RU2737856C1 - Bacillus pumilus strain producing wide-spectrum antibiotic amikumacin - Google Patents

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to the biotechnology. Strain Bacillus pumilus VKM V-3464D, produced by screening method of oral microbiota of Siberian brown bear Ursus arctos collaris, has the ability to synthesize wide-spectrum amikumacin A antibiotic.EFFECT: invention provides enlarging the arsenal of antibiotic producing strains.1 cl, 1 tbl, 1 ex, 2 dwg

Description

The invention relates to the field of biotechnology, namely to a strain producing a broad-spectrum antibiotic amicumacin A in high yield. The invention makes it possible to expand the range of strains for the production of antibiotics and can be used for the subsequent production of amicumacin A.

The increase in the number of cases of infection with resistant strains of microorganisms is currently an avalanche in nature, challenging the sustainable development of mankind and creating the danger of a return to the times when infectious diseases were the main cause of death. To solve the problem of antibiotic resistance, it is fundamentally important to search for new antibiotics, antibiotic-producing strains and to search for new targets for previously discovered antibiotics. In 1982, AI-77-B was isolated from Bacillus pumilus AI-77, which showed good gastroprotective activity and did not possess anticholinergic (cholinolytic) or antihistaminic properties. The antibiotic amikumacin A (isolated substance AI-77-B) belongs to heterocyclic compounds of the oxycoumarin class (Fig. 1). Previously, it was shown that the relationship of gastric ulcer therapy with the antibiotic action of amikumacin A in relation to Helicobacter pilory , an agent causing gastric ulcer (Pinchuk IV et al., 2001). Later, it was shown that amicumacin A is effective against MRSA strains (Lama A., 2012). It was found that amikumacin A has antitumor activity (US 5925671, WO 1996027594), antiviral activity (US 20080161324, US 200100009970). The mechanism of action of amikumacin A is based on specific inhibition of the ribosome (YS Polikanov et al., 2014); later it was shown that the antibiotic acts on both prokaryotic and eukaryotic ribosomes. The use of this substance as a therapeutic agent is limited due to its poor oral absorption (Maddimsetti, Venkateswara, Rao et al. 2014). This fact requires the preparation of stable analogs of amikumacin A. Chemical synthesis is extremely difficult due to the presence of five stereo centers (Fig. 1). At the same time, the modification of natural amicumacin seems to be a more feasible task. This makes it promising to search for producer strains for its subsequent efficient production and purification. Amikumacin A and its close analogs are formed by bacilli isolated from various sources, mainly B. pumilus (Itoh J. et al., 1981, 1982; Berrue F. et al., 2009, Terekhov et al. 2018) and B. subtilis (Li Y. et al, 2012; Azumi M., 2008), as well as unidentified representatives of the genus Bacillus (CA2214776). The most detailed description of the producer of amikumacin A is given in the work of Itoh J. et al., 1981 and patent RU 2627187, which we have chosen as a prototype. In this article, the B. pumilus strain forms a mixture of antibiotics, of which amikumacin A is the main component. In RU 2627187, the content of amicumacin A in the culture fluid formed as a result of biosynthesis is 80-110 μg / ml.

Thus, the technical problem solved by the present invention is to identify a bacterial strain capable of synthesizing the antibiotic amicumacin A with increased efficiency.

To solve the technical problem posed, a new amikumacin A producer strain isolated from a sample of the oral microbiota of the Siberian bear Ursus arctos collaris is proposed using high-throughput screening technology. The strain was identified on the basis of genetic, biotechnological, cultural-morphological and physiological-biochemical characteristics. The claimed strain B. pumilus AmiA_125 deposited in the All-Russian collection of microorganisms, collection number VKM: B-3464D.

The technical result achieved by the present invention consists in the high productivity of the B. pumilus AmiA_125, VKM: B-3464D strain, capable of synthesizing amicumacin A at a level of 145-170 μg / ml in the culture medium, which makes it possible to expand the range of strains for antibiotic production.

Description of the present invention

Generic and specific name of the strain. The producing strain of the antibiotic amikumacin A was assigned to Bacillus pumilus and deposited on November 25, 2019 in the All-Russian collection of microorganisms (VKM) of the Institute of Biochemistry and Physiology of Microorganisms named after V.I. G.K. Scriabin RAS, located at the address: 142290, Russia, Pushchino, Moscow Region, Prospect Nauki, 3 (IBFM RAS - a separate subdivision of the Federal Research Center "Pushchino Scientific Center for Biological Research RAS (FRC PSCBI RAS)) , with the designation AmiA_125 assigned during the deposit and the VKM number: В-3464D.

The origin of the strain (source of isolation, pedigree). The original strain was isolated from a sample of the oral microbiota of the Siberian bear Ursus arctos collaris using high-throughput screening technology (Terekhov et al. 2017, 2018, patent RU2656216) to search for activity that suppresses the growth of the pathogenic microorganism Staphylococcus aureus . As a result of selection, the AmiA_125 strain was obtained, stably retaining the antibiotic production after more than 30 passages. With submerged cultivation on a nutrient medium, the following composition: 50 g / L glucose, 10 g / L bacto-tryptone, 10 g / L yeast extract, 2 g / L sodium chloride, 3 g / L YNB, 1 g / L ammonium hydroxide , 1 g / l of calcium carbonate and 8.2 g / l of sodium phosphate pH 7.5, strain B. pumilus AmiA_125, VKM: B-3464D forms 145-170 μg / ml amikumacin A, which exceeds the level of biosynthesis of the prototype strain declared in patent RU 2627187.

Genetic and physiological and biochemical characteristics. Species genotaxonomic identification was performed using the nucleotide sequence of the 16S rRNA gene. The target gene was amplified by polymerase chain reaction (PCR), sequenced, and the obtained sequence was compared with similar sequences of typical strains of the genus Bacillus stored in the GenBank database (www.ncbi.nlm.nih.gov). The coincidence of the 16S rRNA gene sequence of the VKM: B-3464D strain with similar gene sequences of typical strains of the Bacillus pumilus species is more than 98% (Table 1), which confirms the belonging of the claimed strain to the B. pumilus species. The sequence of the 16S rRNA gene of strain VKM: B-3464D was deposited in GenBank under number MN650262.1. Strain VKM: B-3464D is a facultative anaerobic auxotroph, cultivated on a minimal synthetic medium and does not require the introduction of amino acids or biotin for growth, unlike some other bacilli. Utilizes sucrose, glucose, fructose, practically does not hydrolyze starch.

Cultural and morphological characteristics. Gram-positive, endospore-forming, mobile, straight rods, 1.2 × 4.6 μm. On agar medium Lurie-Bertrand containing 10 g / l bacto-tryptone, 5 g / l yeast extract, 10 g / l sodium chloride, 1% agar-agar at a temperature of 30 ° C strain VKM: B-3464D form beige large matte colonies with an uneven edge, exopigment is absent. On the second day of cultivation on an agar medium, the formation of endospores characteristic of bacteria of the genus Bacillus begins. Strain VKM: B-3464D grows well in the temperature range 22-37 ° C, forming large colonies; no growth at 60 ° C.

Biotechnological characteristics (cultivation conditions; name and properties of the beneficial substance produced by the strain; level of activity (productivity)). Strain VKM: B-3464D forms the antibiotic amikumacin A (Fig. 1) when cultivated in a medium of 50 g / l glucose, 10 g / l bacto-tryptone, 10 g / l yeast extract, 2 g / l sodium chloride, 3 g / l YNB, 1 g / l ammonium hydroxide, 1 g / l calcium carbonate and 8.2 g / l sodium phosphate, pH 7.5. Flasks with a volume of 750 ml with 50 ml of medium were inoculated with a suspension of cells in the amount of 10 ⁶ cells / flask. The cultivation was carried out under aeration on a rotary shaker with a rotation speed of 220 rpm at 30 ° C for 24 hours. The productivity of the strain means the content of amikumacin A per unit volume of the culture fluid formed as a result of biosynthesis during the cultivation of the strain B. pumilus AmiA_125, VKM: B-3464D and is 145-170 μg / ml amikumacin A, which exceeds the level of biosynthesis of the prototype strain.

Determination of the antimicrobial spectrum and the minimum inhibitory concentration (MIC) of amicumacin A. Amicumacin A purified by HPLC to a purity of more than 99% was used as a standard. From FIG. 2, it follows that amicumacin is effective against gram-positive bacteria, namely S. aureus strains, regardless of the presence or absence of methicillin resistance, Macrococcus caseolyticus, Weissella confusa, Staphylococcus warneri, Staphylococcus xylosus, Aerococcus viridans, Enterococcus viridans, Lactocococus, Enterococus casseliflavus , as well as the gram-negative bacteroids Bacteroides vulgatus . In all these cases, the MIC was less than 10 μg / ml.

The invention is illustrated by the following graphic materials:

FIG. 1. Chemical structure of amikumacin A.

FIG. 2. Determination of the antimicrobial spectrum and the minimum inhibitory concentration of amikumacin A.

Table 1. Comparison of the 16S rRNA gene sequence (GenBank: MN650262.1) of the VKM: B-3464D strain with similar gene sequences of typical Bacillus pumilus strains.

Table 1

Sequence Description room Identity,% Bacillus pumilus 16S ribosomal RNA gene, partial sequence DQ105967.1 99.793 Bacillus pumilus strain ATCC 7061 16S ribosomal RNA gene, partial sequence AY876289.1 99.721 Bacillus pumilus strain 001T 16S ribosomal RNA gene, partial sequence FJ477095.1 98.295

The present invention is further illustrated by examples that serve to illustrate it and do not limit the scope of the present invention set forth in the claims.

EXAMPLE 1

In 12 Erlenmeyer flasks with a volume of 750 ml, 50 ml of the medium of the following composition is added: 50 g / l glucose, 10 g / l bacto-tryptone, 10 g / l yeast extract, 2 g / l sodium chloride, 3 g / l YNB, 1 g / l ammonium hydroxide, 1 g / l calcium carbonate and 8.2 g / l sodium phosphate pH 7.5 and sterilized for 45 min in an autoclave. From the cell suspension of the night culture with the VKM: B-3464D strain, 10 ⁶ cells are added to each flask. The flasks are placed on a rotary shaker with a rotation speed of 220 rpm at a temperature of 30 ° C.

After 24 hours, the culture liquid from 12 flasks is combined, the total volume of the culture liquid is 0.56 l. The cells from the culture fluid are precipitated by centrifugation in a Beckman Avanti JCE centrifuge (Beckman Coulter, USA). The supernatant is filtered and ethyl acetate is added in a ratio of 1: 1 = v: v. The extract is evaporated to dryness and dissolved in dimethyl sulfoxide (DMSO). The resulting preparation was applied to HPLC Zorbax C8 (Agilent) and fractionated using a mixture of buffer 1 (20 mM NH ₄ OAc pH 5.0, 5% AcCN), buffer 2 (20 mM NH ₄ OAc pH 5.0, 80% AcCN) at a flow rate of 5 ml / min. Final purification is performed using a Symmetry C18 HPLC column (Waters) with a mixture of buffers 1 and 2 at a rate of 1 ml / min, and using a linear gradient of 0-3 min (100% buffer 1, 0% buffer 2), 3-4 min (0 –30% buffer 2), 4–11 min (30–37% buffer 2), 11–12 min (37–100% buffer 2). Detection is carried out at a wavelength of 315 nm. The eluate is evaporated, lyophilized and dissolved in methanol. The quantitative determination of purified amikumacin A is carried out spectrophotometrically using the extinction coefficient, ε _{315 nm, MeOH} = 4380 M ^-1 cm ^-1 . The productivity of the strain is calculated by the formula

P (mg / L) = A _AmiA × V _AmiA × 423.47 × 1000 / (4380 × 0.56), where

A _AmiA × - value of optical density for spectrophotometric determination of concentration at a wavelength of 315 nm

V _AmiA × - sample volume after cleaning,

423.47 - molecular weight of amicumacin A, g / mol,

0.56 - volume of culture liquid after production, l,

4380 - extinction coefficient at a wavelength of 315 nm, M ^-1 cm ^-1 .

As a result of three successive developments, the average productivity of the B. pumilus AmiA_125, VKM: B-3464D strain under cultivation conditions in Erlenmeyer flasks is 158 mg / l.

Claims (1)

Strain Bacillus pumilus AmiA_125, VKM B-3464D, obtained by screening the oral microbiota of the Siberian brown bear Ursus arctos collaris , capable of synthesizing a broad-spectrum antibiotic amicumacin A.

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