JPH02474A - Production of l-histidine - Google Patents

Abstract

PURPOSE:To advantageously obtain L-histidine by culturing microorganism of Corynebacterium or Brevibacterium containing specific recombinant DNA, generating and accumulating L-histidine in cultured substance. CONSTITUTION:(A) A DNA fragment derived from Corynebacterium mycetoma or Brevibacterium mycetoma and containing gene having activity of transforming a microorganism exhibiting histidine requirement to a histidine non-requirement strain and (B) a recombinant DNA which is recombinant with vector DNA capable of autonomic replicating in said mycetomas and at least one of A and B components are extraneous to host strain are prepared. Next, a host strain selected from microorganism of Corynebacterium or Brevibacterium is transformed using said recombinant DNA. Then, resultant transformed strain is cultured in medium, L-histidine is generated and accumulated in cultured substance, thus gathered.

Description

[Detailed description of the invention] The present invention relates to a novel method for gene expression.

More specifically, the present invention uses a recombinant DNA that is a recombinant of a DNA fragment containing at least one gene and a vector DNA, and in which at least one of both DNAs is foreign to the host strain. This invention relates to a method for gene expression, which comprises culturing a transformed strain obtained by transforming a host strain selected from microorganisms belonging to the genus Bacterium or Brevibacterium in a medium to express the trait of the gene. .

Recombinant gene technology has been established using Escherichia coli as a host, and to date it has been possible to produce bebutides and vaccines such as somatostatin, insulin, human growth hormone, human interferon-α, human interferon-β, and mouth ulcer vaccine. It has been shown. Although Escherichia coli is considered to be sufficient as a host for the expression of these highly bioactive peptides and vaccines in many cases, higher productivity, extracellular secretion, and glycosylation are required, or the contamination of intracellular toxins is avoided. Therefore, yeast and Bacillus subtilis have also been developed as hosts.

When producing physiologically active substances such as peptides and proteins, it is sufficient to use strains for which recombinant DNA techniques such as those mentioned above have already been established or for which the basics are in place.
When using recombinant DNA techniques to improve the industrial productivity of substances such as amino acids, nucleic acids, vitamins, and antibiotics, it is necessary to devise ways to apply the techniques to the respective producing bacteria that have been used conventionally.

Corynebacterium glutamicum was the first microorganism to be used for the industrial production of amino acids. Since then, Coryneform bacteria, including the genus Corynebacterium, have been used to produce glutamic acid, lysine, alanine, histidine, tryptophan, thyronone, phenylalanine, threonine, and amino acids. Industrial production of amino acids such as leucine, valine, leucine, glutamine, proline, and arginine has been developed, and today most amino acids are produced by microorganisms.

Therefore, the establishment of the 411 DNA technique in these microorganisms is considered to be extremely important for improving amino acid production in the future.

Recombinant DNA techniques include, for example, (1) fragmentation of DNA containing the target gene with restriction enzymes, (2) linearization by single cleavage of vector DNA with the same restriction enzyme, and (3) above (1) and (2). Annealing and DNA by mixing the products of! Creation of recombinant DNA by ligation using J gauze (4) Introduction of the above recombinant DNA into the host strain (transformation) (5) Selection of recombinants containing the target gene and purification of the selected clones It depends on each stage. The efficiency of creating the recombinant stock obtained in this way can be said to be the product of each of the above steps, so it is necessary to have a means to verify each step, know the efficiency of each step, and improve it. However, it is not possible to obtain a transformed strain capable of expressing the target gene. Furthermore, even if a transformed strain containing recombinant DNA containing the target gene is obtained in this way, there are various barriers to expressing the gene if the gene is foreign to the host strain. It is known that [“Chemistry and Biology” 18.110~
(1978)) its expression is very difficult.

Recombinant D using a microorganism belonging to the genus Corynebacterium or Brevibacterium as a host and containing a target gene or vector foreign to the host.
Until now, there has been no known example of expressing the trait of the target gene by introducing NA.

In recombinant DNA techniques using microorganisms belonging to the genus Corynebacterium or Brevibacterium as hosts,
It is necessary to construct a vector system that autonomously replicates in these microorganisms, has a selectable phenotype, and can be used for cloning multiple genes, and to establish an efficient transformation system. It is necessary to establish a method to eliminate barriers.

The present inventors previously constructed a plasmid vector that replicates autonomously in microorganisms belonging to the genus Corynebacterium or Brevibacterium and has a selectable phenotype and a suitable loning site, while developing a highly efficient transformation system. developed
9), 56-58187 (Unexamined Japanese Patent Publication No. 57-1864)
92), 56-65777 (Unexamined Japanese Patent Publication No. 57-186)
489) ).

Therefore, the present inventors used the already known in vitro DN8 modification technique (U, S,
Patent 4,237,224), DNAvfr containing a foreign gene involved in amino acid biosynthesis
When the fragments were ligated and the developed transformation system was used to transform Corynebacterium glutamicum strain 122 or its derivatives, the foreign gene was expressed in the host, producing useful amino acids, etc. The present invention was completed based on the discovery that the present invention can be used to increase the production of substances.

The present invention will be explained in detail below.

The present invention uses recombinant DNA, which is a recombinant of a DNA fragment containing one type of gene and a vector DNA, and in which at least one of both DNAs is foreign to the host strain, to produce Corynebacterium spp. Alternatively, the present invention provides a method for culturing a transformed strain obtained by transforming a host strain selected from microorganisms belonging to the genus Brevibacterium in a medium, and expressing the trait of the gene.

DNA fragments containing genes used in the present invention include DNA fragments derived from eukaryotes, prokaryotes, viruses, batatteriophages, or plasmids and containing at least one complete gene.

Examples of genes derived from eukaryotes include genes encoding bebutides such as interferon, inulin, and growth hormone in mammals, particularly humans. Genes derived from prokaryotes include bacteria, especially Escherichia, Corynebacterium, Brevibacterium,
Genes derived from bacterial strains belonging to the genus Bacillus or Staphylococcus include genes involved in cellular metabolism and synthetic activity. Alternatively, it refers to the synthesis of antibiotics, etc., and the metabolic system involved in the synthesis, and in the present invention, biosynthetic activity of amino acids, particularly glutamic acid, lysine, threonine, histidine, or tryptophan, is preferably mentioned.

Furthermore, when the amino acid composition of the target bebutide, protein, etc. is known, the corresponding DNA can also be synthesized and used. Examples of DNA synthesis methods include K, I
takura et ll 5science 191.
1056 (+977)>.

The vector used in the present invention must be compatible with the host bacterium (c
It must be able to reproduce autonomously. As a specific example, the present inventors have collected pcGl from a microorganism belonging to the genus Corynebacterium, or produced it by inducing the collected microorganism [Patent Application No. 1810-1983]
1 (Japanese Patent Application No. 57-134500) ), ρCG2 [Patent Application No. 56-133557 (Japanese Patent Application No. 58-35197)
), pCG4 [Japanese Patent Application No. 56-58186
7-183799) ), pCε53, pCε54, p
Examples include cGll, pcBloL +)εthrl, and the like.

Bacterial strains harboring these plasmids have been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology and the American Type Culture Collection under the following deposit numbers.

Plasmid FERlJ-P 8TCCp
CG 1 5865
3] 808p CG 2
5954 31832p CG
4 5939
31830p CE54
39019p CG 11
39022p
CBlot
39020pεthr 1
39021 preferably ρ
CGII, pCE54 is used. pcGll is a LL plasmid that was previously disclosed by the present inventors [Japanese Patent Application No. 56-18101 (Japanese Unexamined Patent Publication No. 57-134500)].
Corynebacterium glutamicum 225-57 (^
Corynebacterium glutamicum 225-250 (TCC31830, FERM-P
Streptomycin and/or spectinomycin resistance (
This is a plasmid in which a BamHI fragment containing the Sm''/5pec'' gene is joined using the same cohesive ends of both.

ρCG11 is a plasmid with a molecular weight of 6.8 Kb and has Bgi'll and Pst I as single restriction sites.
It gives a phenotype of m''/5pec'.

pEC54 can be created as follows.

First, p(:G2
, ^TCC31832) from cultured bacterial cells, patent application 1982-1
33557 (Japanese Unexamined Patent Publication No. 58-35197), pG^22 is concentrated and isolated from cultured cells of E. coli harboring it by a commonly used method. Both plasmid DNAs are digested with a restriction enzyme having one cut point in each molecule, such as pst.
After complete digestion with I and linearization, T4 phage D was used to generate a conjugate molecule in which both DNA molecules were linked with the same cohesive end protruding as a single strand from both ends of the plasmid molecule.
Let NA ligase act. Obtaining a recombinant plasmid in which both plasmid molecules are conjugated and linked from this DNA mixture is carried out by first obtaining the characteristics of Corynebacterium or Brevibacterium species selected for drug resistance derived from pG^22. This is achieved by isolating transformed strains and analyzing the plasmids possessed by these transformed strains.

Transformation with a DNA hybrid can be carried out using a transformation method using protoplasts of Corynebacterium and Brevibacterium species previously disclosed by the present inventors [Patent Application No. 5]
6-58187 (Japanese Unexamined Patent Publication No. 57-186492) and Japanese Patent Application No. 55-65777 (Unexamined Japanese Patent Application No. 57-186489)
) can be implemented. The drug used for selection is p
Among the drug resistance genes derived from c^22, pGA22
Tetracycline (Tc), chloramphenicol (Cm), or kanamycin (Km) corresponding to resistance genes other than the ampicillin resistance gene, which is inserted and inactivated because it serves as a linkage site with the ampicillin resistance gene, may be used. The transformed strain is prepared in a DNA-free system and treated with drugs (usually tetracycline 0.4-1.6 g/ml, chloramphenicol 2.5-5 g/ml) that do not allow the recipient protoplasts to revert to normal cells and proliferate. mIJ6 and kanamycin 100-
Either isolate the reverting colonies on hypertonic agar containing 800x/ml) or - scrape them after non-selectively repopulating them to normal cells on regeneration medium and mix this resuspension with normal recipient cells. Concentrations of drugs in which cells cannot grow (usually tetracycline 0.5-4 g/i + chloramphenicol 2-15 g/ml and kanamycin 2-2
5q/it) by isolating colonies growing on an agar medium containing 5q/it). Tetracycline, chloramphenicol or kanamycin resistance (Tc',
Among the transformed strains selected by Cm" and Km", some have also acquired other drug resistance traits derived from pG^22.

Plasmid DNA possessed by the thus obtained transformed strain
A was filed by the present inventors in Japanese Patent Application No. 55-18101 (Japanese Unexamined Patent Publication No.
7-134500) and Japanese Patent Application No. 56-65777 (Japanese Unexamined Patent Publication No. 57-186489), the DNA fragments can be isolated and purified from cultured bacterial cells, and the DNA fragments produced by digestion with various restriction enzymes can be subjected to agarose gel electrophoresis. The structure can be known by the usual method of analysis.

pCE5 is a plasmid isolated from one of the transformed strains.
It is 4.

pCE54 is a plasmid approximately 14.5 Kb in size with EcoRI, Sal I SSm as single restriction sites.
a I 5Xho 1 etc., Tc", C+n"
, gives the phenotype of C.

Xho I is located in the Km'' gene, and selection by so-called insertional inactivation (a phenomenon in which expression of the relevant phenotype is prevented by insertion of a DNA fragment) is also possible.

Collection of plasmids from plasmid-carrying strains is described, for example, in Japanese Patent Application No. 56-18101 (Japanese Unexamined Patent Publication No. 57-134500).
), 56-58186 (Unexamined Japanese Patent Publication No. 57-183799)
and 56133557 (Japanese Unexamined Patent Publication No. 58-35197
) may be carried out according to the method described in .

A recombinant of a DNA fragment containing a gene and a vector DNA can be produced by making full use of known in vitro recombinant DNA techniques.

In vitro DNA recombination is usually performed by cutting and recombining donor DNA containing the gene of interest and vector DNA. DNA can be easily cut using restriction enzymes. Restriction enzymes used for in vitro recombination recognize and cleave specific base sequences on all double-stranded DNA, regardless of biological species.

The base sequence differs depending on the type of restriction enzyme.

Therefore, by using an appropriate restriction enzyme, the gene of interest can be excised as a single DNA fragment without impairing its expression function. Donor D cleaved by the same restriction enzyme
The end structures of the cut fragments of NA and vector DNA have the same structure; some restriction enzymes give cohesive ends with a single strand protruding, and other restriction enzymes give blunt ends. As long as both ends are cleaved with the same restriction enzyme, the cut fragments of donor DNA and vector DNA can be ligated using T4 phage DNA ligase.

Even when both DNAs are cut with different restriction enzymes, for example,
Cohesive ends can be repaired with DNA polymerase to make double strands and blunt ends before ligation, terminal transferase can be used to add a complementary homopolymer to create sticky ends before ligation, or some type of Synthetic oligonucleotide linkers containing restriction enzyme cleavage sites can be ligated and then internally cut to create cohesive ends before ligation. By these ligation methods, a recombinant of a DNA fragment containing the gene of interest and a vector DNA fragment is generated.

In addition to the desired recombinant, other recombinants are generated by the ligase reaction, but in order to obtain the desired recombinant, this DN
Corynebacterium or Brevibacterium species are directly transformed using the mixed solution A, and a transformed strain endowed with genetic traits derived from the genetic information of the target gene is selected and isolated, and the cultured bacterial cells are isolated. This can be achieved by extraction and isolation from Instead of directly transforming Corynebacterium or Brevibacterium species, the gene of interest can be transformed into host-vector systems of other microorganisms, such as E. coli.
After that, a recombinant with a vector of a Corynebacterium or Brevibacterium species is produced in vitro, and then a Corynebacterium or Brevibacterium species is transformed in the same manner as above. Recombinants can also be obtained by selectively separating transformed strains.

For recombinant production, the descriptions in the following documents can be widely applied.

S,N,Cohen,v,L al U,S,P
Atent 4.237, 224, Genetic Manipulation Experimental Method [edited by Yasutaka Takagi, Kodansha Sun Entific (1)
980) ), Method in Enzymol
ogy68, Recomb+nant DN, A e
Dited by Ray Wu, Academic
Press 1979 As the host microorganism of the present invention, any strain belonging to the genus Corynebacterium or Brevibacterium and having the ability to take up DNA may be used. Preferably, the present inventors first filed Japanese Patent Application No. 5,615,1464 (Japanese Unexamined Patent Application No. 5,615,1464
8-56678) is used. Specific examples of bacterial strains include the following strains.

Deposit number FεRIJ-P ATC [: Corynebacterium glutamicum L-155946
31834 Corynebacterium hakieris L-1
03594731866 Previbacterium daylicolumn L-204594831867 Previbacterium lactoparmentum L-312594931
868 Transformation of host microorganisms with recombinant DNA
This process is carried out in the following steps:) preparation of protoplasts from cultured cells, 2) transformation of protoplasts with recombinant DNA, and 3) regeneration of protoplasts into normal cells and selection of transformed strains. Examples of specific methods are shown below.

■) Preparation of protoplasts from cultured cells Protoplast formation is performed by growing microorganisms under conditions that make them sensitive to the cell wall lytic enzyme 19zozyme, and then treating the cultured cells with lysozyme in a hypertonic solution to dissolve and remove the cell wall. . Various cell wall synthesis inhibitors are used to convert microorganisms into lysozyme-sensitive cells. For example, microbial cells can be made sensitive to lysozyme by adding penicillin at a concentration that does not inhibit or semi-inhibit growth in the middle of the logarithmic growth phase of microbial culture and allowing the culture to grow for several generations.

The medium used at this time may be any medium that allows microorganisms to grow, such as nutrient medium NB (medium containing 20 g of powdered broth and 5 g of yeast extract in 11 parts of pure water, adjusted to pH 7.2) or semi-synthetic medium SSM [glucose 10g,
'IH, CI! 4 g, urea 2 g, yeast extract I
g, to) 12PO41g, KJPOs 3g1! , I
gCfi・6H200,4gS peso4'7H2o
10mg, Mn5Oa'4-61'1z00,
2 mg, ZnSO44H200,9mgq Cu5
O<・5HzOo, 4 mgsNa2B40t'1(
ILOo,o 9mg, (NHJg!J(hOz<'
4Ha00,04 mg, biotin 30■, thiamine hydrochloride lff1g in water lI! A culture medium containing 100% of the total number of microorganisms and adjusted to a pH of 7.2] is used.

Microorganisms are inoculated into this medium and cultured with shaking.

Measure the absorbance (00) at 660 nm using a colorimeter, and add penicillin to the culture medium at a concentration of 0.1 to 2.0 units/ml at the beginning of the logarithmic growth phase (OD = 0.1 to 0.4). Add penicillins such as G. The culture is further continued, and when the OD increases to 0.3 to 0.5, the cells are harvested and washed with S3M medium. The cells are then cultured in a suitable hypertonic medium, such as PFM medium (medium containing 0.4 M sucrose and 200,01 M MgC12.6H in a 33M 2-fold dilution solution, adjusted to pH 7.0 to 8.5) or RC.
G medium [glucose 5g1 casein hydrolyzate 5g, yeast extract 25g1, 2HPO43.5g, KH2PO
41.5g, lJgcL・6H200,41gpes
ot・7Hz010mg, 1JnsO4・4-6Hz
0 2 mg, ZnSOs・711.0 0.9 m
g, Cu5O<4L00.4 mg, Na2LOi
-10LIIIO, 09mg, (NH4)aMOtOy
a・4N 200.04 mg, biotin 30d1 thiamine hydrochloride 2II 1g, disodium succinate 1.35
resuspended in a medium containing 1 liter of water and adjusted to pH 7.0 to 8.5. Add this cell suspension to a final concentration of 0.2-1.
Add lysozyme to 0mg/ml and add 30~3
React at 7°C. Protoplast formation progresses as the reaction time progresses, and its progress can be observed using an optical microscope. The time required for most of the cells to become protoplasts under a microscope varies depending on the concentration of penicillin added during follicle culture and the concentration of lysozyme used, but is 3 to 24 hours under the above conditions.

The generated protoplasts undergo rupture array under hypotonic conditions, so
The degree of protoplast formation can be indirectly determined by the degree of remaining normal cells that survive under hypotonic conditions. Normally, the residual level of normal cells can be suppressed to about 10-4 of normal cells treated with lysozyme.

The protoplasts prepared in this manner have the ability to form colonies (regenerate) on an appropriate hypertonic agar medium. The agar medium is a nutrient medium, a semi-synthetic medium, or a synthetic medium supplemented with several types of amino acids, with 0.3 to 0.8 M disodium succinate and 0.5 to 6% polyvinylpyrrolidone (110.000 or 40.000 molecules). ')
Those containing the following are preferably used.

Usually, semi-synthetic medium RCGP medium CRCG medium contains 3% polyvinylpyrrolidone (molecules 110.000> and 1.4%
A medium supplemented with agar, pH 7.2) can be used. Cultivation is preferably carried out at 25-35°C.

The number of culture days required for the appearance of regenerated colonies varies depending on the strain, but it takes 10 to 14 days for the colonies to reach the size of one colony.

The regeneration of protoplasts in RCGP medium varies depending on the bacterial species, the concentration of penicillin added during culture, and the concentration of lysozyme treatment, but the regeneration rate is 101 per normal cell sample treated with lysozyme.
The efficiency is ~10-4.

2) Transformation of protoplasts with recombinant DNA Uptake of recombinant DNA into protoplasts involves mixing protoplasts and recombinant DNA in a hypertonic solution that allows cells to maintain a protoplast state, and adding a DNA uptake-mediated effect to this. This is carried out by adding a divalent metal cation to polyethylene glycol (PEG, average molecular weight 1,540 to 6,000) or polyvinyl alcohol (PVA, degree of polymerization 500 to 1,500). Stabilizers that provide hypertonic conditions may be those commonly used to maintain protoplasts of microorganisms, such as sucrose or disodium succinate. P
Usable concentration ranges for EG and PVA are 5-60% and 1-20% final concentration, respectively. Divalent metal cations include Ca'', Mg'', Mn'', and B at final concentrations of 1 to 100 mM.
a"Sr"2 etc. are effective and can be used alone or in combination. The temperature of the treatment is preferably 0 to 25°C.

3) Regeneration of protoplasts back to normal cells and selection of transformed strains Regeneration of protoplasts transformed with recombinant DNA is similar to the regeneration of protoplasts described above, using disodium succinate and polypyrrolidone. Protoplasts are spread on a hypertonic agar medium (for example, RCGP medium) and cultured at a temperature at which normal cells can grow, generally 25 to 35°C. The transformed strain is donor D
It can be obtained by selecting for the trait that a gene derived from NA imparts to the fungus. Selection based on the acquisition of characteristic traits may be carried out simultaneously with regeneration on a hypertonic agar medium, or it may be carried out on a normal hypotonic agar medium after non-selective regeneration and then the regenerated normal cells are collected. good.

When using a lysozyme-sensitive strain shown as a specifically preferred host strain in the present invention, the transformation is carried out in the steps described above except for directly treating cells that have been cultured and grown without penicillin treatment in step 1) above. The same steps as 1) to 3) may be performed. When using lysozyme-sensitive microorganisms, the transformed strain is 10-4 to 10-4 per regenerating microorganism.
Obtained at a high frequency of 10-6.

The transformed strain can express the characteristics of the introduced recombinant DNA by culturing it in a conventional nutrient medium.

If the recombinant DNA has properties derived from genetic DNA or vector DNA, the culture medium may be supplemented with a drug depending on the properties.

Collection of useful substances such as amino acids produced by the method for expressing the characteristics of the present invention is carried out by a conventional method of collecting these substances from a fermentation liquid.

The present invention has made it possible to increase or provide new productivity for amino acids, nucleic acids, vitamins, antibiotics, enzymes, peptides, and proteins in microorganisms of the genus Corynebacterium and Brevibacterium. It has also become possible to improve the production method by enhancing the metabolic activity of microorganisms, increasing their substrate utilization, providing new metabolic activity, and providing new substrate utilization.

A further feature of the present invention is that a heterologous gene or exogenous recombinant DNA has been successfully expressed in a microorganism of the genus Corynebacterium or Brevibacterium. Specifically, the threonine operon of the large intestine, the phosphoenolpyruvate carboxylase <ppc> gene, the gene ptlBll(1(in egg+ns) expressed in Bacillus subtilis and Staphylococcus
K, M, et al, Proc, Natl^
cad, Sci,, [1, S, A, h 1423
(1978)), a gene involved in lysine biosynthesis from Corynebacterium glutamicum, and an anthranilate synthase gene from Brevibacterium flavum were expressed in Corynebacterium genus bacteria.

All of the exemplified genes were simply introduced in the form of a ligation to a Corynebacterium glutamicum plasmid, and no special manipulation was performed to express them in Corynebacterium glutamicum. In addition, DN containing the gene
No matter which direction the A fragment is ligated to the Corynebacterium glutamicum plasmid, it will be expressed in Corynebacterium glutamicum. It is clear that it has the ability to accurately recognize the starting point of translation and carry out transcription and translation. Considering that all genes have similar sites at the base sequence level that are necessary for accurate initiation of transcription and translation, Corynebacterium glutamicum
It is easily inferred that transcription/translation initiation sites of genes other than the exemplified genes can also be recognized and expressed.

The so-called glutamate-producing bacteria, which have a high glutamate production ability, have the same main mycological properties (although they have been given different names by different researchers due to their industrial importance) Even their names vary, such as the genus Corynebacterium and Brevibacterium. However, these bacterial groups are the same species because the amino acid composition of their cell walls and the base composition of their DNA are uniform. Furthermore, it has recently been revealed that there is more than 70-80% DNA homology between these bacterial species, making it clear that they are very closely related microorganisms. niomitsu, Y, :Report of
theFermentat+ve Re5earch
Ins [1tute, No, 55. 1 (19
8Q), and 5uzuki, , Kanek
o, T,, and Komagata.

K, : Int, J, 5yst, Bacte
riol,,31. 131 (1981)]. In this specification, since hosts that can be used in recombinant DNA experiments are regulated, the usefulness of the present invention was shown using a derived strain of Corynebacterium glutamicum L-22 as a host. It can be easily inferred that this method can be applied directly to all production bacteria. recombinant DNA
In order for the plasmid to be stably maintained and expressed in these bacterial species, slight differences depending on the characteristics of the host bacteria such as DNA homology are not a problem;
11 and a function that enables expression of the introduced gene. However, the fact that these bacterial species share both of these functions was previously disclosed by the present inventors [Patent Application No.
Plasmid pCG4, which is isolated from Corynebacterium glutamicum 225-250 and has a streptomycin and/or spectinomycin resistance gene, is used to isolate Corynebacterium and Brevibacterium species, etc. This is clear from the fact that it can be similarly reproduced in glutamic acid-producing bacteria, and its resistance gene is expressed [Japanese Patent Application No. 56-58187 (Japanese Unexamined Patent Publication No. 57-186492)].

Therefore, host bacteria to which the present invention can be applied include not only Corynebacterium glutamicum but also all glutamic acid-producing bacteria including the genus Corynebacterium and the genus Brevibacterium.

Examples of the present invention are shown below.

Example 1. Cloning of the gene involved in lysine biosynthesis of the lysine-producing bacterium Corynebacterium glutamicum ATCC21543 in Corynebacterium glutamicum and production of lysine by Corynebacterium glutamicum using the expression of the gene: (1) Corynebacterium glutamicum Bacterium glutamicum^TCC
Chromosomal DNA of 21543 and vector pcG11!
! : Corynebacterium glutamicum ^TCC130
The chromosomal DNA of Corynebacterium glutamicum^TCC21543, a lysine-producing mutant strain derived from 32 and resistant to the lysine analog S-(2-aminoethyl)-cysteine (hereinafter abbreviated as AEC), was determined as follows. Extract and isolate.

40 Qml semi-synthetic medium SSM [Glucose 20g1 (
NH4) 2S0410 g, urea 3 g, yeast extract I
g, KH, Po, 1 g, ! JgCL-6L00
．． 4 g, Fe50. -711,010mg1! Jn
SO4-4-611, Oo, 2 mg, Zn5O+
4H*00.9mg, CuS mouth 4'5H200,421
g, Na, B4O1-1011200,09mg,
(Nl(4) iM(h口7.・4HffO0,04
■, Piotin 30μg 1 thiamine hydrochloride lag 1 water
The seed culture was inoculated into a medium prepared by adding threonine to a concentration of 100 g/ml, and cultured with shaking at 30°C. 660 on Tokyo Photoden Colorimeter
The absorbance at nm (0 mouth) is measured, and when the ODo reaches 2, Beninrin G is added to the culture medium to a concentration of 0.5 units/m I. Continue culturing and OD
Grow until approximately 0.6.

Collect bacterial cells from the culture solution and add to TBS buffer [0.03M tris(hydroxymethyl)aminomethane (hereinafter abbreviated as Tris), 0.005M DTA, 0.05M NaC.
/: After washing with pH 8,0), lysozyme solution (25%
Shoutang, 0. IMNaCl, 0.05M) Lis, 0.8
mg 7m I lysozyme: pH 8.0 or less) l
Suspend in Qml and react at 37°C for 4 hours. From the collected bacteria, Saito et al.
Top: Biochim.

[1iophys, Acta, 72, 619 (1
Polymeric chromosomal DNA is isolated according to 963).

On the other hand, ρCGII used as a vector plasmid is
Induction method for 22 strains of Corynebacterium glutamicum LAI (13 pcGll-bearing strains LA103/pCG
11 (ATCC39 (122)) as follows.

3 with 400 mINB medium (medium containing 20 g of powdered bouillon and 5 g of yeast extract in 11 parts of water and adjusted to pF 17.2).
Culture with shaking at 0°C until the OD reaches approximately 0.7.

After collecting the bacteria and washing with TES buffer, lysozyme solution I
Suspend in Q+nl and react at 37°C for 2 hours. Add 12.4ml of 5M NaC and 0.5MEDT^ (pH
8,5) Add 4.4 ml of a solution consisting of 0.6+++l, 4% sodium lauryl sulfate and 0.7 M NaCl in sequence, mix gently, and then place in ice water for 15 hours.

Transfer the entire lysate to a centrifuge tube and incubate at 4°C for 60 min.
Centrifuge at 0 x g and collect the supernatant.

Add polyethylene glycol (PEG) 6.000 (manufactured by Gyudon Chemical Co., Ltd.) equivalent to 10% by weight to this, mix gently to dissolve, and then place in ice water. After lO hours 1
．． Collect the pellet by centrifugation at 500 x g for 10 minutes. Gently redissolve the pellet in TES buffer for 5 minutes and then add 1.5 mg/ml ethidium bromide to 2. Add Qml, add cesium chloride to this and gently dissolve to adjust the density to 1.580. Add this solution to 1
Ultracentrifuge at 05.000 x g for 48 hours at 18°C. The circular DNA covalently closed by this density gradient centrifugation is found as a high-density band in the lower part of the centrifugation tube by irradiation with ultraviolet light.

J) CGII DNA is separated by extracting this band from the side of the centrifuge tube with a syringe. Next, the fractionated solution was diluted with an equal volume of isopropyl alcohol solution [volume ff
i percentage 90% isopropyl alcohol, 10% TES
Ethidium bromide is extracted and removed by treatment five times with Himeiboshi solution (this mixed solution contains a saturated amount of cesium chloride), and then dialyzed against TBS buffer.

(2) Corynebacterium glutamicum ATCC
Cloning of genes involved in lysine biosynthesis of 21543 Reaction solution for restriction enzyme BglU (10mM Tris-HCl, 7mM Tris-HCl, 7mM
M MgCL , 60mM NaCj! , 7mM2
-Mercaptoethanol, pH 7.5) 6 units of BgII [(Takara Shuzo Co., Ltd.!) was added to 60 tll, and the mixture was heated at 37°C.
After reacting for 60 minutes at 65°C, the reaction is stopped by heating at 65°C for 10 minutes. On the other hand, Corynebacterium glutamicum ATCC
Restriction enzyme Ram containing chromosomal DNA 8■ of 21543)
I I reaction solution (lomM l-lith hydrochloric acid, 7mM Mg
CL.

100mM NaCl,2IIIM2-mercaptoethanol, 0.01% bovine serum albumin, pH 8,0)
Add 4 units of BamHI to 140 ml and incubate at 37°C for 6
After reacting for 0 minutes, the reaction is stopped by heating at 65° C. for 10 minutes.

Mix the raindrops and add T41J gauze buffer (Tris-HCl 660ml, MgCL 66mM, dithiothreitol 100ml, pH 7,6) 40A+
', ATP (5mM) 40m, T4 ligase (Takara Shuzo Co., Ltd., 1 unit/ρ) 0.3jtI! and) 12012
hj! and react at 12°C for 16 hours. This mixture was saturated with 400% phenol in TBS buffer. d for 2
Extract twice and dialyze against TBS buffer to remove phenol.

This ligase reaction mixture was applied to ΔEC-sensitive LP derived from Corynebacterium glutamicum strain 122.
4 strains are used for transformation.

Transformation is performed using protoplasts of the LP4 strain.

A seed culture of LP4 strain is inoculated into NB medium and cultured with shaking at 30°C. When the OD reached 6, the cells were collected and the cells were
CGP medium [glucose 5g.

casamino acid 5g, yeast extract 2.5g, HPo.

3.5 g, KHiPOn 1.5 gSMgCj!
2・6t+, o O, 41g, FeSO4'7Hz
010mg, Mn5O*・4”6H202ff1g,
2nSOa'7Hz0 0.9 mg, (NHJa
Mo70*s・4H200.04mg, biotin 30J
tg, thiamine hydrochloride 2 mg, disodium succinate 135 g, polyvinylpyrrolidone (molecular weight 1000
0) About 10' cells/m in a solution (pH 7.6) containing 1 mg/ml of Norizozyme in a medium containing 30 g in 11 parts of water]
The suspension was transferred to an L-shaped test tube and subjected to gentle shaking reaction at 30°C for 5 hours to form protoplasts.

Take 5 ml of this protoplast bacterial solution Q into a small test tube 2
Centrifuge at 500 x g for 5 minutes and mix with T SMC buffer (10mM magnesium chloride, 30mM calcium chloride, 5QmM), 4QQ + nM sucrose, pH 7,
5) Resuspend in 1 ml, centrifuge and wash, then resuspend in 1 ml of TSIJC buffer Q. This bacterial solution contains 2 times higher concentration of TS.
! 1:1 of AC buffer and the above ligase reaction DNA mixture
Mixture 1004 is added and mixed, and then 3 ml of solution Q containing 20% PEG 6.000 in TSMC buffer is added and mixed. After 3 minutes, add RCGP medium (pH 7,2
), remove the supernatant by centrifugation at 2.500 × g for 5 min, suspend the sedimented protoplasts in 1 ml of RCGP medium, and then centrifuge at 2.500 × g for 5 min.
1 containing Svectinomycin 400 Jtg/ml
Plate on CGP agar medium (RCGP medium containing 1.4% agar, pH 7.2) and culture at 30°C for 7 days.

The entire amount of bacteria grown on the agar medium is collected, washed with physiological saline, and then suspended in 1 ml of physiological saline.

This bacterial solution was mixed with threonine 2mg/ml, A[iC2+n
g/ml and streptomycin 12.5 ■/m I
Minimal agar medium M1 containing the equivalent [glucose 10 g 1
NH4H2PO41g, KCj! 0.2 g
S', 4gSO4・7H200,2g, Fe5o
44t+2o 10mg,! JnSOn・4～611
20062mg% In5L・TN 200-9
mg-, CuSO4・5820o, 4 mg 5N
a2B40i'1OH200.09mg, (NHs
) Jot mouth 2.・4H200, 04mg, biotin 50
A medium containing 2.5 mg of xSp-aminobenzoic acid, 1 mg of thiamine hydrochloride, and 16 g of agar in 2 parts of water and adjusted to pH 7.2] was recoated and cultured at 30°C for 3 days. Strains resistant to ABC, svectinomycin and streptomycin are obtained from the colonies that appear.

The plasmid possessed by these transformed strains is the above-mentioned p
It is isolated in the same manner as cGll was isolated. Using these plasmid DNA 1■, it was completely digested with the restriction enzyme ECORI having a cleavage site on pcGll, and then analyzed by agarose gel electrophoresis, and the molecular weight was determined from the sum of the generated fragments. The molecular weight is calculated using the restriction enzyme old ndlI [
The calculation is based on a standard curve drawn by the migration distance of each fragment of known molecular weight produced by digestion. The plasmid pAec 5 obtained from one of the transformed strains is a molecule! 10.7K
In b, a 3.9 Kb D was added to the Bgf■ cleavage site of pcGll.
This is a recombinant plasmid into which an NA fragment has been inserted.

The protoplasts of the P4 strain were transformed using pAec5 DNA in the same manner as above, and the transformed strain selected for subectinomain resistance was pAec5, which was determined by the cleavage pattern of EcoRI, which conferred EC resistance to the gene. They carry the same plasmid. That is, it is clear that the gene governing the ABC resistance trait of Corynebacterium glutamicum^TCC21543 has been cloned into pAec5. The pAec 5 nursery strain is included in the American Type Culture Collection.
ynebacter iua+glutamicun
It has been deposited as K17 ATCC39032.

(3) Lysine production by pAec 5 strain P4 strain derived from Corynebacterium glutamicum strain 122 pAec 5 strain (ATCC39032)
A lysine production test will be conducted on non-holding strains. One platinum loop of bacteria grown on NB agar medium was added to 5mM production medium PI (
Glucose 100g, 0JHs)2so<24,5
g, ni82P04 1 g, lJgso, ・71(
200.4g.

Fe50<-711z010mg, Mn5O*-4-
682010mg, biotin 50g, thiamine hydrochloride 20hg, calcium pantothenate 500g, nicotinic acid 500g, soybean hydrolyzate 10g 1 calcium carbonate 30g in 11 parts water, adjusted to pH 1, 2] in a test tube containing the bacteria. and culture with shaking at 30°C for 75 hours.

After culturing, the amount of L-'J gin produced in the medium was measured by a colorimetric method using an acidic-copper ninhydrin reaction.
Shown in the table.

Table 1: Bacterial strain L-lysine production (ig/ml
) P-4 Example 2. Cloning of a gene involved in threonine biosynthesis in Escherichia coli and production of threonine by Corynebacterium glutamicum using the expression of the gene: (11) Cloning of a DNA fragment containing the Escherichia coli threonine operon and production of threonine by Corynebacterium glutamicum Introduction: Cloning is carried out in an E. coli host vector system. pG^22 used as a vector was prepared by the method used by Ahn et al. who created this plasmid [^n, G, et al.
al: J, Bacter+o1. ,
Iil, 400 (1979)], E. coli harboring this plasmid is isolated from cultured cells of 112 substrains. High-molecular chromosomal DNA, which serves as donor DNA, was extracted from cultured E. coli strains of 12 strains (ATCC23740) using Smith's phenol extraction method [S+n1th, M, G
, : ! Jethod 1nEnzy-+nolo
gy, 12. part A, 545 (1967)
).

Restriction enzyme old nd containing pGA22 plasmid DNA 4■
III reaction solution (lom!J) Liss hydrochloric acid, 7mM! J
gCl 2°60mM NaCR, pt(7,5) 6
0.4 units of old ndll (manufactured by Takara Shuzo Co., Ltd., 6 units/
4) was added and reacted at 37°C for 30 minutes, then heated at 65°C for 10 minutes to stop the reaction. There are two Hi positions in pGA22.
nd[[There is a cleavage site, but under the same conditions Hindl
As a result of agarose gel electrophoresis of the 11-digested sample, it was confirmed that it had been cleaved into - fragments. Separately,
Restriction enzyme Hindl containing 1 g of chromosome ON^8J] 1 reaction solution 140 minutes Add 4 units of old ndlI and react at 37°C for 60 minutes, then heat at 65°C for 10 minutes to stop the reaction.

Mix the image chemical compound, add 40 g of T4 ligase buffer, ^T
P (5mM) 40it1, T4 ligase 0.3ttt
Add t and H,0120d and react at 12° C. for 16 hours.

This mixture was mixed with phenol 400 saturated with TESI buffer.
Extract twice with 4 and dialyze against TES buffer to remove phenol.

This ligase reaction mixture was applied to E. coli strain 112 substrain GT-.
3 (J, Bacteriol, 117. 13
3-143 (1974)) (homoserine and diaminopimelic acid auxotrophy).

Competent cells (strains with DNA uptake ability) of the GT-3 strain were obtained using the method of Dagert et al.
11. , et LL: Gene, 6.
23 (1979)). i.e. 100xr
Inoculate 5 g ml of a culture medium (medium containing 10 g of Batatotributone and 5 g of yeast extract in 11 parts of water and adjusted to pH 7.2) supplemented with diaminopimelic acid so that the amount of
Culture at 37°C until the temperature reaches 000.6. Cool the culture solution with ice water for 10 minutes and collect the bacteria by centrifugation. Cooled 0,1
Resuspend in 20ml of M calcium chloride and place at 0°C for 20 minutes.

Resentrifuge the cells and add 0.5 m
1 and leave at 0°C for 18 hours.

200 mL of the above ligase reaction mixture was added to 400 ml of calcium chloride-treated bacterial solution, mixed, kept at 0°C for 10 minutes, and then heated at 37°C for 5 minutes. Next, 9 ml of L medium is added and cultured with shaking at 37°C for 2 hours. After centrifugal washing twice with physiological saline, M9 minimal agar medium (glucose 2 g, NH*C) supplemented with kanamycin equivalent to 12.5 q/ml.
7!1 g, NaJP mouth 46g1Kl12PO43g
SMgSO4・7N200. l gSCaCL・2H
2015a+g, a medium containing 4IIIg of thiamine hydrochloride and 15g of agar in water iz and adjusted to pH 7.2>
and cultured at 37°C for 3 days. The only colony that appeared was ampicillin 254/ml, chloramphenicol 25 u/ml I or kanamycin 25
It has been confirmed that it contains 1/ml and grows even on agar medium.

From the cultured cells of this transformed strain, pG^22 was obtained in the above (1).
Plasmid DNA is isolated by the same method used to isolate plasmid DNA. This plasmid DNA was analyzed by restriction enzyme digestion and agarose gel electrophoresis. As a result, pGH
It has the structure shown as 2. The DNA fragment inserted into pG^22 is an E. coli Obesun-containing DNA fragment 1 that has already been converted to Co55art, P, eL.
al: ! Jolec, Gen.

pGH2 has the same restriction enzyme cleavage site as Genet, Edenyo 39 (1979)].
is confirmed to contain a threonine operon.

Next, a recombinant of pcGll and pGH2 is created. First, pcGll and pGH2 were converted to Bgi and Bam, respectively.
Digest completely with HI under appropriate conditions. Each plasmid DNA2
Mix the digest containing Jig and add 40.0ml of T4 ligase buffer to a total volume of 1200ml. d, ATP (5mM
)40JJI! , T4 ligase 0.2ρ and H,01
Add 20m and react at 12°C for 16 hours. The mixture is extracted twice with 400 ml of phenol saturated with TBS buffer and dialyzed against TBS buffer to remove phenol. Subsequently, Corynebacterium glutamicum strain A201 (LA103) was prepared in the same manner as in Example 1 (1) using 100AI+, a 1:1 mixture of twice as concentrated TSMC buffer and the ligase reaction mixture as donor DNA. After transforming the protoplasts of the derived strain, homoserine, and leucine auxotroph), they are spread on an RCGP agar medium and cultured at 30° C. for 6 days to regenerate and proliferate. The bacteria grown on the entire surface of the agar medium are collected, centrifuged and washed with physiological saline, then reapplied onto a minimal agar medium M1 supplemented with 50 μl/ml of leucine, and cultured at 30° C. for 3 days. From the colonies that appeared, strains capable of growing on NB agar medium containing 12.5 μg/ml of kanamycin or 10 hg/ml of spectinomycin were obtained.

Plasmids are isolated from these transformed strains by ethidium bromide and cesium chloride density gradient centrifugation as described in Example 1 (1).

Using 0.5 g of these plasmid DNAs, the DNA fragments generated by single digestion with various restriction enzymes and double digestion with two types of restriction enzymes were analyzed by agarose gel electrophoresis, and the molecular weight and each restriction enzyme cleavage site in the plasmid molecule were determined. identify - the plasmid obtained from the strain pEth
Restriction enzyme Pst I, Eco
The structure characterized by the RI and Xho 1 cleavage sites is shown in FIG. pEthr 1 was found to have a structure in which a 3amHI fragment containing the threonine operon of pGH2 was bound to pcGll.

As a result of re-transforming Corynebacterium glutamicum LA103 strain using pH:thr I DNA in the same manner as above, homoserine non-auxotrophy and kanamycin and spectinomycin resistance traits were introduced in combination, and these transformations The strain carries a plasmid identical to pEthr 1 characterized by various restriction enzyme cleavage modes. LA due to deletion of homoserine dehydrogenase
The reason why the homoserine auxotrophy of strain 103 is restored to homoserine non-auxotrophy by plEthr 1 is due to the expression of homoserine dehydrogenase located on the E. coli nureonine operon.

(2) p[! Creation of thr 1-bearing strain Corynebacterium glutamicum LA-1, a threonine-producing bacterium derived from 122 strains of Corynebacterium glutamicum
06 (methionine requirement, ABC resistance, α-amino-β
-Hydroxyvaleric acid resistant) pEthr 1 strain is
Obtained by transforming LA-106 protoplasts with pEthr1.

For protoplasts, LA-106 strain is cultured in a semi-synthetic medium SSM supplemented with methionine equivalent to 10 hg/ml, and the cells grown to an OD of approximately 0.6 are treated in the same process as in Example 1 (2). Prepared by: Transformation was carried out in the same manner as in Example 1 (2), and spectinomycin 40
A transformed strain is obtained by selection on an RCGP agar medium containing 0 ml/ml.

The pEthrl-carrying strain is Corynebacter i in the American Type Culture Collection.
umglutamicum K19^TCC39034
It has been deposited as.

(3) Production of threonine by pEthr l strain pEthr of LA-106 strain obtained as above
A threonine production test is carried out on the strain possessing 1 strain (ATCC39034>) and the strain not possessing the strain.One platinum loop of the bacteria produced on the NB agar medium was added to 5+nl of the production medium P2 (glucose 10
0 g, (NHs)zso< 20g, KIL, P
o, 0.5 g.

K2HPO40.5g, Mg5O*'7LOig,
peso4・7H2゜10mg, Mn5On・4~6
H201010ll1 contains 100 grams of biotin, 20 g of calcium carbonate, 100 mg of methionine in 1 liter of water, pH 7
The cells were inoculated into a test tube containing a culture medium adjusted to 2, and cultured with shaking at 30°C for 75 hours.

After culturing, the culture solution was subjected to paper chromatography to develop ninhydrin color, and the amount of L-threonine produced was measured by colorimetry. The results are shown in Table 2.

Table 2 LA-106 6,1 LA-106/pEthr 1 13.4 Example 3 Glutamic acid production by Corynebacterium glutamicum carrying a recombinant plasmid containing the phosphoenolpyruvate carboxylase (PPC) gene of Escherichia coli Production: (1) PPC gene of E. coli (this gene is Gl
Cloning of a DNA fragment containing a gene involved in glutamic acid biosynthesis known to convert u- to Glu+ and introduction into Corynebacterium glutamicum: Cloning was carried out into the E. coli host-vector system. Implemented.

pBR322 used as a vector was incubated with Escherichia coli using the same method used to prepare pGA22 in Example 1 (1).
Isolate from cultured bacterial cells of substrains. The high-molecular chromosomal DNA serving as the donor DNA was injected into Escherichia coli strain 112 (1) in Example 2 (1).
ATCC 23740) is used.

pBR322 plasmid DNA34 and chromosomal DNA
Reaction solution for restriction enzyme 5all (1QmM) containing 9 grams of Lis-HCl, ? +nM! JgCI12.100mM Na
C.R.

7TIIM 2-Mercaptoethanol, 0.01%
Bovine serum albumin, pt17.5 > 200AI+
5all IO small units (manufactured by Takara Shuzo Co., Ltd.) were added to 37°C.
After reacting for 60 minutes at 65°C, the reaction is stopped by heating at 65°C for 10 minutes. Add 4 liters of 741J gauze buffer to this mixed digest.
0β, ATP (5 mM), 0.4 ml of T 4 ligase and 120 ml of water are added, and the mixture is reacted at 12° C. for 16 hours. The mixture is extracted twice with 400 m of phenol saturated with TBS [i-liquid] and dialyzed against TES buffer to remove phenol.

This ligase reaction mixture was injected into E. coli and 12 substrains PPC.
2CGIansdorff, N., Genetic
s, 51. 167 (1965)] (arg-,t
hr-, 1eu-, has-, Ttu-, PPC-.

ST'). Competent cells of the PPC2 strain are cultured in a medium supplemented with 2 mg/ml glutamic acid, and prepared in the same manner as in Example 2 (1) to obtain competent cells of the 6T-3 strain. The transformation was carried out using the ligase reaction mixture 200JL1 described in Example 2 (1).
). Add 9 ml of L medium and culture with shaking at 37° C. for 2 hours to express the expression. Then, with physiological saline
After centrifugation and washing, the mixture is spread on M9 minimal agar medium supplemented with 50 ttg/ml each of arginine, threonine, leucine, and histidine, and cultured at 37°C for 3 days. The colonies that appear are replicated onto an agar medium containing 25 M/ml of ampicillin or 25 M/ml of tetracytalline, cultured at 37°C for 24 hours, and those resistant to ampicillin and sensitive to tetracytalline are selected.

Plasmid DNA is isolated from the cultured cells of these transformed strains in the same manner as described above. As a result of analysis of the plasmid ppcl obtained from one of the transformed strains by restriction enzyme digestion and agarose gel electrophoresis, it was found that the Sal of p8R322
It turned out to be a recombinant plasmid with a genome size of 8.8 and b, in which a DNA fragment of 4.4 and b was inserted into the l cleavage site.

This pPC1 plasmid was used to transform PPC2 strain in the same manner as above, and all of the transformed strains selected for ampicillin resistance were glutamate-nonrequiring and had the same structure as pPC1, which was characterized by the restriction enzyme cleavage pattern. is held. This indicates that the E. coli PPC gene has been cloned onto the PPC1 plasmid.

The cloned PPC gene was transferred to Corynebacterium
pcGll and pPC1 for introduction into Glutamicum
A recombinant is prepared in host E. coli.

ρCGII and pPCl plasmid DNA
Reaction buffer for restriction enzyme Pst I containing silk [20mM Tris-HCl, 10mM! JgCf 2.5Qm former NH,)
2SO, 0.01% bovine serum albumin, I) 87.
Add 4 units of Pstl (manufactured by Takara Shuzo Co., Ltd.) to 53200n, react at 30°C for 60 minutes, and then heat at 65°C for 10 minutes to stop the reaction. Add 40ml of 741J gasse buffer, ATP (5d) 40A1+, T4 to this reaction mixture.
Add 0.2 d of ligase and 120 heads of water, and
Allow to react for 6 hours. After extracting phenol in the same manner as above, phenol is removed by dialysis. This ligase reaction mixture 100. d was used to transform PPC2 strain in the same manner as above.

Isolate the plasmid from the resulting colony using the method described above,
Its size was examined by agarose gel electrophoresis. A plasmid with a size of about 15 to 15 Kb was selected, and the PPC2 strain was transformed again with the plasmid to confirm the presence of the PPC gene. Analysis of the plasmid pEppc 1 obtained from one of the previously transformed strains by restriction enzyme digestion and agarose gel electrophoresis revealed that the genome size was 15, in which pcctt and pPcl were conjugated and linked at the Pstl cleavage sites of both.
．． 6 was clearly identified as the recombinant plasmid of b. Using the pBppcl plasmid DNA thus prepared in Escherichia coli, the LP4 strain derived from Corynebacterium glutamicum L~22 strain is transformed. Transformation was performed in the same manner as in Example 1 (2), and the transformed strain was RCG containing 401 bcg/ml of spectinomycin.
Obtained from colonies growing on P agar medium.

Plasmids isolated from these transformants are digested with the restriction enzymes 5alt or Pst T, or with both, and analyzed by agarose gel electrophoresis to confirm that they retain pεp9C1.

pBppc 1 nursery strain is included in the American Type Medical Record+-- Collection of Corynebacteru
inglutamiqua+ K-18^TCC'3
It has been deposited as No. 9033.

(2) Production of glutamic acid by pEppc 1 strain: LP4 strain derived from Corynebacterium glutamicum 122 strain pEppc 1 strain (^TCC3)
9033) and a non-possessing strain were tested for glutamic acid production. The bacteria grown on the NB agar medium were collected, washed with physiological saline, and then added to 5 ml of the production medium P3C Glucose 50.
g, (NH4)2S04 3 g, urea 3 g, K
HiPOs O, 5g, K2HPO40, 5g, Mg
5On・7HzOo, 5g 5FeSOs'7H,
010mg, Mn5O<・4~6H2o 1011g
, biotin 3, thiamine hydrochloride 50 Lcg, and phenol red 10 mg in pure water lβ and adjusted to pH 7.2] were inoculated into test tubes and cultured with shaking at 30°C. During culture, add 20% urea solution by 0.21111 to 3
2 times and cultured for 40 hours. After culturing, the culture p solution was subjected to paper chromatography, and after coloring with ninhydrin,
The amount of L-glutamic acid produced was measured by colorimetry.

The results are shown in Table 3.

Table 3 P-4 10.1 Example 4. Brevibacterium flavum^TCC+
Cloning and expression of the anthranilate synthase gene of 4067 in Corynebacterium glutamicum: Chromosomal DNA of Brevibacterium flavum ATCC 14067 was produced using jJR in the same manner as in Example 1 (1).

pCε53 used as a vector is isolated from cultured cells of 122 strains of Corynebacterium glutamicum in the same manner as in Example 1 (1) for isolating pcc;11. pCε53 is a plasmid p of Corynebacterium glutamicum previously disclosed by the present inventors.
CG1 [Patent application 1981-18101 (Japanese Patent Application No. 57-13
4500) : ] and E. coli plasmid pGA22 E
An, G, et al: J0 acteriol
, 140.400 (1979)]. For details, there is only one 8g on pCGI! ■There are two B locations on the cleavage site and pG^22.
The amH [amH cleavage site, which is not within the tetracycline resistance gene] and the lamHI cleavage site, were ligated using the same cohesive ends of both restriction enzymes.

pCε53 contains selection markers such as the kanamycin resistance gene derived from pGA22, and has one cleavage site for the restriction enzyme Sal I.

Restriction enzyme Sal I reaction solution 20 containing pCE53 plasmid DNA 3R and chromosomal DNA 9q prepared above
Add 5all in lO units to 0 d, react at 37°C for 60 minutes, and then heat at 65°C for 10 minutes to stop the reaction. This mixed digest was added with T4 ligase buffer 404, A
T P (5111M) 40Al! , added T4 ligase 0.4m and H,0120JIjl, 12
React for 16 hours at °C. The mixture is extracted with 400 m of phenol saturated with TBS buffer and dialyzed against TEStl buffer to remove the phenol.

This ligase reaction mixture is subjected to transformation.

An anthranilic acid auxotrophic mutant strain LA105 (anthranilic acid synthase-deficient mutant strain) derived from Corynebacterium glutamicum strain 122 is used as a recipient bacterium to be transformed. Anthranilic acid auxotrophic mutants are obtained by selecting bacteria that cannot grow on M1 agar medium but can grow on M1 agar medium supplemented with anthranilic acid (equivalent to 30 g/ml) through conventional mutation treatment. Ru. L.A.
Preparation and transformation of protoplasts of strain 105 are carried out in the same manner as in Example 1 (2), except that the growth medium NB contains anthranilic acid equivalent to 1 OOJ 1 g/ml. The transformed strain was treated with kanamycin 200 g/ml.
Colonies are selected as growing on RCGP agar medium containing the corresponding amount. A transformed strain that can grow on M1 agar medium is obtained from the colonies that appear.

Plasmid DNA is isolated from the cultured cells of these transformed strains in the same manner as described above. Analysis of plasmid pTrp2-3 obtained from one of the transformed strains by digestion with various restriction enzymes and agarose gel electrophoresis revealed that the 5alfDNAΔ cleavage fragment of b was inserted at approximately 7.1 into the unique 5ail cleavage site of pcE53. It turned out to be a plasmid.

When the LA105 strain was re-transformed using pTrp 2-3 in the same manner, tryptophan was 100 g/m
RCGP containing l and kanamycin 400 g/ml
Colonies growing on agar medium simultaneously become anthranilic acid non-auxotrophic and they carry a plasmid identical to pTrp 2-3 as determined by Sall's cleavage pattern.

The above results indicate that approximately 7. l to b Sa
l [) The NA section contains Brevibacterium flavum^
It is shown that the gene encoding the anthranilate synthase of TCC14067 exists and is expressed in the Corynebacterium glutamicum LA 105 strain.

The pTrp 2-3 stock is American type.
Corynebacter in culture collection
+umgglutamicum K20^TCC3903
It has been deposited as No. 5.

The same treatment as above was performed using plasmid pCE52, and Brevibacterium flavum ATC (1:1406
A plasmid pTrp 4-3 containing a gene encoding anthranilate synthase 7 is obtained.

pEc52 is a plasmid pcG1 of Corynebacterium glutamicum that the present inventors previously disclosed (Japanese patent application No. 5
6-18101 (JP 57-134500)) and Escherichia coli plasmid ρG^22 [^n, G, et-a
L: J, Bacteriol.

140, 400 (1979)]. For details, see the Bgl■ cleavage site, which has only one site on pCGl, and the Bal, which has two sites on ρG^22.
1ll (l cleavage site, [lamHl cleavage site in the tetracycline resistance gene) is ligated using the same cohesive ends of both restriction enzymes. ρCε52 is ρG
It has a selection marker such as the kanamycin resistance gene derived from ^22, and has one cleavage site for the restriction 6r element 5all.

pcE52 is isolated from 122 cultured strains of Corynebacterium glutamicum harboring pCε52 in the same manner as in the method used to isolate p[G11 in Example 1 (1).

” As in Section 1, 36 strains of tryptophan-producing Corynebacterium glutamicum (FBRM BP-451
) is transformed with pTrp4-3. The obtained transformed strain was stored in the American Type Culture Collection as Corynebacterium glutami.
It has been deposited with cum as 31°^TCC39280.

20. Corynebacterium glutamicum carrying pTrp 2-3. ^TCC39035 and pTrp 4-
3 shares held are 31. The L-)ributophane production test according to TCC 39280 is conducted as follows.

The bacterial strain was cultured in NB liquid medium at 30°C for 16 hours with shaking, and 5 ml of bacterial solution Q was added to 5 mM production medium P4C blackstrap molasses 100
g/J! , (NH4) isO < 20 g/I!
, Kl (zPO* 0.5 g/lSK, 11 questions 4
0.5g/i', MgSO4・7H200, 25g/
f, CaCO320g/1, pl+'7.2
) in a test tube and cultured with shaking at 30°C for 96 hours.

After culturing, the culture v solution is subjected to vapor chromatography, and after ninhydrin color development, the amount of L-1 butophane produced is determined by colorimetry.

As controls, LA-105 strain and Shi^R-1 strain are treated in the same manner.

The results are shown in Table 4.

Table 4 LA-105 LA-105/pTrp 2-3 (20.^TCC3
9035) 0.34LAR-10,48 L^R-1/pTrp 4-3(ni31.^TCC39
280) 1.12 Example 5. Construction of pCB101: Separation of (+) ρCGII and 10 ρCGI
I is a Corynebacterium that carries this plasmid.
Glutamicum LA103/ρCGII (^TCC390
22) was grown in 400ml NB medium until the OD reached approximately 0.8, and from the cultured cells, pCG
Isolate in the same manner as 2 was isolated.

pUollo is based on the method of Gryczan et al. [Gryczan et al.
.

T, J, et al, 2 J,
Bacteriol,, 134. 318 (1978
)], Bacillus subtilis BR1s'/p[IB''' (Proc, Na
tl.

^cad, Sci, USA, 75.1423 (1
It is isolated from the cultured bacterial cells of 978)).

(2) In vitro recombination of ρCGII and puetto Restriction enzyme 8gfn reaction buffer (10a+M) containing 2■ of the pcGll plasmid DNA prepared above! jsu hydrochloric acid, 7mM MgCj! 2.60a+M NaCL
7mM 2-1 Captoethanol, I) H7,5
) 2 units of Bgj for 100ρ! II (manufactured by Zenshuzo Co., Ltd.)
6 units/JdI) and reacted at 37°C for 60 minutes. In addition, a restriction enzyme containing 2ttg of pUB110 plasmid DNA [1amHI reaction buffer (10mM), Lis-HCl, 7nM MgCL, loOmMNaCl,
2111M mercaptoethanol bovine serum albumin, pH 8.0) l O OJIII with 2 units of Ba
mHI (manufactured by Zenshuzo Co., Ltd., 6 units/JIJ1) was added,
React for 60 minutes at 7°C.

Mix both restriction enzyme digests and add 40% T41J gauze buffer.
ρ, ATP (5a+M)40IdI, T4 ligase 0, 2, and H,0120m are added and reacted at 12°C for 16 hours. The mixture is extracted twice with phenol 4004 saturated with TES buffer to remove the phenol which has been dialyzed against T8SM buffer.

(3) 2x higher concentration of TSMCr than pc8101! 1. Corynebacterium glutamicum LA 103 was transformed using the 1:1 mixture 1004 of the new solution and the above ligase reaction mixture as donor DNA in the same manner as in Example 1 (3), and a kanamycin-resistant strain was obtained. select. Treat the emerging colonies with kanamycin 1 2. 5 Itg/ml or 100x/ml of subectinomain were replicated on an NB agar medium containing 100x/ml of subectinomain, and 3 double-resistant transformants grown by culturing at 30°C for 2 days were arbitrarily selected and placed on the same agar medium. Purify above. These three strains were grown in a 400ρNB medium until the OD reached approximately 0.8, and after harvesting, the cultured cells were used in Example 1 (1).
Plasmids are isolated by ethidium bromide-cesium chloride density gradient centrifugation as described. Plasmid DNA of 30-35M can be obtained from any of the transformed strains.

These plasmid DNAs were analyzed by restriction enzyme digestion and agarose gel electrophoresis in the same manner as in Example 1 (3), and the molecular weight and restriction enzymes Pst I, EcoR I, Hinc
Identify the breakpoints of ll and Bgi. The plasmids of the three strains all have a structure in which pcGll and pUBllO are harmoniously linked, and two of them have the structure shown as pcBlol in FIG. 2, but the other type has the binding direction in the opposite direction.

Transformants with either plasmid are ρCGII
It has a subectinomycin resistance trait derived from pU8110 and a kanamycin resistance trait derived from pU8110.

The kanamycin-resistant transformant obtained by retransforming Corynebacterium glutamicum LA103 using these plasmid DNAs simultaneously acquired spectinomycin resistance and was characterized by various restriction enzyme cleavage patterns. Contains the same plasmid as the donated plasmid.

Example 6.

Corynebacterium glutamicum, Corynebacterium no. Production of L-histidine by Brevibacterium lactofermentum: (1) Corynebacterium glutamicum [156
Preparation of strain chromosomal DNA and plasmid pcG11; 1.
2. 4-) Chromosome D of Corynebacterium glutamicum strain C156 (FεRM OP-453) which is resistant to lyazole-3-alanine and has histidine-producing ability.
N8 is prepared in the same manner as in Example 1 (1).

On the other hand, pcGll used as a vector plasmid is
Corynebacterium glutamicum strain 122 induced-like LA103 with pcG11 strain LA 103 /IIC
Example 1 (I) from G I l (^TCC39022)
Isolate in the same manner.

(2) Corynebacterium glutamicum C156
Cloning of genes involved in histidine biosynthesis of strains; pCG l 1 plasmid DNA3 prepared in 1.
Restriction enzyme Bg7 containing Bg and 9 μg of the above chromosomal DNA
! Reaction solution for II [10mM] Squirrel (pH 7.5)
), 7mM MgCL, 60mM Race, 7
mM2-mercaptoethanol) 200d in lO units [1gj! II (manufactured by Zenshuzo Co., Ltd.) and heated to 60°C at 37°C.
After reacting for a minute, the reaction is stopped by heating at 65° C. for 10 minutes. This mixed digest was added with T4 ligase buffer (Tris 2
00mM.

MgClz 66mM, dithiothreitol 100
mM, 987.6) 40m, 5ml TP solution 40d
, T4 ligase (manufactured by Takara Shuzo Co., Ltd., 1 unit/111) 0.3
Add tdl and 120 ρ of water and react at 12° C. for 16 hours.

The T4'll gauze reaction mixture is used to transform Corynebacterium glutamicum strain 1833 (histidine auxotrophic, lysozyme sensitive).

Transformation is performed using protoplasts of the 11133 strain. Protoplasts are prepared in the same manner as in Example 1 (2).

Transfer 0.5ml of protoplast suspension into a small test tube.25
Centrifuge for 5 min at 00 × g and add 73MC buffer (1
0d magnesium chloride, 30ml + calcium chloride, 5
0mM) Squirrel, 400mM sucrose, pH 7.5>
After resuspending in 1 ml and washing by centrifugation, add TSIJC buffer 0.
．． Resuspend in 1 ml. Add to this suspension a double concentration of 73M.
Add 100 pieces of a 1:1 mixture of C buffer and the above ligase reaction DNA mixture and mix, then add 0.81 of a solution containing 20% PE G6.000 in TSIIC buffer and mix. After 3 minutes, RCGP medium (p) 17.2)
Add 2 ml and centrifuge at 2.500Xg for 5 minutes to remove the supernatant and collect the sedimented protoplasts.
ml of RCGP medium, then 0.2 ml was added to RCGP agar medium containing 400 μl/ml of spectinomycin (
RCGP medium containing 1.4% agar, pH 7.2)
and culture at 30°C for 7 days.

Spectinohuicin-resistant colonies grown on the selection plate were collected, and after centrifugal washing twice with physiological saline,
The mixture is spread on a minimal agar medium M1 containing 100 g/mI of subectinomycin and cultured at 30°C for 2 days to select transformants that are resistant to svectinomycin and are not auxotrophic for histidine.

A plasmid is isolated from one of the transformed strains by ethidium bromide/cesium chloride density gradient centrifugation as described in Example 1 (1). DNA produced by single digestion with various restriction enzymes and double digestion with two types of restriction enzymes
The fragments are analyzed by agarose gel electrophoresis to identify the restriction enzyme cleavage mode of this plasmid DNA. This plasmid was named pP418. pPH8 is 8gI of pcGll! This structure has a DNA fragment of approximately 10.6 Kb inserted into the fI cleavage site.

Furthermore, using pPH8DNA, strain H33 [LH33
Parent strain (histidine auxotrophy, lysozyme resistance): FE
RlJ BP-452], all of the transformed strains selected as spectinomycin-resistant strains were non-histidine auxotrophic.

From these results, it is clear that the gene involved in histidine biosynthesis of the histidine-producing bacterium Cl56 strain has been cloned.

Cloning of genes involved in histidine production can also be performed from the beginning using strain 833 as a host strain.

(3) Production of L-histidine by a Corynebacterium glutamicum strain carrying pPH8: Corynebacterium glutamicum L-103 strain (FER
P-5947, ATCC31866) to pPH8D
Transformed with NA, spectinomycin 400Jig/
Transformants resistant to the same drug are selected on an RCGP agar medium containing ml. After purifying the obtained transformant, plasmid isolation and structural analysis were performed in the same manner as above, and it was confirmed that the plasmid had the same structure as pPH3.

pPH8 carrying strain Corynebacterium glutamicum LA
103/pPH8 is American type culture: Corynebacter in IL/section
iumglutamicum K32. ^TCC392
It has been deposited as No. 81.

Corynebacterium glutamicum L^103/pcG
11 (ATCC39022) and the same LA103/p
P) L-histidine production test of 1 g (ATCC39281) is conducted as follows.

Each of the above bacteria, cultured overnight at 30°C on NB agar medium, was cultured in 5 ml of production medium P5 (12% molasses) supplemented with 200 g/ml of arginine and methionine.
(as sugar), KH, PO, 0.2%, K2HPO40
,1%, 11g 5 mouths, 7H200,05%, NaCj
! 0.25%, (N)1. ) 2SO42.3%,
Inoculate in 0.2% urea, 2% CaCL, pH 1.4 (adjusted with 7 ml). After culturing at 30°C for 75 hours,
Colorimetric method using sulfanilic acid (Pauly) reagent to measure the amount of L-histidine produced in the medium CH, Pauly.

1 (oppe-3aylers; Z, Physi
olo, Chem, 42.508 (1904)
, 94.284 (1915)).

The results are shown in Table 5.

Table 5 LAI03/pcG11 0LA103/p
pH8 (K32) 2.6 (4) p P H
Production of L-histidine by Corynebacterium no\-culis, Brevibacterium flavum and Brevibacterium lactofermentum harboring 8: Corynebacterium herculis^TCC13868
, Brevibacterium flavum^TCC14067 and Brevibacterium lactofermentum ATC
In order to cause C13869 to carry plasmid pPH8, transformation is performed using each strain as a recipient strain.

Each strain was grown in 53M medium, and when OD660nf11 reached 0.2, 0.3 units/ml of penicillin G was added.
Add so that Cultivation is continued, and when 00660 nm increases to 0.6, bacteria are harvested and protoplasts are formed in the same manner as described in the culture soil in RCGP medium containing 1 mg/ml lysozyme. Transformation is performed using pPH8 according to the method described above, and the transformed strain is selected as a colony growing on an RCGP agar medium containing 400 μl/ml of spectinomycin.

Plasmid DNA was prepared from the cultured cells of the purified spectinomycin-resistant transformant according to the description in JP-A-57-183799 and JP-A-57-134500.
It is confirmed by restriction enzyme cleavage that it has the same structure as PH8. From the above, plasmid pH13, which is a derivative of plasmid pcG11, can be replicated in Corynebacterium herkyulis, Brevibacterium flavum, and Brevibacterium lactofermentum, and plasmid pcG11 is widely used in these bacterial species. It turns out that it can be used.

33 for Corynebacterium h-quinilis, 34 for Brevibacterium flavum, and 35 for Brevibacterium lactofermentum, which are pPH8-bearing strains.
TCC39282, 39283 and 39 are in the American Type Culture Collection, respectively.
It has been deposited as No. 284.

L-histidine production tests using these strains are conducted as follows.

One platinum loopful of each of the pPH8-carrying strain and its parent strain, which were cultured overnight at 30° C. on NB agar medium, is inoculated into 5 ml of production medium P5. After shaking culture at 30°C for 75 hours,
L-histidine production in the medium is determined colorimetrically by the Bosey method. The results are shown in Table 6.

From the above, the genes involved in histidine production derived from Corynebacterium glutamicum are
In addition to Glutamicum, it was expressed in Corynebacterium herculis, Brevibacterium flavum, and Brevibacterium lactofermentum, and it was clear that it contributed to the production of histidine.

[Brief explanation of the drawing]

FIG. 1 shows the restriction enzyme map of plasmid pGH2. Figure 2 shows restriction enzyme maps of old and old Plasmid C. FIG. 3 shows a flowchart for the construction of plasmid pEthr1. Patent applicant (102) Kyowa Hakko Kogyo Co., Ltd. No. 6
Table Diagram: Furinidae BIIFTIII/8N] I Genus ■ namI4X/RqIX1

Claims (1)

[Claims] DNA that belongs to the genus Corynebacterium or Brevibacterium and contains a gene that has the activity of converting a microorganism that exhibits a histidine auxotrophy and is derived from a species of the genus Corynebacterium or the genus Brevibacterium into a strain that does not require a histidine auxotrophy.
Cultivating a microorganism carrying a recombinant DNA of the fragment and a vector DNA capable of autonomous replication in a Corynebacterium or Brevibacterium species in a medium, producing and accumulating L-histidine in the culture, A method for producing L-histidine, which comprises collecting L-histidine from the culture.