JP3142349B2 - Composite plasmid vector and transformed microorganism - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a novel composite plasmid vector, and more particularly, to plasmids pRC001 and pR001.
C002, derived from a plasmid selected from pRC003 and pRC004, a DNA region capable of replication in Rhodococcus bacteria,
The present invention relates to a novel composite plasmid vector containing a plasmid DNA region capable of replicating in Escherichia coli and a DNA region containing a drug resistance gene, and a microorganism transformed thereby.

[0002]

BACKGROUND OF THE INVENTION Microorganisms belonging to the genus Rhodococcus are known as microbial catalysts for hydrating or hydrolyzing nitriles to produce the corresponding amides or acids (EP-A-188316, Nos. 204555 and 348901) and Rhodococcus rhodochr
It is known that microorganisms belonging to ous have extremely high nitrile hydration activity (European Patent Publication No. 3079).
No. 26). Under such circumstances, the development of a host vector system of the genus Rhodococcus has long been expected. However, for strains belonging to the genus Rhodococcus, development of vectors suitable for using these microorganisms as hosts has been delayed, and strains in which plasmids have been found in the genus Rhodococcus are Rhodococcus sp.H13-A strains (J. Bacteriol., 170 ,
638-645 (1988)), Rhodococcus erythropolis (rhodoch
rous) ATCC 12674 strain (Mol.Gen. Genet., 211 , 148-154
(1988)) and Rhodoco, which we filed a patent earlier.
It is only a few strains including ccusrhodochrous ATCC 4276 etc. (Japanese Patent Application No. 2-270377). Therefore, further Rhodococ
There is a strong demand for the development of new vectors for breeding and improving industrially usable microorganisms from strains belonging to the genus cus.

[0003]

[Problems to be Solved by the Invention] Industrially useful Rhodoc
Cyclic plasmids obtained from bacteria belonging to the genus occus have the potential to genetically improve the genus Bacillus, but most are latent plasmids that do not have a drug resistance gene that can serve as a marker for the plasmid. Therefore,
By introducing a gene that can serve as a marker into such a latent plasmid, it is possible to construct a vector plasmid, but such an example is only one example.
(J. Bacteriol., 170, 638-645 (1988)). We consider the circular plasmids pRC001, pRC002, pRC003 and pRC0.
The present inventors have found that an industrially useful plasmid vector can be created by introducing a drug resistance gene which can be a marker, a cloning site and a gene region necessary for replication in Escherichia coli into 04, and completed the present invention. .

[0004]

Means for Solving the Problems The present invention provides (A) a plasmid region selected from plasmids pRC001, pRC002, pRC003 and pRC004 and capable of replicating and growing in a cell strain belonging to the genus Rhodococcus, A) A composite plasmid vector comprising a plasmid DNA region capable of replicating and growing in Escherichia coli cells and (C) a DNA region containing a drug resistance gene.

The plasmids pRC001, pRC002, pRC003 and
Rhodococcu
As long as replication is possible in bacteria of the s-genus, the plasmid may be the entire plasmid or a single fragment. In addition,
The above plasmids pRC001, pRC002, pRC003 and pRC004 are respectively Rhodococcus rhodochrous ATCC 4276, ATCC14
349, ATCC 14348 and IFO 3338 strains, and their restriction maps are shown in FIG.

[0006] Plasmids capable of replicating and growing in E. coli cells include, for example, pHSG299, pHSG298, pUC19, pU
C18 or the like can be used, and the DNA region of these plasmids may be the entire plasmid or a single fragment as long as it can replicate and grow in E. coli cells. Further, as the drug resistance gene, a kanamycin resistance gene, an ampicillin resistance gene and the like are suitably used, and are expressed in Rhodococcus bacteria and Escherichia coli as hosts, can impart drug resistance to the host, and can be used between both genera. The type of drug is not limited as long as the presence of the plasmid is suggested by the drug resistance, and one or more drugs may be present.

[0007] The composite plasmid vector of the present invention having the above-mentioned characteristics is Rhodococcus spp.
A strain derived from coccus rhodochrous ATCC 12674 can be used as a host, and Escherichia coli can be a K-12 strain. Therefore, it is possible to obtain transformants of those microorganisms using the composite plasmid vector.

[0008]

Next, the present invention will be described in more detail with reference to examples. However, the following examples do not limit the technical scope of the present invention.

[0009]

Example 1 (1) Plasmid pRC001, pRC002, pRC003 or pRC0
Preparation of Composite Plasmid Vectors pK1, pK2, pK3 and pK4 Consisting of Plasmid 04 and Plasmid pHSG299 As shown in FIG.
pK2, pK3 and pK4 were created. Plasmid pRC001, p
Restriction enzyme ClaI (5 units) was added to each of RC002, pRC003 and pRC004 (1 μg), and the mixture was reacted at 37 ° C. for 1 hour to cut the plasmid DNA.

On the other hand, 0.5 μg of plasmid pHSG299 (a 2.7 kb plasmid expressing kanamycin resistance, which can be purchased as a commercial product from Takara Shuzo Co., Ltd.) contains 0.5 μg of the restriction enzyme AccI (5uni
ts) was added and reacted at 37 ° C. for 1 hour to cut the plasmid DNA. Add 1/10 amount of 1M-Tris-HCl (pH9.0) to the reaction solution,
It was made to react with alkaline phosphatase (1 unit) at 65 ° C. for 1 hour.

[0011] 0.7% of the plasmid solution treated above
After agarose gel electrophoresis, plasmid pRC001, pR
2.6 kb from C002, pRC003 and pRC004, plasmid pHS
A 2.7 kb DNA fraction was cut out from G299. At this time, Hind of lambda phage DNA was used as a size marker.
The size of DNA was calculated using the III digest. Gene cl
DNA was recovered from agarose gel using ean kit (Funakoshi Co., Ltd.) and TE buffer (10 mM Tris-HCl, 1 mM EDTA,
(pH 8.0)). An equal volume of the solution containing each DNA fragment was mixed, and 1 unit of T4 DNA ligase, 1 mM ATP,
Each component was added to 10 mM dithiothreitol and 10 mM MgCl ₂ and reacted at 4 ° C. overnight.

The above reaction mixture was added to a competent cell of Escherichia coli JM105 (Takara Shuzo), and the mixture was allowed to stand at 0 ° C. for 1 hour.
Heat treatment for 2 minutes at 2 ℃, 2xYT medium (0.5% NaCl, 1%)
% Yeast extract, 1.6% tryptone)
Shake for 1 hour. 25 μg / ml kanamycin, 1 mM IP
TG (isopropyl-β-galactopyranoside), and
It was spread on a 2 × YT agar medium containing 0.02% X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) and incubated at 37 ° C. overnight. White colonies were selected from the appeared colonies, and cultured with shaking at 37 ° C. for 8 hours in 2 × YT medium (3 ml) containing 50 μg / ml kanamycin.

The cells were collected by centrifugation at 15,000 rpm for 5 minutes, and 0.35 ml of a STET solution (8% sucrose, 0.
5% TritonX-100, 50mM EDTA, 10mM Tris-HCl (pH8.0))
Suspended in water. Add 25 μl of lysozyme solution (10 mg / ml),
After stirring with Vortex for 3 seconds, it was immersed in boiling water for 50 seconds. The precipitate was removed by centrifugation at 15,000 rpm for 15 minutes to obtain a supernatant. 0.5 ml of a TE-saturated phenol: chloroform (1: 1) solution was added thereto, followed by stirring and centrifugation at 15,000 rpm for 5 minutes to obtain an upper layer. 0.5 ml of diethyl ether
After adding and mixing, the mixture was centrifuged to remove the upper layer. Isopropanol 0.5ml, 2.5M sodium acetate solution (pH4.5) 50μ
The mixture was allowed to stand at -80 ° C for 30 minutes and centrifuged at 15,000 rpm for 10 minutes to obtain a precipitate. It was washed with 70% ethanol, dried under reduced pressure, and dissolved in 0.1 ml of TE buffer.

Using the above-prepared plasmid solution, restriction enzymes HindIII, BamHI, SphI, EcoRI, X
The restriction enzyme cleavage pattern using hoI was examined. The composite plasmids obtained from plasmids pRC001, pRC002, pRC003 or pRC004 all had the same restriction enzyme cleavage sites and were named pK1, pK2, pK3 and pK4, respectively (Table 1).

[0015]

[Table 1]

(2) Composite plasmid pK1, p from Escherichia coli
Separation and purification of K2, pK3 and pK4 The above E. coli transformant (kanamycin resistant strain) was
The cells were cultured using 200 ml of T medium, and the cells were collected by centrifugation. Bacterial cells were washed with 40 ml of TES (10 mM Tris-HCl (pH 8.0), 10 mM N
aCl, 1mM EDTA) buffer, then 8ml of STET solution (50mM
Tris-HCl (pH8.0), 5mM EDTA, 35mM sucrose)
Was added, and 10 mg of lysozyme was added. After shaking at 0 ° C for 5 minutes, 4 ml of 0.25M EDTA (pH 8.0) was added, and the mixture was kept at 0 ° C for 5 minutes while occasionally mixing gently. Return to room temperature and add 2 ml
10% SDS (sodium lauryl sulfate) solution, 5ml 5M N
The aCl solution was added and the mixture was allowed to stand at 4 ° C. for 3 to 12 hours. 6 at 4 ° C
The mixture was centrifuged at 5,000 xg for 1 hour to obtain a supernatant, to which 4.6 ml of 50% polyethylene glycol 6000 was added. The mixture was allowed to stand on ice for 3 hours, and centrifuged at 1000 × g for 5 minutes. The precipitate was dissolved in 7.5 ml of TES buffer, and 8.2 g of CsCl and 15 mg / ml of ethidium bromide solution were added.
0.2 ml was added and mixed. This solution was subjected to a density gradient centrifugation at 130,000 × g for 42 hours. After fractionating the plasmid fraction detected by ultraviolet irradiation, it was treated with n-butanol to remove ethidium bromide. After dialysis against TE buffer,
A purified plasmid fraction was obtained by ethanol precipitation. This was subjected to 0.7% agarose gel electrophoresis, and the presence of the plasmid was confirmed by staining the gel with ethidium bromide. (3) Introduction of composite plasmid into bacteria belonging to the genus Rhodococcus The cells in the logarithmic growth phase of Rhodococcus rhodochrous ATCC 12674 were collected by centrifugation, washed three times with ice-cold sterile water, and then treated with 15% PEG6000 (polyethylene glycol 6000). )
The cells were suspended in the solution (the cell concentration was 10 ⁹ cells / ml or more). The above shuttle plasmid vector pK1, pK2, pK3 or pK4D
NA2 ^-2 µg and 10 µl of the cell suspension were mixed and cooled on ice.
A mixed solution of DNA and bacterial cells was placed in the chamber 11 for the Shimadzu cell fusion device SSH-1, cooled on ice, and then subjected to electric pulse treatment with a pulse width of 500 μs and an electric field intensity of 14 kv / cm.

The electric pulse treatment solution is allowed to stand for 10 minutes under ice cooling,
After heat treatment at 37 ° C for 5 minutes, add 1 ml of MY medium,
Shake time. It was spread on MY agar medium containing 50 µg / ml kanamycin and cultured at 25 ° C for 3 to 6 days. It was confirmed that the emerged colonies were clearly kanamycin resistant by applying them to a separately prepared MY agar medium containing kanamycin.

The plasmid vector pK1, p
Transformed microorganisms transformed with a recombinant vector containing K2, pK3 or pK4 have been deposited with the Research Institute of Microorganisms and Technology, respectively, as follows. R.rhodochrous ATCC 12674 / pK1
No.R.rhodochrous ATCC 12674 / pK2
No.R.rhodochrous ATCC 12674 / pK3
No.R.rhodochrous ATCC 12674 / pK4
No. Hereinafter, pK4 was further studied. (4) Recovery and purification of composite plasmid vector from Rhodococcus Rhodococcus rhodochrous ATCC 12674 / pK4 strain
Culture was carried out in 1 MY medium (containing 50 μg / ml kanamycin). Penicillin G when OD660 = 0.15-0.2
After adding 0.5 U / ml and culturing to OD660 = 1.0, the cells were recovered by centrifugation. After washing the cells with 40 ml of TES buffer, 11 ml of 50 mM Tris-HCl (pH 8) -12.5% sucrose-100 mM
The suspension was suspended in NaCl-1 mg / ml lysozyme and shaken at 37 ° C. for 3 hours. Add 0.6 ml of 0.5 M EDTA solution, 2.4 ml of 5 M N
An aCl solution and 4.4 ml of 4% SDS-0.7M NaCl were sequentially added, mixed gently, and allowed to stand on ice for 18 hours. 65,000xg at 4 ℃
After centrifugation for 1 hour, a supernatant was obtained, to which 4.6 ml of 50% polyethylene glycol 6000 was added. Let stand on ice for 3 hours, 1,000x
Centrifuged at g for 5 minutes. The precipitate was dissolved in 5 ml of TES buffer, 7.5 g of CsCl, 1.5 mg / ml ethidium bromide-TE.
2 ml of S buffer was added and mixed. 130,00 hours for 42 hours
It was subjected to 0xg density gradient centrifugation.

After fractionating the plasmid fraction detected by irradiation with ultraviolet light, it was treated with n-butanol to remove ethidium bromide. After dialysis against a TE buffer, a purified plasmid fraction was obtained by ethanol precipitation. This was subjected to 0.7% agarose gel electrophoresis, and the presence of the plasmid was confirmed by staining the gel with ethidium bromide. (5) Comparison of composite plasmid pK4 derived from Escherichia coli and pK4 derived from Rhodococcus 1) Measurement of molecular weight A part of the plasmid was subjected to 0.7% agarose gel electrophoresis. At this time, E. coli plasmid is used as a size marker.
pUC18, pUC118, pBR322 (2.69kb, 3.16kb, 4.36k, respectively)
b) was run simultaneously. As a result, the pK4 plasmid
Escherichia coli and Rhodococcus
It was 5.3 kb. 2) Specificity of cleavage by various restriction enzymes A part of the plasmid was reacted with various restriction enzymes. After the reaction was completed, the reaction solution was subjected to 0.7% agarose gel electrophoresis and 5%
Analysis was performed by acrylamide gel electrophoresis. The size of each restriction enzyme fragment of the plasmid was calculated using HindIII digestion and PstI digestion of lambda phage DNA as size markers. As a result, the pK4 plasmid exhibited the same restriction enzyme cleavage properties as in Table 1. (6) Transformation of Escherichia coli using Rhodococcus-derived composite plasmid pK4 Escherichia coli JM105 was transformed using plasmid pK4 obtained from Rhodococcus rhodochrous ATCC 12674 / pK4 strain. When selected on 2 × YT agar plates containing 50 μg / ml of kanamycin, strains showing kanamycin resistance performance at high frequency were obtained. Plasmids were isolated from 12 of these transformants, and the cleavage patterns of the plasmids were examined. The plasmids obtained from the Rhodococcus transformants were examined.
Same as pK4 (Table 1).

[0020]

Example 2 A plasmid pA3 was prepared as shown in FIG. Cut plasmid pRC003 with restriction enzyme ClaI,
A composite plasmid vector inserted into the AccI site of the pUC19 plasmid was created and named pA3. Introduced into E. coli JM105 strain as in Example 1, 50 μg / ml ampicillin, 1 mM I
White colonies were selected in a 2 × YT medium containing PTG and 0.02% Xgal, amplified, separated and purified, and then confirmed with restriction enzymes. About this plasmid Rhodococcus rhodochrou
s Attempt to transform ATCC 12674 strain revealed that this plasmid imparts resistance to this strain to ampicillin at a concentration of 10 μg / ml. The transformant microorganism obtained here, R. rhodochrous ATCC 12674 / pA
No. 3 was sent to the Institute of Microbial Industry and Technology by the National Institute of Advanced Industrial Science and Technology.
Deposited as No.32. Table 2 shows the restriction enzyme cleavage pattern of plasmid pA3.

[0021]

[Table 2]

[0022]

The composite plasmid vector of the present invention is Rhod
It is useful for breeding and improving industrially usable microorganisms in bacteria belonging to the genus ococcus or Escherichia.

[Brief description of the drawings]

FIG. 1. Plasmids pRC001, pRC002, pRC003 and pRC0
04 restriction map.

FIG. 2: Plasmids pK1, pK2, pK3 and pK4 of the invention
Here is how to create it.

FIG. 3 shows a method for preparing plasmid pA3 of the present invention.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12R 1:19) (72) Inventor Yoshihiro Hashimoto 10-1 Ogurocho, Tsurumi-ku, Yokohama-shi, Kanagawa Nitto Chemical Industry Co., Ltd. Central Research Laboratory (72) Inventor Fujio Yu 10-1 Oguro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa Prefecture Nitto Chemical Industry Co., Ltd. Central Research Laboratory (56) References JP-A-4-148685 (JP, A) JP-A-4-330287 ( JP, A) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/74 BIOSIS (DIALOG) WPI (DIALOG)

Claims (6)

(57) [Claims] 1. A (A) Plasmid pK1, pK2, pK3, in plasmid selected from pK4 Oyo <br/> beauty pA3, Rhodococcus (Rho
a DNA region capable of replicating and growing in a strain cell belonging to the genus dococcus ) , and (B) a plasmid DNA capable of replicating and growing in E. coli cells
And (C) a DNA region containing a drug resistance gene. 2. The plasmid DNA replication region (B) is pHSG
299, pHSG298, pUC19 and pUC18.
A composite plasmid vector as described. 3. A method selected from pK1, pK2, pK3, pK4 and pA3.
2. The composite plasmid vector according to claim 1, which is used . A rhod transformed with the composite plasmid vector according to any one of claims 1 to 3.
Coccus ( Rhodococcus ) or Escherichia ( Esc )
herichia ) A transformed microorganism of a bacterium belonging to the genus. 5. A Rhodococcus (Rhodococcus) bacteria of the genus
Rhodococcus rhodochrou
s ) The transformed microorganism according to claim 4, which is ATCC 12674 strain. 6. Escherichia (Escherichia) bacteria is, claim 4 is E. coli (Escherichia coli) K-12 strain
The transformed microorganism according to the above.