JPS5847497A - Preparation of 5-hydroxymethylcytosine - Google Patents

Abstract

PURPOSE:To collect the titled substance formed and accumulatd in a culture, by cultivating a bacterium belonging to the genus Streptoverticillium capable of producing the titled substance in a medium containing a cytosine derivative. CONSTITUTION:>=0.5mM Cytosine derivative such as cytosine, 5-halogenocytosine, 5-alkylcytidine, etc. shown by the formula (R is H, halogen or lower alkyl) is added to a medium containing a carbon source to be assimilated by a bacterium, nitrogen source, inorganic salt, a very small amount of an effective substance. A bacterium such as Streptoverticillium rimofaciens belonging to the genus Streptoverticillium, capable of producing 5-hydroxymethylcytosine is cultivated in the medium. A mold is removed from the culture, and the desired 5-hydroxymethylcytosine is obtained from the supernatant liquid using an adsorbent, solvent, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing 6-hydroxymethy)vycytosine by a fermentation method. The inventors
As a result of various studies on various products of the genus Yu,
When Streptparticillium bacteria are cultured in a medium,
6-hydroxymethylcytosine is produced and accumulated;
The present invention has been completed by discovering that when a culture medium containing a cytosine derivative represented by the following formula (wherein R represents hydrogen, a halogen atom, or a lower alkyl group) is used, the amount of cytosine produced increases significantly. reached.

[Next 5-1: Doroki V Mechi~Cytosine is Pa V Me) 9
It has been found as an element constituting the molecules of antibiotics such as y*iμ deiomycin, and is only known to exist as a constituent base component of the deoxyribonucleic acid of E. coli-infecting Qi even-numbered phages. ,
It was extremely difficult to obtain a sufficient amount of this. According to the method of the present invention, 6-hydroxymethylcytosine can be accumulated in the culture solution of microorganisms by catalytic formation, so that a sufficient amount of 6-hydroxymethyA/! /) Shin can be obtained. 6 obtained in this way
-Hydroxymethycin is not only important as an intermediate in the production of primary antibiotics, but also serves as a biochemical reagent with an extremely wide range of applications in genetic and cancer research.

As the microorganisms used in the present invention, all Streptparticillium genus bacteria having intracellular or extracellular 5-hydroxymethylcytosine producing activity can be used. For example, Streptparticillium rimofaciens (UFO18692) is an example of VS* that can be effectively used for the purpose of implementing the present invention. This bacterium is described in Bergey's Manual of Determinative Pacteriology, 7th Edition.
of Deter-minatiVe Bacte
Streptomyces limo77x according to the classification criteria in riology7th EcLi, tion)
Streptomyces rimofaci
, ens, ) (Japanese Patent Application Laid-Open No. 5O-126892
), but the classification criteria were subsequently revised, and water bottles were classified as stored per-
Antibiotyphoid fever should belong to the genus Tycilium.
of AntiMotics, Vol. 81, No. 6, pp. 511-618]. This cocoon strain was transferred to the Agency of Industrial Science and Technology, Microbial Research Institute, as i?ERM P Ha-2549, and to the Fermentation Research Institute, Japan, as F0 18692. As The American Type Culture 9 Collection (T
he Ameri, can Type Culture
Collection, U, S, A)4CAT
CCa 1120, respectively. Since the general properties of such bacteria are easily KffiM, various mutant strains can be easily obtained both artificially and naturally. -Kagish that has not lost its hydroxymethylcytosine production activity can be used for the purpose of the present invention. Examples of such mutant strains include Streptparticillium rimofaciens (5treptOVert: LOllium r
imofaciens) (Engineering F0 14125) strain.

The specific method for obtaining this mutant strain is as follows.

An 80 watt ultraviolet light lamp is installed in a sterile box, and the spore suspension of the parent strain is irradiated with ultraviolet light for 90 seconds (wavelength: 2sayX) under 803 while stirring. This is a mutant colony that grew by spreading a suspension of spores that had been irradiated with radiation on a starch-free salt agar medium that is commonly used for the growth of actinomycetes. This mutant strain is characterized by a smooth colony morphology (compared to the parent strain) and a lighter color tone. In addition, using genetic engineering technology, Streptparticillium rimofaciens (Engineering F0 1a6)
The gene governing the 5-hydroxymethylcytosine production activity of 92) was cloned into bacteria other than actinomycetes such as Escherichia coli, or yeast, and these bacteria or yeast were found to have 6-hydroxymethylcytosine production activity. If necessary, these microorganisms can also be used for the purpose of carrying out the present invention.

The culture medium used for the production of 6-hydroxymethycin A/S/) generally contains a return element source that can be assimilated by microorganisms, ff
Sources of nutrients and micronutrients, if necessary.

Trace amounts of active substances such as growth-promoting factors are used.

In other words, as a leg element source, for example, glucose.

vYup loin, honey, starch, dextrin.

Glycerin, sorbitol, etc. are used, and nitrogen sources include, for example, meat extract, soybean flour, corn stay liquor, casein, and peptone.

In addition to natural nitrogen sources such as cotton lees, ammonium salts.

Inorganic substances such as nitrates are used, either alone or in combination.

Furthermore, in order to significantly promote the production of 6-hydroxymethylcytosine, it is preferable to add a yton derivative shown in Compound Engineering to one medium.

Such compounds include compounds in which R in formula (I) is hydrogen, ie, cytosine.

In addition to V-tidine or V-tis) A/acid, nuclear rIat9 containing them is a hydrolyzate thereof or a fungal cell of a rut organism,
Agricultural, livestock, and aquatic resources are used. Compounds in which R is a norogen atom, such as 5-notologenocytosine, 5-halogenocytidine or 6-valogenocytidic acid, are also useful for the purpose of the present invention. When R is a μkyl group, i.e., 5-μkyl F syn, 5-akyl A/V
Tidine or 6-a-iW5/thidic acid can also be used. When adding F:/ii conductor to the culture medium, the addition concentration is usually Q, 5 mM or more, preferably 1mM to 100mM (millimolar concentration type 1 amount/volume).
, more preferably 3mM to 60mM (mimolar concentration 1 weight 1/volume).As for when to add these to the medium, it is most effective to add them before the start of culture, but it is effective to add them continuously during the culture. Alternatively, it may be added intermittently.

As a method for culturing the microorganisms used in the present invention, a surface culture method may be used, but it is usually rational to use a deep aeration agitation culture method. When using the deep culture method, the liquid nature of the medium is preferably neutral to slightly acidic or slightly alkaline, and the culture temperature is 16 to 40°C, especially 1.
It is desirable to maintain the temperature between 24 and 84°C. However, it goes without saying that the culture conditions can be appropriately selected depending on the type of microorganism used, external conditions, etc. so as to obtain preferable results. Normally, if the culture is carried out under such conditions for 4 to 14 days, a significant amount of 5-hydroxymethylcytosine will be accumulated in the culture. To collect 5-hydroxydimethy/I/S/)$/n from the resulting culture, an appropriate combination of separation and collection methods commonly used to collect nucleobases from microbial cultures is used. be done.

For example, first, bacterial cells are removed by means such as filtration or centrifugation. Transfer the resulting supernatant to an appropriate adsorbent.

For example, activity, adsorptive resins, cation exchange resins.

After adsorbing the target substance by contacting it with an adsorbent such as activated alumina, silica gel or molecules, it is then adsorbed with a water-containing liquid such as acetone, methanol, ethanol, propatool, butano, etc. Alternatively, the target substance can be eluted using an acid, a 1v-13y, a buffer solution, an inorganic salt, or an aqueous solution of Arine as a solvent. After performing these separation methods in an appropriate combination, the effective fraction is concentrated and precipitated, and 6-hydroxymethylcytosine can be collected in the free form or in the form of woodcutter.

6-Hydroxymethyfi/￥ton can be used, for example, in the method for producing the antibiotic mildeiomycin (Japanese Patent Application Laid-Open No. 56-8249
No. 5), by including it in the culture medium, micro
Deio! It has the effect of increasing the production amount of isin.

The content of the present invention will be explained in more detail with reference to *Example below, but it goes without saying that this example is just an example of how the present invention can be carried out.

Example 1〉 In a 21-volume slope flask, 1% glucose, #mother extract O1
s%, peptone O 6% (weight/volume) medium 60
0- was adjusted to pH 7 with a 20% aqueous solution of caustic soda, dispensed and sterilized, and then added Stone 1 doparticillium Q.
Streptoverticill
ium rj-mofaci, ens) ENGFO1a
692 (FERM-P Nl 2549. ATC
O81120) was inoculated and cultured on a reciprocating shaker incubator at 28°C for 48 hours. 50111 pot 4
'll with 8% glucose, 2% corn steep liquor, ammonium sulfate o, is, magnesium sulfate 0
．． 05%, 10 flo
! If a culture medium aOg consisting of 8% calcium carbonate (weight/volume) is adjusted to pH 7 with a 20% aqueous solution of caustic soda and sterilized, 500 m of the previously cultured Sakaro flask culture solution is added.
inoculated with l, aeration volume l VVM (ventilation volume per minute per unit liquid volume), agitation II 1 revolution at 16 orpm and 28
The cells were cultured at ℃ for 24 hours and used as a culture medium. 2001 volume fermenter with 12% glucose, a% casein, 0.6% corn steep liquor, Proflo (Trader Oil)
Mill Co. m) 2%, ammonium nitrate 0゜5%, ammonium sulfate +=? A0.5%, sulfuric acid IJNlk0.006%,
A medium 10011 consisting of 0.005% Wt manganese acid, calcium carbonate l-, and some antifoaming agent was prepared, and after adding cytosine 100F to it, it was sterilized, the seed culture solution 101 was transplanted, and the aeration was carried out. Quantity Q, 5VVM.

Culture was carried out for 5 days at a stirring rotation speed of 20 rpm and a temperature of 28°C.

Take aOl from the thus obtained culture solution and add 2 ml of water to it.
ON and 1 g of Hyflo-Spar7al (manufactured by Jimins Manville) were added and filtered to obtain a solution of 461, and concentrated hydrochloric acid was added dropwise to adjust the pH to 2. This solution was passed through a column packed with 6 g of chromatography active solution (manufactured by Shikinichi Pharmaceutical Co., Ltd.). After washing the column with 20e of water, add n-butano-
〃Elute using saturated water 101, collect the effective fraction, 6g
The mixture was passed through a column of ion exchange resin Amberlite R-120 (manufactured by Rohm and Haas) HW, and the column was washed with 201% water and eluted with a% ammonia water. Both effective portions of the eluate were collected and concentrated under reduced pressure to obtain 11 concentrates. This was passed through a column packed with 20 g of molecular sieve and Sephadex G-10 (manufactured by Pharmacia) and eluted with water. The active components were collected and concentrated under reduced pressure to obtain a concentrated solution with a concentration of 0.51, which was then treated with ion exchange resin Amberlite CG-50 (Rohm Φ Anne F9 manufactured by Haas Co., Ltd.) NH
The effective fraction was passed through a column packed with Type 21, and the effective fraction was poured out into a round shape.The effective fraction was concentrated under reduced pressure to 0.151!, and white needle-like crystals were precipitated. −
4 hydroxymethycytosine 51F.

<Example 2> 9! When culturing according to the method of Example 1, the amount of 6-hydroquinemethylcytosine produced was determined when cytosine was not added to the medium and when cytosine was added at various concentrations, and the results shown in Table IK were obtained.

Table 1 When culturing according to the method of Example 1, cytosine was added to the medium, and instead of adding 5-fluoroV-tosine, 5-bromocytosine, 5-g-docytosine, 5-methylcytosine. When cultured with two different concentrations of monohydroxymethylcytosine added to the culture medium, monohydroxymethylcytosine was quantified once it had accumulated in the culture, and the results shown in Table 2 were obtained.

Table 2 Kukuruse@4〉 When culturing according to the method of Example 8, one strain used for culture is Streptparticillium rimofaciens X (St
reptoverticillium rimofac
iens) Engineering F O1a 592 (FERM-P Nagi2
549), Streptparticillium rimofasi:cyp-(Streptovertici
liium rimofaciens) engineering F 0 1
4126 (FERM-P Na 6052-) was used to obtain the results shown in Table 8.

Table 8 6-Hyfurokki V obtained in Example 1.2.1.4 above
Methylcytosine is present in Alegria (Aleg-ria).
) method
Biochemica et Bxophyslc
aActa), Vol. 149, pp. 817-824, (19
The physicochemical properties of the 5-hydroxymethylcytosine sample synthesized based on 1967) and the 5-hydroxymethylcytosine commercially available reagent manufactured by Sigma Co., Ltd. were completely identical. Namely, elemental analysis, paper chromatography Rf
The values, ultraviolet absorption spectrum, and nuclear magnetic resonance spectrum are listed below.

(1) Elemental analysis value Elemental analysis value g C-42,54%, H-5,00%, N
=29, 78% (2) Paper chromatography Paper chromatography Rf values are shown in Table 4.

Table 4 Paper chromatograph Rf value Qin Capacity mixing ratio (3) Ultraviolet absorption spectrum (3) Ultraviolet absorption spectrum (NaoH, MoN) 4C1 was obtained as a solvent, and its maximum absorption wavelength, minimum absorption wavelength, and 250 nm were obtained. Table 6 shows the ratio of absorption values at 280 nm and 260 nm and the ratio of absorption values at 280 nm and 260 nm.

(Margin below) (4) Nuclear Magnetic Resonance Spectrum I values (ppm) obtained from the 130-NMR spectrum measured in heavy water are shown in Table 6.

(Margin below)

Claims (1)

[Claims] (1) Cultivating 6-hydroxymethyA/S/)sin-producing bacteria belonging to the genus Streggedparticillium in a medium, producing and accumulating 6-hydroxymethysA/F)syn in the culture, and collecting it. Characteristic method for producing 6-hydroxymethylcytosine. 2. The production method according to claim 1, which uses a medium containing a compound represented by the formula (wherein R represents hydrogen, halogen, or lower alkyl).