Improved fermentation organisms for ethanol production

Abstract

The present invention relates to a method for producing ethanol comprising saccharifying a cellulose-containing or starch-containing material, and fermenting the saccharified material with a fermenting microorganism to produce ethanol. The fermentation organism is a saccharomyces cerevisiae strain MBG5225 (preserved in the Ilinoi Zhou 61604 United States Agricultural Research Service Patent Culture Collection Center (NRRL) with the preservation number of Y-68199), and a saccharomyces cerevisiae strain MBG5227 (preserved in the Ilinoi Zhou 61604 United States Agricultural Research Service Patent Culture Collection Center (NRRL) with the preservation number of Y-68199) with the preservation number of Y-68199). The Saccharomyces cerevisiae strain MBG5307 (preserved in the Ilinoi Zhou 61604 United States Agricultural Research Service Patent Strain Preservation Center (NRRL) with the preservation number of Y-68201), the Saccharomyces cerevisiae strain MBG5318 (preserved in the Ilinoi Zhou 61604 United States Agricultural Research Service Patent Strain Preservation Center (NRRL) with the preservation number of Y-68201), the Saccharomyces cerevisiae strain MBG5318 (preserved in the Ilinoi Zhou 61604 United the preservation number is Y-68202) or a fermentation organism which has the same or approximately same properties with the saccharomyces cerevisiae MBG5225, MBG5227, MBG5307 or MBG5318.

Description

[0001] Reference to Deposit of Biological Material

[0002] This application contains a reference to a deposit of biological material, which is incorporated herein by reference. Background Art

[0003] The production of ethanol from starch-containing materials is well known in the art. The production of ethanol as a biofuel has become a major industry, with over 25 billion gallons of ethanol produced worldwide in 2021.

[0004] The most commonly used commercial method in the industry, often referred to as the "conventional method", involves liquefying gelatinized starch typically using bacterial α-amylase at high temperature (about 85°C), followed by simultaneous saccharification and fermentation (SSF) typically carried out anaerobically in the presence of glucoamylase and Saccharomyces cerevisiae.

[0005] Yeasts used for the production of ethanol as a fuel, such as in the corn ethanol industry, require several characteristics to ensure the cost-effective production of ethanol. These characteristics include ethanol tolerance, low by-product yield, rapid fermentation, and the ability to limit the amount of residual sugar remaining in the fermentation. Such characteristics have a significant impact on the viability of industrial processes.

[0006] Yeasts of the genus Saccharomyces exhibit many of the characteristics required for ethanol production. In particular, strains of Saccharomyces cerevisiae are widely used in the fuel ethanol industry for ethanol production. Strains of Saccharomyces cerevisiae are widely used in the fuel ethanol industry and can produce high yields of ethanol under the fermentation conditions found, for example, in corn mash fermentation. Examples of such strains are the yeasts used in a commercially available ethanol yeast product called ETHANOL used in the fuel ethanol industry to ferment sugars such as glucose, fructose, sucrose, and maltose to produce ethanol via the glycolytic pathway. These sugars are obtained from sources such as corn and other grains, sugar juice, molasses, grape juice, fruit juice, and starchy root vegetables, and can include the breakdown of cellulosic materials into glucose.

[0007] Strains of Saccharomyces cerevisiae are used in the fuel ethanol industry to ferment sugars such as glucose, fructose, sucrose, and maltose to produce ethanol via the glycolytic pathway. These sugars are obtained from sources such as corn and other grains, sugar juice, molasses, grape juice, fruit juice, and starchy root vegetables, and can include the breakdown of cellulosic materials into glucose.

[0008] Although the strains of Saccharomyces cerevisiae currently used in the fuel ethanol industry are well-suited for ethanol production, due to the increasing demand for ethanol as a fuel and the increasing availability of new strains of corn, there is a growing need to improve the efficiency of ethanol production.

[0009] Therefore, there is a need for new robust yeast strains of the genus Saccharomyces that can improve the efficiency of ethanol production in industrial-scale fermentations.

[0010] In addition, although there have been significant improvements in ethanol production methods over the past several decades, there remains a desire and need to provide methods for producing ethanol from starch-containing materials and yeast that can be used in commercial-scale ethanol processes. SUMMARY OF THE INVENTION

[0011] Methods for producing ethanol from cellulosic or starch-containing materials, and yeasts suitable for such methods, are particularly described herein.

[0012] A first aspect relates to a method for producing a fermentation product from a cellulosic and / or starch-containing material, the method comprising:

[0013] (a) saccharifying the cellulosic or starch-containing material; and

[0014] (b) fermenting the saccharified material of step (a) with a fermenting organism under suitable conditions to produce the fermentation product; wherein the fermenting organism is a Saccharomyces cerevisiae strain (Saccharomyces cerevisiae strain MBG5225) having a deposit number of NRRL Y-68199 or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227) or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307) or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318) or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), deposited in the Agricultural Research Service Patent Culture Collection, NRRL, under the Budapest Treaty, or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5225, Saccharomyces cerevisiae MBG5227, Saccharomyces cerevisiae MBG5307, or Saccharomyces cerevisiae strain MBG5318.

[0015] In one embodiment, the method includes recovering the fermentation product from the fermentation (e.g., by distillation).

[0016] In one embodiment, fermentation and saccharification are carried out simultaneously in simultaneous saccharification and fermentation (SSF). In one embodiment, fermentation and saccharification are carried out sequentially (SHF).

[0017] In one embodiment, the fermentation product is ethanol.

[0018] In one embodiment, step (a) includes contacting the starch- and / or cellulose-containing material with an enzyme composition.

[0019] In one embodiment, step (a) includes saccharifying a starch-containing material. In one embodiment, step (a) includes i) liquefying the starch-containing material using α-amylase at a temperature higher than the initial gelatinization temperature; and ii) saccharifying using glucoamylase.

[0020] In one embodiment, step (a) includes saccharifying a cellulose-containing material. In one embodiment, the cellulose-containing material is pretreated. In one embodiment, the cellulose-containing material comprises bagasse.

[0021] In one embodiment, step (a) includes contacting the cellulose-containing material with an enzyme composition, and wherein the enzyme composition comprises one or more enzymes selected from the group consisting of: cellulase, AA9 polypeptide, hemicellulase, CIP, esterase, expansin, lignin-degrading enzyme, oxidoreductase, pectinase, protease, and swollenin. In one embodiment, the cellulase is one or more enzymes selected from the group consisting of: endoglucanase, cellobiohydrolase, and β-glucosidase. In one embodiment, the hemicellulase is one or more enzymes selected from the group consisting of: xylanase, acetylxylan esterase, ferulic acid esterase, arabinofuranosidase, xylosidase, and glucuronidase.

[0022] In one embodiment, the method results in a fermentation product yield of at least 0.25% (e.g., 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2%, 3%, or 5%).

[0023] In one embodiment, the fermentation is carried out under low oxygen (e.g., anaerobic) conditions.

[0024] In one embodiment, the fermenting organism has one or more of the following properties:

[0025] Under the same method conditions, compared to the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the ethanol yield is increased;

[0026] Under the same method conditions, compared to the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the acetaldehyde production is reduced;

[0027] Under the same method conditions, compared to the Saccharomyces cerevisiae strain Ethanol Compared with (deposited at the National Measurement Institute of Victoria, Australia, deposit number V14 / 007039), the temperature tolerance is increased;

[0028] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol Compared with (deposited at the National Measurement Institute of Victoria, Australia, deposit number V14 / 007039), the production of maltose (DP2) is reduced;

[0029] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol Compared with (deposited at the National Measurement Institute of Victoria, Australia, deposit number V14 / 007039), the production of glycerol is reduced.

[0030] The second aspect relates to a Saccharomyces cerevisiae strain (Saccharomyces cerevisiae strain MBG5225) or its derivatives (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase) deposited at the Patent Culture Depository of the Agricultural Research Service, U.S. (NRRL) under the Budapest Treaty with the deposit number NRRL Y-68199, NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227) or its derivatives (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307) or its derivatives (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318) or its derivatives (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5225, Saccharomyces cerevisiae MBG5227, Saccharomyces cerevisiae MBG5307 or Saccharomyces cerevisiae strain MBG5318.

[0031] In one embodiment, the strain has one or more of the following properties:

[0032] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol Compared with (deposited at the National Measurement Institute of Victoria, Australia, deposit number V14 / 007039), the ethanol yield is increased;

[0033] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol Compared with (deposited at the National Measurement Institute of Victoria, Australia, deposit number V14 / 007039), the production of acetaldehyde is reduced;

[0034] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol Compared with the deposit (deposit number: V14 / 007039) at the National Measurement Institute of Victoria, Australia, the temperature tolerance is increased;

[0035] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol Compared with the deposit (deposit number: V14 / 007039) at the National Measurement Institute of Victoria, Australia, the production of maltose (DP2) is reduced;

[0036] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol Compared with the deposit (deposit number: V14 / 007039) at the National Measurement Institute of Victoria, Australia, the production of glycerol is reduced.

[0037] A third aspect relates to a method for generating derivatives of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318), the method comprising: (a) culturing a first yeast strain with a second yeast strain under conditions that permit DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318) or a derivative thereof; and (b) isolating the hybrid strain; and (c) optionally repeating steps (a) and (b) using the hybrid strain isolated in step (b) as the first yeast strain and / or the second yeast strain.

[0038] A fourth aspect relates to a method of generating a derivative of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225) that exhibits the defined characteristics of Saccharomyces cerevisiae strain MBG5225, NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227) that exhibits the defined characteristics of Saccharomyces cerevisiae strain MBG5227, NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307) that exhibits the defined characteristics of Saccharomyces cerevisiae strain MBG5307, or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318) that exhibits the defined characteristics of Saccharomyces cerevisiae strain MBG5318, the method comprising: (a) providing: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307) or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318) or a derivative thereof; (b) culturing the first yeast strain and the second yeast strain under conditions that permit DNA combination between the first yeast strain and the second yeast strain; and (c) screening or selecting a derivative of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307) or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318).

[0039] In one embodiment, step (c) comprises screening or selecting a hybrid strain that exhibits one or more defined characteristics of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307) or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318). In one embodiment, the method further comprises the step of: (d) repeating steps (a) and (b) using the strain screened or selected in step (c) as the first strain and / or the second strain until a derivative is obtained that exhibits the defined characteristics of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307) or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318).

[0040] In one embodiment, the culturing step (b) comprises: (i) causing the first yeast strain and the second yeast strain to form spores; (ii) hybridizing the germinated spores produced by the first yeast strain with the germinated spores produced by the second yeast strain.

[0041] A fifth aspect relates to a method for producing recombinant derivatives of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318), the method comprising: (a) transforming NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318) or derivatives thereof with one or more expression vectors (e.g., one or more expression vectors encoding glucoamylase and / or α-amylase); and (b) isolating the transformed strain.

[0042] A sixth aspect relates to a Saccharomyces cerevisiae strain produced by any one of the third, fourth, or fifth aspects.

[0043] A seventh aspect relates to a method for producing ethanol, the method comprising incubating a yeast genus strain of the second or sixth aspect with a substrate comprising fermentable sugars under conditions permitting fermentation of the fermentable sugars to produce ethanol.

[0044] An eighth aspect relates to a composition comprising any Saccharomyces cerevisiae strain of the second or sixth aspect and one or more natural and / or non-natural components.

[0045] In one embodiment, the components are selected from the group consisting of surfactants, emulsifiers, gums, swelling agents, and antioxidants.

[0046] In one embodiment, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5225 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection, 1815 University Street, Peoria, IL, USA 61604, under accession number NRRL Y-68199).

[0047] In one embodiment, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5227 (deposited at the Northern Regional Research Center (NRRL) of the Patent Culture Collection Center of the United States Department of Agriculture, 1815 University Street, Peoria, Illinois, USA, with the accession number NRRL Y-68200).

[0048] In one embodiment, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5307 (deposited at the Northern Regional Research Center (NRRL) of the Patent Culture Collection Center of the United States Department of Agriculture, 1815 University Street, Peoria, Illinois, USA, with the accession number NRRL Y-68201).

[0049] In one embodiment, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5318 (deposited at the Northern Regional Research Center (NRRL) of the Patent Culture Collection Center of the United States Department of Agriculture, 1815 University Street, Peoria, Illinois, USA, with the accession number NRRL Y-68202).

[0050] In one embodiment, the Saccharomyces cerevisiae strain is in a viable state, particularly in a dry, paste or compressed state. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 Shows that under non-stress conditions, compared with Ethanol the ethanol yield from fermentation by Saccharomyces cerevisiae strain MBG5225 is increased.

[0052] Figure 2 Shows that compared with Ethanol the glycerol production from fermentation by Saccharomyces cerevisiae strain MBG5225 is decreased.

[0053] Figure 3 Shows that under non-stress conditions, compared with Ethanol the ethanol yield from fermentation by Saccharomyces cerevisiae strain MBG5227 is increased.

[0054] Figure 4 Shows that compared with Ethanol the glycerol production from fermentation by Saccharomyces cerevisiae strain MBG5227 is decreased.

[0055] Figure 5 Shows that under non-stress conditions, compared with Ethanol the ethanol yield from fermentation by Saccharomyces cerevisiae strain MBG5318 is increased.

[0056] Figure 6 Shows that compared with Ethanol Compared with [reference], the glycerol production from the fermentation of Saccharomyces cerevisiae strain MBG5318 is reduced.

[0057] Figure 7 Shows that under non-stress conditions, compared with Ethanol the ethanol yield from the fermentation of Saccharomyces cerevisiae strain MBG5307 is increased.

[0058] Figure 8 Shows that compared with Ethanol the glycerol production from the fermentation of Saccharomyces cerevisiae strain MBG5307 is reduced.

[0059] Figure 9 Shows that under temperature and organic stress conditions, compared with Ethanol the ethanol yield from the fermentation of Saccharomyces cerevisiae strain MBG5225 is increased.

[0060] Figure 10 Shows that under temperature and organic stress conditions, compared with Ethanol the ethanol yield from the fermentation of Saccharomyces cerevisiae strain MBG5227 is increased.

[0061] Figure 11 Shows that under temperature and organic stress conditions, compared with Ethanol the ethanol yield from the fermentation of Saccharomyces cerevisiae strain MBG5318 is increased.

[0062] Definition

[0063] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0064] Allelic variant: The term "allelic variant" means any one of two or more alternative forms of a gene that occupy the same chromosomal locus. Allelic variation occurs naturally through mutation and can result in polymorphism within a population. A gene mutation can be silent (no change in the encoded polypeptide) or can encode a polypeptide with an altered amino acid sequence. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of the gene.

[0065] α-Amylase: The term "α-amylase" means 1,4-α-D-glucan glucanohydrolase (EC.3.2.1.1), which catalyzes the hydrolysis of starch and other linear and branched 1,4-glycosidic oligosaccharides and polysaccharides. α-Amylase activity can be determined using methods known in the art (e.g., the α-amylase assay described in WO2020 / 023411).

[0066] Auxiliary Activity 9: The term "Auxiliary Activity 9" or "AA9" means a polypeptide classified as a lytic polysaccharide monooxygenase (Quinlan et al., 2011, Proc. Natl. Acad. Sci. USA 208:15079-15084; Phillips et al., 2011, ACS Chem. Biol. 6:1399-1406; Lin et al., 2012, Structure 20:1051-1061). According to Henrissat, 1991, Biochem. J. 280:309-316 and Henrissat and Bairoch, 1996, Biochem. J. 316:695-696, AA9 polypeptides were previously classified as glycoside hydrolase family 61 (GH61).

[0067] AA9 polypeptides enhance the hydrolysis of cellulose-containing materials by enzymes with cellulolytic activity. The cellulolytic enhancement activity can be determined by measuring the increase in reducing sugars or the increase in the total amount of cellobiose and glucose resulting from the hydrolysis of cellulose-containing materials by cellulolytic enzymes under the following conditions: 1-50 mg total protein / g cellulose in pretreated corn stover (PCS), where the total protein includes 50%-99.5% w / w cellulolytic enzyme protein and 0.5%-50% w / w AA9 polypeptide protein, at a suitable temperature (e.g., 40°C-80°C, e.g., 50°C, 55°C, 60°C, 65°C, or 70°C), and a suitable pH (e.g., 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5) for 1-7 days, compared to a control hydrolysis with an equal total protein load without cellulolytic enhancement activity (1-50 mg cellulolytic protein / g cellulose in PCS).

[0068] One can use a mixture of 1.5 L (Novozymes A / S, Bagsværd Denmark) and β-glucosidase as a source of cellulolytic activity to assay the AA9 polypeptide enhancement activity, where the β-glucosidase is present at a weight of at least 2%-5% of the cellulase protein load. In one embodiment, the β-glucosidase is Aspergillus oryzae β-glucosidase (e.g., recombinantly produced in Aspergillus oryzae according to WO02 / 095014). In another embodiment, the β-glucosidase is Aspergillus fumigatus β-glucosidase (e.g., as described in WO 02 / 095014, recombinantly produced in Aspergillus oryzae).

[0069] The enhanced activity of the AA9 polypeptide can also be determined as follows: at 40 °C, the AA9 polypeptide is incubated with 0.5% phosphoric acid-swollen cellulose (PASC), 100 mM sodium acetate (pH 5), 1 mM MnSO4, 0.1% gallic acid, 0.025 mg / ml of Aspergillus fumigatus β-glucosidase, and 0.01% X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) for 24 - 96 hours, and then the glucose released from PASC is determined.

[0070] The enhanced activity of the AA9 polypeptide in the high-temperature composition can also be determined according to WO 2013 / 028928.

[0071] The AA9 polypeptide enhances the hydrolysis of cellulose-containing materials catalyzed by enzymes with cellulolytic activity by reducing the amount of cellulase required to achieve the same degree of hydrolysis by preferably at least 1.01-fold, such as at least 1.05-fold, at least 1.10-fold, at least 1.25-fold, at least 1.5-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, or at least 20-fold.

[0072] β-Glucosidase: The term "β-glucosidase" means β-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes the hydrolysis of terminal non-reducing β-D-glucose residues and releases β-D-glucose. The β-glucosidase activity can be determined using p-nitrophenyl-β-D-glucopyranoside as a substrate according to the procedure of Venturi et al., 2002, J. Basic Microbiol. [Journal of Basic Microbiology] 42:55 - 66. One unit of β-glucosidase is defined as the production of 1.0 micromole of p-nitrophenolate anion per minute from 1 mM p-nitrophenyl-β-D-glucopyranoside as a substrate in 50 mM sodium citrate containing 0.01% 20 at 25 °C and pH 4.8.

[0073] β-Xylosidase: The term "β-xylosidase" means β-D-xyloside xylohydrolase (E.C. 3.2.1.37), which catalyzes the exo-hydrolysis of short β(1→4)-oligoxylosides to remove successive D-xylose residues from the non-reducing end. It can be carried out in the presence of 0.01% β-xylosidase activity was determined using 1 mM p-nitrophenyl-β-D-xylopyranoside as a substrate in 100 mM sodium citrate at 20, pH 5, and 40°C. One unit of β-xylosidase is defined as the amount of enzyme that produces 1.0 micromole of p-nitrophenolate anion per minute from 1 mM p-nitrophenyl-β-D-xylopyranoside in 100 mM sodium citrate at 20, pH 5, and 40°C. at 20 in 100 mM sodium citrate from 1 mM p-nitrophenyl-β-D-xylopyranoside.

[0074] Catalase: The term "catalase" means hydrogen peroxide: hydrogen peroxide oxidoreductase (EC 1.11.1.6), which catalyzes the conversion of 2H2O2 to O2 + 2H2O. For the purposes of the present invention, catalase activity was determined according to U.S. Patent No. 5,646,025. One unit of catalase activity is equal to the amount of enzyme that catalyzes the oxidation of 1 micromole of hydrogen peroxide under the assay conditions.

[0075] Cellobiohydrolase: The term "cellobiohydrolase" means 1,4-β-D-glucan cellobiohydrolase (E.C. 3.2.1.91 and E.C. 3.2.1.176), which catalyzes the hydrolysis of 1,4-β-D-glycosidic bonds in cellulose, cellooligosaccharides, or any polymer containing β-1,4-linked glucose, releasing cellobiose from the reducing end (cellobiohydrolase I) or non-reducing end (cellobiohydrolase II) of the chain (Teeri, 1997, Trends in Biotechnology 15: 160-167; Teeri et al., 1998, Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity can be determined according to the procedures described by: Lever et al., 1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem. 170: 575-581.

[0076] Cellulase or cellulolytic enzyme: The term "cellulase" or "cellulolytic enzyme" means one or more (e.g., several) enzymes that hydrolyze cellulosic materials. Such enzymes include one or more endoglucanases, one or more cellobiohydrolases, one or more β-glucosidases, or combinations thereof. Two basic methods for measuring cellulase activity include: (1) measuring total cellulase activity, and (2) measuring individual cellulase activities (endoglucanase, cellobiohydrolase, and β-glucosidase), as described in Zhang et al., 2006, Biotechnology Advances 24:452-481. Total cellulase activity can be measured using insoluble substrates (including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, etc.). The most common total cellulase activity assay is the filter paper assay using Whatman No. 1 filter paper as the substrate. This assay was established by the International Union of Pure and Applied Chemistry (IUPAC) (Ghose, 1987, Pure Appl. Chem. 59:257-68).

[0077] Cellulase activity can be determined by measuring the increase in sugar production / release during hydrolysis of cellulosic materials by one or more cellulases under the following conditions: 1-50 mg cellulase protein / g cellulose in pretreated corn stover (PCS) (or other pretreated cellulosic materials), at a suitable temperature (e.g., 40°C - 80°C, e.g., 50°C, 55°C, 60°C, 65°C, or 70°C), and at a suitable pH (e.g., 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0) for 3-7 days, compared to a control hydrolysis without added cellulase protein. Typical conditions are: 1 ml reaction, washed or unwashed PCS, 5% insoluble solids (dry weight), 50 mM sodium acetate (pH 5), 1 mM MnSO4, 50°C, 55°C, or 60°C, 72 hours, by HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, California, USA) for sugar analysis.

[0078] Coding sequence: The term "coding sequence" or "coding region" means a polynucleotide sequence that specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by a reading frame that usually begins with an ATG start codon or an alternative start codon (such as GTG and TTG) and ends with a stop codon (such as TAA, TAG, and TGA). The coding sequence can be a sequence of genomic DNA, cDNA, synthetic polynucleotide, and / or recombinant polynucleotide.

[0079] Endoglucanase: The term "endoglucanase" means 4-(1,3;1,4)-β-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4), which catalyzes the endohydrolysis of 1,4-β-D-glycosidic bonds in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, mixed β-1,3-1,4 glucans such as cereal β-D-glucan or xyloglucan, and other plant materials containing a cellulose component. Endoglucanase activity can be determined by measuring the decrease in substrate viscosity or the increase in reducing ends determined by reducing sugar assays (Zhang et al., 2006, Biotechnology Advances 24:452 - 481). Endoglucanase activity can also be determined according to the procedure of Ghose, 1987, Pure and Appl. Chem. 59:257 - 268, using carboxymethyl cellulose (CMC) as the substrate at pH 5 and 40 °C.

[0080] Expression: The term "expression" includes any step involved in the production of a polypeptide, including but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be measured - for example, to detect increased expression - by techniques known in the art, such as measuring the levels of mRNA and / or the translated polypeptide.

[0081] Expression vector: The term "expression vector" means a linear or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression.

[0082] Fermentable medium: The term "fermentable medium" or "fermentation medium" refers to a medium containing one or more (e.g., two, several) sugars, such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or soluble oligosaccharides, wherein the medium can be partially converted (fermented) by a host cell into a desired product, such as ethanol. In some cases, the fermentation medium is derived from natural sources, such as sugarcane, starch, or cellulose; and can be from a pretreatment of enzymatic hydrolysis (saccharification) of such sources. The term fermentation medium is understood herein to refer to the medium prior to the addition of the fermentation organism, e.g., the medium resulting from the saccharification process, as well as the medium used in a simultaneous saccharification and fermentation process (SSF).

[0083] Glucoamylase: The term "glucoamylase" (1,4-α-D-glucan glucohydrolase, EC 3.2.1.3) is defined as an enzyme that catalyzes the release of D-glucose from the non-reducing ends of starch or related oligosaccharide and polysaccharide molecules. For the purposes of the present invention, glucoamylase activity can be determined according to procedures known in the art, such as those described in WO2020 / 023411.

[0084] Hemicellulolytic enzyme or hemicellulase: The term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that can hydrolyze hemicellulose materials. See, e.g., Shallom and Shoham, 2003, Current Opinion In Microbiology 6(3):219-228. Hemicellulases are key components in the degradation of plant biomass. Examples of hemicellulases include, but are not limited to: acetylmannan esterase, acetylxylan esterase, arabinase, arabinofuranosidase, coumaric acid esterase, ferulic acid esterase, galactosidase, glucuronidase, glucuronate esterase, mannanase, mannosidase, xylanase, and xylosidase. The substrates of these enzymes (hemicellulose) are a heterogeneous group of branched and linear polysaccharides that are cross-linked into a robust network by hydrogen bonding to cellulose microfibrils in the plant cell wall. Hemicellulose is also covalently attached to lignin, thus forming a highly complex structure together with cellulose. The variable structure and organization of hemicellulose require the concerted action of many enzymes for its complete degradation. The catalytic modules of hemicellulases are glycoside hydrolases (GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases (CEs) that hydrolyze ester bonds of acetic acid or ferulic acid side groups. These catalytic modules can be assigned to GH and CE families based on the homology of their primary sequences. Some families, having generally similar folds, can be further grouped into clans, labeled with letters (e.g., GH-A). The most detailed and up-to-date classification of these and other carbohydrate-active enzymes is available in the Carbohydrate-Active Enzymes (CAZy) database. Hemicellulolytic enzyme activity can be measured according to Ghose and Bisaria, 1987, Pure&AppI.Chem. 59:1739-1752, at a suitable temperature such as 40°C - 80°C, e.g., 50°C, 55°C, 60°C, 65°C or 70°C, and a suitable pH such as 4 - 9, e.g., 5.0, 5.5, 6.0, 6.5 or 7.0.

[0085] Heterologous polynucleotide: The term "heterologous polynucleotide" is defined herein as a polynucleotide that is not native to the host cell; a native polynucleotide in which the coding region has been structurally modified; a native polynucleotide whose expression has been quantitatively altered by manipulation of the DNA by recombinant DNA techniques (e.g., a different (foreign) promoter); or a native polynucleotide in a host cell that has one or more additional copies of the polynucleotide to quantitatively alter expression. A "heterologous gene" is a gene that contains a heterologous polynucleotide.

[0086] Mature polypeptide: The term "mature polypeptide" is defined herein as a biologically active polypeptide in its final form after translation and any post-translational modifications (such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). The mature polypeptide sequence lacks a signal sequence, which can be determined using techniques known in the art (see, for example, Zhang and Henzel, 2004, Protein Science 13:2819-2824). The term "mature polypeptide coding sequence" means a polynucleotide encoding a mature polypeptide.

[0087] Pentose: The term "pentose" means a five-carbon monosaccharide (e.g., xylose, arabinose, ribose, lyxose, ribulose, and xylulose). Pentoses (such as D-xylose and L-arabinose) can be derived, for example, by saccharification of plant cell wall polysaccharides.

[0088] Pretreated corn stover: The term "pretreated corn stover" or "PCS" means a cellulose-containing material obtained from corn stover by thermal and dilute sulfuric acid treatment, alkali pretreatment, neutral pretreatment, or any pretreatment known in the art.

[0089] Protease: The term "protease" is defined herein as an enzyme that hydrolyzes peptide bonds. It includes any enzyme belonging to the EC 3.4 enzyme group (including each of its 13 subclasses). The EC numbers refer to the 1992 Enzyme Nomenclature of the NC-IUBMB in San Diego, California, Academic Press, including Supplements 1-5 published separately in: Eur. J. Biochem. [European Journal of Biochemistry] 223:1-5 (1994); Eur. J. Biochem. [European Journal of Biochemistry] 232:1-6 (1995); Eur. J. Biochem. [European Journal of Biochemistry] 237:1-5 (1996); Eur. J. Biochem. [European Journal of Biochemistry] 250:1-6 (1997); and Eur. J. Biochem. [European Journal of Biochemistry] 264:610-650 (1999). The term "subtilisin" refers to the subgroup of serine proteases according to Siezen et al., 1991, Protein Engng. [Protein Engineering] 4:719-737 and Siezen et al., 1997, Protein Science [Protein Science] 6:501-523. Serine proteases or serine peptidases are subgroups of proteases characterized by having serine at the active site and forming a covalent adduct with the substrate. Additionally, subtilisins (and serine proteases) are characterized by having two active site amino acid residues in addition to serine, namely histidine and aspartic acid residues. Subtilisins can be classified into 6 subclasses, namely, the subtilisin protease family, the thermitase family, the proteinase K family, the lantibiotic peptidase family, the kexin family, and the pyrolysin family. The term "protease activity" means proteolytic activity (EC 3.4). Protease activity can be measured using methods described in the art (e.g., US2015 / 0125925) or using commercially available assay kits (e.g., Sigma-Aldrich).

[0090] Pullulanase: The term "pullulanase" means a starch debranching enzyme (EC 3.2.1.41) having pullulan 6-glucanohydrolase activity, which catalyzes the hydrolysis of α-1,6-glycosidic bonds in pullulan, thereby releasing maltotriose with a reducing carbohydrate end. For the purposes of the present invention, pullulanase activity can be measured according to the PHADEBAS assay or the sweet potato starch assay described in WO 2016 / 087237.

[0091] Sequence identity: The degree of relatedness between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0092] For the purposes described herein, the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. 1970, 48, 443-453) is used to determine the degree of sequence identity between two amino acid sequences, which is implemented as in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., Trends Genet. 2000, 16, 276-277) (preferably version 3.0.0 or later). The optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (the EMBOSS version of BLOSUM62) substitution matrix. The output of "longest identity" marked by Needle (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows:

[0093] (Number of identical residues X 100) / (length of the reference sequence - total number of gaps in the alignment)

[0094] For the purposes described herein, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above) is used to determine the degree of sequence identity between two deoxyribonucleotide sequences, which is implemented as in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, see above) (preferably version 3.0.0 or later). The optional parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (the EMBOSS version of NCBINUC4.4) substitution matrix. The output of "longest identity" marked by Needle (obtained using the -nobrief option) is used as the percentage identity and is calculated as follows:

[0095] (Number of identical deoxyribonucleotides x 100) / (length of the reference sequence - total number of gaps in the alignment)

[0096] Xylanase: The term "xylanase" means 1,4-β-D-xylan-xylohydrolase (E.C. 3.2.1.8), which catalyzes the endohydrolysis of 1,4-β-D-xylosidic linkages in xylan. Xylanase activity can be measured at 37 °C in 0.01% Determined in 200 mM sodium phosphate (pH 6) with 0.2% AZCL-arabinoxylan as substrate at X-100. One unit of xylanase activity is defined as the amount of enzyme that produces 1.0 micromole of azurine per minute from 0.2% AZCL-arabinoxylan as substrate in 200 mM sodium phosphate (pH 6) at 37 °C and pH 6.

[0097] Xylitol dehydrogenase: The term "xylitol dehydrogenase" or "XDH" (AKA D-xylulose reductase) is classified as E.C. 1.1.1.9 and refers to an enzyme that catalyzes the conversion of xylitol to D-xylulose. Xylitol dehydrogenase activity can be determined using methods known in the art (e.g., Richard et al., 1999, FEBS Letters 457, 135 - 138).

[0098] Xylose isomerase: The term "xylose isomerase" or "XI" refers to an enzyme that can catalyze the conversion of D-xylose to D-xylulose in vivo and D-glucose to D-fructose in vitro. Xylose isomerase is also known as "glucose isomerase" and is classified as E.C. 5.3.1.5. Since the structure of this enzyme is very stable, xylose isomerase is a good model for studying the relationship between protein structure and function (Karimaki et al., Protein Eng Des Sel, 2004, 17(12):861 - 869). Xylose isomerase activity can be measured using techniques known in the art (e.g., a coupled enzyme assay using D-sorbitol dehydrogenase as described by Verhoeven et al., 2017, SciRep 7, 46155).

[0099] Xylulokinase: The term "xylulokinase" or "XK" is classified as E.C. 2.7.1.17 and refers to an enzyme that catalyzes the conversion of D-xylulose to D-xylulose 5-phosphate. Xylulokinase activity can be determined using methods known in the art (e.g., Richard et al., 2000, FEBS Microbiol. Letters 190, 39 - 43).

[0100] References herein to "about" a value or parameter include examples that refer to the value or parameter itself. For example, a description of "about X" includes the example "X". When used in combination with a measured value, "about" includes a range that encompasses at least the uncertainty associated with the method of measuring that particular numerical value and can include a range of plus or minus two standard deviations around the given numerical value.

[0101] Similarly, a gene or polypeptide that is "derived from" another gene or polypeptide X includes the gene or polypeptide X.

[0102] As used herein and in the appended claims, the singular forms "a / an," "or," and "the" include plural referents unless the context clearly indicates otherwise.

[0103] It should be understood that the embodiments described herein include "consisting of" and / or "consisting essentially of" the embodiments. As used herein, unless otherwise required by the language of expression or necessary meaning, the word "comprise" or variations such as "comprises" or "comprising" are used in an inclusive sense, i.e., specifying the presence of the stated features, but not excluding the presence or addition of other features in various embodiments. Detailed Description

[0104] Particularly described herein are fermenting organisms and methods for producing fermentation products (such as ethanol) from starch-containing and / or cellulose-containing materials. The applicant has created new Saccharomyces cerevisiae strains that have improved ethanol titers, increased temperature tolerance, increased sugar consumption, a higher ethanol-to-glycerol ratio, and reduced by-products, thereby maximizing product yields. These characteristics are important for the industrial production of corn ethanol.

[0105] In one aspect is a method for producing a fermentation product from a cellulose-containing or starch-containing material, the method comprising:

[0106] (a) saccharifying the cellulose-containing or starch-containing material; and

[0107] (b) fermenting the saccharified material of step (a) with a fermenting organism described herein.

[0108] Steps a) and b) can be carried out sequentially or simultaneously (SSF). In one embodiment, steps a) and b) are carried out simultaneously (SSF). In another embodiment, steps a) and b) are carried out sequentially.

[0109] Fermenting organism

[0110] In one embodiment, the fermenting organism is a Saccharomyces cerevisiae strain deposited with the Patent Culture Depository of the Agricultural Research Service, U.S. Department of Agriculture (NRRL) under the Budapest Treaty and having a deposit number of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5225.

[0111] In one embodiment, the fermenting organism is Saccharomyces cerevisiae strain MBG5227 (Saccharomyces cerevisiae strain MBG5227) deposited with the Agricultural Research Service Patent Culture Collection Center (NRRL) in accordance with the Budapest Treaty under accession number NRRL Y-68200, or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5227.

[0112] In one embodiment, the fermenting organism is Saccharomyces cerevisiae strain MBG5307 (Saccharomyces cerevisiae strain MBG5307) deposited with the Agricultural Research Service Patent Culture Collection Center (NRRL) in accordance with the Budapest Treaty under accession number Y-68201, or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5307.

[0113] In one embodiment, the fermenting organism is Saccharomyces cerevisiae strain MBG5318 (Saccharomyces cerevisiae strain MBG5318) deposited with the Agricultural Research Service Patent Culture Collection Center (NRRL) in accordance with the Budapest Treaty under accession number NRRL Y-68202, or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5318.

[0114] In one embodiment, the fermenting organism has one or more of the following properties:

[0115] Under the same process conditions, compared to Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, accession number V14 / 007039), acetaldehyde production is reduced;

[0116] Under the same process conditions, compared to Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, accession number V14 / 007039), temperature tolerance is increased;

[0117] Under the same process conditions, compared to Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, accession number V14 / 007039), maltose (DP2) production is reduced;

[0118] Under the same process conditions, compared to Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, accession number V14 / 007039), glycerol production is reduced.

[0119] In one embodiment, using the same methods and conditions, such as those described herein, a fermenting organism having substantially the same properties as Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307 or MBG5318 provides an ethanol yield increase of more than 1.0%, such as more than 2.0%, such as more than 2.5%, such as about 2.9% compared to Saccharomyces cerevisiae strain Ethanol (Deposited at the National Measurement Institute, Victoria, Australia, accession number V14 / 007039).

[0120] In one embodiment, under the same method conditions, such as those described herein, compared to Saccharomyces cerevisiae strain Ethanol (Deposited at the National Measurement Institute, Victoria, Australia, accession number V14 / 007039), a fermenting organism having substantially the same properties as Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307 or MBG5318 reduces acetaldehyde production by more than 10%, such as more than 20%, more than 30%, more than 40%, more than 45%, such as between 5% and 60%, such as 30% - 50%.

[0121] In one embodiment, under the same method conditions, such as those described herein, compared to Saccharomyces cerevisiae strain Ethanol (Deposited at the National Measurement Institute, Victoria, Australia, accession number V14 / 007039), a fermenting organism having substantially the same properties as Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307 or MBG5318 has increased temperature tolerance.

[0122] Since the fermentation temperature may fluctuate to some extent, the increased temperature tolerance is an advantage. In the early stage of fermentation, the equipment usually does not actively heat the fermentation. Therefore, the temperature may naturally rise due to the metabolism of the yeast. The equipment can use a heat exchanger to control the early fermentation temperature so that the temperature does not get too high. For most of the year, the equipment can easily control the early temperature, and the peak temperature is typically about 34°C. However, in summer, the cooling water used in the heat exchanger is not cold enough to control the temperature. Therefore, in equipment without a cooler (i.e., a water refrigeration system), the early fermentation temperature can reach above 36.5°C, which can stress the yeast.

[0123] In one embodiment, under the same method conditions, such as those described herein, compared to Saccharomyces cerevisiae strain Ethanol Compared with the deposit (deposit number: V14 / 007039) at the National Measurement Institute of Victoria, Australia, a fermenting organism having substantially the same properties as Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307 or MBG5318 reduces maltose (DP2) production by more than 3%, for example, more than 4%, more than 5%, more than 6%, more than 7%, for example, between 2% and 15%, for example, between 5% and 10%.

[0124] In one embodiment, under the same method conditions, such as those described herein, compared with Saccharomyces cerevisiae strain Ethanol Compared with the deposit (deposit number: V14 / 007039) at the National Measurement Institute of Victoria, Australia, a fermenting organism having substantially the same properties as Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307 or MBG5318 reduces glycerol production by more than 3%, for example, more than 4%, more than 5%, more than 6%, more than 7%, for example, between 2% and 15%, for example, between 5% and 10%.

[0125] The fermenting organism can also be a derivative of Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307 or MBG5318. As used herein, a "derivative" of Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307 or MBG5318 is a strain derived from said strain, for example, by mutagenesis, recombinant DNA technology, mating, cell fusion or cytoduction between yeast strains. A strain derived from Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307 or MBG5318 can be a direct descendant (i.e., the product of mating between Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307 or MBG5318 and another strain or itself), or a distant descendant (which is produced by an initial mating between Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307 or MBG5318 and another strain or itself, followed by a large number of subsequent matings).

[0126] In one embodiment, a derivative of Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307 or MBG5318 is a hybrid strain produced by culturing a first yeast strain together with Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307 or MBG5318 under conditions that allow DNA combination between the first yeast strain and Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307 or MBG5318.

[0127] In one embodiment, derivatives of the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318 exhibit one or more defined characteristics of the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318. The Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318 are respectively used to generate derivatives of the genus Saccharomyces that exhibit one or more defined characteristics of the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318. In this regard, the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318 form the basis for preparing other strains having the defined characteristics of the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318. For example, methods such as classical mating, cell fusion, or cytoplasmic introduction, mutagenesis, or recombinant DNA techniques between yeast strains can be used to respectively derive Saccharomyces genus strains that exhibit one or more defined characteristics of the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318 from the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318.

[0128] In one embodiment, a derivative of the Saccharomyces cerevisiae strain MBG5225 that exhibits one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5225 can be generated by:

[0129] (a) culturing a first yeast strain together with a second yeast strain under conditions that permit DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5225 (or a derivative of the Saccharomyces cerevisiae strain MBG5225);

[0130] (b) screening or selecting a derivative of the Saccharomyces cerevisiae strain MBG5225, such as screening or selecting a derivative that has increased ethanol production in corn mash compared to the first strain;

[0131] (c) optionally repeating steps (a) and (b) using the screened or selected strain as the first yeast strain and / or the second yeast strain until a derivative of the Saccharomyces cerevisiae strain MBG5225 that exhibits one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5225 is obtained.

[0132] In one embodiment, a derivative of the Saccharomyces cerevisiae strain MBG5227 that exhibits one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5227 can be generated by:

[0133] (a) Under conditions that permit DNA combination between a first yeast strain and a second yeast strain, the first yeast strain is co-cultured with the second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5227 (or a derivative of Saccharomyces cerevisiae strain MBG5227);

[0134] (b) Screen or select a derivative of Saccharomyces cerevisiae strain MBG5227, such as a derivative that has increased ethanol production in corn mash compared to the first strain;

[0135] (c) Optionally, repeat steps (a) and (b) using the screened or selected strain as the first yeast strain and / or the second yeast strain until a derivative of Saccharomyces cerevisiae strain MBG5227 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5227 is obtained.

[0136] In one embodiment, a derivative of Saccharomyces cerevisiae strain MBG5307 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5307 can be produced by:

[0137] (a) Under conditions that permit DNA combination between a first yeast strain and a second yeast strain, the first yeast strain is co-cultured with the second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5307 (or a derivative of Saccharomyces cerevisiae strain MBG5307);

[0138] (b) Screen or select a derivative of Saccharomyces cerevisiae strain MBG5307, such as a derivative that has increased ethanol production in corn mash compared to the first strain;

[0139] (c) Optionally, repeat steps (a) and (b) using the screened or selected strain as the first yeast strain and / or the second yeast strain until a derivative of Saccharomyces cerevisiae strain MBG5307 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5307 is obtained.

[0140] In one embodiment, a derivative of Saccharomyces cerevisiae strain MBG5318 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5318 can be produced by:

[0141] (a) Under conditions that permit DNA combination between a first yeast strain and a second yeast strain, the first yeast strain is co-cultured with the second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5318 (or a derivative of Saccharomyces cerevisiae strain MBG5318);

[0142] (b) Screening or selecting derivatives of the Saccharomyces cerevisiae strain MBG5318, e.g., screening or selecting derivatives that have increased ethanol production in corn mash compared to the first strain;

[0143] (c) Optionally, repeating steps (a) and (b) with the screened or selected strain as the first yeast strain and / or the second yeast strain until a derivative of the Saccharomyces cerevisiae strain MBG5318 is obtained that exhibits one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5318.

[0144] If methods such as classical mating, cell fusion, or cytoplast import can be used to combine the DNA of the first yeast strain with the second yeast strain, the first yeast strain can be any strain of yeast. Typically, the first yeast strain is a strain of the genus Saccharomyces. More typically, the first yeast strain is a Saccharomyces cerevisiae strain. Saccharomyces cerevisiae is as defined by Kurtzman (2003) FEMS Yeast Research, Volume 4, pages 233 - 245. The first yeast strain can have desired properties that are intended to be combined with the defined characteristics of the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318. The first yeast strain can be, for example, any Saccharomyces cerevisiae strain, such as, for example, ETHANOL It should also be understood that the first yeast strain can be the Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307, or MBG5318 (or a derivative of the Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307, or MBG5318).

[0145] Culturing the first and second yeast strains under conditions that permit the combination of DNA between the yeast strains. As used herein, "combination of DNA" between yeast strains refers to the combination of all or part of the genomes of the yeast strains. The combination of DNA between yeast strains can be carried out by any method suitable for combining the DNA of at least two yeast cells and can include, for example, a mating method that includes sporulation of the yeast strains to produce haploid cells and subsequent hybridization of compatible haploid cells; cytoplast import; or cell fusion such as protoplast fusion.

[0146] In one embodiment, culturing the first yeast strain with the second yeast under conditions that permit the combination of DNA between the first yeast strain and the second yeast strain, the culturing comprising:

[0147] (i) Causing the first yeast strain and the second yeast strain to form spores;

[0148] (ii) Causing the spores produced by the first yeast strain to germinate and hybridizing them with the spores produced by the second yeast strain.

[0149] In one embodiment, a method for generating a derivative of Saccharomyces cerevisiae strain MBG5225 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5225 includes:

[0150] (a) Providing: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5225 (or a derivative of Saccharomyces cerevisiae strain MBG5225);

[0151] (b) Causing the first yeast strain and the second yeast strain to form spores;

[0152] (c) Germinating the spores of the first yeast strain and hybridizing them with the germinated spores of the second yeast strain;

[0153] (d) Screening or selecting a derivative of Saccharomyces cerevisiae strain MBG5225, such as screening or selecting a derivative that has increased ethanol production in corn mash and / or a higher ethanol yield from glucose during corn mash fermentation compared to the first strain;

[0154] (e) Optionally repeating steps (b) to (d) using the screened or selected strain as the first and / or second yeast strain.

[0155] In one embodiment, a method for generating a derivative of Saccharomyces cerevisiae strain MBG5227 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5227 includes:

[0156] (a) Providing: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5227 (or a derivative of Saccharomyces cerevisiae strain MBG5227);

[0157] (b) Causing the first yeast strain and the second yeast strain to form spores;

[0158] (c) Germinating the spores of the first yeast strain and hybridizing them with the germinated spores of the second yeast strain;

[0159] (d) Screening or selecting a derivative of Saccharomyces cerevisiae strain MBG5227, such as screening or selecting a derivative that has increased ethanol production in corn mash and / or a higher ethanol yield from glucose during corn mash fermentation compared to the first strain;

[0160] (e) Optionally repeating steps (b) to (d) using the screened or selected strain as the first and / or second yeast strain.

[0161] In one embodiment, a method for generating a derivative of Saccharomyces cerevisiae strain MBG5307 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5307 includes:

[0162] (a) Providing: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5307 (or a derivative of Saccharomyces cerevisiae strain MBG5307);

[0163] (b) Causing the first yeast strain and the second yeast strain to form spores;

[0164] (c) Germinating the spores of the first yeast strain and hybridizing them with the germinated spores of the second yeast strain;

[0165] (d) Screening or selecting a derivative of Saccharomyces cerevisiae strain MBG5307, such as screening or selecting a derivative that has increased ethanol production in corn mash and / or a higher ethanol yield from glucose during corn mash fermentation compared to the first strain;

[0166] (e) Optionally repeating steps (b) to (d) using the screened or selected strain as the first and / or second yeast strain.

[0167] In one embodiment, a method for generating a derivative of Saccharomyces cerevisiae strain MBG5318 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5318 includes:

[0168] (a) Providing: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5318 (or a derivative of Saccharomyces cerevisiae strain MBG5318);

[0169] (b) Causing the first yeast strain and the second yeast strain to form spores;

[0170] (c) Germinating the spores of the first yeast strain and hybridizing them with the germinated spores of the second yeast strain;

[0171] (d) Screening or selecting a derivative of Saccharomyces cerevisiae strain MBG5318, such as screening or selecting a derivative that has increased ethanol production in corn mash and / or a higher ethanol yield from glucose during corn mash fermentation compared to the first strain;

[0172] (e) Optionally repeating steps (b) to (d) using the screened or selected strain as the first and / or second yeast strain.

[0173] Methods for forming spores, germinating, and hybridizing yeast strains, and particularly strains of the genus Saccharomyces, are known in the art and are described, for example, in Ausubel, F.M. et al., (1997) Current Protocols in Molecular Biology, Volume 2, pages 13.2.1 to 13.2.5 (John Willey & Sons Inc); Chapter 7, "Sporulation and Hybridisation of yeast" by R.R. Fowell, in "The Yeasts", Volume 1, A.H. Rose and J.S. Harrison (eds.), 1969, Academic Press.

[0174] In one embodiment, the yeast strain can be cultured under conditions that permit cell fusion. Methods for using cell fusion techniques to produce intra- or interspecies hybrids are described, for example, in Spencer et al. (1990), Yeast Technology, Spencer J.F.T. and Spencer D.M. (eds.), Springer Verlag, New York.

[0175] In another embodiment, the yeast strain can be cultured under conditions that permit cytoplasmic import. Methods for cytoplasmic import are described, for example, in Inge-Vechymov et al. (1986) Genetika 22:2625-2636; Johnston (1990), Yeast technology, Spencer J.F.T. and Spencer D.M. (eds.), Springer Verlag, New York.

[0176] In one embodiment, screening or selecting derivatives of Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318 includes screening or selecting derivatives having increased ethanol production compared to a first strain, and / or screening or selecting hybrids having a higher ethanol yield, such as those described in WO 2019 / 161227.

[0177] In one embodiment, a derivative of Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307, or MBG5318 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307, or MBG5318 can be a mutant of Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307, or MBG5318. Methods for producing mutants of yeast genus yeasts, and in particular mutants of Saccharomyces cerevisiae, are known in the art and are described, for example, in Lawrence C. W. (1991) Methods in Enzymology, 194:273 - 281.

[0178] In another embodiment, a derivative of Saccharomyces cerevisiae strain MBG5225 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5225 can be a recombinant derivative of Saccharomyces cerevisiae strain MBG5225. In another embodiment, a derivative of Saccharomyces cerevisiae strain MBG5227 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5227 can be a recombinant derivative of Saccharomyces cerevisiae strain MBG5227. In another embodiment, a derivative of Saccharomyces cerevisiae strain MBG5307 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5307 can be a recombinant derivative of Saccharomyces cerevisiae strain MBG5307. In another embodiment, a derivative of Saccharomyces cerevisiae strain MBG5318 that exhibits one or more defined characteristics of Saccharomyces cerevisiae strain MBG5318 can be a recombinant derivative of Saccharomyces cerevisiae strain MBG5318. A recombinant derivative of Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307, or MBG5318 is a strain produced by introducing nucleic acid into Saccharomyces cerevisiae strain MBG5225, MBG5227, MBG5307, or MBG5318 using recombinant DNA technology. Recombinant methods for introducing nucleic acid into yeast genus yeast cells, and in particular yeast genus strains, are known in the art and are described, for example, in Ausubel, F. M. et al. (1997), Current Protocols in Molecular Biology, Volume 2, pages 13.7.1 to 13.7.7, published by John Wiley & Sons Inc.

[0179] In one embodiment, recombinant derivatives of the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318 have been prepared by genetically modifying the strain (or another derivative thereof) to express a heterologous enzyme (such as the α-amylase and / or glucoamylase described herein (or any enzyme described in WO 2020 / 023411, the content of which is incorporated herein by reference)).

[0180] In one embodiment, a method for generating a recombinant derivative of the Saccharomyces cerevisiae strain MBG5225 (deposited at the Northern Regional Research Center (NRRL) of the United States Department of Agriculture Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois, USA, with accession number NRRL Y-68199) comprises:

[0181] (a) transforming the Saccharomyces cerevisiae strain MBG5225 (or a derivative of the Saccharomyces cerevisiae strain MBG5225) with one or more expression vectors encoding a heterologous enzyme such as glucoamylase and / or α-amylase; and

[0182] (b) isolating the transformed strain.

[0183] In one embodiment, a derivative of the Saccharomyces cerevisiae strain MBG5225 can be prepared by:

[0184] (a) culturing a first yeast strain together with a second yeast strain under conditions allowing DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5225 (or a derivative of the Saccharomyces cerevisiae strain MBG5225); and

[0185] (b) isolating the hybrid strain; and

[0186] (c) optionally repeating steps (a) and (b) using the hybrid strain isolated in step (b) as the first yeast strain and / or a derivative of the Saccharomyces cerevisiae strain MBG5225.

[0187] In one embodiment, a method for generating a recombinant derivative of the Saccharomyces cerevisiae strain MBG5227 (deposited at the Northern Regional Research Center (NRRL) of the United States Department of Agriculture Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois, USA, with accession number NRRL Y-68200) comprises:

[0188] (a) transforming the Saccharomyces cerevisiae strain MBG5227 (or a derivative of the Saccharomyces cerevisiae strain MBG5227) with one or more expression vectors encoding a heterologous enzyme such as glucoamylase and / or α-amylase; and

[0189] (b) isolating the transformed strain.

[0190] In one embodiment, a derivative of the Saccharomyces cerevisiae strain MBG5227 can be prepared by:

[0191] (a) culturing a first yeast strain together with a second yeast strain under conditions that permit DNA combination between the first and second yeast strains, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5227 (or a derivative of the Saccharomyces cerevisiae strain MBG5227); and

[0192] (b) isolating the heterozygous strain; and

[0193] (c) optionally repeating steps (a) and (b) using the heterozygous strain isolated in step (b) as the first yeast strain and / or a derivative of the Saccharomyces cerevisiae strain MBG5227.

[0194] In one embodiment, it is a method for producing a recombinant derivative of the Saccharomyces cerevisiae strain MBG5307 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection, 1815 University Street, Peoria, Illinois 61604, USA, accession number NRRL Y-68201), the method comprising:

[0195] (a) transforming the Saccharomyces cerevisiae strain MBG5307 (or a derivative of the Saccharomyces cerevisiae strain MBG5307) with one or more expression vectors encoding heterologous enzymes such as glucoamylase and / or α-amylase; and

[0196] (b) isolating the transformed strain.

[0197] In one embodiment, a derivative of the Saccharomyces cerevisiae strain MBG5307 can be prepared by:

[0198] (a) culturing a first yeast strain together with a second yeast strain under conditions that permit DNA combination between the first and second yeast strains, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5307 (or a derivative of the Saccharomyces cerevisiae strain MBG5307); and

[0199] (b) isolating the heterozygous strain; and

[0200] (c) optionally repeating steps (a) and (b) using the heterozygous strain isolated in step (b) as the first yeast strain and / or a derivative of the Saccharomyces cerevisiae strain MBG5307.

[0201] In one embodiment, a method for generating a recombinant derivative of Saccharomyces cerevisiae strain MBG5318 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection, 1815 University Street, Peoria, Illinois 61604, USA, under accession number NRRL Y-68202) is provided, the method comprising:

[0202] (a) transforming Saccharomyces cerevisiae strain MBG5318 (or a derivative of Saccharomyces cerevisiae strain MBG5318) with one or more expression vectors encoding heterologous enzymes such as glucoamylase and / or α-amylase; and

[0203] (b) isolating the transformed strain.

[0204] In one embodiment, a derivative of Saccharomyces cerevisiae strain MBG5318 can be prepared by:

[0205] (a) culturing a first yeast strain together with a second yeast strain under conditions permitting DNA combination between the first and second yeast strains, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5318 (or a derivative of Saccharomyces cerevisiae strain MBG5318); and

[0206] (b) isolating the hybrid strain; and

[0207] (c) optionally repeating steps (a) and (b) using the hybrid strain isolated in step (b) as the first yeast strain and / or a derivative of Saccharomyces cerevisiae strain MBG5318.

[0208] In some embodiments, derivatives of Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318 express glucoamylase and / or α-amylase. Derivatives expressing glucoamylase and / or α-amylase have been generated to increase ethanol yield and improve process economics by reducing the cost of the enzymes, since some or all of the essential enzymes required for starch hydrolysis are produced by the yeast organism.

[0209] Composition

[0210] This aspect relates to a formulated yeast composition of the genus Saccharomyces, the yeast composition comprising a yeast strain as described herein and natural and / or non-natural components.

[0211] In one embodiment, it is a composition that contains the Saccharomyces cerevisiae strain MBG5225 (or a derivative of the Saccharomyces cerevisiae strain MBG5225), the Saccharomyces cerevisiae strain MBG5227 (or a derivative of the Saccharomyces cerevisiae strain MBG5227), the Saccharomyces cerevisiae strain MBG5307 (or a derivative of the Saccharomyces cerevisiae strain MBG5307), or the Saccharomyces cerevisiae strain MBG5318 (or a derivative of the Saccharomyces cerevisiae strain MBG5318). The composition can be, for example, cream yeast, compressed yeast, wet yeast, dry yeast, semi-dry yeast, crushed yeast, stabilized liquid yeast, or frozen yeast. Methods for preparing such yeast compositions are known in the art.

[0212] In one embodiment, the Saccharomyces cerevisiae strain is dry yeast, such as active dry yeast or instant yeast. In one embodiment, the Saccharomyces cerevisiae strain is crushed yeast. In one embodiment, the Saccharomyces cerevisiae strain is compressed yeast. In one embodiment, the Saccharomyces cerevisiae strain is cream yeast.

[0213] In one embodiment, it is a composition that contains the yeast of the genus described herein (particularly the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318) and one or more components selected from the group consisting of: surfactants, emulsifiers, gums, swelling agents, and antioxidants and other processing aids.

[0214] Surfactant

[0215] The composition described herein can contain the yeast of the genus described herein (particularly the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318) and any suitable surfactant. In one embodiment, one or more surfactants are anionic surfactants, cationic surfactants, and / or non-ionic surfactants.

[0216] Emulsifier

[0217] The composition described herein can contain the yeast of the genus described herein (particularly the Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, or MBG5318) and any suitable emulsifier. In one embodiment, the emulsifier is a fatty acid ester of sorbitan. In one embodiment, the emulsifier is selected from the group consisting of: sorbitan monostearate (SMS), citric acid esters of mono- and diglycerides, polyglycerol esters, and fatty acid esters of propylene glycol.

[0218] In one embodiment, the composition comprises a yeast of the genus Saccharomyces as described herein (in particular Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307 or MBG5318) and Olindronal SMS, Olindronal SK, or Olindronal SPL, including the compositions described in European Patent No. 1,724,336 (incorporated herein by reference). For active dry yeast, these products are commercially available from Bussetti of Austria.

[0219] Gum

[0220] The composition described herein may comprise a yeast of the genus Saccharomyces as described herein (in particular Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307 or MBG5318) and any suitable gum. In one embodiment, the gum is selected from the group consisting of locust bean gum, guar gum, tragacanth gum, gum arabic, xanthan gum, and gum acacia, particularly for paste, compressed, and dry yeast.

[0221] Swelling agent

[0222] The composition described herein may comprise a yeast of the genus Saccharomyces as described herein (in particular Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307 or MBG5318) and any suitable swelling agent. In one embodiment, the swelling agent is methylcellulose or carboxymethylcellulose.

[0223] Antioxidant

[0224] The composition described herein may comprise a yeast of the genus Saccharomyces as described herein (in particular Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307 or MBG5318) and any suitable antioxidant. In one embodiment, the antioxidant is butylated hydroxyanisole (BHA) and / or butylated hydroxytoluene (BHT), or ascorbic acid (vitamin C), particularly for active dry yeast.

[0225] Method of using starch-containing material

[0226] In some embodiments, the methods described herein produce a fermentation product from a starch-containing material. Starch-containing materials are well known in the art, contain two types of homopolysaccharides (amylose and amylopectin), and are linked by α-(1-4)-D-glycosidic bonds. Any suitable starch-containing starting material can be used. The starting material is typically selected based on the desired fermentation product (such as ethanol). Examples of starch-containing starting materials include cereals, tubers, or grains. In particular, the starch-containing material can be corn, wheat, barley, rye, milo, sago, cassava, tapioca, sorghum, oats, rice, peas, beans, or sweet potatoes, or mixtures thereof. Waxy and non-waxy types of corn and barley are also contemplated.

[0227] In one embodiment, the starch-containing starting material is corn. In one embodiment, the starch-containing starting material is wheat. In one embodiment, the starch-containing starting material is barley. In one embodiment, the starch-containing starting material is rye. In one embodiment, the starch-containing starting material is milo. In one embodiment, the starch-containing starting material is sago. In one embodiment, the starch-containing starting material is cassava. In one embodiment, the starch-containing starting material is tapioca. In one embodiment, the starch-containing starting material is sorghum. In one embodiment, the starch-containing starting material is rice. In one embodiment, the starch-containing starting material is peas. In one embodiment, the starch-containing starting material is beans. In one embodiment, the starch-containing starting material is sweet potatoes. In one embodiment, the starch-containing starting material is oats.

[0228] Methods using a starch-containing material can include conventional methods (e.g., including the liquefaction step described in more detail below) or raw starch hydrolysis methods. In some embodiments using a starch-containing material, the saccharification of the starch-containing material is carried out at a temperature above the initial gelatinization temperature. In some embodiments using a starch-containing material, the saccharification of the starch-containing material is carried out at a temperature below the initial gelatinization temperature.

[0229] Liquefaction

[0230] In embodiments using a starch-containing material, the methods can further include a liquefaction step, which is carried out by subjecting the starch-containing material to α-amylase and optionally protease and / or glucoamylase at a temperature above the initial gelatinization temperature. Other enzymes such as pullulanase and phytase can also be present and / or added during liquefaction. In some embodiments, the liquefaction step is carried out before steps a) and b) of the method.

[0231] The liquefaction step can be carried out for 0.5 - 5 hours, such as 1 - 3 hours, typically about 2 hours.

[0232] The term "initial gelatinization temperature" means the lowest temperature at which gelatinization of the starch-containing material begins. Generally, starch heated in water begins to gelatinize between about 50°C and 75°C; the exact temperature of gelatinization depends on the particular starch and can be readily determined by those skilled in the art. Thus, the initial gelatinization temperature can vary depending on the plant species, the particular variety of the plant species, and the growth conditions. The initial gelatinization temperature of a given starch-containing material can be determined by the temperature at which 5% of the starch granules lose birefringence using the method described by Gorinstein and Lii, 1992, [Starch] 44(12):461 - 466.

[0233] Liquefaction is typically carried out at a temperature in the range from 70°C - 100°C. In one embodiment, the temperature during liquefaction is between 75°C - 95°C, such as between 75°C - 90°C, between 80°C - 90°C, or between 82°C - 88°C, about 85°C.

[0234] The jet cooking step can be carried out before the liquefaction step, for example, at a temperature between 110°C - 145°C, 120°C - 140°C, 125°C - 135°C, or about 130°C for about 1 - 15 minutes, for about 3 - 10 minutes, or about 5 minutes.

[0235] The pH during liquefaction can be between 4 and 7, such as pH 4.5 - 6.5, pH 5.0 - 6.5, pH 5.0 - 6.0, pH 5.2 - 6.2, or about 5.2, about 5.4, about 5.6, or about 5.8.

[0236] In one embodiment, before liquefaction, the method further comprises the following steps:

[0237] i) Preferably reducing the particle size of the starch-containing material by dry milling;

[0238] ii) Forming a slurry comprising the starch-containing material and water.

[0239] The starchy starting material (such as whole grains) can be reduced in particle size, for example, by milling to open up the structure, increase the surface area, and allow for further processing. There are generally two types of methods: wet milling and dry milling. In dry milling, the whole grain is milled and used. Wet milling separates the germ well from the grits (starch granules and proteins). Wet milling is often applied in situations where starch hydrolyzates are used to produce, for example, syrups. Dry milling and wet milling are both well-known in the field of starch processing. In one embodiment, the starchy material is subjected to dry milling. In one embodiment, the particle size is reduced to between 0.05 and 3.0 mm, for example, 0.1 - 0.5 mm, or at least 30%, at least 50%, at least 70%, or at least 90% of the starchy material is suitable to pass through a sieve with a 0.05 to 3.0 mm screen, for example, a 0.1 - 0.5 mm screen. In another embodiment, at least 50%, for example, at least 70%, at least 80%, or at least 90% of the starchy material is suitable to pass through a sieve with a #6 screen.

[0240] The aqueous slurry can contain from 10 - 55 w / w-% dry solids (DS) of the starchy material, for example, 25 - 45 w / w-% dry solids (DS), or 30 - 40 w / w-% dry solids (DS).

[0241] Initially, α-amylase, optionally protease, and optionally glucoamylase can be added to the aqueous slurry to initiate liquefaction (thinning). In one embodiment, only a portion (e.g., about 1 / 3) of these enzymes is added to the aqueous slurry, while the remaining portion (e.g., about 2 / 3) of these enzymes is added during the liquefaction step.

[0242] α-Amylase and glucoamylase for liquefaction can be found in the art, for example, WO2020 / 023411 (the content of which is incorporated herein by reference). Similarly, suitable examples of protease for liquefaction can be found in the art, for example, WO2018 / 222990 (the content of which is incorporated herein by reference).

[0243] Saccharification and fermentation of starch-containing material

[0244] In embodiments using starchy materials, glucoamylase can be present and / or added in the saccharification step a) and / or the fermentation step b) or simultaneous saccharification and fermentation (SSF). The glucoamylase in the saccharification step a) and / or the fermentation step b) or simultaneous saccharification and fermentation (SSF) is typically different from the glucoamylase optionally added in any of the above liquefaction steps. In one embodiment, the glucoamylase is present and / or added together with fungal α-amylase. Suitable glucoamylase for saccharification or SSF can be found in the art, for example, WO2020 / 023411 (the content of which is incorporated herein by reference).

[0245] When saccharification and fermentation are carried out sequentially, the saccharification step a) can be carried out under conditions well known in the art. For example, the saccharification step a) can last up to from about 24 to about 72 hours. In one embodiment, pre-saccharification is carried out. The pre-saccharification is typically carried out at a temperature of 30°C - 65°C, typically about 60°C for 40 - 90 minutes. In one embodiment, in simultaneous saccharification and fermentation (SSF), the pre-saccharification is followed by saccharification during the fermentation process. The saccharification is typically carried out at a temperature from 20°C - 75°C, preferably from 40°C - 70°C, typically about 60°C and typically at a pH between 4 and 5, such as about pH 4.5.

[0246] As is known in the art and as described herein for example, the fermentation is carried out in a fermentation medium. The fermentation medium includes a fermentation substrate, i.e., a carbohydrate source that is metabolized by the fermenting organism. Using the methods described herein, the fermentation medium can contain nutrients and one or more growth stimulants for one or more fermenting organisms. Nutrients and growth stimulants are widely used in the field of fermentation and include nitrogen sources such as ammonia; urea, vitamins, and minerals or combinations thereof.

[0247] Generally, fermenting organisms such as yeast (including Saccharomyces cerevisiae) require a sufficient nitrogen source for proliferation and fermentation. If necessary, many supplementary nitrogen sources can be used and these nitrogen sources are well known in the art. The nitrogen source can be an organic nitrogen source (such as urea, DDG, wet filter cake, or corn mash) or an inorganic nitrogen source (such as ammonia or ammonium hydroxide). In one embodiment, the nitrogen source is urea.

[0248] The fermentation can be carried out under low-nitrogen conditions, for example when using yeast expressing protease. In some embodiments, the fermentation step is carried out under the following conditions: less than 1000 ppm supplementary nitrogen (e.g., urea or ammonium hydroxide), such as less than 750 ppm, less than 500 ppm, less than 400 ppm, less than 300 ppm, less than 250 ppm, less than 200 ppm, less than 150 ppm, less than 100 ppm, less than 75 ppm, less than 50 ppm, less than 25 ppm, or less than 10 ppm supplementary nitrogen. In some embodiments, the fermentation step is carried out without supplementary nitrogen.

[0249] Simultaneous saccharification and fermentation (“SSF”) is widely used in industrial-scale fermentation product production processes, especially in ethanol production processes. When performing SSF, the saccharification step a) and the fermentation step b) are carried out simultaneously. There is no holding stage for saccharification, meaning that the fermenting organism (such as yeast) and one or more enzymes can be added together. However, separate addition of the fermenting organism and one or more enzymes is also contemplated. SSF is typically carried out at a temperature from 25 °C to 40 °C, such as from 28 °C to 35 °C, such as from 30 °C to 34 °C, or about 32 °C. In one embodiment, the fermentation is carried out for 6 to 120 hours, especially 24 to 96 hours. In one embodiment, the pH is between 4 and 5.

[0250] In one embodiment, a cellulolytic enzyme composition is present and / or added in saccharification, fermentation, or simultaneous saccharification and fermentation (SSF). Examples of such cellulolytic enzyme compositions can be found in the “Cellulolytic Enzymes and Compositions” section. The cellulolytic enzyme composition can be present and / or added together with glucoamylase, as disclosed in the “Glucoamylase” section.

[0251] Method of using cellulose-containing material

[0252] In some embodiments, the methods described herein produce fermentation products from cellulosic materials. The major polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third most abundant is pectin. The secondary cell wall produced after cell growth ceases also contains polysaccharides and is strengthened by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of cellobiose anhydride and is thus a linear β-(1-4)-D-glucan, while hemicellulose includes a variety of compounds such as xylan, xyloglucan, arabinoxylan, and mannan with a series of substituents in a complex branched structure. Although cellulose is generally polymorphic, it is found to exist mainly as an insoluble crystalline matrix of parallel glucan chains in plant tissues. Hemicellulose is usually hydrogen-bonded to cellulose and other hemicelluloses, which helps to stabilize the cell wall matrix.

[0253] Cellulose is commonly found, for example, in the stems, leaves, husks, skins, and cobs of plants or the leaves, branches, and wood of trees. The cellulose-containing material can be, but is not limited to: agricultural waste, herbaceous materials (including energy crops), municipal solid waste, pulp and paper mill waste, waste paper, and wood (including forestry waste) (see, for example, Wiselogel et al., 1995, in Handbook on Bioethanol (edited by Charles E. Wyman), pages 105-118, Taylor & Francis, Washington, D.C.; Wyman, 1994, Bioresource Technology 50:3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24 / 25:695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, Advances in Biochemical Engineering / Biotechnology, edited by T. Scheper, volume 65, pages 23-40, Springer-Verlag, New York). It is to be understood herein that the cellulose can be any form of lignocellulose, a plant cell wall material containing lignin, cellulose, and hemicellulose in a mixed matrix. In one embodiment, the cellulose-containing material is any biomass material. In another embodiment, the cellulose-containing material is lignocellulose that comprises cellulose, hemicellulose, and lignin.

[0254] In one embodiment, the cellulose-containing material is agricultural waste, herbaceous materials (including energy crops), municipal solid waste, pulp and paper mill waste, waste paper, or wood (including forestry waste).

[0255] In another embodiment, the cellulose-containing material is Arundo donax, bagasse, bamboo, corncobs, corn fiber, corn stover, Miscanthus, rice straw, switchgrass, or wheat straw.

[0256] In another embodiment, the cellulose-containing material is aspen, eucalyptus, fir, pine, poplar, spruce, or willow.

[0257] In another embodiment, the cellulose-containing material is algal cellulose, bacterial cellulose, cotton linter, filter paper, microcrystalline cellulose (e.g., ) or cellulose treated with phosphoric acid.

[0258] In another embodiment, the cellulosic material is aquatic biomass. As used herein, the term "aquatic biomass" means biomass produced by the process of photosynthesis in an aquatic environment. Aquatic biomass can be algae, emergent plants, floating-leaved plants, or submerged plants.

[0259] The cellulosic material can be used as is or can be pretreated using conventional methods known in the art, as described herein. In a preferred embodiment, the cellulosic material is pretreated.

[0260] Methods using the cellulosic material can be accomplished using conventional methods in the art. Additionally, these methods can be performed using any conventional biomass processing apparatus configured to implement these methods.

[0261] Cellulose pretreatment

[0262] In one embodiment, the cellulosic material is pretreated prior to saccharification.

[0263] In practicing the methods described herein, any pretreatment method known in the art can be used to disrupt the plant cell wall components of the cellulosic material (Chandra et al., 2007, Adv. Biochem. Engin. / Biotechnol. [Advances in Biochemical Engineering / Biotechnology] 108:67 - 93; Galbe and Zacchi, 2007, Adv. Biochem. Engin. / Biotechnol. [Advances in Biochemical Engineering / Biotechnology] 108:41 - 65; Hendriks and Zeeman, 2009, Bioresource Technology [Biological Resource Technology] 100:10 - 18; Mosier et al., 2005, Bioresource Technology [Biological Resource Technology] 96:673 - 686; Taherzadeh and Karimi, 2008, Int. J. Mol. Sci. [International Journal of Molecular Sciences] 9:1621 - 1651; Yang and Wyman, 2008, Biofuels Bioproducts and Biorefining - Biofpr. [Biofuels, Bioproducts and Biorefining] 2:26 - 40).

[0264] The cellulosic material can also be subjected to size reduction, screening, pre - soaking, wetting, washing, and / or conditioning using methods known in the art prior to pretreatment.

[0265] Conventional pretreatment includes, but is not limited to: steam pretreatment (with or without explosion), dilute acid pretreatment, hot water pretreatment, alkali pretreatment, lime pretreatment, wet oxidation, wet explosion, ammonia fiber explosion, organic solvent pretreatment, and biological pretreatment. Additional pretreatment includes ammonia percolation, ultrasound, electroporation, microwave, supercritical CO2, supercritical H2O, ozone, ionic liquid, and γ-radiation pretreatment.

[0266] In one embodiment, the cellulosic material is pretreated prior to saccharification (i.e., hydrolysis) and / or fermentation. The pretreatment is preferably carried out prior to hydrolysis. Alternatively, the pretreatment can be carried out simultaneously with enzymatic hydrolysis to release fermentable sugars such as glucose, xylose, and / or cellobiose. In most cases, the pretreatment step itself causes some conversion of the biomass into fermentable sugars (even in the absence of enzymes).

[0267] In one embodiment, the cellulosic material is pretreated with steam. In steam pretreatment, the cellulosic material is heated to disrupt plant cell wall components including lignin, hemicellulose, and cellulose, so that cellulose and other fractions (e.g., hemicellulose) are accessible to enzymes. The cellulosic material passes through or through a reaction vessel, steam is injected into the reaction vessel to increase the temperature to the desired temperature and pressure, and the steam is maintained therein for the desired reaction time. Steam pretreatment is preferably carried out at 140 °C - 250 °C (e.g., 160 °C - 200 °C or 170 °C - 190 °C), where the optimal temperature range depends on the optional addition of a chemical catalyst. The residence time of steam pretreatment is preferably 1 - 60 minutes, such as 1 - 30 minutes, 1 - 20 minutes, 3 - 12 minutes, or 4 - 10 minutes, where the optimal residence time depends on the temperature and the optional addition of a chemical catalyst. Steam pretreatment allows for a relatively high solid loading, such that the cellulosic material generally only becomes moist during pretreatment. Steam pretreatment is often combined with explosive discharge of the pretreated material, which is called steam explosion, i.e., rapid evaporation to atmospheric pressure and turbulence of the material to increase the accessible surface area by fragmentation (Duff and Murray, 1996, Bioresource Technology 855:1 - 33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. 59:618 - 628; U.S. Patent Application No. 2002 / 0164730). During steam pretreatment, hemicellulose acetyl groups are cleaved, and the resulting acid autocatalytically hydrolyzes the hemicellulose fraction into monosaccharides and oligosaccharides. Lignin is removed only to a limited extent.

[0268] In one embodiment, the cellulosic material is subjected to a chemical pretreatment. The term "chemical treatment" refers to any chemical pretreatment that promotes the separation and / or release of cellulose, hemicellulose, and / or lignin. Such a pretreatment can convert crystalline cellulose to amorphous cellulose. Examples of suitable chemical pretreatment methods include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber / freeze explosion (AFEX), ammonia percolation (APR), ionic liquids, and organic solvent pretreatment.

[0269] Sometimes a chemical catalyst (such as H2SO4 or SO2) (typically 0.3 to 5% w / w) is added before steam pretreatment, which reduces the time and temperature, increases the recovery rate, and improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl. Biochem. Biotechnol [Applied Biochemistry and Biotechnology] 129-132:496-508; Varga et al., 2004, Appl. Biochem. Biotechnol. [Applied Biochemistry and Biotechnology] 113-116:509-523; Sassner et al., 2006, Enzyme Microb. Technol. [Enzyme and Microbial Technology] 39:756-762). In dilute acid pretreatment, the cellulosic material is mixed with dilute acid (typically H2SO4) and water to form a slurry, heated to the desired temperature by steam, and flashed to atmospheric pressure after the residence time. Dilute acid pretreatment can be carried out with many reactor designs, for example, plug flow reactors, countercurrent reactors, or continuous countercurrent shrinking bed reactors (Duff and Murray, 1996, Bioresource Technology [Biological Resource Technology] 855:1-33; Schell et al., 2004, Bioresource Technology [Biological Resource Technology] 91:179-188; Lee et al., 1999, Adv. Biochem. Eng. Biotechnol. [Advances in Biochemical Engineering / Biotechnology] 65:93-115). In a specific embodiment, the dilute acid pretreatment of the cellulosic material is carried out at 180 °C with 4% w / w sulfuric acid for 5 minutes.

[0270] Several pretreatment methods under alkaline conditions can also be used. These alkaline pretreatments include, but are not limited to: sodium hydroxide, lime, wet oxidation, ammonia percolation (APR), and ammonia fiber / freeze blasting (AFEX) pretreatment. Lime pretreatment is carried out with calcium oxide or calcium hydroxide at a temperature of 85 °C - 150 °C and a residence time ranging from 1 hour to several days (Wyman et al., 2005, Bioresource Technology 96:1959 - 1966; Mosier et al., 2005, Bioresource Technology 96:673 - 686). WO 2006 / 110891, WO 2006 / 110899, WO 2006 / 110900 and WO 2006 / 110901 disclose pretreatment methods using ammonia.

[0271] Wet oxidation is a thermal pretreatment that is typically carried out at 180 - 200 °C for 5 - 15 minutes in the presence of an oxidizing agent (such as hydrogen peroxide or overpressure oxygen) (Schmidt and Thomsen, 1998, Bioresource Technology 64:139 - 151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117:1 - 17; Varga et al., 2004, Biotechnol. Bioeng. 88:567 - 574; Martin et al., 2006, J. Chem. Technol. Biotechnol. 81:1669 - 1677). The pretreatment is preferably carried out at 1% - 40% dry matter, such as 2% - 30% dry matter, or 5% - 20% dry matter, and the initial pH often increases due to the addition of bases such as sodium carbonate.

[0272] A modified version of the wet oxidation pretreatment method called wet explosion (a combination of wet oxidation and steam explosion) can handle up to 30% dry matter. In wet explosion, after a certain residence time, an oxidizing agent is introduced during the pretreatment. The pretreatment is then ended by flashing to atmospheric pressure (WO2006 / 032282).

[0273] Ammonia fiber explosion (AFEX) involves treating cellulosic materials with liquid or gaseous ammonia for 5 - 10 minutes at moderate temperatures such as 90 °C - 150 °C and high pressures such as 17 - 20 bar, where the dry matter content can be as high as 60% (Gollapalli et al., 2002, Appl. Biochem. Biotechnol. [Applied Biochemistry and Biotechnology] 98:23 - 35; Chundawat et al., 2007, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 96:219 - 231; Alizadeh et al., 2005, Appl. Biochem. Biotechnol. [Applied Biochemistry and Biotechnology] 121:1133 - 1141; Teymouri et al., 2005, Bioresource Technology [Biological Resource Technology] 96:2014 - 2018). During AFEX pretreatment, cellulose and hemicellulose remain relatively intact. Lignin - carbohydrate complexes are cleaved.

[0274] Organosolv pretreatment delignifies cellulosic materials by extraction with aqueous ethanol (40% - 60% ethanol) at 160 °C - 200 °C for 30 - 60 minutes (Pan et al., 2005, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 90:473 - 481; Pan et al., 2006, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 94:851 - 861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. [Applied Biochemistry and Biotechnology] 121:219 - 230). Sulfuric acid is usually added as a catalyst. In organosolv pretreatment, most of the hemicellulose and lignin are removed.

[0275] Other examples of suitable pretreatment methods are described by Schell et al., 2003, Appl. Biochem. Biotechnol. [Applied Biochemistry and Biotechnology] 105 - 108:69 - 85, and Mosier et al., 2005, Bioresource Technology [Biological Resource Technology] 96:673 - 686, as well as US2002 / 0164730.

[0276] In one embodiment, the chemical pretreatment is carried out as a dilute acid treatment, and more preferably as a continuous dilute acid treatment. The acid is typically sulfuric acid, but other acids such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof can also be used. The weak acid treatment is preferably carried out in a pH range of 1 to 5, such as 1 to 4 or 1 to 2.5. In one embodiment, the acid concentration is preferably in the range of from 0.01 wt.% to 10 wt.% acid, such as 0.05 wt.% to 5 wt.% acid or 0.1 wt.% to 2 wt.% acid. The acid is brought into contact with the cellulosic material and maintained at a temperature preferably in the range of 140°C - 200°C (such as 165°C - 190°C) for a time in the range of from 1 to 60 minutes.

[0277] In another embodiment, the pretreatment is carried out in an aqueous slurry. In a preferred embodiment, the cellulosic material is present in an amount preferably between 10 wt.% - 80 wt.%, such as 20 wt.% - 70 wt.% or 30 wt.% - 60 wt.%, about 40 wt.% during the pretreatment. The pretreated cellulosic material may or may not be washed, or washed using any method known in the art, for example, washed with water.

[0278] In one embodiment, the cellulosic material is subjected to mechanical or physical pretreatment. The term "mechanical pretreatment" or "physical pretreatment" refers to any pretreatment that promotes particle size reduction. For example, such pretreatment can involve different types of grinding or milling (e.g., dry grinding, wet grinding, or vibratory ball milling).

[0279] The cellulosic material can be physically (mechanically) and chemically pretreated. The mechanical or physical pretreatment can be combined with steam / steam explosion, hydrothermolysis, dilute acid or weak acid treatment, high temperature, high pressure treatment, radiation (e.g., microwave radiation), or combinations thereof. In one embodiment, high pressure means a pressure in the range of preferably about 100 to about 400 psi, such as about 150 to about 250 psi. In another embodiment, high temperature means a temperature in the range of about 100°C to about 300°C, such as about 140°C to about 200°C. In a preferred embodiment, the mechanical or physical pretreatment is carried out in a batch process using a steam gun hydrolyzer system, such as the Sunds Hydrolyzer available from Sunds Defibrator AB, Sweden, which uses high pressure and high temperature as defined above. Physical and chemical pretreatments can be carried out sequentially or simultaneously as needed.

[0280] Thus, in one embodiment, the cellulosic material is subjected to a physical (mechanical) or chemical pretreatment, or any combination thereof, to facilitate the separation and / or release of cellulose, hemicellulose, and / or lignin.

[0281] In one embodiment, the cellulosic material is subjected to a biological pretreatment. The term "biological pretreatment" refers to any biological pretreatment that promotes the separation and / or release of cellulose, hemicellulose, and / or lignin from the cellulosic material. Biological pretreatment techniques can involve the application of lignin-dissolving microorganisms and / or enzymes (see, e.g., Hsu, T.-A., 1996, Pretreatment of biomass, in Handbook on Bioethanol: Production and Utilization, Wyman, C.E. ed., Taylor & Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993, Adv. Appl. Microbiol. 39:295-333; McMillan, J.D., 1994, Pretreating lignocellulosic biomass: a review, in Enzymatic Conversion of Biomass for Fuels Production, Himmel, M.E., Baker, J.O. and Overend, R.P. eds., ACS Symposium Series 566, American Chemical Society, Washington, D.C., Chapter 15; Gong, C.S., Cao, N.J., Du, J. and Tsao, G.T., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering / Biotechnology, Scheper, T. ed., Springer-Verlag, Berlin, Heidelberg, Germany, 65:207-241; Olsson and Hahn-Hagerdal, 1996, Enz. Microb. Tech. 18:312-331; and Vallander and Eriksson, 1990, Adv. Biochem. Eng. / Biotechnol. 42:63-95).

[0282] Saccharification and fermentation of cellulose-containing material

[0283] Separate or simultaneous saccharification (i.e., hydrolysis) and fermentation include, but are not limited to: separate hydrolysis and fermentation (SHF); simultaneous saccharification and fermentation (SSF); simultaneous saccharification and co-fermentation (SSCF); hybrid hydrolysis and fermentation (HHF); separated hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF).

[0284] SHF uses separate processing steps to first enzymatically hydrolyze the cellulosic material into fermentable sugars (e.g., glucose, cellobiose, and pentose monomers), and then ferment the fermentable sugars into ethanol. In SSF, the enzymatic hydrolysis of the cellulosic material and the fermentation of sugars into ethanol are combined in one step (Philippidis, G.P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, edited by Wyman, C.E., Taylor & Francis, Washington, D.C., 179 - 212). SSCF involves the co-fermentation of multiple sugars (Sheehan and Himmel, 1999, Biotechnol. Prog. 15:817 - 827). HHF involves a separate hydrolysis step and additionally involves simultaneous saccharification and hydrolysis steps, which can be carried out in the same reactor. The steps in the HHF process can be carried out at different temperatures, i.e., high-temperature enzymatic saccharification followed by SSF at a lower temperature tolerated by the fermenting organisms. It should be understood herein that any method known in the art that includes pretreatment, enzymatic hydrolysis (saccharification), fermentation, or a combination thereof can be used to implement the methods described herein.

[0285] Conventional devices can include fed-batch stirred reactors, batch stirred reactors, continuous flow stirred reactors with ultrafiltration, and / or continuous plug flow column reactors (de Castilhos Corazza et al., 2003, Acta Scientiarum. Technology 25:33-38; Gusakov and Sinitsyn, 1985, Enz. Microb. Technol. 7:346-352), attrition reactors (Ryu and Lee, 1983, Biotechnol. Bioeng. 25:53-65). Additional reactor types include: fluidized beds for hydrolysis and / or fermentation, upflow blanket reactors, immobilized reactors, and extruder-type reactors.

[0286] In the saccharification step (i.e., hydrolysis step), the cellulosic material and / or starchy material (e.g., pretreated) is hydrolyzed to break down cellulose, hemicellulose, and / or starch into fermentable sugars such as glucose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or soluble oligosaccharides. Hydrolysis is carried out enzymatically by, for example, a cellulolytic enzyme composition. The enzymes of these compositions can be added simultaneously or sequentially.

[0287] Enzymatic hydrolysis can be carried out in a suitable aqueous environment under conditions readily determinable by one of ordinary skill in the art. In one embodiment, hydrolysis is carried out under conditions suitable for the activity of one or more enzymes, i.e., under optimal conditions for the one or more enzymes. Hydrolysis can be carried out in a fed-batch or continuous process, where the cellulosic material and / or starchy material is gradually fed into, for example, a hydrolysis solution containing the enzyme.

[0288] Saccharification is typically carried out in a stirred tank reactor or fermenter under controlled pH, temperature, and mixing conditions. Suitable processing times, temperatures, and pH conditions can be readily determined by one of ordinary skill in the art. For example, saccharification can be carried out for up to 200 hours, but is typically carried out preferably for about 12 to about 120 hours, such as about 16 to about 72 hours or about 24 to about 48 hours. The temperature ranges preferably from about 25°C to about 70°C, such as about 30°C to about 65°C, about 40°C to about 60°C, or about 50°C to 55°C. The pH ranges preferably from about 3 to about 8, such as about 3.5 to about 7, about 4 to about 6, or about 4.5 to about 5.5. The dry solids content is preferably about 5 wt.% to about 50 wt.%, such as about 10 wt.% to about 40 wt.%, or about 20 wt.% to about 30 wt.%.

[0289] Saccharification can be carried out using a cellulase composition. Such enzyme compositions are described in the "Cellulase Composition" section below. These cellulase compositions can contain any protein for degrading the cellulosic material. In one embodiment, the cellulase composition contains or further contains one or more (e.g., several) proteins selected from the group consisting of: cellulase, AA9 (GH61) polypeptide, hemicellulase, esterase, expansin, lignin-degrading enzyme, oxidoreductase, pectinase, protease, and swelling factor.

[0290] In another embodiment, the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of: endoglucanase, cellobiohydrolase, and β-glucosidase.

[0291] In another embodiment, the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of: acetylmannan esterase, acetylxylan esterase, arabinase, arabinofuranosidase, coumaric acid esterase, ferulic acid esterase, galactosidase, glucuronidase, glucuronate esterase, mannanase, mannosidase, xylanase, and xylosidase. In another embodiment, the oxidoreductase is one or more (e.g., several) enzymes selected from the group consisting of: catalase, laccase, and peroxidase.

[0292] The enzyme or enzyme composition used in the method of the present invention can exist in any suitable form for use, such as a fermentation broth formulation or cell composition, cell lysate with or without cell debris, semi-purified or purified enzyme preparation, or host cell as a source of the enzyme. The enzyme composition can be a dry powder or granule, dust-free granule, liquid, stabilized liquid or stabilized and protected enzyme. The liquid enzyme preparation can be stabilized according to established methods, such as by adding stabilizers (such as sugars, sugar alcohols or other polyols), and / or lactic acid or another organic acid.

[0293] In one embodiment, the effective amount of the cellulase composition or hemicellulase composition for the cellulosic material is about 0.5 mg to about 50 mg, for example, about 0.5 mg to about 40 mg, about 0.5 mg to about 25 mg, about 0.75 mg to about 20 mg, about 0.75 mg to about 15 mg, about 0.5 mg to about 10 mg, or about 2.5 mg to about 10 mg / g of the cellulosic material.

[0294] In one embodiment, the compound is added at the following molar ratio of the glucosyl unit of cellulose to the compound: about 10 -6 to about 10, for example about 10 -6 to about 7.5, about 10-6 to about 5, about 10 -6 to about 2.5, about 10 -6 to about 1, about 10 -5 to about 1, about 10 -5 to about 10 -1 , about 10 -4 to about 10 -1 , about 10 -3 to about 10 -1 , or about 10 -3 to about 10 -2 . In another embodiment, an effective amount of such a compound is from about 0.1 μM to about 1 M, such as from about 0.5 μM to about 0.75 M, from about 0.75 μM to about 0.5 M, from about 1 μM to about 0.25 M, from about 1 μM to about 0.1 M, from about 5 μM to about 50 mM, from about 10 μM to about 25 mM, from about 50 μM to about 25 mM, from about 10 μM to about 10 mM, from about 5 μM to about 5 mM or from about 0.1 mM to about 1 mM.

[0295] The term "liquor" means the solution phase (aqueous phase, organic phase or a combination thereof) and its soluble contents produced by treating lignocellulosic and / or hemicellulosic materials, or their monosaccharides (e.g., xylose, arabinose, mannose, etc.) in a pulp under the conditions described in WO2012 / 021401. The liquor can be produced by treating the lignocellulosic or hemicellulosic material (or feedstock) by heating and / or pressurizing, optionally in the presence of a catalyst such as an acid, optionally in the presence of an organic solvent, and optionally in combination with physical disruption of the material, and then separating the solution from the residual solids to produce a liquor for enhancing the cellulolysis of AA9 polypeptides (GH61 polypeptides). The degree of enhanced cellulolysis obtainable from the combination of the liquor and the AA9 polypeptide during the hydrolysis of a cellulose substrate by a cellulase preparation is determined by such conditions. Standard methods in the art, such as filtration, precipitation or centrifugation, can be used to separate the liquor from the treated material.

[0296] In one embodiment, an effective amount of the liquor for cellulose is about 10 -6 to about 10 g / g of cellulose, such as about 10 -6 to about 7.5 g, about 10 -6 to about 5 g, about 10 -6 to about 2.5 g, about 10 -6 to about 1 g, about 10 -5 to about 1 g, about 10 -5 to about 10 -1 g, about 10 -4 to about 10 -1 g, about 10 -3from about 0 to about 10 -1 g, or about 10 -3 to about 10 -2 g / g of cellulose.

[0297] In the fermentation step, sugars released from the cellulosic material, for example as a result of a pretreatment and enzymatic hydrolysis step, are fermented to ethanol by a fermenting organism such as the yeast described herein. Hydrolysis (saccharification) and fermentation can be separate or simultaneous.

[0298] Any suitable hydrolyzed cellulosic material can be used in the fermentation step of the methods described herein. Such feedstocks include, but are not limited to, carbohydrates (e.g., lignocellulose, xylan, cellulose, starch, etc.). The material is typically selected based on economics, i.e., the cost per equivalent sugar potential, and recalcitrance to enzymatic conversion.

[0299] Production of ethanol from cellulosic material by a fermenting organism is produced by the metabolism of sugars (monosaccharides). The sugar composition of the hydrolyzed cellulosic material and the ability of the fermenting organism to utilize different sugars have a direct impact on process yield.

[0300] The composition of the fermentation medium and the fermentation conditions depend on the fermenting organism and can be readily determined by those skilled in the art. Typically, fermentation is carried out under conditions known to be suitable for producing the fermentation product. In some embodiments, the fermentation process is carried out under aerobic or microaerophilic conditions (i.e., oxygen concentration less than the oxygen concentration in air) or anaerobic conditions. In some embodiments, the fermentation is carried out under anaerobic conditions (i.e., no detectable oxygen) or in oxygen at less than about 5, about 2.5, or about 1 mmol / L / h. In the absence of oxygen, NADH produced in glycolysis cannot be oxidized by oxidative phosphorylation. Under anaerobic conditions, the fermenting organism can utilize pyruvate or its derivatives as electron and hydrogen acceptors to produce NAD+.

[0301] The fermentation process is typically carried out at a temperature optimal for the cells. For example, in some embodiments, the fermentation process is carried out at a temperature in the range of about 25°C to about 42°C. Generally, the method is carried out at a temperature below about 38°C, below about 35°C, below about 33°C, or below about 38°C, but at least about 20°C, 22°C, or 25°C.

[0302] Fermentation stimulants can be used in the methods described herein to further improve fermentation, and in particular to improve the performance of the fermenting organism, such as increased rate and product yield (e.g., ethanol yield). A "fermentation stimulant" refers to a stimulant for the growth of a fermenting organism (particularly yeast). Preferred fermentation stimulants for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenic acid, niacin, inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and vitamins A, B, C, D, and E. See, for example, Alfenore et al., Improving ethanol production and viability of Saccharomyces cerevisia by a vitamin feeding strategy during fed-batch process, Springer-Verlag (2002), which is hereby incorporated by reference. Examples of minerals include minerals and mineral salts that can supply nutrients containing P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.

[0303] Cellulolytic enzyme and composition

[0304] Cellulolytic enzymes or cellulolytic enzyme compositions can be present and / or added during the saccharification process. A cellulolytic enzyme composition is an enzyme preparation comprising one or more (e.g., several) enzymes that hydrolyze cellulosic materials. Such enzymes include endoglucanases, cellobiohydrolases, β-glucosidases, and / or combinations thereof.

[0305] In some embodiments, the fermenting organism comprises one or more (e.g., several) heterologous polynucleotides encoding enzymes (e.g., endoglucanases, cellobiohydrolases, β-glucosidases, or combinations thereof) that can hydrolyze cellulosic materials. Any enzyme (capable of hydrolyzing cellulosic materials) described or referenced herein is contemplated for expression in the fermenting organism.

[0306] The cellulolytic enzyme can be any cellulolytic enzyme (e.g., endoglucanase, cellobiohydrolase, β-glucosidase) suitable for expression in the fermenting organism and / or methods described herein, such as a naturally occurring cellulolytic enzyme or a variant thereof that retains cellulolytic enzyme activity.

[0307] In some embodiments, when cultured under the same conditions, a fermenting organism comprising a heterologous polynucleotide encoding a cellulase has an increased level of cellulase (e.g., increased endoglucanase, cellobiohydrolase, and / or β-glucosidase) activity compared to a fermenting organism that does not have the heterologous polynucleotide encoding a cellulase. In some embodiments, when cultured under the same conditions, the fermenting organism has an increased cellulase activity level of at least 5%, such as at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500% compared to a fermenting organism that does not have the heterologous polynucleotide encoding a cellulase.

[0308] Exemplary cellulases that can be used with the fermenting organisms and / or methods described herein include bacterial, yeast, or filamentous fungal cellulases, e.g., obtained from any of the microorganisms described or referenced herein, such as those described above under the section related to proteases.

[0309] The cellulase can be of any origin. In one embodiment, the cellulase is derived from a strain of Trichoderma, such as a strain of Trichoderma reesei; a strain of Humicola, such as a strain of Humicola insolens, and / or a strain of Chrysosporium, such as a strain of Chrysosporium lucknowense. In a preferred embodiment, the cellulase is derived from a strain of Trichoderma reesei.

[0310] The cellulase composition can further comprise one or more of the following polypeptides (such as enzymes): an AA9 polypeptide (GH61 polypeptide) having enhanced cellulolytic activity, β-glucosidase, xylanase, β-xylosidase, CBHI, CBHII, or a mixture of two, three, four, five, or six thereof.

[0311] One or more additional polypeptides (e.g., AA9 polypeptide) and / or one or more enzymes (e.g., β-glucosidase, xylanase, β-xylosidase, CBHI, and / or CBHII) can be exogenous to the cellulase composition-producing organism (e.g., Trichoderma reesei).

[0312] In one embodiment, the cellulase composition comprises an AA9 polypeptide having enhanced cellulolytic activity and β-glucosidase.

[0313] In another embodiment, the cellulase composition comprises an AA9 polypeptide having enhanced cellulolytic activity, β-glucosidase, and CBHI.

[0314] In another embodiment, the cellulase composition comprises an AA9 polypeptide having enhanced cellulolytic activity, a β-glucosidase, CBHI, and CBHII.

[0315] Other enzymes, such as endoglucanases, may also be included in the cellulase composition.

[0316] As mentioned above, the cellulase composition may comprise a variety of different polypeptides, including enzymes.

[0317] In one embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises an AA9 (GH61A) polypeptide from Thermoascus aurantiacus having enhanced cellulolytic activity (e.g., WO 2005 / 074656), and an Aspergillus oryzae β-glucosidase fusion protein (e.g., one disclosed in WO2008 / 057637, particularly as shown in SEQ ID NO:59 and 60).

[0318] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further includes an AA9 (GH61A) polypeptide from Thermoascus aurantiacus having enhanced cellulolytic activity (e.g., SEQ ID NO:2 in WO 2005 / 074656) and an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO:2 of WO2005 / 047499).

[0319] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises an AA9 (GH61A) polypeptide from Penicillium emersonii having enhanced cellulolytic activity, particularly one disclosed in WO 2011 / 041397, and an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO:2 of WO2005 / 047499).

[0320] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises an AA9 (GH61A) polypeptide from Penicillium emersonii having enhanced cellulolytic activity, particularly one disclosed in WO 2011 / 041397, and an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO:2 of WO2005 / 047499), or a variant disclosed in WO 2012 / 044915 (incorporated herein by reference), particularly a variant comprising one or more (e.g., all) of the following substitutions: F100D, S283G, N456E, F512Y.

[0321] In one embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, and the Trichoderma reesei cellulase composition further comprises an AA9 (GH61A) polypeptide having enhanced cellulolytic activity (particularly one derived from a Penicillium emersonii strain (e.g., SEQ ID NO:2 in WO 2011 / 041397)), an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO:2 in WO 2005 / 047499) variant having one or more (particularly all) of the following substitutions: F100D, S283G, N456E, F512Y and disclosed in WO 2012 / 044915; Aspergillus fumigatus Cel7A CBH1, such as one disclosed as SEQ ID NO:6 in WO 2011 / 057140 and Aspergillus fumigatus CBH II, such as one disclosed as SEQ ID NO:18 in WO 2011 / 057140.

[0322] In a preferred embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, and the Trichoderma reesei cellulase composition further comprises a hemicellulase or hemicellulose-degrading enzyme composition, such as Aspergillus fumigatus xylanase and Aspergillus fumigatus β-xylosidase.

[0323] In one embodiment, the cellulase composition further comprises xylanase (e.g., derived from a strain of the genus Aspergillus, particularly Aspergillus aculeatus or Aspergillus fumigatus; or the genus Talaromyces, particularly Talaromyces reesei) and / or β-xylosidase (e.g., derived from a strain of the genus Aspergillus, particularly Aspergillus fumigatus, or the genus Talaromyces, particularly Talaromyces emersonii).

[0324] In one embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, and the Trichoderma reesei cellulase composition further comprises a thermoascus AA9 (GH61A) polypeptide having enhanced cellulolytic activity (e.g., WO 2005 / 074656), an Aspergillus oryzae β-glucosidase fusion protein (e.g., one disclosed in WO 2008 / 057637, particularly such as SEQ ID NO:59 and 60), and Aspergillus aculeatus xylanase (e.g., Xyl II in WO 94 / 21785).

[0325] In another embodiment, the cellulase composition comprises a Trichoderma reesei cellulolytic preparation, which further comprises an AA9 (GH61A) polypeptide from Thermoascus aurantiacus having enhanced cellulolytic activity (e.g., SEQ ID NO:2 in WO 2005 / 074656), an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO:2 of WO 2005 / 047499), and an Aspergillus aculeatus xylanase (XylII disclosed in WO 94 / 21785).

[0326] In another embodiment, the cellulase composition comprises a Trichoderma reesei cellulase composition, which further comprises an AA9 (GH61A) polypeptide from Thermoascus aurantiacus having enhanced cellulolytic activity (e.g., SEQ ID NO:2 in WO 2005 / 074656), an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO:2 of WO 2005 / 047499), and an Aspergillus aculeatus xylanase (e.g., XylII disclosed in WO 94 / 21785).

[0327] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises an AA9 (GH61A) polypeptide from Penicillium emersonii having enhanced cellulolytic activity, particularly one disclosed in WO 2011 / 041397, an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO:2 of WO 2005 / 047499), and an Aspergillus fumigatus xylanase (e.g., Xyl III in WO 2006 / 078256).

[0328] In another embodiment, the cellulase composition comprises a Trichoderma reesei cellulase composition, which further comprises an AA9 (GH61A) polypeptide from Penicillium emersonii having enhanced cellulolytic activity, particularly one disclosed in WO 2011 / 041397, an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO:2 of WO 2005 / 047499), an Aspergillus fumigatus xylanase (e.g., XylIII in WO 2006 / 078256), and a CBHI from Aspergillus fumigatus, particularly Cel7A CBH1 disclosed as SEQ ID NO:2 in WO 2011 / 057140.

[0329] In another embodiment, the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic enzyme composition, and the Trichoderma reesei cellulolytic enzyme composition further comprises a Penicillium emersonii AA9 (GH61A) polypeptide having enhanced cellulolytic activity, in particular one disclosed in WO 2011 / 041397, an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO:2 of WO 2005 / 047499), an Aspergillus fumigatus xylanase (e.g., Xyl III in WO 2006 / 078256), CBH I from Aspergillus fumigatus, in particular Cel7A CBH1 disclosed as SEQ ID NO:2 in WO 2011 / 057140, and a CBH II derived from Aspergillus fumigatus, in particular one disclosed as SEQ ID NO:4 in WO 2013 / 028928.

[0330] In another embodiment, the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic enzyme composition, and the Trichoderma reesei cellulolytic enzyme composition further comprises a Penicillium emersonii AA9 (GH61A) polypeptide having enhanced cellulolytic activity (in particular one disclosed in WO2011 / 041397), an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO:2 of WO 2005 / 047499) or a variant thereof having one or more (in particular all) of the following substitutions: F100D, S283G, N456E, F512Y; an Aspergillus fumigatus xylanase (e.g., Xyl III in WO 2006 / 078256), CBH I from Aspergillus fumigatus (in particular Cel7A CBHI disclosed as SEQ ID NO:2 in WO 2011 / 057140), and a CBH II derived from Aspergillus fumigatus (in particular one disclosed in WO2013 / 028928).

[0331] In another embodiment, the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic enzyme composition, and the Trichoderma reesei cellulolytic enzyme composition comprises CBHI (GENSEQP accession number AZY49536 (WO 2012 / 103293)); CBHII (GENSEQP accession number AZY49446 (WO 2012 / 103288)); a β-glucosidase variant (GENSEQP accession number AZU67153 (WO2012 / 44915)), in particular having one or more (in particular all) of the following substitutions: F100D, S283G, N456E, F512Y; and an AA9 (GH61 polypeptide) (GENSEQP accession number BAL61510 (WO2013 / 028912)).

[0332] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, and the Trichoderma reesei cellulase composition comprises CBHI (GENSEQP accession number AZY49536 (WO 2012 / 103293)); CBHII (GENSEQP accession number AZY49446 (WO 2012 / 103288)); GH10 xylanase (GENSEQP accession number BAK46118 (WO2013 / 019827)); and β-xylosidase (GENSEQP accession number AZI04896 (WO2011 / 057140)).

[0333] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, and the Trichoderma reesei cellulase composition comprises CBHI (GENSEQP accession number AZY49536 (WO 2012 / 103293)); CBHII (GENSEQP accession number AZY49446 (WO 2012 / 103288)); and AA9 (GH61 polypeptide; GENSEQP accession number BAL61510 (WO2013 / 028912)).

[0334] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, and the Trichoderma reesei cellulase composition comprises CBHI (GENSEQP accession number AZY49536 (WO 2012 / 103293)); CBHII (GENSEQP accession number AZY49446 (WO 2012 / 103288)), AA9 (GH61 polypeptide; GENSEQP accession number BAL61510 (WO2013 / 028912)), and catalase (GENSEQP accession number BAC11005 (WO2012 / 130120)).

[0335] In one embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, and the Trichoderma reesei cellulase composition comprises CBH I (GENSEQP accession number AZY49446 (WO2012 / 103288)); CBHII (GENSEQP accession number AZY49446 (WO2012 / 103288)), a β-glucosidase variant (GENSEQP accession number AZU67153 (WO2012 / 44915)) having one or more (especially all) of the following substitutions: F100D, S283G, N456E, F512Y; AA9 (GH61 polypeptide; GENSEQP accession number BAL61510 (WO2013 / 028912)), a GH10 xylanase (GENSEQP accession number BAK46118 (WO2013 / 019827)), and a β-xylosidase (GENSEQP accession number AZI04896 (WO2011 / 057140)).

[0336] In one embodiment, the cellulolytic composition is a Trichoderma reesei cellulase preparation, and the Trichoderma reesei cellulase preparation comprises EG I (Swissprot accession number P07981), EGII (EMBL accession number M19373), CBHI (see above); CBH II (see above); a β-glucosidase variant (see above) having the following substitutions: F100D, S283G, N456E, F512Y; AA9 (GH61 polypeptide; see above), a GH10 xylanase (see above); and a β-xylosidase (see above).

[0337] All cellulase compositions disclosed in WO 2013 / 028928 are also contemplated and hereby incorporated by reference.

[0338] The cellulase composition comprises or may further comprise one or more (several) proteins selected from the group consisting of: cellulases, AA9 (i.e., GH61) polypeptides having enhanced cellulolytic activity, hemicellulases, expansins, esterases, laccases, lignin-degrading enzymes, pectinases, peroxidases, proteases, and swelling factors.

[0339] In one embodiment, the cellulase composition is a commercial cellulase composition. Examples of commercial cellulase compositions suitable for the methods of the present invention include: CTec (Novozymes A / S), CTec2 (Novozymes A / S), CTec3 (Novozymes A / S), CELLUCLAST TM (Novozymes A / S), SPEZYMETM CP (Genencor Int.), ACCELLERASE TM 1000, ACCELLERASE 1500, ACCELLERASE TM TRIO (DuPont), NL (DSM); S / L 100 (DSM), ROHAMENT TM 7069W (Röhm GmbH), or GmbH)), or CMAX3 TM (Dyadic International, Inc.). The cellulase composition can be added in an effective amount of from about 0.001 wt.% to about 5.0 wt.% solids, for example, from about 0.025 wt.% to about 4.0 wt.% solids, or from about 0.005 wt.% to about 2.0 wt.% solids.

[0340] Additional enzymes and their compositions can be found in WO 2011 / 153516 and WO 2016 / 045569, the contents of which are incorporated herein.

[0341] Additional polynucleotides encoding suitable cellulases can be obtained from microorganisms of any genus, including those readily available in the UniProtKB database (www.uniprot.org).

[0342] These cellulase coding sequences can also be used to design nucleic acid probes to identify and clone DNA encoding cellulases from strains of different genera or species known in the art.

[0343] Polynucleotides encoding cellulases can also be identified and obtained from other sources, including microorganisms isolated from nature (e.g., soil, compost, water, etc.) known in the art or DNA samples directly obtained from natural materials (e.g., soil, compost, water, etc.).

[0344] Techniques for isolating or cloning polynucleotides encoding cellulases are known in the art.

[0345] In one embodiment, the cellulolytic enzyme has a mature polypeptide sequence having at least 60%, such as at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with any cellulolytic enzyme described or referenced herein (e.g., any endoglucanase, cellobiohydrolase, or β-glucosidase). In one embodiment, the cellulolytic enzyme has a mature polypeptide sequence that differs from any cellulolytic enzyme described or referenced herein by no more than ten amino acids, such as by no more than five amino acids, by no more than four amino acids, by no more than three amino acids, by no more than two amino acids, or by no more than one amino acid. In one embodiment, the cellulolytic enzyme has a mature polypeptide sequence that comprises or consists of: the amino acid sequence of any cellulolytic enzyme described or referenced herein, allelic variants, or fragments thereof having cellulolytic enzyme activity. In one embodiment, the cellulolytic enzyme has one or more (e.g., two, several) amino acid substitutions, deletions and / or insertions. In some embodiments, the total number of amino acid substitutions, deletions and / or insertions does not exceed 10, such as does not exceed 9, 8, 7, 6, 5, 4, 3, 2 or 1.

[0346] In some embodiments, under the same conditions, the cellulolytic enzyme has at least 20%, such as at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the cellulolytic enzyme activity of any cellulolytic enzyme described or referenced herein (e.g., any endoglucanase, cellobiohydrolase, or β-glucosidase).

[0347] In one embodiment, the cellulolytic enzyme coding sequence hybridizes under at least low stringency conditions, such as medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions, to the full-length complementary strand of the coding sequence of any cellulolytic enzyme described or referenced herein (e.g., any endoglucanase, cellobiohydrolase or β-glucosidase). In one embodiment, the cellulolytic enzyme coding sequence has at least 65%, such as at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with the coding sequence of any cellulolytic enzyme described or referenced herein.

[0348] In one embodiment, the polynucleotide encoding the cellulase comprises the coding sequence of any cellulase described or referenced herein (e.g., any endoglucanase, cellobiohydrolase, or β-glucosidase). In one embodiment, the polynucleotide encoding the cellulase comprises a subsequence of the coding sequence of any cellulase described or referenced herein, wherein the subsequence encodes a polypeptide having cellulase activity. In one embodiment, the number of nucleotide residues in the subsequence is at least 75% of the number of the coding sequence, such as at least 80%, 85%, 90%, or 95%.

[0349] The cellulase may also include a fusion polypeptide or a cleavable fusion polypeptide.

[0350] Fermentation product

[0351] The fermentation product can be any substance obtained by fermentation. The fermentation product can be, but is not limited to: alcohols (e.g., arabitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propylene glycol], butanediol, glycerin, sorbitol, and xylitol); alkanes (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane), cycloalkanes (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane), alkenes (e.g., pentene, hexene, heptene, and octene); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); gases (e.g., methane, hydrogen (H2), carbon dioxide (CO2), and carbon monoxide (CO)); isoprene; ketones (e.g., acetone); organic acids (e.g., acetic acid, acetoacetic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylonic acid); and polyketides.

[0352] In one embodiment, the fermentation product is an alcohol. The term "alcohol" encompasses substances containing one or more hydroxyl moieties. The alcohol can be, but is not limited to: n-butanol, isobutanol, ethanol, methanol, arabitol, butanediol, ethylene glycol, glycerol, propanetriol, 1,3-propanediol, sorbitol, xylitol. See, for example, Gong et al., 1999, Ethanol production from renewable resources, in Advances in Biochemical Engineering / Biotechnology, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65:207-241; Silveira and Jonas, 2002, Appl. Microbiol. Biotechnol. 59:400-408; Nigam and Singh, 1995, Process Biochemistry 30(2):117-124; Ezeji et al., 2003, World Journal of Microbiology and Biotechnology 19(6):595-603. In one embodiment, the fermentation product is ethanol.

[0353] In another embodiment, the fermentation product is an alkane. The alkane can be a non-branched or branched alkane. The alkane can be, but is not limited to: pentane, hexane, heptane, octane, nonane, decane, undecane or dodecane.

[0354] In another embodiment, the fermentation product is a cycloalkane. The cycloalkane can be, but is not limited to: cyclopentane, cyclohexane, cycloheptane or cyclooctane.

[0355] In another embodiment, the fermentation product is an alkene. The alkene can be a non-branched or branched alkene. The alkene can be, but is not limited to: pentene, hexene, heptene or octene.

[0356] In another embodiment, the fermentation product is an amino acid. The organic acid can be, but is not limited to: aspartic acid, glutamic acid, glycine, lysine, serine or threonine. See, for example Richard and Margaritis, 2004, Biotechnology and Bioengineering 87(4):501-515.

[0357] In another embodiment, the fermentation product is a gas. The gas can be, but is not limited to: methane, H2, CO2, or CO. See, for example, Kataoka et al., 1997, Water Science and Technology 36(6-7):41-47; and Gunaseelan, 1997, Biomass and Bioenergy 13(1-2):83-114.

[0358] In another embodiment, the fermentation product is isoprene.

[0359] In another embodiment, the fermentation product is a ketone. The term "ketone" encompasses substances containing one or more ketone moieties. The ketone can be, but is not limited to: acetone.

[0360] In another embodiment, the fermentation product is an organic acid. The organic acid can be, but is not limited to: acetic acid, acetone acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, or xylonic acid. See, for example, Chen and Lee, 1997, Appl. Biochem. Biotechnol. 63-65:435-448.

[0361] In another embodiment, the fermentation product is a polyketide.

[0362] Recovery

[0363] Any method known in the art can be used to optionally recover the fermentation product (e.g., ethanol) from the fermentation medium, and the method includes, but is not limited to: chromatography, electrophoresis procedures, differential solubility, distillation, or extraction. For example, alcohols are separated and purified from fermented cellulosic materials by conventional distillation methods. Ethanol with a purity of up to about 96 vol.% can be obtained, which can be used, for example, as fuel ethanol, drinking ethanol (i.e., potable neutral alcoholic beverages), or industrial ethanol.

[0364] In some embodiments of these methods, the recovered fermentation product is substantially pure. For these methods herein, "substantially pure" means that the recovered preparation contains no more than 15% impurities, where impurities mean compounds other than the fermentation product (e.g., ethanol). In one variant, a substantially pure preparation is provided, wherein the preparation contains no more than 25% impurities, or no more than 20% impurities, or no more than 10% impurities, or no more than 5% impurities, or no more than 3% impurities, or no more than 1% impurities, or no more than 0.5% impurities.

[0365] Suitable assays can be performed using methods known in the art to test for ethanol and contaminant production and sugar consumption. For example, ethanol products and other organic compounds can be analyzed by methods such as HPLC (high performance liquid chromatography), GC-MS (gas chromatography - mass spectrometry), and LC-MS (liquid chromatography - mass spectrometry), or other suitable analytical methods using conventional procedures well known in the art. The release of ethanol in the fermentation broth can also be tested using the culture supernatant. By HPLC (Lin et al., Biotechnol. Bioeng. [Biotechnology and Bioengineering] 90:775 - 779 (2005)), for example, a refractive index detector for glucose and alcohols, and a UV detector for organic acids, or other suitable assays and detection methods well known in the art can be used to quantify by-products and residual sugars (e.g., glucose or xylose) in the fermentation medium.

[0366] Biological Material Deposits

[0367] The following biological materials have been deposited under the terms of the Budapest Treaty at the Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL), 1815 University Street, Peoria, Illinois, USA, and the following accession numbers have been given:

[0368]

[0369] In accordance with the provisions of the Budapest Treaty, the following biological materials have been deposited at the National Measurement Institute, Victoria, Australia, and have the following accession numbers:

[0370]

[0371]

[0372] The strain was deposited under conditions that ensure that during the pendency of this patent application, the culture is accessible to persons authorized by the Patent and Trademark Commissioner under 37 C.F.R. § 1.14 and 35 U.S.C. § 122. These deposits represent substantially pure cultures of the deposited strains. Deposits are required in some countries in accordance with foreign patent laws, and a copy of this subject application, or a subsequent text thereof, is to be filed in those countries. However, it should be understood that the availability of the deposits does not constitute a license to practice the subject matter of this invention for uses permitted by the patent rights granted by government action.

[0373] The invention described and claimed herein is not limited to the scope of the specific aspects or embodiments disclosed herein, as these aspects / embodiments are intended to be illustrative of several aspects of the invention. Any equivalent aspects are intended to be within the scope of the 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 case of conflict, the present disclosure, including definitions, will control. All references are hereby incorporated by reference specifically for the description.

[0374] The following examples are provided to illustrate certain aspects / embodiments of the invention, but are not intended to limit in any way the scope of the claimed invention.

[0375] Examples

[0376] Example 1: Production of Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5318 and MBG5307

[0377] The Saccharomyces cerevisiae strains MBG5225, MBG5227, MBG5307, and MBG5318 are derived from breeding and evolution programs (e.g., according to the breeding procedures described in U.S. Patent No. 8,257,959), the goal of which is to improve fermentation performance for characteristics important for the industrial production of corn ethanol. The resulting strains were screened to obtain improved ethanol titers, increased temperature tolerance, increased sugar consumption, higher ethanol-to-glycerol ratios, and reduced by-products, thereby maximizing ethanol yields from corn mash fermentation.

[0378] Example 2: Fermentation of Saccharomyces cerevisiae strain MBG5225 under non-stress conditions

[0379] Saccharomyces cerevisiae strain MBG5225 and Ethanol Fermented under the following conditions:

[0380] Mash: Liquozyme SC 4X

[0381] Solids: 32.7%

[0382] pH: 5.0

[0383] Glucoamylase: Spirizyme Achieve T (Novozymes)

[0384] Glucoamylase dosage: 0.6 AGU / g DS

[0385] Fermentation time: 54 h

[0386] Temperature: 32 °C

[0387] Scale: 80 g conical flask

[0388] As Figure 1 AndFigure 2 As shown, compared with Ethanol the Saccharomyces cerevisiae strain MBG5225 exhibits increased ethanol productivity and reduced glycerol levels.

[0389] Example 3: Fermentation of Saccharomyces cerevisiae strain MBG5227 under non-stress conditions

[0390] The Saccharomyces cerevisiae strains MBG5227 and Ethanol were fermented under the following conditions:

[0391] Mash: Liquozyme SC 4X

[0392] Solids: 32.7%

[0393] pH: 5.0

[0394] Glucoamylase: Spirizyme Achieve T (Novozymes A / S)

[0395] Glucoamylase dosage: 0.6 AGU / g DS

[0396] Fermentation time: 54 h

[0397] Temperature: 32 °C

[0398] Scale: 80 g conical flask

[0399] As Figure 3 and Figure 4 shown, compared with Ethanol the Saccharomyces cerevisiae strain MBG5227 exhibits increased ethanol productivity and reduced glycerol levels.

[0400] Example 4: Fermentation of Saccharomyces cerevisiae strain MBG5318 under non-stress conditions

[0401] The Saccharomyces cerevisiae strains MBG5318 and Ethanol were fermented under the following conditions:

[0402] Mash: Liquozyme SC 4X

[0403] Solids: 32.7%

[0404] pH: 5.0

[0405] Glucoamylase: Spirizyme Achieve T (Novozymes A / S)

[0406] Glucoamylase dosage: 0.6 AGU / g DS

[0407] Fermentation time: 54 h

[0408] Temperature: 32 °C

[0409] Scale: 80 g conical flask

[0410] As Figure 5 and Figure 6 shown, compared with Ethanol the Saccharomyces cerevisiae strain MBG5318 exhibits increased ethanol yield and reduced glycerol levels.

[0411] Example 5: Fermentation of Saccharomyces cerevisiae strain MBG5307 under non-stress conditions

[0412] The Saccharomyces cerevisiae strains MBG5307 and Ethanol were fermented under the following conditions:

[0413] Mash: Liquozyme SC 4X

[0414] Solid: 32.7%

[0415] pH: 5.0

[0416] Glucoamylase: Spirizyme Achieve T (Novozymes A / S)

[0417] Glucoamylase dosage: 0.6 AGU / g DS

[0418] Fermentation time: 54 h

[0419] Temperature: 32 °C

[0420] Scale: 80 g conical flask

[0421] As Figure 7 and Figure 8 shown, compared with Ethanol the Saccharomyces cerevisiae strain MBG5307 exhibits increased ethanol yield and reduced glycerol levels.

[0422] Example 6: Fermentation of Saccharomyces cerevisiae strain MBG5225 under stress conditions

[0423] The Saccharomyces cerevisiae strains MBG5225 and Ethanol were fermented under the following conditions:

[0424] Mash: Liquozyme SC 4X

[0425] Solid 31.9%

[0426] Glucoamylase: Spirizyme Achieve T (Novozymes A / S)

[0427] Glucoamylase dosage: 0.6 AGU / g DS

[0428] Fermentation time: 54 h

[0429] Temperature: 32 °C (7 h) → 35 °C (16 h) → 32 °C (31 h)

[0430] Scale: 80 g conical flask

[0431] As Figure 9 shown, in the presence of organic acids (lactic acid + acetic acid and higher temperature), compared with Ethanol the Saccharomyces cerevisiae strain MBG5225 showed an increased ethanol yield.

[0432] Example 7: Fermentation of Saccharomyces cerevisiae strain MBG5227 under stress conditions

[0433] The Saccharomyces cerevisiae strain MBG5227 and Ethanol were fermented under the following conditions:

[0434] Mash: Liquozyme SC 4X

[0435] Solid 31.9%

[0436] Glucoamylase: Spirizyme Achieve T (Novozymes)

[0437] Glucoamylase dosage: 0.6 AGU / g DS

[0438] Fermentation time: 54 h

[0439] Temperature: 32 °C (7 h) → 35 °C (16 h) → 32 °C (31 h)

[0440] Scale: 80 g conical flask

[0441] As Figure 10 shown, in the presence of organic acids (lactic acid + acetic acid and higher temperature), compared with Ethanol the Saccharomyces cerevisiae strain MBG5227 showed an increased ethanol yield.

[0442] Example 8: Fermentation of Saccharomyces cerevisiae strain MBG5318 under stress conditions

[0443] The Saccharomyces cerevisiae strain MBG5318 and Ethanol were fermented under the following conditions:

[0444] Mash: Liquozyme SC 4X

[0445] Solid 31.9%

[0446] Glucoamylase: Spirizyme Achieve T (Novozymes)

[0447] Glucoamylase dosage: 0.6 AGU / g DS

[0448] Fermentation time: 54 h

[0449] Temperature: 32 °C (7 h) → 35 °C (16 h) → 32 °C (31 h)

[0450] Scale: 80 g conical flask

[0451] As Figure 11 shown, in the presence of organic acids (lactic acid + acetic acid and higher temperature), compared with Ethanol the Saccharomyces cerevisiae strain MBG5318 exhibits an increased ethanol yield.

[0452] The present invention can be further described in the paragraphs numbered below:

[0453] Paragraph [1]. A method for producing a fermentation product from a cellulose-containing and / or starch-containing material, the method comprising:

[0454] (a) saccharifying the cellulose-containing or starch-containing material; and

[0455] (b) fermenting the saccharified material of step (a) with a fermenting organism under suitable conditions to produce the fermentation product; wherein the fermenting organism is a Saccharomyces cerevisiae strain deposited at the Patent Culture Collection of the United States Department of Agriculture, Agricultural Research Service (NRRL) under the Budapest Treaty and having a deposit number of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase) or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5225.

[0456] Paragraph [2]. A method for producing a fermentation product from a cellulose-containing and / or starch-containing material, the method comprising:

[0457] (a) saccharifying the cellulose-containing or starch-containing material; and

[0458] (b) fermenting the saccharified material of step (a) with a fermenting organism under suitable conditions to produce the fermentation product; wherein the fermenting organism is a Saccharomyces cerevisiae strain deposited at the Patent Culture Collection of the United States Department of Agriculture, Agricultural Research Service (NRRL) under the Budapest Treaty and having a deposit number of NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase) or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5227.

[0459] Paragraph [3]. A method for producing a fermentation product from a cellulose-containing and / or starch-containing material, the method comprising:

[0460] (a) Saccharify the cellulose-containing or starch-containing material; and

[0461] (b) Ferment the saccharified material of step (a) with a fermenting organism under suitable conditions to produce the fermentation product; wherein the fermenting organism is a Saccharomyces cerevisiae strain deposited at the Patent Culture Collection of the United States Department of Agriculture, Agricultural Research Service (NRRL) under the Budapest Treaty with the accession number NRRL Y-67971 (Saccharomyces cerevisiae strain MBG5307), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5307.

[0462] Paragraph [4]. A method for producing a fermentation product from a cellulose-containing and / or starch-containing material, the method comprising:

[0463] (a) Saccharify the cellulose-containing or starch-containing material; and

[0464] (b) Ferment the saccharified material of step (a) with a fermenting organism under suitable conditions to produce the fermentation product; wherein the fermenting organism is a Saccharomyces cerevisiae strain deposited at the Patent Culture Collection of the United States Department of Agriculture, Agricultural Research Service (NRRL) under the Budapest Treaty with the accession number NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5318.

[0465] Paragraph [5]. The method according to paragraphs [1]-[4], the method comprising recovering the fermentation product from the fermentation.

[0466] Paragraph [6]. The method according to paragraph [5], wherein recovering the fermentation product from the fermentation comprises distillation.

[0467] Paragraph [7]. The method according to any one of paragraphs [1]-[6], wherein the fermentation and saccharification are carried out simultaneously in simultaneous saccharification and fermentation (SSF).

[0468] Paragraph [8]. The method according to any one of paragraphs [1]-[6], wherein the fermentation and saccharification are carried out sequentially (SHF).

[0469] Paragraph [9]. The method according to any one of paragraphs [1]-[8], wherein the fermentation product is ethanol.

[0470] Paragraph

[10] . The method according to any one of paragraphs [1]-[9], wherein step (a) comprises contacting the starch-containing and / or cellulose-containing material with an enzyme composition.

[0471] Paragraph

[11] . The method according to any one of paragraphs [1] -

[10] , wherein step (a) comprises saccharifying a starch-containing material.

[0472] Paragraph

[12] . The method according to paragraph

[11] , wherein step (a) comprises i) liquefying the starch-containing material using α-amylase at a temperature above the initial gelatinization temperature; and ii) saccharifying using glucoamylase.

[0473] Paragraph

[13] . The method according to any one of paragraphs [1] -

[12] , wherein a protease is added during saccharification or fermentation or SSF.

[0474] Paragraph

[14] . The method according to any one of paragraphs [1] -

[13] , the method further comprising the following steps before the liquefaction step i):

[0475] x) reducing the particle size of the starch-containing material, preferably by dry milling;

[0476] y) forming a slurry comprising the starch-containing material and water.

[0477] Paragraph

[15] . The method according to any one of paragraphs [1] -

[14] , wherein at least 50%, such as at least 70%, at least 80%, at least 90% of the starch-containing material passes through a sieve with a #6 mesh.

[0478] Paragraph

[16] . The method according to any one of paragraphs [1] -

[15] , wherein the pH in liquefaction is between 4 - 7, such as pH 4.5 - 6.5, such as pH 5.0 - 6.5, such as pH 5.0 - 6.0, such as pH 5.2 - 6.2, such as about 5.2, such as about 5.4, such as about 5.6, such as about 5.8.

[0479] Paragraph

[17] . The method according to any one of paragraphs [1] -

[16] , wherein the temperature range in liquefaction is 70°C - 100°C, such as 75°C - 95°C, 75°C - 90°C, 80°C - 90°C, or 82°C - 88°C, such as about 85°C.

[0480] Paragraph

[18] . The method according to any one of paragraphs [1] -

[17] , wherein an injection cooking step is carried out before liquefaction.

[0481] Paragraph

[19] . The method according to paragraph

[18] , wherein the injection cooking is carried out at a temperature of 110°C - 145°C, such as 120°C - 140°C, such as 125°C - 135°C, or about 130°C for about 1 - 15 minutes, such as for about 3 - 10 minutes, or about 5 minutes.

[0482] Paragraph

[20] . The method according to any one of paragraphs [1] -

[19] , wherein the saccharification is carried out at a temperature from 20 °C to 75 °C, such as from 40 °C to 70 °C, such as about 60 °C, and at a pH between 4 and 5.

[0483] Paragraph

[21] . The method according to any one of paragraphs [1] -

[20] , wherein the fermentation or simultaneous saccharification and fermentation (SSF) is carried out at a temperature from 25 °C to 40 °C, such as from 28 °C to 35 °C, such as from 30 °C to 34 °C, or about 32 °C. In one embodiment, the fermentation is carried out for 6 to 120 hours, especially 24 to 96 hours.

[0484] Paragraph

[22] . The method according to any one of paragraphs [1] -

[21] , wherein the starch-containing starting material is whole grains.

[0485] Paragraph

[23] . The method according to any one of paragraphs [1] -

[23] , wherein the starch-containing material is derived from corn, wheat, barley, rye, sorghum, sago, cassava, tapioca, cassava starch, sorghum, oats, rice or potatoes.

[0486] Paragraph

[24] . The method according to any one of paragraphs [1] -

[10] , wherein step (a) comprises saccharifying the cellulose-containing material.

[0487] Paragraph

[25] . The method according to paragraph

[24] , wherein the cellulose-containing material is pretreated.

[0488] Paragraph

[26] . The method according to any one of paragraphs

[24] or

[25] , wherein the cellulose-containing material comprises bagasse.

[0489] Paragraph

[27] . The method according to any one of paragraphs

[24] -

[26] , wherein step (a) comprises contacting the cellulose-containing material with an enzyme composition, and wherein the enzyme composition comprises one or more enzymes selected from the following: cellulase, AA9 polypeptide, hemicellulase, CIP, esterase, expansin, lignin-degrading enzyme, oxidoreductase, pectinase, protease and swelling factor.

[0490] Paragraph

[28] . The method according to paragraph

[27] , wherein the cellulase is one or more enzymes selected from the following: endoglucanase, cellobiohydrolase and β-glucosidase.

[0491] Paragraph

[29] . The method according to paragraph

[27] or

[28] , wherein the hemicellulase is one or more enzymes selected from the following: xylanase, acetylxylan esterase, ferulic acid esterase, arabinofuranosidase, xylosidase and glucuronidase.

[0492] Paragraph

[30] . The method according to any one of paragraphs [1] -

[29] , wherein the method results in a yield of the fermentation product of at least 0.25% (e.g., 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2%, 3% or 5%).

[0493] Paragraph

[31] . The method according to any one of paragraphs [1] -

[30] , wherein the fermentation is carried out under hypoxic (e.g., anaerobic) conditions.

[0494] Paragraph

[32] . The method according to any one of paragraphs [1] -

[31] , wherein the fermenting organism has one or more of the following properties:

[0495] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the ethanol yield is increased;

[0496] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the acetaldehyde production is reduced;

[0497] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the temperature tolerance is increased;

[0498] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the maltose (DP2) production is reduced; and / or

[0499] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the glycerol production is reduced.

[0500] Paragraph

[33] . The method according to any one of paragraphs [1] -

[32] , wherein under the same method conditions, e.g., the conditions described herein, the fermenting organism is more than the Saccharomyces cerevisiae strain Ethanol (Deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039) provides an increase in ethanol production of more than 0.5%, such as more than 1.0%, more than 2.0%, more than 2.5%, such as about 2.9%, such as between 0.5% and 5%, such as between 1% and 3%.

[0501] Paragraph

[34] . The method according to any one of paragraphs [1] -

[33] , wherein under the same method conditions, such as the method conditions described herein, compared with the Saccharomyces cerevisiae strain Ethanol (Deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the fermenting organism reduces acetaldehyde production by more than 10%, such as more than 20%, more than 30%, more than 40%, more than 45%, such as 5% - 60%, such as 30% - 50%.

[0502] Paragraph

[35] . The method according to any one of paragraphs [1] -

[34] , wherein under the same method conditions, such as the method conditions described herein, compared with the Saccharomyces cerevisiae strain Ethanol (Deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the fermenting organism has increased temperature tolerance.

[0503] Paragraph

[36] . The method according to any one of paragraphs [1] -

[35] , wherein under the same method conditions, such as the conditions described herein, compared with the Saccharomyces cerevisiae strain Ethanol (Deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the fermenting organism reduces maltose (DP2) production by more than 3%, such as more than 4%, more than 5%, more than 6%, more than 7%, such as 2% - 15%, such as 5% - 10%.

[0504] Paragraph

[37] . The method according to any one of paragraphs [1] -

[36] , wherein under the same method conditions, such as the conditions described herein, compared with the Saccharomyces cerevisiae strain Ethanol (Deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), the fermenting organism reduces glycerol production by more than 3%, such as more than 4%, more than 5%, more than 6%, more than 7%, such as 2% - 15%, such as 5% - 10%.

[0505] Paragraph

[38] . A Saccharomyces cerevisiae strain deposited with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Laboratory (NRRL), under the Budapest Treaty and having accession number NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5225.

[0506] Paragraph

[39] . A Saccharomyces cerevisiae strain deposited with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Laboratory (NRRL), under the Budapest Treaty and having accession number NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5227.

[0507] Paragraph

[40] . A Saccharomyces cerevisiae strain deposited with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Laboratory (NRRL), under the Budapest Treaty and having accession number NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5307.

[0508] Paragraph

[41] . A Saccharomyces cerevisiae strain deposited with the Agricultural Research Service Patent Culture Collection, Northern Regional Research Laboratory (NRRL), under the Budapest Treaty and having accession number NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5318.

[0509] Paragraph

[42] . The Saccharomyces cerevisiae strain according to any one of paragraphs

[38] -

[41] , wherein the strain has one or more of the following properties:

[0510] Under the same process conditions, compared to the Saccharomyces cerevisiae strain Ethanol (deposited with the National Measurement Institute, Victoria, Australia, accession number V14 / 007039), the ethanol yield is increased;

[0511] Under the same process conditions, compared to the Saccharomyces cerevisiae strain Ethanol (deposited with the National Measurement Institute, Victoria, Australia, accession number V14 / 007039), the acetaldehyde production is reduced;

[0512] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, with the deposit number V14 / 007039), there is an increase in temperature tolerance;

[0513] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, with the deposit number V14 / 007039), there is a decrease in maltose (DP2) production; and / or

[0514] Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, with the deposit number V14 / 007039), there is a decrease in glycerol production.

[0515] Paragraph

[43] . A method for producing a derivative of the Saccharomyces cerevisiae strain MBG5225 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), USA, with the deposit number NRRL Y-68199), the method comprising:

[0516] a. culturing the first yeast strain together with the second yeast strain under conditions that allow DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5225 or a derivative thereof; and

[0517] b. isolating the hybrid strain; and

[0518] c. optionally repeating steps (a) and (b) using the hybrid strain isolated in step (b) as the first yeast strain and / or the second yeast strain.

[0519] Paragraph

[44] . A method for producing a derivative of the Saccharomyces cerevisiae strain MBG5227 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), USA, with the deposit number NRRL Y-68200), the method comprising:

[0520] a. culturing the first yeast strain together with the second yeast strain under conditions that allow DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5227 or a derivative thereof; and

[0521] b. isolating the hybrid strain; and

[0522] c. optionally repeating steps (a) and (b) using the hybrid strain isolated in step (b) as the first yeast strain and / or the second yeast strain.

[0523] Paragraph

[45] . A method for generating a derivative of the Saccharomyces cerevisiae strain MBG5307 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), accession number NRRL Y-68201), the method comprising:

[0524] a. culturing the first yeast strain together with the second yeast strain under conditions that permit DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5307 or a derivative thereof; and

[0525] b. isolating the hybrid strain; and

[0526] c. optionally repeating steps (a) and (b) using the hybrid strain isolated in step (b) as the first yeast strain and / or the second yeast strain.

[0527] Paragraph

[46] . A method for generating a derivative of the Saccharomyces cerevisiae strain MBG5318 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), accession number NRRL Y-68202), the method comprising:

[0528] a. culturing the first yeast strain together with the second yeast strain under conditions that permit DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5318 or a derivative thereof; and

[0529] b. isolating the hybrid strain; and

[0530] c. optionally repeating steps (a) and (b) using the hybrid strain isolated in step (b) as the first yeast strain and / or the second yeast strain.

[0531] Paragraph

[47] . A method for generating a derivative of the Saccharomyces cerevisiae strain MBG5225 that exhibits the defined characteristics of the Saccharomyces cerevisiae strain MBG5225 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), accession number NRRL Y-68199), the method comprising:

[0532] (a) providing:

[0533] (i) a first yeast strain; and

[0534] (ii) a second yeast strain, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5225 or a derivative thereof;

[0535] (b) culturing the first yeast strain and the second yeast strain under conditions that permit combination of the DNA between the first and second yeast strains;

[0536] (c) Screening or selecting a derivative of the Saccharomyces cerevisiae strain MBG5225.

[0537] Paragraph

[48] . The method as described in paragraph

[47] , wherein step (c) comprises screening or selecting a hybrid strain that exhibits one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5225.

[0538] Paragraph

[49] . The method as described in paragraph

[47] , the method comprising an additional step:

[0539] (d) Repeating steps (a) and (b) using the strain screened or selected in step (c) as the first strain and / or the second strain until a derivative is obtained that exhibits the defined characteristics of the Saccharomyces cerevisiae strain MBG5225.

[0540] Paragraph

[50] . The method as described in paragraph

[47] , wherein the culturing step (b) comprises:

[0541] (i) Causing the first yeast strain and the second yeast strain to form spores;

[0542] (ii) Hybridizing the germinated spores produced by the first yeast strain with the germinated spores produced by the second yeast strain.

[0543] Paragraph

[51] . A method for producing a derivative of the Saccharomyces cerevisiae strain MBG5227 that exhibits the defined characteristics of the Saccharomyces cerevisiae strain MBG5227 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), accession number NRRL Y-68200), the method comprising:

[0544] (d) Providing:

[0545] (j) A first yeast strain; and

[0546] (iii) A second yeast strain, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5227 or a derivative thereof;

[0547] (e) Culturing the first yeast strain and the second yeast strain under conditions that permit the combination of DNA between the first and second yeast strains;

[0548] (f) Screening or selecting a derivative of the Saccharomyces cerevisiae strain MBG5227.

[0549] Paragraph

[52] . The method as described in paragraph

[51] , wherein step (c) comprises screening or selecting a hybrid strain that exhibits one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5227.

[0550] Paragraph

[53] . The method as described in paragraph

[51] , the method comprising an additional step:

[0551] (d) Repeat steps (a) and (b) using the strain screened or selected from step (c) as the first strain and / or the second strain until a derivative is obtained that exhibits the defined characteristics of the Saccharomyces cerevisiae strain MBG5227.

[0552] Paragraph

[54] . The method according to paragraph

[51] , wherein the culturing step (b) comprises:

[0553] (i) causing the first yeast strain and the second yeast strain to form spores;

[0554] (ii) hybridizing the germinated spores produced by the first yeast strain with the germinated spores produced by the second yeast strain.

[0555] Paragraph

[55] . A method for producing a derivative of the Saccharomyces cerevisiae strain MBG5307 that exhibits the defined characteristics of the Saccharomyces cerevisiae strain MBG5307 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), accession number NRRL Y-68201), the method comprising:

[0556] (g) providing:

[0557] (k) a first yeast strain; and

[0558] (iv) a second yeast strain, wherein the second yeast strain is the Saccharomyces cerevisiae strain MBG5307 or a derivative thereof;

[0559] (h) culturing the first yeast strain and the second yeast strain under conditions that permit the combination of DNA between the first and second yeast strains;

[0560] (i) screening or selecting a derivative of the Saccharomyces cerevisiae strain MBG5307.

[0561] Paragraph

[56] . The method according to paragraph

[55] , wherein step (c) comprises screening or selecting a hybrid strain that exhibits one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5307.

[0562] Paragraph

[57] . The method according to paragraph

[55] , the method further comprising the step:

[0563] (d) Repeat steps (a) and (b) using the strain screened or selected from step (c) as the first strain and / or the second strain until a derivative is obtained that exhibits the defined characteristics of the Saccharomyces cerevisiae strain MBG5307.

[0564] Paragraph

[58] . The method according to paragraph

[55] , wherein the culturing step (b) comprises:

[0565] (i) causing the first yeast strain and the second yeast strain to form spores;

[0566] (ii) hybridizing the germinated spores produced by the first yeast strain with the germinated spores produced by the second yeast strain.

[0567] Paragraph

[59] . A method for producing a derivative of Saccharomyces cerevisiae strain MBG5318 that exhibits the defining characteristics of Saccharomyces cerevisiae strain MBG5318 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), accession number NRRL Y-68202), the method comprising:

[0568] (j) providing:

[0569] (l) a first yeast strain; and

[0570] (v) a second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5318 or a derivative thereof;

[0571] (k) culturing the first yeast strain and the second yeast strain under conditions that permit the combination of DNA between the first and second yeast strains;

[0572] (l) screening or selecting a derivative of Saccharomyces cerevisiae strain MBG5318.

[0573] Paragraph

[60] . The method according to paragraph

[59] , wherein step (c) comprises screening or selecting a hybrid strain that exhibits one or more of the defining characteristics of Saccharomyces cerevisiae strain MBG5318.

[0574] Paragraph

[61] . The method according to paragraph

[59] , the method comprising an additional step:

[0575] (d) repeating steps (a) and (b) using the strain screened or selected in step (c) as the first strain and / or the second strain until a derivative is obtained that exhibits the defining characteristics of Saccharomyces cerevisiae strain MBG5318.

[0576] Paragraph

[62] . The method according to paragraph

[59] , wherein the culturing step (b) comprises:

[0577] (i) causing the first yeast strain and the second yeast strain to form spores;

[0578] (ii) hybridizing the germinated spores produced by the first yeast strain with the germinated spores produced by the second yeast strain.

[0579] Paragraph

[63] . A method for generating recombinant derivatives of the Saccharomyces cerevisiae strain MBG5225 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), accession number NRRL Y-68199), the method comprising:

[0580] (a) transforming the Saccharomyces cerevisiae strain MBG5225 (or a derivative of the Saccharomyces cerevisiae strain MBG5225) with one or more expression vectors (e.g., one or more expression vectors encoding glucoamylase and / or α-amylase); and

[0581] (b) isolating the transformed strain.

[0582] Paragraph

[64] . A method for generating recombinant derivatives of the Saccharomyces cerevisiae strain MBG5227 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), accession number NRRL Y-68200), the method comprising:

[0583] (a) transforming the Saccharomyces cerevisiae strain MBG5227 (or a derivative of the Saccharomyces cerevisiae strain MBG5227) with one or more expression vectors (e.g., one or more expression vectors encoding glucoamylase and / or α-amylase); and

[0584] (b) isolating the transformed strain.

[0585] Paragraph

[65] . A method for generating recombinant derivatives of the Saccharomyces cerevisiae strain MBG5307 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), accession number NRRL Y-68201), the method comprising:

[0586] (a) transforming the Saccharomyces cerevisiae strain MBG5307 (or a derivative of the Saccharomyces cerevisiae strain MBG5307) with one or more expression vectors (e.g., one or more expression vectors encoding glucoamylase and / or α-amylase); and

[0587] (b) isolating the transformed strain.

[0588] Paragraph

[66] . A method for generating recombinant derivatives of the Saccharomyces cerevisiae strain MBG5318 (deposited at the Agricultural Research Service Patent Culture Collection Center (NRRL), accession number NRRL Y-68202), the method comprising:

[0589] (a) transforming the Saccharomyces cerevisiae strain MBG5318 (or a derivative of the Saccharomyces cerevisiae strain MBG5318) with one or more expression vectors (e.g., one or more expression vectors encoding glucoamylase and / or α-amylase); and

[0590] (b) isolating the transformed strain.

[0591] Paragraph

[67] . A Saccharomyces cerevisiae strain produced by the method according to any one of paragraphs

[43] -

[66] .

[0592] Paragraph

[68] . A method for producing ethanol, the method comprising incubating a Saccharomyces cerevisiae strain according to any one of paragraphs

[38] -

[42] and

[67] with a substrate containing the fermentable sugar under conditions allowing the fermentable sugar to ferment into ethanol.

[0593] Paragraph

[69] . Use of a Saccharomyces cerevisiae strain according to any one of paragraphs

[38] -

[42] and

[67] in ethanol production.

[0594] Paragraph

[70] . Use of the Saccharomyces cerevisiae strain MBG5225 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois, USA, accession number NRRL Y - 68199) in the production of a Saccharomyces strain having substantially the same properties as the Saccharomyces cerevisiae strain MBG5225 or exhibiting one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5225.

[0595] Paragraph

[71] . Use of the Saccharomyces cerevisiae strain MBG5227 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois, USA, accession number NRRL Y - 68200) in the production of a Saccharomyces strain having substantially the same properties as the Saccharomyces cerevisiae strain MBG5227 or exhibiting one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5227.

[0596] Paragraph

[72] . Use of the Saccharomyces cerevisiae strain MBG5307 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois, USA, accession number NRRL Y - 68201) in the production of a Saccharomyces strain having substantially the same properties as the Saccharomyces cerevisiae strain MBG5307 or exhibiting one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5307.

[0597] Paragraph

[73] . Use of the Saccharomyces cerevisiae strain MBG5318 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois 61604, USA, under accession number NRRL Y-68202) in the production of a Saccharomyces cerevisiae strain having substantially the same properties as the Saccharomyces cerevisiae strain MBG5318 or exhibiting one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5318.

[0598] Paragraph

[74] . Use of the Saccharomyces cerevisiae strain MBG5225 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois 61604, USA, under accession number NRRL Y-68199) or a strain having substantially the same properties as the Saccharomyces cerevisiae strain MBG5225 or a derivative thereof in the method according to any one of paragraphs [1] and [5] -

[37] .

[0599] Paragraph

[75] . Use of the Saccharomyces cerevisiae strain MBG5227 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois 61604, USA, under accession number NRRL Y-68200) or a strain having substantially the same properties as the Saccharomyces cerevisiae strain MBG5227 or a derivative thereof in the method according to any one of paragraphs [2] and [5] -

[37] .

[0600] Paragraph

[76] . Use of the Saccharomyces cerevisiae strain MBG5307 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois 61604, USA, under accession number NRRL Y-68201) or a strain having substantially the same properties as the Saccharomyces cerevisiae strain MBG5307 or a derivative thereof in the method according to any one of paragraphs [3] and [5] -

[37] .

[0601] Paragraph

[77] . Use of the Saccharomyces cerevisiae strain MBG5318 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois 61604, USA, under accession number NRRL Y-68202) or a strain having substantially the same properties as the Saccharomyces cerevisiae strain MBG5318 or a derivative thereof in the method according to any one of paragraphs [4] and [5] -

[37] .

[0602] Paragraph

[77] . A composition comprising a Saccharomyces cerevisiae strain according to any one of paragraphs

[38] -

[42] and

[67] , and one or more natural and / or non-natural components.

[0603] Paragraph

[78] . The composition as described in paragraph

[77] , wherein these components are selected from the group consisting of: surfactants, emulsifiers, gums, swelling agents, and antioxidants.

[0604] Paragraph

[79] . The composition as described in paragraph

[77] or

[78] , wherein the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5225 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois 61604, USA, with the accession number NRRL Y-68199).

[0605] Paragraph

[80] . The composition as described in paragraph

[77] or

[78] , wherein the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5227 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois 61604, USA, with the accession number NRRL Y-68200).

[0606] Paragraph

[81] . The composition as described in paragraph

[77] or

[78] , wherein the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5307 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois 61604, USA, with the accession number NRRL Y Y-68201).

[0607] Paragraph

[82] . The composition as described in paragraph

[77] or

[78] , wherein the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5318 (deposited at the Northern Regional Research Center (NRRL), Agricultural Research Service Patent Culture Collection Center, 1815 University Street, Peoria, Illinois 61604, USA, with the accession number NRRL Y-68202).

[0608] Paragraph

[83] . The composition as described in any one of paragraphs

[77] -

[82] , wherein the Saccharomyces cerevisiae strain is in a viable state, particularly in a dry, paste, or compressed state.

Claims

1. A method for producing ethanol from starch-containing materials, the method comprising the following steps: i) liquefying the starch-containing material using α-amylase at a temperature above the initial gelatinization temperature; ii) saccharifying using glucoamylase; iii) fermenting using a fermenting organism; wherein the fermenting organism is: (1) a Saccharomyces cerevisiae strain deposited with the Agricultural Research Service Patent Culture Collection Center (NRRL) under the Budapest Treaty and having a deposit number of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5225; (2) a Saccharomyces cerevisiae strain deposited with the Agricultural Research Service Patent Culture Collection Center (NRRL) under the Budapest Treaty and having a deposit number of NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5227; (3) a Saccharomyces cerevisiae strain deposited with the Agricultural Research Service Patent Culture Collection Center (NRRL) under the Budapest Treaty and having a deposit number of NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5318; or (4) a Saccharomyces cerevisiae strain deposited with the Agricultural Research Service Patent Culture Collection Center (NRRL) under the Budapest Treaty and having a deposit number of NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5318.

2. A yeast strain of the genus Saccharomyces, selected from: (1) a Saccharomyces cerevisiae strain deposited with the Agricultural Research Service Patent Culture Collection Center (NRRL) under the Budapest Treaty and having a deposit number of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5225; (2) a Saccharomyces cerevisiae strain deposited with the Agricultural Research Service Patent Culture Collection Center (NRRL) under the Budapest Treaty and having a deposit number of NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5227; (3) A Saccharomyces cerevisiae strain deposited under the Budapest Treaty at the Agricultural Research Service Patent Culture Collection (NRRL) with the accession number NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5318; and (4) A Saccharomyces cerevisiae strain deposited under the Budapest Treaty at the Agricultural Research Service Patent Culture Collection (NRRL) with the accession number NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5318.

3. The Saccharomyces yeast strain according to claim 1 or 2, wherein the strain comprises one or more of the following properties and defining characteristics: Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, with the deposit number V14 / 007039), the ethanol yield increases; Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, with the deposit number V14 / 007039), the acetaldehyde production is reduced; Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, with the deposit number V14 / 007039), the temperature tolerance is increased; Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039), there is a reduction in the production of maltose (DP2); and Under the same method conditions, compared with the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, with the deposit number V14 / 007039), glycerol production is reduced.

4. The Saccharomyces yeast strain according to any one of claims 1-3, wherein under the same method conditions, said strain is capable of providing an ethanol yield increase of more than 1.0% compared to the Saccharomyces cerevisiae strain Ethanol (deposited at the National Measurement Institute, Victoria, Australia, deposit number V14 / 007039).

5. A method for producing a derivative of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318), the method comprising: (a) Culturing the first yeast strain with the second yeast strain under conditions allowing DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318) or a derivative thereof; and (b) Isolating the hybrid strain.

6. A method for producing a derivative of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225) exhibiting the defining characteristics of Saccharomyces cerevisiae strain MBG5225, NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227) exhibiting the defining characteristics of Saccharomyces cerevisiae strain MBG5227, NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307) exhibiting the defining characteristics of Saccharomyces cerevisiae strain MBG5307, or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318) exhibiting the defining characteristics of Saccharomyces cerevisiae strain MBG5318, the method comprising: (a) Provide: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318) or a derivative thereof; (b) Cultivate the first yeast strain and the second yeast strain under conditions that permit DNA combination between the first yeast strain and the second yeast strain; and (c) Screen for or select a derivative of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318).

7. The method according to claim 6, wherein step (c) comprises screening for or selecting a hybrid strain that exhibits one or more defined characteristics of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318).

8. The method according to claim 6, the method comprising the following additional step: (d) Repeat steps (b) and (c) using the strain screened for or selected from step (c) as the first strain and / or the second strain until a derivative is obtained that exhibits the defined characteristics of NRRL Y-68199 (Saccharomyces cerevisiae strain MBG5225), NRRL Y-68200 (Saccharomyces cerevisiae strain MBG5227), NRRL Y-68201 (Saccharomyces cerevisiae strain MBG5307), or NRRL Y-68202 (Saccharomyces cerevisiae strain MBG5318).

9. The method according to claim 6, wherein the culturing step (b) comprises: (i) Causing the first yeast strain and the second yeast strain to form spores; and (ii) Hybridizing the germinated spores produced by the first yeast strain with the germinated spores produced by the second yeast strain.

10. A yeast genus strain produced by the method according to claim 6.

11. A method for producing ethanol, the method comprising incubating a strain according to claim 2 with a substrate comprising the fermentable sugar under conditions that permit fermentation of the fermentable sugar to produce ethanol.

12. A method for producing distillers' grains, the method comprising: (a) Incubating a yeast genus strain according to claim 2 with a substrate comprising the fermentable sugar under conditions that permit fermentation of the fermentable sugar to produce ethanol and distillers' grains; and (b) Separating the distillers' grains.

13. A distillers' grains produced by the method as described in claim 12.

14. A composition comprising a yeast strain of the genus Saccharomyces as described in claim 2 and one or more natural and / or non-natural components selected from surfactants, emulsifiers, gums, swelling agents, and antioxidants.

15. A method for producing a recombinant derivative of a yeast of the genus Saccharomyces as described in claim 2, the method comprising introducing a nucleic acid into a yeast of the genus Saccharomyces as described in claim 2 using recombinant DNA technology.

16. The method as described in claim 15, wherein the introduced nucleic acid expresses glucoamylase and / or α-amylase.

17. A recombinant-derived yeast strain of the genus Saccharomyces produced by the method as described in claim 15.

18. The yeast strain of the genus Saccharomyces as described in claim 17, wherein the introduced nucleic acid expresses glucoamylase and / or α-amylase.

19. A composition comprising a recombinant-derived yeast strain of the genus Saccharomyces as described in claim 17 and one or more components selected from surfactants, emulsifiers, gums, swelling agents, and antioxidants.

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