JPS6360997B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing 5-hydroxymethylcytosine by fermentation. The present inventors have repeatedly conducted various studies on the metabolic products of Streptoverteicillium bacteria, and have found that when Streptoverteicillium bacteria are cultured in a medium, 5-hydroxymethylcytosine is produced and accumulated. The present inventors have discovered 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, leading to the completion of the present invention. Conventionally, 5-hydroxymethylcytosine has been found as a component of antibiotic molecules such as basimethrin and mildeiomycin.
Since it is only known to exist as a constituent base component of the deoxyribonucleic acid of E. coli infectious T-even phage, it has been extremely difficult to obtain it in sufficient quantities. According to the method of the present invention, 5-hydroxymethylcytosine can be directly produced and accumulated in the culture solution of microorganisms.
5-hydroxymethylcytosine can be obtained easily and in sufficient quantities. The 5-hydroxymethylcytosine thus obtained is not only important as an intermediate for the production of the above-mentioned 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 Streptoverteicillium bacteria having intracellular or extracellular 5-hydroxymethylcytosine producing activity can be used. For example, Streptoverteicillium rimofaciens (IFO13592) is an example of a bacterial species that can be used effectively for the purpose of carrying out the present invention. This bacterium is described in Bergey's Manual of Determinative Bacteriology, 7th edition.
It was identified as Streptomyces rimofaciens according to the classification criteria in Bacteriology 7th Edition (Japanese Unexamined Patent Publication No. 126892/1989), but the classification criteria were subsequently revised and Versey's Manual of Determinative According to the 8th edition of Bacteriology (1974), this bacterium should belong to the genus Streptoverteicillium (The Journal of Antibiotics).
Antibiotics”, Vol. 31, No. 6, pp. 511-518).
This strain was sent to the Institute of Microbiology, Agency of Industrial Science and Technology.
FERM P No.-2549, Fermentation Research Institute Foundation, IFO13592, The American Type Culture Collection (The American Type Culture Collection)
Type Culture Collection, USA)
Each has been deposited as ATCC31120.
Generally speaking, these bacteria easily mutate in their mycological or physiological properties.
Various mutant strains can be easily obtained either artificially or naturally, and these mutant strains can also be used for the purpose of the present invention as long as they have not lost their 5-hydroxymethylcytosine producing activity. An example of such a mutant strain is Streptoverticillium rimofaciens, which has been deposited with the Agency of Industrial Science and Technology as FERM P No.-6052 and with the Fermentation Research Institute as IFO14125, respectively. IFO14125)
Stocks are one example. The specific method for obtaining this mutant strain is as follows. A 30 watt ultraviolet light lamp is installed in a sterile box, and the spore suspension of the parent strain is irradiated with ultraviolet light (wavelength 2537 Å) for 90 seconds while stirring the spore suspension 30 cm below.
This spore suspension irradiated with ultraviolet rays was spread on a starch inorganic salt agar medium commonly used for the growth of actinomycetes, and mutant colonies were obtained. This mutant strain is characterized by a smooth colony morphology and a lighter color than the parent strain. In addition, using genetic engineering technology, we cloned the gene that controls the 5-hydroxymethylcytosine production activity of Streptoverteicillium rimofaciens (IFO13592) into bacteria other than actinomycetes, such as Escherichia coli, or yeast. When these bacteria or yeast are made to have 5-hydroxymethylcytosine producing activity, these microorganisms can also be used for the purpose of carrying out the present invention. The medium used for the production of 5-hydroxymethylcytosine generally contains carbon sources, nitrogen sources and inorganic salts that can be assimilated by microorganisms, and if necessary, trace amounts of effective substances such as micronutrients and growth-promoting factors. That is, as carbon sources, for example, glucose, sucrose, molasses, starch, dextrin, glycerin, sorbitol, etc. are used, and as nitrogen sources, for example, meat extract, soybean flour,
In addition to organic nitrogen sources such as corn steep liquor, casein, peptone, and cottonseed meal, inorganic salts such as ammonium salts and nitrates are used, either alone or in combination. Furthermore, in order to significantly promote the production of 5-hydroxymethylcytosine, it is preferable to add cytosine derivatives shown in the compound to the culture medium.
As such a compound, R in formula ()
is hydrogen, i.e. cytosine,
In addition to cytidine or cytidylic acid, nucleic acids containing them or their hydrolysates, microbial cells, agricultural, livestock and fisheries resources, etc. can be used. Compounds in which R is a halogen atom, such as 5-halogenocytosine, 5-halogenocytidine or 5-halogenocytidylic acid, are also used for the purpose of the invention. When R is an alkyl group, i.e. 5-alkylcytosine, 5-alkylcytidine or 5-
Alkylcytidylic acids and the like are also used. When such cytosine derivatives are added to the medium, the concentration is usually 0.5mM or more, preferably 1mM to 10mM.
(millimolar concentration, weight/volume), more preferably
3mM to 50mM (millimolar concentration, weight/volume) is suitable. Regarding the timing of adding these to the medium, it is most effective to add them before the start of culture, but they may be added continuously or intermittently during culture. 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 desirably maintained at 15 to 40°C, particularly 24 to 34°C. However, it goes without saying that these culture conditions can be appropriately selected depending on the type of microorganism used, external conditions, etc. so as to obtain preferable results. Normally, when cultured under these conditions for 4 to 14 days, a significant amount of 5-hydroxymethylcytosine is accumulated in the culture. In collecting 5-hydroxymethylcytosine from the resulting culture, an appropriate combination of separation and collection means commonly used for collecting nucleic acid bases from microbial cultures is used. For example, first, bacterial cells are removed by means such as filtration or centrifugation. The obtained supernatant liquid is contacted with a suitable adsorbent such as activated carbon, adsorbent resin, cation exchange resin, activated alumina, silica gel, or molecular sieve to adsorb the target substance, and then the target substance is adsorbed, for example, acetone or methanol. The target substance can be eluted using a water-containing solution of a water-soluble organic solvent such as ethanol, propanol, or butanol, or an aqueous solution of an acid, an alkali, a buffer, or an inorganic salt or an organic salt as a solvent. After carrying out a suitable combination of these separation means and concentrating and precipitating the effective fraction, 5-hydroxymethylcytosine can be collected in a free state or a salt state. 5-Hydroxymethylcytosine can be used, for example, in the method for producing the antibiotic mildeiomycin (Japanese Unexamined Patent Application Publication No. 1989-1999).
32495), it has the effect of increasing the production amount of mildeiomycin by including it in the culture medium. The content of the present invention will be explained in more detail with reference to examples below, but it goes without saying that these examples are just examples of embodiments of the present invention. Example 1 500 ml of a medium consisting of 1% glucose, 0.3% yeast extract, and 0.5% peptone (weight/volume) was adjusted to pH 7 with 20% caustic soda diluted solution, then dispensed into a 2-volume Sakaguchi flask, sterilized, and Streptoverticillium rimofaciens IFO13592
(FERM-P No. 2549, ATCC 31120) was inoculated and cultured on a reciprocating shaking incubator at 28°C for 48 hours. Glucose 3 in a 50 volume fermenter
%, corn steep liquor 2%, ammonium sulfate 0.1%, magnesium sulfate 0.05%, Proflo (Trader Oil Mill) 1%, calcium carbonate 0.3% (weight/volume).
30 was adjusted to pH 7 with a 20% caustic soda aqueous solution, sterilized, inoculated with 500 ml of the previously cultured Sakaguchi flask culture, and heated to 28°C at an aeration volume of 1 VVM (aeration volume per minute per unit liquid volume) and a stirring speed of 100 rpm. , and cultured for 24 hours to serve as a seed culture. Glucose in a 200 volume fermenter
12%, casein 3%, corn steep liquor 0.5%, Proflo (Trader Oil Mill) 2%, ammonium nitrate 0.5%, ammonium sulfate 0.5%, ferrous sulfate 0.005%, manganese sulfate 0.005%, calcium carbonate Prepare a medium 100 consisting of 1% and some antifoaming agent, add 100 g of cytosine to it, sterilize it, transplant the above seed culture solution 10, aeration volume 0.5VVM, stirring rotation speed.
The cells were cultured at 200 rpm and a temperature of 28°C for 5 days. A solution of 30 was taken from the culture solution obtained in this way, and 20 of water and 1 kg of Hyflo Super Cell (manufactured by Johns-Manville) were added to it to obtain a solution of 45, and concentrated hydrochloric acid was added dropwise to adjust the pH to 2. . This was passed through a column filled with activated carbon for chromatography (manufactured by Takeda Pharmaceutical Co., Ltd.) No. 6. 20 columns
After washing with water, elution was performed using 10 parts of n-butanol saturated water, and the effective fractions were collected and applied to a column of ion exchange resin Amberlite IR-120 (manufactured by Rohm and Haas) H ⁺ type in part 6. After washing the column with 20 mL of water, it was eluted with 3% aqueous ammonia. Effective fractions of the eluate were collected and concentrated under reduced pressure to obtain a concentrated solution of 1. Pass this through 20 molecular sieves,
The solution was passed through a column packed with Sephadex G-10 (manufactured by Pharmacia) and eluted with water. The active fractions were collected and concentrated under reduced pressure to obtain a 0.5 concentrate.
This was passed through a column filled with ion exchange resin Amberlite CG-50 (manufactured by Rohm and Haas) NH ⁺ ₄ type 2, and the effective fraction was discharged. The effective fraction was concentrated under reduced pressure to a concentration of 0.15% to precipitate white needle-like crystals. This was collected and dried to obtain 5 g of 5-hydroxymethylcytosine. Example 2 When culturing according to the method of Example 1, the amount of 5-hydroxymethylcytosine produced was quantified when cytosine was not added to the medium and when it was added at various concentrations, and the results shown in Table 1 were obtained.

[Table] Example 3 When culturing according to the method of Example 1, instead of adding cytosine to the medium, 5-fluorocytosine, 5-bromocytosine, 5-iodocytosine, and 5-methylcytosine were added to the medium at two concentrations. When the 5-hydroxymethylcytosine accumulated in the culture was quantified, the results shown in Table 2 were obtained.

[Table] Example 4 When culturing according to the method of Example 3, the strain used for culturing was Streptoverticillium rimofaciens .
Streptoverticillium rimofaciens IFO14125 instead of IFO13592 (FERM-PNo.2549)
(FERM-P No. 6052) to obtain the results shown in Table 3.

[Table] The 5-hydroxymethylcytosine obtained in Examples 1, 2, 3, and 4 above was prepared by the method of Alegria [Biochemica et Biophysica Acta].
Acta), Vol. 149, pp. 317-324, (1967)], and the 5-hydroxymethylcytosine sample synthesized based on the 5-hydroxymethylcytosine commercially available reagent manufactured by Sigma Co., Ltd. have completely identical physicochemical properties. did. That is, the elemental analysis, paper chromatography Rf value, ultraviolet absorption spectrum, and nuclear magnetic resonance spectrum are shown below. (1) Elemental analysis value Elemental analysis value; C = 42.54%, H = 5.00%, N =
29.78% (2) Paper chromatography The paper chromatography Rf values are summarized in Table 4.

【table】

[Table] *...Capacity mixing ratio
(3) Ultraviolet absorption spectrum Obtain the spectrum when using 1/10N NaOH and 1/10N HCl as solvents, and calculate the maximum absorption wavelength, minimum absorption wavelength, and 250nm.
Ratio of absorption values at 260nm and 280nm
Table 5 shows the ratio of absorption values at 260 nm.

【table】

[Table] (4) Nuclear magnetic resonance spectrum Table 6 shows the δ values (ppm) obtained from the ¹³ C-NMR spectrum measured in heavy water.

【table】

Claims (1)

[Claims] 1. 5- belonging to the genus Streptoverteicillium
A method for producing 5-hydroxymethylcytosine, which comprises culturing hydroxymethylcytosine-producing bacteria in a medium, producing and accumulating 5-hydroxymethylcytosine in the culture, and collecting the 5-hydroxymethylcytosine. 2 formulas 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).