Improved fermentation organisms for ethanol production

Abstract

The invention relates to a process for producing ethanol, which process comprises saccharifying a cellulosic or starch-containing material and fermenting the saccharified material with a fermenting microorganism to produce ethanol. The fermenting organism is Saccharomyces cerevisiae strain MBG5364 (deposited with the Agricultural Research Service Patent Culture Collection (NRRL) at 314 W. Pierpont Ave., Peoria, IL 61604, USA under Accession No. 68303), Saccharomyces cerevisiae strain MBG5365 (deposited with the Agricultural Research Service Patent Culture Collection (NRRL) at 314 W. Pierpont Ave., Peoria, IL 61604, USA under Accession No. 68304), or a fermenting organism having the same or substantially the same properties as Saccharomyces cerevisiae MBG5364 or MBG5365.

Description

[0001] Citations on the preservation of biological materials This application contains references to the preservation of biological materials, which are incorporated herein by reference. Background Technology

[0002] Ethanol is typically blended into gasoline for use as a transportation fuel. Cellulose materials are used as raw materials in ethanol production methods. Several methods exist in the art for producing cellulose and hemicellulose hydrolysates containing glucose, mannose, xylose, and arabinose. Glucose and mannose are efficiently converted into ethanol in natural anaerobic metabolism. To date, the most efficient ethanol-producing microorganism is yeast—Saccharomyces cerevisiae (Saccharomyces cerevisiae). Saccharomyces cerevisiae However, *Saccharomyces cerevisiae* lacks the enzyme necessary to convert the main sugar, xylose, into xylulose, and therefore cannot utilize xylose as a carbon source. Therefore, genetic engineering of *Saccharomyces cerevisiae* is necessary to express an enzyme that can convert xylose into xylulose. One of the required enzymes is xylose isomerase (EC 5.3.1.5), which converts xylose into xylulose, which is then converted into ethanol during *Saccharomyces cerevisiae* fermentation.

[0003] WO 2003 / 062430 discloses the use of functional Pyrrosia spp. ( Piromyces Xylose isomerase (XI) is introduced into Saccharomyces cerevisiae, through the process of... XKS1 The encoded endogenous xylulose kinase (EC 2.7.1.17) and the enzyme in the non-oxidative portion of the pentose phosphate pathway enable the slow metabolism of xylose and confer the ability of this yeast transformant to grow on xylose.

[0004] U.S. Patent No. 8,586,336 discloses a *Saccharomyces cerevisiae* strain expressing xylose isomerase obtained from bovine rumen fluid. This yeast strain can be used to produce ethanol by culturing under anaerobic fermentation conditions. WO 2016 / 045569 describes a *Saccharomyces cerevisiae* strain CIBTS1260 with improved xylose consumption, glucose consumption, and ethanol production capabilities.

[0005] Despite significant improvements in the process of producing ethanol using cellulose materials, there is still a desire and need for further process improvements, particularly improvements in fermentation kinetics, which would enhance the robustness of fermentation inhibitors. Summary of the Invention

[0006] This article specifically describes the process for producing ethanol from cellulose- or starch-containing materials, and the yeast suitable for such processes.

[0007] The first aspect relates to a method for producing fermentation products from cellulose- and / or starch-containing materials, the method comprising: (a) Saccharification of the cellulose- or starch-containing material; and (b) Fermenting the saccharified material of step (a) under appropriate conditions with a fermenting organism to produce a fermentation product; wherein the fermenting organism is deposited in accordance with the Budapest Treaty at the Agricultural Research Service Patent Culture Collection (NRRL) with accession number NRRL 68303 (Saccharomyces cerevisiae strain MBG5364). Saccharomyces cerevisiae MBG5364), NRRL68304 (Saccharomyces cerevisiae strain MBG5365) Saccharomyces cerevisiae Recombinant strains of Saccharomyces cerevisiae (MBG5365), or derivatives thereof (e.g., expressing heterologous polypeptides such as glucosylamylase and / or α-amylase), or fermentation organisms having substantially the same properties as Saccharomyces cerevisiae MBG5364 or Saccharomyces cerevisiae strain MBG5365.

[0008] In one embodiment, the method includes recovering the fermentation product from the fermentation (e.g., by distillation).

[0009] In one embodiment, fermentation and saccharification are performed simultaneously in simultaneous saccharification and fermentation (SSF). In another embodiment, fermentation and saccharification are performed sequentially (SHF).

[0010] In one embodiment, the fermentation product is ethanol.

[0011] In one embodiment, step (a) includes contacting the starch-containing and / or cellulose-containing material with the enzyme composition.

[0012] In one embodiment, step (a) includes saccharifying the cellulose-containing material. In one embodiment, the cellulose-containing material is pretreated. In one embodiment, the cellulose-containing material comprises bagasse.

[0013] In one embodiment, step (a) includes contacting the cellulose-containing material with an enzyme composition, 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, cellobiase, 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, arabinofuranase, xylosidase, and glucuronidase.

[0014] 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%).

[0015] In one embodiment, fermentation is carried out under low-oxygen (e.g., anaerobic) conditions.

[0016] In one embodiment, the fermenting organism has one or more of the following properties: - Compared with Saccharomyces cerevisiae CIBTS1260 (e.g., 10 to 32 hours), higher ethanol fermentation kinetics at 1 g DWC / L, 32°C, and pH 5.5 (as described in Example 7 of this paper); - Compared with Saccharomyces cerevisiae CIBTS1260, higher xylose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper); - Compared to Saccharomyces cerevisiae CIBTS1260, higher glucose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper).

[0017] The second aspect relates to recombinant Saccharomyces cerevisiae strains, or derivatives thereof (e.g., expressing heterologous polypeptides such as glucosyl amylase and / or α-amylase), deposited in accordance with the Budapest Treaty at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) with accession numbers NRRL 68303 (Saccharomyces cerevisiae strain MBG5364) and NRRL 68304 (Saccharomyces cerevisiae strain MBG5365), or fermentation organisms having substantially the same properties as Saccharomyces cerevisiae MBG5364 or Saccharomyces cerevisiae strain MBG5365.

[0018] In one embodiment, the strain has one or more of the following properties: - Compared with Saccharomyces cerevisiae CIBTS1260 (e.g., 10 to 32 hours), higher ethanol fermentation kinetics at 1 g DWC / L, 32°C, and pH 5.5 (as described in Example 7 of this paper); - Compared with Saccharomyces cerevisiae CIBTS1260, higher xylose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper); - Compared to Saccharomyces cerevisiae CIBTS1260, higher glucose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper).

[0019] In one embodiment, this strain exhibits a higher ethanol yield compared to Saccharomyces cerevisiae CIBTS1260 when fermented for 10 to 30 hours at 1 g DWC / L, 32°C, and pH 5.5 (as described in Example 7 of this document).

[0020] In one embodiment, the strain was able to consume more than 95% of xylose after fermentation for 48 hours under process conditions of 1 g DCW / L, 35°C, and pH 5.5 (as described in Example 3 of this document).

[0021] In one embodiment, the strain was able to consume more than 95% of the glucose after fermentation for 24 hours under process conditions of 1 g DCW / L, 35°C, and pH 5.5 (as described in Example 3 of this document).

[0022] In one embodiment, the strain can provide more than 30 g / L of ethanol, such as more than 40 g / L, more than 45 g / L, or about 47 g / L of ethanol, after fermentation for 48 hours under process conditions of 1 g DCW / L, 35°C, and pH 5.5 (as described in Example 3 of this document).

[0023] In one embodiment, the strain contains a heterologous gene encoding a xylose isomerase. In one embodiment, the strain contains a heterologous gene encoding a pentose transporter, such as the GFX gene (e.g., GFX1 from *Candida intermedia*). In one embodiment, the strain contains a heterologous gene encoding a xylulose kinase (XKS) (e.g., XKS from *Saccharomyces cerevisiae*). In one embodiment, the strain contains a heterologous gene encoding a ribulose 5-phosphate 3-epiisomerase (RPE1) (e.g., RPE1 from *Saccharomyces cerevisiae*). In one embodiment, the strain contains a heterologous gene encoding a ribulose 5-phosphate isomerase (RKI1) (e.g., RKI1 from *Saccharomyces cerevisiae*). In one embodiment, the strain contains a heterologous gene encoding a transketolase (TKL1) and a heterologous gene encoding a transaldolase (TAL1) (e.g., TKL1 and TAL1 from *Saccharomyces cerevisiae*).

[0024] The third aspect relates to a method for producing a derivative of NRRL 68303 (Saccharomyces cerevisiae strain MBG5364) or NRRL 68304 (Saccharomyces cerevisiae strain MBG5365), the method comprising: (a) co-culturing a first yeast strain with a second yeast strain under conditions allowing DNA combination between a first yeast strain and a second yeast strain, wherein the second yeast strain is NRRL 68303 (Saccharomyces cerevisiae strain MBG5364) or NRRL 68304 (Saccharomyces cerevisiae strain MBG5365) or a derivative thereof; and (b) isolating a heterozygous strain; and (c) optionally repeating steps (a) and (b) using the heterozygous strain isolated in step (b) as the first yeast strain and / or the second yeast strain.

[0025] The fourth aspect relates to a method for producing NRRL 68303 (Saccharomyces cerevisiae strain MBG5364) exhibiting the defined characteristics of Saccharomyces cerevisiae strain MBG5364 or NRRL 68304 (Saccharomyces cerevisiae strain MBG5365) exhibiting the defined characteristics of Saccharomyces cerevisiae strain MBG5365, the method comprising: (a) providing: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5364, Saccharomyces cerevisiae strain MBG5365 or a derivative thereof; (b) culturing the first yeast strain and the second yeast strain under conditions allowing DNA combination between the first yeast strain and the second yeast strain; and (c) screening or selecting Saccharomyces cerevisiae strain MBG5364 or a derivative thereof.

[0026] In one embodiment, step (c) includes screening or selecting heterozygous strains exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG5364 or Saccharomyces cerevisiae strain MBG5365. In one embodiment, the method further includes the following step: (d) repeating steps (a) and (b) with the strain screened or selected from step (c) as a first and / or second strain until derivatives exhibiting the defined characteristics of Saccharomyces cerevisiae strain MBG5364 or Saccharomyces cerevisiae strain MBG5365 are obtained.

[0027] In one embodiment, the cultivation step (b) includes: (i) causing the first yeast strain and the second yeast strain to form spores; and (ii) hybridizing the germinating spores produced by the first yeast strain with the germinating spores produced by the second yeast strain.

[0028] The fifth aspect relates to a method for producing a recombinant derivative of NRRL 68303 (Saccharomyces cerevisiae strain MBG5364) or NRRL 68304 (Saccharomyces cerevisiae strain MBG5365), the method comprising: (a) transforming Saccharomyces cerevisiae strain MBG5364 (or a derivative thereof) or Saccharomyces cerevisiae strain MBG5365 (or a derivative thereof) with one or more expression vectors (e.g., one or more expression vectors encoding glucosylamylase and / or α-amylase); and (b) isolating the transformed strain.

[0029] The sixth aspect involves brewer's yeast strains produced by any of the third, fourth, or fifth aspects.

[0030] The seventh aspect relates to a method for producing ethanol, which includes incubating the *Saccharomyces cerevisiae* strain of the second or sixth aspect together with a substrate containing fermentable sugars under conditions that allow fermentable sugars to ferment into ethanol.

[0031] The eighth aspect relates to a composition comprising any of the brewer's yeast strains of the second or sixth aspect, and one or more naturally occurring and / or non-naturally occurring components.

[0032] In one embodiment, these components are selected from the group consisting of surfactants, emulsifiers, gums, swelling agents, and antioxidants.

[0033] In one embodiment, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5364 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, IL, USA, with accession number NRRL 68303).

[0034] In one embodiment, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5365 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, with accession number NRRL 68304).

[0035] In one embodiment, the Saccharomyces cerevisiae strain is in a viable state, particularly in a dried, paste-like, or compressed state. Attached Figure Description

[0036] Figure 1 The plasmid map of pYIE2-mgXI-GXF1-δ carrying the mgXI and GXF expression cassettes is shown.

[0037] Figure 2 The plasmid map of the plasmid used, pSH47-hyg, is shown.

[0038] Figure 3 The spectrum of the generated plasmid pYIE2-XKS1-PPP-δ is shown.

[0039] Figure 4 The fermentation comparison of CIBTS1260 versus BSGX001 in NREL acid-pretreated corn stalk hydrolysate was shown, with 1 g DCW / L yeast inoculation, 35°C, pH 5.5, and 72 hours.

[0040] Figure 5 Comparison of CIBTS1260 and BSGX001 in model culture medium: 2 / L yeast inoculation, 32℃, pH 5.5, 72 hours.

[0041] Figure 6 The fermentation of bagasse hydrolysates produced by cellulase compositions CA and CB with CIBTS1260 after 72 hours of inoculation with 1 g / L yeast is shown as a comparison.

[0042] Figure 7 The percentage decrease in DP2 concentration is shown during fermentation with 1 g / L yeast inoculation, at 35°C and pH 5.5, for 72 hours, using hydrolysates produced by cellulase CA or CB.

[0043] Figure 8 The fermentation rates of yeast reference strain CIBTS1260 and strain MBG5365 were compared at different fermentation temperatures in several biomass hydrolysate media. Fermentation conditions: 34℃; pH 5.5; 130 rpm; inoculum size 1 g CDW / L. Gray: reference strain (CIBTS1260); Black: yeast strain MBG5365.

[0044] Figure 9 The fermentation of yeast reference strain CIBTS1260 and strain MBG5365 on biomass hydrolysate medium at different fermentation temperatures (pH 5.5; 130 rpm; inoculum size 1 g CDW / L) is shown. Gray: reference strain (CIBTS1260); Black: yeast strain MBG5365. Fermentation temperature: Dashed line: 32℃; Dotted line: 34℃; Solid line: 36℃.

[0045] Figure 10 Fermentation of yeast strain MBG5365 on biomass hydrolysate medium at different fermentation pH is shown (34℃; 130 rpm; inoculum size 1 g CDW / L). Initial fermentation pH: 5.0, 5.5, 6.0, 7.0, and 8.0.

[0046] Figure 11 The fermentation yields (g EtOH / theoretical maximum EtOH from glucose and xylose components × 100) and xylose conversion rates (g converted xylose / initial xylose × 100) of reference strains CIBTS1260 and MBG5365 in biomass hydrolysate media under different pH conditions are shown (34℃; 130 rpm; inoculum size 1.25 g CDW / L; acetic acid: 8 g / L). Initial fermentation pH: 4.50; 4.75; 5.0 and 5.5.

[0047] Figure 12 The relative performance of strain MBG5365 (striped column) and reference strain CIBTS1260 (solid column) in ethanol production is shown when using 2G hydrolysate substrate. Fermentation conditions: 33℃; pH 5.5; 130 rpm; inoculum size 1.0 g CDW / L; acetic acid: 8 g / L. Fermentation time = 31 hours.

[0048] Figure 13 The xylose conversion rates (grams of converted xylose / grams of initial xylose) of strain MBG5365 (striped column) and reference strain CIBTS1260 (solid column) on biomass hydrolysate are shown. Fermentation conditions: 33℃; pH 5.5; 130 rpm; inoculum size 1.0 g CDW / L; acetic acid: 8 g / L. Fermentation time = 31 hours.

[0049] definition 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.

[0050] Allelic variants: The term "allelic variant" refers to any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variations arise naturally through mutation and can lead to polymorphism within a population. Gene mutations can be silent (encoding a polypeptide with no change) or can encode a polypeptide with a modified amino acid sequence. Allelic variants of a polypeptide are polypeptides encoded by allelic variants of a gene.

[0051] α-Amylase: The term "α-amylase" refers to 1,4-α-D-glucanase (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., using the α-amylase assay described in WO 2020 / 023411.

[0052] Co-activity 9: The term "co-activity 9" or "AA9" refers to polypeptides classified as soluble polysaccharide monooxygenases (Quinlan et al., 2011). Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 208: 15079-15084; Phillips et al., 2011, ACS Chem. Biol. [ACS Chemical Biology] 6: 1399-1406; Lin et al., 2012, Structure [Structure] 20: 1051-1061). According to Henrissat, 1991, Biochem. J. [Journal of Biochemistry] 280: 309-316 and Henrissat and Bairoch, 1996, Biochem. J. [Journal of Biochemistry] 316:695-696, AA9 polypeptide was previously classified as glycoside hydrolase family 61 (GH61).

[0053] AA9 peptide enhances the hydrolysis of cellulose-containing materials through an enzyme with cellulose-degrading activity. Cellulose-degrading enhancement activity can be determined by measuring the increase in reducing sugars or the total amount of cellobiose and glucose in the cellulose-containing material hydrolyzed by cellulose-degrading enzymes under the following conditions: 1–50 mg total protein / g cellulose in pretreated corn stalks (PCS), wherein the total protein comprises 50%–99.5% w / w cellulose-degrading enzyme protein and 0.5%–50% w / w AA9 peptide protein, for 1–7 days at suitable temperatures (e.g., 40°C–80°C, e.g., 50°C, 55°C, 60°C, 65°C, or 70°C) and 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), compared with control hydrolysis (1–50 mg cellulose-degrading protein / g cellulose in PCS) without cellulose-degrading enhancement activity.

[0054] The AA9 peptide-enhancing activity can be determined using a mixture of CELLUCLAST® 1.5L (Novozymes A / S, Bagsværd, Denmark) and β-glucosidase as a source of cellulolytic activity, wherein the β-glucosidase is present at a protein weight of at least 2%-5% of the cellulase protein. In one embodiment, the β-glucosidase is from Aspergillus oryzae (…). Aspergillus oryzae β-glucosidase (e.g., recombinantly produced in Aspergillus oryzae according to WO 02 / 095014). In another embodiment, the β-glucosidase is Aspergillus fumigatus (…). Aspergillus fumigatusβ-glucosidase (e.g., recombinantly produced in Aspergillus oryzae as described in WO 02 / 095014).

[0055] The enhanced activity of the AA9 peptide can also be determined by incubating the AA9 peptide with 0.5% phosphate-swellable cellulose (PASC), 100 mM sodium acetate (pH 5), 1 mM MnSO4, 0.1% gallic acid, 0.025 mg / ml Aspergillus fumigatus β-glucosidase, and 0.01% TRITON® X-100 (4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol) at 40°C for 24–96 hours, followed by determining the glucose released from the PASC.

[0056] The AA9 peptide enhancement activity of the high-temperature composition can also be determined according to WO 2013 / 028928.

[0057] The AA9 peptide enhances the hydrolysis of cellulose-containing materials catalyzed by enzymes with cellulose-degrading activity by reducing the amount of cellulase required to achieve the same degree of hydrolysis by preferably at least 1.01 times, such as at least 1.05 times, at least 1.10 times, at least 1.25 times, at least 1.5 times, at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 10 times, or at least 20 times.

[0058] β-Glucosidase: The term "β-glucosidase" refers to beta-D-glucoside glucohydrolase (EC 3.2.1.21), which catalyzes the hydrolysis of terminal non-reducing β-D-glucose residues, releasing β-D-glucose. This can be referenced according to Venturi et al., 2002. J. Basic Microbiol. The procedure described in [Journal of Basic Microbiology] 42:55-66 uses p-nitrophenyl-β-D-glucopyranoside as a substrate to determine β-glucosidase activity. One unit of β-glucosidase is defined as the production of 1.0 μmol of p-nitrophenol anion per minute from 1 mM p-nitrophenyl-β-D-glucopyranoside as a substrate in 50 mM sodium citrate containing 0.01% TWEEN® 20 at 25 °C and pH 4.8.

[0059] β-Xylosidase: The term "β-xylosidase" refers to β-D-xyloside xylohydrolase (EC 3.2.1.37), which catalyzes the exolytic hydrolysis of short β(1→4)-xylooligosaccharides to remove consecutive D-xylose residues from their non-reducing ends. β-xylosidase activity can be determined using 1 mM p-nitrophenyl-β-D-xyloside as a substrate in 100 mM sodium citrate containing 0.01% TWEEN® 20 at pH 5 and 40°C. One unit of β-xylosidase is defined as the production of 1.0 μmol of p-nitrophenol anions per minute from 1 mM p-nitrophenyl-β-D-xyloside in 100 mM sodium citrate containing 0.01% TWEEN® 20 at 40°C and pH 5.

[0060] Catalase: The term "catalase" refers to hydrogen peroxide:hydrogen peroxide reductase (EC 1.11.1.6), which catalyzes the conversion of 2 H₂O₂ to O₂ + 2 H₂O. For the purposes of this 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 1 micromolar of hydrogen peroxide under the assay conditions.

[0061] Cellobiose hydrolases: The term "cellobiose hydrolases" refers to 1,4-β-D-glucan-cellobiose hydrolases (EC 3.2.1.91 and EC 3.2.1.176), which catalyze 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 (cellobiose hydrolases I) or the non-reducing end (cellobiose hydrolases II) of the chain (Teeri, 1997). Trends in Biotechnology [Trends in Biotechnology] 15: 160-167; Teeri et al., 1998, Biochem. Soc. Trans. [Journal of the Society for Biochemistry] 26:173-178). Cellobiase activity can be determined according to the procedure described below: Lever et al., 1972. Anal. Biochem. [Analytical Biochemistry] 47: 273-279; van Tilbeurgh et al., 1982, FEBS Letters [Circular of the Federation of European Biochemical Societies] 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS Letters [Circular of the Federation of European Biochemical Societies] 187: 283-288; and Tomme et al., 1988. Eur.J. Biochem.[European Journal of Biochemistry], 170: 575-581.

[0062] Cellulose-degrading enzymes or cellulases: The term “cellulose-degrading enzyme” or “cellulase” refers to one or more (e.g., several) enzymes that hydrolyze cellulose-containing materials. Such enzymes include one or more endoglucanases, one or more cellobiases, one or more β-glucosidases, or combinations thereof. Two basic methods for measuring cellulose-degrading enzyme activity include: (1) measuring total cellulose-degrading enzyme activity, and (2) measuring individual cellulose-degrading enzyme activity (endoglucanase, cellobiase, and β-glucosidase), as described in Zhang et al., 2006. Biotechnology Advances As described in [Advances in Biotechnology] 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 assay for total cellulase activity 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. [Pure and Applied Chemistry] 59: 257-68.

[0063] Cellulase activity can be determined by measuring the increase in sugar production / release during hydrolysis of cellulose-containing materials by one or more cellulases under the following conditions: 1-50 mg cellulase protein / g of cellulose in pretreated corn stalks (PCS) (or other pretreated cellulose-containing 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 with 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, sugar analysis by AMINEX® HPX-87H column chromatography (Bio-Rad Laboratories, Inc., Hercules, California, USA).

[0064] Coding sequence: The term "coding sequence" or "coding region" refers to a polynucleotide sequence that specifies the amino acid sequence of a polypeptide. The boundaries of a coding sequence are generally determined by a read frame, which typically 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). Coding sequences can be sequences of genomic DNA, cDNA, synthetic polynucleotides, and / or recombinant polynucleotides.

[0065] Endoglucanase: The term "endoglucanase" refers to 4-(1,3;1,4)-β-D-glucan-4-glucan hydrolase (EC 3.2.1.4), which catalyzes the endo-hydrolysis of β-1,4-β-D-glycosidic bonds in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichen polysaccharides, mixed β-1,3-1,4-glucans such as cereal β-D-glucan or xyloglucan, and other plant materials containing cellulose components. Endoglucanase activity can be determined by measuring a decrease in substrate viscosity or an increase in reducing ends as determined by reducing sugar assays (Zhang et al., 2006). Biotechnology Advances [Advances in Biotechnology] 24: 452-481). Also see Ghose, 1987. Pure and Appl. Chem. The procedure described in [Pure and Applied Chemistry] 59: 257-268, using carboxymethyl cellulose (CMC) as a substrate, at pH 5 and 40 °C, was used to determine endoglucanase activity.

[0066] 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—using techniques known in the art, such as measuring the level of mRNA and / or translated polypeptide.

[0067] Expression vector: The term "expression vector" refers to a straight or circular DNA molecule that contains a polynucleotide encoding a polypeptide and is operatively linked to a control sequence that provides for its expression.

[0068] Fermentable medium: The term "fermentable medium" or "fermentation medium" refers to a medium containing one or more sugars (e.g., two or more), such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and / or soluble oligosaccharides, wherein the medium can be partially converted (fermented) by host cells into a desired product, such as ethanol. In some cases, the fermentation medium is derived from a natural source, such as sugarcane, starch, or cellulose; and may be derived from a pretreatment of enzymatic hydrolysis (saccharification) of such a source. The term fermentation medium is understood herein to refer to the medium prior to the addition of fermenting organisms, such as media produced by a saccharification process, and media used in simultaneous saccharification and fermentation (SSF) processes.

[0069] Glucoamylase: The term "glucosylase" (1,4-α-D-glucan glucosylhydrolase, 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 this invention, glucosylase activity can be determined according to procedures known in the art, such as those described in WO 2020 / 023411.

[0070] Hemicellulase or hemicellulase: The term "hemicellulase" or "hemicellulase" refers to one or more (e.g., several) enzymes that can hydrolyze hemicellulose materials. See, for example, Sharlom and Shoham, 2003. Current Opinion In Microbiology[Current Views 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, arabinonanase, arabinofuranylase, coumarin esterase, ferulic esterase, galactosidase, glucuronidase, glucuronidase, mannanase, mannosidase, xylanase, and xylosidase. The substrates of these enzymes (hemicellulose) are a heterogeneous group of branched and linear polysaccharides that bind to cellulose microfibers in the plant cell wall via hydrogen bonds, thereby crosslinking them into a robust network. 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 synergistic action of many enzymes to achieve its complete degradation. The catalytic modules of hemicellulases are either glycosidases (GH) that hydrolyze glycosidic bonds, or carbohydrate esterases (CE) that hydrolyze ester bonds on the side groups of acetic acid or ferulic acid. These catalytic modules can be assigned to the GH and CE families based on their primary sequence homology. Some families, with generally similar folds, can be further classified 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. Hemicellulase activity can be found according to Ghose and Bisaria, 1987. Pure& AppI.Chem. [Theoretical and Applied Chemistry] 59: 1739-1752, measurements were taken at suitable temperatures such as 40°C-80°C, for example 50°C, 55°C, 60°C, 65°C or 70°C, and suitable pH such as 4-9, for example 5.0, 5.5, 6.0, 6.5 or 7.0.

[0071] Heteronucleotide: The term “heteronucleotide” 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 is quantitatively altered by manipulating DNA through recombinant DNA techniques, such as different (exogenous) promoters; or a native polynucleotide in the host cell having one or more additional copies of the polynucleotide to quantitatively alter its expression. A “heterogene” is a gene that contains a heteronucleotide.

[0072] Highly stringent conditions: The term "highly stringent conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following a standard DNA blotting procedure at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Vector material is finally washed three times at 65°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0073] Low stringency conditions: The term "low stringency conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following a standard DNA blotting procedure at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Vector material is finally washed three times at 50°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0074] Mature polypeptide: The term "mature polypeptide" is defined herein as a biologically active polypeptide in its final form following translation and any post-translational modifications (such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). Mature polypeptide sequences lack a signal sequence that can be determined using techniques known in the art (see, e.g., Zhang and Henzel, 2004). Protein Science [Protein Science] 13: 2819-2824. The term "mature polypeptide coding sequence" refers to the polynucleotide that encodes a mature polypeptide.

[0075] Medium-tough conditions: The term "medium-tough conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following a standard DNA blotting procedure at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Vector material is finally washed three times at 55°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0076] Medium-high stringent conditions: The term "medium-high stringent conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following a standard DNA blotting procedure at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml of cleaved and denatured salmon sperm DNA, and 35% formamide for 12 to 24 hours. Vector material is finally washed three times at 60°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0077] Pentoses: The term "pentose" refers to a five-carbon monosaccharide (e.g., xylose, arabinose, ribose, lysose, ribulose, and xylulose). Pentoses (e.g., D-xylose and L-arabinose) can be derived, for example, through the saccharification of plant cell wall polysaccharides.

[0078] Pretreated corn stalks: The term “pretreated corn stalks” or “PCS” means cellulose-containing material obtained from corn stalks by heat and dilute sulfuric acid treatment, alkali pretreatment, neutral pretreatment, or any pretreatment known in the art.

[0079] 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). EC numbers are referenced to the 1992 Enzyme Nomenclature of NC-IUBMB, Academic Press, San Diego, California, including Supplements 1-5 published below: 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 "subtilisase" refers to the enzymes described by Siezen et al., 1991. Protein Engng. [Protein Engineering] 4: 719-737 and Siezen et al., 1997, Protein Science [Protein Science] 6:501-523, the serine protease subgroup. Serine proteases, or serine peptidases, are a subgroup of proteases characterized by the presence of a serine residue at their active site, forming a covalent adduct with the substrate. Additionally, subtilisinases (and serine proteases) are characterized by having two additional active site amino acid residues besides serine: histidine and aspartic acid residues. Subtilisinases can be classified into six subclasses: the subtilisin family, the thermophilic protease family, the proteinase K family, the lanethionine antibiotic peptidases family, the Kexin family, and the Pyrolysin family. The term "protease activity" refers to proteolytic activity (EC 3.4). Protease activity can be determined using methods described in the art (e.g., US2015 / 0125925) or using commercially available assay kits (e.g., Sigma-Aldrich).

[0080] Pullulanase: The term "pullulanase" refers to a starch debranching enzyme (EC 3.2.1.41) with pullulan-6-glucan-hydrolyzing activity, which catalyzes the hydrolysis of the α-1,6-glycosidic bond in pullulan, thereby releasing maltotriose with a reducing carbohydrate terminus. For the purposes of this invention, pullulanase activity can be determined according to the PHADEBAS assay or sweet potato starch assay as described in WO 2016 / 087237.

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

[0082] For the purposes of this paper, the Needleman-Wunsch algorithm (Needleman and Wunsch, J. Mol. Biol. [Journal of Molecular Biology] 1970, 48, 443-453) determines the degree of sequence identity between two amino acid sequences using algorithms such as the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al.). Trends Genet. [Trends in Genetics] 2000, 16 This is implemented in the Needle program (version 3.0.0 or later, pp. 276-277). Optional parameters used are a vacancy open penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of "Longest Identity" marked by Needle (obtained using the -nobrief option) is used as the identity percentage and calculated as follows: (Identical residues × 100) / (Length of reference sequence - Total number of vacancies in alignment) For the purposes described herein, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, see above) was used to determine the degree of sequence identity between two deoxyribonucleotide sequences. This algorithm is implemented in the Needleman procedure of the EMBOSS software package (EMBOSS: European Molecular Biology Open Software Suite, Rice et al., 2000, see above) (preferably version 3.0.0 or later). Optional parameters used were a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The Needle-labeled “Longest Identity” output (obtained using the -nobrief option) was used as the identity percentage and calculated as follows: (Identical deoxyribonucleotides × 100) / (Length of reference sequence - Total number of vacancies in alignment) Very High Tough Conditions: The term "very high tough conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following standard DNA blotting procedures at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 50% formamide for 12 to 24 hours. Vector material is finally washed three times at 70°C for 15 minutes each time with 0.2X SSC and 0.2% SDS.

[0083] Very low stringency conditions: The term "very low stringency conditions" refers to pre-hybridization and hybridization for probes of at least 100 nucleotides in length, following standard DNA blotting procedures at 42°C in 5X SSPE, 0.3% SDS, 200 μg / ml cleaved and denatured salmon sperm DNA, and 25% formamide for 12 to 24 hours. Vector material is finally washed three times for 15 minutes each with 0.2X SSC and 0.2% SDS at 45°C.

[0084] Xylanase: The term "xylanase" refers to 1,4-β-D-xylan-xylohydrolase (EC 3.2.1.8), which catalyzes the endo-hydrolysis of the 1,4-β-D-xylosidic bonds in xylan. Xylanase activity can be determined at 37°C in 0.01% TRITON® X-100 and 200 mM sodium phosphate (pH 6) using 0.2% AZCL-arabinosylxylan as a substrate. One unit of xylanase activity is defined as the production of 1.0 μmol of azurine per minute from 0.2% AZCL-arabinosylxylan as a substrate at 37°C, pH 6, and 200 mM sodium phosphate (pH 6).

[0085] Xylitol dehydrogenase: The term "xylitol dehydrogenase" or "XDH" (AKA D-xylitol reductase) is classified as EC1.1.1.9 and refers to an enzyme that catalyzes the conversion of xylitol to D-xylitol. Methods known in the art can be used (e.g., Richard et al., 1999, FEBS Letters [Circular of the Federation of European Biochemical Societies] 457, 135-138) to determine xylitol dehydrogenase activity.

[0086] 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 the conversion of D-glucose to D-fructose in vitro. Xylose isomerase is also known as "glucose isomerase" and is classified as EC5.3.1.5. Due to the high structural stability of this enzyme, xylose isomerase is a good model for studying the relationship between protein structure and function (Karimaki et al., Protein Eng Des Sel [Protein Engineering, Design and Selection], 12004, 17 (12):861-869). Xylose isomerase activity can be determined using techniques known in the art (e.g., using the coenzyme assay of D-sorbitol dehydrogenase, such as Verhoeven et al., 2017, ...). Sci Rep [Scientific Reports] 7,46155 (as described).

[0087] Xylo-ulbucokinase: The term "xylo-ulbucokinase" or "XK" is classified under EC2.7.1.17 and refers to an enzyme that catalyzes the conversion of D-xylo-ulbuco to D-xylo-ulbuco 5-phosphate. Methods known in the art can be used (e.g., Richard et al., 2000). FEBS Microbiol. Letters [European Federation of Microbiological Societies Microbiology Letters] 190, 39-43) to determine xylulose kinase activity.

[0088] This document uses the term "about" to refer to a value or parameter, including an embodiment of that value or parameter itself. For example, a description referring to "about X" includes embodiment "X". When used in conjunction with a measurement, "about" includes a range that covers at least the uncertainty associated with the method of measuring that particular value, and may include a range of two standard deviations positive or negative around a given value.

[0089] Similarly, a gene or polypeptide that is “derived from” another gene or polypeptide X includes that gene or polypeptide X.

[0090] As used herein and in the appended claims, the singular forms “a / an,” “or,” and “the” include plural references unless the context clearly indicates otherwise.

[0091] It should be understood that the embodiments described herein include “consisting of… embodiments” and / or “essentially consisting of… embodiments.” As used herein, the word “comprise” or variations such as “comprises” or “comprising” are used in an inclusive sense, specifying the presence of the described features but not excluding the presence or inclusion of other features in various embodiments, unless otherwise required by the language or necessary meaning. Detailed Implementation

[0092] This article specifically describes recombinant fermentation organisms and methods for producing fermentation products such as ethanol from cellulose- and / or starch-containing materials. The applicant has created a novel *Saccharomyces cerevisiae* strain with improved fermentation kinetics while maintaining fermentation yield. The improved kinetics of the strain are desirable because, for example, it is more robust in the presence of inhibitors, favorable for various biomass pretreatment conditions, and provides shorter fermentation times.

[0093] One aspect is a method for producing fermentation products from cellulose- or starch-containing materials, the method comprising: (a) Saccharification of the cellulose- or starch-containing material; and (b) The saccharified material used in the recombinant fermentation bio-fermentation step (a) described herein.

[0094] Steps a) and b) can be performed sequentially or simultaneously (SSF). In one embodiment, steps a) and b) are performed simultaneously (SSF). In another embodiment, steps a) and b) are performed sequentially.

[0095] Fermented organisms In one embodiment, the fermenting organism is a recombinant strain of *Saccharomyces cerevisiae* deposited in accordance with the Budapest Treaty at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) with accession number NRRL 68303 (*Saccharomyces cerevisiae* strain MBG5364), or a derivative thereof (e.g., expressing heterologous polypeptides such as glucosylamylase and / or α-amylase), or a fermenting organism having substantially the same properties as *Saccharomyces cerevisiae* MBG5364.

[0096] In another embodiment, the fermenting organism is a recombinant strain of Saccharomyces cerevisiae deposited in accordance with the Budapest Treaty at the U.S. Agricultural Research Service Patent Collection Center (NRRL) with accession number NRRL 68304 (Saccharomyces cerevisiae strain MBG5365), or a derivative thereof (e.g., expressing heterologous polypeptides such as glucosylamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5365.

[0097] In one embodiment, the fermenting organism has one or more of the following properties: - Compared with Saccharomyces cerevisiae CIBTS1260 (e.g., 10 to 32 hours), higher ethanol fermentation kinetics at 1 g DWC / L, 32°C, and pH 5.5 (as described in Example 7 of this paper); - Compared with Saccharomyces cerevisiae CIBTS1260, higher xylose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper); - Compared to Saccharomyces cerevisiae CIBTS1260, higher glucose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper).

[0098] In one embodiment, the fermenting organism consumed more than 95% of xylose after fermenting for 48 hours at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this document).

[0099] In one embodiment, the fermenting organism can consume more than 95% of the glucose after fermenting for 24 hours at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this document).

[0100] In one embodiment, this fermenting organism achieves a higher yield of fermentation product (e.g., ethanol) under the same conditions (e.g., fermentation times of 10, 15, 20, 25, or 30 hours) compared to *Saccharomyces cerevisiae* CIBTS1260. In some embodiments, this fermenting organism results in a yield of fermentation product (e.g., ethanol) that is at least 0.25% higher, such as 0.5%, 0.75%, 1.0%, 1.25%, 1.5%, 1.75%, 2%, 3%, or 5%.

[0101] In one embodiment, the fermenting organism, after fermentation for 48 hours at 1 g DWC / L, 35°C, and pH 5.5 (as described in Examples 3 or 7 herein), can provide more than 30 g / L of ethanol, such as more than 40 g / L, more than 45 g / L, or more than 50 g / L.

[0102] In one embodiment, the fermenting organism is *Saccharomyces cerevisiae* MBG5364 (deposited at the American Agricultural Research Service Patent Culture Collection (NRRL) at 61604, Illinois, USA, under accession number NRRL 68303). In another embodiment, the fermenting organism is *Saccharomyces cerevisiae* MBG5365 (deposited at the American Agricultural Research Service Patent Culture Collection (NRRL) at 61604, Illinois, USA, under accession number NRRL 68304).

[0103] In one embodiment, the fermentation organism contains a heterologous gene encoding a xylose isomerase (e.g., the xylose isomerase shown in SEQ ID NO: 13 of WO 2016 / 045569, or an amino acid sequence having at least 80%, such as at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100% sequence identity with SEQ ID NO: 13 of WO 2016 / 045569).

[0104] In one embodiment, the fermenting organism contains a heterologous gene encoding a pentose transporter, such as the GFX gene, particularly GFX1 from *Candida intermedia* (e.g., SEQ ID NO: 18 of WO 2016 / 045569). In one embodiment, the pentose transporter gene has at least 60%, 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 SEQ ID NO: 18 of WO 2016 / 045569.

[0105] In one embodiment, the fermenting organism contains a heterologous (e.g., through overexpression) xylulose kinase gene (XKS), such as an overexpressed XKS gene from Saccharomyces cerevisiae.

[0106] In one embodiment, the fermenting organism contains a heterologous (e.g., by overexpression) ribulose 5-phosphate 3-epiisomerase gene (RPE1), such as an overexpressed RPE1 gene from Saccharomyces cerevisiae.

[0107] In one embodiment, the fermenting organism contains a heterologous (e.g., by overexpression) ribulose-5-phosphate isomerase gene (RKI1), such as an overexpressed RKI1 gene from Saccharomyces cerevisiae.

[0108] In one embodiment, the fermenting organism contains heterologous (e.g., through overexpression) transketolase gene (TKL1) and transaldolase gene (TAL1), such as overexpressed TKL1 and TAL1 genes from Saccharomyces cerevisiae.

[0109] In one embodiment, the fermenting organism has one or more, such as one, two, three, four, five, or all of the following genetic modifications: - Heteroxylose isomerase gene (Ru-XI) obtained from bovine rumen fluid, particularly the one shown in SEQ ID NO: 20 of WO 2016 / 045569, encoding the xylose isomerase shown in SEQ ID NO: 13 of WO 2016 / 045569; - The heteropentose transporter gene (GXF1) from Candida intermedia, especially the one shown in SEQ ID NO: 18 of WO 2016 / 045569; - Heterogeneous xylulokine kinase (XKS) genes, especially from typical strains of Saccharomyces cerevisiae; - Heteroribulose 5-phosphate 3-episomerase gene (RPE1), especially from typical strains of Saccharomyces cerevisiae; - Heteroribulose 5-phosphate isomerase gene (RKI1), especially from typical strains of Saccharomyces cerevisiae; - Heterogeneous transketolase gene (TKL1) and heterogenotransaldehydease gene (TAL1), especially from typical strains of Saccharomyces cerevisiae.

[0110] For example, in one embodiment, the fermentation organism of the present invention has the following genetic modifications: - Heteroxylose isomerase gene (Ru-XI) obtained from bovine rumen fluid, particularly the one shown in SEQ ID NO: 20 of WO 2016 / 045569, encoding the xylose isomerase shown in SEQ ID NO: 13 of WO 2016 / 045569; - Heterogeneous xylulokine kinase (XKS) genes, especially from typical strains of Saccharomyces cerevisiae; - Heteroribulose 5-phosphate 3-episomerase gene (RPE1), especially from typical strains of Saccharomyces cerevisiae; - Heteroribulose 5-phosphate isomerase gene (RKI1), especially from typical strains of Saccharomyces cerevisiae; - Heterogeneous transketolase gene (TKL1) and transaldolase gene (TAL1), especially from typical strains of Saccharomyces cerevisiae.

[0111] The fermenting organism can also be a derivative of *Saccharomyces cerevisiae* strains MBG5364 or MBG5365. As used herein, a “derivative” of *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 is a strain derived from said strain, for example, through mutagenesis, recombinant DNA technology, mating, cell fusion, or cytoduction between yeast strains. Strains derived from *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 can be direct descendants (i.e., products of mating between *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 and another strain or themselves) or distant descendants (produced by initial mating between *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 and another strain or themselves, followed by numerous subsequent matings).

[0112] In one embodiment, the derivative of Saccharomyces cerevisiae strain MBG5364 or MBG5365 is a heterozygous strain produced by culturing the first yeast strain with Saccharomyces cerevisiae strain MBG5364 or MBG5365, under conditions that allow for DNA combination between the first yeast strain and Saccharomyces cerevisiae strain MBG5364 or MBG5365.

[0113] In one embodiment, derivatives of *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 exhibit one or more defined characteristics of *Saccharomyces cerevisiae* strains MBG5364 or MBG5365. *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 are used to generate yeast derivatives exhibiting one or more defined characteristics of *Saccharomyces cerevisiae* strains MBG5364 or MBG5365. In this respect, *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 form the basis for preparing other strains having the defined characteristics of *Saccharomyces cerevisiae* strains MBG5364 or MBG5365. For example, yeast strains exhibiting one or more defined characteristics of *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 can be derived from *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 using methods such as classical mating, cell fusion, or cytoplasmic introduction between yeast strains, mutagenicity, or recombinant DNA techniques.

[0114] In one embodiment, a derivative of Saccharomyces cerevisiae strain MBG5364 exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG5364 can be generated by the following: (a) The first yeast strain is cultured together with the second yeast strain under conditions that allow for DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5364 (or a derivative of Saccharomyces cerevisiae strain MBG5364). (b) Screening or selecting derivatives of Saccharomyces cerevisiae strain MBG5364, for example, screening or selecting derivatives that have increased ethanol production in corn mash compared with the first strain. (c) Optionally repeat steps (a) and (b) using the screened or selected strains as the first yeast strain and / or the second yeast strain until a derivative of Saccharomyces cerevisiae strain MBG5364 exhibiting one or more of the defined characteristics of Saccharomyces cerevisiae strain MBG5364 is obtained.

[0115] In one embodiment, a derivative of Saccharomyces cerevisiae strain MBG5365 exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG5365 can be generated by the following: (a) The first yeast strain is cultured together with the second yeast strain under conditions that allow for DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5365 (or a derivative of Saccharomyces cerevisiae strain MBG5365). (b) Screening or selecting derivatives of Saccharomyces cerevisiae strain MBG5365, for example, screening or selecting derivatives that have increased ethanol production in corn mash compared with the first strain. (c) Optionally repeat steps (a) and (b) using the screened or selected strains as the first yeast strain and / or the second yeast strain until a derivative of Saccharomyces cerevisiae strain MBG5365 exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG5365 is obtained.

[0116] If the DNA of a first yeast strain can be combined with that of a second yeast strain using methods such as classical mating, cell fusion, or cytoplasmic translocation, the first yeast strain can be any strain of yeast. Typically, the first yeast strain is a *Saccharomyces* strain. More typically, the first yeast strain is a *Saccharomyces* strain. *Saccharomyces* is defined as defined by Kurtzman (2003) *FEMS Yeast Research*, Vol. 4, pp. 233-245. The first yeast strain may have desired properties designed to combine with the defining characteristics of *Saccharomyces* strain MBG5364. The first yeast strain can be, for example, any *Saccharomyces* strain, such as, for example, ETHANOL RED®. It should also be understood that the first yeast strain can be *Saccharomyces* strain MBG5364 or MBG5365 (or a derivative of *Saccharomyces* strain MBG5364 or MBG5365).

[0117] The first and second yeast strains are cultured under conditions that allow for DNA combination between yeast strains. As used herein, “DNA combination” between yeast strains refers to the combination of all or part of the genome of a yeast strain. DNA combination between yeast strains can be carried out by any method suitable for combining the DNA of at least two yeast cells, and may include, for example, mating methods involving spore formation of yeast strains to produce haploid cells and subsequent hybridization of compatible haploid cells; cytoplasmic introduction; or cell fusion such as protoplast fusion.

[0118] In one embodiment, the first yeast strain and the second yeast strain are cultured under conditions that allow DNA combination between the first yeast strain and the second yeast strain, the culture comprising: (i) To cause the first yeast strain and the second yeast strain to form spores; (ii) Germinate the spores produced by the first yeast strain and hybridize them with the spores produced by the second yeast strain.

[0119] In one embodiment, a method for generating a derivative of Saccharomyces cerevisiae strain MBG5364 exhibiting one or more defined characteristics of MBG5364 includes: (a) Provide: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5364 (or a derivative of Saccharomyces cerevisiae strain MBG5364). (b) To induce the first yeast strain and the second yeast strain to form spores; (c) Germinate spores of the first yeast strain and hybridize them with germinating spores of the second yeast strain; (d) Screening or selecting derivatives of Saccharomyces cerevisiae strain MBG5364, for example, screening or selecting derivatives that have increased ethanol production in mash compared to the first strain, and / or have a higher ethanol yield from glucose during mash fermentation compared to the first strain. (e) Optionally repeat steps (b) to (d) using the screened or selected strains as the first and / or second yeast strains.

[0120] In one embodiment, a method for generating a derivative of Saccharomyces cerevisiae strain MBG5364 exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG5365 includes: (a) Provide: (i) a first yeast strain; and (ii) a second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5365 (or a derivative of Saccharomyces cerevisiae strain MBG5365). (b) To induce the first yeast strain and the second yeast strain to form spores; (c) Germinate spores of the first yeast strain and hybridize them with germinating spores of the second yeast strain; (d) Screening or selecting derivatives of Saccharomyces cerevisiae strain MBG5365, for example, screening or selecting derivatives that have increased ethanol production in mash compared to the first strain, and / or have a higher ethanol yield from glucose during mash fermentation compared to the first strain. (e) Optionally repeat steps (b) to (d) using the screened or selected strains as the first and / or second yeast strains.

[0121] Methods for enabling yeast strains, particularly those of the genus *Yeast*, to form spores, germinate, and hybridize are known in the art and described, for example, Ausubel, FM et al., (1997) Current Protocols in Molecular Biology, Vol. 2, pp. 13.2.1–13.2.5 (John Willey & Sons Inc.); Chapter 7, “Sporulation and Hybridisation of yeast” by RR Fowell, in *The Yeasts*, Vol. 1, AHRose and JS Harrison (eds.), 1969, Academic Press.

[0122] In one embodiment, the yeast strain can be cultured under conditions that allow cell fusion. Methods using cell fusion techniques to generate intraspecific or interspecific heterozygotes are described, for example, in Spencer et al. (1990), Yeast Technology, Spencer JFT and Spencer DM (eds.), Springer Verlag, New York.

[0123] In another embodiment, the yeast strain can be cultured under conditions that allow for cytoplasmic introduction. Methods for cytoplasmic introduction are described, for example, in Inge-Vechymov et al. (1986) Genetika [Genetics] 22:2625-2636; Johnston (1990), Yeast Technology, Spencer JFT and Spencer DM (eds.), Springer Verlag [Springer Publishing Company], New York.

[0124] In one embodiment, screening or selecting derivatives of Saccharomyces cerevisiae strain MBG5364 or MBG5365 includes screening or selecting derivatives that have increased ethanol production compared to the first strain, and / or screening or selecting heterozygotes with higher ethanol yields, for example, as described in WO 2019 / 161227.

[0125] In one embodiment, a derivative of Saccharomyces cerevisiae strain MBG5364 or MBG5365 exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG5364 or MBG5365 may be a mutant of Saccharomyces cerevisiae strain MBG5364 or MBG5365. Methods for producing yeast mutants, and particularly Saccharomyces cerevisiae mutants, are known in the art and described, for example, in Lawrence CW (1991) Methods in Enzymology, 194: 273-281.

[0126] In another embodiment, a derivative of *Saccharomyces cerevisiae* strain MBG5364 exhibiting one or more defined characteristics of MBG5364 may be a recombinant derivative of MBG5364. In another embodiment, a derivative of *Saccharomyces cerevisiae* strain MBG5365 exhibiting one or more defined characteristics of MBG5365 may be a recombinant derivative of MBG5365. The recombinant derivatives of *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 are strains produced by introducing nucleic acids into *Saccharomyces cerevisiae* strains MBG5364 or MBG5365 using recombinant DNA technology. Recombinant methods for introducing nucleic acids into yeast cells, and especially yeast strains, are known in the art and described, for example, in Ausubel, FM et al. (1997), *Current Protocols in Molecular Biology*, Vol. 2, pp. 13.7.1–13.7.7, published by John Wiley & Sons Inc.

[0127] In one embodiment, a recombinant derivative of Saccharomyces cerevisiae strain MBG5364 or MBG5365 has been prepared by genetically modified strains (or another derivative thereof) to express heterologous enzymes, such as α-amylase and / or glucosylase as described herein (or any enzyme described in WO 2020 / 023411, the contents of which are incorporated herein by reference).

[0128] In one embodiment, it is a method for producing a recombinant derivative of Saccharomyces cerevisiae strain MBG5364 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, under accession number NRRL 68303), the method comprising: (a) Transforming *Saccharomyces cerevisiae* strain MBG5364 (or a derivative thereof) with an expression vector encoding one or more heterologous enzymes, such as glucosylamylase and / or α-amylase; and (b) Isolate the transformed strain.

[0129] In one embodiment, a derivative of the Saccharomyces cerevisiae strain MBG5364 can be prepared by the following: (a) Co-culturing a first yeast strain with a second yeast strain, wherein the second yeast strain is *Saccharomyces cerevisiae* strain MBG5364 (or a derivative thereof), under conditions allowing for DNA combination between the first and second yeast strains; and (b) Isolation of heterozygous strains; and (c) Optionally, steps (a) and (b) may be repeated using the heterozygous strain isolated in step (b) as a derivative of the first yeast strain and / or the Saccharomyces cerevisiae strain MBG5364.

[0130] In one embodiment, it is a method for producing a recombinant derivative of Saccharomyces cerevisiae strain MBG5365 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, under accession number NRRL 68304), the method comprising: (a) Transforming *Saccharomyces cerevisiae* strain MBG5365 (or a derivative thereof) with an expression vector encoding one or more heterologous enzymes, such as glucosylamylase and / or α-amylase; and (b) Isolate the transformed strain.

[0131] In one embodiment, a derivative of the Saccharomyces cerevisiae strain MBG5365 can be prepared by the following: (a) Co-culturing a first yeast strain with a second yeast strain, wherein the second yeast strain is *Saccharomyces cerevisiae* strain MBG5365 (or a derivative of *Saccharomyces cerevisiae* strain MBG5365), under conditions allowing for DNA combination between the first and second yeast strains; and (b) Isolation of heterozygous strains; and (c) Optionally, steps (a) and (b) may be repeated using the heterozygous strain isolated in step (b) as a derivative of the first yeast strain and / or the Saccharomyces cerevisiae strain MBG5365.

[0132] In some embodiments, derivatives of Saccharomyces cerevisiae strains MBG5364 or MBG5365 express glucosylamylase and / or α-amylase. Derivatives expressing glucosylamylase and / or α-amylase have been developed to increase ethanol yield and improve process economics by reducing enzyme costs, since some or all of the essential enzymes required for starch hydrolysis are produced by yeast organisms.

[0133] Composition This aspect relates to a formulated yeast composition comprising the yeast strain described herein and naturally occurring and / or non-naturally occurring components.

[0134] In one embodiment, it is a composition comprising *Saccharomyces cerevisiae* strain MBG5364 (or a derivative of *Saccharomyces cerevisiae* strain MBG5364) or *Saccharomyces cerevisiae* strain MBG5365 (or a derivative of *Saccharomyces cerevisiae* strain MBG5365). The composition may be, for example, paste yeast, compressed yeast, wet yeast, dry yeast, semi-dry yeast, pulverized yeast, stabilized liquid yeast, or frozen yeast. Methods for preparing such yeast compositions are known in the art.

[0135] In one embodiment, the brewer's yeast strain is dry yeast, such as active dry yeast or instant yeast. In one embodiment, the brewer's yeast strain is pulverized yeast. In one embodiment, the brewer's yeast strain is compressed yeast. In one embodiment, the brewer's yeast strain is paste yeast.

[0136] In one embodiment, a composition comprises yeasts of the genus *Saccharomyces* described herein (especially *Saccharomyces cerevisiae* strain MBG5364 or MBG5365) and one or more components selected from the group consisting of surfactants, emulsifiers, gums, swelling agents, antioxidants, and other processing aids.

[0137] surfactants The compositions described herein may comprise yeasts of the genus *Saccharomyces* described herein (especially *Saccharomyces cerevisiae* strains MBG5364 or MBG5365) and any suitable surfactant. In one embodiment, one or more surfactants are anionic surfactants, cationic surfactants, and / or nonionic surfactants.

[0138] emulsifier The compositions described herein may comprise yeasts of the genus *Saccharomyces* described herein (especially *Saccharomyces cerevisiae* strains MBG5364 or MBG5365) and any suitable emulsifier. In one embodiment, the emulsifier is a fatty acid ester of sorbitan. In another embodiment, the emulsifier is selected from the group consisting of: sorbitan monostearate (SMS), citrate of mono- and diglycerides, polyglycerol esters, and fatty acid esters of propylene glycol.

[0139] In one embodiment, the composition comprises yeasts of the genus *Saccharomyces* described herein (especially *Saccharomyces cerevisiae* strains MBG5364 or MBG5365) and *Olindronal* SMS, *Olindronal* SK, or *Olindronal* SPL, including the compositions described in European Patent No. 1,724,336 (incorporated by reference). For active dry yeasts, these products are commercially available from Bussetti AG of Austria.

[0140] gum The compositions described herein may comprise yeasts of the genus *Saccharomyces* described herein (especially *Saccharomyces cerevisiae* strains MBG5364 or MBG5365) and any suitable gums. In one embodiment, the gum is selected from the group consisting of: sophora bean gum, guar gum, astragalus gum, gum arabic, xanthan gum, and gum arabic, particularly for paste, compressed, and dry yeasts.

[0141] swelling agent The compositions described herein may comprise yeasts of the genus *Saccharomyces* described herein (especially *Saccharomyces cerevisiae* strains MBG5364 or MBG5365) and any suitable swelling agent. In one embodiment, the swelling agent is methylcellulose or carboxymethylcellulose.

[0142] antioxidants The compositions described herein may comprise yeasts of the genus *Saccharomyces* described herein (especially *Saccharomyces cerevisiae* strains MBG5364 or MBG5365) 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.

[0143] Methods using cellulose-containing materials In some embodiments, the methods described herein produce fermentation products from cellulose-containing materials. The primary polysaccharide in the primary cell walls of biomass is cellulose, followed by hemicellulose, and then pectin. Secondary cell walls, formed after cell cessation of growth, also contain polysaccharides and are reinforced by polymeric lignin covalently cross-linked with hemicellulose. Cellulose is a homopolymer of dehydrated cellobiose and is therefore a linear β-(1-4)-D-glucan, while hemicellulose comprises a variety of compounds, such as xylan, xyloglucan, arabinoylxylan, and mannan, which have a series of substituents forming complex branched structures. Although cellulose is generally polymorphic, it is found to exist primarily as an insoluble crystalline matrix of parallel glucan chains in plant tissues. Hemicellulose is typically hydrogen-bonded to cellulose and other hemicelluloses, which helps stabilize the cell wall matrix.

[0144] Cellulose is commonly found in, for example, the stems, leaves, shells, bark, and rachis of plants, or the leaves, branches, and wood of trees. Cellulose-containing materials can be, but are 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), pp. 105-118, Taylor & Francis Publishing Group, Washington, D.C.; Wyman, 1994). Bioresource Technology [Bioresource Technology] 50: 3-16; Lynd, 1990, Applied Biochemistry and Biotechnology[Applied Biochemistry and Biotechnology] 24 / 25: 695-719; Mosier et al., 1999, Recent Progress in Bioconversion of Lignocellulosics, Advances in Biochemical Engineering / Biotechnology, T. Scheper (ed.), Vol. 65, pp. 23-40, Springer-Verlag, New York. It should be understood herein that 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, which comprises cellulose, hemicellulose, and lignin.

[0145] In one embodiment, the cellulose-containing material is agricultural waste, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill waste, waste paper, or wood (including forestry waste).

[0146] In another embodiment, the cellulose-containing material is reed, bagasse, bamboo, corn cob, corn fiber, corn stalk, awn, rice straw, willow sorghum, or wheat straw.

[0147] In another embodiment, the cellulose-containing material is aspen, eucalyptus, fir, pine, poplar, spruce, or willow.

[0148] In another embodiment, the cellulose-containing material is seaweed cellulose, bacterial cellulose, cotton linters, filter paper, microcrystalline cellulose (e.g., AVICEL®), or phosphoric acid-treated cellulose.

[0149] In another embodiment, the cellulose-containing material is aquatic biomass. As used herein, the term "aquatic biomass" means biomass produced in an aquatic environment through a photosynthetic process. Aquatic biomass can be algae, emergent plants, floating-leaved plants, or submerged plants.

[0150] The cellulose-containing material can be used as is or pretreated using conventional methods known in the art, as described herein. In a preferred embodiment, the cellulose-containing material has been pretreated.

[0151] Methods using cellulose-containing materials can be performed using methods conventional in the art. Furthermore, these methods can be carried out using any conventional biomass processing apparatus configured to implement them.

[0152] Cellulose pretreatment In one embodiment, the cellulose-containing material is pretreated prior to saccharification.

[0153] In practicing the methods described herein, any pretreatment method known in the art can be used to disrupt the plant cell wall components of cellulose-containing materials (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 [Bioresource Technology] 100: 10-18; Mosier et al., 2005, Bioresource Technology [Bioresource 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 - Biopharmaceuticals. 2: 26-40).

[0154] The cellulose-containing material can also be subjected to particle size reduction, sieving, pre-soaking, wetting, washing and / or conditioning using methods known in the art prior to pretreatment.

[0155] Conventional pretreatment methods include, but are not limited to: steam pretreatment (with or without blasting), dilute acid pretreatment, hot water pretreatment, alkali pretreatment, lime pretreatment, wet oxidation, wet blasting, ammonia fiber blasting, organic solvent pretreatment, and biological pretreatment. Other pretreatment methods include ammonia percolation, ultrasonication, electroporation, microwave treatment, supercritical CO2, supercritical H2O, ozone treatment, ionic liquid treatment, and gamma radiation pretreatment.

[0156] In one embodiment, the cellulose-containing material is pretreated prior to saccharification (i.e., hydrolysis) and / or fermentation. Pretreatment is preferably performed before hydrolysis. Alternatively, 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 converts some of the biomass into fermentable sugars (even in the absence of enzymes).

[0157] In one embodiment, the cellulose-containing material is pretreated with steam. In the steam pretreatment, the cellulose-containing material is heated to break down plant cell wall components, including lignin, hemicellulose, and cellulose, making cellulose and other fractions (e.g., hemicellulose) accessible to enzymes. The cellulose-containing material is passed through or through a reaction vessel, into which steam is injected 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), with the optimal temperature range depending on the optional addition of a chemical catalyst. The residence time for steam pretreatment is preferably 1–60 minutes, e.g., 1–30 minutes, 1–20 minutes, 3–12 minutes, or 4–10 minutes, with the optimal residence time depending on the temperature and the optional addition of a chemical catalyst. Steam pretreatment allows for a relatively high solids loading, so that the cellulose-containing material typically only becomes moist during the pretreatment process. Steam pretreatment is often combined with the explosive discharge of the pretreated material, a phenomenon known as steam explosion, where rapid evaporation to atmospheric pressure and turbulence of the material increase the accessible surface area through breakup (Duff and Murray, 1996). Bioresource Technology [Bioresource Technology] 855: 1-33; Galbe and Zacchi, 2002, Appl. Microbiol. Biotechnol. [Applied Microbiology and Biotechnology] 59: 618-628; U.S. Patent Application No. 2002 / 0164730). During steam pretreatment, the acetyl groups of hemicellulose are cleaved, and the resulting acid autocatalytically hydrolyzes the hemicellulose into monosaccharides and oligosaccharides. Lignin is removed only to a limited extent.

[0158] In one embodiment, the cellulose-containing 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 pretreatment can convert crystalline cellulose into amorphous cellulose. Examples of suitable chemical pretreatment methods include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia cellulose / freeze-explosion (AFEX), ammonia permeation (APR), ionic liquids, and organic solvent pretreatment.

[0159] Sometimes, a chemical catalyst (such as H₂SO₄ or SO₂) (typically 0.3 to 5% w / w) is added prior to steam pretreatment. This catalyst reduces time and temperature, increases recovery, 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 cellulose-containing material is mixed with dilute acid (typically H₂SO₄) and water to form a slurry, heated to the desired temperature by steam, and flashed to atmospheric pressure after a residence time. Dilute acid pretreatment can be performed using many reactor designs, such as plug flow reactors, countercurrent reactors, or continuous countercurrent shrink-bed reactors (Duff and Murray, 1996). Bioresource Technology [Bioresource Technology] 855: 1-33; Schell et al., 2004, Bioresource Technology [Bioresource 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 cellulose-containing material was pretreated with dilute acid at 180°C for 5 minutes using 4% w / w sulfuric acid.

[0160] 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-explosion (AFEX) pretreatment. Lime pretreatment is carried out with calcium oxide or calcium hydroxide at a temperature of 85°C–150°C, with a residence time ranging from 1 hour to several days (Wyman et al., 2005). Bioresource Technology [Bioresource Technology] 96: 1959-1966; Mosier et al., 2005, Bioresource Technology [Bioresource Technology] 96: 673-686). WO 2006 / 110891, WO 2006 / 110899, WO 2006 / 110900 and WO 2006 / 110901 disclose pretreatment methods using ammonia.

[0161] Wet oxidation is a thermal pretreatment typically carried out at 180–200 °C for 5–15 minutes with the addition of an oxidant (such as oxygen peroxide or superpressure oxygen) (Schmidt and Thomsen, 1998). Bioresource Technology [Bioresource Technology] 64: 139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. [Applied Biochemistry and Biotechnology] 117: 1-17; Varga et al., 2004, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 88: 567-574; Martin et al., 2006, J. Chem. Technol. Biotechnol.[Journal of Chemical Technology and Biotechnology] 81: 1669-1677). Pretreatment is preferably carried out with 1%-40% dry matter, for example 2%-30% dry matter, or 5%-20% dry matter, and the initial pH often increases due to the addition of alkali such as sodium carbonate.

[0162] A modified version of the wet oxidation pretreatment method, known as wet blasting (a combination of wet oxidation and steam explosion), can process up to 30% dry matter. In wet blasting, an oxidant is introduced during the pretreatment after a certain residence time. The pretreatment is then terminated by flash to atmospheric pressure (WO 2006 / 032282).

[0163] Ammonia fiber explosion (AFEX) involves treating cellulose-containing materials with liquid or gaseous ammonia for 5-10 minutes at mild 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; Chundawart wait people , 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 [Bioresource Technology] 96: 2014-2018). During AFEX pretreatment, cellulose and hemicellulose remain relatively intact. The lignin-carbohydrate complex is cleaved.

[0164] Organic solvent pretreatment deligninates cellulose-containing materials by extraction with aqueous ethanol (40%-60% ethanol) at 160℃-200℃ 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 organic solvent pretreatment, most of the hemicellulose and lignin are removed.

[0165] Other examples of suitable preprocessing methods are provided by Schell et al., 2003. Appl. Biochem. Biotechnol.[Applied Biochemistry and Biotechnology] 105-108: 69-85, and Mosier et al., 2005. Bioresource Technology It is described in [Bioresource Technology] 96: 673-686 and US 2002 / 0164730.

[0166] In one embodiment, the chemical pretreatment is performed as a dilute acid treatment, and more preferably as a continuous dilute acid treatment. The acid is typically sulfuric acid, but other acids such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof may also be used. The weak acid treatment is preferably carried out in a pH range of 1 to 5, for example, 1 to 4 or 1 to 2.5. In one embodiment, the acid concentration is preferably in the range of 0.01 wt.% to 10 wt.% acid, for example, 0.05 wt.% to 5 wt.% acid or 0.1 wt.% to 2 wt.% acid. The acid is brought into contact with the cellulose-containing material and maintained at a temperature preferably in the range of 140°C to 200°C (e.g., 165°C to 190°C) for a duration in the range of 1 to 60 minutes.

[0167] In another embodiment, pretreatment is carried out in an aqueous slurry. In a preferred embodiment, the cellulose-containing material is present during pretreatment in an amount preferably between 10 wt.% and 80 wt.%, for example, 20 wt.% to 70 wt.% or 30 wt.% to 60 wt.%, such as about 40 wt.%. The pretreated cellulose-containing material may be left unwashed or washed using any method known in the art, for example, washing with water.

[0168] In one embodiment, the cellulose-containing material is subjected to mechanical or physical pretreatment. The terms "mechanical pretreatment" or "physical pretreatment" refer to any pretreatment that promotes a reduction in particle size. For example, such pretreatment may involve different types of grinding or milling (e.g., dry grinding, wet grinding, or vibratory ball milling).

[0169] The cellulose-containing material can be pretreated physically (mechanically) and chemically. Mechanical or physical pretreatment can be combined with steam / steam explosion, hydrothermolysis, dilute 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 preferably in the range of about 100 to about 400 psi, for example, 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, for example, about 140°C to about 200°C. In a preferred embodiment, mechanical or physical pretreatment is carried out in batches using a steam gun hydrolyzer system, such as the Sunds Hydrolyzer available from Sunds Defibrator AB, which uses the high pressure and high temperature as defined above. Physical and chemical pretreatment can be performed sequentially or simultaneously, as needed.

[0170] Therefore, in one embodiment, the cellulose-containing material is subjected to physical (mechanical) or chemical pretreatment, or any combination thereof, to promote the separation and / or release of cellulose, hemicellulose and / or lignin.

[0171] In one embodiment, the cellulose-containing 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 cellulose-containing material. Biological pretreatment techniques may involve the application of lignin-dissolving microorganisms and / or enzymes (see, for example, Hsu, T.-A., 1996, Pretreatment of biomass). Handbook on Bioethanol: Production and Utilization In *The Bioethanol Handbook: Production and Utilization*, edited by Wyman, CE, Taylor & Francis [Taylor & Francis Publishing Group], Washington, DC, 179-212; Ghosh and Singh, 1993. Adv. Appl. Microbiol. [Advances in Applied Microbiology] 39: 295-333; McMillan, JD, 1994, Pretreating lignocellulosic biomass: a review. Enzymatic Conversion of Biomass for Fuels Production[Enzymatic Conversion of Biomass for Fuel Production], edited by Himmel, ME, Baker, JO, and Overend, RP, ACSSymposium Series 566, American Chemical Society, Washington, D.C., Chapter 15; Gong, CS, Cao, NJ, Du, J., and Tsao, GT, 1999, Ethanol Production from Renewable Resources, Advances in Biochemical Engineering / Biotechnology 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. [Enzyme and Microbial Technology] 18: 312-331; and Vallander and Eriksson, 1990, Adv. Biochem. Eng. / Biotechnol. [Biology] Advances in Chemical Engineering / Biotechnology 42: 63-95).

[0172] Saccharification and fermentation of cellulose-containing materials 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); mixed hydrolysis and fermentation (HHF); separate hydrolysis and co-fermentation (SHCF); and hybrid hydrolysis and co-fermentation (HHCF).

[0173] SHF uses separate processing steps to first enzymatically hydrolyze the cellulose-containing material into fermentable sugars (e.g., glucose, cellobiose, and pentose monomers), and then ferment the fermentable sugars into ethanol. In SHF, the enzymatic hydrolysis of the cellulose-containing material and the fermentation of sugars into ethanol are combined in one step (Philippidis, GP, 1996, Cellulose bioconversion technology). Handbook on Bioethanol: Production and Utilization In *The Bioethanol Handbook: Production and Utilization*, edited by Wyman, CE, Taylor & Francis [Taylor & Francis Publishing Group], Washington, DC, 179-212. SSCF involves the co-fermentation of multiple sugars (Sheehan and Himmel, 1999). Biotechnol. Prog.[Advances in Biotechnology] 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 organism. 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 carry out the methods described herein.

[0174] Conventional apparatus may include fed-batch stirred reactors, batch stirred reactors, continuous flow stirred reactors with ultrafiltration capabilities, and / or continuous plug flow column reactors (de Castilhos Corazza et al.). . , 2003, Acta Scientiarum.Technology [Technical Journal] 25: 33-38; Gusakov and Sinitsyn, 1985, Enz. Microb.Technol. [Enzyme and Microbial Technology] 7: 346-352), Wear reactor (Ryu and Lee, 1983, Biotechnol. Bioeng. [Biotechnology and Bioengineering] 25: 53-65). Other reactor types include fluidized bed reactors, upflow blanket reactors, immobilized reactors, and extruder-type reactors for hydrolysis and / or fermentation.

[0175] In the saccharification step (i.e., the hydrolysis step), the cellulose-containing material and / or starch-containing 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 enzymatically promoted by, for example, a composition of cellulases. These enzymes can be added simultaneously or sequentially.

[0176] Enzymatic hydrolysis can be carried out in a suitable aqueous environment under conditions easily determined by those skilled in the art. In one embodiment, hydrolysis is carried out under conditions suitable for the activity of one or more enzymes, i.e., optimal conditions for those enzymes. Hydrolysis can be carried out in a batch-feed or continuous process, wherein the cellulose-containing material and / or starch-containing material is gradually added, for example, to an enzyme-containing hydrolysis solution.

[0177] 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 those skilled in the art. For example, saccharification can last up to 200 hours, but is typically carried out preferably for about 12 to about 120 hours, for example, about 16 to about 72 hours or about 24 to about 48 hours. Temperatures are preferably in the range of about 25°C to about 70°C, for example, about 30°C to about 65°C, about 40°C to about 60°C, or about 50°C to 55°C. pH is preferably in the range of about 3 to about 8, for example, about 3.5 to about 7, about 4 to about 6, or about 4.5 to about 5.5. The dry solids content is preferably about 5 wt.% to about 50 wt.%, for example, about 10 wt.% to about 40 wt.%, or about 20 wt.% to about 30 wt.%.

[0178] Saccharification can be performed using cellulase compositions. Such enzyme compositions are described in the following "Cellulase Compositions" section. These cellulase compositions may contain any protein for degrading the cellulose-containing 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, patulin, lignin-degrading enzyme, oxidoreductase, pectinase, protease, and swelling agent.

[0179] In another embodiment, the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of: endoglucanase, cellobiase, and β-glucosidase.

[0180] In another embodiment, the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of: acetylmannan esterase, acetylxylan esterase, arabinogalactanase, arabinofuranase, coumarate esterase, ferulic esterase, galactosidase, glucuronidase, glucuronidase, 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.

[0181] The enzymes or enzyme compositions used in the methods of this invention can be in any suitable form, such as fermentation broth formulations or cell compositions, cell lysates with or without cell debris, semi-purified or purified enzyme preparations, or host cells as the source of the enzyme. The enzyme composition can be a dry powder or granules, dust-free granules, liquid, stabilized liquid, or stabilized protected enzyme. Liquid enzyme preparations can be stabilized according to established methods, for example, by adding a stabilizer (such as a sugar, sugar alcohol, or other polyol), and / or lactic acid or another organic acid.

[0182] In one embodiment, the effective amount of the cellulase composition or hemicellulose 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.

[0183] In one embodiment, the compound is added to the cellulose glucosyl units in the following molar ratio: approximately 10 -6 Approximately 10, for example, approximately 10 -6 From approximately 7.5, approximately 10 -6 Approximately 5, approximately 10 -6 From approximately 2.5, approximately 10 -6 About 1, about 10 -5 About 1, about 10 -5 To about 10 -1 Approximately 10 -4 To about 10 -1 Approximately 10 -3 To about 10 -1 or about 10 -3 To about 10 -2 In another embodiment, the effective amount of such a compound is about 0.1 μM to about 1 M, for example about 0.5 μM to about 0.75 M, about 0.75 μM to about 0.5 M, about 1 μM to about 0.25 M, about 1 μM to about 0.1 M, about 5 μM to about 50 mM, about 10 μM to about 25 mM, about 50 μM to about 25 mM, about 10 μM to about 10 mM, about 5 μM to about 5 mM, or about 0.1 mM to about 1 mM.

[0184] The term "liquor" refers to a solution phase (aqueous phase, organic phase, or combination thereof) and its soluble contents produced under the conditions described in WO 2012 / 021401 from the treatment of lignocellulose and / or hemicellulose material, or its monosaccharides (e.g., xylose, arabinose, mannose, etc.) in a slurry. A liquid for enhancing the cellulose degradation of the AA9 polypeptide (GH61 polypeptide) can be produced by treating the lignocellulose or hemicellulose material (or raw material) with heat and / or pressure, 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 degradation of the material, followed by separation of the solution from the residual solids. The degree of enhancement in cellulose degradation that can be obtained from the combination of the liquid and the AA9 polypeptide during the hydrolysis of the cellulose substrate by a cellulase preparation is determined by such conditions. The liquid can be separated from the treated material using standard methods in the art, such as filtration, precipitation, or centrifugation.

[0185] In one embodiment, the effective amount of liquid for cellulose is approximately 10. -6 cellulose up to approximately 10 g / g, for example, approximately 10 -6 Approximately 7.5 g, approximately 10 g -6 Approximately 5 g, approximately 10 g -6 Approximately 2.5 g, approximately 10 g -6 About 1 g, about 10 -5 About 1 g, about 10 -5 To about 10 -1 g, approximately 10 -4 To about 10 -1 g, approximately 10 -3 To about 10 -1 g, or about 10 -3 To about 10 -2 cellulose g / g.

[0186] In the fermentation step, for example as a result of pretreatment and enzymatic hydrolysis, the sugars released from the cellulose-containing material are fermented into ethanol by a fermenting organism (such as the yeast described herein). Hydrolysis (saccharification) and fermentation can be separate or simultaneous.

[0187] Any suitable hydrolyzed cellulose-containing material can be used in the fermentation steps described herein. Such raw materials 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 unit sugar potential, and the recalcitrantness to enzymatic conversion.

[0188] The production of ethanol via fermentation using cellulose-containing materials is a process that utilizes the metabolism of sugars (monosaccharides). The sugar composition of the hydrolyzed cellulose-containing material and the fermentation organism's ability to utilize different sugars directly affect the process yield.

[0189] 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 fermentation products. In some embodiments, the fermentation process is carried out under aerobic or microaerophilic conditions (i.e., oxygen concentrations less than those in the air) or anaerobic conditions. In some embodiments, fermentation is carried out under anaerobic conditions (i.e., no detectable oxygen) or oxygen levels 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+.

[0190] The fermentation process is typically carried out at temperatures optimal for the recombinant fungal cells. For example, in some embodiments, the fermentation process is carried out at temperatures ranging from about 25°C to about 42°C. Typically, the method is carried out at temperatures 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.

[0191] Fermentation stimulants can be used in the processes described herein to further improve fermentation, and in particular to improve the performance of fermenting organisms, such as rate increases and product yields (e.g., ethanol yield). “Fermentation stimulant” refers to an agent used to stimulate the growth of fermenting organisms, particularly yeast. Preferred fermentation stimulants for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenic acid, niacin, meso-inositol, thiamine, pyridoxine, para-aminobenzoic acid, folic acid, riboflavin, and vitamins A, B, C, D, and E. For example, see Alfenore et al., Improving ethanol production and viability of Saccharomyces cerevisiae by a vitamin feeding strategy during fed-batch process, Springer-Verlag (2002), which is hereby incorporated by reference. Examples of minerals include minerals and mineral salts that can supply nutrients containing P, K, Mg, S, Ca, Fe, Zn, Mn and Cu.

[0192] Cellulose-degrading enzymes and compositions Cellulase or cellulase compositions may be present and / or added during the saccharification process. Cellulase compositions are enzyme preparations containing one or more (e.g., several) enzymes that hydrolyze cellulose-containing materials. Such enzymes include endoglucanase, cellobiase, β-glucosidase, and / or combinations thereof.

[0193] In some embodiments, the fermentation organism comprises one or more (e.g., several) heteropolynucleotides encoding an enzyme capable of hydrolyzing cellulose-containing materials (e.g., endoglucanase, cellobiase, β-glucosidase, or combinations thereof). Consider any enzyme (capable of hydrolyzing cellulose-containing materials) described or referenced herein for expression in the fermentation organism.

[0194] The cellulase can be any cellulase suitable for expression in the fermentation organisms and / or methods described herein (e.g., endoglucanase, cellobiase, β-glucosidase), such as naturally occurring cellulases or variants thereof that retain cellulase activity.

[0195] In some embodiments, when cultured under identical conditions, fermentation organisms containing heteropolynucleotides encoding cellulases have increased levels of cellulase (e.g., increased levels of endoglucanase, cellobiase, and / or β-glucosidase) activity compared to fermentation organisms without such cellulase. In some embodiments, when cultured under identical conditions, the fermentation organism has an increased level of cellulase activity by at least 5%, for example, 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 fermentation organisms without such cellulase.

[0196] Exemplary cellulases that can be used with the fermentation organisms and / or methods described herein include bacterial, yeast, or filamentous fungal cellulases, such as any microorganisms obtained from the descriptions or references herein, as described above in the section relating to proteases.

[0197] The cellulase can be from any source. In one embodiment, the cellulase is derived from strains of the genus *Trichoderma*, such as *Trichoderma reesei*; strains of the genus *Pyroderma*, such as *Pyroderma utilis*; and / or strains of the genus *Aureospora*, such as *Aureospora loukenowens*. In a preferred embodiment, the cellulase is derived from strains of *Trichoderma reesei*.

[0198] The cellulase composition may further comprise one or more of the following polypeptides (such as enzymes): AA9 polypeptide (GH61 polypeptide) with cellulase-enhancing activity, β-glucosidase, xylanase, β-xylosidase, CBHII, CBHII, or a mixture of two, three, four, five or six thereof.

[0199] Another one or more polypeptides (e.g., AA9 polypeptide) and / or one or more enzymes (e.g., β-glucosidase, xylanase, β-xylosidase, CBH I and / or CBH II) for the production of this cellulase composition of organisms (e.g., Trichoderma reesei) may be exogenous.

[0200] In one embodiment, the cellulase composition comprises an AA9 polypeptide having cellulase-enhancing activity and a β-glucosidase.

[0201] In another embodiment, the cellulase composition comprises an AA9 polypeptide with cellulase-enhancing activity, a β-glucosidase, and CBH I.

[0202] In another embodiment, the cellulase composition comprises an AA9 polypeptide, β-glucosidase, CBH I, and CBH II, which have cellulase-enhancing activity.

[0203] Other enzymes (such as endoglucanase) may also be included in this cellulase composition.

[0204] As mentioned above, the cellulase composition may contain a variety of different polypeptides, including enzymes.

[0205] In one embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises a Thermophilic Ascomycota orange AA9 (GH61A) polypeptide (e.g., WO 2005 / 074656) having cellulase-enhancing activity, 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).

[0206] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises a polypeptide of Ascomycota orangeis AA9 (GH61A) having cellulase-enhancing activity (e.g., SEQ ID NO: 2 in WO 2005 / 074656) and Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO: 2 in WO2005 / 047499).

[0207] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises an Emersonian Penicillium AA9 (GH61A) polypeptide having cellulolytic-enhancing activity, particularly one disclosed in WO 2011 / 041397, and Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO: 2 of WO2005 / 047499).

[0208] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises an Emersonian Penicillium AA9 (GH61A) polypeptide having cellulolytic-enhancing activity, particularly one disclosed in WO 2011 / 041397, and a variant of Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO: 2 of WO2005 / 047499), or a variant disclosed in WO 2012 / 044915 (incorporated hereby by reference), particularly variants comprising one or more (e.g., all) of the following substitutions: F100D, S283G, N456E, F512Y.

[0209] In one embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises an AA9 (GH61A) polypeptide (especially derived from a strain of Penicillium emersonii (e.g., SEQ ID NO: 2 in WO 2011 / 041397)) having cellulase-enhancing activity, a variant of Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO: 2 in WO 2005 / 047499) having one or more (especially all) of the following substitutions: F100D, S283G, N456E, F512Y and disclosed in WO 2012 / 044915; Aspergillus fumigatus Cel7ACBH1, for example, disclosed as SEQ ID NO: 6 in WO 2011 / 057140; and Aspergillus fumigatus CBH II, for example, disclosed as SEQ ID NO: 18 in WO 2011 / 057140.

[0210] In a preferred embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises a hemicellulase or hemicellulase composition, such as Aspergillus fumigatus xylanase and Aspergillus fumigatus β-xylosidase.

[0211] In one embodiment, the cellulase composition further includes xylanase (e.g., strains derived from Aspergillus, especially Aspergillus echinospora or Aspergillus fumigatus; or strains of Basilaria, especially Basilaria resète) and / or β-xylosidase (e.g., strains derived from Aspergillus, especially Aspergillus fumigatus; or strains of Basilaria, especially Basilaria emersonii) Talaromyces emersonii (strains).

[0212] In one embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises a thermophilic ascomycete AA9 (GH61A) polypeptide (e.g., WO 2005 / 074656) having cellulase-enhancing activity, 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 echinospora xylanase (e.g., Xyl II in WO 94 / 21785).

[0213] In another embodiment, the cellulase composition comprises a Trichoderma reesei cellulolytic agent, which further comprises a GH61A polypeptide of Ascomycota orangeus with cellulolytic-enhancing activity (e.g., SEQ ID NO: 2 in WO 2005 / 074656), an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO: 2 in WO 2005 / 047499), and an Aspergillus echinospora xylanase (disclosed in WO 94 / 21785, Xyl II).

[0214] In another embodiment, the cellulase composition comprises a Trichoderma reesei cellulase composition, which further comprises a Thermophilic Ascomycota orange AA9 (GH61A) polypeptide (e.g., SEQ ID NO: 2 in WO 2005 / 074656), Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO: 2 in WO2005 / 047499), and Aspergillus echinospora xylanase (e.g., XylII disclosed in WO 94 / 21785).

[0215] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises an Emersonian Penicillium AA9 (GH61A) polypeptide (especially one disclosed in WO 2011 / 041397) having cellulase-enhancing activity, an Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO: 2 of WO2005 / 047499), and an Aspergillus fumigatus xylanase (e.g., XylIII of WO 2006 / 078256).

[0216] In another embodiment, the cellulase composition comprises a Trichoderma reesei cellulase composition, which further comprises an Emersonian Penicillium AA9 (GH61A) polypeptide having cellulase-enhancing activity, particularly one disclosed in WO 2011 / 041397, Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO: 2 of WO 2005 / 047499), Aspergillus fumigatus xylanase (e.g., Xyl III of WO 2006 / 078256), and CBH I from Aspergillus fumigatus, particularly Cel7A CBH1 disclosed as SEQ ID NO: 2 in WO 2011 / 057140.

[0217] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition, which further comprises an Emersonian Penicillium AA9 (GH61A) polypeptide having cellulolytic-enhancing activity, particularly one disclosed in WO 2011 / 041397, Aspergillus fumigatus β-glucosidase (e.g., SEQ ID NO: 2 of WO 2005 / 047499), Aspergillus fumigatus xylanase (e.g., Xyl III of WO 2006 / 078256), CBH I from Aspergillus fumigatus, particularly Cel7ACBH1 disclosed as SEQ ID NO: 2 in WO 2011 / 057140, and CBH II derived from Aspergillus fumigatus, particularly one disclosed as SEQ ID NO: 4 in WO 2013 / 028928.

[0218] In another embodiment, the cellulase composition is a *Trichoderma reesei* cellulase composition, which further comprises an *Emperor acetamiprid* AA9 (GH61A) polypeptide (particularly one disclosed in WO2011 / 041397) having cellulolytic-enhancing activity, *Aspergillus fumigatus* β-glucosidase (e.g., SEQ ID NO: 2 of WO 2005 / 047499) or a variant thereof, the variant having one or more (particularly all) of the following substitutions: F100D, S283G, N456E, F512Y; *Aspergillus fumigatus* xylanase (e.g., Xyl III of WO 2006 / 078256), CBH I from *Aspergillus fumigatus* (particularly Cel7A CBH I disclosed as SEQ ID NO: 2 in WO 2011 / 057140), and CBH II derived from *Aspergillus fumigatus* (particularly one disclosed in WO 2013 / 028928).

[0219] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition comprising CBH I (GENSEQP Registry No. AZY49536 (WO 2012 / 103293)); CBH II (GENSEQP Registry No. AZY49446 (WO 2012 / 103288)); a β-glucosidase variant (GENSEQP Registry No. AZU67153 (WO 2012 / 44915)), particularly having one or more (particularly all) of the following substitutions: F100D, S283G, N456E, F512Y; and AA9 (GH61 polypeptide) (GENSEQP Registry No. BAL61510 (WO 2013 / 028912)).

[0220] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition comprising CBH I (GENSEQP Registry No. AZY49536 (WO 2012 / 103293)); CBH II (GENSEQP Registry No. AZY49446 (WO 2012 / 103288)); GH10 xylanase (GENSEQP Registry No. BAK46118 (WO 2013 / 019827)); and β-xylosidase (GENSEQP Registry No. AZI04896 (WO 2011 / 057140)).

[0221] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition comprising CBH I (GENSEQP Registry No. AZY49536 (WO 2012 / 103293)); CBH II (GENSEQP Registry No. AZY49446 (WO 2012 / 103288)); and AA9 (GH61 polypeptide; GENSEQP Registry No. BAL61510 (WO 2013 / 028912)).

[0222] In another embodiment, the cellulase composition is a Trichoderma reesei cellulase composition comprising CBH I (GENSEQP Registry No. AZY49536 (WO 2012 / 103293)); CBH II (GENSEQP Registry No. AZY49446 (WO 2012 / 103288)), AA9 (GH61 polypeptide; GENSEQP Registry No. BAL61510 (WO 2013 / 028912)), and catalase (GENSEQP Registry No. BAC11005 (WO 2012 / 130120)).

[0223] In one embodiment, the cellulase composition is a Trichoderma reesei cellulase composition comprising CBHI I (GENSEQP Registry No. AZY49446 (WO 2012 / 103288); CBHII (GENSEQP Registry No. AZY49446 (WO 2012 / 103288)), a β-glucosidase variant (GENSEQP Registry No. AZU67153 (WO 2012 / 44915)), having one or more (particularly all) of the following substitutions: F100D, S283G, N456E, F512Y; AA9 (GH61 polypeptide; GENSEQP Registry No. BAL61510 (WO 2013 / 028912)), GH10 xylanase (GENSEQP Registry No. BAK46118 (WO 2012 / 103288)). 2013 / 019827), and β-xylosidase (GENSEQP accession number AZI04896 (WO 2011 / 057140)).

[0224] In one embodiment, the cellulose-degrading composition is a Trichoderma reesei cellulase preparation comprising EG I (Swissprot accession number P07981), EG II (EMBL accession number M19373), CBHI (see above); CBH II (see above); β-glucosidase variants having the following substitutions (see above): F100D, S283G, N456E, F512Y; AA9 (GH61 polypeptide; see above), GH10 xylanase (see above); and β-xylosidase (see above).

[0225] All cellulase compositions disclosed in WO 2013 / 028928 are also considered and are hereby incorporated by reference.

[0226] The cellulase composition comprises or may further comprise one or more proteins selected from the group consisting of: cellulase, AA9 (i.e., GH61) polypeptide with cellulolytic-enhancing activity, hemicellulase, patulin, esterase, laccase, lignin-degrading enzyme, pectinase, peroxidase, protease, and swelling agent.

[0227] In one embodiment, the cellulase composition is a commercial cellulase composition. Examples of commercial cellulase compositions suitable for use in the methods of the present invention include: CELLIC® CTec (Novozymes), CELLIC® CTec2 (Novozymes), CELLIC® CTec3 (Novozymes), CELLUCLAST™ (Novozymes), SPEZYME™ CP (Genencor Int.), ACCELLERASE™ 1000, ACCELLERASE 1500, ACCELLERASE™ TRIO (DuPont), FILTRASE® NL (DSM); METHAPLUS® S / L 100 (DSM), ROHAMENT™ 7069 W (Röhm GmbH), or ALTERNAFUEL® CMAX3™ (Dyadic International, Inc.). The cellulase composition can be added in an effective amount from about 0.001 wt.% to about 5.0 wt.% of solids, for example, from about 0.025 wt.% to about 4.0 wt.% of solids, or from about 0.005 wt.% to about 2.0 wt.% of solids.

[0228] Other enzymes and their compositions can be found in WO 2011 / 153516 and WO 2016 / 045569 (the contents of which are incorporated herein by reference).

[0229] Additional polynucleotides encoding suitable cellulases can be obtained from any genus of microorganisms, including those readily available in the UniProtKB database (www.uniprot.org).

[0230] 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.

[0231] Polynucleotides encoding cellulases can also be identified and obtained from other sources, including microorganisms isolated from nature known in the art (e.g., soil, compost, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, compost, water, etc.).

[0232] Techniques for isolating or cloning polynucleotides encoding cellulases are known in the art.

[0233] In one embodiment, the cellulase 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 cellulase described or referenced herein. In one embodiment, the cellulase has a mature polypeptide sequence that differs from any cellulase described or referenced herein by no more than ten amino acids, such as no more than five amino acids, four amino acids, three amino acids, two amino acids, or one amino acid. In one embodiment, the cellulase has a mature polypeptide sequence comprising or consisting of the following: the amino acid sequence of any cellulase described or referenced herein, an allelic variant, or a fragment thereof having cellulase activity. In one embodiment, the cellulase 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, for example, not exceeding 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0234] In some embodiments, under the same conditions, the cellulase has at least 20%, for example 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 cellulase activity of any cellulase described or referenced herein (e.g., any endoglucanase, cellobiase, or β-glucosidase).

[0235] In one embodiment, the cellulase coding sequence hybridizes with the full-length complementary strand of the coding sequence of any cellulase described or referenced herein (e.g., any endoglucanase, cellobiase, or β-glucosidase) under at least low stringency conditions, such as medium stringency, medium-high stringency, high stringency, or very high stringency conditions. In one embodiment, the cellulase 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 cellulase described or referenced herein.

[0236] In one embodiment, the polynucleotide encoding the cellulase comprises the coding sequence of any cellulase described or referenced herein (e.g., any endoglucanase, cellobiase, or β-glucosidase). In one embodiment, the polynucleotide encoding the cellulase comprises a subsequence from 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%, such as at least 80%, 85%, 90%, or 95%, of the number of reference coding sequences.

[0237] The cellulase may also include fusion peptides or cleavable fusion peptides.

[0238] Method using starch-containing materials In some embodiments, the methods described herein produce fermentation products from starch-containing materials. Starch-containing materials are well known in the art and contain two types of homopolysaccharides (amylose and amylopectin) 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 starch, sorghum, oats, rice, peas, legumes, or sweet potatoes, or mixtures thereof. Waxy and non-waxy types of corn and barley are also considered.

[0239] 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 West African sorghum. 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 cassava starch. 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 legumes. In one embodiment, the starch-containing starting material is sweet potato. In one embodiment, the starch-containing starting material is oats.

[0240] Methods using starch-containing materials may include conventional methods (e.g., including a liquefaction step described in more detail below) or methods for hydrolyzing crude starch. In some embodiments using starch-containing materials, the saccharification of the starch-containing material is carried out at a temperature above the initial gelatinization temperature. In some embodiments using starch-containing materials, the saccharification of the starch-containing material is carried out at a temperature below the initial gelatinization temperature.

[0241] liquefaction In embodiments using starch-containing materials, these methods may further include a liquefaction step, which is performed by subjecting the starch-containing material to α-amylase and optionally protease and / or glucosylase at a temperature above the initial gelatinization temperature. Other enzymes such as pullulanase and phytase may also be present and / or added during liquefaction. In some embodiments, this liquefaction step is performed prior to steps a) and b) of the method.

[0242] The liquefaction step can take 0.5-5 hours, such as 1-3 hours, typically about 2 hours.

[0243] The term "initial gelatinization temperature" refers to the lowest temperature at which gelatinization of a starch-containing material begins. Typically, starch heated in water begins to gelatinize between approximately 50°C and 75°C; the exact gelatinization temperature depends on the specific starch and can be readily determined by those skilled in the art. Therefore, the initial gelatinization temperature can vary depending on the plant species, the specific variety of the plant species, and the growing conditions. A given initial gelatinization temperature for a starch-containing material can be obtained using Gorinstein and Lii, 1992. Starch / Stärke The method described in [Starch] 44(12): 461-466 is determined by the temperature at which 5% of starch granules lose birefringence.

[0244] Liquefaction is typically carried out at temperatures ranging from 70°C to 100°C. In one embodiment, the temperature during liquefaction is between 75°C and 95°C, such as between 75°C and 90°C, between 80°C and 90°C, or between 82°C and 88°C, such as about 85°C.

[0245] The spray cooking step can be carried out before the liquefaction step, for example, at a temperature between 110°C and 145°C, 120°C and 140°C, 125°C and 135°C, or about 130°C for about 1-15 minutes, about 3-10 minutes, or about 5 minutes.

[0246] 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.

[0247] In one embodiment, prior to liquefaction, the method further includes the following steps: i) Preferably, the particle size of the starch-containing material is reduced by dry grinding; ii) Form a slurry containing the starch-containing material and water.

[0248] The starch-containing 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 allows for good separation of the germ from the coarse flour (starch granules and protein). Wet milling is often used in locations where starch hydrolysates are used to produce, for example, syrups. Both dry and wet milling are well-known in the starch processing industry. In one embodiment, the starch-containing 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 such that at least 30%, at least 50%, at least 70%, or at least 90% of the starch-containing material is suitable for passing 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 starch-containing material is suitable for passing through a sieve with a #6 screen.

[0249] The aqueous slurry may contain starch-containing materials ranging from 10-55 w / w-% dry solids (DS), such as 25-45 w / w-% dry solids (DS) or 30-40 w / w-% dry solids (DS).

[0250] 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 of these enzymes (e.g., about 1 / 3) is added to the aqueous slurry, while the remainder (e.g., about 2 / 3) is added during the liquefaction step.

[0251] α-amylases and glucosylamylases for liquefaction can be found in the art, for example, WO 2020 / 023411 (the contents of which are incorporated herein by reference). Similarly, examples of suitable proteases for liquefaction can be found in the art, for example, WO 2018 / 222990 (the contents of which are incorporated herein by reference).

[0252] Saccharification and fermentation of starch-containing materials In embodiments using starch-containing materials, glucosylamylase may be present and / or added in the saccharification step a) and / or fermentation step b) or simultaneous saccharification and fermentation (SSF). The glucosylamylase in the saccharification step a) and / or fermentation step b) or simultaneous saccharification and fermentation (SSF) is typically different from the glucosylamylase optionally added in any of the liquefaction steps described above. In one embodiment, the glucosylamylase is present and / or added together with fungal α-amylase. Suitable glucosylamylases for saccharification or SSF can be found in the art, for example, WO 2020 / 023411 (the contents of which are incorporated herein by reference).

[0253] When saccharification and fermentation are performed 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 about 24 to about 72 hours. In one embodiment, pre-saccharification is performed. Pre-saccharification is typically carried out for 40 to 90 minutes at a temperature of 30°C to 65°C, typically about 60°C. In one embodiment, in simultaneous saccharification and fermentation (SSF), pre-saccharification is followed by saccharification during fermentation. Saccharification is typically carried out at a temperature of 20°C to 75°C, preferably from 40°C to 70°C, typically about 60°C, and typically at a pH between 4 and 5, such as about pH 4.5.

[0254] Fermentation is carried out in a fermentation medium, as is known in the art and, for example, as described herein. The fermentation medium comprises a fermentation substrate, i.e., a source of carbohydrates to be metabolized by the fermenting organism. Using the methods described herein, the fermentation medium may contain nutrients for one or more fermenting organisms and one or more growth stimulants. 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.

[0255] Typically, fermenting organisms such as yeast (including Saccharomyces cerevisiae) require a sufficient nitrogen source for propagation and fermentation. Many supplemental nitrogen sources can be used if necessary, and these sources are well known in the art. This 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.

[0256] Fermentation can be carried out under low-nitrogen conditions, such as when using yeast expressing proteases. In some embodiments, the fermentation step is carried out under the following conditions: supplemental nitrogen of less than 1000 ppm (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. In some embodiments, the fermentation step is carried out without supplemental nitrogen.

[0257] Simultaneous saccharification and fermentation (“SSF”) is widely used in industrial-scale fermentation product production methods, particularly in ethanol production. When performing SSF, saccharification step a) and fermentation step b) are carried out simultaneously. There is no hold-up phase for saccharification, meaning that the fermentation organism (such as yeast) and one or more enzymes can be added together. However, adding the fermentation organism and one or more enzymes separately is also considered. SSF is typically carried out at temperatures ranging 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, fermentation lasts from 6 to 120 hours, particularly from 24 to 96 hours. In one embodiment, the pH is between 4 and 5.

[0258] In one embodiment, a cellulase composition is present and / or added during saccharification, fermentation, or simultaneous saccharification and fermentation (SSF). Examples of such cellulase compositions can be found in the "Cellulases and Compositions" section. This cellulase composition may be present and / or added together with glucosylamylase, as disclosed in the "Glucoamylase" section.

[0259] Fermentation products Fermentation products can be any substance obtained from fermentation. Fermentation products can be, but are not limited to: alcohols (e.g., arabinitol, n-butanol, isobutanol, ethanol, glycerol, methanol, ethylene glycol, 1,3-propanediol [propylene glycol], butanediol, glycerol, sorbitol, and xylitol); alkanes (e.g., pentane, hexane, heptane, octane, nonane, decane, undecane, and dodecane); cycloalkanes (e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane); alkenes (e.g., pentene, hexene, heptene, and octene); and amino acids (e.g., aspartic acid, glutamic acid, glycine, and 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, acetoic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric 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 xylanic acid); and polyketide compounds.

[0260] In one embodiment, the fermentation product is an alcohol. The term "alcohol" covers substances containing one or more hydroxyl moieties. The alcohol can be, but is not limited to: n-butanol, isobutanol, ethanol, methanol, arabinitol, butanediol, ethylene glycol, glycerol, glycerol, 1,3-propanediol, sorbitol, xylitol. See, for example, Gong et al., 1999, Ethanol production from renewable resources. Advances in Biochemical Engineering / Biotechnology In *Advances in Biochemical Engineering / Biotechnology*, Scheper, T., ed., Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Silvira and Jonas, 2002. Appl. Microbiol. Biotechnol. [Applied Microbiology and Biotechnology] 59: 400-408; Nigam and Singh, 1995, Process Biochemistry [Biochemical Methods] 30(2): 117-124; Ezeji et al., 2003, World Journal of Microbiology and Biotechnology [World Journal of Microbiology and Biotechnology] 19(6): 595-603. In one embodiment, the fermentation product is ethanol.

[0261] In another embodiment, the fermentation product is an alkane. The alkane can be unbranched or branched. The alkane can be, but is not limited to, pentane, hexane, heptane, octane, nonane, decane, undecane, or dodecane.

[0262] In another embodiment, the fermentation product is a cycloalkanes. The cycloalkanes may be, but are not limited to, cyclopentane, cyclohexane, cycloheptane, or cyclooctane.

[0263] In another embodiment, the fermentation product is an olefin. The olefin can be unbranched or branched. The olefin can be, but is not limited to, pentene, hexene, hepten, or octene.

[0264] In another embodiment, the fermentation product is an amino acid. The organic acid may 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 [Biotechnology and Bioengineering] 87(4): 501-515.

[0265] 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 [Water Science and Technology] 36(6-7):41-47; and Gunaseelan, 1997, Biomass and Bioenergy [Biomass and Bioenergy] 13(1-2):83-114.

[0266] In another embodiment, the fermentation product is isoprene.

[0267] In another embodiment, the fermentation product is a ketone. The term "ketone" encompasses substances containing one or more ketone moieties. Ketones can be, but are not limited to, acetone.

[0268] In another embodiment, the fermentation product is an organic acid. The organic acid may be, but is not limited to: acetic acid, acetoic acid, adipic acid, ascorbic acid, citric acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric 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 xylic acid. See, for example, Chen and Lee, 1997. Appl. Biochem. Biotechnol. [Applied Biochemistry and Biotechnology] 63-65:435-448.

[0269] In another embodiment, the fermentation product is a polyketide compound.

[0270] Recycle Fermentation products (e.g., ethanol) can be optionally recovered from fermentation media using any method known in the art, including but not limited to: chromatography, electrophoresis, differential solubility, distillation, or extraction. For example, alcohols can be separated and purified from fermented cellulose material by conventional distillation methods. Ethanol with a purity up to about 96 vol.% can be obtained, which can be used as, for example, fuel ethanol, drinking ethanol (i.e., drinkable neutral alcoholic beverages), or industrial ethanol.

[0271] In some embodiments of these methods, the recovered fermentation product is substantially pure. Regarding these methods herein, "substantially pure" means that the recovered formulation contains no more than 15% impurities, where impurities refer to compounds other than the fermentation product (e.g., ethanol). In one variant, a substantially pure formulation is provided, wherein the formulation 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.

[0272] Suitable assays for the production of ethanol and contaminants, as well as sugar consumption, can be performed using methods known in the art. 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 from fermentation broth can also be tested using culture supernatants. HPLC (Lin et al.) can be used, for example, with refractive index detectors for glucose and alcohols, and UV detectors for organic acids. Biotechnol. Bioeng. [Biotechnology and Bioengineering] 90:775-779 (2005) or quantify byproducts and residual sugars (e.g., glucose or xylose) in the fermentation medium using other suitable assays and detection methods known in the art.

[0273] Preservation of biological materials The following biological material has been deposited in accordance with the terms of the Budapest Treaty at the Northern Regional Research Center of the Patent Culture Collection (NRRL) of the American Agricultural Research Service, located at 1815 University Street, Peoria, Illinois, USA, and is assigned the following accession number: The strain is deposited under the condition that it is accessible to a person authorized by the Patent and Trademark Commission under 37 CFR §1.14 and 35 USC §122 during the pending period of this patent application. These deposits represent substantially pure cultures of the deposited strain. The deposits are required to be provided in accordance with the foreign patent laws of some countries, by filing a copy of the subject matter application or a subsequent text thereof in those countries. However, it should be understood that the availability of the deposits does not constitute a license for practice.

[0274] The invention described and claimed herein is not limited to the specific aspects or embodiments disclosed herein, as these aspects / embodiments are intended to illustrate several aspects of the invention. Any equivalent aspects are intended to be within the scope of the invention. In fact, various modifications to 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 disclosure including the definition shall prevail. All references are specifically incorporated by reference to the description.

[0275] The following examples are provided to illustrate certain aspects / embodiments of the invention, but are not intended to limit the scope of the claimed invention in any way.

[0276] Example Material Cellulose-degrading enzyme composition CA ("CA") The cellulase preparation derived from Trichoderma reesei further comprises GH61A polypeptide (SEQ ID NO:2 in WO 2011 / 041397) with enhanced cellulolytic activity derived from Penicillium emersonii strains; variants F100D, S283G, N456E, and F512Y of Aspergillus fumigatus β-glucosidase (SEQ ID NO:2 in WO 2005 / 047499) disclosed in WO 2012 / 044915; Aspergillus fumigatus Cel7A CBH1 disclosed as SEQ ID NO: 6 in WO 2011 / 057140; and Aspergillus fumigatus CBH II disclosed as SEQ ID NO: 18 in WO 2011 / 057140. In addition, the cellulase preparation CA further comprises 10% cellulase preparation from Trichoderma reesei, and further comprises Aspergillus fumigatus xylanase (SEQ ID NO: 8 in WO 2016 / 045569) and Aspergillus fumigatus β-xylosidase (SEQ ID NO: 9 in WO 2016 / 045569).

[0277] Cellulose-degrading enzyme composition CB ("CB"):The *Trichoderma reesei* cellulase formulation comprises EG I (SEQ ID NO: 21), EG II (SEQ ID NO: 22), and CBH I (SEQ ID NO: 14) of WO 2016 / 045569; CBH II (SEQ ID NO: 15) of WO 2016 / 045569; a β-glucosidase variant of SEQ ID NO: 5 of WO 2016 / 045569 with the following substitutions: F100D, S283G, N456E, F512Y; AA9 (GH61 polypeptide) of SEQ ID NO: 7 of WO 2016 / 045569; GH10 xylanase of SEQ ID NO: 16 of WO 2016 / 045569; and SEQ ID NO: 21 of WO 2016 / 045569. NO: 17 β-xylosidase.

[0278] BSGX001 The method disclosed in U.S. Patent No. 8,586,336-B2 (incorporated herein by reference) was constructed as follows: the host strain Saccharomyces cerevisiae BSPX042 (phenotype: ura3-251, overexpressing XKS1; overexpressing RPE1, RKI1, TAL1 and TKL1, which are genes in PPP; knocking out the aldose reductase gene GRE3; and disrupting the electron transport respiratory chain by deleting the COX4 gene after adaptive evolution) was transformed with the vector pJFE3-RuXI containing the xylose isomerase gene (SEQ ID NO:1 in U.S. Patent No. 8,586,336-B2 or SEQ ID NO: 20 in this document), which encodes RuXI as shown in SEQ ID NO: 2 in U.S. Patent No. 8,586,336-B2.

[0279] MBG5364 and MBG5365 were prepared from CIBTS1260 (see WO 2016 / 045569, the contents of which are incorporated herein by reference) according to the evolution and breeding procedures described in U.S. Patent No. 8,257,959.

[0280] Example 1: Construction of strain CIBTS1000 A diploid Saccharomyces cerevisiae strain was identified as a known effective ethanol producer from glucose. The strain used was Saccharomyces cerevisiae strain CCTCC M94055 from the China Center for Type Culture Collection (CCTCC).

[0281] A xylose isomerase called mgXI was cloned from a metagenomics project indicating that the donor organism was unknown. The isolation and characterization of this xylose isomerase are described in Chinese Patent Application No. 102174549A or US Patent Publication No. 2012 / 0225452.

[0282] A pentose transporter, named GXF, was cloned from *Candida intermedia* using standard methods. This xylose transporter is described in D. Runquist et al. (Runquist D, Fonseca C, Radstrom P, Spencer-Martins I, Hahn-Hägerdal B: “ Expression of the Gxf1 transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae "[Expression of the Candida intermediateis Gxf1 transporter improves the fermentation performance of recombinant xylose-utilizing Saccharomyces cerevisiae] Appl Microbiol Biotechnol 2009, 82: 123-130."

[0283] The xylose isomerase gene was fused with the triose phosphate isomerase (TPI) promoter and TPI terminator from Saccharomyces cerevisiae using standard methods, so that the expression of xylose isomerase in Saccharomyces cerevisiae is controlled by TPI expression signaling.

[0284] The GXF gene was fused with the TPI expression signal in the same manner.

[0285] Insertion of E. coli ( Escherichia coli The two expression cassettes of the cloning vector contain: • This plasmid is the E. coli colE1 replication origin that ensures the plasmid can proliferate in E. coli.

[0286] • A δ (delta) sequence fragment from Saccharomyces cerevisiae.

[0287] • Bleomycin resistance markers from *Streptococcus hirta* were used for the selection of bleomycin-resistant *E. coli* or *Saccharomyces cerevisiae* transformants. A dual promoter was fused to the 5' end of a bleomycin gene consisting of a *Saccharomyces cerevisiae* translation elongation factor (TEF1) promoter and an *E. coli* EM7 promoter. A *Saccharomyces cerevisiae* CYC1 terminator was added to the 3' end of the bleomycin gene. The entire bleomycin expression cassette is flanked by loxP sites to allow deletion of the cassette via Cre-lox recombination (B. Sauer: " Functional expression of the Cre-Lox site specific recombination system in the yeast Saccharomyces cerevisiae. "[Functional expression of the Cre-Lox site-specific recombination system in Saccharomyces cerevisiae] Mol. Cell. Biol. [Molecular Cell Biology] 1987, 7:2087-2096).

[0288] The xylose isomerase / pentose transporter expression plasmid is named pYIE2-mgXI-GXF1-δ, and... Figure 1 As shown in the image.

[0289] The plasmid pYIE2-mgXI-GXF1-δ was initially linearized by XhoI digestion and then transformed into the parental strain *Saccharomyces cerevisiae* CCTCC M94055, subsequently selected for bleomycin resistance transformants. A strain named CIBTS0912 with the plasmid integrated into the δ sequence was isolated. The bleomycin resistance cassette located between the two loxP sites was then deleted via transient CRE recombinase expression, generating strain CIBTS0914.

[0290] The method for achieving transient CRE recombinase expression is similar to that of Prein et al. (Prein B, Natter K, Kohlwein SD). A novel strategy for constructing N-terminal chromosomal fusions to green fluorescent protein in the yeast Saccharomyces cerevisiae [A novel strategy for constructing N-terminal chromosomes fused with green fluorescent protein in the yeast Saccharomyces cerevisiae]”. FEBS Lett. [European Union of Biochemists and Astronautics Bulletin] 2000: 485, 29-34. The standard method described involves transformation with an unstable plasmid expressing the CRE recombinase, followed by removal of the plasmid. In this work, the kanamycin gene in the standard yeast vector pSH47 was replaced with a hygromycin resistance marker, allowing selection using hygromycin rather than kanamycin resistance. A plasmid map using the pSH47-hyg plasmid is available at [link to plasmid map]. Figure 2 The following is shown. A table listing the genetic factors used is shown in Table 1.

[0291] Table 1. The pYIE2-mgXI-GXF1-δ strain CIBTS0914 was digested again with XhoI to increase the copy number of the two expression cassettes, and the bleomycin-resistant strain CIBTS0916 was selected.

[0292] To overexpress genes of the pentose phosphate pathway, expression plasmids carrying selected genes of the pentose phosphate pathway are assembled.

[0293] The genes selected for overexpression are: 1. Xylulose kinase (XKS1).

[0294] 2. Aldolase (TAL1).

[0295] 3. Ribulose-5 phosphate epimerase (RPE1).

[0296] 4. Transketolase (TKL1).

[0297] 5. Ribose-5 phosphate isomerase (RKI1) In addition to these genes, the KanMX selection cassette surrounded by the loxP site is also included as part of the *E. coli*–*Saccharomyces cerevisiae* shuttle vector pUG6 (Güldener U, Heck S, Fielder T, Beinhauer J, Hegemann JH). A new efficient gene disruption cassette for repeated use in Budding yeast. [A novel, highly efficient gene-destruction cassette for repeated use in budding yeast] NAR [Nucleic Acid Research] 1996, 24:2519-24.

[0298] The spectrum of the generated plasmid pYIE2-XKS1-PPP-δ is shown in Figure 3 The following is shown. A table listing the genetic factors used is shown in Table 2.

[0299] Table 2. The plasmid pYIE2-XKS1-PPP-δ was digested with NotI, and the vector elements were removed by agarose gel electrophoresis. The linear fragment containing all expression cassettes was then transformed into CIBTS0916 for double homologous recombination, followed by selection for kanamycin (G418) resistance. Kanamycin-resistant colonies were selected and named CIBTS0931.

[0300] CIBTS0931 contains both bleomycin and kanamycin select markers. Both of them have loxP recombination sites on their flanks.

[0301] To remove the bleomycin and kanamycin resistance markers, the strain was transformed again with the episomal plasmid pSH47-hygs, and transformants were selected on plates containing hygromycin. Subsequently, screening was performed for transformants that had lost bleomycin and kanamycin resistance, followed by screening for strains that had also lost the hygromycin resistance markers. Strain CIBTS1000 was selected, and the strain that had lost the pSH47-hyg plasmid is shown.

[0302] Example 2: Adaptation of strain CIBTS1000 to high xylose uptake and acetate resistance The strain CIBTS1000 was modified to utilize xylose as a carbon source and ferment it into ethanol. However, this xylose utilization was highly inefficient. A well-known way to improve that problem in metabolic engineering is through adaptation. This was also done in this case. The strain CIBTS1000 was sequentially transferred from shake flask to shake flask in a medium containing xylose as the sole carbon source and yeast growth inhibitors known to be present in the hydrolysates of cellulose biomass. Mutations accumulated during these sequential transfers, allowing the strain to grow better under the provided conditions – and thus utilize xylose more effectively.

[0303] In the first round of adaptation, CIBTS1000 was continuously transferred in a shake flask system using YPX medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L xylose) and YPDX (10 g / L yeast extract, 20 g / L peptone, 10 g / L glucose, and 10 g / L xylose). In the second round of adaptation, a continuous transfer was carried out between YPXI (YPX supplemented with 43 mM sodium formate, 50 mM sodium acetate, and 100 mM sodium sulfate) and YPDXI (YPDX supplemented with 43 mM sodium formate, 50 mM sodium acetate, and 100 mM sodium sulfate).

[0304] In the final adaptation, continuous transfer was performed using corn stalk hydrolysate pretreated with NREL dilute acid supplemented with 10 g / l yeast extract, 20 g / l peptone, 10 g / l glucose and 10 g / l xylose (see Example 3).

[0305] The strain named CIBTS1260-J132-F3 was selected as the adaptive strain.

[0306] Example 3: Fermentation of CIBTS1260 and BSGX001 in NREL dilute acid pretreated corn stalk hydrolysates Compare Two strains of *Saccharomyces cerevisiae* (CIBTS1260 and BSGX001) were tested in NREL dilute acid-pretreated corn straw hydrolysate (4% w / w sulfuric acid, 180°C for 5 minutes). Hydrolysate was produced in a 20 kg reactor at 50°C after hydrolysis with 20 mg enzyme protein / g dextran from the cellulase composition CA for 3 days. The dilute acid-pretreated corn straw hydrolysate had a final composition of 63.2 g / L glucose, 44.9 g / L xylose, 0.8 g / L glycerol, and 9.5 g / L acetate. Prior to fermentation, each strain was propagated in a 30°C air shaker at 150 rpm on YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose). After 24 hours of growth, both yeast strains were tested in 50 ml of hydrolysate in 125 ml baffled Erlenmeyer flasks (inoculated with 1 g stem cell weight (DCW) / L yeast). Each flask was sealed with a rubber stopper fitted with an 18-gauge blunt-tipped filling needle and placed in an air shaker at 35°C and 150 rpm. Samples were taken at 24, 48, and 72 hours, and the concentrations of glucose, xylose, and ethanol were determined by HPLC analysis. The results from three replicates of each group were averaged, and... Figure 1 The text presents a comparison of CIBTS1260 versus BSGX001 in NREL acid-pretreated corn stalk hydrolysates inoculated with 1 g / L yeast over 72 hours. (See also...) Figure 4 As shown, after 48 hours of fermentation, strain CIBTS1260 completed complete xylose consumption and produced approximately 47 g / L of ethanol. However, strain BSGX001 took up glucose for ethanol conversion slowly and thus consumed only 3 g / L of xylose. These results indicate that CIBTS1260 improves xylose uptake and utilization for ethanol conversion compared to BSGX001.

[0307] Example 4: Comparison of fermentation performance of CIBTS1260 and BSGX001 in model culture media. The fermentation performance of CIBTS1260 and its precursor BSGX001 was compared. Prior to fermentation, each strain was propagated in YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) at 150 rpm in an air shaker at 30°C. After 24 hours of growth, both yeast strains were tested in YPX medium (5 g / L yeast extract, 5 g / L peptone, and 50 g / L xylose). To test fermentation performance, each strain was inoculated in 50 ml of YPX medium into 125 ml baffled Erlenmeyer flasks (with 2 g DCW / L yeast inoculum). Each flask was sealed with a rubber stopper fitted with an 18-gauge blunt-tipped filling needle, and the flasks were placed in an air shaker at 32°C and 150 rpm. Samples were taken at 24, 48, and 72 hours, and the concentrations of glucose, xylose, and ethanol were determined by HPLC analysis. Average the results from the three replicates in each group, and... Figure 5 The information is provided in the text.

[0308] As in Figure 5 As shown, CIBTS1260 (dashed line) fully utilized all available xylose within 24 hours and produced 21.3 g / L of ethanol. During the 72-hour fermentation period, BSGX001 (solid line) consumed 1.5 g / L of xylose and produced 1.3 g / L of ethanol.

[0309] Example 5: Cellulase composition CA ("CA") with CIBTS1260 and cellulase composition Fermentation of CB ("CB") sugarcane bagasse hydrolysis products CIBTS1260 was used in fermentation tests of bagasse hydrolysates pretreated with dilute acid by Novozymes North America (USA) using NREL. Hydrolysates were produced in a 2L IKA reactor at 50°C after 5 days of hydrolysis with two cellulase compositions (referred to as “CA” and “CB”) at a dose of 6 mg enzyme protein / g dextran. These materials are representative benchmarks for dilute acid-pretreated bagasse hydrolysates with the following final compositions: 40.7 g / L and 58.7 g / L glucose, 42.5 g / L and 44.7 g / L xylose, 0.19 g / L and 0.08 g / L glycerol, and 8.99 g / L and 11.3 g / L acetate, respectively, for “CA” and “CB”. Prior to fermentation, yeast cells were propagated in an air shaker at 30°C and 150 rpm on 2% YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose). After 24 hours of growth, CIBTS1260 was tested in 50 ml of “CA” and “CB” hydrolysates in 125 ml baffled Erlenmeyer flasks (inoculated with 1 g DCW / L yeast). Each flask was sealed with a rubber stopper fitted with an 18-gauge blunt-tipped filling needle and placed in an air shaker at 35°C and 150 rpm. Samples were taken at 24, 48, and 72 hours, and HPLC analysis was used to determine the concentrations of glucose, xylose, ethanol, acetate, and glycerol. The results from three replicates per group were averaged and analyzed. Figure 6 The results are given in the table. During the 72-hour period, more than 95% of the glucose and xylose present in both systems were consumed, with the ethanol yield based on total sugars being 84.1% for the "CA" hydrolysate and 86.4% for the "CB" hydrolysate.

[0310] Example 6: CIBTS1260 and BSGX001 of hydrolysates of corn stalks and sugarcane bagasse pretreated with dilute acid. DP2 reduction during fermentation In a 2 L IKA reactor, corn stalks and bagasse pretreated with dilute acid from the National Renewable Energy Laboratory (NREL) were hydrolyzed at 50 °C for 5 days using a mixture of two enzyme products (referred to as CA and CB) at a dose of 6 mg enzyme protein / g dextran. Prior to fermentation, CIBTS1260 and BSGX001 yeast strains were propagated in YPD medium (10 g / L yeast extract, 20 g / L peptone, and 20 g / L glucose) at 150 rpm in an air shaker at 30 °C. After 24 hours of growth, cells from each strain were harvested by centrifugation and added separately to 50 ml of CA and CB hydrolysates supplemented with 2 g / L urea in 125 ml baffled Erlenmeyer flasks (inoculated with yeast at 1 g DCW / L (stem cell weight / L)). Each flask was sealed with a rubber stopper fitted with an 18-gauge blunt-tipped filling needle and placed in an air shaker at 35 °C and 150 rpm. Samples were taken at 0 and 72 hours and analyzed by HPLC to determine DP2 concentration. The replicates for each group were averaged (n = 3 for CIBTS1260 and n = 2 for BSGX001). Figure 7 As shown, in the same hydrolysis products, fermentation using CIBTS1260 reduced DP2 concentration more significantly than fermentation using BSGX001. The DP2 peak, as measured by HPLC, contains cellobiose and short-chain sugars.

[0311] Example 7: Improved kinetic rates of strain MBG5365 Saccharomyces cerevisiae strains CIBTS1260 and MBG5365 were grown in YPD medium in shake flasks at 32°C for 24 hours. Aliquots were stored in 2 mL cryovials containing 20% ​​glycerol in an ultra-freezing chamber at -80°C.

[0312] Cell propagation for fermentation was carried out in two steps in 500 mL baffled culture flasks containing 100 mL of culture medium, incubated at 32 ± 1 °C and 150 rpm in a shaker. The first-step inoculum was inoculated with 0.05 g cells dry weight / L and stirred for 24 h. At the end of incubation, cell growth was measured by cell dry weight (g / L). Then, an inoculum containing 0.05 g cells dry weight / L (from the first step) was added to a new flask containing culture medium (referred to as "second step") and incubated in a shaker for 19 h. At the end of the second-step incubation, cell growth was measured by cell dry weight (g / L).

[0313] Fermentation assays were performed in 250 mL Schott flasks containing 40 mL of fermentation medium—second-generation fermentation medium obtained from pretreated bagasse. 2GHydrolysates rich in C5 / C6 sugars were prepared. The pH of the medium was adjusted to 5.5, and the medium was inoculated with proliferation medium, followed by incubation in a track-mounted incubator at 32 ± 1 °C and 130 rpm. For inoculation, the medium concentration was adjusted to accommodate different growth rates so that fermentation could begin with the same inoculation amount. Fermentation kinetics were monitored using an ANKOMRF gas production system. After a given fermentation time, samples were taken and analyzed for sugars, ethanol, glycerol, and acetic acid by HPLC (HPX-87H and HPX-87P columns; RID detector).

[0314] Figure 8 Dynamic curves of ethanol fermentation of MBG5365 and reference 2G yeast CIBTS1260 based on gas pressure monitoring are shown, converted to gas mass according to calculations using the ANKOM RF gas production system. Several different fermentation media were used in this assay, exhibiting varying levels of inhibitors (e.g., acetic acid ranging from 0 to 6 g / L; 5-hydroxymethylfurfural ranging from 0 to 0.7 g / L; furfural ranging from 0 to 0.6 g / L) and initial sugar concentrations (total reducing sugars ranging from 0 to 130 g / L). These figures show that MBG5365 has a faster fermentation rate compared to the reference 2G yeast CIBTS1260. A significant advantage in fermentation rate, reaching up to 15 hours, was observed when comparing MBG5365 to the reference. This will significantly impact the fermentation process, thereby improving productivity.

[0315] Example 8: Tolerance of strain MBG5365 to high-temperature fermentation Fermentation assays were performed using 2G hydrolysate rich in C5 and C6 sugars, inoculated with Prop2-derived cells (washed cells) at an initial inoculum of 1.0 g CDW / L. Other conditions included: 40 hours; 130 rpm; pH 5.5; 900 ppm nitrogen and antibiotics. Fermentation was carried out in 250 mL Schottky flasks containing 40 mL of culture medium, inoculated with proliferation medium, and incubated in an incubator at 32°C, 34°C, and 36°C. The fermentation flasks were placed in incubators at different temperatures to examine the robustness of the strain in relation to fermentation temperature levels. Figure 9 As shown, yeast strain MBG5365 exhibited faster fermentation kinetics than the reference strain CIBTS1260 at challenging temperatures (e.g., 36°C). MBG5365 showed good robustness to temperature variations across the range. The reference strain CIBTS1260 showed a slower fermentation rate at higher temperatures (up to 36°C).

[0316] Example 9: Robustness of strain MBG5365 under pH stress Saccharomyces cerevisiae strains CIBTS1260 (reference) and MBG5365 were grown in YPD medium in shake flasks at 32°C for 24 h. Aliquots were stored in 2 mL cryovials containing 20% ​​glycerol in an ultra-freezer at -80°C. Cell propagation for fermentation was carried out in two steps in 500 mL baffled culture flasks containing 100 mL of medium, incubated at 32 ± 1°C and 150 rpm. The first-step inoculum was inoculated with 0.05 g cells dry weight / L and stirred for 24 h. At the end of incubation, cell growth was measured by cell dry weight (g / L). Then, an inoculum containing 0.05 g cells dry weight / L (from the first step) was added to a new flask containing medium (referred to as the second step) and incubated in a shaker for 19 h. At the end of the second-step incubation, cell growth was measured by cell dry weight (g / L).

[0317] Fermentation assays were performed in 250 mL Schott flasks containing 40 mL of fermentation medium (second-generation [2G] C5-C6 sugar-rich hydrolysate obtained from pretreated bagasse), inoculated with washed yeast cells from proliferation medium (1.0 g / L or 1.2 g / L), and incubated in an orbital incubator at 34 ± 1 °C and 130 rpm. Fermentation kinetics were monitored using an ANKOM RF gas production system. Samples were taken at specific time points and analyzed for sugars, ethanol, glycerol, and acetic acid by HPLC (HPX-87H and HPX-87P columns; RID detector).

[0318] Figure 10 The kinetics of 2G fermentation using MBG5365 yeast are shown when the pH of the fermentation medium was adjusted to 5.0, 5.5, 6.0, 7.0, and 8.0. The data show that MBG5365 can maintain its fermentation rate over a wide range of pH conditions, demonstrating its significant robustness in terms of pH parameters. Figure 11 The figure shows the fermentation yield and xylose conversion data of MBG5365 relative to the reference yeast (CIBTS1260) in a 31-hour 2G fermentation. The figure demonstrates that MBG5365 exhibits greater robustness to low pH and high acetic acid content (8 g / L) during fermentation compared to the reference yeast. MBG5365 also demonstrates a significant ability to maintain fermentation performance under inhibitory pH stress.

[0319] Example 4: High tolerance of strain MBG5365 to furan compounds Cell propagation for fermentation was carried out in two steps in 500 mL baffled culture flasks containing 100 mL of culture medium, and incubated in a shaker at 32 ± 1 °C and 150 rpm. The first-step inoculum was inoculated with 0.05 g cells dry weight / L and stirred for 24 h. At the end of incubation, cell growth was measured by cell dry weight (g / L). Then, the inoculum containing 0.05 g cells dry weight / L (from the first step) was added to a new flask containing culture medium (second step), and incubated in a shaker for 19 h. At the end of the second-stage incubation, cell growth was measured by cell dry weight (g / L).

[0320] Fermentation assays were performed in 250 mL Schott flasks containing 40 mL of fermentation medium—a second-generation (2G) C5-C6 sugar-rich hydrolysate obtained from pretreated bagasse (inoculum from secondary proliferation medium; inoculum size 1 g / L). Other culture conditions were: pH 5.5, temperature 33 ± 1 °C, and incubation at 130 rpm in a track-mounted incubator. Samples were taken from the fermenter at 31 hours and analyzed for sugars, ethanol, glycerol, and acetic acid by HPLC (HPX-87H and HPX-87P columns; RID detector). A reference fermentation medium was incorporated with biomass-derived furans, such as 5-hydroxymethylfurfural (HMF) and furfural—known typical 2G fermentation inhibitors.

[0321] Figure 12 The results showed that MBG5365 achieved a relative fermentation yield compared to the reference strain CIBTS1260 when the levels of two furan compounds (5-hydroxymethylfurfural [HMF] and furfural [referred to as 2G inhibitors]) were increased in 2G hydrolysate substrates. Comparing the performance of the two strains, MBG5365 demonstrated greater tolerance to the presence of typical 2G inhibitors in the fermentation of hydrolysates rich in C5 / C6 sugars. Figure 13 The conversion of xylose to 2G ethanol obtained by applying MBG5365 and a reference strain is shown when the levels of furan compounds HMF and furfural in the 2G hydrolysate substrate are increased. Compared to the reference strain, MBG5365 achieved a higher xylose conversion even with increased levels of inhibitors and their combinations. Therefore, MBG5365 is able to maintain its kinetics and performance in the presence of HMF, furfural, or a combination thereof.

[0322] The invention may be further described in the following numbered paragraphs: Paragraph [1]. A method for producing fermentation products from cellulose-containing and / or starch-containing materials, the method comprising: (a) Saccharification of the cellulose- or starch-containing material; and (b) Fermenting the saccharified material of step (a) under appropriate conditions with a fermenting organism to produce a fermentation product; wherein the fermenting organism is a recombinant strain of Saccharomyces cerevisiae deposited in accordance with the Budapest Treaty at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) with accession number NRRL 68303 (Saccharomyces cerevisiae strain MBG5364), or a derivative thereof (e.g., expressing heterologous polypeptides, such as glucosylamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5364.

[0323] Paragraph [2]. A method for producing fermentation products from cellulose-containing and / or starch-containing materials, the method comprising: (a) Saccharification of the cellulose- or starch-containing material; and (b) Fermenting the saccharified material of step (a) under appropriate conditions with a fermenting organism to produce a fermentation product; wherein the fermenting organism is a recombinant strain of Saccharomyces cerevisiae deposited in accordance with the Budapest Treaty at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) with accession number NRRL 68304 (Saccharomyces cerevisiae strain MBG5365), or a derivative thereof (e.g., expressing heterologous polypeptides, such as glucosylamylase and / or α-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5365.

[0324] Paragraph [3]. The method described in paragraph [1] or [2] includes recovering the fermentation product from the fermentation.

[0325] Paragraph [4]. The method described in paragraph [3], wherein recovering the fermentation product from the fermentation includes distillation.

[0326] Paragraph [5]. The method as described in any one of paragraphs [1]-[4], wherein fermentation and saccharification are carried out simultaneously in simultaneous saccharification and fermentation (SSF).

[0327] Paragraph [6]. The method as described in any one of paragraphs [1]-[4], wherein fermentation and saccharification are carried out sequentially (SHF).

[0328] Paragraph [7]. The method as described in any one of paragraphs [1]-[6], wherein the fermentation product is ethanol.

[0329] Paragraph [8]. The method as described in any one of paragraphs [1]-[7], wherein step (a) comprises contacting the starch-containing and / or cellulose-containing material with the enzyme composition.

[0330] Paragraph [9]. The method as described in any one of paragraphs [1]-[7], wherein step (a) comprises saccharifying the cellulose-containing material.

[0331] Paragraph

[10] . The method described in paragraph [9], wherein the cellulose-containing material is pretreated.

[0332] Paragraph

[11] . The method as described in any one of paragraphs [9] or

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

[0333] Paragraph

[12] . The method as described in any one of paragraphs [9]-

[11] , 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 group consisting of cellulase, AA9 polypeptide, hemicellulase, CIP, esterase, patulin, lignin-degrading enzyme, oxidoreductase, pectinase, protease and swelling agent.

[0334] Paragraph

[13] . The method described in paragraph

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

[0335] Paragraph

[14] . The method described in paragraph

[12] or

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

[0336] Paragraph

[15] . The method as described in any one of paragraphs [1]-

[14] , wherein the method results in a yield of 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%).

[0337] Paragraph

[16] . The method as described in any one of paragraphs [1]-

[15] , wherein fermentation is carried out under low-oxygen (e.g., anaerobic) conditions.

[0338] Paragraph

[17] . The fermenting organism has one or more of the following properties as described in any one of paragraphs [1]-

[16] : - Compared with Saccharomyces cerevisiae CIBTS1260 (e.g., 10 to 32 hours), higher ethanol fermentation kinetics at 1 g DWC / L, 32°C, and pH 5.5 (as described in Example 7 of this paper); - Compared with Saccharomyces cerevisiae CIBTS1260, higher xylose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper); - Compared to Saccharomyces cerevisiae CIBTS1260, higher glucose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper).

[0339] Paragraph

[18] . A recombinant Saccharomyces cerevisiae strain, or a derivative thereof (e.g., expressing heterologous polypeptides, such as glucosylamylase and / or α-amylase), deposited in accordance with the Budapest Treaty at the U.S. Agricultural Research Service Patent Collection Center (NRRL) with accession number NRRL 68303 (Saccharomyces cerevisiae strain MBG5364), or a fermentation organism having substantially the same properties as Saccharomyces cerevisiae MBG5364.

[0340] Paragraph

[19] . A recombinant Saccharomyces cerevisiae strain, or a derivative thereof (e.g., expressing heterologous polypeptides, such as glucosylamylase and / or α-amylase), deposited in accordance with the Budapest Treaty at the U.S. Agricultural Research Service Patent Collection Center (NRRL) with accession number NRRL 68304 (Saccharomyces cerevisiae strain MBG5365), or a fermentation organism having substantially the same properties as Saccharomyces cerevisiae MBG5365.

[0341] Paragraph

[20] . The recombinant Saccharomyces cerevisiae strain as described in paragraphs

[18] or

[19] , wherein the strain has one or more of the following properties: - Compared with Saccharomyces cerevisiae CIBTS1260 (e.g., 10 to 32 hours), higher ethanol fermentation kinetics at 1 g DWC / L, 32°C, and pH 5.5 (as described in Example 7 of this paper); - Compared with Saccharomyces cerevisiae CIBTS1260, higher xylose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper); - Compared to Saccharomyces cerevisiae CIBTS1260, higher glucose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper).

[0342] Paragraph

[21] . The recombinant Saccharomyces cerevisiae strain as described in any of paragraphs

[18] -

[20] , wherein the strain has a higher ethanol yield than Saccharomyces cerevisiae CIBTS1260 at 1 g DWC / L, 32°C, and pH 5.5 (as described in Example 7 of this document) for 10 to 30 hours of fermentation.

[0343] Paragraph

[22] . The recombinant Saccharomyces cerevisiae strain as described in any of paragraphs

[18] -

[21] , wherein the strain can consume more than 95% xylose after fermentation for 48 hours under process conditions of 1 g DCW / L, 35°C, and pH 5.5 (as described in Example 3 of this document).

[0344] Paragraph

[23] . The recombinant Saccharomyces cerevisiae strain as described in any of paragraphs

[18] -

[22] , wherein the strain can consume more than 95% of glucose after fermentation for 24 hours under process conditions of 1 g DCW / L, 35°C, and pH 5.5 (as described in Example 3 of this document).

[0345] Paragraph

[24] . The recombinant brewer's yeast as described in any of paragraphs

[18] -

[23] , wherein the strain can provide more than 30 g / L ethanol, such as more than 40 g / L ethanol, such as more than 45 g / L ethanol, or such as about 47 g / L ethanol after fermentation for 48 hours at process conditions of 1 gDCW / L, 35°C, and pH 5.5 (as described in Example 3 of this document).

[0346] Paragraph

[25] . The recombinant Saccharomyces cerevisiae as described in any one of paragraphs

[18] -

[24] contains a heterologous gene encoding xylose isomerase.

[0347] Paragraph

[26] . The recombinant Saccharomyces cerevisiae as described in any of paragraphs

[18] -

[25] contains a heterologous gene encoding a pentose transporter.

[0348] Paragraph

[27] . Recombinant Saccharomyces cerevisiae as described in any of paragraphs

[18] -

[26] , wherein the pentose transporter gene is a GFX gene (e.g., GFX1 from Candida intermedia).

[0349] Paragraph

[28] . The recombinant Saccharomyces cerevisiae as described in any of paragraphs

[18] -

[27] contains a heterologous gene (XKS) encoding xylulokine kinase (e.g., XKS from Saccharomyces cerevisiae).

[0350] Paragraph

[29] . The recombinant Saccharomyces cerevisiae as described in any of paragraphs

[18] -

[28] contains a heterologous gene (RPE1) encoding ribulose 5-phosphate 3-episomerase (e.g., RPE1 from Saccharomyces cerevisiae).

[0351] Paragraph

[30] . The recombinant Saccharomyces cerevisiae as described in any of paragraphs

[18] -

[29] contains a heterologous gene (RKI1) encoding ribulose 5-phosphate isomerase (e.g., RKI1 from Saccharomyces cerevisiae).

[0352] Paragraph

[31] . The recombinant Saccharomyces cerevisiae as described in any of paragraphs

[18] -

[30] contains a heterologous gene (TKL1) encoding a transketolase and a heterologous gene (TAL1) encoding a transaldolase (e.g., TKL1 and TAL1 from Saccharomyces cerevisiae).

[0353] Paragraph

[32] . A method for producing a derivative of Saccharomyces cerevisiae strain MBG5364 (deposited at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) under accession number NRRL 68303), the method comprising: a. Co-culturing the first yeast strain with the second yeast strain, wherein the second yeast strain is *Saccharomyces cerevisiae* strain MBG5364 or a derivative thereof, under conditions allowing for DNA combination between the first and second yeast strains; and b. Isolating heterozygous strains; and c. Optionally, steps (a) and (b) may be repeated using the heterozygous strain isolated in step (b) as the first yeast strain and / or the second yeast strain.

[0354] Paragraph

[33] . A method for producing a derivative of Saccharomyces cerevisiae strain MBG5365 (deposited at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) under accession number NRRL 68304), the method comprising: a. Co-culturing the first yeast strain with the second yeast strain, wherein the second yeast strain is *Saccharomyces cerevisiae* strain MBG5365 or a derivative thereof, under conditions allowing for DNA combination between the first and second yeast strains; and b. Isolating heterozygous strains; and c. Optionally, steps (a) and (b) may be repeated using the heterozygous strain isolated in step (b) as the first yeast strain and / or the second yeast strain.

[0355] Paragraph

[34] . A method for producing a derivative of Saccharomyces cerevisiae strain MBG5364 exhibiting the defined characteristics of Saccharomyces cerevisiae strain MBG5364 (deposited at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) under accession number NRRL 68303), the method comprising: (a) Provide: (i) the first yeast strain; and (ii) A second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5364 or a derivative thereof; (b) Cultivate the first yeast strain and the second yeast strain under conditions that allow the combination of DNA between the first and second yeast strains; (c) Screening or selecting derivatives of Saccharomyces cerevisiae strain MBG5364.

[0356] Paragraph

[35] . The method described in paragraph

[34] , wherein step (c) includes screening or selecting heterozygous strains exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG5364.

[0357] Paragraph

[36] . As described in paragraph

[34] , the method includes the following additional steps: (d) Repeat 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 exhibiting the defined characteristics of the Saccharomyces cerevisiae strain MBG5364 is obtained.

[0358] Paragraph

[37] . The method described in paragraph

[34] , wherein the cultivation step (b) includes: (i) To cause the first yeast strain and the second yeast strain to form spores; (ii) Hybridize the germinating spores produced by the first yeast strain with the germinating spores produced by the second yeast strain.

[0359] Paragraph

[38] . A method for producing a derivative of Saccharomyces cerevisiae strain MBG5365 exhibiting the defined characteristics of Saccharomyces cerevisiae strain MBG5365 (deposited at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) under accession number NRRL 68304), the method comprising: (d) Provide: (j) the first yeast strain; and (iii) A second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5365 or a derivative thereof; (e) The first yeast strain and the second yeast strain are cultured under conditions that allow the combination of DNA between the first and second yeast strains; (f) Screening or selecting derivatives of Saccharomyces cerevisiae strain MBG5365.

[0360] Paragraph

[39] . The method described in paragraph

[38] , wherein step (c) includes screening or selecting heterozygous strains exhibiting one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5365.

[0361] Paragraph

[40] . As described in paragraph

[38] , the method includes the following additional steps: (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 exhibiting the defined characteristics of the Saccharomyces cerevisiae strain MBG5365 is obtained.

[0362] Paragraph

[41] . The method described in paragraph

[38] , wherein the cultivation step (b) includes: (i) To cause the first yeast strain and the second yeast strain to form spores; (ii) Hybridize the germinating spores produced by the first yeast strain with the germinating spores produced by the second yeast strain.

[0363] Paragraph

[42] . A method for producing a recombinant derivative of Saccharomyces cerevisiae strain MBG5364 (deposited at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) under accession number NRRL 68303), the method comprising: (a) Transforming *Saccharomyces cerevisiae* strain MBG5364 (or a derivative thereof) with one or more expression vectors (e.g., one or more expression vectors encoding glucosylamylase and / or α-amylase); and (b) Isolate the transformed strain.

[0364] Paragraph

[43] . A method for producing a recombinant derivative of Saccharomyces cerevisiae strain MBG5365 (deposited at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) under accession number NRRL 68304), the method comprising: (a) Transforming *Saccharomyces cerevisiae* strain MBG5365 (or a derivative thereof) with one or more expression vectors (e.g., one or more expression vectors encoding glucosylamylase and / or α-amylase); and (b) Isolate the transformed strain.

[0365] Paragraph

[44] . A strain of Saccharomyces cerevisiae produced by any one of paragraphs

[32] -

[43] .

[0366] Paragraph

[45] . A method for producing ethanol, the method comprising incubating a strain of Saccharomyces cerevisiae, as described in any one of paragraphs

[18] -

[31] and

[44] , with a substrate containing fermentable sugars under conditions that allow fermentable sugars to ferment to produce ethanol.

[0367] Paragraph

[46] . Use of the Saccharomyces cerevisiae strains as described in any of paragraphs

[18] -

[31] and

[44] in ethanol production.

[0368] Paragraph

[47] . Use of any of the Saccharomyces cerevisiae strains in the production of Saccharomyces genus strains as described in paragraphs

[18] ,

[20] -

[31] and

[44] , which have the defining characteristics of Saccharomyces cerevisiae strain MBG5364 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, under accession number NRRL 68303).

[0369] Paragraph

[48] . Use of any of the Saccharomyces cerevisiae strains in the production of Saccharomyces strains as described in paragraphs

[19] -

[31] and

[44] , which have the defining characteristics of Saccharomyces cerevisiae strain MBG5365 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, under accession number NRRL 68304).

[0370] Paragraph

[49] . Use of Saccharomyces cerevisiae strain MBG5364 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, with accession number NRRL 68303) in the production of yeast strains having substantially the same properties as Saccharomyces cerevisiae strain MBG5364 or yeast strains exhibiting one or more of the defined characteristics of Saccharomyces cerevisiae strain MBG5364.

[0371] Paragraph

[50] . Use of Saccharomyces cerevisiae strain MBG5365 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, under accession number NRRL 68304) in the production of yeast strains having substantially the same properties as Saccharomyces cerevisiae strain MBG5365 or yeast strains exhibiting one or more of the defined characteristics of Saccharomyces cerevisiae strain MBG5365.

[0372] Paragraph

[51] . Use of Saccharomyces cerevisiae strain MBG5364 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, under accession number NRRL 68303) or strains having substantially the same properties as Saccharomyces cerevisiae strain MBG5364 or derivatives thereof in any of the methods described in paragraphs [1]-

[17] .

[0373] Paragraph

[52] . Use of Saccharomyces cerevisiae strain MBG5365 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, under accession number NRRL 68304) or strains having substantially the same properties as Saccharomyces cerevisiae strain MBG5365 or derivatives thereof in any of the methods described in paragraphs [2]-

[16] .

[0374] Paragraph

[53] . A composition comprising a strain of Saccharomyces cerevisiae as described in any one of paragraphs

[18] -

[31] and

[44] , and one or more naturally occurring and / or non-naturally occurring components.

[0375] Paragraph

[54] . The composition as described in paragraph

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

[0376] Paragraph

[55] . The composition as described in paragraph

[53] or

[54] , wherein the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5364 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, with accession number NRRL 68303).

[0377] Paragraph

[56] . The composition as described in paragraph

[53] or

[54] , wherein the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5365 (deposited at the Northern Regional Research Center of the American Agricultural Research Service Patent Culture Collection (NRRL) at 1815 University Street, Peoria, Illinois, USA, with accession number NRRL 68304).

[0378] Paragraph

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

[53] -

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

Claims

1. A method for producing fermentation products from cellulose-containing and / or starch-containing materials, the method comprising: (a) Saccharification of the cellulose- or starch-containing material; as well as (b) Fermenting the saccharified material of step (a) with a fermenting organism under appropriate conditions to produce the fermentation product; The fermenting organisms mentioned above are: (1) A recombinant strain of *Saccharomyces cerevisiae*, or a derivative thereof (e.g., expressing heterologous polypeptides such as glucosylamylase and / or α-amylase), deposited in the U.S. Agricultural Research Service Patent Culture Collection (NRRL) under the Budapest Treaty with accession number NRRL 68303 (*Saccharomyces cerevisiae* strain MBG5364), or a fermentation organism having substantially the same properties as *Saccharomyces cerevisiae* MBG5364; or (2) A recombinant strain of Saccharomyces cerevisiae, or a derivative thereof (e.g., expressing heterologous polypeptides such as glucosylamylase and / or α-amylase), deposited in accordance with the Budapest Treaty at the Patent Culture Collection Center (NRRL) of the U.S. Agricultural Research Service with accession number NRRL 68304 (Saccharomyces cerevisiae strain MBG5365), or a fermentation organism having substantially the same properties as Saccharomyces cerevisiae MBG5365.

2. A recombinant yeast strain selected from the following genus: Saccharomyces cerevisiae strain deposited in accordance with the Budapest Treaty at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) with accession number NRRL 68303 (Saccharomyces cerevisiae strain MBG5364), or derivatives thereof (e.g., expressing heterologous peptides such as glucosyl amylase and / or α-amylase), or fermentation organisms having substantially the same properties as Saccharomyces cerevisiae MBG5364; and Saccharomyces cerevisiae strain MBG5365, deposited in accordance with the Budapest Treaty at the Patent Culture Collection Center (NRRL) of the U.S. Agricultural Research Service with accession number NRRL 68304 (Saccharomyces cerevisiae strain MBG5365), or derivatives thereof (e.g., expressing heterologous polypeptides such as glucosylamylase and / or α-amylase), or fermentation organisms having substantially the same properties as Saccharomyces cerevisiae MBG5365.

3. The recombinant Saccharomyces cerevisiae strain according to claim 2, wherein the strain has one or more of the following properties: - Compared with Saccharomyces cerevisiae CIBTS1260 (e.g., 10 to 32 hours), higher ethanol fermentation kinetics at 1 g DWC / L, 32°C, and pH 5.5 (as described in Example 7 of this paper); - Compared with Saccharomyces cerevisiae CIBTS1260, higher xylose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper); - Compared to Saccharomyces cerevisiae CIBTS1260, higher glucose consumption was observed after 48 hours of fermentation at 1 g DWC / L, 35°C, and pH 5.5 (as described in Example 3 of this paper).

4. The recombinant Saccharomyces cerevisiae strain according to claim 2 or 3, wherein, compared with Saccharomyces cerevisiae CIBTS1260, the strain can achieve a higher ethanol yield when fermented for 10 to 30 hours at 1 g DWC / L, 32°C, and pH 5.5 (as described in Example 7 herein).

5. The recombinant Saccharomyces cerevisiae strain according to any one of claims 2-4, wherein the strain can consume more than 95% xylose after fermentation for 48 hours under process conditions of 1 g DCW / L, 35°C, and pH 5.5 (as described in Example 3 of this document).

6. The recombinant Saccharomyces cerevisiae strain according to any one of claims 2-5, wherein the strain can consume more than 95% of glucose after fermentation for 24 hours under process conditions of 1 g DCW / L, 35°C, and pH 5.5 (as described in Example 3 of this document).

7. The recombinant brewer's yeast according to any one of claims 2-6, wherein the strain, after fermentation for 48 hours at 1 g DCW / L, 35°C, and pH 5.5 (as described in Example 3 herein), can provide more than 30 g / L ethanol, such as more than 40 g / L ethanol, such as more than 45 g / L ethanol, such as about 47 g / L ethanol.

8. The recombinant Saccharomyces cerevisiae according to any one of claims 2-7, comprising a heterologous gene encoding xylose isomerase.

9. The recombinant Saccharomyces cerevisiae according to any one of claims 2-8, comprising a heterologous gene encoding a pentose transporter.

10. The recombinant Saccharomyces cerevisiae according to any one of claims 2-9, wherein the pentose transporter gene is a GFX gene (e.g., GFX1 from Candida intermedia).

11. The recombinant Saccharomyces cerevisiae according to any one of claims 2-10, comprising a heterologous gene (XKS) encoding xylulokine (e.g., XKS from Saccharomyces cerevisiae).

12. The recombinant Saccharomyces cerevisiae according to any one of claims 2-11, comprising a heterologous gene (RPE1) encoding ribulose 5-phosphate 3-epimerase (e.g., RPE1 from Saccharomyces cerevisiae), a heterologous gene (RKI1) encoding ribulose 5-phosphate isomerase (e.g., RKI1 from Saccharomyces cerevisiae), or a heterologous gene (TKL1) encoding transketolase and a heterologous gene (TAL1) encoding transaldolase (e.g., TKL1 and TAL1 from Saccharomyces cerevisiae).

13. A method for producing derivatives of either *Saccharomyces cerevisiae* strain MBG5364 (deposited at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) with accession number NRRL 68303) or *Saccharomyces cerevisiae* strain MBG5365 (deposited at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) with accession number NRRL 68304) exhibiting the defined characteristics of either strain MBG5364 or MBG5365, the method comprising: (a) Provide: (i) First yeast strain; as well as (ii) A second yeast strain, wherein the second yeast strain is Saccharomyces cerevisiae strain MBG5364 or a derivative thereof; (b) Cultivate the first yeast strain and the second yeast strain under conditions that allow the combination of DNA between the first and second yeast strains; (c) Screening or selecting derivatives of Saccharomyces cerevisiae strain MBG5364.

14. The method of claim 134, wherein step (c) comprises screening or selecting heterozygous strains exhibiting one or more defined characteristics of the Saccharomyces cerevisiae strain MBG5364.

15. The method of claim 13, wherein the method comprises the additional step of: (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 exhibiting the defined characteristics of the Saccharomyces cerevisiae strain MBG5364 is obtained.

16. The method of claim 13, wherein the cultivation step (b) comprises: (i) To induce the first yeast strain and the second yeast strain to form spores; (ii) Hybridize the germinating spores produced by the first yeast strain with the germinating spores produced by the second yeast strain.

17. A method for producing recombinant derivatives of *Saccharomyces cerevisiae* strain MBG5364 (deposited at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) under accession number NRRL 68303) or *Saccharomyces cerevisiae* strain MBG5365 (deposited at the U.S. Agricultural Research Service Patent Culture Collection (NRRL) under accession number NRRL 68304), the method comprising: (a) Transforming *Saccharomyces cerevisiae* strain MBG5364 (or a derivative of *Saccharomyces cerevisiae* strain MBG5364) or *Saccharomyces cerevisiae* strain MBG5365 (or a derivative of *Saccharomyces cerevisiae* strain MBG5365) with one or more expression vectors (e.g., one or more expression vectors encoding glucosylamylase and / or α-amylase); and (b) Isolate the transformed strain.

18. A strain of Saccharomyces cerevisiae produced by the method according to any one of claims 13-17.

19. A method for producing ethanol, the method comprising: The *Saccharomyces cerevisiae* strain according to any one of claims 2-12 and 18 is incubated with a substrate containing the fermentable sugar, under conditions that allow the fermentable sugar to ferment into ethanol.

20. A composition comprising a *Saccharomyces cerevisiae* strain according to any one of claims 2-12 and 18, and one or more naturally occurring and / or non-naturally occurring components.

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