JPH0371115B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to plasmid pTA5001(B), which is extremely useful as an acetic acid bacterium vector.

More specifically, the present invention relates to plasmid pTA5001(B), which possesses only one recognition site for the restriction enzyme Xho and is easily transferred to acetic acid bacteria.

Conventionally, regarding plasmids derived from acetic acid bacteria, Acetobacter aceti No. 1023 has a molecular weight of 17 × 10 ⁶ daltons, plasmid pTA5001 (Agric.Biol.
Chem.46(2), 381-389, 1982) and Gluconobacter bacteria (Fermentation Engineering Journal, 61
(1), 15-18, 1983) were reported.

After studying this plasmid pTA5001 in detail, the present inventors learned that it was actually a mixture of two plasmids, and completed the creation of a restriction enzyme cleavage map for the two plasmids and detailed its use. Upon investigation, it was confirmed that each plasmid possesses only one recognition site by the restriction enzyme Xho and is an excellent vector that can be easily transferred into acetic acid bacteria, leading to the completion of the present invention.

The present invention relates to plasmid pTA5001(B), which has a molecular weight of 22.5 Kb and is shown in the restriction enzyme cleavage map of FIG.

Plasmid pTA5001(B) of the present invention and plasmid
pTA5001(A) is also Acetobacter aceti No.
1023, and can be isolated from this bacterium as a mixture of both.

Acetobacter aceti No. 1023 was isolated and identified from acetic acid fermentation mash (Agric.Biol.
Chem., 44(12), 2901-2906, 1980), and has been deposited as FERM P-7122 at FIKEN, containing plasmid pTA5001(A) and plasmid pTA5001(B).

The mycological properties of Acetobacter aceti No. 1023 closely match the description of the mycological properties of Acetobacter aceti in the 8th edition of Virgil, and it is further characterized by having acetic acid resistance and ethanol oxidation ability. Acetobacter aceti No.1023 (Ace ^r , Eth ⁺⁺ )
may also be displayed.

Acetobacter aceti No. 1023 is usually cultured in the following YPG medium, for example, and to detect transformed strains, the YPG medium is supplemented with drugs such as antibiotics to an appropriate concentration, such as ampicillin.
The cells are cultured using the added material at a concentration of 50 μg/ml.

(YPG medium) Yeast extract 0.5% Polypeptone 0.2% Glucose 3.0% Agar (in the case of solid medium) 2.0% PH = 6.5 Acetobacter aceti No. 1023 was cultured in YPG liquid medium with shaking at 30℃ for 24 to 36 hours. Then, the culture solution is centrifuged to collect bacteria. The bacterial cells are thoroughly washed with a buffer solution, suspended in the buffer solution, lysozyme is added thereto, and the cells are lysed. A surfactant and salt are added to the lysate, and after being allowed to stand, it is centrifuged. Polyethylene glycol is added to the supernatant, and after being allowed to stand, it is centrifuged to obtain a precipitate. This precipitate is dissolved in a buffer solution, ethidium bromide is added, cesium chloride is further added, the density is adjusted to 1.57, and density gradient centrifugation is performed. After centrifugation, the centrifugation tube is irradiated with 365 nm ultraviolet light using an ultraviolet lamp to separate the band that appears below the chromosome band.

The band obtained here contains plasmid
pTA5001(A) and plasmid pTA5001(B) are mixed.

As a result of restriction enzyme analysis, it was revealed for the first time that the two mixed plasmids were a mixture of two types of plasmids with approximately the same molecular weight.

The molecular weight of plasmid pTA5001(B) is 22.5Kb,
The restriction enzyme cleavage map is shown in FIG.

The meanings of the abbreviations shown in the drawings are as follows.

E: EcoR: Escherichia coli RY13 source restriction enzyme S: Sal: Streptomyces albus G source restriction enzyme X: Xho: Xanthomonas holcicola source restriction enzyme Plasmid pTA5001(B) of the present invention is cleaved at only one site by Xho This cleavage site is very characteristic, and it is extremely easy to introduce other plasmid fragments or chromosomal fragments into this cleavage site.

Plasmid pTA5001(B) of the present invention is suitable for use as an acetic acid bacterium vector.

That is, the plasmid pTA5001(B) into which a plasmid fragment or chromosome fragment has been introduced can be easily transferred to acetic acid bacteria (Acetobacter spp., Gluconobacter spp.), and can be used for transformation and/or It provides a new and innovative method for material production.

Next, examples of the present invention will be shown.

Example 1, Preparation of DNA receptor cells Acetobacter aceti No. 1023, FERM P-
From Acetobacter aceti 10-8 ( ^Acer , Eth ⁺⁺ , Pro ^- ), a proline auxotrophic (Pro ^- ) parent strain obtained from 7122 by mutation treatment with nitrosoguanidine (NTG) at a concentration of 100 μg/ml. Acetobacter aceti 10-80S1 (Ace ^ss , Eth ^- ), a strain with streptomycin resistance (Str ^r ), has reduced or deleted acetic acid tolerance and ethanol oxidation ability (Ace ^ss , Eth - ) obtained through natural mutation. Eth- ^{, Pro-} , Str ^r )
was inoculated into 100 ml YPG liquid medium in a 500 ml Sakaguchi flask, and cultured with shaking at 30°C for 20 hours.

The culture solution is centrifuged at 0° C. and 6000×g for 10 minutes to collect bacteria. Bacterial cells are 100mM NaCl and 5mM
_5mM Tris-HCl buffer (PH
Wash twice with 0.5 times the volume of 7.6). again at 0℃
Centrifuge at 6000 xg for 5 minutes to collect bacteria.

This bacterial cell was mixed with 0.4 times the volume of _CaCl2 solution ( _100mMCaCl2 , 250mM KCl, 5mM _MgCl2 ,
5mM Tris-HCl, PH7.6) was added and incubated at 0°C.
After standing for 30 minutes, centrifuge at 0°C and 6000 xg for 5 minutes to collect bacteria.

Add 0.004 times the volume of the above CaCl ₂ solution to the bacterial cells.
A DNA receptor cell suspension was prepared.

Example 2, plasmid pTA5001(B) and plasmid
Isolation of pTA5001(A) contaminants Acetobacter aceti No.1023, FERM P-
7122 was inoculated into 40 ml of YPG medium and cultured with shaking at 30°C overnight.

Thereafter, it was subcultured to a new YPG medium 4 at a concentration of 1% and further cultured with shaking at 30°C for 36 hours.

After bacterial collection, add TE buffer (20mM EDTA, 50mM
The bacterial cells were washed twice with Tris-HCl (pH 8.0).

Add 2ml of TES buffer (50mM Tris-HCl, 20mM EDTA, 25% sucrose, PH8.0) per 2g of the obtained wet bacterial cells to suspend the cells, and add 4ml of lysozyme solution (0.25M Tris-HCl, lysozyme). 2
%, PH8.0) was further added, and the mixture was allowed to stand at 0°C for 5 minutes. Next, add 4ml of 0.25MEDTA solution (PH8.0),
After standing at 0°C for 5 minutes, the mixture was reacted at 37°C for 20 minutes. After the reaction, 3 ml of 10% sodium lauryl sulfate was added and left to stand at 37°C for 20 minutes, then 5 ml of 5M saline was added and left to stand at 0°C overnight. Centrifugation was performed at 48,200×g for 60 minutes, and the supernatant was collected. Next, polyethylene glycol 6000 was added to this supernatant to a final concentration of 10%, and after standing at 4°C overnight,
Centrifugation was performed at 3000×g for 10 minutes to obtain a precipitate. After dissolving this precipitate in 7ml of UC buffer (50mM Tris-HCl, 5mM EDTA, 50mM NaCl, PH7.8), ethidium bromide was added to a final concentration of 500μg/ml, and then cesium chloride was added. The density was adjusted to 1.57. This solution
Density gradient centrifugation was performed at 15° C. and 100,200×g for 40 hours. After centrifugation, the centrifugation tube was irradiated with 365 nm ultraviolet light using an ultraviolet lamp, and the band appearing below the chromosome band was separated as a plasmid fraction. The fractionated solution was then treated with isopropanol to remove ethidium bromide, and then treated with TE buffer (10mM Tris-HCl, 1mM
Dialysis was performed against EDTA, pH 7.5). This was used as a plasmid mixed solution.

Two circular plasmids were found mixed in the resulting plasmid mixed solution, and as a result of analysis using restriction enzymes, it was determined that the plasmids were pTA5001(A) shown in Figure 1 and plasmid pTA5001(B) shown in Figure 2. It became clear.

That is, at least a 5-fold excess of restriction enzyme (EcoR) was added to the plasmid mixed solution prepared above.
and Sal were manufactured by Takara Shuzo, and Xho was manufactured by Bethesda Research. ) were reacted according to a conventional method under optimal conditions for each restriction enzyme. After the reaction, it was analyzed by vertical agarose gel electrophoresis. That is, using a 1% agarose gel, Tris acetate buffer (40mM Tris, 20mM acetic acid, 2mM
Electrophoresis was performed in EDTA, pH 8.1). The gel was then stained with 1 μg/ml ethidium bromide. This gel was irradiated with ultraviolet rays, the number of generated fragments was determined, and the molecular weight of each fragment was calculated from the migration distance of each fragment. The molecular weight was calculated based on a standard line created from the migration distance of each fragment of known molecular weight generated by Hind cleavage of lambda phage DNA that was electrophoresed simultaneously on the same agarose.

pTA5001(A) and pTA5001( The restriction enzyme cleavage map shown in Figures 1 and 2 of B) was determined.

Example 3, plasmid pTA5001(A) and plasmid
Use of pTA5001(B) as a vector In the plasmid mixed solution (DNA amount 10 μg) obtained in Example 2, pACYC177, an Escherichia coli drug-resistant vector (kanamycin resistance and ampicillin resistance; Journal of Bacteriology, 134
(3), 1141-1156, 1978) plasmid obtained from Escherichia coli C600
Add pACYC177 (shown in Figure 3; DNA amount: 2 μg) and react with at least a 5-fold excess of the restriction enzyme Xho using a conventional method under optimal conditions. After the reaction is complete, add an equal amount of phenol and stir vigorously. After inactivating the restriction enzymes, ether extraction was performed sufficiently to remove phenol, and twice the volume of ethanol was added and the mixture was kept at -80°C for 1 hour.
The DNA was precipitated by centrifugation at 15,000 rpm for 5 minutes, and the ethanol was removed by vacuum drying. Next, the precipitate was dissolved in water, and then T4 was added using a conventional method.
The DNA ligase reaction was carried out at 21°C for 2 hours, and the precipitate obtained by ethanol precipitation and vacuum drying was added to 0.1 ml of TE buffer in the same manner as above.
A solution containing the chimeric plasmid was obtained.

Each chimeric plasmid contains plasmid pACYA177. However, the kanamycin resistance site of plasmid pACYA177
Since there is an Xho cleavage point and it is cleaved at that point, kanamycin resistance will not be expressed, and only ampicillin resistance will be expressed.

DNA receptor cell suspension obtained in the following Example 1
A 0.2 ml volume was prepared, each of the chimera plasmid-containing solutions mentioned above was added thereto, and the chimera plasmid was directly introduced while gently stirring at 0°C for 90 minutes.

The resulting solution containing the chimeric plasmid-introduced bacterial cells was transferred to 3 ml of YPG medium, cultured with shaking at 30°C for 6 hours, and then supplemented with ampicillin at 50 μg/ml.
The cells were cultured on the added YPG medium (solid) at 30°C for 5 days, and 9 colonies were obtained. These are 10−
They were named 80S1-A1~-A9TO. Of these, 10
-80S1-A1 with ampicillin added at 30μg/ml
When cultured in a YPG liquid medium with shaking at 30°C for 24 hours, the plasmids were separated and analyzed according to the method of Example 2. A large plasmid was obtained. This plasmid is different from the previously introduced chimeric plasmid.
It was recognized as a chimeric plasmid in which pTA5001(A) and pACYA177 were linked via the Xho cleavage site. Furthermore, the fact that Acetobacter aceti 10-80S1 does not have ampicillin resistance, but 10-80S1-A1 does have ampicillin resistance, confirms that the chimeric plasmid was introduced and transformation was performed.

Similarly, at least 10−80S1−A2 to −A6
It was confirmed that a chimeric plasmid in which pTA5001(B) and pACYA177 were linked via the restriction enzyme Xho cleavage site had been introduced.

In addition, when the chimeric plasmids of 10-80S1-A1 to -A6 were reintroduced into 10-80S1 in the same manner as described above, the amount of chimeric plasmids of 10-80S1-A1 to -A6 was calculated per 1 μg DNA.
Approximately 10 ₅ ampicillin-resistant transformants were obtained.

[Brief explanation of drawings]

Figure 1 shows the restriction enzyme cleavage map of plasmid pTA5001(A), and Figure 2 shows the restriction enzyme cleavage map of plasmid pTA5001(B).
Figure 3 shows the restriction enzyme cleavage map of the plasmid.
The restriction enzyme cleavage map of pACYC177 is shown. E...EcoR cleavage site, S...Sal cleavage site, X...Xho cleavage site,
Km...Kanamycin resistance gene, Am...Ampicillin resistance gene.

Claims (1)

[Claims] 1. Plasmid pTA5001(B), which has a molecular weight of 22.5 Kb and is shown in the restriction enzyme map below. E: EcoR: Escherichia coli RY13 source restriction enzyme S: Sal: Streptomyces albus G source restriction enzyme X: Xho: Xanthomonas holcicola source restriction enzyme.