JPS609489A - Transformation of acetic acid bacteria - Google Patents

Abstract

PURPOSE:To transform acetic acid bacteria, by introducing plasmid, its fragment, and a chromosome fragment to acetic acid bacteria in a solution containing one or more of an alkali metal, and an alkaline earth metal. CONSTITUTION:In a solution containing one or more selected from an alkali metal and an alkaline earth metal, plasmid, its fragment, and a chromosome fragment are introduced into acetic acid bacteria collected in a range from middle phase of logarithmic multiplication to the resting phase, so that acetic acid bacteria are transformed. The solution of an alkali metal or an alkaline earth metal contains preferably one metal selected from lithium, rubidium, magnesium, and calcium, especially preferably 1-50mM magnesium and/or 50-100mM calcium. The solution has 6-7pH.

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 acetic acid bacteria by introducing gene-related DNA.

Conventionally, the transformation of acetic acid bacteria has been carried out using a method using conjugative transfer (Journal of Bacteriology).
y, 145 (11, 358468, 1981) are completely unknown.

Based on the idea that if acetic acid bacteria could be genetically transformed, a better acetic acid bacterium could be obtained.The inventors conducted an iJt study and found that all plasmids, plasmid fragments, and chromosomal fragments contained acetic acid. By confirming the introduction into bacteria, we were able to complete the present invention.

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

The DNA used for transformation in the present invention is
Any plasmid, plasmid fragment, chromosome fragment, etc. may be used. As the plasmid, any plasmid isolated from the bacterial body or various chimeric plasmids derived from the plasmid can be used. Furthermore, as the plasmid fragments and chromosome fragments, plasmid fragments generated by bacteriolysis or mechanical treatment can be used.

In addition, as the acetic acid bacteria to be transformed in the present invention, all bacteria of the genus Acetobacter and Gluconobacter are targeted;
is shown.

Acetobacter acetiN [11023 was isolated from acetic acid fermented mash, and was produced using plasmid pTA5, which will be described later.
Contains 001 prisoner and plasmid pTA5001(B)! , has been deposited as FERM P-7122 at the Mana Institute of Technology.

Acetobacter aceti NcL1026 matches well with the description of the bacteriological properties of Acetobacter aceti in Parsi's 8th edition in terms of Dutch properties, and is further characterized by having acetic acid resistance and ethanol oxidation ability.
cter aceti Nn 1023 (Acer.

Eth++) may be displayed.

Acetobacter aceti Nn1021 is usually cultured, for example, in the YPG medium described below, and for the detection of transformed strains, a drug such as an antibiotic is added to the YPG medium at an appropriate concentration, such as ampicillin at 50 r/min. ml of the culture medium or the minimal medium described below.

(YPG medium) Yeast extract 0.5% Polypeptone 0.2% Glucose 3.0% Agar (in the case of solid medium) 2.0% T111=65 (Minimum medium) K2HPO40, former Ti KH2PO40,05% MgSO4・7H ,,,O [1,025% KCA O,
01% caCA2-2H200,01% FeC1,=5" 6H200,0005%Na-Glutamate 0.4Glucose 6.0% Agar (in case of solid medium) 2.0% pH=6.5 Acetobacter acetiNnl [ ] 23 is a YPG liquid medium, cultured with shaking at 60 ° C. for 24 to 66 hours, and the culture solution is centrifuged to collect the bacteria. The bacterial cells are thoroughly washed with a buffer solution, and PWPIA is carried out in the buffer solution. This includes lysozyme5
and lysed. Polyethylene glycol is added to the lysate, and after standing still, it is centrifuged to obtain a precipitate 1. This precipitate is dissolved in a buffer solution, ethidium bromide is added, and cesium chloride is further added to reduce the density to 1.57. Perform density gradient centrifugation. After centrifugation, the centrifuge tube is irradiated with ultraviolet light at 565 nm using an ultraviolet lamp, and the band appearing below the chromosome band is fractionated.

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

As a result of analysis using restriction enzymes, it was revealed for the first time that the two mixed plasmids were a mixture of 24Iji plasmids having approximately the same molecular weight.

Plasmid pTA5.001 (the molecular weight of Al is 23.5
Kb, and the restriction enzyme cleavage map is shown in FIG.

In addition, the molecular weight of plasmid pTA 5001 (Bl is 22.5 Kb, and the restriction enzyme cleavage map is shown in FIG. 2).

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

E: EcoRI: Escherichia c
Restriction enzyme S from Oli Y13 source: 5alI: Streptomyces alb
us G source restriction enzyme X: Xho I: Xhanthomonas
holcicola source restriction enzyme plasmid pTA5001 (A) and plasmid pTA
5001 (Bl is only one by Xho I)
It is very unique in that it is cleaved only at one site, and it is extremely easy to introduce other plasmid fragments or chromosomal fragments into this cleavage site to create chimeric plasmids.

Plasmid pTA5001(A) and plasmid pTA
5001 (BTU) is used alone or as a mixture as an acetic acid bacterium vector.

That is, a chimeric plasmid in which a cilasmid fragment or a chromosome fragment is introduced into cilasmid pTA 5001 (A) and/or plasmid pTA 50 [11 (B)] can be effectively used for the transformation of the present invention.

In addition, the parent strain Acetobacter acetiyo 1026,
Acetobacter aceti 10-8 (Acer.

Eth++, Pro"'), and Acetobacter, which is a strain with reduced acetic acid resistance and ethanol oxidation ability obtained through natural mutation, Acess, Eth-), and streptomycin-resistant (Strr).
Transformation can be carried out as donor.

In this case, the preparation of chromosome fragments is basically carried out according to the plasmid preparation method, but the parent strain Acetobacter
The buffer solution in which the cells of acetiNn1023 are suspended does not contain sucrose, and after adding a surfactant, it is heated at 65°C for 5 minutes without adding salt, followed by phenol treatment, ethanol precipitation, and vacuum drying. The resulting DNA is again subjected to cesium chloride-ethidium bromide density gradient ultracentrifugation in the same manner as in the plasmid preparation method, and the resulting DNA band is fractionated.

Here, the DNA band identified (4I) contains chromosome fragments and/or cilasmid fragments, and if this is added to the mutant strain-containing solution and subjected to DNA-specific treatment, chromosome fragments and/or plasmid fragments can be detected. Transformation occurs when the IJr fragment enters the bacterial cell and the fragment is integrated or recombined within the mutant strain.In this way, the proline synthesis genes of the parent strain can be easily transferred to the mutant strain. It is transferred to the plant and the traits are transformed.

Transformation of acetic acid bacteria can thus take place in solution with plasmids and/or plasmid fragments and/or chromosomal fragments.

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

Calcium chloride is used at a concentration of 50 to 100 mM, magnesium chloride is used at a concentration of about 10 to 50 mM, and P1 is preferably made slightly acidic at a pH of 6 to 7, and the bacterial cells to be transformed are immersed therein.

Furthermore, it is preferable to use cells to be transformed that have been collected from the middle of the logarithmic growth phase to the early stationary phase.

A plasmid and/or a plasmid fragment and/or a chromosome fragment is added to the solution-treated h body while it is immersed in the solution.

Introduction of the DNA is completed by gentle stirring at O''C for about 1.5 hours.

Microbial cells that had undergone DNA introduction treatment were added with l-1' minimal medium (solid) or drugs such as antibiotics at an appropriate concentration, for example ampicillin at a concentration of 5oγ/ml. 60°C15 in YPG medium (solid)
Culture for ~10 days and isolate colonies.

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

During transformation, in the case of a plasmid or chimeric plasmid, it is taken into the cell and that's it! However, in the case of plasmid fragments or chromosomal fragments, recombination occurs within the cells after they are taken into cells, and the expression of the trait is thought to occur.

Next, examples of the present invention will be shown.

Example I Preparation of DNA receptor cells Acetobacter acetito h1025 (Acet.

Eth", pro+, str'), FERM
1) Acetobacter aceti 1O-80 isolated from -7122 by NTG mutation treatment and natural mutation treatment
S, 1 (AcesS, Eth"-, pro-, 5tr
10011YPG containing r) in a 500-Sakaro flask.
It was inoculated into a liquid medium and cultured with shaking at 30°C for 20 hours.

The culture solution was heated to 6,000X at 0°C. Centrifuge for 10 minutes to collect bacteria. The bacterial cells were treated with 100 mM Na (J and 5
Wash twice with 0.5 volumes of 5 mM) Lis-HCl buffer (Pt17,6) containing mM MgCJ2. Centrifuge again at 0°C and 6,000X for 5 minutes to collect bacteria.

This bacterial cell was treated with 0.4 times the volume of Ca CA2 solution (100
mM CaC, #2.250 mM KCL, 5 mM
MgCA2.5mM Tris-HCA, pl(7,
6) is added and centrifuged at 0'O and 6.0OOX for 5 minutes to collect bacteria.

A 0.004-fold volume of the above Ca CZ2 solution was cooled on the bacterial cells to obtain a suspension of DNA-receiving bacterial cells.

Example 2 Isolation of plasmid pTA5001(A) and plasmid pTA5001(Bl) Acetobacter acetiNnl 023.
The cells were inoculated into Qml YPGm soil and cultured overnight at 30°C with shaking.

The cells were subcultured at 1% in YPG medium 4i, which seems to be extractable, and cultured with shaking at 60°C for 36 hours.

After harvesting, add TE buffer (20mM EDTA, 50mM
The two-mouthed body was washed with Tris-HCl (pl-18,0).

7 ml of TBS buffer (
Add 50mM Tris-HCl, 20mM EDTA, 25% sucrose, pH 8.0) to suspend the bacterial cells, and add 4ml of lysozyme i (0.25MIJS-HCl, 2% lysozyme,
(pH 8.0) was further added, and the mixture was allowed to stand at 0.0 for 5 minutes. Next, 0.25 M EDTA solution (Pt48.0) was added four times, and after standing at 0°C for 5 minutes, the mixture was reacted at 37°C for 20 minutes. After the reaction, 5 trtt of 10% sodium lauryl sulfate was added, and after standing at 37°C for 20 minutes, 5M saline solution of 5- was added, and the mixture was left standing at O''O overnight.

Centrifugation was performed at 48.200Xr for 60 minutes, and the supernatant was collected. Next, polyethylene glycol 6.000 was added to this supernatant to a final concentration of 10%, left to stand at 4°C overnight, and then centrifuged at 3,000Xf for 10 minutes.
A precipitate was obtained. This precipitate was added to 7 ml of UC buffer (5 ml).
0mM Tris-HCl, 5+nM EDTA.

After dissolving in 50 mM Na (J, Pll y, 8), ethidium bromide was added to give a final concentration of 500 μf/ml, and cesium chloride was further added to adjust the density to 1.57. This solution was heated at 15°C for 10
Close gradient centrifugation was performed at 0.200Xf for 40 hours. After centrifugation, incubate the centrifuge tube with an ultraviolet lamp at 565°C.
By irradiating with nanometer ultraviolet light, a band appearing below the chromosome band was separated as a plasmid fraction. Next, the two fractions were treated with isopronominol to remove ethidium bromide, and then TE buffer (10mM
) Lis-HCl, 1mM El) TA, pH 7,5). This was used as a plasmid mixed solution.

Two circular plasmids were mixed in the obtained plasmid mixed solution, and as a result of analysis using restriction enzymes, it was confirmed that they were plasmid pTA 5001 shown in Figure 1 and plasmid pTA 5001<Bl shown in Figure 2. It became clear.

That is, with respect to the plasmid mixed solution prepared above, at least a 5-fold excess of restriction enzymes (Eco® and 5a) was added.
II was used from Takara Shuzo Co., Ltd., and Xhol 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), 20mM acetic acid,
2mM EDTA.

, i+s, 1). The gel was then washed with 1 μJ of ethidium bromide! - Stained by soaking in 7ml solution.

This gel was exposed to ultraviolet light (tf<(), 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 a known valley fragment of the molecule M produced by HindIII digestion of lambda phage DNA that was electrophoresed simultaneously on the same agarose.

Based on the number of lli fragments and molecular weight of each fragment obtained by using each restriction enzyme alone and each fragment obtained by treatment using a combination of two or more of each restriction enzyme, pTA500
Figures 1 and 2 of 1 prisoner and pTA 50 o 1 (B)
The restriction enzyme cleavage map shown in the figure was determined.

Example 3 Plasmid pTA5001 prisoner and plasmid p
TA 5001 (use of Bl as a vector) Plasmid mixed solution obtained in Example 2 (DNA amount: 10
μ? ) contains pACYC, an E. coli drug-resistant vector.
177 (Kanamycin resistance and ampicillin resistance; J
our own of f3acteriology,
134(3), 1141-1156. Bscherichia coli C60 with 1978)
Add the plasmid I) ACYC177 (shown in Figure 6, DNA amount 2μ?) obtained from 0) and react with at least a 5-fold excess of the restriction enzyme XboI by a conventional method under optimal conditions. After completing the two reactions, An equal amount of phenol is vigorously stirred,
After inactivating the restriction enzymes by stirring, the mixture was further extracted with ether to remove phenol, and twice the amount of ethanol was added and kept at -80'C for 1 hour.15)
The DNA was precipitated by centrifugation in OOOXf for 5 minutes, and the ethanol was removed by vacuum drying.Then, the precipitate was dissolved in water, and the reaction with T4 DNA ligase was carried out at 21°C for 2 min. The precipitate obtained by performing ethanol precipitation and vacuum drying in the same manner as above was dissolved in 1 ml of TB buffer solution to obtain a solution containing the chimeric plasmid.

Each chimeric plasmid is a plasmid pA.
Contains CYC177. However, plasmid pA
There is an XhoI cleavage point in the kanamycin resistance site of CYC177, and since this point is cleaved, kanamycin resistance is not expressed, and only ampicillin resistance is expressed.

Example 4 Preparation of chromosome fragment solution The lysis solution prepared by using the T]13 buffer instead of the Tgs buffer in the method shown in Example 2 before adding 5M saline was heated at 65°C for 5 minutes. Then, add 6 ounces of water.
After adding twice the amount, add the same amount of water-saturated phenol to 11
Stir vigorously. 7. D 00x yCentrifuge for 10 minutes to separate phenol, separate the upper layer of phenol (aqueous solution), add twice the volume of ethanol to the separated aqueous solution, and leave for -80"02 hourst&7+00・[]:
) After centrifuging for 30 minutes and vacuum drying the resulting precipitate, the resulting DNA band was dissolved in UC relaxation solution, and after removing ethidium bromide, it was dialyzed against TE buffer. A chromosome fragment solution was obtained by doing this.

The chromosome fragment solution contains chromosome fragments and plasmid fragments, and during transformation, dilution or Il.
i! It was used either closed or as is.

Example 5 Transformation Example 1 using chimeric plasmid
Prepare 0.2 ml of the DNA acceptor suspension obtained in Example 3, and add the chimera plasmid-containing solution U obtained in Example 3.
Direct introduction of the chimeric plasmid was carried out by adding .ltoreq.rnt and gently stirring at 0.degree. C. for 90 minutes.

The solution containing the chimera plasmid-introduced m body obtained here was
ml of YPG medium and cultured at 60°C for 6 hours.
The cells were cultured on a medium (solid) at 60° C. for 5 days, and 9 colonies were obtained. These were named 1O-8081-A1--A9. Of these, 10-8081-AI and ampicillin at 60 r/m! 50"01 with added YPG liquid medium
After 4 hours of cinnabar culture for 24 hours, the plasmid was isolated according to the method of Example 2. As a result, plasmid pTA5001 (
In addition to a mixture of Al and plasmid pTA 5001 (B), a plasmid with a slightly larger molecular weight than these was obtained. This plasmid was recognized as a chimeric plasmid in which pTA5001(A) and pAcYc 177 were ligated via the XhoI cleavage site among the previously introduced chimeric plasmids. Also, Acetobacter aceti 10-80
81 does not have ampicillin resistance, but 10-8081-
Since AI has ampicillin resistance, it was confirmed that a chimeric plasmid was introduced and transformation was performed.

Similarly, at least 10-8081-A2--A6
It was confirmed that a chimeric plasmid in which pTA 50 D 1 (Bl and pAcYcl 77 were ligated via the restriction enzyme XhoI cleavage site) had been introduced.

In addition, when the chimera plasmid of 10-8081-Al--A was introduced into io-5osi again in the same manner as described above, each chimera plasmid of 10-8081-Al--A had 1μ)D. Approximately 105 ampicillin-resistant transformants were obtained based on the amount of NA.

Example 6 Transformation using chromosomal fragments Acetobacter Q aceti 10-8081 (AceSS) obtained in Example 1
, Bth-, Pro-, 5trr) was prepared, and 0.2 ml of DNA receptor cell suspension of Acetobacter aceti Nu 1023 (Acer) obtained in Example 4 was prepared.

Add 0.11 chromosomal fragment solution of Eth”) and heat at 0°C.
Stir gently for 60 minutes at
The mixture was subjected to heat shock at 0.degree. C. for 6 minutes, and then gently stirred at 0.degree. C. for 60 minutes to introduce DNA.

The reaction material was minimal medium (solid, streptomycin 5011
f/ml addition) for 30 days for 0.7 to 10 days, and grown colonies were detected.

As a control, the same buffer solution containing no DNA was added, the cells were treated in the same way, cultured in the same medium, and grown colonies were detected. A case in which no DNA receptor was added was also used as a control.

The results are shown in the following table.

As is clear from the table above, it can be seen that the chromosome fragment and/or cilasmid fragment were introduced in the solution, and transformation occurred.

All of the obtained transformed strains were A. aceti1o-ansi (A, ce
s8. Rth, pro”, 5irr).

Example 7 A comparison was made between the case in which heat shock was performed at 42° C. for 6 minutes in Example 6 and the case in which heat shock was not performed.

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

The results are shown in the following table.

From this table, under these conditions, almost no effect of heat shock was observed.

Example 8 Transformation was carried out in the same manner as in Example 6, except that the salt solution of the active cell suspension was replaced with 100 mM lithium, sodium, potassium, rubidium, cesium, magnesium, and calcium chlorides, and the colonies were transformed. Detected.

The results are shown in the following table.

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

Example 9 The effective cell concentration of γ-shu liquid was adjusted to Na('J, KC
Transformation was performed in the same manner as in Example 6 except that the concentrations of J, Mg CA 2 and Ca CA 2 were changed, and colonies were detected.

The results are shown in FIG.

From Figure 4, in the case of magnesium, it is 10-50mM.

It can be seen that in the case of calcium, 50 to 100mM is preferable, and in the case of sodium and potassium, 100mM or more is preferable.

Example 10 Pll of active cell suspension in salt solution 6. O~9.5
Transformation was performed in the same manner as in Example 6, except that a 100 mM calcium chloride solution was used, and colonies were detected.

The results are 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 carried out in the same manner as in Example 6, except that effective cells were collected from the culture solution at each stage of the entire cultivation period, and colonies were detected.

The results are shown in Figure dl)6.

From FIG. 6, it can be seen that bacteria collected from the middle of the logarithmic growth phase to the early stationary phase have an excellent transformation rate.

[Brief explanation of drawings]

Figure 1 shows the restriction enzyme cleavage map of plasmid pTA 5001CAl, and Figure 2 shows the restriction enzyme cleavage map of plasmid pTA 5001CAl.
Figure 3 shows the restriction enzyme cleavage map of plasmid pAcYc177. E...EcoRI cleavage site, S...5alH
Cutting site by X... Cutting site by XhoI, K
m...Kanamycin resistance gene, Am...Ampicillin resistance gene. FIG. 4 is a graph showing the relationship between changes in the concentration of various salts and transformation rate in Example 9, and FIG.
31110 is a graph showing the relationship between the change in pH of the calcium chloride solution and the conversion rate of 2D, and FIG.
Collection timing and shape of effective cells (1
1 is a graph showing the ratio of 1' to 1 conversion rate. Agent: Patent attorney: 1) Parents and men hate each other. Hateful and angry book Sara・Suk @ book legal fat dumb

Claims (1)

[Claims] (1) Introducing a plasmid and/or a plasmid fragment and/or a chromosome fragment into an acetic acid bacterium in a solution containing at least one metal selected from alkali metals and alkaline earth metals. A method for transforming acetic acid bacteria characterized by: (2) The method for transforming acetic acid bacteria according to claim 1, wherein the solution contains one selected from lithium, rubidium, magnesium, and calcium. (5) The method for transforming acetic acid bacteria according to claim 1, wherein the solution contains at least 10 to 50 mM magnesium and/or 50 to 100 mM calcium. (4) The method for transforming acetic acid bacteria according to claim 1, wherein the Pti of the solution is 6 to 7. (5) The method for transforming acetic acid bacteria according to claim 1, characterized in that the acetic acid bacteria are collected from a mid-logarithmic growth phase to an early stationary phase.