JPH05244953A - Plasmid pnc500 - Google Patents

Abstract

PURPOSE:To obtain a new plasmid useful as a vector for nocardiform bacteria. CONSTITUTION:The objective plasmid pNC500, having about 7.2kb molecular weight, expressed by a restriction enzyme cleavage map shown in the figure without being cleaved with restriction enzymes SphI, HindIII, KpnI and XbI. This plasmid is obtained by separating a plasmid mixture solution separated from Nocardia corallina B-276 (FERM P-4094) according to electrophoresis, cutting out a gel containing only the plasmid PNC500 and affording the plasmid from the resultant gel.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a plasmid pNC500 which is extremely useful as a vector for Nocardiophorum bacteria.

[0002]

2. Description of the Related Art Nocardiophorum bacteria such as Nocardia and Rhodococcus have the ability to decompose linear hydrocarbons, alicyclic compounds, aromatic compounds, halogen compounds, lignin and the like and to produce physiologically active substances. Utilization for the production of useful substances, decomposition of persistent substances, etc. is under consideration. For example, Nocardia Coralina ( Nocard
ia corallina) has the ability to epoxidize various olefins, and can be widely used as an intermediate for the production of organic chemical products such as optically active epoxides used in ferroelectric liquid crystals, synthetic resins, pharmaceuticals, agricultural chemicals, etc. It is used for the production of trifluoropropene oxide (TFPO).

Further, for Nocardiophyllum bacteria, the improvement of breeding by recombinant DNA technology is behind that of Escherichia coli and the like, and development of a host-vector system is strongly desired. Previously, Journal of Bacteriology (J. Bacteriol. ) 170 , 638 (1988), Applied and Environmental Microbiology
(Appl. Environ, Microbiol.) 56 , 2818 (1990), plasmid (Plasmid) 23 , 242 (1990), Journal of Japan Society for Agricultural Chemistry 65 ,
617 (1991), etc., describes the host-vector system. However, these hosts are limited to a part of the genus Rhodococcus, and the production of useful substances and breeding of degrading bacteria of persistent substances used in a wider range of Nocardiophorum bacteria are carried out. It is desired to develop a vector that can be used for

[0004]

Therefore, the present inventor conducted research to develop a DNA recombination technique for Nocardiophorum bacteria, particularly Nocardia coralina, which is an epoxide-producing bacterium. The present invention has been completed as a result of further research based on this.

[0005]

The present invention has a molecular weight of about
7.2 kb, with restriction enzymes SphI, Hind III, Kpn I and XbaI
The present invention relates to a plasmid pNC500 which is not cleaved by and is shown in the restriction enzyme development map below. Examples of the microorganism having the plasmid of the present invention include Nocardia coralina, and specifically, Nocardia coralina B-276 isolated as a propylene-assimilating bacterium.
This microorganism has been deposited at the Institute of Microbial Science and Technology of the Agency of Industrial Science and Technology as Microorganism Research Institute 4094 (FERM-P-4094).

Next, a method for obtaining the plasmid pNC500 from Nocardia coralina will be described. Nocardia
To isolate the plasmid pNC500 from Coralina B-276, first crush the fungus. Nocardiophyllum bacteria are less likely to be lysed by lysozyme when cultured in a normal medium. Therefore, cultivate it in a medium containing glycine and use penicillin G
By treating with, it is possible to obtain a bacterium that is easily lysed by a lytic enzyme. As the medium used in the present invention, a commonly used NBG medium having the following composition and containing about 1% of glycine is used. (NBG medium) Nutrient Broth No.2 (Oxoid) 2.5% Glucose 1% Nocardia coralina B-276 was cultured at 30 ° C until the late logarithmic growth phase, and penicillin G was added to 7.5 units / ml for several hours. After culturing, it is lysed with a lytic enzyme (eg, lysozyme, achromobacter peptidase, etc.). Circular plasmid DNA can be isolated from the lysate by a known method [Biokimica e Biophysica].
Actor (Biochimca. Et Biophysica. Acta.), 383, 457-4
63 (1975)].

Among the plasmid DNAs obtained here,
It contains 3 to 4 circular plasmids with a higher molecular weight than the plasmid pNC500. The plasmid pNC500 can be obtained by excising a gel containing only the plasmid pNC500 from the plasmid mixture separated by size by agarose gel electrophoresis and recovering from the gel. The plasmid pNC500 has a molecular weight of about 7.2 kilobase pairs and the restriction enzyme cleavage map is shown in Chemical formula 2.

[0008]

[Chemical 2]

The plasmid pNC500 is sensitive to the restriction enzymes shown in Table 1.

[0010]

[Table 1] Number of pNC500 cleavage sites by restriction enzymes Number of restriction enzyme cleavage sites Sph I 0 Hind III 0 Kpn I 0 Xba I 0 BamH I 1 Bgl II 1 Spe I 1 EcoR I 3 Pst I 2 Sac I 2 Above The name of the restriction enzyme is the abbreviation of the restriction enzyme obtained from the following bacterial species. Sph I Streptomyces phaeochromogenes Hind III Haemophilus influenzae Rd Kpn I Klebsiella pneumoniae OK8 Xba I Xanthomonas badrii (ATCC 11672) BamH I Bacillus subtilis MT-2 (pBamHI RM22) Bgl II Bacillus globigii Spe I SphaeroATCCtan Pst I Providencia stuartii 1641 pPst101 Sac I Streptonyces achromogenes (ATCC 12767)

The number of restriction enzyme cleavage sites is determined by the number of separable fragments obtained by completely digesting the plasmid pNC500 in the presence of an excess of restriction enzyme and subjecting the digested product to 0.7% agarose gel electrophoresis. The molecular weight was the same agarose fragment of known molecular weight obtained by digesting Escherichia coli lambda phage DNA with the restriction enzyme Hind III [J. Mol. Biol., 98 , 551-564 (1975)]. The molecular weight of each fragment of the digested plasmid pNC500 was calculated based on the standard curve drawn by the migration distance on gel, and when multiple fragments were generated, they were calculated by addition.
The restriction enzyme cleavage map of plasmid pNC500 is analyzed by agarose gel electrophoresis for each fragment obtained by using each restriction enzyme alone and each fragment obtained by the treatment using two or more kinds of each restriction enzyme in combination. Created by

The plasmid of the present invention has this cleavage site with various restriction enzymes, which indicates that this plasmid can be modified to develop many useful vectors. It is also possible to transform a host by incorporating a target gene, for example, a gene encoding an enzyme such as monooxygenase, into this plasmid or its derivative, and introducing this into a suitable host microorganism. The plasmid pNC500 can be advantageously used as a vector for Nocardia coralina and other Nocardiophorum bacteria which are capable of producing useful substances such as optically active epoxide and TFPO and capable of decomposing hardly decomposable substances. That is, by cloning the genetic information from Nocardia coralina and other Nocardiophorum bacteria using the plasmid of the present invention, and introducing it into the microorganism of interest, it is possible to increase the productivity of useful substances and persistent substances. Can provide enhanced resolution of

In the above-mentioned use, a method for producing a recombinant plasmid containing a target gene is known per se. For example, Scientific American (Sci
entific American) 233 (1) 24-33 (1975).

In order to introduce the plasmid of the present invention into host microbial cells, a method of transfecting the cells by converting the cells into protoplasts or spheroplasts [J. Bocteriol, 170 , 638 (198
8)], Method of transferring plasmid by electric pulse [Appl. Environ. Microbio., 56 , 2818 (1990)]
Etc. are known. Further, by using the plasmid of the present invention as a vector, a host microorganism introduced with a plasmid incorporating a gene required for production or decomposition of a target substance is cultured by a method known per se to accumulate or decompose the target substance. Can be

[0015]

Example 1 Nocardia corallina B-276
Isolation of the plasmid pNC500 from Nocardia coralina B-276 (Accession No. FERM-P-4094, Research Institute for Microbial Technology, Institute of Industrial Science and Technology, Japan) , 30
The cells were shake-cultured at ℃ for 21 hours. 7.5un in culture at this point
Add Penicillin G to a concentration of its / ml, and
Culture was continued at 30 ° C for 3 hours. Collect the cells from the culture
After washing with TE buffer (0.025M Tris (hydroxymethyl) aminomethane (Tris), 0.025M EDTA: pH 8.0), lysate (0.3M sucrose, 0.025M Tris, 0.025M EDTA, 2mg /
ml lysozyme, 2 mg / ml achromopeptidase, 50 μg / ml
RNase: pH 8.0] was suspended in 20 ml and reacted at 30 ° C. for 2 hours. Add 2% sodium lauryl sulfate and 0.3M to the reaction mixture.
10 ml of a solution of sodium hydroxide was added, mixed well and placed in a 55 ° C. water bath for 1 hour. 4 in this lysate
Add 1 ml of phenol / chloroform solution,
Mix well until the whole becomes cloudy (1 minute). After centrifugation at 17,000 xg for 30 minutes at 4 ° C, the upper layer was taken. Again, an equal amount of a phenol-chloroform (1: 1) solution was added thereto, mixed well, and centrifuged to separate the upper layer.

An equal amount of diethyl ether was added to this solution, and the mixture was gently stirred and then allowed to stand for a while, the upper layer of diethyl ether was discarded, and the lower layer was added with diethyl ether again for extraction. After extraction with ether, 0.1 volume of 3M sodium acetate and 2 volume of ethanol were added to the lower layer, and the deposited precipitate was collected by centrifugation and vacuum dried at -20 ° C.
It was dissolved in TE buffer. This was subjected to 0.7% agarose gel electrophoresis (60V, 5h), and the smallest plasmid band containing pNC500 was cut out. The excised gel was put in a dialysis membrane together with a small amount of TE buffer and electrophoresed at 60 V for 2 hours, and then a current was passed in the opposite direction for 1 minute, and then the solution of the dialysis membrane was recovered. To this solution, 0.1 volume of 3M sodium acetate and 2 volumes of ethanol were added, and the deposited precipitate was collected by centrifugation and vacuum dried at -20 ° C to obtain plasmid pNC500.

Further, a DNA fragment obtained by single-digesting, double-digesting or triple-digesting pNC500 with various restriction enzymes was subjected to agarose gel electrophoresis, and the molecular weight was determined from its mobility [method-in-enzymology ( Method
in Enzymology), Vol. 12, Part B, pages 361 to 377, 1968 Academic Press, New York (USA)], but it was about 7.2 kb after tens of observations. In this case, the reaction conditions of the enzyme followed the conditions defined by the supplier. The molecular weight was determined based on the standard mobility pattern of the fragment obtained by digesting λDNA with the restriction enzyme HindIII. From these results, the above-mentioned restriction enzyme cleavage map was determined.

[0018]

INDUSTRIAL APPLICABILITY The plasmid of the present invention has a cleavage site by various restriction enzymes, and it is possible to modify this plasmid to develop many useful vectors used in Nocardiophorum bacteria. .. In addition, a gene can be incorporated into this plasmid, and this can be introduced into Nocardiophorum to transform this host, thereby producing a useful substance such as an optically active epoxide or decomposing a hardly decomposable substance.

Claims (1)

[Claims] 1. A restriction enzyme Sph having a molecular weight of about 7.2 kb.
The plasmid pNC500 which is not cleaved by I, Hind III, Kpn I and Xba I and is shown in the restriction enzyme development map below. [Chemical 1]