JPS5953831B2 - A new microorganism carrying a new plasmid - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a novel microorganism possessing a novel plasmid useful as a vector for recombinant DNA experiments using hyperthermophilic bacteria as a host. This invention relates to a novel Thermus flavus carrying a novel plasmid characterized by the restriction enzyme cleavage map shown in the figure.

Conventionally, recombinant DNA experiments have been widely conducted mainly in systems using E. coli as a host, and great results have been achieved, such as the mass production of insulin, interferon, human growth hormone, etc. using E. coli.

The host-vector system of E. coli is almost complete.
In addition to E. coli, host-vector systems have been developed using yeast, Bacillus subtilis, etc., and ways to apply them are being considered.

However, all of the above bacteria have a growth temperature of 30°C to 37°C.
The problem is that it is a mesophilic bacterium.

On the other hand, thermophilic bacteria are broadly divided into moderate thermophiles, which have an upper limit of growth temperature between 55°C and 75°C, and highly thermophilic bacteria, which have an upper limit of growth temperature of 75°C or higher. It is known that the enzymes and biological components that it contains are excellent in heat resistance and solvent resistance, especially in terms of the application of thermostable enzymes derived from thermophilic bacteria and thermostable biological functions to industrial processes such as bioreactors. It is attracting attention from

Therefore, 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 most effective means for this purpose. No development research has been conducted to date.

Moreover, regarding the search for plasmid DNA, which should form the basis of vector development research, only the following two reports are known of research using hyperthermophilic bacteria as a material.

(1) Isolation of extrachromosomal DNA from highly thermophilic bacteria Hishinuma, F., Tanaka, T., and Sakaguchi, K. J. G.
en, Microb, LO4, 193-199 (
(1978) (2) Physical properties of a plasmid (pTTl) isolated from Thermus thermophilus Eberhard, M.D., Vasquez, C.O., Valenzuela, P., Vicuna, R., and Yuderevitzk, A.
.

Plasmid, 6, 1-6 (1981)
The plasmids described in the above two reports are all 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 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 1.0 megadaltons from Thermus flavus that showed kanamycin resistance.

As shown in the above-mentioned restriction enzyme cleavage map, this plasmid has an extremely small molecular weight and has specific cleavage points for several types of restriction enzymes (hereinafter, this plasmid will be abbreviated as pNHKlol).

The abbreviations of the restriction enzymes shown in the figure are as follows.

BamHI represents an enzyme derived from Bacillus amyloliquefaciens, KpnI represents an enzyme derived from Klebsiella pneumoniae, and BstNI represents an enzyme derived from Bacillus stearothermophilus.

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, pNHKlol is clearly different from known plasmids in molecular weight and restriction enzyme cleavage pattern, 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 marker derived from the host chromosome to a so-called cryptic plasmid whose properties are unknown.

It would be extremely convenient to use pNHKlol in this case.

This is because, first, pNHKlol is a plasmid that can replicate in hyperthermophiles, and second, it has a much smaller molecular weight than known cryptogenic plasmids derived from other hyperthermophiles. This plasmid has the advantage that essential regions such as the replication origin region and genes involved in replication can be analyzed much more easily than other plasmids with larger molecular weights. Because there is.

Furthermore, as is clear from the figure, pNHKlol is Kpn
It also has cleavage sites for restriction enzymes such as I and BamHI at specific and limited positions.

This is advantageous in that when pNHKlol is used as a vector, it can significantly retain the introduction site for the heterologous gene to be inserted.

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, by cloning the enzyme gene of thermophilic bacteria, which has excellent properties such as heat resistance and solvent resistance, into a thermophilic bacterial host using this plasmid as a vector and mass producing it, it will be possible to apply it to bioreactors, etc. This is possible and is expected to be applied to industrial processes.

pNHKlol was obtained from Thermus flavus TK10, a highly thermophilic bacterium newly isolated by the present inventors from hot spring water.
The strain was grown in Thermus medium (Difco Yeast Extract 0.4%, Polypeptone (Otama Nutrition) 0.8%, NaC
This can be achieved by lysing the bacterial cells obtained by growing them to the late logarithmic stage using 10.2%) by treating them with lysozyme and SDS, but this strain is novel in that it possesses this plasmid.

In addition, Thermus flavus TKIO strain is an aerobic bacillus that is negative for Durham staining, produces yellow pigment, has a DNA GC content of about 70%, and has an optimal growth temperature of 70°C, but pN
It is a novel microorganism that has not been previously recognized as possessing HKlol.

This strain was isolated from hot spring water as a kanamycin-resistant strain, but it was also resistant to erythromycin and streptomycin, and sensitive to ampicillin, taloramphenicol, neomycin, and tetracycline.

In addition, this strain has been deposited as Microtechnology Research Institute No. 6750.

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

Example 1 (Screening of bacterial strains) 1 ml of hot spring water from a hot spring in Shizuoka area was mixed with Thermus medium (Tifco Yeast Extract 0.4%, Polypeptone (Otama Nutrition) 0.8%, NaCl 0.2%) 100%
Thermus flavus TKIO was extracted from one of the colonies grown on a Thermus agar plate containing kanamycin (10 Mg/ml) after shaking culture at 70°C for about 18 hours.
strain (Feikoken Bibori No. 6750) was obtained.

Example 2 Plasmid pNHK101 of Thermus flavus'
Isolation from TKIO strain Thermus flavus TKIO strain (Feikoken Bacterial Serial No. 6750)
100 ml of Thermus medium (Difco yeast extract 0.4%,
Polypeptone (Otama Nutrition) 0.8%, 'NaC10,
2% pE(7,5) and cultured with shaking at 70°C for 16 to 18 hours.

This culture solution is inoculated into Thermus medium containing 10 μg/ml of kanamycin No. 11, and cultured at 40° C. for 5 hours.

The bacterial cells were collected by centrifugation and treated with TES (20mMTri
s-HCI, 5mM EDTA.

After washing with 100 mM NaCl If (7,5), each 4 g wet weight of bacterial cells is suspended in 10 ml of TES containing 25% sucrose.

2 ml of lysozyme (10 mg/ml), 0.25 M-
Add 4 ml of EDTA (pH 8, O) and incubate for 10 minutes at 0°C.
Let stand for 1 minute, then incubate at 37°C for 10 minutes.

Add 2 ml of 110% SDS to this cell mixture, 5 ml of 5.
Add M-NaCl and leave at 4°C for 15 to 18 hours.

This is centrifuged by ultracentrifugation at 2800 rpm for 1 hour to obtain a supernatant.

To this supernatant, add 10% polyethylene glycol 6000 (
W/V), left at 0°C for 2 to 3 hours, 2200 rpm
, obtain 1 precipitate by centrifugation for 2 minutes.

This precipitate was dissolved in 15 ml of TES, and CsC1 and ethidium bromide were added to adjust the density to 1.61-1.6.
Adjust to 2.

The sample is centrifuged in an equilibrium density gradient for 30-40 hours at 3800 rpm.

The resulting plasmid DNA bands were collected, ethidium bromide was removed with isoamyl alcohol, and then TE
N (20mM Tris-HCI, 1mM
D.T.A.

A plasmid solution is obtained by dialysis against 20mM NaCl).

This plasmid solution is a mixture of pNHKlol and pNHK102 with a molecular weight of approximately 9 megadal I.N.
Pure pNHKlol can be obtained by subjecting it to electrophoresis using a DNA chromatography method (manufactured by Biotech, Inc.), cutting out the resulting band corresponding to pNHKlol, and recovering the DNA.

DNA was recovered from the low melting point agarose gel according to the following procedure.

The cut out gel slices are kept warm at 65°C to thaw, 5QmM Tris-HCI buffer (pH 8,0) containing twice the amount of 0 and 5mM EDTA is added thereto, and the gel slices are transferred to 37°C and kept warm.

To this was added an equal volume (7) of 0.1M Tris-HCI buffer (
Add phenol saturated with Ilf(8,0) and mix;
After centrifugation (3000-5000 rpm, 5 minutes), the upper aqueous layer is separated.

After performing phenol extraction once more and removing phenol from the aqueous layer with ether, 1710 volumes of 3M ammonium acetate solution are added and ethanol precipitation is performed with 3 volumes of ethanol.

The obtained precipitate was dissolved in TEN to prepare a plasmid solution.

Procedure for characterizing pNHKlol The molecular weight of pNHKlol is determined by its superhelical structure (sup
The DNA was obtained by agarose gel electrophoresis and polyacrylamide gel electrophoresis of the DNA of the 5-structure (Ercoiled 5structure) and fragments cut with restriction enzymes.

The molecular weight marker at this time was pBR322DNA (2,6
7md), Co1EI DNA (4,2md) and Lambda DNA HindIII fragment (14,6,
5,84,4,05°2.67,1,30.1,17.
0.34md), EcoRI-digested fragment of lambda DNA (13,7,4,74,3,73,3,48°3.02
．． 2.13md), φX 174DNA HaeIII
Decomposition fragment (0,836,0,666,0,539,0,
373,0,192,0,174゜0.167,0,1
45.0, 120.0.073.0.044md) was 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, SeChem agarose was used at 0.
．． used at a concentration of 5% or 0.7% and at a constant voltage of 1.5 μ/cm of gel length in a horizontal gel electrophoresis chamber.
It lasted 17 hours.

For polyacrylamide gel electrophoresis, polyacrylamide/bisacrylamide manufactured by Seikagaku Kogyo Co., Ltd. was used.
The gel was run in a vertical slab gel electrophoresis chamber with a cross-linking degree of 30:1, using a constant voltage of 10 V per cm of gel length for 2-3 hours.

The plasmids of hyperthermophilic bacteria are as shown in the table above, but pNHKlol and the others are clearly different as described above, and pNHKlol is a novel plasmid that has not been previously recognized.

[Brief explanation of the drawings] The drawing shows a restriction enzyme cleavage map of pNHKlol, in which BamHI is an enzyme derived from Bacillus amyloliquefaciens, KpnI is an enzyme derived from Klebsiella pneumoniae, and BstNI is an enzyme derived from Bacillus stearothermo. The enzymes derived from P. fils are shown.

Claims (1)

[Claims] 1. A novel Thermus flavus TKIO strain carrying a plasmid with a molecular weight of 1.0 megadaltons and characterized by the restriction enzyme map shown in the figure.