WO2021022097A1

WO2021022097A1 - Over-expression of adh5p for increased ethanol production by yeast

Info

Publication number: WO2021022097A1
Application number: PCT/US2020/044359
Authority: WO
Inventors: Courtney BEMENT; Daniel Joseph Macool; Yehong Jamie Wang; Quinn Qun Zhu
Original assignee: Danisco Us Inc
Priority date: 2019-08-01
Filing date: 2020-07-30
Publication date: 2021-02-04
Also published as: BR112022001859A2

Abstract

Described are compositions and methods relating to modified yeast that over-expresses Adh5p and harbor a heterologous phosphoketolase pathway. The yeast demonstrates increased ethanol production from glucose compared to parental cells. Such yeast is particularly useful for large-scale ethanol production from starch substrates.

Description

OVER-EXPRESSION OF ADH5P FOR INCREASED

ETHANOL PRODUCTION BY YEAST

TECHNICAL FIELD

[01] The present compositions and methods relate to modified yeast cells that over- expresses the protein Adh5p and harbor a heterologous phosphoketolase pathway. The yeast cells demonstrate increased ethanol production from glucose compared to parental cells.

Such yeast cells are particularly useful for large-scale ethanol production from starch substrates.

BACKGROUND

[02] First-generation yeast-based ethanol production converts sugars into fuel ethanol.

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

[03] Ethanol production in engineered yeast cells with a heterologous phosphoketolase (PKL) pathway is higher than in a parental strain without a PKL pathway (see, e.g.,

WO2015148272; Miasnikov et al ). The PKL pathway consists of phosphoketolase (PKL) and phosphotransacetylase (PTA) to channel carbon flux away from the glycerol pathway and toward the synthesis of acetyl-coA. Two supporting enzymes, acetaldehyde dehydrogenase (AADH) and acetyl-coA synthase (ACS), can help the PKL pathway be more effective.

[04] There is an ongoing need to improve the PKL pathway to further increase ethanol production yield.

SUMMARY

[05] The present compositions and methods relate to modified yeast that over-expresses Adh5p and harbor a heterologous phosphoketolase pathway. 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 Adh5p polypeptides compared to the parental cells, wherein the modified cells and the parental cells both further comprising an heterologous

phosphoketolase pathway, wherein the modified cells produce during fermentation more ethanol from glucose compared to the amount of ethanol produced by otherwise identical parental yeast cells.

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 an Adh5p polypeptide to a level above that of the parental cell grown under equivalent conditions.

3. In some embodiments of the modified cells of paragraph 1, the genetic alteration comprises the introduction of an expression cassette for expressing an Adh5p polypeptide.

4. In some embodiments of the modified cells of any of paragraphs 1-3, the amount of increase in the expression of the Adh5p polypeptide is at least about 500%, at least about 1,000%, at least about 5,000%, or at least about 10,000%, compared to the level expression in the parental cells grown under equivalent conditions.

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

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

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

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

9. In another aspect, a method for increased production of alcohol from yeast cells grown on a carbohydrate substrate is provided, comprising: introducing into parental yeast cells comprising a phophoketolase pathway a genetic alteration that increases the production of Adh5p polypeptides compared to the amount produced in the parental cells.

10. In some embodiments of the method of paragraph 9, the cells having the introduced genetic alteration are the modified cells are the cells of any of paragraphs 1-8.

11. In some embodiments of the method of paragraph 9 or 10, the increased production of alcohol is at least 0.2%, at least 0.5%, at least 0.8%. 12. In some embodiments of the method of any of paragraphs 9-11, Adh5p polypeptides are over-expressed by at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold.

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

DETAILED DESCRIPTION

I. Definitions

[07] 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.

[08] 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.

[09] 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.

[010] 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.

[Oil] 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.

[012] 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.

[013] 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).

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

[015] For example, PILEUP is a useful program to determine sequence homology levels. PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pair-wise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, (Feng and Doolittle (1987) J. Mol. Evol. 35:351-60). The method is similar to that described by Higgins and Sharp ((1989) CABIOS 5: 151-53). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps. Another example of a useful algorithm is the BLAST algorithm, described by Altschul et al. ((1990) J. Mol. Biol. 215:403-10) and Karlin et al. ((1993) Proc. Natl. Acad. Sci. USA 90:5873-87). One particularly useful BLAST program is the WU-BLAST-2 program (see, e.g., Altschul et al. (1996) Meth. Enzymol. 266:460-80). Parameters“W,”“T,” and“X” determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word-length (W) of 11, the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff ( 1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M'5, N'-4, and a comparison of both strands.

[016] 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 al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:

Gap opening penalty: 10.0

Gap extension penalty: 0.05

Protein weight matrix: BLOSUM series

DNA weight matrix: IUB

Delay divergent sequences %: 40

Gap separation distance: 8

DNA transitions weight: 0.50

List hydrophilic residues: GPSNDQEKR

Use negative matrix: OFF

Toggle Residue specific penalties: ON

Toggle hydrophilic penalties: ON

Toggle end gap separation penalty OFF

[017] 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).

[018] 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.”

[019] 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.

[020] As used herein,“over-expressing a polypeptide,”“increasing the expression of a polypeptide,” and similar terms, refer to expressing a polypeptide at higher-than-normal levels compared to those observed with parental or“wild-type cells that do not include a specified genetic modification.

[021] 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).

[022] 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.

[023] 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, a signal transducer, a receptor, a transporter, a transcription factor, a translation factor, a co-factor, 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.

[024] 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 CRISPR, RNAi, antisense, or any other method that abolishes gene expression. A gene can be disrupted by deletion or genetic manipulation of non-adjacent control elements. As used herein,“deletion of a gene,” refers to its removal from the genome of a host cell. Where a gene includes control elements (e.g. , enhancer elements) that 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. Deletion of a gene also refers to the deletion a part of the coding sequence, or a part of promoter immediately or not immediately adjacent to the coding sequence, where there is no functional activity of the interested gene existed in the engineered cell.

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

[026] 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, a signal transducer, a receptor, a transporter, a transcription factor, a translation factor, a co-factor, 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. [027] 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.

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

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

[030] 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:

°c degrees Centigrade

AA a-amylase

AADH acetaldehyde dehydrogenases

ADH alcohol dehydrogenase

bp base pairs

DNA deoxyribonucleic acid

ds or DS dry solids

EC enzyme commission

EtOH ethanol

g or gm gram

g/L grams per liter

GA glucoamylase

H2O water

HPLC high performance liquid chromatography

hr or h hour

kg kilogram

M molar

mg milligram

min minute mL or ml milliliter

mM millimolar

N normal

nm nanometer

PCR polymerase chain reaction

PKL phosphoketolase

ppm parts per million

PTA phosphotransacetylase

D relating to a deletion

mg microgram

mL and pi microliter

mM micromolar

II. Modified yeast cells over-expressing Adh5p

[031] Described are modified yeast cells and methods of use, thereof, involving a genetic alteration resulting in an increased in cellular Adh5p polypeptides compared to corresponding (i.e., otherwise-identical) parental cells harboring a heterologous phosphoketolase pathway.

[032] Engineered yeast cells having a heterologous PKL pathway have been previously described in WO2015148272 (Miasnikov et al.) and are commercially available. These cells express heterologous phosphoketolase (PKL), phosphotransacetylase (PTA) and acetylating acetyl dehydrogenase (AADH), optionally with other enzymes, to channel carbon flux away from the glycerol pathway and toward the synthesis of acetyl-CoA, which is then converted to ethanol.

[033] in Saccharomyces cerevisiae, there are five alcohol dehydrogenase genes, ADH1, ADH2, ADH3, ADH4 and ADH5. All of them are involved in ethanol metabolism. Adhip, encoded by ADH1 (YQL086C), is the enzyme primarily involved in catalyzing the reduction of acetaldehyde to produce ethanol (Ciriacy, M. (1979) Mol. Gen. Genet. 176: 427-31).

Adhlp is constitutively expressed; however, its biosynthesis can be completely or partially repressed by growth under conditions of extreme aerobiosis or during growth on

nonfermentabie substrates (Denis, C. L, et al, (1983) J. Biol. ( hem 258: 1165-71). Adh2p, encoded by ADH2 (YMR303C), is a glucose-repressibie alcohol dehydrogenase that catalyzes the reverse reaction of oxidizing ethanol to acetaldehyde (Mortimer, R.K. and Schild, D.D. (1985) Genetic map of Saccharomyces cerevisiae , edition 9. Microbiol Rev 49:181-213). Adh3p, encoded by ADH3 (YMR083W), is a nuclear-encoded mitochondria ADH (Young, E.T. and Pilgrim, D. (1985). Mol Cell Biol 5:3024-34). Adh4p, encoded by ADH4 (YGL256W), is a zinc-dependent alcohol dehydrogenase (Yuan, D.S. (2000) Genetics 156:45-58). Adh5p, encoded by Adh5p (YBR145W), is a paralog of Adhl that arose from the whole genome duplication, and is also involved in ethanol production (Smith, et al. (2004). Mol Cell. Biol 24:3874-84).

[034] Amino acid sequence comparisons show that Adh2P and Adh3p are more

homologous to AdhlP than Adh5p. Over-expression of Adh 1p has no effect in the aforementioned engineered yeast cells having a heterologous PKL pathway in terms of ethanol production (data not shown). It has now been shown that increased expression of Adh5p in these yeast results in increased production of alcohol compared to otherwise identical parental yeast harboring the PKL pathway but not over-expressing Adh5p.

[035] In some embodiments, the increase in the amount of Adh5p polypeptides produced by modified cells is an increase of at least 500%, at least 1,000%, at least 5,000%, or at least 10,000%, or more, compared to the amount of Adh5p polypeptides produced by parental cells grown under the same conditions.

[036] In some embodiments, the increase in the amount of Adh5p polypeptides produced by the modified cells is at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more, compared to the amount of Adh5p polypeptides produced by parental cells grown under the same conditions.

[037] In some embodiments, the increase in the strength of the promoter used to control expression of the Adh5p polypeptides produced by the modified cells is at least 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, or more, compared to strength of the native promoter controlling Adh5p expression.

[038] In some embodiments, the increase in ethanol production by the modified cells is an increase of at least 0.2%, at least 0.5%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0%, at least 1.1%, at least 1.2%, at least 1.3%, at least 1.4%, at least 1.5%, or more, compared to the amount of ethanol produced by parental cells grown under the same conditions.

[039] Preferably, increased Adh5p expression is achieved by genetic manipulation using sequence-specific molecular biology techniques, as opposed to chemical mutagenesis, which is generally not targeted to specific nucleic acid sequences. However, chemical mutagenesis is not excluded as a method for making modified yeast cells.

[040] In some embodiments, the present compositions and methods involve introducing into yeast cells a nucleic acid capable of directing the over-expression, or increased expression, of a Adh5p polypeptide. Particular methods include but are not limited to (i) introducing an exogenous expression cassette for producing the polypeptide into a host cell, optionally in addition to an endogenous expression cassette, (ii) substituting an exogenous expression cassette with an endogenous cassette that allows the production of an increased amount of the polypeptide, (iii) modifying the promoter of an endogenous expression cassette to increase expression, (iv) increase copy number of the same or different cassettes for over-expression of Adh5p, and/or (v) modifying any aspect of the host cell to increase the half-life of the Adh5 mRNA and/or polypeptide in the host cell.

[041] In some embodiments, the parental cell that is modified already includes a gene of interest, such as a gene encoding a selectable marker, carbohydrate-processing enzyme, or other polypeptide. In some embodiments, a gene of introduced is subsequently introduced into the modified cells.

[042] In some embodiments, the parental cell that is modified already includes an engineered pathway of interest, in addition to the PKL pathway, to increases ethanol production, or any other pathway to increase alcohol production.

[043] The amino acid sequence of the exemplified Adh5p polypeptide is shown, below, as SEQ ID NO: 2:

[045] In some embodiments of the present compositions and methods, the amino acid sequence of the Adh5p polypeptide that is over-expressed in modified yeast cells has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or even at least about 99% identity, to SEQ ID NO: 2.

[046] In further embodiments, the amino acid sequence of the Adh5p polypeptide corresponds to, or has, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or even at least about 99% identity, to a functionally or structurally equivently molecule, or a homolog of the Adh5p polypeptide.

III. Combination of increased Adh5p production with other mutations that affect alcohol production

[047] In some embodiments, in addition to expressing increased amounts of Adh5p polypeptides in combination with a heterologous PKL pathway, the present modified yeast cells include additional beneficial modifications.

[048] The modified cells may further include mutations that result in attenuation of the native glycerol biosynthesis pathway and/or reuse glycerol pathway, which are known to increase alcohol production. Methods for attenuation of the glycerol biosynthesis pathway in yeast are known and include reduction or elimination of endogenous NAD-dependent glycerol 3-phosphate dehydrogenase (GPD) or glycerol phosphate phosphatase activity (GPP), for example by disruption of one or more of the genes GPD 1. 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 ). Methods to enhance the reuse glycerol pathway by over expression of glycerol dehydrogenase (GCY1) and dihydroxyacetone kinase (DAK1) to convert glycerol to dihydroxyacetone phosphate (Zhang et al. (2013) J. Ind. Microbiol. Biotechnol. 40: 1153-60).

[049] 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 Ac-CoA. This partially reduces the undesirable effect of acetate on the growth of yeast cells and may further contribute to an improvement in alcohol yield. Increasing acetyl-CoA synthase activity may be accomplished by introducing a heterologous acetyl-CoA synthase gene into cells, increasing the expression of an endogenous acetyl-CoA synthase gene and the like.

[050] In some embodiments the modified cells may further include a heterologous gene encoding a protein with NAD⁺-dependent acetylating acetaldehyde dehydrogenase activity and/or a heterologous gene encoding a pyruvate-formate lyase. The introduction of such genes in combination with attenuation of the glycerol pathway is described, e.g., in U.S. Patent No. 8,795,998 (Pronk et al ). In some embodiments of the present compositions and methods the yeast expressly lacks a heterologous gene(s) encoding an acetylating acetaldehyde dehydrogenase, a pyruvate-formate lyase or both. [051] In some embodiments, the present modified yeast cells may further over-express a sugar transporter-like (STL1) polypeptide to increase the uptake of glycerol (see, e.g.,

Ferreira et al. (2005) Mol. Biol. Cell. 16:2068-76; Duskova et al. (2015) Mol. Microbiol. 97:541-59 and WO 2015023989 Al) to increase ethanol production and reduce acetate.

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

[053] In some embodiments, the modified yeast cells comprising a butanol biosynthetic pathway further comprise a modification in a polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In some embodiments, the yeast cells comprise a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In some embodiments, the polypeptide having pyruvate decarboxylase activity is selected from the group consisting of: PDC1, PDC5, PDC6, and combinations thereof. In some embodiments, the yeast cells further comprise a deletion, mutation and/or substitution in one or more endogenous polynucleotides encoding FRA2, ALD6, ADH1, GPD2, BDH1, DLS1, DPB3, CPR1, MAL23C, MNN4, PAB1, TMN2,

HAC1, PTC1, PTC2, OSM1, GIS1, CRZ1, HUG1, GDS1, CYB2P, SFC1, MVB12, LDB10, C5SD, GIC1, GIC2 and/or YMR226C. In some embodiments, the yeast cells over-express one or more of these polynucleotides.

IV. Combination of increased expression Adh5p with other beneficial mutations

[054] In some embodiments, in addition to increased expression of Adh5p polypeptides, in combination with the PKL pathway and other genetic modifications that benefit alcohol production and/or acetate reduction, the present modified yeast cells further include any number of additional genes of interest encoding proteins of interest. Additional genes of interest may be introduced before, during, or after genetic manipulations that result in the increased production of Adh5p polypeptides. Proteins of interest, include selectable markers, carbohydrate-processing enzymes, and other commercially -relevant polypeptides, including but not limited to an enzyme selected from the group consisting of a dehydrogenase, a transketolase, a phosphoketolase, a transladolase, an epimerase, a phytase, a xylanase, a b- glucanase, a phosphatase, a protease, an a-amylase, a b-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.

V. Use of the modified yeast for increased alcohol production

[055] The present compositions and methods include methods for increasing alcohol production and/or reducing glycerol 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 alcohol production, the present yeast can also be used for the production of potable alcohol, including wine and beer.

VI. Yeast cells suitable for modification

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

VII. Substrates and products

[057] Alcohol production from a number of carbohydrate substrates, including but not limited to com 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. [058] Alcohol fermentation products include organic compound having a hydroxyl functional group (-OH) is bound to a carbon atom. Exemplary alcohols include but are not limited to methanol, ethanol, «-propanol, isopropanol, «-butanol, isobutanol, «-pentanol, 2- pentanol, isopentanol, and higher alcohols. The most commonly made fuel alcohols are ethanol, and butanol.

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

EXAMPLES

Example 1

Materials and methods

Liquefact preparation:

[060] Liquefact (com mash slurry) was prepared by adding 600 ppm of urea, 0.124 SAPU/g ds acid fungal protease, 0.33 GAU/g ds variant Trichoderma reesei glucoamylase and 1.46 SSCU/g ds Aspergillus kawachii a-amylase, adjusted to a pH of 4.8 with sulfuric acid.

AnKom assays:

[061] 300 mL 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 55 hours.

HPLC analysis:

[062] Samples of the cultures from AnKom assays were collected in Eppendorf tubes by centrifugation for 12 minutes at 14,000 RPM. The supernatants were filtered using 0.2 mM PTFE filters and then used for HPLC (Agilent Technologies 1200 series) analysis with the following conditions: Bio-Rad Aminex HPX-87H columns, running at a temperature of 55°C with a 0.6 ml/min isocratic flow in 0.01 N H₂SO₄ and a 2.5 ml injection volume. Calibration standards were used for quantification of the of acetate, ethanol, glycerol, glucose and other molecules. Unless otherwise indicated, all values are reported in g/L.

RNA-Seq analysis:

[063] RNA was prepared from individual samples according to the TRIzol method (Life- Tech, Rockville, MD). The RNA was then cleaned up with Qiagen RNeasy Mini Kit (Qiagen, Germantown, MD). The cDNA from total mRNA in individual samples was generated using Applied Biosystems High Capacity cDNA Reverse Transcription kit (Thermo Fisher Scientific, Wilmington, Delaware). The prepared cDNA of each sample was sequenced using the shotgun method, and then quantified with respect to individual genes. The results are reported as reads per kilobase ten million transcripts (RPKIOM), and used to quantify the amount of each transcript in a sample.

Example 2

Expression of ADH in yeast

[064] To understand the regulation of ADH in yeast, RNA-Seq analysis was performed on two strains of Saccharomyces cerevisiae, namely (i) FERMAX™ Gold (Martrex Inc., Minnesota, USA; herein abbreviated“FG”), a standard strain used for ethanol production and (ii) an FG strain engineered with the PKL pathway (herein abbreviated“FG-PKL”) described in as described in WO2015148272. RNA-Seq was performed as described in Example 1 and the results are summarized in Table 1. Expression levels are expressed as reads per kilobase ten million transcripts (RPKIOM).

Table 1. RNA-Seq analysis of ADH expression in FG during fermentation

Table 2. RNA-Seq analysis of ADH expression in FG-PKL during fermentation

[065] The data demonstrated that the Adh5p is expressed at much lower level than its paralog ADH1 during fermentation in both FG and FG-PKL strains. Adh5p expressed at the highest levels at 12 hr in both FG and FG-PKL strains, and then expressed at lower level at 24 hrs, 36 hrs and 48 hrs of fermentation.

Example 3

Promoter selection for increased expression of Adh5p

[066] As shown in Tables 3 and 4, the nicotinamidase gene (PNC1) expressed at similar level as Adh5p gene at 6 hr, and then increased its expression at about 6, 20, 35, 60 times of Adh5p at 12hr, 24 hr., 36 hr and 48 hr time point, respectively, in both FG and FG-PKL strains. Therefore, the PNC1 promoter was selected to drive the over-expression of Adh5p CDS.

Table 3. Transcription profiles of PNC1 gene in strains FG and during fermentation

Table 4. Transcription profiles of PNC1 gene in strains FG-PKL during fermentation

Example 4

Preparation of an Adh5p expression cassettes

[067] The ADH5 gene (YBR145W locus) of Saccharomyces cerevisiae was codon optimized and then synthesized to generate ADH5s (SEQ ID NO: 1) shown, below:

[068] The amino acid sequence of the Adh5ps polypeptide is shown, below, as SEQ ID NO: 1:

[069] The PNC1 promoter (YGL037C locus; SEQ ID NO: 3) was linked to the coding optimized coding sequence of ADH5s along with the ADH1 terminator (YOL086C locus; SEQ ID NO: 4) to generate the PNCl ::ADH5::AdhlTer expression cassette that was individually introduced at downstream of JIP5 locus (YPR169W) of FG and FG-PKL strains. The expected insertion of the ADH5 expression cassette in the two parental strains was confirmed by PCR.

[070] The nucleic acid sequence of the PNC1 promoter shown, below, as SEQ ID NO: 3:

Example 5

Ethanol production by Adh5p over-expressing yeast

[072] One each of PCR positive strain over-expressing Adh5ps under the control of the PNC1 promoter, and their parental strain FG and FG-PKL, were tested in an Ankom assay containing 50 g liquefact as described in Example 1. Fermentations were performed at 32°C for 55 hours. Samples from the end of fermentation were analyzed by HPLC analyses. The experiments were repeated several times and a typical result is summarized in Table 3. The data are the average of duplicate samples of each strain. Table 3. HPLC results from FG and FG-PKL over-expressing Adh5ps under the control of PNC1 promoter

[073] There was no effect of ethanol production by over-expression of Adh5ps in FG wild type strain, however, it resulted in a 0.8% increase of ethanol production in FG-PKL strain, with the expression cassette of PNCl ::ADH5::AdhlTer.

[074] These results demonstrated that Adh5ps over-expression is beneficial for increasing ethanol production in industrial ethanol production yeast engineered with PKL pathway.

Claims

CLAIMS What is claimed is:

1. Modified yeast cells derived from parental yeast cells, the modified cells comprising a genetic alteration that causes the modified cells to produce an increased amount of Adh5p polypeptides compared to the parental cells, wherein the modified cells and the parental cells both further comprising an heterologous phosphoketolase pathway, wherein the modified cells produce during fermentation more ethanol from glucose compared to the amount of ethanol produced by otherwise identical parental yeast cells.

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 an Adh5p polypeptide to a level above that of the parental cell grown under equivalent conditions.

3. The modified cells of claim 1, wherein the genetic alteration comprises the introduction of an expression cassette for expressing an Adh5p polypeptide.

4. The modified cells of any of claims 1-3, wherein the amount of increase in the expression of the Adh5p polypeptide is at least about 500%, at least about 1,000%, at least about 5,000%, or at least about 10,000%, compared to the level expression in the parental cells grown under equivalent conditions.

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

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

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

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

9. A method for increased production of alcohol from yeast cells grown on a carbohydrate substrate, comprising: introducing into parental yeast cells comprising a phophoketolase pathway a genetic alteration that increases the production of Adh5p polypeptides compared to the amount produced in the parental cells.

10. The method of claim 9, wherein the cells having the introduced genetic alteration are the modified cells are the cells of any of claims 1-8.

11. The method of claim 9 or 10, wherein the increased production of alcohol is at least 0.2%, at least 0.5%, at least 0.8%.

12. The method of any of claims 9-11, wherein Adh5p polypeptides are over-expressed by at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold.