctggct acgctggcca cactcgcagc tagtgtgcct 60ctagaggagc ggcaagcttg ctcaagcgtc tggggccaat gtggtggcca gaattggtcg 120ggtccgactt gctgtgcttc cggaagcaca tgcgtctact ccaacgacta ttactcccag 180tgtcttcccg gcgctgcaag ctcaagctcg tccacgcgcg ccgcgtcgac gacttctcga 240gtatccccca caacatcccg gtcgagctcc gcgacgcctc cacctggttc tactactacc 300agagtacctc cagtcggatc gggaaccgct acgtattcag gcaacccttt tgttggggtc 360actccttggg ccaatgcata ttacgcctct gaagttagca gcctcgctat tcctagcttg 420actggagcca tggccactgc tgcagcagct gtcgcaaagg ttccctcttt tatgtggcta 480gatactcttg acaagacccc tctcatggag caaaccttgg ccgacatccg caccgccaac 540aagaatggcg gtaactatgc cggacagttt gtggtgtatg acttgccgga tcgcgattgc 600gctgcccttg cctcgaatgg cgaatactct attgccgatg gtggcgtcgc caaatataag 660aactatatcg acaccattcg tcaaattgtc gtggaatatt ccgatatccg gaccctcctg 720gttattgagc ctgactctct tgccaacctg gtgaccaacc tcggtactcc aaagtgtgcc 780aatgctcagt cagcctacct tgagtgcatc aactacgccg tcacacagct gaaccttcca 840aatgttgcga tgtatttgga cgctggccat gcaggatggc ttggctggcc ggcaaaccaa 900gacccggccg ctcagctatt tgcaaatgtt tacaagaatg catcgtctcc gagagctctt 960cgcggattgg caaccaatgt cgccaactac aacgggtgga acattaccag ccccccatcg 1020tacacgcaag gcaacgctgt ctacaacgag aagctgtaca tccacgctat tggacctctt 1080cttgccaatc acggctggtc caacgccttc ttcatcactg atcaaggtcg atcgggaaag 1140cagcctaccg gacagcaaca gtggggagac tggtgcaatg tgatcggcac cggatttggt 1200attcgcccat ccgcaaacac tggggactcg ttgctggatt cgtttgtctg ggtcaagcca 1260ggcggcgagt gtgacggcac cagcgacagc agtgcgccac gatttgactc ccactgtgcg 1320ctcccagatg ccttgcaacc ggcgcctcaa gctggtgctt ggttccaagc ctactttgtg 1380cagcttctca caaacgcaaa cccatcgttc ctg 141354471PRTTrichoderma reesei 54Met Ile Val Gly Ile Leu Thr Thr Leu Ala Thr Leu Ala Thr Leu Ala 1 5 10 15 Ala Ser Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly 20 25 30 Gln Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly 35 40 45 Ser Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly 50 55 60 Ala Ala Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg 65 70 75 80 Val Ser Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly 85 90 95 Ser Thr Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr 100 105 110 Ser Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr 115 120 125 Ala Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met 130 135 140 Ala Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu 145 150 155 160 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 165 170 175 Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 180 185 190 Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu 195 200 205 Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 210 215 220 Thr Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu 225 230 235 240 Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr 245 250 255 Pro Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr 260 265 270 Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala 275 280 285 Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala 290 295 300 Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu 305 310 315 320 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr 325 330 335 Ser Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu 340 345 350 Tyr Ile His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn 355 360 365 Ala Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly 370 375 380 Gln Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly 385 390 395 400 Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val 405 410 415 Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ala 420 425 430 Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala 435 440 445 Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr 450 455 460 Asn Ala Asn Pro Ser Phe Leu 465 470 551377DNATrichoderma reesei 55atggcgccct cagttacact gccgttgacc acggccatcc tggccattgc ccggctcgtc 60gccgcccagc aaccgggtac cagcaccccc gaggtccatc ccaagttgac aacctacaag 120tgtacaaagt ccggggggtg cgtggcccag gacacctcgg tggtccttga ctggaactac 180cgctggatgc acgacgcaaa ctacaactcg tgcaccgtca acggcggcgt caacaccacg 240ctctgccctg acgaggcgac ctgtggcaag aactgcttca tcgagggcgt cgactacgcc 300gcctcgggcg tcacgacctc gggcagcagc ctcaccatga accagtacat gcccagcagc 360tctggcggct acagcagcgt ctctcctcgg ctgtatctcc tggactctga cggtgagtac 420gtgatgctga agctcaacgg ccaggagctg agcttcgacg tcgacctctc tgctctgccg 480tgtggagaga acggctcgct ctacctgtct cagatggacg agaacggggg cgccaaccag 540tataacacgg ccggtgccaa ctacgggagc ggctactgcg atgctcagtg ccccgtccag 600acatggagga acggcaccct caacactagc caccagggct tctgctgcaa cgagatggat 660atcctggagg gcaactcgag ggcgaatgcc ttgacccctc actcttgcac ggccacggcc 720tgcgactctg ccggttgcgg cttcaacccc tatggcagcg gctacaaaag ctactacggc 780cccggagata ccgttgacac ctccaagacc ttcaccatca tcacccagtt caacacggac 840aacggctcgc cctcgggcaa ccttgtgagc atcacccgca agtaccagca aaacggcgtc 900gacatcccca gcgcccagcc cggcggcgac accatctcgt cctgcccgtc cgcctcagcc 960tacggcggcc tcgccaccat gggcaaggcc ctgagcagcg gcatggtgct cgtgttcagc 1020atttggaacg acaacagcca gtacatgaac tggctcgaca gcggcaacgc cggcccctgc 1080agcagcaccg agggcaaccc atccaacatc ctggccaaca accccaacac gcacgtcgtc 1140ttctccaaca tccgctgggg agacattggg tctactacga actcgactgc gcccccgccc 1200ccgcctgcgt ccagcacgac gttttcgact acacggagga gctcgacgac ttcgagcagc 1260ccgagctgca cgcagactca ctgggggcag tgcggtggca ttgggtacag cgggtgcaag 1320acgtgcacgt cgggcactac gtgccagtat agcaacgact actactcgca atgcctt 137756459PRTTrichoderma reesei 56Met Ala Pro Ser Val Thr Leu Pro Leu Thr Thr Ala Ile Leu Ala Ile 1 5 10 15 Ala Arg Leu Val Ala Ala Gln Gln Pro Gly Thr Ser Thr Pro Glu Val 20 25 30 His Pro Lys Leu Thr Thr Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val 35 40 45 Ala Gln Asp Thr Ser Val Val Leu Asp Trp Asn Tyr Arg Trp Met His 50 55 60 Asp Ala Asn Tyr Asn Ser Cys Thr Val Asn Gly Gly Val Asn Thr Thr 65 70 75 80 Leu Cys Pro Asp Glu Ala Thr Cys Gly Lys Asn Cys Phe Ile Glu Gly 85 90 95 Val Asp Tyr Ala Ala Ser Gly Val Thr Thr Ser Gly Ser Ser Leu Thr 100 105 110 Met Asn Gln Tyr Met Pro Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser 115 120 125 Pro Arg Leu Tyr Leu Leu Asp Ser Asp Gly Glu Tyr Val Met Leu Lys 130 135 140 Leu Asn Gly Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro 145 150 155 160 Cys Gly Glu Asn Gly Ser Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly 165 170 175 Gly Ala Asn Gln Tyr Asn Thr Ala Gly Ala Asn Tyr Gly Ser Gly Tyr 180 185 190 Cys Asp Ala Gln Cys Pro Val Gln Thr Trp Arg Asn Gly Thr Leu Asn 195 200 205 Thr Ser His Gln Gly Phe Cys Cys Asn Glu Met Asp Ile Leu Glu Gly 210 215 220 Asn Ser Arg Ala Asn Ala Leu Thr Pro His Ser Cys Thr Ala Thr Ala 225 230 235 240 Cys Asp Ser Ala Gly Cys Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Lys 245 250 255 Ser Tyr Tyr Gly Pro Gly Asp Thr Val Asp Thr Ser Lys Thr Phe Thr 260 265 270 Ile Ile Thr Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu 275 280 285 Val Ser Ile Thr Arg Lys Tyr Gln Gln Asn Gly Val Asp Ile Pro Ser 290 295 300 Ala Gln Pro Gly Gly Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser Ala 305 310 315 320 Tyr Gly Gly Leu Ala Thr Met Gly Lys Ala Leu Ser Ser Gly Met Val 325 330 335 Leu Val Phe Ser Ile Trp Asn Asp Asn Ser Gln Tyr Met Asn Trp Leu 340 345 350 Asp Ser Gly Asn Ala Gly Pro Cys Ser Ser Thr Glu Gly Asn Pro Ser 355 360 365 Asn Ile Leu Ala Asn Asn Pro Asn Thr His Val Val Phe Ser Asn Ile 370 375 380 Arg Trp Gly Asp Ile Gly Ser Thr Thr Asn Ser Thr Ala Pro Pro Pro 385 390 395 400 Pro Pro Ala Ser Ser Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser Thr 405 410 415 Thr Ser Ser Ser Pro Ser Cys Thr Gln Thr His Trp Gly Gln Cys Gly 420 425 430 Gly Ile Gly Tyr Ser Gly Cys Lys Thr Cys Thr Ser Gly Thr Thr Cys 435 440 445 Gln Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu 450 455 571254DNATrichoderma reesei 57atgaacaagt ccgtggctcc attgctgctt gcagcgtcca tactatatgg cggcgccgtc 60gcacagcaga ctgtctgggg ccagtgtgga ggtattggtt ggagcggacc tacgaattgt 120gctcctggct cagcttgttc gaccctcaat ccttattatg cgcaatgtat tccgggagcc 180actactatca ccacttcgac ccggccacca tccggtccaa ccaccaccac cagggctacc 240tcaacaagct catcaactcc acccacgagc tctggggtcc gatttgccgg cgttaacatc 300gcgggttttg actttggctg taccacagat ggcacttgcg ttacctcgaa ggtttatcct 360ccgttgaaga acttcaccgg ctcaaacaac taccccgatg gcatcggcca gatgcagcac 420ttcgtcaacg aggacgggat gactattttc cgcttacctg tcggatggca gtacctcgtc 480aacaacaatt tgggcggcaa tcttgattcc acgagcattt ccaagtatga tcagcttgtt 540caggggtgcc tgtctctggg cgcatactgc atcgtcgaca tccacaatta tgctcgatgg 600aacggtggga tcattggtca gggcggccct actaatgctc aattcacgag cctttggtcg 660cagttggcat caaagtacgc atctcagtcg agggtgtggt tcggcatcat gaatgagccc 720cacgacgtga acatcaacac ctgggctgcc acggtccaag aggttgtaac cgcaatccgc 780aacgctggtg ctacgtcgca attcatctct ttgcctggaa atgattggca atctgctggg 840gctttcatat ccgatggcag tgcagccgcc ctgtctcaag tcacgaaccc ggatgggtca 900acaacgaatc tgatttttga cgtgcacaaa tacttggact cagacaactc cggtactcac 960gccgaatgta ctacaaataa cattgacggc gccttttctc cgcttgccac ttggctccga 1020cagaacaatc gccaggctat cctgacagaa accggtggtg gcaacgttca gtcctgcata 1080caagacatgt gccagcaaat ccaatatctc aaccagaact cagatgtcta tcttggctat 1140gttggttggg gtgccggatc atttgatagc acgtatgtcc tgacggaaac accgactagc 1200agtggtaact catggacgga cacatccttg gtcagctcgt gtctcgcaag aaag 125458418PRTTrichoderma reesei 58Met Asn Lys Ser Val Ala Pro Leu Leu Leu Ala Ala Ser Ile Leu Tyr 1 5 10 15 Gly Gly Ala Val Ala Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Ile 20 25 30 Gly Trp Ser Gly Pro Thr Asn Cys Ala Pro Gly Ser Ala Cys Ser Thr 35 40 45 Leu Asn Pro Tyr Tyr Ala Gln Cys Ile Pro Gly Ala Thr Thr Ile Thr 50 55 60 Thr Ser Thr Arg Pro Pro Ser Gly Pro Thr Thr Thr Thr Arg Ala Thr 65 70 75 80 Ser Thr Ser Ser Ser Thr Pro Pro Thr Ser Ser Gly Val Arg Phe Ala 85 90 95 Gly Val Asn Ile Ala Gly Phe Asp Phe Gly Cys Thr Thr Asp Gly Thr 100 105 110 Cys Val Thr Ser Lys Val Tyr Pro Pro Leu Lys Asn Phe Thr Gly Ser 115 120 125 Asn Asn Tyr Pro Asp Gly Ile Gly Gln Met Gln His Phe Val Asn Glu 130 135 140 Asp Gly Met Thr Ile Phe Arg Leu Pro Val Gly Trp Gln Tyr Leu Val 145 150 155 160 Asn Asn Asn Leu Gly Gly Asn Leu Asp Ser Thr Ser Ile Ser Lys Tyr 165 170 175 Asp Gln Leu Val Gln Gly Cys Leu Ser Leu Gly Ala Tyr Cys Ile Val 180 185 190 Asp Ile His Asn Tyr Ala Arg Trp Asn Gly Gly Ile Ile Gly Gln Gly 195 200 205 Gly Pro Thr Asn Ala Gln Phe Thr Ser Leu Trp Ser Gln Leu Ala Ser 210 215 220 Lys Tyr Ala Ser Gln Ser Arg Val Trp Phe Gly Ile Met Asn Glu Pro 225 230 235 240 His Asp Val Asn Ile Asn Thr Trp Ala Ala Thr Val Gln Glu Val Val 245 250 255 Thr Ala Ile Arg Asn Ala Gly Ala Thr Ser Gln Phe Ile Ser Leu Pro 260 265 270 Gly Asn Asp Trp Gln Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser Ala 275 280 285 Ala Ala Leu Ser Gln Val Thr Asn Pro Asp Gly Ser Thr Thr Asn Leu 290 295 300 Ile Phe Asp Val His Lys Tyr Leu Asp Ser Asp Asn Ser Gly Thr His 305 310 315 320 Ala Glu Cys Thr Thr Asn Asn Ile Asp Gly Ala Phe Ser Pro Leu Ala 325 330 335 Thr Trp Leu Arg Gln Asn Asn Arg Gln Ala Ile Leu Thr Glu Thr Gly 340 345 350 Gly Gly Asn Val Gln Ser Cys Ile Gln Asp Met Cys Gln Gln Ile Gln 355 360 365 Tyr Leu Asn Gln Asn Ser Asp Val Tyr Leu Gly Tyr Val Gly Trp Gly 370 375 380 Ala Gly Ser Phe Asp Ser Thr Tyr Val Leu Thr Glu Thr Pro Thr Ser 385 390 395 400 Ser Gly Asn Ser Trp Thr Asp Thr Ser Leu Val Ser Ser Cys Leu Ala 405 410 415 Arg Lys 59702DNATrichoderma reesei 59atgaagttcc ttcaagtcct ccctgccctc ataccggccg ccctggccca aaccagctgt 60gaccagtggg caaccttcac tggcaacggc tacacagtca gcaacaacct ttggggagca 120tcagccggct ctggatttgg ctgcgtgacg gcggtatcgc tcagcggcgg ggcctcctgg 180cacgcagact ggcagtggtc cggcggccag aacaacgtca agtcgtacca gaactctcag 240attgccattc cccagaagag gaccgtcaac agcatcagca gcatgcccac cactgccagc 300tggagctaca gcgggagcaa catccgcgct aatgttgcgt atgacttgtt caccgcagcc 360aacccgaatc atgtcacgta ctcgggagac tacgaactca tgatctggct tggcaaatac 420ggcgatattg ggccgattgg gtcctcacag ggaacagtca acgtcggtgg ccagagctgg 480acgctctact atggctacaa cggagccatg caagtctatt cctttgtggc ccagaccaac 540actaccaact acagcggaga tgtcaagaac ttcttcaatt atctccgaga caataaagga 600tacaacgctg caggccaata tgttcttagc taccaatttg gtaccgagcc cttcacgggc 660agtggaactc tgaacgtcgc atcctggacc gcatctatca ac 70260234PRTTrichoderma reesei 60Met Lys Phe Leu Gln Val Leu Pro Ala Leu Ile Pro Ala Ala Leu Ala 1 5 10 15 Gln Thr Ser Cys Asp Gln Trp Ala Thr Phe Thr Gly Asn Gly Tyr Thr 20 25 30 Val Ser Asn Asn Leu Trp Gly Ala Ser Ala Gly Ser Gly Phe Gly Cys 35 40 45 Val Thr Ala Val Ser Leu Ser Gly Gly Ala Ser Trp

His Ala Asp Trp 50 55 60 Gln Trp Ser Gly Gly Gln Asn Asn Val Lys Ser Tyr Gln Asn Ser Gln 65 70 75 80 Ile Ala Ile Pro Gln Lys Arg Thr Val Asn Ser Ile Ser Ser Met Pro 85 90 95 Thr Thr Ala Ser Trp Ser Tyr Ser Gly Ser Asn Ile Arg Ala Asn Val 100 105 110 Ala Tyr Asp Leu Phe Thr Ala Ala Asn Pro Asn His Val Thr Tyr Ser 115 120 125 Gly Asp Tyr Glu Leu Met Ile Trp Leu Gly Lys Tyr Gly Asp Ile Gly 130 135 140 Pro Ile Gly Ser Ser Gln Gly Thr Val Asn Val Gly Gly Gln Ser Trp 145 150 155 160 Thr Leu Tyr Tyr Gly Tyr Asn Gly Ala Met Gln Val Tyr Ser Phe Val 165 170 175 Ala Gln Thr Asn Thr Thr Asn Tyr Ser Gly Asp Val Lys Asn Phe Phe 180 185 190 Asn Tyr Leu Arg Asp Asn Lys Gly Tyr Asn Ala Ala Gly Gln Tyr Val 195 200 205 Leu Ser Tyr Gln Phe Gly Thr Glu Pro Phe Thr Gly Ser Gly Thr Leu 210 215 220 Asn Val Ala Ser Trp Thr Ala Ser Ile Asn 225 230 61726DNATrichoderma reesei 61atgaaggcaa ctctggttct cggctccctc attgtaggcg ccgtttccgc gtacaaggcc 60accaccacgc gctactacga tgggcaggag ggtgcttgcg gatgcggctc gagctccggc 120gcattcccgt ggcagctcgg catcggcaac ggagtctaca cggctgccgg ctcccaggct 180ctcttcgaca cggccggagc ttcatggtgc ggcgccggct gcggtaaatg ctaccagctc 240acctcgacgg gccaggcgcc ctgctccagc tgcggcacgg gcggtgctgc tggccagagc 300atcatcgtca tggtgaccaa cctgtgcccg aacaatggga acgcgcagtg gtgcccggtg 360gtcggcggca ccaaccaata cggctacagc taccatttcg acatcatggc gcagaacgag 420atctttggag acaatgtcgt cgtcgacttt gagcccattg cttgccccgg gcaggctgcc 480tctgactggg ggacgtgcct ctgcgtggga cagcaagaga cggatcccac gcccgtcctc 540ggcaacgaca cgggctcaac tcctcccggg agctcgccgc cagcgacatc gtcgagtccg 600ccgtctggcg gcggccagca gacgctctat ggccagtgtg gaggtgccgg ctggacggga 660cctacgacgt gccaggcccc agggacctgc aaggttcaga accagtggta ctcccagtgt 720cttcct 72662242PRTTrichoderma reesei 62Met Lys Ala Thr Leu Val Leu Gly Ser Leu Ile Val Gly Ala Val Ser 1 5 10 15 Ala Tyr Lys Ala Thr Thr Thr Arg Tyr Tyr Asp Gly Gln Glu Gly Ala 20 25 30 Cys Gly Cys Gly Ser Ser Ser Gly Ala Phe Pro Trp Gln Leu Gly Ile 35 40 45 Gly Asn Gly Val Tyr Thr Ala Ala Gly Ser Gln Ala Leu Phe Asp Thr 50 55 60 Ala Gly Ala Ser Trp Cys Gly Ala Gly Cys Gly Lys Cys Tyr Gln Leu 65 70 75 80 Thr Ser Thr Gly Gln Ala Pro Cys Ser Ser Cys Gly Thr Gly Gly Ala 85 90 95 Ala Gly Gln Ser Ile Ile Val Met Val Thr Asn Leu Cys Pro Asn Asn 100 105 110 Gly Asn Ala Gln Trp Cys Pro Val Val Gly Gly Thr Asn Gln Tyr Gly 115 120 125 Tyr Ser Tyr His Phe Asp Ile Met Ala Gln Asn Glu Ile Phe Gly Asp 130 135 140 Asn Val Val Val Asp Phe Glu Pro Ile Ala Cys Pro Gly Gln Ala Ala 145 150 155 160 Ser Asp Trp Gly Thr Cys Leu Cys Val Gly Gln Gln Glu Thr Asp Pro 165 170 175 Thr Pro Val Leu Gly Asn Asp Thr Gly Ser Thr Pro Pro Gly Ser Ser 180 185 190 Pro Pro Ala Thr Ser Ser Ser Pro Pro Ser Gly Gly Gly Gln Gln Thr 195 200 205 Leu Tyr Gly Gln Cys Gly Gly Ala Gly Trp Thr Gly Pro Thr Thr Cys 210 215 220 Gln Ala Pro Gly Thr Cys Lys Val Gln Asn Gln Trp Tyr Ser Gln Cys 225 230 235 240 Leu Pro 631446DNAThielavia terrestris 63atggctcaga agctccttct cgccgccgcc cttgcggcca gcgccctcgc tgctcccgtc 60gtcgaggagc gccagaactg cggttccgtc tggagccaat gcggcggcat tggctggtcc 120ggcgcgacct gctgcgcttc gggcaatacc tgcgttgagc tgaacccgta ctactcgcag 180tgcctgccca acagccaggt gactacctcg accagcaaga ccacctccac caccaccagg 240agcagcacca ccagccacag cagcggtccc accagcacga gcaccaccac caccagcagt 300cccgtggtca ctaccccgcc gagtacctcc atccccggcg gtgcctcgtc aacggccagc 360tggtccggca acccgttctc gggcgtgcag atgtgggcca acgactacta cgcctccgag 420gtctcgtcgc tggccatccc cagcatgacg ggcgccatgg ccaccaaggc ggccgaggtg 480gccaaggtgc ccagcttcca gtggcttgac cgcaacgtca ccatcgacac gctgttcgcc 540cacacgctgt cgcagatccg cgcggccaac cagaaaggcg ccaacccgcc ctacgcgggc 600atcttcgtgg tctacgacct tccggaccgc gactgcgccg ccgccgcgtc caacggcgag 660ttctccatcg cgaacaacgg ggcggccaac tacaagacgt acatcgacgc gatccggagc 720ctcgtcatcc agtactcaga catccgcatc atcttcgtca tcgagcccga ctcgctggcc 780aacatggtga ccaacctgaa cgtggccaag tgcgccaacg ccgagtcgac ctacaaggag 840ttgaccgtct acgcgctgca gcagctgaac ctgcccaacg tggccatgta cctggacgcc 900ggccacgccg gctggctcgg ctggcccgcc aacatccagc cggccgccaa cctcttcgcc 960gagatctaca cgagcgccgg caagccggcc gccgtgcgcg gcctcgccac caacgtggcc 1020aactacaacg gctggagcct ggccacgccg ccctcgtaca cccagggcga ccccaactac 1080gacgagagcc actacgtcca ggccctcgcc ccgctgctca ccgccaacgg cttccccgcc 1140cacttcatca ccgacaccgg ccgcaacggc aagcagccga ccggacaacg gcaatgggga 1200gactggtgca acgttatcgg aactggcttc ggcgtgcgcc cgacgacaaa caccggcctc 1260gacatcgagg acgccttcgt ctgggtcaag cccggcggcg agtgcgacgg cacgagcaac 1320acgacctctc cccgctacga ctaccactgc ggcctgtcgg acgcgctgca gcctgctccg 1380gaggccggca cttggttcca ggcctacttc gagcagctcc tgaccaacgc caacccgccc 1440ttttaa 144664481PRTThielavia terrestris 64Met Ala Gln Lys Leu Leu Leu Ala Ala Ala Leu Ala Ala Ser Ala Leu 1 5 10 15 Ala Ala Pro Val Val Glu Glu Arg Gln Asn Cys Gly Ser Val Trp Ser 20 25 30 Gln Cys Gly Gly Ile Gly Trp Ser Gly Ala Thr Cys Cys Ala Ser Gly 35 40 45 Asn Thr Cys Val Glu Leu Asn Pro Tyr Tyr Ser Gln Cys Leu Pro Asn 50 55 60 Ser Gln Val Thr Thr Ser Thr Ser Lys Thr Thr Ser Thr Thr Thr Arg 65 70 75 80 Ser Ser Thr Thr Ser His Ser Ser Gly Pro Thr Ser Thr Ser Thr Thr 85 90 95 Thr Thr Ser Ser Pro Val Val Thr Thr Pro Pro Ser Thr Ser Ile Pro 100 105 110 Gly Gly Ala Ser Ser Thr Ala Ser Trp Ser Gly Asn Pro Phe Ser Gly 115 120 125 Val Gln Met Trp Ala Asn Asp Tyr Tyr Ala Ser Glu Val Ser Ser Leu 130 135 140 Ala Ile Pro Ser Met Thr Gly Ala Met Ala Thr Lys Ala Ala Glu Val 145 150 155 160 Ala Lys Val Pro Ser Phe Gln Trp Leu Asp Arg Asn Val Thr Ile Asp 165 170 175 Thr Leu Phe Ala His Thr Leu Ser Gln Ile Arg Ala Ala Asn Gln Lys 180 185 190 Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe Val Val Tyr Asp Leu Pro 195 200 205 Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Phe Ser Ile Ala 210 215 220 Asn Asn Gly Ala Ala Asn Tyr Lys Thr Tyr Ile Asp Ala Ile Arg Ser 225 230 235 240 Leu Val Ile Gln Tyr Ser Asp Ile Arg Ile Ile Phe Val Ile Glu Pro 245 250 255 Asp Ser Leu Ala Asn Met Val Thr Asn Leu Asn Val Ala Lys Cys Ala 260 265 270 Asn Ala Glu Ser Thr Tyr Lys Glu Leu Thr Val Tyr Ala Leu Gln Gln 275 280 285 Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly 290 295 300 Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala Asn Leu Phe Ala 305 310 315 320 Glu Ile Tyr Thr Ser Ala Gly Lys Pro Ala Ala Val Arg Gly Leu Ala 325 330 335 Thr Asn Val Ala Asn Tyr Asn Gly Trp Ser Leu Ala Thr Pro Pro Ser 340 345 350 Tyr Thr Gln Gly Asp Pro Asn Tyr Asp Glu Ser His Tyr Val Gln Ala 355 360 365 Leu Ala Pro Leu Leu Thr Ala Asn Gly Phe Pro Ala His Phe Ile Thr 370 375 380 Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln Arg Gln Trp Gly 385 390 395 400 Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly Val Arg Pro Thr Thr 405 410 415 Asn Thr Gly Leu Asp Ile Glu Asp Ala Phe Val Trp Val Lys Pro Gly 420 425 430 Gly Glu Cys Asp Gly Thr Ser Asn Thr Thr Ser Pro Arg Tyr Asp Tyr 435 440 445 His Cys Gly Leu Ser Asp Ala Leu Gln Pro Ala Pro Glu Ala Gly Thr 450 455 460 Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala Asn Pro Pro 465 470 475 480 Phe 6529DNATrichoderma reesei 65aacgttaatt aaggaatcgt tttgtgttt 296629DNATrichoderma reesei 66agtactagta gctccgtggc gaaagcctg 296731DNASaccharomyces cerevisiae 67ttgaattgaa aatagattga tttaaaactt c 316825DNASaccharomyces cerevisiae 68ttgcatgcgt aatcatggtc atagc 256926DNASaccharomyces cerevisiae 69ttgaattcat gggtaataac tgatat 267032DNASaccharomyces cerevisiae 70aaatcaatct attttcaatt caattcatca tt 327111DNAAspergillus niger 71gtactaaaac c 117211DNAAspergillus niger 72ccgttaaatt t 117345DNAAspergillus niger 73ggatgctgtt gactccggaa atttaacggt ttggtcttgc atccc 457414DNAAspergillus niger 74atgcaattta aact 147514DNAAspergillus niger 75cggcaattta acgg 147644DNAAspergillus niger 76ggtattgtcc tgcagacggc aatttaacgg cttctgcgaa tcgc 447729DNAHumicola insolens 77aagcttaagc atgcgttcct cccccctcc 297832DNAHumicola insolens 78ctgcagaatt ctacaggcac tgatggtacc ag 327932DNATrichoderma reesei 79ctgcagaatt ctacaggcac tgatggtacc ag 328036DNATrichoderma reesei 80accgcggact gcgcatcatg cgttcctccc ccctcc 368129DNATrichoderma reesei 81aaacgtcgac cgaatgtagg attgttatc 298217DNATrichoderma reesei 82gatgcgcagt ccgcggt 178329DNATrichoderma reesei 83aaacgtcgac cgaatgtagg attgttatc 298436DNATrichoderma reesei 84ggagggggga ggaacgcatg atgcgcagtc cgcggt 368529DNATrichoderma reesei 85aaacgtcgac cgaatgtagg attgttatc 298632DNATrichoderma reesei 86ctgcagaatt ctacaggcac tgatggtacc ag 328746DNAAspergillus oryzae 87atagtcaacc gcggactgcg catcatgaag cttggttgga tcgagg 468826DNAAspergillus oryzae 88actagtttac tgggccttag gcagcg 268926DNATrichoderma reesei 89gtcgactcga agcccgaatg taggat 269045DNATrichoderma reesei 90cctcgatcca accaagcttc atgatgcgca gtccgcggtt gacta 459157DNAAspergillus oryzae 91atgaagcttg gttggatcga ggtggccgca ttggcggctg cctcagtagt cagtgcc 579219PRTAspergillus oryzae 92Met Lys Leu Gly Trp Ile Glu Val Ala Ala Leu Ala Ala Ala Ser Val 1 5 10 15 Val Ser Ala 9342DNAAspergillus oryzae 93tgccggtgtt ggcccttgcc aaggatgatc tcgcgtactc cc 429428DNAAspergillus oryzae 94gactagtctt actgggcctt aggcagcg 289563DNAHumicola insolens 95atgcgttcct cccccctcct ccgctccgcc gttgtggccg ccctgccggt gttggccctt 60gcc 639621PRTHumicola insolens 96Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro 1 5 10 15 Val Leu Ala Leu Ala 20 9730DNATrichoderma reesei 97acgcgtcgac cgaatgtagg attgttatcc 309842DNATrichoderma reesei 98gggagtacgc gagatcatcc ttggcaaggg ccaacaccgg ca 429920DNATrichoderma reesei 99cccaagctta gccaagaaca 2010029DNATrichoderma reesei 100gggggaggaa cgcatgggat ctggacggc 2910130DNAAspergillus oryzae 101gccgtccaga tccccatgcg ttcctccccc 3010220DNAAspergillus oryzae 102ccaagcttgt tcagagtttc 201033294DNAAspergillus oryzae 103atgcgttcct cccccctcct ccgctccgcc gttgtggccg ccctgccggt gttggccctt 60gccgctgatg gcaggtccac ccgctactgg gactgctgca agccttcgtg cggctgggcc 120aagaaggctc ccgtgaacca gcctgtcttt tcctgcaacg ccaacttcca gcgtatcacg 180gacttcgacg ccaagtccgg ctgcgagccg ggcggtgtcg cctactcgtg cgccgaccag 240accccatggg ctgtgaacga cgacttcgcg ctcggttttg ctgccacctc tattgccggc 300agcaatgagg cgggctggtg ctgcgcctgc tacgagctca ccttcacatc cggtcctgtt 360gctggcaaga agatggtcgt ccagtccacc agcactggcg gtgatcttgg cagcaaccac 420ttcgatctca acatccccgg cggcggcgtc ggcatcttcg acggatgcac tccccagttc 480ggtggtctgc ccggccagcg ctacggcggc atctcgtccc gcaacgagtg cgatcggttc 540cccgacgccc tcaagcccgg ctgctactgg cgcttcgact ggttcaagaa cgccgacaat 600ccgagcttca gcttccgtca ggtccagtgc ccagccgagc tcgtcgctcg caccggatgc 660cgccgcaacg acgacggcaa cttccctgcc gtccagatcc ccatgcgttc ctcccccctc 720ctccgctccg ccgttgtggc cgccctgccg gtgttggccc ttgccaagga tgatctcgcg 780tactcccctc ctttctaccc ttccccatgg gcagatggtc agggtgaatg ggcggaagta 840tacaaacgcg ctgtagacat agtttcccag atgacgttga cagagaaagt caacttaacg 900actggaacag gatggcaact agagaggtgt gttggacaaa ctggcagtgt tcccagactc 960aacatcccca gcttgtgttt gcaggatagt cctcttggta ttcgtttctc ggactacaat 1020tcagctttcc ctgcgggtgt taatgtcgct gccacctggg acaagacgct cgcctacctt 1080cgtggtcagg caatgggtga ggagttcagt gataagggta ttgacgttca gctgggtcct 1140gctgctggcc ctctcggtgc tcatccggat ggcggtagaa actgggaaag tttctcacca 1200gatccagccc tcaccggtgt actttttgcg gagacgatta agggtattca agatgctggt 1260gtcattgcga cagctaagca ttatatcatg aacgaacaag agcatttccg ccaacaaccc 1320gaggctgcgg gttacggatt caacgtaagc gacagtttga gttccaacgt tgatgacaag 1380actatgcatg aattgtacct ctggcccttc gcggatgcag tacgcgctgg agtcggtgct 1440gttatgtgct cttacaacca aatcaacaac agctacggtt gcgagaatag cgaaactctg 1500aacaagcttt tgaaggcgga gcttggtttc caaggcttcg tcatgagtga ttggaccgct 1560caacacagcg gcgtaggcgc tgctttagca ggtctggata tgtcgatgcc cggtgatgtt 1620accttcgata gtggtacgtc tttctggggt gcaaacttga cggtcggtgt ccttaacggt 1680acaatccccc aatggcgtgt tgatgacatg gctgtccgta tcatggccgc ttattacaag 1740gttggccgcg acaccaaata cacccctccc aacttcagct cgtggaccag ggacgaatat 1800ggtttcgcgc ataaccatgt ttcggaaggt gcttacgaga gggtcaacga attcgtggac 1860gtgcaacgcg atcatgccga cctaatccgt cgcatcggcg cgcagagcac tgttctgctg 1920aagaacaagg gtgccttgcc cttgagccgc aaggaaaagc tggtcgccct tctgggagag 1980gatgcgggtt ccaactcgtg gggcgctaac ggctgtgatg accgtggttg cgataacggt 2040acccttgcca tggcctgggg tagcggtact gcgaatttcc catacctcgt gacaccagag 2100caggcgattc agaacgaagt tcttcagggc cgtggtaatg tcttcgccgt gaccgacagt 2160tgggcgctcg acaagatcgc tgcggctgcc cgccaggcca gcgtatctct cgtgttcgtc 2220aactccgact caggagaagg ctatcttagt gtggatggaa atgagggcga tcgtaacaac 2280atcactctgt ggaagaacgg cgacaatgtg gtcaagaccg cagcgaataa ctgtaacaac 2340accgttgtca tcatccactc cgtcggacca gttttgatcg atgaatggta tgaccacccc 2400aatgtcactg gtattctctg ggctggtctg ccaggccagg agtctggtaa ctccattgcc 2460gatgtgctgt acggtcgtgt caaccctggc gccaagtctc ctttcacttg gggcaagacc 2520cgggagtcgt atggttctcc cttggtcaag gatgccaaca atggcaacgg agcgccccag 2580tctgatttca cccagggtgt tttcatcgat taccgccatt tcgataagtt caatgagacc 2640cctatctacg agtttggcta cggcttgagc tacaccacct tcgagctctc cgacctccat 2700gttcagcccc tgaacgcgtc ccgatacact cccaccagtg gcatgactga agctgcaaag 2760aactttggtg aaattggcga tgcgtcggag tacgtgtatc cggaggggct ggaaaggatc 2820catgagttta tctatccctg gatcaactct accgacctga aggcatcgtc tgacgattct 2880aactacggct gggaagactc caagtatatt cccgaaggcg ccacggatgg gtctgcccag 2940ccccgtttgc ccgctagtgg tggtgccgga ggaaaccccg gtctgtacga ggatcttttc 3000cgcgtctctg tgaaggtcaa gaacacgggc aatgtcgccg gtgatgaagt tcctcagctg 3060tacgtttccc taggcggccc gaatgagccc aaggtggtac tgcgcaagtt tgagcgtatt 3120cacttggccc cttcgcagga ggccgtgtgg acaacgaccc ttacccgtcg tgaccttgca 3180aactgggacg tttcggctca ggactggacc

gtcactcctt accccaagac gatctacgtt 3240ggaaactcct cacggaaact gccgctccag gcctcgctgc ctaaggccca gtaa 32941041097PRTAspergillus oryzae 104Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro 1 5 10 15 Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20 25 30 Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro 35 40 45 Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50 55 60 Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln 65 70 75 80 Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr 85 90 95 Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 100 105 110 Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln 115 120 125 Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130 135 140 Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe 145 150 155 160 Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu 165 170 175 Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180 185 190 Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val 195 200 205 Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp 210 215 220 Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Met Arg Ser Ser Pro Leu 225 230 235 240 Leu Arg Ser Ala Val Val Ala Ala Leu Pro Val Leu Ala Leu Ala Lys 245 250 255 Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp 260 265 270 Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala Val Asp Ile Val 275 280 285 Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 290 295 300 Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser Val Pro Arg Leu 305 310 315 320 Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu Gly Ile Arg Phe 325 330 335 Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val Ala Ala Thr 340 345 350 Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala Met Gly Glu Glu 355 360 365 Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala Ala Gly Pro 370 375 380 Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu Ser Phe Ser Pro 385 390 395 400 Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile 405 410 415 Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr Ile Met Asn Glu 420 425 430 Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly Tyr Gly Phe Asn 435 440 445 Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys Thr Met His Glu 450 455 460 Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ala 465 470 475 480 Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Glu Asn 485 490 495 Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly 500 505 510 Phe Val Met Ser Asp Trp Thr Ala Gln His Ser Gly Val Gly Ala Ala 515 520 525 Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Thr Phe Asp Ser 530 535 540 Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly Val Leu Asn Gly 545 550 555 560 Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ala 565 570 575 Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr Pro Pro Asn Phe 580 585 590 Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His Asn His Val Ser 595 600 605 Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp Val Gln Arg Asp 610 615 620 His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser Thr Val Leu Leu 625 630 635 640 Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu Lys Leu Val Ala 645 650 655 Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly Ala Asn Gly Cys 660 665 670 Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala Trp Gly Ser 675 680 685 Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln 690 695 700 Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala Val Thr Asp Ser 705 710 715 720 Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln Ala Ser Val Ser 725 730 735 Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr Leu Ser Val Asp 740 745 750 Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp Lys Asn Gly Asp 755 760 765 Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn Thr Val Val Ile 770 775 780 Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp Tyr Asp His Pro 785 790 795 800 Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly 805 810 815 Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala Lys 820 825 830 Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr Gly Ser Pro Leu 835 840 845 Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp Phe Thr 850 855 860 Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys Phe Asn Glu Thr 865 870 875 880 Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Leu 885 890 895 Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg Tyr Thr Pro Thr 900 905 910 Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu Ile Gly Asp Ala 915 920 925 Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile His Glu Phe Ile 930 935 940 Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser Ser Asp Asp Ser 945 950 955 960 Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu Gly Ala Thr Asp 965 970 975 Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly Ala Gly Gly Asn 980 985 990 Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val Lys Val Lys Asn 995 1000 1005 Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu Tyr Val Ser 1010 1015 1020 Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe Glu 1025 1030 1035 Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr Thr 1040 1045 1050 Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 1055 1060 1065 Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser 1070 1075 1080 Ser Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 1085 1090 1095 1053294DNAAspergillus oryzae 105atgcgttcct cccccctcct ccgctccgcc gttgtggccg ccctgccggt gttggccctt 60gccgctgatg gcaggtccac ccgctactgg gactgctgca agccttcgtg cggctgggcc 120aagaaggctc ccgtgaacca gcctgtcttt tcctgcaacg ccaacttcca gcgtatcacg 180gacttcgacg ccaagtccgg ctgcgagccg ggcggtgtcg cctactcgtg cgccgaccag 240accccatggg ctgtgaacga cgacttcgcg ctcggttttg ctgccacctc tattgccggc 300agcaatgagg cgggctggtg ctgcgcctgc tacgagctca ccttcacatc cggtcctgtt 360gctggcaaga agatggtcgt ccagtccacc agcactggcg gtgatcttgg cagcaaccac 420ttcgatctca acatccccgg cggcggcgtc ggcatcttcg acggatgcac tccccagttc 480ggtggtctgc ccggccagcg ctacggcggc atctcgtccc gcaacgagtg cgatcggttc 540cccgacgccc tcaagcccgg ctgctactgg cgcttcgact ggttcaagaa cgccgacaat 600ccgagcttca gcttccgtca ggtccagtgc ccagccgagc tcgtcgctcg caccggatgc 660cgccgcaacg acgacggcaa cttccctgcc gtccagatcc ccatgcgttc ctcccccctc 720ctccgctccg ccgttgtggc cgccctgccg gtgttggccc ttgccaagga tgatctcgcg 780tactcccctc ctttctaccc ttccccatgg gcagatggtc agggtgaatg ggcggaagta 840tacaaacgcg ctgtagacat agtttcccag atgacgttga cagagaaagt caacttaacg 900actggaacag gatggcaact agagaggtgt gttggacaaa ctggcagtgt tcccagactc 960aacatcccca gcttgtgttt gcaggatagt cctcttggta ttcgtttctc ggactacaat 1020tcagctttcc ctgcgggtgt taatgtcgct gccacctggg acaagacgct cgcctacctt 1080cgtggtcagg caatgggtga ggagttcagt gataagggta ttgacgttca gctgggtcct 1140gctgctggcc ctctcggtgc tcatccggat ggcggtagaa actgggaagg tttctcacca 1200gatccagccc tcaccggtgt actttttgcg gagacgatta agggtattca agatgctggt 1260gtcattgcga cagctaagca ttatatcatg aacgaacaag agcatttccg ccaacaaccc 1320gaggctgcgg gttacggatt caacgtaagc gacagtttga gttccaacgt tgatgacaag 1380actatgcatg aattgtacct ctggcccttc gcggatgcag tacgcgctgg agtcggtgct 1440gtcatgtgct cttacaacca aatcaacaac agctacggtt gcgagaatag cgaaactctg 1500aacaagcttt tgaaggcgga gcttggtttc caaggcttcg tcatgagtga ttggaccgct 1560catcacagcg gcgtaggcgc tgctttagca ggtctggata tgtcgatgcc cggtgatgtt 1620accttcgata gtggtacgtc tttctggggt gcaaacttga cggtcggtgt ccttaacggt 1680acaatccccc aatggcgtgt tgatgacatg gctgtccgta tcatggccgc ttattacaag 1740gttggccgcg acaccaaata cacccctccc aacttcagct cgtggaccag ggacgaatat 1800ggtttcgcgc ataaccatgt ttcggaaggt gcttacgaga gggtcaacga attcgtggac 1860gtgcaacgcg atcatgccga cctaatccgt cgcatcggcg cgcagagcac tgttctgctg 1920aagaacaagg gtgccttgcc cttgagccgc aaggaaaagc tggtcgccct tctgggagag 1980gatgcgggtt ccaactcgtg gggcgctaac ggctgtgatg accgtggttg cgataacggt 2040acccttgcca tggcctgggg tagcggtact gcgaatttcc catacctcgt gacaccagag 2100caggcgattc agaacgaagt tcttcagggc cgtggtaatg tcttcgccgt gaccgacagt 2160tgggcgctcg acaagatcgc tgcggctgcc cgccaggcca gcgtatctct cgtgttcgtc 2220aactccgact caggagaagg ctatcttagt gtggatggaa atgagggcga tcgtaacaac 2280atcactctgt ggaagaacgg cgacaatgtg gtcaagaccg cagcgaataa ctgtaacaac 2340accgttgtca tcatccactc cgtcggacca gttttgatcg atgaatggta tgaccacccc 2400aatgtcactg gtattctctg ggctggtctg ccaggccagg agtctggtaa ctccattgcc 2460gatgtgctgt acggtcgtgt caaccctggc gccaagtctc ctttcacttg gggcaagacc 2520cgggagtcgt atggttctcc cttggtcaag gatgccaaca atggcaacgg agcgccccag 2580tctgatttca cccagggtgt tttcatcgat taccgccatt tcgataagtt caatgagacc 2640cctatctacg agtttggcta cggcttgagc tacaccacct tcgagctctc cgacctccat 2700gttcagcccc tgaacgcgtc ccgatacact cccaccagtg gcatgactga agctgcaaag 2760aactttggtg aaattggcga tgcgtcggag tacgtgtatc cggaggggct ggaaaggatc 2820catgagttta tctatccctg gatcaactct accgacctga aggcatcgtc tgacgattct 2880aactacggct gggaagactc caagtatatt cccgaaggcg ccacggatgg gtctgcccag 2940ccccgtttgc ccgctagtgg tggtgccgga ggaaaccccg gtctgtacga ggatcttttc 3000cgcgtctctg tgaaggtcaa gaacacgggc aatgtcgccg gtgatgaagt tcctcagctg 3060tacgtttccc taggcggccc gaatgagccc aaggtggtac tgcgcaagtt tgagcgtatt 3120cacttggccc cttcgcagga ggccgtgtgg acaacgaccc ttacccgtcg tgaccttgca 3180aactgggacg tttcggctca ggactggacc gtcactcctt accccaagac gatctacgtt 3240ggaaactcct cacggaaact gccgctccag gcctcgctgc ctaaggccca gtaa 32941061097PRTAspergillus oryzae 106Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro 1 5 10 15 Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20 25 30 Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro 35 40 45 Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50 55 60 Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln 65 70 75 80 Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr 85 90 95 Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 100 105 110 Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln 115 120 125 Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130 135 140 Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe 145 150 155 160 Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu 165 170 175 Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180 185 190 Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val 195 200 205 Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp 210 215 220 Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Met Arg Ser Ser Pro Leu 225 230 235 240 Leu Arg Ser Ala Val Val Ala Ala Leu Pro Val Leu Ala Leu Ala Lys 245 250 255 Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp 260 265 270 Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala Val Asp Ile Val 275 280 285 Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 290 295 300 Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser Val Pro Arg Leu 305 310 315 320 Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu Gly Ile Arg Phe 325 330 335 Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val Ala Ala Thr 340 345 350 Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala Met Gly Glu Glu 355 360 365 Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala Ala Gly Pro 370 375 380 Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro 385 390 395 400 Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile 405 410 415 Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr Ile Met Asn Glu 420 425 430 Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly Tyr Gly Phe Asn 435 440 445 Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys Thr Met His Glu 450 455 460 Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ala 465 470 475 480 Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Glu Asn 485 490 495 Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly 500 505 510 Phe Val Met Ser Asp Trp Thr Ala His His Ser Gly Val Gly Ala Ala 515 520 525 Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Thr Phe Asp Ser 530 535 540 Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly Val Leu Asn Gly 545 550 555 560 Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ala 565 570 575 Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr Pro Pro Asn Phe 580 585 590 Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His Asn His Val Ser 595 600 605 Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp Val Gln Arg Asp 610 615 620 His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser Thr Val Leu Leu 625 630 635 640 Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu Lys Leu Val Ala 645 650 655 Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly Ala Asn Gly Cys 660 665 670

Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala Trp Gly Ser 675 680 685 Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln 690 695 700 Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala Val Thr Asp Ser 705 710 715 720 Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln Ala Ser Val Ser 725 730 735 Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr Leu Ser Val Asp 740 745 750 Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp Lys Asn Gly Asp 755 760 765 Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn Thr Val Val Ile 770 775 780 Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp Tyr Asp His Pro 785 790 795 800 Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly 805 810 815 Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala Lys 820 825 830 Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr Gly Ser Pro Leu 835 840 845 Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp Phe Thr 850 855 860 Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys Phe Asn Glu Thr 865 870 875 880 Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Leu 885 890 895 Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg Tyr Thr Pro Thr 900 905 910 Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu Ile Gly Asp Ala 915 920 925 Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile His Glu Phe Ile 930 935 940 Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser Ser Asp Asp Ser 945 950 955 960 Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu Gly Ala Thr Asp 965 970 975 Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly Ala Gly Gly Asn 980 985 990 Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val Lys Val Lys Asn 995 1000 1005 Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu Tyr Val Ser 1010 1015 1020 Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe Glu 1025 1030 1035 Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr Thr 1040 1045 1050 Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 1055 1060 1065 Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser 1070 1075 1080 Ser Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 1085 1090 1095 10720DNAAspergillus oryzae 107ggactgcgca gcatgcgttc 2010830DNAAspergillus oryzae 108agttaattaa ttactgggcc ttaggcagcg 3010928DNAThermoascus aurantiacus 109atgtcctttt ccaagataat tgctactg 2811026DNAThermoascus aurantiacus 110gcttaattaa ccagtataca gaggag 2611119PRTThielavia terrestrisMISC_FEATURE(1)..(1)X=I,L,M, OR VMISC_FEATURE(3)..(6)X=ANY AMINO ACIDMISC_FEATURE(8)..(8)X=ANY AMINO ACIDMISC_FEATURE(10)..(10)X=I, L, M, OR VMISC_FEATURE(11)..(11)X=ANY AMINO ACIDMISC_FEATURE(13)..(13)X=ANY AMINO ACIDMISC_FEATURE(14)..(14)X=E OR QMISC_FEATURE(15)..(18)X=ANY AMINO ACIDMISC_FEATURE(19)..(19)X=H, N, OR Q 111Xaa Pro Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa 11220PRTThielavia terrestrisMISC_FEATURE(1)..(1)X=I, L, M, OR VMISC_FEATURE(3)..(7)X=ANY AMINO ACIDMISC_FEATURE(9)..(9)X=ANY AMINO ACIDMISC_FEATURE(11)..(11)X=I, L, M, OR VMISC_FEATURE(12)..(12)X=ANY AMINO ACIDMISC_FEATURE(14)..(14)X=ANY AMINO ACIDMISC_FEATURE(15)..(15)X=E OR QMISC_FEATURE(16)..(19)X=ANY AMINO ACIDMISC_FEATURE(20)..(20)X=H, N, OR Q 112Xaa Pro Xaa Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa 20 1139PRTThielavia terrestrisMISC_FEATURE(2)..(2)X=ANY AMINO ACIDMISC_FEATURE(5)..(7)X=ANY AMINO ACIDMISC_FEATURE(8)..(8)X=Y OR WMISC_FEATURE(9)..(9)X=A, I, L, M, OR V 113His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5 11410PRTThielavia terrestrisMISC_FEATURE(2)..(3)X=ANY AMINO ACIDMISC_FEATURE(6)..(8)X=ANY AMINO ACIDMISC_FEATURE(9)..(9)X=Y OR WMISC_FEATURE(10)..(10)X=A, I, L, M, OR V 114His Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5 10 11511PRTThielavia terrestrisMISC_FEATURE(1)..(1)X=E OR QMISC_FEATURE(2)..(2)X=ANY AMINO ACIDMISC_FEATURE(4)..(5)X=ANY AMINO ACIDMISC_FEATURE(7)..(7)X=ANY AMINO ACIDMISC_FEATURE(8)..(8)X=E, H, Q, OR NMISC_FEATURE(9)..(9)X=F, I, L, OR VMISC_FEATURE(10)..(10)X=ANY AMINO ACIDMISC_FEATURE(11)..(11)X=I, L, OR V 115Xaa Xaa Tyr Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 1169PRTThielavia terrestrisMISC_FEATURE(2)..(2)X=ANY AMINO ACIDMISC_FEATURE(5)..(7)X=ANY AMINO ACIDMISC_FEATURE(8)..(8)X=Y OR WMISC_FEATURE(9)..(9)X=A, I, L, M, OR V 116His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5 11710PRTThielavia terrestrisMISC_FEATURE(2)..(3)X=ANY AMINO ACIDMISC_FEATURE(6)..(8)X=ANY AMINO ACIDMISC_FEATURE(9)..(9)X=Y OR WMISC_FEATURE(10)..(10)X=A, I, L, M, OR V 117His Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5 10 11811PRTThielavia terrestrisMISC_FEATURE(1)..(1)X=E OR QMISC_FEATURE(2)..(2)X=ANY AMINO ACIDMISC_FEATURE(4)..(5)X=ANY AMINO ACIDMISC_FEATURE(7)..(7)X=ANY AMINO ACIDMISC_FEATURE(8)..(8)X=E, H,Q, OR NMISC_FEATURE(9)..(9)X=F, I, L, OR VMISC_FEATURE(10)..(10)X=ANY AMINO ACIDMISC_FEATURE(11)..(11)X=I, L, OR V 118Xaa Xaa Tyr Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 1199PRTThielavia terrestrisMISC_FEATURE(2)..(2)X=ANY AMINO ACIDMISC_FEATURE(5)..(7)X=ANY AMINO ACIDMISC_FEATURE(8)..(8)X=Y OR WMISC_FEATURE(9)..(9)X=A, I, L, M, OR V 119His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5 12010PRTThielavia terrestrisMISC_FEATURE(2)..(3)X=ANY AMINO ACIDMISC_FEATURE(6)..(8)X=ANY AMINO ACIDMISC_FEATURE(9)..(9)X=Y OR WMISC_FEATURE(10)..(10)X=A, I, L, M, OR V 120His Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5 10 12111PRTThielavia terrestrisMISC_FEATURE(1)..(1)X=E OR QMISC_FEATURE(2)..(2)X=ANY AMINO ACIDMISC_FEATURE(4)..(5)X=ANY AMINO ACIDMISC_FEATURE(7)..(7)X=ANY AMINO ACIDMISC_FEATURE(8)..(8)X=E, H, Q, OR NMISC_FEATURE(9)..(9)X=F, I, L, OR VMISC_FEATURE(10)..(10)X=ANY AMINO ACIDMISC_FEATURE(11)..(11)X=I, L, OR V 121Xaa Xaa Tyr Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 1229PRTThielavia terrestrisMISC_FEATURE(2)..(2)X=ANY AMINO ACIDMISC_FEATURE(5)..(7)X=ANY AMINO ACIDMISC_FEATURE(8)..(8)X=Y OR WMISC_FEATURE(9)..(9)X=A, I, L, M, OR V 122His Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5 12310PRTThielavia terrestrisMISC_FEATURE(2)..(3)X=ANY AMINO ACIDMISC_FEATURE(6)..(8)X=ANY AMINO ACIDMISC_FEATURE(9)..(9)X=Y OR WMISC_FEATURE(10)..(10)X=A, I, L, M, OR V 123His Xaa Xaa Gly Pro Xaa Xaa Xaa Xaa Xaa 1 5 10 12411PRTThielavia terrestrisMISC_FEATURE(1)..(1)X=E OR QMISC_FEATURE(2)..(2)X=ANY AMINO ACIDMISC_FEATURE(4)..(5)X=ANY AMINO ACIDMISC_FEATURE(7)..(7)X=ANY AMINO ACIDMISC_FEATURE(8)..(8)X=E, H, Q, OR NMISC_FEATURE(9)..(9)X=F, I, L, OR VMISC_FEATURE(10)..(10)X=ANY AMINO ACIDMISC_FEATURE(11)..(11)X=I, L, OR V 124Xaa Xaa Tyr Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa 1 5 10 12519PRTThielavia terrestrisMISC_FEATURE(1)..(1)X=I, L, M, OR VMISC_FEATURE(3)..(6)X=ANY AMINO ACIDMISC_FEATURE(8)..(8)X=ANY AMINO ACIDMISC_FEATURE(10)..(10)X=I, L, M, OR VMISC_FEATURE(11)..(11)X=ANY AMINO ACIDMISC_FEATURE(13)..(13)X=ANY AMINO ACIDMISC_FEATURE(14)..(14)X=E OR QMISC_FEATURE(15)..(17)X=ANY AMINO ACIDMISC_FEATURE(19)..(19)X=H, N, OR Q 125Xaa Pro Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Ala Xaa 12620PRTThielavia terrestrisMISC_FEATURE(1)..(1)X=I, L, M, OR VMISC_FEATURE(3)..(7)X=ANY AMINO ACIDMISC_FEATURE(9)..(9)X=ANY AMINO ACIDMISC_FEATURE(11)..(11)X=I, L, M, OR VMISC_FEATURE(12)..(12)X=ANY AMINO ACIDMISC_FEATURE(14)..(14)X=ANY AMINO ACIDMISC_FEATURE(15)..(15)X=E OR QMISC_FEATURE(16)..(18)X=ANY AMINO ACIDMISC_FEATURE(20)..(20)X=H, N, OR Q 126Xaa Pro Xaa Xaa Xaa Xaa Xaa Gly Xaa Tyr Xaa Xaa Arg Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Ala Xaa 20 1275PRTArtificial SequenceARTIFICIAL PRIMERMISC_FEATURE(2)..(2)X = E OR D 127Ile Xaa Asp Gly Arg 1 5 1285PRTArtificial SequenceARTIFICIAL PRIMER 128Asp Asp Asp Asp Lys 1 5 1296PRTArtificial SequenceARTIFICIAL PRIMER 129Leu Val Pro Arg Gly Ser 1 5 1307PRTArtificial SequenceARTIFICIAL PRIMER 130Glu Asn Leu Tyr Phe Gln Gly 1 5 1318PRTArtificial SequenceARTIFICIAL PRIMER 131Leu Glu Val Leu Phe Gln Gly Pro 1 5

Claims

1-170. (canceled)

171. A cellulolytic protein composition, comprising: (a) a GH61 polypeptide having cellulolytic enhancing activity; (b) a beta-glucosidase; and (c) cellulolytic enzymes selected from the group consisting of a cellobiohydrolase I, a cellobiohydrolase II, and an endoglucanase I; wherein the GH61 polypeptide having cellulolytic enhancing activity is selected from the group consisting of: (a) a GH61 polypeptide comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; (b) a GH61 polypeptide encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C. for high stringency and 70.degree. C. for very high stringency; (c) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13; and (d) a GH61 polypeptide comprising the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; wherein the beta-glucosidase is selected from the group consisting of: (a) a beta-glucosidase comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105; (b) a beta-glucosidase encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 104 or SEQ ID NO: 106, or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C. for high stringency and 70.degree. C. for very high stringency; (c) a beta-glucosidase encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 104 or SEQ ID NO: 106; and (d) a beta-glucosidase comprising the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105; wherein the cellobiohydrolase I is selected from the group consisting of: (a) a cellobiohydrolase I comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 52; (b) a cellobiohydrolase I encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 51 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C. for high stringency and 70.degree. C. for very high stringency; (c) a cellobiohydrolase I encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 51; and (d) a cellobiohydrolase I comprising the mature polypeptide of SEQ ID NO: 52; wherein the cellobiohydrolase II is selected from the group consisting of: (a) a cellobiohydrolase II comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 54; (b) a cellobiohydrolase II encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 53 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C. for high stringency and 70.degree. C. for very high stringency; (c) a cellobiohydrolase II encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 53; and (d) a cellobiohydrolase II comprising the mature polypeptide of SEQ ID NO: 54; and wherein the endoglucanase I is selected from the group consisting of: (a) an endoglucanase I comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 56; (b) an endoglucanase I encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 55 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C. for high stringency and 70.degree. C. for very high stringency; (c) an endoglucanase I encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 55; and (d) an endoglucanase I comprising the mature polypeptide of SEQ ID NO: 56.

172. The cellulolytic protein composition of claim 171, further comprising cellulolytic enzymes selected from the group consisting of an endoglucanase II, an endoglucanase III, and an endoglucanase V; wherein the endoglucanase II is selected from the group consisting of: (a) an endoglucanase II comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 58; (b) an endoglucanase II encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 57 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C. for high stringency and 70.degree. C. for very high stringency; (c) an endoglucanase II encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 57; and (d) an endoglucanase II comprising the mature polypeptide of SEQ ID NO: 58; wherein the endoglucanase III is selected from the group consisting of: (a) an endoglucanase III comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 60; (b) an endoglucanase III encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 59 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C. for high stringency and 70.degree. C. for very high stringency; (c) an endoglucanase III encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 59; and (d) an endoglucanase III comprising the mature polypeptide of SEQ ID NO: 60; and wherein the endoglucanase V is selected from the group consisting of: (a) an endoglucanase V comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 62; (b) an endoglucanase V encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 61 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 .mu.g/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C. for high stringency and 70.degree. C. for very high stringency; (c) an endoglucanase V encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 61; and (d) an endoglucanase V comprising the mature polypeptide of SEQ ID NO: 62.

173. The cellulolytic protein composition of claim 171, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

174. The cellulolytic protein composition of claim 171, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14.

175. The cellulolytic protein composition of claim 171, wherein the beta-glucosidase comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105.

176. The cellulolytic protein composition of claim 171, wherein the beta-glucosidase comprises the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105.

177. The cellulolytic protein composition of claim 171, wherein the cellobiohydrolase I comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 52.

178. The cellulolytic protein composition of claim 171, wherein the cellobiohydrolase I comprises the mature polypeptide of SEQ ID NO: 52.

179. The cellulolytic protein composition of claim 171, wherein the cellobiohydrolase II comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 54.

180. The cellulolytic protein composition of claim 171, wherein the cellobiohydrolase II comprises the mature polypeptide of SEQ ID NO: 54.

181. The cellulolytic protein composition of claim 171, wherein the endoglucanase I comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 56.

182. The cellulolytic protein composition of claim 171, wherein the endoglucanase I comprises the mature polypeptide of SEQ ID NO: 56.

183. The cellulolytic protein composition of claim 171, wherein the GH61 polypeptide having cellulolytic enhancing activity comprises the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; the beta-glucosidase comprises the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105; the cellobiohydrolase I comprises the mature polypeptide of SEQ ID NO: 52; the cellobiohydrolase II comprises the mature polypeptide of SEQ ID NO: 54; and the endoglucanase I comprises the mature polypeptide of SEQ ID NO: 56.

184. The cellulolytic protein composition of claim 172, wherein the endoglucanase II comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 58.

185. The cellulolytic protein composition of claim 172, wherein the endoglucanase II comprises the mature polypeptide of SEQ ID NO: 58.

186. The cellulolytic protein composition of claim 172, wherein the endoglucanase III comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 60.

187. The cellulolytic protein composition of claim 172, wherein the endoglucanase III comprises the mature polypeptide of SEQ ID NO: 60.

188. The cellulolytic protein composition of claim 172, wherein the endoglucanase V comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 62.

189. The cellulolytic protein composition of claim 172, wherein the endoglucanase V comprises the mature polypeptide of SEQ ID NO: 62.

190. The cellulolytic protein composition of claim 172, wherein the endoglucanase II comprises the mature polypeptide of SEQ ID NO: 58; the endoglucanase III comprises the mature polypeptide of SEQ ID NO: 60; and the endoglucanase V comprises the mature polypeptide of SEQ ID NO: 62.

191. A method for degrading a cellulose-containing material, comprising: treating the cellulose-containing material with the cellulolytic protein composition of claim 171.

192. The method of claim 191, further comprising recovering the degraded cellulose-containing material.

193. The method of claim 162, wherein the degraded cellulose-containing material is a sugar selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.

194. A method for producing a fermentation product, comprising: (a) saccharifying a cellulose-containing material with the cellulolytic protein composition of claim 171; (b) fermenting the saccharified cellulose-containing material of step (a) with one or more (several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

195. The method of claim 194, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.

196. The method of claim of 194, wherein the fermentation product is an alcohol, organic acid, ketone, aldehyde, amino acid, or gas.