CS246704B1 - Microorganism production strain Claviceps purpurea CP 7/5/35 CC-2/85 - Google Patents

Abstract

The solution concerns a new production strain of the microorganism Claviceps purpures CP 7/5/35, producing clavine alkaloids, which differs from previous strains in higher production of agroclavine and elymoclavine, limited production of extracellular glucans, intensive conidiation and higher germination of spores. The strain is deposited in the collection of microorganisms of the Institute of Microbiology in Prague under number CC—2/85.

Description

The present invention relates to a new production strain of Claviceps purpures CP 7/5/35, producing clavin alkaloids, which differs from the previous strains in higher production of agroclavin and elymoclavin, limited production of extracellular glucans, intensive conidiation and higher germination spores. The strain is deposited in the collection of microorganisms of the Institute of Microbiology in Prague under number CC — 2/85.

The invention relates to a new production strain of the microorganism Claviceps purpurea CP 7/5/35, which produces agroclavin, elymoclavin and trace amounts of other clavin alkaloids. The sample of the strain is deposited in the collection of microorganisms of the Institute of Microbiology of the Czechoslovak Academy of Sciences in Prague under No. CC — 2/85.

The strain is characterized by high submersible production of pharmaceutically important alkaloids of agroclavin (Abe M. et al., Jap. Pat. 178 336) of structural formula I

0) and elymoclavin (Abe M. et. Al., US Pat;

The strain differs from previously patented strains of the same origin

a) double production of alkaloids (measured on day 10 of fermentation),

b) intensive conidia formation,

(c) increased germination capacity of conidia;

d) low levels of extracellular glucans that make alkaloid extracts more difficult by promoting the formation of a solvent emulsion with the culture fluid,

e) higher resistance of alkaloid production to the phosphate of the medium throughout the cultivation.

(II)

Decrease in production of alkaloids ⁶ on soil CS2 tem (Pi)

Strain x Pi

CP 7/5/35 35

CP 7 21 (Desc.

in AO 199 986) fortified phospháCultivation (days)

21 x Pi 5 x Pi 10 x Pi

42 31

34 26 ^§ % control (soil CS2)

The mutant strain of C. purpurea CP 7/5/35 was obtained from the parent strain of C. purpurea CP 7 (deposited in the collection of microorganisms MBU CSAV, Prague) by mutation with UV light. An aqueous suspension of conidia from four-week agar cultures of strain CP 7 on T2 broth (Spala C., In: Genetics of Industrial Microorganisms, Elsevier, RAmsterdam 1973, p. 393) was exposed to UV light to survive 20-40% of conidia. Samples of the irradiated suspension were then sown after appropriate dilution in Petri dishes with T2 soil and incubated for 3 weeks at 24 + 1 ° C. In each case 50 to 100 colonies which did not differ in appearance from the parental strain were individually isolated on sloping T2 agar broths and after 3 to 4 weeks cultivation tested for alkaloid yield as described in AO 199 986, except that vegetative inoculum cultivation and fermentation lasted 10 days. The alkaloid content of the fermentation broth was determined by colorimetric methods (GT Banks et al., J. Gen. Microbiol. 82, 345 (1974). For cultures exceeding the parent strain production, the composition of the alkaloid mixture was determined by thin-layer chromatography. alkaloids were used for the next mutation, a mixed suspension of conidia with an equal representation of both strains was exposed to UV light C. purpurea strain CP 7/5/35 is the result of three mutation steps:

Production day 10 (mg / L)

CP 7 1100

I 1200

II 1990

CP 7/5/35 2,440

Morphological and growth characteristics of strain CP 7/5/35

246

(a) Cultivation on T 2 agar medium

In cultivation on T2, strain CP 7 / / 5/35, like the parent strain CP 7, forms an off-white, airy mycelium with sporophores bearing conidia heads connected by a slime of polysaccharide nature [Pazoutova et al. Can.

J. Microbiol. 23, 1182 (1977)]. Conidiation is fully developed after 12 to 14 days. The red-brown pigment accompanying the formation of alkaloids is deposited in the agar. When washing conidia from agar cultures, the CP 7/5 / / 35 strain shows twice the conidia yields in cultures less than 30 days compared to the CP 7 strain.

The results of the determination of germination of conidia by sowing on T2 soil show that conidia of strain CP 7/5/35 show approximately 10 times higher germination than conidia of parental strain.

bj Submerged cultivation

Submerged cultivation of the CP 7/5/35 strain and the parental strain CP 7 was performed on a rotary shaker and in a laboratory fermenter on the media described in AO 199 986. The parental strain CP 7 formed glucans during submersive cultivation, producing maximum between 7 and 11 day of fermentation. Then they were partially degraded. The amount of polysaccharides reached up to 35 g / L. The CP 7/5/35 mutant strain usually does not form these polysaccharides under conditions allowing maximum alkaloid formation.

The piano mixture on day 21 of the submersible culture of CP 7/5/35 strain contains 10-30% elymoclavin, 65-90% agroclavin, 1-5% chanoclavin-I and traces of some other clavin alkaloids.

It is apparent from the above characteristics that the new strain Clavices purpurea CP 7/5/35, obtained after exposure to UV light, differs from the parent strain C. purpurea CP 7 in the following characteristics:

(a) twice the yield of alkaloids on day 10 of cultivation

(b) increased germination of conidia

(c) increased ability to conidise on T2

(d) limited production of glucans under conditions allowing maximum production of alkaloids

θ) higher alkaloid productivity resistance to phosphate.

The mutant C. purpurea, designated CP 7/5/35, provides a key section of the agroclavin and elymoclavin fermentation, which are suitable substrates for the semi-synthetic preparation of both known and novel ergot alkaloid derivatives.

Claims (1)

Subject A production strain of Claviceps purpurea CP 7/5/35 CC 2/85 producing, under deep cultivation conditions, agroYNALEZ of clavins and elymoclavins with limited extracellular glucan formation.