CN116724117A

CN116724117A - Improved fermenting organisms for ethanol production

Info

Publication number: CN116724117A
Application number: CN202180054628.8A
Authority: CN
Inventors: R·N·梅达; P·J·L·贝尔
Original assignee: Microbiogen Pty Ltd; Novozymes AS
Current assignee: Microbiogen Pty Ltd; Novozymes AS
Priority date: 2020-09-04
Filing date: 2021-09-03
Publication date: 2023-09-08

Abstract

The present application relates to a process for producing ethanol comprising saccharifying cellulosic or starch-containing material and fermenting the saccharified material with a fermenting microorganism to produce ethanol. The fermenting organism is a Saccharomyces cerevisiae strain MBG5151 (deposited under accession number Y-67971 at the American national institute of agricultural research and patent Collection (NRRL) of 61604, illinois), a Saccharomyces cerevisiae strain MBG5248 (deposited under accession number Y-68015 at the American national institute of agricultural research and patent collection (NRRL) of 61604, illinois), or a fermenting organism having the same or substantially the same properties as Saccharomyces cerevisiae MBG5151 or MBG 5248.

Description

Improved fermenting organisms for ethanol production

Reference to preservation of biological Material

The present application contains references to biological material preservation, which are incorporated herein by reference.

Background

Ethanol is typically blended into gasoline for use as a transportation fuel. Cellulosic materials are used as feedstock in ethanol production processes. There are several methods in the art for making cellulose and hemicellulose hydrolysates containing glucose, mannose, xylose and arabinose. Glucose and mannose are efficiently converted to ethanol during natural anaerobic metabolism. Up to now, the most effective ethanol producing microorganism is the yeast Saccharomyces cerevisiae (Saccharomyces cerevisiae). However, saccharomyces cerevisiae lacks the enzymes necessary to convert the primary sugar, xylose, to xylulose, and therefore xylose cannot be utilized as a carbon source. For this purpose, saccharomyces cerevisiae needs to be genetically engineered to express enzymes that can convert xylose to xylulose. One of the enzymes required is xylose isomerase (e.c. 5.3.1.5), which converts xylose to xylulose, which is then converted to ethanol during fermentation of saccharomyces cerevisiae.

WO 2003/062430 discloses the introduction of a functional Piromyces (Piromyces) Xylose Isomerase (XI) into saccharomyces cerevisiae, which slowly metabolizes xylose and confers the ability to grow on xylose on the yeast transformant by enzymes of the non-oxidized part of the endogenous xylulokinase (EC 2.7.1.17) and pentose phosphate pathways encoded by XKS 1.

U.S. patent No. 8,586,336 discloses a strain of saccharomyces cerevisiae expressing xylose isomerase obtained from rumen fluid of cattle. The yeast strain can be used for producing 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 functions.

Although the process of producing ethanol using cellulosic materials is significantly improved, it is still desirable and necessary to provide improved processes, particularly improved fermentation kinetics, which are beneficial for increasing the robustness of fermentation inhibitors.

Disclosure of Invention

Described herein, inter alia, are processes for producing ethanol from cellulose-containing or starch-containing materials, and yeast suitable for use in such processes.

A first aspect relates to a method of producing a fermentation product from cellulose-and/or starch-containing material, the method comprising:

(a) Saccharifying the cellulose-or starch-containing material; and

(b) Fermenting the saccharified material of step (a) with a fermenting organism under suitable conditions to produce a fermentation product; wherein the fermenting organism is a recombinant strain of Saccharomyces cerevisiae deposited under the Budapest Treaty (Budapest treatment) at the American national institute of agricultural research service patent Collection (Agricultural Research Service Patent Culture Collection) (NRRL) and deposited under accession number NRRL Y-67971 (Saccharomyces cerevisiae strain MBG 5151), NRRL Y-68015 (Saccharomyces cerevisiae strain MBG 5248), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and/or an alpha-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5151 or Saccharomyces cerevisiae strain MBG 5248.

In one embodiment, the method comprises recovering the fermentation product from the fermentation (e.g., by distillation).

In one embodiment, the fermentation and saccharification are carried out simultaneously in Simultaneous Saccharification and Fermentation (SSF). In one embodiment, fermentation and Saccharification (SHF) are performed sequentially.

In one embodiment, the fermentation product is ethanol.

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

In one embodiment, step (a) comprises saccharifying the cellulose-containing material. In one embodiment, the cellulose-containing material is pre-treated. In one embodiment, the cellulose-containing material comprises bagasse.

In one embodiment, 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: cellulases, AA9 polypeptides, hemicellulases, CIPs, esterases, expansins (expansins), ligninases, oxidoreductases, pectinases, proteases and swollenins. In one embodiment, the cellulase is one or more enzymes selected from the group consisting of: endoglucanases, cellobiohydrolases and beta-glucosidase. In one embodiment, the hemicellulase is one or more enzymes selected from the group consisting of: xylanase, acetylxylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.

In one embodiment, 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%).

In one embodiment, the fermentation is performed under low oxygen (e.g., anaerobic) conditions.

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

higher ethanol fermentation kinetics at 1gDWC/L, 32 ℃, pH 5.5 (as described in example 7 herein) compared to saccharomyces cerevisiae CIBTS1260 (e.g., 10 to 32 hours);

higher xylose consumption after 48 hours of fermentation at 1g DWC/L, 35 ℃, pH 5.5 (as described in example 3 herein) compared to saccharomyces cerevisiae CIBTS 1260;

higher glucose consumption after fermentation at 1g DWC/L, 35 ℃, pH 5.5 (as described in example 3 herein) for 48 hours compared to saccharomyces cerevisiae CIBTS 1260.

The second aspect relates to a recombinant Saccharomyces cerevisiae strain deposited with Budapest at about the American national institute of agricultural research and services patent culture Collection (NRRL) under accession number NRRL Y-67971 (Saccharomyces cerevisiae strain MBG 5151), NRRL Y-68015 (Saccharomyces cerevisiae strain MBG 5248), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and/or alpha-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG5151 or Saccharomyces cerevisiae strain MBG 5248.

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

In one embodiment, the strain is capable of higher ethanol yields than Saccharomyces cerevisiae CIBTS1260 when fermented at 1gDWC/L at 32℃and pH 5.5 (as described in example 7 herein) for 10 to 30 hours.

In one embodiment, the strain is capable of consuming greater than 95% xylose after 48 hours of fermentation under process conditions of 1g DCW/L, 35 ℃, pH 5.5 (as described in example 3 herein).

In one embodiment, the strain is capable of consuming greater than 95% glucose after fermentation at 1g of DCW/L for 24 hours under process conditions of 35 ℃ and pH 5.5 (as described in example 3 herein).

In one embodiment, the strain is capable of providing greater than 30g/L ethanol, such as greater than 40g/L ethanol, such as greater than 45g/L ethanol, such as about 47g/L ethanol, after fermentation at 1g of DCW/L for 48 hours under process conditions of 35℃and pH 5.5 (as described in example 3 herein).

In one embodiment, the strain comprises a heterologous gene encoding xylose isomerase. In one embodiment, the strain comprises a heterologous gene encoding a pentose transporter, such as a GFX gene (e.g., GFX1 from candida intermedia). In one embodiment, the strain comprises a heterologous gene (XKS) encoding xylulokinase (e.g., XKS from saccharomyces cerevisiae). In one embodiment, the strain comprises a heterologous gene (RPE 1) encoding a ribulose 5 phosphate 3-epimerase (e.g., RPE1 from saccharomyces cerevisiae). In one embodiment, the strain comprises a heterologous gene (RKI 1) encoding ribulose 5 phosphate isomerase (e.g., RKI1 from Saccharomyces cerevisiae). In one embodiment, the strain comprises a heterologous gene (TKL 1) encoding a transketolase and a heterologous gene (TAL 1) encoding a transaldolase (e.g., TKL1 and TAL1 from saccharomyces cerevisiae).

A third aspect relates to a method of producing a derivative of NRRL Y-67971 (Saccharomyces cerevisiae strain MBG 5151) or NRRL Y-68015 (Saccharomyces cerevisiae strain MBG 5248), the method comprising: (a) Culturing the first yeast strain with a second yeast strain under conditions that allow DNA combination between the first and second yeast strains, wherein the second yeast strain is NRRL Y-67971 (saccharomyces cerevisiae strain MBG 5151) or NRRL Y-68015 (saccharomyces cerevisiae strain MBG 5248) or a derivative thereof; and (b) isolating the 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.

A fourth aspect relates to a method of producing a derivative of NRRL Y-67971 (Saccharomyces cerevisiae strain MBG 5151) exhibiting the defined characteristics of Saccharomyces cerevisiae strain MBG5151 or NRRL Y-68015 (Saccharomyces cerevisiae strain MBG 5248) exhibiting the defined characteristics of Saccharomyces cerevisiae strain MBG5248, 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 MBG5151, saccharomyces cerevisiae strain MBG5248, or derivatives thereof; (b) Culturing the first yeast strain and the second yeast strain under conditions that allow for DNA combination between the first yeast strain and the second yeast strain; (c) The derivatives of Saccharomyces cerevisiae strain MBG5151 or Saccharomyces cerevisiae strain MBG5248 are selected or selected.

In one embodiment, step (c) comprises screening or selecting for heterozygous strains exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG5151 or Saccharomyces cerevisiae strain MBG 5248. In one embodiment, the method further comprises the steps of: (d) Repeating steps (a) and (b) with the strain selected or selected from step (c) as the first and/or second strain until a derivative exhibiting the defined characteristics of s.cerevisiae strain MBG5151 or s.cerevisiae strain MBG5248 is obtained.

In one embodiment, the culturing step (b) comprises: (i) Sporulation of the first yeast strain and the second yeast strain; (ii) The germinated spores produced by the first yeast strain are hybridized with the germinated spores produced by the second yeast strain.

A fifth aspect relates to a method of producing a recombinant derivative of NRRL Y-67971 (Saccharomyces cerevisiae strain MBG 5151) or NRRL Y-68015 (Saccharomyces cerevisiae strain MBG 5248), the method comprising: (a) Transforming saccharomyces cerevisiae strain MBG5151 (or a derivative thereof) or saccharomyces cerevisiae strain MBG5248 (or a derivative thereof) with one or more expression vectors (e.g., one or more expression vectors encoding glucoamylase and/or alpha-amylase); and (b) isolating the transformed strain.

A sixth aspect relates to a strain of saccharomyces cerevisiae produced by any of the third, fourth or fifth aspects.

A seventh aspect relates to a method of producing ethanol, the method comprising incubating a saccharomyces strain of the second or sixth aspect with a substrate comprising a fermentable sugar under conditions that allow the fermentable sugar to ferment to ethanol.

An eighth aspect relates to a composition comprising any of the saccharomyces cerevisiae strains of the second or sixth aspects, and one or more naturally occurring and/or non-naturally occurring components.

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

In one embodiment, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5151 (deposited under accession number NRRL Y-67971 at American agricultural research service patent Collection North regional research center (Northern Regional Research Center (NRRL)) of street 1815, pi Aorui, ill..

In one embodiment, the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5248 (deposited under accession number NRRL Y-68015 at the American agricultural research service patent Collection North regional research center (NRRL) of street 1815, university Pi Aorui, ill. A.).

In one embodiment, the saccharomyces cerevisiae strain is in a viable state, in particular in a dry, pasty or compressed state.

Drawings

FIG. 1 shows a plasmid map of plasmid pYIE2-mgXI-GXF 1-delta containing mgXI and GXF expression cassettes.

FIG. 2 shows a plasmid map of a plasmid using pSH 47-hyg.

FIG. 3 shows a diagram of the resulting plasmid pYIE2-XKS 1-PPP-delta.

FIG. 4 shows a comparison of fermentation of CIBTS1260 versus BSGX001 in NREL acid pretreated corn stover hydrolysate at 35℃for 72 hours with 1g DCW/L yeast inoculation, pH 5.5.

Fig. 5 shows a comparison of CIBTS1260 and BSGX001 in model medium: 2/L yeast inoculation, 32 ℃, pH 5.5, 72 hours.

FIG. 6 shows a fermentation comparison of bagasse hydrolysate produced by cellulose degrading enzyme composition CA and cellulose degrading enzyme composition CB together with CIBTS1260 after 72 hours, inoculated with 1g/L yeast.

FIG. 7 shows the percent decrease in DP2 concentration during fermentation of hydrolysates produced with cellulases CA or CB at 1g/L yeast inoculation at 35℃and pH 5.5 for 72 hours.

FIG. 8 shows dynamic curves of MBG5147-MBG5151 fermentation with CIBTS 1260.

Definition of the definition

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

Allelic variants: the term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation occurs naturally through mutation and can lead to polymorphisms within a population. The gene mutation may be silent (no change in the encoded polypeptide) or may encode a polypeptide having an altered amino acid sequence. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene.

Alpha-amylase: the term "alpha amylase" means a 1, 4-alpha-D-glucan hydrolase (ec.3.2.1.1) that catalyzes the hydrolysis of starch and other linear and branched 1, 4-glycosidic oligosaccharides and polysaccharides. The alpha-amylase activity may be determined using methods known in the art (e.g., using the alpha amylase assay described in WO 2020/023411).

Auxiliary activity 9: the term "helper activity 9" or "AA9" means a polypeptide classified as a soluble polysaccharide monooxygenase (Quinlan et al, 2011, proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ]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. [ J. Biochem ]280:309-316 and Henrissat and Bairoch,1996, biochem. J. [ J. Biochem ]316:695-696, AA9 polypeptides were previously classified as glycoside hydrolase family 61 (GH 61).

The AA9 polypeptide enhances hydrolysis of cellulose-containing material by enzymes having cellulolytic activity. Cellulolytic enhancing activity may be determined by measuring an increase in reducing sugar or an increase in the total amount of cellobiose and glucose from hydrolysis of a cellulose-containing material by a cellulolytic enzyme under the following conditions: 1-50mg total protein per gram of cellulose in the Pretreated Corn Stover (PCS), wherein the total protein comprises 50% -99.5% w/w cellulolytic enzyme protein and 0.5% -50% w/w AA9 polypeptide protein, compared to a control hydrolysis of an equivalent total protein load (1-50 mg cellulolytic protein per gram of cellulose in the PCS) without cellulolytic enhancing activity) at a suitable temperature (e.g., 40-80 ℃, e.g., 50 ℃, 55 ℃, 60 ℃, 65 ℃, or 70 ℃) and a suitable pH (e.g., 4-9, e.g., 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 8.5) for 1-7 days.

Can be used1.5L (Novozymes A/S), buerger' S gasDenmark) and a beta-glucosidase as a source of cellulolytic activity to determine AA9 polypeptide enhancing activity, wherein the beta-glucosidase is present at a weight of at least 2% -5% of the protein loaded by the cellulase protein. In one embodiment, the beta-glucosidase is an Aspergillus oryzae (Aspergillus oryzae) beta-glucosidase (e.g., recombinantly produced in Aspergillus oryzae according to WO 02/095014). In another embodiment, the beta-glucosidase is an Aspergillus fumigatus (Aspergillus fumigatus) beta-glucosidase (recombinantly produced in Aspergillus oryzae, e.g., as described in WO 02/095014).

The AA9 polypeptide enhancing activity can also be determined by: AA9 polypeptide was reacted with 0.5% phosphate swellable cellulose (PASC), 100mM sodium acetate (pH 5), 1mM MnSO at 40 ℃ ₄ 0.1% gallic acid, 0.025mg/ml Aspergillus fumigatus beta-glucosidase, and 0.01%X-100 (4- (1, 3-tetramethylbutyl) phenyl-polyethylene glycol) was incubated for 24-96 hours, followed by determination of glucose released from PASC.

AA9 polypeptide enhancing activity of the high temperature compositions may also be determined according to WO 2013/028928.

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

Beta-glucosidase: the term'Beta-glucosidase "means beta-D-glucosidase glucohydrolase (beta-D-glucoside glucohydrolase) (E.C.3.2.1.21), which catalyzes the hydrolysis of terminal non-reducing beta-D-glucose residues and liberates beta-D-glucose. Can be prepared according to Venturi et al 2002,J.Basic Microbiol journal of basic microbiology]The procedure of 42:55-66 uses p-nitrophenyl-beta-D-glucopyranoside as substrate to determine beta-glucosidase activity. One unit of beta-glucosidase is defined as containing 0.01% at 25 ℃, pH 4.820.0. Mu. Moles of p-nitrophenyl-beta-D-glucopyranoside per minute are produced from 1mM p-nitrophenyl-beta-D-glucopyranoside as substrate in 50mM sodium citrate.

Beta-xylosidase: the term "beta-xylosidase" means a beta-D-xylosidase (beta-D-xyloside xylohydrolase) (e.c. 3.2.1.37) that catalyzes the exohydrolysis of short beta (1→4) -xylooligosaccharides to remove consecutive D-xylose residues from the non-reducing end. Can be contained in an amount of 0.01%20, using 1mM p-nitrophenyl-beta-D-xyloside as substrate, beta-xylosidase activity was determined at pH 5, 40 ℃. One unit of beta-xylosidase is defined as containing 0.01% of ++A at 40℃and pH 5 >20.0. Mu. Moles of p-nitrophenyl-beta-D-xyloside per minute were produced from 1mM p-nitrophenyl-beta-D-xyloside in 100mM sodium citrate.

Catalase: the term "catalase" means hydrogen peroxide, hydrogen peroxide oxidoreductase (EC 1.11.1.6), which catalyzes 2H ₂ O ₂ Conversion to O ₂ +2H ₂ O. For the purposes of the present invention, catalase activity was determined according to U.S. patent No. 5,646,025. One unit of catalase activity is equivalent to the amount of enzyme that catalyzes the oxidation of 1 micromole of hydrogen peroxide under the assay conditions.

Cellobiohydrolase: the term "cellobiohydrolase" means a 1, 4-beta-D-glucan cellobiohydrolase (E.C.3.2.1.91 and E.C.3.2.1.176) that catalyzes the hydrolysis of 1, 4-beta-D-glycosidic linkages in cellulose, cellooligosaccharide, or any polymer containing beta-1, 4-linked glucose, releasing cellobiose from the reducing (cellobiohydrolase I) or non-reducing (cellobiohydrolase II) end of the chain (Teeri, 1997,Trends in Biotechnology [ Biotechnology trends ]15:160-167; teeri et al 1998, biochem. Soc. Trans. [ society of biochemistry ] 26:173-178). Cellobiohydrolase activity can be determined according to the procedure described by: lever et al, 1972, anal. Biochem [ analytical biochemistry ]47:273-279; van Tilbeurgh et al, 1982,FEBS Letters [ European society of Biochemical Association flash report ]149:152-156; van Tilbeurgh and Claeyssens,1985,FEBS Letters [ European society of Biochemical Association flash ]187:283-288; and Tomme et al, 1988, eur. J.biochem. [ J.European biochemistry ],170:575-581.

Cellulolytic enzymes or cellulases: the term "cellulolytic enzyme" or "cellulase" means one or more (e.g., several) enzymes that hydrolyze cellulose-containing material. Such enzymes include one or more endoglucanases, one or more cellobiohydrolases, one or more beta-glucosidase, or a combination thereof. Two basic methods for measuring cellulolytic enzyme activity include: (1) Measuring total cellulolytic enzyme activity, and (2) measuring individual cellulolytic enzyme activities (endoglucanase, cellobiohydrolase, and beta-glucosidase) as described in Zhang et al, 2006,Biotechnology Advances [ progress of biotechnology ] 24:452-481. The total cellulolytic enzyme activity may be measured using insoluble substrates including Whatman No. 1 filter paper, microcrystalline cellulose, bacterial cellulose, algal cellulose, cotton, pretreated lignocellulose, and the like. The most common total cellulolytic activity assay is a filter paper assay using a Waterman No. 1 filter paper as a substrate. The 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).

By measuring under the following conditionsDuring hydrolysis of cellulose-containing material by one or more cellulolytic enzymes, the increase in production/release of sugar determines cellulolytic enzyme activity: 1-50mg cellulolytic enzyme protein/g cellulose in Pretreated Corn Stover (PCS) (or other pretreated cellulose-containing material) at a suitable temperature (e.g., 40-80 ℃, e.g., 50 ℃, 55 ℃, 60 ℃, 65 ℃, or 70 ℃) 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, as compared to control hydrolysis without cellulolytic enzyme protein addition. Typical conditions are: 1ml of reacted, washed or unwashed PCS,5% insoluble solids (dry weight), 50mM sodium acetate (pH 5), 1mM MnSO ₄ 50 ℃, 55 ℃ or 60 ℃ for 72 hours byHPX-87H column chromatography (Bio-Rad Laboratories, inc.), heracles, calif., U.S.A.) was used for sugar analysis.

Coding sequence: the term "coding sequence" or "coding region" means a polynucleotide sequence that specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which typically begins with an ATG start codon or alternative start codon (e.g., GTG and TTG) and ends with a stop codon (e.g., TAA, TAG and TGA). The coding sequence may be the sequence of genomic DNA, cDNA, synthetic polynucleotides, and/or recombinant polynucleotides.

Endoglucanases: the term "endoglucanase" means a 4- (1, 3;1, 4) -beta-D-glucan 4-glucanohydrolase (e.c. 3.2.1.4) which catalyzes the endohydrolysis of the 1, 4-beta-D-glycosidic bond in cellulose, cellulose derivatives (such as carboxymethyl cellulose and hydroxyethyl cellulose), lichenin, mixed beta-1, 3-1,4 glucan such as cereal beta-D-glucan or xyloglucan and beta-1, 4 bonds in other plant material containing cellulosic components. Endoglucanase activity can be determined by measuring a decrease in the viscosity of the substrate or an increase in the reducing end as determined by a reducing sugar assay (Zhang et al 2006,Biotechnology Advances [ Biotechnology progress ] 24:452-481). Endoglucanase activity can also be determined according to the procedure of Ghose,1987,Pure and Appl.Chem [ pure vs. applied chemistry ]59:257-268, using carboxymethyl cellulose (CMC) as substrate at pH 5, 40 ℃.

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. The expression can be measured-e.g., to detect increased expression-by techniques known in the art, such as measuring the level of mRNA and/or translated polypeptide.

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

Fermentable medium: the term "fermentable medium" or "fermentation medium" refers to a medium comprising one or more (e.g., two, several) sugars, such as glucose, fructose, sucrose, cellobiose, xylose, xylulose, arabinose, mannose, galactose, and/or soluble oligosaccharides, wherein the medium is capable of being partially converted (fermented) by a host cell 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 pretreatment of enzymatic hydrolysis (saccharification) of such sources. The term fermentation medium is understood herein to mean the medium prior to the addition of the fermenting organism, for example the medium resulting from the saccharification process, as well as the medium used in the simultaneous saccharification and fermentation process (SSF).

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

Hemicellulolytic enzymes or hemicellulases: the term "hemicellulolytic enzyme" or "hemicellulase" means one or more (e.g., several) enzymes that can hydrolyze hemicellulose material. See, for example, shallom and Shoham,2003,Current Opinion In Microbiology [ current point of microbiology ]6 (3): 219-228. Hemicellulases are key components in plant biomass degradation. Examples of hemicellulases include, but are not limited to: acetylmannanase, acetylxylan esterase, arabinanase, arabinofuranosidase, coumarase, feruloyl esterase, galactosidase, glucuronidase, mannanase, mannosidase, xylanase, and xylosidase. Substrates (hemicellulose) for these enzymes are heterogeneous groups of branched and linear polysaccharides that bind to cellulose microfibrils in the plant cell wall via hydrogen bonds, cross-linking them into a robust network. Hemicellulose is also covalently attached to lignin, forming a highly complex structure with cellulose. The variable structure and organization of hemicellulose requires the synergistic action of many enzymes to fully degrade it. The catalytic module of hemicellulases is a Glycoside Hydrolase (GH) that hydrolyzes glycosidic linkages, or a Carbohydrate Esterase (CE) that hydrolyzes ester linkages of acetic acid or ferulic acid side groups. These catalytic modules can be assigned to the GH and CE families based on their primary sequence homology. Some families, having generally similar folds, may be further categorized as clans (clans), labeled with letters (e.g., GH-a). The most detailed and up-to-date classifications of these and other carbohydrate-active enzymes are available in the carbohydrate-active enzyme (CAZy) database. Hemicellulose decomposing enzyme activity can be measured according to Ghose and Bisaria,1987, pure & Appli. Chem. [ theory and applied chemistry ]59:1739-1752 at a suitable temperature, e.g., 40 ℃ to 80 ℃, e.g., 50 ℃, 55 ℃, 60 ℃, 65 ℃, or 70 ℃, and a suitable pH, e.g., 4-9, e.g., 5.0, 5.5, 6.0, 6.5, or 7.0.

Heterologous polynucleotide: the term "heterologous polynucleotide" is defined herein as a polynucleotide that is not native to the host cell; a natural polynucleotide wherein the coding region has been structurally modified; natural polynucleotides whose expression is quantitatively altered by manipulation of DNA by recombinant DNA techniques (e.g., different (exogenous) promoters); or a native polynucleotide in a host cell having one or more additional copies of the polynucleotide to quantitatively alter expression. A "heterologous gene" is a gene comprising a heterologous polynucleotide.

High stringency conditions: the term "high stringency conditions" means prehybridization and hybridization in 5 XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42℃for 12 to 24 hours following standard southern blotting procedures for probes at least 100 nucleotides in length. The carrier material was finally washed three times, 15 minutes each, using 0.2 XSSC, 0.2% SDS at 65 ℃.

Low stringency conditions: the term "low stringency conditions" means prehybridization and hybridization in 5 XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide at 42℃for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. The carrier material was finally washed three times, 15 minutes each, using 0.2 XSSC, 0.2% SDS at 50 ℃.

Mature polypeptide: the term "mature polypeptide" is defined herein as a polypeptide having biological activity in its final form after translation and any post-translational modifications (e.g., N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.). Mature polypeptide sequences lack signal sequences, which 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" means a polynucleotide encoding a mature polypeptide.

Moderately stringent conditions: the term "moderately stringent conditions" means prehybridization and hybridization in 5XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide at 42℃for 12 to 24 hours following standard southern blotting procedures for probes at least 100 nucleotides in length. The carrier material was finally washed three times, 15 minutes each, using 0.2 XSSC, 0.2% SDS at 55 ℃.

Medium-high stringency conditions: the term "medium-high stringency conditions" means prehybridization and hybridization in 5XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 35% formamide at 42 ℃ for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. The carrier material was finally washed three times, 15 minutes each at 60℃using 0.2 XSSC, 0.2% SDS.

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

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

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 refer to the enzyme nomenclature of 1992 of NC-IUBMB of San Diego, california, academic Press, including the journal 1-5 published in the following, respectively: eur.J.biochem. [ J.European biochemistry ]223:1-5 (1994); eur.J.biochem. [ J.European biochemistry ]232:1-6 (1995); eur.J.biochem. [ J.European biochemistry ]237:1-5 (1996); eur.J.biochem. [ J.European biochemistry ]250:1-6 (1997); and Eur.J.biochem. [ J.European biochemistry ]264:610-650 (1999). The term "subtilase" refers to the serine protease subgroup according to Siezen et al, 1991,Protein Engng [ protein engineering ]4:719-737 and Siezen et al, 1997,Protein Science [ protein science ] 6:501-523. Serine proteases or serine peptidases are a subset of proteases characterized by serine at the active site forming a covalent adduct with a substrate. In addition, subtilases (and serine proteases) are characterized by having two active site amino acid residues, i.e., histidine and aspartic acid residues, in addition to serine. Subtilases may be divided into 6 sub-classes, i.e. subtilisin family, thermophilic protease family, proteinase K family, lanthionine antibiotic peptidase family, kexin family and Pyrolysin family. The term "protease activity" means proteolytic activity (EC 3.4). Protease activity may be measured using methods described in the art (e.g., US 2015/0125955) or using commercially available assay kits (e.g., sigma Aldrich).

Pullulanase: the term "pullulanase" means a starch debranching enzyme (EC 3.2.1.41) having pullulan 6-glucan-hydrolase activity, which catalyzes the hydrolysis of alpha-1, 6-glycosidic bonds in pullulan, thereby releasing maltotriose having a reduced carbohydrate end. For the purposes of the present invention, pullulanase activity may be determined according to the PHADEBAS assay or sweet potato starch assay described in WO 2016/087237.

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

For the purposes of this description, the degree of sequence identity between two amino acid sequences is determined using the Nedel-crafts (Needleman-Wunsch) algorithm (Needleman and Wunsch, J.mol. Biol. [ journal of molecular biology ]1970,48,443-453) as implemented in the Needle program of the EMBOSS software package (EMBOSS: the European Molecular Biology Open Software Suite [ European molecular biology open software suite ]), rice et al, trends Genet [ genetic Trends ]2000,16,276-277), preferably version 3.0.0 or an updated version. The optional parameters used are gap opening penalty of 10, gap extension penalty of 0.5, and EBLOSUM62 (the emoss version of BLOSUM 62) substitution matrix. The output of the "longest identity" of the Needle label (obtained using the-nobrief option) is used as the percent identity and is calculated as follows:

(identical residues X100)/(Length of reference sequence-total number of gaps in alignment)

For the purposes described herein, the degree of sequence identity between two deoxyribonucleotide sequences is determined using the Nidleman-Wen application algorithm (Needleman and Wunsch,1970, supra), as implemented in the Nidel program of the EMBOSS software package (EMBOSS: european molecular biology open software suite, rice et al, 2000, supra), preferably version 3.0.0 or newer versions. The optional parameters used are gap opening penalty 10, gap extension penalty 0.5, and EDNAFULL (the EMBOSS version of NCBI NUC 4.4) substitution matrix. The output of the "longest identity" of the Needle label (obtained using the-nobrief option) is used as the percent identity and is calculated as follows:

(identical deoxyribonucleotide x 100)/(Length of reference sequence-total number of gaps in alignment)

Very high stringency conditions: the term "very high stringency conditions" means prehybridization and hybridization in 5XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50% formamide at 42 ℃ for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. The carrier material was finally washed three times, 15 minutes each at 70℃using 0.2 XSSC, 0.2% SDS.

Very low stringency conditions: the term "very low stringency conditions" means prehybridization and hybridization in 5XSSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 25% formamide at 42 ℃ for 12 to 24 hours following standard southern blotting procedures for probes of at least 100 nucleotides in length. The carrier material was finally washed three times, 15 minutes each, using 0.2 XSSC, 0.2% SDS at 45 ℃.

Xylanase: the term "xylanase" means a 1, 4-beta-D-xylan-xylose hydrolase (1, 4-beta-D-xylan-xylohydrolase) (e.c. 3.2.1.8) that catalyzes the endo-hydrolysis of 1, 4-beta-D-xyloside bonds in xylan. Xylanase activity may be at 0.01% at 37%X-100 and 200mM sodium phosphate (pH 6) was determined using 0.2% AZCL-arabinoxylans as substrates. One unit of xylanase activity was defined as the production of 1.0 micromole azurin (azurin) per minute from 0.2% AZCL-arabinoxylan as substrate in 200mM sodium phosphate (pH 6) at 37 ℃, pH 6.

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

Xylose isomerase: the term "xylose isomerase" or "XI" means an enzyme that can catalyze the in vivo conversion of D-xylose to D-xylulose, and the in vitro conversion of D-glucose to D-fructose. Xylose isomerase is also known as "glucose isomerase" and is classified as e.c.5.3.1.5. As the structure of the enzyme is very stable, xylose isomerase is a good model for studying the relationship between protein structure and function (Karimaki et al, protein Eng Des Sel [ protein engineering, design and selection ],12004,17 (12): 861-869). Xylose isomerase activity may be determined using techniques known in the art (e.g., a coupled enzyme assay using D-sorbitol dehydrogenase, as described by Verhoeven et al, 2017, sci Rep [ science report ]7,46155).

Xylulokinase: the term "xylulokinase" or "XK" is classified as e.c.2.7.1.17 and means an enzyme that catalyzes the conversion of D-xylulose to D-xylulose 5-phosphate. Xylulokinase activity can be determined using methods known in the art (e.g., richard et al, 2000,FEBS Microbiol.Letters, european society of microbiology, proc. Microbiol. Fast., 190,39-43).

Reference herein to "about" a value or parameter includes reference to embodiments of the value or parameter itself. For example, a description referring to "about X" includes embodiment "X". When used in combination with a measurement, the term "about" includes a range that encompasses at least the uncertainty associated with the method of measuring the particular value, and may include a range of plus or minus two standard deviations around the given value.

Likewise, reference to a gene or polypeptide "derived from" another gene or polypeptide X includes the gene or polypeptide X.

As used herein and in the appended claims, the singular forms "a," "an," "or" and "the" include plural referents unless the context clearly dictates otherwise.

It should be understood that the embodiments described herein include "consisting of the … … embodiment" and/or "consisting essentially of the … … embodiment. As used herein, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments, except as may be required by the express language or necessary implication.

Detailed Description

Recombinant fermenting organisms and methods for producing fermentation products, such as ethanol, from cellulose-containing and/or starch-containing material are described herein, among others. The applicant has created new strains of saccharomyces cerevisiae with improved fermentation kinetics while maintaining fermentation yields. Strains with improved kinetics are desirable because, for example, such strains can be more robust in the presence of inhibitors, facilitate a variety of biomass pretreatment conditions, and provide shorter fermentation times.

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

(a) Saccharifying the cellulose-or starch-containing material; and

(b) Fermenting the saccharified material of step (a) with a recombinant fermenting organism described herein.

Steps a) and b) may 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.

Fermenting organism

In one embodiment, the fermenting organism is a recombinant strain of Saccharomyces cerevisiae deposited as Budapest strip at about the American national institute of agricultural research patent Collection (NRRL) under accession number NRRL Y-67971 (Saccharomyces cerevisiae strain MBG 5151), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and/or alpha-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG 5151.

The applicant has produced strain NRRL Y-67971 (saccharomyces cerevisiae strain MBG 5151) from saccharomyces cerevisiae CIBTS1260 (see WO 2016/045569, the contents of which are incorporated herein by reference) by the evolutionary and breeding procedures described in us patent No. 8,257,959. As shown in the examples below, strain MBG5151 provides faster kinetics than CIBTS1260, while maintaining similar ethanol titers.

In another embodiment, the fermenting organism is a recombinant strain of Saccharomyces cerevisiae deposited as Budapest strip at about the American national institute of agricultural research patent Collection (NRRL) under accession number NRRL Y-68015 (Saccharomyces cerevisiae strain MBG 5248), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and/or alpha-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG 5248.

In one embodiment, the fermenting organism is capable of consuming greater than 95% xylose after 48 hours of fermentation at 1g DWC/L at 35 ℃, pH 5.5 (as described in example 3 herein).

In one embodiment, the fermenting organism is capable of consuming greater than 95% glucose after 24 hours of fermentation at 1g DWC/L at 35 ℃ at pH 5.5 (as described in example 3 herein).

In one embodiment, the fermenting organism is capable of higher fermentation product (e.g., ethanol) yields under the same conditions (e.g., 10, 15, 20, 25, or 30 hours of fermentation) as Saccharomyces cerevisiae CIBTS 1260. In some embodiments, the fermenting organism results in a yield of the 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%.

In one embodiment, the fermenting organism is capable of providing greater than 30g/L ethanol, such as greater than 40g/L ethanol, such as greater than 45g/L ethanol, such as greater than 50g/L ethanol, after fermentation at 1g DWC/L at 35℃and pH 5.5 (as described in examples 3 or 7 herein) for 48 hours.

In one embodiment, the fermenting organism is Saccharomyces cerevisiae MBG5151 (deposited under accession number NRRL Y-67971, american agricultural research services patent bacterial Collection (NRRL) of 61604, illinois, U.S.A.). In another embodiment, the fermenting organism is Saccharomyces cerevisiae MBG5248 (deposited under accession number NRRL Y-68015, american national institute of agriculture research and services patent culture Collection (NRRL) at 61604, illinois, U.S.A.A.A.A.A.As..

In one embodiment, the fermenting organism comprises a heterologous gene encoding a xylose isomerase (e.g., a xylose isomerase as 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).

In one embodiment, the fermenting organism comprises a heterologous gene encoding a pentose transporter, such as the GFX gene, in particular 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 to SEQ ID NO. 18 of WO 2016/045569.

In one embodiment, the fermenting organism comprises a heterologous (e.g., by over-expression) xylulokinase gene (XKS), such as an over-expressed XKS gene from saccharomyces cerevisiae.

In one embodiment, the fermenting organism comprises a heterologous (e.g., by over-expression) ribulose 5 phosphate 3-epimerase gene (RPE 1), such as an over-expressed RPE1 gene from saccharomyces cerevisiae.

In one embodiment, the fermenting organism comprises a heterologous (e.g., by over-expression) ribulose 5 phosphate isomerase gene (RKI 1), such as an over-expressed RKI1 gene from Saccharomyces cerevisiae.

In one embodiment, the fermenting organism comprises a heterologous (e.g., by over-expression) transketolase gene (TKL 1) and a transaldolase gene (TAL 1), such as over-expressed TKL1 gene and TAL1 gene from saccharomyces cerevisiae.

In one embodiment, the fermenting organism has one or more, such as one, two, three, four, five or all of the following genetic modifications:

heterologous xylose isomerase gene (Ru-XI) obtained from bovine rumen fluid, in particular 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;

heterologous pentose transporter genes from Candida intermedia (GXF 1), in particular the one shown in SEQ ID NO:18 of WO 2016/045569;

heterologous xylulokinase gene (XKS), in particular from a typical strain of saccharomyces cerevisiae;

Heterologous ribulose 5 phosphate 3-epimerase gene (RPE 1), in particular from a typical strain of saccharomyces cerevisiae;

-a heterologous ribulose 5 phosphate isomerase gene (RKI 1), in particular from a typical strain of saccharomyces cerevisiae;

heterologous transketolase gene (TKL 1) and heterologous transaldolase gene (TAL 1), in particular from typical strains of saccharomyces cerevisiae.

For example, in one embodiment, the fermenting organism of the invention has the following genetic modifications:

heterologous xylose isomerase gene (Ru-XI) obtained from bovine rumen fluid, in particular the one shown in SEQ ID NO:20 of WO2016/045569, encoding the xylose isomerase shown in SEQ ID NO:13 of WO 2016/045569;

heterologous transketolase gene (TKL 1) and transaldolase gene (TAL 1), in particular from typical strains of saccharomyces cerevisiae.

The fermenting organism may also be a derivative of the Saccharomyces cerevisiae strain MBG5151 or MBG 5248. As used herein, a "derivative" of saccharomyces cerevisiae strain MBG5151 or MBG5248 is a strain derived from said strain, such as by mutagenesis, recombinant DNA technology, mating, cell fusion or cytoplasmic induction (cytoreduction) between yeast strains. The strain derived from the saccharomyces cerevisiae strain MBG5151 or MBG5248 may be a direct progeny (i.e., the product of a mating between the saccharomyces cerevisiae strain MBG5151 or MBG5248 and another strain or itself), or a distant progeny (which results from an initial mating between the saccharomyces cerevisiae strain MBG5151 or MBG5248 and another strain or itself followed by a number of subsequent mating).

In one embodiment, the derivative of saccharomyces cerevisiae strain MBG5151 or MBG5248 is a hybrid strain produced by culturing the first yeast strain with saccharomyces cerevisiae strain MBG5151 or MBG5248 under conditions that allow DNA combination between the first yeast strain and saccharomyces cerevisiae strain MBG5151 or MBG 5248.

In one embodiment, the derivative of saccharomyces cerevisiae strain MBG5151 or MBG5248 exhibits one or more defined characteristics of saccharomyces cerevisiae strain MBG5151 or MBG 5248. Saccharomyces cerevisiae strain MBG5151 or MBG5248 is used to produce derivatives of Saccharomyces exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG5151 or MBG 5248. In this respect, the Saccharomyces cerevisiae strain MBG5151 or MBG5248 forms the basis for the preparation of other strains having the defined characteristics of the Saccharomyces cerevisiae strain MBG5151 or MBG 5248. For example, a Saccharomyces strain exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG5151 or MBG5248 may be derived from Saccharomyces cerevisiae strain MBG5151 or MBG5248 using methods such as classical mating, cell fusion, or cytoplasmic introduction, mutagenesis, or recombinant DNA techniques between the yeast strains.

In one embodiment, derivatives of saccharomyces cerevisiae strain MBG5151 exhibiting one or more defined characteristics of saccharomyces cerevisiae strain MBG5151 may be produced by:

(a) Culturing the first yeast strain with a second yeast strain under conditions that allow DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is saccharomyces cerevisiae strain MBG5151 (or a derivative of saccharomyces cerevisiae strain MBG 5151);

(b) Screening or selecting a derivative of saccharomyces cerevisiae strain MBG5151, such as screening or selecting a derivative having increased ethanol production in corn mash as compared to the first strain;

(c) Optionally repeating steps (a) and (b) with the selected or selected strain as the first and/or second yeast strain until a derivative of the saccharomyces cerevisiae strain MBG5151 exhibiting one or more defined characteristics of the saccharomyces cerevisiae strain MBG5151 is obtained.

In one embodiment, derivatives of saccharomyces cerevisiae strain MBG5248 exhibiting one or more defined characteristics of saccharomyces cerevisiae strain MBG5248 can be produced by:

(a) Culturing the first yeast strain with a second yeast strain under conditions that allow DNA combination between the first and second yeast strains, wherein the second yeast strain is saccharomyces cerevisiae strain MBG5248 (or a derivative of saccharomyces cerevisiae strain MBG 5248);

(b) Screening or selecting a derivative of saccharomyces cerevisiae strain MBG5248, such as screening or selecting a derivative having increased ethanol production in corn mash as compared to the first strain;

(c) Optionally repeating steps (a) and (b) with the selected or selected strain as the first and/or second yeast strain until a derivative of saccharomyces cerevisiae strain MBG5248 is obtained that exhibits one or more defined characteristics of saccharomyces cerevisiae strain MBG 5248.

If it is possible to use, for example, warpThe DNA of the first yeast strain is combined with the second yeast strain by classical mating, cell fusion or cytoplasmic transfer, and the first yeast strain may be any strain of yeast. Typically, the first yeast strain is a saccharomyces strain. More typically, the first yeast strain is a saccharomyces cerevisiae strain. Saccharomyces cerevisiae was as described by Kurtzman (2003) FEMS Yeast Research [ FEMS Yeast research]Volume 4, pages 233-245. The first yeast strain may have desirable properties that are intended to be combined with the defined characteristics of the saccharomyces cerevisiae strain MBG 5151. The first yeast strain may be, for example, any Saccharomyces cerevisiae strain, such as, for example, ETHANOLIt is also understood that the first yeast strain may be Saccharomyces cerevisiae strain MBG5151 or MBG5248 (or derivatives of Saccharomyces cerevisiae strain MBG5151 or MBG 5248).

Culturing the first and second yeast strains under conditions that allow for DNA combining between the yeast strains. As used herein, "combination of DNA" between yeast strains refers to a combination of all or part of the genome of a yeast strain. The DNA combination between yeast strains may be performed by any method suitable for combining DNA of at least two yeast cells, and may include, for example, a mating method comprising sporulation of a yeast strain to produce haploid cells, and subsequent crossing of compatible haploid cells; cytoplasmic introduction; or cell fusion such as protoplast fusion.

In one embodiment, the first yeast strain is cultured with the second yeast under conditions that allow for DNA combination between the first yeast strain and the second yeast strain, the culturing comprising:

(i) Sporulation of the first yeast strain and the second yeast strain;

(ii) Spores produced by the first yeast strain are germinated and hybridized with spores produced by the second yeast strain.

In one embodiment, a method of producing a derivative of saccharomyces cerevisiae strain MBG5151 exhibiting one or more defined characteristics of saccharomyces cerevisiae strain MBG5151, 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 MBG5151 (or a derivative of saccharomyces cerevisiae strain MBG 5151);

(b) Sporulation of the first yeast strain and the second yeast strain;

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

(d) Screening or selecting derivatives of saccharomyces cerevisiae strain MBG5151, such as screening or selecting derivatives having increased ethanol production in corn mash compared to the first strain, and/or having higher ethanol yield from glucose during fermentation of corn mash compared to the first strain;

(e) Optionally repeating steps (b) to (d) with the selected or selected strain as the first and/or second yeast strain.

In one embodiment, a method of producing a derivative of saccharomyces cerevisiae strain MBG5151 that exhibits one or more defined characteristics of saccharomyces cerevisiae strain MBG5248, 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 MBG5248 (or a derivative of saccharomyces cerevisiae strain MBG 5248);

(b) Sporulation of the first yeast strain and the second yeast strain;

(d) Screening or selecting derivatives of saccharomyces cerevisiae strain MBG5248, such as screening or selecting derivatives having increased ethanol production in corn mash compared to the first strain, and/or having higher ethanol yield from glucose during fermentation of corn mash compared to the first strain;

Methods for sporulation, germination and hybridization of yeast strains, and in particular Saccharomyces strains, are known in the art and are described, for example, in Ausubel, F.M. et al, (1997) Current Protocols in Molecular Biology [ the handbook of contemporary molecular biology ], vol.2, pages 13.2.1 to 13.2.5 (John Willey & Sons Inc [ John Willi father ]); chapter 7, "Sporulation and Hybridisation of yeast [ sporulation and hybridization of Yeast ]" is described by R.R.Fowell, at "The Yeasts [ Yeast ]" volume 1, A.H. Rose and J.S. Harrison (editorial), 1969,Academic Press [ academic Press ].

In one embodiment, the yeast strain can be cultured under conditions that allow cell fusion. Methods for producing intraspecies or interspecific hybrids using cell fusion techniques are described, for example, in Spencer et al (1990), yeast Technology [ Yeast Technology ], spencer JFT and Spencer DM (editors), springer Verlag [ Schpraringer publishing Co., N.Y.).

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-Vechylov et al (1986) Genetika [ genetics ]22:2625-2636; johnston (1990), yeast technology, spencer JFT and Spencer DM (editions), springer Verlag, springs, new York.

In one embodiment, screening or selecting for derivatives of saccharomyces cerevisiae strain MBG5151 or MBG5248 comprises screening or selecting for derivatives having increased ethanol production compared to the first strain, and/or screening or selecting for hybrids having higher ethanol yields, e.g., as described in WO 2019/161227.

In one embodiment, the derivative of saccharomyces cerevisiae strain MBG5151 or MBG5248 that exhibits one or more defined characteristics of saccharomyces cerevisiae strain MBG5151 or MBG5248, respectively, may be a mutant of saccharomyces cerevisiae strain MBG5151 or MBG 5248. Methods for producing Saccharomyces mutants, and in particular Saccharomyces cerevisiae mutants, are known in the art and are described, for example, in Lawrence C.W. (1991) Methods in Enzymology [ methods of enzymology ], 194:273-281.

In another embodiment, the derivative of saccharomyces cerevisiae strain MBG5151 exhibiting one or more defined characteristics of saccharomyces cerevisiae strain MBG5151 may be a recombinant derivative of saccharomyces cerevisiae strain MBG 5151. In another embodiment, the derivative of saccharomyces cerevisiae strain MBG5248 exhibiting one or more defined characteristics of saccharomyces cerevisiae strain MBG5248 may be a recombinant derivative of saccharomyces cerevisiae strain MBG 5248. Recombinant derivatives of Saccharomyces cerevisiae strain MBG5151 or MBG5248 are strains produced by introducing nucleic acids into Saccharomyces cerevisiae strain MBG5151 or MBG5248 using recombinant DNA techniques. Recombinant methods for introducing nucleic acids into Saccharomyces yeast cells, and in particular Saccharomyces strains, are known in the art and are described, for example, in Ausubel, F.M. et al (1997), current Protocols in Molecular Biology [ the handbook of contemporary molecular biology experiments ], vol.2, pages 13.7.1 to 13.7.7, published by John Wiley & Sons Inc [ John Willi father company ].

In one embodiment, recombinant derivatives of Saccharomyces cerevisiae strain MBG5151 or MBG5248 have been prepared by genetically modifying a strain (or another derivative thereof) to express a heterologous enzyme, such as an alpha-amylase and/or glucoamylase as described herein (or any of the enzymes described in WO 2020/023411, the contents of which are incorporated herein by reference).

In one embodiment, is a method of producing a recombinant derivative of saccharomyces cerevisiae strain MBG5151 (deposited under accession No. NRRL Y-67971 at the american agricultural research services patent bacterial deposit north regional research center (NRRL) at street 1815, pi Aorui, il, a), the method comprising:

(a) Transforming Saccharomyces cerevisiae strain MBG5151 (or derivatives of Saccharomyces cerevisiae strain MBG 5151) with one or more expression vectors encoding heterologous enzymes such as glucoamylase and/or alpha-amylase; and

(b) Isolating the transformed strain.

In one example, derivatives of saccharomyces cerevisiae strain MBG5151 may be prepared by:

(a) Culturing the first yeast strain with a second yeast strain under conditions that allow DNA combination between the first yeast strain and the second yeast strain, wherein the second yeast strain is saccharomyces cerevisiae strain MBG5151 (or a derivative of saccharomyces cerevisiae strain MBG 5151); and

(b) Isolating the heterozygous strain; and

(c) Optionally repeating steps (a) and (b) using the heterozygous strain isolated in step (b) as a derivative of the first yeast strain and/or the saccharomyces cerevisiae strain MBG 5151.

In one embodiment, is a method of producing a recombinant derivative of saccharomyces cerevisiae strain MBG5248 (deposited under accession No. NRRL Y-68015 by the american national institute of agriculture research service patent bacterial deposit north regional research center (NRRL) at street 1815, pi Aorui, il) comprising:

(a) Transforming saccharomyces cerevisiae strain MBG5248 (or derivatives of saccharomyces cerevisiae strain MBG 5248) with one or more expression vectors encoding heterologous enzymes such as glucoamylase and/or alpha-amylase; and

(b) Isolating the transformed strain.

In one example, derivatives of saccharomyces cerevisiae strain MBG5248 can be prepared by:

(a) Culturing the first yeast strain with a second yeast strain under conditions that allow DNA combination between the first and second yeast strains, wherein the second yeast strain is saccharomyces cerevisiae strain MBG5248 (or a derivative of saccharomyces cerevisiae strain MBG 5248); and

(b) Isolating the heterozygous strain; and

(c) Optionally repeating steps (a) and (b) using the heterozygous strain isolated in step (b) as a derivative of the first yeast strain and/or the saccharomyces cerevisiae strain MBG 5248.

In some embodiments, the derivative of saccharomyces cerevisiae strain MBG5151 or MBG5248 expresses glucoamylase and/or alpha-amylase. Derivatives expressing glucoamylase and/or alpha-amylase have been produced to increase ethanol yield and improve process economics by reducing enzyme costs because some or all of the necessary enzymes required to hydrolyze starch are produced by yeast organisms.

Composition and method for producing the same

The present aspect relates to a formulated Saccharomyces yeast composition comprising a yeast strain as described herein and naturally occurring and/or non-naturally occurring components.

In one embodiment, is a composition comprising Saccharomyces cerevisiae strain MBG5151 (or a derivative of Saccharomyces cerevisiae strain MBG 5151) or Saccharomyces cerevisiae strain MBG5248 (or a derivative of Saccharomyces cerevisiae strain MBG 5248). The composition may be, for example, a cream yeast, a compressed yeast, a wet yeast, a dry yeast, a semi-dry yeast, a crushed yeast, a stabilized liquid yeast or a frozen yeast. Methods of preparing such yeast compositions are known in the art.

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

In one embodiment is a composition comprising a Saccharomyces yeast described herein (particularly Saccharomyces cerevisiae strain MBG5151 or MBG 5248) and one or more components selected from the group consisting of: surfactants, emulsifiers, gums, swelling agents, antioxidants and other processing aids.

Surface active agent

The compositions described herein may comprise Saccharomyces yeasts described herein (particularly Saccharomyces cerevisiae strain MBG5151 or MBG 5248) and any suitable surfactant. In one embodiment, the one or more surfactants are anionic surfactants, cationic surfactants, and/or nonionic surfactants.

Emulsifying agent

The compositions described herein may comprise Saccharomyces yeasts described herein (particularly Saccharomyces cerevisiae strain MBG5151 or MBG 5248) and any suitable emulsifier. In one embodiment, the emulsifier is a fatty acid ester of sorbitan. In one embodiment, the emulsifier is selected from the group consisting of: sorbitan Monostearate (SMS), citric acid esters of mono-diglycerides, polyglycerol esters, fatty acid esters of propylene glycol.

In one embodiment, the composition comprises Saccharomyces yeast described herein (particularly Saccharomyces cerevisiae strain MBG5151 or MBG 5248) and Olindronal SMS, olindronal SK, or Olindronal SPL, including the compositions referred to in European patent No. 1,724,336 (incorporated herein by reference). For active dry yeasts, these products are commercially available from Bussetti company (Bussetti) in Austria.

Gums

The compositions described herein may comprise Saccharomyces yeasts described herein (particularly Saccharomyces cerevisiae strain MBG5151 or MBG 5248) and any suitable gum. In one embodiment, the gum is selected from the group consisting of: locust bean gum, guar gum, tragacanth, acacia, xanthan gum and gum arabic, especially for pasty, compressed and dry yeasts.

Swelling agent

The compositions described herein may comprise Saccharomyces yeasts described herein (particularly Saccharomyces cerevisiae strain MBG5151 or MBG 5248) and any suitable swelling agent. In one embodiment, the swelling agent is methylcellulose or carboxymethylcellulose.

Antioxidant agent

The compositions described herein may comprise Saccharomyces yeasts described herein (particularly Saccharomyces cerevisiae strain MBG5151 or MBG 5248) and any suitable antioxidant. In one embodiment, the antioxidant is Butylated Hydroxyanisole (BHA) and/or Butylated Hydroxytoluene (BHT), or ascorbic acid (vitamin C), especially for active dry yeasts.

Method of using cellulose-containing material

In some embodiments, the methods described herein produce a fermentation product from a cellulose-containing material. The primary polysaccharide in the primary cell wall of biomass is cellulose, the second most abundant is hemicellulose, and the third most abundant is pectin. The secondary cell wall produced after cell growth has ceased also contains polysaccharides and is reinforced by polymeric lignin covalently cross-linked to hemicellulose. Cellulose is a homopolymer of anhydrocellobiose and thus is a linear beta- (1-4) -D-glucan, whereas hemicellulose includes a variety of compounds such as xylans, xyloglucans, arabinoxylans, and mannans having a series of substituents in a complex branched structure. Although cellulose is generally polymorphic, it is found to exist in plant tissue primarily as an insoluble crystalline matrix of parallel glucan chains. Hemicellulose is often hydrogen bonded to cellulose and other hemicelluloses, which help stabilize the cell wall matrix.

Cellulose is commonly found in, for example, stems, leaves, hulls, bark and cobs of plants or leaves, branches and wood of trees. The cellulose-containing material may be, but is not limited to: agricultural waste, herbaceous material (including energy crops), municipal solid waste, pulp and paper mill waste, waste paper, and wood (including forestry waste) (see, e.g., wiselogel et al, 1995, in Handbook on Bioethanol [ bioethanol handbook ] (Charles E.Wyman editions), pages 105-118, taylor & Francis [ Taylor-Francis publishing group ], washington, techno, 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 [ recent advances in bioconversion of lignocellulose ], advances in Biochemical Engineering/Biotechnology [ advances in bioengineering/Biotechnology ], T.Scheper, vol.65, pages 23-40, springer-Verlag, new York [ New York Springs publishing ]). It is understood herein that the cellulose may be any form of lignocellulose, 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.

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).

In another embodiment, the cellulose-containing material is arundo donax, bagasse, bamboo, corncob, corn fiber, corn stover, miscanthus, rice straw, switchgrass, or wheat straw.

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

In another embodiment, the cellulose-containing material is alginate, bacterial cellulose, cotton linter, filter paper, microcrystalline cellulose (e.g.,) Or phosphoric acid treated cellulose. />

In another embodiment, the cellulose-containing material is aquatic biomass (aquatics). As used herein, the term "aquatic biomass" means biomass produced by a photosynthesis process in an aquatic environment. The aquatic biomass may be algae, emerging plants, floating leaf plants, or submerged plants.

The cellulose-containing material may be used as is or may be pretreated using conventional methods known in the art, as described herein. In a preferred embodiment, the cellulose-containing material is pre-treated.

Methods of using cellulose-containing materials may be accomplished using methods conventional in the art. Further, the methods may be performed using any conventional biomass processing apparatus configured to perform the methods.

Cellulose pretreatment

In one embodiment, the cellulose-containing material is pre-treated prior to saccharification.

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. [ Biochemical engineering/Biotechnology advances ]108:67-93; galbe and Zacchi,2007, adv. Biochem. Engin./Biotechnol. [ Bioengineering/Biotechnology advances ]108:41-65; hendriks and Zeeman,2009,Bioresource Technology [ biological resource technologies ]100:10-18; mosier et al, 2005,Bioresource Technology [ biological resource technologies ]96:673-686; taherezadeh and Karimi,2008, int. Mol. Sci. [ International journal of molecular science ]9:1621-1651; yang and Wyman,2008,Biofuels Bioproducts and Biorefining-Biofpr. [ biofuel, biological products and biorefinery ] 2:26-40).

The cellulose-containing material may also be reduced in particle size, sieved, presoaked, wetted, washed and/or conditioned prior to pretreatment using methods known in the art.

Conventional pretreatment includes, but is not limited to: steam pretreatment (with or without blasting), dilute acid pretreatment, hot water pretreatment, alkaline pretreatment, lime pretreatment, wet oxidation, wet blasting, ammonia fiber blasting, organic solvent pretreatment, and biological pretreatment. Additional pretreatment includes ammonia diafiltration, ultrasound, electroporation, microwaves, supercritical CO ₂ Supercritical H ₂ O, ozone, ionic liquids, and gamma radiation pretreatment.

In one embodiment, the cellulose-containing material is pre-treated prior to saccharification (i.e., hydrolysis) and/or fermentation. The pretreatment is preferably carried out before the hydrolysis. Alternatively, the pretreatment may be performed concurrently with enzymatic hydrolysis to release fermentable sugars such as glucose, xylose, and/or cellobiose. In most cases, the pretreatment step itself results in some conversion of the biomass to fermentable sugars (even in the absence of enzymes).

In one embodiment, the cellulose-containing material is pre-treated with steam. In steam pretreatment, the cellulose-containing material is heated to destroy plant cell wall components, including lignin, hemicellulose, and cellulose, so that the cellulose and other fractions (e.g., hemicellulose) are accessible to enzymes. The cellulose-containing material passes through or across a reaction vessel into which steam is injected to increase the temperature to the required temperature and pressure and to maintain the steam therein for the desired reaction time. The steam pretreatment is preferably performed at 140 ℃ -250 ℃ (e.g., 160 ℃ -200 ℃ or 170 ℃ -190 ℃), wherein the optimal temperature range depends on the optional addition of chemical catalyst. The residence time of the steam pretreatment is preferably from 1 to 60 minutes, for example from 1 to 30 minutes, from 1 to 20 minutes, from 3 to 12 minutes, or from 4 to 10 minutes, with the optimum residence time depending on the temperature and the optional addition of chemical catalyst. Steam pretreatment allows for relatively high solids loadings such that the cellulose-containing material generally only becomes wet during pretreatment. Steam pretreatment is often combined with explosive discharge (explosive discharge) of pretreated material, known as steam explosion, i.e., rapid flash to atmospheric pressure and turbulence of the material to increase the accessible surface area by disruption (Duff and Murray,1996,Bioresource Technology [ biological resource technology ]855:1-33; galbe and Zacchi,2002, appl. Microbiol. Biotechnol. [ applied microbiology and biotechnology ]59:618-628; U.S. patent application Ser. No. 2002/0164730). During steam pretreatment, hemicellulose acetyl groups are cleaved and the resulting acid autocatalyzes the partial hydrolysis of hemicellulose into mono-and oligosaccharides. Lignin is removed only to a limited extent.

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 may convert crystalline cellulose to amorphous cellulose. Examples of suitable chemical pretreatment methods include, for example, dilute acid pretreatment, lime pretreatment, wet oxidation, ammonia fiber/freeze blasting (AFEX), ammonia diafiltration (APR), ionic liquids, and organic solvent pretreatment.

Chemical catalysts (e.g. H) are sometimes added prior to steam pretreatment ₂ SO ₄ Or SO ₂ ) (typically 0.3 to 5% w/w) which 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 pretreated with dilute acid (typically H ₂ SO ₄ ) Mixing with water to form a slurry, heating with steam to Desired temperature, and flash to atmospheric pressure after residence time. Dilute acid pretreatment can be performed with a number of reactor designs, for example, plug flow reactors, countercurrent reactors or continuous countercurrent packed bed reactors (Duff and Murray,1996,Bioresource Technology [ biological resource technology]855:1-33; schell et al, 2004,Bioresource Technology [ biological resource technology]91:179-188; lee et al 1999, adv. Biochem. Eng. Biotechnol. [ progress of biochemical engineering/biotechnology ]]65:93-115). In a particular embodiment, the dilute acid pretreatment of the cellulose-containing material is performed at 180 ℃ using 4% w/w sulfuric acid for 5 minutes.

Several pretreatment methods under alkaline conditions may also be used. These alkaline pretreatments include, but are not limited to: sodium hydroxide, lime, wet oxidation, ammonia diafiltration (APR), and ammonia fiber/freeze burst (AFEX) pretreatment. Lime pretreatment with calcium oxide or hydroxide at temperatures of 85℃to 150℃and residence times ranging from 1 hour to several days (Wyman et al, 2005,Bioresource Technology [ Bioresource technologies ]96:1959-1966; mosier et al, 2005,Bioresource Technology [ Bioresource technologies ] 96:673-686). WO 2006/110891, WO 2006/110899, WO 2006/110900 and WO 2006/110901 disclose pretreatment methods using ammonia.

Wet oxidation is a thermal pretreatment that is typically carried out at 180-200 ℃ for 5-15 minutes with the addition of an oxidizing agent (e.g., peroxide or overpressure of oxygen) (Schmidt and Thomsen,1998,Bioresource Technology [ bioresource technology ]64:139-151; palonen et al, 2004, appl. Biochem. Biotechnol. [ applied biochemistry & biotechnology ]117:1-17; varga et al, 2004, biotechnol. Bioeng. [ biotechnology & bioengineering ]88:567-574; martin et al, 2006, j. Chem. Technology. Biotechnol. [ journal of chemical technology & biotechnology ] 81:1669-1677). The pretreatment is preferably performed at 1% -40% dry matter, for example 2% -30% dry matter, or 5% -20% dry matter, and the initial pH is often increased due to the addition of a base such as sodium carbonate.

A modification of the wet oxidation pretreatment method known as wet blasting (combination of wet oxidation and steam explosion) is capable of handling up to 30% of dry matter. In wet blasting, after a certain residence time, an oxidizing agent is introduced during the pretreatment. The pretreatment is then ended by flashing to atmospheric pressure (WO 2006/032682).

The Ammonia Fiber Explosion (AFEX) involves treating a cellulose-containing material with liquid or gaseous ammonia at a mild temperature, such as 90 ℃ to 150 ℃ and at an elevated pressure, such as 17 to 20 bar, for 5 to 10 minutes, wherein the dry matter content can be up to 60% (Gollapalli et al, 2002, appl. Biochem. Biotechnolol. [ applied biochemistry and biotechnology ]98:23-35; chundawat et al, 2007, biotechnol. Bioengineering. [ biotechnology and biotechnology ]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, the cellulose and hemicellulose remain relatively intact. The lignin-carbohydrate complex is cleaved.

Organic solvent pretreatment cellulose-containing material was delignified by extraction with aqueous ethanol (40% -60% ethanol) at 160 ℃ -200 ℃ for 30-60 minutes (Pan et al 2005, biotechnol. Bioeng [ biotech & bioengineering ]90:473-481; pan et al 2006, biotechnol. Bioeng. [ biotech & bioengineering ]94:851-861; kurabi et al 2005, appl. Biochem. Biotechnol. [ applied biochemistry & biotechnology ] 121:219-230). Sulfuric acid is typically added as a catalyst. In the organic solvent pretreatment, most of hemicellulose and lignin are removed.

Other examples of suitable pretreatment methods are described by Schell et al, 2003, appl. Biochem. Biotechnol. Applied biochemistry 105-108:69-85, and Mosier et al, 2005,Bioresource Technology [ Bioresource technology ]96:673-686, and US 2002/0164730.

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 may be used, such as acetic acid, citric acid, nitric acid, phosphoric acid, tartaric acid, succinic acid, hydrogen chloride, or mixtures thereof. 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 from 0.01wt.% to 10wt.% acid, for example 0.05wt.% to 5wt.% acid or 0.1wt.% to 2wt.% acid. The acid is contacted with the cellulose-containing material and maintained at a temperature preferably in the range 140 ℃ -200 ℃ (e.g. 165 ℃ -190 ℃) for a time in the range from 1 to 60 minutes.

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

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

The cellulose-containing material may be physically (mechanically) and chemically pretreated. The mechanical or physical pretreatment may be combined with steam/steam explosion, hydropyrolysis (hydropyrolysis), dilute acid or weak acid treatment, high temperature, high pressure treatment, radiation (e.g., microwave radiation), or combinations thereof. In one embodiment, high pressure means a pressure in the range of preferably about 100 to about 400psi, such as about 150 to about 250 psi. In another embodiment, the high Wen Yizhi temperature is in the range of about 100 ℃ to about 300 ℃, such as about 140 ℃ to about 200 ℃. In a preferred embodiment, the mechanical or physical pretreatment is performed in a batch process using a steam gun Hydrolyzer system, such as a cisco Hydrolyzer (underwriter) available from cisco (Sunds Defibrator AB), sweden, using high pressure and high temperature as defined above. Physical and chemical pretreatment may be performed sequentially or simultaneously, as needed.

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

In one embodiment, the cellulose-containing material is subjected to a biological pretreatment. The term "biological pretreatment" refers to any biological pretreatment that facilitates 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-solubilizing microorganisms and/or enzymes (see, e.g., hsu, t.—a.,1996,Pretreatment of biomass [ pretreatment of biomass ], in Handbook on Bioethanol: production and Utilization [ handbook of bioethanol: production and utilization ], wyman, c.e. editions, taylor & Francis [ Taylor-franciss publishing group ], washington ad hoc, DC,179-212; ghosh and Singh,1993, adv. Appl. Microbiol. [ application microbiology progress ]39:295-333; mcmillan, j.d.,1994,Pretreating lignocellulosic biomass:a review [ pretreatment of lignocellulosic biomass: in Enzymatic Conversion of Biomass for Fuels Production [ enzymatic conversion of biomass for fuel production ], himmel, m.e., baker, j.o. and overtend, r.p. editions, ACS Symposium Series [ the american society of chemistry series ]566,American Chemical Society [ american society ], washington ad hoc, 15; gong, C.S., cao, N.J., du, J., and Tsao, G.T.,1999,Ethanol production from renewable resources [ ethanol production from renewable resources ], scheper, T.editions, springer-Verlag [ Schpringer publishing company ], berlin, heideburg, 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. [ Biotechnology ] 42:63-95).

Saccharification and fermentation of cellulose-containing materials

Separate or simultaneous saccharification (i.e., hydrolysis) and fermentation includes, but is not limited to: separate Hydrolysis and Fermentation (SHF); simultaneous Saccharification and Fermentation (SSF); simultaneous saccharification and co-fermentation (SSCF); mixed hydrolysis and fermentation (HHF); isolated hydrolysis and co-fermentation (SHCF); hybrid hydrolysis and co-fermentation (HHCF).

SHF uses separate processing steps to first enzymatically hydrolyze the cellulose-containing material to fermentable sugars (e.g., glucose, cellobiose, and pentose monomers), and then ferment the fermentable sugars to ethanol. In SSF, the enzymatic hydrolysis of the cellulose-containing material and the fermentation of sugar into ethanol are combined in one step (Philippidis, g.p.,1996,Cellulose bioconversion technology [ cellulose bioconversion technology ], wyman, c.e. editions, taylor & Francis [ Taylor-franciss publishing group ], washington ad hoc, DC,179-212 in Handbook on Bioethanol: production and Utilization [ bioethanol handbook: production and utilization ]. SSCF involves co-fermentation of a variety of sugars (Seehan and Himmel,1999, biotechnol. Prog. [ Biotechnology progress ] 15:817-827). HHF involves a separate hydrolysis step and additionally involves simultaneous saccharification and hydrolysis steps, which may be performed in the same reactor. The steps in the HHF process can be performed at different temperatures, i.e. high temperature enzymatic saccharification, followed by SSF at lower temperatures tolerated by the fermenting organism. It is to be understood herein that any method known in the art comprising pretreatment, enzymatic hydrolysis (saccharification), fermentation, or combinations thereof, may be used to implement the methods described herein.

Conventional apparatus may include fed batch stirred reactors, continuous flow stirred reactors with ultrafiltration, and/or continuous plug flow column reactors (de Castilhos Corazza et al 2003,Acta Scientiarum.Technology [ technical journal ]25:33-38; gusakov and Sinitsyn,1985, enz. Microb. Technology [ enzyme and microorganism technology ] 7:346-352), attrition reactors (Ryu and Lee,1983, biotechnol. Bioeng. [ biotechnology and bioengineering ] 25:53-65). Additional reactor types include: fluidized bed, upflow blancet, immobilized, and extruder type reactors for hydrolysis and/or fermentation.

In the saccharification step (i.e., 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 facilitated by enzymes such as cellulolytic enzyme compositions. The enzymes of these compositions may be added simultaneously or sequentially.

The enzymatic hydrolysis may be carried out in a suitable aqueous environment under conditions readily determinable by one skilled in the art. In one embodiment, the hydrolysis is performed under conditions suitable for the activity of the one or more enzymes, i.e. optimal conditions for the one or more enzymes. The hydrolysis can be performed as a fed batch or continuous process, wherein the cellulose-containing material and/or starch-containing material is fed gradually, e.g. into a hydrolysis solution containing enzymes.

Saccharification is typically carried out in a stirred tank reactor or fermenter under controlled pH, temperature, and mixing conditions. Suitable treatment times, temperatures and pH conditions can be readily determined by one skilled in the art. For example, saccharification may last up to 200 hours, but is typically carried out preferably for about 12 to about 120 hours, e.g., about 16 to about 72 hours or about 24 to about 48 hours. The temperature is preferably in the range of about 25 ℃ to about 70 ℃, such as about 30 ℃ to about 65 ℃, about 40 ℃ to about 60 ℃, or about 50 ℃ to 55 ℃. The 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 from about 5wt.% to about 50wt.%, e.g., from about 10wt.% to about 40wt.%, or from about 20wt.% to about 30wt.%.

Saccharification may be performed using a cellulolytic enzyme composition. Such enzyme compositions are described in the "cellulolytic enzyme compositions" section below. The cellulolytic enzyme compositions may comprise any protein useful for degrading the cellulose-containing material. In one embodiment, the cellulolytic enzyme composition comprises or further comprises one or more (e.g., several) proteins selected from the group consisting of: cellulases, AA9 (GH 61) polypeptides, hemicellulases, esterases, expansins, lignin-degrading enzymes, oxidoreductases, pectinases, proteases, and swollenins.

In another embodiment, the cellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of: endoglucanases, cellobiohydrolases, and beta-glucosidase.

In another embodiment, the hemicellulase is preferably one or more (e.g., several) enzymes selected from the group consisting of: acetyl mannase, acetyl xylan esterase, arabinanase, arabinofuranosidase, coumarase, feruloyl esterase, galactosidase, 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.

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

In one embodiment, the effective amount of the cellulolytic enzyme composition or hemicellulose cellulolytic enzyme composition to the cellulose-containing material is about 0.5mg to about 50mg, e.g., about 0.5mg to about 40mg, about 0.5mg to about 25mg, about 0.75mg to about 20mg, about 0.75mg to about 15mg, about 0.5mg to about 10mg, or about 2.5mg to about 10mg/g of the cellulose-containing material.

In one embodiment, the compound is added in the following molar ratio of such a compound to glucosyl units of cellulose: about 10 ^-6 To about 10, e.g. about 10 ^-6 To about 7.5, about 10 ^-6 To about 5, about 10 ^-6 To about 2.5, about 10 ^-6 To about 1, about 10 ^-5 To about 1, about 10 ^-5 To about 10 ^-1 About 10 ^-4 To about 10 ^-1 About 10 ^-3 To about 10 ^-1 Or about 10 ^-3 To about 10 ^-2 . In another embodiment, such a combinationAn effective amount of the agent is about 0.1. Mu.M to about 1M, for example about 0.5. Mu.M to about 0.75M, about 0.75. Mu.M to about 0.5M, about 1. Mu.M to about 0.25M, about 1. Mu.M to about 0.1M, about 5. Mu.M to about 50mM, about 10. Mu.M to about 25mM, about 50. Mu.M to about 25mM, about 10. Mu.M to about 10mM, about 5. Mu.M to about 5mM, or about 0.1mM to about 1mM.

The term "liquid (liquor)" means the solution phase (aqueous phase, organic phase or combination thereof) and its soluble content resulting from the treatment of lignocellulosic and/or hemicellulose material, or monosaccharides thereof (e.g. xylose, arabinose, mannose, etc.) in a slurry under the conditions as described in WO 2012/021401. The liquid used to enhance cellulolytic decomposition of the AA9 polypeptide (GH 61 polypeptide) may be produced by treating a lignocellulosic or hemicellulose material (or feedstock) 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 disruption of the material, and then separating the solution from the residual solids. The extent to which cellulolytic enhancement is obtainable from a combination of liquid and AA9 polypeptide during hydrolysis of a cellulosic substrate by a cellulolytic enzyme preparation is determined by such conditions. The liquid may be separated from the treated material using methods standard in the art, such as filtration, precipitation or centrifugation.

In one embodiment, the effective amount of liquid for cellulose is about 10 ^-6 To about 10g/g cellulose, e.g. about 10 ^-6 To about 7.5g, about 10 ^-6 To about 5g, about 10 ^-6 To about 2.5g, about 10 ^-6 To about 1g, about 10 ^-5 To about 1g, about 10 ^-5 To about 10 ^-1 g. About 10 ^-4 To about 10 ^-1 g. About 10 ^-3 To about 10 ^-1 g. Or about 10 ^-3 To about 10 ^-2 g/g cellulose.

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

Any suitable hydrolyzed cellulose-containing material may be used in the fermentation step in which the methods described herein are performed. Such materials include, but are not limited to, carbohydrates (e.g., lignocellulose, xylan, cellulose, starch, etc.). The materials are typically selected based on economics, i.e., cost per unit of carbohydrate potential, and the difficulty in degrading the enzyme-induced transition.

Ethanol production by fermenting organisms using cellulose-containing materials results from the metabolism of sugars (monosaccharides). The sugar composition of the hydrolyzed cellulose-containing material and the ability of the fermenting organism to utilize different sugars have a direct impact on the process yield.

The composition of the fermentation medium and the fermentation conditions depend on the fermenting organism and can be readily determined by a person 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 performed under aerobic or microaerophilic conditions (i.e., oxygen concentration less than that in air) or anaerobic conditions. In some embodiments, fermentation is performed under anaerobic conditions (i.e., no detectable oxygen) or in less than about 5, about 2.5, or about 1mmol/L/h of oxygen. In the absence of oxygen, NADH produced in glycolysis cannot be oxidized by oxidative phosphorylation. Under anaerobic conditions, the fermenting organism can utilize pyruvic acid or a derivative thereof as an electron and hydrogen acceptor to produce nad+.

The fermentation process is typically carried out at a temperature optimal for recombinant fungal cells. For example, in some embodiments, the fermentation process is conducted at a temperature in the range of about 25 ℃ to about 42 ℃. Typically, the process is conducted at a temperature of less than about 38 ℃, less than about 35 ℃, less than about 33 ℃, or less than about 38 ℃, but at least about 20 ℃, 22 ℃, or 25 ℃.

Fermentation stimulators may be used in the processes described herein to further improve fermentation, and in particular to improve performance of the fermenting organism, such as rate increase and product yield (e.g., ethanol yield). "fermentation stimulator" refers to a stimulator for the growth of fermenting organisms, particularly yeast. Preferred fermentation stimulators for growth include vitamins and minerals. Examples of vitamins include multivitamins, biotin, pantothenic acid, niacin, myo-inositol, thiamine, pyridoxine, para-amino benzoic acid, folic acid, riboflavin, and vitamins A, B, C, D and E. See, for example, alfenore et al, improving ethanol production and viability of Saccharomyces cerevisia by a vitamin feeding strategy during fed-batch process [ improving ethanol production and Saccharomyces cerevisiae viability by a vitamin feeding strategy during a fed-batch process ], springer-Verlag [ Schpraringer Press ] (2002), which is hereby incorporated by reference. Examples of minerals include minerals and mineral salts that can be supplied to contain P, K, mg, S, ca, fe, zn, mn and Cu nutrients.

Cellulolytic enzymes and compositions

Cellulolytic enzymes or cellulolytic enzyme compositions may be present and/or added during saccharification. Cellulolytic enzyme compositions are enzyme preparations that comprise one or more (e.g., several) enzymes that hydrolyze cellulose-containing material. Such enzymes include endoglucanases, cellobiohydrolases, beta-glucosidase, and/or combinations thereof.

In some embodiments, the fermenting organism comprises one or more (e.g., several) heterologous polynucleotides encoding an enzyme (e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, or a combination thereof) that hydrolyzes cellulose-containing material. Any enzyme (hydrolyzable cellulose-containing material) described or referenced herein is contemplated for expression in a fermenting organism.

The cellulolytic enzyme may be any cellulolytic enzyme suitable for expression in a fermenting organism and/or method described herein (e.g., endoglucanase, cellobiohydrolase, beta-glucosidase), such as a naturally-occurring cellulolytic enzyme or a variant thereof that retains cellulolytic enzyme activity.

In some embodiments, a fermenting organism comprising a heterologous polynucleotide encoding a cellulolytic enzyme has an increased level of cellulolytic enzyme (e.g., increased endoglucanase, cellobiohydrolase, and/or beta-glucosidase) activity when cultured under the same conditions as a fermenting organism without the heterologous polynucleotide encoding a cellulolytic enzyme. In some embodiments, the fermenting organism has a level of cellulolytic enzyme activity that is increased by at least 5%, e.g., at least 10%, at least 15%, at least 20%, at least 25%, at least 50%, at least 100%, at least 150%, at least 200%, at least 300%, or at least 500%, compared to a fermenting organism that does not have a heterologous polynucleotide encoding a cellulolytic enzyme when cultured under the same conditions.

Exemplary cellulolytic enzymes that may be used with the fermenting organisms and/or methods described herein include bacterial, yeast, or filamentous fungal cellulolytic enzymes, e.g., obtained from any microorganism described or referenced herein, as described above in the section relating to proteases.

The cellulolytic enzyme may be of any origin. In one embodiment, the cellulolytic enzyme is derived from a strain of trichoderma, such as a strain of trichoderma reesei; strains of the genus Humicola, such as the strain of Humicola insolens, and/or strains of the genus Chrysosporium, such as the strain of Chrysosporium ovale Lu Kenuo. In a preferred embodiment, the cellulolytic enzyme is derived from a strain of Trichoderma reesei.

The cellulolytic enzyme composition may further comprise one or more of the following polypeptides (e.g., enzymes): AA9 polypeptides (GH 61 polypeptides), β -glucosidase, xylanase, β -xylosidase, CBH I, CBH II, or mixtures of two, three, four, five or six thereof having cellulolytic enhancing activity.

The additional one or more polypeptides (e.g., AA9 polypeptides) and/or one or more enzymes (e.g., β -glucosidase, xylanase, β -xylosidase, CBH I, and/or CBH II) may be exogenous to the cellulolytic enzyme composition-producing organism (e.g., trichoderma reesei).

In one embodiment, the cellulolytic enzyme composition comprises an AA9 polypeptide having cellulolytic enhancing activity and a beta-glucosidase.

In another embodiment, the cellulolytic enzyme composition comprises an AA9 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, and CBH I.

In another embodiment, the cellulolytic enzyme composition comprises an AA9 polypeptide having cellulolytic enhancing activity, a beta-glucosidase, CBH I, and CBH II.

Other enzymes (e.g., endoglucanases) may also be included in the cellulolytic enzyme composition.

As mentioned above, the cellulolytic enzyme composition may comprise a variety of different polypeptides, including enzymes.

In one embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition further comprising a thermoascus orange AA9 (GH 61A) polypeptide having cellulolytic enhancing activity (e.g., WO 2005/074656), and an aspergillus oryzae beta-glucosidase fusion protein (e.g., one disclosed in WO 2008/057637, particularly as shown in SEQ ID NOs: 59 and 60).

In another embodiment, the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic enzyme composition that further comprises an Thermoascus aurantiacus AA9 (GH 61A) polypeptide having cellulolytic enhancing activity (e.g., SEQ ID NO:2 of WO 2005/074656) and Aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO:2 of WO 2005/047499).

In another embodiment, the cellulolytic enzyme composition is a Trichoderma reesei cellulolytic enzyme composition further comprising an Penicillium emerald AA9 (GH 61A) polypeptide having cellulolytic enhancing activity, particularly one of those disclosed in WO 2011/0410197, and Aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO:2 of WO 2005/047499).

In another embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition further comprising an eimeria amoena AA9 (GH 61A) polypeptide having cellulolytic enhancing activity, in particular one of the polypeptides disclosed in WO 2011/0410197, and aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO:2 of WO 2005/047499), or a variant disclosed in WO 2012/044915 (incorporated herein by reference), in particular a variant comprising one or more (e.g., all) of the following substitutions: f100D, S283G, N456E, F512Y.

In one embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic composition further comprising an AA9 (GH 61A) polypeptide having cellulolytic enhancing activity, especially one derived from a strain of penicillium emersonii (e.g., SEQ ID NO:2 in WO 2011/0410197)), a variant of aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO:2 in WO 2005/047499), the variant having one or more (especially all) of the following substitutions: f100D, S283G, N456E, F512Y and is disclosed in WO 2012/044915; aspergillus fumigatus Cel7A CBH1, for example one disclosed as SEQ ID NO:6 in WO 2011/057140 and Aspergillus fumigatus CBH II, for example one disclosed as SEQ ID NO:18 in WO 2011/057140.

In a preferred embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition further comprising a hemicellulase or a hemicellulolytic enzyme composition, such as aspergillus fumigatus xylanase and aspergillus fumigatus β -xylosidase.

In one embodiment, the cellulolytic enzyme composition further comprises a xylanase (e.g., a strain derived from Aspergillus, especially Aspergillus aculeatus or Aspergillus fumigatus; or a strain of Penicillium, especially Lei Saishi Penicillium) and/or a beta-xylosidase (e.g., a strain derived from Aspergillus, especially Aspergillus fumigatus, or Penicillium, especially Emersen Penicillium (Talaromyces emersonii)).

In one embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition further comprising a thermoascus AA9 (GH 61A) polypeptide having cellulolytic enhancing activity (e.g., WO 2005/074656), an aspergillus oryzae beta-glucosidase fusion protein (e.g., one of those disclosed in WO 2008/057637, particularly as set forth in SEQ ID NOs 59 and 60), and an aspergillus aculeatus xylanase (e.g., xyl II in WO 94/21785).

In another embodiment, the cellulolytic enzyme composition comprises a trichoderma reesei cellulolytic preparation further comprising an orange thermophilic ascomycete GH61A polypeptide having cellulolytic enhancing activity (e.g., SEQ ID NO:2 in WO 2005/074656), aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO:2 in WO 2005/047499), and aspergillus aculeatus xylanase (Xyl II disclosed in WO 94/21785).

In another embodiment, the cellulolytic enzyme composition comprises a Trichoderma reesei cellulolytic enzyme composition further comprising a Thermoascus aurantiacus AA9 (GH 61A) polypeptide having cellulolytic enhancing activity (e.g., SEQ ID NO:2 of WO 2005/074656), aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO:2 of WO 2005/047499), and Aspergillus aculeatus xylanase (e.g., xyl II disclosed in WO 94/21785).

In another embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition further comprising an emerald AA9 (GH 61A) polypeptide having cellulolytic enhancing activity (particularly one of those disclosed in WO 2011/0410197), aspergillus fumigatus beta-glucosidase (e.g., SEQ ID NO:2 of WO 2005/047499), and aspergillus fumigatus xylanase (e.g., xyl III of WO 2006/078256).

In another embodiment, the cellulolytic enzyme composition comprises a trichoderma reesei cellulolytic enzyme composition further comprising an emerald AA9 (GH 61A) polypeptide having cellulolytic enhancing activity, particularly one of the polypeptides disclosed in WO 2011/0410197, aspergillus fumigatus beta-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.

In another embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition further comprising an eimeria reesei AA9 (GH 61A) polypeptide having cellulolytic enhancing activity, particularly one disclosed in WO 2011/0410197, aspergillus fumigatus beta-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 Cel7A CBH1 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.

In another embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition further comprising an emerald AA9 (GH 61A) polypeptide having cellulolytic enhancing activity (particularly one disclosed in WO 2011/0410197), aspergillus fumigatus beta-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., xylIII in WO 2006/078256), CBH I from Aspergillus fumigatus (especially Cel7A CBH I disclosed as SEQ ID NO:2 in WO 2011/057140), and CBH II derived from Aspergillus fumigatus (especially one disclosed in WO 2013/028928).

In another embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition comprising CBH I (genreq ep accession No. AZY49536 (WO 2012/103293)); CBH II (genreq p accession number AZY49446 (WO 2012/103288)); beta-glucosidase variant (genreq ep accession AZU67153 (WO 2012/44915)), in particular with one or more (in particular all) of the following substitutions: f100D, S283G, N456E, F512Y; AA9 (GH 61 polypeptide) (genreq qp accession No. BAL61510 (WO 2013/028912)).

In another embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition comprising CBH I (genreq ep accession No. AZY49536 (WO 2012/103293)); CBH II (genreq p accession number AZY49446 (WO 2012/103288)); GH10 xylanase (GENSEQP accession number BAK46118 (WO 2013/019827)); beta-xylosidase (genreq qp accession No. AZI04896 (WO 2011/057140)).

In another embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition comprising CBH I (genreq ep accession No. AZY49536 (WO 2012/103293)); CBH II (genreq p accession number AZY49446 (WO 2012/103288)); AA9 (GH 61 polypeptide; genreq qp accession No. BAL61510 (WO 2013/028912)).

In another embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition comprising CBH I (genreq ep accession No. AZY49536 (WO 2012/103293)); CBH II (gensamqp accession number AZY49446 (WO 2012/103288)), AA9 (GH 61 polypeptide; gensamqp accession number BAL61510 (WO 2013/028912)), and catalase (gensampp accession number BAC11005 (WO 2012/130120)).

In one embodiment, the cellulolytic enzyme composition is a trichoderma reesei cellulolytic enzyme composition comprising CBH I (genreq p accession No. AZY49446 (WO 2012/103288)), CBH II (genreq p accession No. AZY49446 (WO 2012/103288)), β -glucosidase variant (genreq ep accession No. AZU67153 (WO 2012/44915)), with one or more (especially all) of the following substitutions F100D, S283G, N E, F Y, AA9 (GH 61 polypeptide; genreq ep accession No. BAL61510 (WO 2013/028912)), GH10 xylanase (genreq ep accession No. BAK46118 (WO 2013/019827)), and β -xylosidase (gensp accession No. AZI04896 (WO 2011/057140)).

In one embodiment, the cellulolytic composition is a trichoderma reesei cellulolytic enzyme preparation comprising EG I (Swissprot accession No. P07981), EG II (EMBL accession No. M19373), CBH I (see above); CBH II (see above); beta-glucosidase variants with the following substitutions (see above): f100D, S283G, N456E, F512Y; AA9 (GH 61 polypeptides; see above), GH10 xylanases (see above); and beta-xylosidase (see above).

All cellulolytic enzyme compositions disclosed in WO 2013/028928 are also contemplated and hereby incorporated by reference.

The cellulolytic enzyme composition comprises or may further comprise one or more (several) proteins selected from the group consisting of: cellulases, AA9 (i.e., GH 61) polypeptides having cellulolytic enhancing activity, hemicellulases, expansins, esterases, laccases, lipolytic enzymes, pectinases, peroxidases, proteases, and swollenins.

In one embodiment, the cellulolytic enzyme composition is a commercial cellulolytic enzyme composition. Examples of commercial cellulolytic enzyme compositions suitable for use in the methods of the invention include:CTec (novelin company),CTec2 (novelin corporation), ->CTec3 (novelin corporation), cellucast ^TM (Norwechat Co.) SPEZYME ^TM CP (Genencor int) ACCELLERASE, jenkinidae international company ^TM 1000、ACCELLERASE 1500、ACCELLERASE ^TM TRIO (DuPont)),>NL (dieschmann corporation (DSM));S/L100 (Dissman Co., ltd.), ROHAMENT ^TM 7069W (Rohm company ()>GmbH)), or->CMAX3 ^TM (union international company (Dyadic International, inc.). May be present in an amount of from about 0.001wt.% to about 5.0wt.% solids, for example, about 0.025wt.% to about 4.0wt.% solids, or aboutAn effective amount of 0.005wt.% to about 2.0wt.% solids is added to the cellulolytic enzyme composition.

Additional enzymes and compositions thereof can be found in WO 2011/153516 and WO 2016/045569 (the contents of which are incorporated herein).

Additional polynucleotides encoding suitable cellulolytic enzymes may be obtained from microorganisms of any genus, including those readily available in the UniProtKB database (www.uniprot.org).

These cellulolytic enzyme coding sequences may also be used to design nucleic acid probes to identify and clone DNA encoding cellulolytic enzymes from different genus or species of strains known in the art.

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

Techniques for isolating or cloning polynucleotides encoding cellulolytic enzymes are known in the art.

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

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

In one embodiment, the cellulolytic enzyme coding sequence hybridizes under at least low stringency conditions, e.g., medium stringency conditions, medium-high stringency conditions, or very high stringency conditions, with the full-length complement of the coding sequence from any cellulolytic enzyme described or referenced herein (e.g., any endoglucanase, cellobiohydrolase, or β -glucosidase). In one embodiment, the cellulolytic enzyme coding sequence has at least 65%, e.g., 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 to the coding sequence of any cellulolytic enzyme described or referenced herein.

In one embodiment, the polynucleotide encoding the cellulolytic enzyme comprises the coding sequence of any of the cellulolytic enzymes described or referenced herein (e.g., any endoglucanase, cellobiohydrolase, or beta-glucosidase). In one embodiment, the polynucleotide encoding the cellulolytic enzyme comprises a subsequence from the coding sequence of any of the cellulolytic enzymes described or referred to herein, wherein the subsequence encodes a polypeptide having cellulolytic enzyme 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.

The cellulolytic enzyme may also comprise a fusion polypeptide or a cleavable fusion polypeptide.

Method of using starch-containing material

In some embodiments, the methods described herein produce a fermentation product from starch-containing material. Starch-containing materials are well known in the art and contain two types of homopolysaccharides (amylose and amylopectin) and are linked by alpha- (1-4) -D-glycosidic linkages. Any suitable starch-containing starting material may be used. The starting materials are generally selected based on the desired fermentation product (e.g., ethanol). Examples of starch-containing starting materials include cereals, tubers or grains. In particular, the starch-containing material may be corn, wheat, barley, rye, western sorghum (milo), sago, tapioca (casstra), tapioca (tapioca), sorghum, oat, rice, pea, legume, or sweet potato, or mixtures thereof. Waxy (waxy type) and non-waxy (non-waxy type) corn and barley are also contemplated.

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 western african sorghum. In one embodiment, the starch-containing starting material is sago. In one embodiment, the starch-containing starting material is tapioca. In one embodiment, the starch-containing starting material is tapioca 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 pea. 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 oat.

The method of using the starch-containing material may include conventional methods (e.g., including a liquefaction step described in more detail below) or a crude starch hydrolysis method. In some embodiments using starch-containing material, saccharification of the starch-containing material occurs at a temperature above the initial gelatinization temperature. In some embodiments using starch-containing material, saccharification of the starch-containing material occurs at a temperature below the initial gelatinization temperature.

Liquefaction process

In embodiments using starch-containing material, the methods may further comprise a liquefaction step by subjecting the starch-containing material to an alpha-amylase and optionally a protease and/or glucoamylase at a temperature above the initial gelatinization temperature. Other enzymes such as pullulanase and phytase may also be present and/or added to the liquefaction. In some embodiments, the liquefaction step is performed before steps a) and b) of the method.

The liquefaction step may be carried out for 0.5 to 5 hours, such as 1 to 3 hours, such as typically about 2 hours.

The term "initial gelatinization temperature" means the lowest temperature at which gelatinization of the starch-containing material begins. Typically, starch heated in water begins to gelatinize between about 50 ℃ and 75 ℃; the exact temperature of gelatinization depends on the specific starch and can be readily determined by one skilled in the art. Thus, the initial gelatinization temperature may vary depending on the plant species, the particular variety of plant species, and the growth conditions. The initial gelatinization temperature of a given starch-containing material may be that set forth in the application of Gorinstein and Lii,1992,[ starch ]]44 (12) temperature at which 5% of the starch particles lose birefringence, as determined by the method described in 461-466.

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

The jet cooking step may be performed prior to the liquefaction step, for example, at a temperature of between 110 ℃ and 145 ℃, 120 ℃ and 140 ℃, 125 ℃ and 135 ℃, or about 130 ℃ for about 1 to 15 minutes, about 3 to 10 minutes, or about 5 minutes.

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

In one embodiment, the method further comprises the steps of, prior to liquefying:

i) Reducing the particle size of the starch-containing material, preferably by dry milling;

ii) forming a slurry comprising the starch-containing material and water.

The starch-containing starting material (e.g., whole grain) may be reduced in particle size, for example, by milling, to open the structure, increase surface area, and allow for further processing. There are generally two types of methods: wet milling and dry milling. In dry milling, whole grains are milled and used. Wet milling provides good separation of the germ from the meal (starch granules and protein). Wet milling is often used in applications where starch hydrolysates are used to produce, for example, syrups (location). Both dry and wet milling are well known in the starch processing arts. 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 3.0mm, such as 0.1-0.5mm, or at least 30%, at least 50%, at least 70%, or at least 90% of the starch-containing material is adapted to pass through a screen having a 0.05 to 3.0mm screen, such as 0.1-0.5mm screen. In another embodiment, at least 50%, such as at least 70%, at least 80%, or at least 90% of the starch-containing material is suitable for passing through a screen having a #6 screen.

The aqueous slurry may comprise from 10-55w/w-% Dry Solids (DS), e.g. 25-45w/w-% Dry Solids (DS), or 30-40w/w-% Dry Solids (DS) of starch-containing material.

Initially, an alpha-amylase, optionally a protease and optionally a glucoamylase may be added to the aqueous slurry to begin liquefaction (thinning). In one embodiment, only a portion (e.g., about 1/3) of the enzymes are added to the aqueous slurry, while the remaining portion (e.g., about 2/3) of the enzymes are added during the liquefaction step.

Alpha-amylase and glucoamylase for liquefaction may be found in the art, for example in WO 2020/0234411 (the contents of which are incorporated herein by reference). Similarly, examples of suitable proteases for liquefaction may be found in the art, for example in WO 2018/222990 (the contents of which are incorporated herein by reference).

Saccharification and fermentation of starch-containing material

In embodiments where starch-containing material is used, glucoamylase may be present and/or added in saccharification step a) and/or fermentation step b) or Simultaneous Saccharification and Fermentation (SSF). The glucoamylase of saccharification step a) and/or fermentation step b) or Simultaneous Saccharification and Fermentation (SSF) is typically different from the glucoamylase optionally added in any of the liquefaction steps described above. In one embodiment, the glucoamylase is present and/or added with a fungal alpha-amylase. Suitable glucoamylases for saccharification or SSF can be found in the art, for example in WO 2020/0234411 (the contents of which are incorporated herein by reference).

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

Fermentation is performed in a fermentation medium as known in the art and, for example, as described herein. The fermentation medium comprises a fermentation substrate, i.e. a carbohydrate source that is metabolized by the fermenting organism. Using the methods described herein, the fermentation medium can comprise nutrients for one or more fermenting organisms and one or more growth stimulators. Nutrients and growth stimulators are widely used in the fermentation field and include nitrogen sources, such as ammonia; urea, vitamins and minerals or combinations thereof.

In general, fermenting organisms such as yeast (including Saccharomyces cerevisiae) require a sufficient nitrogen source for proliferation and fermentation. Many supplemental nitrogen sources can be used if necessary and are well known in the art. The nitrogen source may be an organic nitrogen source (such as urea, DDG, wet cake or corn mash) or an inorganic nitrogen source (such as ammonia or ammonium hydroxide). In one embodiment, the nitrogen source is urea.

The fermentation may be performed under low nitrogen conditions, for example when using a yeast expressing the protease. In some embodiments, the fermentation step is performed under the following conditions: less than 1000ppm supplemental nitrogen (e.g., urea or ammonium hydroxide), such as less than 750ppm, less than 500ppm, less than 400ppm, less than 300ppm, less than 250ppm, less than 200ppm, less than 150ppm, less than 100ppm, less than 75ppm, less than 50ppm, less than 25ppm, or less than 10ppm supplemental nitrogen. In some embodiments, the fermentation step is performed without supplementation of nitrogen.

Simultaneous saccharification and fermentation ("SSF") is widely used in industrial-scale fermentation product production processes, particularly ethanol production processes. When SSF is performed, saccharification step a) and fermentation step b) are performed simultaneously. The absence of a holding stage for saccharification means that the fermenting organism (e.g., yeast) can be added along with one or more enzymes. However, separate addition of fermenting organisms and one or more enzymes is also contemplated. SSF is typically performed at a temperature of from 25 ℃ to 40 ℃, such as from 28 ℃ to 35 ℃, such as from 30 ℃ to 34 ℃, or about 32 ℃. In one embodiment, the fermentation is carried out for 6 hours to 120 hours, especially 24 hours to 96 hours. In one embodiment, the pH is between 4 and 5.

In one embodiment, the cellulolytic enzyme composition is present and/or added in saccharification, fermentation, or Simultaneous Saccharification and Fermentation (SSF). Examples of such cellulolytic enzyme compositions may be found in the "cellulolytic enzymes and compositions" section. The cellulolytic enzyme composition may be present and/or added with a glucoamylase, as disclosed in the "glucoamylase" section.

Fermentation product

The fermentation product may be any material resulting from fermentation. The fermentation product may be, but is not limited to: alcohols (e.g., arabitol, 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); amino acids (e.g., aspartic acid, glutamic acid, glycine, lysine, serine, and threonine); gas (e.g., methane, hydrogen (H) ₂ ) Carbon dioxide (CO) ₂ ) And carbon monoxide (CO)); isoprene; ketones (e.g., acetone); organic acids (e.g., acetic acid, acetonic acid, adipic acid, ascorbic acid, citric acid, 2, 5-dione-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, oxaloacetic acid, propionic acid, succinic acid, and xylitol acid); and polyketides.

In one embodiment, the fermentation product is an alcohol. The term "alcohol" encompasses materials containing one or more hydroxyl moieties. The alcohol may be, but is not limited to: n-butanol, isobutanol, ethanol, methanol, arabitol, butanediol, ethylene glycol, glycerol, 1, 3-propanediol, sorbitol, xylitol. See, e.g., gong et al, 1999,Ethanol production from renewable resources [ ethanol production from renewable resources ], scheper, t., editions, springer-Verlag Berlin Heidelberg, germany [ septoria berlin heidburg, germany ],65:207-241 in Advances in Biochemical Engineering/Biotechnology [ biochemical engineering/Biotechnology progression ]; silveira and Jonas,2002, appl. Microbiol. Biotechnol. [ applied microbiology and Biotechnology ]59:400-408; nigam and Singh,1995,Process Biochemistry [ biochemistry method ]30 (2): 117-124; ezeji et al, 2003,World Journal of Microbiology and Biotechnology J.Wolmicrobiology and Biotechnology 19 (6): 595-603. In one embodiment, the fermentation product is ethanol.

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

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

In another embodiment, the fermentation product is an olefin. The olefins may be unbranched or branched olefins. The olefins may be, but are not limited to: pentene, hexene, heptene or octene.

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, e.g., richard and Margaritis,2004,Biotechnology and Bioengineering [ Biotechnology and bioengineering ]87 (4): 501-515.

In another embodiment, the fermentation product is a gas. The gas may be, but is not limited to: methane, H ₂ 、CO ₂ Or CO. See, e.g., kataoka et al, 1997,Water Science and Technology [ Water science and technology]36 (6-7) 41-47; gunaseelan,1997,Biomass and Bioenergy [ Biomass and bioenergy ]]13(1-2):83-114。

In another embodiment, the fermentation product is isoprene.

In another embodiment, the fermentation product is a ketone. The term "ketone" encompasses materials containing one or more ketone moieties. The ketone may be, but is not limited to: acetone.

In another embodiment, the fermentation product is an organic acid. The organic acid may be, but is not limited to: acetic acid, adipic acid, ascorbic acid, citric acid, 2, 5-dione-D-gluconic acid, formic acid, fumaric acid, glucaric acid, gluconic acid, glucuronic acid, glutaric acid, 3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid, malonic acid, oxalic acid, propionic acid, succinic acid, or xylonic acid. See, e.g., chen and Lee,1997, appl. Biochem. Biotechnol. [ applied biochemistry and biotechnology ]63-65:435-448.

In another embodiment, the fermentation product is a polyketide.

Recovery of

The fermentation product (e.g., ethanol) may optionally be recovered from the fermentation medium using any method known in the art, including but not limited to: chromatography, electrophoresis procedure, differential solubility, distillation or extraction. For example, alcohols are separated and purified from fermented cellulosic material by conventional distillation methods. Ethanol may be obtained in a purity of up to about 96vol.%, which can be used, for example, as fuel ethanol, potable ethanol (i.e., drinkable neutral alcoholic beverages), or industrial ethanol.

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

Suitable assays may be performed to test the production of ethanol and contaminants and for sugar consumption using methods known in the art. For example, the ethanol product, as well as other organic compounds, may 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 fermentation broth may also be tested for ethanol release by the culture supernatant. Byproducts and residual sugars (e.g., glucose or xylose) in fermentation media can be quantified by HPLC using, for example, refractive index detectors for glucose and alcohols, and UV detectors for organic acids (Lin et al, biotechnol. Bioeng [ biotechnology and bioengineering ] 90:775-779 (2005)), or using other suitable assays and detection methods well known in the art.

Preservation of biological materials

The following biomaterials have been deposited under the terms of the budapest treaty at the american national institute of agricultural research services patent bacterial deposit (NRRL) northern regional research center, university street 1815, pi Aorui, illinois, usa, and the following accession numbers are given:

the strain was preserved under the following conditions: ensuring that the cultures are available to persons authorized by the patent and trademark committee in accordance with 37c.f.r. ≡1.14 and 35u.s.c. ≡122 during the pendency of this patent application. These deposits represent a substantially pure culture of the deposited strain. There is a need to provide a deposit under the foreign patent statutes of some countries in which copies of the subject application, or subsequent text, are filed. However, it should be understood that the availability of the deposit does not constitute a license for practice.

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

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

Examples

Material

Cellulose decomposing enzyme composition CA“CA”): the cellulolytic enzyme preparation derived from Trichoderma reesei further comprises a GH61A polypeptide having cellulolytic enhancing activity derived from a strain of Penicillium emersonii (SEQ ID NO:2 in WO 2011/0410197), variant F100D, S283G, N456E, F512Y of Aspergillus fumigatus beta-glucosidase disclosed in WO 2012/044915 (SEQ ID NO:2 in WO 2005/047499); 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. Furthermore, the cellulolytic enzyme preparation CA further comprises 10% of a cellulolytic enzyme preparation from Trichoderma reesei, further comprising Aspergillus fumigatus xylanase (SEQ ID NO:8 in WO 2016/045569) and Aspergillus fumigatus beta-xylosidase (SEQ ID NO:9 in WO 2016/045569).

Cellulolytic enzyme composition CB ("CB"): the Trichoderma reesei cellulolytic enzyme preparation comprises EG I of SEQ ID NO:21 in WO 2016/045569, EG II of SEQ ID NO:22 in WO 2016/045569, CBH I of SEQ ID NO:14 in WO 2016/045569; CBH II of SEQ ID NO. 15 of WO 2016/045569; the β -glucosidase variant of SEQ ID NO. 5 of WO 2016/045569 has the following substitutions: f100D, S283G, N456E, F512Y; AA9 of SEQ ID NO. 7 in WO 2016/045569 (GH 61 polypeptide), GH10 xylanase of SEQ ID NO. 16 in WO 2016/045569; beta-xylosidase of SEQ ID NO. 17 of WO 2016/045569.

BSGX001Disclosed in U.S. patent No. 8,586,336-B2 (incorporated herein by reference) and constructed as follows: the host Saccharomyces cerevisiae strain BSPX042 (phenotype: ura3-251, over-expression of XKS1; over-expression of RPE1, RKI1, TAL1 and TKL1, these are genes in PPP; knockout of aldose reductase gene GRE3; and disruption of the electron transporting respiratory chain by deletion of COX4 gene after adaptation evolution) was transformed with vector pJFE3-RuXI inserted with xylose isomerase gene encoding RuXI shown in SEQ ID NO:2 in U.S. Pat. No. 8,586,336-B2 or SEQ ID NO:20 herein.

MBGs 5147, MBG5148, MBG5149, MBG5150, MBG5151 were prepared from CIBTS1260 (see WO 2016/045569, the contents of which are incorporated herein by reference) according to the evolution and breeding program described in U.S. Pat. No. 8,257,959.

Example 1: construction of Strain CIBTS1000

Diploid saccharomyces cerevisiae strains are identified that are known to be efficient ethanol producers from glucose. Saccharomyces cerevisiae strain CCTCC M94055 from China Center for Type Culture Collection (CCTCC) was used.

Xylose isomerase, termed mgXI, was cloned from a metagenomic project indicating that the donor organism was unknown. The isolation and characterization of this xylose isomerase is described in chinese patent application No. 102174549a or U.S. patent publication No. 2012/0225452.

Pentose transporter called GXF was cloned from candida intermedia using standard methods. The xylose transporter is described in D.Runquist et al (Runquist D, fonseca C, radstrom P, spencer-Martins I, hahn-B: "Expression of the Gxf1transporter from Candida intermedia improves fermentation performance in recombinant xylose-utilizing Saccharomyces cerevisiae" [ expression of the Candida intermedia Gxf1transporter improved fermentation performance by recombinant xylose of Saccharomyces cerevisiae ] ]Appl Microbiol Biotechnol [ applying microbiology and biotechnology ]]2009,82:123-130)。

The xylose isomerase gene was fused to a Triose Phosphate Isomerase (TPI) promoter and TPI terminator from saccharomyces cerevisiae using standard methods such that expression of xylose isomerase in saccharomyces cerevisiae was controlled by TPI expression signals.

The GXF gene was fused to the TPI expression signal in the same manner.

These two expression cassettes inserted into E.coli (Escherichia coli) cloning vectors comprise:

e.coli colE1 origin of replication ensuring that the plasmid can proliferate in E.coli.

Delta sequence fragments from Saccharomyces cerevisiae.

Bleomycin resistance markers from streptoverticillium indicum for selection of bleomycin resistant E.coli or Saccharomyces cerevisiae transformants. The double promoter was fused to the 5' end of the bleomycin gene consisting of the Saccharomyces cerevisiae translational elongation factor (TEF 1) promoter and the 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 so that the expression cassette can be deleted by Cre-Lox recombination (B.Sauer: "Functional expression of the Cre-Lox site specific recombination systemin the yeast Saccharomyces cerevisiae." [ functional expression of Cre-Lox site specific recombination system in Saccharomyces cerevisiae ] mol.cell.biol. [ molecular cell biology ]1987, 7:2087-2096).

This xylose isomerase/pentose transporter expression plasmid is called pYIE2-mgXI-GXF 1-delta and is shown in FIG. 1.

This plasmid pYIE2-mgXI-GXF 1-delta was initially linearized by XhoI digestion and then transformed into the parent strain Saccharomyces cerevisiae CCTCC M94055, which was subsequently selected for bleomycin resistant transformants. A strain called CIBTS0912 having a plasmid integrated into the delta sequence was isolated. The bleomycin resistance cassette located between the two loxP sites was then deleted by transient CRE recombinase expression, yielding strain CIBTS0914.

Transient CRE recombinase expression was achieved in a manner similar to the standard method described by 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 to green fluorescent proteins in Saccharomyces cerevisiae ]".FEBS Lett. [ European society of biochemistry "2000:485, 29-34.) ], transformed with an unstable plasmid expressing CRE recombinase, and then the plasmid was cured again. In this work, the kanamycin gene of the yeast standard vector pSH47 was replaced with a hygromycin resistance marker, allowing selection using hygromycin instead of kanamycin resistance. A plasmid map of the plasmid using pSH47-hyg is shown in FIG. 2. The table listing the genetic factors used is shown in table 1.

Table 1.

The XhoI digested pYIE2-mgXI-GXF 1-delta transformant strain CIBTS0914 was again used to increase the copy number of both expression cassettes, and bleomycin resistant strain CIBTS0916 was selected.

To overexpress the genes of the pentose phosphate pathway, an expression plasmid containing the genes of the selected pentose phosphate pathway is assembled.

The genes selected for overexpression were:

1. xylulokinase (XKS 1).

2. Transaldolase (TAL 1).

3. Ribulose 5 phosphate epimerase (RPE 1).

4. Transketolase (TKL 1).

5. Ribose 5 phosphate isomerase (RKI 1)

In addition to these genes, kanMX selection cassettes surrounded by loxP sites were also included as part of E.coli-Saccharomyces cerevisiae shuttle vector pUG6 (Guldener U, heck S, fielder T, bennhauer J, hegemann JH. "A new efficient gene disruption cassette for repeated use in budding yeast. [ a novel efficient gene disruption cassette for use in budding yeast reuse ]" NAR [ nucleic acids research ]1996, 24:2519-24).

A map of the resulting plasmid pYIE2-XKS 1-PPP-delta is shown in FIG. 3. The table listing the genetic factors used is shown in table 2.

Table 2.

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Plasmid pYIE2-XKS 1-PPP-delta was digested with NotI and these vector elements were removed by agarose gel electrophoresis. The linear fragments containing all the expression cassettes were then transformed into CIBTS0916 for double homologous recombination, followed by selection for kanamycin (G418) resistance. Kanamycin resistant colonies were selected and designated CIBTS0931.

CIBTS0931 contains both the bleomycin selectable marker and the kanamycin selectable marker. Both of them are flanked by loxP recombination sites.

To remove bleomycin and kanamycin resistance markers, the strain was reconverted with the additional plasmid pSH47-hygs and transformants were selected on hygromycin containing plates. Subsequently, screening was performed for transformants that had lost bleomycin and kanamycin resistance, and after that, screening was performed for strains that also lost hygromycin resistance markers. Strain CIBTS1000 was selected and shown to have lost plasmid pSH47-hyg.

Example 2: adaptation of strain CIBTS1000 to high xylose uptake and acetate resistance

The strain CIBTS1000 was modified so that it could utilize xylose as a carbon source and ferment it to ethanol. However, this xylose utilization is very inefficient. One well known way to improve that problem in the field of metabolic engineering is to use adaptation. This is also the case in this case. The strain CIBTS1000 was transferred continuously from shake flask to shake flask in a medium comprising xylose as sole carbon source and a yeast growth inhibitor known to be present in cellulosic biomass hydrolysate. The mutations accumulate in these successive transfers, allowing the strain to grow better under the conditions provided-and thus to make better use of xylose.

In the first round of adaptation, CIBTS1000 was transferred continuously in a shake flask system using YPX medium (10 g/L yeast extract, 20g/L peptone, and 20g/L xylose) and YPDX (10 g/L yeast extract, 20g/L peptone, 10g/L glucose, and 10g/L xylose)

In the second round of adaptation, successive transfers were performed in YPXI (YPX supplemented with 43mM sodium formate, 50mM sodium acetate, and 100mM sodium sulfate) and YPDXI (YPDX supplemented with 43mM sodium formate, 50mM sodium acetate, and 100mM sodium sulfate).

In the last round of adaptation, NREL dilute acid pretreated corn stover hydrolysate supplemented with 10g/l yeast extract, 20g/l peptone, 10g/l glucose and 10g/l xylose (see example 3) was subjected to continuous transfer.

A strain designated CIBTS1260-J132-F3 was selected as the adaptive strain.

Example 3: fermentation of CIBTS1260 and BSGX001 in NREL dilute acid pretreated corn stover hydrolysate Comparison of

Two s.cerevisiae strains (CIBTS 1260 and BSGX 001) were tested in NREL dilute acid pretreated corn stover hydrolysate (4% w/w sulfuric acid at 180℃for 5 min). Hydrolysis with 20mg of enzyme protein per g of glucan of cellulolytic enzyme composition CA for 3 days at 50℃in a 20kg reactor yielded a hydrolysate. The dilute acid pretreated corn stover hydrolysate had a final composition of 63.2g/L glucose, 44.9g/L xylose, 0.8g/L glycerol and 9.5g/L acetate. Each strain was propagated on YPD medium (10 g/L yeast extract, 20g/L peptone and 20g/L glucose) at 150rpm in a 30℃air shaker prior to fermentation. After 24 hours of growth, the two yeast strains were tested in 50ml of hydrolysate in 125ml of baffled Erlenmeyer flasks (yeast inoculation at 1g Dry Cell Weight (DCW)/L). Each flask was sealed using a rubber stopper fitted with an 18 gauge blunt fill needle and the flasks were placed in an air shaker at 35 ℃,150 rpm. Samples were taken at 24, 48 and 72 hours and analyzed via HPLC for determining the concentration of glucose, xylose and ethanol. The results of 3 replicates per group were averaged and are presented in figure 1, which shows a comparison of CIBTS1260 versus BSGX001 in NREL acid pretreated corn stover hydrolysate inoculated with 1g/L yeast over 72 hours. As shown in FIG. 4, CIBTS1260 strain completed complete xylose consumption by 48 hours fermentation and produced approximately 47g/L ethanol. However, the BSGX001 strain ingests glucose slowly for ethanol conversion and therefore consumes only 3g/L xylose. These results indicate that CIBTS1260 results in improved xylose uptake and utilization for conversion to ethanol compared to BSGX 001.

Example 4: comparison of CIBTS1260 and BSGX001 in Pattern Medium for fermentation Performance

The fermentation performance of CIBTS1260 and its precursor BSGX001 was compared. Each strain was propagated on YPD medium (10 g/L yeast extract, 20g/L peptone and 20g/L glucose) at 150rpm in a 30℃air shaker prior to fermentation. After 24 hours of growth, the two yeast strains were assayed in YTX medium (5 g/L yeast extract, 5g/L peptone and 50g/L xylose). To test fermentation performance, each strain was inoculated in 50ml of YPX medium in 125ml of a baffled Erlenmeyer flask (inoculated with 2g DCW/L yeast). Each flask was sealed using a rubber stopper fitted with an 18 gauge blunt fill needle and the flasks were placed in an air shaker at 32 c, 150 rpm. Samples were taken at 24, 48 and 72 hours and analyzed via HPLC for determining the concentration of glucose, xylose, and ethanol. The results of 3 replicates per group were averaged and are given in figure 5.

As shown in FIG. 5, CIBTS1260 (dotted line) has fully utilized all available xylose within 24 hours and produced 21.3g/L ethanol. BSGX001 (solid line) consumed 1.5g/L xylose during the 72 hour fermentation time, and the resulting ethanol concentration was 1.3g/L.

Example 5: cellulolytic enzyme composition CA ("CA") and cellulolytic enzyme composition with CIBTS1260 Fermentation of CB ("CB") bagasse hydrolysate

CIBTS1260 was used in fermentation tests of bagasse hydrolysate pretreated with NREL dilute acid produced by North American North American Co (Novozymes North America, USA). Hydrolysis with a dose of 6mg enzyme protein/g dextran of two cellulolytic enzyme compositions (referred to as "CA" and "CB") in a 2L IKA reactor at 50℃for 5 days resulted in a hydrolysate. These materials are representative benchmarks for dilute acid pretreated bagasse hydrolysates with the following final compositions: for "CA" and "CB",40.7g/L and 58.7g/L glucose, 42.5g/L and 44.7g/L xylose, 0.19g/L and 0.08g/L glycerol, and 8.99g/L and 11.3g/L acetate, respectively. Yeast were propagated in 2% YPD medium (10 g/L yeast extract, 20g/L peptone and 20g/L glucose) at 150rpm in a 30℃air shaker prior to fermentation. After 24 hours of growth, CIBTS1260 was tested in 50ml of "CA" and "CB" hydrolysates in 125ml of baffled Erlenmeyer flasks (inoculated with 1g DCW/L yeast). Each flask was sealed using a rubber stopper fitted with an 18 gauge blunt fill needle and the flasks were placed in an air shaker at 35 ℃,150 rpm. Samples were taken at 24, 48 and 72 hours and analyzed via HPLC for determining the concentration of glucose, xylose, ethanol, acetate and glycerol. The results of 3 replicates per group were averaged and are given in figure 6. Over a 72 hour period, greater than 95% of the glucose and xylose present in both systems was consumed, with an ethanol yield of 84.1% for the "CA" hydrolysate and 86.4% for the "CB" hydrolysate, based on total sugars.

Example 6: CIBTS1260 and BSGX001 of bagasse hydrolysate of dilute acid pretreated corn stover and sugarcane DP2 reduction during fermentation

The corn stover and bagasse were pretreated with dilute acid from the National Renewable Energy Laboratory (NREL) in a 2L IKA reactor at 50 ℃ using a dose of 6mg enzyme protein/g glucan of a mixture of two enzyme products, called CA and CB, for 5 days. Prior to fermentation, CIBTS1260 and BSGX001 yeasts were propagated on YPD medium (10 g/L yeast extract, 20g/L peptone and 20g/L glucose) at 150rpm with a 30℃air shaker. After 24 hours of growth, cells from each strain were harvested via centrifugation and added to 50ml of CA and CB hydrolysate (inoculated with 1g dcw/L (dry cell weight/L) of yeast) supplemented with 2g/L urea in 125ml baffled Erlenmeyer flasks, respectively. Each flask was sealed using a rubber stopper fitted with an 18 gauge blunt fill needle and the flasks were placed in an air shaker at 35 ℃,150 rpm. Samples were taken at 0 hours and 72 hours and analyzed via HPLC for determination of DP2 concentration. The repeated results for each group are averaged (n=3 for CIBTS1260 and n=2 for BSGX 001). As shown in fig. 7, fermentation using CIBTS1260 reduced DP2 concentration more than fermentation using BSGX001 in the same hydrolysate. The DP2 peak, as measured on HPLC, contains cellobiose and short chain sugars.

Example 7: fermentation comparison of strains MBG5147-MBG5151 with CIBTS1260

Saccharomyces cerevisiae strains CIBTS1260, MBG5147, MBG5148, MBG5149, MBG5150 and MBG5151 were transferred from inclined tubes to PDA plates at 32℃for incubation for 24 to 48 hours. Isolated colonies were grown in YPD medium in shake flasks at 32℃for 24 hours and aliquots were stored in 2mL freezer tubes containing 20% glycerol at-80 ℃.

Cell proliferation for fermentation was performed in two steps in 500mL baffled flasks containing 100mL of medium and incubated at 32℃in a shaker at 150 rpm. The first step medium was inoculated with 1 freezer tube and after 16 hours transferred to a second flask. At the end of the incubation, cell growth was measured by DO at 600nm in a spectrophotometer and converted to dry cell weight in g/L.

Fermentation was performed using C5-liquor obtained from pretreated bagasse in 250mL Schott flasks containing 50mL pH 5.5 medium, inoculated with propagation medium and incubated in an orbital incubator at 32℃and 110 rpm. For inoculation, the medium concentration was adjusted to account for the different growth rates in order to start fermentation at the same cell spacing (1 g/L). Fermentation kinetics were monitored by the ANKOM RF gas production system, after 48 hours of fermentation, samples were taken and analyzed for sugar, ethanol, glycerol and acetic acid by HPLC (column HPX87-H, RID detector) and xylose by the xylose kit (Megazyme Corp.).

FIG. 8 shows dynamic curves of MBG5147-MBG5151 and CIBTS1260 fermentations based on pressure monitoring and converted to gas mass according to the calculations of the ANKOM RF gas production system. Table 3 shows residual sugars, ethanol titres, ethanol yields and xylose consumed. The data shows that MBG5151 ferments faster than the remaining test strains, including CIBTS 1260.

Table 3.

The invention may be further described in the following numbered paragraphs:

paragraph [1] a process for producing a fermentation product from cellulose-and/or starch-containing material, the process comprising:

(a) Saccharifying the cellulose-or starch-containing material; and

(b) Fermenting the saccharified material of step (a) with a fermenting organism under suitable conditions to produce a fermentation product; wherein the fermenting organism is a recombinant strain of Saccharomyces cerevisiae deposited with Budapest at about the American national institute of agricultural research and service patent Collection (NRRL) under accession number NRRL Y-67971 (Saccharomyces cerevisiae strain MBG 5151), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and/or alpha-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG 5151.

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

(a) Saccharifying the cellulose-or starch-containing material; and

(b) Fermenting the saccharified material of step (a) with a fermenting organism under suitable conditions to produce a fermentation product; wherein the fermenting organism is a recombinant strain of Saccharomyces cerevisiae deposited with Budapest at about the American national institute of agricultural research and services patent culture Collection (NRRL) under accession number NRRL Y-68015 (Saccharomyces cerevisiae strain MBG 5248), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and/or alpha-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG 5248.

The method of paragraph [3] or [2], comprising recovering the fermentation product from the fermentation.

Paragraph [4] the method of paragraph [3], wherein recovering the fermentation product from the fermentation comprises distillation.

The method of any one of paragraphs [5] to [4], wherein the fermenting and saccharifying are performed simultaneously in Simultaneous Saccharification and Fermentation (SSF).

The method of any one of paragraphs [6] to [4], wherein the fermenting and saccharifying are performed Sequentially (SHF).

The method of any one of paragraphs [7] to [6], wherein the fermentation product is ethanol.

The method of any one of paragraphs [8] to [7], wherein step (a) comprises contacting the starch-containing and/or cellulose-containing material with an enzyme composition.

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

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

The method of any of paragraphs [11] or [10], wherein the cellulose-containing material comprises bagasse.

The method of any one of paragraphs [12] - [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: cellulases, AA9 polypeptides, hemicellulases, CIPs, esterases, expansins, ligninases, oxidoreductases, pectinases, proteases, and swellins.

The method of paragraph [13]. The cellulase of paragraph [12], wherein the cellulase is one or more enzymes selected from the group consisting of: endoglucanases, cellobiohydrolases and beta-glucosidase.

The method of paragraph [14] or [13], wherein the hemicellulase is one or more enzymes selected from the group consisting of: xylanase, acetylxylan esterase, feruloyl esterase, arabinofuranosidase, xylosidase and glucuronidase.

The method of any of paragraphs [15]. The method of paragraphs [1] to [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%).

The method of any one of paragraphs [16] to [15], wherein the fermentation is performed under low oxygen (e.g., anaerobic) conditions.

The method of any one of paragraphs [17] [1] to [16], the fermenting organism having one or more of the following properties:

higher ethanol fermentation kinetics at 1g DWC/L, 32 ℃, pH 5.5 (as described in example 7 herein) compared to saccharomyces cerevisiae CIBTS1260 (e.g., 10 to 32 hours);

Paragraph [18] A recombinant Saccharomyces cerevisiae strain deposited as Budapest strip at about the American national institute of agricultural research and service patent Collection (NRRL) under accession number NRRL Y-67971 (Saccharomyces cerevisiae strain MBG 5151), or a derivative thereof (e.g., expressing a heterologous polypeptide, such as glucoamylase and/or alpha-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG 5151.

Paragraph [19] A recombinant Saccharomyces cerevisiae strain deposited as Budapest strip at about the American national institute of agricultural research and service patent Collection (NRRL) under accession number NRRL Y-68015 (Saccharomyces cerevisiae strain MBG 5248), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and/or alpha-amylase), or a fermenting organism having substantially the same properties as Saccharomyces cerevisiae MBG 5248.

The recombinant saccharomyces cerevisiae strain of paragraph [20] or [19], wherein the strain has one or more of the following properties:

Paragraph [21] the recombinant saccharomyces cerevisiae strain of any of paragraphs [18] to [20], wherein the strain is capable of higher ethanol yield when fermented at 1g DWC/L, 32 ℃, pH 5.5 (as described in example 7 herein) for 10 to 30 hours compared to saccharomyces cerevisiae CIBTS 1260.

Paragraph [22] the recombinant saccharomyces cerevisiae strain of any of paragraphs [18] to [21], wherein the strain is capable of consuming greater than 95% xylose after fermentation for 48 hours under process conditions of 1g DCW/L, 35 ℃, pH 5.5 (as described in example 3 herein).

Paragraph [23] the recombinant saccharomyces cerevisiae strain of any of paragraphs [18] to [22], wherein the strain is capable of consuming greater than 95% glucose after fermentation for 24 hours under process conditions of 1g DCW/L, 35 ℃, pH 5.5 (as described in example 3 herein).

Paragraph [24] the recombinant saccharomyces cerevisiae of any of paragraphs [18] to [23], wherein the strain is capable of providing greater than 30g/L ethanol, such as greater than 40g/L ethanol, such as greater than 45g/L ethanol, such as about 47g/L ethanol after fermentation at 1g DCW/L for 48 hours under process conditions of 35 ℃ at pH 5.5 (as described in example 3 herein).

Paragraph [25] the recombinant Saccharomyces cerevisiae of any one of paragraphs [18] to [24], comprising a heterologous gene encoding a xylose isomerase.

The recombinant saccharomyces cerevisiae of any of paragraphs [26] to [25], comprising a heterologous gene encoding a pentose transporter.

Paragraph [27] the recombinant s.cerevisiae of any of paragraphs [18] to [26], wherein the pentose transporter gene is a GFX gene (e.g., GFX1 from Candida intermedia).

Paragraph [28] the recombinant saccharomyces cerevisiae of any of paragraphs [18] to [27], comprising a heterologous gene (XKS) encoding a xylulokinase (e.g., XKS from saccharomyces cerevisiae).

Paragraph [29] the recombinant saccharomyces cerevisiae of any of paragraphs [18] to [28], comprising a heterologous gene (RPE 1) encoding ribulose 5 phosphate 3-epimerase (e.g., RPE1 from saccharomyces cerevisiae).

Paragraph [30] the recombinant saccharomyces cerevisiae of any of paragraphs [18] to [29], comprising a heterologous gene (RKI 1) encoding ribulose 5 phosphate isomerase (e.g., RKI1 from saccharomyces cerevisiae).

Paragraph [31] the recombinant s.cerevisiae of any of paragraphs [18] to [30], comprising a heterologous gene (TKL 1) encoding a transketolase and a heterologous gene (TAL 1) encoding a transaldolase (e.g., TKL1 and TAL1 from s.cerevisiae).

Paragraph [32]. A method of producing a derivative of Saccharomyces cerevisiae strain MBG5151 (deposited under accession number NRRL Y-67971, american type of agricultural research service patent collection (NRRL)), the method comprising:

a. culturing a first yeast strain with a second yeast strain under conditions that allow DNA combination between the first and second yeast strains, wherein the second yeast strain is saccharomyces cerevisiae strain MBG5151 or a derivative thereof; and

b. Isolating the 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.

Paragraph [33]. A method of producing a derivative of Saccharomyces cerevisiae strain MBG5248 (deposited under accession number NRRL Y-68015 at the American national institute of agricultural research and service patent bacterial Collection (NRRL)), the method comprising:

a. culturing a first yeast strain with a second yeast strain under conditions that allow DNA combination between the first and second yeast strains, wherein the second yeast strain is saccharomyces cerevisiae strain MBG5248 or a derivative thereof; and

b. isolating the heterozygous strain; and

Paragraph [34] A method of producing a derivative of Saccharomyces cerevisiae strain MBG5151 exhibiting the defined characteristics of Saccharomyces cerevisiae strain MBG5151 (deposited under accession number NRRL Y-67971) at the American national institute of Electrical and Engineers patent culture Collection (NRRL), 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 MBG5151 or a derivative thereof;

(b) Culturing the first yeast strain and the second yeast strain under conditions that allow combining DNA between the first and second yeast strains;

(c) Screening or selecting derivatives of Saccharomyces cerevisiae strain MBG 5151.

The method of paragraph [35] the method of paragraph [34], wherein step (c) comprises screening or selecting for heterozygous strains exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG 5151.

Paragraph 36 the method of paragraph 34, the method comprising the further steps of:

(d) Repeating steps (a) and (b) with the strain selected or selected from step (c) as the first and/or second strain until a derivative exhibiting the defined characteristics of saccharomyces cerevisiae strain MBG5151 is obtained.

Paragraph [37]. The method of paragraph [34], wherein the culturing step (b) comprises:

(i) Sporulation of the first yeast strain and the second yeast strain;

(ii) The germinated spores produced by the first yeast strain are hybridized with the germinated spores produced by the second yeast strain.

Paragraph [38] A method of producing a derivative of Saccharomyces cerevisiae strain MBG5248 exhibiting the defined characteristics of Saccharomyces cerevisiae strain MBG5248 (deposited under accession number NRRL Y-68015) at the American society for agricultural research service patent bacterial culture Collection (NRRL), the method comprising:

(d) Providing:

(j) A first yeast strain; and

(iii) A second yeast strain, wherein the second yeast strain is saccharomyces cerevisiae strain MBG5248 or a derivative thereof;

(e) Culturing the first yeast strain and the second yeast strain under conditions that allow combining DNA between the first and second yeast strains;

(f) Screening or selecting derivatives of Saccharomyces cerevisiae strain MBG 5248.

Paragraph [39] the method of paragraph [38], wherein step (c) comprises screening or selecting for heterozygous strains exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG 5248.

Paragraph [40] the method of paragraph [38], the method comprising the further steps of:

(d) Repeating steps (a) and (b) with the strain selected or selected from step (c) as the first and/or second strain until a derivative exhibiting the defined characteristics of saccharomyces cerevisiae strain MBG5248 is obtained.

Paragraph [41] the method of paragraph [38], wherein the culturing step (b) comprises:

(i) Sporulation of the first yeast strain and the second yeast strain;

Paragraph [42]. A method of producing a recombinant derivative of Saccharomyces cerevisiae strain MBG5151 (deposited under accession number NRRL Y-67971 at the American national institute of Electrical and Engineers patent culture Collection (NRRL)), the method comprising:

(a) Transforming s.cerevisiae strain MBG5151 (or derivatives of s.cerevisiae strain MBG 5151) with one or more expression vectors (e.g., one or more expression vectors encoding glucoamylase and/or alpha-amylase); and

(b) Isolating the transformed strain.

Paragraph [43] A method for producing a recombinant derivative of Saccharomyces cerevisiae strain MBG5248 (deposited under accession number NRRL Y-68015 at the American national institute of agricultural research and patent bacterial Collection (NRRL)), comprising:

(a) Transforming s.cerevisiae strain MBG5248 (or derivatives of s.cerevisiae strain MBG 5248) with one or more expression vectors (e.g., one or more expression vectors encoding glucoamylase and/or alpha-amylase); and

(b) Isolating the transformed strain.

Paragraph [44] A Saccharomyces cerevisiae strain produced by the method of any one of paragraphs [32] to [43].

Paragraph [45] A method of producing ethanol, the method comprising incubating the Saccharomyces cerevisiae strain of any of paragraphs [18] to [31] and [44] with a substrate comprising a fermentable sugar under conditions that allow the fermentable sugar to ferment to ethanol.

The use of the Saccharomyces cerevisiae strain according to any one of paragraphs [18] to [31] and [44] in ethanol production.

The use of the saccharomyces cerevisiae strain of any of paragraphs [18], [20] - [31] and [44] in the production of a saccharomyces strain having the defined characteristics of saccharomyces cerevisiae strain MBG5151 (deposited under accession No. NRRL Y-67971 at the national center for agricultural research services patent and bacterial collection northern area, university street 1815, pi Aorui, illinois, usa).

The use of the saccharomyces cerevisiae strain according to any of paragraphs [19] to [31] and [44] in the production of a saccharomyces strain having the defined characteristics of saccharomyces cerevisiae strain MBG5248 (deposited under accession No. NRRL Y-68015 at the national center for agricultural research service patent and bacterial deposit northern regional research center (NRRL) at street 1815 at university Pi Aorui, illinois).

Paragraph [49] use of Saccharomyces cerevisiae strain MBG5151 (deposited under accession number NRRL Y-67971 at the American agricultural research service patent Collection North area research center (NRRL) at street 1815, university of Pi Aorui, illinois) in the production of a Saccharomyces strain having substantially the same properties as Saccharomyces cerevisiae strain MBG5151 or exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG 5151.

Paragraph [50] use of Saccharomyces cerevisiae strain MBG5248 (deposited under accession number NRRL Y-68015 at the American agricultural research service patent Collection North area center (NRRL) at street 1815, university Pi Aorui, ill.) in the production of a Saccharomyces strain having substantially the same properties as Saccharomyces cerevisiae strain MBG5248 or exhibiting one or more defined characteristics of Saccharomyces cerevisiae strain MBG 5248.

Use of saccharomyces cerevisiae strain MBG5151 (deposited under accession No. NRRL Y-67971 at the american national institute of agriculture research service patent bacterial deposit north regional center (NRRL) at street 1815, pi Aorui, il) or a strain having substantially the same properties as saccharomyces cerevisiae strain MBG5151 or a derivative thereof in the method of any of paragraphs 1-17.

Paragraph [52] use of saccharomyces cerevisiae strain MBG5248 (deposited under accession number NRRL Y-68015 at the national center for research and development of patent and research collection of regional north areas (NRRL) at university street 1815, pi Aorui a, il) or a strain having substantially the same properties as saccharomyces cerevisiae strain MBG5248 or a derivative thereof in the method of any of paragraphs [2] to [16 ].

Paragraph [53] A composition comprising the Saccharomyces cerevisiae strain of any of paragraphs [18] to [31] and [44], and one or more naturally occurring and/or non-naturally occurring components.

Paragraph 54 the composition of paragraph 53 wherein the components are selected from the group consisting of: surfactants, emulsifiers, gums, swelling agents and antioxidants.

The composition of paragraph [55]. The composition of paragraph [53] or [54], wherein the Saccharomyces cerevisiae strain is Saccharomyces cerevisiae strain MBG5151 (deposited under accession number NRRL Y-67971 at the American agricultural research service patent culture Collection North regional research center (NRRL) of university street 1815, pi Aorui, ill.).

The composition of paragraph [56] or [54], wherein the saccharomyces cerevisiae strain is saccharomyces cerevisiae strain MBG5248 (deposited under accession No. NRRL Y-68015 at the american national institute of agriculture research service patent culture collection (NRRL) northern regional collection of university, pi Aorui, il).

The composition of any one of paragraphs [57] to [56], wherein the saccharomyces cerevisiae strain is in a viable state, particularly in a dry, pasty or compressed state.

PCT

(originally in a spreadsheet)

(this table is not part of the International application and is not regarded as an International application table)

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Claims

1. A method of producing a fermentation product from cellulose-and/or starch-containing material, the method comprising:

(a) Saccharifying the cellulose-or starch-containing material; and

(b) Fermenting the saccharified material of step (a) with a fermenting organism under suitable conditions to produce a fermentation product;

wherein the fermenting organism is:

(1) Recombinant strains of saccharomyces cerevisiae deposited with the budapest strip at about the american national institute of agriculture research service patent collection (NRRL) under accession No. NRRL Y-67971 (saccharomyces cerevisiae strain MBG 5151), or derivatives thereof (e.g., expressing heterologous polypeptides such as glucoamylase and/or alpha-amylase), or fermenting organisms having about the same properties as saccharomyces cerevisiae MBG 5151; or alternatively

(2) Recombinant strains of saccharomyces cerevisiae deposited with the budapest strip at about the american national institute of agriculture research service patent collection (NRRL) under accession No. NRRL Y-68015 (saccharomyces cerevisiae strain MBG 5248), or derivatives thereof (e.g., expressing heterologous polypeptides such as glucoamylase and/or alpha-amylase), or fermenting organisms having substantially the same properties as saccharomyces cerevisiae MBG 5248.

2. A recombinant saccharomyces yeast strain selected from the group consisting of:

a saccharomyces cerevisiae strain deposited with the budapest strip at about the american national institute of agriculture research service patent collection (NRRL) under accession No. NRRL Y-67971 (saccharomyces cerevisiae strain MBG 5151), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and/or alpha-amylase), or a fermenting organism having substantially the same properties as saccharomyces cerevisiae MBG 5151; and

a strain of saccharomyces cerevisiae deposited with the budapest strip at about the american national institute of agriculture research service patent collection (NRRL) under accession No. NRRL Y-68015 (saccharomyces cerevisiae strain MBG 5248), or a derivative thereof (e.g., expressing a heterologous polypeptide such as glucoamylase and/or alpha-amylase), or a fermenting organism having substantially the same properties as saccharomyces cerevisiae MBG 5248.

3. The recombinant saccharomyces cerevisiae strain according to claim 2 wherein the strain has one or more of the following properties:

4. A recombinant saccharomyces cerevisiae strain according to claim 2 or 3, wherein the strain is capable of higher ethanol yield compared to saccharomyces cerevisiae CIBTS1260 when fermented at 1g DWC/L at 32 ℃ at pH 5.5 (as described in example 7 herein) for 10 to 30 hours.

5. The recombinant saccharomyces cerevisiae strain according to any of claims 2-4 wherein the strain is capable of consuming more than 95% xylose after 48 hours of fermentation under process conditions of 1g DCW/L, 35 ℃, pH 5.5 (as described in example 3 herein).

6. The recombinant saccharomyces cerevisiae strain according to any of claims 2-5 wherein the strain is capable of consuming more than 95% glucose after fermentation for 24 hours under process conditions of 1g DCW/L, 35 ℃, pH 5.5 (as described in example 3 herein).

7. The recombinant saccharomyces cerevisiae according to any of claims 2-6 wherein the strain is capable of providing more than 30g/L ethanol, such as more than 40g/L ethanol, such as more than 45g/L ethanol, such as about 47g/L ethanol after fermentation under process conditions of 1g DCW/L, 35 ℃, pH 5.5 (as described in example 3 herein) for 48 hours.

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

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

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

12. The recombinant saccharomyces cerevisiae according to any of claims 2-11 comprising a heterologous gene (RPE 1) encoding a ribulose 5 phosphate 3-epimerase (e.g., RPE1 from saccharomyces cerevisiae), a heterologous gene (RKI 1) encoding a ribulose 5 phosphate isomerase (e.g., RKI1 from saccharomyces cerevisiae), or a heterologous gene (TKL 1) encoding a transketolase and a heterologous gene (TAL 1) encoding a transaldolase (e.g., TKL1 and TAL1 from saccharomyces cerevisiae).

13. A method of producing a derivative of saccharomyces cerevisiae strain MBG5151 (deposited under accession No. NRRL Y-67971) or saccharomyces cerevisiae strain MBG5248 (deposited under accession No. NRRL Y-68015) that exhibits defined characteristics of saccharomyces cerevisiae strain MBG5151 or MBG5248, respectively, the method comprising:

(a) Providing:

(i) A first yeast strain; and

14. The method of claim 134, wherein step (c) comprises screening or selecting for heterozygous strains exhibiting one or more of the defined characteristics of saccharomyces cerevisiae strain MBG 5151.

15. The method according to claim 13, comprising the further step of:

16. The method of claim 13, wherein the culturing step (b) comprises:

(i) Sporulation of the first yeast strain and the second yeast strain;

17. A method of producing a recombinant derivative of saccharomyces cerevisiae strain MBG5151 (deposited with the american national institute of agriculture service patent collection (NRRL) under accession No. NRRL Y-67971) or saccharomyces cerevisiae strain MBG5248 (deposited with the american national institute of agriculture service patent collection (NRRL) under accession No. NRRL Y-68015), the method comprising:

(a) Transformation of Saccharomyces cerevisiae strain MBG5151 (a derivative of Saccharomyces cerevisiae strain MBG 5151) or Saccharomyces cerevisiae strain MBG5248 (or a derivative of Saccharomyces cerevisiae strain MBG 5248) with one or more expression vectors (e.g., one or more expression vectors encoding glucoamylase and/or alpha-amylase); and

(b) Isolating the transformed strain.

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

19. A method of producing ethanol, the method comprising incubating a saccharomyces cerevisiae strain according to any of claims 2-12 and 18 with a substrate comprising fermentable sugars under conditions that allow the fermentable sugars to ferment to ethanol.

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