JPS6221512B2 - - Google Patents

Description

[Detailed Description of the Invention] The present invention relates to a recombinant method using an anaerobic thermophilic bacterium as a host.
This article concerns a novel microorganism that possesses a novel plasmid useful as a vector for DNA experiments, and more specifically, a novel plasmid with a molecular weight of approximately 5.0 megadaltons and characterized by the restriction enzyme cleavage map shown in the figure. This relates to the new Thermoanaerobacter.

Up until now, recombinant DNA experiments have been widely conducted mainly using E. coli as a host system, 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 for E. 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.

Anaerobic thermophilic bacteria that decompose cellulose, hemicellulose, starch, etc. under high-temperature conditions of 50 to 80°C to produce alcohols, organic acids, methane, etc. can be used as efficient biocatalysts for biomass conversion, or as heat-resistant, It is attracting attention as a source of useful enzymes with excellent stability. Therefore, breeding of anaerobic thermophilic bacteria is considered to be important, but research to develop a host-vector system for anaerobic thermophilic bacteria, which is considered to be one of the effective means for this purpose, has not been conducted to date. Not yet. Moreover, there is no known research using anaerobic thermophilic bacteria as a material for searching for plasmid DNA, which should form the basis of vector development research. Therefore, the present inventors searched for a plasmid from anaerobic thermophilic bacteria that has a selection marker (a marker indicating that the plasmid is present in the host) and has a small molecular weight. As a result, we successfully isolated a plasmid with a molecular weight of approximately 5.0 megadaltons from Thermoanaerobacter that showed resistance to erythromycin.

As shown in the above-mentioned restriction enzyme cleavage map, this plasmid has a small molecular weight and has specific cleavage points for several types of restriction enzymes (hereinafter referred to as
This plasmid is abbreviated as pSH49).

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

EcoR is an enzyme derived from Esialicia coli, Cla
is an enzyme derived from Karyophanone latum, Pst
is an enzyme derived from Providencia stuartei,
Hpa is an enzyme derived from Haemophilus parainfluenzae, Hpa is an enzyme derived from Haemophilus parainfluenzae, and Pvu is an enzyme derived from Haemophilus parainfluenzae.
vulgaris-derived enzyme and Hind indicate the enzyme derived from Haemophilus influenzae, respectively.

In order for plasmid DNA to be used as a vector,
It is essential that the plasmid has the ability to autonomously reproduce within the host and a selection marker (a marker indicating that the plasmid is present within the host). However, when considering bacteria such as anaerobic thermophiles, whose growth environment is hot springs and the like 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 cryptographic plasmid whose properties are unknown. It would be extremely convenient to use pSH49 in this case. This is because, first, pSH49 is a plasmid that can replicate in anaerobic thermophiles, and second,
This is because, since it has a small molecular weight, it has the advantage that essential regions of the plasmid, such as the replication origin region, genes involved in replication, etc., can be easily analyzed.

Furthermore, as is clear from the figure, pSH49 is an EcoR
, Hpa, and other restriction enzymes have cleavage sites at specific and limited positions. This thing is
When pSH49 is used as a vector, it is advantageous in that it can significantly retain the introduction site for the heterologous gene to be inserted.

By using this plasmid as a vector, it will become possible to breed anaerobic thermophiles that have high biomass conversion efficiency and produce useful chemical products.

pSH49 was obtained by growing Thermoanaerobacter No. 49, an anaerobic thermophilic bacterium newly isolated by the present inventors from hot spring water, in YPG medium (Difco Yeast Extract 0.2%, Polypeptone (Daigo Nutrients) 0.4%).
%, NaCl 0.2%, Glucose 1%, Na ₂ CO ₃ 0.3
%, L-cysteine 0.2%, PH7.5) until the late logarithmic growth stage.
This can be achieved by lysing the bacteria with SDS treatment, but this strain is novel in that it possesses this plasmid.

In addition, the mycological properties of Thermoanaerobacter No. 49 are shown in the table, and it is a novel microorganism that has not been previously recognized in that it possesses pSH49. This bacterial strain was isolated from hot spring water as an erythromycin-resistant strain.

Table (Taxonomic properties) Optimum growth temperature: 70℃ Growth temperature range: 50-78℃ Morphology: 0.3-0.4 x 1 ~ rods with periflagella
4 μm Spore-forming property: None Gram staining; Undefined GC content: 34% Main products: Availability of acetic acid, lactic acid sugar: Glucose, lactose, maltose, xylose, starch Based on the above mycological properties, it has been designated as the “Archive of Microbiota”. Archives of
Microbiology, 128-129, Bd., 1980-81'', it was recognized as Thermoanaerobacter.

However, considering the fact that it cannot be said to be any conventional species since it possesses the above-mentioned novel plasmid, the species was named the new Thermoanaerobacter No. 49.

This strain has been deposited as Microtechnical Research Institute No. 7495.

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, Shizuoka Prefecture was mixed with YPG medium (Difco Yeast Extract 0.2%, Daigo Nutrient Polypeptone 0.4%, Glucose 1%,
NaCl 0.2%, Na ₂ CO ₃ 0.3%, L-cysteine 0.2
%, PH7.5) in addition to 10 ml under carbon dioxide gas anaerobic conditions,
After culturing at 70℃ for about 18 hours, add 50μ of erythromycin.
Thermoanaerobacter No. 49 from one of the colonies grown in a YPG roll tube containing g/ml
(Feikoken Bibori No. 7495) was obtained.

Example 2 Plasmid pSH49 of Thermoanaerobacter No.
Isolation from Thermoanaerobacter No. 49
7495) from biologically pure culture medium.
Inoculate YPG medium and store at 70℃ under carbon dioxide gas anaerobic conditions for 12 days.
Incubate for hours. This culture solution is inoculated into YPG medium No. 1, and cultured for 12 hours under anaerobic conditions at 70°C. Bacterial cells were collected by centrifugation and mixed with TES (20mM Tris-HCl, 5mM EDTA,
After washing with 100mMNaCl, PH7.5), bacterial cell wet weight 4g
Suspend in 10 ml of TES containing 25% sucrose to which 10 mM of sodium azide has been added. Lysozyme (10
Add 3 ml of 0.25 M EDTA and 2 ml of 0.25 M EDTA to 0°C.
Leave for 20 minutes. 10 to 300μ of this cell mixture
% diethylpyrocarbonate ethanol solution, 16%
ml of alkaline SDS solution (1% SDS, 0.2N
Add 12 ml of sodium acetate (3M, PH4.8) and leave at 0°C for 2 hours. This at 7000rpm,
Centrifuge for 1 hour to obtain supernatant. This supernatant
Add 100 ml of 95% ethanol, let stand at -20°C for 2 hours, and centrifuge at 7000 rpm for 10 minutes to obtain a precipitate. This precipitate is washed with 50 ml of 95% ethanol and dried under reduced pressure, then dissolved in 14 ml of TES, and the density is adjusted to 1.58 by adding CsCl and ethidium bromide. This sample is subjected to density gradient centrifugation at 38000 rpm for 30-40 hours. The resulting plasmid DNA bands were collected, ethidium bromide was removed with isoamyl alcohol, and then TEN (20mM Tris-HCl, 1mM
A plasmid solution is obtained by dialysis against EDTA, 20mM NaCl).

Procedure for characterizing pSH49 The molecular weight of pSH49 was obtained by agarose gel electrophoresis and polyacrylamide gel electrophoresis of its supercoiled DNA and fragments cleaved with restriction enzymes. The molecular weight marker in this case is pBR322DNA.
(2.67md), ColEIDNA (4.2md) and Lambda DNA
Hind decomposition fragment (14.6, 5.84, 4.05, 2.67,
1.30, 1.17, 0.34md), EcoR degradation fragment of lambda DNA (13.7, 4.74, 3.73, 3.48, 3.02, 2.13md),
Hae-digested fragments of φ×174 DNA (0.836, 0.666,
0.539, 0.373, 0.192, 0.174, 0.167, 0.145,
0.120, 0.073, 0.044md) were used. Cleavage with restriction enzymes was performed 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. Agarose gel electrophoresis was carried out using SeaChem agarose at a concentration of 0.5% or 0.7% in a horizontal gel electrophoresis chamber at a constant voltage of 1.5 V per cm of gel length for 15 to 17 hours.

Polyacrylamide gel electrophoresis was performed using polyacrylamide/bisacrylamide manufactured by Seikagaku Kogyo Co., Ltd., using a 5% concentration gel with a degree of crosslinking of 30:1 in a vertical slab gel electrophoresis chamber at 10 V per 1 cm of gel length. This was carried out for 2 to 3 hours using a constant voltage of .

Until now, there has been no known example of a plasmid being detected in an anaerobic thermophile, and pSH49 possessed by the anaerobic thermophile Thermoanaerobacter No. 49 is a completely new plasmid.

[Brief explanation of the drawing]

Figure 1 shows the restriction enzyme cleavage map of pSH49, and EcoR in the figure is an enzyme derived from E. coli.
Cla is an enzyme derived from Karyophanone latum.
Pst is an enzyme derived from Providencia stuartei, Hpa is an enzyme derived from Haemophilus parainfluenzae, Hpa is an enzyme derived from Haemophilus parainfluenzae, Pvu is an enzyme derived from Proteus vulgaris, and Hind is an enzyme derived from Haemophilus influenzae. are shown respectively.

Claims (1)

[Claims] 1. A novel Thermoanaerobacter No. 1 having a molecular weight of approximately 5.0 megadaltons, which possesses a plasmid that is not cleaved by restriction enzymes BamH and Bgl and is characterized by the restriction enzyme cleavage map shown in the figure.
49.