JPH08154693A - Production of taxane type diterpene - Google Patents

Abstract

PURPOSE: To obtain the subject compound useful for treating ovarian, mammary, pulmonary cancers, etc., with good productivity by culturing a tissue, a cell, etc., of a plant capable of producing a taxane type diterpene in a culture medium containing a saccharide and nitrate ions at specific concentrations under specified conditions and recovering the resultant product. CONSTITUTION: A tissue or a cell or both of a plant capable of producing a taxane type diterpene (e.g. Taxus breviflolia) are transplanted into a culture medium containing a sucrose, glucose or fructose at 2-50g/l concentration thereof, nitrate ions such as potassium nitrate, sodium nitrate or calcium nitrate at 2-50mmol/l concentration thereof in culturing the tissue or the cell or both of plant capable of producing the taxane type diterpene. A nutrient source solution daily containing the saccharide at 0.2-5g/l concentration or the nitrate ions at 0.2-5mmol/l concentration based on the initial volume of the culture medium or both is continuously or intermittently added to the culture medium to carry out the culture. The resultant product is recovered from the prepared cultured product to thereby afford the objective taxane type diterpene (e.g. taxol) useful as a therapeutic agent for ovarian, mammary, pulmonary cancers, etc.

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,
Moreover, it is difficult to easily obtain a large amount of taxol because the tree will die when the bark is peeled off. If taxol or the precursor of taxol, baccatin III (b
If taxane-type diterpenes such as accatin III) can be produced using tissue culture, a large amount of taxol can be easily obtained without cutting trees, which is advantageous.

[0003] For the taxol production method using the cultured cells of the plant up to now, see
s brevifolia NUTT) A taxol production method using cultured cells has been patented in the United States (US Patent No. 5019504), but the taxol production amount is described as 1 to 3 mg / l, which is insufficient for industrial production. Is. Further, the productivity of taxol in cell culture is unstable, and even if cells with high productivity are temporarily obtained by selection, it is difficult to maintain the content by subculture (ERM Wickremesine et al.,
World Congress on Cell and Tissue Culture (199
2)].

As a prior art method for producing taxol, a semisynthesis method from taxol biosynthetic precursor baccatin III is disclosed in US Pat. No. 5,015,744 to Holton et al. By using the tissue culture method of a plant, a semi-synthetic raw material such as baccatin III can be produced, and it can also be used for taxol production by the semi-synthetic method. On the other hand, in the culture of plant tissues or cells that is generally carried out, the nutrients and plant hormones necessary for the growth of the tissues or cells are collectively charged at the start of the culture (batch culture method), and the operation is complicated. Due to problems such as the above-mentioned problems, it is rare to perform the medium addition of the nutrient source (fed-batch culture method) or the renewal of the medium (perfusion culture) that combines the medium addition of the nutrient source and the withdrawal of the culture solution. In addition, when plant cells are exposed to a high concentration of nutrients and plant hormones, hyperosmolarity causes protoplast separation, or physiological excess damage to specific components leads to growth arrest and necrosis. Therefore, the concentration of the medium components used in the batch culture method is inevitably low to some extent, and there is an upper limit to the density of tissues or cells that can be transplanted.

However, when the tissue or cells are transplanted and cultured at such a low density, even if the culture is completed, the incubator will have more tissue in view of the permissible density in terms of culture engineering considering aeration and stirring. Or, there is still room for culturing cells, and it cannot be said that the incubator is economically used. In order to solve such a problem, a fed-batch culture method and a perfusion culture method have been developed for culturing microorganisms and animal cells, but for culturing plant tissues or cells, Japanese Patent Application Laid-Open No. 62-1751.
There are only a few examples in the culture of buttercup Coptis cells disclosed in Japanese Patent Publication No. 69, the culture of Solanaceae Zboisia roots disclosed in Japanese Patent Laid-Open No. 2-303431, and the like, which are the subject of the present invention. Regarding the culture of tissues or cells of taxane-type diterpene-producing plants, for example, Taxus genus Taxus plants, the above problems have not been considered at all.

The fed-batch culture method and the perfusion culture method are both known culture process technologies from ancient times, but when used for culturing plant tissues or cells, plant cells have special properties different from those of microorganisms and the like. Therefore, it is often impossible to apply the method itself, or it is necessary to develop extremely advanced technology. For example, plant hormones and phosphate sources are known to be taken up by plant cells more rapidly than other medium components, and calcium and magnesium must be present at a certain concentration in the medium unless they reach a certain level. Cannot capture these. Furthermore, many of the secondary metabolites are known to start production during the period from the logarithmic growth phase where the cell growth rate begins to decrease to the stationary phase, making addition of nutrient sources more complicated. I was there.

Under these circumstances, in the case of culturing plant tissues or cells, the batch culture method is actually applied in favor of the above-mentioned low economic efficiency in using an incubator.

[0008]

An object of the present invention is to provide a method for efficiently producing a taxane-type diterpene by culturing plant tissue, particularly by culturing high-density tissue or cells.

[0009]

As a result of earnest studies, the present inventors have found that when culturing plant tissues and / or cells that produce taxane-type diterpenes, sugar and / or nitrate in the medium at the start of the culture When the concentration of ions is within a specific range and the culture is started by adding sugar and / or nitrate ions at a rate within a specific range, high density culturing of the tissue and / or cells becomes possible. It was found that the production amount of the taxane-type diterpene was dramatically improved, and the present invention was completed.

That is, according to the present invention, a medium having a sugar concentration of 2 to 50 g / l and / or a nitrate ion concentration of 2 to 50 mmol / l is used for culturing a tissue and / or cell of a plant producing a taxane-type diterpene. Tissue and / or cells, and then 1 to the initial volume of the medium
0.2-5 g / l sugar and / or 0.2-5 m per day
A method for producing a taxane-type diterpene, which comprises continuously or intermittently adding a nutrient source solution containing mol / l nitrate ion and culturing, and recovering a taxane-type diterpene from the obtained culture.

The present invention will be described in detail below. The taxane-type diterpene to be the subject of the production method of the present invention,
There is no particular limitation as long as it is a diterpene having a taxane skeleton, for example, taxol, 10-deacetyltaxol,
7-epitaxol, baccatin III, 10-deacetylbaccatin III, 7-epibaccatin III, cephalomannine, 10-deacetylcephalomannin, 7-epicephalomanin, taxakifine and its analogs, taxane 1a and its analogs, Examples include xylosyl cephalomannine and xylosyl taxol.

The taxane-type diterpene-producing plants used in the production method of the present invention include, for example, common yew (Taxus baccata LINN) and yew (T. cuspidata SIE).
B.etZUCC), Carabok (T. cuspidata SIEB.et ZUCC v
ar. nana REHDER), T. brevifolia
NUTT), Canadian yew (T. canadiensis MARSH), Chinese yew (T. chinensis), and T. media. Among these, yew and T.media are particularly preferable in terms of the magnitude of the effect obtained by applying the present invention.

According to the present invention, the culture of the plant tissue and / or cells can be carried out by a conventionally known method except that the culture is carried out under the condition of supplying specific sugar and / or nitrate ions. As the method for supplying sugar in the present invention, the concentration in the medium at the start of culture is 2 to 50 g / l.
It is necessary to make it 10 to 30 g / l. In addition, the rate of addition of sugar during culture is
0 per day based on the initial volume of medium at the start of culture.
It is necessary to set it to 2-5 g / l, and especially 0.5-
It is preferably 5 g / l. The addition of this sugar may be carried out at a constant rate as long as it is within the above range, or it may be appropriately changed. As a method to change appropriately,
In order to maintain the sugar concentration in the medium within a preset constant concentration range, a method of increasing or decreasing the addition rate within the range of the addition rate according to the sugar demand of the cells changing with the progress of culture is exemplified. To be done.

As a method for supplying nitrate ions in the present invention, the concentration in the medium at the start of culture is 2 to 50 mmol /
It is necessary to set it as 1 and especially 10-30 mmol
/ L is preferable. Further, the rate of addition of nitrate ions during the culturing needs to be 0.2 to 5 mmol / l per day with respect to the initial volume of the medium at the start of the culturing, and especially 0.5 to 5 mmol / l. Preferably. The addition of this nitrate ion may be carried out at a constant rate as long as it is within the above range, or it may be appropriately changed. As a method of appropriately changing, in order to maintain the nitrate ion concentration in the medium in a preset constant concentration range, depending on the nitrate ion demand of cells changing with the course of culture, addition within the range of the addition rate A method of increasing or decreasing speed is illustrated.

The addition of the sugar and / or nitrate ion described above
It may be carried out continuously or intermittently. In addition, when the addition is performed intermittently, the number of times of addition per day is not limited, and there may be days when the addition is not performed. In the present invention, the sugar and / or nitrate ion may be added at any time from immediately after the start of the culture until the end of the culture, but it may be added during the logarithmic growth phase of the cells. It is particularly effective in increasing the growth rate of T.
When subculture is carried out every day, it is particularly preferable to carry out the culture during the period from 2 days to 14 days after the start of culture.

The present invention is effective when the cell density becomes high and a sufficient cell growth rate cannot be expected in batch culture. Therefore, when the method of the present invention is applied, the initial stage of the tissue or cells of the plant concerned. The density is preferably 50 g fresh weight / l or more, but even when the initial density is less than 50 g fresh weight / l, by applying the method of the present invention, the growth rate is increased. The effect of can be obtained.

As the sugar used in the present invention, a sugar source generally used in plant tissue culture can be used, examples of monosaccharides include glucose, fructose and galactose, and examples of disaccharides include sucrose. Examples include sugar (sucrose), maltose, and lactose, and examples thereof include polysaccharides such as starch. In the present invention,
Of these, sucrose, glucose and fructose are preferably used, and sucrose is most preferably used.

As a source of the nitrate ion used in the present invention, a nitrate compound generally used in plant tissue culture can be used, and sodium nitrate, potassium nitrate,
Examples thereof include calcium nitrate and ammonium nitrate. In the present invention, it is most preferable to use at least one selected from sodium nitrate, potassium nitrate and calcium nitrate.

The types of the above-mentioned sugar source and nitrate compound can be appropriately changed during the culture period, and if the concentrations of sugar and nitrate ion are within the above ranges, a plurality of sugars and / or nitrates are mixed. It is also possible to use it. When culturing tissues or cells of taxane-type diterpene-producing plants such as Taxus plants, even if they deviate from the above supply condition range,
In order to maximize the growth rate of cells and to keep the productivity of taxane-type diterpenes at a high level without significantly reducing the growth rate of tissues or cells or causing necrosis of tissues or cells, the above-mentioned supply is necessary. It is necessary to add sugar and / or nitrate ions under the conditions.

Generally, among the components of the medium used for culturing plant tissues or cells, sugar is a component contained in a relatively large amount in terms of moles, and compared with other large components such as nitrogen source. Since the rate of uptake into cells is low,
It is known that the osmotic pressure of the medium is greatly affected for a long time after cell transplantation. It is also known that when the osmotic pressure of the medium is high, the cells undergo cytoplasmic separation, and thus the growth rate of the cells decreases, and the sugar concentration in the medium during culture maximizes the growth rate of the cells. It can be pointed out that strict management is required for this.

On the other hand, nitrate ions are taken up by cells and then stored in the nitrate state, or converted into nitrate nitrogen by nitrate reductase and nitrite reductase induced by nitrate nitrogen. The activity of reductase is inhibited by ammonium ion. However, if the regulation of nitrate reduction becomes abnormal due to some cause, ammonia nitrogen or its assimilation products may excessively accumulate in the cells, and their toxicity may cause necrosis of the cells. It can be said that the nitrate ion concentration in the medium is also a factor to be strictly controlled in maximizing the rate.

In addition, in carrying out the intermediate addition of the nutrient source according to the present invention, at the same time as or before or after the addition of the added nutrient source solution, the culture is carried out at a volume within a range that does not substantially change the total volume of the medium. A method of extracting the medium from the container can be used in combination. The withdrawal of the medium can be carried out continuously or intermittently, and its speed or frequency can be appropriately changed.

There is no particular limitation on the speed or volume of the medium to be added on the way and the medium to be extracted correspondingly, but the density of the tissue and / or cells to be cultured becomes high, and the accumulation of certain growth inhibitory substances. When such a problem is observed, increase the rate or volume of the nutrient source addition and medium withdrawal to increase the efficiency of replacing the medium in the incubator with a fresh medium, and try to solve these problems. It is possible.

When the present invention is carried out by using a culture tank such as a tank, this medium is usually extracted by a cell outflow which is different from the supply port provided for the intermediate addition of the nutrient solution. It is preferable to carry out through a medium withdrawal port in which a filter for blocking is installed. Under such circumstances, the present inventors have proposed that the tissue and / or cells of a taxane-type diterpene-producing plant are supplied with sugar and / or nitrate ions under a specific range of conditions, and It was found that the growth rate of cells, particularly the growth rate of the tissue and / or cells when the density of the tissue and / or cells per culture medium at the start of culture is higher than usual, thereby increasing the growth rate per incubator. It was also found that the production efficiency of the taxane-type diterpene was improved. So far, no case has been reported in which production of taxane-type diterpenes was induced by supplying sugar and / or nitrate ions under a specific range of conditions, and the secondary metabolites were produced by the method of the present invention. It would have been unexpected to increase productivity.

The medium used for the tissue culture in the present invention is a conventionally known medium used for the tissue culture of plants, such as Murashige Scoog (1962) [Mu
Rashige &Skoog's Medium, Rinsmeier Scoog
(1965) Medium of [Linsmaier Skoog], Woody
Plant Medium (1981) 〔Woody Plant Mediu
m], B-5 medium of Gamborg, M of Mitsui
-9 medium and the like.

The above-mentioned sugars and plant hormones may be added to these media, and further inorganic components, vitamins, amino acids and the like may be added if necessary. Examples of plant hormones include indoleacetic acid (IAA) and naphthaleneacetic acid (NA).
A) Auxins such as 2,4-dichlorophenoxyacetic acid (2,4-D) and cytokinins such as kinetin, zeatin and dihydrozeatin are used.

Examples of the inorganic component include phosphorus, potassium, calcium, magnesium, sulfur, iron, manganese, zinc, boron, copper, molybdenum, chlorine, sodium, iodine and cobalt in addition to the above-mentioned nitrogen source. , These components are, for example, potassium nitrate, sodium nitrate, calcium nitrate, potassium chloride, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, calcium chloride, magnesium sulfate, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate. , Zinc sulfate, boric acid, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, cobalt chloride and the like.

Examples of vitamins include biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pantothenic acid, inositol and nicotinic acid. As the amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added.

Generally, each of the above-mentioned components comprises about 0.01 to about 10 μM of plant hormones, about 0.1 μM to about 100 mM of inorganic components,
About 0.1 to about 100 mg / min of vitamins and amino acids
Used at a concentration of l. In the tissue culture in the present invention, the root of the plant, the growing point, leaves, stems, seeds, pollen,
Tissue pieces or cells such as anthers and sepals, or cultured cells obtained by tissue-culturing these with the above-mentioned medium or other conventional medium can be used.

The present invention also relates to Agrobacterium tumefacien
s or Agrobacterium rhizogenes can also be applied to tumor cells and / or hairy roots obtained by infecting plant tissue. The productivity of the taxane-type diterpene can be further enhanced by using the production method of the present invention in combination with a method of culturing in the presence of various taxane-type diterpene production-promoting substances.

Examples of the taxane-type diterpene production promoter include Japanese Patent Application Nos. 6-104211, 6-104212 and 6
-104213, etc .; jasmonic acids; coronatine, or coronatine-producing bacteria or culture solution or culture extract thereof; compounds containing heavy metals disclosed in Japanese Patent Application No. 6-201150 , Complex ions containing heavy metals and heavy metal ions; Japanese Patent Application No. 6-201
Amines disclosed in Japanese Patent No. 151; Japanese Patent Application No. 6-
Anti-ethylene agents disclosed in 252528 are included.

Examples of the jasmonic acids include those represented by the general formula (I):

[0033]

Embedded image

[In the formula, R ^1a , R ^1b , R ^1c , R ^1d , R ^1e and R ^1f are each 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. Representation; R
² , R ³ , R ⁴ , R ⁵ and R ^6a each 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 ^6b is hydroxyl or -O
Represents a carbohydrate residue; R ⁷ is a hydroxyl group, OM (where
M is an alkali metal atom, an alkaline earth metal atom or NH
Represents ₄ . ), NHR ⁸ (wherein R ⁸ represents a hydrogen atom, an acyl group having 1 to 6 carbon atoms, an alkyl group having 1 to 6 carbon atoms or an amino acid residue), OR ⁹ (wherein R ⁹ is carbon. Or an alkyl group having 1 to 6 carbon atoms) or an alkyl group having 1 to 6 carbon atoms; n represents an integer of 1 to 7; and the 5-membered ring is between adjacent ring member carbon atoms. May form a double bond. ] The compound shown by the general formula (I
I):

[0035]

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[In the formula, R ^1a , R ^1b , R ^1c , R ^1d , R ^1e and R ^1f are each 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. Representation; R
² , R ³ , R ⁴ , R ⁵ and R ⁶ each 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. ), NHR ⁸ (wherein R ⁸ is a hydrogen atom, an acyl group having 1 to 6 carbon atoms, or an alkyl group having 1 to 6 carbon atoms). Represents an alkyl group 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 an alkyl group having 1 to 6 carbon atoms; n is 1 to
Represents an integer of 7; the 5-membered ring may form a double bond between adjacent ring-member carbon atoms. ] The compound shown by
And the general formula (III):

[0037]

Embedded image

[In the formula, R ^1a , R ^1b , R ^1c , R ^1d , R ^1e and R ^1f are each 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. Representation; R
² , R ³ , R ⁴ , R ⁵ and R ⁶ each 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. ), NHR ⁸ (wherein R ⁸ is a hydrogen atom, an acyl group having 1 to 6 carbon atoms, or an alkyl group having 1 to 6 carbon atoms). Represents an alkyl group 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 an alkyl group having 1 to 6 carbon atoms; n is 1 to
Represents an integer of 7; the 5-membered ring may form a double bond between adjacent ring-member carbon atoms. ] The compound shown by these is mentioned.

In the above general formulas (I), (II) and (III),
And R^1a, R^1b, R^1c, R^1d, R ^1e, R^1f, R², R
³, R^Four, R^Five, R⁶, R^6a, R⁷, R⁸Or R⁹Table
Examples of the alkyl group having 1 to 6 carbon atoms include
Group, ethyl group, n-propyl group, isopropyl group, n
-Butyl group, isobutyl group, sec-butyl group, t-butyl group
Group, n-pentyl group, and n-hexyl group.

In the above formulas (I), (II) and (III),
And R^1a, R^1b, R^1c, R^1d, R ^1eOr R^1fRepresented by
Examples of the alkoxy group having 1 to 6 carbon atoms include
Si group, ethoxy group, n-propoxy group, isopropoxy
Group, n-butoxy group, isobutoxy group, sec-butoxy group
Group, t-butoxy group, n-pentyloxy group, n-hexyl
Examples include a siloxy group.

When R ⁷ is OM, examples of the alkali metal atom or alkaline earth metal atom represented by M include sodium, potassium and calcium. When R ⁷ is NHR ⁸ , the acyl group having 1 to 6 carbon atoms represented by R ⁸ may be linear or branched and includes, for example, formyl group, acetyl group, propionyl group, butyryl group, valeryl. Group, a hexanoyl group, and an acryloyl group.

In the case where R ⁷ is NHR ⁸ , R ⁸
Examples of the amino acid residue represented by are an isoleucyl group, a tyrosyl group, and a tryptophyll group. R ⁷ is OR
⁹ is a carbohydrate residue represented by R ⁹ ,
In the above general formula (I), when R ^6b is an —O-carbohydrate residue, examples of the carbohydrate residue include a glucopyranosyl group.

Further, the above general formulas (I), (II) and (II)
In the compound represented by 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)

[0044]

[Chemical 4]

(Compound B)

[0046]

Embedded image

(Compound C)

[0048]

[Chemical 6]

(Compound D)

[0050]

[Chemical 7]

The following compounds may be mentioned as specific examples of the compound represented by the general formula (II). (Compound E)

[0052]

Embedded image

(Compound F)

[0054]

[Chemical 9]

(Compound G)

[0056]

[Chemical 10]

(Compound H)

[0058]

[Chemical 11]

Specific examples of the compound represented by the general formula (III) include the compounds shown below. (Compound I) 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 J) R ^1a , R ^1b , R ^1c , R ^1d. , R ^1e , R ^1f , R ² , R ³ , R
⁴ , R ⁵ , R ⁶ : H R ⁷ : -OH n: 1 In the compound represented by the general formula (III), R ^1a , R ^1
A compound in which ^1b , R ^1c , R ^1d , R ^1e or R ^1f is a hydroxyl group,
Alternatively, specific examples of the compound in which a double bond is formed between adjacent ring member carbon atoms in the 5-membered ring include the compounds shown below. (Compound K)

[0060]

[Chemical 12]

(Compound L)

[0062]

[Chemical 13]

(Compound M)

[0064]

Embedded image

(Compound N)

[0066]

[Chemical 15]

The compound represented by the above general formula (I), (II) or (III)
Preferred compounds include^1a, R^1b, R
^1c, R^1d, R^1e, R^1f, R², R³, R^Four, R^FiveAnd R
⁶Is a hydrogen atom, and R⁷Is a hydroxyl group or a methoxy group
C¹-C²-C³-C^Four-C^Five-C⁶Side chain consisting of
Does not contain a double bond, or C¹And C², C ²
And C³Or C³And C^FourCompounds containing double bonds between
You can

The above-mentioned general formula (I) used in the present invention,
There are various types of jasmonic acids represented by (II) or (III).
Stereoisomers (cis trans isomer, optical isomer) exist
However, even if each isomer is used alone,
It may be used in the form. The above jasmonic acids are all tachy
It is effective in improving the productivity of sun-type diterpenes.
Also in the above general formulas (I), (II) and (III),
R^1a, R^1b, R ^1c, R^1d, R^1e, R^1f, R², R³, R
^Four, R^FiveAnd R⁶Is a hydrogen atom, and R ⁷Is a hydroxyl group or
A methoxy group, n is 1, and C³And C^FourBetween two
Tuberonic acid, which is a compound containing a heavy bond, or tube
Methyl ronate, cucurbic acid or methyl cucurbitate,
And jasmonic acid or methyl jasmonate improve productivity
It is particularly preferable from the viewpoint of the magnitude of the effect on.

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
Year).

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 the endogenous jasmonic acids by the cultured cells or the cultured tissue, a culture of a microorganism or an extract thereof, a heat-treated product, a plant extract or the like can be added to the medium, and the like.
Specifically, MJ Mueller et al., Proc. Natl. Acad. Sc
The method of adding a mold cell wall fraction described in iUSA, 90 (16), 7490-7494 (1993) can be exemplified. In addition, it is possible to increase the production amount of endogenous jasmonic acid by mechanically or partially injuring the cultured cells or tissue to be used, by ultraviolet rays, heat, etc., and specifically, RACleeman et al., Proc. Nat
The method of mechanically destroying a part of cells described in I. Acad. Sci. USA, 89 ( 11), 4938-4941 (1989) 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. Among the jasmonic acids, the compound represented by the above formula (I) or (III) is
Since the α-position of the 5-membered ring carbonyl group causes epimerization by acid, alkali or heat, it tends to be a stable trans type rather than an unstable cis type. In equilibrium experiments with natural or synthetic jasmonic acid, 90% trans type and 1 cis type
It exists in the state of 0%. Generally, the cis type is considered to have stronger activity, but the jasmonic acids used in the present invention include all stereoisomeric compounds represented by the above formula (I) or (III) and a mixture thereof. .

When using jasmonic acids, it is necessary to adjust the concentration in the medium to 0.01 to 1000 μM, and it is particularly preferable to adjust the concentration of jasmonic acids to the range of 0.1 to 500 μM. Examples of coronatines that can be used as a taxane-type diterpene production promoter include compounds represented by the general formula (IV):

[0073]

Embedded image

Or general formula (V):

[0075]

[Chemical 17]

[Wherein R ¹⁰ is a hydroxyl group, OR ¹¹ (wherein R ¹¹ represents an alkyl group having 1 to 6 carbon atoms or a carbohydrate residue), OM ¹ (wherein M ¹ is Represents an alkali metal atom, an alkaline earth metal atom or NH ₄ ) or NR ^12a R ^12b (wherein R ^12a and R ^12b are each independently a hydrogen atom, an acyl group having 1 to 6 carbon atoms, or 1 carbon atom).
~ 6 alkyl group, amino acid residue or general formula (VI):

[0077]

Embedded image

(Where R¹³Is a hydrogen atom, hydroxyl group, charcoal
Alkyl groups having 1 to 6 primes and alkoxy groups having 1 to 6 carbons
Or the following formula-CO-R¹⁶ (In the formula, R¹⁶Is a hydroxyl group, OM²(Where M²Is
Lucari metal atom, alkaline earth metal atom or NH_FourThe table
You. ), NR^17aR^17b(Where R^17aAnd R
^17bAre each independently a hydrogen atom or an acyl group having 1 to 6 carbon atoms.
Group, alkyl group having 1 to 6 carbon atoms or amino acid residue
You. ) Or OR¹⁸(Where R¹⁸Is a carbon number of 1 to 6
It represents a rukyl group or a carbohydrate residue. ) Represents. )
Represents a group represented by R ^14a, R^14b, R^15aAnd R^15bIs
Independently hydrogen atom, hydroxyl group, C1-6 al
It represents a kill group or an alkoxy group having 1 to 6 carbon atoms. )
Represents a group represented by ) Represents; R^19a, R^19b, R^20a,
R^20b, R^{twenty one}, R^{twenty two}, R^23a, R^23b, R^24a,
R^{twenty four b}, R^25a, R^25b, R²⁶And R²⁸Are each German
Vertically hydrogen atom, hydroxyl group, alkyl group having 1 to 6 carbon atoms, or
Represents an alkoxy group having 1 to 6 carbon atoms; R²⁷Is the hydrogen source
Child, alkyl group having 1 to 6 carbon atoms or carbohydrate residue
A 5-membered ring and a 6-membered ring in the formula are
A double bond may be formed. ] And other compounds
You can

In the above general formulas (IV), (V) and (VI), R ¹¹ , R ^12a , R ^12b , R ¹³ , R ^14a , R ^14b ,
R ^15a , R ^15b , R ^17a , R ^17b , R ¹⁸ , R ^19a , R
^19b , R ^20a , R ^20b , R ²¹ , R ²² , R ^23a , R ^23b ,
The ^{R 24a, R 24b, R 25a} , R 25 b, an alkyl group having 1 to 6 carbon atoms represented by R ^26, or R ^27, for example a methyl group, an ethyl group, n- propyl group, an isopropyl radical, n
-Butyl group, isobutyl group, sec-butyl group, t-butyl group, n-pentyl group, neopentyl group, t-pentyl group, n-hexyl group, isohexyl group and the like.

[0080] In the general formula (IV), (V) and ^{(VI), R 13, R} 14a, R 14b, R 15 a, R 15b,
R ^19a , R ^19b , R ^20a , R ^20b , R ²¹ , R ²² ,
Examples of the alkoxy group having 1 to 6 carbon atoms represented by R ^23a , R ^23b , R ^24a , R ^24b , R ^25a , R ^25b , or R ²⁶ include a methoxy group, an ethoxy group, an n-propoxy group, and isopropoxy group. Group, n-butoxy group, isobutoxy group, sec-butoxy group, t-butoxy group, n-pentyloxy group, neopentyloxy group, t-pentyloxy group, n-hexyloxy group, isohexyloxy group and the like. To be

When R ¹⁰ or R ¹⁶ is OM ¹ or OM ² , examples of the alkali metal atom or the alkaline earth metal atom represented by M ¹ or M ² include sodium, potassium and calcium. To be R ¹⁰ or R
^{In the case where 16} is NR ^12a NR ^12b or NR ^17a R ^17b , the acyl group having 1 to 6 carbon atoms represented by R ^12a , R ^12b , R ^17a or R ^17b may be straight chain or branched chain. Well, for example, formyl group, acetyl group, propionyl group, butyryl group, valeryl group, hexanoyl group, acryloyl group and the like can be mentioned.

R ¹⁰ or R ¹⁶ is NR ^12a NR ^12b or N
In the case of R ^17a R ^17b , R ^12a , R ^12b , R
Examples of the amino acid residue represented by ^17a or R ^17b include isoleucyl group, valyl group, glutamyl group, lysyl group and the like. When R ¹⁰ or R ¹⁶ is OR ¹¹ or OR ¹⁸ , examples of the carbohydrate residue represented by R ¹¹ or R ¹⁸ include a glucopyranosyl group.

In the general formula (V), examples of the carbohydrate residue represented by R ²⁷ include a glucopyranosyl group. As preferred compounds of coronatine,
The following formula (VII):

[0084]

[Chemical 19]

Coronatine represented by: or the following formula (VIII):

[0086]

Embedded image

The coronafasic acid represented by Also, as coronatine-producing bacteria, Pseudomonas
The genus Xanthomonas is known. Specific examples of the genus Pseudomonas include P. syringae, P. glycinea, and P.
tabaci, P. aptata, P. coronafaciens, P. phaseoli
Examples include cola, P. mori, P. helianthi, and the like. The genus Xanthomonas includes X. campestris and X. citr.
Examples include i, X. cucurbitae, X. phaseoli, and X. pruni.

The growth of coronatin-producing bacteria can be carried out in a general bacterial culture medium or a minimal medium. Examples of the coronatine-producing bacterium culture medium used in the present invention include those obtained by aseptic filtration of the culture medium in which these fungi are cultivated. As the coronatine-producing bacterium culture extract, for example, a culture solution obtained by culturing those fungi is autoclaved (120 ° C. for 15 minutes), or a culture solution of those fungi is treated with an organic solvent such as ethyl acetate under acidic conditions. The extracted ones, or the fractions further containing coronatine and coronafasic acid, which are partially purified by a Sephadex LH 20 column or the like, can be mentioned.

Compounds containing heavy metals, complex ions containing heavy metals and heavy metals in heavy metal ions that can be used as a taxane-type diterpene production promoting substance are not particularly limited as long as they are heavy metals belonging to the copper group or the iron group. However, silver is preferably used as the metal belonging to the copper group, and cobalt is preferably used as the metal belonging to the iron group. Furthermore,
When silver or cobalt is used, it is preferably used in the form of a compound containing the heavy metal, a complex ion containing the metal, or the metal ion. Further, these compounds and the like may be used alone or in combination.

Examples of compounds containing silver include silver nitrate, silver sulfate, silver fluoride, silver chlorate, and the like.
Or silver perchlorate, or silver acetate, or silver sulfite, or silver hexafluorophosphate (V), or silver tetrafluoroborate, or silver diamine (I) sulfate, or potassium diaminosilver (I), etc. The compounds can be exemplified. Among these, silver nitrate, silver sulfate and the like can be exemplified as suitable compounds.

Examples of complex ions containing silver include [Ag
(S ₂ O ₃ ) ₂ ] ^3- , or [Ag (S ₂ O ₃ ) ₃ ] ^5- , or [Ag (NH ₃ ) ₂ ]
⁺ , Or [Ag (CN) ₂ ] ^- , or [Ag (CN) ₃ ] ^2- , or [Ag
Examples thereof include complex ions such as (SCN) ₂ ] ^- , or [Ag (SCN) ₄ ] ^3- . Among these, [Ag (S ₂ O ₃ ) ₂ ]
^3- , [Ag (S ₂ O ₃ ) ₃ ] ^{5-, and the} like can be illustrated as suitable complex ions.

As the compound containing cobalt, for example, cobalt chloride, cobalt nitrate, cobalt sulfate, cobalt fluoride, or cobalt perchlorate,
Or cobalt bromide, or cobalt iodide, or cobalt selenate, or cobalt thiocyanate, or cobalt acetate, or ammonium cobalt sulfate, or potassium cobalt (II) sulfate, or hexaammine cobalt (III) chloride, or pentaammine aqua Cobalt (III) chloride, or nitropentaamminecobalt (III) chloride, or dichlorotetraamminecobalt (III) chloride hemihydrate, or dinitrotetraamminecobalt (III) chloride, or carbonatotetraamminecobalt (III) Chloride, or ammonium tetranitrodiamminecobaltate (III), or sodium hexanitrocobaltate (III), or tris (ethylenediamine) cobalt (III) chloride trihydrate, or dichlorobis (ethylenediamine) cobalt. Ortho (III) chloride, or potassium tris (oxalato) cobalt (III) trihydrate, or potassium hexacyanocobalt (III), or (ethylenediaminetetraacetato) cobalt (III) dihydrate, Alternatively, hydridotetracarbonylcobalt (I), dicarbonyl (cyclopentadienyl) cobalt (I), octacarbonyldicobalt (0), hexacarbonyl (acetylene) dicobalt (0), bis (cyclopentadienyl) ) Cobalt (I), or compounds such as (cyclopentadienyl) (1,5-cyclooctadiene) cobalt (I) can be exemplified. Among these, cobalt chloride, cobalt nitrate, cobalt sulfate and the like can be exemplified as suitable compounds.

The complex ions containing cobalt include pentaammine aquacobalt ion, nitropentaamminecobalt ion, dichlorotetraamminecobalt ion, dinitrotetraamminecobalt ion, carbonatotetraamminecobalt ion, and tetranitrodiamminecobalt ion. Or hexanitrocobalt ion, tris (ethylenediamine) cobalt ion, dichlorobis (ethylenediamine) cobalt ion, tris (oxalato) cobalt ion, hexacyanocobalt ion, or (ethylenediaminetetraacetato) cobalt ion or other complex ions Can be illustrated.

Of the above heavy metals, silver-containing compounds, silver-containing complex ions, or silver ions are preferably used in a medium at a concentration of 10 ^-8 M to 10 ^-1 M, and particularly preferably 10 ^-7 M. M ~
It is more preferable to adjust to the range of 10 ^-2 M. Further, compounds containing cobalt, complex ions containing cobalt, or cobalt ions, the concentration in the medium is preferably 10 ^-6 ~ 10 ^-1 M, particularly in the range of 10 ^-5 ~ 10 ^-2 M More preferably.

As a taxane-type diterpene production promoter
And amines that can be used as an amine
, But as such amines, monoamines
Alternatively, any of the polyamines can be used, but especially
Preference is given to using polyamines. In addition,
In the case of mines, some hydrogen atoms in the alkyl group are replaced by hydroxyl groups.
Optionally substituted mono-, di- or trialkylamines,
For example, methylamine, ethylamine, dimethylamine,
Diethylamine, triethylamine, diethanolamine
Or triethanolamine, or their salts; or
The polymethylene part may be interrupted by an imino group,
H of the amino group may be substituted with a lower alkyl group
Polymethylenediamine, such as putrescine, cadaveri
, Spermidine, spermine, ethylenediamine, N,
N-diethyl-1,3-propanediamine, diethylene tria
Min, triethylenetetramine, or salts thereof;
Or cyclic alkyl amines such as cyclopentylami
Amine, cyclohexylamine, or their salts, or
Is a cyclic amine such as mesenamine or piperazine, or
These include salts. Preferred among these amines
For example, putrescine [NH₂(CH₂)_FourNH₂],
Cadaverine [NH₂(CH₂)_FiveNH₂], Spermidine [NH₂(CH₂)
₃NH (CH₂)_FourNH₂], Spermine [NH₂(CH₂)₃NH (CH₂)_FourNH (CH
₂)₃NH₂], Ethylenediamine [NH₂(CH₂)₂NH₂], N, N-Di
Ethyl-1,3-propanediamine [(C₂H_Five)₂N (CH₂)₃N
H₂], Diethylenetriamine [NH₂(CH₂)₂NH (CH₂)₂N
H₂] And other polyamines, or salts thereof are exemplified.
be able to.

The amines have a concentration of 10 ⁻⁸ in the medium.
It is preferable to be M to 10 ^-1 M, and among these, 10 ^-7 is particularly preferable.
It is more preferable to adjust to the range of M to 10 ^-2 M. As an anti-ethylene agent that can be used as a taxane-type diterpene production-promoting substance, it is possible to inhibit the ethylene biosynthesis mechanism of a culture and / or store it in the culture or in an incubator containing the culture. There is no particular limitation as long as it is a substance that removes ethylene existing in the gas phase or medium.

As a method for inhibiting the ethylene biosynthesis mechanism, for example, the activity of an enzyme which catalyzes the conversion of S-adenosylmethionine to 1-aminocyclopropane-1-carboxylic acid is inhibited, or 1-aminocyclo A method of inhibiting the activity of an enzyme that catalyzes the conversion of propane-1-carboxylic acid to ethylene is exemplified, and examples of the compound having the former function include aminooxyacetic acid, acetylsalicylic acid, rhizobitoxine, aminoethoxyvinylglycine, and methoxy. Vinylglycine, α-aminoisobutyric acid,
2,4-dinitrophenol etc. can be mentioned.
Further, it may be a salt, ester, amino acid derivative or carbohydrate derivative of the compounds exemplified above.

As the salt, for example, sodium, potassium, calcium, magnesium salt, and as the ester,
For example, methyl, ethyl, propyl, butyl ester, amino acid derivatives such as glycine, methionine,
Examples of the phenylalanine derivative and the carbohydrate derivative include glucose and maltose derivatives. Examples of the compound having the latter function include gallic acid, and salts, esters, amino acid derivatives and carbohydrate derivatives of the compound (Hiroto Hyodo, 1987 Autumn Meeting of the Horticultural Society, Symposium Abstract, p. 1
22, Shin Kuraishi, Plant Hormone, The University of Tokyo Press, p.111].

Examples of salts include sodium, potassium, calcium, magnesium salts, esters such as methyl, ethyl, propyl and butyl esters, amino acid derivatives such as glycine, methionine, phenylalanine derivatives and carbohydrate derivatives. Examples include glucose and maltose derivatives.

[0100] Further, examples of the substance that removes ethylene stored in the culture or existing in the gas phase or the medium in the incubator containing the culture include 1,5-cyclooctadiene and Mention may be made of isothiocyanic acid and salts, esters (for example, allyl isothiocyanate, benzyl isothiocyanate), amino acid derivatives and carbohydrate derivatives of such compounds (Megumi Munakata, Chemical Regulation of Plants, 29 (1), 89-93 (1994)). ].

Here, salts include, for example, sodium, potassium, calcium, magnesium salts, esters such as methyl, ethyl, propyl, butyl, allyl ester, and amino acid derivatives such as glycine,
Examples of the methionine, phenylalanine derivative, and carbohydrate derivative include glucose and maltose derivative, but the salt, ester, amino acid derivative, and carbohydrate derivative of the substance according to the present invention are not limited to the compounds.

The anti-ethylene agent has a concentration of 10 ⁻⁸ in the medium.
It is necessary to adjust the concentration of M to 10 ^-1 M, and it is particularly preferable to adjust the concentration of the anti-ethylene agent in the range of 10 ^-7 M to 10 ^-2 M. When culturing in the presence of a taxane-type diterpene production-promoting substance as described above, it is effective to add jasmonic acid during the logarithmic growth phase or stationary phase of the cultured cells. It is preferable to add jasmonic acids at the time of transition to the stage. The timing of the treatment for increasing the production of endogenous jasmonic acids is also the same. For example,
When cells are transplanted every 21 days, the 7th to 16th days are the appropriate period of the addition of jasmonic acid or the treatment for increasing the production amount of endogenous jasmonic acid, and the logarithmic growth phase, for example, 7th to 14th day. Immediately after the transplantation, the appropriate time is reached when the cells are transplanted. In addition, the addition of jasmonic acids and the treatment for increasing the production amount of endogenous jasmonic acids are performed at a time,
It may be divided into a plurality of times or continuously.

It is effective to add the coronatine, or the coronatin-producing bacterium or its culture solution or culture extract, during the logarithmic growth phase or stationary phase of the cultured cells. It is preferable to add it at the time of transition. For example, when cells are transplanted every 21 days, the 7th to 16th days correspond to the appropriate period of addition of coronatine, a coronatin-producing bacterium, or a culture solution or extract thereof. Further, as a method of addition, a predetermined amount may be added at once, or may be added several times and sequentially added.

Compounds containing heavy metals, complex ions containing heavy metals, or heavy metal ions are effectively added at the start of the culture or from the time when the cultured cells transition from the logarithmic growth phase to the stationary phase, and particularly, It is preferable to add it at the start of culture. Further, the addition of the compound or ion may be performed at once or may be performed in several times. It is effective to add amines by the time cells transition from the logarithmic growth phase to the stationary phase, and it is particularly preferable to add them at the start of culture. Moreover, the said compound may be performed at once, and may be divided into several times and may be performed.

It is effective to add the anti-ethylene agent during the period from the logarithmic growth phase to the transition to the stationary phase, and it is particularly preferable to add it immediately after the transition to the stationary phase. Moreover, the said compound may be performed at once, and may be divided into several times and may be performed. In addition, as the taxane-type diterpene production promoting substance, a cyclic polysaccharide such as cyclodextrin described in Japanese Patent Application No. 6-291783 may be used.

Furthermore, the manufacturing method of the present invention is described in Japanese Patent Application No. 6-1.
No. 46826, which is a fluid medium in which the oxygen concentration in the gas phase in the incubator is controlled to be less than the oxygen concentration in the atmosphere from the initial stage of the culture, or the culture medium is brought into contact with tissues or cells. It can be used in combination with a method of controlling the dissolved oxygen concentration in the medium from the initial stage of culture under the condition of being less than the saturated dissolved oxygen concentration at that temperature.

[0107] Here, the initial stage of culture refers to the start of culture or the 7th day after the start of culture, and the oxygen concentration in the gas phase in the incubator or the dissolved oxygen concentration in the fluid medium in contact with tissues or cells. The control is preferably performed from the start of culture. The period of control may be controlled to the conditions throughout the entire culture period, or may be controlled only for a part of the entire culture period, and is not particularly limited, but during the entire culture period. It is preferable to control for at least 3 days.

The oxygen concentration in the gas phase in the incubator is 4 to
It is necessary to control to 15%, and it is particularly preferable to control to 6 to 12%. Further, the dissolved oxygen concentration in the fluid medium is 1 of the saturated dissolved oxygen concentration value at that temperature.
To 75%, especially 10 to
It is preferable to control to 75%. The production method of the present invention is described in Japanese Patent Application Nos. 5-284893 and 6-104213.
It can also be used in combination with the method disclosed in the specification, in which cells are divided into a plurality of layers according to the difference in specific gravity and the cells contained in at least one layer are cultured.

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. 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 continuously changes (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 formed layer is usually 1.00 to 1.20 g / ml, preferably 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, these plurality of layers can be individually cultured, but two or more layers of the selected plurality of layers are mixed and cultured. You can also Cultured cells with 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, but may vary depending on the cells to be cultivated and the conditions of the culture, and are not necessarily limited to this range. It is not something that will be done. 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 the taxane-type diterpene high-producing cultured cells, after culturing the cells of all the fractionated layers for a certain period of time, the taxane-type diterpene concentration contained in the cells of each layer is measured, and Among these, it is desirable to select a layer containing highly taxane-type diterpene-producing cells.

Also, by preparing a centrifuge medium having a specific gravity such as 1.07 g / ml and centrifuging by the above-mentioned method, the cultured cells are divided into a plurality of layers due to the difference in specific gravity. Can be divided. Further, it is possible to combine some or all of the methods described in the above-mentioned prior patents with the production method of the present invention.

The taxane-type diterpenes can be separated from the cultures such as the cultured tissues, cultured 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 preferable example of the tissue culture in the present invention.

First, plant pieces of plants belonging to the genus Taxus, for example, plant pieces collected from roots, growing points, leaves, stems, seeds, etc. are sterilized, and then solidified with Woody Plant Medium solidified with gellan gum. Place it at 14-
After about 60 days, a part of the tissue piece is turned into callus. 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, Woody plant liquid medium and grown. The growth rate is further increased in the liquid medium. In the present invention, the stabilized callus or cells constituting the callus is cultured in a solid medium or a liquid medium under the conditions of supplying sugar and / or nitrate ions within the above-mentioned specific range.

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. because the growth rate is high. The culture period is 14
~ 42 days are preferred. When a liquid medium is used in the culture in the present invention, the cultured cells are separated from the medium by a method such as decantation or filtration after the culture is completed, and the target taxane-type diterpene is separated from the cultured cells and / or the medium by an organic solvent. It can be separated by a method such as extraction.

[0117]

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) Woody plant medium to which naphthalene acetic acid was added at a concentration of 10 ^-5 M (Table 1)
2% in solid medium (gellan gum 0.25% by weight) in advance
A part of the stem of Taxus baccata LINN, which had been sterilized with an antiformin solution or a 70% ethanol solution, was placed on a bed and cultivated at 25 ° C. in the dark to obtain Taxus callus. Next, 1 g of this callus (fresh weight) was transferred to an Erlenmeyer flask containing 20 ml of Woody Plant liquid medium to which the above components were added at the same concentration, and swirling culture (amplitude 25 mm, 100 rpm) on a rotary shaker and planting every 21 days. Next, the growth rate of the callus was increased.

250 g fresh weight of liquid cultured cells (corresponding to 20 g dry weight) thus obtained was added to 1 liter of a standard Woody plant liquid medium (however, sucrose concentration: 20 g).
/ L, nitrate ion concentration: 14.7 mmol, α-naphthalene acetic acid: 10 ⁻⁵ M) were transferred to an aeration and stirring culture tank (internal volume: 2 liters; FIG. 1) and stirred at 25 ° C. in the dark. The culture was started while aerating air at a speed of 40 rpm and an aeration rate of 0.1 liter, and during the period from the 2nd day to the 14th day of the culture, the sucrose addition amount of 200 g / l was 0.1% per day. It was continuously added so as to have a concentration of 1 to 8 g / l, and aerating and stirring culture was performed for 21 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 rate. The taxane yield was measured by extracting taxane-type diterpenes from the obtained dry callus and culture medium using methanol or the like, and quantifying the taxane-type diterpenes by comparison with standard taxol using high performance liquid chromatography. The results are shown in Table 2.

(Example 2) The same procedure as in Example 1 was carried out except that the sucrose solution was intermittently added. The results are shown in Table 3. (Example 3) In Example 1, during the period from the 2nd day to the 14th day of culturing, 200 mmol / l of sodium nitrate solution was continuously added so that the amount added per day was 0.1 to 8 mmol / l. It carried out like Example 1 except adding (sucrose was not added on the way). The results are shown in Table 4.

Example 4 Example 4 was carried out in the same manner as in Example 3 except that the sodium nitrate solution was intermittently added. The results are shown in Table 5. (Example 5) Example 1 is the same as Example 1 except that the intermediate addition of sodium nitrate described in Example 3 is also used.
It carried out similarly to. The results are shown in Table 6.

(Example 6) In Example 5, the addition rate of sucrose added midway was 2 g / l per day, and the addition rate of sodium nitrate added midway was 2 mmol / l per day. The same procedure as in Example 5 was carried out except that the plant species from which the cells originated was T. media. The results are shown in Table 7.

(Example 7) In Example 5, the addition rate of sucrose added midway was 2 g / l per day and the addition rate of sodium nitrate added midway was 2 mmol / l per day. The procedure was as in Example 5, except that the medium used at the start contained 200 μM methyl jasmonate. The results are shown in Table 7.

(Example 8) In Example 5, the addition rate of sucrose added midway was 2 g / l per day, and the addition rate of sodium nitrate added midway was 2 mmol / l per day. The procedure was as in Example 5, except that the medium used at the start contained 2 μM coronatine. The results are shown in Table 7.

(Example 9) In Example 5, the addition rate of sucrose added midway was 2 g / l per day, and the addition rate of sodium nitrate added midway was 2 mmol / l per day. Example 5 except that a gas obtained by mixing equal amounts of air and nitrogen is used as the gas to be used.
It carried out similarly to. The results are shown in Table 7.

(Example 10) In Example 5, the addition rate of sucrose added midway was 2 g / l per day,
In addition, the addition rate of sodium nitrate added midway was 2 mmol / l per day, and 20 μ was measured on the 7th day after the start of culture
Example 5 was repeated except that M spermidine was added. The results are shown in Table 7.

Example 11 In Example 5, the addition rate of sucrose added midway was 2 g / l per day,
And the addition rate of sodium nitrate added midway is 2 mmol / l per day, except that the medium used at the start of the culture contains 2 mmol of [Ag (S ₂ O ₃ ) ₂ ] ^3- .
It carried out like Example 5. The results are shown in Table 7.

(Example 12) In Example 5, the addition rate of sucrose added midway was 2 g / l per day,
The procedure was carried out in the same manner as in Example 5, except that the rate of addition of sodium nitrate added midway was 2 mmol / l and the medium used at the start of the culture contained 20 μM cobalt chloride. The results are shown in Table 7.

Example 13 In Example 5, the addition rate of sucrose added midway was 2 g / l per day,
The procedure was performed in the same manner as in Example 5, except that the addition rate of sodium nitrate added during the process was 2 mmol / l per day and the medium used at the start of the culture contained 20 μM acetylsalicylic acid. The results are shown in Table 7.

Example 14 In Example 5, the nutrient source solution added midway was a solution of the standard Woody plant liquid medium having a sucrose concentration of 20 g / l and a nitrate ion concentration of 20 mmol / l (nitric acid). The rate of addition of sucrose added midway is 2 g / l and the rate of addition of nitrate ion midway added is 2 mmol / l per day, which is the same rate as the addition of the nutrient solution. Example 5 except that the culture solution was continuously withdrawn through an outlet provided with a 100-mesh stainless steel filter different from the nutrient source inlet (medium renewal rate in the incubator: 10% / day) It carried out similarly to. Table 8 shows the results.

(Example 15) In Example 14, except that the plant species from which the cultured cells were derived was T. media.
The same procedure as in Example 14 was carried out. Table 8 shows the results. (Example 16) The procedure of Example 14 was repeated, except that the medium used at the start of culture contained 200 µM methyl jasmonate. The results are shown in Table 8
Shown in

Example 17 The procedure of Example 14 was repeated, except that the medium used at the start of culture contained 2 μM coronatine. The results are shown in Table 8
Shown in (Example 18) The procedure of Example 14 was repeated, except that a gas obtained by mixing equal amounts of air and nitrogen was used as the gas to be aerated. Table 8 shows the results.

Example 19 The procedure of Example 14 was repeated, except that 20 μM spermidine was added 7 days after the start of culture. Table 8 shows the results. (Example 20) Example 14, the medium used at the start of culture _{[Ag (S 2 O 3)} 2] 3 of 2 mmol ^- except that it contains, was prepared as in Example 14. Table 8 shows the results.

Example 21 The procedure of Example 14 was repeated, except that the medium used at the start of the culture contained 20 μM cobalt chloride. Table 8 shows the results. (Example 22) The procedure of Example 14 was repeated, except that the medium used at the start of culture contained 20 µM of acetylsalicylic acid. Table 8 shows the results.

Example 23 In Example 20, after the culture was completed, the cells obtained were collected by filtration, freeze-dried, and the dry weight thereof was measured to determine the growth rate. The taxane-type diterpene was extracted from the obtained dry callus and culture medium using methanol or the like, and the taxane-type diterpene yield was measured by comparative quantification with standard taxol, baccatin III and cephalomannine using high performance liquid chromatography. The results are shown in Table 9.

(Comparative Example 1) The same procedure as in Example 1 was carried out except that the sucrose concentration in the medium at the start of culture was 20 to 85 g / l and sucrose was not added midway. did. Table 2 shows the results. (Comparative Example 2) In Example 3, except that the nitrate ion concentration at the initial stage of culture was set to 14.7 to 79.7 mmol / l (adjusted with sodium nitrate), and the solution containing the nitrate ion was not added midway. It carried out like Example 3. The results are shown in Table 3.

(Comparative Example 3) In Example 20, except that the nutrient source was not added midway and the medium was not withdrawn.
It carried out like Example 20. Table 8 shows the results.

[0138]

[Table 1]

[0139]

[Table 2]

[0140]

[Table 3]

[0141]

[Table 4]

[0142]

[Table 5]

[0143]

[Table 6]

[0144]

[Table 7]

[0145]

[Table 8]

[0146]

[Table 9]

[0147]

INDUSTRIAL APPLICABILITY According to the present invention, a taxane-type diterpene-producing plant tissue and / or cells are cultured under a condition of supplying sugar and / or nitrate ions in a specific range, whereby a large amount of taxane-type diterpene is obtained. It became possible to obtain it easily and easily.

[Brief description of drawings]

FIG. 1 is a diagram showing an example of a culture device used in performing tissue culture according to the present invention.

[Explanation of symbols]

 1 Air supply pipe 2 Nitrogen supply pipe 3 Culture tank 4 Oxygen-containing gas supply port 5 Dissolved oxygen concentration meter 6 Dissolved oxygen concentration control device 7 Gas exhaust pipe 8 Valve 9 Oxygen flow control valve 10 Air filter 11 Stirrer blade 12 Nutrient source supply port 13 Supplemental nutrient source 14 Opening with a filter that discharges only the culture solution 15 Culture solution discharge pipe 16 Culture filtrate tank

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Claims (11)

[Claims] 1. When culturing tissue and / or cells of a plant that produces a taxane-type diterpene, a sugar concentration of 2 is used.
~ 50g / l and / or nitrate ion concentration is 2-50mm
Tissues and / or cells were transplanted into a medium that was ol / l, and thereafter, 0.2 times per day relative to the initial volume of the medium.
It is possible to recover a taxane-type diterpene from the obtained culture by continuously or intermittently adding a nutrient source solution containing ~ 5 g / l sugar and / or 0.2-5 mmol / l nitrate ion. A method for producing a taxane-type diterpene. 2. The sugar concentration of the medium at the start of culture is 10 to 30.
The method for producing a taxane-type diterpene according to claim 1, wherein the concentration of nitrate ion in the medium at the start of culture is 10 to 30 mmol / l. 3. The production of the taxane-type diterpene according to claim 1, wherein the nutrient source solution containing sugar and / or nitrate ions is added during the period from 2 days to 14 days after the start of culture. Method. 4. The method for producing a taxane-type diterpene according to claim 1, wherein the tissue and / or cell density of the plant at the start of culture is 50 g fresh weight / l or more based on the medium volume. 5. The method for producing a taxane-type diterpene according to claim 1, wherein the sugar used is at least one selected from the group consisting of sucrose, glucose and fructose. 6. The method for producing a taxane-type diterpene according to claim 1, wherein the source of the nitrate ion is at least one selected from the group consisting of potassium nitrate, sodium nitrate and calcium nitrate. 7. 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. 8. The yew plant is the Taxus (Taxus).
baccata LINN), yew (T. cuspidata SIEB.et ZUC
C), Karaboku (T. cuspidata SIEB.et ZUCC var. Nan
a REHDER), T. brevifolia NUT
T), Canadian yew (T. canadiensis MARSH), Chinese yew (T. chinensis), and at least one kind selected from the group consisting of T. media, The taxane-type diterpene according to claim 7, characterized by the above-mentioned. Method. 9. The taxane-type diterpene is taxol,
10-deacetyltaxol, 7-epitaxole, baccatin III, 10-deacetylbaccatin III, 7-
2. At least one member selected from the group consisting of epibaccatin III, cephalomannine, 10-deacetylcephalomanin, 7-epicephalomanin, taxifin, taxane 1a, xylosyl cephalomannine and xylosyl taxol. A method for producing the taxane-type diterpene described. 10. When adding a nutrient solution, culture is performed while renewing the medium by separating and extracting the same volume of medium from the tissue and / or cells, and the medium and / or the medium collected by extraction during the culture. Alternatively, the taxane-type diterpene may be recovered from the culture obtained at the end of the culture. 11. The medium renewal rate is 10 to 10 per day.
It is 0%, The manufacturing method of the taxane-type diterpene of Claim 10 characterized by the above-mentioned.