JP7731066B2 - Crop yield improver and method for improving crop yield - Google Patents

Description

This disclosure relates to a crop yield enhancer, which is a natural metabolite that contributes to improving crop yield, a method for producing the crop yield enhancer, and a method for improving crop yield.

As the world's population grows and demands increased food production, there is a need to develop crop yield improvement technologies that will enable efficient production of high-quality crops on limited arable land. In particular, in recent years, with the aim of preventing global warming and reducing the environmental impact, there has been a desire to utilize naturally derived substances that consume less fossil energy in the manufacturing process and have a low environmental impact when applied (hereinafter also referred to as application or use).

For example, methods for improving crop yields using naturally occurring substances have been disclosed, such as 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 that have been disclosed include applying a composition containing the natural metabolite adenosine to plants (Patent Document 4), and applying a fertilizer containing an algae cell extract to plants (Patent Document 5).

JP 2018-11600 A Special Publication No. 63-501286 JP 2014-073993 A Patent No. 5943844 Patent No. 3143872

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

However, the 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 less effective at improving 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.

The present disclosure therefore provides a crop yield enhancer containing a naturally occurring substance (hereinafter also referred to as a crop yield enhancer) that has the effect of improving crop yield, and a method for producing the crop yield enhancer simply and efficiently. The present disclosure also provides a crop yield improvement method that can effectively improve crop yield.

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

Furthermore, one aspect of the present disclosure provides a method for producing a crop yield improver, which 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 eliminated, and causing the modified cyanobacterium to secrete a secretory substance involved in improving crop yield.

Furthermore, a method for improving crop yield according to one aspect of the present disclosure involves applying the crop yield improver to crops.

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 yield-improving effects on multiple crop species. Furthermore, the method for improving crop yield 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.

FIG. 1 is a flowchart showing an example of a method for producing a crop yield improver according to an embodiment. FIG. 2 is a schematic diagram showing the cell surface of a cyanobacterium. FIG. 3 is a transmission electron microscope image of an ultrathin section of the modified cyanobacterium of Example 1. FIG. 4 is an enlarged image of the dashed area A in FIG. FIG. 5 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 2. FIG. 6 is an enlarged image of the dashed area B in FIG. FIG. 7 is a transmission electron microscope image of an ultrathin section of the modified cyanobacterium of Comparative Example 1. FIG. 8 is an enlarged view of the dashed line area C in FIG. 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). FIG. 10 is a graph showing the relative average plant weight per plant of lettuce grown in Examples 3 to 5 and Comparative Example 2. FIG. 11 shows the state of representative strains from Examples 3 to 5 and Comparative Example 2. FIG. 12 is a graph showing the relative average weight per plant of spinach cultivated in Examples 6 to 8 and Comparative Example 3. FIG. 13 shows the state of representative strains from Examples 6 to 8 and Comparative Example 3. FIG. 14 is a graph showing the average number of fruits per plant of the tomatoes cultivated in Example 9 and Comparative Example 4. FIG. 15 is a graph showing the average fruit weight per plant of the tomatoes cultivated in Example 9 and Comparative Example 4. FIG. 16 is a graph showing the average sugar content per plant of the tomatoes cultivated in Example 9 and Comparative Example 4. FIG. 17 shows the state of typical fruits in Example 9 and Comparative Example 4.

(Findings that form 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.

The following prior art techniques have been disclosed as techniques for improving crop yields:

For example, Patent Document 1 discloses a method of applying a microbial strain or a culture of said microbial strain that has plant growth-promoting activity to a plant or to the plant's surroundings (e.g., soil). It has been reported that 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.

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 specified conditions, and then applying the composition to plants. It has been reported that this method promotes the vegetative growth of tomatoes when the composition is added to a nutrient solution used in hydroponic plant cultivation.

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

For example, Patent Document 3 discloses a method in which natural metabolites (naturally occurring substances involved in metabolism) such as organic acids are added to soil to chelate metal ions such as iron in the soil, thereby improving the availability of the metal ions to plants.

For example, Patent Document 4 discloses a method of applying a composition containing the natural metabolite adenosine as a main component to plants.

For example, Patent Document 5 discloses a method of applying a fertilizer containing an algae cell extract to plants. More specifically, the cell extract is obtained by treating cyanobacteria with an aqueous solvent (e.g., water) at 60°C or higher.

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, these complicated processes are not necessary, but the effects vary depending on the combination of the microorganism species used, the target plant species, and the properties of the soil, making them less versatile and resulting in unstable effects on improving crop yields. Given these circumstances, there is a need for the development of naturally occurring substances that can be produced using simple processes using cheaper raw materials and that are effective in improving yields for multiple crop species.

The present inventors have discovered that applying at least one of the natural metabolites pyroglutamic acid and aminobenzoic acid 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 enhancer containing at least one of pyroglutamic acid and aminobenzoic acid using cyanobacteria (also known as blue-green bacteria or blue-green algae).

Cyanobacteria are a group of true bacteria that decompose water through photosynthesis to produce oxygen and use the energy obtained to fix _CO2 from the air. Some cyanobacteria species can also fix nitrogen ( _N2 ) from the air. Because cyanobacteria can obtain most of the raw materials (i.e., nutrients) and energy necessary for bacterial growth from air, water, and light, they can be cultured using inexpensive raw materials and a simple process. Furthermore, cyanobacteria are known to have characteristics such as rapid growth and high light utilization efficiency. Furthermore, compared to other algae species, they are easier to genetically manipulate. Therefore, active research and development is being conducted on the production of substances using cyanobacteria, a type of photosynthetic microorganism.

For example, examples of substance production using cyanobacteria include the production of fuels such as ethanol, isobutanol, alkanes, and fatty acids (Patent Document 6: Japanese Patent No. 6341676). Research and development is also being conducted into 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.

The inventors have therefore discovered that partially detaching the outer membrane that covers the cyanobacterial cell wall from the cell wall makes it easier for compounds, proteins, and intracellular metabolites produced within the cyanobacterial cell to be secreted outside the cell. Through this process, the 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, because procedures such as extraction (e.g., disrupting the cell) are not required, the physiological activity of the crop-yield-enhancing substances is less likely to be lost. Therefore, a crop yield enhancer containing the secretions can effectively improve crop yields.

Therefore, the method for producing a crop yield improver disclosed herein allows for the simple and efficient production of a substance that has the effect of enhancing the production of multiple crop species. Furthermore, the crop yield improver disclosed herein can effectively promote plant growth. Furthermore, the use of the crop yield improver disclosed herein allows for the effective enhancement of crop production.

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

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

The crop yield improver according to one aspect of the present disclosure can improve the yield of multiple crop species.

One aspect of the present disclosure provides a method for producing a crop yield enhancer, comprising 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 eliminated, and causing the modified cyanobacterium to secrete a secretory substance containing the crop yield enhancer.

As a result, in the modified cyanobacteria, 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 metabolic products (hereinafter 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 crop yield enhancers. Furthermore, because the extraction processes for the intracellularly produced substances are not required, 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 intracellularly secreted substance has been collected. This eliminates the need to prepare new modified cyanobacteria each time a crop yield improver is produced. Therefore, the method for producing a crop yield improver according to one aspect of the present disclosure allows for the simple and efficient production of a crop yield improver.

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.

As a result, in the modified cyanobacterium, for example, (i) the function of at least one of an SLH domain-containing outer membrane protein that binds to the cell wall and an enzyme that catalyzes the reaction of modifying bound glycans on the surface of the cell wall with pyruvate (i.e., a cell wall-pyruvate-modifying enzyme) is suppressed or lost, or (ii) the expression of at least one of an SLH domain-containing outer membrane protein and a 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 modified cyanobacterium, the reduced bond between the outer membrane and the cell wall makes it easier for the outer membrane to partially detach from the cell wall, which makes it easier for intracellularly produced substances such as proteins and metabolites to leak out of the cell, as described above. This improves the secretion productivity of the modified cyanobacterium, which secretes the crop yield enhancer produced within the bacterial cell outside the bacterial cell. Therefore, according to a 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.

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 having an amino acid sequence that is 50% or more identical to any of these SLH domain-retaining outer membrane proteins.

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 above or a protein with an amino acid sequence that is 50% or more identical to any of these SLH domain-retaining outer membrane proteins is suppressed or lost, or (ii) the expression of any of the SLH domain-retaining outer membrane proteins set forth in SEQ ID NOs: 1 to 3 above or a protein with an amino acid sequence that is 50% or more identical to any of these SLH domain-retaining outer membrane proteins is suppressed. Therefore, in the modified cyanobacterium, (i) the function of the SLH domain-retaining outer membrane protein in the outer membrane or a protein with a function equivalent to an SLH domain-retaining outer membrane protein is suppressed or lost, or (ii) the expression level of the SLH domain-retaining outer membrane protein in the outer membrane or a protein with a function equivalent to an SLH domain-retaining outer membrane protein is reduced. As a result, in the modified cyanobacteria, the binding domain (e.g., the SLH domain) that binds the outer membrane to the cell wall has a reduced binding amount and binding strength to the cell wall, 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.

In the 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.

As a result, in the modified cyanobacterium, for example, (i) the function of any of the cell wall-pyruvate modifying enzymes shown 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 shown 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. As a result, covalently bonded glycans on the surface of the cell wall are less likely to be modified with pyruvate, thereby reducing the amount and strength of binding of the glycans 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 pyruvic acid, weakening the binding force between the cell wall and the outer membrane and making it easier for the outer membrane to partially detach from the cell wall. This makes it easier for 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.

For example, in a 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.

As a result, in the modified cyanobacteria, the expression of proteins involved in the binding between the cell wall and 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 outer membrane (i.e., the amount and strength of binding). As a result, in the modified cyanobacteria, 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 enhancer produced intracellularly. This eliminates the need for extraction processes for the intracellularly produced substances, such as disrupting the cells, and therefore reduces the physiological activity and yield of the intracellularly produced substances. This also reduces the physiological activity and yield of the crop yield enhancer produced intracellularly, making it possible to produce a crop yield enhancer with improved crop yield enhancement effects. Furthermore, because the extraction process for the intracellularly produced substances is no longer necessary, the modified cyanobacteria can be repeatedly used to produce the crop yield enhancer after the substances are recovered. Therefore, there is no need 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 aspect of the present disclosure, a crop yield improver with improved crop yield improvement effects can be produced simply and efficiently.

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 binding between the outer membrane and the cell wall may be at least one of a gene encoding an SLH domain-containing outer membrane protein and a gene encoding a cell wall-pyruvate modifying enzyme.

As a result, in the modified cyanobacterium, at least one gene encoding an SLH domain-retaining outer membrane protein and at least one gene encoding a cell wall-pyruvate-modifying enzyme 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 and the cell wall-pyruvate-modifying enzyme is suppressed, or (ii) the function of at least one of the SLH domain-retaining outer membrane protein and the cell wall-pyruvate-modifying enzyme is suppressed or lost. This reduces the bond (i.e., the amount and strength of the bond) between the SLH domain of the SLH domain-retaining 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 in areas where the bond between the outer membrane and the cell wall is weakened. As a result, in the modified cyanobacterium, the outer membrane is more likely to partially detach from the cell wall due to the reduced bond between the outer membrane and the cell wall, making it easier for proteins and metabolic products produced within the cell to leak out of the cell. This also makes it easier for the crop yield enhancer produced within the bacterial cells to leak out of the bacterial cells. Therefore, according to a 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 the efficient production of the crop yield enhancer.

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 nucleotide sequence shown in SEQ ID NO: 7, nies970_09470 consisting of the nucleotide sequence shown in SEQ ID NO: 8, anacy_3458 consisting of the nucleotide sequence shown in SEQ ID NO: 9, or a gene having a nucleotide sequence that is 50% or more identical to any of these genes.

As a result, in the modified cyanobacterium, a gene encoding any of the SLH domain-containing outer membrane proteins set forth in SEQ ID NOS: 7 to 9, or a gene that is 50% or more identical to the nucleotide sequence of any of these genes, is deleted or inactivated. Therefore, in the modified cyanobacterium, (i) the expression of any of the SLH domain-containing outer membrane proteins or proteins functionally equivalent to any of these proteins is suppressed, or (ii) the function of any of the SLH domain-containing outer membrane proteins or proteins functionally equivalent to any of these proteins is suppressed or lost. As a result, in the modified cyanobacterium, the binding amount and strength of the binding domain (e.g., the SLH domain) that binds the outer membrane to the cell wall is reduced, making the outer membrane more likely to partially detach from the cell wall. This makes it easier for proteins and metabolic products 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 aspect of the present disclosure, the crop yield improver produced within the cells of the modified cyanobacterium is more likely to leak out of the cells, allowing for efficient production of the crop yield improver.

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 nucleotide sequence shown in SEQ ID NO: 10, synpcc7942_1529 consisting of the nucleotide sequence shown in SEQ ID NO: 11, anacy_1623 consisting of the nucleotide sequence shown in SEQ ID NO: 12, or a gene having a nucleotide sequence that is 50% or more identical to any of these genes.

As a result, in the modified cyanobacterium, a gene encoding any one of the cell wall-pyruvate-modifying enzymes shown in SEQ ID NOs: 10 to 12 above, or a gene that is 50% or more identical 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 with 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 with a function equivalent to any one of these enzymes is suppressed or lost. This makes it difficult for covalently bonded glycans on the surface of the cell wall to be modified with pyruvate, thereby reducing the amount and strength of binding between the glycans on the cell wall and the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane. As a result, in the modified cyanobacterium, the amount of glycans that bind the cell wall to the outer membrane is reduced, weakening the binding strength between the cell wall and the outer membrane and making it easier for the outer membrane to partially detach from the cell wall. This makes it easier for proteins and metabolic products produced within the bacterial cells to leak out of the bacterial cells, and therefore makes it easier for crop yield improvers produced within the bacterial cells to leak out of the bacterial cells. Therefore, according to a method for producing a crop yield improver according to one aspect of the present disclosure, crop yield improvers produced within the bacterial cells of the modified cyanobacterium are more likely to leak out of the bacterial cells, allowing for efficient production of the crop yield improver.

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

According to a method for improving crop yield according to one aspect of the present disclosure, by applying a crop yield enhancer with improved crop yield-improving effects to crops, it is possible to effectively improve crop yields.

The following describes the embodiments in detail, with reference to the drawings.

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 scope of this disclosure. Furthermore, among the components in the following embodiments, any components that are not recited in an independent claim that represents a superordinate concept are described as optional components.

Furthermore, the figures are not necessarily strict illustrations. In each figure, substantially identical components are designated by the same reference numerals, and duplicate explanations may be omitted or simplified.

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

In addition, in this specification, the terms "bacterium" and "cell" both refer to a single individual cyanobacterium.

(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 proteins of interest and the nucleotide sequences of the genes encoding those proteins can be obtained from these databases.

[1. Crop yield improvers]
First, a description will be given of a crop yield enhancer according to the present embodiment. 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, thickening stems or trunks, and increasing 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.

Note that plants include not only crops grown in fields, but also garden trees, flowers, lawns, and roadside trees, as well as forest trees that receive little or no fertilization. In other words, in this specification, crops are synonymous with plants as defined herein. Furthermore, improving quality means that the quality of the parts of a plant that are used (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.

Cyanobacteria (i.e., parent cyanobacteria) and modified cyanobacteria will be discussed later.

As described above, the secreted product contains a crop yield enhancer containing at least one of pyroglutamic acid and aminobenzoic acid. In other words, 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).

Crop yield-enhancing substances other than pyroglutamic acid and aminobenzoic acid include, for example, organic matter-degrading enzymes such as peptidases, nucleases, or 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 modified cyanobacterium may be a mixture of these crop yield-enhancing substances.

[2. Method for producing 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 a method for producing a crop yield improver according to the present embodiment.

The method for producing a crop yield enhancer according to this embodiment includes the steps of: preparing modified cyanobacteria (i.e., parent cyanobacteria) in which the function of a protein involved in binding between the outer membrane and the cell wall is suppressed or eliminated (step S01); and causing the modified cyanobacteria to secrete a secretory product containing the crop yield enhancer (step S02).

In step S01, the modified cyanobacteria are prepared. Preparing the modified cyanobacteria means 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 cyanobacteria to produce the modified cyanobacteria, restoring the modified cyanobacteria from a freeze-dried form or glycerol stock of the modified cyanobacteria, or recovering the modified cyanobacteria that have finished secreting a secretion product containing a crop yield enhancer in step S02.

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 lost, so proteins and metabolic products produced within the bacterial cell are more likely to be secreted outside the outer membrane (i.e., extracellularly). These intracellularly produced substances include a crop yield enhancer 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.

Cyanobacteria can generally be cultured using liquid culture or a modified method thereof using BG-11 medium (see Table 2). Therefore, modified cyanobacteria can also be cultured in a similar manner. Furthermore, the culture period for 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. Furthermore, the culture method may be, for example, aerated agitation culture or shaking culture.

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 include crop yield enhancers 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 bodies), and the culture supernatant may be recovered. According to the method for producing a crop yield enhancer of this embodiment, secretions containing intracellularly produced substances involved in crop yield improvement (i.e., crop yield enhancers) are secreted outside the cells of the modified cyanobacteria, eliminating the need to disrupt the cells to recover the crop yield enhancers. Therefore, the modified cyanobacteria remaining after recovery of the crop yield enhancers can be repeatedly used to produce the crop yield enhancer.

The method for recovering the crop yield enhancer secreted into the culture solution is not limited to the above example. The crop yield enhancer may be recovered from the culture solution while culturing the modified cyanobacteria. For example, a permeable membrane that allows proteins to pass through may be used, and the crop yield enhancer that has permeated the membrane may be recovered. In this way, the crop yield enhancer can be recovered from the culture solution while culturing the modified cyanobacteria, eliminating the need for a process to remove the modified cyanobacterial cells from the culture solution. This allows for easier and more efficient production of the crop yield enhancer.

In addition, by eliminating the need to recover and disrupt the bacterial cells from the culture medium, the damage and stress suffered by the modified cyanobacteria can be reduced. As a result, the productivity of the modified cyanobacteria in secreting crop yield-enhancing substances is less likely to decrease, allowing the modified cyanobacteria to be used for a longer period of time.

As described above, by using the modified cyanobacteria of this embodiment, crop yield enhancers can be obtained easily and efficiently.

The following explains cyanobacteria and modified cyanobacteria.

[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.

Figure 2 is a schematic diagram of the cell surface of a cyanobacterium. As shown in Figure 2, the cell surface of a cyanobacterium is composed of, from the inside out, the plasma membrane (also called the inner membrane 1), peptidoglycan 2, and the outer membrane 5, a lipid membrane that forms the outermost layer of the cell. Peptidoglycan 2 is covalently bound to glycan chains 3 composed of glucosamine, mannosamine, and the like, and these covalently bound glycan chains 3 are bound to pyruvate (Non-Patent Document 3: Jurgens and Weckesser, 1986, J. Bacteriol., 168:568-573). In this specification, the peptidoglycan 2 and the covalently bound glycan 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 are present that are involved in protein degradation or the formation of three-dimensional structures, lipid or nucleic acid degradation, and the uptake of extracellular nutrients.

SLH domain-containing outer membrane proteins (e.g., Slr1841 in the figure) consist of a C-terminal region embedded in the lipid membrane (also known as 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 pass through the outer membrane, while SLH domain 7 functions to bind to the cell wall 4 (Non-Patent Document 5: Kowata et al., 2017, J. Bacteriol., 199:e00371-17). For the SLH domain 7 to bind to the cell wall 4, the covalently linked glycan 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.

The enzyme that catalyzes the pyruvate modification of covalently linked glycan 3 in peptidoglycan 2 (hereafter 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 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.

In cyanobacteria, _CO2 fixed by photosynthesis is converted into precursors of various amino acids and intracellular molecules through multistep enzymatic reactions. These raw materials are used to synthesize proteins and metabolic products within the cytoplasm of cyanobacteria. Some of these proteins and metabolic products function within the cytoplasm, while others are transported from the cytoplasm to the periplasm where they function. However, no cases of cyanobacteria actively secreting proteins and metabolic products outside the cell have been reported to date.

Because cyanobacteria have a high photosynthetic capacity, they do not necessarily need to take in organic matter from the outside as nutrients. Therefore, cyanobacteria have very small amounts of 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 take up inorganic ions necessary for growth, cyanobacteria have many ion channel proteins in their outer membrane 5 that allow permeation of only inorganic ions, such as SLH domain-containing outer membrane protein 6 (e.g., Slr1841) in Figure 2. 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.

As such, cyanobacteria have very few channels in the outer membrane 5 that allow the passage of organic substances such as proteins, making it difficult to actively secrete proteins and metabolic products produced within the bacterial cell.

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

In the modified cyanobacteria of this embodiment, the function of a protein (hereinafter also referred to as a binding-associated protein) involved in the binding between the outer membrane 5 and the cell wall 4 in cyanobacteria is suppressed or eliminated. As a result, the binding between the outer membrane 5 and the cell wall 4 (e.g., the amount and strength of binding) is partially reduced in the modified cyanobacteria, making it easier for the outer membrane 5 to partially detach from the cell wall 4. This improves the secretion productivity of intracellularly produced substances, which secrete proteins and metabolic products produced intracellularly outside 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."

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. 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 amount and strength of binding) 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 areas where these bonds are weakened. When the outer membrane 5 is partially detached from the cell wall 4, substances produced within the modified cyanobacterium, particularly proteins and metabolic products present in the periplasm, are more likely to leak out of the cell (outside the outer membrane 5). This improves the secretion productivity of the modified cyanobacterium, which secretes crop yield-enhancing substances produced within the cell.

The following provides a more detailed description of a cyanobacterium that has 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.

The type of cyanobacterium (i.e., the 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 may be any type of cyanobacterium. For example, the parent cyanobacterium may be of the genus Synechocystis, Synechococcus, Anabaena, or Thermosynechococcus, and may be Synechocystis sp. PCC 6803, Synechococcus sp. PCC 7942, or Thermosynechococcus elongatus BP-1.

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 the chromosomal DNA or plasmids can be confirmed in the NCBI database and Cyanobase mentioned above.

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 a plasmid).

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. If the parent cyanobacterium is of the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc.; if the parent cyanobacterium is of the genus Calothrix, it may be A0A1Z4R6U0_9CYAN; if the parent cyanobacterium is of the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc.; if the parent cyanobacterium is of the genus Crocosphaera, it may be B1WRN6_CROS5, etc.; and if the parent cyanobacterium is of the genus Pleurocapsa, it may be K9TAE4_9CYAN, etc.

More specifically, the SLH domain-retaining 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. It may also be a protein whose amino acid sequence is 50% or more identical to these SLH domain-retaining outer membrane proteins 6.

As a result, in the modified cyanobacterium, for example, (i) the function of any of the SLH domain-retaining outer membrane proteins 6 set forth in SEQ ID NOs: 1 to 3 above or a protein having 50% or more identical amino acid sequence to any of these SLH domain-retaining outer membrane proteins 6 may be suppressed or lost, or (ii) the expression of any of the SLH domain-retaining outer membrane proteins 6 set forth in SEQ ID NOs: 1 to 3 above or a protein having 50% or more identical amino acid sequence to any 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 cyanobacteria, 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 the outer membrane 5 more likely to detach partially from the cell wall 4. As a result, in the modified cyanobacteria, substances produced within the cell are more likely to leak out of the cell, and therefore crop yield-improving substances produced within the cell are also more likely to leak out of the cell.

In general, if a protein's amino acid sequence is 30% or more identical, it is said that the protein has a high degree of homology in its three-dimensional structure and is therefore likely to have the same function as that protein. Therefore, an SLH domain-retaining outer membrane protein 6 whose function is suppressed or eliminated may be, for example, a protein or polypeptide consisting of 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 even more preferably 90% or more identical to the amino acid sequence of any of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOS: 1 to 3 above, and that has the function of binding to covalently bound glycans 3 on the cell wall 4.

Also, 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 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 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 if the parent cyanobacterium is of the genus Nostoc, it may be CsaB (NCBI access ID: WP_099068528.1), etc., and if the parent cyanobacterium is of the genus Crocosphaera, it may be CsaB (NCBI access ID: WP_012361697.1), etc., and if the parent cyanobacterium is of the genus Pleurocapsa, it may be CsaB (NCBI access ID: WP_036798735), etc.

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

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 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 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 glycan 3 on the surface of the cell wall 4 is less likely to be modified with pyruvate, thereby reducing the amount and strength of binding between the glycan 3 on the cell wall 4 and the SLH domain 7 of the SLH domain-containing outer membrane protein 6 in the outer membrane 5. Therefore, in the modified cyanobacterium according to this embodiment, the covalently bonded sugar chains 3 on the surface of the cell wall 4 are less likely to be modified with pyruvic acid, weakening the binding force between the cell wall 4 and the outer membrane 5 and making it easier for the outer membrane 5 to partially detach from the cell wall 4. As a result, in the modified cyanobacterium, substances produced within the cell are more likely to leak out of the cell, and therefore crop yield-improving substances produced within the cell are also more likely to leak out of the cell.

Furthermore, as mentioned above, if a protein's amino acid sequence is 30% or more identical, it is said to be highly 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 consisting of 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 even more preferably 90% or more identical to the amino acid sequence of any of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 above, and that has the function of catalyzing the reaction of modifying the covalently bonded glycan 3 of peptidoglycan 2 in the cell wall 4 with pyruvate.

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

In addition, inhibiting or eliminating the function of cell wall-pyruvate modifying enzyme 9 means inhibiting or eliminating the function of the protein to modify covalently bound glycans 3 on cell walls 4 with pyruvate.

Means for inhibiting or eliminating the function of these proteins are not particularly limited, as long as they are means commonly used for inhibiting or eliminating protein function. Such means may include, for example, deleting or inactivating the gene encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9, inhibiting the transcription of these genes, inhibiting the translation of the transcription products of these genes, or administering an inhibitor that specifically inhibits these proteins.

In this embodiment, the modified cyanobacterium has an outer membrane 5 and a cell wall 4. As a result, in the modified cyanobacterium, the expression of a protein involved in the binding between the cell wall 4 and the outer membrane 5 is suppressed, or the function of the protein is suppressed or lost, resulting in a partial reduction in the binding (i.e., the amount and strength of binding) between the cell wall 4 and the outer membrane 5. As a result, the outer membrane 5 is more likely to partially detach from the cell wall 4 in the modified cyanobacterium, making it easier for intracellularly produced substances such as proteins and metabolic products to leak out of the outer membrane 5, i.e., outside the cell. Therefore, the modified cyanobacterium has improved productivity in secreting crop yield-enhancing substances produced intracellularly, thereby eliminating the need for extraction processes for intracellularly produced substances, such as disrupting the cell cells, and therefore reducing the likelihood of a decrease in the physiological activity of the intracellularly produced substances and a decrease in yield. This reduces the risk of a decrease in the physiological activity of the crop yield improver produced within the fungal cells, as well as a decrease in yield, making it possible to produce 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 cyanobacteria can be used repeatedly to produce the crop yield improver even after the substance has been recovered.

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. As a result, 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 it easier for the outer membrane 5 to detach from the cell wall 4 in areas where the bond 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, making it easier for the outer membrane 5 to partially detach from the cell wall 4, which makes 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 bacterial cell of the modified cyanobacterium to leak out of the bacterial cell.

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

For example, the gene encoding 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.; if the parent cyanobacterium is of the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc.; if the parent cyanobacterium is of the genus Calothrix, it may be A0A1Z4R6U0_9CYAN, etc.; if the parent cyanobacterium is of the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc.; if the parent cyanobacterium is of the genus Crocosphaera, it may be B1WRN6_CROS5, etc.; and if 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.

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

As a result, in the modified cyanobacterium, a gene encoding any one of the SLH domain-containing outer membrane proteins 6 shown in SEQ ID NOs: 7 to 9, or a gene having 50% or more identity to the base 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 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 metabolic products 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.

As mentioned above, it is said that if a protein's amino acid sequence is 30% or more identical, it is likely to have the same function as that protein. Therefore, if the base sequence of a gene encoding a protein is 30% or more identical, it is likely that a protein with the same function as 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 base sequence that is 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more identical to the base sequence of any of the genes encoding the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOS: 7 to 9 above, and may be a gene encoding a protein or polypeptide that has the function of binding to the covalently bound glycan 3 of the cell wall 4.

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 if the parent cyanobacterium is of the genus Nostoc, it may be csaB (NCBI access ID: WP_099068528.1), etc., and if the parent cyanobacterium is of the genus Crocosphaera, it may be csaB (NCBI access ID: WP_012361697.1), etc., and if 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.

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. It may also be a gene with a nucleotide sequence that is 50% or more identical to these genes.

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 above, or a gene that is 50% or more identical to the base sequence of a gene encoding any one of these enzymes, is deleted or inactivated. As a result, in the modified cyanobacterium, (i) the expression of any one of the cell wall-pyruvate-modifying enzymes 9 above or a protein with 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 above or a protein with a function equivalent to any one of these enzymes is suppressed or lost. As a result, the covalently bonded glycan 3 on the surface of the cell wall 4 becomes less susceptible to modification with pyruvate, thereby reducing the amount and strength of binding between the glycan 3 on the cell wall 4 and 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 that bind the cell wall 4 to the outer membrane 5 that are modified with pyruvic acid is reduced, weakening the binding strength between the cell wall 4 and the outer membrane 5 and making it easier for the outer membrane 5 to partially detach from the cell wall 4. This makes it easier for proteins and metabolic products 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.

As mentioned above, if the base sequence of a gene encoding a protein is 30% or more identical, it is highly likely that a protein with equivalent function to that 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 consisting of a base sequence that is 40% or more, preferably 50% or more, more preferably 60% or more, even more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more identical to the base sequence of any of the genes encoding cell wall-pyruvate modifying enzyme 9 shown in SEQ ID NOs: 10 to 12 above, and that encodes a protein or polypeptide that has the function of catalyzing a reaction that modifies covalently bonded glycans 3 of peptidoglycan 2 in cell wall 4 with pyruvate.

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.

In this embodiment, the protein involved in 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.

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

Means for deleting or inactivating the gene may include, 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.

Methods for inhibiting transcription of the above-mentioned genes may include, 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). Specific methods for introducing the above-mentioned mutations or substituting or inserting a base sequence may be, for example, ultraviolet irradiation, site-specific mutagenesis, or homologous recombination.

In addition, the means for inhibiting translation of the transcription product of the above gene may be, for example, RNA (ribonucleic acid) interference.

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

As a result, in the modified cyanobacteria produced by the above 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 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 secretion productivity of crop yield-enhancing substances.

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 medium can then be recovered; therefore, the crop yield enhancer in the culture medium can be recovered while the modified cyanobacteria are being cultured. Therefore, the modified cyanobacteria obtained by this production method can be used to efficiently produce microbiological crop yield enhancers. Therefore, the method for producing modified cyanobacteria in this embodiment can provide highly efficient modified cyanobacteria that can be reused after the crop yield enhancer is recovered.

6. Methods for Improving Crop Yield
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.

The above-mentioned crop yield enhancer may be used as is, or after being concentrated or diluted. When applying the crop yield enhancer to crops, the concentration and application method may be determined appropriately depending on the type of crop, soil properties, and purpose. The crop yield enhancer may be, for example, the modified cyanobacterium culture medium 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. Furthermore, the crop yield enhancer may be applied to plants by, for example, spraying, irrigating, or mixing with the plant or soil. More specifically, a few milliliters per plant may be applied to the base of the plant approximately once a week.

The following examples provide a detailed explanation of the modified cyanobacteria, methods for producing the modified cyanobacteria, and methods for producing crop yield enhancers of the present disclosure, but the present disclosure is in no way limited to the following examples.

In the following examples, two types of modified cyanobacteria were produced by suppressing the expression of the slr1841 gene, which encodes an SLH domain-containing outer membrane protein (Example 1), and the slr0688 gene, which encodes a cell wall-pyruvate modifying enzyme (Example 2), as methods for partially detaching the outer membrane of cyanobacteria from the cell wall. 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 these examples is Synechocystis sp. PCC 6803 (hereinafter simply referred to as "cyanobacteria").

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.

(1) Construction of a cyanobacterial mutant strain with suppressed slr1841 gene expression. We used CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference to silence slr1841 gene expression 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 the cyanobacterium.

The mechanism by which gene expression is suppressed using this method is as follows:

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

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

Below, we will explain in detail how to introduce each of the two genes mentioned above into the chromosomal DNA of cyanobacteria.

(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.

1 μg of the resulting pUC19-dCas9 plasmid was mixed with cyanobacterial culture medium (cell concentration 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 had occurred between the psbA1 gene on the chromosomal DNA and the psbA1 upstream fragment region and psbA1 downstream fragment region on the pUC19-dCas9 plasmid. This resulted in a Synechocystis dCas9 strain with the dCas9 cassette inserted into the psbA1 gene region. The composition of the BG-11 medium used is shown in Table 2.

(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 called the protospacer on the sgRNA gene, allowing the sgRNA to specifically bind to the target gene. The protospacer sequence used in this example is shown in Table 3.

In the Synechocystis LY07 strain, the sgRNA gene (excluding the protospacer region) and the kanamycin resistance marker gene are linked and inserted into the slr2030-slr2031 gene on the chromosomal DNA. Therefore, 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.

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

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

The pUC19-slr1841_sgRNA plasmid was introduced into 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 strain (hereinafter also referred to as the slr1841-suppressed strain) in which slr1841_sgRNA was inserted into the slr2030-slr2031 gene on the chromosomal DNA.

(1-3) Suppression of the slr1841 gene The promoter sequences of the dCas9 gene and slr1841_sgRNA gene were designed so that their 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.

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.

(2) Construction of a cyanobacterial modified strain in which expression of the slr0688 gene is suppressed Using the same procedure as in (1-2) above, an sgRNA gene containing a protospacer sequence (SEQ ID NO: 22) complementary to the slr0688 gene (SEQ ID NO: 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) slr0688_sgRNA, (iii) a kanamycin resistance marker gene, and (iv) a 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.

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

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

Next, the strains obtained in Examples 1, 2, and Comparative Example 1 were subjected to observation of the cell surface state and protein secretion productivity tests. Details are explained below.

(3) Observation of the state of the cell surface of the bacterial strains 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.

(3-1) Culturing of Bacterial Strains The slr1841-suppressed strain of Example 1 was inoculated into BG-11 medium containing 1 μg/mL aTc to give an initial cell concentration (OD730) of 0.05, and cultured with shaking for 5 days under conditions of a light intensity of 100 μmol/ ^m2 /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.

(3-2) Preparation of Ultrathin Sections of 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 for two days at -80°C 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 a transmission electron microscope specimen 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.

(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.

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 area A in Figure 3. Figure 4(a) is an enlarged TEM image of the dashed-line area A in Figure 3, and Figure 4(b) is a depiction of the enlarged TEM image of Figure 4(a).

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

To confirm the state of the cell surface in more detail, the dashed line area A was magnified and observed. As shown in Figures 4(a) and 4(b), areas where the outer membrane had partially peeled off (dotted line areas a1 and a2 in the figures) were confirmed. Furthermore, a large area of the outer membrane was confirmed near the dashed line area a1. This area is where the bond between the outer membrane and the cell wall has weakened, and it is thought that the outer membrane expanded outward and bent as the culture medium penetrated through the outer membrane into the periplasm.

Next, we will explain the slr0688-suppressed strain of Example 2. Figure 5 is a TEM image of the slr0688-suppressed strain of Example 2. Figure 6 is an enlarged image of the dashed-line area B in Figure 5. Figure 6(a) is an enlarged TEM image of the dashed-line area B in Figure 5, and Figure 6(b) is a depiction of the enlarged TEM image of Figure 6(a).

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

To confirm the state of the cell surface in more detail, the dashed line area B was magnified and observed. As shown in Figures 6(a) and 6(b), a portion of the outer membrane that was significantly bent (dotted dashed line area b1 in the figure) and a portion where the outer membrane had partially peeled off (dotted dashed line areas b2 and b3 in the figure) were confirmed. Furthermore, portions where the outer membrane had detached from the cell wall were confirmed near each of the dashed dashed line areas b1, b2, and b3.

Next, we will explain the control strain of Comparative Example 1. Figure 7 is a TEM image of the control strain of Comparative Example 1. Figure 8 is an enlarged image of the dashed line area C in Figure 7. Figure 8(a) is an enlarged TEM image of the dashed line area C in Figure 7, and Figure 8(b) is a depiction of the enlarged TEM image of Figure 8(a).

As shown in Figure 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 stacked 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 bent, as was the case in Examples 1 and 2.

(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 cyanobacterium, and the mixture was stirred. After ultrafiltration, the mixture was subjected to measurement.

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

[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
[Aneion 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

(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 greater. 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 HMT (Human Metabolome Technologies, Inc.) metabolite library. 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 100 μM calibration limit. The major identified metabolites are listed in Table 4.

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 is partially detached from the cell wall, making it easier for intracellular metabolites, including crop yield-enhancing substances such as pyroglutamic acid and aminobenzoic acid, to leak outside the outer membrane (i.e., outside the fungus).

(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. 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.

(5-1) Culturing 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.

(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 resulting culture supernatant was filtered using a 0.22 μm pore-size membrane filter 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 content of the extracellular secreted protein of the slr1841-suppressed strain of Example 1 were calculated. Protein content of three cultures of the strains of Example 2 and Comparative Example 1 was also quantified under similar conditions, and the average and standard deviation of the protein content of the three cultures were calculated.

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

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 Comparative Example 1.

Although data is not shown here, the absorbance (730 nm) of the culture medium was measured and the amount of secreted protein per 1 g of dry cell weight (mg protein/g cell dry weight) was calculated. The amount of secreted protein per 1 g of dry cell weight (mg protein/g cell dry weight) was 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.

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 of the slr1841-suppressed strain of Example 1, because the amount and strength of binding between the outer membrane and cell wall were reduced more than in the slr1841-suppressed strain of Example 1.

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

(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. The method is described below.

(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. After adjusting the protein concentration to 1 μg/mL, dithiothreitol (DTT) was added to a final concentration of 10 mM and the mixture was incubated at 50°C for 30 minutes. Subsequently, iodoacetamide (IAA) was added to a final concentration of 30 mM and the mixture was 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 the mixture was incubated at room temperature for 10 minutes. 400 ng of trypsin was added and the mixture was incubated overnight at 37°C to obtain peptide fragments. 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.

(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.

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

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

Software: Scaffold DIA
Database: UniProtKB/Swiss Prot database (Synechocystis sp. PCC 6803)
Fragmentation: HCD
Precursor Tolerance: 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

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.

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 gap 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.

(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.

(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/ ^m2 /s from a white light source, 16 hours of light, and 8 hours of darkness, and the lettuce was cultivated at room temperature (22°C) for 35 days.

(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), or (3) a secretion product of the modified cyanobacteria (Example 5) was added to the hydroponic culture solution once a week in an amount equivalent to 5 mL per plant. After harvest, 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 in Example 1 and the slr0688-suppressed strain in Example 2.

(Comparative Example 2)
The same procedures as in Examples 3 to 5 were carried out except that water was used instead of the above (1) to (3). After harvesting, the weight of the plants was measured, and the average value and standard deviation (SD) were calculated.

(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. In addition, Figure 11 includes photographs of representative plants to visually show the condition of each plant in Examples 3 to 5 and Comparative Example 2.

As shown in Figure 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% higher than that in Comparative Example 2. The average weight of each lettuce head grown in Example 4 was approximately 15% higher than that in Comparative Example 2. The average weight of each lettuce head grown in Example 5 was approximately 29% higher than that in Comparative Example 2.

Furthermore, as shown in Figure 11, the lettuce plants grown in Examples 3 to 5 did not show any particularly noticeable changes in the overall shape of the plants compared to Comparative Example 2. In other words, although the lettuce plants grown 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, stem and leaf size were good, resulting in a well-balanced appearance.

(7-2) Spinach cultivation test Spinach seeds were sown in cultivation pots (12 cm x 10 cm) filled with commercially available potting soil. Cultivation was carried out for 35 days at 22°C, with a photon flux density of 200 μmol/m ² /s from a white light source, under 12 hours of light and 12 hours of darkness. 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 seeds were thinned out to ensure that the size of the plants in each pot was uniform.

(Examples 6, 7, and 8)
During the cultivation period, 5 mL of each of the following solutions was added to the base of spinach plants once a week: (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). After harvesting, the plants were weighed, and the mean and standard deviation (SD) were calculated for each of (1) to (3). The modified cyanobacteria were the slr1841-suppressed strain in Example 1 and the slr0688-suppressed strain in Example 2.

(Comparative Example 3)
The same procedures as in Examples 6 to 8 were carried out except that water was used instead of the above (1) to (3). After harvesting, the weight of the plants was measured, and the average value and standard deviation (SD) were calculated.

(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.

As shown in Figure 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 approximately 24% higher than that in Comparative Example 3. The average weight of each spinach stalk cultivated in Example 7 was approximately 25% higher than that in Comparative Example 3. The average weight of each spinach stalk cultivated in Example 8 was approximately 39% higher than that in Comparative Example 3.

Furthermore, as shown in Figure 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 leaf discoloration), and the leaves were thick and lush, as well as the stems and leaves were large, and the appearance was well-balanced.

(7-3) Tomato Cultivation Test. First, commercial potting soil was placed in cultivation planters (22 cm x 16 cm) and three tomato seeds were sown per planter. Cultivation was carried out for 150 days under conditions of 16 hours of light and 10 hours of darkness at a room temperature of 23°C and a photon flux density of 250 μmol/ ^m² /s from a white light source. During this period, each pot was watered with 500 mL of distilled water every two days. Approximately one week after the start of cultivation, the plants were thinned out when the cotyledons had developed to ensure uniform plant size in each planter. Additionally, 500 mL of a commercially available chemical fertilizer (a 500-fold dilution of the original solution containing 6% total nitrogen, 10% water-soluble phosphate, 5% water-soluble potassium, 0.05% water-soluble magnesium, 0.001% water-soluble manganese, and 0.005% water-soluble boron) was applied to each planter every 50 days.

Example 9
After adjusting the planter size as described above, 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 tomatoes 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.

(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.

(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.

Figure 17 also includes photographs of representative fruits to visually demonstrate the condition of the fruits in Example 9 and Comparative Example 4.

As shown in Figure 14, the average number of fruits harvested per plant in Example 9 increased by approximately 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 the same. Normally, the higher the fruit yield per plant, the lower the fruit weight tends to be. However, in Example 9, despite the increased number of fruits harvested, the average fruit weight was the same as in Comparative Example 4.

Furthermore, as shown in Figure 16, the average sugar content (Brix sugar content) of the fruit per plant harvested in Example 9 and Comparative Example 4 was also equivalent. Furthermore, as shown in Figure 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. Normally, 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 equivalent to those of Comparative Example 4.

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

(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.

The present disclosure provides a crop yield enhancer containing at least one of pyroglutamic acid and aminobenzoic acid. It also provides a modified cyanobacterium with improved productivity in secreting a crop yield enhancer. Furthermore, by culturing the modified cyanobacterium of the present disclosure, the above-described substance can be efficiently produced, and by adding the substance to soil or a hydroponic solution, for example, crop yield can be improved.

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

Claims (2)

including modified cyanobacterial secretions containing pyroglutamic acid and p-aminobenzoic acid;
Crop yield enhancer . The crop yield improver according to claim 1 is used on a crop.
Methods for improving crop yields.