JP2678995B2 - Method for producing tryptophan synthase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing tryptophan synthase,
More specifically, it contains the tryptophan synthase gene.
A coryneform bacterium transformed with a plasmid comprising a DNA region, a DNA region containing a promoter and an operator capable of controlling the expression of the gene, and a DNA region having an autonomous growth ability and a stabilizing function in the coryneform bacterium. It relates to a method for producing tryptophan synthase by culturing in a medium.

Coryneform bacteria including Brevibacterium are industrially useful microorganisms that produce amino acids, organic acids, purine nucleotides, etc., but breeding improvement of strains by the introduction of recombinant DNA technology is performed by Escherichia coli, etc. However, there is a strong demand for the development of an industrially useful vector having excellent stability, which uses a coryneform bacterium as a host.

Generally, regarding the stability of the constructed plasmid in the host,
Heretofore, various genetic instability such as loss of plasmid from host and lack of inserted gene during culturing has been reported, and countermeasures against it have been investigated.

For example, a method of stabilizing a property of a microorganism containing a plasmid by incorporating a plasmid into which a chromosomal gene DNA fragment encoding a property that does not depend on streptomycin of the genus Escherichia is incorporated into a streptomycin-dependent mutant strain of the genus Escherichia Has been proposed (see 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.

In addition, various studies have been conducted so far on cloning of a DNA region containing a gene controlling the biosynthesis of tryptophan synthase, but a vector plasmid into which a DNA region containing a gene controlling the biosynthesis of tryptophan synthase is introduced. Is, for example, the Journal of General Microbi
As described in ology vol118, p253 (1980),
There are problems with stability in the host, and it is thought that there are many difficulties in applying it to the industrial production of tryptophan.

The present inventors previously discovered that a DNA fragment having a stabilizing function was present on a plasmid pBY503 derived from Brevibacterium statinis IFO12144 capable of replicating and propagating in a coryneform bacterium, and the DNA fragment was unstable. We have proposed a method for stabilization by binding to various plasmid vectors (Japanese Patent Application No. 185428/1988).

Therefore, the present inventors have used a DNA fragment responsible for the stabilizing function of the above plasmid, to stabilize the plasmid responsible for the tryptophan synthase gene, the results of examining a method for producing a stable and large amount of tryptophan synthase The present invention has been completed.

Thus, according to the present invention, a coryneform bacterium having the ability to produce tryptophan synthase, in particular, a DNA containing a gene controlling at least the biosynthesis of tryptophan synthase.
Region, and a coryneform bacterium transformed with a plasmid comprising a DNA region having an autonomous growth ability and a stabilizing function in the coryneform bacterium, cultivated in a medium, and collecting tryptophan synthase from the culture. A method for producing a characteristic tryptophan synthase is provided.

"A DNA region containing a gene controlling the biosynthesis of tryptophan synthase" constituting the above-mentioned plasmid according to the present invention
(Hereinafter, the term "T region" may be abbreviated to include a region containing a gene controlling the biosynthesis of L-tryptophan from indole and L- or DL-serine.
The region includes, for example, a DNA region (a) containing the tryptophan synthase gene in the tryptophan operon, that is, a DNA region containing trpA and trpB, and a promoter capable of controlling the expression of the gene derived from the tryptophan operon and An example is one in which the DNA region (b) containing the operator is bound. Practically, those derived from Escherichia coli are preferably used as these T regions, and the source of this T region is, for example, Escherichia coli ATCC23282, Escherichia coli ATCC2343.
7, Escherichia coli ATCC23461 etc. are advantageously used.

The basic procedure for preparing the T region from these source microorganisms is to infect a chromosomal gene with phage φ80 and then induce it to prepare a large amount of the phage in which the tryptophan operon is incorporated into the phage DNA. Next, the phage DNA is extracted, the tryptophan operon DNA fragment is cut out using restriction enzymes BamH I, EcoR I, etc., and this DNA fragment is further digested with restriction enzyme Hinc II to obtain trpA
And a DNA fragment containing the trpB gene is obtained. Next, a Sa1 I linker was added to the DNA fragment, inserted into the Sa1 I site of plasmid pDR720 (Falmatia), cut out with EcoR I, and then EcoR I of plasmid PUC9 (Falmatia).
After inserting into the site, excision with BamHI
A DNA fragment in which trpB is bound to a tryptophan promoter and an operator, that is, a T region can be prepared.

On the other hand, "DNA having a stabilizing function in coryneform bacteria
The “region” (hereinafter sometimes abbreviated as “S region”) includes
A region having a function of stabilizing a plasmid capable of replicative growth in a coryneform bacterium is included, and examples thereof include Brevibacterium stationis.
Plasmid pBY carried by IFO12144 (FERM P-10136)
503 (For details of this plasmid, see Japanese Patent Application No. 62-25379).
(See the specification of the Japanese application), and a DNA fragment having a size of about 7.4 kb obtained by cleaving it with a restriction enzyme such as Kpn I can be mentioned. The molecular weights of the DNA fragment obtained by digestion with various restriction enzymes are shown in Table 1 below.

A vector plasmid for incorporating the above-described DNA region (T region) containing a gene that controls the biosynthesis of tryptophan synthase and a DNA region (S region) having a stabilizing function in coryneform bacteria , There is no particular limitation as long as it can be replicated and propagated in a coryneform sterilization, and examples thereof include the following. As a typical example, a plasmid pCRY disclosed in Japanese Patent Application No. 63-193659 is disclosed.
-2, pCRY-3, etc. can be illustrated.

(1) pAM330 Refer to JP-A-58-67699 (2) pAM1519 Refer to JP-A-58-77895 (3) pAJ655 Refer to JP-A-58-192900 (4) pAJ611 Same as above (5) pAJ18244 Same as above ( 6) pCG1 Refer to JP-A-57-134500 (7) pCG2 Refer to JP-A-58-35197 (8) pCG4 Refer to JP-A-47-183799 (9) pCG11 Same as above (10) pCRY-2 Special See Japanese Patent Application No. 63-13659 (11) pCRY-3 Id. As a method for constructing the above plasmid vectors pCRY- and pCRY-3, Brevibacterium flavum MJ233
(FERM P-3068) and Brevibacterium statinis IFO12144 (FERM P-10136) plasmid pBY50.
2 and pBY5039 DNA were extracted and the restriction enzymes Hind III or Kpn I
The treatment gives 4.1 kb and 6 kb DAN fragments. Next, the DNA fragment and plasmid pHSG398 (manufactured by Takara Shuzo Co., Ltd.) were ligated with the same restriction enzyme to obtain pCRY-2 and pCRY.
RY-3 can be produced (see Japanese Patent Application No. 63-13659).

Introduction of "T region" and "S region" into such vector plasmid is carried out by, for example, first introducing vector plasmid pCRY-
3 is partially cleaved with the restriction enzyme Kpn I, and p
7.4 obtained by complete digestion of BY503 DNA with Kpn I
The "S region" of kb was ligated by ligation enzyme treatment, and then the above plasmid was cleaved by complete digestion with BamHI,
It can be carried out by binding the above-mentioned "T region" thereto by a ligation enzyme treatment.

Examples of the coryneform bacterium that can be transformed with the above-described vector plasmid into which the T region and S region have been introduced include, for example, Brevibacterium flavum.
vum) strains derived from MJ233. When using this strain as a host, pBY502 possessed by this strain (see
63-36787), the transformation may be difficult.
Is desirably removed. As a method for removing such a plasmid, for example, it is possible to spontaneously delete by repeating subculture, or it is possible to artificially remove [Bact. Rev. 36 p361- 405
(1972)].

An example of the method for artificially removing the above plasmid is as follows.

The concentration of acridine orange (concentration: 0.2-) that incompletely inhibits the growth of the host Brevibacterium flavum MJ233
50 μg / ml) or ethidium bromide (concentration: 0.2-50)
(μg / ml) etc., inoculate to a medium containing about 10 cells per ml, and for about 24 hours at 35 ° C while incompletely inhibiting the growth.
Incubate with After diluting the culture solution, apply it on an agar medium and incubate at 35 ° C for about 2 days. Plasmids are extracted from each of the emerged colonies independently to select a strain from which the plasmid has been removed. By this operation, a strain derived from Brevibacterium flavum MJ233 from which pBY502 has been removed can be obtained.

As a method for transforming the plasmid into the strain derived from Brevibacterium flavum MJ233 thus obtained, as is known for Escherichia coli and Erwinia carotovora [Calvin, NM and Hanawa
lt, PC, Journal of Bacteriology, 170 , 2796 (1988); I
to, K., Nishida.T and Izaki, K, Agricultural and Biol
ogical Chemistry, 52 , 293 (1988)], and it is possible to introduce a plasmid by applying a pulse wave to a DNA recipient bacterium.

Brevibacterium flavum capable of producing tryptophan synthase obtained by transformation by the above method
The culture of the MJ233-derived strain can be carried out by a method known per se, for example, as described below.

First, as the carbon source used in the medium, for example, glucose, ethanol, methanol, molasses, etc. can be used, and as the nitrogen source, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. can be used alone or in combination.

As the inorganic salt, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition to this, nutrients such as various vitamins such as peptone, meat extract, yeast extract, corn steep liquor, casamino acid, and biotin can be added to the medium and used.

Culture is carried out under aerobic conditions such as aeration, shaking, and the like.
The cultivation temperature is 20 to 40 ° C, preferably 25 to 35 ° C. The pH during the culturing is adjusted to 5 to 10, preferably around 7 to 8. The pH during the culturing can be adjusted by adding an acid or an alkali.

The carbon source concentration at the start of culture is preferably 1 to 5% by volume, more preferably 2 to 3% by volume. Culture period is 1
~ 7 days, optimal period is 1-3 days.

The tryptophan synthase fraction can be obtained by collecting the cells from the thus obtained culture and treating them by a method known per se.

The content of the present invention has been generally described above, but it will be described more concretely by way of examples. However, the following examples are given only as an aid to gain a concrete recognition of the present invention, and the scope of the present invention is not limited thereby.

Example 1 Construction and properties of composite plasmid pCRY-3 consisting of plasmid PBY503 and plasmid pHSG398 (A) Preparation of plasmid pBY503 Plasmid pBY503 is a newly isolated plasmid from Brevibacterium statinis IFO12144FERM P-10136 with a molecular weight of about 10 mega. Dalton's plasmid, Japanese Patent Application No. 62-
The plasmid described in the specification of No. 252379 was prepared as follows.

Semi-synthetic medium A medium [urea 2g, (NH ₄ ) ₂ SO ₄ 7g, K ₂ HPO
₄ 0.5g, KH ₂ PO ₄ 0.5g, MgSO ₄ 0.5g, FeSO ₄ / 7H ₂ O 6mg, Mn
SO ₄・ 4-6H ₂ O 6mg, yeast extract 2.5g, casamino acid 5g,
Biotin 200 μg, thiamine hydrochloride 200 μg, glucose 20
g, pure water 1] 1, Brevibacterium stationis IFO12144 was cultured until the late logarithmic growth phase, and the bacterial cells were collected. The obtained cells were suspended in 20 ml of a buffer solution containing 25 mg of lysozyme [25 mM tris (hydroxymethyl) aminomethane, 10 mM EDTA, 50 mM glucose] at 37 ° C.
For 1 hour. After the reaction, alkali-SDS solution [0.2N
40 ml of NaOH, 1% (w / v) SDS] was added, mixed gently and allowed to stand at room temperature for 15 minutes. Next, to this reaction solution, potassium acetate solution [60 ml of 5M potassium acetate solution, 11.5 ml of acetic acid,
30 ml of a mixed solution of 28.5 ml of pure water was added and mixed well, and then allowed to stand in ice water for 15 minutes.

Transfer the total amount of lysate to a centrifuge tube, 15,000 x for 10 minutes at 4 ℃.
g was centrifuged to obtain a supernatant.

An equal volume of phenol-chloroform solution (phenol / chloroform 1/1 mixture solution) was added to the suspension to suspend it, and the suspension was transferred to a centrifuge tube and centrifuged at room temperature for 5 minutes at 15,000 × g to recover an aqueous layer. . Add 2 volumes of ethanol to the aqueous layer, leave at -20 ° C for 1 hour, and then at 4 ° C for 10 minutes, 15,000 xg
And the precipitate was recovered.

After drying the precipitate under reduced pressure, TE buffer [Tris 10 mM, EDTA 1 m
The pH was adjusted to 8.0 with M and HCl] and dissolved in 2 ml. To the solution, 15 ml of a cesium chloride solution [a solution of 170 g of cesium chloride dissolved in 100 ml of a TE buffer having a concentration of 5 times] and 10 mg / ml of ethidium bromide solution ml were added to adjust the density to 1.392 g / ml. This solution was centrifuged at 126,000 for 42 hours at 116,000 × g.

Plasmid pBY-503 is found as a lower band in the centrifuge tube by UV irradiation. By removing this band from the side of the centrifuge tube with a syringe, the plasmid pBY
A fraction containing -503 was obtained.

This fraction was then diluted with an equal volume of isoamyl alcohol for 4 hours.
It was treated once to extract and remove ethidium bromide, and then dialyzed against TE buffer. The dialyzing solution containing the plasmid pBY-503 thus obtained was added with a 3M sodium acetate solution to a final concentration of 30 mM, and then 2-fold amount of ethanol was added, and the mixture was allowed to stand at -20 ° C for 1 hour. 15,000 x this solution
DNA was precipitated by centrifugation of g.
50 μg of 503 was obtained.

(B) Preparation of plasmid pHSG398 The plasmid pHSG398 replicates in Escherichia coli and expresses chloramphenicol resistance with a molecular weight of about 1.4.
It is a mega dalton plasmid and can be purchased from Takara Shuzo as a commercial product.

(C) Construction of composite plasmid pCRY-3 0.5 μg of plasmid pHSG398 (Takara Shuzo) was digested with restriction enzyme Kp
The plasmid DNA was completely decomposed by reacting n I (5 units) at 37 ° C. for 1 hour.

2 μg of the plasmid pBY503 prepared in the above (A) was reacted with the restriction enzyme Kpn I (1 unit) at 37 ° C. for 30 minutes to partially decompose the plasmid DNA.

Both plasmid DNA degradation products were mixed and heat-treated at 65 ° C. for 10 minutes to inactivate the restriction enzyme, and then the components in the inactivation solution were each brought to a final concentration of 50 mM Tris buffer pH 7.
6, 10mM MgCl ₂ , 10mM dithiothreitol, 1mM ATP and T ₄ ligase Each unit was strengthened to 1unit, 16 ℃
It was kept warm for 15 hours. This solution was used to transform Escherichia coli JM109 competent cells (Takara Shuzo).

The transformants were 30 μg / ml (final concentration) chloramphenicol 4 100 μg / ml (final concentration) IPTG (isopropyl-β
-D-thiogalactopyranoside), 100 μg / ml (final concentration) X-gal (5-bromo-4-chloro-3indolyl-β-D-galactopyranoside) in 1 medium (tryptone 10 g, yeast) Extract 5g, NaCl 5g, pure water 1, pH7.2)
It was cultured at 37 ° C. for 24 hours and obtained as a growing strain. Of these growing strains, those that had grown in white colonies were selected, and the plasmids were subjected to the alkaline-SDS method [T. Maniatis,
EFFritsch, J. Sambrook, "Molecular cloning" (198
2) See p90-91].

(D) Transformation of composite plasmid into coryneform bacterium Transformation was performed using the electric pulse method. Brevibacterium flavum MJ233 (FERM P-3068) plasmid-free strain was cultivated in 100 ml of the above medium A until the early logarithmic growth phase, penicillin G was added to 1 unit / ml, and the mixture was further cultivated with shaking for 2 hours. Collect the cells by centrifugation and collect the cells in a 20 ml pulse solution (272 mM Sucrose, 7 mM).
Wash with KH ₂ PO ₄ , 1 mM MgCl ₂ : pH 7.4). The bacterial cells were collected by centrifugation and suspended in 5 ml of the pulse solution,
0.75 ml of cells and 50 ml of the DNA solution obtained in the above (C) are mixed and left standing on ice for 20 minutes. Using Gene Pulser (manufactured by Bio-Rad), it is set to 2500 V and 25 μFD, and after application of a pulse, it is allowed to stand in ice for 20 minutes. The total amount is 3m
1 of the above-mentioned A medium and after culturing at 30 ° C. for 1 hour, the cells are inoculated into the A agar medium containing 3 μg / ml (final concentration) of chloramphenicol and cultured at 30 ° C. for 2 to 3 days. From the emerged chloramphenicol resistant strain, a plasmid was obtained using the method described in the above item (A). The molecular weight of this plasmid was measured with various restriction enzymes. The results are shown in Table 2 below.

The plasmid characterized by the above restriction enzymes was designated as "pC
It was named RY-3 ".

Brevibacterium flavum MJ233GE102 transformed with the composite plasmid pCRY-3 was transferred to the Institute of Microbial Technology, Institute of Industrial Science and Technology, 1-3-1 East, Yatabe-cho, Tsukuba-gun, Ibaraki Prefecture, dated January 8, 1988. Deposited under the number: Microindustrial Research Institute No. 9802 (FERM P-9802).

Example 2 Cloning of DNA Fragment Responsible for Stabilizing Function into Plasmid pCRY-3 (a) Preparation of DNA Fragment Responsible for Stabilizing Function Plasmid pBY503 was prepared from Brevibacterium statinis IFO12144 in the section (A) of Example 1. Prepared by the method described. The restriction enzyme Kpn I was added to 20 μg of this plasmid DNA.
(20 units) was reacted at 37 ° C. for 2 hours to completely decompose the plasmid DNA. The degraded product was separated by 0.8% agarose gel electrophoresis, and a DNA fragment fraction of about 7 kb was cut out from the gel. About 5 μg of DNA extracted and purified from the fraction
I got

(B) DNA having a stabilizing function for plasmid pCRY-3
Cloning of fragment 1 μg of plasmid pCRY-3DNA obtained in Example 1 was allowed to react with restriction enzyme Kpn I (5 units) at 37 ° C. for 15 minutes to obtain plasmid D.
2μ of the DNA fragment prepared in (A) above by partially degrading NA
g, and heat treated at 65 ° C. for 10 minutes to inactivate the restriction enzyme, and the final concentration of the nodes in the inactivating solution was 50 mM Tris buffer pH 7.6, 10 mM MgCl ₂ , 10 mM dithiothreath, respectively. Each component was fortified so that it became tall, 1 mM ATP and T ₄ ligase (1 unit), and incubated at 16 ° C for 15 minutes. The solution was used to transform Escherichia coli HB101 competent cells (Takara Shuzo).

The transformant was L medium (trypton 10 g, yeast extract 5 g, N) containing 30 μg / ml (final concentration) of chloramphenicol.
The cells were cultured in aCl5g, pure water 1, pH 7.2) at 37 ° C for 24 hours to obtain a growing strain. From each of the grown strains, the plasmid was added to the alkaline-SDS method [T. Maniatis, EFFritsch, .Jsa.
mbrook, "Molecular clonig" (1982) p90-91].

(C) Transformation of plasmid into coryneform sterilization Transformation was carried out using the electric pulse method described in item (D) of Example 1. From the emerged chloramphenicol resistant strain, a plasmid was obtained using the above method. The results of measuring the molecular weight of this plasmid with various restriction enzymes are shown in Table 3 below.
Shown in

The plasmid characterized by the above restriction enzymes was designated as "pCRY-
I named it 31 ".

Example 3 Preparation of DNA Region Controlling Biosynthesis of Tryptophan Synthase Gene (A) Preparation of Phage φ80pt E. coli K-12 strain (IFO3301) was added to 100 ml of L medium (the composition is the same as above). Inoculated and shaken at 37 ° C for about 4 hours with 0.2 ml of culture and Phage φ80 (ATCC11456a-B1)
0.1 ml of the aqueous solution (10 ⁵ / ml) is mixed with L medium soft agar (L medium + agar) and then overlaid on the L medium agar plate. When the plate is cultured at 37 ° C. for about 5 hours, plaque (bacteriolytic plaque) is generated, and when the culture is continued at 37 ° C. for 2 to 3 days, a growing colony of Phage φ80 lysogen is generated in the plaque. The lysogens were mixed with L medium at 37 ° C for 4
After culturing for a period of time, spread it on the same L medium agar plate as above, and then induce lysogen pharage by UV irradiation (400 to 800 ergs / mm ² , 10 to 20 seconds) to generate Φ80pt (forge DNA containing tryptophan operon). ) Is prepared.

(B) Preparation of tryptophan operon fraction E. coli K-12 strain (IFO3301) was inoculated into 1 medium (the composition is the same as above), and cultured at 37 ° C. for about 3 hours with shaking. 10 ml of 25% (w / v) glucose solution in logarithmic growth phase
And, the Φ80pt solution was added at a concentration of 10 ¹¹ / ml (moi20), and the mixture was shaken for 5 hours, and then chloroform was added in a standard manner to prepare a large amount of Φ80pt [T.Maniatis, EFFritsch , Sambroom; “Molecula
r clonig "(1982) p.76-80 Cold Spring Harbor Labor
see atory].

Next, add the obtained φ80pt solution to Tris buffer (pH
After dialysis with 7.8), the DNA is extracted by the phenol method [see "Molecular cloning" p.85 above].
Extract and purify A, add BamHI restriction enzyme to
After reacting for minutes, a tryptophan operon gene fraction was obtained.

(C) Preparation of pBR322trp The tryptophan operon fraction obtained in the above (B) was reacted with restriction enzymes Sa1 I and Xho I at 37 ° C. for 1 hour and then heat-treated at 65 ° C. for 5 minutes to remove the restriction enzyme. After inactivation, the plasmid pBR322 similarly treated with the restriction enzyme Sa1 I was added and mixed. Then, each component in the inactivating solution was strengthened so that the final concentration of each component was 50 mM Tris buffer pH 7.6, 10 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM ATP and 1 unit of T ₄ ligase, and at 16 ° C. DNA was bound by incubating for 15 hours.

The E. coli K-12 strain (tryptophan-requiring mutant strain, ATCC23718) was transformed by the conventional method using the recombined DNA, and the transformant [Trp-requiring loss, that is, the trpA and trpB genes on the plasmid were used to produce Synthesizable, minimal medium (K ₂ HPO ₄ 7g, KH ₂ PO ₄ 2g, MgSO ₄ 7H ₂ O 0.1g
g, (NH ₄ ) ₂ SO ₄ 1 g, glucose 2 g, pure water 1). This strain was liquid-cultured according to a conventional method, and the plasmid pBR322trp was separated and purified from the culture solution.

(D) Cloning of trpAB fraction The plasmid pBR322trp obtained in the above (C) was digested with restriction enzymes Sac II and Sa1 I at 37 ° C. for 1 hour to obtain a fraction containing the trpAB gene. Then, using the restriction enzyme Hinc II, 37
Partial digestion at ℃ gave the minimum fraction containing trpAB. This fragment was mixed with Sa1 I linker, are bonded through T ₄ DNA ligase to obtain a trpAB fraction with Sa1 I sites at both ends.

Then, pDR720 (manufactured by Pharmacia) was reacted with a restriction enzyme Sa1 I at 37 ° C. for 1 hour and heat-treated at 65 ° C. for 5 minutes to inactivate the restriction enzyme, and then the above trpAB fraction was added and mixed.

The final concentration of the components in the inactivating solution was 50 mM Tris buffer pH 7.6, 10 mM MgCl ₂ , 10 mM dithiothreitol,
DNA was bound by strengthening each component so as to be 1 unit of 1 mM ATP and T ₄ ligase and incubating at 16 ° C. for 15 hours.

The E. coli K-12 strain (tryptophan-requiring mutant strain, ATCC23718) was transformed by the conventional method using the recombined DNA, and the transformant [Trp-requiring loss, that is, the trpA and trpB genes on the plasmid were used to produce Synthesizable, minimal medium (K ₂ HPO ₄ 7g, KH ₂ PO ₄ 2g, MgSO ₄ 7H ₂ O 0.1g
g, (NH ₄ ) ₂ SO ₄ 1 g, glucose 2 g, pure water 1). This strain was liquid-cultured according to a conventional method, and the plasmid pDR720-trpAB was separated and purified from the culture solution.

(E) Recloning of trpAB gene into pUC-9 The pDR720-trpAB fraction obtained in the above (D) was used as a restriction enzyme Eco I.
The reaction was carried out at 37 ° C for 1 hour at I and heat treatment at 65 ° C for 5 minutes to inactivate. Similarly, pUC-9 (Pharmacia) was treated with the restriction enzyme EcoRI and ligated in the same manner as above. The re-ligated DNA was transformed as described above. From the obtained strains, the one in which the trpAB fraction was inserted into pUC-9 was selected, and the plasmid pUC-9trpAB was extracted according to a conventional method.

Example 4 Construction of plasmid pCRY-31-trp1 (A) Preparation of plasmid pCRY-31trp1 10 μg of plasmid pUC-9trpAB prepared in Example 3 and 3 μg of plasmid pCRY31 prepared in Example 1 were used at 37 ° C. with a restriction enzyme BamHI. Cleavage is carried out by reacting for 1 hour, and the restriction enzyme is deactivated by heating at 65 ° C. for 10 minutes.
50mM Tris buffer pH 7.6, 10mM MgCl ₂ , 10mM dithiothreitol, 1mM ATP and T ₄ ligase Each unit was strengthened to be 1unit, and kept at 16 ° C for 15 hours.
DNA was bound, and this solution was used to transform Escherichia coli HB101 competent cells (Takara Shuzo).

The transformant was L medium (trypton 10 g, yeast extract 5 g, N) containing 30 μg / ml (final concentration) of chloramphenicol.
The cells were cultured in aCl5g, pure water 1, pH 7.2) at 37 ° C for 24 hours to obtain a growing strain. From each of the grown strains, the plasmid was added to the alkaline-SDS method [T. Maniatis, EFFritsch, J. Sa.
mbrook, "Molecular cloning" (1982) p.90-91]
Extracted.

(B) Transformation of plasmid into coryneform bacterium Transformation was performed using the electric pulse method described in Example 1 (D). From the emerged chloramphenicol resistant strain, a plasmid was obtained by the method described in the above (A). The results of measuring the molecular weight of the plasmid with various restriction enzymes are shown in Table 4 below.

The plasmid characterized by the above restriction enzymes was designated as "pCRY-
31-trp1 ". The plasmid pCRY-31-trp
Brevibacterium flavum M transformed with 1
J233GE1004 is located at 1-1, Higashi 1-chome, Yatabe-cho, Tsukuba-gun, Ibaraki prefecture.
May, 1988, at the Institute of Microbial Technology, Institute of Industrial Science and Technology.
Contract number on the 26th: Micro Engineering Research Institute, No. 10035 (FERM P-1
0035) has been deposited.

Example 5 Stability of plasmid pCRY-31 trp1 100 ml of the above-mentioned A medium was dispensed into a 500 ml Erlenmeyer flask,
A strain sterilized at 120 ° C for 15 minutes was inoculated with the transformant obtained in Example 4 (B), shake-cultured at 30 ° C for 24 hours, and then similarly prepared as A medium. Dispense 100 ml into a 500 ml Erlenmeyer flask and sterilize at 120 ° C for 15 minutes.
Transplant at a rate of 50 cells per cell at the same temperature of 30 ℃
At 24 hours, shaking culture was performed. Then, the cells were collected by centrifugation and the cells were washed, and then chloramphenicol 5 μg / ml
A fixed amount of the medium A added and the plate medium prepared as an A medium without addition are smeared, and after culturing at 30 ° C. for 1 day, growing colonies are counted.

As a result, it was confirmed that the number of colonies grown on the medium with and without chloramphenicol was the same, and that all the colonies growing on medium A were able to grow on the medium with chloramphenicol, that is, the high stability of the plasmid was confirmed. did.

Example 6 Method for producing tryptophan synthase and production of L-tryptophan using this bacterium Medium (0.4% urea, 1.4% ammonium sulfate, KH ₂ PO ₄₀ .
5%, K ₂ HPO ₄ 0.05%, MgSO ₄ / 7H ₂ O 0.05%, CaCl ₂ / 2H _{2
O 2ppm, FeSO 4 · 7H 2} O 2ppm, MnSO 4 · 4~6H 2 O 2pp
_{m, ZnSO 4 · 7H 2 O} 2ppm, NaCl2ppm, biotin 200 [mu] g /
, Thiamine / HCl 100 μg /, tryptone 0.1%, yeast extract 0.1%) 100 ml was dispensed into a 500 ml Erlenmeyer flask and sterilized (pH 7.0 after sterilization), and then the plasmid pCRY-3 of the present invention.
A strain (FERM P-10035) retaining 1-trp1 was inoculated, glucose was aseptically added to a final concentration of 2% (w / v), and shake culture was carried out at 30 ° C. for 15 hours.

Next, the main culture medium (ammonium sulfate 2.3%, KH ₂ PO
_{4 0.05%, K 2 HPO 4} 0.05%, MgSO 4 · 7H 2 O 0.05%, FeSO 4
・ 7H ₂ O 20ppm, MnSO ₄・ nH ₂ O 20ppm, biotin 200μg /
, Thiamin / HCl 100 μg /, tryptone 0.3%, yeast extract 0.3%) in a 2-volume aeration stirring tank,
After sterilization (120 ° C., 20 minutes), glucose was aseptically added so that the final concentration was 2% (w / v), and 20 ml of the culture was further added, and the rotation speed was 1000 rpm, the aeration rate was 1 vvm, and the temperature was 33.
Culturing was performed at pH 7.6 for 18 hours.

Glucose was intermittently added about every 1 to 2 hours so that the concentration of the culture medium did not exceed 2% by weight.

After culturing, collect the cells by centrifugation from 400 ml of the culture,
The bacterial cells washed twice with demineralized distilled water were added to the reaction mixture [Indole
5g, DL-serine 20g, pyridoxal-5'-phosphate 10m
g, KCl2g, distilled water 1000ml (adjusted to pH8.0 with 5N-KOH)]
After being suspended in 1000 ml of the above, the suspension was charged into a 2-volume aeration and stirring tank, and reacted at a rotation speed of 300 rpm, a temperature of 37 ° C., and a pH of 8.0 for 10 hours. After completion of the reaction, a supernatant was prepared from the reaction solution by centrifugation (6000 rpm, 15 minutes, 4 ° C.), and L-tryptophan in the supernatant was measured by liquid chromatography.

In addition, 500 ml of the reaction completed liquid was passed through a column of an ammonia type strongly acidic ion exchange resin (“Diaion SK-1B”, manufactured by Mitsubishi Kasei Co.), then eluted with an alkaline solution and concentrated to L.
-Precipitated crude crystals of tryptophan.

As a result, the production concentration of L-tryptophan was 3 g /
The purified amount was 1 g. For comparison, plasmid pC
As a result of carrying out the reaction by the above-mentioned method using the host cells that do not retain RY-31-trp1, the production concentration of L-tryptophan was 0.05 g /.

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Claims (7)

(57) [Claims] 1. A DNA region (a) containing at least a tryptophan synthase gene and a DNA region (b) containing a promoter and an operator capable of controlling the expression of the gene.
And Brevibacterium stationis (Brevibacteri
um stationis) IFO12144 (FERM P-10136) possesses an autonomous growth ability and a stabilizing function in coryneform bacterial cells containing a DNA fragment of about 7.4 kb obtained by excising the plasmid pBY503 possessed by Kpn I. A tryptophan synthase obtained by culturing a coryneform bacterium capable of producing tryptophan synthase transformed with a plasmid comprising a DNA region (c), in a medium, and collecting tryptophan synthase from the culture. Method for producing synthase. 2. A DN containing a tryptophan synthase gene.
A region (a) is trpA derived from Escherichia coli chromosome
The method according to claim 1, which is a DNA region containing trpB and trpB. 3. The method according to claim 1, wherein the DNA region (b) containing a promoter and an operator capable of controlling the expression of the tryptophan synthase gene is derived from the tryptophan operon. 4. The plasmid is Brevibacterium flavum MJ233GE1004 (FERM P
-10035) is the plasmid pCRY-31 trp1 carried by the method according to claim 1. 5. The method according to claim 1, wherein the coryneform bacterium is a strain derived from Brevibacterium flavum MJ233. 6. A DNA region (a) containing at least a tryptophan synthase gene and a DNA region (b) containing a promoter and an operator capable of controlling the expression of the gene.
And Brevibacterium stationis (Brevibacteri
um stationis) IFO12144 (FERM P-10136) possesses an autonomous growth ability and a stabilizing function in coryneform bacterial cells containing a DNA fragment of about 7.4 kb obtained by excising the plasmid pBY503 possessed by Kpn I. A coryneform bacterium capable of producing tryptophan synthase, which is transformed with a plasmid comprising a DNA region (c). 7. A DNA region (a) containing at least a tryptophan synthase gene, and a DAN region (b) containing a promoter and an operator capable of controlling the expression of the gene.
And Brevibacterium stationis (Brevibacteri
um stationis) IFO12144 (FERM P-10136) possesses an autonomous growth ability and a stabilizing function in coryneform bacterial cells containing a DNA fragment of about 7.4 kb obtained by excising the plasmid pBY503 possessed by Kpn I. Brevibacterium flavum MJ233GE1004 (FERM P-1003 transformed with a plasmid comprising a DNA region (c)
Five).