JPS61247394A - Production of l-tryptophan - Google Patents

Abstract

PURPOSE:To produce tryptophan synthase, by reacting indole with L-serine, etc., in the presence of a culture of a microorganism, transformed with a specific plasmid and belonging to the genus Escherichia. CONSTITUTION:A microorganism transformed with a plasmid containing a DNA fragment containing a gene governing the biosynthesis of tryptophan synthase, DNA fragement containing a gene governing the multiplication control system derived from F factor and DNA fragment containing a gene governing the autonomous growth ability derived from Co1E1 based plasmid is obtained. Indole is reacted with L- or DL-serine in the presence of the culture of the above-mentioned microorganism or a treated material thereof to produce the aimed L-tryptophan.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing Z-)ligt7a/, and more specifically, the present invention relates to a method for producing Z-)ligt7a/, and more specifically, it uses a microorganism that produces tryptophan synthase at high speed and high efficiency, or a processed product thereof to produce indole and L- Alternatively, it is a method for producing L-)ligtofa/ from DL-serine.

Hitherto, several methods have been proposed for producing L-tryptophan from indole and L- or DL-serine by an enzymatic method using tryptophan synthase-producing microorganisms or their processed products (for example, JP-A-57 -87
92, JP-A-58-16692, etc.).

However, the microorganisms used in these conventionally proposed methods have low productivity of tryptophan synthase, which increases the production cost of L-)ridetophan.
At present, it is mainly used as a raw material for high value-added pharmaceuticals (
The drawback is that it can only be used as an infusion (for example, for infusion).

Therefore, the present inventors conducted intensive research in search of microorganisms with high production efficiency of tryptophan synthase, and as a result,
Contains a DNA fragment that contains a gene that controls the biosynthesis of tryptophan synthase, a DNA fragment that contains a gene that controls the growth control distribution system derived from the F factor plasmid, and a DNA fragment that contains the gene that controls the ability to autonomously reproduce from a Co1El system plasmid. A microorganism belonging to the genus Escherichia that has been transformed with a plasmid of
The above-mentioned microorganism belonging to the genus Escherichia is a DNA fragment having a promoter function capable of expressing trpA and trpB and both genes, and a DNA fragment having an operator function capable of regulating the expression of this promoter function by a trigtophan regressor. They discovered that tryptophan synthase is produced in significant amounts within bacterial cells, and have completed the present invention.

Therefore, according to the present invention, a DNA fragment containing a gene governing the biosynthesis of tryptophan synthase and a DNA fragment containing a gene governing the growth control distribution system derived from the F factor plasmid are combined.
indole and L in the presence of a culture of a microorganism belonging to the genus Escherichia transformed with a plasmid containing the DNA fragment and a DNA fragment containing the gene controlling ColE1 type GraN11+ -regulated reproduction ability, or a processed product thereof.
- or DL-serine is provided.

"DNA containing the gene governing the biosynthesis of trypto-7 ansynthase" constituting the plasmid used in the present invention
"A fragment" (hereinafter sometimes abbreviated as "T fragment") means a DNA fragment containing a gene that controls the biosynthesis of L-tryptophan from indole and L- or DL-serine, and the T fragment used in the present invention The fragments include, in particular, DNA fragments (hereinafter referred to as "tryptophan promoters") that have promoter functions that can express trpA and trpB, which are genes in the tryptophan operon that control the biosynthesis of tryptophan 7-ansynthase. DNA fragment containing a DNA fragment (hereinafter sometimes referred to as a "tryptophan operator") that has an operator function that can restrict the promoter function as necessary.
NA fragments are included. As such a T fragment, one derived from Escherichia coli is preferably used practically. The source of this T fragment is not particularly limited as long as it has a tryptophan operon, but Escherichia coli AT'CC
23282, Escherichia coli ATCC23431,
Escherichia coli ATCC23461, IFO330
1, ATCC10798, ATCCa23562, etc. are advantageously used.

A detailed method for preparing DNA fragments in which the trpA and trpD fragments are combined with the tryptophan promoter and tryptophan operator suitable for the purpose of the present invention from these source microorganisms is shown in Reference Example 1 (B) below.
The basic procedure is to infect Escherichia coli, which has a tryptophan operon in its chromosomal gene, with 7age φ80, induce the infection, and prepare a large amount of 7age that incorporates the tryptophan operon into the phage DNA. Next, 7Age D
Extract the NA, excise the tryptophan operon DNA fragment using restriction enzymes BamHI, EcoRI, etc., and further partially cleave this DNA fragment with the restriction enzyme Hi fLc II to obtain a DNA containing the trpA and trpB genes.
A fragment is obtained. Also, trypto7a/operon DN
When the A fragment is treated with the restriction enzyme PτwM, a DNA fragment containing the promoter and operator is obtained. By linking both of the resulting DNA fragments with a DNA ligase, a DNA fragment in which trpA and trpB fragments are linked to a tryptophan promoter and a tryptophan operator is obtained.
NA fragments can be prepared.

One feature of the plasmid used in the present invention is that the T fragment described above is combined with a DNA fragment containing a gene that controls the growth control distribution system of a plasmid derived from the F factor plasmid. For example, the F factor plasmid is shown in the genetic map of FIG. 1 on page 98 of "Proteins, Nucleic Acids, and Enzymes", Vol. 27, No. 1 (1982), and Ec.

It is a known plasmid with a molecular weight of 94.5kb (62 The plasmid is present in a large number of copies, and the plasmid has a mechanism for precise transmission into each daughter cell after cell division. The mechanism of increasing at a pace is called stringent growth control). This st-ringat>t growth control function in the F factor plasmid is caused by rnin.
It has already been determined that i-F, which has a molecular weight of 9, is carried by a fragment of Fk b that can autonomously reproduce.
It is also known that it can be cut out by I.

However, a "DNA fragment containing a gene controlling the proliferation control distribution system derived from the F factor plasmid" (hereinafter sometimes abbreviated as "F fragment") is a DNA fragment that is used to accurately transfer the F factor plasmid to daughter cells as described above. A typical example of such an F fragment is approximately 9. l
The m1ni-F fragment with a molecular weight of k b is mentioned.

Furthermore, the "DNA fragment containing the gene responsible for autonomous replication derived from the C0tEl-based plasmid" (hereinafter sometimes abbreviated as "S fragment") used in combination with the above T fragment and F fragment has a copy number of 1. It refers to a DNA fragment containing a gene that controls the autonomous replication of Co1El-based plasmids, which have 20 to 30 cell chromosomes, and a typical example of this S fragment is plasmid pBR3, which has a length of about 4.3 kb.
22-derived S fragment, as well as plasmid p
There is an S fragment derived from BR325 etc.

The plasmid used in the present invention is limited to having the above-mentioned three essential DNA fragments, T fragment, F fragment, and S fragment, as well as DNA fragments carrying other genetic information, such as ampicillin, which is an antibiotic resistance marker. Although it may further include a DNA fragment containing a resistance gene, a DNA fragment containing a kanamycin resistance gene, etc., one typical example consists essentially of three DNA fragments: a T fragment, an F fragment, and an S fragment, and has a molecular weight of Approximately 9.0 megadalt/(approximately 13.6kb
tT
It was named "Y-3". In this specification, the molecular weight of a plasmid is a value measured by agarose gel electrophoresis.

The sensitivity (number of recognition sites) of this plasmid pMTY-3 to the following restriction enzymes and the length (kb) of fragments degraded by the restriction enzymes are shown in the table below.

EC0RI 2 1G, 7. λ9Bamlll
2 6.9. a7SL0Ltl 3
LQ, 6. λ9. Q, IPstl 5
5.9,40. L8. L5. α4Aval
13.6 Plasmid pMTY-3 having the characteristics described above can be produced, for example, as follows.

First, a DNA fragment (T fragment) in which a tryptophan promoter and a tryptonoa/operator are linked to trpA and trpE fragments is prepared using, for example, Escherichia coli that has a tryptophan operon in its chromosomal gene, such as E.
schtrichia coli K-12 (IFO3
301, ATCC10798, ATCC523562)
For example, phage φ80 (ATCC1
1456α-B1) and utilized the lysogenization and induction phenomena to prepare a large amount of phage that had incorporated the tryptophan operon into the phage DNA [R. M. Denney, C. Yanofsky; Journal Op.
Bacteriology (R,M.

Dain#y, C, Yanofsky; J, B
actgriol,).

11B1.505 (1974)], then the conventional method [E.F.

Sambroolc;
oning”) (19a2) p, to4-16
5. Cold Spring Harbor Laboratory (
Co1d Spring Harbor Laborat
Extract the 77-di DNA according to the method (see [Reference)] and use a restriction enzyme, such as BatnHI.

Tryptophan operon DNA using p:coRI etc.
Excise the fragment and further treat this DNA fragment with restriction enzyme Hi.
Partial cleavage with nal yields a DNA fragment containing the trpA and trpB genes, and treatment of the tryptophan operon DNA fragment with the restriction enzyme pvxT1 yields a DNA fragment containing the promoter and operator. Next, both of the obtained DNA fragments were combined with T14 phage-derived T.
4 Combine with DNA ligase and further E clo RI
When linkers are mixed and ligated using T4 DNA ligase, a DNA fragment containing a promoter and an operator is attached to the trpA and trpB fragments, and Ec is attached to both ends.

A T fragment with an R1 site is obtained.

On the other hand, the m1ni-F fragment can be prepared using microorganisms carrying the F factor plasmid, such as Escherichia coli (E. coli).
b) x-t 2 strains (ATCC15153, ATCC62
3589, ATCC 123590), etc. ← by a method known per se, for example, B.
Bacteriology CP.

Osgry, D.L., LeBlanc,
S, P”alkow; J.

Bact, 116 1064 (1973), etc. 1) Remove the F factor plasmid by the method described in the literature, and then use the restriction enzyme EaoRI to reduce the molecular weight to approximately 9.1k.
It can be prepared by cutting out the m1ni-F fragment of b.

On the other hand, it is convenient to use plasmid pBR322, which is a typical CQIEt plasmid, as a source of a DNA fragment containing a gene that controls the autonomous replication of the C0IE1 plasmid.

The 9m1ni-F fragment prepared as described above was treated with restriction enzymes EcoRI and BainagaHl, combined with plasmid pBR322 treated with the same restriction enzymes, and treated with T4 DNA ligase to transform plasmid pBR322 into plasmid pBR322.
A plasmid incorporating the nitt-F fragment is created. This plasmid was then cleaved with p:coRI,
Combined with the T fragment prepared as above, T4D
The target plasmid pMTY-3 can be obtained by binding the plasmids using NAI) gauze.

A specific method for preparing plasmid pMTY-3 will be described in more detail in Reference Example 1 below.

The plasmid pMT Y-3 prepared in this way has a large number of copies and has the excellent properties of being stable and less likely to be shed when passed on to daughter cells during cell division of the host.

Examples of microorganisms (hosts) belonging to the genus Escherichia that can be transformed with the above-mentioned plasmids include Escherichia coli (Escherichia coli αcoli).
Examples include K-12 type microorganisms, and among them, strains that do not have L-tryptophan degrading activity (that is, trigtophanase-deficient mutant strains) are preferred.

The above-mentioned plasmids can be introduced into these host microorganisms using methods known per se, such as M, λ (andet).

A, Bigα; J, Mol, BioL, 5
It can be carried out by the method described in literature such as Yu, 159 (1970).

The host microorganism transformed in this way is cultured by a method known per se to sufficiently produce and accumulate triglyto-7a/synthase within the microbial cell, and then indole
and L- or DL-serine to produce L-) IJ gutophane.

Cultivation can be carried out by a method known per se; for example, a natural or synthetic medium containing carbon sources, nitrogen sources, inorganic salts, etc. similar to those used for the cultivation of ordinary microorganisms can be used. .

Thus, various carbohydrates can be used as carbon sources, such as glucose, glycerol, fructose, sucrose, molasses, etc., and as nitrogen sources, for example tryptone, yeast extract, corn steep liquor, casein hydrolyzate, etc. Natural organic nitrogen sources such as nitrogen can be used. Many natural organic nitrogen sources can be used as carbon sources as well as nitrogen sources.

Examples of inorganic salts include potassium hydrogen phosphate,
Potassium dihydrogen acid, magnesium sulfate, sodium chloride, ferrous sulfate, calcium chloride, manganese chloride, etc. can be used.

In addition, other nutrients useful for the growth of microorganisms (eg, vitamins, amino acids, etc.) may be added to the medium.

Furthermore, it has been found that indole acrylic acid or its salt can be added to the culture medium, which can promote the growth of cultured microorganisms and significantly improve the production amount of tryptophan synthase. . Indole acrylic acid or its salt may be added at the beginning of the culture, or may be added during the culture. If added midway through, it is desirable to add at the latest the end of the logarithmic growth phase of the microorganism. The addition may be made at once, or may be made in multiple portions or continuously.

The amount of indole acrylic acid or its salt added is not strictly limited, but in general, it can be added to the medium to a final concentration of at least 25 μl, preferably 80 to 400 μl. It is advantageous to add to a final concentration in the range, more preferably in the range from 100 to 2 ooμg/-.

In addition, examples of the salt of indole acrylic acid include alkali metal salts of indole acrylic acid such as sodium indole acrylate.

Here, the "final concentration" refers to the total concentration of indoleacrylic acid or its salt added to the medium by the end of culture.

Furthermore, in the method of the present invention, it is desirable to use glucose as at least a part of the carbon source, and the amount used is generally about
10 to 600 times the mole, preferably 50 to 400 times the mole,
More preferably, a range of 100 to 300 times the mole is appropriate.

The culture time varies depending on the type of microorganism, other culture conditions, etc., but when culturing is carried out by adding indole acrylic acid or its salt at the beginning of culture, it is usually about 24 to about 120 hours.
In addition, when indole acrylic acid or its salt is added during the culture and culture is carried out, the time can be generally about 1 to about 48 hours.

Further, the culture temperature can generally be in the range of about 20 to about 50°C, and the pH of the culture medium is generally adjusted to the range of 5 to 9, preferably in the range of about 6 to about 8.

Furthermore, the culture is preferably carried out under aerobic conditions such as shaking or aeration.

When using cultured microorganism η cells for the enzyme reaction,
The bacterial cells can be used as they are, but they can also be used as a crushed product obtained by crushing the bacterial cells by ultrasonication, an extract obtained by further extracting the crushed material with water, or the extract further extracted with ammonium sulfate, etc. It can also be used in the form of a crudely purified product obtained by treatment to precipitate the enzyme component, and furthermore, the cells or the treated product can also be used after being immobilized if necessary.

The reaction between indole and L- or DL-serine in the presence of the microbial cells or their processed material can be carried out in the same way as a normal enzymatic reaction, e.g.
Alternatively, it is carried out in a solvent such as water (pH 7.0 to 9.0) at a temperature of about 20 to about 50°C, preferably about 30 to about 40°C, for usually about 10 to about 72 hours.

There is no particular limit to the amount of indole and L- or DL-serine used in the reaction, but generally each is used at 0, F to 20
It is suitable to use a concentration range of % (wt/vol). In addition, the amount of the bacterial cells or their processed material to be used is not particularly limited, but is generally 1 to 10% (
wt/τoj).

Separation and purification of L-tricytophan from the reaction solution after the reaction can be carried out by methods known per se, such as adsorption and desorption treatment using ion exchange resins, activated carbon, etc.

According to the method of the present invention described above, a DNA fragment containing a gene governing the biosynthesis of tryptophan synthase and a D
In the presence of a culture of a microorganism belonging to the genus Escherichia transformed with a plasmid, for example, plasmid pMTY-3, having an NA fragment and a DNA fragment containing a gene governing the autonomous replication ability of a Co IEI-based plasmid, or a method for treating the same, By reacting indole with L- or DL-serine, L
- When producing tricytophan, the production rate of tryptophan can be significantly increased, which is extremely advantageous industrially.

Next, the method of the present invention will be explained in more detail with reference to Examples.

Reference Example 1 Construction of niblasmid pMTY-3 (A) Preparation of phage φ80pt Escherichia coli (E. coli) f-12 strain (IFO330
1) IQQ-L medium (Bacto) Rizoton 1
011 yeast extract 5Jil, NaCl 51I, glucose 1111 water 11; pB7.2) and incubated at 37°C.
0.2- of the culture shaken for about 4 hours and phage φ8
0(,47'C('11456α-B1) aqueous solution (10
After mixing 0 and F- in L medium soft agar (L medium + agar droplet), the mixture is layered on an L medium agar plate. When the plate is cultured at 37° C. for about 5 hours, plaques (lytic plaques) are formed, and when the culture is continued at 37° C. for an additional 2 to 3 days, growing colonies of 7-age φ80 lysogenic bacteria are formed in the plaques. After culturing the lysogenic bacteria in L medium at 37°C for 4 hours, they were plated on the same L medium agar plate as above, and then irradiated with ultraviolet light (400 to 800 #days/w"
, 10-20 seconds) by induction of lysogen 7 age)
Prepare Φ80pt (7age DNA containing tricytophane operon).

(B) trpA and tdelB fragments combined with tryptophan promoter and operator DNA fragments
Preparation of fragments Escherichia coli (E, coLi) strain f-12 (110330
1) was inoculated into 1 liter of L medium (composition is the same as above), cultured with shaking at about 37°C for about 3 hours, and in the logarithmic growth phase 2 slp<
w/ν) Add 10-glucose solution and the phage φ80pt solution prepared above at a concentration of 10ml/-. After continuing shaking for 5 hours, add cycloform in the usual manner and add the phage φ80pt solution. Prepared in large quantities [T. Maniatis, E.F.
1atis.

E, F, Frltsch, Sambrook;
"Molecular Cloning") (19
82) p, 76-80. =x-Led°・Gooseberry/G・Harle(-・Laboratory-(Cold Spring)
Harbor Laboratory) Reference]
.

Next, the obtained phage φ80pt solution was added to Tris buffer (
After dialysis at pH 7, 8), DNA was extracted using the phenol method.
Extraction method [Above “Molecular Cloning” (Mo
1ecular Craning”) p.

The phage DNA was extracted and purified according to [Reference 85], and the restriction enzyme BamHI was added thereto and reacted at 30°C for 30 minutes to obtain a tryptophan operon DNA fragment.

Next, this tryptophan operon DNA fragment of 10 μm
was partially treated with the restriction enzyme HincN at 37°C for 5 minutes to obtain a 2 μ9 DNA fragment containing the trpA and trpB genes.
was prepared. In addition, 20 μy of the trypto7 anoberon DNA fragment was treated with the restriction enzyme pvwU at 37°C for 1 hour to generate a DNA fragment containing the promoter and operator gene region.
4 μl of NA fragment α was prepared. Both of the obtained DNA fragments were mixed and linked by reaction at 12°C for 24 hours using T4 DNA ligase, and then E co RI linker 0,
Mix 5 μl and incubate for 2 hours at 12°C using T4 DNA ligase.
The mixture was reacted for 4 hours and recombined. Furthermore, this recombinant was treated with the restriction enzyme EcoRI at 37°C for 1 hour, and the tr
A T fragment was prepared in which the promoter and operator DNA fragments were linked to the pA and trpB fragments, and the readhesive end was the EaoR1 site.

(C) Construction of plasmid pMTY-3 Plasmid pM
To construct TY-3, as shown in the attached FIG. 1, 7) ER322-mtniFf lasmid is first constructed.

The m1nt-F fragment was prepared using conventional methods [Mukai T, Matsupara
K, Takagi, Y; Proceedings of the National Academy of Sciences x (Mxka
i, T., Matsxbara, K., Takags,
Y; proc, Natl, Aaad, Sc
i,) U, S, A, 7 0 2884 (
1973)], and this m1fLi-F
A 1 μm fragment is treated with restriction enzyme f3amEI at 37° C. for 1 hour. On the other hand, restriction enzymes EcoRI and BamHI are allowed to act on 2 pl of separately prepared plasmid pBR322 DNA of Amp"<ampicillin resistance) and Tc (tetraticline resistance) at 37°C for 1 hour. The post-treated products are mixed, and T4 DNA ligase is added for 12 hours. The DNA was allowed to react for 24 hours at ℃ to recombine.The recombined DNA was transformed into Escherichia coli strain C600 (ATCC623738).1.
Amp" and Tcs (tetracycline sensitive) were selected. Plasmid DNA was extracted from these strains.
was separated and purified, and its molecular weight was measured by agarose dal electrophoresis, and the result was approximately 1 a7 k bopBR322-m.
The 1x iF plasmid was collected.

Next, as shown in Figure 2, p 73 R322-rrL
(n SF plasmid and the T fragment prepared in <E) above are ligated to construct pMTY-3.

That is, pBR322-m1niF7” lasmid DN
After treating A2μm with restriction enzyme Eco'RI at 37°C for 1 hour, trpA and trpA prepared in (B) above were added.
1 μl of a DNA fragment in which the promoter and operator DNA fragments were bound to the pB fragment was mixed, and T4 DNA ligase was applied at 12° C. for 24 hours to cause religation. The recombined DNA was used to transform Escherichia coli strain 112 (tryptophan auxotrophic mutant, ATCC23'11B) according to a conventional method, and the transformed strain (loss of Trp auxotrophy; that is, trpA and Citryptophan biosynthesis is possible by the tτpB gene, and minimal medium (
K, HPO47#XKH, P0.2 F, Mr;ts
O4・7H200,11% (NH4)! S041
, L glucose 211 and a strain capable of growing on pure water 11). This strain was cultured in liquid according to a conventional method, and plasmid pMTY-3 having the restriction enzyme map shown in FIG. 2 was isolated and purified from the culture solution.

The transformed strain carrying this plasmid pMTY-3 is
As Escherichia Co') K12 YKzoa9, it was sent to the Institute of Microbial Technology, Agency of Industrial Science and Technology, Higashi 1-1-3, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, on November 27, 1980, with accession number: Microtechnology Research Institute No. 7957. (FERN P-'!9
5T).

Example 1 A minimal medium of 501 rIt having the following composition was dispensed into a 500ml Erlenmeyer flask and sterilized at 120°C for 15 minutes.
12YK2009 (FERNP-7957) was inoculated,
After culturing with shaking at 37°C for 1 day, L medium 50- of the above composition prepared in the same manner was dispensed into a 500-capacity Erlenmeyer flask,
The cells were sterilized at 120°C for 15 minutes, inoculated with 2 Wtt, and cultured with shaking at 37°C for 5 hours.

In the composition of the minimal medium, HPO47, OI! xn, po, Z Oy (NH,)! 50. L Ot9MQSO,・
7H,00,I 11 Glucose zOg Water 11 Cells were collected using centrifugation without pH adjustment and transported to India.
51. 100 m&)lith buffer (containing DL-serine xQ, O9k and pyridoxal phosphate o, s my)
pH 7,8) Suspended at 50-37℃ with shaking for 15 minutes.
A time reaction was performed. After the reaction was completed, the reaction product was diluted 10 times with water, and the supernatant obtained by centrifugation was analyzed by high performance liquid chromatography for L-)lipidan, which was found to be 3.51 NI/-. The formation of was observed.

After the reaction, 500 g of the 10-fold diluted solution of 50'-
After adsorbing L-tryptophan through a column (manufactured by Mitsubishi Kasei), it was eluted with an alkaline solution and concentrated to precipitate crude crystals of L-)ridetofa/. This was washed with acetone and dried to obtain 1.2 g of L-tryptophan crystals.

Example 2 50W1t of minimal medium was dispensed into a 500-volume Erlenmeyer flask and sterilized at 120°C for 15 minutes to transform the transformed strain Escherichia coli A'-12YK2009 (FERN
-PI 957) and cultured with shaking at 37°C for 1 day, preculture 2- was inoculated in the same manner as in Example 1 into 50 tnt of L medium containing indole acrylic acid at a concentration of 100 μi/1 Plt. , and cultured with shaking at 37°C for 5 hours. After the bacterial cells were collected by centrifugation, a reaction was carried out in the same manner as in Example 1. After the reaction was completed, the produced L-) lipdoan was analyzed in the same manner as in Example 1. Generation of aO+y/- was observed.

Using the same procedure as in Example 1, from the reaction-completed solution 50-
) When Lipdoan was purified and recovered, zI was found as crystals.
Z of I, -) Lipdoan was obtained.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the construction process of plasmid 7)BF222-rrttsiF, and FIG. 2 is a diagram showing the construction process of plasmid pMTY-3.

Claims (1)

[Scope of Claims] 1. A DNA fragment containing a gene governing the biosynthesis of tryptophan synthase, a DNA fragment containing a gene governing the growth control distribution system derived from the F factor plasmid, and a DNA fragment containing the gene governing the autonomous proliferation ability derived from the ColE1-based plasmid. L characterized by reacting indole with L- or DL-serine in the presence of a culture of a microorganism belonging to the genus Escherichia transformed with a plasmid containing a DNA fragment containing a gene or a processed product thereof. - A method for producing tryptophan. 2. The method according to claim 1, wherein the DNA fragment containing the gene responsible for the biosynthesis of tryptophan synthase contains at least trpA and trpB and a DNA fragment having a promoter function capable of expressing both of these genes. 3. The DNA fragment containing the gene governing the biosynthesis of tryptophan synthase contains at least trpA and trpB, a DNA fragment having a promoter function capable of expressing both genes, and a DNA fragment having an operator function capable of controlling the promoter function. The method according to claim 2. 4. The method according to any one of claims 1 to 3, wherein the DNA fragment containing the gene governing the biosynthesis of tryptophan synthase is derived from Escherichia coli. 5. The method according to any one of claims 1 to 4, wherein the DNA fragment containing the gene governing the growth control distribution system derived from the F factor plasmid is a mini-F fragment. 6. The method according to any one of claims 1 to 5, wherein the DNA fragment containing the gene controlling autonomous replication ability derived from the ColE1-based plasmid is derived from plasmid pBR322. 7. The plasmid has a molecular weight of about 9.0 megadaltons and contains the restriction enzymes EcoRI, BamHI and salI.
7. The method according to any one of claims 1 to 6, wherein the plasmid pMTY-3 has two cleavage sites, two cleavage sites, and three cleavage sites, respectively. 8. A microorganism belonging to the genus Escherichia has a plasmid p-
E. coli K-1 transformed with MTY-3
8. The method according to claim 7, wherein the microorganism is a 2-system microorganism. 9. The method according to claim 8, wherein the Escherichia coli K-12 microorganism is a tryptophan auxotrophic mutant. 10. The method according to claim 8 or 9, wherein the Escherichia coli K-12 microorganism is a strain that does not exhibit L-tryptophan degrading activity. 11. The method according to any one of claims 1 to 10, wherein the culture is obtained by culturing the microorganism in a medium to which indole acrylic acid or a salt thereof is added. 12. At least 25 μg of indole acrylic acid or its salt by the end of the logarithmic growth phase of the microorganism at the latest.
12. The method according to claim 11, wherein the addition is carried out to give a final concentration of /ml.