JPH0363356B2 - - Google Patents

Description

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

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

Conventionally, regarding plasmids derived from acetic acid bacteria, Acetobacter aceti No. 1023 has a 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 further completed the creation of restriction enzyme cleavage maps for the two plasmids and detailed their uses. Upon further investigation, we realized that each plasmid possesses only one recognition site for the restriction enzyme Xho I and is an excellent vector that can be easily transferred into acetic acid bacteria. I've reached it.

The present invention relates to plasmid pTA5001(A), which has a molecular weight of 23.5 Kb and is shown in the restriction enzyme cleavage map of FIG.

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

Acetobacter aceti No. 1023 was identified as a unit from acetic acid fermentation mash (Agric.Biol.
Chem., 44(12), 1901-2906, 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 (for fixed 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 disperse the bands below the chromosome bands.

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(A) is 23.5Kb,
The restriction enzyme cleavage map is shown in FIG.

The meanings of the abbreviations shown in FIG. 1 are as follows.

E: coR I: Escherichia coli RY13 source restriction enzyme S: Sal I: Streptomyces albus G source restriction enzyme It is very distinctive because it is cut at only one site, and it is extremely easy to introduce other plasmid fragments or chromosome fragments at this cut site.

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

That is, the plasmid pTA5001(A) 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 of acetic acid bacteria 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
-7122 by mutation treatment with nitrosoguanidine (NTG) at ^a concentration of 100 μg/ml.
Acetic acid tolerance and ethanol oxidation ability obtained by natural mutation from aceti-10-8 (Ace ^r , Eth ⁺⁺ , Pro ^- ) are reduced or deleted (Ace ^ss , Eth ^- ), and streptomycin resistance (Str ^r Acetobacter aceti 10-80S1 (Ace ^ss , Eth ^- , Pro ^- ,
100ml YPG containing Str ^r ) in a 500ml plate-necked flask.
It was inoculated into a liquid medium 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). 0℃6000 again
Centrifuge at ×g for 5 minutes to collect bacteria.

Add 0.4 times the volume of CaCl ₂ solution (100mM
_CaCl2 , 250mM KCl, 5mM _MgCl2 , 5mM
Tris-HCl, pH 7.6) was added and left to stand at 0°C for 30 minutes, followed by centrifugation 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 of plasmid pTA5001(A)
Isolation of contaminant (B) Acetobacter aceti No.1023, FERM P-
7122 was inoculated into 40 ml of YPG medium and cultured with shaking at 30°C overnight.

Thereafter, the cells were subcultured into fresh YPG medium 4 at 1% and cultured with shaking at 30°C.

After harvesting, 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 4 ml of 0.25M EDTA solution (PH8.0) and heat at 0℃.
After being allowed to stand for 5 minutes at 37°C, the reaction mixture was allowed to react for 20 minutes at 37°C.
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, 3000x
A precipitate was obtained by centrifugation at g for 10 minutes. This precipitate was mixed with 7 ml of UC buffer (50mM Tris-HCl,
After dissolving in 5mM EDTA, 50mM NaCl, PH7.8), ethidium bromide was added to give a final concentration of 500 μg/ml, and cesium chloride was further added to adjust the density to 1.57. Add this solution to 15
Density gradient centrifugation was performed at 100,000 x g for 40 hours at °C. 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
Dialyzed against EDTA, PH7.5). 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.
I and Sal I were used from Takara Shuzo Co., Ltd., and Xho I was used from Bethesda Research Co., Ltd. ) 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 EDTA, PH8.1). after that,
The gel was stained by soaking it in a 1 μg/ml solution of 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 is the Hind value of lambda phage DNA that was run simultaneously on the same agarose.
Calculations were made based on a standard line created from the migration distance of each fragment of known molecular weight produced by cleavage.

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 pTA5001
Use of (B) as a vector In the plasmid mixed solution (DNA amount 10 μg) obtained in Example 2, pACY177, an Escherichia coli drug-resistant vector (kanamycin resistance and ampicillin resistance; Journal of Bacteriology, 134(3) ),
1141−1156, 1978)
Plasmid pACYC177 (3rd generation) obtained from coli C600)
As shown in the figure. 2 μg of DNA) and react with at least 5 times excess of restriction enzyme After that, thoroughly perform ether extraction to remove phenol, add twice the amount of ethanol, hold at -80℃ for 1 hour, and centrifuge at 15,000 rpm for 5 minutes to precipitate the DNA. After further vacuum drying to remove ethanol, the precipitate was dissolved and a reaction using T4 DNA ligase was carried out at 21°C for 2 hours in the usual manner, followed by ethanol precipitation and vacuum drying in the same manner as above. The precipitate obtained was dissolved in 0.1 ml of TE buffer to obtain a solution containing the chimeric plasmid.

Each chimeric plasmid contains plasmid pACYC177. However, the kanaimycin resistance site of plasmid pACYC177
Since there is an xho I breakpoint and this point is cleaved, kanamycin resistance is not expressed, and only ampicillin resistance is expressed.

DNA receptor cell suspension obtained in the following Example 1
0.2 ml of the solution was prepared, and 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 to -A9. Kotouchi, 10−
Ampicillin was added to 80S1-A1 at 30 μg/ml.
When cultured with shaking in YPG liquid medium at 30°C for 24 hours, the plasmids were separated and analyzed according to the method of Example 2. A plasmid with a large molecular weight was obtained. This plasmid is one of the previously introduced chimeric plasmids.
It was recognized as a chimeric plasmid in which pTA5001(A) and pACYC177 were linked via the Xho I cleavage site. In addition, Acetobacter aceti 10-80S1 does not have ampicillin resistance, but 10-80S1-A1 has ampicillin resistance, confirming that a chimeric plasmid was introduced and transformation was performed. .

Similarly, at least 10−80S1−A2 to −A6
pTA5001(B) and pTACYC177 are restriction enzyme Xho I
It was confirmed that the chimeric plasmid linked via the 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 transformed strains resistant to ampicillin 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 pTCYC177 is shown. E...cleavage site by EcoR I, S...Sal I
cleavage site by X...... cleavage site by Xho I,
Km...Kanamycin resistance gene, Am...Ampicillin resistance gene.

Claims (1)

[Claims] 1. Plasmid pTA5001(A), which has a molecular weight of 23.5 kb and is shown in the restriction enzyme map below. However, E means EcoR I, S means Sal I, and X means Xho I.