WO1999002648A1

WO1999002648A1 - A taxanes-producing fungus, a method of isolating the fungus and a method of preparing taxanes by using the fungus

Info

Publication number: WO1999002648A1
Application number: PCT/KR1998/000181
Authority: WO
Inventors: Moon-Jong Noh; Jae-Gwon Yang; Sung-Bo Shim
Original assignee: Kolon Industries, Inc.
Priority date: 1997-07-11
Filing date: 1998-06-26
Publication date: 1999-01-21
Also published as: KR100428634B1; KR19990009824A

Abstract

A fungus named Pestalotia heterocornis, which produces taxanes including taxol and a method of isolating the fungus are provided. Furthermore, a method of preparing taxanes by using the fungus is provided.

Description

A TAXANES-PRODUCING FUNGUS,

A METHOD OF ISOLATING THE FUNGUS AND

A METHOD OF PREPARING TAXANES BY USING THE FUNGUS

CROSS REFERENCE TO RELATED APPLICATION

This application is based on application No. 97-32358 filed in the

Korean Industrial Property Office on July 11 , 1997, the content of which is

incorporated hereinto by reference.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The present invention relates to a method of preparing taxanes by

using fungi and, more particularly, to a taxanes-producing fungus obtained

from a tree of the genus Taxus, Yew tree or soil where the tree naturally

grows, a method of isolating fungi from the soil and a method of preparing

taxanes by using the fungi.

(b) Description of the Related Art

Taxol, a taxane analog (taxanes), is a terepenoid-based compound

extracted from the trunk bark of the Yew tree and has structural formula 1. [formula 1]

Taxol is approved from FDA as an antileukemic and antitumor agent

and shows an excellent antitumor activity especially in breast and ovarian

cancers.

Wani et al, "Journal of the American Chemical Society", Vol. 93, May

1971 , No. 9, pages 2325-2327, reports on the structure of taxol and its

potential use as an antileukemic and tumor inhibitory compound. This

publication further discusses an alcohol extraction procedure for producing

taxol.

Taxol is known to be extracted from the stem bark of different

species of the Taxus, Yew tree. Unfortunately, yields are extremely low, no

more than about 100 mg per kg of the bark in the extraction process and the

yew tree is scarce. Therefore, the supplies of taxol are extremely short for

acceding to the demands.

In order to solve these disadvantages, a chemical method for

preparing taxol is disclosed in U. S. patent No. 4,924,011. However, the

chemical method is multi-stepped and totally non-economically feasible. It is reported that one literature reports that an embryo of seed of

Taxus cuspidata which naturally grows in Korea contains taxol 200 times

more than the maximum taxol in the previously known Yew tree.

Recently, a method of isolating Taxomyces andreanae, a taxol-

producing mold, from the inner bark of Yew tree, is disclosed in PCT/US

93/03416 of Anderea stierle, plant pathologist, of Montana in U. S. A. The

mold produce a small quantity of taxol, 1-2 microgram/ liter.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a taxanes-

producing fungus named Pestalotia heterocomis, the fungus obtaining from

a tree of the genus Taxus or soil where the tree grow.

It is another object of the present invention to provide a method of

isolating fungi from soil where a tree of the genus Taxus naturally grow.

It is still another object of the present invention to provide a method

of easily preparing taxanes, particularly, taxol, by using fungi with high yield.

To accomplish these and other objects, the present invention

includes a fungus named Pestalotia heterocomis for producing taxane,

particularly, taxol.

A method of isolating the fungus named Pestalotia heterocomis,

from soil where a tree of the genus Taxus grows, includes the steps of

obtaining soil where the tree of the genus Taxus grows, treating the soil with

50 to 95% alcohol to disinfect the soil and filtering the disinfected soil to obtain residue, adding a sterilized saline solution to the residue and shaking

the solution. The method further includes the steps of culturing a portion of

supernatant in the mixed solution on a agar medium until fungal growth

occurs and transferring the fungal culture to a fungal growth lab medium,

with subsequent growth of the fungal culture, obtaining a portion of the

culture medium containing the fungal culture, thoroughly grinding the fungal

culture, adding a chromatographic solvent to the ground fungal culture,

obtaining chromatography of the fungal culture solution, quantitative

analyzing taxane, taxol and baccatin in the culture and comparing the

chromatography with at least one taxane standards selected from taxol,

baccatin or cephlomannine.

The present invention further provides a method of preparing

taxanes comprising the steps of exposing fungi named Pestalotia

heterocomis to a nutrient medium capable of supporting growth of the fungi,

providing culturing condition for the medium containing the fungi, which are

capable of producing growth and reproducing of the fungi and isolating or

concentrating the taxane from the culture media or the fungi.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention, and many of the

attendant advantages thereof, will be readily apparent as the same becomes

better understood by reference to the following detailed description when

considered in conjunction with the accompanying drawings, wherein: FIG. 1 shows the high-performance liquid chromatography ("HPLC")

spectrum of a taxol produced according to the present invention;

FIG. 2 shows the HPLC spectrum of a taxol standards;

FIG. 3a is a photograph of top portion showing growth condition of

Pestalotia heterocomis mycelium isolating by the method of the present

invention;

FIG. 3b is a photograph of reverse portion showing growth condition

of Pestalotia heterocomis mycelium isolating by the method of the present

invention; and

FIG. 4 is a photograph of spore of Pestalotia heterocomis isolating

by the method of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Small quantity of soil is obtained from Yew tree forest or elsewhere

within 1 meter radius of the root of a Yew tree. The soil is treated with 50 to

95% alcohol such as ethanol as a disinfectant for 10 to 30 seconds and

filtered. The residue is added with a sterilized saline solution and shaken.

A supernatant is partially taken from the shaken solution and placed on an

agar medium. Then, the supernatant on the agar medium is respectively

cultured in incubators keeping 15^°C , 20 ^°C and 25 "C under the light-dark

condition cycled with 12 hours and under the dark condition of 24 hours until

fungal growth occurs, for example after 2 to 5 days. At this time,

contamination due to foreign materials should be prevented. The residue is continuously cultured on agar media so as to obtain a

culture in pure form. Once the organism is obtained in a pure form, it is

ultimately transferred to one or more one lab medium. The lab medium may

be Sabouraud dextrose agar (DIFCO 0382-17-9) medium, Potato dextrose

agar (DIFCO 0013-17-6) medium or Czapec dox agar (DIFCO) medium.

The resulting culture is a fungus named Pestalotia heterocomis.

The fungus, Pestalotia heterocomis, was deposited with Korea

Research Institute of Bioscience and Biotechnology Korean Collection for

Type Cultures located in #52, Oun-dong, Yusong-ku, Taejon 305-333,

Republic of Korea and given Accession number KCTC 0340BP.

It is easily known that such fungus may be also obtained from a Yew

tree using the aforementioned procedure.

In order to demonstrate whether the fungus produces taxanes,

particularly, taxol, the following experiments can be performed.

The fungus is cultured on a nutrient medium including amino acid by

using a shaking fermenter for 2 weeks. The cultivation is carried out at 15^°C

under various shaking rates of 50 rpm, 100 rpm, 150 rpm and 200 rpm,

respectively. Furthermore, the cultivation is carried out at 20 ^°C , 25 ^°C and

30 ^°C under the same shaking rates. The nutrient medium may include

benzoic acid, a benzoic acid metabolite precursor or sodium benzoate.

When the cultivation is completed, the mycelium culture is thoroughly

ground by a waring blender and extraction is carried out by a

dichloromethane solvent. At this time, the volume of the dihloromethane solvent is equal to that of the mycelium culture. The grinding and extracting

steps may repeat 1 to 3 times. In the process, the dichloromethane layer is

formed and evaporated under a pressure at 25 to 35 ^°C.

The evaporated residue is then chromatographed on a 5 X 10cm

plate of silica gel as a stationary phase. A mobile phase used in the

chromatographic procedure is chloroform/acetonitril 7:3 v/v and

hexane/ethylacetate 1 :2 v/v. The chromatography of the residue is

compared with those of taxol, baccatin and cephalomannine as standards.

The taxol, baccatin and cephalomannine absorb short wave ultra violet of

254nm and react with the vanillin-sulfuric acid spray to produce an intense

blue coloration fading to gray.

Instead, the evaporated residue may be dissolved in methanol for

HPLC (high-performance liquid chromatography) and subjected to the HPLC

analysis. The analyzed result (retention time) is compared with standard

taxol peak.

A portion of the chromatographed culture, which do not produce

taxol, is discarded.

When the evaporated residue turns out to be taxol, the previously

prepared ground culture is extracted three times with dichloromethane. The

extract is evaporated under a pressure at 25 to 35 ^°C . The evaporated

residue is dissolved in 20 % of a methanol buffer solution having composition

of the Table 1. Thereafter, a portion of the obtained solution is subjected to

enzyme immunoassay analysis by TA 01 (specific to taxane), TA 02 (specific to taxol) and TA 03 (specific to baccatin) kits of immune system for quantity

detection of Hawaii Biotechnology Group Inc. to inspect taxol and derivatives

thereof.

A mass analyzer is used for exact inspection. The evaporated

solution is dissolved in ethanol for HPLC and subjected to preliminary thin

layer chromatography on a plate from Merck, Co. by using hexane/

ethylacetate of 1 : 2 v/v as an eluting solvent. The band absorbing short

ultraviolet of 254nm and reacting with vaniline-sulfuric acid spray is

separated from the plate and eluted with dimethylene chloride. The eluted

material is evaporated under a pressure and the residue is dissolved in 1ml

of acetonitrile for HPLC to analyze it with mass analyzer.

Furthermore, to dispel the notion that the medium itself contains

taxol and taxane, E. coli and another type molds are cultured under same

condition and same experiments are carried out.

The present invention is further explained in more details with

reference to the following examples. The examples is not intended to limit

the present invention.

Example 1

Small quantity of soil was obtained from Yew tree forest with high

humidity within 1m radius of roots of Yew tree. The soil was dipped into

70% of ethanol as a disinfectant for 30 seconds. The disinfected solution

was filtered. The residue was washed two times with sterilized distilled

water to remove the remaining ethanol and a sterilized saline solution was added thereto and shaken. Then, the solution was allowed to stand for 1

hour. Thereafter, 100ml of supernatant was taken from the solution and

uniformly applied to Sabouraud dextrose agar medium and potato dextrose

agar medium. The media were cultured in incubators of 15^°C , 20 ^°C , 25 ^°C

under the light-dark condition cycled with 12 hours and under the dark

condition of 24 hours, respectively. After two days, molds appeared on the

media were subcultured on Sabouraud dextrose agar medium, potato

dextrose agar medium and Czapec dox agar medium, respectively.

Thereafter, the fungal cultures were subcultured for one month.

The resulting fungus is named Pestalotia heterocomis. As shown in

Figs. 3a and 3b, the fungus had white mycelium and was irregularly gathered

and grown on potato dextrose agar medium. The reverse portion of the

grown mycelium exhibited the salmon color. As shown in Fig. 4, spores of

the fungus had shapes tapered to both end points with or without curvature.

Each of the spores was composed of five cells each with four septates, and

3 to 5 of appendages. Three cells on a center of the spore appeared to be

slightly brown. In contrast, the cell on one end of the spore had one

appendage, while the cell on the opposite end had 2 to 3 of appendages.

Example 2

In order to culture the primarily subcultured fungi on a large scale,

they were cultured on growth lab medium for 3 to 4 days. The fungi on

growth lab medium was cultured on a fermenter for 20 days. The

composition of growth medium was as follows: Glucose 1g/liter

Fructose 3g/liter

Sucrose 6g/liter

Sodium acetate 1g/liter

Bactosoyton 1g/liter

Thiamine 1mg/liter

Miotin 1 mg/liter

Pyridoxal 1 mg/liter

Calcium pantothenate 1 mg/liter

Phenylalanine 5mg/liter

Sodium benzoic acid 100mg/liter

Magnesium sulfate 3.6mg/liter

Potassium nitrate 6.5mg/liter

1 M potassium phosphate 1 m/literl

Copper nitrate 1 mg/liter

Zinc sulfate 2.5mg/liter

Manganese chloride 5mg/liter

Ferrous chloride 2mg/liter

Zinc 9.38mg/liter

Manganese 18.75mg/liter

Magnesium 9.38mg/liter

Small quantity of the usual element

The obtained culture was thoroughly ground with a waring blender and an equal volume of dichloromethane was added to the ground culture.

At this time, non-aqueous material was separated from the aqueous material

and a dichloromethane layer was collected. The separation step repeated

further twice. The dichloromethane layer was concentrated under a

pressure at 25 to 35 ^°C . In order to demonstrate whether the concentrated

material contains taxol, the material was analyzed by using HPLC with micro

Bondapak column, C18. As shown in Figs. 1a and 1b, the result of the

analysis exhibited a taxol peak with a retention time of 14.5 minutes (870

seconds), which value is characteristic of standard taxol. Therefore, the

extract of the fungus turned out taxol but, for more accuracy, enzyme

immunoassay analysis was performed. The concentrated solution was

dissolved in 50ml of a 20% methanol buffer solution having the composition

of the Table 1 and quantitative analyzed with TA 01 , TA 02 and TA 03 kits

from Hawaii Biotechnology.

Table 1

The concentrated solution exhibited reactions identical to those of the standard taxol. Therefore, the extracts obtained from the fungal culture

turned out to contain taxol and derivatives thereof. 30 micrograms of taxol

per 1 liter of the culture were produced with the cultivation for 3 weeks.

To dispel the notion that the medium itself contains the taxol, E. coli

and another mold (Mucor hiemalis f. luteus (Linndemann) Schipper) were

cultured on the medium under the same conditions. However, the resulting

cultures did not produce taxol.

The concentrated solution was dissolved in methanol for HPLC and

subjected to pre-thin layer chromatography on a pre-thin layer

chromatography plate (20 x 20, 2mm, Merck) by using hexane/ ethylacetate

1 : 2 v/v as eluting solvent. The band absorbing short wave light and

reacting with vaniline sulfuric spary was separated from the plate and eluted

with dimethylene chloride. The eluent was concentrated under a pressure

at low temperature. The residue was dissolved in 1ml of acetonitrile for

HPLC and analyzed with mass analyzer. As a result, the band represented

molecular ion (M+) at a m/e value of 854 and the main peaks represented

MNa+ at a m/e value of 876 and MK+ at a m/e value of 893.

As described above, the method of the present invention can

produce a taxanes-producing fungus. The fungus can be used for

preparing taxanes, particularly, taxol, with good yield.

While the present invention has been described in detail with

reference to the preferred embodiments, those skilled in the art will

appreciate that various modifications and substitutions can be made thereto without departing from the spirit and scope of the present invention as set

forth in the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A fungus named Pestalotia Heterocomis, for producing

taxanes including taxol.

2. The fungus of claim 1 wherein the fungus is obtained from a

tree of the genus Taxus or soil where the tree grows.

3. A method of isolating a fungus named Pestalotia

heterocomis, from soil where a yew tree grows, comprising the steps of:

(1) obtaining a portion of soil where the tree of the genus Taxus

grows;

(2) treating the soil with 50 to 95% alcohol to disinfect the soil and

filtering the disinfected soil to obtain residue;

(3) adding a sterilized saline solution to the residue and shaking the

solution;

(4) culturing a portion of supernatant in the mixed solution on agar

medium until fungal growth occurs;

(5) transferring the fungal culture to a growth lab medium, with

subsequent growth of the fungal culture;

(6) obtaining a portion of the culture medium containing the fungal

culture, thoroughly grinding the fungal culture and adding a chromatographic

solvent to the ground fungal culture;

(7) obtaining chromatography of the fungal culture solution; and

(8) quantitative analyzing taxane, taxol and baccatin in the culture

and comparing the chromatography with at least taxane standards selected from the group consisting of taxol, baccatin and cephalomannine.

4. The method of claim 3 further comprising, after growing step

(4), the steps of (4A) placing the growth mycelium on agar for mycelium

culture and subculturing the growth mycelium on agar for mycelium culture

until a culture in pure form is obtained.

5. The method of claim 3 further comprising, after quantitative

analyzing and comparing steps (8), the steps of discarding a taxol non-

producing culture.

6. A method of preparing taxanes comprising the steps of:

(1) exposing fungi to a nutrient medium capable of supporting growth of the fungi;

(2) providing culturing condition for the medium containing the fungi,

which are capable of producing growth and reproduction of the fungi; and

(3) isolating or concentrating the taxanes from the culture medium or

the fungi.

7. The method of claim 6 wherein the taxanes is taxol.

8. The method of claim 6 wherein the nutrient medium

comprises a compound selected from the group consisting of benzoic acid,

benzoic acid metabolism precursor and sodium benzoate.