RU2110576C1 - Method of selection of high-producing strain - a producer of oxytetracycline and the strain streptomyces rimosus - a producer of oxytetracycline - Google Patents

Abstract

FIELD: microbiology, antibiotics. SUBSTANCE: selection of high-producing strain-producers of oxytetracycline is carried out by culturing the strain-producer of species Streptomyces rimosus, in part, the strain Streptomyces rimosus M-1 (VKPM S-1512) producing oxytetracycline. Culturing is carried out on solid nutrient medium in the presence of kanamycin. Kanamycin-resistant mutants are selected and after their culturing on liquid nutrient medium in the presence of L-serine strain variants are selected. In these variants oxytetracycline biosynthesis is inhibited by L-serine. EFFECT: improved method of selection. 2 cl, 2 tbl

Description

 The invention relates to the field of biotechnology and relates to a microbiological method for producing an antibiotic oxytetracycline.

 Oxytetracycline is a broad-spectrum antibiotic and is currently widely used as a starting material for the production of chemical derivatives of oxytetracycline used in medicine.

 The described strains producing oxytetracycline do not differ in the high level of antibiotic biosynthesis, and therefore, the search for more productive strains is of considerable practical interest.

 It is indicated, for example, that known strains producing oxytetracycline of the species Streptomyces rimosus under conditions of controlled fermentation with a fractional supply of a carbon source can form up to 14 g / l of oxytetracycline [1].

 The physiological strain closest to the one proposed is the oxytetracycline producing strain Streptomyces rimosus R83, which accumulates up to 12 g / l of antibiotic under laboratory conditions [2].

 This strain is sensitive to the presence of L-serine in the medium and reduces the level of antibiotic formation by 43-59% at a concentration of L-serine of 250-500 mg / L.

 Existing methods for selecting highly productive antibiotic-producing strains are based mainly on a direct assessment of the level of antibiotic formation in the studied strains, which is a very time-consuming task [3].

 However, methods are described for selecting promising antibiotic producers for signs other than the level of antibiotic formation that correlate with the level of antibiotic biosynthesis.

 Known, for example, are methods of selecting productive strains producing streptomycin and oleandomycin, which are based on the correlation of morphological characters (spore formation, pigmentation, etc.) with productivity [4,5].

 However, these methods were relatively ineffective for the selection of highly productive strains [3].

 The closest analogue to the method of selection of strains producing oxytetracycline is a method based on the selection of antibiotic-forming variants based on the morphology, pigmentation and sporulation of the colonies and their sensitivity to oxytetracycline [6]. However, this method is suitable only for the selection of productive clones from a population of a particular strain, but not for the selection of highly productive mutants.

 The purpose of the invention is to find a producer strain of oxytetracycline, characterized by an increased level of antibiotic formation, and to develop a faster method for the selection of highly productive strains.

 It was experimentally established that there is a clear correlation between the sensitivity of strains of Streptomyces rimosus culture to the inhibitory effect of L-serine on the biosynthesis of oxytetracycline and the intensity of antibiotic formation in the absence of this amino acid in the nutrient medium. In other words, the stronger L-serine inhibits the biosynthesis of oxytetracycline in a particular strain of Streptomyces rimosus, the higher the biosynthesis of oxytetracycline in this strain on commonly used nutrient media that do not contain this amino acid, which is confirmed, in particular, by the data in Table 1.

 Note to table 1. Fermentation was carried out under the conditions described below in example 1. The accumulation of the antibiotic in the presence of L-serine is shown as a percentage of the level of oxytetracycline biosynthesis by each strain in the absence of L-serine. The duration of fermentation in a synthetic medium was 3 days., In a complex medium in the presence of L-serine - 3 days. In the absence of L-serine - 9 days. The synthetic medium had the following composition (g / l): glucose (40.0). ammonium sulfate (4.0), casein hydrolyzate (1.0), yeast extract (0.5), disubstituted potassium phosphate (0.5), magnesium sulfate (0.5), calcium carbonate (3.0), distilled water (rest). The composition of the complex medium is shown in example 2. For strain R83, published data [2].

 The proposed method for selecting highly productive oxytetracycline producing strains of the Streptomyces rimosus species consists in plating the initial strain on a dense standard agar medium and selecting spontaneous kanamycin-resistant mutants that modify the regulation of oxytetracycline biosynthesis.

 Of the isolated kanamycin-resistant mutants, in turn, variants are selected in which the biosynthesis of oxytetracycline in a liquid medium is most suppressed by L-serine.

 As a result, producer strains with a high level of oxytetracycline biosynthesis are selected.

 In the implementation of the proposed method, a strain of Streptomyces rimosus M-1, resistant to kanamycin at a concentration of 30 mg / l, in which the level of oxytetracycline biosynthesis was suppressed by 50-60% in the presence of L-serine at a concentration of 50 mg / l was selected.

 The productivity of oxytetracycline in the selected strain averages 18.5 g / l with periodic fermentation on ordinary nutrient media for 9 days.

 With successive transfers every 2 months. on solid CP-6 medium, for 1 year, the selected strain retained the main morphological and physiological characteristics, in particular, resistance to kanamycin, sensitivity to L-serine, and the level of antibiotic formation.

 According to the main characteristics, the strain of Streptomyces rimosus M-1 does not differ from typical representatives of this species and has the following characteristics.

Morphological features
Spore-carriers are straight, sometimes bent at the end, monopodially branching; spores are oval.

With growth on agarized media at a temperature of 28-29 ^o C for 12 days has the following morphological characteristics.

 Medium with corn extract CP-6. The colonies are rounded, flat, radially folded, with a diameter of 6-8 mm. Aerial mycelium is well developed, in the center of the colony is dark gray, and along the edges of light gray. Brown substrate mycelium.

 Pea environment. The colonies are rounded, slightly convex, radially folded with a small crater, with a diameter of 6-7 mm. Aerial mycelium is dark brown. The center of the colony is sporulating, the edge (rim) is non-sporulating.

 Oatmeal environment. The colonies are rounded, slightly raised, with a crater with a diameter of 6-7 mm. Aerial mycelium in the center of the colony is gray, non-sporulating at the edges. The substrate mycelium is dark brown with a light brown rim.

 Waxman’s environment. The colonies are rounded, flat, in the center a small bulge with a diameter of 2-3 mm. Aerial mycelium is absent. The substrate mycelium is yellow.

 Wednesday Chapek. The colonies are membranous, with a diameter of 4-5 mm. Aerial mycelium is dark brown with a light rim. The substrate mycelium is light brown.

 Gauze-1 medium (with glucose). The colonies are membranous, in the center brown, colorless at the edges, with a diameter of 2-3 mm. Aerial mycelium is absent. The substrate mycelium is light brown with a yellow rim.

 Wednesday Gause-2 (with starch). The colonies are rounded, flat, with a point crater, with a diameter of 2-3 mm. Aerial mycelium is poorly developed. The substrate mycelium is dark brown.

 Wednesday Gauze-2. The colonies are rounded, slightly convex, radially folded, with a crater with a diameter of 6-7 mm. Aerial mycelium is dark gray with a light gray rim. The substrate mycelium is dark brown.

Physiological and biochemical properties
It ferments glucose, maltose, fructose, mannitol, sorbitol, inositol, arabinose, weakly xylose, does not ferment sucrose, lactose, dulcite.

 The reaction to sucrose inversion is negative.

 Starch hydrolyzes intensively.

 Thin gelatin for 4 days.

 Milk peptones for 6 days.

 Nitrate does not recover.

 The accumulation of oxytetracycline during cultivation under ordinary conditions is 18.0 - 19.0 g / l.

 The strain is deposited in the All-Russian collection of industrial microorganisms under registration number VKPM S-1512.

 Example 1. The original strain of Streptomyces rimosus VNIIA 1525, seeded on a dense standard agar medium containing (g / l): starch (30.0), corn extract (10.0), ammonium sulfate (4.0), chalk (5 , 0), sodium chloride (5.0), tap water (up to 1 l) and 30 mg / l kanamycin, after which spontaneous mutants resistant to kanamycin are selected.

 Of the selected 97 kanamycin-resistant mutants, in turn, variants are selected in which the biosynthesis of oxytetracycline in a liquid medium of the following composition (g / l): glucose (40.0), ammonium sulfate (4.0), casein hydrolyzate (1.0) , yeast extract (0.5), disubstituted potassium phosphate (0.5), magnesium sulfate (0.5), calcium carbonate (3.0), distilled water (rest) supplemented with L-serine at a concentration of 50 mg / l , is most suppressed by L-serine. The effect of inhibiting the biosynthesis of oxytetracycline was observed for 3 days. culturing each strain on medium containing and not containing L-serine.

 Three options were selected, the degree of inhibition of the biosynthesis of oxytetracycline in which L-serine was 50-60%.

 The selected options were tested for the ability to biosynthesize oxytetracycline under optimal conditions on a fermentation medium of the following composition (g / l): corn flour (120.0), corn extract (14.0 by dry weight), ammonium sulfate (14.0), chalk (14.0), cobalt chloride (0.0003), soybean oil (20.0), tap water (up to 1 l).

 The producing activity of the selected culture variants of Streptomyces rimosus, see table 2.

 Note to table 2. The degree of inhibition of biosynthesis was determined in a synthetic medium, and productivity in a complex.

 Thus, 3 strains of Streptomyces rimosus were isolated, including M-1, the biosynthetic activity of which was significantly higher than that of the original strain.

 Moreover, as a result of preliminary selection on the basis of sensitivity to the inhibition of oxytetracycline biosynthesis by L-serine, the total volume and duration of the study were significantly reduced: the accepted method for identifying highly active producers of oxytetracycline would require culturing not 3, but 97 strains resistant to kanamycin for 9 days.

Example 2. A suspension of culture of a strain of Streptomyces rimosus M-1, grown on agar medium CP-6, seeded inoculum medium of the following composition (g / l): starch (30.0), corn extract (10.0), ammonium sulfate (4 , 0), chalk (5, 0), sodium chloride (5, 0), tap water (up to 1 l). The pH of the medium is 7.1. The inoculum is grown at a temperature of 28-29 ^o C in flasks with a capacity of 750 ml, filled with 50 ml of medium, on a circular shaker for 48 hours

An inoculum in an amount of 5 vol.% Is seeded with a fermentation medium of the following composition (g / l): corn flour (120.0), corn extract (14.0 by dry weight), ammonium sulfate (14.0), chalk (14.0 ), cobalt chloride (0.0003), soybean oil (20.0), tap water (up to 1 liter). The pH of the medium is 7.1. Fermentation is carried out at a temperature of 28-29 ^o C in flasks with a capacity of 750 ml, filled with 50 ml of medium, on a circular shaker (230-240 rpm) for 9 days.

 After 3 days. soybean oil is added to the fermentation medium at the rate of 2% of the volume of the culture fluid.

 The oxytetracycline content in the culture fluid is 18.9 g / l.

 Thus, the above examples confirm the practical feasibility of the invention and its advantages over the known technical solutions.

 In the patent and available scientific and technical literature, no information was found that a connection is known between the level of oxytetracycline biosynthesis in Streptomyces rimosus strains and their sensitivity to the antibiotic-inhibiting effect of L-serine. No description was also found in accessible sources of information of the producer strain of oxytetracycline identical to the one proposed.

 Therefore, the proposal meets all the requirements for the invention.

Sources of information
1. Patent GDR N 205.930, M. cl. C 12 P 15/20, 1982.

2. Parada l. L. Grown Inhibition of Streptomyces species by L-serine and Its effect on tetracycline biosynthesis .- "Appl. Environ. MIcrobiol", 1982, Vol. 41, N 3, p. 366-370,
3. Alikhanyan C. I. Selection of industrial microorganisms ".- M .: Nauka, 1968.

 4. Waksman S.A., Harris D.A. Streptomycin-producing capability of different strains of Str.griseus.- "Proc. Soc. Exp. Biol. A. Med.", 1949, Vol. 71, p. 232.

5. Zhdanova N. I., Alikhanyan S. I. The effect of stabilizing selection in the selection of the producer of oleandomycin. - "Proceedings of the USSR Academy of Sciences, biological series", 1965, N 1, p. 138,
6. Gravius B. et al. Genetic instability and strain degeneration in Str. rimosus. - "Appl. Environ. Microbiol.", 1993, vol. 59, No. 7, p. 2220-2228. (prototype).

Claims (2)

 1. A method for selecting a highly productive strain - producer of oxytetracycline, characterized in that the producer strain of the species Streptomyces rimosus is cultured on a solid nutrient medium with kanamycin, kanamycin-resistant mutants are selected and, after their cultivation on a liquid nutrient medium with L-serine, variants are selected whose biosynthesis of oxytetracycline suppressed by L-serine.  2. The strain Streptomyces rimosus VKPM S-1512 - producer of oxytetracycline.

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