JP3421357B2 - Novel plasmid pSY1 derived from lactococci and its derivatives - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION The present invention has been widely recognized for its usefulness and safety as a cheese-producing bacterium, Lactoc.
occus lactis subsp. 1 actis
(Hereinafter, sometimes abbreviated as Lc. Lactis), which is a novel circular double-stranded DNA plasmid pSY1
And derivatives thereof and microorganisms transformed with these plasmids.

[0002]

2. Description of the Related Art Lactic acid bacteria are extremely useful microorganisms that have been used for a long time in the production of many fermented foods. If it becomes possible to apply the gene recombination technology that has been rapidly developed in recent years to this lactic acid bacterium, it is expected that its usefulness will be further increased. In fact, some lactic acid bacteria, such as Lacto
coccus lactis (Reference 1), Strept
ococcus salivalis subsp.
thermophilus (reference 2) or Lact
Obacillus casei (Reference 3) and the like have already reported a highly efficient host-vector system, and it is in the process of being attempted to be applied to industry.

In order to apply the gene recombination technology to microorganisms used for food production such as lactic acid bacteria, it is necessary that the safety of the vector used for its transformation is established. As such a vector, one that is naturally retained by microorganisms in foods that have been eaten by humans for a long time and whose safety has been confirmed historically is desirable. On the other hand, cheese has a long history as a fermented food, and its safety is guaranteed. Therefore, the microorganism Lc. lact
The plasmid contained in is is useful as a vector for a microorganism for food production.

[0004]

The present inventors have found that Lc. l
When several strains of Actis were searched for their plasmids, the strain Lc. A plasmid having a relatively small size of about 2.8 kb was found in the Lactis M-128C strain (Deposit of Microindustry, Deposit No. FERM P-12650). This plasmid has the restriction map of FIG. 1, and contains HindIII, SphI, PstI, Sa
II, XbaI, BamHI, SmaI, KpnI, S
It has no recognition site for acI. This plasmid is called pSY
It was named 1.

The trait carried by pSY1 is unknown (crypti
Since it was c), when an erythromycin (erythromycin, sometimes abbreviated as Em in the following) resistance gene was ligated as a selectable marker and transformation was attempted, microorganisms of three genera of Gram-positive bacteria, namely Baci
illus subtilis, Lc. lactis, and Lactobacillus delbruecki
i subsp. bulgaricus and Lacto
bacillus delbrückiisubs
p. lactis (Lb.bulgar respectively
icus, Lb. (sometimes abbreviated as lactis), a transformant expressing the selection marker was successfully obtained. Furthermore, E. coli Esc, which is a gram-negative bacterium
It was possible to obtain a transformed Kataga strain expressing the above-mentioned selection marker also in herichia coli.

This indicates that the plasmid pSY1 of the present invention has a broad host range among Gram-positive and negative bacteria. In particular, Lb. bulgaricus, L
b. Despite the efforts of many researchers, there are still no reports of transformation in the two Lactis subspecies.
b. It is clear that the pSY1 of the present invention is also useful as a vector for delbrüeckii species lactobacilli. The present invention has been completed based on such findings.

The preparation of pSY1 may be carried out according to the method described in Reference 7. That is, a known method in which the M-128C strain is cultured in Lysis medium, and then the cells are collected and lysed with lactic acid bacteria.
For example, lysis is performed using an enzyme such as lysozyme. From the obtained lysate, the plasmid can be separated and purified by a commonly used method such as phenol extraction and cesium chloride density gradient centrifugation in the presence of ethidium bromide.

A method for transforming a microorganism by introducing a recombinant plasmid having a selectable marker gene inserted into pSY1 is a calcium chloride method, a protoplast-polyethylene glycol method, known to those skilled in the art, depending on the characteristics of the target microorganism. The best method may be adopted from the electroporation method and the like, and it is not particularly limited, but when the host is lactic acid bacteria, the electroporation method is preferable.

The gene involved in the autonomous replication ability of pSY1 is the plasmid DNA of the present invention, like other plasmids.
It is considered that the plasmid of the present invention is deficient in a region not involved in replication in the plasmid of the present invention or in which another DNA is inserted and added to the plasmid of the present invention. It is considered to have a similar function. For example, as described in Example 9 below,
Even if a plasmid containing only about 1700 bp of a part of pSY1 is used, Lc. Replication in lactis can be performed. Therefore, the present invention is not limited to pSY1 itself, but a derivative plasmid obtained by modifying this, a selectable marker gene such as Em resistance, and L
It also includes a recombinant plasmid in which a sequence of a gene responsible for a trait to be introduced into a host such as monolactate dehydrogenase is inserted into the plasmid of the present invention.

PSY1 for the selection of transformed strains
As a marker to be added to the above, genes such as various antibiotic resistances used in the art may be appropriately used, but when the transformation organism is used for the purpose of using for food production, safety is confirmed. It is desirable to use the existing marker. An example of such a safe marker is Lc., Which is already approved as a food additive in Europe and America. la
an antimicrobial peptide produced by a particular strain of ctis,
Nisin production and resistance gene (Reference 4), Lactococcus. Examples include lactis-derived thymidine synthase gene (Reference 5).

As a new trait that can be introduced into a host using pSY1 as a vector, for example, a lactose-utilizing gene group can be introduced into a host that is not lactose-utilizing to allow growth in a lactose-containing medium such as milk, or a protease. It is possible to change the flavor of fermented products by introducing genes such as peptidase. In addition, if necessary, a nucleotide sequence involved in expression control such as a promoter may be incorporated into the plasmid of the present invention.

[0012]

EXAMPLES The present invention will be described below with reference to examples.

Example 1 Preparation of plasmid pSY1 Lactococcus lactis subsp.
Lactis M-128C strain (Meiji Dairy Health Science Research Institute holding strain; Microtech Lab deposit deposit deposit number FERM P
-12650; hereinafter sometimes abbreviated as M-128C strain), a pSY1 plasmid DNA was prepared by a method according to the method of Anderson and McKay (Reference 7). That is, the M-128C strain was inoculated into 500 ml of Lysis medium at an initial concentration of 1%, and the cells were incubated at 32 ° C for 4 hours.
Lysozyme and SD were used for the cells obtained by culturing for a period of time.
From S lysis to crude purification of plasmid DNA was performed.

The obtained crude plasmid DNA preparation was RNase-treated according to a conventional method (Reference 8), and then
The DNA band corresponding to pSY1 was cut out by agarose gel electrophoresis, and the GENEC of BIO101 was cut out.
The product recovered using the LEAN DNA purification kit was used as a purified pSY1 standard (about 5 μg).

Various restriction enzymes for pSY1 (Takara Shuzo Co., Ltd.)
The base fragment length of the obtained fragment was determined by agarose gel electrophoresis (Reference 8), and pSY1 was Ec.
It is a circular double-stranded DNA plasmid that has one recognition site for each of oRI, HaeIII, ClaI, and ScaI. The total length of pSY1 is about 2800 base pairs (bp).
Met. Also, HindII, SphI, PstI, S
alI, XbaI, BamHI, SmaI, KpnI,
There was no recognition site for SacI. In Figure 1, pSY
The restriction enzyme map of 1 is shown. Tables 1 and 2 (continuation of Table 1 (part 1)) and Table 3 (continuation of Table 1 (part 2)) show the entire base sequence of pSY1 determined by a conventional method.

[0016]

[Table 1]

[0017]

[Table 2]

[0018]

[Table 3]

Example 2 Selectable marker Em for pSY1
Addition of resistance gene The plasmid p8Em1 (see Japanese Patent Application No. 3-183922), which has an erythromycin resistance gene derived from pAMβ1 (Reference 9) as a cassette having a length of about 1.1 kb,
It was prepared according to Reference 8 from a transformant of E. coli TG1 containing Em1. p8Em1 was cut with EcoRI and pS
Ligation reaction was performed with Y1 digested with EcoRI. The reaction solution after the ligation reaction was subjected to Bacillus sub according to the method of Chang et al. (Reference 10).
DM3 containing 25 μg / ml erythromycin using transformation of the tilis 207-25 strain (Reference 11).
An Em-resistant transformant was obtained by selection on a medium plate (Reference 10).

A plasmid was prepared from the obtained transformant and its restriction enzyme cleavage pattern was analyzed. The transformant contained one of the plasmids having the restriction enzyme maps shown in FIG. 2A and FIG. 2B. The plasmid of A in FIG. 2 was named pSYE1 and the plasmid of B in FIG. 2 was named pSYE2.

Similarly, p8Em1 and pSY1 are set to Ha.
After cutting with eIII or ClaI and ligation, B. subtilis 207
-25 strain could be transformed.

As described above, pAM1 is added to pAMβ.
Recombinant plasmid p ligated with the Em resistance gene derived from 1
SYE1 and pSYE2 (both length is about 3.9k
b) could be obtained. It was also shown that pSY1 can function as a plasmid replicon in Bacillus subtilis.

Example 3 Lactococcus la
ctis subsp. Transformation of Lactis Lc. lactis subsp. IL1403 strain as lactis (Alin Ch
Distributed by Dr. Opin; Lc. lactis IL
1403 strain), and pS
By introducing YE2, Lc. l
We succeeded in obtaining a transformed strain of actis. Details are described below.

The transformed B. subtilis obtained in Example 2 s
The recombinant plasmid pSYE2 was derived from the U.subtilis 207-25 strain by the method of Anderson et al. (Reference 7).
Was prepared according to.

Then, pSYE2 was transformed into Lc. Lactis IL1403 strain was introduced.
The transformant was selected and obtained on a BL agar medium (Eiken Kagaku) plate containing 25 μg / ml of Em.

These transformants were treated with 25 μg / ml of E.
A plasmid was prepared from cells cultured in a medium containing 1% (w / v) glucose (LCMG medium) in LCM medium containing m (Reference 6) by the method of Anderson et al. (Reference 7). When the restriction enzyme site of the obtained plasmid was examined, it had the same restriction enzyme recognition site as the plasmid pSYE2 used for transformation. From this, p
SY1 contains Lc. It was shown that it is possible to function as a plasmid replicon even in Lactis.

Example 4 Escherichia coli Escherichia
Transformation of E. coli TG1 as Escherichia coli
By introducing pSY2 into the strain (Amersham),
We succeeded in obtaining a transformed strain of E. coli showing Em resistance. Details are described below.

The Lc. lactis
subsp. The pSYE2 plasmid was prepared from the transformant of Lactis IL1403 strain using the method of Reference 7. Escherichia coli TG1 strain was transformed with the obtained pSYE2 plasmid by the well-known calcium chloride method (Reference 8), and L containing Em of 500 μg / ml was transformed.
B agar medium (reference 8) was selected.

Plasmids were prepared from the cells by culturing these transformants in LB medium (Reference 8) containing 500 μg / ml of Em with 1% (w / v) glucose added according to a conventional method. . When the restriction enzyme recognition site of the obtained plasmid was examined, it had the same restriction enzyme recognition site as the plasmid pSYE2 used for transformation.
This indicates that pSY1 can function as a plasmid replicon not only in Gram-positive bacteria such as Bacillus subtilis and lactococcus, but also in Gram-negative bacteria such as Escherichia coli, indicating that pSY1 of the present invention is useful. .

Example 5 Lactobacillus
delbrückii subsp. Lactobacillus lactis transformed Lb. delbrückii subsp. lact
By introducing pSY2 into the ATCC12315 strain as is, Lb. delbrück
ii subsp. We succeeded in obtaining a transformed strain of Lactis. Details are described below.

The Lc. lactis
subsp. The pSYE2 plasmid was prepared from the transformant of Lactis IL1403 strain using the method of Reference 7. The obtained pSYE2 plasmid was transformed into Japanese Patent Application No. 3-
183922 according to the method of Lb. lactis ATC
The C12315 strain was transformed by the electroporation method. Transformants were selected on LB agar plates containing 25 μg / ml Em.

These transformants were treated with Em25 μg / ml.
From cells cultured in LCMG medium containing
A plasmid was prepared by the method of N. et al. (Reference 7). When the restriction enzyme recognition site of the obtained plasmid was examined,
It had the same restriction enzyme recognition site as the plasmid pSYE2 used for transformation. From this fact, pSY1 was identified as Lb. delbrückii subsp. l
It was shown that it is possible to function as a plasmid replicon even in actis.

Example 6 Lactobacillus
delbrückii subsp. bulgari
cus transformation Lb. delbrückii subsp. burg
aricus M-878 strain (Meiji dairy health science research institute holding stock; micromachine deposit deposit deposit deposit number FERM P
-11978) -derived BG (-) A strain by introducing pSY2 into Lb. delbrue
ckii subsp. bulgaricus transformant was successfully obtained. Details are described below.

Example 3 as a recombinant plasmid vector
Lc. lactis subsp. lac
tis IL1403 strain (hereinafter referred to as IL1403 strain)
The pSYE2 plasmid prepared by the method according to Reference 7 from the transformant strain was used.

As a host, skim milk powder medium (hereinafter referred to as SM medium; 10% skim milk powder, 01% yeast extract; 120)
The BG (-) A strain, which is one strain derived from M-878, which spontaneously lost the plasmid pBUL1 during the subculture by autoclave sterilization at 7 ° C for 7 minutes, was used.

The BG (-) A strain was inoculated into an F-LCMG medium and precultured at 37 ° C for 15 hours. F-LCMG medium is 1% glucose added to LCM medium and autoclaved at 121 ° C. for 15 minutes, and then sodium formate 5 mg
/ Ml was added, the pH was adjusted to 5.5 with HCl, and the medium was sterilized with a membrane filter (pore size 0.04 μm). Centrifuge the culture solution (3,000 rpm, 5 minutes)
20 mM Tris-HCl buffer (pH 7.0)
After washing once with F-L, it was kept at 42 ℃ in advance.
The cells were inoculated into a CMG medium at an initial concentration (OD ₆₆₀ nm) of 0.25 and main-cultured at 42 ° C. for 2 hours.

The culture broth was centrifuged in the same manner as above to give 20 mM.
After washing three times with Tris-HCl buffer (pH 7.0), electroporation buffer (hereinafter referred to as EB;
0.3 M raffinose, 2 mM K ₂ HPO ₄ (pH 7.
After washing once with 0) containing 1 mM MgCl _2, it was suspended in EB so that the turbidity (OD ₆₆₀ nm) was 50. This suspension (referred to as cell suspension) was kept at 45 ° C. for 30 minutes before electroporation.

Transformation by the electroporation method was carried out using Gene Pulser (registered trademark) manufactured by Bio-Rad, USA. Recombinant plasmid pSYE2 (about 1 μg / ml TE buffer) 1-2 prepared from IL1403
Into a 0.2 cm cuvette, 80 μl of the cell suspension was added, and an electric pulse (1.5 kV / 0.2) was added.
(cm, 25 μF, 200Ω) was applied once.

After the electric pulse was applied, the whole cell suspension in the cuvette was inoculated into an expression culture medium (referred to as EXBG) and cultured at 37 ° C. for 2 hours to recover the damage of the cell due to the electric pulse. . EXBG medium was a 1: 1 (v / v) mixture of a solution containing 0.3M raffinose, 2% casamino acid and 50 mM MgCl ₂ (sterilization filtration with a 0.45 μm pore membrane filter) and SM medium. It is a culture medium.

After culturing in EXBG medium, the number of transformants was 2
Litmus milk containing 5 μg / ml Em (Difco
Company) selected on the plate.

These transformants were treated with Em 25 μg / ml.
After culturing in LCMG medium containing
When a plasmid was prepared by the method of Son et al. (Reference 7), a plasmid showing the same electrophoretic mobility as the plasmid pSYE2 used for transformation was obtained. From this fact, Lb. delbrückii subsp. bu
It was shown that it is possible to function as a plasmid replicon even in lgaricus. Lb. del
It is clear that pSY1 of the present invention is useful as a vector for Brueckii lactobacillus.

Example 7 Lb. delbrueckii
subsp. Introduction of L-lactate dehydrogenase gene-incorporated pSY1 into Lactis and transformed Lactococcus Streptococcus salivar
ius subsp. thermophilus M-
Gene encoding L-lactate dehydrogenase from 192 strain (Meiji Dairy Health Science Research Institute owned strain)
-251172; hereinafter referred to as ST-LDH), a restriction enzyme SspI fragment (about 1.2 kb) was added to pUC11.
PU8ST9 plasmid obtained by inserting it into the HincII recognition site of 8 plasmid (Taboo Shuzo Co., Ltd.)
183922) is cleaved with restriction enzymes BamHI and KpnI, and subjected to agarose gel electrophoresis to give ST-LD.
The portion containing the DNA fragment containing H was excised and then isolated using GENECLEAN DNA purification kit. This DNA and pSYE2 obtained in Example 2 were combined with B
What was cleaved with amHI and KpnI was ligated. Lc. lactis IL
When the 1403 strain was transformed, a transformant carrying the plasmid pSYEL29 as shown in FIG. 3 was obtained.

The pSYEL29 plasmid was prepared by the method described in Reference 7, and Lb. de
lbrueckii subsp. lactis AT
It was introduced into strain CC12315. As a result, transformants showing Em resistance were obtained, but these retained a plasmid having the same restriction map as the introduced plasmid.

These transformants were cultured in a nonfat dry milk medium, the culture solution was diluted with distilled water, and the lactic acid in the centrifugal supernatant was measured with a lactic acid measurement kit (F kit L-lactic acid; Boehringer). However, L-lactate, which is not originally produced by this host bacterium, was detected in almost the same amount as D-lactate.

Furthermore, when the transformant was crushed and the cell extract thereof was examined, L-lactate dehydrogenase activity which was not detected in this host bacterium was detected. When this L-lactate dehydrogenase was purified and the N-terminal amino acid was examined, ST-LDH
Was the same as From the above results, pSY1 was used as a replicon for Lb. delbrückii subsp.
It was shown that it is possible to express the heterologous gene ST-LDH in Lactis.

Example 8 Ligation of pSY1 and E. coli plasmid The p8Em1 plasmid prepared in the same manner as in Example 2 was cleaved with the restriction enzyme HindIII, and a 1.1 kb DNA fragment corresponding to the Em resistant gene was subjected to agarose gel electrophoresis. It was excised from the latter gel and isolated using the GENECLEAN DNA purification kit. The fragment was ligated with a fragment obtained by blunting the ends using a DNA planting kit (Takara Shuzo Co., Ltd.) and a fragment obtained by similarly blunting the ends after cutting pUC118 with restriction enzymes EcoRI and SphI. E. coli TG1 strain was transformed with
By selecting at m500 μg / ml, the plasmid p8Em3 shown in FIG. 4A was prepared. Similarly, p8E
m1 cleaved with the restriction enzyme EcoRI and blunted,
The plasmid p8Em5 shown in FIG. 4B was prepared by using pUC118 digested with restriction enzymes HindIII and SacI and blunted.

P8Em3 and p8Em5 were prepared from the TG1 transformants containing the respective plasmids by a conventional method. PS prepared by the method described in Example 1
The Y1 plasmid was cleaved with the restriction enzyme ScaI to give p8Em.
After ligation with 3 digested with the restriction enzyme SmaI, the reaction solution was treated with T by the same method as in Example 4.
The G1 strain was transformed, and the transformed strain was selected on an LB agar medium containing 50 μg / ml of ampicillin (hereinafter sometimes abbreviated as Ap). The transformant obtained is shown in FIG.
It had the plasmid pE3SY1 as shown in C. Similarly, the pSY1 plasmid was digested with the restriction enzyme ScaI and the p8Em5 was digested with the restriction enzyme HindIII, and then ligated with the blunt-ended one to prepare the plasmid pE5SY1 as shown in FIG. 4D. These transformants were Em resistant (500 μm).
It also had g / m1).

The pE3SY1 and pE5SY1 plasmids were prepared from the TG1 transformants according to a conventional method (Reference 8), and Lc. 1 actis
The IL1403 strain was transformed, and the transformed strain was transformed into E strain.
Selection was performed on BL agar plates containing m25 μg / ml. The plasmid retained by the obtained transformant,
All had the same restriction map as the plasmid prepared from TG1. From the above, it was revealed that by combining pSY1 with an E. coli plasmid, E. coli can be used as a shuttle vector that can be selected by Ap and Em, and lactococcus by Em.

Example 9 Estimation of Region Required for Replication of pSY1 by Deletion Method The pE3SY1 plasmid obtained in Example 8 was digested with restriction enzymes BamHI and PstI to prepare a DNA deletion kit for kilosequencing (Takara Shuzo Co., Ltd.). ) Was used to make the deletion. The reaction solution was transformed into Escherichia coli TG1 strain in the same manner as in Example 4 to obtain a series of deletion plasmids in which the pSY1 portion was deleted in various lengths. These deletion plasmids were prepared from a transformant of TG1 and then transformed into Lc. lactis IL1403
Transformed into strains and selected on BL agar plates containing 25 μg / ml Em. When a plasmid carried by a transformant showing Em resistance was prepared and its restriction enzyme recognition site was examined, it was found that at the shortest obtained, the position shown in FIG. 5, that is, the unique HaeIII recognition site was detected. The deletion occurred to the immediate vicinity of. Similarly, pE5SY1
When the plasmid was cleaved with the restriction enzymes BamHI and KpnI and deleted, the shortest transformant obtained with the IL1403 strain was about 100 bp EcoRI from the unique ClaI recognition site shown in FIG. A deletion up to the position of was occurring. From the above, pSY1
It was found that the region required for replication of at least the region of about 1.7 kb indicated by the bold line in FIG.

References 1. Holo, H. and IF Nes, (1989) Appl. Environ. Mi
crobiol. 55 (12) 3119-3123. 2. Mercenier, A., (1990) FEMS Microbiol. Rev., 87,
61-77. 3. Chassy, BM and JLFlickinger, (1987) FEMS Mi
crobiol, Lett., (44) 173-177. 4. Kaletta, C. and KD Entian, (1989) J. Bactelio
l., 171, 1597-1601. 5. Ross, P. et al., (1990) Appl. Environ. Microbio
l., 56, 2164-2169. 6. Efthymiou, C, et al., (1962) J. Infect. Dis., 1
10, 258-267. 7. Anderson, D. and LL Mckay, (1983) Appl. Envir
on. Microbiol. 46, 549-552. 8. Manistis, T, et al., (1982) Molecular Cloning:
A Laboratory Manual, Cold Spring Harbor Laborator
y, Cold spring Harbor, New York. 9.LeBlanc, DJ and LN Lee, (1984) J. Bacterio
l., 157, 445-453. 10. Chang, S. and SN Cohen, (1979) Mol. Gen. Gen
et, 168, 111-. 11. Yamane K. et al., (1984) J. Biochem, 96, 1849-1.
858.

[0051]

The plasmid pSY1 of the present invention is a cheese-producing bacterium Lactococcus lactis su.
bsp. Since it was isolated from a strain of Lactis, its safety has been confirmed historically.
Moreover, the genus Bacillus and Lactococcus
Genus, and Lac, which has been extremely difficult to transform in the past
2 of Tobacillus delbrueckii species
It is capable of replicating not only important bacterial species such as lactis and bulgaricus in the food industry, but also Escherichia coli, which is the most widely used microbial host, and has the ability to transform those bacteria. . In addition, the overall size is about 2.8 kb, which is relatively small, and various handling in gene recombination can be easily performed. From the features listed above,
The plasmid of the present invention is suitable as a vector for a microorganism for food production, and it is expected that a novel useful microorganism can be bred using the plasmid of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS The abbreviations and the official names of the restriction enzymes used in the following drawings are shown below. B
m: BamHI, Cl: ClaI, Ec: EcoRI,
Hd: HindIII, III: HaeIII, Kp: Kpn
I, Ml: MluI, Sa: SacI, Sc: Sca
I, Ss: SspI, Sp: SphI

FIG. 1 is a restriction map of pSY1. The recognition site of each restriction enzyme is shown in kb units with ScaI as a reference (pSY1 is circular, but shown linearly with ScaI as a reference). In addition, HindIII, SphI, Pst
I, Sa1I, XbaI, BamHI, SmaI, Kp
There were no recognition sites for nI and SacI.

FIG. 2 is a restriction map of pSY1 derivatives, pSYE1 (A in FIG. 2) and pSYE2 (B in FIG. 2).
In the figure, the double line indicates the sequence of pSY1 and the thick arrow indicates pAMβ1
The erythromycin resistant gene (and its transcription direction) of origin is shown.

FIG. 3 is a restriction map of plasmid pSYEL29. Vertical stripe arrows in the figure are Streptococcus s.
alivarius subsp. thermophi
lus M-192 strain L-lactate dehydrogenase (ST-L
DH) gene (and its transcription direction).

FIG. 4: Plasmids p8Em3, p8Em5, pE3S
The restriction map of Y1 and pE5SY1 is shown (both of which are circular, but TCGC in pUC118
It is shown linearly based on the first T of GCGTTC). In the figure, the solid line represents the sequence derived from pUC118, the double line represents the sequence of pSY1, the bold arrow represents the erythromycin resistance gene derived from pAMβ1 (and its transcription direction), and the thin arrow represents the ampicillin resistance gene derived from pUC118 (and its transcription direction). Show.

FIG. 5 is an estimation of the essential replication region of pSY1. In the figure, the arrow indicates the deletion direction, and the bold line indicates the range presumed to be the replication essential region. Although pSY1 has a ring shape, it is shown as a straight line with ScaI as a reference.

─────────────────────────────────────────────────── ─── Continued Front Page (51) Int.Cl. ⁷ Identification Code FI C12R 1:19) C12N 15/00 ZNAA (C12N 1/21 C12R 1:01) (C12N 1/21 C12R 1: 225) (C12N 15/09 ZNA C12R 1:01 1:19) (56) Reference JP-A-4-211384 (JP, A) European Patent Application Publication 228726 (EP, A 1) Applied Environmental Microbiology, 1990, Vol. 56, No. 1, p. 202-209 FEMS Microbiology Letters, 1991, Vol. 78, p. 207-212 Applied Environmental Microbiology, 1984, Vol. 48, No. 4, p. 726 −731 (58) Fields investigated (Int.Cl. ⁷ , DB name) C12N 15/00-15/90 C12N 1/21 BIOSIS / WPI (DIALOG) PubMed JISST file (JOIS)

Claims (17)

(57) [Claims] 1. A circular double-stranded plasmid pSY1: having the following characteristics: (a) The number of base pairs of about 2.8 kbp shown in FIG. 1 below, and unique EcoRI, HaeIII, ClaI, and S.
It has a caI recognition site; [FIG. 1] (B) HindIII, SphI, PstI, SalI,
Does not have XbaI, BamHI, SmaI and KpnI recognition sites; (c) Lactobacillus lactobacillus delbr
It has a replication region in Gram-positive bacteria including Ueckii species and Gram-negative bacteria including Escherichia coli; and (d) It has the nucleotide sequence of SEQ ID NO: 1 shown in Table 1, Table 2 and Table 3. 2. Lactococcus lactis
subsp. The circular double-stranded plasmid pSY1 according to claim 1, which is derived from the Lactis M-128 C strain (accession number: FERM P-12650). 3. A unique HaeIII in the direction of the right arrow of pSY1 shown in FIG. 2 below from the full length of pSY1 according to claim 1.
An autonomously replicable circular double-stranded plasmid represented by the remaining approximately 1700 bp thick line region deleted up to the immediate vicinity of the recognition site and up to approximately 100 bp past the unique ClaI recognition site in the direction of the left arrow. [Fig. 2] 4. A replication region of the plasmid according to claim 3,
And a recombinant plasmid comprising at least one selectable marker gene. 5. A plasmid pSY shown in FIG. 3A below.
E1 or the plasmid pSYE2 shown in Figure 3B below.
The recombinant plasmid according to claim 4, which is [Figure 3] 6. The recombinant plasmid according to claim 4, which contains a foreign gene and a region relating to the expression control of the foreign gene. 7. A plasmid pSYE shown in FIG. 4 below.
The recombinant plasmid according to claim 6, which is L29. [Figure 4] 8. A transformant containing the recombinant plasmid according to any one of claims 4 to 7. 9. The transformant Bacillus subti according to claim 4, which contains pSYE2.
lis. 10. The transformant Lactococcus l according to any one of claims 4 to 7, which contains pSYE2.
actis subsp. lactis. 11. The transformant Lactobacillus according to any one of claims 4 to 7, which contains pSYE2.
delbrückii subsp. lacti
s. 12. The transformant Lactobacillus according to claim 4, which contains pSYE2.
delbrückii subsp. bulgar
icus. 13. The transformant Escherichia c according to claim 4, which contains pSYE2.
oli. 14. A plasmid pSY shown in FIG. 5 below.
Lactobacillus delb including EL29
rueckii subsp. lactis ATC
C 12315 strain. [Figure 5] 15. A shuttle vector having the replication region of claim 3, a replication region in E. coli cells, and a selection marker. 16. The method according to claim 1, which is pE3SY1 shown in FIG. 6C below or pE5SY1 shown in FIG. 6D below.
The shuttle vector according to item 5. [Figure 6] 17. A Lactococcus lactis containing the circular double-stranded plasmid pSY1 according to claim 1.
subsp. Lactis M-128C strain (accession number: FERM P-12650).