JPS61260892A - Production of l-phenylalanine - Google Patents

Abstract

PURPOSE:To obtain efficiently the titled substance, by using a strain containing a recombinant DNA of a DNA fragment containing a gene taking part in the synthesis of chorismate mutase and prephenate dehydratase of specied of the genus Escherichia and a vector DNA. CONSTITUTION:A microorganism, holding a recombinant DNA of a DNA fragment containing a gene taking part in the synthesis of chorismate mutase and prephenate dehydratase of a microorganism belonging to the genus Escherichia and a vector DNA, and belonging to the genus Corynebacterium, Brevibacterium of Microbacterium, is cultivated. The vector is a plasmid obtained from pCG1, pCG4, pCE51, etc., derived from a microorganism belonging to the genus Corynebacterium. The host microorganism is Corynebacterium glutamicum, Corynebacterium acetoacidophilum, etc. The aimed L-phenylalanine is recovered from the resultant culture by a well-known method.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention relates to DNA containing genes of Escherichia species involved in the synthesis of colismate mutase (hereinafter abbreviated as CM) and prephenate dehydratase (hereinafter abbreviated as PD).
The recombinant DNA of the fragment and vector DNA is held in a microorganism belonging to the genus Corynebacterium, Brevibacterium, or Microbacterium, and the microorganism is cultured in a medium to obtain L- The present invention relates to a method for producing L-phenylalanine, which comprises collecting phenylalanine. Therefore, the present invention provides bioindustry v
-, mRegarding the industrial field, particularly the field of producing L-phenylalanine useful in the pharmaceutical and food industries.

[Prior art] Methods for producing L-phenylalanine by fermentation using microorganisms belonging to the genus Lineobacterium, Brevibacterium or Microbacterium include tyrosine auxotrophic mutations, resistant mutations to analogues of phenylalanine, or these. A method using a bacterial strain that also has mutations is known [Journal of the Japanese Society of Agricultural Chemistry, 50. (1) p
, R79 (1976)). Furthermore, a method has also been described in which the genes of enzymes involved in phenylalanine biosynthesis of these microorganisms are cloned using recombinant DNA technology, and a strain created using this is used (Japanese Patent Application Laid-Open No. 1983-1999).
No. 24192).

Problems to be Solved by the Invention In recent years, as the demand for phenylalanine has increased, there has been a strong desire to improve the method for producing phenylalanine. In view of this situation, the present inventors have made effective use of recombinant DNA technology,
We conducted extensive research to create an excellent phenylalanine-producing bacterium.

Means to Solve the Problems If a strain of Escherichia species containing a DNA fragment containing a gene involved in L-phenylalanine biosynthesis and a gene involved in the synthesis of PD and a vector DNA is recombinant, It was discovered that the productivity of L-phenylalanine was improved, and the present invention was completed.

The present invention will be explained in detail below.

According to the present invention, a Corynebacterium, Brevibacterium or microorganism containing a recombinant DNA of a DNA fragment containing the genes of enzymes CM and PD involved in phenylalanine biosynthesis of a Escherichia species and a vector DNA. By culturing microorganisms belonging to the genus Bacterium in a medium, producing and accumulating L-phenylalanine in the culture, and collecting 17-enylalanine from the culture,
L-phenylalanine can be produced.

As the microorganism belonging to the genus Corynebacterium, Brevibacterium, or Microbacterium to be used as the host microorganism, all microorganisms known as coryneform glutamate-producing bacteria can be used, but the following strains are preferably used. used.

Corynebacterium glutamicum ^TCC1303
2 Corynebacterium acetoacidophyllum ATCC
13870 Corynebacterium herculis ^TCC1386
8 Corynebacterium Lilium ^TCC159
90 Brevibacterium divaricolumn ^TCC14
020 Brevibacterium flavum ^TCC1
4067 Brevibacterium imariophyllum^TC
C14068 Brevibacterium lactofermentum ATCC1
3869 Brevibacterium thiogetaris ATCC1924
0 Microbacterium ammonia film ATCC1
5354 As the host microorganism, a strain that does not have the ability to produce L-phenylalanine can be used, but preferably a strain that has the ability to produce L-phenylalanine is used. A strain capable of producing L-phenylalanine can be constructed by a tyrosine-requiring mutation, a phenylalanine analog resistance mutation, or a known method of sharing these mutations, as described above.

In the present invention, the microorganism that serves as the source of CM and PD genes may be any microorganism that belongs to the genus Escherichia and has reactivation of CM and FD. Stock JA194 (
Proceedings of the National Academy of Sciences (Proc, Natl, ^c
ad, Sci, ), 94. 487 (1977
) ) can be used. The 112 strains of Escherichia coli have a gene product with both CM and PD enzyme activities encoded by a single gene called phe^ [Journal of Bacteriology 02I (J, B
acteriol, ) 150.1130 (1982)
) is advantageous in that it can be cloned as a single DNA fragment.

The chromosomal DNA that is the source of the phe^ gene is CM-
A conventional method [Biochem, Biophy] involves treating cultured microorganisms with PD activity with lysozyme and a surfactant to lyse them, removing proteins, and then precipitating them with ethanol.
s,^cta) 72.619 (1963)). As a vector, any plasmid that can be replicated in a microorganism belonging to the genus Escherichia may be used, but for example, the frequently used pBR322 (Gene, 2.95 (1975)) pA
cYc1? ? , pAcYc184 (both Journal of Bacteriology 02 ICJ, Bacteri
ol,) 131.1141 (1978)), pG^
22 [Journal of Bacteriology 02i (J, B
acteriol, ) 140.400 (1979))
etc. can be used. In vitro recombination between chromosomal DNA and plasmid is performed by cutting both with restriction enzymes and then using D.
Conventional methods such as treatment with NA'J gauze, or treatment of the cut fragments with terminal transferase, DNA polymerase, etc. and ligation by applying DNA ligase [Methods in Enzymology 4]
ds in Enzymology) 68 (1979
) ) can be implemented.

The pheA gene was transferred to the vector plasmid by transforming 112 phenylalanine auxotrophic mutant strains of Enerysia coli deficient in the pheA gene using this binding reaction product and selecting the transformants that were non-phenylalanine auxotrophic. can be cloned into. The transformation method used here is, for example, a method in which recipient bacterial cells are treated with calcium chloride at low temperature to incorporate DNA into the cells [Journal of Molecular Biology (J, Mo1. Biol, ) 53. 159 (1
970); Gene, 6.23(1
979)). In this way, the phe^ gene is processed using the restriction enzymes EcoRI and Hind
Approximately 4.2 kilobases (Kb) of D cut out by lli
It can be cloned into vector pBR322 as an NA fragment. On this DNA fragment 4.2b, ar exists adjacently on the chromosome of 112 strains of Escherichia coli.
oF gene and tyr^ gene [Microbiological
Microbiol, Rev., (2
) 180 (1983)), but in addition ρ
By cutting out the heA gene with another restriction enzyme, recombining it with the vector, transforming the pheA-deficient strain in the same manner as above, and selecting a transformed strain that does not require phenylalanine, I) NA containing only the phe^ gene can be obtained. You can get fragments.

pheA gene-containing DN cloned in this way
The A fragment and a vector plasmid of a microorganism belonging to the genus Corynebacterium, Brevibacterium or Microbacterium are recombined in vitro by cutting with a restriction enzyme and ligating with DNA ligase to produce a microorganism belonging to the genus Corynebacterium, Brevibacterium or Microbacterium. A recombinant plasmid that can be replicated in Microbacterium can be produced.

The vector plasmid is not particularly limited as long as it can replicate in the above-mentioned microorganisms, but for example, pccl (Japanese Patent Laid-Open No. 57-1345
00), pCG2 (JP-A-58-35197>, pC
G4, pCGll (both JP-A-57-183799)
) pCE54, pcBlol (both published in Japanese Unexamined Patent Publication No. 58-1
05999), pCE 51 (Patent application 1986-341)
97 > and.

pCE52, pCE53 (both from Molecular and General Genetics (Mol, Gen.

Genet, ) 196. 175 (1984)) etc. are used.

The recombinant DNA of the DNA fragment containing the ρheA gene and the vector plasmid is transduced into CM or PD by normal mutagenesis using an in vitro recombination mixture.
It can be obtained by directly transforming a phenylalanine-auxotrophic mutant strain of the genus Corynebacterium, Brevibacterium or Microbacterium that lacks enzymatic activity, and selecting a transformed strain that has become non-auxotrophic for phenylalanine. In particular, when using a shuttle vector such as pcE51 that can replicate in both Escherichia coli and microorganisms of the genus Corynebacterium, Brevibacterium, or Microbacterium, it is recommended to use a pheA-deficient mutant strain of Escherichia coli as described above. Due to complementarity, phe^
The gene can also be subcloned onto a shuttle vector and subsequently introduced into a microorganism belonging to the genus Corynebacterium, Brevibacterium or Microbacterium. The transformation method for introducing recombinant DNA into these microorganisms is carried out by a method using protoplasts developed by the present inventors (see JP-A-57-186492 and JP-A-57-186489 and the following Examples). can do.

When the chromosomal DNA of Escherichia coli having the wild-type pheA gene is used as a source as described above, the wild-type pheA gene can be used as a recombinant DNA and the chromosomal DNA of the genus Corynebacterium, Brevibacterium, or Microbacterium can be used as a source. Can be introduced into microorganisms. However, wild-type p
It is known that the synthesis of the gene product CM-PD encoded by the heA gene is suppressed by phenylalanine produced in Escherichia coli, and that CMPD activity is inhibited by the phenylalanine produced [Journal of Batatteriology ( J, Bacteriol;
) 150, 1130 (1982); European Journal of Biochemistry (εur,
J.Biochem,) 86, 165 (1978))
. Therefore, the mutant p released from this suppression and inhibition
It is better to introduce the he^ gene into the Corynebacterium genus,
It is preferable because it can impart high phenylalanine production ability to microorganisms of the genus Brevibacterium or Microbacterium.

To introduce the mutant phe^ gene, use the chromosomal DNA of an Escherichia coli mutant strain that has the desired mutated pheA gene as a source and recombine it in the same manner as the method used to obtain the wild-type phe human gene. This is accomplished by creating a body plasmid. Alternatively, microorganisms carrying a recombinant plasmid containing the wild-type pheA gene were inv.
It is also possible to create a recombinant plasmid containing the mutant phe^ gene by mutating it in vivo or by inducing the mutation in vitro and transforming it, which can then be used.

A recombinant plasmid containing the wild type or mutant pheA gene can be introduced into a microorganism of the genus Corynebacterium, Brevibacterium or Microbacterium by the transformation method using protoplasts as described above. L-phenylalanine production using these recombinant plasmid-carrying strains can be carried out by a culture method used for L-phenylalanine production by conventional fermentation methods.

That is, if the transformed strain is cultured in a normal medium containing carbon sources, nitrogen sources, inorganic substances, amino acids, vitamins, etc. under aerobic conditions while controlling temperature, pH, etc., Since L-phenylalanine is produced and accumulated, it is collected.

Carbon sources include glucose, glycerol, fructose, shakerose, maltose, mannose, starch,
Starch hydrolyzate, carbohydrates such as molasses, polyalcohols, various organic acids such as pyruvic acid, fumaric acid, lactic acid, and acetic acid can be used. Furthermore, depending on the assimilation ability of microorganisms, hydrocarbons, alcohols, etc. may also be used. In particular, blackstrap molasses is preferably used.

Nitrogen sources include ammonia or various inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium carbonate, ammonium acetate, or urea and other nitrogen-containing substances, as well as peptones, NZ-amines, meat extracts, yeast extracts, corn stew.・Liquor, casein hydrolyzate, fish! Various materials can be used, including nitrogen-containing organic substances such as -%-ruol or its digested product, and wax hydrolyzate.

Further, as inorganic substances, potassium hydrogen phosphate, potassium dihydrogen phosphate, ammonium sulfate, ammonium chloride, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, etc. are used. Vitamins, amino acid sources, etc. necessary for the growth of microorganisms do not need to be added as long as they are supplied to the medium by the other medium components as described above.

Cultivation is performed under aerobic conditions such as shaking culture or aerated agitation culture. Generally, the culture temperature is preferably 20 to 40°C. It is desirable to maintain the pH of the medium near neutrality. L-7 enylalanine accumulates in the medium during a culture period of usually 1 to 5 days. After culturing, the bacterial cells are removed and L is removed from the culture solution using known methods such as activated carbon treatment and ion exchange resin treatment.
- Phenylalanine is recovered.

By using a Corynebacterium, Brevibacterium, or Microbacterium strain carrying a recombinant DNA containing the phe^ gene, L-7 enylalanine can be produced in high yield. Specifically, such microorganisms include Corynebacterium glutamitum K-51 (FERM BP-76).
8), Corynebacterium glutamitum K-52 (F
ERMBP-769), and its host microorganism Corynebacterium glutamitum K-50 (FERMBP-
767) and has been deposited with the Institute of Microbial Technology (Feikoken), Agency of Industrial Science and Technology.

Examples of the present invention are shown below.

Example 1゜(1) Preparation of plasmid DNA containing phe^ containing the CM and PD genes of Escherichia coli As disclosed by the present inventors in JP-A-60-34197, the wild type pheA of Escherichia coli was The gene is 112 substrains of Escherichia coli JA194 [Proceedings of the National Academy of Op Science (
Proc, Natl.

^cad, Sci, ), 94.487 (1977)
) was used as a source, and pBR322 was used as a vector to generate approximately 4.2 Kb EcoRi-Hindl [7
It was cloned as one fragment. This recombinant plasmid pBaroF1 contains EcoRI,
The DNA cloned by Prowski et al.
Fragment [Procedure of the National Academy of Sciences x (Proc, Natl.
, ^cad, Sci,) USA, 75.4271 (
DNA cloned by comparison with 1978))
Along with the pheA gene, there is another gene aroP on the fragment.
and tyr8.

Therefore, D containing only the pheA gene from p8aroF1
To subclon the NA fragment, first p[! ar
oF1 was isolated from cultured cells of Escherichia coli strain A33248 (the method for producing this strain is described in JP-A-60-34197) by the following method.

40 Qml LB medium containing 100 g/ml ampicillin (10 g bactodryptone, 5 g yeast extract, 1 g glucose and 5 g sodium chloride in water, p
The seed culture was inoculated into a medium (adjusted to H7.2), cultured with shaking at 37°C, and grown to late logarithmic stage. After collecting bacteria from the culture solution, the bacterial cells were collected using the method of Nichi et al. [Journal of Bacteriology 4 (J, Bacteriol), 121.
354 (1975)). The obtained lysate was centrifuged at 28.00 rpm and 4° C. for 1 hour, and the supernatant was collected.

115 volumes of 50% (w/v) polyethylene glycol (PEG) 6000 aqueous solution was added to the top layer, mixed gently, and then left overnight at 4°C. The resulting precipitate was stored at 4°C for 3
．． OOOrpm, collected by centrifugation for 5 minutes and diluted with 5mM TE buffer (10mM Tris-HCl, 1mM E
Dissolved in DTA-Na2, pH 7.5), 1.5 ■/m
Add 1 ml of ethidium bromide at a concentration of T.
E buffer was used to make exactly 7.5mM. In this solution, C5
Cj! After adding 7.875g and completely dissolving, 105g
．． Centrifugation was performed at OOOXg120°C for 40 hours. Remove the plasmid band detected under ultraviolet irradiation with a syringe,
Ethidium bromide was extracted three times with Improper Tools containing 5% (V/V) TEIfr solution, followed by dialysis against TE buffer overnight at 4°C, and the dialysate was purified with plasmid DNA.
It was used as solution A.

(2) Subcloning of phe^ gene From the plasmid pBaroPI DNA prepared above, phe
A DNA fragment of the gene portion is obtained and ligated to the Escherichia coli vector pBR322.

Restriction enzyme reaction solution containing 3 g of plasmid pBaroFI DNA (10mM Tris-HCI, 6mM
MgCl2.100mM NaCl, pH7,5)10
5 units each of EcoRI and BamHI (manufactured by Takara Shuzo Co., Ltd.) were added to 0m and reacted at 37°C for 60 minutes. In addition, a restriction enzyme reaction solution containing pBR322 DNA 3 shoulder (
Same as above) EC0RI and 3amH at 100m and 5am each
I was added and reacted at 37°C for 60 minutes. After reaction, 65
The reaction was stopped by heating at °C for 10 minutes. Mix both reaction solutions and add T4 ligase buffer (660mM Tris
-HCI, 66mM MgC1z, 100mM dithiothreitol, pH 7,6) 40d, ATP (5a
+M) 40JII2, T4 ligase (manufactured by Takara Shuzo Co., Ltd., 1
Unit/JIIt) 0.4 Pa and 120 scraps of H2O were added, and the mixture was reacted at 4°C for 24 hours. After the reaction, the reaction was stopped by heating at 65° C. for 15 minutes.

This ligase reaction mixture is subjected to transformation.

As a recipient strain for transformation, one 12 strains of Escherichia coli lacking the phe^ gene, GS245 (pi
e＾905. araD139. Δ1acU169
．． Δgly^.

str^, thi) (Gy (Gene),
14.63. (1981)] is used. An overnight culture at 37°C was inoculated into LB medium, and M. Dagert
[Gene et al.

6, 23. (1979)) competent cells were prepared.

0.2111 fluids containing 10"/nl of competent cells
50 scraps of the ligase reaction mixture were added to No. 1 and left to stand for 10 minutes under water cooling. Next, after heating at 37°C for 5 minutes, LB medium 211 was added, and the mixture was incubated at 37°C for 90 to 120 minutes.

After that, the bacterial cells were centrifugally washed twice with physiological saline, and 50.1
M9 plate medium containing 1/II glycine (glucose 2g
5NH4CI21 g, NatHPO46g, KHzP
O* 3g5Mg504・7H700,1g5CaC
12.2H2015*, 4* of thiamine hydrochloride, and 15 g of agar in 11 parts of water and adjusted to pH 7.2). Each colony grown on M9 plate medium was diluted with ampicillin 100 at 7 mM.

It was spread on an LB plate medium containing tetracycline 20/a+1, and colonies that grew on the ampicillin-containing medium and did not grow on the tetracycline-containing medium were selected.

Plasmid DNA was isolated in the same manner as described above from the thus selected transformant strain, which grew on the M9 plate medium containing glycine and was resistant to ampicillin and sensitive to progeny tracytalline. pP in the plasmid obtained from one of the transformed strains.
After digesting HEAl with various restriction enzymes, analysis by agarose gel electrophoresis revealed that EcoRI and Ba of pBR322
Approximately 1,8 Kb of EcoRI-Ba is present in the region between mHI.
It turned out to be a plasmid into which an mHI cut DNA fragment had been inserted (see Figure 1).

(3) phe^ gene shuttle vector pcE5
From the plasmid pPHEAI DNA prepared above, p
A DNA fragment of the heA gene portion is obtained and ligated to Escherichia coli and Corynebacterium glutamicum shuttle vector pcE51.

pcE51 is derived from the Corynebacterium glutamicum plasmid pcG1 (Japanese Patent Application Laid-Open No. 134500/1983), for which the present inventors previously applied for a patent, and the Escherichia coli plasmid pGA22 (Journal of Batteriology).
J, Bacteriol, ) 14Q.

400, (1979)). For details, see JP-A-58-1 by the present inventors.
42804, the Bgl II cleavage site, which exists only at one location on pcGl, and the BamHI fragment of pGA22 containing the kanamycin resistance gene are ligated using the same cohesive ends of both restriction enzymes.

Restriction enzyme reaction solution (10mM Tris-HCl, 5mM
gC12, loOmM NaCj! , pH 7,5)
5 units of 5cai and NruI (manufactured by Zenshuzo Co., Ltd.) were added to each of the 100 samples and reacted at 37°C for 60 minutes. Also, pc
Plasmid pcE51 D prepared in the same manner as above from B. coli J^194 strain carrying E51
Restriction enzyme F containing 3 blocks of NA, coRV reaction solution (lQ
mM Tris-HCI, 6mM MgCl*,
150mM NaCl, pH 7.5) Add 5 units of EcoRV [manufactured by Zenshuzo Co., Ltd.] to 100J11 and incubate at 37℃ for 6 hours.
The reaction was allowed to proceed for 0 minutes.

Add T4 ligase buffer 40d, ATP (5mM) 4
0m, T4 ligase (manufactured by Zenshuzo Co., Ltd. 1 unit/d) 0.4
111 and HiO120m were added, and the mixture was reacted at 4°C for 24 hours.

Using this ligase reaction mixture, Escherichia co1 JGs strain 245 was transformed in the same manner as described above, and 50 cells were transformed.
Plate on M9 plates containing g/ml glycine. M
Each colony grown on 9 plates was treated with ampicillin 10
Colonies that grew in the kanamycin-containing medium and could not grow in the ampicillin- and tetracycline-containing medium were selected.

Plasmid DNA was isolated from the thus selected transformed strain in the same manner as described above. After digesting the plasmid pEpheA1 obtained from one of the transformed strains with various restriction enzymes,
As a result of analysis by agarose gel electrophoresis, the EcoRV cleavage site of pcE51 has approximately 2. It turned out to be a plasmid into which a Sea 1-NruI cut DNA fragment containing the Bb phe^ gene was inserted (see Figure 1).

(4) Transformation of Corynebacterium glutamicum with pEpheA1 and acquisition of phenylalanine analog resistant plasmid pEphe^22 The pBphe^1 DNA prepared above was subjected to transformation. Corynebacterium glutamicum L-15 ATCC31834, a zozyme-sensitive mutant strain derived from Corynebacterium glutamicum AT CC31830, was used as the recipient strain for transformation. L-15
A seed culture of the strain was inoculated into an NB medium (medium containing 20 g of powdered broth and 5 g of yeast extract in 11 parts of water and adjusted to pH 7.2) and cultured with shaking at 30°C. When the OD l) reached 6, the cells were collected and transferred to RCGP medium [5 g of glucose, 5 g of casamino acids, 2.5 g of yeast extract, 2 HP.
Oa 3.5g5KHiPOa 1.5g.

MgCA2・6H200,41g5FeSOs・7Hz
0 10■, Mn3O4・4~6H1O2■, Zn5
O,・7H200,9mg, (NH4)@MOvO2s
l 4HzOO, 04mg, piotine 30■, thiamine hydrochloride 2■, 135g disodium conolinate, polyvinylpyrrolidone (molecular weight 10.000) 30g in 11 parts water] A solution containing 1*g/ml Norizozyme (pH 7.6) to approximately 109 cells/ml, transferred to an L-shaped test tube, and subjected to gentle shaking reaction at 30° C. for 5 hours to form protoplasts.

Transfer 5 ml of this protoplast suspension Q to a small test tube, centrifuge at 2.500Xg for 5 minutes, and TSMC 1l buffer (13mM magnesium chloride, 30+nM calcium chloride, 50mM), 400mM sucrose, pH 7.5).
After resuspending to 1mM and washing by centrifugation, TSMC8t buffer Q,
Resuspend to 1mM. Add 73MC to this suspension at twice the concentration.
Add 100 scraps of a 1:1 mixture of buffer and the above pBphe^l DNA mixture and mix, then add 20% polyethylene glycol 6.000 (PEG) in 73MC1I solution.
6.000) was added and mixed.

After 3 minutes, add RCGP medium (pH 7, 2) and
．． The supernatant was removed by centrifugation at 500×g for 5 minutes, and the precipitated protoplasts were suspended in 1 mM RCGP medium and gently shaken at 30° C. for 2 hours. Kanamycin 2 was added to 0.21111 of this protoplast suspension.
00x/ml on RCGP agar medium (RCGP medium containing 1.4% agar, pH 7,2), and
Shake at 0°C for 7 days.

Plasmid DNA was isolated from cultured cells of kanamycin-resistant colonies grown on selection plates in the same manner as described above. As a result of analyzing the plasmid obtained from one transformed strain by agarose gel electrophoresis after digestion with various restriction enzymes, it was found that p
It was found to be the same plasmid as EpheAl.

Next, the 115 strain harboring pEpheAl was grown in NB medium containing 10 μl/ml of kanamycin until late logarithmic growth. After centrifugally washing the bacterial cells twice with 50 mM Tris-maleic acid buffer (pH 6.0), N-methyl-N'-
The cells were treated for 30 minutes at room temperature in 50 mM) lis-maleate buffer (pH 6,0) containing 400 M/ml of nitro-N-nitrosoguanidine. 50111M of treated bacterial cells
)! After centrifugal washing twice with J.sucereic acid buffer (pH 6.0), the washed bacterial cells were washed with N containing 10 μ/ml of kanamycin.
The cells were cultured in medium B at 30°C for 16 hours, and plasmid DNA was isolated in the same manner as described above.

Using the isolated plasmid, strain L-15 was transformed in the same manner as described above. Kanamycin-resistant colonies grown on RCC, P agar medium containing 200 u/ml of kanamycin were collected, and after centrifugal washing twice with physiological saline, p-fluorophenylalanine 0.8 μ/ml or m-fufue-luluori was collected. Minimal agar medium ml containing 0.1■/ml of ranin
(Glucose 10g - (NHi)tsOn 4g, K
Cl 0.2g, Mg5Oa・7H200,2g, F
e SOa ・7 Hz 101g, MnSO4
・4～6HzO0,2mg, Zn5On・7HzOO,
9mg, Cu5Oa.5HiO0.4g, Na.

B*O-+'1O8200,09mg, (NH4)I
IM off24.4H 200.04 mg, piotin 50, p-aminobenzoic acid 2.5 + g, thiamine hydrochloride 1 The cells were cultured for 3 days.

From the colonies that appeared, p-fluorophenylalanine 1.0 7ml Sm-fluorophenylalanine 1.0
Colonies that could grow on M1 agar medium containing 1/ml alone and on NB agar medium containing kanamycin Log/ml were selected. The plasmid isolated from the - strain was named pEpheA22.

L-15 strain and pEpheAl or pEph
The L-15 strain carrying eA22 was compared for its resistance to phenylalanine analogs. L-15 strain contains p-fluorophenylalanine at 0.05 mg/if
pEpheA cannot grow on M1 agar medium containing pEpheA.
1 or pEpheA22, and 115 strains have pEpheA22.
- Fluorophenylalanine 0.2■/ml each
, also grew on M1 agar medium containing 1.2q/1. Similarly, the 115 strains harboring pEpheAl or pEpheA22 had also acquired resistance to m-fluorophenylalanine.

(5) p E p h e A l or pEp
Production of phenylalanine using heA22 stock strain Corynebacterium glutamitum K-50, a phenylalanine-producing strain, was cultured with shaking in NB medium at 30°C for 16 hours, and the seed culture was grown in 33M medium [glucose 20g,
(NH4)tsOn 10g1 urea 3g, yeast extract 1g, KHzPOiIg, Mg(J!z・6H*OO,
4g, Fe50°・7H2010M, MnSO4' 4
~6H200,2mg, ZnSOs・7H200,9
mg5CuSO4' 5HiOO,4g+gS Naz
BsOt40HtO0,09mg5 (NHa)sM
Oto2*' 4H*00.04■, biotin 30%, thiamine hydrochloride 1■ in water 11 I) H7,2
culture medium] and cultured with shaking at 30°C. O
When D reached 0.2, penicillin G was added at a concentration of 0.5 units/ml. The culture was continued, and when the OD reached approximately 0.6, the cells were harvested and added to a solution containing 1 μ/ml lysozyme in RCGP medium (pH 7.6).
The cells were suspended at 0.09 cells/ml, transferred to an L-shaped test tube, and shaken at 30° C. for 14 hours to form protoplasts.

5 ml of this protoplast suspension Q was placed in a small test tube, centrifuged at 2.500 x g for 5 minutes, resuspended in 1 ml of TSMC buffer, centrifuged and washed, then TSMC buffer Q,
Resuspend in 1 ml. This suspension was added with twice the concentration of TSM (
jlfi liquid and pEpheAl or pephe^22
A 1:1 mixture of DNA 1004 was added and mixed, followed by transformation via PEG6.000 in the same manner as in Example 1 (4). After expression, 0.2 ml was added with kanamycin 2.
0 ON/1111 on an RCGP agar medium,
The cells were cultured at 30°C for 10 days.

Among the colonies that appeared, a strain capable of growing on NB agar medium containing 10 ml of kanamycin was obtained.

Transformed strains that have become resistant to kanamycin are injected into 400mlS.
Culture with shaking in SM medium, and when the OD reached 0.2, penicillin G was added at a concentration of 0.5 units/ml.
The cells were further cultured to an OD of approximately 0.6, and plasmids were isolated from the collected cells in the same manner as in Example 1 (1). Analysis of these plasmids by agarose gel electrophoresis after restriction enzyme digestion revealed that pEpheAl or pH:ph
It turned out to be the same plasmid as e^22. pEpheAl or pBph obtained in this way
The transformed strains harboring e^22 were each derived from Corynebacterium glutamitum K-51 (FERM BP-7
68), and Corynebacterium glutamitum K-
52 (FERM BP-769) and has been deposited with the Institute of Fine Technology.

Corynebacterium glutamitum K-50゜K-51
, and Ni-52 were tested for L-phenylalanine production. Seed cultures Q and 5a+I, which were cultured with shaking at 30°C for 16 hours in NB medium, were mixed with 5 ml of production medium P4 (blackstrap molasses 200
g, (NHa)asOt20g, KH2PO40.5g
,,2HP0゜0.5g5MgSO4・78i○ 0
．． 25 g of NZ7, 72.5 g of NZ7, and 520 g of CaCO were inoculated into a test tube containing a medium containing 11 parts of water and adjusted to pH 7.2, and cultured with shaking at 30°C for 72 hours.

After culturing, the culture solution was subjected to paper chromatography, and after coloring with ninhydrin, it was placed colorimetrically to measure the amount of L-phenylalanine produced.

Table 11 shows the amount of L-phenylalanine produced together with the control strain Corynebacterium glutamitum K-50.

Example 2゜(1) Production of phenylalanine by transformed strains of various coryneform glutamate-producing bacteria Corynebacterium herkyulis ATCC13868
, Brevibacterium flavum ΔTCC14067,
and Brevibacterium lactofermentum AT
CC13655 and Microbacterium ammoniaphilum ATCC15354 were cultured in the presence of penicillin G in the same manner as in Example L (5), and the cells were treated with lysozyme to form protoplasts. This protoplast was transformed with pBphe^22 via PEG6,000 in the same manner as in Example 1 (4) to obtain a kanamycin-resistant transformant.

Example 1 from penicillin-treated bacterial cells of transformed strains of each strain.
Plasmids were isolated in the same manner as in (5). After digesting these plasmids with restriction enzymes and analyzing them by agarose gel electrophoresis, it was found that the transformed strain had the same plasmid as pEphe^22.

An L-phenylalanine production test was conducted using these pEphe^22-carrying transformed strains and each parent strain under the same conditions as in Example 1 (5).

The results are shown in Table 1.

Table 1-33 (2) Kozukiichi According to the present invention, CM of microorganisms belonging to the genus Escherichia
Corynebacterium sp.
It is possible to impart or improve the productivity of L-phenylalanine in Brevibacterium or Microbacterium species.

[Brief explanation of drawings]

FIG. 1 shows the restriction enzyme cleavage map of pBphe^1 and its production process. Arrows indicate the direction of gene transcription. Molecular weights of plasmids are expressed in kilopaces (b). Figure 1

Claims (5)

[Claims] (1) A Corynebacterium, Brevibacterium, or Microbacterium containing a recombinant DNA of a DNA fragment containing a gene involved in the synthesis of chorismate mutase and prephenate dehydratase of a microorganism belonging to the Escherichia genus and vector DNA. 1. A method for producing L-phenylalanine, which comprises culturing a microorganism belonging to the genus Phenyl in a medium, producing and accumulating L-phenylalanine in the culture, and collecting L-phenylalanine from the culture. (2) The vector is pCG1, pCG2, pCG4, pCG11, pCG1, pCG2, pCG4, pCG11, or pCG derived from a microorganism belonging to the genus Corynebacterium.
The method according to claim 1, wherein the method is selected from CE51, pCB52, pC853, pCB101 and plasmids derived therefrom. (3) A DNA fragment containing genes involved in the synthesis of chorismate mutase and prephenate dehydratase derived from a microorganism belonging to the genus Escherichia confers resistance to phenylalanine analogs to the genus Corynebacterium, Brevibacterium, or Microbacterium. A recombinant DNA characterized in that it is a DNA fragment that can be added to a microorganism to which it belongs. (4) Corynebacterium, Brevibacterium, or Microbacterium containing a recombinant DNA of a DNA fragment containing a gene involved in the synthesis of chorismate mutase and prephenate dehydratase of a microorganism belonging to the Escherichia genus and vector DNA. A microorganism belonging to the genus Umbrella. (5) Corynebacterium glutamitum K-51 (6
) Corynebacterium glutamitum K-52