Modified yeast cells, method for reducing glycerol and acetate production from cultured yeast cells, and method for producing a fermentation product.

Description

1 / 42 “MODIFIED YEAST CELLS, METHOD FOR REDUCING GLYCEROL AND ACETATE PRODUCTION FROM CULTIVATED YEAST CELLS AND METHOD FOR PRODUCING A FERMENTATION PRODUCT” CROSS-REFERENCE ON RELATED REQUEST

[0001] This application claims priority over Provisional Application No. US 63 / 492,252, filed March 27, 2023, the contents of which are incorporated herein in their entirety by reference. INCORPORATION FOR REFERENCE PURPOSES OF THE SEQUENCE LISTING

[0002] This application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file titled NB42202WOPCTSeqList.xml, created on March 7, 2024, which is 25628 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. FIELD OF TECHNIQUE

[0003] The present compositions and methods refer to modified yeast cells that heterologously express the RuBisCo enzyme from a particular species of iron-oxidizing bacteria. The modified yeast cells demonstrate reduced accumulation of glycerol and acetate during fermentation, while maintaining high ethanol production, making them useful for large-scale ethanol production from starch substrates, where glycerol and acetate represent undesirable byproducts. BACKGROUND

[0004] First-generation yeast-based ethanol production converts sugars into fuel ethanol. Ethanol production Petition 870250087544, dated 09 / 26 / 2025, page 47 / 94 2 / 42 of the annual fuel produced by yeast is approximately 90 billion liters worldwide (Gombert, AK and van Maris, AJ (2015) Curr. Opin. Biotechnol. 33:81-86). It is estimated that about 70% of the cost of ethanol production is raw material. These figures have remained virtually unchanged for about a decade. Given that the volume of ethanol production is so large, even small yield improvements have a massive economic impact on the industry.

[0005] Ribulose-1,5-bisphosphate carboxylase-oxygenase, commonly known as RuBisCo, is an enzyme involved in the first major step of carbon fixation, in which atmospheric carbon dioxide is converted by plants and other photosynthetic organisms into energy-rich molecules such as glucose. The enzyme catalyzes the carboxylation of ribulose-1,5-bisphosphate (also known as RuBP) and is an essential component of the mechanism by which autotrophic bacteria, algae, and land plants fix CO2 in organic biomass via the Calvin-Benson-Basham pentose phosphate reductive pathway.

[0006] The heterologous expression of a proteobacterial RuBisCo of Thiobacillus dentrificans and bacterial protein chaperones in Saccharomyces cerevisiae has been described (see, in general, documents no. WO2014129898, WO2017216136, WO2018114762, WO2019063542, WO2019063543 and WO2020043497). Nevertheless, there is a need to improve the efficiency of ethanol production and reduce the production of undesirable byproducts, particularly genetically modified yeasts that tend to produce a higher amount of acetate. BRIEF DESCRIPTION

[0007] The present compositions and methods refer to modified yeast cells heterologously expressing RuBisCo from the iron-oxidizing chemolithotrophic bacterium Gallionella capsiferriformans. The cells of Petition 870250087544, dated 09 / 26 / 2025, page 48 / 94 3 / 42 Modified yeast cells demonstrate a reduction in glycerol and acetate accumulation during fermentation using a starch substrate. The aspects and achievements of the modified yeast cells and methods are described in the following independently numbered paragraphs: 1. In one aspect, modified yeast cells derived from parental yeast cells are provided, the modified cells comprising a genetic alteration that causes the modified cells to produce the ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCo) enzyme from Gallionella capsiferriformans (GcRuBisCo), wherein the modified cells produce during fermentation a reduced amount of glycerol and / or acetate compared with the amount of glycerol and acetate produced by identical parental cells that produce the RuBisCo enzyme from Thiobacillus dentrificans under identical fermentation conditions, and wherein the modified yeast cells and the parental yeast cells both additionally comprise an exogenous gene encoding a phosphoribulokinase; 2. In some embodiments of the modified cells of paragraph 1, the genetic alteration comprises the introduction into the parental cells of a nucleic acid capable of directing the expression of Gc RuBisCo; 3. In some embodiments of the modified cells from paragraph 2, the genetic alteration comprises the introduction of an expression cassette to target the expression of GcRuBisCo; 4. In some embodiments, the modified cells of any of paragraphs 1-3 additionally comprise exogenous genes encoding chaperone proteins; 5. In some embodiments of the modified cells from paragraph 4, the chaperone proteins are GroES and GroEL from Escherichia coli; 6. In some embodiments, the modified cells additionally comprise an exogenous gene encoding a glycerol transporter, a Petition 870250087544, dated 09 / 26 / 2025, page 49 / 94 4 / 42 glycerol dehydrogenase and dihydroxyacetone kinase; 7. In some embodiments of the modified cells from any of paragraphs 1-6, the cells additionally comprise one or more genes of the phosphoketolase pathway; 8. In some embodiments, the modified cells of any of paragraphs 1-7 additionally comprise an alteration in the glycerol pathway and / or the acetyl-CoA pathway; 9. In some embodiments, the modified cells of any of paragraphs 1-8 additionally comprise an alternative route for the production of ethanol; 10. In some embodiments, the modified cells of any of paragraphs 1-9 additionally comprise an exogenous gene encoding a molecular chaperone; 11. In some embodiments of the modified cells of any of paragraphs 1-10, the cells additionally comprise an exogenous gene encoding a carbohydrate-processing enzyme; 12. In some embodiments of the modified cells from any of paragraphs 1-11, the cells are of a Saccharomyces spp; 13. In another aspect, a method is provided for reducing the production of glycerol and acetate from yeast cells grown on a carbohydrate substrate, comprising: introducing into parental yeast cells a genetic alteration that causes the modified cells to produce ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCo) from Gallionella capsiferriformans; 14. In some embodiments of the method in paragraph 13, the modified cells are the yeast cells from any of paragraphs 1-12.

[0008] These and other aspects and achievements of the modified methods and cells will be evident from the description, which includes any Petition 870250087544, dated 09 / 26 / 2025, page 50 / 94 5 / 42 Drawings / Figures attached. Each of the aspects and realizations described in this document may be used together, unless explicitly or clearly excluded from the context of the realization or aspect. DETAILED DESCRIPTION I. Definitions

[0009] Before describing the modified yeast cells and methods in detail, the following terms are defined for clarity. Undefined terms should be considered consistent with their common meanings as used in the relevant art.

[0010] Definitions of terms may appear throughout the descriptive report. It should be understood that this disclosure is not limited to the particular realizations described, as they may, of course, vary. It should also be understood that terminology used herein is for the purpose of describing only particular realizations and is not intended to be exhaustive.

[0011] It should be noted that, as used in this document and the accompanying claims, the singular forms of “a”, “an”, “the” and “the” include plural referents unless the context clearly indicates otherwise. For example, a, an, “the” and “the” includes at least one and one or more.

[0012] The terms comprising, includes, and comprised by, as used in this document, are synonymous with "that includes, includes, or that contains, contains, has, that has" and their grammatical variants, are inclusive or unrestricted, and do not exclude additional members, elements, or steps of the method not recited. The terms comprising, includes, and comprised by, that includes, includes, or that contains, contains, has, that has" and grammatical variants thereof also include the term consisting of. Petition 870250087544, dated 09 / 26 / 2025, p. 51 / 94 6 / 42

[0013] As used in this document, the term “alcohol” refers to an organic compound in which a hydroxyl functional group (-OH) is attached to a saturated carbon atom.

[0014] As used in this document, the terms “yeast cells,” “yeast strains,” or simply “yeast” refer to organisms of the phyla Ascomycota and Basidiomycota. Exemplary yeast is budding yeast from the order Saccharomycetales. Specific examples of yeast are Saccharomyces spp., including, but not limited to, S. cerevisiae. Yeast includes organisms used for the production of fuel alcohol, as well as organisms used for the production of potable alcohol, including specialized and patented yeast strains used to produce flavored beers, wines, and other fermented beverages.

[0015] As used in this document, the terms “genetically modified yeast cells, variant yeast cells, modified yeast cells” or similar terms refer to yeast that includes genetic modifications and characteristics described in this document. Variant / modified yeast does not include naturally occurring yeast.

[0016] As used in this document, 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 in this document, and all sequences are presented in an N-terminal to C-terminal direction. The polymer may comprise modified amino acids and may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been Petition 870250087544, dated 09 / 26 / 2025, p. 52 / 94 7 / 42 modified naturally or by intervention; for example, by the formation of a disulfide bond, glycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification, such as conjugation with a labeling component. Also included in the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art.

[0017] As used in this document, the expressions “substantially similar” and “substantially identical”, in the context of at least two nucleic acids or polypeptides, typically mean that a polynucleotide or polypeptide comprises a sequence that has at least about 70% identity, at least about 75% identity, at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 91% identity, at least about 92% identity, at least about 93% identity, at least about 94% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or even at least about 99% identity, or more, compared with the reference (i.e., wild-type) sequence.The percentage of 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: Penalty for opening a loophole: 10.0; Penalty for extending the gap: 0.05; Protein weight matrix: BLOSUM series; DNA weight matrix: IUB; Percentage of divergent sequence delay: 40; Petition 870250087544, dated 09 / 26 / 2025, page 53 / 94 8 / 42 Gap separation distance: 8; Weight of DNA transitions: 0.50; List of hydrophilic residues: GPSNDQEKR; Negative usage matrix: DISABLED; Alternating specific waste penalties: ENABLED; Alternating hydrophilic penalties: ENABLED; End-gap separation penalty toggle DISABLED.

[0018] 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, when the two peptides differ only by a conservative substitution. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize with each other under stringent conditions (e.g., within a range of medium to high stringency).

[0019] As used in this document, the term “gene” is synonymous with the term “allele” with reference to a nucleic acid that encodes and directs the expression of a protein or RNA. The vegetative forms of filamentous fungi are generally haploid, therefore a single copy of a specified gene (i.e., a single allele) is sufficient to confer a specified phenotype. The term “allele” is generally preferred when an organism contains more than one similar gene, in which case each different similar gene is called a distinct “allele”.

[0020] As used in this document, the term “express a polypeptide” and similar terms refer to the cellular process. Petition 870250087544, dated 09 / 26 / 2025, page 54 / 94 9 / 42 of producing a polypeptide using the cell's translation machinery (e.g., ribosomes).

[0021] As used in this document, an “expression cassette” refers to a fragment of DNA that includes a promoter and an amino acid coding region and a terminator (i.e., promoter::amino acid coding region::terminator) and other nucleic acid sequences necessary to allow the encoded polypeptide to be produced in a cell. Expression cassettes can be exogenous (i.e., introduced into a cell) or endogenous (i.e., already present in a cell).

[0022] As used in this document, the terms “wild type” and “native” are used interchangeably and refer to genes, proteins, or strains found in nature, or not intentionally modified for the benefit of the yeast currently described.

[0023] As used in this document, the term “protein of interest” refers to a polypeptide that is desired to be expressed in the modified yeast. This protein may be an enzyme, a substrate-binding protein, an active surface protein, a structural protein, a selectable marker, or the like, and may be expressed. The protein of interest is encoded by an endogenous gene or a heterologous gene (i.e., gene of interest) with respect to the parental strain. The protein of interest may be expressed intracellularly or as a secreted protein.

[0024] The term “heterologous,” when used in reference to a polynucleotide, gene, polypeptide, or enzyme, refers to a polynucleotide, gene, polypeptide, or enzyme not normally found in the host organism. “Heterologous” also includes a native coding region or part thereof that is reintroduced into the organism of origin in a form different from the corresponding native gene, for example, not in its natural location in the organism’s genome. The polynucleotide or gene Petition 870250087544, dated 09 / 26 / 2025, p. 55 / 94 10 / 42 A heterologous gene can be introduced into the host organism by, for example, gene transfer. A heterologous gene may include a native coding region that is a portion of a chimeric gene that includes non-native regulatory regions that are reintroduced into the native host. Foreign genes may comprise native genes inserted into a non-native organism or chimeric genes.

[0025] As used in this document, the terms “genetically modified”, genetic alteration, genetic modification and similar terms are used interchangeably and refer to the alteration / change of a nucleic acid sequence. The alteration may include, but is not limited to, a substitution, deletion, insertion or chemical modification of at least one nucleic acid in the nucleic acid sequence.

[0026] As used in this document, “aerobic fermentation” refers to cultivation and production processes in the presence of oxygen.

[0027] As used in this document, “anaerobic fermentation” refers to cultivation and production processes in the absence of oxygen.

[0028] As used in this document, the singular articles “a”, “an”, “the” and “the” encompass the plural, unless the context clearly indicates otherwise. All references cited in this document are incorporated herein in their entirety by reference. The following abbreviations / acronyms have the following meanings, unless otherwise specified: The following meanings, unless otherwise specified: °C Degrees Celsius; base pairs; Petition 870250087544, dated 09 / 26 / 2025, p. 56 / 94 11 / 42 CO2 carbon dioxide; DNA deoxyribonucleic acid; ds or DS dry solids; EC enzyme committee; EtOH ethanol; g or gm grams; GcRuBisCo RuBisCo from Gallionella capsiferriformans; g / l grams per liter; H2O water; High-performance liquid chromatography (HPLC); hr or h hour; kg kilogram; M molar; mg milligram; minute; mL or ml milliliter; millimolar; Normal N; nm nanometer; PCR stands for polymerase chain reaction; ppm parts per million; RuBisCo ribulose-1,5-bisphosphate carboxylase-oxygenase; RuBP ribulose-1,5-bisphosphate; Δ related to a deletion; μ9 microgram; μL and μI microliter; μM micromolar.

[0029] When a range of values ​​is provided, it is understood that Petition 870250087544, dated 09 / 26 / 2025, page 57 / 94 12 / 42 each intervening value, up to one-tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated value or intervening value of that stated range is covered in this disclosure. The upper and lower limits of these smaller ranges may be independently included in or excluded from the range, and each range in which any, neither, or both limits are included in the smaller ranges is also covered in this disclosure, subject to any limit specifically excluded in the stated range. When the stated range includes one or both limits, the ranges that exclude either or both of these included limits are also included in this disclosure.

[0030] Numerical values ​​and ranges may be presented in this document with the numerical value preceded by the term "approximately". The term "approximately" is used in this document to provide literal support for the exact number it precedes, as well as a number that is close to or approximately the number the term precedes. When determining whether a number is close to or approximately a specifically quoted number, the unquoted close or approximate number may be a number that, in the context in which it is presented, provides the substantial equivalent of the specifically quoted number. For example, with respect to a numerical value, the term "approximately" refers to a range of -10% to +10% of the numerical value, unless the term is specifically defined in the context. All values ​​and ranges may implicitly include the term "approximately", except where the context clearly dictates otherwise.

[0031] All publications, including patent documents, scientific articles and databases cited in this application are incorporated in their entirety by reference for any purpose, as if Petition 870250087544, dated 09 / 26 / 2025, p. 58 / 94 13 / 42 each individual publication is incorporated individually by reference. Nothing in this document should be construed as an admission that such publications constitute the prior art of the claims appended to this document. If a definition set forth in this document is contrary to, or otherwise inconsistent with, a definition set forth in the patents, applications, published applications and other publications that are incorporated herein by reference, the definition set forth in this document shall prevail over the definition that is incorporated herein by reference. II. Modified yeast cells expressing G. RuBisCo. capsiferrifirmans

[0032] Ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCo; EC 4.1.1.39) catalyzes the carboxylation of ribulose-1,5-bisphosphate (RuBP). RuBisCo is involved in the first major step of carbon fixation, whereby atmospheric carbon dioxide is converted by plants and other photosynthetic organisms into energy-rich molecules such as glucose.

[0033] The present modified yeast cells and methods are based on the unexpected observation that RuBisCo from a particular Gallionella sp., namely Gallionella capsiferrifirmans, when heterologously expressed in high-ethanol-producing parental yeast cells, reduces the accumulation of glycerol and acetate after fermentation, compared to the amounts obtained with a corresponding parental strain that instead produces RuBisCo from Thiobacillus dentrificans.

[0034] In some embodiments, G. capsiferriformans RuBisCo (in the present document, “GcRuBisCo”) is expressed (or produced) in cells modified by the introduction into a parental yeast cell of a nucleic acid capable of directing the expression of GcRuBisCo. In some embodiments, GcRuBisCo has the sequence established by SEQ ID NO: 1. Petition 870250087544, dated 09 / 26 / 2025, page 59 / 94 14 / 42 Specific methods include, but are not limited to, (i) introducing additional copies of an endogenous expression cassette to increase polypeptide production in a host cell, (ii) introducing exogenous expression cassette(s) to increase polypeptide production in a host cell, (iii) replacing an endogenous cassette with an exogenous expression cassette that allows for the production of a greater amount of the polypeptide, (iv) modifying or replacing the promoter of an endogenous expression cassette to increase expression, and / or (v) modifying any aspect of the host cell to increase the half-life of the polypeptide in the host cell. In some embodiments, an exogenous expression cassette is introduced into a parental yeast cell to produce Gc RuBisCo in the yeast cell.

[0035] In some embodiments, a gene encoding a RuBisCo enzyme that is substantially similar to GcRuBisCo is expressed. Substantially similar RuBisCo enzymes are those that provide advantages similar to GcRuBisCo, compared to RuBisCo from Thiobacillus denitrificans. In some embodiments, the amino acid sequence of the RuBisCo enzyme 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 more identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RuBisCo enzyme has at least 60% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 1.In some embodiments, the amino acid sequence of the RuBisCo enzyme has at least 70% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the enzyme... Petition 870250087544, dated 09 / 26 / 2025, pp. 60 / 94 15 / 42 RuBisCo has at least 75% identity compared to a polypeptide with the amino acid sequence SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RuBisCo enzyme has at least 80% identity compared to a polypeptide with the amino acid sequence SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RuBisCo enzyme has at least 85% identity compared to a polypeptide with the amino acid sequence SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RuBisCo enzyme has at least 90% identity compared to a polypeptide with the amino acid sequence SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RuBisCo enzyme has at least 95% identity compared to a polypeptide with the amino acid sequence SEQ ID NO: 1.In some embodiments, the amino acid sequence of the RuBisCo enzyme has at least 96% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RuBisCo enzyme has at least 97% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RuBisCo enzyme has at least 98% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RuBisCo enzyme has at least 99% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 1.In a particular embodiment, the amino acid sequence of the RuBisCo enzyme has 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 more, in comparison. Petition 870250087544, dated 09 / 26 / 2025, pp. 61 / 94 16 / 42 with a polypeptide having the amino acid of SEQ ID NO: 1. In some embodiments, the amino acid sequence of the RuBisCo enzyme has the amino acid sequence of SEQ ID NO: 1.

[0036] The amino acid sequence of GcRuBisCo is shown below as SEQ ID NO: 1: MDQSNRYANLNLKEEDLIKNGKHLLVAYKLIPAKGHGFLEVAAH VAAESSTGTNVEVSTTDDFTRGVDALVYEIDETAFGDDIVKGGGLFKVAYPVE LFDPNLTDGTYNISHMWSLILGNNQGMGDHQGLRMLDFLVPEMMVRKFDGP SANISNLWKVLGRSETDGGYIAGTIIKPKLGLRPEPFAKACYDFWLGGDFIKND EPQANQPFCPMEVVMPKVAEAMDRAQQETGQAKLFSANITADYYKEMIHRG DFVLETFAKYNSASHVAFLVDGFVTGPAGVTTCRREFPDTFLHFHRAGHGAV TSYKSPMGMDPLCYMKLVRLMGASGMHTGTMGYGKMEGHGKETVLAYMLE RDECQGPYFYQKWYGMKATTPIISGGMNALRLPGFFQNLGHGNVINTCGGG AFGHIDSPAAGGISLGQAYDCWKSGSDPIEYAKTHKEFARAFESFPKDGDKLF AGWREKLGVHK.

[0037] In some embodiments, the nucleic acid sequence of RuBisCo 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 more identity, compared to the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the nucleic acid sequence of GcRuBisCo is the nucleic acid sequence of SEQ ID NO: 2.

[0038] Preferably, the expression of GcRuBisCo in yeast cells is achieved by genetic manipulation using sequence-specific molecular biology techniques, as opposed to chemical mutagenesis, which is generally not targeted at specific nucleic acid sequences. However, chemical mutagenesis is not excluded as a method to produce Petition 870250087544, dated 09 / 26 / 2025, pp. 62 / 94 17 / 42 as present modified yeast cells. In some embodiments, genetic manipulation includes the use of an established gene-editing technique, for example, CRISPR / Cas editing, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs). In some embodiments, CRISPR / Cas9 editing is used. Compositions and methods for fungal genome modification by CRISPR / Cas9 systems are described and well known in the art (e.g., see PCT Publications No. WO2016 / 100571, WO2016 / 100568, WO2016 / 100272, WO2016 / 100562 and the like).

[0039] In some embodiments, the parental yeast cells are genotypically identical to the modified yeast cells. In some embodiments, the parental yeast cells are genotypically identical to the modified yeast cells with respect to the metabolic pathways involved in ethanol production. In some embodiments, the parental yeast cells, before and after modification as described herein, express Thiobacillus denitrificans RuBisCo. In some embodiments, the parental yeast cells, before modification as described herein, expressed Thiobacillus denitrificans RuBisCo.

[0040] In some embodiments, the parental yeast cells are already genetically modified, either using standard molecular biology techniques, by chemical mutagenesis, by mating, by selection and adaptation to stress, or by other methods, to improve ethanol production in starch liquefaction with moderate to high dissolved solids. Examples of these yeast cells are commercially available and include those marketed under the names SYNERXIA® (IFF, USA), EBOOST® (DSM, NE), TRANSFERM® (Lallemand, CA), and INNOVA® (Novozymes, DK).

[0041] It is understood that reference to a parental yeast cell or strain is made to describe the contribution of expression of Petition 870250087544, dated 09 / 26 / 2025, pp. 63 / 94 18 / 42 GcRuBisCo for a desirable phenotype. The yeast selected for modification by GcRuBisCo expression is likely already a high-ethanol-producing yeast that includes additional genetic modifications aimed at achieving the same phenotype, and it may be difficult to determine the individual contribution of each genetic mutation.

[0042] In some embodiments, the reduction in glycerol after fermentation by modified yeast cells expressing GcRuBisCo is a decrease of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, or even at least 10% or more, compared to the amount of glycerol produced by parental yeast cells grown under the same conditions.

[0043] In some embodiments, the reduction of acetate after fermentation by modified yeast cells expressing Gc RuBisCo is a reduction of at least 5%, at least 10%, at least 15%, or even at least 20% or more, compared to the amount of acetate produced by identical parental yeast cells grown under the same conditions.

[0044] In some embodiments, the decrease in acetate produced by the modified yeast cells is a decrease of at least 1%, at least 2%, at least 3%, at least 4%, at least 5% or more, compared to the amount of acetate produced by parental yeast cells growing under the same conditions.

[0045] In some embodiments, the reduction in the amount of residual glucose after fermentation by modified yeast cells expressing GcRuBisCo is a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or even at least 60% or more, compared to the amount of residual glucose present. Petition 870250087544, dated 09 / 26 / 2025, pp. 64 / 94 19 / 42 after fermentation by an identical parental yeast grown under the same conditions.

[0046] In some embodiments, the amount of ethanol produced in the yeast expressing Gc RuBisCo is approximately the same or even greater, for example, at least 0.5%, at least 1.0%, at least 2.0%, or even at least 30% or more, compared to the amount of ethanol produced by the identical parental yeast grown under the same conditions. In some embodiments, the amount of ethanol produced in the yeast expressing Gc RuBisCo is approximately the same or even greater, for example, at least 0.5%, at least 1.0%, at least 2.0%, or even at least 3.0% or more, compared to the amount of ethanol produced by the identical parental yeast grown under the same conditions. III. Yeast cells modified with additional modifications associated with RuBisCo

[0047] Yeast cells containing genes encoding RuBisCo and phosphoribulokinase (EC 2.7.1.19) have been described (document no. WO2014 / 129898). Introduction of one or more genes encoding a NAD+-linked glycerol dehydrogenase (EC 1.1.1.6 or EC 1.1.1.72) and one or more genes encoding a dihydroxyacetone kinase (EC 2.7.1.28 or EC 2.7.1.29) appears to further increase ethanol production (document no. WO2018114762A1).

[0048] In some embodiments, the present modified yeast cells, in addition to expressing GcRuBisCo, additionally include a gene for the heterologous expression of phosphoribulokinase and / or dihydroxyacetone kinase, optionally in combination with genes encoding a glycerol dehydrogenase. The yeast may additionally include a modification in a gene involved in glycerol synthesis, as described in more detail below. The yeast cells may optionally express a gene Petition 870250087544, dated 09 / 26 / 2025, pp. 65 / 94 20 / 42 heterolog that encodes a glycerol transporter, a glycerol exporter, and / or a glycerol kinase.

[0049] In some embodiments, yeast cells additionally include a gene encoding a molecular chaperone. Chaperones can originate from prokaryotes. In specific embodiments, the chaperone is Escherichia coli GroEL and / or GroES. In some embodiments, the amino acid sequence of GroEL 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 more identity compared to a polypeptide that has the amino acid of SEQ ID NO: 3. In some embodiments, the amino acid sequence of GroEL has at least 60% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 3.In some embodiments, the amino acid sequence of GroEL has at least 65% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 3. In some embodiments, the amino acid sequence of GroEL has at least 70% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 3. In some embodiments, the amino acid sequence of GroEL has at least 75% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 3. In some embodiments, the amino acid sequence of GroEL has at least 80% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 3. In some embodiments, the amino acid sequence of GroEL has at least 85% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 3. In some embodiments, the amino acid sequence of GroEL has at least 90% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 3.In some cases. Petition 870250087544, dated 09 / 26 / 2025, pp. 66 / 94 In 21 / 42 realizations, the amino acid sequence of GroEL has at least 95% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 3. In some realizations, the amino acid sequence of GroEL is the amino acid of SEQ ID NO: 3. In some realizations, the nucleic acid sequence of GroEL 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 more identity compared to the nucleic acid sequence of SEQ ID NO: 4.In some embodiments, the amino acid sequence of GroES 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 more identity compared to a polypeptide that has the amino acid of SEQ ID NO: 5. In some embodiments, the amino acid sequence of GroES has at least 60% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 5. In some embodiments, the amino acid sequence of GroES has at least 65% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 5. In some embodiments, the amino acid sequence of GroES has at least 70% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 5.In some embodiments, the amino acid sequence of GroES has at least 75% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 5. In some embodiments, the amino acid sequence of GroES has at least 80% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 5. In some embodiments, the amino acid sequence of GroES has at least 85% identity. Petition 870250087544, dated 09 / 26 / 2025, p. 67 / 94 22 / 42 identity compared to a polypeptide that has the amino acid of SEQ ID NO: 5. In some embodiments, the amino acid sequence of GroES has at least 90% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 5. In some embodiments, the amino acid sequence of GroES has at least 95% identity compared to a polypeptide that has the amino acid of SEQ ID NO: 5. In some embodiments, the amino acid sequence of GroES is the amino acid of SEQ ID NO: 5. In some embodiments, the nucleic acid sequence of GroES 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 more identical to the nucleic acid sequence of SEQ ID NO: 6. IV. Yeast cells modified with genes from an exogenous PKL pathway.

[0050] The expression of GcRuBisCo (optionally with associated genes) can be combined with the expression of genes in the PKL pathway to reduce the production of high amounts of acetate associated with the introduction of an exogenous PKL pathway in yeast.

[0051] Genetically modified yeast cells possessing a heterologous PKL pathway have been previously described (document no. WO2015148272). These cells express heterologous phosphoketolase (PKL), phosphotransacetylase (PTA), and acetylase-acetylase (AADH), optionally with other enzymes, to channel carbon flow in the opposite direction to the glycerol pathway and towards acetyl-CoA synthesis, which is then converted to ethanol. Such modified cells have increased ethanol production capacity in a fermentation process compared to otherwise identical parental yeast cells. Petition 870250087544, dated 09 / 26 / 2025, pp. 68 / 94 23 / 42 V. Yeast cells modified with other mutations that affect alcohol production.

[0052] In some embodiments, in addition to expressing Gc RuBisCo, the modified yeast cells present may additionally include mutations that result in attenuation of the native glycerol biosynthesis pathway and / or glycerol reuse pathway, which are known to increase alcohol production. Methods for attenuation of the glycerol biosynthesis pathway in yeast are known and include reduction or elimination of endogenous NAD-dependent glycerol 3-phosphate dehydrogenase (GPD) or glycerol phosphate phosphatase (GPP) activity, for example, by disrupting one or more of the GPD1, GPD2, GPP1 and / or GPP2 genes. See, for example, U.S. Patent documents No. 9,175,270 (Elke et al.), 8,795,998 (Pronk et al.) and 8,956,851 (Argyros et al.). Methods to enhance glycerol pathway reuse through overexpression of glycerol dehydrogenase (GCY1) and dihydroxyacetone kinase (DAK1) to convert glycerol to dihydroxyacetone phosphate (Zhang et al. (2013) J.Ind. Microbiol. Biotechnol. 40:1153-60). In some embodiments, DAK1 is a heterologous DAK1. In some embodiments, DAK1 is an exogenous DAK1. In some embodiments, the amino acid sequence of DAK1 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 more identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of DAK1 has at least 60% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of DAK1 has at least 65% identity compared to a polypeptide that has. Petition 870250087544, dated 09 / 26 / 2025, p. 69 / 94 24 / 42 the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of DAK1 has at least 70% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of DAK1 has at least 75% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of DAK1 has at least 80% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of DAK1 has at least 85% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of DAK1 has at least 90% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 11.In some embodiments, the amino acid sequence of DAK1 has at least 95% identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 11. In some embodiments, the amino acid sequence of DAK1 is the amino acid sequence of SEQ ID NO: 11. In some embodiments, the nucleic acid sequence of DAK1 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 more identity compared to the nucleic acid sequence of SEQ ID NO: 12.

[0053] The modified yeast may additionally exhibit increased acetyl-CoA synthase (also called acetyl-CoA ligase) activity (EC 6.2.1.1) to remove (i.e., capture) acetate produced by chemical or enzymatic hydrolysis of acetyl phosphate (or present in the yeast culture medium for any other reason) and convert it to Ac-CoA. This Petition 870250087544, dated 09 / 26 / 2025, pp. 70 / 94 25 / 42 partially reduces the undesirable effect of acetate in yeast cell culture and may additionally contribute to improved alcohol yield. Increased acetyl-CoA synthase activity can be achieved by introducing a heterologous acetyl-CoA synthase gene into cells, thus increasing the expression of an endogenous acetyl-CoA synthase gene and similar effects.

[0054] In some embodiments, the modified cells may additionally include a heterologous gene encoding a protein with NAD+-dependent acetylating acetaldehyde dehydrogenase activity and / or a heterologous gene encoding a pyruvate-formate lyase. The introduction of such genes in combination with attenuation of the glycerol pathway is described, for example, in US Patent No. 8,795,998 (Pronk et al.). In some embodiments of the present modified yeast cells and methods, the yeast explicitly lacks a heterologous gene (or genes) encoding an acetylating acetaldehyde dehydrogenase, a pyruvate-formate lyase, or both.

[0055] In some embodiments, the present modified yeast cells may additionally overexpress a sugar transporter-like polypeptide (STL1) to increase glycerol uptake (see, for example, Ferreira et al. (2005) Mol. Biol. Cell. 16:2068-76; Dusková et al. (2015) Mol. Microbiol. 97:541-59 and document no. WO2015023989 A1) to increase ethanol production and reduce acetate. In some embodiments, STL1 is a heterologous STL1. In some embodiments, STL1 is an exogenous STL1. In some embodiments, the amino acid sequence of STL1 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 more identity compared to a polypeptide that has the sequence Petition 870250087544, dated 09 / 26 / 2025, pp. 71 / 94 26 / 42 of the amino acids of SEQ ID NO: 13. In some embodiments, STL1 is an exogenous STL1. In some embodiments, the amino acid sequence of STL1 has the amino acid sequence of SEQ ID NO: 13. In some embodiments, the nucleic acid sequence of STL1 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 more identity compared to the nucleic acid sequence of SEQ ID NO: 14.

[0056] In some embodiments, the modified yeast cells present may additionally overexpress a glycerol dehydrogenase (GldA). In some embodiments, GldA is a heterologous GldA. In some embodiments, GldA is an exogenous GldA. In some embodiments, the amino acid sequence of GldA 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 more identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 7. In some embodiments, the amino acid sequence of GldA has the amino acid sequence of SEQ ID NO: 7.In some embodiments, the nucleic acid sequence of GldA 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 more identity, compared to the nucleic acid sequence of SEQ ID NO: 8.

[0057] In some embodiments, the modified yeast cells present may additionally overexpress a Petition 870250087544, dated 09 / 26 / 2025, pp. 72 / 94 27 / 42 phosphoribulokinase (PRK). In some embodiments, the PRK is a heterologous PRK. In some embodiments, the PRK is an exogenous PRK. In some embodiments, the amino acid sequence of the PRK 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 more identity compared to a polypeptide that has the amino acid sequence of SEQ ID NO: 9. In some embodiments, the amino acid sequence of the PRK has the amino acid sequence of SEQ ID NO: 9.In some embodiments, the nucleic acid sequence of PRK 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 more identity, compared to the nucleic acid sequence of SEQ ID NO: 10.

[0058] In some embodiments, yeast cells additionally 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, ISN1, RSF2, and TDA9. In some embodiments, yeast cells, for example, modified yeast cells, additionally comprise a deletion, mutation, overexpression, and / or substitution in one or more endogenous polynucleotides encoding DLS1, CPR1, and PAB1. In some embodiments, yeast cells, for example, modified yeast cells, overexpress Petition 870250087544, dated 09 / 26 / 2025, pp. 73 / 94 28 / 42 additionally PAB1. In some embodiments, yeast cells, for example, modified yeast cells, additionally express reduced amounts or do not express DLS1. In some embodiments, yeast cells, for example, modified yeast cells, additionally include a deletion of the YJL065c gene. In some embodiments, yeast cells, for example, modified yeast cells, additionally express reduced amounts or do not express CPR1. In some embodiments, yeast cells, for example, modified yeast cells, additionally include a deletion of the YDR155c gene. VI. Yeast cells modified with other beneficial mutations

[0059] In some embodiments, in addition to expressing GcRuBisCo and optionally harboring other mutations that benefit alcohol production, the present modified yeast cells additionally include any number of additional genes of interest encoding proteins of interest. Additional genes of interest may be introduced before, during, or after genetic manipulations that result in the expression of GcRuBisCo.Proteins of interest include selectable markers, carbohydrate processing enzymes, and other commercially relevant polypeptides, including, but not limited to, an enzyme selected from the group consisting of a dehydrogenase, a transketolase, a phosphoketolase, a transladolase, an epimerase, a phytase, a xylanase, a β-glucanase, a phosphatase, a protease, an α-amylase, a β-amylase, a glucoamylase, a pullulanase, an isoamylase, a cellulase, a trehalase, a lipase, a pectinase, a polyesterase, a cutinase, an oxidase, a transferase, a reductase, a hemicellulase, a mannanase, an esterase, an isomerase, a pectinase, a lactase, a peroxidase, and a laccase. Proteins of interest can be secreted, glycosylated, and otherwise modified. Petition 870250087544, dated 09 / 26 / 2025, pp. 74 / 94 29 / 42 VII. Use of modified yeast for improved alcohol production

[0060] The present modified yeast cells and methods include methods for improving alcohol production and / or reducing glycerol production in fermentation reactions. In some embodiments, the present modified yeast cells and methods include methods for increasing alcohol production and reducing glycerol production in fermentation reactions. In some embodiments, the present modified yeast cells and methods include methods for reducing glycerol production in fermentation reactions. In some embodiments, the present modified yeast cells and methods include methods for increasing alcohol production and / or reducing acetate production in fermentation reactions. In some embodiments, the present modified yeast cells and methods include methods for increasing alcohol production and reducing acetate production in fermentation reactions.In some embodiments, the present modified yeast cells and methods include methods for reducing acetate production in fermentation reactions. In some embodiments, the present modified yeast cells and methods include methods for increasing alcohol production and reducing acetate and glycerol production in fermentation reactions. In some embodiments, the present modified yeast cells and methods include methods for reducing acetate and glycerol production in fermentation reactions. In some embodiments, the present modified yeast cells and methods include methods for increasing the rate of alcohol production. In some embodiments, the increase in rate occurs early in the fermentation reactions. Such methods are not limited to a particular fermentation process.The present genetically modified yeast is designed to be a direct substitute for conventional yeast or commercially available yeasts with high ethanol production. Although primarily intended for the production of fuel alcohol, the present... Petition 870250087544, dated 09 / 26 / 2025, pp. 75 / 94 30 / 42 yeast can also be used for the production of potable alcohol, including wine, beer, spirits and the like.

[0061] Processes for producing alcohols, for example ethanol, from carbohydrate substrates are generally well known. The conversion of a carbohydrate substrate into a fermentation product, such as alcohol, typically involves several processing steps and reagents, each of which is important for maximizing conversion rate, efficiency, yield, consistency, concentration, and / or yield. By way of example, a common production process for producing ethanol by fermentation of a carbohydrate substrate (see, for example, Section IX below) typically involves processes that include, but are not limited to, grinding or milling of raw material, liquefaction, saccharification, fermentation, and distillation, and reagents such as enzymes and additional components, for example, microorganisms and / or chemicals, may be added during a unit operation to facilitate the reaction.For example, during alcohol production, liquefaction involves diluting a must through partial hydrolysis to decrease its viscosity. Enzymes, such as alpha-amylases, can be used to facilitate the thinning process. During saccharification, the complex carbohydrates in the diluted must are converted into monosaccharides. Again, enzymes, such as glucoamylases, can be used to facilitate the conversion process. The resulting sugars are then converted by yeast into ethanol during the fermentation process. It is also possible to include enzymes, such as glucoamylases, fungal alpha-amylase, and trehalase, to control the glucose profile during fermentation. Reagents, such as enzymes, can also be used in the milling and distillation stages to facilitate the process. In some cases, the saccharification and fermentation stages can be combined into a single simultaneous saccharification and fermentation (SSF) stage.Other well-known processes include the hydrolysis of... Petition 870250087544, dated 09 / 26 / 2025, pp. 76 / 94 31 / 42 Raw starch (RSH), which involves grinding starch-containing material to produce a carbohydrate substrate and then performing SSF below the initial gelatinization temperature. Enzymes, for example, fungal acid amylases, glucoamylases, can also be used in such processes to produce fermentation products.

[0062] In some embodiments, the modified yeast described herein is used in a process to produce a fermentation product. Thus, in one aspect, a method is provided for producing a fermentation product, including the fermentation of a carbohydrate substrate with a modified yeast cell described herein. In some embodiments, the method includes a liquefaction step, optionally including an alpha-amylase. In some embodiments, the method includes a saccharification step, optionally including a glucoamylase. In some embodiments, the fermentation product is ethanol and / or butanol. In some embodiments, the fermentation product is ethanol.

[0063] As described above, the modified yeast cells described in this document are capable of increasing ethanol production during fermentation. 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% more compared to the amount of ethanol produced by the parent cells 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% more compared to the amount of ethanol produced by the parent cells under the same fermentation conditions. In some embodiments, the increase in ethanol is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% more compared to the amount of ethanol produced by the parent cells under Petition 870250087544, dated 09 / 26 / 2025, pp. 77 / 9432 / 42 under the same fermentation conditions. In some embodiments, the ethanol increase is in a range of about 0.1% to 15% compared to the amount of ethanol produced by the parent cells under the same fermentation conditions. In some embodiments, the ethanol increase is in a range of about 0.5% to 15% compared to the amount of ethanol produced by the parent cells under the same fermentation conditions. In some embodiments, the ethanol increase is in a range of about 1% to 15% compared to the amount of ethanol produced by the parent cells under the same fermentation conditions. In some embodiments, the ethanol increase is in a range of about 0.1% to 10% compared to the amount of ethanol produced by the parent cells 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 cells 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 cells 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 cells under the same fermentation conditions. The terms “increased,” “raised,” “enhanced,” “superior to,” “improved,” “more,” and the like are used interchangeably in this document.

[0064] In some embodiments, the modified yeast cells described herein produce a reduced amount of acetate compared to parent cells under the same fermentation conditions. In some embodiments, the acetate reduction 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%, 25%, Petition 870250087544, dated 09 / 26 / 2025, pp. 78 / 94 33 / 42%, 35%, 40%, 45%, or 50% less compared to the amount of acetate produced by the parent cells under the same fermentation conditions. In some embodiments, the acetate reduction is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, or 30% less compared to the amount of acetate produced by the parent cells under the same fermentation conditions. In some embodiments, the acetate reduction is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, or 25% less compared to the amount of acetate produced by the parent cells under the same fermentation conditions.In some embodiments, the acetate reduction is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% less compared to the amount of acetate produced by the parent cells under the same fermentation conditions. In some embodiments, the acetate reduction is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% less compared to the amount of acetate produced by the parent cells under the same fermentation conditions. In some embodiments, the acetate reduction is in a range of about 0.1% to 30% compared to the amount of acetate produced by the parent cells under the same fermentation conditions. In some embodiments, the reduction in acetate is in a range of about 1% to 30% compared to the amount of acetate produced by the parent cells under the same fermentation conditions.In some realizations, the reduction in acetate is in a range of about 5% to 30% compared to the amount of acetate produced by the parental cells under the same conditions. Petition 870250087544, dated 09 / 26 / 2025, pp. 79 / 94 34 / 42 fermentation conditions. In some embodiments, the acetate reduction is in a range of about 10% to 25% compared to the amount of acetate produced by the parent cells under the same fermentation conditions. In some embodiments, the acetate reduction is in a range of about 5% to 10% compared to the amount of acetate produced by the parent cells under the same fermentation conditions. In some embodiments, the acetate reduction is in a range of about 1% to 5% compared to the amount of acetate produced by the parent cells under the same fermentation conditions. The terms “decreased”, “reduced”, “less”, “less than” and the like are used interchangeably in this document.

[0065] In some embodiments, the modified yeast cells described herein produce a reduced amount of glycerol compared to parent cells under the same fermentation conditions. In some embodiments, the glycerol reduction is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, or 20% less compared to the amount of glycerol produced by parent cells under the same fermentation conditions. In some embodiments, the glycerol reduction is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, or 15% less compared to the amount of glycerol produced by the parent cells under the same fermentation conditions.In some embodiments, the glycerol reduction is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% less compared to the amount of glycerol produced by the parent cells under the same fermentation conditions. In some embodiments, the glycerol reduction is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% less compared to the amount of glycerol produced by the parent cells under the same conditions. Petition 870250087544, dated 09 / 26 / 2025, pages 80 / 94 35 / 42 fermentation. In some embodiments, the glycerol reduction is in a range of about 0.1% to 15% compared to the amount of glycerol produced by the parent cells under the same fermentation conditions. In some embodiments, the glycerol reduction is in a range of about 1% to 15% compared to the amount of glycerol produced by the parent cells under the same fermentation conditions. In some embodiments, the glycerol reduction is in a range of about 1% to 10% compared to the amount of glycerol produced by the parent cells under the same fermentation conditions. In some embodiments, the glycerol reduction is in a range of about 5% to 10% compared to the amount of glycerol produced by the parent cells under the same fermentation conditions.

[0066] In some embodiments, the modified yeast cells described herein produce a reduced amount of residual glucose compared to parent cells under the same fermentation conditions. In some embodiments, the reduction in residual glucose is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% less compared to the amount of residual glucose produced by parent cells under the same fermentation conditions. In some embodiments, the reduction in residual glucose is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, or 70% less compared to the amount of residual glucose produced by the parent cells under the same fermentation conditions.In some embodiments, the reduction in residual glucose is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 30%, 40%, 50%, or 60% less compared to the amount of residual glucose produced by the parent cells under the same fermentation conditions. In some. Petition 870250087544, dated 09 / 26 / 2025, pp. 81 / 94 In some embodiments, the reduction in residual glucose is in the range of about 1% to 70% compared to the amount of residual glucose produced by the parent cells under the same fermentation conditions. In some embodiments, the reduction in residual glucose is in the range of about 10% to 70% compared to the amount of residual glucose produced by the parent cells under the same fermentation conditions. In some embodiments, the reduction in residual glucose is in the range of about 20% to 70% compared to the amount of residual glucose produced by the parent cells under the same fermentation conditions. In some embodiments, the reduction in residual glucose is in the range of about 30% to 70% compared to the amount of residual glucose produced by the parent cells under the same fermentation conditions.In some embodiments, the reduction in residual glucose is in the range of about 40% to 70% compared to the amount of residual glucose produced by the parent cells under the same fermentation conditions. In some embodiments, the reduction in residual glucose is in the range of about 50% to 65% compared to the amount of residual glucose produced by the parent cells under the same fermentation conditions.

[0067] In some embodiments, the modified yeast cells described herein produce ethanol at a higher rate compared to the rate of ethanol produced by parent cells under the same fermentation conditions. In some embodiments, the rate increase is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% faster compared to the rate of ethanol produced by parent cells under the same fermentation conditions. In some embodiments, the rate increase occurs early in the fermentation conditions, for example, between about 0 and about 22 hours in the fermentation condition.

[0068] In some embodiments, the modified yeast described in Petition 870250087544, dated 09 / 26 / 2025, pp. 82 / 94 37 / 42 of this document produces a greater amount of ethanol and a smaller amount of glycerol, as described herein. In some embodiments, the modified yeast described herein produces a greater amount of ethanol and a smaller amount of acetate, as described herein. In some embodiments, the modified yeast described herein produces a greater amount of ethanol and a smaller amount of glycerol and a smaller amount of acetate, as described herein. In some embodiments, the modified yeast described herein produces a reduced amount of glycerol and a reduced amount of acetate, as described herein. In some embodiments, the modified yeast described herein produces ethanol at a faster rate, as described herein. VIII. Suitable yeast cells for modification

[0069] Yeasts are unicellular eukaryotic microorganisms classified as members of the kingdom Fungi and include organisms from the phyla Ascomycota and Basidiomycota. Yeasts that can be used in alcohol production include, but are not limited to, Saccharomyces spp., including S. cerevisiae, as well as Pichia, Issatchenkia, Kluyveromyces, Lachancea, and Schizosaccharomyces spp. In some embodiments, the yeast is S. cerevisiae.

[0070] Numerous yeast strains are commercially available, many of which have been selected or genetically modified for desired characteristics such as high alcohol production and rapid growth rate. In some embodiments, yeast is already genetically modified, either using standard molecular biology techniques, by chemical mutagenesis, by mating, by stress selection and adaptation, or by other methods, to improve ethanol production in plants. Petition 870250087544, dated 09 / 26 / 2025, pp. 83 / 94 38 / 42 of fuel ethanol. IX. Substrates and products

[0071] The production of alcohol from a variety of carbohydrate substrates, which include, but are not limited to, corn starch, sugarcane, cassava and molasses, is well known, given the countless variations and improvements made to enzymatic and chemical conditions and mechanical processes. It is believed that the modified yeast cells and methods are fully compatible with such substrates and conditions.

[0072] Alcohol fermentation products include organic compounds that have a hydroxyl functional group (-OH) that is bonded to a carbon atom. Exemplary alcohols include, but are not limited to, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, n-pentanol, 2-pentanol, isopentanol, and higher alcohols. The most commonly produced fuel alcohols are ethanol and butanol.

[0073] These and other aspects and realizations of the present yeast strains and methods will be apparent to the skilled person in view of the present description. The following examples are intended to illustrate further, but not limited to, the modified yeast cells and methods. EXAMPLES Example 1 Materials and methods Liquefaction preparation:

[0074] The liquefaction (fluid corn must paste) was prepared by adding 600 ppm urea, 0.124 SAPU / g ds of acid fungal protease, 0.33 GAU / g ds of Trichoderma reesei variant glucoamylase and 1.46 SSCU / g ds of Aspergillus kawachii α-amylase, adjusted to a pH of 4.8 with sulfuric acid. ANKOM Essays:

[0075] 300 pl of concentrated yeast culture for one day to Petition 870250087544, dated 09 / 26 / 2025, pages 84 / 94 39 / 42 others were added to each of several ANKOM bottles filled with 50 g of prepared liquefaction (see above) at a final OD of 0.3. The bottles were then incubated at 32 °C with shaking at 150 RPM for 55 hours. HPLC Analysis:

[0076] Samples of the ANKOM assay cultures were collected in Eppendorf tubes by centrifugation for 12 minutes at 14000 RPM. The supernatants were filtered using 0.2 pM PTFE filters and then used for HPLC analysis (Agilent Technologies 1200 series) under the following conditions: Bio-Rad Aminex HPX-87H columns, operating temperature of 55°C. Isocratic flow rate of 0.6 ml / min of 0.01 N H2SO4, 2.5 µl injection volume. Calibration standards were used for quantification of acetate, ethanol, glycerol, glucose, and other molecules. All values ​​are reported in g / l. Example 2 Generation of yeast strains expressing RuBisCo from G. capsiferriformans

[0077] The G5308 strain contains the gene that encodes the RuBisCo gene of the proteobacterium Thiobacillus denitrificans, along with genes that encode the GroEL chaperones from Escherichia coli (SEQ ID NOs: 3 and 4) and GroES (SEQ ID NOs: 5 and 6), phosphoribulose kinase from Spinacia oleracea (PRK1; SEQ ID NOs: 9 and 10), and a glycerol reuptake cassette consisting of the glycerol transporter STL1 from Zygosaccharomyces rouxii (SEQ ID NOs: 13 and 14), dihydroxyacetone kinase DAK1 from Saccharomyces cerevisiae (SEQ ID NOs: 11 and 12), and glycerol dehydrogenase GldA from E. coli (SEQ ID NOs: 7 and 8).

[0078] Using CRISPR-mediated integration, a gene encoding the CbbM / RuBisCo enzyme from Gallionella capsiferriformans (GenBank accession number CP002159; in this document “GcRuBisCo”) was introduced into the parental yeast strain at the CbbM gene site of T. Petition 870250087544, dated 09 / 26 / 2025, pp. 85 / 94 40 / 42 denitrificans. In summary, a guide RNA was designed, specific to the existing CbbM of T. denitrificans, which was then replaced in situ by a codon-optimized version of Gc RuBisCo for functional expression in S. cerevisiae using the existing S. cerevisiae TDH3 promoter (GenBank CP020129) and the S. cerevisiae CTC1 terminator (GenBank KM407505.1) remaining in the T. denitrificans CbbM gene genome.

[0079] The amino acid sequence of GcRuBisCo is shown below as SEQ ID NO: 1: MDQSNRYANLNLKEEDLIKNGKHLLVAYKLIPAKGHGFLEVAAH VAAESSTGTNVEVSTTDFTRGVDALVYEIDETAFGDDIVKGGGLFKVAYPVE LFDPNLTDGTYNISHMWSLILGNNQGMGDHQGLRMLDFLVPEMMVRKFDGP SANISNLWKVLGRSETDGGYIAGTIIKPKLGLRPEPFAKACYDFWLGDGFIKND EPQANQPFCPMEVVMPKVAEAMDRAQQETGQAKLFSANITADYYKEMIHRG DFVLETFAKYNSASHVAFLVDGFVTGPAGVTTCRREFPDTFLHFHRAGHGAV TSYKSPMGMDPLCYMKLVRLMGASGMHTGTMGYGKMEGHGKETVLAYMLE RDECQGPYFYQKWYGMKATTPIISGGMNALRLPGFFQNLGHGNVINTCGGG AFGHIDSPAAGGISLGQAYDCWKSGSDPIEYAKTHKEFARAFESFPKDGDKLF AGWREKLGVHK.

[0080] The optimized coding region of the nucleic acid sequence of the gene encoding GcRuBisCo is shown below as SEQ ID NO: 2: ATGGACCAATCCAACAGATACGCCAATTTGAACTTGAAGGAAGA AGACTTGATCAAGAACGGTAAGCACTTATTGGTTGCTTACAAGCTAATTCCAG CCAAAGGTCACGGCTTCTTGGAAGTCGCTGCCCACGTTGCTGCCGAATCTTC CACTGGTACCAACGTCGAAGTTTCTACTACCGACGATTTCACCAGAGGTGTC GACGCTTTGGTTTACGAAATCGACGAAACTGCTTTTGGTGACGATATCGTCAA GGGTGGTGGCTTGTTCAAGGTTGCTTACCCAGTCGAATTGTTCGACCCAAAC TTGACCGATGGTACTTACAACATTTCTCACATGTGGTCCTTGATCTTAGGCAA Petição 870250087544, de 26 / 09 / 2025, pág. 86 / 94 41 / 42 CAATCAAGGTATGGGCGATCATCAAGGTTTGCGTATGTTGGACTTCTTGGTTC CAGAAATGATGGTCAGAAAGTTTGATGGTCCTTCTGCCAACATTTCCAATTTAT GGAAGGTCTTGGGTAGATCCGAAACAGACGGTGGCTACATTGCTGGTACCAT TATCAAACCAAAGTTGGGTTTACGTCCAGAACCTTTTGCTAAGGCCTGCTACG ACTTCTGGTTAGGTGGCGACTTCATCAAGAACGACGAACCTCAAGCTAACCAA CCATTTTGTCCAATGGAAGTCGTTATGCCAAAGGTTGCTGAAGCTATGGACAG AGCTCAACAGGAAACTGGTCAAGCCAAGTTGTTCTCTGCTAACATTACTGCTG ACTATTACAAGGAAATGATTCACAGAGGTGACTTTGTTCTAGAAACTTTTGCCA AGTACAACTCTGCTTCCCACGTTGCTTTCTTGGTCGATGGTTTCGTTACTGGT CCAGCTGGTGTTACCACTTGTAGACGTGAATTTCCAGATACCTTCTTGCACTT TCACAGAGCTGGTCACGGTGCTGTTACCTCCTACAAGTCTCCAATGGGTATG GATCCATTGTGTTACATGAAGTTGGTCAGATTGATGGGTGCTTCTGGTATGCA TACCGGTACTATGGGTTACGGCAAGATGGAAGGTCACGGCAAGGAAACCGTC TTGGCTTACATGTTGGAAAGAGACGAATGTCAAGGTCCATACTTCTACCAAAA GTGGTACGGTATGAAGGCTACCACTCCAATCATTTCTGGTGGCATGAACGCTT TGAGACTACCAGGTTTCTTTCAAAACTTGGGTCACGGCAACGTCATCAATACT TGTGGTGGCGGTGCCTTTGGTCACATCGACTCTCCAGCTGCCGGTGGCATCT CCTTGGGTCAAGCTTACGACTGTTGGAAGTCTGGTTCCGATCCAATCGAATACGCCAAGACCCACAAGGAATTCGCTAGAGCCTTCGAATCCTTTCCAAAGGATG GCGACAAGTTGTTCGCTGGCTGGAGAGAAAAGTTGGGTGTTCACAAGTAA.

[0081] When grown in an SSF process, yeast cells expressing GcRuBisCo (G5529) showed a significant reduction in glycerol and acetate combined with no change (or even a moderate increase) in ethanol production (Table 1). Table 1. Analysis of the end of fermentation of yeast expressing different RuBisCo enzymes. Strain Source of RuBisCo Ethanol Glucose Glycerol Acetate G5308 T. denitrificans 151.2 2.71 8.23 ​​0.49 G5529 G. capsiferriformans 152.2 0.98 7.46 0.40 Petition 870250087544, dated 09 / 26 / 2025, pp. 87 / 94 42 / 42

[0082] Glycerol production was reduced by 9.26% and acetate production was reduced by 19.47% after fermentation with yeast cells expressing GcRuBisCo, compared to fermentation with yeast cells expressing the RuBisCo enzyme from T. denitrificans. Residual glucose also decreased by more than 60%.

[0083] The scope of the present invention is not limited to the particular embodiments disclosed, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the compositions and methods described will become apparent from the description and teachings contained herein. Such variations may be practiced without departing from the true scope and spirit of the disclosure and are encompassed within the scope of the present disclosure. Although the invention may be described in combination with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. In fact, it is intended that various modifications of the described modes for implementing the invention that are obvious to those skilled in molecular biology or related fields are within the scope of the following claims. Petition 870250087544, dated 09 / 26 / 2025, pages 88 / 94

Claims

1 / 4 CLAIMS 1. MODIFIED YEAST CELLS derived from parental yeast cells, wherein the modified yeast cells are characterized by comprising a genetic alteration that causes the modified cells to produce a ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCo) enzyme from Gallionella capsiferriformans (GcRuBisCo).

2. Modified yeast cells, according to claim 1, characterized by additionally comprising a gene encoding a phosphoribulokinase.

3. MODIFIED YEAST CELLS derived from parental yeast cells, wherein the modified cells are characterized by comprising a genetic alteration that causes the modified cells to produce the ribulose-1,5-bisphosphate carboxylase oxygenase (RuBisCo) enzyme from Gallionella capsiferriformans (GcRuBisCo), wherein the modified cells produce during fermentation a reduced amount of glycerol and / or acetate compared to the amount of glycerol and acetate produced by identical parental cells that produce the RuBisCo enzyme from Thiobacillus dentrificans under identical fermentation conditions, and wherein the modified yeast cells and the parental yeast cells both additionally comprise an exogenous gene encoding a phosphoribulokinase.

4. MODIFIED YEAST CELLS, according to any one of claims 1 to 3, characterized in that the genetic alteration comprises the introduction into the parent cells of a nucleic acid capable of directing the expression of Gc RuBisCo.

5. MODIFIED YEAST CELLS, according to any one of claims 1 to 4, characterized in that the genetic alteration comprises the introduction of an expression cassette to direct the expression of Gc RuBisCo.

6. MODIFIED YEAST CELLS, according to any one of claims 1 to 5, characterized in that GcRuBisCo comprises an amino acid sequence established by SEQ ID NO: 1 or an amino acid sequence that has at least 70% identity with the sequence established by SEQ ID NO:

1.

7. Modified yeast cells, according to any one of claims 1 to 6, characterized by additionally comprising exogenous genes encoding chaperone proteins.

8. Modified yeast cells, according to claim 7, characterized in that the chaperone proteins are GroES and GroEL from Escherichia coli.

9. MODIFIED YEAST CELLS, according to claim 8, characterized in that GroES comprises an amino acid sequence established by SEQ ID NO: 5 or an amino acid sequence that has at least 70% identity with SEQ ID NO: 5 and / or GroEL comprises an amino acid sequence established by SEQ ID NO: 3 or an amino acid sequence that has at least 70% identity with SEQ ID NO:

3.

10. MODIFIED YEAST CELLS, according to any one of claims 1 to 9, characterized by additionally comprising an exogenous gene encoding a glycerol transporter, a glycerol dehydrogenase and dihydroxyacetone kinase.

11. MODIFIED YEAST CELLS, according to any one of claims 1 to 10, wherein the cells are characterized by additionally comprising one or more genes of the phosphoketolase pathway.

12. MODIFIED YEAST CELLS, according to Petition 870250087544, dated 09 / 26 / 2025, pp. 90 / 94 3 / 4, any of claims 1 to 11, characterized by further comprising an alteration in the glycerol pathway and / or the acetyl-CoA pathway.

13. MODIFIED YEAST CELLS, according to any one of claims 1 to 12, characterized by further comprising an alternative route for the production of ethanol.

14. MODIFIED YEAST CELLS, according to any one of claims 2 to 13, characterized in that the phosphoribulokinase comprises an amino acid sequence established by SEQ ID NO: 9 or an amino acid sequence that has at least 70% identity with SEQ ID NO:

9.

15. MODIFIED YEAST CELLS, according to any one of claims 1 to 14, wherein the cells are further characterized by comprising an exogenous gene encoding a carbohydrate-processing enzyme.

16. MODIFIED YEAST CELLS, according to any one of claims 1 to 15, wherein the cells are characterized as being of Saccharomyces spp.

17. METHOD FOR REDUCING GLYCEROL AND ACETATE PRODUCTION IN YEAST CELLS CULTIVATED ON A CARBOHYDRATE SURFACE characterized by comprising: introducing into parental yeast cells a genetic alteration that causes the modified yeast cells to produce ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCo) from Gallionella capsiferriformans.

18. METHOD, according to claim 17, characterized in that the modified yeast cells are the modified yeast cells as defined in any one of claims 1 to 16.

19. METHOD FOR PRODUCING A FERMENTATION PRODUCT characterized by comprising fermenting a carbohydrate substrate with a modified yeast cell, as defined in any of claims 1 to 16, under conditions to produce a fermentation product.

20. METHOD, according to claim 20, characterized in that the fermentation product is ethanol. Petition 870250087544, dated 09 / 26 / 2025, pp. 92 / 94