KR102255306B1 - Genetically engineered yeast cell producing lactate comprising acetaldehyde dehydrogenase, method of producing yeast cell and method of producing lactate using the same - Google Patents

Abstract

Compared to the parent cell, a genetically engineered yeast having lactate-producing ability has increased activity of an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA and an enzyme that catalyzes the conversion of pyruvate to lactate. It provides a cell, a method for producing the same, and a method for producing lactate using the cell.

Description

Genetically engineered yeast cell producing lactate comprising acetaldehyde dehydrogenase, method of producing yeast cell and method of producing lactate using the same}

It relates to a genetically engineered yeast cell capable of producing lactate, a method for producing the same, and a method for producing lactate using the cell.

Lactate is an organic acid that is widely used in various industrial fields such as food, pharmaceutical, chemical, and electronics. Lactate is a colorless, odorless, and low volatility substance that is well soluble in water. Because lactate is not toxic to humans, it is used as a flavoring agent, acidulant, and preservative. It is also an environmentally friendly alternative polymer material, and is a raw material for polylactic acid (PLA), a biodegradable plastic. PLA is technically a ring-open polymerization polyester resin converted to lactide, a dimer for polymer polymerization, and can be processed in a variety of films, sheets, fibers, and injection molding. Therefore, PLA is a bioplastic that can widely replace conventional general-purpose petrochemical plastics such as polyethylene (PE), polypropylene (PP), polyethylene terephthalate (PET), and polystyrene (PS), and the demand has been greatly increasing in recent years. In addition, lactate has a very high reactivity because it has a hydroxyl group and a carboxyl group at the same time, and thus it is easy to convert to industrially important compounds such as lactate ester, acetaldehyde, and propylene glycol. It is attracting attention as a raw material.

Currently, lactate is industrially produced by a petrochemical synthesis process and a biotech fermentation process. The petrochemical synthesis process is produced by oxidizing ethylene derived from crude oil, making lactonitrile by adding hydrogen cyanide through acetaldehyde, distilling and purifying, and hydrolyzing using hydrochloric acid or sulfuric acid. In addition, the biotechnological fermentation process can produce lactate using renewable carbohydrates such as starch, sucrose, maltose, glucose, fructose, and xylose as a substrate. Therefore, even according to such a conventional technique, there is a demand for a strain capable of efficiently producing lactate and a method for producing lactate using the same. In response to such needs, a method for producing lactate using microorganisms has been recently developed. However, in the case of microorganisms, it is limited to mass-produce only one substance due to homeostasis. In the process of researching to solve this problem, the present invention was completed.

One aspect provides a genetically engineered yeast cell capable of effectively producing lactate.

Another aspect provides a method of producing genetically engineered yeast cells capable of effectively producing lactate.

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

As used herein, the term "increase in activity", or "increased activity" may refer to a detectable increase in the activity of a cell, protein, or enzyme. "Increase in activity", or "increased activity" refers to cells, proteins, or enzymes that do not have a given genetic modification (eg, native or "wild-type). A cell, protein, or enzyme of the same type), such as a cell, protein, or enzyme of the same type, may exhibit a higher level of activity of a modified (eg, genetically engineered) cell, protein, or enzyme. "Cell activity" may refer to the activity of a specific protein or enzyme in a cell. For example, the activity of the modified or engineered cell, protein, or enzyme is about 5% or more than that of the same type of unmanipulated cell, protein, or enzyme, e.g., wild-type cell, protein, or enzyme. , At least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 50%, at least about 60%, at least about 70%, or at least about 100%. The activity of a specific protein or enzyme in a cell is about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 30% of the activity of the same protein or enzyme in a parent cell, e.g., an unmanipulated cell. Or more, about 50% or more, about 60% or more, about 70% or more, or about 100% or more. Cells with increased activity of proteins or enzymes can be identified using any method known in the art. The cell having the increased activity may have a genetic modification that increases the activity of one or more enzymes or polypeptides compared to a cell having no genetic modification.

On the other hand, the term "decrease in activity" or "decreased activity" as used herein refers to a lower enzyme or polypeptide than that measured in a parent cell (eg, a cell that has not been genetically engineered). Represents cells with activity. In addition, "decrease in activity" or "decreased activity" refers to an isolated enzyme or polypeptide having a lower activity than the original or wild-type enzyme or polypeptide. Show. Decreased or decreased activity includes no activity. For example, the activity of converting enzymes from substrates to products for modified (eg, genetically engineered) cells or enzymes does not have the modification of cells or enzymes, such as parental cells or “wild-type” "Compared to the enzyme converting activity of cells or enzymes of, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 55% or more, about 60% or more, about 70% or more, about 75% or more , About 80% or more, about 85% or more, about 90% or more, about 95% or more, or about 100% reduction. Reduced activity of enzymes or cells can be ascertained using any method known in the art. The decrease in activity is compared to cells having an unmodified gene, for example, a parent cell or a wild-type cell, when the activity of the enzyme is absent or decreased even when the enzyme is expressed, the gene encoding the enzyme is not expressed or originally genetically modified even if the gene encoding the enzyme is expressed. This includes cases in which the expression level is decreased compared to a gene that is not. The cell having the reduced activity may have a genetic modification that reduces the activity of one or more enzymes or polypeptides compared to a cell having no genetic modification.

The term “parent cell” refers to an original cell, eg, a cell that has not been genetically engineered of the same type relative to the engineered yeast cell. For a particular genetic modification, the "parent cell" is a cell that does not have the specific genetic modification, but may be the same for other conditions. Thus, the parent cell produces a genetically engineered yeast cell with increased activity of a given protein (e.g., a protein having a sequence identity of about 95% or more with an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA). However, it may be a cell used as a starting material.

The term “disruption” refers to a genetic modification that causes the expression of a referenced gene to be reduced. The disruption is genetic modification (hereinafter referred to as "inactivation" of the gene) or genetic modification (hereinafter, the gene is expressed at a reduced level) to prevent the expression of the mentioned gene. Is called "attenuation"). The inactivation includes not only not expressing the functional product of the gene, but also expressing it, but not expressing the functional product. The attenuation includes a decrease in the expression level of the functional product of the gene. That is, the attenuation includes a decrease in the expression level of a functional product even if the net expression level of a gene increases. Here, the functional product of a gene means that the product of the gene (eg, enzyme) possesses a biochemical or physiological function (eg, enzyme activity) in a parent cell or a wild-type cell. Thus, the disruption includes functional disruption of the gene. The genetic modification includes a modification to introduce a polynucleotide encoding a polypeptide, a substitution, addition, insertion, or deletion of one or more nucleotides for the genetic material of the parent cell, or a chemical modification to the genetic material of the parent cell. Such genetic modifications include those for heterologous, homologous, or heterologous and homogeneous polypeptides for the referenced species and for functional fragments thereof. do. In addition, the genetic modification includes modification of non-coding regulatory regions that alter the expression of genes or operons. The non-coding region includes a 5'-non coding sequence and/or a 3'-non coding sequence.

The disruption of the gene can be achieved by genetic manipulation such as homologous recombination, directed mutagenesis, or molecular evolution. When a cell contains a plurality of identical genes, or two or more paralogs of genes, one or more genes can be disrupted. For example, in the genetic modification, a vector containing a partial sequence of a gene is transformed into a cell, and the cell is cultured to cause the sequence to undergo homologous recombination with an endogenous gene of the cell, thereby destroying the gene, Cells in which homologous recombination has occurred can be selected using a selection marker.

The term "gene" as used herein refers to a nucleic acid fragment expressing a specific protein, and 5'-non-coding sequence and/or 3'-non-coding sequence sequence) may or may not include a regulatory sequence.

The "sequence identity" of a nucleic acid or a polypeptide used in the present invention refers to the degree of identity of bases or amino acid residues between sequences after aligning both sequences so as to be matched as much as possible in a specific comparison region. Sequence identity is a value measured by optimally aligning and comparing two sequences in a specific comparison region, and a part of the sequence within the comparison region may be added or deleted by comparison with a reference sequence. The percent sequence identity is, for example, comparing two optimally aligned sequences across the comparison region, determining the number of positions in which the same amino acid or nucleic acid appears in both sequences to obtain the number of matched positions. And dividing the number of matched positions by the total number of positions within the comparison range (ie, range size), and multiplying the result by 100 to obtain a percentage of sequence identity. The percent of sequence identity may be determined using a known sequence comparison program, and examples of the program include BLASTN (NCBI), CLC Main Workbench (CLC bio), MegAlign ^TM (DNASTAR Inc), and the like.

Different levels of sequence identity can be used to identify different species of polypeptides or polynucleotides having the same or similar functions or activities. For example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% Sequence identity including more than, 98% or more, 99% or more, or 100%, etc. may be used as a reference.

The term “exogenous” as used herein means that a referenced molecule or a referenced activity has been introduced into a host cell. Molecules can be introduced as non-chromosomal genetic material, such as plasmids, or the introduction of an encoding nucleic acid into the host genetic material, such as by insertion into the host chromosome. With respect to the expression of an encoding nucleic acid, the term "exogenous" refers to the introduction of the encoding nucleic acid into an individual in an expressible form. With regard to biosynthetic activity, the term “exogenous” refers to the activity introduced into the host parent cell. Its source can be, for example, a homologous or heterologous encoding nucleic acid that expresses the stated activity after introduction into the host parent cell. Therefore, the term “endogenous” refers to the mentioned molecule or activity present in the host cell. Similarly, with respect to the expression of an encoding nucleic acid, the term “endogenous” refers to the expression of an encoding nucleic acid contained within an individual. The term "heterologous" refers to a molecule or activity from an origin other than the stated species and the term "homologous" refers to a molecule or activity from a host parent cell. Thus, exogenous expression of an encoding nucleic acid can use either or both of a heterologous or homologous encoding nucleic acid.

In addition, the term "genetic engineering" or "genetically engineered" as used herein refers to the act of introducing one or more genetic modifications to a cell, or a cell made thereby. Represents.

The term "lactate" as used herein means "lactic acid" or a salt thereof.

In one aspect, compared to the parental cell, the activity of the enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA and the activity of the enzyme that catalyzes the conversion of pyruvate to lactate are increased. Engineered yeast cells are provided.

In the above aspect, the enzyme catalyzing the conversion of acetaldehyde to acetyl-CoA may be an acetylated acetaldehyde dehydrogenase (A-ALD) belonging to EC 1.2.1.10.

One type of enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA is a bifunctional aldolase-dehydrogenase complex related to 4-hydroxy-2-ketovalerate catabolism. complex). These bifunctional enzymes catalyze the last two steps of the meta-cleavage pathway of catechol, an intermediate in many bacterial species in the breakdown of phenol, toluene, naphthalene, biphenyl, and other aromatic compounds (Powlowski. and Shingler (1994) Biodegradation 5, 219-236). 4-hydroxy-2-ketovalerate is first converted to pyruvate and acetaldehyde by 4-hydroxy-2-ketovalerate aldolase, and then acetaldehyde is converted to Acetly-CoA by A-ALD. Is converted. An example of this type of A-ALD is Pseudomonas sp. CF600 (Genbank No: CAA43226) (Shingler et al (1992) J. Bacteriol. 174:71 1-24). E. coli MhpF protein (Ferrandez et al (1997) J. Bacteriol. 179:2573-2581, Genbank No: NP_414885) is a homologue to DmpF. Another type of enzyme that catalyzes the conversion of the acetaldehyde to acetyl-CoA is a protein that catalyzes the reversible conversion of acetly-CoA and acetaldehyde derived from strictly or facultative anaerobic microorganisms, but alcohol dehydrogenase activity It may not have. Examples of this type of protein may be those reported by Clostridium kluyveri (Smith et al (1980) Arch. Biochem. biophys. 203:663-675). A-ALD is annotated in the genome of Clostridium kluyveri DSM 555 (Genbank No: EDK33116). The homologous protein AcdH was identified in the genome of Lactobacillus plantarum (Genbank No: NP_784141). Another example of this type of protein is the gene product of Clostridium beijerinckii NRRL B593 (Toth et al (1999) App.. Environ. Microbiol. 65: 4973-4980, Genbank No: AAD31841). An example of the A-ALD is MhpF derived from E. coli or a functional homologue thereof, such as E. coli, and EutE derived from S.typhimurium (e.g., EutE gene having the nucleotide sequence of SEQ ID NO: 53 and the EutE protein having the amino acid sequence of SEQ ID NO: 54), or Pseudomonas sp. It may be dmpF derived from CF600. A-ALD may be NAD(P)+ dependent. The A-ALD may have an activity to catalyze the following reactions.

Acetaldehyde + CoA (coenzyme A) + NAD+ <=> acetyl-CoA + NADH + H+

The A-ALD may be expressed without forming a complex with other proteins. The yeast cell may be, for example, one that does not contain an exogenous enzyme belonging to EC 4.1.3.39 or its gene.

The A-ALD may be derived from E. coli. In E. coli, the A-ALD gene, that is, mhpF may be one of the units consisting of a transcription unit of mhpA, mhpB, mhpC, mhpD, mhpE and mhpF. In general, in other microorganisms, MhpE and MhpF exist as a single complex, but in E. coli, MhpF may exist alone and exhibit activity. At this time, the enzyme MhpF, which catalyzes the conversion of acetaldehyde to acetyl-CoA, may have 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 1.

The yeast cells may contain an exogenous gene encoding an enzyme that catalyzes the conversion of the acetaldehyde to acetyl-CoA. The A-ALD exogenous gene may be expressed in an amount sufficient to increase the activity of an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA in the yeast cell compared to the parent cell. The A-ALD exogenous gene may be one encoding an amino acid sequence having 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 1. The A-ALD exogenous gene may have 95% or more sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3. SEQ ID NO: 2 is the nucleotide sequence of the A-ALD gene derived from E. coli. The A-ALD exogenous gene can be changed to a sequence that has been changed to a codon suitable for expression in yeast cells, and to a sequence having an optimized codon. This codon change can be appropriately made within a range that does not change the amino acid sequence of the protein. SEQ ID NO: 3 is an example of a nucleotide sequence having an optimized codon in yeast cells of the A-ALD gene derived from E. coli.

The exogenous gene may be introduced into the parent cell through an expression vector. In addition, the exogenous gene may be introduced into the parent cell in the form of a linear polynucleotide. In addition, the exogenous gene may be expressed from an expression vector (eg, a plasmid) in a cell. In addition, the exogenous gene may be expressed by being inserted into a genetic material (eg, chromosome) in a cell for stable expression. In addition, the exogenous gene may be appropriately regulated by an exogenous promoter linked to the gene to be operable. The promoter may be a promoter derived from ccw12, pdc1, tef1 or pgk1 gene.

In the above aspect, the enzyme catalyzing the conversion of pyruvate to lactate may be lactate dehydrogenase (LDH) belonging to EC 1.1.2.27 or EC 1.1.1.28. The LDH may be NAD(P)H-dependent. In addition, the LDH may act on D-lactate and/or L-lactate. The LDH may have 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 6. SEQ ID NO: 6 is Japanese purple (Pelodiscus sinensis japonicus ) LDH.

The yeast cell may contain an exogenous gene encoding an enzyme that catalyzes the conversion of the pyruvate to lactate. The LDH exogenous gene may be expressed in an amount sufficient to increase the activity of an enzyme that catalyzes the conversion of pyruvate to lactate in the yeast cell compared to the parent cell. The LDH exogenous gene may be one encoding an amino acid sequence having 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 6. The LDH exogenous gene may have 95% or more sequence identity with the nucleotide sequence of SEQ ID NO: 7. The LDH exogenous gene can be changed to a sequence with an optimized codon, a sequence that has been changed to a codon suitable for expression in yeast cells. This codon change can be appropriately made within a range that does not change the amino acid sequence of the protein. SEQ ID NO: 7 is Japanese purple (Pelodiscus sinensis japonicus ) is the LDH gene.

The LDH exogenous gene may be included in the genome of the cell. The LDH exogenous gene may be one encoding an enzyme acting on L-Ldh, D-Ldh, or both. Thus, the yeast cells can produce L-lactic acid or D-lactic acid or a racemic mixture thereof, or a salt thereof. The LDH exogenous gene may include those derived from bacteria, yeasts, fungi, and animals such as rodents, mammals, amphibians (amphibian) and sprouts (Sauropsida). The LDH exogenous gene is Japanese ( Pelodiscus sinensis) japonicus ), platypus ( Ornithorhynchus anatinus ), Bottlenose Dolphin ( Tursiops truncatus ), Norwegian House Mouse ( Rattus norvegicus ), or frogs ( Xenopus laevis ). Lactate dehydrogenase derived from Japanese larva, lactate dehydrogenase derived from platypus, lactate dehydrogenase derived from bottlenose dolphin, and lactate dehydrogenase derived from Norwegian house rats, respectively. It may have an amino acid sequence of SEQ ID NOs: 6, 37, 38, and 39. The lactate dehydrogenase is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more of the amino acid sequences of SEQ ID NOs: 6, 37, 38, and 39, respectively. , 98% or more, 99% or more, or 100% sequence identity. The gene encoding the lactate dehydrogenase is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more of the nucleotide sequence of SEQ ID NO: 7, 40, 41, or 42, It may have a sequence identity of 97% or more, 98% or more, 99% or more, or 100%.

The LDH exogenous gene may be expressed from a vector containing it. The vector may include an origin of replication, a promoter, a polynucleotide encoding LDH, and a terminator. The replication initiation point may include a yeast autonomous replication sequence (ARS). The yeast self-replicating sequence may be stabilized by a yeast centrometric sequence (CEN). The promoter may be selected from the group consisting of a CCW12 promoter, a CYC promoter, a TEF1 promoter, a PGK1 promoter, a GPD promoter, and an ADH promoter. The CCW12 promoter, CYC promoter, TEF1 promoter, PGK1 promoter, GPD promoter, and ADH promoter may have nucleotide sequences of SEQ ID NOs: 9, 43, 11, 12, 44, and 45, respectively. The terminator may be selected from the group consisting of phosphoglycerate kinase 1 (PGK1), cytochrome c transcription (CYC1), and GAL1. The CYC1 terminator may have a nucleotide sequence of SEQ ID NO: 46. The vector may further include a selection marker.

The yeast cell may contain a single LDH gene, a plurality of LDH genes, for example, 2 to 10 copies. The yeast cells are, for example, 1 to 10, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 2 to 10, 2 to 8, 2 to 7, 2 To 6, 2 to 5, 2 to 4, or 2 to 3 copies of the LDH gene. When the yeast cell contains a plurality of LDH genes, each gene may include a copy of the same gene or a copy of two or more different LDH genes. Multiple copies of the exogenous LDH gene may be included at the same locus, or at several loci within the genome of the host cell.

Examples of the yeast cells may include one or more exogenous LDH genes in which at least one of an endogenous PDC gene, a CYB2 gene, a GPD gene, a GPP gene, and an ALD gene is replaced by an exogenous LDH gene, respectively. .

In the above aspect, the yeast cell may have a reduced activity of an enzyme that catalyzes the conversion of acetaldehyde to ethanol compared to the parent cell. The enzyme catalyzing the conversion of acetaldehyde to ethanol may be an alcohol dehygrogenase (ADH) belonging to EC 1.1.1.1. Examples of ADH include ADH1, ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7. The alcohol dehydrogenase may be NADH dependent. The ADH1 gene and the ADH1 protein may have a nucleotide sequence of SEQ ID NO: 55 and an amino acid sequence of SEQ ID NO: 56, respectively.

The yeast cell may be a result of disruption of an endogenous gene encoding an enzyme that catalyzes the conversion of the acetaldehyde to ethanol. The yeast cells may be destroyed to be expressed to a sufficient degree to reduce the activity of an enzyme that catalyzes the conversion of acetaldehyde to ethanol compared to the parent cell.

Another example of the yeast cell is that the activity of the enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA and the activity of the enzyme that catalyzes the conversion of pyruvate to lactate are increased compared to the parent cell, compared to the parent cell. The activity of the enzyme that catalyzes the conversion of acetaldehyde to ethanol is reduced, and may be a genetically engineered yeast cell having lactate-producing ability.

Yeast cells are Saccharomyces, Candida, Schizosaccharomyces, Kluyveromyces, Pichia, Isachenkia, and Hansenula. Hansenula) may be a species belonging to any one of the genus. Species of the genus Saccharomyces include, for example, S. cerevisiae, S. bayanus, S. boulardii, and Saccharomyces. S. bulderi, S. cariocanus, S. cariocus, S. chevalieri, Saccharo S. dairenensis, S. ellipsoideus, S. eubayanus, S. exiguus, Saccharomyces florentinus, Saccharomyces kluyveri, S. martiniae, Saccharomyces monacensis , Saccharomyces norbensis, Saccharomyces paradoxus, S. pastorianus, Saccharomyces spencerorum (S. spencerorum), Saccharomyces turicensis, Saccharomyces unisporus, S. uvarum, or Saccharomyces zonatus (S. turicensis) .zonatus). Candida species are, for example, Candida albicans (C. albicans), Candida ascalaphidarum (C. ascalaphidarum), Candida amphixiae (C. amphixiae), Candida antarctica (C. antarctica), Candida. Argentea, Candida atlantica, Candida atmosphaerica, Candida blattae, Candida bromeliacearum (C. bromeliacearum) , Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydali, C. corydali, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida glabrata C. fermentati, C. guilliermondii, C. haemulonii, Candida insectamens, C. insectorum, Candida inter Intermedia, C. jeffresii, C. kefyr, C. krusei, C. lusitaniae, Candida lysophila (C. jeffresii) lyxosophila), Candida maltosa (C. maltosa), Candida marina (C. marina), Candida membranifaciens (C. membranifaciens), Candida milleri (C. milleri), Candida oleophila (C. oleophila) , Candida oregonensis, Candida paracillosis (C. pa) rapsilosis), Candida cuercitrusa (C. quercitrusa), Candida rugosa (C. rugosa), Candida sake (C. sake), Candida shehatea (C. shehatea), Candida temnochilae (C. temnochilae), Candida tenuis (C. tenuis) , Candida seae (C. theae), Candida tolerans (C. tolerans), Candida tropicalis (C. tropicalis), Candida tsuchiyae (C. tsuchiyae), Candida sinolaborantium (C. sinolaborantium), Candida Sojae (C. sojae), Candida subhashii (C. subhashii), Candida viswanathii (C. viswanathii), Candida utilis (C. utilis), or Candida ubatubensis (C. ubatubensis). I can. The species of the genus Schizosaccharomyces are, for example, S. pombe, S. japonicus, and Schizosaccharomyces octosporus (Schizosaccharomyces). S. octosporus), or S. cryophilus. Species of the genus Kluyveromyces are, for example, K. aestuarii, K. africanus, K. bacillisporus, and K. bacillisporus. ), K. blattae, K. dobzhanskii, K. hubeiensis, K. lactis ), K. lodderae, K. marxianus, K. nonfermentans, K. lodderae (K.) piceae), Kluyveromyces sinensis, Kluyveromyces cermotorerans (K. thermotolerans), Kluyberomyces walty (K. waltii), Kluyberomyces wikerhami (K. wickerhamii), or K. yarrowii. The species of the genus Pichia are, for example, P. anomala, P. heedii, P. guilliermondii, P. guilliermondii, and P. kluyveri), P. membranifaciens, P. norvegensis, P. ohmeri, P. pastoris, P. membranifaciens, P. membranifaciens, P. (P. methanolica), or P. subpelliculosa. The species of the genus Issachenkia may be, for example, I. orientalis. The species of the genus Hansenula include, for example, H. subpelliculosa, H. anomala, H. polymorpha, and H. H. holstii Wick), or H. capsulata Wick.

In the above aspect, the yeast cells contain an enzyme catalyzing the conversion of pyruvate to acetaldehyde, an enzyme catalyzing the conversion of lactate to pyruvate, and dihydroxyacetone phosphate (DHAP) into glycerol-3-phosphate ( G3P), an enzyme that catalyzes the conversion of glycerol-3-phosphate (G3P) to glycerol, an enzyme that catalyzes the conversion of acetaldehyde to acetate, or a combination thereof. Can be.

In the yeast cell, the enzyme catalyzing the conversion of pyruvate to acetaldehyde belongs to EC 4.1.1.1, and the enzyme catalyzing the conversion of lactate to pyruvate is EC 1.1.2.4 or EC 1.1. 2.3, the enzyme catalyzing the conversion of DHAP to G3P belongs to EC 1.1.1.8, and the enzyme catalyzing the conversion of glycerol-3-phosphate (G3P) to glycerol belongs to EC 3.1.3.21. , The enzyme that catalyzes the conversion of acetaldehyde to acetate may belong to EC 1.2.1.3, EC 1.2.1.4, or EC 1.2.1.5.

In the yeast cell, a gene encoding an enzyme that catalyzes the conversion of pyruvate to acetaldehyde, a gene encoding an enzyme that catalyzes the conversion of lactate to pyruvate, dihydroxyacetone phosphate (DHAP) is glycerol- A gene encoding an enzyme that catalyzes the conversion to 3-phosphate (G3P), a gene encoding an enzyme that catalyzes the conversion of glycerol-3-phosphate to glycerol, a gene encoding an enzyme that catalyzes the conversion of acetaldehyde to acetate , Or the combination may be disrupted.

The enzyme catalyzing the conversion of pyruvate to acetaldehyde may be pyruvate decarboxylase (PDC). The PDC is a sequence of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of the amino acid sequence of SEQ ID NO: 47 Can have identity. The PDC gene is of the nucleotide sequence of SEQ ID NO: 48 and 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%. It may have sequence identity. PDCs include PDC1 (SEQ ID NO: 48), PDC5, and PDC6. The PDC may catalyze the conversion of pyruvate to acetaldehyde under anaerobic or aerobic conditions. The PDC gene may be destroyed by substitution with an LDH gene. The yeast cell may be a PDC gene attenuated (attenuation). The yeast cell may also be one or more of the PDC1 gene, the PDC5 gene, and the PDC6 gene have been attenuated. In addition, the yeast cell may be a gene encoding one or two of PDC1, PDC5, and PDC6 is inactivated. For example, the PDC1 gene, the PDC5 gene, the PDC6 gene, the PDC1 gene and the PDC5 gene, the PDC1 gene and the PDC6 gene, or the PDC5 gene and the PDC6 gene may be inactivated.

The enzyme catalyzing the conversion of lactate to pyruvate may be lactate cytochrome-c oxidoreductase (CYB2). The enzyme that catalyzes the conversion of lactate to pyruvate may be cytochrome c-dependent. The CYB2 may belong to EC 1.1.2.4, which acts on D-lactate, or EC 1.1.2.3, which acts on L-lactate. The enzyme that catalyzes the conversion of lactate to pyruvate is the amino acid sequence of SEQ ID NO: 48 and 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98 % Or more, 99% or more, or 100% sequence identity. The gene encoding the enzyme that catalyzes the conversion of lactate to pyruvate is the nucleotide sequence of SEQ ID NO: 49 and 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97 % Or more, 98% or more, 99% or more, or 100% sequence identity. The CYB2 gene may be destroyed by substitution by the LDH gene.

The enzyme catalyzing the conversion of DHAP to G3P may be NAD-dependent glycerol-3-phosphate dehydrogenase (NAD-dependent glycerol-3-phosphate dehydrogenase: GPD). The GPD may be a NAD ⁺ -dependent enzyme. An example of the GPD is cytosolic glycerol-3-phosphate dehydrogenase, and may be an enzyme that catalyzes the reduction of dihydroxyacetone phosphate (DHAP) to glycerol-3-phosphate using oxidation of NADH to NAD+. . The GPD may belong to EC 1.1.1.8. Examples of the GPD include GPD1 and GPD2. The GPD is a sequence of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of the amino acid sequence of SEQ ID NO: 51 It may be of identity. The gene encoding the GPD is the nucleotide sequence of SEQ ID NO: 52 and 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or It may have 100% sequence identity. The GPD gene may be destroyed by substitution with the LDH gene.

The enzyme that catalyzes the conversion of glycerol-3-phosphate to glycerol may be glycerol phosphate phosphatase (GPP). GPP may be GPP1 and GPP2 from S. cerevisiae. The GPP may have a nucleotide sequence of SEQ ID NO: 57 and an amino acid sequence of SEQ ID NO: 58. The GPP gene may be destroyed by substitution with the LDH gene.

The enzyme catalyzing the conversion of acetaldehyde to acetate may be acetaldehyde dehydrogenase (ALD). ALD can be NAD(P)+ dependent. The ALD is a sequence of 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of the amino acid sequence of SEQ ID NO: 60 Can have identity. The ALD gene is 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% of the nucleotide sequence of SEQ ID NO: 61. It may have sequence identity. ALD includes ALD6 (also referred to as ALD1), ALD2, ALD3, ALD4, and ALD5. The ALD gene may be destroyed by substitution with the LDH gene. The yeast cell may be the ALD gene is attenuated (attenuation). In addition, the yeast cell may be one or more of the ALD6 gene, ALD2 gene, and ALD3 gene attenuated. In addition, the yeast cell may be a gene encoding one or two of the ALD6 gene, ALD2 gene, and ALD3 gene is inactivated (inactivation).

Another aspect includes introducing into yeast cells a gene encoding an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA and a gene encoding an enzyme that catalyzes the conversion of pyruvate to lactate; And destroying a gene encoding an enzyme that catalyzes the conversion of acetaldehyde to ethanol.

In the method for producing the lactate-producing yeast, the gene encoding the enzyme catalyzing the conversion of acetaldehyde to acetyl-CoA in the yeast cell and the enzyme encoding the enzyme catalyzing the conversion of pyruvate to lactate The steps to introduce the gene are as follows. In this step, "yeast cells", "an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA", "a gene encoding an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA", and "pyruvate. The enzymes that catalyze conversion to lactate", and "genes encoding enzymes that catalyze the conversion of pyruvate to lactate" are as described above. An example of "an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA" may be MhpF derived from E. coli.

“The gene encoding the enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA” and the “gene encoding the enzyme that catalyzes the conversion of pyruvate to lactate” can be introduced into the yeast cells simultaneously or separately. have.

"The gene encoding the enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA" and the "gene encoding the enzyme that catalyzes the conversion of pyruvate to lactate" are also introduced, respectively, to obtain the endogenous (endogenous) of the yeast cells. ) It may be inserted into genetic material (eg, chromosome). In this case, the both genes may be inserted at one or more positions of specific genes of the endogenous genetic material (eg, chromosome) of the yeast cell to destroy the one or more genes. The specific gene is a gene encoding an enzyme that catalyzes the conversion of pyruvate to acetaldehyde, a gene encoding an enzyme that catalyzes the conversion of lactate to pyruvate, and dihydroxyacetone phosphate (DHAP) into glycerol-3. -Encoding a gene encoding an enzyme that catalyzes the conversion to phosphate (G3P), a gene encoding an enzyme that catalyzes the conversion of glycerol-3-phosphate to glycerol, and an enzyme that catalyzes the conversion of acetaldehyde to ethanol It contains a gene that does. These specific genes include PCD, CYB2, GPD, GPP, and ADH genes. When the specific gene is an ADH gene, the two steps may be performed simultaneously.

Both genes may be inserted at the same or different positions of the endogenous genetic material of the yeast cell. In addition, both genes may be present in the yeast cell without being inserted into the endogenous genetic material of the yeast cell. In this case, both genes may be included in a vector such as a plasmid and located separately from the endogenous genetic material of the yeast cell.

Both genes are introduced and expressed in yeast cells in an expressible form, and the respective gene products "an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA" and "an enzyme that catalyzes the conversion of pyruvate to lactate" It may be to produce "in yeast cells. The expressible form may be a structure in which both genes are operably linked to their expression control sequences. For example, it is operably linked to one or more of an exogenous enhancer, an operator, a promoter, and a transcription terminator, so that it is itself capable of expression in yeast cells or yeast It may be connected to the regulatory sequence of the cell to be expressible. The promoter may be selected from the group consisting of a CCW12 promoter, a CYC promoter, a TEF1 promoter, a PGK1 promoter, a GPD promoter, and an ADH promoter. The CCW12 promoter, CYC promoter, TEF1 promoter, PGK1 promoter, GPD promoter, and ADH promoter may have nucleotide sequences of SEQ ID NOs: 9, 43, 11, 12, 44, and 45, respectively. The terminator may be selected from the group consisting of phosphoglycerate kinase 1 (PGK1), cytochrome c transcription (CYC1), and GAL1. The CYC1 terminator may have a nucleotide sequence of SEQ ID NO: 46. The vector may further include a selection marker.

The introducing step may be performed by a method of introducing genetic material into yeast cells known in the art (R. Danile Gietz et al., Biotechniques 30:816-831, April 2001). The introduction method includes the spheroplast method, intact yeast cell transformation, and electroporation. Intact yeast cell transformation method may be the use of specific monovalent alkali cations (Na+, K+, Rb+, Cs+, and Li+) in combination with PEG to promote DNA uptake such as plasmids by yeast cells. For example, it may be to apply a heat shock to an aqueous solution containing PEG, LiAc, carrier ssDNA and plasmid DNA. The electroshock method involves providing an electric pulse in a mixed medium containing yeast cells and DNA, such as plasmid DNA.

Thus, the step of introducing is a contact of a gene encoding an enzyme catalyzing the conversion of acetaldehyde to acetyl-CoA and a gene encoding an enzyme catalyzing the conversion of pyruvate to lactate with yeast cells in an appropriate liquid medium. It may include a step of. The yeast cells may be spheroplasts, or intact yeast cells. The liquid medium may vary depending on the transformation method selected. The liquid medium can be, for example, water, an aqueous solution, or a buffer. The aqueous medium may include monovalent alkali cations (any one or more of Na+, K+, Rb+, Cs+, and Li+), and PEG. The liquid medium may contain a carrier ssDNA. The liquid medium may be an aqueous solution containing PEG, LiAc, and carrier ssDNA.

The contact may be made while applying thermal shock or electric pulse. The thermal shock may be cultured at about 40 to about 45°C, for example, about 42°C. The electric pulse may be applied in an electroporation cuvette or between electrodes in a petri dish. The parameters of electric shock, field strength (kV/cm), capacitance (uF), and resistance, may differ depending on the specifics of the cell preparation. Therefore, transformation efficiency may vary depending on the yeast strain. For a given yeast strain, one of ordinary skill in the art can examine the parameters of the pulses for different cells and select appropriate parameters to ensure that a sufficient number of transformants are obtained.

In the step of introducing, both genes may be included in a vector together with a homologous sequence with an intrinsic genetic material of a parental yeast cell. The homologous sequence is complementary to a target sequence present in the endogenous genetic material of the parental yeast cell, and thus may be substituted with the target sequence by homologous recombination. The target sequence is a gene encoding an enzyme that catalyzes the conversion of pyruvate to acetaldehyde, a gene encoding an enzyme that catalyzes the conversion of lactate to pyruvate, and dihydroxyacetone phosphate (DHAP) into glycerol-3. -Encoding a gene encoding an enzyme that catalyzes the conversion to phosphate (G3P), a gene encoding an enzyme that catalyzes the conversion of glycerol-3-phosphate to glycerol, and an enzyme that catalyzes the conversion of acetaldehyde to ethanol It contains a gene that does. These specific genes include PCD, CYB2, GPD, GPP, and ADH genes. The vector may include two sequences homologous to the 5'end region and the 3'end region of the target sequence. In this case, the step of introducing may include culturing the yeast cells under selection pressure during or after the contacting. The selection pressure represents a substance or condition that allows only cells in which homologous recombination has occurred can be selected. The selection pressure includes incubation in the presence of antibiotics. In this case, the vector may include a gene encoding an enzyme that degrades antibiotics.

In the method for producing the yeast producing lactate, the step of destroying a gene encoding an enzyme that catalyzes the conversion of acetaldehyde to ethanol; is as follows.

The disrupting step may comprise contacting a polynucleotide comprising a homologous sequence with a gene encoding an enzyme catalyzing the conversion of yeast cells and acetaldehyde to ethanol in an appropriate liquid medium. The homologous sequence may be homologous to all or part of the gene. The homologous sequence may be homologous to the coding region or the expression control region of the gene. The polynucleotide comprising a sequence homologous to the gene may be linked to another gene, for example, a gene encoding an enzyme involved in promoting lactate biosynthesis such as the A-ALD gene and the LDH gene. The polynucleotide including the gene and the homologous sequence may be included in a vector such as a plasmid. The homologous sequence can be used to replace a gene encoding an enzyme that catalyzes the conversion of acetaldehyde to ethanol by homologous recombination. The vector may include two sequences homologous to the 5'end region and the 3'end region of the target sequence. In this case, the step of destroying may include culturing the yeast cells under selection pressure during or after the contacting. The selection pressure represents a substance or condition that allows only cells in which homologous recombination has occurred can be selected. The selection pressure includes incubation in the presence of antibiotics. In this case, the vector may include a gene encoding an enzyme that degrades antibiotics.

In addition, in the disrupting step, the contacting step, unless otherwise stated, "a gene encoding an enzyme that catalyzes the conversion of yeast cells and acetaldehyde to acetyl-CoA, as mentioned in the step of introducing, and pyruvate. As described in "step of contacting a gene encoding an enzyme that catalyzes the conversion of to lactate in an appropriate liquid medium". The destroying step and the introducing step may be performed simultaneously.

The method for producing the lactate-producing yeast includes a gene encoding an enzyme that catalyzes the conversion of pyruvate to acetaldehyde in the yeast cell, a gene encoding an enzyme that catalyzes the conversion of lactate to pyruvate, and A gene encoding an enzyme catalyzing the conversion of hydroxyacetone phosphate to glycerol-3-phosphate, a gene encoding an enzyme catalyzing the conversion of glycerol-3-phosphate to glycerol, or a combination thereof. I can. Here, "gene encoding an enzyme that catalyzes the conversion of pyruvate to acetaldehyde", "a gene encoding an enzyme that catalyzes the conversion of lactate to pyruvate", "dihydroxyacetone phosphate to glycerol-3-phosphate. The gene encoding an enzyme that catalyzes conversion to glycerol", and "a gene encoding an enzyme catalyzing the conversion of glycerol-3-phosphate to glycerol" are as described above.

The disrupting step includes yeast cells and "gene encoding an enzyme that catalyzes the conversion of pyruvate to acetaldehyde", "gene encoding an enzyme that catalyzes the conversion of lactate to pyruvate", and "dihydroxyacetone phosphate. A gene encoding an enzyme that catalyzes the conversion of glycerol-3-phosphate", and "a gene encoding an enzyme that catalyzes the conversion of glycerol-3-phosphate to glycerol" Contacting the nucleotides in a suitable liquid medium. The homologous sequence may be homologous to all or part of the gene. The homologous sequence may be homologous to the coding region or the expression control region of the gene. The polynucleotide comprising a sequence homologous to the gene may be linked to another gene, for example, a gene encoding an enzyme involved in promoting lactate biosynthesis such as the A-ALD gene and the LDH gene. The polynucleotide including the gene and the homologous sequence may be included in a vector such as a plasmid. The homologous sequence may be used to replace the one or more genes by homologous recombination. The vector may include two sequences homologous to the 5'end region and the 3'end region of the target sequence. In this case, the step of destroying may include culturing the yeast cells under selection pressure during or after the contacting. The selection pressure represents a substance or condition that allows only cells in which homologous recombination has occurred can be selected. The selection pressure includes incubation in the presence of antibiotics. In this case, the vector may include a gene encoding an enzyme that degrades antibiotics.

In addition, in the disrupting step, the contacting step, unless otherwise stated, "a gene encoding an enzyme that catalyzes the conversion of yeast cells and acetaldehyde to acetyl-CoA, as mentioned in the step of introducing, and pyruvate. As described in "step of contacting a gene encoding an enzyme that catalyzes the conversion of to lactate in an appropriate liquid medium".

Another aspect is the step of culturing the yeast cells to produce lactate; And it provides a method for producing lactate comprising the step of recovering the lactate from the culture.

The cultivation may be carried out in a medium containing a carbon source, for example, glucose. The medium used for culturing yeast cells can be any conventional medium suitable for the growth of host cells, such as minimal or complex medium containing appropriate supplements. Suitable media are available from commercial vendors or can be prepared according to known manufacturing methods. The medium used for the culture may be a medium capable of satisfying the requirements of specific yeast cells. The medium may be a medium selected from the group consisting of a carbon source, a nitrogen source, a salt, a trace element, and a combination thereof.

In order to obtain lactate from the genetically engineered yeast cells, culture conditions can be appropriately adjusted. The cells can be cultured under aerobic conditions for proliferation. Thereafter, the cells may be cultured in microaerobic conditions or anaerobic conditions to produce lactate. The term "anaerobic conditions" refers to an oxygen-free environment. The term "microaerobic conditions" when used by reference to culture or growth conditions, the concentration of dissolved oxygen (DO) in the medium is greater than 0% of the saturation for dissolved oxygen in the liquid medium. This means that it is kept below about 10%. Microaerobic conditions also include growing or resting cells in liquid medium or on solid agar plates in sealed chambers maintained in an atmosphere with less than 1% oxygen. The concentration of oxygen can be maintained, for example, _{by sparging the culture with an N 2} /CO ₂ mixture or other suitable non-oxygen gas. The oxygen condition includes maintaining a dissolved oxygen (DO) concentration of 0% to 10%, for example 0 to 8%, 0 to 6%, 0 to 4%, or 0 to 2%.

The term "culture conditions" means conditions for culturing yeast cells. Such culture conditions may be, for example, carbon sources, nitrogen sources, or oxygen conditions used by yeast cells. The carbon sources available to yeast cells include monosaccharides, disaccharides or polysaccharides. The carbon source may be glucose, fructose, mannose, or galactose. The nitrogen source that can be used by yeast cells may be an organic nitrogen compound or an inorganic nitrogen compound. Examples of nitrogen sources may be amino acids, amides, amines, nitrates, or ammonium salts.

Separation of lactate from culture can be performed by conventional methods known in the art. This separation method may be centrifugation, filtration, ion exchange chromatography or crystallization. For example, the culture medium may be centrifuged at a low speed to remove biomass, and the resulting supernatant may be separated through ion exchange chromatography.

According to the yeast cell according to one aspect, it can be used to efficiently produce lactate.

According to the method for producing lactate-producing yeast cells according to one aspect, it is possible to efficiently produce lactate-producing yeast cells.

According to the method for producing lactate according to an aspect, it is possible to efficiently produce lactate.

1 is a diagram showing a pJ1214-mhpF vector cleavage diagram.
2 is a diagram showing a p416-ldh-HPH vector cleavage diagram.
3 is a diagram showing some metabolic pathways related to conversion of pyruvate to lactate generated through a corresponding process according to an embodiment.

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

Example One: mhpF Genes and/or EutE Gene containing S.cerevisiae

The S. cerevisiae strain used in this example was prepared as follows.

(One) mhpF Gene expression strain production

(1.1) S.cerevisiae CEN . PK2 -1D( Δ adh1 :: ldh , mhpF ) Of the manufacture

(1.1.1) MhpF A vector for gene transfer pJ1214 - MhpF Manufacture of

In order to express the MhpF gene (SEQ ID NO: 2) derived from E. coli from a vector in S. cerevisiae, an optimized MhpF gene (SEQ ID NO: 3) (DNA2.0 Inc., USA) was synthesized as a codon frequently used in S. cerevisiae. I did. The synthesized MhpF gene has a TEF1 promoter sequence (SEQ ID NO: 11) linked to the 5'end, so that transcription is regulated by the TEF1 promoter. The synthesized TEF1 promoter-MhpF gene containing pJ1214-mhpF (DNA2.0 Inc., USA) was provided. pJ1214 (DNA2.0 Inc., USA) contains the URA3 marker as an expression vector for S. cerevisiae and contains the 2um Ori sequence.

1 is a diagram showing a cleavage diagram of the pJ1214-mhpF vector. In Figure 1, P TEF represents the TEF promoter. The nucleotide sequence of the pJ1214-mhpF vector is shown in SEQ ID NO: 8.

(1.1.2) adh1 Preparation of cassette for gene deletion

In order to prepare a vector containing the'ldh cassette', S. cerevisiae CEN . By the primer set of the PK2 -1D genomic DNA as a template, and SEQ ID NO: 13 and 14. PCR using as primers, and amplifying CCW12 gene promoter (promoter) (hereinafter also referred to as "P CCW12" or "CCW12 promoter") The CCW12 gene promoter amplification product (SEQ ID NO: 9) and the synthesized ldh gene (SEQ ID NO: 7) (DNA2.0 Inc., USA) were digested with SacI/XbaI and BamHI/SalI, respectively, and pRS416 vector digested with the same enzyme (ATCC87521 ^TM ) and connected to each other. The pRS416 vector is a yeast centromere shuttle plasmid with a T7 promoter, ampicillin resistance in bacteria, a URA3 cassette (selective marker) in yeast, and a restriction enzyme cloning site.

Further, a "HPH cassette" sequence (SEQ ID NO: 17) was amplified by PCR using pCEP4 plasmid (invitrogen, Cat. no. V044-50) as a template and primer sets of SEQ ID NOs: 15 and 16 as primers. The amplified "HPH cassette" and the pRS416 vector were cut using SacI enzyme, respectively, and ligated to each other to prepare a vector p416-ldh-HPH containing a structure in which the'ldh cassette' and the "HPH cassette" are operably linked. . 2 is a diagram showing a p416-ldh-HPH vector cleavage diagram. In Figure 2. "P CCW12" and "C2 LDH" denote the CCW12 promoter and LDH orf, respectively. pCEP4 plasmid is an episomal mammalian expression vector using cytomegalovirus (CMV) immediate early enhance/promoter for high level transcription of a recombinant gene inserted into a multiple cloning site. pCEP4 has a hygromycin B resistance gene for stable selection in transfected cells. Here, the'ldh cassette' refers to a region that allows the ldh gene to be expressed because it contains the ldh gene and its regulatory region. The ldh gene was allowed to be transcribed under the CCW12 promoter. In addition, the'hygromycin B phosphotransferase (HPH) cassette' includes a hygromycin B resistance gene and a regulatory region thereof, and thus represents a region that allows the hygromycin B resistance gene to be expressed.

The adh1 deletion cassette was prepared by PCR using the p416-ldh-HPH vector as a template and primer sets of SEQ ID NOs: 4 and 5 as primers. In SEQ ID NO: 4 and SEQ ID NO: 5, sequences 1-51 are homologous recombination with a homologous sequence in the S. cerevisiae genome to represent a portion to be substituted with the adh1 gene.

The cassette for adh1 deletion was later published in S.cerevisiae CEN . In the PK2-1D genome, the gene encoding alcohol dehydrogenase (adh1) is replaced with the ldh-HPH cassette, which inactivates adh1 and is used to introduce the ldh gene.

(1.1.3) S.cerevisiae CEN . PK2 -1D( Δ adh1 :: ldh , mhpF ) Of the manufacture

S. cerevisiae CEN . In order from the PK2 -1D the substitution adh1 gene in ldh gene, the "adh1 deletion cassette" produced in (1.1.2) S.cerevisiae CEN. Introduced by the PK2 -1D strain transformed with heat shock (heat shock transformation), and Hi hygromycin 200ug / mL comprises a YPD medium (Yeast extract 1 (w / v )%, peptone 1 (w / v)%, and Glucose 2 (w/v)%) incubated at 30° C. for 3 days to replace the adh1 gene on the chromosome with the ldh gene, S.cerevisiae CEN . A PK2 -1D ( Δadh1::ldh) strain was prepared.

In addition, S.cerevisiae CEN obtained the pJ1214-mhpF vector for MhpF expression prepared in (1.1.1) . PK2 -1D ( Δadh::ldh) strain introduced by heat shock transformation (heat shock transformation), S. cerevisiae CEN . PK2 -1D ( Δadh1::ldh, mhpF) strain was prepared. Specifically, after thermal shock the strain, Minimal Ura-drop out medium (Yeast nitrogen base without amino acids (Sigma-Aldrich: Cat. no. Y0626) 6.7 g/L, and Yeast synthetic drop-out without uracil (Sigma-Aldrich: Cat. no. Y1501) 1.9 g/L, glucose 2 (w/v)%) was prepared by culturing at 30° C. for 3 days. To analyze the genotype of the prepared strain, the genome of the prepared strain was used as a template, and whether adh1 gene was deleted and the ldh gene was introduced by PCR used as a primer set of SEQ ID NOs: 18 and 19, and SEQ ID NOs: 20 and 21 It was confirmed whether the mhpF gene was introduced by PCR used as a primer set of.

As a result, S. cerevisiae CEN . It was confirmed that the PK2 -1D ( Δadh1::ldh, mhpF) strain was prepared.

(1.2) S. cerevisiae CEN . Preparation of mutant strains of PK2 -1D ( Δadh1:: ldh , mhpF)

(1.2.1) pdc1 , cyb2 And gpd1 Of a vector for fruiting

The amplification product amplified by PCR using p416-ldh-HPH as a template and primer sets of SEQ ID NOs: 22 and 23 as a primer was cut with SacI for the deletion cassette for pyruvate decarboxylase 1 (Pdc1), This was ligated with the pUC57-Ura3HA vector (DNA2.0 Inc.: SEQ ID NO: 24) cut with the same enzyme to prepare pUC57-ura3HA-ldh. Next, a cassette for pdc1 deletion was prepared by PCR using pUC57-ura3HA-ldh as a template and primer sets of SEQ ID NOs: 25 and 26 as primers. In SEQ ID NOs: 25 and 26, sequences 1-42 and 1-44 are homologous recombination with the homologous sequence of S. cerevisiae, respectively, and represent a portion to be substituted with the pdc1 gene.

In addition, L-lactate cytochrome-C oxidoreductase (L-lactate cytochrome-c oxidoreductase, cyb2) gene deletion cassette uses the deletion vector pUC57-ura3HA-ldh as a template, and the primers of SEQ ID NOs: 29 and 30 The set was amplified by PCR using the primers. In SEQ ID NO: 29 and SEQ ID NO: 30, sequences 1-45 are homologous recombination with the homologous sequence of S.

In addition, the cassette for deletion of the glycerol-3-phosphate dehydrogenase1 (gpd1) gene uses the deletion vector pUC57-ura3HA-ldh as a template, and the primer sets of SEQ ID NOs: 33 and 34 are primers. Amplified by PCR used as. Sequences 1-50 in SEQ ID NO: 34 represent a portion to be replaced with the gpd1 gene by recombination with the homologous sequence of the chromosome of S. cerevisiae.

(1.2.2.) S. cerevisiae CEN . Preparation of mutant strains of PK2 -1D ( Δadh1:: ldh , mhpF)

First, S. cerevisiae CEN . In order to replace the pdc1 gene with the ldh gene from PK2 -1D ( Δadh1::ldh, mhpF), the "pdc1 deletion cassette" produced in (1.2.1) was obtained from S.cerevisiae CEN . PK2 -1D ( Δadh1::ldh,mhpF) was introduced by heat shock transformation, and after heat shock, the pdc1 gene on the chromosome was ldh by incubating at 30° C. in Ura-drop out medium, a selection marker, for 3 days. Replaced by gene. In order to analyze the genotype of the prepared strain, the genome of the prepared strain was used as a template, and the deletion of the pdc1 gene was confirmed by PCR used as a primer set of SEQ ID NOs: 27 and 28.

As a result, S. cerevisiae CEN . It was confirmed that the PK2 -1D ( Δadh1::ldh, mhpF, Δpdc1::ldh) strain was prepared.

Next, S.cerevisiae CEN . In order to replace the cyb2 gene with ldh from the PK2 -1D ( Δadh1::ldh, mhpF) strain, the "cyb2 deletion cassette" produced in (1.2.1) was used in the strain by heat shock transformation. It was introduced and cultured for 3 days at 30° C. in Ura-drop out medium, which is a selection marker, after heat shock, and the cyb2 gene on the chromosome was replaced with the ldh gene. In order to analyze the genotype of the prepared strain, it was confirmed whether the cyb2 gene was deleted by PCR using the genome of the prepared strain as a template and used as a primer set of SEQ ID NOs: 31 and 32.

As a result, S. cerevisiae CEN . It was confirmed that the PK2 -1D ( Δadh1::ldh, mhpF, Δcyb2::ldh) strain was prepared.

Next, S.cerevisiae CEN . In order to replace the gpd1 gene with ldh from the PK2 -1D ( Δadh1::ldh, mhpF) strain, the "gpd1 deletion cassette" produced in (1.2.1) was used in the strain by heat shock transformation. After the introduction, after heat shock, the gpd1 gene on the chromosome was replaced with the ldh gene by incubating at 30° C. in Ura-drop out medium, which is a selection marker, for 3 days. In order to analyze the genotype of the prepared strain, the genome of the prepared strain was used as a template, and the deletion of the gpd1 gene was confirmed by PCR used as a primer set of SEQ ID NOs: 35 and 36.

As a result, S. cerevisiae CEN . It was confirmed that the PK2 -1D ( Δadh1::ldh, mhpF, Δgpd1:ldh) strain was prepared.

Next, S.cerevisiae CEN . In order to replace the cyb2 gene with ldh from the PK2 -1D ( Δadh1::ldh, mhpF, Δpdc1::ldh) strain, the "cyb2 deletion cassette" produced in (1.2.1) was used for heat shock transformation (heat shock transformation), and after heat shock, the cyb2 gene on the chromosome was replaced with the ldh gene by incubating at 30° C. in Ura-drop out medium, which is a selection marker, for 3 days. In order to analyze the genotype of the prepared strain, it was confirmed whether the cyb2 gene was deleted by PCR using the genome of the prepared strain as a template and used as a primer set of SEQ ID NOs: 31 and 32.

As a result, S. cerevisiae CEN . It was confirmed that PK2 -1D ( Δadh1::ldh, mhpF, Δpdc1::ldh, Δcyb2::ldh) strain was prepared.

Next, S.cerevisiae CEN . In order to replace the gpd1 gene with ldh from the PK2 -1D ( Δadh1::ldh, mhpF, Δpdc1::ldh) strain, the "gpd1 deletion cassette" produced in (1.2.1) was used for heat shock transformation (heat shock transformation), and after heat shock, the gpd1 gene on the chromosome was replaced with the ldh gene by incubating for 3 days at 30° C. in Minimal Ura-drop out medium, which is a selection marker. In order to analyze the genotype of the prepared strain, the genome of the prepared strain was used as a template, and the deletion of the gpd1 gene was confirmed by PCR used as a primer set of SEQ ID NOs: 35 and 36.

As a result, S. cerevisiae CEN . It was confirmed that PK2 -1D ( Δadh1::ldh, mhpF, Δpdc1:ldh, Δgpd1:ldh) strain was prepared.

Next, S.cerevisiae CEN . In order to replace the gpd1 gene with ldh from the PK2 -1D (Δadh1:: ldh, mhpF, Δcyb2::ldh) strain, the "gpd1 deletion cassette" produced in (1.2.1) was used for heat shock transformation (heat shock transformation), and after heat shock, the gpd1 gene on the chromosome was replaced with the ldh gene by incubating for 3 days at 30° C. in Minimal Ura-drop out medium, which is a selection marker. In order to analyze the genotype of the prepared strain, the genome of the prepared strain was used as a template, and the deletion of the gpd1 gene was confirmed by PCR used as a primer set of SEQ ID NOs: 35 and 36.

As a result, S. cerevisiae CEN . It was confirmed that the PK2 -1D ( Δadh1::ldh,mhpF,Δcyb2::ldh,Δgpd1::ldh) strain was prepared.

Next, S.cerevisiae CEN . In order to replace the gpd1 gene with ldh from the PK2 -1D ( Δadh1::ldh, mhpF, Δpdc1::ldh, Δcyb2::ldh) strain, the "gpd1 deletion cassette" produced in (1.2.1) was added to the strain. It was introduced by heat shock transformation, and after heat shock, the gpd1 gene on the chromosome was replaced with the ldh gene by incubating at 30° C. in Ura-drop out medium, which is a selection marker, for 3 days. In order to analyze the genotype of the prepared strain, the genome of the prepared strain was used as a template, and the deletion of the gpd1 gene was confirmed by PCR used as a primer set of SEQ ID NOs: 35 and 36.

As a result, S. cerevisiae CEN . It was confirmed that PK2 -1D ( Δadh1::ldh, mhpF, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh) strains were prepared.

(1.2.3) MhpF Preparation of a control strain in which the gene was not introduced

In addition, the following control strain was prepared through the same procedure as (1.2.2.), except that the same starting strain was used and the MhpF gene was not introduced:

S. cerevisiae CEN . PK2 -1D( Δadh1::ldh,Δpdc1::ldh,Δcyb2::ldh,Δgpd1::ldh)

S. cerevisiae CEN . PK2 -1D( Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh)

S. cerevisiae CEN . PK2 -1D( Δadh1::ldh, Δpdc1::ldh, Δgpd1::ldh)

S. cerevisiae CEN . PK2 -1D( Δadh1::ldh, Δcyb2::ldh, Δgpd1::ldh)

S. cerevisiae CEN . PK2 -1D( Δadh1::ldh, Δpdc1::ldh)

S. cerevisiae CEN . PK2 -1D( Δadh1::ldh, Δcyb2::ldh)

S. cerevisiae CEN . PK2 -1D( Δadh1::ldh, Δgpd1::ldh)

(2) mhpF And EutE Preparation of gene expression strain

(2.1) S. cerevisiae CEN . PK2 -1D (ㅿ pdc1 :: ldh ,ㅿ cyb2 :: ldh ,ㅿ gpd1 :: ldh ,ㅿadh1::ldh, mhpF ) Of the manufacture

(2.1.1) mhpF Construction and introduction of vectors to insert

S. cerevisiae CEN . To PK2 -1D (DELTA pdc1 :: ldh, DELTA cyb2 :: ldh, DELTA gpd1 :: ldh, DELTA adh1 :: ldh) from the path to enhance the transition from acetaldehyde to acetyl-CoA, it was inserted MhpF gene.

The MhpF gene of pJ1214-mhpF and the'HIS3 cassette' were ligated to the'pUC19 vector' (NEB, N3041) using a SalI restriction enzyme, respectively, to prepare a pUC19-His-MhpF vector (SEQ ID NO: 59). The HIS3 cassette is an amplification product obtained by amplifying pRS413 (ATCC8758) by PCR using a primer of SEQ ID NO: 62 and a primer of SEQ ID NO: 63 as a template. In the pUC19-His-MhpF vector, mhpF is expressed under the GPD promoter.

The prepared pUC19-His-MhpF vector was used as a template, and a cassette for mhpF insertion was obtained by PCR using primers of SEQ ID NOs: 64 and 65 to which the leu2 homologous recombination sequence and promoter were bound as a primer. Here, the homologous recombination leu2 is a region that cannot express a functional protein because it has been mutated in the parent strain.

(2.1.2) S. cerevisiae CEN . PK2 -1D (ㅿ pdc1 :: ldh ,ㅿ cyb2 :: ldh , ㅿ gpd1 :: ldh ,ㅿadh1::ldh, mhpF ) Of the manufacture

(2.1.1) the mhpF insertion cassette produced in S. cerevisiae CEN . PK2 -1D was introduced (DELTA pdc1 :: ldh, DELTA cyb2 :: ldh, DELTA gpd1 :: ldh, DELTA adh1 :: ldh).

Introduction was performed by general heat shock transformation, and after transduction histidine drop out medium (Yeast nitrogen base without amino acids (Sigma-Aldrich: Cat. no. Y0626) 6.7 g/L, and Yeast synthetic drop) Cells were cultured in -out without histidine (Sigma-Aldrich: Cat. no. Y1751) 1.9 g/L, glucose 2 (w/v)%) so that the Leu2 ORF on the chromosome was replaced with the cassette.

In order to confirm the introduction of the mhpF gene into the Leu2 locus for the resulting strain, gene deletion and gene introduction were confirmed by PCR using the genome of the cell as a template and primer sets of SEQ ID NOs: 66 and 77 as primers. . As a result, S. cerevisiae CEN . The PK2 -1D (DELTA pdc1 :: ldh, DELTA cyb2 :: ldh, DELTA gpd1 :: ldh, DELTA adh1 :: ldh, mhpF) was prepared.

(2.2) S. cerevisiae CEN . PK2 -1D (ㅿ pdc1 :: ldh ,ㅿ cyb2 :: ldh , ㅿ gpd1 :: ldh ,ㅿadh1::ldh, mhpF , ㅿ ald6 ) Of the manufacture

(2.2.1) ald6 Creation and introduction of vectors for fruiting

Acetaldehyde dehydrogenase 6 (acetaldehyde dehydrogenase 6: ald6) gene deletion cassette is a deletion vector pUC57-ura3HA as a template, SEQ ID NO: 68 And the primer set of 69 was amplified by PCR using a primer. The sequences of SEQ ID NOs: 68 and 69 are recombined with the homologous sequence of the chromosome of S.

(2.2.2) S. cerevisiae CEN . PK2 -1D (ㅿ pdc1 :: ldh ,ㅿ cyb2 :: ldh , ㅿ gpd1 :: ldh ,ㅿa d h1:: ldh , mhpF , ㅿ ald6 ) Strain production

S. cerevisiae CEN . In order to delete the ald6 gene from the PK2 -1D ( Δadh1::ldh, mhpF, Δpdc1:ldh, Δcyb2:ldh, Δgpd1:ldh) strain, the "ald6 deletion cassette" produced in (2.2.1) was added to the strain. It was introduced by heat shock transformation and cultured for 3 days at 30° C. in Minimal Ura-drop out medium, which is a selective marker, after heat shock, to delete the ald6 gene on the chromosome. In order to analyze the genotype of the prepared strain, the genome of the prepared strain was used as a template, and it was confirmed whether the ald6 gene was deleted by PCR used as a primer set of SEQ ID NOs: 70 and 71.

As a result, S. cerevisiae CEN . It was confirmed that PK2 -1D ( Δadh1::ldh, mhpF, Δpdc1:ldh, Δcyb2:ldh, Δgpd1:ldh, Δald6) strain was prepared.

(2.3) S. cerevisiae CEN . PK2 -1D (ㅿ pdc1 :: ldh ,ㅿ cyb2 :: ldh ,ㅿ gpd1 :: ldh ,ㅿadh1::ldh, mhpF ,ㅿ ald6 , EutE ) Of the manufacture

(2.3.1) EutE Creation and introduction of vectors to introduce

(2.3.1.1). leaven Purpose ( dual function ) Overexpression vector pCS - Ex1 Making of

PCR was performed using a primer combination of SEQ ID NO: 72 and SEQ ID NO: 73 from pRS426GPD vector, which is widely used as a yeast overexpression vector, to obtain a 689 bp DNA fragment (GPD promoter). This DNA fragment was mixed with a pCtB1 vector treated with KpnI (Genbank Accession Number KJ922019) and cloned using an In-fusion kit (Clonetech, cat. 639650), and then the TOP10 strain (Invitrogen, cat. C4040-06). In colonies formed by plating and culturing the strain on LB agar medium (Bacto Tryptone 10g/L, Yeast Extract 5g/L, NaCl 10g/L, and Bacto Agar 15g/L) containing kanamycin at a concentration of 50ug/ml after introduction. Plasmid DNA was isolated, and among them, a plasmid having the same plasmid sequence as SEQ ID NO: 74 was identified. As a result, a yeast two-purpose overexpression vector, pCS-Ex1 vector was obtained. Here, this purpose refers to the purpose of expression after gene genome insertion and the purpose of gene expression on a vector.

(2.3.1.2) produced in E. coli, yeast attention ever eutE gene overexpression vector

From the genomic DNA of the E. coli MG1655 strain, PCR was performed using a primer combination of SEQ ID NO: 75 and SEQ ID NO: B76 to obtain a 1447 bp DNA fragment, that is, the EutE gene. This DNA fragment was mixed with the pCS-Ex1 vector treated with KpnI and SacI and cloned using an In-fusion kit (Clonetech cat. 639650), and then the TOP10 strain (Invitrogen cat. C4040-), an Escherichia coli strain for cloning, through a conventional method (Invitrogen cat. C4040- 06). After introduction, the strain was plated on LB agar medium containing kanamycin at a concentration of 50 ug/ml, and the plasmid DNA was isolated from colonies formed by cultivation, and a plasmid having the same plasmid sequence as SEQ ID NO: 77 was identified. As a result, an MD1040 vector, which is a yeast dual purpose Escherichia coli eutE gene overexpression vector, was obtained.

(2.3.2) Production of yeast overexpressing E. coli eutE gene

PCR was performed using a primer combination of SEQ ID NO: 78 and SEQ ID NO: 89 from the prepared MD1040 vector to obtain a DNA fragment of 3985 bp. This piece is S. cerevisiae CEN . PK2 -1D (DELTA pdc1 :: ldh, DELTA cyb2 :: ldh, DELTA gpd1 :: ldh, DELTA adh1 :: ldh, mhpF, DELTA ald6) in SD-URA minimal medium agar did not contain the uracil was introduced into the Medium (Yeast nitrogen base without amino acids (Sigma-Aldrich: Cat. no. Y0626) 6.7 g/L, Yeast synthetic drop-out without uracil (Sigma-Aldrich: Cat. no. Y1501) 1.9 g/L, D-glucose 20g/L, and Bacto Agar 20g/L). Among the colonies formed after 3 days, colonies capable of identifying a 4357bp DNA fragment were selected when PCR was performed using a primer combination of SEQ ID NO: 80 and SEQ ID NO: 81. In the genomic DNA of the natural strain, a DNA fragment of 2300 bp can be obtained by performing PCR using a primer combination of SEQ ID NO: 80 and SEQ ID NO: 81. The obtained clones were inoculated in YPD medium (Bacto Peptone 20g/L, Yeast Extract 10g/L, and D-glucose 20g/L) and cultured by stirring at 30° C. at 230 rpm, and then reverse selection medium containing 5-FOA ( Yeast nitrogen base without amino acids (Sigma-Aldrich: Cat. no. Y0626) 6.7 g/L, Yeast synthetic drop-out without uracil(Sigma-Aldrich: Cat. no. Y1501) 1.9 g/L, Uracil 0.1 g/L , D-glucose 20g/L, 5-fluoroorotic acid (5-FOA) 1g/L, and Bacto Agar 20g/L). Among the colonies formed after 3 days, colonies capable of identifying a 2963 bp DNA fragment were selected when PCR was performed using a combination of SEQ ID NO. C3 and SEQ ID NO. C4 primers.

(3) of the produced strain Lactate Confirmation of production characteristics

Each S. cerevisiae strain prepared in (1) and (2) was cultured in a medium to confirm lactate production. Specifically, 500ml shake-flask containing 5% by weight of glucose (Minimal Ura drop-out media) [Yeast nitrogen base without amino acids (Sigma-Aldrich: Cat. no. Y0626) 6.7 g/L and Yeast Synthetic drop-out without uracil(Sigma-Aldrich: Cat. no. Y1501) 1.9 g/L] or 50 ml of YPD medium were inoculated with fresh cells obtained from the culture plate so _{that the OD 600 value was 1, and 30°C} The seeds were incubated under aerobic conditions for 10 hours while stirring at 240 rpm. Thereafter, the seed culture was added to a 2L fermenter containing 1L of the same medium to be 1.0 OD ₆₀₀ , and stirred at 350rmp while injecting air at a flow rate of 100ml/min at 30℃ for 40 hours. During incubation.

Acetaldehyde generated during cultivation collects volatilized acetaldehyde using a cold-water trap, and GS-MS (Agilent 7890/5973 GC-MS equipped with a 30m length, 0.25mm id, 0.25 um film thickeness, fused silica capillary) column (DB-5MS, Agilent)). _{In addition, the OD 600} value was measured for cell growth during culture using a spectrophotometer. In addition, the culture supernatant from the flask culture and fermentation tank (bioreactor) was analyzed using high performance liquid chromatography (HPLC). After filtering the culture supernatant using a 0.45um syringe filter, L-lactate, glucose, acetate, glycerol, and ethanol were added to the HPLC instrument (Waters e2695 Separation Module instrument equipped with a Waters 2414 Differential Refractometer and a Waters 2998 Photodiode Array. Detector (Waters, Milford, MA)). The HPLC column was an Aminex HPX-87H Organic Acid Analysis Column (300mmx7.8mm; Bio-Rad) equilibrated with 2.5mM _{H 2} SO _{4 in water at a flow rate of 0.5 mL/min at 60°C.}

In control experiments, wild-type S. :ldh) was used.

Table 1 shows the lactate and acetaldehyde measured after 10 hours of aerobic culture and 40 hours of microaerobic culture as a seed culture of the strain prepared in (1) above in Minimal Ura drop-out media. The cell concentration at the time of measurement was 8.0 OD ₆₀₀ .

Strain Lactate (g/l) Acetaldehyde (g/l) Control 42.1 3.76 Control (+mhpF) 48.9 2.47

In Table 1, the control group represents S.cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh). As shown in Table 1, S. cerevisiae containing the foreign mhpF gene increased lactate production by 16.5% compared to the control group, and the production of acetaldehyde, a toxic substance, decreased by 34.3%, compared to the strain that did not (control). In addition, acetaldehyde, a toxic substance, decreased by about 34.31%. Therefore, S. cerevisiae containing the foreign mhpF gene has unexpectedly remarkably high lactate productivity compared to strains that do not (control), and can be effectively used to produce lactate.

Table 2 shows the lactate measured after 10 hours of aerobic culture and 40 hours of microaerobic culture as a seed culture of the strain prepared in (2) above in YPD medium. The cell concentration at the time of measurement was 8.0 OD ₆₀₀ .

Strain Lactate (g/l) Lactate yield (%) Control 103 82.4 Control (+mhpF) 106 83.9 Control (+mhpF,Δald6) 113 86.0 Control (+mhpF,Δald6,+EutE) 136 90.8

In Table 2, the control group represents S.cerevisiae CEN.PK2-1D (Δadh1::ldh, Δpdc1::ldh, Δcyb2::ldh, Δgpd1::ldh). As shown in Table 2, S.cerevisiae containing the foreign mhpF gene, S.cerevisiae containing the foreign mhpF gene and having the ald6 gene deleted, and S.cerevisiae containing the foreign mhpF gene and the EutE gene and having the ald6 gene deleted, are: Compared to the control group, lactate production increased by 2.9%, 9.7%, and 32%, respectively. Therefore, S. cerevisiae including the foreign mhpF gene and/or the foreign EutE gene has unexpectedly and significantly higher lactate productivity compared to the strain (control) that does not, and can be effectively used to produce lactate.

3 is a diagram showing some metabolic pathways related to conversion of pyruvate to lactate generated through a corresponding process according to an embodiment. In FIG. 3, MhpF _EC represents E. coli (EC)-derived exogenous A-ALD.

<110> Samsung Electronics Co. Ltd <120> A microorganism producing Lactate comprising Acetaldehyde dehydrogenase originated from E.coli <130> PN106995KR <160> 81 <170> KopatentIn 2.0 <210> 1 <211> 316 <212> PRT <213> Escherichia coli <400> 1 Met Ser Lys Arg Lys Val Ala Ile Ile Gly Ser Gly Asn Ile Gly Thr 1 5 10 15 Asp Leu Met Ile Lys Ile Leu Arg His Gly Gln His Leu Glu Met Ala 20 25 30 Val Met Val Gly Ile Asp Pro Gln Ser Asp Gly Leu Ala Arg Ala Arg 35 40 45 Arg Met Gly Val Ala Thr Thr His Glu Gly Val Ile Gly Leu Met Asn 50 55 60 Met Pro Glu Phe Ala Asp Ile Asp Ile Val Phe Asp Ala Thr Ser Ala 65 70 75 80 Gly Ala His Val Lys Asn Asp Ala Ala Leu Arg Glu Ala Lys Pro Asp 85 90 95 Ile Arg Leu Ile Asp Leu Thr Pro Ala Ala Ile Gly Pro Tyr Cys Val 100 105 110 Pro Val Val Asn Leu Glu Ala Asn Val Asp Gln Leu Asn Val Asn Met 115 120 125 Val Thr Cys Gly Gly Gln Ala Thr Ile Pro Met Val Ala Ala Val Ser 130 135 140 Arg Val Ala Arg Val His Tyr Ala Glu Ile Ile Ala Ser Ile Ala Ser 145 150 155 160 Lys Ser Ala Gly Pro Gly Thr Arg Ala Asn Ile Asp Glu Phe Thr Glu 165 170 175 Thr Thr Ser Arg Ala Ile Glu Val Val Val Gly Gly Ala Ala Lys Gly Lys 180 185 190 Ala Ile Ile Val Leu Asn Pro Ala Glu Pro Pro Leu Met Met Arg Asp 195 200 205 Thr Val Tyr Val Leu Ser Asp Glu Ala Ser Gln Asp Asp Ile Glu Ala 210 215 220 Ser Ile Asn Glu Met Ala Glu Ala Val Gln Ala Tyr Val Pro Gly Tyr 225 230 235 240 Arg Leu Lys Gln Arg Val Gln Phe Glu Val Ile Pro Gln Asp Lys Pro 245 250 255 Val Asn Leu Pro Gly Val Gly Gln Phe Ser Gly Leu Lys Thr Ala Val 260 265 270 Trp Leu Glu Val Glu Gly Ala Ala His Tyr Leu Pro Ala Tyr Ala Gly 275 280 285 Asn Leu Asp Ile Met Thr Ser Ser Ala Leu Ala Thr Ala Glu Lys Met 290 295 300 Ala Gln Ser Leu Ala Arg Lys Ala Gly Glu Ala Ala 305 310 315 <210> 2 <211> 951 <212> DNA <213> Escherichia coli <400> 2 atgagtaagc gtaaagtcgc cattatcggt tctggcaaca ttggtaccga tctgatgatt 60 aaaattttgc gtcacggtca gcatctggag atggcggtga tggttggcat tgatcctcag 120 tccgacggtc tggcgcgcgc cagacgtatg ggcgtcgcca ccacccatga aggggtgatc 180 ggactgatga acatgcctga atttgctgat atcgacattg tatttgatgc gaccagcgcc 240 ggtgctcatg tgaaaaacga tgccgcttta cgcgaagcga aaccggatat tcgcttaatt 300 gacctgacgc ctgctgccat cggcccttac tgcgtgccgg tggttaacct cgaggcgaac 360 gtcgatcaac tgaacgtcaa catggtcacc tgcggcggcc aggccaccat tccaatggtg 420 gcggcagttt cacgcgtggc gcgtgttcat tacgccgaaa ttatcgcttc tatcgccagt 480 aaatctgccg gacctggcac gcgtgccaat atcgatgaat ttacggaaac cacttcccga 540 gccattgaag tggtgggcgg cgcggcaaaa gggaaggcga ttattgtgct taacccagca 600 gagccaccgt tgatgatgcg tgacacggtg tatgtattga gcgacgaagc ttcacaagat 660 gatatcgaag cctcaatcaa tgaaatggct gaggcggtgc aggcttacgt accgggttat 720 cgcctgaaac agcgcgtgca gtttgaagtt atcccgcagg ataaaccggt caatttaccg 780 ggcgtggggc aattctccgg actgaaaaca gcggtctggc tggaagtcga aggcgcagcg 840 cattatctgc ctgcctatgc gggcaacctc gacattatga cttccagtgc gctggcgaca 900 gcggaaaaaa tggcccagtc actggcgcgc aaggcaggag aagcggcatg a 951 <210> 3 <211> 954 <212> DNA <213> Artificial Sequence <220> <223> S. cevisiae optimized MhpF <400> 3 atgtcaaagc gaaaagtagc tatcataggt tcaggtaata ttggtactga tttgatgatc 60 aaaatcctga gacatggcca gcacttggag atggccgtca tggttggtat cgacccacaa 120 tccgatggct tagctagagc taggagaatg ggtgttgcca caactcacga aggggttatt 180 ggcttaatga acatgccaga atttgcagac atcgatatag tttttgatgc tactagtgca 240 ggggcacatg tgaaaaacga cgcggcttta agagaagcca agccagatat tagattaatt 300 gatcttaccc ctgctgctat aggtccttac tgcgttcctg tagttaacct tgaagctaat 360 gtggaccagt tgaacgtgaa tatggttaca tgtggtggcc aagctaccat accaatggtt 420 gctgctgtct ctagagtggc cagagtacat tatgccgaga tcattgcgtc tatcgcatct 480 aagtctgccg gtcctggaac aagggctaac atcgatgagt tcactgagac aacctctaga 540 gctatcgaag tagtaggagg cgcagcaaaa ggtaaagcga tcattgtttt gaatcctgcc 600 gaaccacctt tgatgatgag agatacggtc tacgtgctat cagatgaagc ttcccaggat 660 gacattgaag ctagcattaa tgagatggca gaagccgttc aagcatacgt gccaggatat 720 agactcaaac aaagagtcca atttgaggtc attccacaag acaagccagt taatctccca 780 ggggtcggtc aattctcagg actaaaaact gctgtttggt tagaagtaga aggagctgct 840 cattacctac cagcctacgc cggtaatttg gatataatga catcttccgc tcttgcaaca 900 gcagaaaaga tggcacaaag tctggcccgt aaggcaggag aagcggcata ataa 954 <210> 4 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> primer 1 <400> 4 acaatatttc aagctatacc aagcatacaa tcaactatct catatacaat gggccgcaaa 60 ttaaagcctt cgagc 75 <210> 5 <211> 75 <212> DNA <213> Artificial Sequence <220> <223> primer 2 <400> 5 aatcataaga aattcgctta tttagaagtg tcaacaacgt atctaccaac gactaaaggg 60 aacaaaagct ggagc 75 <210> 6 <211> 332 <212> PRT <213> Pelodiscus sinensis japonicus <400> 6 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 Pro 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> 7 <211> 999 <212> DNA <213> Pelodiscus sinensis japonicus <400> 7 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> 8 <211> 5999 <212> DNA <213> Artificial Sequence <220> <223> pJ1214-mhpF vector <400> 8 atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt ttgttttaca 60 aaaatgaagc acagattctt cgttggtaaa atagcgcttt cgcgttgcat ttctgttctg 120 taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg catttttgtt 180 ctacaaaatg aagcacagat gcttcgttca ggtggcactt ttcggggaaa tgtgcgcgga 240 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 300 ccctgatatt ggtcagaatt ggttaattgg ttgtaacact gacccctatt tgtttatttt 360 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 420 aatattgaaa aaggaagaat atgagtattc aacatttccg tgtcgccctt attccctttt 480 ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 540 ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 600 tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 660 tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 720 actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 780 gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 840 acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 900 gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 960 acgagcgtga caccacgatg cctgtagcga tggcaacaac gttgcgcaaa ctattaactg 1020 gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 1080 ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatccg 1140 gagccggtga gcgtggttct cgcggtatca tcgcagcgct ggggccagat ggtaagccct 1200 cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1260 agatcgctga gataggtgcc tcactgatta agcattggta actcatgacc aaaatccctt 1320 aacgtgagtt acgcgcgcgt cgttccactg agcgtcagac cccgtagaaa agatcaaagg 1380 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc 1440 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac 1500 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttagccca 1560 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt 1620 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc 1680 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg 1740 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc 1800 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac 1860 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct 1920 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc 1980 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt 2040 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac 2100 cgctcggggt cgtgcaggta tagcttcaaa atgtttctac tcctttttta ctcttccaga 2160 ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca cccaagcaca gcatactaaa 2220 tttcccctct ttcttcctct agggtgtcgt taattacccg tactaaaggt ttggaaaaga 2280 aaaaagtgac cgcctcgttt ctttttcttc gtcgaaaaag gcaataaaaa tttttatcac 2340 gtttcttttt cttgaaaatt tttttttttg atttttttct ctttcgatga cctcccattg 2400 atatttaagt taataaacgg acttcaattt ctcaagtttc agtttcattt ttcttgttct 2460 attacaactt tttttacttc ttgctcatta gaaagaaagc atagcaatct aatctaagtt 2520 taaaatgtca aagcgaaaag tagctatcat aggttcaggt aatattggta ctgatttgat 2580 gatcaaaatc ctgagacatg gccagcactt ggagatggcc gtcatggttg gtatcgaccc 2640 acaatccgat ggcttagcta gagctaggag aatgggtgtt gccacaactc acgaaggggt 2700 tattggctta atgaacatgc cagaatttgc agacatcgat atagtttttg atgctactag 2760 tgcaggggca catgtgaaaa acgacgcggc tttaagagaa gccaagccag atattagatt 2820 aattgatctt acccctgctg ctataggtcc ttactgcgtt cctgtagtta accttgaagc 2880 taatgtggac cagttgaacg tgaatatggt tacatgtggt ggccaagcta ccataccaat 2940 ggttgctgct gtctctagag tggccagagt acattatgcc gagatcattg cgtctatcgc 3000 atctaagtct gccggtcctg gaacaagggc taacatcgat gagttcactg agacaacctc 3060 tagagctatc gaagtagtag gaggcgcagc aaaaggtaaa gcgatcattg ttttgaatcc 3120 tgccgaacca cctttgatga tgagagatac ggtctacgtg ctatcagatg aagcttccca 3180 ggatgacatt gaagctagca ttaatgagat ggcagaagcc gttcaagcat acgtgccagg 3240 atatagactc aaacaaagag tccaatttga ggtcattcca caagacaagc cagttaatct 3300 cccaggggtc ggtcaattct caggactaaa aactgctgtt tggttagaag tagaaggagc 3360 tgctcattac ctaccagcct acgccggtaa tttggatata atgacatctt ccgctcttgc 3420 aacagcagaa aagatggcac aaagtctggc ccgtaaggca ggagaagcgg cataataaat 3480 catgtaatta gttatgtcac gcttacattc acgccctccc cccacatccg ctctaaccga 3540 aaaggaagga gttagacaac ctgaagtcta ggtccctatt tattttttta tagttatgtt 3600 agtattaaga acgttattta tatttcaaat ttttcttttt tttctgtaca gacgcgtgta 3660 cgcatgtaac attatactga aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt 3720 aatttgcggc ccctcacctg cacgcaaaaa gcttttcaat tcaattcatc attttttttt 3780 tattcttttt tttgatttcg gtttctttga aatttttttg attcggtaat ctccgaacag 3840 aaggaagaac gaaggaagga gcacagactt agattggtat atatacgcat atgtagtgtt 3900 gaagaaacat gaaattgccc agtattctta acccaactgc acagaacaaa aaccagcagg 3960 aaacgaagat aaatcatgtc gaaagctaca tataaggaac gtgctgctac tcatcctagt 4020 cctgttgctg ccaagctatt taatatcatg cacgaaaagc aaacaaactt gtgtgcttca 4080 ttggatgttc gtaccaccaa ggaattactg gagttagttg aagcattagg tcccaaaatt 4140 tgtttactaa aaacacatgt ggatatcttg actgattttt ccatggaggg cacagttaag 4200 ccgctaaagg cattatccgc caagtacaat tttttactct tcgaagatag aaaatttgct 4260 gacattggta atacagtcaa attgcagtac tctgcgggtg tatacagaat agcagaatgg 4320 gcagacatta cgaatgcaca cggtgtggtg ggcccaggta ttgttagcgg tttgaagcag 4380 gcggcagaag aagtaacaaa ggaacctaga ggccttttga tgttagcaga attgtcatgc 4440 aagggctccc tatctactgg agaatatact aagggtactg ttgacattgc gaaaagcgac 4500 aaagattttg ttatcggctt tattgctcaa agagacatgg gtggaagaga tgaaggttac 4560 gattggttga ttatgacacc cggtgtgggt ttagatgaca agggagatgc attgggtcaa 4620 cagtatagaa ccgtggatga tgttgtctct acaggatctg acattattat tgttggaaga 4680 ggactatttg caaagggaag ggatgctaag gtagagggtg aacgttacag aaaagcaggc 4740 tgggaagcat atttgagaag atgcggccag caaaactaaa aaactgtatt ataagtaaat 4800 gcatgtatac taaactcaca aattagagct tcaatttaat tatatcagtt attacccacg 4860 ctatgatcca atatcaaagg aaatgatagc attgaaggat gagactaatc caattgagga 4920 gtggcagcat atagaacagc taaagggtag tgctgaagga agcatacgat accccgcatg 4980 gaatgggata atatcacagg aggtactaga ctacctttca tcctacataa atagacgcat 5040 ataagtacgc atttaagcat aaacacgcac tatgccgttc ttctcatgta tatatatata 5100 caggcaacac gcagatatag gtgcgacgtg aacagtgagc tgtatgtgcg cagctcgcgt 5160 tgcattttcg gaagcgctcg ttttcggaaa cgctttgaag ttcctattcc gaagttccta 5220 ttctctagaa agtataggaa cttcagagcg cttttgaaaa ccaaaagcgc tctgaagtcg 5280 cactttcaaa aaaccaaaaa cgcaccggac tgtaacgagc tactaaaata ttgcgaatac 5340 cgcttccaca aacattgctc aaaagtatct ctttgctata tatctctgtg ctatatccct 5400 atataaccta cccatccacc tttcgctcct tgaacttgca tctaaactcg acctctacat 5460 tttttatgtt tatctctagt attactcttt agacaaaaaa attgtagtaa gaactattca 5520 tagagtgaat cgaaaacaat acgaaaatgt aaacatttcc tatacgtagt atatagagac 5580 aaaatagaag aaaccgttca taattttctg accaatgaag aatcatcaac gctatcactt 5640 tctgttcaca aagtatgcgc aatccacatc ggtatagaat ataatcgggg atgcctttat 5700 cttgaaaaaa tgcacccgca gcttcgctag taatcagtaa acgcgggaag tggagtcagg 5760 ctttttttat ggaagagaaa atagacacca aagtagcctt cttctaacct taacggacct 5820 acagtgcaaa aagttatcaa gagactgcat tatagagcgc acaaaggaga aaaaaagtaa 5880 tctaagatgc tttgttagaa aaatagcgct ctcgggatgc atttttgtag aacaaaaaag 5940 aagtatagat tctttgttgg taaaatagcg ctctcgcgtt gcatttctgt tctgtaaaa 5999 <210> 9 <211> 292 <212> DNA <213> Artificial Sequence <220> <223> CCW12 promoter <400> 9 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 <210> 10 <211> 955 <212> DNA <213> Artificial Sequence <220> <223> PDC1 promoter <400> 10 agggtagcct ccccataaca taaactcaat aaaatatata gtcttcaact tgaaaaagga 60 acaagctcat gcaaagaggt ggtacccgca cgccgaaatg catgcaagta acctattcaa 120 agtaatatct catacatgtt tcatgagggt aacaacatgc gactgggtga gcatatgttc 180 cgctgatgtg atgtgcaaga taaacaagca agacagaaac taacttcttc ttcatgtaat 240 aaacacaccc cgcgtttatt tacctatctc taaacttcaa caccttatat cataactaat 300 atttcttgag ataagcacac tgcacccata ccttccttaa aaacgtagct tccagttttt 360 ggtggttctg gcttccttcc cgattccgcc cgctaaacgc ataattttgt tgcctggtgg 420 catttgcaaa atgcataacc tatgcattta aaagattatg tatgctcttc tgacttttcg 480 tgtgatgagg ctcgtggaaa aaatgaataa tttatgaatt tgagaacaat tttgtgttgt 540 tacggtattt tactatggaa taatcaatca attgaggatt ttatgcaaat atcgtttgaa 600 tatttttccg accctttgag tacttttctt cataattgca taatattgtc cgctgcccgt 660 ttttctgtta gacggtgtct tgatctactt gctatcgttc aacaccacct tattttctaa 720 ctattttttt tttagctcat ttgaatcagc ttatggtgat ggcacatttt tgcataaacc 780 tagctgtcct cgttgaacat aggaaaaaaa aatatataaa caaggctctt tcactctcct 840 tggaatcaga tttgggtttg ttccctttat tttcatattt cttgtcatat tcttttctca 900 attattatct tctactcata acctcacgca aaataacaca gtcaaatcaa tcaaa 955 <210> 11 <211> 401 <212> DNA <213> Artificial Sequence <220> <223> TEF1 promoter <400> 11 atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg actccgcgca 60 tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc tttcttcctc 120 tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga ccgcctcgtt 180 tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt tcttgaaaat 240 tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt taataaacgg 300 tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc 360 ttgctcatta gaaagaaagc atagcaatct aatctaagtt t 401 <210> 12 <211> 798 <212> DNA <213> Artificial Sequence <220> <223> PGK1 promoter <400> 12 ctttcctctt tttattaacc ttaattttta ttttagattc ctgacttcaa ctcaagacgc 60 acagatatta taacatctgc ataataggca tttgcaagaa ttactcgtga gtaaggaaag 120 agtgaggaac tatcgcatac ctgcatttaa agatgccgat ttgggcgcga atcctttatt 180 ttggcttcac cctcatacta ttatcagggc cagaaaaagg aagtgtttcc ctccttcttg 240 aattgatgtt accctcataa agcacgtggc ctcttatcga gaaagaaatt accgtcgctc 300 gtgatttgtt tgcaaaaaga acaaaactga aaaaacccag acacgctcga cttcctgtct 360 tcctattgat tgcagcttcc aatttcgtca cacaacaagg tcctagcgac ggctcacagg 420 ttttgtaaca agcaatcgaa ggttctggaa tggcgggaaa gggtttagta ccacatgcta 480 tgatgcccac tgtgatctcc agagcaaagt tcgttcgatc gtactgttac tctctctctt 540 tcaaacagaa ttgtccgaat cgtgtgacaa caacagcctg ttctcacaca ctcttttctt 600 ctaaccaagg gggtggttta gtttagtaga acctcgtgaa acttacattt acatatatat 660 aaacttgcat aaattggtca atgcaagaaa tacatatttg gtcttttcta attcgtagtt 720 tttcaagttc ttagatgctt tctttttctc ttttttacag atcatcaagg aagtaattat 780 ctacttttta caacaaat 798 <210> 13 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer 3 <400> 13 cgagctcttc gcggccacct acgccgctat c 31 <210> 14 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 gctctagata ttgatatagt gtttaagcga at 32 <210> 15 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Primer 5 <400> 15 cggccatggc gggagctcgc atgcaag 27 <210> 16 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Primer 6 <400> 16 cgggatatca ctagtgagct cgctccgc 28 <210> 17 <211> 2321 <212> DNA <213> Artificial Sequence <220> <223> HPH cassette <400> 17 gccgggagag ctcgcatgca agtaacctat tcaaagtaat atctcataca tgtttcatga 60 gggtaacaac atgcgactgg gtgagcatat gttccgctga tgtgatgtgc aagataaaca 120 agcaaggcag aaactaactt cttcttcatg taataaacac accccgcgtt tatttaccta 180 tctctaaact tcaacacctt atatcataac taatatttct tgagataagc acactgcacc 240 cataccttcc ttaaaaacgt agcttccagt ttttggtggt tccggcttcc ttcccgattc 300 cgcccgctaa acgcatattt ttgttgcctg gtggcatttg caaaatgcat aacctatgca 360 tttaaaagat tatgtatgct cttctgactt ttcgtgtgat gaggctcgtg gaaaaaatga 420 ataatttatg aatttgagaa caattttgtg ttgttacggt attttactat ggaataatca 480 atcaattgag gattttatgc aaatatcgtt tgaatatttt tccgaccctt tgagtacttt 540 tcttcataat tgcataatat tgtccgctgc ccctttttct gttagacggt gtcttgatct 600 acttgctatc gttcaacacc accttatttt ctaactattt tttttttagc tcatttgaat 660 cagcttatgg tgatggcaca tttttgcata aacctagctg tcctcgttga acataggaaa 720 aaaaaatata taaacaaggc tctttcactc tccttgcaat cagatttggg tttgttccct 780 ttattttcat atttcttgtc atattccttt ctcaattatt attttctact cataacctca 840 cgcaaaataa cacagtcaaa tcctcgagat gaaaaagcct gaactcaccg cgacgtctgt 900 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg 960 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa 1020 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc 1080 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat 1140 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt 1200 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag 1260 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat 1320 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag 1380 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt 1440 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat 1500 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa 1560 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg 1620 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct 1680 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg 1740 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg 1800 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg 1860 acgccccagc actcgtccgg atcgggagat gggggaggct aactgaggat ccgtagatac 1920 attgatgcta tcaatcaaga gaactggaaa gattgtgtaa ccttgaaaaa cggtgaaact 1980 tacgggtcca agattgtcta cagattttcc tgatttgcca gcttactatc cttcttgaaa 2040 atatgcactc tatatctttt agttcttaat tgcaacacat agatttgctg tataacgaat 2100 tttatgctat tttttaaatt tggagttcag tgataaaagt gtcacagcga atttcctcac 2160 atgtagggac cgaattgttt acaagttctc tgtaccacca tggagacatc aaaaattgaa 2220 aatctatgga aagatatgga cggtagcaac aagaatatag cacgagccgc ggagcgagct 2280 cggccgcact agtgatatcc cgcggccatg gcggccggga g 2321 <210> 18 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer 7 <400> 18 tgctgtcttg ctatcaag 18 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer 8 <400> 19 caggaaagag ttactcaag 19 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer 9 <400> 20 atgtcaaagc gaaaagtagc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer 10 <400> 21 atttattatg ccgcttctcc 20 <210> 22 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Primer 11 <400> 22 gaaacagcta tgaccatg 18 <210> 23 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer 12 <400> 23 gacatgacga gctcgaattg ggtaccggcc gc 32 <210> 24 <211> 4173 <212> DNA <213> Artificial Sequence <220> <223> pUC57-Ura3HA vector <400> 24 gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc ttgtctgtaa 60 gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg 120 ggctggctta actatgcggc atcagagcag attgtactga gagtgcacca tatgcggtgt 180 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccattc gccattcagg 240 ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg ccagctggcg 300 aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc ccagtcacga 360 cgttgtaaaa cgacggccag tgaattcgag ctcggtacct cgcgaatgca tctagatatc 420 ggatcccgac gagctgcacc gcggtggcgg ccgtatcttt tacccatacg atgttcctga 480 ctatgcgggc tatccctatg acgtcccgga ctatgcagga tcctatccat atgacgttcc 540 agattacgct gctcagtgcg gccgcctgag agtgcaccat accacagctt ttcaattcaa 600 ttcatcattt tttttttatt cttttttttg atttcggttt ctttgaaatt tttttgattc 660 ggtaatctcc gaacagaagg aagaacgaag gaaggagcac agacttagat tggtatatat 720 acgcatatgt agtgttgaag aaacatgaaa ttgcccagta ttcttaaccc aactgcacag 780 aacaaaaacc tgcaggaaac gaagataaat catgtcgaaa gctacatata aggaacgtgc 840 tgctactcat cctagtcctg ttgctgccaa gctatttaat atcatgcacg aaaagcaaac 900 aaacttgtgt gcttcattgg atgttcgtac caccaaggaa ttactggagt tagttgaagc 960 attaggtccc aaaatttgtt tactaaaaac acatgtggat atcttgactg atttttccat 1020 ggagggcaca gttaagccgc taaaggcatt atccgccaag tacaattttt tactcttcga 1080 agacagaaaa tttgctgaca ttggtaatac agtcaaattg cagtactctg cgggtgtata 1140 cagaatagca gaatgggcag acattacgaa tgcacacggt gtggtgggcc caggtattgt 1200 tagcggtttg aagcaggcgg cagaagaagt aacaaaggaa cctagaggcc ttttgatgtt 1260 agcagaattg tcatgcaagg gctccctatc tactggagaa tatactaagg gtactgttga 1320 cattgcgaag agcgacaaag attttgttat cggctttatt gctcaaagag acatgggtgg 1380 aagagatgaa ggttacgatt ggttgattat gacacccggt gtgggtttag atgacaaggg 1440 agacgcattg ggtcaacagt atagaaccgt ggatgatgtg gtctctacag gatctgacat 1500 tattattgtt ggaagaggac tatttgcaaa gggaagggat gctaaggtag agggtgaacg 1560 ttacagaaaa gcaggctggg aagcatattt gagaagatgc ggccagcaaa actaaaaaac 1620 tgtattataa gtaaatgcat gtatactaaa ctcacaaatt agagcttcaa tttaattata 1680 tcagttatta ccctatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca 1740 tcaggaaatt gtagcggccg cgaatttgag cttatctttt acccatacga tgttcctgac 1800 tatgcgggct atccctatga cgtcccggac tatgcaggat cctatccata tgacgttcca 1860 gattacgcta ctagcggggg gcccggtgac gggcccgtcg actgcagagg cctgcatgca 1920 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt 1980 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc 2040 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc 2100 cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 2160 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 2220 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 2280 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 2340 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 2400 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 2460 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 2520 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 2580 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 2640 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 2700 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 2760 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 2820 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 2880 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 2940 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 3000 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 3060 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 3120 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 3180 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 3240 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 3300 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 3360 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 3420 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 3480 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 3540 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 3600 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 3660 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 3720 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 3780 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 3840 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 3900 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 3960 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 4020 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 4080 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 4140 atcacgaggc cctttcgtct cgcgcgtttc ggt 4173 <210> 25 <211> 62 <212> DNA <213> Artificial Sequence <220> <223> Primer 13 <400> 25 gcttataaaa ctttaactaa taattagaga ttaaatcgct taaggtttcc cgactggaaa 60 gc 62 <210> 26 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> Primer 14 <400> 26 ctactcataa cctcacgcaa aataacacag tcaaatcaat caaaccagtc acgacgttgt 60 aaaa 64 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer 15 <400> 27 ggacgtaaag ggtagcctcc 20 <210> 28 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer 16 <400> 28 gaagcggacc cagacttaag cc 22 <210> 29 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Primer 17 <400> 29 ccgaaatgat tccctttcct gcacaacacg agatctttca cgcatccagt cacgacgttg 60 taaaa 65 <210> 30 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> Primer 18 <400> 30 aaagtagcct taaagctagg ctataatcat gcatcctcaa attctaggtt tcccgactgg 60 aaagc 65 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer 19 <400> 31 cgcaagaacg tagtatccac atgcc 25 <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer 20 <400> 32 ggatatttac agaacgatgc g 21 <210> 33 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Primer 21 <400> 33 ccctatgtct ctggccgatc acgcgccatt gtccctcaga aacaaatcaa ccagtcacga 60 cgttgtaaaa 70 <210> 34 <211> 70 <212> DNA <213> Artificial Sequence <220> <223> Primer 22 <400> 34 tagaagcaac tgtgccgaca gcctctgaat gagtggtgtt gtaaccaccc aggtttcccg 60 actggaaagc 70 <210> 35 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Primer 23 <400> 35 tcaatgagac tgttgtcctc ctact 25 <210> 36 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer 24 <400> 36 tacatccttg tcgagccttg ggca 24 <210> 37 <211> 332 <212> PRT <213> Ornithorhynchus anatinus <400> 37 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 Pro 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> 38 <211> 332 <212> PRT <213> Tursiops truncatus <400> 38 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 Pro 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> 39 <211> 332 <212> PRT <213> Rattus norvegicus <400> 39 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 Pro 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> 40 <211> 1140 <212> DNA <213> Ornithorhynchus anatinus <400> 40 ttccaagatg gccggcgtca aggaacagct gatccagaat cttctcaaag aggagtacgc 60 ccctcaaaat aagatcaccg tggttggagt tggtgctgtg ggcatggcct gtgccatcag 120 catcttgatg aaggatttgg ctgatgagct cgcccttgtt gatgtcattg aggataagct 180 gaagggagaa atgatggatc ttcagcatgg cagccttttc ctcaggactc caaagatcgt 240 ctctggcaaa gactacagcg tgactgccaa ctccaagctg gttatcatca ccgccggggc 300 ccgtcagcag gagggagaga gccgtctgaa tctggtccag cgcaatgtca acatctttaa 360 attcatcatt cccaacgttg tcaagtacag ccccaactgc aagctgcttg tggtgtccaa 420 tccagtggat attttgacct acgtggcctg gaagatcagt ggcttcccca agaaccgagt 480 tatcggaagc ggctgcaatc tggattctgc ccgcttccgc tatctgatgg gagagaggct 540 gggcatccac tccacaagct gtcacggctg ggtcatcgga gaacacggag actctagtgt 600 tcccgtgtgg agcggggtga acgttgccgg tgtctctctg aagaacctgc accccgattt 660 gggaactgat gcagacaagg agcagtggaa ggatgttcat aagcaggtgg ttgacagtgc 720 ctacgaggtc atcaaactga agggctacac ctcctgggcc atcggcctct cggtagccga 780 tctggcagaa agcatcgtga agaatcttag gcgggtgcac cccatttcca ccatgattaa 840 gggcctgtac gggatcaaag atgaagtctt cctcagcgtc ccctgtgtct tgggccagaa 900 cggcatctcg gacgtggtga agataaccct gaagtccgag gaggaggctc atctgaagaa 960 gagcgcagac accctgtggg gaattcagaa ggaactgcag ttttaaggct tttcaacatc 1020 ctagctgtct actgggtaac ggtagttagg ggattgggta tcccccactt ttgaagtagg 1080 ttagctgtct actgggtaac ggtagttagg ggattgggta tcccccactt ttgaagtagg 1140 1140 <210> 41 <211> 1527 <212> DNA <213> Tursiops truncatus <400> 41 acgtgtactc ccgattcctt tcggttctaa gtccaatatg gcaactgtca aggatcagct 60 gattcagaat cttcttaagg aagaacatgt cccccagaat aagattacag tggttggtgt 120 tggtgctgtt ggcatggcct gtgccatcag tatcttaatg aaggacttgg cagatgaact 180 tgctcttgtt gatgtcatag aagacaaact gaagggagag atgatggatc tccaacatgg 240 cagccttttc cttagaacac caaaaatcgt ctctggcaaa gactatagtg tgacagcaaa 300 ctccaagctg gttattatca cagctggggc acgtcagcaa gagggagaaa gccgtcttaa 360 tttggtccaa cgtaatgtga acatctttaa attcatcgtt cctaatattg taaaatacag 420 cccacactgc aagttgcttg ttgtttccaa tccagtggat atcttgacct atgtggcttg 480 gaagataagc ggctttccca aaaaccgtgt tattggaagt ggttgcaatt tggattcagc 540 ccggttccgt tacctcatgg gggaaaggct gggagttcac ccattaagct gtcatggatg 600 gatccttggg gagcatggag actctagtgt gcctgtatgg agtggagtga atgttgctgg 660 tgtctccctg aagaatctgc accccgaatt aggcactgat gccgataagg aacattggaa 720 agcaattcac aaacaggtgg ttgacagtgc ttatgaggtg atcaaactga aaggctacac 780 atcctgggcc gttggactat ctgtggcaga tttggcagaa agtataatga agaatcttag 840 gcgggtgcat ccgatttcca ccatgattaa gggtttgtat ggaataaaag aggatgtctt 900 ccttagtgtt ccttgcatct tgggacagaa tggaatctca gatgttgtga aagtgactct 960 gactcctgag gaacaggcct gtttgaagaa gagtgcagat acactttggg ggatccagaa 1020 agagctgcag ttttaaagtc taatatcata ccacttcact gtctaggcta caataggatt 1080 ttagttggag gttgtgcata ttgtccttta tatctgatct gtgactaaag cagtaatgtt 1140 aagacagcct aggaaaaaca tcaatttcct aacattagca ataggaatgg ttcataaaac 1200 cctgcagctg tatcctgatg ctgcatggca cttatcttgt gttgtcctaa attggttcgt 1260 gtaaaatagt tctacttcct caagaggtac cactgacagt gttgcagatg ctgcagttgc 1320 ccttcaaacc agatgtgtat ttaactctgt gttatataac ttctggttcc tttagccaag 1380 atgcctagtc caactttttt ctctccaatt aatcacattc tgggattgat tataaatcca 1440 gtattgcatg tcttgtgcat aactgttcta aagaatctta ttttatgtac tatatgtatc 1500 agaatagtat acattgccat gtaatgt 1527 <210> 42 <211> 1609 <212> DNA <213> Rattus norvegicus <400> 42 gtgtgctgga gccactgtcg ccgatctcgc gcacgctact gctgctgctc gcccgtcgtc 60 ccccatcgtg cactaagcgg tcccaaaaga ttcaaagtcc aagatggcag ccctcaagga 120 ccagctgatt gtgaatcttc ttaaggaaga acaggtcccc cagaacaaga ttacagttgt 180 tggggttggt gctgttggca tggcttgtgc catcagtatc ttaatgaagg acttggctga 240 tgagcttgcc cttgttgatg tcatagaaga taagctaaag ggagagatga tggatcttca 300 gcatggcagc cttttcctta agacaccaaa aattgtctcc agcaaagatt atagtgtgac 360 tgcaaactcc aagctggtca ttatcaccgc gggggcccgt cagcaagagg gagagagccg 420 gctcaatttg gtccagcgaa acgtgaacat cttcaagttc atcattccaa atgttgtgaa 480 atacagtcca cagtgcaaac tgctcatcgt ctcaaaccca gtggatatct tgacctacgt 540 ggcttggaag atcagcggct tccccaaaaa cagagttatt ggaagtggtt gcaatctgga 600 ttcggctcgg ttccgttacc tgatgggaga aaggctggga gttcatccac tgagctgtca 660 cgggtgggtc ctgggagagc atggcgactc cagtgtgcct gtgtggagtg gtgtgaacgt 720 cgccggcgtc tccctgaagt ctctgaaccc gcagctgggc acggatgcag acaaggagca 780 gtggaaggat gtgcacaagc aggtggttga cagtgcatac gaagtgatca agctgaaagg 840 ttacacatcc tgggccattg gcctctccgt ggcagacttg gccgagagca taatgaagaa 900 ccttaggcgg gtgcatccca tttccaccat gattaagggt ctctatggaa tcaaggagga 960 tgtcttcctc agcgtcccat gtatcctggg acaaaatgga atctcagatg ttgtgaaggt 1020 gacactgact cctgacgagg aggcccgcct gaagaagagt gcagataccc tctggggaat 1080 ccagaaggag ctgcagttct aaagtcttcc cagtgtccta gcacttcact gtccaggctg 1140 cagcagggtt tctatggaga ccacgcactt ctcatctgag ctgtggttag tccagttggt 1200 ccagttgtgt tgaggtggtc tgggggaaat ctcagttcca cagctctacc ctgctaagtg 1260 gtacttgtgt agtggtaacc tggttagtgt gacaatccca ctgtctccaa gacacactgc 1320 caactgcatg caggctttga ttaccctgtg agcctgctgc attgctgtgc tacgcaccct 1380 caccaaacat gcctaggcca tgagttccca gttagttata agctggctcc agtgtgtaag 1440 tccatcgtgt atatcttgtg cataaatgtt ctacaggata ttttctgtat tatatgtgtc 1500 tgtagtgtac attgcaatat tacgtgaaat gtaagatctg catatggatg atggaaccaa 1560 ccactcaagt gtcatgccaa ggaaaacacc aaataaacct tgaacagtg 1609 <210> 43 <211> 289 <212> DNA <213> Artificial Sequence <220> <223> CYC promoter <400> 43 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> 44 <211> 655 <212> DNA <213> Artificial Sequence <220> <223> GPD promoter <400> 44 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> 45 <211> 1468 <212> DNA <213> Artificial Sequence <220> <223> ADH promoter <400> 45 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> 46 <211> 252 <212> DNA <213> Artificial Sequence <220> <223> CYC1 terminator <400> 46 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> 47 <211> 563 <212> PRT <213> Saccharomyces cerevisiae <400> 47 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 Pro Ser Ile 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 Pro 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> 48 <211> 1692 <212> DNA <213> Saccharomyces cerevisiae <400> 48 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 ggtgatgttg tcattgctga aaccggtacc tccgctttcg gtatcaacca aaccactttc 1200 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> 49 <211> 591 <212> PRT <213> Saccharomyces cerevisiae <400> 49 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 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 Ala 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> 50 <211> 1776 <212> DNA <213> Saccharomyces cerevisiae <400> 50 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> 51 <211> 391 <212> PRT <213> Saccharomyces cerevisiae <400> 51 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 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 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> 52 <211> 1176 <212> DNA <213> Saccharomyces cerevisiae <400> 52 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> 53 <211> 1404 <212> DNA <213> E.coli <400> 53 atgaatcaac aggatattga acaggtggtg aaagcggtac tgctgaaaat gcaaagcagt 60 gacacgccgt ccgccgccgt tcatgagatg ggcgttttcg cgtccctgga tgacgccgtt 120 gcggcagcca aagtcgccca gcaagggtta aaaagcgtgg caatgcgcca gttagccatt 180 gctgccattc gtgaagcagg cgaaaaacac gccagagatt tagcggaact tgccgtcagt 240 gaaaccggca tggggcgcgt tgaagataaa tttgcaaaaa acgtcgctca ggcgcgcggc 300 acaccaggcg ttgagtgcct ctctccgcaa gtgctgactg gcgacaacgg cctgacccta 360 attgaaaacg caccctgggg cgtggtggct tcggtgacgc cttccactaa cccggcggca 420 accgtaatta acaacgccat cagcctgatt gccgcgggca acagcgtcat ttttgccccg 480 catccggcgg cgaaaaaagt ctcccagcgg gcgattacgc tgctcaacca ggcgattgtt 540 gccgcaggtg ggccggaaaa cttactggtt actgtggcaa atccggatat cgaaaccgcg 600 caacgcttgt tcaagtttcc gggtatcggc ctgctggtgg taaccggcgg cgaagcggta 660 gtagaagcgg cgcgtaaaca caccaataaa cgtctgattg ccgcaggcgc tggcaacccg 720 ccggtagtgg tggatgaaac cgccgacctc gcccgtgccg ctcagtccat cgtcaaaggc 780 gcttctttcg ataacaacat catttgtgcc gacgaaaagg tactgattgt tgttgatagc 840 gtagccgatg aactgatgcg tctgatggaa ggccagcacg cggtgaaact gaccgcagaa 900 caggcgcagc agctgcaacc ggtgttgctg aaaaatatcg acgagcgcgg aaaaggcacc 960 gtcagccgtg actgggttgg tcgcgacgca ggcaaaatcg cggcggcaat cggccttaaa 1020 gttccgcaag aaacgcgcct gctgtttgtg gaaaccaccg cagaacatcc gtttgccgtg 1080 actgaactga tgatgccggt gttgcccgtc gtgcgcgtcg ccaacgtggc ggatgccatt 1140 gcgctagcgg tgaaactgga aggcggttgc caccacacgg cggcaatgca ctcgcgcaac 1200 atcgaaaaca tgaaccagat ggcgaatgct attgatacca gcattttcgt taagaacgga 1260 ccgtgcattg ccgggctggg gctgggcggg gaaggctgga ccaccatgac catcaccacg 1320 ccaaccggtg aaggggtaac cagcgcgcgt acgtttgtcc gtctgcgtcg ctgtgtatta 1380 gtcgatgcgt ttcgcattgt ttaa 1404 <210> 54 <211> 467 <212> PRT <213> E.coli <400> 54 Met Asn Gln Gln Asp Ile Glu Gln Val Val Lys Ala Val Leu Leu Lys 1 5 10 15 Met Gln Ser Ser Asp Thr Pro Ser Ala Ala Val His Glu Met Gly Val 20 25 30 Phe Ala Ser Leu Asp Asp Ala Val Ala Ala Ala Lys Val Ala Gln Gln 35 40 45 Gly Leu Lys Ser Val Ala Met Arg Gln Leu Ala Ile Ala Ala Ile Arg 50 55 60 Glu Ala Gly Glu Lys His Ala Arg Asp Leu Ala Glu Leu Ala Val Ser 65 70 75 80 Glu Thr Gly Met Gly Arg Val Glu Asp Lys Phe Ala Lys Asn Val Ala 85 90 95 Gln Ala Arg Gly Thr Pro Gly Val Glu Cys Leu Ser Pro Gln Val Leu 100 105 110 Thr Gly Asp Asn Gly Leu Thr Leu Ile Glu Asn Ala Pro Trp Gly Val 115 120 125 Val Ala Ser Val Thr Pro Ser Thr Asn Pro Ala Ala Thr Val Ile Asn 130 135 140 Asn Ala Ile Ser Leu Ile Ala Ala Gly Asn Ser Val Ile Phe Ala Pro 145 150 155 160 His Pro Ala Ala Lys Lys Val Ser Gln Arg Ala Ile Thr Leu Leu Asn 165 170 175 Gln Ala Ile Val Ala Ala Gly Gly Pro Glu Asn Leu Leu Val Thr Val 180 185 190 Ala Asn Pro Asp Ile Glu Thr Ala Gln Arg Leu Phe Lys Phe Pro Gly 195 200 205 Ile Gly Leu Leu Val Val Thr Gly Gly Glu Ala Val Val Glu Ala Ala 210 215 220 Arg Lys His Thr Asn Lys Arg Leu Ile Ala Ala Gly Ala Gly Asn Pro 225 230 235 240 Pro Val Val Val Asp Glu Thr Ala Asp Leu Ala Arg Ala Ala Gln Ser 245 250 255 Ile Val Lys Gly Ala Ser Phe Asp Asn Asn Ile Ile Cys Ala Asp Glu 260 265 270 Lys Val Leu Ile Val Val Asp Ser Val Ala Asp Glu Leu Met Arg Leu 275 280 285 Met Glu Gly Gln His Ala Val Lys Leu Thr Ala Glu Gln Ala Gln Gln 290 295 300 Leu Gln Pro Val Leu Leu Lys Asn Ile Asp Glu Arg Gly Lys Gly Thr 305 310 315 320 Val Ser Arg Asp Trp Val Gly Arg Asp Ala Gly Lys Ile Ala Ala Ala 325 330 335 Ile Gly Leu Lys Val Pro Gln Glu Thr Arg Leu Leu Phe Val Glu Thr 340 345 350 Thr Ala Glu His Pro Phe Ala Val Thr Glu Leu Met Met Pro Val Leu 355 360 365 Pro Val Val Arg Val Ala Asn Val Ala Asp Ala Ile Ala Leu Ala Val 370 375 380 Lys Leu Glu Gly Gly Cys His His Thr Ala Ala Met His Ser Arg Asn 385 390 395 400 Ile Glu Asn Met Asn Gln Met Ala Asn Ala Ile Asp Thr Ser Ile Phe 405 410 415 Val Lys Asn Gly Pro Cys Ile Ala Gly Leu Gly Leu Gly Gly Glu Gly 420 425 430 Trp Thr Thr Met Thr Ile Thr Thr Pro Thr Gly Glu Gly Val Thr Ser 435 440 445 Ala Arg Thr Phe Val Arg Leu Arg Arg Cys Val Leu Val Asp Ala Phe 450 455 460 Arg Ile Val 465 <210> 55 <211> 1047 <212> DNA <213> S.cerevisiae <400> 55 atgtctatcc cagaaactca aaaaggtgtt atcttctacg aatcccacgg taagttggaa 60 tacaaagata ttccagttcc aaagccaaag gccaacgaat tgttgatcaa cgttaaatac 120 tctggtgtct gtcacactga cttgcacgct tggcacggtg actggccatt gccagttaag 180 ctaccattag ttggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240 aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300 tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360 acccacgacg gttctttcca agaatacgct accgctgacg ctgttcaagc cgctcacatt 420 cctcaaggta ctgacttggc tgaagtcgcc ccagttttgt gtgctggtat caccgtctac 480 aaggctttga agtctgctaa cttgatggcc ggtcactggg ttgctatctc cggtgctgct 540 ggtggtctag gttctttggc tgttcaatac gccaaggcta tgggttacag agtcttgggt 600 attgacggtg gtgaaggtaa ggaagaatta ttcagatcca tcggtggtga agtcttcatt 660 gacttcacta aggaaaagga cattgtcggt gctgttctaa aggccactga cggtggtgct 720 cacggtgtca tcaacgtttc cgtttccgaa gccgctattg aagcttccac cagatacgtt 780 agagctaacg gtaccaccgt tttggtcggt atgccagctg gtgccaagtg ttgttctgat 840 gtcttcaacc aagtcgtcaa gtccatctct attgttggtt cttacgtcgg taacagagct 900 gacaccagag aagctttgga cttcttcgcc agaggtttga tcaagtctcc aatcaaggtt 960 gtcggcttgt ctaccttgcc agaaatttac gaaaagatgg aaaagggtca aatcgttggt 1020 agatacgttg ttgacacttc taaataa 1047 <210> 56 <211> 348 <212> PRT <213> S.cerevisiae <400> 56 Met Ser Ile Pro Glu Thr Gln Lys Gly Val Ile Phe Tyr Glu Ser His 1 5 10 15 Gly Lys Leu Glu Tyr Lys Asp Ile Pro Val Pro Lys Pro Lys Ala Asn 20 25 30 Glu Leu Leu Ile Asn Val Lys Tyr Ser Gly Val Cys His Thr Asp Leu 35 40 45 His Ala Trp His Gly Asp Trp Pro Leu Pro Val Lys Leu Pro Leu Val 50 55 60 Gly Gly His Glu Gly Ala Gly Val Val Val Gly Met Gly Glu Asn Val 65 70 75 80 Lys Gly Trp Lys Ile Gly Asp Tyr Ala Gly Ile Lys Trp Leu Asn Gly 85 90 95 Ser Cys Met Ala Cys Glu Tyr Cys Glu Leu Gly Asn Glu Ser Asn Cys 100 105 110 Pro His Ala Asp Leu Ser Gly Tyr Thr His Asp Gly Ser Phe Gln Gln 115 120 125 Tyr Ala Thr Ala Asp Ala Val Gln Ala Ala His Ile Pro Gln Gly Thr 130 135 140 Asp Leu Ala Gln Val Ala Pro Ile Leu Cys Ala Gly Ile Thr Val Tyr 145 150 155 160 Lys Ala Leu Lys Ser Ala Asn Leu Met Ala Gly His Trp Val Ala Ile 165 170 175 Ser Gly Ala Ala Gly Gly Leu Gly Ser Leu Ala Val Gln Tyr Ala Lys 180 185 190 Ala Met Gly Tyr Arg Val Leu Gly Ile Asp Gly Gly Glu Gly Lys Glu 195 200 205 Glu Leu Phe Arg Ser Ile Gly Gly Glu Val Phe Ile Asp Phe Thr Lys 210 215 220 Glu Lys Asp Ile Val Gly Ala Val Leu Lys Ala Thr Asp Gly Gly Ala 225 230 235 240 His Gly Val Ile Asn Val Ser Val Ser Glu Ala Ala Ile Glu Ala Ser 245 250 255 Thr Arg Tyr Val Arg Ala Asn Gly Thr Thr Val Leu Val Gly Met Pro 260 265 270 Ala Gly Ala Lys Cys Cys Ser Asp Val Phe Asn Gln Val Val Lys Ser 275 280 285 Ile Ser Ile Val Gly Ser Tyr Val Gly Asn Arg Ala Asp Thr Arg Glu 290 295 300 Ala Leu Asp Phe Phe Ala Arg Gly Leu Val Lys Ser Pro Ile Lys Val 305 310 315 320 Val Gly Leu Ser Thr Leu Pro Glu Ile Tyr Glu Lys Met Glu Lys Gly 325 330 335 Gln Ile Val Gly Arg Tyr Val Val Asp Thr Ser Lys 340 345 <210> 57 <211> 753 <212> DNA <213> S.cerevisiae <400> 57 atgcctttga ccacaaaacc tttatctttg aaaatcaacg ccgctctatt cgatgttgac 60 ggtaccatca tcatctctca accagccatt gctgctttct ggagagattt cggtaaagac 120 aagccttact tcgatgccga acacgttatt cacatctctc acggttggag aacttacgat 180 gccattgcca agttcgctcc agactttgct gatgaagaat acgttaacaa gctagaaggt 240 gaaatcccag aaaagtacgg tgaacactcc atcgaagttc caggtgctgt caagttgtgt 300 aatgctttga acgccttgcc aaaggaaaaa tgggctgtcg ccacctctgg tacccgtgac 360 atggccaaga aatggttcga cattttgaag atcaagagac cagaatactt catcaccgcc 420 aatgatgtca agcaaggtaa gcctcaccca gaaccatact taaagggtag aaacggtttg 480 ggtttcccaa ttaatgaaca agacccatcc aaatctaagg ttgttgtctt tgaagacgca 540 ccagctggta ttgctgctgg taaggctgct ggctgtaaaa tcgttggtat tgctaccact 600 ttcgatttgg acttcttgaa ggaaaagggt tgtgacatca ttgtcaagaa ccacgaatct 660 atcagagtcg gtgaatacaa cgctgaaacc gatgaagtcg aattgatctt tgatgactac 720 ttatacgcta aggatgactt gttgaaatgg taa 753 <210> 58 <211> 250 <212> PRT <213> S.cerevisiae <400> 58 Met Pro Leu Thr Thr Lys Pro Leu Ser Leu Lys Ile Asn Ala Ala Leu 1 5 10 15 Phe Asp Val Asp Gly Thr Ile Ile Ile Ser Gln Pro Ala Ile Ala Ala 20 25 30 Phe Trp Arg Asp Phe Gly Lys Asp Lys Pro Tyr Phe Asp Ala Glu His 35 40 45 Val Ile His Ile Ser His Gly Trp Arg Thr Tyr Asp Ala Ile Ala Lys 50 55 60 Phe Ala Pro Asp Phe Ala Asp Glu Glu Tyr Val Asn Lys Leu Glu Gly 65 70 75 80 Glu Ile Pro Glu Lys Tyr Gly Glu His Ser Ile Glu Val Pro Gly Ala 85 90 95 Val Lys Leu Cys Asn Ala Leu Asn Ala Leu Pro Lys Glu Lys Trp Ala 100 105 110 Val Ala Thr Ser Gly Thr Arg Asp Met Ala Lys Lys Trp Phe Asp Ile 115 120 125 Leu Lys Ile Lys Arg Pro Glu Tyr Phe Ile Thr Ala Asn Asp Val Lys 130 135 140 Gln Gly Lys Pro His Pro Glu Pro Tyr Leu Lys Gly Arg Asn Gly Leu 145 150 155 160 Gly Phe Pro Ile Asn Glu Gln Asp Pro Ser Lys Ser Lys Val Val Val 165 170 175 Phe Glu Asp Ala Pro Ala Gly Ile Ala Ala Gly Lys Ala Ala Gly Cys 180 185 190 Lys Ile Val Gly Ile Ala Thr Thr Phe Asp Leu Asp Phe Leu Lys Glu 195 200 205 Lys Gly Cys Asp Ile Ile Val Lys Asn His Glu Ser Ile Arg Val Gly 210 215 220 Glu Tyr Asn Ala Glu Thr Asp Glu Val Glu Leu Ile Phe Asp Asp Tyr 225 230 235 240 Leu Tyr Ala Lys Asp Asp Leu Leu Lys Trp 245 250 <210> 59 <211> 5779 <212> DNA <213> Artificial Sequence <220> <223> pUC19-His-MhpF <400> 59 tcgacctgca ggcatgcaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat 60 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 120 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 180 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 240 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 300 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 360 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 420 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 480 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 540 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 600 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 660 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 720 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 780 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 840 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 900 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 960 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 1020 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 1080 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 1140 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1200 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 1260 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 1320 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 1380 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 1440 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 1500 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 1560 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 1620 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 1680 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 1740 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 1800 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 1860 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 1920 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 1980 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 2040 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 2100 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 2160 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 2220 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt 2280 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 2340 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt 2400 aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg 2460 cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag gctgcgcaac 2520 tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga 2580 tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa 2640 acgacggcca gtgaattcga gctcagttta tcattatcaa tactcgccat ttcaaagaat 2700 acgtaaataa ttaatagtag tgattttcct aactttattt agtcaaaaaa ttagcctttt 2760 aattctgctg taacccgtac atgcccaaaa tagggggcgg gttacacaga atatataaca 2820 tcgtaggtgt ctgggtgaac agtttattcc tggcatccac taaatataat ggagcccgct 2880 ttttaagctg gcatccagaa aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc 2940 aaccatcagt tcataggtcc attctcttag cgcaactaca gagaacaggg gcacaaacag 3000 gcaaaaaacg ggcacaacct caatggagtg atgcaacctg cctggagtaa atgatgacac 3060 aaggcaattg acccacgcat gtatctatct cattttctta caccttctat taccttctgc 3120 tctctctgat ttggaaaaag ctgaaaaaaa aggttgaaac cagttccctg aaattattcc 3180 cctacttgac taataagtat ataaagacgg taggtattga ttgtaattct gtaaatctat 3240 ttcttaaact tcttaaattc tacttttata gttagtcttt tttttagttt taaaacacca 3300 gaacttagtt tcgacggatt ctagaactag tggatccatg tcaaagcgaa aagtagctat 3360 cataggttca ggtaatattg gtactgattt gatgatcaaa atcctgagac atggccagca 3420 cttggagatg gccgtcatgg ttggtatcga cccacaatcc gatggcttag ctagagctag 3480 gagaatgggt gttgccacaa ctcacgaagg ggttattggc ttaatgaaca tgccagaatt 3540 tgcagacatc gatatagttt ttgatgctac tagtgcaggg gcacatgtga aaaacgacgc 3600 ggctttaaga gaagccaagc cagatattag attaattgat cttacccctg ctgctatagg 3660 tccttactgc gttcctgtag ttaaccttga agctaatgtg gaccagttga acgtgaatat 3720 ggttacatgt ggtggccaag ctaccatacc aatggttgct gctgtctcta gagtggccag 3780 agtacattat gccgagatca ttgcgtctat cgcatctaag tctgccggtc ctggaacaag 3840 ggctaacatc gatgagttca ctgagacaac ctctagagct atcgaagtag taggaggcgc 3900 agcaaaaggt aaagcgatca ttgttttgaa tcctgccgaa ccacctttga tgatgagaga 3960 tacggtctac gtgctatcag atgaagcttc ccaggatgac attgaagcta gcattaatga 4020 gatggcagaa gccgttcaag catacgtgcc aggatataga ctcaaacaaa gagtccaatt 4080 tgaggtcatt ccacaagaca agccagttaa tctcccaggg gtcggtcaat tctcaggact 4140 aaaaactgct gtttggttag aagtagaagg agctgctcat tacctaccag cctacgccgg 4200 taatttggat ataatgacat cttccgctct tgcaacagca gaaaagatgg cacaaagtct 4260 ggcccgtaag gcaggagaag cggcataata aatcctcgag tcatgtaatt agttatgtca 4320 cgcttacatt cacgccctcc ccccacatcc gctctaaccg aaaaggaagg agttagacaa 4380 cctgaagtct aggtccctat ttattttttt atagttatgt tagtattaag aacgttattt 4440 atatttcaaa tttttctttt ttttctgtac agacgcgtgt acgcatgtaa cattatactg 4500 aaaaccttgc ttgagaaggt tttgggacgc tcgaaggctt taatttgcgg ccggtaccca 4560 attcgagctc ggtacccggg gatcctctag agtcgacaat tcccgtttta agagcttggt 4620 gagcgctagg agtcactgcc aggtatcgtt tgaacacggc attagtcagg gaagtcataa 4680 cacagtcctt tcccgcaatt ttctttttct attactcttg gcctcctcta gtacactcta 4740 tattttttta tgcctcggta atgattttca tttttttttt tcccctagcg gatgactctt 4800 tttttttctt agcgattggc attatcacat aatgaattat acattatata aagtaatgtg 4860 atttcttcga agaatatact aaaaaatgag caggcaagat aaacgaaggc aaagatgaca 4920 gagcagaaag ccctagtaaa gcgtattaca aatgaaacca agattcagat tgcgatctct 4980 ttaaagggtg gtcccctagc gatagagcac tcgatcttcc cagaaaaaga ggcagaagca 5040 gtagcagaac aggccacaca atcgcaagtg attaacgtcc acacaggtat agggtttctg 5100 gaccatatga tacatgctct ggccaagcat tccggctggt cgctaatcgt tgagtgcatt 5160 ggtgacttac acatagacga ccatcacacc actgaagact gcgggattgc tctcggtcaa 5220 gcttttaaag aggccctact ggcgcgtgga gtaaaaaggt ttggatcagg atttgcgcct 5280 ttggatgagg cactttccag agcggtggta gatctttcga acaggccgta cgcagttgtc 5340 gaacttggtt tgcaaaggga gaaagtagga gatctctctt gcgagatgat cccgcatttt 5400 cttgaaagct ttgcagaggc tagcagaatt accctccacg ttgattgtct gcgaggcaag 5460 aatgatcatc accgtagtga gagtgcgttc aaggctcttg cggttgccat aagagaagcc 5520 acctcgccca atggtaccaa cgatgttccc tccaccaaag gtgttcttat gtagtgacac 5580 cgattattta aagctgcagc atacgatata tatacatgtg tatatatgta tacctatgaa 5640 tgtcagtaag tatgtatacg aacagtatga tactgaagat gacaaggtaa tgcatcattc 5700 tatacgtgtc attctgaacg aggcgcgctt tccttttttc tttttgcttt ttcttttttt 5760 ttctcttgaa ctcgacggg 5779 <210> 60 <211> 500 <212> PRT <213> S. cerevisiae <400> 60 Met Thr Lys Leu His Phe Asp Thr Ala Glu Pro Val Lys Ile Thr Leu 1 5 10 15 Pro Asn Gly Leu Thr Tyr Glu Gln Pro Thr Gly Leu Phe Ile Asn Asn 20 25 30 Lys Phe Met Lys Ala Gln Asp Gly Lys Thr Tyr Pro Val Glu Asp Pro 35 40 45 Ser Thr Glu Asn Thr Val Cys Glu Val Ser Ser Ala Thr Thr Glu Asp 50 55 60 Val Glu Tyr Ala Ile Glu Cys Ala Asp Arg Ala Phe His Asp Thr Glu 65 70 75 80 Trp Ala Thr Gln Asp Pro Arg Glu Arg Gly Arg Leu Leu Ser Lys Leu 85 90 95 Ala Asp Glu Leu Glu Ser Gln Ile Asp Leu Val Ser Ser Ile Glu Ala 100 105 110 Leu Asp Asn Gly Lys Thr Leu Ala Leu Ala Arg Gly Asp Val Thr Ile 115 120 125 Ala Ile Asn Cys Leu Arg Asp Ala Ala Ala Tyr Ala Asp Lys Val Asn 130 135 140 Gly Arg Thr Ile Asn Thr Gly Asp Gly Tyr Met Asn Phe Thr Thr Leu 145 150 155 160 Glu Pro Ile Gly Val Cys Gly Gln Ile Ile Pro Trp Asn Phe Pro Ile 165 170 175 Met Met Leu Ala Trp Lys Ile Ala Pro Ala Leu Ala Met Gly Asn Val 180 185 190 Cys Ile Leu Lys Pro Ala Ala Val Thr Pro Leu Asn Ala Leu Tyr Phe 195 200 205 Ala Ser Leu Cys Lys Lys Val Gly Ile Pro Ala Gly Val Val Asn Ile 210 215 220 Val Pro Gly Pro Gly Arg Thr Val Gly Ala Ala Leu Thr Asn Asp Pro 225 230 235 240 Arg Ile Arg Lys Leu Ala Phe Thr Gly Ser Thr Glu Val Gly Lys Ser 245 250 255 Val Ala Val Asp Ser Ser Glu Ser Asn Leu Lys Lys Ile Thr Leu Glu 260 265 270 Leu Gly Gly Lys Ser Ala His Leu Val Phe Asp Asp Ala Asn Ile Lys 275 280 285 Lys Thr Leu Pro Asn Leu Val Asn Gly Ile Phe Lys Asn Ala Gly Gln 290 295 300 Ile Cys Ser Ser Gly Ser Arg Ile Tyr Val Gln Glu Gly Ile Tyr Asp 305 310 315 320 Glu Leu Leu Ala Ala Phe Lys Ala Tyr Leu Glu Thr Glu Ile Lys Val 325 330 335 Gly Asn Pro Phe Asp Lys Ala Asn Phe Gln Gly Ala Ile Thr Asn Arg 340 345 350 Gln Gln Phe Asp Thr Ile Met Asn Tyr Ile Asp Ile Gly Lys Lys Glu 355 360 365 Gly Ala Lys Ile Leu Thr Gly Gly Glu Lys Val Gly Asp Lys Gly Tyr 370 375 380 Phe Ile Arg Pro Thr Val Phe Tyr Asp Val Asn Glu Asp Met Arg Ile 385 390 395 400 Val Lys Glu Glu Ile Phe Gly Pro Val Val Thr Val Ala Lys Phe Lys 405 410 415 Thr Leu Glu Glu Gly Val Glu Met Ala Asn Ser Ser Glu Phe Gly Leu 420 425 430 Gly Ser Gly Ile Glu Thr Glu Ser Leu Ser Thr Gly Leu Lys Val Ala 435 440 445 Lys Met Leu Lys Ala Gly Thr Val Trp Ile Asn Thr Tyr Asn Asp Phe 450 455 460 Asp Ser Arg Val Pro Phe Gly Gly Val Lys Gln Ser Gly Tyr Gly Arg 465 470 475 480 Glu Met Gly Glu Glu Val Tyr His Ala Tyr Thr Glu Val Lys Ala Val 485 490 495 Arg Ile Lys Leu 500 <210> 61 <211> 1503 <212> DNA <213> S.cerevisiae <400> 61 atgactaagc tacactttga cactgctgaa ccagtcaaga tcacacttcc aaatggtttg 60 acatacgagc aaccaaccgg tctattcatt aacaacaagt ttatgaaagc tcaagacggt 120 aagacctatc ccgtcgaaga tccttccact gaaaacaccg tttgtgaggt ctcttctgcc 180 accactgaag atgttgaata tgctatcgaa tgtgccgacc gtgctttcca cgacactgaa 240 tgggctaccc aagacccaag agaaagaggc cgtctactaa gtaagttggc tgacgaattg 300 gaaagccaaa ttgacttggt ttcttccatt gaagctttgg acaatggtaa aactttggcc 360 ttagcccgtg gggatgttac cattgcaatc aactgtctaa gagatgctgc tgcctatgcc 420 gacaaagtca acggtagaac aatcaacacc ggtgacggct acatgaactt caccacctta 480 gagccaatcg gtgtctgtgg tcaaattatt ccatggaact ttccaataat gatgttggct 540 tggaagatcg ccccagcatt ggccatgggt aacgtctgta tcttgaaacc cgctgctgtc 600 acacctttaa atgccctata ctttgcttct ttatgtaaga aggttggtat tccagctggt 660 gtcgtcaaca tcgttccagg tcctggtaga actgttggtg ctgctttgac caacgaccca 720 agaatcagaa agctggcttt taccggttct acagaagtcg gtaagagtgt tgctgtcgac 780 tcttctgaat ctaacttgaa gaaaatcact ttggaactag gtggtaagtc cgcccatttg 840 gtctttgacg atgctaacat taagaagact ttaccaaatc tagtaaacgg tattttcaag 900 aacgctggtc aaatttgttc ctctggttct agaatttacg ttcaagaagg tatttacgac 960 gaactattgg ctgctttcaa ggcttacttg gaaaccgaaa tcaaagttgg taatccattt 1020 gacaaggcta acttccaagg tgctatcact aaccgtcaac aattcgacac aattatgaac 1080 tacatcgata tcggtaagaa agaaggcgcc aagatcttaa ctggtggcga aaaagttggt 1140 gacaagggtt acttcatcag accaaccgtt ttctacgatg ttaatgaaga catgagaatt 1200 gttaaggaag aaatttttgg accagttgtc actgtcgcaa agttcaagac tttagaagaa 1260 ggtgtcgaaa tggctaacag ctctgaattc ggtctaggtt ctggtatcga aacagaatct 1320 ttgagcacag gtttgaaggt ggccaagatg ttgaaggccg gtaccgtctg gatcaacaca 1380 tacaacgatt ttgactccag agttccattc ggtggtgtta agcaatctgg ttacggtaga 1440 gaaatgggtg aagaagtcta ccatgcatac actgaagtaa aagctgtcag aattaagttg 1500 taa 1503 <210> 62 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 62 cctcctgagt cgacaattcc cgttttaaga g 31 <210> 63 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 63 cgaccgtggt cgacccgtcg agttcaagag 30 <210> 64 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 64 tatatatttc aaggatatac cattctaatg tctgccccta agaagatcgt gctgcaaggc 60 gattaag 67 <210> 65 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 65 gagaatcttt ttaagcaagg attttcttaa cttcttcggc gacagcatcg gctcgtatgt 60 tgtgtgg 67 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 66 gtttcgtcta ccctatgaac 20 <210> 67 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 67 ccaataggtg gttagcaatc g 21 <210> 68 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 68 caagaaacat ctttaacata cacaaacaca tactatcaga atacccagtc acgacgttgt 60 aaaa 64 <210> 69 <211> 65 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 69 gtattttgtg tatatgacgg aaagaaatgc aggttggtac attacaggtt tcccgactgg 60 aaagc 65 <210> 70 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 70 gcatcgggaa cgtatgtaac attg 24 <210> 71 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 71 tgacgtaaga ccaagtaag 19 <210> 72 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 72 ctatagggcg aattggctag cttatcatta tcaatactcg ccatttcaaa gaata 55 <210> 73 <211> 66 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 73 ctcgaggggg ggcccggtac ctcgaaacta agttctggtg ttttaaaact aaaaaaaaga 60 ctaact 66 <210> 74 <211> 6110 <212> DNA <213> Artificial Sequence <220> <223> pCS-Ex1 vector <400> 74 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtacgcatt taagcataaa cacgcactat gccgttcttc tcatgtatat 180 atatatacag gcaacacgca gatataggtg cgacgtgaac agtgagctgt atgtgcgcag 240 ctcgcgttgc attttcggaa gcgctcgttt tcggaaacgc tttgaagttc ctattccgaa 300 gttcctattc tctagctaga aagtatagga acttcagagc gcttttgaaa accaaaagcg 360 ctctgaagac gcactttcaa aaaaccaaaa acgcaccgga ctgtaacgag ctactaaaat 420 attgcgaata ccgcttccac aaacattgct caaaagtatc tctttgctat atatctctgt 480 gctatatccc tatataacct acccatccac ctttcgctcc ttgaacttgc atctaaactc 540 gacctctaca ttttttatgt ttatctctag tattactctt tagacaaaaa aattgtagta 600 agaactattc atagagtgaa tcgaaaacaa tacgaaaatg taaacatttc ctatacgtag 660 tatatagaga caaaatagaa gaaaccgttc ataattttct gaccaatgaa gaatcatcaa 720 cgctatcact ttctgttcac aaagtatgcg caatccacat cggtatagaa tataatcggg 780 gatgccttta tcttgaaaaa atgcacccgc agcttcgcta gtaatcagta aacgcgggaa 840 gtggagtcag gcttttttta tggaagagaa aatagacacc aaagtagcct tcttctaacc 900 ttaacggacc tacagtgcaa aaagttatca agagactgca ttatagagcg cacaaaggag 960 aaaaaaagta atctaagatg ctttgttaga aaaatagcgc tctcgggatg catttttgta 1020 gaacaaaaaa gaagtataga ttctttgttg gtaaaatagc gctctcgcgt tgcatttctg 1080 ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt 1140 ttgttttaca aaaatgaagc acagattctt cgttggtaaa atagcgcttt cgcgttgcat 1200 ttctgttctg taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg 1260 catttttgtt ctacaaaatg aagcacagat gcttcgttaa tgtgctgcaa ggcgattaag 1320 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta 1380 atacgactca ctatagggcg aattggctag cttatcatta tcaatactcg ccatttcaaa 1440 gaatacgtaa ataattaata gtagtgattt tcctaacttt atttagtcaa aaaattagcc 1500 ttttaattct gctgtaaccc gtacatgccc aaaatagggg gcgggttaca cagaatatat 1560 aacatcgtag gtgtctgggt gaacagttta ttcctggcat ccactaaata taatggagcc 1620 cgctttttaa gctggcatcc agaaaaaaaa agaatcccag caccaaaata ttgttttctt 1680 caccaaccat cagttcatag gtccattctc ttagcgcaac tacagagaac aggggcacaa 1740 acaggcaaaa aacgggcaca acctcaatgg agtgatgcaa cctgcctgga gtaaatgatg 1800 acacaaggca attgacccac gcatgtatct atctcatttt cttacacctt ctattacctt 1860 ctgctctctc tgatttggaa aaagctgaaa aaaaaggttg aaaccagttc cctgaaatta 1920 ttcccctact tgactaataa gtatataaag acggtaggta ttgattgtaa ttctgtaaat 1980 ctatttctta aacttcttaa attctacttt tatagttagt ctttttttta gttttaaaac 2040 accagaactt agtttcgagg taccgggccc cccctcgagg tcgacggtat cgataagctt 2100 gatatcgaat tcctgcagcc cgggggatcc actagttcta gagcggccgc caccgcggtg 2160 gagctcggtt ctgcttatcc ttacgacgtg cctgactacg cctgaacccg atgcaaatga 2220 gacgatcgtc tattcctggt ccggttttct ctgccctctc ttctattcac tttttttata 2280 ctttatataa aattatataa atgacataac tgaaacgcca cacgtcctct cctattcgtt 2340 aacgcctgtc tgtagcgctg ttactgaagc tgcgcaagta gttttttcac cgtataggcc 2400 ctctttttct ctctctttct ttctctcccg cgctgatctc ttcttcgaaa cacagagtgc 2460 accataccac cttttcaatt catcattttt tttttattct tttttttgat ttcggtttcc 2520 ttgaaatttt tttgattcgg taatctccga acagaaggaa gaacgaagga aggagcacag 2580 acttagattg gtatatatac gcatatgtag tgttgaagaa acatgaaatt gcccagtatt 2640 cttaacccaa ctgcacagaa caaaaacctc caggaaacga agataaatca tgtcgaaagc 2700 tacatataag gaacgtgctg ctactcatcc tagtcctgtt gctgccaagc tatttaatat 2760 catgcacgaa aagcaaacaa acttgtgtgc ttcattggat gttcgtacca ccaaggaatt 2820 actggagtta gttgaagcat taggtcccaa aatttgttta ctaaaaacac atgtggatat 2880 cttgactgat ttttccatgg agggcacagt taagccgcta aaggcattat ccgccaagta 2940 caatttttta ctcttcgaag acagaaaatt tgctgacatt ggtaatacag tcaaattgca 3000 gtactctgcg ggtgtataca gaatagcaga atgggcagac attacgaatg cacacggtgt 3060 ggtgggccca ggtattgtta gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc 3120 tagaggcctt ttgatgttag cagaattgtc atgcaagggc tccctatcta ctggagaata 3180 tactaagggt actgttgaca ttgcgaagag cgacaaagat tttgttatcg gctttattgc 3240 tcaaagagac atgggtggaa gagatgaagg ttacgattgg ttgattatga cacccggtgt 3300 gggtttagat gacaagggag acgcattggg tcaacagtat agaaccgtgg atgatgtggt 3360 ctctacagga tctgacatta ttattgttgg aagaggacta tttgcaaagg gaagggatgc 3420 taaggtagag ggtgaacgtt acagaaaagc aggctgggaa gcatatttga gaagatgcgg 3480 ccagcaaaac taatcatgta attagttatg tcacgcttac attcacgccc tccccccaca 3540 tccgctctaa ccgaaaagga aggagttaga caacctgaag tctaggtccc tatttatttt 3600 tttatagtta tgttagtatt aagaacgtta tttatatttc aaatttttct tttttttctg 3660 tacagacgcg tgtacgcatg taacattata ctgaaaacct tgcttgagaa ggttttggga 3720 cgctcgaagg ctttaatttg cgtctgtagc gctgttactg aagctgcgca agtagttttt 3780 tcaccgtata ggccctcttt ttctctctct ttctttctct cccgcgctga tctcttcttc 3840 gaaacatcat gaataaaaag aaaaaggaaa tcaagaaaaa aaagccataa tttatcccac 3900 attttttttt attgtcgctg ttcacaccgc ataacgaaga tattggctag ctaaccagct 3960 tttgttccct ttagtgaggg ttaatttcga gcttggcgta atcatggtca tagctgtttc 4020 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 4080 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 4140 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 4200 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 4260 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 4320 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 4380 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 4440 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 4500 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 4560 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 4620 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 4680 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 4740 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 4800 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg 4860 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 4920 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 4980 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 5040 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 5100 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 5160 ctgacatcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg aatcgggagc 5220 ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct cttcagcaat 5280 atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc ggccacagtc 5340 gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg catcgccatg 5400 ggtcacgacg agatcctcgc cgtcgggcat gctcgccttg agcctggcga acagttcggc 5460 tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac cggcttccat 5520 ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg 5580 atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct cggcaggagc 5640 aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc agtcccttcc 5700 cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga 5760 tagccgcgct gcctcgtctt gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa 5820 aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc agccgattgt 5880 ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag aacctgcgtg 5940 caatccatct tgttcaattc gagtgcattc aacatcagcc atactcttcc tttttcaata 6000 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta 6060 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac 6110 <210> 75 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 75 accagaactt agtttcgaga aacaatgaat caacaggata ttgaacaggt ggtga 55 <210> 76 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 76 gtaaggataa gcagaaccgt taaacaatgc gaaacgcatc gactaataca 50 <210> 77 <211> 7413 <212> DNA <213> Artificial Sequence <220> <223> MD1040 vector <400> 77 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggttg agtacgcatt taagcataaa cacgcactat gccgttcttc tcatgtatat 180 atatatacag gcaacacgca gatataggtg cgacgtgaac agtgagctgt atgtgcgcag 240 ctcgcgttgc attttcggaa gcgctcgttt tcggaaacgc tttgaagttc ctattccgaa 300 gttcctattc tctagctaga aagtatagga acttcagagc gcttttgaaa accaaaagcg 360 ctctgaagac gcactttcaa aaaaccaaaa acgcaccgga ctgtaacgag ctactaaaat 420 attgcgaata ccgcttccac aaacattgct caaaagtatc tctttgctat atatctctgt 480 gctatatccc tatataacct acccatccac ctttcgctcc ttgaacttgc atctaaactc 540 gacctctaca ttttttatgt ttatctctag tattactctt tagacaaaaa aattgtagta 600 agaactattc atagagtgaa tcgaaaacaa tacgaaaatg taaacatttc ctatacgtag 660 tatatagaga caaaatagaa gaaaccgttc ataattttct gaccaatgaa gaatcatcaa 720 cgctatcact ttctgttcac aaagtatgcg caatccacat cggtatagaa tataatcggg 780 gatgccttta tcttgaaaaa atgcacccgc agcttcgcta gtaatcagta aacgcgggaa 840 gtggagtcag gcttttttta tggaagagaa aatagacacc aaagtagcct tcttctaacc 900 ttaacggacc tacagtgcaa aaagttatca agagactgca ttatagagcg cacaaaggag 960 aaaaaaagta atctaagatg ctttgttaga aaaatagcgc tctcgggatg catttttgta 1020 gaacaaaaaa gaagtataga ttctttgttg gtaaaatagc gctctcgcgt tgcatttctg 1080 ttctgtaaaa atgcagctca gattctttgt ttgaaaaatt agcgctctcg cgttgcattt 1140 ttgttttaca aaaatgaagc acagattctt cgttggtaaa atagcgcttt cgcgttgcat 1200 ttctgttctg taaaaatgca gctcagattc tttgtttgaa aaattagcgc tctcgcgttg 1260 catttttgtt ctacaaaatg aagcacagat gcttcgttaa tgtgctgcaa ggcgattaag 1320 ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgaattgta 1380 atacgactca ctatagggcg aattggctag cttatcatta tcaatactcg ccatttcaaa 1440 gaatacgtaa ataattaata gtagtgattt tcctaacttt atttagtcaa aaaattagcc 1500 ttttaattct gctgtaaccc gtacatgccc aaaatagggg gcgggttaca cagaatatat 1560 aacatcgtag gtgtctgggt gaacagttta ttcctggcat ccactaaata taatggagcc 1620 cgctttttaa gctggcatcc agaaaaaaaa agaatcccag caccaaaata ttgttttctt 1680 caccaaccat cagttcatag gtccattctc ttagcgcaac tacagagaac aggggcacaa 1740 acaggcaaaa aacgggcaca acctcaatgg agtgatgcaa cctgcctgga gtaaatgatg 1800 acacaaggca attgacccac gcatgtatct atctcatttt cttacacctt ctattacctt 1860 ctgctctctc tgatttggaa aaagctgaaa aaaaaggttg aaaccagttc cctgaaatta 1920 ttcccctact tgactaataa gtatataaag acggtaggta ttgattgtaa ttctgtaaat 1980 ctatttctta aacttcttaa attctacttt tatagttagt ctttttttta gttttaaaac 2040 accagaactt agtttcgaga aacaatgaat caacaggata ttgaacaggt ggtgaaagcg 2100 gtactgctga aaatgcaaag cagtgacacg ccgtccgccg ccgttcatga gatgggcgtt 2160 ttcgcgtccc tggatgacgc cgttgcggca gccaaagtcg cccagcaagg gttaaaaagc 2220 gtggcaatgc gccagttagc cattgctgcc attcgtgaag caggcgaaaa acacgccaga 2280 gatttagcgg aacttgccgt cagtgaaacc ggcatggggc gcgttgaaga taaatttgca 2340 aaaaacgtcg ctcaggcgcg cggcacacca ggcgttgagt gcctctctcc gcaagtgctg 2400 actggcgaca acggcctgac cctaattgaa aacgcaccct ggggcgtggt ggcttcggtg 2460 acgccttcca ctaacccggc ggcaaccgta attaacaacg ccatcagcct gattgccgcg 2520 ggcaacagcg tcatttttgc cccgcatccg gcggcgaaaa aagtctccca gcgggcgatt 2580 acgctgctca accaggcgat tgttgccgca ggtgggccgg aaaacttact ggttactgtg 2640 gcaaatccgg atatcgaaac cgcgcaacgc ttgttcaagt ttccgggtat cggcctgctg 2700 gtggtaaccg gcggcgaagc ggtagtagaa gcggcgcgta aacacaccaa taaacgtctg 2760 attgccgcag gcgctggcaa cccgccggta gtggtggatg aaaccgccga cctcgcccgt 2820 gccgctcagt ccatcgtcaa aggcgcttct ttcgataaca acatcatttg tgccgacgaa 2880 aaggtactga ttgttgttga tagcgtagcc gatgaactga tgcgtctgat ggaaggccag 2940 cacgcggtga aactgaccgc agaacaggcg cagcagctgc aaccggtgtt gctgaaaaat 3000 atcgacgagc gcggaaaagg caccgtcagc cgtgactggg ttggtcgcga cgcaggcaaa 3060 atcgcggcgg caatcggcct taaagttccg caagaaacgc gcctgctgtt tgtggaaacc 3120 accgcagaac atccgtttgc cgtgactgaa ctgatgatgc cggtgttgcc cgtcgtgcgc 3180 gtcgccaacg tggcggatgc cattgcgcta gcggtgaaac tggaaggcgg ttgccaccac 3240 acggcggcaa tgcactcgcg caacatcgaa aacatgaacc agatggcgaa tgctattgat 3300 accagcattt tcgttaagaa cggaccgtgc attgccgggc tggggctggg cggggaaggc 3360 tggaccacca tgaccatcac cacgccaacc ggtgaagggg taaccagcgc gcgtacgttt 3420 gtccgtctgc gtcgctgtgt attagtcgat gcgtttcgca ttgtttaacg gttctgctta 3480 tccttacgac gtgcctgact acgcctgaac ccgatgcaaa tgagacgatc gtctattcct 3540 ggtccggttt tctctgccct ctcttctatt cacttttttt atactttata taaaattata 3600 taaatgacat aactgaaacg ccacacgtcc tctcctattc gttaacgcct gtctgtagcg 3660 ctgttactga agctgcgcaa gtagtttttt caccgtatag gccctctttt tctctctctt 3720 tctttctctc ccgcgctgat ctcttcttcg aaacacagag tgcaccatac caccttttca 3780 attcatcatt ttttttttat tctttttttt gatttcggtt tccttgaaat ttttttgatt 3840 cggtaatctc cgaacagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata 3900 tacgcatatg tagtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca 3960 gaacaaaaac ctccaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg 4020 ctgctactca tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa 4080 caaacttgtg tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag 4140 cattaggtcc caaaatttgt ttactaaaaa cacatgtgga tatcttgact gatttttcca 4200 tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg 4260 aagacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat 4320 acagaatagc agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg 4380 ttagcggttt gaagcaggcg gcagaagaag taacaaagga acctagaggc cttttgatgt 4440 tagcagaatt gtcatgcaag ggctccctat ctactggaga atatactaag ggtactgttg 4500 acattgcgaa gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg 4560 gaagagatga aggttacgat tggttgatta tgacacccgg tgtgggttta gatgacaagg 4620 gagacgcatt gggtcaacag tatagaaccg tggatgatgt ggtctctaca ggatctgaca 4680 ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac 4740 gttacagaaa agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaatcat 4800 gtaattagtt atgtcacgct tacattcacg ccctcccccc acatccgctc taaccgaaaa 4860 ggaaggagtt agacaacctg aagtctaggt ccctatttat ttttttatag ttatgttagt 4920 attaagaacg ttatttatat ttcaaatttt tctttttttt ctgtacagac gcgtgtacgc 4980 atgtaacatt atactgaaaa ccttgcttga gaaggttttg ggacgctcga aggctttaat 5040 ttgcgtctgt agcgctgtta ctgaagctgc gcaagtagtt ttttcaccgt ataggccctc 5100 tttttctctc tctttctttc tctcccgcgc tgatctcttc ttcgaaacat catgaataaa 5160 aagaaaaagg aaatcaagaa aaaaaagcca taatttatcc cacatttttt tttattgtcg 5220 ctgttcacac cgcataacga agatattggc tagctaacca gcttttgttc cctttagtga 5280 gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 5340 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 5400 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 5460 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 5520 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 5580 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 5640 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 5700 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 5760 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 5820 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 5880 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 5940 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 6000 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 6060 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 6120 aagtggtggc ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg 6180 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 6240 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 6300 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 6360 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 6420 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacat cagaagaact 6480 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca 6540 cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg 6600 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc 6660 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct 6720 cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat 6780 gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct 6840 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc 6900 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga 6960 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt 7020 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt 7080 cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct 7140 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat 7200 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa 7260 ttcgagtgca ttcaacatca gccatactct tcctttttca atattattga agcatttatc 7320 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 7380 gggttccgcg cacatttccc cgaaaagtgc cac 7413 <210> 78 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 78 aatcttgtgc tattgcagtc ctcttttata tacagtataa tacgactcac tatagggcg 59 <210> 79 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 79 atgcgaattg cgtaattcac ggcgataacg tagtattaat taaccctcac taaagggaac 60 60 <210> 80 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 80 gcccacaact tatcaagtg 19 <210> 81 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 81 ttataagaca agcgcaggg 19

Claims (23)

Compared to the parent cell, the activity of the enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA and the activity of the enzyme that catalyzes the conversion of pyruvate to lactate As a genetically engineered yeast cell having an increased lactate-producing ability, the enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA is an acetylated acetaldehyde dehydrogenase belonging to EC 1.2.1.10. dehydrogenase: A-ALD), and the enzyme catalyzing the conversion of pyruvate to lactate is lactate dehydrogenase (LDH) belonging to EC 1.1.2.27 or EC 1.1.1.28, and Yeast cells in which the activity of an enzyme that catalyzes the conversion of acetaldehyde to ethanol is reduced compared to the parent cell. delete delete The yeast cell of claim 1, wherein the enzyme catalyzing the conversion of acetaldehyde to acetyl-CoA is MhpF, DmpF, or EutE. The yeast cell of claim 1, wherein the enzyme catalyzing the conversion of acetaldehyde to acetyl-CoA has 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or 54. The method according to claim 1, wherein the yeast cell is an exogenous gene encoding an enzyme that catalyzes the conversion of the acetaldehyde to acetyl-CoA and an exogenous gene encoding an enzyme that catalyzes the conversion of the pyruvate to lactate. Yeast cells containing one or more of the genes (exogenous gene). The yeast cell of claim 6, wherein the exogenous gene encoding an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA encodes an amino acid sequence having 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 1 or 54. . The yeast of claim 6, wherein the exogenous gene encoding an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA has 95% or more sequence identity with the nucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 53. cell. The yeast cell of claim 1, wherein the enzyme catalyzing the conversion of pyruvate to lactate has 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 6. The yeast cell of claim 6, wherein the exogenous gene encoding an enzyme that catalyzes the conversion of pyruvate to lactate encodes an amino acid sequence having 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 6. The yeast cell of claim 10, wherein the exogenous gene encoding an enzyme that catalyzes the conversion of pyruvate to lactate has 95% or more sequence identity with the nucleotide sequence of SEQ ID NO: 7. The yeast cell of claim 1, wherein the yeast cell has a gene encoding an enzyme that catalyzes the conversion of the acetaldehyde to ethanol. The yeast cell of claim 1, wherein the enzyme catalyzing the conversion of acetaldehyde to ethanol is an alcohol dehygrogenase (ADH) belonging to EC 1.1.1.1. The method according to claim 1, wherein the yeast is Saccharomyces, Candida, Schizosaccharomyces, Kluyveromyces, Pichia, Isschenkia. Yeast cells that belong to any one selected from the group consisting of, And Hansenula. The method of claim 1, wherein the enzyme catalyzes the conversion of pyruvate to acetaldehyde, the enzyme catalyzes the conversion of lactate to pyruvate, and dihydroxyacetone phosphate (DHAP) is converted to glycerol-3-phosphate (G3P). An enzyme that catalyzes the conversion of glycerol-3-phosphate (G3P) to glycerol, an enzyme that catalyzes the conversion of acetaldehyde to acetate, or a yeast cell in which the activity of a combination thereof is reduced. The method of claim 15, wherein the enzyme catalyzing the conversion of pyruvate to acetaldehyde belongs to EC 4.1.1.1, and the enzyme catalyzing the conversion of lactate to pyruvate is EC 1.1.2.4 or EC 1.1.2.3. The enzyme catalyzing the conversion of DHAP to G3P belongs to EC 1.1.1.8, and the enzyme catalyzing the conversion of glycerol-3-phosphate (G3P) to glycerol belongs to EC 3.1.3.21, and The enzyme that catalyzes the conversion of acetaldehyde to acetate belongs to EC 1.2.1.4. Yeast cells. The method of claim 15, wherein the enzyme that catalyzes the conversion of pyruvate to acetaldehyde is pyruvate decarboxylase (PDC), and the enzyme that catalyzes the conversion of lactate to pyruvate is lactate citchrome. -c oxidoreductase (lactate cytochrome-c oxidoreductase: CYB2), and the enzyme that catalyzes the conversion of DHAP to G3P is NAD-dependent glycerol-3-phosphate dehydrogenase (NAD-dependent glycerol-3-phosphate). dehydrogenase: GPD), and the enzyme that catalyzes the conversion of glycerol-3-phosphate to glycerol is glycerol phosphate phosphatase (GPP), and the enzyme that catalyzes the conversion of acetaldehyde to acetate is acetaldehyde dehydrogena. The first (acetaldehyde dehydrogenase) is a yeast cell. The method of claim 15, wherein the gene encoding an enzyme catalyzing the conversion of pyruvate to acetaldehyde, a gene encoding an enzyme catalyzing the conversion of lactate to pyruvate, dihydroxyacetone phosphate (DHAP) in glycerol A gene encoding an enzyme that catalyzes the conversion to -3-phosphate (G3P), a gene encoding an enzyme that catalyzes the conversion of glycerol-3-phosphate to glycerol, and an enzyme that catalyzes the conversion of acetaldehyde to acetate. A yeast cell in which the encoding gene, or a combination thereof, has been disrupted. Introducing into yeast cells a gene encoding an enzyme that catalyzes the conversion of acetaldehyde to acetyl-CoA and a gene encoding an enzyme that catalyzes the conversion of pyruvate to lactate; And destroying a gene encoding an enzyme that catalyzes the conversion of acetaldehyde to ethanol, comprising: catalyzing the conversion of acetaldehyde to acetyl-CoA. The enzyme is acetylating acetaldehyde dehydrogenase (A-ALD) belonging to EC 1.2.1.10, and the enzyme catalyzing the conversion of pyruvate to lactate is EC 1.1.2.27 or EC 1.1.1.28 Lactate dehydrogenase (LDH) belonging to, and the enzyme catalyzing the conversion of acetaldehyde to ethanol is alcohol dehydrogenase (ADH) belonging to EC 1.1.1.1. . The method of claim 19, wherein a gene encoding an enzyme that catalyzes the conversion of pyruvate to acetaldehyde in the yeast cell, a gene encoding an enzyme that catalyzes the conversion of lactate to pyruvate, and dihydroxyacetone phosphate in glycerol. Genes encoding enzymes that catalyze the conversion to -3-phosphate, genes encoding enzymes that catalyze the conversion of glycerol-3-phosphate to glycerol, genes encoding enzymes that catalyze the conversion of acetaldehyde to acetate , Or destroying the combination thereof. delete Culturing the yeast cells of claim 1 to produce lactate; And
A method of producing lactate comprising the step of recovering lactate from the culture. The method of claim 22, wherein the culturing is performed under microaerobic conditions.

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