The method of obtaining l-lysine (options) and recombinant dna used for its implementation (options)

 

(57) Abstract:

The invention relates to biotechnology and for the production of L-lysine. The method of obtaining L-lysine provides for the development of coryneform bacteria transformed with recombinant DNA (options), presented in the description in the form of the sequence listing. Recombinant DNA contains a) a DNA sequence encoding aspartokinase, in which the inhibition of the feedback type L-lysine and L-threonine essentially desensitized, and b) a DNA sequence encoding dihydrodipicolinate. Recombinant DNA autonomously replicate in cells coryneform bacteria. Growing the transformed bacteria is carried out in a medium suitable for producing and accumulating L-lysine in the culture of bacteria, and L-lysine are extracted from the culture. The use of the invention allows to improve L-isinprogress ability and growth rate of the producer. 8 S. p. f-crystals, 14 tab., table 1.

This invention relates to a method for producing L-lysine by cultivation of the microorganism obtained by modification coryneform bacteria used to ferment obtain amino acids, or so on, with the help of pic is as an additive to the feed, usually get the enzymatic method using L-lysine-producing mutant strain belonging to coryneform bacteria. Various currently known L-lysine-producing bacteria are bacteria that are created through artificial mutations from wild-type strains belonging to coryneform bacteria.

As for coryneform bacteria described vector plasmid, which autonomously replicated in bacterial cells and is a marker gene of resistance to the drug (see United States Patent No. 4514502), and the method of introducing the gene into bacterial cells (for example, Japanese Patent Laid-open No. 2-207791). Also described the possibility of breeding L-threonine or L-isoleucine-producing bacteria using the above-described method (see United States Patent No. 4452890 and 4442208). As for the breeding of L-lysine-producing bacteria, there is a method in which a gene involved in the biosynthesis of L-lysine, is introduced into a vector plasmid for amplification of this gene in bacterial cells (for example, Japanese Patent Laid-open No. 56-160997).

Known genes for the biosynthesis of L-lysine include, for example, gene dihydrodipicolinate (Japanese Patent Laid-open No. 7-75578) and gene diaminomaleonitrile (Japanese Patent Laid-open No. 60-87788), gene dihydrodipicolinate (Japanese Patent Laid-open No. 6-55149) and gene diaminomaleonitrile (Japanese Patent Laid-open No. 60-62994, in which gene amplification affect the productivity of L-lysine).

As for the enzymes involved in the biosynthesis of L-lysine, a known case for the enzyme, which is inhibited by the type of feedback used in the form of the wild type. In this case, the productivity of L-lysine is improved by introduction of the gene of the enzyme with this mutation, in which inhibition of the type of feedback desencibiliziruta. These known genes specifically includes, for example, gene aspartokinase (International Publication Pamphlet of WO 94/25605).

As described above, some successful results were obtained by amplification of genes for biosynthesis of L-lysine or the introduction of mutant genes. For example, coryneform bacterium, which holds a mutant gene aspartokinase with desensitized joint inhibition by lysine and threonine, produces a significant amount of L-lysine (approximately 25 g/l). However, this bacterium is suffering from reduced growth rate compared with the bacterium, not holding the mutant gene aspartokinase. It was also reported that produa aspartokinase (Applied and Environmental Microbiology. 57(6), 1746-1752 (1991)). However, this bacterium is suffering from an additional reduction in the rate of growth.

As for gene dihydrodipicolinate, it was shown that the activity dihydrodipicolinate increases in coryneform bacteria, which was introduced this gene, however, was not reported effects on the productivity of L-lysine (Japanese Patent Laid-open No. 7-75578).

Currently not aware of any case for coryneform bacteria, when someone has been a significant improvement in the yield of L-lysine without limitation of growth by combining multiple genes for the biosynthesis of L-lysine. Not reported a single case, which was also intended to improve growth by amplifying the gene for the biosynthesis of L-lysine.

Description of the invention

The purpose of this invention is to improve L-lysine-producing ability and growth rate coryneform bacteria through the use of genetic materials DNA sequences coding (each) aspartokinase (hereinafter referred to as "AK", provided that the gene encoding the protein of AK, hereinafter referred to as "lysC, if necessary), dihydrodipicolinate (hereinafter called "DDPR", provided that the gene encoding the protein DDPR, hereinafter referred to as the DDPS, hereinafter referred to as "dapA", if necessary), diaminomaleonitrile (hereinafter called "DDC", provided that the gene encoding the protein DDC, hereinafter referred to as lysA, if necessary), and diaminopimelate (hereinafter called "DDH", provided that the gene encoding the protein DDH, hereinafter referred to as "ddh, if necessary), which are important enzymes of the biosynthesis of L-lysine in the cell coryneform bacteria.

When the target substance enzymatic get by using a microorganism, the receiving rate and the yield of the target substance relative to the input material is extremely important. The target substance can be obtained very cheaply by increasing the rate of production per unit of enzymatic equipment. Therefore, in industrial scale, it is essential that the enzymatic release and the rate of production were compatible with each other. This invention offers a solution to the problems described above for enzyme for L-lysine using coryneform bacteria.

The principle of this invention is based on the fact that the growth coryneform bacteria can be improved and the rate of production of L-lysine can be improved by strengthening when obedie simply referred to as "AK mutant type", if you must), in which the joint inhibition by lysine and threonine is desensitized compared with the case where lysC amplified separately, and that the rate of production of L-lysine can be further improved step by step through the strengthening of dapA, lysA and ddh.

Namely, this invention is based on recombinant DNA autonomously replicable in cells coryneform bacteria containing the DNA sequence encoding aspartokinase for which inhibition of the type of feedback being desensitized, and the DNA sequence encoding dihydrodipicolinate. This invention provides recombinant DNA, optionally containing a DNA sequence encoding dihydrodipicolinate, in addition to each of the above-described DNA sequences. This invention provides recombinant DNA, optionally containing a DNA sequence encoding diaminopimelate, in addition to the three above described DNA sequences. This invention provides recombinant DNA, optionally containing a DNA sequence encoding diaminopimelate, in addition to the four op is rmow bacterium, holding aspartokinase, in which the inhibition of the feedback type L-lysine and L-threonine is substantially desensitized, and containing amplified DNA encoding dihydrodipicolinate. This invention provides coryneform bacterium, optionally containing amplified DNA encoding dihydropyrimidinase in the above coryneform bacteria. This invention provides coryneform bacterium, optionally containing amplified DNA encoding diaminopimelate in the above coryneform bacteria, in addition to the three above-described DNA. This invention provides coryneform bacterium, optionally containing amplified DNA encoding diaminopimelate in the above coryneform bacteria, in addition to the four above-described DNA.

In another embodiment, this invention provides a method of obtaining L-lysine, providing the stage of culturing any of the above coryneform bacteria in a suitable medium, producing and accumulating L-lysine in the culture of this bacterium, and collecting L-lysine from the culture.

Coryneform bacteria, referred to in this invention, are a group of Linyi sticks, not having acid resistance and spore-forming ability. These coryneform bacteria include bacteria belonging to the genus Corinebacterium, bacteria belonging to the genus Brevibacterium, classified up to the present time in the genus Brevibacterium, but United now in the genus Corinebacterium, and bacteria belonging to the genus Brevibacterium, closely related bacteria belonging to the genus Corinebacterium.

This invention will be explained in detail below.

<1> Obtaining genes for the biosynthesis of L-lysine used in this invention

Genes for the biosynthesis of L-lysine used in this invention, are respectively obtaining chromosomal DNA from bacteria as donor DNA, constructing a library of chromosomal DNA using plasmid vector or etc., selection of the strain of holding the desired gene, and retrieval of the selected strain of recombinant DNA, which was inserted into the gene. The donor DNA for the gene for the biosynthesis of L-lysine used in this invention is not specifically limited, provided that the desired gene for biosynthesis of L-lysine expresses the enzyme protein that functions in the cells coryneform bacteria. However, the donor Dvornik bacteria, have a known sequence. Therefore, they can be obtained by carrying out the amplification in accordance with the method of polymerase chain reaction (PCR; see White, T. J. et al. Trends Genet.. 5, 185 (1989)).

Each of the genes for the biosynthesis of L-lysine used in this invention can be obtained in accordance with defined methods described in the examples below.

(1) Obtaining a mutant lysC

The DNA fragment containing mutant lysC, can be obtained from the mutant strain, in which the synergistic inhibition by the type of feedback activity AK L-lysine and L-threonine is substantially desensitized (International Publication Pamphlet of WO 94/25605). This mutant strain can be obtained, for example, from a group of cells derived from wild-type strain coryneform bacteria subjected to mutation treatment by application to a conventional mutation treatment, such as irradiation with ultraviolet light and processing mutating agent such as N-methyl-N'-nitro-N-nitrosoguanidine. The activity of AK can be measured using the method described Miyajima, R. et al. in The Journal of Biochemistry(1968), 63(2), 139-148. The most preferred mutant strain is producing L-lysine bacterium AJ3445 ( present name Corinebacterium glutamicum).

Alternatively, mutant lysC can also be obtained mutation treatment in vitro plasmid DNA containing the wild-type lysC. In another aspect of known specific information about mutations for desensitization synergistic inhibition by the type of feedback AK L-lysine and L-threonine ((International Publication Pamphlet of WO 94/25605). Therefore, mutant lysC can also be obtained from the wild-type lysC on the basis of this information, for example, by way of site-specific mutagenesis.

The fragment containing the lysC, can be isolated from coryneform bacteria by obtaining chromosomal DNA in accordance with the method of Saito and Miura (H. Saito and K. Miura, Biochim. Biophys. Acta,72,619 (1963) ), and amplification of lysC in accordance with the method of polymerase chain reaction (PCR; see White, T. J. et al., Trends Genet., 5, 185 (1989)).

Examples of DNA primers are single-stranded DNA 23-Mer and 21-Mer having the nucleotide sequence shown in SEQ ID 1 and SEQ ID 2 in the List of sequences for amplification, for example, district approximately 1643 p. N., lysC coding, on the basis of sequence, known for Corinebacterium glutamicum (see Molecular Microbiology(1991), 5 (5) 1197-1204; Mol. Gen. Genet. (1990), 224, 317-324). DNA can be synthesized in accordance with the conventional method in which may be carried out using the DNA thermal cycler model PJ2000, produced by Takara Shuzo, and using DNA polymerase Taq according to the method prescribed by the provider.

Preferably, lysC, amplificatory using PCR, was Legerova with vector DNA autonomously replicable in cells of E. coli and/or coryneform bacteria, for the production of recombinant DNA, and the recombinant DNA is introduced into E. coli cells in advance. This facilitates subsequent operations. A vector autonomously replicable in cells of E. coli, is preferably a plasmid vector, which preferably autonomously replicate in host cells, including, for example, pUC19, pUC18, pBR322, pHSG299, pHSG399, pHSG398, and RSF 1010.

When the DNA fragment, which can allow the Autonomous plasmids to replicate in coryneform bacteria introduced into these vectors, they can be used as a so-called Shuttle vector autonomously replicable in E. coli and in coryneform bacteria.

Such Shuttle vectors are vectors. Microorganisms that holds each of the vectors, and the number of deposits in the international media for deposition are shown in parentheses.

RNs: Escherichia coli AJ12617 (FERM BP-3532)

pAJ655: Escherichia coii AJ11882 (FERM BP-136)

Corinebacterium glutamicum SR8201 (ATCC 39135)

pAJ440: Bacillus subtilis AJ11901 (FERM BP-140)

These vectors can be obtained from the deposited microorganisms in the following way. Cells collected at the logarithmic growth phase, were literally using lysozyme and LTOs with subsequent separation from the lysate by centrifugation at 30,000 g to obtain a supernatant, which was added to the glycol and then fractionally and purified by centrifugation to equilibrium density gradient of cesium chloride/ethidium bromide.

E. coli can be transformed by the introduction of plasmids for example, according to the method of D. M. Morrison (Methods in Enzymology, 68, 326 (1979)) or the way in which recipient cells are treated with calcium chloride to increase permeability for DNA (Mandel, M. and Higa, A., J. Mol. Biol., 53, 159 ( 1970)).

Wild-type LysC get in the allocation of lysC containing AK strain wild type, whereas lysC separated from containing AK mutant strain in accordance with the method described above.

An example of the nucleotide sequence of the DNA fragment containing the lysC shown in SEQ ID 3 in the List of sequences. Amino acid sequence-subunit protein AK wild-type decoded from the nucleotide sequence shown in SEQ ID 4 in the pic is 5. Amino acid sequence-subunit protein AK wild-type decoded from the nucleotide sequence of the DNA shown in SEQ ID 6 in the List of sequences with DNA. Only the amino acid sequence shown in SEQ ID 7. In each of these subunits GTG used as the initiator codon and the corresponding amino acid is represented by methionine. However, this representation refers to methionine, valine or formylmethionine.

Mutant lysC used in this invention are not particularly limited, provided that it encodes AK, for which a synergistic inhibition of the feedback type L-lysine and L-threonine is desensitized. However, mutant lysC presents for example a gene having a mutation in which 279-th residue of alanine in counting from N-Terminus is replaced by the amino acid residue different from alanine and non-acidic amino acid, in the subunit, and the 30-th residue is replaced by alanine amino acid residue different from alanine and non-acidic amino acid-subunit in the amino acid sequence AK wild type. Amino acid sequence AK wild type includes the amino acid sequence is required, shown in SEQ ID 7 in the List of sequences, as-subunit.

Amino acid residues other than alanine, and other than acidic amino acid, are preferably residues of threonine, arginine, cysteine, phenylalanine, Proline, serine, tyrosine and valine.

The codon corresponding to the substituted amino acid residue is not particularly limited with respect to its type, provided that it encodes that amino acid residue. It is assumed that the amino acid sequence available AK wild-type and may be slightly different depending on differences in bacterial species and bacterial strains. AK that have the mutation, based on, for example, by replacement, deletion or insertion of one or several amino acid residues in one or more positions that do not have values for the activity of the enzyme described above may also be used for this invention. Other AK that have the mutation, based on, for example, by replacement, deletion or insertion of one or more other amino acid residues, can also be used provided that they do not have a significant impact on the activity of AK and desensitization of the synergistic ingibiruet with mutant lysC in strain AJ12036 (FERM BP-734) as the wild-type strain Brevibacterium lactofermentum, was deposited on 10 April 1992 under the number FERM P-12918 in National Institute of Bioscience and Human of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan), transferred to international deposition based on Budapest Treaty on 10 February 1995 and deposited under the number FERM BP-4999.

(2) Obtaining dapB

The DNA fragment containing dapB, can be obtained from the chromosome coryneform bacteria using PCR. The donor DNA is not specifically limited, however, in the example of the strain Brevibacterium lactofermentum ATCC 13869.

The DNA sequence encoding DDPR, known for Brevibacterium lactofermentum (Journal of Bacteriology, 175 (9), 2743-2749 (1993)), on the basis of this DNA sequence can be obtained from the DNA primers for PCR. Examples of such primers DNA are DNA 23-mers, respectively having the nucleotide sequence shown in SEQ ID 8 and SEQ ID 9 in the List of sequences. DNA synthesis, PCR, preparation of plasmid containing the received dapB, can be performed as described above for lysC.

The nucleotide sequence of the DNA fragment containing dapB, and amino acid sequence, which is decoded from this nucleotide sequence are presented in amino acid sequence, this invention is equivalent to use the DNA fragments encoding amino acid sequences, are basically the same as shown in SEQ ID 11, namely amino acid sequence having a mutation, for example, by replacement, deletion or insertion of one or several amino acids, provided that there is no significant effect on the activity of the DDPR.

The transformed strain J13107, obtained by introducing the plasmid pCRDAPB containing dapB obtained in the Example described below, the strain E. coli JM109 was deposited with international may 26, 1995 under the Deposit number FERM BP-5114 at the National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on the basis of the Budapest Treaty.

(3) Obtaining dapA

The DNA fragment containing dapA, can be obtained from the chromosome coryneform bacteria using PCR. The donor DNA is not particularly limited, but an example of a strain of Brevibacterium lactofermentum ATCC 13869.

The DNA sequence encoding DDPS, known for Corinebacterium glutamicum (see Nucleic Acids Research. 18 (21), 6421 (1990), EMBL accession No. X53993), on the basis of this sequence can be prepared DNA primers for PCR. Specific premaratne in SEQ ID 12, and SEQ ID 13 List of sequences. DNA synthesis, PCR, preparation of plasmid containing dapA, can be performed as described for lysC above.

The nucleotide sequence of the DNA fragment containing dapA and amino acid sequence, which is decoded from this nucleotide sequence shown as examples in SEQ ID 14. Only the amino acid sequence shown in SEQ ID 15. In addition to DNA fragments encoding this amino acid sequence, this invention is equivalent to use the DNA fragments encoding amino acid sequences, are basically the same as the amino acid sequence shown in SEQ ID 15, namely amino acid sequence having a mutation, based on, for example, to substitute, division or insertions of one or several amino acids, provided that there is no significant effect on the activity of the DDPS.

The transformed strain J13106, obtained by introducing the plasmid pCRDAPA containing dapA obtained in the Example described below, the strain E. coli JM 109 was deposited from may 26, 1995 under the Deposit number FERM BP-5113 in National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on otorinolaringol bacteria using PCR. The donor DNA is not particularly limited, but an example of a strain of Brevibacterium lactofermentum ATCC 13869.

In coryneform bacteria lysA forms an operon together with argS (genome arginyl-tRNA-synthase) and lysA is in the course of transcription from argS. Expression of lysA is regulated by a promoter located against the course of transcription from argS (see Journal of Bacteriology, Nov., 7356-7362 (1993)). DNA sequences of these genes are known to Corinebacterium glutamicum (see Molecular Microbiology, 4(11), 1819-1830 (1990); Molecular and General Genetics, 212, 112-119 (1988)), on the basis of these sequences can be prepared DNA primers for PCR. These DNA primers are given as specific examples in the form of a 23-Mer DNA, respectively having the nucleotide sequence shown in SEQ ID 16 in the List of sequences (corresponding to nucleotides 11-33 in the nucleotide sequence described in Molecular Microbiology, 4(11), 1819-1830 (1990)), and SEQ ID 17 (corresponding to nucleotides 1370-1392 in the nucleotide sequence described in Molecular and General Genetics, 212, 112-119 (1988)). DNA synthesis, PCR, preparation of plasmid containing the received lysA, can be performed as described above for lysC.

In the Example described later, the DNA fragment containing the promoter, argS and lysA, isparent DNA where lysA Legerova directly in the course of transcription from the promoter.

The nucleotide sequence of the DNA fragment containing the argS and lysA, and amino acid sequence, which is decoded from this nucleotide sequence shown as examples in SEQ ID 18. An example of the amino acid sequence encoded argS, shown in SEQ ID 19, and an example of the amino acid sequence encoded by lysA shown in SEQ ID 20. In addition to DNA fragments encoding this amino acid sequence, this invention is equivalent to use the DNA fragments encoding amino acid sequences, are basically the same as the amino acid sequence shown in SEQ ID 20, namely amino acid sequence having a mutation, based on, for example, to substitute, division or insertions of one or several amino acids, provided that there is no significant effect on the activity of DDC.

(5) Obtaining ddh

The DNA fragment containing ddh can be obtained from a chromosome coryneform bacteria using PCR. The donor DNA is not particularly limited, but an example of a strain of Brevibacterium lactofermentum ATCC 13869.

Gene DDH known for Corinebacterium glutamicum (ecificatio examples of such primers are given in the form of DNA 20-Mer, respectively having the nucleotide sequence shown in SEQ ID 21 and SEQ ID 22 in the List of sequences. DNA synthesis, PCR, preparation of plasmid containing the received ddh can be performed as described above for lysC.

The nucleotide sequence of the DNA fragment containing ddh, and amino acid sequence, which is decoded from this nucleotide sequence shown as examples in SEQ ID 23. Only the amino acid sequence shown in SEQ ID 24. In addition to DNA fragments encoding this amino acid sequence, this invention is equivalent to use the DNA fragments encoding amino acid sequences, are basically the same as the amino acid sequence shown in SEQ ID 24, namely amino acid sequence having a mutation, based on, for example, to substitute, division or insertions of one or several amino acids, provided that there is no significant effect on the activity of DDH.

<2> Recombinant DNA and coryneform bacterium of the present invention

Coryneform bacterium of the present invention holds aspartokinase (mutant AK), whose inhibition by type obligationto, strengthened. In a preferred embodiment, coryneform bacterium of the present invention is coryneform bacterium, in which DNA (dapA), encoding dihydrodipicolinate, further strengthened. In a more preferred embodiment, coryneform bacterium of the present invention is coryneform bacterium, in which DNA (lysA), encoding diaminopimelate, further strengthened. In a more preferred embodiment, coryneform bacterium of the present invention is coryneform bacterium, in which DNA (ddh), encoding diaminopimelate, further strengthened.

The term "enhanced" DNA refers here to the fact that the intracellular activity of the enzyme encoded by this DNA is increased, for example, by increasing the number of copies of the gene, using a strong promoter, using a gene encoding an enzyme with high specific activity, or a combination of these means.

Coryneform bacteria, retaining mutant AK, can be coryneform bacteria that produce mutant aspartokinase as a result of mutations, or coryneform bacteria that are transformed by the introduction of a mutant for example, following producing lysine wild-type strains:

Corinebacterium acetoacidophilum renowned 13870;

Corinebacterium acetoglutamicum ATCC 15806;

Corinebacterium callunae ATCC 15991;

Corinebacterium glutamicum ATCC 13032;

(Brevibacterium divaricatum ATCC 14020;

(Brevibacterium lactofermentum ATCC 13869;

(Corinebacterium lilium ATCC 15990;

(Brevibacterium flavum ATCC 14067;

Corinebacterium melassecola ATCC 17965;

Brevibacterium saccharolyticum ATCC 14066;

Brevibacterium immariophilum ATCC 14068;

Brevibacterium roseum ATCC 13825;

Brevibacterium thiogenitalis ATCC 19240;

Microbacterium ammoniaphilum ATCC 15354;

Corinebacterium thermoaminogenes AJ12340 (FERM BP-1539).

Other bacterial strains, except as described here, is used as the host, are, for example, mutant strains capable of producing L-lysine produced from the above-mentioned strains. Such artificial mutant strains include S-(2-amino-ethyl)-cysteine (hereinafter abbreviated as "AES")-resistant mutant strains (Brevibacterium lactofermentum AJ11082 (NRRL B-1147), Japanese Patent Publication Nos. 56-1914, 56-1915, 57-14157, 57-14158, 57-30474, 58-10075, 59-4993, 62-35840, 62-24074, 62-36673, 5-11958, 7-112437 and 7-112438); mutant strains that require amino acids such as L-homoserine, for their growth (Japanese Patent Publication Nos. 48-28078 and 56-6499), mutant strains that are resistant to AES and require amino acids such as L-leucine, L-homoserine, L-Proline, L-serine, L-arginine, L-alanine and L-is caprolactam, -aminophenylacetate, the analog of aspartate, sulfa-lekarstvennym means Hinode and N-eurolatin; L-lysine-producing mutant strains which are resistant to inhibitors okelatutulabiviz or enzymes of the respiratory system (Japanese Patent Laid-open Nos. 50-53588, 50-31093, 52-102498, 53-9394, 53-86089, 55-9783, 55-9759, 56-32995 and 56-39778, and Japanese Patent Publication Nos. 53-43591 and 53-1833); L-lysine-producing mutant strains which require Inositol or acetic acid (Japanese Patent Laid-open Nos. 55-9784 and 56-8692); L-lysine-producing mutant strains which exhibit sensitivity to ftorirovannogo acid or a temperature of not less than 34o(Japanese Patent Laid-open Nos. 55-9783 and 53-86090); and L-lysine-producing mutant strains belonging to the genus Brevibacterium or Corinebacterium which are resistant to glycol and produce L-lysine (United States Patent No. 4411997).

In a typical embodiment, for amplification of genes for the biosynthesis of L-lysine in the host, as described above, the genes introduced into the host using plasmid vectors, transposon or phage vector or similar When the introduction is expected to strengthen to some extent even when using a vector with a low kopiosto. However, it is preferable to use a type of vector with high mapinstance, made from coryneform bacteria described in International Publication of Pamphlets WO02/02627 and WO93/18151, European Patent Publication No. 445385, Japanese Patent Laid-open No. 6-46867, Vertes, A. A. et al., Mol. Environ., 11, 739-746 (1994), Bonamy, C., et al., Mol. Environ. , 14, 571-581 (1994), Vertes, A. A. et al., Mol. Gen. Genet,, 245, 397-405 (1994), Jagar, W. et al. , FEMS Microbiology Letters, 126, 1-6 (1995), Japanese Patent Laid-open No. 7-107976, Japanese Patent Laid-open No. 7-327680 etc.

The present invention is not required to mutant lysC was definitely enhanced. You can use strains that have mutations in lysC on the chromosomal DNA, or in which the mutant lysC included in the chromosomal DNA. Alternatively, mutant lysC can be entered using a plasmid vector. On the other hand, dapA, dapB, lysA and ddh preferably amplified for the efficient production of L-lysine.

Each gene lysC, dapA, dapB, lysA and ddh can be successfully introduced into the host by using respectively different vectors. Alternatively, two, three, four or five of these types of genes can be introduced together with a single vector. Using different vectors, genes can be entered in any order, however it is preferable to use vectors that have a stable exchange mechanism and trapping in the host and are able to coexist with each drugery dapB, get, for example, the introduction of coryneform bacterium-host a recombinant DNA containing mutant lysC and dapB, autonomously replicable in cells coryneform bacteria.

Coryneform bacterium, optionally containing dapA, except mutant lysC and dapB receive, for example, the introduction of coryneform bacterium-host a recombinant DNA containing mutant lysC, dapB and dapA, autonomously replicable in cells coryneform bacteria.

Coryneform bacterium, optionally containing mutant lysC, dapB and dapA receive, for example, the introduction of coryneform bacterium-host a recombinant DNA containing lysC, dapB, dapA and lysA, autonomously replicable in cells coryneform bacteria.

Coryneform bacterium, optionally containing reinforced ddh, except mutant lysC, dapB, dapA and lysA receive, for example, the introduction of coryneform bacterium-host a recombinant DNA containing lysC, dapB, dapA, lysA and ddh, autonomously replicable in cells coryneform bacteria.

The above-mentioned recombinant DNA can be obtained, for example, by embedding each of the genes involved in the biosynthesis of L-lysine, in a vector, such as plasmid vectors, transposon or phage vector, as described by according to the electric pulse method ( Sugimoto et al. , Japanese Patent Laidopen No. 2-207791). Amplification of the gene using transposon can be accomplished by introduction of a plasmid carrying the transposon in cell host and inducing transposition of the transposon.

<3> Method of obtaining L-lysine

L-lysine can be produced efficiently by culturing in a suitable medium coryneform bacteria containing amplified genes for the biosynthesis of L-lysine as described above, producing and accumulating L-lysine in the culture of this bacterium, and collecting L-lysine from the culture.

The example used environment is an ordinary medium containing a carbon source, a nitrogen source, inorganic ions, and optionally other organic components.

As a carbon source, it is possible to use sugars such as glucose, fructose, sucrose, molasses and starch hydrolysis; and organic acids such as fumaric acid, citric acid and succinic acid.

As the nitrogen source can be used inorganic ammonium salts such as ammonium sulfate, ammonium chloride and ammonium phosphate, organic nitrogen, such as hydrolyzed soy; ammonia gas; and aqueous ammonia.

As nutrients give is such as vitamin B1and L-homoserine or yeast extract or etc., also add, if you want, potassium phosphate, magnesium sulfate, iron ion, manganese ion, etc.

The cultivation is preferably carried out under aerobic conditions for about 30-90 hours. The cultivation temperature is preferably regulated at 25-37oC, and the pH is preferably adjustable from 5 to 8 during cultivation. To bring the pH it is possible to use inorganic or organic, acidic or alkaline substances or gaseous ammonia or similar L-lysine can be collected from the culture by combining the usual way with ion exchange resins and other known methods.

A brief description of the drawings.

Fig. 1 illustrates the process of constructing plasmids p399AKYB and raw containing mutant lysC.

Fig. 2 illustrates the process of constructing plasmids pDPRB containing dapB and Brevi.-ori. (the replication origin, origin).

Fig. 3 illustrates the process of constructing plasmids pDPSB containing dapA and Brevi.-ori.

Fig.4 illustrates the process of constructing plasmids p299LYSA containing lysA.

Fig. 5 illustrates the process of constructing plasmids pLYSAB containing lysA and Brevi.-ori.

Fig. 8 illustrates the process of constructing the plasmid RSV containing mutant lysC, dapB and Brevi.-ori.

Fig. 9 illustrates the process of constructing plasmids Rav containing dapA, dapB and Brevi.-ori.

Fig. 10 illustrates the process of constructing plasmids p399DL containing ddh and lysA.

Fig. 11 illustrates the process of constructing the plasmid pDL containing ddh, lysA and Brevi.-ori.

Fig. 12 illustrates the process of constructing the plasmids RSV containing mutant lysC, dapA, dapB and Brevi.-ori.

Fig. 13 illustrates the process of constructing plasmids pCABL containing mutant lysC, dapA, dapB, lysA and Brevi.-ori.

Fig. 14 illustrates the process of constructing plasmids pCABDL containing mutant lysC, dapA, dapB, ddh, lysA and Brevi.-ori.

Description of the preferred options

The invention will be more specifically explained below with reference to Examples.

Example 1: Obtaining gene lysC wild-type and mutant lysC gene from Brevibacterium lactofermentum

<1> Getting lysC wild-type and mutant lysC and receipt containing plasmids

As a donor chromosomal DNA used a strain of Brevibacterium lactofermentum ATCC 13869 and L-lysine-producing mutant strain AJ34445 (FERM P-1944), the EN with the advent of significant desensitization from joint inhibition by lysine and threonine (Journal of Biochemistry, 68, 701-710 (1970)).

The DNA fragment containing the lysC, amplified from chromosomal DNA in accordance with the method of PCR (polymerase chain reaction; refer to White, T. J. et al. Trends Genet., 5, 185 (1989)). As for the DNA primers used for amplification, single-stranded DNA 23-Mer and 21-Mer having the nucleotide sequence shown in SEQ ID 1 and SEQ ID 2, was synthesized to amplify a region of approximately 1643 p. N. for lysC-based sequence, known for Corinebacterium glutamicum (see Molecular Microbiology(1991), 5(5), 1197-1204; and Mol. Gen. Genet. (1990), 224, 317-324). DNA was synthesized according to an ordinary method using DNA synthesizer model 380 manufactured by Applied Biosystems, and using phospholidine method (see Tetrahegron Letters(1981), 22, 1859).

Gene is amplified using PCR using a DNA thermal cycler model PJ2000 produced by Takara Shuzo, and using DNA polymerase Taq according to the method recommended by the supplier. Amplificatory fragment of the gene 1643, etc. ad was confirmed by agarose gel electrophoresis. After that, the piece cut from the gel, purified according to an ordinary method and were digested his restrictase Nrul (produced by Takara Shuzo) and EcoRI (manufactured by Takara Shuzo).

pHSG399 (see Takeshita, S. et al., Gene, (1987), 62, 63-74) COI is Takara Shuzo) and EcoRI (manufactured by Takara Shuzo) and ligated with the amplified fragment lysC. DNA is ligated by means of a set for ligating DNA (manufactured by Takara Shuzo) in accordance with the recommended method. So were the resulting plasmid, in which fragments of lysC, amplificatoare of the chromosomes Brevibacterium lactofermentum, were legirovanyh with pHSG399, respectively. A plasmid containing the lysC from ATS 13869 (wild-type strain), was designated as p399AKY, and a plasmid containing lysC from AJ3463 (L-lysine-producing bacteria) was designated as RAC.

The DNA fragment (hereinafter called "Brevi.-ori."), having the ability to do a plasmid autonomously replicable in bacteria belonging to the genus Corinebacterium, was introduced in p399AKY and RAC, respectively, to obtain the plasmid carrying lysC, autonomously replicable in bacteria belonging to the genus Corinebacterium. Brevi.-ori. was obtained from the plasmid vector RNC containing Brevi. -ori. and autonomously replicable in cells such as Escherichia coli, and bacteria belonging to the genus Corinebacterium. RNC designed splitting RNs using Kpnl (produced by Takara Shuzo) and Bam HI (produced by Takara Shuzo), extraction of the fragment Brevi.-ori. and legirovaniem it with pHSG298, also split Kpnl and BamHI (see Japanese Patent Laid-open No. 5-7491). RNC gives the host resistance to kanamycin. Escherichia coli, holding RNC, identified as Escherichia d Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan).

pHK4 was digested with restrictase Kpnl and BamHI and the split edge was a small mistake. The formation of blunt ends was performed with a set for blunting DNA (manufactured by Takara Shuzo) in accordance with the proposed method. After the formation of blunt ends phosphorylated BamHI linker (produced by Takara Shuzo) ligated to obtain modifications to the DNA fragment corresponding to Brevi.-ori.-part of that could be cut from pHK4 splitting only using BamHI. This plasmid was digested BamHI and the resulting DNA fragment Brevi.-ori. ligated with p399AKY and RAC, also already split BamHI, respectively, to obtain plasmids, each of which contains the gene lysC, autonomously replicable in bacteria belonging to the genus Corinebacterium.

A plasmid containing the gene for wild-type lysC originating from p399AKY, was designated as p399AKYB, and a plasmid containing mutant lysC gene, originating from RAC, was designated as raw. The process of designing raw and p399AKYB shown in Fig. 1. Strain AJ12691, obtained by introducing the plasmid raw with mutant lysC in the wild-type strain

Brevibacterium lactofermentum (strain AJ12036, FERM BP-734), was deposited on 10 April 1992 under the Deposit number FERM P-12918 in National Institute of Bioscience and Human in an international Deposit based on Budapest Treaty on 10 February 1995 under the Deposit number FERM BP-4999.

<2> determination of the nucleotide sequences lysC wild-type and mutant lysC from Brevibacterium lactofermentum

Plasmid p399AKY containing lysC wild-type and plasmid RAC containing mutant lysC, received from the respective transformants to determine the nucleotide sequences lysC wild-type and mutant lysC. Determination of the nucleotide sequences was performed according to the method of Sanger et al. (for example, F. Sanger et al., Proc. Natl. Acad. Sci., 74, 5463 (1977)).

The nucleotide sequence of wild-type lysC encoded p399AKY shown in SEQ ID 3 in the List of sequences. On the other hand, the nucleotide sequence of mutant lysC, encoded RAC, had only the mutation of a single nucleotide, i.e., 1051 initial G was replaced by And in SEQ ID 3 compared with wild-type lysC. It is known that lysC Corinebacterium glutamicum has two subunits (,), encoded in the same reading frame identical to the DNA chain (see Kalinowski, J. et al., Molecular Microbiology(1991), 5(5), 1197-1204). Based on the assessment of homology, it is assumed that sequenced here gene also has two subunits (,), encoded in the same reading frame identical to the DNA chain.

Amino acid sequence-subunit protein AK wild type, which is decoded from nuclearblast shown in SEQ ID 5.

Amino acid sequence-subunit protein AK wild type, which is decoded from the nucleotide sequence of the DNA shown in SEQ ID 6 together with the DNA sequence. Only the amino acid sequence shown in SEQ ID 7. In each of the subunits GTG is used as the initiator codon, and the corresponding amino acid is represented by methionine. However, this representation refers to methionine, valine or formylmethionine.

On the other hand, a mutation in a sequence of mutant lysC means the appearance of the replacement amino acid residue, so 279-th residue alanine-subunit is replaced by a threonine residue, and the 30-th residue alanine-subunit is replaced by a threonine residue in the amino acid sequences AK wild-type (SEQ ID 5, SEQ ID 7).

Example 2: Getting dapB from Brevibacterium lactofermentum

<1> Getting dapB and construction of plasmids containing dapB

As a donor chromosomal DNA used the wild-type strain Brevibacterium lactofermentum ATCC 13869. Chromosomal DNA was obtained from strain ATCC 13869 according to a conventional method. The DNA fragment containing dapB, amplified from chromosomal DNA using PCR. As DNA primers used for amplification, ciavarella, respectively, for amplification of the district is approximately 2.0 T. p. N. encoding DDPR, on the basis of sequence, known for Brevibacterium lactofermentum (see Journal of Bacteriology, 157(9), 2743-2749 (1993)). DNA synthesis, PCR was performed as described in Example 1. pCR-Script (produced by Invitrogen) was used as cloning vector for the amplified fragment of the gene 2001 p. N., ligated with the amplified fragment dapB. Thus constructed plasmid, in which a fragment of dapB 2001 p. N. , amplificatory from the chromosome of Brevibacterium lactofermentum, ligated with pCR-Script. The plasmid obtained as described above, which had dapB originating from ATCC 13869 was identified as pCRDAPB. Transformatsii strain AJ13107 obtained by the introduction pCRDAPB in E. coli strain JM109 was internationally deposited from may 26, 1995 under the Deposit number FERM BP-5114 at the National Institute of Bioscience and Human Technology of Agency of Industrial Science and Technology of Ministry of International Trade and Industry (postal code: 305, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on the basis of the Budapest Treaty.

Fragment 1101 p. N., containing the structural gene DDPR, were extracted by splitting pCRDAPB EcoRI and SphI. This fragment is ligated with pHSG399, split HincII and SphI to obtain plasmids. The obtained plasmid was designated as p399DPR.

Brevi. -ori. introduced in the received p399DPR for constructing plasmids carrying dapB, Autonomous replic who was a small mistake. The formation of blunt ends was performed using the kit for blunting DNA (manufactured by Takara Shuzo) according to the proposed method. After the formation of blunt ends phosphorylated BamHI linker (produced by Takara Shuzo) ligated to obtain modifications to the DNA fragment corresponding to Brevi. -ori. -parts that could be cut from RNC splitting only BamHI. This plasmid was digested BamHI and the resulting DNA fragment Brevi. -ori. ligated with p399DPR also split BamHI, to obtain a plasmid containing dapB, Autonomous replicated in coryneform bacteria. The obtained plasmid was designated as pDPRB. The process of designing pDPRB shown in Fig.2.

<2> determination of the nucleotide sequence of the dapB from Brevibacterium lactofermentum

Plasmid DNA was obtained from a strain J13107 holding p399DPR, and its nucleotide sequence was determined as described in Example 1. A specific nucleotide sequence and decoded from her amino acid sequence shown in SEQ ID 10. Only the amino acid sequence shown in SEQ ID 11.

Example 3: Getting dapA from Brevibacterium lactofermentum

<1> Getting dapA and construction of plasmid containing dapA

Strain wild type Brevibac accordance with a customary method. The DNA fragment containing dapA, amplified from chromosomal DNA using PCR. As DNA primers used for amplification were synthesized accordingly DNA 20-Mer having the nucleotide sequence shown in SEQ ID 12, and SEQ ID 13 in the List of sequences for the amplification of the district of approximately 1.5 T. p. N., encoding DDPS, on the basis of sequence, known for Corinebacterium. glutamicum (see Nucleic Acids Research, 18(21), 6421 (1990); EMBL accession No. X53993). DNA synthesis and PCR were performed as described in Example 1. pCR1000 (produced by Invitrogen, see Bio/Technology, 9, 657-663 (1991)) was used as cloning vector for the amplified fragment of the gene 1411 p. N., ligated with the amplified fragment dapA. Ligation of DNA was performed using the kit for ligating DNA (manufactured by Takara Shuzo) according to the recommended method. It was constructed a plasmid in which the fragment dapA 1411 p. N., amplificatory from the chromosome of Brevibacterium lactofermentum was Legerova with pCR1000. The plasmid obtained as described here, with dapA, originating from ATS 13869, was designated as pCRDAPA.

The transformed strain AJ3106 obtained by the introduction pCRDAPA in the E. coli strain was internationally deposited on may 26, 1995 under Deposit n, igashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan) on the basis of the Budapest Treaty.

Brevi. -ori. introduced in the received pCRDAPA for constructing plasmids carrying dapA, Autonomous replicated in coryneform bacteria. RNC were digested with restrictase CRP and BamHI (produced by Takara Shuzo) and split the edge was a small mistake. The formation of blunt ends was performed using the kit for blunting DNA (manufactured by Takara Shuzo) according to the proposed method. After the formation of blunt ends phosphorylated linker Smal (produced by Takara Shuzo) ligated to obtain modifications to the DNA fragment corresponding to Brevi.-ori.-part of that could be cut from RNC splitting only Smal. This plasmid was digested Smal and the obtained DNA fragment Brevi.-ori. ligated with pCRDAPA also split Smal, to obtain a plasmid containing dapA, Autonomous replicated in coryneform bacteria. This plasmid was named pDPSB. The process of designing pDPSB(Kmr) shown in Fig.3.

<2> determination of the nucleotide sequence of the dapA from Brevibacterium lactofermentum

Plasmid DNA was obtained from a strain J13106 carrying pCRDAPA, and its nucleotide sequence was determined as described in Example 1. A specific nucleotide sequence and decoded from the Q ID 15.

Example 4: Obtaining lysA from Brevibacterium lactofermentum

<1> Getting lysA and construction of plasmids containing lysA

The wild-type strain Brevibacterium lactofermentum ATCC 13869 was used as donor chromosomal DNA. Chromosomal DNA was obtained from strain ATCC 13869 in accordance with a customary method. The DNA fragment containing argS, lysA and the promoter of the operon containing them, amplified from chromosomal DNA in accordance with the PCR. As DNA primers used for amplification were used synthetic DNA 23-Mer having the nucleotide sequence shown in SEQ ID 16 and SEQ ID 17 in the List of sequences, respectively, for amplification of the district is approximately 3.6, etc. as called for arginyl-t-RNA synthase and DDC, on the basis of sequence, known for Corinebacterium glutamicum (see Molecular Microbiology, 4(11), 1819-1830 (1990); Molecular and General Genetics, 212, 112-119 (1988)). DNA synthesis and PCR were performed as described in Example 1. pHSG399 was used as cloning vector for amplification of a fragment of the gene 3579 p. N. pHSG399 was digested with restriction enzyme Smal (produced by Takara Shuzo), which is ligated with the DNA fragment containing amplificatory lysA. The plasmid obtained as described above, which had lysA originating from ATCC 13869 was identified as p399LYSA.

led by Takara Shuzo). This DNA fragment is ligated with pHSG299, split and kpni restriction sites BamHI. The obtained plasmid was designated as p299LYSA. The process of designing p299LYSA shown in Fig.4.

Brevi. -ori. introduced in the received p299LYSA for constructing plasmids carrying lysA, Autonomous replicated in coryneform bacteria. RNC were digested with restrictase and kpni restriction sites BamHI and split edge was a small mistake. The formation of blunt ends was performed using the kit for blunting DNA (manufactured by Takara Shuzo) according to the proposed method. After the formation of blunt ends phosphorylated linker Cloned (produced by Takara Shuzo) ligated to obtain modifications to the DNA fragment corresponding to Brevi.-ori.-part of that could be cut from RNC splitting only kpni restriction sites. This plasmid was digested Cloned and the resulting DNA fragment Brevi.-ori. ligated with p299LYSA also split kpni restriction sites, to obtain a plasmid containing lysA, Autonomous replicated in coryneform bacteria. The obtained plasmid was named pLYSAB. The process of designing pLYSAB shown in Fig. 5.

<2> determination of the nucleotide sequence of the lysA from Brevibacterium lactofermentum

Plasmid DNA p299LYSA received and its nucleotide sequence was determined as described in Example 1. Certain 8. In relation to the nucleotide sequence of the amino acid sequence encoded argS, and the amino acid sequence encoded by lysA shown in SEQ ID 19 and SEQ ID 20, respectively.

Example 5: Receiving ddh from Brevibacterium lactofermentum

Gene ddh received the ddh gene amplification from chromosomal DNA of Brevibacterium lactofermentum ATCC 13869 according to the PCR method using two oligonucleotide primers (SEQ ID 21 and SEQ ID 22) obtained based on the known nucleotide sequence of a gene ddh Corinebacterium glutamicum (Ishino, S. et al., Nucleic Acids Res., 15, 3917 (1987) ). Received amplificatory the DNA fragment was digested Eat and Aval and split edge was a small mistake. Then this fragment was incorporated into the Smal site pMW119 obtaining plasmids pDDH.

Then pDDH was digested SalI and EcoRI followed by the formation of blunt ends. The resulting fragment ligated with pUC18 cleaved Smal. Thus obtained plasmid was designated as pUC18DDH.

Brevi. -ori. introduced in the received pUC18DDH for constructing plasmids carrying ddh, Autonomous replicated in coryneform bacteria. RNC were digested with restrictase and kpni restriction sites BamHI and split edge was a small mistake. The formation of blunt ends was performed using the kit for DNA blunting the ker > PST (produced by Takara Shuzo) ligated thus, to be inserted in the website > PST pHSG299. A plasmid constructed was designated as RRC. Then pUC18DDH were digested XbaI and kpni restriction sites and the resulting fragment ligated with RRC, split and kpni restriction sites XbaI. It was constructed plasmid containing ddh, Autonomous replicated in coryneform bacteria. This plasmid was named pPK4D. The process of designing pPK4D shown in f IG. 6.

Example 6: Construction of plasmids containing a combination of mutant lysC and dapA

The plasmids containing mutant lysC, dapA, and start replication coryneform bacteria, designed from the plasmid pCRDAPA containing dapA and plasmids raw containing mutant lysC and Brevi.-ori. p399AK9B completely destroyed SalI and then a small mistake and ligated with the EcoRI linker to construct a plasmid in which the SalI site was modified in the EcoRI site. The obtained plasmid was designated as RAC 9BSE. Mutant lysC and Brevi.-ori. carved in the form of a single piece of partial degradation p399AK9BSE using EcoRI. This fragment is ligated with pCRDAPA, split EcoRI. The obtained plasmid was designated as pCRCAB. This plasmid autonomously replicated in E. coli and coryneform bacteria, and it confers resistance to kanamycin owner, and this plasmid contained the comb is containing a series combination of mutant lysC and dapB

The plasmids containing mutant lysC and dapB, designed from the plasmid RAC with mutant lysC, and plasmids p399DPR with dapB. Fragment 1101 p. N. , containing the structural gene DDPR, were extracted by splitting p399DPR EcoRI and SphI. This fragment is ligated with RAC, split SalI and then dull and additionally split SphI, to construct a plasmid containing a combination of mutant lysC and dapB. This plasmid was designated as 399DDPR.

Then Brevi. -ori. introduced in the received p399AKDDPR. Plasmid RNC containing Brevi.-ori., were digested with restriction enzyme CRP (produced by Takara Shuzo) and split the edge was a small mistake. The formation of blunt ends was performed with a set for blunting DNA (manufactured by Takara Shuzo) in accordance with the recommended method. After the formation of blunt ends phosphorylated BamHI linker (produced by Takara Shuzo) ligated to obtain modifications to the DNA fragment corresponding to Brevi.-ori.-parts could be cut by splitting only BamHI. This plasmid was digested BamHI and the resulting DNA fragment with Brevi.-ori. ligated with p399AKDDPR, split BamHI to construct plasmids containing mutant lysC and dapB, Autonomous replicated in coryneform bacteria. The constructed plasmid is smidi, containing a combination of dapA and dapB

Plasmid pCRDAPA containing dapA, were digested and kpni restriction sites EcoRI for extraction of DNA fragment containing dapA, which is ligated with a vector plasmid pHSG399, split and kpni restriction sites EcoRI. The obtained plasmid was designated as p399DPS.

On the other hand, plasmid pCRDAPB containing dapB, were digested SacII and EcoRI for the extraction of DNA fragment of 2.0 T. p. N., containing the region encoding DDPR, which is ligated with p399DPS, split SacII and EcoRI, to construct a plasmid containing a combination of dapA and dapB. The obtained plasmid was designated as raw.

Then in raw introduced Brevi.-ori. pHK4 containing Brevi.-ori., were digested with restriction enzyme BamHI (manufactured by Takara Shuzo), and the split edge was a small mistake. The formation of blunt ends was performed using the kit for blunting DNA (manufactured by Takara Shuzo) according to the proposed method. After the formation of blunt ends phosphorylated linker Cloned (produced by Takara Shuzo) ligated to obtain modifications to the DNA fragment corresponding to Brevi. -ori. -parts that could be cut from pHK4 splitting only kpni restriction sites. This plasmid was digested Cloned and the resulting DNA fragment Brevi. -ori. ligated with raw, split kpni restriction sites, to construct a plasmid containing dapA and dapB, autodestruirse Rav shown in Fig.9.

Example 9: Construction of plasmids containing a combination of ddh and lysA

Plasmid pUC18DDH containing ddh, was digested EcoRI and XbaI for the extraction of DNA fragment containing ddh. Fragment ddh ligated with plasmid p399LYSA containing lysA, split BamHI and XbaI, zatuplenie split edges after splitting. The obtained plasmid was designated as p399DL. The process of designing p399DL shown in Fig. 10.

Then in p399DL introduced Brevi.-ori. RNC were digested XbaI and BamHI and the split edge was a small mistake. After the formation of blunt ends phosphorylated XbaI linker ligated to obtain modifications to the DNA fragment corresponding to Brevi. -ori. -parts that could be cut from RNC splitting only XbaI. This plasmid was digested XbaI and the resulting DNA fragment Brevi. -ori. ligated with p399DL, split also XbaI, to construct a plasmid containing ddh and lysA, Autonomous replicated in coryneform bacteria. The constructed plasmid was designated as a pDL. The process of constructing the pDL shown in Fig. 11.

Example 10: Construction of plasmids containing a combination of mutant lysC, dapA and dapB

p399DPS destroyed EcoRI and SphI with the formation of blunt ends, followed by extraction of the fragment of the gene dapA. This fragment is ligated lysC and dapA.

Plasmid pCRDAPB containing dapB was digested EcoRI and the ends were a small mistake with subsequent splitting SacI for the extraction of DNA fragment of 2.0 T. p. N., containing dapB. Plasmid RSA containing dapA and mutant lysC, SpeI digested and ends was a small mistake, and then were digested SacI and ligated with the extracted fragment dapB to obtain plasmids containing mutant lysC, dapA and dapB. This plasmid was designated as RSA.

Then in RSV introduced Brevi.-ori. Plasmid RNC containing Brevi.-ori. , were digested with restriction enzyme BamHI (manufactured by Takara Shuzo) and split the edge was a small mistake. The formation of blunt ends was performed using the kit for blunting DNA (manufactured by Takara Shuzo) according to the proposed method. After the formation of blunt ends phosphorylated linker Cloned (produced by Takara Shuzo) ligated to obtain modifications to the DNA fragment corresponding to Brevi.-ori.-part of that could be cut from RNC splitting only kpni restriction sites. This plasmid was digested Cloned and the resulting DNA fragment Brevi.-ori. ligated with RSV, split also kpni restriction sites, to construct a plasmid containing a combination of mutant lysC, dapA and dapB, Autonomous replicated in coryneform bacteria. The constructed plasmid was designated as raw. The process is tantoco lysC, dapA, dapB and lysA

Plasmid p299LYSA containing lysA, were digested and kpni restriction sites BamHI and the ends were a small mistake, and then was extracted fragment of the lysA gene. This fragment is ligated with raw, split HpaI (produced by Takara Shuzo), and the end was a small mistake for constructing plasmids containing a combination of mutant lysC, dapA, dapB and lysA, Autonomous replicated in coryneform bacteria. The constructed plasmid was designated as pCABL. The process of designing pCABL shown in Fig. 13. It should be noted that the fragment of the gene lysA injected into the HpaI site in the DNA fragment containing the gene dapB in pCABL, however, the HpaI site of localized against the course of transcription from the promoter for the gene dapB (number of nucleotides 611-616 in SEQ ID 10), and dapB gene is not disconnected.

Example 12: Construction of plasmids containing a combination of mutant lysC, dapA, dapB, ddh and lysA

pHSG299 were digested XbaI and Cloned, ligated with p399DL containing ddh and lysA, split XbaI and kpni restriction sites. The constructed plasmid was designated as p299DL. p299DL were digested XbaI and kpni restriction sites and the ends were a small mistake. After the formation of blunt ends were extracted DNA fragment containing ddh and lysA. This DNA fragment is ligated with the plasmid RSV containing a combination of mutant lysC, dapA and dapB, split HpaI, and the end was a small mistake to construct the plasmid, Triavna plasmid was designated as pCABDL. The process of designing pCABDL shown in Fig.14.

Example 13: the Introduction of plasmids containing genes for the biosynthesis of L-lysine, L-lysine-producing bacterium Brevibacterium lactofermentum.

The plasmid containing the gene for the biosynthesis of L-lysine, constructed as described above, namely p399KK9B(Cmr), pDPSB(Kmr), pDPRB (Cmr), pLYSAB(Cmr), pPK4D(Cmr), pCRCAB(Kmr), pAB(Cmr), pCB(Cmr), pDL(Cmr), pCAB(Cmr), pCABL(Cmrand pCABDL (Cmr), was introduced in L-lysine-producing bacterium AJ11082 (NRRL B-11470) Brevibacterium lactofermentum, respectively. Strain AJ11082 is resistant to AES. Plasmids were introduced according to the electric pulse method (Sugimoto et al. , Japanese Patent Laid-open No.2-207791). Transformants were selected on the basis of markers of resistance to drug who possess the appropriate plasmids. Transformants were selected in complete medium containing 5 μg/ml of chloramphenicol, with the introduction of a plasmid containing the gene for resistance to chloramphenicol, or transformants were selected in complete medium containing 25 μg/ml kanamycin, with the introduction of a plasmid containing the gene for resistance to kanamycin.

Example 14: Receiving L-lysine

Each of the transformants obtained in Example 13, were cultured in the medium for prela the following structure.

[Wednesday for producing L-lysine]

The following components, in addition to calcium carbonate (1 l) was dissolved with bringing pH 8.0 with KOH. The medium was sterilized at 115oC for 15 minutes, and then thereto was added calcium carbonate, sterilized separately in hot air in a dry condition.

Glucose - 100 g

(NH4)SO4- 55 grams

KN2RHO41 g

gSO47H2About 1 g

Biotin - 500 mcg

Thiamine - 2000 mcg

FeSO47H2O - 0.01 g

MnSO47H2O - 0.01 g

Nicotinamide 5 mg

Hydrolyzed protein(Mamao) - 30 ml

Calcium carbonate - 50 g

Each of the different types of transformants and the parental strain were inoculable in an atmosphere having the composition described above, for carrying out cultivation at 31,5oWith reciprocating swing. The amount of produced L-lysine after 40 and 72 hours of cultivation and growth after 72 hours (OD562shown in the table. In this table lysC* indicates a mutant lysC. Growth was quantified by measuring OD at 560 nm after a 101-fold dilution.

As shown in the table, with separate amplification of mutant lysC, dapA or dapB the amount of produced L-lysine was more the TSS however, the amount of produced L-lysine was lower than the quantity produced by the parent strain after 40 hours of cultivation. That is, the rate of production of L-lysine was decreased under cultivation within a short period. Similarly, when the amplification of mutant lysC and dapA or dapA and dapB combined amount of produced L-lysine was greater than the number produced by the parent strain after 72 hours of cultivation, however, the amount of produced L-lysine was less than the number produced by the parent strain, after 40 hours of cultivation. Thus, the rate of production of L-lysine decreases.

On the other hand, when a separate amplification lysC or ddh or joint reinforcement lysC and ddh amount of produced L-lysine was greater than the number produced by the parent strain after 40 hours of cultivation, however, the amount of produced L-lysine was less than the number produced by the parent strain after a long period of cultivation due to the decrease in growth.

On the contrary, in the case of strain, in which dapB was reinforced with mutant lysC, growth was improved, the rate of production of L-lysine successful the compulsory period of cultivation. In the case of strain, which simultaneously increased three genes, mutant lysC, dapA and dapB, the productivity of L-lysine was further improved. As the rate of production of L-lysine, and the cumulative number of L-lysine was improved step by step by successful amplification lysA and ddh.

Industrial applicability

According to this invention is the ability of producing L-lysine coryneform bacteria can be improved and can be also improved the growth rate.

The rate of production of L-lysine can be improved and productivity can be improved in coryneform producing L-lysine bacteria by enhancing dapB with mutant lysC. The rate of production of L-lysine and productivity can be further improved by successful amplification dapA, lysA and ddh in addition to the above genes.

The list of sequences is given at the end of the description.

1. Recombinant DNA autonomously replicable in cells coryneform bacteria containing: (a) the DNA sequence encoding aspartokinase, in which the inhibition of the feedback type L-lysine and L-threonine essentially desensitized, and including the sequence of the research Institute 1051 changed to A; and (b) a DNA sequence encoding dihydrodipicolinate and including a nucleotide sequence 311-1213 in the nucleotide sequence represented by SEQ ID NO: 14.

2. Recombinant DNA autonomously replicable in cells coryneform bacteria containing: (a) the DNA sequence encoding aspartokinase, in which the inhibition of the feedback type L-lysine and L-threonine essentially desensitized, and comprising a nucleotide sequence 217-1479 in the nucleotide sequence represented by SEQ ID NO: 3, in which the nucleotide G at position 1051 changed to A; and (b) a DNA sequence encoding dihydrodipicolinate and including a nucleotide sequence 311-1213 in the nucleotide sequence represented by SEQ ID NO: 14; and (C) DNA sequence, encoding dihydrodipicolinate and including a nucleotide sequence 730-1473 in the nucleotide sequence represented by SEQ ID NO: 10.

3. Recombinant DNA autonomously replicable in cells coryneform bacteria containing: (a) the DNA sequence encoding aspartokinase, in which the inhibition of the feedback type L-lysine and L-trainingfacility, presented at SEQ ID NO: 3, in which the nucleotide G at position 1051 changed to A; (b) a DNA sequence encoding dihydrodipicolinate and including a nucleotide sequence 311-1213 in the nucleotide sequence represented by SEQ ID NO: 14; (C) a DNA sequence encoding dihydrodipicolinate and including a nucleotide sequence 730-1473 in the nucleotide sequence represented by SEQ ID NO: 10; and (d) the DNA sequence encoding diaminopimelate and including a nucleotide sequence 533-2182 in the nucleotide sequence represented by SEQ ID NO: 18.

4. Recombinant DNA autonomously replicable in cells coryneform bacteria containing: (a) the DNA sequence encoding aspartokinase, in which the inhibition of the feedback type L-lysine and L-threonine essentially desensitized, and comprising a nucleotide sequence 217-1479 in the nucleotide sequence represented by SEQ ID NO: 3, in which the nucleotide G at position 1051 changed to A; (b) a DNA sequence encoding dihydrodipicolinate and including a nucleotide sequence 311-1213 in the nucleotide sequence is the sequence of nucleotides 730-1473 in nucleotide sequence, presented at SEQ ID NO: 10; (d) the DNA sequence encoding diaminopimelate and including a nucleotide sequence 533-2182 in the nucleotide sequence represented by SEQ ID NO: 18; and (e) a DNA sequence encoding diaminopimelate and including a nucleotide sequence 61-1020 in the nucleotide sequence represented by SEQ ID NO: 23.

5. The method of obtaining L-lysine, wherein coryneform bacteria transformed with recombinant DNA described in paragraph 1, are cultivated in an environment suitable for producing and accumulating L-lysine in the culture of bacteria, and L-lysine are extracted from the culture.

6. The method of obtaining L-lysine, wherein coryneform bacteria transformed with recombinant DNA, as described in paragraph 2, are cultivated in an environment suitable for producing and accumulating L-lysine in the culture of bacteria, and L-lysine are extracted from the culture.

7. The method of obtaining L-lysine, wherein coryneform bacteria transformed with recombinant DNA, as described in paragraph 3, were cultured in a medium suitable for producing and accumulating L-lysine in the culture of bacteria, and L-is ' series, transformed by the recombinant DNA described in paragraph 4, are cultivated in an environment suitable for producing and accumulating L-lysine in the culture of bacteria, and L-lysine are extracted from the culture.

 

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The invention relates to biotechnology, can be used for saccharification of starchy raw materials in various food industries that require highly active enzyme preparations that are resistant to acidic pH values
The invention relates to the field of veterinary Virology

The invention relates to the production of forage product and can be used to obtain a protein-carbohydrate feed for fattening cattle and other farm Skoura

The invention relates to the field of Virology and can be used in the production of vaccines
The invention relates to molecular biology, and in particular to methods of DNA extraction, and can be used in laboratory and research practice for separation of high molecular weight DNA or their fragments (macrorestriction) from the yeast and gram-positive microorganisms suitable for further analysis using the technique of pulse electrophoresis, as well as in the formulation of polymerase chain reaction (PCR) and cloning

The invention relates to purified and dedicated not naturally occurring RNA ligands to vascular endothelial growth factor (VEGF) (indicated oligonucleotide sequence)
The invention relates to biotechnology, in particular genetic engineering

The invention relates to the field of animal husbandry

The invention relates to Phytopathology, in particular to a method of determining fungal pathogens using specific primers in the polymerization reaction

The invention relates to the field of biotechnology, genetic engineering, immunology, and can be used for serodiagnosis of HTLV-I/II

The invention relates to genetic engineering
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