JP3024766B2 - Lactobacillus with tryptophan-producing ability - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to Lactobacillus casei.
casel; hereinafter also referred to as "lactobacillus") a gene involved in tryptophan biosynthesis, a shuttle vector used for introducing a foreign gene into lactobacillus, and a tryptophan synthesis gene using the shuttle vector system. Lactobacillus strains transformed by the method.

More specifically, DNA containing a tryptophan synthesis gene derived from lactobacillus, plasmid pAMβ1-1 lacking a part of plasmid pAMβ1 derived from Streptococcus faecalis DS5 strain, or at least a part of pAMα1 and Escherichia coli-derived And a shuttle vector comprising a shuttle vector, a plasmid having a tryptophan synthesis gene-containing DNA linked thereto, and a tryptophan synthesis gene introduced using such a plasmid. The present invention relates to a Lactobacillus strain that has acquired tryptophan-producing ability.

[Problems to be Solved by the Prior Art and the Invention] Lactobacillus is an important material widely used in industrial fields such as yogurt, lactic acid beverages, dairy products such as cheese, feed production, and lactic acid production by fermentation. Fungus,
If this bacterium can be artificially modified so as to express desired properties and bred, the industrial merit will be extremely large, and therefore, the genetic engineering method as a means of breeding is extremely difficult from this viewpoint. It should be an important means.

Also, tryptophan is one of the essential amino acids of animals, and many studies have been made on its industrial production method. Therefore, if tryptophan can be produced by a bacterium of the genus Lactobacillus, industrial benefits will be extremely large.Moreover, when live bacteria having this tryptophan-producing ability are consumed by animals and humans as dairy products or fungal preparations, If tryptophan can be produced in vivo, it is not only unnecessary to supplement tryptophan from outside the body, but also indole, which is a harmful substance produced in the intestine, can be converted into useful tryptophan, improving the health of livestock and human health. Even above, the benefits are considered significant.

Therefore, development of a lactobacillus having such a useful trait has been desired. On the other hand, since a stable method for introducing a foreign gene into lactobacillus has not been established, a technical technique aimed at artificially modifying this lactic acid spot using genetic engineering techniques. In various attempts,
There were almost no results to watch. Recently, however, the present inventors have developed a method for stably introducing foreign DNA into lactobacilli (Japanese Patent Application No. 62-294199 and Fumio Imamot).
o (Kyoto Pharmaceutical University) eta1., Techno J
See apan, vol. 22, No. 3, pp. 8-18. ). By using this method, it became possible to introduce foreign DNA into lactobacillus. With regard to the vector used at that time, development of a useful vector, especially using genetic engineering techniques, was newly performed. There has been a demand for the development of good vectors for producing lactobacilli having useful traits.

[Means for Solving the Problems] In view of this situation, the present inventors have introduced a tryptophan synthesis gene into a lactobacillus to obtain a strain imparted with tryptophan-producing ability, and used the strain at that time. Research was conducted to obtain good and useful shuttle vectors.

The present inventors have succeeded in obtaining a DNA fragment containing a gene involved in tryptophan biosynthesis from the chromosomal DNA of Lactobacillus casei RNL7 strain and succeeded in elucidating the nucleotide sequence thereof. Then, when introducing such a tryptophan synthesis system gene and other useful genes into lactobacilli, a good shuttle vector required is constructed, and then the tryptophan synthesis system gene is bound to these shuttle vectors, Recently, using the above-described gene transfer method developed by the present inventors, a tryptophan synthesis gene was introduced into Lactobacillus casei requiring tryptophan, and a strain having tryptophan-producing ability was successfully produced. Completed the invention.

Examples of the shuttle vector of the present invention include plasmid pAMβ1 derived from Streptococcus faecalis DS5 strain and pAMβ lacking a part thereof.
1-1 and a plasmid vector derived from Escherichia coli, for example, pB
PBLβ15, PBLβ21, pBLβ22, PBLβ23, pBLβ24, PBLβ2 which are plasmids obtained by binding R322 or pUC19, etc.
5 and the like. Further, a plasmid pAMα1 derived from DS5 strain of Streptococcus faecalis and a plasmid vector derived from Escherichia coli such as pACYC177, pHY300PLK (see JP-A-60-248184), can be used.

The DNA fragment in which the tryptophan synthesis gene derived from the lactobacillus of the present invention is present is obtained by culturing lactobacillus, removing DNA from the obtained cells, cutting this DNA with the restriction enzyme HindIII, and cutting this DNA. ligated using DNA and Hind III at the cleaved plasmid vector pBR322 and the appropriate ligases from E. coli, such appropriate means plasmid thus obtained, for example, E. coli suspended in CaCl ₂ solution trp gene mutant strains, such as E. coli mH3trp
C9830 strain was obtained, and a transformant of Escherichia coli was obtained in this way, and only the required transformant was selectively taken out using the ampicillin resistance and tryptophan-producing ability of the transformant of Escherichia coli as an index. It can be obtained by culturing and removing the plasmid from the cultured cells.

The shuttle vector of the present invention comprises a Streptococcus
Plasmid pAMβ1-1 derived from S.ficusalis strain DS5 and a plasmid vector derived from Escherichia coli such as pBR322 or pUC19, or Streptococcus faecalis DS5.
A plasmid pAMα1 derived from the strain and a plasmid vector derived from Escherichia coli such as pACYC177 are ligated, and the thus obtained plasmid is introduced into Escherichia coli by an appropriate means, and the thus obtained transformant of Escherichia coli is transformed. It can be obtained by further culturing this and removing the plasmid from the cultured cells.

The transformed lactobacillus having the tryptophan synthesis gene of the present invention contains the tryptophan synthesis gene obtained by binding the above-described tryptophan synthesis gene-containing DNA fragment to the shuttle vector using an appropriate ligase. Plasmids are prepared, for example, in Japanese Patent Application No. 62-29.
No. 4199, that is, by introduction into lactobacilli by electroporation.

That is, the lactobacillus is suspended in a buffer, the above-mentioned plasmid is added to the suspension, mixed, and a pulse current is applied to the suspension to introduce the above-described plasmid into the lactobacillus.

The lactobacilli thus electroporated are then subjected to a known selection process to select the resulting transformants. Transformants are selected based on, for example, resistance to various antibiotics.

Next, the present invention will be described more specifically with reference to examples. It should be noted that these examples are disclosed for explaining the present invention, and the present invention is not limited by the description.

Example 1 Cloning of Lactobacillus casei tryptophan synthesis gene (1) Extraction and purification of Lactobacillus casei DNA The following medium composition: tomato juice extract ^* 400 ml glucose 20 g soluble starch 0.5 g yeast extract (yeast extract) ) (Difco) 6 g polypeptone (large five nutrient) 15 g monosodium glutamate _{2g Tween80 1g CH 3 COONa · 3H} 2 O 10g KH 2 PO 4 4g NaCl 5g DL- threonine 2g distilled water 600 ml * tomato juice extract is commercially available tomato juice [ Del Monte, Inc.], add an equal volume of distilled water and add
After heating at 0 ° C. for 1 hour and further at 100 ° C. for 5 minutes, the mixture was cooled and adjusted separately for water-insoluble matters.

The resulting medium was adjusted to pH 6.8 with NaOH and then sterilized at 120 ° C. for 15 minutes using an autoclave.

At the time of use, this medium was prepared by dissolving 3.4 g of sodium ascorbate and 200 mg of L-cysteine in distilled water to make 10 ml, and adding 10 ml of a sterilized solution.

To 500 ml of the obtained medium was separately inoculated 5 ml of a culture solution of Lactobacillus casei RNL7 strain, which had been left to stand overnight at 37 ° C in this medium, and left to stand at 37 ° C. When A ₆₀₀ = 0.3, the cells were collected by centrifugation. The cells were treated with 10 mM Tris-HCl buffer (pH 8.0 or less, abbreviated as TE) containing 1 mM EDTA.
Washed twice and then suspended in 3 ml of TE containing 25% (w / v) sucrose. Add lysozyme solution (20mg / ml-0.25M Tris-C
l, pH 8.0), and the mixture was kept at 37 ° C for 60 minutes. to this
3m 100mM Tris-HCl buffer (pH 9.0) containing 120mM EDTA
1,10% sodium lauryl sulfate (SDS) 1.5ml and Protei
0.2 ml of nase K (4 mg / ml-distilled water) was added, the mixture was further kept at 37 ° C. for 60 minutes, and then 4 ml of 100 mM Tris-HCl (pH 9.0) was added.
To this solution was added TE-saturated phenol and shaken gently.
The mixture was centrifuged to separate the supernatant fraction. 100mM for this
4 ml of Tris-HCl (pH 9.0) was added and dialyzed against TE.
After dialysis, RNaseA was added to a concentration of 10 μg / ml, and the mixture was incubated at 37 ° C. for 30 minutes. Then, Proteinase K was added to a concentration of 50 μg / ml, and the mixture was further incubated at 37 ° C. for 30 minutes. To this solution was added SDS in an amount of 0.5%, and the mixture was washed three times with a TE-saturated phenol-chloroform-isoamyl alcohol 25: 24: 1 mixed solution. Further, after washing once with a mixed solution of chloroform and isoamyl alcohol 24: 1, ultracentrifugation was performed with a cesium chloride-ethidium bromide concentration gradient to obtain purified DNA.

(2) Ligation 1 μg of the DNA obtained in (1) was added to 50 mM NaCl, 10 mM Tris
-HCl (pH7.5), dissolved in 20μ of a solution containing 10mM MgCl ₂ , 1mM mercaptoethanol, added with 1μ of Hind III, 37 ℃
For 2 hours. After adding 1 μg of 0.5 M EDTA, the mixture was washed three times with a 25: 24: 1 mixed solution of TE-saturated phenol-chloroform-isoamyl alcohol, and then once with chloroform-isoamyl alcohol 24: 1. Got
1/10 volume of 3M CH ₃ COONa aqueous solution and 2.2
Add twice the volume of ethanol, leave at -80 ° C for 10 minutes, and then add
After centrifugation at 000 rpm for 5 minutes, a precipitate of DNA was obtained. This D
The NA precipitate was washed with 70% ethanol, dried under vacuum and then dissolved in sterile distilled water.

1 μg of pBR322 is added to 50 mM NaCl, 10 mM Tris-HCl (pH 7.
5) Solution 2 containing 10 mM MgCl ₂ and 1 mM mercaptoethanol
In 0μ, 1μ of Hind III was added and incubated at 37 ° C for 2 hours. After adding 1 μg of 0.5 M EDTA, TE saturated sum phenol-chloroform-isoamyl alcohol 25:24:
The mixture was washed three times with one mixed solution, and then once with chloroform-isoamyl alcohol 24: 1. To the solution containing the obtained DNA, 1/10 volume of 3M CH ₃ COONa aqueous solution and 2.2 volume of ethanol were added, and the mixture was allowed to stand at −80 ° C. for 10 minutes and then at 15,000 rpm.
After centrifugation for minutes, a precipitate of DNA was obtained. The DNA precipitate is washed with 70% ethanol, dried under vacuum, and then
Was dissolved. To this, TE25μ, 1M Tris-HCl (pH 8.
8) 2 μl was added, and E. coli alkaline phosphatase was further added, followed by incubation at 65 ° C. for 30 minutes. The mixture was washed three times with a 25: 24: 1 mixed solution of TE-saturated phenol-chloroform-isoamyl alcohol, and further washed once with chloroform-isoamyl alcohol 24: 1. 1/10 times the amount of 3M in the obtained aqueous layer
CH ₃ COONa and 2.2 volumes of ethanol were added, and the mixture was allowed to stand at −80 ° C. for 10 minutes to insolubilize the DNA, followed by centrifugation at 15,000 rpm for 5 minutes to obtain a precipitate of DNA. The precipitate was washed with 70% ethanol, dried under vacuum, and dissolved in sterile distilled water.

Lactobacillus casei DNA50 cut with Hind III
Ligation buffer (50 mM Tris-HCl, pH 7.50) containing ~ 500 ng and 50-500 ng of pBR322 cut with Hind III.
4,10 mM MgCl ₂ , 10 mM dithiothreitol, 1 mM spermidine, 2 mM ATP, 0.1 mg / ml bovine serum albumin) 10μ
Was added with 1 μl of T4 DNA ligase and incubated at 12 ° C. for 12 to 16 hours. After washing three times with TE-saturated phenol-chloroform-isoamyl alcohol 25: 24: 1 and then once with chloroform-isoamyl alcohol 24: 1
The DNA was insolubilized by adding 1/10 volume of 3M CH ₃ COONa aqueous solution and 2.2 volume of ethanol, left at −80 ° C. for 10 minutes, and centrifuged at 15,000 rpm for 5 minutes to obtain a DNA precipitate. The precipitate was washed with 70% ethanol, dried under vacuum, and dissolved in 10 µ of sterile distilled water.

(3) Transformation of Escherichia coli E. coli mH3trpC9830 strain was inoculated into 5 ml of L-broth (composed of 1% bactotryptone, 0.5% yeast extract, 0.5% NaCl and 0.1% glucose) in a test tube and incubated at 37 ° C. and cultured with shaking until the a ₅₅₀ = 0.3. The cells were collected by centrifugation at 3000 rpm for 10 minutes at 4 ° C. The cells were suspended in 2.5 ml of 0.1 M MgCl ₂ and centrifuged at 4 ° C., 3000 rpm for 10 minutes to collect the cells. The cells were suspended in 0.1 M CaCl ₂ and allowed to stand on ice for 20 minutes. The cells were collected again by centrifugation at 4 ° C. and 3000 rpm for 10 minutes, and the cells were suspended in 0.5 ml of 0.1 M CaCl ₂ to prepare competent cells.

100 µ of competent cells were added to 10 µ of the DNA obtained in (2), the mixture was allowed to stand at 0 ° C for 1 hour, and then kept at 42 ° C for 10 minutes. This was left at room temperature, and after the liquid temperature was returned to room temperature, L
-1 ml of broth was added, and the mixture was cultured at 37 ° C for 1 hour. Then, cells were collected by centrifugation at room temperature at 3000 rpm for 10 minutes. The cells were further washed twice with sterile saline, and then suspended in 1 ml of physiological saline. This bacterial suspension was added to Vogel-Bonner agar medium (MgSO ₄ .7H ₂ O 0.02%, citric acid-hydrate 0.2%, KH ₂ PO ₄ 1%, NaNH ₄ HPO ₄₎ supplemented with 25 μg / ml ampicillin.・ 4H ₂ O0.35
%, Glucose 0.5%, casamino acid 0.2%) and cultured at 37 ° C. for 24 hours. The transformant which formed a colony on this agar medium was examined for the possessed plasmid. This plasmid was designated as pTLC100.

The construction process of this plasmid pTLC100 is shown in FIG.

(4) Cloned Lactobacillus casei DN
Properties of A (a) Transformation of competent E. coli W3110 trp mutant cells prepared in the same manner as in (3) using the obtained plasmid is shown in (3). The characteristics of the colonies on Vogel-Bonner agar medium containing 25 μg / ml of ampicillin shown in (3) were examined in the same manner as described above. The results are shown in the table below.

From the above results and the result of (3), the cloned Lactobacillus casei DNA was obtained from trpC, trpF, t of E. coli.
Shows complementarity to rpB and trpA mutations.

(B) Next, the obtained plasmid was cut with various restriction enzymes, and the behavior on electrophoresis was examined to create a restriction enzyme map.

Add one or more restriction enzymes to 20 μl of buffer containing 0.1 to 0.3 μg of pTLC100 and incubate at 37 ° C for 2 hours or, if necessary, incubate with one type of restriction enzyme and then change the buffer further. Was added and incubated. After incubating 0.
1 μM of 5M EDTA was added, and electrophoresis was performed on 0.8% agarose gel and 1.2% agarose gel, and the number and mobility of DNA bands were examined. The restriction enzymes and buffers used are shown below.

As a result, a restriction map shown in FIG. 2 was prepared.

(C) The obtained Lactobacillus casei DNA was cut with various restriction enzymes, and this was subcloned into pUC9 or pUC19. Using the subcloned DNA fragment, the entire nucleotide sequence of DNA was determined by the dideoxy method using a DNA sequencing kit manufactured by Takara Shuzo Co., Ltd. FIG. 3 shows the determined entire nucleotide sequence. The DNA determined here has a sequence size of 6468 bases and D
The position of each gene on NA is trpD506 → 1531, trpC1528 → 23
10, trpF2462 → 3061, trpB3045 → 4265, trpA4252 → 5052, unknown gene X6325 → 5666, unknown gene Y402 → 509.

The positional relationship of each gene of this DNA and the direction of the gene are the fourth.
Shown in the figure.

Example 2 Construction of shuttle vector pBLβ15 (1) Plasmid pAMβ lacking the tra region of pAMβ1
1-1 Buffer containing 0.3 μg (100 mM NaCl, 50 mM Tri
s-HCl, pH 7.5, 10 mM MgCl ₂ , 1 mM mercaptoethanol
20 μl of EcoR I 0.125 unit was added and incubated at 37 ° C. for 1 hour. 1 μg of 0.5 M EDTA was added to stop the incubation, and the treated solution was washed three times with a TE-saturated phenol-chloroform-isoamyl alcohol mixture, and once with a chloroform-isoamyl alcohol 24: 1 mixture.
To the obtained aqueous layer, 1/10 volume of 3M CH ₃ COONa and 2.2 volumes of ethanol were added to insolubilize DNA, left at −80 ° C. for 10 minutes, and then centrifuged at 15,000 rpm for 5 minutes to precipitate DNA. I got
The precipitate was washed with 70% ethanol, dried under vacuum, and dissolved in sterile distilled water.

The buffer composition is 100 mM NaCl, 50 mM Tris-HCl (pH 7.
5) a 10 mM MgCl _2, 1 mM mercaptoethanol, restriction enzymes in the same conditions as in except that it is EcoR I shown in Example 1- (2) to give the EcoR I cleavage of pBR322 .

Next, pAMβ1-1 and pBR322 were ligated using a ligation kit manufactured by Takara Shuzo Co., Ltd. EcoRI cut pAMβ1-1 0.05-0.2 μg, pBR3
22 Buffer (100 mM Tris-HCl,
pH 7.6, 5mM MgCl ₂ ) 5μ into A according to the protocol
30 µ of solution and 5 µ of solution B were added, and a ligation reaction was performed at 16 ° C for 30 minutes to 1 hour. Next, the strain used was E. coli H
Competent cells were prepared under the same conditions as in Example 1- (3) except that B101 was used. Add 100 μl of competent cells to 20 μl of the ligation reaction solution, and add
After standing for an hour, the mixture was kept at 42 ° C. for 10 minutes. This was left at room temperature, and the temperature of the solution was returned to room temperature.
Incubated at ℃ for 1 hour. This culture was added to L-broth at 25 μg /
The mixture was applied to the surface of an agar medium prepared by adding 1.5 ml of agarose to which ml of ampicillin was added, and cultured at 37 ° C. overnight to obtain colonies.

(2) Each of the obtained colonies was treated with 50 μg / ampicillin /
L-broth containing ml was inoculated and cultured with shaking at 37 ° C overnight.
1.5 ml of the culture solution was taken and centrifuged at 15000 rpm for 1 minute to collect the cells. The cells were treated with a buffer containing 5 mg / ml lysozyme (50 mM glucose, 25 mM Tris-HCl pH 8.0, 10 mM EDT
A) Suspend in 100μ, leave at room temperature for 5 minutes, then
250 μL of 0.2N NaOH containing 5% sodium lauryl sulfate
The mixture was gently shaken and left at room temperature for 5 minutes. 3M CH
150 μl of ₃ COOK (pH 4.8) was added, and the mixture was gently shaken and left at room temperature for 5 minutes. After centrifugation at 15000 rpm for 5 minutes, a supernatant fraction was obtained. After adding 0.6 volumes of isopropyl alcohol to the supernatant to insolubilize the DNA and leaving it to stand at room temperature for 15 minutes, 15,000 rpm
For 5 minutes to obtain a precipitate of DNA. 70
After washing with ethanol and drying under vacuum, it was dissolved in sterile distilled water.

(3) The resulting plasmid was subjected to electrophoresis on a 0.8 agarose gel to select a plasmid having a size of about 22.5 kb. These plasmids were further cut with EcoRI and electrophoresed on a 0.8% agarose gel to obtain a pBR322-derived plasmid. A plasmid in which a 4.4 kb band and two bands derived from pAMβ1-1 (a 5 kb band containing a replication initiation region and a 13 kb band containing an erythromycin resistance region) (a total of 3 bands) were recognized was named pBLβ15.

The construction process of this plasmid pBLβ15 is shown in FIG.

Example 3 Preparation of shuttle vector pBLβ21 (1) Preparation of DNA fragment containing erythromycin resistance gene and DNA fragment containing replication initiation region from pAMβ1-1 Buffer containing 100 μg of pBLβ15 (100 mM NaCl, 50 mM T
Ava I75 unit and EcoR I75 unit were added to 100 μm of ris-HCl, pH 7.5, 10 mM MgCl ₂ , 1 mM mercaptoethanol, and incubated at 37 ° C. for 1 hour. Further the same amount of enzyme was added and the incubation was continued for another hour. 0.5M ED
After adding 20 μ of TA and stopping the incubation, 0.8% agarose gel electrophoresis was performed. Gel containing a band of 5 kb EcoRI-EcoRI fragment (hereinafter referred to as fragment-1) containing the replication initiation region of pAMβ1-1 and a band of 4 kb AvaI-EcoRI fragment containing the erythromycin resistance gene of pAMβ1-1 Cut out each gel containing, place in a dialysis bag, and perform electrophoresis again.
The DNA fragment was eluted. Solution containing each DNA fragment (approx.
3 ml) was washed four times with TE-saturated phenol and then shaken with 2-butanol to concentrate the solution containing DNA to about 400μ. This solution was washed three times with ether, and 1/10 volume of 3M CH ₃ COONa and 2.2 volumes of ethanol were added.
Was insolubilized, left at −80 ° C. for 10 minutes, and then centrifuged at 15,000 rpm for 5 minutes to obtain a DNA precipitate. After washing with 70% ethanol, it was dried under vacuum and dissolved in 20 µ of sterile distilled water.

About 0.5 μg of the above Ava I-EcoR I fragment, dATP, dCTP, dGTP, d
Buffer containing 0.1 mM of each TTP (7 mM Tris-HCl, pH 7.5, 0.1 mM
EDTA, 20mM NaCl, 7mM MgCl ₂ ) Klenow enzyme 1 in 50μ
μ (2 units) was added, and the mixture was incubated at 37 ° C. for 1 hour to blunt the ends.
2). After washing three times with a mixed solution of TE-saturated phenol-chloroform-isoamyl alcohol 25: 24: 1, then washing once with chloroform-isoamyl alcohol 24: 1, and then 1/10
Add 2 volumes of 3M CH ₃ COONa and 2.2 volumes of ethanol and DNA
Was insolubilized, left at -80 ° C for 10 minutes, and then centrifuged at 15000 rpm for 5 minutes to precipitate DNA. The precipitate was washed with 70% ethanol, dried under vacuum and dissolved in sterile distilled water.

The relationship between plasmid pBLβ15 and fragments 1 and 2 is shown in FIG.

(2) An Nru I digest of pBR322 was obtained in the same manner as in Example 1- (2) except that the restriction enzyme was Nru I. Then, the DNA to be used is fragment-2 and N of pBR322.
An ampicillin-resistant colony was obtained in the same manner as described in Example 2- (1) except that it was a ruI digest, and then plasmid was isolated from each colony in the same manner as in Example 2- (2). Was purified.

(3) The above-obtained plasmid was subjected to 0.8% agarose gel electrophoresis to select a plasmid having a size of about 8.4 kb, further cut with Hind III, and subjected to agarose gel electrophoresis to clone fragment-2 into pBR322. I confirmed that This plasmid was designated as pBLβ18.

(4) The restriction enzyme is EcoRI and the buffer is 100 mM NaC.
An EcoRI cut product of pBLβ18 was obtained in the same manner as described in Example 1- (2) except that it was composed of 1,50 mM Tris-Cl (pH 7.5), 10 mM MgCl ₂ , and 1 mM mercaptoethanol. Was. Next, the DNA to be used was EcoRI of fragment-1 and pBLβ18.
An ampicillin-resistant colony was obtained in the same manner as described in Example 2- (1) except that it was a cut product, and then the plasmid was purified from each colony in the same manner as in Example 2- (2). did.

(5) The plasmid obtained above was subjected to 0.8% agarose gel electrophoresis to select a plasmid having a size of about 12.4 kb. This plasmid was digested with Hind III, Kpn I, Pvu II and the like, and it was confirmed from the behavior of the band on agarose gel electrophoresis that fragment-1 was cloned. This plasmid was designated as pBLβ21.

The construction process of plasmids pBLβ18 and pBLβ21 and their restriction maps are shown in FIG.

Example 4 Preparation of Shuttle Vectors pBLβ22 and pBLβ23 (1) A Hinc II digest of pUC19 was obtained in the same manner as described in Example 1- (2) except that the restriction enzyme was Hinc II. . Next, the strain to be used is E. coli JM105, and the DNA to be used is fragment-2 and a Hinc II digest of pUC19,
The composition of the agar medium was Xgal (5-bromo-4-chloro-3-
Indolyl-β-D-galactopyranoside) 20 μg / ml,
An ampicillin-resistant white colony was obtained in the same manner as in Example 2- (1), except that it was an L-broth agar medium supplemented with 25 μg / ml ampicillin and 1.5% agar. . Next, the plasmid of each colony was purified in the same manner as in Example 2- (2).

(2) The plasmid obtained above was subjected to 0.8% agarose gel electrophoresis to select a plasmid having a size of about 6.7 kb. These plasmids were then replaced with Hind III, Eco
The DNA was digested with RI, PstI and a combination thereof, and the cloned DNA was examined. As a result, two types of plasmids having different insertion directions of fragment-2 were obtained. These plasmids were named pBLβ19 and pBLβ20.

The construction process of plasmids pBLβ19 and pBLβ20 and their restriction maps are shown in FIG.

(3) An EcoRI digest of pBLβ19 was obtained in the same manner as in Example 3- (3) except that the plasmid used was pBLβ19. The DNA to be subsequently used was fragment-1 and pBLβ19.
An ampicillin-resistant colony was obtained in the same manner as in Example 2- (1) except that it was an EcoRI cut product. Next, the plasmid of each colony was purified in the same manner as in Example 2- (2).

(4) The plasmid obtained above was subjected to 0.8% agarose gel electrophoresis to select a plasmid having a size of about 11.7 kb. These plasmids were then replaced with Kpn I, Hind I
After digestion with II, EcoRI and a combination thereof, 0.8% agarose gel electrophoresis was carried out, and the behavior was examined to obtain two kinds of plasmids having different directions of fragment-1. These plasmids were named pBLβ22 and pBLβ23.

The construction process of plasmids pBLβ22 and pBLβ23 and their restriction maps are shown in FIGS. 8 and 9 (A) and (B).

Example 5 Preparation of Shuttle Vectors pBLβ24, pBLβ25 The fragment of pBLβ20 was prepared in the same manner as in Examples 4- (3) and 4- (4) except that the plasmid used was pBLβ20.
Two types of plasmids having different insertion directions were obtained. These plasmids were named pBLβ24 and pBLβ25.

Construction process of plasmid pBLβ24 and plasmid pBL25,
8 and 9 (C) and (D).

Example 6 Transformation of Lactobacillus casei with pHY300PLK From the Lactobacillus casei medium shown in Example 1
A medium excluding Tween80 and DL-threonine was used for culturing the bacteria. This plasmid pHY300PLK is disclosed in JP-A-60-248.
No. 184.

0.2 ml of overnight culture of Lactobacillus casei JCM1053 strain
Was inoculated into 10 ml of the above medium in a test tube, and the mixture was allowed to stand until A ₆₀₀ = 0.2. The cells were centrifuged at 4 ° C. and 3000 rpm for 10 minutes to collect the cells. The cells were washed twice with 8 mM sodium phosphate buffer (pH 7.0) containing 0.4 M sucrose,
Suspended in 0.1 ml of the above buffer. PH to 100μ of cell suspension
An aqueous solution containing 1 μg of Y300PLK (50 μm) and a 40% polyethylene glycol 2000 aqueous solution (250 μm) were added, stirred, and allowed to stand at 0 ° C. for 10 minutes. Then, using a Generader manufactured by Bio-Rad Inc., electricity was supplied under the conditions of 2 kV and 1 μF, and the mixture was allowed to stand again at 0 ° C. for 10 minutes. The cells were collected by centrifugation at 4 ° C. at 15000 rpm for 2 minutes, suspended in 1 ml of the above medium, and cultured at 37 ° C. for 3 hours. 0.3 ml of this culture was applied to an agar medium containing 10 μg / ml of tetracycline and 1.5% agar added to the above medium, and anaerobically cultured at 37 ° C. for 3 days. As a result, 560 tetracycline-resistant colonies per agar medium were obtained. Obtained. When the same experiment was performed without adding pHY300PLK, tetracycline-resistant colonies were not obtained.

Example 7 Transformation of Lactobacillus casei with shuttle vector pBLβ21 Lactobacillus casei IAM1045 strain was transformed under the same conditions except that the plasmid of Example 6 was changed to pBLβ21 and tetracycline in the agar medium was changed to erythromycin. 673 erythromycin resistant colonies were obtained per agar medium. When the same experiment was performed without adding pBLβ21, erythromycin-resistant colonies were not obtained.

Example 8 Transformation of Lactobacillus casei with Shuttle Vector pBLβ25 Transformation was performed under the same conditions as in Example 7 except that the plasmid of Example 7 was changed to pBLβ25, and 1120 erythromycin-resistant colonies were obtained. When the same experiment was performed without adding pBLβ25, erythromycin-resistant colonies were not obtained.

Example 9 Cloning of Lactobacillus casei trp gene into pBL25 (1) Buffer containing 20 μg of pTLC100 (50 mM NaCl, 10 mM
400 μl of M Tris-HCl (pH 7.5), 10 mM MgCl ₂ , 1 mM mercaptoethanol) was added with 30 units of Hind III, and incubated at 37 ° C. for 1 hour. An additional 30 units of the enzyme were added and incubation was continued at 37 ° C. for 1 hour. 0.5M EDTA20
After adding μ and stopping the incubation, 0.8% agarose gel electrophoresis was performed. Lactobacillus casei
A gel containing a band of a 6.47 kb DNA fragment containing the trp gene was cut out, placed in a dialysis bag, and further subjected to electrophoresis to elute the DNA fragment. The solution (about 3 ml) containing the DNA fragment is
Washed four times with E-saturated phenol and then shaken with 2-butanol to concentrate the DNA containing solution to about 400μ. After washing this solution three times with ether, 1/10 volume of 3M
The DNA was insolubilized by adding CH ₃ COONa and a 2.2-fold amount of ethanol, allowed to stand at −80 ° C. for 10 minutes, and then centrifuged at 15,000 rpm for 5 minutes to obtain a DNA precipitate. After washing with 70% ethanol, it was dried under vacuum and dissolved in 20μ of sterile distilled water (obtained
The DNA fragment is called fragment-3. ).

A buffer containing about 0.5 μg of this DNA fragment, 0.1 mM each of dATP, dCTP, dGTP, and dTTP (7 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 20 mM
2 μL (4 units) of Klenow enzyme was added to 30 μL of NaCl, 7 mM MgCl ₂ ), and the mixture was incubated at 37 ° C. for 1 hour to make the DNA ends blunt. After washing three times with a 25: 24: 1 mixed solution of TE-saturated phenol-chloroform-isoamyl alcohol, and then once with chloroform-isoamyl alcohol 24: 1, 3M CH ₃ COONa and 2.2 volumes of ethanol were added to insolubilize the DNA, left at −80 ° C. for 10 minutes, and centrifuged at 15,000 rpm for 5 minutes to precipitate the DNA. The precipitate was washed with 70% ethanol, dried under vacuum and dissolved in sterile distilled water.

(2) The plasmid used is pBLβ25, the restriction enzyme is SmaI, and the buffer composition is 20 mM KCl, 10 mM Tris.
A SmaI digest of pBLβ25 was obtained in the same manner as in Example 1- (2) except that -HCl (pH 8.0) was 10 mM MgCl ₂ and 1 mM mercaptoethanol. Then use
Example 2- (1) except that the DNA was a DNA containing the Lactobacillus casei trp gene cut out from pTLC100 with HindIII and blunt-ended by (1) above, and a SmaI digest of pBLβ25. The ampicillin-resistant colonies were obtained in the same manner as described in (1), and the plasmid was purified from each colony in the same manner as in Example 2- (2).

(3) The resulting plasmid was subjected to 0.8% agarose gel electrophoresis to obtain a plasmid having a size of about 18 kb. This was further cut with several types of restriction enzymes, and agarose gel electrophoresis was performed to confirm that the Lactobacillus casei trp gene had been cloned, and further confirmed the direction in which the gene was inserted. The resulting plasmid was named pTLC150.

 The construction process of plasmid pTLC150 is shown in FIG.

Example 10 Acquisition of Lactobacillus casei JCM1053 strain of trp ⁺ (1) Transformation of Lactobacillus casei under the same conditions except that the plasmid of Example 6 was changed to pTLC150 and tetracycline in the agar medium was changed to erythromycin 155 erythromycin-resistant colonies were obtained per agar medium. Tween 80 and DL- from the medium of Example 1.
The resulting colonies were inoculated into a medium containing 3 μg / ml of erythromycin except threonine, and incubated overnight at 37 ° C.
The culture was allowed to stand at 0 ° C. 3 ml of the culture solution was centrifuged at 15000 rpm for 1 minute to collect the cells, washed once with 800 μm of 30 mM Tris-HCl (pH 3.0) containing 50 mM NaCl and 5 mM EDTA, and then washed with a lysozyme solution (10 mg / ml-50 mM glucose). , 10mM EDTA, 25
The strain was suspended in 100 μm of mM Tris-HCl (pH 8.0). Three
After incubating at 7 ° C for 45 minutes, 250 µm of a solution composed of 0.2 M NaCl and 1% SDS was added, and the mixture was allowed to stand at room temperature for 10 minutes. Then 0.3
Add MCH ₃ COOK (pH 4.8) and let stand on ice for 5 minutes, then
The supernatant was obtained by centrifugation at m for 5 minutes. RNase (400μ / m
l) 2 μm was added and the mixture was incubated at 37 ° C. for 10 minutes, then 2 μl of Proteinase K (3 mg / ml) was added, and the mixture was further incubated for 20 minutes. After washing twice with a TE-saturated phenol-chloroform-isoamyl alcohol mixture 25: 24: 1, and once with chloroform-isoamyl alcohol 24: 1, ethanol was added twice as much as the aqueous layer to insolubilize DNA, and the DNA was insoluble at -80. After standing at 1 ° C. for 1 hour, the mixture was centrifuged at 15000 rpm for 5 minutes to obtain a precipitate of DNA.
After washing the precipitate with 70% ethanol, it was dried under vacuum and dissolved in sterile distilled water. When this was subjected to 0.8% agarose gel electrophoresis, a plasmid band showing the same mobility as that of pTLC150 was detected. In addition, the obtained colonies were treated with 10 μg / ml of erythromycin and 10 μg of anthranilic acid.
When the cells were applied to the medium shown in the following table containing μg / ml, growth was observed. For comparison, Lactobacillus casei JCM1053 carrying the vector pBLβ25 was unable to grow on the same medium. That is, the Lactobacillus casei JCM1053 strain became non-requiring tryptophan by having a tryptophan gene derived from the Lactobacillus casei RNL7 strain. Lactobacillus casei JCM1053 strain, which became non-requiring tryptophan, was replaced with Lactobacillus casei RNL398.
(Microbial Laboratory No. 10725).

Example 11 Cloning of Lactobacillus casei trp gene into pHY300PLK Example 1 except that the plasmid used was pHY300PLK
-Hind II of pHY300PLK in the same manner as described in (2)
A cut of I was obtained. Next, the DNA to be used is the same as that of Example 2 except that the fragment-3 shown in Example 9 and the HindIII digest of pHY300PLK were used, and the strain used was E. coli mH3trpAE1.
-Ampicillin resistant colonies were obtained in the same manner as described in (1). The obtained colonies were further subjected to Example 1-
Ampicillin 2 was added to the Vogel-Bonner agar medium shown in (3).
5 μg / ml and tetracycline 10 μg / ml, indole 10
The resulting agar medium was inoculated to a concentration of μg / ml, and colonies that were resistant to ampicillin and tetracycline and were trp ⁺ were selected. From these colonies, the plasmid was purified by the same method as in Example 2- (2), and the plasmid was confirmed by the same method as shown in Example 9- (3). The resulting plasmid was named pTLC156.

 The construction process of plasmid pTCL156 is shown in FIG.

Example 12 Acquisition of trp ⁺ Lactobacillus casei JCM1053 strain An operation of introducing pTLC156 into this strain was performed under the same conditions except that the plasmid of Example 6 was changed to pTLC156. The culture solution after the introduction operation was centrifuged at 15000 rpm for 1 minute to collect the cells, and the cells were washed twice with physiological saline. The cells are suspended in 300 μ of physiological saline, and 100 μ of the suspension is indole 10 μ
g / ml and 10 μg / ml of tetracycline was applied to the agar medium shown in Example 10 and anaerobically cultured at 37 ° C. for 3 days. As a result, 2600 tetracycline-resistant trp ⁺ colonies were obtained per agar medium. Was. Further, the plasmid was confirmed in the same manner as in Example 10, and a plasmid having the same size as pTLC156 was detected. Lactobacillus casei JCM1053 strain, which became non-requiring tryptophan, was named Lactobacillus casei RNL420 (Microtechnical Laboratories No. 10724).

Example 13 RN was added to the medium obtained by adding the anthranilic acid or indole that is a tryptophan biosynthesis precursor to the medium shown in Example 10.
When the L398 strain and the RNL420 strain were applied and anaerobically cultured at 37 ° C. for 2 days, the growth status was as shown in the following table.

On the other hand, L. casei JCM having pBLβ25 or pHY300PLK
The 1053 strain could not grow on the above medium, and could grow only on the medium shown in Example 10 with the addition of tryptophan.

[Brief description of the drawings]

FIG. 1 shows the construction process of plasmid pTLC100 and a restriction map of the plasmid. FIG. 2 shows a simple restriction map of Lactobacillus casei DNA. Fig. 3- (1) to (5) show Cratobacillus casei DN
1 shows the entire nucleotide sequence of A. FIG. 4 shows trpA, B, C, F on Lactobacillus casei DNA.
2 shows the positional relationship between the D and D genes and the unknown genes X and Y. FIG. 5 shows the construction process of plasmid pBLβ15. FIG. 6 shows the relationship between plasmid pBLβ15 and fragments 1 and 2. FIG. 7 shows the construction process of plasmids pBLβ18 and pBLβ21. FIG. 8 shows plasmids pBLβ19, pBLβ20, pBLβ22, pBLβ
23 shows the construction process of pBLβ24 and pBL25. 9 (A), (B), (C) and (D)
1 shows a restriction map of plasmids pBLβ22, pBLβ23, pBLβ24 and pBLβ25. FIG. 10 shows the construction process of plasmid pTLC150 and its restriction enzyme map. FIG. 11 shows the construction process of plasmid pTLC156 and its restriction enzyme map.

Continuation of the front page (56) References JP-A-1-171487 (JP, A) Gene, 27 (1984) p. 55-65 Nac. Aci. Res. 9 [24] (1981) p. 6647-6668 J.C. Nol. Biol. 156 [2] (1982) p. 245-256 Bacteriol. 159 [3] (1984) p. 905-912 (58) Fields surveyed (Int. Cl. ⁷ , DB name) BIOSIS (DIALOG) WPI (DIALOG) Genbank / EMBL / DDBJ / Geneseq JISCT file (JOIS)

Claims (4)

(57) [Claims] 1. A DNA containing a tryptophan synthesis gene derived from Lactobacillus, comprising the following base sequence: I) a DNA having the nucleotide sequence at positions 506 to 1531 (trpD), or a DNA that hybridizes to this DNA under stringent conditions and encodes a functional trpD protein derived from Lactobacillus; ii) 1528 to DNA having the nucleotide sequence of position 2310 (trpC), or a DNA that hybridizes to this DNA under stringent conditions and encodes a functional trpC protein derived from Lactobacillus; iii) positions 2462 to 3061 (trpF) Or a DNA that hybridizes to this DNA under stringent conditions and encodes a functional trpF protein derived from Lactobacillus; iv) has a nucleotide sequence at positions 3045 to 4265 (trpB) DNA or a DNA that hybridizes to this DNA under stringent conditions and encodes a trpB protein derived from Lactobacillus; and v) a salt at positions 4252 to 5052 (trpA). DNA having the sequence or the DNA to hybridize under stringent conditions, and functional lactobacilli derived those encoding trpA proteins; from at least one consisting of DNA. 2. A DN comprising the nucleotide sequence of claim 1.
A. 3. An Escherichia coli-lactobacillus shuttle vector obtained by combining a region encoding the erythromycin resistance gene of the plasmid pAMβ1-1 derived from Streptococcus faecalis DS5 strain and a replication initiation region with a plasmid derived from Escherichia coli, An Escherichia coli-lactobacillus shuttle vector further comprising the DNA according to claim 1. [4] A lactobacillus transformed using the shuttle vector according to [3].