KR20170065242A - Genetically engineered yeast cell producing acetoin and method of producing acetoin using the same - Google Patents

Abstract

Genetically engineered yeast cells having acetone producing ability and a method for producing acetone using the same.

Description

Genetically engineered yeast cells having an ability to produce acetone and methods for producing acetone using the same,

Genetically engineered yeast cells having acetone producing ability and a method for producing acetone using the same.

Acetone is a buttery flavoring that is widely used in foods, cosmetics, tobacco, and detergents, and can also be used as an insecticide by acting as a pesticide attractant. Because of the diverse use of acetone and the potential for mass production, acetone is included in 30 platform chemicals that can be produced from biomass by the US Department of Energy.

Currently, most of acetone is produced by chemical synthesis. However, when it is used in cosmetics or foods, preference for use as natural flavor is increasing, and production through bio-processing is attracting attention.

Many recent microbial strain-based studies have been based on bacteria, but most of them have limitations in application to industrial scale due to pathogenicity, lack of acidity, osmotic pressure and high resistance to glucose. Accordingly, there is a demand for a method of producing acetone with high efficiency and high yield by using a microorganism generally considered to be Generally Recognized As Safe (GRAS).

One aspect provides genetically engineered yeast cells capable of efficiently producing acetol.

Another aspect provides a method of producing acetone using genetically engineered yeast cells.

One aspect provides genetically engineered yeast cells with acetone-producing ability.

The yeast cells may have an increased activity of acetolactate synthase and acetolactate decarboxylase as compared to the parent cells.

The term "parent cell" may refer to an original cell, e. G., A genetically untreated cell of the same type for a manipulated microorganism. The parent cell is a cell that does not have a particular genetic modification, but may be the same for other things. Thus, the parent cells of the invention can be cells used as a starting material or starting material in producing genetically engineered microorganisms with increased activity of a given protein.

The term "genetic engineering" or "genetically engineered" as used herein also refers to the act of introducing or making one or more genetic modifications to a cell .

As used herein, the term " increase in activity ", or "increased activity" refers to the activity of an endogenous protein or enzyme that does not have or has a given genetically untreated parental , The activity of the same type of protein or enzyme may have higher activity. Cells with increased activity of proteins or enzymes can be identified using any method known in the art. The cell or microorganism having the increased activity may be one having a genetic modification that increases the activity of one or more enzymes or polypeptides relative to a cell or microorganism that does not have a genetic modification.

As used herein, the term "acetoin" is used interchangeably with 3-hydroxybutanone or acetyl methyl carbinol, and the molecular formula of C ₄ H ₈ O ₂ ≪ / RTI > The acetone may comprise (R) -acetone.

The term "acetolactate synthase (ALS)" (used interchangeably with acetohydroxy acid synthase (AHAS)) in the present invention refers to branched chains such as leucine, valine and isoleucine As a regulatory enzyme for the amino acid biosynthesis pathway, it may be an enzyme that synthesizes one molecule of carbon dioxide and acetolactate from two molecules of pyruvic acid, respectively, and is widely known to exist throughout microorganisms or plants. The acetolactate synthase may comprise an enzyme (e.g., an isoenzyme or a homologue) having an activity similar thereto even if the enzyme has a different name, for example, Bacillus subtilis , Acetolactate synthase II encoded by ilvB or ilvN derived from Escherichia coli, acetolactate synthase II encoded by ilvGMEDA derived from Escherichia coli, or ilvI derived from Escherichia coli Or acetolactate synthase III encoded by ilvH. In addition, in addition to Escherichia coli, Saccharomyces cerevisiae, Bacillus anthracis , Haemophilus influenzae, influenzae , Salmonella Typhimurium , Thermotoga maritima), Corynebacterium glutamicum (Corynebacterium glutamicum), Mycobacterium tuberculosis (Mycobacterium tuberculosis), or Streptomyces thinner MONET system (Streptomyces cinnamonensis ). < / RTI > In addition, plant-derived Arabidopsis ( Arabidopsis thaliana , Gossypium hirsutum , Helianthus annuus , or Brassica napus. < / RTI > Also, the acetolactate synthase is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95% identical to the amino acid sequence of SEQ ID NO: , About 97% or more, about 98% or more, or about 99% or more of the sequence homology. As used herein, "homology" means the degree to which a given polynucleotide sequence corresponds and may be expressed as a percentage. In the present specification, its homologous sequence having the same or similar activity as a given polynucleotide sequence is indicated as "% homology ". For example, standard software for calculating parameters such as score, identity and similarity, specifically BLAST 2.0, or by sequential hybridization experiments under defined stringent conditions, , And the appropriate hybridization conditions to be defined can be determined by methods well known to those skilled in the art.

The term "acetolactate decarboxylase (ALD)" in the present invention may mean an enzyme that produces acetoin by removing carbon dioxide from acetolactate. The acetolactate dicarboxylase may include an enzyme (e.g., an isoenzyme or a homologue) having similar activity to an enzyme having a different name, for example, a Bacillus subtilis the origin of the scan alsD, Lactobacillus del Brewer key (Lactobacillus delbrueckii subsp. lactis) derived aldB, Breda ratio Bacillus brevis (Brevibacillus brevis , Enterobacter aerogenes , Leuconostoc lactis , Saccharomyces cerevisiae. It may be an acetolactate dicarboxylate derived from Staphylococcus aureus. Also, the acetolactate dicarboxylase may have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92% and at least about 95% identity to the amino acid sequence of SEQ ID NO: , At least about 97%, at least about 98%, or at least about 99% sequence homology.

The yeast cell may be a yeast cell in which the activity of NADH oxidase is additionally increased.

In the present invention, the term "NADH oxidase" may mean an enzyme that mediates a reaction to produce water and NAD ⁺ using oxygen and NADH as a substrate. The NADH oxidizing enzyme may include an enzyme (e.g., an isoenzyme or a homologue) having an activity similar thereto even if the enzyme has a different name, for example, nox1, nox3, nox4, And noxE derived from Lactococcus lactis. In addition, it is also possible to use a mixture of Enterococcus sp., Lactobacillus sp., Desulfovibrio sp. sp . ), Clostridium sp . , Streptococcus sp . , And the like. The NADH oxidase may have an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92%, at least about 95% , 97% or more, about 98% or more, or about 99% or more.

Wherein the yeast cell comprises at least one selected from the group consisting of an exogenous gene encoding acetolactate synthase, an exogenous gene encoding acetolactate dicarboxylase, and an exogenous gene encoding NADH oxidase . The term "exogenous" may refer to the introduction of a referenced molecule or a referenced activity into a host cell. Molecules may be introduced into the host genetic material, for example, by encoding nucleic acid, such as by insertion into a host chromosome, or as a non-chromosomal genetic material, such as a plasmid. In connection with the expression of a coding nucleic acid, the term "exogenous" indicates that the coding nucleic acid has been introduced into a form capable of expression into the subject. With respect to biosynthesis activity, the term "exogenous" refers to the activity introduced into the host cell. The source may be, for example, a homologous or heterologous coding nucleic acid which is introduced into the host cell and expresses the activity mentioned. Thus, the term "endogenous" refers to the mentioned molecule or activity present in the host cell. Similarly, in connection with the expression of a coding nucleic acid, the term "endogenous" refers to the expression of a coding nucleic acid contained within an individual. The term " heterologous "refers to a molecule or activity from a different source than the species mentioned, and the term" homologous "refers to a molecule or activity from a host cell. Thus, the exogenous expression of the coding nucleic acid may utilize either or both of heterologous or homologous coding nucleic acids.

The exogenous gene may be expressed in the yeast cell in an amount sufficient to increase the activity of the enzyme mentioned relative to the parent cell. The exogenous gene encoding the acetolactate synthase, the exogenous gene encoding acetolactate dicarboxylase, and the homologous gene of the exogenous gene encoding the NADH oxidase are derived from different microorganisms, but the proteins they encode ≪ RTI ID = 0.0 > and / or < / RTI > The exogenous gene encoding acetolactate synthase, the exogenous gene encoding acetolactate dicarboxylase, and the exogenous gene encoding NADH oxidase are about 70% identical to the amino acid sequences of SEQ ID NOS: 2, 4, and 17, respectively. , Greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 92%, greater than about 95%, greater than about 97%, greater than about 98%, or greater than about 99% ≪ / RTI > The exogenous gene encoding acetolactate synthase, the exogenous gene encoding acetolactate dicarboxylase, and the exogenous gene encoding NADH oxidase are about 70% identical to the nucleotide sequences of SEQ ID NOS: 1, 3, and 16, respectively. , Greater than about 75%, greater than about 80%, greater than about 85%, greater than about 90%, greater than about 92%, greater than about 95%, greater than about 97%, greater than about 98%, or greater than about 99% Lt; / RTI > Such an exogenous gene can be changed to a sequence having a codon suitable for expression in a microorganism, a sequence having an optimized codon. This codon change can be made appropriately within a range that does not change the amino acid sequence of the protein.

The exogenous gene may be introduced into the parent cell through an expression vector. In addition, the exogenous gene may be introduced into the parent cell in the form of a linear polynucleotide. In addition, the exogenous gene may be expressed from an expression vector (e.g., a plasmid) in a cell. In addition, the exogenous gene may be inserted into a genetic material (e.g., a chromosome) in a cell for stable expression. The vector may comprise a cloning start point, a promoter, a polynucleotide encoding the enzyme, and a terminator. The origin of replication may comprise an autonomous replication sequence (ARS). The yeast self-replication sequence can be stabilized by a centrometric sequence (CEN). The promoter may be selected from the group consisting of TDH3 promoter, TEF promoter, and FBA1 promoter. The terminator may be selected from the group consisting of CYC1, GPM1, and FBA1. The vector may further comprise a selection marker.

The yeast cell may contain a single gene, a plurality of genes, for example, 2 to 10 copies. The yeast cell may be cultured in a culture medium containing, for example, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 10, 2 to 8, To 6, 2 to 5, 2 to 4, or 2 to 3 copies of the enzyme. When the yeast cell contains a plurality of genes, each gene may be a copy of the same gene or a copy of two or more different genes. Multiple copies of the exogenous gene may be contained in the same locus or multiple loci within the genome of the host cell.

The term "homolog, " used in connection with the original enzyme or gene of the first family or species in the present invention, refers to a native enzyme of the first or subspecies, by functional, structural or genetic analysis Or a second or different species of an enzyme or gene that is determined to be an enzyme or gene of a second family or species corresponding to the gene. Homologs may have functional, structural, or genetic similarities. Techniques for easily cloning enzymes or homologs of genes using gene probes and PCR are well known. Identification of the cloned sequences as homologues can be confirmed by functional assays and / or by genomic mapping of the genes.

As used herein, polynucleotides include "genes" and nucleic acid molecules as used herein can be understood to include "vectors" or "plasmids". Thus, the term "gene" (aka, "structural gene") refers to a polynucleotide that encodes a particular sequence of amino acids, including all or part of one or more proteins or enzymes, (Non-transcribed) DNA sequence, e.g., a promoter sequence, that determines the conditions under which the gene is transcribed. The transcribed region of the gene may include a coding sequence as well as an untranslated region, including an intron, a 5'-untranslated region (UTR), and a 3'-UTR.

In addition, the yeast cells may contain an alcohol dehydrogenase, a glycerol-3-phosphate dehydrogenase, or a 2,3-butanediol dehydrogenase, Lt; RTI ID = 0.0 > activity. ≪ / RTI >

As used herein, the term "decrease in activity" or "decreased activity" refers to a cell having a lower enzyme or polypeptide activity than that measured in a parent cell (e.g., . In addition, "decrease in activity" or "decreased activity" refers to an isolated enzyme or polypeptide having lower activity than the original or wild-type enzyme or polypeptide . Activity reduction or reduced activity includes those that are not active, for example, inactivation. The cell with reduced activity may be one that has a genetic modification that reduces the activity of one or more enzymes or polypeptides as compared to cells that do not have a genetic modification.

The microorganism having reduced activity of the alcohol dehydrogenase, glycerol-3-phosphate dehydrogenase or 2,3-butanediol dehydrogenase may be one in which the endogenous gene encoding the protein is removed or disrupted have. The term " deletion "or" disruption "refers to a genetic modification that results in decreased expression of the gene. Such destruction can include "inactivation" of a gene or "attenuation" of a gene. The inactivation includes not only the expression of the functional product of the gene but also the expression of the functional product but not the functional product. The attenuation includes a reduction in the expression level of the functional product of the gene. That is, the attenuation may include a decrease in the expression level of the functional product even when the net expression amount of the gene is increased. Here, the functional product of a gene refers to a gene having a biochemical or physiological function (for example, an enzyme activity) of a product (e.g., an enzyme) of the gene in a cell or a wild-type cell. Thus, the removal or destruction involves functional deletion or functional disruption of the gene.

The step of removing or destroying may include 1) deletion of part or all of the gene encoding the protein, 2) modification of the expression control sequence so that the expression of the gene is decreased, 3) A modification of the sequence, or 4) a combination thereof. A method of deleting a part or all of the polynucleotides encoding the protein can be performed, for example, by transforming a cassette for gene deletion into a parent cell using a Cre / loxP recombination system, Into a polynucleotide encoding an intrinsic target protein in the chromosome with a polynucleotide or marker gene in which some nucleic acid sequences have been deleted. The "part" may be, for example, 1 to 700, 1 to 500, 1 to 300, 1 to 100, or 1 to 50 depending on the kind of the polynucleotide. In addition, the method of modifying the expression control sequence so that the expression of the nucleotide is decreased may include a step of mutating the nucleic acid sequence, deletion, insertion, non-conservative or conservative substitution or a combination thereof in the expression control sequence to further weaken the activity of the expression control sequence Or by replacing it with a nucleic acid sequence having a weaker activity. The expression control sequence includes a promoter, an operator sequence, a sequence encoding a ribosome binding site, and a sequence regulating termination of transcription and translation. In addition, a method of modifying a polynucleotide sequence on a chromosome, which encodes the protein, may be performed by deletion, insertion, non-conservative or conservative substitution of a polynucleotide sequence or a combination thereof to induce a mutation in the sequence to further weaken the activity of the protein , Or by replacing with an improved polynucleotide sequence to have weaker activity.

The term "alcohol dehydrogenase" in the present invention may mean an enzyme that promotes the interconversion between alcohol and aldehyde or ketone by oxidation of NADH. The alcohol dehydrogenase may include an enzyme having an activity similar to that of the enzyme but may include, for example, ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, ADH7 or SFA1. The alcohol dehydrogenase has at least about 70% amino acid sequence identity, at least about 75% amino acid sequence identity, at least about 80% amino acid sequence identity, at least about 85% amino acid sequence identity, at least about 90% amino acid sequence identity, at least about 92% amino acid sequence identity, , At least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence homology. The alcohol dehydrogenase gene comprises at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92% of the nucleotide sequence of SEQ ID NO: 24, 26, 28, 30, , At least about 95%, at least about 97%, at least about 98%, or at least about 99% sequence homology.

The term "glycerol-3-phosphate dehydrogenase" in the present invention may mean an enzyme that promotes the conversion of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate (G3P). The glycerol-3-phosphate dehydrogenase may contain an enzyme having similar activity, even though the enzyme has a different name, for example, GPD1 or GPD2. The glycerol-3-phosphate dehydrogenase may have an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92% About 95% or more, about 97% or more, about 98% or more, or about 99% or more of the polypeptide. The glycerol-3-phosphate dehydrogenase gene comprises a nucleotide sequence having at least about 70%, about 75%, about 80%, about 85%, about 90%, about 92% , About 95% or more, about 97% or more, about 98% or more, or about 99% or more.

To the present invention the term "2,3-butane diol dehydrogenase (2,3-butnaediol dehydrogense) (BDH )" is the acetonitrile, NADH, and H ⁺ in a substrate, 2,3-butane diol and NAD ⁺ , Which belongs to the oxidoreductase family. The 2,3-butanediol dehydrogenase may contain an enzyme (for example, an isoenzyme or a homolog) having similar activity even if the enzyme has a different name, for example, BDH1 from Saccharomyces cerevisiae, Paenibacillus < RTI ID = 0.0 > polymyxa ), BDH99 :: 67, Bacillus subtilis, Enterococcus faecium ) Enterococcus Durans can be a 2,3-butanediol dehydrogenase derived from Mycobacterium sp . Lactobacillus lactis . The 2,3-butanediol dehydrogenase may have an amino acid sequence that is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92% About 95% or more, about 97% or more, about 98% or more, or about 99% or more of the polypeptide. Wherein the 2,3-butanediol dehydrogenase gene comprises a nucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 92% , About 95% or more, about 97% or more, about 98% or more, or about 99% or more.

In the production of genetically engineered yeast cells having increased production of acetone, the activity of acetolactate synthase, acetolactate dicarboxylate, or NADH oxidase is increased or the gene encoding the enzyme is introduced, alcohol dehydrogenase Reduction of the activity of the enzyme, glycerol-3-phosphate dehydrogenase or 2,3-butanediol dehydrogenase, or removal or destruction of the gene encoding the enzyme may be performed simultaneously or separately. In one embodiment, the genetically engineered yeast cells are prepared by removing genes encoding alcohol dehydrogenase, glycerol-3-phosphate dehydrogenase, 2,3-butanediol dehydrogenase, or a combination thereof in a parent cell, Lactate synthase, acetolactate dicarboxylase, NADH oxidase, or a combination thereof.

The yeast cell is a strain belonging to the genus Saccharomyces as MY access (Saccharomyces), inclusive Vero My process (Kluyveromyces), Pichia (Pichia), Hanse Cronulla (Hansenula), my process to Xi Kosaka (Zygosaccharomyce s) or Candida (Candida) Lt; / RTI > In addition, the saccharide with my process (Saccharomyces) in a Saccharomyces sensu My process streak soil (Saccharomyces sensustricto ) cluster. Saccharomyces The strains belonging to the sensustricto cluster include, for example, S. cerevisiae , S. bayanus , S. paradoxus , romayi process may be non-catheter (S. mikatae), or a saccharide as MY-ku laundry process Havre lots (S. kudriavzevii).

The acetone production pathway of genetically engineered yeast cells capable of effectively producing acetone will be described with reference to Fig. Figure 1 is a schematic representation of an acetone production path and a competition path according to one embodiment.

As shown in FIG. 1, yeast cells according to one embodiment have increased activity of acetolactate synthase and acetolactate dicarboxylase, as compared with the parent cells, and thus, acetone can be effectively produced. Further, in the production of acetone, in order to suppress the production of by-products and further increase the production of acetone, the yeast cell may be additionally blocked from the competitive metabolic pathway of the acetone production pathway. The competitive metabolic pathway may be an ethanol and glycerol synthesis metabolic pathway, as shown in Figure 1, and the competitive metabolic pathway may be achieved by reducing the activity of an alcohol dehydrogenase or a glycerol-3-phosphate dehydrogenase. In addition, the production of acetone can be further increased by eliminating the metabolic pathway for converting acetone to 2,3-butanediol. In addition, a process for reducing the cofactor imbalance can be additionally performed. As shown in the following reaction, the cell produces two molecules of pyruvic acid from glucose through the corresponding action, while consuming two molecules of NAD ⁺ to generate two molecules of NADH. Thus, NADH (excess) and NAD ⁺ (deficiency) may occur in the acetone synthesis pathway.

Therefore, by increasing the activity of NADH oxidase and oxidizing NADH, the imbalance of cofactor (NAD ⁺ / NADH) can be solved.

Another aspect provides a method of producing acetone comprising culturing a genetically engineered yeast cell having acetone producing ability relative to the parent cell, and isolating the acetone from the culture.

The above-mentioned "genetically engineered yeast cell having an ability to produce acetone" is as described above.

The term "cultivation" of the present invention may mean a series of actions in which the cells are grown in an appropriate artificially controlled environmental condition in order to produce acetol from the yeast cells. The method for culturing the cells in the present invention can be carried out by using methods well known in the art. Specifically, the culture may be continuously cultured in a batch process, an injection batch, or a repeated batch or batch fed batch process. The medium used for the culture may be one containing one or more substrates which can be metabolized to acetone and may be cultured under aerobic conditions in a conventional medium containing a suitable carbon source, nitrogen source, amino acid, And the like, while meeting the requirements of a particular strain. The carbon source that can be used is glucose as the main carbon source and may also be used in combination with sugars and carbohydrates such as xylose, sucrose, lactose, fructose, maltose, starch and cellulose, soybean oil, sunflower oil, castor oil, The same oils and fats, fatty acids such as palmitic acid, stearic acid, linoleic acid, alcohols such as glycerol, ethanol, and organic acids such as acetic acid. These materials may be used individually or as a mixture. Nitrogen sources that may be used include inorganic sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Amino acids such as glutamic acid, methionine and glutamine, and organic substances such as peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolyzate, fish or their decomposition products, defatted soybean cake or decomposition products thereof . These nitrogen sources may be used alone or in combination. The medium may include potassium phosphate, potassium phosphate and the corresponding sodium-containing salts as a source. Potassium which may be used include potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts. As the inorganic compound, sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate and calcium carbonate may be used. Finally, in addition to these materials, essential growth materials such as amino acids and vitamins can be used.

Typically, the cells can be grown in a suitable medium at a temperature ranging from about 20 < 0 > C to about 37 < 0 > C. In the present invention, the growth medium may comprise, for example, a yeast nitrogen base, ammonium sulfate, and dextrose as a carbon / energy source, such as broth or most of the Saccharomyces cerevisiae strain For example, a YPD medium in which peptone, yeast extract and dextrose are blended in an optimal ratio for growth. Other defined or synthetic growth media may also be used, and suitable media for growth of particular microorganisms are known to those skilled in the art of microbiology or fermentation.

In addition, the separation may be to separate from the culture, for example, the cultured microorganism, the cell, the culture medium, the culture medium, or a combination thereof.

The biologically produced acetoin may be isolated from the culture medium using methods known in the art. Such a separation method may be centrifugation, filtration, ion exchange chromatography or crystallization. For example, the culture can be centrifuged at low speed to remove the biomass, and the resulting supernatant can be separated through ion exchange chromatography.

According to one aspect, yeast cells can be used to efficiently produce acetone.

According to one aspect of the method for producing acetone, acetone can be produced with high efficiency and high yield.

Figure 1 is a schematic representation of an acetone production path and a competition path according to one embodiment.
FIG. 2 is a diagram showing the open sequence of a vector for expression of an acetone synthase gene according to an embodiment. FIG.
(In Figure 2, there is a red underline with a misspelled mark.
FIG. 3 is a graph showing production yields of metabolites of S. cerevisiae strains increased in acetone production capacity according to an embodiment.
FIG. 4 is a graph showing the production amount of metabolites of S. cerevisiae strain increased in acetone production capacity according to an embodiment.
FIG. 5 is a graph showing production yields of metabolites of S. cerevisiae strain increased in acetone production ability according to one embodiment.
FIG. 6 is a graph showing the yield of metabolites of S. cerevisiae strain increased in acetone production capacity according to one embodiment.
FIG. 7 is a diagram showing production of metabolites in a fed-batch culture of S. cerevisiae strain with increased acetone producing ability according to an embodiment.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

Example  One. Acetone Production capacity Increased S. cerevisiae  Production of strain

One. Acetone  Synthetic gene expression S. cerevisiae  Production of strain

(1.1) Production of plasmid for introducing acetolactate synthase, acetolactate dicarboxylase and / or NADH oxidase gene

(1.1.1) Production of p413-SD plasmid for alsS and alsD gene introduction

To introduce the acetone synthetic gene, acetolactate synthase and acetolactate decarboxylase gene introduction plasmids were prepared.

Acetolactate synthase ah agent Bacillus subtilis (Bacillus subtilis) Origin of alsS (nucleotide sequence and amino acid sequence of SEQ ID NO: 2 of SEQ ID NO: 1), acetolactate dicarboxylate raise to the Bacillus subtilis (Bacilus subtilis) (Nucleotide sequence of SEQ ID NO: 3, amino acid sequence of SEQ ID NO: 4) was used. Specifically, the alsS gene and alsD were obtained by PCR using the Bacillus subtilis genomic DNA as a template (alsS gene: primer set of SEQ ID NOs: 5 and 6, alsD gene: primer set of SEQ ID NOs: 7 and 8).

Further, the alsS gene obtained by PCR was cloned into p413GPD plasmid vector [ HIS3 , P _TDH3 (SEQ ID NO: 9) _, T _CYC1 (SEQ ID NO: 10)] (Mumberg et al., 1995) using BamHI and XhoI restriction enzymes p413G-alsS-C. The alsD gene was cloned _into the p414P _TEF1- T _GPM1 plasmid vector [ TRP1 , P _TEF1 (SEQ ID NO: 11), T _GPM1 (SEQ ID NO: 12)] (Kim and Hahn, 2015) using BamHI and XhoI restriction enzymes and p414T-alsD -G.

Finally, in order to express all of the necessary genes using one vector, the previously cloned p414T-alsD-G vector was used as a template and the primer set of SEQ ID NOS: 13 and 15 was used to introduce the MluI restriction enzyme sequence at the 5 ' PCR product 'P _{TEF1 -} alsD- T _GPM1 ' having an AscI-NotI-MluI sequence at the terminus of the gene was obtained. This was treated with MluI restriction enzyme and cloned into p413GPD plasmid vector treated with BssHII restriction enzyme and designated as p413-D. Additional cloning is done using the AscI, NotI restriction enzyme sites of the vector and the MluI (or MauBI) and NotI restriction sites of the PCR products. Since AscI, MluI, and MauBI restriction enzymes form the same sticky end, they can be adhered to each other. After adhesion, they are no longer recognized by each enzyme. Therefore, a new AscI restriction enzyme site Additional cloning is possible. In order to further clone the alsS gene, PCR was carried out using the p413G-alsS-C vector as a template and the MauBI restriction enzyme sequence at the 5 'end and the AscI-NotI-MluI sequence at the 3' end using the primer set of SEQ ID NOs: Product 'P _TDH3 - alsS -T _CYC1 ' Respectively. This was cloned into p413-D vector treated with MauBI and NotI restriction enzymes and treated with AscI and NotI restriction enzymes to obtain p413-SD vector.

(1.1.2) Construction of p413-SDN plasmid for the introduction of alsD, alsD, and noxE genes

To further improve the unbalance of the cofactor (NAD ⁺ / NADH) that may occur in the acetone synthesis pathway, we intend to further express NADH oxidase.

As the NADH oxidase, noxE (nucleotide sequence of SEQ ID NO: 16, amino acid sequence of SEQ ID NO: 17) derived from Lactococcus lactis was used. The noxE gene was obtained by PCR (primer set of SEQ ID NOS: 18 and 19) as the template DNA of Lactococcus lactis. This PCR product was cloned into the p414P _FBA1- T _FBA1 vector [ TRP1 , P _FBA1 (SEQ ID NO: 20), T _FBA1 (SEQ ID NO: 21)] (Kim and Hahn, 2015) using HindIII and XhoI restriction enzymes to obtain p414F-noxE -F.

The same method as in (1.1.1) of Example 1 was used to add noxE to p413-SD, which ultimately overexpresses the acetone synthesis pathway. PCR product _{PFBA1 -} noxE- T having the MluI restriction enzyme sequence at the 5 'end and the AscI-NotI-MluI sequence at the 3' end using the p414F-noxE-F vector as a template and the primer sets of SEQ ID NOs: _FBA1 '. This was treated with MluI and NotI restriction enzymes and cloned into p413-SD vector treated with AscI and NotI restriction enzymes to finally complete p413-SDN vector.

FIG. 2 is a diagram showing the open sequence of a vector for expression of an acetone synthase gene according to an embodiment. FIG.

(1.2) alsS, and alsD gene expression S. cerevisiae  Production of strain

In order to overexpress the alsS and alsD genes in the S. cerevisiae strain CEN.PK2-1C ( MATa ura3-52 trp1-289 leu2-3,112 his3Δ1 MAL2-8C SUC2 ) (Euroscarf, Germany) ) Was introduced into the above strain by a chemical transformation method using lithium acetate. Thereafter, the transformant was cultured in SC-H medium (amino acid addition except 20 g / L glucose, 6.7 g / L YNB and 1.92 g / L histidine), and the strain transformed with the gene was selected, S. cerevisiae WT [SD].

2. Acetoin synthetic gene expression and alcohol dehydrogenase, glycerol 3-phosphate dehydrogenase and / or 2,3-butanediol dehydrogenase gene deficiency S. cerevisiae  Production of strain

In this example, the production of acetone was inhibited by inhibiting the production of by-products by further blocking the competitive metabolic pathway in the acetone synthesis pathway. Saccharomyces cerevisiae is a strain that produces ethanol as a major metabolite and grows. In the case of strains in which the acetone synthesis pathway is introduced, glycerol together with ethanol is produced as a major competitive metabolite. Thus, by inhibiting the metabolic pathway for the synthesis of ethanol and glycerol, acetone production can be promoted. (ADH1, ADH2, ADH3, ADH4, ADH5, and SFA1) using NADH and alcohol dehydrogenase (ADH6 and ADH7) using NADPH are present in the Saccharomyces cerevisiae do. There are also glycerol-3-phosphate dehydrogenases (GPD1 and GPD2) that convert dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate using NADH as a cofactor.

ADH2 (the nucleotide sequence of SEQ ID NO: 26, the amino acid sequence of SEQ ID NO: 27), ADH3 (the nucleotide sequence of SEQ ID NO: 28, the amino acid sequence of SEQ ID NO: ADH4 (the nucleotide sequence of SEQ ID NO: 30, the amino acid sequence of SEQ ID NO: 31), ADH5 (the nucleotide sequence of SEQ ID NO: 32, the amino acid sequence of SEQ ID NO: 33), GPD1 (the nucleotide sequence of SEQ ID NO: , The amino acid sequence of SEQ ID NO: 35), and GPD2 (the nucleotide sequence of SEQ ID NO: 36, the amino acid sequence of SEQ ID NO: 37).

(BDH1) (nucleotide sequence of SEQ ID NO: 38, amino acid sequence of SEQ ID NO: 39, amino acid sequence of SEQ ID NO: 39, amino acid sequence of SEQ ID NO: ) Was prepared.

(2.1) Production of cassette for alcohol dehydrogenase, glycerol 3-phosphate dehydrogenase and / or 2,3-butanediol dehydrogenase gene deficiency

Mutants lacking the ADH1 to ADH5, GPD1, and GPD2 genes used the Cre / loxP recombination system. Cassettes for gene deletion were prepared using plasmids pUG27 (plasmid containing loxP - his5 ⁺ - loxP deficient cassette, Euroscarf, Germany) or pUG72 (plasmid containing loxP-URA3-loxP deficient cassette, Euroscarf, Germany) PCR amplification. SEQ ID NOS: 40 and 41 (ADH1), SEQ ID NOS: 42 and 43 (ADH2), SEQ ID NOS: 44 and 45 (ADH3), SEQ ID NOS: 46 and 47 (ADH4), SEQ ID NO: 48 And 49 (ADH5), SEQ ID NOS: 50 and 51 (GPD1), and SEQ ID NOS: 52 and 53 (GPD2), respectively.

Mutations in which the BDH1 gene was deleted use a homologous recombination system. The BDH1 gene deletion cassette was prepared by using primers (a combination of SEQ ID NOS: 54 and 55) homologous to the upper 300 bp and lower 282 bp positions of the BDH1 gene with the genomic DNA of the bdh1Δ (BY4741 bdh1Δ :: KanMX6, Euroscarf) And amplified by PCR.

(2.2) S. cerevisiae  Production of strain

(2.2.1) alsS, and alsD gene expression, ADH 1 to 5 gene deletion, and GPD 1 and 2 gene deletion  S. cerevisiae  Production of strain

S. cerevisiae strain CEN.PK2-1C (MATa ura3 leu2 -3,112 -289 -52 trp1 his3 △ 1 MAL2-8C SUC2) (Euroscarf, Germany) ADH1 prepared in the above step (2.1) to the ADH5, GPD1 and GPD2 gene-deficient The cassette was introduced by a chemical transformation method using lithium acetate. The transformant was cultured in SC medium (20 g / L glucose, 6.7 g / L YNB, a suitable amino acid additive) to select strains transformed with the gene. (ADH1), SEQ ID NOS: 58 and 59 (ADH2), SEQ ID NOS: 60 and 61 (ADH3), SEQ ID NOS: 62 and 63 (ADH4), SEQ ID NOS: 64 and 64 A combination of SEQ ID NO: 65 (ADH5), SEQ ID NOs: 66 and 67 (GPD1), and SEQ ID NOs: 68 and 69 (GPD2) In order to remove the selectable markers of the gene-deficient strains, pSH63 ( TRP1 , Cre recombinase under the control of GAL1 promoter, Euroscarf, Germany) expressing Cre recombinase was transformed and a defective strain with selective marker gene was prepared , And the finally obtained strain was named S. cerevisiae adh1-5Δgpd1Δgpd2Δ. Then, the p413-SD vector prepared in (1.1.1) of Example 1 was transformed into the above strain, and the finally obtained strain was named S. cerevisiae adh1-5Δgpd1Δgpd2Δ [SD].

(2.2.2) alsS, and alsD gene expression, ADH 1 to 5 gene deletion, GPD 1 and 2 gene deletion, and BDH1 gene deletion  S. cerevisiae  Production of strain

The cassette for ADH1 to ADH5, GPD1 and GPD2 gene deletion and the cassette for BDH1 gene deletion prepared in (2.1) above were transformed in the same manner as in (2.2.1), and the plasmids prepared in (1.1.1) The p413-SD plasmid was transformed in the same manner as in (2.2.1) above. The combination of SEQ ID NOs: 70 and 71 was used as a primer for confirming the deletion of the BDH1 gene, and the finally obtained strain was named S. cerevisiae adh1-5Δgpd1Δgpd2Δbdh1Δ [SD].

(2.2.3) alsS, alsD, and noxE gene expression, ADH 1 to 5 gene deletion, GPD 1 and 2 gene deletion, and BDH1 gene deletion  S. cerevisiae  Production of strain

The cassette for ADH1 to ADH5, GPD1 and GPD2 gene deletion and the cassette for BDH1 gene deletion prepared in (2.1) above were transformed in the same manner as in (2.2.1) The p413-SDN plasmid was transformed in the same manner as the above (2.2.1), and the finally obtained strain was named S. cerevisiae adh1-5Δgpd1Δgpd2Δbdh1Δ [SDN].

Example 2 S. cerevisiae  Confirmation of increased acetone productivity of the strain

One. alsS , And alsD  Gene expression S. cerevisiae Strain Acetone  Increase productivity

Expression of Acetone Synthesis Gene To confirm the increase in acetone productivity of S. cerevisiae strain, the strain prepared in (1.2) of Example 1 was cultured in an acetone production medium.

Specifically, the acetone production medium was SC-H (50 g / L glucose, 6.7 g / L YNB, 1.92 g / L amino acid addition except histidine) and YPD5 (50 g / L glucose, 10 g / L yeast extract , 20 g / L bacto-peptone) was used. Cell culture was carried out at 30 rpm at 170 rpm using a shaking incubator. When SC-H medium was used, the initial inoculated cell concentration was fixed at OD ₆₀₀ = 0.3 and proceeded to a 10 mL medium in a 100 mL Erlenmeyer flask. When YPD5 medium was used, the initial inoculated cell concentration was fixed at OD ₆₀₀ = 10 and proceeded to 25 mL medium in a 250 mL Erlenmeyer flask. To analyze the metabolites, 1 mL of the culture solution was centrifuged to obtain a supernatant, which was then filtered through a 0.22 mu m filter to conduct HPLC analysis. UltiMate 3000 HPLC system (Thermo fishers scientific) was used and a BioRad Aminex HPX-87H column and a refractive index detector (RI detector) were used. The mobile phase used was 5 mM sulfuric acid, the flow rate was 0.6 mL / min, and the temperature was set at 60 DEG C, and the production amount of the metabolite was confirmed. The results are shown in FIG.

FIG. 3 is a graph showing production yields of metabolites of S. cerevisiae strains increased in acetone production capacity according to an embodiment.

As shown in FIG. 3, the S. cerevisiae WT [SD] strain expressing the acetone synthetic gene consumed 50 g / L of glucose and contained 9.3 g / L of acetone, 2.1 g / Butanediol, 7.9 g / L of ethanol, and 3.8 g / L of glycerol.

2. alsS, and alsD gene expression, ADH 1 to 5 gene deletion, and GPD 1 and 2 gene deletion  S. cerevisiae  Confirmation of increased acetone productivity of the strain

The metabolism of S. cerevisiae adh1-5Δgpd1Δgpd2Δ [SD] was confirmed in the same manner as described above, and the results are shown in FIG.

FIG. 4 is a graph showing the production amount of metabolites of S. cerevisiae strain increased in acetone production capacity according to an embodiment.

As shown in FIG. 4, the strain S. cerevisiae adh1-5Δgpd1Δgpd2Δ [SD] consumes 50 g / L of glucose and produces 5.9 g / L of acetone. It can also be seen that as a by-product, 9.3 g / L of 2,3-butanediol was produced.

3. alsS, and alsD gene expression, ADH 1 to 5 gene deletion, GPD 1 and 2 gene deletion, and BDH1 gene deletion  S. cerevisiae  Confirmation of increased acetone productivity of the strain

The metabolism of S. cerevisiae adh1-5? Gpd1? Gpd2? Bdh1? [SD] was confirmed in the same manner as described above, and the results are shown in Fig.

FIG. 5 is a graph showing production yields of metabolites of S. cerevisiae strain increased in acetone production ability according to one embodiment.

As shown in FIG. 5, the S. cerevisiae adh1-5Δgpd1Δgpd2Δbdh1Δ [SD] strain consumes 50 g / L of glucose and produces 15.43 g / L of acetone. As a by-product, 0.18 g / L of 2,3-butanediol was produced, and other by-products were hardly produced.

4. alsS, alsD, and noxE gene expression, ADH 1-5 gene defects, GPD 1 and 2 gene defects, and BDH1 gene deficiency  S. cerevisiae  Confirmation of increased acetone productivity of the strain

The metabolism of S. cerevisiae adh1-5? Gpd1? Gpd2? Bdh1? [SDN] was confirmed in the same manner as described above, and the results are shown in Fig.

FIG. 6 is a graph showing the yield of metabolites of S. cerevisiae strain increased in acetone production capacity according to one embodiment.

As shown in FIG. 6, the strain S. cerevisiae adh1-5Δgpd1Δgpd2Δbdh1Δ [SDN] consumes 50 g / L of glucose and produces 20.1 g / L of acetone. In addition, almost no by-products were produced, and the time required for consuming 50 g / L of glucose was significantly shortened to 48 hours.

In addition, the metabolism of S. cerevisiae adh1-5Δgpd1Δgpd2Δbdh1Δ [SDN] by fed-batch culture was confirmed.

Specifically, the cells were cultured using the YPD5 medium as described above, and the carbon source was added by adding an infusion solution composed of 80% glucose before the consumption of glucose. The metabolism of S. cerevisiae adh1-5Δgpd1Δgpd2Δbdh1Δ [SDN] was confirmed in the same manner as described above, and the results are shown in FIG.

FIG. 7 is a diagram showing production of metabolites in a fed-batch culture of S. cerevisiae strain with increased acetone producing ability according to an embodiment.

As shown in Fig. 7, the strain S. cerevisiae adh1-5Δgpd1Δgpd2Δbdh1Δ [SDN] contained glycerol (0.2 g / L), ethanol (0.4 g / L), and 2,3- (0.9 g / L) was produced at a slight level and acetone (80.8 g / L) was overproduced.

<110> SNU R & DB Foundation <120> Genetically engineered yeast cell producing acetoin and          method of producing acetoin using the same <130> PN111922 <160> 71 <170> Kopatentin 2.0 <210> 1 <211> 1713 <212> DNA <213> Bacillus subtilis alsS <400> 1 atgacaaaag caacaaaaga acaaaaatcc cttgtgaaaa acagaggggc ggagcttgtt 60 gttgattgct tagtggagca aggtgtcaca catgtatttg gcattccagg tgcaaaaatt 120 gatgcggtat ttgacgcttt acaagataaa ggacctgaaa ttatcgttgc ccggcacgaa 180 caaaacgcag cattcatggc ccaagcagtc ggccgtttaa ctggaaaacc gggagtcgtg 240 ttagtcacat caggaccggg tgcctctaac ttggcaacag gcctgctgac agcgaacact 300 gaaggagacc ctgtcgttgc gcttgctgga aacgtgatcc gtgcagatcg tttaaaacgg 360 acacatcaat ctttggataa tgcggcgcta ttccagccga ttacaaaata cagtgtagaa 420 gttcaagatg taaaaaatat accggaagct gttacaaatg catttaggat agcgtcagca 480 gggcaggctg gggccgcttt tgtgagcttt ccgcaagatg ttgtgaatga agtcacaaat 540 acgaaaaacg tgcgtgctgt tgcagcgcca aaactcggtc ctgcagcaga tgatgcaatc 600 cggcatgaaa 660 ggcggaagac cggaagcaat taaagcggtt cgcaagcttt tgaaaaaggt tcagcttcca 720 tttgttgaaa catatcaagc tgccggtacc ctttctagag atttagagga tcaatatttt 780 ggccgtatcg gtttgttccg caaccagcct ggcgatttac tgctagagca ggcagatgtt 840 gttctgacga tcggctatga cccgattgaa tatgatccga aattctggaa tatcaatgga 900 gaccggacaa ttatccattt agacgagatt atcgctgaca ttgatcatgc ttaccagcct 960 gatcttgaat tgatcggtga cattccgtcc acgatcaatc atatcgaaca cgatgctgtg 1020 aaagtggaat ttgcagagcg tgagcagaaa atcctttctg atttaaaaca atatatgcat 1080 gaaggtgagc aggtgcctgc agattggaaa tcagacagag cgcaccctct tgaaatcgtt 1140 aaagagttgc gtaatgcagt cgatgatcat gttacagtaa cttgcgatat cggttcgcac 1200 gccatttgga tgtcacgtta tttccgcagc tacgagccgt taacattaat gatcagtaac 1260 ggtatgcaaa cactcggcgt tgcgcttcct tgggcaatcg gcgcttcatt ggtgaaaccg 1320 ggagaaaaag tggtttctgt ctctggtgac ggcggtttct tattctcagc aatggaatta 1380 gagacagcag ttcgactaaa agcaccaatt gtacacattg tatggaacga cagcacatat 1440 gacatggttg cattccagca attgaaaaaa tataaccgta catctgcggt cgatttcgga 1500 aatatcgata tcgtgaaata tgcggaaagc ttcggagcaa ctggcttgcg cgtagaatca 1560 ccagaccagc tggcagatgt tctgcgtcaa ggcatgaacg ctgaaggtcc tgtcatcatc 1620 gatgtcccgg ttgactacag tgataacatt aatttagcaa gtgacaagct tccgaaagaa 1680 ttcggggaac tcatgaaaac gaaagctctc tag 1713 <210> 2 <211> 570 <212> PRT <213> Bacillus subtilis alsS <400> 2 Met Thr Lys Ala Thr Lys Glu Gln Lys Ser Leu Val Lys Asn Arg Gly   1 5 10 15 Ala Glu Leu Val Val Asp Cys Leu Val Glu Gln Gly Val Thr His Val              20 25 30 Phe Gly Ile Pro Gly Ala Lys Ile Asp Ala Val Phe Asp Ala Leu Gln          35 40 45 Asp Lys Gly Pro Glu Ile Ile Val Ala Arg His Glu Gln Asn Ala Ala      50 55 60 Phe Met Ala Gln Ala Val Gly Arg Leu Thr Gly Lys Pro Gly Val Val  65 70 75 80 Leu Val Thr Ser Gly Pro Gly Ala Ser Asn Leu Ala Thr Gly Leu Leu                  85 90 95 Thr Ala Asn Thr Glu Gly Asp Pro Val Val Ala Leu Ala Gly Asn Val             100 105 110 Ile Arg Ala Asp Arg Leu Lys Arg Thr His Gln Ser Leu Asp Asn Ala         115 120 125 Ala Leu Phe Gln Pro Ile Thr Lys Tyr Ser Val Glu Val Gln Asp Val     130 135 140 Lys Asn Ile Pro Glu Ala Val Thr Asn Ala Phe Arg Ile Ala Ser Ala 145 150 155 160 Gly Gln Ala Gly Ala Phe Val Ser Phe Pro Gln Asp Val Val Asn                 165 170 175 Glu Val Thr Asn Thr Lys Asn Val Arg Ala Val Ala Ala Pro Lys Leu             180 185 190 Gly Pro Ala Ala Asp Asp Ala Ile Ser Ala Ala Ila Ala Lys Ile Gln         195 200 205 Thr Ala Lys Leu Pro Val Val Leu Val Gly Met Lys Gly Gly Arg Pro     210 215 220 Glu Ala Ile Lys Ala Val Arg Lys Leu Leu Lys Lys Val Gln Leu Pro 225 230 235 240 Phe Val Glu Thr Tyr Gln Ala Gly Thr Leu Ser Arg Asp Leu Glu                 245 250 255 Asp Gln Tyr Phe Gly Arg Ile Gly Leu Phe Arg Asn Gln Pro Gly Asp             260 265 270 Leu Leu Leu Glu Gln Ala Asp Val Val Leu Thr Ile Gly Tyr Asp Pro         275 280 285 Ile Glu Tyr Asp Pro Lys Phe Trp Asn Ile Asn Gly Asp Arg Thr Ile     290 295 300 Ile His Leu Asp Glu Ile Ile Ala Asp Ile Asp His Ala Tyr Gln Pro 305 310 315 320 Asp Leu Glu Leu Ile Gly Asp Ile Pro Ser Thr Ile Asn His Ile Glu                 325 330 335 His Asp Ala Val Lys Val Glu Phe Ala Glu Arg Glu Gln Lys Ile Leu             340 345 350 Ser Asp Leu Lys Gln Tyr Met His Glu Gly Glu Gln Val Pro Ala Asp         355 360 365 Trp Lys Ser Asp Arg Ala His Pro Leu Glu Ile Val Lys Glu Leu Arg     370 375 380 Asn Ala Val Asp Asp His Val Thr Val Thr Cys Asp Ile Gly Ser His 385 390 395 400 Ala Ile Trp Met Ser Arg Tyr Phe Arg Ser Tyr Glu Pro Leu Thr Leu                 405 410 415 Met Ile Ser Asn Gly Met Gln Thr Leu Gly Val Ala Leu Pro Trp Ala             420 425 430 Ile Gly Ala Ser Leu Val Lys Pro Gly Glu Lys Val Val Ser Ser Ser         435 440 445 Gly Asp Gly Gly Phe Leu Phe Ser Ala Met Glu Leu Glu Thr Ala Val     450 455 460 Arg Leu Lys Ala Pro Ile Val His Ile Val Trp Asn Asp Ser Thr Tyr 465 470 475 480 Asp Met Val Ala Phe Gln Gln Leu Lys Lys Tyr Asn Arg Thr Ser Ala                 485 490 495 Val Asp Phe Gly Asn Ile Asp Ile Val Lys Tyr Ala Glu Ser Phe Gly             500 505 510 Ala Thr Gly Leu Arg Val Glu Ser Pro Asp Gln Leu Ala Asp Val Leu         515 520 525 Arg Gln Gly Met Asn Ala Glu Gly Pro Val Ile Ile Asp Val Pro Val     530 535 540 Asp Tyr Ser Asp Asn Ile Asn Leu Ala Ser Asp Lys Leu Pro Lys Glu 545 550 555 560 Phe Gly Glu Leu Met Lys Thr Lys Ala Leu                 565 570 <210> 3 <211> 768 <212> DNA <213> Bacillus subtilis alsD <400> 3 atgaaacgag aaagcaacat tcaagtgctc agccgtggtc aaaaagatca gcctgtgagc 60 cagatttatc aagtatcaac aatgacttct ctattagacg gagtatatga cggagatttt 120 gaactgtcag agattccgaa atatggagac ttcggtatcg gaacctttaa caagcttgac 180 ggagagctga ttgggtttga cggcgaattt taccgtcttc gctcagacgg aaccgcgaca 240 ccggtccaaa atggagaccg ttcaccgttc tgttcattta cgttctttac accggacatg 300 acgcacaaaa ttgatgcgaa aatgacacgc gaagactttg aaaaagagat caacagcatg 360 ctgccaagca gaaacttatt ttatgcaatt cgcattgacg gattgtttaa aaaggtgcag 420 acaagaacag tagaacttca agaaaaacct tacgtgccaa tggttgaagc ggtcaaaaca 480 cagccgattt tcaacttcga caacgtgaga ggaacgattg taggtttctt gacaccagct 540 tatgcaaacg gaatcgccgt ttctggctat cacctgcact tcattgacga aggacgcaat 600 tcaggcggac acgtttttga ctatgtgctt gaggattgca cggttacgat ttctcaaaaa 660 atgaacatga atctcagact tccgaacaca gcggatttct ttaatgcgaa tctggataac 720 cctgattttg cgaaagatat cgaaacaact gaaggaagcc ctgaataa 768 <210> 4 <211> 255 <212> PRT <213> Bacillus subtilis alsD <400> 4 Met Lys Arg Glu Ser Asn Ile Gln Val Leu Ser Arg Gly Gln Lys Asp   1 5 10 15 Gln Pro Val Ser Gln Ile Tyr Gln Val Ser Thr Met Thr Ser Leu Leu              20 25 30 Asp Gly Val Tyr Asp Gly Asp Phe Glu Leu Ser Glu Ile Pro Lys Tyr          35 40 45 Gly Asp Phe Gly Ily Gly Thr Phe Asn Lys Leu Asp Gly Glu Leu Ile      50 55 60 Gly Phe Asp Gly Glu Phe Tyr Arg Leu Arg Ser Asp Gly Thr Ala Thr  65 70 75 80 Pro Val Gln Asn Gly Asp Arg Ser Pro Phe Cys Ser Phe Thr Phe Phe                  85 90 95 Thr Pro Asp Met Thr His Lys Ile Asp Ala Lys Met Thr Arg Glu Asp             100 105 110 Phe Glu Lys Glu Ile Asn Ser Met Leu Pro Ser Arg Asn Leu Phe Tyr         115 120 125 Ala Ile Arg Ile Asp Gly Leu Phe Lys Lys Val Gln Thr Arg Thr Val     130 135 140 Glu Leu Gln Glu Lys Pro Tyr Val Pro Met Val Glu Ala Val Lys Thr 145 150 155 160 Gln Pro Ile Phe Asn Phe Asp Asn Val Arg Gly Thr Ile Val Gly Phe                 165 170 175 Leu Thr Pro Ala Tyr Ala Asn Gly Ile Ala Val Ser Gly Tyr His Leu             180 185 190 His Phe Ile Asp Glu Gly Arg Asn Ser Gly Gly His Val Phe Asp Tyr         195 200 205 Val Leu Glu Asp Cys Thr Val Thr Ile Ser Gln Lys Met Asn Met Asn     210 215 220 Leu Arg Leu Pro Asn Thr Ala Asp Phe Phe Asn Ala Asn Leu Asp Asn 225 230 235 240 Pro Asp Phe Ala Lys Asp Ile Glu Thr Thr Glu Gly Ser Pro Glu                 245 250 255 <210> 5 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> F primer for alsS <400> 5 ctgaggatcc atgacaaaag caacaaaaga ac 32 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R primer for alsS <400> 6 ctgactcgag ctagagagct ttcgttttca 30 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> F primer for alsD <400> 7 ctgaggatcc atgaaacgag aaagcaacat 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> R primer for alsD <400> 8 ctgactcgag ttattcaggg cttccttcag 30 <210> 9 <211> 644 <212> DNA <213> Artificial Sequence <220> <223> TDH3 promoter <400> 9 tcattatcaa tactcgccat ttcaaagaat acgtaaataa ttaatagtag tgattttcct 60 aactttattt agtcaaaaaa ttagcctttt aattctgctg taacccgtac atgcccaaaa 120 tagggggcgg gttacacaga atatataaca tcgtaggtgt ctgggtgaac agtttattcc 180 tggcatccac taaatataat ggagcccgct ttttaagctg gcatccagaa aaaaaaagaa 240 tcccagcacc aaaatattgt tttcttcacc aaccatcagt tcataggtcc attctcttag 300 cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg ggcacaacct caatggagtg 360 atgcaacctg cctggagtaa atgatgacac aaggcaattg acccacgcat gtatctatct 420 cattttctta caccttctat taccttctgc tctctctgat ttggaaaaag ctgaaaaaaa 480 aggttgaaac cagttccctg aaattattcc cctacttgac taataagtat ataaagacgg 540 taggtattga ttgtaattct gtaaatctat ttcttaaact tcttaaattc tacttttata 600 gttagtcttt tttttagttt taaaacacca gaacttagtt tcga 644 <210> 10 <211> 393 <212> DNA <213> Artificial Sequence <220> <223> CYC1 terminator <400> 10 aaaaagaatc atgattgaat gaagatatta tttttttgaa ttatattttt taaattttat 60 ataaagacat ggtttttctt ttcaactcaa ataaagattt ataagttact taaataacat 120 acattttata aggtattcta taaaaagagt attatgttat tgttaacctt tttgtctcca 180 attgtcgtca taacgatgag gtgttgcatt tttggaaacg agattgacat agagtcaaaa 240 tttgctaaat ttgatccctc ccatcgcaag ataatcttcc ctcaaggtta tcatgattat 300 caggatggcg aaaggatacg ctaaaaattc aataaaaaat tcaatataat tttcgtttcc 360 caagaactaa cttggaaggt tatacatggg tac 393 <210> 11 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> TEF1 promoter <400> 11 atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca 60 tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc 120 tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt 180 tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat 240 tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg 300 tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc 360 ttgctcatta gaaagaaagc atagcaatct aatctaagtt t 401 <210> 12 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> GPM1 terminator <400> 12 gtctgaagaa tgaatgattt gatgatttct ttttccctcc atttttctta ctgaatatat 60 caatgatata gacttgtata gtttattatt tcaaattaag tagctatata tagtcaagat 120 aacgtttgtt tgacacgatt acattattcg tcgacatctt ttttcagcct gtcgtggtag 180 caatttgagg agtattatta attgaatagg ttcattttgc gctcgcataa acagttttcg 240 tcagggacag tatgttggaa tgagtggtaa ttaatggtga catgacatgt tatagcaata 300 accttgatgt ttacatcgta gtttaatgta caccccgcga attcgttcaa gtaggagtgc 360 accaattgca aagggaaaag ctgaatgggc agttcgaata g 401 <210> 13 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Universal MluIF primer <400> 13 gactacgcgt ggaacaaaag ctggagctc 29 <210> 14 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Universal MauBIF primer <400> 14 tgaccgcgcg cgggaacaaa agctggagct c 31 <210> 15 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Universal R primer <400> 15 gactacgcgt gcggccgcta atggcgcgcc atagggcgaa ttgggtacc 49 <210> 16 <211> 1341 <212> DNA <213> Lactococcus lactis noxE <400> 16 atgaaaatcg tagttatcgg tacgaaccac gcaggcattg ctacagcaaa tacattaatt 60 gatcgatatc caggccatga gattgttatg attgaccgta acagtaatat gagttacttg 120 gggtgtggga cagctatttg ggtcggaaga caaattgaaa aaccagatga gctgttttat 180 gccaaagcag aagattttga aaaaaaggga gtaaagatat taacagaaac agaagtttca 240 gaaattgact ttactaataa aatgatttat gccaagtcaa aaactggaga aaagattaca 300 gaaagttatg ataaactcgt tctggcaaca ggttcacgtc caattattcc taacttgcca 360 ggaaaagatc ttaaaggcat tcatttttta aaactttttc aagaagggca agccattgac 420 gaagagtttg ctaagaatga tgtgaaacgg attgctgtga ttggtgctgg ttatattggg 480 acagaaattg ctgaagctgc caaacgtcgt ggaaaagaag tcctactttt tgatgcagaa 540 agtacttcac ttgcttcata ttatgatgaa gagtttgcta aagggatgga tgaaaatctt 600 gcccaacatg gaattgaact ccattttggg gaattagctc aagagtttaa ggcaaatgaa 660 aaaggtcatg tatcacagat tgtaactaat aaatcaactt atgatgttga cctcgttatt 720 aattgtattg gctttacagc caatagtgca ttggctggtg aacatttaga aacctttaaa 780 aatggagcaa tcaaagtgga taaacatcaa caaagtagtg acccagatgt ttctgctgta 840 ggagatgttg ccacaatcta ttctaatgct ttacaagact tcacctacat tgcccttgcc 900 tcaaacgctg ttcgctcagg gattgttgct ggtcataata ttggaggaaa atcaatagag 960 tctgttggtg tacaaggttc taatggaatc tctatttttg gttacaatat gacttctacg 1020 ggcttgtcgg ttaaagctgc gaaaaaaatc ggcctagaag tttcatttag tgattttgaa 1080 gataagcaaa aagcatggtt ccttcatgaa aataatgata gtgtgaaaat tcgtatcgtt 1140 tatgaaacaa aaaatcgcag aattattggt gctcaacttg ctagcaagag tgaaataatt 1200 gcaggaaata ttaatatgtt tagtttagct attcaagaaa agaaaacgat tgatgaatta 1260 gccttacttg atttattctt cttaccacac ttcaatagtc catataatta catgactgtt 1320 gcagctttaa atgcaaaata a 1341 <210> 17 <211> 446 <212> PRT <213> Lactococcus lactis noxE <400> 17 Met Lys Ile Val Val Ile Gly Thr Asn His Ala Gly Ile Ala Thr Ala   1 5 10 15 Asn Thr Leu Ile Asp Arg Tyr Pro Gly His Glu Ile Val Met Ile Asp              20 25 30 Arg Asn Ser Asn Met Ser Tyr Leu Gly Cys Gly Thr Ala Ile Trp Val          35 40 45 Gly Arg Gln Ile Glu Lys Pro Asp Glu Leu Phe Tyr Ala Lys Ala Glu      50 55 60 Asp Phe Glu Lys Lys Gly Val Lys Ile Leu Thr Glu Thr Glu Val Ser  65 70 75 80 Glu Ile Asp Phe Thr Asn Lys Met Ile Tyr Ala Lys Ser Lys Thr Gly                  85 90 95 Glu Lys Ile Thr Glu Ser Tyr Asp Lys Leu Val Leu Ala Thr Gly Ser             100 105 110 Arg Pro Ile Ile Pro Asn Leu Pro Gly Lys Asp Leu Lys Gly Ile His         115 120 125 Phe Leu Lys Leu Phe Gln Glu Gly Gln Ala Ile Asp Glu Glu Phe Ala     130 135 140 Lys Asn Asp Val Lys Arg Ile Ala Val Ile Gly Ala Gly Tyr Ile Gly 145 150 155 160 Thr Glu Ile Ala Glu Ala Ala Lys Arg Arg Gly Lys Glu Val Leu Leu                 165 170 175 Phe Asp Ala Glu Ser Thr Ser Leu Ala Ser Tyr Tyr Asp Glu Glu Phe             180 185 190 Ala Lys Gly Met Asp Glu Asn Leu Ala Gln His Gly Ile Glu Leu His         195 200 205 Phe Gly Glu Leu Ala Gln Glu Phe Lys Ala Asn Glu Lys Gly His Val     210 215 220 Ser Gln Ile Val Thr Asn Lys Ser Thr Tyr Asp Val Asp Leu Val Ile 225 230 235 240 Asn Cys Ile Gly Phe Thr Ala Asn Ser Ala Leu Ala Gly Glu His Leu                 245 250 255 Glu Thr Phe Lys Asn Gly Ala Ile Lys Val Asp Lys His Gln Gln Ser             260 265 270 Ser Asp Pro Asp Val Ser Ala Val Gly Asp Val Ala Thr Ile Tyr Ser         275 280 285 Asn Ala Leu Gln Asp Phe Thr Tyr Ile Ala Leu Ala Ser Asn Ala Val     290 295 300 Arg Ser Gly Ile Val Ala Gly His Asn Ile Gly Gly Lys Ser Ile Glu 305 310 315 320 Ser Val Gly Val Gly Gly Ser Asn Gly Ile Ser Ile Phe Gly Tyr Asn                 325 330 335 Met Thr Ser Thr Gly Leu Ser Val Lys Ala Ala Lys Lys Ile Gly Leu             340 345 350 Glu Val Ser Phe Ser Asp Phe Glu Asp Lys Gln Lys Ala Trp Phe Leu         355 360 365 His Glu Asn Asn Asp Ser Val Lys Ile Arg Ile Val Tyr Glu Thr Lys     370 375 380 Asn Arg Arg Ile Ile Gly Ala Gln Leu Ala Ser Lys Ser Glu Ile Ile 385 390 395 400 Ala Gly Asn Ile Asn Met Phe Ser Leu Ala Ile Gln Glu Lys Lys Thr                 405 410 415 Ile Asp Glu Leu Ala Leu Leu Asp Leu Phe Leu Pro His Phe Asn             420 425 430 Ser Pro Tyr Asn Tyr Met Thr Val Ala Ala Leu Asn Ala Lys         435 440 445 <210> 18 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> F primer for noxE <400> 18 gactaagctt atgaaaatcg tagttatcgg t 31 <210> 19 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> R primer for noxE <400> 19 gactctcgag ttattttgca tttaaagctg ca 32 <210> 20 <211> 821 <212> DNA <213> Artificial Sequence <220> <223> FBA1 pormoter <400> 20 atccaactgg caccgctggc ttgaacaaca ataccagcct tccaacttct gtaaataacg 60 gcggtacgcc agtgccacca gtaccgttac ctttcggtat acctcctttc cccatgtttc 120 caatgccctt catgcctcca acggctacta tcacaaatcc tcatcaagct gacgcaagcc 180 ctaagaaatg aataacaata ctgacagtac taaataattg cctacttggc ttcacatacg 240 ttgcatacgt cgatatagat aataatgata atgacagcag gattatcgta atacgtaata 300 gtggaaaatc tcaaaaatgt gtgggtcatt acgtaaataa tgataggaat gggattcttc 360 tatttttcct ttttccattc tagcagccgt cgggaaaacg tggcatcctc tctttcgggc 420 tcaattggag tcacgctgcc gtgagcatcc tctctttcca tatctaacaa ctgagcacgt 480 aaccaatgga aaagcatgag cttagcgttg ctccaaaaaa gtattggatg gttaatacca 540 tttgtctgtt ctcttctgac tttgactcct caaaaaaaaaaaatctacaa tcaacagatc 600 gcttcaatta cgccctcaca aaaacttttt tccttcttct tcgcccacgt taaattttat 660 ccctcatgtt gtctaacgga tttctgcact tgatttatta taaaaagaca aagacataat 720 acttctctat caatttcagt tattgttctt ccttgcgtta ttcttctgtt cttctttttc 780 ttttgtcata tataaccata accaagtaat acatattcaa a 821 <210> 21 <211> 202 <212> DNA <213> Artificial Sequence <220> <223> FBA1 terminator <400> 21 gttaattcaa attaattgat atagtttttt aatgagtatt gaatctgttt agaaataatg 60 gaatattatt tttatttatt tatttatatt attggtcggc tcttttcttc tgaaggtcaa 120 tgacaaaatg atatgaagga aataatgatt tctaaaattt tacaacgtaa gatattttta 180 caaaagccta gctcatcttt tg 202 <210> 22 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Universal MluIF primer 1 <400> 22 gactacgcgt ggaacaaaag ctggagctc 29 <210> 23 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Universal R primer <400> 23 gactacgcgt gcggccgcta atggcgcgcc atagggcgaa ttgggtacc 49 <210> 24 <211> 1047 <212> DNA <213> Saccharomyces cerevisiae ADH1 <400> 24 atgtctatcc cagaaactca aaaaggtgtt atcttctacg aatcccacgg taagttggaa 60 tacaaagata ttccagttcc aaagccaaag gccaacgaat tgttgatcaa cgttaaatac 120 tctggtgtct gtcacactga cttgcacgct tggcacggtg actggccatt gccagttaag 180 ctaccattag tcggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca acaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ccgacttggc ccaagtcgcc cccatcttgt gtgctggtat caccgtctac 480 aaggctttga agtctgctaa cttgatggcc ggtcactggg ttgctatctc cggtgctgct 540 ggtggtctag gttctttggc tgttcaatac gccaaggcta tgggttacag agtcttgggt 600 attgacggtg gtgaaggtaa ggaagaatta ttcagatcca tcggtggtga agtcttcatt 660 gacttcacta aggaaaagga cattgtcggt gctgttctaa aggccactga cggtggtgct 720 cacggtgtca tcaacgtttc cgtttccgaa gccgctattg aagcttctac cagatacgtt 780 agagctaacg gtaccaccgt tttggtcggt atgccagctg gtgccaagtg ttgttctgat 840 gtcttcaacc aagtcgtcaa gtccatctct attgttggtt cttacgtcgg taacagagct 900 gacaccagag aagctttgga cttcttcgcc agaggtttgg tcaagtctcc aatcaaggtt 960 gtcggcttgt ctaccttgcc agaaatttac gaaaagatgg aaaagggtca aatcgttggt 1020 agatacgttg ttgacacttc taaataa 1047 <210> 25 <211> 348 <212> PRT <213> Saccharomyces cerevisiae ADH1 <400> 25 Met Ser Ile Pro Glu Thr Gln Lys Gly Val Ile Phe Tyr Glu Ser His   1 5 10 15 Gly Lys Leu Glu Tyr Lys Asp Ile Pro Val Pro Lys Pro Lys Ala Asn              20 25 30 Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu          35 40 45 His Ala Trp His Gly Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val      50 55 60 Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val  65 70 75 80 Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly                  85 90 95 Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys             100 105 110 Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln         115 120 125 Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr     130 135 140 Asp Leu Ala Gln Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr 145 150 155 160 Lys Ala Leu Lys Ser Ala Asn Leu Met Ala Gly His Trp Val Ala Ile                 165 170 175 Ser Gly Ala Gly Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys             180 185 190 Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Glu Gly Lys Glu         195 200 205 Glu Leu Phe Arg Ser Ile Gly Gly Glu Val Phe Ile Asp Phe Thr Lys     210 215 220 Glu Lys Asp Ile Val Gly Ala Val Leu Lys Ala Thr Asp Gly Gly Ala 225 230 235 240 His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile Glu Ala Ser                 245 250 255 Thr Arg Tyr Val Arg Ala Asn Gly Thr Thr Val Leu Val Gly Met Pro             260 265 270 Ala Gly Ala Lys Cys Cys Ser Asp Val Phe Asn Gln Val Val Lys Ser         275 280 285 Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu     290 295 300 Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val 305 310 315 320 Val Gly Leu Ser Thr Leu Pro Glu Ile Tyr Glu Lys Met Glu Lys Gly                 325 330 335 Gln Ile Val Gly Arg Tyr Val Val Asp Thr Ser Lys             340 345 <210> 26 <211> 1047 <212> DNA <213> Saccharomyces cerevisiae ADH2 <400> 26 atgtctattc cagaaactca aaaagccatt atcttctacg aatccaacgg caagttggag 60 cataaggata tcccagttcc aaagccaaag cccaacgaat tgttaatcaa cgtcaagtac 120 tctggtgtct gccacaccga tttgcacgct tggcatggtg actggccatt gccaactaag 180 ttaccattag ttggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca agaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ctgacttggc tgaagtcgcg ccaatcttgt gtgctggtat caccgtatac 480 aaggctttga agtctgccaa cttgagagca ggccactggg cggccatttc tggtgctgct 540 gt; attgatggtg gtccaggaaa ggaagaattg tttacctcgc tcggtggtga agtattcatc 660 gacttcacca aagagaagga cattgttagc gcagtcgtta aggctaccaa cggcggtgcc 720 cacggtatca tcaatgtttc cgtttccgaa gccgctatcg aagcttctac cagatactgt 780 agggcgaacg gtactgttgt cttggttggt ttgccagccg gtgcaaagtg ctcctctgat 840 gtcttcaacc acgttgtcaa gtctatctcc attgtcggct cttacgtggg gaacagagct 900 gataccagag aagccttaga tttctttgcc agaggtctag tcaagtctcc aataaaggta 960 gttggcttat ccagtttacc agaaatttac gaaaagatgg agaagggcca aattgctggt 1020 agatacgttg ttgacacttc taaataa 1047 <210> 27 <211> 348 <212> PRT <213> Saccharomyces cerevisiae ADH2 <400> 27 Met Ser Ile Pro Glu Thr Gln Lys Ala Ile Ile Phe Tyr Glu Ser Asn   1 5 10 15 Gly Lys Leu Glu His Lys Asp Ile Pro Val Pro Lys Pro Lys Pro Asn              20 25 30 Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu          35 40 45 His Ala Trp His Gly Asp Trp Pro Leu Pro Thr Lys Leu Pro Leu Val      50 55 60 Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val  65 70 75 80 Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly                  85 90 95 Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys             100 105 110 Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Glu         115 120 125 Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr     130 135 140 Asp Leu Ala Glu Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr 145 150 155 160 Lys Ala Leu Lys Ser Ala Asn Leu Arg Ala Gly His Trp Ala Ala Ile                 165 170 175 Ser Gly Ala Gly Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys             180 185 190 Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Pro Gly Lys Glu         195 200 205 Glu Leu Phe Thr Ser Leu Gly Gly Glu Val Phe Ile Asp Phe Thr Lys     210 215 220 Glu Lys Asp Ile Val Ser Ala Val Val Lys Ala Thr Asn Gly Gly Ala 225 230 235 240 His Gly Ile Ile Asn Val Ser Ser Ser Glu Ala Ala Ile Glu Ala Ser                 245 250 255 Thr Arg Tyr Cys Arg Ala Asn Gly Thr Val Val Leu Val Gly Leu Pro             260 265 270 Ala Gly Ala Lys Cys Ser Ser Asp Val Phe Asn His Val Val Lys Ser         275 280 285 Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu     290 295 300 Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val 305 310 315 320 Val Gly Leu Ser Ser Leu Pro Glu Ile Tyr Glu Lys Met Glu Lys Gly                 325 330 335 Gln Ile Ala Gly Arg Tyr Val Val Asp Thr Ser Lys             340 345 <210> 28 <211> 1128 <212> DNA <213> Saccharomyces cerevisiae ADH3 <400> 28 atgttgagaa cgtcaacatt gttcaccagg cgtgtccaac caagcctatt ttctagaaac 60 attcttagat tgcaatccac agctgcaatc cctaagactc aaaaaggtgt catcttttat 120 gagaataagg ggaagctgca ttacaaagat atccctgtcc ccgagcctaa gccaaatgaa 180 attttaatca acgttaaata ttctggtgta tgtcacaccg atttacatgc ttggcacggc 240 gattggccat tacctgttaa actaccatta gtaggtggtc atgaaggtgc tggtgtagtt 300 gtcaaactag gttccaatgt caagggctgg aaagtcggtg atttagcagg tatcaaatgg 360 ctgaacggtt cttgtatgac atgcgaattc tgtgaatcag gtcatgaatc aaattgtcca 420 gatgctgatt tatctggtta cactcatgat ggttctttcc aacaatttgc gaccgctgat 480 gctattcaag ccgccaaaat tcaacagggt accgacttgg ccgaagtagc cccaatatta 540 tgtgctggtg ttactgtata taaagcacta aaagaggcag acttgaaagc tggtgactgg 600 gttgccatct ctggtgctgc aggtggcttg ggttccttgg ccgttcaata tgcaactgcg 660 atgggttaca gagttctagg tattgatgca ggtgaggaaa aggaaaaact tttcaagaaa 720 ttggggggtg aagtattcat cgactttact aaaacaaaga atatggtttc tgacattcaa 780 gaagctacca aaggtggccc tcatggtgtc attaacgttt ccgtttctga agccgctatt 840 tctctatcta cggaatatgt tagaccatgt ggtaccgtcg ttttggttgg tttgcccgct 900 aacgcctacg ttaaatcaga ggtattctct catgtggtga agtccatcaa tatcaagggt 960 tcttatgttg gtaacagagc tgatacgaga gaagccttag acttctttag cagaggtttg 1020 atcaaatcac caatcaaaat tgttggatta tctgaattac caaaggttta tgacttgatg 1080 gaaaagggca agattttggg tagatacgtc gtcgatacta gtaaataa 1128 <210> 29 <211> 375 <212> PRT <213> Saccharomyces cerevisiae ADH3 <400> 29 Met Leu Arg Thr Ser Thr Leu Phe Thr Arg Arg Val Gln Pro Ser Leu   1 5 10 15 Phe Ser Arg Asn Ile Leu Arg Leu Gln Ser Thr Ala Ala Ile Pro Lys              20 25 30 Thr Gln Lys Gly Val Ile Phe Tyr Glu Asn Lys Gly Lys Leu His Tyr          35 40 45 Lys Asp Ile Pro Val Pro Glu Pro Lys Pro Asn Glu Ile Leu Ile Asn      50 55 60 Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu His Ala Trp His Gly  65 70 75 80 Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val Gly Gly His Glu Gly                  85 90 95 Ala Gly Val Val Val Lys Leu Gly Ser Asn Val Lys Gly Trp Lys Val             100 105 110 Gly Asp Leu Ala Gly Ile Lys Trp Leu Asn Gly Ser Cys Met Thr Cys         115 120 125 Glu Phe Cys Glu Ser Gly His Glu Ser Asn Cys Pro Asp Ala Asp Leu     130 135 140 Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln Phe Ala Thr Ala Asp 145 150 155 160 Ala Ile Gln Ala Ala Lys Ile Gln Gln Gly Thr Asp Leu Ala Glu Val                 165 170 175 Ala Pro Ile Leu Cys Ala Gly Val Thr Val Tyr Lys Ala Leu Lys Glu             180 185 190 Ala Asp Leu Lys Ala Gly Asp Trp Val Ala Ile Ser Gly Ala Ala Gly         195 200 205 Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Thr Ala Met Gly Tyr Arg     210 215 220 Val Leu Gly Ile Asp Ala Gly Glu Glu Lys Glu Lys Leu Phe Lys Lys 225 230 235 240 Leu Gly Gly Glu Val Phe Ile Asp Phe Thr Lys Thr Lys Asn Met Val                 245 250 255 Ser Asp Ile Gln Glu Ala Thr Lys Gly Gly Pro His Gly Val Ile Asn             260 265 270 Val Ser Val Ser Glu Ala Ala Ile Ser Leu Ser Thr Glu Tyr Val Arg         275 280 285 Pro Cys Gly Thr Val Val Leu Val Gly Leu Pro Ala Asn Ala Tyr Val     290 295 300 Lys Ser Glu Val Phe Ser His Val Val Lys Ser Ile Asn Ile Lys Gly 305 310 315 320 Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu Ala Leu Asp Phe Phe                 325 330 335 Ser Arg Gly Leu Ile Lys Ser Pro Ile Lys Ile Val Gly Leu Ser Glu             340 345 350 Leu Pro Lys Val Tyr Asp Leu Met Glu Lys Gly Lys Ile Leu Gly Arg         355 360 365 Tyr Val Val Asp Thr Ser Lys     370 375 <210> 30 <211> 1149 <212> DNA <213> Saccharomyces cerevisiae ADH4 <400> 30 atgtcttccg ttactgggtt ttacattcca ccaatctctt tctttggtga aggtgcttta 60 gaagaaaccg ctgattacat caaaaacaag gattacaaaa aggctttgat cgttactgat 120 cctggtattg cagctattgg tctctccggt agagtccaaa agatgttgga agaacgtggc 180 ttaaacgttg ctatctatga caaaactcaa ccaaacccaa atattgccaa tgtcacagct 240 ggtttgaagg ttttgaagga agaaaactct gaaattgtcg tttccattgg tggtggttct 300 gctcacgaca atgctaaggc cattgcttta ttggctacta acggtgggga aattggagat 360 tatgaaggtg tcaaccaatc taagaaggct gctttaccgc tatttgccat caacactact 420 gctggtactg cttccgagat gaccagattc actattatct ctaatgaaga aaagaaaatc 480 aagatggcca tcattgacaa caacgtcact ccagctgttg ctgtcaacga cccatctacc 540 atgtttggtt tgccacctgc tttgactgct gctactggtc tagatgcttt gactcactgt 600 atcgaagctt acgtttccac cgcctctaac ccaatcaccg atgcttgtgc tttgaagggt 660 attgatttga tcaatgaaag cttggtcgcc gcatacaaag acggtaaaga caagaaggcc 720 agaactgata tgtgttacgc agaatacttg gcaggtatgg ctttcaacaa tgcttctcta 780 ggttatgttc atgcccttgc tcatcaactt ggtggtttct accacttgcc tcatggtgtt 840 tgtaacgctg tcttgttgcc tcatgttcaa gaggccaaca tgcaatgtcc aaaggccaag 900 aagagattag gtgaaattgc cttgcattgc ggtgcttctc aagaagatcc agaagaaacc 960 atcaaggctt tgcacgtttt aaacagaacc atgaacattc caagaaactt gaaagactta 1020 ggtgttaaaa ccgaagattt tgacattttg gctgaacacg ccatgcatga tgcctgccat 1080 ttgactaacc cagttcaatt caccaaagaa caagtggttg ccattatcaa gaaagcctat 1140 gaatattaa 1149 <210> 31 <211> 382 <212> PRT <213> Saccharomyces cerevisiae ADH4 <400> 31 Met Ser Ser Val Thr Gly Phe Tyr Ile Pro Pro Ile Ser Phe Phe Gly   1 5 10 15 Glu Gly Ala Leu Glu Glu Thr Ala Asp Tyr Ile Lys Asn Lys Asp Tyr              20 25 30 Lys Lys Ala Leu Ile Val Thr Asp Pro Gly Ile Ala Ala Ile Gly Leu          35 40 45 Ser Gly Arg Val Gln Lys Met Leu Glu Glu Arg Gly Leu Asn Val Ala      50 55 60 Ile Tyr Asp Lys Thr Gln Pro Asn Pro Asn Ile Ala Asn Val Thr Ala  65 70 75 80 Gly Leu Lys Val Leu Lys Glu Glu Asn Ser Glu Ile Val Val Ser Ile                  85 90 95 Gly Gly Gly Ser Ala His Asp Asn Ala Lys Ala Ile Ala Leu Ala             100 105 110 Thr Asn Gly Gly Glu Ile Gly Asp Tyr Glu Gly Val Asn Gln Ser Lys         115 120 125 Lys Ala Ala Leu Pro Leu Phe Ala Ile Asn Thr Thr Ala Gly Thr Ala     130 135 140 Ser Glu Met Thr Arg Phe Thr Ile Ile Ser Asn Glu Glu Lys Lys Ile 145 150 155 160 Lys Met Ale Ile Ile Asp Asn Asn Ale Val Ale Val Ale Val Asn                 165 170 175 Asp Pro Ser Thr Met Phe Gly Leu Pro Pro Ala Leu Thr Ala Ala Thr             180 185 190 Gly Leu Asp Ala Leu Thr His Cys Ile Glu Ala Tyr Val Ser Thr Ala         195 200 205 Ser Asn Pro Ile Thr Asp Ala Cys Ala Leu Lys Gly Ile Asp Leu Ile     210 215 220 Asn Glu Ser Leu Val Ala Ala Tyr Lys Asp Gly Lys Asp Lys Lys Ala 225 230 235 240 Arg Thr Asp Met Cys Tyr Ala Glu Tyr Leu Ala Gly Met Ala Phe Asn                 245 250 255 Asn Ala Ser Leu Gly Tyr Val His Ala Leu Ala His Gln Leu Gly Gly             260 265 270 Phe Tyr His Leu Pro His Gly Val Cys Asn Ala Val Leu Leu Pro His         275 280 285 Val Gln Glu Ala Asn Met Gln Cys Pro Lys Ala Lys Lys Arg Leu Gly     290 295 300 Glu Ile Ala Leu His Cys Gly Ala Ser Gln Glu Asp Pro Glu Glu Thr 305 310 315 320 Ile Lys Ala Leu His Val Leu Asn Arg Thr Met Asn Ile Pro Arg Asn                 325 330 335 Leu Lys Asp Leu Gly Val Lys Thr Glu Asp Phe Asp Ile Leu Ala Glu             340 345 350 His Ala Met His Asp Ala Cys His Leu Thr Asn Pro Val Gln Phe Thr         355 360 365 Lys Glu Gln Val Val Ala Ile Ile Lys Lys Ala Tyr Glu Tyr     370 375 380 <210> 32 <211> 1056 <212> DNA <213> Saccharomyces cerevisiae ADH5 <400> 32 atgccttcgc aagtcattcc tgaaaaacaa aaggctattg tcttttatga gacagatgga 60 aaattggaat ataaagacgt cacagttccg gaacctaagc ctaacgaaat tttagtccac 120 gttaaatatt ctggtgtttg tcatagtgac ttgcacgcgt ggcacggtga ttggccattt 180 caattgaaat ttccattaat cggtggtcac gaaggtgctg gtgttgttgt taagttggga 240 tctaacgtta agggctggaa agtcggtgat tttgcaggta taaaatggtt gaatgggact 300 tgcatgtcct gtgaatattg tgaagtaggt aatgaatctc aatgtcctta tttggatggt 360 actggcttca cacatgatgg tacttttcaa gaatacgcaa ctgccgatgc cgttcaagct 420 gcccatattc caccaaacgt caatcttgct gaagttgccc caatcttgtg tgcaggtatc 480 actgtttata aggcgttgaa aagagccaat gtgataccag gccaatgggt cactatatcc 540 ggtgcatgcg gtggcttggg ttctctggca atccaatacg cccttgctat gggttacagg 600 gtcattggta tcgatggtgg taatgccaag cgaaagttat ttgaacaatt aggcggagaa 660 atattcatcg atttcacgga agaaaaagac attgttggtg ctataataaa ggccactaat 720 gt; aggtattgta ggcccaatgg tactgtcgtc ctggttggta tgccagctca tgcttactgc 840 aattccgatg ttttcaatca agttgtaaaa tcaatctcca tcgttggatc ttgtgttgga 900 aatagagctg atacaaggga ggctttagat ttcttcgcca gaggtttgat caaatctccg 960 atccacttag ctggcctatc ggatgttcct gaaatttttg caaagatgga gaagggtgaa 1020 attgttggta gatatgttgt tgagacttct aaatga 1056 <210> 33 <211> 351 <212> PRT <213> Saccharomyces cerevisiae ADH5 <400> 33 Met Pro Ser Gln Val Ile Pro Glu Lys Gln Lys Ala Ile Val Phe Tyr   1 5 10 15 Glu Thr Asp Gly Lys Leu Glu Tyr Lys Asp Val Thr Val Pro Glu Pro              20 25 30 Lys Pro Asn Glu Ile Leu Val His Val Lys Tyr Ser Gly Val Cys His          35 40 45 Ser Asp Leu His Ala Trp His Gly Asp Trp Pro Phe Gln Leu Lys Phe      50 55 60 Pro Leu Ile Gly Gly His Glu Gly Ala Gly Val Val Val Lys Leu Gly  65 70 75 80 Ser Asn Val Lys Gly Trp Lys Val Gly Asp Phe Ala Gly Ile Lys Trp                  85 90 95 Leu Asn Gly Thr Cys Met Ser Cys Glu Tyr Cys Glu Val Gly Asn Glu             100 105 110 Ser Gln Cys Pro Tyr Leu Asp Gly Thr Gly Phe Thr His Asp Gly Thr         115 120 125 Phe Gln Glu Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro     130 135 140 Pro Asn Val Asn Leu Ala Glu Val Ala Pro Ile Leu Cys Ala Gly Ile 145 150 155 160 Thr Val Tyr Lys Ala Leu Lys Arg Ala Asn Val Ile Pro Gly Gln Trp                 165 170 175 Val Thr Ile Ser Gly Ala Cys Gly Gly Leu Gly Ser Leu Ala Ile Gln             180 185 190 Tyr Ala Leu Ala Met Gly Tyr Arg Val Ile Gly Ile Asp Gly Gly Asn         195 200 205 Ala Lys Arg Lys Leu Phe Glu Gln Leu Gly Gly Glu Ile Phe Ile Asp     210 215 220 Phe Thr Glu Glu Lys Asp Ile Val Gly Ala Ile Ile Lys Ala Thr Asn 225 230 235 240 Gly Gly Ser His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile                 245 250 255 Glu Ala Ser Thr Arg Tyr Cys Arg Pro Asn Gly Thr Val Val Leu Val             260 265 270 Gly Met Pro Ala His Ala Tyr Cys Asn Ser Asp Val Phe Asn Gln Val         275 280 285 Val Lys Ser Ile Ser Ile Val Gly Ser Cys Val Gly Asn Arg Ala Asp     290 295 300 Thr Arg Glu Ala Leu Asp Phe Phe Ala Arg Gly Leu Ile Lys Ser Pro 305 310 315 320 Ile His Leu Ala Gly Leu Ser Asp Val Pro Glu Ile Phe Ala Lys Met                 325 330 335 Glu Lys Gly Glu Ile Val Gly Arg Tyr Val Val Glu Thr Ser Lys             340 345 350 <210> 34 <211> 1176 <212> DNA <213> Saccharomyces cerevisiae GPD1 <400> 34 atgtctgctg ctgctgatag attaaactta acttccggcc acttgaatgc tggtagaaag 60 agaagttcct cttctgtttc tttgaaggct gccgaaaagc ctttcaaggt tactgtgatt 120 ggatctggta actggggtac tactattgcc aaggtggttg ccgaaaattg taagggatac 180 ccagaagttt tcgctccaat agtacaaatg tgggtgttcg aagaagagat caatggtgaa 240 aaattgactg aaatcataaa tactagacat caaaacgtga aatacttgcc tggcatcact 300 ctacccgaca atttggttgc taatccagac ttgattgatt cagtcaagga tgtcgacatc 360 atcgttttca acattccaca tcaatttttg ccccgtatct gtagccaatt gaaaggtcat 420 gttgattcac acgtcagagc tatctcctgt ctaaagggtt ttgaagttgg tgctaaaggt 480 gtccaattgc tatcctctta catcactgag gaactaggta ttcaatgtgg tgctctatct 540 ggtgctaaca ttgccaccga agtcgctcaa gaacactggt ctgaaacaac agttgcttac 600 cacattccaa aggatttcag aggcgagggc aaggacgtcg accataaggt tctaaaggcc 660 ttgttccaca gaccttactt ccacgttagt gtcatcgaag atgttgctgg tatctccatc 720 tgtggtgctt tgaagaacgt tgttgcctta ggttgtggtt tcgtcgaagg tctaggctgg 780 ggtaacaacg cttctgctgc catccaaaga gtcggtttgg gtgagatcat cagattcggt 840 caaatgtttt tcccagaatc tagagaagaa acatactacc aagagtctgc tggtgttgct 900 gatttgatca ccacctgcgc tggtggtaga aacgtcaagg ttgctaggct aatggctact 960 tctggtaagg acgcctggga atgtgaaaag gagttgttga atggccaatc cgctcaaggt 1020 ttaattacct gcaaagaagt tcacgaatgg ttggaaacat gtggctctgt cgaagacttc 1080 ccattatttg aagccgtata ccaaatcgtt tacaacaact acccaatgaa gaacctgccg 1140 gacatgattg aagaattaga tctacatgaa gattag 1176 <210> 35 <211> 391 <212> PRT <213> Saccharomyces cerevisiae GPD1 <400> 35 Met Ser Ala Ala Ala Asp Arg Leu Asn Leu Thr Ser Gly His Leu Asn   1 5 10 15 Ala Gly Arg Lys Arg Ser Ser Ser Val Ser Leu Lys Ala Ala Glu              20 25 30 Lys Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr Thr          35 40 45 Ile Ala Lys Val Val Ala Glu Asn Cys Lys Gly Tyr Pro Glu Val Phe      50 55 60 Ala Pro Ile Val Gln Met Trp Val Phe Glu Glu Glu Ile Asn Gly Glu  65 70 75 80 Lys Leu Thr Glu Ile Ile Asn Thr Arg His Gln Asn Val Lys Tyr Leu                  85 90 95 Pro Gly Ile Thr Leu Pro Asp Asn Leu Val Ala Asn Pro Asp Leu Ile             100 105 110 Asp Ser Val Lys Asp Val Asp Ile Ile Val Phe Asn Ile Pro His Gln         115 120 125 Phe Leu Pro Arg Ile Cys Ser Gln Leu Lys Gly His Val Asp Ser His     130 135 140 Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Val Gly Ala Lys Gly 145 150 155 160 Val Gln Leu Leu Ser Ser Tyr Ile Thr Glu Glu Leu Gly Ile Gln Cys                 165 170 175 Gly Ala Leu Ser Gly Ala Asn Ile Ala Thr Glu Val Ala Gln Glu His             180 185 190 Trp Ser Glu Thr Thr Val Ala Tyr His Ile Pro Lys Asp Phe Arg Gly         195 200 205 Glu Gly Lys Asp Val Asp His Lys Val Leu Lys Ala Leu Phe His Arg     210 215 220 Pro Tyr Phe His Val Ser Val Ile Glu Asp Val Ala Gly Ile Ser Ile 225 230 235 240 Cys Gly Ala Leu Lys Asn Val Val Ala Leu Gly Cys Gly Phe Val Glu                 245 250 255 Gly Leu Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gln Arg Val Gly             260 265 270 Leu Gly Glu Ile Ile Arg Phe Gly Gln Met Phe Phe Pro Glu Ser Arg         275 280 285 Glu Glu Thr Tyr Gln Glu Ser Ala Gly Val Ala Asp Leu Ile Thr     290 295 300 Thr Cys Ala Gly Gly Arg Asn Val Lys Val Ala Arg Leu Met Ala Thr 305 310 315 320 Ser Gly Lys Asp Ala Trp Glu Cys Glu Lys Glu Leu Leu Asn Gly Gln                 325 330 335 Ser Ala Gln Gly Leu Ile Thr Cys Lys Glu Val His Glu Trp Leu Glu             340 345 350 Thr Cys Gly Ser Val Glu Asp Phe Pro Leu Phe Glu Ala Val Tyr Gln         355 360 365 Ile Val Tyr Asn Asn Tyr Pro Met Lys Asn Leu Pro Asp Met Ile Glu     370 375 380 Glu Leu Asp Leu His Glu Asp 385 390 <210> 36 <211> 1323 <212> DNA <213> Saccharomyces cerevisiae GPD2 <400> 36 atgcttgctg tcagaagatt aacaagatac acattcctta agcgaacgca tccggtgtta 60 tatactcgtc gtgcatataa aattttgcct tcaagatcta ctttcctaag aagatcatta 120 ttacaaacac aactgcactc aaagatgact gctcatacta atatcaaaca gcacaaacac 180 tgtcatgagg accatcctat cagaagatcg gactctgccg tgtcaattgt acatttgaaa 240 cgtgcgccct tcaaggttac agtgattggt tctggtaact gggggaccac catcgccaaa 300 gtcattgcgg aaaacacaga attgcattcc catatcttcg agccagaggt gagaatgtgg 360 gtttttgatg aaaagatcgg cgacgaaaat ctgacggata tcataaatac aagacaccag 420 aacgttaaat atctacccaa tattgacctg ccccataatc tagtggccga tcctgatctt 480 ttacactcca tcaagggtgc tgacatcctt gttttcaaca tccctcatca atttttacca 540 aacatagtca aacaattgca aggccacgtg gcccctcatg taagggccat ctcgtgtcta 600 aaagggttcg agttgggctc caagggtgtg caattgctat cctcctatgt tactgatgag 660 ttaggaatcc aatgtggcgc actatctggt gcaaacttgg caccggaagt ggccaaggag 720 cattggtccg aaaccaccgt ggcttaccaa ctaccaaagg attatcaagg tgatggcaag 780 gatgtagatc ataagatttt gaaattgctg ttccacagac cttacttcca cgtcaatgtc 840 atcgatgatg ttgctggtat atccattgcc ggtgccttga agaacgtcgt ggcacttgca 900 tgtggtttcg tagaaggtat gggatggggt aacaatgcct ccgcagccat tcaaaggctg 960 ggtttaggtg aaattatcaa gttcggtaga atgtttttcc cagaatccaa agtcgagacc 1020 tactatcaag aatccgctgg tgttgcagat ctgatcacca cctgctcagg cggtagaaac 1080 gtcaaggttg ccacatacat ggccaagacc ggtaagtcag ccttggaagc agaaaaggaa 1140 ttgcttaacg gtcaatccgc ccaagggata atcacatgca gagaagttca cgagtggcta 1200 caaacatgtg agttgaccca agaattccca ttattcgagg cagtctacca gatagtctac 1260 aacaacgtcc gcatggaaga cctaccggag atgattgaag agctagacat cgatgacgaa 1320 tag 1323 <210> 37 <211> 440 <212> PRT <213> Saccharomyces cerevisiae GPD2 <400> 37 Met Leu Ala Val Arg Arg Leu Thr Arg Tyr Thr Phe Leu Lys Arg Thr   1 5 10 15 His Pro Val Leu Tyr Thr Arg Arg Ala Tyr Lys Ile Leu Pro Ser Arg              20 25 30 Ser Thr Phe Leu Arg Arg Ser Leu Leu Gln Thr Gln Leu His Ser Lys          35 40 45 Met Thr Ala His Thr Asn Ile Lys Gln His Lys His Cys His Glu Asp      50 55 60 His Pro Ile Arg Arg Ser Asp Ser Ala Val Ser Ile Val His Leu Lys  65 70 75 80 Arg Ala Pro Phe Lys Val Thr Val Ile Gly Ser Gly Asn Trp Gly Thr                  85 90 95 Thr Ile Ala Lys Val Ile Ala Glu Asn Thr Glu Leu His Ser His Ile             100 105 110 Phe Glu Pro Glu Val Arg Met Trp Val Phe Asp Glu Lys Ile Gly Asp         115 120 125 Glu Asn Leu Thr Asp Ile Ile Asn Thr Arg His Gln Asn Val Lys Tyr     130 135 140 Leu Pro Asn Ile Asp Leu Pro His Asn Leu Val Ala Asp Pro Asp Leu 145 150 155 160 Leu His Ser Ile Lys Gly Ala Asp Ile Leu Val Phe Asn Ile Pro His                 165 170 175 Gln Phe Leu Pro Asn Ile Val Lys Gln Leu Gln Gly His Val Ala Pro             180 185 190 His Val Arg Ala Ile Ser Cys Leu Lys Gly Phe Glu Leu Gly Ser Lys         195 200 205 Gly Val Gln Leu Leu Ser Ser Tyr Val Thr Asp Glu Leu Gly Ile Gln     210 215 220 Cys Gly Ala Leu Ser Gly Ala Asn Leu Ala Pro Glu Val Ala Lys Glu 225 230 235 240 His Trp Ser Glu Thr Thr Val Ala Tyr Gln Leu Pro Lys Asp Tyr Gln                 245 250 255 Gly Asp Gly Lys Asp Val Asp His Lys Ile Leu Lys Leu Leu Phe His             260 265 270 Arg Pro Tyr Phe His Val Asn Val Ile Asp Asp Val Ala Gly Ile Ser         275 280 285 Ile Ala Gly Ala Leu Lys Asn Val Val Ala Leu Ala Cys Gly Phe Val     290 295 300 Glu Gly Met Gly Trp Gly Asn Asn Ala Ser Ala Ala Ile Gln Arg Leu 305 310 315 320 Gly Leu Gly Glu Ile Ile Lys Phe Gly Arg Met Phe Phe Pro Glu Ser                 325 330 335 Lys Val Glu Thr Tyr Tyr Gln Glu Ser Ala Gly Val Ala Asp Leu Ile             340 345 350 Thr Thr Cys Ser Gly Gly Arg Asn Val Lys Val Ala Thr Tyr Met Ala         355 360 365 Lys Thr Gly Lys Ser Ala Leu Glu Ala Glu Lys Glu Leu Leu Asn Gly     370 375 380 Gln Ser Ala Gln Gly Ile Ile Thr Cys Arg Glu Val His Glu Trp Leu 385 390 395 400 Gln Thr Cys Glu Leu Thr Gln Glu Phe Pro Leu Phe Glu Ala Val Tyr                 405 410 415 Gln Ile Val Tyr Asn Asn Val Arg Met Glu Asp Leu Pro Glu Met Ile             420 425 430 Glu Glu Leu Asp Ile Asp Asp Glu         435 440 <210> 38 <211> 1149 <212> DNA <213> Saccharomyces cerevisiae BDH1 <400> 5 atgagagctt tggcatattt caagaagggt gatattcact tcactaatga tatccctagg 60 ccagaaatcc aaaccgacga tgaggttatt atcgacgtct cttggtgtgg gatttgtggc 120 tcggatcttc acgagtactt ggatggtcca atcttcatgc ctaaagatgg agagtgccat 180 aaattatcca acgctgcttt acctctggca atgggccatg agatgtcagg aattgtttcc 240 aaggttggtc ctaaagtgac aaaggtgaag gttggcgacc acgtggtcgt tgatgctgcc 300 agcagttgtg cggacctgca ttgctggcca cactccaaat tttacaattc caaaccatgt 360 gatgcttgtc agaggggcag tgaaaatcta tgtacccacg ccggttttgt aggactaggt 420 gtgatcagtg gtggctttgc tgaacaagtc gtagtctctc aacatcacat tatcccggtt 480 ccaaaggaaa ttcctctaga tgtggctgct ttagttgagc ctctttctgt cacctggcat 540 gctgttaaga tttctggttt caaaaaaggc agttcagcct tggttcttgg tgcaggtccc 600 attgggttgt gtaccatttt ggtacttaag ggaatggggg ctagtaaaat tgtagtgtct 660 gaaattgcag agagaagaat agaaatggcc aagaaactgg gcgttgaggt gttcaatccc 720 tccaagcacg gtcataaatc tatagagata ctacgtggtt tgaccaagag ccatgatggg 780 tttgattaca gttatgattg ttctggtatt caagttactt tcgaaacctc tttgaaggca 840 ttaacattca aggggacagc caccaacatt gcagtttggg gtccaaaacc tgtcccattc 900 caaccaatgg atgtgactct ccaagagaaa gttatgactg gttcgatcgg ctatgttgtc 960 gaagacttcg aagaagttgt tcgtgccatc cacaacggag acatcgccat ggaagattgt 1020 aagcaactaa tcactggtaa gcaaaggatt gaggacggtt gggaaaaggg attccaagag 1080 ttgatggatc acaaggaatc caacgttaag attctattga cgcctaacaa tcacggtgaa 1140 atgaagtaa 1149 <210> 39 <211> 382 <212> PRT <213> Saccharomyces cerevisiae BDH1 <400> 6 Met Arg Ala Leu Ala Tyr Phe Lys Lys Gly Asp Ile His Phe Thr Asn   1 5 10 15 Asp Ile Pro Arg Pro Glu Ile Gln Thr Asp Asp Glu Val Ile Ile Asp              20 25 30 Val Ser Trp Cys Gly Ile Cys Gly Ser Asp Leu His Glu Tyr Leu Asp          35 40 45 Gly Pro Ile Phe Met Pro Lys Asp Gly Glu Cys His Lys Leu Ser Asn      50 55 60 Ala Ala Leu Pro Leu Ala Met Gly His Glu Met Ser Gly Ile Val Ser  65 70 75 80 Lys Val Gly Pro Lys Val Thr Lys Val Lys Val Gly Asp His Val Val                  85 90 95 Val Asp Ala Ser Ser Cys Ala Asp Leu His Cys Trp Pro His Ser             100 105 110 Lys Phe Tyr Asn Ser Lys Pro Cys Asp Ala Cys Gln Arg Gly Ser Glu         115 120 125 Asn Leu Cys Thr His Ala Gly Phe Val Gly Leu Gly Val Ile Ser Gly     130 135 140 Gly Phe Ala Glu Gln Val Val Val Ser Ser Gln His His Ile Ile Pro Val 145 150 155 160 Pro Lys Glu Ile Pro Leu Asp Val Ala Ala Leu Val Glu Pro Leu Ser                 165 170 175 Val Thr Trp His Ala Val Lys Ile Ser Gly Phe Lys Lys Gly Ser Ser             180 185 190 Ala Leu Val Leu Gly Ala Gly Pro Ile Gly Leu Cys Thr Ile Leu Val         195 200 205 Leu Lys Gly Met Gly Ala Ser Lys Ile Val Val Ser Glu Ile Ala Glu     210 215 220 Arg Arg Ile Glu Met Ala Lys Lys Leu Gly Val Glu Val Phe Asn Pro 225 230 235 240 Ser Lys His Gly His Lys Ser Ile Glu Ile Leu Arg Gly Leu Thr Lys                 245 250 255 Ser His Asp Gly Phe Asp Tyr Ser Tyr Asp Cys Ser Gly Ile Gln Val             260 265 270 Thr Phe Glu Thr Ser Leu Lys Ala Leu Thr Phe Lys Gly Thr Ala Thr         275 280 285 Asn Ile Ala Val Trp Gly Pro Lys Pro Val Pro Phe Gln Pro Met Asp     290 295 300 Val Thr Leu Gln Glu Lys Val Met Thr Gly Ser Ile Gly Tyr Val Val 305 310 315 320 Glu Asp Phe Glu Glu Val Val Arg Ala Ile His Asn Gly Asp Ile Ala                 325 330 335 Met Glu Asp Cys Lys Gln Leu Ile Thr Gly Lys Gln Arg Ile Glu Asp             340 345 350 Gly Trp Glu Lys Gly Phe Gln Glu Leu Met Asp His Lys Glu Ser Asn         355 360 365 Val Lys Ile Leu Leu Thr Pro Asn Asn His Gly Glu Met Lys     370 375 380 <210> 40 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH1 deletion <400> 40 ttcaagctat accaagcata caatcaacta tctcatatac acagctgaag cttcgtacgc 60                                                                           60 <210> 41 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH1 deletion <400> 41 cttatttaat aataaaaatc ataaatcata agaaattcgc gcataggcca ctagtggat 59 <210> 42 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH2 deletion <400> 42 tacaatcaac tatcaactat taactatatc gtaatacaca cagctgaagc ttcgtacgc 59 <210> 43 <211> 59 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for ADH2 deletion <400> 43 ataatgaaaa ctataaatcg taaagacata agagatccgc gcataggcca ctagtggat 59 <210> 44 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH3 deletion <400> 44 gttaaaacta ggaatagtat agtcataagt taacaccatc cagctgaagc ttcgtacgc 59 <210> 45 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH3 deletion <400> 45 acaaagactt tcataaaaag tttgggtgcg taacacgcta gcataggcca ctagtggat 59 <210> 46 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH4 deletion <400> 46 caagtttaca tttgcaacaa ctaatagtca aataagaaaa cagctgaagc ttcgtacgc 59 <210> 47 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH4 deletion <400> 47 gcacacgcat aattgacgtt tatgagttcg ttcgattttt gcataggcca ctagtggat 59 <210> 48 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> F primer for ADH5 deletion <400> 48 agaaaattat ttaactacat atctacaaaa tcaaagcatc cagctgaagc ttcgtacgc 59 <210> 49 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> R primer for ADH5 deletion <400> 49 taaaaagtaa aaatatattc atcaaattcg ttacaaaaga gcataggcca ctagtggat 59 <210> 50 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for GPD1 deletion <400> 50 cacccccccc ctccacaaac acaaatattg ataatataaa gcagctgaag cttcgtacgc 60                                                                           60 <210> 51 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for GPD1 deletion <400> 51 aagtggggga aagtatgata tgttatcttt ctccaataaa tgcataggcc actagtggat 60                                                                           60 <210> 52 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> F primer for GPD2 deletion <400> 52 tctctttccc tttccttttc cttcgctccc cttccttatc acagctgaag cttcgtacgc 60                                                                           60 <210> 53 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> R primer for GPD2 deletion <400> 53 ggcaacagga aagatcagag ggggaggggg ggggagagtg tgcataggcc actagtggat 60                                                                           60 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for BDH1 deletion <400> 54 gatttgctca cgctactttg 20 <210> 55 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for BDH1 deletion <400> 55 gccatgcttt gttttagacg 20 <210> 56 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH1 deletion <400> 56 caccatatcc gcaatgac 18 <210> 57 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH1 deletion <400> 57 gtgttgtcct ctgaggac 18 <210> 58 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH2 deletion <400> 58 accgggcatc tccaactt 18 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for identification of ADH2 deletion <400> 59 ccatgtctac agtttagagg 20 <210> 60 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH3 deletion <400> 60 atgagcagca gccattttg 19 <210> 61 <211> 21 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for identification of ADH3 deletion <400> 61 tgatggtgat aatgtctctc a 21 <210> 62 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH4 deletion <400> 62 aagaactagt ttttagttcg cg 22 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of ADH4 deletion <400> 63 agaacttccg ttcttctttt 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of ADH5 deletion <400> 64 ctgctatctg cttgtagaag 20 <210> 65 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for identification of ADH5 deletion <400> 65 gaaacgtttg tataggttgt 20 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of GPD1 deletion <400> 66 cgccttgctt ctctcccctt 20 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of GPD1 deletion <400> 67 ccgacagcct ctgaatgagt 20 <210> 68 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of GPD2 deletion <400> 68 tacggaccta ttgccattgt 20 <210> 69 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R primer for identification of GPD2 deletion <400> 69 ttaagggcta tagataacag 20 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F primer for identification of BDH1 deletion <400> 70 gatttgctca cgctactttg 20 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > R primer for identification of BDH1 deletion <400> 71 gccatgcttt gttttagacg 20

Claims (14)

A genetically engineered yeast cell having acetone production ability, wherein the activity of acetolactate synthase and acetolactate decarboxylase is increased compared to the parent cell. The yeast cell according to claim 1, wherein the yeast cell is selected from the group consisting of alcohol dehydrogenase, glycerol-3-phosphate dehydrogenase, 2,3-butanediol dehydrogenase (2,3 -butanediol dehydrogenase) is reduced. The yeast cell according to claim 1, wherein the yeast cell has increased NADH oxidase activity as compared to a parent cell. The yeast cell according to claim 1, wherein the acetolactate synthase has the amino acid sequence of SEQ ID NO: 2. The yeast cell according to claim 1, wherein the acetolactate dicarboxylase has an amino acid sequence of SEQ ID NO: 4. The yeast cell according to claim 1, wherein the yeast cell comprises an exogenous gene encoding acetolactate synthase, and an exogenous gene encoding acetolactate dicarboxylase. 3. The yeast cell according to claim 2, wherein the alcohol dehydrogenase is ADH1, ADH2, ADH3, ADH4, ADH5 or a combination thereof. The yeast cell according to claim 2, wherein the glycerol-3-phosphate dehydrogenase is GPD1, GPD2 or a combination thereof. The yeast cell according to claim 2, wherein the 2,3-butanediol dehydrogenase has the amino acid sequence of SEQ ID NO: 39. [3] The yeast cell according to claim 2, wherein the yeast cell is one wherein the gene encoding the alcohol dehydrogenase, the glycerol-3-phosphate dehydrogenase, or the 2,3-butanediol dehydrogenase is removed or destroyed. 4. The yeast cell according to claim 3, wherein the NADH oxidase is nox1, nox3, nox4 or noxE. The yeast cell according to claim 1, wherein the yeast cell belongs to the genus Saccharomyces . The method according to claim 12, wherein the saccharide with three Levy jiae My process as MY access genus Saccharomyces (Saccharomyces S. cerevisiae , S. bayanus , S. paradoxus , S. mikatae , and Saccharomyces kudryavuzi ( S. cerevisiae ), S. bayanus , S. paradoxus , S. kudriavzevii ). Culturing the yeast cells of any one of claims 1 to 13 in a medium; and
And separating the acetone from the culture.

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