Crop cultivation method and crop cultivation culture supernatant

Abstract

To activate acidic invertase of crops to enhance the crop productivity through the application of cyanobacteria secretion.SOLUTION: Crop cultivation methods involve applying cyanobacteria secretion to crops to activate the acidic invertase of the crops.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present disclosure relates to a method for cultivating agricultural crops. [Background technology]

[0002] As the world population grows and demands increased food production, there is a need to develop technologies for efficient crop production on limited arable land. Approaches aimed at increasing crop yields can be broadly divided into two categories: methods that control environmental factors such as light, temperature, humidity, and soil components to create conditions suitable for crop growth (i.e., environmental control methods), and methods that artificially control plant cellular physiology to promote growth by using genetic engineering or adding physiologically active substances (i.e., cellular physiology control methods).

[0003] One method for controlling cellular physiology is known to be the regulation of invertase activity through genetic engineering (Non-Patent Document 1). Invertase is an enzyme that breaks down sucrose (also known as sucrose) into glucose and fructose. It is deeply involved in the translocation, distribution, and accumulation of sucrose, produced by photosynthesis in leaves, to various organs of the plant. Invertases are broadly classified into neutral invertase and acid invertase based on their optimal pH. There are two types of acid invertase: cell wall invertase, which is localized in the cell wall, and vacuolar invertase, which is localized in the vacuole. Among invertases, acid invertase activity is particularly known to be closely related to crop yield. For example, Non-Patent Document 2 discloses that activating vacuolar invertase through genetic engineering technology can enhance cotton fiber production in cotton. Furthermore, Non-Patent Document 3 discloses that vacuolar invertase activity is essential for rice panicle growth. Furthermore, for example, Non-Patent Documents 4 and 5 disclose that the activation of cell wall invertase in corn and soybean using genetic engineering techniques increases the yield of soybean and corn, and also increases the sugar content of the respective grains. Based on these findings, there is a need for the development of a technology that can improve the productivity of agricultural crops by artificially activating acid invertase. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Roitsch, T. and MC Gonzalez (2004). "Function and regulation of plant invertases: sweet sensations." Trends in Plant Science 9(12): 606-613. [Non-patent document 2] Wang, L., et al. (2010). "Evidence that high activity of vacuolar invertase is required for cotton fiber and Arabidopsis root elongation through osmotic dependent and independent pathways, respectively." Plant Physiology, 154(2):744-756. [Non-Patent Document 3] Morey, S. R., et al. (2018). "Genetic Evidence for the Role of a Rice Vacuolar Invertase as a Molecular Sink Strength Determinant." Rice 11. [Non-Patent Document 4] Tang, X., et al. (2017). "Suppression of extracellular invertase inhibitor gene expression improves seed weight in soybean (Glycine max)." Journal of Experimental Botany 68(3): 469-482. [Non-Patent Document 5] Li, B., et al. (2013). "Constitutive expression of cell wallinvertase genes increases grain yield and starch content in maize." Plant Biotechnology Journal 11(9): 1080-1091. [Non-Patent Document 6] 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. [Non-Patent Document 7] Jurgens and Weckesser, 1986, J. Bacteriol., 168:568-573. [Non-patent document 8] Kojima et al., 2016, Biosci.Biotech. Biochem., 10:1954-1959. [Non-Patent Document 9] Kowata et al., 2017, J. Bacteriol., 199:e00371-17. [Non-Patent Document 10] Kojima et al., 2016, J. Biol. Chem., 291:20198-20209. [Non-Patent Document 11] Mesnage et al., 2000, EMBO J., 19:4473-4484. [Non-Patent Document 12] Yao et al., ACS Synth. Biol., 2016, 5:207-212. Summary of the Invention [Problem to be solved by the invention]

[0005] However, currently, methods for activating acid invertase are limited to genetic engineering, making practical application difficult. Regulatory restrictions on genetic engineering in various countries around the world, as well as the psychological aversion of producers and consumers to applying (hereinafter also referred to as "applying" or "using") genetic engineering to plants, are major obstacles to the social implementation of such technologies. Therefore, there is a need for a method that improves crop productivity by activating acid invertase in agricultural crops using external stimuli (e.g., adding some kind of substance to the plant from outside) without using genetic engineering.

[0006] Therefore, the present disclosure provides a method for cultivating agricultural crops that improves productivity of the crops by applying a cyanobacterial secretion to activate acid invertase in the plants. [Means for solving the problem]

[0007] A method for cultivating agricultural crops according to one embodiment of the present disclosure includes applying a secretion of cyanobacteria to the agricultural crops to activate acid invertase in the agricultural crops. [Effects of the Invention]

[0008] By applying the plant acid invertase activator of the present disclosure to plants, plant acid invertase can be effectively activated, thereby improving the productivity of agricultural crops. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a flowchart showing an example of a method for producing a plant acid invertase activator according to an embodiment. [Figure 2] Figure 2 is a schematic diagram of the cell surface of a cyanobacterium. [Figure 3] FIG. 3 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 1. [Figure 4]FIG. 4 is an enlarged image of the dashed area A in FIG. [Figure 5] FIG. 5 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 2. [Figure 6] FIG. 6 is an enlarged image of the dashed area B in FIG. [Figure 7] FIG. 7 is a transmission electron microscope image of an ultrathin section of the modified cyanobacterium of Comparative Example 1. [Figure 8] FIG. 8 is an enlarged image of the dashed line area C in FIG. [Figure 9] FIG. 9 is a graph showing the amount of protein in the culture medium of the modified cyanobacteria of Example 1, Example 2, and Comparative Example 1 (n=3, error bars=SD). [Figure 10] FIG. 10 is a graph showing the average values ​​of acid invertase activity of spinach cultivated in Example 3 and Comparative Example 2. [Figure 11] FIG. 11 is a graph showing the average value of the dry weight of the above-ground part per plant of spinach cultivated in Example 3 and Comparative Example 2. [Figure 12] FIG. 12 is a graph showing the average values ​​of acid invertase activity of strawberries grown in Example 4 and Comparative Example 3. [Figure 13] FIG. 13 is a graph showing the average number of fruits per plant of the strawberries cultivated in Example 4 and Comparative Example 3. [Figure 14] FIG. 14 is a graph showing the average fruit weight per plant of the strawberries cultivated in Example 4 and Comparative Example 3. [Figure 15] FIG. 15 is a graph showing the average sugar content per plant of the strawberries cultivated in Example 4 and Comparative Example 3. [Figure 16] FIG. 16 shows the state of typical fruits in Example 4 and Comparative Example 3. DETAILED DESCRIPTION OF THE INVENTION

[0010] (Findings that formed the basis of this disclosure) As mentioned in the Background Art section, there is a need for technology to efficiently produce crops within limited arable land. One promising method for achieving this is to activate plant acid invertase.

[0011] The following prior art techniques have been disclosed as techniques for activating plant acid invertase.

[0012] For example, Non-Patent Document 2 reports that cotton fiber elongation is promoted by using a 35S promoter to highly express the cotton vacuolar invertase gene.

[0013] Furthermore, for example, Non-Patent Document 4 reports that cell wall invertase can be activated by suppressing the expression of a gene that inhibits soybean cell wall invertase activity using ribonucleic acid (RNA) interference. Specifically, it has been reported that applying this technology to soybeans increases the weight per soybean kernel, increases the harvest weight per plant, and also increases the sugar content per soybean kernel.

[0014] Furthermore, for example, Non-Patent Document 5 reports that the yield of corn is increased by using the 35S promoter to highly express the corn cell wall invertase gene, and that the sugar content per kernel is also increased.

[0015] However, currently, methods for activating plant acid invertase are limited to genetic engineering techniques, making their practical application difficult. Regulatory restrictions on genetic engineering in various countries around the world, as well as the psychological aversion of producers and consumers to the application of genetic engineering to plants, are major obstacles to the social implementation of such techniques. Therefore, there is a strong demand for methods for activating crop acid invertase by adding some kind of substance to plants from outside, without using genetic engineering. Furthermore, in recent years, with growing awareness of environmental concerns, such as preventing global warming and reducing environmental impact, there is a demand for the use of naturally derived substances that have a low environmental impact when applied. In particular, there is a particular demand for the application of naturally derived substances that consume less fossil energy during their production and therefore have a lower environmental impact.

[0016] The present inventors have focused on cyanobacteria as a microorganism to be used in producing naturally derived substances that contribute to improving agricultural productivity. Cyanobacteria (also known as blue-green bacteria or blue-green algae) are a group of eubacteria that decompose water through photosynthesis to produce oxygen, and use the energy obtained to fix CO2 from the air. Some cyanobacteria species can also fix nitrogen (N2) from the air. As such, cyanobacteria can obtain most of the raw materials (i.e., nutrients) and energy necessary for bacterial growth from air, water, and light, making it possible to culture cyanobacteria using inexpensive raw materials and a simple process.

[0017] Furthermore, cyanobacteria are known to have characteristics such as rapid growth and high light utilization efficiency. Furthermore, compared to other algae species, genetic manipulation is easier. Therefore, active research and development is being conducted on the production of substances using cyanobacteria, a type of photosynthetic microorganism. For example, the production of fuels such as ethanol, isobutanol, alkanes, and fatty acids (Patent Document 1: Japanese Patent No. 6341676) has been reported as examples of substance production using cyanobacteria. Research and development is also being conducted on the production of substances that serve as nutrient sources for living organisms. For example, because proteins can only be synthesized by living organisms, there is a demand for the development of technologies for simple and efficient protein production. Cyanobacteria, which can utilize light energy and atmospheric CO2, are expected to be utilized as one of the biological species used in this technology, and active research and development is being conducted on them (Non-Patent Document 6).

[0018] For example, desired compounds and proteins can be produced within cyanobacterial cells (hereinafter also referred to as intracellular) by arbitrarily modifying the genes of cyanobacteria using the technology described in Non-Patent Document 6. However, the desired compounds and proteins produced within cyanobacterial cells are not easily secreted outside the cells, so it is necessary to disrupt the cyanobacterial cells and extract the desired compounds and proteins produced within the cells.

[0019] The present inventors have discovered that partially detaching the outer membrane covering the cyanobacterial cell wall from the cell wall facilitates the extracellular secretion of desired compounds, proteins, and intracellular metabolites produced within the cyanobacterial cell. Through this process, the present inventors have also discovered that cyanobacterial secretions have the effect of activating acid invertase in multiple crop species. This allows for efficient recovery of the extracellularly secreted plant acid invertase activator (i.e., plant acid invertase activator) without disrupting the cyanobacterial cell. Furthermore, since the physiological activity of the plant acid invertase activator is less likely to be impaired by eliminating the need for extraction or other procedures, a plant acid invertase activator containing the secretion can effectively activate plant acid invertase.

[0020] Therefore, the method for producing a plant acid invertase activator of the present disclosure allows for simple and efficient production of a plant acid invertase activator containing a substance that has the effect of activating acid invertase in multiple crop species. Furthermore, the plant acid invertase activator of the present disclosure can effectively activate plant acid invertase. Furthermore, the method for activating plant acid invertase of the present disclosure allows for effective activation of plant acid invertase by applying the plant acid invertase activator of the present disclosure to plants.

[0021] (Summary of the Disclosure) An outline of one aspect of the present disclosure is as follows.

[0022] A method for producing a plant acid invertase activator according to one aspect of the present disclosure includes the steps 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, and causing the modified cyanobacterium to secrete a secretory product involved in activating plant acid invertase.

[0023] As a result, in the modified cyanobacterium, the bond between the cell wall and the outer membrane (e.g., the amount and strength of the bond) 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 intracellularly produced substances) are more likely to leak out of the outer membrane, i.e., outside the cell. This facilitates extracellular secretion of proteins and metabolites produced within the modified cyanobacterium, eliminating the need for extraction treatments of the intracellularly produced substances, such as disrupting the cell. This allows for simple and efficient production of a plant acid invertase activator containing a secretion product of the modified cyanobacterium. Furthermore, because the extraction treatment of the intracellularly produced substances is unnecessary, a decrease in the physiological activity and yield of the intracellularly produced substances is less likely to occur. Therefore, a decrease in the physiological activity and yield of the substances involved in the activation of plant acid invertase (i.e., plant acid invertase activators), among the intracellularly produced substances of the modified cyanobacterium, is less likely to occur. This improves the effect of the secretion of the modified cyanobacterium on activating plant acid invertase (hereinafter also referred to as plant acid invertase activation effect). Furthermore, because the above-described extraction process of the intracellularly produced substance is unnecessary, the modified cyanobacterium can be repeatedly used to produce the intracellularly produced substance even after the extracellularly secreted intracellularly produced substance is recovered. Therefore, it is not necessary to prepare a new modified cyanobacterium each time a plant acid invertase activator is produced. Therefore, according to the method for producing a plant acid invertase activator according to one embodiment of the present disclosure, a plant acid invertase activator can be produced simply and efficiently.

[0024] For example, in a method for producing a plant acid invertase activator according to one embodiment of the present disclosure, the protein involved in the binding between the outer membrane and the cell wall may be at least one of an SLH (Surface Layer Homology) domain-containing outer membrane protein and a cell wall-pyruvate modifying enzyme.

[0025] As a result, in the engineered cyanobacterium, for example, (i) the function of at least one of the SLH domain-containing outer membrane protein that binds to the cell wall and the enzyme that catalyzes the reaction of modifying the bound glycans on the surface of the cell wall with pyruvate (i.e., cell wall-pyruvate-modifying enzyme) is suppressed or lost, or (ii) the expression of at least one of the SLH domain-containing outer membrane protein and the cell wall-pyruvate-modifying enzyme is suppressed. Therefore, the bond (i.e., the amount and strength of the bond) between the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane and the covalently bound glycans on the surface of the cell wall is reduced. This makes the outer membrane more likely to detach from the cell wall in areas where the bond between the outer membrane and the cell wall is weakened. As a result, in the engineered cyanobacterium, the reduced bond between the outer membrane and the cell wall makes the outer membrane more likely to detach partially from the cell wall, which makes it easier for intracellularly produced substances such as proteins and metabolites to leak out of the cell. This improves the secretion productivity of the engineered cyanobacterium, which secretes the plant acid invertase activator produced intracellularly outside the cell. Therefore, according to the method for producing a plant acid invertase activator according to one embodiment of the present disclosure, the engineered cyanobacterium can be caused to efficiently secrete the plant acid invertase activator, thereby enabling efficient production of a plant acid invertase activator containing the plant acid invertase activator.

[0026] For example, in a method for producing a plant acid invertase activator according to one embodiment 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 having an amino acid sequence that is 50% or more identical to any of these SLH domain-retaining outer membrane proteins.

[0027] As a result, in the modified cyanobacterium, for example, (i) the function of any of the SLH domain-retaining outer membrane proteins set forth in SEQ ID NOS: 1 to 3 or a protein with 50% or more amino acid sequence identity to any of these SLH domain-retaining outer membrane proteins is suppressed or lost, or (ii) the expression of any of the SLH domain-retaining outer membrane proteins set forth in SEQ ID NOS: 1 to 3 or a protein with 50% or more amino acid sequence identity to any of these SLH domain-retaining outer membrane proteins is suppressed. Therefore, in the modified cyanobacterium, (i) the function of an SLH domain-retaining outer membrane protein or a protein with a function equivalent to an SLH domain-retaining outer membrane protein in the outer membrane is suppressed or lost, or (ii) the expression level of an SLH domain-retaining outer membrane protein or a protein with a function equivalent to an SLH domain-retaining outer membrane protein in the outer membrane is reduced. As a result, in the modified cyanobacterium, the binding amount and binding strength of the binding domain (e.g., SLH domain) that binds the outer membrane to the cell wall is reduced, making the outer membrane more likely to partially detach from the cell wall. This facilitates extracellular secretion of the plant acid invertase activator produced within the bacterial cells, thereby facilitating extracellular secretion of the plant acid invertase activator produced within the bacterial cells. Therefore, according to the method for producing a plant acid invertase activator of one aspect of the present disclosure, the plant acid invertase activator produced within the bacterial cells of the modified cyanobacterium can be efficiently produced.

[0028] For example, in a method for producing a plant acid invertase activator according to one embodiment 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 having an amino acid sequence that is 50% or more identical to any of these cell wall-pyruvate modifying enzymes.

[0029] As a result, in the modified cyanobacterium, for example, (i) the function of any of the cell wall-pyruvate-modifying enzymes set forth in SEQ ID NOS: 4 to 6 or a protein whose amino acid sequence is 50% or more identical to that of any of these cell wall-pyruvate-modifying enzymes is suppressed or lost, or (ii) the expression of any of the cell wall-pyruvate-modifying enzymes set forth in SEQ ID NOS: 4 to 6 or a protein whose amino acid sequence is 50% or more identical to that of any of these cell wall-pyruvate-modifying enzymes is suppressed. Therefore, in the modified cyanobacterium, (i) the function of the cell wall-pyruvate-modifying enzyme or a protein with a function equivalent to that enzyme is suppressed or lost, or (ii) the expression level of the cell wall-pyruvate-modifying enzyme or a protein with a function equivalent to that enzyme is reduced. This makes it difficult for covalently bonded sugar chains on the surface of the cell wall to be modified with pyruvate, thereby reducing the amount and strength of binding of the sugar chains on the cell wall to the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane. As a result, in the modified cyanobacterium, the covalently bonded sugar chains on the surface of the cell wall are less likely to be modified with pyruvate, weakening the bond between the cell wall and the outer membrane and making the outer membrane more likely to partially detach from the cell wall. This makes it easier for intracellularly produced substances to leak out of the cells, and therefore makes it easier for the plant acid invertase activator produced within the cells to also leak out of the cells. Therefore, according to a method for producing a plant acid invertase activator according to one aspect of the present disclosure, the plant acid invertase activator produced within the modified cyanobacterium is more likely to be secreted out of the cells, allowing for efficient production of the plant acid invertase activator.

[0030] For example, in a method for producing a plant acid invertase activator according to one embodiment of the present disclosure, a gene that expresses a protein involved in the binding between the outer membrane and the cell wall may be deleted or inactivated.

[0031] As a result, in the modified cyanobacterium, the expression of a protein involved in the binding between the cell wall and the outer membrane is suppressed, or the function of this protein is suppressed or lost, resulting in a partial reduction in the binding between the cell wall and the outer membrane (i.e., the amount and strength of binding). As a result, in the modified cyanobacterium, the outer membrane is more likely to partially detach from the cell wall, making it easier for intracellularly produced substances such as proteins and metabolites to leak out of the outer membrane, i.e., outside the cell. Therefore, the modified cyanobacterium has improved secretion productivity of the plant acid invertase activator produced intracellularly. This eliminates the need for extraction treatments of the intracellularly produced substances, such as disrupting the cells, and therefore reduces the physiological activity and yield of the intracellularly produced substance. Therefore, reduces the physiological activity and yield of the plant acid invertase activator produced intracellularly, making it possible to produce a plant acid invertase activator with improved plant acid invertase activation effect. Furthermore, because the extraction process of the intracellularly produced substance is not required, the engineered cyanobacteria can be repeatedly used to produce a plant acid invertase activator even after the substance is recovered. Therefore, there is no need to prepare a new engineered cyanobacterium each time a plant acid invertase activator is produced. Therefore, the method for producing a plant acid invertase activator according to one embodiment of the present disclosure allows for simple and efficient production of a plant acid invertase activator.

[0032] For example, in a method for producing a plant acid invertase activator according to one embodiment of the present disclosure, the gene expressing a protein involved in the binding between the outer membrane and 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.

[0033] As a result, in the engineered cyanobacterium, at least one gene encoding an SLH domain-containing outer membrane protein and at least one gene encoding a cell wall-pyruvate-modifying enzyme are deleted or inactivated. Therefore, in the engineered cyanobacterium, for example, (i) the expression of at least one of the SLH domain-containing outer membrane protein and the cell wall-pyruvate-modifying enzyme is suppressed, or (ii) the function of at least one of the SLH domain-containing outer membrane protein and the cell wall-pyruvate-modifying enzyme is suppressed or lost. This reduces the bond (i.e., the amount and strength of the bond) between the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane and the covalently bound glycans on the surface of the cell wall. This makes the outer membrane more likely to detach from the cell wall at areas where the bond between the outer membrane and the cell wall is weakened. As a result, in the engineered cyanobacterium, the outer membrane is more likely to detach partially from the cell wall due to the reduced bond between the outer membrane and the cell wall, which makes it easier for proteins and metabolites produced within the cell to leak out of the cell. This facilitates leakage of the plant acid invertase activator produced within the bacterial cells. Therefore, according to the method for producing a plant acid invertase activator according to one embodiment of the present disclosure, the modified cyanobacterium can be caused to efficiently secrete the plant acid invertase activator, thereby enabling efficient production of the plant acid invertase activator.

[0034] For example, in a method for producing a plant acid invertase activator according to one embodiment of the present disclosure, the gene encoding the SLH domain-retaining outer membrane protein may be 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.

[0035] As a result, in the modified cyanobacterium, a gene encoding any one of the SLH domain-containing outer membrane proteins set forth in SEQ ID NOS: 7 to 9, or a gene having 50% or more identity to the nucleotide sequence of any one of these genes, is deleted or inactivated. Therefore, in the modified cyanobacterium, (i) the expression of any one of the SLH domain-containing outer membrane proteins or a protein having a function equivalent to any one of these proteins is suppressed, or (ii) the function of any one of the SLH domain-containing outer membrane proteins or a protein having a function equivalent to any one of these proteins is suppressed or lost. As a result, in the modified cyanobacterium, the binding amount and strength of the binding domain (e.g., the SLH domain) that binds the outer membrane to the cell wall is reduced, making the outer membrane more likely to partially detach from the cell wall. This makes it easier for proteins and metabolites produced within the bacterial cell to leak out of the bacterial cell, and therefore easier for plant acid invertase activators produced within the bacterial cell to also leak out of the bacterial cell. Therefore, according to the method for producing a plant acid invertase activator according to one embodiment of the present disclosure, the plant acid invertase activator produced within the cells of the modified cyanobacterium is more likely to leak out of the cells, allowing the plant acid invertase activator to be produced efficiently.

[0036] For example, in a method for producing a plant acid invertase activator according to one embodiment of the present disclosure, the gene encoding the cell wall-pyruvate modifying enzyme may be 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 having a base sequence that is 50% or more identical to any of these genes.

[0037] As a result, in the modified cyanobacterium, a gene encoding any one of the cell wall-pyruvate-modifying enzymes set forth in SEQ ID NOS: 10 to 12 or a gene having 50% or more identity to the nucleotide sequence of a gene encoding any one of these enzymes is deleted or inactivated. Therefore, in the modified cyanobacterium, (i) the expression of any one of the cell wall-pyruvate-modifying enzymes or a protein having a function equivalent to any one of these enzymes is suppressed, or (ii) the function of any one of the cell wall-pyruvate-modifying enzymes or a protein having a function equivalent to any one of these enzymes is suppressed or lost. This reduces the amount and strength of binding between the cell wall sugar chains and the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane, as covalently bound sugar chains on the cell wall surface are less likely to be modified with pyruvate. As a result, in the modified cyanobacterium, the amount of sugar chains 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 the outer membrane more susceptible to partial detachment from the cell wall. This facilitates the extracellular leakage of proteins and metabolic products produced within the bacterial cells, and therefore facilitates the extracellular leakage of the plant acid invertase activator produced within the bacterial cells. Therefore, according to the method for producing a plant acid invertase activator according to one aspect of the present disclosure, the plant acid invertase activator produced within the modified cyanobacterium cells is easily extracellularly leaked, enabling efficient production of the plant acid invertase activator.

[0038] Furthermore, a plant acid invertase activator according to one embodiment of the present disclosure includes a secretion product of a modified cyanobacterium in which the function of a protein involved in binding between the outer membrane and the cell wall in the cyanobacterium has been suppressed or eliminated.

[0039] As a result, in the modified cyanobacterium, the bond between the cell wall and the outer membrane (i.e., the amount and strength of the bond) is partially reduced, making it easier for the outer membrane to partially detach from the cell wall. Therefore, in the modified cyanobacterium, proteins and metabolites produced intracellularly (i.e., intracellularly produced substances) are more likely to leak out of the outer membrane (i.e., outside the cell). This facilitates extracellular secretion of proteins and metabolites produced intracellularly by the modified cyanobacterium, eliminating the need for extraction treatments of the intracellularly produced substances, such as disrupting the cell. This allows for simple and efficient production of a plant acid invertase activator containing a secretion product of the modified cyanobacterium. Furthermore, because the extraction treatment of the intracellularly produced substances is unnecessary, a decrease in the physiological activity and yield of the intracellularly produced substances is less likely to occur. Therefore, a decrease in the physiological activity and yield of a substance involved in plant acid invertase activation (hereinafter also referred to as a plant acid invertase activator), among the intracellularly produced substances of the modified cyanobacterium, is less likely to occur. This makes it possible to obtain a plant acid invertase activator with improved plant acid invertase activation effect. Therefore, the plant acid invertase activator according to one embodiment of the present disclosure can effectively activate plant acid invertase.

[0040] Furthermore, a method for activating plant acid invertase according to one embodiment of the present disclosure uses the plant acid invertase activator described above in plants.

[0041] According to a method for activating plant acid invertase according to one embodiment of the present disclosure, by applying to a plant a plant acid invertase activator with improved plant acid invertase activation effect, plant acid invertase can be effectively activated.

[0042] Hereinafter, the embodiments will be specifically described with reference to the drawings.

[0043] The embodiments described below are all comprehensive or specific examples. The numerical values, materials, steps, and step orders shown in the following embodiments are merely examples and are not intended to limit the present disclosure. Furthermore, among the components in the following embodiments, components that are not described in the independent claims that represent the highest concepts are described as optional components.

[0044] In addition, the drawings are not necessarily strict illustrations, and in the drawings, substantially the same components are denoted by the same reference numerals, and overlapping descriptions may be omitted or simplified.

[0045] Furthermore, in the following, numerical ranges do not only represent strict meanings, but also include substantially equivalent ranges, for example, measuring the amount (e.g., number or concentration) of a protein or its range.

[0046] In this specification, the term "bacterium" and "cell" both refer to an individual cyanobacterium.

[0047] (Embodiment) Herein, the identity of nucleotide 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 cyanobacterial genes and the proteins they encode is publicly available, for example, in the above-mentioned NCBI database and Cyanobase (http: / / genome.microbedb.jp / cyanobase / ). The amino acid sequences of target proteins and the nucleotide sequences of the genes encoding those proteins can be obtained from these databases.

[0048] [1. Plant acid invertase activator] First, the plant acid invertase activator according to the present embodiment will be described. The plant acid invertase activator contains a secretion involved in the activation of plant acid invertase and has the effect of activating plant acid invertase. As described above, invertase is an enzyme that catabolizes sucrose into reducing sugars such as glucose and fructose in plants. In particular, acid invertase contributes to the utilization of sucrose in plants and its catabolism into reducing sugars, a form of storage sugar. Therefore, by activating plant acid invertase, the plant acid invertase activator according to the present embodiment can promote plant growth and the accumulation of storage sugars in fruits and other crops. Therefore, when used on agricultural crops, the plant acid invertase activator according to the present embodiment can efficiently improve crop production.

[0049] For example, promoting plant growth means increasing the number of leaves, stems, buds, flowers, or fruits of a plant, thickening the stem or trunk, and increasing the height of the plant. By promoting plant growth, the plant body and its roots increase in size, and the number of fruits increases.

[0050] Furthermore, acid invertase contributes to improving plant quality by preventing plant diseases, improving nutrient absorption, increasing fruit sugar content, etc. Therefore, plant acid invertase activators can effectively improve plant quality for multiple crop species by increasing crop yield, increasing crop and fruit volume, increasing fruit sugar content, reducing physiological disorders, and reducing disease damage.

[0051] In addition, plants include crops grown in fields (so-called agricultural crops), as well as garden trees, flowers, lawns, roadside trees, etc., and also forest trees that are rarely fertilized.

[0052] In this embodiment, the plant acid invertase activator comprises a secretion product of a modified cyanobacterium (hereinafter also referred to as the parent cyanobacterium) in which the function of a protein involved in binding between the outer membrane and the cell wall has been suppressed or eliminated. Note that the cyanobacterium (i.e., the parent cyanobacterium) and the modified cyanobacterium will be described later.

[0053] As described above, the secreted products include secreted products involved in the activation of plant acid invertase. The secreted products include proteins and metabolic products produced within the engineered cyanobacterium (i.e., intracellularly produced substances). The intracellularly produced substances include substances involved in the activation of plant acid invertase (i.e., plant acid invertase activators).

[0054] Examples of plant acid invertase activators include organic decomposition enzymes such as peptidases, nucleases, or phosphatases, substances related to DNA metabolism 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 engineered cyanobacteria may also be a mixture of these plant acid invertase activators.

[0055] [2. Method for producing plant acid invertase activator] Next, a method for producing a plant acid invertase activator according to the present embodiment will be described with reference to Fig. 1. Fig. 1 is a flow chart showing an example of the method for producing a plant acid invertase activator according to the present embodiment.

[0056] The method for producing a plant acid invertase activator according to this embodiment includes the steps of: preparing a modified cyanobacterium (i.e., parent cyanobacterium) in which the function of a protein involved in binding between the outer membrane and the cell wall is suppressed or eliminated (step S01); and causing the modified cyanobacterium to secrete a secretion product involved in activating plant acid invertase (step S02). As described above, the secretion product of the modified cyanobacterium includes proteins and metabolic products produced intracellularly by the modified cyanobacterium (i.e., intracellularly produced substances). These intracellularly produced substances include substances involved in activating plant acid invertase (i.e., plant acid invertase activators).

[0057] In step S01, the modified cyanobacteria is prepared. Preparing the modified cyanobacteria refers to adjusting the state of the modified cyanobacteria so that the modified cyanobacteria can secrete a secretion product. Preparing the modified cyanobacteria may involve, for example, genetically modifying a parent cyanobacterium to produce the modified cyanobacterium, restoring the modified cyanobacterium from a freeze-dried form or glycerol stock of the modified cyanobacterium, or recovering the modified cyanobacterium that has finished secreting the plant acid invertase activator in step S02.

[0058] In step S02, the modified cyanobacteria are caused to secrete secretions involved in promoting plant growth. In the modified cyanobacteria of this embodiment, the function of proteins involved in binding the outer membrane to the cell wall in the cyanobacteria (i.e., the parent cyanobacteria) is suppressed or eliminated, so proteins and metabolic products produced within the cell are more likely to be secreted outside the outer membrane (i.e., extracellularly). These intracellularly produced substances also include substances involved in activating plant acid invertase. Therefore, in step S02, the modified cyanobacteria are cultured under specified conditions, thereby causing the intracellularly produced substances involved in activating plant acid invertase to be secreted extracellularly.

[0059] Cyanobacteria can generally be cultured by liquid culture using BG-11 medium (see Table 2) or a modified method thereof. Therefore, modified cyanobacteria may also be cultured in a similar manner. The culture period for cyanobacteria to produce a plant acid invertase activator may be any period that allows sufficient bacterial growth and accumulation of high concentrations of proteins and metabolic products, and may be, for example, 1 to 3 days or 4 to 7 days. The culture method may be, for example, aeration and agitation culture or shaking culture.

[0060] When cultured under the above conditions, the engineered cyanobacteria produce proteins and metabolic products (i.e., intracellularly produced substances) within the cells and secrete the intracellularly produced substances into the culture medium. The intracellularly produced substances include intracellularly produced substances involved in the activation of plant acid invertase (i.e., plant acid invertase activators). To recover the intracellularly produced substances secreted into the culture medium, the culture medium may be filtered or centrifuged to remove solids such as cells (i.e., bacterial cells), and the culture supernatant may be recovered. According to the method for producing a plant acid invertase activator of this embodiment, a secretion containing the intracellularly produced substances involved in the activation of plant acid invertase (i.e., plant acid invertase activators) is secreted outside the cells of the engineered cyanobacteria, eliminating the need to disrupt the cells to recover the plant acid invertase activators. Therefore, the engineered cyanobacteria remaining after recovery of the plant acid invertase activators can be repeatedly used to produce a plant acid invertase activator.

[0061] The method for recovering the plant acid invertase activator secreted into the culture solution is not limited to the above example. The plant acid invertase activator may be recovered from the culture solution while culturing the modified cyanobacterium. For example, a permeable membrane that allows proteins to pass through may be used, and the plant acid invertase activator that has permeated the membrane may be recovered. In this way, the plant acid invertase activator can be recovered from the culture solution while culturing the modified cyanobacterium, eliminating the need for a process to remove the modified cyanobacterial cells from the culture solution. This allows for simpler and more efficient production of the plant acid invertase activator.

[0062] Furthermore, because the need for recovering and disrupting bacterial cells from the culture medium is eliminated, damage and stress to the modified cyanobacterium can be reduced, making it less likely that the productivity of the modified cyanobacterium for secreting plant acid invertase activators will decrease, allowing the modified cyanobacterium to be used for a longer period of time.

[0063] As described above, by using the modified cyanobacterium of this embodiment, a plant acid invertase activator can be obtained simply and efficiently.

[0064] The cyanobacteria and modified cyanobacteria will be described below.

[0065] [3. Cyanobacteria] Cyanobacteria, also known as blue-green algae or blue-green bacteria, are a group of prokaryotes that capture light energy using chlorophyll and use the energy to electrolyze water and generate oxygen through photosynthesis. Cyanobacteria are highly diverse, ranging from unicellular species such as Synechocystis sp. PCC 6803 to multicellular, filamentous species such as Anabaena sp. PCC 7120. They also vary in habitats, from thermophilic species such as Thermosynechococcus elongatus to marine species such as Synechococcus elongatus and freshwater species such as Synechocystis. Many species also possess unique characteristics, such as species that possess gas vesicles and produce toxins, such as Microcystis aeruginosa, and Gloeobacter violaceus, which lacks thylakoids and instead possesses light-harvesting proteins called phycobilisomes in the plasma membrane.

[0066] FIG. 2 is a schematic diagram showing the cell surface of a cyanobacterium. As shown in FIG. 2, the cell surface of a cyanobacterium is composed of, from the inside out, a plasma membrane (also called inner membrane 1), peptidoglycan 2, and an outer membrane 5, which is a lipid membrane that forms the outermost layer of the cell. Sugar chains 3 composed of glucosamine, mannosamine, and the like are covalently bound to peptidoglycan 2, and pyruvate is bound to these covalently bound sugar chains 3 (Non-Patent Document 7). In this specification, peptidoglycan 2 and the covalently bound sugar chains 3 are collectively 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, where various enzymes involved in protein degradation or formation of three-dimensional structures, lipid or nucleic acid degradation, and uptake of extracellular nutrients are present.

[0067] SLH domain-containing 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 Negativicutes class, a group of Gram-negative bacteria (Non-Patent Document 8). The region embedded in the lipid membrane (i.e., outer membrane 5) forms a channel that allows hydrophilic substances to permeate the outer membrane, while the SLH domain 7 functions to bind to the cell wall 4 (Non-Patent Document 9). For SLH domain 7 to bind to the cell wall 4, the covalent sugar chain 3 on peptidoglycan 2 must be modified with pyruvate (Non-Patent Document 10). Examples of genes encoding SLH domain-containing outer membrane protein 6 include slr1841 or slr1908 in Synechocystis sp. PCC 6803 and oprB in Anabaena sp. 90.

[0068] An enzyme that catalyzes the pyruvate modification reaction of covalently bonded glycan 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 Document 11). Among cyanobacteria whose genome sequences have been made public, many species possess genes encoding homologous proteins with amino acid sequence identity of 30% or more to CsaB. Examples include slr0688 possessed by Synechocystis sp. PCC 6803 and syn7502_03092 possessed by Synechococcus sp. 7502.

[0069] In cyanobacteria, CO2 fixed by photosynthesis is converted into precursors of various amino acids and intracellular molecules through multi-step enzymatic reactions. These raw materials are used to synthesize proteins and metabolites within the cytoplasm of cyanobacteria. Some of these proteins and metabolites function within the cytoplasm, while others are transported from the cytoplasm to the periplasm and function there. However, no cases of cyanobacteria actively secreting proteins and metabolites outside the cell have been reported to date.

[0070] Cyanobacteria have a high photosynthetic capacity and do not necessarily require the uptake of organic matter from the outside as nutrients. Therefore, cyanobacteria have very few channel proteins that allow permeation of organic matter, such as organic matter channel protein 8 (e.g., Slr1270) in Figure 2, in their outer membrane 5. For example, in Synechocystis sp. PCC 6803, organic matter channel protein 8, which allows permeation of organic matter, accounts for only about 4% of the total protein mass of the outer membrane 5. On the other hand, to efficiently uptake inorganic ions necessary for growth, cyanobacteria have many ion channel proteins that allow permeation of only inorganic ions, such as SLH domain-containing outer membrane protein 6 (e.g., Slr1841) in Figure 2, in their outer membrane 5. For example, in Synechocystis sp. PCC 6803, ion channel proteins that allow permeation of inorganic ions account for about 80% of the total protein mass of the outer membrane 5.

[0071] As such, in cyanobacteria, there are very few channels in the outer membrane 5 that allow organic substances such as proteins to pass through, so it is thought to be difficult to actively secrete proteins and metabolic products produced within the bacterial cell outside the bacterial cell.

[0072] [4. Modified Cyanobacteria] Next, the modified cyanobacterium of this embodiment will be described with reference to FIG.

[0073] In the modified cyanobacteria of this embodiment, the function of a protein involved in the binding between the outer membrane 5 and the cell wall 4 in cyanobacteria (hereinafter also referred to as a binding-associated protein) is suppressed or eliminated. As a result, the binding between the outer membrane 5 and the cell wall 4 (e.g., the amount and strength of binding) is partially reduced in the modified cyanobacteria, making it easier for the outer membrane 5 to partially detach from the cell wall 4. This improves the secretion productivity of the modified cyanobacteria, which secretes proteins and metabolic products produced intracellularly to the outside of the cell. As described above, the intracellularly produced substances include intracellularly produced substances involved in the activation of plant acid invertase (i.e., plant acid invertase activators). Therefore, the modified cyanobacteria also improves the secretion productivity of the plant acid invertase activators, which secrete the plant acid invertase activators produced intracellularly to the outside of the cell. Furthermore, because there is no need to disrupt the cell to recover the plant acid invertase activators, the modified cyanobacteria can be used repeatedly even after recovering the plant acid invertase activators. In this specification, the production of proteins and metabolites within the bacterial cell by the modified cyanobacterium is referred to as "production," and the secretion of the produced proteins and metabolites outside the bacterial cell is referred to as "secretory production."

[0074] The protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of the SLH domain-containing outer membrane protein 6 and the cell wall-pyruvate-modifying enzyme 9. In this embodiment, the modified cyanobacterium has the function of at least one of the SLH domain-containing outer membrane protein 6 and the cell wall-pyruvate-modifying enzyme 9 suppressed or lost. For example, in the modified cyanobacterium, (i) the function of at least one of the SLH domain-containing outer membrane protein 6 and the cell wall-pyruvate-modifying enzyme 9 may be suppressed or lost, or (ii) at least one of the expression of the SLH domain-containing 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 bound glycan 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 binding amount and binding strength) between the SLH domain 7 of the SLH domain-containing outer membrane protein 6 in the outer membrane 5 and the covalently bound glycan 3 on the surface of the cell wall 4. Therefore, the outer membrane 5 is more likely to detach from the cell wall 4 at the portions where these bonds are weakened. When the outer membrane 5 is partially detached from the cell wall 4, substances produced intracellularly, such as proteins and metabolic products present within the cells of the modified cyanobacterium, particularly in the periplasm, are more likely to leak out of the cells (outside the outer membrane 5). This improves the secretion productivity of the modified cyanobacterium, which secretes the plant acid invertase activating substance produced intracellularly.

[0075] Below, we will explain in more detail about cyanobacteria that have been modified so that the outer membrane 5 is partially detached from the cell wall 4 by suppressing the function of at least one binding-related protein, an SLH domain-containing outer membrane protein 6 and a cell wall-pyruvate-modifying enzyme 9.

[0076] The type of cyanobacterium (i.e., parent cyanobacterium) that serves as the parent microorganism for the modified cyanobacterium of this embodiment before the expression of at least one of the SLH domain-containing outer membrane protein 6 and the cell wall-pyruvate-modifying enzyme 9 is suppressed or eliminated is not particularly limited, and any type of cyanobacterium may be used. For example, the parent cyanobacterium may be of the genus Syenechocystis, Synechococcus, Anabaena, or Thermosynechococcus, and particularly may be Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1.

[0077] The amino acid sequences of the SLH domain-containing 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 locations of these genes on chromosomal DNA or plasmids can be confirmed in the above-mentioned NCBI database and Cyanobase.

[0078] The SLH domain-containing outer membrane protein 6 and cell wall-pyruvate modifying enzyme 9 whose functions are suppressed or lost in the modified cyanobacterium of this embodiment may be from any parent cyanobacterium as long as they are possessed by the parent cyanobacterium, and are not limited by the location of the genes encoding them (e.g., on chromosomal DNA or on a plasmid).

[0079] For example, the SLH domain-containing outer membrane protein 6 may be Slr1841, Slr1908, or Slr0042 when the parent cyanobacterium is of the genus Synechocystis, NIES970_09470 when the parent cyanobacterium is of the genus Synechococcus, Anacy_5815 or Anacy_3458 when the parent cyanobacterium is of the genus Anabaena, A0A0F6U6F8_MICAE when the parent cyanobacterium is of the genus Microcystis, or A0A0F6U6F8_MICAE when the parent cyanobacterium is of the genus Cyanothese. When the parent cyanobacterium is of the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc. When the parent cyanobacterium is of the genus Calothrix, it may be A0A1Z4R6U0_9CYAN, when the parent cyanobacterium is of the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc. When the parent cyanobacterium is of the genus Crocosphaera, it may be B1WRN6_CROS5, etc. When the parent cyanobacterium is of the genus Pleurocapsa, it may be K9TAE4_9CYAN, etc.

[0080] More specifically, the SLH domain-containing outer membrane protein 6 may be, for example, Slr1841 (SEQ ID NO: 1) of Synechocystis sp. PCC 6803, NIES970_09470 (SEQ ID NO: 2) of Synechococcus sp. NIES-970, or Anacy_3458 (SEQ ID NO: 3) of Anabaena cylindrica PCC 7122. Alternatively, the SLH domain-containing outer membrane protein 6 may be a protein having an amino acid sequence that is 50% or more identical to these SLH domain-containing outer membrane proteins 6.

[0081] As a result, in the modified cyanobacterium, for example, (i) the function of any one of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 or a protein having 50% or more identical amino acid sequence to any one of these SLH domain-retaining outer membrane proteins 6 may be suppressed or lost, or (ii) the expression of any one of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 or a protein having 50% or more identical amino acid sequence to any one of these SLH domain-retaining outer membrane proteins 6 may be suppressed. Therefore, in the modified cyanobacterium, (i) the function of the SLH domain-retaining outer membrane protein 6 or a protein having a function equivalent to the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is suppressed or lost, or (ii) the expression amount of the SLH domain-retaining outer membrane protein 6 or a protein having a function equivalent to the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is reduced. As a result, in the modified cyanobacterium, the binding domain (e.g., SLH domain 7) that binds the outer membrane 5 to the cell wall 4 has a reduced binding amount and binding strength with the cell wall 4, making it easier for the outer membrane 5 to partially detach from the cell wall 4. This makes it easier for substances produced intracellularly to leak out of the modified cyanobacterium, and therefore easier for the plant acid invertase activating substance produced intracellularly to leak out of the bacterial cell.

[0082] In general, if a protein has an amino acid sequence that is 30% or more identical to that of the other protein, the homology of the three-dimensional structure of the protein is high, and therefore the protein is likely to have the same function as the other protein. Therefore, the SLH domain-retaining outer membrane protein 6 whose function is suppressed or lost may be, for example, a protein or polypeptide that has 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, and that has the function of binding to covalently bound sugar chains 3 on cell walls 4.

[0083] Furthermore, for example, cell wall-pyruvate modifying enzyme 9 may be Slr0688 or the like when the parent cyanobacterium is of the genus Synechocystis, Syn7502_03092 or Synpcc7942_1529 or the like when the parent cyanobacterium is of the genus Synechococcus, ANA_C20348 or Anacy_1623 or the like when the parent cyanobacterium is of the genus Anabaena, or CsaB when the parent cyanobacterium is of the genus Microcystis. (NCBI access ID: TRU80220), etc., and when the parent cyanobacterium is of the genus Cyanothese, it may be CsaB (NCBI access ID: WP_107667006.1), etc., and when the parent cyanobacterium is of the genus Spirulina, it may be CsaB (NCBI access ID: WP_026079530.1), etc., and when the parent cyanobacterium is of the genus Calothrix, it may be CsaB (NCBI access ID: WP_09665 8142.1), etc., and when the parent cyanobacterium is of the genus Nostoc, it may be CsaB (NCBI access ID: WP_099068528.1), etc., when the parent cyanobacterium is of the genus Crocosphaera, it may be CsaB (NCBI access ID: WP_012361697.1), etc., and when the parent cyanobacterium is of the genus Pleurocapsa, it may be CsaB (NCBI access ID: WP_036798735), etc.

[0084] More specifically, cell wall-pyruvate-modifying enzyme 9 may be, for example, Slr0688 (SEQ ID NO: 4) of Synechocystis sp. PCC 6803, Synpcc7942_1529 (SEQ ID NO: 5) of Synechococcus sp. PCC 7942, or Anacy_1623 (SEQ ID NO: 6) of Anabaena cylindrica PCC 7122. Alternatively, the cell wall-pyruvate-modifying enzyme 9 may be a protein having an amino acid sequence that is 50% or more identical to that of these cell wall-pyruvate-modifying enzymes 9.

[0085] As a result, in the modified cyanobacterium, for example, (i) the function of any of the cell wall-pyruvate-modifying enzymes 9 shown in SEQ ID NOS: 4 to 6 or a protein having an amino acid sequence that is 50% or more identical to that of any of these cell wall-pyruvate-modifying enzymes 9 may be suppressed or lost, or (ii) the expression of any of the cell wall-pyruvate-modifying enzymes 9 shown in SEQ ID NOS: 4 to 6 or a protein having an amino acid sequence that is 50% or more identical to that of any of these cell wall-pyruvate-modifying enzymes 9 may be suppressed. Therefore, in the modified cyanobacterium, (i) the function of the cell wall-pyruvate-modifying enzyme 9 or a protein having a function equivalent to that enzyme is suppressed or lost, or (ii) the expression level of the cell wall-pyruvate-modifying enzyme 9 or a protein having a function equivalent to that enzyme is reduced. As a result, the covalently bonded sugar chains 3 on the surface of the cell wall 4 are less likely to be modified with pyruvate, thereby reducing the amount and strength of binding of the sugar chains 3 on the cell wall 4 to the SLH domain 7 of the SLH domain-containing outer membrane protein 6 in the outer membrane 5. Therefore, in the modified cyanobacterium according to this embodiment, the covalently bonded sugar chains 3 on the surface of the cell wall 4 are less likely to be modified with pyruvic acid, 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, in the modified cyanobacterium, substances produced within the cells tend to leak out of the cells, and therefore the plant acid invertase activating substance produced within the cells also tends to leak out of the cells.

[0086] As mentioned above, it is said that if a protein shares 30% or more of its amino acid sequence with another protein, it is likely to have the same function as that protein. Therefore, the cell wall-pyruvate-modifying enzyme 9 whose function is inhibited or eliminated may be, for example, a protein or polypeptide that has an amino acid sequence that shares 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 identity with the amino acid sequence of any of the cell wall-pyruvate-modifying enzymes 9 shown in SEQ ID NOS: 4 to 6, and that has the function of catalyzing the reaction of modifying the covalently bonded sugar chain 3 of the peptidoglycan 2 in the cell wall 4 with pyruvate.

[0087] In this specification, inhibiting or losing the function of an SLH domain-retaining outer membrane protein 6 means inhibiting or losing the ability of the protein to bind to the cell wall 4, inhibiting or losing the transport of the protein to the outer membrane 5, or inhibiting or losing the ability of the protein to function while embedded in the outer membrane 5.

[0088] In addition, suppressing or eliminating the function of the cell wall-pyruvate modifying enzyme 9 means suppressing or eliminating the function of the protein to modify the covalently bonded sugar chain 3 on the cell wall 4 with pyruvate.

[0089] The means for inhibiting or eliminating the function of these proteins is not particularly limited as long as it is a means commonly used for inhibiting or eliminating protein function, and may be, 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 transcription products of these genes, or administering an inhibitor that specifically inhibits these proteins.

[0090] In this embodiment, the modified cyanobacterium has an outer membrane 5 and a cell wall 4. As a result, in the modified cyanobacterium, the expression of a protein involved in the binding between the cell wall 4 and the outer membrane 5 is suppressed, or the function of the protein is suppressed or lost, resulting in a partial reduction in the binding (i.e., the amount and strength of binding) between the cell wall 4 and the outer membrane 5. As a result, in the modified cyanobacterium, the outer membrane 5 is more likely to partially detach from the cell wall 4, which makes it easier for intracellularly produced substances, such as proteins and metabolic products, to leak out of the outer membrane 5, i.e., outside the cell. Therefore, the modified cyanobacterium has improved productivity in secreting the plant acid invertase activator produced intracellularly. This eliminates the need for extraction processes for intracellularly produced substances, such as disrupting the cell cells, and therefore reduces the likelihood of a decrease in the physiological activity and yield of the intracellularly produced substance. Therefore, the physiological activity and yield of the plant acid invertase activator produced in the bacteria are less likely to decrease, making it possible to produce a plant acid invertase activator with improved plant acid invertase activation effect. Furthermore, because the above-mentioned extraction treatment of the intracellularly produced substance is no longer necessary, the modified cyanobacterium can be used repeatedly to produce the plant acid invertase activator even after the substance has been recovered.

[0091] The gene expressing a protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of a gene encoding an SLH domain-retaining outer membrane protein 6 and a gene encoding a cell wall-pyruvate-modifying enzyme 9. In the modified cyanobacterium, 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 is deleted or inactivated. Therefore, in the modified cyanobacterium, for example, (i) the expression of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate-modifying enzyme 9 is suppressed, or (ii) the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate-modifying enzyme 9 is suppressed or lost. Therefore, the binding (i.e., the binding amount and binding strength) between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalently bound glycan 3 on the surface of the cell wall 4 is reduced. This makes the outer membrane 5 more likely to detach from the cell wall 4 at areas where the binding between the outer membrane 5 and the cell wall 4 is weakened. As a result, in the modified cyanobacterium, the bond between the outer membrane 5 and the cell wall 4 is reduced, which makes it easier for the outer membrane 5 to partially detach from the cell wall 4, thereby facilitating leakage of proteins and metabolic products produced within the bacterial cell. This also makes it easier for the plant acid invertase activating substance produced within the modified cyanobacterium to leak out of the bacterial cell.

[0092] 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 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 genes encoding the cell wall-pyruvate modifying enzyme 9 may be suppressed.

[0093] For example, the gene encoding the SLH domain-containing outer membrane protein 6 may be slr1841, slr1908, or slr0042, etc., when the parent cyanobacterium is of the genus Synechocystis; nies970_09470, etc., when the parent cyanobacterium is of the genus Synechococcus; anacy_5815 or anacy_3458, etc., when the parent cyanobacterium is of the genus Anabaena; A0A0F6U6F8_MICAE, etc., when the parent cyanobacterium is of the genus Microcystis; and A0A0F6U6F8_MICAE, etc., when the parent cyanobacterium is of the genus Cyanothese. A0A3B8XX12_9CYAN, etc., and when the parent cyanobacterium is of the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc., when the parent cyanobacterium is of the genus Calothrix, it may be A0A1Z4R6U0_9CYAN, etc., when the parent cyanobacterium is of the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc., when the parent cyanobacterium is of the genus Crocosphaera, it may be B1WRN6_CROS5, etc., and when the parent cyanobacterium is of the genus Pleurocapsa, it may be K9TAE4_9CYAN, etc. The nucleotide sequences of these genes can be obtained from the above-mentioned NCBI database or Cyanobase.

[0094] More specifically, the gene encoding the SLH domain-containing outer membrane protein 6 may be slr1841 (SEQ ID NO: 7) of Synechocystis sp. PCC 6803, nies970_09470 (SEQ ID NO: 8) of Synechococcus sp. NIES-970, anacy_3458 (SEQ ID NO: 9) of Anabaena cylindrica PCC 7122, or a gene whose amino acid sequence is 50% or more identical to these genes.

[0095] As a result, in the modified cyanobacterium, a gene encoding any one of the SLH domain-containing outer membrane proteins 6 set forth in SEQ ID NOS: 7 to 9, or a gene having 50% or more identity to the nucleotide sequence of any one of these genes, is deleted or inactivated. Therefore, in the modified cyanobacterium, (i) the expression of any one of the SLH domain-containing outer membrane proteins 6 or a protein having a function equivalent to any one of these proteins is suppressed, or (ii) the function of any one of the SLH domain-containing outer membrane proteins 6 or a protein having a function equivalent to any one of these proteins is suppressed or lost. As a result, in the modified cyanobacterium, the binding amount and binding strength of the binding domain (e.g., SLH domain 7) that binds the outer membrane 5 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 bacterial cell to leak out of the bacterial cell, and therefore easier for the plant acid invertase activator produced within the bacterial cell to leak out of the bacterial cell.

[0096] As described above, it is said that if a protein shares 30% or more of its amino acid sequence with another protein, it is likely to have a function equivalent to that protein. Therefore, if the nucleotide sequence of a gene encoding a protein is 30% or more identical to that protein, it is likely that a protein with a function equivalent to that protein will be expressed. Therefore, a gene encoding an SLH domain-retaining outer membrane protein 6 whose function is suppressed or eliminated may be, for example, a gene consisting of a nucleotide sequence that shares 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 even more preferably 90% or more identity with the nucleotide sequence of any of the genes encoding the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOS: 7 to 9, and may be a gene encoding a protein or polypeptide that has the function of binding to the covalently bound sugar chain 3 on the cell wall 4.

[0097] Furthermore, for example, the gene encoding cell wall-pyruvate modifying enzyme 9 may be slr0688 or the like when the parent cyanobacterium is of the genus Synechocystis, syn7502_03092 or synpcc7942_1529 or the like when the parent cyanobacterium is of the genus Synechococcus, ana_C20348 or anacy_1623 or the like when the parent cyanobacterium is of the genus Anabaena, or csaB or the like when the parent cyanobacterium is of the genus Microcystis. (NCBI access ID: TRU80220), etc., and when the parent cyanobacterium is of the genus Cynahothese, it may be csaB (NCBI access ID: WP_107667006.1), etc., and when the parent cyanobacterium is of the genus Spirulina, it may be csaB (NCBI access ID: WP_026079530.1), etc., and when the parent cyanobacterium is of the genus Calothrix, it may be csaB (NCBI access ID: WP_0966 58142.1), etc., and when the parent cyanobacterium is of the genus Nostoc, it may be csaB (NCBI access ID: WP_099068528.1), etc., when the parent cyanobacterium is of the genus Crocosphaera, it may be csaB (NCBI access ID: WP_012361697.1), etc., and when the parent cyanobacterium is of the genus Pleurocapsa, it may be csaB (NCBI access ID: WP_036798735), etc. The nucleotide sequences of these genes can be obtained from the above-mentioned NCBI database or Cyanobase.

[0098] More specifically, the gene encoding cell wall-pyruvate-modifying enzyme 9 may be slr0688 (SEQ ID NO: 10) of Synechocystis sp. PCC 6803, synpcc7942_1529 (SEQ ID NO: 11) of Synechococcus sp. PCC 7942, or anacy_1623 (SEQ ID NO: 12) of Anabaena cylindrica PCC 7122. Alternatively, the gene may be one having a nucleotide sequence that is 50% or more identical to these genes.

[0099] As a result, in the modified cyanobacterium, a gene encoding any one of the cell wall-pyruvate-modifying enzymes 9 shown in SEQ ID NOS: 10 to 12 or a gene having 50% or more identity to the nucleotide sequence of a gene encoding any one of these enzymes is deleted or inactivated. Therefore, in the modified cyanobacterium, (i) the expression of any one of the cell wall-pyruvate-modifying enzymes 9 or a protein having a function equivalent to any one of these enzymes is suppressed, or (ii) the function of any one of the cell wall-pyruvate-modifying enzymes 9 or a protein having a function equivalent to any one of these enzymes is suppressed or lost. This reduces the amount and strength of the binding of the sugar chains 3 on the surface of the cell wall 4 to the SLH domain 7 of the SLH domain-containing outer membrane protein 6 in the outer membrane 5, thereby reducing the amount and strength of the binding of the sugar chains 3 on the cell wall 4 to the SLH domain 7 of the SLH domain-containing outer membrane protein 6 in the outer membrane 5. Therefore, in the modified cyanobacterium according to this embodiment, the amount of sugar chains 3 modified with pyruvate, which is used to bind the cell wall 4 to the outer membrane 5, is reduced, weakening the binding strength between the cell wall 4 and the outer membrane 5 and making the outer membrane 5 more likely to partially detach from the cell wall 4. This makes it easier for proteins and metabolic products produced within the fungal cells to leak out of the fungal cells, and therefore makes it easier for plant acid invertase activating substances produced within the fungal cells to leak out of the fungal cells.

[0100] As described above, if the nucleotide sequence of a gene encoding a protein is 30% or more identical, it is highly likely that a protein with equivalent function to the protein will be expressed. Therefore, a gene encoding cell wall-pyruvate-modifying enzyme 9 whose function is suppressed or eliminated may be, for example, a gene having a nucleotide 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 nucleotide sequence of any of the genes encoding cell wall-pyruvate-modifying enzyme 9 shown in SEQ ID NOS: 10 to 12, and encoding a protein or polypeptide that has the function of catalyzing a reaction to modify covalently bonded sugar chain 3 of peptidoglycan 2 in cell wall 4 with pyruvate.

[0101] [5. Method for producing modified cyanobacteria] Next, a method for producing the modified cyanobacterium of this embodiment will be described. The method for producing the modified cyanobacterium includes a step of suppressing or eliminating the function of a protein involved in the binding between the outer membrane 5 and the cell wall 4 in the cyanobacterium.

[0102] In this embodiment, the protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of an SLH domain-containing outer membrane protein 6 and a cell wall-pyruvate modifying enzyme 9.

[0103] The means for suppressing or eliminating the function of the protein are not particularly limited, but may include, for example, deleting or inactivating the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9, inhibiting the transcription of these genes, inhibiting the translation of the transcription products of these genes, or administering an inhibitor that specifically inhibits these proteins.

[0104] The means for deleting or inactivating the gene may be, for example, introducing a mutation into one or more bases in the base sequence of the gene, substituting or inserting another base sequence into the base sequence, or deleting part or all of the base sequence of the gene.

[0105] The means for inhibiting the transcription of the gene may be, for example, introducing a mutation into the promoter region of the gene, inactivating the promoter by substituting or inserting another base sequence, or CRISPR interference (Non-Patent Document 12), etc. Specific techniques for the above-mentioned introduction of a mutation or substitution or insertion of a base sequence may be, for example, ultraviolet irradiation, site-specific mutagenesis, or homologous recombination.

[0106] Furthermore, the means for inhibiting the translation of the transcription product of the gene may be, for example, RNA interference.

[0107] By using any of the above means, the function of the protein involved in the binding between the outer membrane 5 and the cell wall 4 in cyanobacteria can be suppressed or eliminated to produce a modified cyanobacterium.

[0108] As a result, in the modified cyanobacteria produced by the above-described production method, the bond between the cell wall 4 and the outer membrane 5 (i.e., the amount and strength of the bond) is partially reduced, 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 such as proteins and metabolites are more likely to leak out of the outer membrane 5 (i.e., outside the cell), and substances involved in the activation of plant acid invertase (i.e., plant acid invertase activating substances) are also more likely to leak out of the cell. Therefore, the method for producing modified cyanobacteria in this embodiment can provide modified cyanobacteria with improved secretion productivity of plant acid invertase activating substances.

[0109] Furthermore, in the modified cyanobacteria produced by the production method of this embodiment, the plant acid invertase activator produced within the cells leaks out of the cells, eliminating the need to disrupt the cells to recover the substance. For example, the modified cyanobacteria can be cultured under appropriate conditions and then the plant acid invertase activator secreted into the culture medium can be recovered. This allows the plant acid invertase activator to be recovered from the culture medium while the modified cyanobacteria are being cultured. Therefore, the modified cyanobacteria obtained by this production method can be used to efficiently produce a plant acid invertase activator microbiologically. Therefore, the method for producing modified cyanobacteria of this embodiment can provide a highly efficient modified cyanobacterium that can be reused even after recovery of the plant acid invertase activator.

[0110] [6. Method for activating plant acid invertase] The plant acid invertase activation method according to the present embodiment involves applying the above-described plant acid invertase activator to a plant. As described above, the plant acid invertase activator according to the present embodiment has the effect of activating plant acid invertase, and therefore, by applying the above-described plant acid invertase activator to a plant, plant acid invertase can be effectively activated.

[0111] The plant acid invertase activator may be used as is, or may be concentrated or diluted. When applying (i.e., applying) the plant growth promoter to plants, the concentration and application method of the plant acid invertase activator may be determined appropriately depending on the type of plant, the properties of the soil, and the purpose. The plant acid invertase activator may be, for example, the culture medium of the modified cyanobacterium itself, a solution obtained by removing the modified cyanobacterium cells from the culture medium, or an extract obtained by extracting a desired substance from the culture medium using membrane technology or the like. The desired substance may be an enzyme that decomposes nutrients in soil, a substance that solubilizes insoluble substances in soil (e.g., metals such as iron) (e.g., a substance with a chelating effect), or a substance that improves intracellular physiological activity in plants. Furthermore, the plant acid invertase activator may be applied to plants by, for example, spraying on plants, spraying on soil, irrigation, or mixing, or by mixing into a hydroponic solution. For example, a few milliliters of a plant acid invertase activator per plant may be added to the base of the plant about once a week. [Example]

[0112] The modified cyanobacteria, the method for producing the modified cyanobacteria, and the method for producing the plant acid invertase activator of the present disclosure will be specifically described in the following examples, but the present disclosure is not limited to the following examples in any way.

[0113] In the following examples, two types of modified cyanobacteria were produced by partially detaching the outer membrane of cyanobacteria from the cell wall: suppressing expression of the slr1841 gene, which encodes an SLH domain-retaining outer membrane protein (Example 1), and suppressing expression of the slr0688 gene, which encodes a cell wall-pyruvate modifying enzyme (Example 2). The protein secretion productivity of these modified cyanobacteria was then measured, and the secreted intracellularly produced substances (here, proteins and intracellular metabolites) were identified. The cyanobacterial species used in this example was Synechocystis sp. PCC 6803 (hereinafter simply referred to as "cyanobacteria").

[0114] Example 1 In Example 1, modified cyanobacteria were produced in which the expression of the slr1841 gene, which encodes an SLH domain-containing outer membrane protein, was suppressed.

[0115] (1) Construction of a cyanobacterial mutant in which expression of the slr1841 gene is suppressed We used the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) interference method to silence the slr1841 gene by introducing the gene encoding the dCas9 protein (hereafter referred to as the dCas9 gene) and the slr1841_sgRNA (single-guide ribonucleic acid) gene into the chromosomal DNA of cyanobacteria.

[0116] The mechanism by which gene expression is suppressed by this method is as follows.

[0117] First, a complex is formed between a Cas9 protein (dCas9) lacking nuclease activity and an sgRNA (slr1841_sgRNA) that binds complementarily to the base sequence of the slr1841 gene.

[0118] This complex then recognizes the slr1841 gene on the chromosomal DNA of cyanobacteria and binds specifically to it. This binding creates steric hindrance, inhibiting transcription of the slr1841 gene. As a result, expression of the slr1841 gene in cyanobacteria is suppressed.

[0119] Below, we will specifically explain how to introduce each of the above two genes into the chromosomal DNA of cyanobacteria.

[0120] (1-1) Introduction of dCas9 gene Using the chromosomal DNA of Synechocystis LY07 strain (hereinafter also referred to as LY07 strain) (see Non-Patent Document 12) as a template, the dCas9 gene, an operator gene for dCas9 gene expression control, and a spectinomycin resistance marker gene serving as a marker for gene introduction 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 linked and inserted into the psbA1 gene on the chromosomal DNA, so they can be amplified as a single DNA fragment by PCR. Here, the resulting DNA fragment is referred to as the "psbA1::dCas9 cassette." The psbA1::dCas9 cassette was inserted into the pUC19 plasmid using the In-Fusion PCR cloning method (registered trademark), yielding the pUC19-dCas9 plasmid.

[0121] [Table 1]

[0122] One microgram of the resulting pUC19-dCas9 plasmid was mixed with a cyanobacterial culture medium (cell density OD730 = approximately 0.5), and the pUC19-dCas9 plasmid was introduced into the cyanobacterial cells by natural transformation. Transformed cells were selected by growing them on BG-11 agar medium containing 20 μg / mL spectinomycin. In the selected cells, homologous recombination occurred between the psbA1 gene on the chromosomal DNA and the psbA1 upstream and downstream fragment regions on the pUC19-dCas9 plasmid. This resulted in the generation of Synechocystis dCas9 strains with the dCas9 cassette inserted into the psbA1 gene region. The composition of the BG-11 medium used is shown in Table 2.

[0123] [Table 2]

[0124] (1-2) Introduction of slr1841_sgRNA gene In CRISPR interference, a sequence of approximately 20 bases complementary to the target sequence is introduced into a region of the sgRNA gene called the protospacer, allowing the sgRNA to specifically bind to the target gene. The protospacer sequence used in this example is shown in Table 3.

[0125] [Table 3]

[0126] In the Synechocystis LY07 strain, the sgRNA gene (excluding the protospacer region) is linked to a kanamycin resistance marker gene and inserted into the slr2030-slr2031 gene on the chromosomal DNA. Therefore, by adding a protospacer sequence (SEQ ID NO: 21) complementary to the slr1841 gene (SEQ ID NO: 7) to the primers used to amplify the sgRNA gene by PCR, an sgRNA (slr1841_sgRNA) that specifically recognizes slr1841 can be easily obtained.

[0127] First, using the chromosomal DNA of the LY07 strain as a template, two DNA fragments were amplified by PCR using the primer set slr2030-Fw (SEQ ID NO: 15) and sgRNA_slr1841-Rv (SEQ ID NO: 16) and the primer set sgRNA_slr1841-Fw (SEQ ID NO: 17) and slr2031-Rv (SEQ ID NO: 18) listed in Table 1.

[0128] Next, the mixed solution of the above DNA fragments was used as a template and amplified by PCR using the primers slr2030-Fw (SEQ ID NO: 15) and slr2031-Rv (SEQ ID NO: 18) listed in Table 1. (i) slr2030 gene fragment, (ii) slr1841_sgRNA, (iii) kanamycin resistance marker gene, (iv) slr2031 gene fragment was linked in order to obtain a DNA fragment (slr2030-2031::slr1841_sgRNA). 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.

[0129] The pUC19-slr1841_sgRNA plasmid was introduced into the Synechocystis dCas9 strain using the same method as in (1-1) above, and transformed cells were selected on BG-11 agar medium containing 30 μg / mL kanamycin. This resulted in a Synechocystis dCas9 slr1841_sgRNA transformant (hereinafter referred to as the slr1841-suppressed strain) in which the slr1841_sgRNA was inserted into the slr2030-slr2031 gene on the chromosomal DNA.

[0130] (1-3) Suppression of the slr1841 gene The promoter sequences of the dCas9 gene and the slr1841_sgRNA gene were designed so that expression was induced in the presence of anhydrotetracycline (aTc). In this example, expression of the slr1841 gene was suppressed by adding aTc to the medium at a final concentration of 1 μg / mL.

[0131] Example 2 In Example 2, a modified cyanobacterium in which the expression of the slr0688 gene encoding a cell wall-pyruvate modifying enzyme was suppressed was obtained by the following procedure.

[0132] (2) Construction of a cyanobacterial mutant in which the expression of the slr0688 gene is suppressed Using the same procedure as 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 same conditions as in (1-2) above were used, 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) in which (i) the slr2030 gene fragment, (ii) the slr0688_sgRNA, (iii) the kanamycin resistance marker gene, and (iv) the slr2031 gene fragment were linked in this order was inserted into the pUC19 plasmid using the In-Fusion PCR cloning method (registered trademark) to obtain the pUC19-slr0688_sgRNA plasmid.

[0133] Furthermore, the expression of the slr0688 gene was suppressed by the same procedure as in (1-3) above.

[0134] (Comparative Example 1) In Comparative Example 1, the Synechocystis dCas9 strain was obtained by the same procedure as in Example 1 (1-1).

[0135] Subsequently, the state of the cell surface was observed and a test for protein secretion productivity was carried out for each of the strains obtained in Example 1, Example 2, and Comparative Example 1. Details are explained below.

[0136] (3) Observation of the cell surface of the strain Ultrathin sections were prepared from the modified cyanobacterium Synechocystis dCas9 slr1841_sgRNA strain obtained in Example 1 (i.e., the slr1841-suppressed strain), the modified cyanobacterium Synechocystis dCas9 slr0688_sgRNA strain obtained in Example 2 (hereinafter also referred to as the slr0688-suppressed strain), and the modified cyanobacterium Synechocystis dCas9 strain obtained in Comparative Example 1 (hereinafter referred to as the Control strain), and the state of the cell surface (in other words, the outer membrane structure) was observed using an electron microscope.

[0137] (3-1) Cultivation of strains The slr1841 suppressed strain of Example 1 was inoculated into BG-11 medium containing 1 μg / mL aTc so that the initial bacterial cell concentration OD730 was 0.05, and the light intensity was 100 μmol / m 2 The culture was performed with shaking for 5 days under conditions of 1 / s and 30° C. The slr0688-suppressed strain of Example 2 and the control strain of Comparative Example 1 were also cultured under the same conditions as in Example 1.

[0138] (3-2) Preparation of ultrathin sections of bacterial strains The culture medium obtained in (3-1) above was centrifuged at 2,500 g for 10 minutes at room temperature to recover cells of the slr1841-suppressed strain of Example 1. The cells were then rapidly frozen in liquid propane at -175°C and fixed at -80°C for two days using an ethanol solution containing 2% glutaraldehyde and 1% tannic acid. The fixed cells were dehydrated with ethanol, infiltrated with propylene oxide, and then submerged in a resin (Quetol-651) solution. The cells were then left to stand at 60°C for 48 hours to harden the resin, and the cells were embedded in the resin. The cells in the resin were sliced ​​into 70-nm-thick ultrathin sections using an ultramicrotome (Ultracut). These ultrathin sections were stained with a 2% uranium acetate and 1% lead citrate solution to prepare transmission electron microscopy samples of the slr1841-suppressed strain of Example 1. The same procedure was also carried out for the slr0688 suppressed strain of Example 2 and the control strain of Comparative Example 1, and samples for transmission electron microscopy were prepared.

[0139] (3-3) Observation by electron microscope The ultrathin sections obtained in (3-2) above were observed using a transmission electron microscope (JEOL JEM-1400Plus) at an accelerating voltage of 100 kV. The observation results are shown in Figures 3 to 8.

[0140] First, the slr1841-suppressed strain of Example 1 will be described. Figure 3 is a TEM (Transmission Electron Microscope) image of the slr1841-suppressed strain of Example 1. Figure 4 is an enlarged image of the dashed-line region A in Figure 3. Figure 4(a) is an enlarged TEM image of the dashed-line region A in Figure 3, and Figure 4(b) is a depiction of the enlarged TEM image of Figure 4(a).

[0141] As shown in FIG. 3, in the slr1841 suppressed strain of Example 1, the outer membrane was partially detached from the cell wall (that is, the outer membrane was partially peeled off) and the outer membrane was partially flexed.

[0142] To confirm the state of the cell surface in more detail, we enlarged and observed the dashed-line area A. As shown in Figure 4(a) and Figure 4(b), we were able to confirm areas where the outer membrane had partially peeled off (dotted-line areas a1 and a2 in the figures). We also observed a large area of ​​the outer membrane bending near the dashed-line area a1. This area is thought to be where the bond between the outer membrane and the cell wall was weakened, and the outer membrane expanded outward and bent due to the culture medium penetrating into the periplasm through the outer membrane.

[0143] Next, the slr0688-suppressed strain of Example 2 will be described. Figure 5 is a TEM image of the slr0688-suppressed strain of Example 2. Figure 6 is an enlarged image of the dashed-line region B in Figure 5. Figure 6(a) is an enlarged TEM image of the dashed-line region B in Figure 5, and Figure 6(b) is a depiction of the enlarged TEM image of Figure 6(a).

[0144] As shown in Figure 5, the outer membrane was partially detached from the cell wall and partially bent in the slr0688-suppressed strain of Example 2. It was also confirmed that the outer membrane was partially detached from the cell wall in the slr0688-suppressed strain.

[0145] To confirm the state of the cell surface in more detail, we enlarged and observed the dashed-line area B. As shown in Figures 6(a) and 6(b), we were able to confirm a region where the outer membrane was significantly bent (dotted-dash-line area b1) and a region where the outer membrane had partially peeled off (dotted-dash-line areas b2 and b3). Furthermore, we were able to confirm regions where the outer membrane had detached from the cell wall near the dashed-dash-line areas b1, b2, and b3.

[0146] Next, the control strain of Comparative Example 1 will be described. Fig. 7 is a TEM image of the control strain of Comparative Example 1. Fig. 8 is an enlarged image of the dashed line area C in Fig. 7. Fig. 8(a) is an enlarged TEM image of the dashed line area C in Fig. 7, and Fig. 8(b) is a depiction of the enlarged TEM image of Fig. 8(a).

[0147] 7, the cell surface of the control strain in Comparative Example 1 was intact, with the inner membrane, cell wall, outer membrane, and S layer remaining laminated in that order. In other words, the control strain did not show any 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 sagged, as in Examples 1 and 2.

[0148] (4) Protein secretion productivity test The slr1841-suppressed strain of Example 1, the slr0688-suppressed strain of Example 2, and the control strain of Comparative Example 1 were each cultured, and the amount of protein secreted outside the cells (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. Note that protein secretion productivity refers to the ability to produce a protein by secreting a protein produced inside the cell outside the cell. Specific methods are described below.

[0149] (4-1) Cultivation of strains The slr1841-suppressed strain of Example 1 was cultured in the same manner as in (3-1) above. The culture was carried out three times independently. The strains of Example 2 and Comparative Example 1 were also cultured under the same conditions as the strain of Example 1.

[0150] (4-2) Quantification of extracellularly secreted proteins The culture medium obtained in (4-1) above was centrifuged at 2,500 g for 10 minutes at room temperature to obtain a 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-suppressed strain of Example 1. The total protein content of the filtered culture supernatant was quantified using the BCA (bicinchoninic acid) method. This series of procedures was performed for each of three independently cultured cultures, and the average and standard deviation of the protein amount secreted extracellularly by the slr1841-suppressed strain of Example 1 were calculated. Protein quantification was also performed for three cultures of the strains of Example 2 and Comparative Example 1 under similar conditions, and the average and standard deviation of the protein amount in the three cultures were calculated.

[0151] The results are shown in Figure 9. Figure 9 is a graph showing the protein amounts in the culture solutions of the modified cyanobacteria of Example 1, Example 2, and Comparative Example 1 (n=3, error bars=SD).

[0152] As shown in Figure 9, the amount of protein (mg / L) secreted into the culture supernatant of both the slr1841-suppressed strain of Example 1 and the slr0688-suppressed strain of Example 2 was approximately 25-fold higher than that of the control strain of Comparison Example 1.

[0153] Although the data is not shown here, the absorbance (730 nm) of the culture medium was measured and the amount of secreted protein per gram of dry cell weight (mg protein / g cell dry weight) was calculated. The amount of secreted protein per gram of dry cell weight (mg protein / g cell dry weight) was found to be approximately 36-fold higher for both the slr1841-suppressed strain of Example 1 and the slr0688-suppressed strain of Example 2 compared to the control strain of Comparative Example 1.

[0154] 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, in which expression of the gene encoding the cell wall-pyruvate-modifying enzyme (slr0688) was suppressed, than in the slr1841-suppressed strain of Example 1, in which expression of the gene encoding the SLH domain-retaining outer membrane protein (slr1841) was suppressed. This is thought to be related to the fact that the number of covalently bound glycans on the cell wall surface is greater than the number of SLH domain-retaining outer membrane proteins (Slr1841) in the outer membrane. In other words, the amount of protein secreted in the slr0688-suppressed strain of Example 2 was greater than that in the slr1841-suppressed strain of Example 1, likely 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.

[0155] These results confirmed that inhibiting the function of proteins involved in the binding between the outer membrane and the cell wall partially weakens the binding between the outer membrane and the cell wall of the cyanobacterium, causing the outer membrane to partially detach from the cell wall. It was also confirmed that weakening the binding between the outer membrane and the cell wall makes it easier for proteins produced within the cyanobacterial cell to leak out of the cell. Therefore, the modified cyanobacterium and its production method according to this embodiment significantly improve protein secretion productivity.

[0156] (5) Identification of secreted proteins Subsequently, the secreted proteins contained in the culture supernatant obtained in (4-2) above were identified by LC-MS / MS, as described below.

[0157] (5-1) Sample preparation Eight volumes of cold acetone were added to the culture supernatant, and the mixture was incubated at 20°C for 2 hours. The mixture was then centrifuged at 20,000 g for 15 minutes to obtain a protein precipitate. 100 mM Tris pH 8.5, 0.5% sodium dodecanoate (SDoD) was added to the precipitate, and the protein was dissolved using a closed-loop ultrasonicator. The protein concentration was adjusted to 1 μg / mL, and dithiothreitol (DTT) was added to a final concentration of 10 mM and incubated at 50°C for 30 minutes. Iodoacetamide (IAA) was then added to a final concentration of 30 mM and incubated at room temperature (protected from light) for 30 minutes. To stop the IAA reaction, cysteine ​​was added to a final concentration of 60 mM and incubated at room temperature for 10 minutes. 400 ng of trypsin was added and incubated overnight at 37°C to obtain peptide fragments of the protein. After adding 5% TFA (trifluoroacetic acid), the mixture was centrifuged at 15,000 g for 10 minutes at room temperature to obtain the supernatant. This process removed SDoD. After desalting using a C18 spin column, the sample was dried using a centrifugal evaporator. 3% acetonitrile and 0.1% formic acid were then added, and the sample was dissolved using a closed ultrasonic homogenizer. The peptide concentration was adjusted to 200 ng / μL.

[0158] (5-2)LC-MS / MS analysis The sample obtained in (5-1) above was analyzed using an LC-MS / MS device (UltiMate 3000 RSLCnano LC System) under the following conditions.

[0159] Sample injection amount: 200ng Column: CAPCELL CORE MP 75 μm x 250 mm Solvent: Solvent A is 0.1% formic acid aqueous solution, Solvent B is 0.1% formic acid + 80% acetonitrile Gradient program: 8% B solvent 4 minutes after sample injection, 44% B solvent 27 minutes later, 80% B solvent 28 minutes later, measurement completed 34 minutes later

[0160] (5-3) Data analysis The obtained data was analyzed under the following conditions to identify proteins and peptides and calculate quantitative values.

[0161] Software: Scaffold DIA Database: UniProtKB / Swiss Prot database ( Synechocystis sp. PCC 6803) Fragmentation:HCD Precursor Tolerance: 8 ppm Fragment Tolerance: 10 ppm 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

[0162] Among the identified proteins, the 30 proteins with the highest relative quantitative values ​​that are predicted to have clear enzymatic activity are shown in Table 4.

[0163] [Table 4]

[0164] All six of these proteins were contained in the culture supernatants of the slr1841-suppressed strain of Example 1 and the slr0688-suppressed strain of Example 2. All of these proteins retained periplasmic (referring to the space between the outer and inner membrane) localization signals. These results confirmed that in the modified strains of Examples 1 and 2, partial detachment of the outer membrane from the cell wall facilitates leakage of proteins in the periplasm outside the outer membrane (i.e., outside the bacterial cell). Therefore, it was demonstrated that the modified cyanobacterium of this embodiment has significantly improved protein secretion productivity.

[0165] (6) Identification of secreted intracellular metabolites (6-1) Sample preparation 20 μl of an aqueous solution containing an internal standard adjusted to a concentration of 1,000 μM was added to 80 μl of the culture supernatant of the modified cyanobacteria, and the mixture was stirred, ultrafiltered, and then subjected to measurement.

[0166] (6-2) CE (Capillary electrophoresis)-TOFMS (Time-Of-Flight Mass Spectrometry) analysis In this test, measurements in cation mode and anion mode were carried out under the conditions shown below.

[0167] [Cation Mode] Instrument: 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 [Anechoic mode] Instrument: 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

[0168] (6-3) Data processing Peaks detected by CE-TOFMS were automatically detected using the MasterHands® ver. 2.17.1.11 automatic integration software, with a signal-to-noise ratio of 3 or higher. Metabolites contained in the culture supernatant of the engineered cyanobacteria were searched for based on the mass-to-charge ratio (m / z) and migration time of each metabolite, and compared with the values ​​for all substances registered in the metabolite library of HMT (Human Metabolome Technologies, Inc.). The search tolerances were + / - 0.5 min for migration time and + / - 10 ppm for m / z. The concentration of each identified metabolite was calculated using a calibration limit of 100 μM. The major identified metabolites are listed in Table 5.

[0169] [Table 5]

[0170] All 12 of these intracellular metabolites were contained in the culture supernatant of the slr1841-suppressed strain of Example 1 and the slr0688-suppressed strain of Example 2. Although data is not shown, these metabolites were not contained in the culture supernatant of the control strain of Comparative Example 1. These results confirmed that in the modified strains of Examples 1 and 2, the outer membrane was partially detached from the cell wall, making it easier for intracellular metabolites to leak outside the outer membrane (i.e., outside the bacterial cell).

[0171] (7) Cultivation test and acid invertase activity measurement test Next, the following plant cultivation tests were conducted to evaluate the plant acid invertase activation effect of the secretion product of the modified cyanobacteria (here, the culture supernatant of the modified cyanobacteria). Specifically, a spinach cultivation test was conducted to evaluate the effect on leafy vegetable production. Furthermore, a strawberry cultivation test was conducted to evaluate the effect on fruit production. Each of these cultivation tests is described below.

[0172] (7-1) Spinach cultivation test First, three spinach seeds were sown in a cultivation pot (12 cm × 10 cm) filled with commercially available potting soil. The cultivation was carried out under an indoor temperature of 23°C and a photon flux density of 200 μmol / m 2 The cultivation was carried out at 500 x 1000 dpi under 10 hours of light and 14 hours of darkness for 40 days. During this period, 50 mL of distilled water was supplied to each pot every two days. Approximately one week after the start of cultivation, when the cotyledons had developed, the plants were thinned out to ensure that the size of each plant was uniform.

[0173] Example 3 As described above, after the individual plants in each pot were sized uniformly, 5 mL of the culture supernatant of the modified cyanobacteria (hereafter referred to as the modified cyanobacterial secretion product) was added to the base of the spinach plants once a week. The plants were cultivated for 40 days, and after harvest, the dry weight of the aboveground parts was measured and the mean and standard deviation (SD) were calculated. Acid invertase activity was also measured using the method described below, and the mean and standard deviation (SD) were calculated. The modified cyanobacteria used were the slr1841-suppressed strain in Example 1 and the slr0688-suppressed strain in Example 2.

[0174] (Acid invertase activity measurement) Several leaves approximately 10 cm long were removed from each plant and weighed. The leaves were frozen in liquid nitrogen and then crushed in a mortar. 3 mL of the following extraction buffer was added and homogenized using a glass homogenizer to prepare an extract. 20 μL of this extract was added to 180 μL of acid invertase reaction solution (50 mM sodium acetate, pH 4.3, 0.1 M sucrose) and left to stand at 30°C for 1 hour to allow the extract to react with the acid invertase reaction solution. The reaction was then terminated by incubating at 85°C for 3 minutes. The amount of glucose produced during the reaction was quantified using a commercially available glucose quantification kit. From this value, the amount of glucose produced per hour per 1 g of leaf weight was calculated as acid invertase activity, and the mean and standard deviation (SD) were calculated.

[0175] <Extraction buffer> The extraction buffer has the following composition:

[0176] 100 mM HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)-KOH pH7.4 5mM MgCl2 1mM EDTA(ethylenediaminetetraacetic acid) 1mM EGTA (ethyleneglycol-bis(β-aminoehylether)-tetraacetic acid) 1mM PMSF(phenmethylsulphonyl-fluoride) 5mM DTT (dithiothreitol) 1mL / L Triton X-100 200mL / L glycerol 5mM thiourea

[0177] (Comparative Example 2) The same procedure as in Example 3 was carried out, except that water was used instead of the secretion of the modified cyanobacteria.

[0178] (result) The results of Example 3 and Comparative Example 2 are shown in Figures 10 and 11. Figure 10 is a graph showing the average values ​​of acid invertase activity of spinach cultivated in Example 3 and Comparative Example 2. Figure 11 is a graph showing the average value of dry weight of aboveground parts per plant (referred to as average plant weight) of spinach cultivated in Example 3 and Comparative Example 2.

[0179] As shown in FIG. 10, the acid invertase activity of the spinach cultivated in Example 3 was increased by about 2.3 times compared to Comparative Example 2.

[0180] Furthermore, as shown in FIG. 11, the weight of the spinach plant cultivated in Example 3 was increased by about 1.4 times compared to Comparative Example 1.

[0181] Therefore, it was confirmed that applying the secretions of the modified cyanobacteria to spinach activates the acid invertase in spinach, and that the activation of acid invertase promotes growth and increases the weight of spinach.

[0182] (7-2) Strawberry cultivation test Strawberry seedlings with approximately 9 leaves and a plant length of approximately 7 cm were planted in a cultivation pot (12 cm x 10 cm) filled with commercially available potting soil. The cultivation was carried out under a light temperature of 20°C, a dark temperature of 15°C, and a photon flux density of 200 μmol / m 2 The plants were cultivated at 500 W / s under 14 h light and 10 h dark conditions for 150 days. During this period, each pot was watered with 50 mL of distilled water every other day. Also, once every 50 days, 100 mL of a commercially available chemical fertilizer (a 500-fold dilution of the original solution containing 6% total nitrogen, 10% water-soluble phosphate, 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.

[0183] Example 4 During the cultivation period, 5 mL of the modified cyanobacteria secretion solution per plant was added to the base of the plants once a week. Strawberry fruits were harvested in order of redness and maturity, and the number of fruits harvested was recorded. The weight and sugar content (Brix value) of the harvested fruits were measured, and the mean and standard deviation (SD) were calculated. Acid invertase activity was measured using the same method as in Example 3, except that several fruits were used. Here, the amount of glucose produced per hour by 1 g of fruit was defined as acid invertase activity. The modified cyanobacteria used were the slr1841-suppressed strain in Example 1 and the slr0688-suppressed strain in Example 2.

[0184] (Comparative Example 3) The same procedure as in Example 4 was carried out, except that water was used instead of the secretion of the modified cyanobacteria.

[0185] (result) The results of Example 4 and Comparative Example 3 are shown in Figures 12 to 16. Figure 12 is a graph showing the average acid invertase activity of strawberries grown in Example 4 and Comparative Example 3. Figure 13 is a graph showing the average number of fruits per plant of strawberries grown in Example 4 and Comparative Example 3. Figure 14 is a graph showing the average fruit weight per plant of strawberries grown in Example 4 and Comparative Example 3. Figure 15 is a graph showing the average sugar content per plant of strawberries grown in Example 4 and Comparative Example 3.

[0186] FIG. 16 also shows photographs of representative fruits to visually show the state of the fruits in Example 4 and Comparative Example 3.

[0187] As shown in FIG. 12, the plant acid invertase activity of the strawberries cultivated in Example 4 was increased by about 2.3 times compared to Comparative Example 3.

[0188] Furthermore, as shown in FIG. 13, the average number of fruits harvested per plant in Example 4 was increased by about 1.4 times compared to Comparative Example 3.

[0189] 14, the average fruit weight of the strawberries harvested in Example 4 was not significantly different from that of Comparative Example 3. In other words, although the number of fruits harvested per plant was greater for the strawberries cultivated in Example 4, the average fruit weight was equivalent to that of Comparative Example 3.

[0190] 15, the average sugar content (Brix sugar content) of the strawberries harvested in Example 4 was approximately 1.1 times higher than that of Comparative Example 3. In other words, the strawberries cultivated in Example 4 had a higher average sugar content despite the fact that a larger number of fruits were harvested.

[0191] 16, there was no difference in appearance, such as size, shape, and color, between the strawberries harvested in Example 4 and Comparative Example 3. In other words, although the strawberries cultivated in Example 4 produced a larger number of harvested fruits, the size of the fruits was similar to that of Comparative Example 3.

[0192] Therefore, it was confirmed that applying the secretions of the modified cyanobacteria to strawberries activates the strawberry's acid invertase, and that the activation of acid invertase has effects such as promoting strawberry growth, increasing the number of fruits, maintaining fruit size, and increasing the fruit's sugar content.

[0193] (summary) From the results of the spinach and strawberry cultivation tests and the acid invertase activity measurements, it was confirmed that the plant acid invertase activator according to the present embodiment has effects such as promoting growth, increasing yield, increasing body weight, and increasing the sugar content of fruits in multiple crop species. [Industrial Applicability]

[0194] According to the present disclosure, for example, by adding a plant acid invertase activator substance to soil, plant acid invertase can be activated, thereby enhancing crop production. [Explanation of symbols]

[0195] 1 Intima 2. Peptidoglycan 3. Glycans 4 cell wall 5 Adventitia 6 SLH domain-containing outer membrane proteins 7 SLH Domain 8. Organic Channel Proteins 9. Cell wall-pyruvate modifying enzymes

Claims

1. A method for cultivating agricultural crops, comprising applying a culture supernatant containing a secretion of a modified cyanobacterium to the agricultural crops to activate acid invertase in the agricultural crops, the cyanobacteria is any one of the genus Synechocystis, the genus Synechococcus, the genus Anabaena, or the genus Thermosynechococcus; The modified cyanobacteria was cultured in liquid culture using BG-11 medium or a modified method thereof. How crops are grown.

2. The crop is a leafy vegetable or a fruit. The method for cultivating agricultural crops according to claim 1.

3. A culture supernatant for cultivating agricultural crops used in the method for cultivating agricultural crops according to claim 1, a modified cyanobacterial secretion product, which when applied to the crop activates acid invertase in the crop; the cyanobacteria is any one of the genus Synechocystis, the genus Synechococcus, the genus Anabaena, or the genus Thermosynechococcus; The modified cyanobacteria was cultured in liquid culture using BG-11 medium or a modified method thereof. Culture supernatant for agricultural crop cultivation.

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