JP2001112479A - New plasmid capable of autonomously replicating in coryneform bacterium - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a plasmid useful in the breeding or improvement in Corynebacterium thermoaminogenes belonging to Coryneform bacteria capable of growing at high temperature. SOLUTION: This plasmid is obtained by isolating the plasmid which has a gene encoding the rep protein containing a specific amino acid sequence from Corynebacterium thermoaminogenes FERM BP-1539, FERM BP-1540, FERM BP-1541 and FERM BP-1542.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to Corynebacterium thermoaminogenes.
ogenes). The plasmid of the present invention can be used for breeding a coryneform bacterium used as a bacterium that produces a useful substance such as an L-amino acid.

[0002]

2. Description of the Related Art Amino acids such as L-glutamic acid and L-lysine are mainly used for fermentation using so-called coryneform bacteria belonging to the genus Brevibacterium, Corynebacterium or Microbacterium or mutants thereof. (Amino Acid Fermentation, Gakkai Shuppan Center, pp. 195-215, 1986).

[0003] In the industrial production of amino acid fermentation, increasing the temperature of cultivation is important in addition to improving the yield to sugar, shortening the cultivation time, improving the concentration of accumulated amino acids, and the like, as technical factors that increase economic efficiency. It is considered as something. That is, the cultivation is usually performed at the optimal fermentation temperature, and the optimal temperature for Corynebacterium glutamicum is 31.5 ° C.
When the culture is started, heat of fermentation is generated, so that the culture temperature increases, and the production of amino acids is significantly reduced. Therefore,
In order to optimally maintain the temperature of the culture solution, cooling equipment is required. On the other hand, if the culture temperature is increased, the energy required for cooling can be reduced, and the cooling equipment can be further reduced.

[0004] Among coryneform bacteria, Corynebacterium thermoaminogenes has been isolated as a coryneform bacterium that can grow in a high temperature range (Japanese Patent Application Laid-Open No. 63-240779). Corynebacterium thermoaminogenes is considered to be suitable for high-temperature fermentation, while Corynebacterium glutamicum can grow remarkably at 40 ° C or higher, while it is able to grow at about 40 ° C or higher.

[0005] At present, breeding improvement by DNA recombination technology in Escherichia coli and coryneform bacteria is progressing. In order to improve the breeding of microorganisms by DNA recombination technology, plasmids derived from microorganisms belonging to other species or genera,
Although a high host range vector is often used, a plasmid specific to the microorganism to be bred is generally used.
In particular, when the optimal culture temperature of the microorganism to be bred is different from that of the same or the same genus, it is preferable to use a unique plasmid.

As plasmids derived from coryneform bacteria, pAM330 from Brevibacterium lactofermentum ATCC 13869 (JP-A-58-67669) and pBL1 (Santamaria from Brevibacterium lactofermentum ATCC21798) have been used. R. et al., J. Gen. Microbiol., 130
Pages 2237-2246, 1984), Corynebacterium
PHM1519 from Glutamicum ATCC13058 (Japanese Patent Laid-Open No. 58-77895)
No.), pCG1 from Corynebacterium glutamicum ATCC31808 (JP-A-57-134500), pGA1 from Corynebacterium glutamicum DSM58 (JP-A-9-2603011)
Has been acquired.

[0007] However, no unique plasmid has yet been obtained for Corynebacterium thermoaminogenes.

[0008]

An object of the present invention is to provide a plasmid useful for breeding and improving coryneform bacterium Corynebacterium thermoaminogenes capable of growing at high temperatures.

[0009]

Means for Solving the Problems The present inventors have developed Corynebacterium thermoaminogenes AJ12340 (FERM BP-153).
9), AJ12308 (FERM BP-1540), AJ12309 (FERM BP-1541) and AJ12310 (FERM BP-1542), found that each holds a strain-specific cryptic plasmid,
The plasmid was successfully isolated and identified, and the present invention was completed.

That is, the present invention relates to Corynebacterium
A plasmid which can be isolated from Thermoaminogenes, comprising an amino acid sequence represented by SEQ ID NO: 2 or an amino acid sequence having an amino acid sequence having 90% or more homology with the amino acid sequence.
A plasmid or a derivative thereof containing a gene (rep gene) encoding an ep protein and having a size of about 4.4 Kb or about 6 Kb.

Examples of the plasmid include Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), AJ1
A plasmid having a size of about 4.4 Kb, which can be isolated from 2308 (FERM BP-1540) or AJ12310 (FERM BP-1542), which has a restriction map shown in FIG. Thermoaminogenes AJ12309
(FERM BP-1541) and a plasmid having a size of about 6 Kb and having a restriction map shown in FIG. 2.

[0012] The plasmid may be a plasmid containing a gene encoding a Rep protein having any one of the amino acid sequences shown in SEQ ID NOs: 2, 4 and 6, or an R having the amino acid sequence shown in SEQ ID NO: 8
A plasmid containing a gene encoding the ep protein is exemplified.

[0013]

BEST MODE FOR CARRYING OUT THE INVENTION The plasmid of the present invention is Corynebacterium thermoaminogenes AJ12340 (FERM BP-153).
9), from AJ12308 (FERM BP-1540), AJ12309 (FERM BP-1541) or AJ12310 (FERM BP-1542), alkali method (Biotechnical Experiments, edited by Biotechnology Society of Japan, p. 105, Baifukan, 1992)
Can be isolated according to a conventional method for preparing a plasmid. FERM BP-1539 was founded on March 13, 1987 by the Institute of Biotechnology and Industrial Technology, National Institute of Advanced Industrial Science and Technology (Zip code 305-8566 1-3-1 Higashi, Tsukuba, Ibaraki, Japan)
No.) was transferred from the original deposit deposited under the accession number of FERM P-9277 to an international deposit based on the Budapest Treaty on October 27, 1987, and deposited at the depositary institution. In addition, FERM BP-1540, FERM B
P-1541 and FERM BP-1542 are 198
On March 10, 2007, each of the above depository institutions received FERM P
-9244, FERM P-9245 and FERM P
198 from the original deposit deposited under accession number 9246
It was transferred to the International Deposit under the Budapest Treaty on October 27, 2007 and deposited with the depositary institution.

The present inventors have proposed the above-mentioned Corynebacterium.
Thermoaminogenes AJ12308 (FERM BP-1540), AJ12310 (F
ERM BP-1542), AJ12340 (FERM BP-1539) and AJ12309 (FER
MBP-1541), a unique plasmid was isolated and identified, and pYM1, pYM2, pYM3 and pYM4 were sequentially identified.
It was named. These plasmids carry double-stranded circular DNA in cells of Corynebacterium thermoaminogenes.
Is a plasmid that exists as The base sequence of the rep gene held by pYM1 is shown in SEQ ID NO: 1, the base sequence of the rep gene held by pYM2 is shown in SEQ ID NO: 3, and pYM
SEQ ID NO: 5 shows the nucleotide sequence of the rep gene
And the nucleotide sequence of the rep gene retained by pYM4 is shown in SEQ ID NO: 7. In addition, r carried by these plasmids
The amino acid sequences that can be encoded by the ep gene are shown in SEQ ID NOs: 2, 4, 6, and 8. pYM1, pYM2 and pY
M3 has a size of about 4.4 kb. Also, pYM4
Has a size of about 6 kb.

Table 1 shows the number and size of fragments obtained when pYM1, pYM2 and pYM3 were digested with typical restriction enzymes. Table 2 shows the number and size of fragments obtained when pYM4 was cleaved with a typical restriction enzyme.
Shown in Further, a restriction map of pYM1, pYM2 and pYM3 is shown in FIG. 1, and a restriction map of pYM4 is shown in FIG.

[0016]

Table 1 数 Number of restriction enzyme cleavage positions DNA fragment (Kb) ──── ───────────────────────── BglII 0 − BamHI 2 1.8, 2.6 BstPI 1 4.4 EcoRI 1 4.4 HincII 4 0.3, 0.5, 2.0, 1.6 HindIII 0 − KpnI 0 − NaeI 2 0.1, 4.3 NcoI 1 4.4 NheI 2 1.8, 2.6 PmaCI 1 4.4 SacI 0 − SalI 0 − SacII 3 0.1 1.4 2.9 SmaI 3 0.1 1.8 2.5 SphI 0 − Tth111I 1 4.4 XbaI 0 − ───── ────────────────────────

[0017]

Table 2 数 Number of restriction enzyme cleavage positions DNA fragment (Kb) ──── ───────────────────────── BglII 1 6.0 BamHI 2 3.8, 2.2 BstPI 2 1.2, 4.8 EcoRI 1 6.0 HincII 4 0.3, 0.4, 1.2, 1.7 , 2.4 HindIII 0 − KpnI 0 − NaeI 2 0.1, 5.9 NcoI 3 0.2, 2.8, 3.0 NheI 3 0.1, 2.3, 3.6 PmaCI 0 − SacI 0 − SalI 0 − SacII 5 0.1, 0.2, 0.9, 1.8, 3.0 SmaI 2 0.1 , 5.9 SphI 0 − Tth111I 0 − XbaI 0 − ─────────────────────────────

As a result of determining the nucleotide sequence of the plasmid of the present invention, pYM1, pYM2 and pYM3
368 bp, 4369 bp, and 4369 bp, have almost the same structure, and have 99.9% homology at the nucleotide sequence level. On the other hand, pYM4 has 5967 bp, and the homology with pYM1, pYM2 and pYM3 is about 8
1% but about 4.4 kb excluding an area of about 1.6 kb
Region has very high homology.

Each of these plasmids has a highly homologous rep gene. Amino acid sequence of Rep protein encoded by each rep gene (SEQ ID NOs: 2, 4, 6, and 8), and Rep encoded by rep gene carried by a known coryneform bacterium plasmid
Homology was compared for the amino acid sequences of the proteins.
pYM1, pYM2 and pYM3 have a homology of 99% or more, and homology between pYM2 and pYM4 is 81.91.
%Met. On the other hand, plasmid p of known coryneform bacteria
No homology with AM330, pGA1, pCG1
Was 80% or less. Table 3 shows the results.
As described above, the plasmid of the present invention and the plasmid of a known coryneform bacterium can be distinguished by the homology of the Rep protein.

The homology is described in Takashi, K. and Gotoh,
O., J. Biochem., 92, 1173-1177 (1984).

[0021]

[Table 3] Table 3 Homology of amino acid sequences of Rep proteins coated by various plasmids pYM2 pYM4 pGA1 pCG1 ────────────────────────── pYM2 − 81.91% 68.01% 70.73% pYM4 − − 69.39% 70.23% pGA1 − − − 75.31% pCG1 − − − − ──────────────────────────

Since the plasmid of the present invention can replicate well in coryneform bacterium cells such as Corynebacterium thermoaminogenes, it can be obtained by inserting a foreign gene into any position of these plasmids or derivatives thereof. When a host microorganism is transformed with the recombinant plasmid, the genetic information of the foreign gene can be expressed in the host microorganism.

The following are examples of coryneform bacteria. Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium carnae Corynebacterium glutamicum Corynebacterium thermoaminogenes Corynebacterium lilium (Corynebacterium
(Glutamicum) Corynebacterium meracecora Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium saccharolyticum Brevibacterium inmario film Brevibacterium Um Roseum Brevibacterium flavum (Corynebacterium
Glutamicum) Brevibacterium thiogenitalis

The derivative of the plasmid of the present invention refers to a part of the plasmid of the present invention or a plasmid of the present invention or a plasmid comprising the plasmid of the present invention and another DNA sequence. The term “part” refers to a part including a region essential for autonomous replication of a plasmid. In the plasmid of the present invention, a region essential for autonomous replication of a plasmid (replication control region), that is, a region other than a region containing a replication origin and a gene required for replication, can be removed from a host microorganism even if part or all of the region is removed. And is smaller in size, which is preferable for use as a vector. When a marker gene such as a drug resistance gene is mounted on the plasmid of the present invention or a part thereof, the transformant can be easily detected depending on the phenotype of the marker gene in the transformant. Examples of marker genes usable for the host include a chloramphenicol resistance gene, a kanamycin resistance gene, a streptomycin resistance gene, a tetracycline resistance gene, a trimethoprim resistance gene,
Erythromycin resistance gene and the like.

Further, the plasmid of the present invention or a part thereof is linked to a plasmid capable of autonomously replicating in a bacterium such as Escherichia coli or a part containing a replication control region thereof, so that the plasmids such as coryneform bacteria and Escherichia coli are ligated. By making the shuttle vector autonomously replicable in both bacteria, operations such as preparation of a plasmid and preparation of a recombinant plasmid carrying a target gene can be performed using Escherichia coli. Examples of plasmids capable of autonomous replication in Escherichia coli include, for example, pUC19, pUC18, pBR322, pHSG29
9, pHSG298, pHSG399, pHSG398, RSF1010, pMW119, pMW
118, pMW219, pMW218 and the like.

PYM1, pYM2, pYM3 and pYM
4 itself is characterized by the restriction maps shown in FIGS. 1 and 2; however, the plasmid of the present invention does not necessarily have to have these restriction maps, and any arbitrary restriction enzyme site can be used unless the autonomous replication ability is affected. It may be extinguished. In the plasmid of the present invention, a restriction enzyme site not possessed by pYM1, pYM2, pYM3 and pYM4 may be introduced.

The preparation of the above-mentioned derivatives of the plasmid can be carried out in the same manner as the preparation of a conventionally known cloning vector or expression vector. To make the derivatives, pYM1, pYM2, pYM3 and pYM
It is preferable to determine the base sequence of M4. The nucleotide sequence can be determined by a known method such as the dideoxy method.

In order to insert a foreign gene into the plasmid or derivative thereof of the present invention, it is convenient to insert the plasmid or derivative into a restriction enzyme site. As the restriction enzyme site, one having a single site to be cut is desirable. In order to insert a foreign gene, a plasmid and genomic DNA as a source of the foreign gene are partially or completely digested with a restriction enzyme such that the protruding portion of each cut end has the same sequence, for example, the same restriction enzyme. May be ligated under appropriate conditions. Alternatively, they may be connected at blunt ends.

For preparation of plasmid DNA, cleavage and ligation of DNA, transformation, and the like, usual methods well known to those skilled in the art can be employed. These methods are described in Sambrook, J., Fritsch, EF, and Maniatis,
T., "Molecular Cloning A Laboratory Manual, Second
Edition ", Cold Spring Harbor Laboratory Press,
(1989).

[0030]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the contents of the present invention will be described in detail based on embodiments.

[0031]

Example 1 Corynebacterium thermoaminogenes (FERM BP-1539, FERMBP-1540, FERM BP-1541, FERM BP
-1542) Isolation and characterization of plasmid from Corynebacterium thermoaminogenes FERM BP-1539, FERM B
P-1540, FERMBP-1541, FERM BP-1542 in CM2B liquid medium (B
acto-trypton (Difco) 1%, Bacto-yeast-extract (Difc
o) 1%, NaCl 0.5%, biotin 10μg / L) for 12 hours,
Alkali method (Biological Experiments, edited by the Society of Biotechnology, 105
Page, Baifukan, 1992) to obtain a plasmid DNA fraction. These fractions were subjected to agarose gel electrophoresis (Sambr
ook, J., Fritsch, EF, and Maniatis, T., "Molecu
lar Cloning A Laboratory Manual, Second Edition ",
When analyzed by Cold Spring Harbor Laboratory Press, (1989)), DNA bands were detected in all cases, and it was revealed that the above strains contained plasmids. Plasmids prepared from FERM BP-1540, FERM BP-1542, and FERM BP-1539 were pYM1, pYM2, and pYM2, respectively.
Named YM3. The plasmid prepared from FERM BP-1541 was named pYM4. Plasmid pYM
1, pYM2 and pYM3 had a length of about 4.4 Kb, and plasmid pYM4 had a length of about 6.0 Kb.

The plasmids pYM1, pYM2, pYM3
And pYM4 by using restriction enzymes BglII, BamHI, BstPI, EcoR
I, HincII, HindIII, KpnI, NaeI, NcoI, NheI, PmaC
I, SacI, SacII, SalI, SmaI, SphI, Tth111I, XbaI (T
akara) and the length of the resulting DNA fragment was measured by agarose gel electrophoresis. 0.8% electrophoresis
Was electrophoresed at 100 V / cm for several hours at a constant voltage using an agarose gel. The molecular weight markers include the restriction enzyme HindIII
Λ phage DNA (Takara Shuzo) was used. Table 1 shows the results for pYM1, pYM2 and pYM3. Table 2 shows the results for pYM4. PYM1, pYM created based on these results
FIG. 1 shows a restriction map of YM2 and pYM3.
Is shown in FIG.

PYM1, pYM2, pYM3 and pYM
SEQ ID NOs: 1, 3, 5, and 7 show the results of base sequence No. 4 determined by the dideoxy method.

[0034]

Example 2 Streptococcus faecalis (S. fae
calis) -derived shuttle vector pYM having a Km resistance gene
Construction of FK In the coryneform bacterium, a region required for efficient replication of pYM2 is a region encoding rep and a region upstream of rep.
There are regions that are AT rich and regions involved in downstream copy number.

Therefore, in order to obtain a shuttle vector which can be replicated in coryneform bacteria and E. coli without impairing the replication ability of pYM2, E. coli is located near the BstPI site of pYM2.
A region where autonomous replication is possible and a selection marker were inserted.

First, a vector having a drug resistance gene of Streptococcus faecalis was constructed. The kanamycin resistance gene of Streptococcus faecalis was amplified by PCR from a known plasmid containing the gene. The nucleotide sequence of the kanamycin resistance gene of Streptococcus faecalis has already been elucidated (Trieu-
Cuot, P. And Courvalin, P .: Gene 23 (3), 331-341
(1983)). Based on this sequence, primers having the nucleotide sequences shown in SEQ ID NOs: 16 and 17 were synthesized, and pDG783 (An
ne-Marie Guerout-Fleury et al., Gene, 167, 335-337
(1995)) as a template to amplify a DNA fragment containing the kanamycin resistance gene and its promoter.

After purifying the above DNA fragment with SUPREC02 manufactured by Takara Shuzo, it was completely digested with restriction enzymes HindIII and HincII and blunt-ended. Blunting Kit manufactured by Takara Shuzo Co., Ltd.
Performed by Using this DNA fragment and primers having the nucleotide sequences shown in SEQ ID NOs: 18 and 19, pHSG39 was used.
PCR was performed using 9 (see S. Takeshita et al .: Gene 61, 63-74 (1987)) as a template, and the amplification product obtained was purified and blunt-ended, and the resulting DNA fragment was mixed and ligated. The ligation reaction was performed using DNA ligation kit ver2 manufactured by Takara Shuzo. Using the ligated DNA, competent cells of Escherichia coli JM109 (Takara Shuzo) were transformed, and IPTG (isopropyl-β-D-thiogalactopyranoside) 10 μg / ml, X-Gal (5
-Bromo-4-chloro-3-indolyl-β-D-galactoside)
L medium containing 40 μg / ml and kanamycin 25 μg / ml (Bacto tryptone 10 g / L, Bacto yeast extract 5 g /
L, NaCl 5 g / L, agar 15 g / L, pH 7.2), and after overnight culture, appeared blue colonies were picked and separated into single colonies to obtain transformed strains.

A plasmid was prepared from the transformant using the alkaline method (Biotechnical Experiments, edited by The Biotechnology Society of Japan, p. 105, Baifukan, 1992), and a restriction enzyme map was prepared.
The equivalent to the restriction map shown below was named pK1. This plasmid is stably maintained in Escherichia coli and confers kanamycin resistance on the host. Also, since it contains the lazZ 'gene, it is suitable for cloning vectors.

Next, pYM2 extracted from Corynebacterium thermoaminogenes FERM BP-1542 was used as a template (the entire nucleotide sequence of pYM2 is shown in SEQ ID NO: 9), and FIG.
Primer S1 5'-AAC CAG GGG GAG GGC GCG AGG C-3 '(SEQ ID NO: 10) S3 5'-TCT CGT AGG CTG CAT CCG AGG CGG GG-3' prepared based on the sequence near the BstPI site of middle pYM2 Using (SEQ ID NO: 11), the region containing the replication origin was amplified by Pyrobest-Taq manufactured by Takara Shuzo. The reaction conditions were as follows: 94 ° C for 5 minutes, 98
30 cycles of 20 seconds at 68 ° C and 4 minutes at 68 ° C, and 72
C. for 4 minutes. After the reaction, it was stored at 4 ° C.

The obtained amplified fragment was prepared using Amersham Pharmacia B
After purification with MicroSpin ™ S-400HR columns manufactured by iotech, blunt-ended with DNA Blunting Kit manufactured by Takara Shuzo Co., Ltd., a fragment treated with HincII of pK1 and DNA Ligation Kit.ver.2 manufactured by Takara Shuzo were used. And ligated. Using the ligated DNA, competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo) were transformed to obtain a transformed strain.

From the transformed strain, the alkaline method (Biotechnical Experiments, edited by the Society of Biotechnology, Japan, p. 105, Baifukan 1992)
Prepare a plasmid using, create a restriction map,
A map having the same restriction map as that shown in the lower part of FIG. 3 was named pYMFK. pYMFK is approximately 7.0 Kb in size, can replicate in E. coli and coryneform bacteria, and can confer Km resistance on the host.

[0042]

Example 3 pYMK having Tn903-derived Km resistance gene
Construction of Corynebacterium thermoaminogenes FERM BP-1542
Using pYM2 extracted from the template as a template, primer S1XbaI 5'-GCT CTA GAG CAA CCA GGG GGA GGG CGC GAG
GC-3 '(SEQ ID NO: 12) S3XbaI 5'-GCT CTA GAG CTC TCG TAG GCT GCA TCG GAG
The region containing the replication origin was amplified in the same manner as in Example 2 using GCG GGG-3 ′ (SEQ ID NO: 13).

The obtained amplified fragment was prepared using Amersham Pharmacia B
After purifying with MicroSpinTM S-400HR columns manufactured by iotech, digested with the restriction enzyme XbaI manufactured by Takara Shuzo Co., Ltd., a fragment obtained by completely decomposing pHSG299 manufactured by Takara Shuzo with XbaI, and DNA Ligation Kit.ver. 2. Using the ligated DNA, competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo) were transformed to obtain a transformed strain.

From the transformed strain, the alkaline method (Biotechnical Experiments, edited by the Society of Biotechnology, Japan, p. 105, Baifukan 1992)
Prepare a plasmid using, create a restriction map,
A map having the same restriction map as that shown in the lower part of FIG. 4 was named pYMK. pYMK is approximately 7.0 Kb in size, can replicate in E. coli and coryneform bacteria, and can add Km resistance to the host.

[0045]

Example 4 Construction of shuttle vector pYMC having Cm resistance gene derived from Tn9 Corynebacterium thermoaminogenes FERM BP-1542
Using pYM2 extracted from the template as a template, primer S1XbaI 5'-GCT CTA GAG CAA CCA GGG GGA GGG CGC GAG
GC-3 '(SEQ ID NO: 14) S3XbaI 5'-GCT CTA GAG CTC TCG TAG GCT GCA TCG GAG
Using GCG GGG-3 ′ (SEQ ID NO: 15), a region containing the replication origin was amplified in the same manner as in Example 2.

After purifying the above DNA with MicroSpin ™ S-400 HR columns manufactured by Amersham Pharmacia Biotech, the DNA was treated with restriction enzyme XbaI manufactured by Takara Shuzo Co., Ltd. DNA Lig
Ligation was performed using the ation Kit.ver.2. Using the ligated DNA, competent cells of Escherichia coli JM109 (manufactured by Takara Shuzo) were transformed to obtain a transformed strain.

From the transformed strain, the alkaline method (Biological Engineering Experiment, edited by the Society of Biotechnology, Japan, page 105, Baifukan 1992)
Prepare a plasmid using, create a restriction map,
A map having the same restriction enzyme map as that shown in the lower part of FIG. 5 was named pYMC. pYMC is about 6.6 Kb in size, can replicate in E. coli and coryneform bacteria, and can confer Cm resistance to the host.

[0048]

According to the present invention, a novel plasmid derived from Corynebacterium thermoaminogenes is provided.

[0049]

[Sequence List] SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> Novel plasmid capable of autonomous replication in coryneform bacteria <130> P-7825 <150> JP 11-228391 <151> 1999-08-12 <160> 19 <170> PatentIn Ver. 2.0 <210> 1 <211> 1479 <212> DNA <213> Corynebacterium thermoaminogenes <220> <221> CDS <222> (1) .. (1476) <400> 1 atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 cgg ggt tgg aat cac cag gac gcc gcc gag ttc ctg gcc cac ctg gat 144 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Tla 50 55 60 gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 gcc ggc ggg aac atc gag cac ctt cac ccg cag gtg tac gcc acc ttg 384 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atg gtg aac 432 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480 Pro Leu Ser Gly Lys Cys Gln Leu Ile Ile Asp Pro Val Phe 145 150 155 160 gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 cgg ctg agc cgg tgg ccg ctg cat gtt tct gat gat ccg acc gcg tac 624 Arg Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 tcc tgg cac tgc cag cac aac cga gtc gat att ctt gat gag ctg atg 672 Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met210 215 220 gag gta gcc cgc acg atg acc gga tca aaa aag ccc aga gag cac gct 720 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Glu His Ala 225 230 235 240 cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 g 285 t gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaag 960 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ayr Asn Val Ala Gln 325 330 335 gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 cgt cag acg atg gcc cg gtg cgc gcc tac gtc gcc aaa ggc cag 1104 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152 Pro Thr Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 cga ctc gaa gct gca aac aag cga cgt caca tgg aac aaa tac 1296 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 cg acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 470 475 480 tgc caa atc ctt agg ggg gct cac gcc gta gac aga taa479 Gln Ile Leu Arg Gly Ala His Ala Val Asp Arg 485 490 <210> 2 <211> 492 <212> PRT <213> Corynebacterium thermoaminogenes <400> 2 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 Ser Trp His Cy s Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met 210 215 220 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Glu His Ala 225 230 235 240 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 280 285 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 Arg Leu Glu Al a Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 475 480 Cys Gln Ile Leu Arg Gly Ala His Ala Val Asp Arg 485 490 <210> 3 <211> 1479 <212> DNA <213> Corynebacterium thermoaminogenes <220> <221> CDS <222> (1) .. (1476) <400> 3 atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 cgg ggt tgg aat cac cag gac gcc gca gag ttc ctg gcc cac ctg gat 144 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Tla 50 55 60 gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 gcc ggc ggg aac atc gag cac ctt cac ccg cag gtg tac gcc acc ttg 384 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atg gtg aac 432 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480 Pro Leu Ser Gly Lys Cys Gln Leu Ile Ile Asp Pro Val Phe 145 150 155 160 gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 cgg ctg agc cgg tgg ccg ctg cat gtt ttt gat gat ccg acc gcg tac 624 Arg Ser Arg Trp Pro Leu His Val Phe Asp Asp Pro Thr Ala Tyr 195 200 205 tcc tgg cac tgc cag cac aac cga gtc gat att ctt gat gag ctg atg 672 Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met210 215 220 gag gta gcc cgc acg atg acc gga tca aaa aag ccg aga aag cac gct 720 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 t 285 t gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaag 960 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ayr Asn Val Ala Gln 325 330 335 gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 cgt cag acg atg gcc cg gtg cgc gcc tac gtc gcc aaa ggc cag 1104 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152 Pro Thr Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 cga ctc gaa gct gca aac aag cga cgt caca tgg aac aaa tac 1296 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 cg acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 470 475 480 tgc caa atc ctt agg ggg gct cac gcc gta cac aga taa479 Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 <210> 4 <211> 492 <212> PRT <213> Corynebacterium thermoaminogenes <400> 4 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 Arg Leu Ser Arg Trp Pro Leu His Val Phe Asp Asp Pro Thr Ala Tyr 195 200 205 Ser Trp His Cy s Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met 210 215 220 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 280 285 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 Arg Leu Glu Al a Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 475 480 Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 <210> 5 <211> 1479 <212> DNA <213> Corynebacterium thermoaminogenes <220> <221> CDS <222> (1) .. (1476) <400> 5 atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 cgg ggt tgg aat cac cag gac gcc gcc gag ttc ctg gcc cac ctg gat 144 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Tla 50 55 60 gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 gcc ggc ggg aac atc gag cac ctt cac ccg cag gta tac gcc acc ttg 384 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atc ggg gtg aac 432 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480 Pro Leu Ser Gly Lys Cys Gln Leu Ile Ile Asp Pro Val Phe 145 150 155 160 gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 cgg ctg agc cgg tgg ccg ctg cat gtt tct gat gat ccg acc gcg tac 624 Arg Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 tcc tgg cac tgc cag cac aac cga gtc gat acg ctt gat gag ctg atg 672 Ser Trp His Cys Gln His Asn Arg Val Asp Thr Leu Asp Glu Leu Met210 215 220 gag gta gcc cgc acg atg acc gga tca aaa aag ccg aga aag cac gct 720 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 t 285 t gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaag 960 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ayr Asn Val Ala Gln 325 330 335 gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 cgt cag acg atg gcc cg gtg cgc gcc tac gtc gcc aaa ggc cag 1104 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152 Pro Thr Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 cga ctc gaa gct gca aac aag cga cgt caca tgg aac aaa tac 1296 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 cg acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 470 475 480 tgc caa atc ctt agg ggg gct cac gcc gta cac aga taa479 Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 <210> 6 <211> 492 <212> PRT <213> Corynebacterium thermoaminogenes <400> 6 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 Ser Trp His Cy s Gln His Asn Arg Val Asp Thr Leu Asp Glu Leu Met 210 215 220 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 280 285 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 Arg Leu Glu Al a Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 475 480 Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 <210> 7 <211> 1377 <212> DNA <213> Corynebacterium thermoaminogenes <220> <221> CDS <222> (1) .. (1374) <400> 7 atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 tac tcc aca gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 cgg ggt tgg aat cac cag gac gcc gcc gag ttc ctg gcc cac ctg gat 144 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Tla 50 55 60 gtc aaa gac ccg gag acc gga gaa acc cgc cct aag ctg cac aga gtc 240 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 ggc acc cgg tcg ctt tcc cga tgc cag tac gtc gcg ctg acc cac ccg 288 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 gcc ggc ggg aac atc gag cac ctt cac ccg cag gtc tac gcc acc ttg 384 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 gag cgc tgg gca cgg gtg gag aaa gcg ccg gcc tgg atg gtg aac 432 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 ccg ttg tca gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480 Pro Leu Ser Gly Lys Cys Gln Leu Ile Ile Asp Pro Val Phe 145 150 155 160 gcc gcc gag ggc acc acc agc ccg aac acc cgc ctg cta gcg gcc acc 528 Ala Ala Glu Gly Thr Thr Ser Pro Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 acc gag gaa atg acc cgt atg ttc ggc gct gac cag gca ttt tcc cac 576 Thr Glu Glu Met Thr Arg Met Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 cgg ctg agc cgg tgg ccg ctg cat gta tct gat gat ccg acc gcg tac 624 Arg Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 tcc tgg cac tgc cag cac aac cga gtc gat acg ctt gct gag ctg atg 672 Ser Trp His Cys Gln His Asn Arg Val Asp Thr Leu Ala Glu Leu Met210 215 220 gag gta gcc cgc acg atg acc gga tca aaa aag cca gat agc act gct 720 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Asp Ser Thr Ala 225 230 235 240 cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg agg aaa 768 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 gcc acc gca gaa gcc aaa gcg ctt gct gcc tta gac gcc acg ctg cct 816 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ctg 864 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 g 285 t gca gag ggg cgt gca gcc cgt gat gag acg gcg ttt cgc cat 912 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 gcg ttg acc gtg ggg tat cag ctt aaa gcc gca ggt gaag 960 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 gac acc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008 Asp Thr Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 gcg gtg ggg gct gat ggg cgt gag ccg gat ctg cct gcc atg cgt gat 1056 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 cgt cag acg ttg gcc cgt gtg cgc gcc tac gtc gct aaa ggc cag 1104 Arg Gln Thr Leu Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 ccc acg gtg agc gcc agg agc aca cag acc cag agc agc cgg ggc agg 1152 Thr Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 aaa gcc ctg gcc acc atg gga cgc aga ggc gca gcc acc tcg aat gca 1200 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Ala Ala Thr Ser Asn Ala 385 390 395 400 cgc agg tgg gca gac cca gaa agc gat tac gcc cgc caa act cgg gag 1248 Arg Arg Trp Ala Asp Pro Glu Ser Asp Tyr Ala Arg Gln Thr Arg Glu 405 410 415 cgt tta gcc cga gca atg agc ttc gta cat tca cag acg aga aca 1296 Arg Leu Ala Arg Ala Met Ser Phe Val His Ser Ala Gln Thr Arg Thr 420 425 430 agg gcc gga tcc tgg cct acg ttt ccg agt gca agc gcc acg gtt acg 1344 Arg Ala Gly Ser Trp Pro Thr Phe Pro Ser Ala Ser Ala Thr Val Thr 435 440 445 acc cca cga gca aag aag tcg caa cgg agc tag 1377 Thr Pro Arg Ala Lys Lys Ser Gln Arg Ser 450 455 <210> 8 <211> 458 <212> PRT <213> Corynebacterium thermoaminogenes <400> 8 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Trp 50 55 60 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 Pro Leu Ser Gly Lys Cys Gln Leu Ile Trp Cys Ile Asp Pro Val Phe 145 150 155 160 Ala Ala Glu Gly Thr Thr Ser Pro Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 Thr Glu Glu Met Thr Arg Met Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 Arg Leu Ser Arg Trp Pro Leu His Val Ser Asp Asp Pro Thr Ala Tyr 195 200 205 Ser Trp His Cy s Gln His Asn Arg Val Asp Thr Leu Ala Glu Leu Met 210 215 220 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Asp Ser Thr Ala 225 230 235 240 His Gln Glu Plu Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 280 285 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 Asp Thr Lys Ile Ile Asp Ala Tyr Glu Arg Ala Tyr Asn Val Ala Gln 325 330 335 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 Arg Gln Thr Leu Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 Pro Thr Val Ser Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Ala Ala Thr Ser Asn Ala 385 390 395 400 Arg Arg Trp Ala Asp Pro Glu Ser Asp Tyr Ala Arg Gln Thr Arg Glu 405 410 415 Arg Leu Ala Ar g Ala Met Ser Phe Val His Ser Ala Gln Thr Arg Thr 420 425 430 Arg Ala Gly Ser Trp Pro Thr Phe Pro Ser Ala Ser Ala Thr Val Thr 435 440 445 Thr Pro Arg Ala Lys Lys Ser Gln Arg Ser 450 455 <210> 9 <211> 4369 <212> DNA <213> Corynebacterium thermoaminogenes <220> <221> CDS <222> (1) .. (1476) <400> 9 atg act cta gcg gat tcg cca gga aca tac aca gca gat gcg tgg aat 48 Met Thr Leu Ala Asp Ser Pro Gly Thr Tyr Thr Ala Asp Ala Trp Asn 1 5 10 15 tac tcc act gat ctg ttc gac acc cac cct gag ctg gct tta cgc tcc 96 Tyr Ser Thr Asp Leu Phe Asp Thr His Pro Glu Leu Ala Leu Arg Ser 20 25 30 cgg ggt tgg aat cac cag gac gcc gca gag ttc ctg gcc cac ctg gat 144 Arg Gly Trp Asn His Gln Asp Ala Ala Glu Phe Leu Ala His Leu Asp 35 40 45 cgc agc atg ttt cac ggg tgc ccc acc cgg gat ttc tcc gcg gcc tgg 192 Arg Ser Met Phe His Gly Cys Pro Thr Arg Asp Phe Ser Ala Ala Tla 50 55 60 gtc aaa gac ccg gaa acc gga gaa acc cgc ccc aag ctg cac aga gtt 240 Val Lys Asp Pro Glu Thr Gly Glu Thr Arg Pro Lys Leu His Arg Val 65 70 75 80 ggc acc cgc tca ctt tcc cgg tgc cag tac gtt gcc ctg acc cac ccg 288 Gly Thr Arg Ser Leu Ser Arg Cys Gln Tyr Val Ala Leu Thr His Pro 85 90 95 cag cgc tcc gcg gtg ctg gtc tta gac atc gac atc ccc agc cac cag 336 Gln Arg Ser Ala Val Leu Val Leu Asp Ile Asp Ile Pro Ser His Gln 100 105 110 gcc ggc ggg aac atc gag cac ctt cac ccg cag gtg tac gcc acc ttg 384 Ala Gly Gly Asn Ile Glu His Leu His Pro Gln Val Tyr Ala Thr Leu 115 120 125 gag cgt tgg gca cgg gtg gag aaa gcg ccg gcc tgg atg gtg aac 432 Glu Arg Trp Ala Arg Val Glu Lys Ala Pro Ala Trp Ile Gly Val Asn 130 135 140 ccg ttg tcg gga aag tgc cag ctc atc tgg tgc att gac ccg gtg ttc 480 Pro Leu Ser Gly Lys Cys Gln Leu Ile Ile Asp Pro Val Phe 145 150 155 160 gcc gcc gag ggc acc acc agc tcg aac acc cgc ctg cta gcg gcc acc 528 Ala Ala Glu Gly Thr Thr Ser Ser Asn Thr Arg Leu Leu Ala Ala Thr 165 170 175 acc gag gaa atg acc cgt gtg ttc ggc gct gac cag gca ttt tcc cac 576 Thr Glu Glu Met Thr Arg Val Phe Gly Ala Asp Gln Ala Phe Ser His 180 185 190 cgg ctg agc cgg tgg ccg ctg cat gtt ttt gat gat ccg acc gcg tac 624 Arg Ser Arg Trp Pro Leu His Val Phe Asp Asp Pro Thr Ala Tyr 195 200 205 tcc tgg cac tgc cag cac aac cga gtc gat att ctt gat gag ctg atg 672 Ser Trp His Cys Gln His Asn Arg Val Asp Ile Leu Asp Glu Leu Met210 215 220 gag gta gcc cgc acg atg acc gga tca aaa aag ccg aga aag cac gct 720 Glu Val Ala Arg Thr Met Thr Gly Ser Lys Lys Pro Arg Lys His Ala 225 230 235 240 cac cag gag ttt tcc agc ggt cgg gca cgg atc gaa gcc gcg cgg aaa 768 His Gln Glu Phe Ser Ser Gly Arg Ala Arg Ile Glu Ala Ala Arg Lys 245 250 255 gcc acc gca gag gcc aaa gcg ctt gcc gcc ttg gac gcc acg ctg cct 816 Ala Thr Ala Glu Ala Lys Ala Leu Ala Ala Leu Asp Ala Thr Leu Pro 260 265 270 acg gcg ctg gag gca tca ggc gat ctc att gac ggg gtg cgg gtg ttg 864 Thr Ala Leu Glu Ala Ser Gly Asp Leu Ile Asp Gly Val Arg Val Leu 275 280 t 285 t gca gag ggg cgt gca gcc cgt gat gag aca gcg ttt cgc cat 912 Trp Ala Ala Glu Gly Arg Ala Ala Arg Asp Glu Thr Ala Phe Arg His 290 295 300 gcg ttg acc gtg ggt tat cag ctt aaa gcc gca ggt gaag 960 Ala Leu Thr Val Gly Tyr Gln Leu Lys Ala Ala Gly Glu Arg Leu Lys 305 310 315 320 gat gcc aag atc att gat gcg tat gag cgt gcc tac aac gtc gcc cag 1008 Asp Ala Lys Ile Ile Asp Ala Tyr Glu Arg Ayr Asn Val Ala Gln 325 330 335 gcg gtg gga gct gat ggg cgt gaa ccg gat ctg cct gcc atg cgt gat 1056 Ala Val Gly Ala Asp Gly Arg Glu Pro Asp Leu Pro Ala Met Arg Asp 340 345 350 cgt cag acg atg gcc cg gtg cgc gcc tac gtc gcc aaa ggc cag 1104 Arg Gln Thr Met Ala Arg Arg Val Arg Ala Tyr Val Ala Lys Gly Gln 355 360 365 ccc acg gtc agc gcc agg agc aca cag acc cag agc agt cgg ggc cgg 1152 Pro Thr Ala Arg Ser Thr Gln Thr Gln Ser Ser Arg Gly Arg 370 375 380 aaa gcc ctg gcc acc atg ggc cgc aga ggc ggg caa aaa gcc gct gaa 1200 Lys Ala Leu Ala Thr Met Gly Arg Arg Gly Gly Gln Lys Ala Ala Glu 385 390 395 400 cgc tgg aaa acc gat cct aac ggc aaa tac gcc caa gaa aac cgc caa 1248 Arg Trp Lys Thr Asp Pro Asn Gly Lys Tyr Ala Gln Glu Asn Arg Gln 405 410 415 cga ctc gaa gct gca aac aag cga cgt caca tgg aac aaa tac 1296 Arg Leu Glu Ala Ala Asn Lys Arg Arg Gln Val Ser Trp Asn Lys Tyr 420 425 430 gcg agc acg aat tct ggc tac ggt ttc cga cac gta tgg gcc agc ttg 1344 Ala Ser Thr Asn Ser Gly Tyr Gly Phe Arg His Val Trp Ala Ser Leu 435 440 445 gaa aaa tgc cta cgc gac gag caa atc atg gaa gaa aca ggg ctt tca 1392 Glu Lys Cys Leu Arg Asp Glu Gln Ile Met Glu Glu Thr Gly Leu Ser 450 455 460 cg acc gtg acg cgc cat tgg gtg cac tgc gag agg ctg gcc tgc 1440 Arg Ala Thr Val Thr Arg His Trp Val His Cys Glu Arg Leu Ala Cys 465 470 470 475 480 tgc caa atc ctt agg ggg gct cac gcc gta cac aga taacggcc Cys Gln Ile Leu Arg Gly Ala His Ala Val His Arg 485 490 gtaggggtag cgcttggtcc ctgaagctcc ggctcccatc cctcctcagc actccctccc 1552 cgaggggggg gctcacgccg tagacagata acggttccca ccccgtaggg gtagcgcttg 1612 gtccctgaag ctctcacttc tggctcccct cctggccctc cttgagtgcc cacccataaa 1672 tgcgaaatgc cgtcagcaga caacggttcc cacccctggg gtcctcacaa caggctgcat 1732 cagggctctc gacgcttgct ggcttcatcc atcaactgct gggtgatctc ctcgaacgca 1792 tccttgatcg cgagttcctc gaaatcagcg gcagcttgct cccagtggat ccgttccagc 1852 tccggcagcg ccgcccacag cgccatgcgcc aagtaggttc cacgtgatcg cttagtgcgc 1912 tcggcgagcg cgtccaggcg cc ccggttcca ggcgcacgga gaccacgggg 1972 ccgcgtccgg cggggttctt ctggttggtg gccatgagaa atttcctctc gcttcggtag 2032 ttgtaaacaa tgtttacacc gtgtcgggga gaggggtttt tatttttctc ccgggcactt 2092 tcgagacggg tcatgccgta agcgaggcgc gtggccacac cgcactcggc gacgcaggtg 2152 tcacttgctc cccgactgtc ggccgggaag ggggcgcgca gagcgtccag gagcgccgta 2212 gagcgcctgg gacggttcgt gtggggactt ggtcgcccca cggggcttta atcgcttaaa 2272 aacgcgcaca gcgcatttct tgccacgggc tagcgcgtga ccgctgcgcg ctcacttgct 2332 caggaagaaa atcattcctc gcctaaagcg cttcgcgcgc tcgccctctc cgagggggaa 2392 aactaaccac acacctcatg cactaaagtg ctgatttgca ggtcagcgcg ttttagcgtg 2452 caaaaatagt gcggaaaacg gcgaaaatgg gggcgcgaca atcccctcag tggctcccca 2512 aaattcacct attcacatct gctactggct gacttctttc ccgacaaggg gccctgtgag 2572 ggcgcaggtt gagccacttt tacgtcccgg agatcccttt agggcgtatt cgaggtgtgc 2632 tcagtgaccc gcttcggcgg ggtgggagta gccaaaagtc cgacattttt aacgaacgtt 2692 cgttaaaatg ggggcatgac tcagggacct ttgacctcag aaaccggcgc aatcctgaat 2752 gatcttggcg cagcagaccc tctcgatgtg gctgtcc ggg cacgggagag tgcgcatgtt 2812 ctctctcaag tcgtggagtt tttagagcag atcggccggt ctggggatag cgatttagac 2872 gcggtgtatg agcgtgattg gcagctcgat gcagacacgt tgaccttcat tgcccaggcg 2932 ttggaggggt tggcggacca ggccgaggcg aaggatgccg tgaacgaatg acggatatgt 2992 gtgcccaatg cggtggggaa atcccgcccc ggcctgaccc ccgcggacgc agggcgaagt 3052 attgctcgga tgcttgtcgg gcggcagcga gccgcgaacg cgcgcgccag cgccacgccc 3112 aggaggtcga agccgcgcgt ctccaggccg cactcgatct gaaaaccccg caggagaccc 3172 tggcagaggt agtccaggag cttcaggcca ccacccggat tatccgtgat cgaggggacg 3232 tgccagcgtc gctgcgtccg ctggttaatg ctgcatccga actggtcaac gcagcgcaac 3292 cggttgagga atctaagtca ttccccaacc ggcgagtgcg tcgtgcagtt aaacgaaagt 3352 ttgcgataag cgggtgatgt aactgatgga gatttttacc tgggggtgtc tccagcgagg 3412 tggccaagtc cgattgtgtt gaggattacc ccaaacgtgc ggggattatt caaaatccac 3472 tgtccaaccg cttttccggt taccccgcct ccgatgcagc ctacgagaat agagcccatg 3532 accattgcat tgtggctata tcccgcattt ggatccagcg ccgagaaact ggtgtaggca 3592 ccagcagcgc agcctgcaat gcgagcgcca atgataacca gg gggagggc gcgaggcatt 3652 actcgatttt catctgtggt ctgtcgctga atcgaagcag tgatggcttc ttcaaatgct 3712 tcagggtttg acgtggggtc cgagactgtt gacgcagctt cctgcactgc cttgatgagg 3772 acatcttcag ggatggaatc attgaacatt cctcccagct cagaagtggt ttgaacgtta 3832 gccgaaggga catgcacatc gggggaagcc tgggcggctg gagcaattaa agacagcgac 3892 agtgaagcaa cgagagccgt tacagtggca cgagttttta aatacatgag gcgaacttaa 3952 caaaccattg ataggttgtc gtgcggtaaa gataagaaaa ggataaagat atgaaaacgt 4012 tatttatgaa tctcttaggt gccgcgcttg taggagcggt aatcatggtc ttgacatggt 4072 tatttattga ttttgatgca cctggagcat ggctcggatt ctttattatc accaccatca 4132 gtgattgctg ctttagaagt catccacgga ctttgggaaa aacggcaggg atcttccact 4192 gacaatgatt gataaaacct ggttgaacgg aatacaaaac gcgcaaaata accaggcagt 4252 taaaagaaaa accagataag ctgcaccaat acttgaaaaa tgttgaacgc cccgacagct 4312 gtaactgtcg aggcgtcggc taacccccag tcatcagctg ggagaaagca ctcaaaa 4369 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying replication origin of pYM2 <400> 10 aaccaggggg agggcgcgag gc 22 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying replication origin of pYM2 <400> 11 tctcgtaggc tgcatccgag gcgggg 26 <210> 12 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying replication origin of pYM2 <400> 12 gctctagagc aaccaggggg agggcgcgag gc 32 <210> 13 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying replication origin of pYM2 <400> 13 gctctagagc tctcgtaggc tgcatcggag gcgggg 36 <210> 14 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying replication origin of pYM2 <400> 14 gctctagagc aaccaggggg agggcgcgag gc 32 <210> 15 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying replication origin of pYM2 <400> 15 gctctagagc tctcgtaggc tgcatcggag gcgggg 36 <210> 16 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying kanamycin resistant gene of Streptococcus faecalis <400> 16 cccgttaact gcttgaaacc caggacaata ac 32 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying kanamycin resistant gene of Streptococcus faecalis <400> 17 cccgttaaca tgtacttcag aaaagattag 30 <210> 18 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Escherichia coli cloning vector pHSG399 <400> 18 gatatctacg tgccgatcaa cgtctc 26 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for amplifying Escherichia coli cloning vector pHSG399 <400> 19 aggccttttt ttaaggcagt tattg 25

[Brief description of the drawings]

FIG. 1 is a restriction map of plasmids pYM1, pYM2 and pYM3 of the present invention.

FIG. 2 is a restriction map of plasmid pYM4 of the present invention.

FIG. 3 shows the construction of plasmid pYMFK.

FIG. 4 shows the construction of plasmid pYMK.

FIG. 5 shows the construction of plasmid pYMC.

FIG. 6 shows the construction of plasmid pK1.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Wataru Nakamatsu 1-1, Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Ajinomoto Co., Ltd. Fermentation Research Laboratory (72) Osamu Kurahashi 1 Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa -1 Inside the Ajinomoto Co. Fermentation Research Institute (72) Yoshio Kawahara 1-1, Suzukicho, Kawasaki-ku, Kawasaki-shi, Kanagawa Prefecture Inside the Ajinomoto Co. Fermentation Research Laboratory (72) Shinichi Sugimoto Kawasaki, Kawasaki-shi, Kanagawa 1-1 Suzukicho, Ajinomoto Co., Ltd. Fermentation Research Laboratory

Claims (5)

[Claims] 1. A plasmid that can be isolated from Corynebacterium thermoaminogenes and encodes an amino acid sequence represented by SEQ ID NO: 2 or a Rep protein having an amino acid sequence having 90% or more homology with the amino acid sequence. Gene having a size of about 4.4 Kb or about 6 Kb.
A plasmid which is Kb or a derivative thereof. 2. The plasmid may be Corynebacterium thermoaminogenes AJ12340 (FERM BP-1539), AJ12308.
(FERM BP-1540) or a plasmid having a size of about 4.4 Kb, which can be isolated from AJ12310 (FERM BP-1542),
2. The plasmid or derivative thereof according to claim 1, which has a restriction map shown in FIG. 3. The plasmid is a plasmid having a size of about 6 Kb, which can be isolated from Corynebacterium thermoaminogenes AJ12309 (FERM BP-1541), and has a restriction enzyme map shown in FIG. 2. The plasmid according to 1 or a derivative thereof. 4. The plasmid according to claim 1, which comprises a gene encoding a Rep protein having any one of the amino acid sequences shown in SEQ ID NOs: 2, 4, and 6, or a derivative thereof. 5. The plasmid according to claim 1, which comprises a gene encoding a Rep protein having the amino acid sequence shown in SEQ ID NO: 8, or a derivative thereof.