CA3072306A1 - Increased ethanol production by yeast harboring constitutive transcriptional activator mal alleles - Google Patents

Abstract

Described are compositions and methods relating to modified yeast harboring constitutive transcriptional activator MAL alleles. The yeast produces an increased amount of ethanol, or have an increased rate of ethanol production, which does not appear to be the result of maltose metabolism. Such yeast is particularly useful for ethanol production utilizing starch substrates.

Description

INCREASED ETHANOL PRODUCTION BY YEAST HARBORING
CONSTITUTIVE TRANSCRIPTIONAL ACTIVATOR MAL ALLELES
TECHNICAL FIELD
[01] The present compositions and methods relate to modified yeast harboring constitutive transcriptional activator MAL alleles. The yeast produces an increased amount of ethanol that does not appear to be the result of maltose metabolism. Such yeast is particularly useful for large-scale ethanol production from starch substrates.
BACKGROUND

[02] The first generation of 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 Mar. A.J. (2015) Curr. Op/n. 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 will have massive economic impact across the industry.

[03] Yeast, such as Saccharomyces, are capable of metabolizing a number of mono and di-saccharides, including maltose. Maltose fermentation in Saccharomyces involves at least one of five, apparently-redundant, unlinked MAL loci, referred to as MAL1 , MAL2, MAL3, MAL4 and M4L5 (see, e.g., Needleman, R.B. (1991)Mol. Microbiol. 9:2079-84). As illustrated in Figure 1, each locus includes three genes, encoding (i) a maltose permease, (ii) a maltase and (iii) a transcriptional activator (Kim, J. and. Michels, C.A. (1988) Curr.
Genet. 14:319-23 and Cheng, Q. and Michels (1989) Genetics 123:477-84. The genes are conventionally numbered 1, 2 and 3, respectively, such that the genes at the MAL2 locus, for example, are numbered MAL21, MAL22 and MAL23 , respectively.

[04] Transcription of MAL genes is induced by maltose and repressed by glucose;
however, constitutive mutations have been identified at all MAL loci (Winge, 0. and Roberts, C. (1950) C. R. Tray. Lab. Carlsberg Ser. Physiol. 25:35-81, Kahn, N.A. and Eaton, N.R.
(1971)Mol. Gen. Genet. 112: 317-22; Charronm, J. and Michels, C.A. (1987) Genetics 116 23-31; Zimmerman and Eaton, N.R. (1974)Mol. Gen. Genet. 134 261-271; Rodicio, R.

(1986) Curr. Genet. 11:235-41 and Ten Berge, A.M.A. et al. (1973)Mol. Gen.
Genet. 125:
139-46.

[05] Maltose is present at low levels in commercial-scale, starch-hydrolysates, typically, as an undesirable, DP2-component, which is not likely to be a significant source of additional ethanol. Moreover, transcription ofMAL genes is repressed by high levels of glucose, making it difficult for yeast to utilize maltose under high-glucose conditions, such as those that exist during fuel ethanol production. Deliberate expression of maltose-metabolizing enzymes may in fact slow glucose metabolism and waste carbon, which is unacceptable given the demands of ethanol producers.
SUMMARY

[06] The present compositions and methods relate to modified yeast harboring constitutive transcriptional activator MAL alleles. While such yeast is presumably capable of metabolizing maltose, even in the presence of glucose, the yeast appears to produce an increased amount of ethanol that is not necessarily the result of maltose metabolism. Aspects and embodiments of the compositions and methods are described in the following, independently-numbered, paragraphs.
1. In one aspect, a method for increasing the amount of alcohol produced from fermentation of a starch hydrolysate and/or increasing the rate of production of alcohol from fermentation of a starch hydrolysate is provided, comprising fermenting the starch hydrolysate with modified yeast harboring a constitutive transcriptional activator MAL
allele, where in the modified yeast produces an increased amount of ethanol at the end of fermentation or an increased amount of ethanol over a period of time compared to the amount of ethanol produced by an otherwise-identical parental yeast.
2. In some embodiments of the method of paragraph 1, at least a portion of the increased amount of ethanol cannot be attributed to maltose fermentation based on carbon flux through a maltose metabolic pathway.
3. In some embodiments of the method of paragraph 1 or 2, the level of maltose in the starch hydrolysate at the end of fermentation is about the same as the level of maltose in the starch hydrolysate at the beginning of fermentation, 4. In some embodiments of the method of any of the preceding paragraphs, the amount of maltose in the starch hydrolysate at the beginning of fermentation is no greater than 10 g/L.

5. In some embodiments of the method of any of the preceding paragraphs, the yeast harboring a constitutive transcriptional activator MAL allele further comprises a genetic alteration that introduces a polynucleotide encoding a polypeptide in the phosphoketolase pathway.
6. In some embodiments of the method of any of the preceding paragraphs, the yeast harboring a constitutive transcriptional activator MAL allele further comprises an alteration in the glycerol pathway and/or the acetyl-CoA pathway.

7. In some embodiments of the method of any of the preceding paragraphs, the yeast harboring a constitutive transcriptional activator MAL allele further comprises disruption of a gene encoding DLs1.

8. In some embodiments of the method of any of the preceding paragraphs, the yeast harboring a constitutive transcriptional activator MAL allele comprises an exogenous gene encoding a carbohydrate processing enzyme.

9. In some embodiments of the method of any of the preceding paragraphs, the yeast is a Saccharomyces spp.

10. In another aspect, modified yeast comprising a constitutive transcriptional activator MAL allele, and at least one additional genetic modification not associated with maltose metabolism are provided, which yeast produces during fermentation an increased amount of ethanol at the end of fermentation compared to the amount produced by an otherwise identical parental yeast when grown in a starch hydrolysate.

11. In some embodiments of the modified yeast of paragraph 10, the yeast further comprises a genetic alteration that introduces a polynucleotide encoding a polypeptide in the phosphoketolase pathway.

12. In some embodiments of the modified yeast of paragraph 10 or 11, the yeast further comprises an alteration in the glycerol pathway and/or the acetyl-CoA pathway.

13. In some embodiments of the modified yeast of any of paragraphs 10-12, the yeast further comprises a disruption of a gene encoding DLs1.

14. In some embodiments of the modified yeast of any of paragraphs 10-13, the yeast further comprises an exogenous gene encoding a carbohydrate processing enzyme.

15. In some embodiments of the modified yeast of any of paragraphs 10-14, the yeast is a Saccharomyces spp.
[07] These and other aspects and embodiments of present modified cells and methods will be apparent from the description, including the accompanying Drawings/Figures.

BRIEF DESCRIPTION OF THE DRAWINGS
[08] Figure 1 is a diagram of a typical MAL loci, and its regulation by maltose and glucose [09] Figure 2 depicts a MAL23c expression cassette.
[010] Figure 3 is a map of plasmid pHX19.
[011] Figure 4 is comparison of cumulative CO2 pressure index during fermentation with strains FG and A28.
[012] Figure 5 is a map of plasmid pZKAP1M-(H3C19).
[013] Figure 6 is a map of plasmid pTOPO II-Blunt ura3-loxP-KanMX-loxP-ura3.
[014] Figure 7 is a map of plasmid pGAL-Cre-316.
[015] Figure 8 is comparison of cumulative CO2 pressure index during fermentation with RHY723 and its parent strain GPY10008.
DETAILED DESCRIPTION
I. Definitions

[016] Prior to describing the present yeast 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.

[017] 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.

[018] 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 S. cerevisiae. Yeast include 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.

[019] 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.

[020] 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 are used herein and all sequence are presented from an N-terminal to C-terminal direction. The polymer can comprise modified amino acids, and it can be interrupted by non-amino 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.

[021] 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 cross-reactivity, or determined by their functions.

[022] 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) (i.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).

[023] 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 at. (1984) Nucleic Acids Res. 12:387-95).

[024] 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 at. ((1990)1 Mol. Biol. 215:403-10) and Karlin et at.
((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.

[025] 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 (i.e., wild-type) sequence. Percent sequence identity is calculated using CLUSTAL W algorithm with default parameters. See Thompson et at. (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: GP SNDQEKR

Use negative matrix: OFF
Toggle Residue specific penalties: ON
Toggle hydrophilic penalties: ON
Toggle end gap separation penalty OFF

[026] 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).

[027] 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."

[028] 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.

[029] 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.

[030] As used herein, "overexpressing 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.

[031] As used herein, an "expression cassette" refers to a DNA fragment that includes a promoter, and 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).

[032] 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.

[033] 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.

[034] 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.

[035] 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 function 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 function gene product. A gene can also be disrupted using 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 are 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-adjacent control elements.

[036] As used herein, the terms "genetic manipulation" and "genetic alteration" 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.

[037] 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.

[038] 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.

[039] 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.

[040] 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.

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

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

[043] 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.

[044] 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.

[045] 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:
EC enzyme commission PKL phosphoketolase PTA phosphotransacetylase AADH acetaldehyde dehydrogenases ADH alcohol dehydrogenase Et0H ethanol AA a-amylase GA glucoamylase C degrees Centigrade bp base pairs DNA deoxyribonucleic acid ds or DS dry solids g or gm gram g/L grams per liter H20 water HPLC high performance liquid chromatography hr or h hour kg kilogram molar mg milligram mL or ml milliliter min minute mM millimolar normal nm nanometer PCR polymerase chain reaction ppm parts per million A relating to a deletion tg microgram [EL and 11.1 microliter micromolar II. Modified yeast harboring a constitutive MAL allele

[046] The present inventors have discovered that modified yeast harboring a constitutive transcriptional activator MAL allele produces more ethanol in a starch-hydrolysate-fermentation, and/or produces the same amount of ethanol in less time, than an otherwise identical parental yeast. Remarkably, the additional ethanol produced does not appear to be the result, or at least solely the result, of maltose metabolism, as the small amount of maltose present in the starch hydrolysate remains basically unchanged following fermentation. Yeast harboring constitutive transcriptional activator MAL allele also produces ethanol at a higher rate in a starch-hydrolysate-fermentation than an otherwise identical parental yeast.
Therefore, the constitutive expression of the transcriptional activator MAL
allele appears to produce an unexpected benefit to glucose metabolism in a high-glucose environment.

[047] The presence of a constitutive transcriptional activator MAL allele, optionally in the presence of other genetic modifications, results, in an increase in ethanol production, or rate of ethanol production, of at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0%, at least 1.1%, at least 1.2%, or more. Although such an increase is modest, it amounts to a substantial increase in terms of volume, given the amount of ethanol currently being produced.

[048] The present yeast strains and methods are exemplified using a representative constitutive transcriptional activator M4L23 allele, shown as SEQ ID NO: 1, below:
MG IAKQS CDCCRVRRVKCDRNKPCNRC T QRNLNC TYLQPLKKRGPKS I RAGS LKK IAEVQMV
SMNNNIMTAPVVCKKVPKNL I DQCLRLYHDNLYVI WPMLSYDDLHKLLEENYEDCS TYWFLV
SLSAATLSDLQIE IEYEEGVT FTGEQLCTLCMLSRQFFDDLSNSDI FRIMTYYCLHRCYAQF
ADTRTSYRLSCEAIGL IKIAGFHREE TYE FLP FGEQQLRRKVYYLLLMTERFYAVY IKCVT S
LDTT IAPPLPEVVTDPRLSLES FLEVIRVFTVPGKCFYDALATNCVDDSCTEDSLKRIWNEL
HT T SLDIEPWSYGYVDI S FSRHWIRALAWKLVFQMNGTKFFSNANNAHILVE IAKDMLDDI F
L T PNNLYDVHGPG I PMKS LEVANALVD IVNKYDHNMKLEAWN I LCDVS KFVFS LKHCNHKMF
QRFS TKCQSAL I DLP I SRPLRLNDDSKDEDDI I P

[049] However, it is fully expected that the introduction of other constitutive transcriptional activator MAL alleles will produce similar results. Constitutive transcriptional activator MAL
alleles have been known for more than half-a-century, and are exemplified by those described in, e.g., Winge, 0. and Roberts, C. (1950) C. R. Tray. Lab. Carlsberg Ser.
Physiol. 25:35-81, Kahn, N.A. and Eaton, N.R. (1971)Mol. Gen. Genet. 112: 317-22; Charronm, J.
and Michels, C.A. (1987) Genetics 116 23-31; Zimmerman and Eaton, N.R. (1974)Mol. Gen.
Genet. 134 261-271; Rodicio, R. (1986) Curr. Genet. 11:235-41 and Ten Berge, A.M.A. et al. (1973) Mol. Gen. Genet. 125: 139-46, each of which references is incorporated by reference.

[050] Yeast harboring constitutive transcriptional activator MAL alleles may also include other genetic manipulations that increase alcohol production, particularly modifications that are not associated with enhanced maltose metabolism.
III. Modified yeast harboring a constitutive MAL allele in combination with genes of an exogenous PKL pathway

[051] The presence of constitutive transcriptional activator MAL alleles can be combined with expression of genes in the PKL pathway to increase the growth rate of cells and further increase the production of ethanol.

[052] Engineered yeast cells having a heterologous PKL pathway have been previously described (e.g., W02015148272). These cells express heterologous PKL (EC
4.1.2.9) and PTA (EC 2.3.1.8), 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 harboring a constitutive MAL allele and having reduced D1s1 expression

[053] D1s1, encoded by YJL065c, is a 167-amino acid polypeptide subunit of the yeast chromatin accessibility complex (yCHRAC), which contains Isw2, Itcl, Dpb3-like subunit (D1s1), and Dpb4 (see, e.g., Peterson, C.L. (1996) Curr. Op/n. Genet.
Dev. 6:171-75 and Winston, F. and Carlson, M. (1992) Trends Genet. 8:387-91). Yeast having a genetic alteration that reduces the amount of functional D1s1 in the cell, in the absence of other genetic modifications, exhibit increased robustness in an alcohol fermentation, allowing higher-temperature, and potentially shorter, fermentations (data not shown).

[054] Reduction in the amount of functional D1s1 produced in a cell can be accomplished by disruption of the YA065c gene. Disruption of the YA065c gene can be performed using any suitable methods that substantially prevent expression of a function Y.IL065c gene product, i.e., D1s1. Exemplary methods of disruption as are known to one of skill in the art include but are not limited to: complete or partial deletion of the Y.IL065c gene, including complete or partial deletion of, e.g., the Dlsl-coding sequence, the promoter, the terminator, an enhancer, or another regulatory element; and complete or partial deletion of a portion of the chromosome that includes any portion of the YJL065c gene.

[055] Particular methods of disrupting the Y.IL065c gene include making nucleotide substitutions or insertions in any portion of the Y.IL065c gene, e.g., the Dlsl-coding sequence, the promoter, the terminator, an enhancer, or another regulatory element.
Preferably, deletions, insertions, and/or substitutions (collectively referred 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 the Y.IL065c gene.
V. Additional mutations that affect alcohol production

[056] The present modified yeast may further include, or may expressly exclude, mutations that result in attenuation of the native glycerol biosynthesis pathway, which are known to increase alcohol production. Methods for attenuation of the glycerol biosynthesis pathway in yeast are known and include reduction or elimination of endogenous NAD-dependent glycerol 3-phosphate dehydrogenase (GPD) or glycerol phosphate phosphatase activity (GPP), for example by disruption of one or more of the genes GPD1, GPD2, GPP 1 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.).

[057] The modified yeast may further feature increased acetyl-CoA synthase (also referred to acetyl-CoA ligase) activity (EC 6.2.1.1) to scavenge (i.e., capture) acetate produced by chemical or enzymatic hydrolysis of acetyl-phosphate (or present in the culture medium of the yeast for any other reason) and converts it to acetyl-CoA. This avoids 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. A particularly useful acetyl-CoA
synthase for introduction into cells can be obtained from Methanosaeta concilii (UniProt/TrEMBL Accession No.: WPO13718460). Homologs of this enzymes, including enzymes having at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98% and even at least 99% amino acid sequence identity to the aforementioned acetyl-CoA
synthase from Methanosaeta concilii, are also useful in the present compositions and methods. In other embodiments, the present modified yeast do not have increased acetyl-CoA synthase.

[058] In some embodiments the present modified yeast 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.). However, in most embodiments of the present compositions and methods, the introduction of an acetylating acetaldehyde dehydrogenase and/or a pyruvate-formate lyase is not required because the need for these activities is obviated by the attenuation of the native biosynthetic pathway for making acetyl-CoA that contributes to redox cofactor imbalance. Accordingly, in some embodiments, the present yeast do not have a heterologous gene encoding an NAD+-dependent acetylating acetaldehyde dehydrogenase and/or encoding a pyruvate-formate lyase.

[059] In some embodiments, the present modified yeast further comprises a butanol biosynthetic pathway. In some embodiments, the butanol biosynthetic pathway is an isobutanol biosynthetic pathway. The isobutanol biosynthetic pathway may comprise 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-dihydroxyisovalerate; (c) 2,3-dihydroxyisovalerate to 2-ketoisovalerate; (d) 2-ketoisovalerate to isobutyraldehyde; and (e) isobutyraldehyde to isobutanol. The isobutanol biosynthetic pathway may comprise polynucleotides encoding polypeptides having acetolactate synthase, keto acid reductoisomerase, dihydroxy acid dehydratase, ketoisovalerate decarboxylase, and alcohol dehydrogenase activity.

[060] In some embodiments, the modified yeast comprising a butanol biosynthetic pathway further comprise a modification in a polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. The yeast may 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, and/or substitution in one or more endogenous polynucleotides encoding FRA2, ALD6, ADH1, GPD2, BDH1, and Y1V1R226C. In other embodiments, the present modified yeast cells do not further comprise a butanol biosynthetic pathway.

[061] The present modified yeast may include any number of additional genes of interest encoding protein of interest, including 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

[062] The present yeast, and methods of use, thereof, include methods for increasing alcohol production in fermentation reactions. 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 ethanol production, the present yeast can also be used for the production of potable alcohol, including wine and beer.
VII. Yeast suitable for modification

[063] Yeast is a unicellular eukaryotic microorganism classified as members of the fungus kingdom and includes 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 yeast has been genetically engineered to produce heterologous enzymes, such as glucoamylase or a-amylase.

VIII. Substrates and products

[064] 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. High levels of maltose are not required to benefit from the present compositions and methods. In some embodiments, the concentration is less than about 10 g/L.

[065] These and other aspects and embodiments of the present 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 strains and methods.
EXAMPLES
Example 1 Materials and methods Liquefact preparation:

[066] Liquefact (corn mash slurry) was prepared by adding 600 ppm of urea, 0.124 SAPU/g ds FERMGENTm 2.5x (acid fungal protease), 0.33 GAU/g ds CS4 (a variant Trichoderma glucoamylase) and 1.46 SSCU/g ds AKAA (Aspergillus kawachii a-amylase), adjusted to a pH of 4.8.
Serum vial assays:

[067] 2 mL of YPD in 24-well plates were inoculated with yeast cells and the cultures allowed to grow overnight to an OD between 25-30. 2.5 mL liquefact was transferred to serum vials (Chemglass, Catalog #: CG-4904-01) and yeast was added to each vial to a final OD of about 0.4-0.6. The lids of the vials were installed and punctured with needle (BD, Catalog #305111) for ventilation (to release CO2), then incubated at 32 C with shaking at 200 RPM for 65 hours.

AnKom assays:

[068] 300 L of concentrated yeast overnight culture was added to each of a number ANKOM bottles filled with 50 g prepared liquefact (see above) to a final OD of 0.3. The bottles were then incubated at 32 C with shaking at 150 RPM for 65 hours.
HPLC analysis:

[069] Samples of the cultures from serum vials and AnKom assays were collected in Eppendorf tubes by centrifugation for 12 minutes at 14,000 RPM. The supernatants were filtered using 0.2 M PTFE filters and then used for HPLC (Agilent Technologies 1200 series) analysis with the following conditions: Bio-Rad Aminex HPX-87H
columns, running temperature of 55C. 0.6 ml/min isocratic flow 0.01 N H2504, 2.5 1 injection volume.
Calibration standards were used for quantification of the of acetate, ethanol, glycerol, glucose and other molecules. All values are reported in g/L.
Example 2 Preparation of a constitutive MAL23 allele cassette

[070] A MAL23c expression cassette, consisting of a constitutive allele of MAL23 (SEQ ID
NO: 2) under control of a MAL23c promoter (SEQ ID NO: 3) and MAL23 terminator (SEQ
ID NO: 4), was made using standard procedures. The nucleotide sequences are shown, below, and a representation of the cassette is illustrated in Figure 2:
DNA coding region of the constitutive MAL23 allele (SEQ ID NO: 2):
ATGGGTATTGCGAAACAGTCTTGCGACTGCTGTCGCGTTCGTCGAGTAAAGTGTGACAGGAA
TAAACCATGTAATCGCTGCACTCAGCGCAATTTGAACTGCACTTATCTTCAACCGTTGAAAA
AGAGAGGTCCAATCCAT TAGAGCAGGGCT TAATAGCGGAGTGCAGATGGTG
AGTATGAATAATAATATTATGACCGCTCCGGTGGTATGTAAGAAGGTTCCGAAAAACCTGAT
TGATCAATGTTTGAGGTTGTATCATGATAACTTATATGTAATTTGGCCAATGCTTTCCTACG
ATGATCTTCACAAGCTTTTGGAGGAAAATTATGAGGACTGCAGCACTTATTGGTTTCTGGTA
TCCCTTTCGGCAGCTACTCTTAGCGACTTGCAAATTGAAATAGAGTATGAGGAAGGAGTCAC
TTTTACTGGAGAGCAGTTATGCACTCTTTGCATGTTATCTCGGCAATTCTTTGACGACCTTA
GTAACAGCGACATAT TTCGAATCATGACATACTAT TGT TTGCACCGT TGT TACGCGCAGT TT
GCTGATACAAGAACTTCATACAGACTTTCTTGTGAGGCTATTGGCCTCATCAAGATAGCTGG
ATTCCATCGGGAAGAAACCTATGAATICCTICCCTICGGTGAACAACAACICAGAAGGAAAG
TTTACTATTTACTTCTTATGACAGAGAGATTTTACGCTGTATATATTAAGTGTGTCACGAGC
CTAGATACAACAATAGCGCCACCACTACCAGAGGTTGTAACAGACCCTCGTCTTTCTCTGGA
AAGCTTCCTTGAGGTGATTAGAGTTTTCACTGTACCTGGAAAGTGTTTTTATGATGCTTTGG

CTACTAACTGTGTCGATGATICCTGCACCGAAGACTCTCTAAAAAGGATATGGAACGAACTI
CATACCACATCACTTGATATAGAGCCATGGTCTTATGGCTATGTGGACATTTCATTTTCTCG
ACATTGGATTAGGGCGCTGGCTTGGAAGCTAGTGTTTCAGATGAATGGTACCAAGTTTTTCT
CAAACGCCAATAATGCTCACATATTGGTCGAAATTGCAAAGGATATGCTGGACGACATATTC
TTAACTCCAAACAACCTGTATGATGTACATGGTCCTGGAATACCAATGAAATCATTGGAAGT
AGCCAATGCATTGGTAGATATCGTAAATAAGTATGATCACAATATGAAGTTGGAGGCTTGGA
ATATTTTGTGCGATGTATCCAAGTTCGTTTTCTCCCTGAAACATTGCAATCATAAAATGTTT
CAAAGGITTICAACTAAATGICAGAGTGCTCTAATCGATTIGCCTATCTCTAGACCACTGCG
CCTAAATGATGATTCCAAAGATGAAGACGACATAATTCCTTAA
DNA sequence of the MAL23c promoter (SEQ ID NO: 3):
TATTGTCCGTTCCATTGTATTCAATAACTTAAATGCAACAGAAAATGATTACAGGATTCTTC
TCTCATAGAAATATAAGCAAACT T CAAAT GGAAC T GAGA AATTTCCTTTCTATTGGA
ATTTTACTTTACAAATGACTTTGGCAAAACAGGCATGCGATTGTTGTCGTGTTCGTCGAGTA
AAGTGTGACGGCGAAAAGCCATGTAATCGT TGTCTGCAGCATGAT TTGAAATGTACT TAT TT
ACAACCTTTGAGAAAAAGAGGGCCCAAAAATATTAGATCGAGAAGTTTAAAGAAAATTGCCG
AAACGCAAACGTTCAGTGAGAACAACAACTGTATGACAGCTTTAGAAATATCTATAGGAATC
ATTATATCTTACATGTTATTCTGTTCTGTTGTAGTAACTAATTTCAGAGATCTTTTCGGATG
CTACTACCCCTGTTTATAACGTGTAACTTTAATCTTGAAATTTCGTTTTTTCCCCACAATTT
TTCCGTAGCCTTTCTCGCGAGAATTAATCCGTCGTACAATAATTCGATCTTACTTGTATTTT
CTCCCATGAGCATGCAGACTAATAGGTAGGAAAATAGAACTACTTAGAAACATTCTCCT TAA
GTGTTTTCACCACTAAGCATTTTATATTTAATTGTTAAAAAATATATACTATTGAAGAACCA
CTTTCCTGAAATATCAAGAACAAAAAAGTCTGCACTATGGTCCCGCAATTGATGCATTTGAG
AATTCTTTTAACTCAATAGTAATATGCATTGTTCTTATCTAAAAAATTGCAGGTACCTGCAG
ACTAATCCGGGTCATGATCTGCGCTGCGCCGTCATCCCACCCCGTGCTGCCTGCCACTTGAA
GCTACCCGGGTTTAATAATTCGTTCTTAAGTTCTACAACTTAAATACAGGCAGCTAAAAAAC
TGGGTTCGAGAGTTTTCCACTTTATAGACAAAAATAAAAATACTGCCAGAAAATTTATCATA
TAATAAT
DNA sequence of the MAL23c terminator (SEQ ID NO: 4):
TTTATTGTTCACGCCGTTCACTTATACGAGATAGATATACTGATAGAGTGTGAGTGATATTC
TTAAGTCTTGCTTTTCGAGGGTGTAAGAAGCTATGTTCTTCAGGCGAGATTATTCTACTCCT
GCCTTACTTGTTTGTAATATTTAGTTCTGATGGTCATGATAATTCTATATACAGTTACAT TA
AGTATATACTTAAGCGGGCAGCTTACTAATATAAATTTTGTGGCATTTTTGTTGGGATATGA
GAATCATGTATCGTTGATTTACAAAGCGAATTTACGTTACCAGGAATAGGGAA

[071] A DNA fragment including flanking YDL227C locus sequences was made by amplifying the MAL23c expression cassette with appropriate primers. The fragment was inserted into a plasmid designated pHX19, whichincludes an integrated constitutive MAL23c expression cassette at the Saccharomyces chromosome YDL227C locus, as shown in Figure 3. The functional and structural composition of plasmid pHX19 is described in Table 1.

Table 1. Functional and structural elements of plasmid pHX19 Functional/structural element Description 5' HO Arm fragment of YDL227C 293-bp DNA fragment from S. cerevisiae locus 3' HO Arm fragment of YDL227C 290-bp DNA fragment from S. cerevisiae locus ColE1 replicon, ampicillin Sequences not to be integrated into a yeast genome resistance marker gene, Fl origin of replication, CEN6, ARSH4, His Promoter, His coding region, and His terminator Ma123p::MAL23::MAL23t MAL23c expression cassette Example 3 Generation of yeast with a MAL23c expression cassette

[072] To study the effects of MAL23c in industrial yeast, the wild-type FERMAXTm Gold strain (Martrex, Inc., Chaska, MN, USA), hereafter abbreviated, "FG," was used as a parent to introduce the MAL23c expression cassette at the YDL227C integration site.
Cells were transformed with a 3,362-bp, PCR-amplified DNA fragment using appropriate flanking primers and PFIX 1 plasmid as template. Transformants were selected and a representative member was designated strain A28.
Example 4 Comparison of FG yeast with or without MAL23c

[073] Strain A28, and parental strain FG, were grown in Ankom bottles and their fermentation products analyzed as described in Example 1. Performance in terms of cumulative CO2 pressure index (CPI), indication of the fermentation rate, is shown in Figure 4. The performance in terms of ethanol, glycerol and acetate production is shown in Table 2.
Table 2. FG versus A28 in AnKom assay Increase in Strain Maltotriose Maltose Glucose Glycerol Acetate Ethanol Ethanol FG 2.22 6.90 13.49 17.78 0.91 134.03 A28 2.21 6.86 13.96 17.55 0.88 135.48 1.1%

[074] The performance of the FG parent and strain A28 is similar in terms of the residual amounts of maltotriose, maltose, and glucose, suggesting that the integration of the MAL23c DNA fragment at the YDL227C locus did not affect the maltose metabolism in the glucose based corn mash fermentation. The ethanol titer of the Ma123c expressing strain is higher than the FG control by 1.1%. The CPI data in Figure 4 demonstrates that the fermentation rate of strain A28 is also higher than parent FG strain. The experiment was repeated and the results confirmed.
Example 5 Generation of a plasmid containing a complete PKL pathway

[075] Plasmid pZKAP1m-(H3C19) was designed to integrate four individual polypeptide expression cassettes upstream of the AAP1 locus (YHR047C). The four cassettes were as follows (i) HXT3 promoter: :PKL C::FBA1 terminator; (ii) PGKlpromoter::LpPTA::PGK1 terminator; (iii) TDH3 promoter::eutE A19::ENO terminator; and (iv) PDC1 promoter::AcsAl ::PDC1 terminator, which were designed to express codon-optimized genes encoding phosphoketolase (PKL), derived from Gardnerella vaginalis, phosphotransacetylase (PTA), derived from Lactobacillus plantarum; acylating acetaldehyde dehydrogenase, derived from Desulfospira joergensenii, and acetyl-CoA
synthase, derived from Methanosaeta concilii, respectively. A map of pZKAP1m-(H3C19) is shown in Figure 5. Functional and structural elements are detailed in Table 3.

[076] Table 3. Functional and structural elements of construct pZKAP1m-(H3C19) Functional/structural Description element AAP1-C 522-bp fragment, upstream of AAP 1 LoxP71 LoxP71 site URA3 URA3 gene used as selection marker LoxP66 LoxP66 site AAP1-D 476-bp fragment, upstream of AAP 1 ORI and AP Replication origin and ampicillin resistance gene, these sequences are not part of the DNA fragment integrated into yeast genome AAP1-U 465-bp fragment, upstream of AAP]
PDC1 promoter S. cerevisiae PDC1 promoter X4 Mathanosaeta CDS encoding acetyl-CoA synthase from Methanosaeta concilii acsAl cons/iii, codon-optimized for yeast PDC1 terminator S. cerevisiae PDC1 transcription terminator TDH3 promoter S. cerevisiae TDH3 promoter eutE A19 CDS encoding acylating acetaldehyde dehydrogenase from Desulfospira joergensenii , codon-optimized for yeast EN02 terminator S. cerevisiae EN02 transcription terminator PGK1 promoter S. cerevisiae PGK1 promoter L. plantarum PTA CDS encoding phosphotransacetylase from Lactobacillus plantarum, codon-optimized for yeast PGK1 transcription S. cerevisiae pGK1 transcription terminator terminator HXT3 promoter S. cerevisiae HXT3 promoter PKL C CDS encoding phosphoketolase from Gardnerella vaginalis, codon-optimized for yeast FBA 1 terminator S. cerevisiae FBA1 transcription terminator Example 6 Generation of FG-ura3 yeast

[077] The FG strain was used as the parent strain to make the ura3 auxotrophic strain FG-ura3.
Plasmid pTOPO II-Blunt ura3-loxP-KanMX-loxP-ura3, depicted in Figure 6, was designed to replace the URA3 gene in strain FG with mutated ura3 and URA3-loxP-TEFp-KanMX-TEFt-loxP-URA3 fragment. The functional and structural elements of the plasmid are listed in Table 4.
Table 4. Functional/structural elements of pTOPO II-Blunt ura3-loxP-KanMX-loxP-ura3 Functional/Structural Element Description KanR gene in E. coil Vector sequence pUC origin Vector sequence UR43 3'-flanking region Synthetic DNA identical to S. cerevisiae genomic sequence to URA3 locus loxP66 Synthetic DNA identical to loxP66 consensus TEF1::KanMX4::TEF terminator KanMX expression cassette loxP71 Synthetic DNA identical to loxP71 consensus URA3 5'-flanking region Synthetic DNA identical to the URA3 locus on the S. cerevisiae genome

[078] A 2,018-bp DNA fragment containing the ura3-loxP-KanMX-loxP-ura3 cassette was released from plasmid TOPO II-Blunt ura3-loxP-KanMX-loxP-ura3 by EcoRI
digestion. The fragment was used to transform S. cerevisiae FG cells by electroporation.

[079] Transformed colonies able to grow on media containing G418 were streaked on synthetic minimal plates containing 20 i.tg/mluracil and 2 mg/ml 5-fluoroorotic acid (5-FOA). Colonies able to grow on 5-FOA plates were further confirmed for URA3 deletion by growth of phenotype on SD-Ura plates, and by PCR. The ura3 deletion transformants were unable to grow on SD-Ura plates. A single 1.98-kb PCR fragment was obtained with test primers. In contrast, the same primer pairs generated a 1.3-kb fragment using DNA from the parental FG strain, indicating the presence of the intact ura3 gene. The ura3 deletion strain was named as FG-KanMX-ura3.

[080] To remove the KanMX expression cassette from strain FG-KanMX-ura3, plasmid pGAL-Cre-316, depicted in Figure 7, was used to transform cells of strain FG-KanMX-ura3 by electroporation. The purpose of using this plasmid is to temporary express the Cre enzyme, so that the LoxP-sandwiched KanMX gene will be removed from strain FG-KanMX-ura3 to generate strain FG-ura3. pGAL-Cre-316 is a self-replicating circular plasmid that was subsequently removed from strain FG-ura3. None of the sequence elements from pGAL-cre-316 was inserted into the strain FG-ura3 genome. The functional and structural elements of plasmid pGAL-Cre-316 is listed in Table 5.
Table 5. Functional and structural elements of pGAL-Cre-316.
Functional/structural element Yeast-bacterial shuttle vector pRS316 sequence pBR322 origin of replication S. cerevisiae URA3 gene Fl origin GALp-Cre-ADHt cassette, reverse orientation

[081] The transformed cells were plated on SD-Ura plates. Single colonies were transferred onto a YPG plate and incubated for 2 to 3 days at 30 C. Colonies were then transferred to a new YPD plate for additional days. Finally, cell suspensions from the YPD
plate were spotted on to following plates: YPD, G418 (150 [tg/m1), 5-FOA (2 mg/ml) and SD-Ura.
Cells able to grow on YPD and 5-F0A, and unable to grow on G418 and SD-Ura plates, were picked for PCR confirmation as described, above. The expected PCR product size was 0.4-kb and confirmed the identity of the KanMX (geneticin)-sensitive, ura3-deletion strain, derived from FG-KanMX-ura3. This strain was named as FG-ura3.

Example 7 Generation of yeast with the PKL pathway

[082] The FG-ura3 strain was used as a parent to introduce the PKL pathway.
Cells were transformed with a 14,993-bp Kcal fragment containing the four expression cassettes from pZKAP1m-(H3C19) from Example 5. A transformant with the Kcal fragment integrated upstream of the YHR047C locus was selected and designated as strain GPY10000.

[083] Strains FG and GPY10000 were grown in vial cultures and their fermentation products analyzed as described in Example 1. Performance in terms of ethanol, glycerol and acetate production is shown in Table 6.
Table 6. FG versus G176 in vial assays Strain Transgene(s) expressed Glycerol Acetate Et0H
FG none 17.30 0.70 140.9 GPY10000 PKL pathway 12.94 1.86 146.3

[084] The ethanol titer of the PKL pathway expressing GPY10000 strain was 3.8%
higher than the FG strain. Not surprisingly, the level of acetate was also elevated, which is a known property of yeast harboring the PKL pathway.
Example 8 Generation of yeast with the PKL pathway and reduced production of Dist

[085] Strain GPY10008 was generated by Cas9-mediated deletion of YIL065c (which encodes D1s1) in strain GPY10000. Specifically, a deletion was made from 4-bp before start codon to 10-bp before stop codon of YIL065c.

[086] FG yeast strain GPY10008, and its parent strain G10008, were grown in AnKom bottles and their fermentation products analyzed as described in Example 1.
Performance in terms of ethanol, glycerol and acetate production is shown in Table 7.
Table 7. GPY10000 versus GPY10008 in Ankom assays Strain Transgene(s) expressed Glycerol Acetate Et0H
GPY10000 PKL pathway 12.85 1.80 143.39 GPY10008 PKL pathway + AYJL065c 13.15 1.78 147.23

[087] The ethanol titer of the strain with the YJL065c deletion was 2.7%
higher than the strain without the deletion. Acetate levels were approximately the same.
Example 9 Generation of a yeast combing MAL23c with the PKL pathway and reduced production of D1s1

[088] To study the effects of MAL23c in engineered FG strain with the PKL
pathway and reduced expression of D1s1, the strain GPY10008 was used as a parent to introduce the MAL23c expression cassette, in this case at the 3' region of the YHL041w locus, otherwise as described in Example 3. GPY10008 cells were transformed with a PCR-amplified DNA
fragment containing the MAL23c expression cassette made using the pHX19 plasmid as template and primers that include flanking YHL041w locus sequences.

[089] The new FG yeast strain, designated RHY723, and its parent strain GPY10008 were grown in AnKoms bottles and their fermentation products were analyzed as described in Example 1. Performance in terms of ethanol, glycerol and acetate production is shown in Table 8, and the fermentation rate, represented by the CPI during the fermentation, is illustrated in Figure 8.
Table 8. RHY720 versus GPY10008 in Ankom assays Transgene(s) Strain Maltotriose Maltose Glucose Glycerol Acetate Et0H
expressed PKL pathway GPY10008 1.03 3.24 5.10 14.06 1.58 152.37 + YJL065cd PKL pathway RHY723 + YJL065cd 1.03 3.16 4.24 14.02 1.53 153.63 + MAL23c

[090] The ethanol titer of strain RHY723 was 0.83% higher than strain GPY10008, demonstrating that MAL23c expression increases ethanol production even in yeast that also harbor the PKL pathway and do not express D1s1.

Claims (15)

What is claimed is:

1. A method for increasing the amount of alcohol produced from fermentation of a starch hydrolysate and/or increasing the rate of production of alcohol from fermentation of a starch hydrolysate, comprising fermenting the starch hydrolysate with modified yeast harboring a constitutive transcriptional activator MAL allele, where in the modified yeast produces an increased amount of ethanol at the end of fermentation or an increased amount of ethanol over a period of time compared to the amount of ethanol produced by an otherwise-identical parental yeast.

2. The method of claim 1, wherein at least a portion of the increased amount of ethanol cannot be attributed to maltose fermentation based on carbon flux through a maltose metabolic pathway.

3. The method of claim 1 or 2, wherein the level of maltose in the starch hydrolysate at the end of fermentation is about the same as the level of maltose in the starch hydrolysate at the beginning of fermentation,

4. The method of any of the preceding claims, wherein the amount of maltose in the starch hydrolysate at the beginning of fermentation is no greater than 10 g/L.

5. The method of any of the preceding claims, wherein the yeast harboring a constitutive transcriptional activator M4L allele further comprises a genetic alteration that introduces a polynucleotide encoding a polypeptide in the phosphoketolase pathway.

6. The method of any of the preceding claims, wherein the yeast harboring a constitutive transcriptional activator M4L allele further comprises an alteration in the glycerol pathway and/or the acetyl-CoA pathway.

7. The method of any of the preceding claims, wherein the yeast harboring a constitutive transcriptional activator M4L allele further comprises disruption of a gene encoding DLs1.

8. The method of any of the preceding claims, wherein the yeast harboring a constitutive transcriptional activator M4L allele comprises an exogenous gene encoding a carbohydrate processing enzyme.

9. The method of any of the preceding claims, wherein the yeast is a Saccharomyces spp.

10. Modified yeast comprising a constitutive transcriptional activator MAL
allele, and at least one additional genetic modification not associated with maltose metabolism, which yeast produces during fermentation an increased amount of ethanol at the end of fermentation compared to the amount produced by an otherwise identical parental yeast when grown in a starch hydrolysate.

11. The modified yeast of claim 10, wherein the yeast further comprises a genetic alteration that introduces a polynucleotide encoding a polypeptide in the phosphoketolase pathway.

12. The modified yeast of claim 10 or 11, wherein the yeast further comprises an alteration in the glycerol pathway and/or the acetyl-CoA pathway.

13. The modified yeast of any of claims 10-12, wherein the yeast further comprises a disruption of a gene encoding DLs1.

14. The modified yeast of any of claims 10-13, wherein the yeast further comprises an exogenous gene encoding a carbohydrate processing enzyme.

15. The modified yeast of any of claims 10-14, wherein the yeast is a Saccharomyces spp.