JPH0672B2 - Fermentation method for producing L-phenylalanine or L-tyrosine - Google Patents

Description

TECHNICAL FIELD OF THE INVENTION The present invention has recombinant DNA in which 3-deoxy-D-arabino-hepturonate-7-phosphate synthase (hereinafter referred to as “DS”) is incorporated. The present invention relates to a coryneform bacterium and a method for producing L-phenylalanine or L-tyrosine using the same.

Prior art DS is phosphoenolpyruvate and erythrose 4-
An enzyme that catalyzes the synthesis of 3-deoxy-D-arabino-heptulonic acid-7-phosphate (hereinafter referred to as "DAHP") from phosphoric acid, and DAHP is phenylalanine, tyrosine, or tryptophan via chorismate. Is converted to. In coryneform bacteria, DS activity is known to be synergistically inhibited by phenylalanine and tyrosine, which are the final products, and when breeding aromatic amino acid-producing bacteria, DS activity is released. It is important to select strains that retain DS activity.

On the other hand, breeding of these aromatic amino acid-producing bacteria by the recombinant DNA method is known, but some of them have been known (for example, JP-A-57-208994 and JP-A-57-71397).
JP-A-58-89194, JP-A-58-134994
Etc.), a gene encoding DS of coryneform bacterium (hereinafter referred to as “DS gene”) is not incorporated.

DISCLOSURE OF THE INVENTION Problems to be Solved by the Invention The present invention finds a microorganism having higher productivity of L-phenylalanine or L-tyrosine, and thereby finds a more efficient method for producing L-phenylalanine or L-tyrosine. Especially.

Means for Solving Problems As a result of research to solve the above-mentioned problems,
The coryneform bacterium having a recombinant DNA in which a gene encoding DS that is expressed in a coryneform bacterial cell and ligated to a plasmid vector capable of growing in the coryneform bacterial cell was successfully isolated, and the obtained coryneform It has been found that type bacteria have high productivity of L-phenylalanine or L-tyrosine.

That is, the present invention is obtained from a DNA-donating bacterium belonging to a coryneform bacterium, and is represented by a restriction enzyme map of the inserted gene portion in FIG. 1 or FIG. 2 and represented by 3-deoxy-D-arabino-
D containing the hepturonic acid-7-phosphate synthase gene
The NA fragment is ligated to a vector plasmid capable of autonomously replicating in the coryneform bacterium, and introduced into a DNA recipient bacterium belonging to the coryneform bacterium to cultivate a microorganism capable of producing L-phenylalanine or L-tyrosine. ,
L-phenylalanine or L- accumulated in the culture medium
A method for producing L-phenylalanine or L-tyrosine, which comprises collecting tyrosine.

Further, the present invention is the above-mentioned method for producing L-phenylalanine or L-tyrosine, wherein the DNA-donating bacterium and / or the DNA-accepting bacterium belonging to the coryneform bacterium is resistant to m-fluorophenylalanine.

Coryneform bacteria referred to in the present invention
Is the Bargeys Manual of Determinat.
ive Bacteriology) 8th edition p. 599 (1974), a group of microorganisms, aerobic, gram positive,
It is a non-acidic, non-spore forming bacillus. 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 Inmario Film ATCC
14068 Brevibacterium lactofermentum ATCC
13869 Brevibacterium roseum ATCC
13825 Brevibacterium flavum ATCC
13826 Brevibacterium thiogenitalis ATCC
19240 Corynebacterium acetoside film ATCC
13870 Corynebacterium acetoglutamicum ATCC
15806 Corynebacterium carnae ATCC
15991 Corynebacterium glutamicum ATCC 1
3032, 13060 Corynebacterium Lilium ATCC
15990 Corynebacterium meracecola 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 of isolating the DS gene is as follows. First, a chromosomal gene is extracted from a strain having the DS gene of coryneform bacterium (for example, Biochemical et Biophysica Actor (Bi
ochem.Biophys.Acta) 72, 619, 1963 can be used. ), And cut it with an appropriate restriction enzyme. Then, it was ligated to a plasmid vector capable of growing in coryneform bacterium cells, and the recombinant DNA obtained was used to transform a DS-deficient mutant strain of coryneform bacterium to obtain a strain which possesses DS-forming activity. Isolated and from this D
The S gene can be isolated.

In addition, a similar recombinant DNA was used to transform a wild strain of coryneform bacterium, and a strain that retained the resistance to an aromatic amino acid antagonist having an inhibitory effect on DS activity was isolated. It is also possible to isolate the gene.

Examples of aromatic amino acid antagonists include o-fluorophenylalanine, m-fluorophenylalanine, p-fluorophenylalanine, p-aminophenylalanine, β-3-thienylalanine, 3-aminotyrosine, tyrosine hydroxamate, 5-methyl. There are tryptophan and the like.

As the DNA-donating bacterium, it is more preferable to use a mutant strain in which biosynthesis activity of L-phenylalanine or L-tyrosine or its precursor is enhanced by imparting a mutation such as resistance to the aromatic amino acid antagonist.

The precursor of L-phenylalanine or L-tyrosine referred to herein is DAHP3-dehydroquinic acid, 3-dehydroshikimic acid, shikimic acid, shikimic acid-3-phosphate, 5-enolpyruvylshikimic acid-3-phosphate, Refers to colismic acid, prephenic acid, phenylpyrupic acid, etc.

Examples of such mutant strains include the above-mentioned US Pat.
660,235, U.S. Pat. No. 3,759,970, JP-A-56-64793 and the like.

As the DS gene, a wild type gene can be used, and a mutant gene can also be used. It is particularly preferable that the mutant gene is mutated so as to encode DS having a reduced degree of synergistic inhibition by phenylalanine and tyrosine.

In order to obtain a mutant gene, a DNA-donating bacterium may be subjected to mutation treatment, or the DS gene may be inserted into a vector plasmid, and the resulting recombinant DNA may be introduced into a DNA-accepting bacterium to obtain a transformant strain. Mutation processing may be performed. Furthermore, a mutant gene can also be obtained by subjecting the above recombinant DNA itself to mutation treatment in vitro.

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

The plasmid vector used in the present invention may be any as long as it can grow in coryneform bacterial cells. Specific examples are as follows.

(1) pAM 330 See JP-A-58-67699 (2) pAM 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 Ditto Cleavage of plasmid vector DNA It 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 the chromosomal gene is cleaved, or a chromosomal DNA cleavage fragment and an oligonucleotide having a complementary nucleotide sequence are connected to both ends of the cleaved vector DNA, Then plasmid vector and chromosome D
Subject to ligation reaction with NA fragment.

In order to introduce the recombinant DNA of the chromosomal DNA and the vector plasmid thus obtained into a recipient bacterium belonging to a coryneform bacterium, the Journal of Molecular as reported for Escherichia coli K-12 is used. Biology (J. Mol., Biol.,) 53, 159, 1
970) Treat the recipient cells with calcium chloride and DN
A method of increasing the permeability of A, or as reported for Bacillus subtilis (Gene, 1
Vol. 153, 1977. It is possible by a method of introducing into a growth stage (so-called competent cell) where cells can take up DNA. Alternatively, as is known for Bacillus subtilis, radiofungi and yeast (Molec. Gen., Genet.) 168, 111, 1979.
Year; Nature, 274, 398, 19
1978; Pro-Sweeting of National Academy of Sciences USA (Proc.Natl.Acad.Sei.US)
A), Vol. 75, p. 1929, 1978, it is also possible to introduce a plasmid into a DNA recipient bacterium by converting the DNA recipient bacterium into a protoplast or spheroplast which easily incorporates plasmid DNA.

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 treated with polyethylene glycol or A method of incorporating DNA in the presence of polyvinyl alcohol and a divalent metal ion can of course be used. Similar results can be obtained by a method of promoting the uptake of DNA by adding carboxymethyl cellulose, dextran, Ficoll, Bulonic F68 (Selva Co.) or the like instead of polyethylene glycol or polyvinyl alcohol.

As the L-phenylalanine or L-tyrosine-producing bacterium, a wild strain or a strain transformed with a DS-deficient strain as a host can be used. However, as shown below, the production of L-phenylalanine or L-tyrosine can be further improved by using the host. A strain with high activity may be obtained.

In the case of tyrosine, phenylalanine of the genus Corynebacterium is required.
Mutant strains resistant to aminotyrosine, p-aminophenylalanine, p-fluorophenylalanine, and tyrosine hydroxamate Journal of the Japanese Society of Agricultural Chemistry (Agric.Biol.Che
m.), 37, 2013, (1973), mutant strains resistant to m-fluorophenylalanine of the genus Brevibacterium (Agric. Biol. Chem.), 37
Vol., Page 2327, 1973, etc.

In the case of phenylalanine A mutant strain of the genus Previbacterium that has resistance to m-fluorophenylalanine (Agric.Biol.Che)
m.), 37, 2327, 1973), a mutant strain requiring tyrosine and methionine of the genus Brevibacterium and having resistance to 5-methyltryptophan and p-fluorophenylalanine (JP-A-49-116294), Brevi. Requires tyrosine and methionine of the bacterium,
Mutant strains resistant to methyltryptophan, p-fluorophenylalanine, and sensitive to decoinine (JP-A-56-
64793) Mutant strain that requires tyrosine of the genus Corynebacterium and has resistance to p-fluorophenylalanine and p-aminophenylalanine (Journal of the Japanese Society of Agricultural Chemistry (Agric.Biol.Chem., 38, 157, 1974)).
Etc.

If the following genes are inserted in addition to the DS gene, the productivity of L-phenylalanine or L-tyrosine is often higher.

These genes include 3-dehydroquinate synthase acid gene, 3-dehydroquinate dehydratase gene, shikimate dehydrogenase gene, shikimate kinase gene, 5-enolpyruvyl shikimate-3-phosphate synthase gene, chorismate synthase gene. Etc.

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

L-Phenylalanine or L thus obtained
-L-by culturing a coryneform bacterium capable of producing tyrosine
The method of producing and accumulating phenylalanine or L-tyrosine is not particularly different from the method conventionally used for production of L-phenylalanine or L-tyrosine by coryneform bacteria. That is, as the medium, carbon source, nitrogen source, inorganic ions, further amino acids, if necessary,
It is a normal one containing organic micronutrients such as vitamins. As the carbon source, glucose, sucrose, lactose and the like and starch hydrolysates containing these, whey, molasses and the like are used. As the nitrogen source, ammonia gas, aqueous ammonia, 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 L-phenylalanine or L-tyrosine is substantially stopped.

Example (1) Preparation of chromosomal DNA containing DS gene As a source for preparing chromosomal DNA, phenylalanine has been released from inhibition of DS, and thus has a resistance to m-fluorophenylalanine which is a phenylalanine analog. Phenylalanine-producing bacterium Brevibacterium lactofermentum AJ1957 (FE
RM-P6673) was used.

This fungus was treated with 1 CMG medium (peptone 1 g / dl, yeast extract 1 g / dl, glucose 0.5 g / dl, and NaCl.
0.5 g / dl, adjusted to pH 7.2), and cultured at 30 ° C. for about 3 hours with shaking to collect the bacterial cells in the logarithmic growth phase.

After lysing the cells with lysotil-SDS, 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, Prebibacterium lactofermentum AJ12037 (FERMBP-57, which carries pAJ1844 as a plasmid
7) was inoculated into 100 ml of CMG medium, cultured at 30 ° C. until the late logarithmic growth phase, lysed by lysozyme SDS treatment, and a supernatant was obtained by ultracentrifugation at 30,000 × g for 30 minutes. After phenol treatment, 2 volumes of ethanol was added to precipitate and collect DNA. Add a small amount of TEN buffer (20 mM Tris-HCl group, 20 mM NaCl, 1
After dissolving in mM EDTA (pH 8.0)), it was 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 each kept with restriction endonuclease PatI at 37 ° C. for 1 hour and cleaved. After heating for 10 minutes to 65 ° C., a mixture of both the reaction solution, ATP and dithiothreitol presence was ligated a 24 hour hold to DNA strand 10 ° C. by DNA ligase from _{T 4} phage. Then, the reaction solution was heated at 65 ° C. for 5 minutes, and 2 volumes of ethanol was added to the reaction solution to collect a precipitate of ligated DNA.

(4) Cloning of DS gene As a DNA recipient bacterium, m-fluorophenylalanine-sensitive Prebibacterium lactofermentum AJ
12036 (FERM P-7559, FERM BP-7
34) was used.

As the transformation method, the protoplast transformation method was used. First, the strain was cultured in 5 ml of CMG liquid medium until the beginning of the logarithmic growth phase, and penicillin G was added at 0.6 unit / ml, and then 1.5 After culturing with shaking for a period of time, cells were collected by centrifugation, and the cells were 0.5 ml of SMMP medium (pH 6.5) consisting of 0.5 M sucrose, 20 mM maleic acid, 20 mM magnesium chloride and 3.5% Penassay broth (Difco). Washed with. Then, the cells were suspended in SMMP medium containing 10 mg / ml of lysozyme and protoplastized at 30 ° C. for 20 hours. 6000
After centrifuging at × g for 10 minutes, SMMP the protoplasts
It was washed with and washed again with 0.5 ml of SMMP. The protoplast thus obtained and DNA1 prepared in (3)
After mixing 0 μg in the presence of 5 mM EDTA and adding polyethylene glycol to a final concentration of 30%, the mixture was left at room temperature for 2 minutes to incorporate DNA into protoplasts. The protoplasts were washed with 1 ml of SMMP medium, resuspended in 1 ml of SMMP medium, and cultured at 30 ° C. for 2 hours for expression of traits. This culture solution was adjusted to pH 7.
Plated on 0 protoplast regeneration medium. Protoplast regeneration medium is 12 g of tris (hydroxymethyl) aminomethane, 0.5 g of KCl, 10 g of gucose, 8.1 g of MgCl ₂ .6H ₂ O, 8.1 g of CaCl ₂ per distilled water.
2H ₂ O 2.2 g, peptone 4 g, powdered 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, and these were used as a minimal medium (2% glucose, 1% ammonium sulfate, 0.3% urea) containing 500 μg / ml of m-fluorophenylalanine. , 0.1% potassium dihydrogen phosphate, 0.04% magnesium sulfate heptahydrate, 2 ppm iron ion, 2 ppm manganese ion, 200 μg / cyamine hydrochloride, 50 μg / biotin, chloramphenicol 10 μg / ml, pH 7.0 ,
Agar (1.8%) was replicated to obtain 5 strains of chloramphenicol resistant and m-fluorophenylalanine resistant strains.

(5) Plasmid analysis of transformed strains From these strains, lysates were prepared by the method described in (2), and plasmid DN was analyzed by agarose gel electrophoresis.
When A was detected, vector pAJ184 was detected in all strains.
A plasmid clearly larger than 4 was detected.

Restriction enzyme Ps using 5 strains of plasmid for recombination
When digested with tI, a 6.7 kb D, which is common to the 4 strains
NA insert was observed.

In addition, for the other 1 strain, 3.7 kb, 3.0 kb, 1.7 kb,
1.6kb, 1.4kb, 1.3kb, 0.8kb, 0.7kb,
Nine DNA inserts of 0.4 kb were observed. The 6.7 kb Pat of the former was inserted at the PatI breakpoint of the vector pAJ1844.
The recombinant plasmid containing the I fragment was named pAR-2,
The latter recombinant plasmid having 9 PatI DNA fragments was designated as pAR-1, and a strain carrying pAR-1 was designated as AJ12181.
(FERM-P7948), a strain harboring pAR-2 is AJ12182
(FERM P-7947, FERM BP-917)
I named it. Note that pAR-2 has the restriction enzyme map shown in FIG.

(6) In order to confirm that the gene conferring m-fluorophenylalanine resistance is present on the recombinant plasmid containing the DNA fragment detected by retransformation (5), this plasmid DNA
Brevivacletium lactofermentum AJ12036 was transformed again using pAR-1 and pAR-2.

When 50 chloroamphenicol-resistant colonies were picked up and tested for their sensitivity to m-fluorophenylalanine, resistance was imparted to all of them, and m-fluorophenylalanine resistance was found on the above plasmid. It was revealed that the gene to be added exists.

(7) DS activity of transformant strain The test strain was treated with phenylalanine production medium (glucose 130
_{g, (NH 4) 2 SO} 4 10g, KHP 2 PO 4 1g, MgSO 4 ·
7HP ₂ O1g, fumaric acid 12g, acetic 3 ml, soybean protein acid hydrolyzate "Taste liquid" 50ml, FeSO ₄ · _7H 2 O10m
g, MnSO ₄ .4H ₂ O 10 mg, biotin 50 μg, thiamine hydrochloride 2000 μg and CaCO ₃ 50 g are included in water 1. A lysate was prepared from the cells cultured in 20 ml of pH 7.0) at 30 ° C. for 15 hours by ultrasonic treatment, and the lysate was centrifuged at 32,000 × g for 20 minutes to obtain a supernatant. This supernatant was used as a crude enzyme solution and was composed of 50 mM Tris-hydrochloric acid buffer (pH 7.5), 0.5 mM phosphoenolpyruvate, and 0.5 mM erythrose-4-phosphate.
The reaction is carried out in 0 ml of the reaction solution at 30 ° C. for 10 minutes, and after completion of the reaction, 0.2 ml of trichloroacetic acid is added to inactivate the enzyme.
After removing the denatured protein by centrifugation, 0.25 ml of the resulting supernatant was added with 0.
Add 25 ml of 0.025M Periodic acid and add 30 at 37 ° C.
Incubate for minutes. After 0.5 ml of 2% sodium arsenite was added to stop the oxidation reaction, 2.0 ml of 0.3
% 2-Thiobarbituric acid is added and heated at 100 ° C. for 8 minutes. The absorbance of the reaction solution at 579 nm is measured and DAHP is measured.
Molecular extinction coefficient of 4.5 × 10 ⁴ (Journal of Biological Chemistry (J. Biol. Chem.), 241
Vol. 3, p. 3365, 1966) was used to determine enzyme activity. Further, the activity when either 1 mM of phenylalanine or tyrosine or both of them were added to the reaction solution was also measured, and the activity when not added was expressed as 100%. First
The measurement results are shown in the table.

By introducing the obtained recombinant plasmid, DS
The activity was increased by 2.6 to 14 times, and the activity inhibition pattern by each amino acid was changed from the wild type to the mutant type, and the m-fluorophenylalanine resistance gene encoded by pAR-1 and pAR-2. Is clearly the DS gene. In addition, Pst I possessed by pAR-1 and pAR-2
Since the fragments are completely different, it is considered that there are two or more types of genes encoding proteins having DS activity.

(8) Subcloning of DS gene pAR-1 was cleaved with restriction enzyme PstI, heated at 65 ° C. for 10 minutes, and then left overnight at 4 ° C. in the presence of ATP and dithiothreitol using T4 phage-derived DNA ligase. The DNA strands were then ligated. Then the reaction is carried out at 65 ° C for 10
After heating for 1 minute, 2 volumes of ethanol was added to the reaction solution to collect the ligated DNA precipitate. A small amount of TEN buffer was added to this and used for transformation.

Brevibacterium lactofermentum AJ12036 was transformed using the transformation method shown in (4). Thirty
After regeneration with a regeneration medium containing chloramphenicol 3 µg / ml for 2 weeks at ℃, m-fluorophenylalanine 50
By replicating in a minimal medium containing 0 μg / ml, a large number of strains resistant to chloramphenicol and resistant to m-fluorophenylalanine were obtained. Some of these strains have pAR-1
It had a smaller group of plasmids. The most miniaturized plasmid among these was named pAR-112, and the strain carrying pAR-112 was named AJ12183 (FERM-p7946). PAR
-112 has the restriction map shown in FIG.

(9) Phenylalanine and tyrosine productivity of each transformant Using the above pAR-1, the m-fluorophenylalanine-resistant strain Brevibacterium lactophenylmentum AJ12
184 was transformed by the method described in (4), and a transformant was selected using chloramphenicol resistance as an index. The thus obtained AJ12185 (FERM P-7945, BP
-916D and the above AJ12036, AJ12181, AJ12182, AJ1
When 2183 was cultured and the phenylalanine and tyrosine production ability was examined, the results shown in Table 2 were obtained.

The culturing was carried out by inoculating the test strain into 20 ml of the phenylalanine production medium described in (7) in a 500 ml shoulder flask and shaking at 30 ° C. for 72 hours. After culturing, 2 ml of 6N potassium hydroxide solution was added to precipitate L
-Tyrosine was dissolved and L-phenylalanine and L-tyrosine in the centrifugation supernatant were quantified by liquid chromatography.

In order to obtain AJ12184, it is possible to remove the complex plasmid in the host cell from the deposited AJ12185 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 (bacteriologic debut (Bact.R).
ev.), 36, 361-405, 1972). Examples of other removal operations are as follows. AJ12185 to CMG
Inoculate the liquid medium and incubate at 37 ° C overnight (high temperature treatment),
The culture broth is appropriately diluted and applied to a CMG agar medium containing or not containing chloramphenicol, and 1 to 30 ° C.
Incubate for 3 days. The strain thus isolated as a chloramphenicol sensitive strain is AJ12184.

[Brief description of drawings]

FIG. 1 is a restriction map of plasmid pAR-2. Second
The figure is a map of the regulatory enzyme of plasmid pAR-112. In each figure, Is pAJ1844, Is an inserted gene; E, P, S and B are EcoRI and Pst, respectively.
I, SalI, BamHI are shown.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display location C12R 1:13) Examiner Kazuo Hirata

Claims (4)

[Claims] 1. A DNA fragment containing a 3-deoxy-D-arabino-heptulonic acid-7-phosphate synthase gene represented by restriction enzyme map I obtained from a DNA-donating bacterium belonging to a coryneform bacterium, or a coryneform bacterium. Which can be autonomously replicated in the coryneform bacterium, in which a DNA fragment containing the 3-deoxy-D-arabino-heptulonic acid-7-phosphate synthase gene represented by the restriction map II obtained from a DNA donor bacterium belonging to Connected to the plasmid,
A method for culturing a microorganism having an ability to produce L-phenylalanine or L-tyrosine obtained by being introduced into a DNA recipient bacterium belonging to a coryneform bacterium, and collecting L-phenylalanine or L-tyrosine accumulated in the culture solution. And a method for producing L-phenylalanine or L-tyrosine. 2. A DNA-donating bacterium belonging to a coryneform bacterium is m-
The method for producing L-phenylalanine or L-tyrosine according to claim 1, which is resistant to fluorophenylalanine. 3. The 3-deoxy-D-arabino-heptulonic acid-7-phosphate synthase encoded by the DNA fragment according to claim 1 is L-tyrosine and L-tyrosine.
-The method for producing L-phenylalanine or L-tyrosine according to claim 1, wherein synergistic feedback inhibition by phenylalanine is eliminated. 4. A DNA recipient bacterium belonging to a coryneform bacterium is m-
4. A method for producing L-phenylalanine or L-tyrosine according to claims 1 to 3, which is resistant to fluorophenylalanine.