RU2802575C1 - Streptomyces baarnensis, a new daptomycin producer - Google Patents

Abstract

FIELD: microbiology and biotechnology.

SUBSTANCE: strain of Streptomyces baarnensis VKPM Ac-2200, a daptomycin producer, has been proposed. The use of this strain for production of vancomycin is also proposed. The specified strain is cultivated and vancomycin is extracted from the culture fluid, followed by purification.

EFFECT: group of inventions provides increased productivity of the vancomycin-producing strain, reduced cultivation time, reduced amount of impurities in the vancomycin antibiotic substance, high biochemical activity and increased resistance of the new strain to adverse factors including during storage and cultivation.

3 cl, 2 dwg, 1 tbl, 6 ex

Description

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

BACKGROUND OF THE ART

Daptomycin is an antibiotic from the group of cyclic lipopeptides. Active against clinically significant gram-positive bacteria, including strains resistant to other classes of antimicrobial drugs. Today, in the Russian Federation, daptomycin is used for complicated infections of the skin and soft tissues, for bloodstream infections caused by Staphylococcus aureus , including bacteremia and right-sided endocarditis (Danilov A.I. et al. Daptomycin: possibilities and prospects for use in clinical practice // Reviews on clinical pharmacology and drug therapy. - 2019. - 17 (4): 83-87).

Known from the prior art is the mutant strain Streptomyces roseosporus BS_094 (registration number KACC91589P), obtained by mutation of the wild strain Streptomyces roseosporus KCTC 9568 as the original strain. Strain BS_094 is capable of producing daptomycin with a maximum yield of 2.5 g/l (KR20120058790, 10/01/2010).

Also known is the daptomycin-producing strain Streptomyces griseus, deposited in the General Management Center for the Collection of Microorganism Cultures of China under the number CGMCC17921. The yield of daptomycin after fermentation of this strain can reach approximately 0.0315 g/l. (WO2021000637, 01/07/2021).

The yield of daptomycin after fermentation of the producer strain Streptomyces filamentosus , deposited in the China General Collection Center for Microbiological Cultures under the number CGMCC No. 16016, which was considered by the authors of the present invention as the closest analogue, can reach approximately 3.47 g/l (CN110777084, 02/11/2020 ).

A recombinant daptomycin-producing strain, Streptomyces roseospora L31, is known, deposited under the number CGMCC No.14233 in the General Center for Microbiology of the Committee for the Collection of Microbiological Cultures of China. The yield of daptomycin after strain fermentation can reach approximately 0.074 g/L (CN107267434, 10/20/2017).

A highly productive mutagenized strain of Streptomyces roseosporus WKL-126 is known from the prior art (currently stored in the China General Microbiological Culture Collection Center (CGMCC) under registration number 3731), which has an antibiotic productivity of about 2 g/l (CN102242073, 11/16/2011).

The purpose of the present invention is to obtain a new strain of microorganism that is superior in the level of accumulation of daptomycin in the culture fluid to previously known strains.

The technical result of the claimed invention is an increase in the yield of the antibiotic, a reduction in fermentation time, a decrease in the amount of impurities in the substance, high biochemical activity and increased resistance of the strain to adverse factors and during cultivation.

SUMMARY OF THE INVENTION

In the process of induced mutagenesis and subsequent stepwise selection, a highly productive strain of Streptomyces baarnensis DAP 17-18, producing the antibiotic daptomycin, was isolated from a wild-type strain isolated from a sample of soddy meadow soil (Mordovia).

To enhance the secretion of daptomycin, the wild-type strain was subjected to induced mutagenesis by exposing it to ultraviolet light at low temperatures. The resulting highly productive strain was assigned the internal number DAP 17-18.

Next, strain DAP 17-18 was identified by macro- and micromorphological characteristics, and its genus was confirmed by sequencing of the 16S rDNA gene.

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

The resulting new strain of Streptomyces baarnensis was deposited in the National Bioresource Center All-Russian Collection of Industrial Microorganisms of the National Research Center "Kurchatov Institute" under the number VKPM Ac-2200.

Thus, one of the variants of the present invention is the daptomycin-producing strain Streptomyces baarnensis VKPM Ac-2200. Another embodiment of the present invention is the use of the Streptomyces baarnensis strain VKPM Ac-2200 for the production of the antibiotic daptomycin. Another variant of the present invention is a method for producing the antibiotic daptomycin, which involves cultivating the Streptomyces baarnensis strain VKPM Ac-2200 and purifying daptomycin from the culture liquid.

Terms and Definitions

The following are terms that may be used to describe the present invention. Unless otherwise specified, all technical and technical terms used in the specification have their common meaning in the art.

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

The use of the term "antibiotic" in the context of the present invention means an effect or type of effect 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 baarnensis VKPM Ac-2200 cells separated during centrifugation from the supernatant of the culture liquid 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 two-phase gas-liquid nutrient (or fermentation) medium into which, during deep cultivation, microorganisms secrete metabolic products (including secondary metabolites).

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

The term “strain productivity” in the context of the present invention refers to the amount of antibiotic obtained, calculated per unit volume of culture fluid, during deep cultivation of the strain (according to the present invention, Streptomyces baarnensis VKPM Ac-2200) under bioreactor conditions, expressed in grams of antibiotic per liter of culture fluid.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated by two figures.

In FIG. Figure 1 shows the main stages of screening the Streptomyces baarnensis strain VKPM Ac-2200 and the production of daptomycin from its culture liquid obtained after submerged cultivation.

In FIG. Figure 2 shows a photograph of a fixed specimen stained with methylene blue. Magnification 1000 times.

DISCLOSURE OF INVENTION

According to the present invention, a new daptomycin-producing strain is proposed - the Streptomyces baarnensis strain has been deposited in the National Bioresource Center All-Russian Collection of Industrial Microorganisms of the National Research Center "Kurchatov Institute" under the number VKPM Ac-2200.

Cultural and morphological characteristics of the daptomycin producer strain: aerobic, Gram stain is positive, after 8-10 days of cultivation on a solid medium it forms typical fully sporulating colonies of light pink color. The sole of the colony is dark pink, almost burgundy. The shape of the colony is round, often with a central inverted dome and a round outer ridge (crown), size 4-8 mm.

For storage and preparation of seed material, a medium of the following composition is used, g/l: glucose - 1.0-3.5; sucrose 1.0-2.0; starch 1.0-3.0; malt extract - 5.0-20.0; yeast extract - 2.0-6.0, calcium carbonate - 1.0-1.5; agar-agar - 10.0-30.0; The pH of the medium before sterilization is 5.0-8.0. Cultivation temperature is 25-35°C, duration is 8-10 days.

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 a daptomycin-producing strain from a soil sample.

The objects of the study were samples of sod-meadow soil. Soil samples were collected in the Republic of Mordovia in summer from the upper soil horizons.

To isolate the wild-type daptomycin-producing strain from soil samples, the method of inoculation on solid nutrient media was used. A homogenized soil sample weighing 1 g was placed in a flask with 100 ml of water for injection, then a 1:300 dilution was prepared and inoculated on plates with Gause 1 nutrient medium. Antibiotics were added to the nutrient medium to limit the growth and activity of unwanted microorganisms.

The crops were incubated at temperatures of 20-30°C until visible colonies appeared. A pure culture was developed by successive subcultures to a single colony. Control of the purity of the culture was carried out microscopically.

Example 2. Obtaining a highly productive daptomycin-producing strain.

Through multi-stage selection using mutagenic factors and targeted methods for selecting modifiers of the parental wild strain, a new highly productive strain was obtained - the daptomycin producer DAP 17-18.

To do this, an aqueous suspension of spores and cells of the parent strain, placed at a distance of 55 cm from the lamp, was exposed to UV light with a wavelength of 250 nm (Mineralight lamp) for 20 minutes at a temperature of 0°C. The resulting strain was assigned the internal number DAP 17-18. The result of microscopic research methods is presented in Fig. 2.

Example 3. Identification of strain DAP 17-18 to species using 16s RNA analysis.

Isolation of DNA of strain DAP 17-18 for PCR was carried out according to standard methods (PCR Protocols. A Guide to methods and applications. Innis M, Gelfand D., Sninsky J.p. 14-15).

Conservative primers for generating 16S rDNA were used for sequencing:

8f - AGA GTT TGA TCC TGG CTC AG

926r - CCG TCA ATT CCT TTR AGT TT

Sequencing was carried out on an automatic sequencer AE3000 with reaction modes:

1. 95°C – 3 min

2. 35 cycles

95°C - 30 s

57°C - 30 s

72°C - 1 min 30 s

3. 72°C - 5 min.

Specialized phylogenetic computer programs were used to analyze the sequenced sequences. For stability in reproducing the results, at least three repetitions of PCR reactions were performed.

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

Primary screening using the GenBank database showed that the strain under study belongs to the following systematic groups: Bacteria; Actinobacteria; Streptomycetales; Streptomycetaceae; Streptomyces.

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

Processing of the sequenced sequences was carried out using open access bioinformatics software - the Blast (Basic Local Alignment Search Tool) program, designed incl. to determine the degree of phylogenetic proximity of living organisms.

Based on the results of analysis of the sequences of variable regions of genes encoding 16S rRNA, the tested strain DAP 17-18 turned out to be closest to the species Streptomyces baarnensis.

Example 4. Cultivation technique.

The seed material was prepared by sowing the culture of Streptomyces baarnensis VKPM Ac-2200 in Petri dishes. To do this, the agar medium was seeded with the original seed culture and incubated in a thermostat for 5 to 12 days at a temperature of 25-34°C.

Next, the culture was grown in flasks in a liquid medium at 25-34°C for 24-48 hours on a thermostated rocking unit at 200-300 rpm.

Next, the seed culture was produced in a fermenter with a volume of 5/15 l; for this purpose, the fermenter was seeded by introducing 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 content was monitored by HPLC. When the maximum accumulation of antibiotic in the medium occurred, the fermentation process was stopped.

Example 5. Determination of daptomycin content in culture liquid by HPLC.

Analysis of the daptomycin content in the culture liquid by HPLC was carried out on a C18 column, 100 Å, 250 * 4.6 mm, 5 μm; mobile phase A - 0.1% acetonitrile in 0.1% aqueous solution of trifluoroacetic acid. Mobile phase B: 0.1% isopropyl alcohol and 0.1% mobile phase A in acetonitrile, flow rate -1.5 ml/min; detection wavelength - 214 nm.

The averaged data obtained are presented in Table. 1.

Table 1. Yield of the target product during deep cultivation Strain Strain productivity (g/l culture liquid ±0.2 g/l) in a 5 liter fermenter in a 15 liter fermenter Streptomyces baarnensis VKPM Ac-2200 5.3
5.7
5.4 6.6
6.8
6.4

According to the literature, the yield of daptomycin after fermentation of the Streptomyces filamentosus strain CGMCC No. 16016 can reach approximately 3.47 g/l (CN110777084, 02/11/2020).

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

Example 6 . Method for isolating daptomycin.

After cultivating the microorganism Streptomyces baarnensis VKPM Ac-2200, the biomass was separated from the culture liquid using microfiltration. The culture liquid purified from biomass was transferred to the purification stage, where the ultrafiltration process was carried out, and the waste biomass was disposed of.

The purified solution was passed through a column with hydrophobic sorbent HP-20 twice. Elution fractions were analyzed by HPLC. Fractions containing daptomycin with a purity of at least 90% were combined and transferred portionwise into a rotary evaporator flask. The solvent was removed in vacuum at a temperature not exceeding 35°C. Next, the ultrafiltration process was carried out. The purified daptomycin solution was concentrated using a reverse osmosis system. The composition was cooled and further filtered.

Claims (3)

1. Streptomyces baarnensis strain VKPM Ac-2200 is a producer of daptomycin. 2. Use of the strain according to claim 1 to obtain daptomycin. 3. A method for producing daptomycin, including cultivating the strain according to claim 1 and isolating daptomycin from the culture liquid, followed by purification.