JPH0799975A - Recombinant shuttle vector - Google Patents

Abstract

PURPOSE:To obtain subject vector capable of industrially producing a useful physiologically active substance by gene recombination technology, maintaining both DNA fragments of bacteria of the genus Micromonospora and Escherichia, capable of replicating itself with both the bacteria. CONSTITUTION:Plasmid pSG32 is extracted from a bacterium [e.g. Micromonospora.sp. SG32 (FERM P-11,460)] belonging to the genus Micromonospora, treated with a proper restriction enzyme and linked to a vector such as plasmid pUC119 capable of replicating itself in a bacterium belonging to the genus Escherichia to give the objective recombinant shuttle vector which maintains a DNA fragment containing a gene fragment developed with a bacterium belonging to the genus Micromonospora, a cleft fragment of restriction enzyme BamHI derived from a plasmid capable of replicate itself with a bacterium belonging to the genus Micromonospora and having restriction enzyme BamHI site and a cleft fragment of a plasmid vector capable of replicate itself in a bacterium belonging to the genus Escherichia and can replicate itself with both the bacteria.

Description

Detailed Description of the Invention

[0001]

The present invention relates to a micro monospora (M
The present invention relates to a shuttle vector capable of autonomously replicating in a microorganism belonging to the genus icromonospora and a microorganism belonging to the genus Escherichia. Therefore, the present invention provides a vector for applying the gene recombination technique to a microorganism belonging to the genus Micromonospora.

[0002]

2. Description of the Related Art In recent years, genetic engineering techniques in actinomycetes have rapidly advanced [CurrentTopics i
n Microbiology and Immuno
logy, 96 , 69 (1982)]. The gene manipulation system of actinomycetes is often performed in microorganisms belonging to the genus Streptomyces, and the host-vector system is also the plasmid pIJ41 [Gene, 20 ,
51 (1982)] and plasmid pIJ702 [Jou.
rnal of General Microbiol
Ogy, 129 , 2703 (1983)] and other plasmid-type cloning vectors are often used.

As plasmids derived from strains other than Streptomyces, particularly Micromonospora, plasmid pATM2 [JP-A-57-179196] and plasmid pIM1 [Journal of Antib] are available.
iotics, 37 , 1082 (1984)], plasmid pMZ1 and plasmid pMR2 [JP-A-60-47].
No. 684], plasmid pSCH101 [Abst
racts of the Annual Meeti
ng of the American Societ
y for Microbiology, 86 , 150
(1986)], plasmid pMO101 [JP-A-1-
304887] and plasmid p11725a.
[JP-A-4-229177] and the like have been reported, and cloning vectors derived therefrom have been constructed.

[0004]

The conventionally used actinomycete-derived vector is not capable of autonomous replication when the host belongs to another actinomycete species or genus, and is difficult to be stably retained in the host. , It was difficult to use for host-vector system. In such a case, it was necessary to screen a plasmid capable of stably autonomously replicating in the target host strain.

Microorganisms belonging to the genus Micromonospora are known as strains producing antibiotics such as mycinamycin, gentamicin, sagamicin, etc. Elucidation of the biosynthesis system of these substances is to efficiently produce them or It is very important for the creation of new antibiotics using the above, and for that purpose, it has been desired to establish a gene manipulation system using these microorganisms belonging to the genus Micromonospora as hosts. However, the conventionally used Streptomyces-derived plasmid vector does not stably replicate autonomously in microorganisms belonging to the genus Micromonospora [Journalof Bacteriology, 1
73 , 7004 (1991)], or even when introduced, the transformation efficiency was poor and not practical [Journal
of Antibiotics, 41 , 583 (198)
8)]. Therefore, it stably replicated autonomously in a host belonging to the genus Micromonospora than a plasmid derived from a microorganism belonging to the genus Micromonospora, and was excellent in transformation efficiency, and further useful in cloning and expression of foreign genes. The development of plasmid vectors was expected.

[0006]

In order to develop a useful host-vector system for microorganisms belonging to the genus Micromonospora, the present inventors have developed various plasmids capable of autonomous replication in microorganisms belonging to the genus Micromonospora. investigated. as a result,
First, a plasmid p11725a or a plasmid pTM capable of autonomous replication in a microorganism belonging to the genus Micromonospora
1009 at least a restriction enzyme FbaI and a restriction enzyme K
A shuttle vector using a pnI-cleaved DNA fragment was found (Japanese Patent Application No. 5-43617). However, the shuttle vector thus obtained is difficult to construct as a plasmid smaller than this due to the nature of the plasmid capable of autonomous replication in the microorganisms belonging to the genus Micromonospora used, and the gene used as a shuttle vector is A DNA fragment containing a gene that expresses a genetic trait that can be retained when used in a recombinant technique is 15 k.
It was limited to b or less. Then, as a result of earnest research, a plasmid pSG32 having a restriction enzyme BamHI site derived from Micromonospora sp.
A microorganism belonging to the genus Micromonospora by using a restriction enzyme BamHI cleaved fragment having autonomous replication ability and incorporating a suitable selectable marker, and further ligating with a vector capable of autonomous replication in a microorganism belonging to the genus Escherichia, A more suitable recombinant shuttle vector having autonomous replication ability in both microorganisms belonging to the genus Escherichia was found.

The present invention has been completed based on the above findings, and is autonomously replicated in both a microorganism belonging to the genus Micromonospora and a microorganism belonging to the genus Escherichia, and a genetic trait in a microorganism belonging to the genus Micromonospora. DNA fragment containing a gene that expresses a gene capable of autonomous replication in a microorganism belonging to the genus Micromonospora and having a restriction enzyme BamHI cleavage fragment derived from a plasmid having a restriction enzyme BamHI site and autonomous replication in a microorganism belonging to the genus Escherichia It is a recombinant shuttle vector characterized in that it retains a cleaved fragment of a possible plasmid vector.

The present invention will be described in detail below. The present invention can provide a recombinant shuttle vector capable of autonomous replication in both a microorganism belonging to the genus Micromonospora and a microorganism belonging to the genus Escherichia, and a gene whose genetic trait is expressed in the microorganism belonging to the genus Micromonospora. A recombinant shuttle vector capable of discriminating its presence based on the expression of This vector is a DNA containing a gene whose genetic trait is expressed in a microorganism belonging to the genus Micromonospora.
Fragment and a plasmid-derived restriction enzyme BamHI cleavage fragment capable of autonomous replication in a microorganism belonging to the genus Micromonospora and having a restriction enzyme BamHI site and a cleavage fragment of a plasmid vector capable of autonomous replication in a microorganism belonging to the genus Escherichia Can be constructed by connecting This vector is capable of amplifying a gene derived from actinomycete using a microorganism belonging to the genus Escherichia without using a slow-growing actinomycete, which facilitates gene analysis or in vitro mutation. . Furthermore, amplification is carried out using a microorganism belonging to the genus Escherichia as a host,
The prepared recombinant plasmid can be directly introduced into a microorganism belonging to the genus Micromonospora, amplified in a microorganism belonging to the genus Micromonospora, and further analyzed by the expression of the introduced gene to obtain a gene product such as an enzyme protein. ,
Further, it can be applied to efficient production of secondary metabolites such as antibiotics.

Examples of the plasmid capable of autonomous replication in a microorganism belonging to the genus Micromonospora and having a restriction enzyme BamHI site include a plasmid pSG32 derived from Micromonospora sp. SG32 (FERM P-11460). . This plasmid is disclosed in JP-A-4-051897 or Plasmid, 12 , 1.
9 (1984) for easy separation.

A fragment derived from a plasmid capable of autonomous replication in a microorganism belonging to the genus Micromonospora and having a restriction enzyme BamHI site is digested with an appropriate restriction enzyme so as not to lose the autonomous replication ability of the plasmid. For example, the plasmid pSG32 is a 2.8 kb DNA fragment obtained by digestion with the restriction enzyme BamHI, and the site sensitive to the restriction enzyme is BglII.
2, DNA fragments characterized by PvuII 1, MluI 1, and SphI 1. Furthermore, as an equivalent to the above, this DNA fragment is appropriately digested with a corresponding restriction enzyme using, for example, a restriction enzyme site, and an autonomous replication ability is obtained by a DNA modification considering a translation codon with a DNA polymerase or a ligase by a conventional method. Having minimized DN
A fragment is given.

Further, the plasmid pSG32 does not have a selectable marker for distinguishing its presence, and therefore, in order to establish a host-vector system in a microorganism belonging to the genus Micromonospora, the presence of the vector is suitable. It is desirable to construct a plasmid vector with a different selectable marker. The selectable marker is a good example of a DNA fragment containing a gene whose genetic trait is expressed in a microorganism belonging to the genus Micromonospora. As the selectable marker, a drug resistance gene is particularly preferable, for example, plasmid pI
J702 [Journal of General M
microbiology, 129 , 2703 (198)
3)] A DNA containing a thiostrepton resistance gene derived from
Examples of the fragment include DNA fragments containing drug resistance genes such as neomycin and viomycin.

The drug resistance gene as a selectable marker may be prepared by appropriately cleaving a plasmid vector having the desired drug resistance gene by digestion with a restriction enzyme, and for example, it can be obtained by digesting plasmid pIJ702 with a restriction enzyme FbaI. An example is a DNA fragment containing a 1 kb thiostrepton resistance gene. Examples of the plasmid vector capable of autonomous replication in a microorganism belonging to the genus Escherichia include, for example, plasmid pUC19 [Gene, 3
3 , 103 (1985)], pUC119, plasmid pBR322 [Gene, 22 , 277 (1983)].
Or the plasmid pACYC184 [Journal
of Bacteriology, 134 , 1141
(1978)] and the like. Cleavage fragment of a plasmid vector capable of autonomous replication in a microorganism belonging to the genus Escherichia has a plasmid vector, which is cut with an appropriate restriction enzyme so as not to delete at least the region responsible for autonomous replication ability, or is a selectable marker, for example, A truncated fragment of the region responsible for the autonomous replication ability containing the ampicillin resistance gene may be used. For example, 3.2k obtained by digesting the plasmid pUC119 with the restriction enzyme HincII.
An example is the DNA fragment of b. In this case, when the gene whose genetic trait is expressed in a microorganism belonging to the genus Micromonospora is a selection marker, for example, a drug resistance gene,
If it can be expressed as a selectable marker even in a plasmid vector capable of self-replicating in a microorganism belonging to the genus Escherichia, it can be used as a selectable marker in both hosts of the microorganism belonging to the genus Escherichia and the microorganism belonging to the genus Micromonospora.

To construct the recombinant shuttle vector of the present invention, a DNA fragment containing a gene whose genetic trait is expressed in a microorganism belonging to the genus Micromonospora and a microorganism belonging to the genus Micromonospora are capable of autonomous replication. Restriction enzyme B
A restriction enzyme BamHI digested fragment derived from a plasmid having an amHI site and a digested fragment of a plasmid vector capable of autonomous replication in a microorganism belonging to the genus Escherichia are ligated to each other by using the same digested ends, and then the host microorganism is ligated. Introduced into a transformant, it is possible to autonomously replicate in the microorganism belonging to the genus Micromonospora and the microorganism belonging to the genus Escherichia from the transformant, and its presence can be identified based on the expression of the genetic trait. It is only necessary to collect a possible recombinant shuttle vector.

For example, the end of a 1.1 kb DNA fragment containing a thiostrepton resistance gene obtained by digesting the plasmid pIJ702 with a restriction enzyme FbaI is used as T4D.
Recombinant plasmid pYA101 constructed by ligating a DNA fragment blunted with NA polymerase and a 3.2 kb DNA fragment obtained by digestion with a restriction enzyme HincII of plasmid pUC119 capable of autonomous replication in a microorganism belonging to the genus Escherichia Of the restriction enzyme BamHI and a 2.8 kb DNA fragment cleaved by the restriction enzyme BamHI derived from the plasmid pSG32 capable of autonomous replication in a microorganism belonging to the genus Micromonospora and having a restriction enzyme BamHI site. The recombinant shuttle vector pYA201 (7.0 kb) can be mentioned. In this way, the shuttle vector p suitably obtained
YA201 has sensitive sites for various restriction enzymes as shown in Table 1.

[0015]

[Table 1]

A DNA fragment containing a gene whose genetic trait is expressed in a microorganism belonging to the genus Micromonospora and a restriction enzyme derived from a plasmid capable of autonomous replication in a microorganism belonging to the genus Micromonospora and having a restriction enzyme BamHI site. To bind the BamHI cleavage fragment and the cleavage fragment of a plasmid vector capable of autonomous replication in a microorganism belonging to the genus Escherichia, for example, 50 μl of a ligation reaction solution [6.6 mM magnesium chloride containing 1 unit of a known DNA ligase such as T4 DNA ligase 10m
6 containing M dithiothreitol and 0.8 mM 2'-deoxyadenosine-5'-triphosphate (dATP)
6 mM Tris-HCl buffer (pH 7.6)]
It can be left overnight at ° C.

The plasmid or fragment thus obtained was introduced into a microorganism belonging to the genus Escherichia, such as Escherichia coli (Escherichia coli), which is a host microorganism, as described below, to cause transformation, and a genetic character was expressed. By selecting a transformant, a transformant carrying the target recombinant shuttle vector can be obtained. In addition, a recombinant shuttle vector of interest is collected from the transformant, and introduced into a microorganism belonging to the genus Micromonospora, which is the other host microorganism, by a method of protoplastizing the host microorganism as described below, and the transformation is performed. The birth
By selecting a transformant expressing a genetic trait, a transformant carrying the target recombinant shuttle vector can be obtained. As a method for culturing the above transformant, it may be aerobically cultivated in a medium containing a medium component used for culturing a normal microorganism, and a solid medium or a liquid medium is used as the medium, but particularly for mass production. Liquid media, especially aqueous media, are suitable for this purpose.

Among the medium components, the carbon source is glucose,
Starch, sucrose, molasses, dextrin, etc. are used. Further, peptone, meat extract, soybean powder, casein hydrolyzate and the like are used as the nitrogen source, and cottonseed meal, corn steep liquor, nitrate and ammonia salt can also be used. Other inorganic substances include cations such as sodium, potassium, magnesium, calcium, cobalt, manganese, and iron, and / or
Substances containing anions such as chlorine, sulfuric acid, phosphoric acid and acetic acid can be used. Furthermore, dry yeast or yeast extract can be used as a growth promoting factor of the bacterium. Furthermore, calcium carbonate can be added to the medium in order to adjust the pH of the medium. In addition, in order to suppress foaming in the medium, an appropriate amount of silicone resin, animal or vegetable oil, etc. can be added to the medium.

An example of a medium particularly suitable for culturing the transformant is a medium containing glucose, dextrin, defatted soybean powder, calcium carbonate and cobalt chloride as the medium components. The temperature for culturing the transformant in the medium is preferably in the range of 20 ° C to 37 ° C, particularly 26 ° C to 30 ° C. The number of culture days varies depending on the culture conditions, but is usually 4 to 5 days. As the culturing method, any known culturing method can be used, but the aeration and stirring culturing method in a fermentation tank is particularly suitable for mass production.

To collect the desired recombinant shuttle vector from these transformants, see JP-A-4-229177.
Publication or Plasmid, 12 , 19 (1984)
It may be performed in the same manner as the method described in. The recombinant shuttle vector of the present invention is capable of autonomous replication in a microorganism belonging to the genus Micromonospora, and has the property that its presence can be identified based on the expression of a selectable marker. Also,
The recombinant shuttle vector of the present invention can use a microorganism belonging to the genus Micromonospora as a host.

The microorganism belonging to the genus Micromonospora is not particularly limited, but for example, Micromonospora echinospora
nospora), Micromonospora purpurea (M
micromonospora pururea), Micromonospora sagamiensis (Micromonos)
Pora sagamiensis, Micromonospora gri
sea), Micromonospora Inyoensis (Mic
romonospora inyoensis, Micromonospora dionensis (Micromonosp)
ora zionensis), Micromonospora rho
odorangea), Micromonospora rosaria, Micromonospora laxtris (Micromonos)
Pora lacustris), Micromonospora
Chalsea (Micromonospora chal
cea), Micromonospora olivoster
ospora), Micromonospora griseorub
ida) and the like.

As the host microorganism, a strain in which the restriction system is released is more effective, and such a strain in which the restriction system is released can be obtained by the method of Lomovskaya et al. [Microbio].
logical Reviews, 44 , 206 (19)
80)]. In addition, if plasmid DNA is present in the host microorganism cells at this time, it may affect the transformation efficiency and stable retention with foreign DNA. It is desirable to remove (curing). For example, the A11725CN3 strain obtained by removing the plasmid p11725a from Micromonospora glyceorbida TM1009 can be mentioned as a host microorganism.

In order to introduce the recombinant shuttle vector of the present invention into a host microorganism belonging to the genus Micromonospora, for example, according to the method described in JP-A-4-229177,
A protoplast may be prepared and a vector may be introduced.
The strain carrying the recombinant shuttle vector pYA201 of the present invention is Escherichia coli JM109 / pYA2.
01 (FERM P-13887) has been deposited with the Institute of Biotechnology, Institute of Industrial Science and Technology.

[0024]

EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited thereto.

[0025]

Example 1 Construction of recombinant shuttle vector pYA201 (1) Preparation of plasmid pSG32 Micromonospora sp. SG32 (FERM P-
11460) 10 ml of 172F medium [glucose (Wako Pure Chemical Industries, Ltd.) 10 g, yeast extract (Difco) 5 g, soluble starch (Wako Pure Chemical Industries, Ltd.) 10
g, Kasiton (manufactured by Difco), 2.5 g of magnesium sulfate heptahydrate (manufactured by Wako Pure Chemical Industries, Ltd.), and 2 g of calcium nitrate tetrahydrate (manufactured by Wako Pure Chemical Industries, Ltd.) in distilled water. Adjust the pH to 7.2 and adjust to pH 7.2,
Those that had been autoclaved for a minute (hereinafter, abbreviated as autoclave)] were inoculated and shake-cultured at 28 ° C. for 3 days. Next, 1 ml of this culture was inoculated into 50 ml of 172F medium, and further 2
The culture was performed at 8 ° C. for 3 days with shaking.

The culture solution was centrifuged (4 ° C., 10 minutes, 3,
000 rpm) to collect the cells, and after washing with 10.3% sucrose (manufactured by Wako Pure Chemical Industries, Ltd.) solution, 2 mg / m
10 ml lytic solution containing 1 egg white lysozyme (manufactured by Seikagaku Corporation) [10.3% sucrose, 25 mM
Tris (hydroxymethyl) aminomethane (hereinafter abbreviated as Tris, manufactured by Sigma) -hydrochloric acid buffer solution (pH 8.0), 2
5 mM disodium ethylenediaminetetraacetic acid (EDT
A, manufactured by Dojindo Laboratories)] and incubated at 37 ° C. for 30 minutes. To this, 6 ml of 0.3 N sodium hydroxide containing 2% sodium dodecyl sulfate (SDS, manufactured by Wako Pure Chemical Industries, Ltd.) was added and mixed well, and heated at 55 ° C. for 30 minutes with frequent stirring.

After cooling to room temperature, a 1/5 volume of a phenol / chloroform mixed solution [500 ml of chloroform (manufactured by Wako Pure Chemical Industries, Ltd.), 500 g of phenol (manufactured by Wako Pure Chemical Industries, Ltd.) and 1.0 g of 8- Hydroxyquinoline (manufactured by Nacalai Tesque, Inc.) was mixed well, and then 200 ml of distilled water was added and the lower layer obtained by shaking was used].
Stir vigorously for 2 seconds. Centrifuge (18 ℃, 10 minutes,
3,000 rpm) to the supernatant part obtained by 10
1 volume of 3M sodium acetate solution was added, followed by addition of an equal volume of isopropyl alcohol (manufactured by Wako Pure Chemical Industries, Ltd.) and mixed well, and allowed to stand at room temperature for 10 minutes and then centrifuged again (1
8 ° C., 10 minutes, 3,000 rpm). The resulting pellet was added to TE buffer [10 mM Tris-HCl buffer (p
H8.0), 1 mM EDTA] 8 ml, and ethidium bromide (manufactured by Wako Pure Chemical Industries, Ltd.) to a final concentration of 0.75 mg.
In addition, cesium chloride (manufactured by Nacalai Tesque, Inc.) (8.8 g) was further added.

The solution obtained is treated with 100,000 × at 16 ° C.
g and centrifuged for 16 hours. The circular DNA covalently closed by this density gradient centrifugation was detected as a specific band that fluoresces upon irradiation with an ultraviolet lamp. The band was removed using a needle and ethidium bromide was removed by shaking several times with isopropyl alcohol saturated with TE buffer. The resulting solution is 4
It was dialyzed 3 times at 100 ° C. with 100 volumes of TE buffer. The solution obtained after the dialysis was vigorously stirred with an equal volume of a phenol / chloroform mixed solution and then centrifuged (hereinafter abbreviated as phenol extraction). The obtained supernatant was mixed with 1/10 volume of 3M sodium acetate and an equal volume of isopropyl alcohol, and left at room temperature for 10 minutes. 18 ℃, 5 minutes, 1
The pellet was recovered by centrifugation at 2,000 rpm, further washed with 70% ethanol, and dried under vacuum (hereinafter, abbreviated as isopropyl alcohol precipitation). By this operation, about 40 μg of plasmid pSG32 was obtained. (2) Preparation of DNA fragment containing thiostrepton resistance gene 10 μg of plasmid pIJ702 (ATCC3528)
7) was added to 100 μl of K buffer [20 mM Tris-HCl buffer (pH 8.
5) 10 mM magnesium chloride, 1 mM dithiothreitol, 100 mM potassium chloride]
Incubated for hours. After agarose gel electrophoresis,
An agarose gel containing the target 1.1 kb FbaI-cut DNA fragment was cut out and electroeluted in a dialysis tube. The eluate was extracted with phenol and then precipitated with isopropyl alcohol to collect DNA fragments (hereinafter, referred to as
(Abbreviated as elution recovery of DNA fragment).

40 μl of the T4 DNA polymerase reaction solution [66 mM Tris-hydrochloric acid buffer solution (pH 8.
8), 6.6 mM magnesium chloride, 16.6 mM ammonium sulfate, 10 mM 2-mercaptoethanol,
It was dissolved in 6.6 μM EDTA and 0.0167% bovine serum albumin], 5 units of T4 DNA polymerase (Takara Shuzo) was added, and the mixture was incubated at 37 ° C. for 5 minutes. Further, 2 μl of a nucleotide mixture [2 mM each of 2′-deoxyadenosine-5′-triphosphate (dAT
P), 2'-deoxycytidine-5'-triphosphate (dC
TP), 2'-deoxyguanosine-5'-triphosphate (dGTP) and thymidine-5'-triphosphate (dTT).
P) (both manufactured by Sigma) was added and incubated at 37 ° C. for 15 minutes, and then DNA was recovered by phenol extraction and isopropanol precipitation to obtain a DNA fragment containing a thiostrepton resistant gene having a blunt end. (3) Construction of recombinant plasmid pYA101 100 μl of M buffer [10 mM Tris-HCl buffer (pH 7) containing 10 μg of plasmid pUC119 (3.2 kb, Takara Shuzo) containing 20 units of restriction enzyme HincII (Takara Shuzo) .5) 10 mM magnesium chloride,
1 mM dithiothreitol, 50 mM sodium chloride]
In addition, the mixture was incubated at 37 ° C. for 2 hours. After the reaction solution was extracted with phenol, DNA fragments were recovered by isopropyl alcohol precipitation. Pellet 20 μl of CIAP
Buffer [50 mM Tris-HCl buffer (pH 9.0),
1 mM calf intestinal alkaline phosphatase (CIAP, manufactured by Takara Shuzo)
Was added and incubated at 50 ° C. for 30 minutes. After the reaction, 1 μl of 10% SDS was added and incubated at 70 ° C. for 20 minutes to stop the reaction, and the DNA was recovered by twice phenol extraction and isopropyl alcohol precipitation (hereinafter, abbreviated as CIAP treatment).

HincI of this plasmid pUC119
0.5 μg of a 1.1 kb DNA fragment containing the thiostrepton resistance gene having a blunt end obtained in (2) and 2 μg of the digested product of I was added to 50 μl of a ligation reaction solution [6.
66 mM Tris-HCl buffer (pH 7.6) containing 6 mM magnesium chloride, 10 mM dithiothreitol and 0.8 mM dATP], and 1 unit of T4 DNA ligase (manufactured by Toyobo Co., Ltd.) was added.
The reaction was carried out at 0 ° C overnight.

Using this reaction solution, Escherichia coli JM109 was transformed by the following method (the following method is abbreviated as transformation of Escherichia coli JM109). Fifty
ml LB medium [Bactotryptone (manufactured by Difco) 1
0 g, yeast extract 5 g, sodium chloride 5 g made up to 1 l with distilled water, adjusted to pH 7.2 and sterilized under high pressure]
Escherichia coli JM109 in logarithmic growth phase
Were collected by centrifugation at 3,000 rpm at 4 ° C., and 25 ml of cold TEN buffer [10 mM N-tris (hydroxymethyl) -methyl-2-aminoethanesulfonic acid (TES,
Dojindo Laboratories) (pH 7.2), 50 mM sodium chloride].

The cells were collected by centrifugation, suspended in 25 ml of a cold TEC buffer solution [25 mM TES (pH 7.2), 50 mM calcium chloride], and allowed to stand at 0 ° C. for 15 minutes. The cells were collected by centrifugation and 2 ml of cold TECG buffer [25 mM TES
(PH 7.2), 50 mM calcium chloride, 10% glycerol] to obtain a competent cell suspension.
To 200 μl of this Escherichia coli JM109 competent cell suspension, 20 μl of the above ligation reaction solution was added, and the mixture was left at 0 ° C. for 40 minutes.

After that, heat treatment is performed at 42 ° C. for 90 seconds, and L
2 ml of B medium was added and incubated at 37 ° C. for 60 minutes. 200 μl of this was added to 50 μg / ml of ampicillin sodium (manufactured by Wako Pure Chemical Industries, Ltd.), 0.02% of 5
-Bromo-4-chloro-3-indolyl-β-galactoside (X-Gal, manufactured by Wako Pure Chemical Industries, Ltd.) and 50 mM
Isopropyl-β-D-thio-galactopyranoside (I
LA agar plate containing PTG, manufactured by Wako Pure Chemical Industries, Ltd. [Bacto agar (manufactured by Difco) was added to unsterilized LB medium to a final concentration of 1.5%, and 20 ml each was sterilized under high pressure and then dispensed into a petri dish. ], And cultured overnight at 37 ° C. to obtain ampicillin-resistant transformants. The plasmid carried by the single white colony that appeared was extracted and analyzed according to the following method (the following method is abbreviated as extraction analysis of plasmid). The colonies were transplanted to 2 ml of LB medium containing 50 μg / ml of ampicillin sodium and incubated at 37 ° C.
The cells were cultivated overnight in and collected by centrifugation.

This was suspended in 150 μl of a lytic solution containing 2 mg / ml of egg white lysozyme and incubated at 37 ° C. for 5 minutes. 0.3 containing 2% SDS
Add 90 μl of regular sodium hydroxide and mix well.
Warm for 30 minutes at 5 ° C. After cooling to room temperature, 50
After adding μl of phenol / chloroform mixed solution and stirring vigorously, centrifuge (18 ° C, 5 minutes, 12,000 rp
m) to the supernatant portion obtained by
A sodium acetate solution and an equal volume of isopropyl alcohol were added, mixed well, and allowed to stand at room temperature for 5 minutes. 18 ° C, 5
The pellet obtained by centrifugation at 12,000 rpm for 1 minute was dissolved in 100 μl of TE buffer and then digested with various restriction enzymes to select a recombinant plasmid containing the insert fragment. Further, the recombinant plasmid was analyzed by digestion with various restriction enzymes and agarose gel electrophoresis to obtain a recombinant plasmid pYA101 (4.2 kb). (4) Construction of recombinant shuttle vector pYA201 2 μl of 10 μg of plasmid pSG32 obtained in (1)
It was added to a K buffer solution containing 0 unit of restriction enzyme BamHI (manufactured by Takara Shuzo Co., Ltd.) and incubated at 37 ° C. for 2 hours. After the reaction solution was extracted with phenol, DNA fragments were recovered by isopropyl alcohol precipitation.

10 μg of the recombinant plasmid pYA101 obtained in (3) was added to a K buffer containing 20 units of the restriction enzyme BamHI and incubated at 37 ° C. for 2 hours. After the reaction solution was extracted with phenol, DNA fragments were recovered by isopropyl alcohol precipitation. DNA to CIAP
After treatment, 30 μl of 0.5 μg and 1 μg of the plasmid pSG32 digested with the above restriction enzyme BamHI were treated.
Dissolved in the ligation reaction solution of
DNA ligase was added and the reaction was carried out at 15 ° C overnight.

Escherichia coli JM109 was transformed with this ligation reaction solution, and a plasmid was extracted and analyzed from the obtained ampicillin-resistant transformant.
It was confirmed that the composition was as shown in FIG. 1, and the plasmid was used as a recombinant plasmid vector pYA201 (6.8).
kb). The plasmid vector p in FIG.
Each number in YA201 represents the value (kb) shown below. 1, 0.0 / 7.0 2,0.2 3, 0.2 4, 0.2 5, 0.2 6, 0.2
7, 1.0 8, 1.4 9, 1.4 10, 2.3 11, 2.6 12,3.0 13,
3.0 14, 3.3 15, 4.0 16, 4.0 17, 4.0 18, 4.2

[0037]

Example 2 Plasmid curing of Micromonospora glyceorbida TM1009, transformation and plasmid isolation (1) Micromonospora glyceorvida TM1009
Plasmid curing of Micromonospora Griseorubida TM1009 10
After inoculating into 172F medium (ml) and culturing at 28 ° C for 3 days to completely grow, the bacterial solution was aseptically homogenized with a glass homogenizer, and 100 µl of the obtained bacterial suspension was used.
Was inoculated into 10 ml of fresh 172F medium.

After repeating this operation 5 times, the bacterial solution was treated with M agar medium [soluble starch 20 g, polypeptone 5 g, calcium carbonate (manufactured by Wako Pure Chemical Industries, Ltd.) 1.0 g, iron (II) sulfate heptahydrate. (Wako Pure Chemical Industries, Ltd.) 40 mg and Bactoagar 22 g were sterilized under high pressure with distilled water to 900 ml, and separately sterilized with 7.5% "Essan meat".
(Manufactured by mixing 100 ml of Ajinomoto Co., Inc.) to grow colonies.

The grown colonies were separated with a sterilized toothpick, and fresh M agar medium and thiostrepton 10 μm were used.
The strain was transferred to M agar medium containing g / ml and cultured at 28 ° C. for 7 days to obtain a thiostrepton-sensitive strain. An attempt was made to isolate a plasmid from the resulting 6 strains of thiostrepton sensitive strain according to the method of (1) of Example 1, but no plasmid was detected. One of these strains was designated Micromonospora glyceorbida A11725CN3. (2) Micromonospora Griseorubida A11725
Preparation of CN3 Protoplasts Micromonospora glyceorbida A11725CN3
Was inoculated into 10 ml of 172F medium and shake-cultured at 28 ° C. for 3 days. Next, 2.5 ml of this culture solution was added to 50 ml of 172 containing 0.1% glycine (manufactured by Wako Pure Chemical Industries, Ltd.).
The F medium was inoculated and further shake-cultured at 28 ° C. for 3 days.

The culture solution was centrifuged (4 ° C., 10 minutes, 3,000
The cells were collected at 0 rpm), washed with a 10.3% sucrose solution, and then 10 ml of L-containing 2 mg / ml egg white lysozyme and 1 mg / ml achromopeptidase (manufactured by Wako Pure Chemical Industries, Ltd.). Medium [sucrose 10.3 g, potassium sulfate 25 mg and trace metal element liquid [zinc chloride 40 mg, iron (III) chloride hexahydrate 20]
0 mg, calcium chloride dihydrate 10 mg, manganese (II) chloride tetrahydrate 10 mg, sodium tetraborate decahydrate 10 mg, ammonium molybdate tetrahydrate 10 mg made up to 1 liter with distilled water. ] Make 0.2 ml to 88 ml with distilled water and sterilize under high pressure. Separately, autoclaved 1 ml of 0.5% monopotassium phosphate, 1 ml of 3.68% calcium chloride dihydrate, 0.25 ml of 1M magnesium chloride and 10 ml of 0.25M TE.
S (pH 7.2) added. ], And incubated at 37 ° C. for 2 hours. 4 ℃, 2 minutes, 1,000
The supernatant centrifuged at rpm was filtered through glass wool.

The filtrate was centrifuged (18 ° C., 10 minutes, 3,
000 rpm) to recover the protoplasts, and to collect P-medium [sucrose 10.3 g, potassium sulfate 25
mg, magnesium chloride hexahydrate 203 mg and trace metal element solution 0.2 ml are made up to 80 ml with distilled water and sterilized under high pressure. Separately, autoclaved 1 ml of 0.5% monopotassium phosphate, 10 ml of 3.68% calcium chloride dihydrate and 10 ml of 0.25 M TES (pH 7.
2) added. ], And then suspended in 1 ml of P-medium to obtain a protoplast suspension. (3) Micromonospora Griseorubida A11725
Transformation of CN3 To approximately 1 μg of a plasmid vector pYA201 prepared from a transformant of Escherichia coli JM109, 200 μl of a protoplast suspension of Micromonospora glyceorvida A11725CN3 was added, and further 500 μl of a PEG solution [polyethylene glycol 2000 (manufactured by Nacalai Tesque) ) 25 g is dissolved in unsterilized P-medium to make 100 ml, and autoclaved. ] Was gently mixed, left at room temperature for 1 minute, and then diluted 2-fold with P-medium.

Ten regenerated agar MR0.1S [34.3 g of sucrose, 20 g of soluble starch, 5 g of polypeptone, 10 g of magnesium chloride hexahydrate, 10 g of magnesium sulfate hexahydrate, iron (II) sulfate heptahydrate] 40m
g, 10 ml of trace amount metal element liquid to 690 ml with distilled water, 22.0 g of Bacto agar is added and mixed, and sterilized under high pressure. Separately autoclaved 100 ml of 7.5% "
"Essan meat", 10 ml of 0.5% monopotassium phosphate, 100 ml of 3.68% calcium chloride dihydrate and 100 ml of 0.25 M TES (pH 7.2).
Add and mix quickly, sprinkle 20 ml on each plate,
1.5 ml of soft agar medium (sucrose 34.3 g, magnesium chloride hexahydrate 10.12 g).
And 6.5 g of Bacto agar is made into 820 ml with distilled water and sterilized under high pressure. Separately, autoclaved 80 ml of 3.
68% calcium chloride dihydrate and 100 ml of 0.
25 M TES (pH 7.2) was added], and the whole was spread and spread, and cultured at 28 ° C. for 4 days.

Each plate was overlaid with 2.5 ml of soft agar medium containing 200 μg / ml of thiostrepton, and further 2
It was cultured at 8 ° C for 14 days. As a result, 250 thiostrepton-resistant regenerated strains of the plasmid vector pYA201 appeared. 10 of the regenerated strains obtained
The cells were transferred to M agar medium containing μg / ml thiostrepton and cultured at 28 ° C. for 7 days. When a plasmid possessed by the growing bacteria was collected and analyzed by the same method as the method shown in (1) of Example 1, the plasmid vector pYA201 was stably retained.

Further, one transformant strain having each plasmid vector was treated with thiostrepton at 10 μg / m 2.
Substituting was repeated in M agar medium containing 1 of each of the plasmids, but the plasmids were stably retained in the strains after 10 generations. Since the shuttle vector thus obtained is as small as 7.0 kb, a large molecule, for example, 40 kb
It can be used for a gene recombination technique in which a DNA fragment containing a gene expressing the above-mentioned genetic trait is retained.

[0045]

The microorganisms belonging to the genus Micromonospora are
It is known as a bacterium that produces many antibiotics or useful physiologically active substances. By clarifying the biosynthesis system of these useful substances in these microorganisms belonging to the genus Micromonospora, it is possible to efficiently produce useful substances. Becomes The recombinant shuttle vector obtained by the present invention is 7.0 k
Since it is as small as b, it can be used in a gene recombination technique in which a DNA fragment containing a gene capable of expressing a large molecule inheritance up to 40 kb is retained, and many useful biosynthetic systems for antibiotics or physiologically active substances It contributes to the analysis of or the analysis of biosynthetic genes.

Furthermore, since the recombinant shuttle vector obtained in the present invention is capable of autonomous replication in a microorganism belonging to the genus Micromonospora and is stable, it is a host-vector in a microorganism belonging to the genus Micromonospora. It is possible to provide a system and to provide a useful means in industrial production using various genetic manipulations such as antibiotics and physiologically active substances.

[Brief description of drawings]

FIG. 1 is a diagram showing a construction method of a recombinant plasmid vector pYA201 and a restriction enzyme map.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Reference number within the agency FI Technical indication (C12N 15/09 C12R 1:29) C12R 1:19) (C12N 15/00 A C12R 1:29)

Claims (7)

[Claims] 1. A DNA fragment containing a gene capable of autonomously replicating in both a microorganism belonging to the genus Micromonospora and a microorganism belonging to the genus Escherichia and expressing a genetic trait in the microorganism belonging to the genus Micromonospora, and the genus Micromonospora. Restriction enzyme BamHI capable of autonomous replication in a microorganism belonging to the group and having a restriction enzyme BamHI site
A recombinant shuttle vector, which holds a cleaved fragment and a cleaved fragment of a plasmid vector capable of autonomous replication in a microorganism belonging to the genus Escherichia. 2. The recombinant shuttle vector according to claim 1, wherein the DNA fragment containing a gene whose genetic character is expressed in a microorganism belonging to the genus Micromonospora is a DNA fragment containing a drug resistance gene. 3. The recombinant shuttle vector according to claim 2, wherein the drug resistance gene is a thiostrepton resistance gene. 4. The recombinant shuttle vector according to claim 1, wherein the plasmid capable of autonomous replication in a microorganism belonging to the genus Micromonospora and having a restriction enzyme BamHI site is plasmid pSG32. 5. A restriction enzyme BamHI digested fragment derived from a plasmid which is capable of autonomous replication in a microorganism belonging to the genus Micromonospora and has a restriction enzyme BamHI site is obtained from at least plasmid pSG32 by digestion with restriction enzyme BamHI. It is a DNA fragment of 8 kb, and the sensitive sites for restriction enzymes are BglII 2, PvuI.
The recombinant shuttle vector according to claim 1, which is a DNA fragment characterized by I 1, MluI 1, or SphI 1 or an equivalent thereof. 6. The recombinant shuttle vector according to claim 1, wherein the cleaved fragment of the plasmid vector capable of autonomous replication in a microorganism belonging to the genus Escherichia is derived from the plasmid pUC119. 7. The recombinant shuttle vector according to claim 1, wherein the recombinant shuttle vector is plasmid pYA201.