JPH0833490A - Production of taxane-type diterpene - Google Patents

Abstract

PURPOSE:To produce a taxane-type diterpene useful e.g. as a treating agent for ovarian cancer, mammary cancer, pulmonary cancer, etc., by culturing cells or tissues of a plant capable of producing a taxane-type diterpene in the presence of a specific jasmonic acid and recovering the compound from the cultured product. CONSTITUTION:This taxane-type diterpene (e.g. taxol) is produced in high productivity by culturing the cells or tissues of a plant capable of producing a taxane- type diterpene (e.g. Taxus brevifolia) in the presence of a jasmonic acid compound of, formula [R<1a>, R<1b>, R<1c>, R<1d> and R<1f> are each H, OH, a l-6C alkyl or a l-6C alkoxy; R<2> to R<6> are each H or a 1-6C alkyl; the side chain bonded to C<1> may contain one or more double bonds; R<7> is OH, OM (M is an alkali metal, an alkaline earth metal or NH4), NR<8a>R<8b> (R<8a> and R<8b> are each H, a 1-6C acyl, a 1-6C alkyl or an amino acid residue), OR<9> (R<9> is a 1-6C alkyl or a carbohydrate residue) or a l-6C alkyl; (n) is 1-7] (e.g. methyl jasmonate) and recovering the compound from the cultivation product.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing a taxane-type diterpene containing taxol which is useful as a therapeutic agent for ovarian cancer, breast cancer, lung cancer and the like.

[0002]

2. Description of the Related Art Taxol, which is useful as a therapeutic drug for ovarian cancer, breast cancer, lung cancer, etc., is a taxane-type diterpene isolated and identified from Taxus brevifolia NUTT, which is a Taxus genus plant. Yes, with complex ester groups associated with activity. It has been reported that taxol is present in every part of the yew plant, and its content is highest in the bark.
Currently, taxol is collected from natural or cultivated plants, but yew plants are slow growing plants that take more than 10 years to grow to a height of 20 cm above the ground, and when bark is peeled off, they are trees. It is difficult to obtain a large amount of taxol because it will die. If taxol and / or taxane-type diterpenes such as baccatin III, which is a precursor of taxol and a raw material for the semi-synthesis method described below, can be produced by using tissue culture, the trees should be felled. It is advantageous because a large amount of taxol can be easily obtained.

Regarding the taxol production method using the cultured cells of the plant up until now, the taxi (Taxus
brevifolia NUTT) The production method using cultured cells has been patented in the United States (US Pat. No. 5019504), but its taxol production is stated to be 1 to 3 mg / l, which is insufficient for industrial production. . Further, in the conventional tissue culture technology, the productivity of taxol by cell culture is unstable, and cells with high productivity are obtained primarily by selection, but it is not possible to maintain the content by subculturing. Difficult [ERMWickreme
sine et al., World Congress on Cell and Tissue Cul
ture (1992)].

On the other hand, as a prior art of the taxol production method, a semi-synthesis method from taxol biosynthesis precursor baccatin III is disclosed in US Pat. No. 5,015,744 to Holton et al. If a plant tissue culture method is used, a semi-synthetic raw material such as baccatin III can be produced, and it can be used for taxol production by the semi-synthetic method.

[0005]

An object of the present invention is to provide a simple method for producing taxane-type diterpenes by culturing plant tissues.

[0006]

[Means for Solving the Problems] As a result of earnest research, the present inventors have conducted tissue culture by adding jasmonic acid to a tissue culture medium of a cultured cell or cultured tissue of a plant that produces a taxane-type diterpene. By accelerating the production of jasmonic acids (endogenous jasmonates) produced by the cultured cells or the cultured tissues themselves by subjecting the cultured cells or the cultured tissues to the special treatment described below, the taxane-type diterpene in the culture Finding that productivity improves,
The present invention has been completed.

That is, the present invention relates to a plant cell or tissue producing a taxane-type diterpene, represented by the general formula (I):

[0008]

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[In the formula, R ^1a , R ^1b , R ^1c , R ^1d , R ^1e and R ^1f are each independently a hydrogen atom, a hydroxyl group or a carbon number of 1
It represents 6 alkyl group or an alkoxy group having 1 to 6 carbon ^{atoms; R 2, R 3, R} 4, R 5 and R ⁶ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; C ¹
Side chain consisting of ^{-C 2 -C 3 -C 4 -C 5} -C 6 may contain one or more double bonds; R ⁷ is a hydroxyl group, OM (wherein, M is Represents an alkali metal atom, an alkaline earth metal atom or NH ₄ ), NR ^8a R ^8b (wherein R ^8a and R ^8b are each independently a hydrogen atom, an acyl group having 1 to 6 carbon atoms, and a carbon number of 1). To an alkyl group having from 6 to 6 or an amino acid residue), OR ⁹ (wherein R ⁹ represents an alkyl group having 1 to 6 carbons or a carbohydrate residue) or an alkyl group having 1 to 6 carbons. Represents; n represents an integer of 1 to 7; the 5-membered ring may form a double bond between adjacent ring member carbon atoms. ] The taxane-type diterpene is cultivated in the presence of jasmonic acid, and the taxane-type diterpene is recovered from the obtained culture.

The taxane-type diterpene to which the production method of the present invention is applied is not particularly limited as long as it is a diterpene having a taxane-type skeleton, and for example, it has the following general formula (II) or general formula (III) as a basic skeleton. In the compound [general formula (II) or general formula (III), a part of the ring may be a double bond. ] Can be mentioned.

[0011]

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[0012]

[Chemical 4]

Specific examples of the compound having a skeleton represented by the general formula (II) include taxol, 10-deacetyltaxol,
7-epitaxol, baccatin III, 10-deacetylbaccatin III, 7-epibaccatin III, cephalomannine, 10-deacetylcephalomanin, 7-epicephalominine, baccatin VI, taxane 1a, xylosyl cephalomannin, xylosyl taxol , Taxol C,
10-deacetyl taxol C etc. can be illustrated. Specific examples of the compound having the skeleton of the general formula (III) include taxin I, taxin II, taxin I, taxin II,
Examples include taxagifin.

Examples of plants producing taxane-type diterpenes used in the production method of the present invention include, for example, yew (Taxus baccata LINN) and yew (T. cuspidata SI
EB.et ZUCC), Karaboku (T. cuspidata SIEB.et ZUCC
var.nana REHDER), Taihei Yew (T. brevifol
ia NUTT), Canadian yew (T. canadiensis MARSH), Chinese yew (T. chinensis), and T. media. Among these, the yew and T. med
ia is particularly preferred.

The tissue culture of the plant is conventionally known except that the jasmonic acid represented by the general formula (I) according to the present invention is added or a treatment for promoting the production of endogenous jasmonic acid is performed. This can be done by the method described. The jasmonic acids to which the present invention is directed are compounds represented by the above general formula (I).

In the general formula (I), R ^1a , R ^1b , R
^1c , R ^1d , R ^1e , R ^1f , R ² , R ³ , R ⁴ , R ⁵ ,
1 to ⁶ carbon atoms represented by R ⁶ , R ⁷ , R ^8a , R ^8b or R ⁹ .
Examples of the alkyl group of 6 include a methyl group, an ethyl group,
Examples thereof include n-propyl group, isopropyl group, n-butyl group, isobutyl group, sec-butyl group, t-butyl group, n-pentyl group and n-hexyl group.

In the general formula (I), R ^1a , R ^1b , R
Examples of the alkoxy group having 1 to 6 carbon atoms represented by ^1c , R ^1d , R ^1e or R ^1f include a methoxy group, an ethoxy group and n.
-Propoxy group, isopropoxy group, n-butoxy group,
Isobutoxy group, sec-butoxy group, t-butoxy group, n
Examples include -pentyloxy group and n-hexyloxy group.

When R ⁷ is OM, examples of the alkali metal atom or the alkaline earth metal atom represented by M include sodium, potassium and calcium. In the case where R ⁷ is NR ^8a R ^8b , the acyl group having 1 to 6 carbon atoms represented by R ^8a or R ^8b may be straight chain or branched chain, for example, formyl group, acetyl group,
Examples thereof include a propionyl group, a butyryl group, a valeryl group, a hexanoyl group, and an acryloyl group.

In the case where R ⁷ is NR ^8a R ^8b , R
Examples of the amino acid residue represented by ^8a or R ^8b include an isoleucyl group, a tyrosyl group and a tryptophyll group. When R ⁷ is OR ⁹ , the carbohydrate residue represented by R ⁹ includes a glucopyranosyl group.

Further, in the compound represented by the general formula (I), the 5-membered ring may form a double bond between adjacent ring member carbon atoms.

Specific examples of the compound represented by the general formula (I) include the compounds shown below.

(Compound A) R ^1a , R ^1b , R ^1c , R ^1d , R ^1e , R ^1f , R ² , R ³ , R
⁴ , R ⁵ , R ⁶ : H Double bond formation between C ³ and C ⁴ R ⁷ : -OH or -OCH ₃ n: 1 to 3

(Compound B) R ^1a , R ^1b , R ^1c , R ^1d , R ^1e , R ^1f , R ² , R ³ , R
⁴ , R ⁵ , R ⁶ : HR R ⁷ : -OH n: 1

In the compound represented by the general formula (I), R ^1a , R ^1b , R ^1c , R ^1d , R ^1e, or R ^1f is a hydroxyl group, or between the ring member carbon atoms adjacent to each other in the 5-membered ring. Specific examples of the compound in which the double bond is formed by
For example, the compounds shown below may be mentioned. (Compound C)

[0025]

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(Compound D)

[0027]

[Chemical 6]

(Compound E)

[0029]

[Chemical 7]

(Compound F)

[0031]

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Compounds represented by the above general formula (I)
R is preferably^1a, R ^1b, R^1c, R^1d, R
^1e, R^1f, R², R³, R^Four, R^FiveAnd R⁶Is a hydrogen atom
And R⁷Is a hydroxyl group or a methoxy group, and C¹-C
²-C³-C^Four-C^Five-C⁶Chain consisting of a double bond
Not included, or C¹And C², C²And C³, C ³
And C^FourCompounds that form a double bond between
It

The jasmonic acids represented by the general formula (I) used in the present invention have various stereoisomers (cis trans isomers and optical isomers), and each isomer is used alone. Alternatively, they may be used in the form of a mixture.

Among the above jasmonic acids, in the general formula (I), R ^1a , R ^1b , R ^1c , R ^1d , R ^1e ,
R ^1f , R ² , R ³ , R ⁴ , R ⁵ and R ⁶ are hydrogen atoms, R ⁷ is a hydroxyl group or a methoxy group, n is 1 and a double bond between C ³ and C ^4. Particularly preferred is jasmonic acid or methyl jasmonate, which is a compound that forms the above-mentioned compound, from the viewpoint of the degree of the effect on productivity improvement.

These jasmonic acids are prepared synthetically or by extraction from plants (H. Yamane et a
L., Agric. Biol. Chem., 44 , 2857-2864 (1980)). On the other hand, jasmonic acid is a plant hormone-like substance that induces various reactions involved in growth promotion, tissue maturation, and expression of disease resistance. Volume 4, pages 523-531 (1990)
It is described in.

Therefore, the jasmonic acids can be produced by the cultured cells or tissue to be used, in addition to being added from the outside of the culture system. As a method of promoting the production of this endogenous jasmonic acid by the cultured cells or the cultured tissue, it is possible to exemplify the addition of the culture of the microorganism or its extract, the heat-treated product or the plant extract to the medium. MJ Mueller et al., Proc. Natl. Aca
d. Sci. USA, 90 (16), 7490-7494 (1993),
A method of adding the mold cell wall fraction can be exemplified. Furthermore, it is also possible to increase the production amount of endogenous jasmonic acid by mechanically or partially damaging the cultured cells or tissue to be used with ultraviolet rays, heat, etc., specifically, RACleeman et al., Pro
c. Natl. Acad. Sci. USA, 89 (11), 4938-4941 (198
The method for mechanically destroying a part of cells described in 9) can be exemplified.

Since jasmonic acids are poorly soluble in water, they are usually dissolved in an organic solvent such as ethanol or methanol, or a surfactant, and then added to the medium. The free form of jasmonic acid may be used as it is, or may be neutralized with an alkali to form a salt.

Since the α-position of the 5-membered ring carbonyl group of the compound represented by the above formula (I) undergoes epimerization by acid, alkali or heat, it tends to be a stable trans type rather than an unstable cis type. In equilibrium experiments using natural or synthetic jasmonic acid, 90% of trans type and 10% of cis type exist. Generally, the cis type is more active, but the jasmonic acids that can be used in the present invention include all stereoisomeric compounds represented by the above formula (I) and a mixture thereof.

The concentration of jasmonic acid in the medium is 0.
It is necessary to adjust the concentration to 01 to 1000 μM, and it is particularly preferable to adjust the concentration of jasmonic acids to the range of 0.1 to 500 μM.

It is a German patent publication [DE 4122208 C1] that induction of secondary metabolites by adding jasmonic acids to plant cell cultures, but adding medium in tissue culture of taxane-type diterpene-producing plants An example of tissue culture in the presence of jasmonic acids as a product has not been reported, and a taxane-type diterpene having a completely different biosynthetic pathway or biosynthetic control mechanism from the secondary metabolite disclosed in the patent is reported. It was unexpected that the production amount was increased by the method of the present invention.

In the above formula (I) used in the present invention,
Jasmon structurally similar to the indicated jasmonic acids
Or methyl jasmon is effective in inducing taxol production
There is a description in WO 93/17121
It However, these compounds are
Unlike the acids, in the above formula (I), the formula:-(CH ₂)
_n-CO-R⁷Have a carboxylic acid group etc.
However, these taxol-inducing activities were low.
(Comparative Example 9 described later).

The medium used in the present invention is a conventionally known medium used for tissue culture of plants, for example, Murashige & Skoog (1962) [Murashige & Skoo].
g] medium, Rinsmeier Scoog (1965) [Lin
smaier Skoog] medium, Woody Plant Medium (1981) [Woody Plant Medium] medium, Gamborg B-5 medium, Mitsui M-9 medium and the like.

Plant hormones may be added to these media, and if necessary, carbon sources, inorganic components, vitamins, amino acids and the like may be added. As the carbon source, disaccharides such as sucrose, maltose and lactose, monosaccharides such as glucose, fructose and galactose, starch, or a mixture of two or more of these sugar sources in an appropriate ratio can be used.

Examples of the inorganic component include phosphorus, nitrogen, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt. These components are, for example, Potassium nitrate, sodium nitrate, calcium nitrate,
Potassium chloride, potassium monohydrogen phosphate, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, copper sulfate, sodium molybdate, It can be added as a compound such as molybdenum trioxide, potassium iodide, or cobalt chloride.

Examples of plant hormones include auxins such as indoleacetic acid (IAA), naphthaleneacetic acid (NAA) and 2,4-dichlorophenoxyacetic acid (2,4-D), and cytokinins such as kinetin, zeatin and dihydrozeatin. Kinds are used. Examples of vitamins include biotin,
Thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pantothenic acid, inositol, nicotinic acid and the like are used.

As the amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added. Generally, each of the above components has a carbon source of about 1 to
It is used at a concentration of about 30 g / l, an inorganic component of about 0.1 μM to about 100 mM, a plant hormone of about 0.01 to about 10 μM, and vitamins and amino acids of about 0.1 to about 100 mg / l, respectively.

In the present invention, either a liquid medium or a solid medium containing 0.1 to 1% of agar, gellan gum or the like can be used, but the liquid medium is usually preferable. In the tissue culture of the present invention, the plant root, growing point, leaves,
Tissue pieces or cells such as stems, seeds, pollen, anthers and sepals, or cultured cells obtained by subjecting these to tissue culture in the above-mentioned medium or other conventional medium can be used.

When these tissues or cells are cultured in the presence of jasmonic acids, cultured tissues or cells with high taxane-type diterpene productivity can be obtained as compared with the case of no addition or no treatment.

The taxane-type diterpenes can be separated from the cultures such as culture tissues, culture cells, and media obtained as described above by extraction with an organic solvent such as methanol. Further, a taxane-type diterpene can be continuously recovered during the culture by coexisting an appropriate adsorbent or an organic solvent in the medium.

The following method is mentioned as a preferred example of the tissue culture in the present invention. First, after sterilizing plant pieces of plants belonging to the genus Taxus, for example, roots, growing points, leaves, stems, seeds, etc., they are placed on a solid medium of Woody Plant Medium solidified with gellan gum. Allow a part of the tissue piece to callus at 10-35 ℃ for 14-60 days. When the callus thus obtained is subcultured, the growth rate gradually increases and a stable callus is obtained. The term "stabilized callus" as used herein refers to one in which a part of the callus does not differentiate into shoots or roots during culture and retains the state of the callus and has a uniform cell growth rate.

The stabilized callus is transferred to a liquid medium suitable for growth, for example, a liquid medium of Woody Plant Medium and allowed to grow. The growth rate is further increased in the liquid medium. In the present invention, the stabilized callus or the cells constituting the callus is cultured in a solid medium or a liquid medium in the presence of jasmonic acids. The stabilized callus or cells constituting the callus may be divided into a plurality of layers depending on the difference in specific gravity, and the cells contained in at least one layer may be cultured in a medium containing jasmonic acids.

As a method for separating cells by specific gravity, generally, a density gradient is prepared using a medium for centrifugation,
A method in which cells are layered and then centrifuged is known.
Ficoll and Percoll (both Phar and
macia LKB Biotechnology), sucrose, cesium chloride, etc. are used. In Example 7, the density gradient was created using Ficoll, but there is no particular limitation as long as it does not damage the cells.

There is no particular limitation on the number of layers forming the density gradient. The specific gravity difference between the layers is not particularly limited, and the specific gravity differences may be the same or different. Therefore, the definition of the density gradient includes the case where the gradient changes continuously (the number of layers forming the density gradient is infinite, and the difference in specific gravity between layers is close to 0). By forming a density gradient in this way, cells can be layered and centrifuged to separate cells into a plurality of layers due to the difference in specific gravity.

The specific gravity of the layer to be formed is usually 1.00 to 1.20 g / m
l, preferably in the range of 1.03 to 1.11 g / ml. As the layer to be cultured, at least one layer may be selected, or all layers may be selected and cultured. When a plurality of layers are selected and cultured, the plurality of layers can be individually cultured, or two or more layers of the selected plurality of layers can be mixed and cultured. .

Cultured cells having a high taxane-type diterpene-producing ability are usually obtained by culturing cells contained in a layer having a specific gravity of 1.07 or less. However, the cells may vary depending on the cells to be cultivated and the conditions of the culturing. It is not limited to the range. Further, it is recognized that the cells in the layer having a high specific gravity tend to have a higher taxane-type diterpene content simply by fractionating the cells by the difference in the specific gravity. Therefore, in order to more reliably obtain taxane-type diterpene high-producing cultured cells, after culturing the cells of all the fractionated layers for a certain period of time,
It is desirable to measure the concentration of taxane-type diterpene contained in the cells of each layer and select a layer containing cells that highly produce taxane-type diterpene from them.

In the present invention, a culture medium having a specific gravity such as 1.07 g / ml may be prepared and centrifuged by the above-mentioned method, so that the cultured cells may have different specific gravities. It can be divided into multiple layers.

It is effective that the jasmonic acids are added during the growth phase or stationary phase of the cultured cells. Above all, the addition of jasmonic acids during the transition from the growth phase to the stationary phase is effective for the method of the present invention. preferable. Also,
The same applies to the timing of the treatment for increasing the production of endogenous jasmonic acids. For example, when cells are transplanted every 21 days, the 7th to 16th days are the appropriate period of the treatment for adding jasmonic acids or increasing the production amount of endogenous jasmonic acids. The addition of the jasmonic acids and the treatment for increasing the production amount of the endogenous jasmonic acids may be performed once or may be performed in multiple times.

The culture temperature of the tissue culture in the present invention is preferably about 10 to about 35 ° C., particularly about 23 to about 28 ° C., since the growth rate is high. The culture period is 14
~ 42 days are preferred.

When a liquid medium is used in the culture of the present invention, after the culture is completed, the cultured cells are separated from the medium by a method such as decantation or filtration, and the desired taxane-type diterpene is isolated from the cultured cells and / or the medium. It can be separated by a method such as extraction with an organic solvent.

[0060]

EXAMPLES The present invention will be described in more detail below with reference to examples and comparative examples, but the scope of the present invention is not limited to these examples.

(Example 1) 2% antiformin solution or 70% ethanol solution was added in advance to Woody Plant Medium solid medium (gellan gum 0.25% by weight) to which naphthalene acetic acid was added so as to have a concentration of 10 ^-5 M. A part of the stem of Taxus baccata LINN, which had been sterilized by the above method, was placed and cultured at 25 ° C. in the dark to obtain Taxus callus. Next, 1g of this callus (fresh weight)
The above components were transferred to an Erlenmeyer flask containing 20 ml of liquid Woody Plant Medium, which was added at the same concentration, and swirling culture (amplitude 25 mm, 120 rpm) on a rotary shaker,
Planting was performed every 21 days to accelerate the growth rate of the callus. 1 g of the cultured cells (fresh weight) thus obtained was transferred to an Erlenmeyer flask containing 20 ml of Liquid Woody Plant Medium to which the above-mentioned components were added at the same concentration, and shake-cultured at 25 ° C. for 14 days. On the 14th day of culturing, methyl ester of jasmonic acid as jasmonic acid (in the formula (III), R
^1a , R ^1b , R ^1c , R ^1d , R ^1e , R ^1f , R ² , R ³ ,
Compounds in which R ⁴ , R ⁵ and R ⁶ are hydrogen atoms, R ⁷ is a methoxy group, n is 1 and which contains a double bond between C ³ and C ⁴ , 90% trans type, (Cis type 10%) is added so that the final concentration is 0.01 to 1000 μM, and further 7
Cultured for a day. After completion of the culture, the Taxus vulgaris cultured cells were collected by filtration, lyophilized, and the dry weight thereof was measured to determine the growth weight of the cultured cells per 1 L of the liquid medium. The taxane-type diterpene was extracted from the obtained dry callus with methanol or the like, and the taxane-type diterpene yield was measured by comparative quantification with standard products taxol, cephalomannine and baccatin III using high performance liquid chromatography. Table 1 shows the results.

Comparative Example 1 The same operation as in Example 1 was carried out except that jasmonic acid methyl ester was not added. Table 1 shows the results.

(Example 2) In Example 1, jasmonic acid methyl ester was sequentially added from the 7th day of the culture every other day for a total of 4 times (the final concentration per time was 25 µM, total 100 µM).
The same operation as in the example was carried out except that M) was performed. Table 1 shows the results.

Example 3 The same operation as in Example 1 was carried out except that 100 μM of jasmonic acid methyl ester was added to the first day of the culture and the culture was continued for 20 days. Table 1 shows the results.

Example 4 The same operation as in Example 1 was carried out except that 100 μM of jasmonic acid methyl ester was added on the 7th day of culture in Example 1 and the culture was continued for 14 days. Table 1 shows the results.

Example 5 In Example 1, as jasmonic acids, jasmonic acid (in the formula (I), R ^1a ,
R ^1b , R ^1c , R ^1d , R ^1e , R ^1f , R ² , R ³ , R ⁴ , R
⁵ and R ⁶ are hydrogen atoms, R ⁷ is a hydroxyl group, and n
Is 1 and the compound contains a double bond between C ³ and C ⁴ , the trans-type is 90%, the cis-type is 10%), and the final concentration is 0.
The same operation as in the example was carried out except that the addition amount was adjusted to 01 to 1000 μM. The results are shown in Table 2.

Comparative Example 2 The same operation as in Example 5 was carried out except that jasmonic acid was not added. The results are shown in Table 2.

[0068]

[Table 1]

[0069]

[Table 2]

Example 6 In Example 1, the taxane-type diterpenes present in the medium before, 100, and 100 μM of jasmonic acid methyl ester were analyzed, and the results are shown in FIG. And 2 are shown. On day 7 of culture, about 50% of taxol and about 70% of baccatin III were leaked to the medium.

Comparative Example 3 The procedure of Example 6 was repeated except that the jasmonic acid methyl ester was not added. The results are shown in FIGS. 1 and 2.

(Example 7) The cells having an increased growth rate obtained in Example 1 were first separated into cell clumps having a size of 250 to 840 µm with a stainless mesh.
Next, using Ficoll, a medium having a specific gravity of 1.07 (g / ml) was prepared, and the cells were overlaid and centrifuged at 700 rpm for 6 minutes. The cells were separated into two layers according to the difference in specific gravity. 1.
The cells contained in the layer of 07 g / ml or less were fractionated and washed with 2% sucrose solution at least 3 times to wash away Ficoll. After washing
1 g of cells (fresh weight) was transferred to an Erlenmeyer flask containing 20 ml of Liquid Woody Plant Medium and cultured at 25 ° C. for 14 days with shaking. On day 14 of culture, add jasmonic acid methyl ester to a final concentration of 250 μM, and add 7
Cultured for a day. After the culture was completed, the same operation as in Example 1 was performed. Table 3 shows the results. The productivity of taxane-type diterpenes could be significantly improved by combining the selection of cells with a specific gravity and the addition of jasmonic acid methyl ester.

Comparative Example 4 The procedure of Example 7 was repeated except that jasmonic acid methyl ester was not added. Table 3 shows the results.

(Example 8) 250 µM of jasmonic acid methyl ester was added to the cultured cells of Taxus communis (day 14 of culture) obtained by the same method as in Example 1 and cultured for 7 days. After the completion of the culture, the same operation as in the example was performed. The results are shown in Table 4.

Comparative Example 5 The same operation as in Example 8 was carried out except that jasmonic acid methyl ester was not added. The results are shown in Table 4.

Example 9 In Example 8, T. media
The same operation as in the Example was carried out except that the cultured cells of 1. were used. The results are shown in Table 4.

Comparative Example 6 The same operation as in Example 9 was performed except that jasmonic acid methyl ester was not added. The results are shown in Table 4.

[0078]

[Table 3]

[0079]

[Table 4]

Example 10 Cultured cells of T. media were divided into cell clumps, which were then individually grown and selected using taxol productivity as an index. The same procedure as in Example 9 was performed using the obtained taxol-producing cells. The results are shown in Table 5.

Comparative Example 7 The procedure of Example 10 was repeated except that jasmonic acid methyl ester was not added. The results are shown in Table 5.

[0082]

[Table 5]

(Example 11) 2% antiformin solution or 70% ethanol solution was previously added to Woody Plant Medium solid medium (gellan gum 0.25% by weight) to which naphthalene acetic acid was added at a concentration of 10 ^-5 M. A part of the stem of Taxus baccata LINN, which had been sterilized by the above method, was placed and cultured at 25 ° C. in the dark to obtain Taxus callus. Next, 1g of this callus (fresh weight)
Woody plant containing the above ingredients added at the same concentration
Transfer to medium Erlenmeyer flask containing 20 ml of liquid medium,
Spin culture on a rotary shaker (amplitude 25 mm, 120 rp
m) and planted every 21 days to accelerate the growth rate of the callus.

1 g of the cultured cells thus obtained (new
Fresh weight) to which Woody
Triangular frustum containing 20 ml of plant medium liquid medium
The resulting mixture was transferred to a strain and cultured at 25 ° C. for 14 days with shaking. Day 14 of culture
Methyl ester of jasmonic acid (formula
In (III), R^1a, R^1b, R^1c, R^1d, R^1e,
R ^1f, R², R³, R^Four, R^FiveAnd R⁶Is a hydrogen atom
R⁷Is a methoxy group, n is 1, C³And C
^FourA compound containing a double bond between, trans form 90
%, Cis type 10%), the final concentration will be 10-250 μM
Then, the cells were added and cultured for 7 days.

After completion of the culture, the Taxus vulgaris cultured cells were collected by filtration, lyophilized, and the dry weight thereof was measured to determine the growth weight of the cultured cells per liter of the liquid medium. The taxane-type diterpene was extracted from the obtained dry callus with methanol or the like, and the taxane-type diterpene yield was measured by comparative quantification with the standard products taxin I and taxicin I using high performance liquid chromatography. Table 6 shows the results.

Comparative Example 8 The same operation as in Example 11 was carried out except that the methyl ester of jasmonic acid was not added. Table 6 shows the results.

[0087]

[Table 6]

Comparative Example 9 The same operation as in Example 1 was carried out except that jasmon was added in place of the methyl ester of jasmonic acid so that the final concentration thereof was 0.1 to 1000 μM. The results are shown in Table 7.

Comparative Example 10 The same operation as in Comparative Example 9 was carried out except that Jasmon was not added. The results are shown in Table 7.

[0090]

[Table 7]

[0091]

INDUSTRIAL APPLICABILITY According to the present invention, a large amount of taxane-type diterpenes can be easily obtained by tissue-culturing taxane-type diterpene-producing plants in the presence of jasmonic acids.

[Brief description of drawings]

FIG. 1 is a graph showing changes in taxol yield in a medium after addition of 100 μM methyl jasmonate.

FIG. 2 is a graph showing changes in baccatin III yield in the medium after addition of 100 μM methyl jasmonate.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁶ Identification code Office reference number FI technical display location C12R 1:91)

Claims (8)

[Claims] 1. A plant cell or tissue producing a taxane-type diterpene is represented by the general formula (I): [In the formula, R ^1a , R ^1b , R ^1c , R ^1d , R ^1e and R ^1f each independently represent a hydrogen atom, a hydroxyl group, an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms. ; R ² , R
³ , R ⁴ , R ⁵ and R ⁶ each independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms; C ¹ -C ² -C ³
Side chain consisting of -C ⁴ -C ⁵ -C ⁶ may contain one or more double bonds; R ⁷ is a hydroxyl group, OM
(Here, M represents an alkali metal atom, an alkaline earth metal atom, or NH _4. ), NR ^8a R ^8b (wherein R ^8a and R ^8b are each independently a hydrogen atom, a carbon number of 1 to 6). An acyl group, an alkyl group having 1 to 6 carbon atoms or an amino acid residue), OR ⁹ (wherein R ⁹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue) or 1 carbon atom ~ 6
Represents an alkyl group; n represents an integer of 1 to 7;
The member ring may form a double bond between adjacent ring member carbon atoms. ] In the presence of jasmonic acid shown in,
A method for producing a taxane-type diterpene, which comprises recovering a taxane-type diterpene from the obtained culture. 2. The taxane-type diterpene is taxol, 10
-Deacetyltaxol, 7-Epitaxol, Baccatin III, 10-Deacetylbaccatin III, 7-Epibaccatin III, Cephalomannine, 10-Deacetylcephalomanin, 7-Epicephalomannin, Baccatin VI, Taxane 1a, Xylosyl cephalo Manin, xylosyl taxol, taxol C, 10-deacetyl taxol C
2. The method for producing a taxane-type diterpene according to claim 1, which is at least one selected from the group consisting of: 3. The taxane-type diterpene is taxixin I,
The method for producing a taxane-type diterpene according to claim 1, wherein the method is at least one selected from taxixin II, taxin I, taxin II, and taxifin. 4. The method for producing a taxane-type diterpene according to claim 1, wherein jasmonic acids are added during the logarithmic growth phase or stationary phase of the cultured cells. 5. The method for producing a taxane-type diterpene according to claim 1, wherein the jasmonic acid is added to the culture medium in a plurality of times or continuously. 6. The method for producing a taxane-type diterpene according to claim 1, wherein the plant producing the taxane-type diterpene is a Taxus genus plant. 7. The method for producing a taxane-type diterpene according to claim 1, wherein the concentration of jasmonic acids in the tissue culture medium is 0.01 to 1000 μM. 8. A plant cell that produces a taxane-type diterpene is divided into a plurality of layers according to the difference in specific gravity, and at least 1.
The method for producing a taxane-type diterpene according to claim 1, wherein cells contained in one layer are cultured.