KR20220037729A - Novel microorganism capable of producing tubulysin and use of the same - Google Patents

Abstract

The present invention relates to Archangium gephyra MEHO_003 and Archangium gephyra MEHO_004, which are novel strains capable of producing tubulysin, and uses thereof.

Description

Novel microorganism capable of producing tubulysin and use of the same}

The present invention relates to novel strains capable of producing tublycin, Archangium gephyra MEHO 003, Archangium gephyra MEHO 004, and their uses. it's about

Myxobacteria are Gram-negative bacteria that produce various secondary metabolic physiologically active substances. So far, 118 types of substances have been isolated from myxobacteria, and more than 600 new substances including derivatives have been isolated (Gerth et al., 2003). ; Schaberle et al., 2014, Hyun & Cho, 2018). Among the physiologically active substances derived from slime bacteria, there are many substances that inhibit the action of the eukaryotic cytoskeleton. Substances that act on the cytoskeleton have an effect of inhibiting cell proliferation, so there is a high potential for development as an anticancer drug. Among the substances that inhibit the action of the cytoskeleton, the most well-known substance is epothilone (Gerth et al., 1996), which showed excellent effects on cancer cells showing multi-drug resistance. It has been approved by the US FDA and sold under the product name Ixempra (Stein, 2010).

Tubulysin is another substance that inhibits the action of the cytoskeleton and shows superior anticancer activity than epothilone, and many studies are being conducted (Sasse et al., 2000; Murray et al., 2015; Reddy et al., 2018; Szigetvari et al. al., 2018). Epothilone promotes the polymerization of tubulin (Gerth et al., 1996), but tubulysin promotes the degradation of the tubulin polymer (Steinmetz et al., 2004).

Tubulysin as described above has a very high development value as an anticancer agent, but a tubulysin-producing strain has not been secured in Korea to date. Therefore, for research and development of tubulysin, it is urgent to secure domestic tubulysin-producing strains.

Korean Patent Publication No. 10-2015-0119086

The present invention has been derived from the above needs, and one aspect of the present invention is to provide a novel tublycin producing strain.

In one aspect of the present invention, it is an object of the present invention to provide a novel strain having an excellent tublycin production yield.

In order to solve the above problems, the present invention provides means for solving the problems as follows.

One aspect of the present invention provides a novel arcangium gephyra MEHO 003 (Archangium gephyra MEHO 003) strain.

In one aspect of the present invention, the strain provides an accession number of KCTC14258BP, Archangium gephyra MEHO 003 ( Arangium gephyra MEHO 003) strain.

Another aspect of the present invention provides an Arcangium gephyra MEHO 004 (Archangium gephyra MEHO 004) strain.

In another aspect of the present invention, the strain provides an accession number KCTC14259BP, Archangium gephyra MEHO 004 ( Arangium gephyra MEHO 004) strain.

In one or another aspect of the present invention, the strain provides a strain producing tublycin.

In one or another aspect of the present invention, the tublycin is any one or more selected from tublysin A and B, it provides a strain.

Another aspect of the present invention provides a composition comprising any one of the above strains, a lysate thereof, a culture thereof, or an extract thereof as an active ingredient.

In another aspect of the present invention, the composition provides a food composition or a pharmaceutical composition, the composition.

In another aspect of the present invention, the pharmaceutical composition provides a pharmaceutical composition for anticancer, the composition.

In another aspect of the present invention, the cancer is stomach cancer, lung cancer, liver cancer, colorectal cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, sclerosis, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, Thyroid cancer, kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer (including leukemia, multiple myeloma, myelodysplastic syndrome), lymphoma (including Hodgkin's disease and non-Hodgkin's lymphoma), psoriasis, or fibroadenoma to provide.

Another aspect of the present invention provides a method for producing tublysin (tubulysin) comprising the step of culturing any one of the above strains.

In another aspect of the present invention, the method provides a method for producing tublysin (tubulysin), further comprising the step of extracting after culturing.

Another aspect of the present invention provides a method for preparing an anticancer pharmaceutical composition comprising the step of culturing any one of the above strains.

In another aspect of the present invention, there is provided a method for producing a pharmaceutical composition for anticancer, comprising the step of adding the strain, its lysate, its culture or its extract to the pharmaceutical composition.

The strain according to one aspect of the present invention has the ability to produce tublycin.

The strain according to one aspect of the present invention has excellent tublycin production efficiency.

1 shows Cystobacter sp. The composition of the tublycin biosynthesis gene family of SBCb004 (Chai et al., Chem. Biol. 17: 296-309, 2010).
Figure 2 is HPLC and absorption spectrum analysis of the culture extract of MEHO_003 strain, TAM1446 is a chemically synthesized tubullysin. The peak material at 22.58 minutes and 25.21 minutes of the MEHO_003 strain culture extract showed an absorption spectrum very similar to that of TAM1446, so it was highly probable that it was tubulysin.
3 is a form of feeder cells and fruiting bodies of strain MEHO_003.
4 is a phylogenetic tree of strain MEHO_003 and related slime bacteria.
5 is a liquid culture result of the dispersion mutant MEHO_004 of A. gephyra MEHO_003 strain.
6 is a mass spectrometry of the substance MT003 produced by strain MEHO_003.
7 is a mass spectrometry of the substance MT004 produced by strain MEHO_003.
8 is a double mass spectrometry analysis of the substance MT003 produced by the MEHO_003 strain, and the double mass spectrometry results of the substance MT003 were compared with the previously reported results of tubulysin A. (Source of the results of double mass spectrometry of Tubulysin A: https://mona.fiehnlab.ucdavis.edu/spectra/display/CCMSLIB00000478582).
9 is a structure of material MT003 reconstituted based on double mass spectrometry results.
10 and 11 show the results of a reproducibility test of MT003's cytotoxicity test.

Unless otherwise specified, all numbers, values, and/or expressions expressing ingredients, reaction conditions, and amounts of ingredients used herein refer to a variety of measures that may occur in obtaining such values, among others, in which such numbers are inherently different. Since they are approximations reflecting uncertainty, it should be understood as being modified by the term “about” in all cases. Also, where the disclosure discloses numerical ranges, such ranges are continuous and inclusive of all values from the minimum to the maximum inclusive of the range, unless otherwise indicated. Furthermore, when such ranges refer to integers, all integers inclusive from the minimum to the maximum inclusive are included, unless otherwise indicated.

In this specification, when a range is described for a variable, the variable will be understood to include all values within the stated range including the stated endpoints of the range. For example, a range of “5 to 10” includes the values of 5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc. It will be understood to include any value between integers that are appropriate for the scope of the recited range, such as 5.5, 6.5, 7.5, 5.5 to 8.5 and 6.5 to 9, and the like. Also for example, ranges from "10% to 30%" include values of 10%, 11%, 12%, 13%, etc. and all integers up to and including 30%, as well as 10% to 15%, 12% to It will be understood to include any subranges such as 18%, 20% to 30%, etc., as well as any value between reasonable integers within the scope of the recited ranges, such as 10.5%, 15.5%, 25.5%, and the like.

Hereinafter, various aspects of the present invention will be described in detail.

One aspect of the present invention provides a novel arcangium gephyra MEHO 003 (Archangium gephyra MEHO 003) strain.

In one aspect of the present invention, the strain provides an accession number of KCTC14258BP, Archangium gephyra MEHO 003 ( Arangium gephyra MEHO 003) strain.

Another aspect of the present invention provides an Arcangium gephyra MEHO 004 (Archangium gephyra MEHO 004) strain.

In another aspect of the present invention, the strain provides an accession number KCTC14259BP, Archangium gephyra MEHO 004 ( Arangium gephyra MEHO 004) strain.

In one or another aspect of the present invention, the strain provides a strain producing tublycin.

In one or another aspect of the present invention, the tublycin is any one or more selected from tublysin A and B, it provides a strain.

Another aspect of the present invention provides a composition comprising any one of the above strains, a lysate thereof, a culture thereof, or an extract thereof as an active ingredient.

As used herein, the disrupted strain may refer to a product obtained by crushing the strain itself by chemical or physical force.

As used herein, the culture may refer to a material containing all substances contained in the culture medium of the strain, for example, may refer to a material containing a metabolite or secretion resulting from the culture of the strain, or a lysate thereof, The strain itself may also be included in the culture.

As used herein, the extract may refer to a product obtained by extracting the strain itself, a lysate of the strain, a culture thereof, or a mixture thereof using an extraction solvent.

In another aspect of the present invention, the composition provides a food composition or a pharmaceutical composition, the composition.

The pharmaceutical composition according to another aspect of the present invention may be in various oral or parenteral dosage forms. In the case of formulation, it is prepared using commonly used diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, soft or hard capsules, etc., and such solid preparations include at least one excipient in one or more compounds, for example, starch, calcium carbonate, sucrose Alternatively, it is prepared by mixing lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, internal solutions, emulsions, syrups, etc. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. there is. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin, and the like can be used.

The pharmaceutical dosage form of the composition according to another aspect of the present invention may be used in the form of a pharmaceutically acceptable salt thereof, and may be used alone or in combination with other pharmaceutically active compounds as well as in an appropriate group. The salt is not particularly limited as long as it is pharmaceutically acceptable, and for example, hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrofluoric acid, hydrobromic acid, formic acid acetic acid, tartaric acid, lactic acid, citric acid, fumaric acid, maleic acid, succinic acid, methanesulfonic acid , benzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, etc. can be used.

The pharmaceutical composition according to another aspect of the present invention may be administered parenterally or orally as desired, and may be administered in an amount of 0.1 to 500 mg, specifically 1 to 100 mg per 1 kg of body weight per day, 1 to several times. It can be administered in divided doses. The dosage for a specific patient may vary depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate, severity of disease, and the like.

The pharmaceutical composition according to another aspect of the present invention may be prepared according to a conventional method for oral formulations such as powders, granules, tablets, soft or hard capsules, suspensions, emulsions, syrups, drinks, aerosols, etc., skin such as ointments, creams, etc. It can be formulated and used in any form suitable for pharmaceutical preparations, including external preparations, suppositories, injections, and sterile injection solutions, and specifically formulated in the form of injections or external preparations for skin.

The pharmaceutical composition according to another aspect of the present invention may be administered to mammals such as rats, mice, livestock, and humans by various routes such as parenteral and oral administration, and all modes of administration can be expected, for example, , oral, transdermally, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The pharmaceutical composition according to another aspect of the present invention may be administered by various routes that can be easily applied by those skilled in the art.

In another aspect of the present invention, the formulation of the food composition is not particularly limited, but may be formulated as, for example, tablets, granules, powders, liquids such as drinks, caramel, gels, bars, and the like. In addition to the active ingredient, the food composition of each dosage form can be appropriately selected and formulated by those skilled in the art without difficulty depending on the dosage form or purpose of use in addition to the active ingredient, and a synergistic effect may occur when applied simultaneously with other raw materials.

In the food composition according to another aspect of the present invention, the determination of the dosage of the active ingredient is within the level of those skilled in the art, and the daily dosage thereof is, for example, 0.1 mg/kg/day to 5000 mg/kg/day, more Specifically, it may be 50 mg/kg/day to 500 mg/kg/day, but is not limited thereto, and may vary depending on various factors such as the age, health status, and complications of the subject to be administered.

The food composition according to another aspect of the present invention is, for example, chewing gum, caramel products, candy, ice cream, various foods such as confectionery, soft drinks, mineral water, beverage products such as alcoholic beverages, health including vitamins and minerals It may be functional foods. In addition to the above, the food composition according to another aspect of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the functional food compositions according to another aspect of the present invention may include natural fruit juice, fruit juice, and pulp for the production of fruit juice drinks and vegetable drinks. These components may be used independently or in combination. The proportion of these additives is not so important, but is generally included in the range of 0 to about 20 parts by weight per 100 parts by weight of the composition herein.

In another aspect of the present invention, the pharmaceutical composition provides a pharmaceutical composition for anticancer, the composition.

In another aspect of the present invention, the cancer is stomach cancer, lung cancer, liver cancer, colorectal cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, sclerosis, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, Thyroid cancer, kidney cancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, blood cancer (including leukemia, multiple myeloma, myelodysplastic syndrome), lymphoma (including Hodgkin's disease and non-Hodgkin's lymphoma), psoriasis, or fibroadenoma to provide.

Another aspect of the present invention provides a method for producing tublysin (tubulysin) comprising the step of culturing any one of the above strains.

In another aspect of the present invention, the method provides a method for producing tubulysin (tubulysin), further comprising the step of extracting after culturing.

Another aspect of the present invention provides a method for preparing an anticancer pharmaceutical composition comprising the step of culturing any one of the above strains.

In another aspect of the present invention, there is provided a method for producing a pharmaceutical composition for anticancer, comprising the step of adding the strain, its lysate, its culture or its extract to the pharmaceutical composition.

Hereinafter, the present invention will be described in detail.

Tubulysin was first reported by a German research group as produced by the Mycobacterium Angiococcus disciformis An d48 and Archangium gephyra Ar 315 (Sasse et al., J. Antibiot. 53: 879-885, 2000), It has also been reported to be produced by Cystobacter sp. (Chai et al., Chem. Biol. 17: 296-309, 2010). The tublycin biosynthetic gene family includes Angiococcus disciformis An d48 and Cystobacter sp. It has been identified and reported from SBCb004 (Sandmann et al., Chem. Biol. 11: 1071-1079, 2004; Chai et al., Chem. Biol. 17: 296-309, 2010) ( FIG. 1 ).

The present invention is to provide a strain that produces the tublysin as described above.

Hereinafter, the present invention will be described in detail by way of Examples and Experimental Examples. However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples.

Example

Example 1. Strain selection, characterization, identification and deposit

(1) Selection of strains with tublycin biosynthesis gene

To date, 11 families are known to the slime bacteria, and the genera Angiococcus, Archangium, and Cystobacter are all slime bacteria belonging to the family Cystobacteraceae. Hoseo University Slime Bacteria Bank has about 2,600 isolated slime bacteria in Korea. Of these, 81 slime bacteria belonging to the Cystobacteraceae family have been confirmed to be viable. Strain 81 was isolated by Hoseo University Mycobacterium Bank using the method reported by Park et al.

In the present invention, strains having a tublycin biosynthesis gene group were searched for by using polymerase chain reaction (PCR), targeting 81 strains belonging to the Cystobacteraceae family owned by the Mycobacterium Bank of Hoseo University. For this, a PCR primer specifically detecting only the tublycin biosynthesis gene was required. However, since these primers are not known, in the present invention, the previously reported Angiococcus disciformis An d48 and Cystobacter sp. After arranging the nucleotide sequence (GenBank accession number: AJ620477, GU002154.1) of the tublysine biosynthesis gene group of SBCb004, the tublysine biosynthesis gene tubB-tubC region was specifically detected using the conserved sequence. A pair of PCR primers H403 and H404 were designed and prepared. H403 (5'-GTGCAGAAYCTGCGGCTCTT-3') is located at the end of the tubB gene, and H404 (5'-AGCTGCTCCTGGAACCACA-3') is located at the beginning of the tubC gene. The expected size of the PCR product to be produced using the two primers was 1,399 bp.

As a result of PCR amplification of the genomic DNA of 81 strains using the prepared primer pair, PCR products with a size of about 1.4 kb were specifically amplified in 8 strains. As a result of determining and analyzing the DNA base sequence of the obtained PCR product, it was confirmed that all of the PCR products had the end of the tubB gene and the front of the tubC gene, and the previously reported Cystobacter sp. It was confirmed that SBCb004 has 83% or more similarity to tubB and tubC genes, which are tublycin biosynthesis genes.

(2) Selection of tublycin-producing strains

8 strains having a tublycin biosynthesis gene were cultured in CYS medium to prepare a culture extract. CYS medium is 0.5% casein lysate (casitone), 0.1% yeast extract, 0.3% soluble starch, 0.1% MgSO47HO, 0.05% CaCl22H2O, 50 mM HEPES (pH 7.6), 0.1% trace element solution, 0.5 μg/ml cyanocobalamin (Vitamin B12), and the trace element solution was 100 mg MnCl2·4H2O, 20 mg CoCl2, 10 mg CuSO4, 10 mg Na2MoO4·2H2O, 20 mg ZnCl2, 5 mg LiCl, 5 mg SnCl2·2H2O per 1 liter. mg H3BO3, 20 mg KBr, 20 mg KI, 8 g EDTA Na-Fe3+salt (trihydrate). The strain was cultured at 30°C. The culture extract was prepared as follows. The strains were cultured with shaking in 50 ml CYS medium containing 1% Amberlite XAD-16 resin (Sigma, USA) for 7 days, and then the resin was recovered. The resin was washed 3 times with 10 ml of sterile tertiary distilled water, extracted 3 times with 3 ml of 100% methanol, and dried using a rotary evaporator. After putting 8 ml of sterile tertiary distilled water and ethyl acetate solution mixed in a 1:1 ratio, the ethyl acetate layer was recovered, dried with a rotary evaporator, and dissolved in 1 ml of 100% methanol.

The prepared culture extract was analyzed using an Agilent 1260 VL Infinity Series HPLC system (Agilent Technologies, USA) equipped with a Zorbax SB-C18 column (4.6×150 mm, 5 μm). Synthetic tublycin TAM1446 was used as a control material. Distilled water containing 0.1% formic acid was used for mobile phase A, and acetonitrile containing 0.1% formic acid was used for mobile phase B. The flow rate is 0.5 ml/min, 30% solvent B for 0-5 minutes, 30% to 60% solvent B gradient for 5-25 minutes, 60% to 100% solvent B gradient for 25-30 minutes, 30-35 minutes Eluted with 100% solvent B, 30% solvent B for 35-40 minutes. The eluent was detected using a photo diode array (PAD) detector.

Derivatives belonging to the same substance group tend to show similar delay times and absorption spectra in HPLC analysis. By analyzing the culture extracts of 8 strains, it was investigated whether there is a strain producing a material with a delay time and absorption spectrum similar to that of synthetic tublycin TAM1446. As a result, it was analyzed that the culture extract of one strain produced a material exhibiting almost the same absorption spectrum as the synthetic tublycin TAM1446 (FIG. 2). In the present invention, this strain was named MEHO_003, and the 25.21 minute peak material detected in the culture extract analysis of the MEHO_003 strain was named MT003, and the 22.58 minute peak material was named MT004 (FIG. 2). Subsequent analysis was performed on the MEHO_003 strain and the MT003 and MT004 fractions.

(3) Biological characteristics of the selected strain

Feeder cells of the MEHO_003 strain were 0.6 to 1 μm thick and 5 to 15 μm long, and were in the form of long, pointed and slender bacilli belonging to the suborder Cystobacterineae (Fig. 3). It grew well in WCE medium (10 mM morpholinepropanesulphonic acid (MOPS, pH 7.6), 0.1% CaCl27H2O, 1.5% agar) smeared with live Escherichia coli as a lytic mycobacterium. A fruiting body unique to Archangium gephyra was formed (FIG. 3).

(4) Identification of strains through 16S rRNA gene sequencing

For accurate identification of the strain, phylogenetic analysis was performed by the 16S rRNA gene sequence. After culturing the MEHO_003 strain in VY/3 medium (0.5% baker's yeast, 0.1% CaCl2·2H2O, 10 mM 3-[N-morpholino] propanesulfonic acid (MOPS) (pH 7.6), 0.5 μg/ml cyanocobalamine), the cells DNA was extracted from the PCR (primers: 27F, 5′-GAGTTTGATCCTGGCTCAG-3′ and 1525R, 5′-AGAAAGGAGGTGATCCAGCC-3′/30 cycles: 94°C 30 sec, 55°C 30 sec, 72° C 2 min) to amplify and purify the 16S rRNA gene region, and to analyze the nucleotide sequence at Macrogen Co., Ltd.

As a result, the nucleotide sequence of the 16S rRNA gene of the MEHO_003 strain was analyzed for 1,498 bp as shown in SEQ ID NO: 2 attached separately.

As a result of phylogenetic analysis with other bacteria, the nucleotide sequence of the 16S rRNA gene of the MEHO_003 strain was known to show 99.79% homology with the 16S rRNA gene sequence (GenBank accession number: CP011509) of the standard strain DSM 2261 of Archangium gephyra. It was analyzed as the most similar among bacteria (FIG. 4).

The 16S rRNA gene sequence of the MEHO_003 strain is most similar to the standard strain of Archangium gephyra (FIG. 4), and since it formed a typical fruiting body of Archangium gephyra on WCE medium (FIG. 3), in the present invention, the strain is Archangium gephyra) was finally identified. The nucleotide sequence of the tubB-tubC gene region of strain MEHO_003 was shown in SEQ ID NO: 1.

(5) Separation of variance mutants

The slime bacteria show the characteristics of cells growing together in liquid culture. These characteristics are an obstacle to mass culture using a fermenter, and make it impossible to genetically manipulate for strain improvement. Therefore, in this study, the strain MEHO_003 was successively subcultured in the CYS medium, and the dispersed and growing strain in the liquid medium was isolated and named Archangium gephra MEHO_004.

A photograph of liquid culture of the dispersion mutant was shown in FIG. 5 .

(6) Deposit of microbial strains

The above-described Archangium gephra MEHO_003 and MEHO_004 strains were deposited at the Korean Collection for Type Culture (KCTC) on July 28, 2020. The accession number of the MEHO_003 strain was KCTC 14258BP, and the accession number of the MEHO_004 strain was KCTC 14259BP.

Example 2. Mass spectrometry of substances MT003 and MT004 produced by strain MEHO_003

The MT003 and MT004 peak material fractions produced by Archangium gephra MEHO_003 were separated and UPLC-MS analysis was requested to the Gyeonggi Bio Center of the Gyeonggi Provincial Institute of Economic Sciences. UPLC-MS analysis was performed using an Accelar UHPLC (Thermo Scientific, USA) equipped with a Waters BEH C18 column (2.1×150 mm, 1.7 μm). Distilled water containing 0.1% formic acid was used for mobile phase A, and acetonitrile containing 0.1% formic acid was used for mobile phase B. The solvent flow rate was 0.4 ml/min, 20% solvent B gradient for 0-1 min, 20-80% solvent B gradient for 1-20 min, 80-100% solvent B gradient for 20-24 min, 100% B solvent for 24-27 min. Eluted with solvent conditions.

The division result was the same as in FIGS. 6 and 7 .

As a result of the analysis, MT003 had an m/z value of [M+H]+ of 844.4487, which was very similar to the theoretical molecular weight (844.4525) of tublysin A (FIG. 6), and MT004 had an m/z value of [M+H]+ The value was 830.4334, very similar to the theoretical molecular weight of tublysin B (830.4368) (FIG. 7). When all the chemical formulas with possible molecular weights within Delta (ppm) ±5 presented in the analysis result were analyzed, other substances produced in the myxobacteria other than tublycin A and B were not found. The m/z values were very similar, the absorbance spectrum of the material was very similar to that of synthetic tublysin TAM1446, and it was analyzed that both materials had a molecular weight very similar to that of tublysin at the same time. It was very likely that it was A and tubularysin B.

Example 3. Dual mass spectrometry of material MT003

As a result of mass spectrometry, MT003 and MT004 were highly likely to be tubularisin A and tubullysin B, so the structure of MT003 was analyzed by tandem mass spectromegtry.

Double mass spectrometry was commissioned by the Korea Basic Science Institute and a 15T FT-ICR MS was used.

As a result, the fragmentation aspect of MT003 was confirmed to be very similar to the previously known tubularisin A (FIG. 8), and the structure reconstructed based on the molecular weight of the fragments was also analyzed to be tubularisin A (FIG. 9). Therefore, in the present invention, it was concluded that the substance MT003 produced by the strain MEHO_003 is tubularysin A and that the substance MT004 is tubullysin B.

Experimental Example 1. Analysis of tubularysin production yield by Archangium gephra MEHO_004

go. experimental method

After culturing Archangium gephra MEHO_003 and the dispersion mutant MEHO_004 in 50 ml CYS medium for 7 days, a culture extract was prepared. The prepared culture extract was analyzed using an Agilent 1260 VL Infinity Series HPLC system equipped with a Zorbax SB-C18 column (4.6×150 mm, 5 μm).

me. Experiment result

When quantitative analysis by HPLC using TAM1466, a synthetic tublycin, as a standard material, the wild-type MEHO_003 strain produced 0.32 mg of tublysin A (MT003) and 0.16 mg of tublysin B (MT004) per liter, and dispersion mutants The MEHO_004 strain was analyzed to produce 0.85 mg of tublysin A (MT003) and 0.49 mg of tublysin B (MT004) per liter. Therefore, it was found that the dispersion mutant MEHO_004 produced 2.7 times more tublysin A and 3.1 times more tublysin B than the wild-type strain MEHO_003.

Experimental Example 2. Confirmation of the cancer cell inhibitory effect of MT003

In order to confirm the cancer cell inhibitory effect of MT003, MTT assay was performed.

1. Materials and Equipment

1) Materials: Complete media, DPBS, FBS, 1XTrypsin-EDTA, Penicillin-Streptomycin Solution, 96 well plate, 100X20mm cell culture dish, MTT solution (2mg/ml), reservoir, and DMSO were prepared.

2) Clean bench and ELISA were prepared for the equipment.

2. Sample Preparation

(1) Sample: MT003, solvent

(2) Sample Stock solvent: 10% DMSO + 90% PBS

(3) Sample concentration: 100nM ... 1pM (1/10 serial dilution)

3. MTT assay

1) Incubate for 24 hours after seeding.

2) Treat the prepared sample after suctioning the existing media.

-> Use 2 white tips overlapping for media suction

3) Incubate for an additional 72 hours.

4) After sample suction, 2 mg/ml MTT solution is diluted 2:8 with SF media for the cultured cells, followed by incubation for 2-3 hours.

-> Dissolve MTT of powder in DPBS at a concentration of 2mg/ml and store it in the refrigerator

-> MTT : SF = 2 : 8 ratio, turn off the fluorescent lamp of the clean bench during manufacturing and MTT treat

5) Confirm that Formazan is formed under a microscope.

6) After removing the MTT solution, 100 μl of DMSO (purity 95%) is added to lyse the cell, and after shaking for 30 seconds using a microplate, it is measured at 570 nm.

-> Depending on the density of cells, formazan may not mix well, so shake the plate edges with your hands and shake thoroughly. (Exact time, number of times not set)

-> Shaking of the microplate is performed orbital for 30 seconds.

4. Experimental results

The experimental results were as shown in FIGS. 10 and 11 .

MT003 cell efficacy confirmed strong cytotoxicity in PANC-1 (pancreatic cancer) compared to WI-38 (normal cells).

When evaluating the efficacy of MT003 and the reference drug Paclitaxel, superiority of MT003 was confirmed.

IC50 of PANC-1: MT003-0.311nM=311pM // PTX-1.749nM=1749pM

As mentioned above, although the present invention has been described through Examples, Experimental Examples, and Manufacturing Examples, those of ordinary skill in the art to which the present invention pertains will realize that the present invention may be embodied in other specific forms without changing its technical spirit or essential features. You will understand that you can. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

Korea Institute of Bioscience and Biotechnology KCTC14258BP 20200728 Korea Institute of Bioscience and Biotechnology KCTC14259BP 20200728

<110> Mecox curemed Hoseo University <120> Novel microorganism capable of producing tubulysin and use of the same <130> DP-2020-0413 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 1399 <212> DNA <213> Artificial Sequence <220> <223> tubB-tubC region of MEHO_003 <400> 1 gtgcagaacc tgcggctctt cgagctgttc cacgcctcgg tcgtcctggc ccaggccccc 60 gccgagcagg aggcggcccc gctgctcggg cgcgtgcggc agcgcatgct gctcgatgag 120 cggctgtatg tggaccccgc cttcttcccg gccctggtgg ggcagatgcc cctgctcggc 180 gcggtggacg tgcggctcaa gcggggcagc ggcggcaacg agatgaaccg cttccgctac 240 gacgtgatgt tgtggcgctc gcccgtggcg gaagcccccg ccgccgggcc gccctccctg 300 gactgggaga gcgagggact gtcgctggag cgcgtgcggc gcatgctgga ggagcggccc 360 tcgggactgc tgctgcgccg ggtgcccaac gcccgcacgg ccgatgatgc ctcgcggctg 420 gcgctcctcc gctcgcgcgg cacggcctcc agcgtgggcc gattgatggc gctgccggcg 480 gtggccgcct gtgagccgga ggcgctgtgc cgcctggccg agtccatggg cctgcggtgt 540 cacctcgact ggagtgccga gggggaagag ggccgcttcg acgcgctgct gacctccggc 600 cccgcacggt gggtgcccgc cgcaacgccc cggcggccct ggcgcgacta cgccaacgcg 660 cccctgctgg ccagccagcg ccaggccctg ctcggtgagc tgcgcacccg gctggatcac 720 accctgcccg agtacatggt gccctccgcc ttcgtgctgc tctccgagct gccacgcaag 780 cccacgggga agctggacgt gcccgccctg cctcctcccg agccggtgcg ggccgagcac 840 gcggcgactc atcaggcacc gcgcaccccc accgagcgga agctcgccga gctgtggcgg 900 cgcgtgctcg gcgtgcagcg cgtgggggcc aacgacgact tcttccagct cggaggccac 960 tcgctgctgg ccacgcggct gctgtcactc atccgcggcg agctgggcct ggagctgccc 1020 ctgcgcgtgc tcttcgagca atccaccctg gccaccatgg cgggctgcat ggacgcacgc 1080 agttggctca ccggaccggc cccggctccg caagcgggcg aggagggcga gatttgaacc 1140 cgggagaact gctcgcgcat gccgcgggat tgggcgtgcg gctgtgggtg gagaacgggc 1200 ggctgcggtt ccacgccccc accggggtga tgaaccccga gttgcagtcc cggctggccg 1260 cctcacgcgc cgagctcatc tccctgctgc aacaactcca gggtccccag acggccgccg 1320 cacccatgcc ccgggtgtct cgcgagggcc cgctggagct ctccttcgcc cagcagcggc 1380 tgtggttcca ggagcagct 1399 <210> 2 <211> 1498 <212> DNA <213> Artificial Sequence <220> <223> 16S rRNA of MEHO_003 <400> 2 aacgaacgct ggcggcgtgc ctaacacat caagtcgagc gcgaatggag caatcctagt 60 agagcggcgc acgggtgcgt aacacgtgga taatctgcct gggtgtctgg gataaccagt 120 cgaaagattg gctaataccg gataagcccc cgggagcttc ggctcttgag ggaaaaggtg 180 gcctctgtat acaagctatc acatccagat gagtccgcgg cccatcagct agttggcggg 240 gtaatggccc accaaggcaa cgacgggtag ctggtctgag aggacgatca gccacactgg 300 aactgagaca cggtccagac tcctacggga ggcagcagtg gggaattttg cgcaatgggc 360 gaaagcctga cgcagcaacg ccgcgtgtgt gatgaaggtc ttcggattgt aaagcacttt 420 cgaccgggac gaaaaccctg aggctaacat cctcaggctt gacggtaccg ggagaagaag 480 caccggctaa ctctgtgcca gcagccgcgg taatacagag ggtgcaagcg ttgttcggaa 540 ttattgggcg taaagcgcgt gtaggcggct ttgcaagtcg ggtgtgaaag ccctcagctc 600 aactgaggaa gtgcgcccga aactgcagag cttgagtgcc ggagagggtg gcggaattcc 660 ccaagtagag gtgaaattcg tagatatggg gaggaacacc ggtggcgaag gcggccacct 720 ggacggtaac tgacgctgag acgcgaaagc gtggggagca aacaggatta gataccctgg 780 tagtccacgc cgtaaacgat gagaactagg tgtcgtgggt gttgaccccc gcggtgccgt 840 agctaacgca ttaagttctc cgcctgggaa gtacggtcgc aagactaaaa ctcaaaggaa 900 ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgacgcaa cgcgcagaac 960 cttacctggt cttgacatcc ttggaatcct tcagagatga gggagtgccc gcaagggaac 1020 caagagacag gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1080 cgcaacgagc gcaaccctcg cctttagttg ccgcgcaagc ggatctctag agggactgcc 1140 ggtgttaaac cggaggaagg tggggatgac gtcaagtcct catggccttt atgaccaggg 1200 ctacacacgt gctacaatgg ccggtacaac gcgtcgccaa cccgcgaggg ggagctaatc 1260 gcataaaacc ggtctcagtt cagattggag tctgcaactc gactccatga aggcggaatc 1320 gctagtaatc gcggatcagc acgccgcggt gaatacgttc ccgggccttg tacacaccgc 1380 ccgtcacacc atgggagtcg attgctccag aagtcatctc accaagaggt gcccaaggag 1440 tggtcggtaa ctggggtgaa gtcgtaacaa ggtagccgta ggggaacctg cggctgga 1498

Claims (14)

Arcangium gephyra MEHO 003 (Archangium gephyra MEHO 003) strain.
Arcangium gephyra MEHO 004 (Archangium gephyra MEHO 004) strain.
The method of claim 1,
The strain is an accession number KCTC14258BP, Archangium gephyra MEHO 003 ( Arangium gephyra MEHO 003) strain.
3. The method of claim 2,
The strain is an accession number KCTC14259BP, Archangium gephyra MEHO 004 ( Arangium gephyra MEHO 004) strain.
The method of claim 1,
The strain is a strain producing tublysin.
6. The method of claim 5,
The tublycin is at least one selected from tublycin A and B, the strain.
A composition comprising the strain of any one of claims 1 to 6, a lysate thereof, a culture thereof, or an extract thereof as an active ingredient.
8. The method of claim 7,
The composition is a food composition or a pharmaceutical composition, composition.
9. The method of claim 8,
The pharmaceutical composition is a pharmaceutical composition for anticancer, composition.
9. The method of claim 8,
The cancer is stomach cancer, lung cancer, liver cancer, colorectal cancer, small intestine cancer, pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer, sclerosis, uterine cancer, cervical cancer, head and neck cancer, esophageal cancer, thyroid cancer, parathyroid cancer, kidney cancer, sarcoma, prostate Cancer, urethral cancer, bladder cancer, blood cancer (including leukemia, multiple myeloma, myelodysplastic syndrome), lymphoma (including Hodgkin's disease, non-Hodgkin's lymphoma), psoriasis, or fibroadenoma.
Claims 1 to 6, comprising the step of culturing the strain of any one of claims, tublysin (tubulysin) production method.
12. The method of claim 11,
The method further comprises the step of extracting after culturing, the production method of tubulysin (tubulysin).
A method for producing a pharmaceutical composition for anticancer comprising the step of culturing the strain of any one of claims 1 to 6.
14. The method of claim 13,
A method for producing a pharmaceutical composition for anticancer, comprising the step of adding a strain, a lysate thereof, a culture thereof or an extract thereof to a pharmaceutical composition.

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