JPH0669368B2 - Method for transforming acetic acid bacterium - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for transforming acetic acid bacteria.

More specifically, the present invention relates to a method for transforming an acetic acid bacterium by introducing a gene-related DNA.

Conventionally, a method using conjugal transfer has been used for transformation of acetic acid bacteria (Journal of Bacteriology, 145 (1), 358-368, 19).
Other than 81), it is in a state that is completely unknown.

The present inventors have conducted diligent research from the idea that better transformants can be obtained by gene manipulation in acetic acid bacteria, and when plasmids, plasmid fragments, and chromosomal fragments are all transformed into acetic acid bacteria. The present invention was completed by confirming the introduction of

The present invention is characterized by introducing a plasmid and / or a plasmid fragment and / or a chromosomal fragment into an acetic acid bacterium in a solution containing at least one metal selected from alkali metals and alkaline earth metals. The present invention relates to a method for transforming a bacterium.

The DNA used for transformation in the present invention may be a plasmid, a plasmid fragment, a chromosome fragment, or the like. As the plasmid, a plasmid as it is separated from bacterial cells, various chimeric plasmids derived from the plasmid, or the like can be used. Further, as the plasmid fragment or the chromosome fragment, a plasmid fragment produced by lysis or mechanical treatment can be used.

In addition, as the acetic acid bacterium to be transformed in the present invention, all bacteria of the genus Acetobacter and Gluconobacter are targeted, but as an exemplifying bacterium of the DNA-supplying bacterium and the transformant in the present invention, Acetobacter aceti No. .1023 is shown.

Acetobacter aceti No. 1023 was isolated from the acetic acid fermentation mash, and the plasmid pTA5001 (A) described below was used.
And FER to Micro Incorporated with the plasmid pTA5001 (B) included.
Deposited as MP-7122.

Acetobacter aceti No. 1023 is well characterized in that it is in good agreement with the description of the bacteriological characteristics of Acetobacter aceti in Vergii 8th edition, and has acetic acid resistance and ethanol oxidation ability. Acetobacter aceti N
It may be displayed as o.1023 (Acer, Eth ⁺⁺ ).

Acetobacter aceti No. 1023 is usually cultured in, for example, the following YPG medium, and Y is used for the detection of transformants.
To ensure that the PG medium has an appropriate concentration of drugs such as antibiotics,
For example, ampicillin is added to a concentration of 50γ / ml or the following minimal medium is used for culturing.

(YPG medium) (for solid medium) yeast extract 0.5% polypeptone 0.2% glucose 3.0% agar 2.0% pH = 6.5 (minimal _{medium) K 2 HPO 4 0.01% KH} 2 PO 4 0.05% MgSO 4 · 7H 2 O 0.025 % KCl 0.01% CaCl ₂・ 2H ₂ O 0.01% FeCl ₃・ 6H ₂ O 0.0005% Na-glutamate 0.4% Glucose 3.0% Agar (in solid medium) 2.0% pH = 6.5 Acetobacter aceti No. 1023 is a YPG liquid 30 ℃ in medium
The cells are cultivated with shaking for 24 to 36 hours, and the culture solution is centrifuged to collect the cells. The cells are thoroughly washed with a buffer solution, suspended in the buffer solution, lysozyme is added thereto, and lysed. A surfactant and salt are added to the lysate, which is allowed to stand and then centrifuged, and polyethylene glycol is added to the supernatant, which is then allowed to stand and centrifuged to obtain a precipitate. This precipitate is dissolved in a buffer solution, ethidium bromide is added, cesium chloride is further added to adjust the density to 1.57, and density gradient centrifugation is performed. After centrifuging, centrifuge tube with UV lamp 365nm
The band appearing under the chromosome band is collected by the ultraviolet irradiation of.

The band obtained here contains a mixture of plasmid pTA5001 (A) and plasmid pTA5001 (B).

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

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

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

Meanings of the abbreviations shown in FIGS. 1 and 2 are as follows.

E: EcoRI: Escherichia coli RY13 source restriction enzyme S: SalI: Streptomyces albus G source restriction enzyme X: Xho I: Xhanthomonas holcicola source restriction enzyme plasmid pTA5001 (A) and plasmid pTA5001 (B)
Is extremely characteristic in that it is cleaved at only one site by XhoI, and it is extremely easy to construct a chimeric plasmid by introducing another plasmid fragment or a chromosomal fragment into this cleavage site. Is.

Plasmid pTA5001 (A) and plasmid pTA5001 (B)
Are used alone or as a mixture thereof as an acetic acid bacterium vector.

That is, plasmid pTA5001 (A) and / or plasmid p
A chimeric plasmid obtained by introducing a plasmid fragment or a chromosome fragment into TA5001 (B) is effectively used for the transformation of the present invention.

Also, the parent strain, Acetobacter aceti No. 1023, FER
Nitrosoguanidine (N concentration of 100 μg / ml from MP-7122)
TG) obtained proline auxotrophy Te cowpea to mutagenesis (Pro ^-)
Acetobacter aceti 10-8 (Acer, Eth
^++, Pro ^-), and now cowpea acetate tolerance and ethanol oxidation ability obtained by the deleted drop deleted to natural variation (Acess, Et
h ^-), and Acetobacter aceti 10-80S1 a strain of streptomycin resistance ^{(Strr) (Acess, Eth -} , Pr
o ^-, Strr) and as DNA receptors, the parent strain may be implemented transformants as donor chromosome fragments.

In this case, the preparation of the chromosome fragment is basically carried out according to the method for preparing the plasmid, but the parent strain Acetobacter
The sucrose was not contained in the buffer solution for suspending the bacterial cells of ACETI No. 1023, and after the addition of the surfactant, the mixture was heated at 65 ° C for 5 minutes without adding sodium chloride, followed by phenol treatment, ethanol precipitation, and vacuum drying. The prepared DNA is again subjected to cesium chloride-ethidium bromide density gradient ultracentrifugation in the same manner as in the plasmid preparation method, and the generated DNA band is fractionated.

The DNA band obtained here contains a chromosomal fragment and / or a plasmid fragment, and if this is added as it is to the mutant strain-containing liquid to perform a DNA introduction treatment, the chromosomal fragment and / or the plasmid fragment will enter the bacterial cells. The transformation of the transformant occurs when the fragment is incorporated or the recombination occurs in the mutant strain. In this way, the proline synthetic gene of the parent strain is easily transferred to the mutant strain and the trait is transformed.

Thus, the transformation of the bacterium Acetobacter trait is brought about in solution by the plasmid and / or the plasmid fragment and / or the chromosomal fragment.

In the present invention, first, the bacterial cells are treated with the solution. The solution used here is preferably a salt solution containing one selected from alkali metals and alkaline earth metals. Examples thereof include chlorides of lithium, sodium, potassium, rubidium, cesium, magnesium and potassium.

50-100 mM for calcium chloride, 1 for magnesium chloride
It is advisable to dip the cells to be transformed into a slightly acidic pH of 6 to 7 at about 0 to 50 mM.

The cells to be transformed are preferably those collected from the mid-logarithmic growth phase to the early stationary phase.

To the cells treated with the solution, the plasmid and / or the plasmid fragment and / or the chromosomal fragment is added in the solution while being immersed in the solution.

The introduction of DNA is completed by gently stirring at 0 ° C for about 1.5 hours.

To the cells that had been subjected to the DNA introduction treatment, a minimal medium (solid) or a drug such as an antibiotic was added to an appropriate concentration, for example, ampicillin was added to a concentration of 50γ / ml.
Cultivate in YPG medium (solid) at 30 ° C. for 5 to 10 days to separate colonies.

When the traits of the obtained bacterial cells were confirmed, it was found that various traits that did not exist were introduced.

In the case of transformation, in the case of a plasmid or a chimeric plasmid, the trait is directly expressed by the cell, but in the case of a plasmid fragment or a chromosomal fragment, recombination occurs in the cell after being taken up by the cell, and the trait is expressed. It is thought to occur.

Next, examples of the present invention will be described.

Example 1 Preparation of DNA Receptor Bacteria Acetobacter aceti No. 1023 (Acer, Eth ⁺⁺ , pro ⁺ , str
s), Acetobacter aceti 10-80S1 (Ace isolated from FERM P-7122 by NTG mutation treatment and natural mutation treatment).
^{ss, Eth -, pro -,} strr) was placed in a 500ml Sakaguchi flask 100m
lYPG liquid medium was inoculated and shake-cultured at 30 ° C. for 20 hours.

The culture solution is centrifuged at 6,000 xg at 0 ° C for 10 minutes to collect the cells. The cells are 5 mM containing 100 mM NaCl and 5 mM MgCl _2.
Wash twice with 0.5 volume of Tris-HCl buffer (pH 7.6). Centrifuge again at 6,000 xg for 5 minutes at 0 ° C to collect the cells.

The cells contained 0.4 times the volume of CaCl ₂ solution (100 mM CaCl ₂ , 2
50 mM KCl, 5 mM MaCl ₂ , 5 mM Tris-HCl, pH 7.6) are added, and the cells are collected by centrifugation at 6,000 xg for 5 minutes at 0 ° C.

The CaCl ₂ solution in an amount of 0.004 times the volume was added to the cells to give a DNA-receptor cell suspension.

Example 2 Plasmid pTA5001 (A) and plasmid pTA50
Isolation of 01 (B) mixture Acetobacter aceti No. 1023, FERM P-7122 40 ml Y
The cells were inoculated into a PG medium and shake-cultured overnight at 30 ° C.

Subsequent transfer to 1 liter of new YPG medium 4l at 30%
The cells were shake-cultured for 36 hours.

After harvesting, TE buffer (20 mM EDTA, 50 mM Tris-HCl, pH 8.
The cells were washed twice with 0).

7 ml of TES buffer solution (50 mM Tris-HCl, 20 mM EDTA, 25% sucrose, pH 8.0) was added to 2 g of the obtained wet cells, and the cells were suspended, and 4 ml of lysozyme solution (0.25 M Tris-hydrochloride, lysozyme) was added. 2%, pH8.0) was further added, and the mixture was allowed to stand at 0 ° C for 5 minutes.
Next, 4 ml of 0.25 M EDTA solution (pH 8.0) was added, and the mixture was allowed to stand at 0 ° C for 5 minutes and then reacted at 37 ° C for 20 minutes. After the reaction, 3% of 10%
Sodium lauryl sulfate was added, and after standing at 37 ° C for 20 minutes,
5 ml of 5M saline was added, and the mixture was left standing at 0 ° C overnight.

The supernatant was collected by centrifugation at 48,200 xg for 60 minutes.
Next, add 6,000 polyethylene glycol to the supernatant to a final concentration of 10%, leave it at 4 ° C overnight, and
A precipitate was obtained by centrifugation at 00 xg for 10 minutes. This precipitate was added to 7 ml of UC buffer (50 mM Tris-HCl, 5 mM EDTA, 50 mM Na
Cl, pH 7.8), ethidium bromide was added to a final concentration of 500 μg / ml, and cesium chloride was further added to adjust the density to 1.57. 15 this solution
Density gradient centrifugation was performed for 40 hours at 100,200 xg at 0 ° C. After centrifugation, centrifuge tube with UV lamp at 365 nm
The band appearing under the chromosomal band was fractionated as a plasmid fraction by irradiating it with the ultraviolet light. Then, the fraction solution was treated with isopropanol to remove ethidium bromide, and then TE buffer (10 mM Tris-HCl, 1 mM
It was dialyzed against DTA, pH 7.5). This was used as a plasmid mixed solution.

In the obtained plasmid mixed solution, two circular plasmids are mixed, and as a result of analysis with restriction enzymes, it is the plasmid pTA5001 (A) shown in FIG. 1 and the plasmid pTA5001 (B) shown in FIG. Became clear.

That is, at least a 5-fold excess of restriction enzymes (EcoRI and SalI manufactured by Takara Shuzo Co., Ltd., XhoI manufactured by Bethesda Research Co., Ltd. was used) was added to the plasmid-mixed solution prepared above according to a conventional method. The reaction was carried out under optimum conditions of restriction enzyme. After the reaction, analysis was carried out by vertical agarose gel electrophoresis. That is, 1% agarose gel was used to run in a Tris acetate buffer (40 mM Tris, 20 mM acetic acid, 2 mM EDTA, pH 8.1). Then, the gel was dipped in a 1 μg / ml solution of endium bromide for staining. Irradiate this gel with ultraviolet light, determine the number of generated fragments, and from the migration distance of each fragment,
The molecular weight of each was calculated. The molecular weight was calculated based on the standard line created from the migration distance of each fragment of known molecular weight generated by Hind III cleavage of lambda phage DNA co-migrated on the same agarose.

PTA5001 (A) and pTA5001 (PTA5001 (A) and pTA5001 (from each fragment obtained by using various restriction enzymes alone and the fragment number and molecular weight of each fragment obtained by the treatment using a combination of two or more kinds of each restriction enzyme) The restriction enzyme cleavage maps shown in FIGS. 1 and 2 of B) were determined.

Example 3 Plasmid pTA5001 (A) and plasmid pTA50
Use of 01 (B) as a vector The plasmid mixed solution obtained in Example 2 (DNA amount 10 μm
In g), pACYC 177, which is an E. coli drug resistance vector.
(Kanamycin resistance and ampicillin resistance; Journal of
Plasmid pAC obtained from Escherichia coli C600 carrying Bacteriology, 134 (3), 1441- (1156,1978)
YC 177 (shown in FIG. 3; DNA amount: 2 μg) was added, and at least a 5-fold excess of the restriction enzyme XhoI was reacted under the optimum conditions by a standard method. After the reaction was completed, an equal amount of phenol was added and vigorously stirred. After inactivating the restriction enzyme, further extraction with ether was carried out sufficiently to remove the phenol, and then twice the amount of ethanol was added and the mixture was kept at -80 ° C for 1 hour.
After centrifuging at 00 × g for 5 minutes to precipitate the DNA, and further vacuum drying to remove ethanol, the precipitate was dissolved in water, and the reaction with T4 DNA ligase was carried out by a conventional method. The mixture was incubated at 0 ° C. for 2 hours, and ethanol precipitation and vacuum drying were carried out in the same manner as above, and the precipitate thus obtained was dissolved in 0.1 ml of TE buffer to obtain a chimera plasmid-containing solution.

Each chimeric plasmid is plasmid pACY
Contains C 177. However, there is an XhoI cleavage point at the kanamycin resistance site of plasmid pACYC 177, and because it is cleaved, kanamycin resistance does not develop, but ampicillin resistance only.

Example 4 Preparation of Chromosome Fragment Solution In the method described in Example 2, TE was used instead of TES buffer.
Prepare the lysate before adding 5M saline prepared using the buffer solution.
After heating at 65 ° C for 5 minutes and adding 3 times the volume of sterilized water,
After adding an equal volume of water-saturated phenol and stirring vigorously7,
Centrifuge at 000 xg for 10 minutes to separate the phenol, separate the upper aqueous solution layer of the phenol, and collect 2 of the collected aqueous solution.
7,000 × g after adding double amount of ethanol and leaving at -80 ℃ for 2 hours
Centrifugation was carried out for 30 minutes, the obtained precipitate was vacuum dried, dissolved in UC buffer, and DNA obtained by cesium chloride-ethidium bromide density gradient centrifugation in the same manner as in Example 2 again. The band was separated, ethidium bromide was removed, and then dialyzed against TE buffer to obtain a chromosome fragment solution.

Stain pairs and plasmid fragments were mixed in the chromosome fragment solution, and were appropriately diluted or concentrated or used as they were at the time of transformation.

Example 5 Transformation using chimeric plasmid 0.2 ml of the suspension of the DNA recipient cells obtained in Example 1 was prepared, 0.1 ml of the solution containing the chimeric plasmid obtained in Example 3 was added thereto, and the mixture was incubated at 0 ° C. The chimeric plasmid was directly introduced while gently stirring for 90 minutes.

A solution containing the chimeric plasmid-introduced bacterial cells obtained here is
After transfer to YPG medium (1) and shaking culture at 30 ° C. for 6 hours, it was cultured at 30 ° C. for 5 days on YPG medium (solid) supplemented with ampicillin 50γ / ml to obtain 9 strains of colonies. These were named 10-80S1-Al to -A9. Of these, 10-80S1-Al was added to ampicillin at 30γ / ml in YPG liquid medium at 30 ℃, 2
After shaking culture for 4 hours and separating the plasmid according to the method of Example 2, a plasmid having a slightly higher molecular weight than the plasmid pTA5001 (A) and the plasmid pTA5001 (B) was obtained. This plasmid is pTA5001 (A) among the previously introduced chimeric plasmids.
And pACYC 177 were recognized as a chimeric plasmid in which they were linked via the XhoI cleavage site. Also, Acetobacter aceti
From the fact that 10-80S1 does not have ampicillin resistance and 10-80S1-A1 has ampicillin resistance, it was confirmed that the chimeric plasmid was introduced and transformation was performed.

Similarly, at least 10-80S1-A2 to -A6 is pTA5001.
It was confirmed that a chimeric plasmid in which (B) and pACYC 177 were linked via a restriction enzyme XhoI cleavage site was introduced.

In addition, the chimeric plasmid possessed by 10-80S1-A1 ~ -A6
Introduced into -80S1 by the same method as above, 10-80S1-A
In each of the chimeric plasmids 1 to -A6, about 10 ⁵ transformants resistant to ampicillin were obtained in terms of the amount of 1 μg DNA.

Example 6 Transformation Using Chromosome Fragment Acetobacter aceti 10-80S1 (Aces obtained in Example 1
s, Eth ^-, Pro ^-, prepared DNA recipient cell suspension 0.2ml of Strr), Acetobacter aceti No.1 to this obtained in Example 4
Add 0.1 ml of 023 (Acer, Eth ⁺⁺ ) chromosome fragment solution,
Stir gently for 30 minutes at 40 ° C, then mix the reaction at 42 ° C.
The mixture was heat-shocked for 3 minutes and further gently stirred at 0 ° C. for 60 minutes to introduce DNA.

The reaction product is minimal medium (solid, streptomycin 50 μg /
The cells were cultured at 30 ° C. for 7 to 10 days, and grown colonies were detected.

As a control, the same buffer containing no DNA was added, treated in the same manner, and similarly cultured in the same medium, and grown colonies were detected. A control was also used when no DNA-accepting cells were added.

The results are shown in the table below.

As is clear from the above table, it can be seen that the chromosome fragment and / or the plasmid fragment was introduced into the solution to cause the transformation.

The resulting transformant not all requests proline Natsuta Acetobacter aceti ^{10-80S1 (Acess, Eth -, pro} +, s
trr).

Example 7 In Example 6, the case of performing heat shock at 42 ° C. for 3 minutes was compared with the case of not performing heat shock.

The comparison method is exactly the same as in Example 4.

The results are shown in the table below.

From this table, under this condition, the influence of heat shock was hardly recognized.

Example 8 Transformation was performed in the same manner as in Example 6 except that the salt solution of the suspension of effective cells was replaced with 100 mM of chlorides of lithium, sodium, potassium, rubidium, cesium, magnesium and calcium, and colonies were formed. Detected.

The results are shown in the table below.

Various alkali metals and alkaline earth metals are effective,
Among them, lithium, rubidium, magnesium and calcium showed better effects.

Example 9 A suspension of effective cells was prepared by adding a salt solution to NaCl, KCl, MgCl ₂ , CaCl _2.
The same transformation as in Example 6 was carried out except that the concentration was changed, and colonies were detected.

The result is shown in FIG.

From Fig. 4, it is 10 to 50 mM for magnesium, 50 to 100 mM for calcium, and 100 for sodium and potassium.
It turns out that mM or more is preferable.

Example 10 Transformation was performed in the same manner as in Example 6 except that a 100 mM calcium chloride solution having a pH of 6.0 to 9.5 was used as a salt solution of a suspension of effective cells, and colonies were detected.

The result is shown in FIG.

From FIG. 5, it can be seen that the pH of the 100 mM calcium chloride solution has high activity on the acidic side.

Example 11 Transformation was performed in the same manner as in Example 6 except that effective cells were collected from the culture solution at each stage of the entire culture period, and colonies were detected.

The result is shown in FIG.

From FIG. 6, it can be seen that the cells collected from the mid-logarithmic growth phase to the early resting phase have an excellent transformation rate.

[Brief description of drawings]

FIG. 1 shows the restriction enzyme cleavage map of plasmid pTA5001 (A), FIG. 2 shows the restriction enzyme cleavage map of plasmid pTA5001 (B), and FIG. 3 shows the restriction enzyme cleavage map of plasmid pACYC177. E ... EcoRI cleavage site, S ... SalI cleavage site, X ... XhoI cleavage site, Km ... Kanamycin resistance gene, Am ... Ampicillin resistance gene. FIG. 4 is a graph showing the relationship between the concentration change of various salts and the transformation rate in Example 9, and FIG.
6 is a graph showing the relationship between the change in pH of the calcium chloride solution and the transformation rate in FIG. 6, and FIG. 6 is a graph showing the relationship between the collection time of effective cells and the transformation rate in Example 11.

Claims (1)

[Claims] 1. Use of an acetic acid bacterium collected from the mid-logarithmic growth phase to the early stationary phase in a solution containing at least 10 to 50 mM magnesium and / or 50 to 100 mM calcium and having a pH of 6 to 7. A method for transforming an acetic acid bacterium, which comprises introducing a plasmid and / or a plasmid fragment and / or a chromosomal fragment into an acetic acid bacterium.