Reference to a Sequence Listing

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

Background of the Invention Field of the Invention

The present invention relates to processes for increasing the yield of fermentable sugars during saccharification of a lignocellulosic material.

Description of the Related Art

Biomass feedstocks for the production of ethanol and other chemicals are complex in composition, comprising cellulose, hemicellulose, lignin, and other constituents.

Lignocellulose, the world's largest renewable biomass resource, is composed mainly of lignin, cellulose, and hemicellulose. Cellulose is a polymer of glucose linked by beta-1 ,4- bonds. Many microorganisms produce enzymes that hydrolyze beta-linked glucans. These enzymes include endoglucanases, cellobiohydrolases, and beta-glucosidases. Endoglucanases digest the cellulose polymer at random locations, opening it to attack by cellobiohydrolases. Cellobiohydrolases sequentially release molecules of cellobiose from the ends of the cellulose polymer. Cellobiose is a water-soluble beta-1 ,4-linked dimer of glucose. Beta-glucosidases hydrolyze cellobiose to glucose.

A large part of hemicellulose is xylan. Xylans are polysaccharides formed from 1 ,4-β- glycoside-linked D-xylopyranoses. Xylanases (e.g., endo-1 ,4-beta-xylanase, EC 3.2.1.8) hydrolyze internal β-1 ,4-xylosidic linkages in xylan to produce smaller molecular weight xylose and xylo-oligomers. Beta-xylosidases catalyze the exo-hydrolysis of short beta (1→4)-xylooligosaccharides to remove successive D-xylose residues from non-reducing termini

Beta-xylosidases of glycoside hydrolase family 3 catalyze hydrolysis of (1→4)-beta- D-xylans, e.g., xylobiose, to remove successive D-xylose residues from the non-reducing termini by a retaining mechanism of action. Retaining beta-xylosidases cleave xylobiose via a 2-step mechanism where a glycosyl-enzyme intermediate forms in the first step. Cleavage of this intermediate with water results in hydrolysis of xylobiose to two xylose molecules.

Beta-xylosidases of glycoside hydrolase family 43 catalyze hydrolysis of (1→4)-beta- D-xylans, e.g., xylobiose, to remove successive D-xylose residues from the non-reducing termini by an inverting mechanism of action. Inverting beta-xylosidases (GH43 family) cleave via a 1-step mechanism resulting in the hydrolysis of xylobiose to two xylose molecules.

After enzymatic saccharification of lignocellulose to glucose and xylose, the glucose and xylose can then be fermented into biofuel such as ethanol using suitable fermenting microorganisms. The formation of primeverose, 6-O-beta-D-xylopyranosyl-beta-D- glucopyranose, reduces the amount of free glucose and xylose for fermentation. There is a need in the art for reducing formation of primeverose.

The present invention relates to processes for increasing the yield of fermentable sugars during saccharification of a lignocellulosic or hemicellulosic material.

Summary of the Invention

The present invention relates to processes for reducing production of primeverose during saccharification of a lignocellulosic material, the process comprising: saccharifying the lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.

The present invention also relates to processes for saccharifying a lignocellulosic material, comprising: treating the lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta- xylosidase.

The present invention also relates to processes for producing a fermentation product, the process comprising: (a) saccharifying a lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase; (b) fermenting the saccharified lignocellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

The present invention also relates to processes for fermenting a lignocellulosic material, the process comprising: fermenting the lignocellulosic material with one or more fermenting microorganisms, wherein the lignocellulosic material is saccharified with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.

Brief Description of the Figures

Figure 1 shows a restriction map of plasmid pl_F02.

Figure 2 shows a restriction map of plasmid pEbZn58.

Figure 3 shows a chromatogram from Dionex ion chromatography analysis using a PA-10 column of reaction mixtures containing different concentrations of Geobacillus stearothermophilus GH43 beta-xylosidase incubated with xylo-oligomers and glucose. The chromatogram depicts the signal in nanocoulombs (nC) and retention time in minutes.

Figure 4 shows a chromatogram from Dionex ion chromatography analysis using a

PA-10 column of reaction mixtures containing different concentrations of Talaromyces emersonii GH3 beta-xylosidase incubated with xylo-oligomers and glucose. The axes are labelled as in Figure 3.

Figure 5 shows a chromatogram from Dionex ion chromatography analysis with a PA-10 column of reaction mixtures containing different concentrations of a combination of a Talaromyces emersonii GH3 beta-xylosidase and a Geobacillus stearothermophilus GH43 beta-xylosidase incubated with xylo-oligomers and glucose. The concentration shown is the total concentration of beta-xylosidase in the assay which is a 50:50 mix of T. emersonii GH3 beta-xylosidase and G. stearothermophilus GH43 beta-xylosidase. The axes are labelled as in Figure 3.

Definitions

Acetylxylan esterase: The term "acetylxylan esterase" means a carboxylesterase (EC 3.1.1.72) that catalyzes the hydrolysis of acetyl groups from polymeric xylan, acetylated xylose, acetylated glucose, alpha-napthyl acetate, and p-nitrophenyl acetate. Acetylxylan esterase activity can be determined using 0.5 mM p-nitrophenylacetate as 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 μηιοΐβ of p-nitrophenolate anion per minute at pH 5, 25°C.

Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

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

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

Auxiliary Activity 9 polypeptide: The term "Auxiliary Activity 9 polypeptide" or "AA9 polypeptide" means a polypeptide classified as a lytic polysaccharide monooxygenase (Quinlan et ai, 201 1 , Proc. Natl. Acad. Sci. USA 108: 15079-15084; Phillips et ai, 201 1 , ACS Chem. Biol. 6: 1399-1406; Li et ai, 2012, Structure 20: 1051-1061). AA9 polypeptides were formerly classified into the glycoside hydrolase Family 61 (GH61) according to Henrissat, 1991 , Biochem. J. 280: 309-316, and Henrissat and Bairoch, 1996, Biochem. J. 316: 695-696.

AA9 polypeptides enhance the hydrolysis of a cellulosic material by an enzyme having cellulolytic activity. Cellulolytic enhancing activity can be determined by measuring the increase in reducing sugars or the increase of the total of cellobiose and glucose from the hydrolysis of a cellulosic material by cellulolytic enzyme under the following conditions: 1-50 mg of total protein/g of cellulose in pretreated corn stover (PCS), wherein total protein is comprised of 50-99.5% w/w cellulolytic enzyme protein and 0.5-50% w/w protein of an AA9 polypeptide for 1-7 days at a suitable temperature, such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C and a suitable pH, such as 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, compared to a control hydrolysis with equal total protein loading without cellulolytic enhancing activity (1-50 mg of cellulolytic protein/g of cellulose in PCS).

AA9 polypeptide enhancing activity can be determined using a mixture of

CELLUCLAST™ 1.5L (Novozymes A/S, Bagsvasrd, Denmark) and beta-glucosidase as the source of the cellulolytic activity, wherein the beta-glucosidase is present at a weight of at least 2-5% protein of the cellulase protein loading. In one aspect, the beta-glucosidase is an Aspergillus oryzae beta-glucosidase (e.g., recombinantly produced in Aspergillus oryzae according to WO 02/095014). In another aspect, the beta-glucosidase is an Aspergillus fumigatus beta-glucosidase (e.g., recombinantly produced in Aspergillus oryzae as described in WO 02/095014).

AA9 polypeptide enhancing activity can also be determined by incubating an AA9 polypeptide with 0.5% phosphoric acid swollen cellulose (PASC), 100 mM sodium acetate pH 5, 1 mM MnS0 ₄ , 0.1 % gallic acid, 0.025 mg/ml of Aspergillus fumigatus beta- glucosidase, and 0.01 % TRITON® X-100 (4-(1 , 1 ,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24-96 hours at 40°C followed by determination of the glucose released from the PASC.

AA9 polypeptide enhancing activity can also be determined according to WO 2013/028928 for high temperature compositions.

AA9 polypeptides enhance the hydrolysis of a cellulosic material catalyzed by enzyme having cellulolytic activity by reducing the amount of cellulolytic enzyme required to reach the same degree of hydrolysis preferably at least 1.01-fold, e.g., at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4- fold, at least 5-fold, at least 10-fold, or at least 20-fold.

The AA9 polypeptide can be used in the presence of a soluble activating divalent metal cation according to WO 2008/151043 or WO 2012/122518, e.g., manganese or copper.

The AA9 polypeptide can also be used in the presence of a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquor obtained from a pretreated lignocellulosic 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).

Beta-glucosidase: The term "beta-glucosidase" means a beta-D-glucoside glucohydrolase (E.C. 3.2.1.21) that catalyzes the hydrolysis of terminal non-reducing beta-D- glucose residues with the release of beta-D-glucose. Beta-glucosidase activity can be determined using p-nitrophenyl-beta-D-glucopyranoside as substrate according to the procedure of Venturi et al., 2002, J. Basic Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as 1.0 μηιοΐβ of p-nitrophenolate anion produced per minute at 25°C, pH 4.8 from 1 mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium citrate containing 0.01 % TWEEN® 20.

Beta-xylosidase: The term "beta-xylosidase" means a beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the exo-hydrolysis of short beta (1→4)- xylooligosaccharides to remove successive D-xylose residues from non-reducing termini. Beta-xylosidase activity can be determined using 1 mM p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate containing 0.01 % TWEEN® 20 at pH 5, 40°C. One unit of beta-xylosidase is defined as 1.0 μηιοΐβ of p-nitrophenolate anion produced per minute at 40°C, pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside in 100 mM sodium citrate containing 0.01 % TWEEN® 20.

cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.

Catalase: The term "catalase" means a hydrogen-peroxide:hydrogen-peroxide oxidoreductase (E.C. 1.11.1.6 or E.C. 1.11.1.21) that catalyzes the conversion of two hydrogen peroxides to oxygen and two waters.

Catalase activity can be determined by monitoring the degradation of hydrogen peroxide at 240 nm based on the following reaction:

2H ₂ 0 ₂ → 2H ₂ 0 + 0 ₂

The reaction is conducted in 50 mM phosphate pH 7 at 25°C with 10.3 mM substrate (H2O2). Absorbance is monitored spectrophotometrically within 16-24 seconds, which should correspond to an absorbance reduction from 0.45 to 0.4. One catalase activity unit can be expressed as one μηιοΐβ of H2O2 degraded per minute at pH 7.0 and 25°C.

Cellobiohydrolase: The term "cellobiohydrolase" means a 1 ,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176) that catalyzes the hydrolysis of 1 ,4- beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1 ,4-linked glucose containing polymer, releasing cellobiose from the reducing end (cellobiohydrolase I) or 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). Cellobiohydrolase activity can be determined according to the procedures described by Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et ai, 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.

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

Cellulolytic enzyme activity can be determined by measuring the increase in production/release of sugars during hydrolysis of a cellulosic material by cellulolytic enzyme(s) under the following conditions: 1-50 mg of cellulolytic enzyme protein/g of cellulose in pretreated corn stover (PCS) (or other pretreated cellulosic material) for 3-7 days at a suitable temperature such as 40°C-80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, and a suitable pH, such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0, compared to a control hydrolysis without addition of cellulolytic enzyme protein. Typical conditions are 1 ml reactions, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate pH 5, 1 mM MnS0 ₄ , 50°C, 55°C, or 60°C, 72 hours, sugar analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc.).

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

Control sequences: The term "control sequences" means nucleic acid sequences necessary for expression of a polynucleotide encoding a polypeptide of interest. Each control sequence may be native (i.e., from the same gene) or heterologous or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide or native or foreign to each other. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter, signal peptide sequence, and transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

Dissolved Oxygen Saturation Level: The saturation level of oxygen is determined at the standard partial pressure (0.21 atmosphere) of oxygen. The saturation level at the standard partial pressure of oxygen is dependent on the temperature and solute concentrations. In an embodiment where the temperature during hydrolysis is 50°C, the saturation level would typically be in the range of 5-5.5 mg oxygen per kg slurry, depending on the solute concentrations. Hence, a concentration of dissolved oxygen of 0.5 to 10% of the saturation level at 50°C corresponds to an amount of dissolved oxygen in a range from 0.025 ppm (0.5 x 5/100) to 0.55 ppm (10 x 5.5/100), such as, e.g., 0.05 to 0.165 ppm, and a concentration of dissolved oxygen of 10-70% of the saturation level at 50°C corresponds to an amount of dissolved oxygen in a range from 0.50 ppm (10 x 5/100) to 3.85 ppm (70 x 5.5/100), such as, e.g., 1 to 2 ppm. In an embodiment, oxygen is added in an amount in the range of 0.5 to 5 ppm, such as 0.5 to 4.5 ppm, 0.5 to 4 ppm, 0.5 to 3.5 ppm, 0.5 to 3 ppm, 0.5 to 2.5 ppm, or 0.5 to 2 ppm.

Endoglucanase: The term "endoglucanase" means a 4-(1 ,3; 1 ,4)-beta-D-glucan 4- glucanohydrolase (E.C. 3.2.1.4) that catalyzes endohydrolysis of 1 ,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, beta-1 ,4 bonds in mixed beta-1 ,3-1 ,4 glucans such as cereal beta-D-glucans or xyloglucans, and other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring reduction in substrate viscosity or increase in reducing ends determined by a reducing sugar assay (Zhang et al., 2006, Biotechnology Advances 24: 452-481). Endoglucanase activity can also be determined using carboxymethyl cellulose (CMC) as substrate according to the procedure of Ghose, 1987, Pure andAppl. Chem. 59: 257-268, at pH 5, 40°C.

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

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

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

Fragment: The term "fragment" means a polypeptide having one or more (e.g., several) amino acids absent from the amino and/or carboxyl terminus of a polypeptide, wherein the fragment has enzyme activity. In one aspect, a fragment contains at least 85%, at least 90%, or at least 95% of the number of amino acids of the polypeptide.

Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or

"hemicellulase" means one or more (e.g., several) enzymes that hydrolyze hemicellulose of a lignocellulosic material. See, for example, 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, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The substrates for these enzymes, hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & Appl. Chem. 59: 1739-1752, at a suitable temperature such as 40°C- 80°C, e.g., 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, or 80°C, and a suitable pH such as 4-9, e.g., 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0.

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

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

Laccase: The term "laccase" means a benzenediol:oxygen oxidoreductase (E.C. 1.10.3.2) that catalyzes the following reaction: 1 ,2- or 1 ,4-benzenediol + 02 = 1 ,2- or 1 ,4- benzosemiquinone + 2 H2O.

Laccase activity can be determined by the oxidation of syringaldazine (4,4 ^' -

[azinobis(methanylylidene)]bis(2,6-dimethoxyphenol)) to the corresponding quinone 4,4 ^' - [azobis(methanylylidene])bis(2,6-dimethoxycyclohexa-2,5-dien -1-one) by laccase. The

The reaction is conducted in 23 mM MES pH 5.5 at 30°C with 19 μΜ substrate (syringaldazine) and 1 g/L polyethylene glycol (PEG) 6000. The sample is placed in a spectrophotometer and the change in absorbance is measured at 530 nm every 15 seconds up to 90 seconds. One laccase unit is the amount of enzyme that catalyzes the conversion of 1 μηιοΐβ syringaldazine per minute under the specified analytical conditions.

Lignocellulosic material : The term "lignocellulosic material" means any material containing cellulose, hemicellulose, and lignin. The predominant polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third is pectin. The secondary cell wall, produced after the cell has stopped growing, also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose.

Cellulose is a homopolymer of anhydrocellobiose and thus a linear beta-(1 -4)-D- glucan, while hemicelluloses include a variety of compounds, such as xylans, xyloglucans, arabinoxylans, and mannans in complex branched structures with a spectrum of substituents. Although generally polymorphous, cellulose is found in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains.

Hemicelluloses usually hydrogen bond to cellulose, as well as to other hemicelluloses, which help stabilize the cell wall matrix.

Lignocellulose is generally found, for example, in the stems, leaves, hulls, husks, and cobs of plants or leaves, branches, and wood of trees. The lignocellulosic material can be, but is not limited to, agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, and wood (including forestry residue) (see, for example, Wiselogel et al., 1995, in Handbook on Bioethanol (Charles E. Wyman, editor), pp. 105-1 18, Taylor & Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, in Advances in Biochemical Engineering/Biotechnology, T. Scheper, managing editor, Volume 65, pp. 23-40, Springer- Verlag, New York).

Hemicelluloses include xylan, glucuronoxylan, arabinoxylan, glucomannan, and xyloglucan. These polysaccharides contain many different sugar monomers. Sugar monomers in hemicellulose can include xylose, mannose, galactose, rhamnose, and arabinose. Hemicelluloses contain most of the D-pentose sugars. Xylose is in most cases the sugar monomer present in the largest amount, although in softwoods mannose can be the most abundant sugar. Xylan contains a backbone of beta-(1-4)-linked xylose residues. Xylans of terrestrial plants are heteropolymers possessing a beta-(1-4)-D-xylopyranose backbone, which is branched by short carbohydrate chains. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides, composed of D- xylose, L-arabinose, D- or L-galactose, and D-glucose. Xylan-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-67. Hemicellulosic material is also known herein as "xylan-containing material".

In one aspect, the lignocellulosic material is any biomass material.

In an embodiment, the lignocellulosic material is agricultural residue, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill residue, waste paper, or wood (including forestry residue).

In another embodiment, the lignocellulosic material is arundo, bagasse, bamboo, corn cob, corn fiber, corn stover, miscanthus, rice straw, sugar cane straw, switchgrass, or wheat straw.

In another embodiment, the lignocellulosic material is aspen, eucalyptus, fir, pine, poplar, spruce, or willow.

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

The lignocellulosic material may be used as is or may be subjected to pretreatment, using conventional methods known in the art, as described herein. In a preferred aspect, the lignocellulosic material is pretreated.

Mature polypeptide: The term "mature polypeptide" means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. In one aspect, the mature polypeptide is amino acids 21 to 541 of SEQ ID NO: 30 based on the SignalP 3.0 (Bendtsen et al., 2004, J. Mol. Biol. 340: 783-795) that predicts amino acids 1 to 20 of SEQ ID NO: 30 are a signal peptide. In another aspect, the mature polypeptide is amino acids 27 to 375 of SEQ ID NO: 31 based on the SignalP 3.0 that predicts amino acids 1 to 26 of SEQ ID NO: 31 are a signal peptide. In another aspect, the mature polypeptide is amino acids 38 to 1347 of SEQ ID NO: 32 based on the SignalP 3.0 that predicts amino acids 1 to 37 of SEQ ID NO: 32 are a signal peptide. In another aspect, the mature polypeptide is amino acids 19 to 340 of SEQ ID NO: 33 based on the SignalP 3.0 that predicts amino acids 1 to 18 of SEQ ID NO: 33 are a signal peptide. In another aspect, the mature polypeptide is amino acids 21 to 348 of SEQ ID NO: 34 based on the SignalP 3.0 that predicts amino acids 1 to 20 of SEQ ID NO: 34 are a signal peptide. In another aspect, the mature polypeptide is amino acids 19 to 340 of SEQ ID NO: 35 based on the SignalP 3.0 that predicts amino acids 1 to 18 of SEQ ID NO: 35 are a signal peptide. In another aspect, the mature polypeptide is amino acids 21 to 350 of SEQ ID NO: 36 based on the SignalP 3.0 that predicts amino acids 1 to 20 of SEQ ID NO: 36 are a signal peptide. In another aspect, the mature polypeptide is amino acids 19 to 350 of SEQ ID NO: 37 based on the SignalP 3.0 that predicts amino acids 1 to 18 of SEQ ID NO: 37 are a signal peptide. In another aspect, the mature polypeptide is amino acids 17 to 574 of SEQ ID NO: 38 based on the SignalP 3.0 that predicts amino acids 1 to 16 of SEQ ID NO: 38 are a signal peptide. In another aspect, the mature polypeptide is amino acids 21 to 445 of SEQ ID NO: 39 based on the SignalP 3.0 that predicts amino acids 1 to 20 of SEQ ID NO: 39 are a signal peptide.

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

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

Peroxidase: The term "peroxidase" means an enzyme that converts a peroxide, e.g., hydrogen peroxide, to a less oxidative species, e.g., water. It is understood herein that a peroxidase encompasses a peroxide-decomposing enzyme. The term "peroxide- decomposing enzyme" is defined herein as a donor: peroxide oxidoreductase (E.C. number 1.11.1.x, wherein x=1-3, 5, 7-19, or 21) that catalyzes the reaction reduced substrate(2e ^_ ) + ROOR'→ oxidized substrate + ROH + R'OH; such as horseradish peroxidase that catalyzes the reaction phenol + H2O2→ quinone + H2O, and catalase that catalyzes the reaction H2O2 + H2O2 → O2 + 2H2O. In addition to hydrogen peroxide, other peroxides may also be decomposed by these enzymes.

Peroxidase activity can be determined by measuring the oxidation of 2,2'-azino-bis(3- ethylbenzthiazoline-6-sulfonic acid (ABTS) by a peroxidase in the presence of hydrogen peroxide as shown below. The reaction product ABTS _0X forms a blue-green color which can be quantified at 418 nm.

H2O2 + 2ABTS _red + 2H ⁺ → 2H ₂ 0 + 2ABTSox

The reaction is conducted in 0.1 M phosphate pH 7 at 30°C with 1.67 mM substrate (ABTS), 1.5 g/L TRITON® X-405, 0.88 mM hydrogen peroxide, and approximately 0.040 unit enzyme per ml. The sample is placed in a spectrophotometer and the change in absorbance is measured at 418 nm from 15 seconds up to 60 seconds. One peroxidase unit can be expressed as the amount of enzyme required to catalyze the conversion of 1 μηιοΐβ of hydrogen peroxide per minute under the specified analytical conditions.

Pretreated lignocellulosic material: The term "pretreated lignocellulosic material" means a lignocellulosic material derived from biomass by treatment with heat and dilute sulfuric acid, alkaline pretreatment, neutral pretreatment, or any pretreatment known in the art.

Pretreated corn stover: The term "Pretreated Corn Stover" or "PCS" means a lignocellulosic material derived from corn stover by treatment with heat and dilute sulfuric acid, alkaline pretreatment, neutral pretreatment, or any pretreatment known in the art.

Primeverose: The term "primeverose" means the compound 6-O-beta-D- xylopyranosyl-beta-D-glucopyranose with the following structure or a derivative thereof:

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

For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The parameters used are a gap open 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 -nobrief option) is used as the percent identity and is calculated as follows:

(Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment) Variant: The term "variant" means a polypeptide having enzyme activity comprising an alteration, i.e., a substitution, insertion, and/or deletion, at one or more (e.g., several) positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding an amino acid adjacent to and immediately following the amino acid occupying a position

Xylan-containing material: The term "xylan-containing material" means any material comprising a plant cell wall polysaccharide containing a backbone of beta-(1-4)- linked xylose residues. Xylans of terrestrial plants are heteropolymers possessing a beta-

(1-4)-D-xylopyranose backbone, which is branched by short carbohydrate chains. They comprise D-glucuronic acid or its 4-O-methyl ether, L-arabinose, and/or various oligosaccharides, composed of D-xylose, L-arabinose, D- or L-galactose, and D-glucose.

Xylan-type polysaccharides can be divided into homoxylans and heteroxylans, which include glucuronoxylans, (arabino)glucuronoxylans, (glucurono)arabinoxylans, arabinoxylans, and complex heteroxylans. See, for example, Ebringerova et al., 2005, Adv. Polym. Sci. 186: 1-

67.

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

Xylan degrading activity or xylanolytic activity: The term "xylan degrading activity" or "xylanolytic activity" means a biological activity that hydrolyzes xylan-containing material. The two basic approaches for measuring xylanolytic activity include: (1) measuring the total xylanolytic activity, and (2) measuring the individual xylanolytic activities (e.g., endoxylanases, beta-xylosidases, arabinofuranosidases, alpha-glucuronidases, acetylxylan esterases, feruloyi esterases, and alpha-glucuronyl esterases). Recent progress in assays of xylanolytic enzymes was summarized in several publications including Biely and Puchard, 2006, Journal of the Science of Food and Agriculture 86(1 1): 1636-1647; Spanikova and Biely, 2006, FEBS Letters 580(19): 4597-4601 ; Herrmann et ai, 1997, Biochemical Journal 321 : 375-381.

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

Xylan degrading activity can be determined by measuring the increase in hydrolysis of birchwood xylan (Sigma Chemical Co., Inc.) by xylan-degrading enzyme(s) under the following typical conditions: 1 ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic protein/g of substrate, 50 mM sodium acetate pH 5, 50°C, 24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide (PHBAH) assay as described by Lever, 1972, Anal. Biochem. 47: 273-279.

Xylanase: The term "xylanase" means a 1 ,4-beta-D-xylan-xylohydrolase (E.C.

3.2.1.8) that catalyzes the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans. Xylanase activity can be determined with 0.2% AZCL-arabinoxylan as substrate in 0.01 % TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 μηιοΐβ of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL- arabinoxylan as substrate in 200 mM sodium phosphate pH 6.

Reference to "about" a value or parameter herein includes aspects that are directed to that value or parameter per se. For example, description referring to "about X" includes the aspect "X".

As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that the aspects of the invention described herein include "consisting" and/or "consisting essentially of" aspects.

Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Detailed Description of the Invention

The present invention relates to processes for reducing production of primeverose during saccharification of a lignocellulosic material, the process comprising: saccharifying the lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase. In one aspect, the processes further comprise recovering the saccharified lignocellulosic material.

The present invention also relates to processes for saccharifying a lignocellulosic material, comprising: treating the lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta- xylosidase. In one aspect, the processes further comprise recovering the saccharified lignocellulosic material. Soluble products from the degradation of the lignocellulosic material can be separated from insoluble lignocellulosic material using methods known in the art such as, for example, centrifugation, filtration, or gravity settling.

The present invention also relates to processes for producing a fermentation product, the process comprising: (a) saccharifying a lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase; (b) fermenting the saccharified lignocellulosic material with one or more (e.g., several) fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

The present invention also relates to processes for fermenting a lignocellulosic material, the process comprising: fermenting the lignocellulosic material with one or more fermenting microorganisms, wherein the lignocellulosic material is saccharified with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase. In one aspect, the fermenting of the lignocellulosic material produces a fermentation product. In another aspect, the processes further comprise recovering the fermentation product from the fermentation.

Primeverose forms as a transglycosylation product when a retaining beta-xylosidase (i.e., GH3 family beta-xylosidase) acts on xylobiose. Retaining beta-xylosidases cleave xylobiose via a 2-step mechanism where a glycosyl-enzyme intermediate forms in the first step. Cleavage of this intermediate with water results in hydrolysis of xylobiose to two xylose molecules. Alternatively, cleavage of the glycosyl-enzyme intermediate with another sugar molecule (i.e., glucose) results in the formation of a transglycosylation product (primeverose) where a new sugar linkage is generated. Inverting beta-xylosidases (GH43 family) cleave via a 1-step mechanism and thus transglycosylation products do not form. Consequently, hydrolysis of xylobiose with an enzyme composition comprising a GH43 beta-xylosidase in the presence of other sugars prevents the production of primeverose and only results in the hydrolysis product of two xylose molecules. In contrast, hydrolysis of xylobiose with an enzyme composition comprising a GH3 beta-xylosidase in the presence of other sugars produces primeverose.

In one aspect, the amount of primeverose produced in the processes of the present invention is reduced at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, and most preferably at least 100%. The amount of primeverose can be determined by Dionex ion chromatography using a PA- 10 column and pulsed amperometry detection (IC-PAD, Dionex Corporation) using CHROMELEON™ Software (Dionex Corporation) as described in Example 5.

In another aspect, the amount of fermentable sugars in the processes of the present invention is increased at least 0.1 %, at least 0.2%, at least 0.5%, at least 1 %, at least 2.5%, at least 5%, or at least 10% from saccharification of the lignocellulosic material. The the amount of fermentable sugars can be determined by AM IN EX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc.).

Processing of Lignocellulosic Material

The processes of the present invention can be used to saccharify a lignocellulosic material to fermentable sugars and to convert the fermentable sugars to many useful fermentation products, e.g., fuel (ethanol, n-butanol, isobutanol, biodiesel, jet fuel) and/or platform chemicals (e.g., acids, alcohols, ketones, gases, oils, and the like). The production of a desired fermentation product from the lignocellulosic material typically involves pretreatment, enzymatic hydrolysis (saccharification), and fermentation.

The processing of the lignocellulosic material according to the present invention can be accomplished using methods conventional in the art. Moreover, the processes of the present invention can be implemented using any conventional biomass processing apparatus configured to operate in accordance with the invention. Hydrolysis (saccharification) and fermentation, separate or simultaneous, include, but are not limited to, separate hydrolysis and fermentation (SHF); simultaneous saccharification and fermentation (SSF); simultaneous saccharification and co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF); and direct microbial conversion (DMC), also sometimes called consolidated bioprocessing (CBP). SHF uses separate process steps to first enzymatically hydrolyze the lignocellulosic material to fermentable sugars, e.g., glucose, cellobiose, and pentose monomers, and then ferment the fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of the lignocellulosic material and the fermentation of sugars to ethanol are combined in one step (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, 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 carried out in the same reactor. The steps in an HHF process can be carried out at different temperatures, i.e., high temperature enzymatic saccharification followed by SSF at a lower temperature that the fermentation strain can tolerate. DMC combines all three processes (enzyme production, hydrolysis, and fermentation) in one or more (e.g., several) steps where the same organism is used to produce the enzymes for conversion of the lignocellulosic material to fermentable sugars and to convert the fermentable sugars into a final product (Lynd et al., 2002, Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein that any method known in the art comprising pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination thereof, can be used in the practicing the processes of the present invention.

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

Pretreatment. In practicing the processes of the present invention, any pretreatment process known in the art can be used to disrupt plant cell wall components of the lignocellulosic material (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 Technology 100: 10-18; Mosier et al., 2005, Bioresource Technology 96: 673-686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci. 9: 1621-1651 ; Yang and Wyman, 2008, Biofuels Bioproducts and Biorefining-Biofpr. 2: 26-40). The lignocellulosic material can also be subjected to particle size reduction, sieving, pre-soaking, wetting, washing, and/or conditioning prior to pretreatment using methods known in the art.

Conventional pretreatments include, but are not limited to, steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organosolv pretreatment, and biological pretreatment. Additional pretreatments include ammonia percolation, ultrasound, electroporation, microwave, supercritical CO2, supercritical H2O, ozone, ionic liquid, and gamma irradiation pretreatments.

The lignocellulosic material can be pretreated before hydrolysis and/or fermentation.

Pretreatment is preferably performed prior to the hydrolysis. Alternatively, the pretreatment can be carried out simultaneously with enzyme hydrolysis to release fermentable sugars, such as glucose, xylose, and/or cellobiose. In most cases the pretreatment step itself results in some conversion of biomass to fermentable sugars (even in absence of enzymes).

Steam Pretreatment. In steam pretreatment, the lignocellulosic material is heated to disrupt the plant cell wall components, including lignin, hemicellulose, and cellulose to make the cellulose and other fractions, e.g., hemicellulose, accessible to enzymes. The lignocellulosic material is passed to or through a reaction vessel where steam is injected to increase the temperature to the required temperature and pressure and is retained therein for the desired reaction time. Steam pretreatment is preferably performed at 140-250°C, e.g., 160-200°C or 170-190°C, where the optimal temperature range depends on optional addition of a chemical catalyst. Residence time for the steam pretreatment is preferably 1-60 minutes, e.g., 1-30 minutes, 1-20 minutes, 3-12 minutes, or 4-10 minutes, where the optimal residence time depends on the temperature and optional addition of a chemical catalyst. Steam pretreatment allows for relatively high solids loadings, so that the lignocellulosic material is generally only moist during the pretreatment. The steam pretreatment is often combined with an explosive discharge of the material after the pretreatment, which is known as steam explosion, that is, rapid flashing to atmospheric pressure and turbulent flow of the material to increase the accessible surface area by fragmentation (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent Application No. 2002/0164730). During steam pretreatment, hemicellulose acetyl groups are cleaved and the resulting acid autocatalyzes partial hydrolysis of the hemicellulose to monosaccharides and oligosaccharides. Lignin is removed to only a limited extent.

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

A chemical catalyst such as H2SO4 or SO2 (typically 0.3 to 5% w/w) is sometimes added prior to steam pretreatment, which decreases the time and temperature, increases the recovery, and improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. 113-1 16: 509- 523; Sassner et ai, 2006, Enzyme Microb. Technol. 39: 756-762). In dilute acid pretreatment, the lignocellulosic material is mixed with dilute acid, typically H2SO4, and water to form a slurry, heated by steam to the desired temperature, and after a residence time flashed to atmospheric pressure. The dilute acid pretreatment can be performed with a number of reactor designs, e.g., plug-flow reactors, counter-current reactors, or continuous counter-current shrinking bed reactors (Duff and Murray, 1996, Bioresource Technology 855: 1-33; Schell et al., 2004, Bioresource Technology 91 : 179-188; Lee et al., 1999, Adv. Bi∞hem. Eng. Biotechnol. 65: 93-115).

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

Lime pretreatment is performed with calcium oxide or calcium hydroxide at temperatures of 85-150°C and residence times 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.

Wet oxidation is a thermal pretreatment performed typically at 180-200°C for 5-15 minutes with addition of an oxidative agent such as hydrogen peroxide or over-pressure of oxygen (Schmidt and Thomsen, 1998, Bioresource 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). The pretreatment is performed preferably at 1-40% dry matter, e.g., 2-30% dry matter or 5-20% dry matter, and often the initial pH is increased by the addition of alkali such as sodium carbonate.

A modification of the wet oxidation pretreatment method, known as wet explosion (combination of wet oxidation and steam explosion) can handle dry matter up to 30%. In wet explosion, the oxidizing agent is introduced during pretreatment after a certain residence time. The pretreatment is then ended by flashing to atmospheric pressure (WO 2006/032282).

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

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

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 U.S. Published Application 2002/0164730.

In one aspect, the chemical pretreatment is preferably carried out as a dilute acid treatment, and more preferably as a continuous dilute acid treatment. The acid is typically sulfuric acid, but other acids can also be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof. Mild acid treatment is conducted in the pH range of preferably 1-5, e.g., 1-4 or 1-2.5. In one aspect, the acid concentration is in the range from preferably 0.01 to 10 wt. % acid, e.g., 0.05 to 5 wt. % acid or 0.1 to 2 wt. % acid. The acid is contacted with the lignocellulosic material and held at a temperature in the range of preferably 140-200°C, e.g., 165-190°C, for periods ranging from 1 to 60 minutes.

In another aspect, pretreatment takes place in an aqueous slurry. In preferred aspects, the lignocellulosic material is present during pretreatment in amounts preferably between 10-80 wt. %, e.g., 20-70 wt. % or 30-60 wt. %, such as around 40 wt. %. The pretreated lignocellulosic material can be unwashed or washed using any method known in the art, e.g., washed with water.

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

The lignocellulosic material can be pretreated both physically (mechanically) and chemically. Mechanical or physical pretreatment can be coupled with steaming/steam explosion, hydrothermolysis, dilute or mild acid treatment, high temperature, high pressure treatment, irradiation (e.g., microwave irradiation), or combinations thereof. In one aspect, high pressure means pressure in the range of preferably about 100 to about 400 psi, e.g., about 150 to about 250 psi. In another aspect, high temperature means temperature in the range of about 100 to about 300°C, e.g., about 140 to about 200°C. In a preferred aspect, mechanical or physical pretreatment is performed in a batch-process using a steam gun hydrolyzer system that uses high pressure and high temperature as defined above, e.g., a Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden. The physical and chemical pretreatments can be carried out sequentially or simultaneously, as desired.

Accordingly, in a preferred aspect, the lignocellulosic 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.

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 lignocellulosic material. Biological pretreatment techniques can involve applying lignin-solubilizing microorganisms and/or enzymes (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol. 39: 295-333; McMillan, J. D., 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds., ACS Symposium Series 566, American Chemical Society, Washington, DC, chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering/Biotechnology, Scheper, T., ed., Springer- Verlag Berlin Heidelberg, Germany, 65: 207-241 ; Olsson and Hahn-Hagerdal, 1996, Enz. Microb. Tech. 18: 312-331 ; and Vallander and Eriksson, 1990, Adv. Biochem. Eng./Biotechnol. 42: 63-95).

Saccharification. In the hydrolysis step, also known as saccharification, the lignocellulosic material, e.g., pretreated, is hydrolyzed to break down cellulose and/or hemicellulose to fermentable sugars, such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides. The hydrolysis is performed enzymatically by one or more enzyme compositions in one or more stages. The hydrolysis can be carried out as a batch process or series of batch processes. The hydrolysis can be carried out as a fed batch or continuous process, or series of fed batch or continuous processes, where the lignocellulosic material is fed gradually to, for example, a hydrolysis solution containing an enzyme composition. In an embodiment, the saccharification is a continuous saccharification in which a lignocellulosic material and a cellulolytic enzyme composition are added at different intervals throughout the saccharification and the hydrolysate is removed at different intervals throughout the saccharification. The removal of the hydrolysate may occur prior to, simultaneously with, or after the addition of the lignocellulosic material and the enzyme composition.

Enzymatic hydrolysis is preferably carried out in a suitable aqueous environment under conditions that can be readily determined by one skilled in the art. In one aspect, hydrolysis is performed under conditions suitable for the activity of the enzymes(s), i.e., optimal for the enzyme(s).

The saccharification is generally performed in stirred-tank reactors or fermentors under controlled pH, temperature, and mixing conditions. Suitable process time, temperature and pH conditions can readily be determined by one skilled in the art. For example, the total saccharification time can last up to 200 hours, but is typically performed for preferably about 4 to about 120 hours, e.g., about 12 to about 96 hours or about 24 to about 72 hours. The temperature is in the range of preferably about 25°C to about 80°C, e.g., about 30°C to about 70°C, about 40°C to about 60°C, or about 50°C to about 55°C. The pH is in the range of preferably about 3 to about 9, e.g., about 3.5 to about 8, about 4 to about 7, about 4.2 to about 6, or about 4.3 to about 5.5.

The dry solids content is in the range of preferably about 5 to about 50 wt. %, e.g., about 10 to about 40 wt. % or about 20 to about 30 wt. %.

In one aspect, the saccharification is performed in the presence of dissolved oxygen at a concentration of at least 0.5% of the saturation level.

In an embodiment of the invention the dissolved oxygen concentration during saccharification is in the range of at least 0.5% up to 30% of the saturation level, such as at least 1 % up to 25%, at least 1 % up to 20%, at least 1 % up to 15%, at least 1 % up to 10%, at least 1 % up to 5%, and at least 1 % up to 3% of the saturation level. In a preferred embodiment, the dissolved oxygen concentration is maintained at a concentration of at least 0.5% up to 30% of the saturation level, such as at least 1 % up to 25%, at least 1 % up to 20%, at least 1 % up to 15%, at least 1 % up to 10%, at least 1 % up to 5%, and at least 1 % up to 3% of the saturation level during at least 25% of the saccharification period, such as at least 50% or at least 75% of the saccharification period. When the enzyme composition comprises an oxidoreductase the dissolved oxygen concentration may be higher up to 70% of the saturation level.

Oxygen is added to the vessel to achieve the desired concentration of dissolved oxygen during saccharification. Maintaining the dissolved oxygen level within a desired range can be accomplished by aeration of the vessel, tank or the like by adding compressed air through a diffuser or sparger, or by other known methods of aeration. The aeration rate can be controlled on the basis of feedback from a dissolved oxygen sensor placed in the vessel/tank, or the system can run at a constant rate without feedback control. In the case of a hydrolysis train consisting of a plurality of vessels/tanks connected in series, aeration can be implemented in one or more or all of the vessels/tanks. Oxygen aeration systems are well known in the art. Accordingly, any suitable aeration system may be used. Commercial aeration systems are designed by, e.g., Chemineer, Derby, England, and built by, e.g., Paul Mueller Company. Fermentation. The fermentable sugars obtained from the hydrolyzed lignocellulosic material can be fermented by one or more (e.g., several) fermenting microorganisms capable of fermenting the sugars directly or indirectly into a desired fermentation product. "Fermentation" or "fermentation process" refers to any fermentation process or any process comprising a fermentation step. Fermentation processes also include fermentation processes used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. The fermentation conditions depend on the desired fermentation product and fermenting organism and can easily be determined by one skilled in the art.

In the fermentation step, sugars, released from the lignocellulosic material as a result of the pretreatment and enzymatic hydrolysis steps, are fermented to a product, e.g., ethanol, by a fermenting organism, such as yeast. Hydrolysis (saccharification) and fermentation can be separate or simultaneous.

Any suitable hydrolyzed lignocellulosic material can be used in the fermentation step in practicing the present invention. The material is generally selected based on economics, i.e. , costs per equivalent sugar potential, and recalcitrance to enzymatic conversion.

The term "fermentation medium" is understood herein to refer to a medium before the fermenting microorganism(s) is(are) added, such as, a medium resulting from a saccharification process, as well as a medium used in a simultaneous saccharification and fermentation process (SSF).

"Fermenting microorganism" refers to any microorganism, including bacterial and fungal organisms, suitable for use in a desired fermentation process to produce a fermentation product. The fermenting organism can be hexose and/or pentose fermenting organisms, or a combination thereof. Both hexose and pentose fermenting organisms are well known in the art. Suitable fermenting microorganisms can ferment, i.e., convert, sugars, such as glucose, xylose, xylulose, arabinose, maltose, mannose, galactose, and/or oligosaccharides, directly or indirectly into the desired fermentation product. Examples of bacterial and fungal fermenting organisms producing ethanol are described by Lin et al., 2006, Appl. Microbiol. Biotechnol. 69: 627-642.

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

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

Examples of bacteria that can efficiently ferment hexose and pentose to ethanol include, for example, Bacillus coagulans, Clostridium acetobutylicum, Clostridium thermocellum, Clostridium phytofermentans, Geobacillus sp., Thermoanaerobacter saccharolyticum, and Zymomonas mobilis (Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, DC, 179-212).

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

Commercially available yeast suitable for ethanol production include, e.g., BIO-FERM® AFT and XR (Lallemand Specialities, Inc., USA), ETHANOL RED® yeast (Lesaffre et Compagnie, France), FALI® (AB Mauri Food Inc., USA), FERMIOL® (Rymco International AG, Denmark), GERT STRAND™ (Gert Strand AB, Sweden), and SUPERSTART™ and THERMOSACC® fresh yeast (Lallemand Specialities, Inc., USA).

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

The cloning of heterologous genes into various fermenting microorganisms has led to the construction of organisms capable of converting hexoses and pentoses to ethanol (co- fermentation) (Chen and Ho, 1993, Appl. Biochem. Biotechnol. 39-40: 135-147; Ho et ai, 1998, Appl. Environ. Microbiol. 64: 1852-1859; Kotter and Ciriacy, 1993, Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et ai, 1995, Appl. Environ. Microbiol. 61 : 4184-4190; Kuyper et ai, 2004, FEMS Yeast Research 4: 655-664; Beall et ai, 1991 , Biotech. Bioeng. 38: 296-303; Ingram et ai, 1998, Biotechnol. Bioeng. 58: 204-214; Zhang et ai, 1995, Science 267: 240-243; Deanda et ai, 1996, Appl. Environ. Microbiol. 62: 4465-4470; WO 03/062430).

In another aspect, the fermenting organism comprises one or more polynucleotides encoding one or more cellulolytic enzymes, hemicellulolytic enzymes, and accessory enzymes described herein.

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

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

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

A fermentation stimulator can be used in combination with any of the processes described herein to further improve the fermentation process, and in particular, the performance of the fermenting microorganism, such as, rate enhancement and ethanol yield. A "fermentation stimulator" refers to stimulators for growth of the fermenting microorganisms, in particular, yeast. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenate, nicotinic acid, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B, C, D, and E. See, for example, Alfenore et al., Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process, Springer-Verlag (2002), which is hereby incorporated by reference. Examples of minerals include minerals and mineral salts that can supply nutrients comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

Fermentation products: A fermentation product can be any substance derived from the fermentation. The fermentation product can be, without limitation, an alcohol (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1 ,3- propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); an alkane (e.g. , pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), a cycloalkane (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane), an alkene {e.g., pentene, hexene, heptene, and octene); an amino acid {e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); a gas {e.g., methane, hydrogen (H ₂ ), carbon dioxide (CO2), and carbon monoxide (CO)); isoprene; a ketone {e.g., acetone); an organic acid {e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D- gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); and polyketide.

In one aspect, the fermentation product is an alcohol. The term "alcohol" encompasses a substance that contains one or more hydroxyl moieties. The alcohol can be, but is not limited to, n-butanol, isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol, glycerin, glycerol, 1 ,3-propanediol, sorbitol, xylitol. See, for example, 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): 1 17-124; Ezeji et al., 2003, World Journal of Microbiology and Biotechnology 19(6): 595-603.

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

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

In another aspect, the fermentation product is an alkene. The alkene may be an unbranched or a branched alkene. The alkene can be, but is not limited to, pentene, hexene, heptene, or octene.

In another 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.

In another 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.

In another aspect, the fermentation product is isoprene.

In another aspect, the fermentation product is a ketone. The term "ketone" encompasses a substance that contains one or more ketone moieties. The ketone can be, but is not limited to, acetone.

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

In another aspect, the fermentation product is polyketide.

Recovery. The fermentation product(s) can be optionally recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For example, alcohol is separated from the fermented lignocellulosic material and purified by conventional methods of distillation. Ethanol with a purity of up to about 96 vol. % can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol, i.e., potable neutral spirits, or industrial ethanol. GH43 Beta-Xylosidases

In the processes of the present invention, any GH43 beta-xylosidase may be used. The GH43 beta-xylosidase can be obtained from any source, especially microorganisms of any genus. For purposes of the present invention, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In a preferred aspect, the GH43 beta-xylosidase obtained from a given source is secreted extracellularly.

The GH43 beta-xylosidase may be a bacterial beta-xylosidase. For example, the GH43 beta-xylosidase may be a gram positive bacterial beta-xylosidase such as a Bacillus, Clostridium, Corynebacterium, Enterococcus, Geobacillus, Lactobacillus, Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, or Streptomyces beta-xylosidase, or a Gram negative bacterial beta-xylosidase such as an E. coli, Campylobacter, Flavobacterium, Fusobacterium, Helicobacter, llyobacter, Neisseria, Pseudomonas, Salmonella, or Ureaplasma beta-xylosidase.

In a preferred aspect, the GH43 beta-xylosidase is a Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus cereus, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus polymyxa, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Geobacillus stearothermophilus, Lactobacillus plantarum, Streptococcus equisimilis, Streptococcus pyogenes, Streptococcus uberis, or Streptococcus equi subsp. Zooepidemicus beta-xylosidase. Bacterial GH43 beta-xylosidases can be obtained from Alkaliphilus metalliredigens (UniProt A6TTC7), Bacillus halodurans (UniProt Q9K6P5), Bacillus pumilus (UniProt P07129), Bacillus subtilis (UniProt P94489), Bacteroides ovatus (UniProt P49943), Bifidobacterium adolescentis (UniProt A1A0H6), Bifidobacterium animalis (SwissProt B2ECL1), Butyrivibrio fibrisolvens (UniProt P45982), Caldicellulosiruptor saccharolyticus (UniProt 030426), Caulobacter crescentus (SwissProt Q9A9J1), Cellvibrio japonicus (UniProt B3PDB2), Clostridium stercorarium (UniProt Q76EC8), Enterobacter sp. (UniProt V5J3M5), Geobacillus stearothermophilus (UniProt Q09LX0 and SwissProt Q09LX0), Geobacillus thermoleovorans (UniProt Q2I2N4), Lactobacillus brevis (UniProt Q03N89), Niveispirillum irakense (UniProt Q9LAE3), Prevotella bryantii (SwissProt P48791), Selenomonas ruminantium (UniProt 052575), Sphingobacterium sp. HP455 (UniProt R4P2Z4), and Thermobifida fusca (UniProt Q47PG8).

The GH43 beta-xylosidase may also be a fungal beta-xylosidase, and more preferably a yeast beta-xylosidase such as a Candida, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia beta-xylosidase; or more preferably a filamentous fungal beta-xylosidase such as an Acremonium, Agaricus, Alternaria, Aspergillus, Aureobasidium, Botryospaeria, Ceriporiopsis, Chaetomidium, Chrysosporium, Claviceps, Cochliobolus, Coprinopsis, Coptotermes, Corynascus, Cryphonectria, Cryptococcus, Diplodia, Exidia, Filibasidium, Fusarium, Gibberella, Holomastigotoides, Humicola, Irpex, Lentinula, Leptospaeria, Magnaporthe, Melanocarpus, Meripilus, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Phanerochaete, Piromyces, Poitrasia, Pseudoplectania, Pseudotrichonympha, Rhizomucor, Rhizopus, Schizophyllum, Scytalidium, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Trichophaea, Verticillium, Volvariella, or Xylaria beta-xylosidase.

In a preferred aspect, the GH43 beta-xylosidase is a Saccharomyces carlsbergensis,

Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, or Saccharomyces oviformis beta- xylosidase.

In another preferred aspect, the GH43 beta-xylosidase is an Acremonium cellulolyticus, Aspergillus aculeatus, Aspergillus awamori, Aspergillus fischeri, Aspergillus flavus, Aspergillus foetidus, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae (Swiss-Prot Accession number P78581), Aspergillus usamii, Aspergillus ustus, Aspergillus versicolor, Chrysosporium keratinophilum, Chrysosporium lucknowense, Chrysosporium tropicum, Chrysosporium merdarium, Chrysosporium inops, Chrysosporium pannicola, Chrysosporium queenslandicum, Chrysosporium zonatum, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium solani, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Fusarium verticil/iodides, Gibberella zeae, Humicola grisea, Humicola insolens, Humicola lanuginosa, Irpex lacteus, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Paecilomyces variotii, Penicillium charlesii, Penicillium chrysogenum, Penicillium expansum, Penicillium funiculosum, Penicillium javanicum, Penicillium notatum, Penicillium oxalicum, Penicillium purpurogenum, Penicillium restrictum, Penicillium variabile, Phanerochaete chrysosporium, Rasamsonia emersonii, Rhizopus oryzae, Talaromyces emersonii, Thermoascus aurantiacus, Thielavia achromatica, Thielavia albomyces, Thielavia albopilosa, Thielavia australeinsis, Thielavia fimeti, Thielavia microspora, Thielavia ovispora, Thielavia peruviana, Thielavia spededonium, Thielavia setosa, Thielavia subthermophila, Thielavia terrestris, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, or Trichoderma viride beta-xylosidase.

Filamentous fungal GH43 beta-xylosidases can be obtained from Aspergillus fumigatus (SwissProt Q4X0K2 and SwissProt A0A0J5SK50), Aspergillus oryzae (UniProt Q2URT3), Cochliobolus carbonum (UniProt 093912), Fusarium graminearum (UniProt I 1 S3S2), Fusarium oxysporum (UniProt J9NHC4), Fusarium verticillioides (UniProt W7MVU8, SwissProt W7MCN3, and SwissProt W7MN47), Humicola insolens (UniProt V9TT49 and V9TNS0), Paecilomyces thermophila (UniProt F1APW0), Penicillium herquei (UniProt Q870E8), Penicillium purpurogenum (UniProt K7WBC5), Rasamsonia emersonii (SwissProt A0A0F4YX49), and Thermomyces lanuginosus (UniProt G9B187).

In an embodiment, the GH43 beta-xylosidase is selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to the polypeptide of

SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39;

(b) a polypeptide comprising the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39;

(c) a variant of the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions; and

(d) a fragment of the polypeptide of (a), (b), or (c) that has GH43 beta-xylosidase activity. In one aspect, the GH43 beta-xylosidase has a sequence identity to the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23,

24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36,

37, 38, or 39 of 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%, which have GH43 beta-xylosidase activity. In another aspect, the GH43 beta-xylosidase differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39.

In another aspect, the GH43 beta-xylosidase comprises or consists of the amino acid sequence of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,

25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39 or an allelic variant thereof; or is a fragment thereof having GH43 beta-xylosidase activity. In another aspect, the polypeptide comprises or consists of the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25,

26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39.

In another embodiment, the present invention relates to variants of the polypeptide of

SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37,

38, or 39 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions. In one aspect, the number of amino acid substitutions, deletions and/or insertions introduced into the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39 is up to 10, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. The amino acid changes may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the groups of basic amino acids (arginine, lysine and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine and valine), aromatic amino acids (phenylalanine, tryptophan and tyrosine), and small amino acids (glycine, alanine, serine, threonine and methionine). Amino acid substitutions that do not generally alter specific activity are known in the art and are described, for example, by H. Neurath and R.L. Hill, 1979, In, The Proteins, Academic Press, New York. Common substitutions are Ala/Ser, Val/lle, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that the physico-chemical properties of the polypeptides are altered. For example, amino acid changes may improve the thermal stability of the polypeptide, alter the substrate specificity, change the pH optimum, and the like.

Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for GH43 beta-xylosidase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152- 2156; WO 95/17413; or WO 95/22625. Other methods that can be used include error-prone PCR, phage display {e.g., Lowman et al., 1991 , Biochemistry 30: 10832-10837; U.S. Patent 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).

Mutagenesis/shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide.

It will be understood that for the aforementioned species the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

Strains of these species are readily accessible to the public in a number of culture collections, such as the American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

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

Beta-xylosidases also include fused polypeptides or cleavable fusion polypeptides in which another polypeptide is fused at the N-terminus or the C-terminus of the beta- xylosidase or fragment thereof. A fused polypeptide is produced by fusing a nucleotide sequence (or a portion thereof) encoding another polypeptide to a nucleotide sequence (or a portion thereof) encoding a beta-xylosidase. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator.

A fusion polypeptide can further comprise a cleavage site. Upon secretion of the fusion protein, the site is cleaved releasing the beta-xylosidase from the fusion protein. Examples of cleavage sites include, but are not limited to, a Kex2 site that encodes the dipeptide Lys-Arg (Martin et al. , 2003, J. Ind. Microbiol. Biotechnol. 3: 568-76; Svetina et al. , 2000, J. Biotechnol. 76: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et ai, 1995, Biotechnology 13: 498-503; and Contreras et ai, 1991 , Biotechnology 9: 378-381), an lle-(Glu or Asp)-Gly-Arg site (SEQ ID NO: 51), which is cleaved by a Factor Xa protease after the arginine residue (Eaton et ai, 1986, Biochem. 25: 505-512); an Asp- Asp-Asp-Asp- Lys site (SEQ ID NO: 52), which is cleaved by an enterokinase after the lysine (Collins-Racie et ai, 1995, Biotechnology 13: 982-987); a His- Tyr-Glu site or His-Tyr-Asp site, which is cleaved by Genenase I (Carter et ai, 1989, Proteins: Structure, Function, and Genetics 6: 240-248); a Leu-Val-Pro-Arg-Gly-Ser site (SEQ ID NO: 53), which is cleaved by thrombin after the Arg (Stevens, 2003, Drug Discovery World 4: 35-48;; a Glu-Asn-Leu-Tyr-Phe-Gln-Gly site (SEQ ID NO: 54), which is cleaved by TEV protease after the Gin (Stevens, 2003, supra); and a Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro site (SEQ ID NO: 55), which is cleaved by a genetically engineered form of human rhinovirus 3C protease after the Gin (Stevens, 2003, supra).

Enzyme Compositions

In the processes of the present invention, the enzyme composition may comprise any protein involved in the processing of a lignocellulosic material to fermentable sugars, e.g., glucose and xylose.

In one aspect, the enzyme composition comprises or further comprises one or more

(e.g., several) proteins selected from the group consisting of a cellulase, an AA9 polypeptide, a hemicellulase, an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin. In another aspect, the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase. In another aspect, the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, and a xylanase. In another aspect, the oxidoreductase is preferably one or more (e.g., several) enzymes selected from the group consisting of a catalase, a laccase, and a peroxidase.

In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) cellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) cellulolytic enzymes and one or more (e.g., several) hemicellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises one or more (e.g., several) enzymes selected from the group of cellulolytic enzymes and hemicellulolytic enzymes. In another aspect, the enzyme composition comprises or further comprises an endoglucanase. In another aspect, the enzyme composition comprises or further comprises a cellobiohydrolase. In another aspect, the enzyme composition comprises or further comprises a beta-glucosidase. In another aspect, the enzyme composition comprises or further comprises an AA9 polypeptide. In another aspect, the enzyme composition comprises or further comprises an endoglucanase and an AA9 polypeptide. In another aspect, the enzyme composition comprises or further comprises a cellobiohydrolase and an AA9 polypeptide. In another aspect, the enzyme composition comprises or further comprises a beta-glucosidase and an AA9 polypeptide. In another aspect, the enzyme composition comprises or further comprises an endoglucanase and a cellobiohydrolase. In another aspect, the enzyme composition comprises or further comprises an endoglucanase I, an endoglucanase II, or a combination of an endoglucanase I and an endoglucanase II, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. In another aspect, the enzyme composition comprises or further comprises an endoglucanase and a beta-glucosidase. In another aspect, the enzyme composition comprises or further comprises an endoglucanase I, an endoglucanase II, or a combination of an endoglucanase I and an endoglucanase II, and a beta-glucosidase. In another aspect, the enzyme composition comprises or further comprises a beta-glucosidase and a cellobiohydrolase. In another aspect, the enzyme composition comprises or further comprises a beta-glucosidase and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. In another aspect, the enzyme composition comprises or further comprises an endoglucanase, an AA9 polypeptide, and a cellobiohydrolase. In another aspect, the enzyme composition comprises or further comprises an endoglucanase I, an endoglucanase II, or a combination of an endoglucanase I and an endoglucanase II, an AA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. In another aspect, the enzyme composition comprises or further comprises an endoglucanase, a beta-glucosidase, and an AA9 polypeptide. In another aspect, the enzyme composition comprises or further comprises a beta-glucosidase, an AA9 polypeptide, and a cellobiohydrolase. In another aspect, the enzyme composition comprises or further comprises a beta-glucosidase, an AA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. In another aspect, the enzyme composition comprises or further comprises an endoglucanase, a beta-glucosidase, and a cellobiohydrolase. In another aspect, the enzyme composition comprises or further comprises an endoglucanase I, an endoglucanase II, or a combination of an endoglucanase I and an endoglucanase II, a beta-glucosidase, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II. In another aspect, the enzyme composition comprises or further comprises an endoglucanase, a cellobiohydrolase, a beta-glucosidase, and an AA9 polypeptide. In another aspect, the enzyme composition comprises or further comprises an endoglucanase I, an endoglucanase II, or a combination of an endoglucanase I and an endoglucanase II, a beta-glucosidase, an AA9 polypeptide, and a cellobiohydrolase I, a cellobiohydrolase II, or a combination of a cellobiohydrolase I and a cellobiohydrolase II.

In another aspect, the enzyme composition comprises or further comprises an acetylmannan esterase. In another aspect, the enzyme composition comprises or further comprises an acetylxylan esterase. In another aspect, the enzyme composition comprises or further comprises an arabinanase {e.g., alpha-L-arabinanase). In another aspect, the enzyme composition comprises or further comprises an arabinofuranosidase (e.g., alpha-L- arabinofuranosidase). In another aspect, the enzyme composition comprises or further comprises a coumaric acid esterase. In another aspect, the enzyme composition comprises or further comprises a feruloyl esterase. In another aspect, the enzyme composition comprises or further comprises a galactosidase (e.g., alpha-galactosidase and/or beta- galactosidase). In another aspect, the enzyme composition comprises or further comprises a glucuronidase (e.g., alpha-D-glucuronidase). In another aspect, the enzyme composition comprises or further comprises a glucuronoyl esterase. In another aspect, the enzyme composition comprises or further comprises a mannanase. In another aspect, the enzyme composition comprises or further comprises a mannosidase (e.g., beta-mannosidase). In another aspect, the enzyme composition comprises or further comprises a xylanase. In an embodiment, the xylanase is a Family 10 xylanase. In another embodiment, the xylanase is a Family 1 1 xylanase.

In another aspect, the enzyme composition comprises or further comprises an esterase. In another aspect, the enzyme composition comprises or further comprises an expansin. In another aspect, the enzyme composition comprises or further comprises a ligninolytic enzyme. In an embodiment, the ligninolytic enzyme is a manganese peroxidase. In another embodiment, the ligninolytic enzyme is a lignin peroxidase. In another embodiment, the ligninolytic enzyme is a H ₂ 02-producing enzyme. In another aspect, the enzyme composition comprises or further comprises a pectinase. In another aspect, the enzyme composition comprises or further comprises an oxidoreductase. In an embodiment, the oxidoreductase is a catalase. In another embodiment, the oxidoreductase is a laccase. In another embodiment, the oxidoreductase is a peroxidase. In another aspect, the enzyme composition comprises or further comprises a protease. In another aspect, the enzyme composition comprises or further comprises a swollenin.

In the processes of the present invention, the enzyme(s) can be added prior to or during saccharification, saccharification and fermentation, or fermentation.

One or more (e.g., several) components of the enzyme composition may be native proteins, recombinant proteins, or a combination of native proteins and recombinant proteins. For example, one or more (e.g., several) components may be native proteins of a cell, which is used as a host cell to express recombinantly one or more (e.g., several) other components of the enzyme composition. It is understood herein that the recombinant proteins may be heterologous (e.g., foreign) and/or native to the host cell. One or more (e.g., several) components of the enzyme composition may be produced as monocomponents, which are then combined to form the enzyme composition. The enzyme composition may be a combination of multicomponent and monocomponent protein preparations. The enzymes used in the processes of the present invention may be in any form suitable for use, such as, for example, a fermentation broth formulation or a cell composition, a cell lysate with or without cellular debris, a semi-purified or purified enzyme preparation, or a host cell as a source of the enzymes. The enzyme composition may be a dry powder or granulate, a non-dusting granulate, a liquid, a stabilized liquid, or a stabilized protected enzyme. Liquid enzyme preparations may, for instance, be stabilized by adding stabilizers such as a sugar, a sugar alcohol or another polyol, and/or lactic acid or another organic acid according to established processes.

The optimum amounts of the enzymes depend on several factors including, but not limited to, the mixture of cellulolytic enzymes and/or hemicellulolytic enzymes, the lignocellulosic material, the concentration of lignocellulosic material, the pretreatment(s) of the lignocellulosic material, temperature, time, pH, and inclusion of a fermenting organism (e.g., for Simultaneous Saccharification and Fermentation).

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

In another aspect, an effective amount of a GH43 beta-xylosidase to the lignocellulosic material is about 0.01 to about 50.0 mg, e.g., about 0.01 to about 40 mg, about 0.01 to about 30 mg, about 0.01 to about 20 mg, about 0.01 to about 10 mg, about 0.01 to about 5 mg, about 0.025 to about 1.5 mg, about 0.05 to about 1.25 mg, about 0.075 to about 1.25 mg, about 0.1 to about 1.25 mg, about 0.15 to about 1.25 mg, or about 0.25 to about 1.0 mg per g of the lignocellulosic material.

In another aspect, an effective amount of a GH43 beta-xylosidase to cellulolytic or hemicellulolytic enzyme is about 0.005 to about 1.0 g, e.g., about 0.01 to about 1.0 g, about 0.15 to about 0.75 g, about 0.15 to about 0.5 g, about 0.1 to about 0.5 g, about 0.1 to about 0.25 g, or about 0.05 to about 0.2 g per g of cellulolytic or hemicellulolytic enzyme.

The polypeptides having cellulolytic enzyme activity or hemicellulolytic enzyme activity as well as other proteins/polypeptides useful in the degradation of the lignocellulosic material, e.g., AA9 polypeptides can be derived or obtained from any suitable origin, including, archaeal, bacterial, fungal, yeast, plant, or animal origin. The term "obtained" also means herein that the enzyme may have been produced recombinantly in a host organism employing methods described herein, wherein the recombinantly produced enzyme is either native or foreign to the host organism or has a modified amino acid sequence, e.g., having one or more (e.g., several) amino acids that are deleted, inserted and/or substituted, i.e., a recombinantly produced enzyme that is a mutant and/or a fragment of a native amino acid sequence or an enzyme produced by nucleic acid shuffling processes known in the art. Encompassed within the meaning of a native enzyme are natural variants and within the meaning of a foreign enzyme are variants obtained by, e.g., site-directed mutagenesis or shuffling.

Each polypeptide may be a bacterial polypeptide. For example, each polypeptide may be a Gram-positive bacterial polypeptide having enzyme activity, or a Gram-negative bacterial polypeptide having enzyme activity.

Each polypeptide may also be a fungal polypeptide, e.g., a yeast polypeptide or a filamentous fungal polypeptide.

Chemically modified or protein engineered mutants of polypeptides may also be used.

One or more (e.g., several) components of the enzyme composition may be a recombinant component, i.e., produced by cloning of a DNA sequence encoding the single component and subsequent cell transformed with the DNA sequence and expressed in a host (see, for example, WO 91/17243 and WO 91/17244). The host can be a heterologous host (enzyme is foreign to host), but the host may under certain conditions also be a homologous host (enzyme is native to host). Monocomponent cellulolytic proteins may also be prepared by purifying such a protein from a fermentation broth.

In one aspect, the one or more (e.g., several) cellulolytic enzymes comprise a commercial cellulolytic enzyme preparation. Examples of commercial cellulolytic enzyme preparations suitable for use in the present invention include, for example, CELLIC® CTec (Novozymes A/S), CELLIC® CTec2 (Novozymes A/S), CELLIC® CTec3 (Novozymes A/S), CELLUCLAST™ (Novozymes A/S), NOVOZYM™ 188 (Novozymes A/S), SPEZYME™ CP (Genencor Int.), ACCELLERASE™ TRIO (DuPont), FILTRASE® NL (DSM); METHAPLUS® S/L 100 (DSM), ROHAMENT™ 7069 W (Rohm GmbH), or ALTERNAFUEL® CMAX3™ (Dyadic International, Inc.). The cellulolytic enzyme preparation is added in an amount effective from about 0.001 to about 5.0 wt. % of solids, e.g., about 0.025 to about 4.0 wt. % of solids or about 0.005 to about 2.0 wt. % of solids.

Examples of bacterial endoglucanases that can be used in the processes of the present invention, include, but are not limited to, Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO 93/15186; U.S. Patent No. 5,275,944; WO 96/02551 ; U.S. Patent 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).

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: 1 1-22), Trichoderma reesei Cel5A endoglucanase II (Gen Bank: M 19373), 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 13: 219-228, GenBank:Z33381), Aspergillus aculeatus endoglucanase (Ooi et al., 1990, Nucleic Acids Research 18: 5884), Aspergillus kawachii endoglucanase (Sakamoto et al., 1995, Current Genetics 27: 435-439), Fusarium oxysporum endoglucanase (GenBank:L29381), Humicola grisea var. thermoidea endoglucanase (GenBank:AB003107), Melanocarpus 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), Trichoderma reesei strain No. VTT-D-80133 endoglucanase (GenBank:M 15665), and Penicillium pinophilum endoglucanase (WO 2012/062220).

Examples of cellobiohydrolases useful in the present invention include, but are not limited to, Aspergillus aculeatus cellobiohydrolase II (WO 201 1/059740), Aspergillus fumigatus cellobiohydrolase I (WO 2013/028928), Aspergillus fumigatus cellobiohydrolase II (WO 2013/028928), 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 2006/074435), Trichoderma reesei cellobiohydrolase I, Trichoderma reesei cellobiohydrolase II, and Trichophaea saccata cellobiohydrolase II (WO 2010/057086).

Examples of beta-glucosidases useful in the present invention include, but are not limited to, beta-glucosidases from Aspergillus aculeatus (Kawaguchi et al., 1996, Gene 173:

287-288), Aspergillus fumigatus (WO 2005/047499), Aspergillus niger (Dan et al., 2000, J.

Biol. Chem. 275: 4973-4980), Aspergillus oryzae (WO 02/095014), Penicillium brasilianum

IBT 20888 (WO 2007/019442 and WO 2010/088387), Thielavia terrestris (WO

2011/035029), and Trichophaea saccata (WO 2007/019442).

Other useful endoglucanases, cellobiohydrolases, and beta-glucosidases are disclosed in numerous 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.

In the processes of the present invention, any AA9 polypeptide can be used as a component of the enzyme composition.

Examples of AA9 polypeptides useful in the processes of the present invention include, but are not limited to, AA9 polypeptides from Thielavia terrestris (WO 2005/074647, WO 2008/148131 , and WO 201 1/035027), Thermoascus aurantiacus (WO 2005/074656 and WO 2010/065830), Trichoderma reesei (WO 2007/089290 and WO 2012/149344), Myceliophthora thermophila (WO 2009/085935, WO 2009/085859, WO 2009/085864, WO 2009/085868, and WO 2009/033071), Aspergillus fumigatus (WO 2010/138754), Penicillium pinophilum (WO 201 1/005867), Thermoascus sp. (WO 2011/039319), Penicillium sp. {emersoniO (WO 2011/041397 and WO 2012/000892), Thermoascus crustaceous (WO 2011/041504), Aspergillus aculeatus (WO 2012/030799), Thermomyces lanuginosus (WO 2012/113340, WO 2012/129699, WO 2012/130964, and WO 2012/129699), Aurantiporus alborubescens (WO 2012/122477), Trichophaea saccata (WO 2012/122477), Penicillium thomii (WO 2012/122477), Talaromyces stipitatus (WO 2012/135659), Humicola insolens (WO 2012/146171), Malbranchea cinnamomea (WO 2012/101206), Talaromyces leycettanus (WO 2012/101206), and Chaetomium thermophilum (WO 2012/101206), Talaromyces thermophilus (WO 2012/129697 and WO 2012/130950), Acrophialophora fusispora (WO 2013/043910), and Corynascus sepedonium (WO 2013/043910).

In one aspect, the AA9 polypeptide is used in the presence of a soluble activating divalent metal cation according to WO 2008/151043 or WO 2012/122518, e.g., manganese or copper.

In another aspect, the AA9 polypeptide is used in the presence of a dioxy compound, a bicylic compound, a heterocyclic compound, a nitrogen-containing compound, a quinone compound, a sulfur-containing compound, or a liquor obtained from a pretreated lignocellulosic 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).

In one aspect, such a compound is added at a molar ratio of the compound to glucosyl units of cellulose of about 10 ^"6 to about 10, e.g., 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 about 0.1 μΜ to about 1 M, e.g., about 0.5 μΜ to about 0.75 M, about 0.75 μΜ to about 0.5 M, about 1 μΜ to about 0.25 M, about 1 μΜ to about 0.1 M, about 5 μΜ to about 50 mM, about 10 μΜ to about 25 mM, about 50 μΜ to about 25 mM, about 10 μΜ to about 10 mM, about 5 μΜ to about 5 mM, or about 0.1 mM to about 1 mM.

The term "liquor" means the solution phase, either aqueous, organic, or a combination thereof, arising from treatment of a lignocellulose and/or hemicellulose material in a slurry, or monosaccharides thereof, e.g., xylose, arabinose, mannose, etc., under conditions as described in WO 2012/021401 , and the soluble contents thereof. A liquor for cellulolytic enhancement of an AA9 polypeptide can be produced by treating a lignocellulose or hemicellulose material (or feedstock) by applying heat and/or pressure, optionally in the presence of a catalyst, e.g., acid, optionally in the presence of an organic solvent, and optionally in combination with physical disruption of the material, and then separating the solution from the residual solids. Such conditions determine the degree of cellulolytic enhancement obtainable through the combination of liquor and an AA9 polypeptide during hydrolysis of a cellulosic substrate by a cellulolytic enzyme preparation. The liquor can be separated from the treated material using a method standard in the art, such as filtration, sedimentation, or centrifugation.

In one aspect, an effective amount of the liquor to cellulose is about 10 ^"6 to about 10 g per g of cellulose, e.g. , about 10 ^"6 to about 7.5 g, about 10 ^"6 to about 5 g, about 10 ^"6 to about 2.5 g, about 10 ^"6 to about 1 g, about 10 ^"5 to about 1 g, 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 per g of cellulose.

In one aspect, the one or more (e.g., several) hemicellulolytic enzymes comprise a commercial hemicellulolytic enzyme preparation. Examples of commercial hemicellulolytic enzyme preparations suitable for use in the present invention include, for example, SHEARZYME™ (Novozymes A/S), CELLIC® HTec (Novozymes A/S), CELLIC® HTec2 (Novozymes A/S), CELLIC® HTec3 (Novozymes A/S), VISCOZYME® (Novozymes A/S), ULTRAFLO® (Novozymes A/S), PULPZYME® HC (Novozymes A/S), MULTIFECT® Xylanase (Genencor), ACCELLERASE® XY (Genencor), ACCELLERASE® XC (Genencor), ECOPULP® TX-200A (AB Enzymes), HSP 6000 Xylanase (DSM), DEPOL™ 333P (Biocatalysts Limit, Wales, UK), DEPOL™ 740L. (Biocatalysts Limit, Wales, UK), and DEPOL™ 762P (Biocatalysts Limit, Wales, UK), ALTERNA FUEL 100P (Dyadic), and ALTERNA FUEL 200P (Dyadic).

Examples of xylanases useful in the processes of the present invention include, but are not limited to, xylanases from Aspergillus aculeatus (GeneSeqP:AAR63790; WO 94/21785), Aspergillus fumigatus (WO 2006/078256), Penicillium pinophilum (WO 2011/041405), Penicillium sp. (WO 2010/126772), Thermomyces lanuginosus (GeneSeqP:BAA22485), Talaromyces thermophilus (GeneSeqP:BAA22834), Thielavia terrestris NRRL 8126 (WO 2009/079210), and Trichophaea saccata (WO 2011/057083).

The hemicellulase may be added in an amount effective to hydrolyze hemicellulose, such as, in amounts from about 0.001 to 0.5 wt. % of total solids (TS), more preferably from about 0.05 to 0.5 wt. % of TS.

Xylanases may be added in amounts of 0.001-1.0 g/kg DM (dry matter) substrate, preferably in the amount of 0.005-0.5 g/kg DM substrate, and most preferably from 0.05-0.10 g/kg DM substrate.

Examples of acetylxylan esterases useful in the processes of the present invention include, but are not limited to, acetylxylan esterases from Aspergillus aculeatus (WO 2010/108918), Chaetomium globosum (UniProt: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).

Examples of feruloyi esterases (ferulic acid esterases) useful in the processes of the present invention include, but are not limited to, feruloyi esterases form Humicola insolens DSM 1800 (WO 2009/076122), Neosartorya fischeri (UniProt:A1 D9T4), Neurospora crassa (UniProt:Q9HGR3), Penicillium aurantiogriseum (WO 2009/127729), and Thielavia terrestris (WO 2010/053838 and WO 2010/065448).

Examples of arabinofuranosidases useful in the processes of the present invention include, but are not limited to, arabinofuranosidases from Aspergillus niger (GeneSeqP:AAR94170), Humicola insolens DSM 1800 (WO 2006/1 14094 and WO 2009/073383), and M. giganteus (WO 2006/1 14094).

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

Examples of oxidoreductases useful in the processes of the present invention include, but are not limited to, Aspergillus lentilus catalase, Aspergillus fumigatus catalase, Aspergillus niger catalase, Aspergillus oryzae catalase, Humicola insolens catalase, Neurospora crassa catalase, Penicillium emersonii catalase, Scytalidium thermophilum catalase, Talaromyces stipitatus catalase, Thermoascus aurantiacus catalase, Coprinus cinereus laccase, Myceliophthora thermophila laccase, Polyporus pinsitus laccase, Pycnoporus cinnabarinus laccase, Rhizoctonia solani laccase, Streptomyces coelicolor laccase, Coprinus cinereus peroxidase, Soy peroxidase, and Royal palm peroxidase.

The polypeptides having enzyme activity used in the processes of the present invention may be produced by fermentation of the above-noted microbial strains on a nutrient medium containing suitable carbon and nitrogen sources and inorganic salts, using procedures known in the art (see, e.g., Bennett, J.W. and LaSure, L. (eds.), More Gene Manipulations in Fungi, Academic Press, CA, 1991). Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). Temperature ranges and other conditions suitable for growth and enzyme production are known in the art (see, e.g., Bailey, J.E., and Ollis, D.F., Biochemical Engineering Fundamentals, McGraw-Hill Book Company, NY, 1986).

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

The enzyme composition may comprise an enzyme as the major enzymatic component, e.g., a mono-component composition. Alternatively, the enzyme composition may comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of a cellulase, a hemicellulase, an AA9 polypeptide, a cellulose inducible protein (CIP), a catalase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin. The compositions may also comprise or further comprise 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, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase.

The enzyme composition may be prepared in accordance with methods known in the art and may be in the form of a liquid or a dry composition. The compositions may be stabilized in accordance with methods known in the art.

The enzyme composition may be a fermentation broth formulation or a cell composition. The fermentation broth product further comprises additional ingredients used in the fermentation process, such as, for example, cells (including, the host cells containing the gene encoding a polypeptide of interest which are used to produce the polypeptide), cell debris, biomass, fermentation media and/or fermentation products. In some embodiments, the composition is a cell-killed whole broth containing organic acid(s), killed cells and/or cell debris, and culture medium.

The term "fermentation broth" refers to a preparation produced by cellular fermentation that undergoes no or minimal recovery and/or purification. For example, fermentation broths are produced when microbial cultures are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis {e.g., expression of enzymes by host cells) and secretion into cell culture medium. The fermentation broth can contain unfractionated or fractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises the spent culture medium and cell debris present after the microbial cells (e.g. , filamentous fungal cells) are removed, e.g., by centrifugation. In some embodiments, the fermentation broth contains spent cell culture medium, extracellular enzymes, and viable and/or nonviable microbial cells.

In an embodiment, the fermentation broth formulation and cell compositions comprise a first organic acid component comprising at least one 1-5 carbon organic acid and/or a salt thereof and a second organic acid component comprising at least one 6 or more carbon organic acid and/or a salt thereof. 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 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.

In one aspect, the composition contains an organic acid(s), and optionally further contains killed cells and/or cell debris. In one embodiment, the killed cells and/or cell debris are removed from a cell-killed whole broth to provide a composition that is free of these components.

The fermentation broth formulations or cell compositions may further comprise a preservative and/or anti-microbial (e.g., bacteriostatic) agent, including, but not limited to, sorbitol, sodium chloride, potassium sorbate, and others known in the art.

The fermentation broth formulations or cell compositions may further comprise multiple enzymatic activities, such as one or more (e.g., several) enzymes selected from the group consisting of a cellulase, a hemicellulase, an AA9 polypeptide, a cellulose inducible protein (CIP), a catalase, an esterase, an expansin, a laccase, a ligninolytic enzyme, a pectinase, a peroxidase, a protease, and a swollenin. The fermentation broth formulations or cell compositions may also comprise or further comprise 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, e.g., an alpha-galactosidase, alpha-glucosidase, aminopeptidase, amylase, beta-galactosidase, beta-glucosidase, carbohydrase, carboxypeptidase, catalase, cellobiohydrolase, cellulase, chitinase, cutinase, cyclodextrin glycosyltransferase, deoxyribonuclease, endoglucanase, esterase, glucoamylase, invertase, laccase, lipase, mannosidase, mutanase, oxidase, pectinolytic enzyme, peroxidase, phytase, polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase, or xylanase. The cell-killed whole broth or composition may contain the unfractionated contents of the fermentation materials derived at the end of the fermentation. Typically, the cell-killed whole broth or composition contains the spent culture medium and cell debris present after the microbial cells (e.g., filamentous fungal cells) are grown to saturation, incubated under carbon-limiting conditions to allow protein synthesis (e.g., expression of cellulase and/or glucosidase enzyme(s)). In some embodiments, the cell-killed whole broth or composition contains the spent cell culture medium, extracellular enzymes, and killed filamentous fungal cells. In some embodiments, the microbial cells present in the cell-killed whole broth or composition can be permeabilized and/or lysed using methods known in the art.

A whole broth or cell composition as described herein is typically a liquid, but may contain insoluble components, such as killed cells, cell debris, culture media components, and/or insoluble enzyme(s). In some embodiments, insoluble components may be removed to provide a clarified liquid composition.

The whole broth formulations and cell compositions may be produced by a method described in WO 90/15861 or WO 2010/096673.

Methods of Production

Methods of producing a polypeptide having enzyme activity, comprise (a) cultivating a cell, which in its wild-type form is capable of producing the polypeptide, under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

Alternatively, methods of producing a polypeptide having enzyme activity or cellulolytic enhancing activity, comprise (a) cultivating a recombinant host cell under conditions conducive for production of the polypeptide; and (b) recovering the polypeptide.

In the production methods, the cells are cultivated in a nutrient medium suitable for production of the polypeptide using methods well known in the art. For example, the cell may be cultivated by shake flask cultivation, and small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermentors performed in a suitable medium and under conditions allowing the polypeptide to be expressed and/or isolated. The cultivation takes place in a suitable nutrient medium comprising carbon and nitrogen sources and inorganic salts, using procedures known in the art. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted into the medium, it can be recovered from cell lysates.

The polypeptides having enzyme activity can be detected using the methods described herein or methods known in the art. The resulting broth may be used as is with or without cellular debris or the polypeptide may be recovered using methods known in the art. For example, the polypeptide may be recovered from the nutrient medium by conventional procedures including, but not limited to, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptides may be purified by a variety of procedures known in the art including, but not limited to, chromatography (e.g., ion exchange, affinity, hydrophobic, chromatofocusing, and size exclusion), electrophoretic procedures (e.g., preparative isoelectric focusing), differential solubility (e.g., ammonium sulfate precipitation), SDS- PAGE, or extraction (see, e.g., Protein Purification, J.-C. Janson and Lars Ryden, editors, VCH Publishers, New York, 1989) to obtain substantially pure polypeptides.

The present invention is further described by the following examples that should not be construed as limiting the scope of the invention. Examples

Strain

Bacillus subtilis strain IH14 was used as an expression host for the Geobacillus stearothermophilus GH43 polypeptide.

Media and Reagents

LB medium was composed of 10 g of tryptone, 5 g of yeast extract, 5 g of NaCI, and deionized water to 1 liter.

MRS medium was composed of 55 g of DIFCO™ Lactobacilli MRS powder and deionized water to 1 liter.

Spizizen II medium was composed of 6 g of KH2PO4, 14 g of K2HPO4, 2 g of (NH ₄ )2S0 ₄ , 1 g of NasCeHsOy, 0.2 g of MgS0 -7H ₂ 0, 5 g of glucose, 0.2 g of casein hydrolysate, 1 g of yeast extract, 50 mg of tryptophan, 0.055 g of CaC , 0.24 g of MgC , 0.761 g of EGTA, and deionized water to 1 liter.

TBAB + CM plates were composed of 33 g of Tryptose blood agar base (TBAB), 0.5 ml of 5 mg of chloramphenicol, and deionized water to 1 liter.

2XYT + Amp plates were composed of 16 g of tryptone, 10 g of yeast extract, 5 g of NaCI, 15 g of Bacto agar, 1 ml of 100 mg/ml ampicillin stock solution, and deionized water to 1 liter.

Example 1 : Preparation of Talaromyces emersonii GH3 beta-xylosidase

Talaromyces emersonii GH3 beta-xylosidase (SEQ ID NO: 40 [DNA sequence] and SEQ ID NO: 41 [deduced amino acid sequence]; P4UE) was prepared recombinantly according to Rasmussen et al., 2006, Biotechnology and Bioengineering 94: 869-876 using Aspergillus oryzae Jal_355 as a host (WO 2003/070956).

The harvested broth was sterile filtered using a 0.22 μηι polyethersulfone membrane (Millipore). The filtered broth was concentrated and buffer exchanged with 50 mM sodium acetate pH 5.0 using a tangential flow concentrator (Pall Filtron) equipped with a 10 kDa polyethersulfone membrane at approximately 20 psi. Desalted material was examined on 8- 16% CRITERION™ SDS-PAGE gels (Bio-Rad Laboratories, Inc.) stained with GELCODE® Blue Stain Reagent (Thermo Fisher Scientific). The protein was >90% pure as judged by SDS-PAGE. Protein concentration was determined using a BCA Protein Assay Kit (Thermo Fisher Scientific) in which bovine serum albumin was used as a protein standard.

Example 2: Construction of expression vector pEbZn58

Plasmid pEbZn58 was constructed as described below for expression of the Geobacillus stearothermophilus GH43 polypeptide (SEQ ID NO: 1 [deduced amino acid sequence]). Plasmid pLF02 (Figure 1 ; SEQ ID NO: 42 [vector DNA sequence]) containing a Clostridium thermocellum dockerin gene and 6xHis tag for purification was digested with Kpn I and Xba I to linearize the plasmid and remove an 84 bp multiple cloning site. Plasmid pLF02 is a shuttle vector for transformation into B. subtilis via double cross-over of the crylllA stabilizer sequence and pelB locus. The digested plasmid was purified using a PCR Purification Kit (QIAGEN Inc.) according to the manufacturer's instructions.

The Geobacillus stearothermophilus GH43 beta-xylosidase coding sequence (SEQ ID NO: 43 [genomic DNA sequence]) was optimized for expression in Bacillus subtilis using GeneArt® GeneOptimzer® software (SEQ ID NO: 44 [B. subtilis optimized DNA sequence]) and synthesized by Life Technologies. The optimized sequence was amplified by PCR using the primers shown in Table 1. Bold letters represent coding sequence. The remaining sequences are homologous to insertion sites of pLF02.

Table 1

Fifty picomoles of each of the primers listed in Table 1 were used in a PCR containing 100 ng of G. stearothermophilus GH43 beta-xylosidase synthetic DNA, 1X HF Buffer (Thermo Fisher Scientific), 2 μΙ of a blend of dATP, dTTP, dGTP, and dCTP, each at 10 mM, and 1 μΙ of Phusion polymerase (Thermo Fisher Scientific) in a final volume of 100 μΙ. The amplification reaction was performed in a thermocycler programmed for 1 cycle at 98°C for 30 seconds; 30 cycles each at 98°C for 5 seconds, 62°C for 30 seconds, and 72°C for 45 seconds; and a final elongation at 72°C for 10 minutes. The heat block then went to a 4°C soak cycle.

The reaction product was isolated by 1.0% agarose gel electrophoresis using 40 mM Tris base-20 mM sodium acetate-1 mM disodium EDTA (TAE) buffer where a 1602 bp band was excised from the gel and extracted using an Agarose Gel Purification Kit (Clontech Laboratories, Inc.) according to the manufacturer's instructions.

The homologous ends of the 1602 bp PCR product and the digested pLF02 were joined together using an IN-FUSION™ HD Cloning Kit (Clontech Laboratories, Inc.). A total of 100 ng of the 1602 bp PCR product and 100 ng of the Kpn \IXba I digested plasmid pLF02 were used in a reaction containing 2 μΙ of 5X IN-FUSION™ reaction buffer (Clontech Laboratories, Inc.) and 1 μΙ of IN-FUSION™ enzyme (Clontech Laboratories, Inc.) in a final volume of 10 μΙ. The reaction was incubated for 15 minutes at 50°C and then placed on ice. A 2.5 μΙ volume of the reaction was used to transform E. coli STELLAR® Cells (Stratagene) according to the manufacturer's instructions. E. coli transformants were selected on 2XYT + Amp plates. Plasmid DNA from several of the resulting E. coli transformants was prepared using a BIOROBOT® 9600 (QIAGEN Inc.). The Geobacillus stearothermophilus GH43 polypeptide coding sequence insert was confirmed by DNA sequencing with a Model 377 XL Automated DNA Sequencer (Applied Biosystems Inc.) using dye-terminator chemistry (Giesecke et al., 1992, J. Virol. Methods 38: 47-60). Sequencing primers used for verification of the gene insert and sequence are shown in Table 2.

Table 2

A plasmid containing the correct Geobacillus stearothermophilus GH43 polypeptide coding sequence was selected and designated pEbZn58 (Figure 2).

Example 3: Expression of the Geobacillus stearothermophilus GH43 beta-xylosidase in Bacillus subtil is strain IH14

Bacillus subtilis strain SM025 (U.S. Patent No. 8,580,536) was modified to generate Bacillus subtilis strain IH14 via the deletion of the native GH11 xylanase (xynA) gene, insertion of a spectinomycin resistance marker from transposon Tn554 (Murphy et ai, 1985, EMBO Journal 4 (12): 3357-3365), replacement of the pectate lyase (pelB) gene with a triple promoter (Pam_4i99/Phort consensus amyo/PcryiiiA /crylllA stabilizer sequence triple promoter) (U.S. Patent No. 8,268,586), replacement of the native GH5 endoglucanase (bgIC) gene with a neomycin marker from plasmid pUB110 (McKenzie et ai, 1986, Plasmid 15 (2): 93-103), and an extra copy of the B. subtilis prsA gene driven by the PamL4199/Phort consensus amyQ/PcryiiiA/cryl llA stabilizer sequence triple promoter.

B. subtilis strain I H 14 was made competent using the method described by Anagnostopoulos and Spizizen, 1961 , Journal of Bacteriology 81 : 741-746. Once cells were deemed competent by measuring their log-phase growth the entire culture was centrifuged at 3836 x g for 10 minutes. The supernatant was removed and 18 ml of the removed supernatant was mixed with 2 ml of glycerol. The cell pellet was resuspended in this supernatant/glycerol mixture, distributed in 0.5 ml aliquots, and stored at -80°C.

Plasmid pEbZn58 was linearized with Sal I. A 0.5 ml volume of Spizizen II medium

(containing 2 mM EGTA) was added to 0.5 ml of the frozen competent B. subtilis I H 14 cells and thawed at room temperature for 5 minutes. The 1 ml volume was divided into two 1.5 ml microfuge tubes and 10 μΙ of linearized pEbZn58 were added to one aliquot of cells. Then 0.5 ml of LB medium and 0.2 μg/ml chloramphenicol were added. The tube was incubated at 37°C for 30 minutes. Aliquots of 100 μΙ were plated on TBAB + CM plates using glass beads. The plates were incubated overnight at 37°C. Multiple colonies were re-streaked onto TBAB + CM plates and allowed to grow overnight. The re-streaked plates were used to inoculate 3 ml of LB medium, which was incubated at 37°C for 6 hours with shaking at 300 rpm. Five hundred μΙ volumes of the culture were inoculated into 125 ml plastic non-baffled shake flasks containing 50 ml of MRS medium. The shake flasks were incubated at 37°C with shaking at 250 rpm. Following growth for 72 hours, the cultures were transferred to centrifuge bottles and the cells were pelleted at 3,234 ^χ g for 20 minutes at 4°C. The supernatants were then filtered through a 0.22 μηι EMD Millipore STERICUP™ Sterile Vacuum Filter Unit (EMD Millipore) and stored at -20°C until purification.

Example 4: Purification of the Geobacillus stearothermophilus GH43 beta-xylosidase

The sterile filtered supernatant from Example 3 was adjusted to pH 8 with 50% NaOH and applied to a NUVIA™ IMAC column (Bio-Rad Laboratories, Inc.) equilibrated with 50 mM Tris-HCI pH 8.0, 150 mM NaCI, 20 mM CaCI ₂ , 10 mM imidazole. The column was washed extensively with equilibration buffer and then washed with 20 mM Tris-HCI pH 8.0. Bound proteins were eluted with an imidazole gradient (20 column volumes) of 0 M imidazole to 250 mM imidazole in 20 mM Tris-HCI pH 8.0. Eluted protein was pooled and applied to a CAPTO™ Q column (GE Healthcare) equilibrated with 20 mM Tris-HCI pH 8. Bound proteins were eluted with a salt gradient (15 column volumes) of 0 M NaCI to 1 M NaCI in 20 mM Tris-HCI pH 8.0. Fractions were examined on 8-16% CRITERION™ STAIN- FREE™ SDS-PAGE gels. GH43 beta-xylosidase containing fractions were pooled and judged to be >90% pure by SDS-PAGE. Protein concentration was determined using a BCA Protein Assay Kit in which bovine serum albumin was used as a protein standard.

Example 5: Assay of the Talaromyces emersonii GH3 beta-xylosidase and Geobacillus stearothermophilus GH43 beta-xylosidase with glucose and xylo-oligomers

The Talaromyces emersonii GH3 beta-xylosidase and Geobacillus stearothermophilus GH43 beta-xylosidase were assayed for the degradation of xylo- oligomers in the presence of glucose. Assays were conducted in 0.3 ml 96 well round bottom polypropylene plates (Corning). Reactions were initiated by the addition of 10 μΙ of enzyme to 90 μΙ of substrate (100 mg/ml glucose, 50 mg/ml xylo-oligosaccharides (Cascade Analytical Reagents and Biochemicals), 50 mM sodium acetate pH 5, 0.01 % TWEEN® 20 (Sigma-Aldrich). GH3 beta-xylosidase and GH43 beta-xylosidase were dosed at 0.0750, 0.0188, and 0.0023 mg/ml final concentration in the assay. No enzyme controls were also run by the addition of 10 μΙ of water to 90 μΙ of substrate. The reactions were incubated at 50°C for 24 hours. Following hydrolysis, samples were filtered with a 0.45 μηι Multiscreen 96-well filter plate (Millipore) and filtrates analyzed for carbohydrate content as described below.

Hydrolysate samples were diluted 1 :50 with 10 mM NaOH prior to analysis. The hydrolysis samples were analyzed by Dionex ion chromatography with pulsed amperometry detection (IC-PAD, Dionex Corporation) using CHROMELEON™ Software (Dionex Corporation). Chromatographic separation was obtained using a PA-10 column and elution was achieved with an isocratic gradient of 13 mM NaOH, 2.5 mM sodium acetate for 20 minutes, followed by a linear gradient from 13 to 50 mM NaOH for 10 minutes, and then a linear gradient for 20 minutes from 0.5 to 62.5 mM sodium acetate and 75 mM NaOH. Finally, a linear gradient to 100 mM NaOH and 250 mM sodium acetate over 10 minutes was run. Carbohydrate standards were obtained from Sigma Aldrich, dissolved in water at 1 mg/ml, and diluted to 100 μg/ml with 10 mM NaOH.

The results shown in Figure 3 demonstrated that incubation of the G. stearothermophilus GH43 beta-xylosidase with xylo-oligomers and glucose resulted in the degradation of xylobiose and xylo-oligomers and the concomitant formation of xylose. No other reaction products were generated. The formation of xylose and removal of xylobiose and xylo-oligomers occurred in a dose-dependent manner with respect to the amount of GH43 beta-xylosidase added to the reaction. A strikingly different result was obtained when the T. emersonii GH3 beta-xylosidase was incubated with xylo-oligomers and glucose. As shown in Figure 4, the GH3 beta-xylosidase degraded xylo-oligomers and xylobiose and also generated xylose. However, it also generated two other products with retention times of 33 minutes and 39.5 minutes. The 33 minute product was identified as primeverose (6-Ο-β- D-xylopyranosyl-D-glucose) by 2D NMR spectroscopy. The identity of the 39.5 minute product is unknown but has a retention time suggesting it is oligomeric in nature. Since monomeric glucose was incorporated into a disaccharide with xylose by the action of GH3 beta-xylosidase it appears that primeverose (and likely the unknown product) forms through a transglycosylation mechanism. Furthermore, since GH43 beta-xylosidase does not form these products it is suggested that GH43 beta-xylosidase does not form transglycosylation products during the degradation of xylo-oligomers in the presence of glucose and only generates the hydrolysis product xylose.

A mixture (50:50) of the G. stearothermophilus GH43 beta-xylosidase and T. emersonii GH3 beta-xylosidase did not prevent production of primeverose. As shown in Figure 5, the mixture of the GH43 beta-xylosidase and GH3 beta-xylosidase can degrade xylo-oligomers and xylobiose and generate xylose. However, the mixture of the two beta- xylosidases failed to prevent production of primeverose and the unknown product.

The present invention is further described by the following numbered paragraphs: Paragraph 1. A process for reducing production of primeverose during saccharification of a lignocellulosic material, the process comprising: saccharifying the lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.

Paragraph 2. The process of paragraph 1 , wherein the lignocellulosic material is pretreated before saccharification.

Paragraph 3. The process of paragraph 1 or 2, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein, an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.

Paragraph 4. The process of paragraph 3, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

Paragraph 5. The process of paragraph 3, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, and a glucuronidase.

Paragraph 6. The process of any one of paragraphs 1-5, further comprising recovering the saccharified lignocellulosic material.

Paragraph 7. A process for saccharifying a lignocellulosic material, comprising: treating the lignocellulosic material with an enzyme composition comprising a GH43 beta- xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.

Paragraph 8. The process of paragraph 7, wherein the lignocellulosic material is pretreated.

Paragraph 9. The process of paragraph 7 or 8, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein, an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.

Paragraph 10. The process of paragraph 9, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

Paragraph 1 1. The process of paragraph 9, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, and a glucuronidase.

Paragraph 12. The process of any one of paragraphs 7-11 , further comprising recovering the saccharified lignocellulosic material.

Paragraph 13. The process of paragraph 12, wherein the saccharified lignocellulosic material is a sugar.

Paragraph 14. The process of paragraph 13, wherein the sugar is selected from the group consisting of glucose, xylose, mannose, galactose, and arabinose.

Paragraph 15. A process for producing a fermentation product, the process comprising: (a) saccharifying a lignocellulosic material with an enzyme composition comprising a GH43 beta-xylosidase, wherein the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase; (b) fermenting the saccharified lignocellulosic material with one or more fermenting microorganisms to produce the fermentation product; and (c) recovering the fermentation product from the fermentation.

Paragraph 16. The process of paragraph 15, wherein the lignocellulosic material is pretreated. Paragraph 17. The process of paragraph 15 or 16, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein, an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin.

Paragraph 18. The process of paragraph 17, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

Paragraph 19. The process of paragraph 17, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, and a glucuronidase.

Paragraph 20. The process of any one of paragraphs 15-19, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation.

Paragraph 21. The process of any one of paragraphs 15-20, 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 polyketide.

Paragraph 22. A process for fermenting a lignocellulosic material, the process comprising: fermenting the lignocellulosic material with one or more fermenting microorganisms, wherein the lignocellulosic material is saccharified with an enzyme composition comprising a GH43 beta-xylosidase, wherein the enzyme composition comprising the GH43 beta-xylosidase increases the amount of fermentable sugars from saccharification of the lignocellulosic material by reducing the amount of primeverose produced compared to an enzyme composition comprising a GH3 beta-xylosidase in place of the GH43 beta-xylosidase.

Paragraph 23. The process of paragraph 22, wherein the fermenting of the lignocellulosic material produces a fermentation product.

Paragraph 24. The process of paragraph 23, further comprising recovering the fermentation product from the fermentation.

Paragraph 25. The process of paragraph 23 or 24, 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 polyketide.

Paragraph 26. The process of any one of paragraphs 22-25, wherein the lignocellulosic material is pretreated before saccharification.

Paragraph 27. The process of any one of paragraphs 22-26, wherein the enzyme composition further comprises one or more enzymes selected from the group consisting of a cellulase, an AA9 polypeptide, a hemicellulase, a cellulose inducible protein, an esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a pectinase, a protease, and a swollenin. Paragraph 28. The process of paragraph 27, wherein the cellulase is one or more enzymes selected from the group consisting of an endoglucanase, a cellobiohydrolase, and a beta-glucosidase.

Paragraph 29. The process of paragraph 27, wherein the hemicellulase is one or more enzymes selected from the group consisting of a xylanase, an acetylxylan esterase, a feruloyl esterase, an arabinofuranosidase, and a glucuronidase.

Paragraph 30. The process of any one of paragraphs 1-29, wherein the GH43 beta- xylosidase is selected from the group consisting of.

(a) a polypeptide having at least 60% sequence identity to the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24,

25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39;

(b) a polypeptide comprising the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39;

(c) a variant of the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39 comprising a substitution, deletion, and/or insertion at one or more positions; and

(d) a fragment of the polypeptide of (a), (b), or (c) that has GH43 beta-xylosidase activity.

Paragraph 31. The process of paragraph 30, wherein the GH43 beta-xylosidase has at least 60%, 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 beta-xylosidase of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39.

Paragraph 32. The process of paragraph 30, wherein the GH43 beta-xylosidase comprises or consists of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39.

Paragraph 33. The process of paragraph 30, wherein the GH43 beta-xylosidase is a variant of the polypeptide of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39 comprising a substitution, deletion, and/or insertion at one or more positions.

Paragraph 34. The process of paragraph 30, wherein the GH43 beta-xylosidase is a fragment of the polypeptide the GH43 beta-xylosidase of SEQ ID NO: 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, or 29, or the mature polypeptide of SEQ ID NO: 30, 31 , 32, 33, 34, 35, 36, 37, 38, or 39 that has GH43 beta- xylosidase activity.

Paragraph 35. The process of any one of paragraphs 1-34, wherein the amount of primeverose produced is reduced at least 20%, preferably at least 40%, more preferably at least 60%, even more preferably at least 80%, and most preferably at least 100%.

Paragraph 36. The process of any one of paragraphs 1-34, wherein the amount of fermentable sugars is increased at least 0.1 %, at least 0.2%, at least 0.5%, at least 1 %, at least 2.5%, at least 5%, or at least 10% from saccharification of the lignocellulosic material.

The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Various references are cited herein, the disclosures of which are incorporated by reference in their entireties.