CN111201313B - Increased ethanol production by yeast harboring a constitutive transcriptional activator MAL allele - Google Patents

Abstract

Compositions and methods involving modified yeast with a constitutive transcriptional activator MAL allele are described. The yeast produces an increased amount of ethanol, or has an increased rate of ethanol production that does not appear to be a result of maltose metabolism. Such yeast are particularly useful for ethanol production using starch substrates.

Description

Increased ethanol production by yeast with a constitutive transcriptional activator MAL allele

Technical Field

The compositions and methods of the present invention relate to modified yeasts having a constitutive transcriptional activator MAL allele. The yeast produces an increased amount of ethanol that does not appear to be a result of maltose metabolism. Such yeasts are particularly useful for large-scale ethanol production from starch substrates.

Background Art

The first generation of yeast-based ethanol production converts sugar into fuel ethanol. About 90 billion liters of fuel ethanol are produced by yeast every year worldwide (Gombert, A.K. and van Maris. A.J. (2015) Curr. Opin. Biotechnol. [Biotechnol. New View] 33: 81-86). It is estimated that about 70% of the cost of ethanol production is raw materials. Because the production volume is so large, even a small increase in yield will have a huge economic impact on the industry.

Yeast, such as Saccharomyces, are able to metabolize many monosaccharides and disaccharides, including maltose. Maltose fermentation in Saccharomyces involves at least one of five seemingly redundant, unlinked MAL loci (referred to as MAL1, MAL2, MAL3, MAL4, and MAL5) (see, e.g., Needleman, R.B. (1991) Mol. Microbiol. 9:2079-84). As shown in Figure 1, each locus includes three genes encoding (i) maltose permease, (ii) maltase and (iii) transcription activator (Kim, J. and Michels, C.A. (1988) Curr. Genet. [Current Genetics] 14:319-23 and Cheng, Q. and Michels (1989) Genetics [Genetics] 123:477-84. The genes are conventionally numbered 1, 2 and 3, respectively, so that, for example, the genes at the MAL2 locus are numbered MAL21, MAL22 and MAL23, respectively.

Transcription of the MAL gene is induced by maltose and repressed by glucose; however, constitutive mutations have been identified at all MAL loci (Winge, O. and Roberts, C. (1950) C. R. Trav. Lab. Carlsberg Ser. Physiol. 25:35-81, Kahn, N. A. and Eaton, N. R. (1971) Mol. Gen. Genet. 112:317-22; Charronm, J. and Michels, C. A. (1987) Genetics 116:23-31; Zimmerman and Eaton, N. R. (1974) Mol. Gen. Genet. 134:134-135). 261-271; Rodicio, R. (1986) Curr. Genet. [Current Genetics] 11: 235-41 and Ten Berge, A. M. A. et al. (1973) Mol. Gen. Genet. [Molecular Genetics and General Genetics] 125: 139-46.

Maltose is present at low levels in commercial-scale starch hydrolysates and is typically an undesirable DP2 component that is unlikely to be a significant source of additional ethanol. In addition, transcription of the MAL gene is repressed by high levels of glucose, making it difficult for yeast to utilize maltose under high sugar conditions (such as that present during fuel ethanol production). Intentionally expressing maltose metabolizing enzymes may actually slow glucose metabolism and waste carbon, which is unacceptable given the needs of ethanol producers.

Summary of the invention

The compositions and methods of the present invention relate to modified yeasts having a constitutive transcriptional activator MAL allele. Although such yeasts may be able to metabolize maltose, even in the presence of glucose, the yeasts appear to produce increased amounts of ethanol, which is not necessarily the result of maltose metabolism. Aspects and embodiments of the compositions and methods are described in the following independently numbered paragraphs.

1. In one aspect, a method for increasing the amount of alcohol produced from the fermentation of a starch hydrolysate and/or increasing the rate of alcohol production from the fermentation of a starch hydrolysate is provided, the method comprising fermenting the starch hydrolysate with a modified yeast having a constitutive transcriptional activator MAL allele, wherein in the modified yeast, the amount of ethanol produced at the end of the fermentation is increased, or the amount of ethanol produced over a period of time is increased, compared to the amount of ethanol produced by an otherwise identical parent yeast.

2. In some embodiments of the method as described in paragraph 1, at least a portion of the increased amount of ethanol cannot be attributed to maltose fermentation based on carbon flux through the maltose metabolic pathway.

3. In some embodiments of the method as described in paragraph 1 or 2, the maltose level in the starch hydrolysate at the end of fermentation is approximately the same as the maltose level in the starch hydrolysate at the beginning of fermentation.

4. In some embodiments of the method of any one of the preceding paragraphs, the amount of maltose in the starch hydrolysate does not exceed 10 g/L at the start of fermentation.

5. In some embodiments of the method of any of the preceding paragraphs, the yeast having a constitutive transcriptional activator MAL allele further comprises a genetic alteration that introduces a polynucleotide encoding a polypeptide in the phosphoketolase pathway.

6. In some embodiments of the method of any of the preceding paragraphs, the yeast having a constitutive transcriptional activator MAL allele further comprises an alteration in the glycerol pathway and/or the acetyl-CoA pathway.

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

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

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

10. In another aspect, a modified yeast is provided, comprising a constitutive transcriptional activator MAL allele and at least one additional genetic modification unrelated to maltose metabolism, which, when grown on starch hydrolysate, produces an increased amount of ethanol at the end of fermentation compared to the amount produced by an otherwise identical parent yeast during fermentation.

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

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

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

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

15. In some embodiments of the modified yeast of any of paragraphs 10-14, the cell is a Saccharomyces species.

These and other aspects and embodiments of the modified cells and methods of the invention will be clear from the description including the drawings/figures.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a diagram of a typical MAL locus and its regulation by maltose and glucose

Figure 2 depicts the MAL23c expression cassette.

Figure 3 is a map of plasmid pHX19.

FIG. 4 is a comparison of the cumulative CO ₂ stress index during fermentation with strains FG and A28.

Figure 5 is a map of plasmid pZKAP1M-(H3C19).

Figure 6 is a map of plasmid pTOPO II-Blunt ura3-loxP-KanMX-loxP-ura3.

Figure 7 is a map of plasmid pGAL-Cre-316.

FIG. 8 is a comparison of the cumulative CO ₂ pressure index during fermentation with RHY723 and its parent strain GPY10008.

DETAILED DESCRIPTION

I. Definition

Before describing the yeast and methods of the present invention in detail, the following terms are defined for the sake of clarity. Undefined terms shall be given the common meanings used in the relevant art.

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

As used herein, the term "yeast cell", yeast strain, or simply "yeast" refers to an organism from the phylum Ascomycota and the phylum Basidiomycota. An exemplary yeast is a budding yeast from the order Saccharomycetales. A specific example of yeast is a species of the genus Saccharomycetes, including but not limited to S. cerevisiae. Yeast includes organisms for the production of fuel alcohols and organisms for the production of drinkable alcohols, including specialty and proprietary yeast strains for the preparation of uniquely flavored beer, wine, and other fermented beverages.

As used herein, the phrases "engineered yeast cells," "variant yeast cells," "modified yeast cells," or similar phrases refer to yeast that include the genetic modifications and features described herein. Variant/modified yeast does not include naturally occurring yeast.

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

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

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

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

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

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

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

As used herein, the term "gene" is synonymous with the term "allele" and refers to a nucleic acid that encodes and directs the expression of a protein or RNA. The vegetative form of filamentous fungi is usually haploid, so a single copy of a given gene (i.e., a single allele) is sufficient to confer a specific phenotype. When an organism contains more than one similar gene, the term "allele" is usually preferred, in which case each different similar gene is referred to as a different "allele."

As used herein, "constitutive" expression refers to the production of a polypeptide encoded by a particular gene under essentially all typical growth conditions, as opposed to "conditional" expression which requires the presence of a specific substrate, temperature, etc. to induce or activate expression.

As used herein, the term "expressing a polypeptide" and similar terms refer to the cellular process by which the cell's translation machinery (eg, ribosomes) produces a polypeptide.

As used herein, "overexpressing a polypeptide," "increasing expression of a polypeptide," and similar terms refer to expressing a polypeptide at a higher than normal level than observed in a parent or "wild-type cell" that does not include the specified genetic modification.

As used herein, "expression cassette" refers to a DNA fragment comprising a promoter, an amino acid coding region and a terminator (i.e., promoter::amino acid coding region::terminator) and other nucleic acid sequences required to allow the production of the encoded polypeptide in a cell. The expression cassette can be exogenous (i.e., introduced into a cell) or endogenous (i.e., present in a cell).

As used herein, the terms "fused" and "fusion" with respect to two DNA fragments (eg, a promoter and a coding region for a polypeptide) refer to the physical bonding of the two DNA fragments resulting in a single molecule.

As used herein, the terms "wild type" and "native" are used interchangeably and refer to a gene, protein or strain found in nature, or which has not been intentionally modified for the advantage of the presently described yeast.

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

As used herein, "destruction of gene" refers to any genetic or chemical operation (i.e., mutation) that substantially prevents cells from producing functional gene products (e.g., proteins) in host cells. Exemplary destruction methods include complete or partial (including polypeptide coding sequence, promoter, enhancer or additional regulatory elements) deletion or mutagenesis of any part of the gene, wherein mutagenesis encompasses substitution, insertion, deletion, inversion, and combinations and variations thereof, and any of these mutations substantially prevent the generation of functional gene products. RNAi, antisense or any other method for eliminating gene expression can also be used to destroy genes. Genes can be destroyed by the deletion or genetic manipulation of non-adjacent control elements. As used herein, "gene deletion" refers to the removal of the gene from the genome of the host cell. When a gene includes a control element (e.g., enhancer element) that is not immediately adjacent to a gene coding sequence, the deletion of a gene refers to the deletion of a coding sequence and optionally adjacent enhancer elements (e.g., including but not limited to promoter and/or terminator sequences), but the deletion of non-adjacent control elements is not required.

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

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

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

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

As used herein, "attenuation of a pathway" or "attenuation of flux through a pathway" (i.e., a biochemical pathway) refers broadly to any genetic or chemical manipulation that reduces or completely prevents the flux of a biochemical substrate or intermediate through a metabolic pathway. Attenuation of a pathway can 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, replacement of wild-type alleles of these genes with mutant forms encoding enzymes with reduced catalytic activity or increased Km values, modification of promoters or other regulatory elements that control expression of one or more genes, engineering the enzymes or mRNAs encoding these enzymes for reduced stability, misdirecting enzymes into cellular compartments that are less likely to interact with substrates and intermediates, use of interfering RNAs, and the like.

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

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

As used herein, the expression "end of fermentation" refers to the stage of fermentation when the economic advantage of continued fermentation producing a small amount of additional alcohol is outweighed by the cost of continued fermentation in terms of fixed and variable costs. In a more general sense, "end of fermentation" refers to the point at which fermentation no longer produces significant amounts of additional alcohol, i.e., no more than about 1% additional alcohol.

As used herein, the expression "carbon flux" refers to the turnover rate of carbon molecules through a metabolic pathway. Carbon flux is regulated by enzymes involved in a metabolic pathway, such as a glucose metabolic pathway and a maltose metabolic pathway.

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

EC Enzyme Commission

PKL Phosphoketolase

PTA phosphotransacetylase

AADH acetaldehyde dehydrogenase

ADH alcohol dehydrogenase

EtOH

AA α-amylase

GA Glucoamylase

℃ Celsius

bp base pair

DNA Deoxyribonucleic acid

ds or DS dry solid

g or gm gram

g/L grams/liter

_H2O Water

HPLC High Performance Liquid Chromatography

hr or h hours

kg kilogram

M Molar

mg milligram

mL or ml milliliters

min

mM millimolar

N Normal

nm nanometer

PCR polymerase chain reaction

ppm parts per million

Δ is associated with deletion

μg microgram

μL and μl microliter

μM micromolar

II. Modified Yeast with a Constitutive MAL Allele

The present inventors have found that modified yeasts with constitutive transcriptional activator MAL alleles produce more ethanol in starch hydrolysate fermentation, and/or produce the same amount of ethanol in a shorter time, compared to otherwise identical parent yeasts. It is noteworthy that the additional ethanol produced does not appear to be the result of maltose metabolism, or at least not the result of only maltose metabolism, because the small amount of maltose present in the starch hydrolysate remains essentially unchanged after fermentation. Yeasts with constitutive transcriptional activator MAL alleles also produce ethanol at a higher rate in starch hydrolysate fermentation, compared to otherwise identical parent yeasts. Therefore, constitutive expression of the transcriptional activator MAL allele appears to have unexpected benefits for glucose metabolism in a high sugar environment.

The presence of the constitutive transcriptional activator MAL allele, optionally in the presence of other genetic modifications, results in an increase in ethanol production, or an increase in the rate of ethanol production of at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1.0%, at least 1.1%, at least 1.2% or more. Although such increases are small, given the amount of ethanol currently produced, this is equivalent to a substantial increase in volume.

The yeast strains and methods of the present invention use a representative constitutive transcription activator MAL23 allele as an example, as shown in the following SEQ ID NO: 1:

However, it is entirely expected that the introduction of other constitutive transcriptional activators, MAL alleles, will produce similar results. The constitutive transcriptional activator MAL allele has been known for more than half a century and is exemplified by, for example, Winge, O. and Roberts, C. (1950) C.R. Trav. Lab. Carlsberg Ser. Physiol. 25:35-81, Kahn, N.A. and Eaton, N.R. (1971) Mol. Gen. Genet. 112:317-22; Charronm, J. and Michels, C.A. (1987) Genetics 116 23-31; Zimmerman and Eaton, N.R. (1974) Mol. Gen. Genet. 134 261-271; Rodicio, R. (1986) Curr. Genet. 11:235-41 and Ten. Berge, A. M. A. et al. (1973) Mol. Gen. Genet. 125: 139-46, each of which is incorporated herein by reference.

Yeast possessing a constitutive transcriptional activator MAL allele may also include other genetic manipulations to increase alcohol production, particularly modifications unrelated to enhanced maltose metabolism.

III. Modified yeast with a constitutive MAL allele combined with exogenous PKL pathway genes

The presence of the constitutive transcriptional activator MAL allele can combine with the expression of genes in the PKL pathway to increase the growth rate of cells and further increase ethanol production.

Engineered yeast cells with heterologous PKL pathways have been previously described (e.g., WO 2015148272). These cells express heterologous PKL (EC 4.1.2.9) and PTA (EC 2.3.1.8), optionally with other enzymes, to direct channel carbon flux away from the glycerol pathway, and toward the synthesis of acetyl-CoA, which is then converted into ethanol. Compared to otherwise identical parent yeast cells, such modified cells can increase ethanol production during fermentation.

IV. Modified yeast having a constitutive MAL allele and having reduced Dls1 expression

Dls1, encoded by YJL065c, is a 167 amino acid polypeptide subunit of the ISW2 yeast chromatin accessibility complex (yCHRAC), which contains Isw2, Itc1, a Dpb3-like subunit (Dls1), and Dpb4 (see, e.g., Peterson, C.L. (1996) Curr. Opin. Genet. Dev. 6:171-75 and Winston, F. and Carlson, M. (1992) Trends Genet. 8:387-91). In the absence of other genetic modifications, yeast with genetic alterations that reduce the amount of functional Dls1 in the cell exhibit increased robustness in alcohol fermentation, which allows higher temperatures and potentially shorter fermentations (data not shown).

Reducing the amount of functional Dls1 produced in the cell can be accomplished by destroying the YJL065c gene. The destruction of the YJL065c gene can be performed using any suitable method that substantially prevents the expression of the functional YJL065c gene product (i.e., Dls1). Exemplary destruction methods known to those skilled in the art include, but are not limited to, complete or partial deletion of the YJL065c gene, including complete or partial deletion of, for example, a Dls1 coding sequence, a promoter, a terminator, an enhancer, or other regulatory elements; and complete or partial deletion of a portion of a chromosome comprising any portion of the YJL065c gene.

The specific method of destroying the YJL065c gene is included in any part of the YJL065c gene (for example, Dls1 coding sequence, promoter, terminator, enhancer or another regulatory element) and nucleotide substitution or insertion. Preferably, deletion, insertion and/or substitution (collectively referred to as mutation) are carried out by genetic manipulation using sequence-specific molecular biology techniques, which is contrary to chemical mutagenesis, which does not usually target specific nucleic acid sequences. Nevertheless, in theory, chemical mutagenesis can still be used to destroy the YJL065c gene.

V. Additional mutations affecting alcohol production

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

The modified yeast may further be characterized by increased acetyl-CoA synthase (also known as acetyl-CoA ligase) activity (EC 6.2.1.1) to scavenge (i.e., capture) acetic acid produced by chemical or enzymatic hydrolysis of acetyl-phosphate (or present in the culture medium of the yeast for any other reason) and convert it into acetyl-CoA. This avoids the adverse effects of acetic acid on yeast cell growth and may further contribute to the increase in alcohol production. Increasing acetyl-CoA synthase activity can be achieved by introducing a heterologous acetyl-CoA synthase gene into the cell, increasing the expression of an endogenous acetyl-CoA synthase gene, etc. A particularly useful acetyl-CoA synthase for introduction into a cell can be obtained from Methanosaeta concilii (UniProt/TrEMBL Accession No.: WP_013718460). Homologs of the enzyme, including enzymes having at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, and even at least 99% amino acid sequence identity to the above-mentioned acetyl-CoA synthase from Methanogenum consiliense, can also be used in the compositions and methods of the present invention. In other embodiments, the modified yeast of the present invention does not have increased acetyl-CoA synthase.

In some embodiments, the modified yeast of the present invention may further include a heterologous gene encoding a protein having NAD+-dependent acetylated acetaldehyde dehydrogenase activity and/or a heterologous gene encoding pyruvate formate lyase. For example, the introduction of such genes combined with glycerol pathway attenuation is described in U.S. Patent No. 8,795,998 (Pronk et al.). However, in most embodiments of the compositions and methods of the present invention, it is not necessary to introduce acetylated acetaldehyde dehydrogenase and/or pyruvate formate lyase, because the need for these activities is eliminated by attenuating the natural biosynthetic pathway for preparing acetyl-CoA that causes an imbalance in redox cofactors. Therefore, in some embodiments, the yeast of the present invention does not have a heterologous gene encoding NAD+-dependent acetylated acetaldehyde dehydrogenase and/or a heterologous gene encoding pyruvate formate lyase.

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

In some embodiments, the modified yeast comprising a butanol biosynthetic pathway further comprises a modification in a polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. The yeast may comprise a deletion, a mutation and/or a 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 a combination thereof. In some embodiments, the yeast cell further comprises a deletion, a mutation and/or a substitution in one or more endogenous polynucleotides encoding FRA2, ALD6, ADH1, GPD2, BDH1, and YMR226C. In other embodiments, the modified yeast cell of the present invention does not further comprise a butanol biosynthetic pathway.

The modified yeast of the present invention can 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 enzymes selected from the group consisting of dehydrogenases, transketolases, phosphoketolase, transaldolase, epimerase, phytase, xylanase, β-glucanase, phosphatase, protease, α-amylase, β-amylase, glucoamylase, pullulanase, isoamylase, cellulase, trehalase, lipase, pectinase, polyesterase, cutinase, oxidase, transferase, reductase, hemicellulase, mannanase, esterase, isomerase, pectinase, lactase, peroxidase, and laccase. The protein of interest can be secreted, glycosylated, and otherwise modified.

VI. Use of modified yeast for increased alcohol production

Yeast of the present invention and methods of use thereof include methods for increasing alcohol production in fermentation reactions. Such methods are not limited to specific fermentation processes. It is expected that the engineered yeast of the present invention is a "drop-in" substitute for conventional yeast in any alcohol fermentation facility. Although primarily intended for fuel ethanol production, the yeast of the present invention can also be used to produce drinkable alcohol, including wine and beer.

VII. Yeast suitable for modification

Yeast is a unicellular eukaryotic microorganism classified as a member of the fungi kingdom, and includes organisms from the Ascomycota and Basidiomycota. Yeast that can be used for alcohol production includes but is not limited to Saccharomyces species, including Saccharomyces cerevisiae, and Kluyveromyces, Lachancea, and Schizosaccharomyces species. Many yeast strains are commercially available, many of which have been selected or genetically engineered to obtain desired characteristics, such as high ethanol production, fast growth rate, etc. Some yeasts have been genetically engineered to produce heterologous enzymes, such as glucoamylase or alpha-amylase.

VIII. Substrates and Products

The production of alcohol from many carbohydrate substrates (including but not limited to corn starch, sugar cane, cassava and molasses) is well known, as are the numerous variations and improvements in enzyme conditions and chemical conditions and mechanical methods. It is believed that the compositions and methods of the present invention are fully compatible with such substrates and conditions. High levels of maltose do not need to benefit from the compositions and methods of the present invention. In certain embodiments, the concentration is less than about 10 g/L.

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

Examples

Example 1

Materials and Methods

Liquefaction preparation:

Liquefact (corn mash slurry) was prepared by adding 600 ppm urea, 0.124 SAPU/g ds FERMGEN ^™ 2.5x (acid fungal protease), 0.33 GAU/g ds CS4 (Trichoderma glucoamylase variant), and 1.46 SSCU/g ds AKAA (Aspergillus kawachii alpha-amylase), adjusted to pH 4.8.

Serum bottle assay:

Yeast cells were inoculated into 2 mL of YPD in a 24-well plate and the culture was grown overnight to an OD between 25-30. 2.5 mL of the liquefact was transferred to a serum bottle (Chemglass, catalog number: CG-4904-01) and yeast was added to each vial to a final OD of approximately 0.4-0.6. The cap of the vial was installed and pierced with a needle (BD, catalog number: 305111) for ventilation (to release _CO2 ) and then incubated at 32°C with shaking (200 RPM for 65 hours).

AnKom assay:

300 μL of concentrated yeast overnight culture was added to each of the ANKOM bottles filled with 50 g of prepared liquefact (see above) to reach a final OD of 0.3. The bottles were then incubated at 32°C with shaking (at 150 RPM) for 65 hours.

HPLC analysis:

Samples from serum bottles and cultures for AnKom assay were collected in Eppendorf tubes by centrifugation at 14,000 RPM for 12 minutes. The supernatant was filtered using a 0.2 μM PTFE filter and then used for HPLC (Agilent Technologies 1200 series) analysis under the following conditions: Bio-Rad Aminex HPX-87H column, operating temperature was 55°C. 0.6 ml/min isocratic flow rate, 0.01 NH ₂ SO ₄ , 2.5 μl injection volume. Calibration standards were used for quantification of acetic acid, ethanol, glycerol, glucose and other molecules. All values are reported in g/L.

Example 2

Preparation of constitutive MAL23 allele cassette

A MAL23c expression cassette was prepared using standard procedures, consisting of a constitutive allele of MAL23 (SEQ ID NO: 2) under the control of a MAL23c promoter (SEQ ID NO: 3) and a MAL23 terminator (SEQ ID NO: 4). The nucleotide sequence is shown below, and the representation of the cassette is shown in Figure 2:

DNA coding region of the constitutive MAL23 allele (SEQ ID NO: 2):

DNA sequence of MAL23c promoter (SEQ ID NO: 3):

DNA sequence of MAL23c terminator (SEQ ID NO: 4):

By amplifying the MAL23c expression cassette with appropriate primers, a DNA fragment comprising the flanking YDL227C locus sequence was prepared. The fragment was inserted into a plasmid designated pHX19, which comprises an integrated constitutive MAL23c expression cassette at the YDL227C locus on the Saccharomyces chromosome, as shown in Figure 3. The functional and structural components of plasmid pHX19 are described in Table 1.

Table 1. Functional and structural elements of plasmid pHX19

Example 3

Generation of yeast with MAL23c expression cassette

To investigate the role of MAL23c in industrial yeast, the wild-type FERMAX ^™ Gold strain (Martrex, Inc., Chaska, Minnesota, USA), hereinafter referred to as "FG", was used as a parent to introduce the MAL23c expression cassette at the YDL227C integration site. The cells were transformed with a 3,362-bp PCR amplified DNA fragment using appropriate flanking primers and pHX19 plasmid as a template. Transformants were selected and a representative member was named strain A28.

Example 4

Comparison of FG yeast with or without MAL23c

Strain A28 and parent strain FG were grown in Ankom bottles and their fermentation products were analyzed as described in Example 1. The performance with respect to the cumulative _CO2 pressure index (CPI), which indicates the fermentation rate, is shown in Figure 4. The performance with respect to ethanol, glycerol and acetic acid production is shown in Table 2.

Table 2. FG relative to A28 in the AnKom assay

In terms of residual amounts of maltotriose, maltose and glucose, the performance of the FG parent and strain A28 were similar, indicating that integration of the MAL23c DNA fragment at the YDL227C locus would not affect maltose metabolism in glucose-based corn mash fermentation. The ethanol titer of the Mal23c expressing strain was 1.1% higher than that of the FG control. The CPI data in Figure 4 demonstrated that the fermentation rate of strain A28 was also higher than that of the parent FG strain. The experiment was repeated and the results confirmed.

Example 5

Generation of plasmids containing the complete PKL pathway

Plasmid pZKAP1m-(H3C19) was designed to integrate four separate polypeptide expression cassettes upstream of the AAP1 locus (YHR047C). The four cassettes are as follows: (i) HXT3 promoter::PKL C::FBA1 terminator; (ii) PGK1 promoter::LpPTA::PGK1 terminator; (iii) TDH3 promoter::eutE A19::ENO terminator; and (iv) PDC1 promoter::AcsA1::PDC1 terminator, and the four cassettes are designed to express codon-optimized genes encoding phosphoketolase (PKL) derived from Gardnerella vaginalis, phosphotransacetylase (PTA) derived from Lactobacillus plantarum, acetylating acetaldehyde dehydrogenase derived from Desulfospira joergensenii, and acetyl-CoA synthase derived from Methanobacterium consiliense, respectively. The map of pZKAP1m-(H3C19) is shown in Figure 5. The functional and structural elements are listed in detail in Table 3.

Table 3. Functional and structural elements of construct pZKAP1m-(H3C19)

Example 6

Generation of FG-ura3 yeast

FG strain was used as a parent strain to prepare ura3 auxotrophic strain FG-ura3. Plasmid pTOPO II-Blunt ura3-loxP-KanMX-loxP-ura3 (depicted in Figure 6) was designed to replace the URA3 gene in strain FG with mutant ura3 and URA3-loxP-TEFp-KanMX-TEFt-loxP-URA3 fragments. The functional and structural elements of the plasmid are listed in Table 4.

Table 4. Functional/structural elements of pTOPO II-Blunt ura3-loxP-KanMX-loxP-ura3

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

The transformed colonies that can grow on a medium containing G418 are streaked on synthetic mini-plates containing 20 μg/ml uracil and 2mg/ml 5-fluoroorotic acid (5-FOA). The URA3 deletion of the colonies that can grow on the 5-FOA plate is further confirmed by phenotypic growth on the SD-Ura plate and by PCR. The ura3 deletion transformant cannot grow on the SD-Ura plate. A single 1.98-kb PCR fragment is obtained with the test primers. In contrast, using DNA from the parental FG strain, the same primer pair generates a 1.3-kb fragment, thereby indicating the presence of the complete ura3 gene. The ura3 deletion strain is named FG-KanMX-ura3.

In order to remove the KanMX expression cassette from strain FG-KanMX-ura3, plasmid pGAL-Cre-316 (as depicted in Figure 7) was used to transform the cells of strain FG-KanMX-ura3 by electroporation. The purpose of using this plasmid is to temporarily express the Cre enzyme so that the LoxP sandwich KanMX gene will be removed from strain FG-KanMX-ura3 to generate strain FG-ura3. pGAL-Cre-316 is a self-replicating circular plasmid subsequently removed from strain FG-ura3. All sequence elements from pGAL-cre-316 are not inserted into the strain FG-ura3 locus. The functions and structural elements of plasmid pGAL-Cre-316 are listed in Table 5.

Table 5. Functional and structural elements of pGAL-Cre-316.

Functional/structural elements Yeast-bacteria shuttle vector pRS316 sequence pBR322 origin of replication Saccharomyces cerevisiae URA3 gene F1 starting point GALp-Cre-ADHt cassette, reverse orientation

The transformed cells are plated on SD-Ura plates. Single colonies are transferred to YPG plates and incubated at 30°C for 2 to 3 days. The colonies are then transferred to new YPD plates for a few more days. Finally, the cell suspension from the YPD plate is spotted on the following plates: YPD, G418 (150 μg/ml), 5-FOA (2 mg/ml) and SD-Ura. Cells that can grow on YPD and 5-FOA and cannot grow on G418 and SD-Ura plates are selected for PCR confirmation as described above. The expected PCR product size is 0.4-kb, and the identity of the KanMX (Geneticin) sensitivity ura3 deletion strain derived from FG-KanMX-ura3 is confirmed. This strain is named FG-ura3.

Example 7

Generation of yeast with PKL pathway

The FG-ura3 strain was used as a parent to introduce the PKL pathway. The cells were transformed with a 14,993-bp KasI fragment containing four expression cassettes from pZKAP1m-(H3C19) from Example 5. Transformants with the KasI fragment integrated upstream of the YHR047C locus were selected and named strain GPY10000.

Strains FG and GPY10000 were grown in vial cultures and their fermentation products were analyzed as described in Example 1. The performance with respect to ethanol, glycerol and acetic acid production is shown in Table 6.

Table 6. FG versus G176 in vial assays

Strains One or more transgenes expressed glycerin Acetic acid EtOH FG none 17.30 0.70 140.9 GPY10000 PKL pathway 12.94 1.86 146.3

The ethanol titer of the GPY10000 strain expressing the PKL pathway was 3.8% higher than that of the FG strain. Not surprisingly, the levels of acetate were also elevated, a known property of yeast with the PKL pathway.

Example 8

Generation of yeast with PKL pathway and reduced Dls1 production

Strain GPY10008 was generated by Cas9-mediated deletion of YJL065c (which encodes Dls1) in strain GPY10000. Specifically, the deletion was made from 4-bp before the start codon to 10-bp before the stop codon of YJL065c.

FG yeast strain GPY10008 and its parent strain G10008 were grown in AnKom bottles and their fermentation products were analyzed as described in Example 1. The performance with respect to ethanol, glycerol and acetic acid production is shown in Table 7.

Table 7. GPY10000 vs. GPY10008 in the Ankom assay

Strains One or more transgenes expressed glycerin Acetic acid EtOH GPY10000 PKL pathway 12.85 1.80 143.39 GPY10008 PKL pathway + ΔYJL065c 13.15 1.78 147.23

The ethanol titer of the strain with the YJL065c deletion was 2.7% higher than that of the strain without the deletion. Acetate levels were roughly the same.

Example 9

Generation of yeast with combined MAL23c and PKL pathways and reduced Dls1 production

To investigate the role of MAL23c in an engineered FG strain with a PKL pathway and reduced Dls1 expression, strain GPY10008 was used as a parent to introduce the MAL23c expression cassette, in this case, in the 3′ region of the YHL041w locus, as otherwise described in Example 3. GPY10008 cells were transformed with a PCR-amplified DNA fragment containing the MAL23c expression cassette, prepared using the pHX19 plasmid as a template and primers including flanking YHL041w locus sequences.

The new FG yeast strain named RHY723 and its parent strain GPY10008 were grown in AnKoms bottles and their fermentation products were analyzed as described in Example 1. The performance with respect to ethanol, glycerol and acetic acid production is shown in Table 8 and the fermentation rate expressed as CPI during the fermentation is shown in Figure 8.

Table 8. RHY720 versus GPY10008 in the Ankom assay

The ethanol titer of strain RHY723 was 0.83% higher than that of strain GPY10008, demonstrating that expression of MAL23c increased ethanol production even in yeast that also contains the PKL pathway and does not express Dls1.

Claims (11)

1. A method for increasing the amount of alcohol produced from and/or increasing the rate of alcohol produced from fermentation of a starch hydrolysate, the method comprising fermenting the starch hydrolysate with a modified yeast having a constitutive transcriptional activator MAL23 allele encoding an amino acid sequence as set forth in SEQ ID No. 1 and lacking the YJL065c gene, wherein in the modified yeast the amount of alcohol produced at the end of the fermentation is increased, or the amount of alcohol produced over a period of time is increased, as compared to the amount of alcohol produced by an otherwise identical parent yeast.

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

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

4. A process according to claim 3, wherein the amount of maltose in the starch hydrolysate at the beginning of the fermentation is not more than 10g/L.

5. The method of claim 1, wherein the modified yeast further comprises a genetic alteration that introduces a polynucleotide encoding a polypeptide in a phosphoketolase pathway.

6. The method of any one of claims 1-5, wherein the yeast is a Saccharomyces (Saccharomyces) species.

7. A modified yeast comprising a constitutive transcriptional activator MAL23 allele and lacking the YJL065c gene, the constitutive transcriptional activator MAL23 allele encoding an amino acid sequence set forth in SEQ ID NO:1, the yeast producing an increased amount of ethanol at the end of fermentation as compared to an otherwise identical parent yeast during fermentation when grown in starch hydrolysate.

8. The modified yeast of claim 7, wherein the yeast further comprises a genetic alteration that introduces a polynucleotide encoding a polypeptide in a phosphoketolase pathway.

9. The modified yeast of claim 7, wherein the yeast further comprises a change in the glycerol pathway and/or the acetyl-coa pathway.

10. The modified yeast of claim 7, wherein the yeast further comprises an exogenous gene encoding a carbohydrate processing enzyme.

11. The modified yeast of any one of claims 7-10, wherein the yeast is a saccharomyces species.