JPS62122592A - Production of mildiomycin and mildiomycin-producing microorganism - Google Patents

Abstract

PURPOSE:To produce a large amount of mildiomycin in high efficiency on an industrial scale, by culturing a mildiomycin-producing microbial strain belonging to Streptoverticillium genus in a medium containing N-methyl compound, etc. CONSTITUTION:A mildiomycin-producing strain is converted to be resistant to serine hydroxamate or canavanine to obtain Streptomyces liquefaciens strain having improved mildiomycin-productivity and the following properties. Morphology, forms aerial mycelium; spore, oviform or cylindrical; growth temperature, 15-38 deg.C; inert to liquefaction of gelatin; assimilates inositol and does not assimilate erythritol; etc. The strain is cultured in a medium containing a carbon source, a nitrogen source, etc., and, if necessary, >=3mM of an N-methylated compound having a molecular weight of 50-1,000 or 0.1-1.0wt% 5- hydroxymethylcytosine at 15-40 deg.C for 4-14 days under slightly alkaline condition in an aerated deep tank fermentor to obtain mildiomycin.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing mildiomycin by fermentation.

(Prior Art) Mildiomycin is a compound having the structure of the formula:
It was first discovered as antibiotic B-98891 by culturing a mildiomycin-producing bacterium belonging to the genus ``B-98891,'' and its properties, especially its action characteristics, have attracted attention for its preventive, therapeutic, and acaricidal effects on powdery mildew on a wide range of crops. Therefore, it is desired to increase the production of mildiomycin, and mildiomycin-producing bacteria are treated with N-methyl compounds.
Medium containing at least 5-mM (mmol/9) or 5-
A fermentation method of culturing in a medium containing hydroxymethylcytosine has been attempted (Japanese Patent Publications No. 50-126892, No. 51-9718, No. 56-5100, No. 56-3249).
5; The Journal of Antibiotics"T
he Journal of Antibiotics
'Volume 31, No. 6, pp. 511-518 and No. 519
-524 pages (1978); Journal of the Japanese Society of Pesticides (Journal Pesticide Science)
l of Pe5ticide 5science 4th
Vol. 3, pp. 349-353 (1979); Tetrahedron Vol. 37, No. 1
317-1327 (1981), etc.).

(The problem that the invention is trying to solve) However,
In the conventional fermentation method for producing mildiomycin, the mildiomycin-producing bacteria used had a limited ability to produce mildiomycin. With something that
It cannot be said that it is sufficient for industrially producing large amounts of mildiomycin.

(Means for Solving the Problems) As a result of intensive research to improve the mildiomycin-producing ability of mildiomycin-producing bacteria, the present inventors have developed mildiomycin-producing bacteria into serine hydroxa having the structure of the formula. Canavanine “canav” has a mate or flame structure.
It was unexpectedly discovered that mildiomycin production ability was improved by making it resistant to ``an inine'', and the production efficiency of mildiomycin became higher even in culture in a conventional medium.Based on this, the present invention was developed. completed.

That is, the present invention:

(1) Mildiomycin-producing bacteria belonging to the genus Streptoverticillium and resistant to serine hydroxamate or canavanine are cultured in a medium, mildiomycin is produced and accumulated in the culture, and mildiomycin is collected. (2) The method for producing mildiomycin according to item (1), which comprises culturing in a medium containing 3mM or more of the N-methyl compound; (3) The method for producing mildiomycin according to item (1) or (2), which comprises culturing in a medium containing 5-hydroxymethylcytosine; (4) having resistance to serine hydroxamate or canavanine; Streptoverticillium rimofaciens “Strep” which also has the ability to produce mildiomycin
toverticilSium rimofacie
ns”.

Regarding.

In the method for producing mildiomycin of the present invention, a mildiomycin-producing bacterium (hereinafter referred to as "hekisei") that belongs to the genus Streptoverticillium and is resistant to serine hydroxamate or canavanine is used. For example, a heavy enclosure exhibits the following properties.

[1] Morphological Characteristics The hyphae form aerial hyphae on normal classification media and exhibit axillary branching. Each branch on the whorl is straight.

Loops and hooks are also rarely observed on certain synthetic agar media, such as glucose-asparagine agar.

The shape of the spore is oval or cylindrical, and its size is 0.
6-0.8X O, 7-1.3μ. Spores are usually 3
From 1 to 16 pieces are chained together, and smooth or warty protrusions are observed on the surface. No formation of sporangia, flagellated spores, sclerotia, etc. is observed on ordinary classification media.

[2 plants] Growth status on classification medium The findings on the classification medium of the heavy box are as shown in Table 1.

Note that unless otherwise specified, the culture findings were observed at 28°C for 21 days. Furthermore, the symbols in parentheses in the table are color name symbols according to Color Harmony Manual (4th edition) (Container Corporation of America).

[3] Physiological properties (1) Growth temperature range: It grows in the range of 15 to 38°C, but better growth and attachment of aerial mycelia are observed at 28 to 36°C.

(2) Liquefaction of gelatin (cultivation at 24°C for 28 days) No liquefaction was observed.

(3) Starch hydrolysis: Positive (4) Nitrate reduction: Bacto Nitrate Broth (l5P-No, 8)
(5) Coagulation of skim milk: Positive Peptonization: Positive (6) Production of melanin-like pigments Positive (Peptone/Yeast/Iron agar medium) Negative (Dyrosine agar medium) (7) Carbon source availability ( Prudham-Gottlieb agar) i) Commonly used carbon sources inositol, D-galactose, D-glucose, maltose, D-mannose, starch, glycerin, sodium acetate, sodium succinate, sodium citrate ii) Moderately used carbon sources Carbon source D-Fructose, Mt. Trehalose) Unutilized carbon source Erythritol, Adonitol, D-Sorbitol, D
- Mannitol, dulcitol, D-xyrose-arabinose, L-trunpause, rhamnose, melibiose, thu-rose, lactose, raffinose, salicin, nisculin, inulin (8) Chromogenic effect on protein-containing medium: weak (9)
Crack formation on broth agar medium: No growth (10) Resistance to serine hydroxamate or canavanine: Minimum concentration to inhibit growth is 40 μg for serine hydroxamate
/ml or 100 μg/ml for canavanine.
It is larger than ml.

[4] Other properties Journal of Fermentation Technology
Technology” Volume 63, No. 1, No. 17
- The specific activity for producing 5-hydroxymethylcytosine was 0.18 nm by the enzymatic method described on page 21 (1985).
ol/min/mg - Larger than protein.

As mold closures having such properties, Streptoverticillium rimofaciens is used, and more specifically, Streptoverticillium rimofaciens C, -182 (resistant to serine hydroxamate), Streptoverterticillium rimofaciens, etc. Cirium rimofaciens CV-48 (resistant to serine hydroxamate and canavanine), Streptoverticillium
limofaciens CV-16 (resistant to canavanine)
etc. are used. Streptoberticillium above
rimofaciens C5-182 was deposited with the Institute for Fermentation Foundation (IFO) on June 28, 1985 (C.E. 1985) under the accession number IFO-14448.
It has been deposited with the International Research Institute (FRI) since July 8, 1985, under the accession number FERM P-8334. Also, Streptoverticillium rimofaciens C
V-48 has been deposited with IFO since June 28, 1985 under accession number IFO-14449, and has
(Accession number F'ERMP- from July 8, 1985 to I
It has been deposited as No. 8333. In addition, Streptoverticillium limofanens CV-16 is an IF
Accession number IFO-145 from July 15, 1986 to O.
27, and was further deposited at FRI on Z 2, 1986.
Accession number FERM p-? It has been deposited as G7.

In the culture in the method for producing mildiomycin of the present invention, a medium containing a carbon source that can be assimilated by microorganisms, a nitrogen source that can be digested, inorganic salts, etc. is generally used. Further, trace amounts of effective substances such as micronutrients 1, growth promoting substances, precursor substances, etc. may be added to the medium as necessary. Common carbon sources that can be assimilated by microorganisms include starch and dextrin.

In addition to glucose, maltose, sucrose, corn syrup, starch syrup, etc., organic acids such as acetic acid and phosphoric acid, fats and oils, and polyhydric alcohols such as glycerin can be used alone or in combination.Digestion Possible nitrogen sources include inorganic nitrogen compounds such as various ammonium salts, nitrates, and urea, as well as natural organic substances such as yeast extract, casein, meat extract, cottonseed meal, corn staple liquor, and soybean meal, either singly or in combination. In addition, examples of inorganic salts include iron.

Magnesium, manganese, cobalt, copper, sodium.

Salts such as potassium, calcium, and zinc are used as appropriate and necessary. The microorganisms used are amino acids.

When specific nutrients such as vitamins and nucleobases are required, the required nutrients can be added as appropriate. The concentration of these additives is not particularly limited as long as the object of the present invention is achieved, and is appropriately selected from the range of concentration usually used in general fermentation methods.

Furthermore, in order to improve the production of mildiomycin, it is preferable to include an N-methyl compound in the medium. Such N-methyl compounds contain 1 in the molecule.
A compound having one or more nitrogen atoms substituted with 1 to 4 methyl groups is used. Among them, for example, those having one nitrogen atom substituted with a methyl group in the molecule are preferable, and in particular, trimethylammonio, which has a nitrogen atom substituted with three methyl groups, is preferable.
quaternary ammonium salts with the group + -N (Co3)3 are preferred. Also, substances that can be converted to >N-CH3 in the culture medium, such as N,N-methylenebisacrylamide,
-Used as a methyl compound. The molecular weight of the N-methyl compound is usually 50 to 1000, preferably 90 to 130.
It is. It is used regardless of whether it is water-soluble or water-insoluble, but
Easily water-soluble N-methyl compounds are easily available. Specific examples include N-methyl acid amide, N-methylamino compound, N-methylamine, N-methylammonium, and N,N-methylenebisacrylamide.

Examples of the N-methyl acid amide include N,N-tmethylacetamide, N-methylacrylamide, and N-methylacrylamide.
Examples of methylamino compounds include N-methylurea, I, 1,
3.3-Tetramethylurea, 2-dimethylaminoethanol, etc., N-methylamines include trimethylamine, dimethylamine, etc., and N-methylammoniums include lecithin, choline, betaine, etc.

Tetramethylammonium and the like are advantageously used. N
- Methylammonium Good results are obtained especially with choline, betaine, tetramethylammonium. These N-methyl compounds may be used alone or in combination of two or more. In addition, a large amount of a substance containing an N-methyl compound, such as sugar beet molasses containing betaine, soybean flour containing lecithin, or chicken eggs containing choline and lecithin, is added to the medium, so that it contains 3Il1M or more as an N-methyl compound. The methods of the present invention include methods of The concentration of the N-methyl compound contained in the medium may be appropriately selected at li1'rM which does not inhibit the growth of the microorganism used, and is usually contained at 3+++M or more. Desirably 4+++M to 200mM 1 More preferably 7 to 5 (addition of 1mM is effective, 20
If it exceeds 0mM, the rate of progress of the effect will slow down.

In addition, the amount of natural products containing N-methyl compounds added may be selected so that the concentration of the N-methyl compounds contained falls within the above-mentioned range, but the amount exceeding the amount of conventional natural products added may be selected. Therefore, if the natural product is added alone, the growth of microorganisms will be significantly affected by the influence of other ingredients, and the production of mildiomycin may be inhibited.
In that case, it is preferable to use an N-methyl compound in combination. The easiest and most effective way to add these N-methyl compounds to the medium is before inoculating the medium with microorganisms, but they can also be added as appropriate during cultivation.

Furthermore, in the present invention, in order to facilitate the production of mildiomycin, 5-hydroxymethylcytosine or a compound that produces mildiomycin may be present in the culture medium. When adding 5-hydroxymethylcytosine or when adding a compound that produces 5-hydroxymethylcytosine in the medium, the concentration of 5-hydroxymethylcytosine is 0.01 to 1.5% as 5-hydroxymethylcytosine.
0% (weight/volume) is preferable, more preferably 0% (weight/volume)
．． 03-0°5% (weight/volume). The most effective time to add these to the medium is before the start of culture, but they may also be added at some point during culture.

Although culture may be carried out by surface culture, it is usually more reasonable to use deep aeration culture. When using deep culture method,
The liquid nature of the medium is preferably slightly acidic or slightly alkaline, and the culture temperature is desirably maintained at 15-40°C, particularly 24°C-34°C. However, it goes without saying that these culture conditions should be selected as appropriate to obtain preferable results depending on the type of bacterial strain used, external conditions, etc. Normally, if cultured for 4 to 14 days, a culture solution containing a significant amount of mildiomycin can be obtained, but in order to collect mildiomycin from the culture solution, it is necessary to collect the metabolites of the microorganism from the culture solution. Conventional separation and collection means are used as appropriate. For example, mildiomycin is a water-soluble basic substance and is mainly contained in the culture solution, so the bacterial cells are first removed by means such as filtration or centrifugation. The obtained supernatant liquid is brought into contact with an appropriate 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, such as acetone or methanol. The target substance can be eluted using a water-containing solution of a water-soluble organic solvent such as ethanol, propatool, or butanol, or an aqueous solution of acid, alkali, buffer, or inorganic salt or organic salt as a solvent. After carrying out an appropriate combination of these separation means and then concentrating and pulverizing the effective fraction, mildiomycin can be collected in a free state or a salt state.

Mildiomycin-producing bacteria belonging to the genus Streptoverticillium and resistant to serine hydroxamate or canavanine used in the method for producing mildiomycin of the present invention are, for example, mildiomycin-producing bacteria belonging to the genus Streptoverticillium. It can be obtained by mutating bacteria to be resistant to serine hydroxamate or canavanine. Mildiomycin-producing bacteria belonging to the genus Streptoverticillium that can be subjected to mutation induction include:
For example, Streptoverticillium rimofaciens E5-24-5 (hereinafter referred to as the "parent strain") is frequently used. The above parent stock was listed at IFO in 1982 (1987).
It has been deposited with the accession number IFO-14125 since June 12, 1981, and has been further deposited with the FRl as the accession number FERMP-6052 since June 24, 1981. Methods for inducing mutations to serine hydroxamate or canavanine resistance include, for example, irradiating spores or hyphae of mildiomycin-producing bacteria belonging to the genus Streptoverticillium with ultraviolet light; A method of contacting a bacterium with N-methyl N'-nitro N-nitrosoguanidine having the structure of the formula, a method of contacting a bacterium that produces mildeiomycin belonging to the genus Streptoverticillium with a D-4-amino- A method of growing in a medium containing 3-isoxazolidone (common name "cycloserine"), a method of using a combination of these methods, etc. are used.

For example, in a mutation induction method in which spores or hyphae of a mildiomycin-producing bacterium belonging to the genus Streptoverticillium are irradiated with ultraviolet rays, an ultraviolet lamp of 10 to 100 watts, preferably 30 to 60 watts, is used to induce spores or hyphae of a parent strain. Alternatively, it is better to irradiate the mycelium with ultraviolet light. The wavelength of ultraviolet rays is usually 130 to 350 nm, preferably 1
90-290 nm is used.

The irradiation time is usually 30 seconds to 5 minutes, preferably 60 seconds to 9 minutes.
It is 0 seconds. The spores or hyphae of the parent strain may be suspended in, for example, sterilized water and then subjected to ultraviolet irradiation. Specifically, a suspension of spores of the parent strain in sterile water is placed 30 cm below a 30-watt ultraviolet light lamp installed in a sterile box, and mutations are induced by irradiation with ultraviolet light (wavelength 2537A) for 90 seconds while stirring. You may.

In the mutation induction method, a mildiomycin-producing bacterium belonging to the genus Streptoverticillium is brought into contact with N-methyl N'-nitro N-nitrosoguanidine [IV].
It is preferable to bring the compound [IV] into contact with spores of mildiomycin-producing bacteria and single cells of aerial hyphae. The contact may be carried out by any method as long as the two come into contact, but it is usually carried out by mixing or the like. The temperature at the time of contact is
The temperature is 0°C to 50°C, preferably 106C to 40°C. The contact time is usually 10 seconds to 3 hours, preferably 10 minutes to 2 hours. Spores and single cells of aerial hyphae of mildiomycin-producing bacteria may be used by being dispersed in a suitable dispersion medium, such as sucrose 1-9% (w/v), sodium glutamate 1-6% (w/v). v), Tween 80 (manufactured by Kao Atlas Co., Ltd.) A dispersion medium consisting of 0.001 to 0.1% (w/v) and sterile water was used by dispersing it in a dispersion medium that had been sterilized by steam at 110 to 130°C for 10 to 30 minutes. It's okay. Compound [IV] may also be used after being dissolved in a buffer such as trisaminomethane-hydrochloric acid buffer (pif 7.5). The temperature at which compound [IV] is dissolved in the buffer is 1.0 to 50 degrees.
00 μg/ml, preferably 100 to 200 μg/ml.

Furthermore, in a mutation induction method in which a mildiomycin-producing bacterium belonging to the genus Streptoverticillium is grown in a medium containing cycloserine [V], the mildiomycin-producing bacterium is grown at 10 to 100 μg/ml of cycloserine [V], preferably 20 to 100 μg/ml. It is preferable to culture in a medium containing 60 μg/ml. Other culture conditions may be appropriately selected from those described above for culturing mildiomycin-producing bacteria resistant to serine hydroxamate or canavanine.

Mildiomycin-producing bacteria that are resistant to serine hydroxamate or canavanine are selected from among the mutant strains obtained by such a mutation induction method. Such selection can be carried out according to methods for selecting resistant bacteria that are commonly used in the field of actinomycetes. serine hydroxamate (40~
200 μg/ml, preferably 60-120 μg/ml)
Alternatively, it may be carried out by coating or transplanting on a medium containing canavanine (100 to 300 μg/ml, preferably 110 to 240 μg/ml) and isolating a growing strain. In addition, the suspension, mixture, or bacterial flora obtained after the mutation induction treatment was treated with aminopterin (2 to 50 μg/m
1 (preferably 5 to 20 μg/ml) and allowed to grow, a part of the agar (agar block) is cut out and the antibacterial activity of the agar piece is assayed by a conventional method (test bacteria, Rhodotorula rubra) L,
By selecting a compound with strong antibacterial activity, it is possible to use it as a guideline to more reliably select an excellent mildiomycin producing plant having serine hydroxamate resistance. Mirdinama 7 Tsu Nodefu P which is resistant to serine hydroxamate or canavanine mentioned above! li
By comparing the two results, it is possible to obtain a mildiomain-producing bacterium with enhanced resistance to serine hydroxamate or canavanine. In addition, by mutating serine hydroxamate-resistant mildiomycin-producing bacteria or canavanine-resistant mildiomycin-producing bacteria using the above method, mildiomycin-producing bacteria that are resistant to both serine hydroxamate and canavanine can be produced. You can do it by getting . All of these bacteria also belong to the genus Streptoverticillium and fall within the range of mildiomycin-producing bacteria that are resistant to serine hydroxamate or canavanine.

The mildiomycin obtained by the above-mentioned method of the present invention can be safely used, for example, as an active ingredient in powdery mildew control agents or fungicidal film acaricides for plants (Japanese Patent Publications No. 56-37795 and No. 58-21886).

(Function) Belongs to the genus Streptoverticillium in the present invention,
Mildiomycin-producing bacteria that are resistant to serine hydroxamate or canavanine not only have excellent resistance to serine hydroxamate or canavanine, but also have high activity in 5-hydroxymethylcytosine biosynthesis. There is.

Experimental Example 1 2% sucrose, 0.5% soluble starch, 0.12% ammonium nitrate, 0.25% dibasic potassium phosphate.

Magnesium sulfate 0.005%, Casamino acid 0.002
5%.

An agar medium (pH 7.0) consisting of 0.0025% yeast extract and 2.0% powdered agar (expressed as a percentage W/V) was prepared by adding serine hydroxamate and canavanine at various concentrations. After sterilization at 120°C for 15 minutes, 20ml of each was poured into petri dishes, and after cooling, spores of the following test bacterial strains were applied and cultured at 28°C for 10 days to reach the growth limit concentration of M, [, C (minimum growth inhibitory concentration) was measured and summarized in Table 2.

Test strain: (1) Streptoverticillium rimofaciens E5-24-5 (2) Streptoverticillium rimofaciens C5-182 (3) Streptoverticillium rimofaciens CV-48 (4) Streptoverticillium rimofaciens CV-48 Lium limofanens CV-16 Table 2 Experimental Example 2 Glucose 2.5%, Corn Stapliquor 2%.

A medium consisting of 1.0% cottonseed germ powder (trade name: Proflo, manufactured by Trader Oil), 0.3% ammonium sulfate, 0.05% magnesium sulfate, and 0.3% calcium carbonate (percentages are weight/volume). Construction, 20% (w/v
) Adjust the pH to 7.0 by dropping an aqueous solution of caustic soda.
Adjust to. Dispense 25 ml of this medium into 200 ml Erlenmeyer flasks (with urethane stoppers), and
Steam sterilize under conditions of 1 minute. In this, Streptoverticillium rimofaciens E5-24-5゜C3182
, CVR48, and CVR-16 spores were inoculated and incubated at 28°C.
The mixture is cultured in a rotary shaker incubator at 200 rpm (rotation time: 7 minutes) for 20 hours to obtain a seed culture. Next, glucose 11%, sorbitol 3.0%, casein 3.0%, cornstarch liquor 0.5%, proflo 2,
0%, ammonium sulfate 0.5%, ammonium nitrate 0
．． 5%, choline chloride 0.1%, manganese sulfate 0.005
%, ferrous sulfate 0.005%, copper sulfate o, ooa% (percentages are weight/volume).
(W/V) pH by dropping aqueous caustic soda solution
Adjust to 7.0. Add 1% calcium carbonate to this medium.
(W/V) at 6 degrees Celsius, mix well, and then dispense 25 ml portions into 200 ml Erlenmeyer flasks (with urethane stoppers), steam sterilize at 120°C for 20 minutes, and prepare the fermentation medium. do. The above seed culture solution was transplanted into one fermentation medium (25 ml) at a ratio of 2 ml, and the mixture was incubated at 28°C for 3 ml.
Culture in a rotary shaking incubator at 200 rpm for ~5 days. After culturing, Journal of Fermente
John Technology'Journal of Fer
mentation Technology” Volume 63,
According to the method described on page 17 (1985), a cell-free extract was prepared, an enzyme reaction was performed, and 5-hydroxymethylcytosine was measured, and the 5-hydroxymethylcytosine production activity shown in Table 3 was obtained.

Table 3 E5-24-5 0.17 03 182 0.24 CV -480,27 CV -160,19 m Average values during 3-day, 4-day and 5-day culture (Example) Example 1 (1) Streptococcus Verticillium rimofaciens E5-24-5 (r'ERM P-6052, IFO
-14125), spores and aerial mycelia were scraped off from a slant culture of
Dispersion medium consisting of 0.01% Tween 80 (120℃,
Steam sterilized for 15 minutes) into 30ml,
Grind well using a Potter homogenizer. The ground material is filtered using Toyo Roshi No. 101 (manufactured by Toyo Roshi Co., Ltd.) to obtain a bacterial suspension consisting of spores and aerial mycelium fragments as a filtrate (25 ml).

(2) Add N-methyl N' to the bacterial suspension obtained in (1).
-Nitro-N-nitrosoguanidine at 2 mg/m in trisaminomethane-hydrochloric acid buffer (0.1 M, pH 7.5)
Add an equivalent amount of the solution obtained by dissolving the solution to a concentration of 2
Shake for 90 minutes at 8°C. The mixture thus obtained contained cycloserine at a concentration of 30 μg/ml,
Sucrose 2.0%, soluble starch 0.5%, ammonium nitrate 0.2%, dibasic potassium phosphate 0.1%.

Potassium chloride 0.05%, magnesium sulfate 0.05%
Agar plate medium (pH 7,
0). After static culture at 28° C. for 14 days, 100 morphologically normal colonies with attached aerial mycelia were isolated from among the colonies that appeared. Each of these colonies is ground with a sterile glass rod, about 10 ml of sterile water is added, and a filtrate is obtained by filtration using Toyo Filter Paper N001 inch. This filtrate was applied to an agar plate medium containing no cycloserine and incubated at 28°C for 10 minutes.
After statically culturing for several days, each colony that appeared was placed on a slanted agar medium (medium composition is the same as the agar plate medium described above).
port to. By statically culturing at 28°C for 10 days, the grown aerial mycelium and spores were scraped off from the slant medium, suspended, ground, and filtered with filter paper using the method described in (1) above to obtain a suspension of 100 species of bacteria. A suspension was prepared.

(3) 10 μg of the 100 types of bacterial suspension obtained in (2) above
The cells were plated on the upper surface of an agar plate containing 1/ml of aminopterin, and cultured at 28° C. for 10 days.

From this agar plate, use cork polar to cut agar pieces (6 mm in diameter).
, about 4 mm in height) was cut out and used as an antibacterial assay medium (
Test bacteria, Rhodotorula rubra, broth agar medium.

pH 7.0) and incubated at 30°C overnight.
Five types of bacterial suspensions showed relatively large inhibition circles with diameters of 14 mm or more.

(4) The 100 types of bacterial suspension obtained in (2) above were diluted with sterile water until the number of viable bacteria was approximately IX IQ'/ml, and 20 μσ of this bacterial suspension was mixed with 40 μg/ml of serine Spread on agar plate medium containing samate at 28℃ for 14 hours.
Incubate for 1 day. Of the 100 types of bacterial suspensions, 7 types were able to grow, including all 5 types of bacterial suspensions that showed large halos in (3) above. Among these five bacterial suspensions, one mutant strain that showed the largest halo in (3) above was isolated as Streptoverticillium rimofaciens C1-18.

Example 2 Streptoverticillium 1 Nomofaciens E5-
1 by the same method as (1) and (2) of Example 1 above using Cl-18 obtained in Example 1 instead of 24-5.
Fifty kinds of bacterial suspensions were prepared. Among these, Example 1 (
Streptoverticillium sumofaciens C2125 was grown in the same manner as 3) and (4) (with the exception that an agar plate containing 1 g/ml of serine hydroxamate was used at 50°C). separated.

Example 3 Streptoverticillium rimofaciens E5~
200 in the same manner as in Example 1 (1) and (2) using cl-125 obtained in Example 2 instead of 24-5.
A suspension of seeds was prepared. Among these, Example I (3)
And in the same manner as (4) (but using an agar plate medium containing 601°C g/ml serine hydroxamate as the agar plate medium in the method of 4)), Streptoverticillium rimov 7she, R, / Cs 182 (FERM P-8334, I
FO-14448) was isolated.

Example 4 Streptoverticillium rimofaciens E 5
A suspension of 300 types of bacteria was prepared in the same manner as in Example 1 (1) and (2) using c-182 obtained in Example 3 instead of -244. Among these, in the same manner as (3) and (4) of Example 1 (except in method 4), an agar plate medium containing 80 μg/ml of serine human loxamate was used as the agar plate medium), Streptoverticillium・Remofaciens C, -257 was isolated.

Example 5 Streptoverticillium rimofaciens E 5
Using 04-257 obtained in Example 4 instead of -24-5, 100 bacterial suspensions obtained in the same manner as in Example 1 (1) and (2) were added to 190 μg/n+1 of canavanine. The mixture is applied onto an agar plate medium containing the following, and cultured stationary at 28°C for 14 days. Colonies that emerge from a suspension of one type of bacteria are mixed with 2.0% sucrose and 0.5% soluble starch.

Ammonium nitrate 0.12%, dipotassium phosphate 0.2
5%.

Magnesium sulfate 0.005%, Casamino acid 0.002
5%.

It was transplanted onto an agar slant medium (pH 7.0) consisting of 0.0025% yeast extract and 2.0% agar (percentages are weight/volume) and statically cultured at 28°C for 10 days. Mutant strain C resistant to canavanine
V-48 (FERM P-8333, IFO-14
449) was obtained.

Example 6 2.5% glucose, 2% cornstarch liquor.

Consisting of cottonseed germ powder (trade name: Proflo, manufactured by Trader Oil) 1.0%, ammonium sulfate 0.3%, magnesium sulfate 0.05%, calcium carbonate 0.3% (percentages are weight/volume) Prepare a medium and add 20% (w/v
) Adjust the pH to 7.0 by dropping an aqueous solution of caustic soda.
Adjust to. Transfer this medium to a 200+ nl Erlenmeyer flask (
Dispense 25ml each into 1206C1 (with urethane stopper).
Steam sterilize for 15 minutes. This was inoculated with spores of Streptoverticillium rimofaciens E5-24-5゜RR C3-182, CV-48 and heated to 28°C.
, for 20 hours at 20 rpm (rotation for 7 minutes) in a rotary shaking incubator to obtain each seed culture.

Next, glucose 11% (w/v), sorbitol 3
．． 0%.

Casein 3.0%, cornstarch liquor 0.5%,
Proflo 2.0%, ammonium sulfate 0.5%, ammonium nitrate 05%, choline chloride 1%, manganese sulfate 0.005%, ferrous sulfate 0.005%, copper sulfate 0.0
A medium containing 0.03% (percentages are weight/volume) was prepared, and the pfl was adjusted to 7°0 by dropping a 20% (w/v) aqueous solution of caustic soda. Add calcium carbonate to this medium to a concentration of 1% (W/v), mix thoroughly, and then add 25 ml each into 200 ml Erlenmeyer flasks (
(with urethane stopper) and steam sterilized at 120°C for 20 minutes to obtain a fermentation medium. The above seed culture solution was transferred to one fermentation medium at a ratio of 2 ml, and the mixture was heated at 28°C for 1 hour.
Culture is carried out in a rotary shaking culture machine at 20 rpm for 0 days.

After culturing, the Journal of Fermentation Technology “Journal of Fermenta”
tionTechnology” Volume 62, page 537 (
Mildiomycin is quantified by high performance liquid chromatography according to the method described in (1984). We conducted a production test of mildiomycin using this method,
The results shown in Table 4 were obtained.

Table 4 Example 7 Strepto verticillium under the same conditions as Example 6.
Limofascens CV-48 was subjected to culture medium 1, the fermentation finished liquid 1Q was collected, a supernatant was obtained by centrifugation, and the ion exchange resin Amberlite IRC-50 (H+
- type) (manufactured by Rohm & Haas) 2.512 was passed through a column packed with the solution. After washing the column with water, 0.
Elute using 2.5Q of 5% (w/v) ammonia water,
The active fraction (quantified by the method shown in Example 6) was passed through a column of activated carbon for chromatography (manufactured by Ota Pharmaceutical Co., Ltd.) 25On+1 to be adsorbed. The active components eluted using a mixed solvent of acetone and water (1.2512 mm) were collected and concentrated, and 500 ml of methanol was added to the concentrated solution to precipitate the original mildiomycin. 6.5 g of crude mildiomycin powder obtained after filtration was dissolved in water and passed through a 125 ml column of Amberlite CG-50 (l (''-type) (manufactured by Rohm and Haas). It was washed with 50ml of water, and then eluted with 1.25ml of 0.5% (W/V) aqueous ammonia.The active fractions were collected, passed through a 250ml column of activated carbon for adsorption, and acetone-0, Fractional elution was performed using 1.25Q of 1N formic acid (2+8), and the active fraction was concentrated.

Methanol was added to the concentrated solution to precipitate mildeiomycin. This was collected by centrifugation, dried under reduced pressure,
5.5 g of rudiomycin formate was obtained. Among the physical and chemical properties of this substance, melting point, optical rotation (C1,0, H2O
and C1,0,0,1N-HCl ultraviolet absorbance coefficients are from the literature [The Journal of Antibiotics “
The Journal of'Antibio
tics” Volume 31, No. 6, Page 523, 1978
It completely matched the value listed in [Year].

Example 8 Streptoverticillium rimofaciens E5-
24-5, N-methyl N' to nitro-N-nitrosoguanidine was added to the bacterial suspension obtained in the same manner as in Example 1 (1) using trisaminomethane-hydrochloric acid buffer (
Add an equivalent amount of a solution obtained by dissolving it in 0.1M I)H7,5) to a concentration of 2 mg/ml, and shake at 28°C for 90 minutes. 300μ of the mixture thus obtained
It was coated on an agar plate medium containing g/ml of Shiichi canavanine (medium composition is the same as in Example 1 (2)), and incubated at 28°C for 14 hours.
Culture in vitro. Each colony that appeared was transplanted onto a slanted agar medium (medium composition is the same as the agar plate medium), and
External culture was performed at 8° C. for 10 days, and 14 mutant strains having canavanine resistance were obtained.

These canavanine-resistant mutant strains were isolated from their parent strain (E5-2
Streptoverticillium rimofaciens CV-16 (FERM P- 1g741
．． rFo-1452r). Table 5 shows the amounts of mildiomycin produced by the parent strain and mutant strain CVR-16.

From this table, it is clear that the mutant strain CV'16 has a superior ability to produce mildiomycin compared to the parent strain.

Example 9 Strepto verticillium was grown under the same conditions as in Example 6.
S. rimofaciens CVR-16 was cultured, the fermentation-finished liquid IR was collected, and mildiomycin was isolated and purified in the same manner as in Example 7. As a result, 3.1 g of mildiomycin formate was obtained. . Among the physical and chemical properties of this product, the melting point, optical rotation, and ultraviolet absorbance coefficient at 271 nm and 280 nm completely agreed with the values in the literature (described in Example 7).

(Effects of the Invention) The method for producing mildiomycin of the present invention, which involves culturing mildiomycin-producing bacteria that belong to the genus Streptoverticillium and are resistant to serine hydroxamate or canavanine, can produce mildiomycin with high efficiency. This is an advantageous industrial fermentation method for producing large amounts of mildiomycin.

Claims (4)

[Claims] (1) Mildiomycin-producing bacteria belonging to the genus Streptoverticillium and resistant to serine hydroxamate or canavanine are cultured in a medium, mildiomycin is produced and accumulated in the culture, and mildiomycin is collected. A method for producing mildiomycin, characterized by: (2) The method for producing mildiomycin according to claim (1), which comprises culturing in a medium containing 3 mM or more of the N-methyl compound. (3) The method for producing mildiomycin according to claim (1) or (2), which comprises culturing in a medium containing 5-hydroxymethylcytosine. (4) Streptoverticillium rimofaciens that is resistant to serine hydroxamate or canavanine and has the ability to produce mildiomycin.