JPH04158792A - Production of uracil-based compound - Google Patents

Abstract

PURPOSE:To obtain a uracil-based compound useful as a raw material for drugs, a biochemical reagent, etc., practically and advantageously by culturing a specific bacterium to form and accumulate a uracil-based compound and then collecting the compound. CONSTITUTION:A bacterium (e.g. Brevibacterium ammoniagenes 214-17) belonging to the genus Brevibacterium, having pyrimidine analog resistance, capable of forming and accumulating a uracil-based compound, is subjected to spinner culture in a medium containing a carbon source, a nitrogen source, various kinds of metallic ions, etc., and optionally an amino acid, vitamins, etc., and the uracil-based compound is formed and accumulated and isolated by precipitation method to give the objective compound.

Description

Detailed Description of the Invention [Industrial Field of Application] The present invention provides uracil, uridine or 5'-U1 nodyl M (hereinafter referred to as
The present invention relates to a method for producing these compounds (collectively referred to as "uracil compounds") by fermentation, and to microorganisms belonging to the genus Brevibacterium used in the production method.

[Prior Art] Conventionally, methods for producing uracil compounds by fermentation using microorganisms belonging to the genus Brevibacterium include:
The only method that uses microorganisms resistant to purine analogs has been reported (see Japanese Patent Publication No. 57-3047θ).

[Problems to be Solved by the Invention] However, the above-mentioned conventional method produced a low amount of uracil-based compounds, and was not practically satisfactory.

Therefore, one object of the present invention is to provide a practical method for producing uracil compounds using microorganisms belonging to the genus Brevibacterium.

Means for Solving CSa] The present inventors have conducted various studies on a method for producing uracil-based compounds by fermentation, and found that uracil-based compounds that belong to the genus Brevibacterium, have pyrimidine analog resistance, and The present invention was completed by successfully breeding a group of microorganisms that have the ability to specifically produce and accumulate compounds.

That is, the present invention involves culturing a microorganism that belongs to the genus Brevibacterium, has resistance to pyrimidine analogs, and has the ability to produce and accumulate uracil compounds, and produces and accumulates uracil compounds. The present invention relates to a method for producing a uracil compound, which is characterized by collecting the compound.

The present invention also relates to a microorganism that belongs to the genus Brevibacterium, has resistance to pyrimidine analogs, and has the ability to produce and accumulate uracil compounds, which is used in the production of the uracil compounds.

The present invention will be explained in detail below.

As used herein, the term "uracil compound" refers to uracil, uridine, or 5°-uridylic acid as described above, and in the present invention, at least one of these exemplified compounds is accumulated in the culture solution. If so, it is assumed that uracil-based compounds have accumulated.

[Pyrimidine analog J is uracil, cytosine, 4
Substances having a structure similar to pyrimidine bases such as thymine, such as 5-fluorouracil, 8-azauracil,
2-thiouranyl, etc., or these mean glueboside and ribotide.

"Pyrimidine analog resistance" refers to the property of a mutant strain that can grow even in a medium containing at least one type of pyrimidine analog at a concentration at which the parent strain cannot grow.

The microorganisms used in the present invention are not particularly limited as long as they have the following characteristics A to C.

A: Belongs to the genus Brevibacterium B: Has resistance to pyrimidine analogs C: Has the ability to multiply uracil compounds A specific microorganism that satisfies these characteristics is Brevibacterium ammoniagenes (Brevibacterium ammonium).
oniagenes) 214-17, 14-27, etc.

The above-mentioned microorganism uses, for example, Brevibacterium ammoniagenes ATCC 8872 as a parent strain, and the parent strain undergoes usual mutation treatment (for example, ultraviolet irradiation treatment, N-methyl-N
A method of screening for strains that have resistance to pyrimidine analogs and the ability to accumulate uracil compounds from among mutant strains obtained by subjecting them to N-nitrosoguanidine (NTG) treatment, etc. can be obtained. A specific method for obtaining the above-mentioned microorganisms will be described in Example 1 below.

The thus obtained microorganism capable of producing a uracil compound may be cultured by a method known per se, and the uracil compound may be collected from the cultured product.

The culture medium used for culture may contain a carbon source, a nitrogen source, various metal ions, etc., and if necessary, micronutrients such as various amino acids, nucleic acids, vitamins, etc. can be used. . Specifically, as a carbon source,
Glucose, maltose, galactose, blackstrap molasses, starch hydrolyzate, etc., 1g of nitrogen is ammonia, various inorganic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium carbonate, or various organic nitrogens such as urea, corn nut liquor, yeast extract, etc. etc. can also be used.

For culturing the microorganism, a liquid culture III method under aerobic conditions such as shaking culture or aerated agitation culture may be employed.

The culture conditions may be appropriately selected from a pH range of 5.0 to 8.5 and a temperature range of 23 to 40°C.

The culture time is usually about 3 to 6 days under the above culture conditions, but the final judgment may be based on the accumulated amount of uracil compounds.

Cultivation 1! After the evacuation, uracil compounds can be collected from the culture by methods commonly used to isolate uracil compounds (for example, precipitation, membrane filtration, ion exchange chromatography, adsorption chromatography, etc.). ) may be used in combination as appropriate.

Examples are shown below to specifically explain the present invention.
l Brevibacterium ammoniagenes ATCC887
2 was treated with 300 μg/ml of NTO for 5 minutes, then plated on an agar plate containing the following basal medium supplemented with 100 μg/sl of 5-fluorouracil, and cultured at 30° C. for 5 days. The colonies that appeared were isolated and inoculated into test tubes containing 10 ml of the following production medium, and cultured with shaking at 28°C for 6 days. After completion of the culture, the centrifuged supernatant of the culture solution was analyzed, and Brevibacterium ammoniagenes 214-17 was found to be a microorganism with a high ability to produce uracil compounds.
and Brevibacterium ammoniagenes 14=2
I chose 7. The microbiological characteristics of these microorganisms were the same as the parent strain, except that they had resistance to pyrimidine analogs and produced and accumulated uracil compounds. Table 1 shows the resistance to various pyrimidine analogs, and Table 2 shows the amount of uracil compounds produced when the test tubes containing production medium 101 were inoculated and cultured with shaking at 28°C for 6 days.

Table I * +: Growth, -: No growth Table ■ Base n-style glucose 10g Vitamin Assay Casaminoand (manufactured by Difco) 1g Ammonium chloride 3g Magnenium sulfate lXl0-'g Ferrous sulfate
lXl0-2g manganese sulfate
lXl0-2g copper sulfate
lXl0-”g carnoum pantothenate 1
xlO-2g Thiamine Hydrochloride 5X10
-”g biotin 5X10-5g
25cM potassium phosphate buffer (pH 7) 000ml agar
20gU muglucose 1. OOg (separate full
) Corn stew liquor - 20g Yeast extract 2g Urea
10g Magneium Sulfate Ig O, 1M Potassium Phosphate Buffer (pH 7) 000ii Example 2 Glucose 4 g/dl, Peptone 2 g/dl, Yeast Extract 0. 5 g/dl, original slip 0. 3 g/dl,
KH2POa0, 15g/dl, K2HP Ox
0. 05g/dl, Mg5O+・7820 0. 0
Brevibacterium ammoniagenes 14-27 was inoculated into a test tube containing 10+1 Kashiwa medium with a composition of 5 g/dl (pH 7.0 of the medium before sterilization) and cultured with shaking at 30°C for 24 hours.

This seed culture II solution was inoculated into a 3001 Erlenmeyer flask with a baffle plate containing fermentation medium 201 shown below.
72 on a rotary shaker (200 rpm) at 0°C.
As a result of culturing for a period of time, an average of 5. 7mg/ml
Uranol, 0. 3q/rll uridine, 8. 1x
/ml of 5'-uridylic acid was produced.

Glucose 12 g/dl, (N Ha) aS 0
4 g/di.

Fe5Oa 7H200,001g/di, M
nSO4・4H200,0001g/dl, KH
2PO40, 2g/dI, KH2O40, Ig/d
i, urea 0, 2 g/dl, thiamine/HC
l2.5■/d1゜Biotin 200μg/dl,
M g S Oa・7H200, 05g/i11.
β-alanine 100μg/ml, L-cysteine/HCI 20g/ml, meat extract 0, 2g
#11. Ca C033g/di, (The pH of the medium before sterilization was adjusted to 7.2 using aqueous ammonia) 1 liter of the culture solution was adjusted to pH 5.0 with hydrochloric acid, heated at 80°C for 20 minutes, and centrifuged at 3000 rpm for 10 minutes. The supernatant obtained by separation was washed with Dowex 1x2 (200-40
The solution was passed through a column filled with chloroform resin (manufactured by Dow Chemical Company). After washing with water, 0.0IN salt Wi
The solution and the 0.05N saline solution were sequentially passed through the flask, and each fraction was collected and concentrated under reduced pressure. ,! # Decolorize the condensate with activated carbon and concentrate under reduced pressure to obtain 4.2 g of uranol, 0.2 g of uridine,
116 g of crude crystals of 5'-uridyl were obtained.

As a control, similarly cultured Brevibacterium
No production of uracil compounds was observed in the culture solution of Ammoniagenes ATCCE3872 (parent strain).

[Effect of the invention] The culture performed in Example 2 was performed using the conventional method
This experiment was conducted under exactly the same conditions as Example 1 described in No. 0476. Comparing the productivity of the uracil compound in this example with the productivity of the uracil compound described in Example 1 of Japanese Patent Publication No. 57-30478 in terms of the number of moles produced in the medium, the following table ① shows As can be seen, the method of the present invention exhibits 2.8 times higher productivity than the conventional method described in Example 1 of Japanese Patent Publication No. 57-30478, and is an extremely useful method. This difference in productivity is due to the microorganism used, and the microorganism of the present invention is useful for producing uracil compounds by fermentation.

Table ■ [Deposit of microorganisms] Brevibacterium ammoniagenes 214-17 and Brevibacterium ammoniagenes 14-27 obtained in Example 1 were entrusted to the Institute of Microbial Technology, Agency of Industrial Science and Technology on October 11, 1990.
The accession numbers have been given as FAIKEN BIKUYO No. 11759 and FEIKER No. 11760, respectively.

Patent applicant (677) Yamasa Soy Sauce Co., Ltd. Procedural Amendment (
Spontaneous) November 27, 1990 1, Indication of this case 1990 Patent Application No. 285077 2, Name of the invention Postal code 288\- Address 2-10 Shinseicho, Choshi City, Chiba Prefecture Telephone 0479-22
-0095 (Representative) 4. Addresses of "Shiraki" and "Renju" in the column of the inventor of the application to be amended, and column 5 of claims of the specification, Contents of the amendment 1) Please submit the application as attached. correct.

2. The scope of claims is amended as shown in the attached sheet.

Attachment 2, Claims 1) Cultivating a microorganism that belongs to the genus Brevibacterium, has resistance to pyrimidine analogs, and has the ability to produce and accumulate uracil compounds, and produces and accumulates uracil compounds. A method for producing a uracil compound, which comprises collecting a uracil compound.

2) A microorganism belonging to the genus Brevibacterium that is used in the production of the uracil compound according to claim 1, has resistance to pyrimidine analogs, and has the ability to produce and accumulate uracil compounds.

Claims (1)

[Scope of Claims] 1) A microorganism that belongs to the genus Brevibacterium, has pyrimidine analog resistance, and has the ability to produce and accumulate uracil compounds is cultured to produce and accumulate uracil compounds, and the produced and accumulated uracil 1. A method for producing a uracil-based compound, the method comprising collecting a uracil-based compound. 2) A microorganism belonging to the genus Brevibacterium that is used in the production of the uracil compound according to claim 1, has resistance to pyrimidine analogs, and has the ability to produce and accumulate uracil compounds.