Method for producing fertilizer containing KODA and fertilizer containing KODA

Abstract

To provide a technique of improving efficiency of producing a KODA-containing fertilizer.SOLUTION: A method of producing a KODA-containing fertilizer includes reacting α-linolenic acid derived from an α-linolenic acid supply source to produce KODA, in the presence of 9-lipoxygenase and allene oxide synthase derived from enzyme-supplying microorganisms selected from the group consisting of (a) a combination of a first transgenic microorganism including a 9-lox gene cording 9-lipoxygenase as a foreign gene and a second transgenic microorganism including an aox gene cording allene oxide synthase as a foreign gene, and (b) a transgenic microorganism including a 9-lox gene cording 9-lipoxygenase and an aox gene cording allene oxide synthase as a foreign gene.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing a KODA-containing fertilizer and a KODA-containing fertilizer.

Background Art

[0002] KODA (9,10-α-ketolinoic acid) is known as a plant hormone having a plant growth regulating action such as a flower bud formation promoting action (Patent Document 1). Further, it has been reported that KODA is a physiologically active substance that imparts resistance to various growth environment stresses and environmental fluctuations to plants (Non-Patent Document 1).

[0003] Although KODA is biosynthesized in plants, it has been reported that its biosynthesis amount is extremely small (Non-Patent Document 2). In order to increase the production amount of KODA, it has been reported that an Aouki kusa strain with high KODA productivity was obtained by screening (Patent Document 1). However, when KODA is produced by hydroponically cultivating the Aouki kusa strain and collecting the culture solution, the production amount per unit area is small and it is necessary to extract KODA from the cells, which is laborious and costly. In addition, a method for chemically synthesizing KODA has also been reported, but it is necessary to go through many reaction steps and the final yield is very low at 0.81% (Non-Patent Document 3).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide a technique for enhancing the production efficiency of a KODA-containing fertilizer.

Means for Solving the Problems

[0007] According to a first aspect of the present invention, (a) A combination of a first transformed microorganism containing a 9-lox gene encoding 9-lipoxygenase as a foreign gene and a second transformed microorganism containing an aox gene encoding allene oxide synthase as a foreign gene, and (b) A transformed microorganism containing the 9-lox gene encoding 9-lipoxygenase and the aox gene encoding allene oxide synthase as foreign genes A method for producing a KODA-containing fertilizer is provided, which includes reacting α-linolenic acid derived from an α-linolenic acid source in the presence of 9-lipoxygenase and allene oxide synthase derived from an enzyme-supplying microorganism selected from the group consisting of

[0008] According to the second aspect of the present invention, a KODA-containing fertilizer produced by the above method is provided.

Advantages of the Invention

[0009] According to the present invention, a technique for enhancing the production efficiency of KODA-containing fertilizers is provided.

Brief Description of the Drawings

[0010]

Figure 1

Modes for Carrying Out the Invention

[0011] Hereinafter, embodiments of the present invention will be described. The embodiments described below are more specific forms of any of the above aspects. The matters described below can be incorporated into each of the above aspects alone or in combination.

[0012] According to a preferred embodiment, the method for producing a KODA-containing fertilizer is (a) A combination of a first transformed microorganism containing the 9-lox gene encoding 9-lipoxygenase as a foreign gene and a second transformed microorganism containing the aox gene encoding allene oxide synthase as a foreign gene, and (b) A transformed microorganism containing the 9-lox gene encoding 9-lipoxygenase and the aox gene encoding allene oxide synthase as foreign genes An enzyme-supplying microorganism selected from the group consisting of, an α-linolenic acid source, and are mixed in soil, and in the soil, in the presence of 9-lipoxygenase and allene oxide synthase derived from the enzyme-supplying microorganism, α-linolenic acid derived from the α-linolenic acid source is reacted to produce KODA in the soil.

[0013] In the following description, first, the "enzyme-supplying microorganism" and the "α-linolenic acid source" will be described, and then the "steps of mixing and reaction" will be described.

[0014] (Enzyme-supplying microorganism) The enzyme-supplying microorganism is (a) A combination of a first transformed microorganism containing a 9-lox gene encoding 9-lipoxygenase as a foreign gene and a second transformed microorganism containing an aox gene encoding allene oxide synthase as a foreign gene, and (b) A transformed microorganism containing a 9-lox gene encoding 9-lipoxygenase and an aox gene encoding allene oxide synthase as foreign genes selected from the group consisting of. The "enzyme-supplying microorganism" is preferably a microorganism that does not synthesize α-linolenic acid. That is, the "enzyme-supplying microorganism" preferably excludes microorganisms that synthesize α-linolenic acid. Whether a microorganism corresponds to a microorganism that synthesizes α-linolenic acid or a microorganism that does not synthesize α-linolenic acid can be confirmed by lipid analysis using gas chromatography. Since α-linolenic acid is one of the constituent lipids of biological membranes, when a microorganism does not synthesize α-linolenic acid, it does not affect the composition of the biological membrane. Therefore, a microorganism that does not synthesize α-linolenic acid is excellent in that it can express foreign genes (that is, the 9-lox gene and / or the aox gene) without affecting the growth of the microorganism.

[0015] A preferred enzyme-supplying microorganism is an alga. That is, a preferred enzyme-supplying microorganism is (a) A combination of a first transformed alga containing the 9-lox gene encoding 9-lipoxygenase as a foreign gene and a second transformed alga containing the aox gene encoding allene oxide synthase as a foreign gene, and (b) A transformed alga containing the 9-lox gene encoding 9-lipoxygenase and the aox gene encoding allene oxide synthase as foreign genes It is selected from the group consisting of. As described above, the above-mentioned "preferred enzyme-supplying microorganism" is preferably an alga that does not synthesize α-linolenic acid. That is, the above-mentioned "preferred enzyme-supplying microorganism" preferably excludes algae that synthesize α-linolenic acid.

[0016] The "alga" that is the preferred enzyme-supplying microorganism refers to microalgae. Microalgae are, for example, photosynthetic eukaryotes, which are single-celled organisms or their colonies. Microalgae are, for example, single-celled green algae such as Chlamydomonas reinhardtti and Botryococcus, single-celled red algae such as Cyanidioschyzon merolae, diatoms such as Phaeodactylum, or their colonies. Microalgae do not have to be eukaryotes and may be prokaryotes that perform photosynthesis, such as bacteria such as cyanobacteria.

[0017] The enzyme-supplying microorganism is not limited to algae and may be, for example, Escherichia coli, Bacillus subtilis, yeast, etc. However, since algae can fix carbon, using algae can reduce the culture cost. Also, by using algae, the fertilizer produced can be used as green manure.

[0018] The enzyme-supplying microorganism can be prepared by incorporating a foreign gene (that is, the 9-lox gene and / or the aox gene) into the host microorganism.

[0019] As the "9-lox gene", the 9-lox gene derived from higher plants (e.g., Lemna paucicostata) can be used. Also, as the "aox gene", the aox gene derived from higher plants (e.g., Lemna paucicostata) can be used. The nucleotide sequences of the 9-lox gene and aox gene derived from Lemna paucicostata are described in Patent Document 1 (International Publication No. 2011 / 11841).

[0020] The integration of a foreign gene into a host microorganism can be carried out by cloning the foreign gene into an expression construct (i.e., an expression vector) and introducing the resulting recombinant vector into the host microorganism using genetic recombination techniques. The promoter contained in the expression construct may be a promoter that induces constitutive expression of the foreign gene, or a promoter that induces conditional-specific expression of the foreign gene (e.g., a temperature-sensitive promoter or a nutrient-responsive promoter, etc.). In the case of the latter promoter, since the period during which the foreign gene is expressed can be limited, the influence on the growth of the microorganism can be minimized.

[0021] When using "the combination of (a) a first transformed microorganism containing a 9-lox gene as a foreign gene and a second transformed microorganism containing an aox gene as a foreign gene" as the enzyme supply microorganism, it is preferable to prepare the first transformed microorganism and the second transformed microorganism so that the 9-lox gene and the aox gene have the same expression level. For example, it is preferable to prepare the first transformed microorganism and the second transformed microorganism using the same expression construct so that the foreign genes are arranged under the control of the same promoter.

[0022] The expression levels of the 9-lox gene in the first transformed microorganism and the aox gene in the second transformed microorganism can be confirmed by immunoblot analysis or the like. When it is found by immunoblot analysis or the like that the expression levels of the 9-lox gene in the first transformed microorganism and the aox gene in the second transformed microorganism are different, it is desirable to correct the amount of cells used and adjust so that the two enzymes produced by gene expression are present in approximately the same number of molecules.

[0023] When using "(b) a transformed microorganism containing the 9-lox gene and the aox gene as foreign genes" as the enzyme supply microorganism, it is preferable to prepare the transformed microorganism so that the 9-lox gene and the aox gene have the same expression level. For example, it is preferable to prepare the transformed microorganism by arranging the 9-lox gene and the aox gene under the control of one promoter so that they are transcribed at the same expression level.

[0024] (α-linolenic acid source) The α-linolenic acid source is preferably a plant, more preferably a waste plant. As long as the plant contains α-linolenic acid, it may be the whole plant body or a part of the plant body such as a plant organ or tissue.

[0025] As the waste plant, for example, plant parts remaining after harvesting and shipping agricultural crops (such as vegetables such as corn, tomatoes, and spinach), agricultural crops that cannot be shipped, weeds, etc. can be used. Among them, it is preferable to use the soft leaf part of the tissue. For example, it is preferable to use the remaining leaves after vegetable harvesting.

[0026] The α-linolenic acid source is not limited to plants, and any substance that can supply α-linolenic acid may be used. By using plants, the fertilizer produced can be used as green manure. In addition, by using waste plants, it is possible to reduce the manufacturing cost of the KODA-containing fertilizer.

[0027] (Steps of mixing and reaction) Mix an enzyme - supplying microorganism and an α - linolenic acid source in soil, and in the soil, react α - linolenic acid derived from the α - linolenic acid source in the presence of 9 - lipoxygenase and allene oxide synthase derived from the enzyme - supplying microorganism to produce KODA in the soil.

[0028] In this step, microorganisms in the soil decompose the enzyme - supplying microorganism and supply 9 - lipoxygenase (9 - LOX) and allene oxide synthase (AOS) into the soil. In addition, in this step, microorganisms in the soil decompose the α - linolenic acid source containing α - linolenic acid and supply α - linolenic acid into the soil. As a result, the synthesis of KODA occurs in the soil.

[0029] The reaction pathway for synthesizing KODA from α - linolenic acid is shown in FIG. 1. As shown in FIG. 1, α - linolenic acid is converted to 9 - hydroperoxylinolenic acid by the action of 9 - lipoxygenase (9 - LOX), which is converted to 9,10 - allene oxide by the action of allene oxide synthase (AOS), and this is auto - converted to synthesize 9,10 - α - ketol linolenic acid (KODA).

[0030] As the soil, preferably, soil used for growing crops, more preferably, nutrient - rich soil used for growing crops is used.

[0031] The mixing step can be carried out, for example, by adding 0.1 - 10 parts by mass of an enzyme - supplying microorganism (preferably an enzyme - supplying alga) and 0.5 - 50 parts by mass of an α - linolenic acid source (preferably a plant, more preferably a waste plant) to 100 parts by mass of soil and mixing them. The mass ratio of the enzyme - supplying microorganism (preferably an enzyme - supplying alga) to the α - linolenic acid source (preferably a plant, more preferably a waste plant) is not particularly limited, and can be, for example, 1:5.

[0032] The reaction process can be carried out, for example, by leaving the obtained mixture outdoors with a roof or indoors at an atmospheric temperature (e.g., 0 to 30 °C) for the period required for KODA to be produced in the soil. The reaction process can be carried out, for example, over a period of 1 to 14 days.

[0033] As the reaction conditions, conditions suitable for aerobic decomposition of the enzyme-supplying microorganisms and the α-linolenic acid source by microorganisms in the soil, and conditions suitable for the KODA-synthesizing enzymes (9-LOX and AOS) to function can be adopted.

[0034] During the reaction process, it is preferable to stir the soil regularly. For example, it is preferable to stir the soil at intervals of 1 to 3 days. This supplies oxygen to the aerobic microorganisms in the soil, and as a result, it is possible to promote the decomposition of the enzyme-supplying microorganisms and the α-linolenic acid source by the aerobic microorganisms in the soil.

[0035] Also, during the reaction process, it is preferable to regularly add moisture to the soil in order to adjust the moisture content of the soil. The moisture content of the soil is preferably adjusted to be, for example, 5 to 50% by mass, which is the moisture content adopted in a general composting reaction. Here, the moisture content refers to the value on a wet basis (i.e., the value obtained by dividing the mass of the moisture by the total mass of the soil containing the moisture).

[0036] In a preferred embodiment, although the enzyme-supplying microorganisms and the α-linolenic acid source are mixed in the soil, it is also possible to extract the content containing the KODA-synthesizing enzyme from the enzyme-supplying microorganisms and use the obtained extract instead of the enzyme-supplying microorganisms, and / or extract the content containing α-linolenic acid from the α-linolenic acid source and use the obtained extract instead of the α-linolenic acid source.

[0037] (Fertilizer containing KODA) By the above method, KODA is produced in the soil, and as a result, a KODA-containing fertilizer is manufactured. That is, according to another aspect, a KODA-containing fertilizer manufactured by the above method is provided. The KODA-containing fertilizer manufactured by the above method contains soil, KODA, the decomposition product of the enzyme-supplying microorganism, and the decomposition product of the α-linolenic acid supply source.

[0038] When algae are used as the enzyme-supplying microorganism in the above method, the KODA-containing fertilizer contains soil, KODA, the decomposition product of algae, and the decomposition product of the α-linolenic acid supply source. Also, when a plant (preferably a waste plant) is used as the α-linolenic acid supply source in the above method, the KODA-containing fertilizer contains soil, KODA, the decomposition product of the enzyme-supplying microorganism, and the decomposition product of the plant (preferably a waste plant). Further, when algae are used as the enzyme-supplying microorganism and a plant (preferably a waste plant) is used as the α-linolenic acid supply source in the above method, the KODA-containing fertilizer contains soil, KODA, the decomposition product of algae, and the decomposition product of the plant (preferably a waste plant).

[0039] (Effect) According to the method for manufacturing a KODA-containing fertilizer of the present invention, by reacting the KODA synthase (9-LOX and AOS) derived from the enzyme-supplying microorganism with the KODA synthetic substrate (α-linolenic acid) derived from the α-linolenic acid supply source, a KODA-containing fertilizer can be manufactured only through the three reaction steps shown in FIG. 1. Therefore, a KODA-containing fertilizer can be manufactured inexpensively and simply. Also, since the method of the present invention can produce KODA only through the three reaction steps shown in FIG. 1, a fertilizer containing a large amount of KODA can be manufactured. Therefore, the method of the present invention is excellent in that the production efficiency of the KODA-containing fertilizer is high.

[0040] According to a preferred embodiment, a method for producing a KODA-containing fertilizer involves mixing an enzyme-supplying microorganism and an α-linolenic acid source in soil. In a preferred embodiment, due to the decomposition action of microorganisms in the soil, KODA synthase (9-LOX and AOS) is supplied into the soil from the enzyme-supplying microorganism (preferably algae), and due to the decomposition action of microorganisms in the soil, a KODA synthesis substrate (α-linolenic acid) is supplied into the soil from the α-linolenic acid source (preferably a plant, more preferably a waste plant). Therefore, it is possible to produce a KODA-containing fertilizer without performing crushing operations on algal cells or plant cells, or extraction and purification operations on enzymes or substrates. Thus, according to a preferred embodiment, a KODA-containing fertilizer can be produced more inexpensively and simply.

[0041] Since the KODA-containing fertilizer is expected to be used in the agricultural field, it is difficult to put into practical use unless it is produced inexpensively and simply. As described above, the method of the present invention can produce a KODA-containing fertilizer inexpensively and simply, so it is suitable for practical use.

[0042] Also, when algae are used as the enzyme-supplying microorganism in a preferred embodiment, the KODA-containing fertilizer can contain decomposition products of algae (which contain nutrient sources such as nitrogen and phosphorus), so it is possible to produce a fertilizer (green manure) with an even higher nutritional value. In addition, when algae are used as the enzyme-supplying microorganism, no carbon source is required for culturing the algae when preparing the enzyme-supplying microorganism, so the culturing cost can be suppressed.

[0043] Also, when a plant is used as the α-linolenic acid source in a preferred embodiment, the KODA-containing fertilizer can contain decomposition products of the plant (which contain nutrient sources such as nitrogen and phosphorus), so it is possible to produce a fertilizer (green manure) with an even higher nutritional value. In particular, when a waste plant is used as the α-linolenic acid source, it is not necessary to produce a plant when preparing the α-linolenic acid source, and it is possible to significantly reduce the production cost of the KODA-containing fertilizer.

Claims

1. (a) A combination of a first transformed microorganism containing a 9-lox gene encoding 9-lipoxygenase as a foreign gene and a second transformed microorganism containing an aox gene encoding allene oxide synthase as a foreign gene, and (b) A transformed microorganism containing a 9-lox gene encoding 9-lipoxygenase and an aox gene encoding allene oxide synthase as foreign genes An enzyme-supplying microorganism selected from the group consisting of, An α-linolenic acid source Are mixed in soil, and in the soil, α-linolenic acid derived from the α-linolenic acid source is reacted in the presence of 9-lipoxygenase and allene oxide synthase derived from the enzyme-supplying microorganism to produce KODA in the soil. A method for producing a KODA-containing fertilizer, comprising:

2. The method according to claim 1, wherein the enzyme-supplying microorganism is algae.

3. The method according to claim 1 or 2, wherein the α-linolenic acid source is a plant.

4. The method according to any one of claims 1 to 3, wherein the α-linolenic acid source is a waste plant.

5. A KODA-containing fertilizer produced by the method according to any one of claims 1 to 4.

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