WO2011055991A2

WO2011055991A2 - A novel myxobacteria strain and a method for preparing epothilone using the same

Info

Publication number: WO2011055991A2
Application number: PCT/KR2010/007741
Authority: WO
Inventors: Bu Soo Park; Chong Jin Park; Mee Jeong Lee; Yong Soon Hwang
Original assignee: Samyang Genex Corporation
Priority date: 2009-11-05
Filing date: 2010-11-04
Publication date: 2011-05-12
Also published as: WO2011055991A3; KR20110049304A

Abstract

Sorangium cellulosum SYC248 strain capable of producing a high concentration of epothilone with antibiotic/anticancer activities isolated from Korean soil and having a high growth rate, and a method for preparing epothilone using the strains are provided.

Description

A NOVEL MYXOBACTERIA STRAIN AND A METHOD FOR PREPARING EPOTHILONE USING THE SAME

The present invention relates to a novel myxobacteria strain and a method for preparing epothilone using the same. More specifically, the present invention relates to novel Sorangium cellulosum strains capable of producing a high concentration of epothilone with antibiotic/anticancer activities isolated from Korean soil and having a high growth rate, and a method for preparing epothilone using the strains.

Myxobacteria are a group of Gram-negative, unicellular anaerobic bacteria that live in the soil. Myxobacteria are rod-shaped bacillus with a thickness of 0.6 to 1.0 ㎛ and a length of about 3 to 10 ㎛, which are known as strains that form characteristic structures, so-called “fruiting bodies” due to life cycles different from other Gram-negative bacteria and produce various physiological substances such as antibiotics or antifungal ingredients [5]. 12 genera and 40 species of myxobacteria are known to date. Myxobacteria are included among the delta group of proteobacteria [1].

Sorangium cellulosum, a slime bacterium that belongs to the group Myxobacteria produces a physiological substance called “epothilone” whose antifungal activity was first identified by Gerhard Hofle and his colleagues [2]. Epothilone, via tubulin polymerization assay, was later discovered to exhibit antitumor activity and since then has been widely researched as a potential antitumor agent for treating cancers. Like anticancer agents such as Taxol (ingredient name: paclitaxel) or Taxotere ® which are the most commonly used for cancer treatment, epothilone is known to stabilize microtubules and inhibit cell division to suppress proliferation of cancer cells [3].

The chemical structure of epothilone produced by Sorangium cellulosum, So ce 90 strain, was disclosed in Hofle et al. 1996, Epothilone A and B-novel 16-membered macrolides with cytotoxic activity: isolation, crystal structure, and conformation in solution, Angew, Chem, Int, Ed. Engl. 35(13/14): 1567-1569. Epothilones A (R=H) and B (R=CH₃) are found to have the following structures and exhibit a wide range of cytotoxicity to eukaryotic cells, and remarkable activity and selectivity to breast and colon cancer cell lines [6].

The desoxy counterparts of epothilones A and B also known as epothilone C(R=H) and D(R=CH₃) were chemically synthesized by a de novo method, but are also produced in small amount in cultures of Sorangium cellulosum. The structure of the counterparts is as follows [4].

Like epothilone A or B, epothilones C and D exhibit cytotoxic action, immunosuppression or inhibitory activities to malignant tumors. Of these, epothilone B exhibits the most potent efficacious.

Such epothilone has potent anticancer activity comparable to the antitumor agent Taxol, which is widely used all over the world. In particular, epothilone B was found to exhibit about 2000- to 5000-fold higher inhibitory activity, as compared to Taxol, and thus is attracting a great deal of attention as a next-generation antitumor agent. In addition, epothilone B also exhibits superior activity even to Taxol-resistant cancer cells and has the possibility of application as an antitumor agent. However, it is extremely difficult to produce a high concentration of epothilone using technologies known to date [7,8]. Conventional methods for producing microorganism strains are difficult to put into practical application due to extremely complicated procedures for isolating or screening microorganisms, considerably low growth rate of strains and extremely low epothilone production. Preparation methods using chemical synthesis require high preparation costs and a long period of time and are thus difficult to put into practical application [9]. Epothilone has been noted as a novel anticancer agent comparable to the powerful Taxol, but cannot be practically applicable since a method for producing high-concentration epothilone has not yet been developed.

In particular, most of the microorganisms that have been discovered hardly produce epothilones, and the difficulty in screening Sorangium cellulosum for producing epothilone has limited the utilization of Sorangium cellulosum. In addition, a known strain of Sorangium cellulosum has the longest doubling time (that is, 16 hours) amongst Myxobacteria and thus has low growth rate. For this reason, there was a limitation to application of Sorangium cellulosum to gene manipulation for preparing epothilones with improved production efficiency and high concentration. Therefore, a great deal of research has been conducted to develop methods suitable for preparing high-concentration epothilone.

As a result of extensive research to develop methods for preparing epothilones at a high concentration, the inventors of the present invention discovered that specific Myxobacteria strains isolated from Korean soil samples have high growth rate and useful produce epothilones at a high concentration and thus completed the present invention.

Therefore, the present invention has been made in view of the above problems, and it is one object of the present invention to provide novel Sorangium cellulosum strains which are isolated from Korean soil, have a high growth rate, are capable of producing a high concentration of epothilone upon fermentation and can be rapidly cultured, thus exhibiting high production efficiency and economical efficiency.

It is another object of the present invention to provide a method for preparing epothilone using the Sorangium cellulosum strains.

In accordance with one aspect of the present invention, provided is a Sorangium cellulosum SYC248 strain isolated from the Korean soil, the strain having a rod-shaped shape, forming smooth swarms of colonies, capable of producing a high concentration of epothilones A and B and having a high growth rate.

In accordance with another aspect of the present invention, provided is a Sorangium cellulosum strain deposited under accession number KCCM11007P at the Korean Culture Center of Microorganisms in Korean Federation of Culture Collections.

In accordance with yet another aspect of the present invention, provided is a method for preparing epothilone using the Sorangium cellulosum SYC248 strain.

Hereinafter, isolation of Sorangium cellulosum strains and a method for preparing epothilone using the same according to the present invention will be described in detail.

Collection of soil samples and isolation of strains

The soil containing animal droppings, decayed leaves, manure and the like known as a habitat is suitable for collecting Sorangium cellulosum strains. A soil sample was collected from Korean soil having these characteristics and Myxobacteria, in particular, the species Sorangium cellulosum, is isolated from various bacteria present in the soil sample. Isolation of Sorangium cellulosum strains is carried out based upon the unique cellulose degrading properties of the genus of Sorangium. The isolated Sorangium cellulosum strains may degrade paper such as cellulose filter paper and grow, and may be yellow, orange, red, reddish brown or black in color. Myxobacteria have the ability to decompose biomacromolecules and thus lyses, or decompose cells or proteins of other living organisms such as dead or living bacteria or yeast to produce amino acids and peptides and thereby use the same as nutrients. Accordingly, cells or proteins of other living organisms such as dead or living bacteria or yeast may serve as a nutrition source in the process of isolating pure Myxobacteria. In the isolation process, in a minimal medium containing only an E. coli suspension without other nutrients, only cells capable of decomposing the medium can grow. The cells capable of performing functions such as proteolysis and bacteriolysis are Myxobacteria. Accordingly, Myxobacteria, in particular, the species Sorangium cellulosum, are purely isolated in the afore-mentioned manner.

Identification of strains

The base sequences of 16s rDNA of the selected cellulose-decomposing strains are analyzed and are then compared with those of So ce 56 strains of Sorangium cellulosum using BLAST. A novel Sorangium cellulosum strain having 99% homology is identical to the 16s rDNA sequence [10] of So ce 56 strains of Sorangium cellulosum.

The 16s rDNA base sequence of Sorangium cellulosum strains identified according to the present invention is shown in FIG. 3 (Sequence Listing No. 1).

Culturing of strains and preparation of epothilone

The purely isolated Sorangium cellulosum strains are proliferated in an agar medium and then cultured in a liquid medium. Then, the proliferated strains are further cultured, proliferated and are then finally cultured in a fermenter containing a medium with a XAD-16 resin. After the sequential proliferative culturing and production culturing, the resin separated from the culture medium is extracted in an alcohol solvent, the alcohol extract is concentrated, and epothilone is prepared via a series of pre-treatment and isolation processes.

Specifically, the isolated strains, Sorangium cellulosum, are proliferated in an agar medium and then cultured in a sterilized tube containing a liquid proliferation medium for 5 to 7 days. Then, the proliferated strains are further proliferated in a flask for 5 to 7 days and then finally cultured in a 5L fermenter containing a production culture medium with an XAD-16 resin for about 14 days.

After the sequential proliferative culturing and production culturing are completed, the resin separated from the culture medium is extracted using an alcohol solvent such as methanol, ethanol or isopropanol, the alcohol solution is concentrated and extracted in a solvent such as ethyl acetate, or alkyl halide, e.g., dichloromethane, carbon tetrachloride, chloroform or 1,2-dichloroethane, and cells and impurities are removed using celite (Junsei, Celite 545) as an absorbent. The culture medium extract free of the cells and impurities is subjected to MPLC and Pre-HPLC to isolate epothilones A and B, and whether or not epothilone is purely isolated at a purity of 95% or higher is verified.

Whether or not Sorangium cellulosum concentrates contain epothilones A and B may be determined by TLC, HPLC, LC-MASS or NMR analysis. As mentioned in Examples below, the Sorangium cellulosum strains according to the present invention are verified to produce epothilone A and epothilone B.

The present invention provides a novel Sorangium cellulosum strain with a high growth rate isolated from Korean soil samples, and a method for preparing epothilone, as an antitumor agent, exhibiting anticancer activities against various human-derived cancer cells using the same, thus being useful for the biomedicine industry.

In particular, the strain according to the present invention has a doubling time of 8 to 10 hours, which is considerably shorter than that of conventional strains (commonly, 16 hours), thus advantageously having a high proliferation rate.

FIG. 1 shows the petri dish in which a soil sample is cultured in the ST21CXKA agar medium for 2 to 3 weeks according to Example 2 of the present invention;

FIG. 2 shows the petri dish in which the separated strains is cultured in the VY2 agar medium according to Example 2 of the present invention;

FIG. 3 shows 16s rDNA sequences of strains according to Example 3 of the present invention;

FIG.4 shows the liquid culture of strains according to Example 4 of the present invention;

FIG.5 shows TLC analysis results of epothilones obtained according to Example 5 of the present invention;

FIGS. 6a to 6c show HPLC analysis results of epothilones obtained according to Example 5 of the present invention;

FIGS. 7a to 7b show LC-MS analysis results to measure the molecular weight of epothilones obtained according to Example 5 of the present invention (7a: epothilone A, 7b: epothilone B); and

FIGS. 8a to 8d show NMR analysis results to analyze the structure of epothilones obtained according to Example 5 of the present invention (8a: ¹H-NMR-Data of epothilone A, 8b: ¹H-NMR-Data of epothilone B, 8c: - ¹³C-NMR-Data of epothilone A, and 8d: ¹³C-NMR-Data of epothilone B).

Now, the present invention will be described in more detail with reference to the following examples, preparation examples and experimental examples. These examples are provided only to illustrate the present invention and should not be construed as limiting the scope and spirit of the present invention.

Example 1: Collection of soil sample

100g of soil samples was collected from soil containing decayed leaves or manure as a common habitat of Myxobacteria which is rich in molds such as Aquifoliaceae, ginseng, oak, acacia tree, strawberry beds, grape beds, cocoa and/or plantations and th like, in six regions including Seogwipo of Jeju-do, Yuseong-gu of Daejeon, Jeongeup of Jeolla-do, Gangneung of Gangwon-do, in Korea.

Example 2: Isolation of Myxobacteria, and pure-isolation and culturing of Sorangium cellulosum strains

As Sorangium cellulosum has a cellulose-degrading enzyme, this property was utilized in isolation of Sorangium cellulosum strains according to the present invention.

The liquid medium A and liquid medium B having the composition shown in Table 1 below were sterilized, respectively and mixed, 25 mg/L of cycloheximide was added thereto, 250 mg/L of kanamycin sulfate was added thereto, and the resulting mixture was homogenized using a stirrer to prepare an ST21CXKA agar medium.

Table 1

Medium Soulion A (pH 7.2)
Components	Conc. (g/L)
K₂HPO₄	1
Yeast Extract	0.02
Agar	15

Medium Solution B (pH 7.2)
Components	Conc. (g/L)
KNO ₃	1
MgSO₄-7H₂O	1
CaCl₂-2H₂O	1
FeCl₃	0.2
MnSO₄-7H₂O	0.1

The ST21CXKA agar medium thus obtained was poured into a petri dish and allowed to harden. Sterile paper was placed on the agar medium. Once the paper was adsorbed, the soil sample was seeded on the agar medium and incubated at 30℃ for 2 weeks. As a result, swarms of orange Myxobacteria could be seen on the agar medium (FIG. 1).

Only relatively clear orange swarms were transferred to a WAT agar medium having the composition shown in Table 2, and cultured at 30℃ for 2 to 3 days. The series of transfer and culturing processes was repeated once more.

Table 2

Components	Conc. (g/L)
CaCl₂-2H₂O	1
Agar	15

The pH was adjusted to 7.2 using potassium hydroxide (KOH), E. coli suspension was divided into several 15㎕ portions, and the suspension portions were smeared on petri dishes and then sufficiently dried. After the E. coli suspensions were dried, orange Myxobacteria colonies in the ST21CXKA medium were placed in a WAT medium to culture for pure-isolation.

In order to improve the growth rate of the isolated cells, sufficiently grown strains were transferred to a VY/2 agar medium having the composition shown in Table 3 below, liquid-cultured at 30℃ for 2 weeks and was then used for testing the production of epothilone and storing the same. The activity of the cells could be maintained over one month in the VY/2 agar medium (FIG. 2).

Table 3

Components	Conc. (g/L)
Bakers Yeast	5
CaCl₂-2H₂O	1
Vitamin B12	0.5mg
Agar
	15

Example 3: Identification of Sorangium cellulosum strains

The base sequence of 16s rDNA of Sorangium cellulosum strains (FIG. 2) stored in Example 2 were analyzed and identified. The base sequence of 16s rDNA is shown in FIG. 4 and Sequence Listing No. 1.

The forward primer used herein is as follows:

5'-AGAGTTTGATCCTGGCTCAG-3'

The reverse primer used herein as follows:

5'-TCTTTCCTCCACTAGGTCGG-3'

The strain, SYC248 (Scientific name: Sorangium cellulosum) is a rod-shaped Gram-negative bacteria that predominantly lives in the soil, forms fruiting bodies, when nutrients are scarce, grows in living yeast medium and has an optimal growth temperature of 30℃ and an optimal growth pH of 7.2. Also, SYC248 strains turn orange or red, or blackish orange on the agar medium. Table 4 shows morphological properties of the strain, SYC248 strains according to the present invention.

Table 4

Characteristic	SYC248
Gram-stain	Gram negative
Morphology	Rod
Colony Morphology	Swarm
Colony Surface	Smooth
Colony Color	Orange

Example 4: Liquid-culturing of Sorangium cellulosum strains

When the strains according to the present invention were cultured in the VY2 agar medium for 3 to 4 days, large orange colonies were observed. The colonies were inoculated in a 15 mL tube containing proliferation medium and cultured at 30℃ for 7 days. The cells were homogeneously dispersed to some extent and then sub-cultured 2 to 3 times (FIG. 4). Then, the cells were proliferated in a proliferation medium in a 500 mL shaking flask at 30℃ for 5 to 7 days and thus grew completely homogeneous. At this time, colonies were orange in color. The proliferated cells were inoculated at a ratio of 10% (v/v) in a production medium, and in order to facilitate collection of epothilone, an XAD-16 resin was added to the production medium and cultured in a 3L triangular flask or a 5L fermenter for 14 days. The compositions of proliferation and production media are shown in Tables 5 and 6, respectively.

Table 5

Growth Medium
Components	Conc. (g/L)
Powdered skim milk	4
Soybean cake	4
Starch	10
Yeast Extract	2
Glycerol	5
CaCl₂-2H₂O	1
MgSO₄-7H₂O	1
EDTA-Fe(III)	0.008
HEPES	12
pH	7.2(KOH Solution)

Table 6

Production Medium
Components	Conc. (g/L)
Powered skim milk	4
Soybean cake	4
Starch	15
Yeast Extract	2
Glycerol	10
CaCl₂-2H₂O	1
MgSO₄-7H₂O	1
EDTA-Fe(III)	0.008
XAD-16 resin	20
HEPES	12
pH	7.2(KOH solution)

Most of Sorangium cellulosum strains producing epothilones produce epothilones A, B, C, D, E and F and very small amounts of other epothilone variants. Strain No. 4 of Table 7 in which only epothilones A and B are present as major products exhibited the most excellent production efficiency. In terms of the production efficiency, epothilone A was produced in an amount of 3 to 5 mg/L and epothilone B was produced in an amount of 1 to 3 mg/L. Epothilones produced from screened strains according to the present invention are briefly shown in Table 7 below.

Table 7

Strain	Produced Epothilone type
1	A, B, C, D
2	A, C
3	B, D
4	A, B
5	C, D

The strain No. 4 shown in Table 7 was deposited on May 12, 2009 under the accession number “KCCM11007P” at the Korean Culture Center of Microorganisms in Korean Federation of Culture Collections.

Measurement of doubling time

30 ml of samples were collected from the 5L fermenter at respective hours and then centrifuged at a rate of 5,000 rpm, and OD (optical density) of the culture solution was measured at a wavelength of 660 nm to compare the growth rate of strains.

The doubling time taken by which the packed cell volume (PCV) of the strains according to the present invention reached 1.5 mL from 0.75 ml was 8 to 10 hours and the doubling time taken by which the initial OD of the strains according to the present invention reached 0.2 from 0.1 was 8 to 10 hours. It was demonstrated that these doubling times are considerably shorter than the case of conventional strains (16 hours).

Example 5: Extraction and purification of epothilone

Extraction using a solvent was as follows:

The resin was harvested (collected) from the culture solution obtained in Example 4 using a filter paper and a sieve. The adsorbed epothilone was extracted from the harvested resin using methanol. The extraction of epothilone was carried out by adding 5 to 10 mL of methanol, based on 25 mL of the culture solution (under the condition of 5-fold concentration (v/v) for one hour). The methanol extract was concentrated and was further extracted with the equivalent volume ratio of ethyl acetate to remove impurities. Also, the extracted solvent was concentrated at 40℃, 200 rpm.

The column purification was performed as follows.

Cell masses and impurities were removed. The column used herein was 90 X 100 mm and was made of a celite 545 resin. The purification was performed using methanol ranging from 0% to 100% at a unit of 10% (v/v) in order to remove water-soluble substances, media and metabolites.

Then, the second purification method was MPLC: The desired compound was isolated from compounds composed of fat-soluble substances by a concentration gradient method. The column used herein was a 30 X 300 mm opened glass column filled with silica. Methanol ranging from 30% to 70% (v/v) was used for concentration gradient method. The size of filled silica was 30 to 63 ㎛. The MPLC apparatus used herein was an organic solvent pump-equipped Yamada product.

The third purification method was HPLC. HPLC was used to isolate epothilones A and B. A Prep-HPLC apparatus (Waters) was used and two types of columns were used as follows: one was a Seshuk Pak C-18 UG120 20 X 250 mm and the other was a Shisheido UG 120 column 20 x 250 mm. In the concentration gradient method, methanol was used in a concentration ranging from 50% to 75% (v/v).

Example 6: Analysis and identification of epothilone

The TLC analysis used for analyzing and identifying epothilone is the rapidest method to identify the production of epothilone and will be described below.

When methanol and dichloromethane (9:1) were used as solvents, 0.75 Rf was obtained, which corresponds to the results reported in the document[2](FIG. 5).

Also, epothilones were verified by HPLC analysis.

HPLC analysis proceeds as follows: methanol and water were used as solvents and were testified using a PDA sensor by a concentration gradient method. The concentration gradient of methanol and water was carried out while the concentration of methanol was changed from 65% to 100% over 40 min. As a result of the analysis, the value between 8 to 13 min was obtained, which was the same as the maximum UV adsorption described in other documents. Epothilones A and B were detected at 211 and 248 nm (FIGS. 6a to 6c).

The LC-MS analysis was as follows: molecular weight was measured using the sample isolated in Examples. As a result of the measurement using a positive ion-type LC-MS, the molecular weight of epothilone A was 493.2 and the molecular weight of epothilone B was 507.2 (FIGS. 7a and 7b). These results correspond to the molecular weights of epothilone reported in the document [2], which demonstrates that the substances produced from the strains isolated in Examples were epothilones.

NMR analysis was performed as follows. 300 MHZ or 400MHZ ¹H and 75MHZ or 100MHZ ¹³C Nuclear Magnetic Resonance (NMR) was used to analyze the structure of the epothilones, anticancer substance of the present invention isolated in Example 5. As a result, it can be determined that the anticancer substances of the present invention in Example 5 have the same structures as epothilones A and B [4] (FIG. 8).

Reference

1. Shimekets, L.and C.R.Woese.1992. A phylogenetic analysis of the myxobacetria: bassis for the classification. Proc. Natl. Acad. Sci. USA. 89: 9949~5463.

2. Gerth, K. and Bedorf, N. et al., 1996. Epothilones A and B: antifungal and cytotoxic compounds from sporangium cellulosum(myxobacteria) production, phsico-chemical and biological properties. The journal of antibiotics. 49:560~563.

3. Bollag, D. and Mcquency, P.et al., 1995.Epothilones, a New class of microtubule-stabilizating agents with a taxol-like mechanism of action.Cancer Research., 55:2325~2333.

4. Ingo H. Hardt. and Heinrich Steinmetz, et al., 2001. New Natural Epothilones from sporangium cellulosum, strains so ce90/B2 and So ce90/D13 : Isolation, Structure Elucidation, and SAR Stuides. Journal of Natural Products.63:No7, 847~856.

5. Holt J.G., Krieg N.R., Sneath P.H.A., Stalery J.T. and Williams S.T. 1994. Bergey's manual of determinative bacteriology, p. 515-525. Williams and Wilkins, Baltimore.

6. Kowalski R.J., Giannakakou P. and Hamel E. 1997. Activities of the microtubule-stabilizing agents epothilones A and B with purified tubulin and in cells resistant to Paclitaxel. The Journal of Biological Chemistry 272(4):2534-2541.

7. Altaha R., Fojo T., Reed E. and Abraham J., 2002. Epothilones: a novel class of non-taxane microtubule-stabilizing agents. Current Pharmaceutical Design 8:1707-1712.

8. Tang L., Shah S., Chung L., Carney J., Katz L, Khosla C. and Julien B. 2000. Cloning and heterologous expression of the epothilone gene cluster. Science 287:640-642.

9. Reichenbach H. and Dworkin M. 1992. The myxobacteria. In: The prokaryotes, 2nd ed. vol. IV (Balows A., Truper H.G., Dworkin M., Harder W. and Schleifer K.H., Springer Verlag, New York, pp. 3416-3487.

10. Weisburg W.G., Barns S.M., Pelletier D.A. and Lane D.J. 1991. 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173(2):697-703.

Claims

A Sorangium cellulosum SYC248 strain isolated from Korean soil, the strain being capable of producing a high concentration of epothilones A and B and exhibiting a rapid growth rate.
The strain according to claim 1, wherein the strain is capable of producing epothilone A in an amount of 3 to 5 mg/L or higher and epothilone B in an amount of 1 to 3 mg/L or higher.
The strain according to claim 1, wherein the strain has a growth rate of doubling time of 8 to 10 hours.
A Sorangium cellulosum strain deposited under the accession number KCCM11007P.
A method for preparing epothilone by culturing the strain according to claim 1.
A method for preparing epothilone by culturing the strain according to claim 4.
The method according to claim 6, wherein the epothilone A is prepared in an amount of 3 mg/L or higher and the epothilone B is prepared in an amount of 1 mg/L or higher.