RU2850337C1 - New strain of oleandromycin producer streptomyces lavenduligriseus - Google Patents

Abstract

FIELD: microbiology; biotechnology.

SUBSTANCE: Streptomyces lavenduligriseus strain VKPM As-2218 is proposed as a producer of oleandomycin, as well as a method for obtaining oleandomycin, which is carried out by culturing the specified strain and isolating the antibiotic oleandomycin from the culture fluid, followed by purification.

EFFECT: increase in the productivity of the oleandomycin-producing strain.

2 cl, 1 dwg, 1 tbl, 6 ex

Description

The invention relates to the field of microbiology and biotechnology and concerns a new strain producing the antibiotic oleandomycin.

LEVEL OF TECHNOLOGY

Oleandomycin belongs to the group of macrolide antibiotics and is produced by fermentation of actinomycete strains such as Streptomyces antibioticus . Oleandomycin has a bacteriostatic effect against gram-positive ( Staphylococcus spp., Streptococcus spp. , including S. pneumoniae; Bacillus anthracis, Corynebacterium diphtheriae ) and some gram-negative microorganisms ( Neisseria gonorrhoeae, Haemophillus influenzae, Bordetella pertussis, Brucella spp., Legionella spp. ), as well as intracellular pathogens ( Mycoplasma spp., Chlamydia spp., Treponema spp. and Rickettsia spp.) (Geppe N.A. et al. The role of macrolide antibiotics in the treatment of respiratory infections in children // Consilium medicum (pediatrics). - 2012. - No. 3).

The main producer of oleandomycin is Streptomyces antibioticus ATCC 11891, which is characterized by the ability to produce several antibiotics of different classes (Haque, SF et al. "Physicochemical Properties of Non-aromatic Broad-spectrum Antibiotic of Streptomyces antibioticus Sr15.4". Microbiological Research . - 1998. - 153 (2): 153-156; US 2757123).

Oleandomycin is also used as an additive in animal feed (US 4239750).

A method for the biosynthesis of oleandomycin by Streptomyces antibioticus is known from the prior art, which allows for the production of up to 0.45 g/l of the antibiotic (GB 840829).

A strain of Streptomyces antibioticus LS-OL 32-280 (SU 169193), a high-yield oleandomycin producer, is known. This strain was considered by the authors of the present invention as the closest analogue.

A method for the biosynthesis of oleandomycin by Streptomyces antibioticus strain ATCC 11891 is also known from the prior art (Vilches, C. et al. Biosynthesis of oleandomycin by Streptomyces antibioticus : influence of nutritional conditions and development of resistance // Journal of General Microbiology. - 1990. - 136(8), 1447-1454), which allows obtaining up to 5.4 μg/ml of oleandomycin.

The purpose of the present invention is to obtain a new strain of microorganism that exceeds previously known strains in terms of oleandomycin accumulation.

The technical result of the claimed invention is an increase in the productivity of the new strain.

ESSENCE OF THE INVENTION

A wild-type strain producing the antibiotic oleandomycin was isolated from a sample of sod-podzolic soil (Mordovia).

To enhance oleandomycin secretion, the wild-type strain was subjected to induced mutagenesis by exposing it to ultraviolet light at low temperatures. The resulting high-yielding strain was assigned the internal designation LD-0623.

Further, strain LD-0623 was identified by macro- and micromorphological characteristics, and its generic affiliation was confirmed by sequencing the 16S rRNA gene.

Based on the results of the analysis of the sequences of variable regions of the genes encoding 16S rRNA, the tested strain LD-0623 turned out to be the closest to the species Streptomyces lavenduligriseus.

The newly obtained strain of Streptomyces lavenduligriseus was deposited in the National Bioresource Center All-Russian Collection of Industrial Microorganisms of the Kurchatov Institute Research Center under the number VKPM Ac-2218.

Thus, one embodiment of the present invention is the oleandomycin-producing strain Streptomyces lavenduligriseus VKPM Ac-2218. Another embodiment of the present invention is the use of the Streptomyces lavenduligriseus VKPM Ac-2218 strain for the production of oleandomycin. Yet another embodiment of the present invention is a method for producing the antibiotic oleandomycin, which comprises cultivating the Streptomyces lavenduligriseus VKPM Ac-2218 strain and isolating oleandomycin from the culture fluid, followed by purification (Fig. 1).

TERMS AND DEFINITIONS

Below are terms that may be used to describe the present invention. Unless otherwise specified, all technical and specialized terms used in the description have their generally accepted meanings in the art.

The term "antibiotic" includes any molecule that can inhibit the growth, destroy, or cause the death of microorganisms, but is not lethal to the patient at the concentration ranges and dosages intended for administration. According to the present invention, such antibiotics can be classified as bactericidal (i.e., directly causing the death of the microorganism) or bacteriostatic (i.e., preventing the division and/or reproduction of the microorganism). In the context of the present invention, the antibiotic is not toxic to the patient at the concentration ranges and dosages administered.

The use of the term "antibiotic" in the context of the present invention means an effect or type of action in the treatment or prevention of infections caused by gram-negative and/or gram-positive bacteria.

The term "biomass" in the context of the present invention refers to the total mass of Streptomyces lavenduligriseus VKPM Ac-2218 cells separated during centrifugation from the supernatant of the culture fluid obtained as a result of cultivation, including in shaking flasks or bioreactors (for example, during battery cultivation).

The term “culture liquid” in the context of the present invention means a gas-liquid nutrient (or fermentation) medium into which microorganisms secrete metabolic products (including secondary metabolites) during the process of submerged cultivation.

The term "bioreactor" in the context of the present invention refers to a vessel or device in which the process of submerged cultivation of microorganisms is carried out to produce a target product (in the context of the present invention, the antibiotic oleandomycin).

The term "productivity of a strain" in the context of the present invention refers to the amount of the antibiotic oleandomycin obtained, calculated per unit volume of culture fluid, during submerged cultivation of the strain under bioreactor conditions, expressed in grams of oleandomycin per liter of culture fluid.

When using the term “approximately”, “approximately”, “about” in a description, it should be considered that it characterizes plus or minus ten percent of the specified value.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated by Fig. 1.

Fig. 1 shows the main stages of screening the Streptomyces lavenduligriseus strain VKPM Ac-2218 and the production of oleandomycin from its culture fluid obtained after submerged cultivation.

DISCLOSURE OF THE INVENTION

According to the present invention, a new strain producing oleandomycin, the Streptomyces lavenduligriseus strain, is proposed, deposited in the National Bioresource Center All-Russian Collection of Industrial Microorganisms of the Kurchatov Institute National Research Center under the number VKPM Ac-2218.

The cultural and morphological characteristics of the oleandomycin-producing strain according to the present invention are as follows: aerobic; Gram staining is positive; when cultivated on a solid medium of the composition specified below, it forms colonies 5-6 mm in diameter, folded, with an uneven edge, and slightly convex. Aerial mycelium is light gray to gray; substrate mycelium is dark, almost black. It intensively secretes a dark brown pigment into the medium.

For storage and preparation of seed material, the following medium is used, g/l: liquid corn extract (by technical weight) - 10.0; potato starch - 10.0; CaCO ₃ - 5.0; NH ₄ SO ₄ - 3.0; NaCl - 3.0; agar - 30.0 (pH 7.1±0.1).

Cultivation conditions for isolating single colonies are 8-12 days at a temperature of 27-32°C.

EXAMPLES

The invention is illustrated by the following illustrative examples, which do not limit the claimed scope of protection.

Example 1. Method for selective isolation of oleandomycin-producing strain from a soil sample

The study subjects were sod-podzolic soil samples. The soil samples were collected in the Republic of Mordovia during the summer from the upper soil horizons.

To isolate the wild-type oleandomycin-producing strain from soil samples, we used a solid nutrient medium culture method. A homogenized 1-gram soil sample was placed in a flask with 100 ml of sterile tap water, then diluted 1:300 and plated on Gause 1 nutrient medium. Broad-spectrum antibiotics were added to the nutrient medium to limit the growth and activity of unwanted microorganisms.

The cultures were incubated at 20-30°C until visible colonies appeared. A pure culture was obtained by successive subcultures until a single colony was formed. Culture purity was verified microscopically.

Example 2. Obtaining a highly productive oleandomycin-producing strain

By means of multi-stage selection using mutagenic factors and directed methods of selection of modifiers of the parent wild strain, a new highly productive oleandomycin-producing strain LD-0623 was obtained.

To do this, an aqueous suspension of spores and cells of the parent strain, placed 55 cm from the lamp, was exposed to UV light at a wavelength of 250 nm (Mineralight lamp) for 20 minutes at a temperature of 0°C. The resulting strain was assigned the internal number LD-0623.

Example 3. Identification of strain LD-0623 to the species level using 16s RNA analysis

Isolation of DNA from strain LD-0623 for PCR was performed using the standard method (PCR Protocols. A Guide to methods and applications. Innis M, Gelfand D., Sninsky J. p. 14-15).

For sequencing, conservative primers were used to produce 16S rDNA:

8f - AGA GTT TGA TCC TGG CTC AG

926r - CCG TCA ATT CCT TTR AGT TT.

Sequencing was performed on an AE3000 automatic sequencer with the following reaction modes:

1. 95°C - 3 min

2. 35 cycles

95°C - 30 sec

57°C - 30 sec

72°C - 1 min 30 sec

3. 72°C - 5 min.

Specialized phylogenetic computer programs were used to analyze the sequenced data. To ensure consistent results, at least three PCR reactions were performed.

Next, PCR electrophoresis of the studied samples was performed in 1.0% agarose gel at an electric field strength of 5 V/cm.

Primary screening in the GenBank database showed that the studied strain belongs to the following systematic groups: Bacteria; Actinomycetota; Actinomycetes; Kitasatosporales; Streptomycetaceae; Streptomyces .

The sequences were aligned with the corresponding sequences of the closest bacterial species available from the GenBank database.

The processing of the sequenced sequences was carried out using open-access bioinformatics software - the Blast (Basic Local Alignment Search Tool) program, designed, among other things, to determine the degree of phylogenetic closeness of living organisms.

Based on the results of sequence analysis of the variable regions of the genes encoding 16S rRNA, the tested strain LD-0623 was found to be most closely related to the species Streptomyces lavenduligriseus. The resulting strain was assigned the VKPM number Ac-2218. This strain demonstrated unexpectedly high resistance to adverse conditions, including storage and cultivation, compared to other known strains of this species.

Example 4. Cultivation technique

The seed was prepared by inoculating Streptomyces lavenduligriseus VKPM Ac-2218 in Petri dishes. The agar medium was inoculated with the initial seed culture and incubated in a thermostat for 5 to 12 days at 25-34°C.

Next, the culture was grown in flasks in a liquid medium at 25-37°C for 100-250 hours on a thermostatically controlled shaking unit at 200-300 rpm.

Next, the seed culture was produced in fermenters with a volume of 15/100 l. For this, the fermenter was seeded by adding the seed culture in an amount of 5-15% of the volume of the medium in the fermenter, with a continuous supply of sterile air during cultivation with stirring for 110-170 hours.

Antibiotic levels were monitored using agar diffusion. When maximum antibiotic accumulation in the medium was reached, the fermentation process was stopped.

Example 5. Determination of oleandomycin content in culture fluid by the agar diffusion method

Equal volumes of working solutions of the standard and test antibiotic samples were added to the wells of each Petri dish. Stock solutions of the standard and test samples were prepared in sterile solvents at a concentration of 1 mg/ml. Working solutions of three or one concentration of the test sample and solutions of three or five concentrations of the standard sample were then prepared from the stock solutions. Working solutions of the test samples were prepared from the stock solutions so that their concentrations did not differ significantly from those of the standard sample solution. The dishes were then incubated at 36±1°C for 16-18 hours.

The diameters of the growth inhibition zones of the test microorganism were measured with an accuracy of 0.1 mm.

Calculation of activity and dispersion analysis during use were carried out in accordance with the article of the General Pharmacopoeia Monograph “Statistical processing of the results of determining the specific pharmacological activity of drugs by biological methods”.

The obtained average data are presented in Table 1.

Table 1. Yield of the target product during submerged cultivation

Strain Productivity of strains (g/l of culture liquid ±0.2 g/l) in a 15-liter fermenter in a 100-liter fermenter Streptomyces lavenduligriseus VKPM Ac-2218 4.85 g/l 5.67 g/l

According to literary data, the productivity of the well-known strain Streptomyces antibioticus LS-OL 32-280 is about 1.3 g/l.

The results of the studies allow us to conclude that the microorganism strain according to the present invention has high biotechnological potential, significantly increased productivity and can be used for the industrial production of the antibiotic oleandomycin.

Example 6. Method for isolating oleandomycin

After culturing the Streptomyces lavenduligriseus VKPM Ac-2218 microorganism, the biomass was separated from the culture fluid by coagulation with sulfuric acid at a temperature of 9±1°C, followed by separation by filtration. The culture fluid, freed from biomass, was transferred to the purification stage, and the spent biomass was disposed of.

The culture fluid, purified from biomass, was extracted with butyl acetate in an alkaline medium at pH 9.8-10.3. This was followed by back-extraction in an acidic medium with phosphoric acid at pH 4.0-5.5, and the resulting solution was clarified with activated carbon. The resulting oleandomycin antibiotic was then washed and dried.

Claims (2)

1. Streptomyces lavenduligriseus strain VKPM Ac-2218 is a producer of oleandomycin. 2. A method for producing oleandomycin, comprising cultivating the strain according to item 1 and isolating the antibiotic oleandomycin from the culture fluid with subsequent purification.