JP2003259878A - Dna encoding lactate dehydrogenase and utilization of the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an effective gene expression system by correcting imperfection of expression in expressing higher eucaryote genes in different kind of organisms such as yeasts comprising saccharomyces cerevisae, etc. <P>SOLUTION: The DNA has characteristics comprising either one of the following (a) and (b). (a) The DNA has a base sequence encoding a protein provided with lactate dehydrogenase activity. (b) The DNA hybridizes with a DNA comprising whole or a part of the base sequence or its complimentary strand in a stringent condition and has a base sequence encoding the protein provided with the lactate dehydrogenase activity. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a technique for efficiently producing lactic acid, and more specifically, to a DNA having a nucleotide sequence encoding a protein having lactate dehydrogenase activity suitable for causing yeast to produce lactic acid, and Regarding the use of DNA.

[0002]

2. Description of the Related Art Advances in recombinant DNA technology have made it possible to express foreign genes in hosts such as microorganisms, molds, plants and animals, and to propagate their transformants.
Techniques for obtaining target gene products have been developed. For example, by culturing yeast or the like, it is possible to produce a large amount of the target gene product by fermentation production.

Up to now, there have been several attempts to produce L-lactic acid by yeast. There is an attempt to produce L-lactic acid by introducing a bovine-derived lactate dehydrogenase (LDH) gene into the yeast Saccharomyces cerevisiae (Eri Adahi et al., "Modification of metabolic
pasthway of Saccaromyces cerevisiae by the express
ion of lactate dehydrogenase and deletion of pyruv
ate genes fo the lactic acid fermentation at low p
H value ", J. Ferment. Bioeng. Vol.86, No.3, 284-28
9, 1988, Danio Porro et al., "Development of metab
olically engineered Saccaromyces cerevisiae cells
for the production of lactic asid ", Biotechnol.Pro
g. Vol.11, 294-298, 1995, Special Table 2001-51658
4 publication). However, in both reports,
High production of L-lactic acid has not been observed.

A gene encoding a protein has the amino acid sequence of the protein
It is encoded by a codon (genetic code) consisting of 3 bases, but the genetic code corresponding to one amino acid is not necessarily one. It is known that genes that are expressed in large quantities have a bias in the genetic code used by different species. Therefore, the gene encoding LDH derived from bovine is a gene consisting of the genetic code frequently used in higher eukaryotes such as mammals, and is not necessarily yeast.
In particular, it is considered not suitable for expression in Saccharomyces cerevisiae.

Further, in order to efficiently express a gene, it is necessary to connect to an optimal expression control system. By adding a sequence suitable for expression to the upstream of the start codon or connecting it to the downstream of a more suitable promoter gene, the target gene can be expressed more efficiently. On the other hand, it is necessary to consider the metabolic system related to the target product in the host.

[0006]

As described above, in order to efficiently produce lactic acid by culturing a transformed microorganism, it is desired that the DNA sequence be efficiently expressed in the host, and efficient expression is possible. It is desired to be controlled by various expression control systems. An object of the present invention is to correct imperfections in expressing higher eukaryotic DNA, for example, the LDH gene of a mammal such as bovine, in a heterologous organism including yeast such as Saccharomyces cerevisiae. To provide a more efficient gene expression system, specifically, an expression system for producing L-lactic acid.

[0007]

The present inventors have found that various types of L
As a result of various attempts to optimize the expression of the DH gene in microorganisms, a DNA sequence suitable for increasing the amount of L-lactic acid produced was found. Then, the above-mentioned problems have been solved by utilizing a DNA construct having such a DNA sequence. That is, according to the present invention,
The following means can be provided.

(1) A DNA having any one of the following characteristics (a) and (b): (A) It encodes a protein having lactate dehydrogenase activity and has the base sequence shown in SEQ ID NO: 1. (B) having a base sequence that encodes a protein having lactate dehydrogenase activity by hybridizing under stringent conditions with a DNA consisting of the whole or a part of the base sequence of SEQ ID NO: 1 or a complementary strand thereof . (2) A DNA having any one of the following characteristics (c) and (d). (C) Encodes a protein having lactate dehydrogenase activity and has the nucleotide sequence set forth in SEQ ID NO: 3. (D) having a base sequence encoding a protein having lactate dehydrogenase activity, which hybridizes with a DNA consisting of the whole or a part of the base sequence of SEQ ID NO: 3 or a complementary strand thereof under stringent conditions . (3) having a Kozak sequence across the start codon,
The DNA according to (1) or (2). (4) A DNA encoding a protein having lactate dehydrogenase activity, which has the following characteristics (e) to (g): (e) Designed using the codon usage in Saccharomyces cerevisiae, (f) Having a Kozak sequence across the start codon,
And (g) the GC content of a plurality of regions separated by 100 bases from the start codon of the coding region is in the range of 20% or more and 40% or less.
A. (5) The protein having the lactate dehydrogenase activity is
Bovine lactate dehydrogenase, or a protein consisting of an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added in the amino acid sequence of bovine lactate dehydrogenase (4 ) DN
A. (6) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids consist of a substituted, deleted, inserted and / or added amino acid sequence, which encodes a protein having lactate dehydrogenase activity, and has a sequence Number 1 or 3
DN consisting of all or part of the nucleotide sequence described in 1.
A DNA that hybridizes with A or its complementary strand under stringent conditions. (7) In the amino acid sequence shown in SEQ ID NO: 2, one or several amino acids consist of a substituted, deleted, inserted and / or added amino acid sequence, which encodes a protein having lactate dehydrogenase activity, and has a sequence A DNA having a base sequence in which the base sequence of the coding region of No. 1 or SEQ ID NO: 3 is modified by the substitution, deletion, insertion and / or addition of the amino acid. (8) A DNA segment comprising the DNA according to any one of (1) to (7), and a promoter segment to which the DNA segment is operably linked.
DNA construct. (9) The DNA construct according to (8), wherein the promoter segment has a pyruvate decarboxylase 1 gene promoter activity derived from Saccharomyces cerevisiae. (10) The DNA construct according to (8) or (9), which comprises a DNA segment for homologous recombination with the host chromosome. (11) A DNA construct comprising a DNA segment comprising the DNA according to any one of (1) to (7) and a DNA segment for homologous recombination with a host chromosome. (12) The DNA construct according to (11), which comprises a DNA segment for homologous recombination so that the DNA segment comprising the DNA according to any one of (1) to (7) can be controlled by a promoter of a host chromosome. (13) The DNA construct according to (11) or (12), wherein the host chromosome promoter is a Pyruvate decarboxylase 1 gene promoter derived from Saccharomyces cerevisiae. (14) The DNA construct according to any one of (8) to (13), further comprising a constituent segment of any vector selected from the group consisting of a plasmid, a bacteriophage, a retrotransposon and an artificial chromosome. DNA construct. (15) A transformant obtained by transforming a host cell with the DNA construct according to any one of (8) to (13). (16) The transformant according to (15), wherein the host cell is any of bacteria, yeast, animal cells, insect cells and plant cells. (17) The transformant according to (15), wherein the host cell is Saccharomyces cerevisiae. (18) The pyruvate decarboxylase 1 gene promoter on the host chromosome or a homologue of the promoter substituted with the promoter, which is operably linked to the downstream of the promoter, according to any one of claims 1 to 7. A transformed Saccharomyces yeast comprising a DNA segment that is DNA. (19) Production of lactic acid, which comprises a step of culturing cells derived from the transformant according to any one of (15) to (18) and a step of separating lactic acid from the culture obtained in the step. Method.

The present invention provides a DNA having a nucleotide sequence encoding a protein having LDH activity optimized for expression in a microorganism, particularly yeast. By using this DNA, a transformant capable of highly producing L-lactic acid can be easily obtained, and thus L-lactic acid can be efficiently produced.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described in detail below. (DNA) The DNA of the present invention is L-lactate dehydrogenase (L
DH) DNA having or having a base sequence encoding a protein having DH activity.
Here, LDH is known as an enzyme that mediates the reaction of producing L-lactic acid from pyruvic acid in the glycolytic system of prokaryotic organisms such as lactic acid bacteria or eukaryotic microorganisms such as mold, or in muscle tissues of higher organisms. . Further, in the present specification, DNA does not matter its origin such as cDNA, genomic DNA, or synthetic DNA. Also, even with a single strand,
It may be a double strand having its complementary strand. It may also contain a natural or artificial nucleotide derivative.

As LDH, there are various homologues depending on the type of organism or even in vivo. The protein having LDH activity in the present invention includes LDH naturally derived as well as LDH artificially synthesized by chemical synthesis or genetic engineering. The LDH is preferably derived from a prokaryote such as a lactic acid bacterium or a eukaryote such as a mold, more preferably derived from a higher eukaryote such as a plant, an animal or an insect, and further preferably, including bovine. It is derived from higher eukaryotes including mammals. Most preferably, it is bovine LDH. Examples of bovine LDH include a protein having the amino acid sequence shown in SEQ ID NO: 2. Furthermore, the LDH in the present invention also includes these homologs of LDH. LDH homologue is a naturally derived LD
In the amino acid sequence of H, a protein having one or several amino acid substitutions, deletions, insertions and / or additions and having LDH activity, and homology between naturally occurring LDH and amino sequence Contains at least 70%, preferably 80% or more and a protein having LDH activity.

The DNA of the present invention can have a nucleotide sequence using the codon usage which is frequently used in the host organism to be transformed. For example, the present DNA can have a nucleotide sequence genetically encoded using the codon usage in the genus Saccharomyces, particularly Saccharomyces cerevisiae. The codon usage in Saccharomyces cerevisiae is shown in FIG. In this codon usage, it is preferable to use the underlined codon as the codon of the amino acid corresponding to it. That is, UUU (Phe), UCU (Ser), UAU
(Tyr), UGU (Cys), UUG (Leu), C
AU (His), CCA (Pro), CAA (Gl
n), AUU (Ile), ACU (Thr), AAU
(Asn), AAA (Lys), AGA (Arg), A
UG (Met), GUU (Val), GCU (Al
a), GAU (Asp), GGU (Gly), GAA
It is preferable to use (Glu). Although these are all shown in the form of base sequences on RNA, D
In the case of NA, U becomes T.

When the DNA encoding LDH is modified based on the codon usage of a heterologous organism such as Saccharomyces to design a DNA having a new base sequence, the codon of the base sequence before modification (natural origin) is 70 % Or more, the codon usage is preferably applied, more preferably 80% or more, further preferably 90% or more, and most preferably the codon usage is applied to all codons. There is.

As a preferred embodiment of the DNA of the present invention, the bovine LDH base sequence (SEQ ID NO: 41) is used as the codon usage (codon usage of Saccharomyces cerevisiae).
The DNA designed by applying can be mentioned. Among them, DNA having the base sequences shown in SEQ ID NO: 1 and SEQ ID NO: 3 or consisting of these base sequences is a preferred embodiment. In the base sequence of the coding region of the base sequence, codons different from the original codons are used for all amino acids except methionine.

In a preferred embodiment, it is preferable to have a Kozak sequence near the start codon of the coding region of the protein having LDH activity (with the start codon sandwiched). Specifically, for DNA, 5'-ANNAT
GG-3 ′ However, N may be any of A, T, C, and G. ) Is preferred (for RNA, T is U for this sequence).
Shall be replaced with. ). For example, the base sequence shown in SEQ ID NO: 3 has a base sequence of 5′-ACAATGG-3 ′ with a start codon (ATG) in between.

Further, it is preferable that the present DNA does not contain a sequence that destabilizes mRNA.
For example, it is preferable not to include a poly A signal sequence, a sequence including a stem-loop structure, a sequence such as TATATA sequence, and a repetitive sequence. For example, 1
It is preferable that the appearance rate of a sequence of about 0 bp is 2% or less in DNA (particularly the protein coding region), and more preferably, it is not contained at all.

Further, it is preferable to design the whole DNA so that there is no difference in the bias of the GC content. For example, it is preferable that the GC content of each of a plurality of regions separated by 100 bases from the 5'side (particularly, the start codon) is in the range of about 20% or more and about 40% or less. Further, it is preferable that the total GC content is about 20% or more and about 40% or less. Natural LDH sequence from bovine (SEQ ID NO: 41)
Has a total GC content of 44.94%, and, for example, in the nucleotide sequences of SEQ ID NOs: 1 and 3,
This is because the total GC content is 31.4% and 32.0%, respectively. More preferably, it is about 25% or more and about 35% or less. Alternatively, the total GC content is 10% or more and 15% or less, compared to the natural DNA sequence,
It is preferable to design to reduce the amount by 13% or more and 14% or less.

As the DNA of the present invention, application of codon usage in the host organism to be introduced, presence of Kozak sequence, suitable GC content (dispersion of GC content, total GC content)
It is preferable to have at least one characteristic of (content). More preferably, it has two or more types, most preferably three types. In addition, the codon usage in the host organism is preferably applied to the present DNA. In particular, when transformation is carried out using the genus Saccharomyces (including cerevisiae species) as a host, it is preferable that the codon usage in the genus Saccharomyces (particularly cerevisiae species) is applied.

Furthermore, it is preferable that the DNA is designed so as not to have a restriction enzyme site inappropriate in the gene cloning step, particularly in the coding region. Specifically, it is preferable not to include sites such as Eco RI, Xho I and Afl II. On the other hand, considering a gene cloning operation, it is preferable to provide a restriction enzyme site useful in operation outside the coding region. For example, Eco
Sites such as RI, Xho I, and Afl II can be located upstream and / or downstream of the coding region. For example, in the base sequences shown in SEQ ID NOS: 1 and 3, Eco RI, Xho within the range from the start codon to the stop codon.
I don't have Afl II site. Also, SEQ ID NO: 3
In the base sequence described in, the upstream side of the start codon,
It has an Eco RI site, and Xho is located downstream of the stop codon.
I, equipped with Afl II.

The DNA of the present invention also includes DNA homologues consisting of the nucleotide sequences shown in SEQ ID NOS: 1 and 3. Examples of homologues include DNAs that hybridize with these DNAs under stringent conditions. That is, any of these DNAs
Is a DNA that hybridizes with the whole or a part thereof or a complementary strand thereof under stringent conditions. Such homologues simultaneously code for proteins with LDH activity. The DNA that hybridizes under stringent conditions is, for example, a DNA in which one or a plurality of continuous sequences of any at least 20, preferably 25, and more preferably at least 30 of the original base sequence are selected. As a probe DNA, hybridization techniques known to those skilled in the art (Current Protocols I Mo
lecular Biology edit. Ausubel et al., (1987) Publi
sh. John Wily & SonsSectoin 6.3-6.4) and the like. Here, the stringent condition is a hybridization temperature of 37 ° C in the presence of 50% formamide, and the more stringent condition is about 42 ° C. More severe conditions may be about 65 ° C. in the presence of formamide.

In addition, in the amino acid sequence of bovine LDH (for example, SEQ ID NO: 2), one or several amino acids are substituted, deleted, inserted and / or added, and have LDH activity. It hybridizes with a DNA encoding the protein and consisting of all or part of the nucleotide sequence encoding the bovine LDH (for example, the nucleotide sequence set forth in SEQ ID NO: 1 or 3) or a complementary strand thereof under stringent conditions. The DNA of the present invention is also included in the DNA of the present invention. Further, in the amino acid sequence encoded by the base sequence described in the coding region of SEQ ID NO: 1 or SEQ ID NO: 3, one or several amino acids consist of an amino acid sequence in which substitution, deletion, insertion and / or addition is carried out, The DNA of the present invention also includes a DNA encoding a protein having LDH activity and having a base sequence in which the base corresponding to the substitution of the amino acid is modified in the coding region of SEQ ID NO: 1 or SEQ ID NO: 3. . The number of mutations in the amino acid sequence is not limited as long as the function of the original protein can be maintained, but is preferably 70% or less of all amino acids, more preferably 30% or less, and further preferably 20%. Within%.

Further, such a DNA homolog contains a base sequence having a homology of at least 80%, preferably 90% or more with respect to the coding region of the base sequence of the original DNA, or the base sequence. DN consisting of
It is preferably A. The homology of the DNA base sequence can be determined by the gene analysis program BLAST or the like.

DNA can be chemically synthesized and is known as a method for synthesizing long-chain DNA by Fujimoto et al. (Hideya Fujimoto, synthetic gene production method, Plant Cell Engineering Series 7 Plant). PCR Experimental Protocol, 19
97, Shujunsha Co., Ltd., p95-100).

The modification of the amino acid sequence is carried out by introducing a site-specific mutation into the amino acid sequence to be modified (Current Protocols I Molecular Biology edit. Ausu
bel etal., (1987) Publish .John Wily & Sons Secto
in 8.1-8.5) and the like, and appropriately introducing substitutions, deletions, insertions, and / or additional mutations. In addition, such modifications are not limited to artificially introduced or synthesized mutations, but also include those that are caused by artificial mutation treatment or are not limited to this and that are also caused by amino acid mutations in the natural world. .

(DNA construct) By transforming a host cell with the DNA of the present invention and expressing a protein encoded by this DNA, L-lactic acid can be produced in the host cell by its LDH activity. it can. For transformation, DN consisting of this DNA
A DNA construct is used that allows the A segment to be expressed in the host cell. The form of the DNA construct for transformation is not particularly limited, and it may be plasmid (DNA), bacteriophage (DNA), retrotransposon (D).
NA), artificial chromosomes (YAC, PAC, BAC, MAC)
Etc.) can be selected and adopted according to the introduction form of the foreign gene (extrachromosomal or intrachromosomal) and the type of host cell. Therefore, the present DNA construct can comprise the constituent segment of the vector of any of these aspects in addition to the present DNA. Preferred prokaryotic vectors, eukaryotic vectors, animal cellular vectors, plant cellular vectors are well known in the art.

The plasmid DNA may be, for example, pRS413, pRS415, pRS416, YC.
Y such as p50, pAUR112 or pAUR123
Cp-type E. coli-yeast shuttle vector, YEp-type E. coli-yeast shuttle vector such as pYES32 or YEp13, pRS403, pRS404, pRS405,
YIp type E. coli-yeast shuttle vector such as pRS406, pAUR101 or pAUR135, plasmid derived from E. coli (pBR322, pBR325, pUC
18, pUC19, pUC119, pTV118N, p
TV119N, pBluescript, pHSG29
8, ColSG such as pHSG396 or pTrc99A
System plasmid, pACYC177 or pACYC184
P1A-based plasmids such as pMW118, pMW11
9, pSC101 such as pMW218 or pMW219
System plasmids), plasmids derived from Bacillus subtilis (for example,
pUB110, pTP5, etc.) and the like. As phage DNA, λ phage (Charo
n4A, Charon21A, EMBL3, EMBL
4, λgt100, gt11, zap), φX174,
M13mp18, M13mp19, etc. can be mentioned. Examples of the retrotransposon include Ty factor and the like. For YAC, pYACC2
And so on.

To prepare the present DNA construct, the present DNA
By cleaving a fragment or the like containing an appropriate restriction enzyme and inserting it into the restriction enzyme site or multi-cloning site of the vector DNA used. This DNA
The first embodiment of the construct comprises a promoter segment operably linked to a DNA segment consisting of the present DNA. That is, the present DNA segment is linked to the downstream side of the promoter so as to be controllable by the promoter.

In the expression of the present DNA, that is, a protein having LDH activity, expression in yeast is preferable, and therefore a promoter expressed in yeast is preferably used. Examples of such promoters include pyruvate decarboxylase gene promoter, gal
1 promoter, gal10 promoter, heat shock protein promoter, MFα1 promoter,
It is preferable to use PH05 promoter, PGK promoter, GAP promoter, ADH promoter, AOX1 promoter and the like. In particular, the pyruvate decarboxylase (1) promoter derived from the genus Saccharomyces is preferable, and it is more preferable to use the pyruvate decarboxylase 1 gene promoter derived from Saccharomyces cerevisiae. This is because these promoters are highly expressed in the ethanol fermentation pathway of the genus Saccharomyces (Cerevisiae). The promoter sequence can be isolated by the PCR amplification method using the genomic DNA of pyruvate decarboxylase 1 gene of Saccharomyces yeast as a template. The nucleotide sequence of the promoter derived from Saccharomyces cerevisiae is shown in SEQ ID NO: 40. The promoter segment in the present DNA construct is composed of a DNA having the base sequence of SEQ ID NO: 40, a base sequence in which one or several bases are deleted, substituted or added in the base sequence, and A DNA having a promoter activity, a DNA prepared from all or part of the nucleotide sequence represented by SEQ ID NO: 40, or a DNA hybridizing with a complementary strand thereof under stringent conditions and having a promoter activity (in other words, , A homologue of the promoter) can be used.

The second DNA construct, which is another embodiment of the present DNA construct, comprises a DNA segment for homologous recombination of the host chromosome in addition to the present DNA. The DNA segment for homologous recombination is a DNA sequence homologous to the DNA sequence near the target site into which the present DNA is to be introduced in the host chromosome. At least one, and preferably two, DNA segments for homologous recombination are provided. For example, two DNA segments for homologous recombination are provided on the chromosome at the upstream and downstream sides of the target site.
It is preferable that the DNA sequence is homologous to NA, and the present DNA is ligated between these DNA segments.

When the present DNA is introduced into the host chromosome by homologous recombination, the present DNA can be introduced controllably by the promoter on the host chromosome. In this case, by introducing the target gene, at the same time, the endogenous gene that should originally be regulated by the promoter can be destroyed, and the foreign DNA can be expressed in place of this endogenous gene. In particular, it is useful when the promoter is a high expression promoter in host cells.

In order to create such an expression system on the host chromosome, a highly expressed gene is targeted in the host chromosome, and the present DNA is introduced downstream of the promoter controlling this gene so as to be controlled by the promoter. It is preferable. When an ethanol-fermenting microorganism such as yeast is used as the host, the pyruvate decarboxylase gene (particularly pyruvate decarboxylase 1 gene) is targeted and LDH activity is controlled under the control of the endogenous pyruvate decarboxylase gene promoter. DNA encoding a protein
Can be introduced. In this case, DN for homologous recombination
The A segment is the LD of the pyruvate decarboxylase 1 gene.
It can be homologous to the structural gene region of H or a sequence in the vicinity thereof (including a sequence near the start codon, a sequence in the upstream region of the start codon, a sequence in the structural gene, etc.). Preferably, a saccharomyces genus (particularly cerevisiae) is used as a host, and a DNA construct targeting the pyruvate decarboxylase 1 gene of this host is used. According to such a DNA construct, the disruption of the pyruvate decarboxylase 1 gene and the replacement of this structural gene portion with LDH can be achieved with one vector. Pyruvate decarboxylase 1
Is an enzyme that mediates the reverse addition reaction of pyruvate to acetaldehyde, and disruption of this gene is expected to suppress ethanol production via acetaldehyde, and LDH that uses pyruvate as a substrate It can be expected that lactic acid production will be promoted.

Even the first DNA construct can be used as a DNA construct for homologous recombination by including a DNA segment for homologous recombination with the host chromosome. In the first DNA construct, the promoter segment in the DNA construct can also be used as the DNA segment for homologous recombination with the host chromosome.
For example, for the host Saccharomyces cerevisiae,
A DNA construct having a promoter on the Saccharomyces cerevisiae host chromosome, for example, a pyruvate decarboxylase 1 gene promoter as a promoter segment constitutes a targeting vector having the gene as a target site. In this case, it is preferable to provide a homologous sequence to the structural gene region of the pyruvate decarboxylase 1 gene.

In the DNA construct, a cis element such as an enhancer, if necessary, in addition to a terminator,
A splicing signal, a poly A addition signal, a selection marker, and a ribosome binding sequence (SD sequence) can be linked. The selection marker is not particularly limited, and various known selection marker genes such as drug resistance gene and auxotrophic gene can be used. For example, dihydrofolate reductase gene, ampicillin resistance gene, neomycin resistance gene and the like can be used.

(Transformation with DNA construct) Once D
Once the NA construct is constructed, it can be transformed into an appropriate host cell by transformation, transfection, conjugation, protoplast fusion, electroporation, lipofection, lithium acetate, particle gun, calcium phosphate precipitation, Agrobacterium. This can be introduced by any of various suitable means such as the Um method, the PEG method, and the direct microinjection method. After the introduction of the DNA construct, the recipient cells are cultured in selective medium.

The host cells are Eshrichia coli, Bacillus s.
Bacteria such as ubtilis, Saccharomyces cerevisiae, Saccharomyces pombe,
Yeasts such as Pichia pastoris,
Insect cells such as sf9 and sf21, COS cells, animal cells such as Chinese hamster ovary cells (CHO cells), and plant cells such as sweet potato and tobacco can be used. Preferable are microorganisms that perform alcohol fermentation such as yeast or acid-resistant microorganisms, for example, yeasts such as Saccharomyces genus such as Saccharomyces cerevisiae. Specifically, Saccharomyces
Cerevisiae IFO2260 strain and the same YPH strain.

In the transformant transformed with the DNA construct, the constituent components of the DNA construct are present on the chromosome or on an extrachromosomal factor (including an artificial chromosome). When the DNA construct is maintained extrachromosomally or when it is integrated into the chromosome by random integration, another enzyme having pyruvate, which is a substrate of LDH, as a substrate, for example, pyruvate decarboxylation is used. The enzyme gene (in Saccharomyces yeast, pyruvate decarboxylase 1 gene) is preferably knocked out by a targeting vector. When the above-mentioned DNA construct capable of achieving homologous recombination is introduced, it is operably linked by the promoter downstream of the desired promoter on the host chromosome or a homolog of the promoter substituted with the promoter. There will be a DNA segment that is the present DNA that has been processed. In a transformant of a yeast of the genus Saccharomyces, on the host chromosome, the pyruvate decarboxylase 1 gene promoter or a homolog of the promoter substituted with the promoter is provided downstream of the DNA so as to be controllable by the promoter. Is preferred. In addition, usually, a selectable marker gene and a part of the disrupted structural gene (site corresponding to the homologous sequence on the DNA construct) are present on the downstream side of the present DNA in the homologous recombinant.

Whether or not the present DNA has been introduced under the desired promoter can be confirmed by PCR or Southern hybridization. For example, it can be confirmed by preparing DNA from the transformant, performing PCR with an introduction site-specific primer, and detecting a band expected in electrophoresis of the PCR product. Alternatively, it can be confirmed by performing PCR with a primer labeled with a fluorescent dye or the like. These methods are well known to those of ordinary skill in the art.

(Production of Lactic Acid) By culturing the transformant obtained by introducing the DNA construct, lactic acid, which is an expression product of the foreign gene, is produced in the culture. Lactic acid can be obtained by performing the step of separating lactic acid from the culture. In addition, in the present invention, the culture includes the culture supernatant, cultured cells or cells, and disrupted cells or cells. In culturing the transformant of the present invention, culture conditions can be selected according to the type of transformant. Such culture conditions are well known to those skilled in the art. A medium for culturing a transformant obtained by using a microorganism such as Escherichia coli or yeast as a host contains a carbon source, a nitrogen source, inorganic salts and the like that can be assimilated by the microorganism, and efficiently cultivates the transformant. As long as the medium can be used, either a natural medium or a synthetic medium can be used. As the carbon source, carbohydrates such as glucose, fructose, sucrose and starch, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol and propanol can be used. As the nitrogen source, it is possible to use ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium salts of inorganic acids or organic acids such as ammonium phosphate or other nitrogen-containing compounds, as well as peptone, meat extract, corn steep liquor and the like. it can. As the inorganic substance, potassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride,
Ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used.

Culturing is usually carried out at 30 ° C. for 6 to 24 hours under aerobic conditions such as shaking culture or aeration stirring culture. The pH is preferably maintained at 2.0 to 6.0 during the culture period. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. During the culture, constituent substances such as ampicillin and tetracycline can be added to the medium, if necessary.

RPM which is generally used as a medium for culturing the transformant obtained by using animal cells as a host
I1640 medium, DMEM medium, or a medium obtained by adding fetal bovine serum or the like to these mediums can be used.
Culturing is usually performed at 37 ° C. for 1 to 30 days in the presence of 5% CO ₂ . If necessary, an antibiotic such as kanamycin or penicillin may be added to the medium during the culture.

After the completion of the culture, in order to separate the gene product from the culture, a usual means for producing a protein or the like can be used. For example, when it is produced in a transformed cell, the gene product can be separated from the cell by disrupting the cell by an ultrasonic disruption treatment, an abrasion treatment, a pressure disruption or the like by a conventional method. In this case, a protease is added as needed. When the gene product is produced in the culture supernatant, the solution is subjected to filtration, centrifugation, etc. to remove solids, and if necessary, treated with prototamine to remove nucleic acids.

Ammonium sulfate, alcohol, acetone and the like are added to these crude extract fractions to fractionate them, and the precipitate is collected to obtain a crude protein solution. This protein solution can be subjected to various chromatography, electrophoresis, etc. to obtain a purified enzyme preparation. For example, Sephadex,
Gel filtration using ultra gel or bio gel, ion exchange chromatography, electrophoresis method using polyacrylamide gel, affinity chromatography, fractionation method using reverse phase chromatography, etc. are appropriately selected or combined. Thus, the purified target gene product can be obtained. The amino acid sequence of the purified gene product can be determined by a known amino acid analysis method. The above-mentioned culture method and purification method are merely examples and are not intended to be limited thereto.

[0043]

EXAMPLES Specific examples of the present invention will be described below, but it is not intended to limit the present invention to the following specific examples.

(Example 1: Design of DNA sequence of L-lactate delipidase enzyme gene) L-lactate dehydrogenase, a protein derived from bovine which is a higher eukaryote, is efficiently used in the genus Saccharomyces cerevisiae. To produce
With respect to the DNA (SEQ ID NO: 41) encoding the amino acid sequence of bovine L-lactate dehydrogenase, a novel gene sequence which is not found in nature was designed using the following items as design guidelines. 1) Codons that are frequently used in Saccharomyces cerevisiae were used. 2) Kozak sequence (ANNATG) across the start codon.
G) was added. 3) The unstable sequences and repetitive sequences of mRNA were eliminated as much as possible. 4) The bias of GC content was made uniform throughout the entire region. 5) It was designed to prevent restriction enzyme sites inappropriate for gene cloning in the designed sequence. 6) EcoRI, XhoI, and AflIII sites, which are useful restriction enzymes, were added to both ends for incorporation into a chromosome-introduced vector. Regarding the frequency of codon usage in yeast, the codon usage of Saccharomyces cerevisiae obtained from the codon usage database (http://www.kazusa.or.jp/codon/) is shown in FIG. In this figure, codons frequently used corresponding to specific amino acids are identified (underlined codons).

In addition to the use of the codon usage in the codon usage shown in FIG. 2, a novel DNA sequence (999 bp) encoding a protein having LDH activity obtained based on the above design guidelines 2) to 5) , LDHKCB
It is called a gene. ) Is shown in SEQ ID NO: 1. In addition, a DNA sequence including the DNA sequence shown in SEQ ID NO: 1 and the upstream side of its start codon and the downstream side of its stop codon (1052 bp)
(Hereinafter referred to as LDHKCB sequence) is shown in SEQ ID NO: 3 and FIG.

In the DNA sequence shown in SEQ ID NO: 1, all amino acids except methionine used codons different from those used in the original DNA sequence. The newly adopted codons were all the codons shown in FIG. In addition, LDH derived from the original cow
FIG. 3 shows the results of computer-aided homology analysis of the gene and the LDHKCB gene. As is clear from FIG. 3, it was found that a large number of substitutions were required over almost the entire DNA sequence.

(Example 2: Total synthesis of LDHKCB sequence)
In this example, the method of Fujimoto et al. Known as a method for synthesizing long-chain DNA (Hideya Fujimoto, synthetic gene production method, Plant Cell Engineering Series 7, plant PCR experiment protocol,
1997, Shujunsha, p95-100). The principle of this method is to prepare an oligonucleotide primer of about 100 MER so that the 3'end has an overlap of about 10 to 12 mer, and use the overlapping region of each oligonucleotide primer to extend the defective portion. , PCR using primers at both ends
It is to amplify by doing. This operation is sequentially repeated to synthesize the target long-chain DNA.
The Gene Amp PCR sys is used for the PCR amplification device.
tem9700 (PE, Applied Biosys
tems) was used. Specifically, first mix two types of oligonucleotide primers to be ligated, and
In the presence of OD-plus-DNA polymerase (Toyobo), 96 ° C, 2 minutes, 68 ° C, 2 minutes, 54 ° C2
DNA extension reaction was performed under the reaction conditions of 72 ° C. for 30 minutes. Next, using 1/10 amount of this sample as a template, in the presence of primers at both ends, after 96 ° C. for 2 minutes, (96 ° C. for 30 seconds →
25 cycles of 55 ° C. for 30 seconds → 72 ° C. for 90 seconds were performed, and then PCR reaction was performed at 4 ° C. The buffer, dNTPmix, etc. in the reaction are DNA polym
The one attached to the erase was used. A series of overlapping PCR methods were sequentially performed according to FIG. 4 to prepare the final target soot gene fragment. Various primers shown in FIG. 4 (BA, B01, BB, B02, BC, B03, BD, B04, BE, B05, B
F, B06, BG, B07, BH, B08, BI, B09, BJ, B10, BK, B1
DNA sequences of all 28 types of primers of 1, BL, B12, BM, B13, BN, and B14 are shown in SEQ ID NOS: 5 to 32, respectively. After confirming the nucleotide sequence of the synthesized LDHKCB sequence, it was treated with a restriction enzyme with EcoRI, and similarly, EcoRI
Enzymatically treated pCR2.1 TOPO Vector
(Invitrogen) was ligated by a conventional method. This vector was designated as pBTOPO-LDHKCB vector.

(Example 3: Construction of vector for introducing yeast chromosome) A yeast chromosome-introducing vector was constructed using the LDHKCB sequence totally synthesized in Example 2. This vector was designated as pBTRP-PDC1-LDHKCB, and its plasmid map is shown in FIG. 1. Isolation of PDC1P Fragment for pBTrp-PDC1-LDHKCB Construction The PDC1P fragment was isolated from the Saccharomyces cerevisiae YPH strain (St
PCR using ratagene's genomic DNA as a template
Isolation was performed by the amplification method.

The genomic DNA of the Saccharomyces cerevisiae YPH strain is a genomic DNA preparation kit, Fast DNA Kit.
(Bio 101) was used and the details were prepared according to the attached protocol. DNA concentration is spectrophotometer Ultro
spec 3000 (Amersham Pharma)
Cia Biotech).

In the PCR reaction, Pyrobest DNA Polymerase (Takara Shuzo), which is highly accurate for the amplified fragment, was used as an amplification enzyme. Saccharomyces cerevisiae YPH strain genomic DNA 50 ng / sample, primer DNA 50 pmol / sample, and Pyrobest DNApolymerase 0.
Two units / sample were prepared in a total reaction volume of 50 μl. PCR amplification device Gene Amp
PCR system 9700 (PE Applied)
DNA amplification was performed by Biosystem.
The reaction conditions of the PCR amplification device were as follows:
25 cycles of 6 ° C. for 30 seconds → 55 ° C. for 30 seconds → 72 ° C. for 90 seconds) and then 4 ° C. The amplified fragment of PDCl primer was subjected to 1% TBE agarose gel electrophoresis to confirm the amplified gene fragment. The primer DNA used in the reaction was synthetic DNA (Sawasde Technology Co., Ltd.), and the DNA sequence of this primer was as follows.

PDCIP-LDH-U (31 mer,
Tm value 58.3 ° C) Addition of restriction enzyme BamH1 site to the end: ATA TAT GGA TCC GCG TTT AT
TTAC CTA TCTC (SEQ ID NO: 33) -PDClP-LDH-D (31mer, Tm value 54.
4 ° C) Add restriction enzyme EcoRI site to the end: ATA
TAT GAA TTC TTT GAT TGA TTT
GAC TGTG (SEQ ID NO: 34)

2. Construction of recombinant vector containing promoter and target gene L-lactate dehydrogenase gene (LDH gene) derived from bovine as a target gene under the control of Saccharomyces cerevisiae-derived bilbate decarboxylase 1 gene (PDCl) promoter sequence It was used.

The chromosome-transferred vector newly constructed for the construction of this recombinant vector was used as pBTrp-PDC1-L.
It was named DHKCB. The details of this vector construction example are described below. The outline of this embodiment is shown in FIGS. However,
The procedure for vector construction is not limited to this.
P is a necessary gene fragment for constructing the vector.
DCL gene promoter fragment (PDCIP) 971
bp and PDCl gene downstream region fragment (PDC1D) 5
As described above, 18 bp is a PC using the genomic DNA of Saccharomyces cerevisiae YPH strain as a template.
Isolation was performed by the R amplification method. The procedure of PCR amplification was as described above, but the following primers were used for amplification of the PDCl gene downstream region fragment. -PDC1D-LDH-U (34mer, Tm value 55.
3 ° C) Add restriction enzyme XhoI site to the end: ATA
TAT CTC GAG GCC AGC TAA CTT
CTT GGTCGA C (SEQ ID NO: 35) -PDCID-LDH-D (31mer, Tm value 54.
4 ° C) Add restriction enzyme ApaI site to the end: ATA
TAT GAA TTC TTT GAT TGA TTT
GAC TGTG (SEQ ID NO: 36)

PDC1P and PD obtained in the above reaction
After each C1D gene amplified fragment was purified by ethanol precipitation treatment, the PDC1P amplified fragment was subjected to restriction enzyme reaction treatment with the restriction enzymes BamHI / EcoRI and the PDC1D amplified fragment with the restriction enzymes XhoI / ApaI. All enzymes used below were manufactured by Takara Shuzo. For a detailed manual of a series of ethanol precipitation and restriction enzyme treatments, see Molecular.
Cloning A Laboratory Manual
l second edition (Maniatis
et al. , Cold Spring Harbor L
laboratory press. 1989).

A series of reaction operations in the construction of the vector were carried out according to a general DNA subcloning method. That is, the restriction enzymes BamHI / EcoRI (Takara Shuzo) and the dephosphorylation enzyme Alkaline Phos.
pBlue that has been subjected to phasease (BAP, Takara Shuzo)
esscriptII SK + vector (Toyobo)
PDC1 amplified by the above PCR method and subjected to restriction enzyme treatment
The P fragment was ligated by T4 DNA ligase reaction (FIG. 6, upper row). For T4 DNA Ligase reaction, LigaFast Rapid DNA Ligat is used.
Ion System (Promega) was used, and the details followed the attached protocol.

Then, the solution subjected to the ligation reaction was used to transform competent cells. Escherichia coli JM109 strain (Toyobo Co., Ltd.) was used as the competent cells, and the details were performed according to the attached protocol. The obtained culture solution was spread on an LB plate containing 100 μg / ml of the antibiotic ampicillin, and cultured overnight. The grown colonies were confirmed by a colony PCR method using a primer DNA of the insert fragment and a restriction enzyme treatment of a plasmid DNA preparation solution by miniprep to isolate the target vector pBPDC1P vector (Fig. 6, middle row). ).

Next, as shown in the middle part of FIG. 6, pBTOPO-L constructed by Toyota Central Research Institute Co., Ltd.
The LDHKCB gene fragment obtained by treating the DHKCB vector with the restriction enzyme EcoRI and the terminal modification enzyme T4 DNA polymerase was treated with the same restriction enzyme EcoRI and the terminal modification enzyme T4 DNA poly.
Subcloning was performed in the merase-treated pBPDC1P vector in the same manner as described above to obtain pBDC.
A PDC1P-LDHKCB vector was prepared (FIG. 6, lower row).

On the other hand, as shown in FIG. 7, the pYLDl vector constructed by Toyota Motor Corporation was treated with the restriction enzymes EcoRI / AatII and the end-modifying enzyme T4.
L obtained by treatment with DNA polymerase
The DH gene (derived from Bifidobacterium longum) was treated with the same restriction enzyme EcoRI and the end-modifying enzyme T.
4 pBPD treated with DNA polymerase
Subcloning was performed in the C1P vector by the same operation as described above to prepare a pBPDC1P-LDH1 vector (FIG. 7). The above pYLDl vector was introduced into E. coli (name: “E.coll pYLD
1 "), the National Institute of Advanced Industrial Science and Technology, Patent and Biological Deposit Center (1-1, Higashi 1-1-chome, Tsukuba-shi, Ibaraki), with the deposit number FERM BP-7423 under the Budabest Treaty (the original deposit date: Heisei 11 (19
October 26, 1999).

Then, as shown in FIG. 8, this vector was treated with XhoI / ApaI, and the amplified PDC1D fragment treated with the same restriction enzymes was ligated to pBPDC1P-.
An LDH vector was prepared (FIG. 8, upper row). Finally pBP
The DCRS-LDHII vector treated with EcoRV was added to the pRS404 vector (Stratagene).
Company) treated with AatII / SspI, T4 DNApol
The Trp marker fragment obtained by the ymerase treatment was ligated to construct a pBTrp-PDCl-LDH vector.

Next, as shown in FIG. 9, pBPDC1P
-LDHKCB vector is treated with a restriction enzyme with ApaI / EcoRI, while pBTrp-PDC1-LDH vector is treated with a fragment containing a Trp marker treated with restriction enzymes ApaI and StuI, and the amplified fragment is ligated, Chromosomal transfer type p which is the final construct
The BTrp-PDC1-LDHKCB vector was constructed.

The constructed chromosome-introduced pBTrp-PDC
The nucleotide sequence was determined to confirm the 1-LDHKCB vector. ABI PRISM 3 as a nucleotide sequence analyzer
10 Genetic Analyzer (PE Appl
ied Blosystems) was used, and the details of the method for preparing the sample, the method for using the equipment, and the like were in accordance with the manual attached to the apparatus. The vector DNA used as a sample was prepared by the alkali extraction method, and this was used as GFX DN.
A Purification kit (Amersha
n Pharmacia Blotech) column purification, then spectrophotometer Ultro spec 3000
(Amershan Pharmacia Blotec
h) was used to measure the DNA concentration.

(Example 4: Transformation of yeast) The method of gene transfer into yeast was carried out by the method of Ito et al. (Ito, H., Y. Fukuda,
K. Murata and A. Kimura, Transformation of intact y
east cells treated with alkali cations J. Bacterio
l. Vol.153, p163-168). That is, a strain deficient in tryptophan synthesizing ability of the host yeast IFO2260 strain (a strain registered in the Institute for Fermentation Research Institute) was cultured in 10 ml YPD medium at 30 ° C. until the logarithmic growth phase to collect bacteria and TE. Washing with buffer was performed. Then 0.5 ml TE buffer and 0.5 ml 0.
After adding 2M lithium acetate and shaking culture at 30 ° C. for 1 hour, the chromosome-introduced vector pBTrp-PDCl-LDHKC constructed by the method of Example 3 was used.
The B vector was treated with restriction enzymes ApaI and SacI (both Takara Shuzo) and added.

This yeast suspension was incubated at 30 ° C. for 30 minutes with shaking, and then 150 μl of 70% polyethylene glycol 400 was added.
0 (Wako Pure Chemical Industries, Ltd.) was added and stirred well. Furthermore, 30 ℃
After shaking culture for 1 hour at 42 ° C., heat shock was applied at 42 ° C. for 5 minutes to wash the cells, and the cells suspended in 200 μl of water were spread on a tryptophan selection medium.

The obtained colonies were streak-cultured in a new tryptophan selective medium, and the presence or absence of gene transfer was confirmed by PCR analysis for the selected strains whose stability was confirmed. Yeast genomic DNA used as a template for PCR is
After culturing the single colony in 2 ml YPD medium with shaking overnight, the cells were collected and 50 mM Tris-HCl 50 was added.
0 μl and glass beads (425-600 μm, Ac
id washed, SIGMA) and add 15 at 4 ° C
Prepared by vortexing for minutes. Ethanol precipitation was performed using the supernatant of this solution, and 5 μl of the prepared genomic DNA dissolved in 50 μl of sterilized water was used as a template.
PCR was performed in a reaction system of 50 μl. As a DNA amplification enzyme, EX Taq DNA Polymerase is used.
(Takara Shuzo), PCR amplification device Gene Amp P
CR system 9700 (PE Applied B
iosystems) was used. The reaction conditions of the PCR amplification device are 96 ° C. for 2 minutes and then (96 ° C. for 30 seconds → 55 ° C. for 3 seconds).
0 seconds → 72 ℃ 90 seconds) 30 cycles, then 4 ℃
And The sequences of the primers used were as follows. LDH-KCB-U: TGG TTG ATG TTA T
GG AAG AT (20mer) (SEQ ID NO: 37) LDH-KCB-D: GAC AAG GTA CAT A
AA ACC CAG (21mer) (SEQ ID NO: 38) PDC1P-U3: GTA ATA AAC ACA CC
C CCG G (19mer) (SEQ ID NO: 39)

Those which had a stable tryptophan synthesizing ability and whose PCR amplification could be confirmed under these primers were judged to be transformants into which the LDHKCB gene was appropriately introduced. In this example, as such transformants, KCB-27 strain and KCB-210 strain were used.
Strains and three strains of KCB-211 strain could be obtained. The structures on the genomic chromosome of these yeast Saccharomyces cerevisiae transformants are shown in FIG.

(Example 5: Measurement of L-lactic acid production in transformants) Fermentation tests were carried out on the three transformants prepared. As a pre-culture, Y with glucose concentration of 2%
After culturing overnight in a PD liquid medium, the cells were collected and washed, and then inoculated into a YPD liquid medium having a glucose concentration of 15% to a bacterial concentration of 1% (0.5 g / 50 ml).
Fermentation was carried out for several days in static culture at 0 ° C. 2 main fermentation broth
It was collected every 4 hours, and the amount of L-lactic acid contained in the solution was assumed. The production amount was measured using a biosensor BF-4 (Oji Scientific Instruments), and the details of the measurement method were in accordance with the attached instruction manual.

The results of measuring the amount of L-lactic acid in the culture solution on the third day of fermentation at a glucose concentration of 15% are shown in FIG. 11 and Table 1.

[Table 1] Transformants into which the LDHKCB gene was introduced (KCB-
27 strains, KCB-210 strain, KCB-211 strain),
Despite the fact that only one copy of the LDHKCB gene was introduced, 4.5 to
5.0% (45.0-50.0 g) of L-lactic acid production was observed. It was clear that this result is a dramatic increase in the production amount as compared with the L-lactic acid production in Saccharomyces cerevisiae reported in the past. Moreover, it is considered that the increase in the production amount is due to the introduction of the LDHKCB gene itself. Furthermore, since one copy can increase the production amount,
It is speculated that a further increase in production can be secured when two or more copies are introduced.

[0068]

EFFECTS OF THE INVENTION According to the present invention, it is possible to provide an expression system capable of highly expressing an exogenous lactate dehydrogenase in a host organism such as yeast.

[Sequence list]                                SEQUENCE LISTING        <110> Kabushiki Kaisha Toyota Chuo Kenkyusho       Toyota Jidosha Kabushiki Kaisha <120> Synthetic DNA and Use thereof <130> 010769 (P01-3850) <140> <141> <160> 41 <170> PatentIn Ver. 2.1 <210> 1 <211> 999 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Modified DNA       coding lactate dehydrogenase <221> CDS <222> (1) .. (996) <400> 1 atg gct act ttg aaa gat caa ttg att caa aat ttg ttg aaa gaa gaa 48 Met Ala Thr Leu Lys Asp Gln Leu Ile Gln Asn Leu Leu Lys Glu Glu   1 5 10 15 cat gtt cca caa aat aaa att act att gtt ggt gtt ggt gct gtt ggt 96 His Val Pro Gln Asn Lys Ile Thr Ile Val Gly Val Gly Ala Val Gly              20 25 30 atg gct tgt gct att tct att ttg atg aaa gat ttg gct gat gaa gtt 144 Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala Asp Glu Val          35 40 45 gct ttg gtt gat gtt atg gaa gat aaa ttg aaa ggt gaa atg atg gat 192 Ala Leu Val Asp Val Met Glu Asp Lys Leu Lys Gly Glu Met Met Asp      50 55 60 ttg caa cat ggt tct ttg ttt ttg aga act cca aaa att gtt tct ggt 240 Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile Val Ser Gly  65 70 75 80 aaa gat tat aat gtt act gct aat tct aga ttg gtt att att act gct 288 Lys Asp Tyr Asn Val Thr Ala Asn Ser Arg Leu Val Ile Ile Thr Ala                  85 90 95 ggt gct aga caa caa gaa ggt gaa tct aga ttg aat ttg gtt caa aga 336 Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu Val Gln Arg             100 105 110 aat gtt aat att ttt aaa ttt att att cca aat att gtt aaa tat tct 384 Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Ile Val Lys Tyr Ser         115 120 125 cca aat tgt aaa ttg ttg gtt gtt tct aat cca gtt gat att ttg act 432 Pro Asn Cys Lys Leu Leu Val Val Ser Asn Pro Val Asp Ile Leu Thr     130 135 140 tat gtt gct tgg aaa att tct ggt ttt cca aaa aat aga gtt att ggt 480 Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg Val Ile Gly 145 150 155 160 tct ggt tgt aat ttg gat tct gct aga ttt aga tat ttg atg ggt gaa 528 Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu Met Gly Glu                 165 170 175 aga ttg ggt gtt cat cca ttg tct tgt cat ggt tgg att ttg ggt gaa 576 Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Ile Leu Gly Glu             180 185 190 cat ggt gat tct tct gtt cca gtt tgg tct ggt gtt aat gtt gct ggt 624 His Gly Asp Ser Ser Val Pro Val Trp Ser Gly Val Asn Val Ala Gly         195 200 205 gtt tct ttg aaa aat ttg cat cca gaa ttg ggt act gat gct gat aaa 672 Val Ser Leu Lys Asn Leu His Pro Glu Leu Gly Thr Asp Ala Asp Lys     210 215 220 gaa caa tgg aaa gct gtt cat aaa caa gtt gtt gat tct gct tat gaa 720 Glu Gln Trp Lys Ala Val His Lys Gln Val Val Asp Ser Ala Tyr Glu 225 230 235 240 gtt att aaa ttg aaa ggt tat act tct tgg gct att ggt ttg tct gtt 768 Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly Leu Ser Val                 245 250 255 gct gat ttg gct gaa tct att atg aaa aat ttg aga aga gtt cat cca 816 Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg Val His Pro             260 265 270 att tct act atg att aaa ggt ttg tat ggt att aaa gaa gat gtt ttt 864 Ile Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Glu Asp Val Phe         275 280 285 ttg tct gtt cca tgt att ttg ggt caa aat ggt att tct gat gtt gtt 912 Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser Asp Val Val     290 295 300 aaa gtt act ttg act cat gaa gaa gaa gct tgt ttg aaa aaa tct gct 960 Lys Val Thr Leu Thr His Glu Glu Glu Ala Cys Leu Lys Lys Ser Ala 305 310 315 320 gat act ttg tgg ggt att caa aaa gaa ttg caa ttt taa 999 Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe                 325 330    <210> 2 <211> 332 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: Modified DNA       coding lactate dehydrogenase <400> 2 Met Ala Thr Leu Lys Asp Gln Leu Ile Gln Asn Leu Leu Lys Glu Glu   1 5 10 15 His Val Pro Gln Asn Lys Ile Thr Ile 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 Val          35 40 45 Ala Leu Val Asp Val Met 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 Asn Val Thr Ala Asn Ser Arg 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 Ile 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 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 Gln Trp Lys Ala 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 His Glu Glu Glu 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> 3 <211> 1052 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Modified DNA       coding lactate dehydrogenase <220> <221> CDS <222> (13) .. (1008) <400> 3 acagaattca ca atg gct act ttg aaa gat caa ttg att caa aat ttg ttg 51               Met Ala Thr Leu Lys Asp Gln Leu Ile Gln Asn Leu Leu                 1 5 10 aaa gaa gaa cat gtt cca caa aat aaa att act att gtt ggt gtt ggt 99 Lys Glu Glu His Val Pro Gln Asn Lys Ile Thr Ile Val Gly Val Gly      15 20 25 gct gtt ggt atg gct tgt gct att tct att ttg atg aaa gat ttg gct 147 Ala Val Gly Met Ala Cys Ala Ile Ser Ile Leu Met Lys Asp Leu Ala  30 35 40 45 gat gaa gtt gct ttg gtt gat gtt atg gaa gat aaa ttg aaa ggt gaa 195 Asp Glu Val Ala Leu Val Asp Val Met Glu Asp Lys Leu Lys Gly Glu                  50 55 60 atg atg gat ttg caa cat ggt tct ttg ttt ttg aga act cca aaa att 243 Met Met Asp Leu Gln His Gly Ser Leu Phe Leu Arg Thr Pro Lys Ile              65 70 75 gtt tct ggt aaa gat tat aat gtt act gct aat tct aga ttg gtt att 291 Val Ser Gly Lys Asp Tyr Asn Val Thr Ala Asn Ser Arg Leu Val Ile          80 85 90 att act gct ggt gct aga caa caa gaa ggt gaa tct aga ttg aat ttg 339 Ile Thr Ala Gly Ala Arg Gln Gln Glu Gly Glu Ser Arg Leu Asn Leu      95 100 105 gtt caa aga aat gtt aat att ttt aaa ttt att att cca aat att gtt 387 Val Gln Arg Asn Val Asn Ile Phe Lys Phe Ile Ile Pro Asn Ile Val 110 115 120 125 aaa tat tct cca aat tgt aaa ttg ttg gtt gtt tct aat cca gtt gat 435 Lys Tyr Ser Pro Asn Cys Lys Leu Leu Val Val Ser Asn Pro Val Asp                 130 135 140 att ttg act tat gtt gct tgg aaa att tct ggt ttt cca aaa aat aga 483 Ile Leu Thr Tyr Val Ala Trp Lys Ile Ser Gly Phe Pro Lys Asn Arg             145 150 155 gtt att ggt tct ggt tgt aat ttg gat tct gct aga ttt aga tat ttg 531 Val Ile Gly Ser Gly Cys Asn Leu Asp Ser Ala Arg Phe Arg Tyr Leu         160 165 170 atg ggt gaa aga ttg ggt gtt cat cca ttg tct tgt cat ggt tgg att 579 Met Gly Glu Arg Leu Gly Val His Pro Leu Ser Cys His Gly Trp Ile     175 180 185 ttg ggt gaa cat ggt gat tct tct gtt cca gtt tgg tct ggt gtt aat 627 Leu Gly Glu His Gly Asp Ser Ser Val Pro Val Trp Ser Gly Val Asn 190 195 200 205 gtt gct ggt gtt tct ttg aaa aat ttg cat cca gaa ttg ggt act gat 675 Val Ala Gly Val Ser Leu Lys Asn Leu His Pro Glu Leu Gly Thr Asp                 210 215 220 gct gat aaa gaa caa tgg aaa gct gtt cat aaa caa gtt gtt gat tct 723 Ala Asp Lys Glu Gln Trp Lys Ala Val His Lys Gln Val Val Asp Ser             225 230 235 gct tat gaa gtt att aaa ttg aaa ggt tat act tct tgg gct att ggt 771 Ala Tyr Glu Val Ile Lys Leu Lys Gly Tyr Thr Ser Trp Ala Ile Gly         240 245 250 ttg tct gtt gct gat ttg gct gaa tct att atg aaa aat ttg aga aga 819 Leu Ser Val Ala Asp Leu Ala Glu Ser Ile Met Lys Asn Leu Arg Arg     255 260 265 gtt cat cca att tct act atg att aaa ggt ttg tat ggt att aaa gaa 867 Val His Pro Ile Ser Thr Met Ile Lys Gly Leu Tyr Gly Ile Lys Glu 270 275 280 285 gat gtt ttt ttg tct gtt cca tgt att ttg ggt caa aat ggt att tct 915 Asp Val Phe Leu Ser Val Pro Cys Ile Leu Gly Gln Asn Gly Ile Ser                 290 295 300 gat gtt gtt aaa gtt act ttg act cat gaa gaa gaa gct tgt ttg aaa 963 Asp Val Val Lys Val Thr Leu Thr His Glu Glu Glu Ala Cys Leu Lys             305 310 315 aaa tct gct gat act ttg tgg ggt att caa aaa gaa ttg caa ttt 1008 Lys Ser Ala Asp Thr Leu Trp Gly Ile Gln Lys Glu Leu Gln Phe         320 325 330 taataactcg agcttggttg aacacgttgc caaggcttaa gtga 1052    <210> 4 <211> 332 <212> PRT <213> Artificial Sequence <223> Description of Artificial Sequence: Modified DNA       coding lactate dehydrogenase <400> 4 Met Ala Thr Leu Lys Asp Gln Leu Ile Gln Asn Leu Leu Lys Glu Glu   1 5 10 15 His Val Pro Gln Asn Lys Ile Thr Ile 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 Val          35 40 45 Ala Leu Val Asp Val Met 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 Asn Val Thr Ala Asn Ser Arg 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 Ile 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 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 Gln Trp Lys Ala 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 His Glu Glu Glu 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> 5 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 acagaattca caatggctac tttgaaagat caattgattc aaaatttgtt gaaagaagaa 60 catgttccac aaaataaaat tactattgtt ggtgttggtg 100    <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 acagaattca caatggctac 20    <210> 7 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 atgataacaa ccacaaccac gacaaccata ccgaacacga taaagataaa actactttct 60 aaaccgacta cttcaacgaa accaactaca ataccttcta 100    <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 8 atgataacaa ccacaaccac 20    <210> 9 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 9 tggttgatgt tatggaagat aaattgaaag gtgaaatgat ggatttgcaa catggttctt 60 tgtttttgag aactccaaaa attgtttctg gtaaagatta 100    <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 10 tggttgatgt tatggaagat 20    <210> 11 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 11 taacaaagac catttctaat attacaatga cgattaagat ctaaccaata ataatgacga 60 ccacgatctg ttgttcttcc acttagatct aacttaaacc 100    <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 12 taacaaagac catttctaat a 21    <210> 13 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 13 tgaatctaga ttgaatttgg ttcaaagaaa tgttaatatt tttaaattta ttattccaaa 60 tattgttaaa tattctccaa attgtaaatt gttggttgtt 100    <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 14 tgaatctaga ttgaatttgg t 21    <210> 15 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 15 taacatttaa caaccaacaa agattaggtc aactataaaa ctgaatacaa cgaacctttt 60 aaagaccaaa aggtttttta tctcaataac caagaccaac 100    <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 16 taacatttaa caaccaacaa a 21    <210> 17 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 17 agagttattg gttctggttg taatttggat tctgctagat ttagatattt gatgggtgaa 60 agattgggtg ttcatccatt gtcttgtcat ggttggattt 100    <210> 18 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 18 agagttattg gttctggttg t 21    <210> 19 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 19 cagaacagta ccaacctaaa acccacttgt accactaaga agacaaggtc aaaccagacc 60 acaattacaa cgaccacaaa gaaacttttt aaacgtaggt 100    <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 20 cagaacagta ccaacctaaa a 21    <210> 21 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 21 ctttgaaaaa tttgcatcca gaattgggta ctgatgctga taaagaacaa tggaaagctg 60 ttcataaaca agttgttgat tctgcttatg aagttattaa 100    <210> 22 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 22 ctttgaaaaa tttgcatcca g 21    <210> 23 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 23 agacgaatac ttcaataatt taactttcca atatgaagaa cccgataacc aaacagacaa 60 cgactaaacc gacttagata atacttttta aactcttctc 100    <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 24 agacgaatac ttcaataatt t 21    <210> 25 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 25 tatgaaaaat ttgagaagag ttcatccaat ttctactatg attaaaggtt tgtatggtat 60 taaagaagat gtttttttgt ctgttccatg tattttgggt 100    <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 26 atgaaaaatt tgagaagagt 20    <210> 27 <211> 100 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 27 gacaaggtac ataaaaccca gttttaccat aaagactaca acaatttcaa tgaaactgag 60 tacttcttct tcgaacaaac ttttttagac gactatgaaa 100    <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 28 gacaaggtac ataaaaccca g 21    <210> 29 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 29 aaaaaatctg ctgatacttt gtggggtatt caaaaagaat tgcaatttta ataactcgag 60    <210> 30 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 30 aaaaaatctg ctgatacttt g 21    <210> 31 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 31 acgttaaaat tattgagctc gaaccaactt gtgcaacggt tccgaattca ct 52    <210> 32 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 32 acgttaaaat tattgagctc g 21    <210> 33 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 33 atatatggat ccgcgtttat ttacctatct c 31    <210> 34 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 34 atatatgaat tctttgattg atttgactgt g 31    <210> 35 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 35 atatatctcg aggccagcta acttcttggt cgac 34    <210> 36 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 36 atatatgaat tctttgattg atttgactgt g 31    <210> 37 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 37 tggttgatgt tatggaagat 20    <210> 38 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 38 gacaaggtac ataaaaccca g 21    <210> 39 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 39 gtaataaaca caccccgcg 19    <210> 40 <211> 971 <212> DNA <213> Saccharomyces cerevisiae <400> 40 aagggtagcc tccccataac ataaactcaa taaaatatat agtcttcaac ttgaaaaagg 60 aacaagctca tgcaaagagg tggtacccgc acgccgaaat gcatgcaagt aacctattca 120 aagtaatatc tcatacatgt ttcatgaggg taacaacatg cgactgggtg agcatatgct 180 ccgctgatgt gatgtgcaag ataaacaagc aagacggaaa ctaacttctt cttcatgtaa 240 taaacacacc ccgcgtttat ttacctatct ttaaacttca acaccttata tcataactaa 300 tatttcttga gataagcaca ctgcacccat accttcctta aaagcgtagc ttccagtttt 360 tggtggttcc ggcttccttc ccgattccgc ccgctaaacg catatttttg ttgcctggtg 420 gcatttgcaa aatgcataac ctatgcattt aaaagattat gtatgctctt ctgacttttc 480 gtgtgatgaa gctcgtggaa aaaatgaata atttatgaat ttgagaacaa ttctgtgttg 540 ttacggtatt ttactatgga ataattaatc aattgaggat tttatgcaaa tatcgtttga 600 atatttttcc gaccctttga gtacttttct tcataattgc ataatattgt ccgctgcccg 660 tttttctgtt agacggtgtc ttgatctact tgctatcgtt caacaccacc ttattttcta 720 actatttttt ttttagctca tttgaatcag cttatggtga tggcacattt ttgcataaac 780 ctagctgtcc tcgttgaaca taggaaaaaa aaatatatta acaaggctct ttcactctcc 840 ttgcaatcag atttgggttt gttcccttta ttttcatatt tcttgtcata ttcctttctc 900 aattattatt ttctactcat aaccacacgc aaaataacac agtcaaatca atcaaagatc 960 ccccaattct c 971    <210> 41 <211> 999 <212> DNA <213> Bovine <400> 41 atggcaactc tcaaggatca gctgattcag aatcttctta aggaagaaca tgtcccccag 60 aataagatta caattgttgg ggttggtgct gttggcatgg cctgtgccat cagtatctta 120 atgaaggact tggcagatga agttgctctt gttgatgtca tggaagataa actgaaggga 180 gagatgatgg atctccaaca tggcagcctt ttccttagaa caccaaaaat tgtctctggc 240 aaagactata atgtgacagc aaactccagg ctggttatta tcacagctgg ggcacgtcag 300 caagagggag agagccgtct gaatttggtc cagcgtaacg tgaacatctt taaattcatc 360 attcctaata ttgtaaaata cagcccaaat tgcaagttgc ttgttgtttc caatccagtc 420 gatattttga cctatgtggc ttggaagata agtggctttc ccaaaaaccg tgttattgga 480 agtggttgca atctggattc agctcgcttc cgttatctca tgggggagag gctgggagtt 540 cacccattaa gctgccatgg gtggatcctt ggggagcatg gtgactctag tgtgcctgta 600 tggagtggag tgaatgttgc tggtgtctcc ctgaagaatt tacaccctga attaggcact 660 gatgcagata aggaacagtg gaaagcggtt cacaaacaag tggttgacag tgcttatgag 720 gtgatcaaac tgaaaggcta cacatcctgg gccattggac tgtcagtggc cgatttggca 780 gaaagtataa tgaagaatct taggcgggtg catccgattt ccaccatgat taagggtctc 840 tatggaataa aagaggatgt cttccttagt gttccttgca tcttgggaca gaatggaatc 900 tcagacgttg tgaaagtgac tctgactcat gaagaagagg cctgtttgaa gaagagtgca 960 gatacacttt gggggatcca gaaagaactg cagttttaa 999

[Brief description of drawings]

FIG. 1 is a diagram showing the nucleotide sequence of fully synthesized DNA encoding a protein having LDH activity.

FIG. 2 is a diagram showing codon usage and usage frequency in Saccharomyces cerevisiae.

FIG. 3 is a diagram showing the results of homology analysis of a bovine LDH base sequence and a modified base sequence thereof.
The upper row is the bovine-derived LDH base sequence, the lower row is the modified base sequence, and the bases different from the bovine-derived base sequence in the modified base sequence are described by the symbols (A, T, C, or G).

FIG. 4 is a diagram showing a primer configuration used in long-chain DNA synthesis by PCR used in Example 1 and synthesis steps (steps 1 to 4).

FIG. 5: Constructed vector pBTrp-PDC1-L
It is a figure which shows the plasmid map of DHKCB.

FIG. 6 is a diagram showing part of the steps of constructing the vector shown in FIG.

FIG. 7 is a diagram showing a part of the construction process of pBTrp-PDC1-LDH.

FIG. 8 is a diagram showing a part of the construction process of pBTrp-PDC1-LDH.

FIG. 9 is a diagram showing a part (final step) of the step of constructing the vector shown in FIG.

FIG. 10 is a diagram showing a structural change in a target site on chromosomal DNA in the transformants obtained in Examples.

FIG. 11 is a diagram showing the amount of lactic acid produced in Examples.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁷ Identification code FI Theme Coat (reference) C12N 5/00 C (72) Inventor Masanai Hirai Nagakute-cho, Aichi-gun, Aichi 1st of 41 Toyota Central Research Institute Co., Ltd. (72) Inventor Takao Imaeda Nagakute-cho, Aichi-gun, Aichi Prefecture, Nagatoji 1 41 of Yokomichi Toyota Central Research Institute Co., Ltd. (72) Tsuyoshi Miyazaki, Aichi-gun, Nagakute-cho, Aichi Prefecture 1st at Yokochi 41 Toyota Central Research Institute Co., Ltd. (72) Inventor Kenro Tokuhiro Aki Prefecture Aichi-gun Nagakute-cho Large-length character 1 1st Yokota 1 Toyota Central Research Institute (72) Inventor Satoshi Saito Toyota Aichi Ichiba, Toyota-machi, Toyota City (72) Inventor Rin Saotome Toyota-ichi, Toyota-cho, Aichi Prefecture Toyota Automobile Co., Ltd. (72) Inventor Noriko Hoya Toyota Town Ichiban, Toyota City, Aichi Prefecture Toyota Motor Co., Ltd. (72) Inventor Yasushi Matsuo Toyota Town Ichiban, Toyota City, Aichi Prefecture F Term (Reference) 4B024 AA01 AA05 BA80 CA02 CA05 DA01 DA02 DA05 DA12 EA04 FA02 4B064 AD33 CA06 CA19 CC24 DA01 DA10 4B065 AA80 AA88 AA90 AB01 AC14 BA02 CA10 CA41 CA44

Claims (19)

[Claims] 1. A DNA having any one of the following characteristics (a) and (b): (A) It encodes a protein having lactate dehydrogenase activity and has the base sequence shown in SEQ ID NO: 1. (B) having a base sequence that encodes a protein having lactate dehydrogenase activity by hybridizing under stringent conditions with a DNA consisting of the whole or a part of the base sequence of SEQ ID NO: 1 or a complementary strand thereof . 2. A DNA having any one of the following characteristics (c) and (d): (C) Encodes a protein having lactate dehydrogenase activity and has the nucleotide sequence set forth in SEQ ID NO: 3. (D) having a base sequence encoding a protein having lactate dehydrogenase activity, which hybridizes with a DNA consisting of the whole or a part of the base sequence of SEQ ID NO: 3 or a complementary strand thereof under stringent conditions . 3. The DNA according to claim 1 or 2, which has a Kozak sequence sandwiching the initiation codon. 4. A DNA encoding a protein having lactate dehydrogenase activity, which comprises the following characteristics (e):
(G): (e) designed using the codon usage in Saccharomyces cerevisiae, (f) having a Kozak sequence flanking the initiation codon,
And (g) at least two of the plurality of regions separated from the start codon by 100 bases each having a GC content in the range of 20% to 40%. 5. The protein having lactate dehydrogenase activity is a bovine lactate dehydrogenase or a bovine lactate dehydrogenase amino acid sequence in which one or several amino acids are substituted or deleted, The DNA according to claim 4, which is a protein consisting of an inserted and / or added amino acid sequence. 6. In the amino acid sequence shown in SEQ ID NO: 2,
1 or several amino acids are substituted, deleted, inserted and / or added to form an amino acid sequence, which encodes a protein having lactate dehydrogenase activity, and has the entire nucleotide sequence of SEQ ID NO: 1 or 3. A DNA which hybridizes with a DNA consisting of a part thereof or a complementary strand thereof under stringent conditions. 7. In the amino acid sequence shown in SEQ ID NO: 2,
A base sequence of the coding region of SEQ ID NO: 1 or SEQ ID NO: 3 comprising a protein having lactate dehydrogenase activity, which comprises an amino acid sequence in which one or several amino acids are substituted, deleted, inserted and / or added. In the above, a DN having a base sequence modified by the substitution, deletion, insertion and / or addition of the amino acid
A. 8. A DNA construct comprising a DNA segment consisting of the DNA according to any one of claims 1 to 7, and a promoter segment to which the DNA segment is operably linked. 9. The DNA according to claim 9, wherein the promoter segment has a pyruvate decarboxylase 1 gene promoter activity derived from Saccharomyces cerevisiae.
Construct. 10. A DN for homologous recombination with a host chromosome.
The DN according to claim 8 or 9, comprising an A segment.
A construct. 11. The DNA according to any one of claims 1 to 7.
And a DNA segment for homologous recombination with a host chromosome. 12. The DNA according to any one of claims 1 to 7.
12. The DNA construct according to claim 11, which comprises a DNA segment for homologous recombination so that the DNA segment consisting of can be controlled by the promoter of the host chromosome. 13. The host chromosome promoter is pyruvate decarboxylase 1 derived from Saccharomyces cerevisiae.
The DNA construct according to claim 11 or 12, which is a gene promoter. 14. The DNA construct according to claim 8, further comprising a constituent segment of any vector selected from the group consisting of a plasmid, a bacteriophage, a retrotransposon and an artificial chromosome. DNA construct. 15. The DN according to any one of claims 8 to 13.
A transformant obtained by transforming a host cell with the A construct. 16. The transformant according to claim 15, wherein the host cell is any one of bacteria, yeast, animal cells, insect cells and plant cells. 17. The transformant according to claim 15, wherein the host cell is Saccharomyces cerevisiae. 18. Pyruvate decarboxylase 1 on the host chromosome
A transformed Saccharomyces yeast comprising a DNA segment consisting of the DNA according to any one of claims 1 to 7, operably linked by the promoter downstream of a gene promoter or a homolog of the promoter substituted with the promoter. . 19. A method for producing lactic acid, which comprises a step of culturing cells derived from the transformant according to claim 15 and a step of separating lactic acid from the culture obtained in the step. Method.