JPH1169991A - Production of taxane-type diterpene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for producing a taxane-type diterpene containing taxol useful as a therapeutic agent for ovarian cancer, breast cancer, lung cancer, etc. SOLUTION: This method for producing a taxane-type diterpene by culturing the cell of a plant for producing the taxane-type diterpene comprises either or both of (a) setting an operation at least once for removing a large cell lump from a cell group by using a sieve and/or a filter during a preculture for the preparation of a culture for the production of the taxane-type diterpene or in the transfer of the cell from the preculture to the following preculture and (b) culturing the culture cell under strongly stirring conditions, so as to increase the ratio of a cell lump in a size range suitable for the production.

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 agent for ovarian cancer, breast cancer, lung cancer, etc., is a taxane-type diterpene isolated and identified from Taxus brevifolia NUTT, a plant of the genus Taxus. And have complex ester groups associated with activity. It has been reported that taxol is present in all parts of the yew tree plant, and its content is highest in bark.
Currently, taxol is taken from natural or cultivated plants, but Taxus 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 if the bark is peeled off. If taxol and a taxane-type diterpene such as baccatin III, which is a precursor of taxol, can be synthesized using cell culture, these can be obtained in large quantities and easily without cutting down trees. It is advantageous.

A method for producing taxol using cultured cells of a plant has been described in detail in the text
(s brevifolia NUTT) Taxol production by cultured cells is patented in the United States (US Patent 5,019,504), but the taxol production is described as 1-3 mg / l, which is insufficient for industrial production . In addition, the productivity of taxol by cell culture is unstable, and cells with high productivity can be obtained primarily by selection, but it is difficult to maintain the content by subculture (ERMWickremesine et al., World
Congress on Cell and Tissue Culture (1992)].

[0004] Under such circumstances, various methods have been attempted to improve the productivity of taxol, such as the Chinese yew Taxus having a high taxol content.
chinensis using cultured cells (US Patent 5,407,
816), a method using a continuous culture process (JP-A-7-25549)
5, EP 683232), a method of adding methyl jasmonate which is a signal transmitting substance to a culture medium (JP-A-8-33490, EP 683232)
And so on. However, none of the above methods has led to practical use, and further improvement in productivity is desired.

On the other hand, as a prior art of taxol production method, a semi-synthesis method from baccatin III or 10-deacetylbaccatin III, which is a precursor of taxol biosynthesis, is known as Holto.
The semi-synthetic taxol has already been approved and used clinically in the United States and Europe, as disclosed in US Pat. No. 5,015,744. If a plant cell culture method is used, semi-synthetic raw materials such as baccatin III can be produced, and can also be used for the production of taxol by the semi-synthetic method.

[0006] In general, plant cultured cells do not become single cells even if they are cultured in a liquid medium with shaking because they have strong cell walls to which individual cells adhere. I do. Woody plants such as Taxus plants used in the present invention have particularly strong cell connections due to the development of secondary cell walls such as lignin. Recent studies on secondary metabolite production in plant cell cultures have shown that secondary metabolites are different for each of these cell masses [Y.
Yamada and Y. Fujita, Handbook of Plant Cell Cultu
re, vol. 1, eds. by DA Evans et al., Macmillan
Publishing Co., New York, pp. 717-728 (1983); Y.H.
ara et al., Planta Med., 55, pp. 151-154 (1989)].
However, there is no report on the relationship between the particle size of the cell mass and the productivity of the taxane-type diterpene.

On the other hand, US Pat. No. 5,344,775 discloses a method for obtaining a small cell mass consisting of 1 to 10 cells of a yew plant by the following processes: (i) α-naphthaleneacetic acid as auxin and 6 as cytokinin -On a support holding a medium containing benzylaminopurine, to obtain a callus mass from a Taxus plant section containing cell meristem tissue, (ii) the obtained callus mass is a liquid medium containing the auxin and the cytokinin And the cell adhesion is weak,
Obtaining a suspension culture composed of a cell mass consisting of 1 to 10 cells; (iii) separating the cells from the obtained suspension culture and holding a medium containing the auxin and the cytokine (Iv) growing the cells obtained in (iii) on the above-mentioned support and forming pseudocallus, ie, a differentiated vascular bundle or organ or an amorphous form that does not contain clearly discernable cell division sites. Since cells consist of cells, have low cell adhesion, and have a soft cell mass, they tend to produce large numbers of single cells and small cell masses when subjected to physical shock, and the doubling time of the whole cell on the growth medium is (i ), A cell population having a higher taxane productivity than the callus obtained in (i) and a higher taxane productivity than the callus obtained in (i). Have been.

However, a small cell mass consisting of 1 to 10 cells (cultured cells of Taxus plants usually have a diameter of 20 to 30 cells)
μm, and the diameter of a cell mass consisting of 10 cells is estimated to be less than 100 μm), and it is extremely difficult to obtain a cell mass capable of growing or producing secondary metabolites. As described above, it is impossible to obtain it unless the culture medium and culture conditions are carefully combined. That is, in cultured cells of many Taxus genus plants including cells used by the present inventors, a small cell mass consisting of 1 to 10 healthy cells can be obtained only by increasing the stirring conditions of the liquid culture. Only the remnants of aged cells that do not have the ability to grow or produce secondary metabolites can be fractionated using a 100 μm pore size sieve.

[0009] Therefore, the separation of the small cell mass found in the above-mentioned US patent has a point in producing a callus that easily releases the small cell mass, and the shaking and stirring conditions for releasing the small cell mass are defined. The basic concept and method are different from the present invention. In other words, the shaking and stirring conditions for releasing the small cell mass composed of 1 to 10 healthy cells described in the above-mentioned U.S. Patent are within the conventional range as read from the specification, and are described in the present invention. The method does not exceed the range of conditions used in conventional cell culture as in the method (1).

[0010]

An object of the present invention is to improve the productivity of taxane-type diterpenes by plant cell culture.

[0011]

Means for Solving the Problems The present inventors have conducted intensive studies to solve the above problems, and as a result, when culturing plant cells that produce taxane-type diterpenes, a cell mass having a small particle size was combined with the cultured cells. As a result, it has been found that the productivity of the taxane-type diterpene is improved, and the present invention has been completed.

That is, the present invention provides a method for producing a taxane-type diterpene by culturing the cells of a taxane-type diterpene-producing plant by increasing the proportion of cell mass in a size range suitable for production by the following steps: ) A large cell mass from a cell group using a sieve and / or a filter during preculture for subjecting to culture for the purpose of producing a taxane-type diterpene, either during cell transplantation from the preculture to the next preculture. (B) culturing the cultured cells under a vigorous stirring condition, or both of them; and a method for producing a taxane-type diterpene.

Here, the size range suitable for production means that the average diameter represented by an intermediate value between the major axis and the minor axis is usually 0.12 mm or more.
1.6 mm or less, preferably 0.12 mm or more and 1.0 mm or less.
In addition, it is preferable to increase the proportion of cell clumps in a size range suitable for the production to 65% or more, particularly 80% or more, based on the total fresh weight of the cells. Hereinafter, the present invention will be described in detail.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION Plants producing taxane-type diterpenes used in the production method of the present invention include Taxus genus plants, for example, Taxus baccata LINN,
Yew (T. cuspidata SIEB.et ZUCC), Caraboku (T.
cuspidata SIEB.et ZUCC var.nana REHDER), Thai yew (T. brevifolia NUTT), Canada yew (T.
Cannadensis MARSH, Chinese yew (T. chinensis), Himalayan yew (T. wallichiana ZUCC), T.media and the like. Among them, Yoichi yew and T.media
Is particularly preferred.

[0015] First, a woody plant medium (hereinafter referred to as WP medium) obtained by sterilizing a plant body of a plant belonging to the genus Taxus, for example, a plant piece collected from a root, a growing point, a leaf, a stem, a seed, or the like, is solidified with gellan gum. Abbreviated as a), and allowed to elapse at about 14 to 60 days at 10 to 35 ° C. to allow some of the tissue pieces to callus. When the callus thus obtained is subcultured, the growth rate gradually increases and a stabilized callus is obtained. Here, the term "stabilized callus" refers to a callus having a property that a portion 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. When the stabilized callus is transferred to a liquid medium suitable for growth, for example, a liquid WP medium and grown, the growth rate is further increased (the preculture at this stage is particularly called growth culture).

In the present invention, one or both of the following two steps are carried out in order to increase the cell mass in a size range suitable for the production of a taxane-type diterpene from the stabilized cells. In the first step, the cultured cells are sieved to remove large cell clumps. In this removal operation, a filter may be used instead of the sieve, or both the sieve and the filter may be combined. The pore size of the sieve is not limited as long as the large cell mass can be removed.However, from the viewpoint of the efficiency of improving the productivity of the target substance, it is preferably 1410 μm or less, particularly 840 μm.
The following are more preferred.

Since the cell mass of a cultured cell of a Taxus genus plant is not spherical, a cell mass having a shorter diameter than the above-mentioned pore sizes can pass through the above-mentioned sieves. The average diameter represented by the median of the major and minor diameters of the cell mass that can pass through each sieve is the pore size of the sieve is 1410.
1.6 mm or less for μm, and 840 μm for sieve pore size
1.0 mm or less. The cell mass having an average diameter of 0.12 mm or less that can pass through a sieve having a pore size of 100 μm has a markedly reduced growth ability and taxane-type diterpene production ability. Either one is fine.

The sieving of the cultured cells is performed during the pre-culture.
Alternatively, it is sufficient to transfer the cells from the preculture to the next preculture, but it is preferable to transfer the cells from the preculture to the next preculture from the viewpoint of working efficiency and economy. In addition, from the culture for the purpose of producing taxane-type diterpene,
It is preferable to perform at least one operation of removing large cell masses during the culture and / or at the time of cell transplantation at the start of the culture. In addition, although the operation of sieving is also possible during the cell transplantation from the preculture to the main culture, the cell amount is large in the preculture immediately before the main culture, and there is a high risk of bacterial contamination during the sieving operation. In addition, the large amount of large-diameter cell mass to be discarded has a disadvantage in that wasteful culture costs and waste disposal costs are increased.

[0019] WO95 / 14103 (EP683232) discloses a method of fractionating cells according to particle size at the time of cell transplantation from preculture to main culture, but this operation is intended to increase the precision of experiments. There is a completely different concept and operation from the method used in the present invention. That is, the effect achieved by the present invention, that is, the effect of increasing the proportion of cell clusters in a size range suitable for the production of taxane-type diterpene by sieving, can be achieved without performing the same operation during subsequent cell transplantation. This operation usually lasts from the second generation to about the tenth generation, so there is no need to perform this operation at the time of cell transplantation from preculture to main culture.

When the operation of sieving the cultured cells is performed during the culturing, the cells are once taken out and sieved, or a sieve or a filtration tank is installed as an accessory in the culturing apparatus, and the cells are automatically sized. The cell mass may be removed. Furthermore, when cells are transplanted to a new medium, a stable small cell mass group can be obtained by performing the above-mentioned sieving periodically. Here, the pre-culture is intended mainly for seed cell growth to be supplied to the main culture, and refers to a culture for increasing the culture scale in a stepwise manner according to an increase in the amount of cells. Do 10 times. In addition, the main culture refers to culture for the purpose of finally producing a taxane-type diterpene that is a target substance.

In the second step, the cells are cultured under a strong stirring condition that does not damage individual cells in order to break up the large cell mass to form a small cell mass. Here, the strong stirring conditions mean that the cell mass having an average diameter of 0.12 mm or more and 1.6 mm or less, preferably 0.12 mm or more and 1.0 mm or less is 65% or more, preferably 80% or more of the total fresh weight of the cells. Is a stirring condition that can be pulverized. Such stirring conditions can be obtained by increasing the rotation speed or changing the shape of the stirring blade, or by installing a baffle in the culture tank. The culture under the stirring conditions can be carried out in either main culture or preculture for producing a taxane-type diterpene. During the culture period, the stirring intensity may be increased only during a specific period, or may be increased over the entire period.

In order to obtain the above stirring conditions by increasing the rotation speed of the stirring blade, for example, a paddle type stirring blade having a blade diameter of 1/2 of the inner diameter of the tank and a blade height of 1/4 of the liquid depth is used. In the case of cells having a specific intercellular adhesive strength, the reaction can be performed at 30 to 180 rpm, preferably 40 to 120 rpm. In order to obtain the above stirring conditions by changing the shape of the stirring blade, for example, a perforated paddle type, a screw type, or a turbine type can be used. In order to obtain the above stirring conditions by installing a baffle in the culture tank, it is possible to extend the overhang distance from the inner wall of the culture tank to the baffle tip and / or increase the number of baffles installed. The cell mass thus obtained is subjected to culture (main culture) for the purpose of producing a taxane-type diterpene.

The taxane-type diterpene to be used in the production method of the present invention is not particularly limited as long as it is a diterpene having a taxane skeleton.
-Deacetyltaxol, 7-epitaxol, baccatin III, 10-deacetylbaccatin III, 7-epibaccatin III, cephalomannine, 10-deacetylcephalomanine, 7-episephalomanin, baccatin VI, taxane
1a, xylosylcephalomanine, xylosyltaxol, taxol C, 10-deacetyltaxol C, taxin I, taxin II, taxin I, taxin II, taxa gifin and the like.

As the medium used for the cell culture of the present invention, conventionally known mediums used for cell culture of plants, for example, Murashige Skoog (1962) [Murashige
&Skoog's medium, Rinsmeier Skoog (1965)
[Linsmaier Skoog] Medium, Woody Plant
Medium (1981) [Woody Plant Medium], B-5 medium of Gamborg, M-9 medium of Fujita et al. Plant hormones can be added to these media, and if necessary, carbon sources, inorganic components, vitamins, amino acids and the like can be added.

As the carbon source, disaccharides such as sucrose, maltose, and lactose, monosaccharides such as glucose, fructose, and galactose, starch, and a mixture of two or more of these sugar sources at 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, cobalt and the like. 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, and 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. Are used. As the vitamins, for example, biotin, thiamine (vitamin B ₁ ), pyridoxine (vitamin B ₆ ), pantothenic acid, inositol, nicotinic acid and the like are used. As amino acids, for example, glycine, phenylalanine, leucine, glutamine, cysteine and the like can be added. Generally, each of the above components has an inorganic component of about 0.1 M to 100 m.
M, carbon source about 1 to about 30 g / l, plant hormones about 0.01 to
About 10 M, about 0.1 each for vitamins and amino acids
Used at a concentration of ~ 100 mg / l.

In the cell culture of the present invention, tissue fragments or cells such as roots, growing points, leaves, stems, seeds, pollen, anthers, sepals or the like of the above-mentioned plants, or these are cultured in the above-mentioned medium or other conventional medium. Cultured cells obtained by culturing can be used. The taxane-type diterpene can be separated from the cultured tissue or cultured cells and / or culture medium obtained as described above by extraction with an organic solvent such as methanol. In addition, a taxane-type diterpene can be continuously collected by allowing an appropriate adsorbent or an organic solvent to coexist in the medium.

In a culture for producing a taxane-type diterpene, a jasmonic acid derivative such as methyl jasmonate is used as a production promoter (Japanese Patent Application Laid-Open No. 8-33490).
And compounds containing heavy metals and / or heavy metal ions described in JP-A-8-56680, amines described in JP-A-8-56681,
Cyclic polysaccharides such as cyclodextrin (JP-A-8-1
No. 40690), the productivity of the taxane-type diterpene can be further increased.

As the culturing temperature in the cell culture of the present invention, usually about 10 to about 35 ° C., and particularly preferably 23 to 28 ° C., is preferable because the growth rate is high. In addition, the culture period, 7-42
Days are preferred. When a liquid medium is used in the culture method of the present invention, the cultured cells are separated from the medium by a method such as decantation or filtration after completion of the culture, and the desired taxane-type diterpene is separated from the cultured cells and / or the medium by an organic solvent. Can be separated by extraction or other methods. Further, as a method for enhancing the effects of the present invention, JP-A-7-135567 and JP-A-8-3349
The method disclosed in the specification of Japanese Patent Publication No. 0-90 may be used in combination with a method of dividing cells into a plurality of layers depending on a difference in specific gravity and culturing cells contained in at least one layer.

The present invention also relates to a method for controlling the oxygen concentration in the gas phase in an incubator from the initial stage of cultivation to a level lower than the oxygen concentration in the atmosphere as described in JP-A-8-9983. This method is used in combination with a method of culturing or controlling the dissolved oxygen concentration in a fluid medium in contact with tissues and / or cells under conditions that are lower than the saturated dissolved oxygen concentration at that temperature from the beginning of the culture. This can improve productivity.

[0032]

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to Examples and Comparative Examples, but the scope of the present invention is not limited to these Examples. [Example 1] (Effect of agitation speed during pre-culture on cell mass fractionation by sieve) (1) Pre-culture Aseptically from seeds of Taxus media sterilized with 2% antiformin solution or 70% ethanol solution, etc. Embryos were removed. A part of the solution was added to a concentration of 10 ^-5 M with naphthalene acetic acid, placed on an autoclave-sterilized solid WP medium (0.25% by weight of gellan gum), and cultured at 25 ° C. in a dark place for callus. I got Next, 4 g of this callus (fresh weight) was added to naphthalene acetic acid at the same concentration as above, and autoclaved in a liquid WP medium (hereinafter referred to as a standard medium).
The callus was transferred to a 300 ml Erlenmeyer flask containing 100 ml, and subjected to vortex culture (amplitude: 25 mm, 100 rpm) on a rotary shaker, and subcultured five times every 14 days to increase the growth rate of the callus. The cells thus obtained are hereinafter referred to as standard cells. The obtained standard cells are transferred to a flask under the same conditions as above, and the shaking speed is increased.
14 on the same rotary shaker set at 170 rpm
Cultured for days.

(2) Screening The obtained cells were sequentially fractionated according to the cell mass size using stainless steel sieves having pore diameters of 1410 μm, 840 μm and 500 μm, and the cell fresh weight ratio of each fraction was measured. On the other hand, as a comparison, the obtained standard cells were not cultured at a shaking speed of 170 rpm, but were sieved in the same manner and fractionated sequentially according to the cell mass size, and the cell fresh weight ratio of each fraction was measured. . Table 1 also shows these results. Shaking speed 170
In the case of pre-culture at rpm, fractionation could be performed by sieving so that the ratio of the cell mass having a small average diameter to the total fresh weight of cells increased.

[0034]

[Table 1]

Example 2 Taxol Productivity of Cells Derived from Each Particle Size Fraction 2 g (fresh weight) of each of the four fraction cells obtained in Example 1 was used as a taxane-type diterpene production promoter. 20 ml of standard medium containing 100 μM methyl jasmonate (100 ml
Transfer to an Erlenmeyer flask)
Shaking culture was performed for 14 days on the same type of rotary shaker set at 100 rpm (main culture). After completion of the culture, the cells were collected by filtration and freeze-dried. Measure the dry weight,
The growth weight of the cultured cells per 1 L of the liquid medium was determined. Taxane type diterpene was extracted from the obtained dried callus using methanol or the like, and the taxol yield was measured by quantitatively comparing with a standard product using high performance liquid chromatography. In addition, a taxane-type diterpene was extracted from the culture filtrate using dichloromethane, and quantified in the same manner as described above. The results are shown in Table 2.

[0036]

[Table 2] According to Table 2, it can be seen that the taxol yield increases as the cells are fractionated using a stainless steel sieve having a smaller pore size.

Example 3 (Effect of Shaking Speed During Preculture on Taxol Production) (1) Preculture 4 g of the standard cells described in Example 1 (fresh weight) was added to standard medium 100
ml (with 300 ml Erlenmeyer flask), 25 ℃, dark place,
Shaking culture was performed for 14 days on a rotary shaker of the same type as described above, with the shaking speed set to 170 rpm. (2) Main culture The obtained cells were separated from the liquid medium by filtration, and 2 g (fresh weight) of the cells were transferred to 20 ml of a standard medium containing 100 μM of methyl jasmonate (in a 100 ml Erlenmeyer flask).
Shaking culture was performed for 14 days on a rotary shaker of the same type as described above, which was set at 25 ° C., in a dark place, and at a shaking speed of 100 rpm. The obtained cells and medium were treated by the method described in Example 2, and the yield of dried cells and the yield of taxol were measured. In addition, the shaking speed of the rotary shaker in the preculture was 100 rp.
Except for changing the m and 80 rpm, the same operation as above was performed, and the yield of dried cells and the yield of taxol were measured. Furthermore, the same procedure as above was performed except that the flask used in the preculture was a flask provided with a fold that served as a baffle at the bottom to increase the stirring intensity (FIG. 1), and the yield of dried cells and the yield of taxol were obtained. Was measured. Table 3 shows the above measurement results.

[0038]

[Table 3] According to Table 3, the cells pre-cultured at a higher shaking rate,
It can be seen that the taxol yield increases.

Example 4 (Effect of stirring speed during main culture on taxol production, and the effect of stirring speed on cell mass particle size) Modified paddle-type stirring blade in which stirring speed was set to 40 rpm, 60 rpm and 80 rpm. Transfer 1 liter of standard medium containing 100 μM methyl jasmonate and 100 g of standard cells (fresh weight) to a jar fermenter with
The cells were cultured for 14 days in the dark at 25 ° C. with aeration at a rate of 0.1 vvm. A part of the obtained cells and the medium were treated by the method described in Example 2, and the yield of dried cells and the yield of taxol were measured. The remaining cells were screened using a sieve with a pore size of 840 μm to determine the percentage of small size cells passing through the sieve. Table 4 shows the results.

[0040]

[Table 4] According to Table 4, the more the cells were cultured at a higher stirring speed,
It can be seen that the taxol yield increases. In addition, the ratio of the small-diameter cells to the total fresh weight of the cells increases in the cells cultured at the higher stirring speed.

[Example 5] (Effect of cell mass fractionation by sieve on taxol production) From the standard cells described in Example 1, the pore diameter was 1410 µm or 84.
After separating the cell mass passing through a 0 μm stainless steel sieve, the cell mass was separated from the liquid medium by filtration, and 4 g of
(Fresh weight) was transferred to a standard medium (100 ml, 300 ml Erlenmeyer flask), and cultured with shaking on a rotary shaker of the same type as described above at 25 ° C., in a dark place and at a shaking speed of 100 rpm. The total amount of the cells obtained was separated from the liquid medium by filtration as it was, and 2 g of the cells were transferred to 20 ml of a standard medium containing 100 μM methyl jasmonate (in a 100 ml Erlenmeyer flask) at 25 ° C., in a dark place, with shaking speed. Was shake-cultured for 14 days on a rotary shaker of the same type as set above at 100 rpm. The obtained cells and medium were treated by the method described in Example 2, and the yield of dried cells and the yield of taxol were measured. Table 5 shows the results. Further, the same treatment as described above was conducted except that the large cell mass was not removed using a sieve, and the yield of dried cells and the yield of taxol were measured. Table 5 also shows the results.

[0042]

[Table 5] According to Table 5, it can be seen that the taxol yield increases as the cells are fractionated using a stainless steel sieve having a smaller pore size during cell transplantation in preculture.

[Example 6] [Particle size distribution of cells by sieving with a pore size of 500 µm or less, and taxol productivity of the obtained fraction] When sieving the standard cells described in Example 1,
The same operation was performed except that fractionation was performed using stainless steel sieves having pore sizes of 500 μm, 250 μm, and 100 μm, and the fresh cell weight ratio of each fraction was measured. Table 6 shows the results.

[0044]

[Table 6]

Microscopic observation of the cells passing through a sieve having a pore size of 100 μm revealed a small amount of cell clusters consisting of 10 or less cells regardless of the shaking speed. Or the remnants of it. These cells were cultured again according to Example 2, but no new cell growth and no production of taxane-type diterpenes were observed. Therefore, even if the fraction separated using a sieve having a pore diameter of 500 μm, which was confirmed to improve the productivity of taxol in Example 2, the fraction passing through the sieve having a pore diameter of 100 μm is the same as the fraction of the taxane-type diterpene of the present invention. Not suitable for production.

[0046]

According to the present invention, a large amount of taxane type diterpene can be easily obtained by cell culture of a taxane type diterpene producing plant.

[Brief description of the drawings]

FIG. 1 shows a view of a flask with a fold that serves as a baffle at the bottom.

Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12R 1:91)

Claims (9)

[Claims] 1. In producing a taxane-type diterpene by culturing cells of a taxane-type diterpene-producing plant, the following steps for increasing the proportion of cell mass in a size range suitable for production: (a) taxane-type diterpene Operation of removing large cell clumps from a cell group using a sieve and / or a filter during preculture for use in culture for the purpose of production of cells, either at the time of cell transplantation from the preculture to the next preculture. At least once; (b) culturing the cultured cells under vigorous stirring conditions; or a method of producing a taxane-type diterpene. 2. The method according to claim 1, wherein the cell mass suitable for the production is a cell mass having an average diameter represented by an intermediate value between a major axis and a minor axis of 0.12 mm or more and 1.6 mm or less. 3. The method according to claim 1, wherein the cell mass suitable for the production is a cell mass having an average diameter represented by an intermediate value of a major axis and a minor axis of 0.12 mm or more and 1.0 mm or less. 4. The method according to claim 1, wherein the ratio of the cell mass in a size range suitable for the production to the total fresh weight of the cells is increased to 65% or more. 5. The method according to claim 1, wherein the ratio of the cell mass in a size range suitable for the production to the total fresh weight of the cells is increased to 80% or more. 6. The method according to claim 6, wherein the operation of the step (a) is performed at least during the past 2 to 10 cultures and / or at the time of cell transplantation at the start of the culture, starting from the culture for producing the taxane-type diterpene. 2. The device according to claim 1, wherein the device is provided once.
The described method. 7. A taxane-type diterpene comprising: taxol,
10-deacetyltaxol, 7-epitaxol, baccatin III, 10-deacetylbaccatin III, 7-epibaccatin III, cephalomanine, 10-deacetylcephalomanine, 7-episephalomanin, baccatin VI, taxane 1a, xylosyl The method according to any one of claims 1 to 6, wherein the method is at least one selected from cephalomannine, xylosyltaxol, taxol C, 10-deacetyltaxol C, taxisin I, taxisin II, taxin I, taxin II, and taxaxifin. 8. The method according to claim 1, wherein the plant producing the taxane-type diterpene is a Taxus genus plant. 9. The method according to claim 8, wherein the Taxus plant is Taxus media or Taxus baccata.