JPH01300890A - Cell capable of highly producing tropane-based alkaloid and production of said cell - Google Patents

Abstract

NEW MATERIAL:The title cell of tropane-based alkaloid-productive plant origin, tolerant of phenylalanine or its analogues. USE:Scopolamine as an anticonvulsant or analgesic and hyostiaine as aparasympatholytic drug. PREPARATION:For example, from the tissues or cells of a tropane-based alkaloid-productive plant (e.g., Duboisia myoporoides), cells tolerant of phenylalanine or its analogues are produced using a phenylalanine- or its analogue-contg. culture medium, and from the resultant tolerant cells, the title cells are selected.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to cells suitable for producing tropane-based alkaloids, such as scopolamine and hyoscyamine, with high production capacity, and a method for obtaining the same.

[Conventional technology]

Scopolamine is used as an antispasmodic, analgesic, and parasympathetic nerve blocker, and hyoscyamine is used as a parasympathetic nerve blocker, each of which is used as a medicine. These compounds are manufactured by extracting them from natural plants, but because they are made from natural materials, there are problems such as their production being affected by the weather and the harvesting period being limited. It has become. Therefore, many studies have been conducted both domestically and internationally to produce these compounds through plant tissue culture.

Regarding production by callus, there is a known example of production by Yamada et al. using henbane callus (Plant Ce1lR
eports, 101-103 (1982)), the scopolamine content was 20 ppm, which was low compared to the content in natural plants. Furthermore, Yamada et al.
We found that significant amounts of scopolamine and hyoscyamine were present in adventitious roots obtained by tissue culture of F. Muell (Plant Ce1l Re).
ports 3.186-188 (1984)), but the amount was still not sufficient. Therefore, we investigated various culture conditions for Zboisia adventitious roots, and decided to increase the ratio of ammonium ions to nitrate ions in the medium to 0.2 or more, and to adjust the dissolved oxygen concentration in the medium to 10 to 65 ppm.
The present applicant has discovered that the productivity of tropane alkaloids can be improved by
Patent applications have been filed as No. 2-6675 and JP-A No. 62-6674, but from an industrial standpoint, it is desired to further improve the productivity.

[Problems to be Solved by the Invention] As described above, when aiming at industrial production of tropane-based alkaloids by tissue culture, it has been an important issue to further increase productivity.

An object of the present invention is to provide a highly tropane-based alkaloid-producing cell suitable for producing tropane-based alkaloids and a method for efficiently selecting and obtaining the same.

[Means to solve the problem]

In general, it is known that among the cells that exhibit resistance to amino acid analogs in plant tissue culture, there are cells that have a high amino acid content.

For example, J. M., Widholm et al. found that carrot cell cultures resistant to ethionine, a compound similar to methionine, contained 10 times more methionine than normal cells. [Can, J., Botany, 54 P1
523 (1976)]. Regarding the occurrence of overproduction of amino acids in such amino acid-resistant strains, see "Mutation and Selection of Plant Cultured Cells" edited by Yasuyuki Yamada (Kodansha Scientific (Biotechnology Series); P3
7 to P38) explain that this is due to an increase in intracellular amino acid concentration in order to antagonize the inhibitory effect of amino acid metabolism by amino acid analogs.

Therefore, the present inventors focused on this phenomenon, and researched a method for efficiently selecting tropane-based alkaloid-producing cells from tissues and cells such as adventitious roots of plants that produce tropane-based alkaloids, and found the following facts. I found out.

That is, by treating tissues or cells with phenylalanine, which is a precursor of tropane-based alkaloids, or its analogs (analog substances), phenylalanine-resistant strains or phenylalanine analog-resistant strains are obtained, and a high proportion of the resistant strains are obtained. They discovered that there are strains that produce tropane-based alkaloids in high amounts, leading to the completion of the present invention.

According to the present invention, tropane alkaloid-producing cells derived from plants producing tropane alkaloids and exhibiting resistance to phenylalanine or phenylalanine analogues,
A strain that is resistant to phenylalanine or phenylalanine analogs is created by using a culture medium containing phenylalanine or phenylalanine analogs from tissues or cells of plants that produce tropane alkaloids, and from the resulting resistant cells, high levels of tropane alkaloids can be obtained. Provided is a method for obtaining cells that are highly productive of tropane-based alkaloids, which is characterized by selecting producing cells.

Specifically, the plant producing the tropane alkaloid used in the present invention is Zboisia myoboroides (Du
boisia myoporoides), Duboisia Ieich 4 (Duboisia Ieich)
ardtii) and other plants of the genus Zboisia, Datura tatula.

Datura arborea
, Datura stramonium
plants of the genus Datura such as onium), plants of the genus Kobolia such as Scopolia japonica (order Scopoles:
mus niger) and Atropa belladonna (Atropa belladonna).
) and other plants of the genus Atropa can be exemplified.

In order to obtain the tropane-based alkaloid highly productive cells of the present invention, first, the cultured tissue of the above plant is cultured in a medium containing phenylalanine or its analogs for a certain period of time or more, and strains exhibiting resistance to phenylalanine or its analogs are obtained. It is essential to create. This will be explained in detail below.

A medium for producing a strain resistant to phenylalanine or its analogs is a medium containing phenylalanine or its analogs as an essential component.

Phenylalanine analogs that can be used in the present invention include:
General formula % Formula % (1) [In the formula, R1 represents a lower alkyl group or a halogen atom, R2 is H or R-C- (R is H or a lower alkyl group)
and R3 is 011. NHZ or OM (M represents a metal ion), and n represents an integer of 1 to 3. ]
Examples include compounds represented by 4-methyl-
Alkylphenylalanine such as phenylalanine, 4-
Examples include halogenated phenylalanines such as chlorophenylalanine and 4-fluorophenylalanine, and amide derivatives such as 4-fluorophenylalanine amide. In the present invention, the carboxyl group in these compounds may form a salt with the same metal ion as the below-mentioned inorganic component constituting the medium, and this salt can also be used as a medium component in the present invention. Furthermore, β-2-thienylalanine and the like can also be used as phenylalanine analogues. Other medium components other than the above-mentioned phenylalanine or its analogs include inorganic components and carbon sources as essential components, to which are added plant hormones and vitamins, and further, amino acids are added as necessary.

In the present invention, the ratio of the above-mentioned phenylalanine or its analogs used in the medium is preferably in the range of 10-'M to 10-1, and within this range, 10-'M to 10-2
A range of 1 is particularly preferred. If the concentration of phenylalanine or its analogs in the medium is higher than this range, all cells will die, making it impossible to obtain a phenylalanine or its analogs-resistant strain. In addition, if the concentration is lower than this range, cells that are not resistant to phenylalanine or its analogs will also grow, making it impossible to obtain a phenylalanine analog-resistant strain.

When obtaining a strain resistant to phenylalanine or its analogues, it is desirable to carry out culture for two or more generations in a medium containing phenylalanine or its analogues, with each generation usually lasting one month.

If the period is shorter than that, the cells cannot acquire resistance to phenylalanine or its analogs.

Regarding the medium components other than the above-mentioned phenylalanine or its analogs in the medium used in the present invention, the inorganic components include nitrogen, phosphorus, potassium, sodium, calcium, magnesium, sulfur, iron, manganese, zinc,
Examples include inorganic salts containing elements such as boron, molybdenum, chlorine, iodine, and cobalt; specific examples include potassium nitrate, sodium nitrate, ammonium nitrate, ammonium chloride, potassium chloride, calcium chloride, potassium monohydrogen phosphate, and phosphoric acid. potassium dihydrogen, magnesium sulfate,
Examples include compounds such as magnesium chloride, sodium sulfate, ferrous sulfate, ferric sulfate, manganese sulfate, copper sulfate, sodium molybdate, molybdenum trioxide, potassium iodide, zinc sulfate, boric acid, and cobalt chloride.

Examples of carbon sources for the medium include carbohydrates such as sucrose and derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

Examples of plant hormones in the medium include naphthalene acetic acid (NAA), indole acetic acid (IAA), p-
Examples include auxins such as chlorophenoxyacetic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), indolebutyric acid (IBA), and derivatives thereof, and cytokinins such as benzyladenine (BA), kinetin, and zeatin. In the present invention, it is usually preferable not to add cytokinins to the culture medium, but when added as necessary, the concentration of cytokinins is usually 10-7M.
It is preferable to use it at a low concentration of (0,02 mg/l) or less.

The vitamins in the medium include biotin, thiamine (
Examples include vitamin B1), pyridoxine (vitamin B6), pyridoxal, pyridoxamine, calcium pantothenate, ascorbic acid (vitamin C), inositol, nicotinic acid, nicotinamide, and riboflavin (vitamin BZ).

Examples of amino acids in the medium include glycine, alanine, glutamic acid, and lysine.

The medium of the present invention usually contains about 0.1 of the inorganic components.
μ to about 100 mM, and about 1 g/2 to about 100 g/41 of the carbon source! , the above-mentioned plant hormones at a concentration of about 0.
01 μH to about 100 μH, and the vitamins
1■/I! , or about 150mg/jl! It is desirable that the amino acids be used in an amount of 0 to about 100 mg/l.

Specifically, the medium of the present invention includes a conventionally known medium used for plant tissue culture, such as Murashige & Skoog ('62)
Skoog] culture medium, Linsmeyer-Skoog (
RM4965) (Linsmaier & Sko
og) medium, white ('63) (White)
medium, Gam-borg “J’s B-5
Medium, Mitsui M-9 medium, Niche Niche (Nitsc)
An example is a medium prepared by adding phenylalanine or a derivative thereof, the above-mentioned carbon source, and a plant hormone, and further adding the above-mentioned vitamins, amino acids, etc. as necessary, to a medium etc. of [hN1Lsch], but the present invention Among these, media prepared using H-H, Linsmeyer-Skoog, or Murashige-Skoog are particularly preferred. The composition of the conventionally known culture medium described above is described, for example, in "New Plant Tissue Culture JP 3B6-P391" by Yukinai, Nakajima, and Furuya, Asakura Shoten, 1979.

The medium that can be used in the present invention is a liquid medium or a solid medium containing 0.5 to 1% of agar, gelatin, etc., but a solid medium may be used to create a strain resistant to phenylalanine or its analogues. is preferable, and it is also preferable to use a liquid medium that does not contain phenylalanine or its analogs when culturing the resistant strain for the production of tropane-based alkaloids and for culturing for maintaining the resistant strain.

In the present invention, the above-mentioned plant tissues or cells used for producing phenylalanine or analogue-resistant cells include tissue pieces or cells such as roots, leaves, stems, seeds, and flower buds of the plants. Other examples include cultured cells or cultured tissues of the plants obtained by conventional tissue culture methods. In the present invention, among these, it is particularly preferable to use adventitious roots obtained by previously tissue culturing plant tissues and tissue culturing them using the medium according to the present invention. Furthermore, cultured cells or cultured tissues obtained by subculturing callus derived from the tissue pieces or cells described above can also be used.

In the present invention, when using adventitious roots, plant tissue pieces are, for example, hairy root disease bacteria (e.g. Agrobacterium rh).
izogenes) and the resulting hair roots can be used (for example, the method proposed in Japanese Patent Application No. 61-89975 filed by the present applicant can also be used). By creating cells that are resistant to substances, measuring the amount of tropane alkaloids produced by the resulting resistant cells, and selecting cells that produce a high amount of tropane alkaloids, cells with high tropane alkaloid and roid production levels are obtained. can be obtained.

By tissue culturing the tropane alkaloid-producing cells according to the present invention in a medium, a culture containing a large amount of tropane alkaloids can be obtained.

The medium used is preferably a niche medium containing IBA 10-'M or a Linsmeyer-Skoog medium.

Specific examples of the tropane alkaloid obtained by the method of the present invention include scopolamine, hyoscyamine, and acetyl compounds of these compounds, and among these, scopolamine and hyoscyamine are preferred.

In the present invention, the method for separating tropane-based alkaloids from cultured cells containing them is, for example, the method used to isolate and purify tropane-based alkaloids from plants containing tropane-based alkaloids, as described in the Pharmacopoeia Act. The usual methods that have been used can be adopted.

〔Example〕

Hereinafter, the method of the present invention will be explained in more detail with reference to Examples.

Example 1 Duboisia mypor grown in our herb garden
otdesR,Br leaves were washed, immersed in 10% antiformin solution for 10 minutes, then washed three times with sterile water, cut into approximately 1 cm pieces, and treated with naphthaleneacetic acid and benzyladenine at 10-'M and 10-'M, respectively. The cells are placed on a Linsmeyer-Skoog agar medium supplemented with hM and cultured at 25°C for 30 days. Adventitious roots that appeared at the same time as callus formation were cut out, and indolebutyric acid was added to 10-
The cells were transplanted into an Etch Niche liquid medium supplemented with a concentration of 'H, and subcultured for 2 years.

1 g (dry weight) of the thus obtained indeterminate m20IT was transplanted into a 9 cm diameter plastic petri dish containing 20-' H.E.C. agar medium supplemented with 0.3 mM parafluorophenylalanine and 10-'M indolebutyric acid. After one month, the growing roots were transplanted to a niche agar medium containing rose fluorophenylalanine and indolebutyric acid at 0.3mM and 10-'M, respectively, and incubated on the same medium for another month. Adventitious roots resistant to parafluorophenylalanine were obtained by culturing. 10 cm (dry weight) of these adventitious roots were transplanted into a 100-volume Erlenmeyer flask containing 20 m/ml of Etch Niche's liquid medium supplemented with indolebutyric acid at a concentration of 10-5M, and cultured for 3 weeks. It was subcultured several times. The obtained adventitious roots were dried and the productivity of tropane alkaloids was measured.

Analysis of tropane alkaloids was conducted as follows.

First, the dried adventitious roots were extracted with 50ml of a basic chloroform-methanol solution. 40 ml of IN sulfuric acid was added to this and the alkaloid layer was transferred to the sulfuric acid layer. Further, 2 m/ml of ammonia water and 40 rnl of chloroform were added to transfer the alkaloids to the chloroform layer, which was concentrated under reduced pressure and the amount of alkaloids was measured using a gas chromatograph.

The distribution of alkaloid production of the phenylalanine analog resistant strains obtained at this time is shown in Figure 1. Note that the gas chromatograph analysis was conducted under the following conditions.

Column: 5ilicone 0V-17 (1%) o
nChromosorb W (Mesh 80-10
0) 3 cylinders φ x 1 m glass column Carrier gas: N2 Column temperature: From 200°, the production amount of tropane alkaloids is 70 μ/1
The above cells were selected as cells with high tropane alkaloid production.

Comparative Example 1 The same procedure as in Example 1 was used except that a medium to which parafluorophenylalanine was not added was used, and the distribution of the alkaloid production amount is shown in FIG.

As is clear from FIGS. 1 and 2, a high proportion of tropane-based alkaloid-producing strains exist among the parafluorophenylalanine-resistant strains.

〔Effect of the invention〕

According to the present invention, novel tropane-based alkaloid-producing cells can be provided, and tropane-based alkaloid-rich producing cells can be efficiently obtained.If the tropane-based alkaloid-producing cells of the present invention are used, Among alkaloids, productivity of scopolamine and hyoscyamine can be particularly increased.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing the distribution of alkaloid production of the phenylalanine analog resistant strain obtained in Example 1, and FIG. 2 is a diagram showing the distribution of alkaloid production of the strain obtained in Comparative Example 1.

Claims (6)

[Claims] (1) Tropane-based alkaloid-producing cells that are derived from plants that produce tropane-based alkaloids and exhibit resistance to phenylalanine or phenylalanine analogs. (2) The cell according to claim 1, wherein the plant that produces the tropane alkaloid is Zboisia myoporoides. (3) Generate cells that are resistant to phenylalanine or phenylalanine analogs from tissue or cells of plants that produce tropane alkaloids using a medium containing phenylalanine or phenylalanine analogs, and use the resulting resistant cells to generate tropane-based alkaloids. A method for obtaining tropane-based alkaloid-high producing cells, which comprises selecting cells with high alkaloid production. (4) Claim 3, characterized in that the plant producing tropane-based alkaloids is Zboisia myopoloides.
Acquisition method described. (5) The method according to claim 3 or 4, wherein the phenylalanine analogue is parafluorophenylalanine. (6) The method according to claim 3 or 4, wherein the phenylalanine analogue is thienylalanine.