JPH02472A - Production of l-glutamic acid - Google Patents

Abstract

PURPOSE:To increase productivity of L-glutamic acid by culturing microorganism having specific recombinant DNA. CONSTITUTION:A DNA fragment belonging to Corynebacterium or Brevibacterium and containing gene coding phosphoenolpyruvic carboxylase derived from Escherichia coli and a vector DNA capable of autonomic replicating in Corynebacterium mycetoma or Brevibacterium mycetoma (e.g., pCG11 ATCC39022) are cut after recognition of specific base sequence parts on double stranded DNA by using restriction enzyme, then connected by T4 phage DNA ligase to obtain a microorganism having recombinant DNA. Next, said microorganism is cultured in medium and L-glutamic acid is generated and accumulated in cultured substance, then L-glutamic acid is gathered from the cultured substance.

Description

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

More specifically, the present invention provides a recombinant product of a DNA fragment containing at least one gene and a vector DNA, and
A transformed strain obtained by transforming a host strain selected from microorganisms belonging to the genus Corynebacterium or Brevibacterium using a recombinant DNA in which at least one of the NAs is foreign to the host strain is used as a medium. The present invention relates to a method for expressing a gene, which is characterized by culturing and expressing the trait of the gene.

Recombinant gene technology is a big deal! established as a bacterial host,
To date, it has been shown that it is possible to produce bebutides and vaccines such as somatostatin, insulin, human growth hormone, human interferon-α, human interferon-β, and mouth-to-mouth disease vaccine. Although Escherichia coli is considered to be sufficient as a host for the expression of these highly bioactive bebutides 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 better 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). In order to improve the industrial productivity of substances such as antibiotic π using recombinant DNA technology, it is necessary to devise ways to apply the same technology to each of the conventionally used producing bacteria.

Corynebacterium glutamicum is an industrial amino acid produced by microorganisms! ! The microorganism that was first used in the production of 2, and has since been used to produce glutamic acid, lysine, alanine, histidine, tryptophan, tyrosine, phenylalanine, threonine, isoleucine, valine, leucine, glutamine, proline, arginine, etc. by coryneform bacteria including the genus Corynebacterium. Industrial production of amino acids has been developed, and today most amino acids are produced by microorganisms.

Therefore, the establishment of recombinant DNA techniques 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 (3) (1) and (2) above. Creation of recombinant DNA by annealing by mixing the products and ligation using DNA ligase (4) Introduction of the above recombinant DNA into a host strain (transformation) (5) Selection of a recombinant containing the target gene and each step of purification of the selected clone. The efficiency of creating a recombinant strain obtained in this way can be said to be the product of each of the above steps, so it is important to prepare a means to verify each step, know the efficiency of each step, and improve it. It is not possible to obtain a transformed strain capable of expressing the target gene. Furthermore, even if a transformed strain having recombinant DNA containing the target gene is obtained in this way, if the gene is foreign to the host strain, various r4 desires may occur during expression of the gene. It is known that there is [“Chemistry and Biology” 18.110~
118 (197g)) its expression is very difficult to achieve.

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 many genes, and to establish an efficient transformation system. Furthermore, it is necessary to establish a method to eliminate the above-mentioned 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 [Special conduit 1981-58186 (Japanese Patent Application Laid-Open No. 57-18379)
9), 56-58187 (Unexamined Japanese Patent Publication No. 5718649)
2), 56-65777 (Unexamined Japanese Patent Publication No. 57-1864)
89> 3° Therefore, the present inventors applied the already known in vitro DNA recombination technique (U, S, Patent 4.237, 224) to the plasmid vector.
When a DNA fragment containing an exogenous gene involved in amino acid biosynthesis was ligated using the DNA fragment, and the developed transformation system was used to transform Corynebacterium glutamicum strain 122 or its derivative strain, We have completed the present invention by discovering that the foreign gene is expressed in the host and can be used to increase the production of useful substances such as amino acids.

The present invention will be explained in detail below.

The present invention is a recombinant product of a DNA fragment containing at least one gene and a vector DNA, and has a small amount of both DNAs (one of which is foreign to the host strain).
The present invention provides a method for culturing a transformed strain obtained by transforming a host strain selected from microorganisms belonging to the genus Corynebacterium or Brevibacterium using A, and expressing the trait of the gene.

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

Examples of genes derived from eukaryotes include genes encoding peptides of mammals, particularly humans, such as interferon, insulin, and growth hormone. Genes derived from prokaryotes include bacteria, especially the genera Et/Elichia, Corynebacterium, Brevibacterium,
Genes derived from strains of bacteria belonging to the genus Bacillus or Staphylococcus, including genes involved in cell metabolism, especially synthetic activity. Cellular metabolic or synthetic activity refers to the synthesis of amino acids, vitamins, nucleic acids, or antibiotics, as well as the metabolic systems involved in the synthesis. Synthetic activity is preferred.

Furthermore, when the amino acid composition of the target peptide, protein, etc. is known, the corresponding DNA can also be synthesized and used. For example, the DNA synthesis method is
akura et LL 5science 19J! ,
1056 (1977).

The vector used in the present invention must be compatible with the host bacterium (c
It must be able to reproduce autonomously with ompat 1ble). 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. 56-181]
01 (JP-A-57-134500) ), ρCG2 [
Patent application No. 56-133557 (Unexamined patent application No. 58-35197)
)], pCG4 [Japanese Patent Application No. 56-58186 (Japanese Unexamined Patent Publication No. 57-183799)), pCE53, pCE54,
pcGll, pCCoI2p[! Examples include thrl.

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 F[! RM-P ATCCp
CG 1 5865 3180Bp
CG 2 5954 31832p C
G 4 5939 31830p CE
54 39019p CG II
39022p CBlot
39020pEthr l
39021 Preferably pcGll, pCE
54 is used. pcall was first disclosed by the present inventors [Japanese Patent Application No. 56-18101 (Japanese Unexamined Patent Publication No. 57-134500)
)) Corynebacterium glutamicum 225-57 (ATCC31808, FER
Plasmid pcIl isolated from M-P5865);
Restriction enzyme Bgj in l! Corynebacterium glutamicum 225-25 at the only cleavage site of II.
0 (ATCC31830, FERM-P5939) of streptomycin and/or spectinomycin resistance (Sm”/5p
This is a plasmid in which the BamH1 fragment containing the ec') gene is joined using the same cohesive ends of both.

pcGll is a plasmid of approximately 6.8 Kb with BgjNI and Pstl as single restriction sites and Sm'
/5 pec”.

pEC54 can be created as follows.

First, pcc2 was extracted from the preserved bacterium Corynebacterium glutamicum 225-218 strain (FERIJ-P5954).
, ATI:C31832>, patent application 1982-
133557 (Japanese Unexamined Patent Publication No. 5835197), pc^22 is isolated from cultured E. coli cells 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.
Apply N A IJ gauze. Obtaining a recombinant plasmid in which the two plasmid molecules are conjugated and linked from this DNA mixture is carried out by first obtaining the traits of the Corynebacterium genus or Brevibacterium spp. 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. 56-65777 (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 with a concentration of drugs (usually tetracycline 0.4-1.6 Jtg/rnl, chloramphenicol 2.5-5 Jtg/ml) that prevents the recipient protoplasts from reverting to normal cells and proliferating. Kanamycin l OO-
Either isolate the reverting colonies on a hypertonic agar medium containing 800 ml/ml) or - scrape them after non-selectively reverting to normal cells on a regeneration medium and transfer this resuspension to recipient bacteria. Concentrations of drugs at which normal cells cannot grow (usually tetracycline 0.5-4μ/1 width, chloramphenicol 2-15μ/ml, and kanamycin 2-25μ)
(μg/ml) by isolating colonies growing on an agar medium containing 1 μg/ml). 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. 56-18101 (Japanese Unexamined Patent Publication No.
7-134500) and Japanese Patent Application No. 56-65777 (Japanese Unexamined Patent Publication No. 57-186489), it is possible to purify single #I from cultured bacterial cells, and further to digest with various restriction enzymes to produce DNA Wt fragments. The structure can be determined by the standard method of analysis using agarose gel electrophoresis.

pCε5 is a plasmid isolated from one of the transformed strains.
It is 4.

pCε54 is a plasmid approximately 14.5 Kb in size containing εcoRI s Sal I, S as a single restriction quality.
There are ma I, Xho l, etc., Tc', C+
++”, giving a phenotype of Km”.

Xhol is present in the crab gene, and selection can be performed by so-called insertional inactivation (a phenomenon in which expression of the relevant phenotype is prevented by insertion of a DNA fragment).

Collection of plasmids from plasmid cultured bacterial 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 56-133557 (Japanese Unexamined Patent Publication No. 58-3519
It may be carried out according to the method described in 7).

Production of a recombinant between a DNA fragment containing a gene and vector DNA can be carried out by making full use of known in vitro recombinant DNA techniques.

In vitro DNAΔ 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 the 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 protruding single strand, while 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 the Corynebacterium or Brevibacterium species, the gene of interest is once cloned in a host-vector system of another microorganism, such as E. coli, and then the Corynebacterium or Brevibacterium species is transformed. A recombinant can be obtained by producing a recombinant with a vector of a bacterial species in a test tube, then transforming a Corynebacterium or Brevibacterium species, and then selecting and separating the transformed strain in the same manner as above. can.

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

S, N, Cohen, ,r LL U, S,
Patent 4.237.224, Genetic Manipulation Experimental Method [written by Yasuyoshi Takagi, Kodansha Sun Entifice/Re (1)
980) ],! Jethod inεnzymolo
gy68, Recombinant DNA ed
ited by Ray 11u, ^cadem+c
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 FERM-P 8TCC Corynebacterium glutamicum L-155946
31834 Corynebacterium 11-curis L-
103594731866 Previbacterium L. deiparicarum L-204594831867 Previbacterium lactofermentum 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 lysozyme, 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 pure water II! and adjusted to pH 7.2) or semi-synthetic medium SSM [glucose 10g
, NH4Cl 4 g, urea 2 g, yeast extract 1
gSKH2PO, 1g, K211P043g, Mg
CL・6H200,4g, Fe5o44u2o
10mg5 MnS<・4-6HJ0, 2 mgS
ZnSO<'7H20o, 9 mg, CuSO4'
511zOo, 4 mg.

Na2B1 mouth i・l0LD 0.0 9ff1g,
(NHs) slJotOz<・4H*00,04
mg, piotine 30■, thiamine hydrochloride 1mg in 1 water
A medium containing A.I and adjusted to pH 1 or 2] is used.

Microorganisms are inoculated into this medium and cultured with shaking.

The absorbance at 660 nm (0) was measured using a colorimeter, and the concentration was adjusted to a concentration of 0.1 to 2.0 units/m I in the culture solution at the beginning of the logarithmic growth phase (OD = 0.1-0.4). A penicillin such as penicillin G is added to the solution. The culture is further continued, and when 00 increases to 0.3 to 0.5, the cells are harvested and washed with 33M medium. The cells are then cultured in a suitable hypertonic medium, such as PFM medium (0.4 M sucrose in 2-fold dilution of SSM, MgC12.6H300, OI
containing M and adjusted to pH 7.0 to 8.5) or RCG medium [glucose 5g 1 casein hydrolyzate 5
g, yeast extract 25 g, KJPo, 3.5 g
, KLPO41,5gSMgC'i・6t+,o
0.41gFe5Os'711z0110l1. M1
1SO4-4~61120 2 mg, ZnSO4'
71120 0.9 ff1g, Cu5O*・51(z
Oo, 4 mg, Na2B40t ・IOL mouth 0, 09
mg, (NHJsMOtO7a・4L00.04
gig, biotin 30g, thiamine hydrochloride 2mg
, containing 1.35 g of disodium succinate in water 11,
Re-suspend in a medium adjusted to pH 7.0-8.5. Lysozyme is added to this cell suspension at a final concentration of 0.2-10/ml and reacted at 30-37°C. Protoplast formation progresses as the reaction time progresses, and its progress can be observed using an optical microscope. The time required for most cells to become protoplasts under the microscope varies depending on the concentration of penicillin added during cell culture and the concentration of lysozyme used, but is 3 to 24 hours under the above conditions.

The generated protoplasts rupture and die 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 0.3-0.8M disodium succinate and 0.5-6% polyvinylpyrrolidone (molecular fftlO,000 or 40,000). ) is preferably used.

Usually, a semi-synthetic medium RCGP medium CRCG medium supplemented with 3% polyvinylpyrrolidone (molecules ff11 (1,000) and 1.4% 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 a size that can be harvested.

Regeneration of protoplasts in RCGP medium varies depending on the bacterial species, concentration of penicillin added during culture, and concentration of lysozyme treatment, but lo-
The efficiency is between 2 and 10-'.

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 certain polyethylene glycol (PEG, average molecular weight: 1.540 to 6.000) or polyvinyl alcohol (PVA, molecular weight: 500 to 1,500) and a divalent metal cation. Stabilizers that provide hypertonic conditions may be those commonly used to maintain protoplasts of microorganisms, such as sucrose or disodium conolinate. Usable concentration ranges for PEG and PVA are 5-60% and 1-20% final concentration, respectively. The two flIf metal cations were used at final concentrations of 1-100 mM (a 4 N-1
Mg", Mn", Ba"5 r +-, etc. are effective and can be used alone or in combination. The treatment temperature is 0 to 2
5°C is preferred.

3) Return of protoplasts to normal cells Regeneration and selection of transformed strains Regeneration of protoplasts transformed with recombinant DNA is carried out using a hypertonic solution containing disodium succinate and polypyrrolidone, similar to the regeneration of protoplasts described above. Spread protoplasts on an agar medium (e.g. RCGP medium),
This is carried out by culturing at a temperature at which normal cells can grow, generally 25 to 35°C. Transformed strain uses donor DNA
It can be obtained by selecting for the traits that genes derived from the bacteria impart to the bacteria. Selection based on the acquisition of this characteristic trait may be carried out simultaneously with regeneration on a hypertonic agar medium.
Alternatively, the regeneration may be performed by first regenerating non-selectively and then collecting regenerated normal cells on an ordinary hypotonic agar medium.

When using a lysozyme-sensitive strain shown as a specifically preferred host strain in the present invention, the transformation is performed in step 1) except for direct lysozyme treatment of cells grown in culture without penicillin treatment in step 1). It may be carried out in the same manner as in 1) to 3). When using lysozyme-sensitive microorganisms, the transformed strain is ~0-4 per regenerating microorganism.
Obtained with a high frequency of 10-'.

The transformed strain can express the characteristics of the introduced recombinant DNA by culturing it in a conventional nutrient medium. Gene DNA or vector DNA in recombinant DNA
When properties derived from A are imparted, drugs may be added to the culture medium 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, bebutides, 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 Escherichia coli, the phosphoenolpyruvate carbokinase (ppc) gene, and the gene pU8110 expressed in Bacillus subtilis and Staphylococcus [Keggins,
! J., et al., Proc., Nat.
lAcad, Sci,, 口, S, ^, 7L 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 Bacterium coli 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, as is well known, all genes have similar sites at the base sequence level that are necessary for accurate initiation of transcription and translation, Corynebacterium glutamicum is an example. It can be easily inferred that the transcription/translation initiation sites of genes other than those of genes recognized are also recognized and expressed.

Although the so-called glutamate-producing bacteria that have a high glutamate production ability have the same main mycological properties, each researcher has assigned various enclaves and genus names due to their industrial importance. Even the genus Corynebacterium and Brevibacterium are diverse. However, it has been pointed out that these bacterial groups are the same bacterial 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, and it is clear that they are very closely related microorganisms (Komatsu, Y.: Report of
theFermentative Re5earch
In5titude, No. 55. 1 (1
980), and 5uzuki, K., an
eko, T,, and omagata.

: Int, J, 5yst, Ba
cte mouth o1. ．． 31. 131 (1981)].

In this specification, since hosts that can be used in recombinant DNA experiments are regulated, the usefulness of the present invention is that the method for inducing Corynebacterium glutamicum-22 as a host is shown, but based on the above facts, glutamic acid It can be easily inferred that this method can be applied directly to all production bacteria. Recombinant D
In order for NA to be stably retained and expressed in these bacterial species, slight differences in host bacterial properties such as DNA homology are not a problem, and these bacterial species must be able to autonomously replicate the plasmid and express the transgene. It suffices if it has a function that enables it. However, the fact that these bacterial species share both of these functions was previously disclosed by the present inventors [Patent Application No. 1983].
Plasmid pCG4, which is isolated from Corynebacterium glutamicum 225-250 and has a streptomycin and/or spectinomycin resistance gene, is used to treat Corynebacterium and Brevibacterium species. , can also replicate in glutamate-producing bacteria, and
The resistance gene is expressed.
(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 Corynebacterium and Brevibacterium species.

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^TCC21543 in Corynebacterium glutamicum and production of lysine by Corynebacterium glutamicum using the expression of the gene: (1) Corynebacterium glutamicum^TC [
:21543 chromosomal DNA and vector pcG11 m
Manufactured by: Corynebacterium glutamicum ^TCC130
The chromosomal DNA of Corynebacterium glutamicum ATCC21543, a lysine-producing mutant strain derived from 32 and resistant to the lysine analog 5-(2-aminoethyl)-cysteine (hereinafter abbreviated as AEC), was extracted as follows. isolate

40 Qml semi-synthetic medium SSM [glucose 20g.

(NH4)zsO* lOg, urea 3g, yeast extract I
g.

II, PL 1 gSJl! 2'6HzOo, 4
gSFeSOs'7Ht010II1g, ! ns
O*・4-6) 1zOo, 2 mg, Zn5On'
7H20o, 9mg%Cu5L'5HaOo, 4 m
g%NaJ40t-IQIL20 o, 09mg,
(NL) sMoJ□, '4LOo, 04 alg, 30 μg of biotin, 1 g of thiamine hydrochloride lff, 11 g of water
Add 1 threonine to the medium containing the
The seed culture is inoculated into a medium supplemented with 0.00 I1g/ml and cultured with shaking at 30°C. 66 on Tokyo Photoden Colorimeter
Measure the absorbance (00) at 0 nm, OD 0.2
At this point, penicillin G is added to the culture solution at a concentration of 0.5 units/ml. The culture is further continued until the OD reaches approximately 0.6.

Collect bacteria from the culture medium and use TESIJ! Street liquid ((1, (
13M tris(hydroxymethyl)aminomethane (hereinafter abbreviated as Tris), 0.005M EDT^, 0.05
After washing with MNaCl (pH 8,0), lysozyme solution (25% SiB sugar, 0.I!J NaCl2.0.0
5M) Lys, 0.8mg 7ml Lysozyme: pH
8.0 or less) 1 (suspend in 1 ml and react at 37°C for 4 hours. From the collected bacterial cells, use the method of Saito et al.
o, f(, et al: 13iochillBIo
phys, ^Cta, 72, 619 (1963)
Isolate high molecular weight chromosomal DNA according to:

On the other hand, pcGll used as a vector plasmid is
PCC of Corynebacterium glutamicum and 11 derived strains of LA103 and 22 strains LA103/IICG
11 (ATCC 39022) as follows.

4QQmlNB medium (medium containing 20g of powdered bouillon and 5g of yeast extract in water and adjusted to pH 7.2)
The cells are cultured with shaking at ℃ until the OD reaches about 0.7. After collecting the bacteria and washing with TES buffer, lysozyme solution IQ
ml and reacted at 37°C for 2 hours. 5 to the reaction solution
M NaC(12,4+++l, 0.5M2DTA(
4.4 ml of a solution consisting of 0.6 ml of 4% sodium lauryl sulfate (pH 8.5) and 0.1 M NaCR were sequentially added, mixed gently, and then placed 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) 6.0 (10 (manufactured by Gyudon Chemical Co., Ltd.) equivalent to 10% by weight to this, mix gently to dissolve, and then place in ice water. After 10 hours, 1
．． Collect pellets by centrifugation at 500 x g for 10 minutes. Gently redissolve the pellet in 5 l of TBS buffer, add 5 mg/ml ethidium bromide 2.01111, add cesium chloride and gently dissolve to adjust the density to 1.580. . Add this solution to 105. (1) Ultracentrifuge at 10xg for 48 hours at 18°C. The circular DNA covalently closed by this density gradient centrifugation is found as a dense band in the lower part of the centrifuge tube by UV irradiation. pcGllDN8 is separated by extracting this band from the side of the centrifuge tube with a syringe.Then, the fractionated solution is added to an equal volume of isopropyl alcohol solution [volume N% 90% isopropyl alcohol, lO%
Ethidium bromide is extracted and removed by treatment five times with TBS buffer (this mixture 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 pcG11 plasmid DNA 3.21543 prepared above. Reaction solution for restriction enzyme Bgj'n containing q (IOmAl l-Lis-HCl, 7mM MgC1x, 60mM NaCj!
, ? +nlJ 2-norcaptoethanol, pH 7,
5) Add 6 units of BgllU (manufactured by Zenshuzo Co., Ltd.) to 60m, react at 37°C for 60 minutes, and then heat at 65°C for 10 minutes to stop the reaction. On the other hand, restriction enzyme BamHI reaction solution containing chromosomal DNA 8xr of Corynebacterium glutamicum^TCC21543 (lomM Tris-HCl, 7
mM MgCi72.100mM NaCj! , 2m
M2-1 Lucaf) x9 Nor, 0.01% bovine serum albumin, pH 8,0) 4 units of BaltlHI were added to 140A1+, and after reacting at 37°C for 60 minutes,
The reaction is stopped by heating at 5° C. for 10 minutes.

Mix the images and add T41J gauze buffer (Tris-HCl 660mM SMgCL 66mM, dithiothreitol 100mM, pH 7.6) 41bd! ,A
TP (5mM) 40m, T4 ligase (manufactured by Zenshuzo Co., Ltd.,
1 unit/1d) Add 0.3d and H,0120m,
React for 16 hours at 12°C. The mixture was extracted twice with phenol 400mM saturated with TES buffer and TBS
Dialyze against buffer to remove phenol.

This ligase reaction mixture was combined with AEC-sensitive LP derived from Corynebacterium glutamicum supra-22 strain.
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 ODo reached 6, the cells were collected and the cells were subjected to RC.
GP medium [glucose 5g 1 casamino acid 5g, yeast extract 2.5g, KJPO43.5g, 12
PO41.5g, MgCL・6H,ro 0.41g,
Fe5Oa'7HJ 11) +g%Mn5Oa・4~
6H202ff1g, Zn5O<4Hzro 0.9 m
g, (NH*)sllot02s-4Hz0 0.
0 4mg5 biotin 30g, thiamine hydrochloride 2■,
A solution containing 1 mg/ml Norizozyme (pH 7.6
) to a concentration of approximately 10' cells/ml, 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 min and add TSIJC buffer (
10+nM magnesium chloride, 30mM calcium chloride, 50mM) Lis, 400mM sucrose, pH 7.5)
After resuspending in lit and centrifuging washing, TSIJC buffer Q,
Resuspend to 1mM. This bacterial solution contains twice as high a concentration of TSIJ.
A 1:1 mixture of Buffer C and the above ligase-reacted DNA mixture room is added and mixed, and then 3 ml of solution Q containing 20% PEG 6.000 in 75MC buffer is added and mixed. After 3 minutes, RCGP medium (pH 7,2)
2a+l was added, the supernatant was removed by centrifugation at 2.500 × g for 5 min, and the precipitated protoplasts were suspended in 1 TIl of RCGP medium before 0.211
R containing 11 and spectinomycin 40011 g/ml
It is spread on CGP agar medium (RCGP medium containing 1.4% agar, pH 7.2) and cultured at 30°C for 7 days.

The free volume grown on the agar medium is collected, washed with physiological saline, and then suspended in 1 mM physiological saline.

This bacterial solution was mixed with threonine 2mg/ml, ^εC2IT1
g/III and streptomycin 12.5 ■/m
Minimal agar medium Ml (glucose 10
g.

Nll, H, Po, 1 g, Cj! 0.2
g% Mg5Os・7H200, 2g S Fe50.
4H2010mg, ■n5Os・4~6H20o,
2mg, 2nS mouth, 4820 Q, 9mg, C
uS mouth, 5L0 0.4 mg.

%a, [1, mouth, 1OJ0 0.0 9 mg, (
NL) sMOv mouth <・4HJO, 04mg, biotin 50■, p-aminobenzoic acid 2.5tags thiamine hydrochloride lag, agar 16g in water B and adjusted to pH 7.2]. Culture 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 DNAs 1, they were completely digested with restriction enzyme 5coRI having a cleavage site on pcGll, and then analyzed by agarose gel electrophoresis, and molecule 1 was identified from the sum of the resulting fragments. The molecular weight is calculated based on a standard curve drawn by the migration distance of each fragment of known molecular weight produced by restriction enzyme old ndI[I digestion of lambda phage DNA that was run simultaneously on the same agarose gel. The plasmid pAec 5 obtained from one of the transformed strains is a molecule! 10.7 b
And pcGll's 8gJ! 3.9 Kb D at the II cleavage site
This is a recombinant plasmid into which an NA fragment has been inserted.

The protoplasts of the LP4 strain were transformed using pAec5 DNA in the same manner as above, and the transformed strain selected for spectinomycin resistance was identical to pAec5, which was determined by the cleavage pattern of εcoRI and was also given ABC resistance. It carries a plasmid. That is, pAec 5 contains Corynebacterium glutamicum^TCC21543
It is clear that the gene governing the ABC resistance trait has been cloned. The pAec 5 nursery strain is included in the American Type Culture Collection.
rynebacterium unglutamicum KI
It has been deposited as T^TCC39032.

(3) Production of lysine by ρ to ec5-carrying strain LP derived from Corynebacterium glutamicum strain 122
A lysine production test will be conducted on 4 strains possessing pAec 5 (ATCC39032) and a strain not possessing pAec 5. Bacteria grown on NB agar medium were added to each loopful of 5mM production medium PI (glucose 100g, (NH4) 15O424.5g,
KH, PO, 1gSugsO, -7N20 0.4 g
.

Fe50<'7112010mg, MnSO4'4~
6Hz 010mg, biotin 50■, thiamine hydrochloride 20hg, calcium pantothenate 500Itg, nicotinic acid 500Ig, soybean hydrolyzate 10g, calcium carbonate 30g in 1 water! The cells were inoculated into test tubes containing a medium containing 100% of the total number of 100% fertilized chloride and adjusted to pH 7.2, and cultured with shaking at 30°C for 75 hours. After culturing, the amount of lysine produced in the medium was measured by a colorimetric method using an acidic-copper ninhydrin reaction, and the results are shown in Table 1.

Table 1 Bacterial strain L-lysine production amount (mg/ml
) P-4 Example 2. Cloning of genes involved in threonine biosynthesis in E. coli and production of threonine by Corynebacterium glutamicum using expression of the genes: (1) DNA containing the E. coli threonine operon
Cloning of the fragment and introduction into Corynebacterium glutamicum: Cloning is carried out in the canine pJJ host vector system. ρG^22 used as a vector was obtained by the method used by Ahn et al., who created this plasmid [^n, G, et a
l: J, Bacteriol, L4Q, 40
0 (1979)), the plasmid was isolated from cultured cells of 112 substrains of E. coli carrying this plasmid. Donation DN
The high molecular weight chromosomal DNA that becomes A is present in 12 strains of E. coli (^T[
:C23740) using Smith's phenol extraction method [Sm+th1M, G, : 1Jethod]
inEnzy-mology, 12. part^
, 545 (1967)).

Restriction enzyme 11+ containing pGA22 plasmid DNA4N
nd ([Reaction solution (IOmM) Lith-hydrochloric acid, 7m!J!
JgCl 2゜60d NaCR, pH7.5) 60
Add 0.4 units of Hindll to ufI [(Takara Shuzo Co., Ltd., 6
unit/waste) was added and reacted at 37℃ for 30 minutes, then at 65℃
The reaction is stopped by heating for 0 minutes. There are two HindllI cleavage sites in pGA22, but H
As a result of examining the Indm-digested sample by agarose gel electrophoresis, it is confirmed that it has been cleaved into - fragments. Separately, approximately 4 strokes of old ndIII were added to 140 scraps of the restriction enzyme old nd11 reaction solution containing 811 g of chromosomal DNA, and the mixture was reacted at 37°C for 60 minutes and then heated at 65°C for 10 minutes to stop the reaction.

Mix the image chemical compound, T4 ligase laxative solution 404, ^T
Add 40m of P (5mM), 0.34m of T4 ligase and 120m of H, and react at 12°C for 16 hours.

This mixture was mixed with phenol 400 saturated with TBS buffer.
d twice and dialyzed against TBS buffer to remove phenol.

This ligase reaction mixture was applied to E. coli strain 112 substrain GT-.
3CJ, 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.
! J, et a top: Gene, 6.23 (19
79): Prepared in 1. That is, L medium supplemented with diaminopimelic acid to a concentration of 10 hg/ml (containing 10 g of batatotributone and 5 g of yeast extract in 1 β of water, pH 7).
, culture medium adjusted to 2) inoculated into 5Qml, 000.6
Incubate at 37°C until Cool the culture solution with ice water for 10 minutes and collect the bacteria by centrifugation. Resuspend in chilled 0.1M calcium chloride 20+111 and place at 0°C for 20 minutes.

Centrifuge the cells again and add 0.5 ml of O.1M lucium chloride.
suspend at 0°C for 18 hours.

200 m of the ligase reaction mixture was added to 400 m of the calcium chloride-treated bacterial solution, mixed, kept at 0°C for 10 minutes, and then heated at 37°C for 5 minutes. Then L medium 91+11
and culture with shaking at 37°C for 2 hours. After centrifugal washing twice with physiological saline, M9 minimal agar medium (glucose 2 g,
NII+CI! 1 g, Na211P046
g1K112P04 3 g, ! JgSL・711
20 o, 1 g, CaCL'2+1. O15mg
, 4 mg of thiamine hydrochloride and 15 g of agar in water if applied to a medium adjusted to p) 17.2 and cultured at 37° C. for 3 days. The only colony that appeared was ampicillin 25 g/it, chloramphenicol 25 μ/m
It was confirmed that it contains 25 g/ml of f or kanamycin and grows on agar medium.

From the cultured cells of this transformed strain, perform the above (1) to obtain 1] GA2.
Plasmid DNA is isolated by the same method used to isolate 2. This plasmid DNA was analyzed by restriction enzyme digestion and agarose gel electrophoresis, and the results are shown in Figure 1 (I).
It has the structure shown as GH2. The DNA fragment inserted into ρG^22 is a previously cloned E. coli operon-containing DNA fragment (Cossart, P., e4
al: ! Jolec, Gen.

Genet, LL5.39 (1979)], pGH2 is confirmed to contain a threonine operon.

Next, a recombinant of pcGll and pG)12 is created. First, pcGll and pGH2 are converted into Bgll and Bgll, respectively.
Digest completely with amHI under appropriate conditions. Each plasmid ON
Mix the digest containing ^2 μg and add 40 d of T4 ligase buffer, ΔT P (5mM
) 40JdI, T4 ligase 0.2μQ and H,01
Add 20m and react at 12°C for 16 hours. The mixture is extracted twice with 400 ρ of phenol saturated with TBS buffer and dialyzed against TBS buffer to remove the phenol. Subsequently, Corynebacterium glutamicum strain LA201 (LA10
After transforming the protoplasts of the derived strains of No. 3, homoserine, and loin auxotrophs), the cells were plated on RCGP agar medium.
Cultivate at 30°C for 6 days to regenerate and proliferate. Collect the bacteria that grew all over the agar medium, and after centrifuging and washing with physiological saline,
The mixture is replated onto minimal agar medium M1 supplemented with leucine 50/+nl and cultured at 30°C for 3 days. A strain that can grow on N8 agar medium containing 12.5 μg/ml of kanamycin or 100 μg/ml of spectinomycin is obtained from the Roro-21 that emerged.

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 -p[,t
It was named hr 1. Restriction enzyme Pstr, Eco
The structure characterized by the RI and Xho 1 cleavage sites is shown in FIG. pEthr 1 is ρ to pcGll
It was found to have a structure containing the G112 threonine operon and a lamHI fragment.

As a result of re-transforming Corynebacterium glutamicum LA103 strain using pEthr I DNA in the same manner as described above, homoserine non-auxotrophy and kanamycin and spectinomycin resistance traits were introduced in combination, and these transformed strains , carries a plasmid identical to pBthr l characterized by various restriction enzyme cleavage modes. LA1 due to deletion of homoserine dehydrogenase
The reason why the homoserine auxotrophy of strain 03 is restored to homoserine non-auxotrophy by pBthr l is due to the expression of homoserine dehydrogenase located on the E. coli threonine operon.

(2) Creation of pH 4hr l strain Corynebacterium glutamicum Shihachi-10, a threonine-producing bacterium derived from Corynebacterium glutamicum Shihachi-12 strain
6 (methionine requirement, ABC resistance, α-amino-β-
The pBthr 1 strain (resistant to hydroxyvaleric acid) is L
Obtained by transforming A-106 protoplasts with pBthr1.

For protoplasts, LA-206 strain was grown in semi-synthetic medium SSM.
Example 1 (2) Cells were cultured in a medium supplemented with methionine equivalent to 10 hg/ml and grown to an OD of approximately 0.6.
Prepared by processing in the same manner as above. Transformation was carried out in the same manner as in Example 1 (2), and spectinomycin 4
Transformants are obtained by selection on an RCGP agar medium containing 00 tag 7m I.

The pEthrl-carrying strain is Corynebacterium in the American Type Culture Collection.
It has been deposited at mgluLamicui as 19^TCC39034.

(3) Production of threonine by pEthr l strain pBthr of LA-106 strain obtained as above
A threonine production test will be conducted on the stock (ATCC39034) and the stock that is not stocked. One platinum loop of bacteria produced on NB agar medium was added to 5mM production medium P2 (glucose 100%
g, (NHi)tsO < 20g, H2PO40
,5g- to 2HPO40,5g S MgS mouth 4'? )
120 1 g, Fe50. -78,011b+g
, Mn5Oi・4-6Hz0 10mg, biotin 1
00 questions, 20g of calcium carbonate 100+g of methionine
The cells were inoculated into a test tube containing a medium containing 11 parts of water and adjusted to 87.2 cm, 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 colorimetric fixation. The results are shown in Table 2.

Table 2 LA-106 6,1 LA-106/p8thr 1 13.4 Example 3 Glutamic acid production by Corynebacterium glutamicum carrying a recombinant plasmid containing the E. coli phosphoenolpyruvate carboxylase (PPC) gene Production: (1) DNA containing the PPC gene of E. coli (this gene is a gene involved in glutamic acid biosynthesis, which is known to convert Glu- into Glu'' in E. coli)
Cloning of the fragment and introduction into Corynebacterium glutamicum: Cloning is performed in the E. coli host-vector system.

p8R322 used as a vector was injected into E. coli with '-1' using the same method used to prepare pG^22 in Example 1 (1).
Two substrains are isolated from cultured cells. The high molecular weight chromosomal DNA used as the donor DNA was prepared from Example 2 (+1 and 112 strains of Escherichia coli (ATCC 23740)).

98R322 plasmid DNA 3g and chromosomal DNA
Reaction solution for restriction enzyme 5ail containing 9d (1QmM) Liss hydrochloric acid, 7mM MgCj! 2.100mM NaC
12.

7mM 2-mercaptoethanol, o.i.% bovine serum albumin, pH 7,5) 5 in lO to 200ρ
alr (manufactured by Takara Shuzo Co., Ltd.) is added, and after reacting at 37°C for 60 minutes, the reaction is stopped by heating at 65°C for 10 minutes. Add 40ρ of t1 buffer for T4 ligase to this mixed digest, and add A T
Add 40p of P (5mM), 0.4p of T4 ligase and 120p of water, and react at 12°C for 16 hours. The mixture is extracted twice with phenol 400 scraps saturated with TES buffer and dialyzed against TBS buffer to remove phenol.

This ligase reaction mixture was injected into E. coli and 12 substrains PPC.
2 (Glansdorff, N., Genetic
s, 51. 157 (1965)) (arg-, t
hr, 1eu-, tus-, Th1-, PPC-.

ST"). Competent cells of strain PPC2 were cultured in a medium supplemented with 2 mg/ml glutamic acid, and competent cells of strain GT-3 were obtained in Example 2 (1). Transformation is carried out in the same manner as in Example 2 (1) using 200ml of the ligase reaction mixture.9ml of L medium is added and cultured with shaking at 37°C for 2 hours for expression. Then, after centrifugal washing twice with physiological saline, the cells were spread on M9 minimal agar medium supplemented with 504/ml each of arginine, threonine, loinine, and histidine, and cultured at 37°C for 3 days.The colonies that appeared were treated with ampicillin 25/ml. ml or tetracytalin 25
Replica onto L agar containing g/m+ and incubate at 37°C.
Culture for 24 hours and select those resistant to ampicillin and sensitive to tetracycline.

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 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 4.4 Kb DNA fragment was inserted into the l cleavage site.

This ppc l plasmid was used to transform PPC2 strain in the same manner as described above, and all of the transformed strains selected for ampicillin resistance were glutamic acid non-auxotrophic and had the same structure as ppc I, which was characterized by the restriction enzyme cleavage mode. It has a plasmid of This indicates that the E. coli PPC gene has been cloned onto the pPC1 plasmid.

The cloned PPC gene was transferred to Corynebacterium
pCG 11 and pPC for introduction into Glutamicum
A recombinant of t is prepared in host E. coli.

2 pcGll and ppc 1 plasmid DNAs each
Reaction buffer for restriction enzyme Pstl (20mM)
Liss hydrochloric acid, 10m1J MgC1x, 50m11 (N
H,)2sO4,0,01% bovine serum albumin, pt
17.5) Add 4 units of PstI (manufactured by Takara Shuzo Co., Ltd.) to 200 tdl, react at 30°C for 60 minutes, and then incubate at 65°C for 1
The reaction is stopped by heating for 0 minutes. This reaction mixture has T
40g of 41J gauze buffer, ΔT P (5mM>
40ttlST 4 ligase 0.2 degrees and water 120ρ
and react at 12°C for 16 hours. After phenol extraction in the same manner as above, phenol is removed by dialysis. This ligase reaction mixture 1ooIi1 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 approximately 15 to 16 b was selected, and the PPC2 strain was transformed again with this plasmid to confirm the presence of the PPC gene. As a result of analysis of the plasmid pεppc 1 obtained from one of the above transformed strains by restriction enzyme digestion and agarose gel electrophoresis, the genome size was 15.
It was clearly shown to be a 6 Kb recombinant plasmid.

Using the peppc t plasmid DNA thus prepared in E. coli, Corynebacterium glucumicum L.
Transform the LP4 strain derived from the -22 strain. Transformation was performed in the same manner as in Example 1 (2), and Transformation II! Strains are obtained from colonies growing on RCGP agar containing 1/400 μl of subectinomycin. Plasmids isolated from these transformed strains were digested with restriction enzyme 5a.
1 by single digestion of il or pst 1 or double digestion of both, and analysis by agarose gel electrophoresis.
1B+11) The possession of Ci is [H].

pEppc 1 Nursery Strain US American Type Umbrella Culture Collection 1 Corynebacter
u imglutamicum K-18^TCC39
It has been deposited as 033.

(2) Production of glutamic acid by pE9pC1 strain: LP4 strain derived from Corynebacterium glutamicum strain 122, pEppc1 strain (ATCC
A glutamic acid production test was carried out using the strain A. 39033). The bacteria grown on the NB agar medium were collected, and after washing with physiological saline, 5n+l of production medium P3 (glucose 50g, (N)14)2SO43g, urea 3gSK
H, PO, 0.5g, K21 (PO40.5g.

! JgSO<・7H20o, 5g, Fe5Os
-7L010mg, ! nsO+・4～68,010m
The cells were inoculated into a test tube containing a medium containing 1 g of biotin, 3 g of biotin, 500 g of thiamine hydrochloride, and 10 mg of phenol red in 1 p of pure water and adjusted to pH 1 or 2, and cultured with shaking at 30°C. During the culture, 20% urea solution was added three times (QJml each) and cultured for 40 hours. After culturing, the cultured solution was subjected to paper chromatography, and after coloring with ninhydrin, L-glutamic acid production was measured by colorimetry.

The results are shown in Table 3.

Table 3 P-4 10.1 Example 4. Brevibacterium flavum ATCC1
Cloning and expression of the anthranilate synthase gene of 4067 in Corynebacterium glutamicum: Chromosomal DNA of Brevibacterium flavum ATCC 14067 is prepared in the same manner as in Example 1 (1).

pCE53 used as a vector is isolated from cultured cells of 122 strains of Corynebacterium glutamicum in the same manner as in Example 1 (1) in which pcGll was used as KLHI. pCε53 is a Corynebacterium glutamicum plasmid pc previously disclosed by the present inventors.
GI C patent application 18101/1982
500) ) and E. coli plasmid pG^22 [static,
G. et al.: J. Bacteriol.
140.400 (1979)] are ligated together. For details, there is only one BgIU cleavage site on pcGl and two Bam cleavage sites on pG^22.
Among the HI cleavage sites, the Bamtl I cleavage site that is not within the tetracycline resistance gene is ligated using the same cohesive ends of both restriction enzymes.

pCE53 has selection markers such as the kanamycin resistance gene derived from pG^22, and has one cleavage site for restriction enzyme 5all.

pCε53 plasmid DNA prepared above 3Jig
Add IO units of Sal I to 200 m of the restriction enzyme Sal I reaction solution containing 9■ of chromosomal DNA and incubate at 37°C.
After reacting for 60 minutes at 65°C, the reaction is stopped by heating at 65°C for 10 minutes. To this mixed digest, 40 Tl gauze buffer, ATP (5d) 40ρ, T4 ligase 0.4
Add d and 1204 H2O and react at 12°C for 16 hours. The mixture is extracted with 400 m of phenol saturated with TBS buffer and dialyzed against TBS buffer to remove the phenol.

This ligase reaction mixture is subjected to form ff conversion.

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 Ml agar medium supplemented with anthranilic acid (equivalent to 30 q/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 is supplemented with anthranilic acid equivalent to 100 g/ml. The transformed strain was treated with kanamycin at 200 μ/m! Colonies are selected as growing on IIcGP agar medium containing the equivalent. 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. As a result of analyzing the plasmid ρTrp 2-3 obtained from one of the transformed strains by digestion with various restriction enzymes and agarose gel electrophoresis, it was found that a 5ale) NA cleavage fragment of b at approximately 7,1 was located at the unique 5all cleavage site of ρCε53. It was found that this was a plasmid with an inserted plasmid.

When LA105 strain was retransformed using pTrp 2-3 in the same manner, tryptophan was 1100 u/m
RCGP containing + and kanamycin 400 lines/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 demonstrate that the 5al of cloned approximately 7.1b
Brevibacterium flavum AT for lDNAΔ cut fragment
It is shown that the gene encoding CC14067 anthranilate synthase exists and is expressed in the Corynebacterium glutamicum LA l 05 strain.

pTrp 2-3 Nursery strain is American type.
Corynebacter in the Culture Collection
iumglutamicum K20^TCC390
It has been deposited as No. 35.

Plasmid pCε52 is subjected to the same treatment as above to obtain plasmid pTrp 4-3, which has a gene encoding the anthranilic acid synthase of Brevibacterium flavum ATCC14067.

piIC52 is a corynebacterium f. previously disclosed by the present inventors.
') Plasmid pcGIC of Rum glutamicum Patent application 18101/1984 (Japanese Patent Application No. 134500/1983)
and Escherichia coli plasmid pG^22 [^n, G,
et a top: J, Bacteriol.

140, 400 (1979)]. For details, there is only one Bgj'II cleavage site on pCG1 and two Bgj'II cleavage sites on pG^22.
The amHI cleavage site is ligated to the BamHI cleavage site within the tetracycline resistance gene using the same cohesive ends of both restriction enzymes. pcE52 is pGA2
It has a selection marker such as the kanamycin resistance gene derived from No. 2, and has one cleavage site for the restriction enzyme Sat I.

pCε52 is isolated from 122 cultured strains of Corynebacterium glutamicum harboring pcIE52 in the same manner as in the method used to isolate pcGll in Example 1 (1).

Similarly to the above, 36 strains of tryptophan-producing Corynebacterium glutamicum (FERII 0P-451
) is transformed with pTrp 4-3. The obtained transformed strain was purchased from American Type Culture Collection.
/ Yonni Corynebacter+um glut
Amicum has been deposited as 31° ATCC 39280.

20. Corynebacterium glutamicum carrying pTrp 2-3. ATCC39035 and pTrp4-
3 shares held are 31. The L-)lyptophan production test according to ATCC 39280 is conducted as follows.

The bacterial strain was cultured with shaking in NB liquid medium at 30°C for 16 hours.
g/1, (NH,) 230420 g/1, K
H2PO, 0.5g/l, 2HPO40.5g/CM
g5O<・7L00.25g/j! , CaCO32
Inoculate a test tube containing 0g/1, pH 1.23, and
Culture with shaking at 0°C for 96 hours.

After culturing, the culture p solution is subjected to paper chromatography, and after coloring with ninhydrin, the amount of L-9-butophane produced is measured by colorimetry.

As controls, LA-105 and LAR-1 strains are treated in the same manner.

The results are shown in Table 4.

Table 4 Bacterial strain L-tryptophan (mg/ml) LA-105 LA-105/pTrp 2-3 (K2O, ATCC
39035) 0.3 4LAR-10,48 LAR-1/pTrp 4-3(K31. ATC
C39280) 1.1 2 Example 5. 11
Preparation of CBIOI: (1) Separation of pcGll and p[lB110 pcG
11 is Corynebacterium glutamicum LA103/pcG11 (ATCC39
022> was grown in 400ml NB medium until the OD reached approximately 0.8, and from the cultured cells, I
] CG2 is isolated in the same manner as CG2 was isolated.

pUBllo was generated using the method of Gryczan et al.
.

T, J, ej al, : J, Bacter.
io11134.318 (1978)], Bacillus subtilis BR"'/p carrying this plasmid
HB"' (Proc, Natl.

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

(2) In vitro recombination of pcGll and pUBllo pcGll plasmid DNA 2t prepared above
8 g of restriction enzyme containing tg (#reaction buffer (10mM ToJS-HCl, ?+nM JCj!2, 60mM N
aC1, 7mM 2-1 Kabutoethanol, pH 7
,5) 100tt! 2 units of Bgf■ (manufactured by Takara Shuzo Co., Ltd., 6 units/h) are added to the mixture and reacted at 37°C for 60 minutes. In addition, restriction enzyme []amHI reaction buffer (10mM) containing 2g of pUB110 plasmid DNA, Lis-HCl, 7mM MgC1*, 10 (1mM NaC1,2
2 units of BamHI in 100 mM mercaptoethanol, 0.01% bovine serum albumin, pH 8.0)
(manufactured by Takara Shuzo Co., Ltd., 6 units/4) and reacted at 37°C for 60 minutes.

Mix both restriction enzyme digests and add 40% T41J gauze buffer.
ρ, A T P (5+++M) 40m, T 4 ligase 0.2J11! and H,0120m, 12
React for 16 hours at °C. This mixture was extracted twice with 400 g of phenol saturated with TESff solution and
Exclude phenol dialyzed against buffer.

(3) Acquisition of pcBIQl Corynebacterium glutamicum LA103 was obtained in the same manner as in Example 1 (3) using 100 JL of a 1:1 mixture of twice as high concentration of TS141Jl and the above ligase reaction mixture as donor DNA. and select kanamycin-resistant strains. Treat emerging colonies with kanamycin 12.5x/fal or spectinomynon 100
Replicas were placed on a NB agar medium containing g/ml, cultured at 30°C for 2 days, and three double-resistant transformants grown were arbitrarily selected and purified on the same agar medium. These three stocks cost 400d.
The cells are grown in NB medium until the OD reaches approximately 0.8, and after harvesting, plasmids are isolated from the cultured cells by ethidium bromide-cesium chloride density gradient centrifugation as described in Example 1 (1). 30 to 3 from any transformed strain.
5 ■ plasmids 011 people are obtained.

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 enzyme Pst I, [! coRr.

1inclI and Bgffll breakpoints are identified.

The plasmids of the three strains all have a structure in which p[:G11 and pUBllo are harmoniously linked, and two of them are shown in Figure 2 as pc
Although this is the structure shown in BIQl, the binding direction of the other types is opposite.

Transformants having any of the plasmids have a spectinomycin resistance trait derived from pcGll and a kanamycin resistance trait derived from pUB110.

The kanamycin-resistant transformant obtained as a result of re-transforming Corynebacterium glutamicum strain C156 using these plasmid DNAs has also acquired spectinomycin resistance and can be cleaved with various restriction enzymes. It carries a plasmid identical to the donor plasmid that has been characterized.

Example 6 Corynebacterium glutamicum, Corynebacterium herkyulis, Brevibacterium flavum and other genes were cloned using the cloning of a gene involved in histidine biosynthesis of Corynebacterium glutamicum Cl56 strain and the expression of the gene. Production of histidine by Brevibacterium lactofermentum: (1) Corynebacterium glutamicum C156
Preparation of strain chromosomal DNA and plasmid pCG 11 = 1
, 2.4-) Chromosomal DNA of Corynebacterium glutamicum Cl56 strain (FERM 0P-453) that is resistant to lyazole-3-alanine and capable of producing histidine
is prepared in the same manner as in Example 1(1).

On the other hand, ρCGII used as a vector plasmid is
Corynebacterium glutamicum strain 122 derived strain L^103 possessing pcG11 strain LA103/pCGll
IIL is separated from (^TCC39022) in the same manner as in Example 1 (I).

(2) Corynebacterium glutamicum C156
Cloning of genes involved in histidine biosynthesis of the strain: Reaction solution C1 (1 mM) for 1 U of restriction enzymes containing 8 g of the pcGll plasmid DNA prepared above and 9 g of the above chromosomal DNA (pH 47.5>), 7 mM Mg
CI 2.60mM NaCC 7mM 2-Mercaptoethanol 3200g + 10 units of Bgj! II (manufactured by Takara Shuzo Co., Ltd.) and reacted at 37°C for 60 minutes, then incubated at 65°C for 1
The reaction is stopped by heating for 0 minutes. In this mixed digest, T
Enoki buffer solution for 4 ligase (Tris 200mM.

11gc R25 (imM, dithiothreitol 100
ffllJ1pH7, 6') 40μs, 5mMAT
40 scraps of P solution, T4 ligase (manufactured by Takara Shuzo Co., Ltd., 1 unit/J
11) Add 0.3 m and 120 m of water and heat at 12°C for 16
Allow time to react.

The T44 ligase reaction mixture is used to transform Corynebacterium glutamicum strain LH33 (histidine auxotrophic, lysozyme sensitive).

Transformation is performed using LH33 strain protoplasts.

Protoplasts are prepared in the same manner as in Example 1 (2).

Pour 5 ml of protoplast suspension Q into a small test tube 25
Centrifuge at 00 × g for 5 min and add TSMtJli solution (
10mM magnesium chloride, 30mM calcium chloride,
501TIM Tris, 400mM sucrose, pH 7.5
) After resuspending in 11111 and washing by centrifugation, TSIJC
Resuspend in 1 ml of Buffer Q. To this suspension, add 2x TSMC buffer and 1 l of the above ligase-reacted DNA mixture.
Add and mix 1:1 mixed solution 100ρ, then TSMC1
1 Solution Q containing 20% PE G6.000 in buffer solution, 3
Add m+ and mix. After 3 minutes, add RCGP medium (pH
7,2) Add 2ω1, remove the supernatant by centrifugation at 2.500Xg for 5 minutes, suspend the precipitated protoplasts in 1ml of RCGP medium, and then add 2ml of spectinomycin 400μ/ml I. RCGP agar medium (RCGP medium containing 1.4% agar, pH
7, 2) and cultured at 30°C for 7 days.

The spectinomycin-resistant colonies grown on the selection plate were collected, and after centrifugal washing twice with physiological saline,
The mixture is spread on minimal agar medium M1 containing 1004 g 7 m I of spectinomycin and cultured at 30° C. for 2 days to select transformants that are resistant to subectinomycin 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 fragments generated by single digestion with various restriction enzymes and double digestion with two types of restriction enzymes are analyzed by agarose gel electrophoresis to identify the mode of restriction enzyme cleavage of this plasmid DNA. This plasmid was named pPl8. pPl8 is Bg of pcGll
This structure has a DNA fragment of approximately 10.6 Kb inserted into the J'II cleavage site.

Furthermore, using pPl8 DNA, strain H33 [LH3
Three parent strains (histidine auxotrophy, lysozyme resistance):
When FERM 0P-452) was re-transformed, all of the transformed strains selected as subectinomain-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.

(■ Production of histidine by Corynebacterium glutamicum strain harboring pPl8: Corynebacterium glutamicum strain LA-103 (FERM P-
5947, ^TC(:31866) to pPl8 DN
Transformed with A, subectinomycin 400g/ml
Transformants resistant to the same drug are selected on an RCGP agar medium containing the same drug. 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 pPl8.

pPl (8 strains Corynebacterium glutamicum L
A103/flPH8 is included in the American Type Culture Collection.
mglutamicum K32. ATCC39281
It has been deposited as.

Corynebacterium glutamicum LA103 /pc
G11 (ATCC39022) and the same LA103/1
The L-histidine production test for 1PH8 (ATCC39281) is conducted as follows.

51 production medium P5C molasses 12% (as sugar), KH2PO, 0.2%, supplemented with 200 g/ml arginine and methionine, one loop of each of the above, grown overnight on NB agar at 30°C. , K2HPO40,1
%, Mg5O<・7L 0.05%, NaCl1
0.25%, (N)1. ) 2SO4 2.3%, urea 0.2%, CaC0 = 2%, pH 7.4 (adjusted with ammonia)]. After culturing at 30° C. for 75 hours, the amount of histidine produced in the medium was measured by colorimetric method 1 using sulfanilic acid (Bauley) reagent: H, Pauly.

Hoppe-Seylers; 1. Physiol
o, Chem, 42.508 (1904), same 94
．． 284 (1915)). The results are shown in Table 5.

Table 5 LA103/I) CGII OLΔ103
/pPH8(K32) 2.6(4) p P
1 Lineobacterium herculis harboring H8,
Production of L-histidine by Brevibacterium flavum and Brevibacterium lactofermentum: Corynebacterium herculis ATCC 13868
, Brevibacterium flavum ATCC14067 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 33M medium, and the OD6600m was 0.
When the concentration reaches 2, penicillin G is added at a concentration of 0.3 units/ml. Continue culturing until 00660nm is 0.
When the number of cells reaches 6, the bacteria are harvested, and protoplasts are formed in the same manner as described above in an RCGP medium containing 1 mg/a+1 lysozyme. Transformation is performed using pPH8 according to the method described above, and transformed strains are selected as colonies growing on RCGP agar medium containing 400 g/ml of subectinomycin.

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 from the restriction enzyme cleavage pattern that the plasmid has the same structure as Pl(8).From the above, plasmid p[:G1
Plasmid pPH3, a derivative of 1, can also be replicated in Corynebacterium herculis, Brevibacterium flavum, and Brevibacterium lactofermentum, indicating that plasmid pcG11 can be widely used in these bacterial species. .

Corynebacterium herculis 33, Brevibacterium flavum 34, and Brevibacterium lactofermentum 35, which are pPH3-bearing strains, were sent to the American Type Culture Collection at ATCC392g2.39283 and 392, respectively.
It has been deposited as No. 84.

A histidine production test using these strains is conducted as follows.

One platinum loop of each pPH8-carrying strain and its parent strain, 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,
The amount of histidine produced in the medium is measured by the Beaucoup method in a colorimetric chamber. The results are shown in Table 6.

Table 6 Bacterial strain L-histidine (mg/ml) From the above, genes involved in histidine production derived from Corynebacterium glutamicum include Corynebacterium herculis, Brevibacterium flavum, and Corynebacterium glutamicum. It was clear that it was expressed in various species of Brevibacterium lactofermentum and contributed to the production of histidine.

[Brief explanation of the drawing]

FIG. 1 shows the restriction enzyme map of plasmid pGH2. Figure 2 shows a restriction enzyme map of plasmid pcfll (II). Figure 3 shows a flowchart for the construction of plasmid p8thr 1. Patent applicant (102) Kyowa Hakko Kogyo Co., Ltd.

Claims (1)

[Claims] A DNA fragment belonging to the genus Corynebacterium or the genus Brevibacterium and containing a gene encoding phosphoenolpyruvate carboxylase derived from Escherichia coli, and a vector DNA capable of autonomous replication in a species of the genus Corynebacterium or the genus Brevibacterium. A method for producing L-glutamic acid, which comprises culturing a microorganism carrying recombinant DNA in a medium, producing and accumulating L-glutamic acid in the culture, and collecting L-glutamic acid from the culture.