CS201752B1 - Strain of microorganism streptomyces galilaeus 947 ccm 3320 - Google Patents

Description

The invention relates to a novel industrial microorganism Streptomyces galilaeus 947, CCM 3320 producing ε-pyrromycinone, which can be used for the synthesis of antitumor anthracycline antibiotics.

The standard Streptomyces galilaeus strain JA 3043 (ZIMET, Jena) produces a mixture of biologically active glycosides ε-pyrromycinone and aclavinone. A mutant strain of Streptomyces galilaeus 947 occurred genetically. by interfering with the block in the glycosidation stage of the biosynthesis of these compounds leading to the accumulation of free aglycons. Ε-pyrromycinone is produced as the main metabolite; minor amounts of aclavinone, ε-pyrromycinone and 7-deoxy- and bisanhydroderivatives of ε-pyrromycinone and aclavinone are formed.

The Streptomyces galilaeus strain 947 was obtained from the standard Streptomyces galilaeus JA 3043 strain by a five-step selection procedure using natural selection and mutagenic effects. The strain was isolated from the nitric acid population and its variability was reduced by natural selection. Nitrous acid was applied as 0.05 M NaNO 2 in 0.2 M acetate buffer (pH 4.0) for 25 min. At this dose of mutagen, 99.93% of conidia in suspension were killed. The altered biosynthetic activity of the mutant strain was determined by thin layer chromatography of submersible culture metabolites.

The characteristics of a mutant strain of Streptomyces galilaeus 947, whose sample of culture is deposited in the Collection of Cultures of Microorganisms of the University of J. E. Purkyně in Brno under the number CCM 3320, are given below.

1. Morphological characteristics

In oat agar (ISP 3), inorganic salt and starch (ISP 4), and yeast and malt extract (ISP 2) cultivation, the mutant strain 947 formed an airy mycelium with sporophores monopodially branched, with open spirals of 4— 6 threads. Aerial mycelium strain 947 was less developed than wild type JA 3043. On asparagine soil with glycerol (ISP 5), on tyrosine agar (ISP 7), on Benett agar (Waksman SA The Actinomycetes. Chronica Botanica, Waltham, Mass., USA) (1950) and on a substrate with a mineral nitrogen source (Gause GF et al., Zur Klassifizierung der Actinomyceten. VEB G. Fischer, Jena, 1958) formed a standard strain of well-developed aerial mycelium, whereas the mutant strain contained only traces of aerial mycelium.

The shape and surface of the spores were not the essence of201752

Table I: Staining of substrate mycelium, aerial mycelium and diffusing pigments in mutant strain Streptomyces galilaeus 947 CCM 3320 and wild type strain Streptomyces galilaeus JA 3043 after 21 days of culture at 28 ° C.

Difundu- eating pig- menty Coloring light Brown 1 1 orange light Brown 1 AND 1 « eo •F* rění -grey -grey -grey O 8 9 ‘Fl) 'fl) ‘Fl) O white- 'fl) white- w with Coll Ό 0) > 05 gray Ό (fl) > 05 Ό (fl) > 05 white 24 £ 3 05 / (fl) 3 O 3 and <9 -G £ tupni 2-5 2-5 T 03 / 2-5 1-5 1 7 fM 7 w bfi OT 05 / (fl) 'fl) with _G Ό > O Ί e - 5 > 9 > fl) O · * » fil 0) AT • 5 > I 1> '8 £ tí >) 43> P vé orai > N • 2 § 1> >> 43 1 ’S O mo nžc O ti mo tle O O WHAT with N G g £ G cream rich žlutoi G kré ora kré kré ora '2 Ο οι is> u (f)> φ a. & kré kré O (fl) Ui Tť 05 / at O AND .9 fí Q < ce e »» 9 ra 43 and A ra 3 9 ra g WHAT 9 what CO rji 10 what fr- CO w it M Ul - O o -y O Q Ui A) O 0 fl) Ui O 0 at U 0 o o o o at u o at I, 2 ie ř » , living íftí p » 0) '>? anžc ú) '>> 3 ... « and «g 9 43 §> g 45 t3 S> § .fl '” N > p 05 £ 3 1 1 Ui O 43 1 l 1 · >> - ( tí Ό) ‘Fl) G Ό Ό (fl) 3 > 05 X / 5 £ m (fl) fr · 05 / Cfl G Ό ! Zba: saints £ > fl) 05 / white * gray white * ! 1 AND | white * ί 9 IN 0 9 at * 3 3 WHAT <0 tft bo * 9 £ dull 1 1 03 / T AND what 1 1 AND 1 05 / 8 ω >> § '4) 1 '4) • W fil υ h and rění > fl) tí 43 brown X? (fl) E not > N C 9 Ui Ό) 1 WHAT O 9 O .a, « tí > £ 3 O > AND 'ř 43 N June olive light Yellowfin krémo dark June light krémo žlutooi šedool O with 8 * 3 and with WITH Ui 6 u _ 9 9 G what 3 10 fr · CO WHAT w ® £ t * fM (fl) 4-> O tfi Ui Ui Ui O O 0 Ui WHAT 0 o o ϋ u o o 0 ϋ O ϋ • rd AND • 1 G agar slado 3 agar !? § anorgi new 9 vý ag new source 9 XJ 05 / H 5b 9 1 ráto ragii SŠ tí • 9 9 9 9 and c3 8ř Oi w 3 •G and E with? 9 O 3 what > □ 9 £ · Jd E > 05 / 1 > • M 3 ; Oat O 3 2 § «O« 1 w , Glycero j Agar p 1 extract Benett 1 Sacharc Glukose Substrates

aerial mycelium little developed differences between mutant and standard strain. Both strains formed spores with a smooth surface.

2. Coloring of cultures

The pigmentation of airborne and substrate mycelium was evaluated according to the Prauser scale (Z. Allg. Mikrobiol., 4:95, 1964) after 2, 4, 7, 14 and 21 days of culture.

a) Coloring of aerial mycelium

The aerial mycelium of both the standard and mutant strains were stained in various shades of gray. White mycelium was observed only in young, undeveloped cultures.

b) Staining of the substrate mycelium

The results obtained after 21 days of culture in both strains are shown in Table I.

c) Coloring of diffusing pigments

Mutant strain 947 and standard strain JA

3043 formed a light brown pigment on oat agar and on Benett's agar and an orange pigment on yeast and malt extract agar.

3. Physiological characteristics

(a) Production of melanin pigments

On the peptone broth with iron and on the tyrosine medium, the mutant strain as well as the standard strain formed an intense black-brown pigment.

(b) Assimilation of carbon sources

The results obtained for both strains after 14 days of cultivation on different carbon sources are shown in Table II.

Table II: Assimilation of carbon sources in mutant strain Streptomyces galilaeus 947 CCM 3320 and in standard strain Streptomyces galilaeus JA 3043 after 14 days of culture

Carbon source Streptomyces galilaeus 947 JA 3043 glycerine 4 ++! +++ d-mannitol + (+) d-sorbitol + + ' 1-arabinose +++ +++ rhamnosa ++ +++ d-xylose +++ H — 1 — h d-fructose +++ +++ d-galactose +++. +++ d-mannose +++ _; +++ glucose +++ | - | —1_ i-inositol +++ · +++. lactose +++ +++ maltose +++ sucrose +++ +++ rafinosa ++ +++ starch +++ +++ inulin +++ +++ '. cellulose (+) (+) dextrin ++ +++

U-) Ů ŮST + weak growth + - (- good growth 4 - j- + very good growth

(c) Liquefaction of gelatin, reduction of nitrates, formation of reducing sugars and degradation of starch.

Both the mutant and wild-type strains liquefied gelatin, reduced nitrates, cleaved sucrose to reducing sugars, and degraded starch.

4. Production of secondary metabolites

As a further criterion for the characterization of the mutant strain Streptomyces galilaeus 947, a chromatographic analysis of the production of secondary metabolites in submersible cultures was used, which is the most conclusive indicator of the difference between the mutant and the standard strain.

(a) Cultivation

Submerged cultures were performed on a reciprocating shaker (frequency 1.6 Hz, amplitude 80 mm) at 28 ° C in 500 ml beakers filled with 60 ml of soil. The inoculation flasks were inoculated with spore inoculum from yeast and malt extract agar culture and cultured for 24 hours on the following composition:

soya flour glucose corn-steep

CaCO ₃

NaCl

2.0% 1.0% 0.25%

0.3%

0.5% pH: 6.5 The fermentation flasks were inoculated with 5% inoculum and cultured for 96 hours on the same composition with increased glucose content (2.5%).

(b) Chromatography

Secondary metabolite production was monitored by thin layer chromatography on silica gel impregnated with NaHCO ₃ (10: 1) in a chloroform-methanol (49: 1) system.

The mycelium was filtered off and extracted with HCl acidified methanol (pH 2.5-3.5), the extract was neutralized with ammonia, filtered and evaporated in vacuo. The residue was dissolved in chloroform and used for chromatography.

A complex strain of ε-pyrromycinone and aclavinone together with the 7-deoxy- and bisanhydroderivatives of these aglycons was detected in the standard Streptomyces galilaeus JA 3043 strain. The mutant strain Streptomyces galilaeus 947 does not produce glycosides, it accumulates free ε-pyrromycinone as the main metabolite and produces minor amounts of aclavinone, εί-pyrromycinone, 7-deoxy- and bisanhydroderivatives of ε-pyrromycinone and aclavinone.

Claims (1)

The microorganism strain Streptomyces galilaeus 947, producing ε-pyrromycinone, whose culture sample is deposited with the Microorganism Culture Collection of the University of J. E. Purkyně, Brno (CCM) under the number CCM 3320.