JP3183722B2 - Method for producing gibberellins - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to the production of one or more mixtures of naturally occurring compounds belonging to gibberellins (hereinafter sometimes abbreviated as gibberellins) starting from ent-kaurene. About the method.

[0002]

BACKGROUND OF THE INVENTION Gibberellins are well known as plant hormones naturally contained in trace amounts in all higher plants, and have a diversifying effect by exerting a strong influence on plant growth. It is a very important compound in the agricultural and horticultural fields. For example, certain gibberellins can double the yield per barley or wheat area, remedy crop damage due to frost damage or lack of pollination, break dormancy, delay aging and fruit dropping It is known.

Gibberellins have the general formula (1):

[0004]

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The compound is based on a tetracyclic ring structure represented by the formula (1). Although the ring number is indicated in the formula (1), the exact three-dimensional structure is not shown at present. is there. Currently, is mainly used in the country is a gibberellin mainly composed of gibberellin A _3, which is produced by the fermentation method, it is used to, such as a particular product of production, such as production of seedless grapes At present, it has not yet been used in the production of many other agricultural products.

As an industrial process for producing gibberellins known to date, Gibberella genus, which belongs to the genus Gibberella and has a gibberellin-producing ability, is known.
The fujikuroi bacteria cultured with sugar as a carbon source, production of Gibberellin A ₃ main components (gibberellin A ₄ as the other components, including gibberellin A ₇₎ plant growth hormone as a mixture was (C19- gibberellin) (N. Takahashi, H. Kitamura, A. Kawa
rada, Y. Seta, S. Tamura & Y. Sumiki: Bull. Agric.
Chem. Soc. Japan, 19, 267 (1955). Furthermore, in order to increase the yield per unit volume of Gibberellin A _3, medium polyoxyalkylene glycol compound (JP 49
-14688 JP) and 1,3-dioxane compound (research and the addition of JP 49-14689 JP) (20-50% yield increase), aluminum medium for the purpose of selectively producing gibberellin A ₄ , Copper and zinc compounds are reported to be effective (Japanese Patent Application Laid-Open No. 58-1).
No. 52499).

Furthermore, soybean oil, reported the production of gibberellin A ₃ by using vegetable oils such as cottonseed oil as a carbon source using resting cells of Gibberella-fujikuroi bacteria (JP 49-133
No. 593), using a plant growth inhibitor, add steviol, an intermediate of the gibberellin biosynthetic pathway, to the culture medium of Gibberella fujikuroi bacterium to obtain plant-derived gibberellin A ₁ ,
Reported the production of A _18, A ₁₉ (JP-A-55-120794
Is known. However, the main gibberellins obtained by these methods are gibberellin A ₁ ,
Limited to A ₃ , A ₄ , A ₇ , A ₁₈ , A _19, etc., and are only a few of the more than 80 currently known gibberellins. Further, among these gibberellins, what is actually industrially applicable only gibberellin A _3. Other gibberellins are considered to have specific physiological activities, however, since the amount of their production is very small, a wide range of biological activity tests cannot be performed, and at present, they are hardly applied. To produce other gibberellins in substantial quantities, change the type of bacteria and perform a fermentation process using a fungus that mainly produces another gibberellin, or produce some component using a microorganism as a starting material substrate. Although a method of converting gibberellins into gibberellins having different physical distributions is conceivable, it is difficult to produce gibberellins for the purpose because the distribution of gibberellins in the product is hard to change even if the type of bacteria is changed. It is also possible to derive a compound having the gibberellin backbone that does not exist in another gibberellin or a natural by chemical conversion from the current tends to gibberellin A ₃ and gibberellin A ₄ available. However, chemical conversion is complicated, and it is not easy to freely change to a desired structure.

On the other hand, Tachibana et al. Use ent-kaurene (formula (2)), which is a component contained in cedar leaves and is located before steviol in the gibberellin positive synthesis pathway, as a starting material, and uses a plant growth inhibitor as a starting material. In addition, gibberellin A ₁₅ and gibberellin A ₁₄ -aldehyde-based gibberellins are cultured for 1 day by culturing gibberella fujikuroi bacteria for 10 days, and gibberellin A ₁ , A ₃ , It has been reported that gibberellins containing A ₉ , A ₁₂ -aldehyde and the like as main components are produced (Journal of the Japan Wood Science Society, vol. 35, No. 8, 761).
(1989)).

[0009]

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In this method, A ₁₅ , A ₁₄ -aldehyde, A
_Because gibberellins, such as ₉ , which were conventionally obtained only in trace amounts, can be obtained as a main component, they are expected to be used as a method for producing a plant hormone having a new physiological activity. According to the report, the bioassay of the gibberellin mixture obtained in this system was performed on dwarf rice (short Ginbozu), and the growth promoting effect was compared with the case of starting from another substrate. the extension ratio in the case when a (1.0), the gibberellin a ₃ is (2.8), the culture product with isosteviol as starting material (1.5), also in kaurenoic acid (2 .7), it was reported that when ent-kaurene was used, it was (4.1). Therefore, it can be said that gibberellins produced using ent-kaurene as a raw material have a very strong plant growth activity.

However, the above report has a drawback that the yield of gibberellin is low and it is difficult to produce an industrially usable amount of gibberellin (using IFO-31251 as a strain of Gibberella fujikuroi). , Concentration of ent-kauren in culture medium 0.15%,
The yield of gibberellin relative to ent-kaurene is reported to be 6.7% at 24 ° C. for 7 days.) Therefore, it is considered that the method reported above can be an industrially useful method in the production of various gibberellins that were conventionally obtained only in trace amounts by improving the above problems. Therefore, an object of the present invention is to mass-produce gibberellins, which were conventionally obtained only in trace amounts, by culturing gibberella fujikuroi in the presence of a plant growth inhibitor using a specific amount of ent-kaurene as a starting material. Another object of the present invention is to develop an industrially useful plant growth promoter using gibberellin mass-produced by the above method.

[0012]

In view of the above problems,
As a result of intensive studies to solve, as a starting material, ent-kaurene extracted from cedar leaves, microorganisms belonging to the genus Gibberella and having gibberellin-producing ability derived from microorganisms, such as gibberella fujikuroi, are trace amounts. In a method for mass-producing gibberellins by culturing in the presence of a plant growth inhibitor, the present invention has been completed by achieving a significant increase in yield by using a predetermined concentration of the substrate kaurene.

That is, the gist of the present invention is to provide a microorganism which belongs to the genus Gibberella and has a gibberezin-producing ability derived from a microorganism with an substrate concentration of 0.001% or more and less than 0.15%.
The present invention relates to a method for producing gibberellin, which comprises culturing in a medium in which kaurene and a trace amount of a plant growth inhibitor are present, and causing the microorganism to produce gibberellins. More specifically, ent- obtained by solvent extraction from cedar leaves
In a method of culturing kauren in a medium in a jar fermenter together with a gibberella fujikuroi bacterium that has been cultured and grown in advance and collected, and adding a small amount of a plant growth inhibitor to promote the growth of hyphae, The cultivation is carried out at a low substrate concentration using a method in which the nitrogen content is reduced to 0% to suppress the growth of the cells and to promote the enzymatic reaction. This method involves the production of plant growth hormones such as gibberellin A ₁₅ , A ₁₄ -aldehyde, A ₉ , which were hitherto difficult to obtain quantitatively.

As a microorganism belonging to the genus Gibberella used in the present invention and having a gibberezin-producing ability derived from the microorganism, specifically, for example, Gibberella fujikuroi is preferably mentioned. Gibberella fujikuroi strains include IFO-6603-6607 and IFO-997.
6-9977, IFO-6605, IFO-30337
And IFO-31251 and the like can be mentioned as known bacterial strains, but are not particularly limited as long as they belong to the same genus.

The ent-kauren used in the present invention is:
The cedar leaves are concentrated after extraction with hexane, the concentrated solution is subjected to steam distillation to remove low-boiling substances, and the distillation residue is purified by a hexane solvent packed with silica gel and then recrystallized with a diethyl ether / methanol mixed solvent. Melting point 4
It is a colorless crystal at 9 ° C. From raw cedar leaves 0.05-0.
1% by weight of ent-kaurene is obtained by the above procedure. The amount of ent-kaurene used in the production method of the present invention is 0.001% to 0.15% of the substrate concentration.
And preferably 0.01% or more and less than 0.15%. If the substrate concentration exceeds 0.15%, the yield of gibberellins from kaurene is low, and if the substrate concentration is less than 0.001%, a device for culturing a certain amount of gibberellin becomes large, and gibberellin is extracted. Therefore, the use of a large amount of solvent is not economically preferable.

The plant growth inhibitor used in the present invention includes 2-isopropyl-4-dimethylamino-5-
Methylphenyl-1-piperidinecarboxylate methyl chloride (trade name AMO-1618), (2-chloroethyl) trimethylammonium chloride (trade name CCC), choline chloride (CC) and the like. The amount of the plant growth inhibitor to be used is 0.0001 to 0.003% by weight, preferably 0.0006 to 0.003% by weight, based on ent-kaurene.

In the present invention, a medium used for culturing a microorganism having a gibberellin-producing ability is NH ₄ NO ₃ , (NH ₄ ) ₆ M, which is a nitrogen component, in order to suppress the growth of cells.
o ₇ O _{by 24} · 4H ₂ without O was added, ICI medium (Cho where the nitrogen content and 0%, K.Y.et a
l. : Plant and Cell Physio
l. , 20 , 75-81 (1979).

The gibberellins in the present invention refer to one or more mixtures of compounds belonging to naturally occurring gibberellins, and specifically, gibberellins A ₁ , A ₃ , A ₄ and A _7. , A ₉ , A ₁₂ , A ₁₂ -aldehyde, A ₁₄ , A ₁₄ -aldehyde, A ₁₅ , A ₁₆ , A _24, etc.
In addition, examples of these precursors include 7-β-hydroxykaurenic acid.

Hereinafter, the production method of the present invention will be described in detail. For example, first, the cells are collected by performing preculture twice for the purpose of growing the cells of Gibberella fujikuroi. As the strain used at this time, for example, IFO-30337 and IFO-31251 can be used.

[0020] The medium used for the preculture of the present invention includes Imperial Chemical Indus.
try ICI medium (Cho, KYe)
t al. : Plant and cell Physs
iol. , 20 , 75-81 (1979). The preparation of the ICI medium was performed by adjusting the A medium (glucose 80.0 g, NH ₄ NO ₃ 1.92 g, KH ₂ PO
_{4 5.0g, MgSO 4 · 7H 2} O1.0g, distilled water 1
000Ml) in B medium (FeSO ₄ · 7H ₂ O0.1
g, CuSO ₄ .5H ₂ O 0.015 g, ZnSO ₄
・ 7H ₂ O 0.0161g, MnSO ₄・ 7H ₂ O
_{0.010g, (NH 4) 6 Mo} 7 O 24 · 4H 2 O
(0.010 g, 100 ml of distilled water). This medium has a nitrogen content of 40%.

Next, in order to convert ent-kaurene (substrate) into gibberellin by an enzymatic reaction, the gibberella fujikuroi bacteria collected as described above are cultured. As this medium, in order to suppress the growth of cells, NH ₄ content of nitrogen was used.
NO _3, it is preferable to use _{(NH 4) 6 Mo 7 O} 24 · 4H 2 O ICI medium (nitrogen content 0%) without addition of such. The medium is placed in a jar fermenter, and culture is performed by adding Gibberella fujikuroi bacteria, ent-kaurene, and a plant growth inhibitor to the medium. As a jar fermenter used for culture, for example, a 2.5-liter and 10-liter culture tank of MBF type manufactured by Tokyo Rika Kikai Co., Ltd. can be used.
Stirring speed is 200-500 rpm, aeration rate is 0.1-5 liters / min, and the number of culture days is arbitrarily determined according to the type and required amount of the target gibberellins, for example, about 1 to 10 days. Can be.

After completion of the cultivation, normal Cho,
K. Y. (Cho, KY et al: Pl
ant and Cell Physiol. , 20 ,
75-81 (1979)), ethyl acetate, methanol, butanol, acetone-water (7
0: 30-40: 60), chloroform-methanol-
It can be extracted as a mixture of one or more gibberellins using a mixed solvent such as acetic acid, that is, gibberellins.

The gibberellins after extraction can be chromatographed.
After separation by methylation, methyl ester-trimethyl ether
(Cho, KY et al: Plant an)
dCell Physiol. ,20, 75-81 (1
979), Fujisawa, S .; et al. : Ag
ric. Biol. Chem. ,49, 27-33 (1
985)) to perform gas chromatography.
-(GC), gas chromatography-mass spectrometry (GC-
MS) or the like. Above
When such an analytical method is used, it is obtained by the production method of the present invention.
Gibberellins are gibberellin A₁, A_Three, A_Four,
A₇, A₉, A₁₂, A₁₂-Aldehyde, A₁₄, A₁₄-A
Rudehid, A_Fifteen, A₁₆, A_{twenty four}, Precursor 7-β-hydroxy
Contains cycaurenic acid, but is produced by the method of the present invention
Gibberellin A among gibberellins_ThreeIs less than 1%
Gibberellin A₉, A₁₄-Aldehyde, A_Fifteen,and
A₁, A_Four, A₇Etc. make up the majority
However, this is made clear by the examples described later. As a result
Is reported by Tachibana et al. (Journal of the Japan Wood Science Society, vol. 35, No.
8,761 (1989))
And gibberellin A _ThreeGibberellins other than
It is effective as a new method.

Further, the di- ene obtained by the production method of the present invention.
Dwelling rice (short silver)
The dropping method using a shaven man (literature: Hisane Yamane, Nobutaka Takahashi;
"Bioassay for bioactive substances", Kodansha, 198
4, p105-116) and those who use uniconazole
Method (literature: T. Nishijima, N. Katsur)
a, Plant Cell. Physiol.30
(5), 623 (1989))
be able to.

As described above, gibberellins obtained by the method of the present invention have a strong effect on the growth of plants and have a diversifying effect. Therefore, they are effective in controlling the yield of crops in the fields of agriculture and horticulture. Can be used for

[0026]

The present invention will be described in more detail with reference to the following Examples and Comparative Examples, but the present invention is not limited to these Examples and the like. Example 1 (1) Preculture of Gibberella fujikuroi bacteria (strains of IFO-30337 and IFO-31251): two 300
60 ml of an ICI solution having a nitrogen content of 40% was placed in each of the ml Erlenmeyer flasks, sterilized by heating, and one loop of each of IFO-30337 and 31251 strains was inoculated. This is 2
The cells were cultured with shaking at 150 rpm at 5 ° C. for 3 days.

(2) 24 ml of the culture obtained in the above (1) is aseptically taken, and a freshly prepared ICI having a nitrogen content of 40% is prepared.
1.2 liters of the solution was added to four 500 ml Erlenmeyer flasks, and cultured at 25 ° C. under the same conditions as (1). At this time, 20 mg of a plant growth inhibitor AMO-1618 was added to promote the growth of hypha.

(3) The culture obtained in the above (2) was aseptically filtered to separate the cells. (4) Main culture using Gibberella fujikuroi bacteria: The cells obtained in (3) were sterilized using a sterilized 2.0 liter ICI solution (nitrogen content: 0%) and 20 mg of AM as a plant growth inhibitor.
It was transferred to a culture tank containing O-1618 and 200 mg of kaurene (as a 2 ml solution of ethyl acetate). The cultivation was performed at 25 ° C., a stirring speed of 300 rpm, and air passed through a sterile filter at 0.1 liter / min for 1 to 10 days. Gibberellins produced by sampling the culture solution over time were extracted and analyzed.

(5) Extraction and analysis of metabolites: After the cells were separated from the culture solution (100 ml) collected as a sample, 150 ml of a saturated aqueous sodium hydrogen carbonate solution was added to the filtrate, and the mixture was extracted with ethyl acetate (150 ml × 3). Extracted. The ethyl acetate layer was dried over sodium sulfate and concentrated under reduced pressure to obtain a neutral part extract (neutral part). The aqueous layer was further adjusted to pH 2.5 with diluted hydrochloric acid, and extracted with ethyl acetate (150 ml × 3) to obtain an acidic neutral substance (acidic portion). Gibberellin is UV by spraying 80% sulfuric acid on TLC and then heating (120 ° C, 10 minutes)
Since blue fluorescence was emitted under irradiation with a lamp (365 nm), this method was applied to the extract, and a gibberellin spot was detected in the acidic portion. The acidic part is the Ch
o) to give methyl ester-trimethylsilyl ether (Me-TMS), analyze by GC and GC-MS, and fractionate a gibberellin-colored fraction from the acidic portion by TLC fractionation. Was analyzed. Me-TMS conversion acidic extraction part or methanol solution of gibberellin fraction TLC fractionated (100
μl) was added with an ether solution of diazomethane to perform esterification, the solvent was distilled off, and the residue was dried under reduced pressure.
Bis-trimethylsilyl acetamide (10 μl), trimethylsilyl chloride (5 μl), pyridine (10 μl)
μl) was added for TMS. GC conditions: Silicone OV-101, capillary column (25m ×
0.25mm, IDO.20mm), 150 → 300 ℃ (5 ℃ / min) Heating, Det &
Inj 270 ℃, carrier gas (He) 0.7ml / m, FIDH ₂ 1.3kg
/ cm ² , Split Ratio 1:30. GC-MS conditions: Performed using Shimadzu QP-1000 under the same conditions as GC. However, helium was used as a carrier gas. Table 1 summarizes the results of analysis of the resulting samples of gibberellins.

[0030]

[Table 1]

From Table 1, it can be seen that lowering the substrate concentration clearly improves the yield. That is, the kaurene substrate concentration was changed from the substrate concentration of 0.15% previously reported by Tachibana et al. To 0.01%, and a new strain of IFO-3033 was used.
When microbial conversion was performed in place of 7, the yield with respect to kaurene increased to 53% in 10 days of culture, and gibberellins could be produced at a high yield. Also, when cultivation was performed using IFO-31251 under the same conditions as above, the yield of gibberellin relative to kaurene increased to 39% after culturing for 3 days. In addition, IFO-30337 strain and IFO-3
Tables 2 and 3 show the composition of each component of gibberellins obtained from the 1251 strain.

[0032]

[Table 2]

[0033]

[Table 3]

From Table 2, it can be seen that the gibberellins produced by this example are gibberellins A ₁ , A ₃ , A ₄ , A ₇ , A ₉ ,
A ₁₂ , A ₁₂ -aldehyde, A ₁₄ , A ₁₄ -aldehyde, A
₁₅ , A ₁₆ , A ₂₄ , and the precursor 7-β-hydroxykaurenic acid, but A ₃ % or less of the formed gibberellins, and gibberellin A ₉ , A ₁₄ -aldehyde, A ₁₅ ,
And A ₁ , A ₄ , A _7, etc. make up the majority. This result was reported by Tachibana et al.
No8,761 (1989)) are those similar to the product distribution of the turned out to be a new method as the preparation of Gibberellins except gibberellin A _3. Example 2 (Addition amount of plant growth inhibitor) Except for using IFO-30337 as a bacterial strain and changing the addition amount of plant growth inhibitor (AMO-1618) to no inhibitor, 20 mg, and 60 mg. Example 1
The yield of gibberellins was measured under the same conditions as described above. Table 4 shows the results.

[0035]

[Table 4]

From these results, it was found that AMO-1618 may be added in an amount of about 20 mg to 2 liters of ICI medium.

Example 3 (Type of Plant Growth Inhibitor) The yield of gibberellins when the type of plant growth inhibitor was changed was measured under the same conditions as in Example 2. Table 5 shows the results.

[0038]

[Table 5]

From these results, it was found that A as a plant growth inhibitor
MO-1618 was found to be preferred.

Example 4 (Effect of Stirring Speed) The effect of the stirring speed of the culture tank on the production of gibberellin was examined. Using IFO-30337 as a strain, the yield of gibberellins was measured under the same conditions as in Example 1 except that the stirring blade was changed to 200 to 80 rpm. Table 6 shows the results.

[0041]

[Table 6]

From this result, it was considered that the stirring speed was not required to be 300 rpm or more.

Example 5 (Effect of Air Amount) The influence of the amount of air blown into the culture tank on the yield of gibberellin was shown in terms of% by weight based on kaurene used. Using IFO-31251 as a strain, the yield of gibberellins was measured under the same conditions as in Example 1 except that the amount of air blown into the culture tank was changed. Table 7 shows the results.

[0044]

[Table 7]

From this result, it is considered that the amount of air blown into the culture tank is sufficient at 0.5 liter / min in a 2 liter system.

Example 6 (Effect of Temperature) The influence of the culture temperature on the gibberellin yield was examined. Using IFO-30337 as a strain, the yield of gibberellins was measured under the same conditions as in Example 1 except that the culture temperature was changed. Table 8 shows the results.

[0047]

[Table 8]

From these results, it is considered that the optimum temperature is around 25 ° C.

Example 7 (Culture under Scale-Up Conditions) A test was performed using a 10-liter culture tank. The amount of the drug, the medium, the amount of air, and the like were all five times those of Example 1 (2 liter scale). All other culture conditions were the same as in Example 1, and the strains were tested using IFO-30337 and IFO-31251 to determine the yield of gibberellins. Table 9 shows the results.

[0050]

[Table 9]

From these results, it can be seen that the same results as in Example 1 can be obtained even in culture under scale-up conditions.

Example 8 (Evaluation of Plant Growth-Promoting Effect) Using the gibberellin obtained in the test of Example 1, the growth-promoting effect was measured on dwarf rice (short-gin bud). The results are shown in Tables 10 to 12.

[0053]

[Table 10]

From these results, it can be seen that gibberellins obtained by the production method of the present invention have a remarkable growth promoting effect on the elongation of the second cotyledon of dwarf rice.

[0055]

[Table 11]

[0056]

[Table 12]

[0057]

By providing the production method of the present invention, firstly, gibberellins which have been produced or obtained only in trace amounts, i.e., gibberellins other than gibberellin A ₃ and gibberellin A ₄ , can be converted into kaurene. Microbial conversion (enzyme reaction) using the starting material (substrate) as a starting material enables production to be performed in good yield. Second, it has become possible to provide a gibberellins showing a gibberellin A ₃ alone when as compared to 1.5 times the plant growth-promoting activity. Gibberellins obtained by the production method of the present invention are useful as plant growth promoters.

──────────────────────────────────────────────────続 き Continuation of the front page (51) Int.Cl. ⁷ Identification code FI C12R 1: 645) (56) References Published by The Japan Wood Research Society “Abstracts of the 42nd Annual Meeting of the Japan Wood Society” (1992), p. . 441, 05201 (58) Fields investigated (Int. Cl. ⁷ , DB name) C12P 17/04 C12P 15/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (2)

(57) [Claims] 1. A microorganism which belongs to the genus Gibberella and has a gibberellin-producing ability derived from the microorganism, has a substrate concentration of 0.001%.
A method for producing gibberellins, comprising culturing in a medium containing less than 0.15% of ent-kaurene and a trace amount of a plant growth inhibitor, and causing the microorganism to produce gibberellins. 2. The gibberellins produced are gibberellin A.
_1, gibberellin A ₃ , gibberellin A ₄ , gibberellin A ₇ ,
Gibberellin A ₉ , Gibberellin A ₁₂ , Gibberellin A ₁₂ -aldehyde, Gibberellin A ₁₄ , Gibberellin A ₁₄ -aldehyde, Gibberellin A ₁₅ , Gibberellin A ₁₆ , Gibberellin A ₂₄
2. The production method according to claim 1, wherein the production method is at least one member selected from the group consisting of 7-β-hydroxykaurenic acid, which is a precursor thereof.