JPS62130693A - Production of l-phenylalanine by fermentation method - Google Patents

Abstract

PURPOSE:To obtain L-phenylalanine in good productivity, by cultivating a coryneform bacterium containing both a plasmid integrated with DS gene and a plasmid integrated with PDT gene. CONSTITUTION:A coryneform bacterium containing both a plasmid (recombinant plasmid pT-AR16, etc.,) integrated with a gene to code 3-deoxy-D-arabino- heptulonic acid-7-phosphate synthase and a plasmid (recombinant plasmid pPH14, etc.,) integrated with a gene to code prehenate dehydrolactose is cultivated. The culture is carried out under an aerobic condition until production and accumulation of L-phenylalanine is substantially stopped.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing L-7 enylalanine by a fermentation method.

(Prior art) When attempting to produce Sim-phenylalanine by fermentation, wild strains hardly produce L-phenylalanine outside the bacterial cells, so a mutation is artificially caused in a wild strain to produce L-phenylalanine. - A method of imparting phenylalanine production ability has been adopted. Conventionally known artificial mutant strains having the ability to produce L-7 enylalanine include mutant strains of the genus Brevibacterium or Corynebacterium that are resistant to phenylalanine antagonists (U.S. Patent No. 3,660,23
5), a mutant strain of Corynebacterium that requires tyrosine and is resistant to phenylalanine antagonists (
U.S. Pat. No. 3,759,790), mutant strains of the genus Prepipacteriwaum and Corynebacterium that are resistant to phenylalanine antagonists and sensitive to decoinine (Japanese Patent Application Laid-open No. 56-64793) are known.

On the other hand, recently, attempts have been reported to utilize genetic recombination technology, which is different from the above-mentioned breeding by artificial mutation, for breeding L-7 enylalanine-producing bacteria. That is, JP-A-56-1890 describes that a mutant strain of Escherichia coli containing a plasmid incorporating a chromoseme DNA fragment of the genus Escherichia involved in phenylalanine synthesis produced L-phenylalanine. Further, JP-A-58-134994 discloses a phenylalanine-producing strain of the genus Pacillus bred by recombinant DNA technology. Furthermore, for the breeding of L-phenylalanine-producing bacteria using coryneform bacteria, a gene encoding 3-deoxy-D-arabino-hebutyronate-7-phosphate synthase (hereinafter referred to as "DS") ( A plasmid containing a plasmid (hereinafter referred to as "rDS gene") inserted (Japanese Patent Application No. 59-245700), and a gene encoding prephenate dehydratase (hereinafter referred to as "rPDT J") (hereinafter referred to as "rPDT gene"). Those with an inserted plasmid (patent application 1983-
66880), etc.

(Problems to be Solved by the Invention) However, the L-phenylalanine-producing ability of the bacteria having the above-mentioned L-phenylalanine-producing ability needs to be further improved.

(Another means to solve the problem) Under the above-mentioned situation, the present inventors developed a coryneform cell that has both a plasmid in which the DS gene is integrated and a plasmid in which the PDT gene is integrated. It has been found that the type bacteria have high productivity of L-phenylalanine.

Coryneform bacterium according to the present invention
acteria) is Percy's Manual Op.
Determinative Bacteriology (Barge's Manual of Determinative
Bacteriology) 8th edition 599 pages (197
4) is a group of microorganisms that are aerobic, Durum-positive, non-acid-fast, and rod-shaped bacteria that do not have spore-forming ability. Among these coryneform bacteria, particularly the coryneform glutamate-producing bacteria described below are most suitable for the present invention.

Examples of wild strains of coryneform glutamate-producing bacteria include the following.

Brepinogacterium diparicatum AT
CC14020 Prepinoculacterium salacalolyticum ATCC14066 Prepinoacterium inmariophyllum ATCC14068 Prepinocuterium lactofermentum ATCC13869 Brevibacterium roserum ATCC
13825 Plebibecterium flavum
ATCC13826 Previpactericum thiornitalis ATCC19240 Corynebacterium
Acetoacidophilum ATCC13870 Corynebacterium acetoglutamicum ATCC15806
Corynebacterium carnae ATC
C15991 Corynebacterium glutamicum
ATCC13032, 13060 Corynebacterium
Lilium ATCCl5990Corynebacterium Melasecola ATCC179
65 Microbacterium ammoniaphyllum A
TCC15354 The coryneform glutamate-producing bacteria of the present invention include, in addition to the above-mentioned wild strains that have glutamate productivity, mutant strains that have glutamate productivity but lose glutamate productivity when grown.

The method for isolating the DS gene involves first extracting the chromosomal gene from a strain of coryneform bacteria that has the DS gene. For example, H, Sat to and K, MiuraBi.
ochem, Biophys, Acta 72+6
19+ (1963) method can be used. ), which is then cut with an appropriate restriction enzyme. Next, the recombinant DNA was ligated to a plasmid vector capable of propagating within coryneform bacteria cells, and the resulting recombinant DNA was used to transform a DS-deficient mutant strain of coryneform bacteria, resulting in a strain that possessed DS-producing activity. This allows the 9DS gene to be isolated.

In addition, we transformed a wild strain of coryneform bacteria using the same recombinant DNA, and isolated a strain that was resistant to aromatic amino acid antagonists that have an inhibitory effect on DS activity. It is also possible to isolate genes.

Examples of aromatic amino acid antagonists include 0-fluorophenylalanine, m-fluorophenylalanine, p-fluorophenylalanine, p-aminophenylalanine, β-3-chenylalanine, 3-aminotyrosine, tyrosine hydroxamate, 5-fluorophenylalanine, Examples include methyltricytophan.

The method for isolating the PDT gene can also be performed in the same manner as the method for isolating the DS gene.

That is, using a PDT-deficient mutant strain of coryneform bacteria, PDT
The PDT gene can be obtained by isolating a bacterial strain that possesses production activity.

It is also possible to isolate the PDT gene by using wild strains of coryneform bacteria and isolating strains that have developed resistance to aromatic amino acid antagonists that have an inhibitory effect on PDT activity. .

As the DNA donor bacterium, it is more preferable to use a mutant strain which has a high biosynthetic activity for aromatic amino acids or their precursors by imparting a mutation such as resistance to the aromatic amino acid antaconist.

The aromatic amino acid precursors mentioned here are DAHP3-dehydroquinic acid, 3-dehydroshikimic acid, shikimic acid, shikimic acid-3-phosphoric acid, 5-enolbilpyrshikimic acid-3.
- Phosphoric acid, chorismic acid, prephenic acid, phenylpyruvic acid, anthranilic acid, indole-3-glycerol phosphoric acid, etc., as described in 056-64793.

As the DS gene or PDT gene, a wild type gene can be used, and a mutant strain gene can also be used. The mutant genes include those mutated to encode DS with a reduced degree of synergistic inhibition by phenylalanine and tyrosine, and genes mutated to encode PDT with a reduced degree of inhibition by phenylalanine. preferable.

To obtain a mutant gene, a DNA donor bacterium may be subjected to mutation treatment, or the DS gene or PDT gene may be inserted into a vector plasmid, and the resulting recombinant DNA may be introduced into a DNA recipient bacterium to obtain the resulting trait. The convertible strain may be subjected to mutation treatment.

Furthermore, a mutant gene can also be obtained by mutating the recombinant DNA itself in vitro.

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

The plasmid vector used in the present invention may be any vector as long as it can proliferate in coryneform bacterial cells. Specific examples include the following.

(1) PAM 330 JP-A-58-676
Reference 99 (2) pHM 1519 JP-A-1983-
77895 reference (3) pAJ 655 JP-A-1975
Reference 8-192900 (4) pAJ 611
Same as above (5) pAJ 184
4 Same as above (6) pcGl
t￥f Kaisho 57-134500 reference (7)
pCG 2 See JP-A-58-35197 (8)
pcc 4 See JP-A-57-183799 (9) pCG 11 Same
Cleavage of the above plasmid vector DNA is performed by
Either use a restriction enzyme that cuts NA in one place, or
This is done by partially cutting with a restriction enzyme that cuts at multiple sites.

The vector DNA is cut by the restriction enzyme used to cut the chromosomal gene, or an oligonucleotide having a complementary base sequence is connected to each end of the cut chromosomal DNA fragment and the cut vector DNA. Then, plasmid vector and chromosome D
Subjected to Rider John reaction with NAN roughment.

In order to introduce the thus obtained recombinant DNA of the chromosomal DNA and the vector plasmid into a recipient bacterium belonging to coryneform bacteria, a method such as that reported for -12 in Escherichia coli (Mandel +M, and
HigaeA, lJ, Mo1. , Biol,
, 53 r 159 (1970) treatment of recipient bacterial cells with calcium chloride to increase DNA permeability, or as reported for Bacillus subtilis (D
Uncan.

C, H, Wilson eG, A, and You
ng, F, E, +G@n@+1.153 (197
7)) The proliferation stage in which cells become capable of taking up DNA (
This can be done by introducing the cells into so-called competent cells. Alternatively, as is known for Bacillus subtilis, actinomycetes and yeasts (Chang e
S, and Choen, S, N, +Mo1e
a, Gen, 5Gsnet, , 168.111 (1
979): Bibb + M, J, + Ward
+ J, M, and Hopwood's O, A
, eNature + 274 + 398 (19
78) : Hlnnen IA, jHleks +
J, B, and Flnk + G, R, * Pro
e, Natl, Acad.

Sci, USA, 751929 (1978)), DNA
The recipient bacteria are transformed into glotograsts or spheroplasts, which easily take up plasmid DNA, and the plasmid is transformed into DNA.
It is also possible to introduce A into recipient bacteria.

In the protocillast method, a sufficiently high frequency can be obtained even with the method used for Bacillus subtilis, and Protoplus Of course, a method in which DNA is injected into the presence of /C polyethylene glycol or polyvinyl alcohol and a divalent metal ion can also be used. Equivalent results can also be obtained by adding carboxyl methylcellulose, dextran, Ficoll, Pluronic F68 (Serpa), etc. to polyethylene glycol or polyvinyl alcohol to promote DNA infiltration.

In this way, a plasmid into which the DS gene or PDT gene is integrated is obtained. These two recombinant plasmids are
It must be able to coexist within the cells of coryneform bacteria. If they cannot coexist, the DS gene and PDT gene can be easily transferred to a Grasmid vector that can coexist.

According to the research of the present inventors, in order for two plasmids to coexist in a coryneform bacterial cell, they can coexist if the plasmids have different replication initiation regions, but they must have the same replication initiation region. Even if they exist, they may coexist if they are significantly different in other areas.

The coryneform bacterial host carrying the two plasmids may be a phenylalanine auxotroph or a wild strain. In order to obtain a transformed strain with an even higher ability to produce phenylalanine, the biosynthetic activity of 7-enylalanine or its precursor was increased by imparting mutations such as list resistance and auxotrophy to the phenylalanine ant. If such a mutant strain is used as a recipient strain, L N .

Furthermore, we have developed mutant strains with improved permeability of phenylalanine to the outside of the bacterial cell, mutant strains with decreased degradability of phenylalanine, and ability to biosynthesize metabolites that are supplied as raw materials for phenylalanine biosynthesis, such as glutamine and phosphoenolpyruvate. Naturally, mutant strains or recombinants with increased efficiencies may also produce good results.

The method for culturing the obtained L-phenylalanine-producing bacteria is not particularly different from the conventional culture method for L-phenylalanine-producing bacteria. That is, the culture medium is a usual one 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., starch hydrolyzate containing these, whey, molasses, etc. are used.

As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, etc. can be used.

Cultivation is carried out under aerobic conditions while adjusting the pH and temperature of the medium as appropriate until the production and accumulation of L-phenylalanine substantially ceases.

In this way, a significant amount of L-7 enylalanine is produced and accumulated in the culture solution. Conventional methods can be applied to collect L-7 enylalanine from the culture solution.

Example (1) Chromosomal DNA containing DS gene and PDT gene
As a source for the preparation of chromosomal DNA, the phenylalanine-producing bacterium Brevibacterium, which has resistance to m-fluorophenylalanine, a phenylalanine analog, is used as a source of chromosomal DNA. Um lactofermentum A
J 11957 (FERM-P6673) was used.

This bacterium was grown in 11 CMG medium (-!pton 1? scraps, yeast extract 11-/ltp, glucose:r-su 0.5?/di
, and NaCtO,51-/ell, voice 7.2
The microorganisms were inoculated into a culture medium (adjusted to 100°C), cultured with shaking at 30°C for about 3 hours, and the cells in the logarithmic growth phase were collected.

After the bacterial cells were lysed with lysozyme/SDS, chromosomal DNA was extracted using a conventional phenol treatment method, and finally 3.5 μm of DNA was obtained.

(2) Preparation of vector DNA Using pAJ 1844 (molecular weight 5.4 megadaltons) as a vector, its DNA was prepared as follows.

First, Brepinogacterium lactofermentum AJ 120 harboring pAJ 1844 as a cilasmid.
37 (FERMBP-577) to 1001nl CMG
After inoculating the medium and culturing at 30°C until the late logarithmic growth phase, the bacterium was lysed by Rhizome sDs treatment and incubated at 30.0°C.
OX, P.

A supernatant was obtained by ultracentrifugation for 30 minutes. After the phenol treatment, diluted ethanol was added to precipitate and collect the DNA.

This was mixed with a small amount of TEN buffer (20mM To!JS hydrochloride, 20mM NaCt, 1mM EDTA (p
After dissolving in H8.
Approximately 15 μm was obtained.

(3) Insertion of chromosomal DNA fragment into vector 10μ of chromosomal DNA obtained in (1)? and the plasmid obtained in (2))'
DNA5μ? and the restriction endonuclease Pstr
Each piece was held at 37°C for 1 hour and cut.

After heating to 65°C for 10 minutes, both reaction solutions were mixed and AT
The DNA was maintained at 10°C for 24 hours using DNA ligase derived from T47age in the presence of P and dithiothreitol.
The chains were connected. The reaction solution was then 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 PDT gene Brevibacterium lactofermentum A with deletion of PDT gene
J 12124 (Brevibacterium lactofermentum AJ12036 as the parent strain, N-methyl-N-
After mutation treatment with nitro N-nitrosoguanidine, a mutant strain requiring K-diphenylalanine for growth was selected. ) was used as the recipient bacterium.

The grotoplast transformation method was used as the transformation method. First, the bacterial strain was cultured in a 5 d CMG liquid medium until the early logarithmic growth phase, and after adding 0.6 units/m of penicillin G, the strain was further cultured with shaking for 1.5 hours, and the bacterial cells were collected by centrifugation. The cells were washed with SMMP medium (pH 6.5) 0.54 consisting of 0.5M Shakrose, 20mM maleic acid, 20mM magnesium chloride, and 3.5% Dlfeo Q. Next, the cells were suspended in SMMP medium containing 10 q/d of lysozyme to form protonorlasts at 30° C. for 20 hours. 6
After centrifugation for 000XiI-110 minutes, protoplasts were washed with SMMP and resuspended in 0.511 Ll of SMMP. Prodobcyst obtained in this way and (3)
DNA prepared with 5μ? in the presence of 5 mM EDTA and polyethylene glycol to a final concentration of 30+
%, and was left at room temperature for 2 minutes to allow the DNA to be incorporated into protoplasts. After washing with SMMP medium 1, add SMMP medium 1
d and cultured at 30°C for 2 hours for expression. This culture solution was applied onto a grotograst regeneration medium at pH 7.0.

Protoplast regeneration medium is 11 parts distilled water and 12 parts citris (hydroxymethyl) aminomethane. , KCl O,5
/-, Gluco-x 10? , MgCl2-6H208
, 1j', CaC22'2H202,2J', Knoton 4iP, powdered yeast extract 4f, cabmino acid (Dlfe
company o) 1 t.

K2HPO40,21II-, sodium succinate 9m13
5F-, Agar 8Ji! - and chloramphenicol 3μ
? /- is included.

After culturing at 30°C for 2 weeks, approximately 25,000 chloramphenicol-resistant colonies appeared, which were then transferred to a minimal medium (2-tiniglucose, 1-ammonium thiosulfate, 0.3 urea, 0.1 To dihydrogen phosphate). Potassium, 0.04
% magnesium sulfate heptahydrate, 2 ppm iron ion, 2
ppm manganese ion, 200μt/1 thiamine hydrochloride, 50μ? /l biotin, chloram 7z2: ff-
A/10 pi/m1. pH7.01 agar 1. '8
%) to obtain 10 strains that were resistant to chloramphenicol and had lost the requirement for phenylalanine.

(5) Plasmid analysis of transformed strains Prepare a lysate using the method described in (2) for these strains, and analyze the plasmid D by agarose gel electrophoresis.
When NA was detected, 9 strains showed pAJ 18 of the vector.
Clearly large cilasmids were also detected in No. 44. One of these cilasmids was named pPHII 14, and when it was cut with the restriction enzyme Pstl used for recombination, a 7.6 kb DNA fragment was detected. The PDT gene resides on this 7.6 kb P+st I fragment. pPHs! No. 14 has the restriction enzyme map shown in FIG.

(6) In order to confirm the presence of the PDT gene on the recombinant cilasmid containing the 7 and 6 kilobase DNA fragments detected in re-transformation test (5), this grasmid DNA pPH14 was used to transform Brevi. Bacterium lactofermentum AJ12
124 was transformed again.

When 10 of the live citachloramphenicol-resistant colonies were picked up and tested for phenylalanine auxotrophy, all of them lost the auxotrophy, indicating that the PDT gene was present on the above recombinant plasmid. It became clear that

(7) PDT activity of the transformed strain
? , (NH4)2So41oノ, KI(2Po41p-
1MgSO4・7H201? , fumaric acid 12), acetic acid 3
ml, soybean protein soybean protein layer water "Taste liquid J 50 d, F
eSO4-7H2010■, MnSO4・4H2010
FW, biotin 50μ! , Sai7my [acid acid 2000
μ? and CaCO350y- are contained in water 11. PH7,
0) Ultrasonicate the bacterial cells cultured at 30°C for 22 hours at 20 m/te, prepare a lysate, and add this to 32 m/te.
Oxy-1 was centrifuged for 20 minutes to obtain a supernatant. This supernatant was used as a crude enzyme solution, and 50mM phosphate buffer (pl(
Using an enzyme reaction solution consisting of 7,2) and 2 mM 7'lefuic acid, the reaction was carried out at 30'C for 10 minutes, and the amount of phenylpyruvic acid produced was quantified by measuring the absorbance at 320 nm. Table 1 shows the measurement results.

Table 1 PDT activity (8) Using 5μ of the recombinant DNA obtained in DS gene cloning (3), (4
Transformation was performed as described in ). As a DNA receptor, m-fluorophenylalanine-sensitive Brevibacterium lactofermenta JJ12036 (
FERM-P7559) was used.

After culturing at 30°C for 2 weeks, approximately 25,000 chloramphenicol-resistant colonies appeared, which were then transferred to a minimal medium containing 500μ of m-fluorophenylalanine and nectar (
Diglucose, 1% ammonium sulfate, 0.3% urea, 0.14% potassium dihydrogen phosphate, 0.04% magnesium sulfate heptahydrate, 2 ppm iron ion, 2 ppm
Mafugan ion, 200 pip/Nothiamine hydrochloride, 50μ? /l biotin, chlorampf x2 = r-le 10
ttP/ml, pH 7.0, agar 1.8%) to obtain five strains resistant to chloramphenicol and m-fluorophenylalanine.

(9) Plasmid analysis of transformed strains Prepare a lysate using the method described in (2) for these strains, and perform plasmid D by Agalow medal electrophoresis.
Pecter's pAJ1 was detected in all strains in which NA was detected.
Obviously large grasmids were also detected in 844.

Restriction enzyme P used for recombination with 5 plasmid strains
When cut with st l, 6.7k is common to 4 of the strains.
A DNA insertion fragment of b was observed.

In addition, one other strain has 3.7kb, 3.0kb, 1
．． 7kb.

1.6kb, 1.4kb, 1.3kb, 0.8k
b, 0.7 kb.

Nine DNA inserts of 0.4 kb were observed.゛The recombinant plasmid containing the former 6.7 kb Pat I fragment at the Pat I cut point of vector pAJ 1844 was named pAR-2, and the latter 9 Pst I DN
The recombinant plasmid containing the A fragment was named pAR-1, and the strain carrying pAR-1 was designated AJ 12181 (FER
M-P7948), AJ1 strain carrying pAR-2
2182 (FERM-P7947).

(g) To confirm the presence of a gene conferring m-fluorophenylalanine resistance on the recombinant plasmid containing the DNA fragment detected by retransformation (9), this plasmid DNA was
Brevibacterium lactofermentum AJ 12036 was transformed again using A pAR-1 and pAR-2.

Fifty of the colonies resistant to citachloramphenicol were picked and tested for sensitivity to m-fluorophenylalanine, and all of them were found to be resistant to m-fluorophenylalanine. It has become clear that there are genes that confer resistance.

The DS activity test strain of the αme transformed strain was transferred to a phenylalanine production medium (glucose 130
? , (NH4)2S0410? ,KH2PO41,P,
MgSO4・7H201f, fumaric acid 12P, acetic acid 3m
, Soybean protein acid hydrolyzate "Ajijiru J 50 ml, F
eSO4-7H2010■, MnSO44H2010”
? -Biotin 50 ttP, Thiamine Hydrochloride 2000
μ? and CaCOx 501i- in water 11. pH
7,0) Bacterial cells were cultured in 20 ml at 30°C for 15 hours, treated with ultrasound to prepare a lysate, and this was
The supernatant was obtained by centrifugation for 120 minutes. This supernatant was used as a crude enzyme solution for a 1.Ornl reaction consisting of 50 mM) Lis-HCl buffer (pH 7.5), 0.5 mM phosphoenolpyruvate, and 0.5 mM erythrose-4-phosphate. The mixture is reacted in a solution at 30°C for 10 minutes, and after the reaction is complete, 0.24% trichloroacetic acid is added to deactivate the enzyme. After removing the denatured protein by centrifugation, 0.25 d of supernatant + 0.25 d of 0.025 M periodic acid is added and incubated at 37° C. for 30 minutes. After adding 0.5 m of 2-thiosodium arsenite to stop the oxidation reaction, 2.0-0.312-thiobarbic acid was added and heated at 100°C for 8 minutes. 549 nm of reaction solution
The absorbance of DAHP was measured and the molecular extinction coefficient of DAHP was 4.5
10' (Jenaen+R, A, and Ne5
tsr r E, W, r J, Biol, Che
Enzyme activity was determined using M, 241 r3365 (1966)).

In addition, the activity was also measured when 7-enylalanine, tyrosine, or both were added at 1mM each to the reaction solution, and the value when no addition was added was expressed as 10 (l).The measurement results are shown in Table 2.

By introducing the obtained recombinant grasmid, 9. D
S activity increased 2.6 to 14 times, and the activity inhibition by each amino acid also changed from the wild strain type to the mutant strain type. It is clear that the fluorophenylalanine resistance gene is a DS gene. Furthermore, since the Pstl fragments possessed by pAR-1 and pAR-2 are completely different, it is thought that there are two or more types of genes encoding proteins having DS activity.

(b) Transfer of DS gene to pAJ 224 (4
) The PDT gene obtained in (8) and the Ds gene obtained in (8) were obtained using the plasmid vector pAJ 1844, respectively.
is incorporated into. Therefore, since these two plasmids have exactly the same replication initiation region, there is a possibility that they cannot coexist in one cell. So ζ pAJ 18
Plasmid PA that is already known to coexist with 44
J 224 (, #iji 1986-37168) K.

The DS gene was transferred as follows.

pAJ 224 (3,6 kb) was derived from Previpactericum lactofermentum AJ 12196 (FERM-P2O), which carries pAJ224 as a grasmid.
15) A sample was prepared according to the method described in (2).

pAJ 2441μ? and pAR-15 obtained in (9)
μ restriction endonucleases BamHI and 5a
Each was treated with lI at 37°C for 30 minutes to completely cleave.

After heat treatment at 65° C. for 10 minutes, both reaction solutions were mixed and ligated by the method described in (3), and DNA was collected by precipitation. This DN
Trimethoprim-susceptible Brevibacterium lactofermentum AJ 1 was prepared in the same manner as in (4) using A.
After grotoplast transformation using 2036 as recipient bacteria, trimethoprim 100μf/m
As a result of culturing in a grotozolast regeneration medium containing
00 regenerated colonies have appeared. This is trimethoprim 200μf/rrd! The microorganisms were replicated in a minimal medium containing 500 μl/ml of m-fluorophenylalanine and about 100 strains with trimethoprim resistance and m-fluorophenylalanine resistance were obtained. A lysate of these strains was prepared by the method shown in (2), and ciblasmid DNA was detected by agarose gel electrophoresis, and a 7.5 kb cilasmid was detected. This cilasmid was named pT-AR16. pT-AR1
6 has the restriction enzyme cumulative map shown in FIG.

This plasmid DNA was used to confirm the presence of the DS gene on α→ retransformation recombinant Grasmid pT-AR16. Brevibacterium lactofamentum AJ120
36 was transformed again.

Of the resulting trimethoprim-resistant colonies, 1 each
When 0 were picked up and tested for m-fluorophenylalanine resistance, it was found that all of them retained resistance, and it became clear that the DS gene was present on the above recombinant plasmid.

Production of L-phenylalanine by coexistence of α4pPHI4 and pT-AR16 Using the above pPH14, m-fluorophenylalanine-resistant strain Brevibacterium lactofermentum A
J12184 was transformed by the method described in (4),
Transformed strain 12 using chloramphenicol resistance as an indicator
184-PDT strain was obtained.

Similarly, pT-AR16 was transformed into AJ1218.
4 strains, and the transformed strains were selected for trimethoprim resistance to obtain strain 12184-DS.

Furthermore, pT-AR16 was introduced into the 12184-PDT strain, and the resulting 12184-PDT strain and 12184-DS were resistant to chloramphenicol and trimethoprim.
When the AJ12259 strain and AJ12184 strain were cultured and their ability to produce L-phenylalanine and tyrosine was examined, the results shown in Table 3 were obtained.

Cultivation was carried out under the phenylalanine production medium described in (7). After culturing, add 2rnl of 6N potassium hydroxide solution!
was added to dissolve the precipitated L-tyrosine, and L-phenylalanine and L-tyrosine in the centrifuged supernatant were quantified by liquid chromatography.

Table 3 L-phenylalanine and L- of transformed strains
Tyrosine accumulation amount pPH14 and pT-AR16 are deposited AJ12
It can be isolated from the 259 strain using conventional methods.

Also, AJ 12184.12184-DS, 121
84-PDT is easily obtained from AJ 12259 by removing the complex plasmid in the host cell without damaging the host cell. Plasmids can be lost naturally by the host, or they can be removed by a "removal" operation (Bact, Rev, 36. p36
1-405 (1972)).

An example of the removal operation is as follows: In a medium containing acridine orange at a concentration (2-50 y/m/) that completely inhibits host growth, about 104 cells per plate are added. A small amount of the bacterial strain is inoculated to completely inhibit the growth of the host bacteria, and then cultured overnight at 27-37°C (J, Bact
eriol, 88.261 (1964)).

The culture solution is spread on an agar medium and cultured overnight at 27-37°C. Among the colonies that appeared on the medium, chloramphenicol (10 μP/d) or )! j) Grimm (50μ
The strain showing susceptibility to P/ml) is a strain in which the plasmid has been removed, ie, 12184-DS.

12184-PDT and AJ12184.

[Brief explanation of drawings]

FIG. 1 is a restriction enzyme map of plasmid pPH14. FIG. 2 is a regulatory enzyme map of positive pT-AR16. In each figure, the opening is the vector DNA, ■ is the inserted DNA: E, P, S, and B are E, respectively.
coRI # PatI r 5alI + BamH
Indicates I.

Claims (1)

[Claims] Cultivating a coryneform bacterium having a plasmid into which a gene encoding 3-deoxy-D-arabino-hepturonic acid-7-phosphate synthase has been integrated and a plasmid into which a gene encoding prephenate dehydratase has been integrated. Characteristic method for producing L-phenylalanine.