JPH0476678B2 - - Google Patents

Description

[Detailed description of the invention]

INDUSTRIAL APPLICATION FIELD This invention relates to coryneform bacteria having recombinant DNA. BACKGROUND ART L-tryptophan is anthranilic acid produced by anthranilic acid phosphoribosyltransferase,
It is produced by the action of N-(5'-phosphoribosyl)anthranilate isomerase, indole 3-glycerol phosphate synthase, and tryptophan synthase in this order. Hereinafter, N-(5'-phosphoribosyl)anthranilate isomerase will be referred to as PRAI, and indole 3-glycerol phosphate synthase will be referred to as InGP. On the other hand, the breeding of L-tryptophan-producing coryneform bacteria using recombinant DNA method was reported in JP-A-59-156292.
The gene encoding PRAI-InGP (hereinafter referred to as PRAI
−InGP gene) is not integrated therein. Problems to be Solved by the Invention The present invention aims to obtain microorganisms with higher productivity of L-tryptophan, and thereby to obtain L-tryptophan.
The goal is to find a more efficient method for producing tryptophan. Means for Solving the Problems In order to solve the above-mentioned problems, the present inventors conducted research and found that PRAI is expressed in coryneform bacterial cells.
- We succeeded in isolating a coryneform bacterium with recombinant DNA in which the gene encoding InGP is connected to a plasmid vector capable of propagating within coryneform bacterial cells, and the resulting coryneform bacterium is It was found that this method has high productivity. That is, the present invention provides N-(5
- A DNA fragment encoding phosphoribosyl) anthranilate isomerase - indole-3-glycerol phosphate synthase is connected to a vector plasmid that can autonomously replicate within the body of a coryneform-glutamate-producing bacterium, and the DNA fragment is linked to a vector plasmid that can autonomously replicate within the body of a coryneform-glutamate-producing bacterium. - Cultivating a microorganism capable of producing L-tryptophan obtained by introducing it into a DNA recipient microorganism showing resistance to fluorophenylalanine and 5-fluorotryptophan, and collecting L-tryptophan accumulated in the culture solution. This is a method for producing L-tryptophan, which is characterized by the following. Coryneform bacteria (Coryneform) according to the present invention
bactria) is from the Burgesses Manual of
Bargeys Manual of Determinative
Bacteriology), 8th edition, p. 599 (1974), and are aerobic, Gram-positive, non-acid-fast, rod-shaped bacteria that do not have spore-forming ability. Among these coryneform bacteria, the following coryneform glutamate-producing bacteria are particularly preferred in the present invention. Examples of wild strains of coryneform glutamate-producing bacteria include the following.

[Table] The coryneform glutamate-producing bacteria of the present invention include, in addition to the wild strains having glutamate productivity as described above, mutant strains having glutamate productivity or having lost glutamate productivity. The method for isolating the PRAI-InGP gene is to first extract the chromosomal gene from a strain of coryneform bacteria that has the PRAI-InGP gene (for example, H.
Saito and K.Miura Biochem.Biphys.Acta72,
619, (1963) can be used. ), which is then cut with an appropriate restriction enzyme. It is then connected to a plasmid vector capable of propagating within coryneform bacterial cells,
The obtained recombinant DNA was used to develop coryneform bacteria.
A mutant strain lacking the PRAI-InGP gene is transformed and a strain that maintains PRAI-InGP production activity is isolated, from which each PRAI-InGP gene can be isolated. 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 is
Any substance that can grow within coryneform bacterial cells may be used. Specific examples include the following. (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 Same as above Cleavage of plasmid vector DNA
This is done by cutting using a restriction enzyme that cuts at one site or partially cutting using a restriction enzyme that cuts at multiple sites. When the vector DNA cuts the chromosomal gene,
A ligation reaction between the plasmid vector and the chromosomal DNA fragment is performed by connecting oligonucleotides having complementary base sequences to both ends of the chromosomal DNA cleavage fragment and the cleaved vector DNA, which are cleaved by the restriction enzyme used, and then ligation reaction between the plasmid vector and the chromosomal DNA fragment. attached to. In order to introduce the thus obtained recombinant DNA of chromosomal DNA and vector plasmid into a recipient bacterium belonging to coryneform bacteria, E.
As reported for coli K-12 (Mandel.M. and Higa.A., J.Mol., Biol., 53 ,
159 (1970)) to increase DNA permeability by treating recipient bacterial vacuoles with calcium chloride, or as reported for Bacillus subtilis (Duncan, CH, Wilson, GA and Young, F.
E., Gene. 1 , 153 (1977)) This is possible by introducing cells into a proliferation stage (so-called competent cells) at which they can take up DNA. Alternatively, as is known for Bacillus subtilis, actinomycetes and yeasts (Chang, S. and
Choen, S. N., Molec. Gen., Genet., 168 ,
111 (1979); Bibb, Mj, Ward, JMand
Hopwood, OA, Nature, 274 , 398 (1978);
Hinnen, A., Hicks, JBand Fink, G.R.
Proc.Natl.Acad.Sei.USA, 75 , 1929 (1978)),
It is also possible to transform the DNA recipient bacteria into protoplasts or spheroplasts, which readily take up plasmid DNA, and to introduce the plasmid into the DNA recipient bacteria. In the protoplast method, a sufficiently high frequency can be obtained using the method used for Patillus subtilis, and polyethylene glycol or polyethylene glycol or Of course, a method of incorporating DNA in the presence of polyvinyl alcohol and divalent metal ions can also be used. By adding carboxymethyl cellulose, dextran, Ficoll, Brulonik F68 (Selva), etc. instead of polyethylene glycol or polyvinyl alcohol, DNA
Equivalent results can also be obtained by methods that promote the uptake of As an L-tryptophan-producing bacterium, PRAI-
A strain transformed with an InGP-deficient strain as a host can be used, but a strain with higher L-tryptophan productivity may be obtained by using the following hosts. Specifically, a mutant strain of Brevibacterium that requires phenylalanine and tyrosine and is resistant to 5-methyltryptophan (I. Shiio, H. Sato, M.
Nakagawa., Agric.Biol.Ctem. 36 , 2315
(1972)), require phenylalanine from Brevibacterium, m-fluorophenylalanine,
Mutant strain resistant to 5-fluorotryptophan (I.Shiio, S.Sugimoto, M.Nakagawa.
Agrie.Biol.Chem., 39 , 627 (1975)), a mutant strain of Brevibacterium that requires tyrosine and is resistant to 5-fluorotryptophan and azaserine, phenylalanine of Corynebacterium,
requires tyrosine, 5-methyltryptophan,
4-methyltryptophan, 6-fluorotryptophan, tryptophan hydroxamate, p
- Mutant strains resistant to fluorophenylalanine, tyrosine hydroxamate, and phenylalanine hydroxamate (H. Hagino, K.
Nakayama., Agric.Biol.Chem., 39 , 345
(1975)) etc. Most preferred are microorganisms that belong to coryneform glutamic acid producing bacteria and are resistant to m-fluorophenylalanine and 5-fluorotryptophan. The method of culturing the thus obtained coryneform bacteria capable of producing L-tryptophan to produce and accumulate L-tryptophan is different from the method conventionally used for producing L-tryptophan using coryneform bacteria. There are no major differences.
That is, the medium is a conventional 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 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-tryptophan substantially ceases. Example (1) Preparation of chromosomal DNA containing PRAI-InGP gene Brevibacterium lactofamentum
AJ12036 (FERM-P7559) was mixed with 1 CMG medium (containing peptone 1g/dl, yeast extract 1g/dl, glucose 0.5g/dl, and NaCl 0.5g/dl, PH
7.2), cultured with shaking at 30°C for about 3 hours, and the cells in the logarithmic growth phase were collected. After lysing the bacterial cells with lysozyme/SDS, the chromosomes were treated with normal phenol.
The DNA was extracted and purified, and 3.5 mg of DNA was finally obtained. (2) Preparation of vector DNA Using pAJ1844 (molecular weight 5.4 megadaltons) as a vector, its DNA was prepared as follows. First, Brevibacterium lactofamentum carrying pAJ1844 as a plasmid.
AJ12037 (FERM-P7234) was inoculated into 100 ml of CMG medium, cultured at 30°C until the late logarithmic growth phase, and then lysed by lysozyme SDS treatment.
A supernatant was obtained by ultracentrifugation at 30,000×g for 30 minutes.
After the phenol treatment, 2 volumes of ethanol were added to precipitate and collect the DNA. Add a small amount of this to TEN
Buffer (20mM Tris Hydrochloride, 20mM NaCl,
Dissolved in 1mM EDTA (PH8.0), separated by agarose gel electrophoresis, and excised to create pAJ1844.
Approximately 15 μg of plasmid DNA was obtained. (3) Insertion of chromosomal DNA fragment into vector 10μg of chromosomal DNA obtained in (1) and 5μg of plasmid DNA obtained in (2) were combined with restriction endonuclease.
Each was held at 37°C for 1 hour and cut with PstI. After heating to 65 °C for 10 min, both reactions were mixed and treated with _T4 in the presence of ATP and dithiothreitol.
10℃ using Phage-derived DNA ligase
The DNA strands were allowed to connect for 24 hours. Then, the reaction solution was heated at 65°C for 5 minutes, and 2
Double the volume of ethanol was added to collect the ligated DNA precipitate. (4) Creation of colony bank Brevibacterium lactofamentum AS60 lacking anthranilate synthase (Brevibacterium lactofamentum
AJ12036 was used as the parent strain and was mutated with N-methyl-N-nitro-N-nitrosoguanidine to select a mutant strain that requires anthranilic acid for growth. ) was used as the recipient bacterium. The protoplast transformation method was used as the transformation method. First, the strain was cultured in 5 ml of CMG liquid medium until the early logarithmic growth phase, and penicillin G was added at 0.6 units/ml.
After the addition, the cells were cultured with shaking for another 1.5 hours, and the cells were collected by centrifugation.
Washed with 0.5 ml of SMMP medium (PH6.5). then
The cells were suspended in SMMP medium containing 10 mg/ml of lysozyme and cultured into protoplasts at 30°C for 20 hours.
After centrifugation at 6000×g for 10 minutes, the protoplasts were washed with SMMP and resuspended in 0.5 ml of SMMP. Protoplasts obtained in this way and (3)
Mix 10 μg of the DNA prepared in 5mMEDTA in the presence of
After adding the DNA to a concentration of 30%, it was left at room temperature for 2 minutes to incorporate the 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. This culture solution was spread on a protoplast regeneration medium at pH 7.0. Protoplast regeneration medium contains 12 parts of tris(hydroxymethyl)aminomethane per 1 part of distilled water.
g, KCl 0.5 g, glucose 10 g, MgCl ₂ .
_6H2O8.1g , _CaCl2・_2H2O2.2g , peptone 4
g, powdered yeast extract 4g, casamino acids (Difco
) 1g, K ₂ HPO ₄ 0.2g, sodium succinate 135g, agar 8g and chloramphenicol
Contains 3μg/ml. After culturing at 30°C for two weeks, 50,000 chloramphenicol-resistant colonies appeared, which were all collected to create a colony bank. (5) Selection of clones in which tryptophan biosynthesis system genes have been amplified Appropriately diluted colony bank (approximately
10 ³ -10 ⁴ /ml) and ampicillin-resistant plasmid pUC8 (Messing, J., Gene 19 , 259 (1982)).
A mutant strain of E. coli that requires tryptophan for growth was grown on minimal medium (2% glucose, 1%
Ammonium sulfate, 0.3% urea, 0.1% potassium dihydrogen phosphate, 0.04% magnesium sulfate heptahydrate, 2ppm iron ion, 2ppm manganese ion,
200μg/thiamine hydrochloride, 50μg/biotin, 10μg/ml chloramphenicol, 100μ
g/ml ampicillin, 3% casamino acids (Difco
Co., Ltd.), 100mg/anthranilic acid, PH7.0, agar 1.8%). The tryptophan biosynthesis system gene is amplified,
Escherichia coli auxotrophic for tryptophan proliferates around the transformed strain that has accumulated tryptophan. Ampicillin in the medium is then degraded by β-lactamase encoded by plasmid pUC8 carried by E. coli, and the transformed strain further proliferates to form colonies. On the other hand, a transformed strain that does not accumulate tryptophan does not allow E. coli to proliferate around it. Therefore, since ampicillin is not degraded, they cannot proliferate and do not form colonies. Based on this idea, E. coli and an appropriately diluted colony bank were mixed, applied to a minimal medium, and incubated at 30°C.
The cells were cultured for 40 days. Pick up a colony with E. coli growing around it, isolate a single colony,
The plasmid was isolated by the method used in (2).
This plasmid was named pAJ234. pAJ234 was clearly larger than vector plasmid pAJ1844, and it was thought that a tryptophan biosynthesis system gene had been inserted therein. (6) Identification of tryptophan biosynthesis system genes possessed by pAJ234 (6)-1 Identification of anthranilate phosphoribosyltransferase (PRT) gene and tryptophan synthase (TS) gene PRT gene, TSA subunit gene (hereinafter referred to as TSA Brevibacterium lactofamentum strains lacking the TSB subunit gene (hereinafter referred to as TSB gene)
T13 (NRRLB-1534T), Brevibacterium lactofamentum No. 21, Brevibacterium lactofamentum B5, pAJ234.
Transformation was performed using the method described in (4). When 10 of the chloramphenicol-resistant colonies that emerged after culturing in regeneration medium at 30°C for one week were tested for tryptophan auxotrophy, all of them had lost their tryptophan auxotrophy. PRT gene on plasmid,
The existence of TSA and TSB genes has been revealed. (6)-2 Identification of PRAI-InGP gene Escherichia coli trpC- deficient strain CGSC No.5889 ( trpC 60. pyrF
287, hisG 1, Iac Z53, rpsL 8, λ ^- )
pAJ234 was introduced. pAJ234 was transformed by treating DNA recipient bacterial cells with calcium chloride to increase DNA permeability, and each of the resulting chloramphenylcol-resistant colonies
We caught 10 fish and examined their tryptophan requirements. In both cases, the requirement was lost, and it became clear that the PRAI-InGP gene was present on the above recombinant plasmid. (7) Subcloning of PRAI-InGP gene When pAJ234 was cut with the restriction enzyme PstI used for recombination, 2.1kb, 2.0kb, 1.75kb, 1.2kb,
Six types of inserted PstI fragments of 0.8 kb and 0.5 kb were detected. Of these, a 2.0 kb PstI fragment was fractionated,
Linked to the PstI site of pAJ1844, Brevibacterium lactofamentum T13, B5, No. 21
and E. coli CGSC No. 5889 ( trpC ). As a result, the recombinant plasmid containing the 2.0 kb PstI fragment abolished the requirement for T13, but no.
21, B5, and E. coli CGSC No. 5889 were not eliminated. Next, add this 2.0kb Pst fragment to
Pst was cut with Bgl and the 1.6kb Pst-Bgl fragment was
, ligated to pAJ224 cut with BamHI and T13
When transformed, this requirement disappeared. Therefore, the PRT gene is a 1.6kb Pst-Bgl
It is thought that it exists on fragments. Next, the 2.1 kb. Pst fragment was ligated to the Pst site of pAJ1844 and transformed into Brevibacterium lactofamentum B5. All of the colonies that formed on the regeneration medium containing chloramphenicol lost their tryptophan auxotrophy. was. Plasmids were prepared from representative colonies and again B5 and Brevibacterium lactofamentum T13, No. 21, E. coli CGSC No. 5889
( trpC ) was transformed. As a result, the requirement for B5 disappeared, but the requirement for T13, No. 21, and E. coli did not disappear. Therefore, it is considered that the TSB gene exists on the 2.1 kb. Pst fragment. Further fractionated 2.0kb
and a 2.1kb Pst fragment and cut with Pst
pAJ1844 was ligated by method (3), and E.
We obtained a recombinant plasmid that eliminates the auxotrophy of coliCGSC No.5889. This plasmid is 2.0kb.
It has a 2.1 kb Pst fragment, and when transformed into T13 and B5, both requirements were abolished. Next, in order to subclon the PRAI-InGP gene, we used a recombinant plasmid containing 2.0 kb and 2.1 kb Pst fragments to
The 2 kb. Sst-EcoRI fragment was fractionated and treated with SstI and EcoRI.
pUC19 (Messing, J., et al.,
Gene, 33 103-119, 1985) and arranged to enable transcription from the lac promoter. Or about 2.6kb Sst-Hind in Figure 1
The fragments were fractionated and cut with Sst and Hind.
pUC18 (Messing, J., et Al., Gene, 33 , 103
−119 (1985)) and positioned to enable transcription from the lac promoter.
No.5889 was transformed. As a result, Sst−
Recombinant plasmids containing the EcoRI fragment or the Sst-Hind fragment abolished the auxotrophy of E. coli. From the above results, the PRT gene, PRAI-InGP
It is thought that the TSB gene is present on the 2.0 kb and 2.1 kb Pst fragments at the positions shown in FIG. The plasmid pAJ224 used for subcloning the PRT gene was constructed as follows. pAJ224 was constructed from vector plasmid pAJ228 having trimethoprim resistance. pAJ228
The construction process is as follows. Brevibacterium lactofamentum
Chromosomal DNA was prepared from the trimethoprim-resistant mutant strain AJ12146 (FERM-P7672) mutagenized from AJ12036 by the method described in (1). On the other hand, Brevibacterium lactofamentum carrying plasmid vector pAM330
By the method described in (2) from ATCC13869
pAM330 was prepared. Next, 20 μg of chromosomal DNA and 10 μg of pAM330 were each treated with restriction endonuclease Mbo at 37° C. for 30 minutes to partially cleave them. 65℃,
After heat treatment for 10 minutes, both reaction solutions were mixed, and the DNA was ligated with _T4 phage-derived DNA ligase at 10°C for 24 hours in the presence of ATP and dithiothreitol.
A chain ligation reaction was performed. After heat treatment at 65° C. for 5 minutes, twice the volume of ethanol was added to the reaction solution, and the DNA after the completion of the ligation reaction was precipitated and collected. After dissolving an appropriate amount of the DNA precipitate in TEN buffer, trimethoprim-sensitive AJ12036 was used as a recipient strain for transformation. The method described in (4) was used as the transformation method. Trimethoprim (Sigma) 25μg/
After culturing for one week at 30°C in regeneration medium containing ml,
Approximately 100 colonies appeared, and these were replicated onto a minimal medium containing 50 μg/ml of trimethoprim to obtain one strain resistant to trimethoprim. When the plasmid DNA of this strain was detected, a plasmid clearly larger than the vector pAM330 was detected. This plasmid was named pAJ228 and again
AJ12036 was transformed. When 10 of the resulting trimethoprim-resistant colonies were picked and plasmid DNA was detected by agarose gel electrophoresis, a plasmid of the same size as pAJ228 was present in all of them. It was revealed that a gene expressing trimethoprim resistance was present on the above recombinant plasmid. The construction of pAJ224 from pAJ228 is as shown in FIG. BamHI,
A synthetic oligonucleotide linker as shown in Figure 1 with SalI and PstI cleavage sites was added to T ₄
After ligation using Phage-derived DNA ligase, the DNA was cleaved with the restriction enzyme BamHI. The resulting DNA mixture was subjected to agarose gel electrophoresis to separate and extract a DNA fragment of approximately 2.9 kb, and the DNA fragment was recovered by ethanol precipitation. The DNA fragment thus obtained has a structure in which PatI, SalI, and BamHI cleavage sites are lined up at both ends of the 2.9 kb DNA containing the HK gene. pAJ211 is 2.9kb in the PatI site of pAJ1844.
Contains the HK (homoserine kinase) gene
AJ12079 (FERM
−P7237). On the other hand, pAJ228 was partially digested with the restriction enzyme MboI and then heat-treated at 65°C for 10 minutes. The DNA fragment containing the HK gene obtained by the method described above was added to this reaction solution, and the DNA strands were ligated using T ₄ DNA ligase. After the reaction, heat treatment was performed at 65°C for 5 minutes, and the precipitated DNA was collected by adding 2 times the volume of ethanol. Brevibacterium sensitive to trimethoprim and lacking the HK gene was collected using the same method as in (4).・Lactofamentum
After protoplast transformation using AJ12078 as recipient bacteria, trimethoprim
As a result of culturing in a protoplast regeneration medium containing 100 μg/ml, approximately 200 regenerated colonies appeared. This was replicated to a minimal medium containing 200 μg/ml of trimethoprim and free of threonine, which is a substance required by the recipient bacteria, to obtain four strains that were resistant to trimethoprim and had lost their threonine requirement. A lysate was prepared from these strains by the method shown in (2), and plasmid DNA was detected by agarose gel electrophoresis.
Plasmids of 12.2kb, 9.6kb, and 6.5kb were detected. Among these, the plasmid with the smallest molecular weight of 6.5 kb was named pAJ212. The strain harboring pAJ212 was named 12078-HK strain. pAJ212 was extracted from Brevibacterium lactofamentum AJ12196 as pAJ224 by removing the HK gene by cutting with PstI.
(FERM-P8015). (8) Transfer of PRAI-InGP gene to a vector capable of replicating in Brevibacterium (8)-1 Construction of pAJ82 Add 0.25 units of restriction enzyme HPa to 5 μg of pAJ43 (JP-A-192900) at 37°C. The reaction was allowed to proceed for 5 minutes. On the other hand, pUB110 (Gryczan, TJetal J.
Bacteriol., 134 , 318 (1978)) was reacted with 5 units of Hpa at 37°C for 120 minutes. After mixing both, connect them using method (3) to form AJ12036.
Transformation was performed using the method described in (4). Colonies produced on regeneration medium containing 3 μg/ml of chloramphenicol and 100 μg/ml of kanamycin were isolated. When a plasmid was isolated from the representative strain and transformed into AJ12036 again, all colonies that appeared on the regeneration medium were resistant to kanamycin and chloramphenicol. This plasmid was named pAJ82. The restriction enzyme cleavage map is shown in FIG. (8)-2 Transfer of PRAI-InGP gene to pAJ82 SstI-EcoR carrying PRAI-InGP gene
A recombinant plasmid consisting of the fragment and pUC19
After cutting with EcoR and ligating to the EcoR site located within the chromium fecol resistance gene of pAJ82, m-fluorophenylalanine and 5
- The fluorotryptophan-resistant strain Brevibacterium lactofamentum M247 was transformed by the method described in (4). This strain
AJ12258 (FERM, P-8470). Strains that had become susceptible to chloramphenicol were isolated from the colonies that had grown on a regeneration medium containing 100 μg/ml of kanamycin, and the plasmids were extracted. This plasmid was named pAJ235 (Figure 4). pAJ235 with restriction enzyme EcoR, Hind
, PstI, Bgl, etc., resulting in approximately 2.0kb of PRAI−
It was confirmed that a DNA fragment containing the InGP gene had been inserted. (9) Tryptophan production ability of transformed strain AJ12258 (FERM P) obtained in (8)-2
When the tryptophan production ability of -8470 was investigated, the results shown in Table 1 were obtained. Culture is performed using tryptophan production medium (glucose
130g, (NH ₄ ) ₂ SO ₄ 25g, fumaric acid 12g, acetic acid 3ml, KH ₂ PO ₄ 1g, MnSO ₄・4H ₂ O 10mg,
MgSO ₄ 7H ₂ O 1g, d-bithione 50μg, thiamine hydrochloride 200μg, methionine 400mg, tyrosine 650mg, soybean protein acid hydrolyzate "Ajiri" 50
ml, 50g of CaCO ₃ in 1 part of water, PH6.5. )20ml
The test bacterial strain was inoculated into a 500 ml Sakaguchi flask and incubated at 30°C for 72 hours with shaking. After culturing, L-tryptophan in the centrifuged supernatant was determined by a bioassay method using Leuconostoc mesenteroides ATCC8042 as a quantitative bacterial strain.

[Table] In order to obtain M247, deposited AJ12258 is required.
(FERM P-8470), it is possible to remove complex plasmids in host cells without damaging the host cells. That is, the plasmid may be lost naturally from the host, or it may be removed by a "removal" operation (Bact.Rev., 36 , p. 361-405).
(1972)). Examples of other removal operations are as follows.
AJ12258 (FERM-P8470) was inoculated into a CMG liquid medium, cultured overnight at 37°C (high temperature treatment), the culture solution was diluted appropriately, applied to a CMG agar medium containing or not containing kanamycin, and incubated at 37°C. Culture for 1 to 3 days. The strain that will be distributed as a susceptible strain is M247.

[Brief explanation of the drawing]

Figure 1 shows the PRT gene, PRAI-InGP gene,
FIG. 2 is a diagram showing restriction enzymes, a map, and the position of each gene of a DNA fragment in which the TSB gene is present. Figure 2 shows the construction procedure of PAJ224. Figure 3 shows the restriction enzyme map of PAJ82. Figure 4 shows the PRAI-InGP gene.
Procedure for transferring to PAJ82.

Claims (1)

[Claims] 1 Belongs to coryneform-glutamic acid producing bacteria
N-(5-phosphoribosyl)anthranilate isomerase-indole-obtained from DNA donor bacteria
Encodes 3-glycerol phosphate synthase
The DNA fragment is connected to a vector plasmid that can autonomously replicate within the body of a coryneform glutamate producing bacterium, and m-fluorophenylalanine and 5-
L-tryptophan, which is characterized by culturing a microorganism capable of producing L-tryptophan obtained by introducing it into a DNA recipient bacterium that is resistant to fluorotryptophan, and collecting L-tryptophan accumulated in the culture solution. manufacturing method.