KR20150146274A - Yeast cell with reduced pentose phosphate pathway and method for producing lactate using the same - Google Patents

Abstract

A polypeptide that converts 6-phosphogluconate to libulose 5-phosphate versus genetically engineered cells, a polypeptide that converts xylulose 5-phosphate to sedo heptulos 7-phosphate, sedoheptulose 7-phosphate A yeast cell having lactate-producing ability, and a lactate using the same, wherein the activity of a polypeptide which converts to erythrose 4-phosphate, or a combination thereof, is reduced.

Description

A yeast cell having reduced pentose phosphate pathway and a method for producing lactate using the yeast cell and a reduced pentose phosphate pathway,

A polypeptide that converts 6-phosphogluconate to libulose 5-phosphate versus genetically engineered cells, a polypeptide that converts xylulose 5-phosphate to sedo heptulos 7-phosphate, sedoheptulose 7-phosphate A yeast cell producing lactate, wherein the activity of a polypeptide which converts to erythrose 4-phosphate, or a combination thereof, is reduced, and a method for producing lactate using the same.

Lactate is an organic acid widely used in various industries such as food, pharmaceutical, chemical, and electronics. Lactate is a colorless, odorless and low volatile substance that is well soluble in water. Lactate is not toxic to the human body and is used as a flavoring agent, an acidifier, a preservative, etc. It is also an environmentally friendly alternative polymer material and is a raw material of polylactic acid (PLA) which is a biodegradable plastic.

PLA is technically a ring-open polymerization polyester resin which is converted into dimer lactide for polymer polymerization and can be processed in various ways such as film, sheet, fiber, injection and the like. Therefore, PLA has been in great demand recently as a bioplastics that can widely replace conventional general petrochemical plastics such as polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), and polystyrene (PS).

In addition, since lactate has both a hydroxyl group and a carboxyl group at the same time, its reactivity is very high, and thus it is easy to convert it into an industrially important compound such as lactate ester, acetaldehyde, propylene glycol and the like, And is attracting attention as a raw material.

At present, lactate is produced industrially by petrochemical synthesis process and biotechnological fermentation process. The petrochemical synthesis process is produced by oxidizing ethylene derived from crude oil, making lactonitrile by hydrogenation of cyanide via acetaldehyde, purifying by distillation, and hydrolyzing using hydrochloric acid or sulfuric acid. In addition, the biotechnological fermentation process can produce lactate using a regenerable carbohydrate such as starch, sucrose, maltose, glucose, fructose, xylose as a substrate.

Therefore, even with these conventional techniques, there is a demand for a strain capable of efficiently producing lactate and a lactate production method using the same.

One aspect includes polypeptides that convert 6-phosphogluconate to ribulose-5-phosphate versus genetically engineered cells, polypeptides that convert xylulose 5-phosphate to sedoheptulose 7-phosphate, sedoheptuloses 7 A yeast cell having a lactate producing ability with reduced activity of a polypeptide which converts phosphate to erythrose 4-phosphate, or a combination thereof.

Another aspect provides a method of producing lactate using the yeast cell.

As used herein, the term "activity reduction" of an enzyme or polypeptide refers to a decrease in activity or a decrease in the activity of a cell, or an isolated enzyme or polypeptide compared to an activity level measured in a comparable cell of the same species or its native polypeptide . The activity of the enzyme or polypeptide is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40% above the same biochemical activity by the untreated enzyme or polypeptide, , At least about 50%, at least about 55%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85% % ≪ / RTI > The activity of a specific enzyme or polypeptide in the cell may be about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, , At least about 50%, at least about 60%, at least about 70%, or at least about 100%. The activity of the reduced enzyme or polypeptide can be determined using any method known in the art. The decreased activity of the enzyme or polypeptide may be due to the removal or destruction of the gene encoding the enzyme or polypeptide. The term "deletion" or "disruption" of the gene means that part or all of the gene, or its promoter, gene, gene, gene, Substitution, deletion, or insertion of one or more bases into a gene, all or part of the regulatory factors such as the terminator region. Removal or destruction of the gene can be accomplished through genetic manipulation such as homologous recombination, mutagenesis, or molecular evolution. If the cell contains multiple identical genes or contains two or more different polypeptide homologous paralogs, one or more of the genes may be removed or destroyed.

As used herein, the term "increased activity" of an enzyme or polypeptide may be an increase to an extent sufficient for the enzyme or polypeptide to exhibit activity and may be determined by measuring the cell or isolated polypeptide in a comparable cell of the same species or its native polypeptide Lt; RTI ID = 0.0 > activity level. ≪ / RTI > The activity of the enzyme or polypeptide is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 50% greater than the same biochemical activity by the untreated enzyme or polypeptide, , About 60% or more, about 70% or more, or about 100% or more. The activity of a specific enzyme or polypeptide in the cell may be about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% or more, , Greater than 50%, greater than about 60%, greater than about 70%, or greater than about 100%. Cells with increased activity of the polypeptide can be identified using any method known in the art.

Increases in activity of enzymes or polypeptides can be achieved by increased expression or increased specific activity. The increase in inactivity may be due to enzyme engineering by mutation or random mutagenesis of a specific amino acid of the active domain of the enzyme or polypeptide. The expression increase may be due to the introduction of a polynucleotide encoding an enzyme or polypeptide into the cell, an increase in the number of copies, or a variation in the regulatory region of the polynucleotide. A polynucleotide that is introduced externally or has an increased number of copies may be endogenous or exogenous. The endogenous gene refers to a gene existing on a genetic material contained in a microorganism. The exogenous gene refers to a gene that is integrated into the cell from outside, and the introduced gene may be homologous or heterologous to the host cell to be introduced.

"Heterologous" may mean a foreign that is not native.

The term "copy number increase" may be by introduction or amplification of the gene, or by genetic manipulation of a gene that is not present in the untreated cell. The introduction of the gene may be mediated by a vehicle such as a vector. The introduction may be a transient introduction in which the gene is not integrated into the genome, or it may be inserted into the genome. Such introduction can be achieved, for example, by introducing a vector into which the polynucleotide encoding the desired polypeptide is inserted into the cell, and then replicating the vector in the cell or integrating the polynucleotide into the genome.

The manipulation of the gene used in the present invention can be performed by a molecular biological method known in the art (Roslyn M. Bill, Recombinant Protein Production in Yeast: Methods and Protocols (2012), Sambrook et al., Molecular cloning, A laboratory manual: Cold Spring Habor Laboratory (1989), R Daniel Gietz et al., Nature protocols (2007)) and Quick and easy yeast transformation using the LiAc / SS carrier DNA / PEG method.

The term "gene" means a nucleic acid fragment capable of producing an expression product, e.g., mRNA or protein, by one or more of transcription and translation, and includes a coding region or a 5'- Regulatory sequences such as a 5'-non coding sequence and a 3'-non coding sequence.

"Cell,""strain," or "microorganism" are interchangeable and may include yeast, bacteria, or fungi.

"Sequence identity" of a polypeptide or polynucleotide of the present invention means the same degree of amino acid residues or bases between sequences after aligning both sequences to a maximum in a particular comparison region. Sequence identity is a value measured by optimally aligning two sequences in a specific comparison region, and a part of the sequence in the comparison region may be added or deleted in comparison with a reference sequence. The percent sequence identity can be determined, for example, by comparing two optimally aligned sequences across the comparison region, determining the number of positions at which the same amino acid or nucleic acid appears in both sequences to obtain the number of matched positions Dividing the number of matched positions by the total number of positions in the comparison range (i.e., range size), and multiplying the result by 100 to obtain a percentage of sequence identity. The percentage of the sequence identity can be determined using a known sequence comparison program, and examples thereof include BLASTN (NCBI), CLC Main Workbench (CLC bio), MegAlign (DNASTAR Inc) and the like.

Different levels of sequence identity can be used to identify polypeptides or polynucleotides having the same or similar functions or activities of different species. For example, at least 50 percent, at least 55 percent, at least 60 percent, at least 65 percent, at least 70 percent, at least 75 percent, at least 80 percent, at least 85 percent, at least 90 percent, at least 95 percent, at least 96 percent, Or more, 98% or more, 99% or more, 100% or the like.

As used herein, the term "non-engineered cell" refers to a polypeptide that converts 6-phosphogluconate to ribulose 5-phosphate, xylulose 5-phosphate to sedoheptulose 7-phosphate Means that the activity of a polypeptide that converts, a polypeptide that converts Sedoheptulose 7-phosphate to erythrosine 4-phosphate, or a combination thereof, is reduced in genetic engineering. "Genetical engineering" includes artificially altering the constitution or structure of a genetic material of a cell. The untreated cell may be a parent strain used for genetically manipulating to reduce the activity of Gnd2, Tal1, Tlk2.

As used herein, the term lactate is interpreted to include not only salts of lactic acid, but also lactic acid, anionic forms, solvates, polymorphs or combinations thereof. The salt of lactic acid may be, for example, an inorganic acid salt, an organic acid salt or a metal salt. The inorganic acid salt may be a hydrochloride, a bromate, a phosphate, a sulfate or a disulfide. Organic acid salts include those derived from organic acids such as formate, acetate, acetate, propionate, lactate, oxalate, tartrate, malate, maleate, citrate, fumarate, besylate, camylate, eddylate, trifluoroacetate, , Gluconate, methanesulfonate, glycolate, succinate, 4-toluenesulfonate, galurouronate, ebonate, glutamate or aspartate. The metal salt may be a calcium salt, a sodium salt, a magnesium salt, a strontium salt or a potassium salt.

One aspect is that a polypeptide that converts 6-phosphogluconate to ribulose 5-phophate compared to genetically engineered cells, sedoheptulose 7-phosphate ), A polypeptide that converts sedoheptulose 7-phosphate into erythrose 4-phosphate, or a polypeptide that converts sedoheptulose 7-phosphate into sedoheptulose 7-phosphate, or a polypeptide that converts sedoheptulose 7-phosphate into erythrose 4-phosphate. Lt; RTI ID = 0.0 > lactate < / RTI > production ability.

The yeast cell may comprise one or more genetic modifications that weaken the flow of the pentose phosphate pathway. The genetic modification may result in an attenuated flow through an oxidative partial pentose phosphate pathway, a non-oxidizing partial pentose phosphate pathway, or a combined pathway thereof.

The yeast cell comprises a polypeptide that converts 6-phosphogluconate to ribulose 5-phosphate, a polypeptide that converts xylulose 5-phosphate to sedoheptulose 7-phosphate, a sedoheptulose 7-phosphate to erythrose 4- Phosphate, a polypeptide that converts 6-phosphogluconate to libulose 5-phosphate, and a polypeptide that converts xylulose 5-phosphate to sedoheptulose 7-phosphate, or 6-phosphogluconate, A polypeptide converting to los 5-phosphate and a polypeptide converting sedoheptulose 7-phosphate to erythrosine 4-phosphate.

The yeast cell comprises a polynucleotide encoding a polypeptide that converts the 6-phosphogluconate to a ribulose 5-phosphate, a polynucleotide that encodes a polypeptide that converts xylulose 5-phosphate to sedo heptulose 7-phosphate, A polynucleotide encoding a polypeptide that converts sedoheptulose 7-phosphate to erythrose 4-phosphate, or a combination thereof, may be removed or destroyed.

The yeast cell may be one in which a polynucleotide encoding a polypeptide that converts 6-phosphogluconate to ribulose 5-phophate has been removed or destroyed. The polypeptide that converts the 6-phosphogluconate to ribulose 5-phophate may be an enzyme belonging to EC 1.1.1.44. The polypeptide which converts the 6-phosphogluconate into ribulose 5-phophate is converted to 6-phosphogluconate in the presence of the oxidative NADP of the pentose phosphate pathway. Which catalyzes the reduction to ribulose 5-phophate. A polypeptide which converts the 6-phosphogluconate to a ribulose 5-phophate, for example, phosphogluconate dehydrogenase, and may be GND1 or GND2. The polypeptide converting the 6-phosphogluconate into a ribulose 5-phophate may have at least 60%, at least 70%, at least 80% , At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. For example, GND2 may have the amino acid sequence of SEQ ID NO: 1. For example, GND1 may have the amino acid sequence of SEQ ID NO: 3. The gene encoding the polypeptide that converts the 6-phosphogluconate into ribulose 5-phophate is an amino acid sequence having 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or 3 A polynucleotide sequence encoding a sequence, or a polynucleotide sequence of SEQ ID NO: 2 or 4. The gene may be gndl or gnd2. For example, gnd2 may be one having the polynucleotide sequence of SEQ ID NO: 2. For example, gndl may have the polynucleotide sequence of SEQ ID NO: 4.

The yeast cell may be one in which a polynucleotide encoding a polypeptide that converts sedoheptulose 7-phosphate to sedoheptulose 7-phosphate has been removed or destroyed. The polypeptide that converts the xylulose 5-phosphate into sedo heptulos [epsilon] -phosphate may be an enzyme in the oxidative portion of the pentose phosphate pathway. The polypeptide that converts the xylulose 5-phosphate into sedo heptulos 7-phosphate is D-ribose 5-phosphate + D-xylulose 5-phosphate. Can catalyze a reversible reaction to sedoheptulose 7-phosphate + D-glyceraldehyde 3-phosphate. The polypeptide which converts the xylulose 5-phosphate into sedo heptuloses 7-phosphate may belong to EC 2.2.1.1. The polypeptide which converts the xylulose 5-phosphate into sedoheptulose 7-phosphate is a transketolase, glycolaldehyde transferase or sedoheptolose-7-phosphate: D-glyceraldehyde-3- phosphate glycol aldehyde transferase And may be TKL1, or TKL2. The polypeptide converting the xylulose 5-phosphate into sedo heptulos 7-phosphate has 60% or more, or 70% or more, 80% or more, 90% or more, 95% or more, 96% or more , At least 97%, at least 98%, or at least 99% sequence identity. For example, TKL2 may have the amino acid sequence of SEQ ID NO: 5. For example, TKL1 may have the amino acid sequence of SEQ ID NO: 7. The gene encoding the polypeptide that converts the xylulose 5-phosphate into sedo heptuloses 7-phosphate comprises a polynucleotide sequence encoding an amino acid sequence having at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 5 or 7, The polynucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 8. For example, tkl2 may have the polynucleotide sequence of SEQ ID NO: 6. For example, tkll may be the polynucleotide sequence of SEQ ID NO: 8.

The yeast cell may be one in which a polynucleotide encoding a polypeptide that converts sedoheptulose 7-phosphate to erythrose 4-phosphate has been removed or destroyed. The polypeptide that converts the Sedoheptulose 7-phosphate to the erythrose 4-phosphate may be an enzyme in the non-oxidizing portion of the pentose phosphate pathway. The polypeptide that converts the Sedoheptulose 7-phosphate to erythrose 4-phosphate is a mixture of sedoheptulose 7-phosphate and D-glyceraldehyde 3-phosphate It is possible to catalyze the reversible reaction to 4-phosphate (erythrose 4-phosphate) and D-fructose 6-phosphate. The polypeptide that converts the Sedoheptulose 7-phosphate to erythrose 4-phosphate may belong to EC 2.2.1.2. Such as transaldolase, dihydroxyacetone transferase, dihydroxyacetone synthase, formaldehyde transketolase, or sedoheptulose-7, which converts the Sedoheptulose 7-phosphate to erythrose 4-phosphate -Phosphate: D-glyceraldehyde-3-phosphate glycerol transferase, and may be TAL1. The polypeptide which converts the sedoheptulose 7-phosphate into erythrose 4-phosphate has 60% or more, or 70% or more, 80% or more, , At least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. For example, TAL1 may have the amino acid sequence of SEQ ID NO: 9. The gene encoding the polypeptide that converts the sedoheptulose 7-phosphate into erythrose 4-phosphate has an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: Or a polynucleotide sequence of SEQ ID NO: 10. For example, tal1 may have the polynucleotide sequence of SEQ ID NO: 10.

The yeast cell is Saccharomyces as MY access (Saccharomyces), Cluj Vero My process (Kluyveromyces), Candida (Candida), blood teeth (Pichia), Chen Escherichia (Issatchenkia), debari Oh, my process (Debaryomyces), my process to Xi Kosaka director (Zygosaccharomyces), it may be belonging to the rest research Caro mouses three (Shizosaccharomyces) or saccharose as MY Cobb sheath (Saccharomycopsis). The genus Saccharomyces is, for example, S. cerevisiae , S. bayanus , S. boulardii , S. cerevisiae, S. bulderi , S. cariocanus , S. cariocus , S. chevalieri , and S. cerevisiae, In the case of S. dairenensis , S. ellipsoideus , S. eubayanus , S. exiguus , S. florentinus , S. kluyveri , S. martiniae , S. monacensis , and Sacharinia sp . romayi access Nord Ben systems (S. norbensis), reading in my process parameters Sakae's (S. paradoxus), four Romayi process Pas thoria Taunus (S. pastorianus), a saccharide as MY ROOM process Spencer (S. spencerorum), my process Turi sensor system as Saccharomyces (S. turicensis), my process as Saccharomyces Uni loose spokes (S. unisporus), My access to the Saccharomyces can be Uva Room (S. uvarum), or Saccharomyces access to My Bluetooth Jonas (S. zonatus).

The yeast cell may have lactate production ability. The yeast cell may have the activity of a polypeptide that converts pyruvate to lactate. The yeast cell may have increased activity of the polypeptide converting pyruvate to lactate. The polypeptide that converts pyruvate to lactate may be lactate dehydrogenase (LDH). The lactate dehydrogenase may be an NAD (P) -dependent enzyme. The lactate dehydrogenase may also be stereo-specific and can produce both L-lactate alone or D-lactate, or both L-lactate and D-lactate. The NAD (P) -independent enzyme may be an enzyme classed as EC 1.1.1.27 which acts on L-lactate, or EC 1.1.1.28 which acts on D-lactate.

The lactate-producing yeast cell may have increased activity of lactate dehydrogenase. The yeast cell comprises a polynucleotide encoding at least one lactate dehydrogenase, wherein the gene may be exogenous. The polynucleotide encoding the lactate dehydrogenase may include those derived from bacteria, yeast, fungi, mammals, or reptiles. The polynucleotide Lactobacillus helveticus (Lactobacillus helveticus), L. Bulgaria kusu (L. bulgaricus), John L. Sony (L. johnsonii), L. Planta room (L. plantarum), Japanese grow (Pelodiscus sinensis japonicus ), platypus ( Ornithorhynchus anatinus ), bottlenose dolphins ( Tursiops truncatus , Norwegian Rattus norvegicus , and frog Xenopus laevis ). < / RTI > Lactate dehydrogenase derived from Japanese Zara, lactate dehydrogenase derived from platypus, lactate dehydrogenase derived from bottlenose dolphin, and lactate dehydrogenase derived from Norwegian rats are At least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity with the amino acid sequence of SEQ ID NO: 11, 12, 13, Of the amino acid sequence of SEQ ID NO: 1. In one example, the polynucleotide encoding the lactate dehydrogenase may be a polynucleotide encoding an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NOs: 11, 12, 13, and 14. Or the polynucleotide encoding the lactate dehydrogenase comprises a polynucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 11, 12, 13, or 14, or a polynucleotide sequence of SEQ ID NO: Nucleotide sequence.

The polynucleotide encoding the lactate dehydrogenase may be contained in a vector. The vector may comprise a cloning start point, a promoter, a polynucleotide encoding a lactate dehydrogenase, 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 promoters of CYC (cytochrome c), translation elongation factor (TEF), GPD, and ADH gene. have. The promoters of the CYC (cytochrome c), translation elongation factor (TEF), GPD, and ADH genes may be those having the nucleotide sequences of SEQ ID NOS: 33, 34, 35 and 36, respectively. The terminator may be selected from the group consisting of phosphoglycerate kinase 1 (PGK1), cytochrome c 1 (CYC1), and a terminator of the galactokinase 1 (GALl) gene. The CYC1 terminator may be one having the nucleotide sequence of SEQ ID NO: 37. The vector may further comprise a selection marker. Polynucleotides encoding lactate dehydrogenase may be included at specific locations in the yeast cell genome. The specific position may include a locus of a gene to be removed or destroyed, such as PDC and CYB2. When a polynucleotide encoding a lactate dehydrogenase functions to produce an active protein in a cell, the polynucleotide is considered to be "functional" in the cell.

The yeast cell may comprise a polynucleotide encoding a single lactate dehydrogenase, or a polynucleotide encoding from 2 to 10 copies of a plurality of lactate dehydrogenases. The yeast cell may comprise a polynucleotide encoding, for example, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to 3 copies of a lactate dehydrogenase. When the yeast cell comprises a polynucleotide encoding a plurality of lactate dehydrogenases, each polynucleotide may be a combination of polynucleotides that encode the same or two or more different lactate dehydrogenases. Multiple copies of a polynucleotide encoding an exogenous lactate dehydrogenase may be contained in the same locus or multiple loci within the genome of the host cell, and the promoter or terminator of each copy may be the same or different.

In addition, the yeast cell may be selected from the group consisting of a polypeptide converting pyruvate to acetaldehyde, a polypeptide converting lactate into pyruvate, a polypeptide converting dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, an external mitochondrial NADH dehydrogenase The activity of external mitochondrial NADH dehydrogenase, the mitochondrial pyruvate transporter, or a combination thereof may be decreased.

The yeast cell may be one in which a polynucleotide encoding a polypeptide that converts pyruvate to acetaldehyde has been removed or destroyed. Polypeptides that convert pyruvate to acetaldehyde may be enzymes classified as EC 4.1.1.1. The polypeptide that converts the pyruvate to acetaldehyde may be, for example, pyruvate decarboxylase and may be PDC1. The polypeptide converting from the pyruvate to acetaldehyde is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% 0.0 > 99% < / RTI > sequence identity. The gene encoding the polypeptide converting from pyruvate to acetaldehyde may be a polynucleotide sequence encoding an amino acid sequence having a sequence identity of 95% or more with the amino acid sequence of SEQ ID NO: 16, or a polynucleotide sequence of SEQ ID NO: 17 have. The gene may be pdc1. For example, pdc1 may have the polynucleotide sequence of SEQ ID NO: 17.

The yeast cell may be one in which a polynucleotide encoding a polypeptide that converts lactate to pyruvate has been removed or destroyed. The polypeptide that converts the lactate to pyruvate may be a cytochrome c-dependent enzyme. The polypeptide that converts the lactate to pyruvate may be an enzyme classed as EC 1.1.2.4, which acts on D-lactate, or EC 1.1.2.3, which acts on L-lactate. The polypeptide that converts the lactate to pyruvate may be lactate cytochrome-c oxidoreductase and may be CYB2 (CAA86721.1), CYB2A, CYB2B, DLD1, and the like. Wherein the polypeptide converting the lactate into a pyruvate is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% 0.0 > 99% < / RTI > sequence identity. The gene encoding the polypeptide converting lactate into pyruvate may be a polynucleotide sequence encoding an amino acid sequence having a sequence identity of 95% or more with the amino acid sequence of SEQ ID NO: 14, or a polynucleotide sequence of SEQ ID NO: 15 have.

The yeast cell may be one in which a polynucleotide encoding a polypeptide that converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate has been removed or destroyed. The polypeptide that converts the dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate may be a cytosolic glycerol-3-phosphate dehydrogenase and may be oxidized to NAD + or NADP + by NADH or NADP to form dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate. The polypeptide may belong to EC 1.1.1.8. The cytosolic glycerol-3-phosphate dehydrogenase may be GPDl. Wherein said cytosolic glycerol-3-phosphate dehydrogenase comprises a polypeptide having an amino acid sequence which is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97% Or 99% or more sequence identity to the amino acid sequence shown in SEQ ID NO. The gene encoding the cytosolic glycerol-3-phosphate dehydrogenase comprises a polynucleotide sequence encoding an amino acid sequence having a sequence identity of 95% or more with the amino acid sequence of SEQ ID NO: 16, or a polynucleotide sequence having a polynucleotide sequence of SEQ ID NO: Lt; / RTI >

The yeast cell may be one in which the polynucleotide encoding the external mitochondrial NADH dehydrogenase is removed or destroyed. The external mitochondrial NADH dehydrogenase is described in EC. 1.6.5.9 or EC. It may be an enzyme classified as 1.6.5.3. The external mitochondrial NADH dehydrogenase may be a type II NADH: ubiquinone oxidoreductase (type II NADH). The external mitochondrial NADH dehydrogenase may be located on the outer surface of the inner mitochondrial membrane facing the cytoplasm. The exogenous mitochondrial NADH dehydrogenase may be an enzyme that catalyzes the oxidation of cytosolic NADH to NAD +. The external mitochondrial NADH dehydrogenase may be one which re-oxidizes the cytosolic NADH formed by the process. The external mitochondrial NADH dehydrogenase may be providing the cytosolic NADH to the mitochondrial respiratory chain. The NADH dehydrogenase may be NDEl, NDE2, or a combination thereof. The external mitochondrial NADH dehydrogenase may be distinguished from the internal mitochondrial NADH dehydrogenase NDI1 located within the mitochondria. The amino acid sequence of the foreign mitochondrial NADH dehydrogenase is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% 0.0 > 99% < / RTI > sequence identity. For example, NDE1 and NDE2 may be those having the amino acid sequences of SEQ ID NOs: 18 and 19, respectively. The gene coding for the external mitochondrial NADH dehydrogenase is a polynucleotide sequence encoding an amino acid sequence having a sequence identity of 95% or more with the amino acid sequence of SEQ ID NO: 18 or 19, respectively, or a polynucleotide sequence of SEQ ID NO: 20 or 21 Lt; / RTI > For example, the nde1 may be the sequence of SEQ ID NO: 20, and the nde2 may be the sequence of the polynucleotide of SEQ ID NO: 21.

The yeast cell may be one in which the polynucleotide encoding the mitochondrial pyruvate transporter (MPC) has been removed or destroyed. The mitochondrial pyruvate transporter (MPC) may be a polypeptide which is present in the inner membrane of mitochondria and serves to transfer cytoplasmic pyruvate to the mitochondria. The mitochondrial pyruvate transporter may be MPC1, MPC2, MPC3, or a combination thereof. For example, the mitochondrial pyruvate transporter is at least about 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% , 99% or more amino acid sequence identity. Specifically, the mitochondrial pyruvate transporter may have an amino acid sequence having an amino acid sequence identity of 95% or more with SEQ ID NOS: 22, 23, and 24. MPC1, MPC2, and MPC3 may be those having the amino acid sequences of SEQ ID NOS: 22, 23, and 24, respectively. The polynucleotide encoding the mitochondrial pyruvate transporter may be a polynucleotide encoding a protein having an amino acid sequence identity of 95% or more with SEQ ID NO: 22, 23, or 24. For example, the polynucleotide sequence of SEQ ID NO: 25, 26, or 27 may be included. SEQ ID NOS: 25, 26, and 27 may be polynucleotides encoding MPC1, MPC2, and MPC3, respectively.

Also for example, the yeast cell may be a polypeptide that converts 6-phosphogluconate to libulose 5-phosphate, a polypeptide that converts xylulose 5-phosphate to sedoheptulose 7-phosphate, sedoheptulose 7- The activity of a polypeptide that converts to 4-phosphate, or a combination thereof, is decreased; A polynucleotide encoding a polypeptide that converts pyruvate to acetaldehyde, a polynucleotide that encodes a polypeptide that converts lactate into pyruvate, a polypeptide that converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate A polynucleotide encoding an exogenous mitochondrial NADH dehydrogenase, a polynucleotide encoding a mitochondrial pyruvate transporter (MPC), or a combination thereof, is removed or destroyed; Or a yeast cell in which a polynucleotide encoding a polypeptide that converts pyruvate to lactate is included or added. The yeast cell may be Saccharomyces cerevisiae.

Another aspect provides a composition for use in producing lactate, including the yeast cells described above.

Another aspect provides a method of producing lactate, comprising culturing the yeast cells described above.

The culture may be performed according to a suitable culture medium and culture conditions known in the art. As a conventional technician, the medium and the culture conditions can be easily adjusted according to the selected microorganism. Cultivation methods may include, for example, batch, continuous and fed-batch cultivation. The bacterial cells are as described above.

The medium may comprise various carbon sources, nitrogen sources and trace element components.

The carbon source may be selected from, for example, carbohydrates such as glucose, sucrose, lactose, fructose, maltose, starch and cellulose, fats such as soybean oil, sunflower oil, castor oil, coconut oil, fatty acids such as palmitic acid, stearic acid, linoleic acid, Glycerol and alcohols such as ethanol, organic acids such as acetic acid, and / or combinations thereof. The culture can be performed, for example, using glucose as a carbon source. The nitrogen source may be selected from the group consisting of organic nitrogen sources such as, for example, peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL) and soybean wheat, and urea, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate An inorganic nitrogen source, and / or combinations thereof. The medium can include, for example, metal salts such as potassium dihydrogenphosphate, dipotassium hydrogenphosphate and the corresponding sodium-containing salts, magnesium sulfate or iron sulfate as a source of phosphorus. In addition, amino acids, vitamins, and suitable precursors and the like may be included in the medium. The medium or the individual components may be added to the culture medium batchwise or continuously.

In addition, during culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added to the microorganism culture medium in an appropriate manner to adjust the pH of the culture medium. In addition, bubble formation can be suppressed by using a defoaming agent such as fatty acid polyglycol ester during the culture.

The cells may be cultured under aerobic, microorganism, or anaerobic conditions. The microorganism condition means a culture condition in which a lower level of oxygen is dissolved into the medium than the oxygen level in the atmosphere. The low level of oxygen can be, for example, 0.1% to 10%, 1% to 9%, 2% to 8%, 3% to 7%, or 4 to 6% of the saturation dissolved oxygen concentration to the atmosphere . The microorganism condition may be, for example, a concentration of dissolved oxygen in the medium of 0.9 ppm to 3.6 ppm. The incubation temperature may be, for example, 20 캜 to 45 캜 or 25 캜 to 40 캜. The incubation period can be continued until the desired lactate is obtained as desired. The method of producing the lactate may include recovering or separating the lactate from the culture.

Recovery of the lactate from the culture can be carried out using conventional separation and purification methods. The recovery may be performed by centrifugation, ion exchange chromatography, filtration, precipitation, extraction, distillation, or a combination thereof. For example, the culture may be centrifuged to remove biomass, and the resulting supernatant may be separated by ion exchange chromatography.

According to one aspect of yeast cells, lactate can be produced at a high concentration and a high yield.

According to a method of producing lactate according to one aspect, lactate can be produced at a high concentration and a high yield.

FIG. 1 is a view showing a lactate production pathway of yeast cells having lactate-producing ability according to one embodiment.
Fig. 2 shows the p416-CCW12p-LDH vector.
Figure 3 is a diagram showing the pUC19-HIS3 vector.
Figure 4 is a diagram showing the pUC19-CCW12p-LDH-HIS3 vector.
Figure 5 is a diagram showing the pUC57-ura3HA vector.
Figure 6 is a diagram showing the pUC57-CCW12-LDH-ura3HA vector.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  One. Lactate  Production of strain for high-efficiency production and production of expression vector

1.1 pdc1 , CyB2 , And gpd1  The gene is deleted ldh  Gene-introduced Sakaromayses Selivijia  Production of strain

Using lactose as a lactate-producing strain, the major byproducts were obtained by using Saccharomyces cerevisiae CEN.PK2-1D (MATαura3-52; trp1-289; leu2-3,112; his3Δ1; MAL2-8C; SUC2, EUROSCARF accession number: 30000B) (NAD-dependent glycerol-3-phosphate dehydrogenase), which is a major enzyme of glycerol biosynthesis, is a major enzyme of alcohol fermentation, pyruvate decarboxylase (pdc1) gene for blocking ethanol and glycerol production pathway. L-lactate cytochrome-c oxidoreductase 2 (cyb2) gene, which is a lactate-degrading enzyme, is destroyed, and the gene locus of L-lactate cytochrome c oxidoreductase 2 (KCTC 12415BP), in which the lactate dehydrogenase gene was introduced, was used as CEN.PK2-1D Δpdc1 :: ldh Δcyb2 :: ldhΔgpd1 :: ldh (KCTC 12415BP).

1.1.1 L- LDH  Overexpression vector production

38 and 39 using the genomic DNA of Saccharomyces cerevisiae CEN.PK2-1D as the template for L-LDH overexpression, and the PCR fragment of CCW12 promoter obtained by PCR using the primers of SEQ ID NOS: 38 and 39 was amplified with SacI and XbaI And then introduced into p416-GPD (http://www.atcc.org/products/all/87360.aspx) in which the GPD promoter was digested with SacI and XbaI to prepare p416-CCW12p.

Thereafter, PCR was performed using the genomic DNA of L-ldh (SEQ ID NO: 11) derived from Pelodiscus sinensis japonicus as a template and the primers of SEQ ID NOs: 40 and 41, p416-CCW12p was digested with BamHI and SalI and ligated to prepare p416-CCW12p-LDH. The L-ldh expression vector has a yeast self-replicating sequence / yeast mobility sequence of SEQ ID NO: 32, a CCW12 promoter of SEQ ID NO: 76, and a CYC1 terminator of SEQ ID NO: 37, Derived polynucleotide encoding L-ldh. Fig. 2 shows the p416-CCW12p-LDH vector.

1.1.2 L- ldh  Generation of chromosome-introduced vector

L-ldh was additionally introduced into the genome of the strain KCTC12415BP to increase the production of lactate through redox balance enhancement or glycolysis pathway engineering. To introduce the L-ldh gene into the genome of KCTC12415BP, a gene introduction vector was constructed as follows.

PCR was carried out using the above prepared p416-CCW12p-LDH as a template and the primers of SEQ ID NOS: 42 and 43, and then the obtained PCR fragment and pUC19-HIS3 (Appl Environ Microbiol. 2002 May; 68 (5): 2095-100, FIG. 3). The vector was digested with SacI and ligated to produce pUC19-CCW12p-LDH-HIS3 (FIG. PCR was performed using the primers of SEQ ID NOs: 44 and 45 using the above pUC19-CCW12p-LDH-HIS3 as a template and a cassette for insertion at the TRP1 site was constructed. The expression cassette containing L-ldh can be inserted at the genetic side of TRP1, and in this case, L-ldh can be inserted while the TRP1 gene is deleted. The L-ldh inserted mutant strain was constructed as follows.

KCTC12415BP strain was plated on solid medium of YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and cultured for 24 hours at 30 ° C. Colonies were inoculated into 10 ml of YPD liquid medium Gt; 30 C < / RTI > for a period of time. The fully grown culture was inoculated 1% (v / v) in 50 ml of YPD liquid medium in a 250 ml flask and cultured at 230 rpm in a 30 ° C incubator.

After about 4-5 hours, when the OD ₆₀₀ reached about 0.5, the cells were centrifuged at 4,500 rpm for 10 minutes to suspend the cells in a 100 mM lithium acetate solution. Cells were then centrifuged at 4,500 rpm for 10 min to resuspend the cells in a 1 M lithium acetate solution to which 15% glycerol had been added.

For expression of L-ldh simultaneously with TRP1 deletion, the expression cassette containing L-ldh was mixed with 50% polyethylene glycol, single stranded carrier DNA, and then added to 100 μl of the resuspension. Respectively. The cultures were then plated on a minimal solid medium (YSD, 6.7 g / L yeast nitrogen base without amino acids, 1.4 g / L amino acid dropout mix (-his)) without histidine and cultured at 30 ° C for over 24 hours. Ten colonies (mutants) formed on the plate were selected and transferred to a YSD (-his) solid medium and cultured in a YSD (-his) liquid medium to obtain a commercial kit (Gentra Puregene Cell kit, Qiagen, USA ) Was used to isolate genomic DNA. PCR was carried out using the primers of SEQ ID NOS: 46 and 47 in order to confirm TRP1 deletion using the genomic DNA of the separated mutant strain as a template, and the obtained PCR product was subjected to electrophoresis to obtain L-ldh expression cassette insert Respectively. Thus, a strain of? Trp1 :: ldh (KCTC12415BP? Trp1 :: ldh) was obtained.

1.2 nde1  And nde2 Deficient Sakaromayses Selivijia  Production of strain

1.2.1 nde1  Production of gene deletion cassette

Preparation of pUC57-CCW12p-LDH-ura3HA PCR was carried out using the p416-CCW12p-LDH of Example 1.1.1 as a template and the primers of SEQ ID NOS: 42 and 43, and then the obtained PCR fragment and pUC57- The vector was digested with SacI and ligated to produce pUC57-CCW12p-LDH-ura3HA (Fig. 6).

In order to prepare the nde1 gene deletion cassette, PCR was performed using the prepared pUC57-CCW12p-LDH-ura3HA as a template and the primers of SEQ ID NOs: 48 and 49 to prepare a nde1 gene deletion cassette.

1.2.2 nde1 This fruitful Sakaromayses Selivijia  Production of strain

A mutant strain in which nde1 was deleted from the above-mentioned Δtrp1 :: ldh strain (KCTC12415BPΔtrp1 :: ldh) was prepared as follows.

The Δtrp1 :: ldh strain (KCTC12415BPΔtrp1 :: ldh) was plated on solid medium of YPD (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and cultured at about 30 ° C. for about 24 hours , Colonies were inoculated in about 10 ml of YPD liquid medium and incubated at about 30 ° C for about 18 hours. The fully grown culture was inoculated in about 50 ml of YPD liquid medium containing 250 ml flask, and cultured at about 30 캜 in an incubator at about 230 rpm at 1% (v / v). After about 4-5 hours, when the OD ₆₀₀ reached about 0.5, the cells were centrifuged at about 4,500 rpm for about 10 minutes to suspend the cells in a solution of about 100 mM of lithium acetate. Thereafter, the cells were centrifuged at about 4,500 rpm for about 10 minutes to obtain cells, and the cells were resuspended in a solution of about 1 M of lithium acetate to which about 15% glycerol had been added

To remove the nde1 gene, the nde1 gene-deleted cassette prepared above was mixed with 50% polyethylene glycol, single stranded carrier DNA, and then added to 100 μl of the water-soluble cell suspension, And reacted for 1 hour. The culture was then plated on a minimal solid medium (YSD, 6.7 g / L yeast nitrogen base without amino acids, 1.4 g / L amino acid dropout mix (-ura)) and cultured at 30 ° C for over 24 hours. 10 colonies formed on the plates were selected and transferred to a minimal solid medium without addition of uracil and cultured in a liquid medium of the same composition and genomic DNA was isolated from the strain using a commercial kit (Gentra Puregene Cell kit, Qiagen, USA) . PCR was carried out using the primers of SEQ ID NOS: 50 and 51 in order to confirm deletion of nde1 using the genomic DNA of the above-mentioned mutant strain as a template, and the obtained PCR product was subjected to electrophoresis to confirm deletion of nde1. As a result, KCTC12415BPΔtrp1 :: ldhΔnde1 + ura3 was obtained.

In order to delete additional genes using the gene deletion vector, the URA3 gene, which is a selection marker used for deletion of nde1, was removed using the URA3 pop-out method. (KCTC12415BPΔtrp1 :: ldhΔnde1 + ura3) was inoculated in about 10 ml of YPD liquid medium and incubated at 30 ° C. for about 18 hours. Then, 5-FOA (YSD, 6.7 g / L yeast nitrogen base without amino acids, 1.4 g / acid dropout mix, 1 μg / L 5-Fluoroorotic Acid) and cultured at 30 ° C for about 24 hours. Ten colonies (URA3 pop-out strains) formed on the plate were selected and transferred to a 5-FOA solid medium and cultured in a YPD liquid medium. Using a commercial kit (Gentra Puregene Cell kit, Qiagen, USA) Genomic DNA was isolated. PCR was carried out using the primers of SEQ ID NOS: 50 and 51 in order to confirm the URA3 deletion using the genomic DNA of the separated URA3 pop-out strain as a template, and the obtained PCR products were subjected to electrophoresis to remove URA3 Respectively. As a result, the strain? Da1 (KCTC12415BP? Trp1 :: ldh ?nde1) was obtained.

1.2.3 nde2  Production of deletion cassette of gene

The vector pUC57-CCW12p-LDH-ura3HA prepared in Example 1.2.1 above was used as a vector for destroying the nde2 gene in order to delete the nde2 gene by a homologous recombination method. In order to prepare the nde2 gene deletion cassette, PCR was performed using the prepared pUC57-CCW12p-LDH-ura3HA as a template and the primers of SEQ ID NOs: 52 and 53 to prepare a nde2 gene deletion cassette.

1.2.4 nde1  And nde2 This fruitful Sakaromayses Selivijia  Production of strain

A mutant strain in which the nde2 deletion was performed in the above-mentioned Δnde1 strain (KCTC12415BPΔtrp1 :: ldhΔnde1) was prepared as follows.

The? De1 strain was treated with a lithium acetate solution in the same manner as in Example 1.2.2 to obtain a competent cell.

For the removal of the nde2 gene, the nde2 gene deletion cassette prepared in 1.2.3 was mixed with 50% polyethylene glycol, single stranded carrier DNA, and then added to 100 μl of the above water-soluble cell suspension to obtain the product of Example 1.2. 2 to obtain colonies from the minimum solid medium without uracil. Ten colonies (mutant strains) formed on the plate were selected and transferred to a minimal solid medium without addition of uracil, and cultured in a liquid medium of the same composition. Then, the strain was cultured in a commercial kit (Gentra Puregene Cell kit, Qiagen, USA) DNA was isolated. PCR was performed using the primers of SEQ ID NOs: 54 and 55 to confirm deletion of nde2 using the genomic DNA of the above-mentioned mutant strain as a template, and then PCR products obtained were subjected to electrophoresis to confirm deletion of nde2. As a result, KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2 + ura3 was obtained.

For the additional gene deletion, URA3 pop-out method in the same manner as in Example 1.2.2 was used to transform the URA3 gene, which is a selectable marker used for deletion of nde2, into Saccharomyces cerevisiae KCTC12415BP Δtrp1 :: ldhΔnde1Δnde2 + ura3 . PCR was carried out using the primers of SEQ ID NOS: 54 and 55 in order to confirm the URA3 deletion using the genomic DNA of the separated URA3 pop-out strain as a template, and the obtained PCR product was subjected to electrophoresis to confirm the URA3 deletion Respectively. As a result, the strain ≥ 1 de1Δnde2 (KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2) was obtained.

1.3. mpc1  And mpc2 Deficient Sakaromayses Selivijia  Production of strain

1.3.1 mpc1  Production of gene-deleted cassettes

In order to delete the mpc1 gene by a homologous recombination method, PCR was performed using the pUC57-ura3HA of Example 1.2.1 as a template and the primers (mpc1_del_F, mpc1_del_R) of SEQ ID NOS: 56 and 57 as follows to prepare an mpc1 gene deletion cassette Respectively.

1.3.2 mpc1 This fruitful Sakaromayses Selivijia  Production of strain

A mutant strain in which mpc1 was deleted from the above-mentioned strain Δnde1Δnde2 (KCTC12415BPΔtrp1 :: ldh Δnde1Δnde2) was prepared as follows.

The? -Dione?? 2 strain of Example 1.2 was treated with a lithium acetate solution in the same manner as in Example 1.2.2 to obtain a water-soluble cell suspension.

For mpc1 gene deletion, the mpc1 gene deletion cassette prepared in Example 1.3.1 was mixed with 50% polyethylene glycol, single stranded carrier DNA, and then added to 100 μl of the above water-soluble cell resuspension, .2 to obtain colonies from the minimal solid medium without uracil. Ten colonies (mutant strains) formed on the plate were selected and transferred to a minimal solid medium without addition of uracil, and cultured in a liquid medium of the same composition. Then, the strain was cultured in a commercial kit (Gentra Puregene Cell kit, Qiagen, USA) DNA was isolated. PCR was carried out using the primers (mpc1_up_F, mpc1_down_R) of SEQ ID NOs: 58 and 59 in order to confirm deletion of mpc1 using the genomic DNA of the above mutant strain as a template, and the obtained PCR products were electrophoresed to remove mpc1 Respectively. As a result, KCTC12415BPΔtrp1 :: ldh Δnde1Δnde2Δmpc1 + ura3 was obtained.

In addition, URA3 gene, which is a selection marker used for deletion of mpc1, was inserted into Saccharomyces cerevisiae KCTC12415BP Δtrp1 :: ldhΔnde1Δnde2 Δmpc1 + < / RTI > PCR was performed using the primers of SEQ ID NOs: 58 and 59 to confirm the deletion of URA3 using the genomic DNA of the separated URA3 pop-out strain as a template. Then, the obtained PCR product was subjected to electrophoresis to confirm the URA3 deletion Respectively. As a result, a strain of? Mpc1 (KCTC12415BP? Trp1 :: ldhΔnde1Δnde2Δmpc1) was obtained.

1.3.3 mpc2  Production of gene-deleted cassettes

In order to delete the mpc2 gene by the homologous recombination method, PCR was performed using the pUC57-ura3HA of Example 1.2.1 as a template and the primers (mpc2_del_F, mpc2_del_R) of SEQ ID NOS: 60 and 61 as follows to prepare an mpc2 gene deletion cassette Respectively.

1.3.4 mpc1  And mpc2 Deficient Sakaromayses Selivijia  Production of strain

In order to prepare a strain of mutant Δmpc1Δmpc2 in which mpc1 and mpc2 were simultaneously deleted, the Δmpc1 strain (KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2Δmpc1) obtained in Example 1.3.2 was treated with a lithium acetate solution in the same manner as in Example 1.2.2 To obtain a competent cell resuspension.

For the mpc2 gene deletion, the mpc2 gene deletion cassette prepared in Example 1.3.3 was mixed with 50% polyethylene glycol, single stranded carrier DNA, and then added to 100 μl of the above water-soluble cell suspension to obtain the product of Example 1.2 .2 to obtain colonies from the minimal solid medium without uracil. Ten colonies (mutant strains) formed on the plate were selected, transferred to a minimal solid medium without addition of uracil, and cultured in a liquid medium of the same composition. The genomic DNA was purified from the strain using a commercial kit (Gentra Puregene Cell kit, Qiagen, USA) . PCR was carried out using the primers (mpc2_up_F, mpc2_down_R) of SEQ ID NOS: 62 and 63 in order to confirm the deletion of mpc2 using the genomic DNA of the separated mutant strain as a template, and the obtained PCR products were electrophoresed to remove mpc2 Respectively. As a result, KCTC12415BPΔtrp1 :: ldh Δnde1Δnde2Δmpc1Δmpc2 + ura3 strain was obtained.

In addition, the URA3 gene, which is a selection marker, was removed from the KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2Δmpc1Δmpc2 + ura3 strain using the URA3 pop-out method in the same manner as in Example 1.2.2. PCR was carried out using the primers of SEQ ID NOs: 62 and 63 to confirm the deletion of URA3 using the genomic DNA of the separated URA3 pop-out strain as a template, and the obtained PCR product was subjected to electrophoresis to remove the URA3 deletion Respectively. As a result, a strain of? Mpc1? Mpc2 (KCTC12415BP? Trp1 :: ldh ?? ed1Δnde2? Mpc1? Mpc2) was obtained.

Example  2. gnd2 Deficient Sakaromayses Selivijia  Production of strain

2.1 gnd2  Production of gene-deleted cassettes

In order to delete the gnd2 gene by the homologous recombination method, PCR was performed using pUC57-ura3HA of Example 1.2.1 as a template and the primers (gnd2_del_F, gnd2_del_R) of SEQ ID NOS: 64 and 65 as follows to prepare a gnd2 gene deletion cassette Respectively.

2.2 gnd2 Deficient Sakaromayses Selivijia  Production of strain

A mutant strain in which mpc1 was deleted from the above Δmpc1Δmpc2 strain (KCTC12415BPΔtrp1 :: ldh Δnde1Δnde2Δmpc1Δmpc2) was prepared as follows.

The? Mpc1? Mpc2 strain of Example 1 was treated with a lithium acetate solution in the same manner as in Example 1.2.2 to obtain a water-soluble cell suspension.

For the gnd2 gene deletion, the gnd2 gene deletion cassette prepared in Example 2.1 was mixed with 50% polyethylene glycol, single stranded carrier DNA, and then added to 100 μl of the above water-soluble cell suspension to obtain Example 1.2.2 To obtain colonies from the minimal solid medium without uracil. Ten colonies (mutant strains) formed on the plate were selected and transferred to a minimal solid medium without addition of uracil, and cultured in a liquid medium of the same composition. Then, the strain was cultured in a commercial kit (Gentra Puregene Cell kit, Qiagen, USA) DNA was isolated. PCR was performed using the primers (gnd2_up_F, gnd2_down_R) of SEQ ID NOS: 66 and 67 to confirm deletion of gnd2 using the genomic DNA of the above mutant strain as a template, and the obtained PCR products were subjected to electrophoresis to obtain gnd2 deletion Respectively. As a result, KCTC12415BPΔtrp1 :: ldh Δnde1Δnde2Δmpc1 Δmpc2 Δgnd2 + ura3 was obtained.

For the additional gene deletion, the URA3 gene, which is a selection marker used for deletion of gnd2, was cloned into Saccharomyces cerevisiae KCTC12415BP < RTI ID = 0.0 > Δtrp1 :: ldhΔnde1Δnde2 Δmpc1 Δmpc2 And removed from? Gnd2 +? 3. PCR was performed using the primers of SEQ ID NOS: 66 and 67 in order to identify the URA3 deletion using the genomic DNA of the separated URA3 pop-out strain as a template, and the obtained PCR product was subjected to electrophoresis to confirm the URA3 deletion Respectively. As a result, strain Δgnd2 (KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2Δmpc1Δmpc2Δgnd2) was obtained.

Example  3. tal1 This fruitful Sakaromayses Selivijia  Production of strain

3.1 tal1  Production of gene-deleted cassettes

In order to delete the tal1 gene by a homologous recombination method, PCR was performed using pUC57-ura3HA of Example 1.2.1 as a template and primers (tal1_del_F, tal 1_del_R) of SEQ ID NOS: 68 and 69, Respectively.

3.2 tal1 This fruitful Sakaromayses Selivijia  Production of strain

A mutant strain in which tal1 was deleted from the above-mentioned Δgnd2 strain (KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2 Δmpc1Δmpc2Δgnd2) was prepared as follows.

The? Gnd2 strain of Example 2 was treated with a lithium acetate solution in the same manner as in Example 1.2.2 to obtain a water-soluble cell suspension.

For the removal of the tal1 gene, the tal1 gene-deleted cassette prepared in Example 3.1 was mixed with 50% polyethylene glycol, single stranded carrier DNA, and then added to 100 μl of the above water-soluble cell suspension, To obtain colonies from the minimal solid medium without uracil. Ten colonies (mutant strains) formed on the plate were selected and transferred to a minimal solid medium without addition of uracil, and cultured in a liquid medium of the same composition. Then, the strain was cultured in a commercial kit (Gentra Puregene Cell kit, Qiagen, USA) DNA was isolated. PCR was performed using primers (SEQ ID NOs: 70 and 71) of SEQ ID NOs: 70 and 71 to confirm tal1 deletion using the genomic DNA of the above-mentioned mutant strain as a template, and the obtained PCR products were electrophoresed to obtain tal1 deletion Respectively. As a result, KCTC12415BPΔtrp1 :: ldh Δnde1Δnde2Δmpc1Δmpc2Δtal1 + ura3 was obtained.

The URA3 gene, which is a selectable marker used for deletion of tal1, was amplified using Saccharomyces cerevisiae KCTC12415BP Δtrp1 :: ldhΔnde1Δnde2Δmpc1 Δmpc2Δtal1 + Δmpc2Δtal1 using the URA3 pop-out method in the same manner as in Example 1.2.2 for additional gene deletion. < / RTI > PCR was carried out using the primers of SEQ ID NOS: 70 and 71 in order to confirm the URA3 deletion using the genomic DNA of the separated URA3 pop-out strain as a template, and the obtained PCR product was subjected to electrophoresis to confirm the URA3 deletion Respectively. As a result, a strain of? Gnd2? Alt1 (KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2Δmpc1 Δmpc2Δgnd2Δtal1) was obtained.

Example  4. tkl2 This fruitful Sakaromayses Selivijia  Production of strain

4.1 tkl2  Production of gene-deleted cassettes

In order to delete the tkl2 gene by the homologous recombination method, PCR was performed using the pUC57-ura3HA of Example 1.2.1 as a template and the primers (tkl2_del_F, tkl2_del_R) of SEQ ID NOS: 72 and 73 to prepare a tkl2 gene deletion cassette Respectively.

4.2 tkl2 Deficient Sakaromayses Selivijia  Production of strain

A mutant strain in which tkl2 was deleted from the above Δgnd2 strain (KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2 Δmpc1Δmpc2Δgnd2) was prepared as follows.

The? Gnd2 strain of Example 2 was treated with a lithium acetate solution in the same manner as in Example 1.2.2 to obtain a water-soluble cell suspension.

For the Tkl2 gene deletion, the tkl2 gene deletion cassette prepared in Example 4.1 was mixed with 50% polyethylene glycol, single stranded carrier DNA, and then added to 100 μl of the above water-soluble cell resuspension, To obtain colonies from the minimal solid medium without uracil. Ten colonies (mutant strains) formed on the plate were selected and transferred to a minimal solid medium without addition of uracil, and cultured in a liquid medium of the same composition. Then, the strain was cultured in a commercial kit (Gentra Puregene Cell kit, Qiagen, USA) DNA was isolated. PCR was carried out using primers (tkl2_up_F, tkl2_down_R) of SEQ ID NOs: 74 and 75 in order to confirm tkl2 deletion using the genomic DNA of the above mutant strain as a template, and the obtained PCR products were electrophoresed to remove tkl2 deletion Respectively. As a result, KCTC12415BPΔtrp1 :: ldh Δnde1Δnde2Δmpc1Δmpc2Δtkl2 + ura3 was obtained.

The URA3 gene, which is a selection marker used for deletion of tkl2, was amplified by PCR using Saccharomyces cerevisiae KCTC12415BP Δtrp1 :: ldhΔnde1Δnde2Δmpc1Δmpc2 Δtkl2 + < / RTI > PCR was carried out using the primers of SEQ ID NOs: 74 and 75 in order to confirm the deletion of URA3 using the genomic DNA of the separated URA3 pop-out strain as a template, and the obtained PCR product was subjected to electrophoresis to confirm the URA3 deletion Respectively. As a result,? Gnd2? Tkl2 strain KCTC12415BP? Trp1 :: ldhΔnde1Δnde2Δmpc1Δmpc2Δgnd2Δtkl2 was obtained.

Example  5. gnd2  Using a gene-deficient strain Lactate  production

Each of the Δmpc1Δmpc2 and Δgnd2 strains prepared in Examples 1 and 2 was plated on a YPD solid medium and cultured at 30 ° C. for 24 hours or more. Then, the cells were inoculated into a 50 ml YPD liquid medium containing 40 g / L glucose. Lt; / RTI > for 16 hours. The fermentation was carried out by measuring the cell concentration in the culture medium of 50 ml using the spectrophotometer at an absorbance of 5.0 to 600 nm, centrifuging to discard the supernatant, resuspending the cells, and adding 80 g / L of glucose Lt; RTI ID = 0.0 > 50ml < / RTI > YPD liquid medium.

Fermentation conditions were incubated at 30 ° C for more than 24 hours in a stirred incubator maintained at about 90 rpm. Samples were periodically taken from the flask during fermentation, and the collected samples were centrifuged at about 13,000 rpm for about 10 minutes, and the concentration of lactate in the supernatant was analyzed by liquid chromatography (HPLC).

As shown in Table 1, the Δgnd2 strain increased the L-lactate productivity from 33.6 g / L to 35.0 g / L compared to the control Δmpc1Δmpc2 strain, and the yield increased from 41.5% to 43.2%. Thus, lactate production and yield were increased in strains deficient in gnd gene.

Strain name characteristic O D ₆₀₀ LA
(g / L) yield
(%) ? Mpc1? Mpc2 KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2Δmpc1Δmpc2 10.7 33.6 41.5 Δgnd2 strain KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2Δmpc1Δmpc2Δgnd2 11.5 35.0 43.2

50 ml flask, incubation for about 24 hours, LA: lactate

Example  6. gnd2 , tal1 , tkl2 , Or a strain in which a gene of the combination thereof is deleted Each  Used Lactate  production

Each of the Δgnd2, Δgnd2Δtal1 and Δgnd2Δtkl2 strains prepared in Examples 2, 3 and 4 were plated on a YPD solid medium, cultured at 30 ° C. for 24 hours or more, and inoculated into a 50 ml YPD liquid medium containing 40 g / L glucose And cultured under aerobic conditions at 30 DEG C for 16 hours. The fermentation was carried out by measuring the cell concentration in the culture medium of 50 ml using the spectrophotometer at an absorbance of 5.0 to 600 nm, centrifuging to discard the supernatant, resuspending the cells, and adding 80 g / L of glucose Lt; RTI ID = 0.0 > 50ml < / RTI > YPD liquid medium.

Fermentation conditions were incubated at 30 ° C for more than 24 hours in a stirred incubator maintained at about 90 rpm. Samples were periodically taken from the flask during fermentation, and the collected samples were centrifuged at about 13,000 rpm for about 10 minutes, and the concentration of lactate in the supernatant was analyzed by liquid chromatography (HPLC).

As shown in Table 2, the Δgnd2Δtal1 strain increased the L-lactate productivity from 33.0 g / L to 35.7 g / L compared to the Δgnd2 strain, and the yield increased from 42.0% to 45.9%. Lactate productivity increased from 33.0 g / L to 35.0 g / L in Δgnd2Δtkl2 strain compared to Δgnd2 strain, and the yield increased from 42.0% to 45.3%. Thus, lactate production and yield were increased in strains deficient in tal1 gene or tkl2.

Strain name characteristic O D ₆₀₀ LA
(g / L) yield
(%) Δgnd2 strain KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2Δmpc1Δmpc2Δgnd2 12.2 33.0 42.0 Δgnd2 Δtal1 Lt; 11.6 35.7 45.9 Δgnd2 Δtkl2 KCTC12415BPΔtrp1 :: ldhΔnde1Δnde2Δmpc1Δmpc2Δgnd2Δtkl2 12.1 35.0 45.3

50 ml flask, incubation for about 24 hours, LA: lactate

Korea Biotechnology Research Institute KCTC12415BP 20130530

<110> Samsung Electronics Co. Ltd. <120> Yeast cell with reduced pentose phosphate pathway and method for          producing lactate using the same <130> PN1015351 <160> 76 <170> Kopatentin 2.0 <210> 1 <211> 492 <212> PRT <213> Saccharomyces cerevisiae <400> 1 Met Ser Lys Ala Val Gly Asp Leu Gly Leu Val Gly Leu Ala Val Met   1 5 10 15 Gly Gln Asn Leu Ile Leu Asn Ala Ala Asp His Gly Phe Thr Val Val              20 25 30 Ala Tyr Asn Arg Thr Gln Ser Lys Val Asp Arg Phe Leu Ala Asn Glu          35 40 45 Ala Lys Gly Lys Ser Ile Gly Ala Thr Ser Ile Glu Asp Leu Val      50 55 60 Ala Lys Leu Lys Lys Pro Arg Lys Ile Met Leu Leu Ile Lys Ala Gly  65 70 75 80 Ala Pro Val Asp Thr Leu Ile Lys Glu Leu Val Pro His Leu Asp Lys                  85 90 95 Gly Asp Ile Ile Ile Asp Gly Gly Asn Ser His Phe Pro Asp Thr Asn             100 105 110 Arg Arg Tyr Glu Glu Leu Thr Lys Gln Gly Ile Leu Phe Val Gly Ser         115 120 125 Gly Val Ser Gly Gly Glu Asp Gly Ala Arg Phe Gly Pro Ser Leu Met     130 135 140 Pro Gly Gly Ser Ala Glu Ala Trp Pro His Ile Lys Asn Ile Phe Gln 145 150 155 160 Ser Ile Ala Lys Ser Asn Gly Glu Pro Cys Cys Glu Trp Val Gly                 165 170 175 Pro Ala Gly Ser Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu             180 185 190 Tyr Gly Asp Met Gln Leu Ile Cys Glu Ala Tyr Asp Ile Met Lys Arg         195 200 205 Ile Gly Arg Phe Thr Asp Lys Glu Ile Ser Glu Val Phe Asp Lys Trp     210 215 220 Asn Thr Gly Val Leu Asp Ser Phe Leu Ile Glu Ile Thr Arg Asp Ile 225 230 235 240 Leu Lys Phe Asp Asp Val Asp Gly Lys Pro Leu Val Glu Lys Ile Met                 245 250 255 Asp Thr Ala Gly Gln Lys Gly Thr Gly Lys Trp Thr Ala Ile Asn Ala             260 265 270 Leu Asp Leu Gly Met Pro Val Thr Leu Ile Gly Glu Ala Val Phe Ala         275 280 285 Arg Cys Leu Ser Ala Ile Lys Asp Glu Arg Lys Arg Ala Ser Lys Leu     290 295 300 Leu Ala Gly Pro Thr Val Pro Lys Asp Ala Ile His Asp Arg Glu Gln 305 310 315 320 Phe Val Tyr Asp Leu Glu Gln Ala Leu Tyr Ala Ser Lys Ile Ile Ser                 325 330 335 Tyr Ala Gln Gly Phe Met Leu Ile Arg Glu Ala Ala Arg Ser Tyr Gly             340 345 350 Trp Lys Leu Asn Asn Pro Ala Ile Ala Leu Met Trp Arg Gly Gly Cys         355 360 365 Ile Ile Arg Ser Val Phe Leu Ala Glu Ile Thr Lys Ala Tyr Arg Asp     370 375 380 Asp Pro Asp Leu Glu Asn Leu Leu Phe Asn Glu Phe Phe Ala Ser Ala 385 390 395 400 Val Thr Lys Ala Gln Ser Gly Trp Arg Arg Thr Ile Ala Leu Ala Ala                 405 410 415 Thr Tyr Gly Ile Pro Thr Pro Ala Phe Ser Thr Ala Leu Ala Phe Tyr             420 425 430 Asp Gly Tyr Arg Ser Glu Arg Leu Pro Ala Asn Leu Leu Gln Ala Gln         435 440 445 Arg Asp Tyr Phe Gly Ala His Thr Phe Arg Ile Leu Pro Glu Cys Ala     450 455 460 Ser Ala His Leu Pro Val Asp Lys Asp Ile His Ile Asn Trp Thr Gly 465 470 475 480 His Gly Gly Asn Ile Ser Ser Ser Thr Tyr Gln Ala                 485 490 <210> 2 <211> 1479 <212> DNA <213> Saccharomyces cerevisiae <400> 2 atgtcaaagg cagtaggtga tttaggctta gttggtttag ccgtgatggg tcaaaatttg 60 atcttaaacg cagcggatca cggatttacc gtggttgctt ataataggac gcaatcaaag 120 gtagataggt ttctagctaa tgaggcaaaa ggaaaatcaa taattggtgc aacttcaatt 180 gaggacttgg ttgcgaaact aaagaaacct agaaagatta tgcttttaat caaagccggt 240 gctccggtcg acactttaat aaaggaactt gtaccacatc ttgataaagg cgacattatt 300 atcgacggtg gtaactcaca tttcccggac actaacagac gctacgaaga gctaacaaag 360 caaggaattc tttttgtggg ctctggtgtc tcaggcggtg aagatggtgc acgttttggt 420 ccatctttaa tgcctggtgg gtcagcagaa gcatggccgc acatcaagaa catctttcaa 480 tctattgccg ccaaatcaaa cggtgagcca tgctgcgaat gggtggggcc tgccggttct 540 ggtcactatg tgaagatggt acacaacggt atcgagtacg gtgatatgca gttgatttgc 600 gaggcttacg atatcatgaa acgaattggc cggtttacgg ataaagagat cagtgaagta 660 tttgacaagt ggaacactgg agttttggat tctttcttga ttgaaatcac gagggacatt 720 ttaaaattcg atgacgtcga cggtaagcca ttggtggaaa aaattatgga tactgccggt 780 caaaagggta ctggtaaatg gactgcaatc aacgccttgg atttaggaat gccagtcact 840 ttaattgggg aggctgtttt cgctcgttgt ttgtcagcca taaaggacga acgtaaaaga 900 gcttcgaaac ttctggcagg accaacagta ccaaaggatg caatacatga tagagaacaa 960 tttgtgtatg atttggaaca agcattatac gcttcaaaga ttatttcata tgctcaaggt 1020 ttcatgctga tccgcgaagc tgccagatca tacggctgga aattaaacaa cccagctatt 1080 gctctaatgt ggagaggtgg ctgtataatc agatctgtgt tcttagctga gattacgaag 1140 gcttataggg acgatccaga tttggaaaat ttattattca acgagttctt cgcttctgca 1200 gttactaagg cccaatccgg ttggagaaga actattgccc ttgctgctac ttacggtatt 1260 ccaactccag ctttctctac tgctttagcg ttttacgacg gctatagatc tgagaggcta 1320 ccagcaaact tgttacaagc gcaacgtgat tattttggcg ctcatacatt tagaatttta 1380 cctgaatgtg cttctgccca tttgccagta gacaaggata ttcatatcaa ttggactggg 1440 cacggaggta atatatcttc ctcaacctac caagcttaa 1479 <210> 3 <211> 489 <212> PRT <213> Saccharomyces cerevisiae <400> 3 Met Ser Ala Asp Phe Gly Leu Ile Gly Leu Ala Val Met Gly Gln Asn   1 5 10 15 Leu Ile Leu Asn Ala Ala Asp His Gly Phe Thr Val Cys Ala Tyr Asn              20 25 30 Arg Thr Gln Ser Lys Val Asp His Phe Leu Ala Asn Glu Ala Lys Gly          35 40 45 Lys Ser Ile Gly Ala Thr Ser Ile Glu Asp Phe Ile Ser Lys Leu      50 55 60 Lys Arg Pro Arg Lys Val Met Leu Leu Val Lys Ala Gly Ala Pro Val  65 70 75 80 Asp Ala Leu Ile Asn Gln Ile Val Pro Leu Leu Glu Lys Gly Asp Ile                  85 90 95 Ile Ile Asp Gly Gly Asn Ser His Phe Pro Asp Ser Asn Arg Arg Tyr             100 105 110 Glu Glu Leu Lys Lys Lys Gly Ile Leu Phe Val Gly Ser Gly Val Ser         115 120 125 Gly Gly Glu Glu Gly Ala Arg Tyr Gly Pro Ser Leu Met Pro Gly Gly     130 135 140 Ser Glu Glu Ala Trp Pro His Ile Lys Asn Ile Phe Gln Ser Ile Ser 145 150 155 160 Ala Lys Ser Asp Gly Glu Pro Cys Cys Glu Trp Val Gly Pro Ala Gly                 165 170 175 Ala Gly His Tyr Val Lys Met Val His Asn Gly Ile Glu Tyr Gly Asp             180 185 190 Met Gln Leu Ile Cys Glu Ala Tyr Asp Ile Met Lys Arg Leu Gly Gly         195 200 205 Phe Thr Asp Lys Glu Ile Ser Asp Val Phe Ala Lys Trp Asn Asn Gly     210 215 220 Val Leu Asp Ser Phe Leu Val Glu Ile Thr Arg Asp Ile Leu Lys Phe 225 230 235 240 Asp Asp Val Asp Gly Lys Pro Leu Val Glu Lys Ile Met Asp Thr Ala                 245 250 255 Gly Gln Lys Gly Thr Gly Lys Trp Thr Ala Ile Asn Ala Leu Asp Leu             260 265 270 Gly Met Pro Val Thr Leu Ile Gly Glu Ala Val Phe Ala Arg Cys Leu         275 280 285 Ser Ala Leu Lys Asn Glu Arg Ile Arg Ala Ser Lys Val Leu Pro Gly     290 295 300 Pro Glu Val Pro Lys Asp Ala Val Lys Asp Arg Glu Gln Phe Val Asp 305 310 315 320 Asp Leu Glu Gln Ala Leu Tyr Ala Ser Lys Ile Ile Ser Tyr Ala Gln                 325 330 335 Gly Phe Met Leu Ile Arg Glu Ala Ala Ala Thr Tyr Gly Trp Lys Leu             340 345 350 Asn Asn Pro Ala Ile Ala Leu Met Trp Arg Gly Gly Cys Ile Ile Arg         355 360 365 Ser Val Phe Leu Gly Gln Ile Thr Lys Ala Tyr Arg Glu Glu Pro Asp     370 375 380 Leu Glu Asn Leu Leu Phe Asn Lys Phe Phe Ala Asp Ala Val Thr Lys 385 390 395 400 Ala Gln Ser Gly Trp Arg Lys Ser Ile Ala Leu Ala Thr Thr Tyr Gly                 405 410 415 Ile Pro Thr Pro Ala Phe Ser Thr Ala Leu Ser Phe Tyr Asp Gly Tyr             420 425 430 Arg Ser Glu Arg Leu Pro Ala Asn Leu Leu Gln Ala Gln Arg Asp Tyr         435 440 445 Phe Gly Ala His Thr Phe Arg Val Leu Pro Glu Cys Ala Ser Asp Asn     450 455 460 Leu Pro Val Asp Lys Asp Ile His Ile Asn Trp Thr Gly His Gly Gly 465 470 475 480 Asn Val Ser Ser Thr Tyr Gln Ala                 485 <210> 4 <211> 1470 <212> DNA <213> Saccharomyces cerevisiae <400> 4 atgtctgctg atttcggttt gattggtttg gccgtcatgg gtcaaaattt gatcttgaac 60 gctgctgacc acggtttcac tgtttgtgct tacaacagaa ctcaatccaa ggtcgaccat 120 ttcttggcca atgaagctaa gggcaaatct atcatcggtg ctacttccat tgaagatttc 180 atctccaaat tgaagagacc tagaaaggtc atgcttttgg ttaaagctgg tgctccagtt 240 gacgctttga tcaaccaaat cgtcccactt ttggaaaagg gtgatattat catcgatggt 300 ggtaactctc acttcccaga ttctaataga cgttacgaag aattgaagaa gaagggtatt 360 cttttcgttg gttctggtgt ctccggtggt gaggaaggtg cccgttacgg tccatctttg 420 atgccaggtg gttctgaaga agcttggcca catattaaga acatcttcca atccatctct 480 gctaaatccg acggtgaacc atgttgcgaa tgggttggcc cagccggtgc tggtcactac 540 gtcaagatgg ttcacaacgg tattgaatac ggtgatatgc aattgatttg tgaagcttat 600 gacatcatga agagattggg tgggtttacc gataaggaaa tcagtgacgt ttttgccaaa 660 tggaacaatg gtgtcttgga ttccttcttg gtcgaaatta ccagagatat tttgaaattc 720 gacgacgtcg acggtaagcc attagttgaa aaaatcatgg atactgctgg tcaaaagggt 780 actggtaagt ggactgccat caacgccttg gatttgggta tgccagttac tttgattggt 840 gaagctgtct ttgcccgttg tctatctgct ttgaagaacg agagaattag agcctccaag 900 gtcttaccag gcccagaagt tccaaaagac gccgtcaagg acagagaaca atttgtcgat 960 gatttggaac aagctttgta tgcttccaag attatttctt acgctcaagg tttcatgttg 1020 atccgtgaag ctgctgctac ttatggctgg aaactaaaca accctgccat cgctttgatg 1080 tggagaggtg gttgtatcat tagatctgtt ttcttgggtc aaatcacaaa ggcctacaga 1140 gaagaaccag atttggaaaa cttgttgttc aacaagttct tcgctgatgc cgtcaccaag 1200 gctcaatctg gttggagaaa gtcaattgcg ttggctacca cctacggtat cccaacacca 1260 gccttttcca ccgctttgtc tttctacgat gggtacagat ctgaaagatt gccagccaac 1320 ttactacaag ctcaacgtga ctactttggt gctcacactt tcagagtgtt gccagaatgt 1380 gcttctgaca acttgccagt agacaaggat atccatatca actggactgg ccacggtggt 1440 aatgtttctt cctctacata ccaagcttaa 1470 <210> 5 <211> 681 <212> PRT <213> Saccharomyces cerevisiae <400> 5 Met Ala Gln Phe Ser Asp Ile Asp Lys Leu Ala Val Ser Thr Leu Arg   1 5 10 15 Leu Leu Ser Val Asp Gln Val Glu Ser Ala Gln Ser Gly His Pro Gly              20 25 30 Ala Pro Leu Gly Leu Ala Pro Val Ala His Val Ile Phe Lys Gln Leu          35 40 45 Arg Cys Asn Pro Asn Asn Glu His Trp Ile Asn Arg Asp Arg Phe Val      50 55 60 Leu Ser Asn Gly His Ser Cys Ala Leu Leu Tyr Ser Met Leu His Leu  65 70 75 80 Leu Gly Tyr Asp Tyr Ser Ile Glu Asp Leu Arg Gln Phe Arg Gln Val                  85 90 95 Asn Ser Arg Thr Pro Gly His Pro Glu Phe His Ser Ala Gly Val Glu             100 105 110 Ile Thr Ser Gly Pro Leu Gly Gln Gly Ile Ser Asn Ala Val Gly Met         115 120 125 Ala Ile Ala Gln Ala Asn Phe Ala Ala Thr Tyr Asn Glu Asp Gly Phe     130 135 140 Pro Ile Ser Asp Ser Tyr Thr Phe Ala Ile Val Gly Asp Gly Cys Leu 145 150 155 160 Gln Glu Gly Val Ser Ser Glu Thr Ser Ser Leu Ala Gly His Leu Gln                 165 170 175 Leu Gly Asn Leu Ile Thr Phe Tyr Asp Ser Asn Ser Ile Ser Ile Asp             180 185 190 Gly Lys Thr Ser Tyr Ser Phe Asp Glu Asp Val Leu Lys Arg Tyr Glu         195 200 205 Ala Tyr Gly Trp Glu Val Met Glu Val Asp Lys Gly Asp Asp Asp Met     210 215 220 Glu Ser Ile Ser Ser Ala Leu Glu Lys Ala Lys Leu Ser Lys Asp Lys 225 230 235 240 Pro Thr Ile Ile Lys Val Thr Thr Thr Ile Gly Phe Gly Ser Leu Gln                 245 250 255 Gln Gly Thr Ala Gly Val His Gly Ser Ala Leu Lys Ala Asp Asp Val             260 265 270 Lys Gln Leu Lys Lys Arg Trp Gly Phe Asp Pro Asn Lys Ser Phe Val         275 280 285 Val Pro Gln Glu Val Tyr Asp Tyr Tyr Lys Lys Thr Val Val Glu Pro     290 295 300 Gly Gln Lys Leu Asn Glu Glu Trp Asp Arg Met Phe Glu Glu Tyr Lys 305 310 315 320 Thr Lys Phe Pro Glu Lys Gly Lys Glu Leu Gln Arg Arg Leu Asn Gly                 325 330 335 Glu Leu Pro Glu Gly Trp Glu Lys His Leu Pro Lys Phe Thr Pro Asp             340 345 350 Asp Asp Ala Leu Ala Thr Arg Lys Thr Ser Gln Gln Val Leu Thr Asn         355 360 365 Met Val Gln Val Leu Pro Glu Leu Ile Gly Gly Ser Ala Asp Leu Thr     370 375 380 Pro Ser Asn Leu Thr Arg Trp Glu Gly Ala Val Asp Phe Gln Pro Pro 385 390 395 400 Ile Thr Gln Leu Gly Asn Tyr Ala Gly Arg Tyr Ile Arg Tyr Gly Val                 405 410 415 Arg Glu His Gly Met Gly Ala Ile Met Asn Gly Ile Ser Ala Phe Gly             420 425 430 Ala Asn Tyr Lys Pro Tyr Gly Gly Thr Phe Leu Asn Phe Val Ser Tyr         435 440 445 Ala Ala Gly Ala Val Ale Leu Ale Ala Leu Ser Gly Asn Pro Val Ile     450 455 460 Trp Val Ala Thr His Asp Ser Ile Gly Leu Gly Glu Asp Gly Pro Thr 465 470 475 480 His Gln Pro Ile Glu Thr Leu Ala His Leu Arg Ala Ile Pro Asn Met                 485 490 495 His Val Trp Arg Pro Ala Asp Gly Asn Glu Thr Ser Ala Ala Tyr Tyr             500 505 510 Ser Ala Ile Lys Ser Gly Arg Thr Pro Ser Val Val Ala Leu Ser Arg         515 520 525 Gln Asn Leu Pro Gln Leu Glu His Ser Ser Phe Glu Lys Ala Leu Lys     530 535 540 Gly Gly Tyr Val Ile His Asp Val Glu Asn Pro Asp Ile Ile Leu Val 545 550 555 560 Ser Thr Gly Ser Glu Val Ser Ile Ser Ile Asp Ala Ala Lys Lys Leu                 565 570 575 Tyr Asp Thr Lys Lys Ile Lys Ala Arg Val Val Ser Leu Pro Asp Phe             580 585 590 Tyr Thr Phe Asp Arg Gln Ser Glu Glu Tyr Arg Phe Ser Val Leu Pro         595 600 605 Asp Gly Val Pro Ile Met Ser Phe Glu Val Leu Ala Thr Ser Ser Trp     610 615 620 Gly Lys Tyr Ala His Gln Ser Phe Gly Leu Asp Glu Phe Gly Arg Ser 625 630 635 640 Gly Lys Gly Pro Glu Ile Tyr Lys Leu Phe Asp Phe Thr Ala Asp Gly                 645 650 655 Val Ala Ser Arg Ala Glu Lys Thr Ile Asn Tyr Tyr Lys Gly Lys Gln             660 665 670 Leu Leu Ser Pro Met Gly Arg Ala Phe         675 680 <210> 6 <211> 2046 <212> DNA <213> Saccharomyces cerevisiae <400> 6 atggcacagt tctccgacat tgataaactt gcggtttcca ctttaagatt actttccgtt 60 gaccaggtgg aaagcgcaca atctggccac ccaggtgcac cactaggatt ggcaccagtt 120 gcccatgtaa ttttcaagca actgcgctgt aaccctaaca atgaacattg gatcaataga 180 gacaggtttg ttctgtcgaa cggtcactca tgcgctcttc tgtactcaat gctccatcta 240 ttaggatacg attactctat cgaggacttg agacaattta gacaagtaaa ctcaaggaca 300 ccgggtcatc cagaattcca ctcagcggga gtggaaatca cttccggtcc gctaggccag 360 ggtatctcaa atgctgttgg tatggcaata gcgcaggcca actttgccgc cacttataac 420 gaggatggct ttcccatttc cgactcatat acgtttgcta ttgtagggga tggttgctta 480 caagagggtg tttcttcgga gacctcttcc ttagcgggac atctgcaatt gggtaacttg 540 attacgtttt atgacagtaa tagcatttcc attgacggta aaacctcgta ctcgttcgac 600 gaagatgttt tgaagcgata cgaggcatat ggttgggaag tcatggaagt cgataaagga 660 gacgacgata tggaatccat ttctagcgct ttggaaaagg caaaactatc gaaggacaag 720 ccaaccataa tcaaggtaac tactacaatt ggatttgggt ccctacaaca gggtactgct 780 ggtgttcatg ggtccgcttt gaaggcagat gatgttaaac agttgaagaa gaggtggggg 840 tttgacccaa ataaatcatt tgtagtacct caagaggtgt acgattatta taagaagact 900 gttgtggaac ccggtcaaaa acttaatgag gaatgggata ggatgtttga agaatacaaa 960 accaaatttc ccgagaaggg taaagaattg caaagaagat tgaatggtga gttaccggaa 1020 ggttgggaaa agcatttacc gaagtttact ccggacgacg atgctctggc aacaagaaag 1080 acatcccagc aggtgctgac gaacatggtc caagttttgc ctgaattgat cggtggttct 1140 gccgatttga caccttcgaa tctgacaagg tgggaaggcg cggtagattt ccaacctccc 1200 attacccaac taggtaacta tgcaggaagg tacattagat acggtgtgag ggaacacgga 1260 atgggtgcca ttatgaacgg tatctctgcc tttggtgcaa actacaagcc ttacggtggt 1320 acctttttga acttcgtctc ttatgctgca ggagccgtta ggttagccgc cttgtctggt 1380 aatccagtca tttgggttgc aacacatgac tctatcgggc ttggtgagga tggtccaacg 1440 caccaaccta ttgaaactct ggctcacttg agggctattc caaacatgca tgtatggaga 1500 cctgctgatg gtaacgaaac ttctgctgcg tattattctg ctatcaaatc tggtcgaaca 1560 ccatctgttg tggctttatc acgacagaat cttcctcaat tggagcattc ctcttttgaa 1620 aaagccttga agggtggcta tgtgatccat gacgtggaga atcctgatat tatcctggtg 1680 tcaacaggat cagaagtctc catttctata gatgcagcca aaaaattgta cgatactaaa 1740 aaaatcaaag caagagttgt ttccctgcca gacttttata cttttgacag gcaaagtgaa 1800 gaatacagat tctctgttct accagacggt gttccgatca tgtcctttga agtattggct 1860 acttcaagct ggggtaagta tgctcatcaa tcgttcggac tcgacgaatt tggtcgttca 1920 ggcaaggggc ctgaaattta caaattgttc gatttcacag cggacggtgt tgcgtcaagg 1980 gctgaaaaga caatcaatta ctacaaagga aagcagttgc tttctcctat gggaagagct 2040 ttctaa 2046 <210> 7 <211> 680 <212> PRT <213> Saccharomyces cerevisiae <400> 7 Met Thr Gln Phe Thr Asp Ile Asp Lys Leu Ala Val Ser Thr Ile Arg   1 5 10 15 Ile Leu Ala Val Asp Thr Val Ser Lys Ala Asn Ser Gly His Pro Gly              20 25 30 Ala Pro Leu Gly Met Ala Pro Ala Ala His Val Leu Trp Ser Gln Met          35 40 45 Arg Met Asn Pro Thr Asn Pro Asp Trp Ile Asn Arg Asp Arg Phe Val      50 55 60 Leu Ser Asn Gly His Ala Val Ala Leu Leu Tyr Ser Met Leu His Leu  65 70 75 80 Thr Gly Tyr Asp Leu Ser Ile Glu Asp Leu Lys Gln Phe Arg Gln Leu                  85 90 95 Gly Ser Arg Thr Pro Gly His Pro Glu Phe Glu Leu Pro Gly Val Glu             100 105 110 Val Thr Thr Gly Pro Leu Gly Gln Gly Ile Ser Asn Ala Val Gly Met         115 120 125 Ala Met Ala Gln Ala Asn Leu Ala Ala Thr Tyr Asn Lys Pro Gly Phe     130 135 140 Thr Leu Ser Asp Asn Tyr Thr Tyr Val Phe Leu Gly Asp Gly Cys Leu 145 150 155 160 Gln Glu Gly Ile Ser Ser Glu Ala Ser Ser Leu Ala Gly His Leu Lys                 165 170 175 Leu Gly Asn Leu Ile Ala Ile Tyr Asp Asp Asn Lys Ile Thr Ile Asp             180 185 190 Gly Ala Thr Ser Ile Ser Phe Asp Glu Asp Val Ala Lys Arg Tyr Glu         195 200 205 Ala Tyr Gly Trp Glu Val Leu Tyr Val Glu Asn Gly Asn Glu Asp Leu     210 215 220 Ala Gly Ile Ala Lys Ala Ile Ala Gln Ala Lys Leu Ser Lys Asp Lys 225 230 235 240 Pro Thr Leu Ile Lys Met Thr Thr Thr Ile Gly Tyr Gly Ser Leu His                 245 250 255 Ala Gly Ser Ser Val His Gly Ala Pro Leu Lys Ala Asp Asp Val             260 265 270 Lys Gln Leu Lys Ser Lys Phe Gly Phe Asn Pro Asp Lys Ser Phe Val         275 280 285 Val Pro Gln Glu Val Tyr Asp His Tyr Gln Lys Thr Ile Leu Lys Pro     290 295 300 Gly Val Glu Ala Asn Asn Lys Trp Asn Lys Leu Phe Ser Glu Tyr Gln 305 310 315 320 Lys Lys Phe Pro Glu Leu Gly Ala Glu Leu Ala Arg Arg Leu Ser Gly                 325 330 335 Gln Leu Pro Ala Asn Trp Glu Ser Lys Leu Pro Thr Tyr Thr Ala Lys             340 345 350 Asp Ser Ala Val Ala Thr Arg Lys Leu Ser Glu Thr Val Leu Glu Asp         355 360 365 Val Tyr Asn Gln Leu Pro Glu Leu Ile Gly Gly Ser Ala Asp Leu Thr     370 375 380 Pro Ser Asn Leu Thr Arg Trp Lys Glu Ala Leu Asp Phe Gln Pro Pro 385 390 395 400 Ser Ser Gly Ser Gly Asn Tyr Ser Gly Arg Tyr Ile Arg Tyr Gly Ile                 405 410 415 Arg Glu His Ala Met Gly Ala Ile Met Asn Gly Ile Ser Ala Phe Gly             420 425 430 Ala Asn Tyr Lys Pro Tyr Gly Gly Thr Phe Leu Asn Phe Val Ser Tyr         435 440 445 Ala Ala Gly Ala Val Ale Leu Ser Ala Leu Ser Gly His Pro Val Ile     450 455 460 Trp Val Ala Thr His Asp Ser Ile Gly Val Gly Glu Asp Gly Pro Thr 465 470 475 480 His Gln Pro Ile Glu Thr Leu Ala His Phe Arg Ser Leu Pro Asn Ile                 485 490 495 Gln Val Trp Arg Pro Ala Asp Gly Asn Glu Val Ser Ala Ala Tyr Lys             500 505 510 Asn Ser Leu Glu Ser Lys His Thr Pro Ser Ile Ile Ala Leu Ser Arg         515 520 525 Gln Asn Leu Pro Gln Leu Glu Gly Ser Ser Ile Glu Ser Ala Ser Lys     530 535 540 Gly Gly Tyr Val Leu Gln Asp Val Ala Asn Pro Asp Ile Ile Leu Val 545 550 555 560 Ala Thr Gly Ser Glu Val Ser Seru Ser Val Glu Ala Ala Lys Thr Leu                 565 570 575 Ala Ala Lys Asn Ile Lys Ala Arg Val Val Ser Leu Pro Asp Phe Phe             580 585 590 Thr Phe Asp Lys Gln Pro Leu Glu Tyr Arg Leu Ser Val Leu Pro Asp         595 600 605 Asn Val Pro Ile Met Ser Val Glu Val Leu Ala Thr Thr Cys Trp Gly     610 615 620 Lys Tyr Ala His Gln Ser Phe Gly Ile Asp Arg Phe Gly Ala Ser Gly 625 630 635 640 Lys Ala Pro Glu Val Phe Lys Phe Phe Gly Phe Thr Pro Glu Gly Val                 645 650 655 Ala Glu Arg Ala Gln Lys Thr Ile Ala Phe Tyr Lys Gly Asp Lys Leu             660 665 670 Ile Ser Pro Leu Lys Lys Ala Phe         675 680 <210> 8 <211> 2043 <212> DNA <213> Saccharomyces cerevisiae <400> 8 atgactcaat tcactgacat tgataagcta gccgtctcca ccataagaat tttggctgtg 60 gacaccgtat ccaaggccaa ctcaggtcac ccaggtgctc cattgggtat ggcaccagct 120 gcacacgttc tatggagtca aatgcgcatg aacccaacca acccagactg gatcaacaga 180 gatagatttg tcttgtctaa cggtcacgcg gtcgctttgt tgtattctat gctacatttg 240 actggttacg atctgtctat tgaagacttg aaacagttca gacagttggg ttccagaaca 300 ccaggtcatc ctgaatttga gttgccaggt gttgaagtta ctaccggtcc attaggtcaa 360 ggtatctcca acgctgttgg tatggccatg gctcaagcta acctggctgc cacttacaac 420 aagccgggct ttaccttgtc tgacaactac acctatgttt tcttgggtga cggttgtttg 480 caagaaggta tttcttcaga agcttcctcc ttggctggtc atttgaaatt gggtaacttg 540 attgccatct acgatgacaa caagatcact atcgatggtg ctaccagtat ctcattcgat 600 gaagatgttg ctaagagata cgaagcctac ggttgggaag ttttgtacgt agaaaatggt 660 aacgaagatc tagccggtat tgccaaggct attgctcaag ctaagttatc caaggacaaa 720 ccaactttga tcaaaatgac cacaaccatt ggttacggtt ccttgcatgc cggctctcac 780 tctgtgcacg gtgccccatt gaaagcagat gatgttaaac aactaaagag caaattcggt 840 ttcaacccag acaagtcctt tgttgttcca caagaagttt acgaccacta ccaaaagaca 900 attttaaagc caggtgtcga agccaacaac aagtggaaca agttgttcag cgaataccaa 960 aagaaattcc cagaattagg tgctgaattg gctagaagat tgagcggcca actacccgca 1020 aattgggaat ctaagttgcc aacttacacc gccaaggact ctgccgtggc cactagaaaa 1080 ttatcagaaa ctgttcttga ggatgtttac aatcaattgc cagagttgat tggtggttct 1140 gccgatttaa caccttctaa cttgaccaga tggaaggaag cccttgactt ccaacctcct 1200 tcttccggtt caggtaacta ctctggtaga tacattaggt acggtattag agaacacgct 1260 atgggtgcca taatgaacgg tatttcagct ttcggtgcca actacaaacc atacggtggt 1320 actttcttga acttcgtttc ttatgctgct ggtgccgtta gattgtccgc tttgtctggc 1380 cacccagtta tttgggttgc tacacatgac tctatcggtg tcggtgaaga tggtccaaca 1440 catcaaccta ttgaaacttt agcacacttc agatccctac caaacattca agtttggaga 1500 ccagctgatg gtaacgaagt ttctgccgcc tacaagaact ctttagaatc caagcatact 1560 ccaagtatca ttgctttgtc cagacaaaac ttgccacaat tggaaggtag ctctattgaa 1620 agcgcttcta agggtggtta cgtactacaa gatgttgcta acccagatat tattttagtg 1680 gctactggtt ccgaagtgtc tttgagtgtt gaagctgcta agactttggc cgcaaagaac 1740 atcaaggctc gtgttgtttc tctaccagat ttcttcactt ttgacaaaca acccctagaa 1800 tacagactat cagtcttacc agacaacgtt ccaatcatgt ctgttgaagt tttggctacc 1860 acatgttggg gcaaatacgc tcatcaatcc ttcggtattg acagatttgg tgcctccggt 1920 aaggcaccag aagtcttcaa gttcttcggt ttcaccccag aaggtgttgc tgaaagagct 1980 caaaagacca ttgcattcta taagggtgac aagctaattt ctcctttgaa aaaagctttc 2040 taa 2043 <210> 9 <211> 335 <212> PRT <213> Saccharomyces cerevisiae <400> 9 Met Ser Glu Pro Ala Gln Lys Lys Gln Lys Val Ala Asn Asn Ser Leu   1 5 10 15 Glu Gln Leu Lys Ala Ser Gly Thr Val Val Val Ala Asp Thr Gly Asp              20 25 30 Phe Gly Ser Ile Ala Lys Phe Gln Pro Gln Asp Ser Thr Thr Asn Pro          35 40 45 Ser Leu Ile Leu Ala Ala Ala Lys Gln Pro Thr Tyr Ala Lys Leu Ile      50 55 60 Asp Val Ala Val Glu Tyr Gly Lys Lys His Gly Lys Thr Thr Glu Glu  65 70 75 80 Gln Val Glu Asn Ala Val Asp Arg Leu Leu Val Glu Phe Gly Lys Glu                  85 90 95 Ile Leu Lys Ile Val Pro Gly Arg Val Ser Thr Glu Val Asp Ala Arg             100 105 110 Leu Ser Phe Asp Thr Gln Ala Thr Ile Glu Lys Ala Arg His Ile Ile         115 120 125 Lys Leu Phe Glu Gln Glu Gly Val Ser Lys Glu Arg Val Leu Ile Lys     130 135 140 Ile Ala Ser Thr Trp Glu Gly Ile Gln Ala Ala Lys Glu Leu Glu Glu 145 150 155 160 Lys Asp Gly Ile His Cys Asn Leu Thr Leu Leu Phe Ser Phe Val Gln                 165 170 175 Ala Val Ala Cys Ala Glu Ala Gln Val Thr Leu Ile Ser Pro Phe Val             180 185 190 Gly Arg Ile Leu Asp Trp Tyr Lys Ser Ser Thr Gly Lys Asp Tyr Lys         195 200 205 Gly Glu Ala Asp Pro Gly Val Ile Ser Val Lys Lys Ile Tyr Asn Tyr     210 215 220 Tyr Lys Lys Tyr Gly Tyr Lys Thr Ile Val Met Gly Ala Ser Phe Arg 225 230 235 240 Ser Thr Asp Glu Ile Lys Asn Leu Ala Gly Val Asp Tyr Leu Thr Ile                 245 250 255 Ser Pro Ala Leu Leu Asp Lys Leu Met Asn Ser Thr Glu Pro Phe Pro             260 265 270 Arg Val Leu Asp Pro Val Ser Ala Lys Lys Glu Ala Gly Asp Lys Ile         275 280 285 Ser Tyr Ile Ser Asp Glu Ser Lys Phe Arg Phe Asp Leu Asn Glu Asp     290 295 300 Ala Met Ala Thr Glu Lys Leu Ser Glu Gly Ile Arg Lys Phe Ser Ala 305 310 315 320 Asp Ile Val Thr Leu Phe Asp Leu Ile Glu Lys Lys Val Thr Ala                 325 330 335 <210> 10 <211> 1008 <212> DNA <213> Saccharomyces cerevisiae <400> 10 atgtctgaac cagctcaaaa gaaacaaaag gttgctaaca actctctaga acaattgaaa 60 gcctccggca ctgtcgttgt tgccgacact ggtgatttcg gctctattgc caagtttcaa 120 cctcaagact ccacaactaa cccatcattg atcttggctg ctgccaagca accaacttac 180 gccaagttga tcgatgttgc cgtggaatac ggtaagaagc atggtaagac caccgaagaa 240 caagtcgaaa atgctgtgga cagattgtta gtcgaattcg gtaaggagat cttaaagatt 300 gttccaggca gagtctccac cgaagttgat gctagattgt cttttgacac tcaagctacc 360 attgaaaagg ctagacatat cattaaattg tttgaacaag aaggtgtctc caaggaaaga 420 gtccttatta aaattgcttc cacttgggaa ggtattcaag ctgccaaaga attggaagaa 480 aaggacggta tccactgtaa tttgactcta ttattctcct tcgttcaagc agttgcctgt 540 gccgaggccc aagttacttt gatttcccca tttgttggta gaattctaga ctggtacaaa 600 tccagcactg gtaaagatta caagggtgaa gccgacccag gtgttatttc cgtcaagaaa 660 atctacaact actacaagaa gtacggttac aagactattg ttatgggtgc ttctttcaga 720 agcactgacg aaatcaaaaa cttggctggt gttgactatc taacaatttc tccagcttta 780 ttggacaagt tgatgaacag tactgaacct ttcccaagag ttttggaccc tgtctccgct 840 aagaaggaag ccggcgacaa gatttcttac atcagcgacg aatctaaatt cagattcgac 900 ttgaatgaag acgctatggc cactgaaaaa ttgtccgaag gtatcagaaa attctctgcc 960 gatattgtta ctctattcga cttgattgaa aagaaagtta ccgcttaa 1008 <210> 11 <211> 332 <212> PRT <213> Pelodiscus sinensis japonicus <400> 11 Met Ser Val Lys Glu Leu Leu Ile Gln Asn Val His Lys Glu Glu His   1 5 10 15 Ser His Ala His Asn Lys Ile Thr Val Val Gly Val Gly Ala Val Gly              20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Leu          35 40 45 Ala Leu Val Asp Val Ile Glu Asp Lys Leu Arg Gly Glu Met Leu Asp      50 55 60 Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser Gly  65 70 75 80 Lys Asp Tyr Ser Val Thr Ala His Ser Lys Leu Val Ile Ile Thr Ala                  85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln Arg             100 105 110 Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Val Val Lys Tyr Ser         115 120 125 Pro Asp Cys Met Leu Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr     130 135 140 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys His Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu                 165 170 175 Lys Leu Gly Ile His Ser Leu Ser Cys His Gly Trp Ile Ile Gly Glu             180 185 190 His Gly Asp Ser Ser Val Val Val Trp Ser Gly Val Asn Val Ala Gly         195 200 205 Val Ser Leu Lys Ala Leu Tyr Pro Asp Leu Gly Thr Asp Ala Asp Lys     210 215 220 Glu His Trp Lys Glu Val His Lys Gln Val Val Asp Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val                 245 250 255 Ala Asp Leu Ala Glu Thr Val Met Lys Asn Leu Arg Arg Val His Pro             260 265 270 Ile Ser Thr Met Val Lys Gly Met Tyr Gly Val Ser Ser Asp Val Phe         275 280 285 Leu Ser Val Pro Cys Val Leu Gly Tyr Ala Gly Ile Thr Asp Val Val     290 295 300 Lys Met Thr Leu Lys Ser Glu Glu Glu Glu Lys Leu Arg Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe                 325 330 <210> 12 <211> 332 <212> PRT <213> Ornithorhynchus anatinus <400> 12 Met Ala Gly Val Lys Glu Gln Leu Ile Gln Asn Leu Leu Lys Glu Glu   1 5 10 15 Tyr Ala Pro Gln Asn Lys Ile Thr Val Val Gly Val Gly Ala Val Gly              20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Leu          35 40 45 Ala Leu Val Asp Val Ile Glu Asp Lys Leu Lys Gly Glu Met Met Asp      50 55 60 Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser Gly  65 70 75 80 Lys Asp Tyr Ser Val Thr Ala Asn Ser Lys Leu Val Ile Ile Thr Ala                  85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln Arg             100 105 110 Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Val Val Lys Tyr Ser         115 120 125 Pro Asn Cys Lys Leu Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr     130 135 140 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu                 165 170 175 Arg Leu Gly Ile His Ser Thr Ser Cys His Gly Trp Val Ile Gly Glu             180 185 190 His Gly Asp Ser Ser Val Val Val Trp Ser Gly Val Asn Val Ala Gly         195 200 205 Val Ser Leu Lys Asn Leu His Pro Asp Leu Gly Thr Asp Ala Asp Lys     210 215 220 Glu Gln Trp Lys Asp Val His Lys Gln Val Val Asp Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val                 245 250 255 Ala Asp Leu Ala Glu Ser Ile Val Lys Asn Leu Arg Arg Val His Pro             260 265 270 Ile Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Asp Glu Val Phe         275 280 285 Leu Ser Val Pro Cys Val Leu Gly Gln Asn Gly Ile Ser Asp Val Val     290 295 300 Lys Ile Thr Leu Lys Ser Glu Glu Glu Ala His Leu Lys Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe                 325 330 <210> 13 <211> 332 <212> PRT <213> Tursiops truncatus <400> 13 Met Ala Thr Val Lys Asp Gln Leu Ile Gln Asn Leu Leu Lys Glu Glu   1 5 10 15 His Val Pro Gln Asn Lys Ile Thr Val Val Gly Val Gly Ala Val Gly              20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Leu          35 40 45 Ala Leu Val Asp Val Ile Glu Asp Lys Leu Lys Gly Glu Met Met Asp      50 55 60 Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser Gly  65 70 75 80 Lys Asp Tyr Ser Val Thr Ala Asn Ser Lys Leu Val Ile Ile Thr Ala                  85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln Arg             100 105 110 Asn Val Asn Ile Phe Lys Phe Ile Val Pro Asn Ile Val Lys Tyr Ser         115 120 125 Pro His Cys Lys Leu Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr     130 135 140 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu                 165 170 175 Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Ile Leu Gly Glu             180 185 190 His Gly Asp Ser Ser Val Val Val Trp Ser Gly Val Asn Val Ala Gly         195 200 205 Val Ser Leu Lys Asn Leu His Pro Glu Leu Gly Thr Asp Ala Asp Lys     210 215 220 Glu His Trp Lys Ala Ile His Lys Gln Val Val Asp Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Val Gly Leu Ser Val                 245 250 255 Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro             260 265 270 Ile Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Glu Asp Val Phe         275 280 285 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Val Val     290 295 300 Lys Val Thr Leu Thr Pro Glu Glu Gln Ala Cys Leu Lys Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe                 325 330 <210> 14 <211> 332 <212> PRT <213> Rattus norvegicus <400> 14 Met Ala Ala Leu Lys Asp Gln Leu Ile Val Asn Leu Leu Lys Glu Glu   1 5 10 15 Gln Val Pro Gln Asn Lys Ile Thr Val Val Gly Val Gly Ala Val Gly              20 25 30 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Leu          35 40 45 Ala Leu Val Asp Val Ile Glu Asp Lys Leu Lys Gly Glu Met Met Asp      50 55 60 Leu Gln His Gly Ser Leu Phe Leu Lys Thr Pro Lys Ile Val Ser Ser  65 70 75 80 Lys Asp Tyr Ser Val Thr Ala Asn Ser Lys Leu Val Ile Ile Thr Ala                  85 90 95 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln Arg             100 105 110 Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Val Val Lys Tyr Ser         115 120 125 Pro Gln Cys Lys Leu Leu Ile Val Ser Asn Pro Val Asp Ile Leu Thr     130 135 140 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu                 165 170 175 Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Val Leu Gly Glu             180 185 190 His Gly Asp Ser Ser Val Val Val Trp Ser Gly Val Asn Val Ala Gly         195 200 205 Val Ser Leu Lys Ser Leu Asn Pro Gln Leu Gly Thr Asp Ala Asp Lys     210 215 220 Glu Gln Trp Lys Asp Val His Lys Gln Val Val Asp Ser Ala Tyr Glu 225 230 235 240 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val                 245 250 255 Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro             260 265 270 Ile Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Glu Asp Val Phe         275 280 285 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Val Val     290 295 300 Lys Val Thr Leu Thr Pro Asp Glu Glu Ala Arg Leu Lys Lys Ser Ala 305 310 315 320 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe                 325 330 <210> 15 <211> 999 <212> DNA <213> Pelodiscus sinensis japonicus <400> 15 atgtccgtaa aggaactact tatacaaaac gtccataagg aggagcattc tcacgctcac 60 aataagataa cagttgtagg agtaggtgca gtaggtatgg catgtgctat ttcgatatta 120 atgaaagact tggctgatga actagccttg gttgatgtga ttgaggataa gttacgtgga 180 gaaatgttag atttgcaaca tggttcattg ttcttgagaa cccccaaaat tgtctcgggt 240 aaggattatt cagtcactgc tcattctaaa ctggttatca ttacagcagg tgcaagacag 300 caagaagggg agagcagact aaatctggtt caacgtaatg tcaacatctt caagtttatc 360 atcccgaacg tagtaaaata cagtccagac tgcatgttgc ttgttgtgag taatccagtt 420 gacatcttaa cctatgttgc gtggaaaatc agtgggtttc caaaacatag ggtgattggc 480 tcaggatgca accttgatag cgccaggttt aggtatctaa tgggagaaaa attaggtatt 540 cactccttat cttgtcatgg ctggataata ggcgaacatg gtgattcttc ggtacctgtt 600 tggtccgggg ttaatgtggc tggtgttagt ttaaaagcat tatatcctga cctgggtact 660 gatgccgata aagaacattg gaaagaagtg cacaaacaag tggttgattc tgcttacgaa 720 gttattaaac ttaagggcta cacttcttgg gctataggtc tatcagtagc tgatttggca 780 gaaaccgtta tgaaaaattt aagaagagtc cacccaattt ccacgatggt caagggtatg 840 tacggtgtta gctctgacgt cttcttatct gttccttgtg ttttgggata tgcgggaatt 900 acagacgtcg tgaagatgac attgaaatca gaggaagagg aaaaactaag aaagtcagcc 960 gatactctgt ggggcattca aaaggaattg cagttttaa 999 <210> 16 <211> 563 <212> PRT <213> Saccharomyces cerevisiae <400> 16 Met Ser Glu Ile Thr Leu Gly Lys Tyr Leu Phe Glu Arg Leu Lys Gln   1 5 10 15 Val Asn Val Asn Thr Val Phe Gly Leu Pro Gly Asp Phe Asn Leu Ser              20 25 30 Leu Leu Asp Lys Ile Tyr Glu Val Glu Gly Met Arg Trp Ala Gly Asn          35 40 45 Ala Asn Glu Leu Asn Ala Ala Tyr Ala Ala Asp Gly Tyr Ala Arg Ile      50 55 60 Lys Gly Met Ser Cys Ile Ile Thr Thr Phe Gly Val Gly Glu Leu Ser  65 70 75 80 Ala Leu Asn Gly Ile Ala Gly Ser Tyr Ala Glu His Val Gly Val Leu                  85 90 95 His Val Val Gly Val Ser Ser Ser Ser Ala Gln Ala Lys Gln Leu Leu             100 105 110 Leu His His Thr Leu Gly Asn Gly Asp Phe Thr Val Phe His Arg Met         115 120 125 Ser Ala Asn Ile Ser Glu Thr Thr Ala Met Ile Thr Asp Ile Ala Thr     130 135 140 Ala Pro Ala Glu Ile Asp Arg Cys Ile Arg Thr Thr Tyr Val Thr Gln 145 150 155 160 Arg Pro Val Tyr Leu Gly Leu Pro Ala Asn Leu Val Asp Leu Asn Val                 165 170 175 Pro Ala Lys Leu Leu Gln Thr Pro Ile Asp Met Ser Leu Lys Pro Asn             180 185 190 Asp Ala Glu Ser Glu Lys Glu Val Ile Asp Thr Ile Leu Ala Leu Val         195 200 205 Lys Asp Ala Lys Asn Pro Val Ile Leu Ala Asp Ala Cys Cys Ser Arg     210 215 220 His Asp Val Lys Ala Glu Thr Lys Lys Leu Ile Asp Leu Thr Gln Phe 225 230 235 240 Pro Ala Phe Val Thr Pro Met Gly Lys Gly Ser Ile Asp Glu Gln His                 245 250 255 Pro Arg Tyr Gly Gly Val Tyr Val Gly Thr Leu Ser Lys Pro Glu Val             260 265 270 Lys Glu Ala Val Glu Ser Ala Asp Leu Ile Leu Ser Val Gly Ala Leu         275 280 285 Leu Ser Asp Phe Asn Thr Gly Ser Phe Ser Tyr Ser Tyr Lys Thr Lys     290 295 300 Asn Ile Val Glu Phe His Ser Asp His Met Lys Ile Arg Asn Ala Thr 305 310 315 320 Phe Pro Gly Val Gln Met Lys Phe Val Leu Gln Lys Leu Leu Thr Thr                 325 330 335 Ile Ala Asp Ala Ala Lys Gly Tyr Lys Pro Val Ala Val Ala Arg             340 345 350 Thr Pro Ala Asn Ala Ala Val Pro Ala Ser Thr Pro Leu Lys Gln Glu         355 360 365 Trp Met Trp Asn Gln Leu Gly Asn Phe Leu Gln Glu Gly Asp Val Val     370 375 380 Ile Ala Glu Thr Gly Thr Ser Ala Phe Gly Ile Asn Gln Thr Thr Phe 385 390 395 400 Pro Asn Asn Thr Tyr Gly Ile Ser Gln Val Leu Trp Gly Ser Ile Gly                 405 410 415 Phe Thr Thr Gly Ala Thr Leu Gly Ala Ala Phe Ala Ala Glu Glu Ile             420 425 430 Asp Pro Lys Lys Arg Val Ile Leu Phe Ile Gly Asp Gly Ser Leu Gln         435 440 445 Leu Thr Val Gln Glu Ile Ser Thr Met Ile Arg Trp Gly Leu Lys Pro     450 455 460 Tyr Leu Phe Val Leu Asn Asn Asp Gly Tyr Thr Ile Glu Lys Leu Ile 465 470 475 480 His Gly Pro Lys Ala Gln Tyr Asn Glu Ile Gln Gly Trp Asp His Leu                 485 490 495 Ser Leu Leu Pro Thr Phe Gly Ala Lys Asp Tyr Glu Thr His Arg Val             500 505 510 Ala Thr Thr Gly Glu Trp Asp Lys Leu Thr Gln Asp Lys Ser Phe Asn         515 520 525 Asp Asn Ser Lys Ile Arg Met Ile Glu Ile Met Leu Pro Val Phe Asp     530 535 540 Ala Pro Gln Asn Leu Val Glu Gln Ala Lys Leu Thr Ala Ala Thr Asn 545 550 555 560 Ala Lys Gln             <210> 17 <211> 1692 <212> DNA <213> Saccharomyces cerevisiae <400> 17 atgtctgaaa ttactttggg taaatatttg ttcgaaagat taaagcaagt caacgttaac 60 accgttttcg gtttgccagg tgacttcaac ttgtccttgt tggacaagat ctacgaagtt 120 gaaggtatga gatgggctgg taacgccaac gaattgaacg ctgcttacgc cgctgatggt 180 tacgctcgta tcaagggtat gtcttgtatc atcaccacct tcggtgtcgg tgaattgtct 240 gctttgaacg gtattgccgg ttcttacgct gaacacgtcg gtgttttgca cgttgttggt 300 gtcccatcca tctctgctca agctaagcaa ttgttgttgc accacacctt gggtaacggt 360 gacttcactg ttttccacag aatgtctgcc aacatttctg aaaccactgc tatgatcact 420 gacattgcta ccgccccagc tgaaattgac agatgtatca gaaccactta cgtcacccaa 480 agaccagtct acttaggttt gccagctaac ttggtcgact tgaacgtccc agctaagttg 540 ttgcaaactc caattgacat gtctttgaag ccaaacgatg ctgaatccga aaaggaagtc 600 attgacacca tcttggcttt ggtcaaggat gctaagaacc cagttatctt ggctgatgct 660 tgttgttcca gacacgacgt caaggctgaa actaagaagt tgattgactt gactcaattc 720 ccagctttcg tcaccccaat gggtaagggt tccattgacg aacaacaccc aagatacggt 780 ggtgtttacg tcggtacctt gtccaagcca gaagttaagg aagccgttga atctgctgac 840 ttgattttgt ctgtcggtgc tttgttgtct gatttcaaca ccggttcttt ctcttactct 900 tacaagacca agaacattgt cgaattccac tccgaccaca tgaagatcag aaacgccact 960 ttcccaggtg tccaaatgaa attcgttttg caaaagttgt tgaccactat tgctgacgcc 1020 gctaagggtt acaagccagt tgctgtccca gctagaactc cagctaacgc tgctgtccca 1080 gcttctaccc cattgaagca agaatggatg tggaaccaat tgggtaactt cttgcaagaa 1140 gt; ccaaacaaca cctacggtat ctctcaagtc ttatggggtt ccattggttt caccactggt 1260 gctaccttgg gtgctgcttt cgctgctgaa gaaattgatc caaagaagag agttatctta 1320 ttcattggtg acggttcttt gcaattgact gttcaagaaa tctccaccat gatcagatgg 1380 ggcttgaagc catacttgtt cgtcttgaac aacgatggtt acaccattga aaagttgatt 1440 cacggtccaa aggctcaata caacgaaatt caaggttggg accacctatc cttgttgcca 1500 actttcggtg ctaaggacta tgaaacccac agagtcgcta ccaccggtga atgggacaag 1560 ttgacccaag acaagtcttt caacgacaac tctaagatca gaatgattga aatcatgttg 1620 ccagtcttcg atgctccaca aaacttggtt gaacaagcta agttgactgc tgctaccaac 1680 gctaagcaat aa 1692 <210> 18 <211> 591 <212> PRT <213> Saccharomyces cerevisiae <400> 18 Met Leu Lys Tyr Lys Pro Leu Leu Lys Ile Ser Lys Asn Cys Glu Ala   1 5 10 15 Ala Ile Leu Arg Ala Ser Lys Thr Arg Leu Asn Thr Ile Arg Ala Tyr              20 25 30 Gly Ser Thr Val Pro Lys Ser Lys Ser Phe Glu Gln Asp Ser Arg Lys          35 40 45 Arg Thr Gln Ser Trp Thr Ala Leu Arg Val Gly Ala Ile Leu Ala Ala      50 55 60 Thr Ser Ser Val Ala Tyr Leu Asn Trp His Asn Gly Gln Ile Asp Asn  65 70 75 80 Glu Pro Lys Leu Asp Met Asn Lys Gln Lys Ile Ser Pro Ala Glu Val                  85 90 95 Ala Lys His Asn Lys Pro Asp Asp Cys Trp Val Val Ile Asn Gly Tyr             100 105 110 Val Tyr Asp Leu Thr Arg Phe Leu Pro Asn His Pro Gly Gly Gln Asp         115 120 125 Val Ile Lys Phe Asn Ala Gly Lys Asp Val Thr Ala Ile Phe Glu Pro     130 135 140 Leu His Ala Pro Asn Val Ile Asp Lys Tyr Ile Ala Pro Glu Lys Lys 145 150 155 160 Leu Gly Pro Leu Gln Gly Ser Met Pro Pro Glu Leu Val Cys Pro Pro                 165 170 175 Tyr Ala Pro Gly Glu Thr Lys Glu Asp Ile Ala Arg Lys Glu Gln Leu             180 185 190 Lys Ser Leu Leu Pro Pro Leu Asp Asn Ile Ile Asn Leu Tyr Asp Phe         195 200 205 Glu Tyr Leu Ala Ser Gln Thr Leu Thr Lys Gln Ala Trp Ala Tyr Tyr     210 215 220 Ser Ser Gly Ala Asn Asp Glu Val Thr His Arg Glu Asn His Asn Ala 225 230 235 240 Tyr His Arg Ile Phe Phe Lys Pro Lys Ile Leu Val Asp Val Arg Lys                 245 250 255 Val Asp Ile Ser Thr Asp Met Leu Gly Ser His Val Val Asp Val Pro Phe             260 265 270 Tyr Val Ser Ala Thr Ala Leu Cys Lys Leu Gly Asn Pro Leu Glu Gly         275 280 285 Glu Lys Asp Val Ala Arg Gly Cys Gly Gln Gly Val Thr Lys Val Pro     290 295 300 Gln Met Ile Ser Thr Leu Ala Ser Cys Ser Pro Glu Glu Ile Ile Glu 305 310 315 320 Ala Ala Pro Ser Asp Lys Gln Ile Gln Trp Tyr Gln Leu Tyr Val Asn                 325 330 335 Ser Asp Arg Lys Ile Thr Asp Asp Leu Val Lys Asn Val Glu Lys Leu             340 345 350 Gly Val Lys Ala Leu Phe Val Thr Val Asp Ala Pro Ser Leu Gly Gln         355 360 365 Arg Glu Lys Asp Met Lys Leu Lys Phe Ser Asn Thr Lys Ala Gly Pro     370 375 380 Lys Ala Met Lys Lys Thr Asn Val Glu Glu Ser Gln Gly Ala Ser Arg 385 390 395 400 Ala Leu Ser Lys Phe Ile Asp Pro Ser Leu Thr Trp Lys Asp Ile Glu                 405 410 415 Glu Leu Lys Lys Lys Thr Lys Leu Pro Ile Val Ile Lys Gly Val Gln             420 425 430 Arg Thr Glu Asp Val Ile Lys Ala Ala Glu Ile Gly Val Ser Gly Val         435 440 445 Val Leu Ser Asn His Gly Gly Arg Gln Leu Asp Phe Ser Arg Ala Pro     450 455 460 Ile Glu Val Leu Ala Glu Thr Met Pro Ile Leu Glu Gln Arg Asn Leu 465 470 475 480 Lys Asp Lys Leu Glu Val Phe Val Asp Gly Gly Val Arg Arg Gly Thr                 485 490 495 Asp Val Leu Lys Ala Leu Cys Leu Gly Ala Lys Gly Val Gly Leu Gly             500 505 510 Arg Pro Phe Leu Tyr Ala Asn Ser Cys Tyr Gly Arg Asn Gly Val Glu         515 520 525 Lys Ala Ile Glu Ile Leu Arg Asp Glu Ile Glu Met Ser Met Arg Leu     530 535 540 Leu Gly Val Thr Ser Ile Glu Leu Lys Pro Asp Leu Leu Asp Leu 545 550 555 560 Ser Thr Leu Lys Ala Arg Thr Val Gly Val Pro Asn Asp Val Leu Tyr                 565 570 575 Asn Glu Val Tyr Glu Gly Pro Thr Leu Thr Glu Phe Glu Asp Ala             580 585 590 <210> 19 <211> 1776 <212> DNA <213> Saccharomyces cerevisiae <400> 19 atgctaaaat acaaaccttt actaaaaatc tcgaagaact gtgaggctgc tatcctcaga 60 gcgtctaaga ctagattgaa cacaatccgc gcgtacggtt ctaccgttcc aaaatccaag 120 tcgttcgaac aagactcaag aaaacgcaca cagtcatgga ctgccttgag agtcggtgca 180 attctagccg ctactagttc cgtggcgtat ctaaactggc ataatggcca aatagacaac 240 gagccgaaac tggatatgaa taaacaaaag atttcgcccg ctgaagttgc caagcataac 300 aagcccgatg attgttgggt tgtgatcaat ggttacgtat acgacttaac gcgattccta 360 ccaaatcatc caggtgggca ggatgttatc aagtttaacg ccgggaaaga tgtcactgct 420 atttttgaac cactacatgc tcctaatgtc atcgataagt atatagctcc cgagaaaaaa 480 ttgggtcccc ttcaaggatc catgcctcct gaacttgtct gtcctcctta tgctcctggt 540 gaaactaagg aagatatcgc tagaaaagaa caactaaaat cgctgctacc tcctctagat 600 aatattatta acctttacga ctttgaatac ttggcctctc aaactttgac taaacaagcg 660 tgggcctact attcctccgg tgctaacgac gaagttactc acagagaaaa ccataatgct 720 tatcatagga tttttttcaa accaaagatc cttgtagatg tacgcaaagt agacatttca 780 actgacatgt tgggttctca tgtggatgtt cccttctacg tgtctgctac agctttgtgt 840 aaactgggaa accccttaga aggtgaaaaa gatgtcgcca gaggttgtgg ccaaggtgtg 900 acaaaagtcc cacaaatgat atctactttg gcttcatgtt cccctgagga aattattgaa 960 gcagcaccct ctgataaaca aattcaatgg taccaactat atgttaactc tgatagaaag 1020 atcactgatg atttggttaa aaatgtagaa aagctgggtg taaaggcatt atttgtcact 1080 gtggatgctc caagtttagg tcaaagagaa aaagatatga agctgaaatt ttccaataca 1140 aaggctggtc caaaagcgat gaagaaaact aatgtagaag aatctcaagg tgcttcgaga 1200 gcgttatcaa agtttattga cccctctttg acttggaaag atatagaaga gttgaagaaa 1260 aagacaaaac tacctattgt tatcaaaggt gttcaacgta ccgaagatgt tatcaaagca 1320 gcagaaatcg gtgtaagtgg ggtggttcta tccaatcatg gtggtagaca attagatttt 1380 tcaagggctc ccattgaagt cctggctgaa accatgccaa tcctggaaca acgtaacttg 1440 aaggataagt tggaagtttt cgtggacggt ggtgttcgtc gtggtacaga tgtcttgaaa 1500 gcgttatgtc taggtgctaa aggtgttggt ttgggtagac cattcttgta tgcgaactca 1560 tgctatggtc gtaatggtgt tgaaaaagcc attgaaattt taagagatga aattgaaatg 1620 tctatgagac tattaggtgt tactagcatt gcggaattga agcctgatct tttagatcta 1680 tcaacactaa aggcaagaac agttggagta ccaaacgacg tgctgtataa tgaagtttat 1740 gagggaccta ctttaacaga atttgaggat gcatga 1776 <210> 20 <211> 391 <212> PRT <213> Saccharomyces cerevisiae <400> 20 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> 21 <211> 1176 <212> DNA <213> Saccharomyces cerevisiae <400> 21 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> 22 <211> 560 <212> PRT <213> Saccharomyces cerevisiae <400> 22 Met Ile Arg Gln Ser Leu Met Lys Thr Val Trp Ala Asn Ser Ser Arg   1 5 10 15 Phe Ser Leu Gln Ser Lys Ser Gly Leu Val Lys Tyr Ala Lys Asn Arg              20 25 30 Ser Phe His Ala Ala Arg Asn Leu Leu Glu Asp Lys Lys Val Ile Leu          35 40 45 Gln Lys Val Ala Pro Thr Thr Gly Val Val Ala Lys Gln Ser Phe Phe      50 55 60 Lys Arg Thr Gly Lys Phe Thr Leu Lys Ala Leu Leu Tyr Ser Ala Leu  65 70 75 80 Ala Gly Thr Ala Tyr Val Ser Tyr Ser Leu Tyr Arg Glu Ala Asn Pro                  85 90 95 Ser Thr Gln Val Pro Gln Ser Asp Thr Phe Pro Asn Gly Ser Lys Arg             100 105 110 Lys Thr Leu Val Ile Leu Gly Ser Gly Trp Gly Ser Val Ser Leu Leu         115 120 125 Lys Asn Leu Asp Thr Thr Leu Tyr Asn Val Val Val Val Ser Pro Arg     130 135 140 Asn Tyr Phe Leu Phe Thr Pro Leu Leu Pro Ser Thr Pro Val Gly Thr 145 150 155 160 Ile Glu Leu Lys Ser Ile Val Glu Pro Val Arg Thr Ile Ala Arg Arg                 165 170 175 Ser His Gly Glu Val His Tyr Tyr Glu Ala Glu Ala Tyr Asp Val Asp             180 185 190 Pro Glu Asn Lys Thr Ile Lys Val Lys Ser Ser Ala Lys Asn Asn Asp         195 200 205 Tyr Asp Leu Asp Leu Lys Tyr Asp Tyr Leu Val Val Gly Val Gly Ala     210 215 220 Gln Pro Asn Thr Phe Gly Thr Pro Gly Val Tyr Glu Tyr Ser Ser Phe 225 230 235 240 Leu Lys Glu Ile Ser Asp Ala Gln Glu Ile Arg Leu Lys Ile Met Ser                 245 250 255 Ser Ile Glu Lys Ala Ala Ser Leu Ser Pro Lys Asp Pro Glu Arg Ala             260 265 270 Arg Leu Leu Ser Phe Val Val Gly Gly Gly Pro Thr Gly Val Glu         275 280 285 Phe Ala Ala Glu Leu Arg Asp Tyr Val Asp Gln Asp Leu Arg Lys Trp     290 295 300 Met Pro Glu Leu Ser Lys Glu Ile Lys Val Thr Leu Val Glu Ala Leu 305 310 315 320 Pro Asn Ile Leu Asn Met Phe Asp Lys Tyr Leu Val Asp Tyr Ala Gln                 325 330 335 Asp Leu Phe Lys Glu Glu Lys Ile Asp Leu Arg Leu Lys Thr Met Val             340 345 350 Lys Lys Val Asp Ala Thr Thr Ile Thr Ala Lys Thr Gly Asp Gly Asp         355 360 365 Ile Glu Asn Ile Pro Tyr Gly Val Leu Val Trp Ala Thr Gly Asn Ala     370 375 380 Pro Arg Glu Val Ser Lys Asn Leu Met Thr Lys Leu Glu Glu Gln Asp 385 390 395 400 Ser Arg Arg Gly Leu Leu Ile Asp Asn Lys Leu Gln Leu Leu Gly Ala                 405 410 415 Lys Gly Ser Ile Phe Ala Ile Gly Asp Cys Thr Phe His Pro Gly Leu             420 425 430 Phe Pro Thr Ala Gln Val Ala His Gln Glu Gly Glu Tyr Leu Ala Gln         435 440 445 Tyr Phe Lys Lys Ala Tyr Lys Ile Asp Gln Leu Asn Trp Lys Met Thr     450 455 460 His Ala Lys Asp Asp Ser Glu Val Ala Arg Leu Lys Asn Gln Ile Val 465 470 475 480 Lys Thr Gln Ser Gln Ile Glu Asp Phe Lys Tyr Asn His Lys Gly Ala                 485 490 495 Leu Ala Tyr Ile Gly Ser Asp Lys Ala Ile Ala Asp Leu Ala Val Gly             500 505 510 Glu Ala Lys Tyr Arg Leu Ala Gly Ser Phe Thr Phe Leu Phe Trp Lys         515 520 525 Ser Ala Tyr Leu Ala Met Cys Leu Ser Phe Arg Asn Arg Val Leu Val     530 535 540 Ala Met Asp Trp Ala Lys Val Tyr Phe Leu Gly Arg Asp Ser Ser Ile 545 550 555 560 <210> 23 <211> 1683 <212> DNA <213> Saccharomyces cerevisiae <400> 23 atgattagac aatcattaat gaaaacagtg tgggctaact cctccaggtt tagcctacag 60 agcaagtcgg ggcttgtgaa atatgccaaa aatagatcgt tccatgcagc aagaaatttg 120 ctagaggaca agaaagtcat tttgcaaaaa gtggcgccca ctactggcgt tgttgcgaag 180 cagtcctttt tcaagagaac tgggaaattt actttgaagg ctttattgta ttctgccctc 240 gcgggtacgg cttacgtttc atactcactt taccgagaag ctaacccttc tacccaagtt 300 cctcaatcgg acacttttcc aaacggttca aagaggaaga ctttggtaat tctgggctcc 360 ggttggggtt ctgtgtcgct tttgaaaaat ttggacacca cgttgtataa tgttgttgtt 420 gtttctccaa gaaattattt tctttttact ccgctattgc catctacccc agttggtacc 480 atcgaattga aatctattgt tgaacctgtc aggactattg ctagaagatc gcacggtgaa 540 gtccattact atgaagctga agcgtacgac gttgatcctg aaaacaaaac aattaaggtc 600 aaatcttccg ctaagaataa cgactacgac ttggacttga aatacgacta tctggttgtc 660 ggtgtgggtg ctcaaccaaa cacttttggt actccgggag tttatgaata ttcttctttc 720 ttgaaggaaa tatccgacgc tcaagagatc agattaaaaa ttatgtccag tattgagaaa 780 gctgcctccc tatctccaaa agatcctgag agagcaagat tgttgagctt tgttgtcgtt 840 ggtggtggtc ccaccggtgt cgaatttgcc gctgaattga gagattatgt tgaccaggac 900 ttgagaaaat ggatgcccga attgagtaaa gaaattaaag tcactttggt ggaggctttg 960 ccaaacattt tgaacatgtt tgacaagtat ctcgttgact atgctcaaga tttattcaaa 1020 gaggaaaaaa tcgatttaag attgaaaaca atggttaaga aagttgacgc taccactata 1080 actgccaaaa ctggcgatgg tgacattgaa aatataccgt atggtgtatt agtttgggct 1140 acaggtaatg cgccaagaga agtgtctaag aacctaatga ctaaattaga ggaacaggac 1200 tcaagacgtg gtttgttgat agataacaaa cttcaacttt tgggtgctaa gggatctatt 1260 tttgctatcg gcgattgtac cttccaccct ggcttgttcc ctaccgctca agttgcccac 1320 caagaaggtg aatacttggc tcagtatttc aagaaagctt ataaaatcga tcaattgaac 1380 tggaaaatga cccatgctaa agacgattca gaagtcgcta gattaaagaa ccaaatagtc 1440 aaaacgcaat cgcaaattga agacttcaag tacaaccata agggtgctct ggcttatatt 1500 ggttcagata aagccattgc tgatcttgcc gttggtgaag ccaaatatag gttagccggc 1560 tcattcacct tcctattctg gaaatctgct tatttggcaa tgtgtctatc ctttagaaac 1620 agagttcttg tcgctatgga ttgggctaaa gtttatttct tgggtagaga ttcatctatc 1680 tag 1683 <210> 24 <211> 545 <212> PRT <213> Saccharomyces cerevisiae <400> 24 Met Leu Pro Arg Leu Gly Phe Ala Arg Thr Ala Arg Ser Ile His Arg   1 5 10 15 Phe Lys Met Thr Gln Ile Ser Lys Pro Phe Phe His Ser Thr Glu Val              20 25 30 Gly Lys Pro Gly Pro Gln Gln Lys Leu Ser Lys Ser Tyr Thr Ala Val          35 40 45 Phe Lys Lys Trp Phe Val Arg Gly Leu Lys Leu Thr Phe Tyr Thr Thr      50 55 60 Leu Ala Gly Thr Leu Tyr Val Ser Tyr Glu Leu Tyr Lys Glu Ser Asn  65 70 75 80 Pro Pro Lys Gln Val Pro Gln Ser Thr Ala Phe Ala Asn Gly Leu Lys                  85 90 95 Lys Lys Glu Leu Val Ile Leu Gly Thr Gly Trp Gly Ala Ile Ser Leu             100 105 110 Leu Lys Lys Leu Asp Thr Ser Leu Tyr Asn Val Thr Val Val Ser Pro         115 120 125 Arg Ser Phe Phe Leu Phe Thr Pro Leu Leu Pro Ser Thr Pro Val Gly     130 135 140 Thr Ile Glu Met Lys Ser Ile Val Glu Pro Val Arg Ser Ile Ala Arg 145 150 155 160 Arg Thr Pro Gly Glu Val His Tyr Ile Glu Ala Glu Ala Leu Asp Val                 165 170 175 Asp Pro Lys Ala Lys Lys Val Met Val Gln Ser Val Ser Glu Asp Glu             180 185 190 Tyr Phe Val Ser Ser Leu Ser Tyr Asp Tyr Leu Val Val Ser Val Gly         195 200 205 Ala Lys Thr Thr Thr Phe Asn Ile Pro Gly Val Tyr Gly Asn Ala Asn     210 215 220 Phe Leu Lys Glu Ile Glu Asp Ala Gln Asn Ile Arg Met Lys Leu Met 225 230 235 240 Lys Thr Ile Glu Gln Ala Ser Ser Phe Pro Val Asn Asp Pro Glu Arg                 245 250 255 Lys Arg Leu Leu Thr Phe Val Val Gly Gly Gly Pro Thr Gly Val             260 265 270 Glu Phe Ala Ala Glu Leu Gln Asp Tyr Ile Asn Gln Asp Leu Arg Lys         275 280 285 Trp Met Pro Asp Leu Ser Lys Glu Met Lys Val Ile Leu Ile Glu Ala     290 295 300 Leu Pro Asn Ile Leu Asn Met Phe Asp Lys Thr Leu Ile Lys Tyr Ala 305 310 315 320 Glu Asp Leu Phe Ala Arg Asp Glu Ile Asp Leu Gln Val Asn Thr Ala                 325 330 335 Val Lys Val Val Glu Pro Thr Tyr Ile Arg Thr Leu Gln Asn Gly Gln             340 345 350 Thr Asn Thr Asp Ile Glu Tyr Gly Met Leu Val Trp Ala Thr Gly Asn         355 360 365 Glu Pro Ile Asp Phe Ser Lys Thr Leu Met Ser Arg Ile Pro Glu Gln     370 375 380 Thr Asn Arg Arg Gly Leu Leu Ile Asn Asp Lys Leu Glu Leu Leu Gly 385 390 395 400 Ser Glu Asn Ser Ile Tyr Ala Ile Gly Asp Cys Thr Ala His Thr Gly                 405 410 415 Phe Phe Pro Thr Ala Gln Val Ala His Gln Glu Gly Glu Tyr Leu Ala             420 425 430 Lys Ile Leu Asp Lys Lys Leu Gln Ile Glu Gln Leu Glu Trp Asp Met         435 440 445 Leu Asn Ser Thr Asp Glu Thr Glu Val Ser Arg Leu Gln Lys Glu Val     450 455 460 Asn Leu Arg Lys Ser Lys Leu Asp Lys Phe Asn Tyr Lys His Met Gly 465 470 475 480 Ala Leu Ala Tyr Ile Gly Ser Glu Thr Ala Ile Ala Asp Leu His Met                 485 490 495 Gly Asp Ser Ser Tyr Gln Leu Lys Gly Met Phe Ala Phe Leu Phe Trp             500 505 510 Lys Ser Ala Tyr Leu Ala Met Cys Leu Ser Ile Arg Asn Arg Ile Leu         515 520 525 Ile Ala Met Asp Trp Thr Lys Val Tyr Phe Leu Gly Arg Asp Ser Ser     530 535 540 Val 545 <210> 25 <211> 1638 <212> DNA <213> Saccharomyces cerevisiae <400> 25 atgctgccca gacttggttt tgcgaggact gctaggtcca tacaccgttt caagatgacc 60 cagatctcta aacctttttt ccattccact gaagttggta agcccggacc acagcagaag 120 ctatcgaaat cttacactgc ggtattcaag aaatggtttg tcagaggttt aaagttaacc 180 ttttacacga cgttggccgg cacattgtat gtgtcatacg agctgtacaa agaatcgaac 240 ccacccaaac aggttcccca atcgaccgct tttgctaatg gtttgaaaaa gaaggagctg 300 gttattttgg gtacaggctg gggcgccata tctcttttga agaaattaga cacgtctttg 360 tataacgtga ccgtggtgtc gccaagaagc ttctttttgt tcacaccgtt attaccctca 420 acgcctgtgg gtacgataga gatgaagtct attgtcgaac cggttagatc gatcgctaga 480 agaacgcctg gagaagttca ctacattgag gcggaagcgt tggacgttga tccaaaggcc 540 aaaaaagtaa tggtgcaatc ggtgtcagag gacgaatatt tcgtttcgag cttaagttac 600 gattatcttg ttgttagtgt aggcgctaaa accactactt ttaacattcc cggggtctat 660 ggcaatgcta acttcttgaa agagattgaa gatgctcaaa atattcgtat gaagttaatg 720 aaaaccatag aacaggcaag ttcatttcct gtgaacgatc cggaaaggaa gcgattatta 780 acgttcgtgg ttgttggagg gggccctacg ggggttgaat ttgccgccga actgcaagat 840 tacatcaatc aagatttgag gaagtggatg cccgacttaa gtaaagaaat gaaggttatc 900 ttaattgaag ccctgcctaa tatcctaaac atgttcgata agacgttgat caagtatgcc 960 gaggaccttt ttgccagaga tgaaattgac ttgcaagtga atactgccgt gaaagtcgta 1020 gagccaacct atatacgcac tctgcaaaac ggccaaacaa acacggatat cgaatacggg 1080 atgctggttt gggccacggg aaatgaacca atcgattttt caaagacact gatgagtaga 1140 ataccggagc aaactaatag gcgtggtctg ttaattaatg acaagttgga gcttctcggt 1200 tctgagaatt cgatttatgc aattggtgat tgtaccgcac acacgggttt ctttcccacg 1260 gcacaagttg cacatcagga aggcgaatac ttggccaaga tcttggataa aaaattacag 1320 atagaacaat tggaatggga catgctcaac agtaccgatg aaactgaggt atcacgtcta 1380 caaaaagagg ttaatttgag gaaatctaag ttggataagt tcaactacaa gcatatgggt 1440 gcccttgcgt acatcggctc tgaaaccgca attgcagatt tgcatatggg cgactcatca 1500 taccagttga aaggtatgtt tgccttcttg ttttggaaat ccgcttattt ggccatgtgt 1560 ctctctatca ggaataggat tttaattgcc atggactgga ccaaagttta ctttcttgga 1620 agggattcct ccgtgtag 1638 <210> 26 <211> 130 <212> PRT <213> Saccharomyces cerevisiae <400> 26 Met Ser Gln Pro Val Gln Arg Ala Ala Ala Arg Ser Phe Leu Gln Lys   1 5 10 15 Tyr Ile Asn Lys Glu Thr Leu Lys Tyr Ile Phe Thr Thr His Phe Trp              20 25 30 Gly Pro Val Ser Asn Phe Gly Ile Pro Ile Ala Ala Ile Tyr Asp Leu          35 40 45 Lys Lys Asp Pro Thr Leu Ile Ser Gly Pro Met Thr Phe Ala Leu Val      50 55 60 Thr Tyr Ser Gly Val Phe Met Lys Tyr Ala Leu Ser Val Ser Pro Lys  65 70 75 80 Asn Tyr Leu Leu Phe Gly Cys His Leu Ile Asn Glu Thr Ala Gln Leu                  85 90 95 Ala Gln Gly Tyr Arg Phe Leu Lys Tyr Thr Tyr Phe Thr Thr Asp Glu             100 105 110 Glu Lys Lys Ala Leu Asp Lys Glu Trp Lys Glu Lys Glu Lys Thr Gly         115 120 125 Lys Gln     130 <210> 27 <211> 393 <212> DNA <213> Saccharomyces cerevisiae <400> 27 atgtctcaac cggttcaacg cgctgcagca cgctcattcc ttcaaaaata catcaataaa 60 gaaactttga aatatatttt cacaacacac ttctggggtc ccgtatcaaa tttcggtatc 120 ccaattgctg ctatatatga tctgaaaaaa gaccctacac taatctctgg cccaatgact 180 tttgctttag ttacctattc aggtgttttc atgaagtatg ctctttcagt atcacccaaa 240 aactacttac tgtttggatg ccaccttatt aatgaaactg cgcaattagc tcaaggctat 300 aggtttctca aatacacgta tttcacaaca gatgaggaga agaaagctct agataaggaa 360 tggaaagaga aagaaaaaac tggtaaacag taa 393 <210> 28 <211> 129 <212> PRT <213> Saccharomyces cerevisiae <400> 28 Met Ser Thr Ser Ser Val Arg Phe Ala Phe Arg Arg Phe Trp Gln Ser   1 5 10 15 Glu Thr Gly Pro Lys Thr Val His Phe Trp Ala Pro Thr Leu Lys Trp              20 25 30 Gly Leu Val Phe Ala Gly Phe Ser Asp Met Lys Arg Pro Val Glu Lys          35 40 45 Ile Ser Gly Ala Gln Asn Leu Ser Leu Leu Ser Thr Ala Leu Ile Trp      50 55 60 Thr Arg Trp Ser Phe Val Ile Lys Pro Arg Asn Ile Leu Leu Ala Ser  65 70 75 80 Val Asn Ser Phe Leu Cys Leu Thr Ala Gly Tyr Gln Leu Gly Arg Ile                  85 90 95 Ala Asn Tyr Arg Ile Arg Asn Gly Asp Ser Ile Ser Gln Leu Cys Ser             100 105 110 Tyr Ile Leu Ser Gly Ala Asp Glu Ser Lys Lys Glu Ile Thr Thr Gly         115 120 125 Arg     <210> 29 <211> 390 <212> DNA <213> Saccharomyces cerevisiae <400> 29 atgtctacat catccgtacg ttttgcattt aggcggttct ggcaaagtga gacaggcccc 60 aagacggtgc atttctgggc tcctactttg aaatggggtc tggttttcgc tggattcagc 120 gatatgaaga gaccggtgga aaaaatttct ggtgctcaaa atttgtcgct gctatctact 180 gt; gtcaactcgt ttctttgtct gaccgctggc tatcaattgg gtagaattgc caactacagg 300 atacggaatg gcgactctat atcgcaattg tgtagctata ttctcagcgg cgccgacgaa 360 agcaaaaagg aaattactac gggcagataa 390 <210> 30 <211> 147 <212> PRT <213> Saccharomyces cerevisiae <400> 30 Met Ser Ala Ser Ala Phe Asn Phe Ala Phe Arg Arg Phe Trp Asn Ser   1 5 10 15 Glu Thr Gly Pro Lys Thr Val His Phe Trp Ala Pro Thr Leu Lys Trp              20 25 30 Gly Leu Val Phe Ala Gly Leu Asn Asp Ile Lys Arg Pro Val Glu Lys          35 40 45 Val Ser Gly Ala Gln Asn Leu Ser Leu Leu Ala Thr Ala Leu Ile Trp      50 55 60 Thr Arg Trp Ser Phe Val Ile Lys Pro Lys Asn Tyr Leu Leu Ala Ser  65 70 75 80 Val Asn Phe Phe Leu Gly Cys Thr Ala Gly Tyr His Leu Thr Arg Ile                  85 90 95 Ala Asn Phe Arg Ile Arg Asn Gly Asp Ser Phe Lys Gln Val Ile His             100 105 110 Tyr Ile Ile Lys Gly Glu Thr Pro Ala Ala Val Ala Ala Lys Gln Thr         115 120 125 Ala Ser Thr Ser Met Asn Lys Gly Val Ile Gly Thr Asn Pro Pro Ile     130 135 140 Thr His Arg 145 <210> 31 <211> 441 <212> DNA <213> Saccharomyces cerevisiae <400> 31 atgtcagcat cagcttttaa ttttgccttt agaagatttt ggaatagtga aacaggccct 60 aaaacagtac acttctgggc cccaactttg aagtgggggc tggtcttcgc agggctaaat 120 gatattaaga ggcctgttga gaaggtatca ggagcacaaa atttatcttt attagcgacg 180 gcactgattt ggacgcgttg gtcgtttgtc atcaagccca agaactatct gttagcttcc 240 gtcaattttt tcctgggttg cactgcaggc taccatctaa caagaattgc taactttagg 300 atacggaacg gtgattcttt taaacaggtt attcactaca taataaaagg ggagactcct 360 gcagccgtcg cagcaaagca aactgcatcc acatcgatga acaaaggtgt gatcggtact 420 aatccgccaa taacgcactg a 441 <210> 32 <211> 1267 <212> DNA <213> Artificial Sequence <220> <223> ARS / CEN <400> 32 gagctccttt catttctgat aaaagtaaga ttactccatt tatcttttca ccaacatatt 60 catagttgaa agttatcctt ctaagtacgt atacaatatt aattaaacgt aaaaacaaaa 120 ctgactgtaa aaatgtgtaa aaaaaaaata tcaaattcat agcagtttca aggaatgaaa 180 actattatga tctggtcacg tgtatataaa ttattaattt taaacccata taatttatta 240 tttttttatt ctaaagttta aagtaatttt agtagtattt tatattttga ataaatatac 300 tttaaatttt tatttttata ttttattact tttaaaaata atgtttttat ttaaaacaaa 360 attataagtt aaaaagttgt tccgaaagta aaatatattt tatagttttt acaaaaataa 420 attattttta acgtattttt tttaattata tttttgtatg tgattatatc cacaggtatt 480 atgctgaatt tagctgtttc agtttaccag tgtgatagta tgattttttt tgcctctcaa 540 aagctatttt tttagaagct tcgtcttaga aataggtggt gtataaattg cggttgactt 600 ttaactatat atcattttcg atttatttat tacatagaga ggtgctttta attttttaat 660 ttttattttc aataatttta aaagtgggta cttttaaatt ggaacaaagt gaaaaatatc 720 tgttatacgt gcaactgaat tttactgacc ttaaaggact atctcaatcc tggttcagaa 780 atccttgaaa tgattgatat gttggtggat tttctctgat tttcaaacaa gaggattatttt 840 atttcatatt tattatattt tttacattta ttttatattt ttttattgtt tggaagggaa 900 agcgacaatc aaattcaaaa tatattaatt aaactgtaat acttaataag agacaaataa 960 cagccaagaa tcaaatactg ggtttttaat caaaagatct ctctacatgc acccaaattc 1020 attatttaaa tttactatac tacagacaga atatacgaac ccagattaag tagtcagacg 1080 cttttccgct ttattgagta tatagcctta catattttct gcccataatt tctggattta 1140 aaataaacaa aaatggttac tttgtagtta tgaaaaaagg cttttccaaa atgcgaaata 1200 cgtgttattt aaggttaatc aacaaaacgc atatccatat gggtagttgg acaaaacttc 1260 aatcgat 1267 <210> 33 <211> 289 <212> DNA <213> Artificial Sequence <220> <223> CYC promoter <400> 33 atttggcgag cgttggttgg tggatcaagc ccacgcgtag gcaatcctcg agcagatccg 60 ccaggcgtgt atatatagcg tggatggcca ggcaacttta gtgctgacac atacaggcat 120 atatatatgt gtgcgacgac acatgatcat atggcatgca tgtgctctgt atgtatataa 180 aactcttgtt ttcttctttt ctctaaatat tctttcctta tacattagga cctttgcagc 240 ataaattact atacttctat agacacgcaa acacaaatac acacactaa 289 <210> 34 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> TEF promoter <400> 34 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> 35 <211> 655 <212> DNA <213> Artificial Sequence <220> <223> GPD promoter <400> 35 agtttatcat tatcaatact cgccatttca aagaatacgt aaataattaa tagtagtgat 60 tttcctaact ttatttagtc aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc 120 ccaaaatagg gggcgggtta cacagaatat ataacatcgt aggtgtctgg gtgaacagtt 180 tattcctggc atccactaaa tataatggag cccgcttttt aagctggcat ccagaaaaaa 240 aaagaatccc agcaccaaaa tattgttttc ttcaccaacc atcagttcat aggtccattc 300 tcttagcgca actacagaga acaggggcac aaacaggcaa aaaacgggca caacctcaat 360 ggagtgatgc aacctgcctg gagtaaatga tgacacaagg caattgaccc acgcatgtat 420 ctatctcatt ttcttacacc ttctattacc ttctgctctc tctgatttgg aaaaagctga 480 aaaaaaaggt tgaaaccagt tccctgaaat tattccccta cttgactaat aagtatataa 540 agacggtagg tattgattgt aattctgtaa atctatttct taaacttctt aaattctact 600 tttatagtta gtcttttttt tagttttaaa acaccagaac ttagtttcga cggat 655 <210> 36 <211> 1468 <212> DNA <213> Artificial Sequence <220> <223> ADH promoter <400> 36 gccgggatcg aagaaatgat ggtaaatgaa ataggaaatc aaggagcatg aaggcaaaag 60 acaaatataa gggtcgaacg aaaaataaag tgaaaagtgt tgatatgatg tatttggctt 120 tgcggcgccg aaaaaacgag tttacgcaat tgcacaatca tgctgactct gtggcggacc 180 cgcgctcttg ccggcccggc gataacgctg ggcgtgaggc tgtgcccggc ggagtttttt 240 gcgcctgcat tttccaaggt ttaccctgcg ctaaggggcg agattggaga agcaataaga 300 atgccggttg gggttgcgat gatgacgacc acgacaactg gtgtcattat ttaagttgcc 360 gaaagaacct gagtgcattt gcaacatgag tatactagaa gaatgagcca agacttgcga 420 gacgcgagtt tgccggtggt gcgaacaata gagcgaccat gaccttgaag gtgagacgcg 480 cataaccgct agagtacttt gaagaggaaa cagcaatagg gttgctacca gtataaatag 540 acaggtacat acaacactgg aaatggttgt ctgtttgagt acgctttcaa ttcatttggg 600 tgtgcacttt attatgttac aatatggaag ggaactttac acttctccta tgcacatata 660 ttaattaaag tccaatgcta gtagagaagg ggggtaacac ccctccgcgc tcttttccga 720 tttttttcta aaccgtggaa tatttcggat atccttttgt tgtttccggg tgtacaatat 780 ggacttcctc ttttctggca accaaaccca tacatcggga ttcctataat accttcgttg 840 gtctccctaa catgtaggtg gcggagggga gatatacaat agaacagata ccagacaaga 900 cataatgggc taaacaagac tacaccaatt acactgcctc attgatggtg gtacataacg 960 aactaatact gtagccctag acttgatagc catcatcata tcgaagtttc actacccttt 1020 ttccatttgc catctattga agtaataata ggcgcatgca acttcttttc tttttttttc 1080 ttttctctct cccccgttgt tgtctcacca tatccgcaat gacaaaaaaa tgatggaaga 1140 cactaaagga aaaaattaac gacaaagaca gcaccaacag atgtcgttgt tccagagctg 1200 atgaggggta tctcgaagca cacgaaactt tttccttcct tcattcacgc acactactct 1260 ctaatgagca acggtatacg gccttccttc cagttacttg aatttgaaat aaaaaaaagt 1320 ttgctgtctt gctatcaagt ataaatagac ctgcaattat taatcttttg tttcctcgtc 1380 attgttctcg ttccctttct tccttgtttc tttttctgca caatatttca agctatacca 1440 agcatacaat caactccaag ctggccgc 1468 <210> 37 <211> 252 <212> DNA <213> Artificial Sequence <220> <223> CYC1 terminator <400> 37 tcatgtaatt agttatgtca cgcttacatt cacgccctcc ccccacatcc gctctaaccg 60 aaaaggaagg agttagacaa cctgaagtct aggtccctat ttattttttt atagttatgt 120 tagtattaag aacgttattt atatttcaaa tttttctttt ttttctgtac agacgcgtgt 180 acgcatgtaa cattatactg aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt 240 taatttgcgg cc 252 <210> 38 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 38 cgagctcttc gcggccacct acgccgctat c 31 <210> 39 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 39 gctctagata ttgatatagt gtttaagcga at 32 <210> 40 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 40 ggatccatgt ccgtaaagga actact 26 <210> 41 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 41 acgcgtcgac ttaaaactgc aattcctttt gaat 34 <210> 42 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 42 gagctcaatt aaccctcact aaaggg 26 <210> 43 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 43 gagctccaaa ttaaagcctt cgagcg 26 <210> 44 <211> 69 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 44 tgagcacgtg agtatacgtg attaagcaca caaaggcagc ttggagtatg gtgctgcaag 60 gcgattaag 69 <210> 45 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 45 aggcaagtgc acaaacaata cttaaataaa tactactcag taataacccg gctcgtatgt 60 tgtgtgg 67 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 46 gccaaatgat ttagcattat c 21 <210> 47 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 47 aaaaggagag ggccaagagg g 21 <210> 48 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 48 atgattagac aatcattaat gaaaacagtg tgggctaact ccagtcacga cgttgtaaaa 60                                                                           60 <210> 49 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 49 ctagatagat gaatctctac ccaagaaata aactttagcc aggtttcccg actggaaagc 60                                                                           60 <210> 50 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 50 actgatcatc atttaaaaat gt 22 <210> 51 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 51 aaggaaaaaa attttcacac ta 22 <210> 52 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 52 atgctgccca gacttggttt tgcgaggact gctaggtcca tacaccgttt ccagtcacga 60 cgttgtaaaa 70 <210> 53 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 53 ctacacggag gaatcccttc caagaaagta aactttggtc aggtttcccg actggaaagc 60                                                                           60 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 54 caggaacata gtagaaagac 20 <210> 55 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 55 taacgcgaat cttccatg 18 <210> 56 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 56 aatgtctcaa ccggttcaac gcgctgcagc acgctcattc cttcaaagag ctgcaccgcg 60 gtggc 65 <210> 57 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 57 ttactgttta ccagtttttt ctttctcttt ccattcctta tctagagacc gggccccccg 60 ctagt 65 <210> 58 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 58 ggacgtggcc tgtaaagtt 19 <210> 59 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 59 agaatatgca tgaacatatc cat 23 <210> 60 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 60 aatgtctaca tcatccgtac gttttgcatt taggcggttc tggcaagagc tgcaccgcgg 60 tggc 64 <210> 61 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 61 ttatctgccc gtagtaattt cctttttgct ttctgcggcg ccgctgagac cgggcccccc 60 gctagt 66 <210> 62 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 62 ctattgcgcg catgacta 18 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 63 tgcattgcct tctattatcc 20 <210> 64 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 64 atgtcaaagg cagtaggtga tttaggctta gttggtttag ccgtgatggg ccagtcacga 60 cgttgtaaaa 70 <210> 65 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 65 ttaagcttgg taggttgagg aagatatatt acctccgtgc ccagtccaat aggtttcccg 60 actggaaagc 70 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 66 atgtcaaagg cagtaggtga 20 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 67 ttaagcttgg taggttgagg 20 <210> 68 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 68 atgtctgaac cagctcaaaa gaaacaaaag gttgctaaca actctctaga ccagtcacga 60 cgttgtaaaa 70 <210> 69 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 69 ttaagcggta actttctttt caatcaagtc gaatagagta acaatatcgg aggtttcccg 60 actggaaagc 70 <210> 70 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 70 tcgaactcgt cacatatacg 20 <210> 71 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 71 gcctaaatta atatttccga 20 <210> 72 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 72 atggcacagt tctccgacat tgataaactt gcggtttcca ctttaagatt ccagtcacga 60 cgttgtaaaa 70 <210> 73 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 73 ttagaaagct cttcccatag gagaaagcaa ctgctttcct ttgtagtaat aggtttcccg 60 actggaaagc 70 <210> 74 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 74 atggcacagt tctccgacat 20 <210> 75 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 75 ttagaaagct cttcccatag 20 <210> 76 <211> 292 <212> DNA <213> Artificial Sequence <220> <223> CCW12 promoter <400> 76 ttcgcggcca cctacgccgc tatctttgca acaactatct gcgataactc agcaaatttt 60 gcatattcgt gttgcagtat tgcgataatg ggagtcttac ttccaacata acggcagaaa 120 gaaatgtgag aaaattttgc atcctttgcc tccgttcaag tatataaagt cggcatgctt 180 gataatcttt ctttccatcc tacattgttc taattattct tattctcctt tattctttcc 240 taacatacca agaaattaat cttctgtcat tcgcttaaac actatatcaa ta 292

Claims (20)

Sedoheptulose 7-phosphate, a polypeptide that converts 6-phosphogluconate to ribulose 5-phophate compared to genetically untreated cells, A polypeptide that converts sedoheptulose 7-phosphate to sedoheptulose 7-phosphate, a polypeptide that converts sedoheptulose 7-phosphate to erythrose 4-phosphate, This reduced yeast cell with lactate producing ability. The polypeptide according to claim 1, wherein the 6-phosphogluconate is converted to ribulose 5-phosphate is prepared according to EC. Yeast cells belonging to 1.1.1.44. The polypeptide according to claim 1, wherein said xylulose 5-phosphate is converted to sedo heptulos [epsilon] -phosphate, A yeast cell belonging to 2.2.1.1. The yeast cell according to claim 1, wherein the polypeptide which converts the Sedoheptulose 7-phosphate to erythrose 4-phosphate belongs to EC 2.2.1.2. The yeast cell according to claim 1, wherein the activity of the polypeptide converting 6-phosphogluconate to ribulose 5-phosphate and the polypeptide converting xylulose 5-phosphate to sedo heptulos 7-phosphate is reduced. The yeast cell according to claim 1, wherein the activity of the polypeptide converting 6-phosphogluconate to ribulose 5-phosphate and the polypeptide converting sedoheptulose 7-phosphate to erythrosine 4-phosphate is reduced. The polynucleotide of claim 1, which is a polynucleotide encoding a polypeptide that converts the 6-phosphogluconate to a ribulose 5-phosphate, a polynucleotide that encodes a polypeptide that converts xylulose 5-phosphate to sedo heptolulose 7-phosphate , A polynucleotide encoding a polypeptide that converts sedoheptulose 7-phosphate to erythrosine 4-phosphate, or a combination thereof, is removed or destroyed. The method of claim 1, wherein the polypeptide converts the 6-phosphogluconate to a ribulose 5-phosphate; A polypeptide that converts xylulose 5-phosphate into sedo heptulos 7-phosphate; And Sedoheptulose 7-phosphate into erythrose 4-phosphate are identified as SEQ ID NOS: 1 or 3; 5 or 7; And a polypeptide having a sequence identity of 95% or more with the amino acid sequence of SEQ ID NO: 9. 3. The method of claim 1, further comprising: encoding a polypeptide that converts the 6-phosphogluconate to a ribulose 5-phosphate; A gene encoding a polypeptide that converts xylulose 5-phosphate into sedo heptulos 7-phosphate; And a gene encoding a polypeptide that converts sedoheptulose 7-phosphate to erythrosine 4-phosphate, may be a polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or 3, 5 or 7; And 9, or a polynucleotide sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 2 or 4; 6 or 8; And ten polynucleotide sequences. The yeast cell according to claim 1, which has activity to convert pyruvate to lactate. The yeast cell according to claim 1, comprising a polynucleotide sequence encoding a lactate dehydrogenase. The yeast cell according to claim 10, wherein the polypeptide having activity to convert pyruvate to lactate has an amino acid sequence having 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 11, 12, 13, 11. The polynucleotide of claim 10, wherein the polynucleotide encoding a polypeptide converting pyruvate to lactate comprises a polynucleotide sequence encoding an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NOs: 11, 12, 13, and 14, Or a polynucleotide sequence of SEQ ID NO: 15. The method of claim 1 further comprising administering to the patient a polypeptide that converts pyruvate to acetaldehyde, a polypeptide that converts lactate to pyruvate, a polypeptide that converts dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate, an extrinsic mitochondrial NADH Wherein the activity of the external mitochondrial NADH dehydrogenase, the mitochondrial pyruvate transporter, or a combination thereof is reduced. The method according to claim 1, further comprising a polynucleotide encoding a polypeptide that converts pyruvate to acetaldehyde, a polynucleotide that encodes a polypeptide that converts lactate to pyruvate, dihydroxyacetone phosphate (DHAP) A polynucleotide encoding a polypeptide that converts to a phosphate, a gene encoding an external mitochondrial NADH dehydrogenase, a polynucleotide encoding a mitochondrial pyruvate transporter, or a combination thereof is removed or destroyed Yeast cells. The method according to claim 1, Mai to as My process (Saccharomyces), Cluj Vero My process (Kluyveromyces), Candida (Candida), blood teeth (Pichia), director Chen Escherichia (Issatchenkia), debari Oh, my process (Debaryomyces), Eisai Kosaka Saccharomyces Zygosaccharomyces , Shizosaccharomyces or Saccharomycopsis yeast cells. A composition for use in producing lactate, comprising the yeast cell of claim 1. A method for producing lactate, comprising culturing the yeast cell of claim 1. 19. The method of claim 18 comprising recovering lactate from the culture. 19. The method according to claim 18, wherein the culture is carried out under micro-or anaerobic conditions.

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