GB2040281A - Preparation of xanthothricin - Google Patents

Abstract

The antibiotic xanthothricin may be prepared by cultivating axanthothricin- producing strain of the microorganism Streptomyces bruneus, particularly Streptomyces brunneus subspecies xanthrothricini, which is available under the accession No. RIA 1568 from the All-Union Research Institute of Antibiotics of the Union of Soviet Socialist Republics.

Description

SPECIFICATION Preparation of xanthothricin The present invention relates to the preparation of xanthothricin (toxoflavin).

It is known that xanthothricin can be produced by the cultivation of Pseudomonas cocovenenans. We have now discovered that it is also produced by certain subspecies of Streptomyces brunneus.

Thus, the present invention consists in a process for preparing xanthothricin, which comprises cultivating axanthothricin-producing strain of Streptomyces brunneus in a nutrient medium therefor.

We have also discovered a new subspecies of Streptomyces brunneus which has a particularly good production of xanthothricin. This mictoorganism is Streptomyces brunneus subspecies xanthothricini. A culture of this microorganism has been isolated from soil in Soviet Central Asia and has been deposited in the culture collection of microorganisms of the All-Union Research institute of Antibiotics (RIA) under the accession No. 1568. This culture collection has been registered by the International Federation of Culture Collections, Register for 1972, entry No. 337.

In describing the microorganisms the Bondartsev Colour Scale (USSR Academy of Sciences Publishing House, 1954) has been used.

The microorganism Streptomyces brunneus xanthothricini has the following characteristics: (I) CULTURAL AND MORPHOLOGICAL FEATURES Straight sporophores arranged monopodially and individually. Spores oblong, elongate, cylindrical with smooth membranes. No sporangia or sclerotia.

1. Saccharose-containing synthetic media Aerial mycelia are well-developed, tomentous, whitish-pink or light pink. Colonies and media have brown or dark brown colour.

2. Glucose-asparagine agar Aerial mycelia are weakly developed, practically absent; whitish-pink colour. Colonies and medium are of light sandy colour, 3. Glycerol-asparagine medium Colonies and medium are brown. Aerial mycelia are weak, velvety, sandy-pink in colour.

4. Starch agar Aerial mycelia are whitish-pink and farinaceous. Colonies and medium are brownish-yellow.

5. Tyrosine-containing agar Aerial mycelia weakly developed, farinaceous, whitish-pink. Colonies and medium colourless.

6. Meat-peptone agar No aerial mycelium. Colonies dark-brown. A dark brown pigment diffuses into the medium.

7. Yeast-malt salt medium Aerial mycelia weakly developed, whitish-pink. Colonies and medium brownish-tan.

8. Oatagar Aerial mycelia weakly developed, sandy colour. Colonies and medium dark-brown.

(II) PHYSIOLOGICAL CHARACTERISTICS Liquifies gelatin, peptonizes milk, inverts sacchrose, hydrolyzes starch, grows poorly on cellulose, reduces nitrates, forms no H2S, tyrosinase negative and forms soluble brown pigment on synthetic media. The culture is aerobic.

Carbon source assimilation pattern Glucose ++ Arabinose ++ Xylose + Saccharose ++ Raffinose ++ Rhamnose + Fructose Inositol Mannitol The culture possesses no bactericidal properties.

Taxonomic studies of Streptomyces brunneus subspecies xanthofhricini were conducted by reference to the following prior art publications: N.A. Krasilnikov "Radiant Fungi", Nauka Publishing House, 1970; G.F.

Gauze "Problems of Classification of Actinomycetes-Antagonists", Medgiz Publishing House, 1957; S.

Waksman and H. Lechecalier "Guide to the Classification and Identification of Actinomycetes and their Antiobiotics", Baltimore, 1953; and S Waksman, "The Actinomycetes", Volume II, Baltimore, 1961.

From its cultural and morphological features and from certain biochemical properties, the new microorgnism may be related to the species Streptomyces brunneus (Krasilnikov 1970) although it differs from this known species in certain biochemical and antibacterial properties. Thus, according to the data obtained by Krasilnikov, Streptomyces brunneus exhibits antibacterial activity against Bacillus idosus and Bacillus subtilis and also inhibits bean root nodule bacteria, such as Azotobacter chroococcum and Azotobacter vinelandii whereas the new strain exhibits no such antagonism.

The results of a comparison between the physiological features of Streptomyces brunneus and those of the new strain, Streptomyces brunneus species xanthothricini are given in the following Table.

TABLE Physiological characteristics Streptomyces Strepto myces brunneus brunneus subspecies xanthothricini Liquefaction of gelatin + + Peptonization of milk + + Sugar inversion + + Starch hydrolysis + + Decomposition of cellulose + + Formation of H2S Formation of tyrosinase Formation of melanoid pigment + + Attitude to sugars Glucose ++ ++ Arabinose ++ Xylose + Raffinose ++ ++ Rhamnose + Saccharose ++ ++ Fructose lnositol Mannitol As can be seen from the data in the Table above, the strain Streptomyces brunneus subspecies xanthothricini differs from Streptomyces brunneus itself in certain properties and, moreover, is capable of producing xanthothricin, which the type strain does not do.

Accordingly, it is concluded that the new microorganism is a subspecies of Streptomyces brunneus and we have named it Streptomyces brunneus subspecies xanthothricini.

The ability of the new microorganism to produce xanthothricin can be increased by a variety of means commonly used for producing variants, for example: irradiation with ultra-violet radiation or X-rays; treatment with chemical reagents, such as N-ethyl-N'-nitro-N-nitrosoguanidine, 5-bromouracil or 2 aminopurine;transformations;transduction; or conjugation.

A culture of Streptomyces brunneus subspecies xanthothricini, when grown on a glucose-containing synthetic medium, gives rise to several variants, which vary in form, the presence or absence of aerial mycelia, colour and xanthothricin-producing ability. These variants have the following cultural and morphological characteristics: Variant 1 Colonies are round, elevated and smooth with an even edge. Aerial mycelia are tomentous and pink.

Substrate mycelium is brown to dark-brown. Forms a soluble brown pigment.

Variant 2 Colonies are smooth, flat with an even edge and covered with a whitish-pink velvety aerial mycelium.

Substrate mycelia are light sandy to brownish. Forms a soluble brown pigment.

Variant3 Colonies are pleated, weakly sporulating. Aerial mycelia are whitish-pink. Substrate mycelia are sandy in colour.

All of these variants are capable of producing xanthothricin in an amount up to 100 llg/ml.

The xanthothricin producing strain of Streptomyces brunneus may be cultivated on nutrient media of a type conventional for this kind of microorganism. In general, such media will contain assimilable sources of carbon, nitrogen and generally inorganic salts. The cultivation is preferably carried out under aerobic conditions.

Preferred sources of assimilable carbon are carbohydrates, such as glucose, saccharose, glycerol or starch. It is also possible to use arabinose, xylose, raffinose, rhamnose and other similar compounds.

Preferred sources of assimilable nitrogen include meat extract, corn extract, yeast extract, peptone, casein hydrolyzate, soy flour, cottonseed flour and corn flour, as well as inorganic and organic nitrogen-containing compounds such as ammonium salts (e.g. the nitrate, sulphate or phosphate) or urea.

The medium may also include mineral salts, such as calcium carbonate, sodium or potassium phosphates, sodium or potassium chloride or salts of magnesium or copper.

In order to obtain an improved yield of xanthothricin, it is best to carry out the culture in two stages under deep fermentation conditions with controlled aeration. For inoculation into the fermenters, vegetative mycelia are used. The medium on which the vegetative mycelia are grown may be the same as or different from that employed for the bio-synthesis of xanthothricin.

Fermentation to produce the xanthothricin is generally and preferably conducted at a temperature of from 20 to 40"C, more preferably from 26 to 29"C over a period which may range from 30 to 100 hours. The rate of aeration is preferably from 0.8 1 to 1 1 v/v per minute.

At the end of the fermentation period, the xanthothricin produced may be recovered by conventional means. For example, the culture broth may be filtered to separate off the mycelia and the filtrate extracted at a suitable pH value with an organic solvent, a chlorinated hydrocarbon being preferred. The xanthothricin may then be crystallized from the resulting extract. However, this sequence and combination of steps may be varied and, if required, a certain of these operations may be repeated.

Xanthothricini produced by the process of the invention comprises crystals melting at 172 - 173C (from ethyl acetate) and having an Revalue of 0.09 on thin layer chromatography on silica gel developed with a 3 2 by volume mixture of ethyl acetate and benzene.

Ultraviolet absorption spectrum (ethanol) Xmax nm(loge): 261 (4.18); 330 shoulder (3.29); 400 (3.59).

Infrared absorption spectrum (KBr) Ymax cm~1: 1706,1680,1615,1540.

Proton magnetic resonance spectrum (CDCl3) ppm: 3.50 (3H, singlet); 4.16 3H, singlet); 8.79 (singlet).

The preparation of xanthoth ricin by cultivating Streptomyces brunneus subspecies xanthothricini is illustrated by the following Examples.

Example 1 Into a series of 750 ml Erlenmeyer flasks were introduced 125 ml portions of a nutrient medium having the following composition (% dry solids by weight): Corn extract 0.25 Calcium carbonate 0.5 Sodium chloride 0.5 Ammonium sulphate 0.35 Glucose 1.5 Starch 2.0 Tap water to 1 litre pH 6.9-7.0.

The medium was sterilized under a pressure of 0.8 atmosphere for 30 minutes, after which it was inoculated with an agar biock of a 10 - 12 day old culture of Streptomyces brunneus su bspecies xanthothricini RIA 1568.

The flasks were placed on circular shakers rotating at a speed of 230 - 240 rpm and incubation was continued for 48 hours at a temperature of 26 - 29"C.

The resulting 48 hour old vegetative mycelia were transferred into 750 ml Erlenmeyer flasks each containing 125 ml of a sterile nutrient medium having the composition described above. Biosynthesis of the antibiotic was then conducted under the same conditions as were employed for growing the inoculation material but for a period of 72 hours.

The mycelia were then separated by filtration from 6 litres of the culture broth containing 90 mcg/ml of xanthothricin. The filtrate was acidified with dilute sulphuric acid to a pH of 5.6 - 6.0 and extracted with chloroform. The extract was dried over anhydrous sodium sulphate and the solvent was evaporated off under vacuum (20 mm Hg) at a temperature of 35"C. The residue was dissolved in 20 ml of isopropanol and allowed to stand for 6 hours at 5"C. The precipitated crystals of xanthothricin were filtered off and a further portion of product was isolated from the mother liquor by evaporation and recrystallization from isopropanol. The total quantity of product obtained was 363 mg, melting point 172 - 173"C.

Example 2 Into a 45 litre fermenter were charged 25 litres of a nutrient medium having the composition specified in Example 1. This medium was sterilized at a temperature of 122 - 124"C for 35 minutes and then inoculated with the inoculation material described in Example 1 in an amount of 2% by volume of the medium.

Fermentation was carried out for 65 - 72 hours. The rate of aeration was 0.8 - 1 air volume per volume of medium per minute and and sperm whale fat was used as an anti-foaming agent.

The mycelia were separated from 20 litres of culture broth and the native liquor was extracted with chloroform. After separation of the chloroform phase, the aqueous phase was acidified to pH 5.0 and extracted with ethyl acetate. The solvents were evaporated from the chloroform and ethyl acetate phases and the residues were combined and subjected to column chromatography through a column containing 300 g of anhydrous silicic acid (particle size 150 - 200 mesh). On elution with ethyl acetate, the fraction with an Rf value of 0.08 (silica gel developed with ethyl acetate) was collected and 962 mg of xanthothricin were isolated from this fraction. The melting point was 172 - 173 C (yellow platelets from ethyl acetate).

Example 3 From 6 litres of a culture broth produced as described in Example 1, the mycelia were separated by filtration. 3.6 kg of ammonium sulphate were added to the solution. The precipitate was filtered off and the filtrate was extracted with chloroform. Subsequent treatment was conducted following the procedure described in Example 1.

Claims (7)

1. A process for preparing xanthothricin which comprises cultivating a xanthothricin-producing strain of Streptomyces brunneus in a nutrient medium therefor.

2. A process according to Claim 1, in which Streptomyces brunneus subspecies xanthothricini is used.

3. A process according to Claim 2, in which the Streptomyces brunneus subspecies xanthothricini is that available under the accession No. RIA 1568.

4. A process according to any one of the preceding Claims, in which the culutre is effected at a temperature of from 26 to 29"C.

5. A process according to any one of the preceding Claims, effected under aerobic conditions.

6. A process according to Claim 1, substantially as hereinbefore described with reference to any one of the foregoing Examples.

7. Xanthothricin when produced by a process according to any one of the preceding Claims.