EP3665266A1 - Production accrue d'éthanol par une levure présentant des allèles mal - Google Patents

Production accrue d'éthanol par une levure présentant des allèles mal

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Publication number
EP3665266A1
EP3665266A1 EP18760106.7A EP18760106A EP3665266A1 EP 3665266 A1 EP3665266 A1 EP 3665266A1 EP 18760106 A EP18760106 A EP 18760106A EP 3665266 A1 EP3665266 A1 EP 3665266A1
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EP
European Patent Office
Prior art keywords
yeast
fermentation
pathway
ethanol
transcriptional activator
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP18760106.7A
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German (de)
English (en)
Inventor
Seung-Pyo Hong
Paula Johanna Maria TEUNISSEN
Quinn Qun Zhu
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Danisco US Inc
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Danisco US Inc
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Publication of EP3665266A1 publication Critical patent/EP3665266A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • compositions and methods relate to modified yeast harboring constitutive transcriptional activator MAL alleles.
  • the yeast produces an increased amount of ethanol that does not appear to be the result of maltose metabolism.
  • Such yeast is particularly useful for large-scale ethanol production from starch substrates.
  • 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.
  • Yeast such as Saccharomyces, are capable of metabolizing a number of mono and di- saccharides, including maltose.
  • Maltose fermentation in Saccharomyces involves at least one of five, apparently-redundant, unlinked MAL loci, referred to as MALI, MAL2, MAL3, MAL4 and MAL5 (see, e.g. , Needleman, R.B. (1991) o/. Microbiol. 9:2079-84).
  • each locus includes three genes, encoding (i) a maltose permease, (ii) a maltase and (iii) a transcriptional activator (Kim, J. and. Michels, C.A.
  • the genes are conventionally numbered 1, 2 and 3, respectively, such that the genes at the MAL2 locus, for example, are numbered MAL21, MAL 22 and MAL23, respectively.
  • MAL genes Transcription of MAL genes is induced by maltose and repressed by glucose
  • Maltose is present at low levels in commercial-scale, starch-hydrolysates, typically, as an undesirable, DP2-component, which is not likely to be a significant source of additional ethanol.
  • transcription oiMAL genes is repressed by high levels of glucose, making it difficult for yeast to utilize maltose under high-glucose conditions, such as those that exist during fuel ethanol production.
  • Deliberate expression of maltose-metabolizing enzymes may in fact slow glucose metabolism and waste carbon, which is unacceptable given the demands of ethanol producers.
  • compositions and methods relate to modified yeast harboring constitutive transcriptional activator MAL alleles. While such yeast is presumably capable of
  • a method for increasing the amount of alcohol produced from fermentation of a starch hydrolysate and/or increasing the rate of production of alcohol from fermentation of a starch hydrolysate comprising fermenting the starch hydrolysate with modified yeast harboring a constitutive transcriptional activator MAL allele, where in the modified yeast produces an increased amount of ethanol at the end of fermentation or an increased amount of ethanol over a period of time compared to the amount of ethanol produced by an otherwise-identical parental yeast.
  • At least a portion of the increased amount of ethanol cannot be attributed to maltose fermentation based on carbon flux through a maltose metabolic pathway.
  • the level of maltose in the starch hydrolysate at the end of fermentation is about the same as the level of maltose in the starch hydrolysate at the beginning of fermentation
  • the amount of maltose in the starch hydrolysate at the beginning of fermentation is no greater than 10 g/L. 5.
  • the yeast harboring a constitutive transcriptional activator MAL allele further comprises a genetic alteration that introduces a polynucleotide encoding a polypeptide in the phosphoketolase pathway.
  • the yeast harboring a constitutive transcriptional activator MAL allele further comprises an alteration in the glycerol pathway and/or the acetyl-CoA pathway.
  • the yeast harboring a constitutive transcriptional activator MAL allele further comprises disruption of a gene encoding DLsl.
  • the yeast harboring a constitutive transcriptional activator MAL allele comprises an exogenous gene encoding a carbohydrate processing enzyme.
  • the yeast is a Saccharomyces spp.
  • modified yeast comprising a constitutive transcriptional activator MAL allele, and at least one additional genetic modification not associated with maltose metabolism are provided, which yeast produces during fermentation an increased amount of ethanol at the end of fermentation compared to the amount produced by an otherwise identical parental yeast when grown in a starch hydrolysate.
  • the yeast further comprises a genetic alteration that introduces a polynucleotide encoding a polypeptide in the phosphoketolase pathway.
  • the yeast further comprises an alteration in the glycerol pathway and/or the acetyl-CoA pathway.
  • the yeast further comprises a disruption of a gene encoding DLsl.
  • the yeast further comprises an exogenous gene encoding a carbohydrate processing enzyme.
  • the yeast is a Saccharomyces spp.
  • Figure 1 is a diagram of a typical MAL loci, and its regulation by maltose and glucose
  • Figure 2 depicts aA4AL23c expression cassette.
  • Figure 3 is a map of plasmid pHX19.
  • Figure 4 is comparison of cumulative CO2 pressure index during fermentation with strains FG and A28.
  • Figure 5 is a map of plasmid pZKAPlM-(H3C19).
  • Figure 6 is a map of plasmid pTOPO ⁇ -Blunt ura3-loxP-KanMX-loxP-ura3.
  • Figure 7 is a map of plasmid pGAL-Cre-316.
  • Figure 8 is comparison of cumulative CO2 pressure index during fermentation with RHY723 and its parent strain GPY10008.
  • alcohol refers to an organic compound in which a hydroxyl functional group (-OH) is bound to a saturated carbon atom.
  • 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.
  • engineered yeast cells refer to yeast that include genetic modifications and characteristics described herein. Variant/modified yeast do not include naturally occurring yeast.
  • polypeptide and protein 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • BLAST BLAST algorithm
  • Altschul et al. ((1990) J. Mol. Biol. 215:403-10) and Karlin et al. ((1993) Proc. Natl. Acad. Sci. USA 90:5873-87).
  • WU-BLAST-2 WU-BLAST-2 program (see, e.g., Altschul et al. (1996) et/?. 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.
  • 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 extension penalty 0.05
  • polypeptides are substantially identical.
  • first polypeptide is immunologically cross-reactive with the second polypeptide.
  • polypeptides that differ by conservative amino acid substitutions are immunologically cross- reactive.
  • 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).
  • 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.”
  • “constitutive” expression refers to the production of a polypeptide encoded by a particular gene under essentially all typical growth conditions, as opposed to “conditional” expression, which requires the presence of a particular substrate, temperature, or the like to induce or activate expression.
  • expressing a polypeptide refers to the cellular process of producing a polypeptide using the translation machinery (e.g., ribosomes) of the cell.
  • translation machinery e.g., ribosomes
  • overexpressing a polypeptide refers 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.
  • 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).
  • the terms "fused” and "fusion" with respect to two DNA fragments, such as a promoter and the coding region of a polypeptide refer to a physical linkage causing the two DNA fragments to become a single molecule.
  • 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.
  • protein of interest refers to a polypeptide that is desired to be expressed in modified yeast.
  • 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.
  • disruption of a gene refers broadly to any genetic or chemical manipulation, i.e., mutation, that substantially prevents a cell from producing a function gene product, e.g., a protein, in a host cell.
  • exemplary methods of disruption include complete or partial deletion of any portion of a gene, including a polypeptide-coding sequence, a promoter, an enhancer, or another regulatory element, or mutagenesis of the same, where mutagenesis encompasses substitutions, insertions, deletions, inversions, and combinations and variations, thereof, any of which mutations substantially prevent the production of a function gene product.
  • a gene can also be disrupted using RNAi, antisense, or any other method that abolishes gene expression.
  • a gene can be disrupted by deletion or genetic manipulation of non-adjacent control elements.
  • deletion of a gene refers to its removal from the genome of a host cell.
  • control elements e.g., enhancer elements
  • 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.
  • 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.
  • 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.
  • yeast cells have been "modified to prevent the production of a specified protein” if they have been genetically or chemically altered to prevent the production of a functional protein/polypeptide that exhibits an activity characteristic of the wild-type protein.
  • modifications include, but are not limited to, deletion or disruption of the gene encoding the protein (as described, herein), modification of the gene such that the encoded polypeptide lacks the aforementioned activity, modification of the gene to affect post-translational processing or stability, and combinations, thereof.
  • 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.
  • anaerobic fermentation refers to growth in the absence of oxygen.
  • end of fermentation refers to the point where a fermentation will no longer produce a significant amount of additional alcohol, i.e., no more than about 1% additional alcohol.
  • 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.
  • modified yeast harboring a constitutive transcriptional activator MAL allele produces more ethanol in a starch-hydrolysate- fermentation, and/or produces the same amount of ethanol in less time, than an otherwise identical parental yeast.
  • the additional ethanol produced does not appear to be the result, or at least solely the result, of maltose metabolism, as the small amount of maltose present in the starch hydrolysate remains basically unchanged following fermentation.
  • Yeast harboring constitutive transcriptional activator MAL allele also produces ethanol at a higher rate in a starch-hydrolysate-fermentation than an otherwise identical parental yeast.
  • the constitutive expression of the transcriptional activator MAL allele appears to produce an unexpected benefit to glucose metabolism in a high-glucose environment.
  • a constitutive transcriptional activator MAL allele results, in an increase in ethanol production, or rate of ethanol production, of at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%), at least 1.0%, at least 1.1%, at least 1.2%, or more. Although such an increase is modest, it amounts to a substantial increase in terms of volume, given the amount of ethanol currently being produced.
  • Constitutive transcriptional activator MAL alleles have been known for more than half-a-century, and are exemplified by those described in, e.g., 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) Mo/. Gen. Genet.
  • Yeast harboring constitutive transcriptional activator MAL alleles may also include other genetic manipulations that increase alcohol production, particularly modifications that are not associated with enhanced maltose metabolism.
  • Engineered yeast cells having a heterologous PKL pathway have been previously described (e.g., WO2015148272). These cells express heterologous PKL (EC 4.1.2.9) and PTA (EC 2.3.1.8), optionally with other enzymes, to channel carbon flux away from the glycerol pathway and toward the synthesis of acetyl-CoA, which is then converted to ethanol.
  • Such modified cells are capable of increased ethanol production in a fermentation process when compared to otherwise-identical parent yeast cells.
  • Dlsl encoded by YJL065c, is a 167-amino acid polypeptide subunit of the ISW2 yeast chromatin accessibility complex (yCHRAC), which contains Isw2, Itcl, Dpb3-like subunit (Dlsl), 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). Yeast having a genetic alteration that reduces the amount of functional Dlsl in the cell, in the absence of other genetic modifications, exhibit increased robustness in an alcohol fermentation, allowing higher- temperature, and potentially shorter, fermentations (data not shown).
  • Reduction in the amount of functional Dlsl produced in a cell can be accomplished by disruption of the YJL065c gene.
  • Disruption of the YJL065c gene can be performed using any suitable methods that substantially prevent expression of a function YJL065c gene product, i.e., Dlsl .
  • Exemplary methods of disruption as are known to one of skill in the art include but are not limited to: complete or partial deletion of the YJL065c gene, including complete or partial deletion of, e.g., the Dlsl-coding sequence, the promoter, the terminator, an enhancer, or another regulatory element; and complete or partial deletion of a portion of the
  • chromosome that includes any portion of the YJL065c gene.
  • Particular methods of disrupting the YJL065c gene include making nucleotide substitutions or insertions in any portion of the YJL065c gene, e.g., the Dlsl-coding sequence, the promoter, the terminator, an enhancer, or another regulatory element.
  • deletions, insertions, and/or substitutions are made by genetic manipulation using sequence-specific molecular biology techniques, as opposed to by chemical mutagenesis, which is generally not targeted to specific nucleic acid sequences. Nonetheless, chemical mutagenesis can, in theory, be used to disrupt the YJL065c gene.
  • 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 GPDl, GPD2, GPP1 and/or GPP2. See, e.g., U.S. Patent Nos. 9,175,270 (Elke et al), 8,795,998 (Pronk et al.) and 8,956,851 (Argyros et al.).
  • 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.
  • acetyl-CoA synthase also referred to acetyl-CoA ligase activity
  • scavenge i.e., capture
  • 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
  • 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.
  • 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 an acetylating acetaldehyde dehydrogenase and/or a pyruvate-formate lyase is not required because the need for these activities is obviated by the attenuation of the native biosynthetic pathway for making acetyl-CoA that contributes to redox cofactor imbalance.
  • the present yeast do not have a heterologous gene encoding an NAD+-dependent acetylating
  • the present modified yeast further comprises a butanol biosynthetic pathway.
  • 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 reductoisom erase, dihydroxy acid dehydratase, ketoisovalerate decarboxylase, and alcohol dehydrogenase activity.
  • 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.
  • the polypeptide having pyruvate decarboxylase activity is selected from the group consisting of: PDC1, PDC5, PDC6, and combinations thereof.
  • 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.
  • the present modified yeast cells do not further comprise a butanol biosynthetic pathway.
  • the present modified yeast may include any number of additional genes of interest encoding protein of interest, including selectable markers, carbohydrate-processing enzymes, and other commercially-relevant polypeptides, including but not limited to an enzyme selected from the group consisting of a dehydrogenase, a transketolase, a phosphoketolase, a transladolase, an epimerase, a phytase, a xylanase, a ⁇ -glucanase, a phosphatase, a protease, an a-amylase, a ⁇ -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
  • 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.
  • Yeast is a unicellular eukaryotic microorganism classified as members of the fungus kingdom and includes organisms from the phyla Ascomycota and Basidiomycota. Yeast that can be used for alcohol production include, but are not limited to, Saccharomyces spp., including S. cerevisiae, as well as Kluyveromyces, Lachancea and Schizosaccharomyces spp. Numerous yeast strains are commercially available, many of which have been selected or genetically engineered for desired characteristics, such as high alcohol production, rapid growth rate, and the like. Some yeast has been genetically engineered to produce
  • heterologous enzymes such as glucoamylase or a-amylase.
  • Alcohol production from a number of carbohydrate substrates including but not limited to corn starch, sugar cane, cassava, and molasses, is well known, as are innumerable variations and improvements to enzymatic and chemical conditions and mechanical processes.
  • the present compositions and methods are believed to be fully compatible with such substrates and conditions. High levels of maltose are not required to benefit from the present compositions and methods.
  • the concentration is less than about 10 g/L.
  • Liquefact corn mash slurry was prepared by adding 600 ppm of urea, 0.124 SAPU/g ds FERMGENTM 2.5x (acid fungal protease), 0.33 GAU/g ds CS4 (a variant Trichoderma glucoamylase) and 1.46 SSCU/g ds AKAA ⁇ Aspergillus kawachii a-amylase), adjusted to a pH of 4.8.
  • a MAL23c expression cassette consisting of a constitutive allele oiMAL23 (SEQ ID NO: 2) under control of a MAL23c promoter (SEQ ID NO: 3) and MAL23 terminator (SEQ ID NO: 4), was made using standard procedures. The nucleotide sequences are shown, below, and a representation of the cassette is illustrated in Figure 2:
  • a DNA fragment including flanking YDL227C locus sequences was made by amplifying the MAL23c expression cassette with appropriate primers.
  • the fragment was inserted into a plasmid designated pHX19, whichincludes an integrated constitutive MAL23c expression cassette at the Saccharomyces chromosome YDL227C locus, as shown in Figure 3.
  • the functional and structural composition of plasmid pHX19 is described in Table 1. Table 1. Functional and structural elements of plasmid pHX19
  • Plasmid pZKAPlm-(H3C19) was designed to integrate four individual polypeptide expression cassettes upstream of the AAPl locus (YHR047C).
  • the four cassettes were as follows (i) HXT3 promoter: :PKL C: :FBA1 terminator; (ii) PGK 1 promoter : :LpPT A: :PGK1 terminator; (iii) TDH3 promoter: :eutE A19: :ENO terminator; and (iv) PDC1
  • :AcsAl : :PDCl terminator which were designed to express codon-optimized genes encoding phosphoketolase (PKL), derived from Gardnerella vaginalis,
  • PTA phosphotransacetylase
  • Lactobacillus plantarum derived from Lactobacillus plantarum
  • acylating acetaldehyde dehydrogenase derived from Desulfospira joergensenii
  • acetyl-CoA synthase derived from Methanosaeta concilii, respectively.
  • a map of pZKAPlm-(H3C19) is shown in Figure 5. Functional and structural elements are detailed in Table 3.
  • HXT3 promoter S. cerevisiae HXT3 promoter
  • PKL C CDS encoding phosphoketolase from Gardnerella vaginalis, codon-optimized for yeast
  • the FG strain was used as the parent strain to make the ura3 auxotrophic strain FG-ura3.
  • the functional and structural elements of the plasmid are listed in Table 4.
  • a 2,018-bp DNA fragment containing the ura3-loxP-KanMX-loxP-ura3 cassette was released from plasmid TOPO II-Blunt ura3-loxP-KanMX-loxP-ura3 by EcoRI digestion. The fragment was used to transform S. cerevisiae FG cells by electroporation. [079] Transformed colonies able to grow on media containing G418 were streaked on synthetic minimal plates containing 20 ⁇ g/ml uracil and 2 mg/ml 5-fluoroorotic acid (5- FOA). Colonies able to grow on 5-FOA plates were further confirmed for URA3 deletion by growth of phenotype on SD-Ura plates, and by PCR.
  • the ura3 deletion transformants were unable to grow on SD-Ura plates. A single 1.98-kb PCR fragment was obtained with test primers. In contrast, the same primer pairs generated a 1.3-kb fragment using DNA from the parental FG strain, indicating the presence of the intact ura3 gene.
  • the ura3 deletion strain was named as FG-KanMX-ura3.
  • plasmid pGAL-Cre-316 depicted in Figure 7, was used to transform cells of strain FG-KanMX-ura3 by electroporation.
  • the purpose of using this plasmid is to temporary express the Cre enzyme, so that the LoxP-sandwiched KanMX gene will be removed from strain FG- KanMX-ura3 to generate strain FG-ura3.
  • pGAL-Cre-316 is a self-replicating circular plasmid that was subsequently removed from strain FG-ura3. None of the sequence elements from pGAL-cre-316 was inserted into the strain FG-ura3 genome.
  • the functional and structural elements of plasmid pGAL-Cre-316 is listed in Table 5.
  • the transformed cells were plated on SD-Ura plates. Single colonies were transferred onto a YPG plate and incubated for 2 to 3 days at 30°C. Colonies were then transferred to a new YPD plate for additional days. Finally, cell suspensions from the YPD plate were spotted on to following plates: YPD, G418 (150 5-FOA (2 mg/ml) and SD-Ura. Cells able to grow on YPD and 5-FOA, and unable to grow on G418 and SD-Ura plates, were picked for PCR confirmation as described, above.
  • the expected PCR product size was 0.4- kb and confirmed the identity of the KanMX (geneticin)-sensitive, wra3-deletion strain, derived from FG-KanMX-ura3. This strain was named as FG-ura3.
  • the FG-ura3 strain was used as a parent to introduce the PKL pathway.
  • Cells were transformed with a 14,993-bp KasI fragment containing the four expression cassettes from pZKAPlm-(H3C19) from Example 5.
  • a transformant with the KasI fragment integrated upstream of the YHR047C locus was selected and designated as strain GPY10000.
  • Strain GPY10008 was generated by Cas9-mediated deletion of YJL065c (which encodes Dlsl) in strain GPY10000. Specifically, a deletion was made from 4-bp before start codon to 10-bp before stop codon of YJL065c.
  • the strain GPY10008 was used as a parent to introduce the MAL23c expression cassette, in this case at the 3' region of the YHL041w locus, otherwise as described in Example 3.
  • GPY10008 cells were transformed with a PCR-amplified DNA fragment containing the MAL23c expression cassette made using the pHX19 plasmid as template and primers that include flanking YHL041w locus sequences.
  • the new FG yeast strain designated RHY723, and its parent strain GPY10008 were grown in An Koms bottles and their fermentation products were analyzed as described in Example 1. Performance in terms of ethanol, glycerol and acetate production is shown in Table 8, and the fermentation rate, represented by the CPI during the fermentation, is illustrated in Figure 8.

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Abstract

L'invention concerne des compositions et des procédés se rapportant à une levure modifiée présentant des allèles MAL d'activateurs transcriptionnels constitutifs. La levure produit une quantité accrue d'éthanol, ou présente un taux accru de production d'éthanol, qui ne semble pas être le résultat du métabolisme du maltose. Une telle levure est particulièrement utile pour la production d'éthanol utilisant des substrats d'amidon.
EP18760106.7A 2017-08-08 2018-08-08 Production accrue d'éthanol par une levure présentant des allèles mal Withdrawn EP3665266A1 (fr)

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EP2173881B1 (fr) * 2007-07-23 2016-09-28 DSM IP Assets B.V. Enzymes productrices d'acétyl-coa dans la levure
EP2060632A1 (fr) 2007-10-29 2009-05-20 Technische Universität Berlin Procédé pour la modification d'une cellule de levure pour la production d'éthanol
UA108853C2 (uk) * 2009-07-10 2015-06-25 Спосіб ферментації галактози
EP2277989A1 (fr) 2009-07-24 2011-01-26 Technische Universiteit Delft Production d'éthanol dépourvu de glycérol par fermentation
BR112013025753A8 (pt) 2011-04-05 2018-06-12 Lallemand Hungary Liquidity Man Llc Métodos para aprimoramento do rendimento de produto e produção em um micro-organismo através da adição de aceptores de elétrons alternativos
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UY37835A (es) 2019-03-29
WO2019032688A1 (fr) 2019-02-14
CN111201313B (zh) 2024-06-14

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