Process for producing fermentation products using cellulolytic enzymes and engineered yeasts

Abstract

The present invention relates to processes for producing fermentation products, such as ethanol, from starch-containing material using a fermentation organism expressing cellobiohydrolase I (CBH1) and cellobiohydrolase II (CBH2) in combination with a GH5 xylanase.

Description

[0001] Reference to the Sequence Listing

[0002] This application contains a sequence listing in computer-readable form, which is hereby incorporated by reference. Technical Field

[0003] The present invention relates to processes for producing fermentation products from starch-containing materials. The present invention also relates to a GH5 xylanase composition for use with a recombinant host cell or fermentation organism suitable for use in the processes of the present invention. Background Art

[0004] Processes for producing fermentation products such as ethanol from starch-containing materials or lignocellulosic materials are well known in the art. Preparation of a starch-containing material such as corn for such fermentation processes typically begins with grinding the corn in a dry milling or wet milling process. The wet milling process involves fractionating the corn into different components, with only the starch fraction entering the fermentation process. The dry milling process involves grinding the corn kernels into a powder and mixing the powder with water and enzymes. Two different types of dry milling processes are commonly used. The most commonly used process is often referred to as the "conventional process" and involves grinding a starch-containing grain and then liquefying the gelatinized starch typically using bacterial α-amylase at high temperature, followed by simultaneous saccharification and fermentation (SSF) in the presence of glucoamylase and a fermentation organism. Another well-known process is often referred to as the "raw starch hydrolysis" process (RSH process) and involves grinding a starch-containing grain and then simultaneously saccharifying and fermenting the granular starch at a temperature below the initial gelatinization temperature, typically in the presence of an acidic fungal α-amylase and glucoamylase.

[0005] In a process for producing ethanol from corn, following an SSF or RSH process, the liquid fermentation product is recovered from the fermented mash (often referred to as "beer mash") by distillation, for example, separating the desired fermentation product (such as ethanol) from other liquids and / or solids. The remaining fraction is called "whole stillage". Whole stillage typically contains about 10% to 20% solids. By centrifugation, for example, the whole stillage is separated into a solid fraction and a liquid fraction. The separated solid fraction is called "wet cake" (or "wet grains"), and the separated liquid fraction is called "thin stillage". The wet cake and thin stillage contain about 35% and 7% solids, respectively. The wet cake (with optional additional dehydration) is used as a component in animal feed or is dried to provide "dry distillers grains" (DDG) used as a component in animal feed. Typically, the thin stillage is evaporated to provide evaporator condensate and a slurry, or alternatively, it can be recycled as a "countercurrent" to the slurry tank. The evaporator condensate can be sent to a methane converter before being discharged and / or can be recycled to the slurry tank as "cooking water". The slurry can be co-mixed into the DDG or added to the wet cake before or during a drying process (which can in turn include one or more dryers) to produce DDGS (dry distillers grains with solubles). The slurry typically contains about 25% to 35% solids. Oil can also be extracted from the thin stillage and / or slurry as a by-product (for biodiesel production), as a feed or food additive or product, or other bioreproducible products.

[0006] Yeasts used for producing ethanol as a fuel, such as in the corn ethanol industry, require several characteristics to ensure the cost of efficient ethanol production. These characteristics include ethanol tolerance, low by-product yield, rapid fermentation, and the ability to limit the amount of residual sugar remaining in the fermentation. Such characteristics have an obvious impact on the feasibility of industrial processes.

[0007] Yeasts of the genus Saccharomyces exhibit many of the characteristics required for ethanol production. In particular, strains of Saccharomyces cerevisiae are widely used in the fuel ethanol industry for ethanol production. Industrial strains of Saccharomyces cerevisiae have the ability to produce high yields of ethanol under the fermentation conditions found, for example, in corn mash fermentation. Examples of such strains are the commercially available product ETHANOL

[0008] Saccharomyces cerevisiae has also been genetically engineered to express α - amylase and / or glucoamylase, thereby increasing the yield and reducing the amount of exogenously added enzymes required during SSF (e.g., WO 2018 / 098381, WO 2017 / 087330, WO2017 / 037614, WO 2011 / 128712, WO 2011 / 153516, US 2018 / 0155744). Yeast has also been engineered to express trehalase, thereby attempting to increase the fermentation yield by breaking down residual trehalose (e.g., WO 2017 / 077504).

[0009] It is well known that cellulase is used to convert lignocellulosic raw materials into ethanol. Once the lignocellulose is converted into fermentable sugars (e.g., glucose), it is easy to ferment these fermentable sugars into ethanol by yeast.

[0010] However, despite the progress made in yeast and cellulase technologies, there remains a hope and a need for processes for producing fermentation products (such as ethanol) from starch - containing materials that can provide higher yields of fermentation products or other advantages compared to conventional processes. Summary of the Invention

[0011] The present invention provides a solution to the above - mentioned problem by fermenting a saccharified starch - containing material with a fermenting organism expressing CBH1 and CBH2 in the presence of a GH5_21 xylanase, which provides an unexpectedly increased fermentation product.

[0012] A first aspect relates to a process for producing a fermentation product from a starch - containing material, the process comprising the steps of:

[0013] (a) liquefying the starch - containing material using α - amylase at a temperature above the initial gelatinization temperature;

[0014] (b) saccharifying the liquefied starch - containing material; and

[0015] (c) fermenting the saccharified starch - containing material using a fermenting organism;

[0016] wherein at least one GH5_21 xylanase is present or added during saccharification or fermentation; and

[0017] wherein the fermenting organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2.

[0018] A second aspect relates to a process for producing a fermentation product from a starch - containing material, the process comprising the steps of:

[0019] (a) saccharifying the starch - containing material at a temperature below the initial gelatinization temperature; and

[0020] (b) Ferment the saccharified starch-containing material using a fermenting organism;

[0021] wherein at least one GH5_21 xylanase is present or added during saccharification or fermentation; and

[0022] wherein the fermenting organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2.

[0023] A third aspect relates to a recombinant host cell comprising a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2. Description of the Drawings

[0024] Figure 1 Shows the results of the final ethanol levels fermented with a yeast strain (YS103-A07) expressing CBH1 and CBH2 compared to a control strain (MeJi797).

[0025] Figure 2 Shows the results of the residual solids fermented with a yeast strain (YS103-A07) expressing CBH1 and CBH2 compared to the control strain MeJi797.

[0026] Figure 3 Shows the plasmid map of HP97.

[0027] Figure 4 Shows the plasmid map of TP40.

[0028] Figure 5 Shows the plasmid map of TH58.

[0029] Figure 6 Shows the plasmid map of pMlBa789.

[0030] Definitions

[0031] Active pentose fermentation pathway: As used herein, a host cell or fermenting organism having an "active pentose fermentation pathway" produces the active enzymes necessary for each reaction of the catalytic metabolic pathway in an amount sufficient to produce a fermentation product (e.g., ethanol) from pentose, and thus when cultured in the presence of pentose under fermentation conditions, the host cell or fermenting organism is capable of producing the fermentation product in a measurable yield. A host cell or fermenting organism having an active pentose fermentation pathway comprises one or more active pentose fermentation pathway genes. As used herein, a "pentose fermentation pathway gene" refers to a gene encoding an enzyme involved in the active pentose fermentation pathway. In some embodiments, the active pentose fermentation pathway is an "active xylose fermentation pathway" (i.e., producing a fermentation product, such as ethanol, from xylose) or an "active arabinose fermentation pathway" (i.e., producing a fermentation product, such as ethanol, from arabinose).

[0032] The active enzymes necessary for each reaction in the catalytically active pentose fermentation pathway can be from the activity of endogenous gene expression, the activity of heterologous gene expression, or a combination of the activities from endogenous and heterologous gene expression.

[0033] α-Amylase: The term "α-amylase" means 1,4-α-D-glucan glucanohydrolase (EC.3.2.1.1), which catalyzes the hydrolysis of starch and other linear and branched 1,4-glycosidic oligosaccharides and polysaccharides. α-Amylase activity can be determined using methods known in the art (e.g., the α-amylase assay described in WO2020 / 023411).

[0034] β-Glucosidase: The term "β-glucosidase" means β-D-glucoside glucohydrolase (E.C.3.2.1.21), which catalyzes the hydrolysis of terminal non-reducing β-D-glucose residues and releases β-D-glucose. β-Glucosidase activity can be determined according to the procedure of Venturi et al., 2002, J. Basic Microbiol. [Journal of Basic Microbiology] 42:55-66 using p-nitrophenyl-β-D-glucopyranoside as a substrate. One unit of β-glucosidase is defined as the production of 1.0 micromole of p-nitrophenolate anion per minute from 1 mM p-nitrophenyl-β-D-glucopyranoside as a substrate in 50 mM sodium citrate containing 0.01% 20 at 25 °C and pH 4.8.

[0035] Catalytic domain: The term "catalytic domain" means the region of the enzyme that contains the catalytic machinery of the enzyme.

[0036] Cellobiohydrolase: The term "cellobiohydrolase" means 1,4-β-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176), which catalyzes the hydrolysis of 1,4-β-D-glycosidic bonds in cellulose, cellooligosaccharides, or any polymer containing β-1,4-linked glucose, 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 al., 1982, FEBS Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

[0037] Coding sequence: The term "coding sequence" or "coding region" means a polynucleotide sequence that specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are generally determined by a reading frame that usually begins with an ATG start codon or an alternative start codon (such as GTG and TTG) and ends with a stop codon (such as TAA, TAG, and TGA). A coding sequence can be a sequence of genomic DNA, cDNA, synthetic polynucleotide, and / or recombinant polynucleotide.

[0038] Control sequence: The term "control sequence" means a nucleic acid sequence necessary for the expression of a polypeptide. A control sequence can be native or foreign to the polynucleotide encoding the polypeptide and can be native or foreign to each other. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, and transcription terminator sequences. For the purpose of introducing specific restriction sites that facilitate the ligation of control sequences to the coding region of a polynucleotide encoding a polypeptide, these control sequences can be provided with linkers.

[0039] Disruption: The term "disruption" means that the coding region and / or control sequence of a reference gene is partially or completely modified (e.g., by deletion, insertion, and / or substitution of one or more nucleotides), such that the expression of the encoded polypeptide is absent (inactivated) or reduced, and / or the enzymatic activity of the encoded polypeptide is absent or reduced. Techniques known in the art can be used to measure the effect of disruption, such as measuring the absence or reduction of enzymatic activity using the cell-free extracts cited herein; or by the absence or reduction of the corresponding mRNA (e.g., reduced by at least 25%, reduced by at least 50%, reduced by at least 60%, reduced by at least 70%, reduced by at least 80%, or reduced by at least 90%); the absence or reduction of the amount of the corresponding polypeptide having enzymatic activity (e.g., reduced by at least 25%, reduced by at least 50%, reduced by at least 60%, reduced by at least 70%, reduced by at least 80%, or reduced by at least 90%); or the absence or reduction of the specific activity of the corresponding polypeptide having enzymatic activity (e.g., reduced by at least 25%, reduced by at least 50%, reduced by at least 60%, reduced by at least 70%, reduced by at least 80%, or reduced by at least 90%). A specific gene of interest can be disrupted by methods known in the art, such as by directed homologous recombination (see Methods in Yeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Stems, Cold Spring Harbor Press (1998)).

[0040] Endogenous gene: The term "endogenous gene" means a gene that is native to a reference host cell or fermenting organism. "Endogenous gene expression" means the expression of an endogenous gene.

[0041] Endoglucanase: The term "endoglucanase" means 4-(1,3;1,4)-β-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4), which catalyzes the endohydrolysis of 1,4-β-D-glycosidic linkages in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, mixed β-1,3-1,4 glucans such as cereal β-D-glucan or xyloglucan, and other plant materials containing a cellulose component. Endoglucanase activity can be determined by measuring the decrease in substrate viscosity or by the increase in the reducing end determined by reducing sugar assays (Zhang et al., 2006, Biotechnology Advances 24:452-481). Endoglucanase activity can also be determined according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59:257-268, using carboxymethyl cellulose (CMC) as the substrate at pH 5 and 40 °C.

[0042] 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 can be measured - for example, to detect increased expression - by techniques known in the art, such as measuring the levels of mRNA and / or the translated polypeptide.

[0043] Expression vector: The term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to control sequences providing for its expression.

[0044] Fermentable medium: The term "fermentable medium" or "fermentation medium" refers to a medium that contains one or more (e.g., two, several) sugars, such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or soluble oligosaccharides, wherein the medium can be partially converted (fermented) by a host cell into a desired product, such as ethanol. In some cases, the fermentation medium is derived from natural sources, such as sugar cane, starch, or cellulose; and can be pretreated by enzymatic hydrolysis (saccharification) from that source. The term fermentation medium is understood herein to refer to the medium prior to the addition of the fermenting organism, such as the medium produced by the saccharification process, and the medium used in a simultaneous saccharification and fermentation process (SSF).

[0045] Fermentation product: The term "fermentation product" means a product produced by a process including fermentation using a fermenting organism. Fermentation products include alcohols (e.g., ethanol, methanol, butanol); organic acids (e.g., citric acid, acetic acid, itaconic acid, lactic acid, succinic acid, gluconic acid); ketones (e.g., acetone); amino acids (e.g., glutamic acid); gases (e.g., H2 and CO2); antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin, B 12 、β - carotene); and hormones. In a preferred embodiment, the fermentation product is ethanol, for example, fuel ethanol; potable ethanol, i.e., neutral potable alcohol; or industrial ethanol or product for the consumer alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry, and tobacco industry. Preferred types of beer include ale, stout, porter, lager, bitter, malt liquor, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer, or light beer. In an embodiment, the fermentation product is ethanol.

[0046] Fermenting organism: A "fermenting organism" refers to any organism suitable for a fermentation process and capable of producing the desired fermentation product, including bacterial and fungal organisms, especially yeast.

[0047] GH5 xylanase: "GH5 xylanase" is an abbreviation for glycoside hydrolase family 5 xylanase, which mainly consists of endo-1,4-β-xylanase (EC 3.2.1.8), and this xylanase catalyzes the endohydrolysis of (1→4)-β-D-xylosidic bonds in xylan.

[0048] GH5_21 xylanase: "GH5_21 xylanase" is an abbreviation for glycoside hydrolase family 5 subfamily 21 endo-β-1,4-xylanase, which has a three-dimensional structure characterized by a (β / α)8 barrel and uses a glutamine residue as a catalytic nucleophile / base.

[0049] Glucoamylase: The term "glucoamylase" (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) is defined as an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligosaccharide and polysaccharide molecules. For the purposes of the present invention, glucoamylase activity can be determined according to procedures known in the art, such as those described in WO 2020 / 023411.

[0050] Hemicellulolytic enzymes or hemicellulases: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that can hydrolyze hemicellulose materials. See, e.g., Shallom and Shoham, 2003, Current Opinion In Microbiology 6(3):219-228. Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to: acetylmannan esterase, acetylxylan esterase, arabinase, arabinofuranosidase, coumaric acid esterase, ferulic acid esterase, galactosidase, glucuronidase, glucuronate esterase, mannanase, mannosidase, xylanase, and xylosidase. The substrates of these enzymes (hemicellulose) are a heterogeneous group of branched and linear polysaccharides that are cross-linked into a robust network by hydrogen bonding to cellulose microfibrils in the plant cell wall. Hemicellulose is also covalently attached to lignin, thus forming a highly complex structure together with cellulose. The variable structure and organization of hemicellulose require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are glycoside hydrolases (GHs) that hydrolyze glycosidic bonds or carbohydrate esterases (CEs) that hydrolyze ester bonds of acetic acid or ferulic acid side groups. These catalytic modules can be assigned to GH and CE families based on the homology of their primary sequences. Some families, having generally similar folds, can be further grouped into clans, labeled with letters (e.g., GH-A). The most detailed and up-to-date classification of these and other carbohydrate-active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activity can be measured according to Ghose and Bisaria, 1987, Pure & AppI.Chem. 59:1739-1752, at a suitable temperature such as 40°C - 80°C, e.g., 50°C, 55°C, 60°C, 65°C, or 70°C, and a suitable pH such as 4 - 9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0.

[0051] Heterologous polynucleotide: The term "heterologous polynucleotide" is defined herein as a polynucleotide that is not native to the host cell; a native polynucleotide in which the coding region has been structurally modified; a native polynucleotide whose expression has been quantitatively altered by manipulation of the DNA by recombinant DNA techniques (e.g., a different (foreign) promoter); or a native polynucleotide in a host cell that has one or more additional copies of the polynucleotide to quantitatively alter expression. A "heterologous gene" is a gene that contains a heterologous polynucleotide.

[0052] High stringency conditions: The term "high stringency conditions" means, for a probe of at least 100 nucleotides in length, prehybridization and hybridization for 12 to 24 hours at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures. The carrier material is finally washed three times for 15 minutes each at 65°C in 0.2X SSC, 0.2% SDS.

[0053] Host cell: The term "host cell" means any cell type that is susceptible to transformation, transfection, transduction, etc. with a nucleic acid construct or expression vector containing a polynucleotide as described herein. The term "host cell" encompasses any progeny of a parental cell that is not identical to the parental cell due to mutations that occur during replication. The term "recombinant cell" is defined herein as a non-naturally occurring host cell that contains one or more (e.g., two, several) heterologous polynucleotides.

[0054] Low stringency conditions: The term "low stringency conditions" means, for a probe of at least 100 nucleotides in length, prehybridization and hybridization for 12 to 24 hours at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml sheared and denatured salmon sperm DNA, and 25% formamide, following standard Southern blotting procedures. The carrier material is finally washed three times for 15 minutes each at 50°C in 0.2X SSC, 0.2% SDS.

[0055] Initial gelatinization temperature: "Initial gelatinization temperature" means the lowest temperature at which starch gelatinization begins. Starch heated in water begins to gelatinize between 50°C and 75°C; the exact temperature of gelatinization depends on the specific starch and can be readily determined by a person skilled in the art. Thus, the initial gelatinization temperature can vary depending on the plant species, the particular variety of the plant species, and the growth conditions. In the context of the present disclosure, the initial gelatinization temperature of a given starch-containing cereal is the temperature at which 5% of the starch granules lose birefringence, using the method described by Gorinstein.S. and Lii.C., Starch / Starke [Starch], Vol. 44(12), pp. 461-466 (1992).

[0056] Mature polypeptide: "Mature polypeptide" means a polypeptide in its final form after translation and any post-translational modifications (such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). The mature polypeptide sequence lacks a signal sequence, which can be determined using techniques known in the art (see, e.g., Zhang and Henzel, 2004, Protein Science 13:2819-2824). The term "mature polypeptide coding sequence" means a polynucleotide that encodes a mature polypeptide.

[0057] Medium stringency conditions: The term "medium stringency conditions" means that for a probe of at least 100 nucleotides in length, following standard Southern blotting procedures, prehybridization and hybridization are carried out for 12 to 24 hours at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml sheared and denatured salmon sperm DNA, and 35% formamide. The carrier material is finally washed three times for 15 minutes each at 55°C with 0.2X SSC, 0.2% SDS.

[0058] Medium-high stringency conditions: The term "medium-high stringency conditions" means that for a probe of at least 100 nucleotides in length, following standard Southern blotting procedures, prehybridization and hybridization are carried out for 12 to 24 hours at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml sheared and denatured salmon sperm DNA, and 35% formamide. The carrier material is finally washed three times for 15 minutes each at 60°C with 0.2X SSC, 0.2% SDS.

[0059] Nucleic acid construct: The term "nucleic acid construct" means a polynucleotide comprising one or more (e.g., two, several) control sequences. The polynucleotide can be single-stranded or double-stranded, and can be isolated from a naturally occurring gene, can be modified to contain segments of nucleic acid in a manner that does not otherwise exist in nature, or can be synthetic.

[0060] Operably linked: The term "operably linked" means a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of a polynucleotide such that the control sequence directs the expression of the coding sequence.

[0061] Protease: The term "protease" is defined herein as an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of its 13 subclasses). The EC numbers refer to the 1992 Enzyme Nomenclature of NC-IUBMB in San Diego, California, Academic Press, including Supplements 1-5 published separately in: Eur. J. Biochem. [European Journal of Biochemistry] 223:1-5 (1994); Eur. J. Biochem. [European Journal of Biochemistry] 232:1-6 (1995); Eur. J. Biochem. [European Journal of Biochemistry] 237:1-5 (1996); Eur. J. Biochem. [European Journal of Biochemistry] 250:1-6 (1997); and Eur. J. Biochem. [European Journal of Biochemistry] 264:610-650 (1999). The term "subtilisin" refers to the subgroup of serine proteases according to Siezen et al., 1991, Protein Engng. [Protein Engineering] 4:719-737 and Siezen et al., 1997, Protein Science [Protein Science] 6:501-523. Serine proteases or serine peptidases are subgroups of proteases characterized by having serine at the active site and forming a covalent adduct with the substrate. Additionally, subtilisins (and serine proteases) are characterized by having two active site amino acid residues in addition to serine, namely histidine and aspartic acid residues. Subtilisins can be divided into 6 subfamilies, namely, the subtilisin family, the thermitase family, the proteinase K family, the lantibiotic peptidase family, the kexin family, and the pyrolysin family. The term "protease activity" means proteolytic activity (EC 3.4). Protease activity can be determined using methods described in the art (e.g., US 2015 / 0125925) or using commercially available assay kits (e.g., Sigma-Aldrich).

[0062] Pullulanase: The term "pullulanase" means a starch debranching enzyme (EC 3.2.1.41) having pullulan 6-glucanohydrolase activity, which catalyzes the hydrolysis of α-1,6-glycosidic bonds in pullulan, thereby releasing maltotriose with a reducing carbohydrate end. For the purposes of the present invention, pullulanase activity can be determined according to the PHADEBAS assay or the sweet potato starch assay described in WO 2016 / 087237.

[0063] Sequence identity: The degree of relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter "sequence identity". For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48:443-453) is used to determine the sequence identity between two amino acid sequences, as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277) (e.g., 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 "longest identity" marked by Needle (obtained using the -nobrief option) is used as the percentage of identity and is calculated as follows:

[0064] (Identical residues × 100) / (Alignment length – total number of gaps in the alignment)

[0065] For the purposes of the present invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, ibid.) is used to determine the sequence identity between two deoxyribonucleotide sequences, as implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, ibid.) (e.g., 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 EDNAFULL (EMBOSS version of NCBINUC4.4) substitution matrix. The output of "longest identity" marked by Needle (obtained using the -nobrief option) is used as the percentage of identity and is calculated as follows:

[0066] (Identical deoxyribonucleotides × 100) / (Alignment length – total number of gaps in the alignment).

[0067] Signal peptide: The term "signal peptide" is defined herein as a peptide that is linked (fused) in-frame to the amino terminus of a bioactive polypeptide and directs the polypeptide into the secretory pathway of the cell. Signal sequences can be determined using techniques known in the art (see, e.g., Zhang and Henzel, 2004, Protein Science 13:2819-2824). The polypeptides described herein can contain any suitable signal peptide known in the art, or any signal peptide described in WO 2021 / 025872 (incorporated herein by reference).

[0068] Thermostable: "Thermostable" means that the enzyme is not denatured or inactivated when the enzyme is used in the liquefaction step of the process of the present invention. In other words, if a thermostable enzyme has a denaturation temperature (Td) that is compatible with the liquefaction temperature and retains its activity at this temperature, then the thermostable enzyme is suitable for liquefaction.

[0069] Trehalase: The term "trehalase" means an enzyme that degrades trehalose into its monomeric monosaccharides (i.e., glucose). Trehalase is classified in EC 3.2.1.28 (α,α-trehalase) and EC 3.2.1.93 (α,α-trehalose phosphate trehalase). The EC classification is based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Descriptions of EC classes can be found on the internet, e.g., at " http: / / www.expasy.org / enzyme / ". Trehalase is an enzyme that catalyzes the following reactions:

[0070] EC 3.2.1.28: α,α-trehalose + H2O 2 D-glucose;

[0071] EC3.2.1.93: α,α-trehalose 6-phosphate + H2O D-glucose + D-glucose 6-phosphate.

[0072] Trehalase activity can be determined according to procedures known in the art.

[0073] Very high stringency conditions: The term "very high stringency conditions" means that for a probe at least 100 nucleotides in length, following standard Southern blotting procedures, prehybridization and hybridization are carried out at 42 °C in 5XSSPE, 0.3% SDS, 200 μg / ml sheared and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. The carrier material is finally washed three times at 70 °C with 0.2X SSC, 0.2% SDS, for 15 minutes each.

[0074] Very low stringency conditions: The term "very low stringency conditions" means that for a probe of at least 100 nucleotides in length, following standard Southern blotting procedures, prehybridization and hybridization are carried out at 42 °C in 5X SSPE, 0.3% SDS, 200 μg / ml sheared and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. The carrier material is finally washed three times at 45 °C with 0.2X SSC, 0.2% SDS for 15 minutes each time.

[0075] Whole stillage: "Whole stillage" includes the material remaining at the end of the distillation process after the recovery of fermentation products such as ethanol.

[0076] Xylanase: "Xylanase" encompasses endo-1,4-β-xylanase (EC 3.2.1.8), which catalyzes the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylan; and glucuronoyl arabinoxylan endo-1,4-β-xylanase (E.C. 3.2.1.136), which catalyzes the endohydrolysis of 1,4-β-D-xylosyl linkages in some glucuronoyl arabinoxylans. The activity of EC 3.2.1.8 xylanase can be determined using birchwood xylan as a substrate. One unit of xylanase is defined as the production of 1.0 micromole of reducing sugar per liter per minute by 2 g of birchwood xylan as a substrate in 50 mM sodium acetate containing 0.01% 2 at 50 °C, pH 5 (measured as glucose equivalents as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem. 47:273 - 279). The activity of EC 3.2.1.136 xylanase can be determined at 37 °C, in 0.01% X - 100 and 200 mM sodium phosphate (pH 6) using 0.2% AZCL - glucuronoxylan as a substrate. One unit of xylanase activity is defined as the production of 1.0 micromole of azurine per minute by 0.2% AZCL - glucuronoxylan as a substrate in 200 mM sodium phosphate (pH 6) at 37 °C, pH 6. Detailed Description

[0077] The present invention relates to a process for producing a fermentation product (such as ethanol) from a starch-containing material using a fermenting organism.

[0078] The work described herein unexpectedly demonstrates that fermenting a starch-containing material with a fermenting organism expressing CBH1 and CBH2 in the presence of a GH5 xylanase results in a significantly higher fermentation product yield and significantly lower residual solids.

[0079] The present invention contemplates the use of a fermenting organism and a GH5 xylanase in saccharification, fermentation, or simultaneous saccharification and fermentation to increase product yields in conventional and raw starch hydrolysis (RSH) ethanol production processes as well as cellulosic ethanol processes.

[0080] I. Process for producing a fermentation product from a gelatinized starch-containing cereal

[0081] One aspect of the present invention relates to a process for producing a fermentation product (e.g., fuel ethanol) from a gelatinized starch-containing cereal, wherein at least one GH5_21 xylanase is present or added during saccharification or fermentation; and wherein the fermenting organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2. This aspect of the process of the present invention contemplates any of the GH5_21, CBH1, and CBH2 enzymes described herein, particularly those demonstrated in the following examples.

[0082] In an embodiment, a process for producing a fermentation product from a starch-containing material, the process comprising the steps of:

[0083] (a) liquefying the starch-containing material using α-amylase at a temperature above the initial gelatinization temperature;

[0084] (b) saccharifying the liquefied starch-containing material;

[0085] (c) fermenting the saccharified starch-containing material using a fermenting organism;

[0086] wherein at least one GH5_21 xylanase is present or added during saccharification or fermentation; and

[0087] wherein the fermenting organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2.

[0088] In an embodiment, the GH5_21 xylanase is present or added during saccharification step (b). In an embodiment, the GH5_21 xylanase is present or added during fermentation step (c). In an embodiment, steps (b) and (c) are carried out simultaneously in simultaneous saccharification and fermentation (SSF). In an embodiment, the GH5_21 xylanase is present or added during SSF. In an embodiment, the GH5_21 xylanase used in saccharification step (b) and / or fermentation step (c) is present or added via in situ expression from a fermenting organism (e.g., yeast).

[0089] In an embodiment, a thermostable endoglucanase is added during the liquefaction step (a). In an embodiment, a thermostable lipase is added during the liquefaction step (a). In an embodiment, a thermostable phytase is added during the liquefaction step (a). In an embodiment, a thermostable protease is added during the liquefaction step (a). In an embodiment, a thermostable pullulanase is added during the liquefaction step (a). In an embodiment, a thermostable xylanase is added during the liquefaction step (a). In a preferred embodiment, a thermostable α-amylase, a thermostable protease and a thermostable xylanase are added during the liquefaction step (a).

[0090] In an embodiment, α-amylase is added during step (b) and / or step (c). In an embodiment, β-glucosidase is added during step (a) and / or step (b). In an embodiment, glucoamylase is added during step (b) and / or step (c). In an embodiment, cellobiohydrolase is added during step (b) and / or step (c). In an embodiment, endoglucanase is added during step (b) and / or step (c). In an embodiment, trehalase is added during step (b) and / or step (c).

[0091] In an embodiment, the fermenting organism is yeast. In an embodiment, during step (b) and / or step (c), the yeast in situ expresses α-amylase. In an embodiment, during step (b) and / or step (c), the yeast in situ expresses glucoamylase. In an embodiment, during step (b) and / or step (c), the yeast in situ expresses α-amylase and glucoamylase.

[0092] Process parameters

[0093] Process parameters for producing a fermentation product, such as process parameters for producing ethanol from a starch-containing cereal (e.g., corn), are well known in the art. See, for example, WO 2006 / 086792, WO 2013 / 082486, WO 2012 / 088303, WO2013 / 055676, WO 2014 / 209789, WO 2014 / 209800, WO 2015 / 035914, WO 2017 / 112540, WO2020 / 014407, WO 2021 / 126966 (each of which is incorporated herein by reference).

[0094] Starch-containing cereal

[0095] Any suitable starch-containing starting cereal can be used. The cereal is selected based on the desired fermentation product. Examples of starch-containing cereals include, but are not limited to, barley, beans, cassava, cereal grains, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof. The starch-containing cereals can also be waxy or non-waxy types of corn and barley. Commercially available starch-containing cereals commonly used include corn, milo, and / or wheat.

[0096] Reduction of cereal grain size

[0097] Before the liquefaction step (a), the size of the starch-containing cereal can be reduced, for example, by dry milling.

[0098] Slurry

[0099] Before the liquefaction step (a), a slurry can be formed that includes the starch-containing cereal (e.g., preferably ground) and water. Alpha-amylase and optionally protease can be added to the slurry. The slurry can be heated to a temperature between the initial gelatinization temperature of the starch-containing cereal to above that initial gelatinization temperature to initiate gelatinization of the starch.

[0100] Jet cooking

[0101] Before adding alpha-amylase during the liquefaction step (a), the slurry can optionally be jet cooked to further gelatinize the starch in the slurry. Jet cooking can be carried out at a temperature in the range from 100 °C to 120 °C for at least 15 minutes.

[0102] Liquefaction temperature

[0103] The temperature used during the liquefaction step (a) can be in the range from 70 °C to 110 °C, such as from 75 °C to 105 °C, from 80 °C to 100 °C, from 85 °C to 95 °C, or from 88 °C to 92 °C. Preferably, the temperature is at least 70 °C, at least 80 °C, at least 85 °C, at least 88 °C, or at least 90 °C.

[0104] Liquefaction pH

[0105] The pH used during the liquefaction step (a) can be in the range from 4 to 6, from 4.5 to 5.5, or from 4.8 to 5.2. Preferably, the pH is at least 4.5, at least 4.6, at least 4.7, at least 4.8, at least 4.9, at least 5.0, or at least 5.1.

[0106] Liquefaction time

[0107] The time for performing the liquefaction step (a) can be in the range of from 30 minutes to 5 hours, from 1 hour to 3 hours, or from 90 minutes to 150 minutes. Preferably, the time is at least 30 minutes, at least about 45 minutes, at least about 60 minutes, at least about 90 minutes, or at least about 2 hours.

[0108] Liquefying enzyme

[0109] The present invention contemplates the use of heat-stable enzymes during the liquefaction step (a). The use of various heat-stable enzymes during the liquefaction step (a) is well known in the art and includes, for example, heat-stable α-amylase, heat-stable glucoamylase, heat-stable endoglucanase, heat-stable lipase, heat-stable phytase, heat-stable protease, heat-stable pullulanase, and / or heat-stable xylanase. The present invention contemplates the use of any heat-stable enzyme in the liquefaction step (a). Guidance for determining the denaturation temperature of candidate heat-stable enzymes to be used in the liquefaction step (a) is provided in the following Materials and Methods section. The following listed published patent applications describe activity assays for determining whether candidate heat-stable enzymes contemplated for use in the liquefaction step (a) will be inactivated at the temperatures contemplated for the liquefaction step (a).

[0110] Examples of suitable heat-stable α-amylases and guidance for their use in the liquefaction step (a) include, but are not limited to, the α-amylases described in the following: WO 1996 / 023873, WO 1996 / 023874, WO 1997 / 041213, WO1999 / 019467, WO 2000 / 060059, WO 2002 / 010355, WO 2002 / 092797, WO 2009 / 149130, WO2009 / 061379, WO 2010 / 115021, WO 2010 / 036515, WO 2011 / 082425, WO 2019 / 113413, WO2019 / 113415, WO 2019 / 197318 (each of which is incorporated herein by reference).

[0111] Examples of suitable heat-stable glucoamylases include, but are not limited to, the glucoamylases described in the following: WO2011 / 127802, WO 2013 / 036526, WO 2013 / 053801, WO 2018 / 164737, WO 2020 / 010101, and WO2022 / 090564 (each of which is incorporated herein by reference).

[0112] Examples of suitable heat-stable endoglucanases include, but are not limited to, the endoglucanases described in WO 2015 / 035914 (which is incorporated herein by reference).

[0113] Examples of suitable thermostable lipases include, but are not limited to, the lipases described in WO 2017 / 112542 and WO 2020 / 014407 (both incorporated herein by reference).

[0114] Examples of suitable thermostable phytases include, but are not limited to, the phytases described in: WO 1996 / 28567, WO 1997 / 33976, WO 1997 / 38096, WO 1997 / 48812, WO 1998 / 05785, WO 1998 / 06856, WO 1998 / 13480, WO 1998 / 20139, WO 1998 / 028408, WO 1999 / 48330, WO 1999 / 49022, WO2003 / 066847, WO 2004 / 085638, WO 2006 / 037327, WO 2006 / 037328, WO 2006 / 038062, WO2006 / 063588, WO 2007 / 112739, WO 2008 / 092901, WO 2008 / 116878, WO 2009 / 129489, and WO2010 / 034835 (each incorporated by reference). Commercially available products containing phytase include BIO-FEEDPHYTASE TM , PHYTASE NOVO TM CT or L, LIQMAX or RONOZYME TM NP, HIPHOS, P5000(CT), NATUPHOS TM NG 5000.

[0115] Examples of suitable thermostable proteases include, but are not limited to, the proteases described in: WO 1992 / 02614, WO 98 / 56926, WO 2001 / 151620, WO 2003 / 048353, WO 2006 / 086792, WO 2010 / 008841, WO 2011 / 076123, WO 2011 / 087836, WO 2012 / 088303, WO 2013 / 082486, WO 2014 / 209789, WO 2014 / 209800, WO 2018 / 098124, WO 2018 / 118815A1, and WO 2018 / 169780 A1 (each incorporated herein by reference).

[0116] Examples of suitable commercially available products containing protease include AVANTEC FORTIVA FORTIVA

[0117] Examples of suitable thermostable pullulanases include, but are not limited to, pullulanases described in WO2015 / 007639, WO 2015 / 110473, WO 2016 / 087327, WO 2017 / 014974, and WO 2020 / 187883, each of which is incorporated herein by reference in its entirety. Suitable commercially available pullulanase products include PROMOZYME 400L, PROMOZYME TM D2 (Novozymes A / S, Denmark), OPTIMAX L-300 (Genencor Int., USA), and AMANO 8 (Amano, Japan).

[0118] Examples of suitable thermostable xylanases include, but are not limited to, xylanases described in WO 2017 / 112540 and WO2021 / 126966, each of which is incorporated herein by reference. Suitable commercially available products containing thermostable xylanase include FORTIVA

[0119] One or more of the enzymes described above are to be used in an effective amount in the process of the present invention. Guidance for determining the effective amount of the enzyme to be used in the liquefaction step (a) and guidance for performing activity assays for determining the activity of those enzymes can be found in the published patent applications cited for each different thermostable liquefying enzyme.

[0120] Saccharification temperature

[0121] Saccharification can be carried out at a temperature in the range from 20 °C to 75 °C, from 30 °C to 70 °C, or from 40 °C to 65 °C. Preferably, the saccharification temperature is at least about 50 °C, at least about 55 °C, or at least about 60 °C.

[0122] Saccharification pH

[0123] Saccharification can occur at a pH in the range from 4 to 5. Preferably, the pH is about 4.5.

[0124] Saccharification time

[0125] Saccharification can last from about 24 hours to about 72 hours.

[0126] Fermentation time

[0127] Fermentation can last from 6 to 120 hours, from 24 hours to 96 hours, or from 35 hours to 60 hours.

[0128] Simultaneous saccharification and fermentation

[0129] SSF can be carried out at a temperature from 25 °C to 40 °C, from 28 °C to 35 °C, or from 30 °C to [temperature value not provided] °C, at a pH from 3.5 to 5, or from 3.8 to 4.3 for 24 to 96 hours, 36 to 72 hours, or from 48 to 60 hours. Preferably, SSF is carried out at about 32 °C, at a pH from 3.8 to 4.5 for 48 to 60 hours.

[0130] Saccharification and / or fermentation enzymes

[0131] The present invention contemplates the use of enzymes during the saccharification step (b) and / or the fermentation step (c). The use of various enzymes during the saccharification step (b) and / or the fermentation step (c) is well known in the art and includes, for example, α-amylase, α-glucosidase, β-amylase, β-glucanase, β-glucosidase, cellobiohydrolase, endoglucanase, glucoamylase, lipase, lytic polysaccharide monooxygenase (LPMO), maltogenic α-amylase, pectinase, peroxidase, phytase, protease, and trehalase.

[0132] The enzymes used in the saccharification step (b) and / or the fermentation step (c) can be added exogenously as a single component or formulated as a composition containing the enzyme. The enzymes used in the saccharification step (b) and / or the fermentation step (c) can also be added via in situ expression from a fermenting organism (e.g., yeast). Examples of suitable yeasts expressing the enzyme include, but are not limited to, the yeasts described herein.

[0133] Examples of suitable α-amylases include, but are not limited to, the α-amylases described in: WO 2004 / 055178, WO 2006 / 069290, WO 2013 / 006756, WO 2013 / 034106, WO 2013 / 044867, WO 2021 / 163011, and WO 2021 / 163030 (each of which is incorporated herein by reference).

[0134] Examples of suitable glucoamylases include, but are not limited to, those described in: WO 1984 / 02921, WO 1992 / 00381, WO 1999 / 28448, WO 2000 / 04136, WO 2001 / 04273, WO 2006 / 069289, WO 2011 / 066560, WO 2011 / 066576, WO 2011 / 068803, WO 2011 / 127802, WO 2012 / 064351, WO2013 / 036526, WO 2013 / 053801, WO 2014 / 039773, WO 2014 / 177541, WO 2014 / 177546, WO2016 / 062875, WO 2017 / 066255, and WO 2018 / 191215, each of which is incorporated herein by reference.

[0135] Examples of suitable compositions comprising α-amylase and glucoamylase include, but are not limited to, those described in WO 2006 / 069290, WO 2009 / 052101, WO 2011 / 068803, and WO 2013 / 006756, each of which is incorporated herein by reference. Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300L; SAN TM SUPER, SAN TM EXTRA L, SPIRIZYME TM PLUS, SPIRIZYME TM FUEL, SPIRIZYME TM B4U, SPIRIZYME TM ULTRA, SPIRIZYME TM EXCEL, SPIRIZYME ACHIEVE, and AMG TM E (from Novozymes); OPTIDEX TM 300, GC480, GC417 (from DuPont-Genencor); AMIGASE TM and AMIGASE TM PLUS (from DSM); G-ZYME TM G900, G-ZYME TM and G990 ZR (from DuPont-Genencor).

[0136] Examples of suitable β-glucanases include, but are not limited to, those described in WO 2021 / 055395, which is incorporated herein by reference.

[0137] Examples of suitable β-glucosidases include, but are not limited to, those described in WO 2005 / 047499, WO 2013 / 148993, WO 2014 / 085439, and WO 2012 / 044915, each of which is incorporated herein by reference.

[0138] Examples of suitable cellobiohydrolases include, but are not limited to, those described in WO 2013 / 148993, WO 2014 / 085439, WO 2014 / 138672, and WO 2016 / 040265, each of which is incorporated herein by reference.

[0139] Examples of suitable endoglucanases include, but are not limited to, those described in WO 2013 / 148993 and WO 2014 / 085439, both of which are incorporated herein by reference.

[0140] Examples of suitable maltogenic α-amylases are described in U.S. Patent Nos. 4,598,048, 4,604,355, and 6,162,628, which are hereby incorporated by reference.

[0141] Examples of suitable lipases include, but are not limited to, those described in WO 2017 / 112533, WO 2017 / 112539, and WO 2020 / 076697, each of which is incorporated herein by reference.

[0142] Examples of suitable LPMOs include, but are not limited to, those described in WO 2013 / 148993, WO 2014 / 085439, and WO2019 / 083831, each of which is incorporated herein by reference.

[0143] Examples of suitable phytases include, but are not limited to, those described in WO 2001 / 62947, which is incorporated herein by reference.

[0144] Examples of suitable pectinases include, but are not limited to, those described in WO 2022 / 173694, which is incorporated herein by reference.

[0145] Examples of suitable peroxidases include, but are not limited to, those described in WO 2019 / 231944, which is incorporated herein by reference.

[0146] Examples of suitable proteases include, but are not limited to, proteases described in WO 2017 / 050291, WO 2017 / 148389, WO 2018 / 015303, and WO 2018 / 015304, each of which is incorporated herein by reference.

[0147] Examples of suitable trehalases include, but are not limited to, trehalases described in WO 2016 / 205127, WO 2019 / 005755, WO 2019 / 030165, and WO 2020 / 023411, each of which is incorporated herein by reference.

[0148] II. Process for producing a fermentation product from ungelatinized starch-containing cereals

[0149] One aspect of the invention relates to a process for producing a fermentation product from ungelatinized starch-containing cereals (i.e., a process of granulated starch - often referred to as a "raw starch hydrolysis" process), wherein at least one GH5_21 xylanase is present or added during saccharification or fermentation; and wherein the fermenting organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2. This process of the invention contemplates any of the GH5_21, CBH1, and CBH2 enzymes described herein, particularly the compositions shown in the following examples.

[0150] In an embodiment, a process for producing a fermentation product from ungelatinized starch-containing cereals comprises the steps of:

[0151] (a) saccharifying the starch-containing cereal at a temperature below the initial gelatinization temperature using glucoamylase and α-amylase to produce fermentable sugars; and

[0152] (b) fermenting the sugars using a fermenting organism to produce a fermentation product;

[0153] wherein at least one GH5_21 xylanase is present or added during saccharification or fermentation; and

[0154] wherein the fermenting organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2.

[0155] In an embodiment, the GH5_21 xylanase is present or added during saccharification step (a). In an embodiment, the GH5_21 xylanase is present or added during fermentation step (b). In an embodiment, steps (a) and (b) are carried out simultaneously in simultaneous saccharification and fermentation (SSF). In an embodiment, the GH5_21 xylanase is present or added during SSF. In an embodiment, the GH5_21 xylanase used in saccharification step (a) and / or fermentation step (b) is present or added via in situ expression from a fermenting organism (e.g., yeast).

[0156] Raw starch hydrolysis (RSH) processes are well known in the art. Those skilled in the art will understand that the process parameters described in Part I above apply to the processes described in this Part, except for the process parameters associated with the liquefaction step (a) that are not carried out in the RSH process, including selection of starchy cereals, reduction of cereal particle size, saccharification temperature, time and pH, conditions for simultaneous saccharification and fermentation, and saccharifying enzymes. The process parameters of exemplary raw starch hydrolysis processes are described in further detail in WO2004 / 106533, which is incorporated herein by reference.

[0157] Examples of α - amylases preferably used in step (a) and / or step (b) include, but are not limited to, those described in WO2004 / 055178, WO 2005 / 003311, WO 2006 / 069290, WO 2013 / 006756, WO 2013 / 034106, WO2021 / 163015, and WO 2021 / 163036, each of which is incorporated herein by reference.

[0158] Examples of glucoamylases preferably used in step (a) and / or step (b) include, but are not limited to, WO 1999 / 28448, WO 2005 / 045018, WO2005 / 069840, WO 2006 / 069289, each of which is incorporated herein by reference.

[0159] Examples of compositions containing α - amylase and glucoamylase preferably used in step (a) and / or step (b) include, but are not limited to, the compositions described in WO 2015 / 031477, which is incorporated herein by reference.

[0160] A. Exemplary fermenting organisms

[0161] Aspects of the present invention relate to fermenting organisms used in combination with GH5 family xylanases, which fermenting organisms comprise a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2. The present invention contemplates the use of any fermenting organism that, when used in combination with a GH5 family xylanase, increases the yield of fermentation products and / or reduces residual solids compared to processes using fermenting organisms lacking a heterologous polynucleotide encoding CBH1 and / or a heterologous polynucleotide encoding CBH2.

[0162] Particularly suitable fermenting organisms are capable of directly or indirectly fermenting sugars (such as arabinose, glucose, maltose, and / or xylose) into (i.e., converting them into) the desired fermentation product (such as ethanol). Examples of fermenting organisms include fungal organisms such as yeasts. Preferred yeasts include strains of the species Saccharomyces, particularly Saccharomyces cerevisiae.

[0163] During fermentation (such as SSF), suitable concentrations of viable fermenting organisms are well known in the art or can be readily determined by a person skilled in the art. In one embodiment, a fermenting organism (such as ethanol-fermenting yeast (e.g., Saccharomyces cerevisiae)) is added to the fermentation medium such that the viable fermenting organism (such as yeast) count per mL of the fermentation medium is from 10 5 to 10 12 , preferably from 10 7 to 10 10 , especially about 5 x 10 7 cells.

[0164] Examples of commercially available yeasts include, for example, RED STAR TM and ETHANOL RED TM yeasts (available from Fermentis / Lesaffre, USA), FALI (available from Fleischmann’s Yeast, USA), SUPERSTART and THERMOSACC TM fresh yeast (available from Ethanol Technology, Wisconsin, USA), BIOFERM AFT and XR (available from North American Bioproducts Corporation, Georgia, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties). Other available yeast strains are available from biological depositories such as the American Type Culture Collection (ATCC) or the German Collection of Microorganisms and Cell Cultures (DSMZ), such as, for example, BY4741 (e.g., ATCC201388); Y108-1 (ATCC PTA.10567) and NRRL YB-1952 (ARS Culture Collection). There are also other Saccharomyces cerevisiae strains suitable as host cells, such as DBY746, [Alpha][Eta]22, S150-2B, GPY55-15Ba, CEN.PK, USM21, TMB3500, TMB3400, VTT-A-63015, VTT-A-85068, VTT-c-79093 and their derivatives, as well as yeast species 1400, 424A (LNH-ST), 259A (LNH-ST) and their derivatives.

[0165] As used herein, a "derivative" of a strain is derived from a reference strain, such as by mutagenesis, recombinant DNA technology, mating, cell fusion, or cytoduction between yeast strains. Those skilled in the art will understand that genetic alterations (including the metabolic modifications exemplified herein) can be described with reference to a suitable host organism and its corresponding metabolic reactions or a suitable source organism for the desired genetic material (such as genes of a desired metabolic pathway). However, given the whole-genome sequencing of a wide variety of organisms and the high level of skill in the field of genomics, those skilled in the art can apply the teachings and guidance provided herein to other organisms. For example, the metabolic alterations exemplified herein can be readily applied to other species by incorporating the same or similar coding nucleic acids from a species different from the reference species.

[0166] The host cell or fermenting organism can be a strain of the genus Saccharomyces, such as a Saccharomyces cerevisiae strain produced using the methods described and involved in U.S. Patent No. 8,257,959-BB. In one embodiment, the recombinant cell is a derivative of the strain Saccharomyces cerevisiae CIBTS1260 (deposited under accession number NRRL Y-50973 at the Agricultural Research Service Culture Collection (NRRL), 61604, Illinois, USA).

[0167] The strain can also be a Saccharomyces cerevisiae strain NMI V14 / 004037 (see, WO 2015 / 143324 and WO2015 / 143317, each incorporated herein by reference), strain numbers V15 / 004035, V15 / 004036, and V15 / 004037 (see, WO 2016 / 153924, incorporated herein by reference), strain numbers V15 / 001459, V15 / 001460, V15 / 001461 (see, WO 2016 / 138437, incorporated herein by reference), strain number NRRL Y67342 (see, WO 2018 / 098381, incorporated herein by reference), strain numbers NRRL Y67549 and NRRL Y67700 (see, WO 2019 / 161227, incorporated herein by reference), or a derivative of any strain described in WO 2017 / 087330 (incorporated herein by reference).

[0168] In one embodiment, the fermenting organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2. Any CBH1 and CBH2 having cellobiohydrolase I and cellobiohydrolase II activity, respectively, can be used in conjunction with the processes described herein and / or expressed by the host cells or fermenting organisms described herein.

[0169] CBH1 or CBH2 can be obtained from a microorganism of any genus. For the purposes of the present disclosure, the term "obtained from" as used herein in connection with a given source shall mean that the polypeptide encoded by the polynucleotide is produced by that source or by a strain that has had inserted into it the polynucleotide from that source. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0170] In one embodiment, CBH1 is Penicillium CBH1, such as Penicillium emersonii CBH1 (e.g., Penicillium emersonii CBH1 of SEQ ID NO:16). In one embodiment, CBH2 is Talaromyces CBH2, such as Talaromyces verruculosus cellobiohydrolase II (e.g., Talaromyces verruculosus cellobiohydrolase II of SEQ ID NO:17). It should be understood that for the foregoing species, the present invention encompasses the perfect and imperfect states, as well as other taxonomic equivalents, such as anamorphs, regardless of their known species names. Those skilled in the art will readily recognize the identity of appropriate equivalents.

[0171] Strains of these species are readily available to the public at many culture collections, such as the American Type Culture Collection (ATCC), the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), the Centraalbureau Voor Schimmelcultures (CBS), and the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0172] The CBH1 or CBH2 coding sequences, or subsequences or fragments thereof, described or referenced herein can be used to design nucleic acid probes to identify and clone DNA encoding CBH1 or CBH2 from strains of different genera or species according to methods well known in the art. In particular, standard Southern blotting procedures can be followed using such probes to hybridize to genomic DNA or cDNA of the cells of interest in order to identify and isolate the corresponding gene therein. Such probes can be significantly shorter than the full sequence, but should be at least 15 nucleotides in length, such as at least 25, at least 35, or at least 70 nucleotides. Preferably, the nucleic acid probe is at least 100 nucleotides in length, such as at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. Typically, the probe is labeled (e.g., with 32 P, 3 H, 35 S, biotin, or avidin) for the detection of the corresponding gene.

[0173] Genomic DNA or cDNA libraries prepared from such other strains can be screened for DNA that hybridizes to the probes described above and encodes CBH1 or CBH2. Genomic DNA or other DNA from such other strains can be separated by agarose or polyacrylamide gel electrophoresis or other separation techniques. The DNA from the library or the separated DNA can be transferred to and immobilized on nitrocellulose or other suitable carrier material. This carrier material is used in Southern blotting to identify clones or DNA that hybridize to the coding sequence or subsequences thereof.

[0174] In one embodiment, the nucleic acid probe is a polynucleotide or a subsequence thereof encoding the mature CBH1 of SEQ ID NO:16 or the mature CBH2 of SEQ ID NO:17 or a fragment thereof.

[0175] For the purposes of the above probes, hybridization means that the polynucleotide hybridizes to the labeled nucleic acid probe, or its full-length complementary strand or a subsequence thereof; the hybridization is carried out under very low to very high stringency conditions. Molecules that hybridize to the nucleic acid probe under these conditions can be detected using, for example, X-ray film. Stringency and wash conditions are defined as above.

[0176] In one embodiment, CBH1 is encoded by a polynucleotide that hybridizes under at least low stringency conditions, such as medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions, to the full-length complementary strand of the coding sequence of SEQ ID NO:16. (Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York). In one embodiment, CBH2 is encoded by a polynucleotide that hybridizes under at least low stringency conditions, such as medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions, to the full-length complementary strand of the coding sequence of SEQ ID NO:17.

[0177] Polypeptides having CBH1 and CBH2 can also be identified and obtained from other sources, including microorganisms isolated from nature (such as soil, compost, water, silage, etc.) or DNA samples obtained directly from natural materials (such as soil, compost, water, silage, etc.). Techniques for directly isolating microorganisms and DNA from natural habitats are well known in the art. Polynucleotides encoding CBH1 or CBH2 can then be derived by similarly screening the genomic or cDNA library of another microorganism or a mixed DNA sample.

[0178] Once a polynucleotide encoding CBH1 or CBH2 has been detected with a suitable probe as described herein, the sequence can be isolated or cloned by using techniques known to those of ordinary skill in the art (see, e.g., Sambrook et al., 1989, supra). Techniques for isolating or cloning polynucleotides encoding polypeptides include isolation from genomic DNA, preparation from cDNA, or combinations thereof. Polynucleotides can be cloned from such genomic DNA, for example, by detecting cloned DNA fragments having common structural features using well-known polymerase chain reaction (PCR) or antibody screening of expression libraries (see, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York). Other nucleic acid amplification procedures can be used, such as ligase chain reaction (LCR), ligation-activated transcription (LAT), and nucleic acid sequence-based amplification (NASBA).

[0179] In one embodiment, CBH1 comprises or consists of the amino acid sequence of SEQ ID NO:16 or its mature polypeptide. In another embodiment, CBH1 is a fragment of CBH1 of SEQ ID NO:16 or its mature polypeptide, wherein, for example, the fragment has CBH1 activity. In one embodiment, the number of amino acid residues in the fragment is at least 75% of the number of amino acid residues in the full-length CBH1, such as at least 80%, 85%, 90% or 95%. In other embodiments, CBH1 may comprise the catalytic domain SEQ ID NO:16.

[0180] In one embodiment, CBH2 comprises or consists of the amino acid sequence of SEQ ID NO:17 or its mature polypeptide. In another embodiment, CBH2 is a fragment of CBH2 of SEQ ID NO:17 or its mature polypeptide, wherein, for example, the fragment has CBH2 activity. In one embodiment, the number of amino acid residues in the fragment is at least 75% of the number of amino acid residues in the full-length CBH2, such as at least 80%, 85%, 90% or 95%. In other embodiments, CBH2 may comprise the catalytic domain SEQ ID NO:17.

[0181] CBH1 and CBH2 can be variants of CBH1 and CBH2 described above (e.g., SEQ ID NO:16, SEQ ID NO:17, or their mature polypeptides). In one embodiment, CBH1 has at least 60% sequence identity with the amino acid sequence of SEQ ID NO:16 or its mature polypeptide, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%. In one embodiment, CBH2 has at least 60% sequence identity with the amino acid sequence of SEQ ID NO:17 or its mature polypeptide, such as at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100%.

[0182] In one embodiment, CBH1 differs from the amino acid sequence of SEQ ID NO:16 or its mature polypeptide by no more than ten amino acids, for example, by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by no more than one amino acid. In one embodiment, CBH2 differs from the amino acid sequence of SEQ ID NO:17 or its mature polypeptide by no more than ten amino acids, for example, by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by no more than one amino acid.

[0183] The properties of the amino acid changes are usually minor, i.e., conservative amino acid substitutions or insertions that do not significantly affect protein folding and / or activity; typically small deletions of 1 to about 30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as amino-terminal methionine residues; small linker peptides of up to about 20 - 25 residues; or small extensions that facilitate purification by altering the net charge or another function (such as a polyhistidine segment, an epitope, or a binding domain).

[0184] Examples of conservative substitutions are within the following groups: basic amino acids (arginine, lysine, and histidine), acidic amino acids (glutamic acid and aspartic acid), polar amino acids (glutamine and asparagine), hydrophobic amino acids (leucine, isoleucine, and valine), aromatic amino acids (phenylalanine, tryptophan, and tyrosine), and small amino acids (glycine, alanine, serine, threonine, and methionine). Amino acid substitutions that generally do not alter specific activity are known in the art and are described, for example, by H. Neurath and R. L. Hill, 1979, The Proteins, Academic Press, New York. The most commonly occurring exchanges are Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Tyr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0185] Alternatively, these amino acid changes have such a nature as to alter the physicochemical properties of the polypeptide. For example, the amino acid changes can improve the thermal stability of an enzyme, alter substrate specificity, change the optimal pH, and so on.

[0186] Essential amino acids 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, a single alanine mutation is introduced at each residue in the molecule, and the activity of the resulting mutant molecule is tested to identify the amino acid residues that are crucial for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271:4699-4708. Active sites or other biological interactions can also be determined by physical analysis of the structure, such as by the following techniques: nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, along with mutagenesis of putative contact-site amino acids (see, e.g., de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64). The identity of essential amino acids can also be inferred from the identity analysis of other cellulases related to the reference enzyme.

[0187] Additional guidance regarding the structure-activity relationships of the cellulases herein can be determined using multiple sequence alignment (MSA) techniques well known in the art. Based on the teachings herein, one of ordinary skill in the art can make similar alignments with any number of CBH1 or CBH2 enzymes described herein or known in the art. Such alignments assist one of ordinary skill in the art in determining potentially relevant domains (e.g., binding domains or catalytic domains) and in determining which amino acid residues are conserved and not conserved among different cellulase sequences. It should be understood in the art that changing amino acids that are conserved at a particular position among the disclosed polypeptides is more likely to result in a change in biological activity (Bowie et al., 1990, Science 247:1306-1310: “Residues that are directly involved in protein functions such as binding or catalysis will certainly be among the most conserved”). In contrast, substituting amino acids that are not highly conserved among the polypeptides is less likely or will not significantly alter biological activity.

[0188] A person skilled in the art can find even additional guidance on structure-activity relationships in the publicly known x-ray crystallography studies in the art.

[0189] Using known mutagenesis, recombination, and / or shuffling methods, followed by relevant screening procedures, single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested, such relevant screening procedures 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 site-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0190] The mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect the activity of the cloned, mutagenized polypeptides expressed by the host cells (Ness et al., 1999, Nature Biotechnology 17:893-896). The mutagenized DNA molecules encoding active CBH1 or CBH2 can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow for the rapid determination of the importance of individual amino acid residues in the polypeptide.

[0191] In one embodiment, the heterologous polynucleotide encoding CBH1 comprises or consists of: the coding sequence of CBH1 of SEQ ID NO:16 or its mature polypeptide. In another embodiment, the heterologous polynucleotide encoding CBH1 comprises a subsequence of the coding sequence of CBH1 of SEQ ID NO:16, wherein the subsequence encodes a polypeptide having CBH1 activity. In another embodiment, the number of nucleotide residues in the coding subsequence is at least 75%, such as at least 80%, 85%, 90% or 95% of the number of the coding sequence. In another embodiment, the heterologous polynucleotide encoding CBH1 comprises a coding sequence having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the coding sequence of CBH1 of SEQ ID NO:16 or its mature polypeptide.

[0192] In one embodiment, the heterologous polynucleotide encoding CBH2 comprises or consists of: the coding sequence of CBH2 of SEQ ID NO:17 or its mature polypeptide. In another embodiment, the heterologous polynucleotide encoding CBH2 comprises a subsequence of the coding sequence of CBH2 of SEQ ID NO:17, wherein the subsequence encodes a polypeptide having CBH2 activity. In another embodiment, the number of nucleotide residues in the coding subsequence is at least 75%, such as at least 80%, 85%, 90% or 95% of the number of the coding sequence. In another embodiment, the heterologous polynucleotide encoding CBH2 comprises a coding sequence having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the coding sequence of CBH2 of SEQ ID NO:17 or its mature polypeptide.

[0193] The reference coding sequence for any relevant aspect or embodiment described herein can be a native coding sequence or a degenerate sequence, such as a codon-optimized coding sequence (e.g., optimized for expression in Saccharomyces cerevisiae or any other host used for production). Codon optimization for expression in yeast cells is known in the art (e.g., US 8,326,547).

[0194] CBH1 and CBH2 can be fusion polypeptides or cleavable fusion polypeptides, wherein another polypeptide is fused at the N-terminus or C-terminus of the enzyme. The fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide with CBH1 or CBH2. Techniques for producing fusion polypeptides are known in the art and include ligating the coding sequences encoding the polypeptides such that they are in frame and expressing the fusion polypeptide under the control of the same one or more promoters and terminators. Inteins can also be used to construct fusion polypeptides, where the fusion is generated post-translationally (Cooper et al., 1993, EMBO J. 12:2575-2583; Dawson et al., 1994, Science 266:776-779).

[0195] In some embodiments, CBH1 or CBH2 is a fusion protein that comprises a signal peptide linked to the N-terminus of the mature polypeptide, such as any of the signal sequences described in WO 2021 / 025872 “Fusion Proteins For Improved Enzyme Expression” (the content of which is hereby incorporated by reference).

[0196] In some embodiments, the host cell and / or fermenting organism comprises one or more heterologous polynucleotides encoding an α-amylase, glucoamylase, protease, and / or cellulase. Examples of α-amylase, glucoamylase, protease, and cellulase suitable for expression in the host cell and / or fermenting organism are described in more detail herein.

[0197] In some embodiments, the host cell and / or fermenting organism comprises one or more heterologous polynucleotides encoding a GH5 xylanase (e.g., GH5_21 xylanase). Examples of GH5 xylanases are described in more detail herein.

[0198] In some embodiments, the host cell and / or fermenting organism comprises an active pentose fermentation pathway. In some embodiments, the host cell and / or fermenting organism comprises an active xylose fermentation pathway. In some embodiments, the host cell and / or fermenting organism comprises an active arabinose fermentation pathway.

[0199] The host cells and fermentation organisms described herein can utilize the following expression vectors, which contain the coding sequences of one or more (e.g., two, several) heterologous genes, and these coding sequences are linked to one or more control sequences that direct expression in a suitable cell under conditions compatible with the one or more control sequences. Such expression vectors can be used in any of the cells and methods described herein. The polynucleotides described herein can be manipulated in a variety of ways to provide expression of the desired polypeptide. Depending on the expression vector, it may be desirable or necessary to manipulate the polynucleotide prior to insertion into the vector. Techniques for modifying polynucleotides using recombinant DNA methods are well known in the art.

[0200] A construct or vector (or constructs or vectors) can be introduced into a cell such that the construct or vector is maintained as a chromosomal integrant or as an autonomously replicating extrachromosomal vector, as described earlier; the construct or vector (or these constructs or vectors) contains one or more (e.g., two, several) heterologous genes.

[0201] A variety of nucleotide and control sequences can be ligated together to generate a recombinant expression vector, which can include one or more (e.g., two, several) convenient restriction sites to allow insertion or substitution of the polynucleotide at such sites. Alternatively, the one or more polynucleotides can be expressed by inserting one or more polynucleotides or nucleic acid constructs containing the sequence into an appropriate vector for expression. When generating an expression vector, the coding sequence is positioned in the vector such that the coding sequence is operably linked to an appropriate control sequence for expression.

[0202] The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can cause expression of the polynucleotide. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0203] The vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that integrates into the genome when introduced into a host cell and replicates with the chromosome into which it has integrated. In addition, a single vector or plasmid or two or more vectors or plasmids (which together contain the total DNA to be introduced into the genome of the cell) or a transposon can be used.

[0204] An expression vector can contain any suitable promoter sequence that is recognized by a cell to express the genes described herein. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter can be any polynucleotide that exhibits transcriptional activity in the selected cell, including mutant, truncated, and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the cell.

[0205] Each heterologous polynucleotide described herein can be operably linked to a promoter that is exogenous to the polynucleotide. For example, in one embodiment, a nucleic acid construct encoding a fusion protein is operably linked to a promoter that is exogenous to the polynucleotide. These promoters can be the same as or have a high degree of sequence identity (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%) to the selected native promoter.

[0206] Examples of suitable promoters for directing transcription of a nucleic acid construct in yeast cells include, but are not limited to, promoters obtained from the genes: enolase (e.g., Saccharomyces cerevisiae enolase or Issatchenkia orientalis enolase (ENO1)), galactokinase (e.g., Saccharomyces cerevisiae galactokinase or Issatchenkia orientalis galactokinase (GAL1)), alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (e.g., Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase or Issatchenkia orientalis alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2 / GAP)), phosphoglyceraldehyde isomerase (e.g., Saccharomyces cerevisiae phosphoglyceraldehyde isomerase or Issatchenkia orientalis phosphoglyceraldehyde isomerase (TPI)), metallothionein (e.g., Saccharomyces cerevisiae metallothionein or Issatchenkia orientalis metallothionein (CUP1)), 3-phosphoglycerate kinase (e.g., Saccharomyces cerevisiae 3-phosphoglycerate kinase or Issatchenkia orientalis 3-phosphoglycerate kinase (PGK)), PDC1, xylose reductase (XR), xylitol dehydrogenase (XDH), L-(+)-lactate-cytochrome c oxidoreductase (CYB2), translation elongation factor-1 (TEF1), translation elongation factor-2 (TEF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and orotidine 5'-phosphate decarboxylase (URA3) genes. Other suitable promoters can be obtained from the Saccharomyces cerevisiae TDH3, HXT7, PGK1, RPL18B, and CCW12 genes. Additional useful promoters for yeast host cells are described by Romanos et al., 1992, Yeast 8:423-488.

[0207] The control sequence may also be a suitable transcription terminator sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3'-end of the polynucleotide encoding the polypeptide. Any terminator functional in the selected yeast cell may be used. The terminator may be the same as or have a high degree of sequence identity with the selected native terminator (e.g., at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99%).

[0208] Suitable terminators for yeast host cells may be obtained from the following genes: enolase (e.g., enolase from Saccharomyces cerevisiae or Issatchenkia orientalis), cytochrome C (e.g., cytochrome C (CYC1) from Saccharomyces cerevisiae or Issatchenkia orientalis), glyceraldehyde-3-phosphate dehydrogenase (e.g., glyceraldehyde-3-phosphate dehydrogenase (gpd) from Saccharomyces cerevisiae or Issatchenkia orientalis), PDC1, XR, XDH, transaldolase (TAL), transketolase (TKL), ribose 5-phosphate-ketol isomerase (RKI), CYB2, and the galactose gene family (especially the GAL10 terminator). Other suitable terminators may be obtained from the Saccharomyces cerevisiae ENO2 or TEF1 genes. Additional useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

[0209] The control sequence may also be an mRNA stabilizer region downstream of the promoter and upstream of the coding sequence of the gene, which increases the expression of the gene.

[0210] Examples of suitable mRNA stabilizer regions are obtained from the Bacillus thuringiensis cryIIIA gene (WO 94 / 25612) and the Bacillus subtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

[0211] The control sequence may also be a suitable leader sequence, which, when transcribed, is the untranslated region of the mRNA important for translation by the host cell. The leader sequence is operably linked to the 5'-end of the polynucleotide encoding the polypeptide. Any leader sequence functional in the selected yeast cell may be used.

[0212] Suitable leaders for yeast host cells are obtained from the following genes: enolase (e.g., enolase from Saccharomyces cerevisiae or Issatchenkia orientalis (ENO-1)), 3-phosphoglycerate kinase (e.g., 3-phosphoglycerate kinase from Saccharomyces cerevisiae or Issatchenkia orientalis), α-factor (e.g., α-factor from Saccharomyces cerevisiae or Issatchenkia orientalis), and alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (e.g., alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae or Issatchenkia orientalis (ADH2 / GAP)).

[0213] The control sequence may also be a polyadenylation sequence; a sequence that is operably linked to the 3'-end of a polynucleotide and that is recognized by a host cell as a signal to add polyadenylate residues to the transcribed mRNA. Any polyadenylation sequence that is functional in the selected host cell can be used. Polyadenylation sequences available for yeast cells are described in Guo and Sherman, 1995, Mol. Cellular Biol. 15:5983-5990.

[0214] The control sequence may also be a signal peptide coding region that encodes a signal peptide linked to the N-terminus of a polypeptide and that directs the polypeptide into the secretory pathway of a cell. The 5'-end of the coding sequence of a polynucleotide may itself contain a signal peptide coding sequence that is naturally linked in the translation reading frame to a coding sequence segment encoding the polypeptide. Alternatively, the 5'-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. In cases where the coding sequence does not naturally contain a signal peptide coding sequence, a foreign signal peptide coding sequence may be required. Alternatively, a foreign signal peptide coding sequence may simply replace the native signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs an expressed polypeptide into the secretory pathway of a host cell can be used. Useful signal peptides for yeast host cells are obtained from the genes of the following: Saccharomyces cerevisiae α-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

[0215] The control sequence may also be a propeptide coding sequence that encodes a propeptide located at the N-terminus of a polypeptide. The resulting polypeptide is referred to as a proenzyme or pro-polypeptide (or in some cases as a zymogen). Pro-polypeptides are generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the pro-polypeptide. Propeptide coding sequences can be obtained from the genes of the following: Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95 / 33836), Rhizomucor miehei aspartic protease, and Saccharomyces cerevisiae α-factor.

[0216] In the case where both a signal peptide sequence and a propeptide sequence are present, the propeptide sequence is located immediately adjacent to the N-terminus of the polypeptide and the signal peptide sequence is located immediately adjacent to the N-terminus of the propeptide sequence.

[0217] It may also be desirable to add regulatory sequences that allow the expression of the polypeptide to be regulated relative to the growth of the host cell. Examples of regulatory systems are those that cause gene expression to be switched on or off in response to chemical or physical stimulants, including the presence of regulatory compounds. Regulatory systems in prokaryotic systems include the lac, tac, and trp operon systems. In yeast, the ADH2 system or the GAL1 system can be used.

[0218] These vectors can contain one or more (e.g., two, several) selectable markers that allow for the convenient selection of cells such as transformed cells, transfected cells, transduced cells, etc. A selectable marker is a gene whose product provides biocide resistance or virus resistance, resistance to heavy metals, prototrophy for auxotrophs, etc. Suitable markers for yeast host cells include, but are not limited to: ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0219] These vectors can contain one or more (e.g., two, several) elements that allow the vector to be integrated into the genome of the host cell or to replicate autonomously in the cell independently of the genome.

[0220] For integration into the genome of the host cell, the vector can rely on the polynucleotide sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous or non-homologous recombination. Alternatively, the vector can contain additional polynucleotides for directing integration into one or more precise positions in one or more of the chromosomes of the host cell genome by homologous recombination. To increase the likelihood of integration at a precise position, the integration element should contain a sufficient number of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000 base pairs, and 800 to 10,000 base pairs, which have a high degree of sequence identity with the corresponding target sequence to enhance the probability of homologous recombination. The integration element can be any sequence homologous to the target sequence within the host cell genome. In addition, the integration element can be a non-coding or coding polynucleotide. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination. Potential integration sites include those described in the art (e.g., see US 2012 / 0135481).

[0221] For autonomous replication, the vector can further contain an origin of replication that enables the vector to replicate autonomously in yeast cells. The origin of replication can be any plasmid replicon that functions in the cell to mediate autonomous replication. The term "origin of replication" or "plasmid replicon" means a polynucleotide that enables a plasmid or vector to replicate in vivo. Examples of origins of replication for use in yeast host cells are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0222] More than one copy of the polynucleotides described herein can be inserted into a host cell to increase polypeptide production. An increased copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the yeast cell genome or by including an amplifiable selectable marker gene together with the polynucleotide, wherein cells containing the amplified copy of the selectable marker gene, and thereby an additional copy of the polynucleotide, can be selected by culturing the cells in the presence of an appropriate selective reagent.

[0223] Procedures for ligating the above-described elements to construct the recombinant expression vectors described herein are well known to those skilled in the art (see, for example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd ed., Cold Spring Harbor, New York).

[0224] Additional procedures and techniques known in the art for preparing recombinant cells for ethanol fermentation are described, for example, in WO 2016 / 045569, the contents of which are hereby incorporated by reference.

[0225] The host cell or fermenting organism can be in the form of a composition that contains the host cell or fermenting organism (e.g., a yeast strain described herein) and naturally occurring and / or non-naturally occurring components.

[0226] The host cell or fermenting organism described herein can be in any viable form, including crushed, dried, including active dry and instant, compressed, paste (liquid) form, etc. In one embodiment, the host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) is dry yeast, such as active dry yeast or instant yeast. In one embodiment, the host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) is crushed yeast. In one embodiment, the host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) is compressed yeast. In one embodiment, the host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) is paste yeast.

[0227] In one embodiment, it is a composition that comprises a host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and one or more components selected from the group consisting of: surfactants, emulsifiers, gums, swelling agents, and antioxidants and other processing aids.

[0228] The composition described herein can comprise a host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable surfactant. In one embodiment, one or more surfactants are anionic surfactants, cationic surfactants, and / or nonionic surfactants.

[0229] The composition described herein can comprise a host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable emulsifier. In one embodiment, the emulsifier is a fatty acid ester of sorbitan. In one embodiment, the emulsifier is selected from the group consisting of: sorbitan monostearate (SMS), citric acid esters of mono- and diglycerides, polyglycerol esters, and fatty acid esters of propylene glycol.

[0230] In one embodiment, the composition comprises a host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and Olindronal SMS, Olindronal SK, or Olindronal SPL, including the compositions described in European Patent No. 1,724,336, which is hereby incorporated by reference in its entirety. These products are commercially available from Bussetti of Austria for active dry yeast.

[0231] The composition described herein can comprise a host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable gum. In one embodiment, the gum is selected from the group consisting of: locust bean gum, guar gum, tragacanth gum, gum arabic, xanthan gum, and gum acacia, particularly for paste, compressed, and dry yeast.

[0232] The composition described herein can comprise a host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable swelling agent. In one embodiment, the swelling agent is methylcellulose or carboxymethylcellulose.

[0233] The composition described herein can comprise a host cell or fermenting organism (e.g., a Saccharomyces cerevisiae strain) described herein and any suitable antioxidant. In one embodiment, the antioxidant is butylated hydroxyanisole (BHA) and / or butylated hydroxytoluene (BHT), or ascorbic acid (vitamin C), especially for active dry yeast.

[0234] The host cells and fermenting organisms described herein may also contain one or more (e.g., two, several) gene disruptions, e.g., to divert sugar metabolism away from unwanted products and towards ethanol. In some embodiments, the recombinant host cells produce a greater amount of ethanol when cultured under the same conditions compared to cells that do not contain the one or more disruptions. In some embodiments, one or more of the endogenous genes that are disrupted are inactivated.

[0235] In certain embodiments, the host cells or fermenting organisms provided herein contain disruptions of one or more endogenous genes that encode enzymes involved in the production of alternative fermentation products (such as glycerol) or other by-products (such as acetate or diols). For example, the cells provided herein may contain disruptions in one or more of the following: glycerol 3-phosphate dehydrogenase (GPD, which catalyzes the reaction of dihydroxyacetone phosphate to glycerol 3-phosphate), glycerol 3-phosphatase (GPP, which catalyzes the conversion of glycerol-3-phosphate to glycerol), glycerol kinase (which catalyzes the conversion of glycerol 3-phosphate to glycerol), dihydroxyacetone kinase (which catalyzes the conversion of dihydroxyacetone phosphate to dihydroxyacetone), glycerol dehydrogenase (which catalyzes the conversion of dihydroxyacetone to glycerol), and aldehyde dehydrogenase (ALD, e.g., which converts acetaldehyde to acetate).

[0236] Model analysis can be used to design additional gene disruptions for optimized pathway utilization. An exemplary computational method for identifying and designing metabolic alterations that favor the biosynthesis of a desired product is the OptKnock computational framework, Burgard et al., 2003, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 84:647-657.

[0237] Host cells or fermenting organisms containing gene disruptions can be constructed using methods well known in the art, including those described herein. A portion of the gene, such as the coding region or control sequences required for the expression of the coding region, can be disrupted. Such control sequences of the gene can be the promoter sequence or a functional part thereof, i.e., a part sufficient to affect the expression of the gene. For example, the promoter sequence can be inactivated so that there is no expression, or the native promoter sequence can be replaced with a weaker promoter to reduce the expression of the coding sequence. Other control sequences that can be modified include, but are not limited to, leaders, propeptide sequences, signal sequences, transcription terminators, and transcriptional activators.

[0238] Host cells and fermenting organisms containing gene disruptions can be constructed by gene deletion techniques to eliminate or reduce the expression of the gene. Gene deletion techniques allow for the partial or complete removal of the gene, thereby eliminating its expression. In such methods, the deletion of the gene is accomplished by homologous recombination using a plasmid that has been constructed to contain the 5' and 3' regions flanking the gene adjacent to each other.

[0239] Host cells or fermenting organisms containing gene disruption can also be constructed by introducing, replacing and / or removing one or more (e.g., two, several) nucleotides in the gene or in its control sequences required for its transcription or translation. For example, nucleotides can be inserted or removed to introduce a stop codon, remove a start codon or shift the reading frame. Such modifications can be accomplished by site-directed mutagenesis or mutagenesis generated by PCR according to methods known in the art. See, e.g., Botstein and Shortle, 1985, Science 229:4719; Lo et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 81:2285; Higuchi et al., 1988, Nucleic Acids Res 16:7351; Shimada, 1996, Meth. Mol. Biol. 57:157; Ho et al., 1989, Gene 77:61; Horton et al., 1989, Gene 77:61; and Sarkar and Sommer, 1990, BioTechniques 8:404.

[0240] Host cells and fermenting organisms containing gene disruption can also be constructed by inserting a disruptive nucleic acid construct into the gene, the disruptive nucleic acid construct containing a nucleic acid fragment homologous to the gene, the fragment being capable of producing repeats of regions of homology and incorporating construct DNA between the repeated regions. Such gene disruption can abolish gene expression if the inserted construct separates the promoter of the gene from the coding region or interrupts the coding sequence such that a non-functional gene product is produced. The disruption construct can simply be a selectable marker gene flanked by 5' and 3' regions homologous to the gene. The selectable marker allows identification of transformants containing the disrupted gene.

[0241] Host cells and fermenting organisms containing gene disruption can also be constructed by a gene transformation process (see, e.g., Iglesias and Trautner, 1983, Molecular General Genetics 189:73-76). For example, in a gene transformation method, the nucleotide sequence corresponding to the gene is mutagenized in vitro to produce a defective nucleotide sequence, which is then transformed into a recombinant strain to produce a defective gene. By homologous recombination, the defective nucleotide sequence replaces the endogenous gene. It may be desirable for the defective nucleotide sequence to also contain a marker for selection of transformants containing the defective gene.

[0242] Methods well known in the art can be used to further construct host cells and fermentation organisms containing gene disruptions by random or site-directed mutagenesis, including but not limited to chemical mutagenesis (see, e.g., Hopwood, The Isolation of Mutants in Methods in Microbiology (J.R. Norris and D.W.Ribbons, eds.) pp. 363-433, Academic Press, New York, 1970). The gene can be modified by subjecting the parental strain to mutagenesis and screening for mutant strains in which the expression of the gene has been reduced or inactivated. The mutagenesis can be site-directed or random, for example, by using a suitable physical or chemical mutagen, using a suitable oligonucleotide, or subjecting the DNA sequence to mutagenesis generated by PCR. In addition, the mutagenesis can be carried out by using any combination of these mutagenesis methods.

[0243] Examples of physical or chemical mutagens suitable for the purposes of the present invention include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), N-methyl-N'-nitrosoguanidine (NTG), o-methylhydroxylamine, nitrous acid, ethyl methanesulfonate (EMS), sodium bisulfite, formic acid, and nucleotide analogs. When using such reagents, the mutagenesis is typically carried out by incubating the parental strain to be mutagenized in the presence of the selected mutagen under suitable conditions and selecting for mutants that exhibit reduced or no expression of the gene.

[0244] Nucleotide sequences homologous or complementary to the genes described herein from other microbial sources can be used to disrupt the corresponding genes in the selected recombinant strains.

[0245] In one embodiment, the gene modification in the host cell and fermentation organism is not marked with a selectable marker. The selectable marker gene can be removed by culturing the mutant in a counter-selection medium. In the case where the selectable marker gene contains repetitive sequences flanking its 5' and 3' ends, when the mutant strain is subjected to counter-selection, these repetitive sequences will facilitate the looping out of the selectable marker gene by homologous recombination. The selectable marker gene can also be removed by introducing a nucleic acid fragment into the mutant strain, which nucleic acid fragment contains the 5' and 3' regions of the defective gene but lacks the selectable marker gene, followed by selection on a counter-selection medium, by homologous recombination. By homologous recombination, the defective gene containing the selectable marker gene is replaced by the nucleic acid fragment lacking the selectable marker gene. Other methods known in the art can also be used.

[0246] B. Exemplary GH5 Xylanases

[0247] Aspects of the invention relate to GH5 family xylanases in combination with a fermenting organism that comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2 to increase hemicellulose fiber solubilization and the production of monomeric arabinose and / or xylose. The invention contemplates the use of any GH5 xylanase that, when used in combination with a fermenting organism, increases the yield of a fermentation product and / or reduces residual solids as compared to a process lacking the GH5 xylanase.

[0248] In embodiments, the xylanase is a GH5 family xylanase.

[0249] In embodiments, the xylanase is a GH5_21 xylanase.

[0250] Exemplary GH5_21 xylanases include, but are not limited to, xylanases from the genus Bacteroides, Belliella, Chryseobacterium, or Sphingobacterium.

[0251] Exemplary GH5_21 xylanases include, but are not limited to, xylanases from the following species: Bacteroides cellulosilyticus CL02Y12C19, Belliella sp.-64282, Chryseobacterium oncorhynchi, or Sphingobacterium sp.-64162.

[0252] Exemplary GH5_21 xylanases include, but are not limited to, xylanases from a bioreactor metagenome, an elephant dung metagenome, a xanthan gum alkaline community O, a xanthan gum alkaline community S, or a xanthan gum alkaline community T.

[0253] Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO:1. In embodiments, the GH_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1 and has xylanase activity.

[0254] The exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:2. In the examples, the GH_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:2 and has xylanase activity.

[0255] The exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:3. In the examples, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:3 and has xylanase activity.

[0256] The exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:4. In the examples, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:4 and has xylanase activity.

[0257] The exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:5. In the examples, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5 and has xylanase activity.

[0258] The exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:6. In the examples, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:6 and has xylanase activity.

[0259] The exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:7. In the examples, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:7 and has xylanase activity.

[0260] The exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:8. In the examples, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:8 and has xylanase activity.

[0261] The exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:9. In the examples, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:9 and has xylanase activity.

[0262] The exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:10. In the examples, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:10 and has xylanase activity.

[0263] The exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO:11. In the examples, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:11 and has xylanase activity.

[0264] Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO:12. In an embodiment, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:12 and has xylanase activity.

[0265] Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO:13. In an embodiment, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:13 and has xylanase activity.

[0266] Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO:14. In an embodiment, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:14 and has xylanase activity.

[0267] Exemplary GH5_21 xylanases have the amino acid sequence of SEQ ID NO:15. In an embodiment, the GH5_21 xylanase has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:15 and has xylanase activity.

[0268] GH5 xylanases (e.g., GH5_21 xylanases) can be administered in pre-saccharification, saccharification, and / or simultaneous saccharification and fermentation at a concentration between 0.0001 - 1 mg EP (enzyme protein) / g DS, such as 0.0005 - 0.5 mg EP / g DS, e.g., 0.001 - 0.1 mg EP / g DS or 0.001 - 0.01 mg EP / g DS.

[0269] In one embodiment, the GH5 xylanase (e.g., GH5_21 xylanase) is present or added via in situ expression from a fermenting organism (e.g., yeast).

[0270] III. Back-end or downstream processing

[0271] A. Recovery of Fermentation Products and Production of Whole Distillers Grains

[0272] After fermentation or SSF, the fermentation product can be separated from the fermentation medium. Any method known in the art can be used to optionally recover the fermentation product (e.g., ethanol) from the fermentation medium, and such methods include, but are not limited to: chromatography, electrophoresis procedures, differential solubility, distillation, or extraction. For example, alcohols are separated and purified from fermented starchy grains by conventional distillation methods.

[0273] Thus, in one embodiment, the method of the present invention further includes distillation to obtain the fermentation product, e.g., ethanol. Fermentation and distillation can be carried out simultaneously and / or separately / sequentially; optionally, followed by one or more process steps for further refining the fermentation product. After the distillation process is completed, the remaining material is considered whole distillers grains.

[0274] As another example, the desired fermentation product can be extracted from the fermentation medium by microfiltration or membrane filtration techniques. 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 alcoholic beverages), or industrial ethanol.

[0275] In some embodiments of these methods, the recovered fermentation product is substantially pure. For these methods herein, "substantially pure" means that the recovered preparation contains no more than 15% impurities, where impurities mean compounds other than the fermentation product (e.g., ethanol). In one variant, a substantially pure preparation is provided, wherein the preparation contains no more than 25% impurities, or no more than 20% impurities, or no more than 10% impurities, or no more than 5% impurities, or no more than 3% impurities, or no more than 1% impurities, or no more than 0.5% impurities.

[0276] Suitable assays known in the art can be used to test for the production of ethanol and contaminants as well as sugar consumption. For example, ethanol products and other organic compounds can be analyzed by methods such as HPLC (high performance liquid chromatography), GC-MS (gas chromatography - mass spectrometry), and LC-MS (liquid chromatography - mass spectrometry), or other suitable analytical methods using conventional procedures well known in the art. The release of ethanol in the fermentation broth can also be tested with the culture supernatant. By HPLC (Lin et al., Biotechnol. Bioeng. [Biotechnology and Bioengineering] 90:775 - 779 (2005)), or using other suitable assays and detection methods well known in the art, the by-products and residual sugars (e.g., glucose or xylose) in the fermentation medium can be quantified, for example, using a refractive index detector for glucose and alcohols, and a UV detector for organic acids.

[0277] B. Separating the whole distillers grains (dehydrating) into distillers grains water and wet cake

[0278] In one embodiment, the whole distillers grains are separated or partitioned into a solid phase and a liquid phase by one or more methods of separating the distillers grains water from the wet cake. Separating the whole distillers grains into distillers grains water and wet cake to remove a large portion of the liquid / water can be achieved using any suitable separation technique (including centrifugation, pressing, and filtration). In a preferred embodiment, separation / dehydration is performed by centrifugation. In the industry, a preferred centrifuge is a sedimentation centrifuge, preferably a high-speed sedimentation centrifuge. An example of a suitable centrifuge is the NX 400 steep cone series from Alfa Laval, which is a high-performance sedimentation centrifuge. In another preferred embodiment, other conventional separation equipment (such as plate / frame filter presses, belt filter presses, screw presses, gravity thickeners, and dehydrators) or similar equipment is used for separation.

[0279] C. Processing of distillers grains water

[0280] Distillers grains water is the term for the supernatant from the centrifugation of the whole distillers grains. Typically, distillers grains water contains 4%-6% dry solids (DS) (mainly proteins, soluble fibers, fine fibers, and cell wall components) and has a temperature of about 60°C - 90°C. The distillers grains water stream can be condensed by evaporation to provide two process streams, including: (i) an evaporator condensate stream, which contains the condensed water removed from the distillers grains water during evaporation; and (ii) a slurry stream, which contains a more concentrated stream of non-volatile dissolved and undissolved solids, such as non-fermentable sugars and oils remaining in the distillers grains water due to the removal of the evaporated water.

[0281] Optionally, oil can be removed from the distillers grains water, or oil can be removed as an intermediate step in the evaporation process, which typically uses a series of several evaporation stages.

[0282] The slurry and / or deoiled slurry can be introduced into a dryer together with the wet grains (from the whole distillers grains separation step) to provide a product called dry distillers grains with solubles, which can also be used as animal feed. In an embodiment, the slurry and / or deoiled slurry are sprayed into one or more dryers to combine the slurry and / or deoiled slurry with the whole distillers grains to produce dry distillers grains with solubles.

[0283] Distillers' water (e.g., optionally hydrolyzed) in the range of 5 vol.% - 90 vol.%, such as in the range of 10% - 80%, such as in the range of 15% - 70%, such as in the range of 20% - 60% can be recycled (as a countercurrent) to step (a). The recycled distillers' water (i.e., the countercurrent) can account for about 1 vol.% - 70 vol.%, preferably 15 vol.% - 60 vol.%, especially about 30 vol.% - 50 vol.% of the slurry formed in step (a). In an embodiment, the process further includes optionally recycling at least a portion of the distillers' water stream to the slurry after having extracted oil from the distillers' water stream.

[0284] D. Drying of the wet cake and production of dried distillers grains and dried distillers grains with solubles

[0285] After separating a wet cake containing about 25 wt.% - 40 wt.%, preferably 30 wt.% - 38 wt.% dry solids from the distillers' water (e.g., by dehydration), it can be dried on a drum dryer, spray dryer, ring dryer, fluidized bed dryer, etc. to produce "dried distillers grains" (DDG). DDG is a valuable feed ingredient for animals (such as livestock, poultry, and fish). Preferably, DDG is provided with a moisture content of less than about 10 wt.% - 12 wt.% to avoid mold and microbial decomposition and increase the shelf life. Additionally, a high moisture content also makes the transportation of DDG more expensive. Preferably, the wet cake is dried under conditions that do not denature the proteins in the wet cake. The wet cake can be blended with the slurry separated from the distillers' water and dried to produce dried distillers grains with solubles (DDGS). A partially dried intermediate product, such as sometimes referred to as modified wet distillers grains, can be produced by partially drying the wet cake and optionally adding the slurry before, during, or after the drying process.

[0286] The invention described and claimed herein is not limited to the scope of the specific embodiments disclosed herein, as these embodiments are intended to be illustrative of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of the invention. Indeed, various modifications of the invention become apparent to those skilled in the art from the foregoing description, in addition to those shown and described herein. Such modifications are also intended to fall within the scope of the appended claims. In case of conflict, the present disclosure, including definitions, will control. Various references are cited herein, the disclosures of which are incorporated herein by reference in their entirety. The invention is further described by the following examples, which should not be construed as limiting the scope of the invention.

[0287] Materials and methods

[0288] The yeast strain MEJI797 is MBG5012 of WO 2019 / 161227, which further expresses Pycnopous sanguineus glucoamylase (SEQ ID NO:4 of WO 2011 / 066576) and a heterozygous Rhizomucor pusillus α-amylase expression cassette (as described in WO 2013 / 006756).

[0289] Liquefying enzyme blend: an exemplary thermostable α-amylase from Bacillus stearothermophilus disclosed in SEQ ID NO:19; an exemplary thermostable protease from Pyrococcus furiosus disclosed in SEQ ID NO:20.

[0290] Saccharifying enzyme blend: an exemplary glucoamylase from Gloeophyllum sepiarium disclosed in SEQ ID NO:22; an exemplary α-amylase from Rhizomucor pusillus disclosed in SEQ ID NO:23.

[0291] Cellulase blend: an exemplary β-glucosidase from Aspergillus fumigatus disclosed in SEQ ID NO:24; an exemplary cellobiohydrolase from Aspergillus fumigatus disclosed in SEQ ID NO:25; an exemplary endoglucanase from Trichoderma reesei disclosed in SEQ ID NO:26.

[0292] Examples

[0293] Example 1: Construction of a yeast strain expressing CBH1 and CBH2

[0294] This example describes the construction of yeast cells expressing CBH1 (SEQ ID NO:16) and CBH2 (SEQ ID NO:17) under the control of the following Saccharomyces cerevisiae promoters, respectively: pSeTDH3 and pPGK1, which are strong constitutive promoters. Three DNA fragments containing a promoter, a gene, and a terminator were designed to allow homologous recombination between these 3 DNA fragments and integration into the X-4 locus of yeast MeJi797. The resulting strain will contain: a fragment with 5’ homology containing a promoter, a gene, and a terminator (left fragment 1); a fragment containing a promoter and a gene (middle fragment 1); a 3’ homology fragment with a terminator integrated into the Saccharomyces cerevisiae genome at the X-4 locus (right fragment 1).

[0295] Construction of the fragment (left fragment 1) with 5’ X-4 homology containing pSeTDH3, P244YG and tPDC6

[0296] The first linear DNA containing 500 bp homology to the X-4 site and the Saccharomyces cerevisiae pSeTDH3 promoter was PCR amplified from HP97 plasmid DNA ( Figure 3 ). Fifty picomoles each of the forward and reverse primers were used for the PCR reaction, which contained 5 ng of plasmid DNA as a template, 1X Platinum SuperFi HF buffer (Thermo Fisher Scientific), and 2 units of SuperFi DNA polymerase in a final volume of 50 μL. PCR was performed in a T100 TM thermal cycler (Bio-Rad Laboratories, Inc.). After thermal cycling, the PCR reaction products were separated by gel and purified using a QIAquick Gel Extraction Kit (Qiagen).

[0297] The second fragment (TL4) containing the pSeTDH3 promoter, AGA2 signal peptide, P244YG gene, and tPDC6 terminator was PCR amplified from the Saccharomyces cerevisiae strain S1130-D03. Fifty picomoles each of the forward and reverse primers were used for the PCR reaction, which contained 5 ng of gDNA as a template, 1X Platinum SuperFi HF buffer (Thermo Fisher Scientific), and 2 units of SuperFi DNA polymerase in a final volume of 50 μL. PCR was performed in a T100 TM thermal cycler (Bio-Rad Laboratories). After thermal cycling, the PCR reaction products were separated by gel and purified using a QIAquick Gel Extraction Kit (Qiagen).

[0298] The two above-described fragments were combined in a SOE PCR. Fifty picomoles each of the forward and reverse primers were used for the PCR reaction, which contained 5 ng each of the above DNA fragments as templates, 1X Platinum SuperFi HF buffer (Thermo Fisher Scientific), and 2 units of SuperFi DNA polymerase in a final volume of 50 μL. PCR was performed in a T100 TM thermal cycler (Bio-Rad Laboratories). After thermal cycling, the PCR reaction products were separated by gel and purified using a QIAquick Gel Extraction Kit (Qiagen). This final fragment was designated RRSOE1.

[0299] Construction of the fragment (middle fragment 1) containing tPDC6, pPGK1, P43VY6 and homology to tADH3

[0300] The first linear DNA containing the terminator tPDC6 and the promoter pPGK1 was PCR amplified from TP40 plasmid DNA (Figure 4 )。Forward and reverse primers at 50 picomoles each were used for the PCR reaction, which contained 5 ng of plasmid DNA as a template, 1X Platinum SuperFi HF buffer (Thermo Fisher Scientific), and 2 units of SuperFi DNA polymerase, with a final volume of 50 μL. In the T100 TM thermal cycler (Bio-Rad Laboratories). After thermal cycling, the PCR reaction products were separated by gel and purified using the QIAquick Gel Extraction Kit (Qiagen).

[0301] A second fragment called TL7 was PCR amplified from the yeast strain S1130 - B11, which contained 50 bp homology to the pPGK1 promoter, the AGA2 signal peptide, the Trichoderma viride CBH2 gene, and 50 bp homology to the tADH3 terminator (see PCT / CN2022 / 102201 filed on June 29, 2022, the content of which is incorporated herein by reference). Forward and reverse primers at 50 picomoles each were used for the PCR reaction, which contained 5 ng of gDNA as a template, 1X Platinum SuperFi HF buffer (Thermo Fisher Scientific), and 2 units of SuperFi DNA polymerase, with a final volume of 50 μL. In the T100 TM thermal cycler (Bio-Rad Laboratories). After thermal cycling, the PCR reaction products were separated by gel and purified using the QIAquick Gel Extraction Kit (Qiagen).

[0302] The above two fragments were combined in SOE PCR. Forward and reverse primers at 50 picomoles each were used for the PCR reaction, which contained 5 ng of each of the above DNA fragments as a template, 1X Platinum SuperFi HF buffer (Thermo Fisher Scientific), and 2 units of SuperFi DNA polymerase, with a final volume of 50 μL. In the T100 TM thermal cycler (Bio-Rad Laboratories). After thermal cycling, the PCR reaction products were separated by gel and purified using the QIAquick Gel Extraction Kit (Qiagen). This final fragment was designated as SOE4.

[0303] Construction of the fragment (right fragment 1) with X-4 3’ homology containing tADH3

[0304] A linear DNA containing 500 bp homology to the X - 4 site and the Saccharomyces cerevisiae tADH3 terminator was PCR amplified from TH58 plasmid DNA( Figure 5)。Forward and reverse primers at 50 picomoles each were used for the PCR reaction, which contained 5 ng of plasmid DNA as template, 1X Platinum SuperFi HF buffer (Thermo Fisher Scientific), and 2 units of SuperFi DNA polymerase, with a final volume of 50 μL. The PCR was carried out in a T100 TM thermal cycler (Bio-Rad Laboratories). After thermal cycling, the PCR reaction products were separated by gel and purified using the QIAquick Gel Extraction Kit (Qiagen).

[0305] Integration of the left, middle and right fragments to generate yeast strain YS103-A07

[0306] Yeast MeJi797 was transformed with 150 ng of each of the left (RRSOE1), middle (SOE4), and right (TH58) integration fragments. To assist in homologous recombination of the left, middle, and right fragments at the genomic X-4 locus, 300 ng of a plasmid containing MAD7 and a guide RNA specific to X-4 (pMlBa789; Figure 6 ) was also used in the transformation. The three linear DNA fragments were combined and transformed into MeJi797 following the yeast electroporation protocol. Transformants were selected on YPD + cloNAT to select those containing the Mad7 plasmid pMlBa789. Transformants were inoculated into one well of a 96-well plate containing YPD medium by either manually picking the transformants onto YPD plates or by using the Q-pix Colony Picking System (Molecular Devices). The plates were grown for 2 days, then glycerol was added to a final concentration of 20%, and the plates were stored at -80 °C until needed. Integration of the CBH1+CBH2 construct was verified by PCR using locus-specific primers and subsequent sequencing.

[0307] Example 2: Evaluation of corn mash fermentation using a yeast strain expressing CBH1 and CBH2 in combination with a GH5_21 xylanase

[0308] The yeast strains Yeast MeJi797 and YS103 - A07 (same as above) were incubated overnight at 32 °C and 150 rpm in 20 mL of YPD medium (6% w / v D - glucose, 2% peptone, 1% yeast extract) in 125 - mL baffled shake flasks. After 24 hours of incubation, the cells were harvested and collected by centrifugation, and these cells were washed in DI water before being resuspended in 5 mL of DI water for dosing. 3 ppm of penicillin and 1000 ppm of urea were supplemented to the industrially obtained liquefied corn mash (where liquefaction was carried out using a Liquefaction Enzyme Blend). Simultaneous saccharification and fermentation (SSF) was carried out via small - scale fermentation. Approximately 4 g of the corn mash was added to 12 - mL conical tubes. 1 x 10 7 cells / g of mash with yeast was dosed to each tube, followed by the addition of 0.42 AGU / g of dry solids of an exogenous saccharifying enzyme blend. In some cases, GH5_21 xylanase (2.5 μg EP / g DS; SEQ ID NO:21) and a cellulase blend (67.5 μg EP / g DS) were dosed to the tubes. Six replicate tube fermentations were carried out for each treatment. The enzyme blend and yeast doses were applied based on the exact weight of the corn slurry in each vial. The tubes were incubated at 32 °C and mixed twice daily via brief vortexing. After 68 hours of fermentation time, the contents of the tubes were diluted 10x and then centrifuged at 3500 rpm for 5 min. The supernatant samples were filtered through a 0.2 - mm syringe filter into vials for HPLC analysis of the final ethanol level. The remaining supernatant was discarded, and the pellet was dried at 50 °C for 3 days. The final pellet was weighed to determine the residual solids.

[0309] The results of the final ethanol levels are shown in Figure 1 . Compared to the control strain MeJi797, the yeast strain YS103 - A07 expressing CBH1 and CBH2 had a significantly higher ethanol yield.

[0310] The results of the residual solids are shown in Figure 2 . Compared to the control strain MeJi797, the yeast strain YS103 - A07 expressing CBH1 and CBH2 showed significantly lower residual solids.

[0311] The present invention is further defined by the following numbered paragraphs:

[0312] Paragraph [1]. A process for producing a fermentation product from a starch - containing material, the process comprising the steps of:

[0313] (a) liquefying the starch - containing material using α - amylase at a temperature above the initial gelatinization temperature;

[0314] (b) saccharifying the liquefied starch-containing material; and

[0315] (c) fermenting the saccharified starch-containing material using a fermenting organism;

[0316] wherein at least one GH5_21 xylanase is present or added during saccharification or fermentation; and

[0317] wherein the fermenting organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2.

[0318] Paragraph [2]. The process according to paragraph [1], wherein the GH5_21 xylanase is present or added during the saccharification step (b).

[0319] Paragraph [3]. The process according to paragraph [1] or [2], wherein the GH5_21 xylanase is present or added during the fermentation step (c).

[0320] Paragraph [4]. The process according to any one of the preceding paragraphs, wherein steps (b) and (c) are carried out simultaneously in simultaneous saccharification and fermentation (SSF).

[0321] Paragraph [5]. The process according to any one of the preceding paragraphs, wherein the GH5_21 xylanase is present or added during SSF.

[0322] Paragraph [6]. The process according to any one of the preceding paragraphs, wherein the GH5_21 xylanase used in the saccharification step (b) and / or the fermentation step (c) is present or added via in situ expression from the fermenting organism.

[0323] Paragraph [7]. The process according to any one of the preceding paragraphs, wherein the α-amylase has the amino acid sequence of SEQ ID NO:18 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:18, and the α-amylase has α-amylase activity.

[0324] Paragraph [8]. The process according to any one of the preceding paragraphs, wherein the α-amylase has the amino acid sequence of SEQ ID NO:19 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:19, and the α-amylase has α-amylase activity.

[0325] Paragraph [9]. The process according to any one of the preceding paragraphs, wherein a thermostable endoglucanase is added during the liquefaction step (a).

[0326] Paragraph

[10] . The process according to any one of the preceding paragraphs, wherein a thermostable lipase is added during the liquefaction step (a).

[0327] Paragraph

[11] . The process according to any one of the preceding paragraphs, wherein a thermostable phytase is added during the liquefaction step (a).

[0328] Paragraph

[12] . The process according to any one of the preceding paragraphs, wherein a thermostable protease is added during the liquefaction step (a).

[0329] Paragraph

[13] . The process according to claim 12, wherein the thermostable protease has the amino acid sequence of SEQ ID NO:20 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:20, and the thermostable protease has protease activity.

[0330] Paragraph

[14] . The process according to any one of the preceding paragraphs, wherein a thermostable pullulanase is added during the liquefaction step (a).

[0331] Paragraph

[15] . The process according to any one of the preceding paragraphs, wherein a thermostable xylanase is added during the liquefaction step (a).

[0332] Paragraph

[16] . The process according to claim 15, wherein the thermostable xylanase has the amino acid sequence of SEQ ID NO:21 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:21, and the thermostable xylanase has xylanase activity.

[0333] Paragraph

[17] . The process according to any one of the preceding paragraphs, wherein a thermostable α - amylase, a thermostable protease and a thermostable xylanase are added during the liquefaction step (a).

[0334] Paragraph

[18] . The process according to any one of the preceding paragraphs, wherein glucoamylase is added during step (b) and / or step (c).

[0335] Paragraph

[19] . The process as described in paragraph

[18] , wherein the glucoamylase has the amino acid sequence of SEQ ID NO: 22 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 22, and the glucoamylase has glucoamylase activity.

[0336] Paragraph

[20] . The process as described in any of the preceding paragraphs, wherein α - amylase is added during step (b) and / or step (c).

[0337] Paragraph

[21] . The process as described in paragraph

[20] , wherein the α - amylase has the amino acid sequence of SEQ ID NO: 23 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 23, and the α - amylase has α - amylase activity.

[0338] Paragraph

[22] . The process as described in any of the preceding paragraphs, wherein β - glucosidase is added during step (a) and / or step (b).

[0339] Paragraph

[23] . The process as described in paragraph

[22] , wherein the β - glucosidase has the amino acid sequence of SEQ ID NO: 24 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 24, and the β - glucosidase has β - glucosidase activity.

[0340] Paragraph

[24] . The process as described in any of the preceding paragraphs, wherein cellobiohydrolase is added during step (b) and / or step (c).

[0341] Paragraph

[25] . The process as described in paragraph

[24] , wherein the cellobiohydrolase has the amino acid sequence of SEQ ID NO: 25 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 25, and the cellobiohydrolase has cellobiohydrolase activity.

[0342] Paragraph

[26] . The process according to any one of the preceding paragraphs, wherein an endoglucanase is added during step (b) and / or step (c).

[0343] Paragraph

[27] . The process according to paragraph

[26] , wherein the endoglucanase has the amino acid sequence of SEQ ID NO:26 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:26, and the endoglucanase has endoglucanase activity.

[0344] Paragraph

[28] . The process according to any one of the preceding paragraphs, wherein a trehalase is added during step (b) and / or step (c).

[0345] Paragraph

[29] . The process according to paragraph

[28] , wherein the trehalase has the amino acid sequence of SEQ ID NO:27 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:27, and the trehalase has trehalase activity.

[0346] Paragraph

[30] . A process for producing a fermentation product from an ungelatinized starch-containing cereal, the process comprising the steps of:

[0347] (a) saccharifying the starch-containing cereal with glucoamylase and α-amylase at a temperature below the initial gelatinization temperature to produce fermentable sugars; and

[0348] (b) fermenting the sugars with a fermenting organism to produce a fermentation product;

[0349] wherein at least one GH5_21 xylanase is present or added during saccharification or fermentation; and

[0350] wherein the fermenting organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2.

[0351] Paragraph

[31] . The process according to paragraph

[30] , wherein the GH5_21 xylanase is present or added during the saccharification step (a).

[0352] Paragraph

[32] . The process according to paragraph

[30] , wherein the GH5_21 xylanase is present or added during the fermentation step (b).

[0353] Paragraph

[33] . The process as described in paragraph

[30] , wherein steps (a) and (b) are carried out simultaneously in simultaneous saccharification and fermentation (SSF).

[0354] Paragraph

[34] . The process as described in paragraph

[33] , wherein the GH5_21 xylanase is present or added during SSF.

[0355] Paragraph

[35] . The process as described in paragraph

[30] , wherein the GH5_21 xylanase used in the saccharification step (a) and / or the fermentation step (b) is present or added via in situ expression from the fermenting organism.

[0356] Paragraph

[36] . The process as described in any of the preceding paragraphs, wherein the at least one GH5_21 xylanase has an amino acid sequence selected from the group consisting of:

[0357] (1) The amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:1;

[0358] (2) The amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:2;

[0359] (3) The amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:3;

[0360] (4) The amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:4;

[0361] (5) The amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5;

[0362] (6) The amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:6;

[0363] (7) The amino acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:7;

[0364] (8) The amino acid sequence of SEQ ID NO:8 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:8;

[0365] (9) The amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:9;

[0366] (10) The amino acid sequence of SEQ ID NO:10 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:10;

[0367] (11) The amino acid sequence of SEQ ID NO: 11 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 11;

[0368] (12) The amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12;

[0369] (13) The amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 13;

[0370] (14) The amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 14; and

[0371] (15) The amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 15.

[0372] Paragraph

[37] . The process according to any one of the preceding paragraphs, wherein the at least one GH5_21 comprises or consists of the following amino acid sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15.

[0373] Paragraph

[38] . The process according to any one of the preceding paragraphs, wherein the at least one GH5_21 is a variant of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15 that contains substitutions, deletions, and / or insertions at one or more (e.g., several) positions.

[0374] Paragraph

[39] . The process according to any one of the preceding paragraphs, wherein the at least one GH5_21 is a fragment of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, or SEQ ID NO:15, and the fragment has GH5_21 activity.

[0375] Paragraph

[40] . The process according to any one of the preceding paragraphs, wherein the at least one GH5_21 is administered in the range of 0.0001 - 1 mg EP (enzyme protein) / g DS, such as 0.0005 - 0.5 mg EP / g DS, such as 0.001 - 0.1 mg EP / g DS or 0.001 - 0.01 mg EP / g DS.

[0376] Paragraph

[41] . The process according to any one of the preceding paragraphs, wherein the heterologous polynucleotide encoding CBH1 is operably linked to a promoter that is foreign to the polynucleotide.

[0377] Paragraph

[42] . The process according to any one of the preceding paragraphs, wherein the CBH1 has the amino acid sequence of SEQ ID NO:16 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:16.

[0378] Paragraph

[43] . The process according to any one of the preceding paragraphs, wherein the CBH1 comprises the amino acid sequence of SEQ ID NO:16 or consists of the amino acid sequence of SEQ ID NO:16.

[0379] Paragraph

[44] . The process according to any one of the preceding paragraphs, wherein the CBH1 is a variant of the amino acid sequence of SEQ ID NO:16 that contains substitutions, deletions, and / or insertions at one or more (e.g., several) positions.

[0380] Paragraph

[45] . The process according to any one of the preceding paragraphs, wherein the CBH1 is a fragment of the amino acid sequence of SEQ ID NO:16, and the fragment has CBH1 activity.

[0381] Paragraph

[46] . The process according to any one of the preceding paragraphs, wherein the heterologous polynucleotide encoding CBH2 is operably linked to a promoter that is foreign to the polynucleotide.

[0382] Paragraph

[47] . The process according to any one of the preceding paragraphs, wherein the CBH2 has the amino acid sequence of SEQ ID NO:17 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:17.

[0383] Paragraph

[48] . The process according to any one of the preceding paragraphs, wherein the CBH2 comprises the amino acid sequence of SEQ ID NO:17 or consists of the amino acid sequence of SEQ ID NO:17.

[0384] Paragraph

[49] . The process according to any one of the preceding paragraphs, wherein the CBH2 is a variant of the amino acid sequence of SEQ ID NO: 17 that contains substitutions, deletions, and / or insertions at one or more (e.g., several) positions.

[0385] Paragraph

[50] . The process according to any one of the preceding paragraphs, wherein the CBH2 is a fragment of the amino acid sequence of SEQ ID NO: 17, and the fragment has CBH2 activity.

[0386] Paragraph

[51] . The process according to any one of the preceding paragraphs, wherein the fermenting organism comprises an active pentose fermentation pathway (e.g., an active xylose fermentation pathway and / or an active arabinose fermentation pathway).

[0387] Paragraph

[52] . The process according to any one of the preceding paragraphs, wherein the fermenting organism comprises a heterologous polynucleotide encoding glucoamylase.

[0388] Paragraph

[53] . The process according to any one of the preceding paragraphs, wherein the fermenting organism comprises a heterologous polynucleotide encoding α - amylase.

[0389] Paragraph

[54] . The process according to any one of the preceding paragraphs, wherein the fermenting organism comprises a heterologous polynucleotide encoding a protease.

[0390] Paragraph

[55] . The process according to any one of the preceding paragraphs, wherein the fermenting organism comprises a heterologous polynucleotide encoding the GH5_21 xylanase.

[0391] Paragraph

[56] . The process according to any one of the preceding paragraphs, wherein the fermenting organism comprises a disruption of an endogenous gene encoding glycerol 3 - phosphate dehydrogenase (GPD).

[0392] Paragraph

[57] . The process according to any one of the preceding paragraphs, wherein the fermenting organism comprises a disruption of an endogenous gene encoding glycerol 3 - phosphatase (GPP).

[0393] Paragraph

[58] . The process according to any one of the preceding paragraphs, wherein the fermenting organism is a yeast cell.

[0394] Paragraph

[59] . The process according to any one of the preceding paragraphs, wherein the fermenting organism is a cell of a species of the genus Saccharomyces, Rhodotorula, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Rhodosporidium, Candida, Yarrowia, Lipomyces, Cryptococcus, or Dekkera.

[0395] Paragraph

[60] . The process according to any one of the preceding paragraphs, wherein the fermenting organism is a Saccharomyces cerevisiae cell.

[0396] Paragraph

[61] . The process according to any one of the preceding paragraphs, wherein the starch-containing material comprises beet, maize, corn, wheat, rye, barley, oats, triticale, rice, sorghum, sweet potato, millet, pearl millet, and / or foxtail millet.

[0397] Paragraph

[62] . The process according to any one of the preceding paragraphs, wherein the starch-containing material comprises corn.

[0398] Paragraph

[63] . The process according to any one of the preceding paragraphs, wherein the fermentation product is ethanol, preferably fuel ethanol.

[0399] Paragraph

[64] . A recombinant host cell comprising a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2.

[0400] Paragraph

[65] . The recombinant host cell according to paragraph

[64] , wherein the heterologous polynucleotide encoding CBH1 is operably linked to a promoter that is exogenous to the polynucleotide.

[0401] Paragraph

[66] . The recombinant host cell according to paragraph

[64] or

[65] , wherein the CBH1 has the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 16.

[0402] Paragraph

[67] . The recombinant host cell according to any one of paragraphs

[64] -

[66] , wherein the CBH1 comprises the amino acid sequence of SEQ ID NO: 16 or consists of the amino acid sequence of SEQ ID NO: 16.

[0403] Paragraph

[68] . The recombinant host cell according to any one of paragraphs

[64] -

[67] , wherein the CBH1 is a variant of the amino acid sequence of SEQ ID NO: 16 that contains substitutions, deletions, and / or insertions at one or more (e.g., several) positions.

[0404] Paragraph

[69] . The recombinant host cell according to any one of paragraphs

[64] -

[68] , wherein the CBH1 is a fragment of the amino acid sequence of SEQ ID NO: 16, and the fragment has CBH1 activity.

[0405] Paragraph

[70] . The recombinant host cell according to any one of paragraphs

[64] -

[69] , wherein the heterologous polynucleotide encoding CBH2 is operably linked to a promoter that is exogenous to the polynucleotide.

[0406] Paragraph

[71] . The recombinant host cell according to any one of paragraphs

[64] -

[70] , wherein the CBH2 has the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO: 17.

[0407] Paragraph

[72] . The recombinant host cell according to any one of paragraphs

[64] -

[71] , wherein the CBH2 comprises the amino acid sequence of SEQ ID NO: 17 or consists of the amino acid sequence of SEQ ID NO: 17.

[0408] Paragraph

[73] . The recombinant host cell according to any one of paragraphs

[64] -

[72] , wherein the CBH2 is a variant of the amino acid sequence of SEQ ID NO: 17 that contains substitutions, deletions, and / or insertions at one or more (e.g., several) positions.

[0409] Paragraph

[74] . The recombinant host cell according to any one of paragraphs

[64] -

[73] , wherein the CBH2 is a fragment of the amino acid sequence of SEQ ID NO: 17, and the fragment has CBH2 activity.

[0410] Paragraph

[75] . The recombinant host cell according to any one of paragraphs

[64] -

[74] , wherein the fermenting organism comprises an active pentose fermentation pathway (e.g., an active xylose fermentation pathway and / or an active arabinose fermentation pathway).

[0411] Paragraph

[76] . The recombinant host cell according to any one of paragraphs

[64] -

[75] , wherein the fermenting organism comprises a heterologous polynucleotide encoding glucoamylase.

[0412] Paragraph

[77] . The recombinant host cell according to any one of paragraphs

[64] -

[76] , wherein the fermenting organism comprises a heterologous polynucleotide encoding α - amylase.

[0413] Paragraph

[78] . The recombinant host cell according to any one of paragraphs

[64] -

[77] , wherein the fermenting organism comprises a heterologous polynucleotide encoding a protease.

[0414] Paragraph

[79] . The recombinant host cell according to any one of paragraphs

[64] -

[78] , wherein the fermenting organism comprises a heterologous polynucleotide encoding a GH5_21 xylanase.

[0415] Paragraph

[80] . The recombinant host cell according to any one of paragraphs

[64] -

[79] , wherein the fermenting organism comprises a disruption of an endogenous gene encoding glycerol 3-phosphate dehydrogenase (GPD).

[0416] Paragraph

[81] . The recombinant host cell according to any one of paragraphs

[64] -

[80] , wherein the fermenting organism comprises a disruption of an endogenous gene encoding glycerol 3-phosphatase (GPP).

[0417] Paragraph

[82] . The recombinant host cell according to any one of paragraphs

[64] -

[81] , wherein the fermenting organism is a yeast cell.

[0418] Paragraph

[83] . The recombinant host cell according to any one of paragraphs

[64] -

[82] , wherein the fermenting organism is a cell of a species of the genus Saccharomyces, Rhodotorula, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Rhodosporidium, Candida, Yarrowia, Lipomyces, Cryptococcus, or Dekkera.

[0419] Paragraph

[84] . The recombinant host cell according to any one of paragraphs

[64] -

[83] , wherein the fermenting organism is a Saccharomyces cerevisiae cell.

Claims

1. A process for producing a fermentation product from a starch-containing material, the process comprising the steps of: (a) liquefying the starch-containing material using α-amylase at a temperature above the initial gelatinization temperature; (b) saccharifying the liquefied starch-containing material; and (c) fermenting the saccharified starch-containing material using a fermentation organism; wherein at least one GH5_21 xylanase is present or added during saccharification or fermentation; and wherein the fermentation organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2.

2. The process according to claim 1, wherein the GH5_21 xylanase is present or added during saccharification step (b).

3. The process according to claim 1 or 2, wherein the GH5_21 xylanase is present or added during fermentation step (c).

4. The process according to any one of the preceding claims, wherein steps (b) and (c) are carried out simultaneously in simultaneous saccharification and fermentation (SSF).

5. The process according to any one of the preceding claims, wherein the GH5_21 xylanase is present or added during SSF.

6. The process according to any one of the preceding claims, wherein the GH5_21 xylanase used in saccharification step (b) and / or fermentation step (c) is present or added via in situ expression from the fermentation organism.

7. A process for producing a fermentation product from an ungelatinized starch-containing cereal, the process comprising the steps of: (a) saccharifying the starch-containing cereal using glucoamylase and α-amylase at a temperature below the initial gelatinization temperature to produce fermentable sugars; and (b) fermenting the sugars using a fermentation organism to produce a fermentation product; wherein at least one GH5_21 xylanase is present or added during saccharification or fermentation; and wherein the fermentation organism comprises a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2.

8. The process according to claim 7, wherein the GH5_21 xylanase is present or added during saccharification step (a).

9. The process according to claim 7, wherein the GH5_21 xylanase is present or added during fermentation step (b).

10. The process according to claim 7, wherein steps (a) and (b) are carried out simultaneously in simultaneous saccharification and fermentation (SSF).

11. The process according to claim 10, wherein the GH5_21 xylanase is present or added during SSF.

12. The process according to claim 7, wherein the GH5_21 xylanase used in saccharification step (a) and / or fermentation step (b) is present or added via in situ expression from the fermentation organism.

13. The process according to any one of the preceding claims, wherein the at least one GH5_21 xylanase has an amino acid sequence selected from the group consisting of: (1) The amino acid sequence of SEQ ID NO:1 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:1; (2) The amino acid sequence of SEQ ID NO:2 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:2; (3) The amino acid sequence of SEQ ID NO:3 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:3; (4) The amino acid sequence of SEQ ID NO:4 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:4; (5) The amino acid sequence of SEQ ID NO:5 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:5; (6) The amino acid sequence of SEQ ID NO:6 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:6; (7) The amino acid sequence of SEQ ID NO:7 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:7; (8) The amino acid sequence of SEQ ID NO:8 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:8; (9) The amino acid sequence of SEQ ID NO:9 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:9; (10) The amino acid sequence of SEQ ID NO:10 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:10; (11) The amino acid sequence of SEQ ID NO:11 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:11; (12) The amino acid sequence of SEQ ID NO:12 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:12; (13) The amino acid sequence of SEQ ID NO:13 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:13; (14) The amino acid sequence of SEQ ID NO:14 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:14; and (15) The amino acid sequence of SEQ ID NO:15 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:

15.

14. The process according to any one of the preceding claims, wherein said CBH1 has the amino acid sequence of SEQ ID NO:16 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:

16.

15. The process according to any one of the preceding claims, wherein said CBH2 has the amino acid sequence of SEQ ID NO:17 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:

17.

16. The process according to any one of the preceding claims, wherein said fermenting organism comprises an active pentose fermentation pathway (e.g., an active xylose fermentation pathway and / or an active arabinose fermentation pathway).

17. The process according to any one of the preceding claims, wherein said fermenting organism comprises a heterologous polynucleotide encoding said GH5_21 xylanase.

18. The process according to any one of the preceding claims, wherein said fermenting organism is a yeast cell.

19. The process according to claim 18, wherein said fermenting organism is a Saccharomyces cerevisiae cell.

20. The process according to any one of the preceding claims, wherein said fermentation product is ethanol, preferably fuel ethanol.

21. A recombinant host cell comprising a heterologous polynucleotide encoding CBH1 and a heterologous polynucleotide encoding CBH2; wherein said CBH1 has the amino acid sequence of SEQ ID NO:16 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:16; and / or wherein said CBH2 has the amino acid sequence of SEQ ID NO:17 or an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence of SEQ ID NO:

17.

22. The recombinant host cell according to claim 21, wherein said fermenting organism is a yeast cell.

23. The recombinant host cell according to claim 22, wherein said fermenting organism is a Saccharomyces cerevisiae cell.

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