JPS6037B2 - Novel plasmid derived from highly thermophilic bacteria - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel plasmid useful as a vector for recombinant DNA experiments using highly thermophilic bacteria, and more specifically, its molecular weight is approximately 54 megadaltons, It concerns a novel plasmid characterized by the restriction enzyme map shown in the figure.

Previously, silk transgenic DNA experiments have been widely studied mainly in systems using Escherichia coli as the host, and great results have been achieved, such as the mass production of insulin, interferon, human growth hormone, etc. using Escherichia coli.

The host-vector system for Escherichia coli has almost been completed, and other host-vector systems have been developed for yeast, Bacillus subtilis, etc., and ways to apply them are being considered. However, there is a problem in that all of the above-mentioned bacteria are medium salt bacteria with a growth temperature of 30 to 3,700 degrees Celsius. On the other hand, thermophilic bacteria are broadly divided into moderate thermophiles, which have an upper limit of growth temperature between 55 qo and 75°C, and highly thermophilic bacteria, which have an upper limit of growth temperature of 75 qo or higher. Enzymes and biological components are known to have excellent heat resistance and solvent resistance, and are attracting attention from the perspective of applying thermostable enzymes derived from thermophilic bacteria and thermostable biological functions to industrial processes such as bioreactors. Therefore, the breeding of thermophilic bacteria is considered to be important, and research on the development of host-vector systems for thermophilic bacteria is considered to be one of the effective means for this purpose, especially for highly thermophilic bacteria. Development research on host-vector systems has not been conducted at all to date.Moreover, in terms of searching for plasmid DNA, which should form the basis of vector development research, research using hyperthermophilic bacteria as materials is limited to the following two methods. (1) Isolation of extrachromosomal DNA from highly thermophilic bacteria Hishinuma, F., Tanari, T. and Sakaguchi, KJ. Gen. Microb., 104, 19
3-199 (1978) t2) Physical properties of a plasmid (pTTI) isolated from Thermus thermophilus Berhard, M.; D. ,/ゞSquez, C. , Noyenzuela, P. , Bikiyuna, R. and Yudelevitsk, A. P1asmid, 6, 1-6 (1981) above 2
All of the plasmids described in the report are so-called cryptic plasmids whose properties are unknown, and their molecular weights are also rather large at around 6 megadaltons.

Therefore, it would be extremely difficult to use them as vectors or to develop vectors using them as raw materials. Therefore, the present inventors searched for a plasmid that has a selection marker (a marker indicating that the plasmid is present in the host) and has a small molecular weight from highly thermophilic bacteria. As a result, we succeeded in isolating a plasmid with a molecular weight of approximately 5.4 megadaltons from Thermus flavus that showed resistance to streptomycin. As shown in the above-mentioned restriction enzyme cleavage map, this plasmid has a rather small molecular weight and has specific cleavage points for several types of restriction enzymes (hereinafter, this plasmid will be abbreviated as pNHS212).

The abbreviations of the restriction enzymes shown in the figure are as follows. M1ul is an enzyme derived from Micrococcus luteus and Bg0' is an enzyme derived from Bacillus globigii, Pst.
1 indicates an enzyme derived from Providencia stuarteii.

Differences from plasmids derived from Thermus bacteria, that is, extreme thermophiles, that have been reported so far are shown in the table below.

As is clear from the table of plasmids derived from hyperthermophiles, pNHS212 is clearly different in molecular weight and restriction enzyme cleavage pattern compared to known plasmids, and is recognized as a novel plasmid.

In order for plasmid DNA to be used as a vector, the plasmid must have the ability to autonomously reproduce within the host and a selection marker (
It is essential that the plasmid has a marker indicating that it is present in the host.

However, when considering bacteria such as highly thermophilic bacteria whose growth environment is hot springs with poor nutritional sources and no antibiotics, it is not easy to obtain plasmids containing drug-resistant genes. Therefore, vector development will have to be carried out by providing a host chromosome-specific marker to a so-called cryptic plasmid whose properties are unknown. It would be extremely convenient to use pNHS212 in this case. This is because, firstly, pNHS212 is a plasmid that can replicate in hyperthermophiles, and secondly, it has a slightly smaller molecular weight than other known cryptogenic plasmids derived from hyperthermophiles. This is because it has the advantage that essential regions of this plasmid, such as the replication origin region and genes involved in replication, can be analyzed more easily than with other plasmids with larger molecular weights. be.
Furthermore, as is clear from the figure, pNHS212 has M1u
It has cleavage sites by restriction enzymes such as Bgm at specific and limited positions.

This is advantageous in that when pNHS212 is used as a vector, a site for introducing a heterologous gene to be inserted can be significantly retained. Now, if a different type of heat-resistant gene is introduced into a thermophilic bacterium using this plasmid as a vector, a reduction in cooling costs in the fermentation industry will be achieved.

In addition, we will use this plasmid as a vector to clone the enzyme gene of a thermophilic bacterium, which has excellent properties such as heat resistance and solvent resistance, into an amphophilic bacterial host and mass-produce it, so that it can be applied to bioreactors, etc. is possible, and is expected to be applied to industrial processes. pNHS212 was obtained from a highly thermophilic bacterium, Thermus nigra, which the present inventors newly isolated from hot spring water.
Flavus TS21 strain in Thermus medium (Difco Yeast Extract 0.4%, Polybeptone (Daigo Nutrition)
0.8%, NaCIO. 2%) until the late logarithmic growth stage, and the box strain obtained is fused by treatment with lysozyme and SDS. In addition, Thermus flavus TS21 strain is an aerobic Gram stain-negative Kajibacterium.
Although it is a strain that produces yellow pigment and has a DNA GC capacity of about 70% and an optimal growth temperature of 70 qo, it is a new microorganism that has not been found in its predecessors in that it possesses pNIJS212.

This bacterial strain was isolated from hot spring water as a streptomycin-resistant strain. In addition, this strain is the 6th strain
It has been deposited as No. 752.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 (Screening of bacterial strains) Approximately 1 ml of hot spring water from Atagawa Onsen in Shizuoka Prefecture Thermas Toji (Difco Yeast Extract 0.4%, Polybeptone (Daigo Nutrition) 0.8%, NaCIO.2%) 10 enemy hl
Thermus flavus TS2 was isolated from one of the colonies grown on a Thermus agar plate containing streptomycin (20ml) after shaking incubation for about 1 hour at 70qC.
One strain (Choken Bacteria No. 6752) was obtained.

Example 2 Isolation of plasmid pNHS212 from Thermus flavus TS21 strain Thermus flavus TS2
Thermus medium (Difco Yeast Extract 0.4%, Polypeptone (Daigo Nutrients) 0.8%, NaCIO .2%, pH 7.5) and cultured at 700° for 16 to 18 hours with shaking.

Add 1 ml of this culture solution to 20 ml of streptomycin.
Thermus soil containing the following was inoculated and cultured at 7000 for 5 hours. The bacterial cells were collected by centrifugation and subjected to TES (2 hMT
ris one day CI, ear hMEDTA, one dish hMNaC1p
After washing with H7.5), bacteria body temperature weight 4 days per day, 1 sail 1 25
% sucrose in TES. Lysozyme (9 of 10)
'ml) to 2 hl, 0.29 -EDTA (pH 8.0
) 4mi, static layer for 10 minutes at 0 °C, followed by 3700
Keep warm for 10 minutes. Add 10% of 2hl to this cell mixture.
SOS, add 5 ml of Waza M-NaCI and heat to 4°C for 15~1
Let stand for 8 hours. This was centrifuged by ultracentrifugation at 2,800 pm for 1 hour to obtain the condition. Add 10% (w'v) polyethylene glycol 6000 to this supernatant and
Stir at 0° C. and centrifuge at 220 pm for 2 minutes to obtain a precipitate. This precipitate was dissolved in 15 ml of TES, and CsCI and ethidium furomide were added to adjust the density to 1.61-1.
Adjust to 62. This sample was heated to 30 to 4
Centrifuge the field for an equilibrium density gradient. Resulting plasmid DN
After collecting the A band and removing ethidium furomide with isoamyl alcohol, TEN (2 hMTriS
1 day CI, 1 MMEDTA, 2 melon hMNaCI)
A plasmid solution is obtained by dialysis. This plasmid solution is a mixture of pNHS212, pNHS211 with a molecular weight of about 3.1 megadaltons, and pNH213 with a molecular weight of about 10 megadaltons. Purified pNHS212 is subjected to electrophoresis, the resulting band corresponding to pNHS212 is cut out, and the DNA is recovered.
NHS212 is obtained. DNA was recovered from the low melting point agarose gel according to the following procedure.

The cut out gel slices were kept warm at 65°C to thaw, and twice the amount of 0. 50hMTrS-H containing cheap MEDTA
Add CI buffer (pH 8.0), transfer to 3700 and keep warm. This was added with an equal volume of 0.1M TrS 1 day CI buffer (p
Add phenol saturated with H8.0), mix, and centrifuge (
3000-500pm for 5 minutes), the upper aqueous layer was separated. After performing phenol extraction once more and removing phenol from the aqueous layer with ether, 1/1 3M ammonium acetate solution is added and ethanol precipitation is performed with 3 parts of ethanol. The obtained precipitate was dissolved in TEN to prepare a plasmid solution. Procedure for characterizing pNHS212 The molecular weight of pNHS212 is determined by its superhelical structure (sup
The DNA was obtained by agarose gel electrophoresis and polyacrylamide gel electrophoresis of the DNA of erco edstmcture) and fragments cut with restriction enzymes.

The molecular weight marker at this time was pBR32 fox NA (2.67
md), ColEIDNA (4.2md) and Lambda D
Hdm decomposition fragment of NA (14.6, 5.84, 4.0
5, 2.67, 1.30, 1.17, 0.34md),
EcoRI-digested fragments of lambda DNA (13.7, 4.7
4, 3.73, 3.48, 3.02, 2.13HD input
? ×17 Fox NA Haem degradation fragment (0.836, 0
．． 666" 0.539, 0.373, 0.192"
0.174, 0.167"0145" 0.120,
0.073, 0.044 md) were used. Cleavage with restriction enzymes was carried out by precipitating DNA from a plasmid DNA solution by ethanol precipitation and dissolving it in an appropriate buffer. Restriction enzymes used were commercially available products from Takara Shuzo and Boehringer Mannheim. For agarose gel electrophoresis, SeaChem's agarose was used at a concentration of 0.5% or 0.7%, and the gel was run in a horizontal gel electrophoresis chamber. It was carried out for 15 to 17 hours at a constant voltage of 1.5 V per skin length.Polyacrylamide gel electrophoresis was performed using polyacrylamide/bisacrylamide manufactured by Seikagaku Corporation, with a 5% concentration of 3.
The gel was run with a cross-linking degree of 0:1 in a vertical slab gel electrophoresis chamber at a constant voltage of 10 V per gel length for 2-3 hours. The plasmids of hyperthermophiles are as shown in the table above, but pNHS212 and the others are clearly different as mentioned above, and pNHS
S212 is a novel plasmid that has not been previously recognized.

[Brief explanation of drawings]

The figure shows a restriction enzyme cleavage map of pNHS212, M1ul in the figure is an enzyme derived from Micrococcus luteus, B
g10' Enzyme from Bacillus globigii, Pstl
indicate enzymes derived from Providencia stuartii, respectively.

Claims (1)

[Scope of Claims] 1. A plasmid derived from a Thermus sp. having a molecular weight of approximately 5.4 megadaltons and characterized by the restriction enzyme map shown in the figure. ▲Contains mathematical formulas, chemical formulas, tables, etc.▼