Yeast to increase ethanol production in highly dissolved solids

Abstract

Compositions and methods are described that relate to modified yeasts with disrupted genes that produce increased amounts of ethanol, and in some cases reduced amounts of acetic acid, compared to otherwise identical parent cells. Such yeasts are particularly useful for large scale production of ethanol from starch substrates with highly dissolved solids.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 385,996, filed December 5, 2022, which is incorporated by reference in its entirety.

[0003] Incorporation by Reference into the Sequence Listing

[0004] This application is submitted with a sequence listing in electronic format. The sequence listing is provided as a file titled NB42003WOPCTSeqList.xml, created on December 1, 2023, and is 37,468 bytes in size. The information in this electronic sequence listing is incorporated by reference in its entirety. Technical Field

[0005] The modified cells and methods of the present invention relate to modified yeasts with disrupted genes that produce increased amounts of ethanol, and in some cases, decreased amounts of acetic acid, compared to otherwise identical parent cells. Such yeasts are particularly useful for large-scale production of ethanol from starch substrates with high dissolved solids. Background Art

[0006] First-generation yeast-based ethanol production converts sugars into fuel ethanol. Yeast-based ethanol production worldwide reaches approximately 90 billion liters annually (Gombert, AK and van Maris, AJ (2015) Curr. Opin. Biotechnol. 33:81-86). It is estimated that approximately 70% of ethanol production costs are due to raw materials. Given such large production volumes, even small increases in yield can have a significant economic impact on the industry.

[0007] In the fuel ethanol industry, fermentation at high dry solids levels is a particularly challenging condition for fermenting organisms. High dry solids impose multiple physiological stresses on fermenting organisms, including osmotic stress, high ethanol stress, high acid stress, etc. Temperature fluctuations during the summer months and other causes of high fermentation temperatures pose additional challenges to fermenting organisms.

[0008] Ethanol production in engineered yeast cells with a heterologous phosphoketolase (PKL) pathway is higher than ethanol production in parent strains without the PKL pathway (see, e.g., Miasnikov et al., WO 2015148272). The PKL pathway consists of phosphoketolase (PKL) and phosphotransacetylase (PTA) to divert carbon flux away from the glycerol pathway and toward the synthesis of acetyl-CoA. Two supporting enzymes, acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthase (ACS), can help make the PKL pathway more efficient. Unfortunately, these engineered strains also produce more acetic acid than the parent yeast. Acetic acid is an undesirable byproduct because it has a negative impact on yeast growth and fermentation. In addition, acetic acid reduces the pH of the remaining water (called backflow) from fermentation and distillation, which is typically reused for liquefaction of subsequent batches of substrate. As a result, ethanol producers must adjust the pH of the backflow (or liquefaction) or increase the amount of fresh water used for liquefaction.

[0009] There is a need to increase ethanol production by fermenting organisms and control acetic acid. Summary of the Invention

[0010] The modified cells and methods of the present invention involve genetic mutations in fermenting organisms that result in increased ethanol production and, in some cases, decreased acetic acid under high dissolved solids and high temperature fermentation conditions. Aspects and embodiments of these compositions and methods are described in the following independently numbered paragraphs.

[0011] 1. In one aspect, modified yeast cells derived from a parent yeast cell are provided, the modified cells comprising genetic alterations that cause the modified cells to produce reduced amounts of polypeptides that cause lack of robustness during fermentation under high dry solids and high temperature fermentation conditions compared to the otherwise identical parent cells, wherein the polypeptides are selected from SKG3, YNR068C, MNN14, PIN3, PEX18, MNN4, RIM20 and / or PAN2 polypeptides.

[0012] 2. In some embodiments of the modified cells of paragraph 1, the modified cells further produce a reduced amount of acetic acid during fermentation compared to the amount of acetic acid produced by otherwise identical parent cells under the same fermentation conditions, and wherein the genetic alteration comprises a disruption of a nucleic acid capable of directing expression of a RIM20 and / or PAN2 polypeptide.

[0013] 3. In some embodiments of the modified cells of paragraph 1 or 2, the cells further comprise one or more genes of the phosphoketolase pathway.

[0014] 4. In some embodiments of the modified cell of paragraph 3, the gene of the phosphoketolase pathway is selected from the group consisting of: phosphoketolase, phosphotransacetylase, and acetylated acetyl dehydrogenase.

[0015] 5. In some embodiments of the modified cells of any of paragraphs 1-4, the cells further comprise an exogenous gene encoding a carbohydrate processing enzyme.

[0016] 6. In some embodiments, the modified cell of any of paragraphs 1-5 further comprises an alteration in the glycerol pathway and / or the acetyl-CoA pathway.

[0017] 7. In some embodiments, the modified cell of any of paragraphs 1-6 further comprises an alternative pathway for producing ethanol.

[0018] 8. In some embodiments of the modified cells of any of paragraphs 1-7, the cells are of Saccharomyces spp.

[0019] 9. In another aspect, a method for increasing the robustness of yeast cells under high dry solids and high temperature fermentation conditions is provided, the method comprising introducing a genetic alteration into a parent yeast cell, the genetic alteration causing the resulting modified cell to produce a reduced amount of a polypeptide selected from SKG3, YNR068C, MNN14, PIN3, PEX18, MNN4, RIM20 and / or PAN2 polypeptides during fermentation.

[0020] 10. In some embodiments of the method of paragraph 9, the genetic alteration further results in the modified cells producing a reduced amount of acetic acid during fermentation compared to the amount of acetic acid produced by the parent cells under the same fermentation conditions, and wherein the genetic alteration comprises disrupting a nucleic acid capable of directing expression of a RIM20 and / or PAN2 polypeptide.

[0021] 11. In one aspect, a method for producing a fermentation product is provided, the method comprising fermenting a carbohydrate substrate with the modified yeast cell of any of paragraphs 1-7 under conditions for producing a fermentation product.

[0022] 12. In some embodiments of the method of paragraph 11, the conditions include a dry solids percentage (DS%) of at least 35% and / or a temperature of at least 34°C.

[0023] 13. In some embodiments of the method of paragraph 11 or paragraph 12, the modified yeast cells produce increased amounts of fermentation product compared to the parent cell under the same conditions.

[0024] 14. In some embodiments of the method of any of paragraphs 11-13, the modified yeast cells produce reduced amounts of acetic acid compared to parent cells under the same conditions.

[0025] 15. In some embodiments of the method of any of paragraphs 11-14, the fermentation product is ethanol.

[0026] These and other aspects and embodiments of the modified cells and methods of the invention will be apparent from the description, including any drawings / figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 High temperature ramp conditions used in fermentations with modified and parent yeast are shown. DETAILED DESCRIPTION

[0028] I. Definition

[0029] Before describing the modified cells and methods of the present invention in detail, the following terms are defined for the sake of clarity. Undefined terms shall be given the ordinary meaning used in the relevant art.

[0030] As used herein, the term "alcohol" refers to an organic compound in which a hydroxyl functional group (-OH) is bonded to a saturated carbon atom.

[0031] As used herein, the terms "yeast cell," "yeast strain," or simply "yeast" refer to organisms from the phyla Ascomycota and Basidiomycota. An exemplary yeast is a budding yeast from the order Saccharomycetales. A specific example of yeast is a species of the genus Saccharomyces, including but not limited to Saccharomyces cerevisiae. Yeast includes organisms used to produce fuel alcohols as well as organisms used to produce drinkable alcohols, including specialty and proprietary yeast strains used to prepare uniquely flavored beer, wine, and other fermented beverages.

[0032] As used herein, the phrases "engineered yeast cells," "variant yeast cells," "modified yeast cells," or similar phrases refer to yeast that include the genetic modifications and features described herein. Variant / modified yeast do not include naturally occurring yeast.

[0033] As used herein, the terms "polypeptide" and "protein" (and their respective plural forms) are used interchangeably to refer to polymers of any length comprising amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes for amino acid residues are used herein, and all sequences are presented in the N-terminal to C-terminal direction. Polymers may contain modified amino acids, and they may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art.

[0034] As used herein, proteins that are functionally and / or structurally similar are considered to be "related proteins" or "homologs". Such proteins can be derived from organisms of different genera and / or species, or organisms of different classes (e.g., bacteria and fungi), or be artificially designed proteins. Related proteins also encompass homologs determined by primary sequence analysis, by secondary or tertiary structure analysis, or by immunological cross-reactivity, or by their function.

[0035] As used herein, the term "homologous protein" refers to a protein having similar activity and / or structure to a reference protein. This is not intended to imply that homologs are necessarily evolutionarily related. Thus, the term is intended to encompass one or more enzymes that are identical, similar, or corresponding (i.e., in terms of structure and function) obtained from different organisms. In some embodiments, it is desirable to identify homologs having similar quaternary, tertiary, and / or primary structures to a reference protein. In some embodiments, a homologous protein induces one or more similar immune responses as a reference protein. In some embodiments, a homologous protein is engineered to produce an enzyme having one or more desired activities.

[0036] The degree of homology between sequences can be determined using any suitable method known in the art (see, e.g., Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol., 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al. (1984) Nucleic Acids Res. 12:387-95).

[0037] In one embodiment, PILEUP is a program that can be used to determine the level of sequence homology. PILEUP uses progressive, pairwise comparisons to create a multiple sequence alignment from a group of related sequences. It can also draw a tree showing the clustering relationship for creating this comparison. PILEUP uses a simplified version of Feng and Doolittle's progressive alignment method (Feng and Doolittle (1987) J.Mol.Evol. [Journal of Molecular Evolution] 35:351-60). This method is similar to the method described by Higgins and Sharp ((1989) CABIOS [Computer Applications in Biological Sciences] 5:151-53). Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted end gap. Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al. ((1990) J. Mol. Biol. 215:403-10) and Karlin et al. ((1993) Proc. Natl. Acad. Sci. USA 90:5873-87). A particularly useful BLAST program is the WU-BLAST-2 program (see, e.g., Altschul et al. (1996) Meth. Enzymol. 266:460-80). The parameters "W", "T", and "X" determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), an alignment (B) of 50, an expectation (E) of 10, M'5, N'-4, and a comparison of both strands.

[0038] As used herein, the phrases "substantially similar" and "substantially identical" in the context of at least two nucleic acids or polypeptides typically mean that the polynucleotides or polypeptides comprise sequences that are at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or even at least about 99% identical, or more identical, compared to a reference (i.e., wild-type) sequence. Percent sequence identity is calculated using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. The default parameters for the CLUSTAL W algorithm are:

[0039] Open Shot Penalty: 10.0

[0040] Gap Extension Penalty: 0.05

[0041] Protein Weight Matrix: BLOSUM Series

[0042] DNA weight matrix: IUB

[0043] Delayed Divergence Sequence %: 40

[0044] Space separation distance: 8

[0045] DNA conversion weight: 0.50

[0046] List hydrophilic residues: GPSNDQEKR

[0047] Use Negativity Matrix: Off

[0048] Toggle special residue penalties: On

[0049] Toggle hydrophilic penalty: On

[0050] Switch to end the gap separation penalty level

[0051] Another indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., within a range of moderate to high stringency).

[0052] As used herein, the term "gene" is synonymous with the term "allele" and refers to a nucleic acid that encodes and directs the expression of a protein or RNA. Vegetative forms of filamentous fungi are typically haploid, so a single copy of a given gene (i.e., a single allele) is sufficient to confer a given phenotype. When an organism contains more than one similar gene, the term "allele" is often preferred, in which case each different similar gene is referred to as a different "allele."

[0053] As used herein, "constitutive" expression refers to the production of a polypeptide encoded by a particular gene under essentially all typical growth conditions, as opposed to "conditional" expression which requires the presence of a specific substrate, temperature, etc. to induce or activate expression.

[0054] As used herein, the term "expressing a polypeptide" and similar terms refer to the cellular process by which the polypeptide is produced using the cell's translation machinery (eg, ribosomes).

[0055] As used herein, "overexpressing a polypeptide," "increasing expression of a polypeptide," and similar terms refer to expressing a polypeptide at a level greater than normal compared to that observed in a parental or wild-type cell that does not include the specified genetic modification.

[0056] As used herein, "expression cassette" refers to a DNA fragment that includes a promoter, an amino acid coding region, and a terminator (i.e., promoter::amino acid coding region::terminator) and other nucleic acid sequences required to allow production of the encoded polypeptide in a cell. The expression cassette can be exogenous (i.e., introduced into the cell) or endogenous (i.e., present in the cell).

[0057] As used herein, the terms "fused" and "fusion" with respect to two DNA segments (eg, a promoter and the coding region of a polypeptide) refer to a physical linkage that results in the two DNA segments becoming a single molecule.

[0058] As used herein, the terms "wild type" and "native" are used interchangeably and refer to a gene, protein, or strain as found in nature, or one that has not been intentionally modified for the advantage of the yeast in question.

[0059] As used herein, the term "target protein" refers to a polypeptide that is desired to be expressed in a modified yeast. Such proteins can be enzymes, substrate binding proteins, surfactant proteins, structural proteins, selectable markers, etc., and can be expressed. The target protein is encoded by an endogenous gene or a heterologous gene (i.e., a target gene) relative to the parent strain. The target protein can be expressed intracellularly or as a secreted protein.

[0060] As used herein, "gene disruption" refers to any genetic or chemical manipulation that substantially prevents cells from producing functional gene products in host cells, such as proteins, i.e., mutations. Exemplary methods of destruction include complete or partial deletion or mutagenesis of any part of a gene (including polypeptide coding sequences, promoters, enhancers, or other regulatory elements), wherein mutagenesis encompasses substitutions, insertions, deletions, inversions, and combinations and variations thereof, any of which substantially prevents the generation of functional gene products. CRISPR, RNAi, antisense, or any other method for eliminating gene expression can also be used to destroy genes. Genes can be destroyed by deletion or genetic manipulation of non-adjacent control elements. As used herein, "gene deletion" refers to removing the gene from the host cell genome. When a gene includes a control element (e.g., an enhancer element) that is not immediately adjacent to a gene coding sequence, the deletion of a gene refers to the deletion of a coding sequence and optionally adjacent enhancer elements (e.g., including but not limited to promoter and / or terminator sequences), but the deletion of non-adjacent control elements is not required. Gene deletion also refers to the deletion of a portion of a coding sequence, or a portion of a promoter that may or may not be immediately adjacent to a coding sequence, wherein the functional activity of the gene of interest is absent in the engineered cell.

[0061] As used herein, the terms "genetic manipulation," "genetic alteration," "genetic engineering," and similar terms are used interchangeably and refer to changes in a nucleic acid sequence. Changes can include, but are not limited to, substitutions, deletions, insertions, or chemical modifications of at least one nucleic acid in a nucleic acid sequence.

[0062] As used herein, a "functional polypeptide / protein" is a protein that has activity (e.g., enzymatic activity, binding activity, surface active properties, etc.) and has not been mutagenized, truncated, or otherwise modified to eliminate or reduce this activity. As noted, a functional polypeptide can be thermostable or thermolabile.

[0063] As used herein, a "functional gene" is a gene that can be used by cellular components to produce an active gene product (typically a protein). Functional genes are the opposite of disrupted genes that have been modified so that they cannot be used by cellular components to produce an active gene product, or have a reduced ability to be used by cellular components to produce an active gene product.

[0064] As used herein, yeast cells have been "modified to prevent the production of a specified protein" if they have been genetically or chemically altered to prevent the production of a functional protein / polypeptide exhibiting an activity characteristic of the wild-type protein. Such modifications include, but are not limited to, deletion or disruption of a gene encoding a protein (as described herein), modification of a gene such that the encoded polypeptide lacks the aforementioned activity, modification of a gene that affects post-translational processing or stability, and combinations thereof.

[0065] As used herein, "attenuation of a pathway" or "attenuation of flux through a pathway" (i.e., a biochemical pathway) broadly refers to any genetic or chemical manipulation that reduces or completely prevents the flux of a biochemical substrate or intermediate through a metabolic pathway. Attenuation of a pathway can be achieved by a variety of well-known methods. Such methods include, but are not limited to: complete or partial deletion of one or more genes, replacement of wild-type alleles of these genes with mutant forms encoding enzymes with reduced catalytic activity or increased Km values, modification of promoters or other regulatory elements that control expression of one or more genes, engineering these enzymes or mRNAs encoding these enzymes for reduced stability, misdirecting enzymes into cellular compartments less likely to interact with substrates and intermediates, use of interfering RNA, and the like.

[0066] As used herein, "aerobic fermentation" refers to growth and production processes in the presence of oxygen.

[0067] As used herein, "anaerobic fermentation" refers to growth and production in the absence of oxygen.

[0068] As used herein, the expression "end of fermentation" refers to the stage of fermentation when the economic advantage of continuing the fermentation to produce a small amount of additional alcohol is outweighed by the cost of continuing the fermentation, in terms of fixed and variable costs. In a more general sense, "end of fermentation" refers to the point at which the fermentation no longer produces significant amounts of additional alcohol, i.e., no more than about 1% additional alcohol.

[0069] As used herein, the expression "carbon flux" refers to the turnover rate of carbon molecules through a metabolic pathway. Carbon flux is regulated by enzymes involved in metabolic pathways (such as the glucose metabolic pathway and the maltose metabolic pathway).

[0070] As used herein, the singular articles "a / an" and "the" include plural referents unless the context clearly dictates otherwise. All references cited herein are hereby incorporated by reference in their entirety. Unless otherwise stated, the following abbreviations / acronyms have the following meanings:

[0071] Unless otherwise stated, the following have the following meanings:

[0072] ℃ degrees Celsius

[0073] AA α-amylase

[0074] AADH acetaldehyde dehydrogenase

[0075] bp base pair

[0076] DNA deoxyribonucleic acid

[0077] ds or DS dry solid

[0078] EC Enzyme Commission

[0079] EtOH

[0080] g or gm gram

[0081] g / L grams per liter

[0082] GA glucoamylase

[0083] H2O water

[0084] HPLC high-performance liquid chromatography

[0085] hr or h hours

[0086] kg kilogram

[0087] M Moore

[0088] mg milligrams

[0089] min minutes

[0090] mL or ml milliliters

[0091] mM millimole

[0092] N equivalent concentration

[0093] nm nanometer

[0094] PCR polymerase chain reaction

[0095] PKL phosphoketolase

[0096] ppm parts per million

[0097] PTA phosphotransacetylase

[0098] Δ is associated with deletion

[0099] μg microgram

[0100] μL and μl microliter

[0101] μM micromolar

[0102] Numerical values ​​and ranges may be presented herein with the term "about" preceding the numerical value. The term "about" is used herein to provide textual support for the exact number that follows it, as well as for numbers that are close to or approximately the number that follows the term. When determining whether a number is close to or approximately the number of a specific narration, the close or approximate unrecited number may be a number that provides a substantial equivalent of the number of the specific narration in the context in which it is presented. For example, with respect to numerical values, the term "about" refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in the context. All values ​​and ranges implicitly include the term "about" unless the context clearly dictates otherwise.

[0103] II. Modified Cells with Reduced Expression of Polypeptides Causing High Dry Solids and High Temperature Intolerance

[0104] Described are modified fermenting organisms with reduced levels of gene expression that result in a lack of robustness under high dry solids and high temperature conditions. In some cases, the modified cells also accumulate reduced amounts of acetic acid. The genes and gene products are briefly described below.

[0105] SKG3 (YLR187W) is a paralog of CAF120. It encodes a protein of unknown function. The green fluorescent protein-SKG3 fusion protein is localized to the cell periphery, cytoplasm, bud, and bud neck. It has been determined that the deletion of SKG3 has little effect on ethanol production under standard (medium) dry solids (DS) conditions (34% DS, 32°C), but provides a significant improvement under high DS conditions (37.8% DS, 32°C) and high temperature conditions (32% DS, 36.6°C ramp).

[0106] YNR068C is a putative protein expressed as a read-through of the BSC5 gene. It has been determined that deletion of the YNR068C gene has little effect on ethanol production under standard (medium) dry solids (DS) conditions (34% DS, 32°C), but provides significant improvements under high DS conditions (37.8% DS, 32°C) and high temperature conditions (32% DS, 36.6°C ramp).

[0107] MNN14 (YJR061W) is a protein required for N-glycan mannosylphosphorylation. It is a paralog of MNN4. N-glycan mannosylphosphorylation is abolished in the MNN4 and MNN14 double mutant. It has been found that deletion of the MNN14 gene increases ethanol production (>1.2%) under standard conditions (34% DS, 32°C) and significantly increases ethanol production under high DS and high temperature conditions (32% DS, 36.6°C ramp).

[0108] PIN3 (YPR154W) is a paralog of LSB1, a negative regulator of actin nucleation promoting factor activity. PIN3 interacts with Las17p and, together with LSB1, synergistically inhibits actin filament nucleation. Its expression level increases in response to heat stress. It has been found that deletion of the PIN3 gene has little effect on ethanol titer under standard conditions (34% DS, 32°C), but significantly improves the performance of the strain under high dry solids conditions (37.8% DS, 32°C) and high temperature conditions (32% DS, 36.6°C ramp).

[0109] PEX18 (YHR160C) is a peroxisomal enzyme. It is required for targeting peroxisomal matrix proteins containing PTS2 to peroxisomes. PEX18 interacts with Pex7p and is primarily responsible for peroxisomal import during growth on oleic acid. Its expression is induced during growth on oleic acid. It has been found that deletion of the PEX18 gene has little effect on ethanol production under standard conditions (34% DS, 32°C), but significantly increases production under high DS conditions (37.8% DS, 32°C) and high temperature conditions (32% DS, 36.6°C ramp).

[0110] MNN4 (YKL201C) is a putative positive regulator of the mannosylphosphotransferase Mnn6p. It is involved in the phosphorylation of mannosyl N-linked oligosaccharides. MNN4 expression increases during the late logarithmic and stationary growth phases. It has a paralog in MNN14. It has been shown that deletion of the MNN4 gene has little effect on ethanol production under standard conditions (34% DS, 32°C) but significantly increases production under high DS conditions (37.8% DS, 32°C) and high temperature conditions (32% DS, 36.6°C ramp).

[0111] RIM20 (YOR275C) is a member of the PalA / AIP1 / Alix family. It is involved in the response to alkaline pH. RIM20 is involved in the proteolytic activation of Rim101p. Deletion of the RIM20 gene has been found to have little effect on ethanol production under standard conditions (34% DS, 32°C) but significantly improves production under high DS conditions (37.8% DS, 32°C) and high temperature conditions (32% DS, 36.6°C ramp). It significantly reduces acetate accumulation under both standard and high DS conditions.

[0112] PAN2 (YGL094C) is the catalytic subunit of the Pan2p-Pan3p poly(A)-ribonuclease complex. This complex controls poly(A)-tail length and regulates the stoichiometry and activity of post-replication repair complexes. We found that deletion of the PAN2 gene had little effect on ethanol production under standard conditions (34% DS, 32°C), but significantly increased production under high dry solids conditions (37.8% DS, 32°C) and high temperature conditions (32% DS, 36.6°C ramp). It reduced acetate under all conditions tested.

[0113] As described above, it has now been determined that yeast cells with genetic modifications that reduce expression of these proteins result in high DS and high temperature tolerance and, in some cases, reduced acetic acid accumulation compared to otherwise identical parental cells.

[0114] The destruction of the gene that is responsible for high dry solids and high temperature tolerance and the reduction of acetic acid in some cases can be produced by the destruction of the gene of the encoding corresponding polypeptide present in the parental strain.Because the destruction of one or more of these genes is the main genetic determinant that gives the beneficial phenotype of modified cell, so in some embodiments, modified cell only needs to comprise the destroyed gene that is responsible for high dry solids and high temperature tolerance and the reduction of acetic acid in some cases, and all other genes can remain intact.In other embodiments, compared with the parental cell of derived modified cell, these modified cells can optionally include other genetic alteration.Although such other genetic alteration is not necessary to give described phenotype, they can give other advantages of modified cell.

[0115] Any suitable method that substantially reduces the expression of the corresponding functional polypeptide can be used to disrupt the genes responsible for high dry solids and high temperature tolerance, and in some cases, reduced acetate. Exemplary disruption methods include, but are not limited to: complete or partial deletion of a gene, including complete or partial deletion of a coding sequence, promoter, terminator, enhancer, or other regulatory elements; and complete or partial deletion of a portion of a chromosome containing any portion of such a gene.

[0116] The ad hoc approach that is used to destroy the gene that is responsible for high dry solids and high temperature tolerance and that acetic acid reduces in some cases is included in any part of such gene encoding sequence, promotor, terminator, enhancer or other regulating and controlling element and carries out nucleotide replacement or insertion.Preferably, disappearance, insertion and / or replacement (being referred to as sudden change) are carried out by the genetic manipulation that uses sequence-specific molecular biology technique, contrary with by chemical mutagenesis, chemical mutagenesis is not targeted specific nucleic acid sequence usually.Nevertheless, chemical mutagenesis still can be used for destroying gene in theory.

[0117] In some embodiments, the reduction in the amount of the functional polypeptide responsible for high dry solids and high temperature tolerance, and in some cases, reduced acetate, is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more compared to the amount of the functional polypeptide produced by an otherwise identical parent cell grown under the same conditions.

[0118] As described below, strains with disruptions in SKG3, YNR068C, MNN14, PIN3, PEX18, MNN4, RIM20, and PAN2 exhibit significant increases in ethanol production (e.g., 2.4%-6.2%) under high DS conditions and / or high temperature ramp conditions. In some embodiments, the increase in ethanol production by the modified strain is at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 6%, or more compared to the amount of acetic acid produced by the parental cells grown under the same conditions.

[0119] As also described below, strains with disruptions in RIM20 and PAN2 exhibit significant reductions in ethanol production (e.g., 3.4%-15%) under high DS conditions and / or high temperature ramp conditions. In some embodiments, the reduction in acetic acid produced by the modified strain is at least 20%, at least 15%, at least 10%, at least 7%, at least 5%, at least 4%, at least 3%, or more, compared to the amount of acetic acid produced by the parental cells grown under the same conditions.

[0120] III. Modified Yeast Cells with Exogenous PKL Pathway Genes

[0121] In some embodiments, disruption of one or more genes that affect fermentation under high solids conditions can be combined with expression of genes in the PKL pathway to reduce the production of elevated amounts of acetic acid associated with introduction of an exogenous PKL pathway into yeast.

[0122] Previously described are yeast cells (WO 2015148272) with engineered heterologous PKL pathways. These cells express heterologous phosphoketolase (PKL), phosphotransacetylase (PTA), and acetylated acetyl dehydrogenase (AADH) (optionally with other enzymes) to divert carbon flux away from the glycerol pathway and toward the synthesis of acetyl-CoA, which is then converted into ethanol. Compared to otherwise identical parent yeast cells, such modified cells can increase ethanol production during fermentation.

[0123] IV. Modified Yeast Cells with Other Mutations Affecting Alcohol Production

[0124] In some embodiments, in addition to disrupting one or more genes that affect fermentation under high solids conditions (optionally with a heterologous PKL pathway), the modified yeast cells of the invention also include additional beneficial modifications.

[0125] The modified cells may further include mutations that result in attenuation of the native glycerol biosynthetic pathway and / or the recycled glycerol pathway, which are known to increase alcohol production. Methods for attenuating the glycerol biosynthetic pathway in yeast are known and include, for example, reducing or eliminating endogenous NAD-dependent glycerol 3-phosphate dehydrogenase (GPD) or phosphoglycerophosphatase (GPP) activity by disrupting one or more of the genes GPD1, GPD2, GPP1, and / or GPP2. See, for example, U.S. Patent Nos. 9,175,270 (Elke et al.), 8,795,998 (Pronk et al.), and 8,956,851 (Argyros et al.). Methods to enhance the reuse glycerol pathway by overexpression of glyceroldehydrogenase (GCY1) and dihydroxyacetone kinase (DAK1) to convert glycerol to dihydroxyacetone phosphate (Zhang et al. (2013) J. Ind. Microbiol. Biotechnol. 40: 1153-60).

[0126] The modified yeast may further be characterized by increased acetyl-CoA synthase (also known as acetyl-CoA ligase) activity (EC 6.2.1.1) to scavenge (i.e., capture) acetic acid produced by chemical or enzymatic hydrolysis of acetyl-phosphate (or present in the yeast culture medium for any other reason) and convert it into acetyl-CoA. This partially reduces the undesirable effects of acetic acid on yeast cell growth and can further contribute to improved alcohol yields. Increasing acetyl-CoA synthase activity can be achieved by introducing a heterologous acetyl-CoA synthase gene into the cells, increasing the expression of an endogenous acetyl-CoA synthase gene, etc.

[0127] In some embodiments, the modified cell may further comprise a gene encoding a gene having NAD +A heterologous gene encoding a protein that is dependent on the activity of acetylating acetaldehyde dehydrogenase and / or a heterologous gene encoding pyruvate formate lyase. For example, the introduction of such genes in combination with glycerol pathway attenuation is described in U.S. Patent No. 8,795,998 (Pronk et al.). In some embodiments of the compositions and methods of the present invention, the yeast is intentionally deficient in one or more heterologous genes encoding acetylating acetaldehyde dehydrogenase, pyruvate formate lyase, or both.

[0128] In some embodiments, the modified yeast cells of the present invention can further overexpress a sugar transporter-like (STL1) polypeptide to increase glycerol uptake (see, e.g., Ferreira et al. (2005) Mol. Biol. Cell. 16:2068-76; et al. (2015) Mol. Microbiol. 97:541-59 and WO 2015023989 A1) to increase ethanol production and reduce acetic acid.

[0129] In some embodiments, the modified yeast cells of the present invention further include a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway is an isobutanol biosynthetic pathway. In some embodiments, the isobutanol biosynthetic pathway comprises a polynucleotide encoding a polypeptide that catalyzes the conversion of a substrate to a product selected from the group consisting of: (a) pyruvate to acetolactate; (b) acetolactate to 2,3-dihydroxyisovalerate; (c) 2,3-dihydroxyisovalerate to 2-ketoisovalerate; (d) 2-ketoisovalerate to isobutyraldehyde; and (e) isobutyraldehyde to isobutanol. In some embodiments, the isobutanol biosynthetic pathway comprises a polynucleotide encoding a polypeptide having acetolactate synthase, ketoacid reductoisomerase, dihydroxyacid dehydratase, ketoisovalerate decarboxylase, and alcohol dehydrogenase activity.

[0130] In some embodiments, the modified yeast cell comprising a butanol biosynthetic pathway further comprises a modification in a polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In some embodiments, the yeast cell comprises a deletion, mutation, and / or substitution in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In some embodiments, the polypeptide having pyruvate decarboxylase activity is selected from the group consisting of PDC1, PDC5, PDC6, and combinations thereof. In some embodiments, the yeast cells further comprise a deletion, mutation, overexpression, and / or substitution in one or more endogenous polynucleotides encoding FRA2, ALD6, ADH1, GPD2, BDH1, DLS1, DPB3, CPR1, MAL23C, MNN4, PAB1, TMN2, HAC1, PTC1, PTC2, OSM1, GIS1, CRZ1, HUG1, GDS1, CYB2P, SFC1, MVB12, LDB10, C5SD, GIC1, GIC2, YMR226C, PHO13, ADH5, MIG1, MIG2, MIG3, JID1, KGD2, ARG7, LEU4, MET2, DAL7, and ISN1.

[0131] V. Modified Yeast Cells with Other Beneficial Mutations

[0132] In some embodiments, in addition to disrupting one or more of the genes that affect fermentation under high solids conditions (optionally with a heterologous PKL pathway and still further optionally in combination with other genetic modifications that are beneficial to alcohol production), the modified yeast cells of the present invention further include any number of additional genes of interest encoding proteins of interest. Additional genes of interest can be introduced before, during, or after genetic manipulations that result in increased production of active MIG3 polypeptides. The protein of interest includes selectable markers, carbohydrate processing enzymes and other commercially relevant polypeptides, including but not limited to enzymes selected from the group consisting of dehydrogenases, transketolases, phosphoketolase, transaldolase, epimerase, phytase, xylanase, β-glucanase, phosphatase, protease, α-amylase, β-amylase, glucoamylase, pullulanase, isoamylase, cellulase, trehalase, lipase, pectinase, polyesterase, cutinase, oxidase, transferase, reductase, hemicellulase, mannanase, esterase, isomerase, pectinase, lactase, peroxidase and laccase. The protein of interest can be secreted, glycosylated and modified in other ways.

[0133] VI. Use of modified yeast for increased alcohol production

[0134] The compositions and methods of the present invention include methods for increasing alcohol production and reducing glycerol production in fermentation substrates in some cases. In certain embodiments, the compositions and methods include methods for increasing alcohol production and reducing acetic acid production in some cases. In certain embodiments, the compositions and methods include methods for increasing alcohol production and reducing acetic acid and glycerol production in some cases. Such methods are not limited to specific fermentation processes. It is expected that the engineered yeast of the present invention is a "drop-in" substitute for conventional yeast in any alcohol fermentation facility. Although primarily used for fuel alcohol production, yeast of the present invention can also be used to produce drinkable alcohol, including wine and beer.

[0135] Methods for producing alcohol (e.g., ethanol) from carbohydrate substrates are generally well-known. The conversion of carbohydrate substrates to fermentation products such as alcohol typically involves multiple processing steps and reagents, each of which is important for maximizing conversion efficiency, flux, consistency, concentration, and / or yield. For example, common production processes for producing ethanol by fermentation of carbohydrate substrates (see, e.g., Part VIII below) typically involve processes including but not limited to milling or grinding raw materials, liquefaction, saccharification, fermentation, and distillation, and reagents (e.g., enzymes and other components, e.g., microorganisms and / or chemicals) can be added during unit operations to promote reaction. For example, during alcohol production, liquefaction includes diluting the mash to reduce its viscosity via partial hydrolysis. Enzymes (e.g., α-amylases) can be used to promote the diluting process. During saccharification, the complex carbohydrates of the diluting mash are converted into monosaccharides. Similarly, enzymes (e.g., glucoamylases) can be used to promote the conversion process. Then during the fermentation process step, the resulting sugars are converted into ethanol by yeast. Enzymes such as glucoamylase, fungal alpha amylase and trehalase can also be included to control the glucose profile during fermentation. Reagents such as enzymes can also be used in the grinding and distillation steps to promote the process. In some cases, the saccharification and fermentation steps can be combined into a single simultaneous saccharification and fermentation (SSF) step. Other well-known processes include raw starch hydrolysis (RSH), which includes grinding starch-containing material to produce a carbohydrate substrate and then performing SSF at a temperature lower than the initial gelatinization temperature. Enzymes (e.g., acid fungal amylase, glucoamylase) can also be used in such a process to produce a fermentation product.

[0136] In some embodiments, the modified yeast described herein is used in a process for producing a fermentation product. Therefore, on the one hand, a method for producing a fermentation product is provided, the method comprising fermenting a carbohydrate substrate with a modified yeast cell as described herein. In some embodiments, the method comprises a liquefaction step, optionally comprising an alpha-amylase. In some embodiments, the method comprises a saccharification step, optionally comprising a glucoamylase. In some embodiments, the fermentation product is ethanol and / or butanol. In some embodiments, the fermentation product is ethanol. In some embodiments, the fermentation product is butanol. In some embodiments, fermentation occurs under conditions for producing a fermentation product. In some embodiments, the conditions comprise a dry solids percentage (DS%) of at least 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In some embodiments, the conditions comprise a DS% of at least 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, or 45%. In some embodiments, the conditions include a DS of at least 37%. In some embodiments, the conditions include a DS in the range of 32% to 45%. In some embodiments, the conditions include a DS in the range of 33% to 45%. In some embodiments, the conditions include a DS in the range of 34% to 45%. In some embodiments, the conditions include a DS in the range of 35% to 45%. In some embodiments, the conditions include a DS in the range of 36% to 45%. In some embodiments, the conditions include a DS in the range of 37% to 45%. In some embodiments, the conditions include a DS in the range of 38% to 45%. In some embodiments, the conditions include a DS in the range of 39% to 45%. In some embodiments, the conditions include a DS in the range of 40% to 45%. In some embodiments, the conditions include a DS in the range of 34% to 40%. In some embodiments, the conditions include a DS in the range of 35% to 40%. In some embodiments, the conditions include a DS in the range of 36% to 40%. In some embodiments, the conditions include a DS in the range of 37% to 40%. In some embodiments, the conditions include a temperature of at least 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, or 75°C. In some embodiments, the conditions include a temperature of at least 32°C. In some embodiments, the conditions include a temperature of at least 34°C. In some embodiments, the conditions include a temperature of at least 35°C. In some embodiments, the conditions include a temperature of at least 36°C. In some embodiments, the conditions include a temperature in the range of 30°C to 75°C. In some embodiments, the conditions include a temperature in the range of 30°C to 70°C. In some embodiments, the conditions include a temperature in the range of 30°C to 65°C.In some embodiments, the conditions include a temperature in the range of 30°C to 60°C. In some embodiments, the conditions include a temperature in the range of 30°C to 55°C. In some embodiments, the conditions include a temperature in the range of 30°C to 50°C. In some embodiments, the conditions include a temperature in the range of 30°C to 45°C. In some embodiments, the conditions include a temperature in the range of 30°C to 40°C. In some embodiments, the conditions include a temperature in the range of 34°C to 45°C. In some embodiments, the conditions include a temperature in the range of 35°C to 45°C. In some embodiments, the conditions include a temperature in the range of 34°C to 40°C. In some embodiments, the conditions include a temperature in the range of 35°C to 40°C. In some embodiments, the conditions are specific to the fermentation step.

[0137] As described above, the modified yeast described herein are capable of increasing ethanol production during fermentation at high DS% and / or high temperature. In some embodiments, high DS% is a DS% of at least 35%. In some embodiments, high DS% is a DS% in the range of 35% to 45%. In some embodiments, high temperature is a temperature of at least 34°C. In some embodiments, high temperature is a temperature in the range of 34°C to 40°C. In some embodiments, high temperature is a temperature of at least 35°C. In some embodiments, high temperature is a temperature in the range of 35°C to 40°C. In some embodiments, the modified yeast described herein produce increased amounts of ethanol under fermentation conditions comprising high DS% and / or high temperature, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produce increased amounts of ethanol under fermentation conditions comprising high DS% and high temperature, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produce increased amounts of ethanol under fermentation conditions comprising high DS% and high temperature, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produce increased amounts of ethanol under fermentation conditions comprising high DS%, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produce increased amounts of ethanol under fermentation conditions comprising high DS%, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produce increased amounts of ethanol under fermentation conditions comprising high temperature, compared to the parent cell under the same fermentation conditions. In some embodiments, the increase in ethanol is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% greater than the amount of ethanol produced by the parent cell under the same fermentation conditions. In some embodiments, the increase in ethanol is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% greater than the amount of ethanol produced by the parent cell under the same fermentation conditions. In some embodiments, the increase in ethanol is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% greater than the amount of ethanol produced by the parent cell under the same fermentation conditions. In some embodiments, the increase in ethanol is in the range of about 0.1% to 15% compared to the amount of ethanol produced by the parent cell under the same fermentation conditions. In some embodiments, the increase in ethanol is in the range of about 0.5% to 15% compared to the amount of ethanol produced by the parent cell under the same fermentation conditions. In some embodiments, the increase in ethanol is in the range of about 1% to 15% compared to the amount of ethanol produced by the parent cell under the same fermentation conditions. In some embodiments, the increase in ethanol is in the range of about 0.1% to 10% compared to the amount of ethanol produced by the parent cell under the same fermentation conditions. In some embodiments, the increase in ethanol is in the range of about 0.5% to 10% compared to the amount of ethanol produced by the parent cell under the same fermentation conditions.In some embodiments, the increase in ethanol is in the range of about 1% to 10% compared to the amount of ethanol produced by the parent cell under the same fermentation conditions. In some embodiments, the increase in ethanol is in the range of about 1% to 5% compared to the amount of ethanol produced by the parent cell under the same fermentation conditions. The terms "increased," "enhanced," "enhanced," "greater than," "increased," "more than," and the like are used interchangeably herein.

[0138] In some embodiments, the modified yeast described herein produce a reduced amount of acetic acid under fermentation conditions comprising a high DS% and / or a high temperature, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produce a reduced amount of acetic acid under fermentation conditions comprising a high DS% and a high temperature, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produce a reduced amount of acetic acid under fermentation conditions comprising a high DS%, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produce a reduced amount of acetic acid under fermentation conditions comprising a high DS%, compared to the parent cell under the same fermentation conditions. In some embodiments, the reduction in acetic acid is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or 50% less than the amount of acetic acid produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in acetate is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, or 30% less than the amount of acetate produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in acetate is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% less than the amount of acetate produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in acetate is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% less than the amount of acetate produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in acetate is in the range of about 0.1% to 30% less than the amount of acetate produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in acetic acid is in the range of about 1% to 30% compared to the amount of acetic acid produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in acetic acid is in the range of about 5% to 30% compared to the amount of acetic acid produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in acetic acid is in the range of about 10% to 25% compared to the amount of acetic acid produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in acetic acid is in the range of about 5% to 10% compared to the amount of acetic acid produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in acetic acid is in the range of about 1% to 5% compared to the amount of acetic acid produced by the parent cell under the same fermentation conditions. The terms "reduced," "decreased," "reduced," "less," "less than," and the like are used interchangeably herein.

[0139] In some embodiments, the modified yeast described herein produces a reduced amount of glycerol under fermentation conditions comprising a high DS% and / or high temperature, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produces a reduced amount of glycerol under fermentation conditions comprising a high DS% and high temperature, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produces a reduced amount of glycerol under fermentation conditions comprising a high DS%, compared to the parent cell under the same fermentation conditions. In some embodiments, the modified yeast described herein produces a reduced amount of glycerol under fermentation conditions comprising a high DS%, compared to the parent cell under the same fermentation conditions. In some embodiments, the reduction in glycerol is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% less than the amount of glycerol produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in glycerol is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% less than the amount of glycerol produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in glycerol is in the range of about 0.1% to 5% compared to the amount of glycerol produced by the parent cell under the same fermentation conditions. In some embodiments, the reduction in glycerol is in the range of about 1% to 5% compared to the amount of glycerol produced by the parent cell under the same fermentation conditions.

[0140] VII. Yeast Cells Suitable for Modification

[0141] Yeast is a unicellular eukaryotic microorganism classified as a member of the fungi kingdom, and includes organisms from the Ascomycota and Basidiomycota. Yeast that can be used for alcohol production includes but is not limited to Saccharomyces species (including Saccharomyces cerevisiae) and Kluyveromyces (Kluyveromyces), Lachancea (Lachancea) and Schizosaccharomyces (Schizosaccharomyces) species. Many yeast strains are commercially available, many of which have been selected or genetically engineered to obtain desired characteristics, such as high alcohol production, fast growth rate, etc. Some yeasts have been genetically engineered to produce heterologous enzymes, such as glucoamylase or α-amylase.

[0142] VIII. Substrates and Products

[0143] The production of alcohol from many carbohydrate substrates (including but not limited to corn starch, sugar cane, cassava and molasses) is well known, as are numerous variations and improvements in enzyme and chemical conditions and mechanical methods. It is believed that the compositions and methods of the present invention are fully compatible with such substrates and conditions.

[0144] Alcohol fermentation products include organic compounds with a hydroxyl functional group (-OH) bonded to a carbon atom. Exemplary alcohols include, but are not limited to, methanol, ethanol, n-propyl alcohol, isopropyl alcohol, n-butanol, isobutyl alcohol, n-amyl alcohol, 2-amyl alcohol, isoamyl alcohol, and higher alcohols. The most commonly prepared fuel alcohols are ethanol and butanols.

[0145] These and other aspects and embodiments of the yeast strains and methods of the invention will be clear to the skilled artisan in view of this specification.The following examples are intended to further illustrate but not limit the compositions and methods.

[0146] Examples

[0147] Example 1

[0148] Genetic manipulation and strain construction

[0149] Methods for disrupting genes in yeast and reducing functional polypeptide levels have been described in, for example, WO2018089333 (US2019276905), WO 2018226573 (US2020131591), WO 2019083879 (US2021179674), WO 2019173225 (US2021040474), WO 2020263732 (US2022251608) and US2020354756. Reference is made to the following amino acid and nucleic acid sequences.

[0150] The amino acid sequence of the SKG3 polypeptide is shown in SEQ ID NO: 1 below:

[0151]

[0152] The amino acid sequence of the putative YNR068C polypeptide is shown in SEQ ID NO:2 below: MRNRQNQKNWTNLRGLLSVEEREKLENLRLELVCMQAANSIPHDPPEISSLETELICLTTNTKDCKPVRFHSDLLLKKHKYNEIKKIFKEILENIEAYRDEFTKNQTKINLLLADDARASLRNRSLDFSDLMPSSIIKDVQVLANMEANVVVMKNALKTKLVGEKSVPVASSPISSIIPRTSRNKKTSPSNYHHSVLSHRKSNEWNQVSSTEYKRTLLLNIKYNDDFKATIVPSFESCLCSRSYFLRVKLHFDKGVGSAEIDIPVQVKNSFI

[0153] The amino acid sequence of MNN14 polypeptide is shown as SEQ ID NO:3 below: MMLSLRRFSMYVLRSLRLHFKKIIITLLTIQLLFITIFVLGGRSSIIDGNWKSFMALFFKPLAYTNRNNNHASFDLRSKDNVAKLYEKMNFDTSGKWIDTYTLKNNLLTVKMGPEKGQVLDSVDELRYYDNDPRLVWSVLLDHLLESDSNEYAFSWYDWANFDSTNKLIALRHTNISCQFVCEGAFDKNVLEMVESEVQEPLFVTNRNKYDESLWYNRVRKVVDSNSVQQAIHDHCMNNDAYSNGTPFELPFIISEISERLRPEVYDLQAKNHLLYSNFTPLSLTVLDSDKDAYRINLKTTDSSKSNIVQTNLLQNYIKRHRNEMVNGDLIFNHTSMFEKFLHHGSTKKRKLDVEALDKTIYAGEYLELSPSDFQFNAKERIIELETRLRSEGLPSHDTHYLRSLKTSVNTSPALQQKYFAEASDITDATADGHHRDRRFFSIGHNLLNDPQEFEARLNSLIRNFQKFVKANGLISWLSHGTLYGYLYDGLKFPWDVDHDLQMPIKHLHYLSQYFNQSLILEDPREGNGRFLLDVGSAITVGVHGNGENNIDARFIDIDSGIYIDITGLSVSSDAAKQYMSKFVEEESSGESFSALIEDYKFDENDYFDEVDGREGLAKYTIHELMEWVNSHPDDFTDAEKNLVTKTYKKELAISRSDYAEKDLSPKQRYLVNEKYNLYNCRNQHFSSLNIISPLRNTMFSGVSAFVPNRPIATLNNEYKVPAKYGLLSFQGKVYLPEFRYWFSFADMKKFANLQLKEPKITRLESPLNDLKFSDISLLITNILKCGFHSVFASLFNSFDSTVYRLKELEIQYDPSLSEEEKSSLLKTLRRGMSKKIKSPEKDPIIYIYERKLWENVEKLLNASNIYNIASQVEKEKGKEFVERSQQVYERNFDGFRLPDGGNSKTVNDLNSKGLNLFGDNKKTSNNIFGSDQKY

[0154] The amino acid sequence of the PIN3 polypeptide is shown in SEQ ID NO: 4 below: MSASLINRSLTNIRTELDFLKGSNVISNDVYDQINKSLPAKWDPANAPRNASPASLEYVEALYQFDPQQDGDLGLKPGDKVQLLEKLSPEWYKGSCNGRTGIFPANYVKPAFSGSNGPSNLPPPPQYKAQELQQIPTQNSAASSYQQQPFPPPSTNYYQQPQQQPQQAPPPQQQQQQQQHQSSHSHLKSFGSKLGNAAIFGAGASIGSDIVNNIF

[0155] The amino acid sequence of PEX18 polypeptide is as follows SEQ ID NO:5 shows: MNSNRCQTNEVNKFISSTEKGPFTGRDNTLSFNKIGSRLNSPPILKDKIELKFLQHSEDLNQSRSYVNIRPRTLEDQSYKFEAPNLNDNETSWAKDFRYNFPKNVEPPIENQIANLNINNGLRTSQTDFPLGFYSQKN FNIASFPVVDHQIFKTTGLEHPINSHIDSLINAEFSELEASSLEEDVHTEEENSGTSLEDEETAMKGLASDIIEFCDNNSANKDVKERLNSSKFMGLMGSISDGSIVLKKDNGTERNLQKHVGFCFQNSGNWAGLEFHDVEDRIA

[0156]

[0157] The amino acid sequence of the RIM20 polypeptide is shown in SEQ ID NO:7 below: MSELLAIPLKRTLEVDFATELSKLIDTTSFQTASFFQSDILKVVDARNNAIAPDISIDGLSALKEYYVILLQLEKKFPNNQIEFTWFQTLSQKSRGTSQYSLQWEKLTIIYNIGCMYSLLALNSNNDAAESLKTSCLYFQNAAGCFKHVLDHQKNLETIPVVDDATLNALTSLMLAQAQECFWFKAVQDKHKDSLIAKLSQQIVDFYCEAINDAQRGKLIRSDWINHLKAKKAYFSAVTYYRIALSFNEKKQFGNVVKALQMGLQFINESTLSSQAKFKTVVESSLKEAQRDNEFIYLQEVPSELPSIKPALMVKPSSSATLLPSIKKDETLFKDLIPIEVMEYCTAYNERQDEYVEQRVTNPLASLNKLLKESLTTFQIPQGLTKVSEAELSHYQASLNNLLINNKNVQVQLDNIEQILNEEAFTDNQLRLKHGTLNWTLPESSTTNTAYYEKLKKLRGYLDEGSAIDKQTNELFQSIDKNLIGSEIRLPESNDPLTNKIKMIIQERNDYIDRTRRKSSEYRILPKIITSYKKNGTVDFEPIFIGHLKYFDEDLRYVNSTKEENIKLIEEVNLSKKNNPGRSGIEPKKMVRIDPRELYIEDLRYSFKLLDEVKENLSAGTAFYENLITSTSNLYNEVQEYDTARRAEKARLDKSLTFEDQ

[0158]

[0159] The nucleic acid sequence of the SKG3 coding region of the YLR187W gene is shown in SEQ ID NO:9 below:

[0160]

[0161] The nucleic acid sequence of the coding region of YNR068C is shown as SEQ ID NO:10 below: ATGAGAAACAGGCAGAATCAAAAGAACTGGACTAATTTAAGAGGTCTATTGTCCGTTGAAGAGAGAGAAAAACTAGAAAATTTACGGCTTGAACTAGTATGTATGCAAGCTGCTAATAGTATTCCACATGACCCACCGGAAATCAGTAGTCTGGAGACGGAATTGATTTGCTTAACAACGAACACAAAAGATTGCAAACCAGTGAGGTTCCACAGTGATTTACTTTTAAAGAAGCACAAATACAATGAAATTAAGAAAATATTTAAGGAAATTTTGGAAAACATTGAAGCATATCGCGATGAGTTCACTAAAAATCAAACAAAAATAAACTTACTTCTTGCCGATGACGCAAGAGCTAGCCTGCGTAATAGATCGCTCGATTTTTCTGATTTAATGCCTTCGAGTATAATTAAGGACGTACAGGTTTTAGCCAATATGGAAGCCAATGTTGTTGTCATGAAAAATGCGTTGAAAACGAAGTTGGTTGGAGAGAAAAGTGTGCCTGTTGCTTCTTCTCCTATATCCAGCATAATTCCTCGAACTTCTAGGAATAAAAAAACCTCCCCATCAAACTATCACCATTCGGTACTTTCACATCGGAAATCAAATGAATGGAATCAGGTCAGTTCAACTGAGTATAAGAGAACACTGTTATTGAATATAAAATATAATGATGATTTTAAAGCTACCATAGTGCCAAGTTTTGAAAGTTGTTTATGCTCGAGGTCATATTTCCTCCGCGTAAAACTTCATTTTGATAAAGGTGTTGGATCTGCTGAAATTGATATCCCAGTTCAAGTTAAAAACTCTTTTATTTGA

[0162] The nucleic acid sequence of the coding region of MNN14 of the YJR_061W gene is shown as SEQ ID NO:11 below:

[0163]

[0164] The nucleic acid sequence of the PIN3 coding region of the YPR154W gene is shown in SEQ ID NO: 12 below:

[0165] ATGTCTGCTTCATTGATTAATCGTTCCTTAACAAACATTAGGACAGAACTGGATTTTCTAAAAGGGTCAAATGTCATTTCAAATGACGTTTACGATCAAATAAATAAGAGCTTGCCGGCAAAATGGGATCCTGCCAATGCACCCCGCAACGCCAGTCCAGCT TCCTTGGAATATGTCGAAGCTCTTTATCAATTTGATCCTCAACAAGATGGTGATTTGGGCTTAAAACCAGGTGACAAGGTCCAACTTTTAGAAAAATTATCTCCAGAGTGGTACAAGGGTAGCTGTAATGGCCGTACCGGTATTTTCCCAGCAAACTATGTC AAGCCAGCTTTCTCTGGGTCTAACGGTCCATCCAATCTTCCACCACCTCCACAGTATAAAGCTCAAGAATTACAACAAATCCCCACGCAAAATAGTGCCGCATCTTATCAACAGCAGCCATTTCCTCCACCTTCCACAAATTATTATCAGCAGCCTCAA CAACAGCCGCAACAAGTCCCTCCTCCCCAACAACAACAACAACAACAACATCAGAGCTCACATAGCCACTTGAAGAGCTTTGGTAGCAAATTGGGTAATGCCGCCATTTTTGGGGCAGGCGCTAGTATTGGGTCAGATATTGTTAATAATATCTTTTAA

[0166] The nucleic acid sequence of the PEX18 coding region of the YHR160C gene is shown in SEQ ID NO: 13 below:

[0167] ATGAATAGTAACCGATGCCAAACGAATGAGGTGAATAAATTTATTAGTAGTACAGAAAAGGGGCCTTTTACGGGCAGGGACAATACGCTCTCTTTTAACAAAATCGGGAGCAGACTGAATTCACCACCGATTCTGAAGGATAAAATTGAGCTGAAATTTCTACAACACTCAGAAGATTTGAATCAATCACGGTCCTACGTAAATATTCGTCCTAGAACCTTAGAGGATCAAAGTTACAAATTTGAAGCGCCAAATCTAAATGACAATGAAACTTCTTGGGCCAAGGATTTTAGATATAACTTCCCTAAGAATGTTGAACCGCCCATCGAAAATCAAATCGCGAATCTTAATATAAACAACGGGCTACGGACATCTCAGACAGATTTTCCCTTAGGCTTTTATTCACAGAAAAACTTTAACATTGCTTCCTTCCCTGTGGTTGACCATCAGATATTCAAGACAACAGGTTTAGAACATCCTATCAACAGCCACATTGATTCTTTAATTAATGCTGAATTTTCGGAACTGGAAGCCAGTAGTTTGGAAGAAGATGTCCATACAGAAGAGGAAAATTCAGGTACGAGTCTGGAAGATGAAGAAACTGCCATGAAAGGTTTGGCTTCCGATATAATTGAGTTTTGCGATAATAATAGTGCCAATAAAGATGTAAAAGAAAGACTAAACAGTTCAAAGTTTATGGGGCTGATGGGCAGCATTAGTGATGGTTCTATAGTTTTAAAGAAGGATAACGGTACAGAAAGAAACCTTCAAAAACACGTAGGTTTTTGTTTTCAGAATTCAGGAAACTGGGCTGGTCTTGAGTTCCATGATGTTGAAGACAGAATTGCTTAA

[0168] The nucleic acid sequence of the MNN4 coding region of the YKL201C gene is shown in the following SEQ ID NO:14:

[0169]

[0170] The nucleic acid sequence of the RIM20 coding region of the YOR275C gene is shown in SEQ ID NO: 15 below:

[0171]

[0172] The nucleic acid sequence of the PAN2 coding region of the YGL094C gene is shown in SEQ ID NO: 16 below:

[0173]

[0174] Yeast strains, relevant gene disruptions, and related sequence identifiers are summarized in Table 1 .

[0175] Table 1. Yeast strains

[0176]

[0177]

[0178] Example 2

[0179] Growth of modified yeast in medium DS fermentation substrate

[0180] Strains with deletions of SKG3, YNR068C, MNN14, PIN3, PEX18, or MNN4 and the parent strain were tested in fermentation assays under medium DS conditions at pH 4.8 with 5.5 g of 32% DS liquefact at an initial OD of 0.3. Gold (Martrex, Inc., Chaska, MN, USA; herein referred to as FG). Fermentation was performed at 32°C for 55 hours. Samples from the end of fermentation (EOF) were analyzed by HPLC, and the results are shown in Table 2. The results are the average of two independent vials of the deletion mutant and six independent vials of FG.

[0181] Table 2. HPLC results from fermentation assays at medium DS

[0182]

[0183] The results showed that under moderate DS conditions, strains with deletions of SKG3, YNR068C, PIN3, and PEX18 produced similar amounts of ethanol compared to the parent FG. Strains with deletions of MNN14 and MNN4 showed an increase in ethanol production of approximately 1% compared to FG.

[0184] Example 3

[0185] Growth of modified yeast in high DS fermentation substrates

[0186] Strains with deletions of SKG3, YNR068C, MNN14, PIN3, PEX18, or MNN4 and the parent FG strain were tested in fermentation under high DS conditions containing 5.5 g of 37.8% DS liquefact at pH 4.8 and an initial OD of 0.3. Fermentation was performed at 32°C for 55 hours. Samples from the end of fermentation (EOF) were analyzed by HPLC, and the results are shown in Table 3. Data are the average of two independent vials for each strain and four independent vials for FG.

[0187] Table 3. HPLC results from fermentation assays at high DS

[0188]

[0189] The results showed that under high DS conditions, strains with each of SKG3, YNR068C, MNN14, PIN3, PEX18 and MNN4 disruptions showed significant increases in ethanol production (2.4%-4.7%) compared to the parent FG strain. Accordingly, less glucose remained at the end of the fermentation compared to FG.

[0190] Example 4

[0191] Growth of modified yeast under temperature ramp conditions

[0192] Strains with deletions of SKG3, YNR068C, MNN14, PIN3, or PEX18 were tested in fermentation assays with the parent FG strain under moderate DS conditions as described in Example 2. To further characterize the variant strains, a temperature ramp ( Figure 1 The EOF samples were analyzed by HPLC and the results are shown in Table 4.

[0193] Table 4. HPLC results from fermentation assays under ramp conditions

[0194]

[0195] The results showed that under high temperature ramp conditions, strains with deletions of SKG3, YNR068C, MNN14, PIN3, PEX18, or MNN4 showed greater than 5% increase in ethanol production compared to the parent FG, with less remaining glucose at the end of fermentation, less glycerol, and more acetic acid.

[0196] Example 5

[0197] Growth of modified yeast in medium DS fermentation substrate

[0198] Modified strains with deletions of RIM20 or PAN2 were tested in vials under medium DS conditions, and the products were analyzed by HPLC as described in Example 2. The results are shown in Table 5. The data shown are the average of two independent vials of the deletion strains and six independent vials of the parent FG.

[0199] Table 5. HPLC results from fermentation assays

[0200]

[0201] The results showed that the strain with the RIM20 deletion had some negative effects on ethanol production, while the strain with the PAN2 deletion showed little difference in ethanol production. Both deletion strains produced a 20% decrease in acetic acid production.

[0202] Example 6

[0203] Growth of modified yeast in high DS fermentation substrates

[0204] Modified strains with deletions of RIM20 or PAN2 were tested in vials under high DS conditions, and the products were analyzed by HPLC as described in Example 3. The results are shown in Table 6. The data shown are the average of two independent vials of the deletion strains and six independent vials of the parent FG.

[0205] Table 6. HPLC results from fermentation assays

[0206]

[0207] The results showed that the strain with the RIM20 deletion produced about 3.4% more ethanol and about 13% less acetic acid than the parent FG strain. Similarly, the strain with the PAN2 deletion produced about 2.4% more ethanol and over 15% less acetic acid.

[0208] Example 7

[0209] Growth of modified yeast under temperature ramp conditions

[0210] The modified strains with deletions of RIM20 or PAN2 were tested in vials under moderate DS temperature ramp conditions and as described in Example 5 and Figure 1 The products were analyzed by HPLC as shown in Table 7. The results are shown in Table 7. The data shown are the average of two independent vials of the deletion strain and six independent vials of the parent FG.

[0211] Table 7. HPLC results from fermentation assays

[0212]

[0213] The results showed that the strain with the RIM20 deletion produced about 6.2% more ethanol and a smaller decrease in acetic acid compared to the parent FG strain. Similarly, the strain with the PAN2 deletion produced about 5.4% more ethanol and more than 8% less acetic acid.

[0214] All references cited herein are incorporated by reference, including the following additional references:

[0215] 1. Conde R,et al.(2003)Screening for new yeast mutants affected inmannosylphosphorylation of cell wall mannoproteins.Yeast 20(14):1189-211PMID:14587103

[0216] 2. Kim YH,et al.(2017) Abolishment of N-glycan mannosylphosphorylationin glyco-engineered Saccharomyces cerevisiae by doubledisruption of MNN4 andMNN14 genes.Appl.Microbiol.Biotechnol.101(7):2979-2989PMID:28101612

[0217] 3. Chernova TA,et al.(2011) Prion induction by the short-lived,stress-induced protein Lsb2 is regulated by ubiquitination and association with theactin cytoskeleton.Mol Cell 43(2):242-52PMID:21777813

[0218] 4. Madania A,et al.(1999) The Saccharomyces cerevisiae homologue ofhuman Wiskott-Aldrich syndrome protein Las17p interacts with the Arp2 / 3complex.Mol Biol Cell 10(10):3521-38PMID:10512884

[0219] 5. Purdue PE,et al.(1998)Pex18p and Pex21p,a novel pair of relatedperoxins essential for peroxisomal targeting by the PTS2 pathway.J Cell Biol143(7):1859-69PMID:9864360

[0220] 6. Stein K,et al.(2002) Interactions of Pex7p and Pex18p / Pex21p withthe peroxisomal docking machinery:implications for the first steps in PTS2protein import.Mol Cell Biol 22(17):6056-69PMID:12167700

[0221] 7. Purdue PE and Lazarow PB(2001) Pex18p is constitutively degradedduring peroxisome biogenesis.J Biol Chem 276(50):47684-9 PMID:11590152

[0222] 8. Raschke WC,et al.(1973) Genetic control of yeast mannanstructure.Isolation and characterization of mannan mutants.J Biol Chem 248(13):4660-6 PMID:4578088

[0223] 9. Odani T,et al.(1996) Cloning and analysis of the MNN4 gene requiredfor phosphorylation of N-linked oligosaccharides in Saccharomycescerevisiae.Glycobiology 6(8):805-10 PMID:9023541

[0224] 10. Odani T,etal.(1997) Mannosylphosphate transfer to cell wall mannanis regulated by the transcriptional level of the MNN4 gene in Saccharomycescerevisiae.FEBS Lett 420(2-3):186-90 PMID:9459307

[0225] 11. Xu W and Mitchell AP(2001) Yeast PalA / AIP1 / Alix homolog Rim20passociates with a PEST-like region and is required for its proteolyticcleavage.J Bacteriol 183(23):6917-23 PMID:11698381

[0226] 12. Xu W,et al.(2004) Multivesicular body-ESCRT components function inpH response regulation in Saccharomyces cerevisiae and Candida albicans.MolBiol Cell 15(12):5528-37 PMID:15371534

[0227] 13. Su SS and Mitchell AP(1993) Identification of functionally relatedgenes that stimulate early meiotic gene expression in yeast.Genetics 133(1):67-77 PMID:8417990

[0228] 14. Boeck R,et al.(1996)The yeast Pan2 protein is required for poly(A)-binding protein-stimulated poly(A)-nuclease activity.J Biol Chem 271(1):432-8 PMID:8550599

[0229] 15. Brown CE and Sachs AB(1998) Poly(A)tail length control inSaccharomyces cerevisiae occurs by message-specific deadenylation.Mol CellBiol 18(11):6548-59 PMID:9774670

[0230] 16. Hammet A,et al.(2002) Posttranscriptional regulation of the RAD5DNA repair gene by the Dun1 kinase and the Pan2-Pan3 poly(A)-nuclease complexcontributes to survival of replication blocks.J Biol Chem 277(25):22469-74PMID:11953437

Claims

1. A modified yeast cell derived from a parent yeast cell, the modified cell comprising a genetic alteration which causes the modified cell to produce a reduced amount of a polypeptide that causes lack of robustness during fermentation under high dry solids and high temperature fermentation conditions compared to the otherwise identical parent cell, wherein the polypeptide is selected from the group consisting of SKG3, YNR068C, MNN14, PIN3, PEX18, MNN4, RIM20 and / or PAN2 polypeptides.

2. The modified cell of claim 1 , wherein the modified cell further produces a reduced amount of acetate during fermentation compared to the amount of acetate produced by an otherwise identical parent cell under identical fermentation conditions, and wherein the genetic alteration comprises a disruption of a nucleic acid capable of directing expression of a RIM20 and / or PAN2 polypeptide.

3. The modified cell of claim 1 or 2, wherein the cell further comprises one or more genes of the phosphoketolase pathway.

4. The modified cell of claim 3, wherein the genes of the phosphoketolase pathway are selected from the group consisting of phosphoketolase, phosphotransacetylase, and acetylated acetyl dehydrogenase.

5. The modified cell of any one of claims 1-4, wherein the cell further comprises an exogenous gene encoding a carbohydrate processing enzyme.

6. The modified cell of any one of claims 1-5, further comprising an alteration in the glycerol pathway and / or the acetyl-CoA pathway.

7. The modified cell of any one of claims 1-6, further comprising an alternative pathway for producing ethanol.

8. The modified cell of any one of claims 1-7, wherein the cell belongs to a Saccharomyces spp.

9. A method for increasing the robustness of yeast cells under high dry solids and high temperature fermentation conditions, the method comprising introducing a genetic alteration into a parent yeast cell, the genetic alteration resulting in the resulting modified cell producing a reduced amount of a polypeptide selected from the group consisting of SKG3, YNR068C, MNN14, PIN3, PEX18, MNN4, RIM20 and / or PAN2 polypeptides during fermentation.

10. The method of claim 9, wherein the genetic alteration further results in the modified cell producing a reduced amount of acetic acid during fermentation compared to the amount of acetic acid produced by the parent cell under the same fermentation conditions, and wherein the genetic alteration comprises disrupting a nucleic acid capable of directing expression of a RIM20 and / or PAN2 polypeptide.

11. A method for producing a fermentation product, the method comprising fermenting a carbohydrate substrate with the modified yeast cell of any one of claims 1 to 7 under conditions for producing a fermentation product.

12. The method of claim 11, wherein the conditions include a dry solids percentage (DS%) of at least 35% and / or a temperature of at least 34°C.

13. The method of claim 11 or claim 12, wherein the modified yeast cell produces an increased amount of fermentation product compared to a parent cell under identical conditions.

14. The method of any one of claims 11-13, wherein the modified yeast cell produces a reduced amount of acetic acid compared to a parent cell under identical conditions.

15. The method of any one of claims 11-14, wherein the fermentation product is ethanol.

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