JPH0695946B2 - Uridine production method - Google Patents

Description

Detailed Description of the Invention

[0001]

[Industrial application] Uracil and uridine are useful as raw materials for the production of anticancer agents and antiviral agents such as 5-fluorouracil and 2 ', 3'-dideoxynucleosides which are pharmaceuticals. The present invention relates to a microorganism used and a production method.

[0002]

2. Description of the Related Art Regarding the production of uracils by fermentation, a method using a uracil analogue resistant strain derived from the genus Micromonospora (Japanese Patent Publication No. 57-18873), a purine analog derived from a bacterium of the genus Brevibacterium A method using a resistant strain (Japanese Patent Publication No. 57-30476), a method using a uracil analog resistant strain derived from a bacterium of the genus Bacillus (JP-A 61-10475, Agricultural Biological Chemistry 52, 1479, (1988)) are known.

According to these methods, bases, nucleosides and nucleotides are mixed in the culture solution, and much labor is required for growing useful bacteria.

[0004]

DISCLOSURE OF THE INVENTION The present invention is intended to provide an industrially advantageous method for producing uridine by a fermentation method, by simply growing a strain producing uridine.

[0005]

[Means for Solving the Problems] The inventors of the present invention have made earnest studies on a simple method for raising uridine-producing bacteria using Bacillus, and found that an orotic acid analog-resistant strain derived from a uridine-degrading strain lacks uridine. It has been found that uridine can be produced in a large amount when thymidine analog resistance is imparted.

[0006] Since the mutant strain can be obtained by positive selection, the uridine-producing strain was successfully bred easily and the present invention was completed.

The microorganism used in the present invention is Bacillus subutilis IFO 13719 [(1
977): ATCC 6051-KJConn, strain Marburg. (ATCC 605
1; NCIB3610; NCTC 3610; NRSmith 744 & 1315) Type
strain], a very common Bacillus subtilis that is easily available can be used as a parent strain. Alternatively, it may be screened from the soil and used as the parent strain.

Further, in order to induce the mutant strain, known mutation operations such as ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment may be carried out. You may select more. In addition, a very general method such as the replica method may be used to select the mutant strain.

As a specific example of the thus obtained bacteria to be used, Bacillus subtilis TL1 (microbe) having uridine-degrading deficiency, orotic acid analog resistance, and thymidine analog resistance with Bacillus subtilis IFO 13719 as a parent strain Institute of Industrial Science, No. 11951) (FERM P-11951).

This strain is no different from the parent strain except that it has uridine-degrading deficiency, orotate analog resistance or thymidine analog resistance, and uridine-producing ability.

The "orotic acid analog" referred to in the present invention means
It has a structure similar to orotic acid (6-carboxyuracil), which is an intermediate in the de novo biosynthetic pathway of uracils, and when added to the medium, it inhibits the growth of microorganisms.
Fluoroorotic acid, 2-thioorotic acid, etc.

[0012] The "orotic acid analog resistant strain" means
It refers to a strain that has been genetically altered so that it can grow in a medium containing a concentration of orotic acid analog that the parent strain cannot grow.

The "uridine-degrading strain" is a strain which is sensitive to 5-fluorouracil selected from mutants resistant to 5-fluorouridine and produces only a trace amount of uridine.

The term "thymidine analog" refers to a compound similar to thymidine, such as 5-bromo-2'-deoxyuridine, 5-chloro-2'-deoxyuridine and trifluorothymidine.

The culture and culture of the strain for uridine production may be carried out by a method which has been generally used conventionally.

For example, glucose, sucrose and the like, and cheap sugars such as molasses, starch and starch hydrolysates can be used as the carbon source.

As the nitrogen source, ammonia, ammonium salts such as ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, urea, peptone,
Hydrolyzed casein, nitrogenous organic substances such as meat extract, amino acids such as alanine, aspartic acid, glutamic acid, etc.
As long as Bacillus subtilis can be assimilated, any may be used.

If necessary, phosphates, magnesium salts, calcium salts, mineral salts and vitamins can be added.

As a culture method, generally used shaking culture or aeration stirring culture may be performed. Culture temperature is 20 ° C
40 ° C. is preferred. The pH during the culture may be adjusted within the range of pH 5.5 to 8.5 using an appropriate neutralizing agent.

As the neutralizing agent, ammonia, aqueous ammonia, sodium hydroxide, potassium hydroxide or calcium carbonate can be used. The culture period may be usually 2 to 7 days.

Also in the collection of uridine, a very general method may be used. For example, cells can be removed from the culture-completed liquid, and uridine can be recovered by a known method such as an adsorption method, a precipitation method, an extraction method or an ion exchange resin treatment method.

[0022]

In the past, it was general to select a fermentation-producing strain of a nucleic acid compound from a strain resistant to a base analog or a nucleoside analog of a salvage synthesis system.

The present invention focuses on orotic acid located in the de novo synthesis system and selects a strain having resistance to its analog to efficiently obtain a target production strain, which is a completely new idea. by.

If uridine has a poor resolution, 5-
Since fluorouridine does not decompose, it has strong growth inhibition.
No fluorouracil is produced. Therefore, the uridine-degrading strain is resistant to 5-fluorouridine and sensitive to 5-fluorouracil.

On the other hand, it is known that a thymidine-requiring mutant strain accumulates uracils (Japanese Patent Publication No. Sho 49-22710).

From this fact, it is suggested that the thymidine compound has a mechanism for regulating the biosynthesis of uridines. It is considered that this regulatory mechanism was released due to thymidine analog resistance, but the details are unknown.

[0027]

EXAMPLES The present invention will be described in more detail below with reference to examples.

Example 1 50 μl using Bacillus subtilis IFO 13719 as a parent strain
After treated with g / ml of NTG for 30 minutes (hereinafter, NTG treatment was performed under the same conditions), smeared onto a medium containing 1 mg / ml of 5-fluorouridine added to the basal medium and cultured at 30 ° C for 5 days From the colonies that appeared, by the replica method, 5-
Bacillus subtilis that cannot grow on the basic medium containing fluorouracil (50 μg / ml) and can grow on the basic medium
UR 1 was selected.

Basic medium Glucose 2.0% Ammonium sulfate 0.3% Magnesium sulfate 0.03% Dipotassium phosphate 0.2% Casamino acid 0.3% Agar 1.5% (pH 7.0)

Thus, the selection was made. Bacillus subtilis UR1 was treated with NTG, and then smeared on a basal medium containing 100 μg / ml of 5-fluoroorotic acid at 30 ° C.
After culturing for 5 days, Bacillus subtilis F3A was selected as a uridine-producing bacterium from the emerged colonies.

Furthermore, Bacillus subtilis F3A NT
The cells were treated with G, applied to a basal medium containing 100 μg / l of 5-bromo-2′-deoxyuridine, and cultured at 30 ° C. for 5 days. From the colonies that appeared, Bacillus subtilis TL1 (Microtechnological Research Institute, 11951) was selected as a strain with high uridine-producing ability.
No.) (FERM P-11951). Table 1 shows the degree of resistance of these strains to various pyrimidine analogs.

Next, Bacillus subtilis TL1 and, as a reference, the mutant strain obtained in the growing process was used as a fermentation medium 5
A 500 ml volume baffled Erlenmeyer flask containing 0 ml was inoculated, shake-cultured at 30 ° C. for 3 days, and uracil or uridine was quantified by HPLC (column; YMC-pack ODS-AQ). The results are shown in Table 2.

[0033]

[Table 1]

* +: With growth-: without growth 5FR; 5-fluorouridine (1000 μg / ml), 5F
U; 5-fluorouracil (50 μg / ml), 5FO; 5-fluoroorotic acid (100 μg / ml), BDU; 5-bromo-
2'-deoxyuridine (100 μg / ml), CDU; 5-chloro-2'-deoxyuridine (100 μg / ml), TFT;
Trifluorothymidine (200 μg / ml)

Fermentation medium Glucose 8.0% Urine 1.0% Dipotassium phosphate 0.2% Magnesium sulfate 0.08% Potassium chloride 0.09% Yeast extract 0.4% Calcium carbonate 2.0% (pH = 7.0)

[0036]

[Table 2] ───────────────────── Storage volume (mg / ml) Strain Uraciluridine ────────────── ───────── Parent stock <0.01 <0.01 UR1 <0.01 0.2 F3A 0.2 2.2 TL1 0.4 4.0 ─────────────────────

Example 2 Bacillus subtilis TL1 was mixed with 1 L of fermentation medium.
The cells were cultured in a 2.6 L jar fermenter at 30 ° C for 3 days. After removing the bacterial cells from the culture solution by centrifugation,
The supernatant was adsorbed on an activated carbon column. It was washed with water and eluted with 50% ethanol containing 1.4% aqueous ammonia. After the eluate was concentrated under reduced pressure, methanol was added in a volume of 9 times. The insoluble matter was removed by filtration and further concentrated to dryness. The solid matter was dissolved in a small amount of water, and ethanol was added under cooling to obtain 3.1 g of uridine crystals.

[0038]

INDUSTRIAL APPLICABILITY The present invention provides a method for simply growing a strain having uridine-producing ability from Bacillus, and efficiently produces only uridine, which is industrially extremely advantageous.

Claims (3)

[Claims] 1. A microorganism belonging to the genus Bacillus, having uridine-degrading deficiency, orotate analog resistance and thymidine analog resistance, and having uridine-producing ability is aerobically cultured and collected from the culture. A method for producing uridine. 2. The method for producing uridine according to claim 1, wherein a mutant strain of Bacillus subtilis is used as the microorganism. 3. The method for producing uridine according to claim 1, wherein Microorganism Research Institute No. 11951 (FERMP-11951) is used as the microorganism.