JP2716506B2 - Method for high-density culture of recombinant DNA strain - Google Patents

Description

The present invention relates to a novel high-density culture method for efficiently culturing a recombinant DNA strain having a plasmid having a tryptophan promoter.

(Prior art and problems) When culturing a recombinant DNA strain having a plasmid having a tryptophan promoter, the indoleacrylic acid is added to the medium to cope with the structural gene introduced downstream of the tryptophan promoter. It is known that the suppression of the generation of oxygen is released (see, for example, JP-A-60-87784). However, since indoleacrylic acid inhibits the growth of microbial cells, cultivate the recombinant DNA strain carrying a plasmid having a tryptophan promoter at high density (producing a significant amount of the target oxygen) at high density. It was difficult.

DISCLOSURE OF THE INVENTION The inventors of the present invention have conducted intensive studies to solve the above-mentioned problems, and as a result, when culturing a recombinant DNA strain having a plasmid having a tryptophan promoter, a culture solution, In the middle to late stages of the logarithmic growth of the culture, the structure introduced into the downstream of the tryptophan promoter by adding indoleacrylic acid and then culturing while maintaining the dissolved oxygen concentration in the medium at 3 ppm or more. The present inventors have found that a large amount of oxygen corresponding to the gene can be produced, and that the recombinant DNA strain can be cultured at a high density in that state, thereby completing the present invention.

 Hereinafter, the present invention will be described in more detail.

The microorganism used in the present invention is not particularly limited as long as it has a plasmid having a tryptophan promoter. For example, Escherichia coli K-12YK2009 (FERM P-79) having trpAtrpB (tryptophan synthase gene) as a structural gene downstream of the tryptophan promoter.
57), Escherichia coli K-12YK2014 (FERM P-832)
8).

In the culture medium used for culturing the microorganism, various carbohydrates such as glucose, glycerol, fructose, sucrose and molasses can be used as the carbon source. As the nitrogen source, for example, ammonium salts such as ammonium chloride, ammonium sulfate, and ammonium phosphate, nitrates such as sodium nitrate, potassium nitrate, and ammonium nitrate, and ammonia are preferably used. As inorganic substances, for example, potassium phosphate, Magnesium sulfate, iron, manganese, zinc and the like are used. Also,
If necessary, nutrients such as vitamins and amino acids can be added.

The cultivation temperature is usually in the range of 20 ° C to 50 ° C, preferably 30 to 40 ° C, and the pH of the medium during culturing is generally in the range of 6 to 9, preferably 7 to 8, Can be adjusted by adding to the medium.

As the alkaline substance, for example, an aqueous solution of ammonia, sodium hydroxide, potassium hydroxide, calcium hydroxide or the like is suitably used.

The culture can be performed aerobically under aeration and stirring conditions.

The timing of adding indole acrylic acid to the medium is preferably from the middle to late logarithmic growth of the culture (usually, 3 to 8 hours of culture), and the amount of addition is not strictly limited, Generally 10 to 300 mg / l, preferably 25 to 200
The range of mg / l is appropriate. After the addition of indole acrylic acid, the culture is performed while maintaining the dissolved oxygen level in the medium at 3 ppm or more. As a method for maintaining the dissolved oxygen level in the culture medium within the above range, for example, a method of passing pressurized air or / and oxygen gas may be mentioned. Upper limit of dissolved oxygen level in culture medium does not inhibit growth of microbial cells
It is about 8 ppm, and a suitable dissolved oxygen concentration range is 3 to 5 ppm.

In the present specification, the dissolved oxygen level (concentration) in the medium is a value measured by a Universal Oxygen Analyzer Type RA manufactured by Oriental Electric Co., Ltd.

According to the method of the present invention described above, a recombinant DNA containing a plasmid having a tryptophan promoter
The strain can be cultured at a high yield while producing a high yield of oxygen corresponding to the gene introduced downstream of the promoter, thereby greatly contributing to industrial use.

 Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1 A 50 ml culture medium having the composition shown in Table 1 below was dispensed into a 500 ml Erlenmeyer flask, sterilized at 120 ° C. for 15 minutes, and added with Escherichia coli K-12YK-2009 (FERM P-7957). After adding 2 ml of a 50% glucose solution sterilized at 120 ° C. for 10 minutes, shaking culture (pre-culture) at 37 ° C. for 1 day, and then sterilized at 120 ° C. for 15 minutes, the same medium After adding 20 ml of a sterilizing agent 50% glucose solution to a 2 liter aeration-stirring tank containing 1000 ml, inoculate 20 ml of the preculture, and then incubate at 37 ° C, pH
The cells were cultured in 7.2 (adjusted with 25% aqueous ammonia). The concentration of glucose in the culture solution during the culture was 0.1 to 2 (wt / vol)%.
Were added sequentially so that The stirring rotation speed was adjusted to 1000 rpm, and the aeration amount was adjusted to the initial 1 VVM. After 6 hours from the start of the culture, indoleacrylic acid was added at a concentration of 100 mg / l, and oxygen gas was mixed with air and aerated, so that the dissolved oxygen in the culture solution was adjusted so as to obtain the working group shown in Table 2 below. Culture was performed while maintaining the level. After 20 hours of cultivation, the culture is terminated and centrifuged (8000 rpm, 20 minutes, 4 minutes) from 500 ml of the culture solution.
C.) and the specific activity of tryptophan synthase was measured. The results are shown in Table 2 as relative values when the cells were cultured in air alone as a control, with 100 being the relative value.
The specific activity of tryptophan synthase was determined by centrifuging the cells after culturing for a certain period of time, collecting the cells, washing once with 100 mM Tris-HCl buffer (pH 7.8), and weighing 200 mg of the wet cells. After adding 1 ml of the same buffer, the cells are disrupted by a cell disrupter (Branson 200), and then centrifuged (12000 rp).
m, 40 minutes, 4 ° C.).
Sky et al. [Method in Enzymology, vol.5, 794 (196
2)].

Table 1 (NH ₄ ) ₂ SO ₄ 3g KH ₂ PO ₄ 5〃 K ₂ HPO ₄ 5MgSO MgSO ₄・ 7H ₂ O 0.4〃 FeSO ₄・ 7H ₂ O 0.1〃 yeast extract 1〃 peptone 1〃 distilled water 1000ml ( pH 7.2) Example 2 The strain was transformed into Escherichia coli K-12YK-2014 (FERM P-
The same experiment as in Example 1 was performed except that 8328) was used. The results are shown in Table 3.

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Claims (4)

(57) [Claims] 1. When culturing a recombinant DNA strain belonging to the genus Escherichia carrying a plasmid having a tryptophan promoter, after adding indoleacrylic acid to the culture solution, the dissolved oxygen concentration in the culture solution is 3 ppm or more. A method for culturing a recombinant DNA strain belonging to the genus Escherichia, wherein the cultivation is carried out while maintaining the strain. 2. The method according to claim 1, wherein the indole acrylic acid is added in a period from the middle to late logarithmic growth of the culture. 3. A method of adding pressurized air or / and oxygen gas to a culture solution to reduce the concentration of dissolved oxygen in the culture solution to 3 ppm.
2. The method according to claim 1, wherein said method is retained. 4. The recombinant DNA strain is Escherichia coli K.
The method according to any one of claims 1 to 3, which is -12YK2009 or Escherichia coli K-12YK2014.