RU2098483C1 - Method of avermectin preparing, strain streptomyces avermitilis - a producer of avermectin - Google Patents

Abstract

FIELD: biotechnology, microbiology, veterinary science. SUBSTANCE: method involves culturing the strain Streptomyces avermitilis VKPM S-1440 on sowing medium containing 0.02-0.4% L-lysine followed by inoculation the nutrient medium with sowing material in the presence of carbon, nitrogen and mineral substance sources. Strain-producer is cultured under optimal conditions and the end product is isolated. EFFECT: improved method of preparing. 2 cl, 3 tbl

Description

 The invention relates to biotechnology and for the production of antibiotics avermectins with high antiparasitic activity and finding application in veterinary medicine for the prevention and treatment of pets and birds.

 Avermectins are the total preparation produced by the Streptomyces avermitilis culture and consisting of structurally related compounds called avermectin A and avermectin B, which, in turn, are also divided into several groups.

 The most active are avermectins B, on the basis of which commercial veterinary drugs are mainly created.

 The ratio of avermectins A and avermectins B biosynthesized by microbial culture depends on the characteristics of the producer strain and on the conditions under which the cultivation process is carried out.

 A method for producing avermectins is known, for example, by growing a producer strain of Streptomyces avermitilis ATCC 31267 or ATCC 31271 or ATCC 31272 on a nutrient medium containing simple and complex organic substrates as carbon and nitrogen sources under optimal conditions for a given culture.

The total content of avermectins in the culture fluid reaches 500 mg / l [1] of which up to 50–70% of the group B avermectins account for the total amount of biosynthetic avermectins [2]
When using other producer strains, the ratio of avermectins produced may be different.

In particular, a method for producing avermectins B is patented, which consists in the use of a mutant strain of Streptomyces avermitilis ATCC 53692, which is grown on a nutrient medium containing assimilable sources of carbon, nitrogen, inorganic salts and a precursor carboxylic acid under optimal conditions. Depending on the precursor acid used, both natural and non-natural group Avermectins can be obtained, in some cases completely free of Group F avermectins. However, the absolute accumulation of Avermectins B in a fermentation medium, as follows from the given examples of the method, does not exceed 100 200 mg / l of culture fluid [3]
A method for producing avermectins is described, which involves the cultivation of mutant strains of Streptomyces avermitilis K475 or K655 on a complex nutrient medium that provides for the accumulation in the culture fluid of up to 1200 mg of avermectin in 1 liter of medium after 7 days of cultivation. The maximum accumulation of avermectin was observed after 10 days of cultivation of the Strepomyces avermitilis K655 strain with the additional introduction of 3% glucose into the nutrient medium by a 5-day culture and reached 2500 mg / l. However, no information on the relative content of individual components of the avermectin complex in the cited work is given [4]
However, there are technological methods that can increase the total yield of avermectins or the proportion of avermectins B in the total amount of biosynthetic avermectins when using producers that form both avermectins of group A and avermectins of group B.

 For example, it was reported that the additional introduction of DL-tryptophan at a concentration of 0.5 mg / ml or DL-methionine at a concentration of 2.5 mg / ml into the fermentation medium stimulates the formation of avermectins by the Streptomyces avermitilis NRRL 8165 culture, while others from the studied 18 amino acids did not show a similar effect.

However, the degree of influence of effective amino acids on the biosynthesis of avermectins is not given, as well as the ratio of the individual components of the avermectin complex. The productivity of the culture without additional amino acids does not exceed 500 mg / l [1]
It was found that when synefungin antimetabolite is introduced into the fermentation medium that inhibits the conversion of avermectins B to avermectins A, the proportion of avermectins B produced by the Streptomyces avermitilis NRRL 8165 culture reaches 77% of the total amount of avermectins.

However, the absolute content of avermectins in the culture fluid in this case remains only at the level of 88 105% of the control, since in the presence of synefungin the synthesis of Avermectins A decreases [5]
Closest to the proposed one can recognize the method of producing avermectin, which consists in the fact that the isolate MA 4990 strain Streptomyces avermitilis ATCC 31271 is grown on a nutrient medium, which includes L-isoleucine at a concentration of 1 g / l, under optimal conditions for this culture.

 The proportion of avermectins B in this case is 69% of the total amount of biosynthetic avermectins.

At the same time, the total accumulation of avermectins in the culture fluid under the influence of L-isoleucine does not increase and its absolute value is not given [6]
The purpose of the invention is to increase the accumulation of avermectins in the culture fluid of the Streptomyces avermitilis culture with a high content of avermectins B in the complex of biosynthetic avermectins.

 The goal was achieved as a result of the fact that the seed of the Streptomyces avermitilis culture is prepared by growing a spore culture of this strain in the presence of L-lysine, and in the optimal embodiment of the invention, the Streptomyces avemitilis 103 actinomycete strain is used as a producer.

 The essence of the proposed method is as follows.

 As a producer of avermectin, a Sterptomyces avermitilis 103 strain obtained from the original Streptomyces avermitilis ATCC 31271 strain by three-stage selection is used.

 Strain Str. avermitilis 103 has all the typical for the species Str. avermitilis signs, the main of which are listed below.

Morphological features
On a medium with soy flour for 14 days forms colonies with a diameter of up to 7 mm of almost round shape. The edge contour is fringed. The colonies are flat, in the center there is a slight folding. The surface of the colony is dry, rough, without dropping. The color of the colonies is cream. The structure of the colonies is homogeneous, the texture is dense, leathery. Brown pigment is released on Wednesday.

 On Wednesday, Hopwood on the 14th day forms colonies of irregular shape with a diameter of up to 6 mm. The contour of the edge of the lobed. The colonies are convex. The surface of the colonies is dry, rough, lowered, warty folding. Colonies of gray with patches of white. The structure of the colonies is heterogeneous, the consistency is leathery. No pigment is formed.

 On Wednesday, Gause with glucose for 14 days forms colonies with a diameter of up to 8 mm of irregular shape with a wavy edge. The relief of the colonies is conical. Some colonies have radial folding. The surface of the colonies is dry, rough, lowered, but there are areas without omission. The color of the colonies is light gray and dark gray. The structure of the colonies is heterogeneous, the consistency is dense. A light brown pigment is released on Wednesday.

 On the YME medium, on the 5th day it forms colonies of two morphological types: I - colonies of white color with a cream shade, well-lowered, II colonies are convex almost round in shape with a smooth edge and slight folding. The color of the colonies is gray, in the center is a lighter area. The diameter of the colonies is 2.0 2.5 mm. The surface of the colonies is dry, rough, undescended. Both types of colonies are brown on the back. Brown pigment is isolated on Wednesday. Colonies are slightly buried in agar. On the 14th day it forms colonies of irregular shape with a wavy edge with a diameter of up to 10 mm. The relief of the colonies is conical. The surface of the colonies is dry, rough, well lowered. Folding gyro. The color of the colonies is gray with white spots. The structure is heterogeneous, the texture is dense.

 On Wednesday, CP-6 on the 5th day forms colonies of an almost round grayish-brown color with a diameter of 2 mm. The relief of the colonies is domed. Folding is poorly expressed. Dark brown pigment will be isolated on Wednesday. The surface of the colonies is dry, rough, omission is absent. On day 11, colonies were almost round in shape with a slightly wavy edge, convex with a radial folding of a grayish-brown color and with a slightly darker center. The surface is dry, rough, without dropping. Emits a dark brown pigment.

 On the YMS medium, on day 5 forms light gray and almost white colonies in approximately equal proportions. The shape of the colonies is crater-shaped, diameter 3.0 - 3.5 mm. The surface is well lowered, folded. The colonies are slightly buried in agar, secrete a dark brown pigment. On the 11th day, the colonies were almost round in shape, with a wavy edge, a diameter of 5.5 6.0 mm, with a convex relief. The color of the colonies is gray, the surface is dry, rough, with gyro folding. On Wednesday, it releases brown pigment.

 Good sporulation is observed on CP-6, minimal Hopwood and YMS environments. Sporophores form spirals in the form of branches of the aerial mycelium. Compact at first spirals open as the culture ages. The number of spores in the chains is no more than 15; spores are usually spherical and oval in shape.

 Physiological signs. In a minimal Hopwood environment, with 1% carbon source it grows using glucose, fructose, mannose, mannitol, inositol, rhamnose, sucrose, lactose, maltose, arabinose and xylose as the sole carbon source.

 Hydrolyzes gelatin and casein, coagulates milk.

 It grows well and forms an air mycelium in the temperature range from 27 to 37 degrees, does not grow at a temperature of 50 degrees.

When cultured on ordinary media, the Str. avermitilis 103 is characterized by a total productivity of 1200–1400 mg of avermectins in 1 liter of culture fluid. This level is maintained after three passages when reseeding the culture from the school on the YMS medium. Under standard cultivation conditions, the proportion of avermectins B in the sum of synthesized avermectins is 40–50%
Grown for 8-10 days at a temperature of 28 + 1 degrees on YMS agar medium, the strain culture is stored under a layer of 40% glycerol at a temperature of 20 degrees for 6 months.

 To store the strain, use a standard agar medium with a pH of 7.5 and containing (g / 100 ml of distilled water): soluble starch (0.5), Difco yeast extract (0.5), Difco malt extract (1.5) , chalk (0.3) and agar-agar (2.0).

 The strain Streptomyces avermitilis 103 was deposited in the All-Russian collection of industrial microorganisms of the State research institute of genetics and breeding of industrial microorganisms under registration number VKPM-S1440.

 Compared to the original, strain 103 is morphologically more stable, forming mainly only 2 types of colonies, while the original strain forms up to 6 types of colonies.

 Strain Str. avermitilis 103 has antibiotic stability and provides a high percentage of fermentations with a high level of antibiotic accumulation, as shown in Table 1.

To obtain seeds of the Streptomyces avermitilis strain 103, a liquid medium containing carbon sources, nitrogen, growth factors, mineral salts and trace elements supplemented with L-lysine in an amount of 0.02 to 0.4% is inoculated with spore material. The preferred concentration of L-lysine is from 0.1 to 0.2%
The cultivation of seed is carried out at a temperature of 28 ± 1 degrees for 48 72 hours

 Fermentation medium is inoculated with seed and fermentation is carried out at a temperature of 28 ± 1 degrees for 6 days.

 The content of avermectins in the culture fluid after completion of fermentation reaches 2200 2500 mg / l, and the proportion of avermectins B is 50 - 56% of this amount.

 The biosynthesis of avermectins in the presence of L-lysine introduced into the inoculum and fermentation medium is shown in Table. 2.

 It is noteworthy that the introduction of L-lysine into the fermentation medium leads to a significant decrease in the overall level of antibiotic formation and the relative content of B. avermectins.

 Comparative data on the effect of L-lysine, additionally introduced into the inoculum, on the biosynthesis of avermectins by some strains of Streptomyces avermitilis, are presented in table. 3.

The presented data indicate the stimulating effect of K-lysine introduced into the seed medium on the productivity of the studied cultures, but the most favorable result is achieved using the Streptomyces avermmitilis 103 strain, which gives the highest response: the formation of avermectins exceeds the level of antibiotic accumulation under control conditions (when using seed grown without lysine) by 76% and at the same time increasing the proportion of group B avermectins from 46% to 56%
The following Examples explain in more detail the essence of the invention.

 Example 1. A spore suspension of strain Streptomyces avermitilis 103 is seeded with 5 flasks with a capacity of 750 ml, filled with 50 ml of inoculum medium containing (wt.): Glucose (2.0), soy flour (1.5), yeast extract (0.5) , L-lysine (0.1) and tap water (up to 100.0). The pH of the medium is 7.3.

 Vegetative inoculum is grown at a temperature of 28 ± 1 degrees on a circular rocking chair (230 250 rpm) for 48 hours

 Inoculum is seeded with 50 flasks with a capacity of 750 ml, filled with 50 ml of a fermentation medium containing (wt.): Glucose (4,5), soy flour (1,4), yeast extract (0.25), sodium chloride (0.1) and tap water (up to 100.0). The pH of the medium is 7.4.

 Fermentation is carried out at a temperature of 28 + 1 degrees on a circular rocking chair (230-250 rpm) for 6 days.

After fermentation, the content of avermectins in the culture fluid was 2520 mg / l, of which 56% was avermectins B
Example 2. A spore suspension of a strain of Streptomyces avermitilis 103 is seeded with 5 flasks with a capacity of 750 ml, filled with 50 ml of inoculum medium containing (wt.): Glucose (1.0), lactose (1.0), cotton flour (1.5), yeast extract (0.5), L-lysine (0.1) and tap water. The pH of the medium is 7.2.

 Vegetative inoculum is grown at a temperature of 28 ± 1 degrees on a circular rocking chair (230 250 rpm) for 48 hours

 Inoculum is seeded with 50 flasks with a capacity of 750 ml, filled with 50 ml of a fermentation medium containing (wt.): Glucose (4,5), cotton flour (1,4), yeast extract (0.25), sodium chloride (0.1) tap water (up to 100.0). The pH value is 7.4.

 Fermentation is carried out at a temperature of 28 ± 1 degrees on a circular rocking chair (230 250 rpm for 5 days.

After fermentation, the content of avermectins in the culture fluid was 2350 mg / l, of which 55% was avermectins B
Significant differences of the proposed method from the prototype method are that
in the proposed method, as an agent to increase the biosynthesis of avermectins, use L-lysine, and not L-isoleucine;
in the proposed method, the stimulator of the formation of avermectins is introduced into the composition of the inoculum, and not the fermentation medium.

 in an optimal embodiment of the proposed method, a Streptomyces avermitilis 103 strain is used as the producer of avermectin, which gives the highest response to the administration of L-lysine and has high genetic stability and high antibiotic stability.

 The distinctive techniques of the proposed method does not obviously follow from a known level of knowledge, since the applicant does not know the sources of information from which it would be possible to draw a conclusion about the stimulating effect of L-lysine on the biosynthesis of avermectins and especially on the biosynthesis of avermectins of group B.

 Using the proposed method and the proposed new strain of the producer of avermectins allows, without changing the technological scheme of production, to increase the overall level of antibiotic formation to the level of maximum productivity known from the literature, with a high content of group B avermectins, without increasing the duration of fermentation.

Sources of information taken into account
1. Burg RW Miller BM Baker EE et al. Avermectins, new family of potent anthelmitic agents: producing organism and fermentation. "Anntimicrobical Agents and Chemotherapy", 1979. Vol. 15, No. 3, p. 361 367
2. US patent 4.310.519, M. cl. A 61 K 31/71, 1982
3. USSR patent 1.806.198, M. cl. C 12 P 17/08, 1993
4. Ikeda H. Kotaki H. Tanaka H. Omura S. Involvent of glucose catabolism in avermectin production by Streptomyces avermitilis. "Antimicrobial Agents and Chemotherapy", 1988, Vol. 32, N 2, p. 282,284
5. Schulman MD Valentino D. Streicher S. Ruby C. Streptomyces avermitilis mutants defective in methylation of avermectins. "Antimicrobial Agents and Chemotherapy" 1987, Vol. 31, N 5, p. 744 749
6. McConn-McCormic PA Monaghan RL Baker EE Goegelman RT Stapley EO Studies of the avermectin fermentation. "Advances in Biotechnology", 1981, Vol. 1, p. 69 74 (prototype) and

Claims (2)

1. A method of producing avermectins, comprising growing a producer of the species Streptomyces avermitilis in a seed medium, inoculating with seed material a nutrient medium containing carbon, nitrogen and mineral sources, culturing the producer under optimal conditions and isolating the product, characterized in that L- is introduced into the seed medium lysine at a concentration of 0.02 0.4%
2. The method according to claim 1, characterized in that as a producer using a strain of Streptomyces avermitilis VKPM S-1440.  3. The strain Streptomyces avermitilis VKPM S-1440 producer of avermectin.

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