JP2008278759A - Synthesis method of cis-isoprenoids using plant tissue culture - Google Patents

Abstract

The present invention provides a novel process for producing high-molecular cis-isoprenoids similar to natural rubber having 40 or more carbon atoms.
A plant such as Indian rubber tree in the presence of a low-molecular-weight isoprenoid compound having 20 or less carbon atoms such as farnesyl diphosphate (15 carbon atoms) and isopentenyl diphosphate (5 carbon atoms). A method for producing a polymer cis-isoprenoid having 40 or more carbon atoms, such as ficaprenol C55 and ficaprenol C60, wherein the tissue culture is performed.
[Selection figure] None

Description

  The present invention relates to a method for synthesizing macromolecular cis-isoprenoids similar to natural rubber from low-molecular-weight isoprenoid compounds using plant tissue culture.

  An isoprenoid compound is a substance that is repeatedly polymerized with a 5 carbon isoprene unit as a basic skeleton. Isoprenol (5 carbon atoms), followed by geraniol (10 carbon atoms), a polymer containing isoprenol as a monomer. ), Farnesol (15 carbon atoms) and geranylgeraniol (20 carbon atoms), as well as other high molecular weight compounds that are promising as pharmaceutically active substances due to their physiological activity. ing.

  A method using a microbial system is known as a method of obtaining a higher molecular weight isoprenoid compound by polymerizing these compounds, using isoprenoid compounds as starting materials, but in plant tissue culture systems, isoprenoid compounds are Since it is difficult to be taken up into plant cells, it has been considered difficult to produce isoprenoid compounds using a tissue culture system.

  However, farinsol (FOH: 15 carbon atoms) from geranyl diphosphate (GPP: 10 carbon atoms) and isopentenyl diphosphate (IPP: 5 carbon atoms) using tissue culture of kuriaji pumpkin ), Geranylgeraniol (GGOH: 20 carbon atoms) has been confirmed to be produced, and it is known that trans-isoprenoid compounds are produced by plant tissue culture systems (Japanese Patent Application 2006- 042177).

  It is known that natural rubber is collected from Indian rubber tree. Natural rubber is a long-chain isoprenoid compound in which isoprene units having 5 carbon atoms are polymerized in cis form. The present invention provides a novel method for synthesizing macromolecular cis-isoprenoids from low-molecular-weight isoprenoid compounds using plant tissue culture.

As a result of various studies to solve the above problems, the present inventors have found that high molecular cis-isoprenoids can be synthesized from low-molecular isoprenoid compounds by tissue culture of plants such as rubber tree. completed.
Accordingly, the present invention provides a method for producing a polymer cis-isoprenoid having 40 or more carbon atoms, wherein tissue culture of a plant is performed in the presence of a low-molecular isoprenoid compound having 20 or less carbon atoms. To do.

  The plant is preferably rubber tree, for example Indian rubber tree. The low molecular weight isoprenoid compound is, for example, farnesyl diphosphate (15 carbon atoms), isopentenyl diphosphate (5 carbon atoms), or both. The molecular cis-isoprenoids are, for example, ficaprenol C55 (carbon atoms 55), ficaprenol C60 (carbon atoms 60), or both.

  The present invention also provides a method for producing ficaprenol C55 and ficaprenol C60, which comprises conducting tissue culture of rubber tree in the presence of farnesyl diphosphate and isopentenyl diphosphate. In this method, the rubber tree is, for example, Indian rubber tree.

Plant In the present invention, the plant for tissue culture is not particularly limited as long as it can synthesize high molecular cis-type isoprenoids from low-molecular isoprenoid compounds by tissue culture, such as rubber tree, especially Indian rubber tree.

Low molecular isoprenoid compound The low molecular isoprenoid compound used in the method of the present invention includes an isoprenoid compound having 20 or less carbon atoms, such as dimethylallyl diphosphate (5 carbon atoms), geranyl diphosphate (carbon atoms). 10), farnesyl diphosphate (15 carbon atoms) and geranylgeranyl diphosphate (20 carbon atoms). These may be used alone or in combination of two or more.

Polymeric cis-isoprenoids Polymeric cis-isoprenoids obtained by the method of the present invention are those having 40 or more carbon atoms, such as those having 40 to 500 carbon atoms. These are usually obtained as a mixture of a plurality of types of polymer cis-isoprenoids. Examples thereof include ficaprenol C55 (55 carbon atoms), ficaprenol C60 (60 carbon atoms), or a mixture thereof.

Tissue culture The tissue culture in the present invention can be carried out according to a conventional method of plant tissue culture. That is, plant pieces are cultured aseptically to first form callus. Next, a low molecular isoprenoid compound as a substrate (precursor) is added to the medium in which the callus is cultured without separation, or after the callus is transferred to a new medium, and further cultured. to continue. This cultivation is carried out on a solid medium or in a liquid medium.

As the medium, a conventional medium used in plant tissue culture may be used. The material of the medium is roughly divided into water, inorganic nutrients, organic nutrients, plant hormones, natural substances, and support materials.
The water is preferably pure water purified by a distiller, an ion exchange resin, a filtration filter, or the like, or a combination thereof.

As an inorganic nutrient, a large amount of nitrogen, phosphorus, potassium, calcium, magnesium and sulfur and various trace components are required. Specific types and amounts of inorganic salts used vary depending on the type of medium.
As organic nutrients, sugar and vitamins are mainly added. Sugar includes sucrose, glucose, fructose and the like, and sucrose is mainly used. As vitamins, in addition to thiamine, pyridoxine, nicotinic acid and the like are also used as supplements. In addition, myo-inositol and amino acids are also added as growth promoting substances.

  As plant hormones, auxin and cytokinin are most commonly used. As auxin, naphthalene acetic acid, indolebutyric acid, 2,4-dichlorophenoxyacetic acid and the like are used, and as cytokinin, benzyladenine, kinetin and the like are often used. These are all artificially synthesized plant growth regulators and are not easily decomposed even when heated in an autoclave.

Natural substances that contain growth-promoting substances such as squeezed coconut milk, banana and tomatoes, and malt extract are used, and when the purpose of culturing cannot be achieved with a synthetic medium alone, or when expectations are greater It is often used for.
Agar is most often used as a support material for solid media.

  The culture is performed under aerobic conditions, for example, by shaking culture, aeration / stirring culture, or the like. The culture is preferably performed in the dark, and the culture temperature is approximately 25 ° C. A specific example is described in the following examples.

Separation and collection of high molecular cis isoprenoids High molecular cis isoprenoids are insoluble in water and accumulate mainly in cultured cells constituting callus. For this reason, it is preferable to separate the polymer cis-isoprenoid by organic solvent extraction. The organic solvent extraction may be performed on a culture solution containing callus, or may be performed on callus after separating the callus from the culture solution. Extraction from the separated callus may be performed using the callus as it is, or may be performed after the callus is dried to reduce moisture. In order to increase the extraction efficiency, the callus may be crushed and then extracted.

  The organic solvent for extraction is not limited as long as it is immiscible with water and dissolves isoprenoids, and hydrocarbons such as pentane and hexane are preferable. The extraction temperature is preferably as high as possible below the boiling point of the extraction solvent used. The organic solvent after extraction is separated from the aqueous phase and callus according to conventional methods such as standing and centrifugation. Insoluble impurities can be removed by washing the separated organic solvent with saturated saline or the like.

Next, the present invention will be described more specifically with reference to examples.
Example 1.
(1) Medium Preparation Medium Composition In this example, an MS (Murashige & Skoog) medium was prepared and used for callus induction and liquid shaking culture. The components of the medium are shown below.

Medium preparation method (1 l of solid or liquid medium)
While adding distilled water to a beaker and stirring with a magnetic stirrer, add predetermined amounts of inorganic nutrients, organic nutrients, and plant hormones shown in Table 1, adjust the pH of the medium to pH 5.7-5.8 with NaOH or HCl, and add distilled water. Was added to adjust the target amount (in the solid medium, agar was added). This medium was dispensed into a 100 ml Erlenmeyer flask every 20 ml, sterilized by autoclaving, and stored.

(2) Induction of callus Leaves of Indian rubber tree were picked and disinfected with 70% ethanol and 1% sodium hypochlorite. Cut the leaves and shoots to a size of 5 mm x 5 mm, place them on a solid MS medium in a clean bench so that various bacteria do not touch the medium, and place them on the solid MS medium. did. Callus formation was observed between 1 week and 1 month. In order to obtain a stable callus, several subcultures were performed.

(3) Reaction between liquid culture and isoprenoids Remove the callus from the solid medium in a clean bench so that no germs enter. Transfer the extracted callus to the liquid medium in a 100 ml Erlenmeyer flask and shake in a dark room at 24 ° C. Dark culture was performed. To 20 ml of the above liquid culture, 0.5 ml of 5 mM farnesyl diphosphate (FPP) and 1.0 ml of 5 mM isopentenyl diphosphate (IPP) are added, and 20 days at 24 ° C., 100 rpm (100 rpm) Shake dark culture.

(4) Extraction of high molecular isoprenoids
The produced polymer isoprenoids were extracted after dark culture with shaking for 20 days. That is, put one culture broth (21.5 ml in total) into a 100 ml separatory funnel, add 15 ml of pentane to this separatory funnel, shake 300 times, let stand for 30 minutes, and separate the water layer into another 100 ml separatory funnel. Moved to the funnel. In a separating funnel containing an aqueous layer, 15 ml of pentane was added and shaken 300 times and allowed to stand for 30 minutes to separate the aqueous layer and the organic layer. This operation was repeated 5 times.

To the separatory funnel containing the combined organic layers, 20 ml of saturated saline was added and shaken gently 30 times, and allowed to stand to separate the organic and aqueous layers cleanly. To the separated organic layer, 20 ml of double-distilled water was further added, shaken 20 times lightly, and allowed to stand to separate the organic layer from the aqueous layer. This operation was repeated twice. The separated organic layer was dehydrated by adding MgSO ₄ (anhydrous). The separated organic layer was filtered through celite and concentrated on a rotary evaporator. The crude yield of the extract was 0.1365 g.

(5) Product confirmation
In order to confirm the analysis product by HPLC, the ficaprenol C55,60 preparation expected as the product was compared with the reaction product obtained in (4) above by HPLC analysis. The results are shown in FIG. 1A shows the analysis result of the ficaprenol sample, FIG. 1B shows the analysis result of the reaction product, and FIG. 1C shows the mixture of the ficaprenol sample and the reaction product (Double injection). These analytical results were consistent, confirming that the product was ficaprenol C55,60.

In order to confirm the analysis product by reversed-phase TLC, the ficaprenol C55,60 preparation expected as a product and the reactive organism obtained in (4) above were combined with reversed-phase TLC ( Comparison was made by thin layer chromatography analysis. Reverse phase LKC18 plate silica gel 60 (WHATMAN) was used as the thin layer plate, and acetone: water (9: 1) mixture was used as the developing solution. The results are shown in FIG. 2 and Table 2 below. The reaction products show Rf values of 0.11 and 0.09, while the reaction products of Rica and Ficaprenol C55 and Ficaprenol C60 are 0.11 and 0.08, respectively. It was confirmed to be a mixture.

The structural formulas of substances related to the present invention are shown below.

FIG. 1 is a diagram showing the results of HPLC analysis of a ficaprenol C55,60 preparation and the reaction products obtained in the examples. A is the analysis result of the ficaprenol sample, B is the analysis result of the reaction product, and C is the analysis result of the mixture (double injection) of the ficaprenol sample and the reaction product. FIG. 2 is a diagram showing the results of reversed-phase TLC (thin layer chromatography) analysis of a ficaprenol C55,60 preparation and the reaction products obtained in the examples.

Claims (7)

  A method for producing a polymer cis-isoprenoid having 40 or more carbon atoms, wherein tissue culture of a plant is performed in the presence of a low-molecular isoprenoid compound having 20 or less carbon atoms.   The method according to claim 1, wherein the plant is rubber tree.   The method according to claim 2, wherein the rubber tree is Indian rubber tree.   The method according to any one of claims 1 to 3, wherein the low-molecular isoprenoid compound is farnesyl diphosphate (15 carbon atoms), isopentenyl diphosphate (5 carbon atoms), or both. .   The said high molecular cis-isoprenoid is the ficaprenol (ficaprenol) C55 (carbon atom number 55), the ficaprenol C60 (carbon atom number 60), or these both. The method described in 1.   A method for producing ficaprenol C55 and ficaprenol C60, wherein tissue culture of rubber tree is performed in the presence of farnesyl diphosphate and isopentenyl diphosphate.   The method according to claim 6, wherein the rubber tree is Indian rubber tree.

Images