JP2023182951A - Method for enhancing sugar content of tomato fruit, agent for enhancing sugar content of tomato fruit, and method for producing the same - Google Patents

Abstract

To provide a method that can enhance the sugar content of tomato fruits without reducing the number of harvested fruits.SOLUTION: A method for enhancing the sugar content of tomato fruits includes applying a secretion from modified cyanobacteria, which are cyanobacteria with inhibited or lost functions of proteins involved in the binding between the outer membrane and the cell wall, to tomatoes from the start of their cultivation until the harvest period of the first inflorescence.SELECTED DRAWING: Figure 1

Description

The present disclosure relates to a method for increasing the sugar content of tomato fruits, an agent for increasing the sugar content of tomato fruits, and a method for producing the same.

With the demand for increased food production as the world's population increases, there is a need for technological development to efficiently produce high-quality agricultural products on limited arable land. Specifically, we aim to shorten the cultivation period by increasing growth, increase yield per cultivation area, improve yield by reducing diseases and substandard products, and improve product quality by increasing fruit size and increasing sugar content. It is hoped that this will become a reality. Among these, tomatoes are produced worldwide for food regardless of region, and in recent years in particular, consumer demands for palatability such as high sugar content of tomato fruits have been increasing. Under these circumstances, there is a need to efficiently produce tomatoes with high sugar content.

For example, as a method for increasing the sugar content of tomato fruits, a method is known in which an aqueous solution containing vitamin C and methionine is applied to tomatoes by foliar spraying (Patent Document 1). Furthermore, for example, a method of increasing the sugar content of tomato fruits by genetic recombination is known (Non-Patent Document 1).

Japanese Patent Application Publication No. 5-170605

Sagor et al. “A novel strategy to produce sweeter tomato fruits with high sugar contents by fruit-specific expression of a single bZIP transcription factor gene”, Plant Biotechnology Journal, 2016, Vol. 14, 1116

However, in general, as the number of fruits per plant increases, the sugar content of tomato fruits tends to decrease, so there is a need for a method that can increase the sugar content of tomato fruits without reducing the number of tomato fruits harvested. .

Therefore, the present disclosure provides a method for increasing the sugar content of tomato fruits and an agent for increasing the sugar content of tomato fruits, which can increase the sugar content of tomato fruits without reducing the number of tomato fruits to be harvested. In addition, the present disclosure provides a method for producing a tomato fruit sugar content increasing agent that can easily and efficiently produce a tomato fruit sugar content increasing agent that can increase the sugar content of tomato fruits without reducing the number of tomato fruits to be harvested. I will provide a.

A method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure is a method for increasing the sugar content of tomato fruits using modified cyanobacteria in which the function of a protein involved in bonding between the outer membrane and the cell wall is suppressed or lost. The bacterial secretion is applied to the tomatoes from the start of tomato cultivation until the time of harvesting the first inflorescence.

According to the method for increasing the sugar content of tomato fruits and the agent for increasing the sugar content of tomato fruits of the present disclosure, it is possible to increase the sugar content of tomato fruits without reducing the yield of tomato fruits. According to the method for producing an agent for increasing the sugar content of tomato fruits of the present disclosure, it is possible to easily and efficiently produce an agent for increasing the sugar content of tomato fruits.

FIG. 1 is a flowchart illustrating an example of a method for producing a sugar content increasing agent for tomato fruits according to an embodiment. FIG. 2 is a diagram schematically showing the cell surface layer of cyanobacteria. FIG. 3 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Example 1. FIG. 4 is an enlarged image of the broken line 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 broken line area B in FIG. FIG. 7 is a transmission electron microscope image of an ultrathin section of the modified cyanobacteria of Comparative Example 1. FIG. 8 is an enlarged image of the broken line area C in FIG. FIG. 9 is a graph showing the amount of protein in the culture supernatants of the modified cyanobacteria of Example 1, Example 2, and Comparative Example 1 (n=3, error bar=SD).

(Findings that formed the basis of this disclosure)
As described in the background technology, there is a need for technology to efficiently produce high-quality agricultural products on limited arable land. In particular, there is a growing demand for tomatoes with a high sugar content, and there is a need for a technology that can increase the sugar content of tomato fruits without reducing the yield of tomatoes. Furthermore, in order to promote crop production, there is a demand for the use of naturally derived substances that have less environmental impact when applied. Among these, materials that consume less fossil energy during production and have less environmental impact are desired.

The following conventional techniques have been disclosed as techniques for improving the sugar content of tomato fruits.

For example, Patent Document 1 discloses that methionine, an amino acid that is a substrate for the production of ethylene, a plant hormone, and vitamin C, which reduces polyols in tomatoes to glucose and fructose, are applied to tomatoes by foliar spraying. A method for increasing the sugar content of tomatoes is disclosed. It has been reported that by using this method, it is possible not only to improve the sugar content of tomatoes but also to improve the disease resistance of tomatoes.

However, fossil fuels are consumed to produce methionine and vitamin C.

For example, Non-Patent Document 1 states that only the ORF of the bZIP transcription factor gene that does not contain the SIRT (sucrose-induced translational repression)-responsive uORF (upstream open reading frame) is under the control of the fruit-specific E8 promoter. A method for transforming tomato plants with a binary vector is disclosed. It has been reported that by using this method, it is possible to produce tomato fruits with sugar content approximately 1.5 times higher than non-genetically modified tomato fruits.

However, there are laws and regulations in countries around the world regarding genetically modified technology, and there is a psychological reluctance among producers and consumers to apply genetically modified technology to plants (hereinafter also referred to as applying or using). Therefore, there is a need for a method that can increase the sugar content of tomato fruits without using genetically modified plants.

Therefore, the present inventors have discovered a method that can produce a substance that is involved in improving the quality of tomato fruits (hereinafter referred to as a substance that improves the quality of tomato fruits) with a low environmental impact, using cheaper raw materials, and a simple process. .

Specifically, the present inventors focused on cyanobacteria as microorganisms used in the production of the substance. Cyanobacteria (also called cyanobacteria or blue-green algae) are a group of eubacteria that decompose water through photosynthesis to produce oxygen, and use the energy obtained to fix _CO2 in the air. Depending on the species, cyanobacteria can also fix nitrogen (N ₂ ) in the air. In this way, cyanobacteria can obtain most of the raw materials (i.e., nutrients) and energy necessary for bacterial growth from air, water, and light, so cyanobacteria can be grown using inexpensive raw materials and simple processes. Cyanobacteria can be cultured.

In addition, cyanobacteria are known to grow quickly and have high light utilization efficiency, and in addition, they are easier to genetically manipulate than other algal species, so cyanobacteria are the most popular photosynthetic microorganisms. Active research and development is being carried out regarding material production. For example, production of fuels such as ethanol, isobutanol, alkanes, and fatty acids (Patent Document 2: Japanese Patent No. 6341676) has been reported as an example of substance production using cyanobacteria. Research and development is also being conducted on the production of substances that serve as nutritional sources for living organisms. For example, since proteins can only be synthesized by living organisms, there is a need for the development of technology to easily and efficiently produce proteins. As one of the biological species used in this technology, the use of cyanobacteria, which can utilize light energy and atmospheric _CO2 , is expected to be utilized, and active research and development is being carried out (Non-patent Document 2: Jie Zhou et al. ., “Discovery of a super-strong promoter enable efficient production of heterologous proteins in cyanobacteria”, Scientific Reports, Nature Research, 2014, Vol.4, Article No.4500).

However, the proteins and intracellular metabolites produced within cyanobacterial cells are difficult to secrete outside the cells, so it is necessary to disrupt the cyanobacterial cells.

Therefore, by partially detaching the outer membrane covering the cyanobacterial cell wall from the cell wall, the present inventors succeeded in secreting proteins and intracellular metabolites produced within the cyanobacterial cell to the outside of the cell. We found that this makes it easier to The present inventors also discovered that even if the outer membrane is detached from the cell wall through genetic modification, the modified cyanobacteria can maintain life activity. Furthermore, the present inventors also discovered that the secretion of the modified cyanobacteria has the effect of increasing the sugar content of tomato fruits. As a result, by culturing the modified cyanobacteria, proteins produced within the bacterial cells and intracellular metabolites are leaked out of the bacterial cells. A substance with high sugar content of fruit can be produced. In addition, since operations such as extraction are not required, the physiological activity of the substance is less likely to be impaired, so the tomato fruit sugar content increasing agent containing the secretion can effectively increase the sugar content of tomato fruit. can be done. Furthermore, by applying secretions of modified cyanobacteria to tomatoes from the time when leaves grow to the time when fruits ripen, and not when the fruits ripen, the yield of tomato fruits can be reduced. It was also discovered that it is possible to increase the sugar content of tomato fruits without reducing the sugar content.

Therefore, according to the method for increasing the sugar content of tomato fruits and the agent for increasing the sugar content of tomato fruits of the present disclosure, it is possible to increase the sugar content of tomato fruits without reducing the yield of tomato fruits. According to the method for producing an agent for increasing the sugar content of tomato fruits of the present disclosure, it is possible to easily and efficiently produce an agent for increasing the sugar content of tomato fruits.

(Summary of this disclosure)
An overview of one aspect of the present disclosure is as follows.

A method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure includes cultivating tomatoes using the secreted material of a modified cyanobacterium in which the function of a protein involved in bonding the outer membrane and the cell wall is suppressed or lost in cyanobacteria. It is applied to the tomatoes from the time of harvest of the first inflorescence.

According to the method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, the secretion can be applied to tomatoes before the number of fruits set on tomato fruits begins to increase in earnest, that is, before the harvest period of the first inflorescence. , can promote the growth of tomato plants before tomato fruits start ripening. Therefore, according to the method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, it is possible to increase the sugar content of tomato fruits of tomato plants. Tomato fruits can be made to have a high sugar content without reducing the number of tomato fruits to be harvested.

For example, in the method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, the secretion may be applied to the tomatoes by foliar spraying.

According to the method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, nutrients necessary for photosynthesis can be directly absorbed into the leaves by spraying secretions of the modified cyanobacteria on the leaves. It is possible to effectively increase the sugar content of tomato fruits without reducing the number of fruits to be harvested.

For example, in the method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, the secretion may be applied to the tomatoes multiple times.

According to the method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, by applying the secretion of the modified cyanobacteria to tomatoes multiple times during the above period, nutrients necessary for photosynthesis are less likely to be insufficient. Photosynthesis occurs stably. Therefore, according to the method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, it is possible to effectively increase the sugar content of tomato fruits without reducing the number of tomato fruits to be harvested.

For example, in the method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, the secretion may be applied to the tomatoes four or more times.

According to the method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, by applying the secretion of the modified cyanobacteria to tomatoes four or more times during the above period, nutrients necessary for photosynthesis are less likely to become insufficient. , photosynthesis takes place stably. Therefore, according to the method for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, it is possible to effectively increase the sugar content of tomato fruits without reducing the number of tomato fruits to be harvested.

Furthermore, the agent for increasing the sugar content of tomato fruits according to one aspect of the present disclosure includes a secreted product of a modified cyanobacterium in which the function of a protein involved in bonding the outer membrane and the cell wall in cyanobacteria is suppressed or lost.

As a result, in the modified cyanobacteria, the bond between the cell wall and the outer membrane (that is, the amount of bond and binding force) is partially reduced, so that the outer membrane becomes partially detached from the cell wall. Therefore, in the modified cyanobacteria, proteins and metabolites produced within the bacterial body (that is, substances produced within the bacterial body) tend to leak out of the outer membrane (that is, outside the bacterial body). This makes it easier for the modified cyanobacteria to secrete the proteins and metabolites produced within the cells to the outside of the cells, thereby eliminating the need for extraction of substances produced within the cells, such as crushing the cells. Therefore, it is possible to easily and efficiently produce an agent for increasing the sugar content of tomato fruits containing secreted secretions of modified cyanobacteria. Furthermore, since the above-mentioned extraction process for intracellularly produced substances is not necessary, a decrease in the physiological activity and yield of the intracellularly produced substances is less likely to occur. Therefore, among the substances produced within the cells of the modified cyanobacteria, a decrease in the physiological activity of substances involved in increasing the sugar content of tomato fruits (hereinafter also referred to as substances that increase the sugar content of tomato fruits) and a decrease in yield are less likely to occur. . Thereby, it is possible to obtain an agent for increasing the sugar content of tomato fruits that has an improved effect of increasing the sugar content of tomato fruits. Therefore, the agent for increasing the sugar content of tomato fruits according to one aspect of the present disclosure can effectively increase the sugar content of tomato fruits.

Further, a method for producing a sugar content increasing agent for tomato fruits according to one embodiment of the present disclosure includes preparing modified cyanobacteria in which the function of a protein involved in bonding the outer membrane and the cell wall is suppressed or lost. and causing the modified cyanobacteria to secrete a secretion that is involved in increasing the sugar content of tomato fruits.

As a result, in the modified cyanobacteria, the bond between the cell wall and the outer membrane (for example, the amount of bond and binding force) is partially reduced, so that the outer membrane becomes partially detached from the cell wall. Therefore, proteins and metabolites produced within the bacterial body (hereinafter also referred to as intracellular substances) tend to leak out of the outer membrane, that is, outside the bacterial body. This facilitates the secretion of proteins and metabolites produced within the cells of the modified cyanobacteria to the outside of the cells, thereby eliminating the need for extraction treatment for substances produced within the cells, such as crushing the cells. Therefore, it is possible to easily and efficiently produce an agent for increasing the sugar content of tomato fruits containing secreted secretions of modified cyanobacteria. Furthermore, since the above-mentioned extraction process for intracellularly produced substances is not necessary, a decrease in the physiological activity and yield of the intracellularly produced substances is less likely to occur. Therefore, among the substances produced within the cells of the modified cyanobacteria, a decrease in the physiological activity of substances involved in increasing the sugar content of tomato fruits (hereinafter also referred to as substances that increase the sugar content of tomato fruits) and a decrease in yield are less likely to occur. . As a result, the secretion of the modified cyanobacteria improves the effect of increasing the sophistication of tomato fruits (hereinafter also referred to as the effect of increasing the sugar content of tomato fruits). In addition, since the above-mentioned extraction process for intracellularly produced substances is not necessary, even after recovering the intracellularly produced substances secreted outside the bacterial cells, the modified cyanobacteria can be repeatedly used to produce intracellularly produced substances. I can do it. Therefore, there is no need to prepare a new modified cyanobacterium each time the tomato fruit sugar content increasing agent is produced. Therefore, according to the method for producing an agent for increasing the sugar content of tomato fruits according to one aspect of the present disclosure, an agent for increasing the sugar content of tomato fruits that has an improved effect of increasing the sugar content of tomato fruits can be easily and efficiently produced. can do.

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

Note that the embodiments described below are all inclusive or specific examples. The numerical values, materials, steps, order of steps, etc. shown in the following embodiments are merely examples, and do not limit the present disclosure. Further, among the constituent elements in the following embodiments, constituent elements that are not described in the independent claims indicating the most significant concept will be described as arbitrary constituent elements.

Further, each figure is not necessarily strictly illustrated. In each figure, substantially the same components are denoted by the same reference numerals, and overlapping explanations may be omitted or simplified.

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

Furthermore, in this specification, both a bacterial body and a cell represent one individual cyanobacterium.

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

[1. High sugar content agent for tomato fruits]
First, the agent for increasing the sugar content of tomato fruits according to the present embodiment will be explained. The agent for increasing the sugar content of tomato fruits includes secretions secreted by modified cyanobacteria that are involved in increasing the sugar content of tomato fruits. Modified cyanobacteria are cyanobacteria (hereinafter also referred to as parent cyanobacteria) in which the functions of proteins involved in binding the outer membrane and cell wall (hereinafter also referred to as binding-related proteins) are suppressed or lost, and are produced using genetic recombination technology. can be obtained by controlling the expression level of genes encoding binding-related proteins. Therefore, the secreted material contains proteins and metabolites produced within the cells of the modified cyanobacteria (hereinafter also referred to as intracellularly produced substances). Note that details of cyanobacteria (so-called parent cyanobacteria) and modified cyanobacteria will be described later.

The agent for increasing the sugar content of tomato fruits according to the present embodiment can, for example, increase the number of leaves, stems, buds, flowers, or fruits of tomato plants, thicken the stems or trunks, and increase the height of tomato plants. It has the effect of increasing the sugar content of tomato fruits (effect of increasing the sugar content of tomato fruits) without reducing the number of tomato fruits harvested due to the growth promoting effect. In addition, for example, the agent for increasing the sugar content of tomato fruits can have various effects related to the growth promotion of tomato plants, such as preventing the occurrence of diseases in tomato plants, improving the absorption rate of nutrients, or improving the intracellular physiology of tomato plants. It may also have effects such as improving activity. In other words, being involved in increasing the sugar content of tomato fruits means that it has the effect of increasing the sugar content of tomato fruits by promoting the growth of tomato plants without reducing the number of tomato fruits to be harvested. Further, the tomato plant growth promoting effect may include that the growth of tomato plants is promoted by the various effects related to the above-mentioned growth promotion of tomato plants. Thereby, the agent for increasing the sugar content of tomato fruits can increase the sugar content of tomato fruits without reducing the yield of tomato fruits.

Substances with high sugar content in tomato fruits include, for example, organic substance degrading enzymes such as peptidase, nuclease, or phosphatase, DNA metabolism-related substances such as adenosine or guanosine, and nucleic acids (e.g., DNA or RNA) such as p-aminobenzoic acid or spermidine. ) Intracellular molecules involved in promoting synthesis, ketone bodies such as 3-hydroxybutyric acid, or organic acids such as gluconic acid. The secretion of the modified cyanobacteria may be a mixture of these tomato fruit sugar-enhancing substances.

[2. Method for producing high sugar content agent for tomato fruits]
Next, a method for producing a sugar content increasing agent for tomato fruits according to the present embodiment will be described with reference to FIG. 1. FIG. 1 is a flowchart showing an example of a method for producing an agent for increasing sugar content of tomato fruits according to the present embodiment.

The method for producing a plant growth promoter according to the present embodiment involves preparing a modified cyanobacterium (that is, a parent cyanobacterium) in which the function of a protein involved in bonding the outer membrane and the cell wall is suppressed or lost. (Step S01); and a step (Step S02) of causing the modified cyanobacteria to secrete a secretion that is involved in increasing the sugar content of tomato fruits. As described above, the secreted material of the modified cyanobacteria includes proteins and metabolites produced within the cells of the modified cyanobacteria (that is, substances produced within the cells). These intracellularly produced substances include substances that are involved in increasing the sugar content of tomato plants (that is, substances that increase the sugar content of tomato fruits). The substance that increases the sugar content of tomato fruits has the effect of increasing the sugar content of tomato fruits (the effect of increasing the sugar content of tomato fruits) without reducing the number of tomato fruits to be harvested due to the growth promoting effect of tomato plants.

In step S01, the modified cyanobacteria described above is prepared. Preparing the modified cyanobacteria means adjusting the state of the modified cyanobacteria so that the modified cyanobacteria can secrete secretions. Preparing modified cyanobacteria may mean, for example, producing modified cyanobacteria by genetically modifying parent cyanobacteria, or by restoring the cells from freeze-dried cells or glycerol stock of modified cyanobacteria. Alternatively, the modified cyanobacteria that have finished secreting the substance increasing the sugar content of tomato fruits in step S02 may be collected.

In step S02, the modified cyanobacteria are caused to secrete secretions that are involved in increasing the sugar content of tomato fruits. As mentioned above, in the modified cyanobacteria, the functions of proteins involved in bonding the outer membrane and cell wall are suppressed or lost in cyanobacteria (i.e., parent cyanobacteria), so the outer membrane and cell wall detach. Cheap. Therefore, proteins and metabolites produced within the cells of the modified cyanobacteria are likely to be secreted outside the outer membrane (that is, outside the cells). These intracellularly produced substances include substances that are involved in increasing the sugar content of tomato fruits. Therefore, in step S02, by culturing the modified cyanobacteria under predetermined conditions, substances produced inside the bacteria that are involved in increasing the sugar content of tomato fruits are secreted outside the bacteria.

Cultivation of cyanobacteria can generally be carried out based on liquid culture using BG-11 medium (see Table 2) or a modified method thereof. Therefore, culturing of modified cyanobacteria may be carried out in the same manner. In addition, the cyanobacteria cultivation period for producing the sugar content increasing agent for tomato fruits may be any period that allows proteins and metabolites to accumulate at a high concentration under conditions where the bacterial cells sufficiently proliferate. For example, it may be for 1 to 3 days, or may be for 4 to 7 days. Moreover, the culture method may be, for example, aeration agitation culture or shaking culture.

By culturing under the above conditions, the modified cyanobacteria produce proteins and metabolites (that is, intracellularly produced substances) within the bacterial cells, and secrete the intracellularly produced substances into the culture solution. When recovering intracellular substances secreted into the culture solution, the culture solution is filtered or centrifuged to remove solids such as cells (i.e., bacterial bodies), and the culture supernatant is recovered. may be collected. According to the method for producing an agent for increasing the sugar content of tomato fruits according to the present embodiment, the secretion containing intracellularly produced substances involved in increasing the sugar content of tomato fruits (that is, substances that increase the sugar content of tomato fruits) is modified. Since it is secreted outside the cyanobacterial cells, there is no need to disrupt the cells to recover the substance. Therefore, the modified cyanobacteria remaining after collecting the tomato fruit sugar content increasing substance can be repeatedly used to produce a tomato fruit sugar content increasing agent.

Note that the method for recovering the plant growth promoting substance secreted into the culture solution is not limited to the above example, and the substance increasing the sugar content of tomato fruits may be recovered from the culture solution while culturing the modified cyanobacteria. . For example, by using a permeable membrane that allows proteins to pass through, the substance with high sugar content of tomato fruits that has passed through the permeable membrane may be recovered. In this way, it is possible to collect the high sugar content substances of tomato fruits in the culture solution while cultivating the modified cyanobacteria, so that there is no need to remove the cells of the modified cyanobacteria from the culture solution. Therefore, the plant growth promoter can be produced more easily and efficiently.

Moreover, since the process of recovering bacterial cells from the culture solution and the process of crushing bacterial cells is not necessary, damage and stress to the modified cyanobacteria can be reduced. Therefore, the productivity of the modified cyanobacteria in secreting substances that increase the sugar content of tomato fruits is less likely to decrease, and the modified cyanobacteria can be used for a longer period of time.

As described above, by using the modified cyanobacteria of the present embodiment, an agent for increasing the sugar content of tomato fruits can be obtained simply and efficiently.

Hereinafter, cyanobacteria (that is, parent cyanobacteria) and modified cyanobacteria will be explained.

[3. cyanobacteria]
Cyanobacteria, also called blue-green algae or cyanobacteria, are a group of prokaryotes that perform photosynthesis while collecting light energy with chlorophyll and electrolyzing water to generate oxygen. Cyanobacteria are highly diverse, and include, for example, unicellular species such as Synechocystis sp. PCC 6803 and filamentous multicellular species such as Anabaena sp. PCC 7120. Regarding the growing environment, there are thermophilic species such as Thermosynechococcus elongatus, marine species such as Synechococcus elongatus, and freshwater species such as Synechocystis. In addition, species such as Microcystis aeruginosa, which has gas vesicles and produces toxins, and Gloeobacter violaceus, which does not have thylakoid but has a protein called phycobilisome, which is a light-harvesting antenna on its plasma membrane, have unique characteristics. There are many species that can be mentioned.

FIG. 2 is a diagram schematically showing the cell surface layer of cyanobacteria. As shown in Figure 2, the cell surface layer of cyanobacteria is composed of, in order from the inside, a plasma membrane (also called inner membrane 1), peptidoglycan 2, and outer membrane 5, which is a lipid membrane that forms the outermost layer of the cell. Ru. Sugar chains 3 composed of glucosamine, mannosamine, etc. are covalently bonded to peptidoglycan 2, and pyruvate is bonded to these covalently bonded sugar chains 3 (Non-Patent Document 3: Jurgens and Weckesser, 1986, J. Bacteriol., 168:568-573). In this specification, the peptidoglycan 2 and the covalent sugar chain 3 are collectively referred to as a cell wall 4. In addition, the gap between the plasma membrane (that is, the inner membrane 1) and the outer membrane 5 is called the periplasm, and is used for protein decomposition or three-dimensional structure formation, lipid or nucleic acid decomposition, or the uptake of extracellular nutrients. There are various enzymes involved.

The SLH domain-retaining outer membrane protein (for example, Slr1841 in the figure) consists of a C-terminal region embedded in the lipid membrane (also referred to as outer membrane 5) and an N-terminal SLH domain 7 that protrudes from the lipid membrane. , is widely distributed in cyanobacteria and bacteria belonging to the Negativicutes class, which is 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 to allow the passage of hydrophilic substances through the outer membrane, while the SLH domain 7 has the function of binding to the cell wall 4 (non-transparent membrane 5). Patent Document 5: Kowata et al., 2017, J. Bacteriol., 199:e00371-17). In order for SLH domain 7 to bind to cell wall 4, covalent sugar chain 3 in peptidoglycan 2 needs to be modified with pyruvate (Non-patent Document 6: Kojima et al., 2016, J. Biol Chem., 291:20198-20209). Examples of genes encoding SLH domain-retaining outer membrane protein 6 include slr1841 or slr1908 held by Synechocystis sp. PCC 6803, or oprB held by Anabaena sp. 90.

The enzyme that catalyzes the pyruvate modification reaction of the covalent sugar chain 3 in peptidoglycan 2 (hereinafter referred to as cell wall-pyruvate modification 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 homologous proteins with amino acid sequence identity of 30% or more with CsaB. Examples include slr0688 held by Synechocystis sp. PCC 6803 or syn7502_03092 held by Synechococcus sp. 7502.

In cyanobacteria, CO ₂ fixed through photosynthesis is converted into precursors of various amino acids and intracellular molecules through multistep enzymatic reactions. Using these as raw materials, proteins and metabolites are synthesized within the cytoplasm of cyanobacteria. Some of these proteins and metabolites function within the cytoplasm, while others are transported from the cytoplasm to the periplasm and function within the periplasm. However, to date, no case of active secretion of proteins and metabolites outside the cell has been reported in cyanobacteria.

Cyanobacteria have a high photosynthetic ability, so they do not necessarily need to take in organic matter from the outside as nutrients. Therefore, cyanobacteria have very few channel proteins in their outer membrane 5 that allow organic substances to pass therethrough, such as the organic substance channel protein 8 (eg, Slr1270) in FIG. For example, in Synechocystis sp. PCC 6803, the organic substance channel protein 8 that allows organic substances to pass therethrough is present in only about 4% of the total protein content of the outer membrane 5. On the other hand, in order for cyanobacteria to take in inorganic ions necessary for growth into cells with high efficiency, only inorganic ions can be permeated by cyanobacteria, such as SLH domain-retaining outer membrane protein 6 (e.g., Slr1841) shown in Figure 2. The outer membrane 5 contains many ion channel proteins that cause For example, in Synechocystis sp. PCC 6803, ion channel proteins that permeate inorganic ions account for about 80% of the total protein content of the outer membrane 5.

As described above, in cyanobacteria, there are very few channels in the outer membrane 5 that allow organic substances such as proteins to permeate, so it is difficult to actively secrete proteins and metabolites produced inside the bacterium to the outside of the bacterium. It is considered.

[4. Modified cyanobacteria]
Next, the modified cyanobacteria in this embodiment will be explained with reference to FIG. 2.

In the modified cyanobacteria of this embodiment, the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 (so-called binding-related protein) is suppressed or lost. More specifically, for example, the modified cyanobacteria is such that the total amount of proteins involved in binding between the outer membrane 5 and the cell wall 4 (i.e., binding-related proteins) is lower than that in the parent strain (i.e., parent cyanobacteria). It is suppressed to 30% or more and 70% or less of the total amount of protein. For example, "the total amount of binding-related proteins is suppressed to 30% of the total amount of the protein in the parent strain" means that 70% of the total amount of the protein in the parent strain has been lost and 30% remains. It means that. As a result, in the modified cyanobacteria, the bond between the outer membrane 5 and the cell wall 4 (for example, the amount of bond and the binding force) is partially reduced, so that the outer membrane 5 is easily partially detached from the cell wall 4. Therefore, the modified cyanobacteria has improved secretion productivity of intracellularly produced substances, such as proteins and metabolites produced within the microbial cells, to the outside of the microbial cells. As described above, the intracellularly produced substances include intracellularly produced substances that are involved in increasing the sugar content of tomato fruits (that is, substances that increase the sugar content of tomato fruits). Therefore, the modified cyanobacteria also improves the secretion productivity of the substance that increases the sugar content of tomato fruits, which is secreted outside the bacterial cells. Furthermore, since there is no need to crush the bacterial cells to recover the substance increasing the sugar content of tomato fruits, the modified cyanobacteria can be used repeatedly even after producing the substance increasing the sugar content of tomato fruits. In this specification, production means that the modified cyanobacteria produce proteins and metabolites within the bacterial cells, and secretion production means that the produced proteins and metabolites are secreted outside the bacterial cells.

The protein involved in the binding between the outer membrane 5 and the cell wall 4 may be, for example, at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9. In the present embodiment, the modified cyanobacterium has, for example, the function of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 suppressed or lost. For example, in the modified cyanobacteria, (i) at least one function of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 may be suppressed or lost, and (ii) the SLH domain that binds to the cell wall 4 may be suppressed or lost. At least one of the expression of the retained outer membrane protein 6 and the expression of an enzyme that catalyzes the pyruvate modification reaction of sugar chains bound to the surface of the cell wall 4 (that is, the cell wall-pyruvate modification enzyme 9) may be suppressed. . This reduces the bond (that is, the bond amount and binding force) between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalent sugar chain 3 on the surface of the cell wall 4. Therefore, the outer membrane 5 easily detaches from the cell wall 4 in areas where these bonds are weakened. By partially detaching the outer membrane 5 from the cell wall 4, substances produced within the cell, such as proteins and metabolites, present in the cell of the modified cyanobacterium, especially in the periplasm, exit the cell (outside the outer membrane 5). It becomes easier to leak. This improves the secretion productivity of the modified cyanobacteria, which secretes the substance that increases the sugar content of tomato fruits produced within the bacterial cells to the outside of the bacterial cells.

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

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

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

Note that the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvic acid modifying enzyme 9, whose functions are suppressed or lost in the modified cyanobacteria according to the present embodiment, can be used in any of the parent cyanobacteria as long as they are possessed by the parent cyanobacteria. They may be of cyanobacteria, and are not limited by the location of the genes encoding them (for example, on chromosomal DNA or on plasmids).

For example, the SLH domain-retaining outer membrane protein 6 may be Slr1841, Slr1908, or Slr0042 when the parent cyanobacterium belongs to the genus Synechocystis, or may be NIES970_09470 when the parent cyanobacterium belongs to the genus Synechococcus. Often, when the parent cyanobacteria is of the genus Anabaena, it may be Anacy_5815 or Anacy_3458, etc. When the parent cyanobacteria is of the genus Microcystis, it may be A0A0F6U6F8_MICAE, etc. When the parent cyanobacteria is of the genus Cyanothece, it may be A0A3B8XX12_9CYAN, etc. When the parent cyanobacterium belongs to the genus Leptolyngbya, it may be A0A1Q8ZE23_9CYAN, etc. When the parent cyanobacterium belongs to the genus Calothrix, it may be A0A1Z4R6U0_9CYAN, and when the parent cyanobacterium belongs to the genus Nostoc, it may be A0A1C0VG86_9NOSO, etc. When the parent cyanobacterium belongs to the genus Crocosphaera, it may be B1WRN6_CROS5, and when the parent cyanobacterium belongs to the genus Pleurocapsa, it may be K9TAE4_9CYAN.

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

As a result, in the modified cyanobacteria, for example, (i) any of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 above or any of these SLH domain-retaining outer membrane proteins 6 and amino acids; The function of the protein whose sequence is 50% or more identical may be suppressed or lost, and (ii) any of the SLH domain-retaining outer membrane proteins 6 shown in SEQ ID NOs: 1 to 3 above, or any of these. The expression of a protein whose amino acid sequence is 50% or more identical to SLH domain-retaining outer membrane protein 6 may be suppressed. Therefore, in the modified cyanobacteria, (i) the function of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 or a protein having a function equivalent to the SLH domain-retaining outer membrane protein 6 is suppressed or lost; (ii) The expression level of the SLH domain-retaining outer membrane protein 6 or a protein having the same function as the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 is reduced. As a result, in the modified cyanobacteria, the binding domain (for example, SLH domain 7) for binding the outer membrane 5 to the cell wall 4 decreases the binding amount and binding force with the cell wall 4, so that the outer membrane 5 binds to the cell wall 4. Partial detachment becomes easier. As a result, in the modified cyanobacteria, substances produced inside the bacteria tend to leak out of the bacteria, and therefore plant growth promoting substances produced inside the bacteria also tend to leak out of the bacteria.

Generally, it is said that if the amino acid sequences of a protein are 30% or more identical, the protein has a high degree of homology in its three-dimensional structure and is therefore likely to have the same function as the protein. Therefore, as the SLH domain-retaining outer membrane protein 6 whose function is suppressed or lost, for example, 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 40% It consists of an amino acid sequence having an identity of preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, even more preferably 90% or more, and It may be a protein or polypeptide that has the function of binding to the covalent sugar chain 3.

Furthermore, for example, the cell wall-pyruvate modifying enzyme 9 may be Slr0688, etc. when the parent cyanobacterium belongs to the genus Synechocystis, and may be Syn7502_03092 or Synpcc7942_1529, etc. when the parent cyanobacterium belongs to the genus Synechococcus. If the cyanobacterium belongs to the genus Anabaena, it may be ANA_C20348 or Anacy_1623, and if the parent cyanobacterium belongs to the genus Microcystis, it may be CsaB (NCBI access ID: TRU80220), or if the parent cyanobacterium belongs to the genus Cyanothece. If the parent cyanobacterium belongs to the genus Spirulina, it may be CsaB (NCBI access ID: WP_026079530.1), etc. If the parent cyanobacterium belongs to the genus Calothrix, it may be CsaB (NCBI access ID: WP_096658142.1), etc., and if the parent cyanobacterium belongs to the genus Nostoc, it may be CsaB (NCBI access ID: WP_099068528.1), etc. , if the parent cyanobacterium belongs to the genus Crocosphaera, it may be CsaB (NCBI access ID: WP_012361697.1), and if the parent cyanobacterium belongs to the genus Pleurocapsa, it may be CsaB (NCBI access ID: WP_036798735), etc. Good too.

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

As a result, in the modified cyanobacteria, for example, (i) any of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOs: 4 to 6 above or any of these cell wall-pyruvate modifying enzymes 9 and the amino acid sequence The function of proteins that are 50% or more identical may be suppressed or lost, and (ii) any of the cell walls shown in SEQ ID NOs: 4 to 6 above - pyruvate modifying enzyme 9 or the cell wall of any of these - Expression of a protein whose amino acid sequence is 50% or more identical to pyruvate modifying enzyme 9 may be suppressed. Therefore, in the modified cyanobacteria, (i) the function of the cell wall-pyruvate modifying enzyme 9 or a protein having an equivalent function to the enzyme is suppressed or lost, or (ii) the function of the cell wall-pyruvate modifying enzyme 9 or the relevant enzyme is suppressed or lost. The expression level of proteins with functions equivalent to enzymes decreases. This makes it difficult for the covalent sugar chains 3 on the surface of the cell wall 4 to be modified with pyruvate, so that the sugar chains 3 on the cell wall 4 interact with the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. The amount of binding and the binding strength are reduced. Therefore, in the modified cyanobacteria according to the present embodiment, the covalently bonded sugar chains 3 on the surface of the cell wall 4 are difficult to be modified with pyruvate, so the binding force between the cell wall 4 and the outer membrane 5 is weakened, and the outer membrane 5 becomes easily partially detached from the cell wall 4. As a result, in the modified cyanobacteria, the substances produced inside the bacteria are likely to leak out of the cells, so that the high sugar content substances of tomato fruits produced inside the bacteria are also likely to leak out of the cells.

Moreover, as mentioned above, if the amino acid sequences of a protein are 30% or more identical, it is said that the protein is likely to have the same function as the protein. Therefore, the cell wall-pyruvate modifying enzyme 9 whose function is suppressed or lost is, for example, an amino acid sequence of any of the cell wall-pyruvate modifying enzymes 9 shown in SEQ ID NOS: 4 to 6 above, and 40% or more of the amino acid sequence, The peptidoglycan of the cell wall 4 preferably consists of an amino acid sequence having an identity of 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, still more preferably 90% or more, and It may be a protein or polypeptide that has a function of catalyzing a reaction that modifies the covalently bonded sugar chain 3 of 2 with pyruvate.

Note that in this specification, suppressing or losing the function of the SLH domain-retaining outer membrane protein 6 means suppressing or losing the ability of the protein to bind to the cell wall 4, and refers to suppressing or losing the ability of the protein to bind to the outer membrane 5. or the ability of the protein to become embedded in the outer membrane 5 and function.

Note that suppressing or losing the function of the cell wall-pyruvic acid modifying enzyme 9 means suppressing or losing the function of the protein to modify the covalent sugar chain 3 of the cell wall 4 with pyruvate.

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

In the present embodiment, the modified cyanobacterium has a gene that expresses a protein involved in binding the outer membrane 5 and the cell wall 4 deleted or inactivated. As a result, in the modified cyanobacteria, the expression of proteins involved in binding between the cell wall 4 and outer membrane 5 is suppressed, or the function of the protein is suppressed or lost, so that the cell wall 4 and outer membrane 5 are bonded. The binding (that is, the binding amount and binding strength) is partially reduced. As a result, in the modified cyanobacteria, the outer membrane 5 tends to be partially detached from the cell wall 4. Therefore, in the modified cyanobacteria, intracellularly produced substances such as proteins and metabolites produced inside the microbial cell are removed from the outer membrane 5. It becomes easier to leak outside, that is, outside the bacterial body. Therefore, the modified cyanobacteria improves the secretion productivity of the substance that increases the sugar content of tomato fruits, which is secreted outside the bacterial cells. This eliminates the need for extraction treatment of intracellularly produced substances, such as crushing the bacterial cells, and thus reduces the possibility of a decrease in the physiological activity of the intracellularly produced substances and a decrease in yield. Therefore, a decrease in the physiological activity and a decrease in yield of the tomato fruit sugar content-increasing substances produced within the bacterial cells are less likely to occur, so we manufacture an agent for increasing the sugar content in tomato fruits that has an improved effect on increasing the sugar content in tomato fruits. can do. In addition, since the above-mentioned extraction treatment for intracellularly produced substances is not necessary, even after the substances are collected, the modified cyanobacteria can be repeatedly used to produce substances with high sugar content in tomato fruits.

The gene that expresses the protein involved in the binding between the outer membrane 5 and the cell wall 4 is, for example, at least one of the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9. There may be. In the modified cyanobacteria, at least one of the genes encoding SLH domain-retaining outer membrane protein 6 and the gene encoding cell wall-pyruvate modifying enzyme 9 has been deleted or inactivated. Therefore, in the modified cyanobacteria, for example, (i) the expression of at least one of the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9 is suppressed, or (ii) the SLH domain-retaining outer membrane protein At least one function of cell wall-pyruvate modifying enzyme 6 and cell wall-pyruvate modifying enzyme 9 is suppressed or lost. Therefore, the bond between the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5 and the covalent sugar chain 3 on the surface of the cell wall 4 (that is, the bond amount and binding force) is reduced. This makes it easier for the outer membrane 5 to detach from the cell wall 4 at the portion where the bond between the outer membrane 5 and the cell wall 4 is weakened. As a result, in the modified cyanobacteria, the binding between the outer membrane 5 and the cell wall 4 is reduced, making it easier for the outer membrane 5 to partially detach from the cell wall 4, so that proteins and metabolites produced within the bacterium are transferred to the bacteria. Easily leaks out of the body. As a result, the tomato fruit high sugar content substances produced within the cells of the modified cyanobacteria also tend to leak out of the cells.

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

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

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

As a result, in the modified cyanobacteria, the gene encoding any of the SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 7 to 9 above, or the base sequence of any of these genes, is 50% or more identical. Genes are deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above-mentioned SLH domain-retaining outer membrane protein 6 or a protein having a function equivalent to any of these proteins is suppressed, or (ii) the above-mentioned The function of any of the SLH domain-retaining outer membrane protein 6 or a protein having a function equivalent to any of these proteins is suppressed or lost. As a result, in the modified cyanobacteria, the binding domain (for example, SLH domain 7) for binding the outer membrane 5 to the cell wall 4 decreases the binding amount and binding force with the cell wall 4, so that the outer membrane 5 is separated from the cell wall 4. Parts become easier to disengage. As a result, the proteins and metabolites produced within the microbial cells tend to leak out of the microbial cells, so that the high sugar content substances of tomato fruits produced within the microbial cells also tend to leak out of the microbial cells.

As mentioned above, if the amino acid sequences of a protein are 30% or more identical, it is said that the protein is likely to have the same function as the protein. Therefore, if the base sequences of genes encoding proteins are 30% or more identical, it is considered that there is a high possibility that a protein having the same function as the protein will be expressed. Therefore, as a gene encoding the SLH domain-retaining outer membrane protein 6 whose function is suppressed or lost, for example, any of the genes encoding the SLH domain-retaining outer membrane protein 6 shown in SEQ ID NOs: 7 to 9 above. A base having an identity of 40% or more, preferably 50% or more, more preferably 60% or more, still more preferably 70% or more, even more preferably 80% or more, still more preferably 90% or more, with the above base sequence. It may also be a gene that encodes a protein or polypeptide that has the function of binding to the covalent sugar chain 3 of the cell wall 4.

Furthermore, for example, the gene encoding cell wall-pyruvate modifying enzyme 9 may be slr0688, etc. when the parent cyanobacterium belongs to the genus Synechocystis, and may be syn7502_03092 or synpcc7942_1529, etc. when the parent cyanobacterium belongs to the genus Synechococcus. If the parent cyanobacterium belongs to the genus Anabaena, it may be ana_C20348 or anacy_1623, and if the parent cyanobacterium belongs to the genus Microcystis, it may be csaB (NCBI access ID: TRU80220), etc. If the parent cyanobacterium belongs to the genus Cyanothece, it may be csaB (NCBI access ID: WP_107667006.1), etc., and if the parent cyanobacterium belongs to the genus Spirulina, it may be csaB (NCBI access ID: WP_026079530.1), etc. , if the parent cyanobacteria belongs to the genus Calothrix, it may be csaB (NCBI access ID: WP_096658142.1), etc., and if the parent cyanobacteria belongs to the genus Nostoc, it may be csaB (NCBI access ID: WP_099068528.1), etc. If the parent cyanobacterium belongs to the genus Crocosphaera, it may be csaB (NCBI access ID: WP_012361697.1), and if the parent cyanobacterium belongs to the genus Pleurocapsa, it may be csaB (NCBI access ID: WP_036798735). etc. may be used. The base sequences of these genes can be obtained from the NCBI database or Cyanobase mentioned above.

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

As a result, in the modified cyanobacteria, the base sequence of the gene encoding any of the cell wall-pyruvic acid modifying enzymes 9 shown in SEQ ID NOs: 10 to 12 above or the gene encoding any of these enzymes is 50% or more. Genes that are identical are deleted or inactivated. Therefore, in the modified cyanobacteria, (i) the expression of any of the above cell wall-pyruvate modifying enzymes 9 or a protein having a function equivalent to any of these enzymes is suppressed, or (ii) the above-mentioned The function of any cell wall-pyruvate modifying enzyme 9 or a protein having a function equivalent to any of these enzymes is suppressed or lost. This makes it difficult for the covalent sugar chains 3 on the surface of the cell wall 4 to be modified with pyruvate, so that the sugar chains 3 on the cell wall 4 interact with the SLH domain 7 of the SLH domain-retaining outer membrane protein 6 in the outer membrane 5. The amount of binding and the binding strength are reduced. Therefore, in the modified cyanobacteria according to the present embodiment, the amount of modification of sugar chains 3 with pyruvate for bonding the cell wall 4 to the outer membrane 5 is reduced, so that the binding strength between the cell wall 4 and the outer membrane 5 is reduced. is weakened, and the outer membrane 5 becomes easily partially detached from the cell wall 4. As a result, the proteins and metabolites produced within the microbial cells tend to leak out of the microbial cells, so that the high sugar content substances of tomato fruits produced within the microbial cells also tend to leak out of the microbial cells.

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

[5. Production method of modified cyanobacteria]
Next, a method for producing modified cyanobacteria in this embodiment will be explained. The method for producing a modified cyanobacterium includes the step of suppressing or losing the function of a protein involved in binding 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 the SLH domain-retaining outer membrane protein 6 and the cell wall-pyruvate modifying enzyme 9.

Means for suppressing or losing protein function include, but are not particularly limited to, deletion or deletion of the gene encoding the SLH domain-retaining outer membrane protein 6 and the gene encoding the cell wall-pyruvate modifying enzyme 9. Whether it is by inactivating these genes, inhibiting the transcription of these genes, inhibiting the translation of the transcripts of these genes, or administering inhibitors that specifically inhibit these proteins. good.

Means for deleting or inactivating the above gene include, for example, introducing a mutation into one or more bases on the base sequence of the gene, replacing the base sequence with another base sequence, or replacing the base sequence with another base sequence. It may be insertion, or deletion of part or all of the base sequence of the gene.

Means for inhibiting the transcription of the above genes include, for example, introducing mutations into the promoter region of the gene, inactivating the promoter by substituting or inserting other base sequences, or CRISPR interference (non-transfer). Patent Document 8: Yao et al., ACS Synth. Biol., 2016, 5:207-212), etc. may be used. Specific methods for the above-mentioned mutagenesis or base sequence substitution or insertion may be, for example, ultraviolet irradiation, site-specific mutagenesis, or homologous recombination.

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

By using any of the above means, a modified cyanobacterium may be produced by suppressing or losing the function of a protein involved in binding between the outer membrane 5 and the cell wall 4 in 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 (that is, the bond amount and binding force) is partially reduced, so that the outer membrane 5 is partially separated from the cell wall 4. It becomes easier to detach. As a result, in the modified cyanobacteria, intracellular substances such as proteins and metabolites produced within the microbial cell tend to leak out of the outer membrane 5 (in other words, outside the microbial cell), which increases the productivity of tomato fruits. Substances that are involved in increasing sugar content (that is, substances that increase sugar content in tomato fruits) also tend to leak out of the bacterial cells. Therefore, according to the method for producing a modified cyanobacterium in this embodiment, it is possible to provide a modified cyanobacterium with improved secretion productivity of a substance increasing the sugar content of tomato fruits.

In addition, in the modified cyanobacteria produced by the production method in this embodiment, the substance that increases the sugar content of tomato fruits produced within the bacterial body leaks out of the bacterial body, so the bacterial body is removed to recover the substance. No need to crush. For example, it is sufficient to cultivate the modified cyanobacteria under appropriate conditions and then recover the substances secreted into the culture solution that increase the sugar content of tomato fruits. It is also possible to recover saccharified substances. Therefore, by using the modified cyanobacteria obtained by the present production method, it is possible to efficiently produce a substance with high sugar content of tomato fruits microbiologically. Therefore, according to the method for producing modified cyanobacteria in the present embodiment, it is possible to provide modified cyanobacteria with high utilization efficiency that can be repeatedly used even after the high sugar content substance of tomato fruits is collected.

[6. Method for increasing sugar content of tomato fruits]
In the method for increasing the sugar content of tomato fruits according to the present embodiment, the secreted product of the modified cyanobacteria described above is applied to tomatoes from the start of tomato cultivation until the time of harvesting the first inflorescence. In addition, in the method for increasing the sugar content of tomato fruits, the secretion may be regularly sprayed on the leaves. Specifically, the secretion may be applied to tomatoes by foliar spraying four or more times between the start of tomato cultivation and the harvesting period of the first inflorescence. As mentioned above, the secretion of the modified cyanobacteria contains substances that are involved in increasing the sugar content of tomato fruits, and since the secretion is collected without disrupting the cells of the modified cyanobacteria, the secretion of the modified cyanobacteria is The physiological activity and yield of the substances involved in increasing the sugar content of tomato fruits are not likely to be reduced. Therefore, the agent for increasing the sugar content of tomato fruits containing the secretion can effectively increase the sugar content of tomato fruits.

The above-mentioned tomato fruit sugar content increasing agent may be used as it is, or may be used after being concentrated or diluted. When applying the tomato fruit sugar content increasing agent to tomato plants, the concentration and application method of the tomato fruit high sugar content increasing agent should be adjusted as appropriate depending on the plant type, soil properties, purpose, etc. You may decide. The agent for increasing the sugar content of tomato fruits may be, for example, the culture solution of the modified cyanobacteria itself, or may be a solution (for example, a culture supernatant) obtained by removing the cells of the modified cyanobacteria from the culture solution. It may also be an extract obtained by extracting a desired substance from the culture solution using membrane technology or the like. The desired substance may be an enzyme that decomposes nutrients in the soil, or a substance that solubilizes insoluble substances (e.g., metals such as iron) in the soil (e.g., a substance with a chelating effect). It may also be a substance that improves the intracellular physiological activity of tomato plants. In addition, the method for applying the sugar content increasing agent for tomato fruits to plants may be, for example, spraying on plants or soil, irrigation, foliar spraying, or mixing with soil or hydroponic solution. More specifically, several milliliters per individual plant may be applied to the base or leaves of the plant once a week or once every two weeks.

Hereinafter, the method for increasing the sugar content of tomato fruits, the agent for increasing the sugar content of tomato fruits, and the manufacturing method thereof of the present disclosure will be specifically explained in Examples, but the present disclosure is not limited to the following Examples in any way. isn't it.

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

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

(1) Construction of a modified cyanobacterial strain in which slr1841 gene expression is suppressed CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) interference method was used as a method for suppressing gene expression. In this method, the expression of the slr1841 gene is suppressed by introducing the gene encoding the dCas9 protein (hereinafter referred to as the dCas9 gene) and the slr1841_sgRNA (single-guide Ribonucleic Acid) gene into the chromosomal DNA of cyanobacteria. I can do it.

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

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

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

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

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

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

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

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

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

Next, by using the mixed solution of the above DNA fragments as a template and using the primers slr2030-Fw (SEQ ID NO: 15) and slr2031-Rv (SEQ ID NO: 18) listed in Table 1 by PCR method, ( A DNA fragment (slr2030-2031::slr1841_sgRNA) was obtained in which i) slr2030 gene fragment, (ii) slr1841_sgRNA, (iii) kanamycin resistance marker gene, and (iv) slr2031 gene fragment were 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 in the same manner as in (1-1) above, and the transformed cells were selected on a BG-11 agar medium containing 30 μg/mL kanamycin. As a result, a transformant Synechocystis dCas9 slr1841_sgRNA strain (hereinafter also referred to as slr1841 suppressed strain) in which slr1841_sgRNA was inserted into the slr2030-slr2031 gene on the chromosomal DNA was obtained.

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

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

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

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

Subsequently, the bacterial strains obtained in Example 1, Example 2, and Comparative Example 1 were subjected to observation of cell surface conditions and protein secretion productivity tests, respectively. The details will be explained below.

(3) Observation of cell surface state of bacterial strains The modified cyanobacterium Synechocystis dCas9 slr1841_sgRNA strain obtained in Example 1 (that is, the slr1841 suppressed strain), the modified cyanobacterium Synechocystis dCas9 slr0688_sgRNA strain obtained in Example 2 (hereinafter referred to as slr0688 suppressed strain) and the modified cyanobacterium Synechocystis dCas9 strain obtained in Comparative Example 1 (hereinafter referred to as Control strain). and adventitial structure) were observed.

(3-1) Cultivation of the strain The slr1841 suppressed strain from Example 1 was inoculated into BG-11 medium containing 1 μg/mL aTc so that the initial cell concentration OD730 = 0.05, and the light intensity was 100 μmol/m ² /s. The cells were cultured with shaking at 30°C for 5 days. Note that 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 the bacterial strain The culture solution obtained in (3-1) above was centrifuged at 2,500g at room temperature for 10 minutes to collect cells of the slr1841 suppressed strain of Example 1. . Cells were then quickly frozen in liquid propane at -175°C, and then fixed at -80°C for 2 days using an ethanol solution containing 2% glutaraldehyde and 1% tannic acid. The fixed cells were dehydrated with ethanol, the dehydrated cells were permeated with propylene oxide, and then submerged in a resin (Quetol-651) solution. Thereafter, the resin was 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 create ultrathin sections. This ultrathin section was stained with a 2% uranium acetate and 1% lead citrate solution to prepare a transmission electron microscopy sample of the slr1841 suppressed strain of Example 1. Note that the same operation was performed for the slr0688 suppressed strain of Example 2 and the Control strain of Comparative Example 1, respectively, to prepare samples for transmission electron microscopy.

(3-3) Observation using an electron microscope The ultrathin section obtained in (3-2) above was 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 explained. FIG. 3 is a TEM (Transmission Electron Microscope) image of the slr1841 suppressed strain of Example 1. FIG. 4 is an enlarged image of the broken line area A in FIG. FIG. 4(a) is an enlarged TEM image of the broken line area A in FIG. 3, and FIG. 4(b) is a diagram depicting the enlarged TEM image of FIG. 4(a).

As shown in Figure 3, in the slr1841 suppressed strain of Example 1, the outer membrane was partially peeled off from the cell wall (that is, the outer membrane was partially peeled off), and the outer membrane was partially bent. there was.

In order to confirm the condition of the cell surface layer in more detail, we enlarged the area A shown by the broken line and found that the outer membrane had partially peeled off, as shown in Figures 4(a) and 4(b). The parts (dotted line areas a1 and a2 in the figure) could be confirmed. Further, a portion where the adventitia was largely bent was confirmed near the dotted line area a1. This region is a region where the bond between the outer membrane and the cell wall is weakened, and it is thought that the culture solution permeated from the outer membrane into the periplasm, causing the outer membrane to swell outward and become bent.

Next, the slr0688 suppressed strain of Example 2 will be explained. FIG. 5 is a TEM image of the slr0688 suppressed strain of Example 2. FIG. 6 is an enlarged image of the broken line area B in FIG. 6(a) is an enlarged TEM image of the broken line area B in FIG. 5, and FIG. 6(b) is a diagram depicting the enlarged TEM image of FIG. 6(a).

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

In order to confirm the state of the cell surface layer in more detail, we enlarged the broken line area B and found that the outer membrane was greatly bent ( The dotted line area b1 in the figure and the parts where the outer membrane had partially peeled off (dot and dot line areas b2 and b3 in the figure) were confirmed. In addition, parts where the outer membrane was detached from the cell wall were confirmed near each of the dotted line areas b1, b2, and b3.

Next, the Control strain of Comparative Example 1 will be explained. FIG. 7 is a TEM image of the Control strain of Comparative Example 1. FIG. 8 is an enlarged image of the broken line area C in FIG. FIG. 8(a) is an enlarged TEM image of the broken line area C in FIG. 7, and FIG. 8(b) is a diagram depicting the enlarged TEM image of FIG. 8(a).

As shown in FIG. 7, the cell surface layer of the Control strain of Comparative Example 1 was well-organized, and the inner membrane, cell wall, outer membrane, and S layer remained laminated in this order. In other words, in the Control strain, as in Examples 1 and 2, the parts where the outer membrane detached from the cell wall, the parts where the outer membrane peeled off from the cell wall (that is, the parts fell off), and the parts where the outer membrane bent were I couldn't see it.

(4) Protein secretion productivity test The slr1841 suppressed strain of Example 1, the slr0688 suppressed strain of Example 2, and the Control strain of Comparative Example 1 were cultured, and the amount of protein secreted outside the cells (hereinafter referred to as secreted (also referred to as protein amount) was measured. The protein secretion productivity of each of the above bacterial strains was evaluated based on the amount of protein in the culture solution. Note that protein secretion productivity refers to the ability to produce proteins by secreting proteins produced within the cells to the outside of the cells. A specific method will be explained below.

(4-1) Culture of strain The slr1841 suppressed strain of Example 1 was cultured in the same manner as in (3-1) above. Culturing was performed independently three times. The strains of Example 2 and Comparative Example 1 were also cultured under the same conditions as the strains of Example 1.

(4-2) Quantification of extracellularly secreted proteins The culture solution obtained in (4-1) above was centrifuged at 2,500 g for 10 minutes at room temperature to obtain a culture supernatant. The obtained culture supernatant was filtered using a membrane filter with a pore size of 0.22 μm to completely remove the cells of the slr1841 suppressed strain of Example 1. The total amount of protein contained in the culture supernatant after filtration was quantified by the BCA (Bicinchoninic Acid) method. This series of operations was performed for each of the three independently cultured cultures, and the average value and standard deviation of the amount of protein secreted extracellularly of the slr1841 suppressed strain of Example 1 was determined. In addition, for the strains of Example 2 and Comparative Example 1, the protein in the three culture solutions was quantified under the same conditions, and the average value and standard deviation of the protein amounts in the three culture solutions were determined.

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

As shown in FIG. 9, the amount of protein secreted into the culture supernatant (mg/ L) was improved by about 25 times.

Although the data are omitted, the absorbance (730 nm) of the culture solution was measured and the amount of secreted protein per 1 g of bacterial cell dry weight (mg protein/g cell dry weight) was calculated. In both of the slr0688 suppressed strain of Example 2, the amount of secreted protein per gram of bacterial cell dry weight (mg protein/g cell dry weight) was approximately 36 times higher than that of the Control strain of Comparative Example 1. Ta.

Furthermore, as shown in FIG. 9, the gene encoding the cell wall-pyruvate modifying enzyme ( The slr0688 suppressed strain of Example 2, in which the expression of slr0688) was suppressed, had a higher amount of protein secreted into the culture supernatant. This is thought to be related to the fact that the number of covalently bonded sugar chains 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, in the slr0688 suppressed strain of Example 2, the binding amount and binding strength between the outer membrane and the cell wall were lower than in the slr1841 suppressed strain of Example 1, so the amount of secreted protein was lower than that of the slr1841 suppressed strain of Example 1. It is thought that the amount exceeded that of stocks.

From the above results, by suppressing the function of proteins related to the bond between the outer membrane and the cell wall, the bond between the outer membrane and the cell wall of cyanobacteria is partially weakened, and the outer membrane partially detaches from the cell wall. I was able to confirm that it was released. It was also confirmed that by weakening the bond between the outer membrane and the cell wall, proteins produced within the cyanobacterial cell can more easily leak out of the cell. Therefore, it was shown that the modified cyanobacteria and the method for producing the same according to the present embodiment significantly improve protein secretion productivity.

(5) Identification of secreted proteins Next, secreted proteins contained in the culture supernatant obtained in (4-2) above were identified by LC-MS/MS. The method will be explained below.

(5-1) Sample preparation Add 8 times the amount of cold acetone to the culture supernatant, leave it at 20°C for 2 hours, and centrifuge at 20,000g for 15 minutes to obtain a protein precipitate. . 100mM Tris pH8.5, 0.5% sodium dodecanoate (SDoD) was added to this precipitate, and the protein was dissolved using a closed ultrasonicator. After adjusting the protein concentration to 1 μg/mL, dithiothreitol (DTT) with a final concentration of 10 mM was added and left at 50° C. for 30 minutes. Subsequently, iodoacetamide (IAA) was added at a final concentration of 30 mM, and the mixture was allowed to stand at room temperature (shielded from light) for 30 minutes. To stop the IAA reaction, cysteine was added at a final concentration of 60 mM, and the mixture was allowed to stand at room temperature for 10 minutes. 400 ng of trypsin was added and left standing at 37°C overnight to fragment the protein into peptide fragments. After adding 5% TFA (Trifluoroacetic Acid), centrifugation was performed at 15,000 g for 10 minutes at room temperature to obtain a supernatant. This work eliminated SDoD. After desalting using a C18 spin column, the sample was dried using a centrifugal evaporator. Thereafter, 3% acetonitrile and 0.1% formic acid were added, and the sample was dissolved using a closed ultrasonic crusher. The peptide concentration was adjusted to 200 ng/μL.

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

Sample injection amount: 200ng
Column: CAPCELL CORE MP 75μm×250mm
Solvent: A solvent is 0.1% formic acid aqueous solution, B solvent is 0.1% formic acid + 80% acetonitrile Gradient program: B solvent 8% after 4 minutes of sample injection, B solvent 44% after 27 minutes, B solvent 80% after 28 minutes, 34 Measurement ends after minutes

(5-3) Data analysis The obtained data were analyzed under the following conditions, and proteins and peptides were identified and quantitative values were calculated.

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

Among the 30 proteins that had the highest relative quantitative values among the identified proteins, those predicted to have clear enzymatic activity are shown in Table 4.

All of these six types of proteins were contained in the culture supernatants of the slr1841 suppressed strain of Example 1 and the slr0688 suppressed strain of Example 2, respectively. All of these proteins retained periplasmic (referring to the space between the outer and inner membrane) translocation signals. This result shows that in the modified strains of Examples 1 and 2, the outer membrane partially detaches from the cell wall, making it easier for proteins in the periplasm to leak out of the outer membrane (i.e., outside the bacterial cell). This was confirmed. Therefore, it was shown that the modified cyanobacterium according to the present embodiment has significantly improved protein secretion productivity.

(6) Identification of secreted intracellular metabolites (6-1) Sample preparation To 80 μl of culture supernatant of modified cyanobacteria, add 20 μl of an aqueous solution with an internal standard substance concentration adjusted to 1,000 μM and stir. After ultrafiltration, it was subjected to measurement.

(6-2) CE (Capillary Electrophoresis)-TOFMS (Time-Of-Flight Mass Spectrometry) Analysis In this test, measurements in cation mode and anion mode were performed under the conditions shown below.

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

(6-3) Data processing
For peaks detected by CE-TOFMS, peaks with a signal/noise ratio of 3 or more were automatically detected using automatic integration software MasterHands (registered trademark) ver. 2.17.1.11. Based on the mass-to-charge ratio (m/z) and electrophoresis time values unique to each metabolite, all detected peaks registered in the HMT (Human Metabolome Technologies, Inc.) metabolite library are analyzed. Metabolites contained in the culture supernatant of the modified cyanobacteria were searched by comparing the substance values. The tolerance for the search was +/-0.5 min for electrophoresis time and +/-10 ppm for m/z. Concentrations were calculated for each identified metabolite as a single point calibration of 100 μM. The major metabolites identified are shown in Table 5.

All of these 12 types of intracellular metabolites were contained in the culture supernatants of the slr1841 suppressed strain of Example 1 and the slr0688 suppressed strain of Example 2, respectively. Although data is not shown, the culture supernatant of the Control strain in Comparative Example 1 did not contain these metabolites. This result shows that in the modified strains of Examples 1 and 2, the outer membrane partially detaches from the cell wall, making it easier for intracellular metabolites to leak out of the outer membrane (i.e., outside the bacterial cell). This was confirmed.

(7) Tomato Cultivation Test Next, in order to evaluate the effect of the modified cyanobacteria secretion (here, the culture supernatant of the modified cyanobacteria) on increasing the sugar content of tomato fruits, the following tomato cultivation test was conducted. The modified cyanobacteria were the slr1841 suppressed strain of Example 1 and the slr0688 suppressed strain of Example 2, and each strain was cultured independently three times in the same manner as in (4-1) above. That is, in Examples 3 and 4, the culture supernatant of the modified cyanobacteria of Example 2 was used as an agent for increasing the sugar content of tomato fruits.

(7-1) Cultivation method In the tomato cultivation test, base fertilizer (nitrogen: 15.6 kg/10a, phosphoric acid: 11.1 kg/10a, potassium: 10.2 kg/10a) was applied in advance to cherry tomato seedlings 50 days after sowing. The plants were planted in soil in a plastic greenhouse with a plant spacing of 40 cm and a row spacing of 30 cm. After planting, management was carried out by removing all side buds, and when the plant grew taller than the 1.8 m tall support pipe, cultivation was continued by switching to diagonal induction. Additionally, additional fertilizer (139 g/m ² of 8-8-8 chemical fertilizer) was applied on the 76th day after sowing.

In addition, fruits were harvested twice a week once the fruits in the first inflorescence matured and began to change color, and the number of fruits harvested and the weight of the fruits were recorded. In addition, one out of every 10 fruits was randomly sampled and the sugar content (Brix value) was measured. Then, the cumulative number of fruits harvested per cherry tomato plant (referred to as the number of fruits), the average weight per fruit, and the average value and standard deviation (SD) of sugar content were determined (see Table 6).

(Example 3)
In Example 3, after planting, a tomato fruit high sugar content agent diluted 20 times with water was applied at a rate of 10 mL per cherry tomato plant by foliar spraying once every two weeks. Specifically, in Example 3, the application of the sugar content increasing agent to tomato fruits was carried out a total of four times from the planting of cherry tomato seedlings (in other words, the start of cherry tomato cultivation) to the harvest period of the first inflorescence.

(Example 4)
In Example 4, after planting, 5 mL of an undiluted tomato fruit sugar content increasing agent per cherry tomato plant was applied by irrigation to the plant base once every two weeks. Specifically, in Example 4, the sugar content increasing agent was applied to tomato fruits four times in total from the planting of cherry tomato seedlings (that is, the start of cherry tomato cultivation) to the harvest period of the first inflorescence. In Example 4, insect damage occurred in the cultivation plots from around 120 days after sowing, so Table 6 shows the results at 122 days after sowing.

(Comparative example 2)
Comparative Example 2 was carried out in the same manner as in Example 3, except that water was used instead of the agent for increasing the sugar content of tomato fruits. This is a very common cultivation method (in other words, a conventional method) for cultivating cherry tomatoes.

(Comparative example 3)
In Comparative Example 3, 5 mL of a tomato fruit high sugar content agent that is not diluted with water is regularly irrigated at the base of the plant once every two weeks until the end of fruit harvest (151 days after sowing). It was applied accordingly. Note that the fruit harvest end date is the end date of the third inflorescence harvest period.

(7-2) Results As shown in Table 6, the average number of fruits per cherry tomato plant cultivated and harvested using the method of Example 3 was higher than that of cherry tomatoes grown using the method of Comparative Example 2 (conventional method). Compared to cherry tomatoes, the sugar content increased by about 22%, and the average sugar content increased by about 6%. Note that the average weight per fruit of the cherry tomatoes grown by the method of Example 3 and the cherry tomatoes grown by the method of Comparative Example 2 was almost the same.

Furthermore, when comparing Example 3 and Comparative Example 3, the average number of fruits per cherry tomato plant grown using the method of Example 3 was about the same as that of cherry tomatoes grown using the method of Comparative Example 3. However, the average sugar content increased by nearly 10%. On the other hand, the average weight per cherry tomato grown by the method of Comparative Example 3 was nearly 10% higher than that of the cherry tomatoes grown by the method of Example 3, but the average sugar content was lower. In addition, in the method of Comparative Example 3, the tomato fruit high sugar content agent is regularly irrigated at the base of the plant once every two weeks during the entire cultivation season, and the application period and application location are different from Example 2. .

In addition, Example 4 differs from Example 3 (foliar spraying) in that the agent for increasing the sugar content of tomato fruits is irrigated at the base of the plant, but when comparing the results 122 days after sowing, it is found that The average number of fruits was about the same as the cherry tomatoes grown using the method of Example 3, and the average weight and sugar content per fruit were also about the same, so almost the same effect as Example 3 was observed. It was confirmed that In addition, in Example 4, when compared with the results of Comparative Example 1 at 122 days after sowing, the average number of fruits per cherry tomato plant was increased by 33.0%.

From the above results, by cultivating cherry tomatoes using the methods of Examples 3 and 4, the number of tomato fruits harvested (number of fruits) increased compared to cherry tomatoes grown using conventional methods (Comparative Example 2). It was confirmed that this method can increase the sugar content of tomato fruits without reducing the average weight per fruit. In addition, by cultivating cherry tomatoes using the methods of Examples 3 and 4, it was possible to reduce the number of tomato fruits harvested compared to when the application was applied throughout the cultivation period without dividing the application period (Comparative Example 3). It was confirmed that it increases the sugar content of fruits.

In addition, in the method for increasing the sugar content of tomato fruits, the agent for increasing the sugar content of tomato fruits of the present disclosure can be applied more easily by foliar spraying in Example 3 than by irrigation in Example 4, and therefore, the agent for increasing the sugar content of tomato fruits of the present disclosure can be applied more efficiently. It is possible to increase the sugar content of tomato fruits.

According to the present disclosure, by culturing modified cyanobacteria, it is possible to easily and efficiently produce an agent for increasing the sugar content of tomato fruits, and the agent for increasing the sugar content of tomato fruits can be produced during the harvest period of the first inflorescence. By applying up to 100% of tomato fruit, it is possible to improve the sugar content of tomato fruits without reducing the number of tomato fruits to be harvested.

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

Claims (6)

Applying a secreted product of a modified cyanobacterium in which the function of a protein involved in bonding the outer membrane and the cell wall in cyanobacteria is suppressed or lost to the tomato from the start of tomato cultivation until the time of harvesting the first inflorescence.
A method for increasing the sugar content of tomato fruits. applying the secretion to the tomatoes by foliar spraying;
The method for increasing the sugar content of tomato fruits according to claim 1. applying the secretion to the tomatoes multiple times;
The method for increasing the sugar content of tomato fruits according to claim 1 or 2. applying the secretion to the tomatoes four or more times;
The method for increasing the sugar content of tomato fruits according to claim 3. Contains the secreted material of modified cyanobacteria in which the function of a protein involved in binding the outer membrane and cell wall in cyanobacteria is suppressed or lost.
Agent for increasing sugar content of tomato fruits. preparing a modified cyanobacterium in which the function of a protein involved in bonding the outer membrane and the cell wall in cyanobacteria is suppressed or lost;
causing the modified cyanobacteria to secrete a secretion that is involved in increasing the sugar content of tomato fruits;
including,
A method for producing a sugar content increasing agent for tomato fruits.

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