CA3073387A1 - Modified yeast comprising glucose-specific, atp-mediated transporters - Google Patents

Abstract

Described are compositions and methods relating to modified yeast expressing exogenous, or increased amounts, of glucose-specific, ATP-mediated transporters. The modified yeast produces an increased amount of ethanol compared to parental cells. Such yeast is particularly useful for large-scale ethanol production from starch substrates.

Description

MODIFIED YEAST COMPRISING GLUCOSE-SPECIFIC, ATP-MEDIATED
TRANSPORTERS
TECHNICAL FIELD
[01] The present compositions and methods relate to modified yeast harboring glucose-specific, ATP-mediated transporters. The modified yeast produces an increased amount of ethanol in a starch-hydrolysate-fermentation compared to otherwise identical yeast. 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 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 will have massive economic impact across the industry.

[03] Biochemically, the conversion of glucose to ethanol and carbon dioxide is redox-neutral, with the maximum theoretical yield being about 51%. However, during yeast growth, there is surplus of NADH that is used to produce glycerol for redox balance and osmotic protection. The current industrial yield of yeast fermentation from corn mash for ethanol production is about 45%, thus, there is opportunity to increase ethanol yield by another 10%, which can translate into an additional nine billion liters of ethanol. Apart from the unavoidable carbon dioxide, yeast biomass and glycerol are the two major by-products of this fermentation process.

[04] Monosaccharides and other simple sugars are the favored carbon and energy sources of Saccharomyces cerevisiae (Leandro, M.J., etal. (2009) FEMS Yeast Res. 9:
511-25).
Many monosaccharide transporters operate by facilitated diffusion to move such molecules along a favorable concentration gradient across the cell membrane, in an energy-independent manner. Other monosaccharide transporters are energy-consuming, allowing the uptake of sugar molecules against a concentration gradient, coupled with the simultaneous movement of a proton. Such transporters usually operate only when limited sugar is present in a growth medium. The uptake of hexoses in S. cerevisiae occurs only through facilitated diffusion (Lagunas, R. (1993) FEMS Microbiol. Rev. 10:229-42) mediated by about twenty transporters, including the Hxt proteins, with different kinetic properties and modes of regulation (Reifenberger, E. etal. (1997) Eur. I Biochem. 245:324-33). Sugar uptake has a significant effect on metabolic flux (Elbing, K.D. etal. (2004) Eur. 1 Biochem. 271:4855-64), impacting cell growth, catabolic repression, fermentation and respiration (Goffrini et al.
(2002)1 Bacteriol. 184:427-32; Barnett, J.A. and Entian, K.D. (2005) Yeast 22:835-94).

[05] Engineering yeast for free energy conservation is one of the strategies to increase ethanol yield (Gombert and van Maris, supra). Yeast growth requires energy in the form of ATP. If the ATP consumption could be increased, it could increase ethanol yield on sugar, because more sugar would have to be converted to ethanol and carbon dioxide to provide the same amount of ATP for cellular maintenance, assuming that the increased fraction of the sugar is converted to ethanol, and not biomass and glycerol. It has been reported that overexpression of vacuole alkaline phosphatase (Ph08) increased ethanol yield (Semkiv, M.V. etal. (2014) BMC Biotechnol. 14:42). However, the challenge with the introduction of such non-stoichiometric ATP drains, especially for industrial implementation, is in the fine tuning between the positive impact and decreased cellular robustness (Gombert and van Mar, supra).

[06] S. cerevisiae strain has been engineered with an ATP-mediated sucrose transporter.
In the engineered strains, the ATP requirement for proton extrusion decreases the anaerobic ATP yield on sucrose, which results in increased ethanol yield (Basso, T.O.
etal. (2011)Met Eng. 13:694-703). However, in the current ethanol production industry, sucrose is not the major substrate in USA.

[07] The opportunity continues to exist for linking the concept of free energy conservation with the increasing sugar uptake to increase ethanol yield.
SUMMARY

[08] This invention provides compositions and methods for increasing ethanol production in yeast by expressing glucose-specific, ATP-mediated transporters. 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 an increased amount of a glucose-specific, ATP-mediated transporter compared to the parental cells, wherein the modified cells produce during fermentation an increased amount of ethanol compared to the amount of ethanol produced by the parental cells under identical fermentation conditions.
2. In some embodiments of the modified cells of paragraph 1, the genetic alteration comprises the introduction into the parental cells of a nucleic acid capable of directing the expression of a glucose-specific, ATP-mediated transporter to a level above that of the parental cell grown under equivalent conditions.
3. In some embodiments of the modified cells of paragraph 1 or 2, the genetic alteration comprises the introduction of an expression cassette for expressing a glucose-specific, ATP-mediated transporter.
4. In some embodiments of the modified cells of paragraph 1 or 2, the genetic alteration comprises the introduction of an exogenous gene encoding a glucose-specific, ATP-mediated transporter.
5. In some embodiments of the modified cells of paragraph 4, the exogenous gene is from an organism selected from the group consisting ofArabidopsis thaliana, Arabidopsis lyrate, Arabis alpine, Brassica rapa, Capsella rubella, Camelina sativa, and Eutreme salsugineus.
6. In some embodiments of the modified cells of paragraph 4 or 5, the exogenous gene is selected from the group consisting ofAtSTP9, AaSTP9S, A1STP9S, BrSTP9S, CrSTP9S, CsSTP9S, and EsSTP9S.
7. In some embodiments of the modified cells of paragraph 1, the genetic alteration comprises the introduction of a stronger promoter in an endogenous gene encoding a glucose-specific, ATP-mediated transporter.
8. In some embodiments of the modified cells of paragraphs 1-7, the amount of increase in ethanol he production is at least about 2% compared to the level in the parental cells grown under equivalent conditions.

9. In some embodiments, the modified cells of paragraphs 1-8 further comprise a genetic alteration that introduces one or more polynucleotides encoding a polypeptide of an exogenous phosphoketolase pathway.

10. In some embodiments of the modified cells of paragraphs 1-9, the cells further comprise an exogenous gene encoding a carbohydrate processing enzyme.

11. In some embodiments the modified cells of any of paragraphs 1-10 further comprise an alteration in the glycerol pathway and/or the acetyl-CoA pathway.

12. In some embodiments of the modified cells of paragraphs 1-11, the cells are of a Saccharomyces spp.

13. In another aspect, a method for increasing the amount of ethanol produced by cells grown on a starch-hydrolysate substrate is provided, comprising: introducing into parental yeast cells a genetic alteration that increases the production of glucose-specific, ATP-mediated transporter polypeptides compared to the amount produced in the parental cells.

14. In some embodiments of the method of paragraph 13, the cells do not produce a significant amount of additional glycerol compared to the amount produced by the parental cells.

15. In some embodiments of the method of paragraph 13 or 14, the increased production of the glucose-specific, ATP-mediated transporter polypeptides increases ATP
consumption in the cells.

16. In some embodiments of the method of any of paragraphs 13-15, the starch-hydrolysate comprises at least 5 g/L glucose.

17. In some embodiments of the method of any of paragraphs 13-16, the cells having the introduced genetic alteration are the modified cells of any of paragraphs 1-12.
[09] These and other aspects and embodiments of present modified cells and methods will be apparent from the description, including the accompanying Figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[010] Figure 1 is a map of an AtSTP9S expression cassette.
[011] Figure 2 is a map of plasmid pZK41Wn-H3SP9.
DETAILED DESCRIPTION
I. Definitions [012] 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.
[013] 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.

[014] 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.
[015] 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.
[016] 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.
[017] 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.

[018] 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).

[019] 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. App!. Math.
2:482;
Needleman and Wunsch (1970)1 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. (1984) Nucleic Acids Res. 12:387-95).

[020] 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)1 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 etal. ((1990) 1 Mol. Biol. 215:403-10) and Karlin etal.
((1993) Proc. Natl. Acad. Sci. USA 90:5873-87). One particularly useful BLAST
program is the WU-BLAST-2 program (see, e.g., Altschul etal. (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.

[021] 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 etal. (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: TUB
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

[022] 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).

[023] 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."

[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, "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.

[026] 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).

[027] 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.

[028] 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.

[029] 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.

[030] 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.

[031] 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.

[032] 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.

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

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

[035] 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 I% additional alcohol.

[036] 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.

[037] 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 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 1-1,g microgram pL and pl microliter pM micromolar U/g units/gram SAPU spectrophotometric acid protease unit SSU soluble starch unit Modified yeast harboring a glucose-specific, ATP-mediated transporter

[038] The present compositions and methods are based on the discovery that modified yeast over-expressing a glucose-specific, ATP-mediated transporter produces more ethanol in a starch-hydrolysate-fermentation than an otherwise identical parental yeast.
Without being limited to a theory, it is believed that the introduction of an ATP-mediated transporter affects both glucose uptake and ATP consumption, resulting in an overall increase in the desirable products of yeast metabolism.

[039] Previous attempts to modify yeast to increase ethanol production have involved monosaccharide transporters that uptake monosaccharide via facilitated diffusion, which is energy (ATP)-independent. In addition, previous studies have shown that over-expression of facilitative yeast monosaccharide transporters increases sugar uptake and the ethanol production rate, but not the ultimate ethanol production yield. Accordingly, the present modified yeast and methods are clearly distinguishable from previously described yeast in that they do involve passive transporters and they affect ethanol yield, not merely rate of production.

[040] In some embodiments of the present composition and methods, the glucose-specific, ATP-mediated transporter is an endogenous transporter caused to be overexpressed by introducing into the parental yeast a nucleic acid sequence encoding an addition copy of the transporter, for example, in the form of an expression cassette. In some embodiments, the glucose-specific, ATP-mediated transporter is an endogenous transporter caused to be overexpressed by modifying the endogenous gene in the parental yeast, such as by introducing a stronger promoter.

[041] In some embodiments of the present composition and methods, the glucose-specific, ATP-mediated transporter is from Arabidopsis thaliana, Arabidopsis lyrate, Arabis alpine, Brassica rapa, Capsella rubella, Camelina sativa, Eutreme salsugineus, or a related organism. In some embodiments, the glucose-specific, ATP-mediated transporter is encoded by a gene selected from AtSTP9, AaSTP9S, A1STP9S, BrSTP9S, CrSTP9S, CsSTP9S, and EsSTP9S. In some embodiments, the glucose-specific, ATP-mediated transporter is AtSTP9, AaSTP9S, A1STP9S, BrSTP9S, CrSTP9S, CsSTP9S, EsSTP9S, a structurally or functionally similar protein or a homologous protein.

[042] In some embodiments, the glucose-specific, ATP-mediated transporter is from Arabidopsis thaliana, for example, the glucose-specific, ATP-mediated transporter having the amino acid sequence of SEQ ID NO: 2. In some embodiments the amino acid sequences of the glucose-specific, ATP-mediated transporter has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% amino acid sequence identity to SEQ ID NO: 2.

[043] In some embodiments, the presence of a glucose-specific, ATP-mediated transporter expression cassette results in an increase in ethanol production, of at least 0.5%, at least 1.0%, at least 1.5%, at least 2.0%, at least 2.1%, at least 2.5%, at least 3.0%, at least 4.0%, at least 4.5%, at least 4.8%, or more. In some embodiments, the presence of a glucose-specific, ATP-mediated transporter expression cassette does not additionally result in a significant increase in glycerol production, for example, a less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or even less than 1% increase in glycerol production. In some embodiments, the presence of a glucose-specific, ATP-mediated transporter expression cassette does not additionally result in a significant increase in acetate production, for example, a less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or even less than 1% increase in acetate production. In some embodiments, the presence of a glucose-specific, ATP-mediated transporter expression cassette results in a decrease in acetate production.

[044] In some embodiments, the present modified yeast and methods are associated with fermentations involving high-dissolved-solids and high glucose concentrations, i.e., conditions under which glucose-specific, ATP-mediated transporters do not typically play a role in yeast metabolism. In some embodiments, the glucose concentration is at least 5 g/L, at least 10 g/L, at least 20 g/L, at least 30 g/L, at least 40 g/L, at least 50 g/L, at least 60 g/L, at least 70 g/L, at least 80 g/L, at least 90 g/L, or even at least 100 g/L.
Peak glucose levels during fermentation are typically 100-120 g/L (10-12% w/v).
III. Additional mutations that affect alcohol production

[045] 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 GPP 2. See, e.g.,U U.S. Patent Nos. 9,175,270 (Elke etal.), 8,795,998 (Pronk etal.) and 8,956,851 (Argyros etal.).

[046] 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.: WP 013718460). 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 does not have increased acetyl-CoA synthase.

[047] 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 etal.). In some embodiments, the present yeast does not have a heterologous gene encoding an NAD+-dependent acetylating acetaldehyde dehydrogenase and/or encoding a pyruvate-formate lyase.

[048] 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.

[049] 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 YMR226C. In other embodiments, the present modified yeast cells do not further comprise a butanol biosynthetic pathway.

[050] The present modified yeast may include any number of additional genes of interest encoding proteins 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 (3-glucanase, a phosphatase, a protease, an a-amylase, a (3-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.
IV. Use of the modified yeast for increased alcohol production

[051] 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.
V. Yeast suitable for modification

[052] 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, a-amylase and cellulase.
VI. Substrates and products

[053] 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.

[054] 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:

[055] Liquefact (corn flour slurry) was prepared by adding 600 ppm of urea, 0.124 SAPU/g ds FERMGEN (acid fungal protease) 2.5x, 0.33 GAU/g ds TrGA (Trichoderma glucoamylase) and 1.46 SSU/g ds AKAA (Asper gillus a-amylase), adjusted to a pH of 5.4.
Serum vial assays:

[056] 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:

[057] 300 [1.1_, 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:

[058] 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 1.1.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 ill injection volume.
Calibration standards were used for quantification of the of acetate, ethanol, glycerol, and glucose. All values are expressed in g/L.

Example 2 Constructs for over-expression of a codon-optimized, glucose-specific, ATP-mediated transporter

[059] The gene for glucose-specific, ATP-mediated transporter 9 from Arabidopsis thaliana (AtSTP9) was codon optimized and then synthesized to generate AtSTP9S. The nucleotide and amino acid sequences of AtSTP9S and its expression product, AtSTP9S, are shown, below, as SEQ ID NO: 1 and SEQ ID NO 2, respectively.

[060] Nucleotide sequence of the AtSTP9S gene (SEQ ID NO: 1):
ATGGCTGGTGGTGCCTTTGTCTCCGAAGGTGGCGGTGGAGGCAACTCTTACGAAGGTGGCGT
TACCGTCTTTGTTATCATGACCTGTATTGTTGCCGCTATGGGAGGTTTGCTATTTGGTTACG
ACTTGGGTATCTCTGGCGGTGTCACCTCTATGGAAGAGTTCTTGTCCAAGTTTTTCCCAGAA
GTTGACAGACAAATGCACGAAGCCAGACGTGAAACTGCTTACTGCAAGTTCGATAACCAATT
GCTACAATTGTTCACCTCTTCCTTGTACTTGGCTGCCTTAGTCTCTTCCTTTGTTGCTTCTG
CTGTCACCAGAAAGTACGGTAGAAAGATTTCCATGTTTGTTGGTGGCGTCGCTTTCTTGATC
GGTTCTTTGTTCAACGCCTTTGCTACCAACGTTGCTATGTTGATCATTGGTAGATTGCTATT
GGGTGTCGGCGTCGGTTTTGCTAATCAATCTACTCCAGTTTACTTGTCCGAAATGGCTCCAG
CCAAGATCAGAGGTGCTTTGAACATCGGTTTTCAAATGGCTATCACCATTGGTATCTTGGTT
GCCAATTTGATCAACTACGGTACTTCTCAAATGGCTAGAAACGGTTGGAGAGTCTCCTTGGG
TTTAGCTGCCGTTCCAGCTGTCGTTATGGTCATCGGTTCCTTTGTCTTGCCAGACACTCCCA
ACTCTATGTTGGAAAGAGGCAAGTACGAACAAGCTAGAGAAATGTTGCAAAAGATTCGTGGT
GCTGACAACGTTGATGAAGAGTTTCAAGACTTGTGTGATGCTTGCGAAGCTGCCAAGAAAGT
CGAAAACCCTTGGAAGAACATCTTTCAACACGCCAAGTACAGACCAGCTTTGGTTTTCTGTT
CTGCTATTCCATTCTTTCAACAGATCACTGGTATCAACGTCATCATGTTTTACGCTCCAGTT
TTGTTCAAGACTTTGGGTTTTGCCGACGATGCTTCTTTGATTTCCGCTGTCATCACTGGTGC
TGTCAATGTTGTCTCTACCTTGGTTTCCATCTACGCTGTTGACAGATACGGTAGACGTATCT
TGTTCTTAGAAGGTGGCATTCAAATGATCATTAGCCAAATCGTTGTCGGTACCTTGATCGGT
ATGAAGTTTGGCACCACTGGTTCTGGCACCTTGACTCCAGCTACAGCCGACTGGATTTTGGC
TTTCATCTGTTTGTACGTTGCTGGATTTGCCTGGTCTTGGGGTCCATTGGGTTGGCTAGTTC
CATCCGAAATCTGTCCATTGGAAATCAGACCAGCTGGTCAAGCCATCAACGTTTCTGTCAAC
ATGTTCTTTACCTTCTTGATTGGTCAATTTTTCTTGACTATGTTGTGTCACATGAAGTTTGG
TTTGTTTTACTTCTTTGGTGGAATGGTTGCTGTCATGACTGTCTTTATCTACTTCTTGTTAC
CAGAAACCAAGGGTGTTCCTATCGAAGAGATGGGCAGAGTCTGGAAGCAACACCCATTCTGG

AAGAGATACATTCCAGACGATGCTGTTATCGGTGGCGGTGAAGAGAACTACGTCAAGGAAGT
TTAA

[061] Amino acid sequence of the AtSTP9S polypeptide (SEQ ID NO: 2):
MAGGAFVSEGGGGGNSYEGGVTVFVIMTCIVAAMGGLL FGYDLGI S GGVT SMEE FL S KFFPE
VDRQMHEARRETAYCKFDNQLLQL FT S S L YLAALVS S FVASAVT RKYGRKI SMFVGGVAFL I
GS L FNAFATNVAML I I GRLLLGVGVGFANQST PVYLSEMAPAKIRGALNIGFQMAIT I GILV
ANL I NYGT S QMARNGWRVS LGLAAVPAVVMVI G S FVL P DT PNSMLERGKYEQAREMLQKIRG
ADNVDEEFQDLCDACEAAKKVENPWKNI FQHAKYRPALVFCSAI PFFQQITGINVIMFYAPV
L FE= GFADDAS L I SAVITGAVNVVSTLVS I YAVDRYGRRIL FLEGGIQMI I S QIVVGTL I G
MKFGT TGS GT LT PATADWI LAFICLYVAG FAWSWGPLGWLVP S E IC PLE IRPAGQAINVSVN
MFFT FL I GQFFLTMLCHMKFGL FY FFGGMVAVMTVFIY FLLPETKGVP IEEMGRVWKQHPFW
KRY I PDDAVIGGGEENYVKEV

[062] An AtSTP9S expression cassette consisting of a synthetic AtSTP9S (SEQ ID
NO: 1) under control of a HXT3 promoter (SEQ ID NO: 3; YDR345C) and FBAlterminator (SEQ
ID NO: 4; YKL060C), was made using standard procedures.

[063] The nucleotide sequence of the HXT3 promoter is shown, below, as SEQ ID
NO 3:
G GAG GAG GAG CAAT GAAAT GAAAG GAAAAAAAAT ACT T T CT TTTT CT T GAAAAAAGAAAAAA
ATTGTAAGATGAGCTATTCGCGGAACATTCTAGCTCGTTTGCATCTTCTTGCATTTGGTTGG
T ITT CAATAGTT CGGTAATAT TAACGGATACCTACTAT TAT C CCCTAGTAGGCT CTT TT CAC
GGAGAAATTCGGGAGTGCTTTTTTTCCGTGCGCATTTTCTTAGCTATATTCTTCCAGCTTCG
CCTGCTGCCCGGTCATCGTTCCTGTCACGTAGTTTTTCCGGATTCGTCCGGCTCATATAATA
CCGCAATAAACACGGAATATCTCGTTCCGCGGATTCGGTTAAACTCTCGGTCGCGGATTATC
ACAGAGAAAGCTTCGTGGAGAATTTTTCCAGATTTTCCGCTTTCCCCGATGTTGGTATTTCC
GGAGGT CATTATACT GACC GCCAT TATAAT GACT GTACAACGACCTT CT GGAGAAAGAAACA
ACT CAATAAC GAT GT GGGACATT GGAGGC CCACT CAAAAAAT CT GGGGACTATAT CC CCAGA
GAATT T CT CCAGAAGAGAAGAAAAGT CAAAGTT TTTTT CACT T GGGGGTT GCATATAAATAC
AGGCGCTGTTTTATCTTCAGCATGAATATTCCATAATTTTACTTAATA

[064] The nucleotide sequence of the FBA1 terminator is shown, below, as SEQ
ID NO 4:
GTTAATT CAAATTAATT GATATAGTTTTT TAAT GAGTATT GAAT CT GT TTAGAAATAAT GGA
ATATTATTTTTATTTATTTATTTATATTATTGGTCGGCTCTTTTCTTCTGAAGGTCAATGAC
AAAAT GATAT GAAG GAAATAAT GAT T T CTAAAAT T T TACAAC GTAAGATAT T TTTACAAAAG

CCTAGCTCATCTTTTGTCATGCACTATTTTACTCACGCTTGAAATTAACGGCCAGTCCACTG
CGGAGTCATTTCAAAGTCATCCTAATCGATCTATCGTTTTTGATAGCTCATTTTGGAG

[065] A representation of the AtSTP9S expression cassette is illustrated in Figure 1.
Plasmid pK41Wn-H3SP9, shown in Figure 2, includes an integrated AtSTP9S
expression cassette downstream of the Saccharomyces chromosome YHL041W locus. The functional and structural composition of plasmid pK41Wn-H3SP9 is described in Table 1.
Table 1. Functional and structural elements of plasmid pZK41Wn- H3SP9 Functional/Structural element Description "YHL041W3" fragment, 78-bp DNA fragment (labeled as YHL041W3' in downstream of YHL041W locus Figure 2) from S. cerevisiae "YHL041WM" fragment, 80-bp DNA fragment (labeled as YHL041WM in downstream of YHL041W locus Figure 2) from S. cerevisiae ColE1 replicon and ampicillin These sequences are not part of the DNA
fragment resistance marker gene integrated into yeast genome "YHL041W5" fragment, 76-bp DNA fragment (labeled as YHL041W5' in downstream of YHL041W locus Figure 2) HXT3 Promoter::AtSTP9s:: Cassette for expression of codon-optimized FBA1 Terminator AtSTP9s encoding ATP-mediated glucose specific transporter 9, derived from A. thaliana

[066] To facilitate the integration of the Swal fragment from plasmid pZK41Wn-into the downstream region of the YHL041W locus in yeast, a plasmid (pYRH426;
not shown) was constructed to express the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) associated protein 9 (Cas9) and an sgRNA specific for downstream of the YHL041W locus specific using the sequence 5'-GCTGATAATACGCTAAACGA-3';
SEQ ID NO: 5).
Example 3 Generation of a yeast strain expressing a glucose-specific, ATP-mediated transporter

[067] The FERMAXO GOLD yeast strain (Martrex, Inc., MD, USA; herein, "FG") was used as a parental strain to introduce the AtSTP9S expression cassette. Cells were transformed with a 2,949-bp Swal fragment containing the AtSTP9S expression cassette from plasmid pZK41Wn-H35P9 (Figure 2) and plasmid pYRH426. One transformant with the Swal fragment from pZK41Wn-H35P9 integrated at the downstream of YHL041W locus was selected and designated as strain G027.

[068] The new FG yeast strain, G027, and its parent strain, FG, 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 3.
Table 3. FG versus G027 in vial assays Strain Transgene(s) Et0H Glycerol Acetate expressed FG none 142.27 17.17 0.93 G027 AtSTP9s 145.19 17.06 0.92

[069] Strain G027 produced about 2% more ethanol than the parental strain and produced similar amounts of glycerol and acetate.

[070] To confirm the performance of the modified yeast, strains G027 and FG
were analyzed in AnKom assays, as described in Example 1. Performance in terms of ethanol, glycerol and acetate production is shown in Table 4.
Table 4. FG versus G027 in AnKom assays Strain Transgene(s) Et0H Glycerol Acetate expressed FG none 138.82 16.01 0.81 G027 AtSTP9s 141.75 16.07 0.82

[071] The increase in ethanol production with the G027 was 2.1% compared to the parent, FG, confirming the results of vial assays (Table 3). The production of glycerol and acetate were similar to the parental yeast.
Example 4 Identification of Homologs of AtSTP9

[072] AtSTP9 encodes a glucose-specific, ATP-mediated transporter that belongs to the major facilitator superfamily. Like other sugar transporters, AtSTP9 shares conserved structures, including twelve transmembrane domains and five sequence motifs (Leandro et al.
(2009) Yeast Res. 9: 511-25). Using the amino acid sequence of AtSTP9 as a query, six homologs with more than 93% identity were found in public databases. The homologs are listed in Table 5.

Table 5. AtSTP9 and homologs from public databases Gene Name Source % Identity with AtSTP9 GenBank Accession No.
AtSTP9 Arabidopsis thaliana (100%) NP 175449 AaSTP9 Arabis alpina 94.4% KFK35926 A1STP9 Arabidopsis lyrate 98.1% 0JJ99073 BrSTP9 Brassica rapa 93.2% XP 009147845 CrSTP9 Capsella rubella 94.8% XM 006305941 CsSTP9 Camelina sativa 95.2% XM 010463575 EsSTP9 Eutreme salsugineum 93.8% XM 006393101 Example 5 Constructs for expressing of codon-optimized, glucose-specific, ATP-mediated transporter homologs

[073] The six selected homologs of AtSTP9 (Table 5) were codon-optimized and then synthesized to generate AaSTP9S, A1STP9S, BrSTP9S, CrSTP9S, CsSTP9S, and EsSTP9S
genes.

[074] As was the case with the AtSTP9S expression cassette, the AtSTP9S
homolog expression cassettes were under the control of HXT3 promoter (SEQ ID NO: 3;
YDR345C) and FBAlterminator (SEQ ID NO: 4; YKL060C) and integrated into a suitable plasmid for propagation.
Example 6 Generation of AtSTP9 homolog strains from industrial yeast FERMAXTm Gold

[075] The FG strain was used as a parent to introduce the AaSTP9S, A1STP9S, BrSTP9S, CrSTP9S, CsSTP9S, and EsSTP9S expression cassettes, as described in Example 2.
Yeast were transformed with Swal fragments that individually contained the AaSTP9S, A1STP9S, BrSTP9S, CrSTP9S, CsSTP9S, and EsSTP9S expression cassettes along with plasmid pYRH426. One transformant from each transformation was selected and designated as strain G304, G286, G293, G296, G300, and G303, respectively.

[076] The new FG yeast strains and the parent strain, FG, 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 G286, G293, G296, G300, G303 and G304 in vial assays Strain Transgene expressed Et0H Glycerol Acetate FG none 135.87 17.21 0.60 G286 AlSTP9S 141.19 18.98 0.81 G293 BrSTP9S 141.49 18.91 0.83 G296 CaSTP9S 138.48 19.42 0.69 G300 CrSTP9S 139.50 18.97 0.72 G303 EsSTP9S 138.24 19.11 0.80 G304 AaSTP9S 138.07 18.91 0.74

[077] Each of the new FG yeast strains produced more ethanol than the FG
parent, which is desirable in terms of performance, however, all the new FG strains produced more glycerol and acetate, which are generally not desirable products.

[078] To confirm the performance of new FG strains, strains G286, G293, G296, G300, G303, G304, along with FG and G027 (from Example 3), were analyzed in AnKom assays, as described in Example 1. Performance in terms of ethanol, glycerol and acetate production is summarized in Table 7.
Table 7. FG versus G027, G286, G293, G296, G300, G303 and G304 in AnKom assays Strain Transgene expressed Et0H Glycerol Acetate FG none 135.51 16.27 0.68 G027 AtSTP9S 138.50 16.43 0.65 G286 AlSTP9S 142.53 18.23 0.75 G293 BrSTP9S 142.27 18.19 0.77 G296 CaSTP9S 142.58 17.06 0.79 G300 CrSTP9S 142.06 18.11 0.76 G303 EsSTP9S 143.16 18.15 0.79 G304 AaSTP9S 142.96 18.20 0.75

[079] As demonstrated previously (e.g., Example 3, Table 4), the increase in ethanol production with the G027 was about 2% compared to the parent, FG, without the production of a significant additional amount of glycerol and/or acetate. The increases in ethanol production with the G286, G293, G296, G300, G303 and G304 strains was more than 4.8%
compared to the parent but was accompanied by the production of significantly more glycerol and acetate.

Claims (17)

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 an increased amount of a glucose-specific, ATP-mediated transporter compared to the parental cells, wherein the modified cells produce during fermentation an increased amount of ethanol compared to the amount of ethanol produced by the parental cells under identical fermentation conditions.

2. The modified cells of claim 1, wherein the genetic alteration comprises the introduction into the parental cells of a nucleic acid capable of directing the expression of a glucose-specific, ATP-mediated transporter to a level above that of the parental cell grown under equivalent conditions.

3. The modified cells of claim 1 or 2, wherein the genetic alteration comprises the introduction of an expression cassette for expressing a glucose-specific, ATP-mediated transporter.

4. The modified cells of claim 1 or 2, wherein the genetic alteration comprises the introduction of an exogenous gene encoding a glucose-specific, ATP-mediated transporter.

5. The modified cells of claim 4, wherein the exogenous gene is from an organism selected from the group consisting of Arabidopsis thaliana, Arabidopsis lyrate, Arabis alpine, Brassica rapa, Capsella rubella, Camelina sativa, and Eutreme salsugineus .

6. The modified cells of claim 4 or 5, wherein the exogenous gene is selected from the group consisting of AtSTP9, AaSTP9S, AlSTP9S, BrSTP9S, CrSTP9S, CsSTP9S, and EsSTP9S.

7. The modified cells of claim 1, wherein the genetic alteration comprises the introduction of a stronger promoter in an endogenous gene encoding a glucose-specific, ATP-mediated transporter.

8. The modified cells of any of claims 1-7, wherein the amount of increase in ethanol he production is at least about 2% compared to the level in the parental cells grown under equivalent conditions.

9. The modified cells of any of claims 1-8, further comprising a genetic alteration that introduces one or more polynucleotides encoding a polypeptide of an exogenous phosphoketolase pathway.

10. The modified cells of any of claims 1-9, wherein the cells further comprise an exogenous gene encoding a carbohydrate processing enzyme.

11. The modified cells of any of claims 1-10, further comprising an alteration in the glycerol pathway and/or the acetyl-CoA pathway.

12. The modified cells of any of claims 1-11, wherein the cells are of a Saccharomyces spp.

13. A method for increasing the amount of ethanol produced by cells grown on a starch-hydrolysate substrate, comprising: introducing into parental yeast cells a genetic alteration that increases the production of glucose-specific, ATP-mediated transporter polypeptides compared to the amount produced in the parental cells.

14. The method of claim 13, wherein the cells do not produce a significant amount of additional glycerol compared to the amount produced by the parental cells.

15. The method of claim 13 or 14, wherein the increased production of the glucose-specific, ATP-mediated transporter polypeptides increases ATP consumption in the cells.

16. The method of any of claims 13-15, wherein the starch-hydrolysate comprises at least 5 g/L glucose.

17. The method of any of claims 13-16, wherein the cells having the introduced genetic alteration are the modified cells of any of claims 1-12.