CS204303B1 - Industrial mutant of fungi claviceps purpurea cp 7/274 ccm f-632 - Google Patents

Description

The invention relates to a novel industrial mutant of the strain Claviceps purpurea CP7 / 274 with high submerged production of pharmaceutically important agroclavin alkaloids (M. Abe et al., Jap. Pat. 178 336) of structural formula I

(I) and elymoclavin (M. Abe et al., US Pat. 2,835,675) of structural formula II

Collection of Microorganisms of University J.

E.

Mutant samples are deposited in Czechoslovak Purkyně Brno under the 6th CCM P-632.

The mutant protected by the invention was obtained from 664 isolates prepared by the mutation treatment of ethyl methanesulfonate (EMS) and UV radiation on the laboratory strain Claviceps purpurea CP7 (Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague 4) as described below.

In the preparation of high-production mutants, all incubations of the cultures were performed in the dark at 24 ° C. CP7 maternal culture spores that have been cultured for 3 to 4 weeks on sloping T2 agar broth (C. Spalla, in Genetics of Industrial Microorganism. Eds. Z. Vanek, Z. Hoslakek, J. Cudlin. Elsevier Amsterdam 1973, p. 393 were suspended in 0.066 M phosphate buffer pH 7.2 and in saline. The resulting suspension always contained 1 to 4 x 10 6 spor.ml ^-1 , of which only 0.5% to 1% germinating. T2 agar broth contained the following components <g.1 ^-1 dest. H ₂ O) j Soharose 100, L-asparagine 10, Ca (NO 3) g 4 H 2 O 1, fermented extract 0.1, KH ₂ PO ₄ 0.25, MgSO 4.7 H ₂ 0 0.25, KCl 0, 12, FeSO ₄ .7H ₂ 0 0.02, ZnSO ₄ .7 H2O 0.015, agar 20, pH 5.2 after autoclaving at 105 ° C for 15 min.

The spore suspension in 0.066 M phosphate buffer pH 7.2 was exposed to 0.02-0.075 M EMS (J. Nešvera, Fol. Microbiol. 18. 352 (1973)) for 19 hours, washed twice with said buffer, plated on petri dishes (0 10 cm) with T2 agar broth enriched with 0.02% casein hydrolyzate (Bacto-Casamirio acids Difoo) and incubated for 2 weeks.

Of a spore suspension in physiological saline solution was transferred to 5 ml per Petri dish (0 10 cm) while moving the irradiation of the UV light source set at 10 J m ^- 2 s ^-1 150 to 1 000 Jm "2. 0.5 ml of the spore suspension was withdrawn at 10 second intervals. After appropriate dilution with saline, the resulting spore suspension was transferred to a. Petri dishes (0 10 cm) with T2 agar broth enriched with 0.02% casein hydrolyzate (Bacto-Casamino acids Difoo). The inoculated broth was incubated for 2 weeks.

Colonies grown from mutagenized spores on agar medium in Petri dishes were individually transferred to slanted T2 agar broth and incubated for 3-4 weeks. The grown cultures were then tested for their ability to form alkaloids under submerged fermetaoe conditions (8s. Certificate No. 199 986).

The alkaloids were determined in a fermentation fluid of 14-day cultures by colorimetry (GT Banks et al., J. Gen. Microbiol. 82, 345 (1974)) and by high performance liquid chromatography (M. Wurst et al., J. Chromate. 150. 477). 1978).

Characteristics of high mutant production:

The ability of the mutant to form an extraoellular mixture of agroclavin and elemoclavin under submerged fermentation conditions is shown in Table 1.

T a b u 1 k a 1

Mutant type Mutagen dose (Jm ^- 2) conc. (M) Total alkaloids (g agroclavin ml ^-1 ) Agroklavin % Elymoclavin % CP7 / 274 uv 800 ¹ , 8 70 30

The CP7 / 274 mutant differs from the parent strain because it is a typical sclerooial strain. It grows poorly (5 to 6 mg dry weight of mycelia.ml ^-1 ) on T2 agar broth as well as in submerged conditions, forming a budding mycelium with enlarged round cells. In the older culture, this myoelium disintegrates into arthrospores of 4 x 12 ju. Fibrous mycelium and conidia are rarely formed. The content of extraoellular glucans is negligible.

The production mutant is stored in Endo tubes on T2 agar broth in the dark at 4 ° C. It is inoculated every 3-4 months and after 30 days incubation at 24 ° C it is used for inoculum preparation or stored as a stock culture at 4 ° C.

The mutant Clavioeps purpurea, designated CP7 / 274, provides a key section of the large-scale production of agroclavin and elemoclavin alkaloids by submerged fermentation.

Claims (1)

Industrial mutant of the fungus Claviceps purpurea CP7 / 274, deposited in the Czechoslovak Collection of Microorganisms at the University of J. E. Purkynš in Brno under the number CCM F-632 with production of agroclavin and elymoclavin alkaloids.