Polypeptides having cellulolytic enhancing activity and polynucleotides encoding same

Abstract

The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional application of U.S. application Ser. No. 13/238,755, filed Sep. 21, 2011, which is a divisional application of U.S. application Ser. No. 12/130,722, filed May 30, 2008, now U.S. Pat. No. 8,044,264, which claims the benefit of U.S. Provisional Application No. 60/941,234, filed May 31, 2007, which applications 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 a deposit of biological material, which deposit is incorporated herein by reference.

BACKGROUND OF THE INVENTION

[0005] 1. Field of the Invention

[0006] The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides.

[0007] 2. Description of the Related Art

[0008] 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.

[0009] 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.

[0010] It would be advantageous in the art to improve the ability to convert cellulosic feedstocks.

[0011] WO 2005/074647 discloses isolated polypeptides having cellulolytic enhancing activity and polynucleotides thereof from Thielavia terrestris.

[0012] WO 2005/074656 discloses an isolated polypeptide having cellulolytic enhancing activity and the polynucleotide thereof from Thermoascus aurantiacus.

[0013] U.S. Published Application Serial No. 2007/0077630 discloses an isolated polypeptide having cellulolytic enhancing activity and the polynucleotide thereof from Trichoderma reesei.

[0014] The present invention provides polypeptides having cellulolytic enhancing activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

[0015] The present invention relates to isolated polypeptides having cellulolytic enhancing activity selected from the group consisting of:

[0016] (a) a polypeptide comprising an amino acid sequence having at least 60% identity to the mature polypeptide of SEQ ID NO: 2;

[0017] (b) a polypeptide encoded by a polynucleotide that hybridizes under at least medium stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii);

[0018] (c) a polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 60% identity to the mature polypeptide coding sequence of SEQ ID NO: 1; and

[0019] (d) a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NO: 2.

[0020] The present invention also relates to isolated polynucleotides encoding polypeptides having cellulolytic enhancing activity, selected from the group consisting of:

[0021] (a) a polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 60% identity to the mature polypeptide of SEQ ID NO: 2;

[0022] (b) a polynucleotide that hybridizes under at least medium stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii);

[0023] (c) a polynucleotide comprising a nucleotide sequence having at least 60% identity to the mature polypeptide coding sequence of SEQ ID NO: 1; and

[0024] (d) a polynucleotide encoding a variant comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the mature polypeptide of SEQ ID NO: 2.

[0025] The present invention also relates to nucleic acid constructs, recombinant expression vectors, recombinant host cells comprising the polynucleotides, and methods of producing a polypeptide having cellulolytic enhancing activity.

[0026] The present invention also relates to methods of inhibiting the expression of a polypeptide in a cell, comprising administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention. The present also relates to such a double-stranded inhibitory RNA (dsRNA) molecule, wherein optionally the dsRNA is a siRNA or a miRNA molecule.

[0027] 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 enzyme composition in the presence of an effective amount of such a polypeptide having cellulolytic enhancing activity, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulose-containing material compared to the absence of the polypeptide having cellulolytic enhancing activity.

[0028] The present invention also relates to methods of producing a fermentation product, comprising: (a) saccharifying a cellulose-containing material with an effective amount of a cellulolytic enzyme composition in the presence of an effective amount of such a polypeptide having cellulolytic enhancing activity, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulose-containing material compared to the absence of the polypeptide having cellulolytic enhancing activity; (b) fermenting the saccharified cellulose-containing material of step (a) with one or more fermentating microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[0029] The present invention also relates to plants comprising an isolated polynucleotide encoding such a polypeptide having cellulolytic enhancing activity.

[0030] The present invention also relates to methods of producing such a polypeptide having cellulolytic enhancing activity, comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding such a polypeptide having cellulolytic enhancing activity under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0031] The present invention further relates to nucleic acid constructs comprising a gene encoding a protein, wherein the gene is operably linked to a nucleotide sequence encoding a signal peptide comprising or consisting of amino acids 1 to 15 of SEQ ID NO: 2, wherein the gene is foreign to the nucleotide sequence.

BRIEF DESCRIPTION OF THE FIGURES

[0032] FIG. 1 shows the cDNA sequence and the deduced amino acid sequence of a Thielavia terrestris NRRL 8126 polypeptide having cellulolytic enhancing activity (SEQ ID NOs: 1 and 2, respectively).

[0033] FIG. 2 shows a restriction map of pTter61F.

[0034] FIG. 3 shows a restriction map of pAILo23.

[0035] FIG. 4 shows a restriction map of pMJ04.

[0036] FIG. 5 shows a restriction map of pCaHj527.

[0037] FIG. 6 shows a restriction map of pMT2188.

[0038] FIG. 7 shows a restriction map of pCaHj568.

[0039] FIG. 8 shows a restriction map of pMJ05.

[0040] FIG. 9 shows a restriction map of pSMai130.

[0041] FIG. 10 shows the DNA sequence and amino acid sequence of an Aspergillus oryzae beta-glucosidase native signal sequence (SEQ ID NOs: 37 and 38).

[0042] FIG. 11 shows the DNA sequence and amino acid sequence of a Humicola insolens endoglucanase V signal sequence (SEQ ID NOs: 41 and 42).

[0043] FIG. 12 shows a restriction map of pSMai135.

DEFINITIONS

[0044] Cellulolytic enhancing activity: The term "cellulolytic enhancing activity" is defined herein as a biological activity which 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 in 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/g of cellulose in PCS, wherein total protein is comprised of 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° 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® 1.5 L (Novozymes A/S, Bagsv.ae butted.rd, Denmark) in the presence of 3% of total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 3% of total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae according to Example 22 of WO 02/095014) of cellulase protein loading is used as the source of the cellulolytic activity.

[0045] 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.

[0046] 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.

[0047] Cellulolytic activity: The term "cellulolytic activity" is defined herein as a biological activity which 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. For CELLUCLAST® (Novozymes A/S, Bagsv.ae butted.rd, Denmark) the assay is carried out at 40° C. in 0.1 M phosphate pH 9.0 buffer for 30 minutes with CMC as substrate (33.3 g/L carboxymethyl cellulose Hercules 7 LFD) and an enzyme concentration of approximately 3.3-4.2 CEVU/ml. The CEVU activity is calculated relative to a declared enzyme standard, such as CELLUZYME® Standard 17-1194 (obtained from Novozymes A/S, Bagsv.ae butted.rd, Denmark).

[0048] For purposes of the present invention, cellulolytic activity is determined by measuring the increase in hydrolysis of a cellulose-containing material by a cellulolytic mixture under the following conditions: 1-10 mg of cellulolytic protein/g of cellulose in PCS for 5-7 day at 50° C. compared to a control hydrolysis without addition of cellulolytic protein.

[0049] 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.

[0050] 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.

[0051] 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 μmole of p-nitrophenol produced per minute at 50° C., pH 5 from 4 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 100 mM sodium citrate, 0.01% TWEEN® 20.

[0052] Family 61 glycoside hydrolase: The term "Family 61 glycoside hydrolase" or "Family GH61" is defined herein as a polypeptide falling into the glycoside hydrolase Family 61 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. A GH61 protein is also referred to as a CEL61 protein.

[0053] 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.

[0054] 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 may be in the form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix.

[0055] 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 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.

[0056] 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).

[0057] 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.

[0058] 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.

[0059] 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.

[0060] Mature polypeptide: The term "mature polypeptide" is defined herein as a polypeptide having cellulolytic enhancing activity that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In a preferred aspect, the mature polypeptide is amino acids 16 to 317 of SEQ ID NO: 2 based on the SignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 2 are a signal peptide.

[0061] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" is defined herein as a nucleotide sequence that encodes a mature polypeptide having cellulolytic enhancing activity. In a preferred aspect, the mature polypeptide coding sequence is nucleotides 46 to 951 of SEQ ID NO: 1 based on the SignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 1 encode a signal peptide.

[0062] Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".

[0063] 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×100)/(Length of Alignment-Total Number of Gaps in Alignment)

[0064] 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×100)/(Length of Alignment-Total Number of Gaps in Alignment)

[0065] Homologous sequence: The term "homologous sequence" is defined herein as a predicted protein that gives an E value (or expectancy score) of less than 0.001 in a tfasty search (Pearson, W. R., 1999, in Bioinformatics Methods and Protocols, S. Misener and S. A. Krawetz, ed., pp. 185-219) with the mature polypeptide of SEQ ID NO: 2.

[0066] 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 of SEQ ID NO: 2; or a homologous sequence thereof; wherein the fragment has cellulolytic enhancing activity. In a preferred aspect, a fragment contains at least 255 amino acid residues, more preferably at least 270 amino acid residues, and most preferably at least 285 amino acid residues, of the mature polypeptide of SEQ ID NO: 2 or a homologous sequence thereof.

[0067] 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 of SEQ ID NO: 1; or a homologous sequence thereof; wherein the subsequence encodes a polypeptide fragment having cellulolytic enhancing activity. In a preferred aspect, a subsequence contains at least 765 nucleotides, more preferably at least 810 nucleotides, and most preferably at least 855 nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 1 or a homologous sequence thereof.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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. 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.

[0073] 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.

[0074] Control sequences: 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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 of the present invention.

[0079] Modification: The term "modification" means herein any chemical modification of the polypeptide consisting of the mature polypeptide of SEQ ID NO: 2; 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.

[0080] Artificial variant: When used herein, the term "artificial variant" means a polypeptide having cellulolytic enhancing activity produced by an organism expressing a modified polynucleotide sequence of the mature polypeptide coding sequence of SEQ ID NO: 1; or a homologous sequence thereof. The modified nucleotide sequence is obtained through human intervention by modification of the polynucleotide sequence disclosed in SEQ ID NO: 1; or a homologous sequence thereof.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Cellulolytic Enhancing Activity

[0081] In a first aspect, the present invention relates to isolated polypeptides comprising or consisting of an amino acid sequence having a degree of identity to the mature polypeptide of SEQ ID NO: 2 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%, at least 97%, at least 98%, or at least 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.

[0082] A polypeptide of the present invention preferably comprises the amino acid sequence of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof having 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 16 to 317 of SEQ ID NO: 2, or an allelic variant thereof; or a fragment thereof having cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises amino acids 16 to 317 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 having 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 16 to 317 of SEQ ID NO: 2 or an allelic variant thereof; or a fragment thereof having cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of amino acids 16 to 317 of SEQ ID NO: 2.

[0083] In a second aspect, the present invention relates to isolated polypeptides having cellulolytic enhancing activity that are encoded by polynucleotides comprising or consisting of nucleotide sequences that hybridize under preferably very low stringency conditions, more 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) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, (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 the mature polypeptide coding sequence of SEQ ID NO: 1 contains at least 100 contiguous nucleotides or preferably at least 200 contiguous nucleotides. Moreover, the subsequence may encode a polypeptide fragment having cellulolytic enhancing activity. In a preferred aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO: 1.

[0084] The nucleotide sequence of SEQ ID NO: 1; or a subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2; or a fragment thereof; may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having cellulolytic enhancing activity from strains of different genera or species according to methods well known in the art. 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 preferably at least 600 nucleotides, more preferably at least 700 nucleotides, even 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 ³²P, ³H, ³⁵S, biotin, or avidin). Such probes are encompassed by the present invention.

[0085] 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.

[0086] 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; the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1; 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.

[0087] 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 46 to 951 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 pTter61F which is contained in E. coli NRRL B-50044, 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 pTter61F which is contained in E. coli NRRL B-50044.

[0088] 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° C. in 5×SSPE, 0.3% SDS, 200 μg/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.

[0089] For long probes of at least 100 nucleotides in length, the carrier material is finally washed three times each for 15 minutes using 2×SSC, 0.2% SDS preferably at 45° C. (very low stringency), more preferably at 50° C. (low stringency), more preferably at 55° C. (medium stringency), more preferably at 60° C. (medium-high stringency), even more preferably at 65° C. (high stringency), and most preferably at 70° C. (very high stringency).

[0090] 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° C. to about 10° C. below the calculated T_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×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.

[0091] For short probes of about 15 nucleotides to about 70 nucleotides in length, the carrier material is washed once in 6×SCC plus 0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5° C. to 10° C. below the calculated T_m.

[0092] In a third aspect, the present invention relates to isolated polypeptides having cellulolytic enhancing activity 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 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 96%, 97%, 98%, or 99%, which encode an active polypeptide. See polynucleotide section herein.

[0093] In a fourth aspect, the present invention relates to 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; 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.

[0094] 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.

[0095] 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.

[0096] 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.

[0097] 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 (i.e., cellulolytic enhancing 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.

[0098] 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).

[0099] 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.

[0100] The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 2, such as amino acids 19 to 317 of SEQ ID NO: 2, 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.

Sources of Polypeptides Having Cellulolytic Enhancing Activity

[0101] A polypeptide having cellulolytic enhancing activity of the present invention 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.

[0102] A polypeptide having cellulolytic enhancing activity of the present invention 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.

[0103] 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.

[0104] In another preferred aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having cellulolytic enhancing activity.

[0105] In another preferred aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having cellulolytic enhancing activity.

[0106] A polypeptide having cellulolytic enhancing activity of the present invention 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, Botryospaeria, 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.

[0107] 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.

[0108] 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, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride polypeptide having cellulolytic enhancing activity.

[0109] In another preferred aspect, the polypeptide is a Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, or Thielavia terrestris polypeptide having cellulolytic enhancing activity.

[0110] 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 polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or fragments thereof, e.g., the mature protein.

[0111] 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.

[0112] 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).

[0113] 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).

[0114] Polypeptides of the present invention 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. 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) of the present invention. 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.

[0115] A fusion polypeptide can further comprise a cleavage site. Upon secretion of the fusion protein, the site is cleaved releasing the polypeptide having cellulolytic enhancing activity from the fusion protein. Examples of cleavage sites include, but are not limited to, a Kex2 site that 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 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 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 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 site, which is cleaved by TEV protease after the Gln (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro site, which is cleaved by a genetically engineered form of human rhinovirus 3C protease after the Gln (Stevens, 2003, supra).

Polynucleotides

[0116] The present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences that encode polypeptides having cellulolytic enhancing activity of the present invention.

[0117] 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 pTter61F which is contained in E. coli NRRL B-50044. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding sequence of SEQ ID NO: 1. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 46 to 951 of SEQ ID NO: 1. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding sequence contained in plasmid pTter61F which is contained in E. coli NRRL B-50044. The present invention also encompasses nucleotide sequences that encode polypeptides 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 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: 1 that encode fragments of SEQ ID NO: 2 that have cellulolytic enhancing activity.

[0118] The present invention also relates to mutant polynucleotides comprising or consisting of at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1, in which the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID NO: 2.

[0119] 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. The cloning of the polynucleotides of the present invention 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. The polynucleotides may be cloned from a strain of Thielavia, or another or related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the nucleotide sequence.

[0120] The present invention also relates to isolated polynucleotides comprising or consisting of nucleotide sequences that have a degree of identity to the mature polypeptide coding sequence of SEQ ID NO: 1 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%, at least 97%, at least 98%, or at least 99% identity, which encode an active polypeptide.

[0121] Modification of a nucleotide sequence encoding a polypeptide of the present invention may be necessary for the synthesis of polypeptides substantially similar to the polypeptide. The term "substantially similar" to the polypeptide refers to non-naturally occurring forms of the polypeptide. These polypeptides may differ in some engineered way from the polypeptide isolated from its native source, e.g., artificial variants that differ in specific activity, thermostability, pH optimum, or the like. The variant sequence may be constructed on the basis of the nucleotide sequence presented as the mature polypeptide coding sequence of SEQ ID NO: 1, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not give rise to another amino acid sequence of the polypeptide encoded by the nucleotide sequence, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991, Protein Expression and Purification 2: 95-107.

[0122] It will be apparent to those skilled in the art that such substitutions can be made outside the regions critical to the function of the molecule and still result in an active polypeptide. Amino acid residues essential to the activity of the polypeptide encoded by an isolated polynucleotide of the invention, and therefore preferably not subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (see, e.g., Cunningham and Wells, 1989, supra). In the latter technique, mutations are introduced at every positively charged residue in the molecule, and the resultant mutant molecules are tested for cellulolytic enhancing activity to identify amino acid residues that are critical to the activity of the molecule. Sites of substrate-enzyme interaction can also be determined by analysis of the three-dimensional structure as determined by such techniques as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et al., 1992, supra; Smith et al., 1992, supra; Wlodaver et al., 1992, supra).

[0123] The present invention also relates to isolated polynucleotides encoding polypeptides of the present invention, which hybridize 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) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, 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 complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO: 1.

[0124] The present invention also relates to isolated polynucleotides obtained by (a) hybridizing a population of DNA under very low, low, medium, medium-high, high, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNA sequence comprising the mature polypeptide coding sequence of SEQ ID NO: 1, or (iii) a full-length complementary strand of (i) or (ii); and (b) isolating the hybridizing polynucleotide, which encodes a polypeptide having cellulolytic enhancing activity. In a preferred aspect, the complementary strand is the full-length complementary strand of the mature polypeptide coding sequence of SEQ ID NO: 1.

Nucleic Acid Constructs

[0125] The present invention also relates to nucleic acid constructs comprising an isolated polynucleotide of the present invention 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.

[0126] An isolated polynucleotide encoding a polypeptide of the present invention may be manipulated in a variety of ways to provide for expression of the polypeptide. 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.

[0127] 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 a polypeptide of the present invention. 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.

[0128] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention, especially in a bacterial host cell, are the promoters obtained from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proceedings of the National Academy of Sciences USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proceedings of the National Academy of Sciences USA 80: 21-25). Further promoters are described in "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242: 74-94; and in Sambrook et al., 1989, supra.

[0129] Examples of suitable promoters for directing the transcription of the nucleic acid constructs of the present invention 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 Daria (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.

[0130] In a yeast host, useful promoters are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP), Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomyces cerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8: 423-488.

[0131] 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.

[0132] 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.

[0133] Preferred terminators for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C(CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

[0134] 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.

[0135] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0136] Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

[0137] 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.

[0138] 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.

[0139] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.

[0140] The control sequence may also be a signal peptide coding sequence 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 sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the secreted polypeptide. Alternatively, the 5' end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. The foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, the foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence 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.

[0141] Effective signal peptide coding sequences for bacterial host cells are the signal peptide coding sequences obtained from the genes for Bacillus NCIB 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0142] Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences 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.

[0143] Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0144] In a preferred aspect, the signal peptide comprises or consists of amino acids 1 to 15 of SEQ ID NO: 2. In another preferred aspect, the signal peptide coding sequence comprises or consists of nucleotides 1 to 45 of SEQ ID NO: 1.

[0145] The control sequence may also be a propeptide coding sequence 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 propeptide 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 sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila laccase (WO 95/33836).

[0146] Where both signal peptide and propeptide sequences are present at the amino terminus of a polypeptide, the propeptide sequence is positioned next to the amino terminus of a polypeptide and the signal peptide sequence is positioned next to the amino terminus of the propeptide sequence.

[0147] 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 prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used. In filamentous fungi, 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

[0148] The present invention also relates to recombinant expression vectors comprising a polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleic acids and control sequences described herein may be joined together to produce a recombinant expression vector that 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 sequence of the present invention 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.

[0149] 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.

[0150] 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.

[0151] The vectors of the present invention 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.

[0152] Examples of bacterial selectable markers are the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers that confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol, or tetracycline resistance. Suitable markers for yeast host cells are ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. 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.

[0153] The vectors of the present invention 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.

[0154] 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.

[0155] 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.

[0156] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

[0157] Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0158] 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.

[0159] More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of the gene product. 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.

[0160] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, supra).

Host Cells

[0161] The present invention also relates to recombinant host cells, comprising an isolated polynucleotide of the present invention, which are advantageously used in the recombinant production of the polypeptides. A vector comprising a polynucleotide of the present invention 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.

[0162] The host cell may be any cell useful in the recombinant production of a polypeptide of the present invention, e.g., a prokaryote or a eukaryote.

[0163] The prokaryotic host cell may be any Gram positive bacterium or a Gram negative bacterium. Gram positive bacteria include, but not limited to, Bacillus, Streptococcus, Streptomyces, Staphylococcus, Enterococcus, Lactobacillus, Lactococcus, Clostridium, Geobacillus, and Oceanobacillus. Gram negative bacteria include, but not limited to, E. coli, Pseudomonas, Salmonella, Campylobacter, Helicobacter, Flavobacterium, Fusobacterium, Ilyobacter, Neisseria, and Ureaplasma.

[0164] The bacterial host cell may be any Bacillus cell. Bacillus cells useful in the practice of the present invention include, but are not limited to, 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, and Bacillus thuringiensis cells.

[0165] In a preferred aspect, the bacterial host cell is a Bacillus amyloliquefaciens, Bacillus lentus, Bacillus licheniformis, Bacillus stearothermophilus or Bacillus subtilis cell. In a more preferred aspect, the bacterial host cell is a Bacillus amyloliquefaciens cell. In another more preferred aspect, the bacterial host cell is a Bacillus clausii cell. In another more preferred aspect, the bacterial host cell is a Bacillus licheniformis cell. In another more preferred aspect, the bacterial host cell is a Bacillus subtilis cell.

[0166] The bacterial host cell may also be any Streptococcus cell. Streptococcus cells useful in the practice of the present invention include, but are not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0167] In a preferred aspect, the bacterial host cell is a Streptococcus equisimilis cell. In another preferred aspect, the bacterial host cell is a Streptococcus pyogenes cell. In another preferred aspect, the bacterial host cell is a Streptococcus uberis cell. In another preferred aspect, the bacterial host cell is a Streptococcus equi subsp. Zooepidemicus cell.

[0168] The bacterial host cell may also be any Streptomyces cell. Streptomyces cells useful in the practice of the present invention include, but are not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0169] In a preferred aspect, the bacterial host cell is a Streptomyces achromogenes cell. In another preferred aspect, the bacterial host cell is a Streptomyces avermitilis cell. In another preferred aspect, the bacterial host cell is a Streptomyces coelicolor cell. In another preferred aspect, the bacterial host cell is a Streptomyces griseus cell. In another preferred aspect, the bacterial host cell is a Streptomyces lividans cell.

[0170] The introduction of DNA into a Bacillus cell may, for instance, be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Molecular General Genetics 168: 111-115), by using competent cells (see, e.g., Young and Spizizen, 1961, Journal of Bacteriology 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, Journal of Molecular Biology 56: 209-221), by electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or by conjugation (see, e.g., Koehler and Thorne, 1987, Journal of Bacteriology 169: 5271-5278). The introduction of DNA into an E. coli cell may, for instance, be effected by protoplast transformation (see, e.g., Hanahan, 1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Dower et al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNA into a Streptomyces cell may, for instance, be effected by protoplast transformation and electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g., Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). The introduction of DNA into a Pseudomonas cell may, for instance, be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may, for instance, be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), by protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios. 68: 189-2070, by electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.

[0171] The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.

[0172] In a preferred aspect, the host cell is a fungal cell. "Fungi" as used herein includes the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (as defined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary of The Fungi, 8th edition, 1995, CAB International, University Press, Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al., 1995, supra, page 171) and all mitosporic fungi (Hawksworth et al., 1995, supra).

[0173] In a more preferred aspect, the fungal host cell is a yeast cell. "Yeast" as used herein includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes). Since the classification of yeast may change in the future, for the purposes of this invention, yeast shall be defined as described in Biology and Activities of Yeast (Skinner, F. A., Passmore, S. M., and Davenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0174] In an even more preferred aspect, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.

[0175] In a most preferred aspect, the yeast host cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis cell. In another most preferred aspect, the yeast host cell is a Kluyveromyces lactis cell. In another most preferred aspect, the yeast host cell is a Yarrowia lipolytica cell.

[0176] In another more preferred aspect, the fungal host cell is a filamentous fungal cell. "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.

[0177] In an even more preferred aspect, the filamentous fungal host cell is an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, 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.

[0178] In a most preferred aspect, the filamentous fungal host cell is an Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger or Aspergillus oryzae cell. In another most preferred aspect, the filamentous fungal host cell is a 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, or Fusarium venenatum cell. In another most preferred aspect, the filamentous fungal host cell is a Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, Phlebia radiata, Pleurotus eryngii, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cell.

[0179] 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

[0180] The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a cell, which in its wild-type form produces the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. In a preferred aspect, the cell is of the genus Thielavia. In a more preferred aspect, the cell is Thielavia terrestris. In a most preferred aspect, the cell is Thielavia terrestris NRRL 8126.

[0181] The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a recombinant host cell, as described herein, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0182] The present invention also relates to methods of producing a polypeptide of the present invention, comprising: (a) cultivating a recombinant host cell under conditions conducive for production of the polypeptide, wherein the host cell comprises a mutant nucleotide sequence having at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1, wherein the mutant nucleotide sequence encodes a polypeptide that comprises or consists of the mature polypeptide of SEQ ID NO: 2; and (b) recovering the polypeptide.

[0183] In the production methods of the present invention, the 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 polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.

[0184] The polypeptides may be detected using methods known in the art that are specific for the polypeptides. These detection methods may include use of specific antibodies, formation of an enzyme product, or disappearance of an enzyme substrate. For example, an enzyme assay may be used to determine the activity of the polypeptide as described herein.

[0185] The resulting polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0186] The polypeptides of the present invention 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.

Plants

[0187] The present invention also relates to plants, e.g., a transgenic plant, plant part, or plant cell, comprising an isolated polynucleotide encoding a polypeptide having cellulolytic enhancing activity of the present invention so as to express and produce the polypeptide in recoverable quantities. The polypeptide may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the recombinant polypeptide may be used as such for improving the quality of a food or feed, e.g., improving nutritional value, palatability, and rheological properties, or to destroy an antinutritive factor.

[0188] The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous (a monocot). Examples of monocot plants are grasses, such as meadow grass (blue grass, Poa), forage grass such as Festuca, Lolium, temperate grass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum, and maize (corn).

[0189] Examples of dicot plants are tobacco, legumes, such as lupins, potato, sugar beet, pea, bean and soybean, and cruciferous plants (family Brassicaceae), such as cauliflower, rape seed, and the closely related model organism Arabidopsis thaliana.

[0190] Examples of plant parts are stem, callus, leaves, root, fruits, seeds, and tubers as well as the individual tissues comprising these parts, e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems. Specific plant cell compartments, such as chloroplasts, apoplasts, mitochondria, vacuoles, peroxisomes and cytoplasm are also considered to be a plant part. Furthermore, any plant cell, whatever the tissue origin, is considered to be a plant part. Likewise, plant parts such as specific tissues and cells isolated to facilitate the utilisation of the invention are also considered plant parts, e.g., embryos, endosperms, aleurone and seeds coats.

[0191] Also included within the scope of the present invention are the progeny of such plants, plant parts, and plant cells.

[0192] The transgenic plant or plant cell expressing a polypeptide of the present invention may be constructed in accordance with methods known in the art. In short, the plant or plant cell is constructed by incorporating one or more (several) expression constructs encoding a polypeptide of the present invention into the plant host genome or chloroplast genome and propagating the resulting modified plant or plant cell into a transgenic plant or plant cell.

[0193] The expression construct is conveniently a nucleic acid construct that comprises a polynucleotide encoding a polypeptide of the present invention operably linked with appropriate regulatory sequences required for expression of the nucleotide sequence in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying host cells into which the expression construct has been integrated and DNA sequences necessary for introduction of the construct into the plant in question (the latter depends on the DNA introduction method to be used).

[0194] The choice of regulatory sequences, such as promoter and terminator sequences and optionally signal or transit sequences is determined, for example, on the basis of when, where, and how the polypeptide is desired to be expressed. For instance, the expression of the gene encoding a polypeptide of the present invention may be constitutive or inducible, or may be developmental, stage or tissue specific, and the gene product may be targeted to a specific tissue or plant part such as seeds or leaves. Regulatory sequences are, for example, described by Tague et al., 1988, Plant Physiology 86: 506.

[0195] For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, and the rice actin 1 promoter may be used (Franck et al., 1980, Cell 21: 285-294, Christensen et al., 1992, Plant Mo. Biol. 18: 675-689; Zhang et al., 1991, Plant Cell 3: 1155-1165). organ-specific promoters may be, for example, a promoter from storage sink tissues such as seeds, potato tubers, and fruits (Edwards & Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues such as meristems (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such as the glutelin, prolamin, globulin, or albumin promoter from rice (Wu et al., 1998, Plant and Cell Physiology 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, Journal of Plant Physiology 152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998, Plant and Cell Physiology 39: 935-941), the storage protein napA promoter from Brassica napus, or any other seed specific promoter known in the art, e.g., as described in WO 91/14772. Furthermore, the promoter may be a leaf specific promoter such as the rbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiology 102: 991-1000, the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Molecular Biology 26: 85-93), or the aldP gene promoter from rice (Kagaya et al., 1995, Molecular and General Genetics 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Molecular Biology 22: 573-588). Likewise, the promoter may inducible by abiotic treatments such as temperature, drought, or alterations in salinity or induced by exogenously applied substances that activate the promoter, e.g., ethanol, oestrogens, plant hormones such as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

[0196] A promoter enhancer element may also be used to achieve higher expression of a polypeptide of the present invention in the plant. For instance, the promoter enhancer element may be an intron that is placed between the promoter and the nucleotide sequence encoding a polypeptide of the present invention. For instance, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to enhance expression.

[0197] The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.

[0198] The nucleic acid construct is incorporated into the plant genome according to conventional techniques known in the art, including Agrobacterium-mediated transformation, virus-mediated transformation, microinjection, particle bombardment, biolistic transformation, and electroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990, Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

[0199] Presently, Agrobacterium tumefaciens-mediated gene transfer is the method of choice for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Molecular Biology 19: 15-38) and can also be used for transforming monocots, although other transformation methods are often used for these plants. Presently, the method of choice for generating transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with the transforming DNA) of embryonic calli or developing embryos (Christou, 1992, Plant Journal 2: 275-281; Shimamoto, 1994, Current Opinion Biotechnology 5: 158-162; Vasil et al., 1992, Bio/Technology 10: 667-674). An alternative method for transformation of monocots is based on protoplast transformation as described by Omirulleh et al., 1993, Plant Molecular Biology 21: 415-428.

[0200] Following transformation, the transformants having incorporated the expression construct are selected and regenerated into whole plants according to methods well-known in the art. Often the transformation procedure is designed for the selective elimination of selection genes either during regeneration or in the following generations by using, for example, co-transformation with two separate T-DNA constructs or site specific excision of the selection gene by a specific recombinase.

[0201] The present invention also relates to methods of producing a polypeptide of the present invention comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide having cellulolytic enhancing activity of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

Removal or Reduction of Cellulolytic Enhancing Activity

[0202] The present invention also relates to methods of producing a mutant of a parent cell, which comprises disrupting or deleting a polynucleotide sequence, or a portion thereof, encoding a polypeptide of the present invention, which results in the mutant cell producing less of the polypeptide than the parent cell when cultivated under the same conditions.

[0203] The mutant cell may be constructed by reducing or eliminating expression of a nucleotide sequence encoding a polypeptide of the present invention using methods well known in the art, for example, insertions, disruptions, replacements, or deletions. In a preferred aspect, the nucleotide sequence is inactivated. The nucleotide sequence to be modified or inactivated may be, for example, the coding region or a part thereof essential for activity, or a regulatory element required for the expression of the coding region. An example of such a regulatory or control sequence may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the nucleotide sequence. Other control sequences for possible modification include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, signal peptide sequence, transcription terminator, and transcriptional activator.

[0204] Modification or inactivation of the nucleotide sequence may be performed by subjecting the parent cell to mutagenesis and selecting for mutant cells in which expression of the nucleotide sequence has been reduced or eliminated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, by use of a suitable oligonucleotide, or by subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing agents.

[0205] Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues.

[0206] When such agents are used, the mutagenesis is typically performed by incubating the parent cell to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and screening and/or selecting for mutant cells exhibiting reduced or no expression of the gene.

[0207] Modification or inactivation of the nucleotide sequence may be accomplished by introduction, substitution, or removal of one or more (several) nucleotides in the gene or a regulatory element required for the transcription or translation thereof. For example, nucleotides may be inserted or removed so as to result in the introduction of a stop codon, the removal of the start codon, or a change in the open reading frame. Such modification or inactivation may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. Although, in principle, the modification may be performed in vivo, i.e., directly on the cell expressing the nucleotide sequence to be modified, it is preferred that the modification be performed in vitro as exemplified below.

[0208] An example of a convenient way to eliminate or reduce expression of a nucleotide sequence by a cell is based on techniques of gene replacement, gene deletion, or gene disruption. For example, in the gene disruption method, a nucleic acid sequence corresponding to the endogenous nucleotide sequence is mutagenized in vitro to produce a defective nucleic acid sequence that is then transformed into the parent cell to produce a defective gene. By homologous recombination, the defective nucleic acid sequence replaces the endogenous nucleotide sequence. It may be desirable that the defective nucleotide sequence also encodes a marker that may be used for selection of transformants in which the nucleotide sequence has been modified or destroyed. In a particularly preferred aspect, the nucleotide sequence is disrupted with a selectable marker such as those described herein.

[0209] Alternatively, modification or inactivation of the nucleotide sequence may be performed by established anti-sense or RNAi techniques using a sequence complementary to the nucleotide sequence. More specifically, expression of the nucleotide sequence by a cell may be reduced or eliminated by introducing a sequence complementary to the nucleotide sequence of the gene that may be transcribed in the cell and is capable of hybridizing to the mRNA produced in the cell. Under conditions allowing the complementary anti-sense nucleotide sequence to hybridize to the mRNA, the amount of protein translated is thus reduced or eliminated.

[0210] The present invention further relates to a mutant cell of a parent cell that comprises a disruption or deletion of a nucleotide sequence encoding the polypeptide or a control sequence thereof, which results in the mutant cell producing less of the polypeptide or no polypeptide compared to the parent cell.

[0211] The polypeptide-deficient mutant cells so created are particularly useful as host cells for the expression of native and/or heterologous polypeptides. Therefore, the present invention further relates to methods of producing a native or heterologous polypeptide, comprising: (a) cultivating the mutant cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide. The term "heterologous polypeptides" is defined herein as polypeptides that are not native to the host cell, a native protein in which modifications have been made to alter the native sequence, or a native protein whose expression is quantitatively altered as a result of a manipulation of the host cell by recombinant DNA techniques.

[0212] In a further aspect, the present invention relates to a method of producing a protein product essentially free of cellulolytic enhancing activity by fermentation of a cell that produces both a polypeptide of the present invention as well as the protein product of interest by adding an effective amount of an agent capable of inhibiting cellulolytic enhancing activity to the fermentation broth before, during, or after the fermentation has been completed, recovering the product of interest from the fermentation broth, and optionally subjecting the recovered product to further purification.

[0213] In a further aspect, the present invention relates to a method of producing a protein product essentially free of cellulolytic enhancing activity by cultivating the cell under conditions permitting the expression of the product, subjecting the resultant culture broth to a combined pH and temperature treatment so as to reduce the cellulolytic enhancing activity substantially, and recovering the product from the culture broth. Alternatively, the combined pH and temperature treatment may be performed on an enzyme preparation recovered from the culture broth. The combined pH and temperature treatment may optionally be used in combination with a treatment with an cellulolytic enhancing inhibitor.

[0214] In accordance with this aspect of the invention, it is possible to remove at least 60%, preferably at least 75%, more preferably at least 85%, still more preferably at least 95%, and most preferably at least 99% of the cellulolytic enhancing activity. Complete removal of cellulolytic enhancing activity may be obtained by use of this method.

[0215] The combined pH and temperature treatment is preferably carried out at a pH in the range of 2-4 or 9-11 and a temperature in the range of at least 60-70° C. for a sufficient period of time to attain the desired effect, where typically, 30 to 60 minutes is sufficient.

[0216] The methods used for cultivation and purification of the product of interest may be performed by methods known in the art.

[0217] The methods of the present invention for producing an essentially cellulolytic enhancing-free product is of particular interest in the production of eukaryotic polypeptides, in particular fungal proteins such as enzymes. The enzyme may be selected from, e.g., an amylolytic enzyme, lipolytic enzyme, proteolytic enzyme, cellulolytic enzyme, oxidoreductase, or plant cell-wall degrading enzyme. Examples of such enzymes include an aminopeptidase, amylase, amyloglucosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, galactosidase, beta-galactosidase, glucoamylase, glucose oxidase, glucosidase, haloperoxidase, hemicellulase, invertase, isomerase, laccase, ligase, lipase, lyase, mannosidase, oxidase, pectinolytic enzyme, peroxidase, phytase, phenoloxidase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transferase, transglutaminase, or xylanase. The cellulolytic enhancing-deficient cells may also be used to express heterologous proteins of pharmaceutical interest such as hormones, growth factors, receptors, and the like.

[0218] It will be understood that the term "eukaryotic polypeptides" includes not only native polypeptides, but also those polypeptides, e.g., enzymes, which have been modified by amino acid substitutions, deletions or additions, or other such modifications to enhance activity, thermostability, pH tolerance and the like.

[0219] In a further aspect, the present invention relates to a protein product essentially free from cellulolytic enhancing activity that is produced by a method of the present invention.

Methods of Inhibiting Expression of a Polypeptide

[0220] The present invention also relates to methods of inhibiting the expression of a polypeptide in a cell, comprising administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention. In a preferred aspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.

[0221] The dsRNA is preferably a small interfering RNA (siRNA) or a micro RNA (miRNA). In a preferred aspect, the dsRNA is small interfering RNA (siRNAs) for inhibiting transcription. In another preferred aspect, the dsRNA is micro RNA (miRNAs) for inhibiting translation.

[0222] The present invention also relates to such double-stranded RNA (dsRNA) molecules, comprising a portion of the mature polypeptide coding sequence of SEQ ID NO: 1 for inhibiting expression of a polypeptide in a cell. While the present invention is not limited by any particular mechanism of action, the dsRNA can enter a cell and cause the degradation of a single-stranded RNA (ssRNA) of similar or identical sequences, including endogenous mRNAs. When a cell is exposed to dsRNA, mRNA from the homologous gene is selectively degraded by a process called RNA interference (RNAi).

[0223] The dsRNAs of the present invention can be used in gene-silencing therapeutics. In one aspect, the invention provides methods to selectively degrade RNA using the dsRNAis of the present invention. The process may be practiced in vitro, ex vivo or in vivo. In one aspect, the dsRNA molecules can be used to generate a loss-of-function mutation in a cell, an organ or an animal. Methods for making and using dsRNA molecules to selectively degrade RNA are well known in the art, see, for example, U.S. Pat. No. 6,506,559; U.S. Pat. No. 6,511,824; U.S. Pat. No. 6,515,109; and U.S. Pat. No. 6,489,127.

Compositions

[0224] The present invention also relates to compositions comprising a polypeptide of the present invention. Preferably, the compositions are enriched in such a polypeptide. The term "enriched" indicates that the cellulolytic enhancing activity of the composition has been increased, e.g., with an enrichment factor of at least 1.1.

[0225] The composition may comprise a polypeptide of the present invention as the major component, e.g., a mono-component composition. Alternatively, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase, pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0226] In a preferred aspect, the composition comprises one or more cellulolytic enzymes and a polypeptide of the present invention, as described herein.

[0227] The additional enzyme(s) may be produced, for example, by a microorganism belonging to the genus Aspergillus, preferably Aspergillus aculeatus, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, or Aspergillus oryzae; Fusarium, preferably 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 sulphureum, Fusarium toruloseum, Fusarium trichothecioides, or Fusarium venenatum; Humicola, preferably Humicola insolens or Humicola lanuginosa; or Trichoderma, preferably Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride.

[0228] The polypeptide compositions may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. For instance, the polypeptide composition may be in the form of a granulate or a microgranulate. The polypeptide to be included in the composition may be stabilized in accordance with methods known in the art.

[0229] Examples are given below of preferred uses of the polypeptide compositions of the invention. The dosage of the polypeptide composition of the invention and other conditions under which the composition is used may be determined on the basis of methods known in the art.

Methods of Processing a Cellulose-Containing Material

[0230] 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 enzyme composition in the presence of an effective amount of a polypeptide having cellulolytic enhancing activity of the present invention, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulose-containing material compared to the absence of the polypeptide having cellulolytic enhancing activity.

[0231] The present invention also relates to methods for producing a fermentation product, comprising: (a) saccharifying a cellulose-containing material with an effective amount of a cellulolytic enzyme composition in the presence of an effective amount of a polypeptide having cellulolytic enhancing activity of the present invention, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulose-containing material compared to the absence of the polypeptide having cellulolytic enhancing activity; (b) fermenting the saccharified cellulose-containing material of step (a) with one or more fermentating microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[0232] 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.

[0233] 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.

[0234] 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 practicing the methods of the present invention.

[0235] 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, O. 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.

[0236] Pretreatment.

[0237] 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 CO₂, supercritical H₂O, and ammonia percolation.

[0238] 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).

[0239] 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° C., more preferably at 160-200° C., and most preferably at 170-190° 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).

[0240] A catalyst such as H₂SO₄ or SO₂ (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).

[0241] 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.

[0242] In dilute acid pretreatment, the cellulose-containing material is mixed with dilute acid, typically H₂SO₄, 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).

[0243] 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).

[0244] Lime pretreatment is performed with calcium carbonate, sodium hydroxide, or ammonia at low temperatures of 85-150° 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.

[0245] Wet oxidation is a thermal pretreatment performed typically at 180-200° 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.

[0246] 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).

[0247] Ammonia fiber explosion (AFEX) involves treating cellulose-containing material with liquid or gaseous ammonia at moderate temperatures such as 90-100° 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).

[0248] Organosolv pretreatment delignifies cellulose-containing material by extraction using aqueous ethanol (40-60% ethanol) at 160-200° 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.

[0249] 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.

[0250] 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° C., preferably 165-195° C., for periods ranging from seconds to minutes to, e.g., 1 second to 60 minutes.

[0251] In another aspect, pretreatment is carried out as an ammonia fiber explosion step (AFEX pretreatment step).

[0252] 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.

[0253] Mechanical Pretreatment: The term "mechanical pretreatment" refers to various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).

[0254] 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.

[0255] 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° C., preferably about 140 to about 235° 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.

[0256] 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.

[0257] 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.

[0258] 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).

[0259] Saccharification.

[0260] 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 enzyme composition comprising an effective amount of a polypeptide having cellulolytic enhancing activity of the present invention. The enzymes components of the composition can also be added sequentially.

[0261] In the methods of the present invention, the cellulolytic enzyme composition may comprise any protein involved in the processing of a cellulose-containing material to glucose, or hemicellulose to xylose, mannose, galactose, and arabinose, their polymers, or products derived from them as described below. In one aspect, the cellulolytic enzyme composition comprises an endoglucanase, a cellobiohydrolase, a beta-glucosidase, or a combination thereof. In another aspect, the cellulolytic enzyme composition further comprises one or more additional enzyme activities to improve the degradation of the cellulose-containing material. Preferred additional enzymes are hemicellulases, esterases (e.g., lipases, phospholipases, and/or cutinases), proteases, laccases, peroxidases, or mixtures thereof.

[0262] The cellulolytic enzyme composition may be a monocomponent preparation, e.g., an endoglucanase, a multicomponent preparation, e.g., endoglucanase(s), cellobiohydrolase(s), and beta-glucosidase(s), or a combination of multicomponent and monocomponent protein preparations. The cellulolytic proteins may have activity, i.e., hydrolyze the cellulose-containing material, either in the acid, neutral, or alkaline pH-range.

[0263] As mentioned above, the cellulolytic proteins used in the present invention may be monocomponent preparations, i.e., a component essentially free of other cellulolytic components. The single component may be a recombinant component, i.e., produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host (see, for example, WO 91/17243 and WO 91/17244). The host cell may be a heterologous host (enzyme is foreign to host) or the host may also be a wild-type host (enzyme is native to host). Monocomponent cellulolytic proteins may also be prepared by purifying such a protein from a fermentation broth.

[0264] The cellulolytic enzyme compositions supplemented with an effective amount of a polypeptide having cellulolytic enhancing activity may be in any form suitable for use in the processes described herein, such as, for example, a crude fermentation broth(s) with or without cells, a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a protected enzyme. Granulates may be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452, and may optionally be coated by process known in the art. Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established process. Protected enzymes may be prepared according to the process disclosed in EP 238,216.

[0265] A polypeptide having cellulolytic enzyme activity may be obtained from microorganisms of any genus. The term "obtained from" means herein that the enzyme may have been isolated from an organism that naturally produces the enzyme as a native enzyme. The term "obtained from" also means herein that the enzyme may have been produced recombinantly in a host organism, wherein the recombinantly produced enzyme is either native or foreign to the host organism or has a modified amino acid sequence, e.g., having one or more amino acids that are deleted, inserted and/or substituted, i.e., a recombinantly produced enzyme that is a mutant and/or a fragment of a native amino acid sequence or an enzyme produced by nucleic acid shuffling processes known in the art. Encompassed within the meaning of a native enzyme are natural variants and within the meaning of a foreign enzyme are variants obtained by chemical or recombinant mutagenesis, such as by site-directed mutagenesis or shuffling. Consequently, chemically modified or protein engineered mutants of cellulolytic proteins may also be used in the present invention. In a preferred aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0266] A polypeptide having cellulolytic enzyme 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 enzyme 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 enzyme activity.

[0267] 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 enzyme activity.

[0268] In another preferred aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having cellulolytic enzyme activity.

[0269] In another preferred aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having cellulolytic enzyme activity.

[0270] The polypeptide having cellulolytic enzyme 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 enzyme activity; or more preferably a filamentous fungal polypeptide such as aan Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, 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 enzyme activity.

[0271] 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 enzyme activity.

[0272] 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 cellulolytic enzyme activity.

[0273] In the methods of the present invention, any endoglucanase(s), cellobiohydrolase(s), and/or beta-glucosidase(s), as well as other cellulolytic proteins, e.g., hemicellulase(s), can be used.

[0274] Examples of bacterial endoglucanases that can be used in 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).

[0275] Examples of fungal endoglucanases that can be used in the present invention, include, but are not limited to, a Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263; GenBank® accession no. M15665); Trichoderma reesei endoglucanase II (Saloheimo et al., 1988, Gene 63:11-22; GenBank® accession no. M19373); Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563; GenBank® accession no. AB003694); Trichoderma reesei endoglucanase IV (Saloheimo et al., 1997, Eur. J. Biochem. 249: 584-591; GenBank® accession no. Y11113); and Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228; GenBank® 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® accession no. L29381); Humicola grisea var. thermoidea endoglucanase (GenBank® accession no. AB003107); Melanocarpus albomyces endoglucanase (GenBank® accession no. MAL515703); Neurospora crassa endoglucanase (GenBank® 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® accession nos. AB047927, AB056667, and AB056668); and Thielavia terrestris (WO 2004/053039; EMBL accession no. CQ827970).

[0276] 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.

[0277] In a preferred aspect, the endoglucanase is a Trichoderma reesei endoglucanase I (CEL7B). In another preferred aspect, the endoglucanase is a Trichoderma reesei endoglucanase II (CEL5A). In another preferred aspect, the endoglucanase is a Trichoderma reesei endoglucanase III (CEL12A). In another preferred aspect, the endoglucanase is a Trichoderma reesei endoglucanase V (CEL45A). In another preferred aspect, the endoglucanase is a Myceliophthora thermophila CEL7 endoglucanase. In another preferred aspect, the endoglucanase is a Chrysosporium lucknowense CEL12 endoglucanase. In another preferred aspect, the endoglucanase is a Chrysosporium lucknowense CEL45 endoglucanase.

[0278] In a more preferred aspect, the Trichoderma reesei endoglucanase I (CEL7B) is the mature polypeptide of SEQ ID NO: 46 or an ortholog or variant thereof. In another more preferred aspect, the Trichoderma reesei endoglucanase II (CEL5A) is the mature polypeptide of SEQ ID NO: 48 or an ortholog or variant thereof. In another more preferred aspect, the Trichoderma reesei endoglucanase III (CEL12A) is the mature polypeptide of SEQ ID NO: 50 or an ortholog or variant thereof. In another more preferred aspect, the Trichoderma reesei endoglucanase V (CEL45A) is the mature polypeptide of SEQ ID NO: 52 or an ortholog or variant thereof. In another more preferred aspect, the Myceliophthora thermophila CEL7 endoglucanase is the mature polypeptide of SEQ ID NO: 54 or an ortholog or variant thereof. In another more preferred aspect, the Chrysosporium lucknowense CEL12 endoglucanase is the mature polypeptide of SEQ ID NO: 56 or an ortholog or variant thereof. In another more preferred aspect, the Chrysosporium lucknowense CEL45 endoglucanase is the mature polypeptide of SEQ ID NO: 58 or an ortholog or variant thereof.

[0279] In another more preferred aspect, the Trichoderma reesei endoglucanase I (CEL7B) is encoded by the mature polypeptide coding sequence of SEQ ID NO: 45 or an ortholog or variant thereof. In another more preferred aspect, the Trichoderma reesei endoglucanase II (CEL5A) is encoded by the mature polypeptide coding sequence of SEQ ID NO: 47 or an ortholog or variant thereof. In another more preferred aspect, the Trichoderma reesei endoglucanase III (CEL12A) is encoded by the mature polypeptide coding sequence of SEQ ID NO: 49 or an ortholog or variant thereof. In another more preferred aspect, the Trichoderma reesei endoglucanase V (CEL45A) is encoded by the mature polypeptide coding sequence of SEQ ID NO: 51 or an ortholog or variant thereof. In another more preferred aspect, the Myceliophthora thermophila CEL7 endoglucanase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 53 or an ortholog or variant thereof. In another more preferred aspect, the Chrysosporium lucknowense CEL12 endoglucanase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 55 or an ortholog or variant thereof. In another more preferred aspect, the Chrysosporium lucknowense CEL45 endoglucanase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 57 or an ortholog or variant thereof.

[0280] The Trichoderma reesei endoglucanase I (CEL7B) can be obtained according to Penttila et al., 1986, Gene 45: 253-263. The Trichoderma reesei endoglucanase II (CEL5A) can be obtained according to Saloheimo et al., 1988, Gene 63:11-22. The Trichoderma reesei endoglucanase III (CEL12A) can be obtained according to Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563. The Trichoderma reesei endoglucanase V (CEL45A) can be obtained according to Saloheimo et al., 1994, Molecular Microbiology 13: 219-228. The Myceliophthora thermophila CEL7 endoglucanase can be obtained according to WO 95/024471. The Chrysosporium lucknowense CEL12 endoglucanase can be obtained according to WO 2001/25468. The Chrysosporium lucknowense CEL45 endoglucanase can be obtained according to WO 2000/20555.

[0281] In another preferred aspect, the cellobiohydrolase is a Trichoderma reesei cellobiohydrolase I (CEL7A). In another preferred aspect, the cellobiohydrolase is a Trichoderma reesei cellobiohydrolase II (CEL6A). In another preferred aspect, the cellobiohydrolase is a Chrysosporium lucknowense CEL7 cellobiohydrolase with a cellulose binding domain. In another preferred aspect, the cellobiohydrolase is a Myceliophthora thermophila CEL7 cellobiohydrolase without a cellulose binding domain. In another preferred aspect, the cellobiohydrolase is a Thielavia terrestris cellobiohydrolase.

[0282] In another more preferred aspect, the Trichoderma reesei cellobiohydrolase I (CEL7A) is the mature polypeptide of SEQ ID NO: 60 or an ortholog or variant thereof. In another preferred aspect, the Trichoderma reesei cellobiohydrolase II (CEL6A) is the mature polypeptide of SEQ ID NO: 62 or an ortholog or variant thereof. In another more preferred aspect, the Chrysosporium lucknowense CEL7 cellobiohydrolase with a cellulose binding domain is the mature polypeptide of SEQ ID NO: 64 or an ortholog or variant thereof. In another more preferred aspect, the Myceliophthora thermophila CEL7 cellobiohydrolase without a cellulose binding domain is the mature polypeptide of SEQ ID NO: 66 or an ortholog or variant thereof. In another more preferred aspect, the Thielavia terrestris cellobiohydrolase is the mature polypeptide of SEQ ID NO: 68 or an ortholog or variant thereof.

[0283] In another more preferred aspect, the Trichoderma reesei cellobiohydrolase I (CEL7A) cellobiohydrolase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 59 or an ortholog or variant thereof. In another more preferred aspect, the Trichoderma reesei cellobiohydrolase II (CEL6A) cellobiohydrolase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 61 or an ortholog or variant thereof. In another more preferred aspect, the Chrysosporium lucknowense CEL7 cellobiohydrolase with a cellulose binding domain is encoded by the mature polypeptide coding sequence of SEQ ID NO: 63 or an ortholog or variant thereof. In another more preferred aspect, the Myceliophthora thermophila CEL7 cellobiohydrolase without a cellulose binding domain is encoded by the mature polypeptide coding sequence of SEQ ID NO: 65 or an ortholog or variant thereof. In another more preferred aspect, the Thielavia terrestris cellobiohydrolase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 67 or an ortholog or variant thereof.

[0284] The Trichoderma reesei cellobiohydrolase I (CEL7A) can be obtained according to Shoemaker et al., 1983, Biotechnology (N.Y.) 1: 691-696. The Trichoderma reesei cellobiohydrolase II (CEL6A) can be obtained according to Terri et al., 1987, Gene 51: 43-52. The Chrysosporium lucknowense CEL7 cellobiohydrolase with a cellulose binding domain can be obtained according to WO 2001/79507. The Myceliophthora thermophila CEL7 cellobiohydrolase without a cellulose binding domain can be obtained according to WO 2003/000941. The Thielavia terrestris cellobiohydrolase can be obtained according to WO 2006/074435.

[0285] In another preferred aspect, the beta-glucosidase is obtained from Aspergillus oryzae. In another preferred aspect, the beta-glucosidase is obtained from Aspergillus fumigatus. In another preferred aspect, the beta-glucosidase is obtained from Penicillium brasilianum, e.g., Penicillium brasilianum strain IBT 20888. In another preferred aspect, the beta-glucosidase is obtained from Aspergillus niger. In another preferred aspect, the beta-glucosidase is obtained from Aspergillus aculeatus.

[0286] In a more preferred aspect, the Aspergillus oryzae beta-glucosidase is the mature polypeptide of SEQ ID NO: 70 or an ortholog or variant thereof. In another more preferred aspect, the Aspergillus fumigatus beta-glucosidase is the mature polypeptide of SEQ ID NO: 72 or an ortholog or variant thereof. In another more preferred aspect, the Penicillium brasilianum beta-glucosidase is the mature polypeptide of SEQ ID NO: 74 or an ortholog or variant thereof. In another more preferred aspect, the Aspergillus niger beta-glucosidase is the mature polypeptide of SEQ ID NO: 76 or an ortholog or variant thereof. In another more preferred aspect, the Aspergillus aculeatus beta-glucosidase is the mature polypeptide of SEQ ID NO: 78 or an ortholog or variant thereof.

[0287] In another more preferred aspect, the Aspergillus oryzae beta-glucosidase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 69 or an ortholog or variant thereof. In another more preferred aspect, the Aspergillus fumigatus beta-glucosidase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 71 or an ortholog or variant thereof. In another more preferred aspect, the Penicillium brasilianum beta-glucosidase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 73 or an ortholog or variant thereof. In another more preferred aspect, the Aspergillus niger beta-glucosidase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 75 or an ortholog or variant thereof. In another more preferred aspect, the Aspergillus aculeatus beta-glucosidase is encoded by the mature polypeptide coding sequence of SEQ ID NO: 77 or an ortholog or variant thereof.

[0288] The Aspergillus oryzae polypeptide having beta-glucosidase activity can be obtained according to WO 2002/095014. 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 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.

[0289] In another preferred aspect, the beta-glucosidase is the Aspergillus oryzae beta-glucosidase variant BG fusion protein of SEQ ID NO: 80. In another preferred aspect, the Aspergillus oryzae beta-glucosidase variant BG fusion protein is encoded by the polynucleotide of SEQ ID NO: 79. In another preferred aspect, the beta-glucosidase is the Aspergillus oryzae beta-glucosidase fusion protein of SEQ ID NO: 82. In another preferred aspect, the Aspergillus oryzae beta-glucosidase fusion protein is encoded by the polynucleotide of SEQ ID NO: 81.

[0290] In a preferred aspect, the cellulolytic enzyme composition comprises a polypeptide having cellulolytic enhancing activity of the present invention; a beta-glucosidase; a Trichoderma reesei cellobiohydrolase I (CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and a Trichoderma reesei endoglucanase I (CEL7B).

[0291] In another preferred aspect, the cellulolytic enzyme composition comprises a polypeptide having cellulolytic enhancing activity of the present invention; a beta-glucosidase; a Trichoderma reesei cellobiohydrolase I (CEL7A), a Trichoderma reesei cellobiohydrolase II (CEL6A), and a Trichoderma reesei endoglucanase I (CEL7B), and further comprises (1) one or more enzymes selected from the group consisting of a Trichoderma reesei endoglucanase II (CEL5A), a Trichoderma reesei endoglucanase V (CEL45A), and a Trichoderma reesei endoglucanase III (CEL12A), and/or further comprises (2) a Thielavia terrestris cellobiohydrolase.

[0292] In another preferred aspect, the cellulolytic enzyme composition comprises a polypeptide having cellulolytic enhancing activity of the present invention; a beta-glucosidase fusion protein of SEQ ID NO: 82; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 60, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 62, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 46.

[0293] In another preferred aspect, the cellulolytic enzyme composition comprises a polypeptide having cellulolytic enhancing activity of the present invention; a beta-glucosidase fusion protein of SEQ ID NO: 82; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 60, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 62, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 46, and further comprises one or more enzymes selected from the group consisting of a Trichoderma reesei endoglucanase II (CEL5A) of the mature polypeptide of SEQ ID NO: 47, a Trichoderma reesei endoglucanase V (CEL45A) of the mature polypeptide of SEQ ID NO: 51, and a Trichoderma reesei endoglucanase III (CEL12A) of the mature polypeptide of SEQ ID NO: 49.

[0294] In another preferred aspect, the cellulolytic enzyme composition comprises a polypeptide having cellulolytic enhancing activity of the present invention; a beta-glucosidase fusion protein of SEQ ID NO: 82; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 60, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 62, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 46, and further comprises a Thielavia terrestris cellobiohydrolase of the mature polypeptide of SEQ ID NO: 68.

[0295] In another preferred aspect, the cellulolytic enzyme composition comprises a polypeptide having cellulolytic enhancing activity of the present invention; a beta-glucosidase fusion protein of SEQ ID NO: 82; a Trichoderma reesei cellobiohydrolase I (CEL7A) of the mature polypeptide of SEQ ID NO: 60, a Trichoderma reesei cellobiohydrolase II (CEL6A) of the mature polypeptide of SEQ ID NO: 62, and a Trichoderma reesei endoglucanase I (CEL7B) of the mature polypeptide of SEQ ID NO: 46, and further comprises (1) one or more enzymes selected from the group consisting of a Trichoderma reesei endoglucanase II (CEL5A) of the mature polypeptide of SEQ ID NO: 47, a Trichoderma reesei endoglucanase V (CEL45A) of the mature polypeptide of SEQ ID NO: 51, and a Trichoderma reesei endoglucanase III (CEL12A) of the mature polypeptide of SEQ ID NO: 49, and/or further comprises (2) a Thielavia terrestris cellobiohydrolase of the mature polypeptide of SEQ ID NO: 68.

[0296] In another preferred aspect, the cellulolytic enzyme composition comprises one or more (several) components selected from the group consisting of a Myceliophthora thermophila CEL7 polypeptide having endoglucanase activity, a Chrysosporium lucknowense CEL12 polypeptide having endoglucanase activity, a Chrysosporium lucknowense CEL45 polypeptide having endoglucanase activity, a Chrysosporium lucknowense CEL7 polypeptide having cellobiohydrolase activity with a cellulose binding domain, and a Myceliophthora thermophila CEL7 polypeptide having cellobiohydrolase activity without a cellulose binding domain. In another preferred aspect, the cellulolytic enzyme composition comprises a Myceliophthora thermophila CEL7 polypeptide having endoglucanase activity, a Chrysosporium lucknowense CEL12 polypeptide having endoglucanase activity, a Chrysosporium lucknowense CEL45 polypeptide having endoglucanase activity, a CEL7 polypeptide having cellobiohydrolase activity with a cellulose binding domain, and a Myceliophthora thermophila CEL7 polypeptide having cellobiohydrolase activity without a cellulose binding domain. In another preferred aspect, the composition above further comprises one or more (several) polypeptides having beta-glucosidase activity.

[0297] The cellulolytic enzyme composition can also be a commercial preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLUCLAST® (available from Novozymes A/S) and NOVOZYM® 188 (available from Novozymes A/S). Other commercially available preparations that may be used include CELLUZYME®, CEREFLO® and ULTRAFLO® (Novozymes A/S), LAMINEX® and SPEZYME® CP (Genencor Int.), ROHAMENT® 7069 W (Rohm GmbH), and FIBREZYME® LDI, FIBREZYME® LBR, or VISCOSTAR® 150 L (Dyadic International, Inc., Jupiter, Fla., USA).

[0298] Other cellulolytic proteins that may be useful in the present invention are described in EP 495,257, EP 531,315, EP 531,372, WO 89/09259, WO 94/07998, WO 95/24471, WO 96/11262, WO 96/29397, WO 96/034108, WO 97/14804, WO 98/08940, WO 98/012307, WO 98/13465, WO 98/015619, WO 98/015633, WO 98/028411, WO 99/06574, WO 99/10481, WO 99/025846, WO 99/025847, WO 99/031255, WO 2000/009707, WO 2002/050245, WO 2002/0076792, WO 2002/101078, WO 2003/027306, WO 2003/052054, WO 2003/052055, WO 2003/052056, WO 2003/052057, WO 2003/052118, WO 2004/016760, WO 2004/043980, WO 2004/048592, WO 2005/001065, WO 2005/028636, WO 2005/093050, WO 2005/093073, WO 2006/074005, WO 2006/117432, WO 2007/071818, WO 2007/071820, WO 2008/008070, WO 2008/008793, U.S. Pat. No. 4,435,307, U.S. Pat. No. 5,457,046, U.S. Pat. No. 5,648,263, U.S. Pat. No. 5,686,593, U.S. Pat. No. 5,691,178, U.S. Pat. No. 5,763,254, and U.S. Pat. No. 5,776,757.

[0299] The cellulolytic proteins used in the methods of the present invention may be produced by fermentation of the above-noted microbial strains on a nutrient medium containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g., Bennett, J. W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press, CA, 1991). 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). Temperature ranges and other conditions suitable for growth and cellulolytic protein production are known in the art (see, e.g., Bailey, J. E., and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

[0300] The fermentation can be any method of cultivation of a cell resulting in the expression or isolation of a cellulolytic protein. Fermentation may, therefore, be understood as comprising shake flask cultivation, or small- 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 cellulolytic protein to be expressed or isolated. The resulting cellulolytic proteins produced by the methods described above may be recovered from the fermentation medium and purified by conventional procedures as described herein.

[0301] 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.

[0302] 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° C. to about 70° C., more preferably about 30° C. to about 65° C., and more preferably about 40° C. to 60° C., in particular about 50° 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 %.

[0303] 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).

[0304] 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.

[0305] 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.

[0306] 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.

[0307] 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).

[0308] Fermentation.

[0309] 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.

[0310] 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).

[0311] 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.

[0312] 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).

[0313] "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 C₆ and/or C₅ fermenting organisms, or a combination thereof. Both C₆ and C₅ 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.

[0314] Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] 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).

[0319] Bacteria that can efficiently ferment hexose and pentose to ethanol include, for example, Zymomonas mobilis and Clostridium thermocellum (Philippidis, 1996, supra).

[0320] 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.

[0321] Commercially available yeast suitable for ethanol production includes, e.g., ETHANOL RED® yeast (available from Fermentis/Lesaffre, USA), FALI® (available from Fleischmann's Yeast, USA), SUPERSTART® and THERMOSACC® fresh yeast (available from Ethanol Technology, WI, USA), BIOFERM® AFT and XR (available from NABC--North American Bioproducts Corporation, GA, USA), GERT STRAND® (available from Gert Strand AB, Sweden), and FERMIOL® (available from DSM Specialties).

[0322] 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.

[0323] 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 TAL1 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).

[0324] 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.

[0325] 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° C. to about 60° C., in particular about 32° C. or 50° C., and at about pH 3 to about pH 8, such as around pH 4-5, 6, or 7.

[0326] 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° C. to about 60° C., more preferably about 25° C. to about 50° C., and most preferably about 32° C. to about 50° C., in particular about 32° C. or 50° 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 10⁵ to 10¹², preferably from approximately 10⁷ to 10¹⁰, especially approximately 2×10⁸ 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.

[0327] 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.

[0328] 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.

[0329] Fermentation Products:

[0330] 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 (H₂), carbon dioxide (CO₂), and carbon monoxide (CO)). The fermentation product can also be protein as a high value product.

[0331] 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.

[0332] 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.

[0333] 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.

[0334] In another preferred aspect, the fermentation product is an aldehyde. In another more preferred aspect, the aldehyde is formaldehyde.

[0335] 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.

[0336] 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 H₂. In another more preferred aspect, the gas is CO₂. 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.

[0337] Recovery.

[0338] 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.

Signal Peptide

[0339] The present invention also relates to nucleic acid constructs comprising a gene encoding a protein, wherein the gene is operably linked to a nucleotide sequence encoding a signal peptide comprising or consisting of amino acids 1 to 15 of SEQ ID NO: 2, wherein the gene is foreign to the nucleotide sequence.

[0340] In a preferred aspect, the nucleotide sequence comprises or consists of nucleotides 1 to 45 of SEQ ID NO: 1.

[0341] The present invention also relates to recombinant expression vectors and recombinant host cells comprising such nucleic acid constructs.

[0342] The present invention also relates to methods of producing a protein comprising (a) cultivating such a recombinant host cell under conditions suitable for production of the protein; and (b) recovering the protein.

[0343] The protein may be native or heterologous to a host cell. The term "protein" is not meant herein to refer to a specific length of the encoded product and, therefore, encompasses peptides, oligopeptides, and proteins. The term "protein" also encompasses two or more polypeptides combined to form the encoded product. The proteins also include hybrid polypeptides that comprise a combination of partial or complete polypeptide sequences obtained from at least two different proteins wherein one or more (several) may be heterologous or native to the host cell. Proteins further include naturally occurring allelic and engineered variations of the above mentioned proteins and hybrid proteins.

[0344] Preferably, the protein is a hormone or variant thereof, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter. In a more preferred aspect, the protein is an oxidoreductase, transferase, hydrolase, lyase, isomerase, or ligase. In an even more preferred aspect, the protein is an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, another lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase or xylanase.

[0345] The gene may be obtained from any prokaryotic, eukaryotic, or other source.

[0346] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.

EXAMPLES

Materials

[0347] Chemicals used as buffers and substrates were commercial products of at least reagent grade.

Media

[0348] PDA plates were composed per liter of 39 grams of potato dextrose agar. NNCYP medium was composed per liter of 5.0 g of NH₄NO₃, 0.5 g of MgSO₄.7H₂O, 0.3 g of CaCl₂, 2.5 g of citric acid, 1.0 g of Bacto Peptone, 5.0 g of yeast extract, 1 ml of COVE trace metals solution, and sufficient K₂HPO₄ to achieve a final pH of approximately 5.4.

[0349] NNCYPmod medium was composed per liter of 1.0 g of NaCl, 5.0 g of NH₄NO₃, 0.2 g of MgSO₄.7H₂O, 0.2 g of CaCl₂, 2.0 g of citric acid, 1.0 g of Bacto Peptone, 5.0 g of yeast extract, 1 ml of COVE trace metals solution, and sufficient K₂HPO₄ to achieve a final pH of approximately 5.4.

[0350] COVE trace metals solution was composed per liter of 0.04 g of Na₂B₄O₇.10H₂O, 0.4 g of CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g of Na₂MoO₂.2H₂O, and 10 g of ZnSO₄.7H₂O.

[0351] LB 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.

[0352] MDU2BP medium was composed per liter of 45 g of maltose, 1 g of MgSO₄.7H₂O, 1 g of NaCl, 2 g of K₂HSO₄, 12 g of KH₂PO₄, 2 g of urea, and 500 μl of AMG trace metals solution, and then the pH was adjusted to 5.0 and filter sterilized with a 0.22 μm filtering unit.

[0353] AMG trace metals solution was composed per liter of 14.3 g of ZnSO₄.7H₂O, 2.5 g of CuSO₄.5H₂O, 0.5 g of NiCl₂.6H₂O, 13.8 g of FeSO₄.H₂O, 8.5 g of MnSO₄.7H₂O, and 3 g of citric acid.

[0354] SOC medium was composed of 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl₂, and 10 mM MgSO₄, and then filter-sterilized glucose was added to 20 mM after autoclaving.

[0355] Freezing medium was composed of 60% SOC and 40% glycerol.

[0356] 2×YT medium was composed per liter of 16 g of tryptone, 10 g of yeast extract, 5 g of NaCl, and 15 g of Bacto agar.

Example 1

Identification of a GH61F Polypeptide from Thielavia Terrestris NRRL 8126

[0357] An agarose plug from a fresh plate of Thielavia terrestris NRRL 8126 grown on NNCYPmod medium supplemented with 1% SIGMACELL® Type 20 cellulose (Sigma Chemical Co., St. Louis, Mo., USA) was inoculated into 50 ml of NNCYPmod medium supplemented with 1% glucose and incubated at 45° C. and 200 rpm for 25 hours. Then fifteen 500 ml flasks and two 250 ml flasks containing 100 ml and 50 ml, respectively, of NNCYPmod medium supplemented with 2% SIGMACELL® Type 20 cellulose were each inoculated with 2 ml of the above culture. The flasks were incubated at 45° C., 200 rpm for 4 days. The cultures were pooled and centrifuged at 3000×g for 10 minutes and the supernatant was filtered through a NALGENE® glass fiber prefilter (Nalge Nunc Int'l, Rochester, N.Y., USA). The filtrate was cooled to 4° C. for storage.

[0358] Two-Dimensional Polyacrylamide Gel Electrophoresis.

[0359] One ml of filtrate was precipitated by adding 100 μl of saturated (4° C.) trichloroacetic acid (TCA) and incubating 10 minutes on ice followed by addition of 9 ml of ice-cold acetone and further incubation on ice for 20 minutes. The precipitated solution was centrifuged at 10,000×g for 10 minutes at 4° C., the supernatant decanted, and the pellet rinsed twice with ice-cold acetone and allowed to air-dry.

[0360] The dried pellet was dissolved in 0.2 ml of isoelectric focusing (IEF) sample buffer. The IEF sample buffer was composed of 9.0 M urea, 3.0% w/v 3-[(3-cholamidopropyl) dimethyl-ammonium]-1-propanesulfonate (CHAPS, Pierce Chemical Co. Rockford, Ill., USA), 1% (v/v) pH 4-7 ampholytes, 1% beta-mercaptoethanol, and 0.005% bromophenol blue in distilled water. Urea stock solution was deionized using AG® 501-X8 (D), 20-5-mesh, mixed bed resin (Bio-Rad, Hercules, Calif., USA). The deionized solution was stored at -20° C. The resulting mixture was allowed to solubilize for several hours with gentle mixing on a LABQUAKE® Shaker (Lab Industries, Berkeley, Calif., USA). The sample buffer-protein mixture was applied to an 11 cm IPG strip (Bio-Rad, Hercules, Calif., USA) in an IPG rehydration tray (Amersham Biosciences, Piscataway, N.J., USA). A 750 μl aliquot of dry-strip cover fluid (Amersham Biosciences, Piscataway, N.J., USA) was layered over the IPG strips to prevent evaporation and allowed to rehydrate for 12 hours while applying 30 volts using an IPGPHOR® Isoelectric Focusing Unit (Amersham Biosciences, Piscataway, N.J., USA) at 20° C. The IPGPHOR® Unit was programmed for constant voltage with a maximum current of 50 μA per strip. After 12 hours of rehydration, the isoelectric focusing conditions were as follows: 1 hour at 200 volts, 1 hour at 500 volts, and 1 hour at 1000 volts. Then a gradient was applied from 1000 volts to 8000 volts for 30 minutes and isoelectric focusing was programmed to run at 8000 volts and was complete when >30,000 volt hours was achieved.

[0361] IPG gel strips were reduced and alkylated before the second dimension analysis by first reducing for 15 minutes with 100 mg of dithiothreitol per 10 ml of SDS-equilibration buffer followed by 15 minutes of alkylation with 250 mg of iodoacetamide per 10 ml of equilibration buffer in the dark. The SDS-equilibration buffer was composed of 50 mM Tris HCl pH 8.8, 6.0 M urea, 2% w/v sodium dodecylsulfate (SDS), 30% glycerol, and 0.002% w/v bromophenol blue. The IPG strips were rinsed quickly in SDS-PAGE running buffer (Invitrogen/Novex, Carlsbad, Calif., USA) and placed on an 11 cm, 1 well 8-16% Tris-Glycine SDS-PAGE gel (Bio-Rad, Hercules, Calif., USA) and electrophoresed using a CRITERION® electrophoresis unit (Bio-Rad, Hercules, Calif., USA) at 50 volts until the sample entered the gel and then the voltage was increased to 200 volts and allowed to run until the bromophenol blue dye reached the bottom of the gel.

[0362] Polypeptide detection. The two dimensional gel was stained with a fluorescent SYPRO® Orange Protein Stain (Molecular Probes, Eugene, Oreg., USA). Fluorescent staining methods were optimized and adapted from Malone et al., 2001, Electrophoresis, 22, 919-932. The SDS-PAGE gel was fixed in 40% ethanol, 2% acetic acid, and 0.0005% SDS on a platform rocker for 1 hour to overnight. Fixing solution was removed and replaced by three repeated wash steps consisting of 2% acetic acid and 0.0005% SDS for 30 minutes each. The gel was stained for 1.5 hours to overnight in the dark with 2% acetic acid, 0.0005% SDS, and 0.02% SYPRO® Orange Protein Stain. Staining and de-staining was further optimized to improve reproducibility and automation on a HOEFER® PROCESSOR PLUS® Staining Unit (Amersham Biosciences, Piscataway, N.J., USA). Images of the fluorescent stained SDS-PAGE gel was obtained by scanning on a MOLECULAR DYNAMICS® STORM® 860 Imaging System (Amersham Biosciences, Piscataway, N.J., USA) using blue fluorescence and 200 μm pixel sizes and a photomultiplier tube gain of 800 V. Images were viewed and adjusted using IMAGEQUANT® software version 5.0 (Amersham Biosciences, Piscataway, N.J., USA). The gel was further visualized on a DARK READER® Blue transilluminator with an orange filter (Clare Chemical Co, Denver, Colo., USA). Observed protein gel spots were excised using a 2 mm ACU-PUNCH® Biopsy Punch (Acuderm Inc., Ft. Lauderdale, Fla., USA) and stored in 96-well plates that were pre-washed with 0.1% trifluoroacetic acid (TFA) in 60% acetonitrile followed by two additional washes with HPLC grade water. The stained two-dimensional gel spots were stored in 25-50 μl of water in the pre-washed plates at -20° C. until digested.

[0363] In-Gel Digestion of Polypeptides for Peptide Sequencing.

[0364] A MULTIPROBE® II Liquid Handling Robot (PerkinElmer Life and Analytical Sciences, Boston, Mass., USA) was used to perform the in-gel digestions. Two dimensional gel spots containing polypeptides of interest were reduced with 50 μl of 10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate pH 8.0 for 30 minutes at room temperature. Following reduction, the gel pieces were alkylated with 50 μl of 55 mM iodoacetamide in 100 mM ammonium bicarbonate pH 8.0 for 20 minutes. The dried gel pieces were allowed to swell in a trypsin digestion solution consisting of 6 ng of sequencing grade trypsin (Promega, Madison, Wis., USA) per μl of 50 mM ammonium bicarbonate pH 8 for 30 minutes at room temperature, followed by an 8 hour digestion at 40° C. Each of the reaction steps described was followed by numerous washes and pre-washes with the appropriate solutions following the manufacturer's standard protocol. Fifty μl of acetonitrile was used to dehydrate the gel between reactions and gel pieces were air dried between steps. Peptides were extracted twice with 1% formic acid/2% acetonitrile in HPLC grade water for 30 minutes. Peptide extraction solutions were transferred to a THERMO-FAST® 96 well skirted PCR low profile plate (ABGene, Rochester, N.Y., USA) that had been cooled to 10-15° C. and covered with a 96-well plate lid (Perkin Elmer Life and Analytical Sciences, Boston, Mass., USA) to prevent evaporation. Plates were further stored at 4° C. until mass spectrometry analysis could be performed.

[0365] Peptide Sequencing by Tandem Mass Spectrometry.

[0366] For peptide sequencing by tandem mass spectrometry, a Q-TOF MICRO® hybrid orthogonal quadrupole time-of-flight mass spectrometer (WATERS® MICRO MASS® MS Technologies, Milford, Mass., USA) was used for LC-MS/MS analysis. The Q-TOF MICRO® mass spectrometer was fitted with an ULTIMATE® capillary and nano-flow HPLC system (Dionex, Sunnyvale, Calif., USA) coupled to a FAMOS® micro autosampler (Dionex, Sunnyvale, Calif., USA) and a SWITCHOS® II column switching device (Dionex, Sunnyvale, Calif., USA) for concentrating and desalting samples. Six μl of the recovered peptide solution from the in-gel digestion was loaded onto a guard column (300 μm ID×5 cm, C18 PEPMAP®, Dionex, Sunnyvale, Calif., USA) fitted in the injection loop and washed with 0.1% formic acid in water at 40 μl per minute for 2 minutes using a SWITCHOS® II pump (Dionex, Sunnyvale, Calif., USA). Peptides were separated on a 75 μm ID×15 cm, C18, 3 μm, 100 Å PEPMAP® nanoflow fused capillary column (Dionex, Sunnyvale, Calif., USA) at a flow rate of 175 nl per minute from a split flow of 175 μl per minute using a NAN-75 calibrator (Dionex, Sunnyvale, Calif., USA). The linear elution gradient was 5% to 60% acetonitrile in 0.1% formic acid applied over a 45 minute period. The column eluent was monitored at 215 nm and introduced into the Q-TOF MICRO® mass spectrometer through an electrospray ion source fitted with the nanospray interface. The mass spectrometer was fully microprocessor controlled using MASSLYNX® software version 3.5 (WATERS® MICROMASS® MS Technologies, Milford, Mass., USA). Data was acquired in survey scan mode and from a mass range of 50 to 2000 m/z with switching criteria for MS to MS/MS to include an ion intensity of greater than 10.0 counts per second and charge states of +2, +3, and +4. Analysis spectra of up to 4 co-eluting species with a scan time of 1.9 seconds and inter-scan time of 0.1 seconds could be obtained. A cone voltage of 65 volts was typically used and the collision energy was programmed to vary according to the mass and charge state of the eluting peptide and in the range of 10 to 60 volts. The acquired spectra were combined, smoothed, and centered in an automated fashion and a peak list generated. The generated peak list was searched against selected databases using PROTEINLYNX® Global Server 1.1 software (WATERS® MICROMASS® MS Technologies, Milford, Mass., USA). Results from the PROTEINLYNXT® searches were evaluated and un-identified proteins were analyzed further by evaluating the MS/MS spectrums of each ion of interest and de novo sequence determined by identifying the y and b ion series and matching mass differences to the appropriate amino acid.

[0367] A 2D gel spot corresponding to an approximate molecular weight of 40 kDa and an approximate isoelectric point of 4.5 was in-gel digested with trypsin and subjected to de novo sequencing as described. A doubly charged tryptic peptide ion of 431.782 m/z was determined to be Gly-Pro-[Ile/Leu]-Ala-Tyr-[Ile-Leu]-Lys (amino acids 98 to 105 of SEQ ID NO: 2). A second doubly charged tryptic peptide ion of 570.976 m/z was determined to be His-Thr-[Ile/Leu]-Thr-Ser-Gly-Pro-Asp-Asp-Val-Met-Asp-Ala-Ser-His-Lys (amino acids 82 to 97 of SEQ ID NO: 2). A third doubly charged tryptic peptide ion of 825.9517 m/z was determined to be Val-Asp-Asp-Ala-[Ile/Leu]-Thr-Asp-Thr-Gly-[Ile/Leu]-Gly-Gly-Gly-Trp-Phe-L- ys (amino acids 107 to 122 of SEQ ID NO: 2)

Example 2

Expressed Sequence Tags (EST) cDNA Library Construction

[0368] A two ml aliquot from a 24-hour liquid culture (50 ml of NNCYPmod supplemented with 1% glucose in a 250 ml flask incubated at 45° C., 200 rpm) of Thielavia terrestris NRRL 8126 was used to seed a 500 ml flask containing 100 ml of NNCYPmod medium supplemented with 2% SIGMACELL® Type 20 cellulose. The culture was incubated at 45° C., 200 rpm for 3 days. The mycelia were harvested by filtration through a Buchner funnel with a glass fiber prefilter (Nalgene, Rochester N.Y., USA), washed twice with 10 mM Tris-HCl-1 mM EDTA pH 8 (TE), and quick frozen in liquid nitrogen.

[0369] Total RNA was isolated using the following method. Frozen mycelia of Thielavia terrestris NRRL 8126 were ground in an electric coffee grinder. The ground material was mixed 1:1 v/v with 20 ml of Fenazol (Ambion, Inc., Austin, Tex., USA) in a 50 ml FALCON® tube. Once the mycelia were suspended, they were extracted with chloroform and three times with a mixture of phenol-chloroform-isoamyl alcohol 25:24:1 v/v/v. From the resulting aqueous phase, the RNA was precipitated by adding 1/10 volume of 3 M sodium acetate pH 5.2 and 1.25 volume of isopropanol. The precipitated RNA was recovered by centrifugation at 12,000×g for 30 minutes at 4° C. The final pellet was washed with cold 70% ethanol, air dried, and resuspended in 500 ml of diethylpyrocarbonate treated water (DEPC-water).

[0370] The quality and quantity of the purified RNA was assessed with a 2100 Bioanalyzer (Agilent Technologies, Inc., Palo Alto, Calif., USA). Polyadenylated mRNA was isolated from 360 μg of total RNA with the aid of a POLY(A)PURIST® MAG Kit (Ambion, Inc., Austin, Tex., USA) according to the manufacturer's instructions.

[0371] To create the cDNA library, a CLONEMINER® Kit (Invitrogen, Carlsbad, Calif., USA) was employed to construct a directional library that does not require the use of restriction enzyme cloning, thereby reducing the number of chimeric clones and size bias.

[0372] To insure the successful synthesis of the cDNA, two reactions were performed in parallel with two different concentrations of mRNA (2.2 and 4.4 μg of poly(A).sup.+ mRNA). The mRNA samples were mixed with a Biotin-attB2-Oligo(dt) primer (CLONEMINER® Kit, Invitrogen, Carlsbad, Calif., USA), 1× first strand buffer (Invitrogen, Carlsbad, Calif., USA), 2 μl of 0.1 M dithiothreitol (DTT), 10 mM of each dNTP, and water to a final volume of 18 and 16 μl, respectively.

[0373] The reaction mixtures were mixed carefully and then 2 and 4 μl of SUPERSCRIPT® reverse transcriptase (Invitrogen, Carlsbad, Calif., USA) were added and incubated at 45° C. for 60 minutes to synthesize the first complementary strand. For second strand synthesis to each first strand reaction was added 30 μl of 5× second strand buffer (Invitrogen, Carlsbad, Calif., USA), 3 μl of 10 mM of each dNTP, 10 units of E. coli DNA ligase (Invitrogen, Carlsbad, Calif., USA), 40 units of E. coli DNA polymerase I (Invitrogen, Carlsbad, Calif., USA), and 2 units of E. coli RNase H (Invitrogen, Carlsbad, Calif., USA) in a total volume of 150 μl. The mixtures were then incubated at 16° C. for two hours. After the two-hour incubation 2 μl of T4 DNA polymerase (Invitrogen, Carlsbad, Calif., USA) were added to each reaction and incubated at 16° C. for 5 minutes to create a bunt-ended cDNA. The cDNA reactions were extracted with a mixture of phenol-chloroform-isoamyl alcohol 25:24:1 v/v/v and precipitated in the presence of 20 μg of glycogen, 120 μl of 5 M ammonium acetate, and 660 μl of ethanol. After centrifugation at 12,000×g for 30 at 4° C. the cDNA pellets were washed with cold 70% ethanol, dried under vacuum for 2-3 minutes, and resuspended in 18 μl of DEPC-water. To each resuspended cDNA sample was added 10 μl of 5× adapted buffer, 10 μg of each of the attB1 adapters (provided with the CLONEMINER® Kit), 7 μl of 0.1 M DTT, and 5 units of T4 DNA ligase.

[0374] Ligation reactions were incubated overnight at 16° C. Excess adapters were removed by size-exclusion chromatography in 1 ml of SEPHACRYL® S-500 HR resin (Amersham Biosciences, Piscataway, N.J., USA). Column fractions were collected according to the kit's instructions and fractions 3 to 14 were analyzed with an AGILENT® 2100 Bioanalyzer to determine the fraction at which the attB1 adapters started to elute. This analysis showed that the adapters started eluting around fraction 10 or 11. For the first library fractions 6 to 11 were pooled and for the second library fractions 4-11 were pooled.

[0375] Cloning of the cDNA was performed by homologous DNA recombination according to GATEWAY® Technology (Invitrogen, Carlsbad, Calif., USA) using BP CLONASE® (Invitrogen, Carlsbad, Calif., USA) as the recombinase. Each BP CLONASE® recombination reaction contained approximately 70 ng of attB-flanked-cDNA, 250 ng of pDONR® 222, 2 μl of 5×BP CLONASE® buffer, 2 μl of TE, and 3 μl of BP CLONASE®. Recombination reactions were incubated at 25° C. overnight.

[0376] Heat-inactivated BP recombination reactions were then divided into 6 aliquots and electroporated into ELECTROMAX® DH10B electrocompetent cells (Invitrogen, Carlsbad, Calif., USA) using a GENE PULSER® II Electroporation System (Bio-Rad, Hercules, Calif., USA) with the following parameters: voltage: 2.0 kV, resistance: 200Ω, capacity 25 μF. Electroporated cells were resuspended in 1 ml of SOC medium and incubated at 37° C. for 60 minutes with constant shaking (200 rpm). After the incubation period, the transformed cells were pooled and mixed 1:1 with freezing medium. A 200 μl aliquot was removed for library titration and then the rest of each library was aliquoted into 1.8 ml cryovials (Wheaton Science Products, Millville, N.J., USA) and stored frozen at -80° C.

[0377] Four serial dilutions of each library were prepared: 1/100, 1/1000, 1/10⁴, 1/10⁵. From each dilution 100 μl were plated onto 150 mm LB plates supplemented with 50 μg of kanamycin per ml and incubated at 37 C overnight. The number of colonies on each dilution plate were counted and used to calculate the total number of transformants in each library.

[0378] The first library was shown to have approximately 5.4 million independent clones and the second library was show to have approximately 9 million independent clones.

Example 3

Template Preparation and Nucleotide Sequencing of cDNA Clones

[0379] Aliquots from both libraries were mixed and plated onto 25×25 cm LB plates supplemented with 50 μg of kanamycin per ml. Individual colonies were arrayed onto 96-well plates containing 100 μl of LB medium supplemented with 50 μg of kanamycin per ml with the aid of a QPix Robot (Genetix Inc., Boston, Mass., USA). Forty-five 96-well plates were obtained for a total of 4320 individual clones. The plates were incubated overnight at 37 C with shaking at 200 rpm. After incubation, 100 μl of sterile 50% glycerol was added to each well. The transformants were replicated with the aid of a 96-pin tool (Boekel, Feasterville, Pa., USA) into secondary, deep-dish 96-well microculture plates (Advanced Genetic Technologies Corporation, Gaithersburg, Md., USA) containing 1 ml of MAGNIFICENT BROTH® (MacConnell Research, San Diego, Calif., USA) supplemented with 50 μg of kanamycin per ml in each well. The primary microtiter plates were stored frozen at -80° C. The secondary deep-dish plates were incubated at 37° C. overnight with vigorous agitation (300 rpm) on a rotary shaker. To prevent spilling and cross-contamination, and to allow sufficient aeration, each secondary culture plate was covered with a polypropylene pad (Advanced Genetic Technologies Corporation, Gaithersburg, Md., USA) and a plastic microtiter dish cover. Plasmid DNA was prepared with a Robot-Smart 384 (MWG Biotech Inc., High Point, N.C., USA) and MONTAGE® Plasmid Miniprep96 Kit (Millipore, Billerica, Mass., USA).

[0380] Sequencing reactions were performed using a BIGDYE® Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems, Inc., Foster City, Calif., USA) with terminator chemistry (Giesecke et al., 1992, Journal of Virology Methods 38: 47-60) and a M13 Forward (-20) sequencing primer shown below.

TABLE-US-00001 (SEQ ID NO: 3) 5'-GTAAAACGACGGCCAG-3'

[0381] The sequencing reactions were performed in a 384-well format with a Robot-Smart 384 (MWG Biotech Inc., High Point, N.C., USA) as well as the terminator removal with a MULTISCREEN® Seq384 Sequencing Clean-up Kit (Millipore, Billerica, Mass., USA). Reactions contained 6 μl of plasmid DNA and 4 μl of sequencing master-mix containing 1 μl of 5× sequencing buffer (Millipore, Billerica, Mass., USA), 1 μl of BIGDYE® terminator (Applied Biosystems, Inc., Foster City, Calif., USA), 1.6 pmoles of M13 forward primer, and 1 μl of water. Single-pass DNA sequencing was performed with an ABI PRISM® 3700 DNA Sequencer (Applied Biosystems, Foster City, Calif., USA).

Example 4

Analysis of DNA Sequence Data of cDNA Clones

[0382] Base calling, quality value assignment, and vector trimming were performed with the assistance of PHRED/PHRAP software (University of Washington, Seattle, Wash., USA). Clustering analysis of the ESTs was performed with a Transcript Assembler v. 2.6.2. software (Paracel, Inc., Pasadena, Calif., USA). Analysis of the EST clustering indicated the presence of 395 independent clusters.

[0383] Sequence homology analysis of the assembled EST sequences against various databases, e.g., PIR, was performed with the Blastx program (Altschul et. al., 1990, J. Mol. Biol. 215:403-410) on a 32-node Linux cluster (Paracel, Inc., Pasadena, Calif., USA) using the BLOSUM 62 matrix (Henikoff, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) From these, 246 had hits to known genes in either the public or private protein databases and 149 had no significant hits against these databases. Among these 246 genes, 13 had hits against known glycosyl hydrolase genes.

Example 5

Identification of cDNA Clones Encoding a Family 61 Polypeptide Having Cellulolytic Enhancing Activity (GH61F)

[0384] A cDNA clone encoding a Family 61 polypeptide having cellulolytic enhancing activity (GH61F) was initially identified by its identity to a Family 61 protein from Neurospora crassa (UniProt Q7S439). This initial analysis indicated that the two proteins were 57.67% identical at the protein level over a 211 amino acid (663 basepairs) stretch.

[0385] After this initial identification clone Tter18A8 was retrieved from the original frozen stock plate and streaked onto a LB plate supplemented with 50 μg of kanamycin per ml. The plate was incubated overnight at 37° C. and the next day a single colony from the plate was used to inoculate 3 ml of LB supplemented with 50 μg of kanamycin per ml. The liquid culture was incubated overnight at 37° C. and plasmid DNA was prepared with a BIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA). Clone Tter08C4 plasmid DNA was sequenced again with BIGDYE® terminator chemistry as described above, using the M13 forward and a Poly-T primer shown below to sequence the 3' end of the clone.

TABLE-US-00002 (SEQ ID NO: 4) 5'-TTTTTTTTTTTTTTTTTTTTTTTVN-3'

[0386] wherein V=G, A, C and N=G, A, C, T

[0387] Blastp homology analysis of the new sequence information indicated that the protein encoded by clone Tter18A8 was similar to a Neurospora crassa hypothetical protein NCU02240.1 (UniRef Q7S439). These proteins were 74% identical over a 316 amino acid stretch.

[0388] Analysis of the deduced protein sequence of clone 18A8 with the Interproscan program (Zdobnov and Apweiler, 2001, Bioinformatics 17: 847-848) showed that the gene encoded by clone 18A8 contained the sequence signature of Family 61 proteins. This sequence signature known as the Pfam pattern PF03443 (Bateman, A. et al., 2002, Nucleic Acids Research 30: 276-280) was found 119 amino acids from the starting amino acid methionine confirming that clone Tter18A8 encodes a Thielavia terrestris Family 61 protein. This analysis also indicated that this protein contains a fungal cellulose binding domain (34 amino acids long) located 283 amino acids from the starting amino acid methionine.

[0389] The cDNA sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) are shown in FIG. 1. The cDNA clone encodes a polypeptide of 317 amino acids. The % G+C content of the cDNA clone of the gene is 64.9% and of the mature protein coding region (nucleotides 46 to 955 of SEQ ID NO: 1) is also 64.9%. Using the SignalP software program (Nielsen et al., 1997, Protein Engineering 10:1-6), a signal peptide of 15 residues was predicted. The predicted mature protein contains 302 amino acids with a molecular mass of 31.14 kDa.

[0390] A comparative alignment of Family 61 sequences was determined using the Clustal W method (Higgins, 1989, supra) using the AlignX module of the vector NTI Advance 10.3 software (Invitrogen, Carlsbad, Calif., USA) with a blosum62mt2 scoring matrix and the following multiple alignment parameters: K-tuple size 1; best diagonals 5; window size 5; gap penalty 5; gap opening penalty 10; gap extension penalty 0.1. The alignment showed that the deduced amino acid sequence of the mature Thielavia terrestris gh61 f gene shares 43% identity to the mature region of the Thielavia terrestris Cel61G polypeptide having cellulolytic enhancing activity (WO 2005/074647).

[0391] Once the identity of clone Tter18A8 was confirmed a 0.5 μl aliquot of plasmid DNA from this clone designated pTter61F (FIG. 2) was transferred into a vial of ONE SHOT® E. coli TOP10 cells (Invitrogen, Carlsbad, Calif., USA), gently mixed, and incubated on ice for 10 minutes. The cells were then heat-shocked at 42° C. for 30 seconds and incubated again on ice for 2 minutes. The cells were resuspended in 250 μl of SOC medium and incubated at 37° C. for 60 minutes with constant shaking (200 rpm). After the incubation period, two 30 μl aliquots were plated onto LB plates supplemented with 50 μg of kanamycin per ml and incubated overnight at 37° C. The next day a single colony was picked and streaked onto a 1.8 ml cryovial containing about 1.5 ml of LB agarose supplemented with 50 μg of kanamycin per ml. The vial was sealed with PETRISEAL® (Diversified Biotech, Boston, Mass., USA) and deposited with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, 1815 University Street, Peoria, Ill., USA, as NRRL B-50044, with a deposit date of May 25, 2007.

Example 6

Cloning of the Family GH61F Gene into an Aspergillus oryzae Expression Vector

[0392] Two synthetic oligonucleotide primers, shown below, were designed to PCR amplify the full-length open reading frame from Thielavia terrestris EST Tter18A8 encoding a Family GH61F polypeptide having cellulolytic enhancing activity. An IN-FUSION® PCR Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) was used to clone the fragment directly into plasmid pAILo2 (WO 2004/099228).

TABLE-US-00003 Forward primer: (SEQ ID NO: 5) 5'-ACTGGATTTACCATGAAGGGCCTCAGCCTCCTCG-3' Reverse primer: (SEQ ID NO: 6) 5'-TCACCTCTAGTTAATTAATTACTGGCATTGCGAGTAATAG-3'

Bold letters represent coding sequence. The remaining sequence contains sequence identity compared with the insertion sites of pAILo2.

[0393] Fifty picomoles of each of the primers above were used in a PCR reaction containing 50 ng of pTter18A8 DNA, 1×Pfx Amplification Buffer (Invitrogen, Carlsbad, Calif., USA), 6 μl of 10 mM blend of dATP, dTTP, dGTP, and dCTP, 2.5 units of PLATINUM® Pfx DNA Polymerase (Invitrogen, Carlsbad, Calif., USA), 1 μl of 50 mM MgSO₄, and 5 μl of 10×pCRx Enhancer Solution (Invitrogen, Carlsbad, Calif., USA) in a final volume of 50 μl. An EPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA) was used to amplify the fragment programmed for one cycle at 98° C. for 2 minutes; and 35 cycles each at 94° C. for 30 seconds, 62.1° C. for 30 seconds, and 68° C. for 1.0 minute. After the 35 cycles, the reaction was incubated at 68° C. for 10 minutes and then cooled at 10° C. until further processed. A 984 bp PCR reaction product was isolated on a 0.8% SEAKEM® GTG® agarose gel (Cambrex Bioproducts, East Rutherford, N.J., USA) using 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer and 0.1 μg of ethidium bromide per ml. The DNA band was visualized with the aid of a DARK READER® (Clare Chemical Research, Dolores, Colo., USA) to avoid UV-induced mutations. The DNA band was excised with a disposable razor blade and purified with an ULTRAFREE®-DA spin cup (Millipore, Billerica, Mass., USA) according to the manufacturer's instructions.

[0394] Plasmid pAILo2 was linearized by digestion with Nco I and Pac I. The fragment was purified by gel electrophoresis and ultrafiltration as described above. Cloning of the purified PCR fragment into the linearized and purified pAILo2 was performed with an IN-FUSION® PCR Cloning Kit. The reaction (20 μl) contained 2 μl of 1×IN-FUSION® Buffer, 2 μl of 1×BSA, 1 μl of IN-FUSION® enzyme (diluted 1:10), 100 ng of pAILo2 digested with Nco I and Pac I, and 100 ng of the Thielavia terrestris gh61 f purified PCR product. The reaction was incubated at room temperature for 30 minutes. A 2 μl sample of the reaction was used to transform E. coli XL10 SOLOPACK® Gold cells (Stratagene, La Jolla, Calif., USA) according to the manufacturer's instructions. After a recovery period, two 100 μl aliquots from the transformation reaction were plated onto 150 mm 2×YT plates supplemented with 100 μg of ampicillin per ml. The plates were incubated overnight at 37° C. Four putative recombinant clones were collected from the selection plates and plasmid DNA was prepared from each one using a BIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA). Clones were analyzed by Pst I restriction digest. Two clones that had the expected restriction digest pattern were then sequenced to confirm that there were no mutations in the cloned insert. Sequencing was performed with an ABI PRISM® 3130×1DNA Sequencer (Applied Biosystems, Foster City, Calif., USA). Clone #3 was selected and designated pAILo23 (FIG. 3).

Example 7

Expression of the Thielavia terrestris Family GH61F Gene in Aspergillus oryzae JaL250

[0395] Aspergillus oryzae JaL250 (WO 99/61651) protoplasts were prepared according to the method of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Five micrograms of pAILo23 (as well as pAILo2 as a plasmid control) were used to transform the Aspergillus oryzae JaL250 protoplasts.

[0396] The transformation of Aspergillus oryzae JaL250 with pAILo22 yielded about 50 transformants. Eight transformants were isolated to individual PDA plates and incubated for five days at 34° C.

[0397] Confluent spore plates were washed with 5 ml of 0.01% TWEEN® 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° C. with constant shaking at 200 rpm. At day five post-inoculation, cultures were centrifuged at 6000×g and their supernatants collected. Seven and a half micro-liters of each supernatant were mixed with an equal volume of 2× loading buffer (10% R-mercaptoethanol) and loaded onto a 1.5 mm 8%-16% Tris-Glycine SDS-PAGE gel and stained with BIO-SAFE® Coomassie Blue G250 (Bio-Rad, Hercules, Calif., USA). SDS-PAGE profiles of the culture broths showed that seven out of eight transformants had a new protein band of approximately 45 kDa. Transformant number 4 was selected for further studies and designated Aspergillus oryzae JaL250AILo23.

Example 8

Fermentation of Aspergillus oryzae JaL250AILo23

[0398] One hundred ml of a shake flask medium was added to a 500 ml shake flask. The shake flask medium was composed per liter of 50 g of sucrose, 10 g of KH₂PO₄, 0.5 g of CaCl₂, 2 g of MgSO₄.7H₂O, 2 g of K₂SO₄, 2 g of urea, 10 g of yeast extract, 2 g of citric acid, and 0.5 ml of trace metals solution. The trace metals solution was composed per liter of 13.8 g of FeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 2.5 g of CuSO₄.5H₂O, and 3 g of citric acid. The shake flask was inoculated with two plugs from a solid plate culture of Aspergillus oryzae JaL250AILo23 and incubated at 34° C. on an orbital shaker at 200 rpm for 24 hours.

[0399] Fifty ml of the shake flask broth were used to inoculate a 3 liter fermentation vessel containing 1.8 liters of a fermentation batch medium composed per liter of 10 g of yeast extract, 24 g of sucrose, 5 g of (NH₄)₂SO₄, 2 g of KH₂PO₄, 0.5 g of CaCl₂.2H₂O, 2 g of MgSO₄.7H₂O, 1 g of citric acid, 2 g of K₂SO₄, 0.5 ml of anti-foam, and 0.5 ml of trace metals solution. Trace metals solution was composed per liter of 13.8 g of FeSO₄.7H₂O, 14.3 g of ZnSO₄.7H₂O, 8.5 g of MnSO₄.H₂O, 2.5 g of CuSO₄.5H₂O, and 3 g of citric acid. Fermentation feed medium was composed of maltose and antifoam. The fermentation feed medium was dosed at a rate of 0 to 4.4 g/l/hr for a period of 185 hours. The fermentation vessel was maintained at a temperature of 34° C. and pH was controlled to a set-point of 6.1+/-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×g to remove the biomass. The supernatant was sterile filtered and stored at 35 to 40° C.

Example 9

Construction of pMJ04 Expression Vector

[0400] Expression vector pMJ04 was constructed by PCR amplifying the Trichoderma reesei exocellobiohydrolase 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: 7) 5'-AACGTTAATTAAGGAATCGTTTTGTGTTT-3' Primer 993428 (sense): (SEQ ID NO: 8) 5'-AGTACTAGTAGCTCCGTGGCGAAAGCCTG-3'

[0401] Trichoderma reesei RutC30 genomic DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN Inc., Valencia, Calif., USA).

[0402] The amplification reactions (50 μl) were composed of 1× ThermoPol Reaction Buffer (New England Biolabs, Beverly, Mass., USA), 0.3 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic DNA, 0.3 μM primer 993429, 0.3 μM primer 993428, and 2 units of Vent DNA polymerase (New England Biolabs, Beverly, Mass., USA). The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94° C., 30 seconds at 50° C., and 60 seconds at 72° C., followed by 25 cycles each for 30 seconds at 94° C., 30 seconds at 65° C., and 120 seconds at 72° C. (5 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 229 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer's instructions.

[0403] The resulting PCR fragment was digested with Pac I and Spe I and ligated into pAILo1 (WO 05/067531) digested with the same restriction enzymes using a Rapid Ligation Kit (Roche, Indianapolis, Ind., USA), to generate pMJ04 (FIG. 4).

Example 10

Construction of pCaHj568

[0404] 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: 9, which encodes the amino acid sequence of SEQ ID NO: 10). 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 142779: (SEQ ID NO: 11) 5'-TTGAATTGAAAATAGATTGATTTAAAACTTC-3' 142780: (SEQ ID NO: 12) 5'-TTGCATGCGTAATCATGGTCATAGC-3'

[0405] An EXPAND® 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).

[0406] 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® PCR System. Primer 140288 introduced an Eco RI site in the PCR fragment.

TABLE-US-00006 140288: (SEQ ID NO: 13) 5'-TTGAATTCATGGGTAATAACTGATAT-3' 142778: (SEQ ID NO: 14) 5'-AAATCAATCTATTTTCAATTCAATTCATCATT-3'

[0407] PCR products were separated on an agarose gel and an 1126 bp fragment was isolated and purified using a Jetquick Gel Extraction Spin Kit.

[0408] The two PCR fragments were fused by mixing and amplified 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.

[0409] 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 casaminoacids, 500 μg of thiamine, and 10 mg of kanamycin. A plasmid from one transformant was isolated and designated pCaHj527 (FIG. 5).

[0410] The NA2-tpi promoter present on pCaHj527 was subjected to site-directed mutagenesis by PCR using an EXPAND® PCR System according to the manufacturer's instructions. Nucleotides 134-144 were converted from GTACTAAAACC (SEQ ID NO: 15) to CCGTTAAATTT (SEQ ID NO: 16) using mutagenic primer 141223 shown below.

TABLE-US-00007 Primer 141223: (SEQ ID NO: 17) 5'-GGATGCTGTTGACTCCGGAAATTTAACGGTTTGGTCTTGCATCCC- 3'

Nucleotides 423-436 were converted from ATGCAATTTAAACT (SEQ ID NO: 18) to CGGCAATTTAACGG (SEQ ID NO: 19) using mutagenic primer 141222 shown below.

TABLE-US-00008 Primer 141222: (SEQ ID NO: 20) 5'-GGTATTGTCCTGCAGACGGCAATTTAACGGCTTCTGCGAATCGC-3'

The resulting plasmid was designated pMT2188 (FIG. 6).

[0411] The Humicola insolens endoglucanase V coding region was transferred from pCaHj170 as a Barn HI-Sal I fragment into pMT2188 digested with Bam HI and Xho I to generate pCaHj568 (FIG. 7). Plasmid pCaHj568 comprises a mutated NA2-tpi promoter operably linked to the Humicola insolens endoglucanase V full-length coding sequence.

Example 11

Construction of pMJ05

[0412] 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 HiEGV-F (sense): (SEQ ID NO: 21) 5'-AAGCTTAAGCATGCGTTCCTCCCCCCTCC-3' HiEGV-R (antisense): (SEQ ID NO: 22) 5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3'

[0413] The amplification reactions (50 μl) were composed of 1× ThermoPol Reaction Buffer, 0.3 mM dNTPs, 10 ng/μl of pCaHj568, 0.3 μM HiEGV-F primer, 0.3 μM HiEGV-R primer, and 2 units of Vent DNA polymerase. The reactions were incubated in an EPPENDORF®MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94° C., 30 seconds at 50° C., and 60 seconds at 72° C., followed by 25 cycles each for 30 seconds at 94° C., 30 seconds at 65° C., and 120 seconds at 72° C. (5 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 937 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.

[0414] The 937 bp purified fragment was used as template DNA for subsequent amplifications using the following primers:

TABLE-US-00010 HiEGV-R (antisense): (SEQ ID NO: 23) 5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3' HiEGV-F-overlap (sense): (SEQ ID NO: 24) 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.

[0415] The amplification reactions (50 μl) were composed of 1× ThermoPol Reaction Buffer, 0.3 mM dNTPs, 1 μl of the purified 937 bp PCR fragment, 0.3 μM HiEGV-F-overlap primer, 0.3 μM HiEGV-R primer, and 2 units of Vent DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94° C., 30 seconds at 50° C., and 60 seconds at 72° C., followed by 25 cycles each for 30 seconds at 94° C., 30 seconds at 65° C., and 120 seconds at 72° C. (5 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 945 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.

[0416] 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® Plant Maxi Kit.

TABLE-US-00011 TrCBHIpro-F (sense): (SEQ ID NO: 25) 5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' TrCBHIpro-R (antisense): (SEQ ID NO: 26) 5'-GATGCGCAGTCCGCGGT-3'

[0417] The amplification reactions (50 μl) were composed of 1× ThermoPol Reaction Buffer, 0.3 mM dNTPs, 100 ng/μl Trichoderma reesei RutC30 genomic DNA, 0.3 μM TrCBHIpro-F primer, 0.3 μM TrCBHIpro-R primer, and 2 units of Vent DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 30 seconds at 94° C., 30 seconds at 55° C., and 120 seconds at 72° C. (5 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 998 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.

[0418] The purified 998 bp PCR fragment was used as template DNA for subsequent amplifications using the primers shown below.

TABLE-US-00012 TrCBHIpro-F: (SEQ ID NO: 27) 5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' TrCBHIpro-R-overlap: (SEQ ID NO: 28) 5'-GGAGGGGGGAGGAACGCATGATGCGCAGTCCGCGGT-3'

[0419] 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.

[0420] The amplification reactions (50 μl) were composed of 1× ThermoPol Reaction Buffer, 0.3 mM dNTPs, 1 μl of the purified 998 bp PCR fragment, 0.3 μM TrCBH1pro-F primer, 0.3 μM TrCBH1pro-R-overlap primer, and 2 units of Vent DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94° C., 30 seconds at 50° C., and 60 seconds at 72° C., followed by 25 cycles each for 30 seconds at 94° C., 30 seconds at 65° C., and 120 seconds at 72° C. (5 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 1017 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.

[0421] 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 TrCBHIpro-F: (SEQ ID NO: 29) 5'-AAACGTCGACCGAATGTAGGATTGTTATC-3' HiEGV-R: (SEQ ID NO: 30) 5'-CTGCAGAATTCTACAGGCACTGATGGTACCAG-3'

[0422] The amplification reactions (50 μl) were composed of 1× ThermoPol Reaction Buffer, 0.3 mM dNTPs, 0.3 μM TrCBH1pro-F primer, 0.3 μM HiEGV-R primer, and 2 units of Vent DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 5 cycles each for 30 seconds at 94° C., 30 seconds at 50° C., and 60 seconds at 72° C., followed by 25 cycles each for 30 seconds at 94° C., 30 seconds at 65° C., and 120 seconds at 72° C. (5 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 1926 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.

[0423] The resulting 1926 bp fragment was cloned into a pCR®-Blunt-II-TOPO® vector (Invitrogen, Carlsbad, Calif., USA) using a ZEROBLUNT® TOPO® 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® 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. 8). Plasmid pMJ05 comprises the Trichoderma reesei cellobiohydrolase I promoter and terminator operably linked to the Humicola insolens endoglucanase V full-length coding sequence.

Example 12

Construction of pSMai130 Expression Vector

[0424] A 2586 bp DNA fragment spanning from the ATG start codon to the TAA stop codon of the Aspergillus oryzae beta-glucosidase full-length coding sequence (SEQ ID NO: 31 for cDNA sequence and SEQ ID NO: 32 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: 33) 5'-ATAGTCAACCGCGGACTGCGCATCATGAAGCTTGGTTGGATCGAGG- 3' Primer 993456: (SEQ ID NO: 34) 5'-ACTAGTTTACTGGGCCTTAGGCAGCG-3'

[0425] The amplification reactions (50 μl) were composed of Pfx Amplification Buffer (Invitrogen, Carlsbad, Calif., USA), 0.25 mM dNTPs, 10 ng of pJaL660, 6.4 μM primer 993467, 3.2 μM primer 993456, 1 mM MgCl₂, and 2.5 units of Pfx DNA polymerase (Invitrogen, Carlsbad, Calif., USA). The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 60 seconds at 94° C., 60 seconds at 55° C., and 180 seconds at 72° C. (15 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 2586 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.

[0426] 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: 35) 5'-GTCGACTCGAAGCCCGAATGTAGGAT-3' Primer 993463: (SEQ ID NO: 36) 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.

[0427] The amplification reactions (50 μl) were composed of Pfx Amplification Buffer, 0.25 mM dNTPs, 100 ng of Trichoderma reesei RutC30 genomic DNA, 6.4 μM primer 993453, 3.2 μM primer 993463, 1 mM MgCl₂, and 2.5 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 60 seconds at 94° C., 60 seconds at 55° C., and 180 seconds at 72° C. (15 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 1000 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.

[0428] 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.

[0429] The amplification reactions (50 μl) were composed of Pfx Amplification Buffer, 0.25 mM dNTPs, 6.4 μM primer 99353, 3.2 μM primer 993456, 1 mM MgCl₂, and 2.5 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 60 seconds at 94° C., 60 seconds at 60° C., and 240 seconds at 72° C. (15 minute final extension).

[0430] 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. 9). 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 13

Construction of pSMai135

[0431] The Aspergillus oryzae beta-glucosidase mature coding region (minus the native signal sequence, see FIG. 10; SEQ ID NOs: 37 and 38 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: 39) 5'-TGCCGGTGTTGGCCCTTGCCAAGGATGATCTCGCGTACTCCC-3' Primer 993727: (SEQ ID NO: 40) 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.

[0432] The amplification reactions (50 μl) were composed of Pfx Amplification Buffer, 0.25 mM dNTPs, 10 ng/μl of pJaI660, 6.4 μM primer 993728, 3.2 μM primer 993727, 1 mM MgCl₂, and 2.5 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 60 seconds at 94° C., 60 seconds at 55° C., and 180 seconds at 72° C. (15 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 2523 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.

[0433] 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. 11, SEQ ID NOs: 41 and 42), using primer 993724 (sense) and primer 993729 (antisense) shown below.

TABLE-US-00017 Primer 993724: (SEQ ID NO: 43) 5'-ACGCGTCGACCGAATGTAGGATTGTTATCC-3' Primer 993729: (SEQ ID NO: 44) 5'-GGGAGTACGCGAGATCATCCTTGGCAAGGGCCAACACCGGCA-3'

[0434] 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.

[0435] 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.

[0436] The amplification reactions (50 μl) were composed of Pfx Amplification Buffer, 0.25 mM dNTPs, 10 ng/μl of pMJ05, 6.4 μM primer 993728, 3.2 μM primer 993727, 1 mM MgCl₂, and 2.5 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 60 seconds at 94° C., 60 seconds at 60° C., and 240 seconds at 72° C. (15 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 1063 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.

[0437] 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.

[0438] The amplification reactions (50 μl) were composed of Pfx Amplification Buffer, 0.25 mM dNTPs, 6.4 μM primer 993724, 3.2 μM primer 993727, 1 mM MgCl₂, and 2.5 units of Pfx DNA polymerase. The reactions were incubated in an EPPENDORF® MASTERCYCLER® 5333 programmed for 30 cycles each for 60 seconds at 94° C., 60 seconds at 60° C., and 240 seconds at 72° C. (15 minute final extension). The reaction products were isolated on a 1.0% agarose gel using TAE buffer where a 3591 bp product band was excised from the gel and purified using a QIAQUICK® Gel Extraction Kit according to the manufacturer's instructions.

[0439] 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. 12). 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 14

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

[0440] Plasmid pSMai135 encoding the mature Aspergillus oryzae beta-glucosidase linked to the Humicola insolens endoglucanase V secretion signal (FIG. 11), 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.

[0441] Trichoderma reesei RutC30 was cultivated at 27° C. and 90 rpm in 25 ml of YP medium supplemented with 2% (w/v) glucose and 10 mM uridine for 17 hours. Mycelia was 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®(Novozymes A/S, Bagsv.ae butted.rd, 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° C. with gentle shaking at 90 rpm. Protoplasts were collected by centrifuging for 7 minutes at 400×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 1×10⁸ protoplasts per ml. Excess protoplasts were stored in a Cryo 1° C. Freezing Container (Nalgene, Rochester, N.Y., USA) at -80° C.

[0442] Approximately 7 μg of pSMai135 digested with Pme I were added to 100 μl of protoplast solution and mixed gently, followed by 260 μl 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° C. for 5-7 days. Transformants were sub-cultured onto COVE2 plates and grown at 28° C.

[0443] Sixty-seven transformants designated SMA135 obtained with pSMai135 were subcultured onto fresh plates containing acetamide and allowed to sporulate for 7 days at 28° C.

[0444] 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° 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×g for 5 minutes in a micro-centrifuge and the supernatants transferred to new tubes. Samples were stored at 4° C. until enzyme assay. The supernatants were assayed for beta-glucosidase activity using p-nitrophenyl-beta-D-glucopyranoside as substrate, as described below.

[0445] Beta-glucosidase activity was determined at ambient temperature using 25 μl aliquots of culture supernatants, diluted 1:10 in 50 mM succinate pH 5.0, in 200 μl 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 μl 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 μmol of p-nitrophenyl per minute per liter at pH 5.0, ambient temperature. Aspergillus niger beta-glucosidase (NOVOZYM® 188, Novozymes A/S, Bagsv.ae butted.rd, Denmark) was used as an enzyme standard.

[0446] A number of the SMA135 transformants showed beta-glucosidase activities several-fold higher than that of Trichoderma reesei RutC30. Transformant SMA135-04 produced the highest beta-glucosidase activity.

[0447] SDS-PAGE was carried out using CRITERION® Tris-HCl (5% resolving) gels (Bio-Rad, Hercules, Calif., USA) with the CRITERION® System (Bio-Rad, Hercules, Calif., USA). Five μl of day 7 supernatants (see above) were suspended in 2× 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× Tris/Glycine/SDS as running buffer (Bio-Rad, Hercules, Calif., USA). The resulting gel was stained with BIO-SAFE® Coomassie Stain.

[0448] Totally, 26 of the 38 Trichoderma reesei SMA135 transformants 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.

Example 15

Fermentation of Trichoderma reesei SMA135-04

[0449] One hundred ml of the following shake flask medium was added to a 500 ml shake flask. The shake flask medium was composed per liter of 20 g of dextrose, 10 g of corn steep solids, 1.45 g of (NH₄)₂SO₄, 2.08 g of KH₂PO₄, 0.36 g of CaCl₂, 0.42 g of MgSO₄.7H₂O, and 0.42 ml of trace metals solution. Trace metals solution was composed per liter of 216 g of FeCl₃.6H₂O, 58 g of ZnSO₄.7H₂O, 27 g of MnSO₄.H₂O, 10 g of CuSO₄.5H₂O, 2.4 g of H₃BO₃, and 336 g of citric acid. The shake flask was inoculated with two plugs from a solid plate culture of Trichoderma reesei SMA135-04 and incubated at 28° C. on an orbital shaker at 200 rpm for 48 hours.

[0450] Fifty ml of the shake flask broth was used to inoculate a 3 liter fermentation vessel containing 1.8 liters of a fermentation batch medium composed per liter of 30 g of cellulose, 4 g of dextrose, 10 g of corn steep solids, 3.8 g of (NH₄)₂SO₄, 2.8 g of KH₂PO₄, 2.64 g of CaCl₂, 1.63 g of MgSO₄.7H₂O, 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 FeCl₃.6H₂O, 58 g of ZnSO₄.7H₂O, 27 g of MnSO₄.H₂O, 10 g of CuSO₄.5H₂O, 2.4 g of H₃BO₃, and 336 g of citric acid. Fermentation feed medium was composed of dextrose and cellulose, which 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° 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×g to remove the biomass. The supernatant was sterile filtered and stored at 35 to 40° C.

Example 16

Characterization of Thielavia terrestris GH61F Polypeptide Having Cellulolytic Enhancing Activity

[0451] Corn stover was pretreated at the U.S. Department of Energy's National Renewable Energy Laboratory (NREL), Golden, Colo., using dilute sulfuric acid. The following conditions were used for the pretreatment: 0.048 g sulfuric acid/g dry biomass at 190° C. and 25% w/w dry solids for around 1 minute. According to NREL, the water-insoluble solids in the pretreated corn stover (PCS) contained 53.2% cellulose, 3.2% hemicellulose and 31.5% lignin. Cellulose and hemicellulose were determined by a two-stage sulfuric acid hydrolysis with subsequent analysis of sugars by high performance liquid chromatography using NREL Standard Analytical Procedure #002. Lignin was determined gravimetrically after hydrolyzing the cellulose and hemicellulose fractions with sulfuric acid using NREL Standard Analytical Procedure #003. Prior to enzymatic hydrolysis, the PCS was washed with a large volume of deionized water to get rid of soluble compounds produced during acid pretreatment.

[0452] The Thielavia terrestris GH61F polypeptide was expressed in Aspergillus oryzae as described in Example 7, and the broth was centrifuged at 9500×g and the supernatant was then filtered through 0.22 μm filter (Millipore, Billerica, Mass., USA). The filtered broth was desalted using an ECONO-PAC® 10DG column (Bio-Rad, Hercules, Calif., USA).

[0453] A Trichoderma reesei cellulase preparation containing an Aspergillus oryzae beta-glucosidase (WO 02/095014), hereinafter called Tr/AoBG, was obtained as described in Example 15.

[0454] Hydrolysis of PCS (45 mg/ml in 50 mM sodium acetate pH 5.0 buffer) was conducted using 96-well deep-well plates (Axygen Scientific, Inc., Union City, Calif., USA) sealed by an ALPS 300® automated lab plate sealer (ABgene Inc., Rochester, N.Y., USA), with a total reaction volume of 1.0 ml. The Thielavia terrestris GH61F polypeptide was tested for its ability to enhance the hydrolytic ability of the Trichoderma reesei cellulase preparation containing the Aspergillus oryzae beta-glucosidase. Hydrolysis of PCS was performed using 2.25, 4.5 and 6.75 mg of the Trichoderma reesei cellulase preparation containing the Aspergillus oryzae beta-glucosidase per gram of cellulose, supplemented with 0.25, 0.5 and 0.75 mg of Thielavia terrestris GH61F polypeptide per gram of cellulose, respectively, in comparison with 2.5, 5.0 and 7.5 mg of the Trichoderma reesei cellulase preparation containing the Aspergillus oryzae beta-glucosidase alone per gram of cellulose, respectively. PCS hydrolysis was performed at 50 and 60° C. in a TS Autoflow CO₂ Water Jacketed Incubator (NuAire Inc., Plymouth, Minn., USA). Reactions were run in triplicates and aliquots taken during the course of hydrolysis. PCS hydrolysis reactions were stopped by mixing a 20 μl aliquot of each hydrolyzate with 180 μl of 0.1 M NaOH (stop reagent). Appropriate serial dilutions were generated for each sample and the reducing sugar content determined using a para-hydroxybenzoic acid hydrazide (PHBAH, Sigma Chemical Co., St. Louis, Mo., USA) assay adapted to a 96 well microplate format as described below. Briefly, a 100 μl aliquot of an appropriately diluted sample was placed in a 96 well conical bottomed microplate. Reactions were initiated by adding 50 μl of 1.5% (w/v) PHBAH in 0.5 M NaOH to each well. Plates were heated uncovered at 95° C. for 10 minutes. Plates were allowed to cool to room temperature (RT) and 50 μl of distilled water added to each well. A 100 μl aliquot from each well was transferred to a flat bottomed 96 well plate and the absorbance at 410 nm measured using a SPECTRAMAX® Microplate Reader (Molecular Devices, Sunnyvale, Calif., USA). Glucose standards (0.1-0.0125 mg/ml diluted with 0.1 M sodium hydroxide) were used to prepare a standard curve to translate the obtained A₄10nm values into glucose equivalents. The resultant equivalents were used to calculate the percentage of PCS cellulose conversion for each reaction.

[0455] The degree of cellulose conversion to reducing sugar (conversion, %) was calculated using the following equation:

Conversion ( % ) = RS ( mg / ml ) × 100 × 162 / ( cellulose ( mg / ml ) × 180 ) = = RS ( mg / ml ) × 100 / ( cellulose ( m g / ml ) × 1.111 ) ##EQU00001##

In this equation, RS is the concentration of reducing sugar in solution measured in glucose equivalents (mg/ml), and the factor 1.111 reflects the weight gain in converting cellulose to glucose.

[0456] Cellulose conversions by the Trichoderma reesei cellulase preparation containing the Aspergillus oryzae beta-glucosidase alone (2.5, 5 and 7.5 mg/g cellulose) or 10% replaced by the Thielavia terrestris GH61F polypeptide (2.25+0.25, 4.5+0.5, 6.75+0.75 mg/g cellulose) are summarized in Table 1.

TABLE-US-00018 TABLE 1 Cellulose conversion by the Trichoderma reesei cellulase preparation containing an Aspergillus oryzae beta-glucosidase alone or supplemented with Thielavia terrestris GH61F polypeptide at 50° C. and 60° C., pH 5.0 for 120 hours. Loading, Test mg/g Temp, Conversion # Name cellulose ° C. at 120 h, % 1 Tr/AoBG 2.5 50 59.1 2 Tr/AoBG + 2.25 + 0.25 50 66.3 T. terrestris GH61F 3 Tr/AoBG 5.0 50 81.8 4 Tr/AoBG + 4.5 + 0.5 50 88.1 T. terrestris GH61F 5 Tr/AoBG 7.5 50 90.7 6 Tr/AoBG + 6.75 + 0.75 50 97.3 T. terrestris GH61F 7 T. terrestris GH61F 0.75 50 1.6 8 Tr/AoBG 2.5 60 30.0 9 Tr/AoBG + 2.25 + 0.25 60 34.9 T. terrestris GH61F 10 Tr/AoBG 5.0 60 44.6 11 Tr/AoBG + 4.5 + 0.5 60 51.0 T. terrestris GH61F 12 Tr/AoBG 7.5 60 55.4 13 Tr/AoBG + 6.75 + 0.75 60 63.7 T. terrestris GH61F 14 T. terrestris GH61F 0.75 60 0.9

[0457] The results shown in Table 1 demonstrated that the Thielavia terrestris GH61F polypeptide enhanced the activity of the Trichoderma reesei cellulase preparation containing the Aspergillus oryzae beta-glucosidase on PCS. The Thielavia terrestris GH61F polypeptide by itself (0.75 mg per g of cellulose) yielded a cellulose conversion after 120 hours of 1.6% at 50° C., and 0.9% at 60° C. Supplementing 0.75 mg of the Thielavia terrestris GH61F polypeptide to 6.75 mg of the Trichoderma reesei cellulase preparation containing the Aspergillus oryzae beta-glucosidase yielded a cellulose conversion higher than that by 7.5 mg of Tr/AoBG, at both 50 and 60° C., indicating the activity of the Trichoderma reesei cellulase preparation containing the Aspergillus oryzae beta-glucosidase on PCS was boosted by the Thielavia terrestris GH61F polypeptide, and that there were synergistic effects between the Trichoderma reesei cellulase preparation containing the Aspergillus oryzae beta-glucosidase and the Thielavia terrestris GH61F polypeptide.

Deposit of Biological Material

[0458] The following biological material has 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 number:

TABLE-US-00019 Deposit Accession Number Date of Deposit E. coli pTter61F NRRL B-50044 May 25, 2007

[0459] The strain has been deposited under conditions that assure that access to the culture 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. §1.14 and 35 U.S.C. §122. The deposit represents a substantially pure culture of the deposited strain. The deposit is 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.

[0460] 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.

Sequence CWU 1

821954DNAThielavia terrestris 1atgaagggcc tcagcctcct cgccgctgcg tcggcagcga ctgctcatac catcttcgtg 60cagctcgagt cagggggaac gacctatccg gtatcctacg gcatccggga ccctagctac 120gacggtccca tcaccgacgt cacctccgac tcactggctt gcaatggtcc cccgaacccc 180acgacgccgt ccccgtacat catcaacgtc accgccggca ccacggtcgc ggcgatctgg 240aggcacaccc tcacatccgg ccccgacgat gtcatggacg ccagccacaa ggggccgacc 300ctggcctacc tcaagaaggt cgatgatgcc ttgaccgaca cgggtatcgg cggcggctgg 360ttcaagatcc aggaggccgg ttacgacaat ggcaattggg ctaccagcac ggtgatcacc 420aacggtggct tccaatatat tgacatcccc gcctgcattc ccaacggcca gtatctgctc 480cgcgccgaga tgatcgcgct ccacgccgcc agcacgcagg gtggtgccca gctctacatg 540gagtgcgcgc agatcaacgt ggtgggcggc tccggcagcg ccagcccgca gacgtacagc 600atcccgggca tctaccaggc aaccgacccg ggcctgctga tcaacatcta ctccatgacg 660ccgtccagcc agtacaccat tccgggtccg cccctgttca cctgcagcgg cagcggcaac 720aacggcggcg gcagcaaccc gtcgggcggg cagaccacga cggcgaagcc cacgacgacg 780acggcggcga cgaccacctc ctccgccgct cctaccagca gccagggggg cagcagcggt 840tgcaccgttc cccagtggca gcagtgcggt ggcatctcgt tcaccggctg caccacctgc 900gcggcgggct acacctgcaa gtatctgaac gactattact cgcaatgcca gtaa 9542317PRTThielavia terrestris 2Met Lys Gly Leu Ser Leu Leu Ala Ala Ala Ser Ala Ala Thr Ala His1 5 10 15Thr Ile Phe Val Gln Leu Glu Ser Gly Gly Thr Thr Tyr Pro Val Ser 20 25 30Tyr Gly Ile Arg Asp Pro Ser Tyr Asp Gly Pro Ile Thr Asp Val Thr 35 40 45Ser Asp Ser Leu Ala Cys Asn Gly Pro Pro Asn Pro Thr Thr Pro Ser 50 55 60Pro Tyr Ile Ile Asn Val Thr Ala Gly Thr Thr Val Ala Ala Ile Trp65 70 75 80Arg His Thr Leu Thr Ser Gly Pro Asp Asp Val Met Asp Ala Ser His 85 90 95Lys Gly Pro Thr Leu Ala Tyr Leu Lys Lys Val Asp Asp Ala Leu Thr 100 105 110Asp Thr Gly Ile Gly Gly Gly Trp Phe Lys Ile Gln Glu Ala Gly Tyr 115 120 125Asp Asn Gly Asn Trp Ala Thr Ser Thr Val Ile Thr Asn Gly Gly Phe 130 135 140Gln Tyr Ile Asp Ile Pro Ala Cys Ile Pro Asn Gly Gln Tyr Leu Leu145 150 155 160Arg Ala Glu Met Ile Ala Leu His Ala Ala Ser Thr Gln Gly Gly Ala 165 170 175Gln Leu Tyr Met Glu Cys Ala Gln Ile Asn Val Val Gly Gly Ser Gly 180 185 190Ser Ala Ser Pro Gln Thr Tyr Ser Ile Pro Gly Ile Tyr Gln Ala Thr 195 200 205Asp Pro Gly Leu Leu Ile Asn Ile Tyr Ser Met Thr Pro Ser Ser Gln 210 215 220Tyr Thr Ile Pro Gly Pro Pro Leu Phe Thr Cys Ser Gly Ser Gly Asn225 230 235 240Asn Gly Gly Gly Ser Asn Pro Ser Gly Gly Gln Thr Thr Thr Ala Lys 245 250 255Pro Thr Thr Thr Thr Ala Ala Thr Thr Thr Ser Ser Ala Ala Pro Thr 260 265 270Ser Ser Gln Gly Gly Ser Ser Gly Cys Thr Val Pro Gln Trp Gln Gln 275 280 285Cys Gly Gly Ile Ser Phe Thr Gly Cys Thr Thr Cys Ala Ala Gly Tyr 290 295 300Thr Cys Lys Tyr Leu Asn Asp Tyr Tyr Ser Gln Cys Gln305 310 315316DNAThielavia terrestris 3gtaaaacgac ggccag 16425DNAThielavia terrestrismisc_feature(24)..(24)V=G, A, C 4tttttttttt tttttttttt tttvn 25534DNAThielavia terrestris 5actggattta ccatgaaggg cctcagcctc ctcg 34640DNAThielavia terrestris 6tcacctctag ttaattaatt actggcattg cgagtaatag 40729DNATrichoderma reesei 7aacgttaatt aaggaatcgt tttgtgttt 29829DNATrichoderma reesei 8agtactagta gctccgtggc gaaagcctg 299918DNAHumicola insolens 9atgcgttcct 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 91810305PRTHumicola insolens 10Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro1 5 10 15Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20 25 30Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro 35 40 45Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50 55 60Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln65 70 75 80Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr 85 90 95Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 100 105 110Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln 115 120 125Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130 135 140Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe145 150 155 160Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu 165 170 175Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180 185 190Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val 195 200 205Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp 210 215 220Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser225 230 235 240Pro Val Asn Gln Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr 245 250 255Ser Ser Pro Pro Val Gln Pro Thr Thr Pro Ser Gly Cys Thr Ala Glu 260 265 270Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr Thr Cys 275 280 285Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys 290 295 300Leu3051131DNASaccharomyces cerevisiae 11ttgaattgaa aatagattga tttaaaactt c 311225DNASaccharomyces cerevisiae 12ttgcatgcgt aatcatggtc atagc 251326DNASaccharomyces cerevisiae 13ttgaattcat gggtaataac tgatat 261432DNASaccharomyces cerevisiae 14aaatcaatct attttcaatt caattcatca tt 321511DNAAspergillus niger 15gtactaaaac c 111611DNAAspergillus niger 16ccgttaaatt t 111745DNAAspergillus niger 17ggatgctgtt gactccggaa atttaacggt ttggtcttgc atccc 451814DNAAspergillus niger 18atgcaattta aact 141914DNAAspergillus niger 19cggcaattta acgg 142044DNAAspergillus niger 20ggtattgtcc tgcagacggc aatttaacgg cttctgcgaa tcgc 442129DNAHumicola insolens 21aagcttaagc atgcgttcct cccccctcc 292232DNAHumicola insolens 22ctgcagaatt ctacaggcac tgatggtacc ag 322332DNATrichoderma reesei 23ctgcagaatt ctacaggcac tgatggtacc ag 322436DNATrichoderma reesei 24accgcggact gcgcatcatg cgttcctccc ccctcc 362529DNATrichoderma reesei 25aaacgtcgac cgaatgtagg attgttatc 292617DNATrichoderma reesei 26gatgcgcagt ccgcggt 172729DNATrichoderma reesei 27aaacgtcgac cgaatgtagg attgttatc 292836DNATrichoderma reesei 28ggagggggga ggaacgcatg atgcgcagtc cgcggt 362929DNATrichoderma reesei 29aaacgtcgac cgaatgtagg attgttatc 293032DNATrichoderma reesei 30ctgcagaatt ctacaggcac tgatggtacc ag 32312771DNAAspergillus oryzae 31ctgttctgct 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 277132861PRTAspergillus oryzae 32Met Lys Leu Gly Trp Ile Glu Val Ala Ala Leu Ala Ala Ala Ser Val1 5 10 15Val Ser Ala Lys Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser 20 25 30Pro Trp Ala Asp Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala 35 40 45Val Asp Ile Val Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr 50 55 60Thr Gly Thr Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser65 70 75 80Val Pro Arg Leu Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu 85 90 95Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn 100 105 110Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala 115 120 125Met Gly Glu Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro 130 135 140Ala Ala Gly Pro Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu145 150 155 160Gly Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185 190Ile Met Asn Glu Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly 195 200 205Tyr Gly Phe Asn Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys 210 215 220Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala225 230 235 240Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245 250 255Gly Cys Glu Asn Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265 270Gly Phe Gln Gly Phe Val Met Ser Asp Trp Thr Ala His His Ser Gly 275 280 285Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val 290 295 300Thr Phe Asp Ser Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly305 310 315 320Val Leu Asn Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val 325 330 335Arg Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr 340 345 350Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His 355 360 365Asn His Val Ser Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp 370 375 380Val Gln Arg Asp His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser385 390 395 400Thr Val Leu Leu Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu 405 410 415Lys Leu Val Ala Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly 420 425 430Ala Asn Gly Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435 440 445Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu 450 455 460Gln Ala Ile Gln Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala465 470 475 480Val Thr Asp Ser Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln 485 490 495Ala Ser Val Ser Leu Val Phe Val Asn Ser Asp Ser Gly Glu Ser Tyr 500 505 510Leu Ser Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp 515 520 525Lys Asn Gly Asp Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn 530 535 540Thr Val Val Ile Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp545 550 555 560Tyr Asp His Pro Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly 565 570 575Gln Glu Ser Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn 580 585 590Pro Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595 600 605Gly Ser Pro Leu Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln 610 615 620Ser Asp Phe Thr Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys625 630 635 640Phe Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr 645 650 655Thr Phe Glu Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg 660 665

670Tyr Thr Pro Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu 675 680 685Ile Gly Asp Ala Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile 690 695 700His Glu Phe Ile Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser705 710 715 720Ser Asp Asp Ser Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu 725 730 735Gly Ala Thr Asp Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly 740 745 750Ala Gly Gly Asn Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val 755 760 765Lys Val Lys Asn Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu 770 775 780Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys785 790 795 800Phe Glu Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr 805 810 815Thr Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 820 825 830Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser Ser 835 840 845Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 850 855 8603346DNAAspergillus oryzae 33atagtcaacc gcggactgcg catcatgaag cttggttgga tcgagg 463426DNAAspergillus oryzae 34actagtttac tgggccttag gcagcg 263526DNATrichoderma reesei 35gtcgactcga agcccgaatg taggat 263645DNATrichoderma reesei 36cctcgatcca accaagcttc atgatgcgca gtccgcggtt gacta 453757DNAAspergillus oryzae 37atgaagcttg gttggatcga ggtggccgca ttggcggctg cctcagtagt cagtgcc 573819PRTAspergillus oryzae 38Met Lys Leu Gly Trp Ile Glu Val Ala Ala Leu Ala Ala Ala Ser Val1 5 10 15Val Ser Ala3942DNAAspergillus oryzae 39tgccggtgtt ggcccttgcc aaggatgatc tcgcgtactc cc 424028DNAAspergillus oryzae 40gactagtctt actgggcctt aggcagcg 284163DNAHumicola insolens 41atgcgttcct cccccctcct ccgctccgcc gttgtggccg ccctgccggt gttggccctt 60gcc 634221PRTHumicola insolens 42Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro1 5 10 15Val Leu Ala Leu Ala 204330DNATrichoderma reesei 43acgcgtcgac cgaatgtagg attgttatcc 304442DNATrichoderma reesei 44gggagtacgc gagatcatcc ttggcaaggg ccaacaccgg ca 42451377DNATrichoderma reesei 45atggcgccct 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 137746459PRTTrichoderma reesei 46Met Ala Pro Ser Val Thr Leu Pro Leu Thr Thr Ala Ile Leu Ala Ile1 5 10 15Ala Arg Leu Val Ala Ala Gln Gln Pro Gly Thr Ser Thr Pro Glu Val 20 25 30His Pro Lys Leu Thr Thr Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val 35 40 45Ala Gln Asp Thr Ser Val Val Leu Asp Trp Asn Tyr Arg Trp Met His 50 55 60Asp Ala Asn Tyr Asn Ser Cys Thr Val Asn Gly Gly Val Asn Thr Thr65 70 75 80Leu Cys Pro Asp Glu Ala Thr Cys Gly Lys Asn Cys Phe Ile Glu Gly 85 90 95Val Asp Tyr Ala Ala Ser Gly Val Thr Thr Ser Gly Ser Ser Leu Thr 100 105 110Met Asn Gln Tyr Met Pro Ser Ser Ser Gly Gly Tyr Ser Ser Val Ser 115 120 125Pro Arg Leu Tyr Leu Leu Asp Ser Asp Gly Glu Tyr Val Met Leu Lys 130 135 140Leu Asn Gly Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro145 150 155 160Cys Gly Glu Asn Gly Ser Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly 165 170 175Gly Ala Asn Gln Tyr Asn Thr Ala Gly Ala Asn Tyr Gly Ser Gly Tyr 180 185 190Cys Asp Ala Gln Cys Pro Val Gln Thr Trp Arg Asn Gly Thr Leu Asn 195 200 205Thr Ser His Gln Gly Phe Cys Cys Asn Glu Met Asp Ile Leu Glu Gly 210 215 220Asn Ser Arg Ala Asn Ala Leu Thr Pro His Ser Cys Thr Ala Thr Ala225 230 235 240Cys Asp Ser Ala Gly Cys Gly Phe Asn Pro Tyr Gly Ser Gly Tyr Lys 245 250 255Ser Tyr Tyr Gly Pro Gly Asp Thr Val Asp Thr Ser Lys Thr Phe Thr 260 265 270Ile Ile Thr Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu 275 280 285Val Ser Ile Thr Arg Lys Tyr Gln Gln Asn Gly Val Asp Ile Pro Ser 290 295 300Ala Gln Pro Gly Gly Asp Thr Ile Ser Ser Cys Pro Ser Ala Ser Ala305 310 315 320Tyr Gly Gly Leu Ala Thr Met Gly Lys Ala Leu Ser Ser Gly Met Val 325 330 335Leu Val Phe Ser Ile Trp Asn Asp Asn Ser Gln Tyr Met Asn Trp Leu 340 345 350Asp Ser Gly Asn Ala Gly Pro Cys Ser Ser Thr Glu Gly Asn Pro Ser 355 360 365Asn Ile Leu Ala Asn Asn Pro Asn Thr His Val Val Phe Ser Asn Ile 370 375 380Arg Trp Gly Asp Ile Gly Ser Thr Thr Asn Ser Thr Ala Pro Pro Pro385 390 395 400Pro Pro Ala Ser Ser Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser Thr 405 410 415Thr Ser Ser Ser Pro Ser Cys Thr Gln Thr His Trp Gly Gln Cys Gly 420 425 430Gly Ile Gly Tyr Ser Gly Cys Lys Thr Cys Thr Ser Gly Thr Thr Cys 435 440 445Gln Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu 450 455471254DNATrichoderma reesei 47atgaacaagt 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 125448418PRTTrichoderma reesei 48Met Asn Lys Ser Val Ala Pro Leu Leu Leu Ala Ala Ser Ile Leu Tyr1 5 10 15Gly Gly Ala Val Ala Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Ile 20 25 30Gly Trp Ser Gly Pro Thr Asn Cys Ala Pro Gly Ser Ala Cys Ser Thr 35 40 45Leu Asn Pro Tyr Tyr Ala Gln Cys Ile Pro Gly Ala Thr Thr Ile Thr 50 55 60Thr Ser Thr Arg Pro Pro Ser Gly Pro Thr Thr Thr Thr Arg Ala Thr65 70 75 80Ser Thr Ser Ser Ser Thr Pro Pro Thr Ser Ser Gly Val Arg Phe Ala 85 90 95Gly Val Asn Ile Ala Gly Phe Asp Phe Gly Cys Thr Thr Asp Gly Thr 100 105 110Cys Val Thr Ser Lys Val Tyr Pro Pro Leu Lys Asn Phe Thr Gly Ser 115 120 125Asn Asn Tyr Pro Asp Gly Ile Gly Gln Met Gln His Phe Val Asn Glu 130 135 140Asp Gly Met Thr Ile Phe Arg Leu Pro Val Gly Trp Gln Tyr Leu Val145 150 155 160Asn Asn Asn Leu Gly Gly Asn Leu Asp Ser Thr Ser Ile Ser Lys Tyr 165 170 175Asp Gln Leu Val Gln Gly Cys Leu Ser Leu Gly Ala Tyr Cys Ile Val 180 185 190Asp Ile His Asn Tyr Ala Arg Trp Asn Gly Gly Ile Ile Gly Gln Gly 195 200 205Gly Pro Thr Asn Ala Gln Phe Thr Ser Leu Trp Ser Gln Leu Ala Ser 210 215 220Lys Tyr Ala Ser Gln Ser Arg Val Trp Phe Gly Ile Met Asn Glu Pro225 230 235 240His Asp Val Asn Ile Asn Thr Trp Ala Ala Thr Val Gln Glu Val Val 245 250 255Thr Ala Ile Arg Asn Ala Gly Ala Thr Ser Gln Phe Ile Ser Leu Pro 260 265 270Gly Asn Asp Trp Gln Ser Ala Gly Ala Phe Ile Ser Asp Gly Ser Ala 275 280 285Ala Ala Leu Ser Gln Val Thr Asn Pro Asp Gly Ser Thr Thr Asn Leu 290 295 300Ile Phe Asp Val His Lys Tyr Leu Asp Ser Asp Asn Ser Gly Thr His305 310 315 320Ala Glu Cys Thr Thr Asn Asn Ile Asp Gly Ala Phe Ser Pro Leu Ala 325 330 335Thr Trp Leu Arg Gln Asn Asn Arg Gln Ala Ile Leu Thr Glu Thr Gly 340 345 350Gly Gly Asn Val Gln Ser Cys Ile Gln Asp Met Cys Gln Gln Ile Gln 355 360 365Tyr Leu Asn Gln Asn Ser Asp Val Tyr Leu Gly Tyr Val Gly Trp Gly 370 375 380Ala Gly Ser Phe Asp Ser Thr Tyr Val Leu Thr Glu Thr Pro Thr Ser385 390 395 400Ser Gly Asn Ser Trp Thr Asp Thr Ser Leu Val Ser Ser Cys Leu Ala 405 410 415Arg Lys49702DNATrichoderma reesei 49atgaagttcc 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 70250234PRTTrichoderma reesei 50Met Lys Phe Leu Gln Val Leu Pro Ala Leu Ile Pro Ala Ala Leu Ala1 5 10 15Gln Thr Ser Cys Asp Gln Trp Ala Thr Phe Thr Gly Asn Gly Tyr Thr 20 25 30Val Ser Asn Asn Leu Trp Gly Ala Ser Ala Gly Ser Gly Phe Gly Cys 35 40 45Val Thr Ala Val Ser Leu Ser Gly Gly Ala Ser Trp His Ala Asp Trp 50 55 60Gln Trp Ser Gly Gly Gln Asn Asn Val Lys Ser Tyr Gln Asn Ser Gln65 70 75 80Ile Ala Ile Pro Gln Lys Arg Thr Val Asn Ser Ile Ser Ser Met Pro 85 90 95Thr Thr Ala Ser Trp Ser Tyr Ser Gly Ser Asn Ile Arg Ala Asn Val 100 105 110Ala Tyr Asp Leu Phe Thr Ala Ala Asn Pro Asn His Val Thr Tyr Ser 115 120 125Gly Asp Tyr Glu Leu Met Ile Trp Leu Gly Lys Tyr Gly Asp Ile Gly 130 135 140Pro Ile Gly Ser Ser Gln Gly Thr Val Asn Val Gly Gly Gln Ser Trp145 150 155 160Thr Leu Tyr Tyr Gly Tyr Asn Gly Ala Met Gln Val Tyr Ser Phe Val 165 170 175Ala Gln Thr Asn Thr Thr Asn Tyr Ser Gly Asp Val Lys Asn Phe Phe 180 185 190Asn Tyr Leu Arg Asp Asn Lys Gly Tyr Asn Ala Ala Gly Gln Tyr Val 195 200 205Leu Ser Tyr Gln Phe Gly Thr Glu Pro Phe Thr Gly Ser Gly Thr Leu 210 215 220Asn Val Ala Ser Trp Thr Ala Ser Ile Asn225 23051726DNATrichoderma reesei 51atgaaggcaa 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 72652242PRTTrichoderma reesei 52Met Lys Ala Thr Leu Val Leu Gly Ser Leu Ile Val Gly Ala Val Ser1 5 10 15Ala Tyr Lys Ala Thr Thr Thr Arg Tyr Tyr Asp Gly Gln Glu Gly Ala 20 25 30Cys Gly Cys Gly Ser Ser Ser Gly Ala Phe Pro Trp Gln Leu Gly Ile 35 40 45Gly Asn Gly Val Tyr Thr Ala Ala Gly Ser Gln Ala Leu Phe Asp Thr 50 55 60Ala Gly Ala Ser Trp Cys Gly Ala Gly Cys Gly Lys Cys Tyr Gln Leu65 70 75 80Thr Ser Thr Gly Gln Ala Pro Cys Ser Ser Cys Gly Thr Gly Gly Ala 85 90 95Ala Gly Gln Ser Ile Ile Val Met Val Thr Asn Leu Cys Pro Asn Asn 100 105 110Gly Asn Ala Gln Trp Cys Pro Val Val Gly Gly Thr Asn Gln Tyr Gly 115 120 125Tyr Ser Tyr His Phe Asp Ile Met Ala Gln Asn Glu Ile Phe Gly Asp 130 135 140Asn Val Val Val Asp Phe Glu Pro Ile Ala Cys Pro Gly Gln Ala Ala145 150 155 160Ser Asp Trp Gly Thr Cys Leu Cys Val Gly Gln Gln Glu Thr Asp Pro 165 170 175Thr Pro Val Leu Gly Asn Asp Thr Gly Ser Thr Pro Pro Gly Ser Ser 180 185 190Pro Pro Ala Thr Ser Ser Ser Pro Pro Ser Gly Gly Gly Gln Gln Thr 195 200 205Leu Tyr Gly Gln Cys Gly Gly Ala Gly Trp Thr Gly Pro Thr Thr Cys 210 215 220Gln Ala Pro Gly Thr Cys Lys Val Gln Asn Gln Trp Tyr Ser Gln Cys225 230 235 240Leu Pro531371DNAMyceliophthora thermophila 53atgggtcgcg gcgctgcttt cctaggcctc gcctcgctcc tcgtgggcgc ggccaaggcc 60cagacgcccg gcgagggcga ggaggtgcac

ccgcagatca cgacgtaccg ctgcaccaag 120gcggacgggt gcgaggagaa gaccaactac atcgtgctgg acgccctatc gcacccggtc 180caccaggtcg acaacccgta caactgcggc gactggggcc agaagcccaa cgagacggcc 240tgcccggacc tcgagtcgtg cgccaggaac tgcatcatgg acccggtctc ggactacggc 300cggcacggtg tctcgaccga cggcacctcg ctgcgcctca agcagctagt cggcggcaac 360gtcgtcagcc cgcgcgtcta cctgctcgac gagaccaagg agcgctacga gatgctcaag 420ctgaccggca acgagttcac ctttgacgtc gacgccacca agctgccctg cggcatgaac 480agcgccctct acctctccga gatggacgcc accggcgccc ggagcgagct caacccgggc 540ggcgccacct ttggcaccgg ctactgcgac gcccagtgct acgtcacccc cttcatcaac 600ggcctcggca acatcgaggg caagggcgcg tgctgcaacg agatggatat ctgggaggcc 660aacgcgcggg cgcagcacat cgcgccgcac ccgtgcagca aggcggggcc gtacctgtgc 720gagggcgccg agtgcgagtt cgacggcgtg tgcgacaaga acggctgcgc ctggaacccg 780taccgggtca acgtgacgga ctactacggc gagggcgccg agttcagggt ggacacgacc 840cggcccttct cggtcgtcac gcagttccgc gccggcggcg acgcgggggg cggcaagctc 900gagagcatct accggctctt cgtccaggac ggcagggtga ttgagtcgta cgtcgtcgac 960aagcccggcc tgcccccgac ggaccgcatg acggacgagt tctgcgccgc caccggcgcc 1020gcccgcttca cggagctcgg cgccatggag gccatgggcg acgccctgac gcgcggcatg 1080gtcctcgccc tcagcatctg gtggagcgag ggcgacaaca tgaactggct cgactcgggc 1140gaggccggcc cctgcgaccc ggacgagggc aacccgtcca acatcatccg cgtccagccc 1200gacccggagg tcgtcttcag caacctgcgc tggggcgaga tcggctcaac ctacgagtcc 1260gccgtcgacg ggcccgtcgg caagggcaag ggcaagggca agggcaaggc tcccgccggc 1320gacggcaacg ggaaggagaa gagcaatggc aagcgcttca ggaggttctg a 137154456PRTMyceliophthora thermophila 54Met Gly Arg Gly Ala Ala Phe Leu Gly Leu Ala Ser Leu Leu Val Gly1 5 10 15Ala Ala Lys Ala Gln Thr Pro Gly Glu Gly Glu Glu Val His Pro Gln 20 25 30Ile Thr Thr Tyr Arg Cys Thr Lys Ala Asp Gly Cys Glu Glu Lys Thr 35 40 45Asn Tyr Ile Val Leu Asp Ala Leu Ser His Pro Val His Gln Val Asp 50 55 60Asn Pro Tyr Asn Cys Gly Asp Trp Gly Gln Lys Pro Asn Glu Thr Ala65 70 75 80Cys Pro Asp Leu Glu Ser Cys Ala Arg Asn Cys Ile Met Asp Pro Val 85 90 95Ser Asp Tyr Gly Arg His Gly Val Ser Thr Asp Gly Thr Ser Leu Arg 100 105 110Leu Lys Gln Leu Val Gly Gly Asn Val Val Ser Pro Arg Val Tyr Leu 115 120 125Leu Asp Glu Thr Lys Glu Arg Tyr Glu Met Leu Lys Leu Thr Gly Asn 130 135 140Glu Phe Thr Phe Asp Val Asp Ala Thr Lys Leu Pro Cys Gly Met Asn145 150 155 160Ser Ala Leu Tyr Leu Ser Glu Met Asp Ala Thr Gly Ala Arg Ser Glu 165 170 175Leu Asn Pro Gly Gly Ala Thr Phe Gly Thr Gly Tyr Cys Asp Ala Gln 180 185 190Cys Tyr Val Thr Pro Phe Ile Asn Gly Leu Gly Asn Ile Glu Gly Lys 195 200 205Gly Ala Cys Cys Asn Glu Met Asp Ile Trp Glu Ala Asn Ala Arg Ala 210 215 220Gln His Ile Ala Pro His Pro Cys Ser Lys Ala Gly Pro Tyr Leu Cys225 230 235 240Glu Gly Ala Glu Cys Glu Phe Asp Gly Val Cys Asp Lys Asn Gly Cys 245 250 255Ala Trp Asn Pro Tyr Arg Val Asn Val Thr Asp Tyr Tyr Gly Glu Gly 260 265 270Ala Glu Phe Arg Val Asp Thr Thr Arg Pro Phe Ser Val Val Thr Gln 275 280 285Phe Arg Ala Gly Gly Asp Ala Gly Gly Gly Lys Leu Glu Ser Ile Tyr 290 295 300Arg Leu Phe Val Gln Asp Gly Arg Val Ile Glu Ser Tyr Val Val Asp305 310 315 320Lys Pro Gly Leu Pro Pro Thr Asp Arg Met Thr Asp Glu Phe Cys Ala 325 330 335Ala Thr Gly Ala Ala Arg Phe Thr Glu Leu Gly Ala Met Glu Ala Met 340 345 350Gly Asp Ala Leu Thr Arg Gly Met Val Leu Ala Leu Ser Ile Trp Trp 355 360 365Ser Glu Gly Asp Asn Met Asn Trp Leu Asp Ser Gly Glu Ala Gly Pro 370 375 380Cys Asp Pro Asp Glu Gly Asn Pro Ser Asn Ile Ile Arg Val Gln Pro385 390 395 400Asp Pro Glu Val Val Phe Ser Asn Leu Arg Trp Gly Glu Ile Gly Ser 405 410 415Thr Tyr Glu Ser Ala Val Asp Gly Pro Val Gly Lys Gly Lys Gly Lys 420 425 430Gly Lys Gly Lys Ala Pro Ala Gly Asp Gly Asn Gly Lys Glu Lys Ser 435 440 445Asn Gly Lys Arg Phe Arg Arg Phe 450 45555744DNAChrysosoporium lucknowense 55atgcagccgt ttctgctctt gttcctctcg tcggtcacgg cggcgagccc cctgacggcg 60ctcgacaagc ggcagcaggc gacgttgtgc gagcagtacg gctactggtc gggcaacggt 120tacgaggtca acaacaacaa ctggggcaag gattcggcct cgggcggcca tcagtgcacc 180tacgtcgaca gcagcagctc cagcggcgtc gcctggcaca cgacctggca gtgggaagga 240ggccagaacc aggtcaagag cttcgccaac tgcggcctgc aggtgcccaa gggcaggacc 300atctcgtcca tcagcaacct gcagacctcc atctcgtggt cctacagcaa caccaacatc 360cgcgccaacg tggcctacga cctcttcacc gcggcagacc cgaaccacgc gaccagcagc 420ggcgactacg agctcatgat ctggctggcg agattcggcg acgtctaccc catcggctcg 480tcccagggcc acgtcaacgt ggccggccag gactgggagc tgtggacggg cttcaacggc 540aacatgcggg tctacagctt cgtagcgccc agcccccgca acagcttcag cgccaacgtc 600aaggacttct tcaactatct ccagtccaac cagggcttcc cggccagcag ccaatacctt 660ctcatcttcc aggcgggcac cgagcccttc accggcggcg agaccaccct taccgtcaac 720aactactctg caagggttgc ttaa 74456247PRTChrysosoporium lucknowense 56Met Gln Pro Phe Leu Leu Leu Phe Leu Ser Ser Val Thr Ala Ala Ser1 5 10 15Pro Leu Thr Ala Leu Asp Lys Arg Gln Gln Ala Thr Leu Cys Glu Gln 20 25 30Tyr Gly Tyr Trp Ser Gly Asn Gly Tyr Glu Val Asn Asn Asn Asn Trp 35 40 45Gly Lys Asp Ser Ala Ser Gly Gly His Gln Cys Thr Tyr Val Asp Ser 50 55 60Ser Ser Ser Ser Gly Val Ala Trp His Thr Thr Trp Gln Trp Glu Gly65 70 75 80Gly Gln Asn Gln Val Lys Ser Phe Ala Asn Cys Gly Leu Gln Val Pro 85 90 95Lys Gly Arg Thr Ile Ser Ser Ile Ser Asn Leu Gln Thr Ser Ile Ser 100 105 110Trp Ser Tyr Ser Asn Thr Asn Ile Arg Ala Asn Val Ala Tyr Asp Leu 115 120 125Phe Thr Ala Ala Asp Pro Asn His Ala Thr Ser Ser Gly Asp Tyr Glu 130 135 140Leu Met Ile Trp Leu Ala Arg Phe Gly Asp Val Tyr Pro Ile Gly Ser145 150 155 160Ser Gln Gly His Val Asn Val Ala Gly Gln Asp Trp Glu Leu Trp Thr 165 170 175Gly Phe Asn Gly Asn Met Arg Val Tyr Ser Phe Val Ala Pro Ser Pro 180 185 190Arg Asn Ser Phe Ser Ala Asn Val Lys Asp Phe Phe Asn Tyr Leu Gln 195 200 205Ser Asn Gln Gly Phe Pro Ala Ser Ser Gln Tyr Leu Leu Ile Phe Gln 210 215 220Ala Gly Thr Glu Pro Phe Thr Gly Gly Glu Thr Thr Leu Thr Val Asn225 230 235 240Asn Tyr Ser Ala Arg Val Ala 24557678DNAChrysosoporium lucknowense 57atgcatctct ccgccaccac cgggttcctc gccctcccgg ccctggccct ggcccagctc 60tcgggcagcg gccagacgac ccggtactgg gactgctgca agccgagctg cgcctggccc 120ggcaagggcc cctcgtctcc ggtgcaggcc tgcgacaaga acgacaaccc gctcaacgac 180ggcggctcca cccggtccgg ctgcgacgcg ggcggcagcg cctacatgtg ctcctcccag 240agcccctggg ccgtcagcga cgagctgtcg tacggctggg cggccgtcaa gctcgccggc 300agctccgagt cgcagtggtg ctgcgcctgc tacgagctga ccttcaccag cgggccggtc 360gcgggcaaga agatgattgt gcaggcgacc aacaccggtg gcgacctggg cgacaaccac 420tttgacctgg ccatccccgg tggcggtgtc ggtattttca acgcctgcac cgaccagtac 480ggcgctcccc cgaacggctg gggcgaccgc tacggcggca tccattccaa ggaagagtgc 540gaatccttcc cggaggccct caagcccggc tgcaactggc gcttcgactg gttccaaaac 600gccgacaacc cgtcggtcac cttccaggag gtggcctgcc cgtcggagct cacgtccaag 660agcggctgct cccgttaa 67858225PRTChrysosoporium lucknowense 58Met His Leu Ser Ala Thr Thr Gly Phe Leu Ala Leu Pro Ala Leu Ala1 5 10 15Leu Ala Gln Leu Ser Gly Ser Gly Gln Thr Thr Arg Tyr Trp Asp Cys 20 25 30Cys Lys Pro Ser Cys Ala Trp Pro Gly Lys Gly Pro Ser Ser Pro Val 35 40 45Gln Ala Cys Asp Lys Asn Asp Asn Pro Leu Asn Asp Gly Gly Ser Thr 50 55 60Arg Ser Gly Cys Asp Ala Gly Gly Ser Ala Tyr Met Cys Ser Ser Gln65 70 75 80Ser Pro Trp Ala Val Ser Asp Glu Leu Ser Tyr Gly Trp Ala Ala Val 85 90 95Lys Leu Ala Gly Ser Ser Glu Ser Gln Trp Cys Cys Ala Cys Tyr Glu 100 105 110Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Ile Val Gln 115 120 125Ala Thr Asn Thr Gly Gly Asp Leu Gly Asp Asn His Phe Asp Leu Ala 130 135 140Ile Pro Gly Gly Gly Val Gly Ile Phe Asn Ala Cys Thr Asp Gln Tyr145 150 155 160Gly Ala Pro Pro Asn Gly Trp Gly Asp Arg Tyr Gly Gly Ile His Ser 165 170 175Lys Glu Glu Cys Glu Ser Phe Pro Glu Ala Leu Lys Pro Gly Cys Asn 180 185 190Trp Arg Phe Asp Trp Phe Gln Asn Ala Asp Asn Pro Ser Val Thr Phe 195 200 205Gln Glu Val Ala Cys Pro Ser Glu Leu Thr Ser Lys Ser Gly Cys Ser 210 215 220Arg225591542DNATrichoderma reesei 59atgtatcgga 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 154260514PRTTrichoderma reesei 60Met Tyr Arg Lys Leu Ala Val Ile Ser Ala Phe Leu Ala Thr Ala Arg1 5 10 15Ala Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu Thr 20 25 30Trp Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser 35 40 45Val Val Ile Asp Ala Asn Trp Arg Trp Thr His Ala Thr Asn Ser Ser 50 55 60Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser Thr Leu Cys Pro Asp65 70 75 80Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala 85 90 95Ser Thr Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe 100 105 110Val Thr Gln Ser Ala Gln Lys Asn Val Gly Ala Arg Leu Tyr Leu Met 115 120 125Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu Gly Asn Glu Phe 130 135 140Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala145 150 155 160Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro 165 170 175Thr Asn Thr Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser Gln 180 185 190Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn Val Glu Gly 195 200 205Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly 210 215 220Ser Cys Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu225 230 235 240Ala Leu Thr Pro His Pro Cys Thr Thr Val Gly Gln Glu Ile Cys Glu 245 250 255Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly Gly Thr 260 265 270Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr 275 280 285Ser Phe Tyr Gly Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys 290 295 300Leu Thr Val Val Thr Gln Phe Glu Thr Ser Gly Ala Ile Asn Arg Tyr305 310 315 320Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn Ala Glu Leu Gly 325 330 335Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu 340 345 350Ala Glu Phe Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln 355 360 365Phe Lys Lys Ala Thr Ser Gly Gly Met Val Leu Val Met Ser Leu Trp 370 375 380Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr Tyr Pro Thr385 390 395 400Asn Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr 405 410 415Ser Ser Gly Val Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys 420 425 430Val Thr Phe Ser Asn Ile Lys Phe Gly Pro Ile Gly Ser Thr Gly Asn 435 440 445Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr Thr Thr 450 455 460Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln465 470 475 480Ser His Tyr Gly Gln Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val 485 490 495Cys Ala Ser Gly Thr Thr Cys Gln Val Leu Asn Pro Tyr Tyr Ser Gln 500 505 510Cys Leu611413DNATrichoderma reesei 61atgattgtcg 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 141362471PRTTrichoderma reesei 62Met Ile Val Gly Ile Leu Thr Thr Leu Ala Thr Leu Ala Thr Leu Ala1 5 10 15Ala Ser Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly 20 25 30Gln Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala Ser Gly 35 40

45Ser Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly 50 55 60Ala Ala Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg65 70 75 80Val Ser Pro Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly 85 90 95Ser Thr Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr Tyr 100 105 110Ser Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr 115 120 125Ala Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met 130 135 140Ala Thr Ala Ala Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu145 150 155 160Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile 165 170 175Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 180 185 190Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu 195 200 205Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 210 215 220Thr Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu225 230 235 240Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr 245 250 255Pro Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr 260 265 270Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala 275 280 285Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala 290 295 300Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu305 310 315 320Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr 325 330 335Ser Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu 340 345 350Tyr Ile His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn 355 360 365Ala Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly 370 375 380Gln Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly385 390 395 400Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val 405 410 415Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ala 420 425 430Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala 435 440 445Pro Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr 450 455 460Asn Ala Asn Pro Ser Phe Leu465 470631648DNAChrysosoporium lucknowensemisc_feature(1162)..(1162)N= A,C,G, OR T 63atgtacgcca agttcgcgac cctcgccgcc cttgtggctg gcgccgctgc tcagaacgcc 60tgcactctga ccgctgagaa ccacccctcg ctgacgtggt ccaagtgcac gtctggcggc 120agctgcacca gcgtccaggg ttccatcacc atcgacgcca actggcggtg gactcaccgg 180accgatagcg ccaccaactg ctacgagggc aacaagtggg atacttcgta ctgcagcgat 240ggtccttctt gcgcctccaa gtgctgcatc gacggcgctg actactcgag cacctatggc 300atcaccacga gcggtaactc cctgaacctc aagttcgtca ccaagggcca gtactcgacc 360aacatcggct cgcgtaccta cctgatggag agcgacacca agtaccagag taagttcctc 420tcgcacccgg ccgccgggag atgatggcgc ccagcccgct gacgcgaatg acacagtgtt 480ccagctcctc ggcaacgagt tcaccttcga tgtcgacgtc tccaacctcg gctgcggcct 540caatggcgcc ctctacttcg tgtccatgga tgccgatggt ggcatgtcca agtactcggg 600caacaaggca ggtgccaagt acggtaccgg ctactgtgat tctcagtgcc cccgcgacct 660caagttcatc aacggcgagg ccaacgtaga gaactggcag agctcgacca acgatgccaa 720cgccggcacg ggcaagtacg gcagctgctg ctccgagatg gacgtctggg aggccaacaa 780catggccgcc gccttcactc cccacccttg caccgtgatc ggccagtcgc gctgcgaggg 840cgactcgtgc ggcggtacct acagcaccga ccgctatgcc ggcatctgcg accccgacgg 900atgcgacttc aactcgtacc gccagggcaa caagaccttc tacggcaagg gcatgacggt 960cgacacgacc aagaagatca cggtcgtcac ccagttcctc aagaactcgg ccggcgagct 1020ctccgagatc aagcggttct acgtccagaa cggcaaggtc atccccaact ccgagtccac 1080catcccgggc gtcgagggca actccatcac ccaggactgg tgcgaccgcc agaaggccgc 1140cttcggcgac gtgaccgact tncaggacaa gggcggcatg gtccagatgg gcaaggccct 1200cgcggggccc atggtcctcg tcatgtccat ctgggacgac cacgccgtca acatgctctg 1260gctcgactcc acctggccca tcgacggcgc cggcaagccg ggcgccgagc gcggtgcctg 1320ccccaccacc tcgggcgtcc ccgctgaggt cgaggccgag gcccccaact ccaacgtcat 1380cttctccaac atccgcttcg gccccatcgg ctccaccgtc tccggcctgc ccgacggcgg 1440cagcggcaac cccaacccgc ccgtcagctc gtccaccccg gtcccctcct cgtccaccac 1500atcctccggt tcctccggcc cgactggcgg cacgggtgtc gctaagcact atgagcaatg 1560cggaggaatc gggttcactg gccctaccca gtgcgagagc ccctacactt gcaccaagct 1620gaatgactgg tactcgcagt gcctgtaa 164864526PRTChrysosoporium lucknowenseMISC_FEATURE(365)..(365)X= ANY AMINO ACID 64Met Tyr Ala Lys Phe Ala Thr Leu Ala Ala Leu Val Ala Gly Ala Ala1 5 10 15Ala Gln Asn Ala Cys Thr Leu Thr Ala Glu Asn His Pro Ser Leu Thr 20 25 30Trp Ser Lys Cys Thr Ser Gly Gly Ser Cys Thr Ser Val Gln Gly Ser 35 40 45Ile Thr Ile Asp Ala Asn Trp Arg Trp Thr His Arg Thr Asp Ser Ala 50 55 60Thr Asn Cys Tyr Glu Gly Asn Lys Trp Asp Thr Ser Tyr Cys Ser Asp65 70 75 80Gly Pro Ser Cys Ala Ser Lys Cys Cys Ile Asp Gly Ala Asp Tyr Ser 85 90 95Ser Thr Tyr Gly Ile Thr Thr Ser Gly Asn Ser Leu Asn Leu Lys Phe 100 105 110Val Thr Lys Gly Gln Tyr Ser Thr Asn Ile Gly Ser Arg Thr Tyr Leu 115 120 125Met Glu Ser Asp Thr Lys Tyr Gln Met Phe Gln Leu Leu Gly Asn Glu 130 135 140Phe Thr Phe Asp Val Asp Val Ser Asn Leu Gly Cys Gly Leu Asn Gly145 150 155 160Ala Leu Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Met Ser Lys Tyr 165 170 175Ser Gly Asn Lys Ala Gly Ala Lys Tyr Gly Thr Gly Tyr Cys Asp Ser 180 185 190Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Glu Ala Asn Val Glu 195 200 205Asn Trp Gln Ser Ser Thr Asn Asp Ala Asn Ala Gly Thr Gly Lys Tyr 210 215 220Gly Ser Cys Cys Ser Glu Met Asp Val Trp Glu Ala Asn Asn Met Ala225 230 235 240Ala Ala Phe Thr Pro His Pro Cys Trp Val Ile Gly Gln Ser Arg Cys 245 250 255Glu Gly Asp Ser Cys Gly Gly Thr Tyr Ser Thr Asp Arg Tyr Ala Gly 260 265 270Ile Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg Gln Gly Asn 275 280 285Lys Thr Phe Tyr Gly Lys Gly Met Thr Val Asp Thr Thr Lys Lys Ile 290 295 300Thr Val Val Thr Gln Phe Leu Lys Asn Ser Ala Gly Glu Leu Ser Glu305 310 315 320Ile Lys Arg Phe Tyr Val Gln Asn Gly Lys Val Ile Pro Asn Ser Glu 325 330 335Ser Thr Ile Pro Gly Val Glu Gly Asn Ser Ile Thr Gln Asp Trp Cys 340 345 350Asp Arg Gln Lys Ala Ala Phe Gly Asp Val Thr Asp Xaa Gln Asp Lys 355 360 365Gly Gly Met Val Gln Met Gly Lys Ala Leu Ala Gly Pro Met Val Leu 370 375 380Val Met Ser Ile Trp Asp Asp His Ala Val Asn Met Leu Trp Leu Asp385 390 395 400Ser Thr Trp Pro Ile Asp Gly Ala Gly Lys Pro Gly Ala Glu Arg Gly 405 410 415Ala Cys Pro Thr Thr Ser Gly Val Pro Ala Glu Val Glu Ala Glu Ala 420 425 430Pro Asn Ser Asn Val Ile Phe Ser Asn Ile Arg Phe Gly Pro Ile Gly 435 440 445Ser Thr Val Ser Gly Leu Pro Asp Gly Gly Ser Gly Asn Pro Asn Pro 450 455 460Pro Val Ser Ser Ser Thr Pro Val Pro Ser Ser Ser Thr Thr Ser Ser465 470 475 480Gly Ser Ser Gly Pro Thr Gly Gly Thr Gly Val Ala Lys His Tyr Glu 485 490 495Gln Cys Gly Gly Ile Gly Phe Thr Gly Pro Thr Gln Cys Glu Ser Pro 500 505 510Tyr Thr Cys Thr Lys Leu Asn Asp Trp Tyr Ser Gln Cys Leu 515 520 525651353DNAMyceliophthora thermophila 65atgaagcagt acctccagta cctcgcggcg accctgcccc tggtgggcct ggccacggcc 60cagcaggcgg gtaacctgca gaccgagact caccccaggc tcacttggtc caagtgcacg 120gccccgggat cctgccaaca ggtcaacggc gaggtcgtca tcgactccaa ctggcgctgg 180gtgcacgacg agaacgcgca gaactgctac gacggcaacc agtggaccaa cgcttgcagc 240tctgccaccg actgcgccga gaattgcgcg ctcgagggtg ccgactacca gggcacctat 300ggcgcctcga ccagcggcaa tgccctgacg ctcaccttcg tcactaagca cgagtacggc 360accaacattg gctcgcgcct ctacctcatg aacggcgcga acaagtacca gatgttcacc 420ctcaagggca acgagctggc cttcgacgtc gacctctcgg ccgtcgagtg cggcctcaac 480agcgccctct acttcgtggc catggaggag gatggcggtg tgtcgagcta cccgaccaac 540acggccggtg ctaagttcgg cactgggtac tgcgacgccc aatgcgcacg cgacctcaag 600ttcgtcggcg gcaagggcaa catcgagggc tggaagccgt ccaccaacga tgccaatgcc 660ggtgtcggtc cttatggcgg gtgctgcgct gagatcgacg tctgggagtc gaacaagtat 720gctttcgctt tcaccccgca cggttgcgag aaccctaaat accacgtctg cgagaccacc 780aactgcggtg gcacctactc cgaggaccgc ttcgctggtg actgcgatgc caacggctgc 840gactacaacc cctaccgcat gggcaaccag gacttctacg gtcccggctt gacggtcgat 900accagcaaga agttcaccgt cgtcagccag ttcgaggaga acaagctcac ccagttcttc 960gtccaggacg gcaagaagat tgagatcccc ggccccaagg tcgagggcat cgatgcggac 1020agcgccgcta tcacccctga gctgtgcagt gccctgttca aggccttcga tgaccgtgac 1080cgcttctcgg aggttggcgg cttcgatgcc atcaacacgg ccctcagcac tcccatggtc 1140ctcgtcatgt ccatctggga tgatcactac gccaatatgc tctggctcga ctcgagctac 1200ccccctgaga aggctggcca gcctggcggt gaccgtggcc cgtgtcctca ggactctggc 1260gtcccggccg acgttgaggc tcagtaccct aatgccaagg tcatctggtc caacatccgc 1320ttcggcccca tcggctcgac tgtcaacgtc taa 135366450PRTMyceliophthora thermophila 66Met Lys Gln Tyr Leu Gln Tyr Leu Ala Ala Thr Leu Pro Leu Val Gly1 5 10 15Leu Ala Thr Ala Gln Gln Ala Gly Asn Leu Gln Thr Glu Thr His Pro 20 25 30Arg Leu Thr Trp Ser Lys Cys Thr Ala Pro Gly Ser Cys Gln Gln Val 35 40 45Asn Gly Glu Val Val Ile Asp Ser Asn Trp Arg Trp Val His Asp Glu 50 55 60Asn Ala Gln Asn Cys Tyr Asp Gly Asn Gln Trp Thr Asn Ala Cys Ser65 70 75 80Ser Ala Thr Asp Cys Ala Glu Asn Cys Ala Leu Glu Gly Ala Asp Tyr 85 90 95Gln Gly Thr Tyr Gly Ala Ser Thr Ser Gly Asn Ala Leu Thr Leu Thr 100 105 110Phe Val Thr Lys His Glu Tyr Gly Thr Asn Ile Gly Ser Arg Leu Tyr 115 120 125Leu Met Asn Gly Ala Asn Lys Tyr Gln Met Phe Thr Leu Lys Gly Asn 130 135 140Glu Leu Ala Phe Asp Val Asp Leu Ser Ala Val Glu Cys Gly Leu Asn145 150 155 160Ser Ala Leu Tyr Phe Val Ala Met Glu Glu Asp Gly Gly Val Ser Ser 165 170 175Tyr Pro Thr Asn Thr Ala Gly Ala Lys Phe Gly Thr Gly Tyr Cys Asp 180 185 190Ala Gln Cys Ala Arg Asp Leu Lys Phe Val Gly Gly Lys Gly Asn Ile 195 200 205Glu Gly Trp Lys Pro Ser Thr Asn Asp Ala Asn Ala Gly Val Gly Pro 210 215 220Tyr Gly Gly Cys Cys Ala Glu Ile Asp Val Trp Glu Ser Asn Lys Tyr225 230 235 240Ala Phe Ala Phe Thr Pro His Gly Cys Glu Asn Pro Lys Tyr His Val 245 250 255Cys Glu Thr Thr Asn Cys Gly Gly Thr Tyr Ser Glu Asp Arg Phe Ala 260 265 270Gly Asp Cys Asp Ala Asn Gly Cys Asp Tyr Asn Pro Tyr Arg Met Gly 275 280 285Asn Gln Asp Phe Tyr Gly Pro Gly Leu Thr Val Asp Thr Ser Lys Lys 290 295 300Phe Thr Val Val Ser Gln Phe Glu Glu Asn Lys Leu Thr Gln Phe Phe305 310 315 320Val Gln Asp Gly Lys Lys Ile Glu Ile Pro Gly Pro Lys Val Glu Gly 325 330 335Ile Asp Ala Asp Ser Ala Ala Ile Thr Pro Glu Leu Cys Ser Ala Leu 340 345 350Phe Lys Ala Phe Asp Asp Arg Asp Arg Phe Ser Glu Val Gly Gly Phe 355 360 365Asp Ala Ile Asn Thr Ala Leu Ser Thr Pro Met Val Leu Val Met Ser 370 375 380Ile Trp Asp Asp His Tyr Ala Asn Met Leu Trp Leu Asp Ser Ser Tyr385 390 395 400Pro Pro Glu Lys Ala Gly Gln Pro Gly Gly Asp Arg Gly Pro Cys Pro 405 410 415Gln Asp Ser Gly Val Pro Ala Asp Val Glu Ala Gln Tyr Pro Asn Ala 420 425 430Lys Val Ile Trp Ser Asn Ile Arg Phe Gly Pro Ile Gly Ser Thr Val 435 440 445Asn Val 450671446DNAThielavia terrestris 67atggctcaga 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 144668481PRTThielavia terrestris 68Met Ala Gln Lys Leu Leu Leu Ala Ala Ala Leu Ala Ala Ser Ala Leu1 5 10 15Ala Ala Pro Val Val Glu Glu Arg Gln Asn Cys Gly Ser Val Trp Ser 20 25 30Gln Cys Gly Gly Ile Gly Trp Ser Gly Ala Thr Cys Cys Ala Ser Gly 35 40 45Asn Thr Cys Val Glu Leu Asn Pro Tyr Tyr Ser Gln Cys Leu Pro Asn 50 55 60Ser Gln Val Thr Thr Ser Thr Ser Lys Thr Thr Ser Thr Thr Thr Arg65 70 75 80Ser Ser Thr Thr Ser His Ser Ser Gly Pro Thr Ser Thr Ser Thr Thr 85 90 95Thr Thr Ser Ser Pro Val Val Thr Thr Pro Pro Ser Thr Ser Ile Pro 100 105 110Gly Gly Ala Ser Ser Thr Ala Ser Trp Ser Gly Asn Pro Phe Ser Gly 115 120 125Val Gln Met Trp Ala Asn Asp Tyr Tyr Ala Ser Glu Val Ser Ser Leu 130 135 140Ala Ile Pro Ser Met Thr Gly Ala Met Ala Thr Lys Ala Ala Glu Val145 150 155 160Ala Lys Val Pro Ser Phe Gln Trp Leu Asp Arg Asn Val Thr Ile Asp 165 170 175Thr Leu Phe Ala His Thr Leu Ser Gln Ile Arg Ala Ala Asn Gln Lys 180 185 190Gly Ala Asn Pro Pro Tyr Ala Gly Ile Phe Val Val Tyr Asp Leu Pro 195 200 205Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu Phe Ser Ile Ala 210 215 220Asn Asn Gly Ala Ala Asn Tyr Lys Thr Tyr Ile Asp Ala Ile Arg Ser225 230 235 240Leu Val Ile Gln Tyr Ser Asp Ile Arg Ile Ile Phe Val Ile Glu Pro 245 250 255Asp Ser Leu Ala Asn Met Val Thr Asn Leu Asn Val Ala Lys Cys Ala 260 265 270Asn Ala Glu Ser Thr Tyr Lys Glu Leu Thr

Val Tyr Ala Leu Gln Gln 275 280 285Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala Gly 290 295 300Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala Asn Leu Phe Ala305 310 315 320Glu Ile Tyr Thr Ser Ala Gly Lys Pro Ala Ala Val Arg Gly Leu Ala 325 330 335Thr Asn Val Ala Asn Tyr Asn Gly Trp Ser Leu Ala Thr Pro Pro Ser 340 345 350Tyr Thr Gln Gly Asp Pro Asn Tyr Asp Glu Ser His Tyr Val Gln Ala 355 360 365Leu Ala Pro Leu Leu Thr Ala Asn Gly Phe Pro Ala His Phe Ile Thr 370 375 380Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr Gly Gln Arg Gln Trp Gly385 390 395 400Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly Val Arg Pro Thr Thr 405 410 415Asn Thr Gly Leu Asp Ile Glu Asp Ala Phe Val Trp Val Lys Pro Gly 420 425 430Gly Glu Cys Asp Gly Thr Ser Asn Thr Thr Ser Pro Arg Tyr Asp Tyr 435 440 445His Cys Gly Leu Ser Asp Ala Leu Gln Pro Ala Pro Glu Ala Gly Thr 450 455 460Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala Asn Pro Pro465 470 475 480Phe692586DNAAspergillus oryzae 69atgaagcttg gttggatcga ggtggccgca ttggcggctg cctcagtagt cagtgccaag 60gatgatctcg cgtactcccc tcctttctac ccttccccat gggcagatgg tcagggtgaa 120tgggcggaag tatacaaacg cgctgtagac atagtttccc agatgacgtt gacagagaaa 180gtcaacttaa cgactggaac aggatggcaa ctagagaggt gtgttggaca aactggcagt 240gttcccagac tcaacatccc cagcttgtgt ttgcaggata gtcctcttgg tattcgtttc 300tcggactaca attcagcttt ccctgcgggt gttaatgtcg ctgccacctg ggacaagacg 360ctcgcctacc ttcgtggtca ggcaatgggt gaggagttca gtgataaggg tattgacgtt 420cagctgggtc ctgctgctgg ccctctcggt gctcatccgg atggcggtag aaactgggaa 480ggtttctcac cagatccagc cctcaccggt gtactttttg cggagacgat taagggtatt 540caagatgctg gtgtcattgc gacagctaag cattatatca tgaacgaaca agagcatttc 600cgccaacaac ccgaggctgc gggttacgga ttcaacgtaa gcgacagttt gagttccaac 660gttgatgaca agactatgca tgaattgtac ctctggccct tcgcggatgc agtacgcgct 720ggagtcggtg ctgtcatgtg ctcttacaac caaatcaaca acagctacgg ttgcgagaat 780agcgaaactc tgaacaagct tttgaaggcg gagcttggtt tccaaggctt cgtcatgagt 840gattggaccg ctcatcacag cggcgtaggc gctgctttag caggtctgga tatgtcgatg 900cccggtgatg ttaccttcga tagtggtacg tctttctggg gtgcaaactt gacggtcggt 960gtccttaacg gtacaatccc ccaatggcgt gttgatgaca tggctgtccg tatcatggcc 1020gcttattaca aggttggccg cgacaccaaa tacacccctc ccaacttcag ctcgtggacc 1080agggacgaat atggtttcgc gcataaccat gtttcggaag gtgcttacga gagggtcaac 1140gaattcgtgg acgtgcaacg cgatcatgcc gacctaatcc gtcgcatcgg cgcgcagagc 1200actgttctgc tgaagaacaa gggtgccttg cccttgagcc gcaaggaaaa gctggtcgcc 1260cttctgggag aggatgcggg ttccaactcg tggggcgcta acggctgtga tgaccgtggt 1320tgcgataacg gtacccttgc catggcctgg ggtagcggta ctgcgaattt cccatacctc 1380gtgacaccag agcaggcgat tcagaacgaa gttcttcagg gccgtggtaa tgtcttcgcc 1440gtgaccgaca gttgggcgct cgacaagatc gctgcggctg cccgccaggc cagcgtatct 1500ctcgtgttcg tcaactccga ctcaggagaa ggctatctta gtgtggatgg aaatgagggc 1560gatcgtaaca acatcactct gtggaagaac ggcgacaatg tggtcaagac cgcagcgaat 1620aactgtaaca acaccgttgt catcatccac tccgtcggac cagttttgat cgatgaatgg 1680tatgaccacc ccaatgtcac tggtattctc tgggctggtc tgccaggcca ggagtctggt 1740aactccattg ccgatgtgct gtacggtcgt gtcaaccctg gcgccaagtc tcctttcact 1800tggggcaaga cccgggagtc gtatggttct cccttggtca aggatgccaa caatggcaac 1860ggagcgcccc agtctgattt cacccagggt gttttcatcg attaccgcca tttcgataag 1920ttcaatgaga cccctatcta cgagtttggc tacggcttga gctacaccac cttcgagctc 1980tccgacctcc atgttcagcc cctgaacgcg tcccgataca ctcccaccag tggcatgact 2040gaagctgcaa agaactttgg tgaaattggc gatgcgtcgg agtacgtgta tccggagggg 2100ctggaaagga tccatgagtt tatctatccc tggatcaact ctaccgacct gaaggcatcg 2160tctgacgatt ctaactacgg ctgggaagac tccaagtata ttcccgaagg cgccacggat 2220gggtctgccc agccccgttt gcccgctagt ggtggtgccg gaggaaaccc cggtctgtac 2280gaggatcttt tccgcgtctc tgtgaaggtc aagaacacgg gcaatgtcgc cggtgatgaa 2340gttcctcagc tgtacgtttc cctaggcggc ccgaatgagc ccaaggtggt actgcgcaag 2400tttgagcgta ttcacttggc cccttcgcag gaggccgtgt ggacaacgac ccttacccgt 2460cgtgaccttg caaactggga cgtttcggct caggactgga ccgtcactcc ttaccccaag 2520acgatctacg ttggaaactc ctcacggaaa ctgccgctcc aggcctcgct gcctaaggcc 2580cagtaa 258670861PRTAspergillus oryzae 70Met Lys Leu Gly Trp Ile Glu Val Ala Ala Leu Ala Ala Ala Ser Val1 5 10 15Val Ser Ala Lys Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser 20 25 30Pro Trp Ala Asp Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala 35 40 45Val Asp Ile Val Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr 50 55 60Thr Gly Thr Gly Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser65 70 75 80Val Pro Arg Leu Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu 85 90 95Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn 100 105 110Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala 115 120 125Met Gly Glu Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro 130 135 140Ala Ala Gly Pro Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu145 150 155 160Gly Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185 190Ile Met Asn Glu Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly 195 200 205Tyr Gly Phe Asn Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys 210 215 220Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala225 230 235 240Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245 250 255Gly Cys Glu Asn Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265 270Gly Phe Gln Gly Phe Val Met Ser Asp Trp Thr Ala His His Ser Gly 275 280 285Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val 290 295 300Thr Phe Asp Ser Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly305 310 315 320Val Leu Asn Gly Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val 325 330 335Arg Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr 340 345 350Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His 355 360 365Asn His Val Ser Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp 370 375 380Val Gln Arg Asp His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser385 390 395 400Thr Val Leu Leu Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu 405 410 415Lys Leu Val Ala Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly 420 425 430Ala Asn Gly Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435 440 445Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu 450 455 460Gln Ala Ile Gln Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala465 470 475 480Val Thr Asp Ser Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln 485 490 495Ala Ser Val Ser Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr 500 505 510Leu Ser Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp 515 520 525Lys Asn Gly Asp Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn 530 535 540Thr Val Val Ile Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp545 550 555 560Tyr Asp His Pro Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly 565 570 575Gln Glu Ser Gly Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn 580 585 590Pro Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595 600 605Gly Ser Pro Leu Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln 610 615 620Ser Asp Phe Thr Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys625 630 635 640Phe Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr 645 650 655Thr Phe Glu Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg 660 665 670Tyr Thr Pro Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu 675 680 685Ile Gly Asp Ala Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile 690 695 700His Glu Phe Ile Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser705 710 715 720Ser Asp Asp Ser Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu 725 730 735Gly Ala Thr Asp Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly 740 745 750Ala Gly Gly Asn Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val 755 760 765Lys Val Lys Asn Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu 770 775 780Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys785 790 795 800Phe Glu Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr 805 810 815Thr Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 820 825 830Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser Ser 835 840 845Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 850 855 860713060DNAAspergillus fumigatus 71atgagattcg 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 306072863PRTAspergillus fumigatus 72Met Arg Phe Gly Trp Leu Glu Val Ala Ala Leu Thr Ala Ala Ser Val1 5 10 15Ala Asn Ala Gln Glu Leu Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro 20 25 30Trp Ala Asp Gly Gln Gly Glu Trp Ala Asp Ala His Arg Arg Ala Val 35 40 45Glu Ile Val Ser Gln Met Thr Leu Ala Glu Lys Val Asn Leu Thr Thr 50 55 60Gly Thr Gly Trp Glu Met Asp Arg Cys Val Gly Gln Thr Gly Ser Val65 70 75 80Pro Arg Leu Gly Ile Asn Trp Gly Leu Cys Gly Gln Asp Ser Pro Leu 85 90 95Gly Ile Arg Phe Ser Asp Leu Asn Ser Ala Phe Pro Ala Gly Thr Asn 100 105 110Val Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Lys Ala 115 120 125Met Gly Glu Glu Phe Asn Asp Lys Gly Val Asp Ile Leu Leu Gly Pro 130 135 140Ala Ala Gly Pro Leu Gly Lys Tyr Pro Asp Gly Gly Arg Ile Trp Glu145 150 155 160Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Val Leu Phe Ala Glu Thr 165 170 175Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180 185 190Ile Leu Asn Glu Gln Glu His Phe Arg Gln Val Gly Glu Ala Gln Gly 195 200 205Tyr Gly Tyr Asn Ile Thr Glu Thr Ile Ser Ser Asn Val Asp Asp Lys 210 215 220Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala225 230 235 240Gly Val Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245 250 255Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265 270Gly Phe Gln Gly Phe Val Met Ser Asp Trp Ser Ala His His Ser Gly 275 280 285Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile 290 295 300Ser Phe Asp Asp Gly Leu Ser Phe Trp Gly Thr Asn Leu Thr Val Ser305 310 315 320Val Leu Asn Gly Thr Val Pro Ala Trp Arg Val Asp Asp Met Ala Val 325 330 335Arg Ile Met Thr Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Arg Ile 340 345 350Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Trp Glu His 355 360 365Ser Ala Val Ser Glu Gly Ala Trp Thr Lys Val Asn Asp Phe Val Asn 370 375 380Val Gln Arg Ser His Ser Gln Ile Ile Arg Glu Ile Gly Ala Ala Ser385 390 395 400Thr Val Leu Leu Lys Asn Thr Gly Ala Leu Pro Leu Thr Gly Lys Glu 405 410 415Val Lys Val Gly Val Leu Gly Glu Asp Ala Gly Ser Asn Pro Trp Gly 420 425 430Ala Asn Gly Cys Pro Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435 440 445Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu

Val Thr Pro Glu 450 455 460Gln Ala Ile Gln Arg Glu Val Ile Ser Asn Gly Gly Asn Val Phe Ala465 470 475 480Val Thr Asp Asn Gly Ala Leu Ser Gln Met Ala Asp Val Ala Ser Gln 485 490 495Ser Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Phe 500 505 510Ile Ser Val Asp Gly Asn Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp 515 520 525Lys Asn Gly Glu Ala Val Ile Asp Thr Val Val Ser His Cys Asn Asn 530 535 540Thr Ile Val Val Ile His Ser Val Gly Pro Val Leu Ile Asp Arg Trp545 550 555 560Tyr Asp Asn Pro Asn Val Thr Ala Ile Ile Trp Ala Gly Leu Pro Gly 565 570 575Gln Glu Ser Gly Asn Ser Leu Val Asp Val Leu Tyr Gly Arg Val Asn 580 585 590Pro Ser Ala Lys Thr Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595 600 605Gly Ala Pro Leu Leu Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln 610 615 620Asp Asp Phe Asn Glu Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys625 630 635 640Arg Asn Glu Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr 645 650 655Thr Phe Gly Tyr Ser His Leu Arg Val Gln Ala Leu Asn Ser Ser Ser 660 665 670Ser Ala Tyr Val Pro Thr Ser Gly Glu Thr Lys Pro Ala Pro Thr Tyr 675 680 685Gly Glu Ile Gly Ser Ala Ala Asp Tyr Leu Tyr Pro Glu Gly Leu Lys 690 695 700Arg Ile Thr Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Glu705 710 715 720Asp Ser Ser Asp Asp Pro Asn Tyr Gly Trp Glu Asp Ser Glu Tyr Ile 725 730 735Pro Glu Gly Ala Arg Asp Gly Ser Pro Gln Pro Leu Leu Lys Ala Gly 740 745 750Gly Ala Pro Gly Gly Asn Pro Thr Leu Tyr Gln Asp Leu Val Arg Val 755 760 765Ser Ala Thr Ile Thr Asn Thr Gly Asn Val Ala Gly Tyr Glu Val Pro 770 775 780Gln Leu Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Arg Val Val Leu785 790 795 800Arg Lys Phe Asp Arg Ile Phe Leu Ala Pro Gly Glu Gln Lys Val Trp 805 810 815Thr Thr Thr Leu Asn Arg Arg Asp Leu Ala Asn Trp Asp Val Glu Ala 820 825 830Gln Asp Trp Val Ile Thr Lys Tyr Pro Lys Lys Val His Val Gly Ser 835 840 845Ser Ser Arg Lys Leu Pro Leu Arg Ala Pro Leu Pro Arg Val Tyr 850 855 860732800DNAPenicillium brasilianum 73tgaaaatgca 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 280074878PRTPenicillium brasilianum 74Met Gln Gly Ser Thr Ile Phe Leu Ala Phe Ala Ser Trp Ala Ser Gln1 5 10 15Val Ala Ala Ile Ala Gln Pro Ile Gln Lys His Glu Pro Gly Phe Leu 20 25 30His Gly Pro Gln Ala Ile Glu Ser Phe Ser Glu Pro Phe Tyr Pro Ser 35 40 45Pro Trp Met Asn Pro His Ala Glu Gly Trp Glu Ala Ala Tyr Gln Lys 50 55 60Ala Gln Asp Phe Val Ser Gln Leu Thr Ile Leu Glu Lys Ile Asn Leu65 70 75 80Thr Thr Gly Val Gly Trp Glu Asn Gly Pro Cys Val Gly Asn Thr Gly 85 90 95Ser Ile Pro Arg Leu Gly Phe Lys Gly Phe Cys Thr Gln Asp Ser Pro 100 105 110Gln Gly Val Arg Phe Ala Asp Tyr Ser Ser Ala Phe Thr Ser Ser Gln 115 120 125Met Ala Ala Ala Thr Phe Asp Arg Ser Ile Leu Tyr Gln Arg Gly Gln 130 135 140Ala Met Ala Gln Glu His Lys Ala Lys Gly Ile Thr Ile Gln Leu Gly145 150 155 160Pro Val Ala Gly Pro Leu Gly Arg Ile Pro Glu Gly Gly Arg Asn Trp 165 170 175Glu Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Ile Ala Met Ala Glu 180 185 190Thr Ile Lys Gly Met Gln Asp Thr Gly Val Ile Ala Cys Ala Lys His 195 200 205Tyr Ile Gly Asn Glu Gln Glu His Phe Arg Gln Val Gly Glu Ala Ala 210 215 220Gly His Gly Tyr Thr Ile Ser Asp Thr Ile Ser Ser Asn Ile Asp Asp225 230 235 240Arg Ala Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg 245 250 255Ala Gly Val Gly Ser Phe Met Cys Ser Tyr Ser Gln Ile Asn Asn Ser 260 265 270Tyr Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ser Glu 275 280 285Leu Gly Phe Gln Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser 290 295 300Gly Val Ser Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp305 310 315 320Thr Glu Phe Asp Ser Gly Leu Ser Phe Trp Gly Ser Asn Leu Thr Ile 325 330 335Ala Ile Leu Asn Gly Thr Val Pro Glu Trp Arg Leu Asp Asp Met Ala 340 345 350Met Arg Ile Met Ala Ala Tyr Phe Lys Val Gly Leu Thr Ile Glu Asp 355 360 365Gln Pro Asp Val Asn Phe Asn Ala Trp Thr His Asp Thr Tyr Gly Tyr 370 375 380Lys Tyr Ala Tyr Ser Lys Glu Asp Tyr Glu Gln Val Asn Trp His Val385 390 395 400Asp Val Arg Ser Asp His Asn Lys Leu Ile Arg Glu Thr Ala Ala Lys 405 410 415Gly Thr Val Leu Leu Lys Asn Asn Phe His Ala Leu Pro Leu Lys Gln 420 425 430Pro Arg Phe Val Ala Val Val Gly Gln Asp Ala Gly Pro Asn Pro Lys 435 440 445Gly Pro Asn Gly Cys Ala Asp Arg Gly Cys Asp Gln Gly Thr Leu Ala 450 455 460Met Gly Trp Gly Ser Gly Ser Thr Glu Phe Pro Tyr Leu Val Thr Pro465 470 475 480Asp Thr Ala Ile Gln Ser Lys Val Leu Glu Tyr Gly Gly Arg Tyr Glu 485 490 495Ser Ile Phe Asp Asn Tyr Asp Asp Asn Ala Ile Leu Ser Leu Val Ser 500 505 510Gln Pro Asp Ala Thr Cys Ile Val Phe Ala Asn Ala Asp Ser Gly Glu 515 520 525Gly Tyr Ile Thr Val Asp Asn Asn Trp Gly Asp Arg Asn Asn Leu Thr 530 535 540Leu Trp Gln Asn Ala Asp Gln Val Ile Ser Thr Val Ser Ser Arg Cys545 550 555 560Asn Asn Thr Ile Val Val Leu His Ser Val Gly Pro Val Leu Leu Asn 565 570 575Gly Ile Tyr Glu His Pro Asn Ile Thr Ala Ile Val Trp Ala Gly Met 580 585 590Pro Gly Glu Glu Ser Gly Asn Ala Leu Val Asp Ile Leu Trp Gly Asn 595 600 605Val Asn Pro Ala Gly Arg Thr Pro Phe Thr Trp Ala Lys Ser Arg Glu 610 615 620Asp Tyr Gly Thr Asp Ile Met Tyr Glu Pro Asn Asn Gly Gln Arg Ala625 630 635 640Pro Gln Gln Asp Phe Thr Glu Ser Ile Tyr Leu Asp Tyr Arg His Phe 645 650 655Asp Lys Ala Gly Ile Glu Pro Ile Tyr Glu Phe Gly Phe Gly Leu Ser 660 665 670Tyr Thr Thr Phe Glu Tyr Ser Asp Leu Arg Val Val Lys Lys Tyr Val 675 680 685Gln Pro Tyr Ser Pro Thr Thr Gly Thr Gly Ala Gln Ala Pro Ser Ile 690 695 700Gly Gln Pro Pro Ser Gln Asn Leu Asp Thr Tyr Lys Phe Pro Ala Thr705 710 715 720Tyr Lys Tyr Ile Lys Thr Phe Ile Tyr Pro Tyr Leu Asn Ser Thr Val 725 730 735Ser Leu Arg Ala Ala Ser Lys Asp Pro Glu Tyr Gly Arg Thr Asp Phe 740 745 750Ile Pro Pro His Ala Arg Asp Gly Ser Pro Gln Pro Leu Asn Pro Ala 755 760 765Gly Asp Pro Val Ala Ser Gly Gly Asn Asn Met Leu Tyr Asp Glu Leu 770 775 780Tyr Glu Val Thr Ala Gln Ile Lys Asn Thr Gly Asp Val Ala Gly Asp785 790 795 800Glu Val Val Gln Leu Tyr Val Asp Leu Gly Gly Asp Asn Pro Pro Arg 805 810 815Gln Leu Arg Asn Phe Asp Arg Phe Tyr Leu Leu Pro Gly Gln Ser Ser 820 825 830Thr Phe Arg Ala Thr Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Ile 835 840 845Glu Ala Gln Asn Trp Arg Val Thr Glu Ser Pro Lys Arg Val Tyr Val 850 855 860Gly Arg Ser Ser Arg Asp Leu Pro Leu Ser Ser Gln Leu Glu865 870 875752583DNAAspergillus niger 75atgaggttca 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 258376860PRTAspergillus niger 76Met Arg Phe Thr Leu Ile Glu Ala Val Ala Leu Thr Ala Val Ser Leu1 5 10 15Ala Ser Ala Asp Glu Leu Ala Tyr Ser Pro Pro Tyr Tyr Pro Ser Pro 20 25 30Trp Ala Asn Gly Gln Gly Asp Trp Ala Gln Ala Tyr Gln Arg Ala Val 35 40 45Asp Ile Val Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr 50 55 60Gly Thr Gly Trp Glu Leu Glu Leu Cys Val Gly Gln Thr Gly Gly Val65 70 75 80Pro Arg Leu Gly Val Pro Gly Met Cys Leu Gln Asp Ser Pro Leu Gly 85 90 95Val Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Met Asn Val 100 105 110Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Lys Ala Met 115 120 125Gly Gln Glu Phe Ser Asp Lys Gly Ala Asp Ile Gln Leu Gly Pro Ala 130 135 140Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly145 150 155 160Phe Ser Pro Asp Pro Ala Leu Ser Gly Val Leu Phe Ala Glu Thr Ile 165 170 175Lys Gly Ile Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile 180 185 190Ala Tyr Glu Gln Glu His Phe Arg Gln Ala Pro Glu Ala Gln Gly Phe 195 200 205Gly Phe Asn Ile Ser Glu Ser Gly Ser Ala Asn Leu Asp Asp Lys Thr 210 215 220Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Ile Arg Ala Gly225 230 235 240Ala Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly 245 250 255Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly 260 265

270Phe Gln Gly Phe Val Met Ser Asp Trp Ala Ala His His Ala Gly Val 275 280 285Ser Gly Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Asp 290 295 300Tyr Asp Ser Gly Thr Ser Tyr Trp Gly Thr Asn Leu Thr Ile Ser Val305 310 315 320Leu Asn Gly Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg 325 330 335Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Trp Thr Pro 340 345 350Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Tyr Lys Tyr Tyr 355 360 365Tyr Val Ser Glu Gly Pro Tyr Glu Lys Val Asn Gln Tyr Val Asn Val 370 375 380Gln Arg Asn His Ser Glu Leu Ile Arg Arg Ile Gly Ala Asp Ser Thr385 390 395 400Val Leu Leu Lys Asn Asp Gly Ala Leu Pro Leu Thr Gly Lys Glu Arg 405 410 415Leu Val Ala Leu Ile Gly Glu Asp Ala Gly Ser Asn Pro Tyr Gly Ala 420 425 430Asn Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly 435 440 445Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln 450 455 460Ala Ile Ser Asn Glu Val Leu Lys His Lys Asn Gly Val Phe Thr Ala465 470 475 480Thr Asp Asn Trp Ala Ile Asp Gln Ile Glu Ala Leu Ala Lys Thr Ala 485 490 495Ser Val Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile 500 505 510Asn Val Asp Gly Asn Leu Gly Asp Arg Arg Asn Leu Thr Leu Trp Arg 515 520 525Asn Gly Asp Asn Val Ile Lys Ala Ala Ala Ser Asn Cys Asn Asn Thr 530 535 540Ile Val Val Ile His Ser Val Gly Pro Val Leu Val Asn Glu Trp Tyr545 550 555 560Asp Asn Pro Asn Val Thr Ala Ile Leu Trp Gly Gly Leu Pro Gly Gln 565 570 575Glu Ser Gly Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro 580 585 590Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gln 595 600 605Asp Tyr Leu Val Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln Glu 610 615 620Asp Phe Val Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg625 630 635 640Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr 645 650 655Phe Asn Tyr Ser Asn Leu Glu Val Gln Val Leu Ser Ala Pro Ala Tyr 660 665 670Glu Pro Ala Ser Gly Glu Thr Glu Ala Ala Pro Thr Phe Gly Glu Val 675 680 685Gly Asn Ala Ser Asp Tyr Leu Tyr Pro Ser Gly Leu Gln Arg Ile Thr 690 695 700Lys Phe Ile Tyr Pro Trp Leu Asn Gly Thr Asp Leu Glu Ala Ser Ser705 710 715 720Gly Asp Ala Ser Tyr Gly Gln Asp Ser Ser Asp Tyr Leu Pro Glu Gly 725 730 735Ala Thr Asp Gly Ser Ala Gln Pro Ile Leu Pro Ala Gly Gly Gly Pro 740 745 750Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr 755 760 765Ile Lys Asn Thr Gly Lys Val Ala Gly Asp Glu Val Pro Gln Leu Tyr 770 775 780Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Ile Val Leu Arg Gln Phe785 790 795 800Glu Arg Ile Thr Leu Gln Pro Ser Glu Glu Thr Lys Trp Ser Thr Thr 805 810 815Leu Thr Arg Arg Asp Leu Ala Asn Trp Asn Val Glu Lys Gln Asp Trp 820 825 830Glu Ile Thr Ser Tyr Pro Lys Met Val Phe Val Gly Ser Ser Ser Arg 835 840 845Lys Leu Pro Leu Arg Ala Ser Leu Pro Thr Val His 850 855 860772583DNAAspergillus aculeatus 77atgaagctca 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 258378860PRTAspergillus aculeatus 78Met Lys Leu Ser Trp Leu Glu Ala Ala Ala Leu Thr Ala Ala Ser Val1 5 10 15Val Ser Ala Asp Glu Leu Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro 20 25 30Trp Ala Asn Gly Gln Gly Glu Trp Ala Glu Ala Tyr Gln Arg Ala Val 35 40 45Ala Ile Val Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr 50 55 60Gly Thr Gly Trp Glu Leu Glu Lys Cys Val Gly Gln Thr Gly Gly Val65 70 75 80Pro Arg Leu Asn Ile Gly Gly Met Cys Leu Gln Asp Ser Pro Leu Gly 85 90 95Ile Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val 100 105 110Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Gln Ala Met 115 120 125Gly Gln Glu Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala 130 135 140Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly145 150 155 160Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile 165 170 175Lys Gly Ile Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile 180 185 190Leu Asn Glu Gln Glu His Phe Arg Gln Val Ala Glu Ala Ala Gly Tyr 195 200 205Gly Phe Asn Ile Ser Asp Thr Ile Ser Ser Asn Val Asp Asp Lys Thr 210 215 220Ile His Glu Met Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly225 230 235 240Val Gly Ala Ile Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly 245 250 255Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly 260 265 270Phe Gln Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser Gly Val 275 280 285Gly Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile Thr 290 295 300Phe Asp Ser Ala Thr Ser Phe Trp Gly Thr Asn Leu Thr Ile Ala Val305 310 315 320Leu Asn Gly Thr Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg 325 330 335Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Tyr Gln Pro 340 345 350Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Lys Tyr Phe 355 360 365Tyr Pro Gln Glu Gly Pro Tyr Glu Lys Val Asn His Phe Val Asn Val 370 375 380Gln Arg Asn His Ser Glu Val Ile Arg Lys Leu Gly Ala Asp Ser Thr385 390 395 400Val Leu Leu Lys Asn Asn Asn Ala Leu Pro Leu Thr Gly Lys Glu Arg 405 410 415Lys Val Ala Ile Leu Gly Glu Asp Ala Gly Ser Asn Ser Tyr Gly Ala 420 425 430Asn Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala 435 440 445Trp Gly Ser Gly Thr Ala Glu Phe Pro Tyr Leu Val Thr Pro Glu Gln 450 455 460Ala Ile Gln Ala Glu Val Leu Lys His Lys Gly Ser Val Tyr Ala Ile465 470 475 480Thr Asp Asn Trp Ala Leu Ser Gln Val Glu Thr Leu Ala Lys Gln Ala 485 490 495Ser Val Ser Leu Val Phe Val Asn Ser Asp Ala Gly Glu Gly Tyr Ile 500 505 510Ser Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Leu Thr Leu Trp Lys 515 520 525Asn Gly Asp Asn Leu Ile Lys Ala Ala Ala Asn Asn Cys Asn Asn Thr 530 535 540Ile Val Val Ile His Ser Val Gly Pro Val Leu Val Asp Glu Trp Tyr545 550 555 560Asp His Pro Asn Val Thr Ala Ile Leu Trp Ala Gly Leu Pro Gly Gln 565 570 575Glu Ser Gly Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn Pro 580 585 590Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gly 595 600 605Asp Tyr Leu Val Arg Glu Leu Asn Asn Gly Asn Gly Ala Pro Gln Asp 610 615 620Asp Phe Ser Glu Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg625 630 635 640Asn Glu Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr Thr 645 650 655Phe Asn Tyr Ser Gly Leu His Ile Gln Val Leu Asn Ala Ser Ser Asn 660 665 670Ala Gln Val Ala Thr Glu Thr Gly Ala Ala Pro Thr Phe Gly Gln Val 675 680 685Gly Asn Ala Ser Asp Tyr Val Tyr Pro Glu Gly Leu Thr Arg Ile Ser 690 695 700Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Lys Ala Ser Ser705 710 715 720Gly Asp Pro Tyr Tyr Gly Val Asp Thr Ala Glu His Val Pro Glu Gly 725 730 735Ala Thr Asp Gly Ser Pro Gln Pro Val Leu Pro Ala Gly Gly Gly Ser 740 745 750Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr 755 760 765Val Lys Asn Thr Gly Arg Val Ala Gly Asp Ala Val Pro Gln Leu Tyr 770 775 780Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe785 790 795 800Asp Arg Leu Thr Leu Lys Pro Ser Glu Glu Thr Val Trp Thr Thr Thr 805 810 815Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Val Ala Ala Gln Asp Trp 820 825 830Val Ile Thr Ser Tyr Pro Lys Lys Val His Val Gly Ser Ser Ser Arg 835 840 845Gln Leu Pro Leu His Ala Ala Leu Pro Lys Val Gln 850 855 860793294DNAAspergillus oryzae 79atgcgttcct 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 3294801097PRTAspergillus oryzae 80Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro1 5 10 15Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20

25 30Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro 35 40 45Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50 55 60Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln65 70 75 80Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr 85 90 95Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 100 105 110Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln 115 120 125Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130 135 140Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe145 150 155 160Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu 165 170 175Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180 185 190Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val 195 200 205Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp 210 215 220Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Met Arg Ser Ser Pro Leu225 230 235 240Leu Arg Ser Ala Val Val Ala Ala Leu Pro Val Leu Ala Leu Ala Lys 245 250 255Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp 260 265 270Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala Val Asp Ile Val 275 280 285Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 290 295 300Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser Val Pro Arg Leu305 310 315 320Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu Gly Ile Arg Phe 325 330 335Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val Ala Ala Thr 340 345 350Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala Met Gly Glu Glu 355 360 365Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala Ala Gly Pro 370 375 380Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro385 390 395 400Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile 405 410 415Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr Ile Met Asn Glu 420 425 430Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly Tyr Gly Phe Asn 435 440 445Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys Thr Met His Glu 450 455 460Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ala465 470 475 480Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Glu Asn 485 490 495Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly 500 505 510Phe Val Met Ser Asp Trp Thr Ala His His Ser Gly Val Gly Ala Ala 515 520 525Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Thr Phe Asp Ser 530 535 540Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly Val Leu Asn Gly545 550 555 560Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ala 565 570 575Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr Pro Pro Asn Phe 580 585 590Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His Asn His Val Ser 595 600 605Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp Val Gln Arg Asp 610 615 620His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser Thr Val Leu Leu625 630 635 640Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu Lys Leu Val Ala 645 650 655Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly Ala Asn Gly Cys 660 665 670Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala Trp Gly Ser 675 680 685Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln 690 695 700Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala Val Thr Asp Ser705 710 715 720Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln Ala Ser Val Ser 725 730 735Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr Leu Ser Val Asp 740 745 750Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp Lys Asn Gly Asp 755 760 765Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn Thr Val Val Ile 770 775 780Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp Tyr Asp His Pro785 790 795 800Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly 805 810 815Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala Lys 820 825 830Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr Gly Ser Pro Leu 835 840 845Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp Phe Thr 850 855 860Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys Phe Asn Glu Thr865 870 875 880Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Leu 885 890 895Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg Tyr Thr Pro Thr 900 905 910Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu Ile Gly Asp Ala 915 920 925Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile His Glu Phe Ile 930 935 940Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser Ser Asp Asp Ser945 950 955 960Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu Gly Ala Thr Asp 965 970 975Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly Ala Gly Gly Asn 980 985 990Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val Lys Val Lys Asn 995 1000 1005Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu Tyr Val Ser 1010 1015 1020Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe Glu 1025 1030 1035Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr Thr 1040 1045 1050Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 1055 1060 1065Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser 1070 1075 1080Ser Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 1085 1090 1095813294DNAAspergillus oryzae 81atgcgttcct 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 3294821097PRTAspergillus oryzae 82Met Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro1 5 10 15Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20 25 30Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln Pro 35 40 45Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50 55 60Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln65 70 75 80Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr 85 90 95Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu 100 105 110Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln 115 120 125Ser Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130 135 140Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe145 150 155 160Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu 165 170 175Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180 185 190Asp Trp Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val 195 200 205Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg Arg Asn Asp 210 215 220Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Met Arg Ser Ser Pro Leu225 230 235 240Leu Arg Ser Ala Val Val Ala Ala Leu Pro Val Leu Ala Leu Ala Lys 245 250 255Asp Asp Leu Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp 260 265 270Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala Val Asp Ile Val 275 280 285Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 290 295 300Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser Val Pro Arg Leu305 310 315 320Asn Ile Pro Ser Leu Cys Leu Gln Asp Ser Pro Leu Gly Ile Arg Phe 325 330 335Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val Ala Ala Thr 340 345 350Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala Met Gly Glu Glu 355 360 365Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala Ala Gly Pro 370 375 380Leu Gly Ala His Pro Asp Gly Gly Arg Asn Trp Glu Ser Phe Ser Pro385 390 395 400Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile Lys Gly Ile 405 410 415Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr Ile Met Asn Glu 420 425 430Gln Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly Tyr Gly Phe Asn 435 440 445Val Ser Asp Ser Leu Ser Ser Asn Val Asp Asp Lys Thr Met His Glu 450 455 460Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly Val Gly Ala465 470 475 480Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Glu Asn 485 490 495Ser Glu Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly 500 505 510Phe Val Met Ser Asp Trp Thr Ala Gln His Ser Gly Val Gly Ala Ala 515 520 525Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Thr Phe Asp Ser 530 535 540Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly Val Leu Asn Gly545 550 555 560Thr Ile Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ala 565 570 575Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys Tyr Thr Pro Pro Asn Phe 580 585 590Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His Asn His Val Ser 595 600 605Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp Val Gln Arg Asp 610 615 620His Ala Asp Leu Ile Arg Arg Ile Gly Ala Gln Ser Thr Val Leu Leu625 630 635 640Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys Glu Lys Leu Val Ala 645 650 655Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly Ala Asn Gly Cys 660 665 670Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala Trp Gly Ser 675 680 685Gly Thr Ala Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln 690 695 700Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala Val Thr Asp Ser705 710 715 720Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln Ala Ser Val Ser 725 730 735Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr Leu Ser Val Asp 740 745 750Gly Asn Glu Gly Asp Arg Asn Asn Ile Thr Leu Trp Lys Asn Gly Asp 755 760 765Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn Thr Val Val Ile 770 775 780Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp Tyr Asp His Pro785 790 795 800Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly 805 810 815Asn Ser Ile Ala Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala Lys 820 825 830Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr Gly Ser Pro Leu 835 840 845Val Lys Asp Ala

Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp Phe Thr 850 855 860Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys Phe Asn Glu Thr865 870 875 880Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Leu 885 890 895Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg Tyr Thr Pro Thr 900 905 910Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu Ile Gly Asp Ala 915 920 925Ser Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile His Glu Phe Ile 930 935 940Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala Ser Ser Asp Asp Ser945 950 955 960Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu Gly Ala Thr Asp 965 970 975Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly Ala Gly Gly Asn 980 985 990Pro Gly Leu Tyr Glu Asp Leu Phe Arg Val Ser Val Lys Val Lys Asn 995 1000 1005Thr Gly Asn Val Ala Gly Asp Glu Val Pro Gln Leu Tyr Val Ser 1010 1015 1020Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe Glu 1025 1030 1035Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr Thr 1040 1045 1050Leu Thr Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 1055 1060 1065Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn Ser 1070 1075 1080Ser Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 1085 1090 1095

Claims

1. A nucleic acid construct comprising a gene encoding a protein operably linked to a nucleotide sequence encoding a signal peptide comprising or consisting of amino acids 1 to 15 of SEQ ID NO: 2, wherein the gene is foreign to the nucleotide sequence encoding the signal peptide.

2. A recombinant expression vector comprising the nucleic acid construct of claim 1.

3. A recombinant host cell comprising the nucleic acid construct of claim 1.

4. A method of producing a protein, comprising: (a) cultivating the recombinant host cell of claim 3 under conditions conducive for production of the protein; and (b) recovering the protein.

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