Polypeptides Having Endoglucanase Activity and Polynucleotides Encoding Same

Abstract

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

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No. 13/764,912 filed on Feb. 12, 2013, the contents of which are fully incorporated herein by reference.

SEQUENCE LISTING

[0003] Applicants also submit herewith a Sequence Listing in the form of a text file, which acts as both the paper copy and the computer readable form of the Sequence Listing.

BACKGROUND OF THE INVENTION

[0004] 1. Field of the Invention

[0005] The present invention relates to polypeptides having endoglucanase activity and 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 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. Since glucose is readily fermented to ethanol by a variety of yeasts while cellobiose is not, any cellobiose remaining at the end of the hydrolysis represents a loss of yield of ethanol. More importantly, cellobiose is a potent inhibitor of endoglucanases and cellobiohydrolases. The accumulation of cellobiose during hydrolysis is undesirable for ethanol production.

[0009] The present invention provides polypeptides having endoglucanase activity and polynucleotides encoding the polypeptides.

SUMMARY OF THE INVENTION

[0010] The present invention relates to isolated polypeptides having endoglucanase activity selected from the group consisting of:

[0011] (a) a polypeptide having at least 78% sequence identity to the mature polypeptide of SEQ ID NO: 2, a polypeptide having at least 94% sequence identity to the mature polypeptide of SEQ ID NO: 4, a polypeptide having at least 76% sequence identity to the mature polypeptide of SEQ ID NO: 6, or a polypeptide having at least 81% sequence identity to the mature polypeptide of SEQ ID NO: 8;

[0012] (b) a polypeptide encoded by a polynucleotide that hybridizes under low, or medium, or medium-high, or high, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, (ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or (ii);

[0013] (c) a polypeptide encoded by a polynucleotide having at least 60% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; or the cDNA sequence thereof;

[0014] (d) a variant of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions; and [0015] (e) a fragment of the polypeptide of (a), (b), (c), or (d) that has endoglucanase activity.

[0016] The present invention also relates to isolated polypeptides comprising a catalytic domain selected from the group consisting of:

[0017] (a) a catalytic domain having at least 60% sequence identity to the catalytic domain of SEQ ID NO: 2 (for example, amino acids 92 to 419 of SEQ ID NO: 2), SEQ ID NO: 4 (for example, amino acids 73 to 386 of SEQ ID NO: 4), SEQ ID NO: 6 (for example, amino acids 67 to 378 of SEQ ID NO: 6), or SEQ ID NO: 8 (for example, amino acids 67 to 378 of SEQ ID NO: 8);

[0018] (b) a catalytic domain encoded by a polynucleotide having at least 60% sequence identity to the catalytic domain coding sequence of SEQ ID NO: 1 (for example, nucleotides 459-564, 621-838, 896-993, 1050-1133, 1184-1345, 1410-1585, and 1639-1778 of SEQ ID NO: 1), SEQ ID NO: 3 (for example, nucleotides 334-459, 532-700, 760-808, 862-872, 931-956, 1019-1093, 1156-1312, 1384-1640, and 1711-1782 of SEQ ID NO: 3), SEQ ID NO: 5 (for example, nucleotides 361-408, 463-556, 613-618, 669-909, 975-1192, 1245-1297, 1362-1471, 1540-1688, and 1753-1769 of SEQ ID NO: 5), or SEQ ID NO: 7 (for example, nucleotides 355-402, 457-550, 607-612, 666-906, 969-1186, 1239-1291, 1353-1462, 1520-1668, and 1727-1743 of SEQ ID NO: 7);

[0019] (c) a variant of a catalytic domain comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the catalytic domain of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and

[0020] (d) a fragment of a catalytic domain of (a), (b), or (c), which has endoglucanase activity.

[0021] The present invention also relates to isolated polynucleotides encoding the polypeptides of the present invention; nucleic acid constructs; recombinant expression vectors; recombinant host cells comprising the polynucleotides; and methods of producing the polypeptides.

[0022] The present invention also relates to processes for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having endoglucanase activity of the present invention. In one aspect, the processes further comprise recovering the degraded or converted cellulosic material.

[0023] The present invention also relates to processes of producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having endoglucanase activity of the present invention; (b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[0024] The present invention also relates to processes of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of a polypeptide having endoglucanase activity of the present invention. In one aspect, the fermenting of the cellulosic material produces a fermentation product. In another aspect, the processes further comprise recovering the fermentation product from the fermentation.

[0025] The present invention also relates to a polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 21 of SEQ ID NO: 2, amino acids 1 to 20 of SEQ ID NO: 4, amino acids 1 to 19 of SEQ ID NO: 6, or amino acids 1 to 19 of SEQ ID NO: 8, which is operably linked to a gene encoding a protein; nucleic acid constructs, expression vectors, and recombinant host cells comprising the polynucleotides; and methods of producing a protein.

SEQUENCES OF THE INVENTION

[0026] Trametes versicolor Strain NN055586_Genomic Nucleotide Sequence (SEQ ID NO: 1):

TABLE-US-00001 1 ATGAAGACTT TCGCAGCCTT GCTTTCCGCT GTCACTCTCG CGCTCTCGGT GCGCGCCCAG GCGGCTGTCT GGAGTCAATG 81 TAAGTGCCGC TGCTTTTCAT TGATACGAGA CTCTACGCCG AGCTGACGTG CTACCGTATA GGTGGCGGTA CAGTAAGTTA 161 TCATAGCATA CCGCCTACAT GCACAAGGAC ATACTGCTGA TCAATTCTCT GTGCAGGGTT GGACGGGCGA GACCACTTGC 241 GTTGCTGGTT CGGTTTGTAC CTCCTTGAGC TCATGTGAGC GACTTTCAAT CCGTCGTCAT TGCTCCTCAT GTATTGACGA 321 TTGGCCTTCA TAGCATACTC TCAATGCGTT CCGGGCTCCG CAACGTCCAG CGCTCCGGCG GCCCCCTCAG CGACAACTTC 401 AGGCCCCGCA CCTCCAGCAT CCACGTGTGC ACCCAACAGC CCGCCCGCGA GCGCAGGCAA ACTGCGCTTC GCGGGTGTCA 481 ACATCTCCGG TTTCGACTTC GGATGCAGCA CGGACGGAAC GTGCTCGGCC AGCGGGGCAT GGCCGCCATT GACCAAGTAC 561 TACGGTCAGC CCCTTTCCTG CAGGGTAGTG GCGAGGGTGT TGACATTTTT TGGACGACAG GCATGGATGG TGCGGGCCAG 641 ATGAAGCACT TTGTCGAGAA CGACGGCTAC AATGTTTTCC GCCTGCCCGT TGGGTGGCAG TTCCTGACGA ACGGTGCCGC 721 CACTGGTGAC ATAGACGAGG TCAACTTCGC CGAGTACGAT GATCTCGTCC AAGCGTGCCT CGACGCAGGC GCGTCGTGCA 801 TCGTTGAGGT TCACAACTAC GCGCGGTTTA ACGGCAAGGT ACGATGCGCA CTTCTGCGAG CCAGCTGACT GTAGCCAACC 881 AAACCTGGCA TACAGATCAT CGGTCAGGGT GGGCCCGCAA ATGACCAGTT CGCCGCCCTT TGGAGCAGTT TTGCCTCCAA 961 GTACGCCGAC AACGACAAGA TCATCTTTGG GATGTCAGTG CCTTTTCCGG ATTCCGCAAA CTGTAGCTCA TGCAATAACC 1041 GACTCACAGT ATGAACGAAC CACATGACGT CCCCGATATC AACCTCTGGG CGGAGAGTGT CCAGGCAGCA GTGACCGCCA 1121 TCCGCCAGGC CGGGTATGCA TGTCAACCCC AAAACCAAAT GCTGCTCACT AATCGGTCCC TAGTGCGACT TCGCAGCTGA 1201 TCCTGCTCCC GGGCAACAAC TGGACGTCTG CGGAGACCTT CGTCTCCAAC GGCTCGGCCG ATGCGCTCAA CAAGGTCACG 1281 AACCCCGACG GCAGCATCAC GAACCTCATC TTCGACGTGC ACAAGTACCT CGACTTCGAC AACTCGTACG TCTATGCCCA 1361 CTTGCCCCGT TGATTCTGCA GGTGAATTGA CGTAATGCGG ATCGCACAGG GGCACGAACG CAGAGTGCAC GACGAACAAC 1441 ATCGACAACG CGTGGGCGCC GCTCGCGCAG TGGCTGCGCT GCAACGGCCG GCAGGCGTTC AACACCGAGA CGGGCGGCGG 1521 GAACGTCGCG TCGTGCGAGA CGTTCATGTG CGAGCAGGTC GCGTTCCAGA CGGCGAACTC GGACGGTGCG TTCTGCGTCC 1601 TCGCGTCGTT CTTCACTCTC GCTGATGATA TCGTGTAGTG TTCTTGGGCT ATGTCGGCTG GGCGGCGGGC AACTTCTACG 1681 AAGGCTACGT GCTGAGCGAG GTCCCGACCC AGAATGCGGA TGGGAGCTGG ACGGACCAGC CGCTTGTTGC GCAGTGCATG 1761 GCGCCCAACG CTTCTCAGTG A

Exons/Introns (in base pairs) of SEQ ID NO: 1:

TABLE-US-00002 Exon 1 1-79 bp Intron 1 80-141 bp Exon 2 142-152 bp Intron 2 153-216 bp Exon 3 217-274 bp Intron 3 275-333 bp Exon 4 334-564 bp Intron 4 565-620 bp Exon 5 621-838 bp Intron 5 839-895 bp Exon 6 896-993 bp Intron 6 994-1049 bp Exon 7 1050-1133 bp Intron 7 1134-1183 bp Exon 8 1184-1345 bp Intron 8 1346-1409 bp Exon 9 1410-1585 bp Intron 9 1586-1638 bp Exon 10 1639-1781 bp

Features (in base pairs) of SEQ ID NO: 1:

TABLE-US-00003 Signal Peptide 1-63 bp Cellulose Binding Module (CBM 1) 64-79, 142-152, 217-274, 334-353 bp Linker 354-458 bp Endoglucanase catalytic site 459-564, 621-838, 896-993, 1050-1133, bp 1184-1345, 1410-1585, 1639-1778 Stop codon 1779-1781 bp

Protein Sequence of Trametes versicolor Strain NN055586_protein (SEQ ID NO: 2):

TABLE-US-00004 1 MKTFAALLSA VTLALSVRAQ AAVWSQCGGT GWTGETTCVA GSVCTSLSSS YSQCVPGSAT 61 SSAPAAPSAT TSGPAPPAST CAPNSPPASA GKLRFAGVNI SGFDFGCSTD GTCSASGAWP 121 PLTKYYGMDG AGQMKHFVEN DGYNVFRLPV GWQFLTNGAA TGDIDEVNFA EYDDLVQACL 181 DAGASCIVEV HNYARFNGKI IGQGGPANDQ FAALWSSFAS KYADNDKIIF GIMNEPHDVP 241 DINLWAESVQ AAVTAIRQAG ATSQLILLPG NNWTSAETFV SNGSADALNK VTNPDGSITN 301 LIFDVHKYLD FDNSGTNAEC TTNNIDNAWA PLAQWLRCNG RQAFNTETGG GNVASCETFM 361 CEQVAFQTAN SDVFLGYVGW AAGNFYEGYV LSEVPTQNAD GSWTDQPLVA QCMAPNASQ

Features of SEQ ID NO: 2 (amino acid positions):

TABLE-US-00005 Signal Peptide 1-21 Cellulose Binding 22-56 Module (CBM 1) Linker 57-91 Endoglucanase catalytic site 92-419

Signal Peptide Sequence of SEQ ID NO: 2:

MKTFAALLSAVTLALSVRAQA

[0027] Trametes versicolor Strain NN055586 Genomic Nucleotide Sequence (SEQ ID NO: 3):

TABLE-US-00006 1 ATGAAGACGG TTATCCTCTC CCTCGCTGCC GCGCTCTTCA GCGCCGCGCC CGTGCTCTCC ACCGCCGTCT GGGGCCAGTG 81 CGGCGTGAGT ACTCGACTCG CGACGCGGTC ACGGGGCCAT ACTCACCATC TGCTCGTGTT CTAGGGCACT GGCTTCTCTG 161 GCGACACGAC CTGCGCCTCC GGCTCTAGCT GCGTCGTAGT CAACCAATGT ATGCCGTCCA CGTCCACCCG CTGACATCCT 241 TTACTGACCA CTCACCCTTG AACAGACTAC TCGCAATGCC AGCCCGGCGC GTCCGCCCCC ACGTCGACTG CCTCGGCCCC 321 CGGCCCTTCC GGCTGCTCGG GCACGCGCAC CAAGTTCAAG CTCTTCGGTG TGAACGAGTC CGGCGCGGAG TTCGGGAACA 401 CCGTCATCCC GGGCGCGCTC GGCACGGACT ACACCTGGCC GTCGCCCACC TCCATCGACG TGCGTACTGT TGTCGGACAT 481 GTCTGACGTA GAAGCAGAGG ATGCTGATGG ATGGACGGTT GGGCGATGCA GTTCTTCCTC GGGCAGGGCT TCAACACCTT 561 CCGCATCCCG TTCCTGATGG AGCGCGTCAG CCCGCCGTCG ACGGGCGGCC TTACTGGCCC GTTCAACAGC ACGTACCTCG 641 ACGGGCTGAA GCAGACTGTT AGCTACATCA CGGGCAAGGG GGGCTTTGCC ATCGTCGACC GTGAGTGCTT ACTCCCAACG 721 TATGCTATTT GGAGAGTGGA GTACTGATCT GGTGTGCAGC GCACAACTTC ATGATCTTCA ACGGCGCGAC GATCACGAGC 801 ACCAGCCAGT AAGTCGCATT ATTTACGGTG GAAGAGTTTT ACTGATATCT ATCGCGTTTA GGTTCCAGGC TTGTACGTAT 881 TCGCGTCGCC ATATACACGA CACCGAGCTC TTTGCTGATG TTGACGACAG GGTGGCAGAA GCTCGCTGCT GAGTTCGTGA 961 GTGTGCTCCA TGGCTACGCG GCCGTGAACG CTCTGGCTGA CAAGATGCCG CCTCCCAGAA AACCGACAAC AACGTCATCT 1041 TCGACCTGAT GAACGAGCCG CACGACATCC CCGCGCAGAC CGTCTTCCAG CTCGTACGTA ACACTTTCCG TATGTCCCAA 1121 GCAACCATGT GTTAAGTGAT CATGATCCCG CGCAGATGCA AGCGGCCGTG AACGGCGTGC GCGCGAGCGG CGCGACGAGC 1201 CAGCTCATCC TCGCCGAGGG CACGAGCTGG ACCGGCGCGT GGACCTGGAC GACGTCGGGC AACAGCGACG CGTTCGGCGC 1281 GATCAAGGAC CCCAACAACA ACATCGCCAT CCGTGCGTCC CCCCCCTCCC CCTTCCTCTT GCCCCCTGCC TACTGACGAA 1361 CACGCCATGG GATTGACACA CAGAGATGCA CCAGTACCTA GACTCGGACG GGTCGGGGAC GTCCCCGATC TGCGTGTCGG 1441 ACACGATCGG GGCGGAGCGG CTGCAGGCGG CGACGCAGTG GCTGCAGCAG ACGGGCCTCA AGGGCTTCCT CGGCGAGATC 1521 GGGACGGGGA ACAACACGCA GTGCGTGACC GCGCTGCAGG GCGCGCTGTG CGAGATGCAG CAGGCGGGCG GGACGTGGCT 1601 CGGCGCGCTC TGGTGGGCGG CGGGGCCGTG GTGGGGAGAC GTGAGTGGCT TTCTGTGCTT ATGTGGGGGA GGGGGAGTGG 1681 GGGCTGACGG TGGTTGTGTT GGACTTGCAG TACTACCAGA GCATCGAGCC CCCGAACGGG GACGCGATCG CTGCGATCCT 1761 CCCGGCGCTC AAGGCGTTCC AGTAG

Exons/Introns (in base pairs) of SEQ ID NO: 3:

TABLE-US-00007 Exon 1 1-84 bp Intron 1 85-144 bp Exon 2 145-208 bp Intron 2 209-265 bp Exon 3 266-459 bp Intron 3 460-531 bp Exon 4 532-700 bp Intron 4 701-759 bp Exon 5 760-808 bp Intron 5 809-861 bp Exon 6 862-872 bp Intron 6 873-930 bp Exon 7 931-956 bp Intron 7 957-1018 bp Exon 8 1019-1093 bp Intron 8 1094-1155 bp Exon 9 1156-1312 bp Intron 9 1313-1383 bp Exon 10 1384-1640 bp Intron 10 1641-1710 bp Exon 11 1711-1785 bp

Features (in base pairs) of SEQ ID NO: 3:

TABLE-US-00008 Signal Peptide 1-60 bp Cellulose Binding 61-84, 145-208, 266-285 bp Module (CBM 1) Linker 286-333 bp Endoglucanase catalytic site 334-459, 532-700, 760-808, 862-872, 931-956, bp 1019-1093, 1156-1312,1384-1640, 1711-1782 Stop codon 1783-1785 bp

Protein Sequence of Trametes versicolor Strain NN055586_protein (SEQ ID NO: 4):

TABLE-US-00009 1 MKTVILSLAA ALFSAAPVLS TAVWGQCGGT GFSGDTTCAS GSSCVVVNQY YSQCQPGASA 61 PTSTASAPGP SGCSGTRTKF KLFGVNESGA EFGNTVIPGA LGTDYTWPSP TSIDFFLGQG 121 FNTFRIPFLM ERVSPPSTGG LTGPFNSTYL DGLKQTVSYI TGKGGFAIVD PHNFMIFNGA 181 TITSTSQFQA WWQKLAAEFK TDNNVIFDLM NEPHDIPAQT VFQLMQAAVN GVRASGATSQ 241 LILAEGTSWT GAWTWTTSGN SDAFGAIKDP NNNIAIQMHQ YLDSDGSGTS PICVSDTIGA 301 ERLQAATQWL QQTGLKGFLG EIGTGNNTQC VTALQGALCE MQQAGGTWLG ALWWAAGPWW 361 GDYYQSIEPP NGDAIAAILP ALKAFQ

Features of SEQ ID NO: 4 (amino acid positions):

TABLE-US-00010 Signal Peptide 1-20 Cellulose Binding 21-57 Module (CBM 1) Linker 58-72 Endoglucanase catalytic site 73-386

Signal Peptide Sequence of SEQ ID NO: 4:

MKTVILSLAAALFSAAPVLS

[0028] Trametes versicolor Strain NN055586_Genomic Nucleotide Sequence (SEQ ID NO: 5):

TABLE-US-00011 1 ATGAAGTACG CAACTGCCTC TCTCGTGGCT GCGGCCACCG TCTCCCAGGT TATGGCGGTA TGCACTCAGG CTAGTACTTC 81 ACATGCTGGA TTATACTCAT TAGCAACGTA GGTGTACCAA CAGTGCGGTG GTATCGGTTT CGGTTAGCAG AATTGCAAGT 161 GGCGCATTTG ACTGCCCTTG CTAACGAACT TGCAGATAGG CCCACTGCTT GTGATGCTCA CTCGGTGTGC ACTGCTATCA 241 ATCCCCGTAT GTCTGTCCTG CTATCATTAC ACAGACATAG TAGGCTCATG CTGGCATGCA GACTACTCCC AGTGCCTCCC 321 GTCGTCTTCC CCCTCGGCGC CCTCTGCCCC CCGCTCCAGC CTGATCCAGC TCGGTGGTGT CAACACTGCG GGATACGACT 401 TCAGCGTTGT ACGTCCTCTG AAACAAAAGT CTTAGACCCA TTGCTCACAG ATATTGACAT AGACTATTGA CGGAAGCTTC 481 ACCGGCACCG GTGTCTCTCC CCCGCCCTCT CAGTATACCC ACTTCTCTAG CCAGGGTGCC AACCTCTTCC GCATTCGTGA 561 GTGTGCCAAG TGTTGAGTAC GGGAATAATA CTGATCATAT TTTTTGCCGT AGCCTTCGGT GCGTTCTCTG TCTAGCGATG 641 TGGTTTCGTT CTCATAGCCC TCCTTTAGCC TGGCAGCTGA TGACGCCGAA CGTCGGCGGA CCCATCAACG AGACGTTCTT 721 CGCCACCTAT GACAAGACCG TCCAGGCTGC GCTCAACTCG GGCTCCAACG TGCACGTCAT CATCGATCTG CACAACTATG 801 CGCGCTGGAA CGGCGCCATC ATCGCTCAAG GCGGCCCGAC CAACGAGCAA TTTGCTTCCA TCTGGACCCA GCTCGCCGCC 881 AAGTACGGCC GCAACAAGCG TATCATCTTG TACGCTCCTT CCTCCCTCTC TTTCTTATGA CATGTATGAG GATAGCTGCT 961 GACGACGTCC GCAGCGGTCT CATGAACGAG CCACACGACC TCCCCAGCGT CCCGACCTGG GTTAAGTCTG TCCAGTTCGT 1041 TGTCAACGCC ATCCGCCATG CCGGCGCGAC GAACTTCCTC CTCCTGCCTG GCTCCAGCTT CTCATCCGCC CAGGCCTTCC 1121 CCACCGAGGC CGGCCCTGAC CTCGTCAAGG TCACTGACCC GCTCGGCGGC ACCCACAAGC TGATCTTCGA TGGTAGGTTT 1201 AGCGTGTTAG AACCCATCCT GTGCCGGACT GACCTCTGCC GCAGTCCACA AGTATCTCGA CAGCGACAAC AGCGGCACTC 1281 ACCCCGACTG CACCACTGTA CGTTGCACCG CCCCGTCTAT CACGAACGCG ACTTCCAGCG ATGCTCATGC ACATTCCATA 1361 GGACAACGTC GACGTGCTGA AGACGCTCGT CCAGTTCCTC AAGCAGAACG GCAACCGCCA GGCGCTCCTC AGCGAGACCG 1441 GCGGAGGAAA CACCACGAGC TGCGAGACTC TGTAAGTGCC AGTGGCCTTG CACGCCGCCC ACTTGGGCTT GTAGTAGCAC 1521 GCTGACGCAC TCCCCGAAGC CTCAACACCG AGCTCTCGTT CGTCAAGTCC GCGTTCCCGA CCCTCGTCGG CTTCTCCGCC 1601 TGGGCCGCCG GCGCGTTCGA CACCAACTAC GTGCTCACGC TCACGCCCAA CGCCGACGGC TCCGACCAGC CCCTCTGGAT 1681 CGACGCCGGT ACGTGTGGCC CTGCCAGTGC TCATTCTGTC CTCTCGATAT AGGTGCTCAC CATTCGCTGC AGTCAAGCCC 1761 AACCTGCCTT GA

Exons/Introns (in base pairs) of SEQ ID NO: 5:

TABLE-US-00012 Exon 1 1-57 bp Intron 1 58-111 bp Exon 2 112-142 bp Intron 2 143-195 bp Exon 3 196-246 bp Intron 3 247-301 bp Exon 4 302-408 bp Intron 4 409-462 bp Exon 5 463-556 bp Intron 5 557-612 bp Exon 6 613-618 bp Intron 6 619-668 bp Exon 7 669-909 bp Intron 7 910-974 bp Exon 8 975-1192 bp Intron 8 1193-1244 bp Exon 9 1245-1297 bp Intron 9 1298-1361 bp Exon 10 1362-1471 bp Intron 10 1472-1539 bp Exon 11 1540-1688 bp Intron 11 1689-1752 bp Exon 12 1753-1772 bp

Features (in base pairs) of SEQ ID NO: 5:

TABLE-US-00013 Signal Peptide 1-57 bp Cellulose Binding 112-142, 196-246, 302-321 bp Module (CBM 1) Linker 322-360 bp Endoglucanase 361-408, 463-556, 613-618, 669-909, 975-1192, catalytic site 1245-1297, 1362-1471, 1540-1688, 1753-1769 bp Stop codon 1770-1772 bp

Protein Sequence of Trametes versicolor Strain NN055586_protein (SEQ ID NO: 6):

TABLE-US-00014 1 MKYATASLVA AATVSQVMAV YQQCGGIGFD RPTACDAHSV CTAINPHYSQ CLPSSSPSAP 61 SAPRSSLIQL GGVNTAGYDF SVTIDGSFTG TGVSPPPSQY THFSSQGANL FRIPFAWQLM 121 TPNVGGPINE TFFATYDKTV QAALNSGSNV HVIIDLHNYA RWNGAIIAQG GPTNEQFASI 181 WTQLAAKYGR NKRIIFGLMN EPHDLPSVPT WVKSVQFVVN AIRHAGATNF LLLPGSSFSS 241 AQAFPTEAGP DLVKVTDPLG GTHKLIFDVH KYLDSDNSGT HPDCTTDNVD VLKTLVQFLK 301 QNGNRQALLS ETGGGNTTSC ETLLNTELSF VKSAFPTLVG FSAWAAGAFD TNYVLTLTPN 361 ADGSDQPLWI DAVKPNLP

Features of SEQ ID NO: 6 (amino acid positions):

TABLE-US-00015 Signal Peptide 1-19 Cellulose Binding 20-53 Module (CBM 1) Linker 54-66 Endoglucanase 67-378 catalytic site

Signal Peptide Sequence of SEQ ID NO: 6:

MKYATASLVAAATVSQVMA

[0029] Trametes versicolor Strain NN055586_Genomic Nucleotide Sequence (SEQ ID NO: 7):

TABLE-US-00016 1 ATGAAGTACG CAACTGCCTC TCTCGTGGCT GCGGCCACTG TCTCCCAAGT TGCGGCGGTA TGTCTTCAGT CTACTATACA 81 TTCTGATCCT TACTTATAGG CAACATAGGT GTACCAGCAG TGCGGCGGTA TCGGCTTCGG TTGGTAGCAG TGCAAGTGAC 161 CTATCTAATT ACCCTTGCTA ACGACCTTGC AGTTGGGCCC ACTGCTTGCG ATGCTCAGTC GGTGTGCACT ACTATCAACG 241 CCTGTATGTC TAGCCTGACA TCATTATACC GACATAAACT GATGTTGGCA TGCAGACTAC TCGCAGTGCC TCCCGACGTC 321 CTCCCCCTCC GCGCCCTCCG CTCCCAGCTC CGGCCTGATC CAGCTCGGTG GTGTCAACAC TGCGGGATAC GACTTCAGCG 401 TTGTACGTCC TCTGAGAGAA TGTTTCCAAA CGCATTACTC AAGGATATTG ACGTAGGCTA CTGATGGAAG CTTCACCGGC 481 ACCGGTGTCT CTCCCCCGCC ATCCCAGTTC ACCCACTTCT CCAGCCAGGG TGCTAACCTC TACCGCATTC GTAAGTGGGC 561 TCAGCATTGC GAAGGGAAGT CGTACTGACT GTATGTTTTA TTGTAGCCTT CGGTGTGCCG TTTCTCTAGC GATGTCGATT 641 GGTCCAATTG CTGACCCTTC CTTAGCATGG CAGCTGATGA CGCCCAACCT CGGCGGACCC ATCAACGAGA CGTTCTTCTC 721 CACCTACGAC CAGACTGTCC AAGCCGCGCT CAACTCGGGC TCAAACGTGC ACGTCATCGT CGACCTGCAC AACTACGCGC 801 GCTGGAACGG CGGTATCATC GCCCAGGGCG GCCCGACCAA CGAGGAGTAC GCGTCCATCT GGACCCACCT CGCCGCCAAG 881 TACGGCTCCA ACGAGCGCAT CATTTTGTAC GCCCGCCCCT CGGTCTTCTG ATACGTTGAA GAGGAGAGCT CACGACGTCT 961 GCCTGCAGCG GTGTCATGAA CGAGCCGCAC GACATCCCCA ACGTCCAGAC CTGGGTCGAC TCCGTCCAGT TCGTCGTCAA 1041 CGCTATCCGC CAGGCCGGCG CGACGAACTT CCTCCTCCTG CCTGGCTCCA GCTTCTCATC CGCCCAGGCC TTCCCCACCG 1121 AGGCCGGCCC TTATCTCGTC AAAGTAACTG ACCCGCTTGG CGGCACCGAC AAGCTGATCT TCGATGGTAA GCCCAGCATC 1201 ATAGAACTCA TTATGTACAC AAGTTAACCC GACCGCAGTC CACAAGTACC TCGACAGCGA CAACAGCGGC ACCCACCCCG 1281 ACTGCACCAC CGTACGTTGC ACCTCCTAAA CTATGCACAC CTCTCCCCGC GATGCTCACG TACATTCCGC AGGACAACGT 1361 GGACGTCCTT AAGACCCTCG TCCAGTTCCT TCAGCAAAAC GGCAACCGTC AGGCGATCCT GAGCGAGACC GGCGGAGGCA 1441 ACACTGCGAG CTGTGAGACT CTGTAAGTCG CGCACACCGC TTACAGTCTT CAAGCATTGC GCTAATGCAG CCCCCGCAGC 1521 CTCAACAACG AGCTCTCGTT CGTCAAGTCC GCGTTCCCGA CCCTCGCCGG CTTCTCAGTC TGGGCTGCTG GCGCGTTCGA 1601 CACCACCTAC GTGCTCACCG TCTCGCCCAA CCCCGATGGC TCCGACCAGC CCCTCTGGAA CGACGCCGGT ACGTGTGATC 1681 CCATGCATCC CGTCCTATCG ATTCAATTGC TCACTGTTCG CTACAGTCAA GCCCAACCTG CCCTGA

Exons/Introns (in base pairs) of SEQ ID NO: 7:

TABLE-US-00017 Exon 1 1-57 bp Intron 1 58-108 bp Exon 2 109-139 bp Intron 2 140-192 bp Exon 3 193-243 bp Intron 3 244-295 bp Exon 4 296-402 bp Intron 4 403-456 bp Exon 5 457-550 bp Intron 5 551-606 bp Exon 6 607-612 bp Intron 6 613-665 bp Exon 7 666-906 bp Intron 7 907-968 bp Exon 8 969-1186 bp Intron 8 1187-1238 bp Exon 9 1239-1291 bp Intron 9 1292-1352 bp Exon 10 1353-1462 bp Intron 10 1463-1519 bp Exon 11 1520-1668 bp Intron 11 1669-1726 bp Exon 12 1727-1746 bp

Features (in base pairs) of SEQ ID NO: 7:

TABLE-US-00018 Signal Peptide 1-57 bp Cellulose Binding 109-139, 193-243, 296-315 bp Module (CBM 1) Linker 316-354 bp Endoglucanase 355-402, 457-550, 607-612, 666-906, 969-1186, catalytic site 1239-1291, 1353-1462, 1520-1668, 1727-1743 bp Stop codon 1744-1746 bp

Protein Sequence of Trametes versicolor Strain NN055586_protein (SEQ ID NO: 8):

TABLE-US-00019 1 MKYATASLVA AATVSQVAAV YQQCGGIGFV GPTACDAQSV CTTINAYYSQ CLPTSSPSAP 61 SAPSSGLIQL GGVNTAGYDF SVATDGSFTG TGVSPPPSQF THFSSQGANL YRIPFAWQLM 121 TPNLGGPINE TFFSTYDQTV QAALNSGSNV HVIVDLHNYA RWNGGIIAQG GPTNEEYASI 181 WTHLAAKYGS NERIIFGVMN EPHDIPNVQT WVDSVQFVVN AIRQAGATNF LLLPGSSFSS 241 AQAFPTEAGP YLVKVTDPLG GTDKLIFDVH KYLDSDNSGT HPDCTTDNVD VLKTLVQFLQ 301 QNGNRQAILS ETGGGNTASC ETLLNNELSF VKSAFPTLAG FSVWAAGAFD TTYVLTVSPN 361 PDGSDQPLWN DAVKPNLP

Features of SEQ ID NO: 8 (amino acid positions):

TABLE-US-00020 Signal Peptide 1-19 Cellulose Binding 20-53 Module (CBM 1) Linker 54-66 Endoglucanase 67-378 catalytic site

Signal Peptide Sequence of SEQ ID NO: 8:

MKYATASLVAAATVSQVAA

DEFINITIONS

[0030] Endoglucanase: The term "endoglucanase" means an endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4) that catalyzes 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. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). For purposes of the present invention, endoglucanase activity is determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40.degree. C.

[0031] In one aspect, the polypeptides of the present invention have at least 20%, e.g., at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 100% of the endoglucanase activity of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0032] Acetylxylan esterase: The term "acetylxylan esterase" means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. For purposes of the present invention, acetylxylan esterase activity is determined using 0.5 mM p-nitrophenylacetate as substrate in 50 mM sodium acetate pH 5.0 containing 0.01% TWEEN.TM. 20 (polyoxyethylene sorbitan monolaurate). One unit of acetylxylan esterase is defined as the amount of enzyme capable of releasing 1 micromole of p-nitrophenolate anion per minute at pH 5, 25.degree. C.

[0033] Allelic variant: The term "allelic variant" means 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.

[0034] Alpha-L-arabinofuranosidase: The term "alpha-L-arabinofuranosidase" means an alpha-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing alpha-L-arabinofuranoside residues in alpha-L-arabinosides. The enzyme acts on alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)- and/or (1,5)-linkages, arabinoxylans, and arabinogalactans. Alpha-L-arabinofuranosidase is also known as arabinosidase, alpha-arabinosidase, alpha-L-arabinosidase, alpha-arabinofuranosidase, polysaccharide alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase, L-arabinosidase, or alpha-L-arabinanase. For purposes of the present invention, alpha-L-arabinofuranosidase activity is determined using 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) per ml of 100 mM sodium acetate pH 5 in a total volume of 200 microliters for 30 minutes at 40.degree. C. followed by arabinose analysis by AMINEX.RTM. HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

[0035] Alpha-glucuronidase: The term "alpha-glucuronidase" means an alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that catalyzes the hydrolysis of an alpha-D-glucuronoside to D-glucuronate and an alcohol. For purposes of the present invention, alpha-glucuronidase activity is determined according to de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of alpha-glucuronidase equals the amount of enzyme capable of releasing 1 micromole of glucuronic or 4-O-methylglucuronic acid per minute at pH 5, 40.degree. C.

[0036] Beta-glucosidase: The term "beta-glucosidase" means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that 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 using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, Extracellular beta-D-glucosidase from Chaetomium thermophilum var. coprophilum: production, purification and some biochemical properties, J. Basic Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0 micromole of p-nitrophenolate anion produced per minute at 25.degree. C., pH 4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01% TWEEN.RTM. 20.

[0037] Beta-xylosidase: The term "beta-xylosidase" means a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1.fwdarw.4)-xylooligosaccharides to remove successive D-xylose residues from non-reducing termini. For purposes of the present invention, one unit of beta-xylosidase is defined as 1.0 micromole of p-nitrophenolate anion produced per minute at 40.degree. C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01% TWEEN.RTM. 20.

[0038] cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic 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, including splicing, before appearing as mature spliced mRNA.

[0039] Cellobiohydrolase: The term "cellobiohydrolase" means a 1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that 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 (Teeri, 1997, Crystalline cellulose degradation: New insight into the function of cellobiohydrolases, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose?, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity is determined according to the procedures described by Lever et al., 1972, 047: 273-279; van Tilbeurgh et al., 1982, FEBS Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters, 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581. In the present invention, the Tomme et al. method can be used to determine cellobiohydrolase activity.

[0040] Cellulosic material: The term "cellulosic material" means any material containing cellulose. The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, 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.

[0041] 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, agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, and wood (including forestry residue) (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 any biomass material. In another preferred aspect, the cellulosic material is lignocellulose, which comprises cellulose, hemicelluloses, and lignin.

[0042] In one aspect, the cellulosic material is agricultural residue. In another aspect, the cellulosic material is herbaceous material (including energy crops). In another aspect, the cellulosic material is municipal solid waste. In another aspect, the cellulosic material is pulp and paper mill residue. In another aspect, the cellulosic material is waste paper. In another aspect, the cellulosic material is wood (including forestry residue).

[0043] In another aspect, the cellulosic material is arundo. In another aspect, the cellulosic material is bagasse. In another aspect, the cellulosic material is bamboo. In another aspect, the cellulosic material is corn cob. In another aspect, the cellulosic material is corn fiber. In another aspect, the cellulosic material is corn stover. In another aspect, the cellulosic material is miscanthus. 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 switchgrass. In another aspect, the cellulosic material is wheat straw.

[0044] In another aspect, the cellulosic material is aspen. In another aspect, the cellulosic material is eucalyptus. In another aspect, the cellulosic material is fir. In another aspect, the cellulosic material is pine. In another aspect, the cellulosic material is poplar. In another aspect, the cellulosic material is spruce. In another aspect, the cellulosic material is willow.

[0045] In another aspect, the cellulosic material is algal cellulose. In another aspect, the cellulosic material is bacterial cellulose. In another aspect, the cellulosic material is cotton linter. In another aspect, the cellulosic material is filter paper. In another aspect, the cellulosic material is microcrystalline cellulose. In another aspect, the cellulosic material is phosphoric-acid treated cellulose.

[0046] In another aspect, the cellulosic material is an aquatic biomass. As used herein the term "aquatic biomass" means biomass produced in an aquatic environment by a photosynthesis process. The aquatic biomass can be algae, emergent plants, floating-leaf plants, or submerged plants.

[0047] 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.

[0048] Cellulolytic enzyme or cellulase: The term "cellulolytic enzyme" or "cellulase" means one or more (e.g., several) enzymes that hydrolyze a cellulosic material. Such enzymes include endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or combinations thereof. The two basic approaches for measuring cellulolytic activity include: (1) measuring the total cellulolytic activity, and (2) measuring the individual cellulolytic activities (endoglucanases, cellobiohydrolases, and beta-glucosidases) as reviewed in Zhang et al., Outlook for cellulase improvement: Screening and selection strategies, 2006, Biotechnology Advances 24: 452-481. Total cellulolytic activity is usually measured using insoluble substrates, including Whatman NQ1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc. The most common total cellulolytic activity assay is the filter paper assay using Whatman NQ1 filter paper as the substrate. The assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem. 59: 257-68).

[0049] For purposes of the present invention, cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose in PCS (or other pretreated cellulosic material) for 3-7 days at a suitable temperature, e.g., 50.degree. C., 55.degree. C., or 60.degree. C., compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate pH 5, 1 mM MnSO.sub.4, 50.degree. C., 55.degree. C., or 60.degree. C., 72 hours, sugar analysis by AMINEX.RTM. HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).

[0050] Coding sequence: The term "coding sequence" means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0051] Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a mature polypeptide of the present invention. Each control sequence may be native (i.e., from the same gene) or foreign (i.e., from a different gene) to the polynucleotide 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 polynucleotide encoding a polypeptide.

[0052] Expression: The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0053] Expression vector: The term "expression vector" means a linear or circular DNA molecule that comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

[0054] Family 61 glycoside hydrolase: The term "Family 61 glycoside hydrolase" or "Family GH61" or "GH61" means a polypeptide falling into the glycoside hydrolase Family 61 according to Henrissat, 1991, A classification of glycosyl hydrolases based on amino-acid sequence similarities, Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316: 695-696. The enzymes in this family were originally classified as a glycoside hydrolase family based on measurement of very weak endo-1,4-beta-D-glucanase activity in one family member. The structure and mode of action of these enzymes are non-canonical and they cannot be considered as bona fide glycosidases. However, they are kept in the CAZy classification on the basis of their capacity to enhance the breakdown of lignocellulose when used in conjunction with a cellulase or a mixture of cellulases.

[0055] Feruloyl esterase: The term "feruloyl esterase" means a 4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that catalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl) groups from esterified sugar, which is usually arabinose in "natural" substrates, to produce ferulate (4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III, cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For purposes of the present invention, feruloyl esterase activity is determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM sodium acetate pH 5.0. One unit of feruloyl esterase equals the amount of enzyme capable of releasing 1 micromole of p-nitrophenolate anion per minute at pH 5, 25.degree. C.

[0056] Fragment: The term "fragment" means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a mature polypeptide; wherein the fragment has endoglucanase activity. In one aspect, a fragment contains at least 20 amino acid residues, e.g., at least 30 to 418 amino acid residues or at least 50 to 400, 80 to 360, 100 to 340, 150 to 300, or 200 to 250, or any number in between, amino acid residues of SEQ ID NO: 2. In another aspect, a fragment contains at least 20 amino acid residues, e.g., at least 30 to 385 amino acid residues or at least 50 to 370, 80 to 350, 100 to 330, 150 to 300, or 200 to 250, or any number in between, amino acid residues of SEQ ID NO: 4. In another aspect, a fragment contains at least 20 amino acid residues, e.g., at least 30 to 386 amino acid residues or at least 50 to 380, 80 to 360, 100 to 330, 150 to 300, or 200 to 250, or any number in between, amino acid residues of SEQ ID NO: 6. In another aspect, a fragment contains at least 20 amino acid residues, e.g., at least 30 to 377 amino acid residues or at least 50 to 370, 80 to 350, 100 to 330, 150 to 300, or 200 to 250, or any number in between, amino acid residues of SEQ ID NO: 8.

[0057] Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom and Shoham, 2003, Microbial hemicellulases. Current Opinion In Microbiology 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The substrates of these enzymes, the hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitable temperature, e.g., 50.degree. C., 55.degree. C., or 60.degree. C., and pH, e.g., 5.0 or 5.5.

[0058] High stringency conditions: The term "high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 65.degree. C.

[0059] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present invention. 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.

[0060] Isolated: The term "isolated" means a substance in a form or environment that does not occur in nature. Non-limiting examples of isolated substances include (1) any non-naturally occurring substance, (2) any substance including, but not limited to, any enzyme, variant, nucleic acid, protein, peptide or cofactor, that is at least partially removed from one or more or all of the naturally occurring constituents with which it is associated in nature; (3) any substance modified by the hand of man relative to that substance found in nature; or (4) any substance modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., multiple copies of a gene encoding the substance; use of a stronger promoter than the promoter naturally associated with the gene encoding the substance). An isolated substance may be present in a fermentation broth sample.

[0061] Low stringency conditions: The term "low stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 50.degree. C.

[0062] Mature polypeptide: The term "mature polypeptide" means 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 one aspect, the mature polypeptide is amino acids 22 to 419 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 21 of SEQ ID NO: 2 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 419 of SEQ ID NO: 2. In another aspect, the mature polypeptide is amino acids 21 to 386 of SEQ ID NO: 4 based on the SignalP program that predicts amino acids 1 to 20 of SEQ ID NO: 4 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 386 of SEQ ID NO: 4. In another aspect, the mature polypeptide is amino acids 20 to 378 of SEQ ID NO: 6 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 6 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 378 of SEQ ID NO: 6. In another aspect, the mature polypeptide is amino acids 20 to 378 of SEQ ID NO: 8 based on the SignalP program that predicts amino acids 1 to 19 of SEQ ID NO: 8 are a signal peptide. In another aspect, the mature polypeptide is amino acids 1 to 378 of SEQ ID NO: 8. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C-terminal and/or N-terminal amino acid) expressed by the same polynucleotide.

[0063] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having endoglucanase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 64 to 1778 of SEQ ID NO: 1 or the cDNA sequence thereof based on the SignalP program (Nielsen et al., 1997, supra) that predicts nucleotides 1 to 63 of SEQ ID NO: 1 encode a signal peptide. In another aspect, the mature polypeptide coding sequence is nucleotides 1 to 1778 of SEQ ID NO: 1 or the cDNA sequence thereof. In another aspect, the mature polypeptide coding sequence is nucleotides 61 to 1782 of SEQ ID NO: 3 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 60 of SEQ ID NO: 3 encode a signal peptide. In another aspect, the mature polypeptide coding sequence is nucleotides 1 to 1782 of SEQ ID NO: 3 or the cDNA sequence thereof. In another aspect, the mature polypeptide coding sequence is nucleotides 112 to 1769 of SEQ ID NO: 5 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 5 encode a signal peptide. In another aspect, the mature polypeptide coding sequence is nucleotides 1 to 1769 of SEQ ID NO: 5 or the cDNA sequence thereof. In another aspect, the mature polypeptide coding sequence is nucleotides 109 to 1743 of SEQ ID NO: 7 or the cDNA sequence thereof based on the SignalP program that predicts nucleotides 1 to 57 of SEQ ID NO: 7 encode a signal peptide. In another aspect, the mature polypeptide coding sequence is nucleotides 1 to 1743 of SEQ ID NO: 7 or the cDNA sequence thereof.

[0064] Catalytic domain: The term "catalytic domain" means the portion of an enzyme containing the catalytic machinery of the enzyme.

[0065] Cellulose binding domain: The term "cellulose binding domain" means the portion of an enzyme that mediates binding of the enzyme to amorphous regions of a cellulose substrate. The cellulose binding domain (CBD) is found either at the N-terminal or at the C-terminal extremity of an enzyme. A CBD is also referred to as a cellulose binding module or CBM. In one embodiment the CBM is amino acids 22 to 56 of SEQ ID NO: 2. In one embodiment the CBM is amino acids 21 to 57 of SEQ ID NO: 4. In one embodiment the CBM is amino acids 20 to 53 of SEQ ID NO: 6. In one embodiment the CBM is amino acids 20 to 53 of SEQ ID NO: 8. The CBM is separated from the catalytic domain by a linker sequence. The linker is in one embodiment amino acids 57 to 91 of SEQ ID NO: 2. The linker is in one embodiment amino acids 58 to 72 of SEQ ID NO: 4. The linker is in one embodiment amino acids 54 to 66 of SEQ ID NO: 6. The linker is in one embodiment amino acids 54 to 66 of SEQ ID NO: 8.

[0066] Medium stringency conditions: The term "medium stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 55.degree. C.

[0067] Medium-high stringency conditions: The term "medium-high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and either 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 60.degree. C.

[0068] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule, either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, which comprises one or more control sequences.

[0069] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs expression of the coding sequence.

[0070] Polypeptide having cellulolytic enhancing activity: The term "polypeptide having cellulolytic enhancing activity" means a GH61 polypeptide that catalyzes the enhancement of the hydrolysis of a cellulosic material by enzyme having cellulolytic activity. For purposes of the present invention, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein/g of cellulose in PCS, wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of a GH61 polypeptide having cellulolytic enhancing activity for 1-7 days at a suitable temperature, e.g., 50.degree. C., 55.degree. C., or 60.degree. C., and pH, e.g., 5.0 or 5.5, compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g of cellulose in PCS). In a preferred aspect, a mixture of CELLUCLAST.RTM. 1.5L (Novozymes NS, Bagsv.ae butted.rd, Denmark) in the presence of 2-3% of total protein weight Aspergillus oryzae beta-glucosidase (recombinantly produced in Aspergillus oryzae according to WO 02/095014) or 2-3% of total protein weight Aspergillus fumigatus beta-glucosidase (recombinantly produced in Aspergillus oryzae as described in WO 2002/095014) of cellulase protein loading is used as the source of the cellulolytic activity.

[0071] The GH61 polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a cellulosic material catalyzed by enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

[0072] Pretreated corn stover: The term "PCS" or "Pretreated Corn Stover" means a cellulosic material derived from corn stover by treatment with heat and dilute sulfuric acid, alkaline pretreatment, or neutral pretreatment.

[0073] Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0074] For purposes of the present invention, the sequence 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 Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

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

For purposes of the present invention, the sequence 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 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:

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

Subsequence: The term "subsequence" means a polynucleotide having one or more (e.g., several) nucleotides absent from the 5' and/or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having endoglucanase activity. In one aspect, a subsequence contains at least 900 nucleotides, e.g., at least 1000 nucleotides or at least 1100 nucleotides of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7.

[0075] Variant: The term "variant" means a polypeptide having endoglucanase activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position.

[0076] Very high stringency conditions: The term "very high stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 70.degree. C.

[0077] Very low stringency conditions: The term "very low stringency conditions" means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42.degree. C. in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 2.times.SSC, 0.2% SDS at 45.degree. C.

[0078] Xylan-containing material: The term "xylan-containing material" means any material comprising a plant cell wall polysaccharide containing a backbone of beta-(1-4)-linked xylose residues. Xylans of terrestrial plants are heteropolymers possessing a beta-(1-4)-D-xylopyranose backbone, which is branched by short carbohydrate chains. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.

[0079] In the processes of the present invention, any material containing xylan may be used. In a preferred aspect, the xylan-containing material is lignocellulose.

[0080] Xylan degrading activity or xylanolytic activity: The term "xylan degrading activity" or "xylanolytic activity" means a biological activity that hydrolyzes xylan-containing material. The two basic approaches for measuring xylanolytic activity include: (1) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl esterases). Recent progress in assays of xylanolytic enzymes was summarized in several publications including Biely and Puchard, Recent progress in the assays of xylanolytic enzymes, 2006, Journal of the Science of Food and Agriculture 86(11): 1636-1647; Spanikova and Biely, 2006, Glucuronoyl esterase--Novel carbohydrate esterase produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601; Herrmann, Vrsanska, Jurickova, Hirsch, Biely, and Kubicek, 1997, The beta-D-xylosidase of Trichoderma reesei is a multifunctional beta-D-xylan xylohydrolase, Biochemical Journal 321: 375-381.

[0081] Total xylan degrading activity can be measured by determining the reducing sugars formed from various types of xylan, including, for example, oat spelt, beechwood, and larchwood xylans, or by photometric determination of dyed xylan fragments released from various covalently dyed xylans. The most common total xylanolytic activity assay is based on production of reducing sugars from polymeric 4-O-methyl glucuronoxylan as described in Bailey, Biely, Poutanen, 1992, Interlaboratory testing of methods for assay of xylanase activity, Journal of Biotechnology 23(3): 257-270. Xylanase activity can also be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM sodium phosphate buffer pH 6 at 37.degree. C. One unit of xylanase activity is defined as 1.0 micromole of azurine produced per minute at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.

[0082] For purposes of the present invention, xylan degrading activity is determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50.degree. C., 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279.

[0083] Xylanase: The term "xylanase" means a 1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For purposes of the present invention, xylanase activity is determined with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100 and 200 mM sodium phosphate buffer pH 6 at 37.degree. C. One unit of xylanase activity is defined as 1.0 micromole of azurine produced per minute at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6 buffer.

DETAILED DESCRIPTION OF THE INVENTION

Polypeptides Having Endoglucanase Activity

[0084] In an embodiment, the present invention relates to isolated polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 2 of at least 78%, e.g., at least 79%, at least 80%, at least 83%, at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have endoglucanase activity. In an embodiment, the present invention relates to isolated polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 4 of at least 94%, e.g., at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have endoglucanase activity. In an embodiment, the present invention relates to isolated polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 6 of at least 76%, e.g., at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 83%, at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have endoglucanase activity. In an embodiment, the present invention relates to isolated polypeptides having a sequence identity to the mature polypeptide of SEQ ID NO: 8 of at least 81%, e.g., at least 82%, at least 83%, at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which have endoglucanase activity. In one aspect, the polypeptides differ by no more than 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9, from the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0085] A polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 or an allelic variant thereof; or is a fragment thereof having endoglucanase activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8. In another aspect, the polypeptide comprises or consists of amino acids 22 to 419 of SEQ ID NO: 2, amino acids 21 to 386 of SEQ ID NO: 4, amino acids 20 to 378 of SEQ ID NO: 6, or amino acids 20 to 378 of SEQ ID NO: 8.

[0086] In another embodiment, the present invention relates to an isolated polypeptide having endoglucanase activity encoded by a polynucleotide that hybridizes under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, (ii) the cDNA sequence thereof, or (iii) the full-length complement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

[0087] The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or a subsequence thereof, as well as the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8, or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having endoglucanase 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 DNA or cDNA of a cell 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 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with .sup.32P, .sup.3H, .sup.35S, biotin, or avidin). Such probes are encompassed by the present invention.

[0088] A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having endoglucanase 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 hybridizes with SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 or a subsequence thereof, the carrier material is used in a Southern blot.

[0089] For purposes of the present invention, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe corresponding to (i) SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; (iii) the cDNA sequence thereof; (iv) the full-length complement thereof; or (v) 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 or any other detection means known in the art.

[0090] In one aspect, the nucleic acid probe is a polynucleotide that encodes the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7; or the cDNA sequence thereof.

[0091] In another embodiment, the present invention relates to an isolated polypeptide having endoglucanase activity encoded by a polynucleotide having a sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or the cDNA sequence thereof, of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0092] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In an embodiment, the number of amino acid substitutions, deletions and/or insertions introduced into the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 is not more than 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9. The amino acid changes may be 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 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 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.

[0093] Examples of conservative substitutions are within the groups 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. Common substitutions 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.

[0094] 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.

[0095] Essential amino acids in a 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 endoglucanase 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 identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

[0096] 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, Biochemistry 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).

[0097] 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.

[0098] The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or the C-terminus of a region of another polypeptide.

[0099] The polypeptide may be a fusion polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide 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 fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

[0100] A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; 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; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995, Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

Sources of Polypeptides Having Endoglucanase Activity

[0101] A polypeptide having endoglucanase activity of the present invention may be obtained from microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0102] The polypeptide may be a Trametes polypeptide.

[0103] In another aspect, the polypeptide is a Trametes versicolor polypeptide, e.g., a polypeptide obtained from Trametes versicolor Strain NN055586, or in another aspect the polypeptide is a polypeptide from a species related to Trametes versicolor, for example from another Trametes species.

[0104] 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.

[0105] 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 (DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0106] The polypeptide may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra).

Catalytic Domains

[0107] The present invention also relates to isolated polypeptides comprising a catalytic domain selected from the group consisting of: [0108] (a) a catalytic domain having at least 60% sequence identity to the catalytic domain of SEQ ID NO: 2 (for example, amino acids 92 to 419 of SEQ ID NO: 2), SEQ ID NO: 4 (for example, amino acids 73 to 386 of SEQ ID NO: 4), SEQ ID NO: 6 (for example, amino acids 67 to 378 of SEQ ID NO: 6), or SEQ ID NO: 8 (for example, amino acids 67 to 378 of SEQ ID NO: 8);

[0109] (b) a catalytic domain encoded by a polynucleotide having at least 60% sequence identity to the catalytic domain coding sequence of SEQ ID NO: 1 (for example, nucleotides 459-564, 621-838, 896-993, 1050-1133, 1184-1345, 1410-1585, and 1639-1778 of SEQ ID NO: 1), SEQ ID NO: 3 (for example, nucleotides 334-459, 532-700, 760-808, 862-872, 931-956, 1019-1093, 1156-1312, 1384-1640, and 1711-1782 of SEQ ID NO: 3), SEQ ID NO: 5 (for example, nucleotides 361-408, 463-556, 613-618, 669-909, 975-1192, 1245-1297, 1362-1471, 1540-1688, and 1753-1769 of SEQ ID NO: 5), or SEQ ID NO: 7 (for example, nucleotides 355-402, 457-550, 607-612, 666-906, 969-1186, 1239-1291, 1353-1462, 1520-1668, and 1727-1743 of SEQ ID NO: 7); [0110] (c) a variant of a catalytic domain comprising a substitution, deletion, and/or insertion of one or more (several) amino acids of the catalytic domain of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8; and [0111] (d) a fragment of a catalytic domain of (a), (b), or (c), which has endoglucanase activity.

[0112] The catalytic domain preferably has a degree of sequence identity to the catalytic domain of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8 of at least 60%, e.g. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In an aspect, the catalytic domain comprises an amino acid sequence that differs by ten amino acids, e.g., by five amino acids, by four amino acids, by three amino acids, by two amino acids, and by one amino acid from the catalytic domain of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8.

[0113] The catalytic domain preferably comprises or consists of the catalytic domain of SEQ ID NO: 2 or an allelic variant thereof; or is a fragment thereof having endoglucanase activity. In another preferred aspect, the catalytic domain comprises or consists of amino acids 92 to 419 of SEQ ID NO: 2.

[0114] The catalytic domain preferably comprises or consists of the catalytic domain of SEQ ID NO: 4 or an allelic variant thereof; or is a fragment thereof having endoglucanase activity. In another preferred aspect, the catalytic domain comprises or consists of amino acids 73 to 386 of SEQ ID NO: 4.

[0115] The catalytic domain preferably comprises or consists of the catalytic domain of SEQ ID NO: 6 or an allelic variant thereof; or is a fragment thereof having endoglucanase activity. In another preferred aspect, the catalytic domain comprises or consists of amino acids 67 to 378 of SEQ ID NO: 6.

[0116] The catalytic domain preferably comprises or consists of the catalytic domain of SEQ ID NO: 8 or an allelic variant thereof; or is a fragment thereof having endoglucanase activity. In another preferred aspect, the catalytic domain comprises or consists of amino acids 67 to 378 of SEQ ID NO: 8.

[0117] In an embodiment, the catalytic domain may be encoded by a polynucleotide that hybridizes under very low stringency conditions, low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, and very high stringency conditions (as defined above) with (i) the catalytic domain coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, (ii) the cDNA sequence contained in the catalytic domain coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or (iii) the full-length complementary strand of (i) or (ii) (J. Sambrook et al., 1989, supra).

[0118] The catalytic domain may be encoded by a polynucleotide having a degree of sequence identity to the catalytic domain coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7 of at least 60%, e.g. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, which encode a polypeptide having endoglucanase activity.

[0119] In one aspect, the polynucleotide encoding the catalytic domain comprises or consists of nucleotides 459 to 1778 of SEQ ID NO: 1 or the cDNA sequence thereof. In particular the polynucleotide encoding the catalytic domain comprises or consists of nucleotides 459-564, 621-838, 896-993, 1050-1133, 1184-1345, 1410-1585, and 1639-1778 of SEQ ID NO: 1.

[0120] In one aspect, the polynucleotide encoding the catalytic domain comprises or consists of nucleotides 334 to 1782 of SEQ ID NO: 3 or the cDNA sequence thereof. In particular the polynucleotide encoding the catalytic domain comprises or consists of nucleotides 334-459, 532-700, 760-808, 862-872, 931-956, 1019-1093, 1156-1312, 1384-1640, and 1711-1782 of SEQ ID NO: 3.

[0121] In one aspect, the polynucleotide encoding the catalytic domain comprises or consists of nucleotides 361 to 1769 of SEQ ID NO: 5 or the cDNA sequence thereof. In particular the polynucleotide encoding the catalytic domain comprises or consists of nucleotides 361-408, 463-556, 613-618, 669-909, 975-1192, 1245-1297, 1362-1471, 1540-1688, and 1753-1769 of SEQ ID NO: 5.

[0122] In one aspect, the polynucleotide encoding the catalytic domain comprises or consists of nucleotides 355 to 1743 of SEQ ID NO: 7 or the cDNA sequence thereof. In particular the polynucleotide encoding the catalytic domain comprises or consists of nucleotides 355-402, 457-550, 607-612, 666-906, 969-1186, 1239-1291, 1353-1462, 1520-1668, and 1727-1743 of SEQ ID NO: 7.

Polynucleotides

[0123] The present invention also relates to isolated polynucleotides encoding a polypeptide of the present invention, as described herein.

[0124] The techniques used to isolate or clone a polynucleotide are known in the art and include isolation from genomic DNA or cDNA, or a combination thereof. The cloning of the polynucleotides from 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), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) may be used. The polynucleotides may be cloned from a strain of Trametes, or a related organism and thus, for example, may be an allelic or species variant of the polypeptide encoding region of the polynucleotide.

[0125] Modification of a polynucleotide encoding a polypeptide of the present invention may be necessary for synthesizing 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., variants that differ in specific activity, thermostability, pH optimum, or the like. The variants may be constructed on the basis of the polynucleotide presented as the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, or SEQ ID NO: 7, or the cDNA sequence thereof, e.g., a subsequence thereof, and/or by introduction of nucleotide substitutions that do not result in a change in the amino acid sequence of the polypeptide, 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.

Nucleic Acid Constructs

[0126] The present invention also relates to nucleic acid constructs comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0127] A polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0128] The control sequence may be a promoter, a polynucleotide that is recognized by a host cell for expression of a polynucleotide encoding a polypeptide of the present invention. The promoter contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the host cell 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.

[0129] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a bacterial host cell are the promoters obtained from the Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus licheniformis penicillinase gene (penP), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus subtilis levansucrase gene (sacB), Bacillus subtilis xyIA and xyIB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trc promoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicolor agarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80: 21-25). Further promoters are described in "Useful proteins from recombinant bacteria" in Gilbert et al., 1980, Scientific American 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99/43835.

[0130] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in a filamentous fungal host cell are promoters obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusarium venenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO 00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor miehei lipase, Rhizomucor miehei aspartic proteinase, 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 modified promoter from an Aspergillus neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus triose phosphate isomerase gene; non-limiting examples include modified promoters from an Aspergillus niger neutral alpha-amylase gene in which the untranslated leader has been replaced by an untranslated leader from an Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant, truncated, and hybrid promoters thereof.

[0131] 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.

[0132] The control sequence may also be a transcription terminator, which is recognized by a host cell to terminate transcription. The terminator is operably linked to the 3'-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the host cell may be used in the present invention.

[0133] Preferred terminators for bacterial host cells are obtained from the genes for Bacillus clausii alkaline protease (aprH), Bacillus licheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA (rrnB).

[0134] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0135] 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.

[0136] The control sequence may also be an mRNA stabilizer region downstream of a promoter and upstream of the coding sequence of a gene which increases expression of the gene.

[0137] Examples of suitable mRNA stabilizer regions are obtained from a Bacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177: 3465-3471).

[0138] The control sequence may also be a leader, a nontranslated region of an mRNA that is important for translation by the host cell. The leader is operably linked to the 5'-terminus of the polynucleotide encoding the polypeptide. Any leader that is functional in the host cell may be used.

[0139] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0140] 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).

[0141] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide 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 may be used.

[0142] Preferred polyadenylation sequences for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-like protease.

[0143] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0144] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and directs the polypeptide into the cell's secretory pathway. The 5'-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a 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 may be used.

[0145] 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 licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus alpha-amylase, 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.

[0146] Effective signal peptide coding sequences for filamentous fungal host cells are the signal peptide coding sequences obtained from the genes for Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicola insolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucor miehei aspartic proteinase.

[0147] 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.

[0148] The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an 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), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

[0149] Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

[0150] It may also be desirable to add regulatory sequences that regulate expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause 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 Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA alpha-amylase promoter, and Aspergillus oryzae glucoamylase promoter may be used. 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 polynucleotide encoding the polypeptide would be operably linked with the regulatory sequence.

Expression Vectors

[0151] 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 nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide 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.

[0152] 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 polynucleotide. 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 vector may be a linear or closed circular plasmid.

[0153] 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.

[0154] The vector preferably contains one or more 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.

[0155] Examples of bacterial selectable markers are Bacillus licheniformis or Bacillus subtilis dal genes, or markers that confer antibiotic resistance such as ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to, 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 Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and a Streptomyces hygroscopicus bar gene.

[0156] The vector preferably contains 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.

[0157] 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 non-homologous recombination. Alternatively, the vector may contain additional polynucleotides 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 contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence 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 polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0158] 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" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0159] 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 pAMR1 permitting replication in Bacillus.

[0160] 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.

[0161] Examples of origins of replication useful in a filamentous fungal cell are AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al., 1987, Nucleic Acids Res. 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.

[0162] More than one copy of a polynucleotide of the present invention may be inserted into a host cell to increase production of a polypeptide. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0163] 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

[0164] The present invention also relates to recombinant host cells, comprising a polynucleotide of the present invention operably linked to one or more control sequences that direct the production of a polypeptide of the present invention. A construct or vector comprising a polynucleotide is introduced into a host cell so that the construct or 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.

[0165] 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.

[0166] The prokaryotic host cell may be any Gram-positive or Gram-negative bacterium. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter, Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

[0167] The bacterial host cell may be any Bacillus cell including, but 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.

[0168] The bacterial host cell may also be any Streptococcus cell including, but not limited to, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

[0169] The bacterial host cell may also be any Streptomyces cell including, but not limited to, Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividans cells.

[0170] The introduction of DNA into a Bacillus cell may be effected by protoplast transformation (see, e.g., Chang and Cohen, 1979, Mol. Gen. Genet. 168: 111-115), competent cell transformation (see, e.g., Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau and Davidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), electroporation (see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or conjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169: 5271-5278). The introduction of DNA into an E. coli cell may 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 be effected by protoplast transformation, electroporation (see, e.g., Gong et al., 2004, Folia Microbiol. (Praha) 49: 399-405), conjugation (see, e.g., Mazodier et al., 1989, J. Bacteriol. 171: 3583-3585), or 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 be effected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol. Methods 64: 391-397) or conjugation (see, e.g., Pinedo and Smets, 2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA into a Streptococcus cell may be effected by natural competence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32: 1295-1297), protoplast transformation (see, e.g., Catt and Jollick, 1991, Microbios 68: 189-207), electroporation (see, e.g., Buckley et al., 1999, Appl. Environ. Microbiol. 65: 3800-3804), or conjugation (see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, any method known in the art for introducing DNA into a host cell can be used.

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

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

[0173] The fungal host cell may be 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, Passmore, and Davenport, editors, Soc. App. Bacteriol. Symposium Series No. 9, 1980).

[0174] The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0175] The fungal host cell may be 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] The filamentous fungal host cell may be 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] For example, the filamentous fungal host cell may be an Aspergillus awamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium merdarium, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporium zonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum, Phanerochaete chrysosporium, 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 238023, Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81: 1470-1474, and Christensen et al., 1988, Bio/Technology 6: 1419-1422. 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, J. Bacteriol. 153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. 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 a Trametes cell. In a more preferred aspect, the cell is a Trametes versicolor cell. In a most preferred aspect, the cell is Trametes versicolor Strain NN055586.

[0180] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

[0181] The host cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors 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, it can be recovered from cell lysates.

[0182] The polypeptide may be detected using methods known in the art that are specific for the polypeptides. These detection methods include, but are not limited to, 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.

[0183] The 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, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

[0184] The polypeptide 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, Janson and Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.

[0185] In an alternative aspect, the polypeptide is not recovered, but rather a host cell of the present invention expressing the polypeptide is used as a source of the polypeptide.

Plants

[0186] The present invention also relates to isolated plants, e.g., a transgenic plant, plant part, or plant cell, comprising a polynucleotide of the present invention so as to express and produce a polypeptide or domain in recoverable quantities. The polypeptide or domain may be recovered from the plant or plant part. Alternatively, the plant or plant part containing the polypeptide or domain 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 utilization of the invention are also considered plant parts, e.g., embryos, endosperms, aleurone and seed 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 the polypeptide or domain 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 expression constructs encoding the polypeptide or domain 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 or domain operably linked with appropriate regulatory sequences required for expression of the polynucleotide in the plant or plant part of choice. Furthermore, the expression construct may comprise a selectable marker useful for identifying plant 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 or domain is desired to be expressed. For instance, the expression of the gene encoding a polypeptide or domain 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, or 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 Cell Physiol. 39: 885-889), a Vicia faba promoter from the legumin B4 and the unknown seed protein gene from Vicia faba (Conrad et al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seed oil body protein (Chen et al., 1998, Plant Cell Physiol. 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 Physiol. 102: 991-1000), the chlorella virus adenine methyltransferase gene promoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldP gene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248: 668-674), or a wound inducible promoter such as the potato pin2 promoter (Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promoter may be induced 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 or domain in the plant. For instance, the promoter enhancer element may be an intron that is placed between the promoter and the polynucleotide encoding a polypeptide or domain. 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] Agrobacterium tumefaciens-mediated gene transfer is a method for generating transgenic dicots (for a review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and for transforming monocots, although other transformation methods may be used for these plants. A method 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 J. 2: 275-281; Shimamoto, 1994, Curr. Opin. Biotechnol. 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 Mol. Biol. 21: 415-428. Additional transformation methods include those described in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which are herein incorporated by reference in their entirety).

[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] In addition to direct transformation of a particular plant genotype with a construct of the present invention, transgenic plants may be made by crossing a plant having the construct to a second plant lacking the construct. For example, a construct encoding a polypeptide or domain can be introduced into a particular plant variety by crossing, without the need for ever directly transforming a plant of that given variety. Therefore, the present invention encompasses not only a plant directly regenerated from cells which have been transformed in accordance with the present invention, but also the progeny of such plants. As used herein, progeny may refer to the offspring of any generation of a parent plant prepared in accordance with the present invention. Such progeny may include a DNA construct prepared in accordance with the present invention. Crossing results in the introduction of a transgene into a plant line by cross pollinating a starting line with a donor plant line. Non-limiting examples of such steps are described in U.S. Pat. No. 7,151,204.

[0201] Plants may be generated through a process of backcross conversion. For example, plants include plants referred to as a backcross converted genotype, line, inbred, or hybrid.

[0202] Genetic markers may be used to assist in the introgression of one or more transgenes of the invention from one genetic background into another. Marker assisted selection offers advantages relative to conventional breeding in that it can be used to avoid errors caused by phenotypic variations. Further, genetic markers may provide data regarding the relative degree of elite germplasm in the individual progeny of a particular cross. For example, when a plant with a desired trait which otherwise has a non-agronomically desirable genetic background is crossed to an elite parent, genetic markers may be used to select progeny which not only possess the trait of interest, but also have a relatively large proportion of the desired germplasm. In this way, the number of generations required to introgress one or more traits into a particular genetic background is minimized.

[0203] The present invention also relates to methods of producing a polypeptide or domain of the present invention comprising (a) cultivating a transgenic plant or a plant cell comprising a polynucleotide encoding the polypeptide or domain under conditions conducive for production of the polypeptide or domain; and (b) recovering the polypeptide or domain.

Compositions

[0204] 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 endoglucanase activity of the composition has been increased, e.g., with an enrichment factor of at least 1.1.

[0205] 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 one or more (e.g., several) enzymes selected from the group consisting of a cellulase, a hemicellulase, GH61 polypeptide, an expansin, an esterase, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin.

[0206] 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.

[0207] 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.

Uses

[0208] The present invention is also directed to the following processes for using the polypeptides having endoglucanase activity, or compositions thereof.

[0209] The present invention also relates to processes for degrading a cellulosic material, comprising: treating the cellulosic material with an enzyme composition in the presence of a polypeptide having endoglucanase activity of the present invention. In one aspect, the processes further comprise recovering the degraded or converted cellulosic material. Soluble products of degradation or conversion of the cellulosic material can be separated from insoluble cellulosic material using a method known in the art such as, for example, centrifugation, filtration, or gravity settling.

[0210] The present invention also relates to processes of producing a fermentation product, comprising: (a) saccharifying a cellulosic material with an enzyme composition in the presence of a polypeptide having endoglucanase activity of the present invention; (b) fermenting the saccharified cellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[0211] The present invention also relates to processes of fermenting a cellulosic material, comprising: fermenting the cellulosic material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic material is saccharified with an enzyme composition in the presence of a polypeptide having endoglucanase activity of the present invention. In one aspect, the fermenting of the cellulosic material produces a fermentation product. In another aspect, the processes further comprise recovering the fermentation product from the fermentation.

[0212] The processes of the present invention can be used to saccharify the cellulosic material to fermentable sugars and to convert the fermentable sugars to many useful fermentation products, e.g., fuel, potable ethanol, and/or platform chemicals (e.g., acids, alcohols, ketones, gases, and the like). The production of a desired fermentation product from the cellulosic material typically involves pretreatment, enzymatic hydrolysis (saccharification), and fermentation.

[0213] The processing of the cellulosic material according to the present invention can be accomplished using methods conventional in the art. Moreover, the processes of the present invention can be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention.

[0214] Hydrolysis (saccharification) and fermentation, separate or simultaneous, include, but are not limited to, separate hydrolysis and fermentation (SHF); simultaneous saccharification and fermentation (SSF); simultaneous saccharification and co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF); and direct microbial conversion (DMC), also sometimes called consolidated bioprocessing (CBP). SHF uses separate process steps to first enzymatically hydrolyze the cellulosic material to fermentable sugars, e.g., glucose, cellobiose, and pentose monomers, and then ferment the fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of the cellulosic material 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 co-fermentation of multiple sugars (Sheehan and Himmel, 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 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, hydrolysis, and fermentation) in one or more (e.g., several) steps where the same organism is used to produce the enzymes for conversion of the cellulosic material to fermentable sugars and to convert the fermentable sugars into a final product (Lynd et al., 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 processes of the present invention.

[0215] 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 et al., 2003, Optimal control in fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum. Technology 25: 33-38; Gusakov and Sinitsyn, 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 et al., 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 et al., 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.

[0216] Pretreatment.

[0217] In practicing the processes of the present invention, any pretreatment process known in the art can be used to disrupt plant cell wall components of the cellulosic material (Chandra et al., 2007, Substrate pretreatment: The key to effective enzymatic hydrolysis of lignocellulosics?, Adv. Biochem. Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment of lignocellulosic materials for efficient bioethanol production, Adv. Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman, 2009, Pretreatments to enhance the digestibility of lignocellulosic biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005, Features of promising technologies for pretreatment of lignocellulosic biomass, Bioresource Technol. 96: 673-686; Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: A review, Int. J. of Mol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key to unlocking low-cost cellulosic ethanol, Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40).

[0218] The cellulosic material can also be subjected to particle size reduction, sieving, pre-soaking, wetting, washing, and/or conditioning prior to pretreatment using methods known in the art.

[0219] Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment, and biological pretreatment. Additional pretreatments include ammonia percolation, ultrasound, electroporation, microwave, supercritical CO.sub.2, supercritical H.sub.2O, ozone, ionic liquid, and gamma irradiation pretreatments.

[0220] 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 enzyme hydrolysis to release fermentable sugars, such as glucose, xylose, and/or cellobiose. In most cases the pretreatment step itself results in some conversion of biomass to fermentable sugars (even in absence of enzymes).

[0221] 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., hemicellulose, accessible to enzymes. The cellulosic 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 performed at 140-250.degree. C., e.g., 160-200.degree. C. or 170-190.degree. C., where the optimal temperature range depends on addition of a chemical catalyst. Residence time for the steam pretreatment is preferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10 minutes, where the optimal residence time depends on temperature range and 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.

[0222] Chemical Pretreatment: The term "chemical treatment" refers to any chemical pretreatment that promotes the separation and/or release of cellulose, hemicellulose, and/or lignin. Such a pretreatment can convert crystalline cellulose to amorphous cellulose. Examples of suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze explosion (AFEX), ammonia percolation (APR), ionic liquid, and organosolv pretreatments.

[0223] A catalyst such as H.sub.2SO.sub.4 or SO.sub.2 (typically 0.3 to 5% 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). In dilute acid pretreatment, the cellulosic material is mixed with dilute acid, typically H.sub.2SO.sub.4, and water to form a slurry, heated by steam to the desired temperature, and after a residence time flashed to atmospheric pressure. The dilute acid pretreatment can be performed with a number of reactor designs, e.g., plug-flow reactors, counter-current reactors, or continuous counter-current shrinking bed reactors (Duff and Murray, 1996, supra; Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

[0224] Several methods of pretreatment under alkaline conditions can also be used. These alkaline pretreatments include, but are not limited to, sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), and ammonia fiber/freeze explosion (AFEX).

[0225] Lime pretreatment is performed with calcium oxide or calcium hydroxide at temperatures of 85-150.degree. C. and residence times from 1 hour to several days (Wyman et al., 2005, Bioresource Technol. 96: 1959-1966; Mosier et al., 2005, Bioresource Technol. 96: 673-686). WO 2006/110891, WO 2006/110899, WO 2006/110900, and WO 2006/110901 disclose pretreatment methods using ammonia.

[0226] Wet oxidation is a thermal pretreatment performed typically at 180-200.degree. C. for 5-15 minutes with addition of an oxidative agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64: 139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). The pretreatment is performed preferably at 1-40% dry matter, e.g., 2-30% dry matter or 5-20% dry matter, and often the initial pH is increased by the addition of alkali such as sodium carbonate.

[0227] 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).

[0228] Ammonia fiber explosion (AFEX) involves treating the cellulosic material with liquid or gaseous ammonia at moderate temperatures such as 90-150.degree. C. and high pressure such as 17-20 bar for 5-10 minutes, where the dry matter content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35; Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141; Teymouri et al., 2005, Bioresource Technol. 96: 2014-2018). During AFEX pretreatment cellulose and hemicelluloses remain relatively intact. Lignin-carbohydrate complexes are cleaved.

[0229] Organosolv pretreatment delignifies the cellulosic material by extraction using aqueous ethanol (40-60% ethanol) at 160-200.degree. C. for 30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94: 851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121: 219-230). Sulphuric acid is usually added as a catalyst. In organosolv pretreatment, the majority of hemicellulose and lignin is removed.

[0230] Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. and Biotechnol. 105-108: 69-85, and Mosier et al., 2005, Bioresource Technology 96: 673-686, and U.S. Published Application 2002/0164730.

[0231] In one aspect, the chemical pretreatment is preferably carried out as a dilute acid treatment, and more preferably as a continuous dilute 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, e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in the range from preferably 0.01 to 10 wt % acid, e.g., 0.05 to 5 wt % acid or 0.1 to 2 wt % acid. The acid is contacted with the cellulosic material and held at a temperature in the range of preferably 140-200.degree. C., e.g., 165-190.degree. C., for periods ranging from 1 to 60 minutes.

[0232] 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 %, e.g., 20-70 wt % or 30-60 wt %, such as around 40 wt %. The pretreated cellulosic material can be unwashed or washed using any method known in the art, e.g., washed with water.

[0233] Mechanical Pretreatment or Physical Pretreatment: The term "mechanical pretreatment" or "physical pretreatment" refers to any pretreatment that promotes size reduction of particles. For example, such pretreatment can involve various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).

[0234] The cellulosic material can be pretreated both physically (mechanically) and chemically. Mechanical or physical pretreatment can be coupled with steaming/steam explosion, hydrothermolysis, dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof. In one aspect, high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi. In another aspect, high temperature means temperatures in the range of about 100 to about 300.degree. C., e.g., about 140 to about 200.degree. C. In a preferred aspect, mechanical or physical pretreatment is performed in a batch-process using a 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. The physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired.

[0235] Accordingly, in a preferred aspect, the cellulosic material is subjected to physical (mechanical) or chemical pretreatment, or any combination thereof, to promote the separation and/or release of cellulose, hemicellulose, and/or lignin.

[0236] 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 and/or enzymes (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993, Physicochemical and biological treatments for enzymatic/microbial conversion of 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).

[0237] Saccharification.

[0238] In the hydrolysis step, also known as saccharification, the cellulosic material, e.g., pretreated, is hydrolyzed to break down cellulose and/or hemicellulose to fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides. The hydrolysis is performed enzymatically by an enzyme composition in the presence of a polypeptide having endoglucanase activity of the present invention. The enzymes of the compositions can be added simultaneously or sequentially.

[0239] 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 one 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 cellulosic material is fed gradually to, for example, an enzyme containing hydrolysis solution.

[0240] 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 120 hours, e.g., about 16 to about 72 hours or about 24 to about 48 hours. The temperature is in the range of preferably about 25.degree. C. to about 70.degree. C., e.g., about 30.degree. C. to about 65.degree. C., about 40.degree. C. to about 60.degree. C., or about 50.degree. C. to about 55.degree. C. The pH is in the range of preferably about 3 to about 8, e.g., about 3.5 to about 7, about 4 to about 6, or about 5.0 to about 5.5. The dry solids content is in the range of preferably about 5 to about 50 wt %, e.g., about 10 to about 40 wt % or about 20 to about 30 wt %.

[0241] The enzyme compositions can comprise any protein useful in degrading the cellulosic material.

[0242] In one aspect, the enzyme composition comprises or further comprises one or more (e.g., several) proteins selected from the group consisting of a cellulase, a GH61 polypeptide having cellulolytic enhancing activity, a hemicellulase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin. In another aspect, the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. In another aspect, the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase.

[0243] In another aspect, the enzyme composition comprises one or more (e.g., several) cellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises one or more (e.g., several) cellulolytic enzymes and one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises one or more (e.g., several) enzymes selected from the group of cellulolytic enzymes and hemicellulolytic enzymes. In another aspect, the enzyme composition comprises an endoglucanase. In another aspect, the enzyme composition comprises a cellobiohydrolase. In another aspect, the enzyme composition comprises a beta-glucosidase. In another aspect, the enzyme composition comprises a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises a cellobiohydrolase and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises a beta-glucosidase and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase and a cellobiohydrolase. In another aspect, the enzyme composition comprises an endoglucanase and a beta-glucosidase. In another aspect, the enzyme composition comprises a cellobiohydrolase and a beta-glucosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase, a beta-glucosidase, and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises a cellobiohydrolase, a beta-glucosidase, and a polypeptide having cellulolytic enhancing activity. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. In another aspect, the enzyme composition comprises an endoglucanase, a cellobiohydrolase, a beta-glucosidase, and a polypeptide having cellulolytic enhancing activity.

[0244] In another aspect, the enzyme composition comprises an acetylmannan esterase. In another aspect, the enzyme composition comprises an acetylxylan esterase. In another aspect, the enzyme composition comprises an arabinanase (e.g., alpha-L-arabinanase). In another aspect, the enzyme composition comprises an arabinofuranosidase (e.g., alpha-L-arabinofuranosidase).

[0245] In another aspect, the enzyme composition comprises a coumaric acid esterase. In another aspect, the enzyme composition comprises a feruloyl esterase. In another aspect, the enzyme composition comprises a galactosidase (e.g., alpha-galactosidase and/or beta-galactosidase). In another aspect, the enzyme composition comprises a glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, the enzyme composition comprises a glucuronoyl esterase. In another aspect, the enzyme composition comprises a mannanase. In another aspect, the enzyme composition comprises a mannosidase (e.g., beta-mannosidase). In another aspect, the enzyme composition comprises a xylanase. In a preferred aspect, the xylanase is a Family 10 xylanase. In another aspect, the enzyme composition comprises a xylosidase (e.g., beta-xylosidase).

[0246] In another aspect, the enzyme composition comprises an esterase. In another aspect, the enzyme composition comprises an expansin. In another aspect, the enzyme composition comprises a laccase. In another aspect, the enzyme composition comprises a ligninolytic enzyme. In a preferred aspect, the ligninolytic enzyme is a manganese peroxidase. In another preferred aspect, the ligninolytic enzyme is a lignin peroxidase. In another preferred aspect, the ligninolytic enzyme is a H.sub.2O.sub.2-producing enzyme. In another aspect, the enzyme composition comprises a pectinase. In another aspect, the enzyme composition comprises a peroxidase. In another aspect, the enzyme composition comprises a protease. In another aspect, the enzyme composition comprises a swollenin

[0247] In the processes of the present invention, the enzyme(s) can be added prior to or during fermentation, e.g., during saccharification or during or after propagation of the fermenting microorganism(s).

[0248] One or more (e.g., several) components of the enzyme composition may be wild-type proteins, recombinant proteins, or a combination of wild-type proteins and recombinant proteins. For example, one or more (e.g., several) components may be native proteins of a cell, which is used as a host cell to express recombinantly one or more (e.g., several) other components of the enzyme composition. One or more (e.g., several) components of the enzyme composition may be produced as monocomponents, which are then combined to form the enzyme composition. The enzyme composition may be a combination of multicomponent and monocomponent protein preparations.

[0249] The enzymes used in the processes of the present invention may be in any form suitable for use, such as, for example, a crude fermentation broth with or without cells removed, a cell lysate with or without cellular debris, a semi-purified or purified enzyme preparation, or a host cell as a source of the enzymes. The enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme. 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 processes.

[0250] The optimum amounts of the enzymes and polypeptides having endoglucanase activity depend on several factors including, but not limited to, the mixture of component cellulolytic enzymes, the cellulosic material, the concentration of cellulosic material, the pretreatment(s) of the cellulosic material, temperature, time, pH, and inclusion of fermenting organism (e.g., yeast for Simultaneous Saccharification and Fermentation).

[0251] In one aspect, an effective amount of cellulolytic or hemicellulolytic enzyme to the cellulosic material is about 0.5 to about 50 mg, e.g., about 0.5 to about 40 mg, about 0.5 to about 25 mg, about 0.75 to about 20 mg, about 0.75 to about 15 mg, about 0.5 to about 10 mg, or about 2.5 to about 10 mg per g of the cellulosic material.

[0252] In another aspect, an effective amount of a polypeptide having endoglucanase activity to the cellulosic material is about 0.01 to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg, about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 to about 1.25 mg, or about 0.25 to about 1.0 mg per g of the cellulosic material.

[0253] In another aspect, an effective amount of a polypeptide having endoglucanase activity to cellulolytic or hemicellulolytic enzyme is about 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g, about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g per g of cellulolytic or hemicellulolytic enzyme. The polypeptides having cellulolytic enzyme activity or hemicellulolytic enzyme activity as well as other proteins/polypeptides useful in the degradation of the cellulosic material, e.g., GH61 polypeptides having cellulolytic enhancing activity (collectively hereinafter "polypeptides having enzyme activity") can be derived or obtained from any suitable origin, including, bacterial, fungal, yeast, plant, or mammalian origin. 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 (e.g., several) 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.

[0254] A polypeptide having 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, Caldicellulosiruptor, Acidothermus, Thermobifidia, or Oceanobacillus polypeptide having 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 enzyme activity.

[0255] In one 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 enzyme activity.

[0256] In another aspect, the polypeptide is a Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus polypeptide having enzyme activity.

[0257] In another aspect, the polypeptide is a Streptomyces achromogenes, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces griseus, or Streptomyces lividans polypeptide having enzyme activity.

[0258] The polypeptide having 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 enzyme activity; or more preferably a filamentous fungal polypeptide such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryosphaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria polypeptide having enzyme activity.

[0259] In one aspect, the polypeptide is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis polypeptide having enzyme activity.

[0260] In another 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 enzyme activity.

[0261] Chemically modified or protein engineered mutants of polypeptides having enzyme activity may also be used.

[0262] One or more (e.g., several) components of the 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.

[0263] In one aspect, the one or more (e.g., several) cellulolytic enzymes comprise a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLIC.TM. CTec (Novozymes NS), CELLIC.TM. CTec2 (Novozymes NS), CELLUCLAST.TM. (Novozymes NS), NOVOZYM.TM. 188 (Novozymes NS), CELLUZYME.TM. (Novozymes NS), CEREFLO.TM. (Novozymes NS), and ULTRAFLO.TM. (Novozymes NS), ACCELERASE.TM. (Genencor Int.), LAMINEX.TM. (Genencor Int.), SPEZYME.TM. CP (Genencor Int.), FILTRASE.RTM. NL (DSM); METHAPLUS.RTM. S/L 100 (DSM). ROHAMENT.TM. 7069 W (Rohm GmbH), FIBREZYME.RTM. LDI (Dyadic International, Inc.), FIBREZYME.RTM. LBR (Dyadic International, Inc.), or VISCOSTAR.RTM. 150L (Dyadic International, Inc.). The cellulase enzymes are added in amounts effective from about 0.001 to about 5.0 wt % of solids, e.g., about 0.025 to about 4.0 wt % of solids or about 0.005 to about 2.0 wt % of solids.

[0264] Examples of bacterial endoglucanases that can be used in the processes 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).

[0265] Examples of fungal endoglucanases that can be used in the present invention, include, but are not limited to, a Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45: 253-263, Trichoderma reesei Cel7B endoglucanase I (GENBANK.TM. accession no. M15665), Trichoderma reesei endoglucanase II (Saloheimo, et al., 1988, Gene 63:11-22), Trichoderma reesei Cel5A endoglucanase II (GENBANK.TM. accession no. M19373), Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64: 555-563, GENBANK.TM. accession no. AB003694), Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology 13: 219-228, GENBANK.TM. accession no. Z33381), Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884), 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.TM. accession no. L29381), Humicola grisea var. thermoidea endoglucanase (GENBANK.TM. accession no. AB003107), Melanocarpus albomyces endoglucanase (GENBANK.TM. accession no. MAL515703), Neurospora crassa endoglucanase (GENBANK.TM. accession no. XM.sub.--324477), Humicola insolens endoglucanase V, Myceliophthora thermophila CBS 117.65 endoglucanase, basidiomycete CBS 495.95 endoglucanase, basidiomycete CBS 494.95 endoglucanase, Thielavia terrestris NRRL 8126 CEL6B endoglucanase, Thielavia terrestris NRRL 8126 CEL6C endoglucanase, Thielavia terrestris NRRL 8126 CEL7C endoglucanase, Thielavia terrestris NRRL 8126 CEL7E endoglucanase, Thielavia terrestris NRRL 8126 CEL7F endoglucanase, Cladorrhinum foecundissimum ATCC 62373 CEL7A endoglucanase, and Trichoderma reesei strain No. VTT-D-80133 endoglucanase (GENBANK.TM. accession no. M15665).

[0266] Examples of cellobiohydrolases useful in the present invention include, but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO 2011/059740), Chaetomium thermophilum cellobiohydrolase I, Chaetomium thermophilum cellobiohydrolase II, Humicola insolens cellobiohydrolase I, Myceliophthora thermophila cellobiohydrolase II (WO 2009/042871), Thielavia hyrcanie cellobiohydrolase II (WO 2010/141325), Thielavia terrestris cellobiohydrolase II (CEL6A, WO 2006/074435), Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, and Trichophaea saccata cellobiohydralase II (WO 2010/057086).

[0267] Examples of beta-glucosidases useful in the present invention include, but are not limited to, beta-glucosidases from Aspergillus aculeatus (Kawaguchi et al., 1996, Gene 173: 287-288), Aspergillus fumigatus (WO 2005/047499), Aspergillus niger (Dan et al., 2000, J. Biol. Chem. 275: 4973-4980), Aspergillus oryzae (WO 2002/095014), Penicillium brasilianum IBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO 2011/035029), and Trichophaea saccata (WO 2007/019442).

[0268] The beta-glucosidase may be a fusion protein. In one aspect, the beta-glucosidase is an Aspergillus oryzae beta-glucosidase variant BG fusion protein (WO 2008/057637) or an Aspergillus oryzae beta-glucosidase fusion protein (WO 2008/057637.

[0269] Other useful 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.

[0270] Other cellulolytic enzymes that may be used in the present invention are described in WO 98/13465, WO 98/015619, WO 98/015633, WO 99/06574, WO 99/10481, WO 99/025847, WO 99/031255, 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. 5,457,046, U.S. Pat. No. 5,648,263, and U.S. Pat. No. 5,686,593.

[0271] In the processes of the present invention, any GH61 polypeptide having cellulolytic enhancing activity can be used.

[0272] Examples of GH61 polypeptides having cellulolytic enhancing activity useful in the processes of the present invention include, but are not limited to, GH61 polypeptides from Thielavia terrestris (WO 2005/074647, WO 2008/148131, and WO 2011/035027), Thermoascus aurantiacus (WO 2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290), Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868), Aspergillus fumigatus (WO 2010/138754), GH61 polypeptides from Penicillium pinophilum (WO 2011/005867), Thermoascus sp. (WO 2011/039319), Penicillium sp. (WO 2011/041397), and Thermoascus crustaceous (WO 2011/041504).

[0273] In one aspect, the GH61 polypeptide having cellulolytic enhancing activity is used in the presence of a soluble activating divalent metal cation according to WO 2008/151043, e.g., manganese sulfate.

[0274] In one aspect, the GH61 polypeptide having cellulolytic enhancing activity is used in the presence of a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquor obtained from a pretreated cellulosic material such as pretreated corn stover (PCS).

[0275] The dioxy compound may include any suitable compound containing two or more oxygen atoms. In some aspects, the dioxy compounds contain a substituted aryl moiety as described herein. The dioxy compounds may comprise one or more (e.g., several) hydroxyl and/or hydroxyl derivatives, but also include substituted aryl moieties lacking hydroxyl and hydroxyl derivatives. Non-limiting examples of the dioxy compounds include pyrocatechol or catechol; caffeic acid; 3,4-dihydroxybenzoic acid; 4-tert-butyl-5-methoxy-1,2-benzenediol; pyrogallol; gallic acid; methyl-3,4,5-trihydroxybenzoate; 2,3,4-trihydroxybenzophenone; 2,6-dimethoxyphenol; sinapinic acid; 3,5-dihydroxybenzoic acid; 4-chloro-1,2-benzenediol; 4-nitro-1,2-benzenediol; tannic acid; ethyl gallate; methyl glycolate; dihydroxyfumaric acid; 2-butyne-1,4-diol; (croconic acid; 1,3-propanediol; tartaric acid; 2,4-pentanediol; 3-ethyoxy-1,2-propanediol; 2,4,4'-trihydroxybenzophenone; cis-2-butene-1,4-diol; 3,4-dihydroxy-3-cyclobutene-1,2-dione; dihydroxyacetone; acrolein acetal; methyl-4-hydroxybenzoate; 4-hydroxybenzoic acid; and methyl-3,5-dimethoxy-4-hydroxybenzoate; or a salt or solvate thereof. The bicyclic compound may include any suitable substituted fused ring system as described herein. The compounds may comprise one or more (e.g., several) additional rings, and are not limited to a specific number of rings unless otherwise stated. In one aspect, the bicyclic compound is a flavonoid. In another aspect, the bicyclic compound is an optionally substituted isoflavonoid. In another aspect, the bicyclic compound is an optionally substituted flavylium ion, such as an optionally substituted anthocyanidin or optionally substituted anthocyanin, or derivative thereof. Non-limiting examples of thebicyclic compounds include epicatechin; quercetin; myricetin; taxifolin; kaempferol; morin; acacetin; naringenin; isorhamnetin; apigenin; cyanidin; cyanin; kuromanin; keracyanin; or a salt or solvate thereof.

[0276] The heterocyclic compound may be any suitable compound, such as an optionally substituted aromatic or non-aromatic ring comprising a heteroatom, as described herein. In one aspect, the heterocyclic is a compound comprising an optionally substituted heterocycloalkyl moiety or an optionally substituted heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted 5-membered heterocycloalkyl or an optionally substituted 5-membered heteroaryl moiety. In another aspect, the optionally substituted heterocycloalkyl or optionally substituted heteroaryl moiety is an optionally substituted moiety selected from pyrazolyl, furanyl, imidazolyl, isoxazolyl, oxadiazolyl, oxazolyl, pyrrolyl, pyridyl, pyrimidyl, pyridazinyl, thiazolyl, triazolyl, thienyl, dihydrothieno-pyrazolyl, thianaphthenyl, carbazolyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzotriazolyl, benzothiazolyl, benzooxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisazolyl, dimethylhydantoin, pyrazinyl, tetrahydrofuranyl, pyrrolinyl, pyrrolidinyl, morpholinyl, indolyl, diazepinyl, azepinyl, thiepinyl, piperidinyl, and oxepinyl. In another aspect, the optionally substituted heterocycloalkyl moiety or optionally substituted heteroaryl moiety is an optionally substituted furanyl. Non-limiting examples of the heterocyclic compounds include (1,2-dihydroxyethyl)-3,4-dihydroxyfuran-2(5H)-one; 4-hydroxy-5-methyl-3-furanone; 5-hydroxy-2(5H)-furanone; [1,2-dihydroxyethyl]furan-2,3,4(5H)-trione; .alpha.-hydroxy-.gamma.-butyrolactone; ribonic .gamma.-lactone; aldohexuronicaldohexuronic acid .gamma.-lactone; gluconic acid 5-lactone; 4-hydroxycoumarin; dihydrobenzofuran; 5-(hydroxymethyl)furfural; furoin; 2(5H)-furanone; 5,6-dihydro-2H-pyran-2-one; and 5,6-dihydro-4-hydroxy-6-methyl-2H-pyran-2-one; or a salt or solvate thereof.

[0277] The nitrogen-containing compound may be any suitable compound with one or more nitrogen atoms. In one aspect, the nitrogen-containing compound comprises an amine, imine, hydroxylamine, or nitroxide moiety. Non-limiting examples of thenitrogen-containing compounds include acetone oxime; violuric acid; pyridine-2-aldoxime; 2-aminophenol; 1,2-benzenediamine; 2,2,6,6-tetramethyl-1-piperidinyloxy; 5,6,7,8-tetrahydrobiopterin; 6,7-dimethyl-5,6,7,8-tetrahydropterine; and maleamic acid; or a salt or solvate thereof.

[0278] The quinone compound may be any suitable compound comprising a quinone moiety as described herein. Non-limiting examples of the quinone compounds include 1,4-benzoquinone; 1,4-naphthoquinone; 2-hydroxy-1,4-naphthoquinone; 2,3-dimethoxy-5-methyl-1,4-benzoquinone or coenzyme Q.sub.0; 2,3,5,6-tetramethyl-1,4-benzoquinone or duroquinone; 1,4-dihydroxyanthraquinone; 3-hydroxy-1-methyl-5,6-indolinedione or adrenochrome; 4-tert-butyl-5-methoxy-1,2-benzoquinone; pyrroloquinoline quinone; or a salt or solvate thereof.

[0279] The sulfur-containing compound may be any suitable compound comprising one or more sulfur atoms. In one aspect, the sulfur-containing comprises a moiety selected from thionyl, thioether, sulfinyl, sulfonyl, sulfamide, sulfonamide, sulfonic acid, and sulfonic ester. Non-limiting examples of the sulfur-containing compounds include ethanethiol; 2-propanethiol; 2-propene-1-thiol; 2-mercaptoethanesulfonic acid; benzenethiol; benzene-1,2-dithiol; cysteine; methionine; glutathione; cystine; or a salt or solvate thereof.

[0280] In one aspect, an effective amount of such a compound described above to cellulosic material as a molar ratio to glucosyl units of cellulose is about 10.sup.-6 to about 10, e.g., about 10.sup.-6 to about 7.5, about 10.sup.-6 to about 5, about 10.sup.-6 to about 2.5, about 10.sup.-6 to about 1, about 10.sup.-5 to about 1, about 10.sup.-5 to about 10.sup.-1, about 10.sup.-4 to about 10.sup.-1, about 10.sup.-3 to about 10.sup.-1, or about 10.sup.-3 to about 10.sup.-2. In another aspect, an effective amount of such a compound described above is about 0.1 .mu.M to about 1 M, e.g., about 0.5 .mu.M to about 0.75 M, about 0.75 .mu.M to about 0.5 M, about 1 .mu.M to about 0.25 M, about 1 .mu.M to about 0.1 M, about 5 .mu.M to about 50 mM, about 10 .mu.M to about 25 mM, about 50 .mu.M to about 25 mM, about 10 .mu.M to about 10 mM, about 5 .mu.M to about 5 mM, ord about 0.1 mM to about 1 mM.

[0281] The term "liquor" means the solution phase, either aqueous, organic, or a combination thereof, arising from treatment of a lignocellulose and/or hemicellulose material in a slurry, or monosaccharides thereof, e.g., xylose, arabinose, mannose, etc., under conditions as described herein, and the soluble contents thereof. A liquor for cellulolytic enhancement of a GH61 polypeptide can be produced by treating a lignocellulose or hemicellulose material (or feedstock) by applying heat and/or pressure, optionally in the presence of a catalyst, e.g., acid, optionally in the presence of an organic solvent, and optionally in combination with physical disruption of the material, and then separating the solution from the residual solids. Such conditions determine the degree of cellulolytic enhancement obtainable through the combination of liquor and a GH61 polypeptide during hydrolysis of a cellulosic substrate by a cellulase preparation. The liquor can be separated from the treated material using a method standard in the art, such as filtration, sedimentation, or centrifugation.

[0282] In one aspect, an effective amount of the liquor to cellulose is about 10.sup.-6 to about 10 g per g of cellulose, e.g., about 10.sup.-6 to about 7.5 g, about 10.sup.-6 to about 5, about 10.sup.-6 to about 2.5 g, about 10.sup.-6 to about 1 g, about 10.sup.-6 to about 1 g, about 10.sup.-6 to about 10.sup.-1 g, about 10.sup.-4 to about 10.sup.-1 g, about 10.sup.-3 to about 10.sup.-1 g, or about 10.sup.-3 to about 10.sup.-2 g per g of cellulose. In one aspect, the one or more (e.g., several) hemicellulolytic enzymes comprise a commercial hemicellulolytic enzyme preparation. Examples of commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, SHEARZYME.TM. (Novozymes NS), CELLIC.TM. HTec (Novozymes NS), CELLIC.TM. HTec2 (Novozymes NS), VISCOZYME.RTM. (Novozymes NS), ULTRAFLO.RTM. (Novozymes NS), PULPZYME.RTM. HC (Novozymes NS), MULTIFECT.RTM. Xylanase (Genencor), ACCELLERASE.RTM. XY (Genencor), ACCELLERASE.RTM. XC (Genencor), ECOPULP.RTM. TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL.TM. 333P (Biocatalysts Limit, Wales, UK), DEPOL.TM. 740L. (Biocatalysts Limit, Wales, UK), and DEPOL.TM. 762P (Biocatalysts Limit, Wales, UK).

[0283] Examples of xylanases useful in the processes of the present invention include, but are not limited to, xylanases from Aspergillus aculeatus (GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO 2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp. (WO 2010/126772), Thielavia terrestris NRRL 8126 (WO 2009/079210), and Trichophaea saccata GH10 (WO 2011/057083).

[0284] Examples of beta-xylosidases useful in the processes of the present invention include, but are not limited to, beta-xylosidases from Neurospora crassa (SwissProt accession number Q7SOW4), Trichoderma reesei (UniProtKB/TrEMBL accession number Q92458), and Talaromyces emersonii (SwissProt accession number Q8X212).

[0285] Examples of acetylxylan esterases useful in the processes of the present invention include, but are not limited to, acetylxylan esterases from Aspergillus aculeatus (WO 2010/108918), Chaetomium globosum (Uniprot accession number Q2GWX4), Chaetomium gracile (GeneSeqP accession number AAB82124), Humicola insolens DSM 1800 (WO 2009/073709), Hypocrea jecorina (WO 2005/001036), Myceliophtera thermophila (WO 2010/014880), Neurospora crassa (UniProt accession number q7s259), Phaeosphaeria nodorum (Uniprot accession number QOUHJ1), and Thielavia terrestris NRRL 8126 (WO 2009/042846).

[0286] Examples of feruloyl esterases (ferulic acid esterases) useful in the processes of the present invention include, but are not limited to, feruloyl esterases form Humicola insolens DSM 1800 (WO 2009/076122), Neosartorya fischeri (UniProt Accession number A1D9T4), Neurospora crassa (UniProt accession number Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO 2010/065448).

[0287] Examples of arabinofuranosidases useful in the processes of the present invention include, but are not limited to, arabinofuranosidases from Aspergillus niger (GeneSeqP accession number AAR94170), Humicola insolens DSM 1800 (WO 2006/114094 and WO 2009/073383), and M. giganteus (WO 2006/114094).

[0288] Examples of alpha-glucuronidases useful in the processes of the present invention include, but are not limited to, alpha-glucuronidases from Aspergillus clavatus (UniProt accession number alcc12), Aspergillus fumigatus (SwissProt accession number Q4WW45), Aspergillus niger (Uniprot accession number Q96WX9), Aspergillus terreus (SwissProt accession number QOCJP9), Humicola insolens (WO 2010/014706), Penicillium aurantiogriseum (WO 2009/068565), Talaromyces emersonii (UniProt accession number Q8X211), and Trichoderma reesei (Uniprot accession number Q99024).

[0289] The polypeptides having enzyme activity used in the processes 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 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).

[0290] The fermentation can be any method of cultivation of a cell resulting in the expression or isolation of an enzyme or protein. Fermentation may, therefore, be understood as comprising shake flask cultivation, or small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the enzyme to be expressed or isolated. The resulting enzymes produced by the methods described above may be recovered from the fermentation medium and purified by conventional procedures.

[0291] Fermentation.

[0292] The fermentable sugars obtained from the hydrolyzed cellulosic material can be fermented by one or more (e.g., several) 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.

[0293] 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, as described herein.

[0294] 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.

[0295] 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).

[0296] "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 hexose and/or pentose fermenting organisms, or a combination thereof. Both hexose and pentose 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, and/or oligosaccharides, directly or indirectly into the desired fermentation product. Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

[0297] Examples of fermenting microorganisms that can ferment hexose sugars include bacterial and fungal organisms, such as yeast. Preferred yeast includes strains of Candida, Kluyveromyces, and Saccharomyces, e.g., Candida sonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

[0298] Examples of fermenting organisms that can ferment pentose sugars in their native state include bacterial and fungal organisms, such as some yeast. Preferred xylose fermenting yeast include strains of Candida, preferably C. sheatae or C. sonorensis; and strains of Pichia, preferably P. stipitis, such as P. stipitis CBS 5773. Preferred pentose fermenting yeast include strains of Pachysolen, preferably P. tannophilus. Organisms not capable of fermenting pentose sugars, such as xylose and arabinose, may be genetically modified to do so by methods known in the art.

[0299] Examples of bacteria that can efficiently ferment hexose and pentose to ethanol include, for example, Bacillus coagulans, Clostridium acetobutylicum, Clostridium thermocellum, Clostridium phytofermentans, Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonas mobilis (Philippidis, 1996, supra).

[0300] Other fermenting organisms include strains of Bacillus, such as Bacillus coagulans; Candida, such as C. sonorensis, C. methanosorbosa, C. diddensiae, C. parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis, and C. scehatae; Clostridium, such as C. acetobutylicum, C. thermocellum, and C. phytofermentans; E. coli, especially E. coli strains that have been genetically modified to improve the yield of ethanol; Geobacillus sp.; Hansenula, such as Hansenula anomala; Klebsiella, such as K. oxytoca; Kluyveromyces, such as K. marxianus, K. lactis, K. thermotolerans, and K. fragilis; Schizosaccharomyces, such as S. pombe; Thermoanaerobacter, such as Thermoanaerobacter saccharolyticum; and Zymomonas, such as Zymomonas mobilis.

[0301] In a preferred aspect, the yeast is a Bretannomyces. In a more preferred aspect, the yeast is Bretannomyces clausenii. In another preferred aspect, the yeast is a Candida. In another more preferred aspect, the yeast is Candida sonorensis. In another more preferred aspect, the yeast is Candida boidinii. In another more preferred aspect, the yeast is Candida blankii. 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 entomophiliia. In another more preferred aspect, the yeast is Candida pseudotropicalis. In another more preferred aspect, the yeast is Candida scehatae. 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 Kluyveromyces. In another more preferred aspect, the yeast is Kluyveromyces fragilis. In another more preferred aspect, the yeast is Kluyveromyces marxianus. In another more preferred aspect, the yeast is Kluyveromyces thermotolerans. 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 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.

[0302] In a preferred aspect, the bacterium is a Bacillus. In a more preferred aspect, the bacterium is Bacillus coagulans. In another preferred aspect, the bacterium is a Clostridium. In another more preferred aspect, the bacterium is Clostridium acetobutylicum. In another more preferred aspect, the bacterium is Clostridium phytofermentans. In another more preferred aspect, the bacterium is Clostridium thermocellum. In another more preferred aspect, the bacterium is Geobacillus sp. In another more preferred aspect, the bacterium is a Thermoanaerobacter. In another more preferred aspect, the bacterium is Thermoanaerobacter saccharolyticum. In another preferred aspect, the bacterium is a Zymomonas. In another more preferred aspect, the bacterium is Zymomonas mobilis.

[0303] Commercially available yeast suitable for ethanol production include, e.g., BIOFERM.TM. AFT and XR (NABC--North American Bioproducts Corporation, GA, USA), ETHANOL RED.TM. yeast (Fermentis/Lesaffre, USA), FALI.TM. (Fleischmann's Yeast, USA), FERMIOL.TM. (DSM Specialties), GERT STRAND.TM. (Gert Strand AB, Sweden), and SUPERSTART.TM. and THERMOSACC.TM. fresh yeast (Ethanol Technology, WI, USA).

[0304] 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.

[0305] The cloning of heterologous genes into various fermenting microorganisms has led to the construction of organisms capable of converting hexoses and pentoses to ethanol (co-fermentation) (Chen and Ho, 1993, Cloning and improving the expression of Pichia stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl. Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Genetically engineered Saccharomyces yeast capable of effectively cofermenting glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae, Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995, Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing the TKL1 and TALI genes encoding the pentose phosphate pathway enzymes transketolase and transaldolase, Appl. Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering of Saccharomyces cerevisiae for efficient anaerobic xylose fermentation: a proof of principle, FEMS Yeast Research 4: 655-664; Beall et al., 1991, Parametric studies of ethanol production from xylose and other sugars by recombinant Escherichia coli, Biotech. Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214; Zhang et al., 1995, Metabolic engineering of a pentose metabolism pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243; Deanda et al., 1996, Development of an arabinose-fermenting Zymomonas mobilis strain by metabolic pathway engineering, Appl. Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose isomerase).

[0306] In a preferred aspect, the genetically modified fermenting microorganism is Candida sonorensis. In another preferred aspect, the genetically modified fermenting microorganism is Escherichia coli. In another preferred aspect, the genetically modified fermenting microorganism is Klebsiella oxytoca. In another preferred aspect, the genetically modified fermenting microorganism is Kluyveromyces marxianus. In another preferred aspect, the genetically modified fermenting microorganism is Saccharomyces cerevisiae. In another preferred aspect, the genetically modified fermenting microorganism is Zymomonas mobilis.

[0307] It is well known in the art that the organisms described above can also be used to produce other substances, as described herein.

[0308] The fermenting microorganism is typically added to the degraded cellulosic material or hydrolysate and the fermentation is performed for about 8 to about 96 hours, e.g., about 24 to about 60 hours. The temperature is typically between about 26.degree. C. to about 60.degree. C., e.g., about 32.degree. C. or 50.degree. C., and about pH 3 to about pH 8, e.g., pH 4-5, 6, or 7.

[0309] In one aspect, the yeast and/or another microorganism are applied to the degraded cellulosic material and the fermentation is performed for about 12 to about 96 hours, such as typically 24-60 hours. In another aspect, the temperature is preferably between about 20.degree. C. to about 60.degree. C., e.g., about 25.degree. C. to about 50.degree. C., about 32.degree. C. to about 50.degree. C., or about 32.degree. C. to about 50.degree. C., and the pH is generally from about pH 3 to about pH 7, e.g., about pH 4 to about pH 7. However, some fermenting organisms, e.g., bacteria, have higher fermentation temperature optima. Yeast or another microorganism is preferably applied in amounts of approximately 10.sup.5 to 10.sup.12, preferably from approximately 10.sup.7 to 10.sup.10, especially approximately 2.times.10.sup.8 viable cell count per ml of fermentation broth. Further guidance in respect of using yeast for fermentation can be found in, e.g., "The Alcohol Textbook" (Editors K. Jacques, T. P. Lyons and D. R. Kelsall, Nottingham University Press, United Kingdom 1999), which is hereby incorporated by reference.

[0310] For ethanol production, following the fermentation the fermented slurry is distilled to extract the ethanol. The ethanol obtained according to the processes of the invention can be used as, e.g., fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol.

[0311] A fermentation stimulator can be used in combination with any of the 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.

[0312] Fermentation Products:

[0313] A fermentation product can be any substance derived from the fermentation. The fermentation product can be, without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane), an alkene (e.g. pentene, hexene, heptene, and octene); an amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); a gas (e.g., methane, hydrogen (H.sub.2), carbon dioxide (CO.sub.2), and carbon monoxide (CO)); isoprene; a ketone (e.g., acetone); 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, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); and polyketide. The fermentation product can also be protein as a high value product.

[0314] 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 n-butanol. In another more preferred aspect, the alcohol is isobutanol. In another more preferred aspect, the alcohol is ethanol. In another more preferred aspect, the alcohol is methanol. In another more preferred aspect, the alcohol is arabinitol. In another more preferred aspect, the alcohol is butanediol. In another more preferred aspect, the alcohol is ethylene glycol. In another more preferred aspect, the alcohol is glycerin. In another more preferred aspect, the alcohol is glycerol. 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 and Jonas, 2002, The biotechnological production of sorbitol, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam and Singh, 1995, Processes for fermentative production of xylitol--a sugar substitute, Process Biochemistry 30(2): 117-124; Ezeji et al., 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.

[0315] In another preferred aspect, the fermentation product is an alkane. The alkane can be an unbranched or a branched alkane. In another more preferred aspect, the alkane is pentane. In another more preferred aspect, the alkane is hexane. In another more preferred aspect, the alkane is heptane. In another more preferred aspect, the alkane is octane. In another more preferred aspect, the alkane is nonane. In another more preferred aspect, the alkane is decane. In another more preferred aspect, the alkane is undecane. In another more preferred aspect, the alkane is dodecane.

[0316] In another preferred aspect, the fermentation product is a cycloalkane. In another more preferred aspect, the cycloalkane is cyclopentane. In another more preferred aspect, the cycloalkane is cyclohexane. In another more preferred aspect, the cycloalkane is cycloheptane. In another more preferred aspect, the cycloalkane is cyclooctane.

[0317] In another preferred aspect, the fermentation product is an alkene. The alkene can be an unbranched or a branched alkene. In another more preferred aspect, the alkene is pentene. In another more preferred aspect, the alkene is hexene. In another more preferred aspect, the alkene is heptene. In another more preferred aspect, the alkene is octene.

[0318] 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 and Margaritis, 2004, Empirical modeling of batch fermentation kinetics for poly(glutamic acid) production and other microbial biopolymers, Biotechnology and Bioengineering 87(4): 501-515.

[0319] In another preferred aspect, the fermentation product is a gas. In another more preferred aspect, the gas is methane. In another more preferred aspect, the gas is H.sub.2. In another more preferred aspect, the gas is CO.sub.2. In another more preferred aspect, the gas is CO. See, for example, Kataoka et al., 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, 1997, Anaerobic digestion of biomass for methane production: A review, Biomass and Bioenergy 13(1-2): 83-114, 1997.

[0320] In another preferred aspect, the fermentation product is isoprene.

[0321] 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.

[0322] 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 and Lee, 1997, Membrane-mediated extractive fermentation for lactic acid production from cellulosic biomass, Appl. Biochem. Biotechnol. 63-65: 435-448. In another preferred aspect, the fermentation product is polyketide.

[0323] Recovery.

[0324] 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.

Signal Peptide

[0325] The present invention also relates to an isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 21 of SEQ ID NO: 2, amino acids 1 to 20 of SEQ ID NO: 4, amino acids 1 to 19 of SEQ ID NO: 6, or amino acids 1 to 19 of SEQ ID NO: 8. The polynucleotide may further comprise a gene encoding a protein, which is operably linked to the signal peptide. The protein is preferably foreign to the signal peptide. In one aspect, the polynucleotide encoding the signal peptide is nucleotides 1 to 63 of SEQ ID NO: 1. In another aspect, the polynucleotide encoding the signal peptide is nucleotides 1 to 60 of SEQ ID NO: 3. In another aspect, the polynucleotide encoding the signal peptide is nucleotides 1 to 57 of SEQ ID NO: 5. In another aspect, the polynucleotide encoding the signal peptide is nucleotides 1 to 57 of SEQ ID NO: 7.

[0326] The present invention also relates to nucleic acid constructs, expression vectors and recombinant host cells comprising such polynucleotides.

[0327] The present invention also relates to methods of producing a protein, comprising (a) cultivating a recombinant host cell comprising such polynucleotide; and (b) recovering the protein.

[0328] 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 polypeptides. The term "protein" also encompasses two or more polypeptides combined to form the encoded product. The proteins also include hybrid polypeptides and fused polypeptides.

[0329] Preferably, the protein is a hormone, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter. For example, the protein may be a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, e.g., an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, alpha-galactosidase, beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase, or beta-xylosidase.

[0330] The gene may be obtained from any prokaryotic, eukaryotic, or other source.

[0331] 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

811781DNATrametes versicolorCDS(1)..(79)sig_peptide(1)..(63)mat_peptide(64)..()Intron(80)..- (141)CDS(142)..(152)Intron(153)..(216)CDS(217)..(274)Intron(275)..(333)CDS- (334)..(564)Intron(565)..(620)CDS(621)..(838)Intron(839)..(895)CDS(896)..(- 993)Intron(994)..(1049)CDS(1050)..(1133)Intron(1134)..(1183)CDS(1184)..(13- 45)Intron(1346)..(1409)CDS(1410)..(1585)Intron(1586)..(1638)CDS(1639)..(17- 78) 1atg aag act ttc gca gcc ttg ctt tcc gct gtc act ctc gcg ctc tcg 48Met Lys Thr Phe Ala Ala Leu Leu Ser Ala Val Thr Leu Ala Leu Ser -20 -15 -10 gtg cgc gcc cag gcg gct gtc tgg agt caa t gtaagtgccg ctgcttttca 99Val Arg Ala Gln Ala Ala Val Trp Ser Gln -5 -1 1 5 ttgatacgag actctacgcc gagctgacgt gctaccgtat ag gt ggc ggt aca 152 Cys Gly Gly Thr gtaagttatc atagcatacc gcctacatgc acaaggacat actgctgatc aattctctgt 212gcag ggt tgg acg ggc gag acc act tgc gtt gct ggt tcg gtt tgt acc 261 Gly Trp Thr Gly Glu Thr Thr Cys Val Ala Gly Ser Val Cys Thr 10 15 20 tcc ttg agc tca t gtgagcgact ttcaatccgt cgtcattgct cctcatgtat 314Ser Leu Ser Ser 25 tgacgattgg ccttcatag ca tac tct caa tgc gtt ccg ggc tcc gca acg 365 Ser Tyr Ser Gln Cys Val Pro Gly Ser Ala Thr 30 35 tcc agc gct ccg gcg gcc ccc tca gcg aca act tca ggc ccc gca cct 413Ser Ser Ala Pro Ala Ala Pro Ser Ala Thr Thr Ser Gly Pro Ala Pro 40 45 50 55 cca gca tcc acg tgt gca ccc aac agc ccg ccc gcg agc gca ggc aaa 461Pro Ala Ser Thr Cys Ala Pro Asn Ser Pro Pro Ala Ser Ala Gly Lys 60 65 70 ctg cgc ttc gcg ggt gtc aac atc tcc ggt ttc gac ttc gga tgc agc 509Leu Arg Phe Ala Gly Val Asn Ile Ser Gly Phe Asp Phe Gly Cys Ser 75 80 85 acg gac gga acg tgc tcg gcc agc ggg gca tgg ccg cca ttg acc aag 557Thr Asp Gly Thr Cys Ser Ala Ser Gly Ala Trp Pro Pro Leu Thr Lys 90 95 100 tac tac g gtcagcccct ttcctgcagg gtagtggcga gggtgttgac attttttgga 614Tyr Tyr 105 cgacag gc atg gat ggt gcg ggc cag atg aag cac ttt gtc gag aac 661 Gly Met Asp Gly Ala Gly Gln Met Lys His Phe Val Glu Asn 110 115 gac ggc tac aat gtt ttc cgc ctg ccc gtt ggg tgg cag ttc ctg acg 709Asp Gly Tyr Asn Val Phe Arg Leu Pro Val Gly Trp Gln Phe Leu Thr 120 125 130 135 aac ggt gcc gcc act ggt gac ata gac gag gtc aac ttc gcc gag tac 757Asn Gly Ala Ala Thr Gly Asp Ile Asp Glu Val Asn Phe Ala Glu Tyr 140 145 150 gat gat ctc gtc caa gcg tgc ctc gac gca ggc gcg tcg tgc atc gtt 805Asp Asp Leu Val Gln Ala Cys Leu Asp Ala Gly Ala Ser Cys Ile Val 155 160 165 gag gtt cac aac tac gcg cgg ttt aac ggc aag gtacgatgcg cacttctgcg 858Glu Val His Asn Tyr Ala Arg Phe Asn Gly Lys 170 175 agccagctga ctgtagccaa ccaaacctgg catacag atc atc ggt cag ggt ggg 913 Ile Ile Gly Gln Gly Gly 180 ccc gca aat gac cag ttc gcc gcc ctt tgg agc agt ttt gcc tcc aag 961Pro Ala Asn Asp Gln Phe Ala Ala Leu Trp Ser Ser Phe Ala Ser Lys 185 190 195 200 tac gcc gac aac gac aag atc atc ttt ggg at gtcagtgcct tttccggatt 1013Tyr Ala Asp Asn Asp Lys Ile Ile Phe Gly Ile 205 210 ccgcaaactg tagctcatgc aataaccgac tcacag t atg aac gaa cca cat gac 1068 Met Asn Glu Pro His Asp 215 gtc ccc gat atc aac ctc tgg gcg gag agt gtc cag gca gca gtg acc 1116Val Pro Asp Ile Asn Leu Trp Ala Glu Ser Val Gln Ala Ala Val Thr 220 225 230 gcc atc cgc cag gcc gg gtatgcatgt caaccccaaa accaaatgct 1163Ala Ile Arg Gln Ala Gly 235 gctcactaat cggtccctag t gcg act tcg cag ctg atc ctg ctc ccg ggc 1214 Ala Thr Ser Gln Leu Ile Leu Leu Pro Gly 240 245 aac aac tgg acg tct gcg gag acc ttc gtc tcc aac ggc tcg gcc gat 1262Asn Asn Trp Thr Ser Ala Glu Thr Phe Val Ser Asn Gly Ser Ala Asp 250 255 260 265 gcg ctc aac aag gtc acg aac ccc gac ggc agc atc acg aac ctc atc 1310Ala Leu Asn Lys Val Thr Asn Pro Asp Gly Ser Ile Thr Asn Leu Ile 270 275 280 ttc gac gtg cac aag tac ctc gac ttc gac aac tc gtacgtctat 1355Phe Asp Val His Lys Tyr Leu Asp Phe Asp Asn Ser 285 290 gcccacttgc cccgttgatt ctgcaggtga attgacgtaa tgcggatcgc acag g ggc 1413 Glyacg aac gca gag tgc acg acg aac aac atc gac aac gcg tgg gcg ccg 1461Thr Asn Ala Glu Cys Thr Thr Asn Asn Ile Asp Asn Ala Trp Ala Pro 295 300 305 310 ctc gcg cag tgg ctg cgc tgc aac ggc cgg cag gcg ttc aac acc gag 1509Leu Ala Gln Trp Leu Arg Cys Asn Gly Arg Gln Ala Phe Asn Thr Glu 315 320 325 acg ggc ggc ggg aac gtc gcg tcg tgc gag acg ttc atg tgc gag cag 1557Thr Gly Gly Gly Asn Val Ala Ser Cys Glu Thr Phe Met Cys Glu Gln 330 335 340 gtc gcg ttc cag acg gcg aac tcg gac g gtgcgttctg cgtcctcgcg 1605Val Ala Phe Gln Thr Ala Asn Ser Asp 345 350 tcgttcttca ctctcgctga tgatatcgtg tag tg ttc ttg ggc tat gtc ggc 1658 Val Phe Leu Gly Tyr Val Gly 355 tgg gcg gcg ggc aac ttc tac gaa ggc tac gtg ctg agc gag gtc ccg 1706Trp Ala Ala Gly Asn Phe Tyr Glu Gly Tyr Val Leu Ser Glu Val Pro 360 365 370 acc cag aat gcg gat ggg agc tgg acg gac cag ccg ctt gtt gcg cag 1754Thr Gln Asn Ala Asp Gly Ser Trp Thr Asp Gln Pro Leu Val Ala Gln 375 380 385 390 tgc atg gcg ccc aac gct tct cag tga 1781Cys Met Ala Pro Asn Ala Ser Gln 395 2419PRTTrametes versicolor 2Met Lys Thr Phe Ala Ala Leu Leu Ser Ala Val Thr Leu Ala Leu Ser -20 -15 -10 Val Arg Ala Gln Ala Ala Val Trp Ser Gln Cys Gly Gly Thr Gly Trp -5 -1 1 5 10 Thr Gly Glu Thr Thr Cys Val Ala Gly Ser Val Cys Thr Ser Leu Ser 15 20 25 Ser Ser Tyr Ser Gln Cys Val Pro Gly Ser Ala Thr Ser Ser Ala Pro 30 35 40 Ala Ala Pro Ser Ala Thr Thr Ser Gly Pro Ala Pro Pro Ala Ser Thr 45 50 55 Cys Ala Pro Asn Ser Pro Pro Ala Ser Ala Gly Lys Leu Arg Phe Ala 60 65 70 75 Gly Val Asn Ile Ser Gly Phe Asp Phe Gly Cys Ser Thr Asp Gly Thr 80 85 90 Cys Ser Ala Ser Gly Ala Trp Pro Pro Leu Thr Lys Tyr Tyr Gly Met 95 100 105 Asp Gly Ala Gly Gln Met Lys His Phe Val Glu Asn Asp Gly Tyr Asn 110 115 120 Val Phe Arg Leu Pro Val Gly Trp Gln Phe Leu Thr Asn Gly Ala Ala 125 130 135 Thr Gly Asp Ile Asp Glu Val Asn Phe Ala Glu Tyr Asp Asp Leu Val 140 145 150 155 Gln Ala Cys Leu Asp Ala Gly Ala Ser Cys Ile Val Glu Val His Asn 160 165 170 Tyr Ala Arg Phe Asn Gly Lys Ile Ile Gly Gln Gly Gly Pro Ala Asn 175 180 185 Asp Gln Phe Ala Ala Leu Trp Ser Ser Phe Ala Ser Lys Tyr Ala Asp 190 195 200 Asn Asp Lys Ile Ile Phe Gly Ile Met Asn Glu Pro His Asp Val Pro 205 210 215 Asp Ile Asn Leu Trp Ala Glu Ser Val Gln Ala Ala Val Thr Ala Ile 220 225 230 235 Arg Gln Ala Gly Ala Thr Ser Gln Leu Ile Leu Leu Pro Gly Asn Asn 240 245 250 Trp Thr Ser Ala Glu Thr Phe Val Ser Asn Gly Ser Ala Asp Ala Leu 255 260 265 Asn Lys Val Thr Asn Pro Asp Gly Ser Ile Thr Asn Leu Ile Phe Asp 270 275 280 Val His Lys Tyr Leu Asp Phe Asp Asn Ser Gly Thr Asn Ala Glu Cys 285 290 295 Thr Thr Asn Asn Ile Asp Asn Ala Trp Ala Pro Leu Ala Gln Trp Leu 300 305 310 315 Arg Cys Asn Gly Arg Gln Ala Phe Asn Thr Glu Thr Gly Gly Gly Asn 320 325 330 Val Ala Ser Cys Glu Thr Phe Met Cys Glu Gln Val Ala Phe Gln Thr 335 340 345 Ala Asn Ser Asp Val Phe Leu Gly Tyr Val Gly Trp Ala Ala Gly Asn 350 355 360 Phe Tyr Glu Gly Tyr Val Leu Ser Glu Val Pro Thr Gln Asn Ala Asp 365 370 375 Gly Ser Trp Thr Asp Gln Pro Leu Val Ala Gln Cys Met Ala Pro Asn 380 385 390 395 Ala Ser Gln 31785DNATrametes versicolorCDS(1)..(84)sig_peptide(1)..(60)mat_peptide(61)..()Intron(85)..- (144)CDS(145)..(208)Intron(209)..(265)CDS(266)..(459)Intron(460)..(531)CDS- (532)..(700)Intron(701)..(759)CDS(760)..(808)Intron(809)..(861)CDS(862)..(- 872)Intron(873)..(930)CDS(931)..(956)Intron(957)..(1018)CDS(1019)..(1093)I- ntron(1094)..(1155)CDS(1156)..(1312)Intron(1313)..(1383)CDS(1384)..(1640)I- ntron(1641)..(1710)CDS(1711)..(1782) 3atg aag acg gtt atc ctc tcc ctc gct gcc gcg ctc ttc agc gcc gcg 48Met Lys Thr Val Ile Leu Ser Leu Ala Ala Ala Leu Phe Ser Ala Ala -20 -15 -10 -5 ccc gtg ctc tcc acc gcc gtc tgg ggc cag tgc ggc gtgagtactc 94Pro Val Leu Ser Thr Ala Val Trp Gly Gln Cys Gly -1 1 5 gactcgcgac gcggtcacgg ggccatactc accatctgct cgtgttctag ggc act 150 Gly Thr 10 ggc ttc tct ggc gac acg acc tgc gcc tcc ggc tct agc tgc gtc gta 198Gly Phe Ser Gly Asp Thr Thr Cys Ala Ser Gly Ser Ser Cys Val Val 15 20 25 gtc aac caa t gtatgccgtc cacgtccacc cgctgacatc ctttactgac 248Val Asn Gln cactcaccct tgaacag ac tac tcg caa tgc cag ccc ggc gcg tcc gcc 297 Tyr Tyr Ser Gln Cys Gln Pro Gly Ala Ser Ala 30 35 40 ccc acg tcg act gcc tcg gcc ccc ggc cct tcc ggc tgc tcg ggc acg 345Pro Thr Ser Thr Ala Ser Ala Pro Gly Pro Ser Gly Cys Ser Gly Thr 45 50 55 cgc acc aag ttc aag ctc ttc ggt gtg aac gag tcc ggc gcg gag ttc 393Arg Thr Lys Phe Lys Leu Phe Gly Val Asn Glu Ser Gly Ala Glu Phe 60 65 70 ggg aac acc gtc atc ccg ggc gcg ctc ggc acg gac tac acc tgg ccg 441Gly Asn Thr Val Ile Pro Gly Ala Leu Gly Thr Asp Tyr Thr Trp Pro 75 80 85 tcg ccc acc tcc atc gac gtgcgtactg ttgtcggaca tgtctgacgt 489Ser Pro Thr Ser Ile Asp 90 agaagcagag gatgctgatg gatggacggt tgggcgatgc ag ttc ttc ctc ggg 543 Phe Phe Leu Gly 95 cag ggc ttc aac acc ttc cgc atc ccg ttc ctg atg gag cgc gtc agc 591Gln Gly Phe Asn Thr Phe Arg Ile Pro Phe Leu Met Glu Arg Val Ser 100 105 110 ccg ccg tcg acg ggc ggc ctt act ggc ccg ttc aac agc acg tac ctc 639Pro Pro Ser Thr Gly Gly Leu Thr Gly Pro Phe Asn Ser Thr Tyr Leu 115 120 125 130 gac ggg ctg aag cag act gtt agc tac atc acg ggc aag ggg ggc ttt 687Asp Gly Leu Lys Gln Thr Val Ser Tyr Ile Thr Gly Lys Gly Gly Phe 135 140 145 gcc atc gtc gac c gtgagtgctt actcccaacg tatgctattt ggagagtgga 740Ala Ile Val Asp 150 gtactgatct ggtgtgcag cg cac aac ttc atg atc ttc aac ggc gcg acg 791 Pro His Asn Phe Met Ile Phe Asn Gly Ala Thr 155 160 atc acg agc acc agc ca gtaagtcgca ttatttacgg tggaagagtt 838Ile Thr Ser Thr Ser Gln 165 ttactgatat ctatcgcgtt tag g ttc cag gct t gtacgtattc gcgtcgccat 892 Phe Gln Ala 170 atacacgaca ccgagctctt tgctgatgtt gacgacag gg tgg cag aag ctc gct 947 Trp Trp Gln Lys Leu Ala 175 gct gag ttc gtgagtgtgc tccatggcta cgcggccgtg aacgctctgg 996Ala Glu Phe ctgacaagat gccgcctccc ag aaa acc gac aac aac gtc atc ttc gac ctg 1048 Lys Thr Asp Asn Asn Val Ile Phe Asp Leu 180 185 atg aac gag ccg cac gac atc ccc gcg cag acc gtc ttc cag ctc 1093Met Asn Glu Pro His Asp Ile Pro Ala Gln Thr Val Phe Gln Leu 190 195 200 gtacgtaaca ctttccgtat gtcccaagca accatgtgtt aagtgatcat gatcccgcgc 1153ag atg caa gcg gcc gtg aac ggc gtg cgc gcg agc ggc gcg acg agc 1200 Met Gln Ala Ala Val Asn Gly Val Arg Ala Ser Gly Ala Thr Ser 205 210 215 cag ctc atc ctc gcc gag ggc acg agc tgg acc ggc gcg tgg acc tgg 1248Gln Leu Ile Leu Ala Glu Gly Thr Ser Trp Thr Gly Ala Trp Thr Trp 220 225 230 235 acg acg tcg ggc aac agc gac gcg ttc ggc gcg atc aag gac ccc aac 1296Thr Thr Ser Gly Asn Ser Asp Ala Phe Gly Ala Ile Lys Asp Pro Asn 240 245 250 aac aac atc gcc atc c gtgcgtcccc cccctccccc ttcctcttgc cccctgccta 1352Asn Asn Ile Ala Ile 255 ctgacgaaca cgccatggga ttgacacaca g ag atg cac cag tac cta gac 1403 Gln Met His Gln Tyr Leu Asp 260 tcg gac ggg tcg ggg acg tcc ccg atc tgc gtg tcg gac acg atc ggg 1451Ser Asp Gly Ser Gly Thr Ser Pro Ile Cys Val Ser Asp Thr Ile Gly 265 270 275 gcg gag cgg ctg cag gcg gcg acg cag tgg ctg cag cag acg ggc ctc 1499Ala Glu Arg Leu Gln Ala Ala Thr Gln Trp Leu Gln Gln Thr Gly Leu 280 285 290 295 aag ggc ttc ctc ggc gag atc ggg acg ggg aac aac acg cag tgc gtg 1547Lys Gly Phe Leu Gly Glu Ile Gly Thr Gly Asn Asn Thr Gln Cys Val 300 305 310 acc gcg ctg cag ggc gcg ctg tgc gag atg cag cag gcg ggc ggg acg 1595Thr Ala Leu Gln Gly Ala Leu Cys Glu Met Gln Gln Ala Gly Gly Thr 315 320

325 tgg ctc ggc gcg ctc tgg tgg gcg gcg ggg ccg tgg tgg gga gac 1640Trp Leu Gly Ala Leu Trp Trp Ala Ala Gly Pro Trp Trp Gly Asp 330 335 340 gtgagtggct ttctgtgctt atgtggggga gggggagtgg gggctgacgg tggttgtgtt 1700ggacttgcag tac tac cag agc atc gag ccc ccg aac ggg gac gcg atc 1749 Tyr Tyr Gln Ser Ile Glu Pro Pro Asn Gly Asp Ala Ile 345 350 355 gct gcg atc ctc ccg gcg ctc aag gcg ttc cag tag 1785Ala Ala Ile Leu Pro Ala Leu Lys Ala Phe Gln 360 365 4386PRTTrametes versicolor 4Met Lys Thr Val Ile Leu Ser Leu Ala Ala Ala Leu Phe Ser Ala Ala -20 -15 -10 -5 Pro Val Leu Ser Thr Ala Val Trp Gly Gln Cys Gly Gly Thr Gly Phe -1 1 5 10 Ser Gly Asp Thr Thr Cys Ala Ser Gly Ser Ser Cys Val Val Val Asn 15 20 25 Gln Tyr Tyr Ser Gln Cys Gln Pro Gly Ala Ser Ala Pro Thr Ser Thr 30 35 40 Ala Ser Ala Pro Gly Pro Ser Gly Cys Ser Gly Thr Arg Thr Lys Phe 45 50 55 60 Lys Leu Phe Gly Val Asn Glu Ser Gly Ala Glu Phe Gly Asn Thr Val 65 70 75 Ile Pro Gly Ala Leu Gly Thr Asp Tyr Thr Trp Pro Ser Pro Thr Ser 80 85 90 Ile Asp Phe Phe Leu Gly Gln Gly Phe Asn Thr Phe Arg Ile Pro Phe 95 100 105 Leu Met Glu Arg Val Ser Pro Pro Ser Thr Gly Gly Leu Thr Gly Pro 110 115 120 Phe Asn Ser Thr Tyr Leu Asp Gly Leu Lys Gln Thr Val Ser Tyr Ile 125 130 135 140 Thr Gly Lys Gly Gly Phe Ala Ile Val Asp Pro His Asn Phe Met Ile 145 150 155 Phe Asn Gly Ala Thr Ile Thr Ser Thr Ser Gln Phe Gln Ala Trp Trp 160 165 170 Gln Lys Leu Ala Ala Glu Phe Lys Thr Asp Asn Asn Val Ile Phe Asp 175 180 185 Leu Met Asn Glu Pro His Asp Ile Pro Ala Gln Thr Val Phe Gln Leu 190 195 200 Met Gln Ala Ala Val Asn Gly Val Arg Ala Ser Gly Ala Thr Ser Gln 205 210 215 220 Leu Ile Leu Ala Glu Gly Thr Ser Trp Thr Gly Ala Trp Thr Trp Thr 225 230 235 Thr Ser Gly Asn Ser Asp Ala Phe Gly Ala Ile Lys Asp Pro Asn Asn 240 245 250 Asn Ile Ala Ile Gln Met His Gln Tyr Leu Asp Ser Asp Gly Ser Gly 255 260 265 Thr Ser Pro Ile Cys Val Ser Asp Thr Ile Gly Ala Glu Arg Leu Gln 270 275 280 Ala Ala Thr Gln Trp Leu Gln Gln Thr Gly Leu Lys Gly Phe Leu Gly 285 290 295 300 Glu Ile Gly Thr Gly Asn Asn Thr Gln Cys Val Thr Ala Leu Gln Gly 305 310 315 Ala Leu Cys Glu Met Gln Gln Ala Gly Gly Thr Trp Leu Gly Ala Leu 320 325 330 Trp Trp Ala Ala Gly Pro Trp Trp Gly Asp Tyr Tyr Gln Ser Ile Glu 335 340 345 Pro Pro Asn Gly Asp Ala Ile Ala Ala Ile Leu Pro Ala Leu Lys Ala 350 355 360 Phe Gln 365 51772DNATrametes versicolorCDS(1)..(57)sig_peptide(1)..(57)Intron(58)..(111)mat_peptide(11- 2)..()CDS(112)..(142)Intron(143)..(195)CDS(196)..(246)Intron(247)..(301)CD- S(302)..(408)Intron(409)..(462)CDS(463)..(556)Intron(557)..(612)CDS(613)..- (618)Intron(619)..(668)CDS(669)..(909)Intron(910)..(974)CDS(975)..(1192)In- tron(1193)..(1244)CDS(1245)..(1297)Intron(1298)..(1361)CDS(1362)..(1471)In- tron(1472)..(1539)CDS(1540)..(1688)Intron(1689)..(1752)CDS(1753)..(1769) 5atg aag tac gca act gcc tct ctc gtg gct gcg gcc acc gtc tcc cag 48Met Lys Tyr Ala Thr Ala Ser Leu Val Ala Ala Ala Thr Val Ser Gln -15 -10 -5 gtt atg gcg gtatgcactc aggctagtac ttcacatgct ggattatact 97Val Met Ala -1 cattagcaac gtag gtg tac caa cag tgc ggt ggt atc ggt ttc g 142 Val Tyr Gln Gln Cys Gly Gly Ile Gly Phe 1 5 10 gttagcagaa ttgcaagtgg cgcatttgac tgcccttgct aacgaacttg cag at 197 Asp agg ccc act gct tgt gat gct cac tcg gtg tgc act gct atc aat ccc c 246Arg Pro Thr Ala Cys Asp Ala His Ser Val Cys Thr Ala Ile Asn Pro 15 20 25 gtatgtctgt cctgctatca ttacacagac atagtaggct catgctggca tgcag ac 303 His tac tcc cag tgc ctc ccg tcg tct tcc ccc tcg gcg ccc tct gcc ccc 351Tyr Ser Gln Cys Leu Pro Ser Ser Ser Pro Ser Ala Pro Ser Ala Pro 30 35 40 cgc tcc agc ctg atc cag ctc ggt ggt gtc aac act gcg gga tac gac 399Arg Ser Ser Leu Ile Gln Leu Gly Gly Val Asn Thr Ala Gly Tyr Asp 45 50 55 60 ttc agc gtt gtacgtcctc tgaaacaaaa gtcttagacc cattgctcac 448Phe Ser Val agatattgac atag act att gac gga agc ttc acc ggc acc ggt gtc tct 498 Thr Ile Asp Gly Ser Phe Thr Gly Thr Gly Val Ser 65 70 75 ccc ccg ccc tct cag tat acc cac ttc tct agc cag ggt gcc aac ctc 546Pro Pro Pro Ser Gln Tyr Thr His Phe Ser Ser Gln Gly Ala Asn Leu 80 85 90 ttc cgc att c gtgagtgtgc caagtgttga gtacgggaat aatactgatc 596Phe Arg Ile atattttttg ccgtag cc ttc g gtgcgttctc tgtctagcga tgtggtttcg 648 Pro Phe 95 ttctcatagc cctcctttag cc tgg cag ctg atg acg ccg aac gtc ggc gga 700 Ala Trp Gln Leu Met Thr Pro Asn Val Gly Gly 100 105 ccc atc aac gag acg ttc ttc gcc acc tat gac aag acc gtc cag gct 748Pro Ile Asn Glu Thr Phe Phe Ala Thr Tyr Asp Lys Thr Val Gln Ala 110 115 120 gcg ctc aac tcg ggc tcc aac gtg cac gtc atc atc gat ctg cac aac 796Ala Leu Asn Ser Gly Ser Asn Val His Val Ile Ile Asp Leu His Asn 125 130 135 tat gcg cgc tgg aac ggc gcc atc atc gct caa ggc ggc ccg acc aac 844Tyr Ala Arg Trp Asn Gly Ala Ile Ile Ala Gln Gly Gly Pro Thr Asn 140 145 150 155 gag caa ttt gct tcc atc tgg acc cag ctc gcc gcc aag tac ggc cgc 892Glu Gln Phe Ala Ser Ile Trp Thr Gln Leu Ala Ala Lys Tyr Gly Arg 160 165 170 aac aag cgt atc atc tt gtacgctcct tcctccctct ctttcttatg 939Asn Lys Arg Ile Ile Phe 175 acatgtatga ggatagctgc tgacgacgtc cgcag c ggt ctc atg aac gag cca 993 Gly Leu Met Asn Glu Pro 180 cac gac ctc ccc agc gtc ccg acc tgg gtt aag tct gtc cag ttc gtt 1041His Asp Leu Pro Ser Val Pro Thr Trp Val Lys Ser Val Gln Phe Val 185 190 195 gtc aac gcc atc cgc cat gcc ggc gcg acg aac ttc ctc ctc ctg cct 1089Val Asn Ala Ile Arg His Ala Gly Ala Thr Asn Phe Leu Leu Leu Pro 200 205 210 215 ggc tcc agc ttc tca tcc gcc cag gcc ttc ccc acc gag gcc ggc cct 1137Gly Ser Ser Phe Ser Ser Ala Gln Ala Phe Pro Thr Glu Ala Gly Pro 220 225 230 gac ctc gtc aag gtc act gac ccg ctc ggc ggc acc cac aag ctg atc 1185Asp Leu Val Lys Val Thr Asp Pro Leu Gly Gly Thr His Lys Leu Ile 235 240 245 ttc gat g gtaggtttag cgtgttagaa cccatcctgt gccggactga cctctgccgc 1242Phe Asp ag tc cac aag tat ctc gac agc gac aac agc ggc act cac ccc gac 1288 Val His Lys Tyr Leu Asp Ser Asp Asn Ser Gly Thr His Pro Asp 250 255 260 tgc acc act gtacgttgca ccgccccgtc tatcacgaac gcgacttcca 1337Cys Thr Thr 265 gcgatgctca tgcacattcc atag gac aac gtc gac gtg ctg aag acg ctc 1388 Asp Asn Val Asp Val Leu Lys Thr Leu 270 275 gtc cag ttc ctc aag cag aac ggc aac cgc cag gcg ctc ctc agc gag 1436Val Gln Phe Leu Lys Gln Asn Gly Asn Arg Gln Ala Leu Leu Ser Glu 280 285 290 acc ggc gga gga aac acc acg agc tgc gag act ct gtaagtgcca 1481Thr Gly Gly Gly Asn Thr Thr Ser Cys Glu Thr Leu 295 300 gtggccttgc acgccgccca cttgggcttg tagtagcacg ctgacgcact ccccgaag c 1540ctc aac acc gag ctc tcg ttc gtc aag tcc gcg ttc ccg acc ctc gtc 1588Leu Asn Thr Glu Leu Ser Phe Val Lys Ser Ala Phe Pro Thr Leu Val 305 310 315 320 ggc ttc tcc gcc tgg gcc gcc ggc gcg ttc gac acc aac tac gtg ctc 1636Gly Phe Ser Ala Trp Ala Ala Gly Ala Phe Asp Thr Asn Tyr Val Leu 325 330 335 acg ctc acg ccc aac gcc gac ggc tcc gac cag ccc ctc tgg atc gac 1684Thr Leu Thr Pro Asn Ala Asp Gly Ser Asp Gln Pro Leu Trp Ile Asp 340 345 350 gcc g gtacgtgtgg ccctgccagt gctcattctg tcctctcgat ataggtgctc 1738Ala accattcgct gcag tc aag ccc aac ctg cct tga 1772 Val Lys Pro Asn Leu Pro 355 6378PRTTrametes versicolor 6Met Lys Tyr Ala Thr Ala Ser Leu Val Ala Ala Ala Thr Val Ser Gln -15 -10 -5 Val Met Ala Val Tyr Gln Gln Cys Gly Gly Ile Gly Phe Asp Arg Pro -1 1 5 10 Thr Ala Cys Asp Ala His Ser Val Cys Thr Ala Ile Asn Pro His Tyr 15 20 25 Ser Gln Cys Leu Pro Ser Ser Ser Pro Ser Ala Pro Ser Ala Pro Arg 30 35 40 45 Ser Ser Leu Ile Gln Leu Gly Gly Val Asn Thr Ala Gly Tyr Asp Phe 50 55 60 Ser Val Thr Ile Asp Gly Ser Phe Thr Gly Thr Gly Val Ser Pro Pro 65 70 75 Pro Ser Gln Tyr Thr His Phe Ser Ser Gln Gly Ala Asn Leu Phe Arg 80 85 90 Ile Pro Phe Ala Trp Gln Leu Met Thr Pro Asn Val Gly Gly Pro Ile 95 100 105 Asn Glu Thr Phe Phe Ala Thr Tyr Asp Lys Thr Val Gln Ala Ala Leu 110 115 120 125 Asn Ser Gly Ser Asn Val His Val Ile Ile Asp Leu His Asn Tyr Ala 130 135 140 Arg Trp Asn Gly Ala Ile Ile Ala Gln Gly Gly Pro Thr Asn Glu Gln 145 150 155 Phe Ala Ser Ile Trp Thr Gln Leu Ala Ala Lys Tyr Gly Arg Asn Lys 160 165 170 Arg Ile Ile Phe Gly Leu Met Asn Glu Pro His Asp Leu Pro Ser Val 175 180 185 Pro Thr Trp Val Lys Ser Val Gln Phe Val Val Asn Ala Ile Arg His 190 195 200 205 Ala Gly Ala Thr Asn Phe Leu Leu Leu Pro Gly Ser Ser Phe Ser Ser 210 215 220 Ala Gln Ala Phe Pro Thr Glu Ala Gly Pro Asp Leu Val Lys Val Thr 225 230 235 Asp Pro Leu Gly Gly Thr His Lys Leu Ile Phe Asp Val His Lys Tyr 240 245 250 Leu Asp Ser Asp Asn Ser Gly Thr His Pro Asp Cys Thr Thr Asp Asn 255 260 265 Val Asp Val Leu Lys Thr Leu Val Gln Phe Leu Lys Gln Asn Gly Asn 270 275 280 285 Arg Gln Ala Leu Leu Ser Glu Thr Gly Gly Gly Asn Thr Thr Ser Cys 290 295 300 Glu Thr Leu Leu Asn Thr Glu Leu Ser Phe Val Lys Ser Ala Phe Pro 305 310 315 Thr Leu Val Gly Phe Ser Ala Trp Ala Ala Gly Ala Phe Asp Thr Asn 320 325 330 Tyr Val Leu Thr Leu Thr Pro Asn Ala Asp Gly Ser Asp Gln Pro Leu 335 340 345 Trp Ile Asp Ala Val Lys Pro Asn Leu Pro 350 355 71746DNATrametes versicolorCDS(1)..(57)sig_peptide(1)..(57)Intron(58)..(108)mat_peptide(10- 9)..()CDS(109)..(139)Intron(140)..(192)CDS(193)..(243)Intron(244)..(295)CD- S(296)..(402)Intron(403)..(456)CDS(457)..(550)Intron(551)..(606)CDS(607)..- (612)Intron(613)..(665)CDS(666)..(906)Intron(907)..(968)CDS(969)..(1186)In- tron(1187)..(1238)CDS(1239)..(1291)Intron(1292)..(1352)CDS(1353)..(1462)In- tron(1463)..(1519)CDS(1520)..(1668)Intron(1669)..(1726)CDS(1727)..(1743) 7atg aag tac gca act gcc tct ctc gtg gct gcg gcc act gtc tcc caa 48Met Lys Tyr Ala Thr Ala Ser Leu Val Ala Ala Ala Thr Val Ser Gln -15 -10 -5 gtt gcg gcg gtatgtcttc agtctactat acattctgat ccttacttat 97Val Ala Ala -1 aggcaacata g gtg tac cag cag tgc ggc ggt atc ggc ttc g gttggtagca 149 Val Tyr Gln Gln Cys Gly Gly Ile Gly Phe 1 5 10 gtgcaagtga cctatctaat tacccttgct aacgaccttg cag tt ggg ccc act 203 Val Gly Pro Thr gct tgc gat gct cag tcg gtg tgc act act atc aac gcc t gtatgtctag 253Ala Cys Asp Ala Gln Ser Val Cys Thr Thr Ile Asn Ala 15 20 25 cctgacatca ttataccgac ataaactgat gttggcatgc ag ac tac tcg cag 306 Tyr Tyr Ser Gln 30 tgc ctc ccg acg tcc tcc ccc tcc gcg ccc tcc gct ccc agc tcc ggc 354Cys Leu Pro Thr Ser Ser Pro Ser Ala Pro Ser Ala Pro Ser Ser Gly 35 40 45 ctg atc cag ctc ggt ggt gtc aac act gcg gga tac gac ttc agc gtt 402Leu Ile Gln Leu Gly Gly Val Asn Thr Ala Gly Tyr Asp Phe Ser Val 50 55 60 gtacgtcctc tgagagaatg tttccaaacg cattactcaa ggatattgac gtag gct 459 Ala act gat gga agc ttc acc ggc acc ggt gtc tct ccc ccg cca tcc cag 507Thr Asp Gly Ser Phe Thr Gly Thr Gly Val Ser Pro Pro Pro Ser Gln 65 70 75 80 ttc acc cac ttc tcc agc cag ggt gct aac ctc tac cgc att c 550Phe Thr His Phe Ser Ser Gln Gly Ala Asn Leu Tyr Arg Ile 85 90 gtaagtgggc tcagcattgc gaagggaagt cgtactgact gtatgtttta ttgtag cc 608 Pro 95 ttc g gtgtgccgtt tctctagcga tgtcgattgg tccaattgct gacccttcct tag 665Phe ca tgg cag ctg atg acg ccc aac ctc ggc gga ccc atc aac gag acg 712Ala Trp Gln Leu Met Thr Pro Asn Leu Gly Gly Pro Ile Asn Glu Thr 100 105 110 ttc ttc tcc acc tac gac cag act gtc caa gcc gcg ctc aac tcg ggc 760Phe Phe Ser Thr Tyr Asp Gln Thr Val Gln Ala Ala Leu Asn Ser Gly 115 120 125 tca aac gtg cac gtc atc gtc gac ctg cac aac tac gcg cgc tgg aac 808Ser Asn Val His Val Ile Val Asp Leu His Asn Tyr Ala Arg Trp Asn 130 135 140 ggc ggt atc atc gcc cag ggc ggc

ccg acc aac gag gag tac gcg tcc 856Gly Gly Ile Ile Ala Gln Gly Gly Pro Thr Asn Glu Glu Tyr Ala Ser 145 150 155 160 atc tgg acc cac ctc gcc gcc aag tac ggc tcc aac gag cgc atc att 904Ile Trp Thr His Leu Ala Ala Lys Tyr Gly Ser Asn Glu Arg Ile Ile 165 170 175 tt gtacgcccgc ccctcggtct tctgatacgt tgaagaggag agctcacgac 956Phe gtctgcctgc ag c ggt gtc atg aac gag ccg cac gac atc ccc aac gtc 1005 Gly Val Met Asn Glu Pro His Asp Ile Pro Asn Val 180 185 cag acc tgg gtc gac tcc gtc cag ttc gtc gtc aac gct atc cgc cag 1053Gln Thr Trp Val Asp Ser Val Gln Phe Val Val Asn Ala Ile Arg Gln 190 195 200 205 gcc ggc gcg acg aac ttc ctc ctc ctg cct ggc tcc agc ttc tca tcc 1101Ala Gly Ala Thr Asn Phe Leu Leu Leu Pro Gly Ser Ser Phe Ser Ser 210 215 220 gcc cag gcc ttc ccc acc gag gcc ggc cct tat ctc gtc aaa gta act 1149Ala Gln Ala Phe Pro Thr Glu Ala Gly Pro Tyr Leu Val Lys Val Thr 225 230 235 gac ccg ctt ggc ggc acc gac aag ctg atc ttc gat g gtaagcccag 1196Asp Pro Leu Gly Gly Thr Asp Lys Leu Ile Phe Asp 240 245 catcatagaa ctcattatgt acacaagtta acccgaccgc ag tc cac aag tac 1249 Val His Lys Tyr 250 ctc gac agc gac aac agc ggc acc cac ccc gac tgc acc acc 1291Leu Asp Ser Asp Asn Ser Gly Thr His Pro Asp Cys Thr Thr 255 260 265 gtacgttgca cctcctaaac tatgcacacc tctccccgcg atgctcacgt acattccgca 1351g gac aac gtg gac gtc ctt aag acc ctc gtc cag ttc ctt cag caa aac 1400 Asp Asn Val Asp Val Leu Lys Thr Leu Val Gln Phe Leu Gln Gln Asn 270 275 280 ggc aac cgt cag gcg atc ctg agc gag acc ggc gga ggc aac act gcg 1448Gly Asn Arg Gln Ala Ile Leu Ser Glu Thr Gly Gly Gly Asn Thr Ala 285 290 295 agc tgt gag act ct gtaagtcgcg cacaccgctt acagtcttca agcattgcgc 1502Ser Cys Glu Thr Leu 300 taatgcagcc cccgcag c ctc aac aac gag ctc tcg ttc gtc aag tcc gcg 1553 Leu Asn Asn Glu Leu Ser Phe Val Lys Ser Ala 305 310 315 ttc ccg acc ctc gcc ggc ttc tca gtc tgg gct gct ggc gcg ttc gac 1601Phe Pro Thr Leu Ala Gly Phe Ser Val Trp Ala Ala Gly Ala Phe Asp 320 325 330 acc acc tac gtg ctc acc gtc tcg ccc aac ccc gat ggc tcc gac cag 1649Thr Thr Tyr Val Leu Thr Val Ser Pro Asn Pro Asp Gly Ser Asp Gln 335 340 345 ccc ctc tgg aac gac gcc g gtacgtgtga tcccatgcat cccgtcctat 1698Pro Leu Trp Asn Asp Ala 350 cgattcaatt gctcactgtt cgctacag tc aag ccc aac ctg ccc tga 1746 Val Lys Pro Asn Leu Pro 355 8378PRTTrametes versicolor 8Met Lys Tyr Ala Thr Ala Ser Leu Val Ala Ala Ala Thr Val Ser Gln -15 -10 -5 Val Ala Ala Val Tyr Gln Gln Cys Gly Gly Ile Gly Phe Val Gly Pro -1 1 5 10 Thr Ala Cys Asp Ala Gln Ser Val Cys Thr Thr Ile Asn Ala Tyr Tyr 15 20 25 Ser Gln Cys Leu Pro Thr Ser Ser Pro Ser Ala Pro Ser Ala Pro Ser 30 35 40 45 Ser Gly Leu Ile Gln Leu Gly Gly Val Asn Thr Ala Gly Tyr Asp Phe 50 55 60 Ser Val Ala Thr Asp Gly Ser Phe Thr Gly Thr Gly Val Ser Pro Pro 65 70 75 Pro Ser Gln Phe Thr His Phe Ser Ser Gln Gly Ala Asn Leu Tyr Arg 80 85 90 Ile Pro Phe Ala Trp Gln Leu Met Thr Pro Asn Leu Gly Gly Pro Ile 95 100 105 Asn Glu Thr Phe Phe Ser Thr Tyr Asp Gln Thr Val Gln Ala Ala Leu 110 115 120 125 Asn Ser Gly Ser Asn Val His Val Ile Val Asp Leu His Asn Tyr Ala 130 135 140 Arg Trp Asn Gly Gly Ile Ile Ala Gln Gly Gly Pro Thr Asn Glu Glu 145 150 155 Tyr Ala Ser Ile Trp Thr His Leu Ala Ala Lys Tyr Gly Ser Asn Glu 160 165 170 Arg Ile Ile Phe Gly Val Met Asn Glu Pro His Asp Ile Pro Asn Val 175 180 185 Gln Thr Trp Val Asp Ser Val Gln Phe Val Val Asn Ala Ile Arg Gln 190 195 200 205 Ala Gly Ala Thr Asn Phe Leu Leu Leu Pro Gly Ser Ser Phe Ser Ser 210 215 220 Ala Gln Ala Phe Pro Thr Glu Ala Gly Pro Tyr Leu Val Lys Val Thr 225 230 235 Asp Pro Leu Gly Gly Thr Asp Lys Leu Ile Phe Asp Val His Lys Tyr 240 245 250 Leu Asp Ser Asp Asn Ser Gly Thr His Pro Asp Cys Thr Thr Asp Asn 255 260 265 Val Asp Val Leu Lys Thr Leu Val Gln Phe Leu Gln Gln Asn Gly Asn 270 275 280 285 Arg Gln Ala Ile Leu Ser Glu Thr Gly Gly Gly Asn Thr Ala Ser Cys 290 295 300 Glu Thr Leu Leu Asn Asn Glu Leu Ser Phe Val Lys Ser Ala Phe Pro 305 310 315 Thr Leu Ala Gly Phe Ser Val Trp Ala Ala Gly Ala Phe Asp Thr Thr 320 325 330 Tyr Val Leu Thr Val Ser Pro Asn Pro Asp Gly Ser Asp Gln Pro Leu 335 340 345 Trp Asn Asp Ala Val Lys Pro Asn Leu Pro 350 355

Claims

1-48. (canceled)

49. A nucleic acid construct comprising a polynucleotide encoding a polypeptide having endoglucanase activity, wherein the polynucleotide is operably linked to one or more heterologous control sequences that direct the production of the polypeptide in an expression host, and wherein the polypeptide having endoglucanase activity has at least 94% sequence identity to the sequence of amino acids 21 to 386 of SEQ ID NO: 4, or is a fragment of the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

50. (canceled)

51. A process for degrading a cellulosic material, said process comprising: (i) treating the cellulosic material with an enzyme composition, wherein the composition comprises a polypeptide having endoglucanase activity; and (ii) recovering the degraded cellulosic material; wherein the polypeptide having endoglucanase activity has at least 94% sequence identity to the sequence of amino acids 21 to 386 of SEQ ID NO: 4, or is a fragment of the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

52. A process for producing a fermentation product, said process comprising: (a) saccharifying a cellulosic material with an enzyme composition, wherein the composition comprises a polypeptide having endoglucanase activity; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation; wherein the polypeptide having endoqlucanase activity has at least 94% sequence identity to the sequence of amino acids 21 to 386 of SEQ ID NO: 4, or is a fragment of the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

53. (canceled)

54. A nucleic acid construct comprising a polynucleotide encoding a polypeptide comprising a catalytic domain, wherein the polynucleotide is operably linked to one or more heteroloqous control sequences that direct the production of the polypeptide in an expression host, wherein the catalytic domain has at least 94% sequence identity to the sequence of amino acids 73 to 386 of SEQ ID NO: 4 or comprises, a fragment of the sequence of amino acids 73 to 386 of SEQ ID NO: 4, and wherein the catalytic domain has endoglucanase activity.

55. The nucleic acid construct of claim 54, wherein the polypeptide further comprising a cellulose binding domain.

56. (canceled)

57. A process for degrading a cellulosic material, said process comprising: (i) treating the cellulosic material with an enzyme composition, wherein the composition comprises a polypeptide having endoqlucanase activity; and (ii) recovering the degraded cellulosic material; wherein the polypeptide having endoqlucanase activity comprises a catalytic domain having at least 94% sequence identity to the sequence of amino acids 73 to 386 of SEQ ID NO: 4, or the polypeptide having endoqlucanase activity comprises a catalytic domain having a fragment of the sequence of amino acids 73 to 386 of SEQ ID NO: 4.

58. A process for producing a fermentation product, said process comprising: (a) saccharifying a cellulosic material with an enzyme composition, wherein the composition comprises a polypeptide having endoqlucanase activity; (b) fermenting the saccharified cellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation; wherein the polypeptide having endoqlucanase activity comprises a catalytic domain having at least 94% sequence identity to the sequence of amino acids 73 to 386 of SEQ ID NO: 4, or the polypeptide having endoqlucanase activity comprises a catalytic domain having a fragment of the sequence of amino acids 73 to 386 of SEQ ID NO: 4.

59. (canceled)

60. The nucleic acid construct of claim 49, wherein the polypeptide having endoglucanase activity has at least 97% sequence identity to the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

61. The nucleic acid construct of claim 49, wherein the polypeptide having endoglucanase activity comprises the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

62. The nucleic acid construct of claim 49, wherein the polypeptide having endoglucanase activity is a fragment of the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

63. The nucleic acid construct of claim 49, wherein the polypeptide having endoglucanase activity is a variant of the sequence of amino acids 21 to 386 of SEQ ID NO: 4, comprising a substitution, deletion and/or insertion of one or more amino acids.

64. A recombinant host cell comprising the nucleic acid construct of claim 49.

65. A method of producing a polypeptide having endoglucanase activity, said method comprising: (a) cultivating the host cell of claim 64 under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

66. A recombinant host cell transformed with a polynucleotide encoding a polypeptide having endoglucanase activity, wherein the polypeptide having endoglucanase activity has at least 94% sequence identity to the sequence of amino acids 21 to 386 of SEQ ID NO: 4, or is a fragment of the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

67. The recombinant host cell of claim 66, wherein the polypeptide having endoglucanase activity has at least 97% sequence identity to the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

68. The recombinant host cell of claim 66, wherein the polypeptide having endoglucanase activity comprises the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

69. A method of producing a polypeptide having endoglucanase activity, said method comprising: (a) cultivating a host cell comprising a polynucleotide encoding a polypeptide having endoglucanase activity under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide; wherein the polypeptide having endoglucanase activity has at least 94% sequence identity to the sequence of amino acids 21 to 386 of SEQ ID NO: 4, or is a fragment of the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

70. The process of claim 51, wherein the polypeptide having endoglucanase activity has at least 97% sequence identity to the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

71. The process of claim 51, wherein the polypeptide having endoglucanase activity comprises the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

72. The process of claim 52, wherein the polypeptide having endoglucanase activity has at least 97% sequence identity to the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

73. The process of claim 52, wherein the polypeptide having endoglucanase activity comprises the sequence of amino acids 21 to 386 of SEQ ID NO: 4.

74. The nucleic acid construct of claim 54, wherein the catalytic domain has at least 97% sequence identity to the sequence of amino acids 73 to 386 of SEQ ID NO: 4.

75. The nucleic acid construct of claim 54, wherein the catalytic domain comprises the sequence of amino acids 73 to 386 of SEQ ID NO: 4.

76. The process of claim 57, wherein the polypeptide having endoglucanase activity comprises a catalytic domain having at least 97% sequence identity to the sequence of amino acids 73 to 386 of SEQ ID NO: 4.

77. The process of claim 57, wherein the polypeptide having endoglucanase activity comprises a catalytic domain having the sequence of amino acids 73 to 386 of SEQ ID NO: 4.

78. The process of claim 58, wherein the polypeptide having endoglucanase activity comprises a catalytic domain having at least 97% sequence identity to the sequence of amino acids 73 to 386 of SEQ ID NO: 4.

79. The process of claim 58, wherein the polypeptide having endoglucanase activity comprises a catalytic domain having the sequence of amino acids 73 to 386 of SEQ ID NO: 4.

80. A recombinant host cell transformed with a polynucleotide encoding a polypeptide having endoglucanase activity, wherein the polypeptide having endoglucanase activity comprises a catalytic domain having at least 94% sequence identity to the sequence of amino acids 73 to 386 of SEQ ID NO: 4, or the polypeptide having endoglucanase activity comprises a catalytic domain having a fragment of the sequence of amino acids 73 to 386 of SEQ ID NO: 4.

81. The recombinant host cell of claim 80, wherein the polypeptide having endoglucanase activity comprises a catalytic domain having at least 97% sequence identity to the sequence of amino acids 73 to 386 of SEQ ID NO: 4.

82. The recombinant host cell of claim 81, wherein the polypeptide having endoglucanase activity comprises a catalytic domain having the sequence of amino acids 73 to 386 of SEQ ID NO: 4.