Modified yeast and method for increasing lysine content in fermentation co-products

Abstract

Described are strains and methods involving genetically engineered yeast cells that overproduce lysine in a tunable manner by altering feedback inhibition of the lysine synthesis pathway via LYS20 and LYS21 homocitrate synthase polypeptides. The yeast can be used in conventional bioethanol production facilities to produce alcohol as well as increased amounts of lysine, thereby improving the quality and commercial value of fermentation products and co-products such as animal feed ingredients.

Description

Technical Field

[0001] The strains and methods of the present invention relate to genetically engineered yeast cells that overproduce lysine in a tunable manner by altering feedback inhibition of the lysine synthesis pathway via LYS20 and LYS21 homocitrate synthase polypeptides. The yeast can be used in conventional bioethanol production facilities to produce alcohol as well as increased amounts of lysine, thereby improving the quality and commercial value of fermentation products and co-products (such as animal feed ingredients). Background Art

[0002] Many countries produce fuel alcohol from fermentable substrates such as corn starch, sugar cane, cassava, and molasses. In the United States alone, fuel ethanol production approached 15 billion gallons in 2015, according to the Renewable Fuels Association (Washington, D.C., USA).

[0003] In addition to producing about 2.8 gallons of ethanol, a bushel (56 pounds) of corn processed in a dry-milling ethanol plant also produces about 17.5 pounds of animal feed. Animal feed is usually in the form of distillers dried grains with solute (DDGS), and represents the part of corn that is depleted of starch plus the biomass of yeast used for fermentation. By unit weight, DDGS is more nutritious to animals than unprocessed corn because it is richer in protein and fat. In addition to DDGS, dry-milling ethanol plants can also produce other protein-rich corn co-products for animal feed applications.

[0004] Lysine is an essential amino acid for most animals and must be supplemented if it is not supplied in sufficient quantities in DDGS to meet feed conversion expectations. Synthetic lysine is expensive and can represent a significant cost in animal feed. There is a need for methods to improve or maintain the production of alcohol from starch-containing feedstocks while increasing the nutritional value of animal feed co-products. Summary of the invention

[0005] Described are compositions and methods involving yeast cells having genetic mutations that increase lysine production in the yeast, resulting in ethanol fermentation products and co-products having increased nutritional value. Aspects and embodiments of the compositions and methods are described in the following independently numbered paragraphs.

[0006] 1. In one aspect, a non-naturally occurring variant homocitrate synthase polypeptide is provided, which has at least 80% amino acid sequence identity to SEQ ID NO: 1 and comprises one or more mutations relative to SEQ ID NO: 1 selected from the group consisting of: F36Y, N38K, N289D R349K, Q352E, V375D, R376T and I380M, wherein the variant homocitrate synthase polypeptide exhibits reduced lysine inhibition compared to an otherwise identical homocitrate synthase polypeptide lacking the one or more mutations.

[0007] 2. In some embodiments of the non-naturally occurring variant homocitrate synthase polypeptide as described in paragraph 1, the one or more mutations are F36Y and N38K.

[0008] 3. In a related aspect, a non-naturally occurring variant homocitrate synthase polypeptide is provided, which has at least 80% amino acid sequence identity to SEQ ID NO:2 and comprises one or more mutations relative to SEQ ID NO:2 selected from the group consisting of: N52D, D125N, R289I, N303D and N393D, wherein the variant homocitrate synthase polypeptide exhibits reduced lysine inhibition compared to an otherwise identical homocitrate synthase polypeptide lacking the one or more mutations.

[0009] 4. In another aspect, a yeast cell is provided, which produces the variant homocitrate synthase polypeptide as described in any one of paragraphs 1-3.

[0010] 5. In some embodiments of the yeast cell of paragraph 4, the cell belongs to a species of the genus Saccharomyces.

[0011] 6. In some embodiments of the yeast cell of paragraph 4 or 5, the cell further comprises one or more genes of a phosphoketolase pathway.

[0012] 7. In some embodiments of the yeast cell of paragraph 6, the gene of the phosphoketolase pathway is selected from the group consisting of: phosphoketolase, phosphotransacetylase, and acetylated acetyl dehydrogenase.

[0013] 8. In some embodiments of the yeast cell of any of paragraphs 4-7, the cell further comprises an exogenous gene encoding a carbohydrate processing enzyme.

[0014] 9. In some embodiments, the yeast cell of any of paragraphs 4-8 further comprises an alteration in the glycerol pathway and / or the acetyl-CoA pathway.

[0015] 10. In some embodiments, the yeast cell of any of paragraphs 4-9 further comprises an alternative pathway for producing ethanol.

[0016] 11. In another aspect, a method for increasing the amount of lysine present in a post-fermentation product from an ethanol production facility is provided, the method comprising: (i) hydrolyzing a starch-containing feedstock with an α-amylase to produce a starch liquefact; (ii) saccharifying the starch liquefact with a glucoamylase to produce glucose; (iii) fermenting the glucose with a modified yeast cell derived from a parent yeast cell, the modified yeast cell comprising a genetic alteration that reduces feedback inhibition of a lysine production pathway as mediated by a homocitrate synthase polypeptide in the lysine production pathway; and (iv) recovering a post-fermentation byproduct that is enriched in lysine compared to a post-fermentation byproduct recovered from an otherwise identical process using the parent yeast.

[0017] 12. In some embodiments of the method of paragraph 11, the post-fermentation product is selected from the group consisting of fermentation broth, whole stillage, thin stillage, dried distiller's grains, dried distiller's grains with solubles, concentrated distiller's grains solubles, or other protein-containing co-products.

[0018] 13. In some embodiments of the method of paragraph 11 or 12, one or more steps (i)-(iv) are combined in a simultaneous or overlapping manner.

[0019] 14. In another aspect, a method for increasing the amount of lysine present in a fermentation product is provided, the method comprising: (i) fermenting glucose or another sugar with a modified yeast cell derived from a parent yeast cell, the modified yeast cell comprising a genetic alteration that reduces feedback inhibition of a lysine production pathway as mediated by a homocitrate synthase polypeptide in the lysine production pathway; and (ii) recovering a fermentation product enriched in lysine compared to a fermentation product recovered from an otherwise identical process using the parent yeast.

[0020] 15. In some embodiments of the method of any of paragraphs 11-14, fermenting the glucose with the modified yeast cell is performed in the further presence of the parent yeast cell or a conventional yeast cell.

[0021] 16. In some embodiments of the method of paragraph 15, fermenting the glucose with the modified yeast cell in the further presence of the parent yeast cell or conventional yeast cell is performed by adding the modified yeast cell and the parent or conventional yeast cell to the fermentor at different times.

[0022] 17. In some embodiments of the method of any of paragraphs 11-16, the homocitrate synthase polypeptide is LYS20 and / or LYS21.

[0023] 18. In some embodiments of the method of paragraph 17, the modified yeast produces an altered amount of LYS20 and / or LYS21 polypeptide compared to an otherwise identical yeast.

[0024] 19. In some embodiments of the method of paragraph 17, the modified yeast produces a variant LYS20 and / or LYS21 polypeptide compared to an otherwise identical yeast.

[0025] 20. In some embodiments of the method of any of paragraphs 11-19, the modified yeast is the yeast of any of paragraphs 1-10.

[0026] 21. In another aspect, a post-fermentation product is provided, which is produced by the method of any one of paragraphs 11-20.

[0027] 22. In another aspect, there is provided a composition or method having the features as described in paragraphs 1 to 22 or any of the features mentioned in the specification.

[0028] These and other aspects and embodiments of the modified cells and methods of the invention will be apparent from the description, including any drawings. DETAILED DESCRIPTION

[0029] I. Overview

[0030] Described are methods involving yeast having genetic mutations that reduce the amount of feedback inhibition in the lysine synthesis pathway via LYS20 and LYS21 homocitrate synthase polypeptides. The yeast can be used in conventional bioethanol production facilities to produce alcohol as well as increased amounts of lysine, thereby improving the quality and commercial value of fermentation products and co-products such as animal feed ingredients.

[0031] II. Definitions

[0032] Before describing the strains and methods of the present invention in detail, the following terms are defined for clarity. Undefined terms shall be consistent with the ordinary meanings of these terms used in the relevant art.

[0033] As used herein, "alcohol" refers to an organic compound in which a hydroxyl functional group (-OH) is bonded to a saturated carbon atom.

[0034] As used herein, the phrase "degree of polymerization" (DP) refers to the number (n) of anhydropyranose glucopyranose units in a given sugar. Examples of DP1 are the monosaccharides glucose and fructose. Examples of DP2 are the disaccharides maltose and sucrose. The meanings of DP1, DP12, DP3, DP4, DP4+, etc. are well known in carbohydrate processing science.

[0035] As used herein, "yeast cell," "yeast strain," or simply "yeast" refers to organisms from the phylum Ascomycota and Basidiomycota. An exemplary yeast is a budding yeast from the order Saccharomycetales. A specific example of a yeast is a species of the genus Saccharomycetales, including but not limited to S. cerevisiae. Yeasts include organisms used to produce fuel alcohols as well as organisms used to produce drinkable alcohols, including specialty and proprietary yeast strains used to make unique flavored beer, wine, and other fermented beverages.

[0036] As used herein, the phrase "variant yeast cell," "modified yeast cell," or similar phrases (see above) refers to yeast that includes the genetic modifications and features described herein. Variant / modified yeast does not include naturally occurring yeast.

[0037] As used herein, the phrase "substantially inactive" or similar phrases means that the specified activity is either undetectable in the mixture or is present in an amount that does not interfere with the intended purpose of the mixture.

[0038] As used herein, the terms "polypeptide" and "protein" (and / or their respective plural forms) are used interchangeably to refer to polymers of any length comprising amino acid residues linked by peptide bonds. Conventional one-letter or three-letter codes for amino acid residues are used herein, and all sequences are presented from the N-terminal to the C-terminal direction. The polymer can be linear or branched, it can contain modified amino acids, and it can be interrupted by non-amino acids. The term also encompasses amino acid polymers modified naturally or by intervention (e.g., by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component). Also included within the definition are, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.) and other modifications known in the art.

[0039] As used herein, proteins that are functionally and / or structurally similar are considered "related proteins". Such proteins may be derived from organisms of different genera and / or species, or even from organisms of different classes (e.g., bacteria and fungi). Related proteins also encompass homologs determined by primary sequence analysis, by secondary or tertiary structure analysis, or by immunological cross-reactivity.

[0040] As used herein, the term "homologous protein" refers to a protein with similar activity and / or structure to a reference protein. This is not intended to mean that homologues are necessarily evolutionarily related. Therefore, the term is intended to encompass one or more enzymes that are identical, similar, or corresponding (i.e., in terms of structure and function) obtained from different organisms. In certain embodiments, it is desirable to identify homologues with similar quaternary, tertiary, and / or primary structures to the reference protein. In certain embodiments, homologous proteins induce similar one or more immune responses as reference proteins. In certain embodiments, homologous proteins are engineered to produce enzymes with one or more desired activities.

[0041] The degree of homology between sequences can be determined using any suitable method known in the art (see, e.g., Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol. 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, Wisconsin); and Devereux et al. (1984) Nucleic Acids Res. 12:387-95).

[0042] For example, PILEUP is a useful program for determining the level of sequence homology. PILEUP uses progressive, pairwise comparisons to create a multiple sequence alignment from a group of related sequences. It can also draw a tree showing the clustering relationship used to create the comparison. PILEUP uses a simplification of the progressive comparison method of Feng and Doolittle (Feng and Doolittle (1987) J.Mol.Evol. [Molecular Evolution Magazine] 35:351-60). The method is similar to the method described by Higgins and Sharp ((1989) CABIOS [Computer Applications in Biology] 5:151-53). Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and a weighted end gap. Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al. ((1990) J. Mol. Biol. 215:403-10) and Karlin et al. ((1993) Proc. Natl. Acad. Sci. USA 90:5873-87). A particularly useful BLAST program is the WU-BLAST-2 program (see, e.g., Altschul et al. (1996) Meth. Enzymol. 266:460-80). The parameters "W", "T", and "X" determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89:10915), alignments (B) of 50, expectation (E) of 10, M'5, N'-4, and a comparison of both strands.

[0043] As used herein, in the context of at least two nucleic acids or polypeptides, the phrases "substantially similar" and "substantially identical" typically mean that a polynucleotide or polypeptide comprises a sequence having 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 higher identity compared to a reference (i.e., wild-type) sequence. The sequence identity percentages are 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:

[0044]

[0045]

[0046] 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 in conservative amino acid substitutions are immunologically cross-reactive. Thus, a polypeptide is substantially identical to a second polypeptide, e.g., where the two peptides differ only in conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions (e.g., within a range of moderate to high stringency).

[0047] As used herein, the term "gene" is synonymous with the term "allele" and refers to a nucleic acid that encodes and directs the expression of a protein or RNA. Vegetative forms of filamentous fungi are typically haploid, so a single copy of a given gene (i.e., a single allele) is sufficient to confer a given phenotype.

[0048] As used herein, the terms "wild type" and "native" are used interchangeably and refer to a gene, protein or strain as found in nature.

[0049] As used herein, the term "target protein" refers to a polypeptide that is desired to be expressed in a modified yeast. Such proteins can be enzymes, substrate binding proteins, surfactant proteins, structural proteins, selective markers, etc., and can be expressed at high levels. The target protein is encoded by a modified endogenous gene or heterologous gene (i.e., target gene) relative to the parent strain. The target protein can be expressed intracellularly or as a secreted protein.

[0050] As used herein, "deletion of a gene" refers to the removal of the gene from the genome of the host cell. When a gene includes a control element (e.g., an enhancer element) that is not immediately adjacent to the gene coding sequence, the deletion of the gene refers to the deletion of the coding sequence, and optionally adjacent enhancer elements (e.g., including but not limited to promoter and / or terminator sequences), but does not require the deletion of non-adjacent control elements.

[0051] As used herein, "disruption of a gene" refers generally to any genetic or chemical manipulation (i.e., mutation) that substantially prevents a cell from producing a functional gene product (e.g., protein) in a host cell. Exemplary methods of disruption include complete or partial deletion of any portion of a gene (including a polypeptide coding sequence, a promoter, an enhancer, or another regulatory element) or mutagenesis thereof, wherein mutagenesis encompasses substitutions, insertions, deletions, inversions, and combinations and variations thereof, any of which substantially prevents the production of a functional gene product. Genes may also be disrupted using RNAi, antisense, or any other method for eliminating gene expression. Genes may be disrupted by deletion or genetic manipulation of non-adjacent control elements.

[0052] As used herein, the terms "genetic manipulation" and "genetic alteration" are used interchangeably and refer to changes / variations in a nucleic acid sequence. The changes may include, but are not limited to, substitution, deletion, insertion or chemical modification of at least one nucleic acid in a nucleic acid sequence.

[0053] As used herein, "major genetic determinant" refers to a gene or genetic manipulation thereof that is necessary and sufficient to confer a particular phenotype in the absence of other genes or genetic manipulations thereof. However, the fact that a particular gene is necessary and sufficient to confer a particular phenotype does not exclude the possibility that additional effects on the phenotype may be achieved through further genetic manipulations.

[0054] As used herein, a "functional polypeptide / protein" is a protein that has activity (e.g., enzymatic activity, binding activity, surface active properties, etc.) and has not been mutagenized, truncated, or otherwise modified to eliminate or reduce this activity. As noted, a functional polypeptide can be thermostable or thermolabile.

[0055] As used herein, a "functional gene" is a gene that can be used by cell components to produce an active gene product (typically a protein). A functional gene is the opposite of a disrupted gene, which is modified so that they cannot be used by cell components to produce an active gene product, or have a reduced ability to be used by cell components to produce an active gene product.

[0056] As used herein, a yeast cell has been "modified to prevent the production of a specified protein" if it has been genetically or chemically altered to prevent the production of a functional protein / polypeptide exhibiting an activity characteristic of a wild-type protein. Such modifications include, but are not limited to, deletion or disruption of a gene encoding a protein (as described herein), genetic modifications that render the encoded polypeptide lacking the aforementioned activity, genetic modifications that affect post-translational processing or stability, and combinations thereof.

[0057] As used herein, "fermentation broth" is the product of an ethanol production facility after fermentation with yeast but before distillation.

[0058] As used herein, "whole stillage" is the byproduct of an ethanol production facility after distillation.

[0059] As used herein, "thin stillage" is the liquid portion of whole stillage after separation of solid materials.

[0060] As used herein, "distillers grains (DG)" is the solid / slurry component of whole stillage.

[0061] As used herein, "distillers dried grains (DDG)" is DG that has been dried.

[0062] As used herein, "distillers dried grains with solubles (DDGS)" is DG that has been dried along with concentrated thin stillage to increase nutritional value.

[0063] As used herein, a "wet" distillation by-product contains at least 20% water by weight.

[0064] As used herein, a "dry" distillation by-product contains less than 20% water by weight.

[0065] As used herein, "aerobic fermentation" refers to growth in the presence of oxygen.

[0066] As used herein, "anaerobic fermentation" refers to growth in the absence of oxygen.

[0067] As used herein, the singular articles "a / an" and "the" include plural referents unless the context clearly indicates otherwise. All references cited herein are hereby incorporated by reference in their entirety. Unless otherwise stated, the following abbreviations / acronyms have the following meanings:

[0068] ℃ Celsius

[0069] DG Distillers Lees

[0070] DDG Dried Distillers Grains

[0071] DDGS Distillers Dried Grains with Dissolved Matter

[0072] DNA deoxyribonucleic acid

[0073] DP degree of polymerization

[0074] DS Dry Solids

[0075] EtOH

[0076] g or gm gram

[0077] g / L grams / liter

[0078] GA Glucoamylase

[0079] GAU / g DS Glucoamylase unit / gram dry solids

[0080] HPLC High Performance Liquid Chromatography

[0081] hr or h hours

[0082] kDa kilodaltons

[0083] M Moore

[0084] mg milligram

[0085] mL or ml milliliters

[0086] ml / min ml / min

[0087] mM millimole

[0088] N equivalent concentration

[0089] na Not applicable

[0090] PCR polymerase chain reaction

[0091] ppm parts per million

[0092] SAPU / g DS Protease Unit / g Dry Solids

[0093] SSCU / g DS Fungal α-amylase unit / g dry solid

[0094] Δ is associated with deletion

[0095] μg microgram

[0096] μL and μl microliter

[0097] μM and μm micromolar

[0098] AEC aminoethylcysteine

[0099] III. Yeast Cells with Reduced Feedback Inhibition of the Lysine Biosynthetic Pathway

[0100] The lysine biosynthetic pathway in Saccharomyces cerevisiae and other yeasts is characterized by product feedback inhibition by the interaction of lysine with two nuclear homocitrate synthase isozymes, called LYS20 and LYS21. The isoforms respond to lysine with different sensitivities. Mutant yeasts with changes in the amino acid sequences of LYS20 and LYS21 have been described, which result in reduced sensitivity to lysine feedback (Feller et al. (1999) Eur. J. Biochem. [European Journal of Biochemistry] 261: 163-170).

[0101] The compositions and methods of the present invention are based on the discovery that yeast desensitized to lysine feedback inhibition represents an effective way to significantly improve the quality of animal feed co-products produced by commercial ethanol production facilities. The ability to substantially increase the amount of lysine in, for example, distillers dried grains with solubles (DDGS) means that less synthetic lysine is needed to supplement animal feed products, thereby saving ranchers and farmers a significant amount of cost.

[0102] Described are mutations in LYS20 and LYS21 that result in up to a greater than 350-fold increase in free lysine content in yeast and can be expected to produce up to a 100-fold increase in lysine in an animal feed co-product (e.g., fermentation broth, whole stillage, thin stillage, distillers dried grains, distillers dried grains with solubles, concentrated distillers dried grains solubles or other protein-containing co-products).

[0103] In some embodiments, desensitization of lysine feedback inhibition is achieved by overexpressing LYS20 and LYS21 so that the cell cannot produce sufficient lysine to inhibit all LYS20 and LYS21 in the nucleus. In a preferred embodiment, yeast is modified to produce variant LYS20 and LYS21 polypeptides. As described herein, variant polypeptides can be selected to adjust or "tune" lysine overproduction for a specific application by selecting specific variant LYS20 and LYS21 variants that allow only a few-fold overproduction of lysine or a few hundred-fold overproduction.

[0104] It is expected that overproduction of lysine will compete with alcohol production, so the ability to choose how much carbon to divert to lysine is important to commercial alcohol producers. Using the yeast of the present invention, producers can choose how much carbon to direct to lysine simply by adding different yeasts in batches to the fermentor. Producers can also choose to add the yeast of the present invention and more conventional yeast to the fermentor at different times or in different ratios to further fine-tune the production of alcohol and lysine.

[0105] In some embodiments of the compositions and methods of the invention, the yeast produces a variant LYS20 polypeptide that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, but less than 100% of the amino acid sequence of SEQ ID NO: 1 and comprises one or more mutations relative to SEQ ID NO: 1 selected from the group consisting of: F36Y, N38K, N289D R349K, Q352E, V375D, R376T, and I380M. In specific embodiments, the mutations are F36Y and N38K. In some embodiments of the compositions and methods of the invention, the yeast produces a variant LYS20 polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, but less than 100% amino acid identity to SEQ ID NO: 2 and comprising one or more mutations relative to SEQ ID NO: 2 selected from the group consisting of N52D, D125N, R289I, N303D, and N393D.

[0106] In some embodiments, the modified cells comprise additional genes or other modifications that increase lysine production.

[0107] IV. Combination of reduced lysine feedback inhibition and mutations favoring alcohol production

[0108] In some embodiments, in addition to having reduced feedback inhibition in the lysine biosynthetic pathway via LYS20 and / or LYS21, the modified yeast cells of the invention further include additional modifications that favor alcohol production.

[0109] In a specific embodiment, the modified yeast cell comprises an artificial or alternative ethanol production pathway resulting from the introduction of a heterologous phosphoketolase (PKL) gene, a heterologous phosphotransacetylase (PTA) gene, and a heterologous acetylated acetyl dehydrogenase (AADH) gene, as described in WO 2015148272 (Miasnikov et al.), which enzymes are introduced to channel carbon flux away from the glycerol pathway and towards the synthesis of acetyl-CoA, which is then converted to ethanol.

[0110] The modified cell may further include a mutation that results in a reduction in the native glycerol biosynthetic pathway, which is known to increase alcohol production. Methods for reducing the glycerol biosynthetic pathway in yeast are known and include, for example, reducing or eliminating endogenous NAD-dependent glycerol 3-phosphate dehydrogenase (GPD) or phosphoglycerol phosphatase (GPP) activity by disrupting one or more of the genes GPD1, GPD2, GPP1 and / or GPP2. See, for example, U.S. Pat. Nos. 9,175,270 (Elke et al.), 8,795,998 (Pronk et al.), and 8,956,851 (Argyros et al.).

[0111] The modified yeast may further be characterized by increased acetyl-CoA synthase (also known as acetyl-CoA ligase) activity (EC 6.2.1.1) to scavenge (i.e., capture) acetate produced by chemical or enzymatic hydrolysis of acetyl-phosphate (or present in the culture medium of the yeast for any other reason) and convert it to Ac-CoA. This avoids the adverse effects of acetate on yeast cell growth and may further contribute to an increase in alcohol production. Increasing acetyl-CoA synthase activity may be achieved by introducing a heterologous acetyl-CoA synthase gene into the cell, increasing the expression of an endogenous acetyl-CoA synthase gene, and the like. A particularly useful acetyl-CoA synthase for introduction into a cell may be obtained from Methanosaetaconcilii (UniProt / TrEMBL accession number: WP_013718460). Homologs of these enzymes, including enzymes having at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, and even at least 99% amino acid sequence identity to the above-mentioned acetyl-CoA synthase from M. consiliense, can also be used in the compositions and methods of the present invention.

[0112] In some embodiments, the modified cell may further include a gene encoding NAD + A heterologous gene for a protein that is dependent on acetylation of acetaldehyde dehydrogenase activity and / or a heterologous gene encoding a pyruvate formate lyase. The introduction of such a gene in combination with attenuation of the glycerol pathway is described, for example, in U.S. Pat. No. 8,795,998 (Pronk et al.).

[0113] In some embodiments, the modified yeast cells of the invention may further overexpress a sugar transporter-like (STL1) polypeptide (see, e.g., Ferreira et al. (2005) Mol Biol Cell 16:2068-76; et al. (2015) Mol Microbiol 97:541-59 and WO 2015023989 A1) to increase ethanol production and reduce acetate.

[0114] In some embodiments, the modified yeast cells of the invention may further overexpress a poly(A) binding protein, such as PAB1, thereby increasing alcohol production and decreasing acetate production.

[0115] In some embodiments, the modified yeast cells of the present invention further comprise a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway is an isobutanol biosynthetic pathway. In some embodiments, the isobutanol biosynthetic pathway comprises a polynucleotide encoding a polypeptide that catalyzes the conversion of a substrate to a product selected from the group consisting of: (a) pyruvate to acetolactate; (b) acetolactate to 2,3-dihydroxyisovalerate; (c) 2,3-dihydroxyisovalerate to 2-ketoisovalerate; (d) 2-ketoisovalerate to isobutyraldehyde; and (e) isobutyraldehyde to isobutanol. In some embodiments, the isobutanol biosynthetic pathway comprises a polynucleotide encoding a polypeptide having acetolactate synthase, ketoacid reductoisomerase, dihydroxyacid dehydratase, ketoisovalerate decarboxylase, and alcohol dehydrogenase activity.

[0116] In some embodiments, the modified yeast cell comprising a butanol biosynthetic pathway further comprises a modification in a polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In some embodiments, the yeast cell comprises a deletion, mutation, and / or substitution in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In some embodiments, the polypeptide having pyruvate decarboxylase activity is selected from the group consisting of: PDC1, PDC5, PDC6, and combinations thereof. In some embodiments, the yeast cell further comprises a deletion, mutation, and / or substitution in one or more endogenous polynucleotides encoding FRA2, ALD6, ADH1, GPD2, BDH1, and YMR226C.

[0117] V. Combination of reduced lysine feedback inhibition with other beneficial mutations

[0118] In some embodiments, in addition to having feedback inhibition in the lysine biosynthetic pathway reduced via LYS20 and / or LYS21, optionally in combination with a genetic modification that is beneficial to alcohol production, the modified yeast cell of the present invention further comprises any number of additional target genes encoding target proteins. Additional target genes may be introduced before, during, or after genetic manipulations that result in reduced feedback inhibition of lysine or increased alcohol production. Target proteins include selective markers, carbohydrate processing enzymes, and other commercially relevant polypeptides, including but not limited to enzymes selected from the group consisting of: dehydrogenases, transketolases, phosphoketolase, transaldolase, epimerases, phytases, xylanases, β-glucanases, phosphatases, proteases, α-amylases, β-amylases, glucoamylases, pullulanases, isoamylases, cellulases, trehalases, lipases, pectinases, polyesterases, cutinases, oxidases, transferases, reductases, hemicellulases, mannanases, esterases, isomerases, pectinases, lactases, peroxidases, and laccases. The protein of interest can be secreted, glycosylated, and modified in other ways.

[0119] VI. Yeast Cells Suitable for Modification

[0120] Yeast is a unicellular eukaryotic microorganism classified as a member of the fungi kingdom, and includes organisms from the Ascomycota and Basidiomycota. Yeast that can be used for alcohol production includes but is not limited to Saccharomyces species, including Saccharomyces cerevisiae, and Kluyveromyces, Lachancea, and Schizosaccharomyces species. Many yeast strains are commercially available, many of which have been selected or genetically engineered to obtain desired characteristics, such as high ethanol production, fast growth rate, etc. Many yeasts have been genetically engineered to produce heterologous enzymes or even include heterologous pathways. It is believed that any yeast with a homologue of LYS20 and / or LYS21 in the lysine biosynthetic pathway is a candidate for the modification.

[0121] VII. Substrates and conditions

[0122] The production of alcohol from many carbohydrate substrates (including but not limited to corn starch, sugar cane, cassava and molasses) is well known, as are the numerous variations and modifications of enzyme and chemical conditions and mechanical methods. It is believed that the compositions and methods of the present invention are fully compatible with such substrates and conditions.

[0123] There are many variations of ethanol production processes, including cold cooking or no cooking, processes involving liquefaction at or below gelatinization temperature, simultaneous saccharification and fermentation, fractionation, etc. It is contemplated that the above processes are compatible with the compositions and methods of the present invention.

[0124] VIII. Fermentation Products and Co-products

[0125] Typical alcohol fermentation products include organic compounds with hydroxyl functional groups (-OH) combined with carbon atoms. Exemplary alcohols include but are not limited to methanol, ethanol, n-propyl alcohol, isopropyl alcohol, n-butanol, isobutyl alcohol, n-pentanol, 2-pentanol, isopentanol and higher alcohols. The most commonly prepared fuel alcohols are ethanol and butanols.

[0126] A valuable byproduct (or co-product) of alcohol production, particularly dry mill ethanol production, is an animal feed product, typically in the form of distillers dried grains (DDG) or, more commonly, distillers dried grains with solubles (DDGS). These animal feed products are in many ways more nutritious than the original feedstock for ethanol production, as they are depleted of carbohydrates but enriched with amino acids from the feedstock and the fermenting organism (i.e., the ethanologen).

[0127] The specific amino acid composition of DDGS or other corn co-products is important to the quality of animal feed because some amino acids are far more important than others. Lysine is an essential amino acid for most farm animals and must be supplemented to maximize feed conversion if it is not adequately provided by DDG, DDGS or other fermentation co-products. Synthetic lysine is expensive and represents a significant cost in animal feed.

[0128] Since yeast represents an important component in post-fermentation products, the amino acid content of yeast significantly affects the amino acid content of fermentation broth, whole stillage, thin stillage, dried distiller's grains, dried distiller's grains with solubles, concentrated distiller's grains solubles or other protein-containing post-fermentation co-products. Substituting the yeast of the present invention for conventional yeast increases the amount of lysine in such post-fermentation products, thereby increasing their value as animal feed products.

[0129] Using the modified yeast of the present invention, an increase in lysine of 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 can be achieved.

[0130] These and other aspects and embodiments of the strains and methods of the invention will be clear to the skilled person in view of this specification.The following examples are intended to further illustrate but not limit the strains and methods.

[0131] Examples

[0132] Example 1: Increased lysine production by yeast expressing variant LYS20 polypeptides

[0133] Using standard molecular biology techniques, error-prone PCR was used to generate thousands of random mutations in the Saccharomyces cerevisiae gene YDL182w encoding the homocitrate synthase polypeptide LYS20. The resulting variant gene library was transformed into Gold yeast (Martrex, Inc., Chaska, Minnesota, USA, referred to herein as "FG"), a well-known strain of Saccharomyces cerevisiae used for fuel ethanol production, in which the native gene encoding LYS20 is deleted, thereby obtaining LYS20 - FG). Transformants were allowed to compete as a pool in minimal growth medium supplemented to 100 mM with increasing concentrations of a lysine toxic analog (aminoethylcysteine; AEC). After selection, the culture was centrifuged to recover viable cells, genomic DNA was extracted, and PCR was performed to amplify the variant gene encoding the variant LYS20 polypeptide. After sequencing the target gene using the Illumina platform, the sequencing reads were mapped to the native sequence using standard mapping tools and mutations were analyzed for relative frequency (see, e.g., Fowler, DM and Fields, S. (2014) Nat. Methods [Natural Methods] 11: 801-07 and Starita, LM and Fields, S. (2015) Cold Spring Harb Protoc. [Cold Spring Harb Guide] 2015: 777-80).

[0134] The process is based on the nucleic acid sequence of YDL182w (LYS20, chr IV: 133437..134723), which is shown in SEQ ID NO: 4 below:

[0135]

[0136] YDL182w encodes homocitrate synthase polypeptide LYS20 (Genbank accession number NP_010099), as shown in SEQ ID NO: 2 below:

[0137]

[0138] For the initial screen, 40% of the mutation space was explored and the enrichment score (E-score) change was determined for each mutation in the dataset. The highest scoring mutations, i.e., the most overrepresented mutations in the yeast population able to grow in the presence of lysine toxic analogs, are summarized in Table 1. Mutations with lower E-scores are not shown.

[0139] Table 1. Average E-score changes for high LYS20 variants tolerant to toxic lysine analogs

[0140] mutation Average E N289 mutation to D, H, I, K, S or T 2.12 Q352 mutation to E, H, K, L or R 1.64 I380 mutated to M, N or T 1.56 V375 mutated to F, G or D 1.52 N38 mutation to D, H, I, K, T or Y 1.51 R376 mutated to T, S or I 1.026

[0141] Genes encoding eight single position variants and one combined variant were individually introduced into the new LYS20 - FG. The transformants were grown for 26 hours in a poor medium (Gasent-Ramírez, JM and Benítez, T. (1997) Applied and Environmental Microbiology 63:4800-4806) containing proline as a nitrogen source. The samples were analyzed for L-lysine content after derivatization with o-phthalaldehyde and measurement of derivatized lysine by HPLC (Agilent Technologies 1260), using an Eclipse Plus C18 column (4.6x150 mm, 3.5 microns), at 40°C in a gradient of phosphate buffer (pH 7.8) and acetonitrile:methanol:water (45:45:10). The calibration standards used for quantification included a known amount of lysine or an amino acid standard mixture (Agilent Technologies) including a known amount of L-lysine.

[0142] As shown in Table 2, yeast expressing the variant LYS20 polypeptides produced a wide range of free intracellular lysine increases ranging from 2.7 to 33 fold compared to the amount produced by the unmodified reference strain.

[0143] Table 2. Free intracellular lysine produced in individual variant LYS20 transformants

[0144]

[0145]

[0146] Four transformants expressing variant LYS20 polypeptides were selected for further analysis. Total protein produced by the strain after 48 hours of growth in poor medium was hydrolyzed by treatment with 6N HCl at 110°C for 24 hours (Zumwalt, RW et al. (1987) J. Assoc. Off. Anal. Chem. [Journal of the Association of Official Analytical Chemists] 70: 147-51). After derivatization with o-phthalaldehyde as described above, the total amino acid composition and in particular the lysine content in the samples were analyzed. Derivatized lysine was measured as above, and the results are summarized in Table 3. An increase in lysine relative to the wild-type FG strain is reported. Yeast expressing variant LYS20 polypeptides produced 1.5 to 2.7 times more protein lysine than the unmodified FG reference strain.

[0147] Table 3. Fold increase in protein lysine produced by LYS20 variant yeast

[0148] mutation Multiple increase FG na FG N38K 2.10 FG N289D 1.50 FG R349K 1.58 FG Q352E 2.69

[0149] Example 2: Increased lysine production by yeast expressing variant LYS21 polypeptides

[0150] For the gene encoding LYS21, the experiments described in Example 1 were partially repeated. Using standard molecular biology techniques, thousands of random mutations were made in the Saccharomyces cerevisiae gene YDL131w encoding the homocitrate synthase polypeptide LYS21. The resulting library was screened as described above.

[0151] The process is based on the nucleic acid sequence of YDL131w (LYS21, chr IV: 227393..228715),

[0152] It is shown in SEQ ID NO:3 below:

[0153]

[0154] YDL131w encodes homocitrate synthase polypeptide LYS21 (Genbank accession number AY692941), as shown in SEQ ID NO: 1 below:

[0155]

[0156]

[0157] Intracellular free lysine content (mM) was measured as described above after 24 h of growth in poor medium and analyzed by HPLC. The results are summarized in Table 4. Yeast expressing variants of LYS21 produced 60 to 350 times more free intracellular lysine compared to the unmodified FG reference strain.

[0158] Free intracellular lysine produced in variant LYS21 transformants

[0159] mutation Lysine Multiple increase FG 0.6 na N52D 65.8 114 D125N 202.7 353 R289I 35.1 61 N303D 65.9 114 N393D 99.1 172

Claims

1. A non-naturally occurring variant homocitrate synthase polypeptide having a mutation relative to SEQ ID NO: 1 of: N52D, D125N, N303D or N393D, wherein the variant homocitrate synthase polypeptide exhibits reduced lysine inhibition compared to an otherwise identical homocitrate synthase polypeptide lacking the mutation.

2. A yeast cell, which produces the variant homocitrate synthase polypeptide according to claim 1.

3. The yeast cell of claim 2, wherein the cell further comprises another non-naturally occurring variant homocitrate synthase polypeptide having mutations relative to SEQ ID NO: 2 of F36Y, N38K, N289D, R349K, Q352E, V375D, R376T and I380M, wherein the variant homocitrate synthase polypeptide exhibits reduced lysine inhibition compared to an otherwise identical homocitrate synthase polypeptide lacking the mutations.

4. The yeast cell of claim 2, wherein the cell further comprises another non-naturally occurring variant homocitrate synthase polypeptide, wherein the mutations of the another variant homocitrate synthase polypeptide relative to SEQ ID NO: 2 are F36Y and N38K, wherein the variant homocitrate synthase polypeptide exhibits reduced lysine inhibition compared to an otherwise identical homocitrate synthase polypeptide lacking the mutations.

5. The yeast cell of claim 2, wherein the cell belongs to a species of the genus Saccharomyces.

6. The yeast cell of claim 2, wherein the cell further comprises one or more genes of a phosphoketolase pathway.

7. The yeast cell of claim 6, wherein the genes of the phosphoketolase pathway are selected from the group consisting of phosphoketolase, phosphotransacetylase, and acetylated acetyl dehydrogenase.

8. The yeast cell of claim 2, wherein the cell further comprises an exogenous gene encoding a carbohydrate processing enzyme.

9. The yeast cell of claim 2, further comprising an alteration in the glycerol pathway and / or the acetyl-CoA pathway.

10. The yeast cell of claim 2, further comprising an alternative pathway for producing ethanol.

11. A method for increasing the amount of lysine present in a post-fermentation product from an ethanol production facility, the method comprising: (i) hydrolyzing a starch-containing raw material with α-amylase to produce a starch liquefact; (ii) saccharifying the starch liquefact with glucoamylase to produce glucose; (iii) fermenting the glucose with a modified yeast cell derived from a parent yeast cell, the modified yeast cell comprising a genetic alteration that reduces feedback inhibition of a lysine production pathway as mediated by a homocitrate synthase polypeptide in the lysine production pathway, wherein the modified yeast cell is a yeast cell according to any one of claims 2 to 10; and (iv) recovering a post-fermentation by-product enriched in lysine compared to a post-fermentation by-product recovered from an otherwise identical process using the parent yeast.

12. The method of claim 11, wherein the post-fermentation product is selected from the group consisting of fermentation broth, whole stillage, thin stillage, distillers dried grains, distillers dried grains with solubles, concentrated distillers dried grains solubles, and other protein-containing co-products.

13. The method of claim 11, wherein one or more steps (i)-(iv) are combined in a simultaneous or overlapping manner.

14. A method for increasing the amount of lysine present in a fermentation product, the method comprising: (i) fermenting glucose or another sugar with a modified yeast cell derived from a parent yeast cell, the modified yeast cell comprising a genetic alteration that reduces feedback inhibition of a lysine production pathway mediated by a homocitrate synthase polypeptide in the lysine production pathway, wherein the modified yeast cell is a yeast cell according to any one of claims 2 to 10; and (ii) recovering a fermentation product enriched in lysine compared to a fermentation product recovered from an otherwise identical process using said parent yeast.

15. The method of claim 11 or 14, wherein fermenting the glucose with the modified yeast cell is performed in the further presence of the parent yeast cell or a conventional yeast cell.

16. The method of claim 15, wherein: Fermentation of the glucose with the modified yeast cells in the further presence of the parent yeast cells or conventional yeast cells is performed by adding the modified yeast cells and parent or conventional yeast cells to the fermentor at different times.

17. The method of claim 11 or 14, wherein the homocitrate synthase polypeptide is LYS21 or LYS20 and LYS21.

18. The method of claim 17, wherein the modified yeast produces an altered amount of a LYS21 polypeptide or a LYS20 and LYS21 polypeptide compared to an otherwise identical yeast.

19. The method of claim 17, wherein the modified yeast produces a variant LYS21 polypeptide or both LYS20 and LYS21 polypeptides compared to an otherwise identical yeast.

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