JPS6339596A - Production of alkaloid - Google Patents

Abstract

PURPOSE:To speed up the growth of hair roots and increase the production of alkaloid thereof, by transforming solanaceous plant cells with a special plasmid and cyultivating the generated roots in a culture medium having a glycine concentration below a specific value. CONSTITUTION:When cells of solanaceous plant (preferably the genus Atropa, Datura, Hyoscyamus or Scopolia) is transformed by an Ri plasmid held by Agrobacterium rhizogenes and the generated roots are cultivated, the glycine concentration in a culture medium is kept at <=20 mumol, preferably <=10 mumol.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention is directed to the production of alkaloids, especially tropane alkaloids, which are physiologically active substances and medicinal ingredients biosynthesized by plants of the Solanaceae family, by culturing the hairy roots of plants of the Solanaceae family. The present invention relates to a method for efficiently manufacturing.

[Conventional technology]

As an industrial production method for plant secondary metabolites using plant cell/tissue culture methods, the hair root disease fungus Agrobacterium spp.
Rhizogenes (Agrobacterium rhizo)
A method is known in which the hairy roots that have grown are cultured by inoculating the hairy roots. This method is based on the following principle. In other words, when Agrobacterium rhizogenes is inoculated into the stems, leaves, roots, etc. of a plant, roots called hairy roots develop from the infected area. These roots are generated by integrating part of the genes of a giant plasmid (Ri plasmid) present in Rhizogenes into the plant's genes, and they grow much faster than normal roots and produce secondary metabolites. It is known that the amount is the same or higher.

Therefore, this method takes advantage of the above properties to inoculate plants containing useful substances in their roots with Agrobacterium rhizogenes, cut out the hairy roots that have developed, culture them in a device such as a tank, and grow the hairs. This is a method to extract useful substances by destroying the roots.

However, according to the method using this hair root disease fungus, the cells are destroyed and the target substance accumulated in the cells is extracted, so separating the cell components and the target substance is complicated.
This method has the drawback of increasing costs, and because it is difficult to take out the growing plant organs continuously, it can only be produced by a batch method.

Therefore, the present inventors developed a production method that does not have the above problems by inoculating a specific plant with the hairy root disease fungus, culturing the hairy roots that have grown, and extracting the alkaloids secreted by the hairy roots into the medium. He developed a method to do this and filed a patent application as Japanese Patent Application No. 47046/1983.

However, when hairy roots are cultured in a normal medium, a problem arises in that the growth rate of the hairy roots and the alkaloid content of the hairy roots are still insufficient.

[Problem that the invention seeks to solve]

Therefore, an object of the present invention is to develop a medium formulation suitable for the growth of hairy roots, to accelerate the growth rate of hairy roots, and to provide a method with high production efficiency of alkaloids.

[Means for solving problems]

Conventional media contain quite a lot of glycine, but
The present invention was made based on the knowledge that the above-mentioned problems can be effectively solved by using a liquid medium in which the concentration of glycine is 20 μmol or less.

That is, the present invention involves transforming Solanaceae plant cells with the R1 plasmid held by Agrobacterium rhizogenes, culturing the resulting hairy roots in a liquid medium with a glycine concentration of 0 to 20 μmol, Provided is a method for producing alkaloids, which comprises extracting alkaloids produced by hairy roots.

The target of the treatment in the present invention is a Solanaceae plant,
In the present invention, it is particularly important to limit the processing target in this manner. That is, the hairy roots of plants of the Solanaceae family secrete alkaloids into the medium. In the present invention, any plant of the Solanaceae family can be used, including plants of the genus Atropa,
It is preferable to use plants selected from the group of plants of the genus Datura, plants of the genus Hyostiamus, and plants of the genus Scoboria. Among these, plants of the genus Datura are particularly preferred, and specifically, plants of the genus Datura are preferred.
a! J), Datura japonicus (D
, inermisJ), D. stramonium (D., metaloidesDC), D. stramonium.
L), D. tatul
a L), Datura (D, alba N),
Kodachi Datura (D, arborea L)
, Kidachi Datura (D, 5uaveol)
ens H) is exemplified, with E. ens H) being particularly preferred.

Agrobacterium rhizogenes that can be used to make these plants produce hairy roots include Agrobacterium rhizogenes 25818 Agrobacterium rhizogenes 15834 Agrobaterium rhizogenes 8196 Agrobacterium rhizogenes
Examples include Rhizogenes 84. Also, in other cases such as E. coli, the R1 plasmid or a part of it is T-DN.
Kan, which introduces A to Shodenshi, can also be used.

When plants are treated with Agrobacterium rhizogenes according to the present invention, a portion of the R1 plasmid (T-DNA) in the Rhizogenes bacteria is introduced into the nuclear DNA of the plant cells (
transformation).

Ri plasmid T- was applied to the stems, roots, leaves, etc. of the Solanaceae plants.
Examples of methods for obtaining transformed hairy roots by introducing DNA include the following methods.

1. Direct inoculation method to individual plants 2. Leaf disc method using leaf discs (L, Comai
etat,, Nature, 317.741 (
1985)) 3. Co-culture method using plant protoplasts (ZoM, Wei et al., Plan
t Ce1l Rep,, 1npress (198
6)) 4. Plant protoplasts and Agrobacterium
Rhizogenes spheroplast method (R.

Hahn et al., Plant Cell R
ep,, Yu, 605, A method of directly injecting the R1 plasmid of Agrobacterium rhizogenes or a part of the T-DNA into cells by a method such as microinjection.The Ri plasmid was introduced using methods 1 to 4 above. In case,
After eating, it is necessary to sterilize Agrobacterium rhizogenes, and the following methods are available.

○ High temperature treatment (40°C) ○ Antibiotic treatment O Transplanting hairy root tips in a fast cycle The following methods are effective for culturing hairy roots obtained by the above method.

In the present invention, the above-mentioned hairy roots are prepared at a glycine concentration of 0 to 0.
It is characterized by culturing in a liquid medium of 20 μmol, preferably 0 to 10 μmol.

In the present invention, for example, a medium conventionally used for tissue culture of plants, that is, an inorganic component and a carbon source are essential components, and at least one or more components selected from plant hormones, vitamins, and amino acids. In a culture medium to which the above-mentioned specific elements are added and other components are added as necessary, the above-mentioned specific elements can be used in specific amounts.

The inorganic components in the above medium include nitrogen, shoulder, potassium,
Calcium, magnesium, sulfur, manganese, zinc,
These include boron, molybdenum, salts, sodium, iodine, cobalt, etc. Specifically, potassium nitrate, sodium nitrate, calcium nitrate, phosphoric acid, etc. Potassium, disodium phosphate, potassium chloride, calcium chloride, magnesium sulfate, sulphate) IJium, ferrous sulfate, ferric sulfate, manganese sulfate, zinc sulfate, boric acid, sodium molybdate, molybdenum dioxide, iodide Examples include potassium and cobalt chloride.

Examples of carbon sources include hydrocarbons such as sucrose, derivatives thereof, organic acids such as fatty acids, and primary alcohols such as ethanol.

Plant hormones include auxins such as indoleacetic acid (IAΔ), naphthaleneacetic acid (NAA), I)-chlorophenoxyisoacetic acid, and 2,4-dichlorophenoxyacetic acid (2,4-D), kinetin, zeatin, and dihydroacetic acid. Cytokinins such as zeatin are exemplified.

Vitamins include biotin, thiamin (vitamin Bl)
Examples include pyridoxine (vitamin B8), pantothenic acid, ascorbic acid (vitamin C), inositol, and nicotinic acid.

Examples of amino acids include glycine, alanine, glutamine, and cysteine.

The concentrations of components other than glycine in the liquid medium can be varied within a wide range. Usually, the inorganic component is about 0.1μM
~about 100mM, carbon source about 1g71-120g/
In addition, the amount of plant hormones can be about 0.01 μM to about 10 μM, and the amount of vitamins and amino acids can be about 0.1 mg/42 to about 100 mg/j2, respectively.

In the present invention, the initial concentration of hairy roots in the liquid medium can be varied within a wide range. Usually, it is desirable to add about 10 mg to about 1 g (fresh weight) of hairy roots per 50 mJ of liquid doubling.

In culturing the hairy roots of the present invention, light is not necessarily necessary, and culturing in the dark is preferable for the biosynthesis of alkaloids such as scopolamine, and the culture temperature is about 0°C to about 35°C, especially about 35°C. C. to about 28.degree. C. is preferred. In other words, below about 10°C, the growth rate of hairy roots is low, and about 35°C.
This is because the growth rate of hairy roots similarly decreases even if the temperature exceeds ℃.

In the present invention, alkaloids can be extracted from the hairy roots cultured as described above by various methods, but it is preferable to extract alkaloids secreted by the hairy roots into the liquid medium.

〔Effect of the invention〕

According to the present invention, since alkaloids can be efficiently produced from Solanaceae plant cells, the method of the present invention is extremely suitable as an industrial method for producing alkaloids.

Next, the present invention will be explained with reference to Examples.

〔Example〕

Example 1 Datura lnnoxia
After sterilizing the seeds of M) with a disinfectant such as a sodium hypochlorite solution, they were sown on a Murashige-Skoog (abbreviated as MS) solid medium, and the Ri plasmid was injected into the stems and leaves of the germinated sterile plants. Agrobacterium rhizogenes (15834) containing Agrobacterium rhizogenes was inoculated.

After 2 to 5 weeks, the hairy roots developed from the inoculation site were cut out and transplanted onto MS solid medium containing carbenicillin Ig/fl, and after 1 to 2 weeks, they were transplanted to a new medium with the same composition.

This operation was repeated 2 to 3 times to obtain sterilized hairy roots.

50 ml of a liquid medium having the composition shown in Table 1 was placed in a 100 mN Erlenmeyer flask and sterilized at 120°C for 15 minutes. 100 mg of the hairy roots described above was added to this liquid medium, and the culture was incubated at 25° C. for 30 days with shaking # (swivel rotation speed: 100 times/min, amplitude: 30 mm).

After culturing, the hairy roots of Aspergillus chinensis were collected by filtration, weighed, and freeze-dried. After drying, it was weighed and ground into powder in a mortar. Next, the powder was extracted with a mixture of chloroform:methanol:ammonia=15:5:1 and filtered to obtain an extract. This was extracted with chloroform using acidic sulfuric acid (pH 2), the aqueous layer was separated, and then the mixture was made alkaline with ammonia (pH 10) and extracted with chloroform. After dehydrating the chloroform with sodium sulfate, it was evaporated to dryness to obtain an alkaloid fraction.

In addition, after measuring the weight of the culture medium, the sulfuric acid acid (ptl)
2) and then extracted using the same method as above.

Scopolamine and atropine in the alkaloid fraction were quantitatively determined by gas chromatography.

The column is ○V -17, (2mX 3mm), the column temperature is 235℃, the carrier gas is nitrogen, and the flow rate is 50ml.
/min detector was used FID.

Both amounts of scopolamine were added to determine the total amount of scopolamine produced per flask. In addition, the amount of atropin produced was determined in the same manner.

The phosphate ion concentration in the medium and the results obtained are summarized in Table 2.

Table 1 As is clear from Table 2, the conventionally used MS liquid medium (however, sucrose at 30 g
In the comparative example in which the culture was carried out with (including β), the amount of total scopolamine produced was extremely small, but it can be seen that according to the present invention, a large amount of scopolamine can be produced. Furthermore, it can be seen that according to the present invention, a large amount of atropine is produced.

Claims (2)

[Claims] (1) Solanaceous plant cells are transformed with the Ri plasmid held by Agrobacterium rhizogenes, and the resulting hairy roots are cultured in a liquid medium with a glycine concentration of 0 to 20 μmol. A method for producing an alkaloid, which comprises extracting an alkaloid produced by. (2) The production method according to claim (1), wherein the Solanaceae plant is selected from the group of plants of the genus Atropa, plants of the genus Darra, plants of the genus Hyostiamus, and plants of the genus Scopolia.