Method for producing plant quality improving agent, plant quality improving agent, and method for improving plant quality

Abstract

To provide a method for producing an agent for increasing plant quality that can easily and efficiently produce the agent for increasing plant quality.SOLUTION: The method for producing an agent for increasing plant quality comprises: a step of preparing a modified cyanobacteria in which the function of the protein involved in the binding between an outer membrane 5 and a cell wall 4 is suppressed or lost in cyanobacteria (step S01); and a step of causing the modified cyanobacteria to secrete the secretions involved in the quality increase of the plant (step S02).SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to a method for producing a plant quality improver, which is a natural metabolite contributing to the improvement of plant quality, a plant quality improver, and a method for using the plant quality improver.

Background Art

[0002] With the demand for increased food production accompanying the growth of the world population, the development of technologies for efficiently producing agricultural crops on limited arable land is required. Specifically, shortening the cultivation period until harvest, increasing the yield per cultivation area, improving the yield by reducing diseases and off-quality products, and improving the quality of agricultural products such as increasing the size and sugar content of fruits are desired. Approaches to achieve these are diverse, but in recent years, from the viewpoints of preventing global warming and reducing environmental impact, the use of bio-based raw materials towards non-fossil resources has been increasing. Among them, the utilization of natural-derived substances with low fossil energy consumption during the manufacturing process and low environmental impact during application (hereinafter also referred to as application or use) is particularly desired.

[0003] For example, as a method for promoting crop production by utilizing natural-derived substances, a method of inoculating a plant with a microorganism that produces a substance contributing to plant growth promotion (hereinafter also referred to as a plant growth promoting substance) or a culture solution of a microorganism (Patent Documents 1, 2, and Non-Patent Document 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 the soil and improve the availability of metal ions to 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 fertilizing a plant with a fertilizer containing an algal cell extract (Patent Document 5) are disclosed. Further, for example, a method of applying a neutral to alkaline water extract of licorice to increase the sugar content of fruits is disclosed (Patent Document 6). Further, a method of applying alginate oligosaccharide to improve the quality of fruits is disclosed (Patent Document 7). Further, for example, a method of reducing diseases such as blossom end rot of tomatoes by applying a calcium supplement containing a pentose derivative is disclosed (Patent Document 8). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Patent Publication No. 2018-11600 [Patent Document 2] Special Publication No. 63-501286 [Patent Document 3] Japanese Patent Publication No. 2014-073993 [Patent Document 4] Patent No. 5943844 [Patent Document 5] Patent No. 3143872 [Patent Document 6] Japanese Patent Publication No. 2015-70823 [Patent Document 7] Japanese Patent Application Publication No. 10-66449 [Patent Document 8] Japanese Patent Application Publication No. 7-89784 [Non-patent literature]

[0005] [Non-Patent Document 1] Jimenez-Gomez et al., “Probiotic activities of Rhizobium laguerreae on growth and quality of spinach”, Scientific reports, 2018, Vol. 8, 295 [Overview of the project] [Problems that the invention aims to solve]

[0006] However, as described above, when using naturally derived substances (hereinafter also referred to as plant quality-enhancing substances) that contribute to improving plant quality, such as promoting plant growth, increasing yield, increasing crop and fruit size, increasing fruit sugar content, reducing physiological disorders, and reducing diseases, the processes for producing the substance by microorganisms, extracting it, or purifying it are complicated and result in excessive costs. On the other hand, if the microorganisms that produce the substance are ingested by the plants, the complicated processes described above are unnecessary, but the effect varies depending on the combination of the microbial species used, the target plant species, and the soil properties, resulting in a lack of versatility and unstable effects. Furthermore, when applying substances that contribute to reducing diseases and increasing fruit sugar content to plants, the effect varies greatly depending on the substance applied, the timing of application according to the growth stage, and the combination of target crop species, resulting in a lack of versatility and unstable effects.

[0007] Therefore, this disclosure provides a simple and efficient method for producing a plant quality improving agent containing a naturally derived substance (hereinafter also referred to as a plant quality improving substance) that has the effect of improving the quality of plants. Furthermore, this disclosure provides a plant quality improving agent and a plant quality improving method that can efficiently improve the quality of plants. [Means for solving the problem]

[0008] A method for producing a plant quality enhancer according to one aspect of the present disclosure includes the steps of preparing modified cyanobacteria in which the function of proteins involved in the binding of the outer membrane to the cell wall is suppressed or lost, and causing the modified cyanobacteria to secrete secretions involved in plant quality enhancer. [Effects of the Invention]

[0009] According to the method for producing a plant quality improver of the present disclosure, a plant quality improver having an effect of improving the quality of plants can be produced simply and efficiently. Further, according to the plant quality improver of the present disclosure, the quality of plants can be effectively improved. Further, according to the plant quality improvement method of the present disclosure, by using the plant quality improver of the present disclosure for plants, the quality of plants can be effectively improved.

Brief Description of the Drawings

[0010] [Figure 1] Figure 1 is a flowchart showing an example of a method for producing a plant quality improver according to an embodiment. [Figure 2] Figure 2 is a diagram schematically showing the cell surface layer of cyanobacteria. [Figure 3] Figure 3 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 1. [Figure 4] Figure 4 is an enlarged image of the broken line area A in Figure 3. [Figure 5] Figure 5 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 2. [Figure 6] Figure 6 is an enlarged image of the broken line area B in Figure 5. [Figure 7] Figure 7 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Comparative Example 1. [Figure 8] Figure 8 is an enlarged image of the broken line area C in Figure 7. [Figure 9] Figure 9 is a graph showing the amount of protein in the culture solution of the modified cyanobacteria of Example 1, Example 2 and Comparative Example 1 (n = 3, error bar = SD). [Figure 10] Figure 10 is a graph showing the average number of fruits per tomato plant cultivated in Example 3 and Comparative Example 2. [Figure 11] Figure 11 is a graph showing the average fruit weight per tomato plant cultivated in Example 3 and Comparative Example 2. [Figure 12] Figure 12 is a graph showing the average sugar content per tomato plant cultivated in Example 3 and Comparative Example 2. [Figure 13] Figure 13 is a diagram showing the states of representative fruits of Example 3 and Comparative Example 2, respectively. [Figure 14] Figure 14 is a graph showing the average number of fruits per strawberry plant cultivated in Example 4 and Comparative Example 3. [Figure 15] Figure 15 is a graph showing the average fruit weight per strawberry plant cultivated in Example 4 and Comparative Example 3. [Figure 16] Figure 16 is a graph showing the average sugar content per strawberry plant cultivated in Example 4 and Comparative Example 3. [Figure 17] Figure 17 is a diagram showing the states of representative fruits of Example 4 and Comparative Example 3, respectively. [Figure 18] Figure 18 is a graph showing the average plant weight per lettuce plant cultivated in Example 5 and Comparative Example 4. [Figure 19] Figure 19 is a graph showing the ratio of lettuce plants with tip burn among the lettuce plants cultivated in Example 5 and Comparative Example 4. [Figure 20] Figure 20 is a diagram showing the states of representative plants of Example 5 and Comparative Example 4, respectively.

Embodiments for Carrying Out the Invention

[0011] (Findings on which the present disclosure is based) As described in the background art, there is a need for technologies to efficiently produce agricultural crops in limited arable land. In addition, in order to improve the quality of plants, the use of natural-derived substances with less environmental impact in application is required. Among them, substances with less fossil energy consumption during the production of such substances and less environmental impact are desired.

[0012] As technologies for improving the quality of plants, the following conventional technologies are disclosed.

[0013] For example, Patent Document 1 discloses a method for applying a microbial strain having plant growth-promoting activity or a culture of such a microbial strain to a plant or the area surrounding a plant (e.g., soil). It has been reported that this method can not only promote plant growth and increase yield, but also prevent the occurrence of pathogenic diseases in plants.

[0014] Furthermore, for example, Patent Document 2 discloses a method for producing a plant growth promoting composition obtained by mixing a bacterial culture solution and an algal culture solution and incubating the mixture under predetermined conditions, and then applying the composition to plants. This method has been reported to promote the vegetative growth of tomatoes when the composition is added to the nutrient solution used in hydroponic cultivation of plants.

[0015] Furthermore, for example, Non-Patent Document 1 discloses a method for applying a type of rhizobia to spinach, more specifically to the roots of spinach, as a plant probiotic bacterium with a plant growth-promoting mechanism. This method has been reported to produce growth-promoting effects, such as an increase in the number and size of leaves of spinach inoculated with the rhizobia.

[0016] Furthermore, for example, Patent Document 3 discloses a method for improving the utilization of metal ions by plants by adding natural metabolites (substances involved in metabolism that exist naturally) such as organic acids to soil, thereby chelating metal ions such as iron in the soil.

[0017] Furthermore, for example, Patent Document 4 discloses a method for applying a composition containing adenosine, a natural metabolite, as a main component to plants.

[0018] Furthermore, for example, Patent Document 5 discloses a method for applying a fertilizer containing algal cell extracts to plants. More specifically, the cell extracts are obtained by treating cyanobacteria with an aqueous solvent (e.g., water) at a temperature of 60°C or higher.

[0019] Furthermore, for example, Patent Document 6 discloses a method for increasing the sugar content of fruit by applying a neutral to alkaline aqueous extract of licorice.

[0020] Furthermore, for example, Patent Document 7 discloses a method for improving fruit quality by applying alginate oligosaccharides.

[0021] Furthermore, for example, Patent Document 8 discloses a method for reducing blossom-end rot and other diseases in tomatoes by applying a calcium supplement containing a pentose derivative.

[0022] However, as mentioned above, when using naturally derived substances to improve the quality of plants, the processes for producing, extracting, or purifying the substance by microorganisms are complicated and result in excessive costs. On the other hand, if the microorganisms that produce the substance are directly ingested by the plants, the complicated processes described above are unnecessary. However, the effect varies depending on the combination of microbial species used, target plant species, and soil properties, lacking versatility and resulting in an unstable effect on improving plant quality. Furthermore, when applying substances to plants that contribute to reducing disease and increasing the sugar content of fruits, the effect varies greatly depending on the substance applied, the timing of application according to the growth stage, and the combination of target crop species, lacking versatility and resulting in an unstable effect. Given these circumstances, there is a need for the development of plant quality-enhancing agents containing naturally derived substances (i.e., plant quality-enhancing substances) that can be produced with cheaper raw materials and simpler processes, and that have plant quality-enhancing effects on multiple crop species, such as increased yield, increased crop and fruit size, increased fruit sugar content, reduced physiological disorders, and reduced disease.

[0023] The inventors focused on cyanobacteria as microorganisms to be used in the production of plant quality enhancers. Cyanobacteria (also called blue-green bacteria or blue algae) are a group of eubacteria that produce oxygen by breaking down water through photosynthesis, and use the energy obtained to fix CO2 from the air. Depending on the species, cyanobacteria can also fix nitrogen (N2) from the air. Thus, since cyanobacteria can obtain most of the raw materials (i.e., nutrients) and energy necessary for cell growth from air, water, and light, cyanobacteria can be cultured using inexpensive raw materials and a simple process.

[0024] Furthermore, cyanobacteria are known to grow rapidly and have high light utilization efficiency, and because they are easier to genetically modify compared to other algal species, active research and development is being conducted on the production of substances using cyanobacteria among photosynthetic microorganisms. For example, the production of fuels such as ethanol, isobutanol, alkanes, and fatty acids (Patent Document 9: Japanese Patent No. 6341676) has been reported as an example of substance production using cyanobacteria. Research and development is also being conducted on the production of substances that serve as nutrients for living organisms. For example, since proteins can only be synthesized by living organisms, there is a need to develop technologies for producing proteins simply and efficiently. Cyanobacteria, which can utilize light energy and CO2 from the atmosphere, are expected to be one of the biological species to be used in such technologies, and active research and development is being conducted on them (Non-Patent Document 2: Jie Zhou et al., “Discovery of a super-strong promoter enable efficient production of heterologous proteins in cyanobacteria”, Scientific Reports, Nature Research, 2014, Vol.4, Article No.4500).

[0025] For example, the technology described in Non-Patent Document 2 above enables the efficient expression of heterologous genes in cyanobacteria. Using this technology, desired compounds and proteins can be produced within the cells of cyanobacteria (hereinafter also referred to as intracellular cells). However, since the desired compounds and proteins produced within the cells of cyanobacteria are not easily secreted outside the cells, it is necessary to lyse the cyanobacterial cells and extract the desired compounds and proteins produced within the cells.

[0026] Therefore, the inventors have found that by partially detaching the outer membrane covering the cell wall of cyanobacteria from the cell wall, desired compounds, proteins, and intracellular metabolites produced within the cyanobacteria cells are more easily secreted outside the cells. In this process, the inventors also discovered that the secretions from cyanobacteria have a quality-enhancing effect on multiple crop species. As a result, plant quality-enhancing substances secreted outside the cells can be efficiently recovered without disrupting the cyanobacteria cells. Furthermore, since operations such as extraction are unnecessary, the physiological activity of the plant quality-enhancing substances is less likely to be impaired, and therefore, a plant quality-enhancing agent containing these secretions can effectively improve plant quality.

[0027] Therefore, according to the method for producing a plant quality enhancer of this disclosure, a plant quality enhancer containing a substance that has a quality-enhancing effect on multiple crop species can be produced simply and efficiently. Furthermore, according to the plant quality enhancer of this disclosure, the quality of plants can be effectively improved. In addition, according to the method for improving plant quality of this disclosure, the quality of plants can be effectively improved by using the plant quality enhancer of this disclosure on plants.

[0028] (Summary of this disclosure) An overview of one aspect of this disclosure is as follows:

[0029] A method for producing a plant quality enhancer according to one aspect of the present disclosure includes the steps of preparing modified cyanobacteria in which the function of proteins involved in the binding of the outer membrane to the cell wall is suppressed or lost, and causing the modified cyanobacteria to secrete secretions involved in improving the quality of plants.

[0030] As a result, in modified cyanobacteria, the binding between the cell wall and the outer membrane (e.g., the amount and strength of the binding) is partially reduced, making it easier for the outer membrane to partially detach from the cell wall. Therefore, proteins and metabolites produced within the cell (hereinafter also referred to as intracellular products) are more easily leaked outside the outer membrane, i.e., outside the cell. This makes it easier for proteins and metabolites produced within the modified cyanobacteria to be secreted outside the cell, eliminating the need for extraction treatments of intracellular products, such as cell lysis. Therefore, plant quality improvement agents containing secretions from modified cyanobacteria can be produced simply and efficiently. Furthermore, since the above-mentioned extraction treatment of intracellular products is unnecessary, a decrease in the physiological activity and yield of intracellular products is less likely to occur. Therefore, a decrease in the physiological activity and yield of substances among the intracellular products of modified cyanobacteria that are involved in improving plant quality (i.e., plant quality improvement substances) is also less likely to occur. As a result, the effect of the secretions from modified cyanobacteria on improving plant quality (hereinafter also referred to as the plant quality improvement effect) is enhanced. Furthermore, since the extraction process of intracellularly produced substances is unnecessary, the modified cyanobacteria can be repeatedly used to produce intracellularly produced substances even after the intracellularly produced substances secreted outside the cell have been recovered. Therefore, it is not necessary to prepare new modified cyanobacteria each time a plant quality-enhancing agent is manufactured. Accordingly, according to one aspect of the present disclosure, a plant quality-enhancing agent with improved plant quality-enhancing effects can be manufactured simply and efficiently.

[0031] For example, in a method for producing a plant quality enhancer according to one aspect of the present disclosure, the protein involved in the binding of the outer membrane to the cell wall may be at least one of an SLH (Surface Layer Homology) domain-retaining outer membrane protein and a cell wall-pyruvate modifying enzyme.

[0032] As a result, in modified cyanobacteria, for example, (i) the function of at least one of the SLH domain-retaining outer membrane proteins that bind to the cell wall and the enzyme that catalyzes the pyruvate modification of the glycans bound to the cell wall surface (i.e., cell wall-pyruvate modifying enzyme) is suppressed or lost, or (ii) the expression of at least one of the SLH domain-retaining outer membrane proteins and cell wall-pyruvate modifying enzymes is suppressed. Therefore, the binding (i.e., the amount and strength of binding) between the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane and the covalent glycans on the cell wall surface is reduced. This makes it easier for the outer membrane to detach from the cell wall in areas where the binding between the outer membrane and the cell wall is weakened. Consequently, in modified cyanobacteria, the binding between the outer membrane and the cell wall is reduced, making it easier for the outer membrane to partially detach from the cell wall, and thus making it easier for intracellular products such as proteins and metabolites produced within the bacterial cell to leak outside the cell, as described above. As a result, the modified cyanobacteria have improved secretory productivity, secreting plant-enhancing substances produced within their cells into the extracellular space. Therefore, according to one aspect of the present disclosure, the modified cyanobacteria can be made to secrete plant-enhancing substances efficiently, thus enabling the efficient production of plant-enhancing agents.

[0033] For example, in a method for producing a plant quality enhancer according to one aspect of the present disclosure, the SLH domain-retaining outer membrane protein may be 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 whose amino acid sequence is 50% or more identical to any of these SLH domain-retaining outer membrane proteins.

[0034] As a result, in modified cyanobacteria, for example, (i) the function of any SLH domain-retaining outer membrane protein shown in SEQ ID NOs: 1-3 above, or a protein whose amino acid sequence is 50% or more identical to any of these SLH domain-retaining outer membrane proteins, is suppressed or lost, or (ii) the expression of any SLH domain-retaining outer membrane protein shown in SEQ ID NOs: 1-3 above, or a protein whose amino acid sequence is 50% or more identical to any of these SLH domain-retaining outer membrane proteins, is suppressed. Therefore, in modified cyanobacteria, (i) the function of SLH domain-retaining outer membrane proteins in the outer membrane or proteins with equivalent function to SLH domain-retaining outer membrane proteins is suppressed or lost, or (ii) the expression level of SLH domain-retaining outer membrane proteins in the outer membrane or proteins with equivalent function to SLH domain-retaining outer membrane proteins is reduced. Consequently, in modified cyanobacteria, the binding domain (e.g., SLH domain) for the outer membrane to bind to the cell wall is reduced in both the amount and strength of the binding to the cell wall, making the outer membrane more easily detached from the cell wall. As a result, intracellularly produced substances are more easily leaked out of the cell, and therefore, plant-enhancing substances produced within the cell are also more easily leaked out. Accordingly, according to the method for producing a plant-enhancing agent according to one aspect of this disclosure, plant-enhancing substances produced within the modified cyanobacteria are more easily secreted out of the cell, thus enabling the efficient production of a plant-enhancing agent.

[0035] For example, in a method for producing a plant quality enhancer according to one aspect of the present disclosure, the cell wall-pyruvate modifying enzyme may be 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; Anacy_1623, consisting of the amino acid sequence shown in SEQ ID NO: 6; or a protein whose amino acid sequence is 50% or more identical to any of these cell wall-pyruvate modifying enzymes.

[0036] As a result, in the modified cyanobacteria, for example, (i) the function of any cell wall-pyruvate modifying enzyme shown in SEQ ID NOs. 4-6 above, or a protein whose amino acid sequence is 50% or more identical to any of these cell wall-pyruvate modifying enzymes, is suppressed or lost, or (ii) the expression of any cell wall-pyruvate modifying enzyme shown in SEQ ID NOs. 4-6 above, or a protein whose amino acid sequence is 50% or more identical to any of these cell wall-pyruvate modifying enzymes, is suppressed. Therefore, in the modified cyanobacteria, (i) the function of cell wall-pyruvate modifying enzymes or proteins with equivalent function is suppressed or lost, or (ii) the expression level of cell wall-pyruvate modifying enzymes or proteins with equivalent function is reduced. As a result, the covalent glycans on the surface of the cell wall are less likely to be modified with pyruvate, thus reducing the amount and strength of binding between the cell wall glycans and the SLH domain of SLH domain-retaining outer membrane proteins in the outer membrane. 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, thus weakening the binding force between the cell wall and the outer membrane, and making it easier for the outer membrane to partially detach from the cell wall. This makes it easier for intracellular products to leak out of the cell, and therefore, plant quality-enhancing substances produced within the cell are also more likely to leak out. Accordingly, according to one aspect of the present disclosure, plant quality-enhancing substances produced within the modified cyanobacteria are more easily secreted outside the cell, thus enabling the efficient production of plant quality-enhancing agents.

[0037] For example, in a method for producing a plant quality enhancer according to one aspect of the present disclosure, the gene that expresses the protein involved in the binding of the outer membrane to the cell wall may be deleted or inactivated.

[0038] As a result, in modified cyanobacteria, the expression of proteins involved in the binding of the cell wall to the outer membrane is suppressed, or the function of these proteins is suppressed or lost, partially reducing the binding (i.e., the amount and strength of the binding) between the cell wall and the outer membrane. Consequently, in modified cyanobacteria, the outer membrane becomes more easily partially detached from the cell wall, making it easier for intracellular products such as proteins and metabolites produced within the cell to leak outside the outer membrane, i.e., outside the cell. Therefore, modified cyanobacteria have improved secretion productivity of plant quality-enhancing substances produced within the cell. This eliminates the need for extraction treatments of intracellular products, such as cell lysis, thus reducing the likelihood of decreased physiological activity and yield of intracellular products. Therefore, it is possible to produce plant quality-enhancing agents with improved plant quality-enhancing effects. Furthermore, since the above-mentioned extraction treatment of intracellular products is unnecessary, the modified cyanobacteria can be repeatedly used to produce plant quality-enhancing substances even after the substances have been recovered. Therefore, it is not necessary to prepare new modified cyanobacteria each time a plant quality-enhancing agent is manufactured. Accordingly, according to the method for manufacturing a plant quality-enhancing agent according to one aspect of this disclosure, a plant quality-enhancing agent with improved plant quality-enhancing effects can be manufactured simply and efficiently.

[0039] For example, in a method for producing a plant quality enhancer according to one aspect of the present disclosure, the gene that expresses the protein involved in the binding of the outer membrane to the cell wall may be at least one of a gene encoding an SLH domain-retaining outer membrane protein and a gene encoding a cell wall-pyruvate modifying enzyme.

[0040] As a result, in the modified cyanobacteria, at least one gene encoding an SLH domain-retaining outer membrane protein and at least one gene encoding a cell wall-pyruvate modifying enzyme are deleted or inactivated. Therefore, in the modified cyanobacteria, for example, (i) the expression of at least one of the SLH domain-retaining outer membrane protein and the cell wall-pyruvate modifying enzyme is suppressed, or (ii) the function of at least one of the SLH domain-retaining outer membrane protein and the cell wall-pyruvate modifying enzyme is suppressed or lost. Consequently, the binding (i.e., the amount and strength of the binding) between the SLH domain of the SLH domain-retaining outer membrane protein in the outer membrane and the covalently bonded glycans on the surface of the cell wall is reduced. This makes it easier for the outer membrane to detach from the cell wall in areas where the binding between the outer membrane and the cell wall is weakened. As a result, in the modified cyanobacteria, the reduced binding between the outer membrane and the cell wall makes it easier for the outer membrane to partially detach from the cell wall, thus making it easier for proteins and metabolites produced within the bacterial cell to leak out of the cell. As a result, plant quality-enhancing substances produced within the microbial cells are more easily released outside the cells. Therefore, according to one embodiment of the present disclosure, the modified cyanobacteria can be efficiently made to secrete plant quality-enhancing substances, thus enabling the efficient production of plant quality-enhancing agents.

[0041] For example, in a method for producing a plant quality enhancer according to one aspect of the present disclosure, the gene encoding the SLH domain-retaining outer membrane protein may be slr1841 consisting of the nucleotide sequence shown in SEQ ID NO: 7, nies970_09470 consisting of the nucleotide sequence shown in SEQ ID NO: 8, anacy_3458 consisting of the nucleotide sequence shown in SEQ ID NO: 9, or a gene whose nucleotide sequence is 50% or more identical to any of these genes.

[0042] As a result, in the modified cyanobacteria, the gene encoding any of the SLH domain-retaining outer membrane proteins shown in Sequence IDs 7-9 above, or a gene that is 50% or more identical in base sequence to any of these genes, is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the SLH domain-retaining outer membrane proteins or proteins with equivalent function to any of these proteins is suppressed, or (ii) the function of any of the SLH domain-retaining outer membrane proteins or proteins with equivalent function to any of these proteins is suppressed or lost. Consequently, in the modified cyanobacteria, the amount and strength of the binding domain (e.g., SLH domain) that the outer membrane uses to bind to the cell wall is reduced, making it easier for the outer membrane to partially detach from the cell wall. This makes it easier for proteins and metabolites produced within the cell to leak out of the cell, and therefore, plant-enhancing substances produced within the cell also leak out of the cell. Therefore, according to the method for producing a plant quality enhancer according to one aspect of this disclosure, the plant quality enhancer produced within the cells of modified cyanobacteria is more easily leaked out of the cells, thus enabling the efficient production of the plant quality enhancer.

[0043] For example, in a method for producing a plant quality enhancer according to one aspect of the present disclosure, the gene encoding the cell wall-pyruvate modifying enzyme may be slr0688 consisting of the nucleotide sequence shown in SEQ ID NO: 10, synpcc7942_1529 consisting of the nucleotide sequence shown in SEQ ID NO: 11, anacy_1623 consisting of the nucleotide sequence shown in SEQ ID NO: 12, or a gene whose nucleotide sequence is 50% or more identical to any of these genes.

[0044] As a result, in the modified cyanobacteria, the gene encoding any of the cell wall-pyruvate modifying enzymes shown in Sequence IDs 10-12 above, or a gene whose base sequence is 50% or more identical to that of a gene encoding any of these enzymes, is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the cell wall-pyruvate modifying enzymes or proteins with equivalent function to any of these enzymes is suppressed, or (ii) the function of any of the cell wall-pyruvate modifying enzymes or proteins with equivalent function to any of these enzymes is suppressed or lost. As a result, the covalent glycans on the surface of the cell wall are less likely to be modified with pyruvate, reducing the amount and strength of the binding of the cell wall glycans to the SLH domain of SLH domain-retaining outer membrane proteins in the outer membrane. Consequently, in the modified cyanobacteria, the amount of pyruvate modification of the glycans that bind the cell wall to the outer membrane is reduced, weakening the binding strength between the cell wall and the outer membrane, and making it easier for the outer membrane to partially detach from the cell wall. As a result, proteins and metabolites produced within the bacterial cells are more likely to leak out of the cells, and therefore, plant-enhancing substances produced within the cells are also more likely to leak out. Accordingly, according to the method for producing a plant-enhancing agent according to one aspect of this disclosure, plant-enhancing substances produced within the cells of modified cyanobacteria are more likely to leak out of the cells, thus enabling the efficient production of plant-enhancing agents.

[0045] Furthermore, a plant quality enhancer according to one aspect of this disclosure includes secretions from modified cyanobacteria in which the function of proteins involved in the binding of the outer membrane to the cell wall in cyanobacteria is suppressed or lost.

[0046] As a result, in modified cyanobacteria, the binding (i.e., the amount and strength of the binding) between the cell wall and the outer membrane is partially reduced, making it easier for the outer membrane to partially detach from the cell wall. Therefore, in modified cyanobacteria, proteins and metabolites produced within the cell (i.e., intracellular products) are more easily leaked out of the outer membrane (i.e., outside the cell). This makes it easier for proteins and metabolites produced within the cell to be secreted outside the cell by modified cyanobacteria, eliminating the need for extraction treatments of intracellular products, such as cell lysis. Therefore, plant quality improvement agents containing secretions from modified cyanobacteria can be produced simply and efficiently. Furthermore, since the above-mentioned extraction treatment of intracellular products is unnecessary, a decrease in the physiological activity and yield of intracellular products is less likely to occur. Therefore, a decrease in the physiological activity and yield of substances involved in plant quality improvement (i.e., plant quality improvement substances) among the intracellular products of modified cyanobacteria is also less likely to occur. As a result, plant quality improvement agents with improved plant quality improvement effects can be obtained. Therefore, a plant quality enhancer according to one aspect of this disclosure can effectively improve the quality of plants.

[0047] Furthermore, a method for improving plant quality according to one aspect of this disclosure involves using the above-mentioned plant quality improving agent on plants.

[0048] According to one aspect of the present disclosure, a method for improving plant quality can be used on plants to effectively improve plant quality by applying a plant quality improving agent with enhanced plant quality-improving effects to the plants.

[0049] The embodiments will be described in detail below with reference to the drawings.

[0050] The embodiments described below are all comprehensive or specific examples. The numerical values, materials, steps, and order of steps shown in the following embodiments are examples only and are not intended to limit this disclosure. Furthermore, among the components in the following embodiments, those components that are not described in the independent claim representing the highest-level concept will be described as optional components.

[0051] Furthermore, the figures are not necessarily strictly accurate. In each figure, substantially identical components are denoted by the same reference numerals, and redundant explanations may be omitted or simplified.

[0052] Furthermore, in the following, numerical ranges do not represent only strict meanings, but also include substantially equivalent ranges, such as measuring the amount of protein (e.g., number or concentration) or its range.

[0053] Furthermore, in this specification, both "bacterial body" and "cell" refer to an individual cyanobacterium.

[0054] (Embodiment) In this specification, the identity of base sequences and amino acid sequences is calculated using the BLAST (Basic Local Alignment Search Tool) algorithm. Specifically, it is calculated by performing pairwise analysis using the BLAST program available on the NCBI (National Center for Biotechnology Information) website (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). Information on cyanobacteria genes and the proteins encoded by those genes is publicly available, for example, in the aforementioned NCBI database and Cyanobase (http: / / genome.microbedb.jp / cyanobase / ). From these databases, the amino acid sequences of the target proteins and the base sequences of the genes encoding those proteins can be obtained.

[0055] [1. Plant quality enhancer] First, the plant quality enhancer according to this embodiment will be described. The plant quality enhancer contains secretions involved in promoting plant growth and has plant growth-promoting effects, such as increasing the number of leaves, stems, buds, flowers, or fruits of a plant, thickening stems or trunks, and increasing height. In addition, the plant quality enhancer has effects such as controlling plant diseases, improving nutrient absorption, and increasing the sugar content of fruits. As a result, the plant quality enhancer can effectively improve the quality of plants for multiple crop species, such as increasing crop yield, increasing the size of crops and fruits, increasing the sugar content of fruits, reducing physiological disorders, and reducing diseases.

[0056] Furthermore, plants include not only crops cultivated in fields, but also garden trees, flowers, lawns, street trees, etc., as well as forest trees that receive little or no fertilization. Moreover, improving quality means that the quality of the usable parts of the plant (fruit, leaves, rhizomes, etc.), such as their composition and appearance, moves in a favorable direction (for example, increased nutritional value, improved taste, etc.). Composition includes, for example, general components such as water, protein, lipids, carbohydrates (e.g., sugars, starch, etc.), and ash; inorganic substances such as sodium, potassium, and calcium; vitamins such as vitamin A, vitamin C, and folic acid; fatty acids, cholesterol, and dietary fiber. Appearance includes measurable items such as shape, color, aroma, skin, fleshiness, thickness, luster, degree of flowering, and degree of physiological disorder, as well as items that are difficult to measure, such as beauty, suppleness, freshness, and visual balance.

[0057] In this embodiment, the plant enhancement agent includes the secretion of modified cyanobacteria in which the function of proteins involved in the binding of the outer membrane and the cell wall in the cyanobacteria (hereinafter also referred to as parent cyanobacteria) is suppressed or lost. Cyanobacteria (i.e., parent cyanobacteria) and modified cyanobacteria will be described later.

[0058] As described above, the secretion contains substances that contribute to improving plant quality. The secretion contains proteins and metabolites (i.e., intracellular products) produced within the cells of modified cyanobacteria. These intracellular products contain substances that contribute to improving plant quality, such as increasing crop yield, increasing crop and fruit size, increasing fruit sugar content, reducing physiological disorders, and reducing disease (i.e., plant quality-enhancing substances).

[0059] Plant quality-enhancing substances include, for example, organic matter-degrading enzymes such as peptidases, nucleases, or phosphatases; DNA metabolism-related substances such as adenosine or guanosine; intracellular molecules involved in promoting nucleic acid (e.g., DNA or RNA) synthesis, such as p-aminobenzoic acid or spermidine; ketone bodies such as 3-hydroxybutyric acid; or organic acids such as gluconic acid. The secretions of modified cyanobacteria may be mixtures of these plant quality-enhancing substances.

[0060] [2. Method for producing plant quality enhancers] Next, a method for producing the plant quality enhancer according to this embodiment will be described with reference to Figure 1. Figure 1 is a flowchart showing an example of a method for producing the plant quality enhancer according to this embodiment.

[0061] The method for producing a plant quality enhancer according to this embodiment includes the steps of: preparing modified cyanobacteria (i.e., parent cyanobacteria) in which the function of proteins involved in the binding of the outer membrane to the cell wall is suppressed or lost (step S01); and causing the modified cyanobacteria to secrete secretions involved in plant quality enhancer (step S02). As described above, the secretions of the modified cyanobacteria include proteins and metabolites (i.e., intracellular products) produced within the cells of the modified cyanobacteria. These intracellular products include substances involved in plant quality enhancer (i.e., plant quality enhancer substances).

[0062] Step S01 involves preparing the modified cyanobacteria. Preparing the modified cyanobacteria means adjusting the state of the modified cyanobacteria so that they can secrete secretions. Preparing the modified cyanobacteria may, for example, involve genetically modifying parent cyanobacteria to create modified cyanobacteria, restoring cells from freeze-dried or glycerol stocks of modified cyanobacteria, or recovering the modified cyanobacteria that have finished secreting plant-enhancing substances in Step S02.

[0063] In step S02, the modified cyanobacteria are made to secrete substances that contribute to improving plant quality. In this embodiment, the modified cyanobacteria have suppressed or lost the function of proteins involved in the binding of the outer membrane to the cell wall in the cyanobacteria (i.e., the parent cyanobacteria), so proteins and metabolites produced within the cell are easily secreted outside the outer membrane (i.e., outside the cell). These intracellularly produced substances include substances that contribute to improving plant quality. Therefore, in step S02, by culturing the modified cyanobacteria under predetermined conditions, intracellularly produced substances that contribute to improving plant quality are secreted outside the cell.

[0064] Cyanobacteria can generally be cultured using liquid culture with BG-11 medium (see Table 2) or a modified version thereof. Therefore, modified cyanobacteria can be cultured in the same manner. The culture period for cyanobacteria to produce plant quality enhancers can be any period during which sufficient cell growth occurs and proteins and metabolites accumulate at high concentrations, for example, 1 to 3 days or 4 to 7 days. The culture method can also be, for example, aerated stirring culture or shaking culture.

[0065] By culturing under the above conditions, the modified cyanobacteria produce proteins and metabolites (i.e., intracellular products) within their cells and secrete these intracellular products into the culture medium. These intracellular products include intracellular products involved in improving plant quality (i.e., plant quality-enhancing products). When recovering the intracellular products secreted into the culture medium, the culture medium may be filtered or centrifuged to remove solid components such as cells (i.e., bacterial cells), and the culture supernatant may be recovered. According to the method for producing a plant quality-enhancing agent of this embodiment, since the secretions containing intracellular products involved in improving plant quality (i.e., plant quality-enhancing products) are secreted outside the cells of the modified cyanobacteria, there is no need to crush the cells to recover the plant quality-enhancing products. Therefore, the modified cyanobacteria remaining after the recovery of the plant quality-enhancing products can be repeatedly used to produce the plant quality-enhancing agent.

[0066] Furthermore, the method for recovering the plant-enhancing substances secreted into the culture medium is not limited to the above example; the plant-enhancing substances in the culture medium may also be recovered while culturing the modified cyanobacteria. For example, by using a permeable membrane that allows proteins to pass through, the plant-enhancing substances that have permeated the membrane may be recovered. In this way, since the plant-enhancing substances in the culture medium can be recovered while culturing the modified cyanobacteria, the process of removing the modified cyanobacteria cells from the culture medium becomes unnecessary. Therefore, plant-enhancing agents can be manufactured more simply and efficiently.

[0067] Furthermore, by eliminating the need for cell recovery and cell disruption from the culture medium, the damage and stress experienced by the modified cyanobacteria can be reduced. As a result, the productivity of the modified cyanobacteria in secreting plant quality-enhancing substances is less likely to decrease, allowing the modified cyanobacteria to be used for a longer period.

[0068] As described above, by using the modified cyanobacteria in this embodiment, a plant quality enhancer can be obtained simply and efficiently.

[0069] The following describes cyanobacteria and modified cyanobacteria.

[0070] [3. Cyanobacteria] Cyanobacteria, also known as blue-green algae or blue-green bacteria, are a group of prokaryotes that perform photosynthesis by using chlorophyll to capture light energy and then electrolyzing water to produce oxygen. Cyanobacteria are highly diverse; for example, in terms of cell shape, there are unicellular species such as Synechocystis sp. PCC 6803 and filamentous species with multiple cells linked together, such as Anabaena sp. PCC 7120. In terms of habitat, there are thermophilic species such as Thermosynechococcus elongatus, marine species such as Synechococcus elongatus, and freshwater species such as Synechocystis. Furthermore, there are many species with unique characteristics, such as Microcystis aeruginosa, which possesses gas vesicles and produces toxins, and Gloeobacter violaceus, which lacks thylakoids but has a protein called a phycobilisome, a light-harvesting antenna, on its plasma membrane.

[0071] Figure 2 is a schematic diagram of the cell surface of cyanobacteria. As shown in Figure 2, the cell surface of cyanobacteria is composed of, from the inside out, the plasma membrane (also called the inner membrane 1), peptidoglycan 2, and the outer membrane 5, which is a lipid membrane that forms the outermost layer of the cell. Peptidoglycan 2 is covalently bonded to sugar chains 3, which consist of glucosamine and mannosamine, and pyruvate is also bound to these covalent sugar chains 3 (Non-patent Literature 3: Jurgens and Weckesser, 1986, J. Bacteriol., 168:568-573). In this specification, peptidoglycan 2 and the covalent sugar chains 3 together are referred to as the cell wall 4. The space between the plasma membrane (i.e., inner membrane 1) and the outer membrane 5 is called the periplasm, and contains various enzymes involved in protein degradation or structural formation, lipid or nucleic acid degradation, or the uptake of extracellular nutrients.

[0072] SLH domain-retaining outer membrane protein 6 (e.g., Slr1841 in the figure) consists of a C-terminal region embedded in the lipid membrane (also called outer membrane 5) and an N-terminal SLH domain 7 protruding from the lipid membrane. It is widely distributed in cyanobacteria and bacteria belonging to the class Negativicutes, a group of Gram-negative bacteria (Non-patent Literature 4: Kojima et al., 2016, Biosci. Biotech. Biochem., 10:1954-1959). The region embedded in the lipid membrane (i.e., outer membrane 5) forms a channel that enables the passage of hydrophilic substances through the outer membrane, while the SLH domain 7 has the function of binding to the cell wall 4 (Non-patent Literature 5: Kowata et al., 2017, J. Bacteriol., 199:e00371-17). For the SLH domain 7 to bind to the cell wall 4, the covalent sugar chain 3 in peptidoglycan 2 must be modified with pyruvate (Non-patent Literature 6: Kojima et al., 2016, J. Biol. Chem., 291:20198-20209). Examples of genes encoding SLH domain-containing outer membrane protein 6 include slr1841 or slr1908 in Synechocystis sp. PCC 6803, or oprB in Anabaena sp. 90.

[0073] The enzyme that catalyzes the pyruvate modification reaction of covalent sugar chain 3 in peptidoglycan 2 (hereinafter referred to as cell wall-pyruvate modifying enzyme 9) was identified in the Gram-positive bacterium Bacillus anthracis and named CsaB (Non-patent Literature 7: Mesnage et al., 2000, EMBO J., 19:4473-4484). In cyanobacteria whose genome sequences are publicly available, many species possess genes encoding homologous proteins with amino acid sequence identity of 30% or more to CsaB. Examples include slr0688 in Synechocystis sp. PCC 6803 and syn7502_03092 in Synechococcus sp. 7502.

[0074] In cyanobacteria, CO2 fixed by photosynthesis is converted into various amino acids and precursors of intracellular molecules through multi-step enzymatic reactions. These are then used as raw materials to synthesize proteins and metabolites within the cyanobacterial cytoplasm. Some of these proteins and metabolites function within the cytoplasm, while others are transported from the cytoplasm to the periplasm and function there. However, there have been no reports to date of cyanobacteria actively secreting proteins and metabolites outside the cell.

[0075] Because cyanobacteria have high photosynthetic capacity, they do not necessarily need to take in organic matter as nutrients from outside. Therefore, cyanobacteria have very few organic matter channel proteins in their outer membrane 5, such as organic matter channel protein 8 (e.g., Slr1270) in Figure 2. For example, in Synechocystis sp. PCC 6803, organic matter channel protein 8, which allows organic matter to pass through, accounts for only about 4% of the total protein content of the outer membrane 5. On the other hand, in order to efficiently take in inorganic ions necessary for growth, cyanobacteria have many ion channel proteins in their outer membrane 5 that allow only inorganic ions to pass through, such as SLH domain-retaining outer membrane protein 6 (e.g., Slr1841) in Figure 2. For example, in Synechocystis sp. PCC 6803, ion channel proteins that allow inorganic ions to pass through account for about 80% of the total protein content of the outer membrane 5.

[0076] Thus, in cyanobacteria, there are very few channels in the outer membrane 5 that allow organic matter such as proteins to pass through, making it difficult for them to actively secrete proteins and metabolites produced within the cell to the outside.

[0077] [4. Modified Cyanobacteria] Next, the modified cyanobacteria in this embodiment will be described with reference to Figure 2.

[0078] In this embodiment, the modified cyanobacteria have suppressed or lost the function of proteins involved in the binding of the outer membrane 5 to the cell wall 4 (hereinafter also referred to as binding-related proteins). As a result, the binding (e.g., amount and strength) between the outer membrane 5 and the cell wall 4 is partially reduced in the modified cyanobacteria, making it easier for the outer membrane 5 to partially detach from the cell wall 4. Therefore, the modified cyanobacteria have improved secretion productivity of intracellular products, which are proteins and metabolites produced within the cell and secreted outside the cell. As mentioned above, intracellular products include intracellular products that contribute to the improvement of plant quality (i.e., plant quality improvement substances). Therefore, the modified cyanobacteria also have improved secretion productivity of plant quality improvement substances, which are produced within the cell and secreted outside the cell. Furthermore, since it is not necessary to crush the cells to recover the plant quality improvement substances, the modified cyanobacteria can be used repeatedly even after the plant quality improvement substances have been recovered. In this specification, the production of proteins and metabolites within the cell by modified cyanobacteria is referred to as "production," and the secretion of the produced proteins and metabolites outside the cell is referred to as "secretory production."

[0079] The proteins involved in the binding of the outer membrane 5 to the cell wall 4 may be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9. In this embodiment, the modified cyanobacteria have, for example, the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 suppressed or lost. For example, in the modified cyanobacteria, (i) the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 may be suppressed or lost, and (ii) the expression of at least one of the SLH domain-retaining outer membrane protein 6 that binds to the cell wall 4 and the expression of the enzyme that catalyzes the pyruvate modification reaction of the linked glycans on the surface of the cell wall 4 (i.e., the cell wall-pyruvate modifying enzyme 9) may be suppressed. This reduces the binding (i.e., the amount and strength of binding) between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalent glycans 3 on the surface of the cell wall 4. Therefore, in areas where these bonds are weakened, the outer membrane 5 becomes more easily detached from the cell wall 4. Partial detachment of the outer membrane 5 from the cell wall 4 makes it easier for intracellular products such as proteins and metabolites present inside the modified cyanobacteria, particularly in the periplasm, to leak out of the cell (outside the outer membrane 5). As a result, the modified cyanobacteria have improved secretory productivity, secreting plant-enhancing substances produced inside the cell to the outside.

[0080] The following describes in more detail a cyanobacterium in which the outer membrane 5 is modified to partially detach from the cell wall 4 by suppressing the function of at least one binding-related protein of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9.

[0081] The type of cyanobacteria (i.e., parent cyanobacteria) that serves as the parent microorganism of the modified cyanobacteria before the suppression or loss of at least one of the expression of SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 in this embodiment is not particularly limited and may be any type of cyanobacteria. For example, the parent cyanobacteria may belong to the genera Synechocystis, Synechococcus, Anabaena, or Thermosynechococcus, and among these, may be Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1.

[0082] The amino acid sequences of the SLH domain-retaining outer membrane protein 6 and the enzyme that catalyzes the cell wall-pyruvate modification reaction (i.e., cell wall-pyruvate modifying enzyme 9) in these parent cyanobacteria, the nucleotide sequences of the genes encoding their binding-related proteins, and the location of these genes on chromosomal DNA or plasmids can be confirmed in the aforementioned NCBI database and Cyanobase.

[0083] Furthermore, the SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 whose function is suppressed or lost in the modified cyanobacteria according to this embodiment may belong to any parent cyanobacteria, as long as they are present in the parent cyanobacteria, and are not limited by the location of the encoding gene (e.g., on chromosomal DNA or on a plasmid).

[0084] For example, if the parent cyanobacteria is of the genus Syenchocystis, the SLH domain-retaining outer membrane protein 6 may be Slr1841, Slr1908, or Slr0042, etc. If the parent cyanobacteria is of the genus Synechococcus, it may be NIES970_09470, etc. If the parent cyanobacteria is of the genus Anabaena, it may be Anacy_5815 or Anacy_3458, etc. If the parent cyanobacteria is of the genus Microcystis, it may be A0A0F6U6F8_MICAE, etc. If the parent cyanobacteria is of the genus Cyanothese, In general, it may be A0A3B8XX12_9CYAN, etc. If the parent cyanobacteria is of the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc. If the parent cyanobacteria is of the genus Calothrix, A0A1Z4R6U0_9CYAN is an example. If the parent cyanobacteria is of the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc. If the parent cyanobacteria is of the genus Crocosphaera, it may be B1WRN6_CROS5, etc. If the parent cyanobacteria is of the genus Pleurocapsa, it may be K9TAE4_9CYAN, etc.

[0085] More specifically, the SLH domain-retaining outer membrane protein 6 may be, for example, Slr1841 (SEQ ID NO: 1) from Synechocystis sp. PCC 6803, NIES970_09470 (SEQ ID NO: 2) from Synechococcus sp. NIES-970, or Anacy_3458 (SEQ ID NO: 3) from Anabaena cylindrica PCC 7122. Alternatively, it may be a protein whose amino acid sequence is 50% or more identical to these SLH domain-retaining outer membrane proteins 6.

[0086] As a result, in the modified cyanobacteria, for example, (i) the function of any of the SLH domain-retaining outer membrane proteins 6 shown in Sequence IDs 1-3 above, or proteins whose amino acid sequence is 50% or more identical to any of these SLH domain-retaining outer membrane proteins 6, may be suppressed or lost, and (ii) the expression of any of the SLH domain-retaining outer membrane proteins 6 shown in Sequence IDs 1-3 above, or proteins whose amino acid sequence is 50% or more identical to any of these SLH domain-retaining outer membrane proteins 6, may be suppressed. Therefore, in the modified cyanobacteria, (i) the function of SLH domain-retaining outer membrane proteins 6 in the outer membrane 5, or proteins having equivalent function to SLH domain-retaining outer membrane proteins 6, is suppressed or lost, or (ii) the expression level of SLH domain-retaining outer membrane proteins 6 in the outer membrane 5, or proteins having equivalent function to SLH domain-retaining outer membrane proteins 6, is reduced. As a result, in modified cyanobacteria, the binding domain (e.g., SLH domain 7) that binds the outer membrane 5 to the cell wall 4 has reduced binding strength and amount, making it easier for the outer membrane 5 to partially detach from the cell wall 4. This makes it easier for intracellular products to leak out of the cell in modified cyanobacteria, and consequently, plant-enhancing substances produced within the cell also leak out more easily.

[0087] Generally, if the amino acid sequences of proteins are 30% or more identical, the homology of the three-dimensional structure of the proteins is high, and it is said that there is a high probability that the protein will have equivalent function. Therefore, the SLH domain-retaining outer membrane protein 6 whose function is suppressed or lost may be, for example, a protein or polypeptide having an amino acid sequence that is 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and still more preferably 90% or more identical to the amino acid sequence of any of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 above, and which has the function of binding to the covalently linked glycans 3 of the cell wall 4.

[0088] Furthermore, for example, cell wall-pyruvate modifying enzyme 9 may be Slr0688, etc., if the parent cyanobacteria is of the genus Syenchocystis; if the parent cyanobacteria is of the genus Synechococcus; if the parent cyanobacteria is of the genus Synechococcus; if the parent cyanobacteria is of the genus Anabaena; if the parent cyanobacteria is of the genus Microcystis; CsaB It may also be (NCBI access ID: TRU80220), and if the parent cyanobacteria is of the genus Cyanothese, it may also be CsaB (NCBI access ID: WP_107667006.1), and if the parent cyanobacteria is of the genus Spirulina, it may also be CsaB (NCBI access ID: WP_026079530.1), and if the parent cyanobacteria is of the genus Calothrix, it may be CsaB (NCBI access ID: WP_09665 It may also be 8142.1), and if the parent cyanobacteria is of the genus Nostoc, it may be CsaB (NCBI access ID: WP_099068528.1), etc. If the parent cyanobacteria is of the genus Crocosphaera, it may be CsaB (NCBI access ID: WP_012361697.1), etc. If the parent cyanobacteria is of the genus Pleurocapsa, it may be CsaB (NCBI access ID: WP_036798735), etc.

[0089] More specifically, cell wall-pyruvate modifying enzyme 9 may be, for example, Slr0688 (SEQ ID NO: 4) from Synechocystis sp. PCC 6803, Synpcc7942_1529 (SEQ ID NO: 5) from Synechococcus sp. PCC 7942, or Anacy_1623 (SEQ ID NO: 6) from Anabaena cylindrica PCC 7122. Alternatively, it may be a protein whose amino acid sequence is 50% or more identical to these cell wall-pyruvate modifying enzymes 9.

[0090] As a result, in the modified cyanobacteria, for example, (i) the function of any cell wall-pyruvate modifying enzyme 9 shown in SEQ ID NOs. 4-6 above, or a protein whose amino acid sequence is 50% or more identical to any of these cell wall-pyruvate modifying enzymes 9, may be suppressed or lost, and (ii) the expression of any cell wall-pyruvate modifying enzyme 9 shown in SEQ ID NOs. 4-6 above, or a protein whose amino acid sequence is 50% or more identical to any of these cell wall-pyruvate modifying enzymes 9, may be suppressed. Therefore, in the modified cyanobacteria, (i) the function of cell wall-pyruvate modifying enzyme 9 or a protein having equivalent function to said enzyme is suppressed or lost, or (ii) the expression level of cell wall-pyruvate modifying enzyme 9 or a protein having equivalent function to said enzyme is reduced. As a result, the covalent glycan 3 on the surface of the cell wall 4 is less likely to be modified with pyruvate, and the amount and strength of the binding of glycan 3 of the cell wall 4 to the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is reduced. Therefore, in the modified cyanobacteria according to this embodiment, the covalent sugar chain 3 on the surface of the cell wall 4 is less likely to be modified with pyruvate, thus weakening the binding force between the cell wall 4 and the outer membrane 5, and making it easier for the outer membrane 5 to partially detach from the cell wall 4. As a result, in the modified cyanobacteria, intracellularly produced substances are more likely to leak out of the cell, and plant quality-enhancing substances produced inside the cell are also more likely to leak out of the cell.

[0091] Furthermore, as mentioned above, if the amino acid sequence of a protein is 30% or more identical, it is highly likely that it will have the same function as the protein in question. Therefore, the cell wall-pyruvate modifying enzyme 9 whose function is suppressed or lost may be, for example, a protein or polypeptide having an amino acid sequence that is 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and still more preferably 90% or more identical to the amino acid sequence of any of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 above, and which has the function of catalyzing the reaction in which the covalent sugar chain 3 of peptidoglycan 2 of the cell wall 4 is modified with pyruvate.

[0092] In this specification, inhibiting or causing loss of function of the SLH domain-retaining outer membrane protein 6 means inhibiting or causing loss of the protein's ability to bind to the cell wall 4, inhibiting or causing loss of the protein's transport to the outer membrane 5, or inhibiting or causing loss of the protein's ability to be embedded in the outer membrane 5 and function.

[0093] Furthermore, inhibiting or losing the function of cell wall-pyruvate modifying enzyme 9 means inhibiting or losing the function of the protein in modifying the covalent sugar chain 3 of the cell wall 4 with pyruvate.

[0094] The means of suppressing or losing the function of these proteins are not particularly limited, as long as they are means commonly used for suppressing or losing the function of proteins. Such means may include, for example, deleting or inactivating the gene encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9, inhibiting the transcription of these genes, inhibiting the translation of the transcripts of these genes, or administering inhibitors that specifically inhibit these proteins.

[0095] In this embodiment, the modified cyanobacteria have an outer membrane 5 and a cell wall 4. As a result, the expression of proteins involved in the binding of the cell wall 4 and the outer membrane 5 is suppressed, or the function of such proteins is suppressed or lost, so the binding (i.e., the amount and strength of the binding) between the cell wall 4 and the outer membrane 5 is partially reduced. Consequently, in the modified cyanobacteria, the outer membrane 5 becomes more easily partially detached from the cell wall 4, so intracellular products such as proteins and metabolites produced within the cell are more easily leaked out of the outer membrane 5, i.e., outside the cell. Therefore, the modified cyanobacteria have improved productivity in secreting plant-enhancing substances produced within the cell. This eliminates the need for extraction treatment of intracellular products, such as cell pulverization, thus reducing the likelihood of decreased physiological activity and yield of intracellular products. Therefore, a plant-enhancing agent with improved plant-enhancing effects can be produced. Furthermore, since the extraction process of the intracellularly produced substances mentioned above is unnecessary, the modified cyanobacteria can be repeatedly used to produce plant quality-enhancing substances even after the substances have been recovered.

[0096] The gene that expresses the protein involved in the binding of the outer membrane 5 to the cell wall 4 may be, for example, at least one of the genes encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9. In modified cyanobacteria, at least one of the genes encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9 is deleted or inactivated. Therefore, in modified cyanobacteria, for example, (i) the expression of at least one of SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 is suppressed, or (ii) the function of at least one of SLH domain-retaining outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 is suppressed or lost. As a result, the binding (i.e., the amount and strength of binding) between the SLH domain 7 of SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalent sugar chain 3 on the surface of the cell wall 4 is reduced. This makes it easier for the outer membrane 5 to detach from the cell wall 4 in areas where the binding between the outer membrane 5 and the cell wall 4 is weakened. As a result, in modified cyanobacteria, the binding between the outer membrane 5 and the cell wall 4 is reduced, making it easier for the outer membrane 5 to partially detach from the cell wall 4. This allows proteins and metabolites produced within the cell to leak out of the cell. Consequently, plant-enhancing substances produced within the modified cyanobacteria also leak out of the cell.

[0097] In this embodiment, in order to suppress or eliminate the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 in cyanobacteria, for example, the transcription of at least one of the genes encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9 may be suppressed.

[0098] For example, the gene encoding SLH domain-retaining outer membrane protein 6 may be slr1841, slr1908, or slr0042 if the parent cyanobacterium is of the genus Syenchocystis, nies970_09470 if the parent is of the genus Synechococcus, anacy_5815 or anacy_3458 if the parent cyanobacterium is of the genus Anabaena, A0A0F6U6F8_MICAE if the parent cyanobacterium is of the genus Microcystis, and Cyanothese if the parent cyanobacterium is of the genus Cyanothese. The gene may be A0A3B8XX12_9CYAN, etc. If the parent cyanobacteria is of the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc. If the parent cyanobacteria is of the genus Calothrix, it may be A0A1Z4R6U0_9CYAN, etc. If the parent cyanobacteria is of the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc. If the parent cyanobacteria is of the genus Crocosphaera, it may be B1WRN6_CROS5, etc. If the parent cyanobacteria is of the genus Pleurocapsa, it may be K9TAE4_9CYAN, etc. The base sequences of these genes can be obtained from the NCBI database or Cyanobase mentioned above.

[0099] More specifically, the gene encoding SLH domain-retaining outer membrane protein 6 may be slr1841 (SEQ ID NO: 7) from Synechocystis sp. PCC 6803, nies970_09470 (SEQ ID NO: 8) from Synechococcus sp. NIES-970, anacy_3458 (SEQ ID NO: 9) from Anabaena cylindrica PCC 7122, or a gene whose amino acid sequence is 50% or more identical to these genes.

[0100] As a result, in the modified cyanobacteria, the gene encoding any of the SLH domain-retaining outer membrane proteins 6 shown in Sequence IDs 7-9 above, or a gene that is 50% or more identical in base sequence to any of these genes, is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the SLH domain-retaining outer membrane proteins 6 or a protein with equivalent function to any of these proteins is suppressed, or (ii) the function of any of the SLH domain-retaining outer membrane proteins 6 or a protein with equivalent function to any of these proteins is suppressed or lost. Consequently, in the modified cyanobacteria, the amount and strength of the binding domain (e.g., SLH domain 7) for the outer membrane 5 to bind to the cell wall 4 is reduced, making it easier for the outer membrane 5 to partially detach from the cell wall 4. This makes it easier for proteins and metabolites produced within the cell to leak out of the cell, and therefore, plant-enhancing substances produced within the cell also leak out of the cell.

[0101] As mentioned above, if the amino acid sequence of a protein is 30% or more identical, it is highly likely that it will have equivalent function to the protein in question. Therefore, if the base sequence of the gene encoding the protein is 30% or more identical, it is highly likely that a protein with equivalent function to the protein in question will be expressed. For this reason, the gene encoding the SLH domain-retaining outer membrane protein 6 whose function is suppressed or lost may be, for example, a gene whose base sequence is 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and still more preferably 90% or more identical to the base sequence of any of the genes encoding the SLH domain-retaining outer membrane protein 6 shown in Sequence ID Nos. 7 to 9 above, and which is also a gene encoding a protein or polypeptide that has the function of binding to the covalent sugar chain 3 of the cell wall 4.

[0102] Furthermore, for example, the gene encoding cell wall-pyruvate modifying enzyme 9 may be slr0688, etc., if the parent cyanobacteria is of the genus Synchocystis; syn7502_03092 or synpcc7942_1529, etc., if the parent cyanobacteria is of the genus Synechococcus; ana_C20348 or anacy_1623, etc., if the parent cyanobacteria is of the genus Anabaena; and csaB, etc., if the parent cyanobacteria is of the genus Microcystis. It may also be (NCBI access ID: TRU80220), and if the parent cyanobacteria is of the genus Cynahothese, it may also be csaB (NCBI access ID: WP_107667006.1), and if the parent cyanobacteria is of the genus Spirulina, it may also be csaB (NCBI access ID: WP_026079530.1), and if the parent cyanobacteria is of the genus Calothrix, it may be csaB (NCBI access ID: WP_0966 It may also be 58142.1), and if the parent cyanobacteria is of the genus Nostoc, it may be csaB (NCBI access ID: WP_099068528.1), if the parent cyanobacteria is of the genus Crocosphaera, it may be csaB (NCBI access ID: WP_012361697.1), and if the parent cyanobacteria is of the genus Pleurocapsa, it may be csaB (NCBI access ID: WP_036798735), etc. The base sequences of these genes can be obtained from the NCBI database or Cyanobase mentioned above.

[0103] More specifically, the gene encoding cell wall-pyruvate modifying enzyme 9 may be slr0688 (sequence number 10) from Synechocystis sp. PCC 6803, synpcc7942_1529 (sequence number 11) from Synechococcus sp. PCC 7942, or anacy_1623 (sequence number 12) from Anabaena cylindrica PCC 7122. Alternatively, it may be a gene whose nucleotide sequence is 50% or more identical to these genes.

[0104] As a result, in the modified cyanobacteria, the gene encoding any of the cell wall-pyruvate modifying enzymes 9 shown in Sequence IDs 10-12 above, or a gene whose base sequence is 50% or more identical to that of a gene encoding any of these enzymes, is deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the cell wall-pyruvate modifying enzymes 9 or a protein having equivalent function to any of these enzymes is suppressed, or (ii) the function of any of the cell wall-pyruvate modifying enzymes 9 or a protein having equivalent function to any of these enzymes is suppressed or lost. As a result, the covalently bonded glycans 3 on the surface of the cell wall 4 are less likely to be modified with pyruvate, and the amount and strength of the binding of glycans 3 of the cell wall 4 to the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is reduced. Therefore, in the modified cyanobacteria according to this embodiment, the amount of pyruvate modification of glycans 3 for the cell wall 4 to bind to the outer membrane 5 is reduced, weakening the binding strength between the cell wall 4 and the outer membrane 5, and making it easier for the outer membrane 5 to partially detach from the cell wall 4. As a result, proteins and metabolites produced within the fungal cells are more likely to leak out of the cells, and consequently, plant-enhancing substances produced within the fungal cells are also more likely to leak out.

[0105] As described above, if the base sequence of a gene encoding a protein is 30% or more identical, it is considered highly likely that a protein with equivalent function to the protein in question will be expressed. Therefore, the gene encoding cell wall-pyruvate modifying enzyme 9 whose function is suppressed or lost may be, for example, a gene encoding a protein or polypeptide that has a base sequence that is 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and still more preferably 90% or more identical to the base sequence of any of the genes encoding cell wall-pyruvate modifying enzyme 9 shown in SEQ ID NOs. 10 to 12 above, and that also has a base sequence that is more preferably 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and still more preferably 90% or more identical, and that has the function of catalyzing the reaction of modifying the covalent sugar chain 3 of peptidoglycan 2 of the cell wall 4 with pyruvate.

[0106] [5. Method for producing modified cyanobacteria] Next, the method for producing modified cyanobacteria in this embodiment will be described. The method for producing modified cyanobacteria includes the step of suppressing or eliminating the function of proteins involved in the binding of the outer membrane 5 and the cell wall 4 in cyanobacteria.

[0107] In this embodiment, the protein involved in the binding of the outer membrane 5 to the cell wall 4 may be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9.

[0108] The means of suppressing or causing loss of protein function are not particularly limited, but may include, for example, deleting or inactivating the gene encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9, inhibiting the transcription of these genes, inhibiting the translation of the transcripts of these genes, or administering inhibitors that specifically inhibit these proteins.

[0109] Means for deleting or inactivating the above-mentioned gene may include, for example, introducing a mutation to one or more bases in the base sequence of the gene, substituting the base sequence with another base sequence or inserting another base sequence, or deleting part or all of the base sequence of the gene.

[0110] Means for inhibiting the transcription of the above gene may include, for example, introducing a mutation into the promoter region of the gene, inactivating the promoter by substitution or insertion of another base sequence, or CRISPR interference (Non-patent Literature 8: Yao et al., ACS Synth. Biol., 2016, 5:207-212). Specific methods for introducing the above mutation, or substitution or insertion of base sequences, may include, for example, ultraviolet irradiation, site-directed mutagenesis, or homologous recombination.

[0111] Furthermore, the means for inhibiting the translation of the transcript of the above gene may be, for example, RNA (ribonucleic acid) interference.

[0112] Modified cyanobacteria may be produced by using any of the above means to suppress or eliminate the function of proteins involved in the binding of the outer membrane 5 and the cell wall 4 in cyanobacteria.

[0113] As a result, the modified cyanobacteria produced by the above manufacturing method have a partially reduced bond (i.e., bond amount and bond strength) between the cell wall 4 and the outer membrane 5, making it easier for the outer membrane 5 to partially detach from the cell wall 4. Consequently, in the modified cyanobacteria, intracellular products such as proteins and metabolites produced within the cell are more likely to leak out of the outer membrane 5 (i.e., out of the cell), and so are substances involved in improving plant quality (i.e., plant quality-enhancing substances). Therefore, the manufacturing method for modified cyanobacteria in this embodiment can provide modified cyanobacteria with improved secretion productivity of plant quality-enhancing substances.

[0114] Furthermore, in the modified cyanobacteria produced by the manufacturing method of this embodiment, the plant-enhancing substances produced within the cells leak out of the cells, eliminating the need to crush the cells to recover these substances. For example, it is possible to cultivate the modified cyanobacteria under appropriate conditions and then recover the plant-enhancing substances secreted into the culture medium, thus allowing for the recovery of plant-enhancing substances in the culture medium while culturing the modified cyanobacteria. Therefore, by using the modified cyanobacteria obtained by this manufacturing method, efficient production of microbiological plant-enhancing substances can be carried out. Accordingly, the manufacturing method of modified cyanobacteria in this embodiment provides modified cyanobacteria with high utilization efficiency that can be repeatedly used even after the plant-enhancing substances have been recovered.

[0115] [6. Plant quality improvement method] The plant quality improvement method according to this embodiment involves using the above-mentioned plant quality improvement agent on plants. As described above, the plant quality improvement agent according to this embodiment is a plant quality improvement agent with improved plant quality improvement effect, and by using the above-mentioned plant quality improvement agent on plants, the quality of the plants can be effectively improved.

[0116] The above-mentioned plant quality enhancer may be used as is, or after being concentrated or diluted. When applying the plant quality enhancer to plants (i.e., applying it), the concentration and application method may be determined appropriately according to the type of plant, the properties of the soil, and the purpose. The plant quality enhancer may be, for example, the culture solution of modified cyanobacteria itself, a solution obtained by removing the cells of modified cyanobacteria from the culture solution, or an extract obtained by extracting a desired substance from the culture solution using membrane technology or the like. The desired substance may be an enzyme that breaks down nutrients in the soil, a substance that solubilizes insoluble substances in the soil (e.g., metals such as iron) (e.g., a substance with a chelating effect), or a substance that improves the intracellular physiological activity of plants. Furthermore, the method of applying the plant quality enhancer to plants may be, for example, spraying it onto the plant, spraying it onto the soil, irrigating it, mixing it, or mixing it into a hydroponic solution. For example, a few milliliters of the plant quality enhancer may be added to the base of each plant about once a week. [Examples]

[0117] The modified cyanobacteria, methods for producing the modified cyanobacteria, and methods for producing the plant quality enhancer described below will be explained in detail in the examples provided, but the disclosure is not limited in any way to the examples provided below.

[0118] In the following examples, two types of modified cyanobacteria were produced by partially detaching the outer membrane of cyanobacteria from the cell wall: suppression of the expression of the slr1841 gene encoding an SLH domain-retaining outer membrane protein (Example 1), and suppression of the expression of the slr0688 gene encoding a cell wall-pyruvate modifying enzyme (Example 2). The protein secretion productivity of these modified cyanobacteria was then measured, and the secreted intracellular products (here, proteins and intracellular metabolites) were identified. The cyanobacterial species used in these examples was Synechocystis sp. PCC 6803 (hereinafter simply referred to as "cyanobacteria").

[0119] (Example 1) In Example 1, modified cyanobacteria were produced in which the expression of the slr1841 gene, which encodes an SLH domain-retaining outer membrane protein, was suppressed.

[0120] (1) Construction of a modified cyanobacterial strain in which the expression of the slr1841 gene is suppressed. As a method for suppressing gene expression, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference was used. In this method, the expression of the slr1841 gene can be 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.

[0121] The mechanism by which gene expression is suppressed by this method is as follows:

[0122] First, a Cas9 protein lacking nuclease activity (dCas9) and an sgRNA (slr1841_sgRNA) that binds complementarily to the nucleotide sequence of the slr1841 gene form a complex.

[0123] Next, this complex recognizes the slr1841 gene on the chromosomal DNA of cyanobacteria and specifically binds to it. This binding causes steric hindrance, inhibiting the transcription of the slr1841 gene. As a result, the expression of the slr1841 gene in cyanobacteria is suppressed.

[0124] The following describes in detail how to introduce each of the two genes mentioned above into the chromosomal DNA of cyanobacteria.

[0125] (1-1) Introduction of the dCas9 gene Using the chromosomal DNA of the Synechocystis LY07 strain (hereinafter also referred to as the LY07 strain) (see Non-Patent Document 8) as a template, the dCas9 gene, an operator gene for regulating the expression of the dCas9 gene, and a spectinomycin resistance marker gene that serves as a marker for gene transfer were amplified by PCR (Polymerase chain reaction) using the primers psbA1-Fw (SEQ ID NO: 13) and psbA1-Rv (SEQ ID NO: 14) listed in Table 1. In the LY07 strain, the above three genes are inserted into the psbA1 gene on the chromosomal DNA in a ligated state, so they can be amplified by PCR as a single DNA fragment. Here, the obtained DNA fragment is referred to as the "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.

[0126] [Table 1]

[0127] One μg of the obtained pUC19-dCas9 plasmid was mixed with cyanobacterial culture medium (cellular concentration OD730 = approximately 0.5), and the pUC19-dCas9 plasmid was introduced into cyanobacterial cells by spontaneous transformation. The transformed cells were selected by growing them on BG-11 agar medium containing 20 μg / mL of spectinomycin. In the selected cells, homologous recombination occurred between the psbA1 gene on chromosomal DNA and the upstream and downstream fragment regions of psbA1 on the pUC19-dCas9 plasmid. This resulted in obtaining a Synechocystis dCas9 strain in which the dCas9 cassette was inserted into the psbA1 gene region. The composition of the BG-11 medium used is shown in Table 2.

[0128] [Table 2]

[0129] (1-2) Introduction of the slr1841_sgRNA gene In CRISPR interference, a sequence of approximately 20 base pairs complementary to the target sequence is introduced into a region called the protospacer on the sgRNA gene, allowing the sgRNA to specifically bind to the target gene. The protospacer sequence used in this example is shown in Table 3.

[0130] [Table 3]

[0131] In the Synechocystis LY07 strain, the sgRNA gene (excluding the protospacer region) and the kanamycin resistance marker gene are linked together and inserted into the slr2030-slr2031 gene on chromosomal DNA. Therefore, by attaching a protospacer sequence (sequence number 21) complementary to the slr1841 gene (sequence number 7) to the primer used when amplifying the sgRNA gene by PCR, it is possible to easily obtain an sgRNA (slr1841_sgRNA) that specifically recognizes slr1841.

[0132] First, using the chromosomal DNA of strain LY07 as a template, two DNA fragments were amplified by PCR using the primer sets slr2030-Fw (sequence number 15) and sgRNA_slr1841-Rv (sequence number 16) listed in Table 1, as well as the sets sgRNA_slr1841-Fw (sequence number 17) and slr2031-Rv (sequence number 18).

[0133] Next, using the mixed solution of the above DNA fragments as a template, the mixture was amplified by PCR using the primers slr2030-Fw (SEQ ID NO: 15) and slr2031-Rv (SEQ ID NO: 18) listed in Table 1 to obtain a DNA fragment (slr2030-2031::slr1841_sgRNA) in which (i) the slr2030 gene fragment, (ii) slr1841_sgRNA, (iii) the kanamycin resistance marker gene, and (iv) the slr2031 gene fragment were sequentially ligated. 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.

[0134] The pUC19-slr1841_sgRNA plasmid was introduced into Synechocystis dCas9 strain using the same method as described in (1-1) above, and transformed cells were selected on BG-11 agar medium containing 30 μg / mL kanamycin. This yielded a transformed Synechocystis dCas9 slr1841_sgRNA strain (hereinafter also referred to as the slr1841 suppressor strain) in which slr1841_sgRNA was inserted into the slr2030-slr2031 genes on the chromosomal DNA.

[0135] (1-3) Suppression of the slr1841 gene The promoter sequences of the dCas9 gene and the slr1841_sgRNA gene described above are designed to induce expression 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 culture medium.

[0136] (Example 2) In Example 2, modified cyanobacteria were obtained in which the expression of the slr0688 gene, which encodes a cell wall-pyruvate modifying enzyme, was suppressed by the following procedure.

[0137] (2) Construction of a modified cyanobacteria strain in which the expression of the slr0688 gene is suppressed. Following the same procedure as described in (1-2) above, an sgRNA gene containing a protospacer sequence (sequence number 22) complementary to the slr0688 gene (sequence number 4) was introduced into the Synechocystis dCas9 strain to obtain the Synechocystis dCas9 slr0688_sgRNA strain. The procedure was carried out under the same conditions as in (1-2) above, except that the set of primers slr2030-Fw (SEQ ID NO: 15) and sgRNA_slr0688-Rv (SEQ ID NO: 19), and the set of primers sgRNA_slr0688-Fw (SEQ ID NO: 20) and slr2031-Rv (SEQ ID NO: 18) listed in Table 1 were used, and a DNA fragment (slr2030-2031::slr0688_sgRNA) consisting of (i) the slr2030 gene fragment, (ii) slr0688_sgRNA, (iii) the kanamycin resistance marker gene, and (iv) the slr2031 gene fragment was sequentially linked and inserted into the pUC19 plasmid using the In-Fusion PCR Cloning Method (registered trademark) to obtain the pUC19-slr0688_sgRNA plasmid.

[0138] Furthermore, the expression of the slr0688 gene was suppressed using the same procedure as described in (1-3) above.

[0139] (Comparative Example 1) In Comparative Example 1, the Synechocystis dCas9 strain was obtained using the same procedure as in Example 1 (1-1).

[0140] Next, the cell surface condition and protein secretion productivity tests were performed on the bacterial strains obtained in Example 1, Example 2, and Comparative Example 1, respectively. Details are described below.

[0141] (3) Observation of the state of the cell surface of the bacterial strain Ultrathin sections were prepared for each of the modified cyanobacterial Synechocystis dCas9 slr1841_sgRNA strain obtained in Example 1 (i.e., the slr1841 suppressor strain), the modified cyanobacterial Synechocystis dCas9 slr0688_sgRNA strain obtained in Example 2 (hereinafter also referred to as the slr0688 suppressor strain), and the modified cyanobacterial Synechocystis dCas9 strain obtained in Comparative Example 1 (hereinafter referred to as the Control strain). The state of the cell surface (in other words, the outer membrane structure) was observed using an electron microscope.

[0142] (3-1) Culture of bacterial strains The slr1841 inhibitory strain from Example 1 was inoculated into BG-11 medium containing 1 μg / mL aTc so that the initial bacterial cell concentration OD730 = 0.05, and the light intensity was 100 μmol / m². 2 The cells were cultured with shaking for 5 days under conditions of 0 / s and 30°C. The slr0688 inhibitory strain from Example 2 and the control strain from Comparative Example 1 were cultured under the same conditions as in Example 1.

[0143] (3-2) Preparation of ultrathin sections of bacterial strains The culture medium obtained in (3-1) above was centrifuged at room temperature at 2,500 g for 10 minutes to collect the cells of the slr1841 inhibitory strain from Example 1. The cells were then rapidly frozen with liquid propane at -175°C and fixed at -80°C for 2 days using an ethanol solution containing 2% glutaraldehyde and 1% tannic acid. The fixed cells were dehydrated with ethanol, and the dehydrated cells were permeated with propylene oxide and then immersed in a resin (Quetol-651) solution. The cells were then left to stand at 60°C for 48 hours to cure the resin, embedding the cells in the resin. The cells in the resin were sectioned to a thickness of 70 nm using an ultramicrotome (Ultracut) to prepare ultrathin sections. These ultrathin sections were stained with a 2% uranium acetate and 1% lead citrate solution to prepare a transmission electron microscope sample of the slr1841 inhibitory strain from Example 1. Similarly, the slr0688-inhibited strain from Example 2 and the control strain from Comparative Example 1 were prepared for transmission electron microscopy by performing the same procedure.

[0144] (3-3) Observation using an electron microscope The ultrathin sections obtained in (3-2) above were observed using a transmission electron microscope (JEOL JEM-1400Plus) under an acceleration voltage of 100kV. The observation results are shown in Figures 3 to 8.

[0145] First, let's describe the slr1841 inhibitory strain of Example 1. Figure 3 is a TEM (Transmission Electron Microscope) image of the slr1841 inhibitory strain of Example 1. Figure 4 is a magnified image of the dashed area A in Figure 3. Figure 4(a) is a magnified TEM image of the dashed area A in Figure 3, and Figure 4(b) is a depiction of the magnified TEM image of Figure 4(a).

[0146] As shown in Figure 3, in the slr1841-suppressed strain of Example 1, the outer membrane was partially detached from the cell wall (i.e., the outer membrane was partially peeled off), and the outer membrane was also partially flexed.

[0147] To examine the state of the cell surface in more detail, we magnified the dashed area A and were able to confirm areas where the outer membrane had partially peeled off (dashed areas a1 and a2 in the figures), as shown in Figures 4(a) and 4(b). In addition, we were able to confirm an area where the outer membrane was significantly bent, adjacent to dashed area a1. This area is where the connection between the outer membrane and the cell wall was weakened, and it is thought that the outer membrane expanded outward and bent because the culture medium penetrated from the outer membrane into the periplasm.

[0148] Next, we will describe the slr0688 inhibitor strain of Example 2. Figure 5 is a TEM image of the slr0688 inhibitor strain of Example 2. Figure 6 is a magnified image of the dashed area B in Figure 5. Figure 6(a) is a magnified TEM image of the dashed area B in Figure 5, and Figure 6(b) is a diagram depicting the magnified TEM image of Figure 6(a).

[0149] As shown in Figure 5, in the slr0688-suppressed strain of Example 2, the outer membrane was partially detached from the cell wall and partially bent. Furthermore, it was confirmed that the outer membrane was partially detached from the cell wall in the slr0688-suppressed strain.

[0150] To examine the state of the cell surface in more detail, we performed a magnified observation of the dashed-line region B, and as shown in Figures 6(a) and 6(b), we were able to identify areas where the outer membrane was significantly bent (dashed-line region b1 in the figure) and areas where the outer membrane had partially peeled off (dashed-line regions b2 and b3 in the figure). In addition, we were able to identify areas where the outer membrane had detached from the cell wall near each of the dashed-line regions b1, b2, and b3.

[0151] Next, we will describe the Control strain of Comparative Example 1. Figure 7 is a TEM image of the Control strain of Comparative Example 1. Figure 8 is a magnified image of the dashed area C in Figure 7. Figure 8(a) is a magnified TEM image of the dashed area C in Figure 7, and Figure 8(b) is a diagram depicting the magnified TEM image of Figure 8(a).

[0152] As shown in Figure 7, the cell surface of the Control strain in Comparative Example 1 was well-organized, maintaining a state in which the inner membrane, cell wall, outer membrane, and S layer were stacked in order. In other words, in the Control strain, there were no areas where the outer membrane had detached from the cell wall, areas where the outer membrane had peeled off from the cell wall (i.e., fallen off), or areas where the outer membrane had bent, as was observed in Examples 1 and 2.

[0153] (4) Protein secretion productivity test The slr1841 inhibitory strain from Example 1, the slr0688 inhibitory strain from Example 2, and the control strain from Comparative Example 1 were each cultured, and the amount of protein secreted extracellularly (hereinafter also referred to as secreted protein amount) was measured. The protein secretion productivity of each of the above strains was evaluated based on the amount of protein in the culture medium. Protein secretion productivity refers to the ability to produce protein by secreting proteins produced inside the cell into the extracellular space. The specific method is described below.

[0154] (4-1) Culture of bacterial strains The slr1841 inhibitory strain from Example 1 was cultured in the same manner as described in (3-1) above. The culture was performed independently three times. The strains from Example 2 and Comparative Example 1 were also cultured under the same conditions as the strain from Example 1.

[0155] (4-2) Quantification of proteins secreted extracellularly The culture medium obtained in (4-1) above was centrifuged at 2,500 g for 10 minutes at room temperature to obtain the culture supernatant. The obtained culture supernatant was filtered using a membrane filter with a pore size of 0.22 μm to completely remove the cells of the slr1841 inhibitor strain from Example 1. The total amount of protein contained in the filtered culture supernatant was quantified by the BCA (Bicinchoninic Acid) method. This series of operations was performed for each of the three independently cultured culture media, and the mean and standard deviation of the amount of protein secreted extracellularly by the slr1841 inhibitor strain from Example 1 was determined. Similarly, the protein quantification of the three culture media for the strains of Example 2 and Comparative Example 1 was performed under the same conditions, and the mean and standard deviation of the protein amounts in the three culture media were determined.

[0156] The results are shown in Figure 9. Figure 9 is a graph showing the amount of protein (n=3, error bars=SD) in the culture medium of modified cyanobacteria from Example 1, Example 2, and Comparative Example 1.

[0157] As shown in Figure 9, both the slr1841-suppressed strain in Example 1 and the slr0688-suppressed strain in Example 2 showed an approximately 25-fold increase in the amount of protein (mg / L) secreted into the culture supernatant compared to the Control strain in Comparative Example 1.

[0158] Although the data is omitted here, the absorbance of the culture medium (730 nm) was measured, and the amount of secreted protein per gram of dry cell weight (mg protein / g cell dry weight) was calculated. In both the slr1841 inhibitory strain in Example 1 and the slr0688 inhibitory strain in Example 2, the amount of secreted protein per gram of dry cell weight (mg protein / g cell dry weight) was approximately 36 times higher compared to the control strain in Comparative Example 1.

[0159] Furthermore, as shown in Figure 9, the amount of protein secreted into the culture supernatant was greater in the slr0688-suppressed strain of Example 2, which suppressed the expression of the gene encoding the cell wall-pyruvate modifying enzyme (slr0688), than in the slr1841-suppressed strain of Example 1, which suppressed the expression of the gene encoding the SLH domain-retaining outer membrane protein (slr1841). This is thought to be related to the fact that there are more covalently bonded glycans on the cell wall surface than there are SLH domain-retaining outer membrane proteins (Slr1841) in the outer membrane. In other words, it is thought that the amount of protein secreted was greater in the slr0688-suppressed strain of Example 2 than in the slr1841-suppressed strain of Example 1 because the amount and strength of binding between the outer membrane and the cell wall were reduced more than in the slr1841-suppressed strain of Example 1.

[0160] The results above confirm that by suppressing the function of proteins involved in the binding of the outer membrane to the cell wall, the binding of the outer membrane to the cell wall of cyanobacteria is partially weakened, and the outer membrane partially detaches from the cell wall. It was also confirmed that the weakening of the binding of the outer membrane to the cell wall makes it easier for proteins produced inside the cyanobacteria cell to leak out of the cell. Therefore, it was demonstrated that the modified cyanobacteria and the method for producing the same according to this embodiment significantly improve protein secretion productivity.

[0161] (5) Identification of secreted proteins Next, the secreted proteins contained in the culture supernatant obtained in (4-2) above were identified by LC-MS / MS. The method is described below.

[0162] (5-1) Sample preparation Eight times the volume of cold acetone was added to the culture supernatant, and the mixture was allowed to stand at 20°C for 2 hours. Afterward, the mixture was centrifuged at 20,000 g for 15 minutes to obtain a protein precipitate. 100 mM Tris pH 8.5 and 0.5% sodium dodecanoate (SDoD) were added to this precipitate, and the protein was dissolved using a closed-type ultrasonic lysator. After adjusting the protein concentration to 1 μg / mL, 10 mM dithiothreitol (DTT) was added, and the mixture was allowed to stand at 50°C for 30 minutes. Subsequently, 30 mM iodoacetamide (IAA) was added, and the mixture was allowed to stand at room temperature (protected from light) for 30 minutes. To stop the reaction with IAA, 60 mM cysteine ​​was added, and the mixture was allowed to stand at room temperature for 10 minutes. 400 ng of trypsin was added, and the mixture was allowed to stand overnight at 37°C to fragment the protein into peptides. After adding 5% TFA (Trifluoroacetic Acid), the sample was centrifuged at 15,000 g for 10 minutes at room temperature to obtain the supernatant. This removed SDoD. After desalting using a C18 spin column, the sample was dried using a centrifugal evaporator. Subsequently, 3% acetonitrile and 0.1% formic acid were added, and the sample was dissolved using a closed-type ultrasonic disruptor. The peptide concentration was adjusted to 200 ng / μL.

[0163] (5-2)LC-MS / MS analysis The samples obtained in (5-1) above were analyzed using an LC-MS / MS instrument (UltiMate 3000 RSLCnano LC System) under the following conditions.

[0164] Sample injection volume: 200 ng Column: CAPCELL CORE MP 75μm×250mm Solvents: Solvent A is a 0.1% formic acid aqueous solution, Solvent B is 0.1% formic acid + 80% acetonitrile Gradient program: 8% solvent B 4 minutes after sample injection, 44% solvent B 27 minutes later, 80% solvent B 28 minutes later, measurement complete at 34 minutes later.

[0165] (5-3) Data Analysis The obtained data was analyzed under the following conditions to identify proteins and peptides and to calculate their quantitative values.

[0166] 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

[0167] Table 4 shows the 30 proteins with the highest relative quantitative values ​​among the identified proteins, those that are expected to have clear enzymatic activity.

[0168] [Table 4]

[0169] All six of these proteins were present in the culture supernatants of the slr1841 inhibitory strain in Example 1 and the slr0688 inhibitory strain in Example 2, respectively. Periplasmic (the space between the outer and inner membranes) translocation signals were preserved in all of these proteins. This result confirmed that in the modified strains of Example 1 and Example 2, the outer membrane partially detaches from the cell wall, making it easier for proteins within the periplasm to leak out of the outer membrane (i.e., outside the bacterial cell). Therefore, the modified cyanobacteria according to this embodiment were shown to have significantly improved protein secretion productivity.

[0170] (6) Identification of secreted intracellular metabolites (6-1) Sample preparation To 80 μl of the culture supernatant of modified cyanobacteria, a 20 μl aqueous solution of an internal standard substance, adjusted to a concentration of 1,000 μM, was added and stirred. After ultrafiltration, the solution was subjected to measurement.

[0171] (6-2) CE (Capillary electrophoresis)-TOFMS (Time-Of-Flight Mass Spectrometry) analysis In this test, measurements in cation mode and anion mode were performed under the following conditions.

[0172] [Cation Mode] Equipment: Agilent CE-TOFMS system Capillary: Fused silica capillary id 50μm×80cm Measurement conditions: 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 50μm×80cm Measurement conditions: 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

[0173] (6-3) Data Processing Peaks detected by CE-TOFMS were automatically identified using the automated integration software MasterHands® ver.2.17.1.11, specifically peaks with a signal-to-noise ratio of 3 or higher. For each detected peak, the specific mass-to-charge ratio (m / z) and electrophoresis time were compared with the values ​​of all substances registered in the HMT (Human Metabolome Technologies, Inc.) metabolite library to search for metabolites present in the culture supernatant of modified cyanobacteria. The acceptable error for the search was + / -0.5 min for electrophoresis time and + / -10 ppm for m / z. The concentration of each identified metabolite was calculated using a 100 μM single-count dose. The major identified metabolites are shown in Table 5.

[0174] [Table 5]

[0175] All 12 of these intracellular metabolites were present in the culture supernatants of the slr1841 inhibitory strain in Example 1 and the slr0688 inhibitory strain in Example 2, respectively. Although the data is not shown, these metabolites were not present in the culture supernatant of the Control strain in Comparative Example 1. This result confirms that in the modified strains of Example 1 and Example 2, intracellular metabolites are more easily leaked outside the outer membrane (i.e., outside the bacterial cell) due to the partial detachment of the outer membrane from the cell wall.

[0176] (7) Plant cultivation experiment Next, to evaluate the plant quality-enhancing effect of the secretions of modified cyanobacteria (in this case, the culture supernatant of modified cyanobacteria), the following plant cultivation trials were conducted. Specifically, tomato and strawberry cultivation trials were conducted to evaluate the effect on fruit production. In addition, lettuce cultivation trials were conducted to evaluate the effect on leafy vegetable production. These cultivation trials are described below.

[0177] (7-1) Tomato cultivation experiment First, commercially available potting soil was placed in a cultivation planter (22cm x 16cm), and three tomato seeds were sown per planter. Cultivation was carried out at an indoor temperature of 23°C and with a white light source with a photosynthetic photon flux density of 250 μmol / m². 2 The experiment was conducted for 150 days under conditions of 16 hours of light and 10 hours of darkness at a rate of / s. During this time, 500 mL of distilled water was supplied to each pot every two days. Approximately one week after the start of cultivation, when the cotyledons had unfolded, the plants were thinned to ensure uniform size in each planter. Additionally, 500 mL of commercially available chemical fertilizer (a 500-fold dilution of a stock solution containing 6% total nitrogen, 10% water-soluble phosphorus, 5% water-soluble potassium, 0.05% water-soluble magnesium, 0.001% water-soluble manganese, and 0.005% water-soluble boron) was applied to each planter once every 50 days.

[0178] (Example 3) As described above, after standardizing the individual plant sizes in each planter, 5 mL of the culture supernatant of the modified cyanobacteria (hereinafter referred to as the secretion of the modified cyanobacteria) was added to the base of each plant once a week. The plants were cultivated for 150 days, during which time the tomatoes were harvested in order as they ripened and turned red, and the number of harvested fruits was recorded. The weight and sugar content (Brix value) of the harvested fruits were also measured, and their mean and standard deviation (SD) were calculated. The modified cyanobacteria used were the slr1841 inhibitor strain from Example 1 and the slr0688 inhibitor strain from Example 2.

[0179] (Comparative Example 2) The procedure was the same as in Example 3, except that water was used instead of the secretions of modified cyanobacteria.

[0180] (result) The results for Example 3 and Comparative Example 2 are shown in Figures 10 to 13. Figure 10 is a graph showing the average number of fruits per tomato plant cultivated in Example 3 and Comparative Example 2. Figure 11 is a graph showing the average fruit weight per tomato plant cultivated in Example 3 and Comparative Example 2. Figure 12 is a graph showing the average sugar content per tomato plant cultivated in Example 3 and Comparative Example 2.

[0181] Furthermore, Figure 13 includes photographs of representative fruits to visually illustrate the state of the fruits in Example 3 and Comparative Example 2.

[0182] As shown in Figure 10, the average number of fruits per plant harvested in Example 3 increased by approximately 67% compared to Comparative Example 2. However, as shown in Figure 11, the average fruit weight per plant harvested in both Example 3 and Comparative Example 2 was comparable. Normally, as the yield of fruits per plant increases, the weight of the fruits tends to decrease, but in Example 3, despite the increased number of fruits harvested, the average fruit weight was comparable to that of Comparative Example 2.

[0183] Furthermore, as shown in Figure 12, the average sugar content (Brix sugar content) of the fruit harvested per plant in Example 3 and Comparative Example 2 were also similar. In addition, as shown in Figure 13, there were no differences in the size, shape, and gloss of the tomatoes harvested in Example 3 and Comparative Example 2. Normally, as the yield of fruit per plant increases, the sugar content of the fruit tends to decrease and the size of the fruit tends to decrease, but in Example 3, despite the larger number of fruits harvested, the average sugar content and size of the fruit were similar to those of Comparative Example 2.

[0184] Therefore, it was confirmed that applying the secretions of modified cyanobacteria to tomatoes could increase yields without reducing fruit quality (i.e., weight, size, and sugar content).

[0185] (7-2) Strawberry cultivation experiment Strawberry seedlings with approximately 9 leaves and a height of about 7 cm were transplanted into commercially available potting soil (12 cm x 10 cm). Cultivation was carried out under a light-period temperature of 20°C, a dark-period temperature of 15°C, and a white light source with a photosynthetic photon flux density of 200 μmol / m². 2 The experiment was conducted for 150 days under conditions of 14 hours of light and 10 hours of darkness at a rate of / s. During this time, 50 mL of distilled water was supplied to each pot every other day. In addition, 100 mL of commercially available chemical fertilizer (a 500-fold dilution of a stock solution containing 6% total nitrogen, 10% water-soluble phosphorus, 5% water-soluble potassium, 0.05% water-soluble magnesium, 0.001% water-soluble manganese, and 0.005% water-soluble boron) was applied to each pot once every 50 days.

[0186] (Example 4) During the cultivation period described above, 5 mL of the modified cyanobacteria secretion was added to the base of each plant once a week. Strawberries were harvested in order of ripening and redness, and the number of harvested fruits was recorded. The weight and sugar content (Brix value) of the harvested fruits were also measured, and their mean and standard deviation (SD) were calculated. The modified cyanobacteria were the slr1841 inhibitor strain from Example 1 and the slr0688 inhibitor strain from Example 2.

[0187] (Comparative Example 3) The procedure was the same as in Example 4, except that water was used instead of the secretions of modified cyanobacteria.

[0188] (result) The results for Example 4 and Comparative Example 3 are shown in Figures 14 to 17. Figure 14 is a graph showing the average number of fruits per strawberry plant cultivated in Example 4 and Comparative Example 3. Figure 15 is a graph showing the average fruit weight per strawberry plant cultivated in Example 4 and Comparative Example 3. Figure 16 is a graph showing the average sugar content per strawberry plant cultivated in Example 4 and Comparative Example 3.

[0189] Furthermore, Figure 17 includes photographs of representative fruits to visually illustrate the state of the fruits in Example 4 and Comparative Example 3.

[0190] As shown in Figure 14, the average number of fruits per plant harvested in Example 4 increased by approximately 47% compared to Comparative Example 3. However, as shown in Figure 15, there was no significant difference in the average fruit weight of the strawberries harvested in Example 4 and Comparative Example 3. In other words, despite the increased number of fruits harvested per plant in Example 4, the average fruit weight was comparable to that of Comparative Example 3.

[0191] Furthermore, as shown in Figure 16, the average sugar content (Brix sugar content) of the strawberries harvested in Example 4 was approximately 10% higher than that of Comparative Example 3. In other words, despite the larger number of fruits harvested, the average sugar content of the strawberries grown in Example 4 was higher.

[0192] Furthermore, as shown in Figure 17, there were no differences in appearance, such as size, shape, and color, between the strawberries harvested in Example 4 and Comparative Example 3. In other words, despite the larger number of fruits harvested in Example 4, the size of the fruits was comparable to that of Comparative Example 3.

[0193] Therefore, it was confirmed that applying the secretions of modified cyanobacteria to strawberries can increase yields while improving fruit quality (in this case, sugar content).

[0194] (7-3) Hydroponic Lettuce Cultivation Experiment For hydroponics, a 500-fold dilution of a commercially available nutrient solution stock containing 6% total nitrogen, 10% water-soluble phosphorus, 5% water-soluble potassium, 0.05% water-soluble magnesium, 0.001% water-soluble manganese, and 0.005% water-soluble boron was used. The lighting conditions were a white light source with a photosynthetic photon flux density of 200 μmol / m². 2 The plants were cultivated at room temperature (22°C) for 35 days under conditions of 16 hours of light and 8 hours of darkness.

[0195] (Example 5) During the cultivation period described above, a 5 mL dose of the modified cyanobacteria secretion per plant was added to the hydroponic culture medium once a week. After harvesting, the plant weight was measured, and the mean and standard deviation (SD) were calculated. Furthermore, the percentage of plants that developed tip burn (a phenomenon in which the tips of the leaves turn black and wither), a physiological disorder commonly seen in hydroponic cultivation, was calculated. The modified cyanobacteria used were the slr1841 inhibitor strain from Example 1 and the slr0688 inhibitor strain from Example 2.

[0196] (Comparative Example 4) The procedure was the same as in Example 5, except that water was used instead of the secretions of modified cyanobacteria.

[0197] (result) The results for Example 5 and Comparative Example 4 are shown in Figures 18 to 20. Figure 18 is a graph showing the average plant weight per lettuce plant cultivated in Example 5 and Comparative Example 4. Figure 19 is a graph showing the percentage of lettuce that developed tip burn among the lettuce plants cultivated in Example 5 and Comparative Example 4.

[0198] Furthermore, Figure 20 includes photographs of representative plants to visually illustrate the condition of each plant in Example 5 and Comparative Example 4.

[0199] As shown in Figure 18, the average weight per lettuce plant harvested in Example 5 was approximately 21% higher than in Comparative Example 4. However, as shown in Figure 19, the percentage of lettuce plants that developed tip burn (also known as the tip burn incidence rate) among the lettuce plants cultivated in Example 5 was approximately 94% lower than in Comparative Example 4. Generally, in hydroponic lettuce cultivation, the faster the plant growth, the higher the tip burn incidence rate tends to be. However, despite the rapid growth of the lettuce plants cultivated in Example 5, the tip burn incidence rate was significantly reduced.

[0200] Furthermore, as shown in Figure 20, the lettuce plants cultivated in Example 5 did not show any particularly noticeable changes in overall shape compared to Comparative Example 4. In other words, the lettuce plants cultivated in Example 5 had an increased average plant weight and faster growth than those in Comparative Example 4, yet their appearance was the same as in Comparative Example 4, and the incidence of tip burn was significantly reduced.

[0201] Therefore, it was confirmed that applying secretions from modified cyanobacteria can increase crop yields while improving crop quality.

[0202] (summary) Based on the results of cultivation trials of tomatoes, strawberries, and lettuce, it was confirmed that the plant quality enhancer according to this embodiment has the effect of improving the quality of plants in multiple crop species, such as increasing yield, maintaining or increasing the sugar content of fruits, and reducing physiological disorders. [Industrial applicability]

[0203] According to this disclosure, it is possible to provide modified cyanobacteria with improved secretion productivity of plant quality-enhancing substances. Furthermore, by culturing the modified cyanobacteria of this disclosure, the above-mentioned substances can be produced efficiently, and by adding these substances to soil and hydroponic solutions, for example, plant quality can be improved. [Explanation of Symbols]

[0204] 1 Intima 2. Peptide Glycan 3. Glycans 4 cell wall 5 Adventitia 6. SLH domain-retaining outer membrane proteins 7 SLH domain 8 Organic Channel Proteins 9 Cell wall-pyruvate modifying enzymes

Claims

1. A step of preparing a modified cyanobacterium in which the function of a protein involved in binding between the outer membrane and the cell wall in cyanobacteria is suppressed or lost; A step of secreting a secretion product involved in improving the quality of plants from the modified cyanobacteria; Including, A method for producing a plant quality enhancer.

2. The protein involved in the binding between the outer membrane and the cell wall is at least one of an SLH (Surface Layer Homology) domain-containing outer membrane protein and a cell wall-pyruvate modifying enzyme. A method for producing the plant quality improving agent according to claim 1.

3. The SLH domain-containing 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-containing outer membrane proteins. A method for producing the plant quality improving agent according to claim 2.

4. 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; 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-pyruvate modifying enzymes; A method for producing the plant quality improving agent according to claim 2.

5. a gene expressing a protein involved in the binding of the outer membrane to the cell wall is deleted or inactivated; A method for producing the plant quality improving agent according to claim 1.

6. The gene expressing a protein involved in the binding between the outer membrane and the cell wall is at least one of a gene encoding an SLH domain-retaining outer membrane protein and a gene encoding a cell wall-pyruvate modifying enzyme. A method for producing the plant quality improving agent according to claim 5.

7. The gene encoding the SLH domain-containing outer membrane protein is slr1841 consisting of the base sequence shown in SEQ ID NO: 7; nies970_09470 consisting of the base sequence shown in SEQ ID NO: 8, anacy_3458 consisting of the base sequence shown in SEQ ID NO: 9, or A gene whose base sequence is 50% or more identical to any of these genes. A method for producing the plant quality improving agent according to claim 6.

8. The gene encoding the cell wall-pyruvate modifying enzyme is slr0688 consisting of the base sequence shown in SEQ ID NO: 10; synpcc7942_1529 consisting of the base sequence shown in SEQ ID NO: 11, anacy_1623 consisting of the base sequence shown in SEQ ID NO: 12, or A gene whose base sequence is 50% or more identical to any of these genes. A method for producing the plant quality improving agent according to claim 6.

9. The secretion of a modified cyanobacterium in which the function of a protein involved in binding between the outer membrane and the cell wall in cyanobacteria is suppressed or lost, Plant quality enhancer.

10. The plant quality improving agent according to claim 9 is used for plants. Method for improving plant quality.

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