Polypeptides having xylanase activity and polynucleotides encoding the same

Abstract

The present invention relates to isolated polypeptides having xylanase activity, catalytic domains, and carbohydrate binding modules, as well as polynucleotides encoding these polypeptides, catalytic domains, or carbohydrate binding modules. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising these polynucleotides, as well as methods for producing and using these polypeptides, catalytic domains, or carbohydrate binding modules.

Description

[0001] Reference to a sequence listing

[0002] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. Background of the Invention Field of the Invention

[0004] The present invention relates to polypeptides having xylanase activity, a catalytic domain, and a carbohydrate binding module, and polynucleotides encoding these polypeptides, catalytic domains, and carbohydrate binding modules. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising these polynucleotides, and methods of producing and using these polypeptides, catalytic domains, and carbohydrate binding modules.

[0005] Related technical notes

[0006] Lignocellulose, the world's largest renewable biomass resource, is primarily composed of lignin, cellulose, and hemicellulose, the latter of which is mostly xylan. Xylanases (e.g., endo-1,4-β-xylanases, EC 3.2.1.8) hydrolyze the internal β-1,4-xylosidic linkages in xylan to produce smaller molecular weight wood sugars and wood-oligomers. Xylan is a polysaccharide formed by the D-xylopyranose linked by 1,4-β-glycosides.

[0007] Cellulose is a polymer of monoglucose covalently linked by β-1,4-links. Many microorganisms produce enzymes that hydrolyze β-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and β-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it up for attack by cellobiohydrolases. Cellobiohydrolases sequentially release cellobiose molecules from the ends of the cellulose polymer. Cellobiose is a water-soluble β-1,4-linked dimer of glucose. β-glucosidases hydrolyze cellobiose into glucose. Once cellulose is converted to glucose, the glucose can be readily fermented into ethanol by yeast.

[0008] The lignocellulose raw material conversion into ethanol has the following advantages, promptly is easy to obtain a large amount of raw materials, avoids the desirability of burning or landfilling materials and the cleanliness of ethanol fuel.Now think that timber, agricultural waste, herbaceous crops, and municipal solid waste are the raw materials for producing ethanol.These materials are mainly composed of cellulose, hemicellulose and lignin.Once cellulose is converted into glucose, just easily glucose is fermented into ethanol by yeast.

[0009] Yoshioka et al., 1981, Agric. Biol. Chem. 45(3):579-586 disclosed the production and characterization of a thermostable xylanase from Talaromyces fusilis YH-50. Yoshioka et al., 1981, Agric. Biol. Chem. 45(11):2425-2432 disclosed the purification and characterization of a thermostable xylanase from Talaromyces fusilis YH-50. Hayashida et al., 1988, Methods In Enzymology 160:675-678 disclosed a Talaromyces fusilis xylanase. WO 02 / 24926 discloses a Talaromyces emersonii GH10 xylanase (GENESEQP: AAU99346).

[0010] There is a need in the art to improve cellulolytic enzyme compositions by supplementing them with additional enzymes to increase the effectiveness and provide cost-effective enzyme solutions for the degradation of lignocellulose.

[0011] The present invention provides polypeptides having xylanase activity and polynucleotides encoding these polypeptides. SUMMARY OF THE INVENTION

[0013] The present invention relates to isolated polypeptides having xylanase activity selected from the group consisting of:

[0014] (a) a polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2 or to the mature polypeptide of SEQ ID NO: 6;

[0015] (b) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or its cDNA sequence, or to the mature polypeptide coding sequence of SEQ ID NO: 5 or its cDNA sequence;

[0016] (c) a variant of the mature polypeptide of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 6, the variant comprising a substitution, deletion, and / or insertion at one or more (e.g., several) positions; and

[0017] (d) A fragment of the polypeptide of (a), (b), or (c), wherein the fragment has xylanase activity.

[0018] The present invention also relates to an isolated polypeptide comprising a catalytic domain selected from the group consisting of:

[0019] (a) a catalytic domain having at least 90% sequence identity to amino acids 24 to 340 of SEQ ID NO: 2 or to amino acids 24 to 341 of SEQ ID NO: 6;

[0020] (b) a catalytic domain encoded by a polynucleotide having at least 90% sequence identity to nucleotides 157 to 1339 of SEQ ID NO: 1, or its cDNA sequence, or at least 90% sequence identity to nucleotides 151 to 1387 of SEQ ID NO: 5, or its cDNA sequence;

[0021] (c) a variant of amino acids 24 to 340 of SEQ ID NO: 2 or amino acids 24 to 341 of SEQ ID NO: 6, the variant comprising a substitution, deletion, and / or insertion at one or more (e.g., several) positions; and

[0022] (d) A fragment of the catalytic domain of (a), (b), or (c), which fragment has xylanase activity.

[0023] The present invention also relates to an isolated polypeptide comprising a carbohydrate binding module selected from the group consisting of:

[0024] (a) a carbohydrate binding moiety having at least 90% sequence identity to amino acids 373 to 406 of SEQ ID NO: 2 or at least 90% sequence identity to amino acids 371 to 406 of SEQ ID NO: 6;

[0025] (b) a carbohydrate binding module encoded by a polynucleotide having at least 90% sequence identity to nucleotides 1436 to 1537 of SEQ ID NO: 1, or its cDNA sequence, or encoded by a polynucleotide having at least 90% sequence identity to nucleotides 1480 to 1587 of SEQ ID NO: 5;

[0026] (c) a variant of amino acids 373 to 406 of SEQ ID NO: 2 or amino acids 371 to 406 of SEQ ID NO: 6, the variant comprising a substitution, deletion, and / or insertion at one or more (e.g., several) positions; and

[0027] (d) A fragment of the carbohydrate binding module of (a), (b), or (c), wherein the fragment has binding activity.

[0028] The present invention also relates to isolated polynucleotides encoding the polypeptides of the present invention; to nucleic acid constructs, recombinant expression vectors, and recombinant host cells comprising these polynucleotides; and to methods of producing these polypeptides.

[0029] The present invention also relates to methods for degrading cellulosic or xylan-containing materials, comprising treating the cellulosic or xylan-containing material with an enzyme composition in the presence of a polypeptide having xylanase activity of the present invention. In one aspect, the methods further comprise recovering the degraded cellulosic or xylan-containing material.

[0030] The present invention also relates to methods for producing a fermentation product, comprising: (a) saccharifying a cellulosic or xylan-containing material with an enzyme composition in the presence of a polypeptide having xylanase activity of the present invention; (b) fermenting the saccharified cellulosic or xylan-containing material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[0031] The present invention also relates to methods for fermenting a cellulosic or xylan-containing material, comprising: fermenting the cellulosic or xylan-containing material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic or xylan-containing material is saccharified with an enzyme composition in the presence of a polypeptide having xylanase activity of the present invention. In one aspect, fermenting the cellulosic or xylan-containing material produces a fermentation product. In another aspect, the methods further comprise recovering the fermentation product from the fermentation.

[0032] The present invention also relates to isolated polynucleotides encoding a signal peptide comprising or consisting of amino acids 1 to 23 of SEQ ID NO: 2, or comprising or consisting of amino acids 1 to 23 of SEQ ID NO: 6, operably linked to a gene encoding a protein, wherein the protein is foreign to the signal peptide; nucleic acid constructs, expression vectors, and recombinant host cells comprising these polynucleotides; and methods of producing the proteins.

[0033] BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Shown are the results of hydrolysis of washed ground sieved alkaline pretreated corncob (GS-APCC) as substrate by Rasamsonia byssochlamydoides GH10 xylanase (P24GTR) supplemented with Talaromyces emersonii GH3 β-xylosidase at pH 4.0 from 50°C to 65°C.

[0035] definition

[0036] Acetylxylan esterase: The term "acetylxylan esterase" means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl autopolymeric xylan, acetylated xylose, acetylated glucose, α-naphthyl acetate, and p-nitrophenyl acetate. Acetylxylan esterase activity is preferably determined using 0.5 mM p-nitrophenyl acetate as a substrate in 50 mM sodium acetate (pH 5.0) containing 0.01% TWEEN™ 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°C.

[0037] 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 from mutations and can lead to polymorphism within a population. Gene mutations can be silent (no change in the encoded polypeptide) or can encode polypeptides with altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

[0038] α-L-arabinofuranosidase: The term "α-L-arabinofuranosidase" means an α-L-arabinofuranoside arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis of terminal non-reducing α-L-arabinofuranoside residues in α-L-arabinosides. The enzyme acts on α-L-arabinofuranosides, α-L-arabinans containing (1,3)- and / or (1,5)-linkages, arabinoxylans, and arabinogalactans. α-L-arabinofuranosidase is also known as arabinosidase, α-arabinosidase, α-L-arabinosidase, α-arabinofuranosidase, polysaccharide α-L-arabinofuranosidase, α-L-arabinofuranoside hydrolase, L-arabinosidase, or α-L-arabinanase. Preferably, 5 mg of medium viscosity wheat arabinoxylan (Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland) in 100 mM sodium acetate (pH 5) per ml is used in a total volume of 200 μl at 40°C for 30 minutes, followed by α-L-arabinofuranosidase activity was determined by arabinose analysis using HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0039] α-Glucuronidase: The term "α-glucuronidase" refers to an α-D-glucuronide glucuronidase (EC 3.2.1.139) that catalyzes the hydrolysis of α-D-glucuronide to D-glucuronic acid esters and alcohols. α-Glucuronidase activity is preferably determined according to de Vries, 1998, J. Bacteriol. 180:243-249. One unit of α-glucuronidase is equal to the amount of enzyme capable of releasing 1 micromole of glucuronic acid or 4-O-methylglucuronic acid per minute at pH 5 and 40°C.

[0040] β-Glucosidase: The term "β-glucosidase" means a β-D-glucoside glucohydrolase (EC 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing β-D-glucose residues and releases β-D-glucose. β-glucosidase activity is preferably determined according to the procedure of Venturi et al., 2002, J. Basic Microbiol. 42:55-66 using p-nitrophenyl-β-D-pyranoside as a substrate. One unit of β-glucosidase is defined as the activity of β-glucosidase in a solution containing 0.01% β-glucosidase at 25°C, pH 4.8. 20% of 50 mM sodium citrate produces 1.0 micromole of p-nitrophenolate anion per minute from 1 mM p-nitrophenyl-β-D-pyranoglucopyranoside as a substrate.

[0041] β-Xylosidase: The term "β-xylosidase" means a β-D-xyloside xylose hydrolase (EC 3.2.1.37) that catalyzes the exohydrolysis of short β(1→4)-xylooligosaccharides to remove consecutive D-xylose residues from the non-reducing ends. β-xylosidase activity was determined in 100 mM sodium citrate at pH 5 and 40°C using 1 mM p-nitrophenyl-β-D-xyloside as a substrate. One unit of β-xylosidase was defined as the activity of 1 mM p-nitrophenyl-β-D-xyloside at 40°C and pH 5 in a solution containing 0.01% 20% of 100 mM sodium citrate produces 1.0 micromole of p-nitrophenolate anion per minute from 1 mM p-nitrophenyl-β-D-xyloside.

[0042] cDNA: The term "cDNA" refers to 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 first, primary RNA transcript is a precursor to mRNA that undergoes a series of processing steps, including splicing, before appearing as mature, spliced mRNA.

[0043] Carbohydrate binding module: The term "carbohydrate binding module" means a region within a carbohydrate-active enzyme that provides carbohydrate binding activity (Boraston et al., 2004, Biochem. J. 383:769-781). Most known carbohydrate binding modules (CBMs) are contiguous amino acid sequences with a discrete fold. Carbohydrate binding modules (CBMs) are typically found at the N-terminus or at the end of the C-terminus of an enzyme. Some CBMs are known to have specificity for cellulose.

[0044] Catalytic domain: The term "catalytic domain" means the region of an enzyme that contains the catalytic machinery of the enzyme.

[0045] Cellobiohydrolase: The term "cellobiohydrolase" means a 1,4-β-D-glucan cellobiohydrolase (EC 3.2.1.91 and EC 3.2.1.176) that catalyzes the hydrolysis of 1,4-β-D-glycosidic linkages in cellulose, cellooligosaccharides, or any polymer containing β-1,4-linked glucose, thereby releasing cellobiose from the reducing end (cellobiohydrolase I) or the non-reducing end (cellobiohydrolase II) of the chain (Teeri, 1997, Trends in Biotechnology 15:160-167; Teeri et al., 1998, Biochem. Soc. Trans. 26:173-178). Preferably, cellobiohydrolase activity is determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47:273-279; 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.

[0046] Cellulolytic enzyme or cellulase: The term "cellulolytic enzyme" or "cellulase" means one or more (e.g., several) enzymes that hydrolyze cellulosic material. Such enzymes include one or more endoglucanases, one or more cellobiohydrolases, one or more β-glucosidases, or a combination thereof. Two basic methods for measuring cellulolytic enzyme activity include: (1) determining total cellulolytic activity, and (2) determining individual cellulolytic activities (endoglucanase, cellobiohydrolase, and β-glucosidase), as reviewed in Zhang et al., 2006, Biotechnology Advances 24:452-481. Total cellulolytic activity is typically measured using insoluble substrates including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, and the like. The most commonly used total cellulolytic activity assay is the filter paper assay using Whatman No. 1 filter paper as the substrate. This assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appl. Chem. 59: 257-68).

[0047] For the purposes of the present invention, preferably, cellulolytic enzyme activity is determined by measuring the increase in hydrolysis of a cellulosic material by one or more cellulolytic enzymes compared to a control hydrolysis to which no cellulolytic enzyme protein is added under the following conditions: 1-50 mg of cellulolytic enzyme protein / g of cellulose in PCS (or other pretreated cellulosic material) at a suitable temperature (e.g., 40°C-80°C, e.g., 50°C, 55°C, 60°C, 65°C, or 70°C) and a suitable pH (e.g., 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5) for 3-7 days. Typical conditions are: 1 ml reaction, washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate (pH 5), 1 mM MnSO4, 50°C, 55°C, or 60°C, 72 hours, by Sugar analysis was performed using an HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

[0048] Cellulosic material: The term "cellulosic material" means any material containing cellulose. The major polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant being hemicellulose, and the third most abundant being pectin. The secondary cell wall, produced after the cells have ceased growing, also contains polysaccharides and is reinforced by polymeric lignin covalently cross-linked with the hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and is therefore a linear β-(1-4)-D-glucan, while hemicellulose includes a variety of compounds such as xylans, xyloglucans, arabinoxylans, and mannans that have a series of substituents present in a complex branched structure. Although cellulose is generally polymorphic, it is found in plant tissues primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicellulose is typically hydrogen bonded to cellulose and other hemicelluloses, which helps to stabilize the cell wall matrix.

[0049] Cellulose is commonly found, for example, in the stems, leaves, husks, bark, and cobs of plants, or the leaves, branches, and wood of trees. The cellulosic material can be, but is not limited to, agricultural waste, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill waste, waste paper, and wood (including forestry waste) (see, e.g., Wiselogel et al., 1995, Handbook on Bioethanol (Charles E. Wyman, ed.), 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 Advances in the Bioconversion of Lignocellulose," Advances in Biochemical Engineering / Biotechnology. Engineering / Biotechnology, T. Scheper, ed., Vol. 65, pp. 23-40, Springer-Verlag, New York. It should be understood that cellulose can be in the form of lignocellulose, a plant cell wall material comprising 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, hemicellulose, and lignin.

[0050] In one aspect, the cellulosic material is agricultural waste, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill waste, waste paper, or wood (including forestry waste).

[0051] In another aspect, the cellulosic material is Arundo donax, bagasse, bamboo, corn cob, corn fiber, corn stover, Miscanthus, rice straw, switchgrass, or wheat straw.

[0052] In another aspect, the cellulosic material is aspen, eucalyptus, fir, pine, poplar, spruce, or willow.

[0053] In another aspect, the cellulosic material is algal cellulose, bacterial cellulose, cotton linters, filter paper, microcrystalline cellulose (e.g., ), or phosphoric acid-treated cellulose.

[0054] In another aspect, the cellulosic material is an aquatic biomass. As used herein, the term "aquatic biomass" refers to biomass produced by photosynthesis in an aquatic environment. The aquatic biomass can be algae, emergent plants, floating-leaved plants, or submerged plants.

[0055] The cellulosic material can be used as is or can be pretreated using conventional methods known in the art, such as described herein. In a preferred aspect, the cellulosic material is pretreated.

[0056] Coding sequence: The term "coding sequence" means a polynucleotide that 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 (e.g., ATG, GTG, or TTG) and ends with a stop codon (e.g., TAA, TAG, or TGA). The coding sequence can be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0057] Control sequences: The term "control sequences" means nucleic acid sequences necessary for the 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 exogenous (i.e., from a different gene) to the polynucleotide encoding the polypeptide, or native or exogenous to each other. Such control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, and transcription terminators. At a minimum, control sequences include promoters, and transcription and translation termination signals. For the purpose of introducing specific restriction enzyme sites that facilitate ligation of these control sequences to the coding region of a polynucleotide encoding a polypeptide, these control sequences may be provided with multiple linkers.

[0058] Endoglucanase: The term "endoglucanase" refers to an endo-1,4-(1,3;1,4)-β-D-glucan 4-glucanohydrolase (EC 3.2.1.4) that catalyzes the endohydrolysis of 1,4-β-D-glucosidic bonds in cellulose, cellulose derivatives (e.g., carboxymethylcellulose and hydroxyethylcellulose), lichenin, β-1,4 bonds in mixed β-1,3 glucans (e.g., cereal β-D-glucans or xyloglucans), and other plant materials containing cellulosic components. Endoglucanase activity can be determined by measuring a decrease in substrate viscosity or an increase in reducing ends as determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24:452-481). Preferably, endoglucanase activity is determined according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59: 257-268, at pH 5, 40°C, using carboxymethylcellulose (CMC) as substrate.

[0059] Expression: The term "expression" includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0060] Expression vector: The term "expression vector" means a linear or circular DNA molecule that includes a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

[0061] Family 10 glycoside hydrolase: The term "Family 10 glycoside hydrolase" or "Family GH10" or "GH10" means a polypeptide belonging to glycoside hydrolase family 10 according to Henrissat, 1991, A classification of glycosyl hydrolases based on amino-acid sequences similarities, Biochem. J. 280:309-316; and Henrissat and Bairoch, 1996, Updating the sequence-based classification of glycosyl hydrolases, Biochem. J. 316:695-696.

[0062] Family 61 glycoside hydrolase: The term "Family 61 glycoside hydrolase" or "Family GH61" or "GH61" means a polypeptide belonging to glycoside hydrolase family 61 according to Henrissat, 1991, Biochem. J. 280:309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316:695-696. The enzymes in this family were originally classified as a glycoside hydrolase family based on very weak endo-1,4-β-D glucanase activity measured in one family member. Recently, GH61 has been classified as a lytic polysaccharide monooxygenase (Quinlan et al., 2011, Proc. Natl. Acad. Sci. USA 208: 15079-15084; Phillips et al., 2011, ACS Chem. Biol. 6: 1399-1406; Lin et al., 2012, Structure 20: 1051-1061).

[0063] Feruloyl esterase: The term "feruloyl esterase" refers to a 4-hydroxy-3-methoxycinnamoyl-saccharide hydrolase (EC 3.1.1.73) that catalyzes the hydrolysis of 4-hydroxy-3-methoxycinnamoyl (feruloyl) groups from esterified sugars, typically arabinose in natural biomass substrates, to produce ferulic acid ester (4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as feruloyl esterase, hydroxycinnamoyl esterase, FAE-III, cinnamate hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. Preferably, feruloyl esterase activity is measured in 50 mM sodium acetate (pH 5.0) using 0.5 mM p-nitrophenyl ferulate as a substrate. One unit of ferulic acid esterase is equal to the amount of enzyme capable of releasing 1 μmol of p-nitrophenolate anion per minute at pH 5 and 25°C.

[0064] Fragment: The term "fragment" means a polypeptide having one or more (e.g., several) amino acids deleted from the amino and / or carboxyl terminus of a mature polypeptide body; wherein the fragment has xylanase activity. In one aspect, a fragment comprises at least 320 amino acid residues of SEQ ID NO: 2 or SEQ ID NO: 6, e.g., at least 340 amino acid residues or at least 360 amino acid residues.

[0065] Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that hydrolyze hemicellulosic materials. See, e.g., Shallom and Shoham, 2003, 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, acetylmannan esterases, acetylxylan esterases, arabinanases, arabinofuranosidases, coumaric acid esterases, ferulic acid esterases, galactosidases, glucuronidases, glucuronidases, mannanases, mannosidases, xylanases, and xylosidases. The substrate for these enzymes, hemicellulose, is a heterogeneous group of branched and linear polysaccharides that can bind to cellulose microfibrils in plant cell walls through hydrogen bonds, cross-linking into a strong network. Hemicellulose is also covalently attached to lignin, thereby forming a highly complex structure together with cellulose. The variable structure and organization of hemicellulose require the synergistic action of many enzymes to make it fully degraded. The catalytic module of hemicellulase is the glycoside hydrolase (GH) that hydrolyzes glycosidic bonds, or the carbohydrate esterase (CE) that hydrolyzes the ester bond of acetic acid or ferulic acid side groups. These catalytic modules can be assigned to GH and CE families based on the homology of their primary sequences. Some families with overall similar folding can be further grouped into clans (e.g., GH-A) with letter marks. The most informative and up-to-date classification of these and other carbohydrate-active enzymes can be obtained in the carbohydrate-active enzyme (Carbohydrate-Active Enzymes) (CAZy) database. The activity of the hemicellulolytic enzyme can be determined according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752 at a suitable temperature (e.g., 50°C, 55°C, or 60°C) and pH (e.g., 4.5, 5.0, or 5.5).

[0066] High stringency conditions: The term "high stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide. The support material is finally washed three times with 2X SSC, 0.2% SDS at 65°C for 15 minutes each.

[0067] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. 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.

[0068] Isolated: The term "isolated" means a substance that is in a form or setting not found 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 components with which it is essentially associated; (3) any substance that has been modified by the hand of man relative to the substance found in nature; or (4) any substance that has been modified by increasing the amount of the substance relative to other components with which it is naturally associated (e.g., recombinant production in a host cell; multiple copies of a gene encoding the substance; and use of a stronger promoter than the promoter naturally associated with the gene encoding the substance).

[0069] Low stringency conditions: The term "low stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 25% formamide. The support material is finally washed three times with 0.2X SSC, 0.2% SDS at 50°C for 15 minutes each.

[0070] Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, based on the prediction by the SignaIP program that amino acids 1 to 23 of SEQ ID NO: 2 are a signal peptide (Nielsen et al., 1997, Protein Engineering 10: 1-6), the mature polypeptide is amino acids 24 to 406 of SEQ ID NO: 2 (P24GTR). In another aspect, based on the prediction by the SignaIP program that amino acids 1 to 23 of SEQ ID NO: 6 are a signal peptide (Nielsen et al., 1997, Protein Engineering 10: 1-6), the mature polypeptide is amino acids 24 to 406 of SEQ ID NO: 6 (P34RRZ). It is known in the art that a host cell can produce a mixture of two or more different mature polypeptides (ie, having different C-terminal and / or N-terminal amino acids) expressed from the same polynucleotide.

[0071] Mature polypeptide coding sequence: The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide having xylanase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 157 to 1537 of SEQ ID NO: 1 (D82RVA), or a cDNA sequence thereof, based on the prediction by the SignalP program (Nielsen et al., 1997, supra) that nucleotides 1 to 156 of SEQ ID NO: 1 encode a signal peptide. In another aspect, the mature polypeptide coding sequence is nucleotides 151 to 1587 of SEQ ID NO: 5 (D24EPN), or a cDNA sequence thereof, based on the prediction by the SignalP program (Nielsen et al., 1997, supra) that nucleotides 1 to 150 of SEQ ID NO: 5 encode a signal peptide.

[0072] Moderate stringency conditions: The term "moderate stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 35% formamide. The support material is finally washed three times with 2X SSC, 0.2% SDS at 55°C for 15 minutes each.

[0073] Medium-high stringency conditions: The term "medium-high stringency conditions" means that for probes of at least 100 nucleotides in length, standard Southern blotting procedures are followed, with prehybridization and hybridization at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 35% formamide. The support material is finally washed three times with 2X SSC, 0.2% SDS at 60°C for 15 minutes each.

[0074] Nucleic acid construct: The term "nucleic acid construct" means a single-stranded or double-stranded nucleic acid molecule that is isolated from a naturally occurring gene, or modified in a manner not originally found in nature to contain a nucleic acid segment, or is synthesized, and includes one or more control sequences.

[0075] 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 the expression of the coding sequence.

[0076] Polypeptide having cellulolytic enhancing activity: The term "polypeptide having cellulolytic enhancing activity" means a GH61 polypeptide that promotes enhanced hydrolysis of a cellulosic material by an enzyme having cellulolytic activity. Preferably, cellulolytic enhancing activity is determined by measuring the increase in reducing sugars or the increase in the total amount of cellobiose and glucose in cellulosic material hydrolyzed by cellulolytic enzymes compared to a control hydrolysis of an equal total protein loading (1-50 mg of cellulolytic protein / g of cellulose in PCS) with no cellulolytic enhancing activity under the following conditions: 1-50 mg of total protein / g of cellulose in pretreated corn stover (PCS), wherein the total protein consists of 50-99.5% w / w cellulolytic enzyme protein and 0.5-50% w / w GH61 polypeptide protein, at a suitable temperature (such as 40°C-80°C, for example 50°C, 55°C, 60°C, 65°C or 70°C) and a suitable pH (such as 4-9, for example 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0 or 8.5) for 1-7 days.

[0077] In one aspect, GH61 polypeptide enhancing activity is determined using the following: using Aspergillus oryzae β-glucosidase (produced recombinantly in Aspergillus oryzae according to WO 02 / 095014) in the presence of a cellulase protein loading of 2%-3% by weight of total protein or Aspergillus fumigatus β-glucosidase (produced recombinantly in Aspergillus oryzae as described in WO 02 / 095014) A mixture of 1.5 L of HYDROZYME(R) (Novozymes A / S, Bagsvaerd, Denmark) served as the source of cellulolytic activity.

[0078] In another aspect, the GH61 polypeptide enhancing activity is determined according to WO 2013 / 028928 for high temperature compositions.

[0079] GH61 polypeptides having cellulolytic enhancing activity enhance the hydrolysis of a cellulosic material catalyzed by an enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to achieve the same degree of hydrolysis, preferably by 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.

[0080] Pretreated corn stover: The term "PCS" or "pretreated corn stover" means a cellulosic material derived from corn stover by heat and dilute sulfuric acid treatment, alkaline pretreatment, neutral pretreatment, or any pretreatment known in the art.

[0081] Sequence identity: The degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity".

[0082] For the 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 3.0.0, 5.0.0 or later). The parameters used are a gap opening penalty of 10, a 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 -unreduced option) is used as the percent identity and is calculated as follows:

[0083] (number of identical residues x 100) / (length of alignment - total number of gaps in the alignment)

[0084] 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) implemented in the Needleman program of the EMBOSS package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, supra) (preferred 5.0.0 version or updated version). The parameters used are gap opening penalty 10, gap extension penalty 0.5, and EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of the " longest consistency " of Needleman annotation (using-non-simplified option to obtain) is used as percentage identity, and is calculated as follows:

[0085] (Number of identical DNA nucleotides x 100) / (length of alignment - total number of gaps in the alignment).

[0086] Subsequence: The term "subsequence" means a polynucleotide having one or more (e.g., several) nucleotides deleted from the 5' and / or 3' end of a mature polypeptide coding sequence; wherein the subsequence encodes a fragment having xylanase activity. In one aspect, the subsequence comprises at least 960 nucleotides of SEQ ID NO: 1 or SEQ ID NO: 5, e.g., at least 1020 nucleotides or at least 1080 nucleotides.

[0087] Variant: The term "variant" means a polypeptide having xylanase activity that comprises an alteration (i.e., substitution, insertion, and / or deletion) at one or more (e.g., several) positions. A substitution means replacing the amino acid occupying a position with a different amino acid; a deletion means removing 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.

[0088] Very high stringency conditions: The term "very high stringency conditions" refers to prehybridization and hybridization for probes of at least 100 nucleotides in length, following standard Southern blotting procedures at 42°C for 12 to 24 hours in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 50% formamide. The support material is finally washed three times with 2X SSC, 0.2% SDS at 70°C for 15 minutes each.

[0089] Very low stringency conditions: The term "very low stringency conditions" refers to prehybridization and hybridization for probes of at least 100 nucleotides in length, following standard Southern blotting procedures at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms / ml sheared and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. The support material is finally washed three times with 0.2X SSC, 0.2% SDS at 45°C for 15 minutes each.

[0090] Xylan-containing material: The term "xylan-containing material" means any material comprising plant cell wall polysaccharides containing a backbone of β-(1-4)-linked xylose residues. The xylans of terrestrial plants are heteropolymers having a β-(1-4)-D-xylopyranose backbone branched by short carbohydrate chains. They include D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and / or different oligosaccharides composed of D-xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-type polysaccharides can be divided into homooxylans and heterooxylans, including glucuronoxylans, (arabinose)glucuronoxylans, (glucuronano)arabinoxylans, arabinoxylans, and complex heterooxylans. See, eg, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67.

[0091] In the method of the present invention, any material containing xylan may be used. In a preferred aspect, the xylan containing material is lignocellulose.

[0092] Xylan degrading activity or xylanolytic activity: The term "xylan degrading activity" or "xylanolytic activity" means biological activity that hydrolyzes xylan-containing material. Two basic methods for measuring xylanolytic activity include: (1) measuring total xylanolytic activity, and (2) measuring individual xylanolytic activities (e.g., endoxylanase, β-xylosidase, arabinofuranosidase, α-glucuronidase, acetylxylan esterase, feruloyl esterase, and α-glucuronyl esterase). Recent advances in the assay of xylanolytic enzymes are summarized in several publications, including Biely and Puchard, 2006, Journal of the Science of Food and Agriculture 86(11): 1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601; Herrmann et al., 1997, Biochemical Journal 321: 375-381.

[0093] Total xylan degrading activity can be measured by determining the reducing sugars formed from different types of xylans, including, for example, oatspelt xylan, beechwood xylan, and larch wood xylan, or by photometric determination of stained xylan fragments released from different covalently stained xylans. The most common assay for total xylanolytic activity is based on the generation of reducing sugars from poly-4-O-methylglucuronoxylan, as described in Bailey et al., 1992, "Interlaboratory testing of methods for assay of xylanase activity," Journal of Biotechnology 23(3): 257-270. Xylanase activity can also be measured at 37°C in the presence of 0.01% The assay was performed in X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) and 200 mM sodium phosphate (pH 6) using 0.2% AZCL-arabinoxylan as a substrate. One unit of xylanase activity was defined as the production of 1.0 μmole of azurin per minute from 0.2% AZCL-arabinoxylan as a substrate in 200 mM sodium phosphate (pH 6) at 37°C.

[0094] Xylan degrading activity is preferably determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc., St. Louis, MO, USA) caused by one or more xylan degrading enzymes under the following typical conditions: 1 ml reaction, 5 mg / ml substrate (total solids), 5 mg xylanolytic protein / g substrate, 50 mM sodium acetate (pH 5), 50°C, 24 hours, sugar analysis using the p-hydroxybenzoic acid hydrazide (PHBAH) assay as described in Lever, 1972, Anal. Biochem 47: 273-279.

[0095] Xylanase: The term "xylanase" means a 1,4-β-D-xylan-xylose hydrolase (EC 3.2.1.8) that catalyzes the endohydrolysis of 1,4-β-D-xylosidic bonds in xylans. Xylanase activity was determined using 0.2% AZCL-arabinoxylan as a substrate in 200 mM sodium phosphate (pH 6) and X-100. One unit of xylanase activity was defined as the production of 1.0 μmole of azurin per minute from 0.2% AZCL-arabinoxylan as a substrate in 200 mM sodium phosphate (pH 6) at 37°C.

[0096] Polypeptides of the 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%, and at least 100% of the xylanase activity of the mature polypeptide of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 6.

[0097] Detailed description of the invention

[0098] Polypeptides with xylanase activity

[0099] In one embodiment, the invention relates to isolated polypeptides having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to the mature polypeptide of SEQ ID NO: 2, wherein the isolated polypeptides have xylanase activity. In one aspect, the polypeptides differ from the mature polypeptide of SEQ ID NO: 2 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids.

[0100] In another embodiment, the invention relates to isolated polypeptides having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to the mature polypeptide of SEQ ID NO: 6, which isolated polypeptides have xylanase activity. In one aspect, these polypeptides differ from the mature polypeptide of SEQ ID NO: 6 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids.

[0101] The polypeptide of the present invention preferably comprises or consists of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 6, or an allelic variant thereof, or a fragment thereof having xylanase activity. In another aspect, the polypeptide comprises or consists of the mature polypeptide of SEQ ID NO: 2. In another aspect, the polypeptide comprises or consists of amino acids 24 to 406 of SEQ ID NO: 2. In another aspect, the polypeptide comprises or consists of amino acids 24 to 406 of SEQ ID NO: 6.

[0102] In another embodiment, the invention relates to an isolated polypeptide having xylanase 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 to: (i) the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 5, (ii) the cDNA sequence of SEQ ID NO: 1 or SEQ ID NO: 5, or (iii) the full-length complement of (i) or (ii) (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY).

[0103] The polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 5, or a subsequence thereof, together with the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6, its mature polypeptide or a fragment thereof, can be used to design nucleic acid probes to identify and clone DNA encoding polypeptides having xylanase activity from strains of different genera or species according to methods well known in the art. Specifically, such probes can be used to hybridize with genomic DNA or cDNA of cells of interest following standard Southern blotting procedures to identify and isolate the corresponding gene therein. Such probes can be significantly 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 probe is typically labeled (e.g., with a 32 P. 3 H. 35 S, biotin, or avidin) to detect the corresponding gene. The present invention covers such probes.

[0104] Genomic DNA or cDNA libraries prepared from such other strains can be screened for DNA that hybridizes with the probes described above and encodes a polypeptide having xylanase activity. Genomic DNA or other DNA from such other strains can be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or separated DNA can be transferred to a suitable support material, such as nitrocellulose. This support material can be used in a Southern blot to identify clones or DNA that hybridize to SEQ ID NO: 1 or SEQ ID NO: 5, the mature polypeptide coding sequence thereof, or a subsequence thereof.

[0105] For the purposes of the present invention, hybridization means hybridization of a polynucleotide to a labeled nucleic acid probe corresponding to: (i) SEQ ID NO: 1 or SEQ ID NO: 5; (ii) the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 5; (iii) the cDNA sequence of SEQ ID NO: 1 or SEQ ID NO: 5, or the mature polypeptide coding sequence thereof; (iv) a full-length complement thereof; or (v) a subsequence thereof; the hybridization being conducted 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.

[0106] In one aspect, the nucleic acid probe is a polynucleotide encoding: the polypeptide of SEQ ID NO: 2; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 1; the mature polypeptide coding sequence thereof; or a cDNA sequence thereof. In another aspect, the nucleic acid probe is a polynucleotide contained in Rasamsonia byssochlamydoides CBS 413.71, wherein the polynucleotide encodes a polypeptide having xylanase activity. In another aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in Rasamsonia byssochlamydoides CBS 413.71.

[0107] In one aspect, the nucleic acid probe is a polynucleotide encoding: the polypeptide of SEQ ID NO: 6; the mature polypeptide thereof; or a fragment thereof. In another aspect, the nucleic acid probe is SEQ ID NO: 5; the mature polypeptide coding sequence thereof; or its cDNA sequence. In another aspect, the nucleic acid probe is a polynucleotide contained in Rasamsonia byssochlamydoides CBS 150.75, wherein the polynucleotide encodes a polypeptide having xylanase activity. In another aspect, the nucleic acid probe is the mature polypeptide coding sequence contained in Rasamsonia byssochlamydoides CBS 150.75.

[0108] In another embodiment, the invention relates to an isolated polypeptide having xylanase activity, which isolated polypeptide is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or its cDNA sequence.

[0109] In another embodiment, the invention relates to an isolated polypeptide having xylanase activity, which isolated polypeptide is encoded by a polynucleotide having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5 or its cDNA sequence.

[0110] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 2 comprising substitutions, deletions, and / or insertions at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the mature polypeptide of SEQ ID NO: 2 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0111] In another embodiment, the present invention relates to variants of the mature polypeptide of SEQ ID NO: 6 comprising substitutions, deletions, and / or insertions at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the mature polypeptide of SEQ ID NO: 6 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0112] These amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small connecting peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding domain.

[0113] Examples of conservative substitutions are within the group consisting 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 generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and RL 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.

[0114] Alternatively, the amino acid change has such a property that it changes the physicochemical properties of the polypeptide. For example, the amino acid change can increase the thermal stability of the polypeptide, change the substrate specificity, change the optimal pH, etc.

[0115] 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 resulting mutant molecules are tested for xylanase activity to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Mutations in putative contact site amino acids can also be combined with physical analysis of the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, to determine the active site of the enzyme or other biological interactions. See, e.g., 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. Essential amino acids can also be identified by inference from alignments with related polypeptides.

[0116] 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 relevant screening procedures, 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).

[0117] Mutagenesis / shuffling methods can be combined with high-throughput automated screening methods to detect the activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). Mutagenized DNA molecules encoding active polypeptides can be recovered from 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.

[0118] In each of the above embodiments, in one aspect, the isolated polypeptide of the invention having xylanase activity has at least 10% more, e.g., at least 15% and at least 20% more, xylanase activity at pH 4.0 and 60° C. or 65° C. as compared to at pH 4.0 and 50° C. In one aspect, xylanase activity is preferably determined according to the protocol described in Example 7 using washed ground sieved alkaline pretreated corncob (GS-APCC) for 72 hours as substrate in 50 mM sodium acetate (pH 4.0 to 5.5) or 50 mM Tris (pH 6.0 to 7.0) buffer containing 1 mM manganese sulfate.

[0119] In each of the above embodiments, in another aspect, the isolated polypeptide of the invention having xylanase activity has at least 10% more, e.g., at least 15% and at least 20% more, xylanase activity at pH 4.0 and 50° C., 55° C., 60° C., or 65° C., respectively, as compared to at pH 6.0 and 50° C., 55° C., 60° C., or 65° C. In one aspect, xylanase activity is preferably determined according to the protocol described in Example 7 using washed ground sieved alkaline pretreated corncob (GS-APCC) for 72 hours as a substrate in 50 mM sodium acetate (pH 4.0 to 5.5) or 50 mM Tris (pH 6.0 to 7.0) buffer containing 1 mM manganese sulfate.

[0120] The polypeptide may be a hybrid polypeptide in which a region of one polypeptide is fused at the N-terminus or C-terminus of a region of another polypeptide.

[0121] The polypeptide can be a fusion polypeptide or a cleavable fusion polypeptide, wherein another polypeptide is fused at the N-terminus or C-terminus of the polypeptide of the present invention. Fusion polypeptides are produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention. Technology for producing fusion polypeptides is known in the art and includes connecting the coding sequences encoding the polypeptides so that they are in frame and expression of the fusion polypeptide is under the control of the same one or more promoters and terminators. Fusion polypeptides can also be constructed using intein technology, wherein the fusion polypeptide is produced after translation (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

[0122] The fusion polypeptide may further include a cleavage site between the two polypeptides. When the fusion protein is secreted, the site is cleaved, thereby releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, those disclosed in the following literature: Martin et al., 2003, Journal of Industrial Microbiology and Biotechnology (J.Ind.Microbiol.Biotechnol.) 3:568-576; Svetina et al., 2000, Journal of Biotechnology (J.Biotechnol.) 76:245-251; Rasmussen-Wilson et al., 1997, Appl.Environ.Microbiol. 63:3488-3493; Ward et al., 2003, Journal of Industrial Microbiology and Biotechnology (J.Ind.Microbiol ... d) 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.

[0123] Sources of polypeptides having xylanase activity

[0124] The polypeptides having xylanase activity of the present invention can be obtained from microorganisms of any genus. For the purposes of the present invention, the term "obtained from" as used herein in conjunction with a given source shall mean that the polypeptide encoded by the polynucleotide is produced by the source or by a strain into which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0125] The polypeptide can be a fungal polypeptide. In one aspect, the polypeptide is a Rasamsonia polypeptide. In another aspect, the polypeptide is a Rasamsonia byssochlamydoides polypeptide. In another aspect, the polypeptide is a Rasamsonia byssochlamydoides CBS 413.71 polypeptide. In another aspect, the polypeptide is a Rasamsonia byssochlamydoides CBS 150.75 polypeptide.

[0126] It will be understood that for the species mentioned above, the present invention encompasses both perfect and imperfect states, as well as other taxonomic equivalents, such as anamorphs, regardless of their known species names. One of ordinary skill in the art will readily recognize the identity of appropriate equivalents. For example, the species Rasamsonia byssochlamydoides is sometimes referred to as Rasamsonia byssochlamydoides, or is referred to by its anamorph Paecilomyces byssochlamydoides.

[0127] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the German Collection of Microorganisms (DSMZ), the Netherlands Center for the Study of Microorganisms (Centraalbureau Voor Schimmelcultures, CBS), and the Agricultural Research Service Northern Regional Research Center (NRRL).

[0128] The above-mentioned probe can be used from other sources, including microorganisms isolated from nature (for example, soil, compost, water, etc.) or directly from natural materials (for example, soil, compost, water, etc.) to identify and obtain the polypeptide. The technology for directly isolating microorganisms and DNA from natural living environments is well known in the art. The polynucleotide encoding the polypeptide can then be obtained by similarly screening the genomic DNA or cDNA library of another microorganism or the mixed DNA sample. Once the polynucleotide encoding the polypeptide is detected with one or more probes, the polynucleotide can be separated or cloned by using technology known to those of ordinary skill in the art (see, for example, Sambrook et al., 1989, the same).

[0129] catalytic domain

[0130] In one embodiment, the present invention also relates to catalytic domains having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to amino acids 24 to 340 of SEQ ID NO: 2. In one aspect, the catalytic domains comprise an amino acid sequence that differs from amino acids 24 to 340 of SEQ ID NO: 2 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. The catalytic domain preferably comprises or consists of amino acids 24 to 340 of SEQ ID NO: 2 or an allelic variant thereof; or is a fragment thereof having xylanase activity.

[0131] In another embodiment, the present invention also relates to catalytic domains having at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to amino acids 24 to 341 of SEQ ID NO: 6. In one aspect, the catalytic domains comprise an amino acid sequence that differs from amino acids 24 to 341 of SEQ ID NO: 6 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. The catalytic domain preferably comprises or consists of amino acids 24 to 341 of SEQ ID NO: 6 or an allelic variant thereof; or is a fragment thereof having xylanase activity.

[0132] In another embodiment, the present invention also relates to a catalytic domain encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to nucleotides 157 to 1339 of SEQ ID NO: 1, or its cDNA sequence. The polynucleotide encoding the catalytic domain preferably comprises or consists of nucleotides 157 to 1339 of SEQ ID NO: 1, or is the sequence contained in Rasamsonia byssochlamydoides strain CBS 413.71.

[0133] In another embodiment, the present invention also relates to a catalytic domain encoded by a polynucleotide having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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% sequence identity to nucleotides 151 to 1387 of SEQ ID NO: 5, or its cDNA sequence. The polynucleotide encoding the catalytic domain preferably comprises or consists of nucleotides 151 to 1387 of SEQ ID NO: 5, or is the sequence contained in Rasamsonia byssochlamydoides strain CBS150.75.

[0134] In another embodiment, the present invention also relates to catalytic domain variants of amino acids 24 to 340 of SEQ ID NO: 2, which include substitutions, deletions, and / or insertions at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the sequence of amino acids 24 to 340 of SEQ ID NO: 2 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or 10.

[0135] In another embodiment, the present invention also relates to catalytic domain variants of amino acids 24 to 341 of SEQ ID NO: 6, which include substitutions, deletions, and / or insertions at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the sequence of amino acids 24 to 341 of SEQ ID NO: 6 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or 10.

[0136] Carbohydrate binding module

[0137] In one embodiment, the present invention also relates to carbohydrate binding moieties having at least 90%, such as 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% sequence identity to amino acids 373 to 406 of SEQ ID NO: 2. In one aspect, the carbohydrate binding moieties comprise an amino acid sequence that differs from amino acids 373 to 406 of SEQ ID NO: 2 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. The carbohydrate binding moiety preferably comprises or consists of amino acids 373 to 406 of SEQ ID NO: 2, or an allelic variant thereof, or is a fragment thereof having carbohydrate binding activity.

[0138] In one embodiment, the present invention also relates to carbohydrate binding moieties having at least 90%, such as 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% sequence identity to amino acids 371 to 406 of SEQ ID NO: 6. In one aspect, the carbohydrate binding moieties comprise an amino acid sequence that differs from amino acids 371 to 406 of SEQ ID NO: 6 by up to 10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids. The carbohydrate binding moiety preferably comprises or consists of amino acids 371 to 406 of SEQ ID NO: 6, or an allelic variant thereof, or is a fragment thereof having carbohydrate binding activity.

[0139] In another embodiment, the present invention also relates to carbohydrate binding modules encoded by polynucleotides having at least 90%, such as 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% sequence identity to nucleotides 1436 to 1537 of SEQ ID NO: 1. The polynucleotide encoding the carbohydrate binding module preferably comprises or consists of nucleotides 1436 to 1537 of SEQ ID NO: 1, or is a sequence contained in Rasamsonia byssochlamydoides CBS 413.71.

[0140] In another embodiment, the present invention also relates to carbohydrate binding modules encoded by polynucleotides having at least 90%, such as 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% sequence identity to nucleotides 1480 to 1587 of SEQ ID NO: 5. The polynucleotide encoding the carbohydrate binding module preferably comprises or consists of nucleotides 1480 to 1587 of SEQ ID NO: 5, or is a sequence contained in Rasamsonia byssochlamydoides CBS 150.75.

[0141] In another embodiment, the present invention also relates to carbohydrate binding module variants of amino acids 373 to 406 of SEQ ID NO: 2, which variants include substitutions, deletions, and / or insertions at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the sequence of amino acids 373 to 406 of SEQ ID NO: 2 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or 10.

[0142] In another embodiment, the present invention also relates to carbohydrate binding module variants of amino acids 371 to 406 of SEQ ID NO: 6, which variants include substitutions, deletions, and / or insertions at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions, and / or insertions introduced into the sequence of amino acids 371 to 406 of SEQ ID NO: 6 is up to 10, e.g., 1, 2, 3, 4, 5, 6, 8, 9, or 10.

[0143] The catalytic domain operably linked to the carbohydrate binding module can be from a hydrolase, isomerase, ligase, lyase, oxidoreductase or transferase, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glucosyltransferase, deoxyribonuclease, endoglucanase, esterase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase or β-xylosidase. The polynucleotide encoding the catalytic domain can be obtained from any prokaryotic, eukaryotic or other source.

[0144] polynucleotides

[0145] The present invention also relates to isolated polynucleotides encoding a polypeptide, catalytic domain or carbohydrate binding module of the invention as described herein.

[0146] The technology for isolating or cloning polynucleotides is known in the art and includes isolating from genomic DNA or cDNA, or a combination thereof. Cloned DNA fragments with common structural features can be detected, for example, by antibody screening using the polymerase chain reaction (PCR) or expression library known to the art, to achieve cloning of polynucleotides from genomic DNA. See, for example, Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York. Other nucleic acid amplification programs such as ligase chain reaction (LCR), ligation activated transcription (LAT) and polynucleotide-based amplification (NASBA) can be used. These polynucleotides can be cloned by Corymphospora strains or related organisms, and therefore, for example, can be alleles or species variants of the polypeptide coding region of the polynucleotides.

[0147] Modification of the polynucleotide encoding the polypeptide of the present invention may be necessary to synthesize polypeptides that are 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 from the polypeptide isolated from its natural source in some engineered manner, for example, variants that differ in specific activity, thermostability, optimal pH, etc. These variants can be constructed based on the polynucleotide represented by the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 5 or its cDNA sequence by introducing nucleotide substitutions that do not change the amino acid sequence of the polypeptide but correspond to the codon usage of the host organism intended for production of the enzyme, or by introducing nucleotide substitutions that may result in a different amino acid sequence. For a general description of nucleotide substitutions, see, for example, Ford et al., 1991, Protein Expression and Purification 2: 95-107.

[0148] Nucleic acid constructs

[0149] 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.

[0150] The polynucleotide can be manipulated in various ways to provide expression of the polypeptide. Depending on the expression vector, manipulation of the polynucleotide prior to its insertion into the vector may be desirable or necessary. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0151] The control sequence can be a promoter, i.e., a polynucleotide that is recognized by the host cell to express a polynucleotide encoding a polypeptide of the present invention. The promoter comprises transcriptional control sequences that mediate expression of the polypeptide. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell, including mutant, truncated, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0152] Examples of suitable promoters for directing transcription of the nucleic acid constructs of the present invention in bacterial host cells are promoters obtained from the following genes: 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 xylA and xylB genes, Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994, Molecular Microbiology). coli lac operon, the E. coli trc promoter (Egon et al., 1988, Gene 69:301-315), the Streptomyces coelicolor agarohydrolase gene (dagA), and the prokaryotic β-lactamase gene (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA 75:3727-3731), and the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Other promoters are described in Gilbert et al., 1980, "Useful proteins from recombinant bacteria," Scientific American, 242: 74-94; and in Sambrook et al., 1989, supra. Examples of tandem promoters are disclosed in WO 99 / 43835.

[0153] 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 alpha-amylase (WO 00 / 56900), Aspergillus oryzae ... miehei) lipase, Rhizomucor miehei aspartic proteinase, Trichoderma reesei β-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, Trichoderma reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei β-xylosidase, and Trichoderma reesei translation elongation factor, with and NA2-tpi promoter (a modified promoter derived from the Aspergillus neutral α-amylase gene in which the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus triose phosphate isomerase gene; non-limiting examples include a modified promoter derived from the Aspergillus niger neutral α-amylase gene in which the untranslated leader sequence is replaced by the untranslated leader sequence of the Aspergillus nidulans or Aspergillus oryzae triose phosphate isomerase gene); and mutant promoters, truncated promoters, and hybrid promoters thereof. Other promoters are described in U.S. Patent No. 6,011,147.

[0154] In yeast hosts, 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 triosephosphate 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.

[0155] The control sequence may also be a transcription terminator that is recognized by the host cell to terminate transcription. The terminator is operably linked to the 3'-end of the polynucleotide encoding the polypeptide. Any terminator that functions in the host cell may be used in the present invention.

[0156] 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).

[0157] Preferred terminators for filamentous fungal host cells are obtained from the genes for Aspergillus nidulans acetamidase, Aspergillus nidulans anthranilate synthase, Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase, Aspergillus oryzae TAKA amylase, Fusarium oxysporum trypsin-like protease, 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 V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei xylanase III, Trichoderma reesei beta-xylosidase, and Trichoderma reesei translation elongation factor.

[0158] 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.

[0159] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of a gene, which increases the expression of the gene.

[0160] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriologry 177: 3465-3471).

[0161] The control sequence can also be a leader sequence, a non-translated mRNA region that is important for translation by the host cell. The leader is operably linked to the 5'-end of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the host cell can be used.

[0162] Preferred leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase.

[0163] 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).

[0164] The control sequence may also be a polyadenylation sequence, a sequence operably linked to the 3'-terminus of the polynucleotide and, when transcribed, recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that functions in the host cell may be used.

[0165] 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.

[0166] Useful polyadenylation sequences for yeast host cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

[0167] The control sequence can also be a signal peptide coding region that encodes a signal peptide that is connected to the N-terminus of the polypeptide and instructs the polypeptide to enter the secretory pathway of the cell. The 5'-end of the coding sequence of the polynucleotide can inherently include a signal peptide coding sequence that is naturally connected to the segment of the coding sequence of the coding polypeptide in the translation reading frame. Alternatively, the 5' end of the coding sequence can include a signal peptide coding sequence that is exogenous to the coding sequence. In the case where the coding sequence does not naturally include a signal peptide coding sequence, an exogenous signal peptide coding sequence may be required. Alternatively, an exogenous signal peptide coding sequence can simply replace the natural signal peptide coding sequence to enhance the secretion of the polypeptide. However, any signal peptide coding sequence that instructs the expressed polypeptide to enter the secretory pathway of the host cell can be used.

[0168] Effective signal peptide coding sequences for bacterial host cells are those obtained from the genes for Bacillus sp. NCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis β-lactamase, Bacillus stearothermophilus α-amylase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prs A. Additional signal peptides are described by Simonen and Palva, 1993, Microbiological Reviews 57: 109-137.

[0169] Effective signal peptide coding sequences for filamentous fungal host cells are those 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.

[0170] 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.

[0171] The control sequence can also be a propeptide coding sequence of the propeptide at the N-terminal end of the polypeptide. The polypeptide generated is called a proenzyme (proenzyme) or propolypeptide (or is called a zymogen (zymogen) in some cases). Propolypeptide is normally inactive and can be converted into a kind of active polypeptide by catalytic cracking or autocatalytic cracking propeptide from the propolypeptide. The propeptide coding sequence can be obtained from the following genes: subtilis alkaline protease (aprE), subtilis neutral protease (nprT), thermophilic myceliophthora laccase (WO 95 / 33836), miehei mucor aspartic proteinase and saccharomyces cerevisiae α-factor.

[0172] Where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is positioned immediately adjacent to the N-terminus of the polypeptide and the signal peptide sequence is positioned immediately adjacent to the N-terminus of the propeptide sequence.

[0173] It may also be desirable to add regulatory sequences that regulate the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory sequences are those that enable the expression of the gene to be turned on or off in response to chemical or physical stimuli (including the presence of regulatory compounds). Regulatory sequences in prokaryotic systems include lac, tac, and trp operon systems. In yeast, the ADH2 system or the GAL1 system can be used. In filamentous fungi, the Aspergillus niger glucoamylase promoter, Aspergillus oryzae TAKA α-amylase promoter, and Aspergillus oryzae glucoamylase promoter, Trichoderma reesei cellobiohydrolase I promoter, and Trichoderma reesei cellobiohydrolase II promoter can be used. Other examples of regulatory sequences are those that allow gene amplification. In eukaryotic systems, these regulatory sequences include dihydrofolate reductase genes amplified in the presence of methotrexate and metallothionein genes amplified with heavy metals. In these cases, the polynucleotide encoding the polypeptide will be operably connected to the regulatory sequence.

[0174] expression vector

[0175] The present invention further relates to the recombinant expression vector comprising polynucleotide of the present invention, promoter and transcription and translation termination signal.Different Nucleotide and control sequence can be linked together to produce a recombinant expression vector, and this recombinant expression vector can comprise one or more convenient restriction enzyme sites to allow the polynucleotide of inserting or replacing this variant of coding at these sites.Alternately, these polynucleotide can be expressed by inserting this polynucleotide or the nucleic acid construct comprising this polynucleotide into the appropriate vector for expression.When producing this expression vector, this encoding sequence is positioned in this vector, so that this encoding sequence is operably connected with this appropriate control sequence for expression.

[0176] The recombinant expression vector can be any vector (e.g., a plasmid or virus) that can be readily subjected to recombinant DNA procedures and that can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0177] The carrier can be an autonomous replicating vector, that is, a carrier existing as an extrachromosomal entity, which replicates independently of chromosomal replication, for example, a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The carrier can include any element to ensure self-replication. Alternatively, the carrier can be a vector that, when introduced into the host cell, is integrated into the genome and replicates with one or more chromosomes into which it has been integrated. In addition, a single vector or plasmid or two or more vectors or plasmids (these vectors or plasmids contain the total DNA to be introduced into the genome of the host cell together) or a transposon can be used.

[0178] The vector preferably contains one or more selectable markers that allow easy selection of transformed, transfected, transduced or similar cells. A selectable marker is a gene whose product provides biocide resistance or viral resistance, heavy metal resistance, prototrophy of auxotrophs, etc.

[0179] Examples of bacterial selectable markers are the dal genes of Bacillus licheniformis or Bacillus subtilis, or markers that confer antibiotic resistance (e.g., ampicillin, chloramphenicol, kanamycin, neomycin, spectinomycin, or tetracycline resistance). Suitable markers for use in yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, adeA (phosphoribosylamidoimidazole-succinylcarboxamide synthase), adeB (phosphoribosylamidoimidazole synthase), amdS (acetamidase), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfate adenosyltransferase), and trpC (anthranilate synthase), as well as their equivalents. Preferred for use in Aspergillus cells are the Aspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and the Streptomyces hygroscopicus bar gene. Preferred for use in Trichoderma cells are the adeA, adeB, amdS, hph and pyrG genes.

[0180] The selectable marker may be a dual selectable marker system as described in WO 2010 / 039889. In one aspect, the dual selectable marker is an hph-tk dual selectable marker system.

[0181] The vector preferably contains one or more elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0182] For being integrated into the host cell genome, this vector can rely on the polynucleotide sequence encoding this polypeptide or be used for being integrated into any other element of this vector in this genome by homologous or non-homologous recombination.Alternatively, this vector can comprise the other polynucleotide at one or more precise positions in one or more chromosomes that are integrated into the host cell genome by homologous recombination.In order to increase the possibility of integrating at precise position, these integrated elements should comprise sufficient number of nucleic acids, for example 100 to 10,000 base pairs, 400 to 10,000 base pairs and 800 to 10,000 base pairs, and these base pairs have a high sequence identity with the corresponding target sequence to improve the possibility of homologous recombination.These integration elements can be any sequence homologous to the target sequence in the genome of the host cell.In addition, these integration elements can be non-coding polynucleotides or encoding polynucleotides.On the other hand, this vector can be integrated into the genome of the host cell by non-homologous recombination.

[0183] For autonomous replication, the vector may further include an origin of replication that enables the vector to replicate autonomously in the host cell in question. The origin of replication can be any plasmid replicator that mediates autonomous replication that functions in the cell. The term "origin of replication" or "plasmid replicator" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0184] Examples of bacterial origins of replication are the origins of replication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permitting replication in E. coli, and the origins of replication of plasmids pUB110, pE194, pTA1060, and pAMβ1 permitting replication in Bacillus.

[0185] 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.

[0186] Examples of origins of replication useful in filamentous fungal cells are AMA1 and ANS1 (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 containing the gene can be accomplished according to the methods disclosed in WO 00 / 24883.

[0187] More than one copy of the polynucleotide of the present invention can be inserted into a host cell to increase the production of the polypeptide. Increased copy numbers 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 together with the polynucleotide, wherein cells comprising an amplified copy of the selectable marker gene and thus additional copies of the polynucleotide can be selected by culturing the cells in the presence of an appropriate selective agent.

[0188] The procedures used to ligate the elements described above to construct the recombinant expression vectors of the present invention are well known to those skilled in the art (see, eg, Sambrook et al., 1989, supra).

[0189] host cells

[0190] The present invention also relates to recombinant host cells comprising polynucleotides of the present invention, operably linked to one or more control sequences that direct the production of polypeptides of the present invention. Constructs or vectors comprising the polynucleotides are introduced into host cells so that the constructs or vectors are maintained as chromosomal integrants or as autonomously replicating extrachromosomal vectors, as described earlier. The term "host cell" encompasses any progeny of a parent cell that differs from the parent cell due to mutations that occur during replication. The selection of host cells depends largely on the gene encoding the polypeptide and its source.

[0191] The host cell can be any cell useful for recombinant production of the polypeptides of the present invention, such as a prokaryotic cell or a eukaryotic cell.

[0192] Prokaryotic host cells can be any Gram-positive or Gram-negative bacteria. Gram-positive bacteria include, but are not limited to, Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Ocean Bacillus, Staphylococcus, Streptococcus and Streptomyces. Gram-negative bacteria include, but are not limited to, Campylobacter, Escherichia coli, Flavobacterium, Fusobacterium, Helicobacter, Silene Bacillus, Neisseria, Pseudomonas, Salmonella and Ureaplasma.

[0193] The bacterial host cell can be any Bacillus cell, including but not limited to Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus laurens, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus brevis, Bacillus stearothermophilus, Bacillus subtilis, and Bacillus thuringiensis cells.

[0194] Introduction of DNA into Bacillus cells can be accomplished 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 David

[0015] Introduction of DNA into E. coli cells can be accomplished 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). Introduction of DNA into Streptomyces cells can be achieved 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). Introduction of DNA into Pseudomonas cells can be achieved 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).Introduction of DNA into Streptococcus cells can be achieved 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 host cells can be used.

[0195] The host cell can also be a eukaryotic cell, such as a mammalian, insect, plant, or fungal cell.

[0196] The host cell may be a fungal cell. "Fungi," as used herein, include Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota, as well as Oomycota and all mitosporic fungi (as defined by Hawksworth et al. in Ainsworth and Bisby's Dictionary of The Fungi, 8th ed., 1995, CAB International, University Press, Cambridge, UK).

[0197] The fungal host cell can be a yeast cell. " Yeast " as used herein includes yeasts that produce ascospore yeasts (Endosporales), basidiomycetes and yeasts that belong to Fungi Deuteromyces (Blastomycetes). Because the classification of yeast may change in the future, for purposes of the present invention, yeast should be defined as described in the Biology and Activities of Yeast (Biology and Activities of Yeast) (Skinner, Passmore and Davenport, eds., Soc. App. Bacteriol. Symposium Series No. 9, 1980) of the Society for Applied Bacteriology.

[0198] The yeast host cell can be a Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia cell, such as a Kluyveromyces lactis, Kluyveromyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, or Yarrowia lipolytica cell.

[0199] The fungal host cell can be a filamentous fungal cell. "Filamentous fungi" includes all filamentous forms of the subphylum Eumycota and Oomycota (as defined by Hawkesworth et al., 1995, supra). Filamentous fungi are generally characterized by a mycelial wall composed of chitin, cellulose, dextran, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation, while carbon catabolism is obligately aerobic. In contrast, vegetative growth of yeasts (such as Saccharomyces cerevisiae) is by budding of a unicellular thallus, while carbon catabolism can be fermentative.

[0200] The filamentous fungal host cell can be an Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceroplastes, Chrysosporium, Coprinus, Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Pyrospora, Mucor, Myceliophthora, Neomycota, Neurospora, Paecilomyces, Penicillium, Pseudomonas, Phlebia, Chytridium, Pleurotus, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Toxoplasma, Trametes, or Trichoderma cell.

[0201] For example, the filamentous fungal host cell can be Aspergillus awamori, Aspergillus foetida, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Bjerkandera adusta, Ceriporiopsis aneurina, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium feces, or Chrysosporium spp. merdarium), rent spore fungus, Chrysosporium queenslandicum, tropical chrysosporium, Chrysosporium zonatum, Coprinus cinereus, Coriolushirsutus, rod-shaped fusarium, cereal fusarium, Kuwei fusarium, broadsword fusarium, graminearum fusarium, graminearum fusarium, heterosporous fusarium, albizia fusarium, sharp fusarium, multi-branched fusarium, pink fusarium, elder fusarium, color fusarium, pseudo-branched fusarium, sulfur-colored fusarium, round fusarium, pseudo-sporic fusarium, embellished fusarium, Humicola insolens, Humicola lanuginosa, rice black mold, thermophilic myceliophthora, rough neurospora, purple-producing Penicillium, Phanerochaete chrysosporium, radiata fusarium, Pleurotus eryngii eryngii), Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride cells.

[0202] Fungal cells can be transformed in a manner known per se by methods involving protoplast formation, protoplast transformation, and cell wall regeneration. Suitable procedures for transforming 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 can be transformed using procedures described by, for example, Becker and Guarente, in Abelson, JN and Simon, MI, eds., Guide to Yeast Genetics and Molecular Biology, Methods in Enzymology, Vol. 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.

[0203] Generation method

[0204] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a cell under conditions conducive for production of the polypeptide, wherein the cell produces the polypeptide in its wild-type form; and optionally (b) recovering the polypeptide. In one aspect, the cell is a Rasamsonia cell. In another aspect, the cell is a Rasamsonia byssochlamydoides cell. In another aspect, the cell is a Rasamsonia byssochlamydoides CBS 413.71 cell. In another aspect, the cell is a Rasamsonia byssochlamydoides CBS 150.75 cell.

[0205] 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 optionally (b) recovering the polypeptide.

[0206] These host cells are cultivated in a nutrient medium suitable for producing the polypeptide using methods known in the art. For example, it is possible to culture by shaking a flask in a suitable medium and under conditions allowing expression and / or separation of the polypeptide, or to culture cells in a laboratory or industrial fermentor tank using a small or large-scale fermentation (including continuous, in batches, fed-batch, or solid-state fermentation). The culture is performed using a program known in the art, and a suitable nutrient medium occurs, the medium including carbon and nitrogen sources and inorganic salts. Suitable nutrient medium can be obtained from commercial suppliers or can be prepared according to disclosed composition (for example, in the catalog of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be directly recovered from the medium. If the polypeptide is not secreted, it can be recovered from the cell lysate.

[0207] The polypeptide can be detected using methods known in the art that are specific for the polypeptide. These detection methods include, but are not limited to, the use of specific antibodies, the formation of an enzyme product, or the disappearance of an enzyme substrate. For example, an enzyme assay can be used to determine the activity of the polypeptide.

[0208] The polypeptide can be recovered using methods known in the art. For example, the polypeptide can be recovered from the nutrient medium by conventional procedures, including, but not limited to, collection, centrifugation, filtration, extraction, spray drying, evaporation, or precipitation. In one aspect, the entire fermentation broth comprising the hybrid polypeptide having xylanase activity of the present invention is recovered.

[0209] The polypeptide can be purified to obtain a substantially pure polypeptide by a variety of procedures known in the art, including, but not limited to, chromatography (e.g., ion exchange chromatography, affinity chromatography, hydrophobic interaction chromatography, chromatofocusing, and size exclusion chromatography), 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, eds., VCH Publishers, New York, 1989).

[0210] plant

[0211] The present invention also relates to isolated plants, such as transgenic plants, plant parts, or plant cells, comprising a polynucleotide for expressing and producing a polypeptide or domain of the present invention in recoverable quantities. The polypeptide or domain can be recovered from the plant or plant part. Alternatively, the plant or plant part comprising the polypeptide or domain can be used as is to improve the quality of food or feed, for example, to improve nutritional value, palatability, and rheological properties, or to eliminate anti-nutritional factors.

[0212] Transgenic plants can be dicots (dicots) or monocots (monocots). Examples of monocots are grasses, such as meadow grasses (bluegrass, Poa); forage grasses, such as Festuca and Lolium; temperate grasses, such as bentgrass (Agrostis); and cereals, such as wheat, oats, rye, barley, rice, sorghum, and maize (corn).

[0213] Examples of dicots are tobacco, legumes (such as lupins, potatoes, sugar beets, peas, beans, and soybeans), and crucifers (family Brassicaceae) (such as cauliflower, rapeseed, and the closely related model organism Arabidopsis thaliana).

[0214] The example of plant part is stem, callus, leaf, root, fruit, seed and tuber and the independent tissue that comprises these parts, for example, epidermis, mesophyll, parenchyma (parenchyme), vascular tissue, meristem.Specific plant cell compartment, as chloroplast, apoplast (apoplast), mitochondria, vacuole, peroxisome and cytoplasm are also considered to plant part.In addition, any vegetable cell, no matter which kind of tissue origin, is all considered to plant part.Similarly, plant part, as separation to contribute to the specific tissue and cell of utilization of the present invention, is also considered to plant part, for example embryo, endosperm, aleurone and seed coat.

[0215] Also included within the scope of the present invention are the progeny of such plants, plant parts, and plant cells.

[0216] Transgenic plants or plant cells expressing the polypeptide or domain can be constructed according to methods known in the art. Briefly, plants or plant cells are 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 plants or plant cells into transgenic plants or plant cells.

[0217] The expression construct is conveniently a nucleic acid construct comprising a polynucleotide encoding a polypeptide or domain operably linked to 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 for identifying plant cells that have incorporated the expression construct, and the DNA sequences necessary for introducing the construct into the plant in question (the latter depending on the method of DNA introduction used).

[0218] The choice of regulatory sequences (such as promoter and terminator sequences and optional signal or transit sequences) is determined, for example, based on when, where, and how the desired polypeptide or domain is expressed (Sticklen, 2008, Nature Reviews 9: 433-443). For example, expression of a gene encoding a polypeptide or domain can be constitutive or inducible, or can be developmental, stage, or tissue specific, and the gene product can be targeted to a specific tissue or plant part, such as seeds or leaves. Regulatory sequences are described, for example, by Tague et al., 1988, Plant Physiology 86: 506.

[0219] For constitutive expression, the 35S-CaMV, maize ubiquitin 1, or rice actin 1 promoter can 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 can be promoters from, for example, storage tissues (e.g., seeds, potato tubers, and fruits) (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24: 275-303), or from metabolic sink tissues (e.g., meristems) (Ito et al., 1994, Plant Mol. Biol. 24: 863-878), seed-specific promoters, for example, glutelin, prolamin, globulin, or albumin promoters from rice (Wu et al., 1998, Plant Cell Physiol. 39: 885-889), the broad bean promoter from legumin B4 and an unknown seed protein gene from broad bean (Conrad et al., 1998, J. Plant Physiol. 39: 885-889). Physiol. 152:708-711), a promoter from a seed oleosin (Chen et al., 1998, Plant Cell Physiol. 39:935-941), a storage protein napA promoter from Brassica napus, or any other seed-specific promoter known in the art, for example, as described in WO 91 / 14772. In addition, 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 can be induced by abiotic treatments, such as changes in temperature, drought, or salinity, or by exogenously applied substances that activate the promoter, such as ethanol, estrogens, plant hormones (such as ethylene, abscisic acid, and gibberellic acid), and heavy metals.

[0220] Promoter enhancer elements can also be used to achieve higher expression of a polypeptide or domain in plants. For example, a promoter enhancer element can be an intron located between the promoter and the polynucleotide sequence encoding the polypeptide or domain. For example, Xu et al., 1993, supra, disclose the use of the first intron of the rice actin 1 gene to enhance expression.

[0221] The selectable marker gene and any other parts of the expression construct can be selected from those available in the art.

[0222] Nucleic acid constructs can be 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).

[0223] Agrobacterium tumefaciens-mediated gene transfer is currently a method used to generate transgenic dicots (for review, see Hooykas and Schilperoort, 1992, Plant Mol. Biol. 19:15-38) and for transformation of monocots, but other transformation methods are also commonly used for these plants. A method used to generate transgenic monocots is particle bombardment (microscopic gold or tungsten particles coated with transforming DNA) of embryonic callus 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 transforming monocotyledons 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 incorporated herein by reference in their entirety).

[0224] After transformation, transformants that have incorporated the expression construct are selected and regenerated into whole plants according to methods well known in the art. Transformation procedures are typically designed for selective elimination of the selection gene during regeneration or in subsequent generations by, for example, co-transformation with two independent T-DNA constructs or site-specific excision of the selection gene using specific recombinases.

[0225] In addition to directly transforming specific plant genotypes with constructs of the present invention, transgenic plants can also be produced by hybridizing a plant with the construct with a second plant lacking the construct. For example, a construct encoding a polypeptide or domain can be introduced into a specific plant variety by hybridization, without always directly transforming the plant of the given variety. Therefore, the present invention not only encompasses plants directly regenerated from cells transformed according to the present invention, but also encompasses the offspring of this type of plant. As used herein, offspring can refer to the offspring of any generation of the parent plant prepared according to the present invention. This type of offspring can include the DNA construct prepared according to the present invention. Hybridization results in cross-pollination of a donor plant line with a starting line, introducing a transgenic plant line. The non-limiting examples of this type of step are described in U.S. Patent number 7,151,204.

[0226] Plants can be generated by backcross transformation methods. For example, plants include plants of genotypes, lines, inbreds, or hybrids known as backcross transformations.

[0227] Can use genetic marker to assist one or more transgenics of the present invention to infiltrate another from one genetic background.The selection that mark assists provides advantage with respect to conventional breeding, is that it can be used for avoiding the mistake that causes by phenotypic variation.In addition, genetic marker can provide the data about improved germplasm relative degree in the individuation offspring of concrete hybridization.For example, when having desired proterties and having the plant of non-agronomy desired genetic background in addition and the hybridization of improved variety parent, can use genetic marker to select not only to have interested proterties, also have the offspring of relatively large ratio desired germplasm.In this way, one or more proterties are infiltrated into the required number of generations of specific genetic background and be minimized.

[0228] The present invention also relates to methods of producing a polypeptide or domain of the present invention, comprising (a) cultivating a transgenic plant or 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.

[0229] Elimination or reduction of xylanase activity

[0230] The present invention also relates to a method for producing a mutant of a parent cell, the method comprising disrupting or deleting a polynucleotide encoding a polypeptide of the present invention or a portion thereof, which results in the mutant cell producing less polypeptide than the parent cell cultured under the same conditions.

[0231] Mutant cells can be constructed using methods well known in the art by reducing or eliminating the expression of the polynucleotide, such as by inserting, destroying, replacing, or lacking. In a preferred aspect, the polynucleotide is inactivated. For example, the polynucleotide to be modified or inactivated can be a coding region or portion thereof required for activity, or a regulatory element required for expression of the coding region. Examples of such regulating or control sequences can be promoter sequences or their functional parts, i.e., parts that are sufficient to affect the expression of the polynucleotide. Other control sequences that can be modified include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, signal peptide sequences, transcription terminators, and transcriptional activators.

[0232] The modification or inactivation of the polynucleotide can be carried out by subjecting the parental cell to mutagenesis and selecting mutant cells in which the expression of the polynucleotide is reduced or eliminated. The mutagenesis can be specific or random, for example, by using a suitable physical or chemical mutagen, by using a suitable oligonucleotide, or by PCR-generated mutagenesis of the DNA sequence. In addition, mutagenesis can be carried out using any combination of these mutagens.

[0233] Examples of physical or chemical mutagens suitable for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), o-methylhydroxylamine, nitrous acid, ethylmethanesulfonic acid (EMS), sodium bisulfite, formic acid and nucleotide analogs.

[0234] When such agents are used, mutagenesis is generally performed by incubating the parental cells to be mutagenized in the presence of the chosen mutagenizing agent under appropriate conditions and screening and / or selecting for mutant cells that exhibit reduced or no expression of the gene.

[0235] The modification of these polynucleotide or inactivation can be completed by inserting, replacing or lacking one or more Nucleotide in gene or in its required regulatory element for transcription or translation.For example, thereby can insert or remove Nucleotide and form the introduction of terminator codon, the removal of start codon or the change of open reading frame.This type of modification or inactivation can be completed by the mutagenesis that site-directed mutagenesis or PCR produce according to methods known in the art.Although in principle, modification can be carried out in vivo, promptly directly on the cell of expressing polynucleotide to be modified, carry out, preferably modify in vitro as exemplified below.

[0236] The example of the method for eliminating or reducing the expression of polynucleotide conveniently is based on gene replacement, gene deletion or gene disruption technology.For example, in gene disruption method, the nucleotide sequence corresponding to endogenous polynucleotide is carried out mutagenesis in vitro to produce the nucleotide sequence of defect, then it is transformed into parental cell to produce defective gene.Through homologous recombination, the nucleotide sequence of this defect replaces endogenous polynucleotide.It is desirable that the polynucleotide of defect also encodes the mark that can be used for selecting wherein these polynucleotide are modified or destroyed transformant.On the one hand, with those selected markers such as described herein, destroy these polynucleotide.

[0237] The present invention also relates to methods of inhibiting expression of a polypeptide having xylanase activity in a cell, comprising administering to the cell or expressing in the cell a double-stranded RNA (dsRNA) molecule, wherein the dsRNA comprises a subsequence of a polynucleotide of the present invention. In a preferred aspect, the dsRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more dinucleotides in length.

[0238] The dsRNA is preferably a small interfering RNA (siRNA) or microRNA (miRNA). In a preferred aspect, the dsRNA is a small interfering RNA for inhibiting transcription. In another preferred aspect, the dsRNA is a microRNA for inhibiting translation.

[0239] The present invention also relates to such double-stranded RNA (dsRNA) molecules, including a portion of the mature polypeptide coding sequence of SEQ ID NO: 1 or SEQ ID NO: 5, for inhibiting expression of the polypeptide in a cell. While the present invention is not limited to any particular mechanism of action, the dsRNA can enter a cell and cause the degradation of single-stranded RNA (ssRNA) of similar or identical sequence, including endogenous mRNA. When cells are exposed to the dsRNA, mRNA from homologous genes is selectively degraded by a process called RNA interference (RNAi).

[0240] The dsRNA of the present invention can be used for gene silencing. In one aspect, the present invention provides methods for selectively degrading RNA using the dsRNAi of the present invention. The process can be performed in vitro, ex vivo, or in vivo. In one aspect, these dsRNA molecules can be used to generate loss-of-function mutations in cells, organs, or animals. Methods for preparing and using dsRNA molecules for selectively degrading RNA are well known in the art; see, for example, U.S. Patent Nos. 6,489,127; 6,506,559; 6,511,824; and 6,515,109.

[0241] The present invention further relates to mutant cells of a parent cell comprising a disruption or deletion in the polynucleotide encoding the polypeptide or its control sequences, or in a silent gene encoding the polypeptide, which results in the mutant cell producing less or no polypeptide compared to the parent cell.

[0242] These polypeptide-deficient mutant cells are particularly useful as host cells for the expression of native and heterologous polypeptides. Accordingly, the present invention further relates to methods for producing a native or heterologous polypeptide, comprising (a) culturing the mutant cell under conditions conducive to production of the polypeptide; and (b) recovering the polypeptide. The term "heterologous polypeptide" refers to a polypeptide that is not native to the host cell, such as a variant of a naturally occurring protein. The host cell may include more than one copy of a polynucleotide encoding the native or heterologous polypeptide.

[0243] Cultivation and purification of the desired product can be performed using methods known in the art.

[0244] The methods of the present invention for producing a substantially xylanase-free product are particularly advantageous in the production of eukaryotic polypeptides, particularly fungal proteins, such as enzymes. Xylanase-deficient cells can also be used to express heterologous proteins of pharmaceutical interest, such as hormones, growth factors, receptors, and the like. The term "eukaryotic polypeptide" includes not only native polypeptides, but also polypeptides, such as enzymes, that have been modified by amino acid substitutions, deletions, or additions, or other such modifications to enhance activity, thermostability, pH tolerance, and the like.

[0245] In another aspect, the invention relates to a protein product produced by the method of the invention that is substantially free of xylanase activity.

[0246] Fermentation broth preparation or cell composition

[0247] The present invention also relates to a fermentation broth formulation or cell composition comprising a polypeptide of the present invention. The fermentation broth product further includes other ingredients used in the fermentation process, such as, for example, cells (including host cells containing genes encoding the polypeptide of the present invention, which host cells are used to produce the polypeptide of interest), cell debris, biomass, fermentation medium and / or fermentation product. In some embodiments, the composition is a cell-killed whole broth containing one or more organic acids, killed cells and / or cell debris, and culture medium.

[0248] Term " fermented liquid " as used herein refers to the preparation of recovery and / or purification produced by cell fermentation, not experiencing or experiencing minimum.For example, when microbial culture is grown to saturation, under carbon restricted condition, hatch to allow protein synthesis (for example, by host cell, carry out the expression of enzyme) and be secreted in cell culture medium, produce fermented liquid.Fermented liquid can be included in the unfractionated or graded content of the fermentation material that obtains when fermentation ends.Typically, fermented liquid is unfractionated and comprises spent culture medium and for example, by the cell debris that exists after centrifugal removal microbial cell (for example, filamentous fungal cell).In certain embodiments, fermented liquid comprises spent cell culture medium, extracellular enzyme and energetic and / or non-vital microbial cell.

[0249] In one embodiment, the fermentation broth formulation and cell composition include a first organic acid component (including at least one 1-5 carbon organic acid and / or its salt) and a second organic acid component (including at least one 6 or more carbon organic acid and / or its salt). In a specific embodiment, the first organic acid component is acetic acid, formic acid, propionic acid, a salt thereof, or a mixture of two or more of the foregoing acids; and the second organic acid component is benzoic acid, cyclohexanecarboxylic acid, 4-methylvaleric acid, phenylacetic acid, a salt thereof, or a mixture of two or more of the foregoing acids.

[0250] In one aspect, the composition comprises one or more organic acids and optionally further comprises killed cells and / or cell debris. In one embodiment, these killed cells and / or cell debris are removed from the whole culture medium of cell killing to provide a composition without these components.

[0251] The fermentation broth formulations or cell compositions may further include a preservative and / or antimicrobial (eg, bacteriostatic) agent, including but not limited to sorbitol, sodium chloride, potassium sorbate, and other agents known in the art.

[0252] The fermentation broth formulations or cell compositions can further include enzyme activities, such as one or more (e.g., several) enzymes selected from the group consisting of cellulases, hemicellulases, esterases, expansins, laccases, ligninolytic enzymes, pectinases, peroxidases, proteases, and swollenins. The fermentation broth formulations or cell compositions can also include one or more (e.g., several) enzymes selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or a transferase, for example, an α-galactosidase, an α-glucosidase, an aminopeptidase, an amylase, a β-galactosidase, a β-glucosidase, a β-xylosidase, a carbohydrase, a carboxypeptidase, a catalase, a cellobiohydrolase, a cellulase, a chitinase, a cutinase, a cyclodextrin glucosyltransferase, a deoxyribonuclease, an endoglucanase, an esterase, a glucoamylase, an invertase, a laccase, a lipase, a mannosidase, a mutase, an oxidase, a pectinolytic enzyme, a peroxidase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, or a xylanase.

[0253] The full culture fluid of this cell killing or compositions can be included in the unfractionated content of the fermentation material that obtains when fermentation stops.Typically, the full culture fluid of this cell killing or compositions comprise spent substratum and at microbial cell (for example, filamentous fungal cell) growth to saturation, hatch under carbon restricted condition to allow protein synthesis (for example, the expression of cellulase and / or one or more beta-glucosidase enzymes) to exist afterwards cell debris.In certain embodiments, the full culture fluid of cell killing or compositions contain spent cell culture medium, extracellular enzyme and the filamentous fungal cell that kills.In certain embodiments, methods known in the art can be used to make the microbial cell permeability and / or the cracking that exist in the full culture fluid of cell killing or compositions.

[0254] The whole culture fluid or cell composition described herein is typically liquid, but may contain insoluble components, such as killed cells, cell debris, culture medium components, and / or one or more insoluble enzymes. In some embodiments, the insoluble components may be removed to provide a clarified liquid composition.

[0255] The whole culture broth formulation and cell composition of the present invention can be produced by the methods described in WO 90 / 15861 or WO 2010 / 096673.

[0256] Examples of preferred uses of the composition of the present invention are given below. The dosage of the composition and other conditions for using the composition can be determined based on methods known in the art.

[0257] Enzyme composition

[0258] The present invention also relates to compositions comprising a polypeptide of the present invention. Preferably, these compositions are enriched in this polypeptide. The term "enriched" indicates that the xylanase activity of the composition has been increased, for example, with an enrichment factor of at least 1.1.

[0259] These compositions can include polypeptides of the present invention as the main enzyme component, such as monocomponent compositions. Alternatively, these compositions can include multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of cellulases, hemicellulases, GH61 polypeptides with cellulolytic enhancing activity, esterases, expansins, laccases, lignin decomposing enzymes, pectinases, peroxidases, proteases, and swollenins. The compositions can also include one or more (e.g., several) enzymes selected from the group consisting of a hydrolase, an isomerase, a ligase, a lyase, an oxidoreductase, or a transferase, for example, an α-galactosidase, an α-glucosidase, an aminopeptidase, an amylase, a β-galactosidase, a β-glucosidase, a β-xylosidase, a carbohydrase, a carboxypeptidase, a catalase, a cellobiohydrolase, a cellulase, a chitinase, a cutinase, a cyclodextrin glucosyltransferase, a deoxyribonuclease, an endoglucanase, an esterase, a glucoamylase, an invertase, a laccase, a lipase, a mannosidase, a mutase, an oxidase, a pectinolytic enzyme, a peroxidase, a phytase, a polyphenol oxidase, a proteolytic enzyme, a ribonuclease, a transglutaminase, or a xylanase.

[0260] These compositions can be prepared according to methods known in the art and can be in the form of liquid or dry compositions. These compositions can be stabilized according to methods known in the art.

[0261] Examples of preferred uses of the composition of the present invention are given below. The dosage of the composition and other conditions for using the composition can be determined based on methods known in the art.

[0262] use

[0263] The invention is also directed to the following processes for using polypeptides having xylanase activity or compositions thereof.

[0264] The present invention also relates to methods for degrading cellulosic or xylan-containing materials, comprising treating the cellulosic or xylan-containing material with an enzyme composition in the presence of a polypeptide having xylanase activity of the present invention. In one aspect, the methods further comprise recovering the degraded cellulosic or xylan-containing material. Soluble products of degradation or conversion of the cellulosic or xylan-containing material can be separated from insoluble cellulosic or xylan-containing material using methods known in the art, such as, for example, centrifugation, filtration, or gravity settling.

[0265] The present invention also relates to methods for producing a fermentation product, comprising: (a) saccharifying a cellulosic or xylan-containing material with an enzyme composition in the presence of a polypeptide having xylanase activity of the present invention; (b) fermenting the saccharified cellulosic or xylan-containing material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

[0266] The present invention also relates to methods for fermenting a cellulosic or xylan-containing material, comprising: fermenting the cellulosic or xylan-containing material with one or more (e.g., several) fermenting microorganisms, wherein the cellulosic or xylan-containing material is saccharified with an enzyme composition in the presence of a polypeptide having xylanase activity of the present invention. In one aspect, fermenting the cellulosic or xylan-containing material produces a fermentation product. In another aspect, the methods further comprise recovering the fermentation product from the fermentation.

[0267] The methods of the present invention can be used to saccharify cellulosic materials or xylan-containing materials into fermentable sugars, and convert the fermentable sugars into a variety of useful fermentation products, such as fuels (ethanol, n-butanol, isobutanol, biodiesel, jet fuel) and / or platform compounds (e.g., acids, alcohols, ketones, gases, oils, etc.). The production of desired fermentation products from cellulosic materials or xylan-containing materials typically involves pretreatment, enzymatic hydrolysis (saccharification), and fermentation.

[0268] Treatment of cellulosic or xylan-containing material according to the present invention may be accomplished using methods conventional in the art. Furthermore, the methods of the present invention may be practiced using any conventional biomass processing equipment configured to operate in accordance with the present invention.

[0269] Separate or simultaneous hydrolysis (saccharification) and fermentation 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), sometimes also referred to as combined bioprocessing (CBP). SHF uses separate processing steps to first enzymatically hydrolyze the cellulosic material into fermentable sugars (e.g., glucose, cellobiose, and pentose monomers), and then ferment the fermentable sugars into ethanol. In SSF, enzymatic hydrolysis of cellulosic material and fermentation of sugars to ethanol are combined in a single step (Philippidis, GP, 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, ed. Wyman, CE, Taylor & Francis, Washington, DC, 179-212). SSCF involves the co-fermentation of multiple sugars (Sheehan and Himmel, 1999, Biotechnol. Prog. 15:817-827). HHF involves a separate hydrolysis step and, in addition, a simultaneous saccharification and hydrolysis step, which can be performed in the same reactor. The steps in the HHF process can be performed 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, wherein the same organism is used to produce the enzymes for converting the cellulosic material into fermentable sugars and the enzymes for converting the fermentable sugars into the final product (Linde et al., 2002, Microbiol. Mol. Biol. Reviews 66: 506-577). It should be understood that any method known in the art including pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination thereof can be used to implement the method of the present invention.

[0270] Conventional apparatuses may include a fed-batch stirred reactor, a stirred-batch reactor, a continuous flow stirred reactor with ultrafiltration, and / or a continuous plug-flow column reactor (de Castilhos Corazza et al., 2003, Acta Scientiarum. Technology 25:33-38; Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7:346-352), a milling reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. 25:53-65). Other reactor types include fluidized bed reactors, upflow blanket reactors, immobilized reactors, and extruder-type reactors for hydrolysis and / or fermentation.

[0271] Preprocessing. In practicing the processes of the present invention, any pretreatment process known in the art to disrupt the plant cell wall components of the cellulosic material or xylan-containing material can be used (Chandra et al., 2007, Adv. Biochem. Engin. / Biotechnol. 108:67-93; Galbe and Zacchi, 2007, Adv. Biochem. Engin. / Biotechnol. 108:41-65; Hendriks and Zeeman, 2009, Bioresource Technol. 100:10-18; Mosier et al., 2005, Bioresource Technol. 96:673-686; Taherzadeh and Karimi, 2008, Int. J. of Molecular Sciences). Mol. Sci. 9: 1621-1651; Yang and Wyman, 2008, Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40.

[0272] The cellulosic or xylan-containing material may also be subjected to particle size reduction, screening, pre-soaking, wetting, washing, and / or conditioning prior to pretreatment using methods known in the art.

[0273] Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkali pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organic solvent pretreatment, and biological pretreatment. Additional pretreatments include ammonia percolation, ultrasound, electroporation, microwaves, supercritical CO2, supercritical H2O, ozone, ionic liquids, and gamma irradiation pretreatment.

[0274] Can be before hydrolysis and / or fermentation, cellulosic material or the material containing xylan is pretreated.Preferably, pretreated before hydrolysis.Alternatively, can be pretreated with enzymatic hydrolysis simultaneously, to release fermentable sugars, for example glucose, xylose and / or cellobiose.In most cases, pretreatment step itself causes biomass to be converted into fermentable sugars (even when there is no enzyme).

[0275] Steam pretreatment. In steam pretreatment, the cellulosic or xylan-containing material is heated to disrupt plant cell wall components, including lignin, hemicellulose, and cellulose, making the cellulose and other fractions, such as hemicellulose, accessible to enzymes. The cellulosic or xylan-containing material is passed through or into a reactor, where steam is injected to increase the temperature to the desired temperature and pressure and to retain the cellulosic or xylan-containing material therein for the desired reaction time. Steam pretreatment is preferably carried out at temperatures between 140°C and 250°C, for example, between 160°C and 200°C or between 170°C and 190°C, with the optimal temperature range depending on the optional addition of a chemical catalyst. The residence time for steam pretreatment is preferably between 1 and 60 minutes, for example, between 1 and 30 minutes, between 1 and 20 minutes, between 3 and 12 minutes, or between 4 and 10 minutes, with the optimal residence time depending on the temperature and the optional addition of a chemical catalyst. Steam pretreatment allows for relatively high solids loadings, so that the cellulosic or xylan-containing material is typically only moist during the pretreatment process. Steam pretreatment is often combined with explosive discharge of the pretreated material, known as steam explosion, i.e., a rapid flash to atmospheric pressure and turbulence 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. 2002 / 0164730). During steam pretreatment, hemicellulose acetyl groups are cleaved, and the resulting acid autocatalyzes the partial hydrolysis of hemicellulose into monosaccharides and oligosaccharides. Lignin is removed only to a limited extent.

[0276] 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 pretreatment can convert crystalline cellulose into amorphous cellulose. Examples of suitable chemical pretreatment processes include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber / freeze expansion (AFEX), ammonia percolation (APR), ionic liquids, and organosolv pretreatment.

[0277] A chemical catalyst (e.g., H2SO4 or SO2) is sometimes added before steam pretreatment (typically 0.3% to 5% w / w), which reduces time and temperature, increases 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 H2SO4) and water to form a slurry, heated to the desired temperature by steam, and flashed to atmospheric pressure after a residence time. A variety of reactor designs can be used for dilute acid pretreatment, such as plug flow reactors, countercurrent reactors, or continuous countercurrent contracting bed reactors (Duff and Murray, 1996, supra; Schell et al., 2004, Bioresource Technology 91: 179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).

[0278] Several pretreatment methods 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 expansion (AFEX) pretreatments.

[0279] Lime pretreatment is performed with calcium oxide or calcium hydroxide at temperatures of 85° C. to 150° C. with residence times ranging from 1 hour to several days (Wyman et al., 2005, Bioresource Technology 96: 1959-1966; Mosier et al., 2005, Bioresource Technology 96: 673-686). WO 2006 / 110891, WO 2006 / 110899, WO 2006 / 110900, and WO 2006 / 110901 disclose pretreatment methods using ammonia.

[0280] Wet oxidation is a thermal pretreatment typically carried out at 180° C. to 200° C. for 5 to 15 minutes with the addition of an oxidizing agent such as hydrogen peroxide or oxygen under pressure (Schmidt and Thomsen, 1998, Bioresource Technology 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). Pretreatment is preferably carried out at 1% to 40% dry matter, for example 2% to 30% dry matter or 5% to 20% dry matter, and the initial pH is usually increased by adding a base such as sodium carbonate.

[0281] A modification of the wet oxidation pretreatment method known as wet explosion (a combination of wet oxidation and steam explosion) is capable of treating up to 30% dry matter. In wet explosion, an oxidizing agent is introduced during pretreatment after a certain residence time. The pretreatment is then terminated by flash evaporation to atmospheric pressure (WO 2006 / 03228).

[0282] Ammonia fiber expansion (AFEX) involves treating cellulosic material with liquid or gaseous ammonia for 5 to 10 minutes at moderate temperatures, such as 90° C. to 150° C., and high pressures, such as 17 to 20 bar, wherein 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 Technology 96: 2014-2018). During AFEX pretreatment, cellulose and hemicellulose remain relatively intact. The lignin-carbohydrate complex is broken down.

[0283] Organosolv pretreatment delignifies the cellulosic material by extracting it with aqueous ethanol (40%-60% ethanol) at 160°C-200°C for 30-60 minutes (Pan et al., 2005, Biotechnol. Bioeng. 90:473-481; Pan et al., 2006, Biotechnol. Bioeng. 94:851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121:219-230). Sulfuric acid is typically added as a catalyst. In organosolv pretreatment, most of the hemicellulose and lignin are removed.

[0284] Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. Biotechnol. 105-108: 69-85, and Mosier et al., 2005, Bioresource Technology 96: 673-686, and US Application No. 2002 / 0164730.

[0285] 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 such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof may also be used. The weak acid treatment is preferably carried out in a pH range of 1 to 5, e.g., 1 to 4 or 1 to 2.5. In one aspect, the acid concentration is preferably in the range of from 0.01 wt.% to 10 wt.% acid, e.g., 0.05 wt.% to 5 wt.% acid or 0.1 wt.% to 2 wt.% acid. The acid is contacted with the cellulosic material or the material containing xylan and maintained at a temperature preferably in the range of 140°C to 200°C, e.g., 165°C to 190°C, for a time in the range of from 1 to 60 minutes.

[0286] In another aspect, the pretreatment is carried out in an aqueous slurry. In a preferred aspect, the cellulosic material or xylan-containing material is present during the pretreatment in an amount preferably between 10 wt.% and 80 wt.%, for example, 20 wt.% and 70 wt.% or 30 wt.% and 60 wt.%, such as about 40 wt.%. The pretreated cellulosic material or xylan-containing material may be unwashed or washed using any method known in the art, for example, with water.

[0287] Mechanical or physical pretreatment: The term "mechanical pretreatment" or "physical pretreatment" refers to any pretreatment that promotes particle size reduction. For example, such pretreatment may involve various types of grinding or milling (e.g., dry milling, wet milling, or vibratory ball milling).

[0288] The cellulosic material or the material containing xylan can be physically (mechanically) and chemically pretreated. Mechanical or physical pretreatment can be combined with: steam / steam explosion, hydrothermolysis (hydrothermolysis), dilute acid or weak acid treatment, high temperature, high pressure treatment, radiation (such as microwave radiation) or its combination. On the one hand, high pressure means preferably about 100 to about 400psi, for example, a pressure in the range of about 150 to about 250psi. On the other hand, high temperature means a temperature in the range of about 100°C to about 300°C, for example, about 140°C to about 200°C. In a preferred aspect, mechanical or physical pretreatment uses a steam gun hydrolyzer system in a batch process, for example, from Sunds Defibrator AB, Sweden's available Sunds Hydrolyzer (Sunds Hydrolyzer) to carry out, which uses high pressure and high temperature as defined above. These physical pretreatments and chemical pretreatments can be carried out sequentially or simultaneously as needed.

[0289] Thus, in a preferred aspect, the cellulosic material or xylan-containing 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.

[0290] 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 or xylan-containing material.Biological pretreatment techniques can involve the use of lignin-solubilizing microorganisms and / or enzymes (see, e.g., Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, CE, ed., Taylor & Francis Publishing Group, Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol. 39:295-333; McMillan, JD, 1994, Pretreating lignocellulosic biomass: a review, Enzymatic Conversion of Biomass for Fuels). 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering / Biotechnology. (Engineering / Biotechnology, Scheper, T., ed., Springer-Verlag, Berlin, Heidelberg, Germany, 65: 207-241); Olsson and Hahn-Hagerdal, 1996, Enz. Microb. Tech. 18: 312-331; and Vallander and Eriksson, 1990, Progress in Biochemical Engineering / Biotechnology 42: 63-95).

[0291] saccharification In the hydrolysis step (also referred to as saccharification), the (e.g., pretreated) cellulosic material or xylan-containing material is hydrolyzed to break down cellulose and / or hemicellulose into fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or soluble oligosaccharides. The hydrolysis is enzymatically promoted by an enzyme composition in the presence of a polypeptide having xylanase activity of the present invention. The enzymes of these compositions can be added simultaneously or sequentially.

[0292] Enzyme hydrolysis is preferably carried out under the condition that is easy to be determined by those skilled in the art in a suitable aqueous environment.On the one hand, hydrolysis is carried out under the activity that is suitable for one or more enzyme components, namely under the condition that is best for these one or more enzyme components.Hydrolysis can be carried out with batch feeding or continuous process, wherein this cellulosic material or the material that contains xylan are gradually mended, for example, in the hydrolysis solution that comprises enzyme.

[0293] Saccharification is typically carried out in a stirred tank reactor or fermentor under controlled pH, temperature and mixing conditions. Suitable processing time, temperature and pH conditions can be easily determined by those skilled in the art. For example, saccharification can last up to 200 hours, but is typically carried out for preferably about 12 to about 120 hours, for example, about 16 to about 72 hours or about 24 to about 48 hours. The temperature is preferably in the range of about 25°C to about 70°C, for example, about 30°C to about 65°C, about 40°C to about 60°C or about 50°C to 55°C. The pH is preferably in the range of about 3 to about 8, for example, about 3.5 to about 7, about 4 to about 6 or about 4.0 to about 5.5. The dry solids content is in the range of about 5 to about 50wt.%, for example, about 10 to about 40wt.%, or about 20 to about 30wt.%.

[0294] These enzyme compositions can include any protein useful for degrading cellulosic or xylan-containing materials.

[0295] In one aspect, the enzyme composition includes or further includes one or more (such as, several) proteins selected from the group consisting of: cellulase, GH61 polypeptide, hemicellulase, esterase, expansin, lignin decomposing enzyme, oxidoreductase, pectinase, protease and swellin. On the other hand, the cellulase is preferably one or more (such as, several) enzymes selected from the group consisting of: endoglucanase, cellobiohydrolase and beta-glucosidase. On the other hand, the hemicellulase is preferably one or more (such as, several) enzymes selected from the group consisting of: acetylmannan esterase, acetylxylan esterase, arabinanase, arabinofuranosidase, coumaric acid esterase, ferulic acid esterase, galactosidase, glucuronidase, glucuronidase, mannanase, mannosidase, xylanase and xylosidase.

[0296] On the other hand, the enzyme composition includes one or more (e.g., several) cellulolytic enzymes. On the other hand, the enzyme composition includes or further includes one or more (e.g., several) hemicellulolytic enzymes. On the other hand, the enzyme composition includes one or more (e.g., several) cellulolytic enzymes and one or more (e.g., several) hemicellulolytic enzymes. On the other hand, the enzyme composition includes one or more (e.g., several) enzymes selected from the group of cellulolytic enzymes and hemicellulolytic enzymes. On the other hand, the enzyme composition includes endoglucanase. On the other hand, the enzyme composition includes cellobiohydrolase. On the other hand, the enzyme composition includes beta-glucosidase. On the other hand, the enzyme composition includes GH61 polypeptide. On the other hand, the enzyme composition includes endoglucanase and GH61 polypeptide. On the other hand, the enzyme composition includes cellobiohydrolase and GH61 polypeptide. On the other hand, the enzyme composition includes beta-glucosidase and GH61 polypeptide. On the other hand, the enzyme composition includes endoglucanase and cellobiohydrolase. On the other hand, the enzyme composition includes an endoglucanase and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. On the other hand, the enzyme composition includes an endoglucanase and a beta-glucosidase. On the other hand, the enzyme composition includes a beta-glucosidase and a cellobiohydrolase. On the other hand, the enzyme composition includes an endoglucanase and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. On the other hand, the enzyme composition includes an endoglucanase, a GH61 polypeptide, and a cellobiohydrolase. On the other hand, the enzyme composition includes an endoglucanase, a GH61 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. On the other hand, the enzyme composition includes an endoglucanase, a beta-glucosidase, and a GH61 polypeptide. On the other hand, the enzyme composition includes beta-glucosidase, GH61 polypeptide and cellobiohydrolase. On the other hand, the enzyme composition includes beta-glucosidase, GH61 polypeptide and cellobiohydrolase I, cellobiohydrolase II or the combination of cellobiohydrolase I and cellobiohydrolase II. On the other hand, the enzyme composition includes endoglucanase, beta-glucosidase and cellobiohydrolase. On the other hand, the enzyme composition includes endoglucanase, beta-glucosidase and cellobiohydrolase I, cellobiohydrolase II or the combination of cellobiohydrolase I and cellobiohydrolase II. On the other hand, the enzyme composition includes endoglucanase, cellobiohydrolase, beta-glucosidase and GH61 polypeptide.In another aspect, the enzyme composition comprises an endoglucanase, a beta-glucosidase, a GH61 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II.

[0297] On the other hand, the enzyme composition includes acetylmannan esterase. On the other hand, the enzyme composition includes acetylxylan esterase. On the other hand, the enzyme composition includes arabinanase (e.g., α-L-arabinanase). On the other hand, the enzyme composition includes arabinofuranosidase (e.g., α-L-arabinofuranosidase). On the other hand, the enzyme composition includes coumaric acid esterase. On the other hand, the enzyme composition includes ferulic acid esterase. On the other hand, the enzyme composition includes galactosidase (e.g., α-galactosidase and / or β-galactosidase). On the other hand, the enzyme composition includes glucuronidase (e.g., α-D-glucuronidase). On the other hand, the enzyme composition includes glucuronidase. On the other hand, the enzyme composition includes mannanase. On the other hand, the enzyme composition includes mannosidase (e.g., β-mannosidase). On the other hand, the enzyme composition includes xylanase. In a preferred aspect, the xylanase is a Family 10 xylanase. In another aspect, the enzyme composition comprises a xylosidase (eg, β-xylosidase).

[0298] In another aspect, the enzyme composition comprises an esterase. In another aspect, the enzyme composition comprises patulin. In another aspect, the enzyme composition comprises a lignin decomposing enzyme. In a preferred aspect, the lignin decomposing enzyme is a manganese peroxidase. In another preferred aspect, the lignin decomposing enzyme is a lignin peroxidase. In another preferred aspect, the lignin decomposing enzyme is a H2O2 generating enzyme. In another aspect, the enzyme composition comprises a pectinase. In another aspect, the enzyme composition comprises an oxidoreductase. In another aspect, the enzyme composition comprises a protease. In another aspect, the enzyme composition comprises an expansin.

[0299] In the methods of the present invention, the one or more enzymes may be added before or during saccharification, saccharification and fermentation, or fermentation.

[0300] One or more (for example, several) components of this enzyme composition can be a combination of native protein, recombinant protein or native protein and recombinant protein.For example, one or more (for example, several) components can be the native protein of the cell of one or more (for example, several) other components of this enzyme composition used as host cell with recombinant expression.It should be understood that recombinant protein can be heterologous (for example, exogenous) and primary for host cell.One or more (for example, several) components of enzyme composition can be generated as single component, then they are combined to form enzyme composition.Enzyme composition can be the combination of multicomponent and single component protein preparation.

[0301] The enzyme used in the method for the invention can be to exist in any form suitable for use, for example as fermentation liquid preparation or cell composition, with or without cell lysate of cell debris, semi-purified or purified enzyme preparation or as the host cell in the source of enzyme.The enzyme composition can be dry powder or granule, non-dust granule, liquid, stabilized liquid or stabilized protected enzyme.Can for example, by adding stabilizer (as sugar, sugar alcohol or other polyols) and / or lactic acid or another organic acid according to the method for having set up, liquid enzyme preparation is stabilized.

[0302] The optimal amount of enzymes and polypeptides having xylanase activity depends on several factors, including, but not limited to, the mixture of cellulolytic and / or hemicellulolytic enzymes, the cellulosic or xylan-containing material, the concentration of the cellulosic or xylan-containing material, one or more pretreatments of the cellulosic or xylan-containing material, temperature, time, pH, and the fermenting organism involved (e.g., for simultaneous saccharification and fermentation).

[0303] In one aspect, the effective amount of a cellulolytic enzyme or hemicellulolytic enzyme 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 / g of cellulosic or xylan-containing material.

[0304] In another aspect, an effective amount of a polypeptide having xylanase activity for cellulosic or xylan-containing 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 / g of cellulosic or xylan-containing material.

[0305] In another aspect, the effective amount of a polypeptide having xylanase activity for a cellulolytic enzyme 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 / g cellulolytic enzyme or hemicellulolytic enzyme.

[0306] Polypeptides having cellulolytic or hemicellulolytic enzyme activity, as well as other proteins / polypeptides useful for degrading cellulosic or xylan-containing materials (hereinafter collectively referred to as "polypeptides having enzymatic activity"), can be derived or obtained from any suitable source, including archaebacteria, bacteria, fungi, yeast, plants, or mammals. The term "obtained" herein also means that the enzyme may have been recombinantly produced in a host organism using the methods described herein, wherein the recombinantly produced enzyme is native or exogenous to the host organism, or has a modified amino acid sequence, for example, one or more (e.g., several) deletions, insertions, and / or substitutions of amino acids, i.e., the recombinantly produced enzyme is a mutant and / or fragment of a native amino acid sequence, or an enzyme produced by nucleic acid shuffling methods known in the art. Natural variants are encompassed within the meaning of native enzymes, while variants obtained, for example, by site-directed mutagenesis or shuffling are encompassed within the meaning of exogenous enzymes.

[0307] The polypeptide having enzyme activity can be a bacterial polypeptide. For example, the polypeptide can be a Gram-positive bacterial polypeptide having enzyme activity, or a Gram-negative bacterial polypeptide having enzyme activity.

[0308] The polypeptide having enzyme activity may also be a fungal polypeptide, and more preferably is a yeast polypeptide having enzyme activity or more preferably is a filamentous fungal polypeptide having enzyme activity.

[0309] Chemically modified or protein engineered mutants of polypeptides having enzymatic activity may also be used.

[0310] One or more (e.g., several) components of the enzyme composition may be recombinant components, i.e., produced by cloning a DNA sequence encoding the single component and subsequently transforming cells with the DNA sequence and expressing it in a host (see, e.g., WO 91 / 17243 and WO 91 / 17244). Preferably, the host is a heterologous host (the enzyme is heterologous to the host), but under certain conditions, the host may also be a homologous host (the enzyme is native to the host). A single-component cellulolytic protein may also be prepared by purifying such a protein from a fermentation broth.

[0311] In one aspect, the one or more (e.g., several) cellulolytic enzymes comprise commercial cellulolytic enzyme preparations. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example: CTec (Novozymes), CTec2 (Novozymes), CTec3 (Novozymes), CELLUCLAST TM (Novozymes), NOVOZYM TM 188 (Novozymes), SPEZYME TM CP (Genencor Int.), ACCELERASE TM TRIO (DuPont), NL (DSM); S / L 100 (DSM), ROHAMENT TM 7069 W(Rohm Corporation( GmbH)), or CMAX3 TM (Dyadic International, Inc.) An effective amount of cellulase is added from about 0.001 to about 5.0 wt. % of solids, such as about 0.025 to about 4.0 wt. % of solids, or about 0.005 to about 2.0 wt. % of solids.

[0312] Examples of bacterial endoglucanases that can be used in the methods of the present invention include, but are not limited to, Acidothermus cellulolyticus endoglucanase (WO 91 / 05039; WO 93 / 15186; U.S. Patent Application No. 5,275,944; WO 96 / 02551; U.S. Patent Application No. 5,536,655, WO 00 / 70031, WO 05 / 093050), Erwinia carotovara endoglucanase (Saarilahti et al., 1990, Gene 90:9-14), Thermobifida fusca endoglucanase III (WO 05 / 093050), and Thermobifida fusca endoglucanase V (WO 05 / 093050).

[0313] Examples of fungal endoglucanases that can be used in the present invention include, but are not limited to: Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45:253-263, Trichoderma reesei Cel7B endoglucanase I (GenBank: M15665); Trichoderma reesei endoglucanase II (Saloheimo et al., 1988, Gene 63:11-22), Trichoderma reesei C ... e15A endoglucanase II (GenBank: M19373); Trichoderma reesei endoglucanase III (Okada et al., 1988, Appl. Environ. Microbiol. 64:555-563, GenBank: AB003694); Trichoderma reesei endoglucanase V (Saloheimo et al., 1994, Molecular Microbiology). endoglucanase (GenBank: L29381); endoglucanase from Fusarium oxysporum (GenBank: AB003107); endoglucanase from Aspergillus aculeatus (Ooi et al., 1990, Nucleic Acids Research 18: 5884); endoglucanase from Aspergillus kawachii (Sakamoto et al., 1995, Current Genetics 27: 435-439); endoglucanase from Fusarium oxysporum (GenBank: L29381); endoglucanase from Humicola grisea var. thermoidea (GenBank: AB003107); endoglucanase from Melanocarpus thunbergii (GenBank: AB003107); endoglucanase from Aspergillus niger ... albomyces) endoglucanase (GenBank: MAL515703); Neurospora crassa endoglucanase (GenBank: XM_324477); Humicola insolens endoglucanase V; Myceliophthora thermophila CBS 117.65 endoglucanase; Thermoascus aurantiacus endoglucanase I (GenBank: AF487830) and Trichoderma reesei strain number VTT-D-80133 endoglucanase (GenBank: M15665).

[0314] Examples of cellobiohydrolases that can be used 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), Penicillium occitanis cellobiohydrolase I (GenBank: AY690482), Talaromyces emersonii cellobiohydrolase I (GenBank: AF439936), Thielavia hyrcanie cellobiohydrolase II (WO 2010 / 141325), Thielavia terrestris cellobiohydrolase II (CEL6A, WO 2010 / 141327), and Thielavia terrestris cellobiohydrolase II (CEL6A, WO 2010 / 141329). 2006 / 074435), Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, and Trichophaea saccharosporium cellobiohydrolase II (WO 2010 / 057086).

[0315] Examples of β-glucosidases suitable for use in the present invention include, but are not limited to, β-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 02 / 095014), Penicillium brasiliensis IBT 20888 (WO 2007 / 019442 and WO 2010 / 088387), Thielavia terrestris (WO 2011 / 035029), and Trichophyton saccharomyces chromis (WO 2007 / 019442).

[0316] Other useful endoglucanases, cellobiohydrolases, and β-glucosidases are disclosed in a number of glycosyl hydrolase families using the classification according to Henrissat, 1991, Biochem. J. 280:309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316:695-696.

[0317] In the methods of the present invention, any GH61 polypeptide having cellulolytic enhancing activity can be used as a component of the enzyme composition.

[0318] Examples of GH61 polypeptides that can be used in the methods of the invention include, but are not limited to, GH61 polypeptides from Thielavia terrestris (WO 2005 / 074647, WO 2008 / 148131, and WO 2011 / 035027), Thermoascus aureus (WO 2005 / 074656 and WO 2010 / 065830), Trichoderma reesei (WO 2007 / 089290), Myceliophthora thermophila (WO 2009 / 085935, WO 2009 / 085859, WO 2009 / 085864, and WO 2009 / 085868), Aspergillus fumigatus (WO 2010 / 138754), Penicillium pinophilum (WO 2011 / 005867), Thermoascus sp. (WO 2011 / 005868), 2011 / 039319), Penicillium (WO 2011 / 041397), Thermoascus crustaceous (WO 2011 / 041504), Aspergillus aculeatus (WO 2012 / 125925), Thermomyces lanuginosus (WO 2012 / 113340, WO 12 / 129699, and WO 2012 / 130964), Aurantiporus alborubescens (WO 2012 / 122477), Trichoderma sphenosporum (WO 2012 / 122477), Penicillium thomsoni (WO 2012 / 122477), Talaromyces stalked (WO 2012 / 135659), Humicola insolens (WO 2012 / 146171), Cladosporium camphora (WO 2012 / 101206), Talaromyces leycettanus (WO 2012 / 101206), and Chaetomium thermophilum (WO 2012 / 101206).

[0319] In one aspect, the GH61 polypeptide is used in the presence of a soluble activating divalent metal cation according to WO 2008 / 151043, such as manganese or copper.

[0320] On the other hand, the GH61 polypeptide is used in the presence of a dioxygen compound, a bicyclic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquid obtained from a pretreated cellulosic material, such as pretreated corn stover (WO 2012 / 021394, WO 2012 / 021395, WO 2012 / 021396, WO 2012 / 021399, WO 2012 / 021400, WO 2012 / 021401, WO 2012 / 021408, and WO 2012 / 021410).

[0321] In one aspect, the compound is added in a molar ratio of about 10 to about 20 glucosyl units of the cellulose. -6 to about 10, for example about 10 -6 to about 7.5, about 10 -6 to about 5, about 10 -6 to about 2.5, about 10 -6 to about 1, about 10 -5 to about 1, about 10 -5 to about 10 -1 , about 10 -4 to about 10 -1 , about 10 -3 to about 10 -1 , or about 10 -3 to about 10 -2 In another aspect, an effective amount of such a compound is from about 0.1 μM to about 1 M, e.g., from about 0.5 μM to about 0.75 M, from about 0.75 μM to about 0.5 M, from about 1 μM to about 0.25 M, from about 1 μM to about 0.1 M, from about 5 μM to about 50 mM, from about 10 μM to about 25 mM, from about 50 μM to about 25 mM, from about 10 μM to about 10 mM, from about 5 μM to about 5 mM, or from about 0.1 mM to about 1 mM.

[0322] The term "liquor" means, under conditions as described herein, a solution phase (aqueous phase, organic phase or a combination thereof) and its soluble contents produced by treating the lignocellulose and / or hemicellulose materials in the slurry, or its monosaccharide (e.g., xylose, arabinose, mannose, etc.). The cellulose decomposition-enhancing liquor for GH61 polypeptides can be prepared by, optionally in the presence of a catalyst (e.g., acid), optionally in the presence of an organic solvent, and optionally in combination with a lignocellulose or hemicellulose material (or raw material) that is physically destroyed, treating the material by applying heat and / or pressure, and then separating the solution from the residual solids to produce the result. In the hydrolysis process of cellulose substrates by a cellulase preparation, the degree of cellulose decomposition enhancement that can be obtained from the combination of liquor and GH61 polypeptides is determined by such conditions. The standard methods of this area can be used, such as filtration, precipitation or centrifugation, and the liquor is separated from the treated material.

[0323] In one aspect, the effective amount of liquid to cellulose is about 10 -6 to about 10 g / g of cellulose, for example about 10 -6 to about 7.5g, about 10 -6 to about 5g, about 10 -6 to about 2.5g, about 10 -6 to about 1g, about 10 -5 to about 1g, about 10 -5 to about 10-1 g, about 10 -4 to about 10 -1 g, about 10 -3 to about 10 -1 g, or about 10 -3 to about 10 -2 g / g of cellulose.

[0324] In one aspect, the one or more (eg, 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), HTec (Novozymes), HTec2 (Novozymes), HTec3 (Novozymes), (Novozymes), (Novozymes), HC (Novozymes), Xylanase (Genencor), XY (Genencor), XC (Genencor), TX-200A (AB Enzymes), HSP 6000 xylanase (DSM), DEPOL TM 333P (Biocatalysts Limit, Wales, UK), DEPOL TM 740L (Biocatalysts Ltd, Wales, UK) and DEPOL TM 762P (Biocatalysts Ltd, Wales, UK), ALTERNAFUEL 100P (Dyadic), and ALTERNAFUEL 200P (Dyadic).

[0325] Examples of xylanases useful in the methods of the 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), Thermomyces lanuginosus GH11 (WO 2012 / 130965), Talaromyces thermophilus GH11 (WO 2012 / 13095), Thielavia terrestris NRRL 8126 (WO 2009 / 079210), and Trichophaea saccharina GH10 (WO 2011 / 057083).

[0326] β-Xylosidases useful in the methods of the invention include, but are not limited to, β-xylosidases from Neurospora crassa (SwissProt: Q7SOW4), Trichoderma reesei (UniProtKB / TrEMBL: Q92458), Talaromyces emersonii (SwissProt: Q8X212), and Talaromyces thermophilus GH11 (WO 2012 / 130965).

[0327] Examples of acetylxylan esterases useful in the methods of the invention include, but are not limited to, acetylxylan esterases from Aspergillus aculeatus (WO 2010 / 108918), Chaetomium globosum (UniProt: Q2GWX4), Chaetomium gracile (GeneSeqP: AAB82124), Humicola insolens DSM 1800 (WO 2009 / 073709), Hypocrea jecorina (WO 2005 / 001036), Myceliophtera thermophila (WO 2010 / 014880), Neurospora crassa (UniProt: q7s259), Phaeosphaeria nodorum (UniProt: Q0UHJ1), and Thielavia terrestris NRRL 8126 (WO 2009 / 042846).

[0328] Examples of feruloyl esterases (ferulic acid esterases) useful in the methods of the present invention include, but are not limited to, feruloyl esterases from Humicola insolens DSM 1800 (WO 2009 / 076122), Neosartorya fischeri (UniProt: A1D9T4), Neurospora crassa (UniProt: Q9HGR3), Penicillium aurantiogriseum (WO 2009 / 127729), and Thielavia terrestris (WO 2010 / 053838 and WO 2010 / 065448).

[0329] Examples of arabinofuranosidases useful in the methods of the invention include, but are not limited to, arabinofuranosidases from Aspergillus niger (GeneSeqP: AAR94170), Humicola insolens DSM 1800 (WO 2006 / 114094 and WO 2009 / 073383), and M. giganteus (WO 2006 / 114094).

[0330] Examples of α-glucuronidases useful in the methods of the present invention include, but are not limited to, α-glucuronidases from Aspergillus clavatus (UniProt: alcc12), Aspergillus fumigatus (SwissProt: Q4WW45), Aspergillus niger (UniProt: Q96WX9), Aspergillus terreus (SwissProt: Q0CJP9), Humicola insolens (WO 2010 / 014706), Penicillium chrysogenum (WO 2009 / 068565), Talaromyces emersonii (UniProt: Q8X211), and Trichoderma reesei (UniProt: Q99024).

[0331] The polypeptides having enzymatic activity used in the methods of the invention can be produced by fermenting the above-mentioned 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, California, 1991). Suitable culture media are available from commercial suppliers or can be prepared according to published compositions (e.g., in the catalog 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, New York, 1986).

[0332] Fermentation can be any method of the cultured cells that causes the expression of enzyme or protein or separation.So, fermentation can be understood to comprise shake flask culture, or in a kind of applicable substratum and under the condition that allows expression or separation of this enzyme, carry out small-scale or large-scale fermentation (comprising continuous fermentation, batch fermentation, batch fed fermentation or solid-state fermentation) in laboratory or industrial fermentor tank.The gained enzyme produced by said method can be reclaimed from fermentation medium and by conventional procedure purification.

[0333] Fermentation . The fermentable sugars obtained from the hydrolyzed cellulosic material or xylan-containing material can be fermented by one or more (e.g., several) fermenting microorganisms that are capable of fermenting the sugars directly or indirectly into the desired fermentation product. "Fermentation" or "fermentation process" refers to any fermentation process or any process that includes a fermentation step. Fermentation methods also include fermentation methods for the consumable alcohol industry (e.g., beer and wine), the dairy industry (e.g., fermented dairy products), the leather industry, and the tobacco industry. Fermentation conditions depend on the desired fermentation product and the fermenting organism, and can be easily determined by one of ordinary skill in the art.

[0334] In the fermentation step, sugars released from the cellulosic material or xylan-containing material as a result of the pretreatment and enzymatic hydrolysis steps are fermented by a fermenting organism (such as yeast) to a product, for example, ethanol. The hydrolysis (saccharification) and fermentation can be separate or simultaneous.

[0335] Any suitable hydrolyzed cellulosic material may be used in practicing the fermentation step of the present invention. The material is generally selected based on economics, ie, cost per equivalent sugar potential, and recalcitrance to enzymatic conversion.

[0336] The term "fermentation medium" is understood herein to mean the culture medium before the addition of one or more fermenting microorganisms, eg, the culture medium resulting from a saccharification process, as well as the culture medium used in a simultaneous saccharification and fermentation process (SSF).

[0337] "Fermenting microorganism" refers to any microorganism suitable for a desired fermentation process to produce a fermentation product, including bacteria and fungal organisms. The fermenting organism can be a hexose and / or pentose fermenting organism, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art. Suitable fermenting microorganisms are capable of fermenting (i.e., converting) 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 that produce ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

[0338] Examples of fermenting microorganisms capable of fermenting hexoses include bacterial organisms and fungal organisms, such as yeast. Yeast includes strains of Candida, Kluyveromyces, and Saccharomyces, such as Candida sonorensis, Kluyveromyces marxianus, and Saccharomyces cerevisiae.

[0339] Examples of fermentative microorganisms that can ferment pentoses in their native state include bacteria and fungal organisms, such as a certain yeast. Yeasts that ferment xylose include strains of the genus Candida, preferably C. sheatae or C. sonorensis; and strains of the genus Pichia, such as Pichia stipitis, like Pichia stipitis CBS 5773. Yeasts that ferment pentoses include strains of the genus Pachysolensis, preferably P. tannophilus. Organisms that cannot ferment pentoses (such as xylose and arabinose) can be genetically modified and ferment pentoses by methods known in the art.

[0340] Examples of bacteria that can efficiently ferment hexose and pentose sugars to ethanol include, e.g., Bacillus coagulans, Clostridium acetobutylicum, Clostridium thermocellum, Clostridium phytofermentans, Geobacillus species, Thermoanaerobacter saccharolyticum, and Zymomonas mobilis (Filippidis, 1996, supra).

[0341] Other fermenting organisms include strains of Bacillus, such as B. coagulans; Candida, such as C. sanarici, C. methanosorbosa, C. diddensiae, Candida parapsilosis, C. naedodendra, C. blankii, C. entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C. utilis, and C. shehatae; Clostridium, such as C. acetobutylicum, C. thermocellum, and C. utilis; Escherichia coli, particularly strains of E. coli that have been genetically modified to improve ethanol production; Geobacillus species; Hansenula, such as Hansenula anomala. 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 saccharolyticus; and Zymomonas, such as Zymomonas mobilis.

[0342] Commercially available yeast suitable for ethanol production include, for example, BIOFERM TMAFT and XR (NABC - North American Bioproducts Corporation, Georgia, USA), ETHANOL RED TM Yeast (Fermentis / Lesaffre, USA), FALI TM (Fleischmann's Yeast, USA), FERMIOL TM (DSM Specialties), GERT STRAND TM (GertStrand AB, Sweden), and SUPERSTART TM and THERMOSACC TM Fresh yeast (Ethanol Technology, Wisconsin, USA).

[0343] In one aspect, the fermenting microorganism has been genetically modified to provide the ability to ferment pentose sugars, such as xylose-utilizing microorganisms, arabinose-utilizing microorganisms, and xylose- and arabinose-co-utilizing microorganisms.

[0344] Cloning of heterologous genes into a variety of fermenting microorganisms has resulted in the construction of organisms capable of converting both hexose and pentose sugars to ethanol (co-fermentation) (Chen and Ho, 1993, Appl. Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Appl. Environ. Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995, Appl. Environ. Microbiol. 61: 4184-4190; Kuyper et al., 2004, FEMS Yeast Biol. (Biotech. Bioeng. 38:296-303; Ingram et al., 1998, Biotech. Bioeng. 58:204-214; Zhang et al., 1995, Science 267:240-243; Deanda et al., 1996, Applied and Environmental Microbiology 62:4465-4470; WO 2003 / 062430).

[0345] It is well known in the art that the organisms described above can also be used to produce other substances, as described herein.

[0346] Typically, a fermentative microorganism is added to the degraded cellulosic material or hydrolysate, and the fermentation is carried out for about 8 to about 96 hours, such as about 24 to about 60 hours. The temperature is typically between about 26° C. and about 60° C., such as about 32° C. or 50° C., and the pH is about pH 3 to about pH 8, such as pH 4 to 5, 6, or 7.

[0347] In one aspect, yeast and / or another microorganism is applied to the degraded cellulosic material and fermented for about 12 to about 96 hours, such as typically 24-60 hours. In another aspect, the temperature is preferably between about 20°C to about 60°C, such as about 25°C to about 50°C, about 32°C to about 50°C, or about 32°C to about 50°C, and the pH is typically from about pH 3 to about pH 7, such as about pH 4 to about pH 7. However, some fermenting organisms (such as bacteria) have higher optimum fermentation temperatures. Yeast or another microorganism is preferably used at about 10% by volume per ml of fermentation broth. 5 to 10 12, preferably from about 10 7 to 10 10 , especially about 2×10 8 Further guidance on fermentation using yeast can be found, for example, in "The Alcohol Textbook" (K. Jacques, TP Lyons and DR Kelsall, eds., Nottingham University Press, United Kingdom 1999), which is incorporated herein by reference.

[0348] Fermentation stimulants can be used in combination with any method described herein to further improve the fermentation process, particularly to improve the performance of fermenting microorganisms, such as, to increase speed and ethanol yield. " Fermentation stimulant " refers to a stimulant for the growth of fermenting microorganisms (particularly yeast). Preferred fermentation stimulants for growth include vitamins and minerals. The example of vitamin includes multivitamins, biotin, pantothenic acid, nicotinic acid, meso-inositol, thiamine, pyridoxine, p-aminobenzoic acid, folic acid, riboflavin and vitamins A, B, C, D and E. For example, see Alfredo et al., Improving ethanol production and viability of Saccharomyces cerevisia by a vitamin feeding strategy during fed-batch process by improving ethanol production and the viability of saccharomyces cerevisia in a vitamin feeding strategy during fed-batch process, Springer (2002), which is incorporated herein by reference. The example of mineral includes minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn and Cu.

[0349] Fermented products:Fermentation products can be any substance obtained by fermentation. Fermentation products can be, without limitation, alcohols (e.g., arabitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol (propylene glycol), butanediol, glycerol, sorbitol, and xylitol); alkanes (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane); cycloalkanes (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane); olefins (e.g., pentene, hexene, heptene, and octene); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, glutamic acid ... , serine, and threonine); gases (e.g., methane, hydrogen (H2), carbon dioxide (CO2), and carbon monoxide (CO)); isoprene; ketones (e.g., acetone); organic acids (e.g., acetic acid, acetic 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 polyketides. The fermentation product can also be a protein as a high-value product.

[0350] In a preferred aspect, the fermentation product is an alcohol. It should be understood that the term "alcohol" encompasses substances containing one or more hydroxyl moieties. The alcohol can be, but is not limited to, n-butanol, isobutanol, ethanol, methanol, arabitol, butanediol, ethylene glycol, glycerin, glycerol, 1,3-propylene glycol, sorbitol, and xylitol. See, e.g., Gong et al., 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, Appl. Microbiol. Biotechnol. 59: 400-408; Nigam and Singh, 1995, Process Biochemistry 30(2): 117-124; Ezeji et al., 2003, World Journal of Microbiology and Biotechnology 19(6): 595-603.

[0351] In another preferred aspect, the fermentation product is an alkane. The alkane can be an unbranched or branched alkane. The alkane can be, but is not limited to, pentane, hexane, heptane, octane, nonane, decane, undecane, or dodecane.

[0352] In another preferred aspect, the fermentation product is a cycloalkane. The cycloalkane can be, but is not limited to, cyclopentane, cyclohexane, cycloheptane or cyclooctane.

[0353] In another preferred aspect, the fermentation product is an olefin. The olefin can be an unbranched or branched olefin. The olefin can be, but is not limited to, pentene, hexene, heptene, or octene.

[0354] In another preferred aspect, the fermentation product is an amino acid. The organic acid can be, but is not limited to, aspartic acid, glutamic acid, glycine, lysine, serine, or threonine. See, for example, Richard and Margaritis, 2004, Biotechnology and Bioengineering 87(4):501-515.

[0355] In another preferred aspect, the fermentation product is a gas. The gas can be, but is not limited to, methane, H2, CO2, or CO. See, for example, Kataoka et al., 1997, Water Science and Technology 36(6-7):41-47; and Gunaseelan, 1997, Biomass and Bioenergy 13(1-2):83-114.

[0356] In another preferred aspect, the fermentation product is isoprene.

[0357] In another preferred aspect, the fermentation product is a ketone. It should be understood that the term "ketone" encompasses substances containing one or more ketone moieties. The ketone may be, but is not limited to, acetone.

[0358] In another preferred aspect, the fermentation product is an organic acid. The organic acid can be, but is not limited to, acetic acid, acetic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, or xylonic acid. See, for example, Chen and Lee, 1997, Biochem. Biotechnol. 63-65: 435-448.

[0359] In another preferred aspect, the fermentation product is a polyketide.

[0360] Recycle One or more fermentation products can optionally be 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, the alcohol can be separated and purified from the fermented cellulosic material by conventional distillation methods. Ethanol having a purity of up to about 96% by volume can be obtained, which can be used, for example, as fuel ethanol, drinking ethanol, i.e., a drinkable neutral spirit, or industrial ethanol.

[0361] signal peptide

[0362] The present invention also relates to an isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 23 of SEQ ID NO: 2, or comprising or consisting of amino acids 1 to 23 of SEQ ID NO: 6. The polynucleotide may further comprise a gene encoding a protein 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 156 of SEQ ID NO: 1. In another aspect, the polynucleotide encoding the signal peptide is nucleotides 1 to 150 of SEQ ID NO: 5.

[0363] The present invention also relates to nucleic acid constructs, expression vectors and recombinant host cells comprising these polynucleotides.

[0364] The present invention also relates to methods of producing a protein, comprising (a) cultivating a recombinant host cell comprising such a polynucleotide; and optionally (b) recovering the protein.

[0365] The protein may be native or heterologous to the host cell. The term "protein" is not intended to refer to a specific length of an encoded product and therefore encompasses peptides, oligopeptides, and polypeptides. The term "protein" also encompasses two or more polypeptides that combine to form the encoded product. These proteins also include hybrid polypeptides and fusion polypeptides.

[0366] Preferably, the protein is a hormone, enzyme, receptor or portion thereof, antibody or portion thereof, or reporter gene. For example, the protein can be a hydrolase, isomerase, ligase, lyase, oxidoreductase, or transferase, such as α-galactosidase, α-glucosidase, aminopeptidase, amylase, β-galactosidase, β-glucosidase, β-xylosidase, saccharidase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

[0367] The gene may be obtained from any prokaryotic, eukaryotic or other source.

[0368] The present invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0369] Examples

[0370] strain

[0371] Rasamsonia byssochlamydoides strains CBS 413.71 and CBS 150.75 were used as sources of GH10 polypeptides having xylanase activity. Aspergillus oryzae strain MT3568 was used to express Rasamsonia byssochlamydoides genes encoding polypeptides having xylanase activity. Aspergillus oryzae MT3568 is an amdS (acetamidase)-disrupted derivative of Aspergillus oryzae JaL355 (WO 02 / 40694), in which the pyrG auxotrophy was restored by disruption of the Aspergillus oryzae acetamidase (amdS) gene.

[0372] Culture media and solutions

[0373] COVE sucrose plates were prepared from 342 g of sucrose, 20 g of agar powder, 20 ml of COVE salt solution, and deionized water to 1 liter. The culture medium was sterilized by autoclaving at 15 psi for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). The culture medium was cooled to 60°C and supplemented with 10 mM acetamide, 15 mM CsCl, and Triton X-100 (50 μl / 500 ml).

[0374] The COVE salt solution was composed of 26 g of MgSO4.7H2O, 26 g of KCl, 26 g of KH2PO4, 50 ml of COVE trace metal solution, and deionized water to make up to 1 liter.

[0375] The COVE trace metal solution consists of 0.04 g of Na2B4O7.10H2O, 0.4 g of CuSO4.5H2O, 1.2 g of FeSO4.7H2O, 0.7 g of MnSO4.H2O, 0.8 g of Na2MoO4.2H2O, 10 g of ZnSO4.7H2O, and deionized water to make up to 1 liter.

[0376] Dap-4C medium consists of 20 g of dextrose, 10 g of maltose, 11 g of MgSO4·7H2O, 1 g of KH2PO4, 2 g of citric acid, 5.2 g of K3PO4·H2O, 0.5 g of yeast extract (Difco), 1 ml of defoamer, 0.5 ml of KU6 trace metal solution, 2.5 g of CaCO3, and deionized water to 1 liter. This medium is sterilized by autoclaving at 15 psi for 15 minutes (Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Prior to use, 3.5 ml of sterile 50% (NH4)2HPO4 and 5 ml of sterile 20% lactic acid are added to every 150 ml of Dap-4C medium.

[0377] The KU6 trace metal solution is composed of 0.13 g of NiCl2, 2.5 g of CuSO4.5H2O, 13.9 g of FeSO4.7H2O, 8.45 g of MnSO4.H2O, 6.8 g of ZnCl2, 3 g of citric acid, and deionized water to make up to 1 liter.

[0378] LB plates were made up of 10 g of Bacto-Tryptone, 5 g of yeast extract, 10 g of sodium chloride, 15 g of Bacto-agar, and deionized water added to 1 liter. The culture medium was sterilized by autoclaving at 15 psi for 15 minutes (Bacteriological Analytical Manual, 8th edition, Revision A, 1998).

[0379] PDA agar plate is made of potato extract, and this potato extract is by the potato (through washing but not peeling) of 300g slices being boiled in water for 30 minutes, and then this culture fluid is decanted or filtered through cheesecloth and is made.Then add distilled water, until the cumulative volume of suspension is one liter, add the dextrose of 20g and the agar powder of 20g subsequently.This culture medium is sterilized (Bacteriological Analytical Manual (Bacteriological Analytical Manual), the 8th edition, revision A, 1998) by autoclaving under 15psi for 15 minutes.

[0380] YP+2% glucose medium consists of 1% yeast extract, 2% peptone, and 2% glucose in deionized water.

[0381] Example 1: Sources of DNA sequence information for Rasamsonia byssochlamydoides CBS 413.71

[0382] The genomic DNA of Rasamsonia byssochlamydoides CBS 413.71 was sequenced by Illumina DNA sequencing in Plan-les-Ouates, Switzerland. The preliminary assembly of the genome was analyzed using GeneMark v2.3c (Georgia Tech's Center for Bioinformatics and Computational Genomics, Atlanta, Georgia, U.S.). The gene model constructed by the software was used as the starting point for detecting the GH10 homologues in the genome. A more accurate gene model was constructed using a plurality of known GH10 protein sequences as guides.

[0383] Example 2: Genomic DNA extraction from Rasamsonia byssochlamydoides CBS 413.71 and CBS150.75

[0384] To obtain genomic DNA for PCR amplification, Rasamsonia byssochlamydoides CBS413.71 was propagated on PDA agar plates by growing for 7 days at 26° C. Spores harvested from the PDA plates were used to inoculate 25 ml of YP + 2% glucose medium in baffled shake flasks and incubated at 26° C. for 72 hours while stirring at 85 rpm.

[0385] To obtain genomic DNA for PCR amplification, Rasamsonia byssochlamydoides CBS150.75 was grown on PDA agar plates by growing for 7 days at 26° C. 50 mg of fungal material was harvested by scraping the surface of the PDA agar plates with a scalpel.

[0386] According to the modified Plant Maxi Kit (Qiagen Danmark, Copenhagen, Denmark) will be separated from the genomic DNA of Rasamsonia byssochlamydoides CBS 413.71. By centrifuging for 2 minutes at 14,000xg, the fungal material from the above culture was harvested. The supernatant was removed and the pellet (0.5g) was frozen in liquid nitrogen with quartz sand and ground into a fine powder in a precooled mortar. The powder was transferred to a 15ml centrifuge tube and 5ml was added to an AP1 buffer (Qiagen Danmark, Copenhagen, Denmark) and 10 μl of RNase A stock solution (100mg / ml) that was preheated to 65°C, followed by a vigorous vortex. After incubation at 65°C for 10 minutes using a regular inverting tube, 1.8 ml of AP2 buffer (Qiagen Denmark, Copenhagen, Denmark) was added to the lysate with gentle mixing and then incubated on ice for 10 minutes. The lysate was then centrifuged at 3000 x g for 5 minutes at room temperature and the supernatant was decanted into a QIASHREDDER placed in a 50 ml collection tube. TM The sample was placed in a Maxi Spin Column (QIAGEN Denmark, Copenhagen, Denmark) and centrifuged at 3000 x g for 5 minutes at room temperature. The flow-through was transferred to a new 50 ml tube and 1.5 volumes of AP3 / E buffer (QIAGEN Denmark, Copenhagen, Denmark) were added followed by vortexing. Fifteen milliliters of sample was transferred to a 50 ml collection tube in a 50 ml tube. The precipitate was added to a large nucleic acid purification column and centrifuged at 3000 x g for 5 minutes at room temperature. The flow-through was discarded and transferred to a 50 ml collection tube. 12 ml of AW buffer (Qiagen Denmark, Copenhagen, Denmark) was added to the large nucleic acid purification column and centrifuged at 3000 x g for 10 minutes at room temperature. After discarding the flow-through, the centrifugation was repeated to dispose of the remaining alcohol. The large nucleic acid purification column was transferred to a new 50 ml test tube and 0.5 ml of AE buffer (Qiagen Denmark, Copenhagen, Denmark) preheated to 70° C. was added. After incubation at room temperature for 5 minutes, the sample was eluted by centrifugation at room temperature for 5 minutes at 3000×g. The elution was repeated with another 0.5 ml of AE buffer and the eluate was combined. The concentration of the DNA obtained was measured at 260 nm using a UV spectrophotometer.

[0387] Using Maxwell 16 instruments according to Maxwell Genomic DNA from Rasamsonia byssochlamydoides CBS 150.75 was isolated using the 16 DNA purification kit protocol (TH. Geyer Danmark Aps, Roskilde, Denmark). The genomic DNA was eluted with 300 μl of elution buffer. The concentration of the harvested DNA was measured using a UV spectrophotometer at 260 nm.

[0388] Example 3: Construction of an Aspergillus oryzae expression vector containing the genomic sequence of Rasamsonia byssochlamydoides strain CBS 413.71 encoding a family GH10 polypeptide having xylanase activity

[0389] Two synthetic oligonucleotide primers shown below were designed to PCR amplify the Rasamsonia byssochlamydoides CBS 413.71 P24GTR gene (SEQ ID NO: 1) and the Rasamsonia byssochlamydoides CBS 150.75 P34RRZ gene (SEQ ID NO: 5) from the genomic DNA prepared in Example 2. TM The fragment was directly cloned into the expression vector pDau109 (WO 2005 / 042735) using the BIOMEDIC cloning kit (BD Biosciences, Palo Alto, CA, USA).

[0390] Primer F-P24GTR

[0391]

[0392] Primer R-P24GTR

[0393] Bold letters represent gene sequences. The underlined sequences are homologous to the insertion site of pDau109.

[0394] use High-fidelity PCR kit ( High-Fidelity PCR Kit (Finnzymes Oy, Espoo, Finland) was used for PCR amplification. PCR consisted of 5 μl of 5X HF buffer (Finnzymes Oy, Espoo, Finland), 0.5 μl of dNTP (10 mM), 0.5 μl of DNA polymerase (0.2 units / μl) (Fazem, Espoo, Finland), 2 μl of primer F-P24GTR (2.5 μM), 2 μl of primer R-P24GTR (2.5 μM), 0.5 μl of Rasamsonia byssochlamydoides genomic DNA (100 ng / μl) and 14.5 μl of deionized water were used in a total volume of 25 μl. PCR was performed using a PTC-200 DNA engine (MJ Research, Waltham, MA, USA) with a program of 1 cycle at 95°C for 2 minutes; 35 cycles of 10 seconds at 98°C, 30 seconds at 60°C, and 2.5 minutes at 72°C; and 1 cycle at 72°C for 10 minutes. The sample was then kept at 12°C until removed from the PCR machine.

[0395] The PCR products were separated by 1.0% agarose gel electrophoresis using 40 mM Tris base, 20 mM sodium acetate, 1 mM disodium EDTA (TAE) buffer, where the 1575 bp product band was excised from the gel and analyzed using ILLUSTRA TM PCR DNA was purified using the Gel Band Purification Kit (GE Healthcare Life Sciences, Brondby, Denmark). TMThe fragments were cloned into Bam HI and Xho I degraded pDau109 using a cloning kit according to the manufacturer's instructions, generating plasmids pP24GTR and pP34RRZ. Cloning the P24GTR and P34RRZ genes into Bam HI-Xho I degraded pDau109 resulted in transcription of the Rasamsonia byssochlamydoides P24GTR or P34RRZ gene under the control of the NA2-tpi promoter. The NA2-tpi promoter is a modified promoter from the gene encoding Aspergillus niger neutral α-amylase, in which the untranslated leader sequence has been replaced with the untranslated leader sequence from the gene encoding Aspergillus nidulans triose phosphate isomerase.

[0396] Plasmids pP24GTR and pP34RRZ were transformed into One TOP10F' chemically competent E. coli cells (Invitrogen, Carlsbad, CA, USA) were plated on LB plates supplemented with 0.1 mg of ampicillin / ml. After incubation at 37°C overnight, colonies were observed growing under selection on the plates. Two colonies transformed with the P24GTR GH10 construct were cultured in LB medium supplemented with 0.1 mg of ampicillin / ml and stained with QIAPREP according to the manufacturer's protocol. Plasmids were isolated using the SpinMiniprep kit (Qiagen, Valencia, CA, USA).

[0397] The isolated plasmids were sequenced using vector primers and P24GTR gene-specific primers to identify representative plasmid-expressing clones free of PCR errors.

[0398] Example 4: Characterization of the Rasamsonia byssochlamydoides genomic sequence encoding a GH10 polypeptide having xylanase activity

[0399] The sequence was sequenced using an Applied Biosystems Model 3700 Automated DNA Sequencer and version 3.1 BIG-DYE TMDNA sequencing of Rasamsonia byssochlamydoides CBS 413.71 P24GTR and Rasamsonia byssochlamydoides CBS 150.75 P34RRZ GH10 genomic clones was performed using terminator chemistry (Applied Biosystems, Inc., Foster City, CA, USA) and a primer walking strategy. The quality of the nucleotide sequence data was carefully checked and all sequences were compared to each other using PHRED / PHRAP software (University of Washington, Seattle, WA, USA).

[0400] The nucleotide sequence and deduced amino acid sequence of the Rasamsonia byssochlamydoides P24GTR xylanase gene are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The coding sequence is 1540 bp, including the stop codon, and is interrupted by four introns of 87 bp (nucleotides 63-149), 88 bp (nucleotides 304-391), 60 bp (nucleotides 525-584), and 84 bp (nucleotides 699-782). The encoded predicted protein is 406 amino acids. A 23-residue signal peptide was predicted using the SignalP program (Nielsen et al., 1997, Protein Eng. 10: 1-6). By sequence similarity, the xylanase was determined to include a carbohydrate binding module (nucleotides 1436-1537). The predicted mature protein contains 383 amino acids, has a predicted molecular weight of 41 kDa, and an isoelectric pH of 4.2.

[0401] The nucleotide sequence and deduced amino acid sequence of the Rasamsonia byssochlamydoides P34RRZ xylanase gene are set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The coding sequence is 1590 bp, including the stop codon, and is interrupted by four introns of 81 bp (nucleotides 63-143), 103 bp (nucleotides 298-400), 67 bp (nucleotides 534-600), and 118 bp (nucleotides 715-832). The encoded predicted protein is 406 amino acids. A 23-residue signal peptide was predicted using the SignalP program (Nielsen et al., 1997, Protein Eng. 10: 1-6). By sequence similarity, the xylanase was determined to include a carbohydrate binding module (nucleotides 1480-1587). The predicted mature protein contains 383 amino acids, has a predicted molecular weight of 41 kDa, and an isoelectric pH of 4.1.

[0402] Comparative pairwise global alignments of amino acid sequences were determined using the Needleman and Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol., 48:443-453), using a gap opening penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 matrix. The alignment showed that the deduced amino acid sequences of the Rasamsonia byssochlamydoides genes encoding the P24GTR and P34RRZ GH10 xylanases each shared 85% identity (excluding gaps) with the deduced amino acid sequence of a predicted GH10 family protein from Rasamsonia emersonii (GENESEQP: AAU99346) with endoxylanase activity.

[0403] Example 5: Expression and purification of Rasamsonia byssochlamydoides GH10 xylanase P24GTR

[0404] The expression plasmids pP24GTR and pP34RRZ were each transformed separately into protoplasts of Aspergillus oryzae MT3568, prepared according to the method of European Patent No. 0238023, pages 14-15. This transformation was performed according to the procedure described in WO 2005 / 042735. Aspergillus oryzae MT3568 is a derivative of JaL355 with a disrupted amdS (acetamidase) gene (WO 02 / 40694), in which pyrG auxotrophy was restored during the knockout of the Aspergillus oryzae amdS gene.

[0405] Transformants were purified from single conidia on COVE sucrose plates before being sporulated on PDA plates. Production of the Rasamsonia byssochlamydoides GH10 polypeptide by the transformants was analyzed from culture supernatants of 1 ml 96-well fixed cultures in YP + 2% glucose medium at 30°C. Expression was verified by SDS-PAGE using E-Page 8% SDS-PAGE 48-well gels (Invitrogen, Carlsbad, CA, USA) and Coomassie Brilliant Blue staining. One transformant resulting from transformation with pP24GTR was selected for further work and designated A. oryzae 69.2. One transformant resulting from transformation with pP34RRZ was selected for further work and designated A. oryzae 46.4.

[0406] For larger scale production, spores of Aspergillus oryzae strains 69.2 and 46.4 were spread onto PDA plates and incubated at 37°C for five days. The spore plates of fusion were washed twice at 20°C to maximize the number of spores collected. The spore suspension of each bacterial strain was then inoculated into twenty-five 500ml flasks containing 100ml Dap-4C culture medium. The culture was incubated at 30°C while incubating at 100rpm. After inoculation, the 4th day, culture fluid was collected by filtering through a bottle cap MF75 Supor MachV 0.2 μm PES filter (Thermo Fisher Scientific, Roskilde, Denmark). The SDS-PAGE analysis of the fresh culture fluid of transformants 69.2 and 46.4 yielded a band of approximately 42kDa. The consistency of these obvious bands and the Rasamsonia byssochlamydoides GH10 polypeptide was confirmed by peptide sequencing.

[0407] The filtered culture broth from a culture of Aspergillus oryzae strain 69.2 was adjusted to pH 7.0 and filtered using a 0.22 μm PES filter (Nalgene Nunc International, Nalgene labware catalog number 595-4520). Ammonium sulfate was added to the filtrate to a concentration of 1.8 M. The filtrate was loaded onto Phenyl Sepharose equilibrated with 1.8 M ammonium sulfate, 25 mM HEPES (pH 7.0). TM 6 Fast Flow column (high sub) (GE Healthcare, Piscataway, NJ, USA). After washing with 1.0 M ammonium sulfate, bound protein was eluted in batches with 25 mM HEPES (pH 7.0). Multiple fractions were collected and analyzed by SDS-PAGE. These fractions were combined and applied to Sephadex equilibrated in 25 mM HEPES (pH 7.0). TM G-25 (medium) (GE Healthcare, Piscataway, NJ, USA) column. The fractions were collected, combined, and applied to SOURCE ELECTRONICS equilibrated with 25 mM HEPES pH 7.0. TM 15Q (GE Healthcare, Piscataway, NJ, USA) column, and bound proteins were eluted with a linear gradient from 0-1000 mM sodium chloride over 20 column volumes. Fractions were collected and analyzed by SDS-PAGE.

[0408] Example 6: Alternative Methods for Producing Rasamsonia byssochlamydoides GH10 Xylanase

[0409] Synthetic genes based on the nucleotide sequences identified as SEQ ID NO: 1 and SEQ ID NO: 5 can be obtained from various vendors, such as Gene Art (GENEART BioPark, Josef-Engert-Str. 11, 93053 Regensburg, Germany) or DNA2.0 (DNA2.0, 1430 O'Brien Drive, Suite E, Menlo Park, CA 94025, USA). Synthetic genes can be designed to incorporate additional DNA sequences, such as restriction enzyme sites or homologous recombination regions, to facilitate cloning into expression vectors.

[0410] Using the two synthetic oligonucleotide primers F-P24GTR and F-P24GTR described above, PCR can be used to amplify the full-length open reading frame of the synthetic gene from SEQ ID NO: 1 or SEQ ID NO: 5. The gene can then be cloned into an expression vector (e.g., as described above) and expressed in a host cell (e.g., Aspergillus oryzae as described above).

[0411] Example 7: Hydrolysis assay of pretreated corncobs

[0412] Corn cob was pretreated with NaOH (0.08 g / g dry weight core) at 15% total dry weight solids (TS) for 60 minutes at 120°C. The resulting material was washed with water until it reached a pH of 8.2 to form washed alkaline pretreated corn cob (APCC). The pH of APCC was adjusted to 5.0 by adding 6M HCl and extensively mixed with water. APCC was ground in a Cosmos ICMG 40 wet multi-utility grinder (EssEmm, Tamil Nadu, India) and autoclaved at 121°C for 45 minutes to prepare ground and sieved alkaline pretreated corn cob (GS-APCC) to a final TS of 3.33%. GS-APCC was hydrolyzed in a total reaction volume of 1.0 using a 2.2 ml deep well plate (Axygen, Union City, California, USA).

[0413] Hydrolysis was performed with 10 mg of GS-APCC total solids / ml in 50 mM sodium acetate (pH 4.0 to 5.5) or 50 mM Tris (pH 6.0 to 7.0) buffer containing 1 mM manganese sulfate and different protein loadings (expressed as mg protein / gram cellulose) of different enzyme compositions. The enzyme compositions were prepared and then added to all wells simultaneously in volumes ranging from 50 μl to 200 μl, with a final volume of 1 ml in each reaction. The plates were then sealed using an ALPS-300™ plate heat sealer (ALPS-300 TM Plates were sealed using a plate heat sealer (Abgene, Epsom, UK), mixed thoroughly, and incubated at the specified temperature for 72 hours. All reported experiments were repeated in triplicate.

[0414] After hydrolysis, 0.45 μm 96-well filter plate ( The samples were filtered using a 96-well filter plate (Millipore, Bedford, MA, USA) and the filtrates were analyzed for sugar content as described below. When not used immediately, the filtered aliquots were frozen at -20°C. The sugar concentration of the samples diluted in 0.005 M H2SO4 was measured by using a 4.6 x 250 mm HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was eluted with 0.05% w / w benzoic acid-0.005 MH2SO4 at 65 °C at a flow rate of 0.6 ml / min and detected by integrating the refractive index ( The glucose of 1100HPLC, Agilent Technologies (Agilent Technologies, Santa Clara, California, the U.S.) is carried out quantitatively, and cellobiose and xylose signal are used.The glucose equivalent of gained is used to calculate the percentage ratio of the cellulose conversion of each reaction.The xylose equivalent of gained is used to calculate the percentage ratio of the xylooligosaccharide conversion of each reaction.

[0415] Glucose, cellobiose, and xylose were measured individually. The measured sugar concentrations were adjusted for the appropriate dilution factor. MICROSOFT EXCEL TM All HPLC data processing was performed using HPLC software (Microsoft, Richland, WA, USA).

[0416] The extent of conversion of xylo-oligosaccharides to xylose was calculated using the following equation: % xylose conversion = xylose concentration / xylose concentration in the restricted digest. To calculate % conversion, a 100% conversion point was set based on the cellulase control (100 mg of Trichoderma reesei cellulase per gram of cellulose supplemented with Penicillium emersonii GH61A polypeptide (WO 2011 / 041397), Aspergillus fumigatus GH10 xylanase (xyn3) (WO 2006 / 078256), and Talaromyces emersonii GH3 β-xylosidase (WO 2003 / 070956)), and all values were divided by this number and then multiplied by 100. The relative activity % for each temperature was calculated using the following equation: % relative activity = (% xylose conversion by xylanase at a specific pH and temperature - % xylose conversion by β-xylosidase at that specific pH and temperature) / (% xylose conversion by the xylanase containing the highest % xylose conversion for that pH and temperature - % xylose conversion by the β-xylosidase containing the highest % xylose conversion for that pH and temperature) x 100.

[0417] Example 8: Preparation of Talaromyces emersonii CBS 393.64 GH3 β-xylosidase

[0418] Using Aspergillus oryzae JaL355 as a host (WO 2003 / 070956), Talaromyces emersonii CBS 393.64 β-xylosidase (GENESEQP: AZI104896) was recombinantly prepared according to Rasmussen et al., 2006, Biotechnology and Bioengineering 94: 869-876. The filtered culture broth was concentrated and desalted into 50 mM sodium acetate (pH 5.0) using a tangential flow concentrator equipped with a 10 kDa polyethersulfone membrane. Microplate BCA was used. TM Protein concentration was determined using a protein assay kit (Thermo Fischer Scientific, Waltham, MA, USA) using bovine serum albumin as a protein standard.

[0419] Example 9: Preparation of Aspergillus fumigatus GH10 xylanase (P4D6)

[0420] Aspergillus fumigatus NN055679 GH10 xylanase (xyn3) (GENESEQP: AEC74753) was recombinantly produced according to WO 2006 / 078256 using Aspergillus oryzae BECh2 (WO 00 / 39322) as host.

[0421] use The filtered culture fluid was desalted and buffer exchanged into 50 mM sodium acetate (pH 5.0) using a 26 / 10 desalting column (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer's instructions. TM Protein concentration was determined using a protein assay kit, with bovine serum albumin as the protein standard.

[0422] Example 10: Effect of Rasamsonia byssochlamydoides GH10 xylanase (P24GTR) supplemented with Talaromyces emersonii GH3 β-xylosidase at pH 4.0 to 7.0 using GS-APCC

[0423] Rasamsonia byssochlamydoides GH10 xylanase (P24GTR) supplemented with Talaromyces emersonii GH3 β-xylosidase (Example 8) was evaluated using washed ground sieved alkaline pretreated corn cob (GS-APCC) as substrate at 50°C, 55°C, 60°C, 65°C from pH 4.0 to 7.0. Aspergillus fumigatus GH10 xylanase (P4D6) supplemented with Talaromyces emersonii GH3 β-xylosidase was also tested for comparison. Xylanax was added to the GS-APCC hydrolysis at 1 mg total protein / g cellulose (β-xylosidase supplemented with 4.0 mg total protein / g cellulose).

[0424] The assay was performed as described in Example 7. 1 ml reactions with GS-APCC (1% total solids) were carried out for 72 hours in 50 mM sodium acetate (pH 4.0 to 5.5) or 50 mM Tris (pH 6.0 to 7.0) containing 1 mM manganese sulfate. All reactions were performed in triplicate and involved a single mix at the start of the hydrolysis.

[0425] The hydrolysis results at pH 4.0 from 50°C to 65°C are shown in Figure 1 The relative activity results for the Rasamsonia byssochlamydoides GH10 xylanase are shown in Table 1, where these results are compared with a temperature of 65°C and a pH of 4.0; and the relative activity results for the Aspergillus fumigatus GH10 xylanase are shown in Table 2, where these results are compared with a temperature of 55°C and a pH of 5.5. Figure 1As shown in, at all temperatures, the Rasamsonia byssochlamydoides GH10 xylanase supplemented with β-xylosidase has a significantly higher activity than the Aspergillus fumigatus GH10 xylanase supplemented with β-xylosidase. In addition, as the temperature increases from 50°C to 65°C, the Rasamsonia byssochlamydoides GH10 xylanase supplemented with β-xylosidase has a gradually increasing activity, while as the temperature increases from 50°C to 65°C, the Aspergillus fumigatus GH10 xylanase supplemented with β-xylosidase has a gradually decreasing activity. As shown in Table 1, the Rasamsonia byssochlamydoides GH10 xylanase has an optimum temperature of 65°C and an optimum pH range of from pH 4.0 to 6.0 at the optimum temperature (greater than 75% relative activity). In contrast, the Aspergillus fumigatus GH10 xylanase had a temperature optimum of 55° C. and an optimum pH range of pH 4.5 to 6.0 at 55° C. (greater than 75% relative activity). Overall, the Rasamsonia byssochlamydoides GH10 xylanase had a higher temperature optimum activity and activity at lower pH than the Aspergillus fumigatus GH10 xylanase.

[0426] Table 1

[0427]

[0428] Table 2

[0429]

[0430]

[0431] Example 11: Specific activity of Rasamsonia byssochlamydoides GH10 xylanase and Aspergillus fumigatus GH10 xylanase on birchwood xylan at pH 5.0 and 50°C

[0432] Determine the specific activity of Rasamsonia byssochlamydoides GH10 xylanase (P24GTR) and Aspergillus fumigatus GH10 xylanase on birchwood xylan (Sigma Chemical Co., St. Louis, MO, USA). A solution of 2 g / L birchwood xylan was prepared in 50 mM sodium acetate buffer (pH 5.0). 10 μl of xylanase was added to 190 μl of 2 g / L birchwood xylan at different enzyme loadings. Controls for separate substrates and separate enzymes were also run. The reaction was incubated at 50°C for 30 minutes and then terminated by adding 50 μl of 0.5 M NaOH to each reaction. The reducing sugars produced were determined using p-hydroxybenzoic acid hydrazide (PHBAH, Sigma, St. Louis, MO, USA) assay adapted for 96-well microplate format as described below. In brief, 100 μ l aliquots of the appropriately diluted sample were placed in a 96-well conical bottom microplate. The reaction was initiated by adding 50 μ l 1.5% (w / v) PHBAH in 2% NaOH to each well. The plate was not covered and heated at 95 ° C for 10 minutes. The plate was allowed to cool to room temperature and 50 μ l Milli Q H2O (Millipore, Bedford, Massachusetts, USA) was added to each well. 100 μ l aliquots from each well were transferred to a flat-bottomed 96-well plate and the absorbance at 410 nm was measured using a SpectraMax microplate reader (Molecular Devices, Sunnyvale, California). Glucose standards (0.1-0.0125 mg / ml, diluted with 0.4% sodium hydroxide) were used to make a standard curve to obtain the A 410nm The values were converted to glucose equivalents. The enzyme loading was plotted against the reducing sugars produced, and the linear range was used to calculate the specific activity of the Rasamsonia byssochlamydoides GH10 xylanase and the Aspergillus fumigatus GH10 xylanase, expressed as μmol glucose equivalents / minute / mg enzyme produced, or IU / mg. The specific activity results are shown in Table 3.

[0433] Table 3. Specific activity against birchwood xylan at pH 5.0 and 50°C

[0434]

[0435] Example 12: Preparation of Rasamsonia emersonii GH10 xylanase (P23CQ3)

[0436] Rasamsonia emersonii GH10 xylanase (GENESEQP: AZI05030) was recombinantly produced and purified according to WO 2011 / 057140 using Aspergillus oryzae (WO 00 / 39322) as a host. Protein concentration was determined using a microplate BCATM protein assay kit with bovine serum albumin as a protein standard.

[0437] Example 13: Specific activity of Rasamsonia byssochlamydoides GH10 xylanase and Rasamsonia emersonii GH10 xylanase on birchwood xylan at pH 4.0 and 60°C

[0438] The specific activity of Rasamsonia byssochlamydoides GH10 xylanase (P24GTR) and Rasamsonia emersonii GH10 xylanase on birchwood xylan (Sigma Chemical Co., Ltd.) was determined as described in Example 11. A solution of 2 g / L birchwood xylan was prepared in 50 mM sodium acetate buffer (pH 4.0), and the reaction was incubated at 60°C for 30 minutes. A standard curve was prepared using xylose standards (0.1-0.0125 mg / ml, diluted in 0.4% sodium hydroxide) to compare the obtained A values to the A values. 410nm The specific activity results are shown in Table 4.

[0439] Table 4. Specific activity on birchwood xylan at pH 4.0 and 60°C.

[0440]

[0441] Example 14: Preparation of natural variant Rasamsonia byssochlamydoides GH10 xylanase P34RRZ

[0442] use The protein solution of Rasamsonia byssochlamydoides GH10 xylanase P34RRZ was desalted and buffer exchanged into 50 mM sodium acetate (pH 5.0) using a 10-DG desalting column (Bio-Rad Laboratories) according to the manufacturer's instructions. TM Protein concentration was determined using a protein assay kit using bovine serum albumin as a protein standard.

[0443] Example 15: Specific activity of the natural variant Rasamsonia byssochlamydoides GH10 xylanase P34RRZ on birchwood xylan at pH 4.0 and 60°C

[0444] The specific activity of the natural variant Rasamsonia byssochlamydoides GH10 xylanase (P34RRZ) on birchwood xylan (Sigma Chemicals Ltd.) was determined as described in Example 13. The specific activity results are shown in Table 5.

[0445] Table 5. Specific activity on birchwood xylan at pH 4.0 and 60°C.

[0446]

[0447] The present invention is further described by the following numbered paragraphs:

[0448] [1] An isolated polypeptide having xylanase activity, the isolated polypeptide being selected from the group consisting of:

[0449] (a) a polypeptide having at least 90% sequence identity to the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6;

[0450] (b) a polypeptide encoded by a polynucleotide having at least 90% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or its cDNA sequence, or to the mature polypeptide coding sequence of SEQ ID NO: 5 or its cDNA sequence;

[0451] (c) a variant of the mature polypeptide of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 4, the variant comprising a substitution, deletion, and / or insertion at one or more positions; and

[0452] (d) A fragment of the polypeptide of (a), (b) or (c), wherein the fragment has xylanase activity.

[0453] [2] The polypeptide of paragraph [1], which has 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% sequence identity to the mature polypeptide of SEQ ID NO: 2.

[0454] [3] The polypeptide of paragraph [1] or [2], which has 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% sequence identity to the mature polypeptide of SEQ ID NO: 6.

[0455] [4] The polypeptide of any of paragraphs [1] to [3], which is encoded by a polynucleotide having 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% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 1 or its cDNA sequence.

[0456] [5] The polypeptide of any of paragraphs [1] to [4], which is encoded by a polynucleotide having 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% sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 5 or its cDNA sequence.

[0457] [6] The polypeptide according to any one of paragraphs [1] to [5], which comprises or consists of SEQ ID NO: 2 or the mature polypeptide thereof.

[0458] [7] The polypeptide of paragraph [6], wherein the mature polypeptide is amino acids 24 to 406 of SEQ ID NO: 2.

[0459] [8] The polypeptide of any one of paragraphs [1] to [5], which comprises or consists of SEQ ID NO: 6 or its mature polypeptide.

[0460] [9] The polypeptide of paragraph [8], wherein the mature polypeptide is amino acids 24 to 406 of SEQ ID NO: 6.

[0461]

[10] The polypeptide of any one of paragraphs [1] to [5], which is a variant of the mature polypeptide of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO: 6, comprising a substitution, deletion, and / or insertion at one or more positions.

[0462]

[11] The polypeptide of paragraph [1], which is a fragment of SEQ ID NO: 2 or a fragment of SEQ ID NO: 6, wherein the fragment has xylanase activity.

[0463]

[12] The polypeptide of any one of paragraphs [1] to

[11] , which is encoded by a polynucleotide contained in Rasamsonia byssochlamydoides CBS 413.71.

[0464]

[13] The polypeptide of any of paragraphs [1] to

[12] , which has at least 10%, such as at least 15% and at least 20% more xylanase activity at pH 4.0 and 60°C or 65°C than at pH 4.0 and 50°C.

[0465]

[14] The polypeptide of any of paragraphs [1] to

[13] , which has at least 10%, such as at least 15% and at least 20% more xylanase activity at pH 4.0 and 50°C, 55°C, 60°C, or 65°C compared to at pH 6.0 and 50°C, 55°C, 60°C, or 65°C, respectively.

[0466]

[15] An isolated polypeptide having xylanase activity, wherein the isolated polypeptide has at least 10%, such as at least 15% and at least 20% more xylanase activity at pH 4.0 and 60°C or 65°C than at pH 4.0 and 50°C.

[0467]

[16] An isolated polypeptide having xylanase activity, which has at least 10%, e.g., at least 15% and at least 20% more xylanase activity at pH 4.0 and 50°C, 55°C, 60°C, or 65°C compared to at pH 6.0 and 50°C, 55°C, 60°C, or 65°C, respectively.

[0468]

[17] An isolated polypeptide comprising a catalytic domain selected from the group consisting of:

[0469] (a) a catalytic domain having at least 60% sequence identity to amino acids 24 to 340 of SEQ ID NO: 2 or at least 60% sequence identity to amino acids 24 to 341 of SEQ ID NO: 6;

[0470] (b) a catalytic domain encoded by a polynucleotide having at least 90% sequence identity to nucleotides 157 to 1339 of SEQ ID NO: 1, or its cDNA sequence, or at least 90% sequence identity to nucleotides 151 to 1387 of SEQ ID NO: 5, or its cDNA sequence;

[0471] (c) a variant of amino acids 24 to 340 of SEQ ID NO: 2 or amino acids 24 to 341 of SEQ ID NO: 6, the variant comprising a substitution, deletion, and / or insertion at one or more (e.g., several) positions; and

[0472] (d) A fragment of the catalytic domain of (a), (b) or (c), which fragment has xylanase activity.

[0473]

[18] The polypeptide of paragraph

[17] , further comprising a carbohydrate binding module.

[0474]

[19] An isolated polypeptide comprising a carbohydrate binding module operably linked to a catalytic domain, wherein the binding domain is selected from the group consisting of:

[0475] (a) a carbohydrate binding moiety having at least 90% sequence identity to amino acids 373 to 406 of SEQ ID NO: 2 or at least 90% sequence identity to amino acids 371 to 406 of SEQ ID NO: 6;

[0476] (b) a carbohydrate binding module encoded by a polynucleotide having at least 90% sequence identity to nucleotides 1436 to 1537 of SEQ ID NO: 1, or its cDNA sequence, or encoded by a polynucleotide having at least 90% sequence identity to nucleotides 1480 to 1587 of SEQ ID NO: 5, or its cDNA sequence;

[0477] (c) a variant of amino acids 373 to 406 of SEQ ID NO: 2 or amino acids 3731 to 406 of SEQ ID NO: 6, the variant comprising a substitution, deletion, and / or insertion at one or more (e.g., several) positions; and

[0478] (d) A fragment of the carbohydrate binding module of (a), (b), or (c), wherein the fragment has binding activity.

[0479]

[20] The polypeptide of paragraph

[19] , wherein the catalytic domain is obtained from a hydrolase, isomerase, ligase, lyase, oxidoreductase or transferase, such as an aminopeptidase, amylase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, α-galactosidase, β-galactosidase, glucoamylase, α-glucosidase, β-glucosidase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenol oxidase, proteolytic enzyme, ribonuclease, transglutaminase, xylanase or β-xylosidase.

[0480]

[21] A composition comprising the polypeptide described in any one of paragraphs [1] to

[20] .

[0481]

[22] An isolated polynucleotide encoding the polypeptide described in any one of paragraphs [1] to

[20] .

[0482]

[23] A nucleic acid construct or expression vector comprising the polynucleotide of paragraph

[22] operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

[0483]

[24] A recombinant host cell comprising the polynucleotide of paragraph

[22] operably linked to one or more control sequences that direct the production of the polypeptide.

[0484]

[25] A method of producing a polypeptide as described in any of paragraphs [1] to

[20] , the method comprising: culturing a cell under conditions conducive for production of the polypeptide, the cell producing the polypeptide in its wild-type form.

[0485]

[26] The method of paragraph

[25] , further comprising recovering the polypeptide.

[0486]

[27] A method for producing a polypeptide having xylanase activity, the method comprising: cultivating the host cell of paragraph

[24] under conditions conducive for production of the polypeptide.

[0487]

[28] The method of paragraph

[27] further comprises recovering the polypeptide.

[0488]

[29] A transgenic plant, plant part or plant cell transformed with a polynucleotide encoding the polypeptide of any one of paragraphs [1] to

[20] .

[0489]

[30] A method for producing a polypeptide having xylanase activity, the method comprising: cultivating the transgenic plant or plant cell of paragraph

[29] under conditions conducive for production of the polypeptide.

[0490]

[31] The method of paragraph

[30] , further comprising recovering the polypeptide.

[0491]

[32] A method for producing a mutant of a parent cell, the method comprising inactivating a polynucleotide encoding a polypeptide as described in any of paragraphs [1]-

[20] , such that the mutant produces less of the polypeptide than the parent cell.

[0492]

[33] A mutant cell produced by the method of paragraph

[32] .

[0493]

[34] The mutant cell as described in paragraph

[33] , further comprising a gene encoding a native or heterologous protein.

[0494]

[35] A method for producing a protein, comprising culturing the mutant cell of paragraph

[33] or

[34] under conditions conducive to production of the protein.

[0495]

[36] The method of paragraph

[35] , further comprising recovering the protein.

[0496]

[37] A double-stranded inhibitory RNA (dsRNA) molecule comprising a subsequence of the polynucleotide of paragraph

[22] , wherein optionally, the dsRNA is an siRNA or miRNA molecule.

[0497]

[38] The double-stranded inhibitory RNA (dsRNA) molecule of paragraph

[37] , which is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more dinucleotides in length.

[0498]

[39] A method of inhibiting expression of a polypeptide having xylanase activity in a cell, the method comprising administering to the cell or expressing in the cell the double-stranded inhibitory RNA (dsRNA) molecule of paragraph

[37] or

[38] .

[0499]

[40] A cell produced by the method of paragraph

[39] .

[0500]

[41] The cell of paragraph

[40] , further comprising a gene encoding a native or heterologous protein.

[0501]

[42] A method of producing a protein, comprising culturing the cell of paragraph

[40] or

[41] under conditions conducive for production of the protein.

[0502]

[43] The method of paragraph

[42] further comprises recovering the protein.

[0503]

[44] An isolated polynucleotide encoding a signal peptide comprising or consisting of amino acids 1 to 23 of SEQ ID NO: 2 or amino acids 1 to 23 of SEQ ID NO: 6.

[0504]

[45] A nucleic acid construct or expression vector comprising a gene encoding a protein operably linked to the polynucleotide of paragraph

[44] , wherein the gene is foreign to the polynucleotide encoding the signal peptide.

[0505]

[46] A recombinant host cell comprising a gene encoding a protein operably linked to the polynucleotide of paragraph

[44] , wherein the gene is foreign to the polynucleotide encoding the signal peptide.

[0506]

[47] A method of producing a protein, the method comprising: culturing a recombinant host cell under conditions conducive for production of the protein, the recombinant host cell comprising a gene encoding the protein operably linked to the polynucleotide of paragraph

[40] , wherein the gene is heterologous to the polynucleotide encoding the signal peptide.

[0507]

[48] The method of paragraph

[47] further comprises recovering the protein.

[0508]

[49] A method for degrading a cellulosic material or a xylan-containing material, the method comprising treating the cellulosic material or the xylan-containing material with an enzyme composition in the presence of a polypeptide having xylanase activity as described in any of paragraphs [1] to

[20] .

[0509]

[50] The method of paragraph

[49] , wherein the cellulosic material or xylan-containing material is pretreated.

[0510]

[51] The method of paragraph

[49] or

[50] , wherein the enzyme composition comprises one or more enzymes selected from the group consisting of cellulase, polypeptides having cellulolytic enhancing activity, hemicellulase, esterase, expansin, laccase, ligninase, pectinase, peroxidase, protease, and swellin.

[0511]

[52] The method of paragraph

[51] , wherein the cellulase is one or more enzymes selected from the group consisting of endoglucanases, cellobiohydrolases, and β-glucosidases.

[0512]

[53] The method of paragraph

[51] , wherein the hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, ferulic acid esterase, arabinofuranosidase, xylosidase, and glucuronidase.

[0513]

[54] The method of any of paragraphs

[49] -

[53] , further comprising recovering the degraded cellulosic material or xylan-containing material.

[0514]

[55] The method of paragraph

[54] , wherein the degraded cellulosic material or xylan-containing material is a sugar.

[0515]

[56] The method of paragraph

[55] , wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose and arabinose.

[0516]

[57] A method for producing a fermentation product, the method comprising:

[0517] (a) saccharifying a cellulosic material or a xylan-containing material with an enzyme composition in the presence of a polypeptide having xylanase activity as described in any of paragraphs [1-16];

[0518] (b) fermenting the saccharified cellulosic material or xylan-containing material with one or more fermenting microorganisms to produce the fermentation product; and

[0519] (c) recovering the fermentation product from the fermentation.

[0520]

[58] The method of paragraph

[57] , wherein the cellulosic material or xylan-containing material is pretreated.

[0521]

[59] The process of paragraph

[57] or

[58] , wherein the enzyme composition comprises one or more enzymes selected from the group consisting of cellulase, polypeptides having cellulolytic enhancing activity, hemicellulase, esterase, expansin, laccase, ligninase, pectinase, peroxidase, protease, and swellin.

[0522]

[60] The method of paragraph

[59] , wherein the cellulase is one or more enzymes selected from the group consisting of endoglucanases, cellobiohydrolases, and β-glucosidases.

[0523]

[61] The method of paragraph

[59] or

[60] , wherein the hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, ferulic acid esterase, arabinofuranosidase, xylosidase, and glucuronidase.

[0524]

[62] The method of any of paragraphs

[57] -

[61] , wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.

[0525]

[63] The method of any of paragraphs

[57] -

[62] , wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or a polyketide.

[0526]

[64] A method for fermenting a cellulosic material or a xylan-containing material, the method comprising: fermenting the cellulosic material or the xylan-containing material with one or more fermenting microorganisms, wherein the cellulosic material or the xylan-containing material is saccharified by an enzyme composition in the presence of a polypeptide having xylanase activity as described in any of paragraphs [1]-

[20] .

[0527]

[65] The method of paragraph

[64] , wherein fermenting the cellulosic material or xylan-containing material produces a fermentation product.

[0528]

[66] The method of paragraph

[65] further comprises recovering the fermentation product from the fermentation.

[0529]

[67] The method of any of paragraphs

[64] -

[66] , wherein the cellulosic material or xylan-containing material is pretreated prior to saccharification.

[0530]

[68] The method of any of paragraphs

[64] -

[67] , wherein the enzyme composition comprises one or more enzymes selected from the group consisting of cellulase, polypeptides having cellulolytic enhancing activity, hemicellulase, esterase, expansin, laccase, ligninase, pectinase, peroxidase, protease, and swellin.

[0531]

[69] The method of paragraph

[68] , wherein the cellulase is one or more enzymes selected from the group consisting of endoglucanases, cellobiohydrolases, and β-glucosidases.

[0532]

[70] The method of paragraph

[68] , wherein the hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, ferulic acid esterase, arabinofuranosidase, xylosidase, and glucuronidase.

[0533]

[71] The method of any of paragraphs

[65] -

[70] , wherein the fermentation product is an alcohol, an alkane, a cycloalkane, an alkene, an amino acid, a gas, isoprene, a ketone, an organic acid, or a polyketide.

[0534]

[72] A whole culture broth formulation or cell culture composition comprising the polypeptide described in any one of paragraphs [1] to

[20] .

[0535]

[73] An enzyme composition comprising a polypeptide having xylanase activity as described in any of paragraphs [1] to

[20] and one or more enzymes selected from the group consisting of cellulase, polypeptide having cellulolytic enhancing activity, hemicellulase, esterase, expansin, laccase, ligninase, pectinase, peroxidase, protease, and swellin.

[0536]

[74] The enzyme composition of paragraph

[73] , wherein the cellulase is one or more enzymes selected from the group consisting of endoglucanases, cellobiohydrolases, and β-glucosidases.

[0537]

[75] The enzyme composition of paragraph

[73] or

[74] , wherein the hemicellulase is one or more enzymes selected from the group consisting of xylanase, acetylxylan esterase, ferulic acid esterase, arabinofuranosidase, xylosidase, and glucuronidase.

[0538] The invention described and claimed herein is not limited to the scope of the specific aspects disclosed herein, as these aspects are intended to be illustrative of several aspects of the invention. It is contemplated that any equivalent aspects are within the scope of the invention. Indeed, various modifications of the invention, other than those shown and described herein, will become clear to those of ordinary skill in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the event of a conflict, the present disclosure, including definitions, will prevail.

Claims

1. An isolated polypeptide having xylanase activity, the isolated polypeptide being selected from the group consisting of: (a) the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 6, wherein the mature polypeptide of SEQ ID NO: 2 is amino acids 24 to 406 of SEQ ID NO: 2, and the mature polypeptide of SEQ ID NO: 6 is amino acids 24 to 406 of SEQ ID NO: 6; (b) a polypeptide encoded by the mature polypeptide coding sequence of SEQ ID NO: 1 or its cDNA sequence, or by the mature polypeptide coding sequence of SEQ ID NO: 5 or its cDNA sequence, wherein the mature polypeptide coding sequence of SEQ ID NO: 1 is nucleotides 157 to 1537 of SEQ ID NO: 1 or its cDNA sequence, and the mature polypeptide coding sequence of SEQ ID NO: 5 is nucleotides 151 to 1587 of SEQ ID NO: 5 or its cDNA sequence.

2. The polypeptide of claim 1, which is the mature polypeptide of SEQ ID NO: 2 or the mature polypeptide of SEQ ID NO:

6.

3. The polypeptide according to claim 1 or 2, which is Rasamsonia byssochlamydoides CBS413.71 or Rasamsonia byssochlamydoides The polynucleotide contained in CBS 150.75 encodes.

4. The polypeptide of claim 1 or 2, having at least 10% more xylanase activity at pH 4.0 and 60°C or 65°C than at pH 4.0 and 50°C.

5. The polypeptide of claim 4, having at least 10% more xylanase activity at pH 4.0 and 50°C, 55°C, 60°C, or 65°C compared to at pH 6.0 and 50°C, 55°C, 60°C, or 65°C, respectively.

6. A composition comprising the polypeptide according to any one of claims 1 to 5.

7. An isolated polynucleotide encoding the polypeptide of any one of claims 1 to 5.

8. A nucleic acid construct or expression vector comprising the polynucleotide of claim 7 operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

9. A recombinant host cell comprising the polynucleotide of claim 7 operably linked to one or more control sequences that direct the production of the polypeptide.

10. A method for producing the polypeptide according to any one of claims 1 to 5, the method comprising: The cells are cultured under conditions conducive for production of the polypeptide, which the cells produce in its wild-type form.

11. The method of claim 10, further comprising recovering the polypeptide.

12. A method for producing a polypeptide having xylanase activity, the method comprising: The host cell of claim 9 is cultured under conditions conducive to production of the polypeptide.

13. The method of claim 12, further comprising recovering the polypeptide.

14. A method of producing a mutant of a parent cell, the method comprising inactivating a polynucleotide encoding a polypeptide according to any one of claims 1 to 5, which results in the mutant producing less of the polypeptide than the parent cell.

15. A mutant cell produced by the method of claim 14.

16. A method for producing a protein, the method comprising: Cultivating the mutant cell of claim 15 under conditions conducive to production of the protein.

17. The method of claim 16, further comprising recovering the protein.

18. An isolated polynucleotide encoding a signal peptide consisting of amino acids 1 to 23 of SEQ ID NO: 2 or amino acids 1 to 23 of SEQ ID NO:

6.

19. A method for degrading a cellulosic material or a xylan-containing material, the method comprising: The cellulosic or xylan-containing material is treated with an enzyme composition in the presence of the isolated polypeptide having xylanase activity of any one of claims 1-5.

20. The method of claim 19, wherein the cellulosic or xylan-containing material is pretreated.

21. The method of claim 20, wherein the enzyme composition comprises one or more enzymes selected from the group consisting of: Cellulases, polypeptides having cellulolytic enhancing activity, hemicellulases, esterases, laccases, ligninolytic enzymes, pectinases, peroxidases, and proteases.

22. The method of claim 21, wherein the cellulase is one or more enzymes selected from the group consisting of: Endoglucanases, cellobiohydrolases, and beta-glucosidases.

23. The method of claim 21, wherein the hemicellulase is one or more enzymes selected from the group consisting of: Xylanases, acetylxylan esterases, feruloyl esterases, arabinofuranosidases, xylosidases, and glucuronidases.

24. The method of any one of claims 19-23, further comprising recovering the degraded cellulosic or xylan-containing material.

25. The method of claim 24, wherein the degraded cellulosic or xylan-containing material is a sugar.

26. The method of claim 25, wherein the sugar is selected from the group consisting of: Glucose, xylose, mannose, galactose and arabinose.

27. A method for producing a fermentation product, the method comprising: (a) saccharifying a cellulosic material or a xylan-containing material with an enzyme composition in the presence of an isolated polypeptide having xylanase activity according to any one of claims 1 to 5; (b) fermenting the saccharified cellulosic material or xylan-containing material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

28. The method of claim 27, wherein the cellulosic or xylan-containing material is pretreated.

29. The method of claim 27, wherein the enzyme composition comprises one or more enzymes selected from the group consisting of: Cellulases, polypeptides having cellulolytic enhancing activity, hemicellulases, esterases, laccases, ligninolytic enzymes, pectinases, peroxidases, and proteases.

30. The method of claim 29, wherein the cellulase is one or more enzymes selected from the group consisting of: Endoglucanases, cellobiohydrolases, and beta-glucosidases.

31. The method of claim 29 or 30, wherein the hemicellulase is one or more enzymes selected from the group consisting of: Xylanases, acetylxylan esterases, feruloyl esterases, arabinofuranosidases, xylosidases, and glucuronidases.

32. The method of claim 27, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.

33. The method of claim 27, wherein the fermentation product is an alcohol, an alkane, an alkene, a gas, isoprene, a ketone, or an organic acid.

34. A method of fermenting a cellulosic material or a xylan-containing material, the method comprising: The cellulosic or xylan-containing material is fermented with one or more fermenting microorganisms, wherein the cellulosic or xylan-containing material is saccharified with an enzyme composition in the presence of the isolated polypeptide having xylanase activity of any one of claims 1-5.

35. The method of claim 34, wherein fermenting the cellulosic or xylan-containing material produces a fermentation product.

36. The method of claim 35, further comprising recovering the fermentation product from the fermentation.

37. The method of any one of claims 34-36, wherein the cellulosic or xylan-containing material is pretreated prior to saccharification.

38. The method of claim 34, wherein the enzyme composition comprises one or more enzymes selected from the group consisting of: Cellulases, polypeptides having cellulolytic enhancing activity, hemicellulases, esterases, laccases, ligninolytic enzymes, pectinases, peroxidases, and proteases.

39. The method of claim 38, wherein the cellulase is one or more enzymes selected from the group consisting of: Endoglucanases, cellobiohydrolases, and beta-glucosidases.

40. The method of claim 38, wherein the hemicellulase is one or more enzymes selected from the group consisting of: Xylanases, acetylxylan esterases, feruloyl esterases, arabinofuranosidases, xylosidases, and glucuronidases.

41. The method of any one of claims 35-36, wherein the fermentation product is an alcohol, an alkane, an alkene, a gas, isoprene, a ketone, or an organic acid.

42. An enzyme composition comprising the isolated polypeptide having xylanase activity of any one of claims 1-5 and one or more enzymes selected from the group consisting of: Cellulases, polypeptides having cellulolytic enhancing activity, hemicellulases, esterases, laccases, ligninolytic enzymes, pectinases, peroxidases, and proteases.

43. The enzyme composition of claim 42, wherein the cellulase is one or more enzymes selected from the group consisting of: Endoglucanases, cellobiohydrolases, and beta-glucosidases.

44. The enzyme composition of claim 42, wherein the hemicellulase is one or more enzymes selected from the group consisting of: Xylanases, acetylxylan esterases, feruloyl esterases, arabinofuranosidases, xylosidases, and glucuronidases.

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