JP3142349B2 - Composite plasmid vector and transformed microorganism - Google Patents
Composite plasmid vector and transformed microorganismInfo
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- JP3142349B2 JP3142349B2 JP4539392A JP4539392A JP3142349B2 JP 3142349 B2 JP3142349 B2 JP 3142349B2 JP 4539392 A JP4539392 A JP 4539392A JP 4539392 A JP4539392 A JP 4539392A JP 3142349 B2 JP3142349 B2 JP 3142349B2
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- plasmid
- rhodococcus
- composite
- plasmid vector
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Description
【0001】[0001]
【産業上の利用分野】本発明は、新規な複合プラスミド
ベクターに関し、更に詳しくは、プラスミドpRC001, pR
C002, pRC003およびpRC004から選ばれるプラスミド由来
の、Rhodococcus属細菌内で複製可能なDNA領域と、
大腸菌内で複製可能なプラスミドDNA領域と、薬剤耐
性遺伝子を含むDNA領域とを含有する新規な複合プラ
スミドベクターおよびそれにより形質転換された微生物
に関する。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】[0002]
【従来の技術】Rhodococcus属に属する微生物は、ニト
リル類を水和または加水分解して対応するアミドまたは
酸を生産するための微生物触媒として知られており(欧
州特許出願公開第188316号明細書、同第204555号明細
書、同第348901号明細書)、またRhodococcus rhodochr
ous に属する微生物が極めて高性能なニトリル水和活性
を有することが知られている(欧州特許出願公開第3079
26号明細書)。このような状況下、Rhodococcus属の宿
主ベクター系の開発が以前から期待されていた。しかし
ながら、Rhodococcus属に属する菌株についてはこれら
の微生物を宿主とするに適したベクターの開発は遅れて
おり、Rhodococcus属においてプラスミドの見い出され
た株はRhodococcus sp. H13-A 株 (J.Bacteriol., 170,
638-645(1988))、Rhodococcus erythropolis (rhodoch
rous) ATCC 12674株 (Mol. Gen. Genet., 211, 148-154
(1988))および本発明者らがさきに特許出願したRhodoco
ccusrhodochrous ATCC 4276 等(特願平2-270377号)を
はじめわずか数株にすぎない。そのため、更にRhodococ
cus属に属する菌株から工業的に利用し得る微生物を育
種、改良するための新しいベクターの開発が強く要望さ
れる。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】[0003]
【発明が解決しようとする課題】工業的に有用なRhodoc
occus属細菌から得られる環状プラスミドは本菌属を遺
伝的に改良するにたる可能性は秘めているもののほとん
どがプラスミドのマーカーとなり得るような薬剤耐性遺
伝子を持っていない潜在性プラスミドである。そこで、
このような潜在性プラスミドにマーカーとなりうる遺伝
子を導入することによりベクタープラスミドの構築が可
能となるが、そのような例はまだ一例あるに過ぎない
(J. Bacteriol., 170, 638-645(1988)) 。本発明者ら
は、環状プラスミドpRC001, pRC002, pRC003およびpRC0
04にマーカーとなり得る薬剤耐性遺伝子、クローニング
サイトおよび大腸菌内で複製するのに必要な遺伝子領域
を導入することにより、工業的に有用なプラスミドベク
ターを創成することができることを見いだし、本発明を
完成した。[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】[0004]
【課題を解決するための手段】本発明は、(A)プラス
ミドpRC001, pRC002, pRC003およびpRC004から選ばれる
プラスミド由来の、Rhodococcus属細菌に属する菌株細
胞内で複製増殖可能なDNA領域と、(B)大腸菌細胞
内で複製増殖可能なプラスミドDNA領域と、(C)薬
剤耐性遺伝子を含むDNA領域とを含むことを特徴とす
る複合プラスミドベクターである。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.
【0005】プラスミドpRC001, pRC002, pRC003および
pRC004の複製増殖可能なDNA領域としてはRhodococcu
s属細菌内で複製が可能であれば、該プラスミドの全体
であってもよく、或いは一断片であってもよい。なお、
上記プラスミドpRC001, pRC002, pRC003およびpRC004は
それぞれ Rhodococcus rhodochrous ATCC 4276、ATCC14
349、ATCC 14348およびIFO 3338株由来であり、その制
限酵素切断地図を図1に示す。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】また、大腸菌細胞内で複製増殖し得るプラ
スミドとしては、例えば、pHSG299,pHSG298, pUC19, pU
C18 等を用いることが可能であり、これらのプラスミド
のDNA領域も、大腸菌細胞内で複製増殖可能であれ
ば、プラスミド全体であってもよく、或いは一断片であ
ってもよい。更に薬剤耐性遺伝子としては、カナマイシ
ン耐性遺伝子、アンピシリン耐性遺伝子などが好適に用
いられるが、宿主とするRhodococcus属細菌および大腸
菌内で発現し、宿主に薬剤耐性を与えることができ、両
菌属間で、薬剤耐性能によりプラスミドの存在が示唆さ
れる限り薬剤の種類は限られるものではなく、また一種
類でも複数存在してもよい。[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】以上のような特性を持つ本発明の複合プラ
スミドベクターは、Rhodococcus属細菌としては、Rhodo
coccus rhodochrous ATCC 12674由来菌株を宿主とする
事が可能であり、また、大腸菌としてはK-12系統株を宿
主とすることが可能である。従って、上記複合プラスミ
ドベクターによるそれら微生物の形質転換体を得ること
ができる。[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】[0008]
【実施例】次に、実施例により本発明を更に具体的に説
明するが、下記の実施例は本発明の技術的範囲を限定す
るものではない。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】[0009]
【実施例1】 (1) プラスミドpRC001, pRC002, pRC003またはpRC0
04とプラスミドpHSG299とからなる複合プラスミドベク
ターpK1, pK2, pK3およびpK4の作成 図2に示したようにして複合プラスミドベクターpK1,
pK2,pK3およびpK4を作成した。プラスミドpRC001, p
RC002, pRC003およびpRC004(1μg)にそれぞれ制限酵
素ClaI(5units)を加え37℃,1時間反応させプラスミ
ドDNAを切断した。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.
【0010】一方、プラスミドpHSG299(カナマイシン
耐性を発現する2.7kbのプラスミドであり、市販品とし
て宝酒造より購入が可能)0.5μgを制限酵素AccI (5uni
ts) を加え37℃,1時間反応させプラスミドDNAを切
断した。反応液に1M-Tris-HCl(pH9.0) を1/10量加え、
アルカリホスファターゼ(1unit)と65℃、1時間反応さ
せた。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】上記の処理を行ったプラスミド液を 0.7%
アガロースゲル電気泳動に供し、プラスミドpRC001, pR
C002, pRC003およびpRC004からは2.6kb、プラスミドpHS
G299からは2.7kbのDNAの画分を切り出した。この
際、サイズマーカーとしてラムダファージDNAのHind
III消化物を用い、DNAのサイズを算出した。Gene cl
ean kit(フナコシ(株))を用いてアガロースゲルより
DNAを回収しTE緩衝液(10mMTris-HCl, 1mMEDTA,
(pH8.0))に溶解した。それぞれのDNA断片を含む液を
等量ずつ混合し、T4DNAリガーゼ1unit, 1mM ATP,
10mM ジチオスレイトール, 10mM MgCl2となるように各
成分を加えて4℃、1夜反応させた。[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.
【0012】大腸菌JM105株のコンピテントセル(宝酒
造製)に上記反応液を加え、0℃、1時間静置後、42
℃、2分間の熱処理を行い、2xYT培地(0.5%NaCl,1
%イーストエキス、1.6%トリプトン)を加えて37℃、
1時間振とうした。 25μg/mlカナマイシン、1mM IP
TG(イソプロピル−β−ガラクトピラノシド)、および
0.02% X-gal(5−ブロモ−4−クロロ−3−インドリ
ル−β−D−ガラクトピラノシド)を含む2xYT寒天培
地に塗布し、37℃、1夜静置培養した。出現したコロニ
ーより白色のコロニーを選択し、50μg/mlカナマイシ
ン入りの2xYT培地(3ml)で37℃にて8時間振とう培
養した。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.
【0013】15,000rpm、5分間の遠心分離により菌体
を回収し、0.35ml STET溶液(8%シュークロース、0.
5% TritonX-100, 50mM EDTA, 10mM Tris-HCl(pH8.0))
に懸濁した。リゾチーム液(10mg/ml)25μlを加え、
Vortexで3秒間攪拌後、沸騰している湯に50秒間浸し
た。15,000rpm、15分間の遠心分離により沈澱を取り除
き上清を得た。これにTE飽和フェノール:クロロホル
ム(1:1)液を0.5ml加え攪拌後、15,000rpm、5分
遠心分離を行い、上層を得た。ジエチルエーテル0.5ml
を加えて混合後、遠心分離を行い上層を除去した。イソ
プロパノール0.5ml、2.5M酢酸ソーダ液 (pH4.5) 50μ
lを加え、−80℃、30分静置後15,000rpm、10分遠心分
離を行い沈澱を得た。70%エタノールで洗浄し、減圧乾
燥させ、0.1mlのTE緩衝液に溶解させた。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.
【0014】このようにして調製した上記プラスミド溶
液を用いて、制限酵素HindIII, BamHI, SphI, EcoRI, X
hoIを用いた制限酵素切断パターンを調べた。プラスミ
ドpRC001, pRC002, pRC003またはpRC004から得られた複
合プラスミドはいずれも同じ制限酵素切断部位を持ちそ
れぞれpK1, pK2, pK3およびpK4と命名した(表1)。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】[0015]
【表1】 [Table 1]
【0016】(2)大腸菌からの複合プラスミドpK1, p
K2, pK3およびpK4の分離精製 上記の大腸菌形質転換体(カナマイシン耐性株)を2xY
T培地200mlを用いて培養し、遠心により菌体を回収し
た。菌体を40mlの TES (10mM Tris-HCl (pH8.0),10mM N
aCl, 1mM EDTA)緩衝液で洗浄後、8mlのSTET溶液(50mM
Tris-HCl(pH8.0), 5mM EDTA, 35mM シュークロース)
を加え、リゾチームを10mg添加した。0℃、5分間振と
う後4ml 0.25M EDTA (pH8.0)を加え、時々緩やかに混
合しながら0℃に5分間保った。室温に戻して、2mlの
10% SDS(ラウリル硫酸ナトリウム)溶液、5mlの5M N
aCl溶液を加えて4℃、3−12時間静置した。4℃にて6
5,000xgで1時間遠心し上清を得、これに50%ポリエチ
レングリコール6000を4.6ml加えた。氷上で3時間静置
し、1000xgで5分遠心した。沈澱物を7.5ml TES緩衝液
に溶解し、CsClを8.2g, 15mg/ml臭化エチジウム溶液を
0.2ml加え混合した。この溶液を42時間130,000xgの密
度勾配遠心分離にかけた。紫外線照射により検出された
プラスミド画分を分取した後、n−ブタノールで処理し
臭化エチジウムを除いた。TE緩衝液に対して透析後、
エタノール沈澱により精製プラスミド画分を得た。これ
を0.7%アガロースゲル電気泳動に供し、ゲルを臭化エ
チジウムで染色することによりプラスミドの存在を確認
した。 (3)複合プラスミドのRhodococcus属細菌への導入 Rhodococcus rhodochrous ATCC 12674株の対数増殖期の
細胞を遠心分離により集菌し、氷冷した滅菌水にて3回
洗浄し、15% PEG6000(ポリエチレングリコール6000)
溶液に懸濁した(菌体濃度109cell/ml以上)。上記の
シャトルプラスミドベクターpK1, pK2, pK3またはpK4D
NA10-2μgと菌体懸濁液10μlを混合し、氷冷した。
島津細胞融合装置SSH-1用のチャンバー11にDNAと菌
体の混合液を入れ、氷冷した後、パルス幅500μs,電
場強度14kv/cmで電気パルス処理を行った。(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.
【0017】電気パルス処理液を氷冷下10分間静置し、
37℃、5分間熱処理後、MY培地 1mlを加え、25℃、3
時間振とうした。50μg/mlカナマイシン入りMY寒天
培地に塗布し25℃、3−6日間培養した。出現したコロ
ニーが明らかにカナマイシン耐性であることを別に作成
したカナマイシン入りMY寒天培地に塗布することによ
り確認した。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.
【0018】ここで得られたプラスミドベクターpK1, p
K2, pK3またはpK4を含む組換えベクターで形質転換され
た形質転換微生物はそれぞれ次の通り工業技術院微生物
工業技術研究所に寄託されている。 R.rhodochrous ATCC 12674/pK1 微工研条寄第3728
号 R.rhodochrous ATCC 12674/pK2 微工研条寄第3729
号 R.rhodochrous ATCC 12674/pK3 微工研条寄第3730
号 R.rhodochrous ATCC 12674/pK4 微工研条寄第3731
号 以下、pK4について更に検討した。 (4)Rhodococcusからの複合プラスミドベクターの回
収、精製 Rhodococcus rhodochrous ATCC 12674/pK4株を、400m
lのMY培地(50μg/mlカナマイシンを含む)にて培
養を行った。OD660=0.15〜0.2の頃にペニシリンG
0.5U/mlを加え、OD660=1.0まで培養後、遠心によ
り菌体を回収した。菌体を40mlのTES緩衝液で洗浄
後、11mlの50mM Tris-HCl (pH8)-12.5% sucrose-100mM
NaCl-1mg/mlリゾチームに懸濁し、37℃にて3時間振
盪した。これに0.6mlの0.5M EDTA溶液、2.4mlの5M N
aCl 溶液、4.4mlの4% SDS-0.7M NaClを順次加え、緩
やかに混合し氷上で18時間静置した。4℃にて65,000xg
1時間遠心し上清を得、これに50%ポリエチレングリコ
ール6000を4.6ml加えた。氷上で3時間静置し、1,000x
g5分遠心した。沈澱物を5mlのTES緩衝液に溶解
し、CsClを7.5g、1.5mg/ml臭化エチジュウム−TE
S緩衝液を2ml加え混合した。この溶液を42時間130,00
0xgの密度勾配遠心分離にかけた。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.
【0019】紫外線照射により検出されたプラスミド画
分を分取した後、n−ブタノールで処理し臭化エチジュ
ウムを除いた。TE緩衝液に対して透析後、エタノール
沈澱により精製プラスミド画分を得た。これを0.7%ア
ガロースゲル電気泳動に供し、ゲルを臭化エチジュウム
で染色することによりプラスミドの存在を確認した。 (5)大腸菌由来の複合プラスミドpK4とRhodococcus由
来の複合プラスミドpK4の比較 1)分子量測定 プラスミドの一部を0.7%アガロースゲル電気泳動に供
した。この際、サイズマーカーとして大腸菌プラスミド
pUC18, pUC118, pBR322(各々2.69kb, 3.16kb, 4.36k
b)を同時に泳動した。その結果、pK4プラスミドは、
大腸菌由来のものと、Rhodococcus由来のものは共に約
5.3kbであった。 2)各種制限酵素による切断特異性 プラスミドの一部を各種制限酵素と反応させ、反応終了
後、反応液を0.7%アガロースゲル電気泳動および5%
アクリルアミドゲル電気泳動により分析した。サイズマ
ーカーとしてはラムダファージDNAのHindIII消化物
およびPstI消化物を用い、プラスミドの各制限酵素断
片のサイズを算出した。その結果、pK4プラスミドは表
1と同様の制限酵素切断特性を示した。 (6)Rhodococcus由来の複合プラスミドpK4を用いた
大腸菌の形質転換 Rhodococcus rhodochrous ATCC 12674/pK4株より得た
プラスミドpK4を用いて大腸菌JM105株を形質転換し
た。カナマイシン50μg/mlを含む2xYT寒天プレート
で選択したところ高頻度でカナマイシン耐性能を示す株
が得られた。これらの形質転換株の内12株よりプラスミ
ドを分離し、制限酵素による切断パターンを調べたとこ
ろ、Rhodococcus の形質転換株から得られたプラスミド
pK4(表1)と同じであった。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】[0020]
【実施例2】図3に示したようにしてプラスミドpA3を
作成した。プラスミドpRC003を制限酵素ClaIで切断し、
pUC19プラスミドのAccI部位に挿入した複合プラスミド
ベクターを作成しpA3と命名した。実施例1と同様に大
腸菌JM105株へ導入し、50μg/mlアンピシリン、1mM I
PTG,0.02% Xgalを含む2xYT培地にて白色コロニーを選
別し、増幅させ、分離精製後、制限酵素による確認を行
った。このプラスミドについてRhodococcus rhodochrou
s ATCC 12674株の形質転換を試みた結果、このプラスミ
ドが本菌株に10μg/ml濃度のアンピシリンに対して耐
性を賦与することが明らかとなった。なお、ここで得ら
れた形質転換体微生物、R.rhodochrous ATCC 12674/pA
3は工業技術院微生物工業技術研究所に微工研条寄第37
32号として寄託している。表2にプラスミドpA3の制限
酵素切断パターンを示す。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】[0021]
【表2】 [Table 2]
【0022】[0022]
【発明の効果】本発明の複合プラスミドベクターはRhod
ococcus属またはEscherichia属に属する細菌において工
業的に利用しうる微生物を育種、改良するために有用で
ある。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.
【図面の簡単な説明】[Brief description of the drawings]
【図1】プラスミドpRC001, pRC002, pRC003およびpRC0
04の制限酵素切断地図。FIG. 1. Plasmids pRC001, pRC002, pRC003 and pRC0
04 restriction map.
【図2】本発明のプラスミドpK1, pK2, pK3およびpK4
の作成方法を示す。FIG. 2: Plasmids pK1, pK2, pK3 and pK4 of the invention
Here is how to create it.
【図3】本発明のプラスミドpA3の作成方法を示す。FIG. 3 shows a method for preparing plasmid pA3 of the present invention.
───────────────────────────────────────────────────── フロントページの続き (51)Int.Cl.7 識別記号 FI C12R 1:19) (72)発明者 橋本 好弘 神奈川県横浜市鶴見区大黒町10番1号 日東化学工業株式会社 中央研究所内 (72)発明者 湯 不二夫 神奈川県横浜市鶴見区大黒町10番1号 日東化学工業株式会社 中央研究所内 (56)参考文献 特開 平4−148685(JP,A) 特開 平4−330287(JP,A) (58)調査した分野(Int.Cl.7,DB名) C12N 15/74 BIOSIS(DIALOG) WPI(DIALOG)──────────────────────────────────────────────────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)
びpA3から選ばれるプラスミド中の、ロドコッカス(Rho
dococcus)属細菌に属する菌株細胞内で複製増殖可能な
DNA領域と、 (B)大腸菌細胞内で複製増殖可能なプラスミドDNA
領域と、 (C)薬剤耐性遺伝子を含むDNA領域とを含むことを
特徴とする複合プラスミドベクター。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.
299,pHSG298,pUC19およびpUC18から選ばれる請求項1
記載の複合プラスミドベクター。2. The plasmid DNA replication region (B) is pHSG
299, pHSG298, pUC19 and pUC18.
A composite plasmid vector as described.
れる請求項1記載の複合プラスミドベクター。3. A method selected from pK1, pK2, pK3, pK4 and pA3.
2. The composite plasmid vector according to claim 1, which is used .
載の複合プラスミドベクターにより形質転換されたロド
コッカス(Rhodococcus)属またはエッシェリヒア(Esc
herichia)属に属する細菌の形質転換微生物。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.
ロドコッカス・ロドクロウス(Rhodococcus rhodochrou
s)ATCC 12674株である請求項4記載の形質転換微生
物。5. A Rhodococcus (Rhodococcus) bacteria of the genus
Rhodococcus rhodochrou
s ) The transformed microorganism according to claim 4, which is ATCC 12674 strain.
が、大腸菌(Escherichia coli)K-12株である請求項4
記載の形質転換微生物。6. Escherichia (Escherichia) bacteria is, claim 4 is E. coli (Escherichia coli) K-12 strain
The transformed microorganism according to the above.
Applications Claiming Priority (2)
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JP3754591 | 1991-03-04 | ||
JP3-37545 | 1991-03-04 |
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JP3142349B2 true JP3142349B2 (en) | 2001-03-07 |
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US7907282B2 (en) | 2003-08-14 | 2011-03-15 | Microptix Technologies, Llc | Integrated sensing module for handheld spectral measurements |
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JP3235934B2 (en) | 1994-08-04 | 2001-12-04 | 三菱レイヨン株式会社 | Kanamycin resistance gene from Rhodococcus bacteria |
JP4733298B2 (en) * | 2001-07-05 | 2011-07-27 | 三菱レイヨン株式会社 | DNA fragment containing a gene having a function relating to autonomous growth of a plasmid |
JP4493011B2 (en) * | 2004-06-11 | 2010-06-30 | 三菱レイヨン株式会社 | Trimethoprim-resistant dihydrofolate reductase and its gene from Rhodococcus spp. |
JP4570075B2 (en) * | 2004-08-12 | 2010-10-27 | 三菱レイヨン株式会社 | Plasmid derived from Rhodococcus bacterium and derivatives thereof, and E. coli-Rhodococcus bacterium shuttle vector |
WO2014038216A1 (en) | 2012-09-10 | 2014-03-13 | 三菱レイヨン株式会社 | Method for producing methacrylic acid and/or ester thereof |
BR112015000069B1 (en) | 2012-09-10 | 2022-06-21 | Mitsubishi Chemical Corporation | Method for producing methacrylic acid ester |
AU2015225348B2 (en) | 2014-03-07 | 2017-06-08 | Mitsubishi Chemical Corporation | Method for producing methacrylic acid ester, and novel methacrylic acid ester synthase |
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