EP4638768A2 - Processes for producing fermentation products using fiber-degrading enzymes with engineered yeast - Google Patents

Abstract

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

Description

PROCESSES FOR PRODUCING FERMENTATION PRODUCTS USING FIBERDEGRADING ENZYMES WITH ENGINEERED YEAST

REFERENCE TO A SEQUENCE LISTING

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

FIELD OF THE INVENTION

The present invention relates to processes for producing fermentation products from starch-containing material. The invention also relates to a GH5 xylanase composition used with a recombinant host cell or fermenting organism suitable for use in a process of the invention.

BACKGROUND OF THE INVENTION

Processes for producing fermentation products, such as ethanol, from a starch or lignocellulose containing material are well known in the art. The preparation of the starch containing material such as corn for utilization in such fermentation processes typically begins with grinding the corn in a dry-grind or wet-milling process. Wet-milling processes involve fractionating the corn into different components where only the starch fraction enters the fermentation process. Dry-grind processes involve grinding the corn kernels into meal and mixing the meal with water and enzymes. Generally, two different kinds of dry-grind processes are used. The most commonly used process, often referred to as a "conventional process," includes grinding the starch-containing grain and then liquefying gelatinized starch at a high temperature using typically a bacterial alpha-amylase, followed by simultaneous saccharification and fermentation (SSF) carried out in the presence of a glucoamylase and a fermentation organism. Another well-known process, often referred to as a "raw starch hydrolysis" process (RSH process), includes grinding the starch-containing grain and then simultaneously saccharifying and fermenting granular starch below the initial gelatinization temperature typically in the presence of an acid fungal alpha-amylase and a glucoamylase.

In a process for producing ethanol from corn, following SSF or the RSH process, the liquid fermentation products are recovered from the fermented mash (often referred to as “beer mash”), e.g., by distillation, which separates the desired fermentation product, e.g., ethanol, from other liquids and/or solids. The remaining fraction is referred to as “whole stillage”. Whole stillage typically contains about 10 to 20% solids. The whole stillage is separated into a solid and a liquid fraction, e.g., by centrifugation. The separated solid fraction is referred to as “wet cake” (or “wet grains”) and the separated liquid fraction is referred to as “thin stillage”. Wet cake and thin stillage contain about 35 and 7% solids, respectively. Wet cake, with optional additional dewatering, is used as a component in animal feed or is dried to provide “Distillers Dried Grains” (DDG) used as a component in animal feed. Thin stillage is typically evaporated to provide evaporator condensate and syrup or may alternatively be recycled to the slurry tank as “backset”. Evaporator condensate may either be forwarded to a methanator before being discharged and/or may be recycled to the slurry tank as “cook water”. The syrup may be blended into DDG or added to the wet cake before or during the drying process, which can comprise one or more dryers in sequence, to produce DDGS (Distillers Dried Grain with Solubles). Syrup typically contains about 25% to 35% solids. Oil can also be extracted from the thin stillage and/or syrup as a by-product for use in biodiesel production, as a feed or food additive or product, or other biorenewable products.

Yeasts which are used for production of ethanol for use as fuel, such as in the corn ethanol industry, require several characteristics to ensure cost-effective production of the ethanol. These characteristics include ethanol tolerance, low by-product yield, rapid fermentation, and the ability to limit the amount of residual sugars remaining in the ferment. Such characteristics have a marked effect on the viability of the industrial process.

Yeast of the genus Saccharomyces exhibit many of the characteristics required for production of ethanol. In particular, strains of Saccharomyces cerevisiae are widely used for the production of ethanol in the fuel ethanol industry. Industrial strains of Saccharomyces cerevisiae have the ability to produce high yields of ethanol under fermentation conditions found in, for example, the fermentation of corn mash. An example of such a strain is the is the commercially available product ETHANOL RED®.

Saccharomyces cerevisae yeast also have been genetically engineered to express alpha-amylase and/or glucoamylase to improve yield and decrease the amount of exogenously added enzymes necessary during SSF (e.g., WO2018/098381, WO2017/087330, WO2017/037614, WO2011/128712, WO2011/153516, US2018/0155744). Yeast have also been engineered to express trehalase in an attempt to increase fermentation yield by breaking down residual trehalose (e.g., WO2017/077504).

Cellulases are well-known for use in the conversion of lignocellulosic feedstocks into ethanol. Once the lignocellulose is converted to fermentable sugars, e.g., glucose, the fermentable sugars are easily fermented by yeast into ethanol.

However, despite the advances in yeast and cellulase technology, there is still a desire and need for providing processes for producing fermentation products, such as ethanol, from starch-containing material that can provide a higher fermentation product yield, or other advantages, compared to a conventional process. SUMMARY OF THE INVENTION

The present invention provides a solution to the above problem by fermenting a saccharified starch-containing material with a fermenting organism that expresses a CBH1 and a CBH2 in the prescence of a GH5_21 xylanase, which provides an unexpected increase in fermentation product.

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

(a) liquefying the starch-containing material at a temperature above the initial gelatinization temperature using an alpha-amylase;

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

(c) fermenting the saccharified starch-containing material using a fermenting organism; 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 a CBH1 and a heterologous polynucleotide encoding a CBH2.

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

(a) saccharifying the starch-containing material at a temperature below the initial gelatination temperature; and

(b) fermenting the saccharified starch-containing material using a fermenting organism; 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 a CBH1 and a heterologous polynucleotide encoding a CBH2.

A third aspect relates to a recombinant host cell comprising a heterologous polynucleotide encoding a CBH1 and a heterologous polynucleotide encoding a CBH2.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the final ethanol level results for fermentation with a yeast strain expressing CBH1 and CBH2 (YS103-A07) as compared to a control strain (MeJi797).

FIG. 2 shows the residual solids results for fermentation with a yeast strain expressing CBH1 and CBH2 (YS103-A07) as compared to control strain MeJi797.

FIG. 3 shows a plasmid map for HP97.

FIG. 4 shows a plasmid map for TP40. FIG. 5 shows a plasmid map for TH58.

FIG. 6 shows a plasmid map for pMIBa789.

DEFINITIONS

Active pentose fermentation pathway: As used herein, a host cell or fermenting organism having an “active pentose fermentation pathway” produces active enzymes necessary to catalyze each reaction of a metabolic pathway in a sufficient amount to produce a fermentation product (e.g., ethanol) from pentose, and therefore is capable of producing the fermentation product in measurable yields when cultivated under fermentation conditions in the presence of pentose. A host cell or fermenting organism having an active pentose fermentation pathway comprises one or more active pentose fermentation pathway genes. A “pentose fermentation pathway gene” as used herein refers to a gene that encodes an enzyme involved in an active pentose fermentation pathway. In some embodiments, the active pentose fermentation pathway is an “active xylose fermentation pathway” (i.e., produces a fermentation product, such as ethanol, from xylose) or an “active arabinose fermentation pathway (i.e., produces a fermentation product, such as ethanol, from arabinose).

The active enzymes necessary to catalyze each reaction in an active pentose fermentation pathway may result from activities of endogenous gene expression, activities of heterologous gene expression, or from a combination of activities of endogenous and heterologous gene expression.

Alpha-amylase: The term “alpha amylase” means an 1,4-alpha-D-glucan glucanohydrolase, EC. 3.2.1.1, which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides. Alpha-amylase activity can be determined using methods known in the art (e.g., using an alpha amylase assay described W02020/023411).

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

Catalytic domain: The term “catalytic domain” means the region of an enzyme containing the catalytic machinery of the enzyme. Cellobiohydrolase: The term “cellobiohydrolase” means a 1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176) that catalyzes the hydrolysis of 1,4- beta-D-glucosidic linkages in cellulose, cellooligosaccharides, or any beta-1,4-linked glucose containing polymer, releasing cellobiose from the reducing end (cellobiohydrolase I) or nonreducing 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.

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

Control sequence: The term “control sequence” means a nucleic acid sequence necessary for polypeptide expression. Control sequences may be native or foreign to the polynucleotide encoding the polypeptide, and native or foreign to each other. Such control sequences include, but are not limited to, a leader sequence, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, and transcription terminator sequence. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the polynucleotide encoding a polypeptide.

Disruption: The term “disruption” means that a coding region and/or control sequence of a referenced gene is partially or entirely modified (such as by deletion, insertion, and/or substitution of one or more nucleotides) resulting in the absence (inactivation) or decrease in expression, and/or the absence or decrease of enzyme activity of the encoded polypeptide. The effects of disruption can be measured using techniques known in the art such as detecting the absence or decrease of enzyme activity using from cell-free extract measurements referenced herein; or by the absence or decrease of corresponding mRNA (e.g., at least 25% decrease, at least 50% decrease, at least 60% decrease, at least 70% decrease, at least 80% decrease, or at least 90% decrease); the absence or decrease in the amount of corresponding polypeptide having enzyme activity (e.g., at least 25% decrease, at least 50% decrease, at least 60% decrease, at least 70% decrease, at least 80% decrease, or at least 90% decrease); or the absence or decrease of the specific activity of the corresponding polypeptide having enzyme activity (e.g., at least 25% decrease, at least 50% decrease, at least 60% decrease, at least 70% decrease, at least 80% decrease, or at least 90% decrease). Disruptions of a particular gene of interest can be generated by methods known in the art, e.g., by directed homologous recombination (see Methods in Yeast Genetics (1997 edition), Adams, Gottschling, Kaiser, and Stems, Cold Spring Harbor Press (1998)).

Endogenous gene: The term “endogenous gene” means a gene that is native to the referenced host cell or fermenting organism. “Endogenous gene expression” means expression of an endogenous gene.

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

Expression: The term “expression” includes any step involved in the production of the 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 levels of mRNA and/or translated polypeptide.

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

Fermentable medium: The term “fermentable medium” or “fermentation medium” refers to a medium comprising 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 is capable, in part, of being converted (fermented) by a host cell into a desired product, such as ethanol. In some instances, the fermentation medium is derived from a natural source, such as sugar cane, starch, or cellulose, and may be the result of pretreating the source by enzymatic hydrolysis

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

Fermentation product: “Fermentation product” means a product produced by a process including fermenting 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, B12, beta-carotene); and hormones. In a preferred embodiment the fermentation product is ethanol, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol or products used in the consumable alcohol industry (e.g., beer and wine), dairy industry (e.g., fermented dairy products), leather industry and tobacco industry. Preferred beer types comprise ales, stouts, porters, lagers, bitters, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer. In an embodiment the fermentation product is ethanol.

Fermenting organism: “Fermenting organism” refers to any organism, including bacterial and fungal organisms, especially yeast, suitable for use in a fermentation process and capable of producing the desired fermentation product.

GH5 xylanase: “GH5 xylanase” is an abbreviation for Glycoside Hydrolase Family 5 xylanase, which consist primarily of endo-1,4- p-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1— >4)-p-D-xylosidic linkages in xylans.

GH5_21 xylanase: “GH5_21 xylanase” is an abbreviation for Glycoside Hydrolase Family 5 subfamily 21 endo-beta-1 , 4-xylanases that possess a three-dimensional structure characterized by a (P / a) 8 barrel and use a glutamine residue as a catalytic nucleophile/base.

Glucoamylase: The term “glucoamylase” (1 ,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) is defined as an enzyme that catalyzes the release of D-glucose from the nonreducing ends of starch or related oligo- and polysaccharide molecules. For purposes of the present invention, glucoamylase activity may be determined according to the procedures known in the art, such as those described in W02020/023411.

Hemicellulolytic enzyme or hemicellulase: The term “hemicellulolytic enzyme” or “hemicellulase” means one or more (e.g., several) enzymes that hydrolyze a hemicellulosic material. See, for example, Shallom and Shoham, 2003, Current Opinion In Microbiology 6(3): 219-228). Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to, an acetylmannan esterase, an acetylxylan esterase, an arabinanase, an arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase, a galactosidase, a glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase, a xylanase, and a xylosidase. The substrates for these enzymes, hemicelluloses, are a heterogeneous group of branched and linear polysaccharides that are bound via hydrogen bonds to the cellulose microfibrils in the plant cell wall, crosslinking them into a robust network. Hemicelluloses are also covalently attached to lignin, forming together with cellulose a highly complex structure. The variable structure and organization of hemicelluloses require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are either glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs), which hydrolyze ester linkages of acetate or ferulic acid side groups. These catalytic modules, based on homology of their primary sequence, can be assigned into GH and CE families. Some families, with an overall similar fold, can be further grouped into clans, marked alphabetically (e.g., GH-A). A most informative and updated classification of these and other carbohydrate active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be measured according to Ghose and Bisaria, 1987, Pure & AppL Chem. 59: 1739-1752, at a suitable temperature such as 40°C-80°C, e.g., 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.

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 structural modifications have been made to the coding region; a native polynucleotide whose expression is quantitatively altered as a result of a manipulation of the DNA by recombinant DNA techniques, e.g., a different (foreign) promoter; or a native polynucleotide in a host cell having one or more extra copies of the polynucleotide to quantitatively alter expression. A “heterologous gene” is a gene comprising a heterologous polynucleotide.

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

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

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

Initial gelatinization temperature: "Initial gelatinization temperature" means the lowest temperature at which gelatinization of the starch commences. Starch heated in water begins to gelatinize between 50 degrees centigrade and 75 degrees C; the exact temperature of gelatinization depends on the specific starch, and can readily be determined by the skilled artisan. Thus, the initial gelatinization temperature may vary according to the plant species, to the particular variety of the plant species as well as with the growth conditions. In the context of this disclosure the initial gelatinization temperature of a given starch-containing grain is the temperature at which birefringence is lost in 5 percent of the starch granules using the method described by Gorinstein. S. and Lii. C, Starch/Starke, Vol. 44 (12) pp. 461-466 (1992).

Mature polypeptide: “Mature polypeptide” means a polypeptide in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. The mature polypeptide sequence lacks a signal sequence, which may 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.

Medium stringency conditions: The term “medium stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 55°C.

Medium-high stringency conditions: The term “medium-high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 60°C. Nucleic acid construct: The term "nucleic acid construct" means a polynucleotide comprises one or more (e.g., two, several) control sequences. The polynucleotide may be single-stranded or double-stranded, and may be isolated from a naturally occurring gene, modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature, or synthetic.

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

Protease: The term “protease” is defined herein as an enzyme that hydrolyses peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of the thirteen subclasses thereof). The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, Academic Press, San Diego, California, including supplements 1-5 published in Eur. J. Biochem. 223: 1-5 (1994); Eur. J. Biochem. 232: 1-6 (1995); Eur. J. Biochem. 237: 1-5 (1996); Eur. J. Biochem. 250: 1-6 (1997); and Eur. J. Biochem. 264: 610- 650 (1999); respectively. The term "subtilases" refer to a sub-group of serine protease according to Siezen et al., 1991, Protein Engng. 4: 719-737 and Siezen et al., 1997, Protein Science 6: 501-523. Serine proteases or serine peptidases is a subgroup of proteases characterised by having a serine in the active site, which forms a covalent adduct with the substrate. Further the subtilases (and the serine proteases) are characterised by having two active site amino acid residues apart from the serine, namely a histidine and an aspartic acid residue. The subtilases may be divided into 6 sub-divisions, i.e. , 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 a proteolytic activity (EC 3.4). Protease activity may be determined using methods described in the art (e.g., US 2015/0125925) or using commercially available assay kits (e.g., Sigma-Aldrich).

Pullulanase: The term “pullulanase” means a starch debranching enzyme having pullulan 6-glucano-hydrolase activity (EC 3.2.1.41) that catalyzes the hydrolysis the a-1,6- glycosidic bonds in pullulan, releasing maltotriose with reducing carbohydrate ends. For purposes of the present invention, pullulanase activity can be determined according to a PHADEBAS assay or the sweet potato starch assay described in WO2016/087237.

Sequence identity: The relatedness between two amino acid sequences or between two nucleotide sequences is described by the parameter “sequence identity”. For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman- Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), e.g., version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100)/(Length of Alignment - Total Number of Gaps in Alignment) For purposes of the present invention, the sequence identity between two deoxyribonucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), e.g., version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The output of Needle labeled “longest identity” (obtained using the - nobrief option) is used as the percent identity and is calculated as follows:

(Identical Deoxyribonucleotides x 100)/(Length of Alignment - Total Number of Gaps in Alignment).

Signal peptide: The term “signal peptide” is defined herein as a peptide linked (fused) in frame to the amino terminus of a polypeptide having biological activity and directs the polypeptide into the cell’s secretory pathway. Signal sequences may be determined using techniques known in the art (See, e.g., Zhang and Henzel, 2004, Protein Science 13: 2819-2824). The polypeptides described herein may comprise any suitable signal peptide known in the art, or any signal peptide described in WO2021/025872 (incorporated herein by reference).

Thermostable: “Thermostable” means the enzyme is not denatured or deactivated when it is used in a liquefaction step of a process of the invention. In other words, a thermostable enzyme is suitable for liquefaction if it has a denaturation temperature (Td) that is compatible with the liquefaction temperature and retains its activity at that temperature.

Trehalase: The term “trehalase” means an enzyme which degrades trehalose into its unit monosaccharides (i.e., glucose). Trehalases are classified in EC 3.2.1.28 (alpha, alpha-trehalase) and EC. 3.2.1.93 (alpha, alpha-phosphotrehalase). The EC classes are based on recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Description of EC classes can be found on the internet, e.g., on “http://www.expasy.org/enzvme/”. Trehalases are enzymes that catalyze the following reactions:

EC 3.2.1.28: Alpha, alpha-trehalose + 2 D-glucose;

EC 3.2.1. 93: Alpha, alpha-trehalose 6-phosphate + H2O <P D-glucose + D-glucose 6- phosphate. Trehalase activity may be determined according to procedures known in the art. Very high stringency conditions: The term “very high stringency conditions” means for probes of at least 100 nucleotides in length, prehybridization and hybridization at 42°C in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide, following standard Southern blotting procedures for 12 to 24 hours. The carrier material is finally washed three times each for 15 minutes using 0.2X SSC, 0.2% SDS at 70°C.

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

Whole Stillage: "Whole stillage" includes the material that remains at the end of the distillation process after recovery of the fermentation product, e.g., ethanol.

Xylanase: “Xylanase” encompasses endo-1,4- p-xylanases (EC 3.2.1.8) that catalyze the endohydrolysis of (1— >4)-p-D-xylosidic linkages in xylans and glucuronoarabinoxylan endo-1 ,4-beta-xylanases (E.C. 3.2.1.136) that catalyze the endohydrolysis of 1,4-beta-D-xylosyl links in some glucuronoarabinoxylans. Activity of EC 3.2.1.8 xylanases can be determined using birchwood xylan as substrate. One unit of xylanase is defined as 1.0 pmole of reducing sugar (measured in glucose equivalents as described by Lever, 1972, A new reaction for colorimetric determination of carbohydrates, Anal. Biochem 47: 273-279) produced per minute during the initial period of hydrolysis at 50° C., pH 5 from 2 g of birchwood xylan per liter as substrate in 50 mM sodium acetate containing 0.01% TWEEN® 2. Activity of EC 3.2.1.136 xylanases can be determined with 0.2% AZCL-glucuronoxylan as substrate in 0.01% TRITON® X-100 and 200 mM sodium phosphate pH 6 at 37°C. One unit of xylanase activity is defined as 1.0 pmole of azurine produced per minute at 37°C, pH 6 from 0.2% AZCL-glucuronoxylan as substrate in 200 mM sodium phosphate pH 6.

DESCRIPTION OF THE INVENTION

The present invention relates to processes of producing fermentation products, such as ethanol from starch-containing material using a fermenting organism.

Work described herein unexpectedly demonstrates that fermenting a starch- containing material with a fermenting organism that expresses a CBH1 and a CBH2 in the prescence of a GH5 xylanase results in significantly higher yield of fermentation product and significantly lower residual solids.

The present invention contemplates using the fermenting organism and GH5 xylanase in saccharification, fermentation, or simultaneous saccharification and fermentation, to improve product yield in conventional and raw-starch hydrolysis (RSH) ethanol production processes, as well as cellulosic ethanol processes.

I. Process for producing a fermentation product from a gelatinized starch- containing grain

An aspect of the invention relates to a process for producing a fermentation product, (e.g., fuel ethanol), from a gelatinized starch-containing grain, 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 a CBH1 and a heterologous polynucleotide encoding a CBH2. This process of the invention contemplates any of the GH5_21 , CBH1 and CBH2 enzymes described herein, especially those demonstrated in the examples below.

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

(a) liquefying the starch-containing material at a temperature above the initial gelatinization temperature using an alpha-amylase;

(b) saccharifying the liquefied starch-containing material;

(c) fermenting saccharified starch-containing material using a fermenting organism; 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 a CBH1 and a heterologous polynucleotide encoding a CBH2.

In an embodiment, the GH5_21 xylanase is present or added during saccharifying step (b). In an embodiment, the GH5_21 xylanase is present or added during fermenting step (c). In an embodiment, steps (b) and (c) are performed simultaneously in a 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 saccharifying step (b) and/or fermenting step (c) is present or added via in situ expression from the fermenting organism (e.g., yeast).

In an embodiment, a thermostable endoglucanase is added during liquefying step (a). In an embodiment, a thermostable lipase is added during liquefying step (a). In an embodiment, a thermostable phytase is added during liquefying step (a). In an embodiment, a thermostable protease is added during liquefying step (a). In an embodiment, a thermostable pullulanase is added during liquefying step (a). In an embodiment, a thermostable xylanase is added during liquefying step (a). In a preferred embodiment, a thermostable alpha-amylase, a thermostable protease and a thermostable xylanase are added during liquefying step (a).

In an embodiment, an alpha-amylase is added during step (b) and/or step (c). In an embodiment, a beta-glucosidase is added during step (a) and/or step (b). In an embodiment, a glucoamylase is added during step (b) and/or step (c). In an embodiment, a cellobiohydrolase is added during step (b) and/or step (c). In an embodiment, an endoglucanase is added during step (b) and/or step (c). In an embodiment, a trehalase is added during step (b) and/or step (c).

In an embodiment, the fermenting organism is yeast. In an embodiment, the yeast expresses an alpha-amylase in situ during step (b) and/or step (c). In an embodiment, the yeast expresses a glucoamylase in situ during step (b) and/or step (c). In an embodiment, the yeast expresses an alpha-amylas and a glucoamylase in situ during step (b) and/or step (c).

Process Parameters

The parameters for processes for producing fermentation products, such as the production of ethanol from starch-containing grain (e.g., corn) are well known in the art. See, e.g., WO 2006/086792, WO 2013/082486, WO 2012/088303, WO 2013/055676, WO 2014/209789, WO 2014/209800, WO 2015/035914, WO 2017/112540, WO 2020/014407, WO 2021/126966 (each of which is incorporated herein by reference).

Starch-containing grain

Any suitable starch-containing starting grain may be used. The grain is selected based on the desired fermentation product. Examples of starch-containing grains, include without limitation, barley, beans, cassava, cereals, corn, milo, peas, potatoes, rice, rye, sago, sorghum, sweet potatoes, tapioca, wheat, and whole grains, or any mixture thereof. The starch-containing grain may also be a waxy or non-waxy type of corn and barley. Commonly used commercial starch-containing grains include corn, milo and/or wheat.

Grain Particle Size Reduction

Prior to liquefying step (a), the particle size of the starch-containing grain may be reduced, for example by dry milling.

Slurry Prior to liquefying step (a), a slurry comprising the starch-containing grain (e.g., preferably milled) and water may be formed. Alpha-amylase and optionally protease may be added to the slurry. The slurry may be heated to between to above the initial gelatinization temperature of the starch-containing grain to begin gelatinization of the starch.

Jet Cook

The slurry may optionally be jet-cooked to further gelatinize the starch in the slurry before adding alpha-amylase during liquefying step (a). Jet cooking can be performed at temperatures ranging from 100 °C to 120 °C for up to at least 15 minutes.

Liquefaction Temperature

The temperature used during liquefying step (a) may 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.

Liquefaction pH

The pH used during liquefying step (a) may 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.

Liquefaction Time

The time for performing liquefying step (a) may range from 30 minutes to 5 hours, from 1 hour to 3 hours, or 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.

Liquefaction Enzymes

The present invention contemplates the use of thermostable enzymes during liquefying step (a). It is well known in the art to use various thermostable enzymes during liquefying step (a), including, for example, thermostable alpha-amylases, thermostable glucoamylases, thermostable endoglucanases, thermostable lipases, thermostable phytase, thermostable proteases, thermostable pullulanases, and/or thermostable xylanases. The present invention contemplates the use of any thermostable enzyme in liquefying step (a). Guidance for determining the denaturation temperature of a candidate thermostable enzyme for use in liquefying step (a) is provided in the Materials & Methods section below. The published patent applications listed below describe activity assays for determining whether a candidate thermostable enzyme contemplated for use in liquefying step (a) will be deactivated at a temperature contemplated for liquefying step (a).

Examples of suitable thermostable alpha-amylases and guidance for using them in liquefying step (a) include, without limitation, the alpha-amylases described in WO 1996/023873, WO 1996/023874, WO 1997/041213, WO 1999/019467, WO 2000/060059, WO 2002/010355, WO 2002/092797, WO 2009/149130, WO 2009/061379, WO 2010/115021 , WO 2010/036515, WO 2011/082425, WO 2019/113413, WO 2019/113415, WO 2019/197318 (each of which is incorporated herein by reference).

Examples of suitable thermostable glucoamylases include, without limitation, the glucoamylases described in WO 2011/127802, WO 2013/036526, WO 2013/053801 , WO 2018/164737, WO 2020/010101 , and WO 2022/090564 (each of which is incorporated herein by reference).

Examples of suitable thermostable endoglucanases include, without limitation, the endoglucanases described in WO 2015/035914 (which is incorporated herein by reference)

Examples of suitable thermostable lipases include, without limitation, the lipases described in WO 2017/112542 and WO 2020/014407 (which are both incorporated herein by reference).

Examples of suitable thermostable phytases include, without limitation, 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, WO 2003/066847, WO 2004/085638, WO 2006/037327, WO 2006/037328, WO 2006/038062, WO 2006/063588, WO 2007/112739, WO 2008/092901 , WO 2008/116878, WO 2009/129489, and WO 2010/034835 (each of which is incorporated by reference). Commercially available phytase containing products include BIO-FEED PHYTASE™, PHYTASE NOVO™ CT or L, LIQMAX or RONOZYME™ NP, RONOZYME® HIPHOS, RONOZYME® P5000 (CT), NATUPHOS™ NG 5000.

Examples of suitable thermostable proteases include, without limitation, 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, WO2018/118815 A1 , and WO2018/169780A1 (each of which is incorporated herein by reference).

Suitable commercially available protease containing products include AVANTEC AMP®, FORTIVA REVO®, FORTIVA HEMI®.

Examples of suitable thermostable pullulanases include, without limitation, the pullulanases described in WO 2015/007639, 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™ D2 (Novozymes A/S, Denmark), OPTIMAX L-300 (Genencor Int. , USA), and AMANO 8 (Amano, Japan).

Examples of suitable thermostable xylanases include, without limitation, the xylanases described in WO 2017/112540 and WO 2021/126966 (each of which is incorporated herein by reference). Suitable commercially available thermostable xylanase containing products include FORTIVA HEM I®.

The enzyme(s) described above are to be used in effective amounts in the processes of the present invention. Guidance for determining effective amounts of enzymes to be used in liquefying step (a) can be found in the published patent applications cited for each of the different thermostable liquefaction enzymes, along with guidance for performing activity assays for determining the activity of those enzymes.

Saccharification Temperature

Saccharification may be performed at temperatures ranging 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.

Saccharification pH

Saccharification may occur at a ph ranging from 4 to 5. Preferably, the pH is about 4.5.

Saccharification Time

Saccharification may last from about 24 hours to about 72 hours.

Fermentation Time

Fermentation may last from 6 to 120 hours, from 24 hours to 96 hours, or from 35 hours to 60 hours.

Simultaneous Saccharification and Fermentation

SSF may be performed at a temperature from 25 °C to 40 °C, from 28 °C to 35 °C, or from 30 °C to °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 performed at about 32 °C, at a pH from 3.8 to 4.5 for from 48 to 60 hours.

Saccharification and/or Fermentation Enzymes

The present invention contemplates the use of enzymes during saccharifying step (b) and/or fermenting step (c). It is well known in the art to use various enzymes during saccharifying step (b) and/or fermenting step (c), including, for example, alpha-amylases, alpha-glucosidases, beta-amylases, beta-glucanases, beta-glucosidases, cellobiohydrolases, endoglucanases, glucoamylases, lipases, lytic polysaccharide monooxygenases (LPMOs), maltogenic alpha-amylases, pectinases, peroxidases, phytases, proteases, and trehalases.

The enzymes used in saccharifying step (b) and/or fermenting step (c) may be added exogenously as mono-components or formulated as compositions comprising the enzymes. The enzymes used in saccharifying step (b) and/or fermenting step (c) may also be added via in situ expression from the fermenting organism (e.g., yeast). Examples of suitable yeast expressing enzymes include, without limitation, the yeast described herein.

Examples of suitable alpha-amylases include, without limitation, the alpha-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).

Examples of suitable glucoamylases include, without limitation, the glucoamylases 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, WO 2013/036526, WO 2013/053801, WO 2014/039773, WO 2014/177541, WO 2014/177546, WO 2016/062875, WO 2017/066255, and WO 2018/191215 (each of which is incorporated herein by reference.

Examples of suitable compositions comprising alpha-amylases and glucoamylases include, without limitation, the compositons described in WO 2006/069290, WO 2009/052101, WO 2011/068803, and WO 2013/006756 (each of which is incorporated by reference herein). Commercially available compositions comprising glucoamylase include AMG 200L; AMG 300 L; SAN™ SUPER, SAN™ EXTRA L, SPIRIZYME™ PLUS, SPIRIZYME™ FUEL, SPIRIZYME™ B4U, SPIRIZYME™ ULTRA, SPIRIZYME™ EXCEL, SPIRIZYME ACHIEVE and AMG™ E (from Novozymes A/S); OPTIDEX™ 300, GC480, GC417 (from DuPont-Genencor); AMIGASE™ and AMIGASE™ PLUS (from DSM); G- ZYME™ G900, G-ZYME™ and G990 ZR (from DuPont-Genencor).

Examples of suitable beta-glucanases include, without limitation, the beta-glucanases described in WO 2021/055395 (which is incorporated herein by reference).

Examples of suitable beta-glucosidases include, without limitation, the betaglucosidases described in WO 2005/047499, WO 2013/148993, WO 2014/085439 and WO 2012/044915 (each of which is incorporated herein by reference). Examples of suitable cellobiohydrolases include, without limitation, the cellobiohydrolases described in WO 2013/148993, WO 2014/085439, WO 2014/138672, and WO 2016/040265 (each of which is incorporated herein by reference).

Examples of suitable endoglucanases include, without limitation, the endoglucanases described in WO 2013/148993 and WO 2014/085439 (both of which are incorporated herein by reference).

Examples of suitable maltogenic alpha-amylases are described in US Patent nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference.

Examples of suitable lipases include, without limitation, the lipases described in WO 2017/112533, WO 2017/112539, and WO 2020/076697 (each of which is incorporated herein by reference).

Examples of suitable LPMOs include, without limitation, the LPMOs described in WO 2013/148993, WO 2014/085439, and WO 2019/083831 (each of which is incorporated herein by reference).

Examples of suitable phytases include, without limitation, the phytases described in WO 2001/62947 (which is incorporated herein by reference).

Examples of suitable pectinases include, without limitation, the pectinases described in WO 2022/173694 (which is incorporated herein by reference).

Examples of suitable peroxidases include, without limitation, the peroxidases described in WO 2019/231944 (which is incorporated herein by reference).

Examples of suitable proteases include, without limitation, the proteases described in WO 2017/050291, WO 2017/148389, WO 2018/015303, and WO 2018/015304 (each of which is incorporated herein by reference).

Examples of suitable trehalases include, without limitation, the trehalases described in WO 2016/205127, WO 2019/005755, WO 2019/030165, and WO 2020/023411 (each of which is incorporated herein by reference).

II. Process for producing a fermentation product from ungelatinized starch- containing grain

An aspect of the invention relates to a process for producing a fermentation product from an ungelatinized starch-containing grain (i.e. , granularized 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 a CBH1 and a heterologous polynucleotide encoding a CBH2. This process of the invention contemplates any of the GH5_21, CBH1 and CBH2 enzymes described herein, especially the compositions demonstrated in the examples below.

In an embodiment, a process for producing a fermentation product from an ungelatinized starch-containging grain comprises the following steps:

(a) saccharifying a starch-containing grain at a temperature below the initial gelatinization temperature using a glucoamylase and an alpha-amylase to produce a fermentable sugar; and

(b) fermenting the sugar using a fermenting organism to produce a fermentation product; 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 a CBH1 and a heterologous polynucleotide encoding a CBH2.

In an embodiment, the GH5_21 xylanase is present or added during saccharifying step (a). In an embodiment, the GH5_21 xylanase is present or added during fermenting step (b). In an embodiment, steps (a) and (b) are performed simultaneously in a 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 saccharifying step (a) and/or fermenting step (b) is present or added via in situ expression from the fermenting organism (e.g., yeast).

Raw starch hydrolysis (RSH) processes are well-known in the art. The skilled artisan will appreciate that, except for the process parameters relating to liquefying step (a) which is not done in a RSH process, the process parameters described in Section I above are applicable to the process described in this section, including selection of the starch- containing grain, reducing the grain particle size, saccharification temperature, time and pH, conditions for simultaneous saccharification and fermentation, and saccharification enzymes. The process parameters for an exemplary raw-starch hydrolysis process are described in further detail in WO 2004/106533 (which is incorporated herein by reference).

Examples of alpha-amylases that are preferably used in step (a) and/or step (b) include, without limitation, the alpha-amylases described in WO 2004/055178, WO 2005/003311 , WO 2006/069290, WO 2013/006756, WO 2013/034106, WO 2021/163015, and WO 2021/163036 (each of which is incorporated by reference herein).

Examples of glucoamylases that are preferably used in step (a) and/or step (b) include, without limitation, WO 1999/28448, WO 2005/045018, W02005/069840, WO 2006/069289 (each of which is incorporated by reference herein). Examples of compositions comprising alpha-amylases and glucoamylase that are preferably used in step (a) and/or step (b) include, without limitation, the compositions described in WO 2015/031477 (which is incorporated by reference herein).

A. Exemplary Fermenting Organisms

Aspects of the invention relate to a fermenting organism that comprises a heterologous polynucleotide encoding a CBH1 and a heterologous polynucleotide encoding a CBH2, used in combination with a GH5 family xylanase. The present invention contemplates using any fermenting organism that, when used in combination with a GH5 family xylanase, increases production of a fermentation product and/or decreases the residual solids compared to processes using fermenting organism that lack a heterologous polynucleotide encoding a CBH1 and/or a heterologous polynucleotide encoding a CBH2.

Especially suitable fermenting organisms are able to ferment, i.e. , convert, sugars, such as arabinose, glucose, maltose, and/or xylose, directly or indirectly into the desired fermentation product, such as ethanol. Examples of fermenting organisms include fungal organisms, such as yeast. Preferred yeast includes strains of Saccharomyces spp., in particular, Saccharomyces cerevisiae.

Suitable concentrations of the viable fermenting organism during fermentation, such as SSF, are well known in the art or can easily be determined by the skilled person in the art. In one embodiment, the fermenting organism, such as ethanol fermenting yeast, (e.g., Saccharomyces cerevisiae) is added to the fermentation medium so that the viable fermenting organism, such as yeast, count per mL of fermentation medium is in the range from 10⁵ to 10¹², preferably from 10⁷ to 10¹⁰, especially about 5x10⁷.

Examples of commercially available yeast includes, e.g., RED STAR™ and ETHANOL RED™ yeast (available from Fermentis/Lesaffre, USA), FALI (available from Fleischmann’s Yeast, USA), SUPERSTART and THERMOSACC™ fresh yeast (available from Ethanol Technology, Wl, USA), BIOFERM AFT and XR (available from NABC - North American Bioproducts Corporation, GA, USA), GERT STRAND (available from Gert Strand AB, Sweden), and FERMIOL (available from DSM Specialties). Other useful yeast strains are available from biological depositories such as the American Type Culture Collection (ATCC) or the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ), such as, e.g., BY4741 (e.g., ATCC 201388); Y108-1 (ATCC PTA.10567) and NRRL YB- 1952 (ARS Culture Collection). Still other S. cerevisiae strains suitable as host cells 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 Saccharomyces sp. 1400, derivatives thereof. As used herein, a “derivative” of strain is derived from a referenced strain, such as through mutagenesis, recombinant DNA technology, mating, cell fusion, or cytoduction between yeast strains. Those skilled in the art will understand that the genetic alterations, including metabolic modifications exemplified herein, may be described with reference to a suitable host organism and their corresponding metabolic reactions or a suitable source organism for desired genetic material such as genes for a desired metabolic pathway. However, given the complete genome sequencing of a wide variety of organisms and the high level of skill in the area 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 readily be applied to other species by incorporating the same or analogous encoding nucleic acid from species other than the referenced species.

The host cell or fermenting organism may be Saccharomyces strain, e.g., Saccharomyces cerevisiae strain produced using the method described and concerned in US patent no. 8,257,959-BB. In one embodiment, the recombinant cell is a derivative of a strain Saccharomyces cerevisiae CIBTS1260 (deposited under Accession No. NRRL Y- 50973 at the Agricultural Research Service Culture Collection (NRRL), Illinois 61604 U.S.A.).

The strain may also be a derivative of Saccharomyces cerevisiae strain NMI V14/004037 (See, WO2015/143324 and WO2015/143317 each incorporated herein by reference), strain nos. V15/004035, V15/004036, and V15/004037 (See, WO 2016/153924 incorporated herein by reference), strain nos. V15/001459, V15/001460, V15/001461 (See, WO2016/138437 incorporated herein by reference), strain no. NRRL Y67342 (See, WO2018/098381 incorporated herein by reference), strain nos. NRRL Y67549 and NRRL Y67700 (See, WO 2019/161227 incorporated herein by reference), or any strain described in WO2017/087330 (incorporated herein by reference).

In one embodiment, the fermenting organisms comprise a heterologous polynucleotide encoding a CBH1 and a heterologous polynucleotide encoding a CBH2. Any CBH1 and CBH2 having cellobiohydrolase I and cellobiohydrolase II activity, respectively, may be used with the processes described herein and/or expressed by the host cells or fermenting organisms described herein.

The CBH1 or CBH2 may be obtained from microorganisms of any genus. For purposes herein, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide from the source has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

In one embodiment, the CBH1 is a Penicillium CBH1, such as a Penicillium emersonii CBH1 (e.g., the Penicillium emersonii CBH1 of SEQ ID NO: 16). In one embodiment, the CBH2 is a Talaromyces CBH2, such as a Talaromyces verruculosus cellobiohydrolase II (e.g., the Talaromyces verruculosus cellobiohydrolase II of SEQ ID NO: 17). It will be understood that for the aforementioned species, the invention encompasses both the perfect and imperfect states, and other taxonomic equivalents, e.g., anamorphs, regardless of the species name by which they are known. Those skilled in the art will readily recognize the identity of appropriate equivalents.

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

The CBH1 or CBH2, coding sequences described or referenced herein, or a subsequence thereof, or a fragment thereof, may be used to design nucleic acid probes to identify and clone DNA encoding a CBH1 or CBH2 from strains of different genera or species according to methods well known in the art. In particular, such probes can be used for hybridization with the genomic DNA or cDNA of a cell of interest, following standard Southern blotting procedures, in order to identify and isolate the corresponding gene therein. Such probes can be considerably shorter than the entire sequence, but should be at least 15, e.g., at least 25, at least 35, or at least 70 nucleotides in length. Preferably, the nucleic acid probe is at least 100 nucleotides in length, e.g., at least 200 nucleotides, at least 300 nucleotides, at least 400 nucleotides, at least 500 nucleotides, at least 600 nucleotides, at least 700 nucleotides, at least 800 nucleotides, or at least 900 nucleotides in length. Both DNA and RNA probes can be used. The probes are typically labeled for detecting the corresponding gene (for example, with ³²P, ³H, ³⁵S, biotin, or avidin).

A genomic DNA or cDNA library prepared from such other strains may be screened for DNA that hybridizes with the probes described above and encodes a CBH1 or CBH2. Genomic or other DNA from such other strains may be separated by agarose or polyacrylamide gel electrophoresis, or other separation techniques. DNA from the libraries or the separated DNA may be transferred to and immobilized on nitrocellulose or other suitable carrier material. In order to identify a clone or DNA that hybridizes with a coding sequence, or a subsequence thereof, the carrier material is used in a Southern blot.

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

For purposes of the probes described above, hybridization indicates that the polynucleotide hybridizes to a labeled nucleic acid probe, or the full-length complementary strand thereof, or a subsequence of the foregoing; under very low to very high stringency conditions. Molecules to which the nucleic acid probe hybridizes under these conditions can be detected using, for example, X-ray film. Stringency and washing conditions are defined as described supra.

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

The polypeptide having CBH1 and CBH2 may also be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, silage, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, silage, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. The polynucleotide encoding a CBH1 or CBH2 may then be derived by similarly screening a genomic or cDNA library of another microorganism or mixed DNA sample.

Once a polynucleotide encoding a CBH1 or CBH2 has been detected with a suitable probe as described herein, the sequence may be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the art (See, e.g., Sambrook et al., 1989, supra). Techniques used to isolate or clone polynucleotides encoding polypeptides include isolation from genomic DNA, preparation from cDNA, or a combination thereof. The cloning of the polynucleotides from such genomic DNA can be affected, e.g., by using the well-known polymerase chain reaction (PCR) or antibody screening of expression libraries to detect cloned DNA fragments with shares structural features (See, e.g., Innis et al., 1990, PCR: A Guide to Methods and Application, Academic Press, New York). Other nucleic acid amplification procedures such as ligase chain reaction (LCR), ligated activated transcription (LAT) and nucleotide sequence-based amplification (NASBA) may be used.

In one embodiment, the CBH1 comprises or consists of the amino acid sequence of SEQ ID NO: 16, or the mature polypeptide thereof. In another embodiment, the CBH1 is a fragment of the CBH1 of SEQ ID NO: 16, or the mature polypeptide thereof, wherein, e.g., the fragment has CBH1 activity. In one embodiment, the number of amino acid residues in the fragment is at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of amino acid residues in referenced full length CBH1. In other embodiments, the CBH1 may comprise the catalytic domain SEQ ID NO: 16.

In one embodiment, the CBH2 comprises or consists of the amino acid sequence of SEQ ID NO: 17, or the mature polypeptide thereof. In another embodiment, the CBH2 is a fragment of the CBH2 of SEQ ID NO: 17, or the mature polypeptide thereof, wherein, e.g., the fragment has CBH2 activity. In one embodiment, the number of amino acid residues in the fragment is at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of amino acid residues in referenced full length CBH2. In other embodiments, the CBH2 may comprise the catalytic domain SEQ ID NO: 17.

The CBH1 and CBH2 may be a variant of a CBH1 and CBH2 described supra (e.g., SEQ ID NO: 16, SEQ ID NO: 17, or the mature polypeptide thereof). In one embodiment, the CBH1 has at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 16 or the mature polypeptide thereof. In one embodiment, the CBH2 has at least 60%, e.g., at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 17, or the mature polypeptide thereof.

In one embodiment, the CBH1 differs by no more than ten amino acids, e.g., 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 one amino acid from the amino acid sequence of SEQ ID NO: 16, or the mature polypeptide thereof. In one embodiment, the CBH2 differs by no more than ten amino acids, e.g., 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 one amino acid from the amino acid sequence of SEQ ID NO: 17, or the mature polypeptide thereof.

The amino acid changes are generally of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and/or activity of the protein; small deletions, typically of one to about 30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to about 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding domain.

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

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

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, single alanine mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The active site or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids (See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64). The identities of essential amino acids can also be inferred from analysis of identities with other cellulases that are related to the referenced enzyme.

Additional guidance on the structure-activity relationship of the cellulases herein can be determined using multiple sequence alignment (MSA) techniques well-known in the art. Based on the teachings herein, the skilled artisan could make similar alignments with any number of CBH1 or CBH2 enzymes described herein or known in the art. Such alignments aid the skilled artisan to determine potentially relevant domains (e.g., binding domains or catalytic domains), as well as which amino acid residues are conserved and not conserved among the different cellulase sequences. It is appreciated in the art that changing an amino acid that is conserved at a particular position between disclosed polypeptides will more likely 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 an amino acid that is not highly conserved among the polypeptides will not likely or significantly alter the biological activity. Even further guidance on the structure-activity relationship for the skilled artisan can be found in published x-ray crystallography studies known in the art.

Single or multiple amino acid substitutions, deletions, and/or insertions can be made and tested using known methods of mutagenesis, recombination, and/or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152- 2156; WO95/17413; or WO95/22625. 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; W092/06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et a/., 1988, DNA 7: 127).

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

In one embodiment, the heterologous polynucleotide encoding the CBH1 comprises or consists of a coding sequence of the CBH1 of SEQ ID NO: 16, or the mature polypeptide thereof. In another embodiment, the heterologous polynucleotide encoding the CBH1 comprises a subsequence of a coding sequence of the CBH1 of SEQ ID NO: 16 wherein the subsequence encodes a polypeptide having CBH1 activity. In another embodiment, the number of nucleotides residues in the coding subsequence is at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of the referenced coding sequence. In another embodiment, the heterologous polynucleotide encoding the CBH1 comprises a coding sequence having at least 60%, e.g., 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 to a coding sequence of the CBH1 of SEQ ID NO: 16, or the mature polypeptide thereof.

In one embodiment, the heterologous polynucleotide encoding the CBH2 comprises or consists of a coding sequence of the CBH2 of SEQ ID NO: 17, or the mature polypeptide thereof. In another embodiment, the heterologous polynucleotide encoding the CBH2 comprises a subsequence of a coding sequence of the CBH2 of SEQ ID NO: 17 wherein the subsequence encodes a polypeptide having CBH2 activity. In another embodiment, the number of nucleotides residues in the coding subsequence is at least 75%, e.g., at least 80%, 85%, 90%, or 95% of the number of the referenced coding sequence. In another embodiment, the heterologous polynucleotide encoding the CBH2 comprises a coding sequence having at least 60%, e.g., 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 to a coding sequence of the CBH2 of SEQ ID NO: 17, or the mature polypeptide thereof.

The referenced coding sequence of any related aspect or embodiment described herein can be the native coding sequence or a degenerate sequence, such as a codon- optimized coding sequence designed for use in a particular host cell (e.g., optimized for expression in Saccharomyces cerevisiae or any other host used for production). Codonoptimization for expression in yeast cells is known in the art (e.g., US 8,326,547).

The CBH1 and CBH2 may be a fused polypeptide or cleavable fusion polypeptide in which another polypeptide is fused at the N-terminus or the C-terminus of the enzyme. A fused polypeptide may be produced by fusing a polynucleotide encoding another polypeptide to a CBH1 or CBH2. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame and that expression of the fused polypeptide is under control of the same promoter(s) and terminator. Fusion proteins may also be constructed using intein technology in which fusions are created post-translationally (Cooper et al. , 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779).

In some embodiments, the CBH1 or CBH2 is a fusion protein comprising a signal peptide linked to the N-terminus of a mature polypeptide, such as any signal sequences described in WO2021/025872 “Fusion Proteins For Improved Enzyme Expression” (the content of which is hereby incorporated by reference).

In some embodiments, the host cells and/or fermenting organisms comprise one or more heterologous polynucleotides encoding an alpha-amylase, glucoamylase, protease and/or cellulase. Examples of alpha-amylase, glucoamylase, protease and cellulases suitable for expression in the host cells and/or fermenting organisms are described in more detail herein.

In some embodiments, the host cells and/or fermenting organisms comprise one or more heterologous polynucleotides encoding a GH5 xylanase (e.g., a GH5_21 xylanase). Examples of GH5 xylanases are described in more detail herein.

In some embodiments, the host cells and/or fermenting organisms comprise an active pentose fermentation pathway. In some embodiments, the host cells and/or fermenting organisms comprise an active xylose fermentation pathway. In some embodiments, the host cells and/or fermenting organisms comprise an active arabinose fermentation pathway. The host cells and fermenting organisms described herein may utilize expression vectors comprising the coding sequence of one or more (e.g., two, several) heterologous genes linked to one or more control sequences that direct expression in a suitable cell under conditions compatible with the control sequence(s). Such expression vectors may be used in any of the cells and methods described herein. The polynucleotides described herein may be manipulated in a variety of ways to provide for expression of a desired polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. The techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

A construct or vector (or multiple constructs or vectors) comprising the one or more (e.g., two, several) heterologous genes may be introduced into a cell so that the construct or vector is maintained as a chromosomal integrant or as a self-replicating extra-chromosomal vector as described earlier.

The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more (e.g., two, several) convenient restriction sites to allow for insertion or substitution of the polynucleotide at such sites. Alternatively, the polynucleotide(s) may be expressed by inserting the polynucleotide(s) or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

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

The vector may be an autonomously replicating vector, i.e. , a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the cell, or a transposon, may be used.

The expression vector may contain any suitable promoter sequence that is recognized by a cell for expression of a gene described herein. The promoter sequence contains transcriptional control sequences that mediate the expression of the polypeptide. The promoter may be any polynucleotide that shows transcriptional activity in the cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the cell.

Each heterologous polynucleotide described herein may be operably linked to a promoter that is foreign to the polynucleotide. For example, in one embodiment, the nucleic acid construct encoding the fusion protein is operably linked to a promoter foreign to the polynucleotide. The promoters may be identical to or share 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%) with a selected native promoter.

Examples of suitable promoters for directing the transcription of the nucleic acid constructs in a yeast cells, include, but are not limited to, the promoters obtained from the genes for enolase, (e.g., S. cerevisiae enolase or /. orientalis enolase (ENO1)), galactokinase (e.g., S. cerevisiae galactokinase or /. orientalis galactokinase (GAL1)), alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (e.g., S. cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase or /. orientalis alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1 , ADH2/GAP)), triose phosphate isomerase (e.g., S. cerevisiae triose phosphate isomerase or /. orientalis triose phosphate isomerase (TPI)), metallothionein (e.g., S. cerevisiae metallothionein or /. orientalis metallothionein (CLIP1)), 3-phosphoglycerate kinase (e.g., S. cerevisiae 3-phosphoglycerate kinase or /. 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 (LIRA3) genes. Other suitable promoters may be obtained from S. 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.

The control sequence may also be a suitable transcription terminator sequence, which is recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3’-terminus of the polynucleotide encoding the polypeptide. Any terminator that is functional in the yeast cell of choice may be used. The terminator may be identical to or share 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%) with the selected native terminator. Suitable terminators for yeast host cells may be obtained from the genes for enolase (e.g., S. cerevisiae or /. orientalis enolase cytochrome C (e.g., S. cerevisiae or /. orientalis cytochrome (CYC1)), glyceraldehyde-3-phosphate dehydrogenase (e.g., S. cerevisiae or /. orientalis glyceraldehyde-3-phosphate dehydrogenase (gpd)), PDC1, XR, XDH, transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI), CYB2, and the galactose family of genes (especially the GAL10 terminator). Other suitable terminators may be obtained from S. cerevisiae ENO2 or TEFI genes. Additional useful terminators for yeast host cells are described by Romanos et al., 1992, supra.

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

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

The control sequence may also be a suitable leader sequence, when transcribed is a non-translated region of an mRNA that is important for translation by the host cell. The leader sequence is operably linked to the 5’-terminus of the polynucleotide encoding the polypeptide. Any leader sequence that is functional in the yeast cell of choice may be used.

Suitable leaders for yeast host cells are obtained from the genes for enolase (e.g., S. cerevisiae or /. orientalis enolase (ENO-1)), 3-phosphoglycerate kinase (e.g., S. cerevisiae or /. orientalis 3-phosphoglycerate kinase), alpha-factor (e.g., S. cerevisiae or /. orientalis alpha-factor), and alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (e.g., S. cerevisiae or /. orientalis alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP)).

The control sequence may also be a polyadenylation sequence; a sequence operably linked to the 3’-terminus of the polynucleotide and, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA. Any polyadenylation sequence that is functional in the host cell of choice may be used. Useful polyadenylation sequences for yeast cells are described by Guo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

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 directs the polypeptide into the cell’s secretory pathway. The 5’-end of the coding sequence of the polynucleotide may inherently contain a signal peptide coding sequence naturally linked in translation reading frame with the segment of the coding sequence that encodes the polypeptide. Alternatively, the 5’-end of the coding sequence may contain a signal peptide coding sequence that is foreign to the coding sequence. A foreign signal peptide coding sequence may be required where the coding sequence does not naturally contain a signal peptide coding sequence. Alternatively, a foreign signal peptide coding sequence may simply replace the natural signal peptide coding sequence in order to enhance secretion of the polypeptide. However, any signal peptide coding sequence that directs the expressed polypeptide into the secretory pathway of a host cell may be used. Useful signal peptides for yeast host cells are obtained from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase. Other useful signal peptide coding sequences are described by Romanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence that encodes a propeptide positioned at the N-terminus of a polypeptide. The resultant polypeptide is known as a proenzyme or propolypeptide (or a zymogen in some cases). A propolypeptide is generally inactive and can be converted to an active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding sequence may be obtained from the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor miehei aspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present, the propeptide sequence is positioned next to the N-terminus of a polypeptide and the signal peptide sequence is positioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. Regulatory systems in prokaryotic systems include the lac, tac, and trp operator systems. In yeast, the ADH2 system or GAL1 system may be used.

The vectors may contain one or more (e.g., two, several) selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Suitable markers for yeast host cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2, MET3, TRP1 , and URA3.

The vectors may contain one or more (e.g., two, several) elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on the polynucleotide’s 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 may contain additional polynucleotides for directing integration by homologous recombination into the genome of the host cell at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements 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 to the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding polynucleotides. On the other hand, the vector may be integrated into the genome of the host cell by non-homologous recombination. Potential integration loci include those described in the art (e.g., See US2012/0135481).

For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the yeast cell. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

More than one copy of a polynucleotide described herein may be inserted into a host cell to increase production of a polypeptide. An increase in the 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 with the polynucleotide where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

The procedures used to ligate the elements described above to construct the recombinant expression vectors described herein are well known to one skilled in the art (see, e.g., Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, New York).

Additional procedures and techniques known in the art for the preparation of recombinant cells for ethanol fermentation, are described in, e.g., WO 2016/045569, the content of which is hereby incorporated by reference.

The host cell or fermenting organism may be in the form of a composition comprising a host cell or fermenting organism (e.g., a yeast strain described herein) and a naturally occurring and/or a non-naturally occurring component. The host cell or fermenting organism described herein may be in any viable form, including crumbled, dry, including active dry and instant, compressed, cream (liquid) form etc. In one embodiment, the host cell or fermenting organism (e.g., a Saccharomyces cerevisiae yeast 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 yeast strain) is crumbled yeast. In one embodiment, the host cell or fermenting organism (e.g., a Saccharomyces cerevisiae yeast strain) is compressed yeast. In one embodiment, the host cell or fermenting organism (e.g., a Saccharomyces cerevisiae yeast strain) is cream yeast.

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

The compositions described herein may comprise a host cell or fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable surfactants. In one embodiment, the surfactant(s) is/are an anionic surfactant, cationic surfactant, and/or nonionic surfactant.

The compositions described herein may comprise a host cell or fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) 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 of sorbitan monostearate (SMS), citric acid esters of monodiglycerides, polyglycerolester, fatty acid esters of propylene glycol.

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

The compositions described herein may comprise a host cell or fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable gum. In one embodiment, the gum is selected from the group of carob, guar, tragacanth, arabic, xanthan and acacia gum, in particular for cream, compressed and dry yeast.

The compositions described herein may comprise a host cell or fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable swelling agent. In one embodiment, the swelling agent is methyl cellulose or carboxymethyl cellulose.

The compositions described herein may comprise a host cell or fermenting organism described herein (e.g., a Saccharomyces cerevisiae yeast strain) and any suitable anti-oxidant. In one embodiment, the antioxidant is butylated hydroxyanisol (BHA) and/or butylated hydroxytoluene (BHT), or ascorbic acid (vitamin C), particular for active dry yeast.

The host cells and fermenting organisms described herein may also comprise one or more (e.g., two, several) gene disruptions, e.g., to divert sugar metabolism from undesired products to ethanol. In some embodiments, the recombinant host cells produce a greater amount of ethanol compared to the cell without the one or more disruptions when cultivated under identical conditions. In some embodiments, one or more of the disrupted endogenous genes is inactivated.

In certain embodiments, the host cell or fermenting organism provided herein comprises a disruption of one or more endogenous genes encoding enzymes involved in producing alternate fermentative products such as glycerol or other byproducts such as acetate or diols. For example, the cells provided herein may comprise a disruption of one or more of glycerol 3-phosphate dehydrogenase (GPD, catalyzes reaction of di hydroxyacetone phosphate to glycerol 3-phosphate), glycerol 3-phosphatase (GPP, catalyzes conversion of glycerol-3 phosphate to glycerol), glycerol kinase (catalyzes conversion of glycerol 3- phosphate to glycerol), dihydroxyacetone kinase (catalyzes conversion of dihydroxyacetone phosphate to dihydroxyacetone), glycerol dehydrogenase (catalyzes conversion of dihydroxyacetone to glycerol), and aldehyde dehydrogenase (ALD, e.g., converts acetaldehyde to acetate).

Modeling analysis can be used to design gene disruptions that additionally optimize utilization of the pathway. One exemplary computational method for identifying and designing metabolic alterations favoring biosynthesis of a desired product is the OptKnock computational framework, Burgard et al., 2003, Biotechnol. Bioeng. 84: 647-657.

The host cells and fermenting organisms comprising a gene disruption may be constructed using methods well known in the art, including those methods described herein. A portion of the gene can be disrupted such as the coding region or a control sequence required for expression of the coding region. Such a control sequence of the gene may be a promoter sequence or a functional part thereof, i.e., a part that is sufficient for affecting expression of the gene. For example, a promoter sequence may be inactivated resulting in no expression or a weaker promoter may be substituted for the native promoter sequence to reduce expression of the coding sequence. Other control sequences for possible modification include, but are not limited to, a leader, propeptide sequence, signal sequence, transcription terminator, and transcriptional activator.

The host cells and fermenting organisms comprising a gene disruption may be constructed by gene deletion techniques to eliminate or reduce expression of the gene. Gene deletion techniques enable the partial or complete removal of the gene thereby eliminating their expression. In such methods, deletion of the gene is accomplished by homologous recombination using a plasmid that has been constructed to contiguously contain the 5' and 3' regions flanking the gene.

The host cells and fermenting organisms comprising a gene disruption may also be constructed by introducing, substituting, and/or removing one or more (e.g., two, several) nucleotides in the gene or a control sequence thereof required for the transcription or translation thereof. For example, nucleotides may be inserted or removed for the introduction of a stop codon, the removal of the start codon, or a frame-shift of the open reading frame. Such a modification may be accomplished by site-directed mutagenesis or PCR generated mutagenesis in accordance with methods known in the art. See, for example, 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.

The host cells and fermenting organisms comprising a gene disruption may also be constructed by inserting into the gene a disruptive nucleic acid construct comprising a nucleic acid fragment homologous to the gene that will create a duplication of the region of homology and incorporate construct DNA between the duplicated regions. Such a gene disruption can eliminate 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 results. A disrupting construct may be simply a selectable marker gene accompanied by 5’ and 3’ regions homologous to the gene. The selectable marker enables identification of transformants containing the disrupted gene.

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

The host cells and fermenting organisms comprising a gene disruption may be further constructed by random or specific mutagenesis using methods well known in the art, including, but not limited to, chemical mutagenesis (see, for example, 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). Modification of the gene may be performed by subjecting the parent strain to mutagenesis and screening for mutant strains in which expression of the gene has been reduced or inactivated. The mutagenesis, which may be specific or random, may be performed, for example, by use of a suitable physical or chemical mutagenizing agent, use of a suitable oligonucleotide, or subjecting the DNA sequence to PCR generated mutagenesis. Furthermore, the mutagenesis may be performed by use of any combination of these mutagenizing methods.

Examples of a physical or chemical mutagenizing agent suitable for the present purpose include ultraviolet (UV) irradiation, hydroxylamine, N-methyl-N'-nitro-N- nitrosoguanidine (MNNG), N-methyl-N’-nitrosogaunidine (NTG) O-methyl hydroxylamine, nitrous acid, ethyl methane sulphonate (EMS), sodium bisulphite, formic acid, and nucleotide analogues. When such agents are used, the mutagenesis is typically performed by incubating the parent strain to be mutagenized in the presence of the mutagenizing agent of choice under suitable conditions, and selecting for mutants exhibiting reduced or no expression of the gene.

A nucleotide sequence homologous or complementary to a gene described herein may be used from other microbial sources to disrupt the corresponding gene in a recombinant strain of choice.

In one embodiment, the modification of a gene in the host cells and fermenting organisms is unmarked with a selectable marker. Removal of the selectable marker gene may be accomplished by culturing the mutants on a counter-selection medium. Where the selectable marker gene contains repeats flanking its 5' and 3' ends, the repeats will facilitate the looping out of the selectable marker gene by homologous recombination when the mutant strain is submitted to counter-selection. The selectable marker gene may also be removed by homologous recombination by introducing into the mutant strain a nucleic acid fragment comprising 5' and 3' regions of the defective gene, but lacking the selectable marker gene, followed by selecting on the counter-selection medium. By homologous recombination, the defective gene containing the selectable marker gene is replaced with the nucleic acid fragment lacking the selectable marker gene. Other methods known in the art may also be used.

B. Exemplary GH5 xylanases

Aspects of the invention relate to GH5 family xylanases in combination with a fermenting organism that comprises a heterologous polynucleotide encoding a CBH1 and a heterologous polynucleotide encoding a CBH2 to increase hemicellulosic fiber solubilization and production of monomeric arabinose and/or xylose. The present invention contemplates using any GH5 xylanase that, when used in combination with the fermenting organisms, increases production of a fermentation product and/or decreases the residual solids compared to processes that lack the GH5 xylanase.

In an embodiment, the xylanase is a GH5 family xylanase.

In an embodiment, the xylanase is a GH5_21 xylanase.

Exemplary GH5_21 xylanases include, without limitation, ones from the genus Bacteroides, Belliella, Chryseobacterium, or Sphingobacterium.

Exemplary GH5_21 xylanases include, without limitation, ones from the species Bacteroides cellulosilyticus CL02Y12C19, Belliella sp-64282, Chryseobacterium sp., Chryseobacterium oncorhynchi, or Sphingobacterium sp-64162.

Exemplary GH5_21 xylanases include, without limitation, ones from bioreactor metagenome, Elephant dung metagenome, Xanthan alkaline community O, Xanthan alkaline community S, or Xanthan alkaline community T.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 1. In an embodiment, 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.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 2. In an embodiment, 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: 2 and has xylanase activity.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 3. 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 to the amino acid sequence of SEQ ID NO: 3 and has xylanase activity.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 4. 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 to the amino acid sequence of SEQ ID NO: 4 and has xylanase activity.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 5. 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 to the amino acid sequence of SEQ ID NO: 5 and has xylanase activity.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 6. 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 to the amino acid sequence of SEQ ID NO: 6 and has xylanase activity.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 7. 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 to the amino acid sequence of of SEQ ID NO: 7 and has xylanase activity.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 8. 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 to the amino acid sequence of SEQ ID NO: 8 and has xylanase activity.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 9. 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 to the amino acid sequence of of SEQ ID NO: 9 and has xylanase activity.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 10. 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 to the amino acid sequence of SEQ ID NO:

10 and has xylanase activity.

An exemplary GH5_21 xylanase has the amino acid sequence of SEQ ID NO: 11. 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 to the amino acid sequence of SEQ ID NO:

11 and has xylanase activity.

An exemplary GH5_21 xylanase has 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 to the amino acid sequence of SEQ ID NO:

12 and has xylanase activity.

An exemplary GH5_21 xylanase has 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 to the amino acid sequence of SEQ ID NO:

13 and has xylanase activity.

An exemplary GH5_21 xylanase has 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 to to the amino acid sequence of SEQ ID NO: 14 and has xylanase activity.

An exemplary GH5_21 xylanase has 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 to the amino acid sequence of SEQ ID NO: 15 and has xylanase activity.

The GH5 xylanase (e.g., GH5_21 xylanase) may be dosed in pre-saccharification, saccharification, and/or simultaneous saccharification and fermentation in a concentration of between 0.0001-1 mg EP (Enzyme Protein)/g DS, e.g., 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.

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

III. Backend or downstream processing

A. Recovery of the fermentation product and production of whole stillage

Subsequent to fermentation or SSF, the fermentation product may be separated from the fermentation medium. The fermentation product, e.g., ethanol, can optionally be recovered from the fermentation medium using any method known in the art including, but not limited to, chromatography, electrophoretic procedures, differential solubility, distillation, or extraction. For example, alcohol is separated from the fermented starch-containing grain and purified by conventional methods of distillation.

Thus, in one embodiment, the method of the invention further comprises distillation to obtain the fermentation product, e.g., ethanol. The fermentation and the distillation may be carried out simultaneously and/or separately/sequentially; optionally followed by one or more process steps for further refinement of the fermentation product. Following the completion of the distillation process, the material remaining is considered the whole stillage.

As another example, the desired fermentation product may be extracted from the fermentation medium by micro 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 spirits, or industrial ethanol.

In some embodiments of the methods, the fermentation product after being recovered is substantially pure. With respect to the methods herein, "substantially pure" intends a recovered preparation that contains no more than 15% impurity, wherein impurity intends compounds other than the fermentation product (e.g., ethanol). In one variation, a substantially pure preparation is provided wherein the preparation contains no more than 25% impurity, or no more than 20% impurity, or no more than 10% impurity, or no more than 5% impurity, or no more than 3% impurity, or no more than 1% impurity, or no more than 0.5% impurity.

Suitable assays to test for the production of ethanol and contaminants, and sugar consumption can be performed using methods known in the art. For example, ethanol product, as well as other organic compounds, can be analyzed by methods such as HPLC (High Performance Liquid Chromatography), GC-MS (Gas Chromatography Mass Spectroscopy) and LC-MS (Liquid Chromatography-Mass Spectroscopy) or other suitable analytical methods using routine procedures well known in the art. The release of ethanol in the fermentation broth can also be tested with the culture supernatant. Byproducts and residual sugar in the fermentation medium (e.g., glucose or xylose) can be quantified by HPLC using, for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids (Lin et al., Biotechnol. Bioeng. 90:775 -779 (2005)), or using other suitable assay and detection methods well known in the art.

B. Separating (Dewatering) Whole Stillage into Thin Stillage and Wet Cake

In one embodiment, the whole stillage is separated or partitioned into a solid and liquid phase by one or more methods for separating the thin stillage from the wet cake. Separating whole stillage into thin stillage and wet cake to remove a significant portion of the liquid/water, may be done using any suitable separation technique, including centrifugation, pressing and filtration. In a preferred embodiment, the separation/dewatering is carried out by centrifugation. Preferred centrifuges in industry are decanter type centrifuges, preferably high-speed decanter type centrifuges. An example of a suitable centrifuge is the NX 400 steep cone series from Alfa Laval which is a high-performance decanter. In another preferred embodiment, the separation is carried out using other conventional separation equipment such as a plate/frame filter presses, belt filter presses, screw presses, gravity thickeners and deckers, or similar equipment.

C. Processing of Thin Stillage

Thin stillage is the term used for the supernatant of the centrifugation of the whole stillage. Typically, the thin stillage contains 4-6 percent dry solids (DS) (mainly proteins, soluble fiber, fine fibers, and cell wall components) and has a temperature of about 60-90 degrees centigrade. The thin stillage stream may be condensed by evaporation to provide two process streams including: (i) an evaporator condensate stream comprising condensed water removed from the thin stillage during evaporation, and (ii) a syrup stream, comprising a more concentrated stream of the non-volatile dissolved and non-dissolved solids, such as non-fermentable sugars and oil, remaining present from the thin stillage as the result of removing the evaporated water.

Optionally, oil can be removed from the thin stillage or can be removed as an intermediate step to the evaporation process, which is typically carried out using a series of several evaporation stages.

Syrup and/or de-oiled syrup may be introduced into a dryer together with the wet grains (from the whole stillage separation step) to provide a product referred to as distillers dried grain with solubles, which also can be used as animal feed. In an embodiment, syrup and/or de-oiled syrup is sprayed into one or more dryers to combine the syrup and/or deoiled syrup with the whole stillage to produce distillers dried grain with solubles.

Between 5-90 vol-%, such as between 10-80%, such as between 15-70%, such as between 20-60% of thin stillage (e.g., optionally hydrolyzed) may be recycled (as backset) to step (a). The recycled thin stillage (i.e. , backset) may constitute from about 1-70 vol.-%, preferably 15-60% vol.-%, especially from about 30 to 50 vol.-% of the slurry formed in step (a). In an embodiment, the process further comprises recycling at least a portion of the thin stillage stream to the slurry, optionally after oil has been extracted from the thin stillage stream.

D. Drying of Wet Cake and Producing Distillers Dried Grains and Distillers Dried Grains with Solubles

After the wet cake, containing about 25-40 wt-%, preferably 30-38 wt-% dry solids, has been separated from the thin stillage (e.g., dewatered) it may be dried in a drum dryer, spray dryer, ring drier, fluid bed drier or the like in order to produce “Distillers Dried Grains” (DDG). DDG is a valuable feed ingredient for animals, such as livestock, poultry and fish. It is preferred to provide DDG with a content of less than about 10-12 wt.-% moisture to avoid mold and microbial breakdown and increase the shelf life. Further, high moisture content also makes it more expensive to transport DDG. The wet cake is preferably dried under conditions that do not denature proteins in the wet cake. The wet cake may be blended with syrup separated from the thin stillage and dried into DDG with Solubles (DDGS). Partially dried intermediate products, such as are sometimes referred to as modified wet distillers grains, may be produced by partially drying wet cake, optionally with the addition of syrup before, during or after the drying process.

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

Materials & Methods

Yeast strain MEJI797 is MBG5012 of WO2019/161227 further expressing a Pycnopous sanguineus glucoamylase (SEQ ID NO: 4 of WO2011/066576) and a hybrid Rhizomucor pusillus alpha amylase expression cassette (as described in WO2013/006756).

Liquefaction Enzyme Blend: exemplary thermostable alpha-amylase from Bacillus stearothermophilus disclosed in SEQ ID NO: 19; exemplary thermostable protease from Pyrococcus furiosus disclosed in SEQ ID NO: 20.

Saccharification Enzyme Blend: exemplary glucoamylase from Gloeophyllum sepiarium disclosed in SEQ ID NO: 22; exemplary alpha-amylase from Rhizomucor pusillus disclosed in SEQ ID NO: 23.

Cellulase Blend: exemplary beta-glucosidase from Aspergillus fumigatus disclosed in SEQ ID NO: 24; exemplary celliobiohydrolase from Aspergillus fumigatus disclosed in SEQ ID NO: 25; exemplary endoglucanase from Trichoderma reesei disclosed in SEQ ID NO: 26.

EXAMPLES Example 1 : Construction of yeast strains expressing a CBH1 and CBH2

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

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

The first linear DNA containing 500 bp homology to the X-4 site and the S. cerevisiae pSeTDH3 promoter was PCR amplified from HP97 plasmid DNA (Figure 3). Fifty pmoles each of forward and reverse primer was used in a PCR reaction containing 5 ng of plasmid DNA as template, 1X Platinum SuperFi HF Buffer (Thermo Fisher Scienctific), and 2 units SuperFi DNA polymerase in a final volume of 50 pL. The PCR was performed in a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc. Following thermocycling, the PCR reaction products gel isolated and cleaned up using the QIAguick Gel Extraction kit (Qiagen).

A second fragment (TL4) containing the pSeTDH3 promoter, AGA2 signal peptide, P244YG gene, and tPDC6 terminator was PCR amplified from a Saccharomyces cerevisiae yeast strain S1130-D03. Fifty pmoles each of forward and reverse primer was used in a PCR reaction containing 5 ng of gDNA as template, 1X Platinum SuperFi HF Buffer (Thermo Fisher Scienctific), and 2 units SuperFi DNA polymerase in a final volume of 50 pL. The PCR was performed in a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc.). Following thermocycling, the PCR reaction products gel isolated and cleaned up using the QIAguick Gel Extraction kit (Qiagen).

The two fragments described above were combined in an SOE PCR. Fifty pmoles each of forward and reverse primer was used in a PCR reaction containing 5 ng of each of the above DNA fragments as template, 1X Platinum SuperFi HF Buffer (Thermo Fisher Scienctific), and 2 units SuperFi DNA polymerase in a final volume of 50 pL. The PCR was performed in a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc.). Following thermocycling, the PCR reaction products gel isolated and cleaned up using the QIAguick Gel Extraction kit (Qiagen). This final piece was designated RRSOE1. Construction of the fragment containing tPDC6, pPGK1, P43VY6, and homology to tADH3 (middle fragment 1)

A first linear DNA containing terminator tPDC6 and promoter pPGK1 was PCR amplified from TP40 plasmid DNA (Figure 4). Fifty pmoles each of forward and reverse primer was used in a PCR reaction containing 5 ng of plasmid DNA as template, 1X Platinum SuperFi HF Buffer (Thermo Fisher Scienctific), and 2 units SuperFi DNA polymerase in a final volume of 50 pL. The PCR was performed in a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc. Following thermocycling, the PCR reaction products gel isolated and cleaned up using the QIAquick Gel Extraction kit (Qiagen).

A second fragment called TL7 containing 50 bp homology to the pPGK1 promoter, AGA2 signal peptide, Talaromyces verruculosus CBH2 gene, and 50 bp homology to the tADH3 terminator was PCR amplified from yeast strain S1130-B11 (See, PCT/CN2022/102201, filed June 29, 2022, the contents of which are incorporated by reference). Fifty pmoles each of forward and reverse primer was used in a PCR reaction containing 5 ng of gDNA as template, 1X Platinum SuperFi HF Buffer (Thermo Fisher Scienctific), and 2 units SuperFi DNA polymerase in a final volume of 50 pL. The PCR was performed in a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc.). Following thermocycling, the PCR reaction products gel isolated and cleaned using the QIAquick Gel Extraction kit (Qiagen).

The two fragments described above were combined in an SOE PCR. Fifty pmoles each of forward and reverse primer was used in a PCR reaction containing 5 ng of each of the above DNA fragments as template, 1X Platinum SuperFi HF Buffer (Thermo Fisher Scienctific), and 2 units SuperFi DNA polymerase in a final volume of 50 pL. The PCR was performed in a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc.). Following thermocycling, the PCR reaction products gel isolated and cleaned up using the QIAquick Gel Extraction kit (Qiagen). This final piece was designated SOE4.

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

The linear DNA containing 500 bp homology to the X-4 site and the S. cerevisiae tADH3 terminator was PCR amplified from TH58 plasmid DNA (Figure 5). Fifty pmoles each of forward and reverse primer was used in a PCR reaction containing 5 ng of plasmid DNA as template, 1X Platinum SuperFi HF Buffer (Thermo Fisher Scienctific), and 2 units SuperFi DNA polymerase in a final volume of 50 pL. The PCR was performed in a T100™ Thermal Cycler (Bio-Rad Laboratories, Inc.). Following thermocycling, the PCR reaction products gel isolated and cleaned up using the QIAquick Gel Extraction kit (Qiagen).

Integration of the left, middle and right-hand fragments to generate yeast strain YS103-A07 The yeast MeJi797 was transformed with the left (RRSOE1), middle (SOE4) and right (TH58) integration fragments using 150ng of each fragment. To aid homologous recombination of the left, middle and right fragments at the genomic X-4 site, a 300ng of a plasmid containing MAD7 and guide RNA specific to X-4 (pMIBa789; Figure 6) was also used in the transformation. The three linear DNA fragments were combined and transformed into MeJi797 following a yeast electroporation protocol. Transformants were selected on YPD+cloNAT to select for transformants that contain the Mad7 plasmid pMIBa789. Transformants were either picked manually by hand onto YPD plates or by using a Q-pix Colony Picking System (Molecular Devices) to inoculate 1 well of 96-well plate containing YPD media. The plates were grown for 2 days then glycerol was added to 20% final concentration and the plates were stored at -80°C until needed. Integration of the CBH1+CBH2 construct was verified by PCR with locus specific primers and subsequent sequencing.

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

Yeast strains yeast MeJi797 and YS103-A07 (supra) were incubated overnight in 20 mL YPD media (6% w/v D-glucose, 2% peptone, 1% yeast extract) in 125 ml baffled shake flasks at 32°C and 150 rpm. Cells were harvested after 24 hours incubation and collected by centrifugation and washed in DI water prior to resuspending in 5 mL DI water for dosing. Industrially obtained liquefied corn mash, where liquefaction was carried out using the Liquefaction Enzyme Blend supplemented with 3 ppm penicillin and 1000 ppm of urea. Simultaneous saccharification and fermentation (SSF) was performed via mini-scale fermentations. Approximately 4 g of corn mash was added to 12 mL conical tubes. Each tube was dosed with 1 x 10⁷ cells/g of mash with yeast followed by the addition of 0.42 AGU/g of dry solids of an exogenous Saccharification Enzyme Blend. In certain instances, tubes were dosed with a GH5_21 xylanase (2.5 ugEP/g DS; SEQ ID NO: 21) and a Cellulase Blend (67.5 ugEP/g DS). Six replicate tube fermentations were conducted for each treatment. The enzyme blend and yeast dosages were administered based on the exact weight of corn slurry in each vial. Tubes were incubated at 32°C and mixed two times per day via brief vortex. After 68 hours fermentation time, contents of the tubes were diluted 10x and then centrifuged @3500 rpm for 5 min. Supernatant samples were filtered with 0.2 mm syringe filters into vials for analysis of final ethanol level via HPLC. The remaining supernatant was discarded, and the pellet was dried for 3 days at 50°C. The final pellet was weighed to determine residual solids. The final ethanol level results are shown in Figure 1. Yeast strain YS103-A07 expressing CBH1 and CBH2 had significantly higher ethanol yield as compared to control strain MeJi797.

The residual solids results are shown in Figure 2. Yeast strain YS103-A07 expressing CBH1 and CBH2 showed significantly lower residual solids as compared to control strain MeJi797.

The invention is further defined by the following numbered paragraphs:

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

(a) liquefying the starch-containing material at a temperature above the initial gelatinization temperature using an alpha-amylase;

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

(c) fermenting saccharified starch-containing material using a fermenting organism; 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 a CBH1 and a heterologous polynucleotide encoding a CBH2.

Paragraph [2], The process of paragraph [1], wherein the GH5_21 xylanase is present or added during saccharifying step (b).

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

Paragraph [4], The process of any of the preceding paragraphs, wherein steps (b) and (c) are performed simultaneously in a simultaneous saccharification and fermentation (SSF).

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

Paragraph [6], The process of any of the preceding paragraphs, wherein the GH5_21 xylanase used in saccharifying step (b) and/or fermenting step (c) is present or added via in situ expression from the fermenting organism. Paragraph [7], The process of any of the preceding paragraphs, wherein the alpha-amylase has the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence that is 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: 18, which has alpha-amylase activity.

Paragraph [8], The process of any of the preceding paragraphs, wherein the alpha-amylase has the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence that is 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: 19, which has alpha-amylase activity.

Paragraph [9], The process of any of the preceding paragraphs, wherein a thermostable endoglucanase is added during liquefying step (a).

Paragraph [10], The process of any of the preceding paragraphs, wherein a thermostable lipase is added during liquefying step (a).

Paragraph [11], The process of any of the preceding paragraphs, wherein a thermostable phytase is added during liquefying step (a).

Paragraph [12]. The process of any of the preceding paragraphs, wherein a thermostable protease is added during liquefying step (a).

Paragraph [13], The process of claim 12, wherein the thermostable protease has the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence that is 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: 20, which has protease activity.

Paragraph [14], The process of any of the preceding paragraphs, wherein a thermostable pullulanase is added during liquefying step (a).

Paragraph [15], The process of any of the preceding paragraphs, wherein a thermostable xylanase is added during liquefying step (a). Paragraph [16], The process of claim 15, wherein the thermostable xylanase has an amino acid sequence of SEQ ID NO: 21 or an amino acid sequence that is 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: 21, which has xylanase activity.

Paragraph [17], The process of any of the preceding paragraphs, wherein a thermostable alpha-amylase, a thermostable protease and a thermostable xylanase are added during liquefying step (a).

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

Paragraph [19], The process of paragraph [18], wherein the glucoamylase has an amino acid sequence of SEQ ID NO: 22 or an amino acid sequence that is 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: 22, which has glucoamylase activity.

Paragraph [20], The process of any of the preceding paragraphs, wherein an alpha-amylase is added during step (b) and/or step (c).

Paragraph [21], The process of paragraph [20], wherein the alpha-amylase has an amino acid sequence of SEQ ID NO: 23 or an amino acid sequence that is 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: 23, which has alpha-amylase activity.

Paragraph [22], The process of any of the preceding paragraphs, wherein a betaglucosidase is added during step (a) and/or step (b).

Paragraph [23], The process of paragraph [22], wherein the beta-glucosidase has an amino acid sequence of SEQ ID NO: 24 or an amino acid sequence that is 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: 24, which has beta-glucosidase activity. Paragraph [24]. The process of any of the preceding paragraphs, wherein a cellobiohydrolase is added during step (b) and/or step (c).

Paragraph [25], The process of paragraph [24], wherein the cellobiohydrolase has an amino acid sequence of SEQ ID NO: 25 or an amino acid sequence that is 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: 25, which has cellobiohydrolase activity.

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

Paragraph [27], The process of paragraph [26], wherein the endoglucanase has an amino acid sequence of SEQ ID NO: 26 or an amino acid sequence that is 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: 26, which has endoglucanase activity.

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

Paragraph [29], The process of paragraph [28], wherein the trehalase has an amino acid sequence of SEQ ID NO: 27 or an amino acid sequence that is 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: 27, which has trehalase activity.

Paragraph [30], A process for producing a fermentation product from an ungelatinized starch-containging grain comprises the following steps:

(a) saccharifying a starch-containing grain at a temperature below the initial gelatinization temperature using a glucoamylase and an alpha-amylase to produce a fermentable sugar; and

(b) fermenting the sugar using a fermenting organism to produce a fermentation product; 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 a CBH1 and a heterologous polynucleotide encoding a CBH2.

Paragraph [31], The process of paragraph [30], wherein the GH5_21 xylanase is present or added during saccharifying step (a).

Paragraph [32], The process of paragraph [30], wherein the GH5_21 xylanase is present or added during fermenting step (b).

Paragraph [33], The process of paragraph [30], wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation (SSF).

Paragraph [34], The process of paragraph [33], wherein the GH5_21 xylanase is present or added during SSF.

Paragraph [35], The process of paragraph [30], wherein the GH5_21 xylanase used in saccharifying step (a) and/or fermenting step (b) is present or added via in situ expression from the fermenting organism.

Paragraph [36], The process of any of the preceding paragraphs, wherein the at least one 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 one 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: 1 ;

(2) the amino acid sequence of SEQ ID NO: 2, or one 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: 2;

(3) the amino acid sequence of SEQ ID NO: 3, or one 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: 3; (4) the amino acid sequence of SEQ ID NO: 4, or one 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: 4;

(5) the amino acid sequence of SEQ ID NO: 5, or one 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: 5;

(6) the amino acid sequence of SEQ ID NO: 6, or one 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: 6;

(7) the amino acid sequence of SEQ ID NO: 7, or one 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: 7;

(8) the amino acid sequence of SEQ ID NO: 8, or one 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: 8;

(9) the amino acid sequence of SEQ ID NO: 9, or one 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 one 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 one 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 I D NO: 11 ;

(12) the amino acid sequence of SEQ ID NO: 12, or one 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 one 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 one 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 one 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.

Paragraph [37], The process of any of the preceding paragraphs, wherein the at least one the at least one GH5_21 comprises or consists 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.

Paragraph [38], The process of any of the preceding paragraphs, wherein the at least one GH5_21 is a variant of the 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 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.

Paragraph [39], The process of any 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, wherein the fragment has GH5_21 activity.

Paragraph [40], The process of any of the preceding paragraphs, wherein the at least one GH5_21 dosed in the range of 0.0001-1 mg EP (Enzyme Protein)/g DS, e.g., 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. Paragraph [41], The process of any of the preceding paragraphs, wherein the heterologous polynucleotide encoding the CBH1 is operably linked to a promoter that is foreign to the polynucleotide.

Paragraph [42]. The process of any of the preceding paragraphs, wherein the CBH1 has the amino acid sequence of SEQ ID NO: 16, or one 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: 16.

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

Paragraph [44], The process of any of the preceding paragraphs, wherein the CBH1 is a variant of the the amino acid sequence of SEQ ID NO: 16 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.

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

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

Paragraph [47], The process of any of the preceding paragraphs, wherein the CBH2 has the amino acid sequence of SEQ ID NO: 17, or one 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: 17.

Paragraph [48], The process of any of the preceding paragraphs, wherein the CBH2 comprises or consists of the amino acid sequence of SEQ ID NO: 17. Paragraph [49], The process of any of the preceding paragraphs, wherein the CBH2 is a variant of the the amino acid sequence of SEQ ID NO: 17 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.

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

Paragraph [51], The process of any 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).

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

Paragraph [53], The process of any of the preceding paragraphs, wherein the fermenting organism comprises a heterologous polynucleotide encoding an alpha-amylase.

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

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

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

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

Paragraph [58], The process of any of the preceding paragraphs, wherein the fermenting organism is a yeast cell. Paragraph [59], The process of any of the preceding paragraphs, wherein the fermenting organism is a Saccharomyces, Rhodotorula, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Rhodosporidium, Candida, Yarrowia, Lipomyces, Cryptococcus, or Dekkera sp. cell.

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

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

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

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

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

Paragraph [65], The recombinant host cell of paragraph [64], wherein the heterologous polynucleotide encoding the CBH1 is operably linked to a promoter that is foreign to the polynucleotide.

Paragraph [66], The recombinant host cell of paragraph [64] or [65], wherein the CBH1 has the amino acid sequence of SEQ ID NO: 16, or one 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: 16.

Paragraph [67], The recombinant host cell of any of paragraphs [64]-[66], wherein the CBH1 comprises or consists of the amino acid sequence of SEQ ID NO: 16. Paragraph [68], The recombinant host cell of any of paragraphs [64]-[67], wherein the CBH1 is a variant of the the amino acid sequence of SEQ ID NO: 16 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.

Paragraph [69], The recombinant host cell of any of paragraphs [64]-[68], wherein the CBH1 is a fragment of the amino acid sequence of SEQ ID NO: 16, wherein the fragment has CBH1 activity.

Paragraph [70], The recombinant host cell of any of paragraphs [64]-[69], wherein the heterologous polynucleotide encoding the CBH2 is operably linked to a promoter that is foreign to the polynucleotide.

Paragraph [71], The recombinant host cell of any of paragraphs [64]-[70], wherein the CBH2 has the amino acid sequence of SEQ ID NO: 17, or one 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: 17.

Paragraph [72], The recombinant host cell of any of paragraphs [64]-[71 ], wherein the CBH2 comprises or consists of the amino acid sequence of SEQ ID NO: 17.

Paragraph [73], The recombinant host cell of any of paragraphs [64]-[72], wherein the CBH2 is a variant of the the amino acid sequence of SEQ ID NO: 17 comprising a substitution, deletion, and/or insertion at one or more (e.g., several) positions.

Paragraph [74], The recombinant host cell of any of paragraphs [64]-[73], wherein the CBH2 is a fragment of the amino acid sequence of SEQ ID NO: 17, wherein the fragment has CBH2 activity.

Paragraph [75], The recombinant host cell of any 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).

Paragraph [76], The recombinant host cell of any of paragraphs [64]-[75], wherein the fermenting organism comprises a heterologous polynucleotide encoding a glucoamylase. Paragraph [77] . The recombinant host cell of any of paragraphs [64]-[76], wherein the fermenting organism comprises a heterologous polynucleotide encoding an alpha-amylase.

Paragraph [78], The recombinant host cell of any of paragraphs [64]-[77], wherein the fermenting organism comprises a heterologous polynucleotide encoding a protease.

Paragraph [79], The recombinant host cell of any of paragraphs [64]-[78], wherein the fermenting organism comprises a heterologous polynucleotide encoding the GH5_21 xylanase.

Paragraph [80], The recombinant host cell of any of paragraphs [64]-[79], wherein the fermenting organism comprises a disruption to an endogenous gene encoding a glycerol 3- phosphate dehydrogenase (GPD).

Paragraph [81], The recombinant host cell of any of paragraphs [64]-[80], wherein the fermenting organism comprises a disruption to an endogenous gene encoding a glycerol 3- phosphatase (GPP).

Paragraph [82], The recombinant host cell of any of paragraphs [64]-[81 ], wherein the fermenting organism is a yeast cell.

Paragraph [83], The recombinant host cell of any of paragraphs [64]-[82], wherein the fermenting organism is a Saccharomyces, Rhodotorula, Schizosaccharomyces, Kluyveromyces, Pichia, Hansenula, Rhodosporidium, Candida, Yarrowia, Lipomyces, Cryptococcus, or Dekkera sp. cell.

Paragraph [84], The recombinant host cell of any of paragraphs [64]-[83], wherein the fermenting organism is a Saccharomyces cerevisiae cell.

Claims

CLAIMS What is claimed is:

1. A process for producing a fermentation product from starch-containing material comprising the steps of:

(a) liquefying the starch-containing material at a temperature above the initial gelatinization temperature using an alpha-amylase;

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

(c) fermenting saccharified starch-containing material using a fermenting organism; 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 a CBH1 and a heterologous polynucleotide encoding a CBH2.

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

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

4. The process of any of the preceding claims, wherein steps (b) and (c) are performed simultaneously in a simultaneous saccharification and fermentation (SSF).

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

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

7. A process for producing a fermentation product from an ungelatinized starch-containging grain comprises the following steps: (a) saccharifying a starch-containing grain at a temperature below the initial gelatinization temperature using a glucoamylase and an alpha-amylase to produce a fermentable sugar; and

(b) fermenting the sugar using a fermenting organism to produce a fermentation product; 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 a CBH1 and a heterologous polynucleotide encoding a CBH2.

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

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

10. The process of claim 7, wherein steps (a) and (b) are performed simultaneously in a simultaneous saccharification and fermentation (SSF).

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

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

13. The process of any of the preceding claims, wherein the at least one 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 one 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: 1;

(2) the amino acid sequence of SEQ ID NO: 2, or one 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: 2; (3) the amino acid sequence of SEQ ID NO: 3, or one 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: 3;

(4) the amino acid sequence of SEQ ID NO: 4, or one 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: 4;

(5) the amino acid sequence of SEQ ID NO: 5, or one 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: 5;

(6) the amino acid sequence of SEQ ID NO: 6, or one 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: 6;

(7) the amino acid sequence of SEQ ID NO: 7, or one 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: 7;

(8) the amino acid sequence of SEQ ID NO: 8, or one 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: 8;

(9) the amino acid sequence of SEQ ID NO: 9, or one 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 one 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 one 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 I D NO: 11 ; (12) the amino acid sequence of SEQ ID NO: 12, or one 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 one 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 one 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 one 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 of any of the preceding claims, wherein the CBH1 has the amino acid sequence of SEQ ID NO: 16, or one 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: 16.

15. The process of any of the preceding claims, wherein the CBH2 has the amino acid sequence of SEQ ID NO: 17, or one 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: 17.

16. The process of any of the preceding claims, wherein the 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 of any of the preceding claims, wherein the fermenting organism comprises a heterologous polynucleotide encoding the GH5_21 xylanase.

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

19. The process of claim 18, wherein the fermenting organism is a Saccharomyces cerevisiae cell.

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

21. A recombinant host cell comprising a heterologous polynucleotide encoding a CBH1 and a heterologous polynucleotide encoding a CBH2; wherein the CBH1 has the amino acid sequence of SEQ ID NO: 16, or one 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: 16; and/or wherein the CBH2 has the amino acid sequence of SEQ ID NO: 17, or one 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: 17.

22. The recombinant host cell of claim 21 , wherein the fermenting organism is a yeast cell.

23. The recombinant host cell of claim 22, wherein the fermenting organism is a Saccharomyces cerevisiae cell.