Crop yield improver and method for improving crop yield

Abstract

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

Description

[Technical Field]

[0001] The present disclosure relates to a crop yield improver that is a natural metabolite that contributes to improving crop yield, a method for producing the crop yield improver, and a method for improving crop yield. [Background technology]

[0002] With the demand for increased food production due to the growing world population, there is a need to develop crop yield improvement technologies to efficiently produce high-quality agricultural crops on limited arable land. In particular, in recent years, from the perspective of preventing global warming and reducing the environmental burden, there has been a desire to utilize naturally derived substances that consume less fossil energy in the manufacturing process and that have a low environmental burden when applied (hereinafter also referred to as application or use).

[0003] For example, methods for improving crop yields using naturally occurring substances have been disclosed, such as a method of inoculating plants with microorganisms or microbial culture solutions that produce substances that contribute to promoting plant growth (Patent Documents 1 and 2 and Non-Patent Document 1). Another method has been disclosed, for example, of adding natural metabolites such as organic acids to soil to chelate metal ions in the soil and improve the availability of metal ions to plants (Patent Document 3). Other methods have also been disclosed, such as a method of applying a composition containing the natural metabolite adenosine to plants (Patent Document 4) and a method of fertilizing plants with a fertilizer containing an algae cell extract (Patent Document 5). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2018-11600 A [Patent Document 2] Special Publication No. 63-501286 [Patent Document 3] Japanese Patent Application Laid-Open No. 2014-073993 [Patent Document 4] Patent No. 5943844 [Patent Document 5] Patent No. 3143872 [Non-patent literature]

[0005] [Non-Patent Document 1] Jimenez-Gomez et al., “Probiotic activities of Rhizobium laguerreae on growth and quality of spinach”, Scientific reports, 2018, Vol. 8, 295 Summary of the Invention [Problem to be solved by the invention]

[0006] However, the effectiveness of microbial materials varies depending on the combination of the microorganism species used, the target plant species, and the properties of the soil, making them less versatile and unstable in their ability to improve crop yields.On the other hand, naturally occurring substances such as natural metabolites are more likely to improve yields on multiple plant species (i.e., plants in general) than microbial materials, but the processes for producing, extracting, or refining these substances are complicated and incur excessive costs.

[0007] Therefore, the present disclosure provides a crop yield improver containing a naturally occurring substance having a crop yield improving effect (hereinafter also referred to as a crop yield improver), a method for producing the crop yield improver simply and efficiently, and a method for improving crop yield effectively. [Means for solving the problem]

[0008] A crop yield improver according to one embodiment of the present disclosure includes at least one of pyroglutamic acid and aminobenzoic acid.

[0009] Furthermore, a method for producing a crop yield enhancer 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 substance involved in improving crop yield.

[0010] Furthermore, a method for improving crop yield according to one aspect of the present disclosure includes applying the crop yield improver to crops. [Effects of the Invention]

[0011] The crop yield improver of the present disclosure can effectively improve crop yield. Furthermore, the method for producing the crop yield improver of the present disclosure can simply and efficiently produce a crop yield improver that exhibits a yield-improving effect on multiple crop species. Furthermore, the crop yield improvement method of the present disclosure can effectively improve crop yield by using (hereinafter also referred to as applying) the crop yield improver of the present disclosure on crops. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a flowchart showing an example of a method for producing a crop yield improver 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 cyanobacterium 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 view 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 relative average plant weight per plant of the lettuce plants grown in Examples 3 to 5 and Comparative Example 2. [Figure 11] FIG. 11 shows the state of representative strains from Examples 3 to 5 and Comparative Example 2. [Figure 12] FIG. 12 is a graph showing the relative average weight per stalk of spinach cultivated in Examples 6 to 8 and Comparative Example 3. [Figure 13] FIG. 13 shows the state of representative strains from Examples 6 to 8 and Comparative Example 3. [Figure 14] FIG. 14 is a graph showing the average number of fruits per plant of the tomatoes cultivated in Example 9 and Comparative Example 4. [Figure 15] FIG. 15 is a graph showing the average fruit weight per plant of the tomatoes cultivated in Example 9 and Comparative Example 4. [Figure 16] FIG. 16 is a graph showing the average sugar content per plant of the tomatoes cultivated in Example 9 and Comparative Example 4. [Figure 17] FIG. 17 shows the state of typical fruits in Example 9 and Comparative Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0013] (Findings that formed the basis of this disclosure) As described in the background art, there is a demand for technologies to improve crop yields in order to efficiently produce agricultural crops within limited cultivated land. Furthermore, in order to improve crop yields, there is a demand for the use of naturally occurring substances that impose a low environmental load when applied. In particular, there is a demand for substances that consume less fossil energy during their production and impose a lower environmental load.

[0014] The following prior art techniques have been disclosed as techniques for improving crop yields.

[0015] For example, Patent Document 1 discloses a method of applying a microbial strain having plant growth-promoting activity or a culture of the microbial strain to a plant or its surroundings (e.g., soil). It has been reported that the use of this method not only promotes plant growth and increases yield, but also makes it possible to prevent the occurrence of pathogenic plant diseases.

[0016] Furthermore, for example, Patent Document 2 discloses a method for producing a plant growth-promoting composition by mixing a bacterial culture solution with an algae culture solution and incubating the mixture under predetermined conditions, and then applying the composition to a plant. It has been reported that this method promotes the vegetative growth of tomatoes when the composition is added to a nutrient solution for hydroponic cultivation of plants.

[0017] Furthermore, for example, Non-Patent Document 1 discloses a method of applying a type of rhizobia to spinach, more specifically to spinach roots, as a plant probiotic bacterium with a plant growth-promoting mechanism. It has been reported that this method achieves growth-promoting effects such as an increase in the number and size of leaves of spinach inoculated with the rhizobia.

[0018] Furthermore, for example, Patent Document 3 discloses a method of adding natural metabolites (substances involved in metabolism that exist in nature) such as organic acids to soil to chelate metal ions such as iron in the soil and improve the availability of the metal ions to plants.

[0019] Furthermore, for example, Patent Document 4 discloses a method of applying a composition containing adenosine, a natural metabolite, as a main component to plants.

[0020] Furthermore, for example, Patent Document 5 discloses a method of applying a fertilizer containing an algae cell extract to a plant. More specifically, the cell extract is prepared by treating cyanobacteria with an aqueous solvent (e.g., water) at 60°C or higher.

[0021] However, when using naturally occurring substances such as those described above, the production, extraction, or purification processes are generally complicated and incur excessive costs. On the other hand, when microorganisms themselves are ingested into plants, complicated processes are not required, but the effect varies depending on the combination of the microorganism species used, the target plant species, and the properties of the soil, so the method lacks versatility and its effect on improving crop yield is unstable. In light of the above circumstances, there is a need for the development of naturally occurring substances that can be produced using cheaper raw materials through simple processes and that have the effect of improving yields in multiple crop species.

[0022] The present inventors have discovered that applying at least one of pyroglutamic acid and aminobenzoic acid, which are natural metabolites, to crops improves crop productivity, i.e., increases the yield of high-quality crops. Furthermore, the present inventors have discovered a method for inexpensively and simply (i.e., efficiently) producing a crop yield improver containing at least one of pyroglutamic acid and aminobenzoic acid using cyanobacteria (also called blue-green bacteria or blue-green algae).

[0023] Cyanobacteria are a group of true bacteria that split 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 cultivate cyanobacteria using inexpensive raw materials and a simple process. Furthermore, cyanobacteria are known to have characteristics such as rapid growth and high light utilization efficiency. In addition, they are easier to genetically manipulate than other algae species, and therefore active research and development is being conducted on the production of substances using cyanobacteria, a type of photosynthetic microorganism.

[0024] For example, as an example of substance production using cyanobacteria, the production of fuels such as ethanol, isobutanol, alkanes, and fatty acids (Patent Document 6: Japanese Patent No. 6341676) has been reported. Research and development is also being conducted on the production of substances that serve as nutrient sources for living organisms. However, because the metabolites and proteins within cyanobacterial cells are difficult to secrete outside the cells, it is necessary to disrupt the cyanobacterial cells and extract the desired metabolites and proteins produced within the cells.

[0025] 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 compounds, proteins, and intracellular metabolites produced within the cyanobacterial cell. Through this process, the present inventors discovered that the cyanobacterial secretions contain pyroglutamic acid and aminobenzoic acid, and that pyroglutamic acid and aminobenzoic acid each have yield-enhancing effects on multiple crop species. This allows for efficient recovery of crop-yield-enhancing substances secreted outside the cyanobacterial cell without disrupting the cell. Furthermore, since procedures such as extraction (e.g., disruption of the cell) are not required, the physiological activity of the crop-yield-enhancing substance is less likely to be lost. Therefore, a crop yield enhancer containing the secretions can effectively improve crop yield.

[0026] Therefore, according to the method for producing a crop yield improver of the present disclosure, a substance having the effect of enhancing the production of multiple crop species can be produced simply and efficiently. Furthermore, the crop yield improver of the present disclosure can effectively promote plant growth. Furthermore, according to the crop yield improver of the present disclosure, by using the crop yield improver of the present disclosure, crop production can be effectively enhanced.

[0027] (Summary of the Disclosure) An outline of one aspect of the present disclosure is as follows.

[0028] A crop yield improver according to one embodiment of the present disclosure includes at least one of pyroglutamic acid and aminobenzoic acid.

[0029] The crop yield improver according to one embodiment of the present disclosure can improve the yield of a plurality of crop species.

[0030] A method for producing a crop yield enhancer 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 containing the crop yield enhancer.

[0031] As a result, in the modified cyanobacterium, the bond between the cell wall and 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. This makes it easier for proteins and metabolites (hereinafter also referred to as intracellularly produced substances) produced within the bacterial cell to leak outside the outer membrane, i.e., outside the bacterial cell. These leaked intracellularly produced substances include a group of substances with crop yield-enhancing effects, characterized by containing pyroglutamic acid and aminobenzoic acid. This eliminates the need for extraction processes for the intracellularly produced substances, such as disrupting the bacterial cell. This allows for the simple and efficient production of a crop yield improver. Furthermore, because the extraction process for the intracellularly produced substances is unnecessary, a decrease in the physiological activity of the intracellularly produced substances and a decrease in yield are less likely to occur. This also reduces a decrease in the physiological activity and yield of substances involved in increasing crop yield among the intracellularly produced substances of the modified cyanobacterium. Furthermore, because the above-described extraction process of the intracellularly produced substance is not required, the modified cyanobacteria can be repeatedly used to produce the intracellularly produced substance even after the extracellularly secreted intracellularly produced substance is recovered. Therefore, there is no need to prepare a new modified cyanobacterium each time a crop yield improver is produced. Therefore, the method for producing a crop yield improver according to one embodiment of the present disclosure allows for simple and efficient production of the crop yield improver.

[0032] For example, in a method for producing a crop yield improver according to one embodiment of the present disclosure, the protein involved in 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.

[0033] 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 modified cyanobacterium, which secretes the crop yield enhancer produced intracellularly outside the fungal body. Therefore, according to the method for producing a crop yield enhancer according to one aspect of the present disclosure, the modified cyanobacterium can be caused to efficiently secrete the crop yield enhancer, thereby enabling efficient production of the crop yield enhancer.

[0034] For example, in a method for producing a crop yield improver 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 whose amino acid sequence is 50% or more identical to any of these SLH domain-retaining outer membrane proteins.

[0035] 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 the extracellular secretion of the crop yield improver produced within the bacterial cells, thereby facilitating the extracellular secretion of the crop yield improver produced within the bacterial cells. Therefore, according to the method for producing a crop yield improver of one aspect of the present disclosure, the crop yield improver produced within the bacterial cells of the modified cyanobacterium can be efficiently produced.

[0036] In a method for producing a crop yield improver 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.

[0037] 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 easier for crop yield improvers produced within the cells to leak out of the cells. Therefore, according to a method for producing a crop yield improver according to one aspect of the present disclosure, the crop yield improver produced within the modified cyanobacterium is more likely to be secreted out of the cells, allowing for efficient production of the crop yield improver.

[0038] For example, in the method for producing a crop yield improver according to one embodiment of the present disclosure, a gene that expresses a protein involved in binding between the outer membrane and the cell wall may be deleted or inactivated.

[0039] As a result, in the modified cyanobacterium, the expression of proteins involved in the binding between the cell wall and the outer membrane is suppressed, or the function of these proteins 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. This improves the secretion productivity of the crop yield improver produced intracellularly. This eliminates the need for extraction processes for the intracellularly produced substances, such as disrupting the cells, and therefore reduces the likelihood of a decrease in the physiological activity and yield of the intracellularly produced substance. This also reduces the likelihood of a decrease in the physiological activity and yield of the intracellularly produced crop yield improver, allowing the production of a crop yield improver with improved crop yield improvement effects. Furthermore, because the extraction process for the intracellularly produced substance is no longer necessary, the modified cyanobacterium can be repeatedly used to produce the crop yield improver after the substance is recovered. Therefore, it is not necessary to prepare a new modified cyanobacterium each time a crop yield improver is produced. Therefore, according to the method for producing a crop yield improver according to one embodiment of the present disclosure, a crop yield improver having an improved crop yield improving effect can be produced simply and efficiently.

[0040] For example, in a method for producing a crop yield improver 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.

[0041] 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 allows the crop yield improver produced within the bacterial cells to be more easily leaked out of the bacterial cells. Therefore, according to the method for producing a crop yield improver according to one aspect of the present disclosure, the modified cyanobacterium can be caused to efficiently secrete the crop yield improver, thereby enabling the crop yield improver to be produced efficiently.

[0042] For example, in a method for producing a crop yield improver 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.

[0043] 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 binding 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 crop yield-improving substances produced within the bacterial cell to leak out of the bacterial cell. Therefore, according to the method for producing a crop yield improver according to one embodiment of the present disclosure, the crop yield improver can be produced efficiently because the crop yield improver produced within the cells of the modified cyanobacterium is more likely to leak out of the cells.

[0044] For example, in a method for producing a crop yield improver 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.

[0045] 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 leakage of proteins and metabolites produced within the bacterial cells, which in turn facilitates leakage of the crop yield improver produced within the bacterial cells. Therefore, according to the method for producing a crop yield improver according to one aspect of the present disclosure, the crop yield improver produced within the modified cyanobacterium bacterial cells is easily leaked outside the bacterial cells, allowing for efficient production of the crop yield improver.

[0046] Furthermore, a method for improving crop yield according to one aspect of the present disclosure involves applying the above-described crop yield improver to crops.

[0047] According to the crop yield improvement method according to one aspect of the present disclosure, by applying to crops a crop yield improver with an improved crop yield improvement effect, the crop yield can be effectively improved.

[0048] Hereinafter, the embodiments will be specifically described with reference to the drawings.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] In this specification, the term "bacterium" and "cell" both refer to an individual cyanobacterium.

[0053] (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.

[0054] [1. Crop yield improver] First, the crop yield enhancer according to the present embodiment will be described. The crop yield enhancer contains at least one of pyroglutamic acid and aminobenzoic acid. The crop yield enhancer is produced intracellularly by modified cyanobacteria (hereinafter also referred to as parent cyanobacteria) in which the function of a protein involved in binding the outer membrane to the cell wall has been suppressed or eliminated, and is secreted extracellularly. In other words, the crop yield enhancer is contained in a secretion of the modified cyanobacterium. The pyroglutamic acid and aminobenzoic acid are preferably contained in the crop yield enhancer at concentrations ranging from about 0.1 μM to about 10 μM. The crop yield enhancer has a plant growth-promoting effect, such as increasing the number of leaves, stems, buds, flowers, or fruits of a plant, thickening the stem or trunk, and increasing the height. The crop yield enhancer also has an effect of improving plant quality (also referred to as a plant quality improving effect), such as preventing plant diseases, improving nutrient absorption, and increasing the sugar content of fruits. As a result, the crop yield enhancer can effectively improve the quality of plants for multiple crop species by increasing crop yield, increasing crop and fruit volume, increasing fruit sugar content, reducing physiological disorders, and reducing disease damage, etc. The crop yield enhancer has a plant growth promoting effect and a plant quality improving effect, and therefore can effectively improve crop yield.

[0055] Plants include not only crops cultivated in fields, but also garden trees, ornamental plants, lawns, roadside trees, and forest trees that receive little or no fertilization. In other words, in this specification, crops are synonymous with the plant as defined herein. Furthermore, improving quality means that the quality of the parts of a plant that are used (e.g., fruit, leaves, rhizomes, etc.), such as its components and appearance, is improved (e.g., increased nutritional value, improved taste, etc.). Components include, for example, general components such as water, protein, lipids, carbohydrates (e.g., sugars, starch, etc.), and ash; inorganic substances such as sodium, potassium, and calcium; vitamins such as vitamin A, vitamin C, and folic acid; fatty acids, cholesterol, and dietary fiber. Furthermore, appearance includes measurable aspects such as shape, color, fragrance, cortex, fleshiness, thickness, gloss, flowering level, and degree of physiological disorders, as well as less measurable aspects such as beauty, suppleness, freshness, and visual balance.

[0056] The cyanobacteria (i.e., the parent cyanobacteria) and the modified cyanobacteria will be described later.

[0057] As described above, the secreted product contains a crop yield enhancer including at least one of pyroglutamic acid and aminobenzoic acid. That is, the modified cyanobacterium secretes a secreted product involved in improving crop yield. The secreted product contains proteins and metabolic products produced within the modified cyanobacterium's cells (i.e., intracellularly produced substances). The intracellularly produced substances include substances involved in improving crop yield (i.e., crop yield-enhancing substances).

[0058] Examples of crop yield improvers other than pyroglutamic acid and aminobenzoic acid include organic decomposition enzymes such as peptidases, nucleases, and phosphatases, intracellular molecules involved in promoting nucleic acid (e.g., DNA or RNA) synthesis such as adenosine or guanosine, or spermidine, ketone bodies such as 3-hydroxybutyric acid, or organic acids such as gluconic acid. The secretion product of the engineered cyanobacterium may be a mixture of these crop yield improvers.

[0059] [2. Manufacturing method of crop yield improver] Next, a method for producing a crop yield improver 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 crop yield improver according to the present embodiment.

[0060] The method for producing a crop yield enhancer in 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 lost (step S01); and causing the modified cyanobacterium to secrete a secretory product containing the crop yield enhancer (step S02).

[0061] 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 secretion product containing the crop yield enhancer in step S02.

[0062] In step S02, the modified cyanobacterium is caused to secrete a secretion containing a crop yield enhancer. In the modified cyanobacterium of this embodiment, the function of a protein involved in binding the outer membrane to the cell wall in the cyanobacterium (i.e., the parent cyanobacterium) 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 include crop yield enhancers containing at least one of pyroglutamic acid and aminobenzoic acid. Therefore, in step S02, the modified cyanobacterium is cultured under specified conditions, whereby intracellularly produced substances involved in improving crop yield, such as pyroglutamic acid and aminobenzoic acid, are secreted extracellularly.

[0063] 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 period for culturing cyanobacteria to produce a crop yield enhancer 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.

[0064] When cultured under the above conditions, the modified 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, for example, at least one of pyroglutamic acid and aminobenzoic acid, and may also contain crop yield improvers other than these substances. 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 crop yield improver of this embodiment, a secretion containing an intracellularly produced substance involved in crop yield improvement (i.e., a crop yield improver) is secreted outside the cells of the modified cyanobacteria, eliminating the need to disrupt the cells to recover the crop yield improver. Therefore, the modified cyanobacteria remaining after recovery of the crop yield improver can be repeatedly used to produce a crop yield improver.

[0065] The method for recovering the crop yield improver secreted into the culture solution is not limited to the above example. The crop yield improver 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 crop yield improver that has permeated the membrane may be recovered. In this way, the crop yield improver 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 a simpler and more efficient production of the crop yield improver.

[0066] Furthermore, because the need to recover and disrupt the bacterial cells from the culture medium is eliminated, damage and stress to the modified cyanobacteria can be reduced, which reduces the productivity of the modified cyanobacteria in secreting the crop yield-enhancing substance and allows the modified cyanobacteria to be used for a longer period of time.

[0067] As described above, by using the modified cyanobacterium of this embodiment, a crop yield enhancer can be obtained simply and efficiently.

[0068] The cyanobacteria and modified cyanobacteria will be described below.

[0069] [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.

[0070] FIG. 2 is a schematic diagram of 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, 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 3: Jurgens and Weckesser, 1986, J. Bacteriol., 168:568-573). In this specification, the 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.

[0071] SLH domain-containing outer membrane proteins (e.g., Slr1841 in the figure) consist 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. They are widely distributed in cyanobacteria and bacteria belonging to the Negativicutes class, a group of Gram-negative bacteria (Non-Patent Document 4: Kojima et al., 2016, Biosci. Biotech. Biochem., 10:1954-1959). The region embedded in the lipid membrane (i.e., outer membrane 5) forms a channel that allows hydrophilic substances to permeate the outer membrane, while the SLH domain 7 functions to bind to the cell wall 4 (Non-Patent Document 5: Kowata et al., 2017, J. Bacteriol., 199:e00371-17). In order for the SLH domain 7 to bind to the cell wall 4, the covalently bound sugar chain 3 in the peptidoglycan 2 must be modified with pyruvate (Non-Patent Document 6: Kojima et al., 2016, J. Biol. Chem., 291:20198-20209). Examples of genes encoding the SLH domain-containing outer membrane protein 6 include slr1841 or slr1908 contained in Synechocystis sp. PCC 6803, and oprB contained in Anabaena sp. 90.

[0072] An enzyme that catalyzes the pyruvate modification reaction of covalently linked 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 7: Mesnage et al., 2000, EMBO J., 19:4473-4484). Among cyanobacteria whose genome sequences have been published, many species possess genes encoding proteins homologous to CsaB with an amino acid sequence identity of 30% or more. Examples include slr0688 possessed by Synechocystis sp. PCC 6803 and syn7502_03092 possessed by Synechococcus sp. 7502.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] [4. Modified Cyanobacteria] Next, the modified cyanobacterium of this embodiment will be described with reference to FIG.

[0077] 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. As a result, the modified cyanobacteria have improved secretion productivity of intracellularly produced substances, which secrete proteins and metabolic products produced intracellularly to the outside of the cell. As described above, intracellularly produced substances include intracellularly produced substances involved in improving crop yields (i.e., crop yield-enhancing substances). Examples of crop yield-enhancing substances include pyroglutamic acid and aminobenzoic acid. Secretions of the modified cyanobacteria include, for example, crop yield enhancers containing at least one of pyroglutamic acid and aminobenzoic acid. Therefore, the modified cyanobacteria also have improved productivity in secreting crop yield-enhancing substances, such as pyroglutamic acid and aminobenzoic acid, produced within the bacterial cells and secreted outside the bacterial cells. Furthermore, because there is no need to disrupt the bacterial cells to recover the crop yield-enhancing substances, the modified cyanobacteria can be used repeatedly even after recovering the crop yield-enhancing substances. In this specification, the production of proteins and metabolites within the bacterial cells by the modified cyanobacteria is referred to as "production," and the secretion of the produced proteins and metabolites outside the bacterial cells is referred to as "secretory production."

[0078] 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 within the cells of the modified cyanobacterium, particularly those present in the periplasm, such as proteins and metabolic products, are more likely to leak out of the cell (outside the outer membrane 5). This improves the secretory productivity of the modified cyanobacterium, which secretes crop yield-improving substances produced within the cells.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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).

[0083] 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.

[0084] 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 that of these SLH domain-containing outer membrane proteins 6.

[0085] 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 within the modified cyanobacterium to leak out of the cells, and therefore easier for crop yield-improving substances produced within the cells to leak out of the cells.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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 the present 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 crop yield-improving substances produced within the cells also tend to leak out of the cells.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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.

[0094] In this embodiment, the modified cyanobacterium has an outer membrane 5 and a cell wall 4. Therefore, 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, the outer membrane 5 is more likely to detach partially from the cell wall 4 in the modified cyanobacterium, which makes it easier for intracellularly produced substances, such as proteins and metabolites, to leak out of the outer membrane 5, i.e., outside the cell. Therefore, the modified cyanobacterium has improved productivity in secreting crop yield-enhancing substances produced intracellularly. This eliminates the need for extraction processes for intracellularly produced substances, such as disrupting the cell body, thereby reducing the physiological activity and yield of the intracellularly produced substances. Therefore, the physiological activity and yield of the intracellularly produced crop yield-enhancing substances are less likely to decrease, making it possible to produce a crop yield enhancer with improved crop yield improvement effects. Furthermore, since the above-mentioned extraction treatment of the intracellularly produced substance is no longer necessary, the modified cyanobacteria can be used repeatedly to produce the crop yield improving substance even after the substance has been recovered.

[0095] 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 making it easier for proteins and metabolic products produced within the bacterial cell to leak out of the bacterial cell. This also makes it easier for crop yield-improving substances produced within the modified cyanobacterium to leak out of the bacterial cell.

[0096] 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.

[0097] 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.

[0098] More specifically, the gene encoding the SLH domain-retaining 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.

[0099] 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 crop yield-improving substances produced within the bacterial cell to leak out of the bacterial cell.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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 easier for crop yield improving substances produced within the fungal cells to leak out of the fungal cells.

[0104] 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.

[0105] [5. Method for producing modified cyanobacteria] Next, a method for producing modified cyanobacteria in this embodiment will be described. The method for producing modified cyanobacteria 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.

[0106] 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.

[0107] 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.

[0108] 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.

[0109] The means for inhibiting 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 8: Yao et al., ACS Synth. Biol., 2016, 5:207-212), etc. Specific techniques for introducing the mutation or substituting or inserting the base sequence may be, for example, ultraviolet irradiation, site-specific mutagenesis, or homologous recombination.

[0110] Furthermore, the means for inhibiting the translation of the transcription product of the gene may be, for example, RNA (ribonucleic acid) interference.

[0111] 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.

[0112] 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 bacterial cell), and therefore substances involved in improving crop yields (i.e., crop yield-enhancing substances), such as pyroglutamic acid and aminobenzoic acid, are also more likely to leak out of the bacterial cell. Therefore, the method for producing modified cyanobacteria in this embodiment can provide modified cyanobacteria with improved productivity for secreting crop yield-enhancing substances.

[0113] Furthermore, in the modified cyanobacteria produced by the production method of this embodiment, the crop yield enhancer 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 the crop yield enhancer secreted into the culture solution can then be recovered. This allows the crop yield enhancer to be recovered from the culture solution while the modified cyanobacteria are being cultured. Therefore, the modified cyanobacteria obtained by this production method can be used to efficiently produce a microbiological crop yield enhancer. Therefore, the method for producing modified cyanobacteria of this embodiment can provide a highly efficient modified cyanobacterium that can be reused after the crop yield enhancer is recovered.

[0114] [6. Methods for improving crop yields] In the method for improving crop yield according to the present embodiment, the above-described crop yield improver is applied to crops. As described above, the crop yield improver according to the present embodiment is a crop yield improver with an improved crop yield improving effect, and therefore, by applying the above-described crop yield improver to crops, the yield of the crop can be effectively improved.

[0115] The above-described crop yield enhancer may be used as is, or may be concentrated or diluted. When applying the crop yield enhancer to crops, the concentration and application method of the crop yield enhancer may be determined appropriately depending on the type of crop, the properties of the soil, and the purpose. The crop yield enhancer may be, for example, a 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, for example, a substance involved in improving crop yield, such as 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 crops. The crop yield enhancer may be applied to plants by, for example, spraying, irrigating, or mixing the plant or soil. More specifically, a few milliliters per plant may be applied to the base of the plant about once a week. [Example]

[0116] The modified cyanobacteria, the method for producing the modified cyanobacteria, and the method for producing the crop yield improver of the present disclosure will be specifically explained in the following examples, but the present disclosure is not limited to the following examples in any way.

[0117] 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").

[0118] 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.

[0119] (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.

[0120] The mechanism by which gene expression is suppressed by this method is as follows.

[0121] 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.

[0122] 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.

[0123] Below, we will specifically explain how to introduce each of the above two genes into the chromosomal DNA of cyanobacteria.

[0124] (1-1) Introduction of dCas9 gene Using the chromosomal DNA of Synechocystis LY07 strain (hereinafter also referred to as LY07 strain) (see Non-Patent Document 8) as a template, the dCas9 gene, an operator gene for regulating dCas9 gene expression, 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.

[0125] [Table 1]

[0126] 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.

[0127] [Table 2]

[0128] (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.

[0129] [Table 3]

[0130] 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.

[0131] 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.

[0132] 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.

[0133] 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.

[0134] (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.

[0135] 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.

[0136] (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.

[0137] Furthermore, the expression of the slr0688 gene was suppressed by the same procedure as in (1-3) above.

[0138] (Comparative Example 1) In Comparative Example 1, the Synechocystis dCas9 strain was obtained by the same procedure as in Example 1 (1-1).

[0139] 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.

[0140] (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.

[0141] (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.

[0142] (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 ​​to a thickness of 70 nm using an ultramicrotome (Ultracut) to prepare ultrathin sections. These ultrathin sections were stained with a 2% uranium acetate and 1% lead citrate solution to prepare 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.

[0143] (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.

[0144] 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).

[0145] 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.

[0146] 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.

[0147] 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).

[0148] 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.

[0149] 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.

[0150] 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).

[0151] 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.

[0152] (4) Identification of secreted intracellular metabolites (4-1) 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.

[0153] (4-2) 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.

[0154] [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

[0155] (4-3) 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 4.

[0156] [Table 4]

[0157] 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, including crop yield-improving substances such as pyroglutamic acid and aminobenzoic acid, to leak out of the outer membrane (i.e., outside the fungus).

[0158] (5) 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.

[0159] (5-1) 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.

[0160] (5-2) Quantification of extracellularly secreted proteins The culture medium obtained in (5-1) 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.

[0161] 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).

[0162] 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.

[0163] 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.

[0164] 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.

[0165] 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.

[0166] (6) Identification of secreted proteins Subsequently, the secreted proteins contained in the culture supernatant obtained in (5-2) above were identified by LC-MS / MS, as described below.

[0167] (6-1) 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.

[0168] (6-2)LC-MS / MS analysis The sample obtained in (6-1) above was analyzed using an LC-MS / MS device (UltiMate 3000 RSLCnano LC System) under the following conditions.

[0169] 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

[0170] (6-3) Data analysis The obtained data was analyzed under the following conditions to identify proteins and peptides and calculate quantitative values.

[0171] 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

[0172] 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.

[0173] [Table 5]

[0174] 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.

[0175] (7) Crop cultivation tests Next, the following crop cultivation tests were conducted to evaluate the crop yield-enhancing effects of pyroglutamic acid, aminobenzoic acid, and the secretion product of the modified cyanobacterium (here, the culture supernatant of the modified cyanobacterium). Specifically, lettuce and spinach cultivation tests were conducted to evaluate the yield-enhancing effects on leafy vegetable production. Furthermore, tomato cultivation tests were conducted to evaluate the yield-enhancing effects on fruit production. Each of these cultivation tests is described below.

[0176] (7-1) Lettuce hydroponic cultivation test The hydroponic culture solution used was a 500-fold dilution of a commercially available nutrient 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. The light conditions were a photon flux density of 200 μmol / m from a white light source. 2 The plants were cultivated at room temperature (22°C) for 35 days under conditions of 16 hours of light and 8 hours of darkness / s.

[0177] (Examples 3, 4, and 5) During the cultivation period, (1) a 1 μM pyroglutamic acid solution (Example 3), (2) a 1 μM aminobenzoic acid solution (Example 4), and (3) a secretion product of the modified cyanobacteria (Example 5) were added to the hydroponic culture solution once a week in an amount of 5 mL per plant. After harvesting, the plant weights were measured, and the mean and standard deviation (SD) were calculated for each of (1) to (3). The modified cyanobacteria were the slr1841-suppressed strain of Example 1 and the slr0688-suppressed strain of Example 2.

[0178] (Comparative Example 2) Except for using water instead of the above (1) to (3), the same procedures as in Examples 3 to 5 were carried out. After harvesting, the weight of the plants was measured, and the average value and standard deviation (SD) were calculated.

[0179] (result) The results of Examples 3 to 5 and Comparative Example 2 are shown in Figures 10 and 11. Figure 10 is a graph showing the relative average plant weight per plant of the lettuce plants grown in Examples 3 to 5 and Comparative Example 2. Figure 11 also includes photographs of representative plants to visually show the condition of each plant in Examples 3 to 5 and Comparative Example 2.

[0180] As shown in FIG. 10, the average weight of each lettuce head harvested in Examples 3 to 5 was increased compared to Comparative Example 2. Specifically, the average weight of each lettuce head grown in Example 3 was approximately 14% greater than that in Comparative Example 2. The average weight of each lettuce head grown in Example 4 was approximately 15% greater than that in Comparative Example 2. The average weight of each lettuce head grown in Example 5 was approximately 29% greater than that in Comparative Example 2.

[0181] 11, the lettuce plants cultivated in Examples 3 to 5 did not show any particularly noticeable changes in the overall shape of the plant compared to Comparative Example 2. In other words, although the lettuce plants cultivated in Examples 3 to 5 grew faster than those in Comparative Example 2, they did not suffer from any noticeable physiological disorders (such as tip burn), and the leaf growth and size of the stems and leaves were good, resulting in a well-balanced appearance.

[0182] (7-2) Spinach cultivation test Spinach seeds were sown in a cultivation pot (12 cm × 10 cm) filled with commercially available potting soil. The cultivation was carried out at 22°C under a white light source with a photon flux density of 200 μmol / m 2 The cultivation was carried out at 500 W / s under 12 hours of light and 12 hours of darkness for 35 days. During this time, 50 mL of distilled water was supplied to each pot every other day. Approximately one week after the start of cultivation, when the cotyledons had developed, the plants were thinned out to ensure that the size of the plants in each pot was uniform.

[0183] (Examples 6, 7, and 8) During the cultivation period, (1) a 1 μM pyroglutamic acid solution (Example 6), (2) a 1 μM aminobenzoic acid solution (Example 7), or (3) a secretion product of the modified cyanobacteria (Example 8) was added to the base of the spinach plants once a week in an amount of 5 mL per plant. After harvesting, the plant weights were measured, and the average and standard deviation (SD) were calculated for each of (1) to (3). The modified cyanobacteria were the slr1841-suppressed strain of Example 1 and the slr0688-suppressed strain of Example 2.

[0184] (Comparative Example 3) Except for using water instead of the above (1) to (3), the same procedures as in Examples 6 to 8 were carried out. After harvesting, the weight of the plants was measured, and the average value and standard deviation (SD) were calculated.

[0185] (result) The results of Examples 6 to 8 and Comparative Example 3 are shown in Figures 12 and 13. Figure 12 is a graph showing the relative average weight per plant of spinach cultivated in Examples 6 to 8 and Comparative Example 3. In addition, Figure 13 shows photographs of representative plants to visually show the condition of each plant in Examples 6 to 8 and Comparative Example 3.

[0186] As shown in FIG. 12, the average weight of each spinach stalk harvested in Examples 6 to 8 was increased compared to Comparative Example 3. Specifically, the average weight of each spinach stalk cultivated in Example 6 was increased by approximately 24% compared to Comparative Example 3. Furthermore, the average weight of each spinach stalk cultivated in Example 7 was increased by approximately 25% compared to Comparative Example 3. Furthermore, the average weight of each spinach stalk cultivated in Example 8 was increased by approximately 39% compared to Comparative Example 3.

[0187] 13, the spinach plants cultivated in Examples 6 to 8 did not show any particularly noticeable changes in the overall shape of the plants compared to Comparative Example 3. In other words, although the spinach plants cultivated in Examples 6 to 8 grew faster than those in Comparative Example 3, they did not suffer from any noticeable physiological disorders (such as fading of leaf color), and the leaves were thick and the stems and leaves were large, and the appearance was well-balanced.

[0188] (7-3) Tomato cultivation test First, commercially available potting soil was placed in a cultivation planter (22 cm x 16 cm), and three tomato seeds were sown per planter. The cultivation was carried out under an indoor temperature of 23°C and a photon flux density of 250 μmol / m from a white light source. 2 The cultivation was carried out under conditions of 16 hours of light and 10 hours of darkness at 1000 x ...

[0189] Example 9 As described above, after the individual plants in each planter were sized uniformly, 5 mL of the modified cyanobacteria secretion solution was added to the base of each plant once a week. The plants were cultivated for 150 days, during which time the tomato 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 also measured, 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.

[0190] Comparative Example 4 The same procedure as in Example 9 was carried out, except that water was used instead of the secretion of the modified cyanobacteria.

[0191] (result) The results of Example 9 and Comparative Example 4 are shown in Figures 14 to 17. Figure 14 is a graph showing the average number of fruits per plant for the tomatoes grown in Example 9 and Comparative Example 4. Figure 15 is a graph showing the average fruit weight per plant for the tomatoes grown in Example 9 and Comparative Example 4. Figure 16 is a graph showing the average sugar content per plant for the tomatoes grown in Example 9 and Comparative Example 4.

[0192] In addition, FIG. 17 shows photographs of representative fruits to visually show the state of the fruits in Example 9 and Comparative Example 4.

[0193] As shown in Figure 14, the average number of fruits harvested per plant in Example 9 increased by about 67% compared to Comparative Example 4. However, as shown in Figure 15, the average weight of fruits harvested per plant in Example 9 and Comparative Example 4 was equivalent. Usually, the greater the fruit yield per plant, the smaller the fruit weight tends to be. However, in Example 9, despite the greater number of fruits harvested, the average fruit weight was equivalent to that of Comparative Example 4.

[0194] 16, the average sugar content (Brix sugar content) of the fruit per plant harvested in Example 9 and Comparative Example 4 was also comparable. Also, as shown in FIG. 17, there was no difference in appearance, such as size, shape, and gloss, between the tomato fruits harvested in Example 9 and Comparative Example 4. Generally, the higher the fruit yield per plant, the lower the sugar content of the fruit and the smaller the size. However, in Example 9, despite the fact that a larger number of fruits were harvested, the average sugar content and size of the fruit were comparable to those of Comparative Example 4.

[0195] Therefore, it was confirmed that application of the modified cyanobacterial secretion to tomatoes can increase yield without reducing fruit quality (i.e., weight, size, and sugar content).

[0196] (summary) From the results of the cultivation tests on lettuce, spinach, and tomato, it was confirmed that the crop yield improver according to the present embodiment has a crop yield improving effect on a plurality of crop species. [Industrial Applicability]

[0197] According to the present disclosure, a crop yield enhancer containing at least one of pyroglutamic acid and aminobenzoic acid can be provided. Furthermore, a modified cyanobacterium with improved productivity in secreting a crop yield enhancer can be provided. Furthermore, by culturing the modified cyanobacterium of the present disclosure, the substance can be efficiently produced, and by adding the substance to soil or a hydroponic solution, for example, crop yield can be improved. [Explanation of symbols]

[0198] 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 producing a cyanobacterial product, comprising: Crop yield enhancer.

2. The crop yield improver according to claim 1 is used on a crop. Methods for improving crop yields.

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