JPH04252190A - Production of uridine - Google Patents

Abstract

PURPOSE:To produce uridine by fermentation method. CONSTITUTION:A variant having resistance to 5-fluorouridine and 5-fluorouracil sensitivity is selected by using Bacillus subtilis as a parent strain, successively provided with resistance to 5-fluoroorotic acid and resistance to 5-bromo-2'- deoxyuridine, grown and aerobically cultured to produce uridine.

Description

[Detailed description of the invention]

0001

[Industrial Application Field] Uracil and uridine are useful as raw materials for the production of anticancer and antiviral agents such as pharmaceuticals such as 5-fluorouracil and 2',3'-dideoxynucleosides, and the present invention is applicable to this fermentation production. Regarding the microorganisms used and the manufacturing method.

0002

[Prior Art] Regarding the production of uracils by fermentation methods, there is a method using a uracil analog resistant strain derived from the genus Micromonospora (Japanese Patent Publication No. 18873/1983);
A method using a purine analog resistant strain derived from a bacterium of the genus Brevibacterium (Japanese Patent Publication No. 57-30476),
A method using a uracil analog resistant strain derived from Bacillus bacteria (Japanese Patent Application Laid-open No. 10475/1983, Agricultural Biological Chemistry 52, 1
479, (1988), etc.) are known.

[0003] These methods involve the presence of bases, nucleosides, and nucleotides in the culture solution, and require a great deal of effort to grow useful bacteria.

0004

SUMMARY OF THE INVENTION The present invention aims to provide an industrially advantageous method for producing uridine by a fermentation method by simply growing a bacterial strain that produces uridine.

[0005]

[Means for Solving the Problems] The present inventors conducted intensive studies on a simple method for growing uridine-producing bacteria using Bacillus bacteria, and found that an orotic acid analog resistant strain derived from a strain deficient in uridine degrading ability We discovered that if thymidine analogue resistance is conferred, significant amounts of uridine can be produced.

[0006] Since mutant strains can be obtained by positive selection, we have succeeded in simply growing a strain that produces uridine, and have completed the present invention.

[0007] The microorganism used in the present invention is Bacillus subtilis.
is) IFO 13719 [(1977): ATC
C 6051-K. J. Conn, strain Ma
rburg. (ATCC 6051; NCIB361
0; NCTC 3610; N. R. Smith 7
44 & 1315) Type strain] can be used as a parent strain. Alternatively, it may be used as a parent strain by screening from soil.

Furthermore, for the induction of mutant strains, ultraviolet irradiation and N-methyl-N'-nitro-N-nitrosoguanidine (
Known mutation operations such as NTG) treatment may be performed,
It may be selected from among natural mutant strains. In addition, a very common method such as the replica method may be used to select a mutant strain.

[0009] Specific examples of the used bacteria obtained in this manner include Bacillus subtilis IFO 13719 as a parent strain with uridine decomposition deficiency, orotic acid analog resistance,
In addition, Bacillus subtilis TL1 (FERM No. 11951) conferred thymidine analog resistance.
P-11951).

This strain is no different from the parent strain except that it lacks the ability to degrade uridine, has resistance to orotic acid analogs or thymidine analogs, and has the ability to produce uridine.

[0011] The "orotic acid analog" referred to in the present invention is
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 culture medium, inhibits the growth of microorganisms. For example,
5-fluoroorotic acid, 2-thioorotic acid, etc.

[0012] Also, "orotic acid analog resistant strain" means
This refers to a strain that has been genetically altered so that it can grow in a medium containing orotic acid analogs at concentrations that the parent strain cannot grow.

[0013] "Uridine-degrading strain" refers to a strain that is sensitive to 5-fluorouracil and produces only a trace amount of uridine, selected from mutant strains resistant to 5-fluorouridine.

[0014] The term "thymidine analog" refers to a compound similar to thymidine, such as 5-bromo-2'-deoxyuridine, 5-chloro-2'-deoxyuridine, and trifluorothymidine.

[0015] The culture medium and cultivation of the bacterial strain for uridine production may be carried out by conventional methods.

For example, glucose, sucrose, etc., and inexpensive carbohydrates such as molasses, starch, or starch hydrolysates can be used as carbon sources.

As the nitrogen source, ammonium salts such as ammonia, ammonium nitrate, ammonium sulfate, ammonium chloride, ammonium phosphate, urea, peptone,
Casein hydrolyzate, nitrogenous organic substances such as meat extract, amino acids such as alanine, aspartic acid, glutamic acid, etc.
Any material that can be assimilated by Bacillus subtilis may be used.

[0018] If necessary, phosphates, magnesium salts, calcium salts, mineral salts, and vitamins can also be added.

[0019] As a culturing method, commonly used dipping culture or aerated agitation culture may be used. Culture temperature is 20℃~4
0°C is preferred. The pH during culturing may be adjusted to a range of pH 5.5 to 8.5 using a suitable neutralizing agent.

As the neutralizing agent, ammonia, aqueous ammonia, sodium hydroxide, potassium hydroxide, calcium carbonate, etc. can be used. The culturing period may normally be carried out for 2 to 7 days.

[0021] Uridine can also be collected using a very common method. For example, uridine can be recovered by removing bacterial cells from the culture solution and using known methods such as adsorption, precipitation, extraction, and ion exchange resin treatment.

[0022]

[Action] Conventionally, bacteria producing nucleic acid compounds by fermentation have generally been selected from strains resistant to base analogs or nucleoside analogs in salvage synthesis systems.

[0023] The present invention focuses on orotic acid, which is located in the de novo synthesis system, and selects a strain that is resistant to its analogues to efficiently obtain the desired production strain, which is a completely new idea. by.

[0024] Furthermore, if the ability to decompose uridine is lost, 5
- Since fluorouridine is not decomposed, 5-fluorouracil, which strongly inhibits growth, is not produced. Therefore, a strain lacking the ability to degrade uridine 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. 49-227).
10).

This fact suggests the existence of a mechanism for regulating the biosynthesis of uridines by thymidine compounds. It is thought that this regulatory mechanism is released due to thymidine analog resistance, but the details are unknown.

[0027]

[Examples] The present invention will be explained in more detail below using examples.

Example 1 Bacillus subtilis IFO 13719 was used as a parent strain and treated with 50 μg/ml NTG for 30 minutes (hereinafter referred to as
NTG treatment was carried out under the same conditions), then 1 mg was added to the basal medium.
5-fluorouracil (50 μg/ml) was plated on a medium supplemented with 5-fluorouridine and cultured at 30°C for 5 days.
Bacillus subtilis UR 1, which could not grow on the basal medium containing ml), was selected because it could grow on the basal medium.

[0029]
Basic medium
glucose
2.0%
Ammonium sulfate 0.3%
Magnesium sulfate 0
．． 03% Dipotassium phosphate 0.2%
Casamino acids
0.3%
Agar 1.5%
(pH 7.0)

[0030] The selection was thus made. After applying NTG treatment to Bacillus subtilis UR1, 1
It was spread on a basal medium supplemented with 00 μg/ml of 5-fluoroorotic acid and cultured at 30° C. for 5 days, and from the colonies that appeared, Bacillus subtilis F3A was selected as a uridine-producing bacterium.

Furthermore, NT to Bacillus subtilis F3A
The cells were treated with G, spread on a basal medium containing 100 μg/l of 5-bromo-2'-deoxyuridine, and cultured at 30° C. for 5 days. Among the colonies that appeared, Bacillus subtilis TL1 (FERM P-11951) was selected as a strain with high uridine production ability. Table 1 shows the degree of resistance of these strains to various pyrimidine analogs.

[0032] Next, Bacillus subtilis TL1 and the mutant strain obtained during the growth process as a reference were placed in a fermentation medium of 50%
ml was inoculated into a 500 ml Erlenmeyer flask with a baffle, cultured under shaking 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]

*+; Growth -; No growth 5FR; 5-fluorouridine (1000 μg/ml)
, 5FU; 5-fluorouracil (50 μg/ml),
5FO; 5-fluoroorotic acid (100μg/ml)
, BDU; 5-bromo-2'-deoxyuridine ( 1
00μg/ml), CDU; 5-chloro-2'-deoxyuridine (100μg/ml), TFT; trifluorothymidine (200μg/ml)

[0035]
Fermentation medium
glucose
8.0%
Urea 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] ──────────
────────────
Accumulation
Amount (mg/ml)
Bacterial strain


Uracil Uridine
──────────────
───────
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 cultured at 30° C. for 3 days in a 2.6 L jar fermenter containing 1 L of fermentation medium. After removing the bacterial cells from the culture medium by centrifugation, the supernatant liquid was adsorbed onto an activated carbon column. Wash with water,
Elution was performed with 50% ethanol containing 1.4% aqueous ammonia. After concentrating the eluate under reduced pressure, 9 volumes of methanol were added. Insoluble matter was removed by filtration, and the mixture was 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 easily cultivating a strain capable of producing uridine from Bacillus bacteria, and also efficiently produces only uridine, which is extremely advantageous industrially.

Claims (3)

[Claims] Claim 1: A microorganism belonging to the genus Bacillus, deficient in uridine degrading ability, orotic acid analog resistance, and thymidine analog resistance, and capable of producing uridine is cultivated aerobically, and the microorganism is collected from the culture. A method for producing uridine. [Claim 2] Bacillus subtilis I as a microorganism
The method for producing uridine according to claim 1, wherein FO 13719 is used. [Claim 3] As a microorganism, Microorganism Research Institute No. 1195
The method for producing uridine according to claim 1, wherein FERM P-11951 of No. 1 is used.