Patent application title: Polypeptides Having Cellulolytic Enhancing Activity And Polynucleotides Encoding Same
Inventors:
Paul Harris (Carnation, WA, US)
Novozymes, Inc. (Davis, CA, US)
Suchindra Maiyuran (Gold River, CA, US)
Suchindra Maiyuran (Gold River, CA, US)
Kimberly Brown (Elk Grove, CA, US)
Assignees:
NOVOZYMES, INC.
IPC8 Class: AC12N924FI
USPC Class:
800298
Class name: Multicellular living organisms and unmodified parts thereof and related processes plant, seedling, plant seed, or plant part, per se higher plant, seedling, plant seed, or plant part (i.e., angiosperms or gymnosperms)
Publication date: 2013-05-09
Patent application number: 20130117892
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.Claims:
1. An isolated polypeptide having cellulolytic enhancing activity,
selected from the group consisting of: (a) a polypeptide comprising an
amino acid sequence having at least 60% identity to the mature
polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4; (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 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature
polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a
full-length complementary strand of (i) or (ii); (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 (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 or SEQ ID NO: 4.
2. The polypeptide of claim 1, comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or a fragment thereof having cellulolytic enhancing activity.
3. The polypeptide of claim 1, which is encoded by the polynucleotide contained in plasmid plasmid pSMai190 which is contained in E. coli NRRL B-50083 or plasmid pSMai192 which is contained in E. coli NRRL B-50085.
4. An isolated polynucleotide comprising a nucleotide sequence that encodes the polypeptide claim 1.
5. A nucleic acid construct comprising the polynucleotide of claim 4 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.
6. A recombinant host cell comprising the nucleic acid construct of claim 5.
7. A method of producing the polypeptide of claim 1, 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.
8. A method of producing the polypeptide of claim 1, comprising: (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
9. A method of producing a mutant of a parent cell, comprising disrupting or deleting a nucleotide sequence encoding the polypeptide of claim 1, which results in the mutant producing less of the polypeptide than the parent cell.
10. A method of producing the polypeptide of claim 1, comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
11. A transgenic plant, plant part or plant cell transformed with a polynucleotide encoding the polypeptide of claim 1.
12. A double-stranded inhibitory RNA (dsRNA) molecule comprising a subsequence of the polynucleotide of claim 4, wherein optionally the dsRNA is a siRNA or a miRNA molecule.
13. A method of inhibiting the expression of a polypeptide having cellulolytic enhancing activity 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 the polynucleotide of claim 4.
14. 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 17 of SEQ ID NO: 2 or amino acids 1 to 15 of SEQ ID NO: 4, wherein the gene is foreign to the nucleotide sequence.
15. A recombinant host cell comprising the nucleic acid construct of claim 14.
16. A method of producing a protein, comprising: (a) cultivating the recombinant host cell of claim 15 under conditions conducive for production of the protein; and (b) recovering the protein.
17. A method for degrading or converting a cellulosic material, comprising: treating the cellulosic material with a cellulolytic enzyme composition in the presence of the polypeptide having cellulolytic enhancing activity of claim 1, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity.
18. The method of claim 17, further comprising recovering the degraded cellulosic material.
19. A method for producing a fermentation product, comprising: (a) saccharifying a cellulosic material with a cellulolytic enzyme composition in the presence of the polypeptide having cellulolytic enhancing activity of claim 1, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity; (b) fermenting the saccharified cellulosic material of step (a) with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
20. A method of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is hydrolyzed with a cellulolytic enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity of claim 1 and the presence of the polypeptide having cellulolytic enhancing activity increases the hydrolysis of the cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent application Ser. No. 12/745,985, now U.S. Pat. No. 8,323,944, which is a 35 U.S.C. 371 national application of PCT/US2008/087402, filed on Dec. 18, 2008, which claims priority from U.S. provisional application Ser. No. 61/014,900, filed on Dec. 19, 2007, and U.S. provisional application Ser. No. 61/014,980 filed on Dec. 19, 2007. The contents of these applications are fully incorporated herein by reference.
REFERENCE TO A SEQUENCE LISTING
[0002] This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.
REFERENCE TO DEPOSIT OF BIOLOGICAL MATERIAL
[0003] This application contains a reference to a deposit of biological material, which deposit is incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] 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.
[0006] 2. Description of the Related Art
[0007] Cellulose is a polymer of the simple sugar glucose linked by beta-1,4-bonds. 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.
[0008] The conversion of lignocellulosic 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.
[0009] It would be advantageous in the art to improve the ability to convert cellulosic feedstocks.
[0010] WO 2005/074647 discloses isolated polypeptides having cellulolytic enhancing activity and polynucleotides thereof from Thielavia terrestris. WO 2005/074656 discloses an isolated polypeptide having cellulolytic enhancing activity and the polynucleotide thereof from Thermoascus aurantiacus. WO 2007/089290 discloses an isolated polypeptide having cellulolytic enhancing activity and the polynucleotide thereof from Trichoderma reesei.
[0011] The present invention relates to polypeptides having cellulolytic enhancing activity and polynucleotides encoding the polypeptides.
SUMMARY OF THE INVENTION
[0012] The present invention relates to isolated polypeptides having cellulolytic enhancing activity selected from the group consisting of:
[0013] (a) a polypeptide comprising an amino acid sequence having at least 60% identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4;
[0014] (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 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary strand of (i) or (ii);
[0015] (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 or SEQ ID NO: 3; and
[0016] (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 or SEQ ID NO: 4.
[0017] The present invention also relates to isolated polynucleotides encoding polypeptides having cellulolytic enhancing activity, selected from the group consisting of:
[0018] (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 or SEQ ID NO: 4;
[0019] (b) a polynucleotide that hybridizes under at least medium stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary strand of (i) or (ii);
[0020] (c) a polynucleotide comprising a nucleotide sequence having at least 60% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3; and
[0021] (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 or SEQ ID NO: 4.
[0022] 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.
[0023] The present invention also relates to methods of inhibiting the expression of a polypeptide having cellulolytic enhancing activity 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.
[0024] The present invention also relates to methods for degrading or converting a cellulosic material, comprising: treating the cellulosic material with a cellulolytic enzyme composition in the presence of such a polypeptide having cellulolytic enhancing activity, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity.
[0025] The present invention also relates to methods of producing a fermentation product, comprising: (a) saccharifying a cellulosic material with a cellulolytic enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity; (b) fermenting the saccharified cellulosic material of step (a) with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
[0026] The present invention also relates to methods of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is hydrolyzed with a cellulolytic enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity of the present invention and the presence of the polypeptide having cellulolytic enhancing activity increases the hydrolysis of the cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity.
[0027] The present invention also relates to plants comprising an isolated polynucleotide encoding a polypeptide having cellulolytic enhancing activity.
[0028] The present invention also relates to methods of producing a polypeptide having cellulolytic enhancing activity, comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide having cellulolytic enhancing activity under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
[0029] 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 17 of SEQ ID NO: 2 or amino acids 1 to 15 of SEQ ID NO: 4, wherein the gene is foreign to the nucleotide sequence.
BRIEF DESCRIPTION OF THE FIGURES
[0030] FIG. 1 shows the genomic DNA sequence and the deduced amino acid sequence of a Myceliophthora thermophila CBS 202.75 GH61A polypeptide having cellulolytic enhancing activity (SEQ ID NOs: 1 and 2, respectively).
[0031] FIG. 2 shows a restriction map of pSMai190.
[0032] FIG. 3 shows the genomic DNA sequence and the deduced amino acid sequence of a Myceliophthora thermophila CBS 202.75 GH61F polypeptide having cellulolytic enhancing activity (SEQ ID NOs: 3 and 4, respectively).
[0033] FIG. 4 shows a restriction map of pSMai192.
[0034] FIG. 5 shows a restriction map of pSMai185.
[0035] FIG. 6 shows a restriction map of pSMai198.
[0036] FIG. 7 shows the effect of Myceliophthora thermophila GH61A and GH61F polypeptides having cellulolytic enhancing activity on enzymatic hydrolysis of pretreated corn stover.
DEFINITIONS
[0037] Cellulolytic enhancing activity: The term "cellulolytic enhancing activity" is defined herein as a biological activity that enhances the hydrolysis of a cellulosic 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 cellulosic material by cellulase 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 cellulase protein/g of cellulose in PCS and 0.5-20% w/w protein of cellulolytic enhancing activity for 1-7 days 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, Bagsvaerd, 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.
[0038] 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 or SEQ ID NO: 4.
[0039] The polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a cellulosic 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.
[0040] Cellulolytic activity: The term "cellulolytic activity" is defined herein as a biological activity which hydrolyzes a cellulosic 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 NS, Bagsvaerd, 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 NS, Bagsvaerd, Denmark).
[0041] For purposes of the present invention, cellulolytic activity is determined by measuring the increase in hydrolysis of a cellulosic 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.
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] Cellulosic material: The cellulosic material can be any material containing cellulose. 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.
[0047] Cellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. The cellulosic material can be, but is not limited to, herbaceous material, agricultural residue, forestry residue, municipal solid waste, waste paper, and pulp and paper mill residue The cellulosic 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. In a preferred aspect, the cellulosic material is lignocellulose.
[0048] In one aspect, the cellulosic material is herbaceous material. In another aspect, the cellulosic material is agricultural residue. In another aspect, the cellulosic material is forestry residue. In another aspect, the cellulosic material is municipal solid waste. In another aspect, the cellulosic material is waste paper. In another aspect, the cellulosic material is pulp and paper mill residue.
[0049] In another aspect, the cellulosic material is corn stover. In another preferred aspect, the cellulosic material is corn fiber. In another aspect, the cellulosic material is corn cob. In another aspect, the cellulosic material is orange peel. In another aspect, the cellulosic material is rice straw. In another aspect, the cellulosic material is wheat straw. In another aspect, the cellulosic material is switch grass. In another aspect, the cellulosic material is miscanthus. In another aspect, the cellulosic material is bagasse.
[0050] In another aspect, the cellulosic material is microcrystalline cellulose. In another aspect, the cellulosic material is bacterial cellulose.
[0051] The cellulosic material may be used as is or may be subjected to pretreatment, using conventional methods known in the art, as described herein. In a preferred aspect, the cellulosic material is pretreated.
[0052] Pre-treated corn stover: The term "PCS" or "Pre-treated Corn Stover" is defined herein as a cellulosic material derived from corn stover by treatment with heat and dilute acid.
[0053] 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.
[0054] 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 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, 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.
[0055] Mature polypeptide: The term "mature polypeptide" is defined herein as a polypeptide 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 18 to 232 of SEQ ID NO: 2 based on the SignalP program (Nielsen et al., 1997, Protein Engineering 10:1-6) that predicts amino acids 1 to 17 of SEQ ID NO: 2 are a signal peptide. In another preferred aspect, the mature polypeptide is amino acids 16 to 235 of SEQ ID NO: 4 based on the SignalP program that predicts amino acids 1 to 15 of SEQ ID NO: 4 are a signal peptide.
[0056] 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 52 to 921 of SEQ ID NO: 1 based on the SignalP program that predicts nucleotides 1 to 51 of SEQ ID NO: 1 encode a signal peptide. In another preferred aspect, the mature polypeptide coding sequence is nucleotides 46 to 851 of SEQ ID NO: 3 based on the SignalP program that predicts nucleotides 1 to 45 of SEQ ID NO: 3 encode a signal peptide.
[0057] Identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "identity".
[0058] 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)
[0059] 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)
[0060] Homologous sequence: The term "homologous sequence" is defined herein as a predicted protein having 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 Myceliophthora thermophila polypeptide having cellulolytic enhancing activity of SEQ ID NO: 2 or SEQ ID NO: 4, or the mature polypeptide thereof.
[0061] 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 SEQ ID NO: 4; or a homologous sequence thereof; wherein the fragment has cellulolytic enhancing activity. In a preferred aspect, a fragment contains at least 185 amino acid residues, more preferably at least 195 amino acid residues, and most preferably at least 205 amino acid residues of the mature polypeptide of SEQ ID NO: 2 or a homologous sequence thereof. In another preferred aspect, a fragment contains at least 190 amino acid residues, more preferably at least 200 amino acid residues, and most preferably at least 210 amino acid residues of the mature polypeptide of SEQ ID NO: 4 or a homologous sequence thereof.
[0062] 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 SEQ ID NO: 3; 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 555 nucleotides, more preferably at least 585 nucleotides, and most preferably at least 615 nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 1 or a homologous sequence thereof. In another preferred aspect, a subsequence contains at least 570 nucleotides, more preferably at least 600 nucleotides, and most preferably at least 630 nucleotides of the mature polypeptide coding sequence of SEQ ID NO: 3 or a homologous sequence thereof.
[0063] 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.
[0064] 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.
[0065] 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% pure, 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.
[0066] 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.
[0067] cDNA: The term "cDNA" is defined herein as a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic cell. cDNA lacks intron sequences that may be 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.
[0068] 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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.
[0073] 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.
[0074] Modification: The term "modification" means herein any chemical modification of the polypeptide consisting of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4; 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.
[0075] 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 SEQ ID NO: 3; 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 SEQ ID NO: 3; or a homologous sequence thereof.
DETAILED DESCRIPTION OF THE INVENTION
Polypeptides Having Cellulolytic Enhancing Activity
[0076] In a first aspect, the present invention relates to isolated polypeptides comprising an amino acid sequence having a degree of identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 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 have 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 or SEQ ID NO: 4.
[0077] 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 18 to 232 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 18 to 232 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 18 to 232 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 18 to 232 of SEQ ID NO: 2.
[0078] A polypeptide of the present invention preferably comprises the amino acid sequence of SEQ ID NO: 4 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: 4. In another preferred aspect, the polypeptide comprises the mature polypeptide of SEQ ID NO: 4. In another preferred aspect, the polypeptide comprises amino acids 16 to 235 of SEQ ID NO: 4, or an allelic variant thereof; or a fragment thereof having cellulolytic enhancing activity. In another preferred aspect, the polypeptide comprises amino acids 16 to 235 of SEQ ID NO: 4. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 4 or an allelic variant thereof; or a fragment thereof having cellulolytic enhancing activity. In another preferred aspect, the polypeptide consists of the amino acid sequence of SEQ ID NO: 4. In another preferred aspect, the polypeptide consists of the mature polypeptide of SEQ ID NO: 4. In another preferred aspect, the polypeptide consists of amino acids 16 to 235 of SEQ ID NO: 4 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 235 of SEQ ID NO: 4.
[0079] In a second aspect, the present invention relates to isolated polypeptides having cellulolytic enhancing activity that are encoded by polynucleotides 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 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (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 or SEQ ID NO: 3 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 or SEQ ID NO: 3.
[0080] The nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or a subsequence thereof; as well as the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; 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, or most preferably at least 800 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.
[0081] 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 SEQ ID NO: 3; or a subsequence thereof; the carrier material is preferably used in a Southern blot.
[0082] 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 or SEQ ID NO: 3; the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3; 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.
[0083] 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 52 to 921 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 pSMai190 which is contained in E. coli NRRL B-50083, 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 pSMai190 which is contained in E. coli NRRL B-50083.
[0084] In another preferred aspect, the nucleic acid probe is the mature polypeptide coding sequence of SEQ ID NO: 3. In another preferred aspect, the nucleic acid probe is nucleotides 46 to 851 of SEQ ID NO: 3. In another preferred aspect, the nucleic acid probe is a polynucleotide sequence that encodes the polypeptide of SEQ ID NO: 4, or a subsequence thereof. In another preferred aspect, the nucleic acid probe is SEQ ID NO: 3. In another preferred aspect, the nucleic acid probe is the polynucleotide sequence contained in plasmid pSMai192 which is contained in E. coli NRRL B-50085, 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 pSMai192 which is contained in E. coli NRRL B-50085.
[0085] 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.
[0086] 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).
[0087] 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 Tm 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.
[0088] 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 Tm.
[0089] 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 or SEQ ID NO: 3 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 encode a polypeptide having cellulolytic enhancing activity. See polynucleotide section herein.
[0090] 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 SEQ ID NO: 4; 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.
[0091] 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.
[0092] 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.
[0093] 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.
[0094] 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.
[0095] 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).
[0096] 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.
[0097] The total number of amino acid substitutions, deletions and/or insertions of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, 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
[0098] A polypeptide 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.
[0099] 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.
[0100] 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.
[0101] In another preferred aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having cellulolytic enhancing activity.
[0102] In another preferred aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having cellulolytic enhancing activity.
[0103] 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.
[0104] 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.
[0105] 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 graminurn, 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, 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, or Trichoderma viride polypeptide having cellulolytic enhancing activity.
[0106] In another preferred aspect, the polypeptide is a Myceliophthora hinnulea, Myceliophthora lutea, Myceliophthora thermophila, or Myceliophthora vellerea polypeptide having cellulolytic enhancing activity.
[0107] In a more preferred aspect, the polypeptide is a Myceliophthora thermophila polypeptide having cellulolytic enhancing activity. In a most preferred aspect, the polypeptide is a Myceliophthora thermophila CBS 202.75 polypeptide having cellulolytic enhancing activity, e.g., the polypeptide comprising the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0108] 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.
[0109] 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).
[0110] 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).
[0111] 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.
[0112] 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
[0113] 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.
[0114] 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 pSMai190 which is contained in E. coli NRRL B-50083. 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 52 to 921 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 pSMai190 which is contained in E. coli NRRL B-50083.
[0115] In a preferred aspect, the nucleotide sequence comprises or consists of SEQ ID NO: 3. In another more preferred aspect, the nucleotide sequence comprises or consists of the sequence contained in plasmid pSMai192 which is contained in E. coli NRRL B-50085. In another preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding sequence of SEQ ID NO: 3. In another preferred aspect, the nucleotide sequence comprises or consists of nucleotides 46 to 851 of SEQ ID NO: 3. In another more preferred aspect, the nucleotide sequence comprises or consists of the mature polypeptide coding sequence contained in plasmid pSMai192 which is contained in E. coli NRRL B-50085.
[0116] The present invention also encompasses nucleotide sequences that encode polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or the mature polypeptide thereof, which differ from SEQ ID NO: 1 or SEQ ID NO: 3, or the mature polypeptide coding sequence thereof, respectively, by virtue of the degeneracy of the genetic code. The present invention also relates to subsequences of SEQ ID NO: 1 or SEQ ID NO: 3 that encode fragments of SEQ ID NO: 2 or SEQ ID NO: 4 that have cellulolytic enhancing activity, respectively.
[0117] 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 or SEQ ID NO: 3, in which the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, respectively.
[0118] 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 Myceliophthora, 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.
[0119] 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 or SEQ ID NO: 3 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 a polypeptide having cellulolytic enhancing activity.
[0120] 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 or SEQ ID NO: 3, 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.
[0121] 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).
[0122] 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 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, 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 or SEQ ID NO: 3.
[0123] 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 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, 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 or SEQ ID NO: 3.
Nucleic Acid Constructs
[0124] 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.
[0125] 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.
[0126] 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.
[0127] 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 (VIIIa-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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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.
[0134] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.
[0135] 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).
[0136] 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.
[0137] 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.
[0138] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Molecular Cellular Biology 15: 5983-5990.
[0139] 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.
[0140] 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. 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.
[0141] 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.
[0142] In a preferred aspect, the signal peptide comprises or consists of amino acids 1 to 17 of SEQ ID NO: 2. In another preferred aspect, the signal peptide coding sequence comprises or consists of nucleotides 1 to 51 of SEQ ID NO: 1.
[0143] In another preferred aspect, the signal peptide comprises or consists of amino acids 1 to 15 of SEQ ID NO: 4. In another preferred aspect, the signal peptide coding sequence comprises or consists of nucleotides 1 to 45 of SEQ ID NO: 3.
[0144] 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).
[0145] 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.
[0146] 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
[0147] 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.
[0148] 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.
[0149] 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.
[0150] 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.
[0151] 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.
[0152] 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.
[0153] 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.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANS1 (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.
[0158] 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.
[0159] 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
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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.
[0166] 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.
[0167] 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.
[0168] 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.
[0169] 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.
[0170] The host cell may also be a eukaryote, such as a mammalian, insect, plant, or fungal cell.
[0171] 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).
[0172] 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).
[0173] In an even more preferred aspect, the yeast host cell is a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell.
[0174] 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.
[0175] 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.
[0176] 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.
[0177] 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.
[0178] 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
[0179] 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 Myceliophthora. In a more preferred aspect, the cell is Myceliophthora thermophila. In a most preferred aspect, the cell is Myceliophthora thermophila CBS 202.75. In another most preferred aspect, the cell is Myceliophthora thermophila CBS 117.65.
[0180] 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.
[0181] 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 or SEQ ID NO: 3, wherein the mutant nucleotide sequence encodes a polypeptide that comprises or consists of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4; and (b) recovering the polypeptide.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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
[0186] 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.
[0187] 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).
[0188] 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.
[0189] 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.
[0190] Also included within the scope of the present invention are the progeny of such plants, plant parts, and plant cells.
[0191] 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.
[0192] 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).
[0193] 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.
[0194] 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 Mol. 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 and 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.
[0195] 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.
[0196] The selectable marker gene and any other parts of the expression construct may be chosen from those available in the art.
[0197] 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).
[0198] 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.
[0199] 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.
[0200] 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
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] 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.
[0209] 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.
[0210] 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.
[0211] 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.
[0212] 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.
[0213] 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.
[0214] 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.
[0215] The methods used for cultivation and purification of the product of interest may be performed by methods known in the art.
[0216] 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.
[0217] 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.
[0218] 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 Having Cellulolytic Enhancing Acitivity
[0219] The present invention also relates to methods of inhibiting the expression of a polypeptide having cellulolytic enhancing activity 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.
[0220] 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.
[0221] 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 or SEQ ID NO: 3 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).
[0222] 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
[0223] 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.
[0224] The composition may comprise a polypeptide of the present invention as the major enzymatic component, e.g., a mono-component composition. Alternatively, the composition may comprise multiple enzymatic activities, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, 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. 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.
[0225] 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.
[0226] 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.
Processing of Cellulosic Material
[0227] The present invention also relates to methods for degrading or converting a cellulosic material, comprising: treating the cellulosic material with a cellulolytic enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity of the present invention. In a preferred aspect, the method further comprises recovering the degraded or converted cellulosic material.
[0228] The present invention also relates to methods of producing a fermentation product, comprising: (a) saccharifying a cellulosic material with a cellulolytic enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity of the present invention; (b) fermenting the saccharified cellulosic material of step (a) with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.
[0229] The present invention also relates to methods of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is hydrolyzed with a cellulolytic enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity of the present invention and the presence of the polypeptide having cellulolytic enhancing activity increases the hydrolysis of the cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity. In a preferred aspect, the fermenting of the cellulosic material produces a fermentation product. In another preferred aspect, the method further comprises recovering the fermentation product from the fermentation.
[0230] The composition comprising the polypeptide having cellulolytic enhancing activity can be in the form of a crude fermentation broth with or without the cells removed or in the form of a semi-purified or purified enzyme preparation or the composition can comprise a host cell of the present invention as a source of the polypeptide having cellulolytic enhancing activity in a fermentation process with the biomass.
[0231] The methods of the present invention can be used to saccharify a cellulosic material 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 cellulosic material typically involves pretreatment, enzymatic hydrolysis (saccharification), and fermentation.
[0232] The processing of cellulosic material according to the present invention can be accomplished using processes conventional in the art. Moreover, the methods of the present invention can be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention.
[0233] Hydrolysis (saccharification) and fermentation, separate or simultanoeus, 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). SHF uses separate process steps to first enzymatically hydrolyze lignocellulose to fermentable sugars, e.g., glucose, cellobiose, cellotriose, and pentose sugars, and then ferment the fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of lignocellulose and the fermentation of sugars to ethanol are combined in one step (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). SSCF involves the cofermentation of multiple sugars (Sheehan, J., and Himmel, M., 1999, Enzymes, energy and the environment: A strategic perspective on the U.S. Department of Energy's research and development activities for bioethanol, Biotechnol. Prog. 15: 817-827). HHF involves a separate hydrolysis separate step, and in addition a simultaneous saccharification and hydrolysis step, which can be carried out in the same reactor. The steps in an HHF process can be carried out at different temperatures, i.e., high temperature enzymatic saccharification followed by SSF at a lower temperature that the fermentation strain can tolerate. DMC combines all three processes (enzyme production, lignocellulose hydrolysis, and fermentation) in one or more steps where the same organism is used to produce the enzymes for conversion of the lignocellulose to fermentable sugars and to convert the fermentable sugars into a final product (Lynd, L. R., Weimer, P. J., van Zyl, W. H., and Pretorius, I. S., 2002, Microbial cellulose utilization: Fundamentals and biotechnology, Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein that any method known in the art comprising pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination thereof can be used in the practicing the methods of the present invention.
[0234] 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: Fluidized bed, upflow blanket, immobilized, and extruder type reactors for hydrolysis and/or fermentation.
[0235] Pretreatment.
[0236] 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. The cellulosic 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 pretreatments.
[0237] The cellulosic 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 cellulosic 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).
[0238] Steam Pretreatment. In steam pretreatment, the cellulosic 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 lignocellulose 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 160-200° C., and most preferably 170-190° C., where the optimal temperature range depends on any 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 temperature range and any addition of a chemical catalyst. Steam pretreatment allows for relatively high solids loadings, so that the cellulosic 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). During steam pretreatment, hemicellulose acetyl groups are cleaved and the resulting acid autocatalyzes partial hydrolysis of the hemicellulose to monosaccharides and oligosaccharides. Lignin is removed to only a limited extent.
[0239] 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 the 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).
[0240] 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.
[0241] In dilute acid pretreatment, the cellulosic 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).
[0242] 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).
[0243] 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.
[0244] 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.
[0245] 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).
[0246] Ammonia fiber explosion (AFEX) involves treating cellulosic 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). AFEX pretreatment results in the depolymerization of cellulose and partial hydrolysis of hemicellulose. Lignin-carbohydrate complexes are cleaved.
[0247] Organosolv pretreatment delignifies cellulosic 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.
[0248] 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.
[0249] 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 cellulosic material and held at a temperature in the range of preferably 160-220° C., and more preferably 165-195° C., for periods ranging from seconds to minutes to, e.g., 1 second to 60 minutes.
[0250] In another aspect, pretreatment is carried out as an ammonia fiber explosion step (AFEX pretreatment step).
[0251] In another aspect, pretreatment takes place in an aqueous slurry. In preferred aspects, the cellulosic 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 cellulosic material can be unwashed or washed using any method known in the art, e.g., washed with water.
[0252] Mechanical Pretreatment: The term "mechanical pretreatment" refers to various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).
[0253] Physical Pretreatment: The term "physical pretreatment" refers to any pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin from cellulosic material. For example, physical pretreatment can involve irradiation (e.g., microwave irradiation), steaming/steam explosion, hydrothermolysis, and combinations thereof.
[0254] 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.
[0255] Combined Physical and Chemical Pretreatment: The cellulosic 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.
[0256] Accordingly, in a preferred aspect, the cellulosic 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.
[0257] 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 cellulosic 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
[0258] Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of cellulosic 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 cellulosic material is hydrolyzed to break down cellulose and alternatively also hemicellulose to fermentable sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, or soluble oligosaccharides. The hydrolysis is performed enzymatically by a cellulolytic enzyme composition comprising a polypeptide having cellulolytic enhancing activity of the present invention, which can further comprise one or more hemicellulolytic enzymes. The enzymes of the compositions can also be added sequentially.
[0261] 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 cellulosic material (substrate) is fed gradually to, for example, an enzyme containing hydrolysis solution.
[0262] 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 %.
[0263] In addition to a polypeptide having cellulolytic enhancing activity of the present invention, the cellulolytic enzyme components of the composition are preferably enzymes having endoglucanase, cellobiohydrolase, and beta-glucosidase activities. In a preferred aspect, the cellulolytic enzyme composition comprises one or more (several) cellulolytic enzymes selected from the group consisting of a cellulase, endoglucanase, cellobiohydrolase, and beta-glucosidase. In another preferred aspect, the cellulolytic enzyme preparation is supplemented with one or more additional enzyme activities selected from the group consisting of hemicellulases, esterases (e.g., lipases, phospholipases, and/or cutinases), proteases, laccases, peroxidases, or mixtures thereof. In the methods of the present invention, the additional enzyme(s) can be added prior to or during fermentation, including during or after propagation of the fermenting microorganism(s).
[0264] The enzymes can be derived or obtained from any suitable origin, including, bacterial, fungal, yeast, plant, or mammalian origin. The term "obtained" means herein that the enzyme may have been isolated from an organism that naturally produces the enzyme as a native enzyme. The term "obtained" also means herein that the enzyme may have been produced recombinantly in a host organism employing methods described herein, 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 recombinantly, such as by site-directed mutagenesis or shuffling.
[0265] The enzymes used in the present invention can be in any form suitable for use in the methods described herein, such as a crude fermentation broth with or without cells or substantially pure polypeptides. The enzyme(s) can be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a protected enzyme(s). Granulates can be produced, e.g., as disclosed in U.S. Pat. Nos. 4,106,991 and 4,661,452, and can optionally be coated by process known in the art. Liquid enzyme preparations can, 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 can be prepared according to the process disclosed in EP 238,216.
[0266] 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 enzymes, 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).
[0267] In a preferred aspect, an effective amount of cellulolytic enzyme(s) to cellulosic 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 cellulosic material.
[0268] In another preferred aspect, an effective amount of a polypeptide having cellulolytic enhancing activity to cellulosic material is about 0.01 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 cellulosic material.
[0269] In another preferred aspect, an effective amount of polypeptide(s) having cellulolytic enhancing activity to cellulosic 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 cellulosic material.
[0270] In another preferred aspect, an effective amount of polypeptide(s) having cellulolytic enhancing activity to cellulolytic enzyme(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 enzyme(s).
[0271] Fermentation.
[0272] The fermentable sugars obtained from the pretreated and hydrolyzed cellulosic 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 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.
[0273] In the fermentation step, sugars, released from the cellulosic 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).
[0274] Any suitable hydrolyzed cellulosic 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. Examples of substrates suitable for use in the methods of present invention, include cellulosic materials, such as wood or plant residues or low molecular sugars DP1-3 obtained from processed cellulosic material that can be metabolized by the fermenting microorganism, and which can be supplied by direct addition to the fermentation medium.
[0275] 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 used in a simultaneous saccharification and fermentation process (SSF).
[0276] "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.
[0277] Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.
[0278] Examples of fermenting microorganisms that can ferment C6 sugars include bacterial and fungal organisms, such as yeast. Preferred yeast includes strains of the Saccharomyces spp., preferably Saccharomyces cerevisiae.
[0279] 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.
[0280] 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.
[0281] 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).
[0282] Bacteria that can efficiently ferment hexose and pentose to ethanol include, for example, Zymomonas mobilis and Clostridium thermocellum (Philippidis, 1996, supra).
[0283] 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.
[0284] 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, Wis., 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).
[0285] 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.
[0286] 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).
[0287] 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.
[0288] It is well known in the art that the organisms described above can also be used to produce other substances, as described herein.
[0289] The fermenting microorganism is typically added to the degraded lignocellulose 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.
[0290] In a preferred aspect, the yeast and/or another microorganism is applied to the degraded lignocellulose 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 bacterial fermenting organisms, for example, have higher fermentation temperature optima. Yeast or another microorganism 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.
[0291] The most widely used process in the art is the simultaneous saccharification and fermentation (SSF) process where there is no holding stage for the saccharification, meaning that yeast and enzyme are added together.
[0292] 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.
[0293] 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.
[0294] Fermentation Products:
[0295] 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 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.
[0296] 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.
[0297] 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.
[0298] 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.
[0299] 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.
[0300] 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.
[0301] Recovery.
[0302] 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, electrophoretic procedures, differential solubility, distillation, or extraction. For example, alcohol is separated from the fermented cellulosic 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.
Cellulolytic Enzyme Compositions
[0303] 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 one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. 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.
[0304] 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.
[0305] 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.
[0306] The enzymes used in the present invention may be in any form suitable for use in the processes described herein, such as, for example, a crude fermentation broth 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.
[0307] 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.
[0308] 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.
[0309] In another preferred aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having cellulolytic enzyme activity.
[0310] In another preferred aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having cellulolytic enzyme activity.
[0311] 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.
[0312] 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.
[0313] 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.
[0314] Chemically modified or protein engineered mutants of cellulolytic proteins may also be used.
[0315] One or more components of the cellulolytic enzyme composition 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 is preferably a heterologous host (enzyme is foreign to host), but the host may under certain conditions also be a homologous host (enzyme is native to host). Monocomponent cellulolytic proteins may also be prepared by purifying such a protein from a fermentation broth.
[0316] Examples of commercial cellulolytic protein preparations suitable for use in the present invention include, for example, CELLUCLAST® (available from Novozymes NS) and NOVOZYM® 188 (available from Novozymes A/S). Other commercially available preparations comprising cellulase that may be used include CELLUZYMET®, 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). The cellulase enzymes are added in amounts effective from about 0.001% to about 5.0% wt. of solids, more preferably from about 0.025% to about 4.0% wt. of solids, and most preferably from about 0.005% to about 2.0% wt. of solids.
[0317] Examples of bacterial endoglucanases that can be used in the methods of the present invention, include, but are not limited to, an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No. 5,536,655, WO 00/70031, WO 05/093050); Thermobifida fusca endoglucanase III (WO 05/093050); and Thermobifida fusca endoglucanase V (WO 05/093050).
[0318] Examples of fungal endoglucanases that can be used in the methods of the present invention, include, but are not limited to, a Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263; GENBANK® 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); Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439); 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); Humicola insolens endoglucanase V (SEQ ID NO: 25); Myceliophthora thermophila CBS117.65 endoglucanase (SEQ ID NO: 27); basidiomycete CBS 495.95 endoglucanase (SEQ ID NO: 29); basidiomycete CBS 494.95 endoglucanase (SEQ ID NO: 31); Thielavia terrestris NRRL 8126 CEL6B endoglucanase (SEQ ID NO: 33); Thielavia terrestris NRRL 8126 CEL6C endoglucanase (SEQ ID NO: 35); Thielavia terrestris NRRL 8126 CEL7C endoglucanase (SEQ ID NO: 37); Thielavia terrestris NRRL 8126 CEL7E endoglucanase (SEQ ID NO: 39); Thielavia terrestris NRRL 8126 CEL7F endoglucanase (SEQ ID NO: 41); Cladorrhinum foecundissimum ATCC 62373 CEL7A endoglucanase (SEQ ID NO: 43); and Trichoderma reesei strain No. VTT-D-80133 endoglucanase (SEQ ID NO: 45; GENBANK® accession no. M15665). The endoglucanases of SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 41, SEQ ID NO: 43, and SEQ ID NO: 45 described above are encoded by the mature polypeptide coding sequence of SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, and SEQ ID NO: 44, respectively.
[0319] Examples of cellobiohydrolases useful in the methods of the present invention include, but are not limited to, Trichoderma reesei cellobiohydrolase I (SEQ ID NO: 47); Trichoderma reesei cellobiohydrolase II (SEQ ID NO: 49); Humicola insolens cellobiohydrolase I (SEQ ID NO: 51), Myceliophthora thermophila cellobiohydrolase II (SEQ ID NO: 53 and SEQ ID NO: 55), Thielavia terrestris cellobiohydrolase II (CEL6A) (SEQ ID NO: 57), Chaetomium thermophilum cellobiohydrolase I (SEQ ID NO: 59), and Chaetomium thermophilum cellobiohydrolase II (SEQ ID NO: 61). The cellobiohydrolases of SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, and SEQ ID NO: 61 described above are encoded by the mature polypeptide coding sequence of SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and SEQ ID NO: 60, respectively.
[0320] Examples of beta-glucosidases useful in the methods of the present invention include, but are not limited to, Aspergillus oryzae beta-glucosidase (SEQ ID NO: 63); Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 65); Penicillium brasilianum IBT 20888 beta-glucosidase (SEQ ID NO: 67); Aspergillus niger beta-glucosidase (SEQ ID NO: 69); and Aspergillus aculeatus beta-glucosidase (SEQ ID NO: 71). The beta-glucosidases of SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, and SEQ ID NO: 71 described above are encoded by the mature polypeptide coding sequence of SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, and SEQ ID NO: 70, respectively.
[0321] 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.
[0322] The beta-glucosidase may be a fusion protein. In one aspect, the beta-glucosidase is the Aspergillus oryzae beta-glucosidase variant BG fusion protein of SEQ ID NO: 73 or the Aspergillus oryzae beta-glucosidase fusion protein of SEQ ID NO: 75. In another aspect, the Aspergillus oryzae beta-glucosidase variant BG fusion protein is encoded by the polynucleotide of SEQ ID NO: 72 or the Aspergillus oryzae beta-glucosidase fusion protein is encoded by the polynucleotide of SEQ ID NO: 74.
[0323] Other endoglucanases, cellobiohydrolases, and beta-glucosidases are disclosed in numerous 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.
[0324] Other cellulolytic enzymes that may be used 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.
[0325] The cellulolytic enzymes 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 enzyme 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).
[0326] The fermentation can be any method of cultivation of a cell resulting in the expression or isolation of a cellulolytic enzyme. 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 enzyme to be expressed or isolated. The resulting cellulolytic enzymes produced by the methods described above may be recovered from the fermentation medium and purified by conventional procedures.
Signal Peptide
[0327] 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 17 of SEQ ID NO: 2 or amino acids 1 to 15 of SEQ ID NO: 4, wherein the gene is foreign to the nucleotide sequence.
[0328] In a preferred aspect, the nucleotide sequence comprises or consists of nucleotides 1 to 51 of SEQ ID NO: 1 or nucleotides 1 to 45 of SEQ ID NO: 3.
[0329] The present invention also relates to recombinant expression vectors and recombinant host cells comprising such nucleic acid constructs.
[0330] 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.
[0331] 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.
[0332] 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.
[0333] The gene may be obtained from any prokaryotic, eukaryotic, or other source.
[0334] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention.
EXAMPLES
Materials
[0335] Chemicals used as buffers and substrates were commercial products of at least reagent grade.
Media
[0336] BA medium was composed per liter of 10 g of corn steep liquor dry matter, 10 g of NH4NO3, 10 g of KH2PO4, 0.75 g of MgSO4.7H2O, 0.1 ml of pluronic, and 0.5 g of CaCO3. The pH was adjusted to 6.5 before autoclaving.
[0337] YEG medium was composed per liter of 20 g of dextrose and 5 g of yeast extract.
[0338] Minimal medium plates were composed per liter of 6 g of NaNO3, 0.52 g of KCl, 1.52 g of KH2PO4, 1 ml of COVE trace elements solution, 20 g of Noble agar, 20 ml of 50% glucose, 2.5 ml of MgSO4.7H2O, and 20 ml of a 0.02% biotin solution.
[0339] 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.
[0340] M410 medium was composed per liter of 50 g of maltose, 50 g of glucose, 2 g of MgSO4.7H2O, 2 g of KH2PO4, 4 g of anhydrous citric acid, 8 g of yeast extract, 2 g of urea, 0.5 g of CaCl2, and 0.5 ml of AMG trace metals solution.
[0341] AMG trace metals 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 FeSO4.7H2O, 8.5 g of MnSO4.7H2O, and 3 g of citric acid.
Example 1
Identification of Family 61 Peptides
[0342] SDS-PAGE Analysis.
[0343] A commercial product was diluted 1:10 with water. Twenty μl was separated on a CRITERION® 8-16% Tris-HCl SDS-PAGE gel according to the manufacturer's suggested conditions (Bio-Rad Laboratories, Inc., Hercules, Calif., USA). PRECISION PLUS PROTEIN® standards (Bio-Rad Laboratories, Inc., Hercules, Calif., USA) were used as molecular weight markers. The gel was stained with BIO-SAFE® Coomassie Stain (Bio-Rad Laboratories, Inc., Hercules, Calif., USA), and visible bands were excised with a razor blade for protein identification analysis.
[0344] In-Gel Digestion of Polypeptides for Peptide Sequencing.
[0345] A MuItiPROBE® II Liquid Handling Robot (PerkinElmer Life and Analytical Sciences, Boston, Mass., USA) was used to perform the in-gel digestions. Gel bands containing protein were reduced with 50 μl of 10 mM dithiothreitol (DTT) in 100 mM ammonium bicarbonate pH 8.0 for 30 minutes. Following reduction, the gel piece was alkylated with 50 μl of 55 mM iodoacetamide in 100 mM ammonium bicarbonate pH 8.0 for 20 minutes. The dried gel piece was allowed to swell in 25 μl of a trypsin digestion solution (6 ng/μl sequencing grade trypsin (Promega, Madison, Wis., USA) in 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 above 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 de-hydrate the gel piece between reactions and the gel piece was 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 96 well skirted PCR type plate (ABGene, Rochester, N.Y., USA) that had been cooled to 10-15° C. and covered with a 96-well plate lid (PerkinElmer Life and Analytical Sciences, Boston, Mass., USA) to prevent evaporation. Plates were further stored at 4° C. until mass spectrometry analysis could be performed.
[0346] Protein Identification.
[0347] For de novo peptide sequencing by tandem mass spectrometry, a Q-TOFMICRO® (Waters Micromass MS Technologies, Milford, Mass., USA), a hybrid orthogonal quadrupole time-of-flight mass spectrometer was used for LC/MS/MS analysis. The Q-TOF MICRO® is fully microprocessor controlled using MASSLYNX® software version 4.1 (Waters Micromass MS Technologies, Milford, Mass., USA). The Q-TOF MICRO® was fitted with an ULTIMATE® capillary and nano-flow HPLC system, which was coupled with a FAMOS® micro autosampler and a SWITCHOS® II column switching device (LCPackings/Dionex, Sunnyvale, Calif., USA) for concentrating and desalting samples. Samples were loaded onto a guard column (300 μm ID×5 cm, PEPMAP® C18) 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. Peptides were separated on a 75 μm ID×15 cm, C18, 3 μm, 100 Å PEPMAP® (LC Packings, San Francisco, Calif., USA) nanoflow fused capillary column at a flow rate of 175 nl/minute from a split flow of 175 μl/minute using a NAN-75 calibrator (Dionex, Sunnyvale, Calif., USA). A step elution gradient of 5% to 80% acetonitrile in 0.1% formic acid was applied over a 45 minute interval. The column eluent was monitored at 215 nm and introduced into the Q-TOF MICRO® through an electrospray ion source fitted with the nanospray interface.
[0348] Data was acquired in survey scan mode and from a mass range of m/z 400 to 1990 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 45 volts was typically used and the collision energy was programmed to be varied according to the mass and charge state of the eluting peptide and in the range of 10-60 volts. The acquired spectra were combined, smoothed, and centered in an automated fashion and a peak list generated. This peak list was searched against selected databases using PROTEINLYNX® Global Server 2.2.05 software (Waters Micromass MS Technologies, Milford, Mass., USA) and PEAKS Studio version 4.5 (SP1) (Bioinformatic Solutions Inc., Waterloo, Ontario, Canada) Results from the PROTEINLYNX® and PEAKS Studio searches were evaluated and un-identified proteins were analyzed further by evaluating the MS/MS spectra of each ion of interest and de novo sequence was determined by identifying the y and b ion series and matching mass differences to the appropriate amino acid.
[0349] Peptide sequences were obtained from several multiply charged ions for the in-gel digested approximately 24 kDa polypeptide gel band. A doubly charged tryptic peptide ion of 871.56 m/z sequence was determined to be [Leu]-Pro-Ala-Ser-Asn-Ser-Pro-Val-Thr-Asp-Val-Thr-Ser-Asn-Ala-[Leu]- -Arg (SEQ ID NO: 5). A doubly charged tryptic peptide ion of 615.84 m/z sequence was determined to be Val-Asp-Asn-Ala-Ala-Thr-Ala-Ser-Pro-Ser-Gly-[Leu]-Lys (SEQ ID NO: 6). A doubly charged tryptic peptide ion of 715.44 m/z sequence was determined to be [Leu]-Pro-Ala-Asp-[Leu]-Pro-Ser-Gly-Asp-Tyr-[Leu]-[Leu]-Arg (SEQ ID NO: 7). A doubly charged tryptic peptide ion of 988.58 m/z sequence was determined to be Gly-Pro-[Leu]-[Gln]-Val-Tyr-[Leu]-Ala-Lys (SEQ ID NO: 8). A double charged tryptic peptide ion of 1272.65 m/z sequence was determined to be Val-Ser-Val-Asn-Gly-[Gln]-Asp-[Gln]-Gly-[Gln]-[Leu]-Lys (SEQ ID NO: 9). [Leu] above may be Ile or Leu and [Gln] above may be Gln or Lys because they could not be distinguished due to equivalent masses.
Example 2
Preparation of Myceliophthora thermophila CBS117.65 cDNA Pool
[0350] Myceliophthora thermophila CBS117.65 was cultivated in 200 ml of BA medium at 30° C. for five days at 200 rpm. Mycelia from the shake flask culture were harvested by filtering the contents through a funnel lined with MIRACLOTH® (CalBiochem, San Diego, Calif., USA). The mycelia were then sandwiched between two MIRACLOTH® pieces and blotted dry with absorbent paper towels. The mycelial mass was then transferred to plastic centrifuge tubes and frozen in liquid nitrogen. Frozen mycelia were stored in a -80° C. freezer until use.
[0351] The extraction of total RNA was performed with guanidinium thiocyanate followed by ultracentrifugation through a 5.7 M CsCl cushion, and isolation of poly(A)+RNA was carried out by oligo(dT)-cellulose affinity chromatography, using the procedures described in WO 94/14953.
[0352] Double-stranded cDNA was synthesized from 5 μg of poly(A)+ RNA by the RNase H method (Gubler and Hoffman, 1983, Gene 25: 263-269, Sambrook et al., 1989, Molecular cloning: A laboratory manual, Cold Spring Harbor lab., Cold Spring Harbor, N.Y., USA). The poly(A)+ RNA (5 μg in 5 μl of DEPC (0.1% diethylpyrocarbonate)-treated water) was heated at 70° C. for 8 minutes in a pre-siliconized, RNase-free EPPENDORF® tube, quenched on ice, and combined in a final volume of 50 μl with reverse transcriptase buffer composed of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM dithiothreitol (DTT) (Bethesda Research Laboratories, Bethesda, Md., USA), 1 mM of dATP, dGTP and dTTP, and 0.5 mM 5-methyl-dCTP (GE Healthcare, Piscataway, N.J., USA), 40 units of human placental ribonuclease inhibitor (RNasin; Promega, Madison, Wis., USA), 1.45 μg of oligo(dT)18-Not I primer (GE Healthcare, Piscataway, N.J., USA), and 1000 units of SuperScript II RNase H reverse transcriptase (Bethesda Research Laboratories, Bethesda, Md., USA). First-strand cDNA was synthesized by incubating the reaction mixture at 45° C. for 1 hour. After synthesis, the mRNA:cDNA hybrid mixture was gel filtrated through a MICROSPIN® S-400 HR spin column (GE Healthcare, Piscataway, N.J., USA) according to the manufacturer's instructions.
[0353] After gel filtration, the hybrids were diluted in 250 μl of second strand buffer (20 mM Tris-HCl, pH 7.4, 90 mM KCl, 4.6 mM MgCl2, 10 mM (NH4)2SO4, 0.16 mM NAD) containing 200 μM of each dNTP, 60 units of E. coli DNA polymerase I (GE Healthcare, Piscataway, N.J., USA), 5.25 units of RNase H (Promega, Madison, Wis., USA), and 15 units of E. coli DNA ligase (Boehringer Mannheim, Manheim, Germany). Second strand cDNA synthesis was performed by incubating the reaction tube at 16° C. for 2 hours and an additional 15 minutes at 25° C. The reaction was stopped by addition of EDTA to a final concentration of 20 mM followed by phenol and chloroform extractions.
[0354] The double-stranded cDNA was precipitated at -20° C. for 12 hours by addition of 2 volumes of 96% ethanol and 0.2 volume of 10 M ammonium acetate, recovered by centrifugation at 13,000×g, washed in 70% ethanol, dried, and resuspended in 30 μl of Mung bean nuclease buffer (30 mM sodium acetate pH 4.6, 300 mM NaCl, 1 mM ZnSO4, 0.35 mM DTT, 2% glycerol) containing 25 units of Mung bean nuclease (GE Healthcare, Piscataway, N.J., USA). The single-stranded hair-pin DNA was clipped by incubating the reaction at 30° C. for 30 minutes, followed by addition of 70 μl of 10 mM Tris-HCl-1 mM EDTA pH 7.5, phenol extraction, and precipitation with 2 volumes of 96% ethanol and 0.1 volume of 3 M sodium acetate pH 5.2 on ice for 30 minutes.
[0355] The double-stranded cDNAs were recovered by centrifugation at 13,000×g and blunt-ended in 30 μl of T4 DNA polymerase buffer (20 mM Tris-acetate, pH 7.9, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT) containing 0.5 mM of each dNTP and 5 units of T4 DNA polymerase (New England Biolabs, Ipswich, Mass., USA) by incubating the reaction mixture at 16° C. for 1 hour. The reaction was stopped by addition of EDTA to a final concentration of 20 mM, followed by phenol and chloroform extractions, and precipitation for 12 hours at -20° C. by adding 2 volumes of 96% ethanol and 0.1 volume of 3 M sodium acetate pH 5.2. After the fill-in reaction the cDNAs were recovered by centrifugation at 13,000×g, washed in 70% ethanol, and dried.
Example 3
Myceliophthora thermophila CBS 202.75 and Myceliophthora thermophila CBS117.65 Genomic DNA Extraction
[0356] Myceliophthora thermophila CBS 202.75 and Myceliophthora thermophila CBS 117.65 strains were grown in 100 ml of YEG medium in a baffled shake flask at 45° C. and 200 rpm for 2 days. Mycelia were harvested by filtration using MIRACLOTH® (Calbiochem, La Jolla, Calif., USA), washed twice in deionized water, and frozen under liquid nitrogen. Frozen mycelia were ground, by mortar and pestle, to a fine powder, and total DNA was isolated using a DNEASY® Plant Maxi Kit (QIAGEN Inc., Valencia, Calif., USA).
Example 4
Molecular Screening of a Family 61 Gene from Myceliophthora thermophila
[0357] Degenerate primers were designed, as shown below, based upon peptide sequences obtained through tandem mass spectrometry as described in Example 1.
TABLE-US-00001 Primer 061562 (CI61A sense): (SEQ ID NO: 10) 5'-GCCTCCAACTCGCCCGTCACNGAYGTNAC-3' Primer 061563 (CI61A anti): (SEQ ID NO: 11) 5'-GAGGTAGTCGCCGGANGGGATRTCNGCNGG-3'
[0358] Fifty picomoles each of Cl61A sense and Cl61A anti primers were used in a PCR reaction composed of 100 ng of Myceliophthora thermophila CBS117.65 cDNA pool, or Myceliophthora thermophila CBS117.65 genomic DNA, 1× ADVANTAGE® GC-Melt LA Buffer (Clontech Laboratories, Inc., Mountain View, Calif., USA), 0.4 mM each of dATP, dTTP, dGTP, and dCTP, and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix (Clontech Laboratories, Inc., Mountain View, Calif., USA) in a final volume of 25 μl. The amplifications were performed using an EPPENDORF® MASTERCYCLER® 5333 (Eppendorf Scientific, Inc., Westbury, N.Y., USA) programmed for 1 cycle at 94° C. for 1 minute; and 30 cycles each at 94° C. for 30 seconds, 56.5° C. for 30 seconds, and 72° C. for 30 seconds, followed by a final extension of 5 minutes at 72° C.
[0359] The reaction products were fractionated by 1% agarose gel electrophoresis in 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer and bands of greater than 400 bp were excised, purified using a MINELUATE® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer's instructions, and subcloned using a TOPO® TA Kit (Invitrogen, Carlsbad, Calif., USA). Plasmid DNA was extracted from a number of E. coli transformants and sequenced. Sequence analysis of the E. coli clones showed that the sequences contained a coding region of a Family 61 gene (gh61a).
[0360] A second gh61 gene was isolated in a separate PCR reaction performed under different conditions. Thirty picomoles each of Cl61A sense and Cl61A anti primers were used in a PCR reaction composed of 200 ng of Myceliophthora thermophila CBS 202.75 genomic DNA, 1× THERMOPOL® Buffer (New England BioLabs, Ipswich, Mass., USA), 0.28 mM each of dATP, dTTP, dGTP, and dCTP, and 1.0 unit of Taq DNA polymerase (New England BioLabs, Ipswich, Mass.) in a final volume of 30 μl. The amplifications were performed using a ROBOCYCLER® 40 (Strategene, La Jolla, Calif., USA) programmed for 1 cycle at 96° C. for 3 minutes; 1 cycle at 72° C. for 3 minutes during which DNA polymerase was added; 30 cycles each at 94° C. for 50 seconds, 52° C. for 50 seconds, and 72° C. for 90 seconds, followed by a final extension of 7 minutes at 72° C.
[0361] The reaction products were fractionated by 1% agarose gel electrophoresis in 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer and a band of greater than 400-500 bp was excised, purified using a QIAEX II® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer's instructions, and subcloned using a TOPO® TA Kit (Invitrogen, Carlsbad, Calif., USA). Plasmid DNA was extracted from a number of E. coli transformants and sequenced. Sequence analysis revealed several clones containing a coding region of one Family 61 gene designated gh61f.
Example 5
Isolation of a Full-Length Family 61 Gene (gh61a) from Myceliophthora thermophila CBS 202.75
[0362] A full-length Family 61 gene (gh61a) from Myceliophthora thermophila CBS 202.75 was isolated using a GENOMEWALKER® Universal Kit (Clontech Laboratories, Inc., Mountain View, Calif., USA) according to the manufacturer's instructions. Briefly, total genomic DNA from Myceliophthora thermophila CBS 202.75 was digested separately with four different restriction enzymes (Dra I, Eco RV, Pvu II, and Stu I) that leave blunt ends. Each batch of digested genomic DNA was then ligated separately to the GENOMEWALKER® Adaptor (Clontech Laboratories, Inc., Mountain View, Calif., USA) to create four libraries. These libraries were then employed as templates in PCR reactions using four gene-specific primers for Myceliophthora thermophila Family 61 gh61a gene. The primers shown below were designed based on the partial Family gh61a gene sequences obtained in Example 4.
TABLE-US-00002 Upstream Region Primers: (SEQ ID NO: 12) MtCeI61A-R1: 5'-CCGTTCGGGCCGTCTTGGTAGATCTTGAACC-3' (SEQ ID NO: 13) MtCeI61A-R2: 5'-CCGATGGAGGGATCCACGCTGAAGGTGAATT-3' Downstream Region Primers: (SEQ ID NO: 14) MtCeI61A-F1: 5'-CAGGTCAAGGCGGGCTCCCAATTCACCTT-3' (SEQ ID NO: 15) MtCeI61A-F2: 5'-ACGGCACGGGAGCCGTGTGGTTCAAGATCTA-3'
[0363] Two primary PCR amplifications were performed, one to isolate the upstream region and the other the downstream region of the Myceliophthora thermophila gh61a gene. Each PCR amplification (25 μl) was composed of 1 μl (approximately 6 ng) of each library as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 μmol of Adaptor Primer 1 (Clontech Laboratories, Inc., Mountain View, Calif., USA), 10 μmol of primer MtCel61A-R1 or primer MtCel61A-F1, 1× ADVANTAGE® GC-Melt LA Buffer, and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix. The amplifications were performed using an EPPENDORF® MASTERCYCLER® 5333 programmed for pre-denaturing at 94° C. for 1 minute; 7 cycles each at a denaturing temperature of 94° C. for 30 seconds; annealing and elongation at 72° C. for 5 minutes; and 32 cycles each at a denaturing temperature of 94° C. for 30 seconds; annealing and elongation 67° C. for 5 minutes, followed by a final extension of 7 minutes at 67° C.
[0364] The secondary amplifications were composed of 1 μl of each primary PCR product as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 pmol of Adaptor Primer 2 (Clontech Laboratories, Inc., Mountain View, Calif., USA), 10 pmol of nested primer MtCel61A-R2 or MtCel61A-F2, 1× ADVANTAGE® GC-Melt LA Buffer, and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix in a final volume of 25 μl. The amplifications were performed using an EPPENDORF® MASTERCYCLER® 5333 programmed for pre-denaturing at 94° C. for 1 minute; 5 cycles each at a denaturing temperature of 94° C. for 30 seconds; annealing and elongation at 72° C. for 5 minutes; and 20 cycles each at a denaturing temperature of 94° C. for 30 seconds; annealing and elongation at 67° C. for 5 minutes, followed by a final extension of 7 minutes at 67° C.
[0365] The reaction products were isolated by 1.0% agarose gel electrophoresis in TAE buffer where a 1.7 kb product band (upstream region) from the Eco RV library and a 1.6 kb band (downstream region) from the Stu I library were excised from the gel, purified using a MINELUTE® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer's instructions. The PCR products were sequenced directly or subcloned using a TOPO® TA Kit and then sequenced.
Example 6
Characterization of the Myceliophthora thermophila CBS 202.75 Genomic Sequence Encoding a Family GH61A Polypeptide Having Cellulolytic Enhancing Activity
[0366] DNA sequencing of the PCR fragment was performed with a Perkin-Elmer Applied Biosystems Model 377 XL Automated DNA Sequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif., USA) using dye-terminator chemistry (Giesecke et al., 1992, Journal of Virology Methods 38: 47-60) and primer walking strategy. Nucleotide sequence data were scrutinized for quality and all sequences were compared to each other with assistance of PHRED/PHRAP software (University of Washington, Seattle, Wash., USA).
[0367] A gene model for the Myceliophthora thermophila GH61A polypeptide having cellulolytic enhancing activity was constructed based on similarity of the encoded protein to homologous glycoside hydrolase Family 61 proteins from Thielavia terrestris (accession numbers GENESEQP:ADM97933, GENESEQP:AEB90517), Chaetomium globosum (UNIPROT:Q2HGH1, UNIPROT:Q2GW98) and Neurospora crassa (UNIPROT:Q7S439). To verify the sequence information obtained for the Myceliophthora thermophila gh61a gene, a further PCR reaction was carried out using a pair of gene specific primers (shown below), which encompass the complete gene.
TABLE-US-00003 Primer MtGH61A-F3: (SEQ ID NO: 16) 5'-ACTGGATTTACCATGAAGTTCACCTCGTCCCTCGCT-3' Primer MtGH61A-R3: (SEQ ID NO: 17) 5'-TCACCTCTAGTTAATTAATTAGCAAGAGACGGGGGCCG-3'
Bold letters represent coding sequence. The remaining sequence is homologous to the insertion sites of pAlLo2 (WO 2004/099228).
[0368] The PCR consisted of fifty picomoles of forward and reverse primers in a PCR reaction composed of 100 ng of Myceliophthora thermophila CBS 202.75 genomic DNA, Pfx Amplification Buffer (Invitrogen, Carlsbad, Calif., USA), 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 1 mM MgCl2, and 2.5 units of Pfx DNA polymerase (Invitrogen, Carlsbad, Calif., USA) in a final volume of 50 μl. The amplification was performed using an EPPENDORF® MASTERCYCLER® 5333 programmed for 1 cycle at 98° C. for 3 minutes; and 30 cycles each at 98° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute, followed by a final extension of 15 minutes at 72° C. The heat block then went to a 4° C. soak cycle.
[0369] The reaction products were isolated by 1.0% agarose gel electrophoresis in TAE buffer and purified using a MINELUTE® Gel Extraction Kit according to the manufacturer's instructions. In order to clone the PCR fragments into pCR®2.1-TOPO® vector (Invitrogen, Carlsbad, Calif., USA), addition of 3' A-overhangs was performed using Taq DNA polymerase (New England Biolabs, Ipswich, Mass., USA).
[0370] A 958 bp Myceliophthora thermophila gh61a gene fragment was cloned into pCR®2.1-TOPO® vector (Invitrogen, Carlsbad, Calif., USA) using a TOPO® TA Cloning Kit to generate pSMai190 (FIG. 2).
[0371] The Myceliophthora thermophila gh61a insert was confirmed by DNA sequencing. E. coli pSMai190 was deposited with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, Peoria, Ill., USA, on Dec. 5, 2007, and assigned accession number B-50083.
[0372] The nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO: 2) of the Myceliophthora thermophila GH61A polypeptide having cellulolytic enhancing activity are shown in FIG. 1. The genomic polynucleotide encodes a polypeptide of 232 amino acids, interrupted by 2 introns of 88 and 137 bp. The % G+C content of the full-length coding sequence and the mature coding sequence are 61.1% and 66.5%, respectively. Using the SignalP software program (Nielsen et al., 1997, Protein Engineering 10:1-6), a signal peptide of 17 residues was predicted. The predicted mature protein contains 215 amino acids with a molecular mass of 22.6 kDa.
[0373] A comparative pairwise global alignment of amino acid sequences was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of EMBOSS with gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that the deduced amino acid sequence of the Myceliophthora thermophila GH61A mature polypeptide shared 76.6% identity (excluding gaps) to the deduced amino acid sequence of a Family 61 glycoside hydrolase protein from Thielavia terrestris (GeneSeqP accession numbers ADM97933).
Example 7
Isolation of a Full-Length Family 61 Gene (gh61f) from Myceliophthora thermophila CBS 202.75
[0374] A full-length Family 61 gene (gh61f) from Myceliophthora thermophila CBS 202.75 was isolated using a GENOMEWALKER® Universal Kit (Clontech Laboratories, Inc., Mountain View, Calif., USA) according to the manufacturer's instructions. Briefly, total genomic DNA from Myceliophthora thermophila CBS 202.75 was digested separately with four different restriction enzymes (Dra I, Eco RV, Pvu II, and Stu I) that leave blunt ends. Each batch of digested genomic DNA was then ligated separately to the GENOMEWALKER® Adaptor (Clontech Laboratories, Inc., Mountain View, Calif., USA) to create four libraries. These libraries were then employed as templates in PCR reactions using gene-specific primers for the Myceliophthora thermophila Family 61 gene (gh61f). The primers shown below were designed based on the partial Family 61 gh61f gene sequences obtained in Example 4.
TABLE-US-00004 Upstream Region Primers: (SEQ ID NO: 18) MtGH61 F-R1: 5'-CCCTTGTGGCTGGCGTCCATGACATCGTC-3' (SEQ ID NO: 19) MtGH61F-R2: 5'-GTGCCTCCAGATGGCCTTGACCGTGGTG-3' Downstream Region Primers: (SEQ ID NO: 20) MtGH61F-F6: 5'-GGCGGCGAGCACTACATGTGAGCCATTCCT-3' (SEQ ID NO: 21) MtGH61F-F7: 5'-TGACGATCTCGCTGACCCGTGCAACAAGTG-3'
[0375] Two primary PCR amplifications were performed, one to isolate the upstream region and the other to isolate the downstream region of the Myceliophthora thermophila gh61f gene. Each PCR amplification (25 μl) was composed of 1 μl (approximately 6 ng) of each library as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 pmol of Adaptor Primer 1 (Clontech Laboratories, Inc., Mountain View, Calif., USA), 10 pmol of primer MtGH61F-R1 or primer MtGH61F-F6, 1× ADVANTAGE®GC-Melt LA Buffer, and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix. The amplifications were performed using an EPPENDORF® MASTERCYCLER® 5333 programmed for pre-denaturing at 94° C. for 1 minute; 7 cycles each at a denaturing temperature of 94° C. for 30 seconds; annealing and elongation at 72° C. for 5 minutes; and 32 cycles each at a denaturing temperature of 94° C. for 30 seconds; annealing and elongation 67° C. for 5 minutes, followed by a final extension of 7 minutes at 67° C.
[0376] The secondary amplifications were composed of 1 μl of each primary PCR product as template, 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 10 pmol of Adaptor Primer 2 (Clontech Laboratories, Inc., Mountain View, Calif., USA), 10 pmol of nested primer MtGH61F-R2 or MtGH61F-F7, 1× ADVANTAGE® GC-Melt LA Buffer, and 1.25 units of ADVANTAGE® GC Genomic Polymerase Mix in a final volume of 25 μl. The amplifications were performed using an EPPENDORF® MASTERCYCLER® 5333 programmed for pre-denaturing at 94° C. for 1 minute; 5 cycles each at a denaturing temperature of 94° C. for 30 seconds; annealing and elongation at 72° C. for 5 minutes; and 20 cycles each at a denaturing temperature of 94° C. for 30 seconds; annealing and elongation at 67° C. for 5 minutes, followed by a final extension of 7 minutes at 67° C.
[0377] The reaction products were isolated by 1.0% agarose gel electrophoresis in TAE buffer where a 1.3 kb PCR product (upstream region) from the Puv II library and a 1.2 kb PCR product (upstream region) from the Puv II library were excised from the gel, purified using a MINELUTE® Gel Extraction Kit (QIAGEN Inc., Valencia, Calif., USA) according to the manufacturer's instructions, and the PCR products were sequenced directly or subcloned using a TOPO® TA Kit and then sequenced.
Example 8
Characterization of the Myceliophthora thermophila Genomic Sequence Encoding a Family GH61F Polypeptide Having Cellulolytic Enhancing Activity
[0378] DNA sequencing of the PCR fragments was performed with a Perkin-Elmer Applied Biosystems Model 377 XL Automated DNA Sequencer (Perkin-Elmer/Applied Biosystems, Inc., Foster City, Calif., USA) using dye-terminator chemistry (Giesecke et al., 1992, Journal of Virology Methods 38: 47-60) and primer walking strategy. Nucleotide sequence data were scrutinized for quality and all sequences were compared to each other with assistance of PHRED/PHRAP software (University of Washington, Seattle, Wash., USA).
[0379] A gene model for the Myceliophthora thermophila GH61F polypeptide having cellulolytic enhancing activity was constructed based on similarity of the encoded protein to homologous glycoside hydrolase Family 61 proteins from Thielavia terrestris (accession numbers GENESEQP:ADM97933, GENESEQP:AEB90517), Chaetomium globosum (UNIPROT:Q2HGH1, UNIPROT:Q2GW98) and Neurospora crassa (UNIPROT:Q7S439). To verify the sequence information obtained for the Myceliophthora thermophila gh61f gene, a further PCR reaction was carried out using gene specific primers (shown below), which encompass the complete gene.
TABLE-US-00005 Primer MtGH61F-F8: (SEQ ID NO: 22) 5'-ACTGGATTTACCATGAAGGCCCTCTCTCTCCTTGCG-3' Primer MtGH61F-R3: (SEQ ID NO: 23) 5'-TCACCTCTAGTTAATTAACTAGCACTTGAAGACGGGCG-3'
Bold letters represent coding sequence. The remaining sequence is homologous to the insertion sites of pAlLo2 (WO 2004/099228).
[0380] The PCR consisted of 50 picomoles of forward and reverse primers in a PCR reaction composed of 100 ng of Myceliophthora thermophila CBS 202.75 genomic DNA, Pfx Amplification Buffer (Invitrogen, Carlsbad, Calif., USA), 0.4 mM each of dATP, dTTP, dGTP, and dCTP, 1 mM MgCl2, and 2.5 units of Pfx DNA polymerase (Invitrogen, Carlsbad, Calif., USA) in a final volume of 50 μl. The amplification were performed using an EPPENDORF® MASTERCYCLER® 5333 programmed for 1 cycle at 98° C. for 3 minutes; and 30 cycles each at 98° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 1 minute, followed by a final extension of 15 minutes at 72° C. The heat block then went to a 4° C. soak cycle.
[0381] The reaction products were isolated by 1.0% agarose gel electrophoresis in TAE buffer and purified using a MINELUTE® Gel Extraction Kit according to the manufacturer's instructions. In order to clone the PCR fragments into pCR®2.1-TOPO® vector (Invitrogen, Carlsbad, Calif., USA), addition of 3' A-overhangs was performed using Taq DNA polymerase (New England Biolabs, Ipswich, Mass., USA).
[0382] An 884 bp Myceliophthora thermophila gh61f gene fragment was cloned into pCR®2.1-TOPO® vector using a TOPO® TA Cloning Kit to generate pSMai192 (FIG. 2).
[0383] The Myceliophthora thermophila gh61f insert was confirmed by DNA sequencing. E. coli pSMai192 was deposited with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center, 1815 University Street, Peoria, Ill., USA, on Dec. 5, 2007, and assigned accession number B-50085.
[0384] The nucleotide sequence (SEQ ID NO: 3) and deduced amino acid sequence (SEQ ID NO: 4) of the Myceliophthora thermophila GH61F polypeptide having cellulolytic enhancing activity are shown in FIG. 1. The genomic polynucleotide encodes a polypeptide of 235 amino acids, interrupted by 2 introns of 62 and 84 bp. The % G+C content of the full-length coding sequence and the mature coding sequence are 64.1% and 65.4%, respectively. 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 220 amino acids with a molecular mass of 23 kDa.
[0385] A comparative pairwise global alignment of amino acid sequences was determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of EMBOSS with gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that the deduced amino acid sequence of the Myceliophthora thermophila GH61F mature polypeptide shared 83.8% identity (excluding gaps) to the deduced amino acid sequence of a Family 61 glycoside hydrolase protein from Chaetomium globosum (UniProt accession number Q2HGH1).
Example 9
Construction of an Aspergillus Oryzae Expression Vector Containing Myceliophthora thermophila CBS 202.75 Genomic Sequence Encoding a Family GH61A Polypeptide Having Cellulolytic Enhancing Activity
[0386] The same 958 bp Myceliophthora thermophila gh61a PCR fragment generated in Example 6 was cloned into Nco I and Pac I digested pAlLo2 (WO 2004/099228) using an Infusion Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) resulting in pSMai185 (FIG. 5) in which transcription of the Myceliophthora thermophila gh61a gene was under the control of a hybrid of promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase (NA2-tpi promoter). The ligation reaction (50 μl) was composed of 1× InFusion Buffer (BD Biosciences, Palo Alto, Calif., USA), 1×BSA (BD Biosciences, Palo Alto, Calif., USA), 1 μl of Infusion enzyme (diluted 1:10) (BD Biosciences, Palo Alto, Calif., USA), 100 ng of pAlLo2 digested with Nco I and Pac I, and 50 ng of the Myceliophthora thermophila gh61a purified PCR product. The reaction was incubated at room temperature for 30 minutes. One μl of the reaction was used to transform E. coli XL10 SOLOPACK® Gold Supercompetent cells (Stratagene, La Jolla, Calif., USA). An E. coli transformant containing pSMai185 was detected by restriction digestion and plasmid DNA was prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA). The Myceliophthora thermophila gh61a insert in pSMai185 was confirmed by DNA sequencing.
Example 10
Construction of an Aspergillus Oryzae Expression Vector Containing Myceliophthora thermophila CBS 202.75 Genomic Sequence Encoding a Family GH61F Polypeptide Having Cellulolytic Enhancing Activity
[0387] The same 884 bp Myceliophthora thermophila gh61f PCR fragment generated in Example 8 was cloned into Nco I and Pac I digested pAlLo2 (WO 2004/099228) using an Infusion Cloning Kit (BD Biosciences, Palo Alto, Calif., USA) resulting in pSMai198 (FIG. 6) in which transcription of the Myceliophthora thermophila gh61f gene was under the control of a hybrid of promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase (NA2-tpi promoter). The ligation reaction (50 μl) was composed of 1× InFusion Buffer (BD Biosciences, Palo Alto, Calif., USA), 1×BSA (BD Biosciences, Palo Alto, Calif., USA), 1 μl of Infusion enzyme (diluted 1:10) (BD Biosciences, Palo Alto, Calif., USA), 100 ng of pAlLo2 digested with Nco I and Pac I, and 50 ng of the Myceliophthora thermophila gh61f purified PCR product. The reaction was incubated at room temperature for 30 minutes. One μl of the reaction was used to transform E. coli XL10 SOLOPACK® Gold Supercompetent cells (Stratagene, La Jolla, Calif., USA). An E. coli transformant containing pSMai198 was detected by restriction digestion and plasmid DNA was prepared using a BIOROBOT® 9600 (QIAGEN Inc., Valencia, Calif., USA). The Myceliophthora thermophila gh61f insert in pSMai198 was confirmed by DNA sequencing.
Example 11
Expression of the Myceliophthora Thermophila Family 61 Glycosyl Hydrolase Genes (GH61A and GH61f) Individually in Aspergillus Oryzae JaL355
[0388] Aspergillus oryzae JaL355 (WO 2002/40694) protoplasts were prepared according to the method of Christensen et al., 1988, Bio/Technology 6: 1419-1422. Three μg of pSMai185 (gh61a) or pSMai198 (gh61f) were transformed individually into Aspergillus oryzae JaL355.
[0389] Twenty transformants were isolated to individual Minimal medium plates from each transformation experiment.
[0390] Confluent Minimal Medium plates of each of the transformants were washed with 5 ml of 0.01% TWEEN® 20 and inoculated separately into 25 ml of M410 medium in 125 ml glass shake flasks and incubated at 34° C., 250 rpm. After 5 days incubation, 5 μl of supernatant from each culture were analyzed on CRITERION® 8-16% Tris-HCl gels with a CRITERION® Cell (Bio-Rad Laboratories, Inc., Hercules, Calif., USA), according to the manufacturer's instructions. The resulting gels were stained with BIO-SAFE® Coomassie Stain. SDS-PAGE profiles of the cultures showed that the majority of the transformants had the expected band sizes: 23 KDa for GH61A and 23 KDa for GH61F.
[0391] One of each high protein expressing GH61A and GH61F transformants were washed with 10 ml of 0.01% TWEEN® 20 and inoculated into a 2 liter Fernbach containing 500 ml of M410 medium to generate broth for characterization of the proteins. The cultures were harvested on day 5 and filtered using a 0.22 μm EXPRESS® Plus Membrane (Millipore, Bedford, Mass., USA).
Example 12
Effect of Myceliophthora Thermophila GH61A and GH61f Polypeptides on Enzymatic Hydrolysis of Pretreated Corn Stover
[0392] Culture broth was prepared as described in Example 11 and concentrated approximately 20-fold using an Amicon ultrafiltration device (Millipore, Bedfored, Mass., 10 kDa polyethersulfone membrane, 40 psi, 4° C.). Protein concentration was estimated by densitometry following SDS-PAGE and Coomassie blue staining. Corn stover was pretreated and prepared as an assay substrate as described in WO 2005/074647 to generate pretreated corn stover (PCS). The base cellulase mixture used to assay enhancing activity was prepared from Trichoderma reesei strain SMA135 (WO 2008/057637).
[0393] Hydrolysis of PCS was conducted using 1.6 ml deep-well plates (Axygen, Santa Clara, Calif.) using a total reaction volume of 1.0 ml and a PCS concentration of 50 mg/ml in 1 mM manganese sulfate-50 mM sodium acetate, pH 5.0. The M. thermophila polypeptides (GH61A and GH61F) were added to the base cellulase mixture at concentrations ranging from 0 to 25% of the protein concentration of the base cellulase mixture. Incubation was at 50° C. for 168 hours. Assays were performed in triplicate. Aliquots were centrifuged, and the supernatant liquid was filtered by centrifugation (MULTISCREEN® HV 0.45 μm, Millipore, Billerica, Mass., USA) at 3000 rpm for 10 minutes using a plate centrifuge (SORVALL® RT7, Thermo Fisher Scientific, Waltham, Mass., USA). When not used immediately, filtered hydrolysate aliquots were frozen at -20° C. Sugar concentrations of samples diluted in 0.005 M H2SO4 with 0.05% w/w benzoic acid were measured after elution by 0.005 M H2SO4 with 0.05% w/w benzoic acid at a flow rate of 0.6 ml/minute from a 4.6×250 mm AMINEX® HPX-87H column (Bio-Rad Laboratories, Inc., Inc., Hercules, Calif., USA) at 65° C. with quantitation by integration of glucose and cellobiose signal from refractive index detection (CHEMSTATION®, AGILENT® 1100 HPLC, Agilent Technologies, Santa Clara, Calif., USA) calibrated by pure sugar samples (Absolute Standards Inc., Hamden, Conn., USA). The resultant equivalents were used to calculate the percentage of cellulose conversion for each reaction. The degree of cellulose conversion to glucose plus cellobiose sugars (conversion, %) was calculated using the following equation:
Conversion(%)=(glucose+cellobiose×1.053)(mg/ml)×100×16- 2/(Cellulose(mg/ml)×180)=(glucose+cellobiose×1.053)(mg/ml).tim- es.100/(Cellulose(mg/ml)×1.111)
In this equation the factor 1.111 reflects the weight gain in converting cellulose to glucose, and the factor 1.053 reflects the weight gain in converting cellobiose to glucose. Cellulose in PCS was determined by a limit digest of PCS to release glucose and cellobiose.
[0394] The results of adding increasing amounts of Myceliopthora thermophila polypeptides to the base cellulase mix are shown in FIG. 7. Addition of the M. thermophila GH61A polypeptide provided a stimulation factor of 1.26 at the 25% addition level. At the same addition percentage, M. thermophila GH61F provided a stimulation factor of 1.13. Stimulation factor is defined as the ratio of conversion observed in the presence of added GH61 protein versus conversion in the absence of added GH61 protein.
Deposits of Biological Material
[0395] The following biological materials have been deposited under the terms of the Budapest Treaty with the Agricultural Research Service Patent Culture Collection (NRRL), Northern Regional Research Center, 1815 University Street, Peoria, Ill., 61604, USA, and given the following accession numbers:
TABLE-US-00006 Deposit Accession Number Date of Deposit E. coli pSMai190 NRRL B-50083 Dec. 5, 2007 E. coli pSMai192 NRRL B-50085 Dec. 5, 2007
[0396] The strains have been deposited under conditions that assure that access to the cultures will be available during the pendency of this patent application to one determined by foreign patent laws to be entitled thereto. The deposits represent substantially pure cultures of the deposited strains. The depositse 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.
[0397] The present invention is further described by the following numbered paragraphs:
[0398] [1] An isolated polypeptide having cellulolytic enhancing activity, selected from the group consisting of:
[0399] (a) a polypeptide comprising an amino acid sequence having at least 60% identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4;
[0400] (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 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary strand of (i) or (ii);
[0401] (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 or SEQ ID NO: 3; and
[0402] (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 or SEQ ID NO: 4.
[0403] [2] The polypeptide of paragraph 1, comprising an amino acid sequence having at least 60% identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0404] [3] The polypeptide of paragraph 2, comprising an amino acid sequence having at least 65% identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0405] [4] The polypeptide of paragraph 3, comprising an amino acid sequence having at least 70% identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0406] [5] The polypeptide of paragraph 4, comprising an amino acid sequence having at least 75% identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0407] [6] The polypeptide of paragraph 5, comprising an amino acid sequence having at least 80% identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0408] [7] The polypeptide of paragraph 6, comprising an amino acid sequence having at least 85% identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0409] [8] The polypeptide of paragraph 7, comprising an amino acid sequence having at least 90% identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0410] [9] The polypeptide of paragraph 8, comprising an amino acid sequence having at least 95% identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0411] [10] The polypeptide of paragraph 1, comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; or a fragment thereof having cellulolytic enhancing activity.
[0412] [11] The polypeptide of paragraph 10, comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.
[0413] [12] The polypeptide of paragraph 10, comprising or consisting of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.
[0414] [13] The polypeptide of paragraph 1, which is encoded by a polynucleotide that hybridizes under at least medium stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary strand of (i) or (ii).
[0415] [14] The polypeptide of paragraph 13, which is encoded by a polynucleotide that hybridizes under at least medium stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary strand of (i) or (ii).
[0416] [15] The polypeptide of paragraph 14, which is encoded by a polynucleotide that hybridizes under at least medium stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, or (iii) a full-length complementary strand of (i) or (ii).
[0417] [16] The polypeptide of paragraph 1, which is encoded by a polynucleotide comprising a nucleotide sequence having at least 60% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0418] [17] The polypeptide of paragraph 16, which is encoded by a polynucleotide comprising a nucleotide sequence having at least 65% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0419] [18] The polypeptide of paragraph 17, which is encoded by a polynucleotide comprising a nucleotide sequence having at least 70% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0420] [19] The polypeptide of paragraph 18, which is encoded by a polynucleotide comprising a nucleotide sequence having at least 75% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0421] [20] The polypeptide of paragraph 19, which is encoded by a polynucleotide comprising a nucleotide sequence having at least 80% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0422] [21] The polypeptide of paragraph 20, which is encoded by a polynucleotide comprising a nucleotide sequence having at least 85% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0423] [22] The polypeptide of paragraph 21, which is encoded by a polynucleotide comprising a nucleotide sequence having at least 90% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0424] [23] The polypeptide of paragraph 22, which is encoded by a polynucleotide comprising a nucleotide sequence having at least 95% identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0425] [24] The polypeptide of paragraph 1, which is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3; or a subsequence thereof encoding a fragment having cellulolytic enhancing activity.
[0426] [25] The polypeptide of paragraph 24, which is encoded by a polynucleotide comprising or consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0427] [26] The polypeptide of paragraph 24, which is encoded by a polynucleotide comprising or consisting of the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3.
[0428] [27] The polypeptide of paragraph 1, wherein the polypeptide is 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 or SEQ ID NO: 4.
[0429] [28] The polypeptide of paragraph 1, which is encoded by the polynucleotide contained in plasmid pSMai190 which is contained in E. coli NRRL B-50083 or plasmid pSMai192 which is contained in E. coli NRRL B-50085.
[0430] [29] The polypeptide of any of paragraphs 1-28, wherein the mature polypeptide is amino acids 18 to 232 of SEQ ID NO: 2 or SEQ ID NO: 4.
[0431] [30] The polypeptide of any of paragraphs 1-29, wherein the mature polypeptide coding sequence is nucleotides 52 to 921 of SEQ ID NO: 1 or nucleotides 46 to 851 of SEQ ID NO: 3.
[0432] [31] An isolated polynucleotide comprising a nucleotide sequence that encodes the polypeptide of any of paragraphs 1-30.
[0433] [32] The isolated polynucleotide of paragraph 31, comprising at least one mutation in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, in which the mutant nucleotide sequence encodes the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, respectively.
[0434] [33] A nucleic acid construct comprising the polynucleotide of paragraph 31 or 32 operably linked to one or more (several) control sequences that direct the production of the polypeptide in an expression host.
[0435] [34] A recombinant expression vector comprising the nucleic acid construct of paragraph 33.
[0436] [35] A recombinant host cell comprising the nucleic acid construct of paragraph 33.
[0437] [36] A method of producing the polypeptide of any of paragraphs 1-30, 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.
[0438] [37] A method of producing the polypeptide of any of paragraphs 1-30, comprising: (a) cultivating a host cell comprising a nucleic acid construct comprising a nucleotide sequence encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
[0439] [38] A method of producing a mutant of a parent cell, comprising disrupting or deleting a nucleotide sequence encoding the polypeptide of any of paragraphs 1-30, which results in the mutant producing less of the polypeptide than the parent cell.
[0440] [39] A mutant cell produced by the method of paragraph 38.
[0441] [40] The mutant cell of paragraph 39, further comprising a gene encoding a native or heterologous protein.
[0442] [41] A method of producing a protein, comprising: (a) cultivating the mutant cell of paragraph 40 under conditions conducive for production of the protein; and (b) recovering the protein.
[0443] [42] The isolated polynucleotide of paragraph 31 or 32, obtained by (a) hybridizing a population of DNA under at least high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, (ii) the cDNA sequence contained in the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, 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.
[0444] [43] The isolated polynucleotide of paragraph 42, wherein the mature polypeptide coding sequence is nucleotides 52 to 921 of SEQ ID NO: 1 or nucleotides 46 to 851 of SEQ ID NO: 3.
[0445] [44] A method of producing a polynucleotide comprising a mutant nucleotide sequence encoding a polypeptide having cellulolytic enhancing activity, comprising: (a) introducing at least one mutation into the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 3, wherein the mutant nucleotide sequence encodes a polypeptide comprising or consisting of the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4; and (b) recovering the polynucleotide comprising the mutant nucleotide sequence.
[0446] [45] A mutant polynucleotide produced by the method of paragraph 44.
[0447] [46] A method of producing a polypeptide, comprising: (a) cultivating a cell comprising the mutant polynucleotide of paragraph 45 encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
[0448] [47] A method of producing the polypeptide of any of paragraphs 1-30, comprising: (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.
[0449] [48] A transgenic plant, plant part or plant cell transformed with a polynucleotide encoding the polypeptide of any of paragraphs 1-30.
[0450] [49] A double-stranded inhibitory RNA (dsRNA) molecule comprising a subsequence of the polynucleotide of paragraph 31 or 32, wherein optionally the dsRNA is a siRNA or a miRNA molecule.
[0451] [50] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph 49, which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.
[0452] [51] A method of inhibiting the expression of a polypeptide having cellulolytic enhancing activity 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 the polynucleotide of paragraph 31 or 32.
[0453] [52] The method of paragraph 51, wherein the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more duplex nucleotides in length.
[0454] [53] 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 17 of SEQ ID NO: 2 or amino acids 1 to 15 of SEQ ID NO: 4, wherein the gene is foreign to the nucleotide sequence.
[0455] [54] A recombinant expression vector comprising the nucleic acid construct of paragraph 53.
[0456] [55] A recombinant host cell comprising the nucleic acid construct of paragraph 53.
[0457] [56] A method of producing a protein, comprising: (a) cultivating the recombinant host cell of paragraph 55 under conditions conducive for production of the protein; and (b) recovering the protein.
[0458] [57] A method for degrading or converting a cellulosic material, comprising: treating the cellulosic material with a cellulolytic enzyme composition in the presence of the polypeptide having cellulolytic enhancing activity of any of paragraphs 1-30, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity.
[0459] [58] The method of paragraph 57, wherein the cellulosic material is pretreated.
[0460] [59] The method of paragraph 57 or 58, wherein the cellulolytic enzyme composition comprises one or more cellulolytic enzymes are selected from the group consisting of a cellulase, endoglucanase, cellobiohydrolase, and beta-glucosidase.
[0461] [60] The method of any of paragraphs 57-59, further comprising treating the cellulosic material with one or more enzymes selected from the group consisting of a hemicellulase, esterase, protease, laccase, and peroxidase.
[0462] [61] The method of any of paragraphs 57-60, further comprising recovering the degraded cellulosic material.
[0463] [62] The method of paragraph 61, wherein the degraded cellulosic material is a sugar.
[0464] [63] The method of paragraph 62, wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.
[0465] [64] A method for producing a fermentation product, comprising:
[0466] (a) saccharifying a cellulosic material with a cellulolytic enzyme composition in the presence of the polypeptide having cellulolytic enhancing activity of any of paragraphs 1-20, wherein the presence of the polypeptide having cellulolytic enhancing activity increases the degradation of cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity;
[0467] (b) fermenting the saccharified cellulosic material of step (a) with one or more fermenting microorganisms to produce the fermentation product; and
[0468] (c) recovering the fermentation product from the fermentation.
[0469] [65] The method of paragraph 64, wherein the cellulosic material is pretreated.
[0470] [66] The method of paragraph 64 or 65, wherein the cellulolytic enzyme composition comprises one or more cellulolytic enzymes selected from the group consisting of a cellulase, endoglucanase, cellobiohydrolase, and beta-glucosidase.
[0471] [67] The method of any of paragraphs 64-66, further comprising treating the cellulosic material with one or more enzymes selected from the group consisting of a hemicellulase, esterase, protease, laccase, and peroxidase.
[0472] [68] The method of any of paragraphs 64-67, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.
[0473] [69] The method of any of paragraphs 64-68, wherein the fermentation product is an alcohol, organic acid, ketone, amino acid, or gas.
[0474] [70] A method of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more fermenting microorganisms, wherein the cellulosic material is hydrolyzed with a cellulolytic enzyme composition in the presence of a polypeptide having cellulolytic enhancing activity of any of paragraphs 1-30 and the presence of the polypeptide having cellulolytic enhancing activity increases the hydrolysis of the cellulosic material compared to the absence of the polypeptide having cellulolytic enhancing activity.
[0475] [71] The method of paragraph 70, wherein the fermenting of the cellulosic material produces a fermentation product.
[0476] [72] The method of paragraph 71, further comprising recovering the fermentation product from the fermentation.
[0477] [73] The method of any of paragraphs 70-72, wherein the cellulosic material is pretreated before saccharification.
[0478] [74] The method of any of paragraphs 70-73, wherein the cellulolytic enzyme composition comprises one or more cellulolytic enzymes selected from the group consisting of a cellulase, endoglucanase, cellobiohydrolase, and beta-glucosidase.
[0479] [75] The method of any of paragraphs 70-74, wherein the cellulolytic enzyme composition further comprises one or more enzymes selected from the group consisting of a hemicellulase, esterase, protease, laccase, and peroxidase.
[0480] [76] The method of any of paragraphs 70-75, wherein the fermentation product is an alcohol, organic acid, ketone, amino acid, or gas.
[0481] 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
1
751924DNAMyceliophthora thermophila 1atgaagttca cctcgtccct cgctgtcctg
gccgctgccg gcgcccaggc tcactgttag 60tcgaccctcg aacccaacac ccccctcccc
ccttttctcc tccatctcct cggcctcact 120tagtagccgc tgacaacgac tagatacctt
ccctagggcc ggcactggtg gctcgctctc 180tggcgagtgg gaggtggtcc gcatgaccga
gaaccattac tcgcacggcc cggtcaccga 240tgtcaccagc cccgagatga cctgctatca
gtccggcgtg cagggtgcgc cccagaccgt 300ccaggtcaag gcgggctccc aattcacctt
cagcgtggat ccctcgatcg gccaccccgg 360ccctctccag ttctacatgg ctaaggtgcc
gtcgggccag acggccgcca cctttgacgg 420cacgggagcc gtgtggttca agatctacca
agacggcccg aacggcctcg gcaccgacag 480cattacctgg cccagcgccg gttcgtgact
tcctccccac tcgctttttt ttttttattt 540tttatttttt tttctttcgg aactcaagaa
tctttctctc tctctcccgt ctttggcctt 600gaacaacact aaaactcttc cttactgtat
taattaggca aaaccgaggt ctcggtcacc 660atccccagct gcatcgatga tggcgagtac
ctgctccggg tcgagcacat cgcgctccac 720agcgccagca gcgtgggcgg cgctcagttc
tacattgcct gcgcccagct ctccgtcacc 780ggcggctccg gcaccctcaa cacgggctcg
ctcgtctccc tgcccggcgc ctacaaggcc 840accgacccgg gcatcctctt ccagctctac
tggcccatcc cgaccgagta catcaacccc 900ggcccggccc ccgtctcttg ctaa
9242232PRTMyceliophthora thermophila
2Met Lys Phe Thr Ser Ser Leu Ala Val Leu Ala Ala Ala Gly Ala Gln 1
5 10 15 Ala His Tyr Thr
Phe Pro Arg Ala Gly Thr Gly Gly Ser Leu Ser Gly 20
25 30 Glu Trp Glu Val Val Arg Met Thr Glu
Asn His Tyr Ser His Gly Pro 35 40
45 Val Thr Asp Val Thr Ser Pro Glu Met Thr Cys Tyr Gln Ser
Gly Val 50 55 60
Gln Gly Ala Pro Gln Thr Val Gln Val Lys Ala Gly Ser Gln Phe Thr 65
70 75 80 Phe Ser Val Asp Pro
Ser Ile Gly His Pro Gly Pro Leu Gln Phe Tyr 85
90 95 Met Ala Lys Val Pro Ser Gly Gln Thr Ala
Ala Thr Phe Asp Gly Thr 100 105
110 Gly Ala Val Trp Phe Lys Ile Tyr Gln Asp Gly Pro Asn Gly Leu
Gly 115 120 125 Thr
Asp Ser Ile Thr Trp Pro Ser Ala Gly Lys Thr Glu Val Ser Val 130
135 140 Thr Ile Pro Ser Cys Ile
Asp Asp Gly Glu Tyr Leu Leu Arg Val Glu 145 150
155 160 His Ile Ala Leu His Ser Ala Ser Ser Val Gly
Gly Ala Gln Phe Tyr 165 170
175 Ile Ala Cys Ala Gln Leu Ser Val Thr Gly Gly Ser Gly Thr Leu Asn
180 185 190 Thr Gly
Ser Leu Val Ser Leu Pro Gly Ala Tyr Lys Ala Thr Asp Pro 195
200 205 Gly Ile Leu Phe Gln Leu Tyr
Trp Pro Ile Pro Thr Glu Tyr Ile Asn 210 215
220 Pro Gly Pro Ala Pro Val Ser Cys 225
230 3854DNAMyceliophthora thermophila 3atgaaggccc tctctctcct
tgcggctgcc tcggcagtct ctgcgcatac catcttcgtc 60cagctcgaag cagacggcac
gaggtacccg gtctcgtacg ggatccggga cccaagctac 120gacggcccca tcaccgacgt
cacatccaac gacgttgctt gcaacggcgg gccgaacccg 180acgaccccct ccagcgacgt
catcaccgtc accgcgggca ccacggtcaa ggccatctgg 240aggcacaccc tccaatccgg
cccggacgat gtcatggacg ccagccacaa gggcccgacc 300ctggcctacc tcaagaaggt
cggcgatgcc accaaggact cgggcgtcgg cggtggctgg 360ttcaagattc aggaggacgg
ctacaacaac ggccagtggg gcaccagcac cgttatctcc 420aacggcggcg agcactacat
gtgagccatt cctccgagag aagaccaaga ctcttgacga 480tctcgctgac ccgtgcaaca
agtgacatcc cggcctgcat ccccgagggt cagtacctcc 540tccgcgccga gatgatcgcc
ctccacgcgg ccgggtcccc cggcggtgcc cagctctacg 600taagcctctg cccttccccc
cttcctcttg atcgaatcgg actgcccacc ccccttttcg 660actccgacta acaccgttgc
cagatggaat gtgcccagat caacatcgtc ggcggctccg 720gctcggtgcc cagctcgacc
gtcagcttcc ccggcgcgta cagccccaac gacccgggtc 780tcctcatcaa catctattcc
atgtcgccct cgagctcgta caccatcccg ggcccgcccg 840tcttcaagtg ctag
8544235PRTMyceliophthora
thermophila 4Met Lys Ala Leu Ser Leu Leu Ala Ala Ala Ser Ala Val Ser Ala
His 1 5 10 15 Thr
Ile Phe Val Gln Leu Glu Ala Asp Gly Thr Arg Tyr Pro Val Ser
20 25 30 Tyr Gly Ile Arg Asp
Pro Ser Tyr Asp Gly Pro Ile Thr Asp Val Thr 35
40 45 Ser Asn Asp Val Ala Cys Asn Gly Gly
Pro Asn Pro Thr Thr Pro Ser 50 55
60 Ser Asp Val Ile Thr Val Thr Ala Gly Thr Thr Val Lys
Ala Ile Trp 65 70 75
80 Arg His Thr Leu Gln Ser Gly Pro Asp Asp Val Met Asp Ala Ser His
85 90 95 Lys Gly Pro Thr
Leu Ala Tyr Leu Lys Lys Val Gly Asp Ala Thr Lys 100
105 110 Asp Ser Gly Val Gly Gly Gly Trp Phe
Lys Ile Gln Glu Asp Gly Tyr 115 120
125 Asn Asn Gly Gln Trp Gly Thr Ser Thr Val Ile Ser Asn Gly
Gly Glu 130 135 140
His Tyr Ile Asp Ile Pro Ala Cys Ile Pro Glu Gly Gln Tyr Leu Leu 145
150 155 160 Arg Ala Glu Met Ile
Ala Leu His Ala Ala Gly Ser Pro Gly Gly Ala 165
170 175 Gln Leu Tyr Met Glu Cys Ala Gln Ile Asn
Ile Val Gly Gly Ser Gly 180 185
190 Ser Val Pro Ser Ser Thr Val Ser Phe Pro Gly Ala Tyr Ser Pro
Asn 195 200 205 Asp
Pro Gly Leu Leu Ile Asn Ile Tyr Ser Met Ser Pro Ser Ser Ser 210
215 220 Tyr Thr Ile Pro Gly Pro
Pro Val Phe Lys Cys 225 230 235
517PRTMyceliophthora thermophila 5Leu Pro Ala Ser Asn Ser Pro Val Thr Asp
Val Thr Ser Asn Ala Leu 1 5 10
15 Arg 613PRTMyceliophthora thermophila 6Val Asp Asn Ala Ala
Thr Ala Ser Pro Ser Gly Leu Lys 1 5 10
713PRTMyceliophthora thermophila 7Leu Pro Ala Asp Leu Pro Ser
Gly Asp Tyr Leu Leu Arg 1 5 10
89PRTMyceliophthora thermophila 8Gly Pro Leu Gln Val Tyr Leu Ala Lys 1
5 912PRTMyceliophthora thermophila 9Val
Ser Val Asn Gly Gln Asp Gln Gly Gln Leu Lys 1 5
10 1029DNAMyceliophthora
thermophilamisc_feature(21)..(21)N=A,C,G, OR T 10gcctccaact cgcccgtcac
ngaygtnac 291130DNAMyceliophthora
thermophilamisc_feature(16)..(16)N=A,C,G, OR T 11gaggtagtcg ccgganggga
trtcngcngg 301231DNAMyceliophthora
thermophila 12ccgttcgggc cgtcttggta gatcttgaac c
311331DNAMyceliophthora thermophila 13ccgatggagg gatccacgct
gaaggtgaat t 311429DNAMyceliophthora
thermophila 14caggtcaagg cgggctccca attcacctt
291531DNAMyceliophthora thermophila 15acggcacggg agccgtgtgg
ttcaagatct a 311636DNAMyceliophthora
thermophila 16actggattta ccatgaagtt cacctcgtcc ctcgct
361738DNAMyceliophthora thermophila 17tcacctctag ttaattaatt
agcaagagac gggggccg 381829DNAMyceliophthora
thermophila 18cccttgtggc tggcgtccat gacatcgtc
291928DNAMyceliophthora thermophila 19gtgcctccag atggccttga
ccgtggtg 282030DNAMyceliophthora
thermophila 20ggcggcgagc actacatgtg agccattcct
302130DNAMyceliophthora thermophila 21tgacgatctc gctgacccgt
gcaacaagtg 302236DNAMyceliophthora
thermophila 22actggattta ccatgaaggc cctctctctc cttgcg
362338DNAMyceliophthora thermophila 23tcacctctag ttaattaact
agcacttgaa gacgggcg 3824923DNAHumicola
insolens 24atgcgttcct 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 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 gcctgtagaa ttc
92325305PRTHumicola insolens 25Met Arg Ser Ser Pro Leu Leu
Arg Ser Ala Val Val Ala Ala Leu Pro 1 5
10 15 Val Leu Ala Leu Ala Ala Asp Gly Arg Ser Thr
Arg Tyr Trp Asp Cys 20 25
30 Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn Gln
Pro 35 40 45 Val
Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50
55 60 Lys Ser Gly Cys Glu Pro
Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln 65 70
75 80 Thr Pro Trp Ala Val Asn Asp Asp Phe Ala Leu
Gly Phe Ala Ala Thr 85 90
95 Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr Glu
100 105 110 Leu Thr
Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln 115
120 125 Ser Thr Ser Thr Gly Gly Asp
Leu Gly Ser Asn His Phe Asp Leu Asn 130 135
140 Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly Cys
Thr Pro Gln Phe 145 150 155
160 Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175 Cys Asp Arg
Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180
185 190 Asp Trp Phe Lys Asn Ala Asp Asn
Pro Ser Phe Ser Phe Arg Gln Val 195 200
205 Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys Arg
Arg Asn Asp 210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Ser Ser Ser Thr Ser Ser 225
230 235 240 Pro Val Asn Gln
Pro Thr Ser Thr Ser Thr Thr Ser Thr Ser Thr Thr 245
250 255 Ser Ser Pro Pro Val Gln Pro Thr Thr
Pro Ser Gly Cys Thr Ala Glu 260 265
270 Arg Trp Ala Gln Cys Gly Gly Asn Gly Trp Ser Gly Cys Thr
Thr Cys 275 280 285
Val Ala Gly Ser Thr Cys Thr Lys Ile Asn Asp Trp Tyr His Gln Cys 290
295 300 Leu 305
261188DNAMyceliophthora thermophila 26cgacttgaaa cgccccaaat gaagtcctcc
atcctcgcca gcgtcttcgc cacgggcgcc 60gtggctcaaa gtggtccgtg gcagcaatgt
ggtggcatcg gatggcaagg atcgaccgac 120tgtgtgtcgg gctaccactg cgtctaccag
aacgattggt acagccagtg cgtgcctggc 180gcggcgtcga caacgctgca gacatcgacc
acgtccaggc ccaccgccac cagcaccgcc 240cctccgtcgt ccaccacctc gcctagcaag
ggcaagctga agtggctcgg cagcaacgag 300tcgggcgccg agttcgggga gggcaattac
cccggcctct ggggcaagca cttcatcttc 360ccgtcgactt cggcgattca gacgctcatc
aatgatggat acaacatctt ccggatcgac 420ttctcgatgg agcgtctggt gcccaaccag
ttgacgtcgt ccttcgacca gggttacctc 480cgcaacctga ccgaggtggt caacttcgtg
acgaacgcgg gcaagtacgc cgtcctggac 540ccgcacaact acggccggta ctacggcaac
atcatcacgg acacgaacgc gttccggacc 600ttctggacca acctggccaa gcagttcgcc
tccaactcgc tcgtcatctt cgacaccaac 660aacgagtaca acacgatgga ccagaccctg
gtgctcaacc tcaaccaggc cgccatcgac 720ggcatccggg ccgccggcgc gacctcgcag
tacatcttcg tcgagggcaa cgcgtggagc 780ggggcctgga gctggaacac gaccaacacc
aacatggccg ccctgacgga cccgcagaac 840aagatcgtgt acgagatgca ccagtacctc
gactcggaca gctcgggcac ccacgccgag 900tgcgtcagca gcaccatcgg cgcccagcgc
gtcgtcggag ccacccagtg gctccgcgcc 960aacggcaagc tcggcgtcct cggcgagttc
gccggcggcg ccaacgccgt ctgccagcag 1020gccgtcaccg gcctcctcga ccacctccag
gacaacagcg acgtctggct gggtgccctc 1080tggtgggccg ccggtccctg gtggggcgac
tacatgtact cgttcgagcc tccttcgggc 1140accggctatg tcaactacaa ctcgatcttg
aagaagtact tgccgtaa 118827389PRTMyceliophthora thermophila
27Met Lys Ser Ser Ile Leu Ala Ser Val Phe Ala Thr Gly Ala Val Ala 1
5 10 15 Gln Ser Gly Pro
Trp Gln Gln Cys Gly Gly Ile Gly Trp Gln Gly Ser 20
25 30 Thr Asp Cys Val Ser Gly Tyr His Cys
Val Tyr Gln Asn Asp Trp Tyr 35 40
45 Ser Gln Cys Val Pro Gly Ala Ala Ser Thr Thr Leu Gln Thr
Ser Thr 50 55 60
Thr Ser Arg Pro Thr Ala Thr Ser Thr Ala Pro Pro Ser Ser Thr Thr 65
70 75 80 Ser Pro Ser Lys Gly
Lys Leu Lys Trp Leu Gly Ser Asn Glu Ser Gly 85
90 95 Ala Glu Phe Gly Glu Gly Asn Tyr Pro Gly
Leu Trp Gly Lys His Phe 100 105
110 Ile Phe Pro Ser Thr Ser Ala Ile Gln Thr Leu Ile Asn Asp Gly
Tyr 115 120 125 Asn
Ile Phe Arg Ile Asp Phe Ser Met Glu Arg Leu Val Pro Asn Gln 130
135 140 Leu Thr Ser Ser Phe Asp
Gln Gly Tyr Leu Arg Asn Leu Thr Glu Val 145 150
155 160 Val Asn Phe Val Thr Asn Ala Gly Lys Tyr Ala
Val Leu Asp Pro His 165 170
175 Asn Tyr Gly Arg Tyr Tyr Gly Asn Ile Ile Thr Asp Thr Asn Ala Phe
180 185 190 Arg Thr
Phe Trp Thr Asn Leu Ala Lys Gln Phe Ala Ser Asn Ser Leu 195
200 205 Val Ile Phe Asp Thr Asn Asn
Glu Tyr Asn Thr Met Asp Gln Thr Leu 210 215
220 Val Leu Asn Leu Asn Gln Ala Ala Ile Asp Gly Ile
Arg Ala Ala Gly 225 230 235
240 Ala Thr Ser Gln Tyr Ile Phe Val Glu Gly Asn Ala Trp Ser Gly Ala
245 250 255 Trp Ser Trp
Asn Thr Thr Asn Thr Asn Met Ala Ala Leu Thr Asp Pro 260
265 270 Gln Asn Lys Ile Val Tyr Glu Met
His Gln Tyr Leu Asp Ser Asp Ser 275 280
285 Ser Gly Thr His Ala Glu Cys Val Ser Ser Thr Ile Gly
Ala Gln Arg 290 295 300
Val Val Gly Ala Thr Gln Trp Leu Arg Ala Asn Gly Lys Leu Gly Val 305
310 315 320 Leu Gly Glu Phe
Ala Gly Gly Ala Asn Ala Val Cys Gln Gln Ala Val 325
330 335 Thr Gly Leu Leu Asp His Leu Gln Asp
Asn Ser Asp Val Trp Leu Gly 340 345
350 Ala Leu Trp Trp Ala Ala Gly Pro Trp Trp Gly Asp Tyr Met
Tyr Ser 355 360 365
Phe Glu Pro Pro Ser Gly Thr Gly Tyr Val Asn Tyr Asn Ser Ile Leu 370
375 380 Lys Lys Tyr Leu Pro
385 281232DNABasidiomycete CBS 495.95 28ggatccactt
agtaacggcc gccagtgtgc tggaaagcat gaagtctctc ttcctgtcac 60ttgtagcgac
cgtcgcgctc agctcgccag tattctctgt cgcagtctgg gggcaatgcg 120gcggcattgg
cttcagcgga agcaccgtct gtgatgcagg cgccggctgt gtgaagctca 180acgactatta
ctctcaatgc caacccggcg ctcccactgc tacatccgcg gcgccaagta 240gcaacgcacc
gtccggcact tcgacggcct cggccccctc ctccagcctt tgctctggca 300gccgcacgcc
gttccagttc ttcggtgtca acgaatccgg cgcggagttc ggcaacctga 360acatccccgg
tgttctgggc accgactaca cctggccgtc gccatccagc attgacttct 420tcatgggcaa
gggaatgaat accttccgta ttccgttcct catggagcgt cttgtccccc 480ctgccactgg
catcacagga cctctcgacc agacgtactt gggcggcctg cagacgattg 540tcaactacat
caccggcaaa ggcggctttg ctctcattga cccgcacaac tttatgatct 600acaatggcca
gacgatctcc agtaccagcg acttccagaa gttctggcag aacctcgcag 660gagtgtttaa
atcgaacagt cacgtcatct tcgatgttat gaacgagcct cacgatattc 720ccgcccagac
cgtgttccaa ctgaaccaag ccgctgtcaa tggcatccgt gcgagcggtg 780cgacgtcgca
gctcattctg gtcgagggca caagctggac tggagcctgg acctggacga 840cctctggcaa
cagcgatgca ttcggtgcca ttaaggatcc caacaacaac gtcgcgatcc 900agatgcatca
gtacctggat agcgatggct ctggcacttc gcagacctgc gtgtctccca 960ccatcggtgc
cgagcggttg caggctgcga ctcaatggtt gaagcagaac aacctcaagg 1020gcttcctggg
cgagatcggc gccggctcta actccgcttg catcagcgct gtgcagggtg 1080cgttgtgttc
gatgcagcaa tctggtgtgt ggctcggcgc tctctggtgg gctgcgggcc 1140cgtggtgggg
cgactactac cagtccatcg agccgccctc tggcccggcg gtgtccgcga 1200tcctcccgca
ggccctgctg ccgttcgcgt aa
123229397PRTBasidiomycete CBS 495.95 29Met Lys Ser Leu Phe Leu Ser Leu
Val Ala Thr Val Ala Leu Ser Ser 1 5 10
15 Pro Val Phe Ser Val Ala Val Trp Gly Gln Cys Gly Gly
Ile Gly Phe 20 25 30
Ser Gly Ser Thr Val Cys Asp Ala Gly Ala Gly Cys Val Lys Leu Asn
35 40 45 Asp Tyr Tyr Ser
Gln Cys Gln Pro Gly Ala Pro Thr Ala Thr Ser Ala 50
55 60 Ala Pro Ser Ser Asn Ala Pro Ser
Gly Thr Ser Thr Ala Ser Ala Pro 65 70
75 80 Ser Ser Ser Leu Cys Ser Gly Ser Arg Thr Pro Phe
Gln Phe Phe Gly 85 90
95 Val Asn Glu Ser Gly Ala Glu Phe Gly Asn Leu Asn Ile Pro Gly Val
100 105 110 Leu Gly Thr
Asp Tyr Thr Trp Pro Ser Pro Ser Ser Ile Asp Phe Phe 115
120 125 Met Gly Lys Gly Met Asn Thr Phe
Arg Ile Pro Phe Leu Met Glu Arg 130 135
140 Leu Val Pro Pro Ala Thr Gly Ile Thr Gly Pro Leu Asp
Gln Thr Tyr 145 150 155
160 Leu Gly Gly Leu Gln Thr Ile Val Asn Tyr Ile Thr Gly Lys Gly Gly
165 170 175 Phe Ala Leu Ile
Asp Pro His Asn Phe Met Ile Tyr Asn Gly Gln Thr 180
185 190 Ile Ser Ser Thr Ser Asp Phe Gln Lys
Phe Trp Gln Asn Leu Ala Gly 195 200
205 Val Phe Lys Ser Asn Ser His Val Ile Phe Asp Val Met Asn
Glu Pro 210 215 220
His Asp Ile Pro Ala Gln Thr Val Phe Gln Leu Asn Gln Ala Ala Val 225
230 235 240 Asn Gly Ile Arg Ala
Ser Gly Ala Thr Ser Gln Leu Ile Leu Val Glu 245
250 255 Gly Thr Ser Trp Thr Gly Ala Trp Thr Trp
Thr Thr Ser Gly Asn Ser 260 265
270 Asp Ala Phe Gly Ala Ile Lys Asp Pro Asn Asn Asn Val Ala Ile
Gln 275 280 285 Met
His Gln Tyr Leu Asp Ser Asp Gly Ser Gly Thr Ser Gln Thr Cys 290
295 300 Val Ser Pro Thr Ile Gly
Ala Glu Arg Leu Gln Ala Ala Thr Gln Trp 305 310
315 320 Leu Lys Gln Asn Asn Leu Lys Gly Phe Leu Gly
Glu Ile Gly Ala Gly 325 330
335 Ser Asn Ser Ala Cys Ile Ser Ala Val Gln Gly Ala Leu Cys Ser Met
340 345 350 Gln Gln
Ser Gly Val Trp Leu Gly Ala Leu Trp Trp Ala Ala Gly Pro 355
360 365 Trp Trp Gly Asp Tyr Tyr Gln
Ser Ile Glu Pro Pro Ser Gly Pro Ala 370 375
380 Val Ser Ala Ile Leu Pro Gln Ala Leu Leu Pro Phe
Ala 385 390 395
301303DNABasidiomycete CBS 495.95 30ggaaagcgtc agtatggtga aatttgcgct
tgtggcaact gtcggcgcaa tcttgagcgc 60ttctgcggcc aatgcggctt ctatctacca
gcaatgtgga ggcattggat ggtctgggtc 120cactgtttgc gacgccggtc tcgcttgcgt
tatcctcaat gcgtactact ttcagtgctt 180gacgcccgcc gcgggccaga caacgacggg
ctcgggcgca ccggcgtcaa catcaacctc 240tcactcaacg gtcactacgg ggagctcaca
ctcaacaacc gggacgacgg cgacgaaaac 300aactaccact ccgtcgacca ccacgaccct
acccgccatc tctgtgtctg gtcgcgtctg 360ctctggctcc aggacgaagt tcaagttctt
cggtgtgaat gaaagcggcg ccgaattcgg 420gaacactgct tggccagggc agctcgggaa
agactataca tggccttcgc ctagcagcgt 480ggactacttc atgggggctg gattcaatac
attccgtatc accttcttga tggagcgtat 540gagccctccg gctaccggac tcactggccc
attcaaccag acgtacctgt cgggcctcac 600caccattgtc gactacatca cgaacaaagg
aggatacgct cttattgacc cccacaactt 660catgcgttac aacaacggca taatcagcag
cacatctgac ttcgcgactt ggtggagcaa 720tttggccact gtattcaaat ccacgaagaa
cgccatcttc gacatccaga acgagccgta 780cggaatcgat gcgcagaccg tatacgaact
gaatcaagct gccatcaatt cgatccgcgc 840cgctggcgct acgtcacagt tgattctggt
tgaaggaacg tcatacactg gagcttggac 900gtgggtctcg tccggaaacg gagctgcttt
cgcggccgtt acggatcctt acaacaacac 960ggcaattgaa atgcaccaat acctcgacag
cgacggttct gggacaaacg aagactgtgt 1020ctcctccacc attgggtcgc aacgtctcca
agctgccact gcgtggctgc aacaaacagg 1080actcaaggga ttcctcggag agacgggtgc
tgggtcgaat tcccagtgca tcgacgccgt 1140gttcgatgaa ctttgctata tgcaacagca
aggcggctcc tggatcggtg cactctggtg 1200ggctgcgggt ccctggtggg gcacgtacat
ttactcgatt gaacctccga gcggtgccgc 1260tatcccagaa gtccttcctc agggtctcgc
tccattcctc tag 130331429PRTBasidiomycete CBS 495.95
31Met Val Lys Phe Ala Leu Val Ala Thr Val Gly Ala Ile Leu Ser Ala 1
5 10 15 Ser Ala Ala Asn
Ala Ala Ser Ile Tyr Gln Gln Cys Gly Gly Ile Gly 20
25 30 Trp Ser Gly Ser Thr Val Cys Asp Ala
Gly Leu Ala Cys Val Ile Leu 35 40
45 Asn Ala Tyr Tyr Phe Gln Cys Leu Thr Pro Ala Ala Gly Gln
Thr Thr 50 55 60
Thr Gly Ser Gly Ala Pro Ala Ser Thr Ser Thr Ser His Ser Thr Val 65
70 75 80 Thr Thr Gly Ser Ser
His Ser Thr Thr Gly Thr Thr Ala Thr Lys Thr 85
90 95 Thr Thr Thr Pro Ser Thr Thr Thr Thr Leu
Pro Ala Ile Ser Val Ser 100 105
110 Gly Arg Val Cys Ser Gly Ser Arg Thr Lys Phe Lys Phe Phe Gly
Val 115 120 125 Asn
Glu Ser Gly Ala Glu Phe Gly Asn Thr Ala Trp Pro Gly Gln Leu 130
135 140 Gly Lys Asp Tyr Thr Trp
Pro Ser Pro Ser Ser Val Asp Tyr Phe Met 145 150
155 160 Gly Ala Gly Phe Asn Thr Phe Arg Ile Thr Phe
Leu Met Glu Arg Met 165 170
175 Ser Pro Pro Ala Thr Gly Leu Thr Gly Pro Phe Asn Gln Thr Tyr Leu
180 185 190 Ser Gly
Leu Thr Thr Ile Val Asp Tyr Ile Thr Asn Lys Gly Gly Tyr 195
200 205 Ala Leu Ile Asp Pro His Asn
Phe Met Arg Tyr Asn Asn Gly Ile Ile 210 215
220 Ser Ser Thr Ser Asp Phe Ala Thr Trp Trp Ser Asn
Leu Ala Thr Val 225 230 235
240 Phe Lys Ser Thr Lys Asn Ala Ile Phe Asp Ile Gln Asn Glu Pro Tyr
245 250 255 Gly Ile Asp
Ala Gln Thr Val Tyr Glu Leu Asn Gln Ala Ala Ile Asn 260
265 270 Ser Ile Arg Ala Ala Gly Ala Thr
Ser Gln Leu Ile Leu Val Glu Gly 275 280
285 Thr Ser Tyr Thr Gly Ala Trp Thr Trp Val Ser Ser Gly
Asn Gly Ala 290 295 300
Ala Phe Ala Ala Val Thr Asp Pro Tyr Asn Asn Thr Ala Ile Glu Met 305
310 315 320 His Gln Tyr Leu
Asp Ser Asp Gly Ser Gly Thr Asn Glu Asp Cys Val 325
330 335 Ser Ser Thr Ile Gly Ser Gln Arg Leu
Gln Ala Ala Thr Ala Trp Leu 340 345
350 Gln Gln Thr Gly Leu Lys Gly Phe Leu Gly Glu Thr Gly Ala
Gly Ser 355 360 365
Asn Ser Gln Cys Ile Asp Ala Val Phe Asp Glu Leu Cys Tyr Met Gln 370
375 380 Gln Gln Gly Gly Ser
Trp Ile Gly Ala Leu Trp Trp Ala Ala Gly Pro 385 390
395 400 Trp Trp Gly Thr Tyr Ile Tyr Ser Ile Glu
Pro Pro Ser Gly Ala Ala 405 410
415 Ile Pro Glu Val Leu Pro Gln Gly Leu Ala Pro Phe Leu
420 425 321580DNAThielavia terrestris
32agccccccgt tcaggcacac ttggcatcag atcagcttag cagcgcctgc acagcatgaa
60gctctcgcag tcggccgcgc tggcggcact caccgcgacg gcgctcgccg ccccctcgcc
120cacgacgccg caggcgccga ggcaggcttc agccggctgc tcgtctgcgg tcacgctcga
180cgccagcacc aacgtttgga agaagtacac gctgcacccc aacagctact accgcaagga
240ggttgaggcc gcggtggcgc agatctcgga cccggacctc gccgccaagg ccaagaaggt
300ggccgacgtc ggcaccttcc tgtggctcga ctcgatcgag aacatcggca agctggagcc
360ggcgatccag gacgtgccct gcgagaacat cctgggcctg gtcatctacg acctgccggg
420ccgcgactgc gcggccaagg cgtccaacgg cgagctcaag gtcggcgaga tcgaccgcta
480caagaccgag tacatcgaca gtgagtgctg ccccccgggt tcgagaagag cgtgggggaa
540agggaaaggg ttgactgact gacacggcgc actgcagaga tcgtgtcgat cctcaaggca
600caccccaaca cggcgttcgc gctggtcatc gagccggact cgctgcccaa cctggtgacc
660aacagcaact tggacacgtg ctcgagcagc gcgtcgggct accgcgaagg cgtggcttac
720gccctcaaga acctcaacct gcccaacgtg atcatgtacc tcgacgccgg ccacggcggc
780tggctcggct gggacgccaa cctgcagccc ggcgcgcagg agctagccaa ggcgtacaag
840aacgccggct cgcccaagca gctccgcggc ttctcgacca acgtggccgg ctggaactcc
900tggtgagctt ttttccattc catttcttct tcctcttctc tcttcgctcc cactctgcag
960ccccccctcc cccaagcacc cactggcgtt ccggcttgct gactcggcct ccctttcccc
1020gggcaccagg gatcaatcgc ccggcgaatt ctcccaggcg tccgacgcca agtacaacaa
1080gtgccagaac gagaagatct acgtcagcac cttcggctcc gcgctccagt cggccggcat
1140gcccaaccac gccatcgtcg acacgggccg caacggcgtc accggcctgc gcaaggagtg
1200gggtgactgg tgcaacgtca acggtgcagg ttcgttgtct tctttttctc ctcttttgtt
1260tgcacgtcgt ggtccttttc aagcagccgt gtttggttgg gggagatgga ctccggctga
1320tgttctgctt cctctctagg cttcggcgtg cgcccgacga gcaacacggg cctcgagctg
1380gccgacgcgt tcgtgtgggt caagcccggc ggcgagtcgg acggcaccag cgacagctcg
1440tcgccgcgct acgacagctt ctgcggcaag gacgacgcct tcaagccctc gcccgaggcc
1500ggcacctgga acgaggccta cttcgagatg ctgctcaaga acgccgtgcc gtcgttctaa
1560gacggtccag catcatccgg
158033396PRTThielavia terrestris 33Met Lys Leu Ser Gln Ser Ala Ala Leu
Ala Ala Leu Thr Ala Thr Ala 1 5 10
15 Leu Ala Ala Pro Ser Pro Thr Thr Pro Gln Ala Pro Arg Gln
Ala Ser 20 25 30
Ala Gly Cys Ser Ser Ala Val Thr Leu Asp Ala Ser Thr Asn Val Trp
35 40 45 Lys Lys Tyr Thr
Leu His Pro Asn Ser Tyr Tyr Arg Lys Glu Val Glu 50
55 60 Ala Ala Val Ala Gln Ile Ser Asp
Pro Asp Leu Ala Ala Lys Ala Lys 65 70
75 80 Lys Val Ala Asp Val Gly Thr Phe Leu Trp Leu Asp
Ser Ile Glu Asn 85 90
95 Ile Gly Lys Leu Glu Pro Ala Ile Gln Asp Val Pro Cys Glu Asn Ile
100 105 110 Leu Gly Leu
Val Ile Tyr Asp Leu Pro Gly Arg Asp Cys Ala Ala Lys 115
120 125 Ala Ser Asn Gly Glu Leu Lys Val
Gly Glu Ile Asp Arg Tyr Lys Thr 130 135
140 Glu Tyr Ile Asp Lys Ile Val Ser Ile Leu Lys Ala His
Pro Asn Thr 145 150 155
160 Ala Phe Ala Leu Val Ile Glu Pro Asp Ser Leu Pro Asn Leu Val Thr
165 170 175 Asn Ser Asn Leu
Asp Thr Cys Ser Ser Ser Ala Ser Gly Tyr Arg Glu 180
185 190 Gly Val Ala Tyr Ala Leu Lys Asn Leu
Asn Leu Pro Asn Val Ile Met 195 200
205 Tyr Leu Asp Ala Gly His Gly Gly Trp Leu Gly Trp Asp Ala
Asn Leu 210 215 220
Gln Pro Gly Ala Gln Glu Leu Ala Lys Ala Tyr Lys Asn Ala Gly Ser 225
230 235 240 Pro Lys Gln Leu Arg
Gly Phe Ser Thr Asn Val Ala Gly Trp Asn Ser 245
250 255 Trp Asp Gln Ser Pro Gly Glu Phe Ser Gln
Ala Ser Asp Ala Lys Tyr 260 265
270 Asn Lys Cys Gln Asn Glu Lys Ile Tyr Val Ser Thr Phe Gly Ser
Ala 275 280 285 Leu
Gln Ser Ala Gly Met Pro Asn His Ala Ile Val Asp Thr Gly Arg 290
295 300 Asn Gly Val Thr Gly Leu
Arg Lys Glu Trp Gly Asp Trp Cys Asn Val 305 310
315 320 Asn Gly Ala Gly Phe Gly Val Arg Pro Thr Ser
Asn Thr Gly Leu Glu 325 330
335 Leu Ala Asp Ala Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp Gly
340 345 350 Thr Ser
Asp Ser Ser Ser Pro Arg Tyr Asp Ser Phe Cys Gly Lys Asp 355
360 365 Asp Ala Phe Lys Pro Ser Pro
Glu Ala Gly Thr Trp Asn Glu Ala Tyr 370 375
380 Phe Glu Met Leu Leu Lys Asn Ala Val Pro Ser Phe
385 390 395 341203DNAThielavia
terrestris 34atgaagtacc tcaacctcct cgcagctctc ctcgccgtcg ctcctctctc
cctcgctgca 60cccagcatcg aggccagaca gtcgaacgtc aacccataca tcggcaagag
cccgctcgtt 120attaggtcgt acgcccaaaa gcttgaggag accgtcagga ccttccagca
acgtggcgac 180cagctcaacg ctgcgaggac acggacggtg cagaacgttg cgactttcgc
ctggatctcg 240gataccaatg gtattggagc cattcgacct ctcatccaag atgctctcgc
ccagcaggct 300cgcactggac agaaggtcat cgtccaaatc gtcgtctaca acctcccaga
tcgcgactgc 360tctgccaacg cctcgactgg agagttcacc gtaggaaacg acggtctcaa
ccgatacaag 420aactttgtca acaccatcgc ccgcgagctc tcgactgctg acgctgacaa
gctccacttt 480gccctcctcc tcgaacccga cgcacttgcc aacctcgtca ccaacgcgaa
tgcccccagg 540tgccgaatcg ccgctcccgc ttacaaggag ggtatcgcct acaccctcgc
caccttgtcc 600aagcccaacg tcgacgtcta catcgacgcc gccaacggtg gctggctcgg
ctggaacgac 660aacctccgcc ccttcgccga actcttcaag gaagtctacg acctcgcccg
ccgcatcaac 720cccaacgcca aggtccgcgg cgtccccgtc aacgtctcca actacaacca
gtaccgcgct 780gaagtccgcg agcccttcac cgagtggaag gacgcctggg acgagagccg
ctacgtcaac 840gtcctcaccc cgcacctcaa cgccgtcggc ttctccgcgc acttcatcgt
tgaccaggga 900cgcggtggca agggcggtat caggacggag tggggccagt ggtgcaacgt
taggaacgct 960gggttcggta tcaggcctac tgcggatcag ggcgtgctcc agaacccgaa
tgtggatgcg 1020attgtgtggg ttaagccggg tggagagtcg gatggcacga gtgatttgaa
ctcgaacagg 1080tatgatccta cgtgcaggag tccggtggcg catgttcccg ctcctgaggc
tggccagtgg 1140ttcaacgagt atgttgttaa cctcgttttg aacgctaacc cccctcttga
gcctacctgg 1200taa
120335400PRTThielavia terrestris 35Met Lys Tyr Leu Asn Leu Leu
Ala Ala Leu Leu Ala Val Ala Pro Leu 1 5
10 15 Ser Leu Ala Ala Pro Ser Ile Glu Ala Arg Gln
Ser Asn Val Asn Pro 20 25
30 Tyr Ile Gly Lys Ser Pro Leu Val Ile Arg Ser Tyr Ala Gln Lys
Leu 35 40 45 Glu
Glu Thr Val Arg Thr Phe Gln Gln Arg Gly Asp Gln Leu Asn Ala 50
55 60 Ala Arg Thr Arg Thr Val
Gln Asn Val Ala Thr Phe Ala Trp Ile Ser 65 70
75 80 Asp Thr Asn Gly Ile Gly Ala Ile Arg Pro Leu
Ile Gln Asp Ala Leu 85 90
95 Ala Gln Gln Ala Arg Thr Gly Gln Lys Val Ile Val Gln Ile Val Val
100 105 110 Tyr Asn
Leu Pro Asp Arg Asp Cys Ser Ala Asn Ala Ser Thr Gly Glu 115
120 125 Phe Thr Val Gly Asn Asp Gly
Leu Asn Arg Tyr Lys Asn Phe Val Asn 130 135
140 Thr Ile Ala Arg Glu Leu Ser Thr Ala Asp Ala Asp
Lys Leu His Phe 145 150 155
160 Ala Leu Leu Leu Glu Pro Asp Ala Leu Ala Asn Leu Val Thr Asn Ala
165 170 175 Asn Ala Pro
Arg Cys Arg Ile Ala Ala Pro Ala Tyr Lys Glu Gly Ile 180
185 190 Ala Tyr Thr Leu Ala Thr Leu Ser
Lys Pro Asn Val Asp Val Tyr Ile 195 200
205 Asp Ala Ala Asn Gly Gly Trp Leu Gly Trp Asn Asp Asn
Leu Arg Pro 210 215 220
Phe Ala Glu Leu Phe Lys Glu Val Tyr Asp Leu Ala Arg Arg Ile Asn 225
230 235 240 Pro Asn Ala Lys
Val Arg Gly Val Pro Val Asn Val Ser Asn Tyr Asn 245
250 255 Gln Tyr Arg Ala Glu Val Arg Glu Pro
Phe Thr Glu Trp Lys Asp Ala 260 265
270 Trp Asp Glu Ser Arg Tyr Val Asn Val Leu Thr Pro His Leu
Asn Ala 275 280 285
Val Gly Phe Ser Ala His Phe Ile Val Asp Gln Gly Arg Gly Gly Lys 290
295 300 Gly Gly Ile Arg Thr
Glu Trp Gly Gln Trp Cys Asn Val Arg Asn Ala 305 310
315 320 Gly Phe Gly Ile Arg Pro Thr Ala Asp Gln
Gly Val Leu Gln Asn Pro 325 330
335 Asn Val Asp Ala Ile Val Trp Val Lys Pro Gly Gly Glu Ser Asp
Gly 340 345 350 Thr
Ser Asp Leu Asn Ser Asn Arg Tyr Asp Pro Thr Cys Arg Ser Pro 355
360 365 Val Ala His Val Pro Ala
Pro Glu Ala Gly Gln Trp Phe Asn Glu Tyr 370 375
380 Val Val Asn Leu Val Leu Asn Ala Asn Pro Pro
Leu Glu Pro Thr Trp 385 390 395
400 361501DNAThielavia terrestris 36gccgttgtca agatgggcca
gaagacgctg cacggattcg ccgccacggc tttggccgtt 60ctcccctttg tgaaggctca
gcagcccggc aacttcacgc cggaggtgca cccgcaactg 120ccaacgtgga agtgcacgac
cgccggcggc tgcgttcagc aggacacttc ggtggtgctc 180gactggaact accgttggat
ccacaatgcc gacggcaccg cctcgtgcac gacgtccagc 240ggggtcgacc acacgctgtg
tccagatgag gcgacctgcg cgaagaactg cttcgtggaa 300ggcgtcaact acacgagcag
cggtgtcacc acatccggca gttcgctgac gatgaggcag 360tatttcaagg ggagcaacgg
gcagaccaac agcgtttcgc ctcgtctcta cctgctcggc 420tcggatggaa actacgtaat
gctcaagctg ctcggccagg agctgagctt cgatgtcgat 480ctctccacgc tcccctgcgg
cgagaacggc gcgctgtacc tgtccgagat ggacgcgacc 540ggtggcagga accagtacaa
caccggcggt gccaactacg gctcgggcta ctgtgacgcc 600cagtgtcccg tgcagacgtg
gatgaacggc acgctgaaca ccaacgggca gggctactgc 660tgcaacgaga tggacatcct
cgaggccaac tcccgcgcca acgcgatgac acctcacccc 720tgcgccaacg gcagctgcga
caagagcggg tgcggactca acccctacgc cgagggctac 780aagagctact acggaccggg
cctcacggtt gacacgtcga agcccttcac catcattacc 840cgcttcatca ccgacgacgg
cacgaccagc ggcaccctca accagatcca gcggatctat 900gtgcagaatg gcaagacggt
cgcgtcggct gcgtccggag gcgacatcat cacggcatcc 960ggctgcacct cggcccaggc
gttcggcggg ctggccaaca tgggcgcggc gcttggacgg 1020ggcatggtgc tgaccttcag
catctggaac gacgctgggg gctacatgaa ctggctcgac 1080agcggcaaca acggcccgtg
cagcagcacc gagggcaacc cgtccaacat cctggccaac 1140tacccggaca cccacgtggt
cttctccaac atccgctggg gagacatcgg ctcgacggtc 1200caggtctcgg gaggcggcaa
cggcggctcg accaccacca cgtcgaccac cacgctgagg 1260acctcgacca cgaccaccac
caccgccccg acggccactg ccacgcactg gggacaatgc 1320ggcggaatcg gggtacgtca
accgcctcct gcattctgtt gaggaagtta actaacgtgg 1380cctacgcagt ggactggacc
gaccgtctgc gaatcgccgt acgcatgcaa ggagctgaac 1440ccctggtact accagtgcct
ctaaagtatt gcagtgaagc catactccgt gctcggcatg 1500g
150137464PRTThielavia
terrestris 37Met Gly Gln Lys Thr Leu His Gly Phe Ala Ala Thr Ala Leu Ala
Val 1 5 10 15 Leu
Pro Phe Val Lys Ala Gln Gln Pro Gly Asn Phe Thr Pro Glu Val
20 25 30 His Pro Gln Leu Pro
Thr Trp Lys Cys Thr Thr Ala Gly Gly Cys Val 35
40 45 Gln Gln Asp Thr Ser Val Val Leu Asp
Trp Asn Tyr Arg Trp Ile His 50 55
60 Asn Ala Asp Gly Thr Ala Ser Cys Thr Thr Ser Ser Gly
Val Asp His 65 70 75
80 Thr Leu Cys Pro Asp Glu Ala Thr Cys Ala Lys Asn Cys Phe Val Glu
85 90 95 Gly Val Asn Tyr
Thr Ser Ser Gly Val Thr Thr Ser Gly Ser Ser Leu 100
105 110 Thr Met Arg Gln Tyr Phe Lys Gly Ser
Asn Gly Gln Thr Asn Ser Val 115 120
125 Ser Pro Arg Leu Tyr Leu Leu Gly Ser Asp Gly Asn Tyr Val
Met Leu 130 135 140
Lys Leu Leu Gly Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Thr Leu 145
150 155 160 Pro Cys Gly Glu Asn
Gly Ala Leu Tyr Leu Ser Glu Met Asp Ala Thr 165
170 175 Gly Gly Arg Asn Gln Tyr Asn Thr Gly Gly
Ala Asn Tyr Gly Ser Gly 180 185
190 Tyr Cys Asp Ala Gln Cys Pro Val Gln Thr Trp Met Asn Gly Thr
Leu 195 200 205 Asn
Thr Asn Gly Gln Gly Tyr Cys Cys Asn Glu Met Asp Ile Leu Glu 210
215 220 Ala Asn Ser Arg Ala Asn
Ala Met Thr Pro His Pro Cys Ala Asn Gly 225 230
235 240 Ser Cys Asp Lys Ser Gly Cys Gly Leu Asn Pro
Tyr Ala Glu Gly Tyr 245 250
255 Lys Ser Tyr Tyr Gly Pro Gly Leu Thr Val Asp Thr Ser Lys Pro Phe
260 265 270 Thr Ile
Ile Thr Arg Phe Ile Thr Asp Asp Gly Thr Thr Ser Gly Thr 275
280 285 Leu Asn Gln Ile Gln Arg Ile
Tyr Val Gln Asn Gly Lys Thr Val Ala 290 295
300 Ser Ala Ala Ser Gly Gly Asp Ile Ile Thr Ala Ser
Gly Cys Thr Ser 305 310 315
320 Ala Gln Ala Phe Gly Gly Leu Ala Asn Met Gly Ala Ala Leu Gly Arg
325 330 335 Gly Met Val
Leu Thr Phe Ser Ile Trp Asn Asp Ala Gly Gly Tyr Met 340
345 350 Asn Trp Leu Asp Ser Gly Asn Asn
Gly Pro Cys Ser Ser Thr Glu Gly 355 360
365 Asn Pro Ser Asn Ile Leu Ala Asn Tyr Pro Asp Thr His
Val Val Phe 370 375 380
Ser Asn Ile Arg Trp Gly Asp Ile Gly Ser Thr Val Gln Val Ser Gly 385
390 395 400 Gly Gly Asn Gly
Gly Ser Thr Thr Thr Thr Ser Thr Thr Thr Leu Arg 405
410 415 Thr Ser Thr Thr Thr Thr Thr Thr Ala
Pro Thr Ala Thr Ala Thr His 420 425
430 Trp Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro Thr Val
Cys Glu 435 440 445
Ser Pro Tyr Ala Cys Lys Glu Leu Asn Pro Trp Tyr Tyr Gln Cys Leu 450
455 460 381368DNAThielavia
terrestris 38accgatccgc tcgaagatgg cgcccaagtc tacagttctg gccgcctggc
tgctctcctc 60gctggccgcg gcccagcaga tcggcaaagc cgtgcccgag gtccacccca
aactgacaac 120gcagaagtgc actctccgcg gcgggtgcaa gcctgtccgc acctcggtcg
tgctcgactc 180gtccgcgcgc tcgctgcaca aggtcgggga ccccaacacc agctgcagcg
tcggcggcga 240cctgtgctcg gacgcgaagt cgtgcggcaa gaactgcgcg ctcgagggcg
tcgactacgc 300ggcccacggc gtggcgacca agggcgacgc cctcacgctg caccagtggc
tcaagggggc 360cgacggcacc tacaggaccg tctcgccgcg cgtatacctc ctgggcgagg
acgggaagaa 420ctacgaggac ttcaagctgc tcaacgccga gctcagcttc gacgtcgacg
tgtcccagct 480cgtctgcggc atgaacggcg ccctgtactt ctccgagatg gagatggacg
gcggccgcag 540cccgctgaac ccggcgggcg ccacgtacgg cacgggctac tgcgacgcgc
agtgccccaa 600gttggacttt atcaacggcg aggtatttct tctctcttct gtttttcttt
tccatcgctt 660tttctgaccg gaatccgccc tcttagctca acaccaacca cacgtacggg
gcgtgctgca 720acgagatgga catctgggag gccaacgcgc tggcgcaggc gctcacgccg
cacccgtgca 780acgcgacgcg ggtgtacaag tgcgacacgg cggacgagtg cgggcagccg
gtgggcgtgt 840gcgacgaatg ggggtgctcg tacaacccgt ccaacttcgg ggtcaaggac
tactacgggc 900gcaacctgac ggtggacacg aaccgcaagt tcacggtgac gacgcagttc
gtgacgtcca 960acgggcgggc ggacggcgag ctgaccgaga tccggcggct gtacgtgcag
gacggcgtgg 1020tgatccagaa ccacgcggtc acggcgggcg gggcgacgta cgacagcatc
acggacggct 1080tctgcaacgc gacggccacc tggacgcagc agcggggcgg gctcgcgcgc
atgggcgagg 1140ccatcggccg cggcatggtg ctcatcttca gcctgtgggt tgacaacggc
ggcttcatga 1200actggctcga cagcggcaac gccgggccct gcaacgccac cgagggcgac
ccggccctga 1260tcctgcagca gcacccggac gccagcgtca ccttctccaa catccgatgg
ggcgagatcg 1320gcagcacgta caagagcgag tgcagccact agagtagagc ttgtaatt
136839423PRTThielavia terrestris 39Met Ala Pro Lys Ser Thr Val
Leu Ala Ala Trp Leu Leu Ser Ser Leu 1 5
10 15 Ala Ala Ala Gln Gln Ile Gly Lys Ala Val Pro
Glu Val His Pro Lys 20 25
30 Leu Thr Thr Gln Lys Cys Thr Leu Arg Gly Gly Cys Lys Pro Val
Arg 35 40 45 Thr
Ser Val Val Leu Asp Ser Ser Ala Arg Ser Leu His Lys Val Gly 50
55 60 Asp Pro Asn Thr Ser Cys
Ser Val Gly Gly Asp Leu Cys Ser Asp Ala 65 70
75 80 Lys Ser Cys Gly Lys Asn Cys Ala Leu Glu Gly
Val Asp Tyr Ala Ala 85 90
95 His Gly Val Ala Thr Lys Gly Asp Ala Leu Thr Leu His Gln Trp Leu
100 105 110 Lys Gly
Ala Asp Gly Thr Tyr Arg Thr Val Ser Pro Arg Val Tyr Leu 115
120 125 Leu Gly Glu Asp Gly Lys Asn
Tyr Glu Asp Phe Lys Leu Leu Asn Ala 130 135
140 Glu Leu Ser Phe Asp Val Asp Val Ser Gln Leu Val
Cys Gly Met Asn 145 150 155
160 Gly Ala Leu Tyr Phe Ser Glu Met Glu Met Asp Gly Gly Arg Ser Pro
165 170 175 Leu Asn Pro
Ala Gly Ala Thr Tyr Gly Thr Gly Tyr Cys Asp Ala Gln 180
185 190 Cys Pro Lys Leu Asp Phe Ile Asn
Gly Glu Leu Asn Thr Asn His Thr 195 200
205 Tyr Gly Ala Cys Cys Asn Glu Met Asp Ile Trp Glu Ala
Asn Ala Leu 210 215 220
Ala Gln Ala Leu Thr Pro His Pro Cys Asn Ala Thr Arg Val Tyr Lys 225
230 235 240 Cys Asp Thr Ala
Asp Glu Cys Gly Gln Pro Val Gly Val Cys Asp Glu 245
250 255 Trp Gly Cys Ser Tyr Asn Pro Ser Asn
Phe Gly Val Lys Asp Tyr Tyr 260 265
270 Gly Arg Asn Leu Thr Val Asp Thr Asn Arg Lys Phe Thr Val
Thr Thr 275 280 285
Gln Phe Val Thr Ser Asn Gly Arg Ala Asp Gly Glu Leu Thr Glu Ile 290
295 300 Arg Arg Leu Tyr Val
Gln Asp Gly Val Val Ile Gln Asn His Ala Val 305 310
315 320 Thr Ala Gly Gly Ala Thr Tyr Asp Ser Ile
Thr Asp Gly Phe Cys Asn 325 330
335 Ala Thr Ala Thr Trp Thr Gln Gln Arg Gly Gly Leu Ala Arg Met
Gly 340 345 350 Glu
Ala Ile Gly Arg Gly Met Val Leu Ile Phe Ser Leu Trp Val Asp 355
360 365 Asn Gly Gly Phe Met Asn
Trp Leu Asp Ser Gly Asn Ala Gly Pro Cys 370 375
380 Asn Ala Thr Glu Gly Asp Pro Ala Leu Ile Leu
Gln Gln His Pro Asp 385 390 395
400 Ala Ser Val Thr Phe Ser Asn Ile Arg Trp Gly Glu Ile Gly Ser Thr
405 410 415 Tyr Lys
Ser Glu Cys Ser His 420 401000DNAThielavia
terrestris 40atgaccctac ggctccctgt catcagcctg ctggcctcgc tggcagcagg
cgccgtcgtc 60gtcccacggg cggagtttca cccccctctc ccgacttgga aatgcacgac
ctccgggggc 120tgcgtgcagc agaacaccag cgtcgtcctg gaccgtgact cgaagtacgc
cgcacacagc 180gccggctcgc ggacggaatc ggattacgcg gcaatgggag tgtccacttc
gggcaatgcc 240gtgacgctgt accactacgt caagaccaac ggcaccctcg tccccgcttc
gccgcgcatc 300tacctcctgg gcgcggacgg caagtacgtg cttatggacc tcctcaacca
ggagctgtcg 360gtggacgtcg acttctcggc gctgccgtgc ggcgagaacg gggccttcta
cctgtccgag 420atggcggcgg acgggcgggg cgacgcgggg gcgggcgacg ggtactgcga
cgcgcagtgc 480cagggctact gctgcaacga gatggacatc ctcgaggcca actcgatggc
gacggccatg 540acgccgcacc cgtgcaaggg caacaactgc gaccgcagcg gctgcggcta
caacccgtac 600gccagcggcc agcgcggctt ctacgggccc ggcaagacgg tcgacacgag
caagcccttc 660accgtcgtca cgcagttcgc cgccagcggc ggcaagctga cccagatcac
ccgcaagtac 720atccagaacg gccgggagat cggcggcggc ggcaccatct ccagctgcgg
ctccgagtct 780tcgacgggcg gcctgaccgg catgggcgag gcgctggggc gcggaatggt
gctggccatg 840agcatctgga acgacgcggc ccaggagatg gcatggctcg atgccggcaa
caacggccct 900tgcgccagtg gccagggcag cccgtccgtc attcagtcgc agcatcccga
cacccacgtc 960gtcttctcca acatcaggtg gggcgacatc gggtctacca
100041336PRTThielavia terrestris 41Met Thr Leu Arg Leu Pro Val
Ile Ser Leu Leu Ala Ser Leu Ala Ala 1 5
10 15 Gly Ala Val Val Val Pro Arg Ala Glu Phe His
Pro Pro Leu Pro Thr 20 25
30 Trp Lys Cys Thr Thr Ser Gly Gly Cys Val Gln Gln Asn Thr Ser
Val 35 40 45 Val
Leu Asp Arg Asp Ser Lys Tyr Ala Ala His Ser Ala Gly Ser Arg 50
55 60 Thr Glu Ser Asp Tyr Ala
Ala Met Gly Val Ser Thr Ser Gly Asn Ala 65 70
75 80 Val Thr Leu Tyr His Tyr Val Lys Thr Asn Gly
Thr Leu Val Pro Ala 85 90
95 Ser Pro Arg Ile Tyr Leu Leu Gly Ala Asp Gly Lys Tyr Val Leu Met
100 105 110 Asp Leu
Leu Asn Gln Glu Leu Ser Val Asp Val Asp Phe Ser Ala Leu 115
120 125 Pro Cys Gly Glu Asn Gly Ala
Phe Tyr Leu Ser Glu Met Ala Ala Asp 130 135
140 Gly Arg Gly Asp Ala Gly Ala Gly Asp Gly Tyr Cys
Asp Ala Gln Cys 145 150 155
160 Gln Gly Tyr Cys Cys Asn Glu Met Asp Ile Leu Glu Ala Asn Ser Met
165 170 175 Ala Thr Ala
Met Thr Pro His Pro Cys Lys Gly Asn Asn Cys Asp Arg 180
185 190 Ser Gly Cys Gly Tyr Asn Pro Tyr
Ala Ser Gly Gln Arg Gly Phe Tyr 195 200
205 Gly Pro Gly Lys Thr Val Asp Thr Ser Lys Pro Phe Thr
Val Val Thr 210 215 220
Gln Phe Ala Ala Ser Gly Gly Lys Leu Thr Gln Ile Thr Arg Lys Tyr 225
230 235 240 Ile Gln Asn Gly
Arg Glu Ile Gly Gly Gly Gly Thr Ile Ser Ser Cys 245
250 255 Gly Ser Glu Ser Ser Thr Gly Gly Leu
Thr Gly Met Gly Glu Ala Leu 260 265
270 Gly Arg Gly Met Val Leu Ala Met Ser Ile Trp Asn Asp Ala
Ala Gln 275 280 285
Glu Met Ala Trp Leu Asp Ala Gly Asn Asn Gly Pro Cys Ala Ser Gly 290
295 300 Gln Gly Ser Pro Ser
Val Ile Gln Ser Gln His Pro Asp Thr His Val 305 310
315 320 Val Phe Ser Asn Ile Arg Trp Gly Asp Ile
Gly Ser Thr Thr Lys Asn 325 330
335 421480DNACladorrhinum foecundissimum 42gatccgaatt
cctcctctcg ttctttagtc acagaccaga catctgccca cgatggttca 60caagttcgcc
ctcctcaccg gcctcgccgc ctccctcgca tctgcccagc agatcggcac 120cgtcgtcccc
gagtctcacc ccaagcttcc caccaagcgc tgcactctcg ccggtggctg 180ccagaccgtc
gacacctcca tcgtcatcga cgccttccag cgtcccctcc acaagatcgg 240cgacccttcc
actccttgcg tcgtcggcgg ccctctctgc cccgacgcca agtcctgcgc 300tgagaactgc
gcgctcgagg gtgtcgacta tgcctcctgg ggcatcaaga ccgagggcga 360cgccctaact
ctcaaccagt ggatgcccga cccggcgaac cctggccagt acaagacgac 420tactccccgt
acttaccttg ttgctgagga cggcaagaac tacgaggatg tgaagctcct 480ggctaaggag
atctcgtttg atgccgatgt cagcaacctt ccctgcggca tgaacggtgc 540tttctacttg
tctgagatgt tgatggatgg tggacgtggc gacctcaacc ctgctggtgc 600cgagtatggt
accggttact gtgatgcgca gtgcttcaag ttggatttca tcaacggcga 660ggccaacatc
gaccaaaagc acggcgcctg ctgcaacgaa atggacattt tcgaatccaa 720ctcgcgcgcc
aagaccttcg tcccccaccc ctgcaacatc acgcaggtct acaagtgcga 780aggcgaagac
gagtgcggcc agcccgtcgg cgtgtgcgac aagtgggggt gcggcttcaa 840cgagtacaaa
tggggcgtcg agtccttcta cggccggggc tcgcagttcg ccatcgactc 900ctccaagaag
ttcaccgtca ccacgcagtt cctgaccgac aacggcaagg aggacggcgt 960cctcgtcgag
atccgccgct tgtggcacca ggatggcaag ctgatcaaga acaccgctat 1020ccaggttgag
gagaactaca gcacggactc ggtgagcacc gagttctgcg agaagactgc 1080ttctttcacc
atgcagcgcg gtggtctcaa ggcgatgggc gaggctatcg gtcgtggtat 1140ggtgctggtt
ttcagcatct gggcggatga ttcgggtttt atgaactggt tggatgcgga 1200gggtaatggc
ccttgcagcg cgactgaggg cgatccgaag gagattgtca agaataagcc 1260ggatgctagg
gttacgttct caaacattag gattggtgag gttggtagca cgtatgctcc 1320gggtgggaag
tgcggtgtta agagcagggt tgctaggggg cttactgctt cttaaggggg 1380gtgtgaagag
aggaggaggt gttgttgggg gttggagatg ataattgggc gagatggtgt 1440agagcgggtt
ggttggatat gaatacgttg aattggatgt
148043440PRTCladorrhinum foecundissimum 43Met Val His Lys Phe Ala Leu Leu
Thr Gly Leu Ala Ala Ser Leu Ala 1 5 10
15 Ser Ala Gln Gln Ile Gly Thr Val Val Pro Glu Ser His
Pro Lys Leu 20 25 30
Pro Thr Lys Arg Cys Thr Leu Ala Gly Gly Cys Gln Thr Val Asp Thr
35 40 45 Ser Ile Val Ile
Asp Ala Phe Gln Arg Pro Leu His Lys Ile Gly Asp 50
55 60 Pro Ser Thr Pro Cys Val Val Gly
Gly Pro Leu Cys Pro Asp Ala Lys 65 70
75 80 Ser Cys Ala Glu Asn Cys Ala Leu Glu Gly Val Asp
Tyr Ala Ser Trp 85 90
95 Gly Ile Lys Thr Glu Gly Asp Ala Leu Thr Leu Asn Gln Trp Met Pro
100 105 110 Asp Pro Ala
Asn Pro Gly Gln Tyr Lys Thr Thr Thr Pro Arg Thr Tyr 115
120 125 Leu Val Ala Glu Asp Gly Lys Asn
Tyr Glu Asp Val Lys Leu Leu Ala 130 135
140 Lys Glu Ile Ser Phe Asp Ala Asp Val Ser Asn Leu Pro
Cys Gly Met 145 150 155
160 Asn Gly Ala Phe Tyr Leu Ser Glu Met Leu Met Asp Gly Gly Arg Gly
165 170 175 Asp Leu Asn Pro
Ala Gly Ala Glu Tyr Gly Thr Gly Tyr Cys Asp Ala 180
185 190 Gln Cys Phe Lys Leu Asp Phe Ile Asn
Gly Glu Ala Asn Ile Asp Gln 195 200
205 Lys His Gly Ala Cys Cys Asn Glu Met Asp Ile Phe Glu Ser
Asn Ser 210 215 220
Arg Ala Lys Thr Phe Val Pro His Pro Cys Asn Ile Thr Gln Val Tyr 225
230 235 240 Lys Cys Glu Gly Glu
Asp Glu Cys Gly Gln Pro Val Gly Val Cys Asp 245
250 255 Lys Trp Gly Cys Gly Phe Asn Glu Tyr Lys
Trp Gly Val Glu Ser Phe 260 265
270 Tyr Gly Arg Gly Ser Gln Phe Ala Ile Asp Ser Ser Lys Lys Phe
Thr 275 280 285 Val
Thr Thr Gln Phe Leu Thr Asp Asn Gly Lys Glu Asp Gly Val Leu 290
295 300 Val Glu Ile Arg Arg Leu
Trp His Gln Asp Gly Lys Leu Ile Lys Asn 305 310
315 320 Thr Ala Ile Gln Val Glu Glu Asn Tyr Ser Thr
Asp Ser Val Ser Thr 325 330
335 Glu Phe Cys Glu Lys Thr Ala Ser Phe Thr Met Gln Arg Gly Gly Leu
340 345 350 Lys Ala
Met Gly Glu Ala Ile Gly Arg Gly Met Val Leu Val Phe Ser 355
360 365 Ile Trp Ala Asp Asp Ser Gly
Phe Met Asn Trp Leu Asp Ala Glu Gly 370 375
380 Asn Gly Pro Cys Ser Ala Thr Glu Gly Asp Pro Lys
Glu Ile Val Lys 385 390 395
400 Asn Lys Pro Asp Ala Arg Val Thr Phe Ser Asn Ile Arg Ile Gly Glu
405 410 415 Val Gly Ser
Thr Tyr Ala Pro Gly Gly Lys Cys Gly Val Lys Ser Arg 420
425 430 Val Ala Arg Gly Leu Thr Ala Ser
435 440 441380DNATrichoderma reesei 44atggcgccct
cagttacact gccgttgacc acggccatcc tggccattgc ccggctcgtc 60gccgcccagc
aaccgggtac cagcaccccc gaggtccatc ccaagttgac aacctacaag 120tgtacaaagt
ccggggggtg cgtggcccag gacacctcgg tggtccttga ctggaactac 180cgctggatgc
acgacgcaaa ctacaactcg tgcaccgtca acggcggcgt caacaccacg 240ctctgccctg
acgaggcgac ctgtggcaag aactgcttca tcgagggcgt cgactacgcc 300gcctcgggcg
tcacgacctc gggcagcagc ctcaccatga accagtacat gcccagcagc 360tctggcggct
acagcagcgt ctctcctcgg ctgtatctcc tggactctga cggtgagtac 420gtgatgctga
agctcaacgg ccaggagctg agcttcgacg tcgacctctc tgctctgccg 480tgtggagaga
acggctcgct ctacctgtct cagatggacg agaacggggg cgccaaccag 540tataacacgg
ccggtgccaa ctacgggagc ggctactgcg atgctcagtg ccccgtccag 600acatggagga
acggcaccct caacactagc caccagggct tctgctgcaa cgagatggat 660atcctggagg
gcaactcgag ggcgaatgcc ttgacccctc actcttgcac ggccacggcc 720tgcgactctg
ccggttgcgg cttcaacccc tatggcagcg gctacaaaag ctactacggc 780cccggagata
ccgttgacac ctccaagacc ttcaccatca tcacccagtt caacacggac 840aacggctcgc
cctcgggcaa ccttgtgagc atcacccgca agtaccagca aaacggcgtc 900gacatcccca
gcgcccagcc cggcggcgac accatctcgt cctgcccgtc cgcctcagcc 960tacggcggcc
tcgccaccat gggcaaggcc ctgagcagcg gcatggtgct cgtgttcagc 1020atttggaacg
acaacagcca gtacatgaac tggctcgaca gcggcaacgc cggcccctgc 1080agcagcaccg
agggcaaccc atccaacatc ctggccaaca accccaacac gcacgtcgtc 1140ttctccaaca
tccgctgggg agacattggg tctactacga actcgactgc gcccccgccc 1200ccgcctgcgt
ccagcacgac gttttcgact acacggagga gctcgacgac ttcgagcagc 1260ccgagctgca
cgcagactca ctgggggcag tgcggtggca ttgggtacag cgggtgcaag 1320acgtgcacgt
cgggcactac gtgccagtat agcaacgact actactcgca atgcctttag
138045459PRTTrichoderma reesei 45Met Ala Pro Ser Val Thr Leu Pro Leu Thr
Thr Ala Ile Leu Ala Ile 1 5 10
15 Ala Arg Leu Val Ala Ala Gln Gln Pro Gly Thr Ser Thr Pro Glu
Val 20 25 30 His
Pro Lys Leu Thr Thr Tyr Lys Cys Thr Lys Ser Gly Gly Cys Val 35
40 45 Ala Gln Asp Thr Ser Val
Val Leu Asp Trp Asn Tyr Arg Trp Met His 50 55
60 Asp Ala Asn Tyr Asn Ser Cys Thr Val Asn Gly
Gly Val Asn Thr Thr 65 70 75
80 Leu Cys Pro Asp Glu Ala Thr Cys Gly Lys Asn Cys Phe Ile Glu Gly
85 90 95 Val Asp
Tyr Ala Ala Ser Gly Val Thr Thr Ser Gly Ser Ser Leu Thr 100
105 110 Met Asn Gln Tyr Met Pro Ser
Ser Ser Gly Gly Tyr Ser Ser Val Ser 115 120
125 Pro Arg Leu Tyr Leu Leu Asp Ser Asp Gly Glu Tyr
Val Met Leu Lys 130 135 140
Leu Asn Gly Gln Glu Leu Ser Phe Asp Val Asp Leu Ser Ala Leu Pro 145
150 155 160 Cys Gly Glu
Asn Gly Ser Leu Tyr Leu Ser Gln Met Asp Glu Asn Gly 165
170 175 Gly Ala Asn Gln Tyr Asn Thr Ala
Gly Ala Asn Tyr Gly Ser Gly Tyr 180 185
190 Cys Asp Ala Gln Cys Pro Val Gln Thr Trp Arg Asn Gly
Thr Leu Asn 195 200 205
Thr Ser His Gln Gly Phe Cys Cys Asn Glu Met Asp Ile Leu Glu Gly 210
215 220 Asn Ser Arg Ala
Asn Ala Leu Thr Pro His Ser Cys Thr Ala Thr Ala 225 230
235 240 Cys Asp Ser Ala Gly Cys Gly Phe Asn
Pro Tyr Gly Ser Gly Tyr Lys 245 250
255 Ser Tyr Tyr Gly Pro Gly Asp Thr Val Asp Thr Ser Lys Thr
Phe Thr 260 265 270
Ile Ile Thr Gln Phe Asn Thr Asp Asn Gly Ser Pro Ser Gly Asn Leu
275 280 285 Val Ser Ile Thr
Arg Lys Tyr Gln Gln Asn Gly Val Asp Ile Pro Ser 290
295 300 Ala Gln Pro Gly Gly Asp Thr Ile
Ser Ser Cys Pro Ser Ala Ser Ala 305 310
315 320 Tyr Gly Gly Leu Ala Thr Met Gly Lys Ala Leu Ser
Ser Gly Met Val 325 330
335 Leu Val Phe Ser Ile Trp Asn Asp Asn Ser Gln Tyr Met Asn Trp Leu
340 345 350 Asp Ser Gly
Asn Ala Gly Pro Cys Ser Ser Thr Glu Gly Asn Pro Ser 355
360 365 Asn Ile Leu Ala Asn Asn Pro Asn
Thr His Val Val Phe Ser Asn Ile 370 375
380 Arg Trp Gly Asp Ile Gly Ser Thr Thr Asn Ser Thr Ala
Pro Pro Pro 385 390 395
400 Pro Pro Ala Ser Ser Thr Thr Phe Ser Thr Thr Arg Arg Ser Ser Thr
405 410 415 Thr Ser Ser Ser
Pro Ser Cys Thr Gln Thr His Trp Gly Gln Cys Gly 420
425 430 Gly Ile Gly Tyr Ser Gly Cys Lys Thr
Cys Thr Ser Gly Thr Thr Cys 435 440
445 Gln Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu 450
455 461545DNATrichoderma reesei 46atgtatcgga
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 tgtaa
154547514PRTTrichoderma reesei 47Met Tyr Arg Lys Leu Ala Val Ile Ser Ala
Phe Leu Ala Thr Ala Arg 1 5 10
15 Ala Gln Ser Ala Cys Thr Leu Gln Ser Glu Thr His Pro Pro Leu
Thr 20 25 30 Trp
Gln Lys Cys Ser Ser Gly Gly Thr Cys Thr Gln Gln Thr Gly Ser 35
40 45 Val Val Ile Asp Ala Asn
Trp Arg Trp Thr His Ala Thr Asn Ser Ser 50 55
60 Thr Asn Cys Tyr Asp Gly Asn Thr Trp Ser Ser
Thr Leu Cys Pro Asp 65 70 75
80 Asn Glu Thr Cys Ala Lys Asn Cys Cys Leu Asp Gly Ala Ala Tyr Ala
85 90 95 Ser Thr
Tyr Gly Val Thr Thr Ser Gly Asn Ser Leu Ser Ile Gly Phe 100
105 110 Val Thr Gln Ser Ala Gln Lys
Asn Val Gly Ala Arg Leu Tyr Leu Met 115 120
125 Ala Ser Asp Thr Thr Tyr Gln Glu Phe Thr Leu Leu
Gly Asn Glu Phe 130 135 140
Ser Phe Asp Val Asp Val Ser Gln Leu Pro Cys Gly Leu Asn Gly Ala 145
150 155 160 Leu Tyr Phe
Val Ser Met Asp Ala Asp Gly Gly Val Ser Lys Tyr Pro 165
170 175 Thr Asn Thr Ala Gly Ala Lys Tyr
Gly Thr Gly Tyr Cys Asp Ser Gln 180 185
190 Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Gln Ala Asn
Val Glu Gly 195 200 205
Trp Glu Pro Ser Ser Asn Asn Ala Asn Thr Gly Ile Gly Gly His Gly 210
215 220 Ser Cys Cys Ser
Glu Met Asp Ile Trp Glu Ala Asn Ser Ile Ser Glu 225 230
235 240 Ala Leu Thr Pro His Pro Cys Thr Thr
Val Gly Gln Glu Ile Cys Glu 245 250
255 Gly Asp Gly Cys Gly Gly Thr Tyr Ser Asp Asn Arg Tyr Gly
Gly Thr 260 265 270
Cys Asp Pro Asp Gly Cys Asp Trp Asn Pro Tyr Arg Leu Gly Asn Thr
275 280 285 Ser Phe Tyr Gly
Pro Gly Ser Ser Phe Thr Leu Asp Thr Thr Lys Lys 290
295 300 Leu Thr Val Val Thr Gln Phe Glu
Thr Ser Gly Ala Ile Asn Arg Tyr 305 310
315 320 Tyr Val Gln Asn Gly Val Thr Phe Gln Gln Pro Asn
Ala Glu Leu Gly 325 330
335 Ser Tyr Ser Gly Asn Glu Leu Asn Asp Asp Tyr Cys Thr Ala Glu Glu
340 345 350 Ala Glu Phe
Gly Gly Ser Ser Phe Ser Asp Lys Gly Gly Leu Thr Gln 355
360 365 Phe Lys Lys Ala Thr Ser Gly Gly
Met Val Leu Val Met Ser Leu Trp 370 375
380 Asp Asp Tyr Tyr Ala Asn Met Leu Trp Leu Asp Ser Thr
Tyr Pro Thr 385 390 395
400 Asn Glu Thr Ser Ser Thr Pro Gly Ala Val Arg Gly Ser Cys Ser Thr
405 410 415 Ser Ser Gly Val
Pro Ala Gln Val Glu Ser Gln Ser Pro Asn Ala Lys 420
425 430 Val Thr Phe Ser Asn Ile Lys Phe Gly
Pro Ile Gly Ser Thr Gly Asn 435 440
445 Pro Ser Gly Gly Asn Pro Pro Gly Gly Asn Pro Pro Gly Thr
Thr Thr 450 455 460
Thr Arg Arg Pro Ala Thr Thr Thr Gly Ser Ser Pro Gly Pro Thr Gln 465
470 475 480 Ser His Tyr Gly Gln
Cys Gly Gly Ile Gly Tyr Ser Gly Pro Thr Val 485
490 495 Cys Ala Ser Gly Thr Thr Cys Gln Val Leu
Asn Pro Tyr Tyr Ser Gln 500 505
510 Cys Leu 481611DNATrichoderma reesei 48atgattgtcg gcattctcac
cacgctggct acgctggcca cactcgcagc tagtgtgcct 60ctagaggagc ggcaagcttg
ctcaagcgtc tggtaattat gtgaaccctc tcaagagacc 120caaatactga gatatgtcaa
ggggccaatg tggtggccag aattggtcgg gtccgacttg 180ctgtgcttcc ggaagcacat
gcgtctactc caacgactat tactcccagt gtcttcccgg 240cgctgcaagc tcaagctcgt
ccacgcgcgc cgcgtcgacg acttctcgag tatcccccac 300aacatcccgg tcgagctccg
cgacgcctcc acctggttct actactacca gagtacctcc 360agtcggatcg ggaaccgcta
cgtattcagg caaccctttt gttggggtca ctccttgggc 420caatgcatat tacgcctctg
aagttagcag cctcgctatt cctagcttga ctggagccat 480ggccactgct gcagcagctg
tcgcaaaggt tccctctttt atgtggctgt aggtcctccc 540ggaaccaagg caatctgtta
ctgaaggctc atcattcact gcagagatac tcttgacaag 600acccctctca tggagcaaac
cttggccgac atccgcaccg ccaacaagaa tggcggtaac 660tatgccggac agtttgtggt
gtatgacttg ccggatcgcg attgcgctgc ccttgcctcg 720aatggcgaat actctattgc
cgatggtggc gtcgccaaat ataagaacta tatcgacacc 780attcgtcaaa ttgtcgtgga
atattccgat atccggaccc tcctggttat tggtatgagt 840ttaaacacct gcctcccccc
ccccttccct tcctttcccg ccggcatctt gtcgttgtgc 900taactattgt tccctcttcc
agagcctgac tctcttgcca acctggtgac caacctcggt 960actccaaagt gtgccaatgc
tcagtcagcc taccttgagt gcatcaacta cgccgtcaca 1020cagctgaacc ttccaaatgt
tgcgatgtat ttggacgctg gccatgcagg atggcttggc 1080tggccggcaa accaagaccc
ggccgctcag ctatttgcaa atgtttacaa gaatgcatcg 1140tctccgagag ctcttcgcgg
attggcaacc aatgtcgcca actacaacgg gtggaacatt 1200accagccccc catcgtacac
gcaaggcaac gctgtctaca acgagaagct gtacatccac 1260gctattggac gtcttcttgc
caatcacggc tggtccaacg ccttcttcat cactgatcaa 1320ggtcgatcgg gaaagcagcc
taccggacag caacagtggg gagactggtg caatgtgatc 1380ggcaccggat ttggtattcg
cccatccgca aacactgggg actcgttgct ggattcgttt 1440gtctgggtca agccaggcgg
cgagtgtgac ggcaccagcg acagcagtgc gccacgattt 1500gactcccact gtgcgctccc
agatgccttg caaccggcgc ctcaagctgg tgcttggttc 1560caagcctact ttgtgcagct
tctcacaaac gcaaacccat cgttcctgta a 161149471PRTTrichoderma
reesei 49Met Ile Val Gly Ile Leu Thr Thr Leu Ala Thr Leu Ala Thr Leu Ala
1 5 10 15 Ala Ser
Val Pro Leu Glu Glu Arg Gln Ala Cys Ser Ser Val Trp Gly 20
25 30 Gln Cys Gly Gly Gln Asn Trp
Ser Gly Pro Thr Cys Cys Ala Ser Gly 35 40
45 Ser Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln
Cys Leu Pro Gly 50 55 60
Ala Ala Ser Ser Ser Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg 65
70 75 80 Val Ser Pro
Thr Thr Ser Arg Ser Ser Ser Ala Thr Pro Pro Pro Gly 85
90 95 Ser Thr Thr Thr Arg Val Pro Pro
Val Gly Ser Gly Thr Ala Thr Tyr 100 105
110 Ser Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn
Ala Tyr Tyr 115 120 125
Ala Ser Glu Val Ser Ser Leu Ala Ile Pro Ser Leu Thr Gly Ala Met 130
135 140 Ala Thr Ala Ala
Ala Ala Val Ala Lys Val Pro Ser Phe Met Trp Leu 145 150
155 160 Asp Thr Leu Asp Lys Thr Pro Leu Met
Glu Gln Thr Leu Ala Asp Ile 165 170
175 Arg Thr Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe
Val Val 180 185 190
Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu Ala Ser Asn Gly Glu
195 200 205 Tyr Ser Ile Ala
Asp Gly Gly Val Ala Lys Tyr Lys Asn Tyr Ile Asp 210
215 220 Thr Ile Arg Gln Ile Val Val Glu
Tyr Ser Asp Ile Arg Thr Leu Leu 225 230
235 240 Val Ile Glu Pro Asp Ser Leu Ala Asn Leu Val Thr
Asn Leu Gly Thr 245 250
255 Pro Lys Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Ile Asn Tyr
260 265 270 Ala Val Thr
Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala 275
280 285 Gly His Ala Gly Trp Leu Gly Trp
Pro Ala Asn Gln Asp Pro Ala Ala 290 295
300 Gln Leu Phe Ala Asn Val Tyr Lys Asn Ala Ser Ser Pro
Arg Ala Leu 305 310 315
320 Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn Gly Trp Asn Ile Thr
325 330 335 Ser Pro Pro Ser
Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu Lys Leu 340
345 350 Tyr Ile His Ala Ile Gly Arg Leu Leu
Ala Asn His Gly Trp Ser Asn 355 360
365 Ala Phe Phe Ile Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro
Thr Gly 370 375 380
Gln Gln Gln Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly 385
390 395 400 Ile Arg Pro Ser Ala
Asn Thr Gly Asp Ser Leu Leu Asp Ser Phe Val 405
410 415 Trp Val Lys Pro Gly Gly Glu Cys Asp Gly
Thr Ser Asp Ser Ser Ala 420 425
430 Pro Arg Phe Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro
Ala 435 440 445 Pro
Gln Ala Gly Ala Trp Phe Gln Ala Tyr Phe Val Gln Leu Leu Thr 450
455 460 Asn Ala Asn Pro Ser Phe
Leu 465 470 502046DNAHumicola insolens 50gccgtgacct
tgcgcgcttt gggtggcggt ggcgagtcgt ggacggtgct tgctggtcgc 60cggccttccc
ggcgatccgc gtgatgagag ggccaccaac ggcgggatga tgctccatgg 120ggaacttccc
catggagaag agagagaaac ttgcggagcc gtgatctggg gaaagatgct 180ccgtgtctcg
tctatataac tcgagtctcc ccgagccctc aacaccacca gctctgatct 240caccatcccc
atcgacaatc acgcaaacac agcagttgtc gggccattcc ttcagacaca 300tcagtcaccc
tccttcaaaa tgcgtaccgc caagttcgcc accctcgccg cccttgtggc 360ctcggccgcc
gcccagcagg cgtgcagtct caccaccgag aggcaccctt ccctctcttg 420gaacaagtgc
accgccggcg gccagtgcca gaccgtccag gcttccatca ctctcgactc 480caactggcgc
tggactcacc aggtgtctgg ctccaccaac tgctacacgg gcaacaagtg 540ggatactagc
atctgcactg atgccaagtc gtgcgctcag aactgctgcg tcgatggtgc 600cgactacacc
agcacctatg gcatcaccac caacggtgat tccctgagcc tcaagttcgt 660caccaagggc
cagcactcga ccaacgtcgg ctcgcgtacc tacctgatgg acggcgagga 720caagtatcag
agtacgttct atcttcagcc ttctcgcgcc ttgaatcctg gctaacgttt 780acacttcaca
gccttcgagc tcctcggcaa cgagttcacc ttcgatgtcg atgtctccaa 840catcggctgc
ggtctcaacg gcgccctgta cttcgtctcc atggacgccg atggtggtct 900cagccgctat
cctggcaaca aggctggtgc caagtacggt accggctact gcgatgctca 960gtgcccccgt
gacatcaagt tcatcaacgg cgaggccaac attgagggct ggaccggctc 1020caccaacgac
cccaacgccg gcgcgggccg ctatggtacc tgctgctctg agatggatat 1080ctgggaagcc
aacaacatgg ctactgcctt cactcctcac ccttgcacca tcattggcca 1140gagccgctgc
gagggcgact cgtgcggtgg cacctacagc aacgagcgct acgccggcgt 1200ctgcgacccc
gatggctgcg acttcaactc gtaccgccag ggcaacaaga ccttctacgg 1260caagggcatg
accgtcgaca ccaccaagaa gatcactgtc gtcacccagt tcctcaagga 1320tgccaacggc
gatctcggcg agatcaagcg cttctacgtc caggatggca agatcatccc 1380caactccgag
tccaccatcc ccggcgtcga gggcaattcc atcacccagg actggtgcga 1440ccgccagaag
gttgcctttg gcgacattga cgacttcaac cgcaagggcg gcatgaagca 1500gatgggcaag
gccctcgccg gccccatggt cctggtcatg tccatctggg atgaccacgc 1560ctccaacatg
ctctggctcg actcgacctt ccctgtcgat gccgctggca agcccggcgc 1620cgagcgcggt
gcctgcccga ccacctcggg tgtccctgct gaggttgagg ccgaggcccc 1680caacagcaac
gtcgtcttct ccaacatccg cttcggcccc atcggctcga ccgttgctgg 1740tctccccggc
gcgggcaacg gcggcaacaa cggcggcaac cccccgcccc ccaccaccac 1800cacctcctcg
gctccggcca ccaccaccac cgccagcgct ggccccaagg ctggccgctg 1860gcagcagtgc
ggcggcatcg gcttcactgg cccgacccag tgcgaggagc cctacatttg 1920caccaagctc
aacgactggt actctcagtg cctgtaaatt ctgagtcgct gactcgacga 1980tcacggccgg
tttttgcatg aaaggaaaca aacgaccgcg ataaaaatgg agggtaatga 2040gatgtc
204651525PRTHumicola insolens 51Met Arg Thr Ala Lys Phe Ala Thr Leu Ala
Ala Leu Val Ala Ser Ala 1 5 10
15 Ala Ala Gln Gln Ala Cys Ser Leu Thr Thr Glu Arg His Pro Ser
Leu 20 25 30 Ser
Trp Asn Lys Cys Thr Ala Gly Gly Gln Cys Gln Thr Val Gln Ala 35
40 45 Ser Ile Thr Leu Asp Ser
Asn Trp Arg Trp Thr His Gln Val Ser Gly 50 55
60 Ser Thr Asn Cys Tyr Thr Gly Asn Lys Trp Asp
Thr Ser Ile Cys Thr 65 70 75
80 Asp Ala Lys Ser Cys Ala Gln Asn Cys Cys Val Asp Gly Ala Asp Tyr
85 90 95 Thr Ser
Thr Tyr Gly Ile Thr Thr Asn Gly Asp Ser Leu Ser Leu Lys 100
105 110 Phe Val Thr Lys Gly Gln His
Ser Thr Asn Val Gly Ser Arg Thr Tyr 115 120
125 Leu Met Asp Gly Glu Asp Lys Tyr Gln Thr Phe Glu
Leu Leu Gly Asn 130 135 140
Glu Phe Thr Phe Asp Val Asp Val Ser Asn Ile Gly Cys Gly Leu Asn 145
150 155 160 Gly Ala Leu
Tyr Phe Val Ser Met Asp Ala Asp Gly Gly Leu Ser Arg 165
170 175 Tyr Pro Gly Asn Lys Ala Gly Ala
Lys Tyr Gly Thr Gly Tyr Cys Asp 180 185
190 Ala Gln Cys Pro Arg Asp Ile Lys Phe Ile Asn Gly Glu
Ala Asn Ile 195 200 205
Glu Gly Trp Thr Gly Ser Thr Asn Asp Pro Asn Ala Gly Ala Gly Arg 210
215 220 Tyr Gly Thr Cys
Cys Ser Glu Met Asp Ile Trp Glu Ala Asn Asn Met 225 230
235 240 Ala Thr Ala Phe Thr Pro His Pro Cys
Thr Ile Ile Gly Gln Ser Arg 245 250
255 Cys Glu Gly Asp Ser Cys Gly Gly Thr Tyr Ser Asn Glu Arg
Tyr Ala 260 265 270
Gly Val Cys Asp Pro Asp Gly Cys Asp Phe Asn Ser Tyr Arg Gln Gly
275 280 285 Asn Lys Thr Phe
Tyr Gly Lys Gly Met Thr Val Asp Thr Thr Lys Lys 290
295 300 Ile Thr Val Val Thr Gln Phe Leu
Lys Asp Ala Asn Gly Asp Leu Gly 305 310
315 320 Glu Ile Lys Arg Phe Tyr Val Gln Asp Gly Lys Ile
Ile Pro Asn Ser 325 330
335 Glu Ser Thr Ile Pro Gly Val Glu Gly Asn Ser Ile Thr Gln Asp Trp
340 345 350 Cys Asp Arg
Gln Lys Val Ala Phe Gly Asp Ile Asp Asp Phe Asn Arg 355
360 365 Lys Gly Gly Met Lys Gln Met Gly
Lys Ala Leu Ala Gly Pro Met Val 370 375
380 Leu Val Met Ser Ile Trp Asp Asp His Ala Ser Asn Met
Leu Trp Leu 385 390 395
400 Asp Ser Thr Phe Pro Val Asp Ala Ala Gly Lys Pro Gly Ala Glu Arg
405 410 415 Gly Ala Cys Pro
Thr Thr Ser Gly Val Pro Ala Glu Val Glu Ala Glu 420
425 430 Ala Pro Asn Ser Asn Val Val Phe Ser
Asn Ile Arg Phe Gly Pro Ile 435 440
445 Gly Ser Thr Val Ala Gly Leu Pro Gly Ala Gly Asn Gly Gly
Asn Asn 450 455 460
Gly Gly Asn Pro Pro Pro Pro Thr Thr Thr Thr Ser Ser Ala Pro Ala 465
470 475 480 Thr Thr Thr Thr Ala
Ser Ala Gly Pro Lys Ala Gly Arg Trp Gln Gln 485
490 495 Cys Gly Gly Ile Gly Phe Thr Gly Pro Thr
Gln Cys Glu Glu Pro Tyr 500 505
510 Ile Cys Thr Lys Leu Asn Asp Trp Tyr Ser Gln Cys Leu
515 520 525 521812DNAMyceliophthora
thermophila 52atggccaaga agcttttcat caccgccgcc cttgcggctg ccgtgttggc
ggcccccgtc 60attgaggagc gccagaactg cggcgctgtg tggtaagaaa gcccggtctg
agtttcccat 120gactttctca tcgagtaatg gcataaggcc caccccttcg actgactgtg
agaatcgatc 180aaatccagga ctcaatgcgg cggcaacggg tggcagggtc ccacatgctg
cgcctcgggc 240tcgacctgcg ttgcgcagaa cgagtggtac tctcagtgcc tgcccaacaa
tcaggtgacg 300agttccaaca ctccgtcgtc gacttccacc tcgcagcgca gcagcagcac
ctccagcagc 360agcaccagga gcggcagctc ctcctcctcc accaccacgc cccctcccgt
ctccagcccc 420gtgactagca ttcccggcgg tgcgaccacc acggcgagct actctggcaa
ccccttctcg 480ggcgtccggc tcttcgccaa cgactactac aggtccgagg tccacaatct
cgccattcct 540agcatgaccg gtactctggc ggccaaggct tccgccgtcg ccgaagtccc
tagcttccag 600tggctcgacc ggaacgtcac catcgacacc ctgatggtcc agactctgtc
ccagatccgg 660gctgccaata atgccggtgc caatcctccc tatgctggtg agttacatgg
cggcgacttg 720ccttctcgtc ccccaccttt cttgacggga tcggttacct gacctggagg
caaaacaaaa 780ccagcccaac ttgtcgtcta cgacctcccc gaccgtgact gcgccgccgc
tgcgtccaac 840ggcgagtttt cgattgcaaa cggcggcgcc gccaactaca ggagctacat
cgacgctatc 900cgcaagcaca tcattgagta ctcggacatc cggatcatcc tggttatcga
gcccgactcg 960atggccaaca tggtgaccaa catgaacgtg gccaagtgca gcaacgccgc
gtcgacgtac 1020cacgagttga ccgtgtacgc gctcaagcag ctgaacctgc ccaacgtcgc
catgtatctc 1080gacgccggcc acgccggctg gctcggctgg cccgccaaca tccagcccgc
cgccgacctg 1140tttgccggca tctacaatga cgccggcaag ccggctgccg tccgcggcct
ggccactaac 1200gtcgccaact acaacgcctg gagtatcgct tcggccccgt cgtacacgtc
ccctaaccct 1260aactacgacg agaagcacta catcgaggcc ttcagcccgc tcctgaacgc
ggccggcttc 1320cccgcacgct tcattgtcga cactggccgc aacggcaaac aacctaccgg
tatggttttt 1380ttcttttttt ttctctgttc ccctccccct tccccttcag ttggcgtcca
caaggtctct 1440tagtcttgct tcttctcgga ccaaccttcc cccaccccca aaacgcaccg
cccacaaccg 1500ttcgactcta tactcttggg aatgggcgcc gaaactgacc gttcgacagg
ccaacaacag 1560tggggtgact ggtgcaatgt caagggcact ggctttggcg tgcgcccgac
ggccaacacg 1620ggccacgacc tggtcgatgc ctttgtctgg gtcaagcccg gcggcgagtc
cgacggcaca 1680agcgacacca gcgccgcccg ctacgactac cactgcggcc tgtccgatgc
cctgcagcct 1740gctccggagg ctggacagtg gttccaggcc tacttcgagc agctgctcac
caacgccaac 1800ccgcccttct aa
181253482PRTMyceliophthora thermophila 53Met Ala Lys Lys Leu
Phe Ile Thr Ala Ala Leu Ala Ala Ala Val Leu 1 5
10 15 Ala Ala Pro Val Ile Glu Glu Arg Gln Asn
Cys Gly Ala Val Trp Thr 20 25
30 Gln Cys Gly Gly Asn Gly Trp Gln Gly Pro Thr Cys Cys Ala Ser
Gly 35 40 45 Ser
Thr Cys Val Ala Gln Asn Glu Trp Tyr Ser Gln Cys Leu Pro Asn 50
55 60 Asn Gln Val Thr Ser Ser
Asn Thr Pro Ser Ser Thr Ser Thr Ser Gln 65 70
75 80 Arg Ser Ser Ser Thr Ser Ser Ser Ser Thr Arg
Ser Gly Ser Ser Ser 85 90
95 Ser Ser Thr Thr Thr Pro Pro Pro Val Ser Ser Pro Val Thr Ser Ile
100 105 110 Pro Gly
Gly Ala Thr Thr Thr Ala Ser Tyr Ser Gly Asn Pro Phe Ser 115
120 125 Gly Val Arg Leu Phe Ala Asn
Asp Tyr Tyr Arg Ser Glu Val His Asn 130 135
140 Leu Ala Ile Pro Ser Met Thr Gly Thr Leu Ala Ala
Lys Ala Ser Ala 145 150 155
160 Val Ala Glu Val Pro Ser Phe Gln Trp Leu Asp Arg Asn Val Thr Ile
165 170 175 Asp Thr Leu
Met Val Gln Thr Leu Ser Gln Ile Arg Ala Ala Asn Asn 180
185 190 Ala Gly Ala Asn Pro Pro Tyr Ala
Ala Gln Leu Val Val Tyr Asp Leu 195 200
205 Pro Asp Arg Asp Cys Ala Ala Ala Ala Ser Asn Gly Glu
Phe Ser Ile 210 215 220
Ala Asn Gly Gly Ala Ala Asn Tyr Arg Ser Tyr Ile Asp Ala Ile Arg 225
230 235 240 Lys His Ile Ile
Glu Tyr Ser Asp Ile Arg Ile Ile Leu Val Ile Glu 245
250 255 Pro Asp Ser Met Ala Asn Met Val Thr
Asn Met Asn Val Ala Lys Cys 260 265
270 Ser Asn Ala Ala Ser Thr Tyr His Glu Leu Thr Val Tyr Ala
Leu Lys 275 280 285
Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly His Ala 290
295 300 Gly Trp Leu Gly Trp
Pro Ala Asn Ile Gln Pro Ala Ala Asp Leu Phe 305 310
315 320 Ala Gly Ile Tyr Asn Asp Ala Gly Lys Pro
Ala Ala Val Arg Gly Leu 325 330
335 Ala Thr Asn Val Ala Asn Tyr Asn Ala Trp Ser Ile Ala Ser Ala
Pro 340 345 350 Ser
Tyr Thr Ser Pro Asn Pro Asn Tyr Asp Glu Lys His Tyr Ile Glu 355
360 365 Ala Phe Ser Pro Leu Leu
Asn Ala Ala Gly Phe Pro Ala Arg Phe Ile 370 375
380 Val Asp Thr Gly Arg Asn Gly Lys Gln Pro Thr
Gly Gln Gln Gln Trp 385 390 395
400 Gly Asp Trp Cys Asn Val Lys Gly Thr Gly Phe Gly Val Arg Pro Thr
405 410 415 Ala Asn
Thr Gly His Asp Leu Val Asp Ala Phe Val Trp Val Lys Pro 420
425 430 Gly Gly Glu Ser Asp Gly Thr
Ser Asp Thr Ser Ala Ala Arg Tyr Asp 435 440
445 Tyr His Cys Gly Leu Ser Asp Ala Leu Gln Pro Ala
Pro Glu Ala Gly 450 455 460
Gln Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr Asn Ala Asn Pro 465
470 475 480 Pro Phe
541725DNATrichoderma reesei 54gagggcagct cacctgaaga ggcttgtaag atcaccctct
gtgtattgca ccatgattgt 60cggcattctc accacgctgg ctacgctggc cacactcgca
gctagtgtgc ctctagagga 120gcggcaagct tgctcaagcg tctggggcca atgtggtggc
cagaattggt cgggtccgac 180ttgctgtgct tccggaagca catgcgtcta ctccaacgac
tattactccc agtgtcttcc 240cggcgctgca agctcaagct cgtccacgcg cgccgcgtcg
acgacttctc gagtatcccc 300cacaacatcc cggtcgagct ccgcgacgcc tccacctggt
tctactacta ccagagtacc 360tccagtcgga tcgggaaccg ctacgtattc aggcaaccct
tttgttgggg tcactccttg 420ggccaatgca tattacgcct ctgaagttag cagcctcgct
attcctagct tgactggagc 480catggccact gctgcagcag ctgtcgcaaa ggttccctct
tttatgtggc tagatactct 540tgacaagacc cctctcatgg agcaaacctt ggccgacatc
cgcaccgcca acaagaatgg 600cggtaactat gccggacagt ttgtggtgta tgacttgccg
gatcgcgatt gcgctgccct 660tgcctcgaat ggcgaatact ctattgccga tggtggcgtc
gccaaatata agaactatat 720cgacaccatt cgtcaaattg tcgtggaata ttccgatatc
cggaccctcc tggttattga 780gcctgactct cttgccaacc tggtgaccaa cctcggtact
ccaaagtgtg ccaatgctca 840gtcagcctac cttgagtgca tcaactacgc cgtcacacag
ctgaaccttc caaatgttgc 900gatgtatttg gacgctggcc atgcaggatg gcttggctgg
ccggcaaacc aagacccggc 960cgctcagcta tttgcaaatg tttacaagaa tgcatcgtct
ccgagagctc ttcgcggatt 1020ggcaaccaat gtcgccaact acaacgggtg gaacattacc
agccccccat cgtacacgca 1080aggcaacgct gtctacaacg agaagctgta catccacgct
attggacctc ttcttgccaa 1140tcacggctgg tccaacgcct tcttcatcac tgatcaaggt
cgatcgggaa agcagcctac 1200cggacagcaa cagtggggag actggtgcaa tgtgatcggc
accggatttg gtattcgccc 1260atccgcaaac actggggact cgttgctgga ttcgtttgtc
tgggtcaagc caggcggcga 1320gtgtgacggc accagcgaca gcagtgcgcc acgatttgac
tcccactgtg cgctcccaga 1380tgccttgcaa ccggcgcctc aagctggtgc ttggttccaa
gcctactttg tgcagcttct 1440cacaaacgca aacccatcgt tcctgtaagg ctttcgtgac
cgggcttcaa acaatgatgt 1500gcgatggtgt ggttcccggt tggcggagtc tttgtctact
ttggttgtct gtcgcaggtc 1560ggtagaccgc aaatgagcaa ctgatggatt gttgccagcg
atactataat tcacatggat 1620ggtctttgtc gatcagtagc tagtgagaga gagagaacat
ctatccacaa tgtcgagtgt 1680ctattagaca tactccgaga aaaaaaaaaa aaaaaaaaaa
aaaaa 172555471PRTTrichoderma reesei 55Met Ile Val Gly
Ile Leu Thr Thr Leu Ala Thr Leu Ala Thr Leu Ala 1 5
10 15 Ala Ser Val Pro Leu Glu Glu Arg Gln
Ala Cys Ser Ser Val Trp Gly 20 25
30 Gln Cys Gly Gly Gln Asn Trp Ser Gly Pro Thr Cys Cys Ala
Ser Gly 35 40 45
Ser Thr Cys Val Tyr Ser Asn Asp Tyr Tyr Ser Gln Cys Leu Pro Gly 50
55 60 Ala Ala Ser Ser Ser
Ser Ser Thr Arg Ala Ala Ser Thr Thr Ser Arg 65 70
75 80 Val Ser Pro Thr Thr Ser Arg Ser Ser Ser
Ala Thr Pro Pro Pro Gly 85 90
95 Ser Thr Thr Thr Arg Val Pro Pro Val Gly Ser Gly Thr Ala Thr
Tyr 100 105 110 Ser
Gly Asn Pro Phe Val Gly Val Thr Pro Trp Ala Asn Ala Tyr Tyr 115
120 125 Ala Ser Glu Val Ser Ser
Leu Ala Ile Pro Ser Leu Thr Gly Ala Met 130 135
140 Ala Thr Ala Ala Ala Ala Val Ala Lys Val Pro
Ser Phe Met Trp Leu 145 150 155
160 Asp Thr Leu Asp Lys Thr Pro Leu Met Glu Gln Thr Leu Ala Asp Ile
165 170 175 Arg Thr
Ala Asn Lys Asn Gly Gly Asn Tyr Ala Gly Gln Phe Val Val 180
185 190 Tyr Asp Leu Pro Asp Arg Asp
Cys Ala Ala Leu Ala Ser Asn Gly Glu 195 200
205 Tyr Ser Ile Ala Asp Gly Gly Val Ala Lys Tyr Lys
Asn Tyr Ile Asp 210 215 220
Thr Ile Arg Gln Ile Val Val Glu Tyr Ser Asp Ile Arg Thr Leu Leu 225
230 235 240 Val Ile Glu
Pro Asp Ser Leu Ala Asn Leu Val Thr Asn Leu Gly Thr 245
250 255 Pro Lys Cys Ala Asn Ala Gln Ser
Ala Tyr Leu Glu Cys Ile Asn Tyr 260 265
270 Ala Val Thr Gln Leu Asn Leu Pro Asn Val Ala Met Tyr
Leu Asp Ala 275 280 285
Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Gln Asp Pro Ala Ala 290
295 300 Gln Leu Phe Ala
Asn Val Tyr Lys Asn Ala Ser Ser Pro Arg Ala Leu 305 310
315 320 Arg Gly Leu Ala Thr Asn Val Ala Asn
Tyr Asn Gly Trp Asn Ile Thr 325 330
335 Ser Pro Pro Ser Tyr Thr Gln Gly Asn Ala Val Tyr Asn Glu
Lys Leu 340 345 350
Tyr Ile His Ala Ile Gly Pro Leu Leu Ala Asn His Gly Trp Ser Asn
355 360 365 Ala Phe Phe Ile
Thr Asp Gln Gly Arg Ser Gly Lys Gln Pro Thr Gly 370
375 380 Gln Gln Gln Trp Gly Asp Trp Cys
Asn Val Ile Gly Thr Gly Phe Gly 385 390
395 400 Ile Arg Pro Ser Ala Asn Thr Gly Asp Ser Leu Leu
Asp Ser Phe Val 405 410
415 Trp Val Lys Pro Gly Gly Glu Cys Asp Gly Thr Ser Asp Ser Ser Ala
420 425 430 Pro Arg Phe
Asp Ser His Cys Ala Leu Pro Asp Ala Leu Gln Pro Ala 435
440 445 Pro Gln Ala Gly Ala Trp Phe Gln
Ala Tyr Phe Val Gln Leu Leu Thr 450 455
460 Asn Ala Asn Pro Ser Phe Leu 465 470
561446DNAThielavia terrestris 56atggctcaga 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
144657481PRTThielavia terrestris 57Met
Ala Gln Lys Leu Leu Leu Ala Ala Ala Leu Ala Ala Ser Ala Leu 1
5 10 15 Ala Ala Pro Val Val Glu
Glu Arg Gln Asn Cys Gly Ser Val Trp Ser 20
25 30 Gln Cys Gly Gly Ile Gly Trp Ser Gly Ala
Thr Cys Cys Ala Ser Gly 35 40
45 Asn Thr Cys Val Glu Leu Asn Pro Tyr Tyr Ser Gln Cys Leu
Pro Asn 50 55 60
Ser Gln Val Thr Thr Ser Thr Ser Lys Thr Thr Ser Thr Thr Thr Arg 65
70 75 80 Ser Ser Thr Thr Ser
His Ser Ser Gly Pro Thr Ser Thr Ser Thr Thr 85
90 95 Thr Thr Ser Ser Pro Val Val Thr Thr Pro
Pro Ser Thr Ser Ile Pro 100 105
110 Gly Gly Ala Ser Ser Thr Ala Ser Trp Ser Gly Asn Pro Phe Ser
Gly 115 120 125 Val
Gln Met Trp Ala Asn Asp Tyr Tyr Ala Ser Glu Val Ser Ser Leu 130
135 140 Ala Ile Pro Ser Met Thr
Gly Ala Met Ala Thr Lys Ala Ala Glu Val 145 150
155 160 Ala Lys Val Pro Ser Phe Gln Trp Leu Asp Arg
Asn Val Thr Ile Asp 165 170
175 Thr Leu Phe Ala His Thr Leu Ser Gln Ile Arg Ala Ala Asn Gln Lys
180 185 190 Gly Ala
Asn Pro Pro Tyr Ala Gly Ile Phe Val Val Tyr Asp Leu Pro 195
200 205 Asp Arg Asp Cys Ala Ala Ala
Ala Ser Asn Gly Glu Phe Ser Ile Ala 210 215
220 Asn Asn Gly Ala Ala Asn Tyr Lys Thr Tyr Ile Asp
Ala Ile Arg Ser 225 230 235
240 Leu Val Ile Gln Tyr Ser Asp Ile Arg Ile Ile Phe Val Ile Glu Pro
245 250 255 Asp Ser Leu
Ala Asn Met Val Thr Asn Leu Asn Val Ala Lys Cys Ala 260
265 270 Asn Ala Glu Ser Thr Tyr Lys Glu
Leu Thr Val Tyr Ala Leu Gln Gln 275 280
285 Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp Ala Gly
His Ala Gly 290 295 300
Trp Leu Gly Trp Pro Ala Asn Ile Gln Pro Ala Ala Asn Leu Phe Ala 305
310 315 320 Glu Ile Tyr Thr
Ser Ala Gly Lys Pro Ala Ala Val Arg Gly Leu Ala 325
330 335 Thr Asn Val Ala Asn Tyr Asn Gly Trp
Ser Leu Ala Thr Pro Pro Ser 340 345
350 Tyr Thr Gln Gly Asp Pro Asn Tyr Asp Glu Ser His Tyr Val
Gln Ala 355 360 365
Leu Ala Pro Leu Leu Thr Ala Asn Gly Phe Pro Ala His Phe Ile Thr 370
375 380 Asp Thr Gly Arg Asn
Gly Lys Gln Pro Thr Gly Gln Arg Gln Trp Gly 385 390
395 400 Asp Trp Cys Asn Val Ile Gly Thr Gly Phe
Gly Val Arg Pro Thr Thr 405 410
415 Asn Thr Gly Leu Asp Ile Glu Asp Ala Phe Val Trp Val Lys Pro
Gly 420 425 430 Gly
Glu Cys Asp Gly Thr Ser Asn Thr Thr Ser Pro Arg Tyr Asp Tyr 435
440 445 His Cys Gly Leu Ser Asp
Ala Leu Gln Pro Ala Pro Glu Ala Gly Thr 450 455
460 Trp Phe Gln Ala Tyr Phe Glu Gln Leu Leu Thr
Asn Ala Asn Pro Pro 465 470 475
480 Phe 581593DNAChaetomium thermophilum 58atgatgtaca agaagttcgc
cgctctcgcc gccctcgtgg ctggcgccgc cgcccagcag 60gcttgctccc tcaccactga
gacccacccc agactcactt ggaagcgctg cacctctggc 120ggcaactgct cgaccgtgaa
cggcgccgtc accatcgatg ccaactggcg ctggactcac 180actgtttccg gctcgaccaa
ctgctacacc ggcaacgagt gggatacctc catctgctct 240gatggcaaga gctgcgccca
gacctgctgc gtcgacggcg ctgactactc ttcgacctat 300ggtatcacca ccagcggtga
ctccctgaac ctcaagttcg tcaccaagca ccagcacggc 360accaatgtcg gctctcgtgt
ctacctgatg gagaacgaca ccaagtacca gatgttcgag 420ctcctcggca acgagttcac
cttcgatgtc gatgtctcta acctgggctg cggtctcaac 480ggcgccctct acttcgtctc
catggacgct gatggtggta tgagcaagta ctctggcaac 540aaggctggcg ccaagtacgg
taccggctac tgcgatgctc agtgcccgcg cgaccttaag 600ttcatcaacg gcgaggccaa
cattgagaac tggacccctt cgaccaatga tgccaacgcc 660ggtttcggcc gctatggcag
ctgctgctct gagatggata tctgggatgc caacaacatg 720gctactgcct tcactcctca
cccttgcacc attatcggcc agagccgctg cgagggcaac 780agctgcggtg gcacctacag
ctctgagcgc tatgctggtg tttgcgatcc tgatggctgc 840gacttcaacg cctaccgcca
gggcgacaag accttctacg gcaagggcat gaccgtcgac 900accaccaaga agatgaccgt
cgtcacccag ttccacaaga actcggctgg cgtcctcagc 960gagatcaagc gcttctacgt
tcaggacggc aagatcattg ccaacgccga gtccaagatc 1020cccggcaacc ccggcaactc
catcacccag gagtggtgcg atgcccagaa ggtcgccttc 1080ggtgacatcg atgacttcaa
ccgcaagggc ggtatggctc agatgagcaa ggccctcgag 1140ggccctatgg tcctggtcat
gtccgtctgg gatgaccact acgccaacat gctctggctc 1200gactcgacct accccattga
caaggccggc acccccggcg ccgagcgcgg tgcttgcccg 1260accacctccg gtgtccctgc
cgagattgag gcccaggtcc ccaacagcaa cgttatcttc 1320tccaacatcc gcttcggccc
catcggctcg accgtccctg gcctcgacgg cagcaccccc 1380agcaacccga ccgccaccgt
tgctcctccc acttctacca ccaccagcgt gagaagcagc 1440actactcaga tttccacccc
gactagccag cccggcggct gcaccaccca gaagtggggc 1500cagtgcggtg gtatcggcta
caccggctgc actaactgcg ttgctggcac tacctgcact 1560gagctcaacc cctggtacag
ccagtgcctg taa 159359530PRTChaetomium
thermophilum 59Met Met Tyr Lys Lys Phe Ala Ala Leu Ala Ala Leu Val Ala
Gly Ala 1 5 10 15
Ala Ala Gln Gln Ala Cys Ser Leu Thr Thr Glu Thr His Pro Arg Leu
20 25 30 Thr Trp Lys Arg Cys
Thr Ser Gly Gly Asn Cys Ser Thr Val Asn Gly 35
40 45 Ala Val Thr Ile Asp Ala Asn Trp Arg
Trp Thr His Thr Val Ser Gly 50 55
60 Ser Thr Asn Cys Tyr Thr Gly Asn Glu Trp Asp Thr Ser
Ile Cys Ser 65 70 75
80 Asp Gly Lys Ser Cys Ala Gln Thr Cys Cys Val Asp Gly Ala Asp Tyr
85 90 95 Ser Ser Thr Tyr
Gly Ile Thr Thr Ser Gly Asp Ser Leu Asn Leu Lys 100
105 110 Phe Val Thr Lys His Gln His Gly Thr
Asn Val Gly Ser Arg Val Tyr 115 120
125 Leu Met Glu Asn Asp Thr Lys Tyr Gln Met Phe Glu Leu Leu
Gly Asn 130 135 140
Glu Phe Thr Phe Asp Val Asp Val Ser Asn Leu Gly Cys Gly Leu Asn 145
150 155 160 Gly Ala Leu Tyr Phe
Val Ser Met Asp Ala Asp Gly Gly Met Ser Lys 165
170 175 Tyr Ser Gly Asn Lys Ala Gly Ala Lys Tyr
Gly Thr Gly Tyr Cys Asp 180 185
190 Ala Gln Cys Pro Arg Asp Leu Lys Phe Ile Asn Gly Glu Ala Asn
Ile 195 200 205 Glu
Asn Trp Thr Pro Ser Thr Asn Asp Ala Asn Ala Gly Phe Gly Arg 210
215 220 Tyr Gly Ser Cys Cys Ser
Glu Met Asp Ile Trp Asp Ala Asn Asn Met 225 230
235 240 Ala Thr Ala Phe Thr Pro His Pro Cys Thr Ile
Ile Gly Gln Ser Arg 245 250
255 Cys Glu Gly Asn Ser Cys Gly Gly Thr Tyr Ser Ser Glu Arg Tyr Ala
260 265 270 Gly Val
Cys Asp Pro Asp Gly Cys Asp Phe Asn Ala Tyr Arg Gln Gly 275
280 285 Asp Lys Thr Phe Tyr Gly Lys
Gly Met Thr Val Asp Thr Thr Lys Lys 290 295
300 Met Thr Val Val Thr Gln Phe His Lys Asn Ser Ala
Gly Val Leu Ser 305 310 315
320 Glu Ile Lys Arg Phe Tyr Val Gln Asp Gly Lys Ile Ile Ala Asn Ala
325 330 335 Glu Ser Lys
Ile Pro Gly Asn Pro Gly Asn Ser Ile Thr Gln Glu Trp 340
345 350 Cys Asp Ala Gln Lys Val Ala Phe
Gly Asp Ile Asp Asp Phe Asn Arg 355 360
365 Lys Gly Gly Met Ala Gln Met Ser Lys Ala Leu Glu Gly
Pro Met Val 370 375 380
Leu Val Met Ser Val Trp Asp Asp His Tyr Ala Asn Met Leu Trp Leu 385
390 395 400 Asp Ser Thr Tyr
Pro Ile Asp Lys Ala Gly Thr Pro Gly Ala Glu Arg 405
410 415 Gly Ala Cys Pro Thr Thr Ser Gly Val
Pro Ala Glu Ile Glu Ala Gln 420 425
430 Val Pro Asn Ser Asn Val Ile Phe Ser Asn Ile Arg Phe Gly
Pro Ile 435 440 445
Gly Ser Thr Val Pro Gly Leu Asp Gly Ser Thr Pro Ser Asn Pro Thr 450
455 460 Ala Thr Val Ala Pro
Pro Thr Ser Thr Thr Thr Ser Val Arg Ser Ser 465 470
475 480 Thr Thr Gln Ile Ser Thr Pro Thr Ser Gln
Pro Gly Gly Cys Thr Thr 485 490
495 Gln Lys Trp Gly Gln Cys Gly Gly Ile Gly Tyr Thr Gly Cys Thr
Asn 500 505 510 Cys
Val Ala Gly Thr Thr Cys Thr Glu Leu Asn Pro Trp Tyr Ser Gln 515
520 525 Cys Leu 530
601434DNAChaetomium thermophilum 60atggctaagc agctgctgct cactgccgct
cttgcggcca cttcgctggc tgcccctctc 60cttgaggagc gccagagctg ctcctccgtc
tggggtcaat gcggtggcat caattacaac 120ggcccgacct gctgccagtc cggcagtgtt
tgcacttacc tgaatgactg gtacagccag 180tgcattcccg gtcaggctca gcccggcacg
actagcacca cggctcggac caccagcacc 240agcaccacca gcacttcgtc ggtccgcccg
accacctcga atacccctgt gacgactgct 300cccccgacga ccaccatccc gggcggcgcc
tcgagcacgg ccagctacaa cggcaacccg 360ttttcgggtg ttcaactttg ggccaacacc
tactactcgt ccgaggtgca cactttggcc 420atccccagct tgtctcctga gctggctgcc
aaggccgcca aggtcgctga ggttcccagc 480ttccagtggc tcgaccgcaa tgtgactgtt
gacactctct tctccggcac tcttgccgaa 540atccgcgccg ccaaccagcg cggtgccaac
ccgccttatg ccggcatttt cgtggtttat 600gacttaccag accgtgattg cgcggctgct
gcttcgaacg gcgagtggtc tatcgccaac 660aatggtgcca acaactacaa gcgctacatc
gaccggatcc gtgagctcct tatccagtac 720tccgatatcc gcactattct ggtcattgaa
cctgattccc tggccaacat ggtcaccaac 780atgaacgtcc agaagtgctc gaacgctgcc
tccacttaca aggagcttac tgtctatgcc 840ctcaaacagc tcaatcttcc tcacgttgcc
atgtacatgg atgctggcca cgctggctgg 900cttggctggc ccgccaacat ccagcctgct
gctgagctct ttgctcaaat ctaccgcgac 960gctggcaggc ccgctgctgt ccgcggtctt
gcgaccaacg ttgccaacta caatgcttgg 1020tcgatcgcca gccctccgtc ctacacctct
cctaacccga actacgacga gaagcactat 1080attgaggcct ttgctcctct tctccgcaac
cagggcttcg acgcaaagtt catcgtcgac 1140accggccgta acggcaagca gcccactggc
cagcttgaat ggggtcactg gtgcaatgtc 1200aagggaactg gcttcggtgt gcgccctact
gctaacactg ggcatgaact tgttgatgct 1260ttcgtgtggg tcaagcccgg tggcgagtcc
gacggcacca gtgcggacac cagcgctgct 1320cgttatgact atcactgcgg cctttccgac
gcactgactc cggcgcctga ggctggccaa 1380tggttccagg cttatttcga acagctgctc
atcaatgcca accctccgct ctga 143461477PRTChaetomium thermophilum
61Met Ala Lys Gln Leu Leu Leu Thr Ala Ala Leu Ala Ala Thr Ser Leu 1
5 10 15 Ala Ala Pro Leu
Leu Glu Glu Arg Gln Ser Cys Ser Ser Val Trp Gly 20
25 30 Gln Cys Gly Gly Ile Asn Tyr Asn Gly
Pro Thr Cys Cys Gln Ser Gly 35 40
45 Ser Val Cys Thr Tyr Leu Asn Asp Trp Tyr Ser Gln Cys Ile
Pro Gly 50 55 60
Gln Ala Gln Pro Gly Thr Thr Ser Thr Thr Ala Arg Thr Thr Ser Thr 65
70 75 80 Ser Thr Thr Ser Thr
Ser Ser Val Arg Pro Thr Thr Ser Asn Thr Pro 85
90 95 Val Thr Thr Ala Pro Pro Thr Thr Thr Ile
Pro Gly Gly Ala Ser Ser 100 105
110 Thr Ala Ser Tyr Asn Gly Asn Pro Phe Ser Gly Val Gln Leu Trp
Ala 115 120 125 Asn
Thr Tyr Tyr Ser Ser Glu Val His Thr Leu Ala Ile Pro Ser Leu 130
135 140 Ser Pro Glu Leu Ala Ala
Lys Ala Ala Lys Val Ala Glu Val Pro Ser 145 150
155 160 Phe Gln Trp Leu Asp Arg Asn Val Thr Val Asp
Thr Leu Phe Ser Gly 165 170
175 Thr Leu Ala Glu Ile Arg Ala Ala Asn Gln Arg Gly Ala Asn Pro Pro
180 185 190 Tyr Ala
Gly Ile Phe Val Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala 195
200 205 Ala Ala Ala Ser Asn Gly Glu
Trp Ser Ile Ala Asn Asn Gly Ala Asn 210 215
220 Asn Tyr Lys Arg Tyr Ile Asp Arg Ile Arg Glu Leu
Leu Ile Gln Tyr 225 230 235
240 Ser Asp Ile Arg Thr Ile Leu Val Ile Glu Pro Asp Ser Leu Ala Asn
245 250 255 Met Val Thr
Asn Met Asn Val Gln Lys Cys Ser Asn Ala Ala Ser Thr 260
265 270 Tyr Lys Glu Leu Thr Val Tyr Ala
Leu Lys Gln Leu Asn Leu Pro His 275 280
285 Val Ala Met Tyr Met Asp Ala Gly His Ala Gly Trp Leu
Gly Trp Pro 290 295 300
Ala Asn Ile Gln Pro Ala Ala Glu Leu Phe Ala Gln Ile Tyr Arg Asp 305
310 315 320 Ala Gly Arg Pro
Ala Ala Val Arg Gly Leu Ala Thr Asn Val Ala Asn 325
330 335 Tyr Asn Ala Trp Ser Ile Ala Ser Pro
Pro Ser Tyr Thr Ser Pro Asn 340 345
350 Pro Asn Tyr Asp Glu Lys His Tyr Ile Glu Ala Phe Ala Pro
Leu Leu 355 360 365
Arg Asn Gln Gly Phe Asp Ala Lys Phe Ile Val Asp Thr Gly Arg Asn 370
375 380 Gly Lys Gln Pro Thr
Gly Gln Leu Glu Trp Gly His Trp Cys Asn Val 385 390
395 400 Lys Gly Thr Gly Phe Gly Val Arg Pro Thr
Ala Asn Thr Gly His Glu 405 410
415 Leu Val Asp Ala Phe Val Trp Val Lys Pro Gly Gly Glu Ser Asp
Gly 420 425 430 Thr
Ser Ala Asp Thr Ser Ala Ala Arg Tyr Asp Tyr His Cys Gly Leu 435
440 445 Ser Asp Ala Leu Thr Pro
Ala Pro Glu Ala Gly Gln Trp Phe Gln Ala 450 455
460 Tyr Phe Glu Gln Leu Leu Ile Asn Ala Asn Pro
Pro Leu 465 470 475
622586DNAAspergillus oryzae 62atgaagcttg 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
258663861PRTAspergillus oryzae 63Met Lys Leu Gly
Trp Ile Glu Val Ala Ala Leu Ala Ala Ala Ser Val 1 5
10 15 Val Ser Ala Lys Asp Asp Leu Ala Tyr
Ser Pro Pro Phe Tyr Pro Ser 20 25
30 Pro Trp Ala Asp Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys
Arg Ala 35 40 45
Val Asp Ile Val Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr 50
55 60 Thr Gly Thr Gly Trp
Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser 65 70
75 80 Val Pro Arg Leu Asn Ile Pro Ser Leu Cys
Leu Gln Asp Ser Pro Leu 85 90
95 Gly Ile Arg Phe Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val
Asn 100 105 110 Val
Ala Ala Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala 115
120 125 Met Gly Glu Glu Phe Ser
Asp Lys Gly Ile Asp Val Gln Leu Gly Pro 130 135
140 Ala Ala Gly Pro Leu Gly Ala His Pro Asp Gly
Gly Arg Asn Trp Glu 145 150 155
160 Gly Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr
165 170 175 Ile Lys
Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180
185 190 Ile Met Asn Glu Gln Glu His
Phe Arg Gln Gln Pro Glu Ala Ala Gly 195 200
205 Tyr Gly Phe Asn Val Ser Asp Ser Leu Ser Ser Asn
Val Asp Asp Lys 210 215 220
Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala 225
230 235 240 Gly Val Gly
Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245
250 255 Gly Cys Glu Asn Ser Glu Thr Leu
Asn Lys Leu Leu Lys Ala Glu Leu 260 265
270 Gly Phe Gln Gly Phe Val Met Ser Asp Trp Thr Ala His
His Ser Gly 275 280 285
Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val 290
295 300 Thr Phe Asp Ser
Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly 305 310
315 320 Val Leu Asn Gly Thr Ile Pro Gln Trp
Arg Val Asp Asp Met Ala Val 325 330
335 Arg Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys
Tyr Thr 340 345 350
Pro Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His
355 360 365 Asn His Val Ser
Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp 370
375 380 Val Gln Arg Asp His Ala Asp Leu
Ile Arg Arg Ile Gly Ala Gln Ser 385 390
395 400 Thr Val Leu Leu Lys Asn Lys Gly Ala Leu Pro Leu
Ser Arg Lys Glu 405 410
415 Lys Leu Val Ala Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly
420 425 430 Ala Asn Gly
Cys Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met 435
440 445 Ala Trp Gly Ser Gly Thr Ala Asn
Phe Pro Tyr Leu Val Thr Pro Glu 450 455
460 Gln Ala Ile Gln Asn Glu Val Leu Gln Gly Arg Gly Asn
Val Phe Ala 465 470 475
480 Val Thr Asp Ser Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln
485 490 495 Ala Ser Val Ser
Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr 500
505 510 Leu Ser Val Asp Gly Asn Glu Gly Asp
Arg Asn Asn Ile Thr Leu Trp 515 520
525 Lys Asn Gly Asp Asn Val Val Lys Thr Ala Ala Asn Asn Cys
Asn Asn 530 535 540
Thr Val Val Ile Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp 545
550 555 560 Tyr Asp His Pro Asn
Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly 565
570 575 Gln Glu Ser Gly Asn Ser Ile Ala Asp Val
Leu Tyr Gly Arg Val Asn 580 585
590 Pro Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser
Tyr 595 600 605 Gly
Ser Pro Leu Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln 610
615 620 Ser Asp Phe Thr Gln Gly
Val Phe Ile Asp Tyr Arg His Phe Asp Lys 625 630
635 640 Phe Asn Glu Thr Pro Ile Tyr Glu Phe Gly Tyr
Gly Leu Ser Tyr Thr 645 650
655 Thr Phe Glu Leu Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg
660 665 670 Tyr Thr
Pro Thr Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu 675
680 685 Ile Gly Asp Ala Ser Glu Tyr
Val Tyr Pro Glu Gly Leu Glu Arg Ile 690 695
700 His Glu Phe Ile Tyr Pro Trp Ile Asn Ser Thr Asp
Leu Lys Ala Ser 705 710 715
720 Ser Asp Asp Ser Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu
725 730 735 Gly Ala Thr
Asp Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly 740
745 750 Ala Gly Gly Asn Pro Gly Leu Tyr
Glu Asp Leu Phe Arg Val Ser Val 755 760
765 Lys Val Lys Asn Thr Gly Asn Val Ala Gly Asp Glu Val
Pro Gln Leu 770 775 780
Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys 785
790 795 800 Phe Glu Arg Ile
His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr 805
810 815 Thr Leu Thr Arg Arg Asp Leu Ala Asn
Trp Asp Val Ser Ala Gln Asp 820 825
830 Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val Gly Asn
Ser Ser 835 840 845
Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 850
855 860 643060DNAAspergillus fumigatus 64atgagattcg
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
306065863PRTAspergillus fumigatus 65Met Arg Phe Gly Trp Leu Glu Val Ala
Ala Leu Thr Ala Ala Ser Val 1 5 10
15 Ala Asn Ala Gln Glu Leu Ala Phe Ser Pro Pro Phe Tyr Pro
Ser Pro 20 25 30
Trp Ala Asp Gly Gln Gly Glu Trp Ala Asp Ala His Arg Arg Ala Val
35 40 45 Glu Ile Val Ser
Gln Met Thr Leu Ala Glu Lys Val Asn Leu Thr Thr 50
55 60 Gly Thr Gly Trp Glu Met Asp Arg
Cys Val Gly Gln Thr Gly Ser Val 65 70
75 80 Pro Arg Leu Gly Ile Asn Trp Gly Leu Cys Gly Gln
Asp Ser Pro Leu 85 90
95 Gly Ile Arg Phe Ser Asp Leu Asn Ser Ala Phe Pro Ala Gly Thr Asn
100 105 110 Val Ala Ala
Thr Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Lys Ala 115
120 125 Met Gly Glu Glu Phe Asn Asp Lys
Gly Val Asp Ile Leu Leu Gly Pro 130 135
140 Ala Ala Gly Pro Leu Gly Lys Tyr Pro Asp Gly Gly Arg
Ile Trp Glu 145 150 155
160 Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Val Leu Phe Ala Glu Thr
165 170 175 Ile Lys Gly Ile
Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr 180
185 190 Ile Leu Asn Glu Gln Glu His Phe Arg
Gln Val Gly Glu Ala Gln Gly 195 200
205 Tyr Gly Tyr Asn Ile Thr Glu Thr Ile Ser Ser Asn Val Asp
Asp Lys 210 215 220
Thr Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala 225
230 235 240 Gly Val Gly Ala Val
Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245
250 255 Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys
Leu Leu Lys Ala Glu Leu 260 265
270 Gly Phe Gln Gly Phe Val Met Ser Asp Trp Ser Ala His His Ser
Gly 275 280 285 Val
Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile 290
295 300 Ser Phe Asp Asp Gly Leu
Ser Phe Trp Gly Thr Asn Leu Thr Val Ser 305 310
315 320 Val Leu Asn Gly Thr Val Pro Ala Trp Arg Val
Asp Asp Met Ala Val 325 330
335 Arg Ile Met Thr Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Arg Ile
340 345 350 Pro Pro
Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Trp Glu His 355
360 365 Ser Ala Val Ser Glu Gly Ala
Trp Thr Lys Val Asn Asp Phe Val Asn 370 375
380 Val Gln Arg Ser His Ser Gln Ile Ile Arg Glu Ile
Gly Ala Ala Ser 385 390 395
400 Thr Val Leu Leu Lys Asn Thr Gly Ala Leu Pro Leu Thr Gly Lys Glu
405 410 415 Val Lys Val
Gly Val Leu Gly Glu Asp Ala Gly Ser Asn Pro Trp Gly 420
425 430 Ala Asn Gly Cys Pro Asp Arg Gly
Cys Asp Asn Gly Thr Leu Ala Met 435 440
445 Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr Leu Val
Thr Pro Glu 450 455 460
Gln Ala Ile Gln Arg Glu Val Ile Ser Asn Gly Gly Asn Val Phe Ala 465
470 475 480 Val Thr Asp Asn
Gly Ala Leu Ser Gln Met Ala Asp Val Ala Ser Gln 485
490 495 Ser Ser Val Ser Leu Val Phe Val Asn
Ala Asp Ser Gly Glu Gly Phe 500 505
510 Ile Ser Val Asp Gly Asn Glu Gly Asp Arg Lys Asn Leu Thr
Leu Trp 515 520 525
Lys Asn Gly Glu Ala Val Ile Asp Thr Val Val Ser His Cys Asn Asn 530
535 540 Thr Ile Val Val Ile
His Ser Val Gly Pro Val Leu Ile Asp Arg Trp 545 550
555 560 Tyr Asp Asn Pro Asn Val Thr Ala Ile Ile
Trp Ala Gly Leu Pro Gly 565 570
575 Gln Glu Ser Gly Asn Ser Leu Val Asp Val Leu Tyr Gly Arg Val
Asn 580 585 590 Pro
Ser Ala Lys Thr Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr 595
600 605 Gly Ala Pro Leu Leu Thr
Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln 610 615
620 Asp Asp Phe Asn Glu Gly Val Phe Ile Asp Tyr
Arg His Phe Asp Lys 625 630 635
640 Arg Asn Glu Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr
645 650 655 Thr Phe
Gly Tyr Ser His Leu Arg Val Gln Ala Leu Asn Ser Ser Ser 660
665 670 Ser Ala Tyr Val Pro Thr Ser
Gly Glu Thr Lys Pro Ala Pro Thr Tyr 675 680
685 Gly Glu Ile Gly Ser Ala Ala Asp Tyr Leu Tyr Pro
Glu Gly Leu Lys 690 695 700
Arg Ile Thr Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Glu 705
710 715 720 Asp Ser Ser
Asp Asp Pro Asn Tyr Gly Trp Glu Asp Ser Glu Tyr Ile 725
730 735 Pro Glu Gly Ala Arg Asp Gly Ser
Pro Gln Pro Leu Leu Lys Ala Gly 740 745
750 Gly Ala Pro Gly Gly Asn Pro Thr Leu Tyr Gln Asp Leu
Val Arg Val 755 760 765
Ser Ala Thr Ile Thr Asn Thr Gly Asn Val Ala Gly Tyr Glu Val Pro 770
775 780 Gln Leu Tyr Val
Ser Leu Gly Gly Pro Asn Glu Pro Arg Val Val Leu 785 790
795 800 Arg Lys Phe Asp Arg Ile Phe Leu Ala
Pro Gly Glu Gln Lys Val Trp 805 810
815 Thr Thr Thr Leu Asn Arg Arg Asp Leu Ala Asn Trp Asp Val
Glu Ala 820 825 830
Gln Asp Trp Val Ile Thr Lys Tyr Pro Lys Lys Val His Val Gly Ser
835 840 845 Ser Ser Arg Lys
Leu Pro Leu Arg Ala Pro Leu Pro Arg Val Tyr 850 855
860 662800DNAPenicillium brasilianum
66tgaaaatgca 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
280067878PRTPenicillium brasilianum 67Met Gln Gly Ser Thr Ile Phe Leu Ala
Phe Ala Ser Trp Ala Ser Gln 1 5 10
15 Val Ala Ala Ile Ala Gln Pro Ile Gln Lys His Glu Pro Gly
Phe Leu 20 25 30
His Gly Pro Gln Ala Ile Glu Ser Phe Ser Glu Pro Phe Tyr Pro Ser
35 40 45 Pro Trp Met Asn
Pro His Ala Glu Gly Trp Glu Ala Ala Tyr Gln Lys 50
55 60 Ala Gln Asp Phe Val Ser Gln Leu
Thr Ile Leu Glu Lys Ile Asn Leu 65 70
75 80 Thr Thr Gly Val Gly Trp Glu Asn Gly Pro Cys Val
Gly Asn Thr Gly 85 90
95 Ser Ile Pro Arg Leu Gly Phe Lys Gly Phe Cys Thr Gln Asp Ser Pro
100 105 110 Gln Gly Val
Arg Phe Ala Asp Tyr Ser Ser Ala Phe Thr Ser Ser Gln 115
120 125 Met Ala Ala Ala Thr Phe Asp Arg
Ser Ile Leu Tyr Gln Arg Gly Gln 130 135
140 Ala Met Ala Gln Glu His Lys Ala Lys Gly Ile Thr Ile
Gln Leu Gly 145 150 155
160 Pro Val Ala Gly Pro Leu Gly Arg Ile Pro Glu Gly Gly Arg Asn Trp
165 170 175 Glu Gly Phe Ser
Pro Asp Pro Val Leu Thr Gly Ile Ala Met Ala Glu 180
185 190 Thr Ile Lys Gly Met Gln Asp Thr Gly
Val Ile Ala Cys Ala Lys His 195 200
205 Tyr Ile Gly Asn Glu Gln Glu His Phe Arg Gln Val Gly Glu
Ala Ala 210 215 220
Gly His Gly Tyr Thr Ile Ser Asp Thr Ile Ser Ser Asn Ile Asp Asp 225
230 235 240 Arg Ala Met His Glu
Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg 245
250 255 Ala Gly Val Gly Ser Phe Met Cys Ser Tyr
Ser Gln Ile Asn Asn Ser 260 265
270 Tyr Gly Cys Gln Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ser
Glu 275 280 285 Leu
Gly Phe Gln Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser 290
295 300 Gly Val Ser Ser Ala Leu
Ala Gly Leu Asp Met Ser Met Pro Gly Asp 305 310
315 320 Thr Glu Phe Asp Ser Gly Leu Ser Phe Trp Gly
Ser Asn Leu Thr Ile 325 330
335 Ala Ile Leu Asn Gly Thr Val Pro Glu Trp Arg Leu Asp Asp Met Ala
340 345 350 Met Arg
Ile Met Ala Ala Tyr Phe Lys Val Gly Leu Thr Ile Glu Asp 355
360 365 Gln Pro Asp Val Asn Phe Asn
Ala Trp Thr His Asp Thr Tyr Gly Tyr 370 375
380 Lys Tyr Ala Tyr Ser Lys Glu Asp Tyr Glu Gln Val
Asn Trp His Val 385 390 395
400 Asp Val Arg Ser Asp His Asn Lys Leu Ile Arg Glu Thr Ala Ala Lys
405 410 415 Gly Thr Val
Leu Leu Lys Asn Asn Phe His Ala Leu Pro Leu Lys Gln 420
425 430 Pro Arg Phe Val Ala Val Val Gly
Gln Asp Ala Gly Pro Asn Pro Lys 435 440
445 Gly Pro Asn Gly Cys Ala Asp Arg Gly Cys Asp Gln Gly
Thr Leu Ala 450 455 460
Met Gly Trp Gly Ser Gly Ser Thr Glu Phe Pro Tyr Leu Val Thr Pro 465
470 475 480 Asp Thr Ala Ile
Gln Ser Lys Val Leu Glu Tyr Gly Gly Arg Tyr Glu 485
490 495 Ser Ile Phe Asp Asn Tyr Asp Asp Asn
Ala Ile Leu Ser Leu Val Ser 500 505
510 Gln Pro Asp Ala Thr Cys Ile Val Phe Ala Asn Ala Asp Ser
Gly Glu 515 520 525
Gly Tyr Ile Thr Val Asp Asn Asn Trp Gly Asp Arg Asn Asn Leu Thr 530
535 540 Leu Trp Gln Asn Ala
Asp Gln Val Ile Ser Thr Val Ser Ser Arg Cys 545 550
555 560 Asn Asn Thr Ile Val Val Leu His Ser Val
Gly Pro Val Leu Leu Asn 565 570
575 Gly Ile Tyr Glu His Pro Asn Ile Thr Ala Ile Val Trp Ala Gly
Met 580 585 590 Pro
Gly Glu Glu Ser Gly Asn Ala Leu Val Asp Ile Leu Trp Gly Asn 595
600 605 Val Asn Pro Ala Gly Arg
Thr Pro Phe Thr Trp Ala Lys Ser Arg Glu 610 615
620 Asp Tyr Gly Thr Asp Ile Met Tyr Glu Pro Asn
Asn Gly Gln Arg Ala 625 630 635
640 Pro Gln Gln Asp Phe Thr Glu Ser Ile Tyr Leu Asp Tyr Arg His Phe
645 650 655 Asp Lys
Ala Gly Ile Glu Pro Ile Tyr Glu Phe Gly Phe Gly Leu Ser 660
665 670 Tyr Thr Thr Phe Glu Tyr Ser
Asp Leu Arg Val Val Lys Lys Tyr Val 675 680
685 Gln Pro Tyr Ser Pro Thr Thr Gly Thr Gly Ala Gln
Ala Pro Ser Ile 690 695 700
Gly Gln Pro Pro Ser Gln Asn Leu Asp Thr Tyr Lys Phe Pro Ala Thr 705
710 715 720 Tyr Lys Tyr
Ile Lys Thr Phe Ile Tyr Pro Tyr Leu Asn Ser Thr Val 725
730 735 Ser Leu Arg Ala Ala Ser Lys Asp
Pro Glu Tyr Gly Arg Thr Asp Phe 740 745
750 Ile Pro Pro His Ala Arg Asp Gly Ser Pro Gln Pro Leu
Asn Pro Ala 755 760 765
Gly Asp Pro Val Ala Ser Gly Gly Asn Asn Met Leu Tyr Asp Glu Leu 770
775 780 Tyr Glu Val Thr
Ala Gln Ile Lys Asn Thr Gly Asp Val Ala Gly Asp 785 790
795 800 Glu Val Val Gln Leu Tyr Val Asp Leu
Gly Gly Asp Asn Pro Pro Arg 805 810
815 Gln Leu Arg Asn Phe Asp Arg Phe Tyr Leu Leu Pro Gly Gln
Ser Ser 820 825 830
Thr Phe Arg Ala Thr Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Ile 835
840 845 Glu Ala Gln Asn Trp Arg Val
Thr Glu Ser Pro Lys Arg Val Tyr Val 850
855 860Gly Arg Ser Ser Arg Asp Leu Pro Leu Ser Ser Gln
Leu Glu 865 870 875
682583DNAAspergillus niger 68atgaggttca 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
258369860PRTAspergillus niger 69Met Arg
Phe Thr Leu Ile Glu Ala Val Ala Leu Thr Ala Val Ser Leu 1 5
10 15 Ala Ser Ala Asp Glu Leu Ala
Tyr Ser Pro Pro Tyr Tyr Pro Ser Pro 20 25
30 Trp Ala Asn Gly Gln Gly Asp Trp Ala Gln Ala Tyr
Gln Arg Ala Val 35 40 45
Asp Ile Val Ser Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr
50 55 60 Gly Thr Gly
Trp Glu Leu Glu Leu Cys Val Gly Gln Thr Gly Gly Val 65
70 75 80 Pro Arg Leu Gly Val Pro Gly
Met Cys Leu Gln Asp Ser Pro Leu Gly 85
90 95 Val Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro
Ala Gly Met Asn Val 100 105
110 Ala Ala Thr Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Lys Ala
Met 115 120 125 Gly
Gln Glu Phe Ser Asp Lys Gly Ala Asp Ile Gln Leu Gly Pro Ala 130
135 140 Ala Gly Pro Leu Gly Arg
Ser Pro Asp Gly Gly Arg Asn Trp Glu Gly 145 150
155 160 Phe Ser Pro Asp Pro Ala Leu Ser Gly Val Leu
Phe Ala Glu Thr Ile 165 170
175 Lys Gly Ile Gln Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile
180 185 190 Ala Tyr
Glu Gln Glu His Phe Arg Gln Ala Pro Glu Ala Gln Gly Phe 195
200 205 Gly Phe Asn Ile Ser Glu Ser
Gly Ser Ala Asn Leu Asp Asp Lys Thr 210 215
220 Met His Glu Leu Tyr Leu Trp Pro Phe Ala Asp Ala
Ile Arg Ala Gly 225 230 235
240 Ala Gly Ala Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly
245 250 255 Cys Gln Asn
Ser Tyr Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly 260
265 270 Phe Gln Gly Phe Val Met Ser Asp
Trp Ala Ala His His Ala Gly Val 275 280
285 Ser Gly Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly
Asp Val Asp 290 295 300
Tyr Asp Ser Gly Thr Ser Tyr Trp Gly Thr Asn Leu Thr Ile Ser Val 305
310 315 320 Leu Asn Gly Thr
Val Pro Gln Trp Arg Val Asp Asp Met Ala Val Arg 325
330 335 Ile Met Ala Ala Tyr Tyr Lys Val Gly
Arg Asp Arg Leu Trp Thr Pro 340 345
350 Pro Asn Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Tyr Lys
Tyr Tyr 355 360 365
Tyr Val Ser Glu Gly Pro Tyr Glu Lys Val Asn Gln Tyr Val Asn Val 370
375 380 Gln Arg Asn His Ser
Glu Leu Ile Arg Arg Ile Gly Ala Asp Ser Thr 385 390
395 400 Val Leu Leu Lys Asn Asp Gly Ala Leu Pro
Leu Thr Gly Lys Glu Arg 405 410
415 Leu Val Ala Leu Ile Gly Glu Asp Ala Gly Ser Asn Pro Tyr Gly
Ala 420 425 430 Asn
Gly Cys Ser Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Gly 435
440 445 Trp Gly Ser Gly Thr Ala
Asn Phe Pro Tyr Leu Val Thr Pro Glu Gln 450 455
460 Ala Ile Ser Asn Glu Val Leu Lys His Lys Asn
Gly Val Phe Thr Ala 465 470 475
480 Thr Asp Asn Trp Ala Ile Asp Gln Ile Glu Ala Leu Ala Lys Thr Ala
485 490 495 Ser Val
Ser Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Tyr Ile 500
505 510 Asn Val Asp Gly Asn Leu Gly
Asp Arg Arg Asn Leu Thr Leu Trp Arg 515 520
525 Asn Gly Asp Asn Val Ile Lys Ala Ala Ala Ser Asn
Cys Asn Asn Thr 530 535 540
Ile Val Val Ile His Ser Val Gly Pro Val Leu Val Asn Glu Trp Tyr 545
550 555 560 Asp Asn Pro
Asn Val Thr Ala Ile Leu Trp Gly Gly Leu Pro Gly Gln 565
570 575 Glu Ser Gly Asn Ser Leu Ala Asp
Val Leu Tyr Gly Arg Val Asn Pro 580 585
590 Gly Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu
Ala Tyr Gln 595 600 605
Asp Tyr Leu Val Thr Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln Glu 610
615 620 Asp Phe Val Glu
Gly Val Phe Ile Asp Tyr Arg Gly Phe Asp Lys Arg 625 630
635 640 Asn Glu Thr Pro Ile Tyr Glu Phe Gly
Tyr Gly Leu Ser Tyr Thr Thr 645 650
655 Phe Asn Tyr Ser Asn Leu Glu Val Gln Val Leu Ser Ala Pro
Ala Tyr 660 665 670
Glu Pro Ala Ser Gly Glu Thr Glu Ala Ala Pro Thr Phe Gly Glu Val
675 680 685 Gly Asn Ala Ser
Asp Tyr Leu Tyr Pro Ser Gly Leu Gln Arg Ile Thr 690
695 700 Lys Phe Ile Tyr Pro Trp Leu Asn
Gly Thr Asp Leu Glu Ala Ser Ser 705 710
715 720 Gly Asp Ala Ser Tyr Gly Gln Asp Ser Ser Asp Tyr
Leu Pro Glu Gly 725 730
735 Ala Thr Asp Gly Ser Ala Gln Pro Ile Leu Pro Ala Gly Gly Gly Pro
740 745 750 Gly Gly Asn
Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val Ser Val Thr 755
760 765 Ile Lys Asn Thr Gly Lys Val Ala
Gly Asp Glu Val Pro Gln Leu Tyr 770 775
780 Val Ser Leu Gly Gly Pro Asn Glu Pro Lys Ile Val Leu
Arg Gln Phe 785 790 795
800 Glu Arg Ile Thr Leu Gln Pro Ser Glu Glu Thr Lys Trp Ser Thr Thr
805 810 815 Leu Thr Arg Arg
Asp Leu Ala Asn Trp Asn Val Glu Lys Gln Asp Trp 820
825 830 Glu Ile Thr Ser Tyr Pro Lys Met Val
Phe Val Gly Ser Ser Ser Arg 835 840
845 Lys Leu Pro Leu Arg Ala Ser Leu Pro Thr Val His 850
855 860 702583DNAAspergillus aculeatus
70atgaagctca 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
258371860PRTAspergillus aculeatus 71Met Lys Leu Ser Trp Leu Glu Ala Ala
Ala Leu Thr Ala Ala Ser Val 1 5 10
15 Val Ser Ala Asp Glu Leu Ala Phe Ser Pro Pro Phe Tyr Pro
Ser Pro 20 25 30
Trp Ala Asn Gly Gln Gly Glu Trp Ala Glu Ala Tyr Gln Arg Ala Val
35 40 45 Ala Ile Val Ser
Gln Met Thr Leu Asp Glu Lys Val Asn Leu Thr Thr 50
55 60 Gly Thr Gly Trp Glu Leu Glu Lys
Cys Val Gly Gln Thr Gly Gly Val 65 70
75 80 Pro Arg Leu Asn Ile Gly Gly Met Cys Leu Gln Asp
Ser Pro Leu Gly 85 90
95 Ile Arg Asp Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val
100 105 110 Ala Ala Thr
Trp Asp Lys Asn Leu Ala Tyr Leu Arg Gly Gln Ala Met 115
120 125 Gly Gln Glu Phe Ser Asp Lys Gly
Ile Asp Val Gln Leu Gly Pro Ala 130 135
140 Ala Gly Pro Leu Gly Arg Ser Pro Asp Gly Gly Arg Asn
Trp Glu Gly 145 150 155
160 Phe Ser Pro Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr Ile
165 170 175 Lys Gly Ile Gln
Asp Ala Gly Val Val Ala Thr Ala Lys His Tyr Ile 180
185 190 Leu Asn Glu Gln Glu His Phe Arg Gln
Val Ala Glu Ala Ala Gly Tyr 195 200
205 Gly Phe Asn Ile Ser Asp Thr Ile Ser Ser Asn Val Asp Asp
Lys Thr 210 215 220
Ile His Glu Met Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly 225
230 235 240 Val Gly Ala Ile Met
Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly 245
250 255 Cys Gln Asn Ser Tyr Thr Leu Asn Lys Leu
Leu Lys Ala Glu Leu Gly 260 265
270 Phe Gln Gly Phe Val Met Ser Asp Trp Gly Ala His His Ser Gly
Val 275 280 285 Gly
Ser Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile Thr 290
295 300 Phe Asp Ser Ala Thr Ser
Phe Trp Gly Thr Asn Leu Thr Ile Ala Val 305 310
315 320 Leu Asn Gly Thr Val Pro Gln Trp Arg Val Asp
Asp Met Ala Val Arg 325 330
335 Ile Met Ala Ala Tyr Tyr Lys Val Gly Arg Asp Arg Leu Tyr Gln Pro
340 345 350 Pro Asn
Phe Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Lys Tyr Phe 355
360 365 Tyr Pro Gln Glu Gly Pro Tyr
Glu Lys Val Asn His Phe Val Asn Val 370 375
380 Gln Arg Asn His Ser Glu Val Ile Arg Lys Leu Gly
Ala Asp Ser Thr 385 390 395
400 Val Leu Leu Lys Asn Asn Asn Ala Leu Pro Leu Thr Gly Lys Glu Arg
405 410 415 Lys Val Ala
Ile Leu Gly Glu Asp Ala Gly Ser Asn Ser Tyr Gly Ala 420
425 430 Asn Gly Cys Ser Asp Arg Gly Cys
Asp Asn Gly Thr Leu Ala Met Ala 435 440
445 Trp Gly Ser Gly Thr Ala Glu Phe Pro Tyr Leu Val Thr
Pro Glu Gln 450 455 460
Ala Ile Gln Ala Glu Val Leu Lys His Lys Gly Ser Val Tyr Ala Ile 465
470 475 480 Thr Asp Asn Trp
Ala Leu Ser Gln Val Glu Thr Leu Ala Lys Gln Ala 485
490 495 Ser Val Ser Leu Val Phe Val Asn Ser
Asp Ala Gly Glu Gly Tyr Ile 500 505
510 Ser Val Asp Gly Asn Glu Gly Asp Arg Asn Asn Leu Thr Leu
Trp Lys 515 520 525
Asn Gly Asp Asn Leu Ile Lys Ala Ala Ala Asn Asn Cys Asn Asn Thr 530
535 540 Ile Val Val Ile His
Ser Val Gly Pro Val Leu Val Asp Glu Trp Tyr 545 550
555 560 Asp His Pro Asn Val Thr Ala Ile Leu Trp
Ala Gly Leu Pro Gly Gln 565 570
575 Glu Ser Gly Asn Ser Leu Ala Asp Val Leu Tyr Gly Arg Val Asn
Pro 580 585 590 Gly
Ala Lys Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ala Tyr Gly 595
600 605 Asp Tyr Leu Val Arg Glu
Leu Asn Asn Gly Asn Gly Ala Pro Gln Asp 610 615
620 Asp Phe Ser Glu Gly Val Phe Ile Asp Tyr Arg
Gly Phe Asp Lys Arg 625 630 635
640 Asn Glu Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr Thr
645 650 655 Phe Asn
Tyr Ser Gly Leu His Ile Gln Val Leu Asn Ala Ser Ser Asn 660
665 670 Ala Gln Val Ala Thr Glu Thr
Gly Ala Ala Pro Thr Phe Gly Gln Val 675 680
685 Gly Asn Ala Ser Asp Tyr Val Tyr Pro Glu Gly Leu
Thr Arg Ile Ser 690 695 700
Lys Phe Ile Tyr Pro Trp Leu Asn Ser Thr Asp Leu Lys Ala Ser Ser 705
710 715 720 Gly Asp Pro
Tyr Tyr Gly Val Asp Thr Ala Glu His Val Pro Glu Gly 725
730 735 Ala Thr Asp Gly Ser Pro Gln Pro
Val Leu Pro Ala Gly Gly Gly Ser 740 745
750 Gly Gly Asn Pro Arg Leu Tyr Asp Glu Leu Ile Arg Val
Ser Val Thr 755 760 765
Val Lys Asn Thr Gly Arg Val Ala Gly Asp Ala Val Pro Gln Leu Tyr 770
775 780 Val Ser Leu Gly
Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe 785 790
795 800 Asp Arg Leu Thr Leu Lys Pro Ser Glu
Glu Thr Val Trp Thr Thr Thr 805 810
815 Leu Thr Arg Arg Asp Leu Ser Asn Trp Asp Val Ala Ala Gln
Asp Trp 820 825 830
Val Ile Thr Ser Tyr Pro Lys Lys Val His Val Gly Ser Ser Ser Arg
835 840 845 Gln Leu Pro Leu
His Ala Ala Leu Pro Lys Val Gln 850 855
860 723294DNAAspergillus oryzae 72atgcgttcct 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 3294731097PRTAspergillus oryzae 73Met
Arg Ser Ser Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro 1
5 10 15 Val Leu Ala Leu Ala Ala
Asp Gly Arg Ser Thr Arg Tyr Trp Asp Cys 20
25 30 Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys
Ala Pro Val Asn Gln Pro 35 40
45 Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe
Asp Ala 50 55 60
Lys Ser Gly Cys Glu Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln 65
70 75 80 Thr Pro Trp Ala Val
Asn Asp Asp Phe Ala Leu Gly Phe Ala Ala Thr 85
90 95 Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp
Cys Cys Ala Cys Tyr Glu 100 105
110 Leu Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val
Gln 115 120 125 Ser
Thr Ser Thr Gly Gly Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130
135 140 Ile Pro Gly Gly Gly Val
Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe 145 150
155 160 Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile
Ser Ser Arg Asn Glu 165 170
175 Cys Asp Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
180 185 190 Asp Trp
Phe Lys Asn Ala Asp Asn Pro Ser Phe Ser Phe Arg Gln Val 195
200 205 Gln Cys Pro Ala Glu Leu Val
Ala Arg Thr Gly Cys Arg Arg Asn Asp 210 215
220 Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Met Arg
Ser Ser Pro Leu 225 230 235
240 Leu Arg Ser Ala Val Val Ala Ala Leu Pro Val Leu Ala Leu Ala Lys
245 250 255 Asp Asp Leu
Ala Tyr Ser Pro Pro Phe Tyr Pro Ser Pro Trp Ala Asp 260
265 270 Gly Gln Gly Glu Trp Ala Glu Val
Tyr Lys Arg Ala Val Asp Ile Val 275 280
285 Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr Thr
Gly Thr Gly 290 295 300
Trp Gln Leu Glu Arg Cys Val Gly Gln Thr Gly Ser Val Pro Arg Leu 305
310 315 320 Asn Ile Pro Ser
Leu Cys Leu Gln Asp Ser Pro Leu Gly Ile Arg Phe 325
330 335 Ser Asp Tyr Asn Ser Ala Phe Pro Ala
Gly Val Asn Val Ala Ala Thr 340 345
350 Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala Met Gly
Glu Glu 355 360 365
Phe Ser Asp Lys Gly Ile Asp Val Gln Leu Gly Pro Ala Ala Gly Pro 370
375 380 Leu Gly Ala His Pro
Asp Gly Gly Arg Asn Trp Glu Gly Phe Ser Pro 385 390
395 400 Asp Pro Ala Leu Thr Gly Val Leu Phe Ala
Glu Thr Ile Lys Gly Ile 405 410
415 Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr Ile Met Asn
Glu 420 425 430 Gln
Glu His Phe Arg Gln Gln Pro Glu Ala Ala Gly Tyr Gly Phe Asn 435
440 445 Val Ser Asp Ser Leu Ser
Ser Asn Val Asp Asp Lys Thr Met His Glu 450 455
460 Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg
Ala Gly Val Gly Ala 465 470 475
480 Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Glu Asn
485 490 495 Ser Glu
Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly 500
505 510 Phe Val Met Ser Asp Trp Thr
Ala His His Ser Gly Val Gly Ala Ala 515 520
525 Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val
Thr Phe Asp Ser 530 535 540
Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly Val Leu Asn Gly 545
550 555 560 Thr Ile Pro
Gln Trp Arg Val Asp Asp Met Ala Val Arg Ile Met Ala 565
570 575 Ala Tyr Tyr Lys Val Gly Arg Asp
Thr Lys Tyr Thr Pro Pro Asn Phe 580 585
590 Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His Asn
His Val Ser 595 600 605
Glu Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp Val Gln Arg Asp 610
615 620 His Ala Asp Leu
Ile Arg Arg Ile Gly Ala Gln Ser Thr Val Leu Leu 625 630
635 640 Lys Asn Lys Gly Ala Leu Pro Leu Ser
Arg Lys Glu Lys Leu Val Ala 645 650
655 Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly Ala Asn
Gly Cys 660 665 670
Asp Asp Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala Trp Gly Ser
675 680 685 Gly Thr Ala Asn
Phe Pro Tyr Leu Val Thr Pro Glu Gln Ala Ile Gln 690
695 700 Asn Glu Val Leu Gln Gly Arg Gly
Asn Val Phe Ala Val Thr Asp Ser 705 710
715 720 Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln
Ala Ser Val Ser 725 730
735 Leu Val Phe Val Asn Ser Asp Ser Gly Glu Gly Tyr Leu Ser Val Asp
740 745 750 Gly Asn Glu
Gly Asp Arg Asn Asn Ile Thr Leu Trp Lys Asn Gly Asp 755
760 765 Asn Val Val Lys Thr Ala Ala Asn
Asn Cys Asn Asn Thr Val Val Ile 770 775
780 Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp Tyr
Asp His Pro 785 790 795
800 Asn Val Thr Gly Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly
805 810 815 Asn Ser Ile Ala
Asp Val Leu Tyr Gly Arg Val Asn Pro Gly Ala Lys 820
825 830 Ser Pro Phe Thr Trp Gly Lys Thr Arg
Glu Ser Tyr Gly Ser Pro Leu 835 840
845 Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp
Phe Thr 850 855 860
Gln Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys Phe Asn Glu Thr 865
870 875 880 Pro Ile Tyr Glu Phe
Gly Tyr Gly Leu Ser Tyr Thr Thr Phe Glu Leu 885
890 895 Ser Asp Leu His Val Gln Pro Leu Asn Ala
Ser Arg Tyr Thr Pro Thr 900 905
910 Ser Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu Ile Gly Asp
Ala 915 920 925 Ser
Glu Tyr Val Tyr Pro Glu Gly Leu Glu Arg Ile His Glu Phe Ile 930
935 940 Tyr Pro Trp Ile Asn Ser
Thr Asp Leu Lys Ala Ser Ser Asp Asp Ser 945 950
955 960 Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro
Glu Gly Ala Thr Asp 965 970
975 Gly Ser Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly Ala Gly Gly Asn
980 985 990 Pro Gly
Leu Tyr Glu Asp Leu Phe Arg Val Ser Val Lys Val Lys Asn 995
1000 1005 Thr Gly Asn Val Ala
Gly Asp Glu Val Pro Gln Leu Tyr Val Ser 1010 1015
1020 Leu Gly Gly Pro Asn Glu Pro Lys Val Val
Leu Arg Lys Phe Glu 1025 1030 1035
Arg Ile His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr Thr
1040 1045 1050 Leu Thr
Arg Arg Asp Leu Ala Asn Trp Asp Val Ser Ala Gln Asp 1055
1060 1065 Trp Thr Val Thr Pro Tyr Pro
Lys Thr Ile Tyr Val Gly Asn Ser 1070 1075
1080 Ser Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys
Ala Gln 1085 1090 1095
743294DNAAspergillus oryzae 74atgcgttcct 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 3294751097PRTAspergillus oryzae 75Met Arg Ser Ser
Pro Leu Leu Arg Ser Ala Val Val Ala Ala Leu Pro 1 5
10 15 Val Leu Ala Leu Ala Ala Asp Gly Arg
Ser Thr Arg Tyr Trp Asp Cys 20 25
30 Cys Lys Pro Ser Cys Gly Trp Ala Lys Lys Ala Pro Val Asn
Gln Pro 35 40 45
Val Phe Ser Cys Asn Ala Asn Phe Gln Arg Ile Thr Asp Phe Asp Ala 50
55 60 Lys Ser Gly Cys Glu
Pro Gly Gly Val Ala Tyr Ser Cys Ala Asp Gln 65 70
75 80 Thr Pro Trp Ala Val Asn Asp Asp Phe Ala
Leu Gly Phe Ala Ala Thr 85 90
95 Ser Ile Ala Gly Ser Asn Glu Ala Gly Trp Cys Cys Ala Cys Tyr
Glu 100 105 110 Leu
Thr Phe Thr Ser Gly Pro Val Ala Gly Lys Lys Met Val Val Gln 115
120 125 Ser Thr Ser Thr Gly Gly
Asp Leu Gly Ser Asn His Phe Asp Leu Asn 130 135
140 Ile Pro Gly Gly Gly Val Gly Ile Phe Asp Gly
Cys Thr Pro Gln Phe 145 150 155
160 Gly Gly Leu Pro Gly Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu
165 170 175 Cys Asp
Arg Phe Pro Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe 180
185 190 Asp Trp Phe Lys Asn Ala Asp
Asn Pro Ser Phe Ser Phe Arg Gln Val 195 200
205 Gln Cys Pro Ala Glu Leu Val Ala Arg Thr Gly Cys
Arg Arg Asn Asp 210 215 220
Asp Gly Asn Phe Pro Ala Val Gln Ile Pro Met Arg Ser Ser Pro Leu 225
230 235 240 Leu Arg Ser
Ala Val Val Ala Ala Leu Pro Val Leu Ala Leu Ala Lys 245
250 255 Asp Asp Leu Ala Tyr Ser Pro Pro
Phe Tyr Pro Ser Pro Trp Ala Asp 260 265
270 Gly Gln Gly Glu Trp Ala Glu Val Tyr Lys Arg Ala Val
Asp Ile Val 275 280 285
Ser Gln Met Thr Leu Thr Glu Lys Val Asn Leu Thr Thr Gly Thr Gly 290
295 300 Trp Gln Leu Glu
Arg Cys Val Gly Gln Thr Gly Ser Val Pro Arg Leu 305 310
315 320 Asn Ile Pro Ser Leu Cys Leu Gln Asp
Ser Pro Leu Gly Ile Arg Phe 325 330
335 Ser Asp Tyr Asn Ser Ala Phe Pro Ala Gly Val Asn Val Ala
Ala Thr 340 345 350
Trp Asp Lys Thr Leu Ala Tyr Leu Arg Gly Gln Ala Met Gly Glu Glu
355 360 365 Phe Ser Asp Lys
Gly Ile Asp Val Gln Leu Gly Pro Ala Ala Gly Pro 370
375 380 Leu Gly Ala His Pro Asp Gly Gly
Arg Asn Trp Glu Ser Phe Ser Pro 385 390
395 400 Asp Pro Ala Leu Thr Gly Val Leu Phe Ala Glu Thr
Ile Lys Gly Ile 405 410
415 Gln Asp Ala Gly Val Ile Ala Thr Ala Lys His Tyr Ile Met Asn Glu
420 425 430 Gln Glu His
Phe Arg Gln Gln Pro Glu Ala Ala Gly Tyr Gly Phe Asn 435
440 445 Val Ser Asp Ser Leu Ser Ser Asn
Val Asp Asp Lys Thr Met His Glu 450 455
460 Leu Tyr Leu Trp Pro Phe Ala Asp Ala Val Arg Ala Gly
Val Gly Ala 465 470 475
480 Val Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr Gly Cys Glu Asn
485 490 495 Ser Glu Thr Leu
Asn Lys Leu Leu Lys Ala Glu Leu Gly Phe Gln Gly 500
505 510 Phe Val Met Ser Asp Trp Thr Ala Gln
His Ser Gly Val Gly Ala Ala 515 520
525 Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Val Thr Phe
Asp Ser 530 535 540
Gly Thr Ser Phe Trp Gly Ala Asn Leu Thr Val Gly Val Leu Asn Gly 545
550 555 560 Thr Ile Pro Gln Trp
Arg Val Asp Asp Met Ala Val Arg Ile Met Ala 565
570 575 Ala Tyr Tyr Lys Val Gly Arg Asp Thr Lys
Tyr Thr Pro Pro Asn Phe 580 585
590 Ser Ser Trp Thr Arg Asp Glu Tyr Gly Phe Ala His Asn His Val
Ser 595 600 605 Glu
Gly Ala Tyr Glu Arg Val Asn Glu Phe Val Asp Val Gln Arg Asp 610
615 620 His Ala Asp Leu Ile Arg
Arg Ile Gly Ala Gln Ser Thr Val Leu Leu 625 630
635 640 Lys Asn Lys Gly Ala Leu Pro Leu Ser Arg Lys
Glu Lys Leu Val Ala 645 650
655 Leu Leu Gly Glu Asp Ala Gly Ser Asn Ser Trp Gly Ala Asn Gly Cys
660 665 670 Asp Asp
Arg Gly Cys Asp Asn Gly Thr Leu Ala Met Ala Trp Gly Ser 675
680 685 Gly Thr Ala Asn Phe Pro Tyr
Leu Val Thr Pro Glu Gln Ala Ile Gln 690 695
700 Asn Glu Val Leu Gln Gly Arg Gly Asn Val Phe Ala
Val Thr Asp Ser 705 710 715
720 Trp Ala Leu Asp Lys Ile Ala Ala Ala Ala Arg Gln Ala Ser Val Ser
725 730 735 Leu Val Phe
Val Asn Ser Asp Ser Gly Glu Gly Tyr Leu Ser Val Asp 740
745 750 Gly Asn Glu Gly Asp Arg Asn Asn
Ile Thr Leu Trp Lys Asn Gly Asp 755 760
765 Asn Val Val Lys Thr Ala Ala Asn Asn Cys Asn Asn Thr
Val Val Ile 770 775 780
Ile His Ser Val Gly Pro Val Leu Ile Asp Glu Trp Tyr Asp His Pro 785
790 795 800 Asn Val Thr Gly
Ile Leu Trp Ala Gly Leu Pro Gly Gln Glu Ser Gly 805
810 815 Asn Ser Ile Ala Asp Val Leu Tyr Gly
Arg Val Asn Pro Gly Ala Lys 820 825
830 Ser Pro Phe Thr Trp Gly Lys Thr Arg Glu Ser Tyr Gly Ser
Pro Leu 835 840 845
Val Lys Asp Ala Asn Asn Gly Asn Gly Ala Pro Gln Ser Asp Phe Thr 850
855 860 Gln Gly Val Phe Ile
Asp Tyr Arg His Phe Asp Lys Phe Asn Glu Thr 865 870
875 880 Pro Ile Tyr Glu Phe Gly Tyr Gly Leu Ser
Tyr Thr Thr Phe Glu Leu 885 890
895 Ser Asp Leu His Val Gln Pro Leu Asn Ala Ser Arg Tyr Thr Pro
Thr 900 905 910 Ser
Gly Met Thr Glu Ala Ala Lys Asn Phe Gly Glu Ile Gly Asp Ala 915
920 925 Ser Glu Tyr Val Tyr Pro
Glu Gly Leu Glu Arg Ile His Glu Phe Ile 930 935
940 Tyr Pro Trp Ile Asn Ser Thr Asp Leu Lys Ala
Ser Ser Asp Asp Ser 945 950 955
960 Asn Tyr Gly Trp Glu Asp Ser Lys Tyr Ile Pro Glu Gly Ala Thr Asp
965 970 975 Gly Ser
Ala Gln Pro Arg Leu Pro Ala Ser Gly Gly Ala Gly Gly Asn 980
985 990 Pro Gly Leu Tyr Glu Asp Leu
Phe Arg Val Ser Val Lys Val Lys Asn 995 1000
1005 Thr Gly Asn Val Ala Gly Asp Glu Val Pro
Gln Leu Tyr Val Ser 1010 1015 1020
Leu Gly Gly Pro Asn Glu Pro Lys Val Val Leu Arg Lys Phe Glu
1025 1030 1035 Arg Ile
His Leu Ala Pro Ser Gln Glu Ala Val Trp Thr Thr Thr 1040
1045 1050 Leu Thr Arg Arg Asp Leu Ala
Asn Trp Asp Val Ser Ala Gln Asp 1055 1060
1065 Trp Thr Val Thr Pro Tyr Pro Lys Thr Ile Tyr Val
Gly Asn Ser 1070 1075 1080
Ser Arg Lys Leu Pro Leu Gln Ala Ser Leu Pro Lys Ala Gln 1085
1090 1095
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