JPH01304885A - Novel cloning vector - Google Patents

Abstract

PURPOSE:To obtain a cloning vector capable of autonomic replication in Streptomyces.sannanensis strain by forming a plasmid derived from a bacterium of the genus streptomyces capable of the autonomic replication in the above- mentioned strain. CONSTITUTION:A recombinant cloning vector which is a recombinant plasmid,, having a DNA fragment containing a gene capable of expressing a genetic character in Streptomyces.sannanensis strain integrated in a vector plasmid capable of autonomic replication in the Streptomyces.sannanensis strain and capable of autonomic replication in the Streptomyces.sannanensis and discriminating the presence of the introduced gene based on the expression thereof is prepared. A DNA fragment containing a drug-tolerant gene is preferred as the above-mentioned DNA fragment. pEN100, pEN200, pEN300, etc., are cited as specific examples of the vector plasmid.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of Industrial Application The present invention is directed to two types of microorganisms belonging to the genus Streptomyces, namely Streptomyces sp.
FERM 0P-1619) and three plasmids pENloO, pEN200. derived from Streptomyces sp^T[:C21274. The present invention relates to a cloning vector in which a DNA fragment containing pEN3Qo or a region necessary for its replication is ligated to a DNA fragment containing an appropriate selection marker. The plasmid of the present invention is a Streptomyces sannanensis (SLr)
Since it can autonomously replicate in bacterial species (Eptomyces 5annanensis), it is expected to establish a new host-vector system in Streptomyces species.

Conventional Techniques Genetic recombination techniques for Streptomyces species have been rapidly developing in recent years [see F. Chater et al.;
Current Topics in Microbiology
And Immunology ([:current topic
sin Microbiology and Immu
nology), 96 +69-95 (1982)
'].

However, vectors that have been reported and frequently used,
For example, ρIJ41 (low copy number vector) [C0J,
Thompson et al.; Gene 20,
5l-62 (1982)] has an extremely narrow host range, and the high copy number vector plJ702 [E, ats
; Journal of General Microbiology (J, Gen.

Microbiol, ) 129.2703-271
4 (1983)] is said to have a relatively wide host range, but there are many actinomycetes such as Streptomyces sannanensis for which it is impossible to introduce this vector, and it is structurally unstable. I'm having some problems.

Problem to be solved by the invention
of Antibiot4x (J, Antibiot
ics) 32(10), 1061. (1979)]
It is known as a production bacterium.

With the advancement of gene recombination technology for actinomycetes, biosynthetic genes for various antibiotics have been cloned and analyzes at the genetic level are progressing. However, since no vector capable of autonomous replication has been developed in Streptomyces sannanensis, the bacterium that produces sannamycin, cloning and analysis of sannamycin biosynthetic genes have not progressed.

In order to elucidate the biosynthetic system of sannamycin, it is desired to develop a host-vector system for Streptomyces sannanensis.

Moreover, since the new vector that makes this possible has a host range that goes beyond the host range of conventionally used vectors, it can be expected to be widely used in the development of host-vector systems for Streptomyces species.

Means for Solving the Problems In order to elucidate the biosynthetic pathway of sannamycin and clone the genes involved in the production of this antibiotic, the present inventor developed various vectors capable of autonomous replication in Streptomyces sannanensis. investigated. As a result, three types of plasmids capable of autonomous replication in Streptomyces sannanensis were obtained from 2:1 microorganisms belonging to the genus Streptomyces. Furthermore, a cloning vector expressing thiopepsin resistance as a vector marker was constructed by incorporating a DNA fragment expressing thiopepsin resistance into the plasmid.

The present invention will be explained in detail below.

The present invention provides a plasmid derived from Streptomyces and capable of autonomous replication in Streptomyces sannanensis.

Specifically, Streptomyces sp. S-'51
4-16 (FεIIM BP-1619>) and plasmid pBN300 derived from Streptomyces sp. TCC21274. These plasmids are plasmids (Plasmids) 12 .

19 (1984).

Plasmid p[! N100 is a circular DNA with a size of 4.6 kilobases (bib), has sensitive sites for restriction enzymes as shown in Table 1, and has [lamH[, C
J! a[.

Not cleaved by HindI[1 right and Pst[.

pi! N100 is a plasmid with a copy number of approximately 160 copies per cell.

Table 1 Restriction enzyme cleavage characteristics of pBNloo Restriction enzyme Cutting location Length of resulting DNA fragment (Kb) Bcj
! I 3 3.1.1.2.0.3B
gI111 1 4.6BstE[I
2 4.3.0.3EcoRI 1
4.6 pnl 3 2.1.
2. G, 0.5I'vu IT 1
4.6Sac 1 1 4.6 SafGI 4 2,7.1,1.0.5
．． 0.4Sstll l' 4.6Xba
I l 4.6 Blasmid PEN2
00 is a circular DNA with a size of 10.7 Kb and has a sensitive site for restriction enzymes as shown in Table 2,
Not cleaved by IJaI, former ndlIl and pstI. ρ[EN200 is a plasmid with a copy number of approximately 40 copies per cell.

Table 2 Restriction enzyme cleavage characteristics of ρ1iN200 []
amH145,8,3,1,IJ, 0.6BcI! I
3 4.5.4.1.2.1BgβI
I 2 8.2, 2.5BstE■
3 7,9.1.9.0.9EcoRl
1 1G, 7Kpnl 3
9.4.0.9.0.4Mβul 3
5.6.3.1.2.0Pvu11 3
9.9.0.6.0.2Sac l 1
10.7 Plasmid ρEN300 has a size of 8
．． It is a 7Kb circular DNA and has sensitive sites for restriction enzymes as shown in Table 3, including BcβI and βII.
, (J!al.

[1coRI, HindIII; not cleaved by Pst I and Pvu II. ρ[EN300
- is a plasmid with a copy number of approximately 10 copies per cell.

Table 3: Cutting characteristics of ρEN300 using restriction enzymes Restriction enzyme Cutting location Length of resulting DNA fragment (Kb) nam
HI l 8.3Kpn 1 1
8.3 1.1! t+I 4 4.2.2.2.1
．． 2.0.70.1 Sma! 18.3 The above three plasmids are plasmids capable of autonomous replication in Streptomyces thannanesis species, but do not have appropriate vector markers that can identify the presence of the plasmids. In order to establish a host-vector system in the Streptomyces sannanesis species, cloning vectors with appropriate vector markers that can identify their presence are preferred. Such a cloning vector can be obtained by utilizing the autonomous replication ability of the plasmid described in 1. and inserting an appropriate vector marker.

Any vector marker may be used as long as it expresses a compelling trait in the Streptomyces thannanesis species and can impart that trait to the host.

Examples include resistance genes for streptomycin, neomycin, thiopeptin, and the like. Also,
If the host is an auxotrophic mutant strain, a gene that complements the auxotrophy may be used.

As a recombinant cloning vector that is capable of autonomous replication in Streptomyces sannanesis and has a vector marker that can identify the presence of the vector, the above-mentioned ρBN100. pEN200 or ρI':N30
The recombinant cloning vector pfEN101.0 utilizes the autonomous replication ability of pfEN101.0 and inserts a thiopeptin resistance gene as a vector marker. pEN201. pEN30
1 is given. The plasmids are pBNIoo, p
Cut EN200 or pBN300 at a restriction enzyme-sensitive site that does not lose autonomous replication ability, and insert plJ7 there.
02 [Journal Gen Microbiology (J, Gen, Microbiol,) 12
9.2703-2714 (1983)) by inserting a DNA fragment containing the thioveptin resistance gene.

Plasmid p[1N101 contains pENloo with restriction enzyme Bcj! This is a circular DNA with a size of 5.7 Kb constructed by partially digesting with ρIJ702 and inserting the thiopectin resistance gene derived from ρIJ702, and has sensitive sites for restriction enzymes as shown in Table 4. Also, BamHI
.

11ind, [II and not cleaved by PstI.

Bcj! I 4 3.1.1.2.1
．． 1. O13Bgi'■ 15.7 BstεII 2 5.4.0.3IJ
pnl to al 1 5,7 EcoRI 1 5.7
3 3.2.2.0.0.5PvuII
2 3.8.1.9Sac!
1 5.7SaiGI 5 2.7
．． 1.7.1.4.0.5.0.4SstII
2 For 4.9.0.8 plasmid p[iN2 old, pEiN200 was partially digested with restriction enzyme BamHI,
A circular DNA with a size of 11.8 and b constructed by inserting the thiopebutin resistance gene derived from ρ1J702.
It has sensitive sites for restriction enzymes as shown in Table 5. Also, it is not cut by Hind[n and Pst[.

Table 5 Restriction enzyme cleavage characteristics of ρBN201 Restriction enzyme Cutting location Length of generated ON^ fragment (Kb) Bam
lll 4 6.8.3.1.1.3.
0.6 Bcj! 1 4 4.5.4.1
．． 2.1.1. IBgJ! ■ 2 8.2
．． 3.60stBII 3,
9.0. 1.9. 0.9Cj! al
l 11.8εcoRI l
11.8Kpnl 3 9.4.
1.5.0.9MJ! ul 3 5.6
．． 4.2.2.0PvuIl 4 7.
6.3,4.0.6.0.2Sacl 1
11.8Sma I 4 3.8.
3.3.2.6.2.1 Plasmid ρεN301 is p
Restriction fermentation of H1N300! Cut with BamHI and plJ7
The size of the 10. It is a circular DNA of IKb, and has sensitive sites for restriction enzymes as shown in Table 6. Also, 13cj! I, BgA'U, BcoR
I, 1lind'l[I and Pst[l are thus not cleaved.

Table 6: Restriction enzyme cleavage characteristics of pεN301 Ram
tll 2 8.3. 1.8CIla
l 1 10.1Kpn I
l 10. IMJul 5
3.2.2.8.2.2.1.2.0.7PvuII
1 10.10.2.0.2.0.1.
0.1 Small 2 6.4.3.7 3 above
The seed plasmid' is a plasmid that can autonomously replicate in the Streptomyces sunnanensis species, and when introduced into a host, it expresses thiopeptin resistance, so the presence of the vector can be easily identified.

pENlol, pEN201, and pBN301 were each introduced into Streptomyces lividans T at 23 days, and each was transferred to Streptomyces lividans εN at the National Institute of Microbiology, Agency of Industrial Science and Technology on December 15, 1988. -101 (FεRM 0P-1620
), Streptomyces lividans εN-201 (F
ER310P-1621), Streptomyces lividans E! It has been deposited as NN-301 (FER0P-1622).

The plasmid of the present invention can be introduced into the following actinomycetes in addition to the Streptomyces sunnanensis species.

(S, caespitosus) Streptomyces griseus (S, griseus)
s) Streptomyces lividans (S, 1ivid
ans) Streptomyces reticuli (S, re
ticuri) Streptomyces venezuela (S,
Venezuela) is more effective. Such restrictions can be lifted using the method of Lomovskaya et al.
logical Review), 44, 206 (+
980):].

To introduce the above plasmid into a host microorganism,
F. Cha'ter et al.'s method [Current Topics in Microbiology and Immunology ((:current Topics in l
Jicrobiology and Immuno
96, 69-95 (1982)],
Protoplasts are prepared and the plasmid is introduced (details are shown in Examples).

Examples are shown below.

Example 1. - Plasmid pEN 100. ρ1EN200 and ρE
Collection of N300 (+) Culture Streptomyces sp^T (:C21274t
or Streptomyces sp. S5-514-16
(FER0P-1619) in 4 ml of YEME medium [yeast extract (Difco Co., Ltd.!a) 3.0 g, Batatopebutone (Difco Co., Ltd. a!ri 5.0 g, malt extract (Difco Co., Ltd.) 3.0 g and glucose 10 .0
Dissolve g in pure water if and adjust to ρ147.2, 120
The medium was autoclaved at 30°C for 20 minutes] and cultured with shaking at 30°C for 2 to 3 days. Then, transfer the entire amount to 500ml of S.

2 Medium [Stabilose K (manufactured by Matsutani Chemical Industry Co., Ltd.) 20 g.

Glucose 5g, yeast extract (Daigo Nutrient Chemical Co., Ltd.!! Ri) 5g, peptone (Kyokuto Pharmaceutical Co., Ltd.) 3g, 112PO
40.2g and Mg5L・7H200.6g in pure water 1
1, adjusted to pH 7.6 and sterilized under high pressure at 120°C for 20 minutes], and cultured with shaking at 30°C for 2 to 3 days to obtain a culture solution.

(2) Isolation of plasmid The culture solution obtained as above was centrifuged (10 minutes,
The cells were collected at 4°C, 12.00 rpm).

40-50 ml of lytic solution containing 5 mg/ml of egg white lysozyme (Seikagaku Corporation) [10.3% sucrose, 25 ml of lysozyme (Hydroxymethyl)]
Aminomethane (hereinafter abbreviated as Tris)-hydrochloric acid buffer (pH
8,0), 25mM disodium ethylenediaminetetraacetate (HOT^)] and incubation for 30 minutes at 37°C, followed by 30ml of 0,3N hydroxide solution containing 2% sodium dodecyl sulfate) +7um solution. was added and left at room temperature for 10 minutes. Next, heat it at 75℃ for 10 minutes, cool it by immersing it in a water bath, and then add three-quarters volume of phenol/chloroform C500g of phenol, 500ml of chloroform, and 800mg of 8-hydroxyquinoline (manufactured by Tokyo Kasei Kogyo Co., Ltd.). After mixing, 200m1
of deionized water and shake well to obtain a lower layer] and stirred vigorously for 30 seconds. Centrifugation (10 minutes,
Collect the upper layer obtained by 15°C, 12,000 rpm), add 1/10 volume of 314 sodium acetate solution, then add an equal volume of isopropyl alcohol containing 2% Triton x 100, and mix well. ,
After leaving at room temperature for 30 minutes, 5℃ at 4℃ and 8.00Orpm.
Centrifuged for minutes.

The resulting pellet was mixed with TE buffer [10mM], J-sulfur acid buffer (pH 8,0), and 1mM EDT^12.
R dissolved in 0ml and heat-treated at 80°C for 10 minutes.
Add N^-degrading enzyme (type 1 to ribonuclease, manufactured by Sigma) to a final concentration of 20 μ/ml and incubate at 37°C.
and incubated for 30 minutes. Then 2ml of 3
After adding M sodium acetate and 20 ml of isopropyl alcohol and mixing, the mixture was left at 4°C for 30 minutes and cooled and centrifuged (5 minutes, 4°C, 8.OOOrpm>) to obtain a pellet. This was added to TE buffer. 8.00 g of cesium chloride was added to 8 ml of a solution in which ethidium bromide was dissolved and ethidium bromide was added to a final concentration of 0.75 mg/ml, and the mixture was centrifuged at 105,000×g and 20° C. for 40 hours.

Through this density gradient centrifugation, a circular DN obtained by covalent bonding
A was detected as a specific band that fluoresced when irradiated with an ultraviolet lamp. Remove this band using a syringe needle and remove the culture ethidium by shaking several times with isoamyl alcohol saturated with 11 buffer, then add 3 volumes of TE buffer and then add 1/10 volume of TE buffer. 3M1W:l'i! ) IJum and equal volumes of isopropyl alcohol were mixed and allowed to stand for 30 minutes under water cooling. 12.00 Orp for 5 minutes at 4°C
The pellet was collected by centrifugation, which was further washed with cold 70% ethanol and smoked dry under vacuum. 0.3m1 T
It was dissolved in E buffer and stored at -20°C. By this operation, pεN100 is 200-300ug, pEN20
0 is 100-150μg and ρEN300 is 50-6
0 μg was obtained.

Example 2゜(1) Obtaining a DNA fragment containing the thiopeptin resistance gene from plJ702 ρ1J7026.5M prepared in the same manner as in Example 1
[Reaction volume: 30 d, M buffer used, reaction at 50°C for 2 hours, composition of M buffer according to "Manual for Genetic Engineering" (Cold Spring Harbor Laboratory) p. 228 (1980) . Come on, phenol/chlorophor!・Add evenly and stir vigorously for 30 seconds, then for 5 minutes. 5-20℃, 12. It was centrifuged at OQ Orpm. The obtained supernatant was further subjected to the same operation and the supernatant was collected in the same manner.

(2) Construction of cloning vector pEN101 The ρεNIQO1,bg obtained in Example 1 was injected into the restriction enzyme port c
It was dissolved in a 50 d reaction solution (using 11 buffer) containing 5 units of Rt O, and reacted at 37° C. for 10 minutes for partial digestion, and then 0.5 M BUT^ was added to stop the reaction. Add 50M phenol/chloroform to this, shake vigorously for 30 seconds, and then heat at 15-20°C for 5 minutes at 12.0°C.
Centrifuge at 0 rpm and remove the supernatant (1). Add 5IL1 sodium monoacetate and 55m isopropyl alcohol, leave to stand for 30 minutes under water cooling, and then heat at 4°C for 5 minutes.
A DNA pellet was obtained by centrifugation at 2.00 rpm. Deionized water was added to make up to 90 animals, 10-IM) Lis-HCl buffer (pH 8.6) was added, and then 1.5 units of alkaline fut-sphatase (Boehringer 1) was added.
(for molecular biology) was added and incubated at 37°C for 45 minutes. After adding 100 m of phenol/chloroform and stirring vigorously for 30 seconds, the mixture was centrifuged at 15-20°C for 5 minutes at 12.00 rpm to obtain a supernatant. Mix the supernatant obtained by performing this operation once again with the supernatant of the DNA fragment containing the thiopectin resistance gene obtained in (1), and add 1/10 volume of 3M acetic acid. Alcohol was added and mixed, and the mixture was allowed to stand for 30 minutes under water cooling. The pellet was centrifuged again at 12.00 Orrpm for 5 minutes at 4°C, and the resulting pellet was washed with cold 70% ethanol9 and dried in vacuum. To this, ligation buffer C6 of 204, 6mM'magnesium chloride, lomM dithiothreitol (manufactured by Gyudon Chemical Co., Ltd.) and 0.8mM
adenosine) IJ phosphate (Sigma 5!l)
) containing 66mM) Lis-HCl buffer (pH 7,6
):] was added, and 1 unit of T4 ligase (manufactured by Boehringer) was added, followed by incubation at 15°C overnight.

To this reaction solution, 804 P-medium 14 [sucrose lO, 3 g, K2SO425 mg, MgCj!
2.61120203 mg and fine metal element liquid 0
．． Mix 2 ml with deionized water to make 90 ml and add 0.25 of lOm I! J TES [N-)lis(hydroxymethyl)-methyl-2-aminoethanesulfonic acid] buffer (p11' 7.2), 0.5 ml of 5M
Calcium chloride, 1 ml of 0.5% KH, P was added. All solutions should be sterilized under high pressure; see below.
-It is called a solution. ] to this, F, Chat
er et al.'s method [Current Topics in Mike P.
Biology and Information Science 96. ．． 69-
95 (1982)] protoplast suspension of Streptomyces lividans TK-23 strain 50
After adding m and gently mixing, the mixture was left at room temperature for 1 minute. 60
0- T-medium [25% volume of polyethylene glycol 1000 in P-solution (Gyudon Kagakusha:!A)
(hereinafter referred to as T-solution) was added and stirred, and then diluted 10 to 1000 times with P-solution.

100 ρ of this diluted solution was added to R5 regeneration agar medium [sucrose 103 g, K2S010.25 g, Mg[R2
・fi fl -010,12g, glucose log
, Casamino Acid (Difco!! Ri Ig, Yeast Extract
(manufactured by Difco) 5g, trace gold 14 element liquid 2ml, T
BS 5.73g.

2.5 g of sodium hydroxide was made into Il with deionized water, and p
Adjusted to H7.2. Divide this into 5 equal parts, add 4.4 g of Batato Agar (manufactured by Difco), and sterilize under high pressure. One of these contains 3 ml of 20% sodium glutamate, 3 ml of solution 2% NaNL and 0.8 ml of 51J C
Add aCR2 and spread 20ml each onto plates.
It was dried for one and a half hours and applied to the prepared product. 30℃
After incubation for 15 hours, add 2.5 ml of soft agar medium Cog's Nutriendobroth (Day 7 Co., Ltd. 5 ml) containing the antibiotic thiopectin at a concentration of 0.5 mg/ml and pre-maintained at 42°C. and 5 g of Batato agar, autoclaved with 1 f! of deionized water] and further cultured at 30°C for 3 to 4 days. The colonies were transferred to a medium and further cultured for 3 to 4 days at 30"C. It is thought that the colonies that have grown carry a recombinant plasmid having a thiopeptin resistance gene, and this plasmid is shown in Example 1. The plasmid was collected using the same method and named plasmid pENlol.

(3) Construction of cloning vector pBN201 pEN2001.5■ obtained in Example 1 was added to the reaction solution (i,
Restriction enzyme ffiDaml in 50uσ (using buffer)
Partial digestion was achieved by incubation with 10.5 units of 10.5 units at 37°C for 10 minutes, and the reaction was stopped with 0.5M EDTA of 5I. Add 50 μQ of phenol/chloroform to this, shake vigorously for 30 seconds, and then heat at 15°C for 5 minutes.
12. The supernatant was obtained by centrifugation at OOOrpm.

Add deionized water to this to make 90uQ, and then add 10μQ.
IM) Lis-HCl buffer (pl+ 8.6); 1.
Add 5 units of alkaline phosphatase and incubate at 37°C.
and incubated for 45 minutes. In addition to this, (1
) Bcβ [DNA
The fragment was ligated in the same manner as in Example 2 (2), and Streptomyces lividans TK23 was transformed.

The plasmid possessed by the obtained transformed strain was isolated and named plasmid pEN2 old.

(・1) Construction of cloning vector ρEN301 1.5 μg of pEN300 obtained in Example 1 was added to the reaction solution of 50 animals (using Ill buffer) with 4 units of restriction enzyme Ba.
The mixture was incubated with m1lI at 37°C for 2 hours to complete digestion. Add 50 heads of phenol/chloroform to this, stir vigorously for 30 seconds, and hold for 5 minutes at 15℃, 12.00O
Centrifugation was performed at rpm to obtain a supernatant. Phenol/chloroform was added again and the same operation was performed to obtain a supernatant. Example 2 (
Perform one crop using the same method as shown in 2), isolate a plasmid from the obtained transformed strain, and use plasmid pEN3.
Named old.

Example 3゜Plasmid pEN100゜PEN200. obtained in Examples 1 and 2. ρB11300. ρεNl0I, pE
N2old and ρEN301 were each digested with various restriction enzymes and analyzed by agarose gel electrophoresis, and restriction enzyme cleavage maps were prepared as shown in FIGS. 1 to 6.

Example 4 Transformation of Streptomyces lividans TK23 with pIENIO1, pEN201 and pENMAR
EN101. 23 into Streptomyces lividans T carrying pEN201 and pEN301, respectively.
A plasmid was generated from the transformed strain using the same method as in Example 1. 1' [and 1 μg of them were used to transform Streptomyces lividans TK23. The method of transformation and regeneration was exactly the same as the method described in Example 2 (2). All of the regenerated transformants exhibiting thiopebutin resistance retained the same structure as the plasmid used for transformation.

From the number of regenerated colonies, the transformation efficiency of each plasmid vector was calculated. The results are shown in Table 7, and all vectors had high transformation efficiency.

Table 7 Transformation Efficiency Example 5 Transformation of Streptomyces sannanensis with ρεNl0I, pHN301 Streptomyces lividans T carrying pENlol, PEN301 was transformed from 23 transformants in the same manner as in Example 1. The plasmids were isolated and 1 part of them was dissolved in 20 d of TE buffer. Pss-medium (
P-solution prepared in the same manner as the P-solution shown in (2) of Example 2, except that the 0.25M TBS buffer solution was adjusted to pH 6.8; hereinafter referred to as Pss-solution) 80-
added. Streptomyces sannanensis protoplast suspension 1 obtained by the same method as used to prepare Streptomyces lividans protoplasts
00ρ was mixed and left at room temperature for 1 minute. Furthermore, 48
A mixture of 30% volume of polyethylene glycol 4.000 (manufactured by Gyudon Kagaku Co., Ltd.) was added to and mixed with the T-solution of 0- and CPss-solution. Appropriately diluted with Pss-solution, this 1004 was used to prepare a regenerated agar medium for Streptomyces sannaensis [103 g of sucrose, 0.5 g of yeast extract (manufactured by Difco).

Peptone (manufactured by Difco) 2.5g, malt extract (manufactured by Difco) 1.5g, casamino acid (manufactured by Difco) O
, Ig, soluble starch (manufactured by Gyudon Kagaku Co., Ltd.) 1
0g, glucose 5.0g, zsOa O, 25g,
1 ml of trace metal element liquid and 5.7 g of TεS are brought to 11 with deionized water and adjusted to pH 6.6. Divide this into 5 equal parts, 4
g of Batato Agar (manufactured by Difco) is added and sterilized under high pressure. 2.8 ml of 5M CaCl in one of these 22 ml
of MgCRa was added, 20 ml of each was spread on a plate, and dried for 120 minutes.

After culturing at 30°C for 20 to 24 hours, 2.5 ml of soft agar medium containing the antibiotic thiopeptin at a concentration of 45J1g/ml, which had been kept at 42°C in advance, was overlaid, and the culture was further cultured at 30°C for 4 to 6 days. did. All of the regenerated transformants exhibiting thiopeptin resistance retained the plasmid used for transformation. A plasmid was extracted from the transformed strain obtained using pENlol, and re-transformed using this. When the transformation efficiency was calculated from the number of regenerated colonies obtained here, 2.7XIO' transformed strains were obtained from 1 ug of plasmid.

When PEN301 was used, the number was 104 to 10S.

Effects of the Invention According to the present invention, it is possible to obtain a rolling vector capable of autonomous replication in Streptomyces sunnanensis species, and to provide a host-vector system in Streptomyces sunnanensis species. Furthermore, it may be used as a vector for a new host range not found in conventional vectors for Streptomyces species.

[Brief explanation of the drawing]

Figures 1 to 6 show pBNloo and pHN2, respectively.
00°PEN300.・ρεNl01. Restriction enzyme cleavage maps of P201 and p[EN301 are shown. In the figure,
A DNA fragment containing a thiopeptin resistance gene exists in the range indicated by the arrow. In addition, each number in the figure represents the following value (b). Figure 1: 1.0.00/4.602.0.17 3
．． 0.7?4, 0.8B 5.1.42 6.1.4
4? , 1.48 g, 1.78 9.2.08!
0.2,28 11.2.55 12.2.5813,
2.85 14.3.00 15.3.3616, 3,
39 17.3.50 18.3.7219.4.04
20.4.43 Figure 2: 1.0.00/10,70 2.0.05
3.1.024, 1.77 5.1.90 6.2.
02? , 2.80 g, 2.95 9.3.1510
, 3.16 11.3.18 12.3.2013.3
,? 5 14.4.50 15.5.0516.5.2
5 17.5.45 18.5.4719, 5.67
20.5.83 21.6.3022.6.45 23
．． 7.95 24.8.1025.9.65 26.
9.75 27.10.55 Figure 3: 1.0.0
0/8.28 2.0.25 3.0.954.1.1
0 5.3.03 6.3.237.3.32
81.43 9.4.2110, 4.51 I
l, 4.68 12. 5.2313, 5.3
5 14. 5.65 15. 5.6716,
7.45 1? , 7.84Figure 4: 1. O,
0O15,702,O,+7 3.0.774.0.8
8 5.1.42 6.1.447.1,48
8.1.78 9.2.0810, 2.2B
11. 2.55 12. 2,7913, 2.8
9 14. 3.32 +5. 3.42+6.
3.65 17. 3.68 18. 3.95
+9. 4.10 20. 4.46 21. 4
．． 4922.4.60 23.4.82 24.5
．． I425, 5.53 Figure 5: 1.0.00/11.80 2.0.05
3.1.024.1.7? 5.1.90 6
．． 2.027.2.80 8.2.95 9.3.
151.0. 3J5 11. 3.95 12.
4.25I3.4.26 14.4.28 15
．． 4J. 1(i, 4.85 17.5.60 +8.6.
1519.6,35 20. 6.55 21.
6.5722.6.77 23.6.93 24.
7.4025.7゜55 26.9.05 27.
9.202B, 10.75 29. 10.85
30. 11.65 Figure 6: 1.0.00/1
0.09 2.0,25 3.0.954.1.10
51.03 6.3.18? , 3.48 8.3.
71 9゜3. Old IQ, 4.24 11. 4.
5B 12. 4.6013.4.65 14.
4.83' 15. 4.8416, 5.04
1? , 5.13 18. 5.2419, 6
．． 02 20. 6.32 2+, 6.492
2.7.04 23.7.16 24.7.462
5.7.49 26.9.26 27.9.65 Patent applicant (102) Kyowa Hakko Kogyo Co., Ltd. Figure 2 Figure 3 Figure 4

Claims (6)

[Claims] (1) A plasmid derived from Streptomyces and capable of autonomous replication in Streptomyces sannanensis. (2) the plasmid has a size of 4.6 kilobases,
The number of sensitive sites to restriction enzymes is Bcl I 3, Bg
lII1, BstEII2, EcoR I 1, Kpn I 3,
PvuII1, SacI1, SalGI4, SstII1
, Xba I 1, plasmid pEN100, 10.
It has a size of 7 kilobases, and the number of sensitive sites for restriction enzymes is BamH I 4, Bcl I 3, Bgl II 2, Bs
tEII3, EcoR I 1, Kpn I 3, Mlu I 3
, Pvu II 3, Sac I 1, Sma I 4, or 8.3 kilobases in size and with a number of sensitive sites for restriction enzymes, BamH I 1,
Kpn I 1, Mlu I 4, SalG I 9, Sma I
1. The plasmid according to claim 1, which is a plasmid selected from plasmid pEN300, which is Sph I 1. (3) A recombinant plasmid in which a DNA fragment containing a gene whose genetic trait is expressed in Streptomyces sannanensis is incorporated into a vector plasmid that can autonomously replicate in Streptomyces sannanensis.
and a recombinant cloning vector that autonomously replicates in Streptomyces sunnanensis species and whose presence can be identified based on the expression of the introduced gene. (4) The cloning vector according to claim 3, wherein the DNA fragment is a DNA fragment containing a drug resistance gene. (5) The vector plasmid is pEN100, pEN2
5. The cloning vector according to claim 3, which is selected from pEN300 and pEN300. (6) The cloning vector is pEN101, pE
The cloning vector according to claims 3 to 5, which is a recombinant cloning vector selected from N201 or pEN301.