EP4630533A1

EP4630533A1 - Increased ethanol production by yeast in high dissolved solids

Info

Publication number: EP4630533A1
Application number: EP23841389.2A
Authority: EP
Inventors: Zhongqiang Chen; Xiaochun FAN; Patricia MAUVAIS; Min QI; Yehong Jamie Wang
Original assignee: Danisco US Inc
Current assignee: Danisco US Inc
Priority date: 2022-12-05
Filing date: 2023-12-04
Publication date: 2025-10-15
Also published as: CN120712342A; MX2025006337A; WO2024123691A1

Abstract

Described are compositions and methods relating to modified yeast with disrupted genes that produces an increased amount of ethanol and, in some cases, a decreased amount of acetate compared to otherwise identical parental cells. Such yeast is particularly useful for large-scale ethanol production from starch substrates with high dissolved solids.

Description

INCREASED ETHANOL PRODUCTION BY YEAST IN HIGH DISSOLVED SOLIDS

CROSS-REFERENCE TO RELATED APPLICATION

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

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

[02] The present application is being filed with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled NB42003WOPCTSeqList.xml, created on December 1, 2023, which is 37,468 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.

TECHNICAL FIELD

[03] The present modified cells and methods relate to modified yeast with disrupted genes that produce an increased amount of ethanol, and, in some cases, a decreased amount of acetate compared to otherwise identical parental cells. Such yeast is particularly useful for large-scale ethanol production from starch substrates with high dissolved solids.

BACKGROUND

[04] First-generation yeast-based ethanol production converts sugars into fuel ethanol. The annual fuel ethanol production by yeast is about 90 billion liters worldwide (Gombert, A.K. and van Maris. A.J. (2015) Curr. Opin. Biotechnol. 33:81-86). It is estimated that about 70% of the cost of ethanol production is the feedstock. Since the production volume is so large, even small yield improvements have massive economic impact across the industry.

[05] In fuel ethanol industry, fermentation under high dry solid levels is a particularly challenging condition for fermenting organisms. High dry solids impart multiple physiological stresses on the fermenting organisms, including osmotic stress, high ethanol stress, high acid stress and the like. Temperature excursions during summer months and other reasons for high fermentation temperatures poses yet a further challenge for fermenting organisms.

[06] Ethanol production in engineered yeast cells with a heterologous phosphoketolase (PKL) pathway is higher than in a parental strain without a PKL pathway (see, e.g., Miasnikov et al. WO2015148272). The PKL pathway consists of phosphoketolase (PKL) and phosphotransacctylasc (PTA) to channel carbon flux away from the glycerol pathway and toward the synthesis of acetyl-coA. Two supporting enzymes, acetaldehyde dehydrogenase (AADH) and acetyl-coA synthase (ACS), can help the PKL pathway be more effective. Unfortunately, the engineered strains also produce more acetate than the parental yeast. Acetate is not a desirable by-product as it has negative effects on yeast growth and fermentation. In addition, acetate reduces the pH of left-over water from fermentation and distillation, referred to as backset, which is typically reused for liquefaction of a subsequent batch of substrate. As a result, ethanol producers must adjust the pH of the backset (or liquefact) or increase the amount of fresh water used for liquefaction.

[07] The need exists to increase ethanol and control acetate produced by fermenting organisms.

SUMMARY

[08] The present modified cells and methods relate to genetic mutations in fermenting organisms that result in increased ethanol production, and in some cases acetate reduction, under high dissolved solids and elevated temperature fermentation conditions. Aspects and embodiments of the compositions and methods are described in the following, independently- numbered paragraphs.

1. In one aspect, modified yeast cells derived from parental yeast cells are provided, the modified cells comprising a genetic alteration that causes the modified cells to produce during fermentation a decreased amount of polypeptides responsible for lack of robustness under high dry solids and elevated temperature fermentation conditions compared to otherwise identical parental cells, wherein the polypeptides are selected from SKG3, YNR068C, MNN14, PIN3, PEX18, MNN4, RIM20 and/or PAN2 polypeptides.

2. In some embodiments of the modified cells of paragraph 1, the modified cells further produce during fermentation a decreased amount of acetate compared to the amount of acetate produced by otherwise identical parental cells under the same fermentation conditions, and wherein the genetic alteration comprises the disruption of a nucleic acid capable of directing the expression of RIM20 and/or PAN2 polypeptides. 3. In some embodiments of the modified cells of paragraph 1 or 2, the cells further comprise one or more genes of the phosphokctolasc pathway.

4. In some embodiments of the modified cells of paragraph 3, the genes of the phosphoketolase pathway are selected from the group consisting of phosphoketolase, phosphotransacetylase and acetylating acetyl dehydrogenase.

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

6. In some embodiments, the modified cells of any of par agraphs 1-5 further comprise an alteration in the glycerol pathway and/or the acetyl-CoA pathway.

7. In some embodiments, the modified cells of any of paragraphs 1-6 further comprise an alternative pathway for making ethanol.

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

9. In another aspect, a method for increasing the robustness of yeast cells under high dry solids and elevated temperature fermentation conditions is provided, comprising introducing into parental yeast cells a genetic alteration that causes the resulting modified cells to produce during fermentation a decreased amount of polypeptides selected from SKG3, YNR068C, MNN14, PIN3, PEX18, MNN4, RIM20 and/or PAN2 polypeptides.

10. In some embodiments of the method of paragraph 9, the genetic alteration that further causes the modified cells to produce during fermentation a decreased amount of acetate compared to the amount of acetate produced by the parental cells under the same fermentation conditions, and wherein the genetic alteration comprises the disruption of a nucleic acid capable of directing the expression of RIM20 and/or PAN2 polypeptides.

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

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

13. In some embodiments of the method of paragraph 11 or paragraph 12, the modified yeast cells produce an increased amount of fermentation product compared to parental cells under the same conditions. 14. In some embodiments of the method of any one of paragraphs 1 1-13, the modified yeast cells produce a decreased amount of acetate compared to parental cells under the same conditions.

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

[09] These and other aspects and embodiments of present modified cells and methods will be apparent from the description, including any accompanying Drawings/Figures.

BRIEF DESCRIPTION OF THE DRAWING

[010] Figure 1 illustrates the elevated temperature ramp conditions used in fermentations with modified and parental yeast.

DETAILED DESCRIPTION

I. Definitions

[Oil] Prior to describing the present modified cells and methods in detail, the following terms are defined for clarity. Terms not defined should be accorded their ordinary meanings as used in the relevant art.

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

[013] As used herein, the terms “yeast cells,” “yeast strains,” or simply “yeast” refer to organisms from the phyla Ascomycota and Basidiomycota. Exemplary yeast is budding yeast from the order Saccharomycetales. Particular examples of yeast are Saccharomyces spp., including but not limited to 5. cerevisiae. Yeast includes organisms used for the production of fuel alcohol as well as organisms used for the production of potable alcohol, including specialty and proprietary yeast strains used to make distinctive-tasting beers, wines, and other fermented beverages.

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

[015] As used herein, the terms “polypeptide” and “protein” (and their respective plural forms) are used interchangeably to refer to polymers of any length comprising amino acid residues linked by peptide bonds. The conventional one-letter or three-letter codes for amino acid residues arc used herein and all sequence arc presented from an N-tcrminal to C-tcrminal direction. The polymer can comprise modified amino acids, and it can be interrupted by nonamino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art.

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

[017] As used herein, the term “homologous protein” refers to a protein that has similar activity and/or structure to a reference protein. It is not intended that homologs necessarily be evolutionarily related. Thus, it is intended that the term encompass the same, similar-, or corresponding enzyme(s) (z.e., in terms of structure and function) obtained from different organisms. In some embodiments, it is desirable to identify a homolog that has a quaternary, tertiary and/or primary structure similar to the reference protein. In some embodiments, homologous proteins induce similar immunological response(s) as a reference protein. In some embodiments, homologous proteins are engineered to produce enzymes with desired activity(ies).

[018] The degree of homology between sequences can be determined using any suitable method known in the art (see, e.g., Smith and Waterman (1981) Adv. Appl. Math. 2:482; Needleman and Wunsch (1970) J. Mol. Biol., 48:443; Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85:2444; programs such as GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI); and Devereux et al. (198' 4) Nucleic Acids Res. 12:387-95). [019] For example, PILEUP is a useful program to determine sequence homology levels. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pair-wise alignments. It can also plot 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. 35:351-60). The method is similar to that described by Higgins and Sharp ((1989) CABIOS 5:151-53). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. 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). One particularly useful BLAST program is the WU-BLAST-2 program (see, e.g., Altschul et al. (1996) Meth. Enzymol. 266:460-80). Parameters “W,” “T,” and “X” determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word-length (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.

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

Gap opening penalty: 10.0

Gap extension penalty: 0.05

Protein weight matrix: BLOSUM series

DNA weight matrix: IUB

Delay divergent sequences %: 40

Gap separation distance: 8 DNA transitions weight: 0.50

List hydrophilic residues: GPSNDQEKR

Use negative matrix: OFF

Toggle Residue specific penalties: ON

Toggle hydrophilic penalties: ON

Toggle end gap separation penalty OFF

[021] Another indication that two polypeptides are substantially identical is that the first polypeptide is immunologically cross-reactive with the second polypeptide. Typically, polypeptides that differ by conservative amino acid substitutions are immunologically cross- reactive. Thus, a polypeptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution. 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 medium to high stringency).

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

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

[024] As used herein, the term “expressing a polypeptide” and similar terms refers to the cellular process of producing a polypeptide using the translation machinery (e.g., ribosomes) of the cell.

[025] As used herein, “over-expressing a polypeptide,” “increasing the expression of a polypeptide,” and similar terms, refer to expressing a polypeptide at higher- than-normal levels compared to those observed with parental or wild-type cells that do not include a specified genetic modification. [026] 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) and other nucleic acid sequence needed to allow the encoded polypeptide to be produced in a cell. Expression cassettes can be exogenous (i.e., introduced into a cell) or endogenous (i.e., extant in a cell).

[027] As used herein, the terms “fused” and “fusion” with respect to two DNA fragments, such as a promoter and the coding region of a polypeptide refer to a physical linkage causing the two DNA fragments to become a single molecule.

[028] As used herein, the terms “wild-type” and “native” are used interchangeably and refer to genes, proteins or strains found in nature, or that are not intentionally modified for the advantage of the presently described yeast.

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

[030] As used herein, “disruption of a gene” 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 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 of the same, where mutagenesis encompasses substitutions, insertions, deletions, inversions, and combinations and variations, thereof, any of which mutations substantially prevent the production of a functional gene product. A gene can also be disrupted using CRISPR, RNAi, antisense, or any other method that abolishes gene expression. A gene can be disrupted by deletion or genetic manipulation of non- adjacent control elements. As used herein, “deletion of a gene,” refers to its removal from the genome of a host cell. Where a gene includes control elements (e.g., enhancer elements) that arc not located immediately adjacent to the coding sequence of a gene, deletion of a gene refers to the deletion of the coding sequence, and optionally adjacent enhancer elements, including but not limited to, for example, promoter and/or terminator sequences, but does not require the deletion of non-adjaccnt control elements. Deletion of a gene also refers to the deletion of a part of the coding sequence, or a part of a promoter immediately or not immediately adjacent to the coding sequence, where there is no functional activity of the interested gene existed in the engineered cell.

[031] As used herein, the terms “genetic manipulation,” “genetic alteration”, “genetic engineering”, and similar terms are used interchangeably and refer to the alteration/change of a nucleic acid sequence. The alteration can include but is not limited to a substitution, deletion, insertion or chemical modification of at least one nucleic acid in the nucleic acid sequence.

[032] As used herein, a “functional polypeptide/protein” is a protein that possesses an activity, such as an enzymatic activity, a binding activity, a surface-active property, or the like, and which has not been mutagenized, truncated, or otherwise modified to abolish or reduce that activity. Functional polypeptides can be thermostable or thermolabile, as specified.

[033] As used herein, “a functional gene” is a gene capable of being used by cellular components to produce an active gene product, typically a protein. Functional genes are the antithesis of disrupted genes, which are modified such that they cannot be used by cellular components to produce an active gene product, or have a reduced ability to be used by cellular components to produce an active gene product.

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

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

[036] As used herein, “aerobic fermentation” refers to growth and production process in the presence of oxygen.

[037] As used herein, “anaerobic fermentation” refers to growth and production in the absence of oxygen.

[038] As used herein, the expression “end of fermentation” refers to the stage of fermentation when the economic advantage of continuing fermentation to produce a small amount of additional alcohol is exceeded by the cost of continuing fermentation in terms of fixed and variable costs. In a more general sense, “end of fermentation” refers to the point where a fermentation will no longer produce a significant amount of additional alcohol, i.e., no more than about 1% additional alcohol.

[039] As used herein, the expression “carbon flux” refers to the rate of turnover of carbon molecules through a metabolic pathway. Carbon flux is regulated by enzymes involved in metabolic pathways, such as the pathway for glucose metabolism and the pathway for maltose metabolism.

[040] As used herein, the singular articles “a,” “an” and “the” encompass the plural referents unless the context clearly dictates otherwise. All references cited herein are hereby incorporated by reference in their entirety. The following abbreviations/acronyms have the following meanings unless otherwise specified:

[041] following meanings unless otherwise specified:

°C degrees Centigrade

AA a-amylase

AADH acetaldehyde dehydrogenases bp base pairs DNA deoxyribonucleic acid ds or DS dry solids

EC enzyme commission

EtOH ethanol g or gm gram g/L grams per liter

GA glucoamylase

H2O water

HPLC high performance liquid chromatography hr or h hour kg kilogram

M molar mg milligram min minute mL or ml milliliter mM millimolar

N normal nm nanometer

PCR polymerase chain reaction

PKL phosphoketolase ppm parts per million

PTA phosphotransacetylase

A relating to a deletion pg microgram pL and pl microliter pM micromolar

[042] Numerical values and ranges may be presented herein with the numerical value being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number can be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. For example, in connection with a numerical value, the term “about” refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in context. All values and ranges implicitly include the term “about” unless the context clearly dictates otherwise.

II. Modified cells with reduced expression of polypeptides responsible for high dry solid and high temperature intolerance

[043] Described are modified fermentation organisms with reduced levels of expression of genes responsible for lack of robustness under high dry solids and high temperature conditions. In some cases, the modified cells also accumulate a reduced amount of acetate. The genes and gene products are described briefly, below.

[044] SKG3 (YLR187W) is the paralog of CAF120. It encodes for a protein with unknown function. A Green fluorescent protein- SKG3 fusion protein localizes to the cell periphery, cytoplasm, bud and bud neck. It has now been determined that deletion of SKG3 has little effect on ethanol production under standard (moderate) dry solids (DS) condition (34% DS, 32°C), but provides a significant improvement under high DS condition (37.8% DS, 32°C) and high temperature condition (32% of DS, 36.6°C ramp).

[045] YNR068C is a putative protein expressed as a readthrough production of the BSC5 gene. It has now been determined that deletion of YNR068C gene has little effect on ethanol production under standard (moderate) dry solids (DS) condition (34% DS, 32°C), but provides a significant improvement under high DS condition (37.8% DS, 32°C) and high temperature condition (32% DS, 36.6°C ramp).

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

[047] PIN3 (YPR154W) is the paralog of LSB1, a negative regulator of actin nucleationpromoting factor activity. It interacts with Lasl7p, and along with LSB1, cooperatively inhibits the nucleation of actin filaments. Its expression levels increase in response to thermal stress. It has now been found that deletion of PIN3 gene has little effect on ethanol titer under standard conditions (34% DS, 32°C), but significantly improves performance of the strain under high dry solid condition (37.8% DS, 32°C) and high temperature condition (32% DS, 36.6°C ramp).

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

[049] MNN4 (YKL201C) is a putative positive regulator of mannosylphosphate transferase Mnn6p. It is involved in manno sylphosphorylation of N-linked oligosaccharides. Its expression increases in late-logarithmic and stationary growth phases. It has a paralog in MNN14. Is has now been shown that deletion of the MNN4 gene has little effect on ethanol production under standard condition (34% of DS, 32°C), but significantly improve production under high DS condition (37.8% DS, 32°C) and high temperature condition (32% DS, 36.6°C ramp).

[050] RIM20 (YOR275C) is a member of PalA/AIPl/Alix family. It is involved in the response to alkaline pH. It is involved in proteolytic activation of RimlOlp. It has now been found that deletion of RIM20 gene has a small effect on ethanol production under standard condition (34% DS, 32°C), but significantly improve production under high DS conditions (37.8% DS, 32°C) and high temperature condition (32% DS, 36.6°C ramp). It significantly reduces acetate accumulation under both standard and high DS conditions.

[051] PAN2 (YGL094C) is the catalytic subunit of the Pan2p-Pan3p poly(A)-ribonuclease complex. The complex acts to control poly(A)-tail length and regulate the stoichiometry and activity of postreplication repair complexes. We found deletion of PAN2 gene has little effect on ethanol production under standard condition (34% DS, 32°C), but significantly improves production under high dry solid condition (37.8% DS, 32°C) and high temperature condition (32% DS, 36.6°C ramp). It reduces acetate under all conditions tested.

[052] As described, above, it has now been determined that yeast cells with genetic modifications that reduce expression of these proteins results in high DS and high temperature tolerance and, in some cases, results in reduced acetate accumulation, compared to otherwise identical parental cells. [053] Disruption of a gene responsible for high dry solid and high temperature tolerance and, in some cases, acetate reduction, can result from disruption of a gene encoding a corresponding polypeptide present in the parental strain. Because disruption of one or more of these genes is a primary genetic determinant for conferring the beneficial phenotype to the modified cells, in some embodiments the modified cells need only comprise a disrupted gene responsible for high dry solid and high temperature tolerance and, in some cases, acetate reduction, while all other genes can remain intact. In other embodiments, the modified cells can optionally include additional genetic alterations compared to the parental cells from which they are derived. While such additional genetic alterations are not necessary to confer the described phenotype, they may confer other advantages to the modified cells.

[054] Disruption of a gene responsible for high dry solid and high temperature tolerance and, in some cases, acetate reduction, can be performed using any suitable methods that substantially reduce expression of a corresponding function polypeptide. Exemplary methods of disruption include but are not limited to: complete or partial deletion of a gene, including complete or partial deletion of a coding sequence, promoter, terminator, enhancer, or another regulatory element; and complete or partial deletion of a portion of the chromosome that includes any portion of such a gene.

[055] Particular methods for disrupting a gene responsible for high dry solid and high temperature tolerance and, in some cases, acetate reduction, include making nucleotide substitutions or insertions in any portion of such a gene-coding sequence, the promoter, the terminator, an enhancer, or another regulatory element. Preferably, deletions, insertions, and/or substitutions (collectively re I erred to as mutations) are made by genetic manipulation using sequence-specific molecular biology techniques, as opposed to by chemical mutagenesis, which is generally not targeted to specific nucleic acid sequences. Nonetheless, chemical mutagenesis can, in theory, be used to disrupt a gene.

[056] In some embodiments, the decrease in the amounts of functional polypeptides responsible for high dry solid and high temperature tolerance and, in some cases, acetate reduction, is a decrease of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%, or more, compared to the amounts of functional polypeptides produced by otherwise identical parental cells growing under the same conditions. [057] As described below, strains with SKG3, YNRO68C, MNN 14, PIN3, PEX18, MNN4, RIM20 and PAN2 disruptions showed significant improvement in ethanol production (e.g., 2.4- 6.2%) under high DS conditions and/or elevated temperature ramp conditions. In some embodiments, the increase in ethanol produced by the modified strains is an increase of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 6% or more, compared to the amount of acetate produced by parental cells growing under the same conditions.

[058] Also as described below, strains with RIM20 and PAN2 disruptions showed significant reduction in ethanol production (e.g., 3.4-15%) under high DS conditions and/or elevated temperature ramp conditions. In some embodiments, the decrease in acetate produced by the modified strains is a decrease of at least 20%, at least 15%, at least 10%, at least 7%, at least 5%, at least 4%, at least 3% or more compared to the amount of acetate produced by parental cells growing under the same conditions.

III. Modified yeast cells with genes of an exogenous PKL pathway

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

[060] Engineered yeast cells having a heterologous PKL pathway have been previously described (WO2015148272). These cells express heterologous phosphoketolase (PKL), phosphotransacetylase (PTA) and acetylating acetyl dehydrogenase (AADH), optionally with other enzymes, to channel carbon flux away from the glycerol pathway and toward the synthesis of acetyl-CoA, which is then converted to ethanol. Such modified cells are capable of increased ethanol production in a fermentation process when compared to otherwise-identical parent yeast cells.

IV. Modified yeast cells with other mutations that affect alcohol production

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

[062] The modified cells may further include mutations that result in attenuation of the native glycerol biosynthesis pathway and/or reuse glycerol pathway, which are known to increase alcohol production. Methods for attenuation of the glycerol biosynthesis pathway in yeast are known and include reduction or elimination of endogenous NAD-dcpcndcnt glycerol 3- phosphate dehydrogenase (GPD) or glycerol phosphate phosphatase activity (GPP), for example by disruption of one or more of the genes GPD1, GPD2, GPP1 and/or GPP2. See, e.g., 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 over expression of glycerol dehydrogenase (GCY1) and dihydroxy acetone kinase (DAK1) to convert glycerol to dihydroxy acetone phosphate (Zhang et al. (2013) J. Ind. Microbiol. Biotechnol. 40:1153-60).

[063] The modified yeast may further feature increased acetyl-CoA synthase (also referred to acetyl-CoA ligase) activity (EC 6.2.1.1) to scavenge (z.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 partially reduces the undesirable effect of acetate on the growth of yeast cells and may further contribute to an improvement in alcohol yield. Increasing acetyl-CoA synthase activity may be accomplished by introducing a heterologous acetyl-CoA synthase gene into cells, increasing the expression of an endogenous acetyl-CoA synthase gene and the like.

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

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

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

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

V. Modified yeast cells with other beneficial mutations

[068] In some embodiments, in addition to disruption of one or more of the genes that affect fermentation under high solids conditions, optionally with a heterologous PKL pathway, and still further optionally in combination with other genetic modifications that benefit alcohol production, the present modified yeast cells further include any number of additional genes of interest encoding proteins of interest. Additional genes of interest may be introduced before, during, or after genetic manipulations that result in the increased production of active MIG3 polypeptides. Proteins of interest, include selectable markers, carbohydrate -processing enzymes, and other commercially-relevant polypeptides, including but not limited to an enzyme selected from the group consisting of a dehydrogenase, a transketolase, a phosphoketolase, a transladolase, an epimerase, a phytase, a xylanase, a 0-glucanase, a phosphatase, a protease, an a-amylase, a 0- amylase, a glucoamylase, a pullulanase, an isoamylase, a cellulase, a trehalase, a lipase, a pectinase, a polyesterase, a cutinase, an oxidase, a transferase, a reductase, a hemicellulase, a mannanase, an esterase, an isomerase, a pectinases, a lactase, a peroxidase and a laccase. Proteins of interest may be secreted, glycosylated, and otherwise-modified.

VI. Use of the modified yeast for increased alcohol production

[069] The present compositions and methods include methods for increasing alcohol production and, in some cases, reducing glycerol production, in fermentation substrates. In some embodiments, the compositions and methods include methods for increasing alcohol production and, in some cases, reducing acetate production. In some embodiments, the compositions and methods include methods for increasing alcohol production and, in some cases, reducing acetate and glycerol production. Such methods are not limited to a particular fermentation process. The present engineered yeast is expected to be a “drop-in” replacement for convention yeast in any alcohol fermentation facility. While primarily intended for fuel alcohol production, the present yeast can also be used for the production of potable alcohol, including wine and beer.

[070] Processes for producing alcohols, e.g., ethanol, from carbohydrate substrates arc generally well known. The conversion of a carbohydrate substrate to a fermentation product such as alcohol typically involves multiple processing steps and reagents, each of which is important for maximizing conversion efficiency, throughput, consistency, concentration, and/or yield. By way of example, a common production process for producing ethanol by fermentation of a carbohydrate substrate (see, e.g., Section VIII below) typically involves processes including, but not limited to, milling or grinding feedstock, liquefaction, saccharification, fermentation, and distillation, and reagents such as enzymes and additional components, e.g., microorganisms and/or chemicals, may be added during a unit operation to facilitate the reaction. For example, during alcohol production, liquefaction includes thinning a mash via partial hydrolysis to lower its viscosity. Enzymes, e.g., alpha- amylases, may be used to facilitate the thinning process. During saccharification, complex carbohydrates of the thinned mash are converted to monosaccharides. Again, enzymes, e.g., glucoamylases, may be used to facilitate the conversion process. The resulting sugars are then converted by yeasts to ethanol during the fermentation process step. It is also possible to include enzymes, e.g., glucoamylases, fungal alpha amylase, and trehalase, to control the glucose profile during fermentation. Reagents, such as enzymes, may also be used in the steps of milling and distillation to facilitate the process. In some cases, the saccharification and fermentation steps may be combined into a single simultaneous saccharification and fermentation (SSF) step. Other well- known processes include raw starch hydrolysis (RSH), which includes milling the starch- containing material to produce a carbohydrate substrate and then performing SSF below the initial gelatinization temperature. Enzymes, e.g., acid fungal amylases, glucoamylases, may also be used in such processes to produce fermentation products.

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

40°C. In some embodiments, the conditions are specific to the fermenting step.

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

[073] In some embodiments, the modified yeast described herein produce a decreased amount of acetate under fermentation conditions including high DS% and/or high temperatures compared to the parental cells under the same fermentation conditions. In some embodiments, the modified yeast described herein produce a decreased amount of acetate under fermentation conditions including high DS% and high temperatures compared to the parental cells under the same fermentation conditions. In some embodiments, the modified yeast described herein produce a decreased amount of acetate under fermentation conditions including high DS% compared to the parental cells under the same fermentation conditions. In some embodiments, the modified yeast described herein produce a decreased amount of acetate under fermentation conditions including high temperatures compared to the parental cells under the same fermentation conditions. In some embodiments, the decrease in acetate is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or 50% less compared to the amount of acetate produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in acetate is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 25%, or 30% less compared to the amount to the amount of acetate produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in acetate is atleast about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% less compared to the amount to the amount of acetate produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in acetate is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% less compared to the amount of acetate produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in acetate is in a range of about 0.1% to 30% compared to the amount to of acetate produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in acetate is in a range of about 1% to 30% compared to the amount of acetate produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in acetate is in a range of about 5% to 30% compared to the amount of acetate produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in acetate is in a range of about 10% to 25% compared to the amount of acetate produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in acetate is in a range of about 5% to 10% compared to the amount of acetate produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in acetate is in a range of about 1% to 5% compared to the amount of acetate produced by parental cells under the same fermentation conditions. The terms “decreased,” “lowered,” “reduced,” “less”, “less than,” and the like are used interchangeably herein.

[074] In some embodiments, the modified yeast described herein produce a decreased amount of glycerol under fermentation conditions including high DS% and/or high temperatures compared to the parental cells under the same fermentation conditions. In some embodiments, the modified yeast described herein produce a decreased amount of glycerol under fermentation conditions including high DS% and high temperatures compared to the parental cells under the same fermentation conditions. In some embodiments, the modified yeast described herein produce a decreased amount of glycerol under fermentation conditions including high DS% compared to the parental cells under the same fermentation conditions. In some embodiments, the modified yeast described herein produce a decreased amount of glycerol under fermentation conditions including high temperatures compared to the parental cells under the same fermentation conditions. In some embodiments, the decrease in glycerol is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% less compared to the amount of glycerol produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in glycerol is at least about 0.1%, 0.5%, 1%, 2%, 3%, 4%, or 5% less compared to the amount of glycerol produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in glycerol is in a range of about 0.1% to 5% compared to the amount to of glycerol produced by parental cells under the same fermentation conditions. In some embodiments, the decrease in glycerol is in a range of about 1% to 5% compared to the amount of glycerol produced by parental cells under the same fermentation conditions.

VII. Yeast cells suitable for modification

[075] Yeasts are unicellular eukaryotic microorganisms classified as members of the fungus kingdom and include organisms from the phyla Ascomycota and Basidiomycota. Yeast that can be used for alcohol production include, but are not limited to, Saccharomyces spp., including .S'. cerevisiae. as well as Kluyveromyces, Lachancea and Schizosaccharomyces spp. Numerous yeast strains are commercially available, many of which have been selected or genetically engineered for desired characteristics, such as high alcohol production, rapid growth rate, and the like. Some yeasts have been genetically engineered to produce heterologous enzymes, such as glucoamylase or a-amylase.

VIII. Substrates and products

[076] Alcohol production from a number of carbohydrate substrates, including but not limited to corn starch, sugar cane, cassava, and molasses, is well known, as are innumerable variations and improvements to enzymatic and chemical conditions and mechanical processes. The present compositions and methods are believed to be fully compatible with such substrates and conditions.

[077] Alcohol fermentation products include organic compounds having a hydroxyl functional group (-OH) is bound to a carbon atom. Exemplary alcohols include but are not limited to methanol, ethanol, u-propanol, isopropanol, n-butanol, isobutanol, n-pentanol, 2-pentanol, isopcntanol, and higher alcohols. The most commonly made fuel alcohols arc ethanol, and butanol.

[078] These and other aspects and embodiments of the present yeast strains and methods will be apparent to the skilled person in view of the present description. The following examples are intended to further illustrate, but not limit, the compositions and methods.

EXAMPLES

Example 1

Genetic manipulation and strain construction

[079] Methods for disrupting genes and reducing the levels of functional polypeptides in yeast have been described, e.g., in WO2018089333 (US2019276905), WO2018226573

(US2020131591), WO2019083879 (US2021179674), WO2019173225 (US2021040474), WO2020263732 (US2022251608) and US2020354756). The following amino acid and nucleic acid sequences are referenced.

[080] The amino acid sequence of the SKG3 polypeptide is shown, below, as SEQ ID NO: 1:

MKRIFSGVKSPKLSAPPKVFKNDESPSTP S SPKFDQGLRSLSASASRLFSNS I STPGSPTLDLP QEHS INGD I SPELVP IVTLLSAQAHRRYHYGIFLILHDLKTDGTPAARQWEECYGVLLGTQLAL WDAKELSDSKNNKNTSTMKKAASRP SFINFTDASVRSLDANDQVI I ASENEKTKKDLDNVLVVS TTLKNRYFLKFKNSKSFKTWNAAIRLSLFEFTALQEAYTGSFLSSRGVKLGD IKVVMADTKFTY EDWVSVRFGTGMPWKRCYAVI SPQSGKKKKNSKGS ICFYENNKKTKKSNIMTTVVDARALYAVY P S SP ILIDTST I IKLEGFVSFDKSEEPQETNLFIMPEKHQGVPGYDT I IRFLIPAMNAFYLYGR PKGLIANRTDPDSLLFALPTLPHI YYLQVDDVLSLTKDKNYIHWSAADWRNNIVQVLQKKLSKG YKGCGNKTVSVS SGMMKSPAI S SAELFEGYDSLPERQMESPQKSKMKSPTLASTDD INSASASV NSHATSVKQTELFVTDNS SKINDSVSAQS SVTTNFKDTFTTPMTSGMLNHENSERSFGSGLKLK I TDSNLENMEDVEAKSANEFSTTPEDKHIHLANAAELSALYDKYSTSPFGKSEANS SPKPQKLE VKDRSKNENRSPYERYVGTSAESKTFEIGNVRESKST INTSLSSPLRVEDSRRSKNEDLGSLKE FEELSQKI SNMGMANI S SEALSDTAENS SFVTDLNLNINNS S SVNLNEEQRVPDFGEENVFDPD

YMEQNQMLETESRYTTDEFDFSDNQDAAS SNYSNGQTNRTVTETLSASDRNDKIPHS SLFTNLN QLTSNGGNYQDREDFSGDQINKPQQSQPLHVKGPQTS SFGYRNSSANS SQPQAPYPVGRPLGKI RTGPLTVQPMQQGGNSSMYSFQSSQHRFHSSQQRQNQSLSFRNNTYGSGNNQNTFHPSPQLQQQ

PQNMRYLNNKLPINDRSPIPQTQHHVPDGRPSLHINTTNRTNPLTAQSGFSQFMPPNSTSTNPY

SS

[081] The amino acid sequence of the putative YNRO68C polypeptide is shown, below, as SEQ ID NO: 2:

MRNRQNQKNWTNLRGLLSVEEREKLENLRLELVCMQAANSIPHDPPEISSLETELICLTT

NTKDCKPVRFHSDLLLKKHKYNEIKKIFKEILENIEAYRDEFTKNQTKINLLLADDARAS LRNRSLDF SDLMP S S I IKDVQVLANMEANVVVMKNALKTKLVGEKS VP VAS SP I S S I IPR TSRNKKTSPSNYHHSVLSHRKSNEWNQVSSTEYKRTLLLNIKYNDDFKATIVPSFESCLC SRSYFLRVKLHFDKGVGSAEIDIPVQVKNSFI

[082] The amino acid sequence of the MNN14 polypeptide is shown, below, as SEQ ID NO: 3:

MMLSLRRFSMYVLRSLRLHFKKIIITLLTIQLLFITIFVLGGRSSIIDGNWKSFMALFFKPLAY TNRNNNHASFDLRSKDNVAKLYEKMNFDTSGKWIDTYTLKNNLLTVKMGPEKGQVLDSVDELRY YDNDPRLVWSVLLDHLLESDSNEYAFSWYDWANFDSTNKLIALRHTNISCQFVCEGAFDKNVLE

MVESEVQEPLFVTNRNKYDESLWYNRVRKVVDSNSVQQAIHDHCMNNDAYSNGTPFELPFIISE

ISERLRPEVYDLQAKNHLLYSNFTPLSLTVLDSDKDAYRINLKTTDSSKSNIVQTNLLQNYIKR HRNEMVNGDLIFNHTSMFEKFLHHGSTKKRKLDVEALDKTIYAGEYLELSPSDFQFNAKERI IE LETRLRSEGLPSHDTHYLRSLKTSVNTSPALQQKYFAEASDITDATADGHHRDRRFFSIGHNLL

NDPQEFEARLNSLIRNFQKFVKANGLISWLSHGTLYGYLYDGLKFPWDVDHDLQMPIKHLHYLS

QYFNQSLILEDPREGNGRFLLDVGSAITVGVHGNGENNIDARFIDIDSGIYIDITGLSVSSDAA

KQYMSKFVEEESSGESFSALIEDYKFDENDYFDEVDGREGLAKYTIHELMEWVNSHPDDFTDAE

KNLVTKTYKKELAISRSDYAEKDLSPKQRYLVNEKYNLYNCRNQHFSSLNIISPLRNTMFSGVS

AFVPNRPIATLNNEYKVPAKYGLLSFQGKVYLPEFRYWFSFADMKKFANLQLKEPKITRLESPL NDLKFSDISLLITNILKCGFHSVFASLFNSFDSTVYRLKELEIQYDPSLSEEEKSSLLKTLRRG MSKKIKSPEKDP I I YI YERKLWENVEKLLNASNI YNI ASQVEKEKGKEFVERSQQVYERNFDGF RLPDGGNSKTVNDLNSKGLNLFGDNKKTSNNIFGSDQKY

[083] The amino acid sequence of the PIN3 polypeptide is shown, below, as SEQ ID NO: 4:

MSASLINRSLTNIRTELDFLKGSNVISNDVYDQINKSLPAKWDPANAPRNASPASLEYVE

ALYQFDPQQDGDLGLKPGDKVQLLEKLSPEWYKGSCNGRTGIFPANYVKPAFSGSNGPSN LPPPPQYKAQELQQIPTQNSAASSYQQQPFPPPSTNYYQQPQQQPQQAPPPQQQQQQQQH

QSSHSHLKSFGSKLGNAAIFGAGASIGSDIVNNIF

[084] The amino acid sequence of the PEX18 polypeptide is shown, below, as SEQ ID NO: 5:

MNSNRCQTNEVNKFISSTEKGPFTGRDNTLSFNKIGSRLNSPPILKDKIELKFLQHSEDLNQSR SYVNIRPRTLEDQSYKFEAPNLNDNETSWAKDFRYNFPKNVEPPIENQIANLNINNGLRTSQTD FPLGFYSQKNFNIASFPVVDHQIFKTTGLEHPINSHIDSLINAEFSELEASSLEEDVHTEEENS GTSLEDEETAMKGLASDIIEFCDNNSANKDVKERLNSSKFMGLMGSISDGSIVLKKDNGTERNL QKHVGFCFQNSGNWAGLEFHDVEDRIA

[085] The amino acid sequence of the MNN4 polypeptide is shown, below, as SEQ ID NO: 6:

MLQRISSKLHRRFLSGLLRVKHYPLRRILLPLILLQIIIITFIWSNSPQRNGLGRDADYLLPNY NELDSDDDSWYSILTSSFKNDRKIQFAKTLYENLKFGTNPKWVNEYTLQNDLLSVKMGPRKGSK LESVDELKFYDFDPRLTWSVVLNHLQNNDADQPEKLPFSWYDWTTFHELNKLI S IDKTVLPCNF LFQSAFDKESLEAIETELGEPLFLYERPKYAQKLWYKAARNQDRIKDSKELKKHCSKLFTPDGH GSPKGLRFNTQFQIKELYDKVRPEVYQLQARNYILTTQSHPLSISIIESDNSTYQVPLQTEKSK NLVQSGLLQEYINDNINSTNKRKKNKQDVEFNHNRLFQEFVNNDQVNSLYKLEIEETDKFTFDK DLVYLSPSDFKFDASKKIEELEEQKKLYPDKFSAHNENYLNSLKNSVKTSPALQRKFFYEAGAV KQYKGMGFHRDKRFFNVDTLINDKQEYQARLNSMIRTFQKFTKANGI I SWLSHGTLYGYLYNGM AFPWDNDFDLQMP IKHLQLLSQYFNQSLILEDPRQGNGRYFLDVSDSLTVRINGNGKNNIDARF IDVDTGLYIDITGLASTSAPSRDYLNSYIEERLQEEHLDINNIPESNGETATLPDKVDDGLVNM ATLNITELRDYITSDENKNHKRVPTDTDLKDLLKKELEELPKSKTIENKLNPKQRYFLNEKLKL YNCRNNHFNSFEELSPLINTVFHGVPALIPHRHTYCLHNEYHVPDRYAFDAYKNTAYLPEFRFW FDYDGLKKCSNINSWYPNIPSINSWNPNLLKEISSTKFESKLFDSNKVSEYSFKNLSMDDVRLI YKNIPKAGFIEVFTNLYNSFNVTAYRQKELEIQYCQNLTFIEKKKLLHQLRINVAPKLSSPAKD PFLFGYEKAMWKDLSKSMNQTTLDQVTKIVHEEYVGKI IDLSESLKYRNFSLFNITFDETGTTL DDNTEDYTPANTVEVNPVDFKSNLNFSSNSFLDLNSYGLDLFAPTLSDVNRKGIQMFDKDPIIV YEDYAYAKLLEERKRREKKKKEEEEKKKKEEEEKKKKEEEEKKKKEEEEKKKKEEEEKKKKEEE EKKKQEEEEKKKKEEEEKKKQEEGEKMKNEDEENKKNEDEEKKKNEEEEKKKQEEKNKKNEDEE KKKQEEEEKKKNEEEEKKKQEEGHSN

[086] The amino acid sequence of the R1M20 polypeptide is shown, below, as SEQ ID NO: 7:

MSELLAIPLKRTLEVDFATELSKLIDTTSFQTASFFQSDILKVVDARNNAIAPDISIDGLSALK EYYVILLQLEKKFPNNQIEFTWFQTLSQKSRGTSQYSLQWEKLTI I YNIGCMYSLLALNSNNDA AESLKTSCLYFQNAAGCFKHVLDHQKNLETIPVVDDATLNALTSLMLAQAQECFWFKAVQDKHK DSLIAKLSQQIVDFYCEAINDAQRGKLIRSDWINHLKAKKAYFSAVTYYRIALSFNEKKQFGNV VKALQMGLQF INE S TLS S QAKFKT VVE S S LKEAQRDNEF I YLQE VP SELF S I KP ALMVKP S S S A TLLPSIKKDETLFKDLIPIEVMEYCTAYNERQDEYVEQRVTNPLASLNKLLKESLTTFQIPQGL TKVSEAELSHYQASLNNLLINNKNVQVQLDNIEQILNEEAFTDNQLRLKHGTLNWTLPESSTTN TAYYEKLKKLRGYLDEGSAIDKQTNELFQS IDKNLIGSEIRLPESNDPLTNKIKMI IQERNDYI DRTRRKSSEYRILPKIITSYKKNGTVDFEPIFIGHLKYFDEDLRYVNSTKEENIKLIEEVNLSK KNNPGRSGIEPKKMVRIDPRELYIEDLRYSFKLLDEVKENLSAGTAFYENLITSTSNLYNEVQE YDTARRAEKARLDKSLTFEDQ

[087] The amino acid sequence of the PAN2 polypeptide is shown, below, as SEQ ID NO: 8:

MNNWQHFFNNPVDLSEHLKKPYFRFDNRDKEITAISFDEKANLIWSGDSYGCISSYDPTFQLYT RYRGHIGGNSVKDILSHRDGILSISEDSLHFANRRGVTKLNLTSIDIAAFSELNTMCYSPHSLK NNIYCGGDNTNWGIASIDLNRGCLDSLLNYSSKVKLMCSNNKVLSIGRQTGTVDLLDPTSNRTI KSFNAHSASISAMDLRDNTLVTVGKSKRFYNLYADPFVNVYDLRTMRQLPPVSFSKGTTMGSGG ADFVQLHPLLPTVMIVASSSGSFDFIDLSNPTLRTQYVHPCQSIKKLCLSPNGDVLGILEADNH LDTWRRSSNNMGMFTNTPEMLAYPDYFNDITSDGPISVDDETYPLSSVGMPYYLDKLLSAWPPV VFKSEGTIPQLTGKSPLPSSGKLKSNLAVISSQNEKLSTQEFPLLRYDRTKYGMRNAIPDYVCL RDIRKQITSGLETSDIQTYTSINKYEVPPAYSRLPLTSGRFGTDNFDFTPFNNTEYSGLDPDVD NHYTNAI IQLYRFIPEMFNFVVGCLKDENFETTLLTDLGYLFDMMERSHGKICSSSNFQASLKS LTDKRQLENGEPQEHLEEYLESLCIRESIEDFNSSESIKRNMPQKFNRFLLSQLIKEEAQTVNH NITLNQCFGLETEIRTECSCDHYDTTVKLLPSLSISGINKTVIKQLNKKSNGQNILPYIEYAMK NVTQKNSICPTCGKTETITQECTVKNLPSVLSLELSLLDTEFSNIRSSKNWLTSEFYGSIIKNK AVLRSTASELKGTSHIFKYELNGYVAKITDNNNETRLVTYVKKYNPKENCFKWLMFNDYLVVEI TEEEALKMTYPWKTPEIIIYCDAEELRKPFFSVDTYSINYDILFRDYFANGIRDTARREYKLLT HDEAPKSGTLVAIDAEFVSLQSELCEIDHQGIRSIIRPKRTALARISIIRGEEGELYGVPFVDD YVVNTNHIEDYLTRYSGILPGDLDPEKSTKRLVRRNVVYRKVWLLMQLGCVFVGHGLNNDFKHI NINVPRNQIRDTAIYFLQGKRYLSLRYLAYVLLGMNIQEGNHDSIEDAHTALILYKKYLHLKEK AIFEKVLNSVYEEGRAHNFKVPETSKG

[088] The nucleic acid sequence of the SKG3-coding region of the YLR187W gene is shown, below, as SEQ ID NO: 9: ATGAAAAGGATTTTCTCTGGTGTGAAGTCCCCAAAGTTATCTGCTCCCCCAAAAGTTTTCAAAA

ATGATGAGAGCCCATCCACTCCGAGCTCTCCCAAATTCGACCAAGGGCTTCGAAGTTTATCAGC

ATCAGCTTCAAGACTCTTCAGTAACTCAATATCCACTCCTGGGAGCCCTACGTTAGATCTTCCG

CAAGAACACTCTATTAATGGTGATATCTCACCAGAACTAGTACCAATCGTTACTTTACTCTCTG

CACAGGCTCATAGGAGATACCATTATGGTATATTCTTGATATTACATGATTTGAAGACAGACGG

AACACCTGCCGCGCGTCAGTGGGAAGAATGCTATGGTGTGTTGCTAGGAACTCAGCTGGCTTTA

TGGGATGCAAAAGAATTATCAGATTCCAAGAATAATAAGAACACATCAACCATGAAAAAAGCCG

CATCGAGACCTTCTTTTATCAATTTTACAGATGCTTCTGTGAGGAGTTTGGACGCTAATGATCA

GGTAATCATTGCATCAGAGAACGAAAAGACAAAGAAAGATCTAGACAATGTTCTTGTTGTTTCA

ACAACTTTAAAAAACAGGTATTTCTTAAAATTCAAAAATTCAAAATCATTCAAAACTTGGAACG

CAGCAATTAGGCTTAGTTTGTTCGAGTTTACGGCGTTGCAAGAGGCCTACACGGGCTCATTTTT

GTCAAGCAGAGGTGTTAAACTTGGTGATATCAAAGTTGTTATGGCAGATACAAAATTCACTTAT

GAGGACTGGGTTAGCGTAAGATTCGGGACAGGTATGCCATGGAAACGTTGTTATGCTGTTATTT

CTCCACAGTCTGGCAAAAAAAAGAAAAATTCGAAAGGTTCAATATGTTTCTATGAAAATAACAA

AAAAACCAAAAAATCAAACATAATGACAACGGTAGTAGATGCACGGGCTTTATATGCCGTTTAT

CCTTCCTCGCCGATATTAATCGACACATCTACAATCATAAAATTGGAAGGTTTTGTCTCTTTTG

ACAAAAGTGAGGAACCTCAAGAAACCAACCTATTTATCATGCCTGAAAAGCATCAAGGTGTTCC

GGGATATGACACTATTATTCGCTTCTTAATCCCAGCTATGAACGCGTTCTACTTATATGGTAGG

CCAAAGGGTTTGATTGCCAACAGAACTGACCCAGACTCCCTGCTGTTTGCCTTACCCACTCTTC

CGCATATATATTATTTGCAGGTGGATGACGTTTTATCATTAACAAAAGACAAAAATTACATACA

TTGGAGTGCTGCTGATTGGAGGAATAACATTGTTCAAGTATTACAGAAAAAATTAAGTAAGGGC

TATAAAGGCTGTGGTAATAAAACTGTATCAGTTTCATCTGGGATGATGAAGTCGCCGGCAATTA

GTTCAGCTGAATTATTTGAAGGGTACGATTCTCTCCCTGAAAGACAAATGGAAAGTCCGCAAAA

ATCTAAGATGAAGTCTCCTACATTAGCATCGACCGATGATATTAATTCCGCTTCTGCTTCCGTA

AACTCACATGCTACCTCAGTAAAACAAACTGAATTATTTGTGACTGATAATTCTTCAAAAATCA

ATGACTCTGTGTCGGCGCAGTCCAGTGTCACTACTAACTTCAAAGATACTTTTACCACCCCAAT

GACATCAGGAATGCTCAACCATGAAAATTCAGAGAGAAGCTTTGGTTCAGGATTAAAACTCAAA

ATCACTGATTCAAATTTGGAAAATATGGAAGACGTTGAAGCGAAATCTGCTAACGAATTCTCTA

CGACACCAGAAGACAAACATATTCACCTCGCCAATGCAGCCGAATTATCCGCCCTTTACGACAA

ATATTCCACATCTCCGTTTGGTAAATCGGAGGCTAATTCAAGCCCTAAACCTCAAAAATTGGAA

GTTAAAGATCGGTCAAAAAATGAAAACAGAAGCCCCTATGAGAGATACGTAGGTACATCCGCTG

AAAGCAAGACATTTGAAATAGGTAACGTCAGAGAGTCGAAAAGTACGATAAATACTTCCCTTTC TTCGCCTTTGAGGGTAGAAGATAGTAGACGTTCAAAAAATGAGGACCTGGGATCTTTGAAAGAG

TTTGAAGAGTTATCTCAAAAGATTAGCAATATGGGAATGGCAAATATTTCTTCAGAAGCTTTAA

GTGACACAGCAGAAAACAGTTCTTTTGTTACGGACCTGAACTTGAATATCAATAACAGTTCATC

GGTCAATCTCAATGAAGAACAACGTGTGCCGGATTTTGGGGAGGAAAACGTATTTGATCCAGAT

TATATGGAACAAAACCAAATGTTGGAAACAGAAAGTAGGTACACTACGGATGAGTTTGACTTTT

CAGATAATCAGGATGCAGCATCCAGTAATTATAGCAATGGGCAAACCAACCGAACAGTAACTGA

AACCCTTTCTGCCAGTGACAGAAATGACAAGATCCCACATTCTTCTTTATTTACAAACTTAAAC

CAGCTTACTTCGAATGGAGGAAACTATCAGGATAGGGAAGATTTCTCTGGTGATCAAATAAACA

AACCTCAACAATCTCAACCACTTCATGTGAAAGGACCACAAACTTCGTCATTTGGTTACAGAAA

TTCTTCAGCCAATAGCTCGCAGCCTCAAGCACCATACCCGGTTGGTCGCCCGTTAGGTAAGATA

AGAACGGGTCCGCTGACCGTCCAGCCTATGCAACAAGGTGGAAACTCATCAATGTATTCTTTCC

AATCTTCGCAACATCGGTTTCATTCGTCGCAACAGCGCCAAAACCAATCACTTTCCTTCAGAAA

CAATACCTATGGAAGTGGTAACAATCAAAATACATTCCATCCATCGCCGCAACTGCAACAACAG

CCTCAAAATATGAGATATTTGAATAATAAGTTACCAATAAATGATAGATCTCCAATACCGCAAA

CACAGCACCATGTACCAGATGGCCGTCCCAGCCTCCATATAAATACCACTAACCGCACAAATCC

GCTTACTGCACAAAGTGGATTTTCTCAATTTATGCCTCCCAATAGCACATCAACCAACCCATAT TCCAGCTGA

[089] The nucleic acid sequence of the YNR068C-coding region is shown, below, as SEQ ID

NO: 10:

ATGAGAAACAGGCAGAATCAAAAGAACTGGACTAATTTAAGAGGTCTATTGTCCGTTGAA

GAGAGAGAAAAACTAGAAAATTTACGGCTTGAACTAGTATGTATGCAAGCTGCTAATAGT

ATTCCACATGACCCACCGGAAATCAGTAGTCTGGAGACGGAATTGATTTGCTTAACAACG

AAC AC AAAAG AT T GC AAACC AG T GAGGT T CC AC AGT GAT T T AC T T T T AAAGAAGC AC AAA

TACAATGAAATTAAGAAAATATTTAAGGAAATTTTGGAAAACATTGAAGCATATCGCGAT

GAGTTCACTAAAAATCAAACAAAAATAAACTTACTTCTTGCCGATGACGCAAGAGCTAGC

CTGCGTAATAGATCGCTCGATTTTTCTGATTTAATGCCTTCGAGTATAATTAAGGACGTA

CAGGTTTTAGCCAATATGGAAGCCAATGTTGTTGTCATGAAAAATGCGTTGAAAACGAAG

TTGGTTGGAGAGAAAAGTGTGCCTGTTGCTTCTTCTCCTATATCCAGCATAATTCCTCGA

ACTTCTAGGAATAAAAAAACCTCCCCATCAAACTATCACCATTCGGTACTTTCACATCGG

AAATCAAATGAATGGAATCAGGTCAGTTCAACTGAGTATAAGAGAACACTGTTATTGAAT ATAAAATATAATGATGATTTTAAAGCTACCATAGTGCCAAGTTTTGAAAGTTGTTTATGC

TCGAGGTCATATTTCCTCCGCGTAAAACTTCATTTTGATAAAGGTGTTGGATCTGCTGAA

ATTGATATCCCAGTTCAAGTTAAAAACTCTTTTATTTGA

[090] The nucleic acid sequence of the MNN14-coding region of the YJR061W gene is shown, below, as SEQ ID NO: 11:

ATGATGTTATCACTGCGCAGGTTCTCCATGTACGTTTTGAGATCTCTGCGGCTTCACTTTAAAA

AGATAATCATTACTCTTCTAACTATCCAGTTACTATTCATTACCATATTTGTATTGGGCGGTCG

CTCGTCGATTATTGACGGTAACTGGAAGTCATTCATGGCGCTCTTTTTCAAACCGCTTGCTTAC

ACTAACAGAAACAACAACCATGCTTCTTTCGATCTGAGATCAAAAGACAACGTAGCCAAACTTT

ACGAAAAAATGAATTTTGATACTTCAGGCAAATGGATCGACACGTACACCTTGAAGAATAATCT

TCTCACTGTGAAAATGGGTCCTGAAAAAGGGCAAGTTCTTGATTCGGTAGATGAATTGAGATAT

TACGATAACGACCCAAGGCTGGTATGGTCAGTTTTACTAGATCACTTATTAGAATCAGATTCCA

ATGAATACGCATTTTCGTGGTACGATTGGGCTAATTTTGACTCTACAAACAAACTCATTGCACT

GAGACACACGAACATATCTTGCCAGTTCGTTTGCGAGGGTGCCTTTGATAAAAATGTGCTAGAA

ATGGTAGAGAGTGAAGTCCAAGAGCCTTTATTCGTCACAAATAGGAATAAATATGACGAATCGC

TCTGGTACAACAGGGTAAGAAAGGTTGTCGATTCTAATTCTGTGCAGCAAGCCATACATGATCA

CTGCATGAATAATGACGCGTATTCCAATGGTACTCCCTTCGAATTGCCTTTTATCATAAGCGAA

ATTTCTGAAAGGTTGAGGCCAGAAGTGTATGACTTACAAGCCAAAAACCACTTGTTATATTCTA

ACTTTACTCCACTGTCATTAACCGTACTGGACAGCGATAAAGATGCATACAGAATCAATTTGAA

GACAACAGACTCTTCCAAATCAAATATAGTACAGACAAATCTACTACAGAATTACATTAAGAGG

CACAGAAATGAAATGGTAAATGGCGACCTCATTTTCAACCACACTTCCATGTTTGAAAAATTTT

TACATCATGGATCCACTAAAAAAAGGAAACTTGACGTTGAAGCGTTGGATAAAACAATATACGC

TGGAGAGTATCTAGAACTATCACCATCTGATTTCCAATTCAATGCAAAAGAGAGGATCATTGAA

TTAGAGACCAGGCTCAGGTCTGAAGGCCTACCATCTCATGATACCCACTATTTACGAAGTTTAA

AGACGTCCGTAAATACGTCCCCTGCATTACAGCAAAAGTATTTCGCAGAGGCCTCTGATATTAC

GGACGCGACTGCCGATGGTCATCATAGAGACAGGCGATTTTTCTCAATCGGACATAATCTCCTA

AATGACCCTCAGGAGTTTGAAGCAAGATTGAATTCTTTGATCAGAAATTTTCAGAAATTTGTTA

AGGCTAACGGATTAATTTCCTGGCTATCGCATGGTACATTGTATGGATATCTATATGATGGTCT

GAAGTTTCCCTGGGATGTCGACCATGATTTACAGATGCCCATTAAACATTTACATTACTTGAGT

CAATATTTCAACCAATCCCTAATATTAGAAGATCCAAGAGAAGGTAATGGAAGATTCTTACTAG

ATGTAGGAAGCGCAATTACGGTAGGAGTTCATGGGAACGGCGAAAACAATATTGATGCTCGTTT CATCGATATTGACTCAGGTATATACATTGACATCACGGGACTTAGCGTTAGTTCCGATGCGGCT

AAACAGTACATGTCCAAATTTGTAGAAGAAGAAAGCTCGGGCGAAAGCTTTTCTGCCCTTATTG

AAGACTATAAGTTTGACGAAAACGACTATTTTGACGAGGTGGATGGTAGAGAAGGTTTAGCTAA

ATATACCATACATGAATTAATGGAATGGGTTAATTCTCATCCAGACGACTTTACGGATGCAGAA

AAGAATTTAGTCACCAAAACATACAAGAAAGAGCTTGCAATTTCGAGAAGCGATTATGCTGAAA

AAGACTTGTCTCCGAAACAAAGGTATTTGGTAAATGAGAAGTATAACCTTTACAATTGTAGAAA

CCAGCATTTTTCCAGTCTAAACATCATATCACCCTTGAGAAATACAATGTTCAGCGGTGTGTCA

GCATTTGTTCCTAATAGGCCCATAGCAACATTGAATAATGAGTATAAAGTTCCGGCAAAATACG

GGCTTTTGTCATTCCAAGGTAAGGTGTATTTACCGGAATTCAGATACTGGTTCTCGTTTGCAGA

CATGAAGAAGTTTGCAAATTTGCAGCTGAAAGAACCCAAGATAACACGACTGGAAAGTCCCTTA

AATGATTTAAAATTCAGCGACATAAGCCTACTGATAACAAACATTTTAAAATGTGGGTTTCACT

CCGTATTTGCCAGCTTATTTAATTCTTTTGACAGTACTGTTTACAGACTCAAAGAGCTTGAAAT

ACAGTATGATCCTAGCTTGAGTGAGGAAGAAAAAAGTAGTCTATTAAAAACTCTACGGCGAGGA

ATGTCAAAAAAAATAAAATCACCAGAAAAAGATCCGATCATATATATATACGAAAGAAAGTTAT

GGGAAAACGTGGAAAAGTTGTTGAATGCGTCAAACATCTACAACATTGCTTCACAAGTTGAGAA

GGAAAAAGGTAAAGAGTTTGTTGAACGGTCCCAGCAAGTATATGAAAGAAACTTTGACGGCTTC

AGACTTCCCGATGGCGGCAACAGTAAGACTGTAAATGATCTGAATTCTAAGGGCTTAAATCTCT

TTGGTGATAATAAGAAAACTTCAAACAATATATTTGGTTCAGACCAAAAATATTAA

[091] The nucleic acid sequence of the PIN3 -coding region of the YPR154W gene is shown, below, as SEQ ID NO: 12:

ATGTCTGCTTCATTGATTAATCGTTCCTTAACAAACATTAGGACAGAACTGGATTTTCTAAAAG

GGTCAAATGTCATTTCAAATGACGTTTACGATCAAATAAATAAGAGCTTGCCGGCAAAATGGGA

TCCTGCCAATGCACCCCGCAACGCCAGTCCAGCTTCCTTGGAATATGTCGAAGCTCTTTATCAA

TTTGATCCTCAACAAGATGGTGATTTGGGCTTAAAACCAGGTGACAAGGTCCAACTTTTAGAAA

AATTATCTCCAGAGTGGTACAAGGGTAGCTGTAATGGCCGTACCGGTATTTTCCCAGCAAACTA

TGTCAAGCCAGCTTTCTCTGGGTCTAACGGTCCATCCAATCTTCCACCACCTCCACAGTATAAA

GCTCAAGAATTACAACAAATCCCCACGCAAAATAGTGCCGCATCATCTTATCAACAGCAGCCAT

TTCCTCCACCTTCCACAAATTATTATCAGCAGCCTCAACAACAGCCGCAACAAGCTCCTCCTCC

CCAACAACAACAACAACAACAACAACATCAGAGCTCACATAGCCACTTGAAGAGCTTTGGTAGC

AAATTGGGTAATGCCGCCATTTTTGGGGCAGGCGCTAGTATTGGGTCAGATATTGTTAATAATA

TCTTTTAA [092] The nucleic acid sequence of the PEX18-coding region of the YHR160C gene is shown, below, as SEQ ID NO: 13:

ATGAATAGTAACCGATGCCAAACGAATGAGGTGAATAAATTTATTAGTAGTACAGAAAAGGGGC

CTTTTACGGGCAGGGACAATACGCTCTCTTTTAACAAAATCGGGAGCAGACTGAATTCACCACC

GATTCTGAAGGATAAAATTGAGCTGAAATTTCTACAACACTCAGAAGATTTGAATCAATCACGG

TCCTACGTAAATATTCGTCCTAGAACCTTAGAGGATCAAAGTTACAAATTTGAAGCGCCAAATC

TAAATGACAATGAAACTTCTTGGGCCAAGGATTTTAGATATAACTTCCCTAAGAATGTTGAACC

GCCCATCGAAAATCAAATCGCGAATCTTAATATAAACAACGGGCTACGGACATCTCAGACAGAT

TTTCCCTTAGGCTTTTATTCACAGAAAAACTTTAACATTGCTTCCTTCCCTGTGGTTGACCATC

AGATATTCAAGACAACAGGTTTAGAACATCCTATCAACAGCCACATTGATTCTTTAATTAATGC

TGAATTTTCGGAACTGGAAGCCAGTAGTTTGGAAGAAGATGTCCATACAGAAGAGGAAAATTCA

GGTACGAGTCTGGAAGATGAAGAAACTGCCATGAAAGGTTTGGCTTCCGATATAATTGAGTTTT

GCGATAATAATAGTGCCAATAAAGATGTAAAAGAAAGACTAAACAGTTCAAAGTTTATGGGGCT

GATGGGCAGCATTAGTGATGGTTCTATAGTTTTAAAGAAGGATAACGGTACAGAAAGAAACCTT

CAAAAACACGTAGGTTTTTGTTTTCAGAATTCAGGAAACTGGGCTGGTCTTGAGTTCCATGATG

TTGAAGACAGAATTGCTTAA

[093] The nucleic acid sequence of the MNN4-coding region of the YKL201C gene is shown, below, as SEQ ID NO: 14:

ATGCTTCAGCGAATATCATCTAAACTTCACAGGCGGTTCTTATCTGGCCTGCTGCGTGTCAAGC

ACTACCCATTAAGGCGCATTCTCCTTCCACTGATTCTACTGCAGATCATCATTATAACGTTTAT

CTGGTCAAATTCACCGCAGCGTAACGGACTTGGGCGGGACGCTGATTACCTTCTACCAAATTAC

AACGAACTTGACAGTGATGATGATTCCTGGTATAGCATCCTGACTTCGTCTTTCAAAAACGATC

GCAAGATCCAGTTCGCTAAGACATTATACGAAAATTTAAAATTCGGCACCAACCCTAAATGGGT

CAATGAATATACTCTGCAAAATGACCTGCTCTCGGTCAAAATGGGCCCTCGAAAGGGCAGTAAG

CTCGAATCCGTGGATGAGTTGAAGTTTTACGACTTCGACCCTCGTCTCACGTGGTCCGTTGTGC

TGAACCATTTGCAAAATAATGACGCAGATCAGCCAGAAAAGTTACCCTTTTCATGGTACGACTG

GACAACCTTCCACGAGCTGAATAAGCTGATTTCCATAGATAAAACTGTTCTGCCCTGCAATTTT

CTTTTCCAGTCCGCTTTCGACAAAGAGTCTTTAGAGGCCATTGAGACAGAGCTCGGCGAACCTT

TGTTCCTATACGAAAGACCAAAGTACGCGCAGAAACTGTGGTACAAGGCCGCTAGAAACCAGGA

CAGAATCAAAGACTCAAAGGAACTAAAAAAGCATTGTTCCAAGCTATTCACTCCAGACGGGCAT

GGCTCTCCTAAGGGTTTAAGATTTAATACGCAATTTCAAATAAAGGAGCTGTATGATAAAGTTA GACCCGAAGTTTACCAATTGCAGGCAAGAAACTACATTTTGACTACACAGTCGCATCCACTATC

CATTTCCATCATCGAATCAGATAATTCCACGTATCAAGTCCCCTTGCAAACTGAAAAATCAAAA

AACTTGGTGCAATCCGGCCTGTTGCAGGAATATATTAATGATAACATTAATTCTACGAACAAGA

GAAAGAAAAATAAACAGGACGTAGAATTCAACCATAACAGGCTTTTCCAGGAATTCGTCAATAA

CGACCAAGTTAACTCCCTATACAAACTGGAAATTGAAGAAACTGATAAATTCACTTTTGATAAA

GATTTGGTTTATTTATCCCCTTCGGATTTCAAGTTCGATGCCTCCAAAAAAATTGAAGAGTTAG

AGGAACAGAAGAAACTCTATCCGGACAAATTTTCCGCTCATAATGAGAATTATCTGAACAGTTT

GAAGAATTCCGTAAAGACAAGCCCTGCATTGCAAAGAAAGTTCTTCTATGAGGCTGGTGCCGTG

AAGCAATATAAAGGTATGGGGTTCCATCGTGACAAGAGGTTCTTCAATGTTGATACATTAATCA

ATGATAAACAAGAATACCAGGCTAGATTGAACTCAATGATCAGAACATTCCAAAAGTTTACTAA

AGCCAACGGCATCATATCTTGGTTGTCTCACGGAACGCTGTACGGCTATCTTTACAATGGAATG

GCTTTCCCTTGGGATAACGATTTCGACTTGCAAATGCCCATTAAGCATTTACAATTGCTCAGTC

AATACTTCAACCAATCTCTTATATTGGAAGACCCAAGACAGGGTAATGGACGTTATTTCCTAGA

CGTCAGCGACTCCTTGACAGTAAGAATTAACGGTAACGGTAAAAACAATATCGATGCAAGATTC

ATTGACGTCGACACCGGCCTTTACATTGATATTACCGGTCTAGCTAGCACTTCTGCCCCTAGTA

GGGATTACTTGAATTCTTATATTGAAGAGCGGTTGCAAGAGGAACATTTGGATATCAATAATAT

CCCTGAATCGAACGGTGAGACCGCTACTTTGCCCGACAAAGTAGATGATGGGTTAGTCAATATG

GCTACACTAAACATCACTGAGCTACGTGATTACATTACCAGCGACGAAAATAAAAATCATAAAA

GAGTCCCCACTGATACTGATTTGAAAGATCTTTTGAAAAAGGAACTGGAAGAGTTACCAAAGTC

TAAGACCATTGAAAACAAGTTGAATCCTAAACAAAGATATTTTCTCAACGAAAAACTTAAACTT

TACAATTGTAGAAACAACCATTTTAACTCGTTCGAGGAACTATCTCCCTTAATCAATACTGTTT

TCCATGGTGTGCCAGCGTTGATTCCTCACAGACATACCTACTGCTTGCACAATGAATATCATGT

ACCTGATAGATATGCATTTGATGCTTACAAAAATACTGCTTATTTGCCCGAATTTAGATTTTGG

TTCGACTATGACGGGTTAAAGAAATGCAGTAATATTAATTCATGGTATCCAAACATCCCCAGTA

TTAATTCATGGAATCCGAACCTCTTGAAAGAAATATCGTCTACGAAATTTGAGTCGAAACTTTT

TGATTCCAACAAAGTCTCTGAATACTCTTTCAAAAACCTATCCATGGATGATGTTCGCTTAATT

TATAAAAATATTCCAAAAGCTGGCTTTATCGAGGTATTTACTAACTTGTACAATTCCTTCAATG

TCACTGCATATAGGCAAAAGGAATTGGAAATTCAATACTGCCAAAACCTGACATTTATTGAAAA

AAAGAAATTATTACATCAATTGCGCATTAATGTTGCTCCTAAGTTAAGCTCCCCTGCAAAGGAC

CCATTTCTTTTTGGTTATGAAAAAGCTATGTGGAAGGATTTATCAAAATCTATGAACCAGACTA

CATTAGATCAAGTTACCAAGATTGTTCATGAAGAATATGTCGGAAAAATTATTGATCTGTCCGA

AAGTTTGAAATACAGGAATTTTTCACTTTTCAACATTACTTTTGATGAAACTGGAACAACTCTA GATGATAACACAGAAGATTATACTCCTGCTAATACTGTTGAAGTAAATCCTGTGGATTTTAAAT

CAAATTTAAACTTTAGTAGCAACTCCTTTTTGGATTTAAATTCATATGGTTTAGACCTTTTTGC

GCCAACTTTATCCGACGTTAACAGAAAGGGTATTCAAATGTTTGATAAGGACCCTATTATTGTA

TACGAGGACTATGCTTATGCCAAGTTACTTGAAGAAAGAAAGCGGAGGGAGAAGAAGAAGAAGG

AGGAAGAGGAGAAGAAGAAGAAGGAAGAAGAGGAAAAGAAGAAGAAGGAAGAAGAAGAAAAGAA

AAAGAAGGAAGAGGAAGAGAAGAAAAAGAAGGAAGAAGAAGAGAAGAAAAAGAAGGAAGAAGAA

GAAAAGAAGAAGCAGGAGGAAGAGGAGAAAAAGAAGAAGGAAGAAGAAGAGAAGAAGAAGCAGG

AAGAAGGAGAAAAGATGAAGAATGAAGATGAAGAAAATAAGAAGAATGAAGATGAAGAAAAGAA

GAAGAACGAAGAAGAGGAAAAAAAGAAGCAGGAAGAGAAAAACAAGAAGAATGAAGATGAAGAA

AAGAAGAAGCAGGAAGAGGAAGAAAAGAAGAAGAACGAAGAAGAGGAAAAAAAGAAGCAGGAGG AGGGGCACAGCAATTAA

[094] The nucleic acid sequence of the RIM20 -coding region of the YOR275C gene is shown, below, as SEQ ID NO: 15:

ATGAGTGAACTGCTTGCCATTCCACTTAAGCGAACTTTAGAAGTGGACTTTGCCACGGAGTTGT

CAAAACTAATTGATACTACTTCCTTTCAAACGGCATCCTTTTTCCAATCAGACATACTCAAAGT

TGTTGATGCAAGAAACAATGCGATTGCACCCGATATCTCAATCGACGGTCTTTCTGCACTGAAG

GAATACTATGTAATATTGTTGCAATTAGAGAAAAAGTTTCCCAATAATCAAATAGAATTTACAT

GGTTTCAAACGCTATCACAAAAATCTCGTGGTACTAGTCAATACTCATTACAATGGGAAAAATT

AACCATTATTTATAATATTGGTTGCATGTATTCACTGTTGGCTCTAAATAGCAACAATGATGCA

GCCGAATCCCTCAAAACGTCATGTCTGTACTTCCAGAATGCCGCCGGATGCTTTAAACATGTTT

TAGACCACCAAAAAAATCTTGAGACAATCCCAGTGGTAGATGACGCCACATTGAACGCTTTAAC

CTCCCTGATGCTAGCACAAGCGCAAGAATGCTTCTGGTTTAAAGCCGTGCAAGATAAACACAAG

GACTCCTTAATCGCGAAGTTGTCACAACAGATCGTGGACTTCTACTGCGAGGCTATTAATGACG

CCCAAAGGGGGAAACTTATTAGAAGTGATTGGATAAACCACTTAAAGGCTAAAAAGGCTTACTT

TAGTGCGGTGACTTATTACAGAATAGCCTTATCTTTTAATGAGAAGAAGCAGTTCGGAAATGTT

GTGAAAGCTCTGCAAATGGGATTGCAATTTATAAATGAATCAACACTATCTTCGCAAGCTAAGT

TTAAAACTGTTGTCGAATCTTCTCTGAAAGAAGCCCAAAGAGACAATGAATTCATCTACTTACA

AGAAGTTCCTTCGGAATTACCATCGATCAAACCAGCTCTCATGGTTAAACCATCTTCTTCAGCA

ACATTGTTGCCTTCTATTAAAAAGGATGAGACTTTATTCAAAGACCTAATCCCTATAGAAGTGA

TGGAATATTGCACAGCATACAATGAAAGGCAAGATGAATACGTTGAACAACGTGTTACCAATCC

CCTTGCTTCCCTAAATAAACTATTGAAGGAGTCCTTGACTACTTTCCAAATACCACAAGGGCTT ACAAAGGTGAGTGAAGCAGAACTGAGTCATTACCAGGCATCACTGAATAATCTTCTCATTAATA

ATAAAAATGTGCAGGTACAACTCGATAACATTGAGCAAATTTTAAATGAAGAAGCATTTACTGA

CAATCAGCTCCGACTAAAACACGGAACCTTAAATTGGACTTTACCCGAATCTTCAACTACTAAC

ACAGCATATTATGAAAAACTAAAAAAGCTGAGAGGATACCTTGATGAAGGTAGTGCGATTGACA

AACAAACAAATGAACTATTTCAAAGCATCGATAAAAATTTGATTGGATCAGAGATCAGGTTACC

GGAATCTAATGATCCTCTAACGAATAAGATTAAGATGATCATACAGGAAAGAAATGATTATATT

GATAGGACGAGGCGGAAATCTTCAGAATATAGAATACTTCCAAAGATCATTACATCGTATAAAA

AAAATGGAACAGTCGACTTTGAACCCATTTTCATAGGCCATTTAAAATACTTTGACGAAGATTT

AAGATACGTTAATAGTACAAAGGAAGAAAATATCAAACTTATTGAGGAAGTAAACCTAAGTAAG

AAGAATAACCCCGGCAGAAGCGGGATTGAACCAAAGAAAATGGTAAGAATAGATCCCAGAGAGC

TATATATTGAAGATCTGAGATATTCCTTCAAACTGCTTGATGAGGTGAAAGAAAATTTAAGTGC

CGGCACAGCATTTTACGAAAATTTAATTACTTCTACCAGCAATCTGTACAATGAGGTACAGGAA

TATGATACAGCAAGAAGAGCAGAAAAAGCTAGGCTTGATAAGAGTTTGACATTTGAAGACCAAT GA

[095] The nucleic acid sequence of the PAN2 -coding region of the Y GL094C gene is shown, below, as SEQ ID NO: 16:

ATGAATAATTGGCAACATTTCTTCAACAATCCAGTTGATCTTTCGGAACATTTGAAGAAGCCAT

ACTTTCGCTTCGATAATAGGGATAAGGAAATTACAGCGATTAGCTTCGATGAGAAGGCAAACTT

AATTTGGAGTGGAGACAGCTATGGTTGCATTTCGTCATATGATCCAACTTTTCAACTTTATACA

AGATATAGGGGCCACATAGGTGGAAATTCCGTGAAGGATATTCTCAGTCATCGGGATGGTATTT

TATCTATTAGTGAAGATTCCTTACACTTTGCTAATAGAAGAGGTGTTACTAAATTGAACCTCAC

TAGCATTGATATTGCTGCATTTAGCGAATTGAACACTATGTGCTATTCTCCTCATTCACTGAAA

AACAATATCTACTGTGGTGGTGACAACACAAATTGGGGAATTGCGTCCATTGACTTGAACAGAG

GTTGCTTAGATTCCCTCTTGAATTACTCATCTAAAGTGAAGTTAATGTGCTCTAATAATAAAGT

TTTGTCTATCGGAAGACAAACAGGGACTGTGGATTTGCTAGATCCAACATCGAATCGTACTATC

AAATCATTTAATGCACACTCTGCATCCATATCCGCTATGGATTTACGGGATAACACCTTGGTTA

CAGTAGGGAAGTCCAAAAGATTTTATAACTTATACGCTGACCCATTTGTGAATGTTTACGACTT

GAGAACAATGCGTCAACTCCCTCCTGTTTCCTTTTCTAAAGGAACAACTATGGGATCTGGAGGC

GCAGATTTTGTTCAATTACATCCTTTGCTTCCTACTGTTATGATCGTCGCCTCAAGTTCTGGTT

CGTTTGATTTCATCGACCTTTCCAATCCAACTTTAAGAACACAATATGTTCATCCTTGCCAGTC

GATTAAAAAGTTATGTTTGTCCCCCAATGGTGACGTATTGGGTATACTAGAAGCTGATAATCAC CTAGATACATGGAGAAGATCATCAAACAACATGGGAATGTTTACCAATACCCCTGAAATGCTAG CATATCCTGATTATTTTAATGACATTACCTCTGACGGCCCAATATCTGTCGACGATGAAACATA TCCATTGAGTTCTGTGGGGATGCCGTACTATCTTGATAAACTTTTGTCTGCATGGCCCCCTGTA GTGTTTAAAAGTGAAGGTACCATACCGCAATTAACAGGTAAGTCACCCTTACCATCGAGCGGCA AATTAAAAAGTAACCTTGCTGTGATCTCGAGCCAAAATGAGAAGTTGAGCACACAAGAATTTCC TTTGTTAAGATATGATCGCACCAAATACGGTATGAGAAATGCTATACCAGATTACGTTTGTCTA AGAGATATAAGGAAACAGATAACAAGCGGTTTAGAAACCAGCGATATACAGACATATACCTCAA TCAACAAGTACGAAGTACCCCCTGCATACAGTAGACTTCCACTGACATCAGGTAGATTTGGTAC TGATAATTTTGATTTTACGCCCTTTAATAACACTGAGTATTCAGGATTGGATCCAGATGTTGAT AATCACTACACAAATGCTATCATACAATTGTATCGCTTTATTCCAGAAATGTTTAATTTCGTTG TTGGGTGTTTGAAAGACGAGAATTTTGAAACAACGTTGCTAACTGATCTAGGCTACCTCTTTGA CATGATGGAAAGATCACATGGAAAAATATGTAGTTCTTCCAATTTTCAGGCGTCATTGAAATCC T T AAC T GAT AAAAGAC AAT T AGAAAACGGT GAACC AC AAGAAC AT T T AGAAGAGT AT T T AGAAT CGCTGTGCATAAGGGAAAGTATCGAGGATTTTAATTCTTCTGAAAGTATTAAACGCAATATGCC TCAAAAATTTAACAGATTCCTGCTCTCGCAACTTATTAAAGAGGAAGCGCAGACAGTCAACCAT AATATCACCCTAAATCAATGCTTTGGTTTGGAAACGGAAATACGAACAGAGTGTAGCTGTGATC ACTACGACACTACCGTCAAACTTCTACCCTCCTTATCAATATCAGGAATCAACAAAACCGTAAT CAAACAATTGAACAAGAAAAGCAATGGACAGAATATTTTGCCTTATATTGAATATGCCATGAAA AATGTAACCCAAAAGAACAGTATTTGCCCAACCTGCGGCAAAACCGAAACTATCACCCAGGAGT GTACTGTCAAGAATTTACCTTCAGTGTTGTCATTAGAATTATCACTATTAGATACCGAATTTTC CAATATAAGGTCGTCGAAAAACTGGTTAACTAGTGAATTTTATGGAAGCATCATTAAAAACAAG GCAGTTCTAAGATCGACGGCGTCCGAATTGAAGGGCACAAGCCACATATTTAAATACGAATTGA ATGGTTACGTGGCTAAAATCACTGATAACAATAACGAGACGCGTCTAGTAACATATGTCAAAAA ATATAATCCAAAAGAGAATTGCTTCAAGTGGCTCATGTTTAATGATTATTTGGTTGTTGAGATA ACAGAGGAAGAGGCGCTTAAAATGACATACCCTTGGAAAACACCAGAAATTATCATATATTGTG ATGCGGAAGAATTACGAAAACCTTTCTTTTCTGTTGATACGTATTCCATCAACTATGACATACT TTTCCGTGATTATTTCGCAAACGGAATAAGAGATACTGCAAGACGTGAATATAAGTTATTAACA CATGATGAGGCACCTAAATCTGGAACCTTGGTTGCCATTGATGCCGAATTTGTCTCATTACAAA GTGAACTATGTGAAATCGATCATCAAGGAATCAGAAGTATTATTCGACCTAAAAGAACTGCTTT GGCCAGAATATCCATTATTAGAGGCGAAGAAGGAGAACTGTATGGAGTACCATTTGTCGATGAT TATGTGGTAAACACGAACCACATAGAAGACTATTTGACAAGATATAGTGGGATTCTTCCTGGTG ACTTGGACCCTGAAAAGAGTACCAAAAGGCTTGTGAGAAGAAACGTTGTATATCGAAAAGTCTG GCTTTTAATGCAGCTCGGATGCGTATTTGTTGGTCATGGTTTGAATAATGACTTCAAACACATT

AATATTAATGTCCCAAGAAACCAAATTCGCGACACTGCCATATATTTTCTACAAGGAAAGAGAT

ATCTTTCATTGCGTTATCTGGCATATGTGTTGTTAGGAATGAATATCCAAGAGGGAAATCACGA

TTCAATTGAAGATGCCCATACTGCCTTGATTCTTTACAAAAAATATCTCCACCTGAAAGAAAAA

GCTATCTTTGAGAAAGTACTGAACAGCGTGTACGAAGAAGGAAGAGCCCATAATTTCAAAGTTC

CAGAAACTTCAAAGGGATAA

[096] Yeast strains, the relevant gene disruption and associated sequence identifiers, are summarized in Table 1.

Table 1. Yeast strains

Example 2

Growth of modified yeast in moderate DS fermentation substrate

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

[098] The results demonstrated that strains with SKG3, YNRO68C, PIN3 and PEX18 deletions produce similar amounts of ethanol compared to parental FG under the moderate DS conditions. Strains with MNN14 and MNN4 deletions show an about 1% increase in ethanol production compared to FG.

Example 3

Growth of modified yeast in high DS fermentation substrate

[099] Strains with SKG3, YNR068C, MNN14, PIN3, PEX18 or MNN4 deletions were tested in fermentations along with the parental FG strain in high DS conditions comprising 5.5g 37.8% DS liquefact at pH4.8 with initial OD 0.3. The fermentations were carried out at 32°C for 55 hours. Samples from the end of fermentation (EOF) were analyzed by HPLC and the results are shown in Table 3. Data are the average of two independent vials for each strain and four independent vials for FG.

Table 3. HPLC results from fermentation assays at high DS

[0100] The results demonstrated that strains with each of SKG3, YNR068C, MNN14, PIN3, PEX18 and MNN4 disruptions showed significant improvement in ethanol production (2.4 - 4.7%) compared to the parental FG strain under high DS conditions. Correspondingly, less glucose remained at the end of fermentation compared to FG.

Example 4

Growth of modified yeast under temperature ramp conditions

[0101] Strains with SKG3, YNR068C, MNN14, PIN3 or PEX18 deletions were tested in fermentation assays together with parental FG strain in the moderate DS conditions described in Example 2. To further characterize the variant strains, fermentations were carried out on a 36.6°C temperature ramp (illustrated in Figure 1) for 55 hours. EOF samples were analyzed by HPLC and the results shown in Table 4.

Table 4. HPLC results from fermentation assays under ramp conditions

[0102] The results demonstrated that strains with SKG3, YNRO68C, MNN14, PIN3, PEX18 or

MNN4 deletions show a greater than 5% increase in ethanol production compared to parental FG under elevated temperature ramp conditions. Less end of fermentation glucose remained as well as less glycerol and greater acetate.

Example 5

Growth of modified yeast in moderate DS fermentation substrate

[0103] Modified strains with RIM20 or PAN2 deletion were tested in vials under moderate DS conditions and the products analyzed by HPLC as described in Example 2. The results are shown in Table 5. Data shown arc the average of two independent vials for deletion strains and six independent vials for parental FG.

Table 5. HPLC results from fermentation assays

[0104] The results demonstrated that the strain with RIM20 deletion had some negative effects on ethanol production, while strain with the PAN2 deletion showed little difference in ethanol production. Both deletion strains produced a 20% decrease in acetate production.

Example 6

Growth of modified yeast in high DS fermentation substrate

[0105] Modified strains with RIM20 or PAN2 deletion were tested in vials under high DS conditions and the products analyzed by HPLC as described in Example 3. The results are shown in Table 6. Data shown are the average of two independent vials for deletion strains and six independent vials for parental FG.

Table 6. HPLC results from fermentation assays

[0106] The results demonstrated that the strain with the RIM20 deletion produced about 3.4% more ethanol and an about 13% less acetate compared to the parental FG strain. Similarly, the strain with the PAN2 deletion produced about 2.4% more ethanol and more than 15% less acetate.

Example 7

Growth of modified yeast under temperature ramp conditions

[0107] Modified strains with RIM20 or PAN2 deletion were tested in vials under moderate DS temperature ramp conditions and the products analyzed by HPLC as described in Example 5 and illustrated in Figure 1. The results are shown in Table 7. Data shown are the average of two independent vials for deletion strains and six independent vials for parental FG.

Table 7. HPLC results from fermentation assays

[0108] The results demonstrated that the strain with the RIM20 deletion produced about 6.2% more ethanol and a small decrease in acetate compared to the parental FG strain. Similarly, strain with the PAN2 deletion produced an about 5.4% increase in ethanol and more than an 8% decrease in acetate production.

[0109] All references cited herein are incorporated by references, including the following additional references: Conde R. et al. (2003) Screening for new yeast mutants affected in mannosylphosphorylation of cell wall mannoprotcins. Yeast 20(14): 1189- 211 PMID: 14587103 Kim YH. et al. (2017) Abolishment of N-glycan mannosylphosphorylation in glycoengineered Saccharomyces cerevisiae by double disruption of MNN4 and MNN14 genes. Appl. Microbiol. Biotechnol. 101(7):2979-2989 PMID: 28101612 Chernova TA, et al. (2011) Prion induction by the short-lived, stress-induced protein Lsb2 is regulated by ubiquitination and association with the actin cytoskeleton. Mol Cell 43(2):242-52 PMID: 21777813 Madania A, et al. (1999) The Saccharomyces cerevisiae homologue of human Wiskott- Aldrich syndrome protein Lasl7p interacts with the Arp2/3 complex. Mol Biol Cell 10(10):3521-38 PMID: 10512884 Purdue PE, et al. (1998) Pexl8p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway. J Cell Biol 143(7): 1859-69 PMID: 9864360 Stein K, et al. (2002) Interactions of Pex7p and Pexl8p/Pex21p with the peroxisomal docking machinery: implications for the first steps in PTS2 protein import. Mol Cell Biol 22(17):6056-69 PMID: 12167700 Purdue PE and Lazaro w PB (2001) Pexl8p is constitutively degraded during peroxisome biogenesis. J Biol Chem 276(50):47684-9 PMID: 11590152 Raschke WC, et al. (1973) Genetic control of yeast mannan structure. Isolation and characterization of mannan mutants. J Biol Chem 248(13):4660-6 PMID: 4578088 Odani T, et al. (1996) Cloning and analysis of the MNN4 gene required for phosphorylation of N-linked oligosaccharides in Saccharomyces cerevisiae. Glycobiology 6(8):8O5-1O PMID: 9023541 Odani T, et al. (1997) Mannosylphosphate transfer to cell wall mannan is regulated by the transcriptional level of the MNN4 gene in Saccharomyces cerevisiae. FEBS Lett 420(2- 3): 186-90 PMID: 9459307 Xu W and Mitchell AP (2001) Yeast PalA/AIPl/Alix homolog Rim20p associates with a PEST-like region and is required for its proteolytic cleavage. J Bacteriol 183(23):6917- 23 PMID: 11698381 Xu W. et al. (2004) Multivesicular body-ESCRT components function in pH response regulation in Saccharomyccs ccrcvisiac and Candida albicans. Mol Biol Cell 15(12):5528-37 PMID: 15371534 Su SS and Mitchell AP (1993) Identification of functionally related genes that stimulate early meiotic gene expression in yeast. Genetics 133( I ):67-77 PMID: 8417990 Boeck R. et al. (1996) The yeast Pan2 protein is required for poly(A)-binding protein- stimulated poly(A)-nuclease activity. J Biol Chem 27 l(l):432-8 PMID: 8550599 Brown CE and Sachs AB (1998) Poly (A) tail length control in Saccharomyces cerevisiae occurs by message-specific deadenylation. Mol Cell Biol 18(11 ):6548-

59 PMID: 9774670 Hammet A, et al. (2002) Posttranscriptional regulation of the RAD5 DNA repair gene by the Dunl kinase and the Pan2-Pan3 poly(A)-nuclease complex contributes to survival of replication blocks. J Biol Chem 277(25):22469-74 PMID: 11953437

Claims

CLAIMS What is claimed is:

1. Modified yeast cells derived from parental yeast cells, the modified cells comprising a genetic alteration that causes the modified cells to produce during fermentation a decreased amount of polypeptides responsible for lack of robustness under high dry solids and elevated temperature fermentation conditions compared to otherwise identical parental cells, wherein the polypeptides arc selected from SKG3, YNRO68C, MNN14, PIN3, PEX18, MNN4, RIM20 and/or PAN2 polypeptides.

2. The modified cells of claim 1, wherein the modified cells further produce during fermentation a decreased amount of acetate compared to the amount of acetate produced by otherwise identical parental cells under the same fermentation conditions, and wherein the genetic alteration comprises the disruption of a nucleic acid capable of directing the expression of RIM20 and/or PAN2 polypeptides.

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

4. The modified cells of claim 3, wherein the genes of the phosphoketolase pathway arc selected from the group consisting of phosphoketolase, phosphotransacetylase and acetylating acetyl dehydrogenase.

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

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

7. The modified cells of any of claims 1 -6, further comprising an alternative pathway for making ethanol.

8. The modified cells of any of claims 1-7, wherein the cells are of a Saccharomyces spp.

9. A method for increasing the robustness of yeast cells under high dry solids and elevated temperature fermentation conditions, comprising introducing into parental yeast cells a genetic alteration that causes the resulting modified cells to produce during fermentation a decreased amount of polypeptides selected from SKG3, YNR068C, MNN14, PIN3, PEX18, MNN4, RIM20 and/or PAN2 polypeptides.

10. The method of claim 9, wherein the genetic alteration that further causes the modified cells to produce during fermentation a decreased amount of acetate compared to the amount of acetate produced by the parental cells under the same fermentation conditions, and wherein the genetic alteration comprises the disruption of a nucleic acid capable of directing the expression of RIM20 and/or PAN2 polypeptides.

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

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

13. The method of claim 11 or claim 12, wherein the modified yeast cells produce an increased amount of fermentation product compared to parental cells under the same conditions.

14. The method of any one of claims 11-13, wherein the modified yeast cells produce a decreased amount of acetate compared to parental cells under the same conditions.

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