JPH08107784A - Transformation of alkalophilic bacterium of genus bacillus - Google Patents

Abstract

PURPOSE: To provide a method for transforming an alkalophilic bacterium of the genus Bacillus at a high conversion rate. CONSTITUTION: (1) A microbial cell of an alkalophilic bacterium of the genus Bacillus is cultured in a liquid culture medium containing 2-8vol.% amino acid mixture solution and (2) the microbial cell is suspended in a buffer solution for preparing a protoplast containing 4-16vol.% amino acid mixture solution, 0.5-2wt.% fish meat extract and 0.09-0.4wt.% potassium sulfate to carry out the lysozyme treatment to prepare the protoplast. (3) An exogenote and a polyethylene glycol solution are then added to the protoplast suspension to thereby make the protoplast take up the exogenote thereinto. (4) The protoplast is then suspended in a buffer solution for preparing the protoplast similar to that described above and cultured to afford a suspension culture solution, which is subsequently applied to a solid culture medium containing 0.5-8vol.% amino acid mixture solution and cultured.

Description

Detailed Description of the Invention

[0001]

The present invention relates to an alkaline bacillus (Bacillus) having an optimum pH for growth in the alkaline region.
s) It relates to a method for transforming an alkalophilic Bacillus bacterium for introducing a foreign gene into the cells of a genus bacterium.

[0002]

2. Description of the Related Art As a host strain for expressing a heterologous gene by genetic engineering technology, it is necessary to select a strain having high protein productivity and reliable safety. And is attracting attention as a useful host strain. Therefore, a method for transforming a bacterium of the genus Bacillus with a foreign gene has been developed for a long time, and the competent cell method [G. A. Wils
on & K. F. Bott, J .; Bacterio
l. , 95, p1439-1449. (1968)],
Protoplast method [S. Chang & S. N. Co
hen, Mol. Gen. Genet. , 168, p1
11-115. (1979)] and the electroporation method [J. B. Luchansky et. al. ,
Molecular Microbiology, 2,
p637-6469. (1983)] and the like are known. In addition, recently, for the alkalophilic Bacillus bacterium, the protoplast transformation method [R. Aono et. a
l. , Biosci. Biotech. Bioche
m. , 57, p1597-1598. (1993)] has been reported.

[0003]

However, according to these methods, even in the case of Bacillus bacteria, the frequency of introduction of a foreign gene is extremely different depending on the species and strain, and in some cases, no transformation occurs. Sometimes. In fact, the present inventors have found that by these methods, Bacillus sp.
lus sp. ) KSM-635 [Microtechnology Research Institute, Article 148
No. 5 (FERM BP-1485)] strain was attempted, but no transformation occurred.

Therefore, an object of the present invention is to provide a novel method for introducing a foreign gene into the cells of an alkalophilic Bacillus bacterium.

[0005]

In such a situation, the present inventor has diligently studied the treatment conditions in each operation step of the protoplast transformation method of an alkaliphilic Bacillus bacterium, and as a result, The inventors have found that transformants of alkalophilic Bacillus bacteria can be obtained at high frequencies by improving the culture method, the protoplast preparation buffer, and the regeneration medium, and completed the present invention.

That is, the present invention provides the following operations (1)-
The present invention provides a method for transforming an alkalophilic Bacillus bacterium, which comprises (4). (1) Amino acid mixture solution of 2 to 8% by volume and final concentration of 0.
PH 7-1 containing sucrose in an amount of 2-1M
Culturing the cells of the alkalophilic Bacillus bacterium in a liquid medium of 0; (2) Amino acid mixture 4 to 16% by volume, fish meat extract 0.
25-2% by weight, potassium sulfate 0.09-0.4% by weight
And the bacterial cells obtained in (1) were suspended in a protoplast preparation buffer having a pH of 7 to 10 containing sucrose in an amount such that the final concentration was 0.25 to 1 M, and the final concentration was suspended in the suspension. To prepare protoplasts by adding lysozyme in an amount of 50 to 200 μg / ml; (3) Add a foreign gene and a polyethylene glycol solution in an amount of 5 to 20% by weight to the protoplast suspension. To incorporate the foreign gene into the protoplasts; (4) The protoplasts are suspended and cultured in the same protoplast preparation buffer as used in step (2), and the resulting suspension culture is , Amino acid mixture 0.5-8
The culture is carried out by coating on a solid medium having a pH of 7 to 10 containing a volume% and a final concentration of 0.5 to 0.9 M sodium succinate.

The method for transforming an alkalophilic Bacillus bacterium according to the present invention comprises the above steps (1) to (4), that is, (1).
It consists of culturing cells for preparing protoplasts, (2) converting the cells into protoplasts, (3) addition of a foreign gene and a protoplast aggregating agent, and (4) regeneration into normal cells. Here, examples of the alkalophilic Bacillus bacterium include Bacillus sp.
KSM-635 (FERM BP-1485) strain and the like can be mentioned.

First, the culture of the protoplast-preparing cells in the operation (1) will be described. In the liquid medium used for this culture, 2 to 8% by volume of the amino acid mixture is added,
The regeneration rate from protoplasts can be increased by adding 0.2 to 1 M sucrose to increase the osmotic pressure. The amino acid mixture used here is preferably one having a composition shown in Table 2 below.

[0009]

[Table 2]

In this liquid medium, fish meat extract 0.25-
2% by weight, potassium monohydrogen phosphate 0.05 to 1% by weight,
It is preferable to add 0.025 to 0.5% by weight of yeast extract, 0.045 to 1% by weight of potassium sulfate, and 0.25 to 2% by weight of glucose from the viewpoint of increasing the regeneration rate from protoplasts.

Since the strain to be used in this liquid medium is alkalophilic, it is necessary to adjust the pH to 7 to 10 using an alkaline compound such as sodium carbonate or sodium hydroxide. Can be 0.15 to 1.2% by weight.

Cultivation of protoplast cells is carried out at a temperature of 30.
From 40 to 40 ° C., preferably 37 ° C., shaking culture is performed, and when the absorbance of the culture solution at the absorbance (OD ₆₀₀ ) at a wavelength of 600 nm reaches 0.05 to 0.2, it is necessary to collect the bacteria from the protoplasts. It is preferable in that the regeneration rate can be increased. The method of collecting the bacteria is not particularly limited, and for example, 10 to 200 ml of the culture solution is centrifuged (6,0 ml).
It is possible to use a method of collecting the bacterial cells by carrying out the treatment at 00 xg for 10 minutes at 4 ° C.

In the protoplast preparation buffer used in the protoplast formation treatment of the bacterial cells in the operation (2), the amino acid mixture solution is 4 to 16% by volume and the fish meat extract is 0.5.
The transformation rate can be increased by adding ˜2% by weight and 0.09 to 0.4% by weight of potassium sulfate. In addition, it is necessary to add sucrose in an amount of 0.25 to 1 M to increase the osmotic pressure of the buffer solution in order to prevent rupture of protoplasts and obtain a high regeneration rate.

The composition of the amino acid mixture used here is preferably as shown in Table 2 above. In addition, this buffer contains phosphate-
0.1 to 2 wt% potassium hydrogen, yeast extract 0.05 to
1% by weight, glucose 0.5-4% by weight, magnesium chloride 0.01-2% by weight and bovine serum albumin (BS
A) It is preferable to add 0.05 to 0.5% by weight from the viewpoint of stabilizing protoplasts.

The pH of this buffer solution in which the cells to be transformed are alkalophilic needs to be 7 to 10.
Can be adjusted in the same manner as the operation (1).

To prepare protoplasts, first, lysozyme treatment is carried out for the purpose of removing the cell wall of bacterial cells. In this treatment, the cells were suspended in the above-mentioned protoplast preparation buffer solution, and the final concentration was 50 to 200 μg / ml.
This is done by adding an amount of lysozyme. From the viewpoint that a high regeneration rate can be obtained, this treatment is preferably carried out at 20 to 42 ° C for 10 to 100 minutes after addition of lysozyme, and particularly at 30 to 37 ° C for 20 to 4 minutes.
It is preferable to hold for 0 minutes. After lysozyme treatment, protoplasts are collected and resuspended in the same protoplast preparation buffer as above. Here, the method for collecting the bacteria is not particularly limited, and for example, centrifugation (1,000 × g,
For example, a method of collecting protoplasts by performing 15 minutes at 4 ° C.) can be used.

The contact reaction between the protoplast and the foreign gene in the operation (3) is carried out by adding the foreign gene to the protoplast suspension and further adding polyethylene glycol (PEG) solution (solvent) in an amount such that the final concentration is 5 to 20% by weight. Can be used as water). Specifically, 10 to 4 equivalent to the protoplast suspension
This is done by adding 0% by weight PEG solution. P
It is preferable to use EG having an average molecular weight of 1,000 to 8,000, since the plasmid can be introduced at high frequency. In addition, it is desirable that the PEG solution contains 0.25 to 1 M sucrose from the viewpoint of preventing rupture of protoplasts and increasing the regeneration rate.

The contact reaction between protoplasts and the foreign gene is preferably carried out for 30 seconds to 10 minutes. In addition, it is preferable to collect the protoplast precipitate immediately after the reaction, for example, adding 5 to 10 times the volume of the reaction solution as described above to the protoplast preparation buffer to stop the contact reaction, and immediately centrifuge (1 1,000 xg, 15 minutes, 4
C.) can be used.

To regenerate normal cells in the operation (4), first, the cells are suspended in the same protoplast preparation buffer as used in the operation (2) and cultured. The culture is preferably shaking culture, for example, 100 to 15 at 20 to 40 ° C.
It is preferable to carry out at a rotation speed of 0 rpm for 1 to 5 hours. Next, the resulting protoplast suspension was treated with a composition for solid medium containing 0.5 to 8% by volume of an amino acid mixture to give a pH of 7 to 10.
And apply to a regeneration medium (solid medium) prepared by adding sodium succinate in an amount to give a final concentration of 0.5 to 0.9 M, and perform culture to regenerate normal cells. . Here, the amino acid mixture preferably has the composition shown in Table 2 above.

In the present invention, a high transformation rate can be obtained by incorporating the above-mentioned specific amino acid mixture in the regeneration medium. A higher transformation rate can be obtained by adding 0.02 to 1% by weight of potassium sulfate and 0.05 to 2% by weight of gelatin.

Further, the regeneration medium has a high concentration (0.5 to 0.9).
A high regeneration rate (5 × 10 ⁶ to 1 × 10 ⁸ pieces / ml) can be obtained by adding M) sodium succinate. That is, the conventional protoplast regeneration method of Bacillus subtilis [S. Ch
ang & S.A. N. Cohen, Mol. Gen. G
enet. , 168, p111-115. (197
9)], 0.3M sodium succinate was added to the alkalophilic Bacillus bacterium KSM-635 (FERM).
BP-1485) had a very low level of protoplast regeneration (5 × 10 ³ cells / ml),
An extremely high regeneration rate can be obtained by adding 0.5-0.9 M sodium succinate.

The regeneration medium used in the present invention further comprises 0.1 to 0.6% by weight of fish meat extract, and 0.
02-0.2 wt%, yeast extract 0.01-0.1 wt%, glucose 0.2-2 wt%, magnesium chloride 0.05-1 wt%, bovine serum albumin (BSA) 0.
It is preferable to add 005 to 0.1% by weight and 0.5 to 2% by weight of agar in order to further increase the regeneration rate.

Since the cells used in this regeneration medium are alkalophilic, it is necessary to adjust the pH to 7-10 using the same alkaline compound as in step (1). When sodium carbonate is used, the amount added is Is 0.1 to 0.6% by weight
Can be

Regeneration of protoplasts can be carried out by applying a protoplast suspension to the above medium and culturing at 30 to 50 ° C. for 2 to 14 days.
A transformant can be obtained by selecting a transformant from colonies of the regenerant that have appeared by such culture. This selection method is not particularly limited, and for example, plasmid DNA containing a drug resistance gene
In the case of carrying out transformation with, for example, it is possible to allow only the transformant to regenerate by containing an appropriate concentration of the drug in the regeneration medium.

Such agents include kanamycin,
Tetracycline, chloramphenicol, erythromycin and the like can be mentioned.

By the transformation method of the present invention, for example, Bacillus sp. KSM-635 (FERM BP-14).
85) When the strain is used as a host and transformed with the plasmid pUB110, 1 × 10 ⁴ to 1 × 10 ⁶ cells / μg D
Transformants can be obtained at a frequency of NA or higher. In addition, since the alkalophilic Bacillus bacterium is used as a bacterium for producing a detergent enzyme and has a high protein-producing ability,
By applying the transformation method of the present invention, various useful proteins can be produced at a high production rate.

[0027]

According to the present invention, it is possible to transform an alkalophilic Bacillus bacterium at a high conversion rate.

[0028]

The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

Example 1 An alkaline cellulase-producing bacterium was transformed with the plasmid pUB110. Unless otherwise specified, “%” in each table means “% by weight”.

(1) Cultivation of cells for protoplast formation: Bacillus sp. KSM-635 (FERM BP-1)
485) was applied to a solid medium (Table 3) and the strain was applied at 30 ° C for 24
Cultured for hours. 1 platinum loop of fungus body in liquid medium (Table 4) 1
A test tube containing 0 ml was inoculated and shake culture was performed at 30 ° C. After 14 hours, 1 ml of the culture medium was added to 100 ml of liquid medium (Table 4).
A Sakaguchi flask containing 100 ml was inoculated and shake-cultured at 37 ° C. When the turbidity (OD ₆₀₀ ) of the culture reached 0.1, the cells were collected by centrifugation (6,000 × g, 10 minutes, 4 ° C.), and 2 ml of the protoplast preparation buffer (Table 5) was collected. ).

[0031]

[Table 3]

[0032]

[Table 4]

[0033]

[Table 5]

(2) Protoplasting process: Operation (1)
The final concentration of the protoplast suspension obtained in
Lysozyme was added so that the concentration would be g / ml, and the mixture was kept warm at 30 ° C for 30 minutes. Prospect formation rate was 99 by microscopy
% After confirming that
Protoplasts were collected by 0xg, 15 min, 4 ° C) and suspended in 1 ml of protoplast preparation buffer.

(3) Contact of protoplasts with DNA: 1 μg (1) in 0.5 ml of the protoplast suspension of the operation (2)
0 μl) of the plasmid pUB110 DNA solution and 0.5 ml of a 30 wt% PEG solution (Table 6) were added, gently stirred, and allowed to stand at room temperature for 1 minute. After this, 5 ml of protoplast buffer was added and mixed, and then the protoplast suspension was centrifuged (1,000 × g, 15 minutes, 4 ° C.) to collect protoplasts.

[0036]

[Table 6]

(4) Regeneration of protoplasts and acquisition of transformants: The precipitate of protoplasts obtained in step (3) was resuspended in 1 ml of protoplast preparation buffer, and
Gently shaken (125 rpm) for 2 hours at 0 ° C. After that, 100 μl of the protoplast suspension was applied to a regeneration medium (Table 7) containing 200 μg / ml of the antibiotic kanamycin and cultured at 30 ° C. for 7 days. As a result, transformants that acquired kanamycin resistance appeared at a frequency of 1 × 10 ⁵ cells / ml on the regeneration medium.

[0038]

[Table 7]

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Claims (13)

[Claims] 1. A method for transforming an alkalophilic Bacillus bacterium, which comprises the following operations (1) to (4): (1) Amino acid mixture solution of 2 to 8% by volume and final concentration of 0.
PH 7-1 containing sucrose in an amount of 2-1M
Culturing the cells of the alkalophilic Bacillus bacterium in a liquid medium of 0; (2) Amino acid mixture 4 to 16% by volume, fish meat extract 0.
25-2% by weight, potassium sulfate 0.09-0.4% by weight
And the bacterial cells obtained in (1) were suspended in a protoplast preparation buffer having a pH of 7 to 10 containing sucrose in an amount such that the final concentration was 0.25 to 1 M, and the final concentration was suspended in the suspension. To prepare protoplasts by adding lysozyme in an amount of 50 to 200 μg / ml; (3) Add a foreign gene and a polyethylene glycol solution in an amount of 5 to 20% by weight to the protoplast suspension. To incorporate the foreign gene into the protoplasts; (4) The protoplasts are suspended in the same protoplast-preparation buffer as used in step (2) and cultured, and the resulting suspension culture is , Amino acid mixture 0.5-8
The culture is carried out by coating on a solid medium having a pH of 7 to 10 containing a volume% and a final concentration of 0.5 to 0.9 M sodium succinate. 2. Operation (1), operation (2) and operation (4)
The transformation method according to claim 1, wherein the amino acid mixture used in (1) has the composition shown in Table 1. [Table 1] 3. Operation (1), operation (2) and operation (4)
3. The transformation method according to claim 1 or 2, wherein the pH adjustment of the medium or the protoplast preparation buffer in (1) is performed by adding an alkali carbonate or an alkali hydroxide. 4. The transformation method according to claim 3, wherein the alkali carbonate is sodium carbonate or sodium hydroxide. 5. The liquid culture medium of the operation (1) further comprises 0.25 to 2% by weight of fish meat extract and 0.0 of potassium monohydrogen phosphate.
5-1% by weight, yeast extract 0.025-0.5% by weight,
0.045 to 1% by weight of potassium sulfate and glucose of 0.
The transformation method according to any one of claims 1 to 4, which comprises 25 to 2% by weight. 6. The culture of the operation (1) is carried out at 30 to 40 ° C.
The transformation method according to any one of claims 1 to 5, wherein the culture is cultivated with shaking until the absorbance at 600 nm of the culture reaches 0.05 to 0.2. 7. The protoplast preparation buffer of step (2) further comprises 0.1 to 2% by weight of potassium monohydrogen phosphate,
Yeast extract 0.05-1% by weight, glucose 0.5-4
The transformation method according to any one of claims 1 to 6, wherein the transformation method comprises 0.01% to 2.0% by weight of magnesium chloride and 0.05 to 0.5% by weight of bovine serum albumin. 8. The transformation method according to any one of claims 1 to 7, wherein the protoplast preparation in step (2) is carried out at 20 to 42 ° C for 10 to 100 minutes after the addition of lysozyme. 9. The polyethylene glycol used in the operation (3) has an average molecular weight of 1,000 to 8,000.
[8] The method for transformation according to any one of [8] to [8]. 10. The transformation method according to claim 1, wherein the polyethylene glycol solution used in the step (3) further contains 0.25 to 1 M sucrose. 11. The suspension culture according to the operation (4) is 20 to 40.
The transformation method according to any one of claims 1 to 10, wherein the culture is carried out with shaking at 100 to 150 rpm. 12. The solid medium of the operation (4) further comprises 0.02 to 1% by weight of potassium sulfate, 0.05 to 2% by weight of gelatin, 0.1 to 0.6% by weight of fish meat extract, and monohydrogen phosphate. 0.02-0.2% by weight potassium, 0.01 yeast extract
.About.0.1 wt%, glucose 0.2 to 2 wt%, magnesium chloride 0.05 to 1 wt%, bovine serum albumin 0.
The transformation method according to any one of claims 1 to 11, which comprises 005 to 0.1% by weight and 0.5 to 2% by weight of agar. 13. The transformation method according to claim 1, wherein the culture in the solid medium of the operation (4) is performed at 30 to 50 ° C. for 2 to 14 days.