RU2801749C1 - New producer strain of vancomycin amycolatopsis keratiniphila - Google Patents

Abstract

FIELD: microbiology; biotechnology.

SUBSTANCE: proposed strain Amycolatopsis keratiniphila VKPM Ac-2192 is a producer of vancomycin. 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 number 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, 1 dwg, 1 tbl, 6 ex

Description

The invention relates to the field of microbiology and biotechnology and concerns a strain-producer of the antibiotic vancomycin.

BACKGROUND OF THE INVENTION

Vancomycin is a branched-chain tricyclic glycosylated nonribosomal peptide antibiotic and is often used to prevent and treat infections caused by various gram-positive bacteria.

Currently, vancomycin is used to treat infections caused by gram-positive microorganisms resistant to β-lactam antibiotics, as well as hypersensitivity or intolerance to β-lactam compounds; pseudomembranous colitis and enterocolitis, not amenable to treatment with metronidazole; Clostridium difficile - associated diarrhea; for the prevention of endocarditis; for the treatment of infections of bones and joints, including osteomyelitis; for anti-infective prophylaxis in traumatology with artificial prosthetics of the joints; for the treatment of sepsis, meningitis, pneumonia, lung abscesses, as well as for some skin diseases (Belousov D.Yu., et al., Comparative characteristics of vancomycin preparations registered in the Russian Federation // Qualitative Clinical Practice. - 2009. - 4).

The main producers of the antibiotic vancomycin are Amycolatopsis orientalis strains (P. Naga Padma; A. Bhaskar Rao; JS Yadav; Gopal Reddy. Optimization of fermentation conditions for the production of glycopeptide antibiotic vancomycin by Amycolatopsis orientalis. - 2002.- 102-103 ), 395-405.). The prior art also known strain producer of vancomycin Amycolatopsis orientalis ( CN101608209, 12/23/2009) and Amycolatopsis orientalis VKPM Ac-1505, selected by the inventors as prototypes.

It should be noted that the bacterial genus Amycolatopsis is the most important genus of actinomycetes and is of particular interest, since its representatives are the most important sources of the formation of various valuable bioactive metabolites, for example, antibiotics, including vancomycin. The genus Amycolatopsis currently continues to be the focus of attention in the search for new active agents for drugs (Kisil OV et al., Looking Back to Amycolatopsis: History of the Antibiotic Discovery and Future Prospects // Antibiotics (Basel).- 2021 Oct 15. -10(10):1254).

A group of researchers described a strainAmycolatopsis keratiniphila subsp. keratiniphilaD2T degrading bovine keratin by-products (Espersen R, et al., Two novel S1 peptidases fromAmycolatopsis keratiniphila subsp. keratiniphila D2T degrading keratinous slaughterhouse by-products. Appl Microbiol Biotechnol. 2020 Mar;104(6):2513-2522).

A highly efficient CRISPR/Cas9-mediated genome editing system in a vancomycin-producing A. keratiniphila HCCB10007 strain is known from the prior art. By removing large fragments of ECO-0501 BGC, it was possible to increase vancomycin production (Hu M, et al., CRISPR/Cas9-mediated genome editing in vancomycin-producing strain Amycolatopsis keratiniphila. Front Bioeng Biotechnol. 2023 Mar 3; 11: 1141176).

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

The technical result of the claimed invention is to increase the productivity of the vancomycin-producing strain, reduce the cultivation time, reduce the 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.

SUMMARY OF THE INVENTION

In the process of induced mutagenesis and subsequent stepwise selection, a highly productive strain V-0509 producing the antibiotic vancomycin was obtained from a wild-type strain isolated from a sample of soddy-podzolic soil (Mordovia).

To increase the secretion of vancomycin, the wild-type strain was subjected to induced mutagenesis as a result of targeted exposure to ultraviolet light at low temperatures. The resulting highly productive strain was assigned the internal number V-0509.

Further, strain V-0509 was identified by macro- and micromorphological features, and the genus was confirmed by sequencing of the 16S rDNA gene.

According to the results of the analysis of the sequences of the variable regions of the genes encoding 16S rRNA, the tested strain was the closest to the species Amycolatopsis keratiniphila.

The resulting new strain of Amycolatopsis keratiniphila was deposited with the National Bioresource Center All-Russian Collection of Industrial Microorganisms of the National Research Center "Kurchatov Institute" under the number VKPM Ac-2192.

Thus, one of the variants of the present invention is the vancomycin producing strain Amycolatopsis keratiniphila VKPM Ac-2192. Another embodiment of the present invention is the use of the Amycolatopsis keratiniphila VKPM Ac-2192 strain for the preparation of the antibiotic vancomycin. Another option of the present invention is a method for producing the antibiotic vancomycin, which consists in culturing a strain of Amycolatopsis keratiniphila VKPM Ac-2192 and isolating vancomycin from the culture fluid, followed by purification (Figure 1).

Terms and Definitions

The following are terms that may be used in describing the present invention. Unless otherwise indicated, all technical and technical terms used in the description have the meaning generally accepted in the art.

The term "antibiotic" includes a molecule that can inhibit the growth, destroy or kill microorganisms, but is not fatal to a patient at the concentration and dosage ranges intended for administration. According to the present invention, these antibiotics can be classified as bactericidal (ie, directly killing 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, in the range of administered concentrations and dosing.

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 Amycolatopsis keratiniphila VKPM Ac-2192 cells separated by centrifugation from the supernatant of the culture liquid obtained as a result of cultivation, including in shake flasks or bioreactors (for example, when culturing in a battery way ).

The term "culture liquid" in the context of the present invention means a gas-liquid nutrient (or fermentation) medium into which, during submerged cultivation, microorganisms excrete 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 submerged cultivation of microorganisms is carried out to produce the target product (in the framework of the present invention, the antibiotic vancomycin).

The term "strain productivity" in the context of the present invention refers to the amount of antibiotic vancomycin obtained, calculated per unit volume of culture fluid, when submerged cultivation of the Amycolatopsis keratiniphila VKPM Ac-2192 strain under bioreactor conditions, expressed in grams of vancomycin per liter of culture fluid.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is further illustrated by FIG. 1.

On FIG. 1 shows the main stages of screening the strain Amycolatopsis keratiniphila VKPM Ac-2192 and the production of vancomycin from its culture liquid obtained after submerged cultivation.

DISCLOSURE OF THE INVENTION

According to the present invention, a new vancomycin-producing strain , the Amycolatopsis keratiniphila strain, deposited at the National Bioresource Center of the All-Russian Collection of Industrial Microorganisms of the National Research Center "Kurchatov Institute" under the number VKPM Ac-2192, is proposed.

Cultural and morphological features of the vancomycin producer strain:

Aerobe, Gram stain - positive; on a dense medium of the following composition, after 5-12 days of cultivation, it forms colonies from white to milky, conical in shape, 3-5 mm in diameter, raised above the agar, with wavy edges and an uneven surface.

For storage and preparation of inoculum, the medium of the following composition is used, g/l: soy peptone - 8.0 - 12.0; soluble starch - 7.0 - 9.0; glucose - 1.0 - 4.0; agar-agar - 18.0 - 20.0; purified water; Medium pH 6.9±0.3.

Conditions of cultivation for isolation of single colonies: - 5-12 days, at a temperature of 25-34°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 Amycolatopsis keratiniphila from Soil Samples.

The objects of the study were samples of soddy-podzolic soil taken in the Republic of Mordovia in the summer from the upper soil horizons.

To isolate the wild-type strain - the producer of vancomycin from soil samples, the method of sowing 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. Broad-spectrum antibiotics were added to the nutrient medium to limit the growth and activity of undesirable microorganisms.

The inoculations were incubated at temperatures of 20 - 30°C until the appearance of visible colonies with a morphology characteristic of this strain. The pure culture was derived by successive passages to a single colony. Culture purity was controlled microscopically.

Example 2. Obtaining a highly productive strain-producer of vancomycin.

By means of multistage selection with the use of mutagenic factors and directed methods of selection of wild-type strain modifiers, a new highly productive strain producing vancomycin V-0509 was obtained.

To do this, UV irradiation with a wavelength of 250 nm (Mineralight lamp) was performed on an aqueous suspension of spores and cells of a wild-type strain placed at a distance of 55 cm from the lamp for 20 minutes at a temperature of 0°C. The resulting strain was assigned the internal number V-0509.

Example 3 Identification of strain V-0509 to species by PCR analysis of 16sRNA.

DNA isolation of strain V-0509 for PCR was performed according to 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 generate 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 reaction modes:

1. 95°C - 3 min.

2. 35 cycles

95°C - 30 sec.

57°C - 30 sec.

72°С - 1 min. 30 sec.

3. 72°C - 5min

For the analysis of sequenced sequences, specialized phylogenetic computer programs were used. For the stability of reproducing the results, at least three repetitions of PCR reactions were performed.

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

According to the GenBank database, the studied strain belonged to the following systematic groups: Bacteria; Actinobacteria; micromonosporales; Micromonosporaceae; Actinoplanes.

The sequences were aligned according to the sequences of the closest bacterial species available in the GenBank database.

The sequenced sequences were also processed using the BLAST (Basic Local Alignment Search Tool) program module, which is designed, among other things, to determine the degree of phylogenetic similarity of microorganisms.

According to the results of the analysis of the sequences of the variable regions of the genes encoding 16S rRNA, the studied strain V-0509 was the closest to the strains of the species Amycolatopsis keratiniphila.

Example 4. Method of cultivation.

The inoculum was prepared by seeding the Amycolatopsis keratiniphila VKPM Ac-2192 culture on Petri dishes. To do this, the agar medium was seeded with the original seed culture, and kept in a thermostat for 5 to 12 days at a temperature of 25-34°C.

Further cultivation was carried out in shake flasks in a liquid medium at 25–34°C for 24–48 hours on a thermostatically controlled shaker at 200–300 rpm.

Next, a seed culture was produced in a fermenter with a volume of 15/100 l; for this, the fermenter was seeded by introducing a seed culture in an amount of 5-15% of the volume of the medium in the fermenter; sterile air was also continuously supplied during cultivation with stirring for 120-170 hours .

Antibiotic content was monitored by HPLC. At the maximum accumulation of the antibiotic, the fermentation process was stopped.

Example 5. Determination of the content of vancomycin in the culture liquid by HPLC.

Analysis of the content of vancomycin 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 -1.1 ml/min; detection wavelength - 254 nm.

The data obtained are presented in Table 1.

Table 1. Yield of the target product during submerged cultivation Strain Productivity of strains (g/l of culture liquid±0.5 g/l) in a 15 liter fermenter in a 100 liter fermenter Amycolatopsis keratiniphila VKPM Ac-2192 16.09
15.60
15.93 17.05
17.59
16.95 Amycolatopsis orientalis
VKPM Ac-1505 9.02
8.45
8.95 8.05
8.95
8.70

Also, according to the literature data, the productivity of the mutant strain Amycolatopsis orientalis was up to 8.9 g/l (CN101608209, 12/23/2009).

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

Example 6 Vancomycin Isolation Procedure.

After the cultivation of the microorganism Amycolatopsis keratiniphila VKPM Ac-2192 was completed, the culture liquid obtained during submerged cultivation in the bioreactor was acidified with a solution of hydrochloric acid to pH values ~ 1.8–2.5, then the biomass was separated by two successive stages of centrifugation. The culture liquid purified from biomass was transferred to the stage of further purification, and the spent biomass was disposed of.

The supernatant was purified from low molecular weight impurities by dialysis through a semipermeable membrane, after which it was passed through an adsorption column. The solution displaced from the column was discarded. Then the column was desorbed with an aqueous ammonia solution with a concentration of 3.3 g/l.

The eluate obtained at the previous stage was applied to a column with a hydrophobic sorbent HP-20. The solution displaced from the column was discarded. The column was washed with purified water. The eluate was collected and filtered.

The resulting solution was again applied to the column with the HP-20 sorbent. The column was washed with purified water. Then the column was eluted with 50% aqueous ethanol with the addition of acetic acid. The control was carried out by HPLC/UV.

Next, the eluate was fractionated, the fractions were analyzed by HPLC. Fractions containing vancomycin with a purity of at least 92% were pooled. Less pure fractions were submitted for re-purification. The purified vancomycin solution was decolorized by stirring for 30 minutes. with 0.5% wt. pyrogen-free coal. Coal was separated by filtration through a layer of silica gel.

Next, the vancomycin solution was filtered and the crystallization process was carried out. The resulting composition was further filtered.

Claims (3)

1. The strain Amycolatopsis keratiniphila VKPM Ac-2192 is a producer of vancomycin. 2. Use of the strain according to claim 1 for the preparation of vancomycin. 3. A method for producing vancomycin, including cultivating the strain according to claim 1 and isolating vancomycin from the culture liquid, followed by purification.