JPH0724591B2 - Method for producing aromatic amino acid using coryneform bacterium having recombinant DNA - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a coryneform bacterium having a recombinant DNA in which a shikimate kinase is incorporated, and a method for producing an aromatic amino acid using the bacterium.

[0002]

2. Description of the Related Art Shikimate kinase (hereinafter referred to as "SK") is an enzyme that catalyzes the reaction of converting shikimic acid into shikimic acid 3-phosphate. Shikimic acid 3-phosphate converts chorismate to Via phenylalanine, tyrosine or tryptophan. On the other hand, breeding of these aromatic amino acid-producing bacteria by the recombinant DNA method is known, but some of them are known (for example, JP-A-57-208994).
JP-A-57-71397, JP-A-58-89194, JP-A-58-134994, etc.) and a gene encoding SK (hereinafter referred to as "SK gene") are not incorporated.

[0003]

DISCLOSURE OF THE INVENTION The present invention is to obtain a microorganism having a higher productivity of aromatic amino acids, thereby finding a more efficient production method of aromatic amino acids.

As a result of research to solve the above-mentioned problems, the present inventors have found that a gene expressed in coryneform bacterial cells and encoding SK is propagated in coryneform bacterial cells. Succeeded in isolating a coryneform bacterium having a recombinant DNA ligated to a plasmid vector
It was found that the obtained coryneform bacterium has a high productivity of aromatic amino acids. That is, the present invention relates to a coryneform bacterial cell containing a DNA fragment derived from a coryneform bacterium, which has a restriction enzyme cleavage pattern shown in FIG. 2 and has a length of about 1.9 kb and contains a gene encoding shikimate kinase. A method for producing an aromatic amino acid, which comprises culturing a coryneform bacterium having a recombinant DNA capable of growing inside. The present invention also relates to a coryneform bacterium having a restriction enzyme cleavage pattern shown in FIG. 3, a length of about 2.9 kb, and containing a gene encoding shikimate kinase and a gene encoding 3-dehydroquinate synthase. Origin DNA
Recombinant D containing fragments and capable of growing in coryneform bacterial cells
A method for producing an aromatic amino acid, which comprises culturing a coryneform bacterium having NA. Further, the present invention is shown in FIG.
A gene having a restriction enzyme cleavage pattern of about 2.9 kb and encoding a shikimate kinase and 3-
A DNA fragment derived from a coryneform bacterium, which contains a gene encoding dehydroquinate synthase, and which contains about 8.
DNA fragment derived from coryneform bacterium, which has a length of 35 kb and has a restriction enzyme PstI at both ends and contains a gene encoding shikimate kinase, a gene encoding 3-dehydroquinate synthase, and a gene encoding shikimate dehydrogenase A method for producing an aromatic amino acid, which comprises culturing a coryneform bacterium having a recombinant DNA capable of growing in a coryneform bacterial cell.

The coryneform bacterium referred to in the present invention (Coryne)
form bacterium is the Bargeys Manual of Determinate Bacteriology.
inactive Biology) 8th edition 5
They are a group of microorganisms defined on page 99 (1974), and are aerobic, gram-positive, non-acidic, and spore-forming bacilli. Among such coryneform bacteria, coryneform glutamic acid-producing bacteria as described below are most preferable in the present invention.

Examples of wild strains of coryneform glutamic acid-producing bacteria include the following. Brevibacterium divalicatum ATCC 14020 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium inmaliofilm ATCC 14068 Brevibacterium lactofermentum ATCC 13869 Brevibacterium roseum ATCC 13825 Brevibacterium flavum 26 ATCC 14038 Corynebacterium acetoside film ATCC 19240 Corynebacterium acetoacetate film ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 Corynebacterium carnae ATCC 15991 Corynebacterium glutamicum ATCC 13032, 13060 Corynebacterium varium 90 Cterium Melacecola ATCC 17965 Microbacterium Ammonia Philum ATCC 15354

The coryneform glutamic acid-producing bacterium of the present invention includes mutant strains having glutamic acid productivity or having lost glutamic acid productivity in addition to the above-mentioned wild strain having glutamic acid productivity.

The method for isolating the SK gene is based on a coryneform cell type.
First, from the strain having the SK gene of fungus, the chromosomal gene
(For example, H. Saito and K. Miu)
raBiochem. Biophys. Acta7
Two, 619, (1963) can be used. ), This
This is cut with an appropriate restriction enzyme. Then, coryneform bacteria
When the plasmid vector which can grow in the cell is ligated,
SK Deletion Mutation of Coryneform Bacteria Using Recombinant Recombinant DNA
Transform the strain and retain the SK-producing activity.
The SK gene can be isolated from the isolated strain.

In order to cleave the chromosomal gene, a wide variety of restriction enzymes can be used by controlling the cleavage reaction time and the like to control the degree of cleavage.

The plasmid vector used in the present invention may be any one so long as it can grow in coryneform bacterial cells. Specific examples are as follows. (1) pAM 330 See JP-A-58-67699 (2) pHM 1519 See JP-A-58-77895 (3) pAJ 655 See JP-A-58-192900 (4) pAJ 611 Same as above (5) pAJ 1844 Same as above (6) pCG 1 See JP-A-57-134500 (7) pCG 2 See JP-A-58-35197 (8) pCG 4 See JP-A-57-183799 (9) pCG 11 Same as above

Cleavage of the plasmid vector DNA is carried out by cutting the DNA with a restriction enzyme that cuts at one site or by partially cutting with a restriction enzyme that cuts at multiple sites.

The vector DNA is cleaved by the restriction enzyme used when cleaving the chromosomal gene, or the chromosomal DNA cleaving fragment and the cleaved vector DN.
Oligonucleotides having complementary nucleotide sequences are connected to both ends of A, and then subjected to a ligation reaction between the plasmid vector and the chromosomal DNA fragment.

To introduce the recombinant DNA of the chromosomal DNA and the vector plasmid thus obtained into a recipient bacterium belonging to a coryneform bacterium, Escherichia coli K-
12 (Mandel, M .;
and Higa, A .; J. Mol. , Biol. ，
53 , 159 (1970) Receptor cells treated with calcium chloride to increase DNA permeability, or as reported for Bacillus subtilis (Dunca).
n, C.I. H. Wilson, G .; A. andYoun
g, F. E. , Gene, 1 , 153 (1977)) by introducing it into a growth stage (so-called competent cell) so that the cells can take up DNA. Alternatively, as is known for Bacillus subtilis, actinomycetes and yeast (Chang, S. and Ch.
oen, S.M. N. Molec. Gen. , Gene
t. , 168 . 111 (1979); Bibb, M .;
J. Ward, J .; M. and Hopwood,
O. A. , Nature, 274 , 398 (197).
8); Hinnen, A .; Hicks, J .; B. an
dFink, G .; R. , Proc. Natl. Aca
d. Sci. USA, 75 1929 (1978)), D
It is also possible to transform the NA recipient bacteria into protoplasts or spheroplasts that readily incorporate the plasmid DNA and introduce the plasmid into the DNA recipient bacteria.

In the protoplast method, a sufficiently high frequency can be obtained even by the method used in the above Bacillus subtilis, and the protoplasts of the genus Corynebacterium or Brevibacterium described in JP-A-57-183799 can be obtained. D in the presence of polyethylene glycol or polyvinyl alcohol and divalent metal ions
Naturally, the method of incorporating NA can also be used. Equivalent results can be obtained by a method of promoting the uptake of DNA by adding carboxymethyl cellulose, dextran, Ficoll, Bluronic F68 (Celva) instead of polyethylene glycol or polyvinyl alcohol.

As the aromatic amino acid-producing bacterium, a strain transformed with a SK-deficient strain as a host can be used.
When a host as shown below is used, a strain with higher aromatic amino acid productivity may be obtained.

In the case of tyrosine, phenylalanine is required for Corynebacterium and 3-
A mutant strain H. aureus resistant to aminotyrosine, p-aminophenylalanine, p-fluorophenylalanine, tyrosine hydroxamate. Hagino, K .; Nakay
ama: Agric. Biol. Chem. , 37 , 2
013 (1973), a mutant strain of S. cerevisiae resistant to m-fluorophenylalanine of the genus Brevibacterium. Sugi
moto, M.M. Nakagawa, T .; Tsuchid
a, I. Shiio. , Agric. Biol. Che
m. , 37 , 2327 (1973).

In the case of tryptophan: A mutant strain requiring phenylalanine and tyrosine of the genus Brevibacterium and having resistance to 5-methyltryptophan (I. Shiio, H. Sato, M. Nakaga).
wa, Agric. Biol. Chem. , 36 , 23
15 (1972)), which requires phenylalanine of the genus Brevibacterium, m-fluorophenylalanine, 5
-Mutant strains resistant to fluorotryptophan (I.
Shiio, S .; Sugimoto, M .; Nakaga
wa, Agric. Biol. Chemi. , 39 , 6
27 (1975)), which requires tyrosine of the genus Brevibacterium, requires a mutant strain having resistance to 5-fluorotryptophan and azaserine, requires phenylalanine and tyrosine of the genus Corynebacterium, and requires 5-methyltryptophan and 4-methyltryptophan. , 6-fluorotryptophan, tryptophan hydroxamate, p-fluorophenylalanine, tyrosine hydroxamate, phenylalanine hydroxamate-resistant mutants (H. Hagino, K. Nakayama, Agri)
c. Biol. Chem. , 39 , 345 (197)
5)) etc.

In case of phenylalanine A mutant strain of Brevibacterium having resistance to m-fluorophenylalanine (S. Sugimoto, M. Na.
kagawa, T .; Tsuchida, I .; Shii
o. , Agric. Biol. Chem. , 37 , 23
27 (1973)), requires tyrosine and methionine of Brevibacterium, and requires 5-methyltryptophan, p.
-A mutant strain having resistance to fluorophenylalanine (JP-A-49-116294), a mutant strain requiring tyrosine and methionine of the Brevibacterium genus, and having resistance to 5-methyltryptophan, p-fluorophenylalanine and decoinine (JP-A No. 56-64793) A mutant strain that requires tyrosine of the genus Corynebacterium and has resistance to p-fluorophenylalanine and p-aminophenylalanine (H. Hagino, K. Nakayama).
a, Agric. Biol. Chem. , 38 , 157
(1974)).

If the following genes are inserted in addition to the SK gene, the productivity of aromatic amino acids is often higher. As shown in the examples, when a gene encoding 3-dehydroquinate synthase, a gene encoding shikimate dehydrogenase is inserted, or 3-deoxy-
D-arabino-heptyuronic acid-7-phosphate (DAH
P) synthase gene, 3-dehydroquinate dehydratase gene, 5-enolpyruvylshikimate-3-phosphate synthase, and chorismate synthase gene.

In addition, when trying to obtain tryptophan-producing bacteria, anthranilate synthase gene, anthranilate phosphoribosyl transferase gene, N-
Better results may be obtained if the (5'-phosphoribosyl) anthranilate inmerase gene, indole-3-glycerol phosphate synthase gene, tryptophan synthase gene, etc. are inserted.

When obtaining a phenylalanine- or tyrosine-producing bacterium, the prephenate dehydratase gene, prephenate transaminase, pretyrosine dehydrogenase gene, tyrosine aminotransferase gene, etc. may be inserted in addition to the SK gene. desirable.

The method of culturing the thus obtained coryneform bacterium having an aromatic amino acid-producing ability to produce and accumulate the aromatic amino acid has been conventionally used for producing the aromatic amino acid by the coryneform bacterium. There is no big difference from the method. That is, the medium is a normal medium containing a carbon source, a nitrogen source, inorganic ions, and if necessary, organic micronutrients such as amino acids and vitamins. As the carbon source, glucose, sucrose, lactose, etc., and a starch hydrolyzate containing them, whey, molasses, etc. are used. As the nitrogen source, ammonia gas, ammonia water, ammonium salt and the like can be used.

Culturing is carried out under aerobic conditions while appropriately adjusting the pH and temperature of the medium until the production and accumulation of aromatic amino acids is substantially stopped.

[0023]

【Example】

(1) Preparation of chromosomal DNA containing SK gene Brevibacterium lactofermentum AJ112
25 (FERM-BP1219) was inoculated into 1 L of CMG medium (containing peptone 1 g / dl, yeast extract 1 g / dl, glucose 0.5 g / dl, and NaCl 0.5 g / dl and adjusted to pH 7.2). Then, shaking culture was carried out at 30 ° C. for about 3 hours to collect cells in the logarithmic growth phase.
After culturing the cells in lysozyme / SDS, the chromosomal DNA was extracted and purified by a usual phenol treatment method to finally obtain 3.5 mg of DNA.

(2) Preparation of vector DNA Using pAJ1844 (molecular weight 5.4 megadalton) as a vector, the DNA was prepared as follows. First, Brevibacterium lactofermentum AJ12037 (F which has pAJ1844 as a plasmid)
ERM-P577) was inoculated into 100 ml of CMG medium, cultured at 30 ° C. until the late logarithmic growth phase, lysed by lysozyme SDS treatment, and 30,000 × g, 3
The supernatant was obtained by 0 minute ultracentrifugation. After phenol treatment, 2 volumes of ethanol was added to precipitate and collect DNA.
Add a small amount of TEN buffer (20 mM Tris hydrochloride, 2
It was dissolved in 0 mM NaCl, 1 mM EDTA (pH 8.0), subjected to agarose gel electrophoresis for separation, and cut out to obtain about 15 μg of pAJ1844 plasmid DNA.

(3) Insertion of Chromosomal DNA Fragment into Vector 10 μg of chromosomal DNA obtained in (1) and 5 μg of plasmid DNA obtained in (2) were used as restriction endonuclease Pst.
Each was kept at 37 ° C. for 1 hour with I and cut. 65
After heating at 0 ° C for 10 minutes, both reaction solutions were mixed, and DN derived from T ₄ phage in the presence of ATP and dithiothreitol.
The DNA strands were ligated with A ligase at 10 ° C. for 24 hours. Then, the reaction solution was heated at 65 ° C. for 5 minutes, and 2 volumes of ethanol was added to the reaction solution to form D
The NA precipitate was collected.

(4) Cloning of SK gene Brevibacterium lactofermentum AJ12157 (Brevibacterium lactofermentum AJ12036 (FERMBP-75) deficient in SK gene
59) was used as a parent strain and subjected to mutation treatment with N-methyl-N-nitro-N-nitrosoguanidine to select 3 mutant amino acids of phenylalanine, tryptophan and tyrosine for growth. ) Was used as a recipient bacterium.

As a transformation method, the protoplast transformation method was used. First, 5 strains
Incubate in ml CMG liquid medium until the beginning of the logarithmic growth phase,
After adding 0.6 unit / ml of penicillin G, the cells were further cultivated with shaking for 1.5 hours, and the bacterial cells were collected by centrifugation, and the bacterial cells were collected with 0.5 M sucrose, 20 mM maleic acid and 2 mM.
SMMP medium (pH 6.5) consisting of 0 mM magnesium chloride and 3.5% Penassay broth (Difco)
It was washed with 0.5 ml. Then, the cells were suspended in SMMP medium containing 10 mg / ml of lysozyme and protoplasted at 30 ° C for 20 hours. After centrifugation at 6000 xg for 10 minutes, the protoplasts were washed with SMMP and resuspended in 0.5 ml SMMP. 5 μm of the protoplast thus obtained and 10 μg of the DNA prepared in (3)
After mixing in the presence of EDTA and adding polyethylene glycol to a final concentration of 30%, the mixture was allowed to stand at room temperature for 2 minutes to incorporate DNA into protoplasts.
After washing this protoplast with 1 ml of SMMP medium, S
Resuspend in 1 ml of MMP medium, 30 ℃ for expression
It was cultured for 2 hours. This culture solution was applied on a protoplast regeneration medium having a pH of 7.0. The protoplast regeneration medium contains 12 g of tris (hydroxymethyl) aminomethane, 0.5 g of KCl, 10 g of glucose and M per liter of distilled water.
_{gCl 2 · 6H 2 O8.1g, CaCl} 2 · 2H 2 O2.2
g, peptone 4 g, powder yeast extract 4 g, casamino acid (Difco) 1 g, K ₂ HPO ₄ 0.2 g, sodium succinate 135 g, agar 8 g, and chloramphenicol 3 μg / ml.

After culturing at 30 ° C. for 2 weeks, about 25,000 chloramphenicol resistant colonies appeared, which were used as a minimum medium (2% glucose, 1% ammonium sulfate, 0.3% urea, 0.1% phosphorus). Potassium dihydrogen acid,
0.04% magnesium sulfate heptahydrate, 2 ppm iron ion, 2 ppm manganese ion, 200 μg / l thiamine hydrochloride, 50 μg / l biotin, chloramphenicol 10 μg / ml, pH 7.0, agar 1.8%) Then, 5 strains were obtained which were resistant to chloramphenicol and had no requirement for phenylalanine, tryptophan, or tyrosine.

(5) Plasmid analysis of transformed strains From these strains, lysates were prepared by the method described in (2), and plasmid D was analyzed by agarose gel electrophoresis.
When NA was detected, vector pAJ1 was detected in all strains.
A plasmid clearly larger than 844 was detected.

When the plasmids of the 5 strains were each digested with the restriction enzyme Pst I used for recombination, a 2.9 kb DNA insert fragment common to all plasmids was observed. Therefore, it is considered to exist on the Pst I DNA fragment of the SK gene of 2.9 kb. A recombinant plasmid in which a 2.9 kb DNA fragment was inserted at the Pst I cleavage point of vector pAJ1844 was named pAJ927, and a recombinant plasmid having Pst I DNA fragments of 1.15 kb and 4.3 kb in addition to the 2.9 kb DNA fragment. Is named pAJ1219, p
A strain that retains AJ927 is AJ12158 FERM-
A strain carrying P7865, pAJ1219 was designated as AJ121.
It was named 59 FERM-P7866.

(6) Retransformation In order to confirm the presence of the SK gene on the recombinant plasmid containing the 2.9 kilobase DNA fragment detected in (5), this plasmid DNA pAJ1219 and p
Brevibacterium lactofermentum AJ12157 was transformed again using AJ927. 10 out of each chloramphenicol resistant colonies
When the auxotrophic properties of all of these were eliminated, it was revealed that the SK gene was present on the above recombinant plasmid.

(7) SK activity of transformant strain The test strain was treated with a tryptophan production medium (glucose 130).
g, (NH ₄ ) ₂ SO ₄ 25 g, KH ₂ PO ₄ 1 g, MgS
O ₄ · 7H ₂ O1g, fumaric acid 12g, acetic 3 ml, soybean protein acid hydrolyzate "Taste liquid" 50ml, MnSO ₄ · 4H ₂
O10mg, biotin 50μg, thiamine hydrochloride 20
00 μg and 50 g CaCO ₃ are contained in 1 l of water. pH
6.5) A lysate was prepared by sonication using the bacterial cells cultured in 20 ml at 30 ° C. for 22 hours.
The supernatant was obtained by centrifugation at 000 × g for 20 minutes. Using this supernatant as a crude enzyme solution, 50 mM Veronal buffer (pH 9.0), 1 mM shikimic acid, 4 mM ATP,
After reaction at 30 ° C. for 30 minutes in a reaction solution containing 5 mM MgCl ₂ and 10 mM NaF, 1 M Tris-
Add 0.2 ml of hydrochloric acid buffer (pH 7.8) and add 100 ° C.
And inactivate the enzyme for 2 minutes. 3.0 ml after cooling
0.5 ml of 1% periodic acid was added to the reaction solution of (the reaction solution appropriately diluted to contain 2 to 10 μg of shikimic acid),
The mixture was allowed to stand at room temperature for 3 hours, 0.5 ml of 1N sodium hydroxide was added thereto, 0.3 ml of 0.1 M glycine was immediately added thereto, and shikimic acid was quantified by measuring the absorbance at 380 mμ. The SK activity was determined by subtracting the amount of shikimic acid after completion of the reaction from the amount of shikimic acid added to the reaction solution. Table 1 shows the measurement results.

[0034]

[Table 1]

(8) Identification of shikimate dehydrogenase gene and 3-dehydroquinate synthase gene on recombinant plasmids pAJ927 and pAJ1219 pAJ927 and pAJ1219 were derived from Escherichia coli K-12 strain AB2834,3 deficient in shikimate dehydrogenase.
-Dehydroquinate synthase deficient strain AB2847 (J.
Pittard et al. Bacterio
l. , 1494, 91 , 1966).

E. coli is a mutant strain of the K-12 strain AB283.
For 4,2847, pAJ92 was prepared by treating DNA recipient cells with calcium chloride to increase DNA permeability.
7, pAJ1219 was transformed, and 10 clones each of the resulting chloramphenylchol-resistant colonies were picked up and examined for phenylalanine, tyrosine and tryptophan requirements. AB28 for both pAJ927 and pAJ1219
All of the 47 requirements were lost, but the AB2834 requirement was not abolished in the pAJ927 transformants and pAJ1
The 219 transformants lost all requirements.

Accordingly, 2.9 kb inserted Pst I of SK, 3-dehydroquinate synthase gene was inserted into pAJ927.
Cloned on a DNA fragment, pAJ121
It was revealed that the SK, 3-dehydroquinate synthase, and shikimate dehydrogenase genes were cloned in 9.

(9) Shikimate Dehydrogenase Activity of Transformed Strains A crude enzyme solution of the wild strains AJ12036 and pAJ1219 transformed strains was prepared by the method described in (7).
0.1 M Tris-hydrochloric acid buffer solution (pH 7.4), 0.8 m
The above-mentioned crude enzyme solution was added to the reaction solution consisting of MNADP and 4 mM shikimic acid, and the increase in absorbance at 340 nm was measured to determine the shikimate dehydrogenase activity. Second result
Shown in the table.

[0039]

[Table 2]

[0040] (10) was completely cut subcloning pAJ927 of SK gene with a restriction enzyme Bgl II, 6
After heating at 5 ° C. for 10 minutes, the DNA strand was ligated by allowing it to stand at 4 ° C. overnight with a DNA ligase derived from T4 phage in the presence of ATP and dithiothreitol. Then, the reaction solution was heated at 65 ° C. for 10 minutes, and 2 volumes of ethanol was added to the reaction solution to collect a precipitate of ligated DNA.
After dissolving in TEN buffer, cleaved with restriction enzyme Bam HI,
After heating at 65 ° C for 10 minutes, 2 volumes of ethanol was added to the reaction solution to collect a DNA precipitate, and a small amount of TE was added to this.
N buffer was added and used for transformation.

Brevibacterium lactofermentum AJ12157 was prepared using the transformation method described in (4).
Was transformed. After regenerating in a regenerating medium containing 3 μg / ml of chloramphenicol at 30 ° C. for 2 weeks, a replica of a minimal medium was performed, and a strain that was resistant to chloramphenicol and had a triple requirement for phenylalanine, tyrosine, and tryptophan disappeared at the same time. Got a lot. All of these strains are pAJ
927 had the small plasmid pAJ912. Note that pAJ927 and pAJ912 have the restriction enzyme cleavage maps shown in FIG.

When AJ12157 was transformed again with pAJ912 and the resulting chloramphenicol resistant colonies were picked up and tested for the requirement of phenylalanine, tyrosine and tryptophan, all of them lost the three requirements at the same time. Therefore, it was revealed that the SK gene was present on the above recombinant plasmid.

Next, pAJ912 was used as a mutant strain of Escherichia coli K-12 strain 3-dehydroquinate synthase deficient strain AB2847.
Transformed into. The resulting chloramphenicol-resistant colonies were picked up and examined for phenylalanine, tyrosine, and tryptophan requirement, but none of them recovered the requirement. Therefore, pAJ912 is considered to have the 3-dehydroquinate synthase gene partially or completely deleted.

(11) Tryptophan-Producing Ability of Each Transformant Using the above pAJ927, pAJ912, and pAJ1219, m-fluorophenylalanine and 5-fluorotryptophan resistant strains Brevibacterium lactofermentum M247 and Corynebacterium glutamicum ( ATCC13060) was transformed by the method described in (4), and a transformant was selected using chloramphenicol resistance as an index. AJ1216 thus obtained
0 (FERM-P7867), AJ12161 (FER
M-P7868), AJ12169 (FERM-P78
69), AJ12170 (FERM-P7870), A
When J12171 (FERM-P7871) was cultured and the tryptophan-producing ability was examined, the results shown in Table 3 were obtained.

The culture was a tryptophan production culture (glucose 130 g, (NH ₄ ) ₂ SO ₄ 25 g, fumaric acid 12
g, acetic acid 3 ml, KH ₂ PO ₄ 1 g, MgSO ₄ .7H ₂ O
10 mg, MnSO ₄ .7H ₂ O 1 g, d-biotin 50
μg, thiamine hydrochloride 2000 μg, methionine 40
0 mg, tyrosine 650 mg, soybean protein acid hydrolyzed liquid "taste liquid" 50 ml, CaCO ₃ 50 g in 1 liter of water,
pH 6.5) 20 ml was put in a 500 ml Sakaguchi flask, the test strain was inoculated, and the mixture was incubated at 30 ° C. for 72 hours.
Performed under shaking. After culturing, the L-tryptophan in the centrifugal supernatant was added to Leuconostoc mescentroides (Leu).
conostoc mesenteroides) AT
It was determined by a bioassay method using CC8042 as a quantitative strain.

[0046]

[Table 3]

In order to obtain AJ12157 and M247, it is possible to remove the complex plasmid in the host cell from the deposited AJ12158 and AJ12160 without damaging the host cell. That is, the plasmid may be naturally lost by the host, or it may be removed by a "removal" operation (Bact. Rev., 36 , p3.
61-405 (1972)). Examples of other removal operations are as follows. CM for AJ12158 and AJ12160
Inoculate G liquid medium and incubate at 37 ℃ overnight (high temperature treatment)
After that, the culture solution is appropriately diluted, applied to CMG agar medium containing no chloramphenicol, and cultured at 30 ° C. for 1 to 3 days. The strains thus isolated as the chloramphenicol sensitive strains are AJ12157 and M247.

[0048]

INDUSTRIAL APPLICABILITY The coryneform bacterium having a recombinant DNA in which a gene expressing SK and encoding SK is connected to a plasmid vector capable of growing in the coryneform bacterial cell was successfully isolated. Have succeeded in providing a more efficient production method of aromatic amino acids using the obtained coryneform bacterium.

[Brief description of drawings]

FIG. 1 shows restriction enzyme digestion maps of plasmids pAJ927 and pAJ912.

FIG. 2 shows a restriction map of the insert fragment of plasmid pAJ912.

FIG. 3 shows a restriction map of the insert fragment of plasmid pAJ927.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12N 15/09 // (C12N 15/09 C12R 1:13) C12R 1:13)

Claims (3)

[Claims] 1. A coryneform bacterial cell having a DNA fragment derived from coryneform bacteria, which has a restriction enzyme cleavage pattern shown in FIG. 2, is about 1.9 kb in length, and contains a gene encoding shikimate kinase. A method for producing an aromatic amino acid, which comprises culturing a coryneform bacterium that retains a recombinant DNA capable of growing inside. 2. A coryneform bacterium derived from a coryneform bacterium having a restriction enzyme cleavage pattern as shown in FIG. 3, a length of about 2.9 kb, and containing a gene encoding shikimate kinase and a gene encoding 3-dehydroquinate synthase. A method for producing an aromatic amino acid, which comprises culturing a coryneform bacterium containing a recombinant DNA capable of growing in a coryneform bacterial cell containing a DNA fragment. 3. A coryneform bacterium derived from a coryneform bacterium, which has the restriction enzyme cleavage pattern shown in FIG. 3, is about 2.9 kb in length, and contains a gene encoding shikimate kinase and a gene encoding 3-dehydroquinate synthase. It contains a DNA fragment inside, is about 8.35 kb in length, and has a restriction enzyme P at both ends.
In a coryneform bacterial cell containing a DNA fragment derived from a coryneform bacterium having a gene encoding shikimate kinase, a gene encoding 3-dehydroquinate synthase and a gene encoding shikimate dehydrogenase, having a stI site, A method for producing an aromatic amino acid, which comprises culturing a coryneform bacterium having a recombinant DNA capable of growing.