JPH0970291A - Amplification of gene using artificial transposon - Google Patents

Abstract

PROBLEM TO BE SOLVED: To amplify a desired gene on a chromosome by creating an artificial transposon having a medicine-resistant gene and a desired gene between both inverted repeats and capable of transferring in a specific bacterial cell and introducing the transposon into a cell and transferring the transposon onto the chromosome. SOLUTION: An artificial transposon having a structure inserting a desired gene comprising a gene which participates in biosynthesis of amino acid, e.g. drug-resistant gene such as a chloramphenicol-resistant gene or a tetracycline- resistant gene and an aspartokinase gene and/or a dihydropicolinic acid synthetase gene into inverted repeats derived from an insertion sequence of a coryneform bacterium and capable of transferring in the coryneform bacterium cell is created and the artificial transposon is introduced into the coryneform bacterium cell and transferred onto chromosome of the chromosome bacterium and a desired gene is introduced onto the chromosome and amplified to provide a coryneform bacterium used for industrial production of amino acid and nucleic acid.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for amplifying a desired gene on the chromosome of a coryneform bacterium using an artificial transposon capable of transposing in a coryneform bacterium, and a coryneform bacterium obtained by the method. If the desired gene is a gene involved in biosynthesis of amino acids, nucleic acids, etc., the resulting coryneform bacteria can be used to produce amino acids, nucleic acids, etc. A method of amplifying a desired gene on a chromosome is important for breeding and improving coryneform bacteria used for industrial production of amino acids and nucleic acids.

[0002]

2. Description of the Related Art Studies for breeding and improving coryneform bacteria and efficiently producing amino acids and nucleic acids have been vigorously carried out. Many genetic engineering techniques have been reported as means for breeding. The genetic engineering technique for breeding coryneform bacteria is the establishment of a transformation method using protoplasts (Ka
tsumata, R., Ozaki, A., Oka, T. and Furuya, A.: J.Bact
eriol., 159, 306-311 (1984), Santamaria, RI, Gil,
JA and Martin, JF: J. Bacteriol., 161, 463-467
(1985)), development of various vectors (Miwa, K., Matsui, H.,
Terabe, M., Nakamori, S., Sano, K. And Momose, H.: Agr
ic.Biol.Chem., 48, 2901-2903 (1984), Katsumata, R.,
Ozaki, A., Oka, T. And Furuya, A.: J.Bacteriol., 15
9, 306-311 (1984), Santamaria, RI, Gil, JA, Mesa
s, JM and Martin, JF: J.Gen.Microbiol., 130, 223
7-2246 (1984), Yeh, P., Oreglia, J., Prevotos, F. and
Scicard, AM: Gene, 47, 301-306 (1986), Patek, M.,
Nesvera, J. And Hochmannova, J.: Appl.Microbiol.Bio
technol., 31, 65-69 (1989)), Development of gene expression control method (Tsuchiya, M. and Morinaga, Y.: Bio / Technology,
6, 428-430 (1988)) and cosmid development (Miwa, K., Ma
tsui, K., Terabe, M., Ito, K., Ishida, M., Takagi, H.,
Nakamori, S. And Sano, K.: Gene, 39, 281-286 (198
5)) and other systems using plasmids and phages have been developed. In addition, gene cloning from coryneform bacteria (Matsui, K., Sano, K. and Ohtsubo, E .: Nucleic Acids
Res., 14, 10113-10114 (1986), Follettie, MT and
Shinskey, AJ: J.Bacteriol., 167,695-702 (1986), M
ateos, LM, Del, RG, Aguilar, A. and Martin, JF:
Nucleic Acids Res., 15, 10598 (1987), Mateos, LM,
Del, RG, Auilar, A. And Martin, JF: Nucleic Acid
s Res., 15, 3922 (1987), Melumbres, M., Mateos, L.
M., Guerrero, C. And Martin, JF: Nucleic Acids Re
s., 16, 9859 (1988), Matsui, K., Miwa, K. and Sano,
K .: Agric.Biol.Chem., 52,525-531 (1988), Peoples,
OP, Liebl, W., Bodis, M., Maeng, PJ, Follettie, M.
T., Archer, JA and Shinskey, AJ: Mol. Microbiol.,
2, 63-72 (1988), Eikmanns, BJ, Follettie, MT, G
riot, MU, Martin, U. and Shinskey, AJ: Mol.Gen.Ge
net., 218, 330-339 (1989), O'Regan, M., Thierbach,
G., Bachmann, B., Vgilleval, D., Lepage, P., Viret, J.
F. and Lemoine, Y.: Gene, 77, 237-251 (1989)) and increased yields of various amino acids (Sano, k., Miwa, K. and Nakam
ori, S.: Agric.Biol.Chem., 51, 597-599 (1987)).

Recently, transposable elements derived from coryneform bacteria have been reported one after another (WO92 / 02627, WO93 / 18151, EP.
0445385, JP-A-6-46867, Vertes, AA, Inu
i, M., Kobayashi, M., Kurusu, Y. and Yukawa, H.: Mol.Mi
crobiol., 11, 739-746 (1994), Bonamy, C., Labarre,
J., Reyer, O. And Leblon, G.: Mol. Microbiol., 14, 57
1-581 (1994), Vertes, AA, Asai, Y., Inui, M., Kobay
ashi, M., Kurusu, Y. and Yukawa, H.: Mol.Gen.Genet.,
245, 397-405 (1994), Jagar, W., Schafer, A., Kalinows.
ki, J. and Puhler, A.: FEMS Microbiology Letters, 12
6, 1-6 (1995), JP-A-7-107976).

The transposable element is a DN capable of transposing on the chromosome.
Fragment A is known to exist in a wide range of organisms from prokaryotes to eukaryotes. For eukaryotes, detailed knowledge has been obtained for maize, drosophila, yeast, etc., and for prokaryotes, Escherichia coli, etc. (Mobile
DNA. American Society for Microbiology, Washingto
n DC (1989)). Bacterial transposable elements are classified into two types, insertion sequences and transposons. The insertion sequence is a DNA fragment having a size of about 760 to 2000 bp, having inverted repeats of about 8 to 20 bp at both ends, and internally encodes transposase, which is an enzyme required for transposition. On the other hand, transposons are transposable elements that have genes such as drug resistance genes that are not directly involved in the transposition function in addition to inverted repeats and transposases, and that the drug resistance gene is sandwiched between two insertion sequences. There are two types, that is, the drug resistance gene is inserted in the insertion sequence. As a characteristic common to both the insertion sequence and the transposon, it is also known that a nucleotide sequence overlap of about 10 bp is observed at the target gene site into which these are introduced (Mobile Genetic E
lements. Academic Press, New York, p.159-221 (198
3)).

Among the currently known transposable elements are the Escherichia coli transposons Tn10, Tn5 and M.
There are some very useful for chromosomal genetic engineering such as u-phage. As an example of using these, by transferring a transposon into a gene on a chromosome,
1) Gene disruption to suppress the expression of the chromosomal gene 2) Expression of the chromosomal gene at the site of introduction by inserting a promoter sequence on the transposon It may be possible to introduce a new gene into a chromosome by loading it on a transposon and carrying out transposition (Mobile DNA. American Society for Microbiology, W
ashington DC, p.879-925 (1989)).

In coryneform bacteria, a transposable element called an insertion sequence was recently found.
A transposon type having a drug resistance gene and the like has not been found. However, artificially inserting a kanamycin resistance gene into a transposon (WO93 / 18151,
JP-A-7-107976, Vertes, AA, Asai, Y., Inu
i, M., Kobayashi, M., Kurusu, Y. and Yukawa, H.: Mol.G
en.Genet., 245, 397-405 (1994)), it has become possible to carry out transposition on the chromosome. Was created here
The artificial transposon has a drug resistance gene sandwiched between two insertion sequences (WO93 / 1
8151) and a drug resistance gene inserted into the insertion sequence (JP-A-7-107976,
Vertes, AA, Asai, Y., Inui, M., Kobayashi, M., Kurus
u, Y. and Yukawa, H.: Mol.Gen.Genet., 245, 397-405
(1994)), but such an artificial transposon has no transfer in multiple copies, or the increase in copy number is not satisfactory, and it has utility value in the amino acid and nucleic acid industries. The technology for gene amplification has not been reached yet.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides an artificial transposon carrying a drug resistance gene and a desired useful gene on the basis of an insertion sequence derived from coryneform bacteria, and uses the artificial transposon to produce an amino acid. It is an object of the present invention to provide a method for amplifying a desired gene on the chromosome of coryneform bacteria used for industrial production of nucleic acid and nucleic acid. Another object of the present invention is to provide a coryneform bacterium in which a desired useful gene is amplified on the chromosome. Furthermore, the present invention uses a coryneform bacterium in which a desired useful gene is amplified on a chromosome,
It is an object to provide a method for producing substances such as amino acids and nucleic acids.

[0008]

[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors have proposed a transposon known in Escherichia coli et al. Chromosomal DNA of coryneform bacteria, focusing on the transfer by taking a structure having a drug resistance gene unrelated to the function
Artificially constructing various transposon-like sequences having a structure in which a drug resistance gene not involved in the transposition function and a desired gene are inserted between the inverted repeats at both ends of the insertion sequence present in the transposon. To efficiently transfer the gene-amplified gene in which multiple copies of the artificial transposon were transferred to the chromosome by appropriately setting the drug resistance gene and its selective concentration. As a result, they have completed the present invention.

That is, the present invention is as follows. (Invention 1) An artificial transposon having a structure in which a drug resistance gene and a desired gene are sandwiched in an inverted repeat and capable of transposition in coryneform bacterial cells is constructed, and the artificial transposon is introduced into coryneform bacterial cells. Then, the transposon is transferred onto the chromosome of the coryneform bacterium, and the desired gene is introduced into the chromosome and amplified. (Invention 2) The method according to Invention 1, wherein the artificial transposon has a structure in which a transposase gene is further sandwiched between inverted repeats. (Invention 3) The method according to Invention 1 or 2, wherein the inverted repeat and transposase gene are derived from the insertion sequence of coryneform bacteria. (Invention 4) The method according to Invention 3, wherein the insertion sequence has a base sequence represented by any one of SEQ ID NOS: 1, 5 and 9 in the sequence listing. (Invention 5) The method according to any one of Inventions 1 to 4, wherein the drug resistance gene is a chloramphenicol resistance gene or a tetracycline resistance gene. (Invention 6) The method according to Invention 1 to 5, wherein the desired gene is a gene involved in amino acid biosynthesis. (Invention 7) The method according to Invention 6, wherein the desired gene is an aspartokinase gene and / or a dihydropicolinate synthase gene. (Invention 8) A coryneform bacterium in which a desired gene is introduced into a chromosome by the method according to any one of Inventions 1 to 7. (Invention 9) A coryneform bacterium in which a gene involved in amino acid biosynthesis has been introduced into a chromosome by the method according to Invention 6, is cultured in a medium, and amino acids are produced and accumulated in the medium,
A method for producing an amino acid, which comprises recovering the amino acid. (Invention 10) The method according to Invention 9, wherein the gene involved in amino acid biosynthesis is an aspartokinase gene and / or a dihydropicolinate synthase gene, and the amino acid is lysine.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The inverted repeats referred to in the present invention are preferably those present at both ends of a transposable element isolated from coryneform bacteria. As the transposable element derived from coryneform bacteria, for example, the insertion sequence described in SEQ ID NOS: 1, 5 and 9 is known (WO93 / 18151), but the insertion sequence IS714 described in SEQ ID NO: 5 is 5 '. The sequence shown in SEQ ID NO: 3 is provided on the side and the sequence shown in SEQ ID NO: 4 is provided on the reverse chain 5 ′ side to form an inverted repeat. The insertion sequence IS719 described in SEQ ID NO: 5 has the sequence described in SEQ ID NO: 7 on the 5 ′ side and the sequence described in SEQ ID NO: 8 on the reverse chain 5 ′ side, forming an inverted repeat. One kind of sequence selected from SEQ ID NOs: 3, 4, 7 and 8 can be arranged on the 5 ′ side and the reverse chain 5 ′ side to form an inverted repeat, or SEQ ID NOs: 3, 4, 7 It is also possible to form two inverted sequences by arranging two kinds of sequences selected from the above and 8 on the 5 ′ side and the reverse 5 ′ side, respectively. The insertion sequence IS903 described in SEQ ID NO: 9 has the sequence described in SEQ ID NO: 11 on the 5 ′ side and the sequence described in SEQ ID NO: 12 on the reverse chain 5 ′ side, forming an inverted repeat. One kind of sequence selected from SEQ ID NOS: 10 and 11 can be arranged on the 5 ′ side and the reverse chain 5 ′ side to form an inverted repeat, or two kinds of sequences of SEQ ID NOS: 10 and 11 can be formed. Inverted repeats can be formed by arranging them on the 5 ′ side and the reverse chain 5 ′ side, respectively. The inverted repeat of the present invention has SEQ ID NO: 1,
It is not limited to those having the sequences described in 5 and 9, but there are other sequences that function in the transposable element.

Examples of the drug resistance gene referred to in the present invention include genes resistant to various drugs such as kanamycin resistance gene, chloramphenicol resistance gene and tetracycline resistance gene, as well as ampicillin and methotrexate resistance gene. Particularly, a drug resistance gene in which the degree of drug resistance and the copy number of the drug resistance gene have a correlation is preferable.

Examples of the desired gene to be amplified include genes involved in biosynthesis of various amino acids and nucleic acids. For example, a glutamate dehydrogenase gene for glutamate biosynthesis, a glutamine synthetase gene for glutamine biosynthesis, an aspartokinase gene for lysine biosynthesis (hereinafter, aspartokinase is referred to as “AK”).
And the aspartokinase gene may be referred to as "lysC"), the dihydrodipicolinate synthase gene (hereinafter, dihydrodipicolinate synthase is referred to as "DDPS",
The dihydrodipicolinate synthase gene may be referred to as "dapA") and the dihydrodipicolinate reductase gene (hereinafter, dihydrodipicolinate reductase may be referred to as "DDPR" and the dihydrodipicolinate reductase gene may be referred to as "dapB"). , Diaminopimelate decarboxylase gene (hereinafter, diaminopimelate decarboxylase may be referred to as “DDC” and diaminopimelate decarboxylase gene may be referred to as “lysA”), diaminopimelate dehydrogenase gene (hereinafter, diaminopimelate dehydrogenase as “DDH”). , The diaminopimelate dehydrogenase gene is referred to as "ddh"), the homoserine dehydrogenase gene for threonine biosynthesis, and acetoline for isoleucine or valine biosynthesis. Droxyate synthase gene, 2-isopropylmalate synthase gene for leucine biosynthesis, glutamate kinase gene for proline and arginine biosynthesis, phosphoribosyl-ATP pyrophosphorylase gene for histidine biosynthesis, Deoxyarabinoheptulonate phosphate (DAHP) synthase genes and nucleic acids for the biosynthesis of aromatic amino acids such as tryptophan, tyrosine and phenylalanine, eg phosphoribosyl pyrophosphate (PRPP) for the biosynthesis of inosinic acid and guanylic acid. ) Amidotransferase gene, inosing anosine kinase gene,
Examples include inosine acid (IMP) dehydrogenase gene and guanylate (GMP) synthase gene. In addition, genes encoding bioactive proteins such as interleukin 2 and interleukin 6 are also included.

The coryneform bacteria referred to in the present invention means Berg.
ey's Manual of Determinative Bacteriology, 8th E
As described in d., p.599 (1974), it is an aerobic gram-positive bacillus, which is a bacterium that has been conventionally classified into the genus Corynebacterium, but was conventionally classified into the genus Brevibacterium. Bacteria currently integrated as Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (198
1)), and Brevibacterium or Microbacterium which are very closely related to the genus Corynebacterium, and are the following microorganisms generally known as L-glutamic acid-producing bacteria. . Corynebacterium acetoacidophilum Corynebacterium acetoglutamicum Corynebacterium carnae Corynebacterium glutamicum Corynebacterium lilium (Corynebacterium
Glutamicum) Corynebacterium meracecora Brevibacterium divaricatum (Corynebacterium glutamicum) Brevibacterium flavum (Corynebacterium)
Brevibacterium inmariophyllum Brevibacterium lactofermentum (Corynebacterium glutamicum) Brevibacterium roseum Brevibacterium saccharoticum Brevibacterium thiogenitalis Brevibacterium ammoniagenes (Corynebacterium Bacteria / Ammoniagenes) Microbacterium / Ammonia Philum Corynebacterium thermogenicness

Specific examples include the following wild strains and various mutant strains derived from them. Corynebacterium acetoacidophilum ATCC
13870 Corynebacterium acetoglutamicum ATCC1
5806 Corynebacterium karnamie ATCC15991 Corynebacterium glutamicum ATCC1302
0 Corynebacterium lilium (Corynebacterium
Glutamicum) ATCC15990 Corynebacterium meracecora ATCC1796
5 Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC14020 Brevibacterium flavum (Corynebacterium
Glutamicum) ATCC14067 Brevibacterium inmariophilum ATCC1
4068 Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC13869 Brevibacterium roseum ATCC13825 Brevibacterium saccharolyticum ATCC1
4066 Brevibacterium thiogenitalis ATCC 192
40 Brevibacterium ammoniagenes (Corynebacterium ammoniagenes) ATCC6871 Microbacterium ammoniaphilum ATCC1
5354 Corynebacterium thermoaminogenes AJ123
40 (FERM BP-1539)

The artificial transposon of the present invention has a structure in which a drug resistance gene and a desired gene are sandwiched between inverted repeats, and is transposable in coryneform bacterial cells.

The transposase of the present invention is considered to have, for example, the amino acid sequences shown in SEQ ID NOS: 2 and 6 in the sequence listing. However, as long as it has transposase activity, one or more amino acid residues may be deleted, inserted, added, substituted or transferred. The transposase gene of the present invention means, for example, from the 130th position to 1437th position in the nucleotide sequence shown in SEQ ID NO: 1 in the Sequence Listing.
The 1st sequence, and the 130th to 1437th sequences among the base sequences shown in SEQ ID NO: 5 of the Sequence Listing. As long as the gene product has transposase activity, one or more bases may be deleted, inserted, added, substituted or transferred. The transposase gene may be inside the artificial transposon. That is, they are sandwiched between inverted repeats and arranged so as not to impair the functions of the drug resistance gene and the desired gene. The transposase gene may be outside the artificial transposon. The artificial transposon may be simultaneously mounted on a vector mounted thereon, or may be mounted on another vector different from the vector.
It may be present on the chromosome of coryneform bacteria.

In order to construct the artificial transposon of the present invention, a transposable element derived from a coryneform bacterium is used as a starting material, which facilitates the construction operation.

The insertion sequence used in the present invention is present on the chromosome of the coryneform bacterium described above,
There is no particular limitation as long as it has a size of about 760 to 2000 bp and has inverted repeats of about 8 to 20 bp at both ends and encodes transposase which is an enzyme required for transposition inside. In order to obtain such an insertion sequence, the method disclosed in WO93 / 18151 may be followed. 1) Coryneform bacteria with plasmid pE
2) pEC701, which was transformed by introducing C701
The strain transformed with Kanamycin is selected as a marker, and 3) Coryneform bacteria having plasmid pEC701 is applied to an agar plate containing isopropyl-β-thiogalactoside (IPTG) to select a grown strain, and 4) Selection is performed. The nucleotide sequence of the expression control region or structural gene region of the chloramphenicol acetyltransferase gene of the plasmid retained by the strain was analyzed, and 5) a DNA fragment containing the insertion sequence was found by finding the sequence inserted into the sequence. Obtainable. In addition, 1) plasmid pEC901 was introduced into coryneform bacteria for transformation, and 2) pEC9.
The strain transformed with 01 was selected using kanamycin resistance as a marker, 3) coryneform bacteria having pEC901 were cultured at 30 ° C., and a strain expressing chloramphenicol resistance even at 30 ° C. was selected and 4) selected. It can also be obtained by analyzing the nucleotide sequence of the cI repressor of the plasmid held by the strain and finding the sequence inserted into the sequence, 5).

Specific examples of the insertion sequence of coryneform bacteria include SEQ ID NOS: 1, 5 and 9 in the Sequence Listing.
3 insertion sequences shown in
14, IS719, IS903. The base sequence shown here is not necessarily strict, including an inverted repeat sequence, a part of the base is replaced with another base, a part of the sequence is deleted,
Even if a new sequence is inserted or added, it can be used for constructing an artificial transposon as long as it has a function as an insertion sequence.

Based on these insertion sequences, various types of artificial transposons can be prepared. The structures of these artificial transposons are shown in FIG.
Among them, the artificial transposon used in the present invention has a structure in which a drug resistance gene and a desired gene to be amplified are inserted inside the insertion sequence. In the case of IS714 shown in SEQ ID NO: 1, there is a restriction enzyme NheI cleavage site at the 37th position to the 42nd position, and even if insertion of a base occurs at this position, the functions of inverted repeat and transposase are not impaired. It is suitable as a position for introducing a gene and a desired gene to be amplified. In the case of IS719 shown in SEQ ID NO: 5, there is a restriction enzyme NheI cleavage site at the 37th position to the 42nd position, and even if insertion of a base occurs at this position, the functions of inverted repeat and transposase are not impaired, so drug resistance It is suitable as a position for introducing a gene and a desired gene to be amplified. In the case of IS903 shown in SEQ ID NO: 9, there is a restriction enzyme XcmI cleavage site at positions 34 to 48, and even if insertion of a base occurs at this position, the functions of inverted repeat and transposase are not impaired, so drug resistance It is suitable as a position for introducing a gene and a desired gene to be amplified.

In the following, using IS714 as an example, a method for constructing an artificial transposon in which a gene resistant to a drug such as kanamycin (neomycin), chloramphenicol, and tetracycline is inserted. A method for constructing an artificial transposon in which a desired gene useful for production (eg, aspartokinase gene) is inserted will be described.

(1) Construction of artificial transposon carrying kanamycin resistance gene Brevibacterium lactofermentum AJ12036 (FERM) which is a wild-type strain of coryneform bacteria.
Plasmid pEC701-I having the sequence of IS714 which is an insertion sequence derived from BP-734)
S14 (see WO93 / 18151) has the restriction enzymes PvuII and Eco
1.6 including IS714 by cutting with RI
A kb fragment is obtained. On the other hand, IS7 is present at the restriction enzyme SalI site of the plasmid pHSC4 (see JP-A-5-7491) having a temperature-sensitive replication origin derived from coryneform bacteria.
The fragment containing 14 is inserted to create the plasmid pHIS714. Another 1.6 kb fragment containing IS714 obtained above is inserted into the SmaI site of pHIS714 to prepare plasmids pHTN7141 and pHTN7142 containing two IS714 fragments in the forward and reverse directions (Fig. 2). Then pHTN7141 and pHTN7
By cleaving 142 with the restriction enzyme PvuII, a fragment containing two sequences of IS714 and a sequence of the temperature-sensitive replication origin of pHSC4 in the coryneform bacterium can be excised. On the other hand, plasmid vector pHS
G298 (Takara Shuzo Co., Ltd.) also has 2 restriction enzyme PvuII sites.
There are several sites, and by cutting with the restriction enzyme PvuII, a 2.3 kb fragment containing the neomycin phosphotransferase gene (which is also a kanamycin resistance gene) can be obtained. Therefore, pHTN714
1 and pHSG298 are cleaved with a restriction enzyme PvuII and then ligated to transform Brevibacterium lactofermentum AJ12036. The plasmid pHTN7143 is obtained from a transformant resistant to kanamycin (FIG. 3).

In a similar manner, the plasmid pHTN7
142 and pHSG298 from plasmid pHTN714
4 is obtained (FIG. 4). pHTN7143 and pHTN71
44 has a structure in which the neomycin phosphotransferase gene is sandwiched between two IS714s. In addition, by the same method, plasmid pHIS71
4 and pHSG298 from plasmid pHIS714K1
And pHIS714K2 are prepared as control (FIG. 5). pHIS714K1 and pHIS714K2 have a reverse relationship in the orientation of the insert fragment containing the neomycin phosphotransferase gene.

Next, in order to miniaturize the artificial transposon, an artificial transposon in which the neomycin phosphotransferase gene is inserted into one IS714 is prepared. In IS714, a restriction enzyme NheI site exists at a position that does not impair the function of transposase.
Then, the plasmid pHIS714 is cleaved with the restriction enzyme NheI to make its ends blunt-ended. On the other hand, the neomycin phosphotransferase gene region is excised from the plasmid pUC4K (Pharmacia Biotech) with the restriction enzyme PstI, and the end thereof is made a blunt end. Ligate both to obtain the desired plasmid pH
TN7145 is obtained (Fig. 6).

(2) Construction of artificial transposon carrying chloramphenicol resistance gene Approximately 1.1 containing chloramphenicol acetyltransferase gene by cutting plasmid vector pHSG398 (Takara Shuzo) with restriction enzyme AccII.
Fragments as large as kb can be obtained. Next this Ac
The cII fragment inserted into the SmaI site of pUC18 (Takara Shuzo) is cloned. The desired clone is selected to obtain the plasmid pUC18-CM. Furthermore pUC
A fragment of about 1.1 kb containing the chloramphenicol acetyltransferase gene excised from 18-CM with EcoRI and HindIII and p constructed previously.
HIS714K2 was ligated with a blunt-ended restriction enzyme NheI site existing at a position not related to the transfer function in IS714, and Escherichia coli was transformed with the chloramphenicol acetyltransferase gene fragment inserted. Select a clone.
From the obtained clone, the target plasmid pHTN7151
Can be obtained (FIG. 7).

(3) Construction of artificial transposon carrying tetracycline resistance gene By digesting plasmid vector pBR322 (manufactured by Takara Shuzo) with restriction enzymes EcoRI and AvaI, a fragment of about 1.4 kb containing the tetracycline resistance gene was obtained. Obtainable. This DNA fragment was ligated with the previously constructed pHIS714K2 blunt-ended with a restriction enzyme NheI site existing at a position not related to the transfer function in IS714, and Escherichia coli was transformed with the tetracycline resistance gene fragment. Select the clone in which was inserted. The target plasmid pHTN7152 can be obtained from the obtained clone (FIG. 8).

(4) Insertion of aspartokinase gene, which is one of lysine biosynthesis genes, into artificial transposon carrying tetracycline resistance gene A good restriction enzyme for inserting aspartokinase gene into pHTN7152 constructed in FIG. Since there is no site, pHTN7156 with a new insertion site is constructed as follows. About 1.4k containing the tetracycline resistance gene by cutting the plasmid vector pBR322 (Takara Shuzo) with the restriction enzymes EcoRI and AvaI.
Fragments as large as b can be obtained. This DNA fragment is ligated with a plasmid vector pHY300PLK (manufactured by Takara Shuzo Co., Ltd.) cleaved with a restriction enzyme SmaI to transform Escherichia coli, and a clone into which a tetracycline resistance gene fragment has been inserted is selected. From the obtained clone, the plasmid pHY300-T
Get C. In addition, pHY300-TC is a restriction enzyme Eco.
A fragment containing the tetracycline resistance gene obtained by digestion with RI and XbaI, and pHIS constructed above
A blunt-ended restriction enzyme NheI site present at a position not related to the transfer function in IS714 of 714K2 is ligated to transform Escherichia coli, and a clone into which a tetracycline resistance gene fragment has been inserted is selected. . The desired plasmid pHTN7156 is obtained from the obtained clone (FIG. 9).

Next, the aspartokinase gene, which is one of the lysine biosynthesis genes, is inserted into the plasmid pHTN7156 as follows. A plasmid p399AK9B containing a lysine-producing mutant strain of the coryneform bacterium Brevibacterium lactofermentum and containing an aspartokinase gene that is desensitized to the concerted inhibition of lysine and threonine (see WO94 / 25605). Is cleaved with the restriction enzyme BamHI and self-ligated to construct pHSG399AK excluding the origin of replication that functions in coryneform bacteria. This pHSG3
By cutting 99AK with restriction enzymes EcoRI and SphI,
An about 1.7 Kb aspartokinase gene fragment was obtained and inserted into the plasmid pHTN7156 having an artificial transposon carrying a tetracycline resistance gene at the blunted restriction enzyme BglII site to construct a plasmid pHTN7156-C ( (Figure 9).

(5) Construction of artificial transposon equipped with a tetracycline resistance gene consisting of a unit in which transposase is separated from transposon A fragment containing the transposase gene of IS714 excised by restriction enzymes NheI and XbaI was obtained from plasmid pHIS714 to obtain a plasmid vector. It is cloned into the XbaI site of pUC19 to construct the plasmid TnpL / pUC19. Next, Tn
By cleaving pL / pUC19 with the restriction enzymes MroI and XbaI, a sequence designed to remove the sequence containing the IS714 stop codon and the 3'inverted repeat (IR), and instead regenerate only the stop codon was synthesized. Insert double-stranded DNA. By this operation, a transposase gene which is not adjacent to IR is obtained. Next, this ORFL / pUC19 was added to the restriction enzymes SmaI and Xb
Approximately 1.5 including the transposase gene, cleaved with aI
Obtain a kb fragment. This transposase gene fragment was cloned into SmaI of the plasmid vector pHY300PLK.
To XbaI are inserted, and then the product is further excised with restriction enzymes EcoRI and KpnI. After the plasmid vector pHSG398 was partially digested with a restriction enzyme PvuII to remove the fragment containing the multicloning site, the transposase gene fragment excised from pHY300PLK was blunt-ended, and
Ligate to construct plasmid pORF1 (FIG. 10).

On the other hand, IS7 cut out from the plasmid pHIS714 by the restriction enzymes NheI and XbaI.
After obtaining a fragment containing 14 transposase genes, the fragment was blunt-ended, and Ps of the plasmid vector pUC19 was obtained.
The tI site is blunt-ended and cloned to construct a plasmid Tnp (Pst) / pUC19. On this Tnp (Pst) / pUC19, partial base substitution in the transposase gene was carried out by U.S.P. S. E. FIG. Mu
It is performed using a tagenesis kit (Pharmacia Biotech). The replaced base is G corresponding to the 288th base of IS714 shown in SEQ ID NO: 1, and this is changed to C. The plasmid with the base substitutions is designated as Tnp (Pst) M / pUC19. Tn
The structure of p (Pst) M / pUC19 is shown in FIG. * Indicates the introduced mutation.

Transposition of transposable elements is regulated in various ways. For example, (Mobile DNA.Ame
rican Society for Microbiology, Washington DC (19
89)). 1) In addition to transposase, transposase inhibitors and repressors are encoded side by side in the transposable element (eg, Tn3). 2) There are two ORFs in frame, of which 3 '
The flanking ORF encodes the inhibitor. Two ORFs
Infrequently, a frame shift of translation occurs, which causes the two ORFs to be continuously translated to generate a transposase (for example, IS1). 3) Another initiation codon (ATG, GTG) exists inside the ORF in which transposase is encoded, and translation is initiated from there to produce an inhibitor (eg, T
n5 (IS50)).

By the way, in IS714, one ORF exists over almost the entire length thereof, and no other long ORF is found. From this fact, IS714, like Tn5, is an ORF encoding transposase.
, While it is possible that the inhibitor will be translated from another start codon present within it. A search for a promoter-like sequence revealed that 2 was found on the IS714 sequence.
It was revealed that the sequence of GTG corresponding to bases 86 to 288 may correspond to the start codon of the inhibitor. That is, the mutation introduced on Tnp (Pst) M / pUC19 is to prevent the translation of the inhibitor.

Tnp (Pst) M / pUC19 was obtained by removing between the restriction enzyme sites SmaI and NaeI on the transposase first half gene on pORF1.
Is ligated to the transposase first half gene fragment obtained by cutting with the restriction enzymes SmaI and NaeI to construct pORF2. Sm of pORF2
When the region between the aI site and the XbaI site was removed to make a blunt end, pBSF2-SD7 (WO92 / 14
A gene fragment containing the tryptophan operon attenuator, which was excised with the restriction enzymes NaeI and HindIII from 832), is blunt-ended and then inserted. The plasmid constructed here is named pORF3. pOR
F3 was digested with restriction enzymes SalI and Bpu1102I to remove the transposase first half gene fragment, and Tnp (Pst) / pUC19 was digested with restriction enzymes SalI.
The first half of the transposase gene fragment obtained by digesting with I and Bpu1102I was ligated to obtain pORF.
4 is constructed (FIG. 11).

After cutting TnpL / pUC19 with SacI, it is digested with BAL31 nuclease at 30 ° C. for 20 minutes to delete the vicinity of the start codon of the transposase gene from the upstream side. Then, the transposase gene was excised using the SphI site, and pHSG398
Is inserted where it was cut with SmaI and SphI. The plasmid constructed in this way was named delTnp5 /
It is named 398. The transposase first half gene fragment obtained by cutting delTnp5 / 398 with restriction enzymes KpnI and HindIII was blunt-ended, and then the plasmid vector pKK233-2 (Pharmacia Biotech) was blunt-ended with NcoI and HindIII. Ligation in place, pTrc-OR
Build F. pTrc-ORF with SspI and Bpu1
A fragment containing the Trc promoter and the transposase first half gene obtained by cutting with 102I was digested with XbaI at pORF3 to make it blunt-ended, and then further cut with Bpu1102I to remove the transposase first half gene fragment. Insert and p
Construct ORF7 (Figure 12).

The transposase first half gene fragment obtained by cleaving delTnp5 / 398 with the restriction enzymes KpnI and HindIII was used as a plasmid vector pU.
Clone between KpnI and HindIII of C18. Bsm of the plasmid (delTnp5 / 18)
In and NaeI are removed, and Tnp (Pst) M / p
The transposase first half gene fragment obtained by cutting UC19 with the restriction enzymes BsmI and NaeI is ligated to construct delTnp5M / 18. delTnp5M / 18 with KpnI and HindIII
The first half of the transposase gene fragment obtained by digestion with pKK233-2 was digested with NcoI and Hi.
It is cut with ndIII and ligated at the blunted end to construct pTrc-TnpM. pTrc-O
PORF8 is constructed from pTrc-TnpM and pORF3 using a method similar to the method for constructing pORF7 from RF (FIG. 13).

Conventional plasmids pORF3 and pOR
Each plasmid for introduction into a coryneform bacterium is constructed using F4, pORF7, and pORF8 as materials. PORF below
3 shows the procedure for constructing pORF41 from 3. First, pH
PH7147 is constructed by cutting IS714 with NheI and SacII and inserting most of the transposase gene into the double-stranded synthetic DNA designed for cloning site construction. pHTN716
0 is digested with a restriction enzyme KpnI to make it blunt-ended, and then further digested with BglI to obtain a gene fragment containing IR on both sides of IS714 and a temperature-sensitive replication origin that functions in coryneform bacteria. In addition, pORF3 was cleaved with the restriction enzyme EarI to make it blunt-ended, and then BglI was further added.
Disconnect with. The pHTN7160-derived fragment is inserted therein to construct pORF41-pre. Then pOR
When F41-pre was cleaved at the EcoR site located at the position sandwiched by IR on both ends of IS714, the restriction enzyme EcoRI was obtained from plasmid vector pBR322.
And blunt-ended tetracycline resistance gene excised using AvaI are inserted to construct pORF41 (FIG. 14).

Using the same method, from pORF4 to pORF31 via pORF31-pre,
From RF7 via pORF71-pre to pORF
71 is constructed from pORF8 via pORF81-pre to construct pORF81. Also, set pORF3 to X
After cutting with baI and EarI, it is blunt-ended and self-ligated to construct pORFC0 (FIG. 1).
5). pORFC2 is constructed from pORFC0 via pORFC2-pre in the same manner as in the case of constructing pORF41 from pORF3.

On these final constructed plasmids, the transposase structural gene, the chloramphenicol resistance gene, the origin of replication that functions in E. coli, the temperature-sensitive origin of replication that functions in coryneform bacteria, and the invertors at both ends derived from IS714. There is a tetracycline resistance gene flanked by ted repeats (IR). However, pORFC
For 2, there is no transposase structural gene.

The IR and tetracycline resistance gene units at both ends of IS714 were replaced with transposon unit Tn.
It is named 7162. While IS714 itself, or Tn7152 described above, has a transposase structural gene in the gene of the transposable region,
Tn7162 is characterized by not having a transposase structural gene in the transposable region. In this case, it is considered that transposition is caused by the transposase expressed from the transposase gene existing outside the unit on the same vector carrying Tn7162 (FIG. 16). Alternatively, it is considered that the transposition is caused by the transposase expressed by the transposase gene existing on the chromosome.

Next, a plasmid for introduction into a coryneform bacterium in which only the transposase gene is mounted without mounting the transposon unit is constructed. Plasmid p
After removing IS714 by cutting HIS714K1 with EcoO109I and MroI, self-ligation is performed to construct pHIS714Kdel. On the other hand, pORF3 is cleaved with the restriction enzyme EarI to make it blunt-ended, and then further cleaved with BglI. PH there
After fragmenting IS714Kdel with a restriction enzyme KpnI to make it blunt-ended, it is further digested with BglI to ligate a fragment containing a temperature-sensitive replication origin that functions in coryneform bacteria to construct pORF40 (FIG. 17). . Performing the same operation, pORF4 to pORF30, pORF7 to pORF70, pORF7
PORF80 from ORF8 and p from pORFC0
Build ORFC1.

In addition to IS714 described above, IS71 having the nucleotide sequences shown in SEQ ID NOS: 5 and 9 of the Sequence Listing, respectively.
9 and IS903 and other insertion sequences derived from coryneform bacteria, the chloramphenicol resistance gene and the tetracycline resistance were inserted into appropriate restriction enzyme sites existing outside the expression control region and structural gene region of the transposase gene in the insertion sequence. An artificial transposon can be constructed by inserting a drug resistance gene such as a gene and a desired gene such as an aspartokinase gene. When there are no appropriate restriction enzyme sites outside the expression control region and structural gene region of the transposase gene, the polymerase chain
A site-specific mutation or gene insertion may be carried out by a reaction method (PCR method) or a synthetic DNA oligonucleotide (adapter) to modify the nucleotide sequence to prepare an appropriate restriction enzyme site in advance.

The artificial transposon thus constructed is
It is introduced into a coryneform bacterium which is a host by mounting it on an appropriate vector such as a plasmid. The plasmid carrying the artificial transposon is not particularly limited, but a plasmid derived from coryneform bacteria may be usually used.
Specifically, pHM1519 (Agric.Biol.Chem., 48,
2901-2903 (1984)), pAM330 (Agric.Biol.Che
m., 48, 2901-2903 (1984)), and a plasmid having a drug resistance gene obtained by improving these. Furthermore, in order to efficiently amplify the introduced artificial transposon on the chromosome, it is preferable to use a plasmid having a temperature-sensitive replication origin as described in (1) above (see JP-A-5-7491). As a method for introducing a plasmid carrying an artificial transposon into coryneform bacteria, the commonly used protoplast method (Gene, 3
9, 281-286 (1985)), electroporation method (B
io / Technology, 7, 1067-1070 (1989)) and the like.

When the artificial transposon is loaded on a temperature-sensitive plasmid and introduced into a coryneform bacterium, the constructed plasmid is transformed into a coryneform bacterium and cultured at 25 ° C. at which the plasmid can be replicated. ~ A few hundred copies of the artificial transposon-laden plasmid were amplified and introduced into the chromosome, then 34
It is only necessary to remove the extra plasmid by culturing at ℃, and this method allows efficient amplification of the gene on the chromosome. A normal plasmid can be used without using the temperature-sensitive plasmid, but it is often difficult to remove an extra plasmid after the introduction into the chromosome. In addition, there is also a method of introducing a DNA fragment containing only an artificial transposon or a plasmid vector that cannot replicate in coryneform bacteria (for example, a plasmid vector that replicates in Escherichia coli) into the chromosome of coryneform bacteria (JP-A-7-107976, Vertes, AA,
Asai, Y., Inui, M., Kobayashi, M., Kurusu, Y. And Yuk
awa, H.: Mol. Gen. Genet., 245, 397-405 (1994)), but this method uses DN within the host cell or outside the host chromosome after transformation.
The A fragment cannot be amplified and the transfer efficiency to the host chromosome is extremely poor.

As a method for selecting a strain in which a desired gene is introduced into a chromosome or a strain in which the gene is further amplified on a chromosome, a method of utilizing the drug resistance degree of a drug resistant gene introduced together with the desired gene is used. Is used. Examples of drug resistance genes used include genes resistant to various drugs such as kanamycin, chloramphenicol and tetracycline resistance genes, as well as ampicillin and methotrexate resistance genes. Most preferred are drug resistance genes whose numbers are interrelated. That is, a strain in which the desired gene is amplified on the chromosome can be obtained from a clone that can grow in the presence of a higher concentration of drug.

A coryneform bacterium was transformed with the constructed plasmid (eg pHTN7156-C) carrying an artificial transposon containing a drug resistance gene such as tetracycline resistance gene and a desired gene such as desensitized aspartokinase gene. After the transfer of the artificial transposon to the host chromosome, the transfer copy number on the chromosome generated by the transfer can be evaluated by the following method. The CM2G (yeast extract 10 g / l, tryptone 10 g / l) containing an appropriate concentration (1 to 20 μg / ml for Tc) of a selective drug such as tetracycline (Tc) in the transformant.
l, glucose 5 g / l and NaCl 5 g / l) After culturing in liquid medium overnight at 25 ° C., appropriately diluting with 0.9% NaCl solution and adding 100 μl to the above CM2G agar medium containing the drug in an appropriate concentration range. Apply one by one. These were cultured at 34 ° C., a few clones were randomly selected from each of the emerged colonies, chromosomal DNA was prepared, completely digested with various appropriate restriction enzymes such as PvuII, and agarose gel electrophoresis was performed. Blot on a filter such as nitrocellulose, nylon, or polyvinylidene difluoride (PVDF). This filter is ³² P
-Southern hybridization is carried out using the labeled tetracycline resistance gene fragment as a probe, and the number of bands that hybridize with the probe is assayed.

The transformant obtained by amplifying the desired gene on the chromosome thus obtained may be cultured according to the methods and conditions generally used. The medium for culturing is an ordinary medium containing a carbon source, a nitrogen source, inorganic ions and the like. In addition, it is desirable to add organic micronutrients such as vitamins and amino acids, if necessary. As the carbon source, carbohydrates such as glucose and sucrose, organic acids such as acetic acid, alcohols such as ethanol, and the like are appropriately used. As the nitrogen source, ammonia gas, ammonia water, ammonium salt, etc. are used. As the inorganic ions, magnesium ions, phosphate ions,
Potassium ion, iron ion, and others are appropriately used as necessary. The culture is carried out under aerobic conditions at a pH of 5.0 to 8.
5. Culturing is carried out for about 1 to 7 days while controlling the temperature within a suitable range from 15 ° C to 37 ° C. As a result of the gene amplification using the artificial transposon, the production efficiency of the target useful substance is increased, and a large amount of the target substance is produced and accumulated inside or outside the cells in the culture. The target substance can be collected from the culture by a known method.

[0047]

EXAMPLES The present invention will now be described in more detail with reference to examples.

[Example 1] Artificial use of IS714 containing kanamycin resistance gene
Construction of transposon Plasmid pEC701- having the sequence of IS714 which is an insertion sequence derived from coryneform bacteria.
By cutting IS14 with restriction enzymes PvuII and EcoRI, a 1.6 kb fragment containing IS714 was obtained.
Brevibacterium lactofermentum AJ126 carrying plasmid pEC701-IS14
84 is the accession number FERM P on March 10, 1992.
-Deposited under 12863 from the Institute of Biotechnology, Institute of Technology, Ministry of International Trade and Industry (1-3, Higashi 1-3-3, Tsukuba, Ibaraki, Japan, 305), under the Budapest Treaty on March 9, 1993. Deposited on the basis of deposit number FER
MBP-4232 has been added. On the other hand, a fragment containing IS714 at the restriction enzyme SalI site existing at a position not involved in replication of the mutant plasmid pHSC4 whose replication function became temperature sensitive was blunt-ended by Klenow fragment treatment, and then inserted by ligation. , Plasmid pHIS714 was prepared (FIG. 2). Escherichia coli AJ12571, which carries the plasmid pHSC4, was developed under the contract number FERM P-11763 on October 11, 1990 under the Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology (Postal code 305, Ibaraki, Japan. It was deposited at Tsukuba City, Higashi 1-3-3), and was transferred to a deposit under the Budapest Treaty on August 26, 1991, and is given the deposit number FERM BP-3524. At the SmaI site of pHIS714, another 1.6 kb fragment containing IS714 previously obtained was blunt-ended by Klenow fragment treatment and then inserted by ligation to prepare plasmids pHTN7141 and pHTN7142 (Fig. 2). ). As a result of analysis by restriction enzyme cleavage, this plasmid pHTN7141 had the same orientation in the fragment containing IS714 inserted at two positions, and the plasmid pHTN7142 had the opposite orientation.

By digesting pHTN7141 and pHTN7142 with the restriction enzyme PvuII, respectively, IS
A fragment containing two sequences of 714 and a sequence of the temperature-sensitive replication origin of pHSC4 in a coryneform bacterium can be excised. On the other hand, the plasmid vector pHSG29
8 (manufactured by Takara Shuzo Co., Ltd.) also has two restriction enzyme PvuII sites, and a 2.3 kb fragment containing the neomycin phosphotransferase gene (kanamycin resistance gene) can be obtained by cutting with the restriction enzyme PvuII. . Therefore, pHTN7141 and pHS
Cleavage of G298 with the restriction enzyme PvuII was followed by ligation to transform Brevibacterium lactofermentum AJ12036. Among the transformants,
The plasmid pHTN7143 was obtained from a strain resistant to kanamycin (Km) 25 μg / ml (FIG. 3). Brevibacterium lactofermentum AJ12826 harboring the plasmid pHTN7143
Institute of Biotechnology, Institute of Technology, Ministry of International Trade and Industry, Ministry of International Trade and Industry, Japan
No. 3) has been deposited under the Budapest Treaty and is given the deposit number FERM BP-4231. In the same manner, plasmid pHTN7144 was obtained from pHTN7142 and pHSG298 (Fig. 4). pHTN7143 and pHTN7144 are two IS714 of IS714.
It consists of a structure in which the neomycin phosphotransferase gene is sandwiched between. By the same method,
Plasmids pHIS714K1 and pHIS714K2 were prepared from plasmids pHIS714 and pHSG298 as control groups (FIG. 5). Where pHIS714K1
And pHIS714K2 have an inverse relationship in the insert fragment containing the neomycin phosphotransferase gene.

Next, in order to miniaturize the artificial transposon, an artificial transposon in which the neomycin phosphotransferase gene was inserted into one IS714 was prepared. In IS714, a restriction enzyme NheI site exists at a position that does not impair the function of transposase. Therefore, the plasmid pHIS714 was used as the restriction enzyme Nhe.
It was cut with I and the end was made blunt. On the other hand, the neomycin phosphotransferase gene region was excised from the plasmid pUC4K (Pharmacia Biotech) with the restriction enzyme PstI, and the end thereof was made a blunt end. Both were ligated and the resulting plasmid was named pHTN7145 (Fig. 6). Escherichia coli AJ carrying plasmid pHTN7145
13128 was deposited on June 29, 1995, at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (Postal Code 305, 1-3 East, Tsukuba City, Ibaraki Prefecture, Japan) with deposit number F
ERM P-15011 is assigned. The strain was transferred to a deposit under the Budapest Treaty on May 14, 1996, and is assigned the deposit number FERM BP-5537.

Evaluation of Transfer Function of Artificial Transposon The transfer function of the artificial transposon thus prepared was evaluated as follows. Plasmid pA carrying the chloramphenicol acetyltransferase gene
Brevibacterium lactofermentum for J43
The strain held in AJ12036 was transformed with the plasmid pHTN7145 carrying an artificial transposon to make it a coexisting state. Escherichia coli AJ11882, which holds pAJ43, was developed under the contract number FERM P-6517 on April 28, 1982 under the contract number FERM P-6517.
Deposited at 1-3, Higashi 1-3, Tsukuba, Ibaraki, Japan.
It was transferred to a deposit under the Budapest Treaty on May 22, 1982, and is given the deposit number FERM BP-136. PHTN7145 and p obtained as described above
CM2G medium containing 25 μg / ml Km of Brevibacterium lactofermentum simultaneously holding AJ43 and 5 μg / ml of chloramphenicol (Cm) (yeast extract 1
0 g / l, tryptone 10 g / l, glucose 5 g / l and Na
Cl2 g / l) at 25 ° C. overnight with shaking, and then the culture solution is appropriately diluted to contain CM2 containing Km of 25 μg / ml and Cm of 5 μg / ml
It was applied to G agar medium and cultured at 34 ° C. Plasmids were extracted from 100 of the emerged colonies, and their sizes were examined by electrophoresis.
The strain had a molecular weight different from that of both pHTN7145 and pAJ43 plasmids, and there was a plasmid having the sum of the molecular weights of pAJ43 and the artificial transposon. When these plasmids were analyzed by restriction enzyme digestion, it was found that the sequence on pHTN7145 was inserted into pAJ43. For one of these strains, the inserted fragment on pAJ43 and pAJ4
The nucleotide sequence near the ligation site with 3 was determined by the dideoxy method. Was confirmed. From these results, a gene not involved in the transposition function (here, neomycin phosphotransferase gene) in one IS714
If you take the transposon structure of the inserted
It was shown that the structure is conserved and transposes like a so-called transposon.

Evaluation of Transfer Frequency of Artificial Transposon pHIS714K1 was used as a control plasmid, and pHTN7143 and pHTN714 equipped with artificial transposons were used.
4 and pHTN7145 were used to transform Brevibacterium lactofermentum AJ12036, and the frequency of transfer of the artificial transposon to the host chromosome was evaluated. After each transformant was cultured in the above CM2G liquid medium containing 25 μg / ml of Km at 25 ° C. overnight,
Appropriately diluted with 0.9% NaCl solution, 100 μl of each was applied to CM2G agar medium containing 25 μg / ml of Km. 34 ° C
The cells were cultured at 25 ° C. and at 25 ° C., the appearance frequency of Km resistant strains at each temperature was measured by the number of colonies, and the value obtained by dividing the number of colonies at 34 ° C. by the number of colonies at 25 ° C. was taken as the transfer frequency.
Table 1 shows the results.

[0053]

[Table 1]

From this result, IS714 (pH
Artificial transposon Tn7143 (mounted on pHTN7143) and Tn71 compared to IS714K1 mounted)
44 (loaded on pHTN7144) had a transfer frequency of 1-2 times at most to its host chromosome, but artificial transposon Tn7145 (loaded on pHTN7145)
It was shown that the type is an artificial transposon that is extremely efficient with a factor of about 11.

Example 2 A chloramphenicol resistance gene using IS714 was prepared.
Construction of an artificial transposon containing the plasmid vector pHSG398 (manufactured by Takara Shuzo Co., Ltd.) was cleaved with a restriction enzyme AccII to obtain about 1.1 containing a chloramphenicol acetyltransferase gene.
A kb sized fragment was obtained. This AccII fragment is called pUC
It was inserted into the SmaI site of 18 (Takara Shuzo) and cloned. That is, Cm 25 μg / ml and ampicillin (A
p) L medium containing 100 μg / ml (tryptone 10 g / l,
A target clone was selected from the Escherichia coli transformant grown in yeast extract 5 g / l and NaCl 5 g / l), and the plasmid was named pUC18-CM. Further, a fragment of about 1.1 kb containing the chloramphenicol acetyltransferase gene, which was excised from pUC18-CM with EcoRI and HindIII, was blunt-ended, and this DNA fragment was transferred to IS714 of pHIS714K2 constructed in Example 1 in IS714. The restriction enzyme NheI site existing at a position not related to the function was ligated with Klenow fragment-blunted one, and Escherichia coli was transformed with Cm25μ.
A colony growing in an L medium containing g / ml and Km of 50 μg / ml was obtained, and a clone in which the chloramphenicol acetyltransferase gene fragment was inserted was selected. The plasmid carried by this clone was named pHTN7151 (FIG. 7). The Escherichia coli AJ13129 carrying the plasmid pHTN7151 was prepared on June 2, 1995.
Institute of Biotechnology, Ministry of International Trade and Industry, Institute of Industrial Science and Technology (Postal code 305, 1-3, East 1-3, Tsukuba, Ibaraki, Japan)
No.) and is given the deposit number FERM P-150012. The shares were transferred to a deposit under the Budapest Treaty on May 14, 1996, and the deposit number is FERM.
BP-5538 is assigned.

Caused by the transfer of an artificial transposon
Evaluation of Copy Number on Chromosome pHTN7151 was used to transform Brevibacterium lactofermentum AJ12036, and the copy number on the chromosome generated by the transfer of the artificial transposon to the host chromosome was evaluated by the following method. The obtained transformant was cultured overnight in the above CM2G liquid medium containing Cm3 μg / ml at 25 ° C., and then appropriately diluted with 0.9% NaCl solution to prepare a CM2G agar medium containing Cm3 μg / ml.
0 μl of each was applied and cultured at 34 ° C., and kanamycin-sensitive clones were selected from the appeared colonies. This clone was cultured in the above CM2G liquid medium containing Cm3 μg / ml at 30 ° C. overnight, and then appropriately diluted with 0.9% NaCl solution to the above CM2G agar medium containing Cm6 μg / ml.
0 μl of each was applied and cultured at 30 ° C., and several clones were randomly selected from the appeared colonies. Chromosomal DNA was prepared from each clone, completely digested with the restriction enzyme PvuII, subjected to agarose gel electrophoresis, and blotted on a polyvinylidene difluoride (PVDF) filter. This filter was subjected to Southern hybridization using a ³² P-labeled chloramphenicol acetyltransferase gene fragment as a probe, and the number of bands hybridizing with the probe was assayed. As a result, it was confirmed that 2 copies of the artificial transposon having the chloramphenicol resistance gene were transferred to the host chromosome in 3 out of 4 randomly selected clones.

[Example 3] Includes a tetracycline resistance gene using IS714
Construction of artificial transposon Plasmid vector pBR322 (Takara Shuzo) was digested with restriction enzymes EcoRI and AvaI to obtain a fragment of about 1.4 kb containing the tetracycline resistance gene. Next, this EcoRI-AvaI digested fragment was treated with T
This DNA was blunt-ended by treatment with 4DNA polymerase
The fragment was ligated with the restriction enzyme NheI site existing at a position not related to the transfer function of pHIS714K2 constructed in Example 1 in IS714 and blunt-ended by Klenow fragment treatment to transform Escherichia coli. , A colony growing in L medium containing 25 μg / ml of Tc was obtained, and a clone in which the tetracycline resistance gene fragment was inserted was selected. The plasmid carried by this clone was named pHTN7152 (FIG. 8).
Escherichia harboring the plasmid pHTN7152
Kori AJ13130 is the Institute of Biotechnology, Ministry of International Trade and Industry, Institute of Biotechnology (Postal code 305) dated June 29, 1995.
It has been deposited at Tsukuba City, Ibaraki Prefecture, Japan, 1-3-1 Higashi, and has been given the deposit number FERM P-15013.
The shares were transferred to a deposit under the Budapest Treaty on May 14, 1996, and the deposit number is FERM BP-5539.
Is given.

Caused by the transfer of an artificial transposon
Evaluation of Copy Number on Chromosome Brevibacterium lactofermentum AJ12036 was transformed with pHTN7152, and the copy number on the chromosome generated by the transfer of the artificial transposon to the host chromosome was evaluated as follows. The transformant was cultured in the above CM2G liquid medium containing 1.5 μg / ml of Tc at 25 ° C. overnight, and then appropriately diluted with 0.9% NaCl solution to obtain Tc in the range of 1.5 μg / ml to 5 μg / ml. 100 μl each was applied to the above CM2G agar medium contained in 1. and cultured at 34 ° C., and several clones that appeared were randomly selected. Chromosomal DNA was prepared from each clone, completely digested with restriction enzyme PvuII, and subjected to agarose gel electrophoresis to obtain polyvinylidene difluoride (PVD
F) Blotted on the filter. This filter was subjected to Southern hybridization using a ³² P-labeled tetracycline resistance gene fragment as a probe, and the number of bands hybridizing with the probe was assayed. As a result, as shown in Table 2, artificial transposons having a tetracycline resistance gene were frequently detected at 2-3 copies. From this, it was shown that the target multicopy transformant can be obtained at high frequency by using the tetracycline resistance gene as the selective drug resistance gene.

[0059]

[Table 2]

Shows resistance to Tc 4 μg / ml, and 3 on the chromosome
Brevibacterium lactofermentum AJ13188, in which a copy of the artificial transposon was detected, was the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, Ministry of International Trade and Industry (Postal Code 305, 1-1 Higashi Tsukuba, Ibaraki, Japan) on May 14, 1996. No. 3) has been deposited under the Budapest Treaty and is given the deposit number FERM BP-5536.

Example 4 Tetracycline resistance gene using IS714 and gene
Structure of the artificial transposon containing a scan Aparthotel kinase gene
An aspartokinase gene, which is one of the lysine biosynthesis genes, was inserted into an artificial transposon carrying a constructed tetracycline resistance gene as follows. Plasmid vector pBR322 (manufactured by Takara Shuzo) was used as restriction enzyme Eco.
By cutting with RI and AvaI, a DNA fragment of about 1.4 kb containing the tetracycline resistance gene was obtained. This EcoRI-AvaI digested fragment was designated as T4DN.
The resulting DNA fragment was blunt-ended by A polymerase treatment and ligated with the plasmid vector pHY300PLK (Takara Shuzo Co., Ltd.) digested with a restriction enzyme SmaI, and Escherichia coli was transformed with the obtained recombinant DNA. A colony growing in an L medium containing 25 μg / ml of Tc was obtained, and a strain into which a recombinant DNA having a tetracycline resistance gene fragment inserted was introduced was selected. The plasmid contained in the strain was named pHY300-TC. Furthermore, pHY300-TC was digested with restriction enzymes EcoRI and XbaI.
The pBR322-derived tetracycline resistance gene-containing fragment obtained by digestion with blunt-ended DNA was blunt-ended by Klenow fragment treatment, and this DNA fragment and the previously constructed restriction enzyme NheI in IS714 of pHIS714K2 were constructed.
The site was ligated with that blunt-ended by Klenow fragment treatment, Escherichia coli was transformed to obtain a colony that grows in L medium containing Tc 25 μg / ml, and a clone in which the tetracycline resistance gene fragment was inserted is selected. did. The plasmid contained in this clone is p
It was named HTN7156 (Fig. 9).

On the other hand, Escherichia coli harboring the plasmid p399AK9B, which is derived from a lysine-producing mutant strain of Brevibacterium lactofermentum and contains the aspartokinase gene that is desensitized to the concerted inhibition of lysine and threonine, Collision AJ12691 (WO94 / 2560
5) was deposited on April 10, 1992 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry, Ministry of International Trade and Industry (1-3, Higashi 1-3-3, Tsukuba City, Ibaraki Prefecture, Japan), with a deposit number of FER
MP-12198 is assigned. The stock is Heisei 7
It was transferred to a deposit under the Budapest Treaty on February 10, 2012, and is given the deposit number FERM BP-4999. This p399AK9B was cleaved with a restriction enzyme BamHI and self-ligated to construct pHSG399AK excluding the origin of replication that functions in coryneform bacteria. This pHSG399AK was digested with restriction enzymes EcoRI and SphI to obtain an approximately 1.7 kb aspartokinase gene fragment, which was blunt-ended by T4 DNA polymerase treatment, and then a plasmid having an artificial transposon carrying a tetracycline resistance gene. pHTN
The restriction enzyme BglII site of 7156 was inserted into the site blunted by Klenow fragment treatment, and the plasmid p
HTN7156-C was constructed (Figure 9). Plasmid p
Escherichia coli AJ13131 transformed with HTN7156-C was Escherichia coli AJ13131, which was dated June 29, 1995. No.) and deposit number FERM P-15014
Is given. The shares were transferred to a deposit under the Budapest Treaty on May 14, 1996, and the deposit number was FER.
MBP-5540 is assigned.

Caused by the transfer of an artificial transposon
Evaluation of copy number on chromosome Using pHTN7156-C, Brevibacterium
Lactofermentum AJ12036 or Brevibacterium lactofermentum AJ3445 was transformed, and the copy number of the transposon on the chromosome generated by the transfer of the artificial transposon to the host chromosome was evaluated. The AJ12036 strain has a wild-type aspartokinase gene on its chromosome, whereas AJ34
Forty-five strains show S-2-aminoethyl-L-cysteine resistance and have an aspartokinase gene that is desensitized to the concerted inhibition of lysine and threonine.

Brevibacterium lactofermentum AJ12036 strain was introduced on March 26, 1984 on the Institute of Biotechnology, Ministry of International Trade and Industry, Institute of Biotechnology (Postal code 305).
It has been deposited at Tsukuba City, Ibaraki Prefecture, Japan, 1-3-1 Higashi, and has been given the deposit number FERM P-7559. The stock was transferred to a deposit under the Budapest Treaty on March 13, 1985, and the deposit number is FERM BP-7.
34 is assigned. Brevibacterium lactofermentum AJ3445 strain was deposited at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry (Postal Code 305, 1-3-1 Higashi Tsukuba, Ibaraki, Japan) on March 2, 1973. Accession number FERM P-1944 is assigned. The shares were transferred to a deposit under the Budapest Treaty on May 17, 1996, and the deposit number is FERM BP-
5541 is added.

First, the transformant was treated with Tc of 0.7 μg / ml.
Containing CM2G medium (yeast extract 10 g / l, tryptone 10 g / l, glucose 5 g / l and NaCl 5 g / l
After culturing in 1) at 25 ° C. overnight, Tc is 1.5 μg / ml to 5 μg / m by appropriately diluting with 0.9% NaCl solution.
100 μl of each of the above CM2G agar medium contained in the range of 1 is applied. Cultivate them at 34 ° C and select several clones at random for each emerging colony.
Replicate to CM2G agar medium containing μg / ml, Km
Sensitive strains were selected. Chromosomal DNA of the selected Km-sensitive strain was prepared, completely digested with restriction enzyme BglII, subjected to agarose gel electrophoresis, and blotted on a polyvinylidene difluoride (PVDF) filter. This filter is labeled with ^32- P aspartokinase gene (from the HindIII site in the latter half of the gene to E
Southern hybridization was carried out using 440 bp up to the coRI site as a probe, and the number of bands hybridizing with the probe was assayed. As a result, AJ120
4 out of 10 strains analyzed when 36 was used as a host, and 8 out of 22 strains analyzed when AJ3445 was used as a host
It was detected that two copies of transposon Tn7156-C were transferred in each strain. From this, it was shown that by using the tetracycline resistance gene as the selective drug resistance gene, it is possible to introduce a multicopy of the useful gene into the chromosome at high frequency.

Aspartoki using an artificial transposon
Evaluation of Lysine Productivity of Strain Transferred with Nase Gene Next, the lysine productivity of the artificial transposon-transferred strain was evaluated. Artificial transposon transfer strain Tc
After applying to the entire surface of CM2G agar medium containing 0.7 μg / ml and culturing at 34 ° C. overnight, one-sixth of the amount of cells was lysine-producing medium (glucose 100 g / l, ammonium sulfate 55 g / l, beans Concentration 50 ml / l, potassium dihydrogen phosphate 1 g / l, magnesium sulfate 1 g /
1, vitamin B1 2mg / l, biotin 0.5mg
/ L, nicotinamide 5 mg / l, iron sulfate 2 mg
/ L, manganese sulphate 2 mg / l, p with potassium hydroxide
Adjusted to H7.5, autoclaved at 115 ° C for 15 minutes, then added calcium carbonate 50g / l), inoculated into 20 ml, and analyzed the lysine content of the culture broth cultured at 30 ° C for 72 hours in a Sakaguchi flask, The lysine productivity of the artificial transposon transfer strain was evaluated. As a result, as shown in Tables 3 and 4, the transfer of Tn7156-C caused an increase in lysine productivity in both cases where AJ12036 was used as the parent strain and AJ3445 was used as the parent strain. Was given. Also, lysine productivity was higher depending on the transfer copy number (1 copy and 2 copies) of the transposon. From this, it was shown that it is possible to improve the amino acid productivity of the strain by utilizing the tetracycline resistance gene as a selective drug resistance gene and introducing a multicopy of a useful gene.

[0067]

[Table 3]

[0068]

[Table 4]

[Example 5] Construction of shuttle vector pVK7 As a cryptic plasmid existing in Brevibacterium lactofermentum, pAM330 is available. pAM330 is Japanese Patent Publication No. 1-1280, USP
4,788,762 and is prepared from Brevibacterium lactofermentum strain ATCC 13869. pAM330 can be used as an origin of replication for shuttle vectors that can grow in coryneform bacteria. PHSG299 is a general-purpose vector for Escherichia coli
(Takara Shuzo) and pAM330 were combined to construct a new shuttle vector. Restriction enzyme Hind for pAM330
It was cut at one site with III, and the cut surface was blunt-ended with T4 DNA polymerase. Further, pHSG299 was cut at one site with a restriction enzyme AvaII, and the cut surface was blunt-ended with T4 DNA polymerase. The obtained fragment was ligated to obtain a plasmid in which pAM330 and pHSG299 were ligated. FIG. 18 shows the process of constructing pVK7.
Shown in pVK7 is replicable in Escherichia coli and coryneform bacteria and confers kanamycin resistance on the host. As a cloning site, pHSG
Of the 299-derived multi-cloning sites, PstI and Sal are used as cloning sites that cut at one site.
It has I, BamHI, KpnI, SacI, and EcoRI.

Construction of Shuttle Vector pVC7 Similar to pVK7, pHSG399 (manufactured by Takara Shuzo), which is a general-purpose vector for Escherichia coli, and pAM330 were ligated to construct a new shuttle vector pVC7. p
Cut AM330 at one site with the restriction enzyme HindIII,
The cut surface was blunt-ended with T4 DNA polymerase.
Further, pHSG399 was cut at one site with a restriction enzyme BsaI and blunt-ended with T4 DNA polymerase in the same manner.
The resulting fragment was ligated and pAM330 and pH
A plasmid to which SG399 was ligated was obtained. Among the obtained plasmids, the process of constructing pVC7 is shown in FIG. pVC7 is replicable in Escherichia coli and coryneform bacteria and confers kanamycin resistance on the host. Further, as a cloning site, pHSG39
Of the 9-derived multiple cloning sites, PstI and Sal are used as cloning sites that cut at one site.
I, BamHI, KpnI, SacI, EcoRI, S
It has maI and HindIII.

Contains dapA, dapB, lysA
Construction of plasmid (1) Acquisition of lysA and construction of plasmid containing it LysA wild type Brevibacterium lactofermentum A
The TCC13869 strain was used as a chromosomal DNA donor. Chromosome DN from ATCC13869 strain according to a conventional method
A was prepared. From chromosomal DNA by PCR, arg
A DNA fragment containing S, lysA and an operon promoter containing them was amplified. As the DNA primer used for amplification, a sequence known in Corynebacterium glutamicum (Molecular Microbiology) is used.
4 (11), 1819-1830 (1990), Molecular and General Geneti
cs 212, 112-119 (1988)).
About 3.6 kb encoding RNA synthase and DDC
In order to amplify the region (1), a 23-mer synthetic DNA having the nucleotide sequences shown in SEQ ID NOs: 13 and 14 of the sequence listing was used. The DNA synthesis and PCR reaction were performed according to a conventional method. That is, the synthesis of DNA is performed by Applied B
DNA synthesizer model 38 manufactured by iosystems
OB, using the phosphoamitite method (Tetrahed
See Ron Letters (1981), 22, 1859). For the PCR reaction, DNA thermal cycler PJ2000 type manufactured by Takara Shuzo Co., Ltd. was used, and Taq DNA polymerase was used to perform gene amplification according to the method specified by the supplier. The sequence of the amplified DNA is shown in SEQ ID NO: 15. PHSG399 was used as a vector for cloning the amplified 3579 bp gene fragment. pHSG
399 was digested with the restriction enzyme SmaI and amplified to ly.
It was ligated with a DNA fragment containing sA. The thus obtained plasmid having lysA derived from ATCC13869 was designated as p399LYSA. Furthermore, p399LY
By cutting SA with KpnI and BamHI,
A DNA fragment containing ysA was obtained. This DNA fragment is
HSG299 was ligated with KpnI and BamHI cut. The resulting plasmid was named p299LYSA. The process of constructing p299LYSA is shown in FIG.
Digestion of p399LYSA with KpnI and BamHI gave a lysA fragment, which was digested with KpnI and BamHI.
It was ligated with pVK7 cut with amHI. This prepared plasmid was named pLYSAm (FIG. 21).

(2) Acquisition of dapA and preparation of plasmid containing it A wild strain AT of Brevibacterium lactofermentum AT
The CC13869 strain was used as a chromosomal DNA donor. Chromosome DN from ATCC13869 strain according to a conventional method
A was prepared. DapA by PCR from chromosomal DNA
Was amplified. The DNA primer used for amplification was a sequence known in Corynebacterium glutamicum (Nucleic Acids Research 18 (21),
6421 (1990), EMBL accession No. X53993) to amplify a region of about 1.5 kb that encodes DDPS based on SEQ ID NOs: 16 and 17 of the sequence listing, each of which is a 23-mer DNA. Was synthesized. The DNA synthesis and PCR reaction were performed according to a conventional method. The sequence of the amplified DNA is shown in SEQ ID NO: 18. 141 amplified
PC was used as a vector for cloning a 1 bp gene fragment.
R1000 (manufactured by Invitrogen; Bio / Technolo
gy 9,657-663 (1991)) and ligated with the amplified dapA fragment. The ligation of DNA was performed by a designated method using a DNA ligation kit (manufactured by Takara Shuzo). In this way, dCR amplified from Brevibacterium lactofermentum chromosome in pCR1000
A plasmid in which the A fragment 1411 bp was inserted was prepared. ATCC13869-derived d obtained in this manner
The plasmid with apA was named pCRDAPA (Figure 22). The transformant AJ13106 strain obtained by introducing pCRDAPA into Escherichia coli was
The Ministry of International Trade and Industry, Ministry of International Trade and Industry, Institute of Biotechnology, Institute of Biotechnology (Postal Code 305 Japan, Ichiro, Ibaraki, 1-3-1 Higashi 1-chome, Japan), with a consignment number of FERM BP-5113, internationally based on the Budapest Treaty. Has been deposited. da
The plasmid pCRDAPA having pA was digested with KpnI and EcoRI to obtain a DNA fragment containing dapA, which was ligated with the vector plasmid pHSG399 digested with KpnI and EcoRI. The resulting plasmid was designated as p399DPS (Fig. 23).

(3) Acquisition of wild type and mutant type lysC and preparation of plasmid containing them Lycobacterium lactofermentum ATCC1
The 3869 strain and the L-lysine-producing mutant strain AJ3445 obtained from the ATCC13869 strain by mutation treatment were used as chromosomal DNA donors. AJ3445 strain is
The mutation virtually eliminates the concerted inhibition of lysC by lysine and threonine (Journal of Biochemis
try 68,701-710 (1970)). PCR method from chromosomal DNA (polymerase chain reaction; White, T, J. et al; Trends
Genet. 5,185 (1989)), DNA containing lysC
The fragment was amplified. The DNA primer used for amplification was a sequence known in Corynebacterium glutamicum (Molecular Microbiology (1991) 5 (5), 1197-120).
4, Mol. Gen. Genet. Single-stranded DNA was synthesized. DNA was synthesized according to a conventional method. The PCR reaction was carried out by gene amplification according to a conventional method. Amplified DN
The sequence of A is shown in SEQ ID NO: 21. Amplified 1643b
After confirming the gene fragment of p by agarose gel electrophoresis, the fragment excised from the gel was purified by a conventional method and digested with restriction enzymes NruI and EcoRI. PHSG399 was used as a vector for cloning gene fragments. pHSG399 is a restriction enzyme SmaI and a restriction enzyme E.
It was cut with coRI and ligated with the amplified lysC fragment. The ligation of DNA was performed by a designated method using a DNA ligation kit (manufactured by Takara Shuzo). In this way, a plasmid in which pHSG399 was ligated with the lysC fragment amplified from the Brevibacterium lactofermentum chromosome was prepared. ATCC1 which is a wild strain
The plasmid having lysC derived from strain 3869 was designated as p39
The plasmid having lysC derived from AJ3445, which is a 9AKY, L-lysine-producing bacterium, was designated as p399AK9 (FIG. 24).

(4) Acquisition of dapB and preparation of a plasmid containing it BREVIbacterium lactofermentum wild strain AT
The CC13869 strain was used as a chromosomal DNA donor. Chromosome DN from ATCC13869 strain according to a conventional method
A was prepared. DapB by PCR from chromosomal DNA
Was amplified. The DNA primer used for amplification was Brevibacterium lactophare 2743-2749 (19
Approximately 2.0k that encodes DDPR based on (see 93))
SEQ ID NOS: 22 and 23 of the sequence listing for amplifying the region of b
Each 23-mer DNA fragment having the nucleotide sequence described in 1. was synthesized. The DNA synthesis and PCR reaction were performed according to a conventional method. The sequence of the amplified DNA is shown in SEQ ID NO: 24. As a vector for cloning the amplified 2001 bp gene fragment, pCR-Script (lnvitr
(manufactured by Ogen) and ligated with the amplified dapB fragment. In this way, dCR amplified from Brevibacterium lactofermentum chromosome was added to pCR-Script.
A plasmid having the apB fragment 2001bp inserted therein was prepared. The plasmid having dapB derived from ATCC13869 thus obtained was designated as pCRDAPB (FIG. 25). The transformant AJ13107 strain obtained by introducing pCRDAPB into Escherichia coli was
The Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology, Institute of Technology (ZIP 305, 1-3-1 Higashi 1-chome, Tsukuba, Ibaraki, Japan) under the FERM BP-5114 consignment number based on the Budapest Treaty. Has been internationally deposited.

(5) Preparation of plasmid containing lysC, dapA and dapB p399DPS was digested with EcoRI and SphI and blunt-ended to obtain a dapA gene fragment. This fragment was ligated with one obtained by digesting p399AK9 with SalI and blunt-ended to give mutant lysC and dapA.
Was constructed to construct a plasmid p399CA. dap
The plasmid pCRDAPB containing B was digested with EcoRI to make it blunt-ended, and then digested with SacI to give dap.
A 2.0 kb DNA fragment containing B was obtained. The plasmid p399CA containing dapA and mutant lysC was sp
After cutting with eI and blunt-ended, it was cut with SacI and ligated with the previously obtained 2.0 kb dapB fragment to obtain a plasmid containing mutant lysC, dapA and dapB. This plasmid was designated as p399CAB (Fig. 26). Next, p399CAB was cleaved with SacII and blunt-ended to obtain dapA and dapB fragments. This fragment was ligated with pLYSAm that had been cut with BamHI and blunt-ended. In this way, it is capable of autonomous growth in coryneform bacteria and has dapA, dapB, l
A plasmid containing ysA was prepared. The prepared plasmid was named pABLm. The process of pABLm construction is shown in FIG.

A plastic containing dapA, dapB, lysA
Brevibacterium Rakutofa Mentamu T of the plasmid
Introduction into n7156-Cint-Y2 The plasmid pABLm containing the prepared dapA, dapB and lysA was introduced into Brevibacterium lactofermentum Tn7156-Cint-Y2. The method for introducing the plasmid is the electric pulse method (Sugimoto et al.
-207791). Selection of transformants was based on the drug resistance marker of the plasmid, the kanamycin resistance gene, and the tetracycline resistance gene amplified on the chromosome. Therefore, 25 μg / ml kanamycin (Km) and 1.5 μg / ml tetracycline (T
Transformants were selected in a complete medium containing c). The transformant was designated as Tn7156-Cint-Y2 / pABLm.

LysC, dapA, dapB, lysA
Brevibacterium of a plasmid containing the
Introduction into mentum wild type strain (1) Brevi. -Ori was introduced. Brevi. Plasmid pHK4 with -ori
Was cleaved with the restriction enzyme BamHI to make the cut surface blunt-ended. The blunt end is a DNA Blunting kit
(Manufactured by Takara Shuzo) was used according to the designated method.
After blunting, a phosphorylated KpnI linker (manufactured by Takara Shuzo Co., Ltd.) was ligated, and pHvi was used for Brevi. The DNA fragment of the -ori portion was modified so that it was excised by cleavage with KpnI only. This plasmid was cut with KpnI and the resulting Brevi. -Ori DNA fragment was ligated to p399CAB, which was also cut with Kpnl, and was capable of autonomous growth in coryneform bacteria and mutant lysC and da.
A plasmid having both pA and dapB was prepared, and p
It was named CAB. The process of construction of pCAB is shown in FIG. pHK4 cleaves pHC4 with KpnI and BamHI, and Brevi. -Extract the ori fragment and repeat K
It is constructed by ligating to pHSG298 digested with pnI and BamHI (see JP-A-5-7491). pHK4 confers kanamycin resistance on the host. Escherichia coli A that retains pHK4
J13136 was entrusted with FERM on August 1, 1995 at the Institute of Biotechnology, Institute of Biotechnology, Ministry of International Trade and Industry (Postal Code 305, 1-3, Higashi 1-chome, Tsukuba, Ibaraki, Japan).
It has been deposited under the Budapest Treaty as BP-5186.

(2) Plasmid p29 containing lysA
9LYSA was cleaved with KpnI and BamHI and blunt-ended to obtain a lysA gene fragment. This fragment was ligated with pCAB cleaved with HpaI to allow autonomous growth in a coryneform bacterium and a mutant l
A plasmid having both ysC, dapA, dapB, and lysA was prepared and named pCABL. pCA
The process of BL construction is shown in FIG. In pCAEL,
The 1ysA gene fragment is inserted at the Hpal site in the DNA fragment containing the dapB gene, and this HpaI site is located upstream of the dapB gene promoter (base numbers 611 to 616 in SEQ ID NO: 24),
The dapB gene is not disrupted. Created lys
Plasmid p containing C, dapA, dapB, lysA
CABL was introduced into Brevibacterium lactofermentum wild strain AJ12036, and transformants were selected in a complete medium containing 5 μg / ml chloramphenicol (Cm). The transformant was named AJ12036 / pCABL.

Culture evaluation of constructed strain Brevibacterium lactofermentum wild strain AJ
12036 transformants AJ12036 / pCABL and Tn
7156-Cint-Y2 / pABLm was cultured in an L-lysine producing medium, and its L-lysine producing ability was evaluated. L-
The composition of the lysine production medium is as shown below. (L-lysine production medium) The following components (in 1 L) other than calcium carbonate were dissolved, and the pH was adjusted to 8.0 with KOH.
After sterilizing at 15 ° C. for 15 minutes, 50 g of dry heat sterilized calcium carbonate is added. Glucose 100g (NH4) 2SO4 55g KH2PO4 1g MgSO4 · 7H2O 1g biotin 500μg thiamine 2000μg FeSO4 / 7H2O 0.01g MnSO4 / 7H2O 0.01g nicotinamide 5mg protein hydrolyzate (bean concentrate) 30ml calcium carbonate 50g parent strain and trait Inoculate the transformant, 31.5 ℃
Reciprocal shaking culture was performed. Table 5 shows the amount of L-lysine produced after 72 hours of culture, growth ( _OD562 ), and stability at the end of culture. Growth is 101 times diluted and then 562 nm
It was quantified by measuring OD. In addition, regarding the stability, the culture medium at the end of the culture was diluted and grown on a complete medium, and the grown colonies were shown by the rate of growth on a plate containing a drug.

[0080]

[Table 5]

As shown above, lys on the chromosome
Even with the C-enhanced strain, lys
As in the case of increasing C, lysine productivity was improved. The stability is 90% for AJ12036 / pCABL, whereas it is 10 for Tn7156-Cint-Y2 / pABLm.
It was 0%.

Example 6 Construction of pHTN7150 Since the artificial transposon Tn7145 carrying a kanamycin resistance gene does not have a good site for inserting a gene encoding dihydrodipicolinate synthase, pHTN7150 having a newly inserted site was constructed. It was constructed as follows. The kanamycin resistance gene was excised from the plasmid vector pUC4K (Pharmacia Biotech) using the restriction enzyme PstI and blunt-ended, and then Sm of pHY300PLK (Takara Shuzo)
It was cloned into the aI site to construct pHY300-KM. Next, the restriction enzyme Eco was added from pHY300-KM.
The fragment containing the kanamycin resistance gene was excised using RI and XbaI to make it blunt-ended, and this fragment was constructed earlier, and the restriction enzyme NheI in IS714 on pHIS714 was constructed.
Insert the site at the blunt end and insert the plasmid p
HTN7150 was constructed. The artificial transposon Tn7150 mounted on pHTN7150 has a kanamycin resistance gene as its marker gene.
The II site can be used as a gene transfer site (Fig. 30).

PHTN7150 and Brevibacterium
A gene encoding dihydrodipicolinate synthase linked kanamycin resistance gene dapA gene derived lactofermentum is gene-lysine biosynthesis system in the artificial transposon pHTN7150 mounted (DDPS) was inserted as follows. The plasmid p399DPS carrying dapA was digested with EcoRI and
The DNA was blunt-ended by treatment with 4DNA polymerase, and phosphorylated BamHI linker (manufactured by Takara Shuzo Co., Ltd.) was ligated to modify the dapA gene so that it could be excised by cutting only BamHI. This plasmid was designated as p399DPS2. 1.4 kb generated by digesting this plasmid with BamHI
A plasmid was constructed in which the dapA fragment of E. coli was ligated to pHTN7150 that had been cut with BglII to generate cohesive ends similar to BamHI. This plasmid was designated as pHTN7150.
It was named A. FIG. 28 shows the construction process of pHTN7150A.
Shown in

Blur of artificial transposon Tn7150A
Translocation to the Bibakuteriumu, Lactobacillus fermentum on the chromosome
A strain in which the artificial transposon (Tn7150A) carrying dapA was transferred to the Brevibacterium lactofermentum AJ12036 strain was transferred using pHTN7150A as described below. The AJ12036 strain was transformed with pHTN7150A, and the obtained transformant was CM2S containing 25 μg / ml of kanamycin (Km).
Liquid medium (Yeast extract 10 g / l, Tryptone 10 g /
1, sucrose 5 g / l, and NaCl 5 g / l)
CM2 containing 25 μg / ml of kanamycin after appropriately culturing in the medium at 25 ° C. overnight and appropriately diluted with 0.9% NaCl solution
It was applied to S agar medium and cultured at 34 ° C. Select a chloramphenicol sensitive strain for the emerged colonies,
Chromosome DN selected from these strains at random
A was prepared and Southern hybridization was carried out using the dapA fragment as a probe to confirm the transfer of the artificial transposon. The strain with the transferred artificial transposon obtained as described above was named AJ12036 :: A.

Construction of pCBLmc and production of strain Using pAM330 and pVC7 which is a shuttle vector of pHSG399, mutant lysC, dapB and lys were used.
The plasmid with A was constructed as follows. d
After SCR treatment of pCRDAPB with apB, T
A blunt-ended plasmid was constructed by treatment with 4 DNA polymerase and a phosphorylated PstI linker (Takara Shuzo) was ligated to the plasmid. The obtained plasmid is designated as pCRDAPB
No. 2. This plasmid was designated BamHI and PstI.
The 2.0 kb dapB fragment generated by digestion with
It was inserted into pVC7 cut with mHI and PstI. This plasmid was named pBmc. p399AK9 carrying lysC was digested with BamHI and EcoRI, and the resulting 1.6 kb lysC fragment was also digested with BamHI and E.
ligated to pBmc cut with coRI, dapB, ly
A plasmid carrying sC was constructed. This plasmid was named pBCmc. p399 equipped with lysA
LYSA was cleaved with EcoRI, blunt-ended with T4 DNA polymerase, ligated with a phosphorylated KpnI linker, and a plasmid modified so that lysA could be excised with KpnI was constructed. This plasmid is p3
It was named 99LYSA2. Kp for p399LYSA2
The 3.6 kb lysA fragment generated by digestion with nI was digested with pBC.
The mc was digested with EcoRI, blunt-ended by treatment with T4 DNA polymerase, and ligated to the fragment into which the phosphorylated KpnI linker had been inserted. Obtained plasmid is pCBL
It was named mc. This plasmid is capable of autonomous replication in Escherichia coli and coryneform bacteria, imparts chloramphenicol resistance to the host, and is mutated lysC and dap.
It is a plasmid that holds both B and lysA. p
The construction process of CBLmc is shown in FIG. The pCBLm constructed as described above was introduced into the AJ12036 :: A strain in which the artificial transposon Tn7150A was transferred on the chromosome. The method for introducing the plasmid was the electric pulse method (Sugimoto et al., JP-A-2-207791). Selection of transformants was carried out with 5 μg / ml of chloramphenicol (C
m) and 25 μg / ml of kanamycin (Km) in the above CM2S medium. The constructed strain is AJ120
It was named 36 :: A / pCBLmc.

Culture evaluation of constructed strains Parent strain and obtained transformant AJ12036 / pCAB
L and AJ12036 :: A / pCBLmc were cultured in an L-lysine production medium, and their lysine production ability was evaluated.
Table 6 shows the results.

[0087]

[Table 6]

As shown above, dap on the chromosome
Even with the A-enhanced strain, lys was detected on the plasmid.
As in the case of increasing C, lysine productivity was improved. The stability is 90% for AJ12036 / pCABL, whereas it is 1 for AJ12036 :: A / pCBLmc.
00%.

[Example 7] No transposase was included in the transposon unit.
Build an artificial transposon

The Tc promoter derived from Escherichia coli
Lanceposase expression plasmid construction plasmid pHIS714 with restriction enzymes NheI and Xb
A fragment containing a gene encoding a transposase excluding the 5'-side inverted repeat (IR) of IS714 was obtained by digesting with aI, and this DNA fragment was introduced into the XbaI site of plasmid vector pUC19 to construct plasmid TnpL / pUC19. did. Further T
By cleaving npL / pUC19 with the restriction enzymes MroI and XbaI, the sequence containing the stop codon of IS714 and the 3'inverted repeat (IR) is removed,
A synthetic double-stranded DNA having the sequence shown below was ligated and inserted therein. 5'-CCGGACAGCTCACCCACAAAATCAATGCACTCTAAAAAGGTACCT-3'SEQ ID NO: 25 3'- TGTCGAGTGGGTGTTTTAGTTACGTGAGATTTTTCCATGGAGATC-5 'SEQ ID NO: 26 As a result, a plasmid URF / urf / URL in which the IR present on TnpL / pUC19 was removed was constructed. Next, this ORFL /
pUC19 was cleaved with restriction enzymes SmaI and XbaI to obtain a gene fragment of about 1.5 kb containing transposase. This transposase gene fragment was inserted into the plasmid vector pHY300PLK (manufactured by Takara Shuzo Co., Ltd.) after removing the region between SmaI and XbaI, and then excised with restriction enzymes EcoRI and KpnI. This EcoR
The transposase gene fragment of I-KpnI was added to T4DN.
The blunt-ended one with A polymerase was used as a restriction enzyme Pvu with a plasmid vector pHSG398 (Takara Shuzo).
It was partially digested with II and ligated to the region where the approximately 0.3 kb fragment containing the multicloning site was removed to construct plasmid pORF1 (FIG. 10).

On the other hand, the previously obtained plasmid pHIS7
The plasmid Tnp (Pst) introduced into the site where the NheI-XbaI digestion fragment of 14 was blunt-ended and the PstI site of the plasmid vector pUC19 was blunt-ended.
/ PUC19 was constructed. This Tnp (Pst) / pU
On C19, partial base substitution in the transposase gene was performed by U.S.E. S. E. FIG. Mutagenesis kit
(Pharmacia Biotech). The replaced base was G, which corresponds to the 288th base on the IS714 sequence, and was changed to C. This is GTG
To GTC, not at the amino acid level. The plasmid with base substitution was Tnp (Ps
t) It was named M / pUC19. When the region between the restriction enzyme sites SmaI and NaeI present in the transposase first half gene on pORF1 was removed, Tnp
(Pst) M / pUC19 is a restriction enzyme SmaI and Nae
Transposase first half gene fragment obtained by cutting with I (including mutation of GTG → GTC)
Was ligated to construct pORF2. pORF
The DNA fragment containing the tryptophan operon attenuator excised from pBSF2-SD7 with the restriction enzymes NaeI and HindIII was blunt-ended after removing the region between the SmaI site and the XbaI site of 2 to make it blunt-ended. Inserted. The plasmid constructed here was named pORF3. Plasmid pBSF2
-SD7 (WO92 / 14832) was introduced into Escherichia coli HB101, and the obtained transformant was named Escherichia coli AJ12448. The stock was purchased from the Ministry of International Trade and Industry, Institute of Industrial Science and Technology, Institute of Biotechnology, on June 1, 1989.
No. 3) deposit number FERM P-10758
Was granted. In addition, it was transferred to an international deposit under the Budapest Treaty on February 19, 1992, and the deposit number was FERM.
BP-3753 is assigned.

Restriction of pORF3 with restriction enzymes SalI and Bpu1
The first half of the transposase gene fragment obtained by cleaving Tnp (Pst) / pUC19 with the restriction enzymes SalI and Bpu1102I was digested with 102I to remove the first half of the transposase gene fragment, and pORF4 was constructed (FIG. 11). ). Also, TnpL
After cleaving / pUC19 with SacI, it is digested with BAL31 nuclease at 30 ° C. for 20 minutes, and the vicinity of the start codon of the transposase gene is deleted from the upstream side. Then, the deleted ends were blunt-ended, the transposase gene fragment was excised using the SphI site, and pHSG398 was inserted at the site cleaved with SmaI and SphI. The plasmid constructed in this way was named delTnp5 / 398. delTnp5
/ 398 is cleaved with restriction enzymes KpnI and HindIII to obtain the transposase first half gene fragment, which is then blunt-ended, and then the plasmid vector pKK233-2
(Pharmacia Biotech) with NcoI and Hin
Ligation was performed by cutting with dIII and blunt-ended to construct pTrc-ORF. pTrc-ORF
A fragment containing the Trc promoter and the transposase first half gene obtained by cutting with SspI and Bpu1102I was digested with XbaI at pORF3 to make it blunt-ended, and then further cut with Bpu1102I to remove the transposase first half gene fragment. PORF7 was constructed by inserting it at the site (Fig. 12).

Further, the transposase first half gene fragment obtained by cleaving delTnp5 / 398 with restriction enzymes KpnI and HindIII was used as a plasmid vector pU.
C18 was cloned between KpnI and HindIII. The region between BsmI and NaeI of the plasmid was removed, and this was combined with the transposase front half gene fragment (replacement type G → C) obtained by cutting Tnp (Pst) M / pUC19 with the restriction enzymes BsmI and NaeI. Were ligated to construct delTnp5M / 18. delTnp5M / 18 with KpnI and Hin
A blunt-ended version of the transposase first half gene fragment obtained by cleaving with dIII was designated as pKK.
233-2 was cleaved with NcoI and HindIII to make it blunt-ended and ligated to pTrc-Tnp.
Constructed M. pORF8 was constructed from pTrc-TnpM and pORF3 using the same method as that used to construct pORF7 from pTrc-Tnp (FIG. 13).

Artificial transposon unit and outside the unit
Coryneform bacteria introduced equipped with a transposase expression system
Construction of plasmids for plasmids pORF3, pORF4, pORF
7. Each plasmid was constructed using pORF8 as a material.
The procedure for constructing pORF3 to pORF41 is shown below. First, pHIS714 was cleaved with NheI and SacII to remove most of the transposase gene, and then double-stranded synthetic DNA of the following sequence was inserted to obtain pHTN.
7160 was constructed. 5'-CTAGCTCGAGATATCAGATCTACTAGTCGACCGC-3 'SEQ ID NO: 27 3'- GAGCTCTATAGTCTAGATGATCAGCTGG-5' SEQ ID NO: 28 pHTN7160 is cleaved with a restriction enzyme KpnI to make it blunt-ended, and then IS is further cleaved with BglI.
A fragment containing an inverted repeat (IR) on both sides of 714 and a temperature-sensitive origin of replication that functions in a coryneform bacterium was obtained. In addition, pORF3 is cleaved with the restriction enzyme EarI to make it blunt-ended, and then further cleaved with BglI. The pHTN7160-derived fragment was inserted therein, and pORF4
1-pre was constructed. Next, when pORF41-pre was cleaved with EcoR ■, T from pBR322 was isolated.
A blunt-ended fragment of EcoRI-AvaI containing the c resistance gene was inserted to construct pORF41 (Fig. 14). Repeating the same method, pORF4 to pORF31
-Pre via pORF31, pORF7 via pORF71-pre via pORF71, pORF71
From RF8 via pORF81-pre to pORF
81 was built. The plasmid pORF81 is harbored in Escherichia coli AJ13208. The strain was internationally deposited on June 3, 1996 at the Institute of Biotechnology, Institute of Industrial Science and Technology, Ministry of International Trade and Industry (1-3, Higashi 1-3, Tsukuba, Ibaraki, Japan, 305), based on the Budapest Treaty.
Accession number FERM BP-5557 is assigned.

Further, pORF3 was cleaved with XbaI and EarI, blunt-ended, and self-ligated to construct pORFC0 containing no transposase gene (FIG. 15). pORFC from pORFC0 in the same way as when pORF41 was constructed from pORF3
PORFC consisting only of transposon unit (without transposase gene) via 2-pre
2 was built. On these finally constructed plasmids, the transposase structural gene, Cm resistance gene, origin of replication functioning in E. coli, temperature-sensitive origin of replication functioning in coryneform bacteria, and IR derived from IS714
There is a Tc resistance gene sandwiched between. However, pOR
There is no transposase structural gene for FC2. The IR and Tc resistance gene units at both ends of IS714 were designated as transposon unit Tn7162.

Live by transposition of transposon unit
Door with a Tc resistance gene on chromosome Jill Ransupozon
Evaluation of unit copy number Among the constructed plasmids, pORF31 and pORF4
A metastasis experiment was performed using 1, pORF81 and pORFC2. The unit considered to be transposed is the transposon unit Tn7162. Using the above plasmid, Brevibacterium lactofermentum AJ1
2036 was transformed and transposon unit Tn7
The copy number of Tn7162 on the chromosome resulting from the transfer of 162 to the host chromosome was evaluated. The method is as follows. The transformant was cultured overnight in the above CM2G liquid medium containing 5 μg / ml of Cm at 25 ° C. and then appropriately diluted with 0.9% NaCl solution to obtain Tc of 1.5 μg / ml to 4 μg / ml. 100 μl each was applied to the CM2G agar medium containing the above range. They were cultured at 34 ° C., and Cm-sensitive clones were selected from the appeared colonies. Put them at 34 ° C
, A few clones were randomly selected from the appeared colonies, chromosomal DNA was prepared, and the restriction enzyme PvuII was prepared.
Completely digested with, subjected to agarose gel electrophoresis, and blotted on a nitrocellulose (or nylon, PVDF) filter. This filter is 32-P
Southern hybridization was performed using a Tc resistance gene fragment labeled with a label or an ECL direct labeling system (manufactured by Amersham) as a probe, and the number of bands hybridizing with the probe was assayed. As a result, as shown in Table 7, it was detected that the transposon unit Tn7162 having the Tc resistance marker gene had high copy transfer at a certain frequency. This result indicates that the expressed transposase gene exists outside the transoposon unit on the plasmid (pORF31, 41,
(In the case of 81), or by the transposase originally present on the chromosome (in the case of pORFC2).

[0097]

[Table 7]

[Example 8] For coryneform bacteria equipped with only a transposase expression system
Doo-run sports Dzong unit on the building and the chromosome of the plasmid
Transfer of

[0100] A clone equipped with only the transposase expression system
Construction of plasmids for line type bacteria Plasmid pHIS714K1 was added to EcoO109I and M
After removing IS714 by cleaving with roI, self-ligation is performed to obtain pHIS714Kd.
constructed el. On the other hand, pORF3 is a restriction enzyme EarI
It was cleaved with to make a blunt end, and then further cleaved with BglI. The pHIS714Kdel was then added to the restriction enzyme KpnI.
The fragment containing the temperature-sensitive origin of replication functioning in coryneform bacterium, which was obtained by further digesting with p.
F40 was constructed (Figure 17). Using a similar method,
pORF30 to pORF4 and pORF7 to pORF7
ORF70, pORF8 to pORF80, pORF8
PORFC1 was constructed from RFC0.

Live by transposition of transposon unit
Door with a Tc resistance gene on chromosome Jill Ransupozon
Evaluation of copy number of unit Among the constructed plasmids, pORF80 and pORFC1
Was used to perform a transfer experiment. The unit considered to be transposed is the transposon unit Tn7162. Brevibacterium lactofermentum A in Example 7
J12036 was transformed with a plasmid carrying the transposon unit Tn7162, showing that the transposon unit Tn7162 transferred to the host chromosome in multiple copies. Using the one-copy chromosomal transfer strain obtained here as a host, the plasmids pORF80 and pO
Southern hybridization analysis of chromosomal DNA was carried out to determine whether Tn7162 on the chromosome is further transferred or replicated when RFC1 is introduced to enhance the transposase activity, and the copy number is evaluated. The method is as follows. The transformant was cultured in the above CM2G liquid medium containing 5 μg / ml of Cm at 25 ° C. overnight, and then appropriately diluted with 0.9% NaCl solution to give Tc of 6 μg / ml to 20 μg / ml.
100 μl each was applied to the CM2G agar medium containing the above range. They were cultured at 34 ° C., and Cm-sensitive clones were selected from the appeared colonies. These Cm
Several clones were randomly selected from susceptible clones, chromosomal DNA was prepared, completely digested with restriction enzyme PvuII, subjected to agarose gel electrophoresis, and blotted on a nitrocellulose (or nylon, PVDF) filter. The Tc resistance gene fragment labeled the filter at ³ 2P label or ECL direct labeling system (Amersham) Southern hybridization as a probe to test the number of bands that hybridized with the probe. As a result, a transposon unit Tn7 having a Tc resistance marker gene
162 was found to have multiple copy transfer, replication at a certain frequency.

[0101]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1453 base pairs Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Brevibacterium lactofermentum (Brevibacteriu)
m lactofermentum) Strain name: AJ12036 Sequence features Characteristic symbol: insertion seq Location: 1..1453 Method of determining features: E Sequence characteristic Feature symbol: Location: 130..1440 Characteristic features Method: Feature of S sequence: Symbol representing a feature: repeat region Location: 1..15 Method of determining feature: S Sequence feature: Symbol representing feature: repeat region Location: 1339..1453 Method of determining feature: Features of S sequence: Symbol representing a feature: -35 region Location: 71..76 Method of determining feature: S Sequence feature: Symbol representing features: -10 region Location: 92..97 Method of determining feature: S Sequence GGCCCTTCCG GTTTTGGGGT ACATCACAGA ACCTGGGCTA GCGGTGTAGA CCCGAAAATA 60 AACGAGCCTT TTGTCAGGGT TAAGGTTTAG GTATCTAAGC TAACCAAACA CCAACAAAAG 120 GCTCTACCC ATG AGC Tla GL GAA CTA GGA CTC ACC ATC ACC GGC GCT TCC GAT GCA GGT 216 Arg Thr Ala Glu Leu Gly Leu Thr Ile Thr Gly Ala Ser Asp Ala Gly 15 20 25 GAT TAC ACC CTG ATC GAA GCA GAC GCA CTC GAC TAT ACC TCC ACC TGC 264 Asp Tyr Thr Leu Ile Glu Ala Asp Ala Leu Asp Tyr Thr Ser Thr Cys 30 35 40 45 CCA GAA TGC TTC CAA CCT GGG GTG TTT CGT CAT CAC ACC CAC CGG ATG 312 Pro Glu Cys Phe Gln Pro Gly Val Phe Arg His His Thr His Arg Met 50 55 60 CTC ATT GAT TTA CCC ATC GTC GGG TTT CCC ACC AAA CTG TTT ATC CGT 360 Leu Ile Asp Leu Pro Ile Val Gly Phe Pro Thr Lys Leu Phe Ile Arg 65 70 75 CTA CCT CGC TAC CGC TGC ACC AAC CCG ACA TGT AAG CAA AAG TAT TTC 408 Leu Pro Arg Tyr Arg Cys Thr Asn Pro Thr Cys Lys Gln Lys Tyr Phe 80 85 90 CAA GCA GAA CTA AGC TGC GCT GAC CAC GGT AAA AAG GTC ACC CAC CGG 456 Gln Ala Glu Leu Ser Cys Ala Asp His Gly Lys Lys Val Thr His Arg 95 100 105 GTC ACC CGC TGG ATT TTG CAA CGC CTT GCT ATT GAC CGG ATG AGT GTT 504 Val Thr Arg Trp Ile Leu Gln Arg Leu Ala Ile Asp Arg Met Ser Val 110 115 120 125 CAC GCA ACT GCG AAA GCA CTT GGG CTA GGG TGG G AT TTA ACC TGC CAA 552 His Ala Thr Ala Lys Ala Leu Gly Leu Gly Trp Asp Leu Thr Cys Gln 130 135 140 CTA GCC CTC GAT ATG TGC CGT GAG CTG GTC TAT AAC GAT CCT CAC CAT 600 Leu Ala Leu Asp Met Cys Arg Glu Leu Val Tyr Asn Asp Pro His His 145 150 155 CTT GAT GGA GTG TAT GTC ATT GGG GTG GAT GAG CAT AAG TGG TCA CAT 648 Leu Asp Gly Val Tyr Val Ile Gly Val Asp Glu His Lys Trp Ser His 160 165 170 AAT AGG GCT AAG CAT GGT GAT GGG TTT GTC ACC GTG ATT GTC GAT ATG 696 Asn Arg Ala Lys His Gly Asp Gly Phe Val Thr Val Ile Val Asp Met 175 180 185 ACC GGG CAT CGG TAT GAC TCA CGG TGT CCT GCC CGG TTA TTA GAT GTC 744 Thr Gly His Arg Tyr Asp Ser Arg Cys Pro Ala Arg Leu Leu Asp Val 190 195 200 205 GTC CCA GGT CGT AGT GCT GAT GCT TTA CGG TCC TGG CTT GGC TCC CGC 792 Val Pro Gly Arg Ser Ala Asp Ala Leu Arg Ser Trp Leu Gly Ser Arg 210 215 220 GGT GAA CAG TTC CGC AAT CAG ATA CGG ATC GTG TCC ATG GAT GGA TTC 840 Gly Glu Gln Phe Arg Asn Gln Ile Arg Ile Val Ser Met Asp Gly Phe 225 230 235 CAA GGC TAC GCC ACA GCA AGT AAA G AA CTC ATT CCT TCT GCT CGT CGC 888 Gln Gly Tyr Ala Thr Ala Ser Lys Glu Leu Ile Pro Ser Ala Arg Arg 240 245 250 GTG ATG GAT CCA TTC CAT GTT GTG CGG CTT GCT GGT GAC AAG CTC ACC 936 Val Met Asp Pro Phe His Val Val Arg Leu Ala Gly Asp Lys Leu Thr 255 260 265 GCC TGC CGG CAA CGC CTC CAG CGG GAG AAA TAC CAG CGT CGT GGT TTA 984 Ala Cys Arg Gln Arg Leu Gln Arg Glu Lys Tyr Gln Arg Arg Gly Leu 270 275 280 285 AGC CAG GAT CCG TTG TAT AAA AAC CGG AAG ACC TTG TTG ACC ACG CAC 1032 Ser Gln Asp Pro Leu Tyr Lys Asn Arg Lys Thr Leu Leu Thr Thr His 290 295 300 AAG TGG TTG AGT CCT CGT CAG CAA GAA AGC TTG GAG CAG TTG TGG GCG 1080 Lys Trp Leu Ser Pro Arg Gln Gln Glu Ser Leu Glu Gln Leu Trp Ala 305 310 315 TAT GAC AAA GAC TAC GGG GTG TTA AAG CTT GCG TGG CTT GCG TAT CAG 1128 Tyr Asp Lys Asp Tyr Gly Val Leu Lys Leu Ala Trp Leu Ala Tyr Gln 320 325 330 GCG ATT ATT GAT TGT TAT CAG ATG GGT AAT AAG CGT GAA GCG AAG AAG 1176 Ala Ile Ile Asp Cys Tyr Gln Met Gly Asn Lys Arg Glu Ala Lys Lys 335 340 345 AAA ATG CGG ACC A TT ATT GAT CAG CTT CGG GTG TTG AAG GGG CCG AAT 1224 Lys Met Arg Thr Ile Ile Asp Gln Leu Arg Val Leu Lys Gly Pro Asn 350 355 360 365 AAG GAA CTC GCG CAG TTG GGT CGT AGT TTG TTT AAA CGA CTT GGT GAT 1272 Lys Glu Leu Ala Gln Leu Gly Arg Ser Leu Phe Lys Arg Leu Gly Asp 370 375 380 GTG TTG GCG TAT TTC GAC GTA GGA GTC TCC AAC GGA CCA GTC GAA GCC 1320 Val Leu Ala Tyr Phe Asp Val Gly Val Ser Asn Gly Pro Val Glu Ala 385 390 395 ATC AAT GGA CGC CTA GAA CAC CTC CGC GGA ATC GCG CTT GGA TTC CGC 1368 Ile Asn Gly Arg Leu Glu His Leu Arg Gly Ile Ala Leu Gly Phe Arg 400 405 410 AAC CTC ACC CAC TAC ATC CTT CGA TGC CTC ATC CAC TCC GGA CAG CTC 1416 Asn Leu Thr His Tyr Ile Leu Arg Cys Leu Ile His Ser Gly Gln Leu 415 420 425 ACC CAC AAA ATC AAT GCA CTC TAA AAACGGAAGA GCC 1453 Thr His Lys Ile Asn Ala Leu 430 435

SEQ ID NO: 2 Sequence length: 436 amino acids Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Protein Origin: Organism: Brevibacterium lactofermentum (Brevibacteriu)
M lactofermentum) Strain name: AJ12036 Sequence Met Lys Ser Thr Gly Asn Ile Ile Ala Asp Thr Ile Cys Arg Thr Ala 1 5 10 15 Glu Leu Gly Leu Thr Ile Thr Gly Ala Ser Asp Ala Gly Asp Tyr Thr 20 25 30 Leu Ile Glu Ala Asp Ala Leu Asp Tyr Thr Ser Thr Cys Pro Glu Cys 35 40 45 Phe Gln Pro Gly Val Phe Arg His His Thr His Arg Met Leu Ile Asp 50 55 60 Leu Pro Ile Val Gly Phe Pro Thr Lys Leu Phe Ile Arg Leu Pro Arg 65 70 75 80 Tyr Arg Cys Thr Asn Pro Thr Cys Lys Gln Lys Tyr Phe Gln Ala Glu 85 90 95 Leu Ser Cys Ala Asp His Gly Lys Lys Val Thr His Arg Val Thr Arg 100 105 110 Trp Ile Leu Gln Arg Leu Ala Ile Asp Arg Met Ser Val His Ala Thr 115 120 125 Ala Lys Ala Leu Gly Leu Gly Trp Asp Leu Thr Cys Gln Leu Ala Leu 130 135 140 Asp Met Cys Arg Glu Leu Val Tyr Asn Asp Pro His His Leu Asp Gly 145 150 155 160 Val Tyr Val Ile Gly Val Asp Glu His Lys Trp Ser His Asn Arg Ala 165 170 175 Lys His Gly Asp Gly Phe Val Thr Val Ile Val Asp Met Thr Gly His 180 185 190 Arg Tyr Asp Ser Arg Cys Pro Ala Arg Leu Leu Asp Val Va l Pro Gly 195 200 205 Arg Ser Ala Asp Ala Leu Arg Ser Trp Leu Gly Ser Arg Gly Glu Gln 210 215 220 Phe Arg Asn Gln Ile Arg Ile Val Ser Met Asp Gly Phe Gln Gly Tyr 225 230 235 240 Ala Thr Ala Ser Lys Glu Leu Ile Pro Ser Ala Arg Arg Val Met Asp 245 250 255 Pro Phe His Val Val Arg Leu Ala Gly Asp Lys Leu Thr Ala Cys Arg 260 265 270 Gln Arg Leu Gln Arg Glu Lys Tyr Gln Arg Arg Gly Leu Ser Gln Asp 275 280 285 Pro Leu Tyr Lys Asn Arg Lys Thr Leu Leu Thr Thr His Lys Trp Leu 290 295 300 Ser Pro Arg Gln Gln Glu Ser Leu Glu Gln Leu Trp Ala Tyr Asp Lys 305 310 315 320 Asp Tyr Gly Val Leu Lys Leu Ala Trp Leu Ala Tyr Gln Ala Ile Ile 325 330 335 Asp Cys Tyr Gln Met Gly Asn Lys Arg Glu Ala Lys Lys Lys Met Arg 340 345 350 Thr Ile Ile Asp Gln Leu Arg Val Leu Lys Gly Pro Asn Lys Glu Leu 355 360 365 Ala Gln Leu Gly Arg Ser Leu Phe Lys Arg Leu Gly Asp Val Leu Ala 370 375 380 Tyr Phe Asp Val Gly Val Ser Asn Gly Pro Val Glu Ala Ile Asn Gly 385 390 395 400 Arg Leu Glu His Leu Arg Gly Ile Ala Leu Gly Phe Arg As n Leu Thr 405 410 415 His Tyr Ile Leu Arg Cys Leu Ile His Ser Gly Gln Leu Thr His Lys 420 425 430 Ile Asn Ala Leu 435

SEQ ID NO: 3 Sequence length: 15 base pairs Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Antisense: NO Origin Biological name: Blephabacterium Lactofermentum (Brevibacteriu
Strain name: AJ12036 Sequence GGCCCTTCCG GTTTTT
Fifteen

SEQ ID NO: 4 Sequence length: 15 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Antisense: YES Origin organism name: Blephabacterium Lactofermentum (Brevibacteriu
M lactofermentum) Strain name: AJ12036 Sequence GGCTCTTCCG TTTTT 15

SEQ ID NO: 5 Sequence length: 1453 base pairs Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin organism name: Brebebacterium lactofermentum ( Brevibacteriu
m lactofermentum) Strain name: AJ12036 Sequence features Characteristic symbol: insertion seq Location: 1..1453 Method of determining features: E Sequence characteristic Feature symbol: Location: 130..1440 Characteristic features Method: Feature of S sequence: Symbol representing a feature: repeat region Location: 1..15 Method of determining feature: S Sequence feature: Symbol representing feature: repeat region Location: 1339..1453 Method of determining feature: Features of S sequence: Symbol representing a feature: -35 region Location: 71..76 Method of determining feature: S Sequence feature: Symbol representing features: -10 region Location: 92..97 Method of determining feature: S Sequence GGCCCTTCCG GTTTTGGGGT ACATCACAGA ACCTGGGCTA GCGGTGTAGA CCCGAAAATA 60 AACGAGCCTT TTGTCAGGGT TAAGGTTTAG GTATCTAAGC TAACCAAACA CCAACAAAAG 120 GCTCTACCC ATG AGC Tla GL GAA CTA GGA CTC ACC ATC ACC GGC GCT TCC GAT GCA GGT 216 Arg Thr Ala Glu Leu Gly Leu Thr Ile Thr Gly Ala Ser Asp Ala Gly 15 20 25 GAT TAC ACC CTG ATC GAA GCA GAC GCA CTC GAC TAT ACC TCC ACC TGC 264 Asp Tyr Thr Leu Ile Glu Ala Asp Ala Leu Asp Tyr Thr Ser Thr Cys 30 35 40 45 CCA GAA TGC TTC CAA CCT GGG GTG TTT CGT CAT CAC ACC CAC CGG ATG 312 Pro Glu Cys Phe Gln Pro Gly Val Phe Arg His His Thr His Arg Met 50 55 60 CTC ATT GAT TTA CCC ATC GTC GGG TTT CCC ACC AAA CTG TTT ATC CGT 360 Leu Ile Asp Leu Pro Ile Val Gly Phe Pro Thr Lys Leu Phe Ile Arg 65 70 75 CTA CCT CGC TAC CGC TGC ACC AAC CCG ACA TGT AAG CAA AAG TAT TTC 408 Leu Pro Arg Tyr Arg Cys Thr Asn Pro Thr Cys Lys Gln Lys Tyr Phe 80 85 90 CAA GCA GAA CTA AGC TGC GCT GAC CAC GGT AAA AAG GTC ACC CAC CGG 456 Gln Ala Glu Leu Ser Cys Ala Asp His Gly Lys Lys Val Thr His Arg 95 100 105 GTC ACC CGC TGG ATT TTG CAA CGC CTT GCT ATT GAC CGG ATG AGT GTT 504 Val Thr Arg Trp Ile Leu Gln Arg Leu Ala Ile Asp Arg Met Ser Val 110 115 120 125 CAC GCA ACT GCG AAA GCA CTT GGG CTA GGG TGG G AT TTA ACC TGC CAA 552 His Ala Thr Ala Lys Ala Leu Gly Leu Gly Trp Asp Leu Thr Cys Gln 130 135 140 CTA GCC CTC GAT ATG TGC CGT GAG CTG GTC TAT AAC GAT CCT CAC CAT 600 Leu Ala Leu Asp Met Cys Arg Glu Leu Val Tyr Asn Asp Pro His His 145 150 155 CTT GAT GGA GTG TAT GTC ATT GGG GTG GAT GAG CAT AAG TGG TCA CAT 648 Leu Asp Gly Val Tyr Val Ile Gly Val Asp Glu His Lys Trp Ser His 160 165 170 AAT AGG GCT AAG CAT GGT GAT GGG TTT GTC ACC GTG ATT GTC GAT ATG 696 Asn Arg Ala Lys His Gly Asp Gly Phe Val Thr Val Ile Val Asp Met 175 180 185 ACC GGG CAT CGG TAT GAC TCA CGG TGT CCT GCC CGG TTA TTA GAT GTC 744 Thr Gly His Arg Tyr Asp Ser Arg Cys Pro Ala Arg Leu Leu Asp Val 190 195 200 205 GTC CCA GGT CGT AGT GCT GAT GCT TTA CGG TCC TGG CTT GGC TCC CGC 792 Val Pro Gly Arg Ser Ala Asp Ala Leu Arg Ser Trp Leu Gly Ser Arg 210 215 220 GGT GAA CAG TTC CGC AAT CAG ATA CGG ATC GTG TCC ATG GAT GGA TTC 840 Gly Glu Gln Phe Arg Asn Gln Ile Arg Ile Val Ser Met Asp Gly Phe 225 230 235 CAA GGC TAC GCC ACA GCA AGT AAA G AA CTC ATT CCT TCT GCT CGT CGC 888 Gln Gly Tyr Ala Thr Ala Ser Lys Glu Leu Ile Pro Ser Ala Arg Arg 240 245 250 GTG ATG GAT CCA TTC CAT GTT GTG CGG CTT GCT GGT GAC AAG CTC ACC 936 Val Met Asp Pro Phe His Val Val Arg Leu Ala Gly Asp Lys Leu Thr 255 260 265 GCC TGC CGG CAA CGC CTC CAG CGG GAG AAA TAC CAG CGT CGT GGT TTA 984 Ala Cys Arg Gln Arg Leu Gln Arg Glu Lys Tyr Gln Arg Arg Gly Leu 270 275 280 285 AGC CAG GAT CCG TTG TAT AAA AAC CGG AAG ACC TTG TTG ACC ACG CAC 1032 Ser Gln Asp Pro Leu Tyr Lys Asn Arg Lys Thr Leu Leu Thr Thr His 290 295 300 AAG TGG TTG AGT CCT CGT CAG CAA GAA AGC TTG GAG CAG TTG TGG GCG 1080 Lys Trp Leu Ser Pro Arg Gln Gln Glu Ser Leu Glu Gln Leu Trp Ala 305 310 315 TAT GAC AAA GAC TAC GGG GTG TTA AAG CTT GCG TGG CTT GCG TAT CAG 1128 Tyr Asp Lys Asp Tyr Gly Val Leu Lys Leu Ala Trp Leu Ala Tyr Gln 320 325 330 GCG ATT ATT GAT TGT TAT CAG ATG GGT AAT AAG CGT GAA GCG AAG AAG 1176 Ala Ile Ile Asp Cys Tyr Gln Met Gly Asn Lys Arg Glu Ala Lys Lys 335 340 345 AAA ATG CGG ACC A TT ATT GAT CAG CTT CGG GTG TTG AAG GGG CCG AAT 1224 Lys Met Arg Thr Ile Ile Asp Gln Leu Arg Val Leu Lys Gly Pro Asn 350 355 360 365 AAG GAA CTC GCG CAG TTG GGT CGT AGT TTG TTT AAA CGA CTT GGT GAT 1272 Lys Glu Leu Ala Gln Leu Gly Arg Ser Leu Phe Lys Arg Leu Gly Asp 370 375 380 GTG TTG GCG TAT TTC GAT GTT GGT GTC TCC AAC GGT CCG GTC GAA GCG 1320 Val Leu Ala Tyr Phe Asp Val Gly Val Ser Asn Gly Pro Val Glu Ala 385 390 395 ATC AAC GGA CGG TTG GAG CAT TTG CGT GGG ATT GCT CTA GGT TTC CGT 1368 Ile Asn Gly Arg Leu Glu His Leu Arg Gly Ile Ala Leu Gly Phe Arg 400 405 410 AAT TTG AAC CAC TAC ATT CTG CGG TGC CTT ATC CAT TCA GGG CAG TTG 1416 Asn Leu Asn His Tyr Ile Leu Arg Cys Leu Ile His Ser Gly Gln Leu 415 420 425 GTC CAT AAG ATC AAT GCA CTC TAA AACAGGAAGA GCC 1453 Val His Lys Ile Asn Ala Leu 430 435

SEQ ID NO: 6 Sequence length: 436 amino acids Sequence type: Amino acid Number of chains: Single strand Topology: Linear Sequence type: Protein Origin organism name: Brevibacterium lactofermentum (Brevibacteriu)
M lactofermentum) Strain name: AJ12036 Sequence Met Lys Ser Thr Gly Asn Ile Ile Ala Asp Thr Ile Cys Arg Thr Ala 1 5 10 15 Glu Leu Gly Leu Thr Ile Thr Gly Ala Ser Asp Ala Gly Asp Tyr Thr 20 25 30 Leu Ile Glu Ala Asp Ala Leu Asp Tyr Thr Ser Thr Cys Pro Glu Cys 35 40 45 Phe Gln Pro Gly Val Phe Arg His His Thr His Arg Met Leu Ile Asp 50 55 60 Leu Pro Ile Val Gly Phe Pro Thr Lys Leu Phe Ile Arg Leu Pro Arg 65 70 75 80 Tyr Arg Cys Thr Asn Pro Thr Cys Lys Gln Lys Tyr Phe Gln Ala Glu 85 90 95 Leu Ser Cys Ala Asp His Gly Lys Lys Val Thr His Arg Val Thr Arg 100 105 110 Trp Ile Leu Gln Arg Leu Ala Ile Asp Arg Met Ser Val His Ala Thr 115 120 125 Ala Lys Ala Leu Gly Leu Gly Trp Asp Leu Thr Cys Gln Leu Ala Leu 130 135 140 Asp Met Cys Arg Glu Leu Val Tyr Asn Asp Pro His His Leu Asp Gly 145 150 155 160 Val Tyr Val Ile Gly Val Asp Glu His Lys Trp Ser His Asn Arg Ala 165 170 175 Lys His Gly Asp Gly Phe Val Thr Val Ile Val Asp Met Thr Gly His 180 185 190 Arg Tyr Asp Ser Arg Cys Pro Ala Arg Leu Leu Asp Val Va l Pro Gly 195 200 205 Arg Ser Ala Asp Ala Leu Arg Ser Trp Leu Gly Ser Arg Gly Glu Gln 210 215 220 Phe Arg Asn Gln Ile Arg Ile Val Ser Met Asp Gly Phe Gln Gly Tyr 225 230 235 240 Ala Thr Ala Ser Lys Glu Leu Ile Pro Ser Ala Arg Arg Val Met Asp 245 250 255 Pro Phe His Val Val Arg Leu Ala Gly Asp Lys Leu Thr Ala Cys Arg 260 265 270 Gln Arg Leu Gln Arg Glu Lys Tyr Gln Arg Arg Gly Leu Ser Gln Asp 275 280 285 Pro Leu Tyr Lys Asn Arg Lys Thr Leu Leu Thr Thr His Lys Trp Leu 290 295 300 Ser Pro Arg Gln Gln Glu Ser Leu Glu Gln Leu Trp Ala Tyr Asp Lys 305 310 315 320 Asp Tyr Gly Val Leu Lys Leu Ala Trp Leu Ala Tyr Gln Ala Ile Ile 325 330 335 Asp Cys Tyr Gln Met Gly Asn Lys Arg Glu Ala Lys Lys Lys Met Arg 340 345 350 Thr Ile Ile Asp Gln Leu Arg Val Leu Lys Gly Pro Asn Lys Glu Leu 355 360 365 Ala Gln Leu Gly Arg Ser Leu Phe Lys Arg Leu Gly Asp Val Leu Ala 370 375 380 Tyr Phe Asp Val Gly Val Ser Asn Gly Pro Val Glu Ala Ile Asn Gly 385 390 395 400 Arg Leu Glu His Leu Arg Gly Ile Ala Leu Gly Phe Arg As n Leu Asn 405 410 415 His Tyr Ile Leu Arg Cys Leu Ile His Ser Gly Gln Leu Val His Lys 420 425 430 Ile Asn Ala Leu 435

SEQ ID NO: 7 Sequence length: 15 base pairs Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Antisense: NO Origin Biological name: Blebibacterium Lactofermentum (Brevibacteriu
M lactofermentum) Strain name: AJ12036 Sequence GGCCCTTCCG GTTTT 15

SEQ ID NO: 8 Sequence length: 15 base pairs Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Genomic DNA Antisense: YES Origin organism name: Brebebacterium Lactofermentum (Brevibacteriu
M lactofermentum) Strain name: AJ12036 Sequence GGCTCTTCCG GTTTT 15

SEQ ID NO: 9 Sequence length: 1279 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded topology: Linear Sequence type: Genomic DNA Origin organism name: Brebebacterium lactofermentum ( Brevibacteriu
m lactofermentum) Strain name: AJ12036 Sequence features Characteristic signature: insertion seq Location: 1..1279 Sequence characteristics Characteristic signature: repeat region Location: 1..14 Method of determining features: S Sequence symbols characteristic features: repeat region present position: 1266..1279 method to determine the characteristics: S sequence GGGACTGACC CCTGTTTGGT GGACACCTTG AAACCAGCAT GATGCTGGAA AGGTAATCTG 60 CCACCATGCC ACGCAAGACC TATACAGAGG AGTTCAAGCG CGATGCCGTC GCCTTGTACG 120 AGAACTCCCC AGAGGCTTCG ATCCAGACCA TCGCCACCGA TCTCGGGGTC AACCGCGCCA 180 CGTTGGCGAA CTGGGTGAAA AAATACGGCA CCGCAGGCTC CCAACGAAAC ACCCTCGCCA 240 GCCTCTGTGA ACGAGGCTGA GCAGATCCGG AAACTGGAAC GGGAAAACGC TCGCTTGAGA 300 GAAGAGCGCG ATATCCTGCG GAAAGCTGCA AAATATTTCG CGGAAGAGAC GAATTGGTGA 360 TCCGCTTCCG GTTCGTTGAT GACGCCTCCA AGACCTACTC GGTCAAGCGG ATATGTGACG 420 TCCTCAAACT CAACAGGTCT TCCTACTATA AATGGAAAAG TACCTGCTCA GCACGCAGGA 480 AACGCCTCAT GTCGACGCGA TCCTCGGGGC TCGAGTCAAG GCTGTCTTCA CCACCGAAAA 540 TGGTTGTTAT GGGGCCAAGC GGATC ACCGC TGAACTCAAA GACCAGGTGG ATCATGACCC 600 CGTAAATCAC AAGCGGGTCG CTCGGGTGAT GCGCTCGTTG AAGCTGTTTG GCTACACAAA 660 TAAACGCAAG GTCACCACCA CTGTGTCGGA TAAAACCAAG ACAGTGTTTC CTGACCTTGT 720 CGGCCGGAAG TTCACCGCTA ATAAGCCAAA TCAGGTGTAC GTCGGGACAT CACGTACCTG 780 CCGATTGCTG ATGGGTCGAA TATGTACCTG GCTACGGTCA TTGACTGCTA TTCCCGCAGG 840 TTGGTGGGCT TTTCTATCGC ACATCACATG CGTACCTCCC TGGTGCAGAC GCGCTGCTGA 900 TGGCTAAGGG CCAGCGCGAA GCTGACGGGG GCGATCTTTC ACTCGGATCA CGGAAGTGTT 960 TACACTTCTC ACGCATTCCA GACACCTGTA AAGACCTGGG ATAAGGCAGT CGATGGGATC 1020 AATCGGCACC AGTGCGACAA TGCCTCGCGG AGTCCTTCAA CGCAGCACTG AAGCGGAAGT 1080 CCTCCAGGAT TCCAAGACAT TCATGAACCA GTTGCGCTGT CGCCGGGACG TCTTCCGCTG 1140 GTGTACCCGC TACAACATGG TGCGCCGGCA TTCCTGGTGT AAATATCTCG CCCTGCGGTG 1200 TTTGAGAAGC GCTGTCCTGC TATCCTGAAA TCTGCTTCCT GATCAAATCC TCCGTGTCTA 1260 CTATCCGGGG GTCGGGCCC 1279

SEQ ID NO: 10 Sequence length: 14 base pairs Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Antisense: NO Origin Biological name: Blephabacterium Lactofermentum (Brevibacteriu
M lactofermentum) Strain name: AJ12036 Sequence GGGACTGACC CCTG 14

SEQ ID NO: 11 Sequence length: 14 base pairs Sequence type: Nucleic acid Number of strands: Double stranded Topology: Linear Sequence type: Genomic DNA Antisense: YES Origin organism name: Brebebacterium Lactofermentum (Brevibacteriu
M lactofermentum) Strain name: AJ12036 Sequence GGGCCCGACC CCCG 14

SEQ ID NO: 12 Sequence length: 8 bases Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear sequence GGTTTATT 8

SEQ ID NO: 13 Sequence length: 23 bases Sequence type: Nucleic acid Number of strands: Single-stranded Topology: Linear Sequence type: Other nucleic acid Synthetic DNA Antisense: NO sequence GTGGAGCCGA CCATTCCGCG AGG 23

SEQ ID NO: 14 sequence length: 23 bases sequence type: nucleic acid number of strands: single-stranded topology: linear sequence type: other nucleic acid synthetic DNA antisense: YES sequence CCAAAACCGC CCTCCACGGC GAA 23

SEQ ID NO: 15 Sequence Length: 3579 base pairs Sequence Type: Nucleic Acid Number of Strands: Double-Stranded Topology: Linear Sequence Type: Genomic DNA Origin: Organism Name: Brebebacterium lactofermentum (Brevibacteriu
m lactofermentum) Strain name: ATCC 13869 Sequence features: Feature symbol: CDS Location: 533..2182 Sequence feature: Feature symbol: CDS Location: 2188..3522 Sequence GTGGAGCCGA CCATTCCGCG AGGCTGCACT GCAACGAGGT CGTAGTTTTG GTACATGGCT 60 TCTGGCCAGT TCATGGATTG GCTGCCGAAG AAGCTATAGG CATCGCACCA GGGCCACCGA 120 GTTACCGAAG ATGGTGCCGT GCTTTTCGCC TTGGGCAGGG ACCTTGACAA AGCCCACGCT 180 GATATCGCCA AGTGAGGGAT CAGAATAGTG CATGGGCACG TCGATGCTGC CACATTGAGC 240 GGAGGCAATA TCTACCTGAG GTGGGCATTC TTCCCAGCGG ATGTTTTCTT GCGCTGCTGC 300 AGTGGGCATT GATACCAAAA AGGGGCTAAG CGCAGTCGAG GCGGCAAGAA CTGCTACTAC 360 CCTTTTTATT GTCGAACGGG GCATTACGGC TCCAAGGACG TTTGTTTTCT GGGTCAGTTA 420 CCCCAAAAAG CATATACAGA GACCAATGAT TTTTCATTAA AAAGGCAGGG ATTTGTTATA 480 AGTATGGGTC GTATTCTGTG CGACGGGTGT ACCTCGGCTA GAATTTCTCC CC ATG 535 Met 1 ACA CCA GCT GAT CTC GCA ACA TTG ATT AAA GAG ACC GCG GTA GAG GTT 583 Thr Pro Ala Asp Leu Ala Thr Leu Ile Lys Glu Thr Ala Val Glu Val 5 10 15 TTG GACC TCC CTC GAT ACT TCT GTT CTT CC G GAG CAG GTA GTT 631 Leu Thr Ser Arg Glu Leu Asp Thr Ser Val Leu Pro Glu Gln Val Val 20 25 30 GTG GAG CGT CCG CGT AAC CCA GAG CAC GGC GAT TAC GCC ACC AAC ATT 679 Val Glu Arg Pro Arg Asn Pro Glu His Gly Asp Tyr Ala Thr Asn Ile 35 40 45 GCA TTG CAG GTG GCT AAA AAG GTC GGT CAG AAC CCT CGG GAT TTG GCT 727 Ala Leu Gln Val Ala Lys Lys Val Gly Gln Asn Pro Arg Asp Leu Ala 50 55 60 65 ACC TGG CTG GCA GAG GCA TTG GCT GCA GAT GAC GCC ATT GAT TCT GCT 775 Thr Trp Leu Ala Glu Ala Leu Ala Ala Asp Asp Ala Ile Asp Ser Ala 70 75 80 GAA ATT GCT GGC CCA GGC TTT TTG AAC ATT CGC CTT GCT GCA GCA GCA 823 Glu Ile Ala Gly Pro Gly Phe Leu Asn Ile Arg Leu Ala Ala Ala Ala 85 90 95 CAG GGT GAA ATT GTG GCC AAG ATT CTG GCA CAG GGC GAG ACT TTC GGA 871 Gln Gly Glu Ile Val Ala Lys Ile Leu Ala Gln Gly Glu Thr Phe Gly 100 105 110 AAC TCC GAT CAC CTT TCC CAC TTG GAC GTG AAC CTC GAG TTC GTT TCT 919 Asn Ser Asp His Leu Ser His Leu Asp Val Asn Leu Glu Phe Val Ser 115 120 125 GCA AAC CCA ACC GGA CCT ATT CAC CTT GGC GGA ACC CG C TGG GCT GCC 967 Ala Asn Pro Thr Gly Pro Ile His Leu Gly Gly Thr Arg Trp Ala Ala 130 135 140 145 GTG GGT GAC TCT TTG GGT CGT GTG CTG GAG GCT TCC GGC GCG AAA GTG 1015 Val Gly Asp Ser Leu Gly Arg Val Leu Glu Ala Ser Gly Ala Lys Val 150 155 160 ACC CGC GAA TAC TAC TTC AAC GAT CAC GGT CGC CAG ATC GAT CGT TTC 1063 Thr Arg Glu Tyr Tyr Phe Asn Asp His Gly Arg Gln Ile Asp Arg Phe 165 170 175 GCT TTG TCC CTT CTT GCA GCG GCG AAG GGC GAG CCA ACG CCA GAA GAC 1111 Ala Leu Ser Leu Leu Ala Ala Ala Lys Gly Glu Pro Thr Pro Glu Asp 180 185 190 GGT TAT GGC GGC GAA TAC ATT AAG GAA ATT GCG GAG GCA ATC GTC GAA 1159 Gly Tyr Gly Gly Glu Tyr Ile Lys Glu Ile Ala Glu Ala Ile Val Glu 195 200 205 AAG CAT CCT GAA GCG TTG GCT TTG GAG CCT GCC GCA ACC CAG GAG CTT 1207 Lys His Pro Glu Ala Leu Ala Leu Glu Pro Ala Ala Thr Gln Glu Leu 210 215 220 225 TTC CGC GCT GAA GGC GTG GAG ATG ATG TTC GAG CAC ATC AAA TCT TCC 1255 Phe Arg Ala Glu Gly Val Glu Met Met Phe Glu His Ile Lys Ser Ser 230 235 240 CTG CAT GAG TTC GGC ACC GAT TTC GAT GTC TAC TAC CAC GAG AAC TCC 1303 Leu His Glu Phe Gly Thr Asp Phe Asp Val Tyr Tyr His Glu Asn Ser 245 250 255 CTG TTC GAG TCC GGT GCG GTG GAC AAG GCC GTG CAG GTG CTG AAG GAC 1351 Leu Phe Glu Ser Gly Ala Val Asp Lys Ala Val Gln Val Leu Lys Asp 260 265 270 AAC GGC AAC CTG TAC GAA AAC GAG GGC GCT TGG TGG CTG CGT TCC ACC 1399 Asn Gly Asn Leu Tyr Glu Asn Glu Gly Ala Trp Trp Leu Arg Ser Thr 275 280 285 GAA TTC GGC GAT GAC AAA GAC CGC GTG GTG ATC AAG TCT GAC GGC GAC 1447 Glu Phe Gly Asp Asp Lys Asp Arg Val Val Ile Lys Ser Asp Gly Asp 290 295 300 305 GCA GCC TAC ATC GCT GGC GAT ATC GCG TAC GTG GCT GAT AAG TTC TCC 1495 Ala Ala Tyr Ile Ala Gly Asp Ile Ala Tyr Val Ala Asp Lys Phe Ser 310 315 320 CGC GGA CAC AAC CTA AAC ATC TAC ATG TTG GGT GCT GAC CAC CAT GGT 1543 Arg Gly His Asn Leu Asn Ile Tyr Met Leu Gly Ala Asp His His Gly 325 330 335 TAC ATC GCG CGC CTG AAG GCA GCG GCG GCG GCA CTT GGC TAC AAG CCA 1591 Tyr Ile Ala Arg Leu Lys Ala Ala Ala Ala Ala Leu Gly Tyr Lys Pro 340 345 350 GAA GGC G TT GAA GTC CTG ATT GGC CAG ATG GTG AAC CTG CTT CGC GAC 1639 Glu Gly Val Glu Val Leu Ile Gly Gln Met Val Asn Leu Leu Arg Asp 355 360 365 GGC AAG GCA GTG CGT ATG TCC AAG CGT GCA GGC ACC GTG GTC ACC CTATA 1687 Gly Lys Ala Val Arg Met Ser Lys Arg Ala Gly Thr Val Val Thr Leu 370 375 380 385 GAT GAC CTC GTT GAA GCA ATC GGC ATC GAT GCG GCG CGT TAC TCC CTG 1735 Asp Asp Leu Val Glu Ala Ile Gly Ile Asp Ala Ala Arg Tyr Ser Leu 390 395 400 ATC CGT TCC TCC GTG GAT TCT TCC CTG GAT ATC GAT CTC GGC CTG TGG 1783 Ile Arg Ser Ser Val Asp Ser Ser Leu Asp Ile Asp Leu Gly Leu Trp 405 410 415 GAA TCC CAG TCC TCC GAC AAC CCT GTG TAC TAC GTG CAG TAC GGA CAC 1831 Glu Ser Gln Ser Ser Asp Asn Pro Val Tyr Tyr Val Gln Tyr Gly His 420 425 430 GCT CGT CTG TGC TCC ATC GCG CGC AAG GCA GAG ACC TTG GGT GTC ACC 1879 Ala Arg Leu Cys Ser Ile Ala Arg Lys Ala Glu Thr Leu Gly Val Thr 435 440 445 GAG GAA GGC GCA GAC CTA TCT CTA CTG ACC CAC GAC CGC GAA GGC GAT 1927 Glu Glu Gly Ala Asp Leu Ser Leu Leu Thr His Asp Arg Glu Gly Asp 45 0 455 460 465 CTC ATC CGC ACA CTC GGA GAG TTC CCA GCA GTG GTG AAG GCT GCC GCT 1975 Leu Ile Arg Thr Leu Gly Glu Phe Pro Ala Val Val Lys Ala Ala Ala 470 475 480 GAC CTA CGT GAA CCA CAC CGC ATT GCC CGC TAT GCT GAG GAA TTA GCT 2023 Asp Leu Arg Glu Pro His Arg Ile Ala Arg Tyr Ala Glu Glu Leu Ala 485 490 495 GGA ACT TTC CAC CGC TTC TAC GAT TCC TGC CAC ATC CTT CCA AAG GTT 2071 Gly Thr Phe His Arg Phe Tyr Asp Ser Cys His Ile Leu Pro Lys Val 500 505 510 GAT GAG GAT ACG GCA CCA ATC CAC ACA GCA CGT CTG GCA CTT GCA GCA 2119 Asp Glu Asp Thr Ala Pro Ile His Thr Ala Arg Leu Ala Leu Ala Ala 515 520 525 GCA ACC CGC CAG ACC CTC GCT AAC GCC CTG CAC CTG GTT GGC GTT TCC 2167 Ala Thr Arg Gln Thr Leu Ala Asn Ala Leu His Leu Val Gly Val Ser 530 535 540 545 GCA CCG GAG AAG ATG TAACA ATG GCT ACA GTT GAA AAT TTC AAT GAA 2214 Ala Pro Glu Lys Met Met Ala Thr Val Glu Asn Phe Asn Glu 550 1 5 CTT CCC GCA CAC GTA TGG CCA CGC AAT GCC GTG CGC CAA GAA GAC GGC 2262 Leu Pro Ala His Val Trp Pro Arg Asn Ala Val Arg Gln Glu A sp Gly 10 15 20 25 GTT GTC ACC GTC GCT GGT GTG CCT CTG CCT GAC CTC GCT GAA GAA TAC 2310 Val Val Thr Val Ala Gly Val Pro Leu Pro Asp Leu Ala Glu Glu Tyr 30 35 40 GGA ACC CCA CTG TTC GTA GTC GAC GAG GAC GAT TTC CGT TCC CGC TGT 2358 Gly Thr Pro Leu Phe Val Val Asp Glu Asp Asp Phe Arg Ser Arg Cys 45 50 55 CGC GAC ATG GCT ACC GCA TTC GGT GGA CCA GGC AAT GTG CAC TAC GCA 2406 Arg Asp Met Ala Thr Ala Phe Gly Gly Pro Gly Asn Val His Tyr Ala 60 65 70 TCT AAA GCG TTC CTG ACC AAG ACC ATT GCA CGT TGG GTT GAT GAA GAG 2454 Ser Lys Ala Phe Leu Thr Lys Thr Ile Ala Arg Trp Val Asp Glu Glu 75 80 85 GGG CTG GCA CTG GAC ATT GCA TCC ATC AAC GAA CTG GGC ATT GCC CTG 2502 Gly Leu Ala Leu Asp Ile Ala Ser Ile Asn Glu Leu Gly Ile Ala Leu 90 95 100 105 GCC GCT GGT TTC CCC GCC AGC CGT ATC ACC GCG CAC GGC AAC AAC AAA 2550 Ala Ala Gly Phe Pro Ala Ser Arg Ile Thr Ala His Gly Asn Asn Lys 110 115 120 GGC GTA GAG TTC CTG CGC GCG TTG GTT CAA AAC GGT GTG GGA CAC GTG 2598 Gly Val Glu Phe Leu Arg Ala Leu Val Gln Asn Gly Val Gly His Val 125 130 135 GTG CTG GAC TCC GCA CAG GAA CTA GAA CTG TTG GAT TAC GTT GCC GCT 2646 Val Leu Asp Ser Ala Gln Glu Leu Glu Leu Leu Asp Tyr Val Ala Ala 140 145 150 GGT GAA GGC AAG ATT CAG GAC GTG TTG ATC CGC GTA AAG CCA GGC ATC 2694 Gly Glu Gly Lys Ile Gln Asp Val Leu Ile Arg Val Lys Pro Gly Ile 155 160 165 GAA GCA CAC ACC CAC GAG TTC ATC GCC ACT AGC CAC GAA GAC CAG AAG 2742 Glu Ala His Thr His Glu Phe Ile Ala Thr Ser His Glu Asp Gln Lys 170 175 180 185 TTC GGA TTC TCC CTG GCA TCC GGT TCC GCA TTC GAA GCA GCA AAA GCC 2790 Phe Gly Phe Ser Leu Ala Ser Gly Ser Ala Phe Glu Ala Ala Lys Ala 190 195 200 GCC AAC AAC GCA GAA AAC CTG AAC CTG GTT GGC CTG CAC TGC CAC GTT 2838 Ala Asn Asn Ala Glu Asn Leu Asn Leu Val Gly Leu His Cys His Val 205 210 215 GGT TCC CAG GTG TTC GAC GCC GAA GGC TTC AAG CTG GCA GCA GAA CGC 2886 Gly Ser Gln Val Phe Asp Ala Glu Gly Phe Lys Leu Ala Ala Glu Arg 220 225 230 GTG TTG GGC CTG TAC TCA CAG ATC CAC AGC GAA CTG GGC GTT GCC CTT 2934 Val Leu Gly Leu Tyr Ser Gln Ile H is Ser Glu Leu Gly Val Ala Leu 235 240 245 CCT GAA CTG GAT CTC GGT GGC GGA TAC GGC ATT GCC TAT ACC GCA GCT 2982 Pro Glu Leu Asp Leu Gly Gly Gly Tyr Gly Ile Ala Tyr Thr Ala Ala 250 255 260 265 GAA GAA CCA CTC AAC GTC GCA GAA GTT GCC TCC GAC CTG CTC ACC GCA 3030 Glu Glu Pro Leu Asn Val Ala Glu Val Ala Ser Asp Leu Leu Thr Ala 270 275 280 GTC GGA AAA ATG GCA GCG GAA CTA GGC ATC GAC GCA CCA ACC GTG CTT 3078 Val Gly Lys Met Ala Ala Glu Leu Gly Ile Asp Ala Pro Thr Val Leu 285 290 295 GTT GAG CCC GGC CGC GCT ATC GCA GGC CCC TCC ACC GTG ACC ATC TAC 3126 Val Glu Pro Gly Arg Ala Ile Ala Gly Pro Ser Thr Val Thr Ile Tyr 300 305 310 GAA GTC GGC ACC ACC AAA GAC GTC CAC GTA GAC GAC GAC AAA ACC CGC 3174 Glu Val Gly Thr Thr Lys Asp Val His Val Asp Asp Asp Lys Thr Arg 315 320 325 CGT TAC ATC GCC GTG GAC GGA GGC ATG TCC GAC AAC ATC CGC CCA GCA 3222 Arg Tyr Ile Ala Val Asp Gly Gly Met Ser Asp Asn Ile Arg Pro Ala 330 335 340 345 CTC TAC GGC TCC GAA TAC GAC GCC CGC GTA GTA TCC CGC TTC GCC GAA 3270 Leu Tyr Gl y Ser Glu Tyr Asp Ala Arg Val Val Ser Arg Phe Ala Glu 350 355 360 GGA GAC CCA GTA AGC ACC CGC ATC GTG GGC TCC CAC TGC GAA TCC GGC 3318 Gly Asp Pro Val Ser Thr Arg Ile Val Gly Ser His Cys Glu Ser Gly 365 370 375 GAT ATC CTG ATC AAC GAT GAA ATC TAC CCA TCT GAC ATC ACC AGC GGC 3366 Asp Ile Leu Ile Asn Asp Glu Ile Tyr Pro Ser Asp Ile Thr Ser Gly 380 385 390 GAC TTC CTT GCA CTC GCA GCC ACC GGC GCA TAC TGC TAC GCC ATG AGC 3414 Asp Phe Leu Ala Leu Ala Ala Thr Gly Ala Tyr Cys Tyr Ala Met Ser 395 400 405 TCC CGC TAC AAC GCC TTC ACA CGG CCC GCC GTC GTG TCC GTC CGC GCT 3462 Ser Arg Tyr Asn Ala Phe Thr Arg Pro Ala Val Val Ser Val Arg Ala 410 415 420 425 GGC AGC TCC CGC CTC ATG CTG CGC CGC GAA ACG CTC GAC GAC ATC CTC 3510 Gly Ser Ser Arg Leu Met Leu Arg Arg Glu Thr Leu Asp Asp Ile Leu 430 435 440 TCA CTA GAG GCA TAACGCTTTT CGACGCCTGA CCCCGCCCTT CACCTTCGCC 3562 Ser Leu Glu Ala 445 GTGGAGGGCG GTTTTGG 3579

SEQ ID NO: 16 Sequence Length: 23 bases Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: NO Sequence GTCGACGGAT CGCAAATGGC AAC
23

SEQ ID NO: 17 sequence length: 23 bases sequence type: nucleic acid number of strands: single-stranded topology: linear sequence type: other nucleic acid synthetic DNA antisense: YES sequence GGATCCTTGA GCACCTTGCG CAG 23

SEQ ID NO: 18 Sequence Length: 1411 base pairs Sequence Type: Nucleic Acid Number of Strands: Double-Stranded Topology: Linear Sequence Type: Genomic DNA Origin: Organism Name: Brebebacterium lactofermentum (Brevibacteriu
m lactofermentum) strain name: characteristics of ATCC 13869 sequence: Symbol indicating the feature: CDS existing position: 311..1213 sequence CTCTCGATAT CGAGAGAGAA GCAGCGCCAC GGTTTTTCGG TGATTTTGAG ATTGAAACTT 60 TGGCAGACGG ATCGCAAATG GCAACAAGCC CGTATGTCAT GGACTTTTAA CGCAAAGCTC 120 ACACCCACGA GCTAAAAATT CATATAGTTA AGACAACATT TTTGGCTGTA AAAGACAGCC 180 GTAAAAACCT CTTGCTCATG TCAATTGTTC TTATCGGAAT GTGGCTTGGG CGATTGTTAT 240 GCAAAAGTTG TTAGGTTTTT TGCGGGGTTG TTTAACCCCC AAATGAGGGA AGAAGGTAAC 300 CTTGAACTCT ATG AGC ACA GGT TTA ACA GCT AAG ACC GGA GTA GAG GTA GAG GTA GAG GTA GAG LAG GLA GLA GLA GTT ACT CCA TTC ACG GAA TCC GGA 397 Phe Gly Thr Val Gly Val Ala Met Val Thr Pro Phe Thr Glu Ser Gly 15 20 25 GAC ATC GAT ATC GCT GCT GGC CGC GAA GTC GCG GCT TAT TTG GTT GAT 445 Asp Ile Asp Ile Ala Ala Gly Arg Glu Val Ala Ala Tyr Leu Val Asp 30 35 40 45 AAG GGC TTG GAT TCT TTG GTT CTC GCG GGC ACC ACT GGT GAA TCC CCA 493 Lys Gly Leu Asp Ser Leu Val Leu Ala Gly Thr Thr Gly Glu Se r Pro 50 55 60 ACG ACA ACC GCC GCT GAA AAA CTA GAA CTG CTC AAG GCC GTT CGT GAG 541 Thr Thr Thr Ala Ala Glu Lys Leu Glu Leu Leu Lys Ala Val Arg Glu 65 70 75 GAA GTT GGG GAT CGG GCG AAC GTC ATC GCC GGT GTC GGA ACC AAC AAC 589 Glu Val Gly Asp Arg Ala Asn Val Ile Ala Gly Val Gly Thr Asn Asn 80 85 90 ACG CGG ACA TCT GTG GAA CTT GCG GAA GCT GCT GCT TCT GCT GGC GCA 637 Thr Arg Thr Ser Val Glu Leu Ala Glu Ala Ala Ala Ser Ala Gly Ala 95 100 105 GAC GGC CTT TTA GTT GTA ACT CCT TAT TAC TCC AAG CCG AGC CAA GAG 685 Asp Gly Leu Leu Val Val Thr Pro Tyr Tyr Ser Lys Pro Ser Gln Glu 110 115 120 125 GGA TTG CTG GCG CAC TTC GGT GCA ATT GCT GCA GCA ACA GAG GTT CCA 733 Gly Leu Leu Ala His Phe Gly Ala Ile Ala Ala Ala Thr Glu Val Pro 130 135 140 ATT TGT CTC TAT GAC ATT CCT GGT CGG TCA GGT ATT CCA ATT GAG TCT 781 Ile Cys Leu Tyr Asp Ile Pro Gly Arg Ser Gly Ile Pro Ile Glu Ser 145 150 155 GAT ACC ATG AGA CGC CTG AGT GAA TTA CCT ACG ATT TTG GCG GTC AAG 829 Asp Thr Met Arg Arg Leu Ser Glu Leu Pro Thr Ile Leu Ala Val Lys 160 165 170 GAC GCC AAG GGT GAC CTC GTT GCA GCC ACG TCA TTG ATC AAA GAA ACG 877 Asp Ala Lys Gly Asp Leu Val Ala Ala Thr Ser Leu Ile Lys Glu Thr 175 180 185 GGA CTT GCC TGG TAT TCA GGC GAT GAC CCA CTA AAC CTT GTT TGG CTT 925 Gly Leu Ala Trp Tyr Ser Gly Asp Asp Pro Leu Asn Leu Val Trp Leu 190 195 200 205 GCT TTG GGC GGA TCA GGT TTC ATT TCC GTA ATT GGA CAT GCA GCC CCC 973 Ala Leu Gly Gly Ser Gly Phe Ile Ser Val Ile Gly His Ala Ala Pro 210 215 220 ACA GCA TTA CGT GAG TTG TAC ACA AGC TTC GAG GAA GGC GAC CTC GTC 1021 Thr Ala Leu Arg Glu Leu Tyr Thr Ser Phe Glu Glu Gly Asp Leu Val 225 230 235 CGT GCG CGG GAA ATC AAC GCC AAA CTA TCA CCG CTG GTA GCT GCC CAA 1069 Arg Ala Arg Glu Ile Asn Ala Lys Leu Ser Pro Leu Val Ala Ala Gln 240 245 250 GGT CGC TTG GGT GGA GTC AGC TTG GCA AAA GCT GCT CTG CGT CTG CAG 1117 Gly Arg Leu Gly Gly Val Ser Leu Ala Lys Ala Ala Leu Arg Leu Gln 255 260 265 GGC ATC AAC GTA GGA GAT CCT CGA CTT CCA ATT ATG GCT CCA AAT GAG 1165 Gly Ile Asn Val Gly Asp Pro Arg Leu Pro Ile Met Ala Pro Asn Glu 270 275 280 285 285 CAG GAA CTT GAG GCT CTC CGA GAA GAC ATG AAA AAA GCT GGA GTT CTA 1213 Gln Glu Glu Leu Glu Ala Leu Arg Glu Asp Met Lys Lys Ala Gly Val Leu 290 295 300 TAAATATGAA GGATTCCGA CCGCAAGGCG GCCCACCAGA 1273 AGCTGGTCAG GAAAACCATC TGGATACCCC TGTCTTTCAG GCACCAGATG CTTCCTCTAA 1333 CCAGAGCGCT GTAAAAGCTG AGACCGCCGG AAACGACAAT CGGGATGCTG CGCAAGGTGC 1393 TCAAGGATCC CAACATTC

SEQ ID NO: 19 Sequence Length: 23 bases Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: NO Sequence TCGCGAAGTA GCACCTGTCA CTT 23

SEQ ID NO: 20 Sequence Length: 21 bases Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: YES Sequence ACGGAATTCA ATCTTACGGC C 21

SEQ ID NO: 21 Sequence length: 1643 base pairs Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin: Organism name: Brebebacterium lactofermentum (Brevibacteriu
m lactofermentum) strain name: ATCC 13869 SEQ TCGCGAAGTA GCACCTGTCA CTTTTGTCTC AAATATTAAA TCGAATATCA ATATACGGTC 60 TGTTTATTGG AACGCATCCC AGTGGCTGAG ACGCATCCGC TAAAGCCCCA GGAACCCTGT 120 GCAGAAAGAA AACACTCCTC TGGCTAGGTA GACACAGTTT ATAAAGGTAG AGTTGAGCGG 180 GTAACTGTCA GCACGTAGAT CGAAAGGTGC ACAAAGGTGG CCCTGGTCGT ACAGAAATAT 240 GGCGGTTCCT CGCTTGAGAG TGCGGAACGC ATTAGAAACG TCGCTGAACG GATCGTTGCC 300 ACCAAGAAGG CTGGAAATGA TGTCGTGGTT GTCTGCTCCG CAATGGGAGA CACCACGGAT 360 GAACTTCTAG AACTTGCAGC GGCAGTGAAT CCCGTTCCGC CAGCTCGTGA AATGGATATG 420 CTCCTGACTG CTGGTGAGCG TATTTCTAAC GCTCTCGTCG CCATGGCTAT TGAGTCCCTT 480 GGCGCAGAAG CTCAATCTTT CACTGGCTCT CAGGCTGGTG TGCTCACCAC CGAGCGCCAC 540 GGAAACGCAC GCATTGTTGA CGTCACACCG GGTCGTGTGC GTGAAGCACT CGATGAGGGC 600 AAGATCTGCA TTGTTGCTGG TTTTCAGGGT GTTAATAAAG AAACCCGCGA TGTCACCACG 660 TTGGGTCGTG GTGGTTCTGA CACCACTGCA GTTGCGTTGG CAGCTGCTTT GAACGCTGAT 720 GTGTGTGAGA TTTACTCGGA CGTTGACGGT GTGTATACCG CTGACCCGCG CATCGTTCCT 780 AATGCACAGA AGCTGGAAAA GCTCAGCTTC GAAGAAATGC T GGAACTTGC TGCTGTTGGC 840 TCCAAGATTT TGGTGCTGCG CAGTGTTGAA TACGCTCGTG CATTCAATGT GCCACTTCGC 900 GTACGCTCGT CTTATAGTAA TGATCCCGGC ACTTTGATTG CCGGCTCTAT GGAGGATATT 960 CCTGTGGAAG AAGCAGTCCT TACCGGTGTC GCAACCGACA AGTCCGAAGC CAAAGTAACC 1020 GTTCTGGGTA TTTCCGATAA GCCAGGCGAG GCTGCCAAGG TTTTCCGTGC GTTGGCTGAT 1080 GCAGAAATCA ACATTGACAT GGTTCTGCAG AACGTCTCCT CTGTGGAAGA CGGCACCACC 1140 GACATCACGT TCACCTGCCC TCGCGCTGAC GGACGCCGTG CGATGGAGAT CTTGAAGAAG 1200 CTTCAGGTTC AGGGCAACTG GACCAATGTG CTTTACGACG ACCAGGTCGG CAAAGTCTCC 1260 CTCGTGGGTG CTGGCATGAA GTCTCACCCA GGTGTTACCG CAGAGTTCAT GGAAGCTCTG 1320 CGCGATGTCA ACGTGAACAT CGAATTGATT TCCACCTCTG AGATCCGCAT TTCCGTGCTG 1380 ATCCGTGAAG ATGATCTGGA TGCTGCTGCA CGTGCATTGC ATGAGCAGTT CCAGCTGGGC 1440 GGCGAAGACG AAGCCGTCGT TTATGCAGGC ACCGGACGCT AAAGTTTTAA AGGAGTAGTT 1500 TTACAATGAC CACCATCGCA GTTGTTGGTG CAACCGGCCA GGTCGGCCAG GTTATGCGCA 1560 CCCTTTTGGA AGAGCGCAAT TTCCCAGCTG ACACTGTTCG TTTCTTTGCT TCCCCGCGTT 1620 CCGCAGGCCG TAAGATTGAA TTC 1643

SEQ ID NO: 22 sequence length: 23 bases sequence type: nucleic acid number of strands: single-stranded topology: linear sequence type: other nucleic acid synthetic DNA antisense: NO sequence GGATCCCCAA TCGATACCTG GAA 23

SEQ ID NO: 23 Sequence Length: 23 bases Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: YES Sequence CGGTTCATCG CCAAGTTTTT CTT 23

SEQ ID NO: 24 Sequence Length: 2001 base pairs Sequence Type: Nucleic Acid Number of Strands: Double-Stranded Topology: Linear Sequence Type: Genomic DNA Origin: Organism Name: Brebebacterium lactofermentum (Brevibacteriu
m lactofermentum) strain name: characteristics of ATCC 13869 sequence: Symbol indicating the feature: CDS existing position: 730..1473 sequence GGATCCCCAA TCGATACCTG GAACGACAAC CTGATCAGGA TATCCAATGC CTTGAATATT 60 GACGTTGAGG AAGGAATCAC CAGCCATCTC AACTGGAAGA CCTGACGCCT GCTGAATTGG 120 ATCAGTGGCC CAATCGACCC ACCAACCAGG TTGGCTATTA CCGGCGATAT CAAAAACAAC 180 TCGCGTGAAC GTTTCGTGCT CGGCAACGCG GATGCCAGCG ATCGACATAT CGGAGTCACC 240 AACTTGAGCC TGCTGCTTCT GATCCATCGA CGGGGAACCC AACGGCGGCA AAGCAGTGGG 300 GGAAGGGGAG TTGGTGGACT CTGAATCAGT GGGCTCTGAA GTGGTAGGCG ACGGGGCAGC 360 ATCTGAAGGC GTGCGAGTTG TGGTGACCGG GTTAGCGGTT TCAGTTTCTG TCACAACTGG 420 AGCAGGACTA GCAGAGGTTG TAGGCGTTGA GCCGCTTCCA TCACAAGCAC TTAAAAGTAA 480 AGAGGCGGAA ACCACAAGCG CCAAGGAACT ACCTGCGGAA CGGGCGGTGA AGGGCAACTT 540 AAGTCTCATA TTTCAAACAT AGTTCCACCT GTGTGATTAA TCTCCAGAAC GGAACAAACT 600 GATGAACAAT CGTTAACAAC ACAGACCAAA ACGGTCAGTT AGGTATGGAT ATCAGCACCT 660 TCTGAATGGG TACGTCTAGA CTGGTGGGCG TTTGAAAAAC TCTTCGCCCC ACGAAAATGA 720 AGGAGCATA ATG GGA ATC AAG GTT GGC GTT CTC GGA G CC AAA GGC CGT 768 Met Gly Ile Lys Val Gly Val Leu Gly Ala Lys Gly Arg 1 5 10 GTT GGT CAA ACT ATT GTG GCA GCA GTC AAT GAG TCC GAC GAT CTG GAG 816 Val Gly Gln Thr Ile Val Ala Ala Val Asn Glu Ser Asp Asp Leu Glu 15 20 25 CTT GTT GCA GAG ATC GGC GTC GAC GAT GAT TTG AGC CTT CTG GTA GAC 864 Leu Val Ala Glu Ile Gly Val Asp Asp Asp Leu Ser Leu Leu Val Asp 30 35 40 45 AAC GGC GCT GAA GTT GTC GTT GAC TTC ACC ACT CCT AAC GCT GTG ATG 912 Asn Gly Ala Glu Val Val Val Asp Phe Thr Thr Pro Asn Ala Val Met 50 55 60 GGC AAC CTG GAG TTC TGC ATC AAC AAC GGC ATT TCT GCG GTT GTT GGA 960 Gly Asn Leu Glu Phe Cys Ile Asn Asn Gly Ile Ser Ala Val Val Gly 65 70 75 ACC ACG GGC TTC GAT GAT GCT CGT TTG GAG CAG GTT CGC GCC TGG CTT 1008 Thr Thr Gly Phe Asp Asp Ala Arg Leu Glu Gln Val Arg Ala Trp Leu 80 85 90 GAA GGA AAA GAC AAT GTC GGT GTT CTG ATC GCA CCT AAC TTT GCT ATC 1056 Glu Gly Lys Asp Asn Val Gly Val Leu Ile Ala Pro Asn Phe Ala Ile 95 100 105 TCT GCG GTG TTG ACC ATG GTC TTT TCC AAG CAG GCT GCC CGC TTC TTC 1104 Ser Ala Val Leu Thr Met Val Phe Ser Lys Gln Ala Ala Arg Phe Phe 110 115 120 125 GAA TCA GCT GAA GTT ATT GAG CTG CAC CAC CCC AAC AAG CTG GAT GCA 1152 Glu Ser Ala Glu Val Ile Glu Leu His His Pro Asn Lys Leu Asp Ala 130 135 140 CCT TCA GGC ACC GCG ATC CAC ACT GCT CAG GGC ATT GCT GCG GCA CGC 1200 Pro Ser Gly Thr Ala Ile His Thr Ala Gln Gly Ile Ala Ala Ala Arg 145 150 155 AAA GAA GCA GGC ATG GAC GCA CAG CCA GAT GCG ACC GAG CAG GCA CTT 1248 Lys Glu Ala Gly Met Asp Ala Gln Pro Asp Ala Thr Glu Gln Ala Leu 160 165 170 GAG GGT TCC CGT GGC GCA AGC GTA GAT GGA ATC CCA GTT CAC GCA GTC 1296 Glu Gly Ser Arg Gly Ala Ser Val Asp Gly Ile Pro Val His Ala Val 175 180 185 CGC ATG TCC GGC ATG GTT GCT CAC GAG CAA GTT ATC TTT GGC ACC CAG 1344 Arg Met Ser Gly Met Val Ala His Glu Gln Val Ile Phe Gly Thr Gln 190 195 200 205 GGT CAG ACC TTG ACC ATC AAG CAG GAC TCC TAT GAT CGC AAC TCA TTT 1392 Gly Gln Thr Leu Thr Ile Lys Gln Asp Ser Tyr Asp Arg Asn Ser Phe 210 215 220 GCA CCA GGT GTC TTG GTG GGT GTG CGC AAC ATT GC A CAG CAC CCA GGC 1440 Ala Pro Gly Val Leu Val Gly Val Arg Asn Ile Ala Gln His Pro Gly 225 230 235 CTA GTC GTA GGA CTT GAG CAT TAC CTA GGC CTG TAAAGGCTCA TTTCAGCAGC 1493 Leu Val Val Gly Leu Glu His Tyr Leu Gly Leu 240 245 GGGTGGAATT TTTTAAAAGG AGCGTTTAAA GGCTGTGGCC GAACAAGTTA AATTGAGCGT 1553 GGAGTTGATA GCGTGCAGTT CTTTTACTCC ACCCGCTGAT GTTGAGTGGT CAACTGATGT 1613 TGAGGGCGCG GAAGCACTCG TCGAGTTTGC GGGTCGTGCC TGCTACGAAA CTTTTGATAA 1673 GCCGAACCCT CGAACTGCTT CCAATGCTGC GTATCTGCGC CACATCATGG AAGTGGGGCA 1733 CACTGCTTTG CTTGAGCATG CCAATGCCAC GATGTATATC CGAGGCATTT CTCGGTCCGC 1793 GACCCATGAA TTGGTCCGAC ACCGCCATTT TTCCTTCTCT CAACTGTCTC AGCGTTTCGT 1853 GCACAGCGGA GAATCGGAAG TAGTGGTGCC CACTCTCATC GATGAAGATC CGCAGTTGCG 1913 TGAACTTTTC ATGCACGCCA TGGATGAGTC TCGGTTCGCT TTCAATGAGC TGCTTAATGC 1973 GCTGGAAGAA AAACTTGGCG ATGAACCG 2001

SEQ ID NO: 25 Sequence Length: 45 bases Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: NO Sequence CCGGACAGCT CACCCACAAA ATCAATGCAC TCTAAAAAGG TACCT 45

SEQ ID NO: 26 Sequence Length: 45 bases Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: YES Sequence CTAGAGGTAC CTTTTTAGAG TGCATTGATT TTGTGGGTGA GCTGT 45

SEQ ID NO: 27 Sequence Length: 34 bases Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: NO Sequence CTAGCTCGAG ATATCAGATC TACTAGTCGA CCGC 34

SEQ ID NO: 28 Sequence Length: 28 bases Sequence Type: Nucleic Acid Number of Strands: Single Strand Topology: Linear Sequence Type: Other Nucleic Acid Synthetic DNA Antisense: YES Sequence GGTCGACTAG TAGATCTGAT ATCTCGAG 28

[Brief description of drawings]

FIG. 1 is a diagram showing structures of various artificial transposons. Kmr represents a neomycin phosphotransferase gene (kanamycin resistance gene), Tnp represents a transposase gene, and a black solid portion represents an inverted repeat sequence.

FIG. 2 Plasmid p carrying an artificial transposon
It is a construction drawing of HTN7141 and pHTN7142.

[Fig. 3] Plasmid p carrying an artificial transposon
It is a construction drawing of HTN7143.

[Fig. 4] Plasmid p carrying an artificial transposon
FIG. 6 is a construction diagram of HTN7144.

FIG. 5: Plasmids pHIS714K1 and pHIS7
It is a construction drawing of 14K2.

[Fig. 6] Plasmid p carrying an artificial transposon
FIG. 6 is a construction diagram of HTN7145.

FIG. 7: Plasmid p carrying an artificial transposon
It is a construction drawing of HTN7151.

FIG. 8: Plasmid p carrying an artificial transposon
FIG. 6 is a construction diagram of HTN7152.

FIG. 9: Plasmid p carrying an artificial transposon
FIG. 6 is a construction diagram of HTN7156-C.

FIG. 10 is a construction diagram of plasmid pORF1.

FIG. 11 is a construction diagram of plasmids pORF3 and pORF4.

FIG. 12 is a construction diagram of plasmid pORF7.

FIG. 13 is a construction diagram of plasmid pORF8.

FIG. 14 is a construction diagram of a plasmid pORF41 carrying a transposon unit.

FIG. 15 is a construction diagram of plasmid pORFC0.

FIG. 16 is a schematic diagram showing the difference in the structure of the insertion sequence, the artificial transposon and the transposon unit. TCr is a tetracycline resistance gene, T
np represents the transposase gene, and the blackened portion represents the inverted repeat sequence (IR). Also,
The part indicated by a dotted line below the schematic diagram of each structure shows a transition region.

FIG. 17 is a construction diagram of plasmid pORF40.

FIG. 18 is a construction diagram of plasmid pVK7.

FIG. 19 is a construction diagram of plasmid pVC7.

FIG. 20 is a construction diagram of plasmid p399LYSA and plasmid p299LYSA.

FIG. 21 is a construction diagram of plasmid pABLm.

FIG. 22 is a construction diagram of plasmid pCRDAPA.

FIG. 23 is a construction diagram of plasmid p399DPS.

FIG. 24 is a construction diagram of plasmid p399AK9.

FIG. 25 is a construction diagram of plasmid pCRDAPB.

FIG. 26 is a construction diagram of plasmid p399CAB and plasmid pCAB.

FIG. 27 is a construction diagram of plasmid pCABL.

FIG. 28 is a construction diagram of plasmid pHTN7150A.

FIG. 29 is a construction diagram of plasmid pCBLmc.

FIG. 30 is a construction diagram of plasmid pHTN7150.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI technical display location C12Q 1/68 9453-4B C12Q 1/68 A (C12N 15/09 ZNA C12R 1:13) (C12N 1/21 C12R 1:13) (C12N 1/21 C12R 1:19) (C12P 13/08 C12R 1:13) (C12N 9/12 C12R 1:13) (C12N 9/12 C12R 1:19) (72 ) Inventor Seiko Hirano 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Production Technology Laboratory (72) Inventor Atsushi Hayakawa 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Production Technology In the laboratory (72) Inventor Masako Izumi 1-1, Suzuki-cho, Kawasaki-ku, Kawasaki-shi, Kanagawa Ajinomoto Co., Inc. Production Technology Laboratory (72) Inventor Masakazu Sugimoto Suzuki, Kawasaki-ku, Kawasaki-shi, Kanagawa 1-1 in the original Co., Ltd., Institute of Industrial Science of taste

Claims (10)

[Claims] 1. An artificial transposon having a structure in which a drug resistance gene and a desired gene are sandwiched in an inverted repeat and capable of transposition in coryneform bacterial cells, and introducing the artificial transposon into coryneform bacterial cells. Then, the transposon is transferred onto the chromosome of the coryneform bacterium, and the desired gene is introduced into the chromosome and amplified. 2. The method according to claim 1, wherein the artificial transposon has a structure in which a transposase gene is sandwiched between inverted repeats. 3. The method according to claim 1 or 2, wherein the inverted repeat and transposase genes are derived from the insertion sequence of coryneform bacteria. 4. The method according to claim 3, wherein the insertion sequence has a base sequence represented by any one of SEQ ID NOS: 1, 5 and 9 in the sequence listing. 5. The method according to claim 1, wherein the drug resistance gene is a chloramphenicol resistance gene or a tetracycline resistance gene. 6. The method according to claim 1, wherein the desired gene is a gene involved in amino acid biosynthesis. 7. The method according to claim 6, wherein the desired gene is an aspartokinase gene and / or a dihydropicolinate synthase gene. 8. A coryneform bacterium in which a desired gene has been introduced into a chromosome by the method according to any one of claims 1 to 7. 9. A method of culturing a coryneform bacterium in which a gene involved in amino acid biosynthesis on a chromosome is introduced into a medium by the method according to claim 6, to collect and accumulate the amino acid in the medium, and to recover the amino acid. Characteristic amino acid production method. 10. The method according to claim 9, wherein the gene involved in amino acid biosynthesis is an aspartokinase gene and / or a dihydropicolinate synthase gene, and the amino acid is lysine.