JPS6221514B2 - - 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 invention relates to a novel plasmid useful as a vector for DNA experiments, and more particularly to a novel plasmid whose molecular weight is approximately 3.3 megadaltons and which is characterized by the restriction enzyme cleavage map shown in the figure.

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 3.3 megadaltons from Thermoanaerobacter that showed neomycin resistance.

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 pSH62).

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

EcoR is an enzyme derived from Eschialsia coli,
Hpa is an enzyme derived from Haemophilus parainfluenzae, Cla is an enzyme derived from Karyophanon latum, Acc is an enzyme derived from Acinetobacter calcoaceticus, Bgl is an enzyme derived from Bacillus globigii, and Hind is an enzyme derived from Haemophilus parainfluenzae.
Each enzyme derived from influenza is shown.

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 pSH62 in this case. This is because, firstly, pSH62 is a plasmid that can replicate in anaerobic thermophiles, and secondly,
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, pSH62 is an EcoR
It has cleavage sites for restriction enzymes such as , Bgl, etc. at specific and limited positions. This thing is
When pSH62 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.

pSH62 was obtained by growing Thermoanaerobacter No. 62, an anaerobic thermophile newly isolated from soil, 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 is achieved by lysing the bacteria by SDS treatment.

In addition, the mycological properties of Thermoanaerobacter No. 62 are shown in the table, and it is a novel microorganism that has not been previously recognized in that it possesses pSH62. This bacterial strain was isolated from soil as a neomycin-resistant strain.

Table (Taxonomic properties) Optimum growth temperature: 66℃ Growth temperature range: 50-74℃ Morphology: 0.3-0.4 x 1 ~ rods with periflagella
4 μm Spore-forming ability; None Gram staining; Undefined GC content; 34% Main products; Availability of ethanol, acetic acid, and lactic sugar; Glucose, lactose, maltose, mannose, xylose, cellobiose, starch More mycological properties ``Archives of Microbiology''
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. 62.

This strain has been deposited as Microtechnical Research Institute No. 7493.

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

Example 1 (Screening of bacterial strains) Approximately 0.5 g of soil from the Ibaraki Prefecture region 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) 10
After culturing for about 18 hours at 70℃ under anaerobic carbon dioxide conditions, one of the colonies grown in a YPG roll tube containing 50 μg/ml of neomycin was infected with Thermoanaerobacter No. issue)
was gotten.

Example 2 Plasmid pSH62 of Thermoanaerobacter No.
Isolation from Thermoanaerobacter No. 62
7493) 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 Na
Add 12 ml of sodium acetate (3M, PH4.8) and leave at 0°C for 2 hours. This at 7000rpm, 1
Centrifuge for an hour to obtain the supernatant. In 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 pSH62 The molecular weight of pSH62 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 fragment of φ×174 DNA (1.836, 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 pSH62 possessed by the anaerobic thermophile Thermoanaerobacter No. 62 is a completely new plasmid.

[Brief explanation of the drawing]

Figure 1 shows the restriction enzyme cleavage map of pSH62, and EcoR in the figure is an enzyme derived from Eschialsia coli.
Hpa is an enzyme derived from Haemophilus parainfluenzae, Cla is an enzyme derived from Karyophanone latum, Acc is an enzyme derived from Acinetobacter calcoaceticus, and Bgl is an enzyme derived from Bacillus globigii. Hind is an enzyme derived from Haemophilus parainfluenzae.
Influenzae-derived enzymes are shown.

Claims (1)

[Scope of Claims] 1. A novel plasmid derived from an anaerobic thermophile that has a molecular weight of approximately 3.3 megadaltons, is not cleaved by the restriction enzymes BamH and Pst, and is characterized by the restriction enzyme cleavage map shown in the figure.