Compositions for degrading cellulosic material

Abstract

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

1 - 170 . (canceled) 
     
     
         171 . A cellulolytic protein composition, comprising: (a) a GH61 polypeptide having cellulolytic enhancing activity; (b) a beta-glucosidase; and (c) cellulolytic enzymes selected from the group consisting of a cellobiohydrolase I, a cellobiohydrolase II, and an endoglucanase I;
 wherein the GH61 polypeptide having cellulolytic enhancing activity is selected from the group consisting of:   (a) a GH61 polypeptide comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14;   (b) a GH61 polypeptide encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13, or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;   (c) a GH61 polypeptide encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, or SEQ ID NO: 13; and   (d) a GH61 polypeptide comprising the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14;   wherein the beta-glucosidase is selected from the group consisting of:   (a) a beta-glucosidase comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105;   (b) a beta-glucosidase encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 104 or SEQ ID NO: 106, or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;   (c) a beta-glucosidase encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 104 or SEQ ID NO: 106; and   (d) a beta-glucosidase comprising the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105;   wherein the cellobiohydrolase I is selected from the group consisting of:   (a) a cellobiohydrolase I comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 52;   (b) a cellobiohydrolase I encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 51 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;   (c) a cellobiohydrolase I encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 51; and   (d) a cellobiohydrolase I comprising the mature polypeptide of SEQ ID NO: 52;   wherein the cellobiohydrolase II is selected from the group consisting of:   (a) a cellobiohydrolase II comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 54;   (b) a cellobiohydrolase II encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 53 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;   (c) a cellobiohydrolase II encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 53; and   (d) a cellobiohydrolase II comprising the mature polypeptide of SEQ ID NO: 54; and   wherein the endoglucanase I is selected from the group consisting of:   (a) an endoglucanase I comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 56;   (b) an endoglucanase I encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 55 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;   (c) an endoglucanase I encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 55; and   (d) an endoglucanase I comprising the mature polypeptide of SEQ ID NO: 56.   
     
     
         172 . The cellulolytic protein composition of  claim 171 , further comprising cellulolytic enzymes selected from the group consisting of an endoglucanase II, an endoglucanase III, and an endoglucanase V;
 wherein the endoglucanase II is selected from the group consisting of:   (a) an endoglucanase II comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 58;   (b) an endoglucanase II encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 57 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;   (c) an endoglucanase II encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 57; and   (d) an endoglucanase II comprising the mature polypeptide of SEQ ID NO: 58;   wherein the endoglucanase III is selected from the group consisting of:   (a) an endoglucanase III comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 60;   (b) an endoglucanase III encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 59 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;   (c) an endoglucanase III encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 59; and   (d) an endoglucanase III comprising the mature polypeptide of SEQ ID NO: 60; and   wherein the endoglucanase V is selected from the group consisting of:   (a) an endoglucanase V comprising an amino acid sequence having at least 95% sequence identity to the mature polypeptide of SEQ ID NO: 62;   (b) an endoglucanase V encoded by a polynucleotide which hybridizes under at least high stringency conditions or at least very high stringency conditions with the mature polypeptide coding sequence of SEQ ID NO: 61 or the full-length complement thereof, wherein high stringency conditions and very high stringency conditions are defined as prehybridization and hybridization at 42° C. in 5×SSPE, 0.3% SDS, 200 μg/ml sheared and denatured salmon sperm DNA, and 25% formamide for high and very high stringencies and washing three times each for 15 minutes using 2×SSC, 0.2% SDS at 65° C. for high stringency and 70° C. for very high stringency;   (c) an endoglucanase V encoded by a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 61; and   (d) an endoglucanase V comprising the mature polypeptide of SEQ ID NO: 62.   
     
     
         173 . The cellulolytic protein composition of  claim 171 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. 
     
     
         174 . The cellulolytic protein composition of  claim 171 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14. 
     
     
         175 . The cellulolytic protein composition of  claim 171 , wherein the beta-glucosidase comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105. 
     
     
         176 . The cellulolytic protein composition of  claim 171 , wherein the beta-glucosidase comprises the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105. 
     
     
         177 . The cellulolytic protein composition of  claim 171 , wherein the cellobiohydrolase I comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 52. 
     
     
         178 . The cellulolytic protein composition of  claim 171 , wherein the cellobiohydrolase I comprises the mature polypeptide of SEQ ID NO: 52. 
     
     
         179 . The cellulolytic protein composition of  claim 171 , wherein the cellobiohydrolase II comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 54. 
     
     
         180 . The cellulolytic protein composition of  claim 171 , wherein the cellobiohydrolase II comprises the mature polypeptide of SEQ ID NO: 54. 
     
     
         181 . The cellulolytic protein composition of  claim 171 , wherein the endoglucanase I comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 56. 
     
     
         182 . The cellulolytic protein composition of  claim 171 , wherein the endoglucanase I comprises the mature polypeptide of SEQ ID NO: 56. 
     
     
         183 . The cellulolytic protein composition of  claim 171 , wherein the GH61 polypeptide having cellulolytic enhancing activity comprises the mature polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, or SEQ ID NO: 14; the beta-glucosidase comprises the mature polypeptide of SEQ ID NO: 103 or SEQ ID NO: 105; the cellobiohydrolase I comprises the mature polypeptide of SEQ ID NO: 52; the cellobiohydrolase II comprises the mature polypeptide of SEQ ID NO: 54; and the endoglucanase I comprises the mature polypeptide of SEQ ID NO: 56. 
     
     
         184 . The cellulolytic protein composition of  claim 172 , wherein the endoglucanase II comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 58. 
     
     
         185 . The cellulolytic protein composition of  claim 172 , wherein the endoglucanase II comprises the mature polypeptide of SEQ ID NO: 58. 
     
     
         186 . The cellulolytic protein composition of  claim 172 , wherein the endoglucanase III comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 60. 
     
     
         187 . The cellulolytic protein composition of  claim 172 , wherein the endoglucanase III comprises the mature polypeptide of SEQ ID NO: 60. 
     
     
         188 . The cellulolytic protein composition of  claim 172 , wherein the endoglucanase V comprises an amino acid sequence having at least 97% sequence identity to the mature polypeptide of SEQ ID NO: 62. 
     
     
         189 . The cellulolytic protein composition of  claim 172 , wherein the endoglucanase V comprises the mature polypeptide of SEQ ID NO: 62. 
     
     
         190 . The cellulolytic protein composition of  claim 172 , wherein the endoglucanase II comprises the mature polypeptide of SEQ ID NO: 58; the endoglucanase III comprises the mature polypeptide of SEQ ID NO: 60; and the endoglucanase V comprises the mature polypeptide of SEQ ID NO: 62. 
     
     
         191 . A method for degrading a cellulose-containing material, comprising: treating the cellulose-containing material with the cellulolytic protein composition of  claim 171 . 
     
     
         192 . The method of  claim 191 , further comprising recovering the degraded cellulose-containing material. 
     
     
         193 . The method of claim  162 , wherein the degraded cellulose-containing material is a sugar selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose. 
     
     
         194 . A method for producing a fermentation product, comprising: (a) saccharifying a cellulose-containing material with the cellulolytic protein composition of  claim 171 ; (b) fermenting the saccharified cellulose-containing material of step (a) with one or more (several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation. 
     
     
         195 . The method of  claim 194 , wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation. 
     
     
         196 . The method of claim of  194 , wherein the fermentation product is an alcohol, organic acid, ketone, aldehyde, amino acid, or gas.