Method for reducing viscosity in saccharification process

Abstract

The present invention relates to compositions that can be used in hydrolyzing biomass such as compositions comprising a polypeptide having glycosyl hydrolase family 61/endoglucanase activity, methods for hydrolyzing biomass material, and methods for reducing viscosity of biomass mixture using a composition comprising a polypeptide having glycosyl hydrolase family 61/endoglucanase activity.

1 . A biomass saccharification mixture comprising:
 a. a biomass material   b. an enzyme composition comprising a glycosyl hydrolase family 61 enzyme having endoglucanase activity, which is:
 i. at least 65% in sequence identity to any one of SEQ ID NO:1-29 and 148; 
 ii. at least 65% in sequence identity to residues 22-344 of SEQ ID NO:27 
 iii. comprises at least one amino acid sequence motifs selected from the group consisting of: SEQ ID NOs: 84-91; 
 iv. comprises one or more sequence motifs selected from the group consisting of: (1) SEQ ID NO:84 and 88; (2) SEQ ID NOs: 85 and 88; (3) SEQ ID NO:86; (4) SEQ ID NO:87; (5) SE ID NO:84, 88 and 89; (6) SEQ ID NOs: 84, 88 and 91; (10) SEQ ID NOs: 85, 88 and 91; (11) SEQ ID NOs: 84, 88, 89 and 91; (12) SEQ ID NOs: 84, 88, 90 and 91; (13) SEQ ID NOs: 85, 88, 89 and 91; and (14) SEQ ID NOs: 85, 88, 90 and 91; 
 v. encoded by a polynucleotide sequence or a complement thereof that is at least 65% sequence identity to SEQ ID NO:30; or 
 vi. encoded by a polynucleotide sequence that hybridizes under high stringency conditions to SEQ ID NO:30 or to a complement thereof; 
   
       wherein said biomass saccharification mixture has a lower viscosity than a biomass saccharification mixture without the glycosyl hydrolyase family 61 enzyme and/or is capable of increasing the level of saccharification in the mixture as compared to the level of saccharification in a mixture having no or a lower level of glycosyl hydrolase family 61 enzyme, wherein the level of saccharification is measured by the yield of fermentable sugar after the mixture is incubated for a period of time sufficient to cause saccharification of the biomass. 
     
     
         2 . (canceled) 
     
     
         3 . The biomass saccharification mixture of  claim 1 , wherein the glycosyl hydrolase family 61 enzyme is derived from a filamentous fungus; optionally wherein the filamentous fungus is one selected from the group:  Trichoderma, Humicola, Fusarium, Aspergillus, Neurospora, Penicillium, Cephalosporium, Achlva, Podospora, Endothia, Mucor, Cochliobolus, Pyricularia, Chrvsosporium, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, 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, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens, Penicillium solitum, Penicillium funiculosum Phanerochaete chrysosporium, Phlebia radiate, Pleurotus ervngii, Talaromvces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, Trichoderma viride, Geosmithia emersonii , or  G. stearothermophilus.    
     
     
         4 - 7 . (canceled) 
     
     
         8 . The biomass saccharification mixture of  claim 1 , wherein the enzyme composition further comprises one or more or all of: (1) a polypeptide having xylanase activity, (2) a polypeptide having beta-xylosidase activity; (3) a polypeptide having L-alpha-arabinofuranosidase activity; and (4) at least one polypeptide having cellobiohydrolase activity and at least one polypeptide having beta-glucosidase activity; 
       optionally wherein:
 a. the polypeptide having xylanase activity is:
 i. a polypeptide encoding a  T. reesei  Xyn3 (SEQ ID NO:76),  T. reesei  Xyn2 (SEQ ID NO:77), an AfuXyn2 (SEQ ID NO:58), and AfuXyn5 (SEQ ID NO:60), or a variant thereof having at least 90% sequence identity thereto; or 
 ii. a polypeptide encoded by a polynucleotide (1) having at least 90% sequence identity to SEQ ID NO:75, 57, or 59; or (2) hybridizes under high stringency conditions to SEQ ID NO: 75, 57, or 59, or to a complement thereof; 
 
 b. the at least one polypeptide having beta-xylosidase activity is:
 i. a polypeptide encoding an Fv3A (SEQ ID NO:36), an Fv43A (SEQ ID NO:44), a Pf43A (SEQ ID NO:38), an Fv43D (SEQ ID NO:62), an Fv39A (SEQ ID NO:42), an Fv43E (SEQ ID NO:40), an Fo43A (SEQ ID NO:52), an Fv43B (SEQ ID NO:46), a Pa51A (SEQ ID NO:48), a Gz43A (SEQ ID NO:50), a  T. reesei  Bxl1 (SEQ ID NO:78), or a variant thereof having at least 90% sequence identity thereto; or 
 ii. a polypeptide encoded by a polynucleotide (1) having at least 90% sequence identity to SEQ ID NO:35, 43, 37, 61, 41, 39, 51, 45, 47, 49, or 159; (2) hybridizes under high stringency conditions to SEQ ID NO: 35, 43, 37, 61, 41, 39, 51, 45, 47, 49, 159, or to a complement thereof; and/or 
 
 c. the at least one polypeptide having L-alpha-arabinofuranosidase activity is:
 i. a polypeptide encoding an Af43A (SEQ ID NO:54), an Fv43B (SEQ ID NO:46), a Pf51A (SEQ ID NO:56), a Pa51A (SEQ ID NO:48), an Fv51A (SEQ ID NO:66), or a variant thereof having at least 90% sequence identity thereto; or 
 ii. a polypeptide encoded by a polynucleotide (1) having at least 90% sequence identity to SEQ ID NO:53, 45, 55, 47, or 65; (2) hybridizes under high stringency conditions to SEQ ID NO: 53, 45, 55, 47, or 65, or to a complement thereof; 
 d. the at least one polypeptide having cellobiohydrolase activity is a polypeptide encoding a  T. reesei  CBH1, Af 7A (SEQ ID NO:150), Af7B (SEQ ID NO:151), Cg7A (SEQ ID NO:152), Cg7B (SEQ ID NO:153), Tt7A (SEQ ID NO:154), Tt7B (SEQ ID NO:155),  T. reesei  CBH2, Tt6A (SEQ ID NO:156), St6A (SEQ ID NO:157), St6B (SEQ ID NO:158), or a variant thereof having at least 90% sequence identity thereto; and/or 
 
 e. the at least one polypeptide having beta-glucosidase activity is:
 i. a polypeptide encoding an Fv3C (SEQ ID NO:100), a Pa3D (SEQ ID NO:94), an Fv3G (SEQ ID NO:96), an Fv3D (SEQ ID NO:98), a Tr3A (SEQ ID NO:102), a Tr3B (SEQ ID NO:104), a Te3A (SEQ ID NO:106), an An3A (SEQ ID NO:108), an Fo3A (SEQ ID NO:110), a Gz3A (SEQ ID NO:112), an Nh3A (SEQ ID NO:114), a Vd3A (SEQ ID NO:116), a Pa3G (SEQ ID NO:118), a Tn3B (SEQ ID NO:119), or a variant thereof having at least 90% sequence identity thereto; or 
 ii. a polypeptide encoded by a polynucleotide (1) having at least 90% sequence identity to SEQ ID NO:99, 93, 95, 97, 101, 103, 105, 107, 109, 111, 113, 115, or 117; (2) hybridizes under high stringency conditions to SEQ ID NO: 99, 93, 95, 97, 101, 103, 105, 107, 109, 111, 113, 115, or 117, or to a complement thereof. 
 
 
     
     
         9 - 11 . (canceled) 
     
     
         12 . The biomass saccharification mixture of  claim 1 , wherein the enzyme composition comprises (1) about 0.1 wt. % to about 50 wt. %, about 1 wt. % to about 20 wt. %, about 5 wt. % to about 15 wt. % of the polypeptide having GH61/endoglucanase activity, referencing the total weight of proteins in the enzyme composition; or (2) about 0.2 mg to about 30 mg, about 0.2 mg to about 20 mg, about 0.5 mg to about 10 mg, or about 1 mg to about 5 mg of the polypeptide having GH61/endoglucanase activity per gram of cellulose, hemicelluloses or a mixture of cellulose and hemicelluloses contained in the biomass material. 
     
     
         13 . The biomass saccharification mixture of  claim 8 , wherein the enzyme composition comprises cellobiohydrolase in an amount that is (1) about 0.1 wt. % to about 80 wt. %, about 5 wt. % to about 70 wt. %, about 10 wt. % to about 60 wt. %, about 20 wt. % to about 50 wt. %, or about 25 wt. % to about 50 wt. % of the total weight of proteins in the enzyme composition; or (2) about 0.2 mg to about 30 mg, about 0.2 mg to about 20 mg, about 0.5 mg to about 10 mg, or about 0.5 mg to about 5 mg per gram of cellulose, hemicelluloses, or a mixture of cellulose and hemicelluloses in the biomass saccharification mixture; and comprises beta-glucosidase in an amount that is (1) about 0.1 wt. % to about 50 wt. %, about 1 wt. % to about 30 wt. %, about 2 wt. % to about 20 wt. %, about 5 wt. % to about 20 wt. %, or about 8 wt. % to about 15 wt. % of the total weight of proteins in the enzyme composition; or (2) about 0.2 mg to about 30 mg, about 0.2 mg to about 20 mg, about 0.5 mg to about 10 mg, or about 0.5 mg to about 5 mg per gram of cellulose, hemicelluloses, or a mixture of cellulose and hemicelluloses in the biomass saccharification mixture. 
     
     
         14 . The biomass saccharification mixture of  claim 8 , wherein:
 a. the enzyme composition comprises (1) about 0.1 wt. % to about 50 wt. %, about 1 wt. % to about 40 wt. %, about 4 wt. % to about 30 wt. %, about 5 wt. % to about 20 wt. %, or about 8 wt. % to about 15 wt. % of the polypeptide having xylanase activity, referencing the total weight of proteins in the enzyme composition; or (2) about 0.2 mg to about 30 mg, about 0.2 mg to about 20 mg, about 0.5 mg to about 10 mg, or about 0.5 mg to about 5 mg of the polypeptide having xylanase activity per gram of cellulose, hemicelluloses, or a mixture of cellulose and hemicelluloses in the biomass saccharification mixture;   b. the enzyme composition comprises (1) about 0.1 wt. % to about 50 wt. %, about 1 wt. % to about 40 wt. %, about 2 wt. % to about 30 wt. %, about 4 wt. % to about 20 wt. %, or about 5 wt. % to about 15 wt. % of the polypeptide having beta-xylosidase activity, referencing the total weight of proteins in the enzyme composition; or (2) about 0.2 mg to about 30 mg, about 0.2 mg to about 20 mg, about 0.5 mg to about 10 mg, or about 0.5 mg to about 5 mg of the polypeptide having beta-xylosidase activity per gram of cellulose, hemicelluloses, or a mixture of cellulose and hemicelluloses in the biomass saccharification mixture; and/or   c. the enzyme composition comprises (1) about 0.1 wt. % to about 50 wt. %, about 0.1 wt. % to about 50 wt. %, about 1 wt. % to about 40 wt. %, about 2 wt. % to about 30 wt. %, about 4 wt. % to about 20 wt. %, or about 5 wt. % to about 15 wt. % of the polypeptide having L-alpha-arabinofuranosidase activity, referencing the total weight of proteins in the enzyme composition; or (2) about 0.2 mg to about 30 mg, about 0.2 mg to about 20 mg, about 0.5 mg to about 10 mg, or about 0.5 mg to about 5 mg of the polypeptide having L-alpha-arabinofuranosidase activity per gram of cellulose, hemicelluloses, or a mixture of cellulose and hemicelluloses in the biomass saccharification mixture.   
     
     
         15 - 16 . (canceled) 
     
     
         17 . The biomass saccharification mixture of  claim 1 , wherein the enzyme composition is a whole cellulase composition, wherein the whole cellulase composition is derived from a host cell expressing a polynucleotide encoding a polypeptide having GH61/endoglucanase activity, optionally wherein the polynucleotide encoding the polypeptide having GH61 family enzyme activity is heterologous to the host cell. 
     
     
         18 - 21 . (canceled) 
     
     
         22 . The biomass saccharification mixture of  claim 17 , wherein the whole cellulase composition is derived from a host cell expressing one or more or all of (1) a polynucleotide encoding a peptide having beta-xylosidase activity; (2) a polynucleotide encoding a polypeptide having xylanase activity; and (3) a polynucleotide peptide having L-alpha-arabinofuranosidase activity; (4) a polynucleotide encoding a polypeptide having cellobiohydrolase activity; and (5) a polynucleotide encoding a polypeptide having beta-glucosidase activity, optionally wherein the polynucleotide of one or more or all of (1) to (5) is heterologous to the host cell. 
     
     
         23 - 24 . (canceled) 
     
     
         25 . The biomass saccharification mixture of  claim 22 , wherein one or more or all of: (1) the gene encoding the polypeptide having GH61/endoglucanase activity; (2) the gene encoding the polypeptide having cellobiohydrolase activity; (3) the gene encoding the polypeptide having beta-glucosidase activity; (4) the gene encoding the polypeptide having beta-xylosidase activity; (5) the gene encoding the polypeptide having xylanase activity; and (6) the gene encoding the polypeptide having L-alpha-arabinofuranosidase activity are integrated into the genetic material of the host cell. 
     
     
         26 . The biomass saccharification mixture of  claim 17 , wherein the host cell is a bacterial host cell, yeast host cell, or a fungal host cell, optionally wherein the host cell is a filamentous fungal host cell, and optionally wherein the filamentous fungal host cell is one selected from a cell of  Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Trichoderma reesei, Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, 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, Bierkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis aneirina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus, Coriolus hirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Mvceliophthora thermophile, Neurospora crassa, Neurospora intermedia, Penicillium purpurogenum, Penicillium canescens, Penicillium solitum, Penicillium funiculosum Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum , or  Trichoderma viride.    
     
     
         27 - 28 . (canceled) 
     
     
         29 . The biomass saccharification mixture of  claim 1 , wherein the saccharification mixture is prepared by first blending the enzyme composition comprising the polypeptide having GH61/endoglucanase activity, followed by mixing the enzyme composition with the biomass. 
     
     
         30 - 31 . (canceled) 
     
     
         32 . The biomass saccharification mixture of  claim 1 , wherein the biomass material is selected from seeds, grains, tubers, plant waste, byproducts of food processing or industrial processing, corn cobs, corn stover, grasses,  Sorghastrum nutans , switchgrass, perennial canes, wood, wood chips, wood processing waste, sawdust, paper, paper waste, pulp, and recycled paper, potatoes, soybean, barley, rye, oats, wheat, beets, sugar cane bagasse and straw. 
     
     
         33 . The biomass saccharification mixture of  claim 1 , wherein the biomass material is subjected to pretreatment with an acid or a base, optionally wherein the pretreated biomass is adjusted to pH of about 4.0 to 6.5 before mixing with the enzyme composition. 
     
     
         34 . 
     
     
         35 . The biomass saccharification mixture of  claim 1 , wherein the biomass material is present in the mixture in an amount of about 5 wt. % to about 60 wt. %, about 10 wt. % to about 50 wt. %, about 15 wt. % to about 40 wt. %, about 15 wt. % to about 30 wt. %, or about 20 wt. % to about 30 wt. %, referring to the amount of biomass material in its solid state relative to the total weight of the mixture. 
     
     
         36 . A method of hydrolyzing a biomass material comprising incubating the biomass saccharification mixture of  claim 1 , under conditions suitable for hydrolyzing the biomass materials in the biomass saccharification mixture and for a sufficient period of time. 
     
     
         37 . The method of  claim 36 , wherein the conditions suitable for hydrolyzing the biomass materials in the biomass saccharification mixture comprises: (1) a pH of about 3.5 to about 7.0; (2) for a duration of about 2 hours or longer; and/or (3) a temperature of about 20° C. to about 75° C. 
     
     
         38 . (canceled) 
     
     
         39 . The method of  claim 36 , wherein at any given time above 2 hours, the amount of fermentable sugars is produced by the biomass saccharification mixture is increased by at least about 5% or at least about 10% as compared to the amount of fermentable sugars produced by a control biomass saccharification mixture comprising the same amount and type of biomass material, and the same composition of enzyme components but in the absence of the GH61/endoglucanase. 
     
     
         40 . (canceled) 
     
     
         41 . The method of  claim 36 , wherein the biomass material is present in an amount of about 10 wt. % to about 50 wt. % in its solid state. 
     
     
         42 . The method of  claim 41 , wherein the viscosity of the biomass saccharification mixture is reduced by at least about 5%, about 10%, about 15%, about 20%, about 25%, or more, as compared to the viscosity of the control biomass saccharification mixture comprising the same amount and type of biomass material, and the same composition of enzyme components but in the absence of the GH61/endoglucanase. 
     
     
         43 . A method of using the composition of  claim 1  to convert a biomass material into fermentable sugars in a merchant enzyme supply model or an on-site bio-refinery model.