JPS63240794A - Production of l-tryptophan - Google Patents

Abstract

PURPOSE:To produce L-tryptophan in higher yield, by cultivating a bacterium having a specific vector from coryneform bacteria having ability capable of producing L-tryptophan from anthranilic acid. CONSTITUTION:A bacterium containing an operon obtained from chromosome of a coryneform bacterium or vector integrating gene domain of part thereof in coryneform bacteria having ability capable of producing L-tryptophan from anthranilic acid is cultivated. The coryneform bacteria include preferably coryneform glutamic acid-producing bacterium.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-) lipdoan using anthranilic acid as a raw material using microorganisms.

As a method for producing L-)ligdoane using anthranilic acid as a raw material, 5-methyl)-IJ of Bacillus subtilis
A method using an artificial mutant strain that is resistant to detophane is known (Japanese Patent Publication No. 1358-1983).

On the other hand, several attempts have recently been reported to utilize genetic recombination technology, which is different from the above-mentioned breeding by artificial mutation, for breeding L-tryptophan-producing bacteria. For example, Appl, Environ Mlcrobiol.

38, (2), 181-190, (1979), a particular mutant strain of Escherichia coli containing grasmids carrying the trp, E472 gene of Escherichia coli was found to have approximately 1.
It has been reported that 3 g/e of L-)ligdoane was produced. In addition, [Japan Society for Fermentation Engineering 1980 Conference Abstracts, p. 170 (1980) J also lists 360 mutant strains of Escherichia coli that contain grasmid that incorporates the tryptophan operon of Escherichia coli.
It has been reported that L-tryptophan of 1/l can be produced.

In contrast to the conventional production method of L-)ligdoane as described above, the present inventors have developed a method for producing coryneforms containing a tryptophan operon obtained from a bacterial chromosome or a vector into which a partial gene region thereof is incorporated. It was discovered that coryneform bacteria produced L-tryptophan from anthranilic acid with high yield.

Coryneform bacterium (Coryneformba) referred to in the present invention
eterlm) is the Percy eManual Op.
Determinative Bacteriolozo (Bargey)
sManual of Determinology we B
acteriology)! 8th edition 599 pages (197
It is a part of the microorganism defined in 4), and is an aerobic, Durum-positive, non-acid-fast, rod-shaped bacterium that does 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.

Brevibacterium diparicatum ATCC14020 Brevibacterium salacalolyticum ATCC14
066 Brevibacterium No. Inmariofilm ATCC14
068 Brevibacterium lactofermentum ATCC1
3869 Brevibacterium roserum ATCC13825
Brevibacterium flavum ATCC13826
Prepipacterium thiodanitalis ATCC19240 Corynebacterium acetoacidophilum ATC01
3870 Corynebacterium acetoglutamicum ATCC15
806 Corynebacterium carnae ATCC15991 Corynebacterium glutamicum ATCC13032,13060 Corynebacterium Lilium ATCC15990
Corynebacterium melasecola ATCC17965 Microbacterium ammoniaphyllum ATCC15
354 The coryneform glutamate-producing bacteria of the present invention include, in addition to the above-mentioned wild strains with glutamate productivity, mutant strains that have glutamate productivity but have lost glutamate productivity.

The tryptophan operon here includes a promoter, an attenuator, a region encoding a leader peptide (trpL), a 7thranylate synthase gene (LrpE, trpG), a phosphoryphosyl anthranilate transferase gene (trpD
), N-(5'-phosphoribosyl) anthranilate isomerase indole-3-glycerol phosphate synthase gene (trpc), and trypto7a/synthase genes (trpB, trpA) are arranged adjacent to each other. A transcription unit that functions as one transcription unit.

The method for isolating each structural gene is to first extract the chromosomal genes from the bacterial tryptophan operon or strains possessing each structural gene, for example, H, 5ai.
to and K, Miura Biochem.

B (ophys, Acta 72, 619 (1963)
method can be used. ), which is then cut with an appropriate restriction enzyme.

Next, it is connected to a vector that can replicate in microbial cells and has bromotor activity, and the resulting recombinant DNA is used to mutate the structural gene of the tributophane biosynthesis system in coryneform bacteria or other microorganisms. Then, we transform a mutant strain in which the # element does not lose its activity and exhibits an auxotrophy for tricyto7, and a strain in which the enzyme activity recovers and increases and the tryptophan auxotrophy disappears. From this, a complex plasmid containing the structural gene can be isolated.

Even with this method, it may be possible to isolate the entire operon by luck, but if the entire operon cannot be isolated (cloned), a portion of each structural gene isolated by the above method or All of them can be labeled with isotopes, used as probes, and isolated by colony hybridization from zone punctures of the chromosomal genes of coryneform bacteria created using cilasmids or phage vectors.

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, etc.

It is more preferable to use a mutant strain that has increased tryptophan biosynthetic activity by imparting a mutation such as tryptophan antagonist resistance as the DNA donor bacterium.

A tributophane antagonist is something that suppresses the growth of coryneform bacteria, but this suppression is likely to be completely or partially released if L-t17gt7an coexists in the medium. It is something. For example, 4-fluoro-tributophane (hereinafter referred to as 4-FT), 5-fluorotributophane (hereinafter referred to as 5-FT), 6-fluoro-tryptophan, 7-fluoro-tryptophan, 4-methyl-tryptophan, 5-fluoro-tryptophan, -Methyltricytophan, 6-methyl-tricytophan, 7-methyl-tricytophan, naphthylalanine, indole acrylic acid, naphthyl acrylic acid, β-(2-pentchenyl)alanine, styrylacetic acid, indole, trictozan, etc. .

As the tryptophan operon, a wild-type one can be used, and a gene of a mutant strain can also be used. Particularly preferred mutant genes are those mutated to reduce the degree of feedback inhibition by each structural gene metrifofhan in the tryptophan operon.

After inserting the resulting recombinant DNA f: into a recipient microorganism, the resulting transformed strain may be subjected to mutation treatment. Furthermore, a mutant gene can also be obtained by mutating the recombinant DNA itself in vitro.

In the present invention, the vector used when the tryptophan operon or a part thereof is used for the production of tryptophan is a vector in a coryneform bacterium or in E, coil,
B. Any substance that can proliferate in subtilim may be used. Specific examples include the following.

(1) pAM 330 JP-A-58-67699
Reference (2) PAM 1519% 1986-
77895 Reference (3) pAJ 655%
Reference 192900 (1972) (4) pAJ 6
11 Same as above (5) pAJ 184
4 Same as above (6) pCG 1 See JP-A-57-134500 (7) pCG 2
%Kasho 58-35197 Reference (8) pCG
4 See JP-A-57-183799 (9)
pCG 11 Same as above α0pcc1
fF open (Mau t in/Aj
i c o ) (11) pBL 100 JP-A
(〃)(6) pBR322 α埠pc194 The vector DNA can be cleaved using a restriction enzyme that cuts the DNA at one site, or partially using a restriction enzyme that cuts the DNA at multiple sites. This is done by

The vector DNA is cleaved with the restriction enzyme used to cleave the chromosomal gene, or oligonucleotides having complementary base sequences are connected to both ends of the chromosomal DNA cleavage fragment and the cleaved companion vector DNA. , then Grasmid vector and chromosome D
Subjected to Rider John reaction with NA fragment.

In order to introduce the recombinant DNA of the chromosomal DNA and the vector obtained in this way into a recipient bacterium belonging to coryneform bacteria, a method such as that reported for Escherichia coi IJK-12 (Mandel, M. and )(1e
e.

A, , J, Mo1. , Blol, , 53 , 15
9 (1970) to increase the permeability of DNA by treating recipient bacterial cells with calcium chloride, or as reported for Bacillus subtilis (Duncan, C.
, H., Wi 1son, Go A.

and Young, F., E., Gon@, Yu, 153 (1977)), this is possible by a method in which cells are introduced into a proliferation stage (so-called competent cells) in which they become capable of taking up DNA. Alternatively, as is known for Bacillus subtilis, actinomycetes and yeasts (Chang, Ss and Choen, S
, N. , Mo1ee, Gen.

Gonet, 16+1111 (1979) :B
lbb 1M, J,, WardlJ, M, and H
opvrood, 0. A, ,Nature *2
74, 398 (1978); Hlnnen, A.
m5H1cka, J, B, andFrink
.

G.R., Proe, Natl., Aead, Sc1. U
SA, 75 1929 (1978)), it is also possible to introduce the recombinant DNA recipient bacteria into protoplasts or spheroplasts that readily take up Grasmid DNA.

In the protogellast method, a sufficiently high frequency can be obtained even with the method used for Bacillus subtilis mentioned above. Of course, a method in which DNA is incorporated in the last step in the presence of polyethylene glycol or polyvinyl alcohol and divalent metal ions can also be used. - Equivalent results can be obtained by adding carboxymethyl cellulose, dextran, Ficoll, Pluronic F68 (Serpa), etc. instead of lyethylene glycol or polyvinyl alcohol to promote DNA uptake.

The receptor for the thus obtained conjugate of the chromosomal DNA fragment and the vector may be any type of Corynebacterium, but it is convenient to use L-tributophane auxotrophs when selecting transformed strains. be.

Of course, more favorable results can be obtained by using a strain with a higher ability to produce L-tryptophan from anthranilic acid, or an L-tributophane-requiring strain derived from a strain with a higher L-tryptophan production ability.

In order to produce L-tributophane using L-)-lipdoane-producing bacteria, the L-)lipdoane-producing bacteria are cultured in a medium containing anthranilic acid.

The medium used is a conventional medium containing a carbonate source, a nitrogen source, inorganic ions, and, if necessary, organic micronutrients such as amino acids and vitamins. Carbon sources include TEHA, glucose,
Sucrose, lactose, etc., starch hydrolyzate containing these, sugars such as whey and molasses, and organic acids such as acetic acid, fumaric acid, citric acid, and pyruvic acid are used. As the nitrogen source, ammonia gas, aqueous ammonia, ammonium salt, etc. can be used.

Anthranilic acid may be added to the medium at the beginning of culture, or
It may be added to the medium after L-tryptophan-producing bacteria have grown to a certain extent. Since anthranilic acid inhibits the growth of microorganisms when its production in the medium exceeds a certain limit, it may be added in small amounts to the medium so that the concentration does not exceed a certain limit.

Cultivation is carried out under aerobic conditions, adjusting the temperature and temperature of the medium as appropriate, until the production and accumulation of L-to+J gutophane substantially ceases.

L-tryptophan produced in the culture solution or reaction solution can be separated and collected by a conventional method.

Example 1 Anthranilate synthase gene, phosphoryl? Cloning of dianthranilate transferase gene and tryptophan synthase β subunit gene 1-1 Preparation of chromosomal DNA containing the tryptophan operon of Brevibacterium lactofermentum AJ112
25 (FERM-P4370) was inoculated into 1 l of CMG medium (containing pegton ILt, yeast extract 117 dt, glucose 0.511/di, and NaCtO,5,P reservoir, adjusted to -7,2). Then, culture with shaking at 30°C for about 3 hours, and collect the cells in the logarithmic growth phase.

After this bacterial cell was lysed with lysozyme/SDS, chromosomal DNA was extracted and purified by a conventional phenol treatment method, and finally 3.5 μm of DNA was obtained.

1-2 Preparation of Vector DNA Using pAJ1844 (molecular weight 5.4 megadaltons) as a vector, its DNA was prepared as follows.

Brevibacterium lactofermentum AJ1203 carrying t f pAJ1844 as a plasmid
7 for 100r! After inoculating Lt in CMG medium and culturing at 30°C until the late logarithmic growth phase, the cells were lysed by lysozyme SDS treatment, and 30. The supernatant was obtained by ultracentrifugation at OOOxg for 30 minutes. After the phenol treatment, 2 volumes of ethanol were added to precipitate and collect the DNA. Add this to a small amount of TE
N buffer (20mM) Liss hydrochloride, 20mM NaC2,
1mM gDTA (P)! 8.0) After dissolving in )
Plasmid fractions were separated by cesium chloride density gradient equilibrium centrifugation and finally pAJ1844
Approximately 200 μl of plasmid DNA was obtained.

1-3 Chromosome I) Insertion of NA fragment into vector 10 μg of chromosomal DNA obtained in 1-1 and 5 μg of plasmid DNA obtained in 1-2 were each kept at 37°C for 1 hour and cut with restriction endonuclease Pstl. did.

After heating to 65° C. for 10 minutes, both reactions were mixed.

In the presence of ATP and dithiothreitol, the T47 aso-derived DNA was kept at 10°C for 24 hours using PCRase.
The NA chains were linked. Then, the reaction solution was heated at 65°C for 5 minutes.
The mixture was heated for a minute, and 2 volumes of ethanol was added to the reaction solution I/c to collect a precipitate of ligated DNA.

1-4 Cloning of anthranilate synthase gene, phosphoribosyl anthranilate transferase gene, and trypto-7 ansynthase β subunit gene Brevibacterium lactofermentum anthranilate synthase-deficient strain AS60, phosphoribosyl anthranilate transferase-deficient strain M 38,)! J gutophane synthase β subunit-deficient strain Ya 30 (both parent strains were isolated from AJ12125 by mutation treatment with N-)f-N-nitro-N-nitroa/'7nison) as a DNA recipient strain. Using.

As the transformation method, the Protogelast trans7omeci, 7 method was used. First, the bacterial strain was cultured in a 5-cmG liquid medium until the early logarithmic growth phase (after adding Nijirin G at 0.6 units/rnl, the strain was further cultured with shaking for 1.5 hours, and the bacterial cells were collected by centrifugation. The bacterial cells were incubated with 0.5M sucrose, 20mM maleic acid, 20mM magnesium chloride, and 3.5M tipenuceipros (Direo
) consisting of SMMP medium (PH6,5 > 0, washed with 5 ml).
The cells were suspended in a medium and incubated at 30°C for 20 hours to form protoplasts. After centrifugation for 10 minutes at 6,000) l, the protogelasts were washed with SMMP and resuspended in smpK in 5 rnl. Nine glotodelast obtained in this way and 1-
10μ9 of the DNA prepared in step 3 was mixed in the presence of 5mM EDTA, and polyethylene glycol was added to a final concentration of 30mM.
%, and leave at room temperature for 2 minutes to allow the DNA to be incorporated into the protogellast. After washing this protogellast with SMMP medium 1M,
1 and cultured at 30°C for 2 hours to ensure gene expression. This culture solution was spread on a -7.0 protogellast regeneration medium. Protocilast regeneration medium contains 129% K of tris(hydroxymethyl)aminomethane per 1j of distilled water.
Cl0.59% glucose 10.9, MgCl2・
6H20B, 1 lI.

CaCl2”2H202, 2g, peptone 41!, powdered yeast extract 4J, casamino acid CDirco) 111%
2HPO40.29, sodium succinate 135g,
Agar 8Ii and chloramphenicol 3μ,! i'/M
t-contains.

After two weeks of incubation at 30°C, approximately 250
00 chloramphenicol resistant colonies appeared, so they were transferred to a minimal medium (2-tiglucose, 1tIb ammonium sulfate, 0.3% urea, 0.1 potassium dihydrogen thiphosphate, 0.04% magnesium sulfate 7 water). Salt, 2Pp
m iron ion%2pPm manganese ion, 200μFl/
1 thiamine hydrochloride, 50μ9A biotin, casamino acid (Dirco) 31/l, chloramphenicol 1
0μji/Ill, PH7.0, agar 1.8%), and chloramphenicol-resistant strains that have lost their tryptophan requirement were 2 strains from the 9th section using AS60 and 1 strain from the 9th section using A38. , 1 was selected from the classification using 430.

We extracted grasmids from the five strains mentioned above, and found that all plasmids were clearly thicker than the vector grasmid pAJ1844 (9 recombinant grasmids obtained from the classification using AS60) were obtained from the classification using ptrpE36, ptrpE4, and Grave38. The obtained recombinant plasmid was transformed into ptrpD385
The recombinant plasmid obtained from the 9th section using A30 was named ptrpB301.

1-5 Re-transformation Recombinant plasmids ptrpE36 and ptr obtained in 1-4
pE4, ptrpD3851, and ptrpB301 each contain the anthranilate synthase gene and phosphoryl? To confirm the presence of the dianthranilate transferase gene and the tryptophan synthase β subunit gene, ptrpE36 and ptrpE4 were transferred to AS60.
ptrpD3851 to ya38, ptrpB301
was transformed again into A30.

Ten of each of the live citachloramphenylcol resistant colonies were picked up and tested for tryptophan requirement. As a result, both have lost their requirement, and p t
rpE36, ptrpE4. has the anthranilate phosphatase gene;
It was revealed that rpB3011c contains the trithophan synthase β subunit gene. However, in the ptrpE4 transformed strain, the degree of loss of auxotrophy and the accumulation of anthranilic acid on the minimal medium
ptrpE4 compared to the transformed strain of E36.
It was suggested that part of the gene for K-hanthranilate synthase may be missing.

1-6 Preparation of a restriction enzyme map of the inserted DNA fragment of recombinant Grasmid
rpE36, ptrpE4, ptrpD3851, pt
rpB301 was prepared and cut with various restriction enzymes according to conventional methods to create a restriction enzyme map of the inserted DNA fragment (Figure 1).
.

Example 2 Brevibacterium lactofermentum trypto 7
Cloning of the entire Ann Oberon strain 41 resistant to 5-fluorotributophan isolated from Brevibacterium lactofermentum AJ11255 by natural mutation
Example 1 from 041 (a strain in which the feedback inhibition of anthranilate synthase by tryptophan was released)
Chromosomal DNA was prepared by the method shown in 9, and restriction enzyme B
Completely cut with amHI or 5alIs or
ssing, J., at al.

Gene, 33, 103-119 (1985)), and L cot i JM109
(Mensing + J, *eta1..Ge
n5.33, 103-119 (1985)) and transformed X-Ga1 (5-bromo-4chloro
-3-1ndolyl-β-galacLoside
) + IPTG (1sopropyl-β-D
-thio -galaetopyranoside
), grating on L agar medium containing ampicillin. After culturing at 37°C for 24 hours, a total of about 1500 white colonies appeared on a nitrocellulose filter. 1.2 of pLrpE36 containing the anthranilate cy/tase gene (trpE) obtained in Example 1
kb.

Glove the Pstr insert fragment of
M., ,Walls, J.: Methods.
in Enzymology + 68 + 379
*AcademicPress Inc, Ne
York (1979)) and restriction enzyme B.
One positive clone was obtained from the division using amHI, and one positive clone was obtained from the nine divisions using the restriction enzyme 5alI. B
Nine recombinant cilasmids obtained from the amHI section were ptrpg9.
The plasmid obtained from the 7.5stI section was transformed into ptrpFJ2
A restriction enzyme cleavage map of the inserted DNA fragment was created using the method shown in Example 1 (Fig. 1).

As a result, ptrpE97, ptrpE36, ptrp
D3851.

It has a PstI fragment that has the same restriction enzyme map as the inserted PstI fragment of ptrpB301, and ptrpE42
is the PmtI fragment of ptrpg36 and ptrpD385
It became clear that it had the same restriction enzyme map as part of the PstI fragment of 1.ft. Also, ptrpE97 and ptrpE97
rpE42 had a common BamHI-5stI fragment.

Example 3 Subcloning of the N-(5-phosphorigesyl)anthranilic acid isomerase indole-3-glycerol phosphate heptase gene (trpC) and )! , 17'
dooransynthase α subunit gene (trpA)
From the comparison of restriction enzyme maps of the inserted DNA fragment of the recombinant grasmid shown in Figure 1, the Vctrpc gene is located between the trpD gene and the LrpB gene.
It was thought that the trpA gene might exist downstream of the gene.

Therefore, we conducted the following experiments to confirm the existence of each gene.

3-1 Subcloning of the trpc gene from the recombinant Grasmid p t rpE97 shown in FIG.
b, the 5stI-EeoRI fragment of 5stIe
pUc19 cut with EcoRI (M@ssing,
J, , @t at, .

Gen5, 33, 103-119.1985) and arranged to enable transcription from the lae promoter. Or 5stI-H of about 2.6kb in Figure 1
The lnd Ill fragment was fractionated and 5stI, Hlnd
Cut at 7'j pU018 (Mess tng,
Jm at ale, Gen@, Ushi, LO3
-419°1985)) and positioned to allow transcription from the lac promoter,
l i CG5CA3889(υIpC601pyr
F287*hlgGl, 1acZ53, r
psL8. λ-) was transformed. As a result, 5stl
-EeoRI fragment or 5sLI -Hlnd II
The recombinant plasmid containing the I fragment abolished the auxotrophy of E. coli.

3-2 Confirmation of existence of trpA gene The NruI-BamHI fragment of Tomozuri 2.4 kb shown in Fig. 1 was fractionated from the recombinant Grasmid ptrpg97.
Small.

Cut with BamHI, ligate to pUc18, position to enable transcription from the lac promoter,
col t CGSCA 5644 (trpA33
, rha-7, λ-). As a result, N
Loss of tryptophan auxotrophy was observed in the transformed strain carrying the recombinant grasmid containing the ru I - Bam HI fragment.

Example 4 Determination of base sequence of tritfan operon ptrpE36, ptrpD3851, and ptrp obtained in Example 1
Grasmids were prepared from each of the transformants B301 and ptrpE97 obtained in Example 2.

pU018 for each Grasmid insert DNA fragment
Alternatively, pUc19 or M13mplO (Messing
, J, andVieira+J,+Gene 19
a 269 (1982))
Chain termination method (Sang@
r, Fa @ta1. ．． Proc, Nat1. Aca
The entire nucleotide sequence of the tributophane operon was determined according to the ninth strategy diagram for nucleotide sequence determination shown in FIG. As a result, the DNA base sequence shown in formula 1 was obtained, and this base sequence includes the RNA polymerase binding site (trp promoter) necessary for the expression of the tryptophan operon of Prepino4cterium Lacto7aermentum, the ligesome binding site, and anthranil. Acid synthase gene (trpE
.

trpG), phosphory? dianthranilate transferase gene (trpD), N-(5-phosphoribosyl) anthranilate isomerase indole-3-glycerol phosphate synthase gene (trp
C), tryptophan synthase gene (trpB,
DNA sequence corresponding to trpA) and termination sequence (
Terminator).

Example 5 Brevibacterium lacto 7 Elmentum AJ1203
Cloning of the tryptophan operon from 6 and AJ12337 As a source of chromosomal DNA 0 disruption, the degree of feedback inhibition by tryptophan of each structural gene in the tryptophan operon was reduced, resulting in resistance to the tryptophan analog 5Pr. Brevibacterium lactofermentum AJ12337, a tributophane-producing strain, and Brevibacterium fermentum A, a strain harboring the wild-type tryptophan operon
J12036 (FERM-P7559),
As vector DNA, pAJ1845 is a shuttle vector between E. eoli and Brevibacterium.
Example 2 using BamHI as a restriction enzyme.
A similar experiment was conducted, and one positive clone was obtained from nine sections using AJ12036 and one positive clone was obtained from the section using AJ12337.

The recombinant grasmid from the AJ12036 classification was transformed into pT
s-1, Grasmid obtained from AJ12337 division was pT
The DNA fragment was named R-1 and a restriction enzyme cleavage map of the inserted DNA fragment was created using the method shown in Example 1.

As a result, both pTs-1 and pTR-1 showed that ptrpE97
The insertion of BamH with the same restriction map as BamHI
It became clear that it had an I fragment. ptrpE
Since pTS-1 and pTR-1 have the entire region of the tryptophan operon, it is thought that pTS-1 and pTR-1 similarly contain the entire region of the tryptophan operon. pTs-1
In order to confirm the difference between the tryptophan operon possessed by pTR-1 and pTR-1, these grasmids were incubated with E. coliC6.
The minimum growth-inhibiting concentration of 5FT on a plate was investigated for the 5FT introduced into the 00 strain, and the results shown in Table 1 were obtained. pT
The strain having R-1 clearly has resistance to high concentrations of 5FT, and it is thought that the degree of feedback inhibition by tryptophan of each structural gene in tryptophan O(Ron) is reduced.

C6005μg~ C600/PTS-15μ! 1 to Example 6 Breeding of tryptophan-producing bacteria from angelanilic acid by amplification of streptophane operon pTR-1, pTs-
1 was used to transform Brevibacterium lactofermentum AJ12036 by the Kita method described in 1-4, and each transformed strain was selected using chloramphenicol resistance as an indicator.

The thus obtained pTR-1 transformed strain AJ12
338 (rERM-p9Z!51) and pTS-
Transformed strain AJ12339 (F'ERM-P q'Z5'2
) was cultured in a medium supplemented with angelanilic acid, and the tryptophan production ability was tested after 4 trials, and the results shown in Table 2 were obtained.

The culture was carried out on a tricytophane production medium (glucose 130f
(NH

Claims (1)

[Claims] A coryneform bacterium containing a vector into which the tryptophan operon or a partial gene region thereof obtained from the chromosome of a coryneform bacterium is integrated, and which has the ability to produce L-tryptophan from anthranilic acid, is cultured, and the product is produced and accumulated in the culture medium. A method for producing L-tryptophan using a microorganism, the method comprising collecting L-tryptophan.