CS248612B1 - Industrial mutant Claviceps fusiformis W1 CC-1/85 - Google Patents

Abstract

The classification concerns a new industrial mutant of the fungus Claviceps fusiformis W1 producing the alkaloid elymoclavine, deposited in the collection of microorganisms of the Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague 4 under 6. CC 1/85. Submersible fermentation of this strain can be increased several times by the production of clavine alkaloids, especially the pharmacologically important alkaloid elyamoclavine.

Description

The invention relates to a new industrial mutant of the Claviceps fusiformis Wi strain producing the pharmaceutically important alkaloid elyaoklavin (M.Abe et al., US Pat. 2835675) of the structural formula

Samples of the mutant are stored in the collection of microorganisms of the Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague 4 under the designation Claviceps fusiformis under no. CC 1/85.

The mutant was obtained from 70 isolates prepared by the mutational action of UV radiation on the laboratory strain Claviceps fusiformis MI525 (Institute of Microbiology, Czech Academy of Sciences, Prague 4) described below.

Preparation of a high-production mutant. All static cultivations of the strain were carried out in the dark at 24+1 °C on T2 agar medium (C.Spalla, in Genetice of Industrial Microorganisms. Eds.

Z.Vaněk, Z.HoStálek, U.Cudlin. Elsevier, Amsterdam 1973, p.393). Soil T2 contained the following components (gL ^-1 5: sucrose 00, L-asparagine i0, Ca(N0^) _2< 4H ₂ 0 i, yeast extract 0.1,

KH ₂ PO ₄ 0.25, MgSO ₄ .7H ₂ 0 0.25, KC1 0.12, FeSO ₄ .7H ₂ O 0.02, ZnSO ₄ .7H ₂ O 0.015, agar 20, dest.

HjO, pH 5.2 after sterilization in an autoclave for 15 min. at 105 °C.

Spores of a 21-day-old parental culture of Claviceps fusiformis HI525, grown on T2 agar medium in Endo tubes, were washed from the surface of the culture with distilled water. 5 ml of the suspension thus obtained containing approximately 1.5 .-10' spores in 1 ml was transferred to a Petri dish (0 10 cm) and irradiated with a UV source set at 10 uJ.m" ² .s"' at a dose of 0.1 mUvm" ² (1 OOO erg).

The irradiated spore suspension was suitably diluted with distilled water and the spores contained in 5 ml of the resulting suspension were sown on the surface of T2 agar medium in a Petri dish (0 10 cm) and incubated for 21 days.

Colonies grown from autogenized spores were then individually transferred to T2 agar slants and incubated for 21 to 28 days.

The grown cultures were tested for their ability to form elymoclavine and other clavine alkaloids under submerged fermentation conditions (Seháček et al., Czechoslovak AO 199986).

Alkaloids were determined in the fermentation broth of 21-day submerged cultures calorimetrically (G.J.Banks et al., J.gen. Mikrobiol 82,345,1974) and by high-performance liquid chromatography (M.Wurst et, al., J.Chromat. 150,477, 1978).

Characteristics of the high-producing mutant. A comparison of the basic characteristics of the high-producing strain W 1 with those of the parental strain MI 525 is shown in Table 1.

a) Cultivation on T 2 agar medium.

Mutant W 1 grows on T 2 agar medium at 24 +Ί °C in a continuous, richly wrinkled layer formed by grey mycelium and oval conidia. It secretes a violet pigment into the medium.

b) Submerged cultivation

Submerged cultivation of the new strain W 1 and the parent strain MI 525 was carried out at a temperature of 24+1 °C on a rotary shaker and in a laboratory fermenter on modified fermentation medium CS2 tis. A0 199986). Submerged cultures of strain W i are characterized by a filamentous, branched multicellular mycelium, which forms numerous micro- and macrocor.idia and at the end of fermentation

240612 disintegrates into blastoarthrospores. Submerged development of strain W 1 is accompanied by the formation of a purple exocellular pigment.

In comparison with the parent strain Claviceps fusiformis WI 525, the mutant W t is characterized by almost three times higher production of olymoclavin and half the content of undesirable exogenous glucans. Under conditions of submerged fermentation, it produces 4.2 g of clavine alkaloids in 1 l of medium, of which 2.5 g of elymoclavin. The accompanying alkaloids are agroclavin and chanoclavin. A comparison of the basic characteristics of the high-production mutant W 1 with the characteristics of the parent strain C.fusiformis W1525 is shown in Table 1. The mutant according to the invention is stored in the dark at 4 °C, protected against drying out of the medium, in the form of 21-day cultures grown on T2 medium slices in Endo tubes at a temperature of 24 + 1 °C. The stored cultures are regularly re-inoculated at three-month intervals.

The high-production mutant Claviceps fusiformis W 1, protected by the invention, provides a key part of the large-scale preparation of elymoclavin by submerged fermentation.

Table 1

Culture on agar medium T2 Submerged culture^

Character

strain W1 strain MT525 strain W1 strain MI525 Increase rich in the social stratum rare in individual colonies good rich Coloration of the aerial mycelium gray brown and white - - Conidia oval oval macroconidia microconidia elongated oval Soluble pigment purple red-brown dark purple brownish pink Exogenous glucans - -1 , 20 40

tg.L-'l

Total alkaloids (g.L”’) 4.2 1.2 Elymokla- vin (g.L-’) 2.5 0.9 § In modified fermentation medium CS2 (Czech copyright certificate 199986)

SUBJECT V Y H i. L SZU

Claims (1)

Industrial mutant Claviceps fusiformis W1 CC - 1/85 producing elymoclavin alkaloid.