JP4570075B2 - Plasmid derived from Rhodococcus bacterium and derivatives thereof, and E. coli-Rhodococcus bacterium shuttle vector - Google Patents

Description

  The present invention relates to a plasmid having a novel replication system and a shuttle vector using the plasmid. More specifically, the present invention relates to an autonomously replicable plasmid having a novel replication system derived from Rhodococcus rhodochrous, and an Escherichia coli-Rhodococcus genus bacterial shuttle vector using the replication system.

  Rhodococcus bacteria are used as microbial catalysts in the conversion reactions of many kinds of organic compounds. For example, it is used in a conversion reaction to an amide compound using a nitrile compound as a substrate, and is currently industrialized (see Non-Patent Document 1).

It is also known that Rhodococcus bacteria have the ability to metabolize and decompose many kinds of compounds such as aromatic compounds and heterocyclic compounds (see Non-Patent Document 2).
As described above, Rhodococcus bacteria have a wide range of catalytic activities for various organic compounds, and thus can be expected to be widely used in industry.

  Against this background, in order to further improve the function of Rhodococcus bacteria as a microbial catalyst, host-vector systems using Rhodococcus bacteria as hosts have been developed. Several types of potential plasmids of Rhodococcus bacteria have already been found. Among them, the nucleotide sequences of plasmids pFAJ2600, pRC4 and pAK22 have been determined (see Non-Patent Documents 3, 4 and 5). These base sequences are relatively high in homology with the replication protein RepA of the plasmid pAL5000 derived from Mycobacterium fortuitum belonging to the family of ColE2 type plasmids. Therefore, it can be presumed that these plasmids have a similar replication system.

Kobayashi M et al., Trends Biotechnol, 10, 402-408 (1992) Bell KS, etc., J Appl Microbiol, 85, 195-210 (1998) De Mot R et al., Microbiology, 143, 3137-3147 (1997) Matsui T et al., Appl Microbiol Biotechnol, 56,196-200 (2001) Kulakov, L.A., et al., Plasmid, 38, 61-69 (1997)

  However, it can be presumed that the plasmids known so far have a similar replication system as described above, and if a plurality of such plasmids are present in the same cell, the plasmids are incompatible. Coexistence between them becomes difficult. Therefore, it becomes difficult to produce several types of proteins in the same cell.

  Accordingly, an object of the present invention is to provide an autonomously replicable plasmid having a novel replication system, and an Escherichia coli-Rhodococcus genus bacterial shuttle vector.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that a latent plasmid derived from Rhodococcus rhodochrous ATCC 21199 strain has a novel replication system and completed the present invention. In addition, an E. coli-Rhodococcus bacterium shuttle vector having a novel replication system was constructed using such a plasmid and an E. coli plasmid, and the present invention was completed.

That is, the present invention is as follows.
(1) A plasmid comprising the nucleotide sequence set forth in SEQ ID NO: 1 or a derivative thereof. (2) A plasmid having a base sequence encoding the amino acid sequence set forth in SEQ ID NO: 2. (3) A plasmid having the base sequence described in SEQ ID NO: 3. (4) A plasmid having the base sequence described in SEQ ID NO: 4. (5) The plasmid according to any one of (1) to (4), capable of autonomous replication in a bacterium belonging to the genus Rhodococcus. (6) It is constructed from the plasmid according to any one of (1) to (5) or a part thereof, and a plasmid or a part thereof capable of autonomous replication in Escherichia coli, and in a Rhodococcus bacterium or in Escherichia coli. A self-replicating shuttle vector.

  ADVANTAGE OF THE INVENTION According to this invention, the plasmid which has a novel replication system which can coexist with the existing plasmid and vector within the bacterium of the genus Rhodococcus, and E. coli-Rhodococcus bacterium shuttle vector can be provided.

  The present invention is an autonomously replicable plasmid having a novel replication system. A plasmid having a base sequence described in SEQ ID NO: 1 or a derivative thereof. Specifically, plasmid pRC032 collected from Rhodococcus rhodochrous ATCC 21199 strain is mentioned. The plasmid pRC032 has the base sequence described in SEQ ID NO: 1. FIG. 1 shows a restriction map of plasmid pRC032.

  The above-mentioned plasmid is confirmed, for example, by electrophoresis or the like from among bacteria of the genus Rhodococcus derived from the natural world and distribution agencies, and a plasmid having a plasmid size of 10 kb or less is selected.

  In order to obtain a plasmid having a replication system different from that of a conventionally known plasmid, the base sequence of the collected plasmid is compared with that of the replication protein RepA of the plasmid pAL5000 derived from Mycobacterium fortuitum belonging to the family of ColE2-type plasmids. If the homology is low, it is judged that the collected plasmid has a replication system dissimilar to previously known plasmids.

  Here, a “derivative” of a plasmid means a plasmid that necessarily contains a replication essential region (replication initiation site and replication protein gene (rep gene)) of a plasmid collected from a strain. For example, the duplication essential area is saved, and the other part is inserted and / or deleted.

  Identification of the replication essential region is performed by preparing a deletion plasmid using various restriction enzymes and removing the sequence fragment digested with the restriction enzymes. In order to facilitate identification of the replication essential region from the deletion plasmid, the plasmid preferably has a drug resistance gene or the like in advance.

Next, the E. coli-Rhodococcus genus shuttle vector of the present invention will be described.
The shuttle vector of the present invention has an essential replication region derived from a plasmid of the genus Rhodococcus and an essential replication region derived from an Escherichia coli plasmid. Preferably, it has a drug resistance gene in addition to the above-mentioned replication essential region. As the plasmid of the genus Rhodococcus, the plasmid pRC032 described above is preferably used. A shuttle vector having a base sequence encoding the amino acid sequence of SEQ ID NO: 2, a base sequence of SEQ ID NOs: 3 and 4, particularly preferably a base sequence of SEQ ID NO: 4 is prepared.

  Examples of the plasmid derived from E. coli used in the present invention include pBR322, pSC101, pACYC184, pACYC177, pTrc99A, pUC18, pUC19, pUC118, pUC119, pHSG298 or pHSG299. Preferred examples include pTrc99A, pUC18, pUC19, pUC118, pUC119, pHSG298, pHSG299, and the like, which are pUC vector derivatives having drug resistance genes. In particular, pHSG298 having a kanamycin resistance gene is preferred.

  The shuttle vector of the present invention can be prepared by ligating the aforementioned Rhodococcus bacterium plasmid, Escherichia coli-derived plasmid and, if necessary, a drug resistance gene. As the drug resistance gene, an antibiotic resistance gene is preferable, and a kanamycin resistance gene is more preferable.

Preferred shuttle vectors of the present invention include pKR321, pKR322, pKR323, pKR324, pKR325, pKR326, pKR327 or pKR328. Any of these shuttle vectors can be prepared using plasmid pHSG298 derived from E. coli and pRC032 derived from Rhodococcus rhodochrous ATCC21199.
For example, shuttle vector pKR321 can be prepared by ligating a fragment obtained by digesting plasmid pHSG298 with BamHI and a fragment obtained by digesting plasmid pRC032 with BamHI. The shuttle vector pKR321 was deposited on May 31, 2004 at the National Institute of Advanced Industrial Science and Technology Patent Biological Depositary Center (1-1-1 Higashi 1-1-1, Tsukuba City, Ibaraki Prefecture). The deposit number is FERM P -20070. The shuttle vectors pKR322, pKR323, pKR324, pKR325, pKR326, pKR327 or pKR328 can be prepared using the plasmid pRC032 and pHSG298 or pKR321.

  The shuttle vector is then introduced into the host cell. Selection of a shuttle vector that functions in any of the Rhodococcus bacteria and E. coli is performed, for example, using the drug resistance of the host cell as an indicator. The method for introducing the host cell is not particularly limited as long as it is a method for introducing DNA into bacteria. Examples thereof include a method using calcium ions and an electroporation method.

  As the host cell into which the shuttle vector is introduced used in the present invention, Rhodococcus rhodochrous ATCC999 strain, ATCC12674 strain, ATCC17895 strain, ATCC15998 strain, ATCC33275 strain, ATCC184 strain, ATCC4001 strain, ATCC4273 strain, ATCC4276 , ATCC9356, ATCC12483, ATCC14341, ATCC14347, ATCC14347, ATCC14350, ATCC15905, ATCC15998, ATCC17041, ATCC19149, ATCC19150, ATCC21243, ATCC29670, ATCC29672, ATCC29675, ATCC13258, ATCC13808 ATCC17043, ATCC19067, ATCC21999, ATCC21291, ATCC21785, ATCC21924, IFO14894, IFO3338, NCIMB11215, NCIMB11216, JCM3202, Rhodococcus globerulus, IFO14531, Rhodococcus globerulus JCM6162, JCM6164, Rhodococcus erythropolis IFO12538, IFO12320, Rhodococc Equi (Rhodococc) us equi) IFO3730 strain or JCM1313 strain. Preferably, Rhodococcus rhodochrous ATCC 12674 strain, Rhodococcus rhodochrous ATCC 19140 strain or Rhodococcus rhodochrous ATCC 17895 strain, particularly preferably Rhodococcus rhodochrous strain 126 . The ATCC strain can be easily obtained from the American Type Culture Collection. The IFO strain can be easily obtained from the Fermentation Research Institute. The NCIMB strains can be easily obtained from the NCIMB Japan Research Center. The JCM strain can be easily obtained from a microbial strain storage facility.

In the preparation of the shuttle vector described above, a replication essential region of a plasmid derived from a Rhodococcus genus bacterium was identified in advance, and this region was linked to an essential replication region derived from Escherichia coli, but first from a genus Rhodococcus genus. An appropriate plasmid region may be ligated with an E. coli-derived replication essential region, and a deletion plasmid may be prepared for the plasmid. By creating and evaluating deletion plasmids, it is possible to optimize the size of the shuttle vector and to identify the replication essential region of the plasmid derived from the genus Rhodococcus.
As described above, a deletion plasmid can also be prepared for plasmid pRC032, and a replication essential region can be identified. SEQ ID NO: 2 is an amino acid sequence of ORF (Open Reading Frame) found in an approximately 2 kb fragment obtained by digestion with restriction enzyme sites XbaI and BglII in FIG. SEQ ID NO: 3 is the base sequence of ORF (Open Reading Frame) found in an approximately 2 kb fragment obtained by digestion with restriction enzyme sites XbaI and BglII in FIG. SEQ ID NO: 4 is the base sequence of an approximately 2 kb fragment obtained by digestion with restriction enzyme sites XbaI and BglII in FIG.

Hereinafter, the present invention will be described in more detail with reference to examples.
[Example 1]
(1) Preparation of plasmid pRC032 from Rhodococcus rhodochrous ATCC21199 strain and determination of nucleotide sequence -yeast extract 3 g / L, KH ₂ PO ₄ 0.5 g / L, K ₂ HPO ₄ 0.5 g / L) was inoculated into a test tube and cultured at 37 ° C. for 24 hours with shaking. 1 ml of the preculture was inoculated into a 500 ml Erlenmeyer flask containing 80 ml of MYK liquid medium, shake-cultured at 30 ° C. for 8 hours, added with 8 ml of 20% glycine, and further cultured with shaking for 3 days. After completion of the culture, the culture solution was transferred to a centrifuge tube and centrifuged at 5000 rpm (about 3700 × g) for 30 minutes to recover the cells. A TENS solution (0.05M-Tris, 0.01M-EDTA, 0.05M-NaCl, 20% sucrose) containing 5 ml of lysozyme (150 mg) was added to the collected cells and reacted at 37 ° C. overnight. Then, it was once frozen at −20 ° C. and thawed at room temperature. To this, 750 μl of 10% SDS was added, immediately stirred vigorously with Vortex, incubated at 37 ° C. for 30 minutes, and placed on ice for 10 minutes. Next, 750 μl of 2N-NaOH was added and immediately stirred with Vortex and placed on ice for 10 minutes. To this was added 4 ml of 3M sodium acetate (pH 4.8), stirred with Voretx, and placed on ice for 30 minutes. The treated bacterial solution was centrifuged at 14000 rpm (about 20,000 × g) for 10 minutes, 10 ml of the supernatant was transferred to another centrifuge tube, 25 ml of cold ethanol was added, and the mixture was centrifuged at 14000 rpm for 30 minutes. Add 1 ml of Sol IV (prepare 2 μl RNase / 7.5 μl Sol IV) to the precipitate, leave it at room temperature for 20 minutes, add 2.5 ml of cold ethanol, centrifuge at 14000 rpm for 30 minutes, and add 5 ml of cold 70% ethanol. Poured and centrifuged at 14000 rpm for 1 minute to remove the supernatant.
After drying the precipitate, 100 μl of sterilized water was added to obtain a plasmid solution. The plasmid solution was analyzed by 0.7% agarose gel electrophoresis to confirm the presence of the plasmid. The plasmid was named pRC032. The base sequence of the plasmid was determined using ABI PRISM 3100. As a result, the base sequence represented by SEQ ID NO: 1 was obtained.

(2) Preparation of restriction enzyme map of plasmid derived from Rhodococcus rhodochrous ATCC 21199 1 μl of plasmid pRC032 solution, 2 μl of 10xH Buffer, 16 μl of sterilized water and 1 μl of restriction enzyme (when combining two restriction enzymes) 1 μl each) was added and reacted at 37 ° C. for 4 hours. The generated DNA fragment was analyzed by agarose gel electrophoresis, and a restriction enzyme map (FIG. 1) was prepared.

[Example 2]
Construction of E. coli-Rhodococcus genus shuttle vector (1) Digestion of plasmid pRC032 with restriction enzyme BamHI Into 20 μl of plasmid pRC032 solution prepared as described above, 3 μl of 10 × H Buffer, 5 μl of sterile water, 2 μl of restriction enzyme BamHI And reacted at 37 ° C. for 4 hours. After completion of the reaction, 3 μl of 3M sodium acetate (pH 5.2) and 75 μl of cold ethanol were added to 30 μl of the reaction solution, and a precipitate was obtained by centrifugation at 15000 rpm (about 21000 × g) for 30 minutes. To this, 200 μl of 70% cold ethanol solution was added and centrifuged at 15000 rpm for 1 minute, and the supernatant was removed to obtain a precipitate. The precipitate was dried, dissolved in 10 μl of sterilized water, 2 μl of 10 × loading buffer (2 μl) was added, and the entire amount was subjected to 0.7% agarose gel electrophoresis. After completion of the electrophoresis, a target band (gel) was cut out by irradiation with a 365 nm ultraviolet lamp, and a DNA fragment was obtained by GFX PCR DNA and Gel Band Purification Kit (Amersham Bioscience). Specifically, it was performed as follows. The excised gel was transferred to a microcentrifuge tube, 160 μl of Capture Buffer was added, and the mixture was incubated at 60 ° C. until the gel was completely dissolved. The entire lysate was transferred to the GFX Column and allowed to stand for 1 minute, and then centrifuged at 15000 rpm for 30 seconds to remove the liquid accumulated in the collection tube. Furthermore, 500 μl of wash buffer was added to the GFX Column, and centrifuged at 15000 rpm for 30 seconds to remove the liquid accumulated in the collection tube. The filter part was placed on a microcentrifuge tube, 50 μl of sterilized water was poured into it, and centrifuged at 15000 rpm for 30 seconds, and a solution of the plasmid pRC032-BamHI fragment was recovered in the microcentrifuge tube.

(2) Digestion of plasmid pHSG298 with restriction enzyme BamHI Restriction enzyme BamHI was performed in the same manner as in (1) above except that 10 µl of plasmid pHSG298 (Takara Bio Inc .: see Fig. 2) was used and the restriction enzyme reaction solution was halved. DNA fragments were recovered by digestion with ethanol and ethanol precipitation. 44 μl of sterilized water was added to the obtained DNA fragment, and SAP (Shrimp Alkaline Phosphatase: Amersham Bioscience) treatment was performed. SAP treatment was performed at 37 ° C. for 15 minutes by adding 5 μl of 10 × SAP Buffer and 1 μl of SAP to the DNA fragment solution. After the treatment, the mixture was incubated at 60 ° C. for 15 minutes to inactivate SAP. 150 μl of sterilized water was added to 50 μl of the SAP-treated DNA fragment solution, and 200 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA) saturated phenol was dispensed and stirred well. This was centrifuged at 15000 rpm (about 21,000 × g) for 2 minutes, and the upper layer (aqueous layer) was transferred to another microcentrifuge tube. Similarly, 200 μl of TE saturated phenol was added, stirred well, centrifuged at 15000 rpm, and then 180 μl of the upper layer was transferred to another microcentrifuge tube. 200 μl of chloroform (including isoamyl alcohol) was added to the obtained upper layer solution and stirred well, followed by centrifugation at 15000 rpm, and 160 μl of the upper layer was transferred to another microcentrifuge tube. 200 μl of chloroform (including isoamyl alcohol) was added again and stirred well, followed by centrifugation at 15000 rpm, and 140 μl of the upper layer was transferred to another microcentrifuge tube.
14 μl of 3M-sodium acetate (pH 5.2) and 350 μl of cold ethanol were added to the recovered solution, and centrifuged at 15000 rpm to obtain a precipitate. To this, 500 μl of 70% cold ethanol solution was poured, centrifuged at 15000 rpm, and the supernatant was removed. The precipitate was dried and then dissolved in 20 μl of sterilized water to obtain a pHSG298-BamHI fragment that had been treated with SAP.

(3) Construction of shuttle vector by binding of pRC032-BamHI fragment and pHSG298-BamHI fragment 0.5 μl of SAP-treated plasmid pHSG298-BamHI fragment solution and 6 μl of pRC032-BamHI fragment solution were added to DNA Ligation Kit ver 2.1 (Takara Bio Inc.) ) Was used for overnight ligation reaction at 4 ° C. Transformation of E. coli was performed as follows. E. coli JM109 strain competent was prepared by the method of Nojima et al. (Hiroshi Nojima. 1991. Highly efficient competent cell preparation method. Edited by Masami Muramura and Hiroto Okayama, Genetic Engineering Handbook, pp 46-51, Yodosha, Tokyo). did. 100 μl of E. coli JM109 strain competent cell was dispensed into a test tube placed on ice, and 5 μl of the Ligation reaction solution was added and allowed to stand for 30 minutes. Next, it was immersed in a constant temperature bath at 42 ° C. for 30 seconds to give a heat shock, and then kept on ice for 2 minutes. 0.4 ml of SOC medium (Bacto-tryptone 20 g / L, Bacto-yeast extract 5 g / L with final concentrations of 1 mM sodium chloride, 2.5 mM potassium chloride, 5 mM magnesium chloride, 5 mM magnesium sulfate, 20 mM glucose) In addition, the cells were cultured for 1 hour in a shaking incubator at 37 ° C. LB agar medium (Bacto-Trypton 10g / L, Bacto-Yeast Extract 5g / L, (Difco), sodium chloride 5g / L, agar 15g / L) And then statically cultured overnight in a 37 ° C. incubator.
The next day, arbitrarily select 6 white colonies that grew on the plate and add 1.5 ml of LB liquid medium (Bacto-Trypton 10 g / L, Bacto-Yeast Extract 5 g / L (above, Difco), sodium chloride 5 g / L). The test tube was inoculated and cultured with shaking at 37 ° C. for 9 hours. After completion of the culture, the cells were transferred to a microcentrifuge tube and the cells were collected by centrifugation. Preparation of the plasmid from the cells was performed using FlexiPrep Kit (Amersham Bioscience).

Specifically, 200 μl of Solution I was added to the cells and suspended well with Vortex, 200 μl of Solution II was added and stirred gently, and then 200 μl of Solution III was added and stirred well. The mixture was centrifuged at 15000 rpm (about 21,000 × g) for 5 minutes, and the supernatant was transferred to another microcentrifuge tube. 420 μl of isopropanol was added thereto, mixed, and allowed to stand at room temperature for 10 minutes, and then centrifuged at 15000 rpm for 10 minutes to obtain a precipitate. 150 μl of Sephaglas FP was added to the precipitate and stirred well, allowed to stand for 1 minute, and centrifuged at 15000 rpm for 15 seconds to obtain a precipitate. To this, 200 μl of Wash Buffer was added and stirred well, followed by centrifugation at 15000 rpm for 15 seconds to obtain a precipitate. 300 μl of 70% ethanol was added to the precipitate and mixed. The supernatant was removed by centrifugation at 15000 rpm for 15 seconds. The precipitate was broken and dried with the lid left open. 50 μl of sterilized water was added and mixed well, and allowed to stand for 5 minutes. Thereafter, the mixture was centrifuged at 15000 rpm for 1 minute, and 45 μl of the supernatant was transferred to another microcentrifuge tube to obtain a purified plasmid.
The purified plasmid thus obtained was confirmed in size by 0.7% agarose gel electrophoresis. At that time, after digestion with several kinds of restriction enzymes, the fragment size was examined by 0.7% agarose gel electrophoresis to determine whether the obtained plasmid was the target one. As a result, target plasmids having different orientations of the inserted fragments were obtained and named shuttle vectors pKR321 and pKR322, respectively (FIG. 3).

[Example 3]
Transformation of ATCC12674 strain using shuttle vector pKR321 Cryopreserved Rhodococcus rhodochrous ATCC12674 competent cells were thawed at room temperature, mixed well, and transferred to a sterile microcentrifuge tube 20 μl. For competent cells, cells in the logarithmic growth phase of ATCC 12674 strain are collected by centrifugation, washed three times with ice-cooled sterilized water, suspended in sterilized water, and stored frozen at -80 ° C. Yes. Next, 0.5 μl of the pKR321 solution was added thereto and left on ice for 10 minutes. The whole amount was transferred to a 0.1 cm electro cuvette (greiner bio-one), and a high voltage of 2.00 kV and 200 HMs was applied with Gene Pulser (BIO-RAD). After standing on ice for 10 minutes, incubate for 10 minutes in a 37 ° C incubator. MYK medium (polypeptone 5 g / L, Bacto-Malt extract 3 g / L, Bacto-yeast extract 3 g / L, KH ₂ PO ₄ 0.5 g / L, K ₂ HPO ₄ 0.5 g / L) was added, and the mixture was statically cultured at 30 ° C. for 5 hours. After completion of the culture, pipette well inside the electro cuvette and add 100 μl of the culture solution to MYK agar medium containing 75 mg / L kanamycin (polypeptone 5 g / L, Bacto-Malt extract 3 g / L, Bacto-yeast extract 3 g / L, KH ₂ PO ₄ 0.5 g / L, K ₂ HPO ₄ 0.5 g / L, agar 15 g / L) and statically cultured in a 30 ° C. incubator for 2 days.
The grown colonies were inoculated into 10 ml of MYK liquid medium containing 50 mg / L kanamycin and pre-cultured at 30 ° C. for 2 days. Next, 0.1 ml of the culture solution was added to a test tube containing 10 ml of MYK (containing 50 mg / L kanamycin) liquid medium, and cultured with shaking for 8 hours. Further, 1 ml of 20% glycine was added and cultured for 16 hours. 1.5 ml of the culture solution was transferred to a microcentrifuge tube and centrifuged at 15000 rpm (about 21,000 × g) for 5 minutes to obtain a precipitate. To this, a TENS solution containing 200 μl of lysozyme (30 mg / ml) was added, reacted at 37 ° C. overnight, once frozen, and then thawed at room temperature. To this, 30 μl of 10% SDS was added and vortexed immediately, incubated at 37 ° C. for 30 minutes, and then placed on ice for 10 minutes. Next, 30 μl of 2N-NaOH was added and stirred immediately and placed on ice for 10 minutes. To this, 160 μl of 3M sodium acetate (pH 4.8) was added and stirred, and placed on ice for 30 minutes. After centrifugation at 15000 rpm for 10 minutes, 360 μl of the supernatant was transferred to another centrifuge tube, 0.9 ml of cold ethanol was added, and the mixture was centrifuged at 15000 rpm for 30 minutes. To the precipitate, 100 μl of SolIV (0.5 μl RNase / 0.5 ml SolIV was prepared) was added and left at 37 ° C. for 1 hour. To this, 250 μl of cold ethanol was added and centrifuged at 15000 rpm for 30 minutes to obtain a precipitate. 500 μl of cold 70% ethanol was poured into the precipitate and centrifuged at 15000 rpm for 1 minute to obtain a precipitate. After drying the precipitate, 20 μl of sterilized water was added to obtain a plasmid solution.
The size of the plasmid thus obtained and the fragment size after cleavage with several restriction enzymes were the same as pKR321. From these results, it was revealed that pKR321 has the ability to replicate in the ATCC12674 strain.

[Example 4]
Preparation of deletion plasmid from pKR321 and identification of replication essential region by transformation of ATCC12674 strain using it (1) Preparation of deletion plasmids pKR323 and pKR324 Using 5 μl of plasmid pKR321 solution, restriction enzymes BamHI and BglII were used. The digestion was carried out in the same manner as in (2) of Example 2 except that the amount of the reaction solution was reduced to 1/4. After completion of the reaction, add 160 μl of sterilized water to make a total volume of 200 μl, dispense 200 μl of TE saturated phenol, stir well with Vortex, centrifuge for 2 minutes at 15000 rpm (about 21,000 × g) ) Was transferred to another microcentrifuge tube. Similarly, 200 μl of TE-saturated phenol was added, stirred well with Vortex, centrifuged at 15000 rpm for 2 minutes, and the upper layer (aqueous layer) was transferred to another microcentrifuge tube. 200 μl of chloroform (including isoamyl alcohol) was added to the obtained upper layer solution, suspended well, then centrifuged at 15000 rpm for 30 seconds, and the upper layer was transferred to another microcentrifuge tube. 200 μl of chloroform (including isoamyl alcohol) was added again and suspended well, followed by centrifugation at 15000 rpm for 30 seconds, and 170 μl of the upper layer was transferred to another microcentrifuge tube. To this, 17 μl of 3M sodium acetate and 425 μl of cold ethanol were added and centrifuged at 15000 rpm for 30 minutes to obtain a precipitate. 500 μl of cold 70% ethanol was added to the precipitate and centrifuged at 15000 rpm for 1 minute, and the supernatant was removed. The precipitate was dried and then dissolved in 10 μl of sterilized water to obtain a BamHI-BglII digested plasmid pKR321 solution. This solution contains three fragments of 4.2 kb, 2.2 kb, and 2.7 kb. Using 5 μl of this solution, a ligation reaction was performed overnight at 4 ° C. using DNA Ligation Kit ver 2.1 (Takara Bio Inc.). E. coli strain JM109 was transformed in the same manner as in Example 2 (3), and a plasmid was prepared from the resulting colonies.
When the plasmid prepared from the obtained colony was examined, this plasmid was obtained by removing the BamHI-BglII fragment of about 2.2 kb from pKR321. This was named plasmid pKR323. A plasmid was also obtained in which the inserted fragment was reversed. This was named plasmid pKR324.

(2) Preparation of deletion plasmids pKR327 and pKR328 5 μl of plasmid pKR323 solution was digested with XbaI and NotI, and an XbaI fragment (about 1.9 kb) was purified by agarose electrophoresis. This fragment and the pHSG298-XbaI fragment that had been treated with SAP were subjected to a ligation reaction overnight at 4 ° C. using DNA Ligation Kit ver 2.1 (Takara Bio Inc.). E. coli strain JM109 was transformed in the same manner as in Example 2 (3), and a plasmid was prepared from the resulting colonies.
When a plasmid prepared from one of the obtained colonies was examined, this plasmid was obtained by removing about 2.3 kb of XbaI-BamHI fragment from pKR323. This was named plasmid pKR327. A plasmid was also obtained in which the inserted fragment was reversed. This was named plasmid pKR328.

(3) Preparation of deletion plasmid pKR329 10 μl of plasmid pKR323 solution was cleaved with SacI to obtain a SacI fragment solution. This solution consists of a mixture of about 1.3 kb, 1.5 kb, 4.1 kb three pieces. Using 1 μl of the solution, a ligation reaction was performed overnight at 4 ° C. using DNA Ligation Kit ver 2.1 (Takara Bio Inc.). E. coli strain JM109 was transformed in the same manner as in Example 2 (3), and a plasmid was prepared from the resulting colonies. When a plasmid prepared from one of the obtained colonies was examined, this plasmid was obtained by removing about 2.8 kb of SacI fragment from pKR323. This was named plasmid pKR329.

(4) Preparation of deletion plasmid pKR325 1 μl of plasmid pKR321 solution was digested with XbaI. After completion of the reaction, XbaI was inactivated by treating the reaction solution at 65 ° C. for 15 minutes, and an XbaI fragment mixture was obtained by ethanol precipitation and dissolved in sterilized water. This solution consists of a mixture of about 0.6 kb, 1.7 kb, and 6.8 kb three pieces.
Using 1 μl of the solution, a ligation reaction was performed overnight at 4 ° C. using DNA Ligation Kit ver 2.1 (Takara Bio Inc.). E. coli strain JM109 was transformed in the same manner as in Example 2 (3), and a plasmid was prepared from the resulting colonies.
When a plasmid prepared from one of the obtained colonies was examined, this plasmid was obtained by removing an about 2.3 kb XbaI fragment from pKR321. This was named plasmid pKR325.

(5) Preparation of deletion plasmid pKR326 5 μl of plasmid pKR325 solution was digested with BamHI and BglII to obtain a BamHI-BglII fragment mixture and dissolved in sterile water. This solution consists of a mixture of two fragments of 2.2 kb and 4.6 kb. Using 5 μl of the solution, a ligation reaction was performed overnight at 4 ° C. using DNA Ligation Kit ver 2.1 (Takara Bio Inc.). E. coli strain JM109 was transformed in the same manner as in Example 2 (3), and a plasmid was prepared from the resulting colonies.
When a plasmid prepared from one of the obtained colonies was examined, this plasmid was obtained by removing the BamHI-BglII fragment of about 2.2 kb from pKR325. This was named plasmid pKR326.

(6) Preparation of deletion plasmid pKR3211 1 μl of plasmid pKR321 solution was digested with XhoI to obtain a XhoI fragment mixture, which was dissolved in sterile water. This solution consists of a mixture of 4 fragments. Using 1 μl of the solution, a ligation reaction was performed overnight at 4 ° C. using DNA Ligation Kit ver 2.1 (Takara Bio Inc.). E. coli strain JM109 was transformed in the same manner as in Example 2 (3), and a plasmid was prepared from the resulting colonies. When a plasmid prepared from one of the obtained colonies was examined, this plasmid was obtained by removing three XhoI fragments of about 2.9 kb in total from pKR321. This was named plasmid pKR3211.

(7) Transformation of ATCC12674 strain using a deletion plasmid Transformation of Rhodococcus rhodochrous ATCC12674 strain using a deletion plasmid was carried out in the same manner as in Example 3. As a result, it was clarified that the region essential for transformation was in a fragment of about 2 kb sandwiched between the XbaI site and the BglII site. FIG. 4 shows the flow of preparation of a deletion plasmid from plasmid pKR321 prepared as described above. FIG. 5 shows the possibility of replication in the Rhodococcus rhodochrous ATCC12674 strain of the deletion plasmid from the plasmid pKR321.

(8) Base sequence of replication essential region The base sequence of the about 2 kb fragment sandwiched between the XbaI site and the BglII site described above was determined using ABI PRISM 3100. As a result, the base sequence shown in SEQ ID NO: 4 was obtained. One ORF (Open Reading Frame) was found in this sequence. As a result of homology search against GenBank for the nucleotide sequence (SEQ ID NO: 3) and amino acid sequence (SEQ ID NO: 2) of the ORF, no significant homology was found, so this is a completely new replication system. It was considered.

[Example 5]
Coexistence of two shuttle vectors pTP014 and pKR326 in bacteria belonging to the genus Rhodococcus The trimethoprim resistant shuttle vector pTP014 was introduced into Rhodococcus rhodochrous ATCC12674 strain in the same manner as in Example 3. As the selective medium, M9-casamino acid agar medium (KH ₂ PO ₄ 3 g / L, Na ₂ HPO ₄ 6 g / L, NaCl 0.5 g / L, NH 4 Cl 1 g / L, glucose 4 g / L) containing trimethoprim (200 μg / ml) L, thiamine-hydrochloride 2 mg / L, MgSO4 (1 mM), casamino acid 2 g / L, agar 15 g / L). A plasmid was prepared from the obtained transformant, and it was confirmed that the plasmid was introduced. pTP014 was deposited on May 26, 2004 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Chuo 6-1-1 Tsukuba City, Ibaraki Prefecture), and the deposit number is FERM P-20065 It is.
Next, cells in the logarithmic growth phase of the transformant were collected by centrifugation, washed three times with ice-cooled sterilized water, and suspended in sterilized water to prepare competent cells. The competent cells were stored frozen at -80 ° C. Transformation was performed using plasmid pKR326 in the same manner as in Example 3 except that the competent cells were used. As the selective medium, MYK agar medium containing 75 mg / l kanamycin was used. Plasmids were prepared from the resulting transformants, and the presence of both plasmids was confirmed.
This shuttle vector was confirmed to be capable of coexisting with the shuttle vector pTP014 considered to have a different replication system in bacteria belonging to the genus Rhodococcus.

  A shuttle vector having a novel replication system capable of mutual replication in E. coli and Rhodococcus bacteria was a vector that could coexist with an existing vector in Rhodococcus bacteria.

It is a restriction map of plasmid pRC032.

It is a restriction enzyme map of plasmid pHSG298.

It is a restriction enzyme map of E. coli-Rhodococcus genus bacterial shuttle vectors pKR321 and pKR322.

It is a figure which shows the flow of preparation of the deletion plasmid from plasmid pKR321.

FIG. 4 is a diagram showing whether replication of a deletion plasmid from the plasmid pKR321 in the Rhodococcus rhodochrous ATCC12674 strain is possible.

Claims (6)

The plasmid according to the following (a) or (b).
(A) A plasmid comprising the nucleotide sequence set forth in SEQ ID NO: 1.
(B) A plasmid comprising a base sequence described in SEQ ID NO: 1, wherein one or several bases other than the replication essential region are deleted, added, inserted or substituted.   A plasmid having a base sequence encoding the amino acid sequence set forth in SEQ ID NO: 2.   A plasmid having the base sequence set forth in SEQ ID NO: 3.   A plasmid having the base sequence set forth in SEQ ID NO: 4.   The plasmid according to any one of claims 1 to 4, which can autonomously replicate in a bacterium of the genus Rhodococcus. A shuttle constructed from the plasmid according to any one of claims 1 to 5 or a part thereof and a plasmid or a part thereof capable of autonomous replication in E. coli and capable of autonomous replication in bacteria of the genus Rhodococcus and in E. coli. vector.

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