WO1995004154A1

WO1995004154A1 - Process for producing taxol and its analogues using microorganisms

Info

Publication number: WO1995004154A1
Application number: PCT/JP1994/001239
Authority: WO
Inventors: Makoto Tahara; Tetsuo Sakamoto; Masamichi TAKAMI; Kenji Takigawa
Original assignee: Nippon Steel Corp
Current assignee: Nippon Steel Corp
Priority date: 1993-07-27
Filing date: 1994-07-27
Publication date: 1995-02-09
Anticipated expiration: 1996-01-27
Also published as: JPH07177879A

Abstract

The present invention relates to Brevibacterium sp. capable of producing a diterpene-like compound having a taxane skeleton, and processes for producing diterpene-like compounds having a taxane skeleton, which comprise the steps of culturing the microorganisms in a medium and recovering the diterpene-like compound having a taxane skeleton from the culture. According to the present invention, diterpene-like compounds having a taxane skeleton can be produced efficiently within a short time.

Description

DESCRIPTION

PROCESS FOR PRODUCING TAXOL AND ITS ANALOGUES USING MICROORGANISMS

TECHNICAL FIELD

The present invention relates to novel microorganisms capable of producing diterpene-like compounds having a taxane skeleton and to processes for producing diterpene-like compounds having a taxane skeleton using microorganisms.

BACKGROUND ART

Taxol is a diterpene contained in plants of the genus Taxus and is sanctioned as an agent for treating ovarian and breast cancers in the United States, Canada, Sweden and other countries. In addition to taxol, its analogues having a taxane skeleton have been developed as new anti-tumor agents.

One of the greatest problems in utilizing taxol as an anti-tumor agent is its limited supply.

Taxol is currently extracted from the bark of Taxus brevifolia NUTT. However, this plant is not suitable for cultivation because of its slow growth and the like, and there is a limitation on gathering this plant which grows naturally mainly on the North American Pacific coast. The chemical synthesis of taxol has been studied at many research institutes in the world and full synthesis was first achieved in 1994. Among various aspects of its chemical structure, the taxane skeleton is particularly difficult to synthesize and its commercial production by synthesis is considered to be unfeasible. Hence, a semisynthesis process comprising the steps of extracting compounds having a taxane skeleton such as baccatin III and 10-deacetylbaccatin III from the leaves of plants of the genus Taxus and chemically modifying them has been studied as an alternative method of taxol production. In the synthesis of novel compounds having a taxane skeleton such as taxotere (EP 253738) which has been examined as an anti-tumor agent in clinical tests, baccatin III, 10-deacetylbaccatin III and the like that are extracted from plants of the genus Taxus are used.

In order to develop novel processes for producing taxol and its analogues having a taxane skeleton, tissue culture of plants of the genus Taxus has been studied in various research institutes in the world (United States Patent No. 5,019,504, W092/13961, WO93/10253, W093/17121 and W093/23555). However, research is still underway and these processes can not yet be developed to substitute for the processes of extraction from plants. It was reported that a novel fungus capable of producing taxol was discovered (Science, Vol.260, pp.214-216, and W093/21338). However, the yield of taxol by the fungus is too low (24-50 ng per liter of culture) for practical use.

DISCLOSURE OF INVENTION

An object of the present invention is to provide novel processes for producing taxol and its analogues having a taxane skeleton such as baccatin III, 10-deacetylbaccatin III and the like which are essential to the synthesis of taxol and taxotere.

As a result of the various studies to overcome the aforementioned problems, the inventors discovered a novel bacterium capable of producing taxol and other compounds having a taxane skeleton, and succeeded in efficiently producing taxol and its analogues having a taxane skeleton from the bacterial culture, thereby accomplishing the present invention.

The present invention provides Brevibacterium sp. which is capable of producing a diterpene-like compound having a taxane skeleton. Brevibacterium sp. capable of producing a diterpene-like compound having a taxane skeleton includes Brevibacterium sp. TA519 strain.

In addition, the present invention provides processes for producing diterpene-like compounds having a taxane skeleton, which comprise the steps of culturing in a medium a microorganism of the genus Brevibacterium capable of producing the diterpene-like compounds having a taxane skeleton and obtaining the diterpene-like compounds having a taxane skeleton from the culture.

The diterpene-like compounds having a taxane skeleton include compounds represented by the following formula (I):

wherein R₁ is a hydrogen atom or an acyl group, R₂ is a hydrogen atom or an acyl group, R₃ is an oxygen atom or the combination of an acetoxyl group and a hydrogen atom, R₄ is a hydrogen atom or a hydroxyl group, R₅ is a hydrogen atom, an acetyl group or a xylosyl group, Ph is a phenyl group, and Ac is an acetyl group.

The acyl group as R₁ in formula (I) includes an acetyl group or a group represented by the following formula (II):

wherein R₆ is a 1-methyl-1-propenyl, phenyl or n-pentyl group, and Ph is a phenyl group.

The acyl group as R₂ in formula (I) includes an acetyl or β -hydrolybutyryl group.

Specific examples of the diterpene-like compounds having a taxane skeleton represented by formula (I) include taxol, cephalomannine, 10-deacetyltaxol, 10-deacetylcephalomannine, 7-xylosyl-10-deacetyltaxol, 7-xylosyl-10-deacetylcephalomannine, 7-xylosyl-10-deacetyltaxol C, 7-xylosyltaxol, 7-xylosylcephalomannine, 7-xylosyltaxol C, 10-(β -hydroxybutyryl)-10-deacetyltaxol, 10-( β -hydroxybutyryl)-10-deacetylcephalomannine, baccatin III, 19-hydroxybaccatin III, 10-deacetylbaccatin III and baccatin VI.

These compounds are represented by the following structural formula.

Taxol R₅ =H R₂ =AC R₆ =C₆ H₅

Cephalomannine R₅ =H R₂ =Ac R₆ =CH₃ CH=C(CH₃ )

7-Xylosyl-10-deacetyltaxol R₅ = β -xylose R₂ =H R₆ =C₆ H₅

7-Xylosyl-10-deacetylceρhalomannine R₅=β -xylose R₂ =H R₆ =CH₃ CH=C(CH₃ ) 7-Xylosyl-10-deacetyltaxol C R₅ = β -xylose R₂ =H R₆ =n-C₅ H₁₁

7-Xylosyltaxol R₅ = β -xylose R₂ =Ac R₆ =C₆ H₅

7-Xylosylcephalomannine R₅ = β -xylose R₂ =Ac R₆ =CH₃ CH=C(CH₃ )

7-Xylosyltaxol C R₅ = β -xylose R₂ =Ac R₆ =n-C₅ H₁₁ 10-Deacetyltaxol R₅ =R₂ =H R₆ =C₆ H₅

10-Deacetylcephalomannine R₅ =R₂ =H R₆ =CH₃ CH-=C ( CH₃ )

10- ( β -Hydroxybutyryl ) -10-deacetyltaxol R₅ =H R₂ =CH₃ CH ( OH ) CH₂ CO R₆ =C₆ H₅ 10- ( β -hydroxybutyryl ) -10-deacetylcephalomannine

R₅ =H R₂ =CH₃ CH ( OH ) CH₂ CO R₆ =CH₃ CH=C ( CH₃ )

Baccatin III R₁ =R₄ =R₅ =H R₂ =Ac R₃ =O

19-Hydroxybaccatin III R₁ =R₅ =H R₂ =Ac R₃ =O R₄ =OH

10-Deacetylbaccatin III R₁ =R₂ =R₄ =R₅ =H R₃ =O

Baccatin VI R₁ =R₂ =R₅ =Ac R₃ = a OAc, β H R₄ =H

The bacteria capable of producing the diterpene-like compounds having a taxane skeleton were isolated from natural sources by the inventors and include the TA519 strain. The TA519 strain has, been depositted with National Institute of Bioscience and Human-Technology, the Agency of Industrial Science and Technology, Japan, with accession number FERM BP-4721.

TA519 strain is capable of producing the diterpene-like compounds having a taxane skeleton such as taxol and the like and has the following mycological properties.

I. Morphological properties

This strain exhibits the following morphological properties when being cultured in a broth agar medium and a broth liquid medium.

1. Shape and size of cells: short rods, 1-1.5μm

2. Polymorphism: - (not observed) 3. Motility: - (not observed)

4. Spore: - (not observed)

5. Gram's stain: positive

II. Culture properties

1. Broth agar plate medium

1) Colony size: 1-1.5 mm

2) Colony shape: circular

3) Colony boss: low convex

4) Colony color: opaque

5) Diffusible pigment: undetectable

2. Broth agar slant medium

same as in the broth agar plate medium.

3. Broth liquid medium

Positive growth

Turbidity in the liquid: moderate

Conditions of the bottom of the liquid: detectable precipitation

4. Litmus milk: alkaline

III. Physicochemical properties

1. Reduction of nitrate: undetectable

2. Denitrification: undetectable

3. MR test : negative

4. VP test: negative

5. Production of indole: negative

6. Production of hydrogen sulfide : trace

7. Hydrolysis of starch: undetectable

8. Utilization of citric acid

1) Koser's medium: detectable

2) Simmon's medium: detectable

9. Production of pigments: undetectable 10. Urease: positive

11. Oxidase: negative

12. Catalase: positive

13. Growth range

1)Temperature: growth detectable at 20°C -37 °C but not at 45°C.

14. Attitude to oxygen: aerobic

15. O-F test: negative

16. Acid and gas production from saccharides: No detectable acid or gas production from the following saccharides.

1) L-Arabinose

2) D-Xylose

3 ) D-Mannose

4 ) D-Fructose

5) D-Galactose

6) Maltose

7) Sucrose

8) Lactose

9) Trehalose

10) D-Sorbitol

11) Glucose

12) Inositol

On the basis of these mycological properties, this strain was identified as a microorganism of the genus Brevibacterium according to the description in Bergey's Manual of Systematic Bacteriology (Noel R. Kreig et al edited, Williams & Wilkins). However, the mycological properties of this strain are not completely consistent with those of any known species of the genus Brevibacterium and therefore the strain is designated as Brevibacterium sp. TA519.

Taxol and its analogues having a taxane skeleton can be obtained by culturing the bacteria of the present invention in a conventional manner for culture of microorganisms. As the medium to be used, any natural or synthetic media containing carbon sources, nitrogen sources, inorganic compounds and trace amounts of other nutrients required by the bacteria to be cultured can be used. As the carbon sources, carbohydrates such as glucose, fructose, galactose, molasses, wort, starch hydrolyzates, cellulose hydrolyzates and the like, and organic acids such as pyruvic acid, acetic acid, fumaric acid, malic acid, citric acid and the like can be used. As the nitrogen sources, various kinds of inorganic salts such as ammonium sulfate, ammonium chloride, ammonium nitrate, ammonium phosphate and the like, ammonium salts of organic acids, amines, peptone, meat extract, casein hydrolyzates, soybean-cake hydrolyzates, various kinds of fermented cells or their digests and the like can be used. As the inorganic compounds, potassium dihydrogenphosphate, dipotassium hydrogenphosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, calcium carbonate and the like can be used. In addition, corn starch, yeast extract, meat extract and the products of decomposition of defatted soybean cake with acids can be used as growth promoters and vitamins may be added as appropriate.

Cultivation may preferably be carried out under aerobic conditions in shake culture or aeration culture at pH's of 5-9 and at temperatures of 20°C -40 °C.

Taxol and its analogues having a taxane skeleton can be isolated from the culture of the microorganism. Briefly, these compounds can be isolated from the culture of the microorganism, the microorganism cells or medium separated from the culture solution, the cells or solid medium in the solid culture, and the like.

Any known method for extracting compounds from cultured microorganisms or medium can be used for extracting taxol and its analogues having a taxane skeleton. They include, for example, a method comprising the steps of separating the microorganism cells from the culture solution in the liquid culture by centrifugation, for example, and then immersing the recovered cells in an organic solvent such as methylene chloride (dichloromethane), methanol, acetone or the like, and a method comprising the steps of subjecting the culture solution in the liquid culture to an ultrasonic treatment and conducting extraction with a water-immiscible organic solvent such as methylene chloride or the like.

Pure taxol and its analogues having a taxane skeleton can be obtained from the extract by any known method including partition between a water-immiscible organic solvent and water, adsorption chromatography, gel filtration chromatography, high performance liquid chromatography, droplet counter current chromatography and the like, as well as methods in which these techniques are combined as appropriate with affinity chromatography using antibodies against taxol and taxane skeletons.

Taxol and its analogues having a taxane skeleton in the extract from the culture or enriched solution can be determined quantitatively by any known method including high performance chromatography, enzyme immunoassays and the like.

BEST MODE FOR CARRING OUT THE INVENTION

The present invention will now be described in more detail with reference to the following examples, which should not be construed to limit the present invention.

Example 1 : Microorganism culture and Taxol production in a Flask

1. Microorganism culture Brevibacterium sp. TA519 strain (FERM BP-4721) was inoculated in a 300 ml flask containing 100 ml of a liquid medium (Tryptic Soy Broth) containing 1.7% pancreatic digest of casein, 0.3% papaic digest of soybean-cake, 0.25% glucose, 0.5% sodium chloride and 0.25% dipotassium hydrogenphosphate, and incubated at 25 °C for 4 days on a rotary shaker at 100rpm. The cultured medium was centrifuged at 10,000 rpm for 30 min, and approximately one gram (fresh weight) of the microorganism cells was obtained as a pellet .

2. Extraction of taxol and the like

One gram of the thus obtained cells was placed in a screw-capped glass centrifuge tube containing 50 ml of methanol and methylene chloride (1:1) solution and, after ultrasonic treatment, subjected to extraction with shaking at room temperature for 16 hr. After the extraction, the tube was centrifuged at 3,000 rpm for 30 min to thereby obtain a cell-free extract. This extract was concentrated under reduced pressure, and residual compounds therein were dissolved in 400 μl of methanol. The resultant solution was diluted in 50 mM phosphate buffer containing 150 mM sodium chloride, 0.25% bovine serum albumin, 0.05% Tween 20 and 0.02% sodium azide.

3. Determination of taxol and the like

After dilution of the resultant solution, the amounts of the taxane skeleton-containing compounds and taxol contained in the cell extract were determined using two kinds of enzyme immunoassay kits (manufactured by Hawaii Biotechnology Group Inc.) employing a polyclonal antibody against a taxane skeleton-containing compound and a monoclonal antibody against taxol, respectively. As a result, it was found that about 600 μ g of taxane skeleton-containing compounds and 400 μ g of taxol are produced per liter of the culture. (Both values are estimated in terms of an amount in which an antibody reacts against taxol . )

When the above values are converted into amounts per gram of dry cell matter, they fall in the range from 0.04 to 0.06%. These values are about the same as the taxol content of the bark of Taxus brevifolia NUTT, which is 0.05%.

The cross-reactivity of the above-mentioned polyclonal antibody (TAO1) or monoclonal antibody (TAO2) against taxol or its analogues is as shown in Table 1 below.

Table 1

IC₅₀ (nM)

Taxane skeleton-containing compounds TAO1 TAP2

Taxol 0 . 5 10

10-Deacetyltaxol 0 . 7 15

7-Epi-10-deacetyltaxol 1. 2 25

7-Xylosyl-10-deacetyltaxol 1. 8 30

Cephalomannine 1. 0 220

7-Epi-10-deacetylbaccatin III 34 -

In Table 1, the value represents the concentration of each compound required to lower the binding of each antibody to taxol in a solid phase by 50% when each antibody is reacted in the presence of a certain amount of taxol (in a solid phase). Thus, it is clearly shown that the reactivity greatly differs between the two antibodies, in particular the reactivity against cephalomannine (the polyclonal antibody is more reactive). In the above enzyme immunoassay, even on the same sample, the more cephalomannine the sample contains, the greater the value determined with the polyclonal antibody becomes.

Example 2: Microorganism Culture and Taxol Production in a 30 1 Fermenter 1. Microorganism culture

1) Seed culture

Brevibacterium sp. TA519 strain (FERM BP-4721) was inoculated in three 500 ml flasks with baffles each containing 200 ml of a liquid medium. The medium was Tryptic Soy Broth, which contained 1.7% pancreatic digest of casein, 0.3% papaic digest of soybean-cake, 0.25% glucose, 0.5% sodium chloride and 0.25% dipotassium hydrogenphosphate. The bacterium was incubated at 25 °C for 7 days. In this way, a seed culture solution to be inoculated in a fermenter was obtained.

2) Culture in a 30 1 fermenter

The thus obtained seed culture solution (450ml) was inoculated in a 30 1 fermenter (Mitsuwa Rikagaku model KMJ-301MGU-2U) containing 18 1 of a liquid medium (Tryptic Soy Broth) containing 1.7% pancreatic digest of casein, 0.3% papaic digest of soybean-cake, 0.25% glucose, 0.5% sodium chloride and 0.25% dipotassium hydrogenphosphate, and incubated at 25°C for 2 days. During the 2-day incubation, the medium pH was adjusted at 7.4 with 2N HCl; the number of revolutions of the agitator blade was set at 300 rpm; and aeration was provided at a rate of 18 l/min. After the incubation, the medium was centrifuged at 13,000 rpm with a continuous-flow centrifuge rotor to thereby obtain a supernatant.

2. Extraction of taxol

A portion (250 ml) of the thus obtained supernatant was adjusted to pH 3.7 with 6N HCl. To this supernatant, one half its volume of butyl acetate was added, and the resultant solution was shaken and agitated for 15 min. The resultant solution was transferred to a separator funnel, and the layer of butyl acetate was recovered and concentrated under reduced pressure. The residual compounds were dissolved in 250 μ l of methanol to thereby obtain an extract sample. 3. Determination of taxol

1) Enzyme immunoassay

A portion of the thus obtained extract sample was analyzed with an enzyme immunoassay kit (manufactured by Hawaii Biotechnology Group Inc.) using a monoclonal antibody against taxol. As a result, it was found that 200 μ g of taxol is produced per liter of the medium.

2) High performance liquid chromatography

The extract sample obtained in 2. above was analyzed by high performance liquid chromatography (HPLC), and the results obtained therefrom were compared with the results of analysis of an authentic taxol standard (500μg/ml methanol). The HPLC analysis conditions were as follows:

- Column: C₁₈ reverse phase column (Tosoh ODS80TM 4.6mm ID 250 mm)

- Column temperature: 35°C

- Mobile phase: A solution of methanol, water and acetonitrile mixed in a ratio of 20:41:39, respectively (isocratic elution).

- Flow rate: 1 ml/min

- Detection: Absorbance at 227 nm and absorption spectrum at an elution peak with photodiode array.

As a result, the extract sample revealed an elution peak at 22.18 min which was nearly the same as the elution time of the authentic taxol standard (approximately 23.0 min). Moreover, the absorption spectrum over the range of 190-400 nm of the extract sample at that elution peak was identical to the spectrum of taxol, having a peak absorption value at 227 nm. The agreement of elution times was confirmed by mixing the extract sample and the standard taxol solution, subjecting the resultant mixture to HPLC analysis, and finding that the taxol elution peaks comigrate. In addition, on the standard taxol, quantitative relationships between the amount injected into the HPLC column and the peak area were dete rmined , and based on the dete rmination , the concentration of taxol in the extract sample was estimated from its peak area . The taxol concentration thus estimated agreed with the results obtained by the enzyme immunoassay .

Example 3

It was conf irmed , according to a method other than antibody reaction , that one of thos e substance s which are pr oduced by Brevibacterium sp . TA519 strain ( FERM BP-4721 ) and reactive to an antibody against a taxane skeleton-containing compound or taxol is taxol . The confirmation procedure was as follws .

1. Microorganism culture

Brevibacterium sp. TA519 strain (FERM BP-4721) was inoculated in a 500 ml flask with baffles containing 200 ml of a liquid medium (Tryptic Soy Broth) containing 1.7% pancreatic digest of casein, 0.3% papaic digest of soybean-cake, 0.25% glucose, 0.5% sodium chloride and 0.25% dipotassium hydrogenphosphate, and incubated at 25 °C for 5 days on a rotary shaker at 100 rpm. The cultured medium was centrifuged at 10,000 rpm for 30 min, and approximately 1.3 g (fresh weight) of the microorganism cells was obtained as a pellet.

2. Extraction of taxol and enzyme immunoassay analyses

All of the thus obtained cells were transferred to a screw-capped glass centrifuge tube containing 50 ml of methanol and methylene chloride (1:1) solution, and after ultrasonic treatment, subjected to extraction with shaking at room temperature for 16 hr. After the extraction, the tube was centrifuged at 3,000 rpm for 30 min to thereby obtain a cell-free extract. This extract was concentrated under reduced pressure, and residual compounds therein were dissolved in 200μl of methanol to thereby obtain an extract sample. A portion of this extract sample was diluted in 50 mM phosphate buffer containing 150 mM sodium chloride, 0.25% bovine serum albumin, 0.05% Tween 20 and 0.02% sodium azide. The diluted extract was analyzed with two kinds of enzyme immunoassay kits (manufactured by Hawaii Biotechnology Group Inc.) using a polyclonal antibody against a taxane skeleton-containing compound and a monoclonal antibody against taxol, respectively. As a result, it was confirmed that substances reactive to the antibodies were contained in the extract sample.

3. High performance liquid chromatography

The extract sample obtained in 2. above was analyzed by high performance liquid chromatography (HPLC), and the results obtained therefrom were compared with the results of analysis of an authentic taxol standard (100μg/ml methanol). The HPLC analysis conditions were as follows:

- Column: C₁₈ reverse phase column (Senshu Pak ODS-1251-SS)

- Column temperature: 35°C

- Flow rate: 1 ml/min

As a result, the extract sample revealed an elution peak at 14.7 min which was nearly the same as the elution time of the authentic taxol standard (approximately 14.5 min). Moreover, the absorption spectrum over the range of 190-400 nm of the extract sample at that elution peak was identical to the spectrum of taxol, having a peak absorption value at 227 nm. The agreement of elution times was confirmed by mixing the extract sample and the standard taxol solution, subjecting the resultant mixture to HPLC analysis, and finding that the taxol elution peaks co-migrate.

4. Liquid chromatography/mass spectrometry

An extract sample obtained by the same procedure as mentioned in 1. and 2. above was analyzed by liquid chromatography/mass spectrometry (LC/MS using Hitachi mass spectrometer model M1200H). The analysis conditions were as follows:

- Column: DEVELOSIL ODS-HG-5 column

- Column temperature: 40°C

- Mobile phase: gradient elution by changing the mixing ratio of acetonitrile and water from 40:60 to 80:20 in 10 min.

- Flow rate: 1 ml/min

- Temperature of mist generator: 190°C

- Temperature of desolventation chamber: 420°C

- Drift voltage: -40 V

First, the authentic taxol standard was analyzed, and it was found that the elution time of taxol was approximately 9.5 min. With respect to the mass spectrum, a large number of ion counts was observed at 525, 792 and 852 m/z (mass/electric charge). Therefore, these three ions (m/z) were selected for SIM (selected ion monitoring) analyses, and the extract sample was subjected to the analyses. As a result, the peaks of the ion counts were observed for all three ions (m/z) at the same elution time (approximately 9.5 min) as that of the standard taxol.

Furtheremore, the relative intensities of these three ion counts for the extract around the taxol elution time were almost identical to those of the authentic taxol.

Example 4: Determination of Baccatins

It was confirmed that one of the substances produced by Brevibacterium sp. TA519 strain (FERM BP-4721) and reactive to an antibody against a taxane skeleton-containing compound is a baccatin. The confirmation procedure was as follows.

1. Microorganism culture

2. Extraction of baccatins and enzyme immunoassay analyses

All of the thus obtained cells were transferred to a screw-capped glass centrifuge tube containing 50 ml of methanol and methylene chloride (1:1) solution, and after ultrasonic treatment, subjected to extraction with shaking at room temperature for 16 hr. After the extraction, the tube was centrifuged at 3,000 rpm for 30 min to thereby obtain a cell-free extract. This extract was concentrated under reduced pressure, and residual compounds therein were dissolved in 200μ 1 of methanol. The resultant solution was further diluted in 50 mM phosphate buffer containing 150 mM sodium chloride, 0.25% bovine serum albumin, 0.05% Tween 20 and 0.02% sodium azide. The resultant dilution was analyzed with an enzyme immunoassay kit (TAO3 manufactured by Hawaii Biotechnology Group Inc.) using a monoclonal antibody against baccatins to thereby examine if baccatins were contained in the extract. As a result, it was found that 90 ng* of baccatins was contained per liter of the medium (* a value estimated in terms of an amount in which the monoclonal antibody reacts against baccatin III).

INDUSTRIAL APPLICABILITY According to the present invention, a diterpene-like compound having a taxane skeleton can be produced highly efficiently. The taxol content of a culture of the microorganisms of the present invention belonging to the genus Brevibacterium is by far greater than that of the fungus described in Science as mentioned earlier in this specification. The taxol content per gram of dry matter of the microorganisms of the present invention is similar to that of the known plant producing taxol. In addition, the culture period of the microorganisms of the present invention required for the production of taxol and the like is several days at most, which is far shorter than that required for the tissue culture of plants of the genus Taxus, i.e. several weeks, and also far shorter than that required for growing and harvesting plants of the genus Taxus, i.e. several years at least.

Claims

1. Brevibacterium sp . capable of producing a diterpene-like compound having a taxane skeleton .

2. Brevibacterium sp. according to Claim 1 , wherein said diterpene-like compound having a taxane skeleton is represented by the fol lowing formula ( I ) :

wherein R₁ is a hydrogen atom or an acyl group, R₂ is a hydrogen atom or an acyl group, R₃ is an oxygen atom or the combination of an acetoxyl group with a hydrogen atom, R₄ is a hydrogen atom or a hydroxyl group, R₅ is a hydrogen atom, an acetyl group or a xylosyl group, Ph is a phenyl group, and Ac is an acetyl group.

3. Brevibacterium sp. according to Claim 1, wherein said diterpene-like compound having a taxane skeleton is at least one compound selected from the group consisting of taxol, cephalomannine, 10-deacetyltaxol, 10-deacetylcephalomannine, 7-xylosyl-10-deacetyltaxol, 7-xylosyl-10-deacetylcephalomannine, 7-xylosyl-10-deacetyltaxol C, 7-xylosyltaxol, 7-xylosylcephalomannine, 7-xylosyltaxol C, 10-(β-hydroxybutyryl)-10-deacetyltaxol, 10-( β-hydroxybutyryl)-10-deacetylcephalomannine, baccatin III, 19-hydroxybaccatin III, 10-deacetylbaccatin III and baccatin VI.

4. Brevibacterium sp. according to Claim 1, wherein said diterpene-like compound having a taxane skeleton is taxol.

5 . Brevibacterium sp . according to Claim 1 which is Brevibacterium sp. TA519 strain .

6 . A process for producing a diterpene-like compound having a taxane ske l eton , whi ch compr ises the steps of culturing in a medium a microorganism of the genus Brevibacterium capable of producing the diterpene-like compound having a taxane skeleton and obtaining the diterpene-like compound having a taxane skeleton from the culture .

7 . The process according to Claim 6 , wherein said diterpene-like compound having a taxane skeleton is represented by the following formula ( I ) :

8. The process according to Claim 6, wherein said diterpene-like compound having a taxane skeleton is at least one compound selected from the group consisting of taxol, cephalomannine, 10-deacetyltaxol, 10-deacetylcephalomannine, 7-xylosyl-10-deacetyltaxol, 7-xylosyl-10-deacetylcephalomannine, 7-xylosyl-10-deacetyltaxol C, 7-xylosyltaxol, 7-xylosylcephalomannine, 7-xylosyltaxol C, 10-( β-hydroxybutyryl)-10-deacetyltaxol, 10-( β-hydroxybutyryl)-10-deacetylcephalomannine, baccatin III, 19-hydroxybaccatin III, 10-deacetylbaccatin III and baccatin VI.

9. The process according to Claim 6, wherein said diterpene-like compound having a taxane skeleton is taxol.

10. The process according to Claim 6, wherein said microorganism of the genus Brevibacterium capable of producing the diterpene-like compound having a taxane skeleton is Brevibacterium sp. TA519 strain.