CS272657B1 - Monensin a producing high-productive strain of microorganism streptomyces cinnamonensis - Google Patents

Abstract

The micro-organism Streptomyces cinnamonensis is used for production of monensin A and B. The new highly productive mutant of this strain is called SMU 21, whose culture sample is stored under number CCM 3977, and it produces monensin A in a concentration of more than 6,000 micrograms/mL as opposed to the initial strain than produced monensin A in a concentration of around 1,000 micrograms/mL. Streptomyces cinnamonensis ACB-NLR-2 also differs from the parent strain as follows:a) the colouring of the aired mycelia with cultivation in a solid agar mediumb) the colouring of the submersion culturesc) the use of fructose for growth during surface cultivation on a solid medium.A mixture of fats is used as a source of carbon during fermentation. Mutagenic action is caused by 8-methoxypsoralene and UV radiation.

Description

The present invention relates to a high-yielding monensin A producing strain of Streptomyces cinnamonensis, wherein fat is used as the carbon source.

Standard strains of Streptomyces cinnamonensis produce a mixture of monensins in which monensins A and B are the main constituents. The proportion of monensins produced in these pigmenting low-production strains can be controlled to a limited extent by using vegetable oils as carbon sources (AO No.218032). The non-pigmenting mutant strain of Streptomyces cinnamonensis KI-1 (AO # 226964) produces about 90% of monensins A upon addition of specific precursors of this substance, i.e. valine or butyrate (AO # 225938).

SUMMARY OF THE INVENTION The present invention is based on a new high-production monensin A Streptomyces cinnamonensis SMU-21 strain utilizing a fat blend as a carbon source for fermentation, which is deposited in the Collection of Microorganisms of the University of O. E. Purkyně (CCM) in Brno under CCM 3977.

The novelty of the strain is that, unlike the parental low-producing strain, producing monensin A on soils containing glucose, resp. some other sugars, the new SMU-21 strain produces more than 6000 yug / mL monensin A in submersible cultures in media containing a fat blend as a carbon source.

The mutant Streptomyces cinnamonensis SMU-21 strain was obtained from a double-resistant mutant Streptomyces cinnamonensis ACM-NLR-2, resistant to 2-amino-3-chlorobutyrate and norleucine, which is stored in the Collection of Microorganisms of the Institute of Microbiology CSAV, Prague. chemical mutants of 8-methoxypsoralen and N-methyl-N'-nitro-N-nitrosoguanidine to the spore suspension and irradiation of the spore suspension with UV light.

To a suspension of spores (conidia concentration 01/05/10 mL ^ ^- '' ·) in 0.1 M phosphate buffer (Na) pH 7.0 was added to an ethanolic solution (96% ethanol), 8-methoxypsoralen, so that the final concentration of this compound was 500 / µg mL ^- ' ^- . The suspension was continuously stirred on an electromagnetic stirrer at 24 ° C. After 15 min. the suspension was exposed to stirring for 20 min. UV radiation (source of Philips 57415 P / 40 15W DHW lamp), corresponding to energy flow

-2 - 7 mJ mm. Percentage of conidia surviving after mutagenic intervention (2.10 30 was determined from the number of colonies grown from conidia after mutagen removal after 5 days of cultivation on agar medium □ composition (X):

yeast extract 0.4 agar with wort extract 2.0 glucose 0.4 agar Oxaid 3.0 water

The spore suspension (conidia concentration 5.10 ^ mL ^-1 , in 0.05 M Tris-maleic acid buffer (Na) pH 9.0 was exposed to N-methyl-N'-nitro-N-nitrosoquanidine at a final concentration of 3 with continuous stirring on an electromagnetic stirrer. mg mL ^- '. the exposure time was 120 min. at 24 ° C. the percentage of surviving conidia (4.10 ^- θ%) mutagenic action was determined in the same manner as after the action of 8-methoxypsoralen and UV-strain variability is obtained after treatment with the chemical The mutant selection was based on the biological activity of the submersible cultures, the ratio of monensins A to 8 produced in the selected strains with high production, the absence of undesirable phenazine derivatives and the ability to utilize fats. cinnamonensis SMU-21 was compared to the parent parental culture after purification Streptomyces cinnamonensis ACQ-NLR-2.

Morfeloflická charakteristika

When cultivated on the soil with malt and yeast extract, the mutant SMU 21 grew in the form of beige-white, wrinkled colonies with a raised center. Colonies were regular, spherical, reaching 4 to 8 mm in size after 5 days of culture. Growing aerial mycelium with straight and undulating sporophores after 6 days of cultivation divided into individual white-white conidia (spores)

CS 272 657 B1 with smooth surface.

While the starting ACM-NLR-2 strain formed long branched fibers during submersive cultivation, the mutant mycelite experienced a point thickening of the fibers after 40 hours of cultivation, and after 50 hours, the mycelium markedly disintegrated into spore-like formations.

Coloring of cultures in cultivations

In surface cultures, the staining of airborne mycelium and the pigment diffusing into the agar medium were observed. In contrast to the parent parent strain ACB-NLR-2, which secreted yellow-white pigments, the SMU-21 mutant was stained with brown-red pigment after 7 days of culture. Airy rnycelium and spores in both strains were beige-white.

In submersive cultures, the medium had an intense dark brown color after 144 hours, unlike the starting strain, where light brown pigments were formed.

Assimilation of carbon sources by the starting strain ACB-NLR-2 and the mutant SMU 21

The ability to utilize different carbon sources in surface cultivation on solid media was tested according to Pridham and Gottlieb (J.Bact. 58: 107, 1948) and Shirling and Gottlieb (Int.J. Syst.Bacteriol. 16: 313, 1966), For comparison The table below shows the use of various carbon sources in surface cultivation by the starting strain ACB-NLR-2 and the SMU 21 strain according to the invention.

Both strains grew equally on glucose, arabinose, xylose, mannitol and rhamnose. Unlike the parent strain, the SMU 21 mutant grew much better on fructose. In contrast, the parent strain grew better on inositol. Growth was not observed (except for control) on sucrose, raffinose and cellulose.

Utilization of various carbon sources by ACB-NLR-2 and SMU 21 strains

carbon source ACB-NLR-1 SMU 21 0 - - D-glucose + + + + L-arabinose + + ++ sucrose - - O-xylose + + i.-inositol ++ + O-mannitol ++ D-fructose + + + + L-rhamnosa + + ruffinosa - - cellulosa - - - does not grow ++ good grow + weak growth +++ very good grow

Submersive cultivation

It was cultured on a rotary shaker at 4.5 Hz with a radius of rotation of 15 mm at 34 ° C in 500 ml 50 ml flat-bottomed flasks while culturing 100 ml of mutant strain inoculum.

The parent parental strain ACD-NLR-2 was cultured in medium containing (%):

glucose 2.5 (inoculum) 5.0 (2nd generation) K ₂ HPO ₄ 0.05 MgS0 ₄ 0.05 kno ₃ 0.1 NaCl 0.05 FeS0 ₄ 0.001

CS 272 657 Bl

Mutant strains were cultured in inoculation medium (%):

soy flour 1.5 yeast extract 0.25 glucose 0.5 dextrin 2,0 CaCO-j 0.1 Production medium it contained C 6); soy flour 3.3 glucose 3.7 NaCl 0,033 FeSO4 0.011 CaCQ-j 0.25 On _? S0 ₄ 0.22 ai ₂ (Sat ₄ ) ₃ 0.07 k ₂ hpo ₄ 0.0075 ascorbic acid 0,001? soybean oil 2.5 ergot oil 0.5 methyl oleate 0.8 HaHOj 0.8

Inoculation flasks of the first vegetative generation were inoculated with a single loop of spores from cultures on sporulated soil, 7-10 days old. The submersive cultivation itself took place in II. vegetative generation; flasks were inoculated with 4% ipocula at 18-14 hours. Cultivation was terminated after 8 days.

At the end of the cultivation, antibiotic activity was monitored in the methanolic extract from submersible cultures by diffusion plate method and the formation of phenazine-type substances autobiographically after thin-layer chromatography. The test organism was Bacillus subtilis.

The ratio of produced monensins A and B was determined in hexane-chloroform extract from cultures. After thin-layer chromatography, the monensin zones were delegated with a vanillin reagent and their intensity measured densitometrically.

Fat consumption was determined in the supernatant after centrifugation of submersible cultures by measuring the saponification number.

It is apparent from the above characteristics that the SMU 21 strain of the invention obtained by multi-stage selection using 8-methoxypsoralen and N-methyl-N'-nitro-M-nitrosoquanidine chemical mutagens and UV-radiation from the parent strain Streptomyces cinnamonensis ACB-NLR-2 they differ in these features;

(a) staining the aerial mycelium when grown on solid agar medium

(b) coloring of submersible cultures

c) utilizing fructose for growth in surface culture on a solid medium

(d) ability to use fats as a carbon source in cultivations

o) high production of monensin A. (Compared to the initial monensin A-producing strain at concentrations around 1000 yug / ml, the SMZ 21 strain of the invention produces more than 6000 / Ug / ml.)

Claims (1)

SUBJECT OF THE INVENTION A monensin A producing strain of Streptomyces cinnamonensis microorganism, designated SMU 21, using a fat blend as a carbon source in fermentation, a culture sample of which is stored in the O.E.Purkyně University Collection of Microorganisms (CCM) under CCM 3977.