JPH06133781A - Production of l-tryptophan - Google Patents

Abstract

PURPOSE:To produce the subject amino acid in high efficiency by reacting indole with serine in the presence of E.co1i containing a plasmid having fragments of genes governing the biosynthesis of tryptophan synthase, a proliferation controlling and distributing system and an autonomous replication capability. CONSTITUTION:A strain of E. coli is transformed by introducing a recombinant plasmid containing a DNA fragment containing a gene governing the biosynthesis of tryptophan synthase and originated from tryptophan operon, a DNA fragment containing a gene governing a proliferation controlling and distributing system and originated from F factor plasmid and a DNA fragment containing a gene governing the autonomous replication capability of ColE1 plasmid. The obtained transformed strain Escherichia coli K-12 YK2004 (FERM P-7838) is cultured in a medium and indole is made to react with DL-serine in the presence of the cultured product or its treated product to obtain L-tryptophan on an industrial scale in high efficiency.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel plasmid having a DNA fragment containing a gene controlling the biosynthesis of tryptophan synthase, and more specifically to tryptophan synthase which can be stably maintained in a host. The present invention relates to a method for producing L-tryptophan using Escherichia coli transformed with a DNA fragment containing a gene controlling the biosynthesis of E. coli, for example, a plasmid having a tryptophan operon.

[0002]

2. Description of the Related Art There have been various studies on cloning of tryptophan operon, but for example, Journal of General.
Microbiology, Vol. 118, 253
As described in (1980), there are problems in stability in the host, and it is considered that there are many difficulties in applying it to industrial production of tryptophan.

On the other hand, in terms of the stability of the plasmid in the host, the most important factor in terms of application is generally the phenomenon of plasmid loss during subculture of the host. Therefore, various attempts have hitherto been made in order to prevent the loss of the plasmid, and for example, a chromosomal gene DNA that controls the property of not depending on streptomycin of the genus Escherichia
A method has been proposed in which a fragment-incorporated plasmid is contained in a streptomycin-dependent mutant of the genus Escherichia to stabilize the properties of a microorganism containing the plasmid (Japanese Patent Laid-Open No. 55-156591). However, this method is not only economically problematic, but also requires complicated functions to be incorporated into the desired plasmid, and therefore it is expected that the plasmid will not be stably distributed to daughter cells during mitotic growth of the host. There is a considerable problem in applying it to the future.

Therefore, the present inventors have succeeded in stably and stably distributing a DNA fragment containing a gene controlling the biosynthesis of tryptophan synthase, for example, tryptophan operon, from a parent cell to a daughter cell. We have conducted extensive research on plasmids that have the enabling function, paying attention to the fact that the fraction containing the gene that controls the growth control distribution system derived from the F factor plasmid greatly contributes to the stabilization of the plasmid. Novel plasmid characterized by having a DNA fragment containing a gene controlling the biosynthesis of tophan synthase and a DNA fragment containing a gene controlling the growth control distribution system of F factor plasmid, particularly a plasmid by the present inventors p
A plasmid called MTY-1 was created and proposed (see Japanese Patent Laid-Open No. 60-2189). However, although this novel plasmid pMTY-1 is useful for producing an unstable gene product in the bacterial cell, it has a small copy number of 1 to 2 and produces a large amount of the gene product in the bacterial cell. Has the drawback of low efficiency.

Therefore, a plasmid having a large copy number in the transformed microbial cell, a high expression efficiency of the trait, and a stable and low dropout, which can be reliably subsequently distributed from a parent cell to a daughter cell, was developed. It is desired to do.

Therefore, the present inventors have noticed that the Escherichia coli ColE1 plasmid has a copy number of several tens per normal cell chromosome, and this time, a DNA containing a gene that controls the autonomous growth ability of the ColE1 plasmid. By combining the fragment, a DNA fragment containing a gene controlling the growth control distribution system derived from the F factor plasmid, and a DNA fragment containing a gene controlling the above-mentioned tryptophan synthase biosynthesis, a large copy number can be obtained. We have succeeded in creating a plasmid that can be stably distributed on passage, and found that L-tryptophan can be efficiently produced by using Escherichia coli transformed with the plasmid, and completed the present invention. Came to do.

[0007]

Therefore, according to the present invention, a DNA fragment containing a gene that controls the biosynthesis of tryptophan synthase derived from tryptophan operon,
A culture of Escherichia coli transformed with a plasmid having a DNA fragment containing a gene controlling a growth control distribution system derived from an F factor plasmid and a DNA fragment containing a gene controlling a autonomous growth ability of a ColE1 plasmid, or a treatment thereof There is provided a method for producing L-tryptophan, which comprises reacting indole with L- or DL-serine in the presence of a substance.

The "DNA fragment containing a gene that controls the biosynthesis of tryptophan synthase" (hereinafter sometimes abbreviated as "T fraction") constituting the plasmid used in the method of the present invention means indole and L-. Or DL-serine to L-
It means a DNA fragment containing a gene controlling the biosynthesis of tryptophan, and the T fraction contains a tryptophan operon or a larger DN containing the tryptophan operon.
A fragment or TrpA which is a gene controlling the biosynthesis of tryptophan synthase in tryptophan operon
DNA fragment in which promoter and operator are bound to and TrpB, or TrpC and Tr to TrpA and TrpB
A DNA fragment in which at least one of pD and TrpE is combined with a promoter and an operator (eg, Trp
A, TrpB, TrpC, TrpD, and TrpE to which a promoter and an operator are bound) and the like. Practically, those derived from Escherichia coli (Escherichia coli) are preferably used as these T fractions. The source of this T fraction is not particularly limited, but Escherichia coli ATCC 23282, Escherichia coli
E. coli ATCC 23437, Escherichia coli ATCC
23461 and the like are advantageously used.

As described above, the plasmid used in the method of the present invention has one feature in that the above T fraction is combined with a DNA fragment containing a gene controlling the growth control distribution system derived from the F factor plasmid. . Factor F plasmids are, for example, "Protein Nucleic Acid Enzymes" Vol. 27, No. 1 (198
2), page 98, with a structure as shown in the genetic map of FIG. 1 and the physical map by EcoRI, the molecular weight is 94.
It is a known 5 kb (62 × 10 ⁶ daughter) plasmid, which is usually present in enterobacteria such as Escherichia coli at a copy number of 1 to 2 per cell chromosome, and therefore, if there are 2 chromosomes per cell, 2 to 4 for F factor plasmid
Only the individual is carried. This plasmid has a mechanism that exactly doubles during cell division and is accurately transmitted to each daughter cell (in this way, the copy number is kept at a low level and the growth and pace of the host are accurately matched). The mechanism to increase the number is called stringent growth control). Such a st in F factor plasmid
It has already been elucidated that the ringent growth control function is borne by a fragment called mini-F capable of autonomous growth with a molecular weight of 9.1 kb.
It is also known that can be excised from the F factor plasmid with the restriction enzyme EcoRI.

The plasmid used in the present invention is this mini.
It utilizes a growth control distribution system carried by -F. Therefore, "DNA fragment containing a gene controlling the growth control distribution system derived from F factor plasmid" may be abbreviated as "F fraction" hereinafter. ) Is a gene DN having a mechanism for accurately transmitting the F factor plasmid as described above to daughter cells.
A, and a typical example of such F fraction is about 9.
There is a mini-F fragment having a molecular weight of 1 kb.

Furthermore, in the plasmid used in the present invention, "Co" used in combination with the above-mentioned T fraction and F fraction is used.
DN containing a gene that controls autonomous growth of 1E1 plasmid
"A fragment" (hereinafter referred to as "S fraction") means a DNA fragment containing a gene that controls autonomous growth of a ColE1 plasmid having a copy number of 20 to 30 per cell chromosome,
A typical example of such an S fraction is the S fraction of plasmid pBR322 having a length of approximately 4.3 kb,
In addition, there are S fractions such as plasmid pBR325.

The plasmid used in the present invention is another gene fraction, for example, an antibiotic resistance marker, as long as it has the three essential gene fractions of the above-mentioned T fraction, F fraction and S fraction. A DNA fragment containing an ampicillin resistance gene, a DNA fragment containing a kanamycin resistance gene, etc. may be further included, but one typical example is substantially composed of three fractions of T fraction, F fraction and S fraction. , A plasmid having a molecular weight of about 13.7 megadalton (about 20.8 kb), which the present inventors named "plasmid pMTY-2". In the present specification, the molecular weight of the plasmid is a value measured by agarose gel electrophoresis.

The plasmid pMTY-2 will be described in more detail below.

The sensitivities of the following restriction enzymes (number of recognition sites) of the plasmid pMTY-2 and the length (kb) of the fragment digested by the restriction enzymes are shown in the following table.

[0015]

[Table 1] Number of restriction enzyme recognition sites Length of fragment (kb) EcoRI 2 11.7, 9.1 BamHI 3 17.8, 2.4, 0.6 Sal I 2 17.0, 3.8 Ava I 1 20.8 Hind III 4 14.4, 3.0, 2.6, 0.8 Pst I 5 14.1, 3.0, 1.8, 1.5, 0.4 The plasmid pMTY-2 used in the present invention having the characteristics as described above can be produced, for example, as follows.

First, DN containing tryptophan operon
The A fragment (T fraction) can be prepared, for example, by Escherichia coli having a tryptophan operon in the chromosomal gene, such as Esc.
herichia coli K-12 (IFO330
1, ATCC10798, ATCC e23562) and the like, for example, Φ80 (ATCC11
456a-B1) and the like, and utilizing the phenomenon of solubilization and induction, a large amount of a phage in which the tryptophan operon was incorporated into the phage DNA was prepared [R. M. De
nney, C.I. Yanofsky; J. Bacteri
ol. , 118 , 505 (1974)], and then [E. F. Fritsch, Sambrook,
"Molecular cloning" (1982)
p. 164-165, Cold Spin Harbo
r Laboratory ”]
A is extracted and a restriction enzyme such as BamHI or EcoRI is extracted.
It can be carried out by cutting out a DNA fragment containing the tryptophan operon using the above.

On the other hand, the mini F fragment can be prepared by, for example, a microorganism having an F factor plasmid, for example, E. coli K-12 strain (ATCC15153,
ATCC e23589, ATCC e23590) and the like by a method known per se, for example, P. Guerry,
D. L. LeBlanc, S .; Falkow; J. Ba
ct. , 116 , 1064 (1973) and the like, and then the F factor plasmid is extracted by a method described in the literature, and then a mini enzyme having a molecular weight of about 9.1 kb is obtained using a restriction enzyme EcoRI.
It can be prepared by cutting out the F fragment.

On the other hand, the source of the DNA fragment containing the gene that controls the autonomous growth of the ColE1 plasmid is Col.
Representative plasmid pBR322 as E1 plasmid
It is convenient to use.

First, the DNA fragment (T fraction) containing the tryptophan operon prepared as described above was added to the T fragment.
The plasmid pB322 was treated with the same restriction enzyme used to excise the fraction, and ligase such as ligase derived from T4 phage was allowed to act on the plasmid pB322.
A plasmid in which the above T fraction is incorporated into R322 is prepared. Next, this plasmid is cleaved with a restriction enzyme EcoRI, combined with the mini F fragment prepared as described above, and ligated with ligase to give the desired plasmid pMTY-2.

The specific method for preparing the plasmid pMTY-2 will be described in detail in Reference Example 1 below.

The thus-prepared plasmid used in the present invention has excellent characteristics that it has a high copy number and is not easily lost when it is inherited by daughter cells during cell division of the host and is stable.

Therefore, this plasmid is highly expected to have industrial application in the production of tryptophan. In the production of tryptophan, the host is transformed with the above-described plasmid. Escherichia coli, Bacillus subtilis, Saccharomyces, etc. can be considered as host bacteria that can be used for this transformation, and among these, Escherichia coli (Escherichia coli) is preferable. Furthermore, it is particularly preferable that these host bacteria are tryptophan-requiring mutant strains.

The introduction of the above-mentioned plasmid into these host bacteria can be carried out by a method known per se, such as M. Mand
el, A.A. Higa; J. Mol. Biol. 53 , 1
59 (1970) and the like.

The host bacterium thus transformed is cultivated by a method known per se to sufficiently produce and accumulate tryptophan synthase in the cells, and then to produce indole and L- or DL-serine. From L-tryptophan. When the cultured bacterial cells are used for the enzymatic reaction, the bacterial cells can be used as they are, but the bacterial cells are crushed by ultrasonic treatment or the like, or the crushed material is further treated with water or the like. It is also possible to use the extracted extract, or a crudely purified product obtained by further treating the extract with ammonium sulfate or the like to precipitate the enzyme component, and further fixing the bacterial cells or these treated products as necessary. It can also be used.

The reaction of indole with L- or DL-serine in the presence of the bacterial cells or a treated product thereof is, for example, 0.1M phosphate buffer solution (pH 7.0 to pH 7.0) in the same manner as a usual enzyme reaction.
9.0) or a solvent such as water (pH 7.0 to 9.0) and the like at a temperature of about 20 to about 50 ° C., preferably about 30 to about 40 ° C. and usually about 10 to about 72 hours.

The amount of indole and L- or DL-serine used during the reaction is not particularly limited, but generally, it is suitable to use each in the concentration range of 0.1 to 20% (wt / vol). is there. The amount of the bacterial cells or the processed product thereof is not particularly limited, but is generally 1 to 10%.
It can be used at a concentration of (wt / vol).

Cultivation of the above-mentioned transformed bacterium varies depending on the kind of the host bacterium, but generally, it can be carried out using a commonly used synthetic or natural medium. Therefore, as the carbon source, various carbohydrates such as glucose, glycerol, fructose, sucrose and molasses can be used. As the nitrogen source, natural organic nitrogen sources such as tryptone, yeast extract, corn steep liquor and casein hydrolyzate can be used. Many natural organic nitrogen sources can be carbon sources as well as nitrogen sources.

The culture can be carried out under aerobic conditions such as shaking culture or aeration-agitation deep culture. The culture temperature is generally 20 to 50 ° C., and the pH of the medium in the medium is preferably maintained near neutral or slightly alkaline. The culture period is usually 1 to 5 days.

Using the bacterial cells obtained by the above-mentioned culture method or a treated product thereof, indole and L- or DL
Separation and purification of L-tryptophan produced in the reaction solution, which is obtained by reacting serine, is carried out by ion exchange resin,
It can be performed by a known method such as adsorption or desorption treatment with activated carbon or the like.

The host strain transformed with the above plasmid can also be used for the production of L-tryptophan by a fermentation method. That is, if a host transformed with the plasmid of the present invention is cultured in a medium containing indole,
L-tryptophan can be produced and accumulated in the medium, and L-tryptophan can be produced by collecting the L-tryptophan.

[0031]

EXAMPLES Reference Example 1 : Construction of plasmid pMTY-2 (A) Preparation of mini F fragment E. coli K-12 strain (ATCC 151)
53) to 1 l of L medium (Bacto tryptone 10
g, yeast extract 5g, NaCl 5g, glucose 1
g, 1 liter of water; pH 7.2) and incubate at about 37 ° C for about 4
After culturing with shaking for a period of time, the cells were collected, treated with lysodium, and sodium dodecyl sulfate (SDS) was added to lyse them. This lysate was centrifuged at 32,000 xg for 40 minutes, the supernatant was fractionated, and then cesium chloride-
After carrying out an ethidium bromide equilibrium density gradient centrifugation treatment, a solution containing the F factor plasmid was collected by dialysis treatment. This solution was subjected to ethanol precipitation to finally collect about 20 μg of F factor plasmid.

Next, in the preparation of the mini F fragment, 5 μg of the above F factor plasmid was taken and E which was a restriction enzyme was used.
The cofactor was allowed to act at 37 ° C. for 1 hour to cleave the F factor plasmid DNA chain to prepare a DNA solution containing a mini F fragment of about 9.1 kb.

(B) Preparation of Phage φ80pt E. coli K-12 strain (IFO3301)
Inoculate 100 ml of L medium (the same composition as above),
0.2 ml of the culture shaken at 37 ° C. for about 4 hours and an aqueous solution of Phage φ80 (ATCC11456a-B1) (1
And 0.1ml of 0 ⁵ months / ml), were mixed into the L medium soft agar (L medium + agar smear), overlaid on L medium agar plates. When the plate was cultured at 37 ° C for about 5 hours, plaques (dissolved spots) were formed, and the plate was further incubated at 37 ° C for 2-3 days
Continuation of the culture in P. plaques gives rise to colonies of Phage φ80 lysogen. The lysogen was mixed with L medium for 3
After culturing at 7 ° C for 4 hours, smearing on the same L medium agar plate as above, then irradiating with ultraviolet light (400 to 800 ergs
/ Mm ^2, to prepare phage DNA) containing phage Fai80pt (tryptophanase Juan operon by 10-20 seconds) due to the induction of lysogenic phage.

(C) Preparation of DNA fragment containing tryptophan operon E. coli K-12 strain (IFO3301)
Inoculate 1 l of medium (composition same as above) to about 37
Cultivated with shaking at ℃ for about 3 hours, 25% (w /
v) Add 10 ml of the glucose solution and the Phage φ80pt solution prepared above at a concentration of 10 ¹¹ pieces / ml (mo.
i 20) Shaking was continued for 5 hours, and chloroform was added in a conventional manner to prepare a large amount of Φ80 pt [T. Maniatis, E .; F. Fritsch, S
ambrook; "Molecular clone
g "(1982) p.76-80 Cold Spri
ng Harbor Laboratory].

The obtained Φ80 pt solution was dialyzed with Tris buffer (pH 7.8) and then subjected to the DNA extraction method by the phenol method [above-mentioned "Molecular Cl"].
oning "p.85], and the purified DNA was extracted and purified, and the restriction enzyme BamHI was added thereto and reacted at 30 ° C. for 30 minutes to give DN containing tryptophan operon.
A fragment was obtained.

(D) Construction of plasmid pMTY-2 DNA containing tryptophan operon obtained in (C) above
The fragment was reacted with restriction enzymes SalI and XhoI at 37 ° C. for 1 hour and then heat-treated at 65 ° C. for 5 minutes to generate the restriction enzyme S.
After inactivating alI and XhoI, the restriction enzyme Sa was similarly added.
The plasmid pBR322 treated with II was added and mixed,
ATP (adenosine triphosphate) and DTT
(Dithiothreitol), and then DNA ligase derived from T4 phage (having DNA binding activity; “T4 DNA ligase” manufactured by Takara Shuzo Co., Ltd.) was added, and then 1
The reaction was carried out at 2 ° C for 24 hours. Then, the mixture was heat-treated at 65 ° C. for 5 minutes to inactivate the DNA ligase, and then Escherichia coli K-12 strain (tryptophan-requiring mutant strain) was used in a conventional manner (SN Cohen: P
roc. Natl. Acad. Sci 69 , 2110
~ 2114, 1972), the transformant [Trp auxotrophy disappeared, that is, biosynthesis became possible by the tryptophan operon on the plasmid, and minimal medium (K ₂ HPO ₄ 7 g, KH ₂ PO ₄ 2 g, MgSO _{4 4} - 7
H ₂ O 0.1 g, (NH ₄ ) ₂ SO ₄ 1 g, glucose 2
g, a strain capable of growing on pure water 1 l)] was obtained. This strain was liquid-cultured according to a conventional method, and the plasmid was purified and separated from the culture solution.

Next, the tryptophan operon-containing plasmid was reacted with the restriction enzyme EcoRI at 37 ° C. for 1 hour and then heat-treated at 65 ° C. for 5 minutes to obtain EcoRI.
Was inactivated, the mini-F fragment obtained in (A) above was added and mixed, ATP and DTT were added, and DNA ligase was further added, followed by reaction at 12 ° C. for 24 hours. Then, the mixture was heat-treated at 65 ° C. for 5 minutes to inactivate the DNA ligase, and then Escherichia coli K-12 strain (tryptophan-requiring mutant strain) was transformed with this reaction solution according to a conventional method to obtain 50 μg of ampicillin. L medium (Bacto-tryptone 10
g, yeast extract 5g, NaCl 5g, glucose 1
g, 1 liter of water: pH 7.2) and inoculated at 37 ° C for 18
After shaking culture for a period of time, the cells are collected by centrifugation and the cells are washed,
A transformant was obtained by smearing on a minimal medium (the composition is the same as above). This strain was liquid-cultured according to a conventional method, and the plasmid was purified and separated from the culture solution.

The resulting plasmid pMTY-2 has the restriction map shown in the attached FIG. Plasmid pMTY
This restriction map of -2 shows the restriction enzymes EcoRI, BamH
After treatment with I, SalI, etc., alone or in combination, it was confirmed by agarose gel electrophoresis. The transformant strain carrying this plasmid pMTY-2 was designated as Escherichia coli K-12 YK2004 at the Institute of Biotechnology, Institute of Biotechnology, Institute of Industrial Science and Technology, 1-3-1 Higashi, Tsukuba, Ibaraki Prefecture, September 7, 1984. Deposited by date: Deposited under Microorganism Research Institute No. 7838 (February 1, 1988)
Transferred to international deposit on 5th; deposit number: FERM BP-
1732).

Reference Example 2 Stability of Transformant Strain 100 ml of the above-mentioned minimal medium was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, and the transformant obtained in Reference Example 1 was planted. After incubating and shaking culture at 37 ° C. for 24 hours, L medium 10 prepared in the same manner
0 ml was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, and subcultured at a rate of 50 cells per ml, and shaking culture was also carried out at 37 ° C. for 24 hours. Then, the cells were collected by centrifugation and the cells were washed, and a fixed amount was spread on an L medium supplemented with ampicillin at a rate of 50 μg / ml and a plate medium prepared as an L medium without addition, and the mixture was incubated at 37 ° C. After culturing for 1 day, the number of growing colonies is counted.

As a result, it was confirmed that the number of colonies grown on the medium with and without addition of ampicillin was the same, and that all the L-medium-grown colonies grew on the minimal medium containing no tryptophan, that is, the high stability of the plasmid was confirmed. did.

Example 1 Production of L-Tryptophan 100 ml of a minimal medium was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, and the transformant Escherichia coli K-12 YK2004 was inoculated. 3
L culture medium 5 prepared in the same manner after shaking culture at 7 ° C for 1 day
Dispense 00 ml into a 5-liter Erlenmeyer flask and
Sterilized for 5 minutes, inoculate 10 ml to 37
The cells were cultured with shaking at 5 ° C for 5 hours. The cells were collected by centrifugation, and 25 g of indole and 100 g of DL-serine were collected.
And pyridoxal phosphate 5 mg, 100 mM
The mixture was suspended in 500 ml of Tris buffer (pH 7.8) and reacted at 37 ° C. for 24 hours while shaking. After the reaction,
After 10 ml of the liquid was added to 10 ml of methanol and vigorously stirred, the supernatant obtained by centrifugation was analyzed for L-tryptophan produced by high performance liquid chromatography. As a result, 22 mg / ml was found to be produced.

An aqueous solution of caustic soda was added to 500 ml of the reaction-terminated liquid to adjust the pH to 10, and crude crystals of L-tryptophan were precipitated through a column of an ammonia-type strongly acidic ion exchange resin (Diaion SK-IB, manufactured by Mitsubishi Kasei). This was washed with acetone and dried to obtain 7.7 g of L-tryptophan crystals.

Example 2 100 ml of the minimal medium was dispensed into a 500 ml Erlenmeyer flask and sterilized at 120 ° C. for 15 minutes, and the transformant Escherichia coli K12 YK2004 was inoculated into the medium.
After incubating at 7 ° C for 1 day with shaking, 10% indole acrylic acid was added.
Similarly to Example 1, 10 ml of the preculture was inoculated into 500 ml of the L medium containing 0 μg / ml, and the mixture was cultured at 37 ° C. for 5 hours with shaking. After the cells were collected by centrifugation, the reaction was carried out in the same manner as in Example 1 to produce L
-Tryptophan analysis showed 40 mg /
Production of ml was observed.

L-tryptophan was purified and recovered in the same manner as in Example 1 from 500 ml of the reaction-finished solution.
As crystals, 13.6 g of L-tryptophan was obtained.

[Brief description of drawings]

FIG. 1 shows the plasmid pMTY-2 used in the present invention.
Is a restriction enzyme map of.

Continuation of front page (51) Int.Cl. ⁵ Identification number Office reference number FI technical display location C12P 13/22 A 8931-4B // (C12N 1/21 C12R 1:19) (C12N 15/11 C12R 1: 19) (C12N 15/60 C12R 1:19) (C12P 13/22 C12R 1:19) (72) Inventor Hideaki Yukawa 576-81 Wakaguri, Ami-cho, Inashiki-gun, Ibaraki

Claims (1)

[Claims] 1. A DN containing a gene that controls the biosynthesis of tryptophan synthase derived from tryptophan operon.
Cultivation of Escherichia coli transformed with a plasmid having an A fragment, a DNA fragment containing a gene controlling the growth control distribution system derived from the F factor plasmid, and a DNA fragment containing a gene controlling the autonomous growth ability of the ColE1 plasmid A process for producing L-tryptophan, which comprises reacting indole with L- or DL-serine in the presence of a product or a treated product thereof.