Aglycosylated enzyme and uses thereof

Abstract

The present invention relates to compositions comprising an aglycosylated polypeptide having cellobiase activity, and methods for producing and using the same.

What is claimed is: 
     
         1 . An aglycosylated polypeptide having cellobiase activity comprising at least 90% identity to SEQ ID NO: 1, or a functional fragment thereof. 
     
     
         2 . The aglycosylated polypeptide of  claim 1 , wherein the polypeptide comprises a Cel3A enzyme from wild-type  T. reesei,  or a functional variant or fragment thereof. 
     
     
         3 . The aglycosylated polypeptide of  claim 2 , wherein the Cel3A enzyme comprises (e.g., consists of) the amino acid sequence SEQ ID NO: 1. 
     
     
         4 . The aglycosylated polypeptide of any one of  claims 1 - 3 , wherein the polypeptide is encoded by a nucleic acid sequence comprising (e.g., consisting of) SEQ ID NO: 2. 
     
     
         5 . The aglycosylated polypeptide of any one of  claims 1 - 4 , wherein the polypeptide comprises a mutation proximal to or at one or more glycosylation sites, wherein the mutation prevents glycosylation at the one or more glycosylation sites. 
     
     
         6 . The aglycosylated polypeptide of  claim 5 , wherein the mutation is at one or more of the threonine at amino acid position 78, the threonine at amino acid position 241, the serine at amino acid position 343, the serine at amino acid position 450, the threonine at amino acid position 599, the serine at amino acid position 616, the threonine at amino acid position 691, the serine at amino acid position 21, the threonine at amino acid position 24, the serine at amino acid position 25, the serine at amino acid position 28, the threonine at amino acid position 38, the threonine at amino acid position 42, the threonine at amino acid position 303, the serine at amino acid position at 398, the at serine amino acid position 435, the serine at amino acid position 436, the threonine at amino acid position 439, threonine at amino acid position 442, the threonine at amino acid position 446, the serine at amino acid position 451, the serine at amino acid position 619, the serine at amino acid position 622, the threonine at amino acid position 623, the serine at amino acid position 626, or the threonine at amino acid position 630 of SEQ ID NO: 1. 
     
     
         7 . The aglycosylated polypeptide of any one of the preceding claims, wherein the aglycosylated polypeptide has increased cellobiase activity as compared to glycosylated Cel3A enzyme from wild-type  T. reesei.    
     
     
         8 . The aglycosylated polypeptide of any one of the preceding claims, wherein the aglycosylated polypeptide has increased substrate recognition, a more active substrate recognition site, or reduced steric hindrance as compared to glycosylated Cel3A enzyme from wild-type  T. reesei.    
     
     
         9 . The aglycosylated polypeptide of any one of the preceding claims, wherein the aglycosylated polypeptide hydrolyzes a carbohydrate such as cellobiose into one or more monosaccharides, e.g., glucose. 
     
     
         10 . The aglycosylated polypeptide of any one of the preceding claims, wherein the cellobiase activity comprises hydrolysis of a beta 1,4 glycosidic linkage of cellobiose. 
     
     
         11 . A nucleic acid sequence encoding the polypeptide of  claims 1 - 10 . 
     
     
         12 . A nucleic acid sequence encoding a Cel3A enzyme or functional variant thereof, wherein the nucleic acid sequence comprises (e.g., consists of) at least 90% identity to SEQ ID NO: 2 or SEQ ID NO: 3. 
     
     
         13 . A nucleic acid molecule comprising a nucleic acid sequence of any of  claims 11  and  12 . 
     
     
         14 . The nucleic acid molecule of  claim 13 , further comprising a promoter, e.g., a promoter for prokaryotic cell expression, e.g., bacterial cell expression, e.g., expression in  E. coli.    
     
     
         15 . The nucleic acid molecule of  claim 14 , wherein the promoter sequence is a constitutive promoter sequence, inducible promoter sequence, or a repressible promoter sequence. 
     
     
         16 . The nucleic acid molecule of  claim 14  or  15 , wherein the promoter is a T7 promoter. 
     
     
         17 . The nucleic acid molecule of any one of  claims 13 - 16 , further comprising a nucleic acid sequence encoding a tag, e.g., a tag for detection and/or purification and/or for linkage to another molecule, e.g., a His tag. 
     
     
         18 . The nucleic acid molecule of any one of  claims 13 - 17 , further comprising a nucleic acid encoding one or more signal sequences, e.g., a secretion signal sequence. 
     
     
         19 . An expression vector comprising the nucleic acid sequence of any of  claims 11 - 18 . 
     
     
         20 . The expression vector of  claim 18 , further comprising a nucleic acid sequence encoding a selection marker, e.g., a kanamycin or an ampicillin marker. 
     
     
         21 . A cell comprising the vector of any one of  claims 13 - 20 . 
     
     
         22 . A prokaryotic cell or bacterial cell comprising the vector of any one of  claims 13 - 20 . 
     
     
         23 . A cell expressing a polypeptide of any one of  claims 1 - 10 . 
     
     
         24 . A prokaryotic cell or bacterial cell expressing a polypeptide of any one of  claims 1 - 10 . 
     
     
         25 . The bacterial cell of  claim 22  or  24  wherein the bacterial cell is impaired for glycosylation. 
     
     
         26 . The bacterial cell of  claim 25 , wherein the bacterial cell is an  E. coli  cell. 
     
     
         27 . The bacterial cell of  claim 26 , wherein the  E. coli  cell is an origami  E. coli  cell. 
     
     
         28 . A method for producing the aglycosylated polypeptide of any one of  claim 1 ,  2 ,  3 ,  4 ,  7 ,  8 ,  9  or  10 , comprising culturing a cell expressing a polypeptide of any one of  claim 1 ,  2 ,  3 ,  4 ,  7 ,  8 ,  9  or  10 , under conditions suitable for the expression of the polypeptide, wherein the cell does not glycosylate the polypeptide, e.g., a bacterial cell, e.g., an  E. coli  cell, e.g., an origami  E. coli  cell. 
     
     
         29 . A method for producing the aglycosylated polypeptide of any of  claim 1 ,  2 ,  3 ,  4 ,  7 ,  8 ,  9  or  10 , comprising treating a polypeptide comprising an amino acid with at least 90% identity to SEQ ID NO: 1 with a deglycosylating enzyme. 
     
     
         30 . The method of  claim 29 , wherein the deglycosylating enzyme is selected from PGNase and EndoH. 
     
     
         31 . A method for producing the aglycosylated polypeptide of any one of  claim 1 ,  2 ,  3 ,  4 ,  7 ,  8 ,  9  or  10 , comprising culturing a cell that comprises a nucleic acid sequence encoding a polypeptide of any one of  claim 1 ,  2 ,  3 ,  4 ,  7 ,  8 ,  9  or  10 , wherein the nucleic acid sequence has one or more mutations which prevent glycosylation of the encoded polypeptide. 
     
     
         32 . A method for culturing a cell expressing the aglycosylated polypeptide of any one of  claim 1 ,  2 ,  3 ,  4 ,  7 ,  8 ,  9  or  10  in the presence of a glycosylation inhibitor, e.g., tunicamycin. 
     
     
         33 . An enzyme mixture comprising a glycosylated polypeptide comprising an amino acid sequence with at least 90% identity to SEQ ID NO: 1 and an aglycosylated polypeptide comprising an amino acid sequence with at least 90% identity to SEQ ID NO: 1, wherein both of the glycosylated polypeptide and the aglycosylated peptide have cellobiase activity. 
     
     
         34 . The enzyme mixture of  claim 33 , wherein the aglycosylated polypeptide is the aglycosylated polypeptide of any one of  claims 1 - 10 . 
     
     
         35 . The enzyme mixture of  claim 33  or  34 , wherein the glycosylated polypeptide and the aglycosylated polypeptide both comprise Cel3A enzyme from wild-type  T. reesei.    
     
     
         36 . The enzyme mixture of any one of  claims 33 - 35 , further comprising at least one additional enzyme derived from a microorganism, wherein the additional enzyme has a biomass-degrading activity of a cellulose based material. 
     
     
         37 . The enzyme mixture of  claim 36 , wherein the additional enzyme is selected from a ligninase, an endoglucanase, a cellobiohydrolase, xylanase, and a cellobiase. 
     
     
         38 . The enzyme mixture of  claim 33  or  34 , wherein the mixture further comprises one or more ligninase, one or more endoglucanase, one or more cellobiohydrolase, one or more xylanase. 
     
     
         39 . The enzyme mixture of any one of  claims 33 - 38 , wherein the ratio between the aglycosylated polypeptide to the remaining enzymes in the mixture is at least 1:32, e.g., 1:32 to 1:300. 
     
     
         40 . The enzyme mixture of any of  claims 33 - 38 , wherein the ratio of the aglycosylated polypeptide to a glycosylated polypeptide is at least 1:32, e.g., 1:32 to 1:300. 
     
     
         41 . A method of producing a product (e.g., a hydrogen, sugar, alcohol, etc.) from a biomass (or converting a biomass to a product) comprising contacting a biomass, e.g., by treatment with an electron beam, with an aglycosylated polypeptide of any one of  claims 1 - 10  and a microorganism (mixture) that produces one or more biomass-degrading enzyme or an enzyme mixture comprising biomass-degrading enzymes under conditions suitable for the production of the sugar product. 
     
     
         42 . A method of producing a product (e.g., a hydrogen, a sugar, an alcohol) from a biomass comprising contacting a biomass with an enzyme mixture of any one of  claims 33 - 40  under conditions suitable for the production of the product. 
     
     
         43 . The method of  claim 41  or  42 , wherein said product is a sugar product. 
     
     
         44 . The method of any one of  claims 41 - 43 , further comprising isolating the sugar product. 
     
     
         45 . The method of  claim 44 , wherein the isolating of the sugar product comprises precipitation, crystallization, chromatography, centrifugation, and/or extraction. 
     
     
         46 . The method of any one of  claims 43 - 45 , wherein the sugar product is glucose and/or xylose. 
     
     
         47 . The method of any one of  claims 41 - 46 , wherein the enzyme mixture comprises at least two of the enzymes selected from the group consisting of B2AF03, CIP1, CIP2, Cel1a, Cel3a, Cel5a, Cel6a, Cel7a, Cel7b, Cel12a, Cel45a, Cel74a, paMan5a, paMan26a, Swollenin, and the enzymes listed in Table 1. 
     
     
         48 . The method of any one of  claims 41 - 47 , wherein the biomass comprises one or more of an agricultural product or waste, a paper product or waste, a forestry product, or a general waste, or any combination thereof; wherein:
 a) an agricultural product or waste comprises sugar cane jute, hemp, flax, bamboo, sisal, alfalfa, hay, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, canola straw, wheat straw, barley straw, oat straw, rice straw, corn cobs, corn stover, corn fiber, coconut hair, beet pulp, bagasse, soybean stover, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, or beeswing, or a combination thereof;   b) a paper product or waste comprises paper, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, cardstock, cardboard, paperboard, or paper pulp, or a combination thereof;   c) a forestry product comprises aspen wood, particle board, wood chips, or sawdust, or a combination thereof; and   d) a general waste comprises manure, sewage, or offal, or a combination thereof.   
     
     
         49 . The method of any one of  claims 41 - 48 , further comprising a step of treating the biomass prior to introducing the microorganism or the enzyme mixture to reduce the recalcitrance of the biomass, wherein the treating comprises bombardment with electrons, sonication, oxidation, pyrolysis, steam explosion, chemical treatment, mechanical treatment, or freeze grinding. 
     
     
         50 . The method of any one of  claims 41 - 49  wherein the microorganism that produces a biomass-degrading enzyme is from species in the genera selected from  Bacillus, Coprinus, Myceliophthora, Cephalosporium, Scytalidium, Penicillium, Aspergillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, Chrysosporium  or  Trichoderma.    
     
     
         51 . The method of any one of  claims 41 - 50 , wherein the microorganism that produces a biomass-degrading enzyme is selected from  Aspergillus, Humicola insolens  ( Scytalidium thermophilum )  Coprinus cinereus, Fusarium oxysporum, Myceliophthora thermophila, Meripilus giganteus, Thielavia terrestris, Acremonium persicinum, Acremonium acremonium, Acremonium brachypenium, Acremonium dichromosporum, Acremonium obclavatum, Acremonium pinkertoniae, Acremonium roseogriseum, Acremonium incoloratum, Acremonium furatum, Chrysosporium lucknowense, Trichoderma viride, Trichoderma reesei,  or  Trichoderma koningii.    
     
     
         52 . The method of any one of  claims 41 - 51 , wherein the microorganism has been induced to produce a biomass-degrading enzyme by combining the microorganism with an induction biomass sample under conditions suitable for increasing production of a biomass-degrading enzyme compared to an uninduced microorganism. 
     
     
         53 . The method of any of  claim 52 , wherein said induction biomass sample comprises paper, paper products, paper waste, paper pulp, pigmented papers, loaded papers, coated papers, filled papers, magazines, printed matter, printer paper, polycoated paper, card stock, cardboard, paperboard, cotton, wood, particle board, forestry wastes, sawdust, aspen wood, wood chips, grasses, switchgrass, miscanthus, cord grass, reed canary grass, grain residues, rice hulls, oat hulls, wheat chaff, barley hulls, agricultural waste, silage, canola straw, wheat straw, barley straw, oat straw, rice straw, jute, hemp, flax, bamboo, sisal, abaca, corn cobs, corn stover, soybean stover, corn fiber, alfalfa, hay, coconut hair, sugar processing residues, bagasse, beet pulp, agave bagasse, algae, seaweed, manure, sewage, offal, agricultural or industrial waste, arracacha, buckwheat, banana, barley, cassava, kudzu, oca, sago, sorghum, potato, sweet potato, taro, yams, beans, favas, lentils, peas, or any combination thereof.