阅读说明：本技术 细胞色素b2在酵母中过表达用于增加乙醇生产 (Cytochrome B2 overexpression in yeast for increasing ethanol production ) 是由 D·J·马库尔 Q·Q·朱 于 2020-03-13 设计创作，主要内容包括：描述了涉及过表达细胞色素B2的经修饰的酵母的组合物和方法。与亲本细胞相比,所述酵母产生增加量的醇。这种酵母对于从淀粉底物大规模生产乙醇特别有用。(Compositions and methods relating to modified yeast overexpressing cytochrome B2 are described. The yeast produces an increased amount of alcohol compared to the parent cell. Such yeasts are particularly useful for large scale production of ethanol from starch substrates.)

1. A modified yeast cell derived from a parent yeast cell, the modified cell comprising a genetic alteration that causes the modified cell to produce an increased amount of a CYB2P polypeptide as compared to the parent cell, wherein the modified cell produces more ethanol during fermentation as compared to the amount of ethanol produced by an otherwise identical parent yeast cell.

2. The modified cell of claim 1, wherein the genetic alteration comprises introducing into the parent cell a nucleic acid capable of directing the expression of a CYB2P polypeptide at a level that is higher than that of a parent cell grown under equivalent conditions.

3. The modified cell of claim 1, wherein the genetic alteration comprises introduction of an expression cassette for expression of a CYB2P polypeptide.

4. The modified cell of any one of claims 1-3, wherein the increased amount of expression of the CYB2P polypeptide is at least about 500% as compared to the expression level in a parent cell grown under equivalent conditions.

5. The modified cell of any one of claims 1-3, wherein the mRNA encoding the CYB2P polypeptide is produced in an amount increased by at least about 1,000% as compared to the level in a parent cell grown under equivalent conditions.

6. The modified cell of any one of claims 1-3, wherein the increased amount of mRNA encoding the CYB2P polypeptide produced is at least about 5,000% as compared to the level in a parent cell grown under equivalent conditions.

7. The modified cell of any one of claims 1-6, wherein the cell further comprises an exogenous gene encoding a carbohydrate processing enzyme.

8. The modified cell of any one of claims 1-7, further comprising a PKL pathway.

9. The modified cell of any one of claims 1-8, further comprising an alteration in a glycerol pathway and/or an acetyl-CoA pathway.

10. The modified cell of any one of claims 1-9, further comprising an alternative pathway for producing ethanol.

11. The modified cell of any one of claims 1-10, wherein the cell belongs to a Saccharomyces species (Saccharomyces spp.).

12. A method for increasing alcohol production by a yeast cell grown on a carbohydrate substrate, the method comprising: introducing into a parent yeast cell a genetic alteration that increases production of a CYB2P polypeptide as compared to the amount produced in the parent cell.

13. The method of claim 12, wherein the cell having the introduced genetic alteration is a modified cell, the modified cell being the cell of any one of claims 1-3 and 7-11.

14. The method of claim 12 or 13, wherein the alcohol production is increased by at least 0.2%, at least 0.5%, at least 0.7%, or at least 1.0%.

15. The method of any one of claims 12-14, wherein CYB2P polypeptide is overexpressed by at least 5-fold, at least 10-fold, at least 50-fold, at least 80-fold, or even at least 100-fold.

Technical Field

This application claims priority from U.S. provisional patent application No. 62/818448, filed on 3, 14, 2019, the disclosure of which is incorporated by reference in its entirety.

The compositions and methods of the invention relate to modified yeast that overexpress cytochrome B2. The yeast produces an increased amount of ethanol compared to its parent cell. Such yeasts are particularly useful for large scale production of ethanol from starch substrates.

Background

The first generation of yeast-based ethanol production converted sugars to fuel ethanol. Annual fuel ethanol production by yeast worldwide is about 900 hundred million liters (Gombert, a.k. and van maris.a.j. (2015) curr. opin.biotechnol. [ state of the art ]33: 81-86). It is estimated that about 70% of the ethanol production cost is feedstock.

Butanol is an important industrial chemical and drop-in fuel (drop-in fuel) component that has a variety of applications, including use as a renewable fuel additive, raw chemicals in the plastic industry, and food grade extractants in the food and fragrance industry. Therefore, there is a high demand for alcohols (such as butanol and isobutanol) and efficient and environmentally friendly production methods.

In view of the world-wide production of large quantities of alcohol, even a small increase in the efficiency of the fermenting organism can produce a huge increase in the amount of alcohol available. Thus, there is a need for organisms that produce alcohols more efficiently.

Disclosure of Invention

The compositions and methods of the invention relate to modified yeast that overexpress cytochrome B2. Aspects and examples of the compositions and methods are described in the following independently numbered paragraphs.

1. In one aspect, a modified yeast cell derived from a parent yeast cell is provided, the modified cell comprising a genetic alteration that causes the modified cell to produce an increased amount of a CYB2P polypeptide as compared to the parent cell, wherein the modified cell produces more ethanol during fermentation as compared to the amount of ethanol produced by an otherwise identical parent yeast cell.

2. In some embodiments of the modified cell of paragraph 1, the genetic alteration comprises introducing into the parent cell a nucleic acid capable of directing the expression of a CYB2P polypeptide at a level higher than that of the parent cell grown under equivalent conditions.

3. In some embodiments of the modified cell of paragraph 1, the genetic alteration comprises introducing an expression cassette for expression of a CYB2P polypeptide.

4. In some embodiments of the modified cell of any of paragraphs 1-3, the increase in expression of the CYB2P polypeptide is at least about 500% as compared to the expression level in a parent cell grown under equivalent conditions.

5. In some embodiments of the modified cell of any of paragraphs 1-3, the mRNA encoding the CYB2P polypeptide is produced in an amount increased by at least about 1,000% as compared to the level in a parent cell grown under equivalent conditions.

6. In some embodiments of the modified cell of any of paragraphs 1-3, the mRNA encoding the CYB2P polypeptide is produced in an amount increased by at least about 5,000% as compared to the level in a parent cell grown under equivalent conditions.

7. In some embodiments of the modified cell of any of paragraphs 1-6, the cell further comprises an exogenous gene encoding a carbohydrate processing enzyme.

8. In some embodiments, the modified cell of any one of paragraphs 1-7 further comprises a PKL pathway.

9. In some embodiments, the modified cell of any of paragraphs 1-8 further comprises an alteration in the glycerol pathway and/or the acetyl-coa pathway.

10. In some embodiments, the modified cell of any of paragraphs 1-9 further comprises an alternative pathway for the production of ethanol.

11. In some embodiments of the modified cell of any one of paragraphs 1-10, the cell is of a Saccharomyces species (Saccharomyces spp.).

12. In another aspect, there is provided a method for increasing alcohol production by a yeast cell grown on a carbohydrate substrate, the method comprising: introducing into a parent yeast cell a genetic alteration that increases production of a CYB2P polypeptide as compared to the amount produced in the parent cell.

13. In some embodiments of the method of paragraph 12, the cell having the introduced genetic alteration is a modified cell, the modified cell being the cell of any one of paragraphs 1-3 and 7-11.

14. In some embodiments of the method of paragraph 12 or 13, the alcohol production is increased by at least 0.2%, at least 0.5%, at least 0.7%, or at least 1.0%.

15. In some embodiments of the method of any one of paragraphs 12-14, the CYB2P polypeptide is overexpressed by at least 5-fold, at least 10-fold, at least 50-fold, at least 80-fold, or even at least 100-fold.

These and other aspects and embodiments of the modified cells and methods of the invention will be apparent from the specification, including any drawings/figures.

Detailed Description

I. Definition of

Before describing the yeast and methods of the present invention in detail, the following terms are defined for the sake of clarity. Undefined terms should be accorded the conventional meaning used in the relevant art.

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

As used herein, the term "yeast cell", "yeast strain", or simply "yeast" refers to organisms from the phyla Ascomycota (Ascomycota) and Basidiomycota (Basidiomycota). An exemplary yeast is a budding yeast from the order Saccharomyces (Saccharomyces). A specific example of a yeast is a saccharomyces species, including but not limited to saccharomyces cerevisiae (s. Yeasts include organisms used to produce fuel alcohols as well as organisms used to produce potable alcohols, including specialty and proprietary yeast strains used to prepare uniquely tasting beer, wine, and other fermented beverages.

As used herein, the phrase "engineered yeast cell," "variant yeast cell," "modified yeast cell," or similar phrases, refers to a yeast that includes the genetic modifications and features described herein. Variant/modified yeasts do not include naturally occurring yeasts.

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

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

As used herein, the term "homologous protein" refers to a protein having similar activity and/or structure as a reference protein. This is not intended to imply that homologs necessarily correlate with evolution. Thus, the term is intended to encompass the same, similar, or corresponding (i.e., in structural and functional aspects) one or more enzymes obtained from different organisms. In some embodiments, it is desirable to identify homologs having similar quaternary, tertiary, and/or primary structures as the reference protein. In some embodiments, the homologous protein acts as a reference protein to induce a similar immune response or responses. In some embodiments, homologous proteins are engineered to produce enzymes having one or more desired activities.

The degree of homology between sequences may be determined using any suitable method known in the art (see, e.g., Smith and Waterman (1981) adv. Appl. Math. [ applied math progress ]2: 482; Needleman and Wunsch (1970) J.mol.biol. [ journal of molecular biology ],48: 443; Pearson and Lipman (1988) Proc.Natl.Acad.Sci.USA [ Proc.Acad.Sci.USA ]85: 2444; Wisconsin Genetics Software Package (Wisconsin Genetics Software Package) (Genetics Computer Group, Madison, Wis.) programs such as GAP, BESTFIT, FASTA and ASTFTA; and Devereux et al (1984) eic Acids Res. Nucleic acid research [ 12: 387. [ 95 ]: 387-95 ]).

For example, PILEUP is a useful program for determining the level of sequence homology. PILEUP creates multiple sequence alignments from a set of related sequences using progressive, pairwise alignments. It may 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 (1987) J. mol. Evol. [ J. molecular evolution ]35: 351-60). The method is similar to that described by Higgins and Sharp ((1989) CABIOS [ computer for biological applications ]5: 151-53). Useful PILEUP parameters include 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. [ journal of molecular biology ]215:403-10) and Karlin et al ((1993) Proc.Natl.Acad.Sci.USA [ Proc. Natl.Acad ]90: 5873-87). One particularly useful BLAST program is the WU-BLAST-2 program (see, e.g., Altschul et al, (1996) meth. enzymol. [ methods of enzymology ]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, a BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA [ Proc. Natl. Acad. Sci. USA ]89:10915) alignment (B) of 50, an expectation (E) of 10, M '5, N' -4, and a comparison of the two strands.

As used herein, the phrases "substantially similar" and "substantially identical" in the context of at least two nucleic acids or polypeptides typically mean that the polynucleotide or polypeptide comprises a sequence that is at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 91% identical, at least about 92% identical, at least about 93% identical, at least about 94% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, or even at least about 99% identical, or more, compared to a reference (i.e., wild-type) sequence. Percentage sequence identity was calculated using the CLUSTAL W algorithm with default parameters. See Thompson et al (1994) Nucleic Acids Res. [ Nucleic Acids research ]22: 4673-one 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 by conservative amino acid substitutions are immunologically cross-reactive. Thus, the polypeptide is substantially identical to the second polypeptide, e.g., where the two peptides differ only by 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., in the range of medium 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 nutritional profile of filamentous fungi is typically haploid, so a single copy (i.e., a single allele) of a given gene is sufficient to confer a given phenotype. The term "allele" is generally preferred when the organism contains more than one similar gene, 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, rather than "conditional" expression, which requires the presence of a particular substrate, temperature, etc. to induce or activate expression.

As used herein, the term "expressing a polypeptide" and similar terms refer to a cellular process that uses the translation machinery (e.g., ribosomes) of a cell to produce the polypeptide.

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

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

As used herein, the terms "fused" and "fusion" with respect to two DNA fragments (e.g., a promoter and a coding region of a polypeptide) refer to a physical linkage that causes the two DNA fragments to become 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 a gene, protein or strain that is not intentionally modified for the benefit of the presently described yeast.

As used herein, the term "protein of interest" refers to a polypeptide that is desired to be expressed in the modified yeast. Such proteins may be enzymes, substrate binding proteins, surface active proteins, structural proteins, selectable markers, signal transducers, receptors, transporters, transcription factors, translation factors, cofactors, and the like, and may be expressed. The protein of interest is encoded by an endogenous gene or a heterologous gene (i.e., the gene of interest) relative to the parent strain. The protein of interest may be expressed intracellularly or as a secreted protein.

As used herein, "disruption of a gene" broadly refers to any genetic or chemical manipulation (i.e., mutation) that substantially prevents a cell from producing a functional gene product (e.g., a protein) in a host cell. Exemplary disruption methods include deletion of any portion of the gene, either completely or partially (including polypeptide coding sequences, promoters, enhancers, or additional regulatory elements), or mutagenesis thereof, wherein mutagenesis encompasses substitutions, insertions, deletions, inversions, and combinations and variations thereof, any of which substantially prevents the production of a functional gene product. Genes can also be disrupted using CRISPR, RNAi, antisense, or any other method of eliminating gene expression. Genes can be disrupted by deletion or genetic manipulation of non-adjacent control elements. As used herein, "deletion of a gene" refers to the removal of the gene from the genome of a host cell. When a gene includes a control element (e.g., an enhancer element) that is not immediately adjacent to the coding sequence of the gene, deletion of the gene refers to deletion of the coding sequence, and optionally adjacent enhancer elements (e.g., including, but not limited to, promoter and/or terminator sequences), but deletion of non-adjacent control elements is not required. A gene deletion also refers to the deletion of a portion of the coding sequence, or a portion of the promoter that is immediately adjacent or not adjacent to the gene coding sequence, wherein the functional activity of the gene of interest is absent from the engineered cell.

As used herein, the terms "genetic manipulation" and "genetic alteration" are used interchangeably and refer to changes/alterations in nucleic acid sequences. Alterations may include, but are not limited to, substitutions, deletions, insertions, or chemical modifications of at least one nucleic acid in a nucleic acid sequence.

As used herein, a "functional polypeptide/protein" is a protein that has an activity (e.g., an enzymatic activity, a binding activity, a surface active property, a signal transducer, a receptor, a transporter, a transcription factor, a translation factor, a cofactor, etc.) and which has not been mutagenized, truncated, or otherwise modified to eliminate or reduce this activity. As noted, the functional polypeptide may be thermostable or thermolabile.

As used herein, a "functional gene" is a gene that can be used by a cellular component to produce an active gene product (typically a protein). Functional genes are counterparts of disrupted genes that are modified such that they are not available to, or have a reduced ability to, be used by cellular components to produce active gene products.

As used herein, a yeast cell has been "modified to prevent production of a given protein" if the yeast cell has been genetically or chemically altered to prevent production of a functional protein/polypeptide that exhibits the active characteristics of the wild-type protein. Such modifications include, but are not limited to, deletions or disruptions of the gene encoding the protein (as described herein), genetic modifications such that the encoded polypeptide lacks 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) broadly refers to any genetic or chemical manipulation that reduces or completely prevents the flux of biochemical substrates or intermediates through a metabolic pathway. Attenuation of pathways can be achieved by various well-known methods. Such methods include, but are not limited to: deletion of one or more genes in whole or in part, substitution 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 controlling the expression of one or more genes, engineering of the enzymes or mrnas encoding these enzymes for reduced stability, misdirecting the enzymes into cellular compartments that are less likely to interact with substrates and intermediates, use of interfering RNAs, etc.

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 fermentation stage when the economic advantage of continuous fermentation to produce small amounts of additional alcohol, in terms of fixed and variable costs, is outweighed by the cost of continuous fermentation. In a more general sense, "end of fermentation" refers to the point at which the fermentation will no longer produce significant additional alcohol, i.e., no more than about 1% additional alcohol, or no more substrate for further alcohol production remains.

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

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

DEG C

AA alpha-amylase

bp base pair

CYB2 cytochrome B2

DNA deoxyribonucleic acid

DS or DS dry solids

EC enzyme Committee

EtOH ethanol

FG FERMAX^TMGold

g or gm gram

g/L

GA glucoamylase

H₂O water

HPLC high performance liquid chromatography

hr or h hours

kg kilogram

M mol

mg of

min for

mL or mL

mM millimole

N equivalent concentration

nm nanometer

PCR polymerase chain reaction

parts per million ppm

STL1 sugar transporter-like polypeptide

Delta is related to deletion

Microgram of μ g

μ L and μ L microliter

Micromolar at μ M

Modified yeast cells with increased expression of Cyb2p

In Saccharomyces cerevisiae cytochrome B2(CYB 2; L-lactate dehydrogenase) is a component of the mitochondrial membrane space (mitogenic interface space). Cytochrome B2 converts L-lactate to pyruvate because it converts NAD + to NADH with a concomitant reverse reaction. CYB2 is essential for lactic acid utilization. Its expression is induced by lactic acid and inhibited by glucose and anaerobic conditions. The compositions and methods of the present invention are based on the following findings: overexpression of CYB2 at appropriate levels may increase ethanol production in saccharomyces.

In some embodiments, the increase in the amount of CYB2P polypeptide produced by the modified cell, as compared to the amount of CYB2P polypeptide produced by a parent cell grown under the same conditions, is an increase of at least 200%, at least 500%, at least 1000%, at least 2,500%, or even at least 5,000%, or more, during fermentation.

In some embodiments, the amount of CYB2P polypeptide produced by the modified cell is increased by at least 2-fold, at least 5-fold, at least 10-fold, at least 25-fold, or even at least 50-fold, or more during fermentation as compared to the amount of CYB2P polypeptide produced by a parent cell grown under the same conditions.

In some embodiments, the strength of the promoter used to control expression of the CYB2P polypeptide produced by the modified cell is increased at least 2 fold, at least 5 fold, at least 10 fold, at least 25 fold, at least 50 fold, at least 70 fold, at least 100 fold, or more compared to the strength of the native promoter controlling expression of CYB2P during fermentation.

In some embodiments, the increase in ethanol produced by the modified cell is an increase of at least 0.2%, at least 0.5%, at least 0.75%, at least 0.9%, at least 1.0%, or more, compared to the amount of ethanol produced by a parent cell grown under the same conditions.

Preferably, increased Cyb2p expression is achieved by genetic manipulation using sequence-specific molecular biology techniques, as opposed to chemical mutagenesis, which generally does not target specific nucleic acid sequences. However, chemical mutagenesis is not excluded as a method for preparing modified yeast cells.

In some embodiments, the compositions and methods of the invention comprise introducing into a yeast cell a nucleic acid capable of directing overexpression or increased expression of a Cyb2p polypeptide. Particular methods include, but are not limited to, (i) introducing an exogenous expression cassette for producing the polypeptide into a host cell, optionally also an endogenous expression cassette, (ii) replacing the exogenous expression cassette with an endogenous cassette that allows for increased production of the polypeptide, (iii) modifying the promoter of the endogenous expression cassette to increase expression, (iv) increasing the copy number of the same or different cassette used for CYB2 overexpression, and/or (v) modifying any aspect of the host cell to increase the half-life of the polypeptide in the host cell.

In some embodiments, the parent cell being modified already includes a gene of interest, e.g., a gene encoding a selectable marker, a carbohydrate processing enzyme, or other polypeptide. In some embodiments, the introduced gene is subsequently introduced into the modified cell.

In some embodiments, the modified parent cell already includes an engineered pathway of interest that increases ethanol production (e.g., the PKL pathway), or any other pathway that increases alcohol production.

The amino acid sequence of an exemplary Saccharomyces cerevisiae Cyb2p polypeptide is shown in SEQ ID NO: 1:

in some embodiments of the compositions and methods of the invention, the amino acid sequence of the Cyb2p polypeptide overexpressed in the modified yeast cell has at least about 70%, at least about 75%, at least about 80%, at least about 85%, 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. 1.

Amino acid sequence search several known Cyb2p molecules were identified within 90% amino acid sequence identity of an exemplary molecule with similar annotations (i.e., SEQ ID NO: 1). In particular embodiments of the compositions and methods of the invention, the amino acid sequence of the Cyb2p polypeptide overexpressed in the modified yeast cell has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or even at least 99% identity to SEQ ID No. 1 and/or one or more of the Cyb2p amino acid sequences referred to in table 1.

TABLE 1 cytochrome B2 proteins from public databases

Modified yeast cells with increased Cyb2p expression in combination with genes of the exogenous PKL pathway

Increased expression of Cyb2p can be combined with gene expression in the PKL pathway to further increase ethanol production. Engineered yeast cells with a heterologous PKL pathway have been previously described in WO 2015148272 (miasanikov et al). These cells express heterologous Phosphoketolases (PKLs), Phosphotransacetylases (PTA), and acetoacetyl dehydrogenases (AADH), optionally with other enzymes, to direct channel carbon flux away from the glycerol pathway and towards the synthesis of acetyl-coa, which is then converted to ethanol. Such modified cells are capable of increasing ethanol production during fermentation as compared to an otherwise identical parent yeast cell.

Combination of increased Cyb2p production with other mutations affecting alcohol production

In some embodiments, the modified yeast cells of the invention include additional modifications that affect ethanol production in addition to expressing increased amounts of the Cyb2p polypeptide, optionally in combination with the introduced exogenous PKL pathway.

The modified cell may further comprise a mutation that results in attenuation of the native glycerol biosynthetic pathway and/or the glycerol reuse pathway, which is 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 phosphoglycerate phosphatase (GPP) activity by disruption of one or more of the genes GPD1, GPD2, GPP1 and/or GPP 2. See, e.g., U.S. Pat. Nos. 9,175,270(Elke et al), 8,795,998(Pronk et al), and 8,956,851(Argyros et al). Method to enhance the reuse of the glycerol pathway by converting glycerol to dihydroxyacetone phosphate by over-expressing glycerol dehydrogenase (GCY1) and dihydroxyacetone kinase (DAK1) (Zhang et al (2013) J.Ind.Microbiol.Biotechnol. [ J.Industrial Microbiol.Biotechnol. ]40: 1153-60).

The modified yeast may be further characterized by an increased acetyl-CoA synthase (also known as 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 convert it to Ac-CoA. This in part reduces the undesirable effects of acetic acid on yeast cell growth and may further contribute to the improvement in alcohol yield. Increasing acetyl-coa synthase activity can be achieved by introducing a heterologous acetyl-coa synthase gene into the cell, increasing expression of an endogenous acetyl-coa synthase gene, and the like.

In some embodiments, the modified cell can further comprise a nucleic acid encoding a polypeptide having NAD⁺A heterologous gene for a protein dependent on acetylacetaldehyde dehydrogenase activity and/or a heterologous gene encoding pyruvate formate lyase. The introduction of such genes in combination with glycerol pathway attenuation is described, for example, in U.S. Pat. No. 8,795,998(Pronk et al). In some embodiments of the compositions and methods of the invention, the yeast is intentionally deficient in a gene encoding an acetylated acetaldehyde dehydrogenase,One or more heterologous genes for pyruvate formate lyase or both.

In some embodiments, the modified yeast cells of the invention can further overexpress a sugar transporter-like (STL1) polypeptide to increase glycerol uptake (see, e.g., Ferreira et al (2005) mol.biol.cell. [ cell molecular biology]16:2068-76；Et al (2015) mol. Microbiol. [ molecular microbiology]97:541-59 and WO 2015023989 a1) to increase ethanol production and decrease acetic acid.

In some embodiments, the modified yeast cell of the invention further comprises 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) pyruvic acid to acetolactic acid; (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 activities.

In some embodiments, the modified yeast cell comprising a butanol biosynthetic pathway further comprises a modification in the polynucleotide encoding a polypeptide having pyruvate decarboxylase activity. In some embodiments, the yeast cell comprises 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 cell further comprises a deletion, mutation, and/or substitution in one or more endogenous polynucleotides encoding FRA2, ALD6, ADH1, GPD2, BDH1, and YMR 226C.

Increased Cyb2p expression in combination with other beneficial mutations

In some embodiments, the modified yeast cells of the invention further comprise any number of additional genes of interest encoding proteins of interest, in addition to expressing increased Cyb2p polypeptide, optionally in combination with other genetic modifications that provide benefits. Additional genes of interest can be introduced before, during or after genetic manipulation that results in increased production of the Cyb2p polypeptide. Proteins of interest include selectable markers, carbohydrate processing enzymes, and other commercially relevant polypeptides, including but not limited to enzymes selected from the group consisting of: dehydrogenases, transketolases, phosphoketolases, transaldolases, epimerases, phytases, xylanases, beta-glucanases, phosphatases, proteases, alpha-amylases, beta-amylases, glucoamylases, pullulanases, isoamylases, cellulases, trehalases, lipases, pectinases, polyesterases, cutinases, oxidases, transferases, reductases, hemicellulases, mannanases, esterases, isomerases, pectinases, peroxidases, and laccases. The protein of interest may be secreted, glycosylated, and otherwise modified.

Use of modified yeast for increased alcohol production

The compositions and methods of the present invention include methods for increasing alcohol production and/or decreasing glycerol production in a fermentation reaction. Such methods are not limited to a particular fermentation process. The engineered yeast of the present invention is expected to be a "drop-in" alternative to conventional yeast in any alcohol fermentation facility. Although primarily used for fuel alcohol production, the yeast of the present invention may also be used for the production of potable alcohols, including wine and beer.

Yeast cells suitable for modification

Yeasts are unicellular eukaryotic microorganisms classified as members of the kingdom fungi and include organisms from the phylum ascomycota and basidiomycota. Yeasts that may be used for alcohol production include, but are not limited to Saccharomyces species, including Saccharomyces cerevisiae, and Kluyveromyces (Kluyveromyces), Lazarachia (Lachancea), and Schizosaccharomyces (Schizosaccharomyces) species. Many yeast strains are commercially available, many of which have been selected or genetically engineered to achieve desired characteristics, such as high ethanol production, rapid growth rates, and the like. Some yeasts have been genetically engineered to produce heterologous enzymes, such as glucoamylases or alpha-amylases.

Substrates and products

The production of alcohols from a number of carbohydrate substrates, including but not limited to corn starch, sugar cane, tapioca and molasses, is well known, as are numerous variations and improvements in enzymatic and chemical conditions and mechanical processes. The compositions and methods of the present invention are believed to be fully compatible with such substrates and conditions.

Alcohol fermentation products include organic compounds having hydroxyl functionality (-OH) bonded to a carbon atom. Exemplary alcohols include, but are not limited to, methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, n-pentanol, 2-pentanol, isopentanol, and higher alcohols. The most commonly produced fuel alcohols are ethanol and butanol.

These and other aspects and embodiments of the yeast strains and methods of the invention 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 of the invention

Example 1

Materials and methods

Preparing a liquefied substance:

the liquefact (corn mash slurry) was prepared by adding 600ppm urea, 0.124SAPU/g ds acid fungal protease, 0.33GAU/g ds variant Trichoderma reesei (Trichoderma reesei) glucoamylase and 1.46SSCU/g ds Aspergillus kawachi alpha-amylase, adjusted to pH 4.8 with sulfuric acid.

AnKom assay:

300 μ L of concentrated yeast overnight culture was added to each of a plurality of ANKOM bottles filled with 50g of the prepared liquefact (see above) to reach a final OD of 0.3. The flasks were then incubated at 32 ℃ for 55 hours with shaking at 150 RPM.

HPLC analysis:

samples from cultures assayed by AnKom were collected in Eppendorf tubes by centrifugation at 14,000RPM for 12 minutes. The supernatant was filtered with a 0.2 μ M PTFE filter and then used for HPLC (Agilent Technologies 1200 series) analysis under the following conditions: Bio-Rad Aminex HPX-87H column, running at 55 ℃ in 0.01NH₂SO₄The isocratic flow rate in (1) was 0.6ml/min and the injection volume was 2.5. mu.l. Calibration standards were used for quantification of acetic acid, ethanol, glycerol, glucose and other molecules. Unless otherwise stated, all values are reported in g/L.

RNA-Seq analysis:

RNA was prepared from individual samples according to the TRIzol method (Life technologies, Inc. (Life-Tech), Rokville, Md.). The RNA was then cleaned using Qiagen RNeasy mini kit (Qiagen, riermann, maryland). cDNA was generated from total mRNA of a single sample using a large-volume cDNA reverse transcription kit (Seimer Feishell science, Wilmington, Del.) from Applied Biosystems (Applied Biosystems). The cDNA of each sample prepared was sequenced using a shotgun method (shotgun method), and then quantified with respect to individual genes. The results are reported as readings of tens of millions of transcripts per kilobase (RPK10M) and are used to quantify the amount of each transcript in a sample.

Example 2

Expression of CYB2 in Yeast

RNA-Seq analysis was performed as described in example 1. As summarized in Table 2, yeast overexpressing STL1 was significantly higher than the parent yeast, i.e., FERMAX, with or without the exogenous Phosphoketolase (PKL) pathway^TMThe level of Gold (martex Inc., mn, usa; abbreviated herein as "FG") expresses CYB 2. Expression levels are expressed as reads of million transcripts per kilobase (RPK 10M).

TABLE 2 RNA-Seq analysis of CYB2 expression in different strains

The results indicate that this gene may be a target for increasing ethanol production in engineered yeast.

Example 3

Promoter selection for overexpression of CYB2

RNA-Seq analysis was performed to identify the promoter for overexpression of CYB2 during fermentation. The analysis was performed as described in example 1. Consistent with the results described in example 2, CYB2 expression in FG was lower within the first 24 hours of fermentation. In contrast, the actin (ACT1) gene was highly expressed at 6, 15 and 24hr into the fermentation. Thus, the ACT1 promoter was chosen for overexpression of CYB2 in yeast.

TABLE 3 transcription profiles of CYB2 and ACT1 in FG during fermentation

Example 4

Preparation of Cyb2p expression cassette

The CYB2 gene of Saccharomyces cerevisiae (YML054C locus, SEQ ID:2) was synthesized to produce CYB2 s. The ACT1 promoter (YFL039C locus; SEQ ID NO:3) and FBA1 terminator (YKL060C locus; SEQ ID NO:4) were operably linked to the coding sequence to produce the ACT1Pro:: CYB2ss:: Fba1Ter expression cassette. At FERMAX^TMThis expression cassette was introduced at position 350000 of chromosome II of Gold (Ma Cui Co., Minn., USA; abbreviated herein as "FG"), the FERMAX^TMGold is a well-known fermenting yeast used in the grain ethanol industry. The desired insertion of the CYB2s expression cassette in both parental strains was confirmed by PCR.

The amino acid sequence of the CYB2 polypeptide is shown as the following SEQ ID NO: 1:

the DNA sequence of the CYB2 coding region is shown in SEQ ID NO: 2:

the ACT1 promoter region for CYB2s overexpression is shown in SEQ ID NO 3:

the terminator region of FBA1 for overexpression of CYB2s is shown in SEQ ID NO 4:

example 5

Alcohol production by yeast overexpressing CYB2

One PCR positive strain overexpressing CYB2s under the control of the ACT1 promoter and its parental strain FG were tested in an Ankom assay containing 50g of liquefact. Fermentation was carried out at 32 ℃ for 65 hours. Samples at the end of fermentation were analyzed by HPLC. The experiment was repeated several times and typical results are summarized in table 4. Data are the average of duplicate samples for each strain.

TABLE 4 HPLC results of FG and FG-CYB2 strains

Overexpression of CYB2s resulted in an increase in ethanol production of about 1.0% in FG yeast, which is considered to be a robust, high ethanol producing yeast for the fuel ethanol industry. These results indicate that CYB2 overexpression is beneficial for increasing ethanol.