阅读说明：本技术 具有改善的醇生产的酵母 (Yeast with improved alcohol production ) 是由 C·E·G·帕扬 M·齐 于 2018-06-04 设计创作，主要内容包括：本文描述了与酵母细胞相关的组合物和方法,所述酵母细胞具有产生增加的醇生产的基因突变。此类酵母非常适合用于醇生产以减少发酵时间和/或增加产量。(Described herein are compositions and methods related to yeast cells having a genetic mutation that results in increased alcohol production. Such yeasts are well suited for alcohol production to reduce fermentation time and/or increase yield.)

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 a reduced amount of a functional Cpr1 polypeptide compared to the parent cell, wherein the modified cell produces an increased amount of ethanol during fermentation compared to the parent cell under equivalent fermentation conditions.

2. The modified cell of claim 1, wherein said genetic alteration comprises a disruption of the YDR155c gene present in said parent cell.

3. The modified cell of claim 2 wherein the disruption of the YDR155c gene is the result of deletion of all or a portion of the YDR155c gene.

4. The modified cell of claim 2, wherein the disruption of the YDR155c gene is the result of deletion of portion of genomic DNA comprising the YDR155c gene.

5. The modified cell of claim 2, wherein the disruption of the YDR155c gene is the result of mutagenesis of the YDR155c gene.

6. The modified cell of any of claims 2-5, wherein the disruption of the YDR155c gene is performed in combination with the introduction of a gene of interest at the genetic locus of the YDR155c gene.

7. The modified cell of any of claims 1-6, wherein the cell does not produce a functional Cpr1 polypeptide.

8. The modified cell of any of claims 1-6, wherein the cell does not produce a Cpr1 polypeptide.

9. The modified cell of any of claims 1-8, wherein the cell further comprises steps an exogenous gene encoding a carbohydrate processing enzyme.

10. The modified cell of any of claims 1-9, further comprising an alteration in the glycerol pathway and/or acetyl-coa pathway at steps.

11. The modified cell of any of claims 1-10, further steps comprising an alternative pathway for the production of ethanol.

12. The modified cell of any of claims 1-11, further comprising steps of disruption of the YJL065 gene present in the parental cell.

13. The modified cell of any of claims 1-12, wherein the cell does not produce a functional Dls1 polypeptide.

14. The modified cell of any of claims 1-13, wherein the cell belongs to the genus Saccharomyces (Saccharomyces) species.

15. The modified cell of any of claims 1-14, wherein the amount of ethanol produced by the modified yeast cell and the parent yeast cell is measured at 24 hours after seeding with a hydrolyzed starch substrate comprising 34% -35% dissolved solids and having a pH of 4.8-5.4.

16. a method for producing a modified yeast cell comprising introducing into a parent yeast cell a genetic alteration that reduces or prevents production of a functional Cpr1 polypeptide compared to the parent cell, thereby producing a modified cell that produces an increased amount of ethanol during fermentation compared to the parent cell under equivalent fermentation conditions.

17. The method of claim 16, wherein the genetic alteration comprises disruption of the YDR155c gene in the parent cell by genetic manipulation.

18. The method of claim 16 or 17, wherein the genetic alteration comprises deletion of the YDR155c gene in the parental cell using genetic manipulation.

19. The method of any of claims 16-18, wherein the disruption of the YDR155c gene is performed in combination with the introduction of a gene of interest at the genetic locus of the YDR155c gene.

20. The method of any of claims 16-19, wherein the disruption of the YDR155c gene is performed in combination with effecting an alteration in the glycerol pathway and/or the acetyl-coa pathway.

21. The method of any of claims 16-20, wherein the disruption of the YDR155c gene is performed in combination with the addition of an alternative pathway for the production of ethanol.

22. The method of any of claims 16-21, wherein the disruption of the YDR155c gene is performed in combination with disrupting a YJL065 gene present in the parental cell.

23. The method of any of claims 16-22, wherein the disrupting of the YDR155c gene is performed in combination with introducing an exogenous gene encoding a carbohydrate processing enzyme.

24. The method of any of claims 16-23, wherein the modified cell is from a saccharomyces species.

25. In embodiments of the method of any of claims 16-24, the amount of ethanol produced by the modified yeast cell and the parent yeast cell is measured at 24 hours after seeding with a hydrolyzed starch substrate comprising 34% -35% dissolved solids and having a pH of 4.8-5.4.

26. A modified yeast cell produced by the method of any of claims 16-25, which is .

Technical Field

The strains and methods of the invention relate to yeast having genetic mutations that result in increased ethanol production. Such yeasts are well suited for alcohol production to reduce fermentation time and/or increase yield.

Background

Many countries produce fuel alcohols from fermentable substrates (e.g., corn starch, sugar cane, tapioca, and molasses). According to the renewable fuels society (washington, d.c.) in the united states alone, 2015 fuel ethanol production is nearly 150 billion gallons.

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

Methods are described that involve yeast cells having modifications that increase alcohol production. Aspects and embodiments of the compositions and methods are described in the following independently numbered paragraphs.

1. In aspects, there is provided a modified yeast cell derived from a parent yeast cell, the modified cell comprising a genetic alteration that causes the modified cell to produce a reduced amount of a functional Cpr1 polypeptide as compared to the parent cell, wherein the modified cell produces an increased amount of ethanol during fermentation as compared to the parent cell under equivalent fermentation conditions.

2. In embodiments of the modified cell of paragraph 1, the genetic alteration comprises a disruption of the YDR155c gene present in the parental cell.

3. In embodiments of the modified cell of paragraph 2, the disruption of the YDR155c gene is the result of a deletion of all or part of the YDR155c gene.

4. In embodiments of the modified cell of paragraph 2, the disruption of the YDR155c gene is the result of a deletion of portion of genomic DNA comprising the YDR155c gene.

5. In embodiments of the modified cell of paragraph 2, the disruption of the YDR155c gene is the result of mutagenesis of the YDR155c gene.

6. In embodiments of the modified cell of any of paragraphs 2-5, the disrupting of the YDR155c gene is performed in combination with introducing a gene of interest at the genetic locus of the YDR155c gene.

7. In embodiments of the modified cell of any of paragraphs 1-6, the cell does not produce a functional Cpr1 polypeptide.

8. In embodiments of the modified cell of any of paragraphs 1-6, the cell does not produce a Cpr1 polypeptide.

9. In embodiments of the modified cell of any of paragraphs 1-8, the cell further comprises an exogenous gene encoding a carbohydrate processing enzyme.

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

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

12. In embodiments, the modified cell of any of paragraphs 1-11 further comprises a step disruption of the YJL065 gene present in the parental cell.

13. In embodiments of the modified cell of any of paragraphs 1-12, the cell does not produce a functional Dls1 polypeptide.

14. In embodiments of the modified cell of any of paragraphs 1-13, the cell belongs to a Saccharomyces (Saccharomyces) species.

15. In embodiments of the modified cell of any of paragraphs 1-14, the amount of ethanol produced by the modified yeast cell and the parent yeast cell is measured at 24 hours after seeding with a hydrolyzed starch substrate comprising 34% -35% dissolved solids and having a pH of 4.8-5.4.

16. In another aspect , a method for producing a modified yeast cell is provided, the method comprising introducing into a parent yeast cell a genetic alteration that reduces or prevents production of a functional Cpr1 polypeptide as compared to the parent cell, thereby producing a modified cell that produces an increased amount of ethanol as compared to the parent cell during fermentation under equivalent fermentation conditions.

17. In embodiments of the method of paragraph 16, the genetic alteration comprises disrupting by genetic manipulation the YDR155c gene in the parental cell.

18. In embodiments of the method of paragraph 16 or 17, the genetic alteration comprises deleting the YDR155c gene in the parental cell using genetic manipulation.

19. In embodiments of the method of any of paragraphs 16-18, the disrupting of the YDR155c gene is performed in combination with introducing a gene of interest at a genetic locus of the YDR155c gene.

20. In embodiments of the method of any of paragraphs 16-19, the disrupting of the YDR155c gene is performed in combination with making an alteration in the glycerol pathway and/or the acetyl-coa pathway.

21. In embodiments of the method of any of paragraphs 16-20, the disrupting of the YDR155c gene is performed in combination with adding an alternative pathway for the production of ethanol.

22. In embodiments of the method of any of paragraphs 16-21, the disrupting of the YDR155c gene is performed in combination with disrupting the YJL065 gene present in the parental cell.

23. In embodiments of the method of any of paragraphs 16-22, the disrupting of the YDR155c gene is performed in combination with introducing an exogenous gene encoding a carbohydrate processing enzyme.

24. In embodiments of the method of any of paragraphs 16-23, the modified cell is from a saccharomyces species.

25. In embodiments of the method of any of paragraphs 16-24, the amount of ethanol produced by the modified yeast cell and the parent yeast cell is measured at 24 hours after seeding with a hydrolyzed starch substrate comprising 34% -35% dissolved solids and having a pH of 4.8-5.4.

26. In another aspect , there is provided a modified yeast cell produced by the method of any of paragraphs 16-25 .

These and other aspects and embodiments of the modified cells and methods herein will be apparent from the specification.

Detailed Description

I. Overview

The compositions and methods of the invention relate to modified yeast cells that exhibit increased ethanol production compared to their parent cells. When used in ethanol production, the modified cells can increase yield and/or shorten fermentation time, thereby increasing the supply of ethanol for worldwide consumption.

Definition of

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

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

As used herein, "butanol" refers to the butanol isomers 1-butanol, 2-butanol, tert-butanol, and/or isobutanol (also referred to as 2-methyl-1-propanol) alone or in mixtures thereof.

As used herein, a "yeast cell" yeast strain, or simply "yeast" refers to an organism from ascomycete (Ascomycota) and basidiomycete (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.

As used herein, the phrase "variant yeast cell," "modified yeast cell," or similar phrases (see above) 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 phrase "substantially inactive" or similar phrases means that the particular activity is not detectable in the mixture or is present in an amount that does not interfere with the intended purpose of the mixture.

As used herein, the terms "polypeptide" and "protein" (and their respective plural forms) are used interchangeably to refer to a polymer of any length comprising amino acid residues joined by peptide bonds, the conventional -letter or three-letter code for amino acid residues is used herein, and all sequences are presented in the N-terminal to C-terminal direction.

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

As used herein, the term "homologous protein" refers to a protein having similar activity and/or structure to a reference protein.

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 (Wisconsins Software Package) (Genetics Computer Group, Madison, Wis Corp.) programs such as GAP, BESTFIT, ThTA and ASTA; and Microson et al (1984) eic Acids Res [ Nucleic acid research ]12: 387. [ Nucleic acid research ] 95).

PILEUP, for example, is a useful program for determining the level of sequence homology PILEUP creates a multiple sequence alignment from sets of related sequences using progressive, pairwise alignments it can also plot trees that show the clustering relationships used to create the alignment PILEUP uses a simplification of the progressive alignment methods of Feng and Doolittle (1987) J. mol. Evol. [ journal of molecular evolution ]35: 351-60.) this method is similar to the methods 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 weighted end gaps- examples of useful algorithms are the Alternal algorithms, as described by Altschul et al ((J. mol. biol. [ biol. ]215: 10) and the methods described by Altschul et al (USA.) see, USA. Sorbet al., USA, 15, USA, 15, USA, 15, USA, 15, USA, 15, USA, 15, USA.

As used herein, the phrases "substantially similar" and "substantially identical" in the context of at least two Nucleic Acids or polypeptides typically means that the polynucleotide or polypeptide comprises a sequence that has at least about 70% congealing , at least about 75% congealing , at least about 80% congealing 0, at least about 85% congealing 1, at least about 90% congealing 2, at least about 91% congealing 3, at least about 92% congealing 4, at least about 93% congealing 5, at least about 94% congealing , at least about 95% congealing , at least about 96% congealing , at least about 97% congealing , at least about 98% congealing , or even at least about 99% congealing , or higher congealing properties as compared to a reference (i.e. wild type) sequence with default parameters the clusta W algorithm calculates the percent congealing properties of the sequence with default parameters.

Typically, the polypeptide is substantially identical to the second polypeptide, e.g., where two peptides differ only by conservative substitutions.another indication that two nucleic acid sequences are substantially identical is that two molecules hybridize to each other under stringent conditions (e.g., within a 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 particular phenotype.

As used herein, the terms "wild-type" and "native" are used interchangeably and refer to a naturally found gene, protein or strain.

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, etc., and can be expressed at high levels. The protein of interest is encoded by a modified 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, "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., 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.

As used herein, "disruption of a gene" broadly refers to any genetic or chemical manipulation (i.e., mutation) that substantially prevents a cell in a host cell from producing a functional gene product (e.g., a protein). 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 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, the terms "genetic manipulation" and "genetic alteration" are used interchangeably and refer to changes/alterations of a nucleic acid sequence.

As used herein, "major genetic determinant" refers to a gene or genetic manipulation thereof that is necessary and sufficient to confer a particular phenotype in the absence of other genes or genetic manipulations thereof, however, a particular gene is necessary and sufficient to confer a particular phenotype, which does not exclude the possibility that additional effects on the phenotype may be achieved by further genetic manipulation of .

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, etc.) and which has not been mutagenized, truncated, or otherwise modified to abolish or reduce that 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 particular 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) refers broadly to any genetic or chemical manipulation that reduces or completely blocks the flux of biochemical substrates or intermediates through a metabolic pathway.

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 singular articles "/ (a/an)" and "the" encompass a plurality of referents unless the context clearly dictates otherwise.

DEG C

AA α -Amylase

bp base pair

DNA deoxyribonucleic acid

Degree of DP polymerization

DS or DS dry solids

EtOH ethanol

g or gm gram

g/L

GA glucoamylase

GAU/g ds glucoamylase units/g dry solids

H₂O water

HPLC high performance liquid chromatography

hr or h hours

kg kilogram

M moles of

mg of

mL or mL

ml/min

mM millimolar

N normal

nm nanometer

PCR polymerase chain reaction

ppm parts per million parts

SAPU/g ds protease units/g dry solids

SSCU/g ds fungus α -Amylase Unit/g Dry solids

Delta is related to deletion

Microgram of μ g

μ L and μ L microliter

Micromolar of μ M

Modified yeast cells with reduced or eliminated Cpr1 activity

In aspects, modified yeast cells are provided that have genetic alterations that result in cells of the modified strain producing reduced amounts of functional Cpr1 polypeptide (alternatively referred to as Cpr1p or YDR155c polypeptide) compared to an otherwise identical parent cell Cpr1 is a peptidyl-prolyl cis-trans isomerase of amino acids that accelerates protein folding, Cpr1 is localized to the nucleus and is believed to have multiple roles in chromatin structure, cell division and transport (Hashimi, H. and Nishikawa, T. (1993) biom. Biophys. acta [ Proc. biochem ]1161: 161-67; Brown, C.R. et al, (2001) J.biol. chem [ journal of biochemistry ].276:48017 and Areval-Roiguez, M. and Heityot [ cell ] Eurka [ 2005 ] Eucell 29: 17.

Applicants have discovered that yeast with genetic alterations that affect Cpr1 function exhibit increased ethanol production in fermentation, which may improve yield, shorten fermentation time, or both. Shorter fermentation times allow the alcohol production facility to perform more fermentations in a given period of time to increase productivity. Shorter fermentation times and higher fermentation temperatures also reduce the risk of contamination during fermentation and, depending on the environmental conditions, reduce the need to cool the fermentation reaction to maintain yeast viability.

Because disruption of the YDR155c gene is the primary genetic determinant conferring an increased ethanol production phenotype to a modified cell, in certain embodiments the modified cell need only comprise the disrupted YDR155c gene, while all other genes may remain intact.

Disruption of the YDR155c gene can be performed using any suitable method that substantially prevents the expression of a functional YDR155c gene product (i.e., Cpr1) exemplary disruption methods as known to those skilled in the art include, but are not limited to, complete or partial deletion of the YDR155c gene, including complete or partial deletion of, for example, the Cpr1 coding sequence, promoter, terminator, enhancer, or another regulatory elements, and complete or partial deletion of the portion of the chromosome containing any portion of the YDR155c gene, specific methods of disrupting the YDR155c gene include nucleotide substitutions or insertions in any portion of the YDR155c gene (e.g., the Cpr1 coding sequence, promoter, terminator, enhancer, or another regulatory element).

Mutations in the YDR155c gene may reduce the efficiency of the YDR155c promoter, reduce the efficiency of the YDR155c enhancer, interfere with splicing or editing of YDR155c mRNA, interfere with translation of YDR155c mRNA, introduce a stop codon into the YDR155c coding sequence to prevent translation of the full-length tdydr 155c protein, alter the coding sequence of the Cpr1 protein to produce a less active or inactive protein or reduce interaction of Cpr1 with other nucleoprotein components or DNA, alter the coding sequence of the Cpr1 protein to produce a less stable protein or target protein for disruption, cause misfolding or mismodification of the Cpr1 protein (e.g., by glycosylation), or interfere with cellular trafficking of the Cpr1 protein in some embodiments of these and other genetic manipulations are used to reduce or prevent expression of functional Cpr1 protein, or reduce or prevent normal biological activity of Cpr 1.

In embodiments, the modified cell herein comprises a genetic manipulation that reduces or prevents expression of a functional Cpr1 protein, or reduces or prevents the normal biological activity of Cpr 1.

In some embodiments, the decrease in the amount of functional Cpr1 polypeptide in the modified cell is a decrease of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more compared to the amount of functional Cpr1 polypeptide in a parent cell grown under the same conditions in some embodiments, the decrease in the expression of functional Cpr1 protein in the modified cell is a decrease of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more compared to the amount of functional Cpr1 polypeptide in a parent cell grown under the same conditions.

In some embodiments of , the increase in alcohol in the modified cell is an increase of at least 1%, at least 2%, at least 3%, at least 4%, at least 5% or more compared to the amount of alcohol produced in a parent cell grown under the same conditions.

Preferably, disruption of the YDR155c gene is performed by genetic manipulation using sequence-specific molecular biology techniques, as opposed to chemical mutagenesis, which generally does not target a particular nucleic acid sequence. However, chemical mutagenesis is not excluded as a method for preparing modified yeast cells.

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

The amino acid sequence of an exemplary Saccharomyces cerevisiae Cpr1 polypeptide is shown in SEQ ID NO: 1:

based on a BLAST search of the NCBI protein database, the Cpr1 polypeptide is 100% identical to at least ten deposits:

TABLE 1 comparison of SEQ ID NO 1 with other Saccharomyces cerevisiae Cpr1 polypeptides

Description of the invention	E value	% homology of	GenBank accession number
				Cpr1p [ Saccharomyces cerevisiae S288c]	2.8E-79	100％	NP_010439.1
Cpr1p [ Saccharomyces cerevisiae VL3 ]]	2.8E-79	100％	EGA87502.1
				Cpr1p [ Saccharomyces cerevisiae AVRI796]	2.8E-79	100％	EGA75445.1
Cpr1p [ Saccharomyces cerevisiae RM11-1a]	2.8E-79	100％	EDV08157.1
				Cpr1p [ Saccharomyces cerevisiae Kyokai No.7]	2.8E-79	100％	GAA22386.1
Cpr1p [ Saccharomyces cerevisiae JAY291]	2.8E-79	100％	EEU04638.1.1
				Cpr1p [ Saccharomyces cerevisiae Foster SO]	2.8E-79	100％	EGA63002.1
Cpr1p [ Saccharomyces cerevisiae YJM789]	2.8E-79	100％	EDN60494.1
				Cpr1p [ Saccharomyces cerevisiae Vin13 ]]	2.8E-79	100％	EGA79484.1
Cpr1p [ Saccharomyces cerevisiae CEN. PK113-7D]	2.8E-79	100％	EIW11359.1

It is contemplated that the compositions and methods of the present invention may be applied to other structurally similar Cpr1 polypeptides, as well as other related proteins, homologs, and functionally similar polypeptides.

In embodiments of the compositions and methods of the invention, the amino acid sequence of the Cpr1 protein altered at the production level has a certain degree of global amino acid sequence identity with the amino acid sequence of SEQ ID No. 1, e.g., 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 with SEQ ID No. 1.

In embodiments of the compositions and methods of the invention, the disrupted YDR155c gene encodes a Cpr1 protein having a particular degree of global amino acid sequence identity to the amino acid sequence of SEQ ID No. 1, e.g., 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.

The amino acid sequence information provided herein readily allows the skilled person to identify the Cpr1 protein and the nucleic acid sequence encoding the Cpr1 protein in any yeast and to generate appropriate disruptions in the YDR155c gene to affect the production of the Cpr1 protein.

In some embodiments, the decrease in the amount of functional Cpr1 polypeptide in the modified cell is a decrease of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more compared to the amount of functional Cpr1 polypeptide in a parent cell grown under the same conditions in some embodiments, the decrease in the expression of functional Cpr1 protein in the modified cell is a decrease of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more compared to the amount of functional Cpr1 polypeptide in a parent cell grown under the same conditions.

In embodiments, the modified cell has an increase in ethanol production of at least 0.2%, at least 0.4%, at least 0.6%, at least 0.8%, at least 1.0%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2.0% or more, as compared to an otherwise identical parent cell.

Modified yeast cells with reduced Cpr1 expression and reduced Dls1 expression

In some embodiments of , the modified yeast cells of the invention express a reduced amount of functional Dls1 polypeptide further in addition to expressing a reduced amount of functional Cpr1 polypeptide.

Dls1, encoded by YJL065c, is a polypeptide subunit of 167 amino acids of the ISW2 yeast chromatin accessibility complex (yCHRAC) that contains ISW2, Itc1, Dpb 3-like subunits (Dls1) and Dpb4 (see, e.g., Peterson, c.l. (1996) curr. opin. genet.dev. [ recent views of genetics and development ]6:171-75 and Winston, f. and Carlson, M. (1992) Trends Genet. [ genetic Trends ]8: 387-91). Applicants have determined that in the absence of other genetic modifications, yeast with genetic alterations that reduce the amount of functional Dls1 in the cells exhibit enhanced robustness in alcoholic fermentation, which allows for higher temperatures and possibly shorter fermentations (data not shown).

The disruption of the YJL065c gene may be performed using any suitable method that substantially prevents expression of the functional YJL065c gene product (i.e., Dls 1). exemplary disruption methods as 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, the Dls1 coding sequence, promoter, terminator, enhancer, or other regulatory elements, and complete or partial deletion of portions of the chromosome containing any portion of the YJL065c gene.

Mutations in the YJL065c gene can reduce the efficiency of the YJL065c promoter, reduce the efficiency of the YJL065c enhancer, interfere with splicing or editing of the YJL065c mRNA, interfere with translation of the YJL065c mRNA, introduce a stop codon into the YJL065c coding sequence to prevent translation of the full-length tYJL065c protein, alter the coding sequence of the Dls1 protein to produce a less active or inactive protein or reduce interactions of Dls1 with other nuclear protein components or DNA, alter the coding sequence of the Dls1 protein to produce a less stable protein or target protein for disruption, cause the Dls1 protein to misfold or be incorrectly folded (e.g., by glycosylation), or interfere with cellular trafficking of the Dls1 protein in some embodiments these and other genetic manipulations are used to reduce or prevent expression of the functional Dls1 protein, or reduce or prevent normal biological activity of Dls 1.

In some embodiments, the modified cell of the invention comprises a genetic manipulation that reduces or prevents expression of a functional Dls1 protein, or reduces or prevents normal biological activity of Dls1, and an additional mutation that reduces or prevents expression of a functional Isw2, Itc1, or Dpb4 protein, or reduces or prevents normal biological activity of an Isw2, Itc1, or Dpb4 protein in some embodiments, the modified cell of the invention comprises a genetic manipulation that reduces or prevents expression of a functional Dls1 protein, or reduces or prevents normal biological activity of a Dls1 protein, but does not have an additional mutation that reduces or prevents expression of a functional Isw2, Itc1, or Dpb4 protein, or reduces or prevents normal biological activity of an Isw2, Itc1, or Dpb4 protein.

The amino acid sequence of an exemplary Saccharomyces cerevisiae Dls1 polypeptide is set forth in SEQ ID NO: 3:

based on such BLAST and Clustal W data, it is apparent that the exemplary Saccharomyces cerevisiae Dls1 polypeptide (SEQ ID NO:3) shares a very high degree of sequence identity with other known Saccharomyces cerevisiae Dls1 polypeptides and Dls1 polypeptides from other Saccharomyces species.

In embodiments of the compositions and methods of the invention, the amino acid sequence of the disrupted Dls1 protein has an overall amino acid sequence identity to the amino acid sequence of SEQ ID No. 3 of 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 .

Preferably, disruption of the YJL065c gene is performed by genetic manipulation using sequence-specific molecular biology techniques, as opposed to chemical mutagenesis, which generally does not target a particular nucleic acid sequence. However, chemical mutagenesis is not excluded as a method for preparing modified yeast cells.

In embodiments, the reduction in the amount of functional Dls1 polypeptide in the modified cell is a reduction of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more as compared to the amount of functional Dls1 polypeptide in a parent cell grown under the same conditions in embodiments, the reduction in the expression of functional Dls1 protein in the modified cell is a reduction of at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99% or more as compared to the amount of functional Dls1 polypeptide in a parent cell grown under the same conditions.

In embodiments, the additional increase in ethanol production in the modified cell is an increase of at least 0.2%, at least 0.4%, at least 0.6%, at least 0.8%, at least 1.0%, at least 1.2%, at least 1.4%, at least 1.6%, at least 1.8%, at least 2.0% or more compared to a cell that only decreases Cpr1 expression.

V. increased Cpr1 expression in combination with other mutations affecting alcohol production

In some embodiments of , in addition to expressing a reduced amount of a functional Cpr1 polypeptide, optionally in combination with reducing expression of a functional Dls1 polypeptide, the modified yeast cells of the invention further comprise additional modifications that affect ethanol production.

In particular embodiments, the modified yeast cell comprises an artificial or alternative pathway resulting from the introduction of a heterologous phosphotransketolase gene and a heterologous phosphotransacetylase gene. Exemplary phosphoketolases can be obtained from Gardnerella vaginalis (Gardnerella vaginalis) (Uniprot/TrEMBL accession No.: WP-016786789). Exemplary phosphotransacetylases are available from Lactobacillus plantarum (UniProt/TrEMBL accession number: WP _ 003641060).

The modified cells of the invention may further comprise steps including mutations that result in attenuation of the native glycerol biosynthetic pathway, which are known to increase alcohol production methods for attenuating glycerol biosynthetic pathways 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 or more of 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).

A modified yeast may be further characterized by 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, which avoids adverse effects of acetate on yeast cell growth, and may further contribute to the improvement of alcohol production increasing acetyl-CoA synthase activity may be achieved by introducing a heterologous acetyl-CoA synthase gene into the cell, increasing expression of an endogenous acetyl-CoA synthase gene, etc. particularly useful acetyl-CoA synthases for introduction into the cell may be obtained from Methanomyces australis (Methanostaconicii) (Uniprot/TrEMBL accession No.: WP _ 013718460.) homologues of this enzyme, including those having at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, and even % amino acid sequence identity to the subject methods of the invention described above.

In embodiments, the modified cell can further comprise steps including encoding a polypeptide having NAD⁺In most embodiments of the compositions and methods of the invention, however, the introduction of acetylacetaldehyde dehydrogenase and/or pyruvate formate lyase is not required because the need for these activities is eliminated by attenuating the natural biosynthetic pathway for the production of acetyl-CoA which leads to an imbalance in redox cofactors.

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

In embodiments, the modified yeast cell comprising a butanol biosynthetic pathway further comprises steps a modification in a polynucleotide encoding a polypeptide having pyruvate decarboxylase activity in embodiments, the yeast cell comprises a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having pyruvate decarboxylase activity in embodiments, the polypeptide having pyruvate decarboxylase activity is selected from the group consisting of PDC1, PDC5, PDC6, and combinations thereof in embodiments, the yeast cell comprises a deletion, mutation, and/or substitution in or more endogenous polynucleotides encoding FRA2, ALD6, ADH1, GPD2, BDH1, and YMR226C in embodiments.

GOI part

Reduced Cpr1 expression in combination with other beneficial mutations

In embodiments, in addition to expressing reduced amounts of the Cpr1 polypeptide, optionally in combination with other genetic modifications conducive to alcohol production, the modified yeast cells of the invention further comprise steps additional genes of interest encoding proteins of interest, additional genes of interest may be introduced before, during or after genetic manipulations that result in reduced expression of the Cpr1 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, amylases, xylanases, β -glucanases, phosphatases, proteases, α -amylases, β -amylases, pullulanases, isoamylases, cellulases, trehalases, lipases, pectinases, polyesterases, cutinases, oxidases, transferases, reductases, hemicellulases, mannanases, esterases, isomerases, lactases, peroxidases, and other enzymes of interest, secreted by way of the desired proteins, and other glycosylating enzymes.

Yeast cells suitable for modification

Yeast are unicellular eukaryotic microorganisms classified as members of the kingdom fungi and include organisms from ascomycetes and basidiomycetes yeasts that can be used for alcohol production include, but are not limited to, Saccharomyces species, including Saccharomyces cerevisiae, as well as Kluyveromyces (Kluyveromyces), Kluyveromyces (Lachancea), and Schizosaccharomyces (Schizosaccharomyces) species numerous yeast strains are commercially available, many of which have been selected or genetically engineered to obtain desired characteristics, such as high ethanol production, rapid growth rates, etc. yeasts have been genetically engineered to produce heterologous enzymes, such as glucoamylase or α -amylase.

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 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 to limit the strains and methods.

Examples of the invention

Example 1 deletion of YDR155c in Saccharomyces cerevisiae

Genetic screening was performed to identify saccharomyces cerevisiae mutants that were able to increase ethanol production after 24 hours of fermentation and identified and selected a number of candidate genes for further steps of the test (data not shown). of the genes selected for further steps of the analysis was YDR155c, whose amino acid sequence encoding cpr 1. cpr1 is provided below as SEQ ID NO: 1:

the YDR155c gene was disrupted by deleting the substantially complete coding sequence of Cpr1, i.e., by deleting the nucleic acid sequence 4 base pairs before the start codon to 10 base pairs before the stop codon of both alleles of saccharomyces cerevisiae using standard yeast molecular biology techniques. All procedures are based on the publicly available nucleic acid sequence of YDR155c, provided below as SEQ ID NO:2(5 'to 3'):

the host yeast used for the preparation of the modified yeast cells is commercially available Fermax^TMGold (martex, Inc., usa), Inc. Deletion of the YDR155c gene was confirmed by colony PCR. The modified yeast is grown in a non-selective medium to remove the plasmid that confers kanamycin resistance for selection of transformants, resulting in a modified yeast that does not require a growth supplement as compared to the parent yeast.

Example 2: ethanol production by modified yeast with reduced expression of Cpr1

FG-155c yeast having a deletion of YDR155c gene was tested against reference yeast (i.e., FERMAX)^TMGold, which is a wild-type of YDR155c gene) compared to the ability to produce ethanol in the liquefact at 32 ℃ and 34 ℃. By adding 600ppm urea, 0.124SAPU/g ds FERGMEN^TM2.5X (acid fungal protease), 0.33GAU/g ds CS4 (Trichoderma reesei) glucoamylase variant) and 1.46SSCU/g ds Aspergillus kawachi (Aspergillus kawachi) α -amylase to make a liquefact at pH 4.8 (i.e., a corn flour slurry with a dry solids (ds) value of 34.2%).

50 g of the liquefact are weighed into 100ml containers and inoculated with fresh overnight cultures from colonies of the modified strain or FG strain at 32 ℃ and 34 ℃. Samples were collected by centrifugation at 24 and 55 hours, filtered through a 0.2 μm filter, and applied to a Bio-Rad Aminex HPX-87H column (which was at 0.01N H) at 55 deg.C₂SO₄Isocratic flow rate in the eluate was 0.6ml/min) the ethanol, glucose, acetate and glycerol content was analyzed by HPLC (Agilent Technologies 1200 series). An injection volume of 2.5 μ l sample was used. Calibration standards for quantification include known amounts of DP4+, DP3,DP2, DP1, glycerol and ethanol. The analysis results are shown in table 2. An increase in ethanol relative to FG strain was reported.

Table 2: analysis of the fermentation broth after 24 and 55 hours of fermentation

Nominal reference value

The yeast with the YDR155cc gene deletion produced significantly more ethanol (i.e., almost 1.2% to 1.7%) than the unmodified reference strain at 32 ℃ and 34 ℃ for 24 hours.

Example 3 reduction of Cpr1 expression in combination with reduction of Dls1 expression

Experiments were performed to determine if reducing the amount of Cpr1 in yeast combined with reducing the amount of Dls1 (encoded by the YJL065c gene) increased further tolerance and alcohol production compared to reducing Cpr1 alone using standard yeast molecular biology techniques, the YDR155c gene was disrupted by deleting essentially the entire coding sequence of Cpr1 in the above-described FG host yeast (FG-65c) that had deleted YJL065c, the ability of yeast with the deletion of YJL065c and the deletion of Cpr1 (FG-65c-155c) to produce ethanol in liquefacts incubated at 32 ℃ compared to the benchmark yeast, by adding 600ppm urea, 0.124SAPU/g feds gen^TM2.5X (acid fungal protease), 0.33GAU/g ds CS4 (Trichoderma reesei glucoamylase variant) and 1.46SSCU/g ds Aspergillus kawachii α -amylase to make a liquefact at pH 4.8 (i.e., a corn flour slurry with a dry solids (ds) value of 34.1%).

50 g of the liquefact were weighed into 250ml containers and inoculated with fresh overnight cultures of colonies from the FG-65c or FG-65c-155c strain and incubated for 24 hours at 32 ℃. Based on CO production over time₂The resulting pressure build-up was recorded using a gas monitoring system (ankham Technology) to record the fermentation rate. Samples were collected by centrifugation, filtered through a 0.2 μm filter, and applied to a Bio-Rad Aminex HPX-87H column (at 0.01 NH) at 55 deg.C₂SO₄Isocratic flow rate in eluent 0.6ml/min) by HPLC: (Agilent technologies 1200 series) analyzed ethanol, glucose, acetate, and glycerol content. An injection volume of 2.5 μ l sample was used. Calibration standards for quantification include known amounts of DP4+, DP3, DP2, DP1, glycerol and ethanol. The analysis results are shown in table 3. An increase in ethanol at 24 hours relative to the FG-65c strain was reported.

TABLE 3 analysis of fermentation broths with FG, FG-65c and FG-65c-155c yeast for 24 hours

Nominal reference value

Yeast with a deletion of gene YDR155c in addition to the deletion of gene DLS1 (YJ065c) produced significantly more ethanol (i.e. up to 2.8%) at 24 hours compared to the strain with the deletion of the DLS1 gene alone. It should be noted that FG-65c yeast eventually produced more ethanol at 48 hours than the wild type FG yeast (data not shown); however, they are relatively "slow starters" and produce less ethanol at 24 hours. Thus, deletion of the YDR155c gene appears to increase the initial growth rate of FG-65c yeast.

Example 4: ethanol production by modified yeast having alternative ethanol pathway

Yeasts having a deletion of the gene YDR155c and expressing the alternative pathway for ethanol production further (i.e. by expressing heterologous phosphoketolase, heterologous phosphotransacetylase and acetylacetaldehyde dehydrogenase as described in international patent application WO 2015/148272 (miasonikov et al)) were tested for their ability to produce ethanol compared to the parent yeast (which includes an alternative ethanol pathway but does not have the deletion of the gene YDR155c in this case the parent yeast was named "GPY 10009" and the modified yeast was named "GPY 10009-155 c". assay conditions and procedures were as described in the previous examples the ethanol, glucose and glycerol content of the samples were analysed and the results are shown in table 4. an increase in ethanol at 24 hours relative to the GPY10009 strain was reported.

TABLE 4 analysis of the fermentation broths after 24 h fermentation at 32 ℃ with FG, GPY10009 and GPY10009-155c

Nominal reference value

At 24 hours, yeast with the deletion of gene YDR155c and also expressing an alternative pathway produced significantly more ethanol (i.e., more than 2%) than the equivalent yeast without the deletion of YDR155 c. It should be noted that GPY10009 yeast eventually produced more ethanol at 48 hours than wild-type FG yeast (data not shown); however, they are relatively "slow starters" and produce less ethanol at 24 hours. Thus, deletion of the YDR155c gene appears to increase the initial growth rate of GPY10009 yeast.

Example 5: ethanol production by glucoamylase-expressing modified yeast

The yeast expressing the CS4 variant of the Trichoderma reesei glucoamylase described above and further steps with a deletion of the gene YDR155c (i.e., SA-155c) and a reference yeast without a deletion of YDR155c (i.e., SYNERXIA) were tested^TMADY, herein "SA", which is a wild-type of YDR155c gene) compared to the capacity to produce ethanol in a liquefact at 32 ℃ for 24 hours. By adding 600ppm urea, 0.124SAPU/g ds FERGMEN^TM2.5x (acid fungal protease), no exogenous CS4 (variant of trichoderma reesei glucoamylase), and 1.46SSCU/g ds aspergillus kawachi α -amylase were added to make a liquefact of pH 4.8 (i.e., a corn flour slurry with a dry solids (ds) value of 34.3%).

5 g of the liquefact were weighed into a 10ml container and inoculated with a fresh overnight culture of colonies from SA or SA-155c strain and incubated for 24 hours at 32 ℃. Samples were collected by centrifugation, filtered through a 0.2 μm filter, and applied to a Bio-Rad Aminex HPX-87H column (at 0.01N H) at 55 deg.C₂SO₄Isocratic flow rate in the eluate was 0.6ml/min) the ethanol, glucose, acetate and glycerol content was analyzed by HPLC (agilent technologies 1200 series). An injection volume of 2.5 μ l sample was used. Calibration standards for quantification include known amounts of DP4+, DP3, DP2, DP1, glycerol and ethanol. Display of analysis resultsIn table 5. An increase in ethanol relative to the SA strain was reported.

TABLE 5 analysis of fermentation broths after fermentation with SA and SA-155c strains

Nominal reference value

At 24 hours, yeast with the gene YDR155c deleted and also expressing glucoamylase produced significantly more ethanol (i.e., about 4%) than the strain without YDR155c deletion.