Decreasing acetate production by overexpressing a mig polypeptide in yeast

Abstract

Compositions and methods involving modified yeast that overexpress a MIG transcriptional regulator polypeptide are described. The yeast produce reduced amounts of acetic acid compared to the parent cell. Such yeast are particularly useful for large scale production of ethanol from starch substrates, where acetic acid is an undesirable end product.

Description

Technical Field

[0001] The compositions and methods of this invention relate to modified yeast overexpressing a MIG transcription factor peptide. This yeast produces reduced amounts of acetic acid compared to parental cells. This yeast is particularly useful for the large-scale production of ethanol from starch substrates, where acetic acid is an undesirable byproduct. Background Technology

[0002] The first generation of yeast-based ethanol production converted sugar into fuel ethanol. Global annual yeast-based fuel ethanol production is approximately 90 billion liters (Gombert, AK, and van Maris. AJ (2015) Curr. Opin. Biotechnol. [New Insights in Biotechnology] 33: 81-86). It is estimated that about 70% of the cost of ethanol production is for feedstock. Because production volumes are so large, even small yield increases can have a significant economic impact on the entire industry.

[0003] Ethanol production is higher in engineered yeast cells with a heterologous phosphoketone (PKL) pathway than in parental strains without a PKL pathway (see, for example, Miasnikov et al., WO 2015148272). The PKL pathway consists of phosphoketone (PKL) and phosphoacetyltransferase (PTA) to divert carbon flux away from the glycerol pathway and toward acetyl-CoA synthesis. Two supporting enzymes, acetaldehyde dehydrogenase (AADH) and acetyl-CoA synthase (ACS), contribute to making the PKL pathway more efficient.

[0004] Unfortunately, these engineered strains also produce more acetic acid than the parent yeast. Acetic acid is an undesirable byproduct because it negatively impacts yeast growth and fermentation. Furthermore, acetic acid lowers the pH of the residual water from fermentation and distillation (known as the countercurrent), which is typically reused for liquefaction of subsequent batches of substrate. As a result, ethanol producers must adjust the pH of the countercurrent (or liquefaction) or increase the amount of fresh water used for liquefaction.

[0005] There is a need to control the amount of acetic acid produced by yeast (especially engineered yeast that tends to produce increased amounts of acetic acid). Summary of the Invention

[0006] The compositions and methods of the present invention relate to modified yeast overexpressing MIG transcription factor peptides. Aspects and examples of these compositions and methods are described in the following independently numbered paragraphs.

[0007] 1. In one aspect, there is provided a modified yeast cell derived from parental yeast cells, the modified cells comprising a genetic alteration that causes the modified cells to produce an increased amount of MIG transcription factor peptide during fermentation compared to otherwise identical parental cells, wherein the modified cells produce a reduced amount of acetic acid during fermentation compared to the amount of acetic acid produced by these otherwise identical parental cells under the same fermentation conditions.

[0008] 2. In some embodiments of the modified cells as described in paragraph 1, the genetic alteration includes the introduction of nucleic acids into these parental cells that are capable of directing the expression of MIG transcriptional regulatory factor peptides at a level higher than that of parental cells grown under equivalent conditions.

[0009] 3. In some embodiments of the modified cells as described in paragraph 1, the genetic alteration includes the introduction of a novel promoter for expressing the MIG transcriptional regulatory factor polypeptide.

[0010] 4. In some embodiments of the modified cells as described in any one of paragraphs 1-3, these cells further comprise one or more genes of the phosphotransketase pathway.

[0011] 5. In some embodiments of the modified cells as described in paragraph 4, the genes for these phosphoketone pathways are selected from the group consisting of: phosphoketonease, phosphoacetyltransferase, and acetylated acetyl dehydrogenase.

[0012] 6. In some embodiments of the modified cells as described in any one of paragraphs 1-5, the expression level of the MIG transcription factor peptide is increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 500%, at least 1,000%, at least 2,000%, or more compared to the amount of MIG transcription factor peptide produced by parental cells grown under the same conditions.

[0013] 7. In some embodiments of the modified cells as described in any one of paragraphs 1-5, the production of mRNA encoding the MIG transcription regulatory factor polypeptide is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more compared to the levels in these parental cells grown under equivalent conditions.

[0014] 8. In some embodiments of the modified cells as described in any one of paragraphs 1-7, the cells further comprise an exogenous gene encoding a carbohydrate processing enzyme.

[0015] 9. In some embodiments, the modified cells as described in any one of paragraphs 1-8 further comprise alterations in the glycerol pathway and / or the acetyl-CoA pathway.

[0016] 10. In some embodiments, the modified cells, as described in any one of paragraphs 1-9, further comprise an alternative pathway for the preparation of ethanol.

[0017] 11. In some embodiments of the modified cells as described in any one of paragraphs 1-10, these cells belong to the species of the genus *Saccharomyces*.

[0018] 12. In some embodiments of the modified cells as described in any one of paragraphs 1-11, the MIG transcription factor polypeptide is MIG1, MIG2, MIG3, or a combination thereof.

[0019] 13. In another aspect, a method for reducing acetic acid production in yeast cells growing on a carbohydrate substrate is provided, the method comprising: introducing a genetic alteration into parent yeast cells that increases the production of MIG transcription factor peptides compared to the amount produced by the parent cells.

[0020] 14. In some embodiments of the method as described in paragraph 13, the cells having the introduced genetic alterations are modified cells, which are cells as described in any one of paragraphs 1-12.

[0021] 15. In some embodiments of the method as described in paragraph 13 or 14, the reduction in acetic acid production is at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, or more.

[0022] 16. In some embodiments of the method as described in any one of paragraphs 13-15, the MIG transcription factor peptide is overexpressed by at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 500%, at least 1,000%, at least 2,000%, or more, compared to the amount produced by parental cells grown under the same conditions and otherwise identical.

[0023] 17. In some embodiments of the method as described in any one of paragraphs 13-16, the overexpression of MIG transcription factor mRNA is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more than the amount produced in these parental cells grown under equivalent conditions.

[0024] These and other aspects of the modified cells and methods of the present invention, as well as embodiments thereof, will be clear from the description which includes any accompanying drawings / figures. Detailed Implementation

[0025] I. Definition

[0026] Before describing the yeast and method of the present invention in detail, the following terms are defined for clarity. Undefined terms shall conform to their conventional meanings as used in the relevant art.

[0027] 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.

[0028] As used herein, the terms "yeast cell," "yeast strain," or simply "yeast" refer to organisms belonging to the phyla Ascomycota and Basidiomycota. Exemplary yeasts are budding yeasts from the order Saccharomycetales. Specific examples of yeast are species of the genus *Saccharomycetes*, including but not limited to *Saccharomyces cerevisiae*. Yeasts include organisms used for the production of fuel alcohols as well as organisms used for the production of drinkable alcohols, including specialized and proprietary yeast strains used to prepare distinctive flavors in beer, wine, and other fermented beverages.

[0029] As used herein, the phrases “engineered yeast cells,” “variant yeast cells,” “modified yeast cells,” or similar phrases refer to yeast that includes the genetic modifications and characteristics described herein. Variant / modified yeast does not include naturally occurring yeast.

[0030] As used herein, the terms “peptide” and “protein” (and their respective plural forms) are used interchangeably and refer to polymers of any length containing amino acid residues linked by peptide bonds. Conventional one- or three-letter codes for amino acid residues are used herein, and all sequences are presented from the N-terminus to the C-terminus. Polymers may contain modified amino acids, and they may be interrupted by non-amino acid components. These terms also cover amino acid polymers that are naturally modified or modified by interventions (e.g., through disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeled component). These definitions also include, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids) and other modifications known in the art.

[0031] As used herein, functionally and / or structurally similar proteins are considered “related proteins” or “homologs.” Such proteins can be derived from organisms of different genera and / or species, or different classes (e.g., bacteria and fungi), or can be artificially engineered proteins. Related proteins also encompass homologs identified by primary sequence analysis, secondary or tertiary structure analysis, immune cross-reactivity, or their function.

[0032] As used herein, the term "homology" refers to a protein that has similar activity and / or structure to a reference protein. This is not intended to imply that homologs are necessarily evolutionarily related. Therefore, the term is intended to cover one or more enzymes that are identical, similar, or corresponding (i.e., in terms of structure and function) from different organisms. In some embodiments, it is desirable to identify homologs that have similar quaternary, tertiary, and / or primary structures to a reference protein. In some embodiments, the homolog serves as a reference protein and induces one or more similar immune responses. In some embodiments, the homolog is engineered to produce an enzyme with one or more desired activities.

[0033] The degree of homology between sequences can be determined using any suitable method known in the art (see, for example, Smith and Waterman (1981) Adv. Appl. Math. [Advances in Applied Mathematics] 2: 482; Needleman and Wunsch (1970) J. Mol. Biol. [Journal of Molecular Biology], 48: 443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 85: 2444; programs in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wisconsin, WI) such as GAP, BESTFIT, FASTA, and TFASTA; and Devereux et al. (1984) Nucleic Acids Res. [Nucleic Acid Research] 12: 387-95).

[0034] For example, PILEUP is a useful procedure for determining the level of sequence homology. PILEUP creates multiple sequence alignments from a set of related sequences using progressive, pairwise alignments. It can also draw a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (Feng and Doolittle (1987) J. Mol. Evol. [Journal of Molecular Evolution] 35: 351-60). This method is similar to that described by Higgins and Sharp ((1989) CABIOS [Computers in Biology] 5: 151-53). Useful PILEUP parameters include a default empty weight of 3.00, a default empty length weight of 0.10, and a weighted terminal empty weight. Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al. ((1990) J. Mol. Biol. [Journal of Molecular Biology] 215: 403-10) and Karlin et al. ((1993) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States of America] 90: 5873-87). A particularly useful BLAST procedure is the WU-BLAST-2 procedure (see, for example, Altschul et al., (1996) Meth. Enzymol. [Enzymological Methods] 266: 460-80). The parameters “W”, “T”, and “X” determine the sensitivity and speed of the alignment. The BLAST procedure uses a word length (W) of 11, a BLOSUM62 score matrix (see, for example, Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences] 89: 10915) alignment (B) of 50, expectation value (E) of 10, M′5, N-4, and a comparison of two chains as default values.

[0035] As used herein, in the context of at least two nucleic acids or polypeptides, the phrases “substantially similar” and “substantially identical” typically mean that the polynucleotide or polypeptide contains a sequence with at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, or even at least approximately 99%, or higher, identity compared to a 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. [Nucleic Acids Research] 22: 4673-4680. The default parameters for the CLUSTAL W algorithm are:

[0036]

[0037]

[0038] Another indication that two polypeptides are substantially identical is that the first polypeptide and the second polypeptide are immunoreactive. Typically, polypeptides differing only in conserved amino acid substitutions are immunoreactive. Therefore, the polypeptides are substantially identical, for example, where the two peptides differ only in conserved 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., in the range of moderate to high stringency).

[0039] As used herein, the term "gene" is synonymous with the term "allele," referring to a nucleic acid that encodes and directs the expression of a protein or RNA. The vegetative form of filamentous fungi is typically haploid, therefore a single copy of a designated gene (i.e., a single allele) is sufficient to confer a designated phenotype. When an organism contains more than one similar gene, the term "allele" is generally preferred, in which case each distinct similar gene is referred to as a different "allele."

[0040] As used in this article, “constitutive” expression refers to the production of a polypeptide encoded by a specific gene under virtually all typical growth conditions, rather than “conditional” expression, which requires the presence of specific substrates, temperatures, etc., to induce or activate expression.

[0041] As used herein, the term "expressed polypeptide" and similar terms refer to the cellular process of producing polypeptides using the cell's translational machinery (e.g., ribosomes).

[0042] As used herein, “overexpressed peptide,” “increased peptide expression,” and similar terms refer to peptide expression at a higher-than-normal level compared to that observed in parental or “wild-type” cells that do not contain the specified genetic modification.

[0043] As used herein, an "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), as well as other nucleic acid sequences required to allow the encoded polypeptide to be produced in the cell. Expression cassettes can be exogenous (i.e., introduced into the cell) or endogenous (i.e., present in the cell).

[0044] As used herein, the terms “fused” and “fused” (e.g., the coding region of a promoter and a polypeptide) refer to the physical bond that causes the two DNA fragments to become a single molecule.

[0045] As used herein, the terms “wild-type” and “natural” are used interchangeably and refer to genes, proteins, or strains found in nature, or genes, proteins, or strains that have not been intentionally modified for the advantages of the yeast described herein.

[0046] As used herein, the term "target protein" refers to a polypeptide that is intended to be expressed in modified yeast. This protein can be an enzyme, substrate-binding protein, surfactant protein, structural protein, selectable marker, etc., and can be expressed. The target protein is encoded by an endogenous or heterologous gene relative to the parent strain (i.e., the target gene). The target protein can be expressed intracellularly or as a secreted protein.

[0047] As used herein, “gene disruption” refers broadly to any genetic or chemical manipulation (i.e., mutation) that substantially prevents a cell from producing a functional gene product (e.g., a protein) in a host cell. Exemplary disruption methods include the complete or partial deletion of any part of a gene (including a polypeptide coding sequence, promoter, enhancer, or other regulatory element), or its mutagenesis, wherein mutagenesis encompasses substitution, insertion, deletion, inversion, and combinations and variations thereof, any of which substantially prevent the production of a functional gene product. Genes may also be disrupted using CRISPR, RNAi, antisense, or any other method of eliminating gene expression. Genes can be disrupted by the deletion or genetic manipulation of non-adjacent control elements. As used herein, “gene deletion” means the removal of the gene from the genome of the host cell. When a gene includes control elements (e.g., enhancer elements) that are not immediately adjacent to the gene coding sequence, gene deletion means the deletion of the 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 part of the coding sequence, or a part of the promoter that is adjacent to or not adjacent to the coding sequence, in which the functional activity of the target gene is absent in engineered cells.

[0048] As used herein, the terms “genetic manipulation,” “genetic alteration,” “genetic engineering,” and similar terms are used interchangeably and refer to changes / modifications of nucleic acid sequences. Changes may include, but are not limited to, substitution, deletion, insertion, or chemical modification of at least one nucleic acid in the nucleic acid sequence.

[0049] As used herein, a “functional peptide / protein” is a protein that possesses activity (e.g., enzymatic activity, binding activity, surface activity, etc.) and has not been mutagenized, truncated, or otherwise modified to eliminate or reduce this activity. As noted, functional peptides may be heat-stable or heat-sensitive.

[0050] 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). A functional gene is the opposite of a disrupted gene, which has been modified so that it cannot be used by cellular components to produce an active gene product, or has a reduced ability to be used by cellular components to produce an active gene product.

[0051] As used herein, yeast cells that have undergone genetic or chemical alterations to prevent the production of functional proteins / peptides exhibiting the activity characteristics of wild-type proteins have been “modified to prevent the production of the specified protein.” Such modifications include, but are not limited to, deletions or disruptions of genes encoding proteins (as described herein), modifications of genes that render the encoded peptides lacking the aforementioned activity, modifications of genes that affect post-translational processing or stability, and combinations thereof.

[0052] As used herein, “pathway attenuation” or “attenuation of flux through a pathway” (i.e., biochemical pathway) refers to any genetic or chemical manipulation that reduces or completely blocks the flux of biochemical substrates or intermediates through metabolic pathways. Pathway attenuation can be achieved through 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 controlling the expression of one or more genes; engineering these enzymes or the mRNA encoding these enzymes for reduced stability; misdirecting enzymes into cellular compartments unlikely to interact with substrates and intermediates; using interfering RNA; etc.

[0053] As used in this article, "aerobic fermentation" refers to the growth and production process in the presence of oxygen.

[0054] As used in this article, "anaerobic fermentation" refers to growth and production in the absence of oxygen.

[0055] As used herein, the term "end of fermentation" refers to the stage of fermentation at which, with respect to both fixed and variable costs, the economic advantage of producing a small amount of additional alcohol from continuous fermentation is outweighed by the costs of continuous fermentation. In a more general sense, "end of fermentation" means the point at which fermentation will no longer produce a significant amount of additional alcohol, i.e., no more than about 1% additional alcohol.

[0056] As used in this article, "carbon flux" refers to the rate at which carbon molecules are turned over through metabolic pathways. Carbon flux is regulated by enzymes involved in metabolic pathways, such as glucose metabolism and maltose metabolism.

[0057] As used herein, the singular articles “a / an” and “the” cover plural indicators unless the context clearly indicates otherwise. All references cited herein are incorporated herein by way of their full text. Unless otherwise stated, the following abbreviations / acronyms have the following meanings:

[0058] Unless otherwise stated, the following meanings apply:

[0059] ℃ Celsius

[0060] AA αμ-amylase

[0061] AADH acetaldehyde dehydrogenase

[0062] bp base pairs

[0063] DNA deoxyribonucleic acid

[0064] ds or DS dry solids

[0065] EC Enzyme Committee

[0066] EtOH (ethanol)

[0067] g or gm grams

[0068] g / L g / L

[0069] GA glucosyl amylase

[0070] H2O water

[0071] HPLC (High Performance Liquid Chromatography)

[0072] hr or h hours

[0073] kg

[0074] M Moore

[0075] mg

[0076] min minutes

[0077] mL or ml

[0078] mM millimole

[0079] N equivalent concentration

[0080] nm Nem

[0081] PCR Polymerase Chain Reaction

[0082] PKL (phosphotransketase)

[0083] ppm (parts per million)

[0084] PTA phosphoryltransferase

[0085] Δ is related to missing values.

[0086] μg micrograms

[0087] μL and μl

[0088] μM micromolar

[0089] II. Modified yeast cells with increased MIG expression

[0090] Modified yeast and methods involving genetic alterations that result in increased production of MIG transcription factor peptides compared to corresponding (i.e., otherwise identical) parental cells are described. MIG transcription factor peptides (or simply MIG transcription factors) belong to the Cys2His2 zinc finger protein family. MIG1 is 504 amino acids long. MIG2 is 383 amino acids long, and MIG3 is 395 amino acids long. While the overall amino acid sequence identity among these transcription factors is less than 30%, they exhibit >70% sequence identity within their DNA-binding domains.

[0091] MIG1 and MIG2 play well-defined roles in glucose-responsive repression of genes involved in gluconeogenesis, aerobic respiration, and alternative carbon source utilization. Less is known about the function of MIG3. A recent study showed that MIG3 plays a role in catabolite repression in some strains and also exerts additional regulatory effects upon exposure to ethanol (Lewis, JA, and Gasch, AP (2012) G3 2: 1607-12). However, prior to this, the association between MIG overexpression and reduced acetic acid production in yeast fermentation for ethanol production had not been established.

[0092] The applicant has discovered that yeast cells overexpressing the transcriptional regulatory peptides MIG1, MIG2, and MIG3 produce reduced amounts of acetic acid during fermentation compared to parental cells with similar characteristics. This reduction in acetic acid is desirable because it adversely affects yeast growth and fermentation and additionally causes a backflow with a lower pH than desired, requiring pH adjustment or the use of fresher water to dilute the backflow.

[0093] In some embodiments, the amount of MIG peptide produced by the modified cells is increased by at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 500%, at least 1,000%, at least 2,000%, or more compared to the amount of MIG peptide produced by parental cells grown under the same conditions.

[0094] In some embodiments, the amount of MIG mRNA produced by the modified cells is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more compared to the amount of MIG mRNA produced by parental cells grown under the same conditions.

[0095] In some embodiments, the strength of the promoter used to control the expression of the MIG peptide produced by the modified cells (based on the amount of mRNA produced) is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, or more compared to the strength of the natural promoter controlling MIG expression.

[0096] In some embodiments, the modified cells produce at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, or more less acetic acid compared to the amount produced by parental cells grown under the same conditions.

[0097] Preferably, the increase in MIG expression was achieved through genetic manipulation using sequence-specific molecular biology techniques, which is the opposite of chemical mutagenesis, which typically does not target specific nucleic acid sequences. However, chemical mutagenesis is not excluded as a method for preparing modified yeast cells.

[0098] In some embodiments, the compositions and methods of the present invention involve introducing nucleic acids into yeast cells capable of directing the overexpression or increased expression of a MIG peptide. Specific methods include, but are not limited to, (i) introducing a foreign expression cassette for producing the peptide into a host cell, optionally with the addition of an endogenous expression cassette, (ii) replacing the foreign expression cassette with an endogenous cassette that allows for the production of an increased amount of the peptide, (iii) modifying the promoter of the endogenous expression cassette to increase expression, and / or (iv) modifying any aspect of the host cell to increase the half-life of the peptide in the host cell.

[0099] In some embodiments, the modified parental cell already contains the target gene, such as a gene encoding a selectable marker, a carbohydrate processing enzyme, or other polypeptide. In some embodiments, the introduced gene is subsequently introduced into the modified cell.

[0100] An exemplary amino acid sequence of the Saccharomyces cerevisiae MIG1 polypeptide is shown in SEQ ID NO: 5 below:

[0101]

[0102] An exemplary amino acid sequence of the Saccharomyces cerevisiae MIG2 polypeptide is shown in SEQ ID NO: 7 below:

[0103]

[0104] An exemplary amino acid sequence of the Saccharomyces cerevisiae MIG3 polypeptide is shown in SEQ ID NO: 1 below:

[0105]

[0106] The NCBI database includes many Saccharomyces cerevisiae MIG peptides. While the amino acid sequence identity among MIG1, MIG2, and MIG3 is highest in the DNA-binding domain, it is expected that native differences throughout the amino acid sequences of MIG1, MIG2, and MIG3 will not affect their function. Furthermore, it is evident that the exemplary Saccharomyces cerevisiae MIG1, MIG2, and MIG3 peptides share sequence identity with peptides from other organisms, and overexpression of functionally and / or structurally similar proteins, homologous proteins, and / or substantially similar or identical proteins is expected to produce similar beneficial results.

[0107] In specific embodiments of the compositions and methods of the present invention, the amino acid sequences of the MIG1, MIG2 and / or MIG3 polypeptides overexpressed in modified yeast cells have at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or even at least about 99% identity with SEQ ID NO: 5, SEQ ID NO: 7 and / or SEQ ID NO: 1, respectively.

[0108] III. Modified yeast cells with increased expression of MIG transcriptional regulators in combination with genes from the exogenous PKL pathway.

[0109] Increased expression of MIG transcriptional regulators can combine with the expression of genes in the PKL pathway to reduce acetic acid production, which is associated with the introduction of exogenous PKL pathways into yeast.

[0110] Engineered yeast cells with a heterologous PKL pathway have previously been described in WO 2015148272 (Miasnikov et al.). These cells express heterologous phosphotransketolase (PKL), phosphotransacetase (PTA), and acetyl-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 to ethanol. Compared to otherwise identical parental yeast cells, such modified cells are able to increase ethanol production during fermentation.

[0111] IV. Combination of increased MIG transcription factor production with other mutations affecting alcohol production

[0112] In some embodiments, in addition to optionally combining increased expression of the MIG peptide with the heterologous PKL pathway, the modified yeast cells of the present invention include additional beneficial modifications.

[0113] The modified cells may further include mutations that weaken the natural glycerol biosynthesis pathway and / or the glycerol reuse pathway, which are known to increase alcohol production. Methods for weakening the glycerol biosynthesis pathway in yeast are known and include, for example, reducing or eliminating endogenous NAD-dependent glycerol 3-phosphate dehydrogenase (GPD) or glycerol phosphate phosphatase (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 overexpressing glycerol dehydrogenase (GCY1) and dihydroxyacetone kinase (DAK1) to convert glycerol to dihydroxyacetone phosphate (Zhang et al. (2013) J. Ind. Microbiol. Biotechnol. 40: 1153-60).

[0114] The modified yeast can be further characterized by increased acetyl-CoA synthase (also known as acetyl-CoA ligase) activity (EC 6.2.1.1) to remove (i.e. capture) acetic acid produced by the chemical or enzymatic hydrolysis of acetyl-phosphate (or present in the yeast culture medium for any other reason) and convert it to acetyl-CoA. This partially reduces the undesirable effects of acetic acid on yeast cell growth and can further contribute to increased alcohol yield. Increased acetyl-CoA synthase activity can be achieved by introducing a heterologous acetyl-CoA synthase gene into the cell, increasing the expression of an endogenous acetyl-CoA synthase gene, etc.

[0115] In some embodiments, the modified cells may further include cells encoding NAD+. + Heterologous genes encoding proteins with acetaldehyde dehydrogenase-dependent activity and / or heterologous genes 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, yeast specifically lacks one or more heterologous genes encoding acetaldehyde dehydrogenase, pyruvate-formate lyase, or both.

[0116] In some embodiments, the modified yeast cells of the present invention may be further overexpressed with a sugar transporter-like (STL1) peptide to increase glycerol uptake (see, for example, Ferreira et al. (2005) Mol. Biol. Cell. [Cell Molecular Biology] 16: 2068-76; et al. (2015) Mol. Microbiol. [Molecular Microbiology] 97: 541-59 and WO2015023989 A1) to increase ethanol production and reduce acetic acid.

[0117] In some embodiments, the modified yeast cells of the present invention further comprise a butanol biosynthesis pathway. In some embodiments, the butanol biosynthesis pathway is an isobutanol biosynthesis pathway. In some embodiments, the isobutanol biosynthesis pathway comprises a polynucleotide encoding a polypeptide that catalyzes the conversion of a substrate 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 biosynthesis pathway comprises a polynucleotide encoding a polypeptide having acetolactate synthase, keto acid reductase, dihydroxy acid dehydratase, ketoisovalerate decarboxylase, and alcohol dehydrogenase activities.

[0118] In some embodiments, the modified yeast cells comprising the butanol biosynthesis pathway further comprise modifications in the polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In some embodiments, the yeast cells contain deletions, mutations, and / or substitutions in the endogenous polynucleotide encoding the 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 deletions, mutations, overexpressions, and / or substitutions 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, and / or YMR226C.

[0119] V. Combination of increased MIG transcription factor expression with other beneficial mutations

[0120] In some embodiments, in addition to optionally combining increased expression of the MIG polypeptide with other gene modifications beneficial to alcohol production, the modified yeast cells of the present invention further comprise any number of additional target genes encoding the target protein. Additional target genes may be introduced before, during, or after genetic manipulations that result in increased production of the active MIG polypeptide. The target protein 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, phosphotransketases, transaldolases, epimerases, phytases, xylanases, β-glucanases, phosphatases, proteases, α-amylases, β-amylases, glucosylamylases, amylopectinases, isoamylases, cellulases, trehalases, lipases, pectinases, polyesterases, cutinases, oxidases, transferases, reductases, hemicellulases, mannanases, esterases, isomerases, pectinases, lactases, peroxidases, and laccases. The target protein may be secreted, glycosylated, and otherwise modified.

[0121] VI. Uses of modified yeast to increase alcohol production

[0122] The compositions and methods of the present invention comprise methods for increasing alcohol production and / or decreasing glycerol production during fermentation reactions. Such methods are not limited to specific fermentation processes. The engineered yeast of the present invention is intended to be a "drop-in" alternative to conventional yeast in any alcohol fermentation facility. While primarily intended for fuel alcohol production, the yeast of the present invention can also be used to produce drinkable alcohols, including wine and beer.

[0123] VII. Yeast cells suitable for modification

[0124] Yeasts are single-celled eukaryotic microorganisms classified as members of the kingdom Fungi, including organisms from the phyla Ascomycota and Basidiomycota. Yeasts that can be used for alcohol production include, but are not limited to, species from the genera *Saccharomyces*, including *Saccharomyces cerevisiae*, as well as species from the genera *Kluyveromyces*, *Lachancea*, and *Schizosaccharomyces*. Many yeast strains are commercially available, and many have been selected or genetically engineered to obtain desired characteristics, such as high alcohol production and rapid growth rates. Some yeasts have been genetically engineered to produce heterologous enzymes, such as glucosylamylase or α-amylase.

[0125] VIII. Substrate and Product

[0126] The production of alcohols from many carbohydrate substrates (including, but not limited to, corn starch, sugarcane, cassava, and molasses) is well known, as are numerous variations and improvements in enzymatic and chemical conditions and mechanical methods. The compositions and methods of the present invention are believed to be fully compatible with such substrates and conditions.

[0127] Alcohol fermentation products include organic compounds having a hydroxyl functional group (-OH) 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 prepared fuel alcohols are ethanol and butanol.

[0128] Based on this specification, these and other aspects of the yeast strains and methods of the present invention, as well as the examples, will be clear to those skilled in the art. The following examples are intended to further illustrate, but not limit, these compositions and methods.

[0129] Example

[0130] Example 1

[0131] Materials and Methods

[0132] Preparation of liquefied substances:

[0133] The liquefied product (corn mash) was prepared by adding 600 ppm urea, 0.124 SAPU / g ds acidic fungal protease, 0.33 GAU / g ds Trichoderma reesei glucoamylase and 1.46 SSCU / g ds Aspergillus kawachii α-amylase, and adjusting the pH to 4.8 with sulfuric acid.

[0134] Serum bottle assay:

[0135] Yeast cells were inoculated into 2 mL of YPD in a 24-well plate and allowed to grow overnight until the OD reached 25–30. 2.5 mL of liquefaction was transferred to a serum vial (Chemglass, catalog number: CG-4904-01), and yeast was added to each vial until the final OD was approximately 0.4–0.6. The vials were capped and punctured with a needle (BD, catalog number 305111) for aeration (to release CO2), and then incubated at 32°C for 65 hours with shaking at 200 RPM.

[0136] AnKom determination:

[0137] 300 μL of concentrated overnight yeast culture was added to each of several ANKOM flasks filled with 50 g of the prepared liquefaction (see above) to achieve a final OD of 0.3. The flasks were then incubated at 32 °C for 65 hours with shaking at 150 RPM.

[0138] HPLC analysis:

[0139] Samples from serum vials and AnKom assays were collected in Eppendorf tubes by centrifugation at 14,000 RPM for 12 min. The supernatant was filtered through a 0.2 μM PTFE filter and then used for HPLC analysis (Agilent Technologies 1200 series) under the following conditions: Bio-Rad Aminex HPX-87H column, operating temperature 55 °C, isocratic flow rate 0.6 mL / min, 0.01 N H₂SO₄, 2.5 μL injection volume. Calibration standards were used for quantification of acetic acid, ethanol, glycerol, glucose, and other molecules. All values ​​are reported in g / L.

[0140] RNA-Seq analysis:

[0141] RNA was prepared from individual samples using the TRIzol method (Life-Tech, Rockville, MD). The RNA was then cleaned using the Qiagen RNeasy Mini Kit (Qiagen, Germantown, MD). cDNA was generated from the total mRNA in each individual sample using the Applied Biosystems Large Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Wilmington, Delaware). The cDNA from each prepared sample was sequenced using the shotgun method and then quantified relative to individual genes. Results were reported as readings per kilobase ten million transcripts (RPK10M) and used to quantify the amount of each transcript in the sample.

[0142] Example 2

[0143] MIG expression in yeast

[0144] To understand the regulation of MIG in yeast, the *Saccharomyces cerevisiae* strain FERMAX was used. TM RNA-Seq analysis was performed on Gold (Martrex Inc., Minnesota, USA; abbreviated as "FG" in this article) (which is the standard strain used for ethanol production). RNA-Seq was performed as described in Example 1, and the results are summarized in Table 1. Expression levels are expressed as readings of 10 million transcripts per kilobase (RPK 10M).

[0145] Table 1. RNA-Seq analysis of MIG expression in FG during fermentation

[0146]

[0147] The results showed that MIG1, MIG2, and MIG3 were expressed at low levels in the FG strain during fermentation. In contrast, MIG3 was expressed at the highest level at approximately 24 hours during fermentation.

[0148] Example 3

[0149] Promoter selection for increasing MIG3 expression

[0150] As shown in Table 2, in strain FG, translation elongation factor 1β (EFB1) was expressed at its highest level at approximately 6 hours during fermentation, and then maintained at a high level for the remainder of fermentation. Therefore, the EFB1 promoter was selected to drive the overexpression of MIG3.

[0151] Table 2. Transcription profile of MIG3 and EFB1 during fermentation

[0152]

[0153] Example 4

[0154] overexpression of MIG3 in yeast

[0155] To overexpress MIG3, the promoter of endogenous MIG3 was exchanged with the EFB1 promoter in the FG parent. PCR confirmed the expected substitution of the MIG3 promoter by the EFB1 promoter. The amino acid sequence of the MIG3 peptide is shown in SEQ ID NO: 1 below:

[0156]

[0157] The DNA sequence of the MIG3 coding region is shown in SEQ ID NO: 2 below:

[0158]

[0159] The DNA sequence of the EFB1 promoter is shown in SEQ ID NO: 3 below:

[0160]

[0161] Example 5

[0162] Alcohol production using yeast overexpressing MIG3

[0163] As described in Example 1, a yeast strain expressing MIG3 (FG-MIG3) and its corresponding parent strain (FG) were tested in vials containing 5.6 g of liquefied material. Fermentation was carried out at 32°C for 55 hours. The samples at the end of fermentation were analyzed by HPLC. The results are summarized in Table 3.

[0164] Table 3. HPLC results from vial determination

[0165]

[0166] Overexpression of MIG3 resulted in a reduction of approximately 24% in acetic acid production, without negatively impacting ethanol production. These results demonstrate that overexpression of MIG3 is beneficial for reducing acetic acid production during yeast fermentation for ethanol production.

[0167] Example 6

[0168] Additional promoter selection for increasing MIG1 and MIG2 expression

[0169] Encouraged by the significant reduction in acetic acid in cells overexpressing MIG3, further experiments were conducted to determine whether a similar reduction in acetic acid could be achieved by overexpressing MIG1 and MIG2 using a promoter weaker than EFB1 (Table 2). Similar to Example 2, FERMAX was used to overexpress MIG1, MIG2, or SUI3 (which encodes the β subunit of eukaryotic initiation factor 2 (eIF-2)). TM RNA-Seq analysis was performed on Gold yeast. The results are summarized in Table 4. As shown above, levels are expressed as readings of ten million transcripts per kilobase (RPK10M). The data confirm that, at FERMAX... TM In the Gold strain, MIG1 and MIG2 were expressed at low levels during fermentation.

[0170] Table 4. FERMAX during fermentation TM RNA-Seq analysis of MIG1, MIG2 and SUI3 in gold

[0171]

[0172] In contrast to the very weak expression of MIG1 and MIG2, the expression level of SUI3 was approximately 5 to 20 times higher than that of MIG1 or MIG2 throughout the fermentation process. Therefore, the SUI3 promoter was chosen to drive the overexpression of MIG1 and MIG2.

[0173] Example 7

[0174] Preparation of MIG1 and MIG2 expression cassettes

[0175] Codon optimization was performed on the MIG1 gene (YGL035C) and MIG2 gene (YGL209W) of *Saccharomyces cerevisiae*, and then MIG1s (SEQ ID: 4 and 5) and MIG2s (SEQ ID: 6 and 7) were synthesized, respectively. The SUI3 promoter (YPL237W locus; SEQ ID NO: 8) was individually ligated to either MIG1 or MIG2, and then ligated to the GPD1 terminator (YDL022W locus; SEQ ID NO: 9) to generate the SUI3Pro::MIGls::GpdlTer and SUI3pro::MIG2s::GpdlTer expression cassettes. These two expression cassettes were introduced downstream of the RPA190 locus (YOR341W) into FERMAX. TM In Gold yeast, the expected insertion of MIG1s and MIG2s expression cassettes in the parental strain was confirmed by PCR.

[0176] The DNA sequence of the codon-optimized MIG1 coding region (MIG1s) is shown in SEQ ID NO: 4 below:

[0177]

[0178] The amino acid sequence of the MIG1 polypeptide is shown in SEQ ID NO: 5 below:

[0179]

[0180] The DNA sequence of the codon-optimized MIG2 coding region (MIG2s) is shown in SEQ ID NO: 6 below:

[0181]

[0182] The amino acid sequence of the MIG2 polypeptide is shown in SEQ ID NO: 7 below:

[0183]

[0184] The DNA sequence of the SUI3 promoter is shown in SEQ ID NO: 8 below:

[0185]

[0186] The DNA sequence of the GPD1 terminator region is shown in SEQ ID NO: 9 below:

[0187] Example 8

[0188] Ethanol and Acetic Acid Production from MIG1 or MIG2 Overexpressing Yeasts

[0189] As described in Example 1, the following strains were tested in the Ankom assay: each of the PCR-confirmed strains overexpressing MIG1 or MIG2 under the control of the SUI1 promoter, and the FG parent strain. Fermentation was carried out at 32°C for 55 hours. The samples at the end of fermentation were analyzed by HPLC. The results are summarized in Table 5.

[0190] Table 5. HPLC results of FG and FG overexpressing MIG1 or MIG2 with SUI3 promoter

[0191]

[0192] The results showed that, in the presence of the SUI3 promoter, overexpression of MIG1 or MIG2 resulted in a reduction of acetic acid by approximately 15% or 36%, and a reduction of glycerol by approximately 3.8% or 6%, respectively; with no negative impact on ethanol yield.

Claims

1. A brewing yeast for reducing growth on carbohydrate substrates ( Saccharomyces cerevisiae A method for producing acetic acid from cells, the method comprising: Introducing a genetic alteration into parental Saccharomyces cerevisiae cells that increases the production of MIG transcription factor peptides compared to the amount produced by the parental Saccharomyces cerevisiae cells, wherein the MIG transcription factor peptide is MIG2, MIG3, or a combination thereof, and wherein the genetic alteration is selected from introducing an EFB1 promoter consisting of the DNA sequence of SEQ ID NO: 3 for expressing the MIG3 transcription factor peptide and a SUI3 promoter consisting of the DNA sequence of SEQ ID NO: 8 for expressing the MIG2 transcription factor peptide.

2. The method of claim 1, wherein the genetic alteration comprises introducing a nucleic acid into the parental Saccharomyces cerevisiae cells, the nucleic acid being capable of directing the expression of the MIG transcriptional regulatory factor polypeptide at a level higher than that of the parental Saccharomyces cerevisiae cells grown under equivalent conditions.

3. The method of any one of claims 1-2, wherein the cell further comprises one or more genes of the phosphotransketase pathway.

4. The method of claim 3, wherein the gene for the phosphoketone pathway is selected from the group consisting of: a gene encoding phosphoketone, a gene encoding phosphoacetyltransferase, and a gene encoding acetylated acetyl dehydrogenase.

5. The method of any one of claims 1-2 and 4, wherein the cell further comprises an exogenous gene encoding a carbohydrate processing enzyme and / or other polypeptide, wherein the carbohydrate processing enzyme and / or other polypeptide is selected from dehydrogenases, oxidases, transferases, reductases, esterases and isomerases.

6. The method of any one of claims 1-2 and 4, wherein the cell further comprises an exogenous gene encoding a carbohydrate processing enzyme and / or other polypeptide, wherein the carbohydrate processing enzyme and / or other polypeptide is selected from transketolase, phosphoketolase, transaldolase, epimerase, phosphatase, protease, α-amylase, β-amylase, glucosylamylase, amylopectinase, isoamylase, cellulase, trehalase, lipase, pectinase, polyesterase, cutinase, hemicellulase, lactase, peroxidase, and laccase.

7. The method of any one of claims 1-2 and 4, wherein the cell further comprises an exogenous gene encoding a carbohydrate processing enzyme and / or other polypeptide, wherein the carbohydrate processing enzyme and / or other polypeptide is selected from phytase, xylanase, β-glucanase and mannanase.

8. The method according to any one of claims 1-2 and 4, further comprising alterations in the glycerol pathway and / or the acetyl-CoA pathway.

9. The method of claim 8, further comprising: a) Reduced or eliminated endogenous NAD-dependent glycerol-3-phosphate dehydrogenase (GPD) activity; b) Overexpression of glycerol dehydrogenase (GCY1) and dihydroxyacetone kinase (DAK1); and / or c) Increased acetyl-CoA synthase activity.

10. The method of claim 9, wherein endogenous NAD-dependent glycerol-3-phosphate dehydrogenase (GPD) activity is reduced or eliminated by disrupting one or more of genes GPD1 and GPD2, and acetyl-CoA synthase activity is increased by introducing a heterologous acetyl-CoA synthase gene.

11. The method according to any one of claims 1-2 and 4, further comprising an alternative route for preparing ethanol.

12. The method according to any one of claims 1-2 and 4, wherein the reduction in acetic acid production is at least 5%.

13. The method according to any one of claims 1-2 and 4, wherein the reduction in acetic acid production is at least 10%.

14. The method according to any one of claims 1-2 and 4, wherein the reduction in acetic acid production is at least 15%.

15. The method of any one of claims 1-2 and 4, wherein the reduction in acetic acid production is at least 20%.

16. The method of any one of claims 1-2 and 4, wherein the reduction in acetic acid production is at least 25%.

17. The method of any one of claims 1-2 and 4, wherein the reduction in acetic acid production is at least 30%.

18. The method of any one of claims 1-2 and 4, wherein the reduction in acetic acid production is at least 35%.

19. The method of any one of claims 1-2 and 4, wherein the MIG transcription factor polypeptide is overexpressed by at least 20% compared to the amount of MIG transcription factor polypeptide produced by parental Saccharomyces cerevisiae cells grown under the same conditions and otherwise identical.

20. The method of any one of claims 1-2 and 4, wherein the MIG transcription factor mRNA is overexpressed at least 2-fold compared to the amount produced in parental Saccharomyces cerevisiae cells grown under equivalent conditions.

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