JP2022102889A - Method for manufacturing crop yield improver, crop yield improver, and crop yield improving method - Google Patents

Abstract

To provide a crop yield improver improving the yield of crops.SOLUTION: A crop yield improver contains at least either of a pyroglutamic acid or an aminobenzoic acid.SELECTED DRAWING: None

Description

The present disclosure relates to a crop yield improver, which is a natural metabolite that contributes to the improvement of crop yield, a method for producing a crop yield improver, and a method for improving crop yield.

With the demand for increased food production due to the increase in the world population, the development of crop yield improvement technology for efficiently producing high-quality agricultural products in limited cultivated land is required. In particular, in recent years, from the viewpoint of preventing global warming and reducing the environmental load, naturally-derived substances that consume less fossil energy in the manufacturing process and have less environmental load in application (hereinafter, also referred to as application or use). Is desired to be utilized.

For example, as a method for improving crop yield using a naturally derived substance, a method of inoculating a plant with a microorganism or a culture solution of a microorganism that produces a substance that contributes to the growth promotion of the plant (Patent Documents 1 and 2 and Non-Patent Documents). 1) is disclosed. Further, for example, a method of adding a natural metabolite such as an organic acid to soil to chelate metal ions in soil to improve the utilization of metal ions by plants is disclosed (Patent Document 3). Further, for example, a method of applying a composition containing adenosine, which is a natural metabolite, to a plant (Patent Document 4) and a method of applying a fertilizer containing an algae cell extract to a plant (Patent Document 5) are disclosed. There is.

Japanese Unexamined Patent Publication No. 2018-11600 Special Table No. 63-501286 Gazette Japanese Unexamined Patent Publication No. 2014-073993 Japanese Patent No. 5943844 Japanese Patent No. 3143872

Jimenez-Gomez et al., “Probiotic activities of Rhizobium laguerreae on growth and quality of spinach”, Scientific reports, 2018, Vol. 8, 295

However, the effect of the microbial material differs depending on the combination of the microbial species used, the target plant species, and the properties of the soil, lacks versatility, and the crop yield improving effect is unstable. On the other hand, naturally derived substances such as natural metabolites are more likely to exert a yield improving effect on a plurality of plant species (that is, plants in general) than microbial materials, but the production, extraction or purification process of the substances is complicated. , Excessive cost is incurred.

Therefore, in the present disclosure, a crop yield improving agent containing a naturally derived substance having a crop yield improving effect (hereinafter, also referred to as a crop yield improving substance) and a crop yield improving agent can be easily and efficiently produced. A method for producing a yield improver is provided. The present disclosure also provides a crop yield improving method capable of effectively improving the crop yield.

The crop yield improver according to one aspect of the present disclosure contains at least one of pyroglutamic acid and aminobenzoic acid.

Further, the method for producing a crop yield improving agent according to one aspect of the present disclosure is the method for producing a crop yield improving agent, in which the function of a protein involved in the binding between the outer membrane and the cell wall in cyanobacteria is suppressed or lost. It comprises the steps of preparing the modified cyanobacteria that have been modified and causing the modified cyanobacteria to secrete the secretions involved in improving the crop yield.

Further, in the crop yield improving method according to one aspect of the present disclosure, the crop yield improving agent is used for a crop.

According to the crop yield improver of the present disclosure, the yield of the crop can be effectively improved. Further, according to the method for producing a crop yield improver of the present disclosure, it is possible to easily and efficiently produce a crop yield improver that exerts a yield improving effect on a plurality of crop species. Further, according to the crop yield improving method of the present disclosure, the yield of the crop can be effectively improved by using the crop yield improving agent of the present disclosure for the crop (hereinafter, also referred to as application).

FIG. 1 is a flowchart showing an example of a method for producing a crop yield improver according to an embodiment. FIG. 2 is a diagram schematically showing the cell surface layer of cyanobacteria. FIG. 3 is a transmission electron microscope observation image of an ultrathin section of the modified cyanobacteria of Example 1. FIG. 4 is an enlarged image of the broken line region A in FIG. FIG. 5 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 2. FIG. 6 is an enlarged image of the broken line region B of FIG. FIG. 7 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Comparative Example 1. FIG. 8 is an enlarged view of the broken line region C of FIG. 7. FIG. 9 is a graph showing the amount of protein (n = 3, error bar = SD) in the culture medium of the modified cyanobacteria of Example 1, Example 2 and Comparative Example 1. FIG. 10 is a graph showing the relative average strain weight per lettuce strain cultivated in Examples 3 to 5 and Comparative Example 2. FIG. 11 is a diagram showing the states of representative strains of Examples 3 to 5 and Comparative Example 2. FIG. 12 is a graph showing the relative average weight per spinach plant cultivated in Examples 6 to 8 and Comparative Example 3. FIG. 13 is a diagram showing the states of representative strains of Examples 6 to 8 and Comparative Example 3. FIG. 14 is a graph showing the average number of fruits per tomato plant cultivated in Example 9 and Comparative Example 4. FIG. 15 is a graph showing the average fruit weight per tomato strain cultivated in Example 9 and Comparative Example 4. FIG. 16 is a graph showing the average sugar content per tomato strain cultivated in Example 9 and Comparative Example 4. FIG. 17 is a diagram showing typical fruit states of Example 9 and Comparative Example 4.

(Findings underlying this disclosure)
As mentioned in the background technology, there is a demand for crop yield improvement technology for efficiently producing crops in limited cultivated land. In addition, in order to improve the yield of crops, it is required to utilize naturally derived substances having a low environmental load in application. Above all, a substance that consumes less fossil energy during the production of the substance and has a smaller environmental load is desired.

The following prior art techniques are disclosed as techniques for improving crop yields.

For example, Patent Document 1 discloses a method of applying a microbial strain having a plant growth promoting activity or a culture of the microbial strain around a plant or a plant (for example, soil). It has been reported that by using this method, it is possible not only to promote the growth of plants and increase the yield, but also to prevent the outbreak of pathogenic diseases of plants.

Further, for example, in Patent Document 2, a plant growth promoting composition obtained by mixing a bacterial culture solution and an algae culture solution and incubating the mixed solution under predetermined conditions is produced, and the composition is used. Methods of application to plants are disclosed. It has been reported that the method promotes the vegetative growth of tomatoes when the composition is added to the vegetative solution of hydroponic cultivation of plants.

Further, for example, Non-Patent Document 1 discloses a method of applying a kind of rhizobia to spinach as a plant probiotic bacterium having a plant growth promoting mechanism, and more specifically to spinach roots. It has been reported that this method has a growth promoting effect such as an increase in the number and size of leaves of spinach inoculated with the rhizobia.

Further, for example, in Patent Document 3, a natural metabolite such as an organic acid (a substance involved in metabolism and a naturally occurring substance) is added to soil to chelate metal ions such as iron in the soil. Then, a method for improving the utilization of metal ions by plants is disclosed.

Further, for example, Patent Document 4 discloses a method of applying a composition containing adenosine, which is a natural metabolite, as a main component to a plant.

Further, for example, Patent Document 5 discloses a method of fertilizing a plant with a fertilizer containing a cell extract of algae. More specifically, the cell extract is treated with cyanobacteria in an aqueous solvent (eg, water) at 60 ° C. or higher.

However, when a naturally occurring substance as described above is used, the production, extraction or purification process thereof is generally complicated, resulting in excessive cost. On the other hand, when the microorganism itself is ingested into a plant, a complicated process is not required, but the effect differs depending on the combination of the microorganism species used, the target plant species, and the properties of the soil, and it lacks versatility. The effect of improving crop yield is unstable. From the above situation, it is desired to develop a naturally-derived substance that can be produced by a simple process using cheaper raw materials and has an effect of improving yield on a plurality of crop species.

The present inventor applies at least one of the natural metabolites pyroglutamic acid and aminobenzoic acid to crops to improve the productivity of the crops, that is, to increase the yield of high-quality crops. I found. Furthermore, the present inventor uses cyanobacteria (also referred to as cyanobacteria or cyanobacteria) to inexpensively and conveniently (i.e.,) obtain a crop yield improver containing at least one of pyroglutamic acid and aminobenzoic acid. Efficiently) found a way to manufacture.

Cyanobacteria are a group of eubacteria that decompose water by photosynthesis to produce oxygen and fix CO ₂ in the air with the obtained energy. Cyanobacteria can also fix nitrogen (N ₂ ) in the air, depending on the species. In this way, cyanobacteria can obtain most of the raw materials (that is, nutrients) and energy necessary for the growth of cells from air, water, and light, so that they are inexpensive raw materials and a simple process. Cyanobacteria can be cultivated. In addition, it is known that cyanobacteria grow quickly and have high light utilization efficiency. In addition, since genetic manipulation is easier than other algae species, cyanobacteria were used among photosynthetic microorganisms. Active research and development is underway regarding material production.

For example, as an example of substance production using cyanobacteria, production of fuels such as ethanol, isobutanol, alcans, and fatty acids (Patent Document 6: Japanese Patent No. 6341676) has been reported. In addition, research and development on the production of substances that are nutrient sources for living organisms are being carried out. However, since the intracellular metabolites and proteins of cyanobacteria are difficult to be secreted extracellularly, it is necessary to disrupt the cells of cyanobacteria to extract the desired metabolites and proteins produced in the cells.

Therefore, the present inventors partially detach the outer membrane covering the cell wall of cyanobacteria from the cell wall, whereby the compounds, proteins and intracellular metabolites produced in the cells of cyanobacteria are removed from the cells. It was found that it is easily secreted into. In this process, the present inventor found that the secretions of cyanobacteria contained pyroglutamic acid and aminobenzoic acid, and that each of pyroglutamic acid and aminobenzoic acid had a yield improving effect on a plurality of crop species. did. As a result, it is possible to efficiently recover the substance having the effect of improving the crop yield secreted outside the cells without crushing the cells of cyanobacteria. In addition, since the physiological activity of the crop yield improving substance is less likely to be impaired by eliminating the need for operations such as extraction (for example, crushing cells), the crop yield improving agent containing the secretion is effective. The yield of crops can be improved.

Therefore, according to the method for producing a crop yield improver of the present disclosure, a substance having an effect of promoting production on a plurality of crop species can be easily and efficiently produced. Further, according to the crop yield improver of the present disclosure, the growth of plants can be effectively promoted. Further, according to the crop yield improver of the present disclosure, the crop production can be effectively promoted by using the crop yield improver of the present disclosure.

(Summary of this disclosure)
The outline of one aspect of the present disclosure is as follows.

The crop yield improver according to one aspect of the present disclosure contains at least one of pyroglutamic acid and aminobenzoic acid.

According to the crop yield improver according to one aspect of the present disclosure, the yield can be improved for a plurality of crop species.

The method for producing a crop yield improver according to one aspect of the present disclosure is the method for producing a crop yield improver, in which the function of a protein involved in the binding between the outer membrane and the cell wall in cyanobacteria is suppressed or lost. It comprises the step of preparing the modified cyanobacteria and the step of causing the modified cyanobacteria to secrete a secretion containing the crop yield improving substance.

As a result, in the modified cyanobacteria, the binding between the cell wall and the outer membrane (for example, the amount of binding and the binding force) is partially reduced, so that the outer membrane is easily detached from the cell wall partially. Therefore, proteins and metabolites produced in the cells (hereinafter, also referred to as substances produced in the cells) tend to leak out of the outer membrane, that is, outside the cells. The leaked intracellularly produced substance includes a group of substances having an effect of improving crop yield, which is characterized by containing pyroglutamic acid and aminobenzoic acid. This eliminates the need for extraction processing of substances produced in the cells, such as crushing the cells. Therefore, the crop yield improving agent can be easily and efficiently produced. Further, since the above-mentioned extraction treatment of the intracellularly produced substance becomes unnecessary, the decrease in the physiological activity and the decrease in the yield of the intracellularly produced substance are less likely to occur. Therefore, among the substances produced in the cells of the modified cyanobacteria, the decrease in the physiological activity and the decrease in the yield of the substance involved in the increase in the yield of the crop are less likely to occur. In addition, since the above-mentioned extraction process of the intracellularly produced substance is not required, even after the intracellularly produced substance secreted outside the bacterial cell is recovered, the modified cyanobacteria are repeatedly used to produce the intracellularly produced substance. Can be done. Therefore, it is not necessary to prepare a new modified cyanobacteria every time the crop yield improving agent is produced. Therefore, according to the method for producing a crop yield improver according to one aspect of the present disclosure, the crop yield improver can be easily and efficiently produced.

For example, in the method for producing a crop yield improver according to one aspect of the present disclosure, the proteins involved in the binding between the outer membrane and the cell wall are SLH (Surface Layer Homology) domain-retaining outer membrane protein and cell wall-pyruvic acid. It may be at least one of the acid modifying enzymes.

Thereby, in the modified cyanobacteria, for example, (i) an enzyme that catalyzes the reaction of modifying the SLH domain-retaining outer membrane protein that binds to the cell wall and the bound sugar chain on the surface of the cell wall with pyruvate (that is, the cell wall-pyruvate modification). At least one function of (enzyme) is suppressed or lost, or (ii) expression of SLH domain-retaining outer membrane protein and at least one cell wall-pyruvate modifying enzyme is suppressed. Therefore, the binding (that is, the amount of binding and the binding force) between the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane and the covalently bound sugar chain on the surface of the cell wall is reduced. This facilitates the detachment of the outer membrane from the cell wall at the portion where the bond between the outer membrane and the cell wall is weakened. As a result, in modified cyanobacteria, the binding between the outer membrane and the cell wall is reduced, which makes it easier for the outer membrane to partially detach from the cell wall. Substances produced in the cells are likely to leak out of the cells. As a result, the modified cyanobacteria improve the secretory productivity of secreting the crop yield improving substance produced in the cells to the outside of the cells. Therefore, according to the method for producing a crop yield improving agent according to one aspect of the present disclosure, the modified cyanobacteria can efficiently secret the crop yield improving substance, so that the crop yield improving agent can be efficiently produced. can.

For example, in the method for producing a crop yield improver according to one aspect of the present disclosure, the SLH domain-retaining outer membrane protein comprises Slr1841 consisting of the amino acid sequence represented by SEQ ID NO: 1 and the amino acid sequence represented by SEQ ID NO: 2. It may be NIES970_09470, Anacy_3458 consisting of the amino acid sequence represented by SEQ ID NO: 3, or a protein having an amino acid sequence that is 50% or more identical to that of any of these SLH domain-retaining outer membrane proteins.

Thereby, in the modified cyanobacteria, for example, (i) any SLH domain-retaining outer membrane protein shown in SEQ ID NOs: 1 to 3 above or any one of these SLH domain-retaining outer membrane proteins and an amino acid sequence can be obtained. Functions of proteins that are 50% or more identical are suppressed or lost, or (ii) any SLH domain-retaining outer membrane protein set forth in SEQ ID NOs: 1-3 above, or any SLH domain thereof. The expression of a protein whose amino acid sequence is 50% or more identical to that of the retained outer membrane protein is suppressed. Therefore, in the modified cyanobacteria, (i) the function of the SLH domain-retaining outer membrane protein or the protein having the same function as the SLH domain-retaining outer membrane protein in the outer membrane is suppressed or lost, or (ii). The expression level of the SLH domain-retaining outer membrane protein or the protein having the same function as the SLH domain-retaining outer membrane protein in the outer membrane is reduced. As a result, in the modified cyanobacteria, the binding domain (for example, SLH domain) for the outer membrane to bind to the cell wall is partially detached from the cell wall because the binding amount and the binding force to bind to the cell wall are reduced. It will be easier. As a result, the substance produced in the cells is easily leaked to the outside of the cells, so that the crop yield improving substance produced in the cells is also easily leaked to the outside of the cells. Therefore, according to the method for producing a crop yield improving agent according to one aspect of the present disclosure, the crop yield improving substance produced in the cells of the modified cyanobacteria is likely to be secreted to the outside of the cells. It can be manufactured efficiently.

In the method for producing a crop yield improver according to one aspect of the present disclosure, the cell wall-pyruvate modifying enzyme is Slr0688 consisting of the amino acid sequence shown in SEQ ID NO: 4, Synpcc7942_1529 consisting of the amino acid sequence shown in SEQ ID NO: 5, and the sequence. It may be Anacy_1623 consisting of the amino acid sequence represented by No. 6, or a protein having an amino acid sequence of 50% or more identical to that of any of these cell wall-pyruvate modifying enzymes.

Thereby, in the modified cyanobacteria, for example, (i) any cell wall-pyruvic acid modifying enzyme shown in SEQ ID NOs: 4 to 6 above or any one of these cell wall-pyruvic acid modifying enzymes and an amino acid sequence of 50%. The function of the same protein is suppressed or lost, or (ii) any cell wall-pyruvic acid modifying enzyme shown in SEQ ID NOs: 4 to 6 above or any of these cell walls-pyruvic acid modification. The expression of proteins having an amino acid sequence that is 50% or more identical to that of the enzyme is suppressed. Therefore, in the modified cyanobacteria, (i) the function of the cell wall-pyruvic acid modifying enzyme or a protein having the same function as the enzyme is suppressed or lost, or (ii) the cell wall-pyruvic acid modifying enzyme or the enzyme is used. The expression level of proteins having the same function is reduced. This makes it difficult for the covalently bound sugar chains on the surface of the cell wall to be modified with pyruvic acid, so that the amount and binding force of the sugar chains on the cell wall to bind to the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane. Is reduced. As a result, in modified cyanobacteria, the covalent sugar chains on the surface of the cell wall are less likely to be modified with pyruvate, so that the binding force between the cell wall and the outer membrane is weakened, and the outer membrane is easily detached from the cell wall. Become. As a result, the substance produced in the cells is easily leaked to the outside of the cells, so that the crop yield improving substance produced in the cells is also easily leaked to the outside of the cells. Therefore, according to the method for producing a crop yield improving agent according to one aspect of the present disclosure, the crop yield improving substance produced in the cells of the modified cyanobacteria is likely to be secreted to the outside of the cells. It can be manufactured efficiently.

For example, in the method for producing a crop yield improver according to one aspect of the present disclosure, the gene expressing the protein involved in the binding between the outer membrane and the cell wall may be deleted or inactivated.

As a result, in the modified cyanobacteria, the expression of the protein involved in the binding between the cell wall and the outer membrane is suppressed, or the function of the protein is suppressed or lost, so that the binding between the cell wall and the outer membrane (that is, that is). , Bonding amount and binding force) is partially reduced. As a result, in modified cyanobacteria, the outer membrane is more likely to be partially detached from the cell wall, so that intracellularly produced substances such as proteins and metabolites produced in the cells are outside the outer membrane, that is, outside the cells. It becomes easy to leak. Therefore, the modified cyanobacteria improve the secretory productivity of the crop yield improving substance produced in the cells. This eliminates the need for extraction processing of the substance produced in the cell, such as crushing the cell, so that the decrease in the physiological activity and the decrease in the yield of the substance produced in the cell are less likely to occur. Therefore, it is difficult for the physiological activity and the yield of the crop yield improving substance produced in the cells to decrease, so that it is possible to produce a crop yield improving agent having an improved crop yield improving effect. Further, since the extraction process of the above-mentioned substance produced in the cells is not required, the modified cyanobacteria can be repeatedly used to produce the substance for improving the crop yield even after the substance is recovered. Therefore, it is not necessary to prepare a new modified cyanobacteria every time the crop yield improving agent is produced. Therefore, according to the method for producing a crop yield improving agent according to one aspect of the present disclosure, it is possible to easily and efficiently produce a crop yield improving agent having an improved crop yield improving effect.

For example, in the method for producing a crop yield improver according to one aspect of the present disclosure, the gene expressing the protein involved in the binding between the outer membrane and the cell wall is a gene encoding an SLH domain-retaining outer membrane protein and a gene. It may be at least one of the genes encoding the cell wall-pyruvate modifying enzyme.

As a result, in the modified cyanobacteria, at least one gene encoding the SLH domain-retaining outer membrane protein and the gene encoding the cell wall-pyruvic acid modifying enzyme is deleted or inactivated. Therefore, in the modified cyanobacteria, for example, the expression of at least one of (i) SLH domain-retaining outer membrane protein and cell wall-pyruvic acid modifying enzyme is suppressed, or (ii) SLH domain-retaining outer membrane protein and cell wall. -At least one function of pyruvate modifying enzyme is suppressed or lost. Therefore, the binding (that is, the amount of binding and the binding force) between the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane and the covalently bound sugar chain on the surface of the cell wall is reduced. This facilitates the detachment of the outer membrane from the cell wall at the portion where the bond between the outer membrane and the cell wall is weakened. As a result, in modified cyanobacteria, the binding between the outer membrane and the cell wall is reduced, which makes it easier for the outer membrane to partially detach from the cell wall, so that proteins and metabolites produced in the cells leak out of the cells. It will be easier to do. As a result, the crop yield improving substance produced in the cells is likely to leak out of the cells. Therefore, according to the method for producing a crop yield improving agent according to one aspect of the present disclosure, the modified cyanobacteria can efficiently secret the crop yield improving substance, so that the crop yield improving agent can be efficiently produced. can.

For example, in the method for producing a crop yield improver according to one aspect of the present disclosure, the gene encoding the SLH domain-retaining outer membrane protein is represented by slr1841 and SEQ ID NO: 8, which consist of the base sequence represented by SEQ ID NO: 7. It may be nies970_09470 consisting of a base sequence, anacy_3458 consisting of the base sequence represented by SEQ ID NO: 9, or a gene having a base sequence 50% or more identical to any of these genes.

As a result, in the modified cyanobacteria, the gene encoding any of the SLH domain-retaining outer membrane proteins shown in SEQ ID NOs: 7 to 9 above or the gene having the same base sequence of 50% or more as the base sequence of any of these genes. Is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) expression of any of the above SLH domain-retaining outer membrane proteins or a protein having the same function as any of these proteins is suppressed, or (ii) the above. The function of any SLH domain-retaining outer membrane protein or a protein having a function equivalent to that of any of these proteins is suppressed or lost. As a result, in the modified cyanobacteria, the binding amount and the binding force for the binding domain (for example, SLH domain) for the outer membrane to bind to the cell wall are reduced, so that the outer membrane is easily partially detached from the cell wall. Become. As a result, proteins and metabolites produced in the cells are likely to leak out of the cells, so that the crop yield improving substance produced in the cells is also likely to leak out of the cells. Therefore, according to the method for producing a crop yield improver according to one aspect of the present disclosure, the crop yield improver produced in the cells of the modified cyanobacteria is likely to leak out of the cells. It can be manufactured efficiently.

For example, in the method for producing a crop yield improver according to one aspect of the present disclosure, the gene encoding the cell wall-pyruvate modifying enzyme is slr0688 consisting of the base sequence represented by SEQ ID NO: 10 and the base represented by SEQ ID NO: 11. It may be synpcc7942_1529 consisting of a sequence, anacy_1623 consisting of the base sequence represented by SEQ ID NO: 12, or a gene having a base sequence 50% or more identical to any of these genes.

Thereby, in the modified cyanobacteria, the base sequence of the gene encoding any cell wall-pyruvic acid modifying enzyme shown in SEQ ID NOs: 10 to 12 above or the gene encoding any of these enzymes is 50% or more identical. The gene is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) expression of any of the above cell wall-pyruvic acid modifying enzymes or a protein having the same function as any of these enzymes is suppressed, or (ii) any of the above. The function of the cell wall-pyruvic acid modifying enzyme or a protein having the same function as any of these enzymes is suppressed or lost. This makes it difficult for the covalently bound sugar chains on the surface of the cell wall to be modified with pyruvic acid, so that the amount and binding force of the sugar chains on the cell wall to bind to the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane. Is reduced. As a result, in modified cyanobacteria, the amount of sugar chains for the cell wall to bind to the outer membrane is reduced by pyruvate, so that the binding force between the cell wall and the outer membrane is weakened, and the outer membrane is partially separated from the cell wall. It becomes easy to leave. As a result, proteins and metabolites produced in the cells are likely to leak out of the cells, so that the crop yield improving substance produced in the cells is also likely to leak out of the cells. Therefore, according to the method for producing a crop yield improver according to one aspect of the present disclosure, the crop yield improver produced in the cells of the modified cyanobacteria is likely to leak out of the cells. It can be manufactured efficiently.

Further, in the crop yield improving method according to one aspect of the present disclosure, the above-mentioned crop yield improving agent is used for crops.

According to the crop yield improving method according to one aspect of the present disclosure, the crop yield can be effectively improved by using the crop yield improving agent having an improved crop yield improving effect on the crop.

Hereinafter, embodiments will be specifically described with reference to the drawings.

It should be noted that all of the embodiments described below show comprehensive or specific examples. The numerical values, materials, steps, the order of steps, etc. shown in the following embodiments are examples, and are not intended to limit the present disclosure. Further, among the components in the following embodiments, the components not described in the independent claim indicating the highest level concept are described as arbitrary components.

In addition, each figure is not necessarily exactly illustrated. In each figure, substantially the same configuration is designated by the same reference numerals, and duplicate description may be omitted or simplified.

Further, in the following, the numerical range does not mean only a strict meaning, but also includes measuring a substantially equivalent range, for example, the amount of protein (for example, number or concentration, etc.) or the range thereof.

Further, in the present specification, both the bacterial cell and the cell represent one cyanobacterial individual.

(Embodiment)
In the present specification, the identity of the base sequence and the amino acid sequence is calculated by the BLAST (Basic Local Alignment Search Tool) algorithm. Specifically, it is calculated by performing pairwise analysis with the BLAST program available on the website of NCBI (National Center for Biotechnology Information) (https://blast.ncbi.nlm.nih.gov/Blast.cgi). To. Information on cyanobacterial genes and proteins encoded by these genes is published, for example, in the NCBI database described above and Cyanobase (http://genome.microbedb.jp/cyanobase/). From these databases, the amino acid sequences of the proteins of interest and the base sequences of the genes encoding those proteins can be obtained.

[1. Crop yield improver]
First, the crop yield improver according to the present embodiment will be described. The crop yield enhancer comprises at least one of pyroglutamic acid and aminobenzoic acid. The crop yield improver is produced in the cells of modified cyanobacteria in which the function of the protein involved in the binding between the outer membrane and the cell wall is suppressed or lost in cyanobacteria (hereinafter, also referred to as parent cyanobacteria). It is secreted to the outside. That is, the crop yield improver is contained in the secretion of modified cyanobacteria. Pyroglutamic acid and aminobenzoic acid should be contained in the crop yield improver in the range of about 0.1 μM or more and about 10 μM or less, respectively. The crop yield enhancer has a plant growth promoting effect, for example, an effect of increasing the number of leaves, stems, buds, flowers, or fruits of a plant, thickening the stem or trunk, and extending the height. In addition, the crop yield improving agent has an effect of improving the quality of plants (also referred to as an effect of improving plant quality), for example, controlling diseases of plants, improving the absorption rate of nutrients, and increasing the sugar content of fruits. As a result, the crop yield improver can improve the quality of plants for multiple crop species, such as increasing crop yield, increasing crop and fruit weight, increasing fruit sugar content, reducing physiological disorders, and reducing diseases. It can be effectively improved. Since the crop yield improver has a plant growth promoting effect and a plant quality improving effect, the crop yield can be effectively improved.

In addition to crops cultivated in fields, plants include garden trees, flowers, lawns, roadside trees, and forest trees that are rarely fertilized. That is, as used herein, crop is synonymous with plant as defined herein. Further, the improvement of quality means that the quality of the parts used by the plant (fruits, leaves, rhizomes, etc.), for example, the components and appearance are preferable (for example, the nutritional value is increased, the taste is improved, etc.). Means that. Ingredients include, for example, water, proteins, lipids, carbohydrates (eg, sugar, starch, etc.), general ingredients such as ash, inorganic substances such as sodium, potassium, and calcium, vitamins such as vitamin A, vitamin C, and folic acid, and fatty acids. , Cholesterol, dietary fiber, etc. In addition, the appearance includes measurable items such as shape, color, scent, cortex, meat quality, thickness, luster, degree of flowering, degree of physiological disorder, beauty, suppleness, freshness, and appearance. Includes items that are difficult to measure, such as balance.

In addition, cyanobacteria (that is, parent cyanobacteria) and modified cyanobacteria will be described later.

As mentioned above, the secretion comprises a crop yield enhancer containing at least one of pyroglutamic acid and aminobenzoic acid. That is, the modified cyanobacteria secrete secretions that are involved in improving crop yields. The secretion contains proteins and metabolites (ie, intracellularly produced substances) produced in the cells of the modified cyanobacteria. The substance produced in the cells includes a substance involved in improving the crop yield (that is, a substance for improving the crop yield).

Crop yield-enhancing substances other than pyroglutamic acid and aminobenzoic acid are, for example, peptidases, nucleases, organic degrading enzymes such as phosphatase, DNA metabolism-related substances such as adenosine or guanosine, or nucleic acids such as spermidin (eg, DNA or RNA). It is an intracellular molecule involved in the promotion of synthesis, a ketone body such as 3-hydroxybutyric acid, or an organic acid such as gluconic acid. The modified cyanobacterial secretions may be a mixture of these crop yield-enhancing substances.

[2. Manufacturing method of crop yield improver]
Subsequently, a method for producing the crop yield improver according to the present embodiment will be described with reference to FIG. 1. FIG. 1 is a flowchart showing an example of a method for producing a crop yield improver according to the present embodiment.

The method for producing a crop yield improver according to the present embodiment prepares modified cyanobacteria in which the function of a protein involved in the binding between the outer membrane and the cell wall is suppressed or lost in cyanobacteria (that is, parent cyanobacteria). (Step S01) and a step (step S02) of causing the modified cyanobacteria to secrete a secretion containing a crop yield improving agent.

In step S01, the above-mentioned modified cyanobacteria are prepared. Preparing the modified cyanobacteria means adjusting the state of the modified cyanobacteria so that the modified cyanobacteria can secrete secretions. Preparing the modified cyanobacteria may be, for example, genetically modifying the parent cyanobacteria to produce the modified cyanobacteria, or restoring the cells from a lyophilized or glycerol stock of the modified cyanobacteria. Alternatively, the modified cyanobacteria that have finished secreting the secretion containing the crop yield improving agent in step S02 may be recovered.

In step S02, the modified cyanobacteria are made to secrete a secretion containing a crop yield improver. The modified cyanobacteria in the present embodiment suppress or lose the function of the protein involved in the binding between the outer membrane and the cell wall in the cyanobacteria (that is, the parent cyanobacteria), so that the protein produced in the cells and the protein produced in the cells are lost. Metabolites are likely to be secreted outside the outer membrane (ie, outside the bacterium). These intracellularly produced substances include crop yield improvers containing at least one of pyroglutamic acid and aminobenzoic acid. Therefore, in step S02, by culturing the modified cyanobacteria under predetermined conditions, substances produced in the cells, such as pyroglutamic acid and aminobenzoic acid, which are involved in improving the crop yield, are secreted outside the cells.

Cyanobacterial culture can generally be carried out on the basis of liquid culture using BG-11 medium (see Table 2) or a modification thereof. Therefore, the culture of modified cyanobacteria may be carried out in the same manner. In addition, the culture period of cyanobacteria for producing a crop yield improver may be a period during which proteins and metabolites can be accumulated at a high concentration under the condition that the cells have sufficiently grown, for example, 1. It may be up to 3 days or 4 to 7 days. Further, the culture method may be, for example, aeration stirring culture or shaking culture.

By culturing under the above conditions, the modified cyanobacteria produce proteins and metabolites (that is, intracellularly produced substances) in the cells, and secrete the intracellularly produced substances into the culture medium. The intracellularly produced substance may contain, for example, at least one of pyroglutamic acid and aminobenzoic acid, and may contain a crop yield improving substance other than these substances. When recovering intracellularly produced substances secreted into the culture medium, solids such as cells (that is, cells) are removed from the culture medium by filtering or centrifuging the culture medium, and the culture supernatant is obtained. May be collected. According to the method for producing a crop yield improving agent according to the present embodiment, a secretion containing an intracellularly produced substance (that is, a crop yield improving substance) involved in the crop yield improvement is secreted extracellularly of the modified cyanobacteria. Therefore, it is not necessary to crush the cells in order to recover the crop yield improving substance. Therefore, the modified cyanobacteria remaining after the recovery of the crop yield improving substance can be repeatedly used to produce the crop yield improving agent.

The method for recovering the crop yield improving substance secreted into the culture solution is not limited to the above example, and the crop yield improving substance in the culture solution may be recovered while culturing the modified cyanobacteria. For example, by using a permeable membrane that allows proteins to permeate, the crop yield improving substance that has permeated the permeable membrane may be recovered. As described above, since the crop yield improving substance in the culture solution can be recovered while culturing the modified cyanobacteria, the treatment for removing the cells of the modified cyanobacteria from the culture solution becomes unnecessary. Therefore, the crop yield improving agent can be produced more easily and efficiently.

In addition, the damage and stress suffered by the modified cyanobacteria can be reduced by eliminating the need for the recovery treatment of the bacterial cells from the culture solution and the crushing treatment of the bacterial cells. Therefore, it becomes difficult to reduce the secretory productivity of the crop yield improving substance of the modified cyanobacteria, and the modified cyanobacteria can be used for a longer period of time.

As described above, by using the modified cyanobacteria in the present embodiment, the crop yield improving agent can be easily and efficiently obtained.

Hereinafter, cyanobacteria and modified cyanobacteria will be described.

[3. Cyanobacteria]
Cyanobacteria, also called cyanobacteria or cyanobacteria, are a group of prokaryotes that collect photoenergy with chlorophyll and electrolyze water with the obtained energy to generate oxygen while photosynthesizing. Cyanobacteria are highly diverse, for example, in cell shape, there are unicellular species such as Synechocystis sp. PCC 6803 and multicellular filamentous species such as Anabaena sp. PCC 7120. As for the habitat, there are thermophilic species such as Thermosynechococcus elongatus, marine species such as Synechococcus elongatus, and freshwater species such as Synechocystis. It also has unique characteristics such as species that have gas vesicles and produce toxins such as Microcystis aeruginosa, and Gloeobacter violaceus that has a protein called phycobilisome that is a focusing antenna on the plasma membrane without thylakoids. There are many species that have.

FIG. 2 is a diagram schematically showing the cell surface layer of cyanobacteria. As shown in FIG. 2, the cell surface layer of cyanobacteria is composed of a plasma membrane (also referred to as inner membrane 1), peptidoglycan 2, and an outer membrane 5, which is a lipid membrane forming the outermost layer of cells, in this order from the inside. To. A sugar chain 3 composed of glucosamine, mannosamine, etc. is covalently bound to peptidoglycan 2, and pyruvate is bound to these covalently bound sugar chains 3 (Non-Patent Document 3: Jurgens and). Weckesser, 1986, J. Bacteriol., 168: 568-573). In the present specification, the peptidoglycan 2 and the covalently bonded sugar chain 3 are collectively referred to as a cell wall 4. The gap between the plasma membrane (that is, the inner membrane 1) and the outer membrane 5 is called periplasm, and is called periplasm, which decomposes proteins or forms a three-dimensional structure, decomposes lipids or nucleic acids, or takes up extracellular nutrients. There are various enzymes involved in.

The SLH domain-retaining outer membrane protein (for example, Slr1841 in the figure) consists of a C-terminal region embedded in a lipid membrane (also referred to as outer membrane 5) and an N-terminal SLH domain 7 protruding from the lipid membrane. , Cyanobacterium and a group of gram-negative bacteria, which are widely distributed in bacteria belonging to the Negativicutes family (Non-Patent Document 4: Kojima et al., 2016, Biosci. Biotech. Biochem., 10: 1954-1959). The regions embedded in the lipid membrane (ie, the outer membrane 5) form channels to allow the outer membrane of hydrophilic substances to permeate, while the SLH domain 7 has the function of binding to the cell wall 4 (non-). Patent Document 5: Kowata et al., 2017, J. Bacteriol., 199: e00371-17). In order for SLH domain 7 to bind to the cell wall 4, the covalent sugar chain 3 in peptidoglycan 2 needs to be modified with pyruvic acid (Non-Patent Document 6: Kojima et al., 2016, J. Biol). . Chem., 291: 20198-20209). Examples of genes encoding SLH domain-retaining outer membrane protein 6 include slr1841 or slr1908 carried by Synechocystis sp. PCC 6803, or oprB carried by Anabaena sp. 90.

The enzyme that catalyzes the pyruvate modification reaction of the covalently bound sugar chain 3 in peptidoglycan 2 (hereinafter referred to as cell wall-pyruvic acid modifying enzyme 9) has been identified in the gram-positive bacterium Bacillus anthracis and is named CsaB. (Non-Patent Document 7: Mesnage et al., 2000, EMBO J., 19: 4473-4484). In cyanobacteria whose genomic base sequence is open to the public, many species carry genes encoding homologous proteins having an amino acid sequence identity of 30% or more with CsaB. Examples include slr0688 held by Synechocystis sp. PCC 6803 or syn7502_03092 held by Synechococcus sp. 7502.

In cyanobacteria, CO ₂ immobilized by photosynthesis is converted into precursors of various amino acids and intracellular molecules through a multi-step enzymatic reaction. Using them as raw materials, proteins and metabolites are synthesized in the cytoplasm of cyanobacteria. Some of these proteins and metabolites function within the cytoplasm, while others are transported from the cytoplasm to the periplasm and function within the periplasm. However, cases of active secretion of proteins and metabolites extracellularly have not been reported in cyanobacteria to date.

Since cyanobacteria have high photosynthetic ability, it is not always necessary to take in organic matter as nutrients from the outside. Therefore, cyanobacteria have very little channel protein in the outer membrane 5 that allows organic matter to permeate, such as the organic channel protein 8 (eg, Slr1270) in FIG. For example, in Synechocystis sp. PCC 6803, the organic channel protein 8 that allows the organic matter to permeate is present in only about 4% of the total protein content of the outer membrane 5. On the other hand, cyanobacteria permeate only inorganic ions like SLH domain-retaining outer membrane protein 6 (for example, Slr1841) in FIG. 2 in order to take up inorganic ions necessary for growth into cells with high efficiency. The outer membrane 5 has a large amount of ion channel proteins to cause. For example, in Synechocystis sp. PCC 6803, ion channel proteins that permeate inorganic ions account for about 80% of the total protein content of the outer membrane 5.

As described above, in cyanobacteria, since there are very few channels for permeating organic substances such as proteins in the outer membrane 5, it is difficult to actively secrete proteins and metabolites produced in the cells to the outside of the cells. It is considered.

[4. Modified cyanobacteria]
Subsequently, the modified cyanobacteria in the present embodiment will be described with reference to FIG.

In the modified cyanobacteria of the present embodiment, the function of the protein involved in the binding between the outer membrane 5 and the cell wall 4 (hereinafter, also referred to as a binding-related protein) is suppressed or lost in the cyanobacteria. As a result, in the modified cyanobacteria, the binding between the outer membrane 5 and the cell wall 4 (for example, the amount of binding and the binding force) is partially reduced, so that the outer membrane 5 is more likely to be partially detached from the cell wall 4. Therefore, the modified cyanobacteria improve the secretory productivity of intracellularly produced substances that secrete proteins and metabolites produced in the cells to the outside of the cells. As described above, the intracellularly produced substance includes an intracellularly produced substance (that is, a crop yield improving substance) that is involved in improving the yield of the crop. The crop yield improving substances are, for example, pyroglutamic acid and aminobenzoic acid. Modified cyanobacterial secretions include, for example, crop yield enhancers containing at least one of pyroglutamic acid and aminobenzoic acid. Therefore, the modified cyanobacteria also improve the secretion productivity of crop yield improving substances that secrete crop yield improving substances such as pyroglutamic acid and aminobenzoic acid produced in the cells to the outside of the cells. In addition, since it is not necessary to crush the cells to recover the crop yield improving substance, the modified cyanobacteria can be used repeatedly even after the crop yield improving substance is recovered. In this specification, the production of modified cyanobacteria in the cells is called production, and the secretion of the produced proteins and metabolites out of the cells is called secretory production.

The protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvic acid modifying enzyme 9. In this embodiment, the modified cyanobacteria suppress or lose the function of at least one protein of, for example, SLH domain-retaining outer membrane protein 6 and cell wall-pyruvic acid modifying enzyme 9. For example, in modified cyanobacteria, (i) the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 may have at least one function suppressed or lost, and (ii) the SLH domain that binds to the cell wall 4. At least one of the expression of the retained outer membrane protein 6 and the enzyme that catalyzes the pyruvate modification reaction of the bound sugar chain on the surface of the cell wall 4 (that is, the cell wall-pyruvate modifying enzyme 9) may be suppressed. .. As a result, the binding (that is, the binding amount and binding force) between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalently bound sugar chain 3 on the surface of the cell wall 4 is reduced. Therefore, the outer membrane 5 is likely to be detached from the cell wall 4 at the portion where these bonds are weakened. By partially detaching the outer membrane 5 from the cell wall 4, intracellularly produced substances such as proteins and metabolites present in the cells of the modified cyanobacteria, especially periplasm, move out of the cells (outside the outer membrane 5). It becomes easy to leak. As a result, the modified cyanobacteria improve the secretory productivity of secreting the crop yield improving substance produced in the cells to the outside of the cells.

Hereinafter, the outer membrane 5 is modified to partially detach from the cell wall 4 by suppressing the functions of at least one binding-related protein of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9. The cyanobacteria will be described more specifically.

Cyanobacteria before suppressing or losing at least one of the expression of SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9, which are the parent microorganisms of the modified cyanobacteria in the present embodiment. , The type of parent cyanobacteria) is not particularly limited and may be any type of cyanobacteria. For example, the parent cyanobacteria may be of the genus Synechocystis, Synechococcus, Anabaena, or Thermosynechococcus, among others Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1. May be good.

The amino acid sequences of the SLH domain-retaining outer membrane protein 6 and the enzyme that catalyzes the cell wall-pyruvate modification reaction (that is, the cell wall-pyruvate modifying enzyme 9) in these parent cyanobacteria, and the genes encoding their binding-related proteins. The base sequence and the position of the gene on the chromosomal DNA or plasmid can be confirmed by the above-mentioned NCBI database and Cyanobase.

The SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 whose functions are suppressed or lost in the modified cyanobacteria according to the present embodiment are any parent as long as they are possessed by the parent cyanobacteria. It may be of cyanobacteria and is not limited by the location of the genes encoding them (eg, on chromosomal DNA or plasmid).

For example, the SLH domain-retaining outer membrane protein 6 may be Slr1841, Slr1908, Slr0042, or the like when the parent cyanobacteria belongs to the genus Syenchocystis, or NIES970_09470 or the like when the parent cyanobacteria belongs to the genus Synechococcus. Often, if the parent cyanobacteria belongs to the genus Anabaena, it may be Anacy_5815 or Anacy_3458, etc., if the parent cyanobacteria belongs to the genus Microcystis, it may be A0A0F6U6F8_MICAE, etc. If the parent cyanobacteria belongs to the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc. If the parent cyanobacteria belongs to the genus Crocosphaera, it may be B1WRN6_CROS5 or the like, and if the parent cyanobacteria belongs to the genus Pleurocapsa, it may be K9TAE4_9CYAN or the like.

More specifically, the SLH domain-retaining outer membrane protein 6 is, for example, Slr1841 (SEQ ID NO: 1) of Synechocystis sp. PCC 6803, NIES970_09470 (SEQ ID NO: 2) of Synechococcus sp. NIES-970, or Anabaena cylindrica PCC. It may be Anacy_3458 (SEQ ID NO: 3) of 7122 or the like. Further, it may be a protein having an amino acid sequence of 50% or more identical to these SLH domain-retaining outer membrane proteins 6.

Thereby, in the modified cyanobacteria, for example, (i) any SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 1 to 3 above or any of these SLH domain-retaining outer membrane proteins 6 and amino acids. The function of the protein having the same sequence of 50% or more may be suppressed or lost, and (ii) any SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 1 to 3 above, or any of these. The expression of a protein having an amino acid sequence of 50% or more identical to that of SLH domain-retaining outer membrane protein 6 may be suppressed. Therefore, in the modified cyanobacteria, (i) the function of the SLH domain-retaining outer membrane protein 6 or the protein having the same function as the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is suppressed or lost, or (Ii) The expression level of the SLH domain-retaining outer membrane protein 6 or the protein having the same function as the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is reduced. As a result, in the modified cyanobacteria, the binding amount and the binding force of the binding domain (for example, SLH domain 7) for the outer membrane 5 to bind to the cell wall 4 are reduced, so that the outer membrane 5 is the cell wall 4 It becomes easier to partially detach from. As a result, in the modified cyanobacteria, the substance produced in the cells is likely to leak out of the cells, so that the crop yield improving substance produced in the cells is also easily leaked out of the cells.

Generally, it is said that if the amino acid sequence of a protein is 30% or more the same, the homology of the three-dimensional structure of the protein is high, and therefore it is highly possible that the protein has the same function as the protein. Therefore, the SLH domain-retaining outer membrane protein 6 whose function is suppressed or lost is, for example, 40% with any amino acid sequence of the SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 1 to 3 above. As described above, the amino acid sequence is preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, still more preferably 90% or more, and the cell wall 4 It may be a protein or polypeptide having a function of binding to the covalent bond type sugar chain 3.

Further, for example, the cell wall-pyruvate modifying enzyme 9 may be Slr0688 or the like when the parent cyanobacteria belongs to the genus Syenchocystis, or may be Syn7502_03092 or Synpcc7942_1529 or the like when the parent cyanobacteria belongs to the genus Synechococcus. If the cyanobacteria belongs to the genus Anabaena, it may be ANA_C20348 or Anacy_1623, etc., if the parent cyanobacteria belongs to the genus Microcystis, it may be CsaB (NCBI access ID: TRU80220), etc., and the parent cyanobacteria belongs to the genus Cyanothese. In the case, it may be CsaB (NCBI access ID: WP_107667006.1) or the like, and if the parent cyanobacteria belongs to the genus Spirulina, it may be CsaB (NCBI access ID: WP_026079530.1) or the like, and the parent cyanobacteria. If is of the genus Calothrix, it may be CsaB (NCBI access ID: WP_096658142.1) or the like, and if the parent cyanobacteria is of the Nostoc genus, it may be CsaB (NCBI access ID: WP_099068528.1) or the like. , If the parent cyanobacteria belongs to the genus Crocosphaera, it may be CsaB (NCBI access ID: WP_012361697.1), etc., and if the parent cyanobacteria belongs to the genus Pleurocapsa, it may be CsaB (NCBI access ID: WP_036798735), etc. May be good.

More specifically, the cell wall-pyruvic acid modifying enzyme 9 is, for example, Slr0688 (SEQ ID NO: 4) of Synechocystis sp. PCC 6803, Synpcc7942_1529 (SEQ ID NO: 5) of Synechococcus sp. PCC 7942, or Anabaena cylindrica PCC 7122. It may be Anacy_1623 (SEQ ID NO: 6) or the like. Further, it may be a protein having an amino acid sequence of 50% or more identical to these cell wall-pyruvic acid modifying enzymes 9.

Thereby, in the modified cyanobacteria, for example, (i) any cell wall-pyruvic acid modifying enzyme 9 shown in SEQ ID NOs: 4 to 6 above or any one of these cell wall-pyruvic acid modifying enzyme 9 and an amino acid sequence can be obtained. The function of proteins that are 50% or more identical may be suppressed or lost (ii) any cell wall set forth in SEQ ID NOs: 4-6 above-pyruvic acid modifying enzyme 9 or any of these cell walls-. The expression of a protein having an amino acid sequence of 50% or more the same as that of pyruvate modifying enzyme 9 may be suppressed. Therefore, in the modified cyanobacteria, (i) the function of the cell wall-pyruvic acid modifying enzyme 9 or a protein having a function equivalent to the enzyme is suppressed or lost, or (ii) the cell wall-pyruvic acid modifying enzyme 9 or the said The expression level of proteins having the same function as enzymes is reduced. As a result, the covalently bound sugar chain 3 on the surface of the cell wall 4 is less likely to be modified with pyruvic acid, so that the sugar chain 3 of the cell wall 4 becomes the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. The amount of binding and the binding force are reduced. Therefore, in the modified cyanobacteria according to the present embodiment, the covalent bond type sugar chain 3 on the surface of the cell wall 4 is less likely to be modified with pyruvate, so that the binding force between the cell wall 4 and the outer membrane 5 is weakened, and the outer membrane is weakened. 5 is more likely to partially detach from the cell wall 4. As a result, in the modified cyanobacteria, the substance produced in the cells is likely to leak out of the cells, so that the crop yield improving substance produced in the cells is also easily leaked out of the cells.

Further, as described above, if the amino acid sequences of the proteins are 30% or more the same, it is said that there is a high possibility that the proteins have the same functions as the proteins. Therefore, the cell wall-pyruvic acid modifying enzyme 9 whose function is suppressed or lost includes, for example, 40% or more of the amino acid sequence of any of the cell wall-pyruvic acid modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 above. It consists of an amino acid sequence having an amino acid sequence of preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, still more preferably 90% or more, and the peptide glycan of the cell wall 4 It may be a protein or polypeptide having a function of catalyzing a reaction of modifying the covalent bond type sugar chain 3 of 2 with pyruvic acid.

In the present specification, suppressing or losing the function of the SLH domain-retaining outer membrane protein 6 means suppressing or losing the binding ability of the protein to the cell wall 4, and to the outer membrane 5 of the protein. The transport of the protein is suppressed or lost, or the ability of the protein to be embedded in the outer membrane 5 to function is suppressed or lost.

In addition, suppressing or losing the function of the cell wall-pyruvic acid modifying enzyme 9 means that the protein suppresses or loses the function of modifying the covalent sugar chain 3 of the cell wall 4 with pyruvic acid.

The means for suppressing or losing the function of these proteins is not particularly limited as long as it is a means usually used for suppressing or losing the function of the protein. The means is, for example, deleting or inactivating the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9, and inhibiting the transcription of these genes. It may be to inhibit the translation of transcripts of these genes, or to administer an inhibitor that specifically inhibits these proteins.

In the present embodiment, the modified cyanobacteria are the outer membrane 5 and the cell wall 4, and thereby the modified cyanobacteria suppresses the expression of the protein involved in the binding between the cell wall 4 and the outer membrane 5, or the present invention. Since the function of the protein is suppressed or lost, the binding between the cell wall 4 and the outer membrane 5 (that is, the binding amount and the binding force) is partially reduced. As a result, in the modified cyanobacteria, the outer membrane 5 is easily partially detached from the cell wall 4. Therefore, in the modified cyanobacteria, the intracellularly produced substances such as proteins and metabolites produced in the cells are contained in the outer membrane 5. It is easy to leak to the outside, that is, to the outside of the bacterial cell. Therefore, the modified cyanobacteria improve the secretory productivity of the crop yield improving substance that secretes the crop yield improving substance produced in the cells to the outside of the cells. This eliminates the need for extraction processing of the substance produced in the cell, such as crushing the cell, so that the decrease in the physiological activity and the decrease in the yield of the substance produced in the cell are less likely to occur. Therefore, it is difficult for the physiological activity and the yield of the crop yield improving substance produced in the cells to decrease, so that it is possible to produce a crop yield improving agent having an improved crop yield improving effect. Further, since the extraction process of the above-mentioned substance produced in the cells is not required, the modified cyanobacteria can be repeatedly used to produce the substance for improving the crop yield even after the substance is recovered.

The gene that expresses the protein involved in the binding between the outer membrane 5 and the cell wall 4 is, for example, at least one of the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9. There may be. In the modified cyanobacteria, at least one gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvic acid modifying enzyme 9 are deleted or inactivated. Therefore, in the modified cyanobacteria, for example, the expression of at least one of (i) SLH domain-retaining outer membrane protein 6 and cell wall-pyruvic acid modifying enzyme 9 is suppressed, or (ii) SLH domain-retaining outer membrane protein. 6 and cell wall-at least one function of pyruvate modifying enzyme 9 is suppressed or lost. Therefore, the binding (that is, the binding amount and binding force) between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalently bound sugar chain 3 on the surface of the cell wall 4 is reduced. As a result, the outer membrane 5 is easily detached from the cell wall 4 at the portion where the bond between the outer membrane 5 and the cell wall 4 is weakened. As a result, in the modified cyanobacteria, the binding between the outer membrane 5 and the cell wall 4 is reduced, so that the outer membrane 5 is more likely to be partially detached from the cell wall 4, so that the proteins and metabolites produced in the cells are bacterial. It becomes easy to leak out of the body. As a result, the crop yield improving substance produced in the cells of the modified cyanobacteria also easily leaks out of the cells.

In this embodiment, for example, SLH domain-retaining outer membrane protein 6 is used to suppress or eliminate at least one function of SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 in cyanobacteria. The encoding gene and the cell wall-the transcription of at least one of the genes encoding the pyruvate modifying enzyme 9 may be suppressed.

For example, the gene encoding the SLH domain-retaining outer membrane protein 6 may be slr1841, slr1908, slr0042, etc. when the parent cyanobacteria belongs to the genus Syenchocystis, or may be nies970_09470, etc. in the case of the genus Synechococcus. Often, if the parent cyanobacteria belongs to the genus Anabaena, it may be anacy_5815 or anacy_3458, etc., if the parent cyanobacteria belongs to the genus Microcystis, it may be A0A0F6U6F8_MICAE, etc. If the parent cyanobacteria is Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc., if the parent cyanobacteria is Calothrix, it may be A0A1Z4R6U0_9CYAN, etc., if the parent cyanobacteria is Nostoc, A0A1C0VG86_9NOSO, etc. If the parent cyanobacteria belongs to the genus Crocosphaera, it may be B1WRN6_CROS5 or the like, and if the parent cyanobacteria belongs to the genus Pleurocapsa, it may be K9TAE4_9CYAN or the like. The nucleotide sequences of these genes can be obtained from the NCBI database described above or Cyanobase.

More specifically, the genes encoding SLH domain-retaining outer membrane protein 6 are slr1841 (SEQ ID NO: 7) of Synechocystis sp. PCC 6803, nies970_09470 (SEQ ID NO: 8) of Synechococcus sp. NIES-970, and Anabaena cylindrica PCC. It may be anacy_3458 (SEQ ID NO: 9) of 7122, or a gene whose amino acid sequence is 50% or more identical to these genes.

Thereby, in the modified cyanobacteria, it is 50% or more identical to the gene encoding any of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 7 to 9 above or the base sequence of any of these genes. The gene is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) expression of any of the above SLH domain-retaining outer membrane proteins 6 or a protein having the same function as any of these proteins is suppressed, or (ii) the above. The function of any of the SLH domain-retaining outer membrane proteins 6 or a protein having a function equivalent to that of any of these proteins is suppressed or lost. As a result, in the modified cyanobacteria, the binding domain (for example, SLH domain 7) for the outer membrane 5 to bind to the cell wall 4 has a reduced amount and binding force to bind to the cell wall 4, so that the outer membrane 5 is attached to the cell wall 4 from the cell wall 4. It becomes easier to partially separate. As a result, proteins and metabolites produced in the cells are likely to leak out of the cells, so that the crop yield improving substance produced in the cells is also likely to leak out of the cells.

As described above, if the amino acid sequence of a protein is 30% or more the same, it is said that there is a high possibility that the protein has the same function as the protein. Therefore, if the base sequence of the gene encoding the protein is 30% or more the same, it is highly likely that a protein having the same function as the protein will be expressed. Therefore, the gene encoding the SLH domain-retaining outer membrane protein 6 whose function is suppressed or lost is, for example, any of the genes encoding the SLH domain-retaining outer membrane protein 6 represented by SEQ ID NOs: 7 to 9 above. A base having the same identity with the base sequence of 40% or more, preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, still more preferably 90% or more. It may be a gene consisting of a sequence and a gene encoding a protein or polypeptide having a function of binding to a covalent bond type sugar chain 3 of the cell wall 4.

Further, for example, the gene encoding the cell wall-pyruvate modifying enzyme 9 may be slr0688 or the like when the parent cyanobacteria belongs to the genus Syenchocystis, or syn7502_03092 or synpcc7942_1529 or the like when the parent cyanobacteria belongs to the genus Synechococcus. If the parent cyanobacteria belongs to the genus Anabaena, it may be ana_C20348 or anacy_1623, etc., and if the parent cyanobacteria belongs to the genus Microcystis, it may be csaB (NCBI access ID: TRU80220), etc. If is Cynahothese, it may be csaB (NCBI access ID: WP_107667006.1), etc., and if the parent cyanobacteria is Spirulina, it may be csaB (NCBI access ID: WP_026079530.1), etc. , If the parent cyanobacteria belongs to the genus Calothrix, it may be csaB (NCBI access ID: WP_096658142.1), etc., and if the parent cyanobacteria belongs to the genus Nostoc, it may be csaB (NCBI access ID: WP_099068528.1), etc. If the parent cyanobacteria belongs to the genus Crocosphaera, it may be csaB (NCBI access ID: WP_012361697.1), etc., and if the parent cyanobacteria belongs to the genus Pleurocapsa, csaB (NCBI access ID: WP_036798735). And so on. The nucleotide sequences of these genes can be obtained from the NCBI database described above or Cyanobase.

More specifically, the gene encoding the cell wall-pyruvic acid modifying enzyme 9 is slr0688 (SEQ ID NO: 10) of Synechocystis sp. PCC 6803, synpcc7942_1529 (SEQ ID NO: 11) of Synechococcus sp. PCC 7942, or Anabaena cylindrica PCC. It may be anacy_1623 (SEQ ID NO: 12) of 7122. Further, it may be a gene whose base sequence is 50% or more identical to these genes.

Thereby, in the modified cyanobacteria, 50% or more of the base sequence of the gene encoding any cell wall-pyruvic acid modifying enzyme 9 shown in SEQ ID NOs: 10 to 12 above or the gene encoding any one of these enzymes. Genes that are identical are deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above cell wall-pyruvic acid modifying enzymes 9 or a protein having the same function as any of these enzymes is suppressed, or (ii) the above. The function of any cell wall-pyruvic acid modifying enzyme 9 or a protein having the same function as any of these enzymes is suppressed or lost. As a result, the covalently bound sugar chain 3 on the surface of the cell wall 4 is less likely to be modified with pyruvic acid, so that the sugar chain 3 of the cell wall 4 becomes the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. The amount of binding and the binding force are reduced. Therefore, in the modified cyanobacteria according to the present embodiment, the amount of the sugar chain 3 for binding the cell wall 4 to the outer membrane 5 is reduced by pyruvate, so that the binding force between the cell wall 4 and the outer membrane 5 is reduced. Is weakened, and the outer membrane 5 is more likely to partially detach from the cell wall 4. As a result, proteins and metabolites produced in the cells are likely to leak out of the cells, so that the crop yield improving substance produced in the cells is also likely to leak out of the cells.

As described above, if the base sequence of the gene encoding the protein is 30% or more the same, it is considered that there is a high possibility that a protein having the same function as the protein will be expressed. Therefore, the gene encoding the cell wall-pyruvate modifying enzyme 9 whose function is suppressed or lost is, for example, any of the genes encoding the cell wall-pyruvate modifying enzyme 9 shown in SEQ ID NOs: 10 to 12 above. From a base sequence having an identity of 40% or more, preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, still more preferably 80% or more, still more preferably 90% or more. It may also be a gene encoding a protein or polypeptide having a function of catalyzing a reaction of modifying a covalently bound sugar chain 3 of the peptide glycan 2 of the cell wall 4 with pyruvate.

[5. Method for producing modified cyanobacteria]
Subsequently, a method for producing modified cyanobacteria in the present embodiment will be described. The method for producing a modified cyanobacteria comprises a step of suppressing or abolishing the function of a protein involved in the binding between the outer membrane 5 and the cell wall 4 in the cyanobacteria.

In this embodiment, the protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvic acid modifying enzyme 9.

The means for suppressing or losing the function of the protein is not particularly limited, but for example, the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9 are deleted or deleted. Even by inactivating, inhibiting the transcription of these genes, inhibiting the translation of transcripts of these genes, or administering inhibitors that specifically inhibit these proteins, etc. good.

The means for deleting or inactivating the above gene is, for example, introduction of a mutation to one or more bases on the base sequence of the relevant gene, substitution of the relevant base sequence with another base sequence, or other base sequences. It may be inserted, or a part or all of the base sequence of the relevant gene may be deleted.

The means for inhibiting transcription of the gene is, for example, introduction of a mutation into the promoter region of the gene, inactivation of the promoter by substitution with another base sequence or insertion of another base sequence, or CRISPR interference method (non-CRISPR interference method). Patent Document 8: Yao et al., ACS Synth. Biol., 2016, 5: 207-212) and the like may be used. The specific method for introducing a mutation or substituting or inserting a base sequence may be, for example, ultraviolet irradiation, site-specific mutagenesis, or a homologous recombination method.

Further, the means for inhibiting the translation of the transcript of the gene may be, for example, RNA (Ribonucleic Acid) interferometry or the like.

By using any of the above means, modified cyanobacteria may be produced by suppressing or abolishing the function of the protein involved in the binding between the outer membrane 5 and the cell wall 4 in cyanobacteria.

As a result, in the modified cyanobacteria produced by the above-mentioned production method, the binding between the cell wall 4 and the outer membrane 5 (that is, the binding amount and the binding force) is partially reduced, so that the outer membrane 5 is partially reduced from the cell wall 4. It becomes easy to detach. As a result, in modified cyanobacteria, intracellularly produced substances such as proteins and metabolites produced in the cells are likely to leak out of the outer membrane 5 (that is, out of the cells), and thus, for example, pyroglutamic acid. And substances involved in crop yield improvement such as aminobenzoic acid (that is, crop yield improving substances) are also likely to leak out of the cells. Therefore, according to the method for producing modified cyanobacteria in the present embodiment, it is possible to provide modified cyanobacteria having improved secretion productivity of a crop yield improving substance.

Further, in the modified cyanobacteria produced by the production method in the present embodiment, the crop yield improving substance produced in the cells leaks out of the cells, so that it is necessary to crush the cells in order to recover the substances. There is no. For example, since the modified cyanobacteria may be cultured under appropriate conditions and then the crop yield improving substance secreted into the culture solution may be recovered, the crop yield improving substance in the culture solution is recovered while culturing the modified cyanobacteria. It is also possible. Therefore, by using the modified cyanobacteria obtained by this production method, it is possible to efficiently produce a microbiological crop yield improving substance. Therefore, according to the method for producing modified cyanobacteria in the present embodiment, it is possible to provide highly efficient modified cyanobacteria that can be used repeatedly even after the crop yield improving substance is recovered.

[6. How to improve crop yield]
In the crop yield improving method according to the present embodiment, the above crop yield improving agent is used for the crop. As described above, since the crop yield improving agent according to the present embodiment is a crop yield improving agent having an improved crop yield improving effect, the crop can be effectively harvested by using the above crop yield improving agent for the crop. Yield can be improved.

The above-mentioned crop yield improver may be used as it is, or may be concentrated or diluted. In applying the crop yield improver to crops, the concentration of the crop yield improver and the method of application may be appropriately determined according to the type of crop, the nature of the soil, the purpose, and the like. The crop yield improving agent may be, for example, a culture solution of modified cyanobacteria itself, or a solution obtained by removing bacterial cells of modified cyanobacteria from the culture solution, and a desired substance may be applied to the membrane from the culture solution. It may be an extract extracted by a technique or the like. The desired substance is, for example, a substance involved in improving the yield of crops, may be an enzyme that decomposes nutrients in soil, and is a substance that solubilizes insoluble substances (for example, metals such as iron) in soil. It may be (for example, a substance having a chelating effect) or a substance that improves the intracellular physiological activity of the crop. Further, the method of applying the crop yield improver to a plant may be, for example, spraying, irrigation, or mixing of the plant or soil. More specifically, several milliliters per plant may be added to the root of the plant about once a week.

Hereinafter, the modified cyanobacteria of the present disclosure, the method for producing the modified cyanobacteria, and the method for producing the crop yield improver will be specifically described in Examples, but the present disclosure is not limited to the following Examples. do not have.

In the following examples, as a method for partially removing the outer membrane of cyanobacteria from the cell wall, suppression of the expression of the slr1841 gene encoding the SLH domain-retaining outer membrane protein (Example 1) and cell wall-pyruvate modification The expression of the slr0688 gene encoding the enzyme was suppressed (Example 2), and two types of modified cyanobacteria were produced. Then, the secretory productivity of the proteins of these modified cyanobacteria was measured, and the secreted intracellularly produced substances (here, proteins and intracellular metabolites) were identified. The cyanobacterial species used in this example is Synechocystis sp. PCC 6803 (hereinafter, simply referred to as "cyanobacteria").

(Example 1)
In Example 1, a modified cyanobacteria in which the expression of the slr1841 gene encoding the SLH domain-retaining outer membrane protein was suppressed was produced.

(1) Construction of a cyanobacterial modified strain in which the expression of the slr1841 gene was suppressed As a gene expression suppression method, a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interferometry was used. In this method, the expression of the slr1841 gene is suppressed by introducing the gene encoding the dCas9 protein (hereinafter referred to as the dCas9 gene) and the slr1841_sgRNA (single-guide Ribonucleic Acid) gene into the chromosomal DNA of cyanobacteria. Can be done.

The mechanism of gene expression suppression by this method is as follows.

First, a Cas9 protein (dCas9) lacking nuclease activity and an sgRNA (slr1841_sgRNA) that complementarily binds to the base sequence of the slr1841 gene form a complex.

This complex then recognizes the slr1841 gene on the cyanobacterial chromosomal DNA and specifically binds to the slr1841 gene. This binding becomes a steric hindrance, which inhibits transcription of the slr1841 gene. As a result, the expression of the cyanobacterial slr1841 gene is suppressed.

Hereinafter, a method for introducing each of the above two genes into the chromosomal DNA of cyanobacteria will be specifically described.

(1-1) Introduction of dCas9 gene
Using the chromosomal DNA of Synechocystis LY07 strain (hereinafter also referred to as LY07 strain) (see Non-Patent Document 8) as a template, the dCas9 gene, the operator gene for controlling the expression of the dCas9 gene, and spectinomycin as a marker for gene transfer. The resistance marker gene was amplified by the PCR (Polymerase Chain Reaction) method using the primers psbA1-Fw (SEQ ID NO: 13) and psbA1-Rv (SEQ ID NO: 14) shown in Table 1. In the LY07 strain, since the above three genes are inserted into the psbA1 gene on the chromosomal DNA in a linked state, they can be amplified as one DNA fragment by the PCR method. Here, the obtained DNA fragment is referred to as "psbA1 :: dCas9 cassette". Using the In-Fusion PCR cloning method (registered trademark), the psbA1 :: dCas9 cassette was inserted into the pUC19 plasmid to obtain the pUC19-dCas9 plasmid.

The obtained pUC19-dCas9 plasmid (1 μg) was mixed with a cyanobacterial culture solution (cell concentration OD730 = about 0.5), and the pUC19-dCas9 plasmid was introduced into the cells of cyanobacteria by natural transformation. Transformed cells were selected by growing on BG-11 agar medium containing 20 μg / mL spectinomycin. In the selected cells, homologous recombination occurs between the psbA1 gene on the chromosomal DNA and the psbA1 upstream fragment region and the psbA1 downstream fragment region on the pUC19-dCas9 plasmid. As a result, a Synechocystis dCas9 strain in which a dCas9 cassette was inserted into the psbA1 gene region was obtained. The composition of the BG-11 medium used is as shown in Table 2.

(1-2) Introduction of slr1841_sgRNA gene
In CRISPR interferometry, sgRNA specifically binds to a target gene by introducing a sequence of about 20 bases complementary to the target sequence into a region called protospacer on the sgRNA gene. The protospacer sequences used in this example are shown in Table 3.

In the Synechocystis LY07 strain, the sgRNA gene (excluding the protospacer region) and the kanamycin resistance marker gene are linked and inserted into the slr2030-slr2031 gene on the chromosomal DNA. Therefore, sgRNA (slr1841_sgRNA) that specifically recognizes slr1841 by adding a protospacer sequence (SEQ ID NO: 21) complementary to the slr1841 gene (SEQ ID NO: 7) to the primer used when amplifying the sgRNA gene by the PCR method. ) Can be easily obtained.

First, using the chromosomal DNA of the LY07 strain as a template, the set of primers slr2030-Fw (SEQ ID NO: 15) and sgRNA_slr1841-Rv (SEQ ID NO: 16) shown in Table 1, and sgRNA_slr1841-Fw (SEQ ID NO: 17) and slr2031- Two DNA fragments were amplified by PCR using the set of Rv (SEQ ID NO: 18).

Subsequently, the mixed solution of the above DNA fragments was used as a template and amplified by the PCR method using the primers slr2030-Fw (SEQ ID NO: 15) and slr2031-Rv (SEQ ID NO: 18) shown in Table 1 to obtain ((SEQ ID NO: 15). A DNA fragment (slr2030-2031 :: slr1841_sgRNA) was obtained in which i) slr2030 gene fragment, (ii) slr1841_sgRNA, (iii) kanamycin resistance marker gene, and (iv) slr2031 gene fragment were sequentially linked. Using the In-Fusion PCR cloning method (registered trademark), slr2030-2031 :: slr1841_sgRNA was inserted into the pUC19 plasmid to obtain the pUC19-slr1841_sgRNA plasmid.

The pUC19-slr1841_sgRNA plasmid was introduced into Synechocystis dCas9 strain by the same method as in (1-1) above, and transformed cells were selected on BG-11 agar medium containing 30 μg / mL kanamycin. As a result, a transformant Synechocystis dCas9 slr1841_sgRNA strain (hereinafter, also referred to as slr1841 inhibitory strain) in which slr1841_sgRNA was inserted into the slr2030-slr2031 gene on the chromosomal DNA was obtained.

(1-3) Suppression of slr1841 gene The promoter sequences of the dCas9 gene and slr1841_sgRNA gene are designed so that their expression is induced in the presence of anhydrotetracycline (aTc). In this example, the expression of the slr1841 gene was suppressed by adding a final concentration of 1 μg / mL aTc to the medium.

(Example 2)
In Example 2, a modified cyanobacteria in which the expression of the slr0688 gene encoding the cell wall-pyruvic acid modifying enzyme was suppressed was obtained by the following procedure.

(2) Construction of a modified cyanobacterial strain in which the expression of the slr0688 gene is suppressed By the same procedure as in (1-2) above, an sgRNA containing a protospacer sequence (SEQ ID NO: 22) complementary to the slr0688 gene (SEQ ID NO: 4). The gene was introduced into the Synechocystis dCas9 strain to obtain the Synechocystis dCas9 slr0688_sgRNA strain. The set of primers slr2030-Fw (SEQ ID NO: 15) and sgRNA_slr0688-Rv (SEQ ID NO: 19) shown in Table 1 and the set of sgRNA_slr0688-Fw (SEQ ID NO: 20) and slr2031-Rv (SEQ ID NO: 18) are used. In-Fusion PCR was used and DNA fragments (slr2030-2031 :: slr0688_sgRNA) in which (i) slr2030 gene fragment, (ii) slr0688_sgRNA, (iii) canamycin resistance marker gene, and (iv) slr2031 gene fragment were linked in order. The procedure was carried out under the same conditions as in (1-2) above, except that the pUC19-slr0688_sgRNA plasmid was obtained by inserting into the pUC19 plasmid using the cloning method (registered trademark).

Furthermore, the expression of the slr0688 gene was suppressed by the same procedure as in (1-3) above.

(Comparative Example 1)
In Comparative Example 1, a Synechocystis dCas9 strain was obtained by the same procedure as in (1-1) of Example 1.

Subsequently, for the strains obtained in Example 1, Example 2 and Comparative Example 1, the state of the cell surface layer was observed and the protein secretion productivity test was carried out, respectively. The details will be described below.

(3) Observation of the state of the cell surface layer of the strain The modified cyanobacteria Synechocystis dCas9 slr1841_sgRNA strain obtained in Example 1 (that is, the slr1841 inhibitory strain) and the modified cyanobacteria Synechocystis dCas9 slr0688_sgRNA strain obtained in Example 2 (hereinafter referred to as “slr1841 inhibitory strain”). Ultra-thin sections of each of the slr0688 inhibitory strain) and the modified cyanobacteria Synechocystis dCas9 strain (hereinafter referred to as Control strain) obtained in Comparative Example 1 were prepared, and the state of the cell surface layer (in other words, in other words) was prepared using an electron microscope. And the outer membrane structure) was observed.

(3-1) Culturing of strains The slr1841 inhibitory strain of Example 1 was inoculated into a BG-11 medium containing 1 μg / mL aTc so that the initial cell concentration was OD730 = 0.05, and the light intensity was 100 μmol / m ² / s. , 30 ° C., shake culture for 5 days. The slr0688-suppressed strain of Example 2 and the Control strain of Comparative Example 1 were also cultured under the same conditions as in Example 1.

(3-2) Preparation of Ultrathin Section of Strain The culture solution obtained in (3-1) above was centrifuged at 2,500 g at room temperature for 10 minutes, and the cells of the slr1841 inhibitory strain of Example 1 were recovered. .. The cells were then snap frozen in liquid propane at -175 ° C and then fixed at -80 ° C for 2 days with an ethanol solution containing 2% glutaraldehyde and 1% tannic acid. The fixed cells were dehydrated with ethanol, and the dehydrated cells were impregnated into propylene oxide and then submerged in a resin (Quetol-651) solution. Then, the cells were allowed to stand at 60 ° C. for 48 hours to cure the resin, and the cells were embedded with the resin. The cells in the resin were sliced to a thickness of 70 nm using an ultramicrotome (Ultracut) to prepare ultrathin sections. This ultrathin section was stained with 2% uranium acetate and 1% lead citrate solution to prepare a transmission electron microscope sample of the slr1841 inhibitory strain of Example 1. The same operation was performed for the slr0688-suppressed strain of Example 2 and the Control strain of Comparative Example 1, and a sample of a transmission electron microscope was prepared.

(3-3) Observation with an electron microscope Using a transmission electron microscope (JEOL JEM-1400Plus), the ultrathin sections obtained in (3-2) above were observed under an acceleration voltage of 100 kV. The observation results are shown in FIGS. 3 to 8.

First, the slr1841 inhibitory strain of Example 1 will be described. FIG. 3 is a TEM (Transmission Electron Microscope) image of the slr1841 inhibitory strain of Example 1. FIG. 4 is an enlarged image of the broken line region A in FIG. FIG. 4A is an enlarged TEM image of the broken line region A of FIG. 3, and FIG. 4B is a diagram depicting an enlarged TEM image of FIG. 4A.

As shown in FIG. 3, in the slr1841 inhibitory strain of Example 1, the adventitia was partially exfoliated from the cell wall (that is, the adventitia was partially exfoliated) and the adventitia was partially flexed. board.

When the broken line region A was magnified and observed in order to confirm the state of the cell surface layer in more detail, the outer membrane was partially peeled off as shown in FIGS. 4 (a) and 4 (b). The part (one-dot dashed line area a1 and a2 in the figure) could be confirmed. In addition, a portion where the outer membrane was greatly bent could be confirmed near the one-dot broken line region a1. This part is a part where the bond between the outer membrane and the cell wall is weakened, and it is considered that the outer membrane is expanded outward and bent because the culture solution permeated into the periplasm from the outer membrane.

Subsequently, the slr0688 inhibitory strain of Example 2 will be described. FIG. 5 is a TEM image of the slr0688 inhibitory strain of Example 2. FIG. 6 is an enlarged image of the broken line region B of FIG. FIG. 6A is an enlarged TEM image of the broken line region B of FIG. 5, and FIG. 6B is a diagram depicting an enlarged TEM image of FIG. 6A.

As shown in FIG. 5, in the slr0688 inhibitory strain of Example 2, the outer membrane was partially exfoliated from the cell wall and the outer membrane was partially bent. In addition, in the slr0688 inhibitory strain, it was confirmed that the adventitia was partially detached from the cell wall.

In order to confirm the state of the cell surface layer in more detail, the dashed line region B was magnified and observed. It was possible to confirm the one-dot broken line region b1) in the figure and the portion where the outer membrane was partially peeled off (one-dot dashed line region b2 and b3 in the figure). In addition, it was confirmed that the outer membrane was detached from the cell wall in the vicinity of each of the one-dot broken line regions b1, b2 and b3.

Subsequently, the Control strain of Comparative Example 1 will be described. FIG. 7 is a TEM image of the Control strain of Comparative Example 1. FIG. 8 is an enlarged image of the broken line region C of FIG. 7. FIG. 8A is an enlarged TEM image of the broken line region C of FIG. 7, and FIG. 8B is a diagram depicting an enlarged TEM image of FIG. 8A.

As shown in FIG. 7, the cell surface layer of the Control strain of Comparative Example 1 was arranged, and the intima, the cell wall, the outer membrane, and the S layer were kept in a laminated state in order. That is, in the Control strain, the portion where the outer membrane was detached from the cell wall, the portion where the outer membrane was detached from the cell wall (that is, peeled off), and the portion where the outer membrane was bent as in Examples 1 and 2 were I couldn't see it.

(4) Identification of secreted intracellular metabolites (4-1) Sample preparation Add 20 μl aqueous solution adjusted to 1,000 μM of the internal standard substance concentration to 80 μl of the culture supernatant of the modified cyanobacteria and stir. After ultrafiltration, it was subjected to measurement.

(4-2) CE (Capillary Electrophoresis) -TOFMS (Time-Of-Flight Mass Spectrometry) Analysis In this test, cation mode and anion mode were measured under the conditions shown below.

[Cation mode]
Equipment: Agilent CE-TOFMS system
Capillary: Fused silica capillary id 50 μm × 80 cm
Measurement condition:
Run buffer: Cation buffer solution (p / n: H3301-1001)
CE voltage: Positive, 30kV
MS ionization: ESI positive
MS scan range: m / z 50-1,000
[Anion mode]
Equipment: Agilent CE-TOFMS system
Capillary: Fused silica capillary id 50 μm × 80 cm
Measurement condition:
Run buffer: Anion buffer solution (p / n: H3301-1001)
CE voltage: Positive, 30kV
MS ionization: ESI negative
MS scan range: m / z 50-1,000

(4-3) Data processing
For the peaks detected by CE-TOFMS, peaks with a signal / noise ratio of 3 or more were automatically detected using the automatic integration software MasterHands (registered trademark) ver.2.17.1.11. All the detected peaks registered in the metabolite library of HMT (Human Metabolite Technologies Co., Ltd.) based on the mass-to-charge ratio (m / z) peculiar to each metabolite and the value of migration time. The metabolites contained in the culture supernatant of the modified cyanobacteria were searched for by comparing with the value of the substance. The tolerance for the search was +/- 0.5 min for migration time and +/- 10 ppm for m / z. Concentrations were calculated for each identified metabolite as a 100 μM check. The major metabolites identified are shown in Table 4.

All of these 12 intracellular metabolites were contained in the culture supernatants of the slr1841 inhibitory strain of Example 1 and the slr0688 inhibitory strain of Example 2, respectively. Although no data are included, the culture supernatant of the Control strain of Comparative Example 1 did not contain these metabolites. Based on this result, in the modified strains of Examples 1 and 2, intracellular metabolites containing crop yield-enhancing substances such as pyroglutamic acid and aminobenzoic acid were released from the outer membrane by partially detaching the outer membrane from the cell wall. It was confirmed that it was easy to leak to the outside of the cell (that is, outside the cells).

(5) Protein secretion productivity test The slr1841 inhibitory strain of Example 1, the slr0688 inhibitory strain of Example 2, and the Control strain of Comparative Example 1 were cultured, and the amount of protein secreted extracellularly (hereinafter, secreted). (Also called the amount of protein) was measured. The secretory productivity of each of the above strains was evaluated based on the amount of protein in the culture medium. The secretory productivity of a protein means the ability to produce a protein by secreting the protein produced inside the cell extracellularly. Hereinafter, a specific method will be described.

(5-1) Culturing of strain The slr1841 inhibitory strain of Example 1 was cultured by the same method as in (3-1) above. The culture was independently performed 3 times. The strains of Example 2 and Comparative Example 1 were also cultured under the same conditions as those of Example 1.

(5-2) Quantification of extracellularly secreted protein The culture solution obtained in (5-1) above was centrifuged at 2,500 g at room temperature for 10 minutes to obtain a culture supernatant. The obtained culture supernatant was filtered using a membrane filter having a pore size of 0.22 μm, and the cells of the slr1841 inhibitory strain of Example 1 were completely removed. The total amount of protein contained in the culture supernatant after filtration was quantified by the BCA (Bicinchoninic Acid) method. This series of operations was performed for each of the three independently cultured culture solutions, and the average value and standard deviation of the amount of protein secreted extracellularly from the slr1841 inhibitory strain of Example 1 were determined. For the strains of Example 2 and Comparative Example 1, the proteins of the three culture broths were quantified under the same conditions, respectively, and the average value and standard deviation of the protein amounts in the three culture broths were obtained.

The results are shown in FIG. FIG. 9 is a graph showing the amount of protein (n = 3, error bar = SD) in the culture medium of the modified cyanobacteria of Example 1, Example 2 and Comparative Example 1.

As shown in FIG. 9, both the slr1841 inhibitory strain of Example 1 and the slr0688 inhibitory strain of Example 2 had the amount of protein secreted in the culture supernatant (mg / mg /) as compared with the Control strain of Comparative Example 1. L) was improved about 25 times.

Although the description of the data is omitted, when the absorbance (730 nm) of the culture solution was measured and the amount of secreted protein (mg protein / g cell dry weight) per 1 g of dry cell weight was calculated, the slr1841 inhibitory strain of Example 1 was calculated. In all of the slr0688-suppressed strains of Example 2, the amount of secreted protein (mg protein / g cell dry weight) per 1 g of dry cell weight was improved by about 36 times as compared with the Control strain of Comparative Example 1. rice field.

In addition, as shown in FIG. 9, the gene encoding the cell wall-pyruvic acid modifying enzyme (slr1841) is higher than that of the slr1841 inhibitory strain of Example 1 in which the expression of the gene encoding the SLH domain-retaining outer membrane protein (slr1841) is suppressed (slr1841). The slr0688-suppressed strain of Example 2 in which the expression of slr0688) was suppressed had a larger amount of protein secreted in the culture supernatant. This may be related to the fact that the number of covalent sugar chains on the cell wall surface is higher than the number of SLH domain-retaining outer membrane proteins (Slr1841) in the outer membrane. That is, since the slr0688-suppressed strain of Example 2 had a lower binding amount and binding force between the outer membrane and the cell wall than the slr1841-suppressed strain of Example 1, the amount of secreted protein suppressed slr1841 of Example 1. It is thought that there were more than stocks.

From the above results, by suppressing the function of the protein related to the binding between the outer membrane and the cell wall, the binding between the outer membrane and the cell wall of cyanobacteria is partially weakened, and the outer membrane is partially removed from the cell wall. I was able to confirm that it was released. It was also confirmed that the weakening of the bond between the outer membrane and the cell wall facilitates the leakage of intracellularly produced cyanobacterial proteins to the outside of the cell. Therefore, it was shown that the modified cyanobacteria according to the present embodiment and the method for producing the same have greatly improved the secretory productivity of the protein.

(6) Identification of secreted protein Subsequently, the secreted protein contained in the culture supernatant obtained in (5-2) above was identified by LC-MS / MS. The method will be described below.

(6-1) Sample preparation Eight times the amount of cold acetone was added to the amount of the culture supernatant, and the mixture was allowed to stand at 20 ° C. for 2 hours and then centrifuged at 20,000 g for 15 minutes to obtain a protein precipitate. .. To this precipitate was added 100 mM Tris pH 8.5, 0.5% sodium dodecanoate (SDoD) and the protein was lysed by a closed ultrasonic crusher. After adjusting the protein concentration to 1 μg / mL, dithiothreitol (DTT) having a final concentration of 10 mM was added, and the mixture was allowed to stand at 50 ° C. for 30 minutes. Subsequently, iodoacetamide (IAA) having a final concentration of 30 mM was added, and the mixture was allowed to stand at room temperature (light-shielded) for 30 minutes. To stop the IAA reaction, cysteine with a final concentration of 60 mM was added and allowed to stand at room temperature for 10 minutes. 400 ng of trypsin was added and allowed to stand overnight at 37 ° C. to fragment the protein into peptide fragments. After adding 5% TFA (Trifluoroacetic Acid), the mixture was centrifuged at 15,000 g at room temperature for 10 minutes to obtain a supernatant. This work removed SDoD. After desalting using a C18 spin column, the sample was dried by a centrifugal evaporator. Then, 3% acetonitrile and 0.1% formic acid were added, and the sample was dissolved using a closed ultrasonic crusher. The peptide concentration was adjusted to 200 ng / μL.

(6-2) LC-MS / MS analysis The sample obtained in (6-1) above was analyzed under the following conditions using an LC-MS / MS device (UltiMate 3000 RSLCnano LC System).

Sample injection amount: 200ng
Column: CAPCELL CORE MP 75 μm × 250 mm
Solvent: A solvent is 0.1% formic acid aqueous solution, B solvent is 0.1% formic acid + 80% acetonitrile Gradient program: B solvent 8% 4 minutes after sample injection, B solvent 44% 27 minutes later, B solvent 80%, 34 minutes later. Measurement ends after minutes

(6-3) Data analysis The obtained data was analyzed under the following conditions to identify proteins and peptides and calculate quantitative values.

Software: Scaffold DIA
Database: UniProtKB / Swiss Prot database (Synechocystis sp. PCC 6803)
Fragmentation: HCD
Precursor Tolerance: 8ppm
Fragment Tolerance: 10ppm
Data Acquisition Type: Overlapping DIA
Peptide Length: 8-70
Peptide Charge: 2-8
Max Missed Cleavages: 1
Fixed Modification: Carbamidomethylation
Peptide FDR: 1% or less

Table 4 shows the 30 types of proteins with the highest relative quantification values that are expected to have clear enzymatic activity.

All of these 6 proteins were contained in the culture supernatants of the slr1841 inhibitory strain of Example 1 and the slr0688 inhibitory strain of Example 2, respectively. All of these proteins retained the periplasmic (pointing to the gap between the adventitia and intima) transition signals. As a result, in the modified strains of Example 1 and Example 2, the protein in the periplasm is likely to leak out of the outer membrane (that is, outside the cells) due to the partial detachment of the outer membrane from the cell wall. I was able to confirm that. Therefore, it was shown that the modified cyanobacteria according to the present embodiment have significantly improved protein secretion productivity.

(7) Crop cultivation test Next, in order to evaluate the crop yield improving effect of pyroglutamic acid, aminobenzoic acid, and the secretion of modified cyanobacteria (here, the culture supernatant of modified cyanobacteria), the following crops A cultivation test was conducted. Specifically, in order to evaluate the effect of improving yield on leafy vegetable production, a cultivation test of lettuce and spinach was carried out. In addition, a tomato cultivation test was conducted to evaluate the effect of improving yield on fruit production. Hereinafter, each of these cultivation tests will be described.

(7-1) Lettuce Hydroponics Test The hydroponic culture solution contains 6% total nitrogen, 10% water-soluble phosphoric acid, 5% water-soluble potassium, 0.05% water-soluble bitter soil, 0.001% water-soluble manganese, and , A 500-fold diluted solution of a commercially available culture solution stock solution containing 0.005% of water-soluble boron was used. The plants were cultivated at room temperature (22 ° C.) for 35 days under the conditions of a white light source, a photon flux density of 200 μmol / m ² / s, a bright condition of 16 hours and a dark condition of 8 hours.

(Examples 3, 4, 5)
During the above cultivation period, one strain of (1) 1 μM pyroglutamic acid aqueous solution (Example 3), (2) 1 μM aminobenzoic acid aqueous solution (Example 4), and (3) modified cyanobacterial secretion (Example 5). An amount of 5 mL per week was added to the culture solution for hydroponic cultivation once a week. After harvesting, the weight of the strain was measured, and the mean value and standard deviation (SD) were obtained for (1) to (3), respectively. The modified cyanobacteria are the slr1841 inhibitory strain of Example 1 and the slr0688 inhibitory strain of Example 2.

(Comparative Example 2)
It was carried out in the same manner as in Examples 3 to 5 except that water was used instead of the above (1) to (3). After harvesting, the strain weight was measured to determine the mean and standard deviation (SD).

(result)
The results of Examples 3 to 5 and Comparative Example 2 are shown in FIGS. 10 and 11. FIG. 10 is a graph showing the relative average strain weight per lettuce strain cultivated in Examples 3 to 5 and Comparative Example 2. Further, FIG. 11 shows photographs of representative strains in order to visually show the states of the strains of Examples 3 to 5 and Comparative Example 2.

As shown in FIG. 10, the average weight of one lettuce strain harvested in Examples 3 to 5 was increased as compared with Comparative Example 2. Specifically, the average weight of one lettuce strain cultivated in Example 3 was increased by about 14% as compared with Comparative Example 2. In addition, the average weight of one lettuce strain cultivated in Example 4 was increased by about 15% as compared with Comparative Example 2. In addition, the average weight of one lettuce strain cultivated in Example 5 was increased by about 29% as compared with Comparative Example 2.

Further, as shown in FIG. 11, the lettuce strains cultivated in Examples 3 to 5 did not have a particularly noticeable change in the shape of the whole strain as compared with Comparative Example 2. That is, although the lettuce strains cultivated in Examples 3 to 5 grew faster than Comparative Example 2, no conspicuous physiological disorders (for example, chip burn) occurred, and the leaves were thickened and the leaves were grown. The size of the stems and leaves was also good, and the appearance was well-balanced.

(7-2) Spinach cultivation test Spinach seeds were sown in cultivation pots (12 cm x 10 cm) containing commercially available potting soil. Cultivation was carried out for 35 days under the conditions of 22 ° C., a photon bundle density of a white light source of 200 μmol / m ² / s, and a light condition of 12 hours and a dark condition of 12 hours. Meanwhile, each pot was supplied with 50 mL of distilled water every other day. Approximately one week after the start of cultivation, the cotyledons were thinned out at the stage of development, and the solid size in each pot was made uniform.

(Examples 6, 7, 8)
During the above cultivation period, one strain of (1) 1 μM pyroglutamic acid aqueous solution (Example 6), (2) 1 μM aminobenzoic acid aqueous solution (Example 7), and (3) modified cyanobacterial secretion (Example 8). An amount of 5 mL per week was added to the roots of cyanobacteria once a week. After harvesting, the weight of the strain was measured, and the mean value and standard deviation (SD) were obtained for (1) to (3), respectively. The modified cyanobacteria are the slr1841 inhibitory strain of Example 1 and the slr0688 inhibitory strain of Example 2.

(Comparative Example 3)
The procedure was the same as in Examples 6 to 8 except that water was used instead of the above (1) to (3). After harvesting, the strain weight was measured to determine the mean and standard deviation (SD).

(result)
The results of Examples 6 to 8 and Comparative Example 3 are shown in FIGS. 12 and 13. FIG. 12 is a graph showing the relative average weight per spinach plant cultivated in Examples 6 to 8 and Comparative Example 3. Further, FIG. 13 shows photographs of representative strains in order to visually show the states of the strains of Examples 6 to 8 and Comparative Example 3.

As shown in FIG. 12, the average weight of one spinach plant harvested in Examples 6 to 8 was increased as compared with Comparative Example 3. Specifically, the average weight of one spinach plant cultivated in Example 6 was increased by about 24% as compared with Comparative Example 3. In addition, the average weight of one spinach plant cultivated in Example 7 was increased by about 25% as compared with Comparative Example 3. In addition, the average weight of one spinach plant cultivated in Example 8 was increased by about 39% as compared with Comparative Example 3.

Further, as shown in FIG. 13, the spinach strains cultivated in Examples 6 to 8 did not have a particularly noticeable change in the shape of the whole strain as compared with Comparative Example 3. That is, although the spinach strains cultivated in Examples 6 to 8 grew faster than in Comparative Example 3, no conspicuous physiological disorders (for example, fading of leaf color) occurred, and the leaves were thickened. In addition, the size of the stems and leaves was good, and the appearance was well-balanced.

(7-3) Tomato cultivation test First, commercially available potting soil was placed in a cultivation planter (22 cm × 16 cm), and 3 tomato seeds were sown per planter. Cultivation was carried out for 150 days under the conditions of an indoor temperature of 23 ° C., a photon flux density of a white light source of 250 μmol / m ² / s, and a light condition of 16 hours and a dark condition of 10 hours. Meanwhile, each pot was supplied with 500 mL of distilled water every two days. Approximately one week after the start of cultivation, the cotyledons were thinned out at the stage of development, and the individual sizes in each planter were adjusted. Also, once every 50 days, use commercially available chemical fertilizers (total nitrogen 6%, water-soluble phosphoric acid 10%, water-soluble potassium 5%, water-soluble bitter soil 0.05%, water-soluble manganese 0.001%, water-soluble boron 0.005%). A 500-fold diluted solution of the containing undiluted solution) was applied in an amount of 500 mL per planter.

(Example 9)
As described above, after adjusting the individual size of each planter, 5 mL of modified cyanobacterial secretion per strain was added to the root once a week. The tomato fruits were cultivated for 150 days, and the tomato fruits were harvested in order from the red mature ones, and the number of harvested fruits was recorded. In addition, the weight and sugar content (Brix value) of the harvested fruits were measured, and their average value and standard deviation (SD) were determined. The modified cyanobacteria are the slr1841 inhibitory strain of Example 1 and the slr0688 inhibitory strain of Example 2.

(Comparative Example 4)
The procedure was the same as in Example 9 except that water was used instead of the secretion of the modified cyanobacteria.

(result)
The results of Example 9 and Comparative Example 4 are shown in FIGS. 14 to 17. FIG. 14 is a graph showing the average number of fruits per tomato plant cultivated in Example 9 and Comparative Example 4. FIG. 15 is a graph showing the average fruit weight per tomato strain cultivated in Example 9 and Comparative Example 4. FIG. 16 is a graph showing the average sugar content per tomato strain cultivated in Example 9 and Comparative Example 4.

Further, FIG. 17 shows photographs of representative fruits in order to visually show the state of the fruits of Example 9 and Comparative Example 4.

As shown in FIG. 14, the average number of fruits per plant harvested in Example 9 was increased by about 67% as compared with Comparative Example 4. However, as shown in FIG. 15, the average fruit weights per plant harvested in each of Example 9 and Comparative Example 4 were equivalent. Normally, the weight of the fruit tends to decrease as the yield of the fruit per strain increases, but in Example 9, the average fruit weight is a comparative example even though the number of fruits harvested is large. It was equivalent to 4.

In addition, as shown in FIG. 16, the average sugar content (Brix sugar content) of the fruits harvested in each of Example 9 and Comparative Example 4 was also the same. Further, as shown in FIG. 17, the tomato fruits harvested in each of Example 9 and Comparative Example 4 had no difference in appearance such as size, shape, and luster. Normally, as the yield of fruit per strain increases, the sugar content of the fruit tends to decrease and the size tends to decrease. However, in Example 9, although the number of fruits harvested is large, the fruit of the fruit tends to be small. The average sugar content and size were also the same as in Comparative Example 4.

Therefore, it was confirmed that when the secretory solution of modified cyanobacteria was applied to tomatoes, the yield could be increased without deteriorating the quality (that is, weight, size, and sugar content) of the fruits.

(summary)
From the results of the cultivation test of lettuce, spinach, and tomato, it was confirmed that the crop yield improving agent according to the present embodiment has a crop yield improving effect on a plurality of crop species.

According to the present disclosure, it is possible to provide a crop yield improver containing at least one of pyroglutamic acid and aminobenzoic acid. In addition, it is possible to provide modified cyanobacteria having improved secretory productivity of crop yield improving substances. Further, by culturing the modified cyanobacteria of the present disclosure, the above-mentioned substance can be efficiently produced, and for example, the yield of crops can be improved by adding the substance to soil or hydroponic solution.

1 Inner membrane 2 Peptidoglycan 3 Sugar chain 4 Cell wall 5 Outer membrane 6 SLH domain-retaining outer membrane protein 7 SLH domain 8 Organic channel protein 9 Cell wall-pyruvic acid modifying enzyme

Claims (10)

Contains at least one of pyrolutamic acid and aminobenzoic acid,
Crop yield improver. The method for producing a crop yield improver according to claim 1.
Steps to prepare modified cyanobacteria in which the function of proteins involved in the binding of the outer membrane to the cell wall is suppressed or lost in cyanobacteria,
A step of causing the modified cyanobacteria to secrete a secretion containing the crop yield improver,
including,
A method for producing a crop yield improver. The protein involved in the binding between the outer membrane and the cell wall is at least one of SLH (Surface Layer Homology) domain-retaining outer membrane protein and cell wall-pyruvic acid modifying enzyme.
The method for producing a crop yield improver according to claim 2. The SLH domain-retaining outer membrane protein is
Slr1841, consisting of the amino acid sequence shown in SEQ ID NO: 1,
NIES970_09470, consisting of the amino acid sequence shown in SEQ ID NO: 2,
Anacy_3458 consisting of the amino acid sequence shown in SEQ ID NO: 3 or
A protein having an amino acid sequence that is 50% or more identical to any of these SLH domain-retaining outer membrane proteins.
The method for producing a crop yield improver according to claim 3. The cell wall-pyruvic acid modifying enzyme
Slr0688, which consists of the amino acid sequence shown in SEQ ID NO: 4.
Synpcc7942_1529, which consists of the amino acid sequence shown in SEQ ID NO: 5.
Anacy_1623 consisting of the amino acid sequence shown in SEQ ID NO: 6 or
A protein having an amino acid sequence that is 50% or more identical to any of these cell wall-pyruvic acid modifying enzymes.
The method for producing a crop yield improver according to claim 3. A gene that expresses a protein involved in the binding between the outer membrane and the cell wall has been deleted or inactivated.
The method for producing a crop yield improver according to claim 2. The gene that expresses the protein involved in the binding between the outer membrane and the cell wall is at least one of the gene encoding the SLH domain-retaining outer membrane protein and the gene encoding the cell wall-pyruvic acid modifying enzyme.
The method for producing a crop yield improver according to claim 6. The gene encoding the SLH domain-retaining outer membrane protein is
Slr1841, consisting of the base sequence shown in SEQ ID NO: 7.
Nies970_09470, which consists of the base sequence shown in SEQ ID NO: 8.
Anacy_3458 consisting of the base sequence shown by SEQ ID NO: 9 or
A gene whose base sequence is 50% or more identical to any of these genes.
The method for producing a crop yield improver according to claim 7. The gene encoding the cell wall-pyruvic acid modifying enzyme is
Slr0688, which consists of the base sequence shown by SEQ ID NO: 10.
Synpcc7942_1529, which consists of the base sequence shown in SEQ ID NO: 11.
Anacy_1623 consisting of the base sequence shown by SEQ ID NO: 12 or
A gene whose base sequence is 50% or more identical to any of these genes.
The method for producing a crop yield improver according to claim 7. The crop yield improver according to claim 1 is used for the crop.
How to improve crop yield.

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