Method for managing production of plant growth promoting substance, method for producing plant growth promoting substance, apparatus for managing production of plant growth promoting substance, and program

Abstract

A method for managing the production of plant growth promoting substance according to the present disclosure includes determining the degree of progress in production of a plant growth promoting substance on the basis of the activity value of lipopolysaccharide contained in a cyanobacterial culture supernatant. The degree of progress is determined, for example, on the basis of time-series data of the activity value of lipopolysaccharide from the start of cyanobacterial culture. The degree of progress may also be determined in accordance with the rate of change of the activity value calculated from the time-series data.

Description

Plant growth promoting substance production management method, plant growth promoting substance production management device, and program

[0001] The present disclosure relates to a production management method for a plant growth-promoting substance, a production method for a plant growth-promoting substance, a production management device for a plant growth-promoting substance, and a program.

[0002] Conventionally, methods for producing substances using photosynthetic microorganisms such as cyanobacteria and algae have been known. It is also known that the productivity of such substances by photosynthetic microorganisms can be improved by using genetically modified strains in which genes involved in the metabolism or biosynthesis of the substances of the photosynthetic microorganisms have been modified.

[0003] For example, Non-Patent Documents 1 to 5 describe that the productivity of substances such as sucrose, isobutanol, fatty acids, amino acids, and proteins can be improved by genetically modified strains.

[0004] Patent Document 1 describes a method for producing a plant growth promoter, which includes a step of causing a modified cyanobacterium to secrete a secretion involved in promoting plant growth.

[0005] International Publication No. 2021 / 132110 International Publication No. 2021 / 173163

[0006] Ducat et al., 2012, Applied and Environmental Microbiology, 2660Atsumi et al., 2009, Nature Biotechnology, Nature Publishing Group, 27: 1177Liu et al., 2011, PNAS, 108: 6899Deshpande et al., 2020, Applied Environmental Microbiology, 86: e02816Gregory et al., 2013, Applied and Environmental Microbiology, 79: 3917 Yu Hirose et al., Photosynthesis research method, Low temperature science, Volume 67, Institute of Low Temperature Science, Hokkaido University, 2008, pp. 9 - 15, ISSN: 1880 - 7593, http: / / hdl.handle.net / 2115 / 39084Ungerer et al., 2016, Scientific Reports, 6: 39681Kojima et al., 2016, Biosci. Biotech. Biochem., 80: 1954Kowata et al., 2017, J. Bacteriol., 199 : e00371-17Kojima et al., 2016, J. Biol. Chem., 291: 20198Mesnage et al., 2000, EMBO J., 19: 4473Heidorn et al., 2011, Methods in Enzymology, 497: 539Briggs et al., 1990, Plant Molecular Biology, 15: 633Cheah et al., 2012, Biotechnol. Prog., 29: 23

[0007] The techniques described in the above documents have room for reexamination from the perspective of easily determining the progress of production of a plant growth-promoting substance in the production of a plant growth-promoting substance involving the cultivation of cyanobacteria. Therefore, the present disclosure provides a method for easily determining the progress of production of a plant growth-promoting substance in the production of a plant growth-promoting substance involving the cultivation of cyanobacteria.

[0008] The present disclosure provides a method for controlling the production of a plant growth-promoting substance, which includes determining the progress of production of the plant growth-promoting substance based on the activity value of lipopolysaccharide contained in a cyanobacterial culture supernatant.

[0009] According to the production control method of the present disclosure, the progress of production of a plant growth-promoting substance can be easily determined in the production of a plant growth-promoting substance involving the cultivation of cyanobacteria.

[0010] FIG. 1 is a flowchart showing an example of a method for controlling the production of a plant growth-promoting substance and a method for producing a plant growth-promoting substance according to the present embodiment. FIG. 2 is a schematic diagram showing an example of a production control device according to the present embodiment. FIG. 3 is a schematic diagram showing an example of vector DNA for gene modification. FIG. 4 is a flowchart showing an example of a method for determining the state of a cyanobacterial culture supernatant according to the present embodiment. FIG. 5 is a schematic diagram showing an example of a determination device according to the present embodiment. FIG. 6 is a diagram showing the results of electrophoresis of DNA fragments amplified from a modified cyanobacterium according to an example. FIG. 7 is a graph showing the relationship between the turbidity of a culture solution and the number of days of culture according to an example. FIG. 8 is a graph showing the relationship between the activity value of lipopolysaccharide in a culture supernatant and the number of days of culture according to an example. FIG. 9 is a graph showing the weight of the above-ground part of spinach to which a culture supernatant according to an example was applied. FIG. 10 is a graph showing the relationship between the turbidity of a culture solution and the number of days of culture. FIG. 11 is a graph showing the relationship between the activity value of lipopolysaccharide in a culture supernatant and the number of days of culture. Figure 12 is a graph showing the relationship between the relative weight of plants to which the culture supernatant was applied and the number of days of culture. Figure 13 is a graph showing the results of growth promotion in plants to which a solution of a polysaccharide fraction of the modified cyanobacterium was applied.

[0011] (Findings that form the basis of the present disclosure) Photosynthetic microorganisms, such as cyanobacteria and algae, are attracting attention as tools for realizing next-generation material production systems with low environmental impact. Material production by photosynthetic microorganisms is carried out in an environment at room temperature and atmospheric pressure, using light as an energy source, water, and carbon dioxide (CO2) from the air. Furthermore, recent advances in genetic engineering technology have made it possible to produce a wide range of chemical compounds using genetically modified photosynthetic microorganisms. For this reason, material production by photosynthetic microorganisms is expected to be a next-generation technology that can contribute to the realization of carbon neutrality.

[0012] For example, in the modified cyanobacteria used in the method for producing a plant growth promoter described in Patent Document 1, the function of a protein involved in binding between the outer membrane and the cell wall is suppressed or lost. In order to confirm whether a sufficient amount of plant growth promoter has been produced by the production method described in Patent Document 1, it is necessary to actually apply the plant growth promoter to a plant and observe the plant's growth process. This method is time-consuming and labor-intensive, and therefore cannot be said to be simple.

[0013] Therefore, the present inventors have conducted extensive research into a method for easily determining the progress of plant growth-promoting substance production in the production of plant growth-promoting substances involving the cultivation of cyanobacteria. As a result, they have newly discovered that the progress of plant growth-promoting substance production can be determined by focusing on a specific substance contained in the cyanobacterial culture supernatant. Based on this new finding, the present inventors have completed the production control method disclosed herein.

[0014] Furthermore, the present inventors have conducted extensive research into a method for easily determining whether a cyanobacterial culture supernatant is in a state capable of exerting the desired plant growth-promoting effect. As a result, they have newly discovered that by focusing on a specific substance contained in the cyanobacterial culture supernatant, it is possible to determine whether the cyanobacterial culture supernatant is in a state capable of exerting the desired plant growth-promoting effect. Based on this new finding, the present inventors have completed the determination method of the present disclosure.

[0015] (Embodiments of the Present Disclosure) Hereinafter, embodiments of the present disclosure will be described with reference to the drawings. Note that the embodiments described below are all comprehensive or specific examples. The numerical values, shapes, materials, components, component placement positions, connection configurations, process conditions, steps, and step order shown in the following embodiments are examples and are not intended to limit the present disclosure. Furthermore, among the components in the following embodiments, components that are not described in the independent claims that represent the highest concept are described as optional components. Note that each figure is a schematic diagram and is not necessarily an exact illustration. In each figure, substantially identical components are assigned the same reference numerals, and duplicated descriptions may be omitted or simplified.

[0016] (Embodiment 1) Hereinafter, embodiment 1 will be specifically described with reference to Figures 1 to 3. In this specification, the homology between base sequences and amino acid sequences is calculated using the Basic Local Alignment Search Tool (BLAST) algorithm. Specifically, it is calculated by performing pairwise analysis using the BLAST program available on the website of the National Center for Biotechnology Information (NCBI). Information regarding the base sequences of genes and the amino acid sequences of proteins encoded by those genes is published, for example, on the above-mentioned NCBI website and can be referenced at any time.

[0017] 1 is a flowchart showing an example of a method for controlling the production of a plant growth-promoting substance and a method for producing a plant growth-promoting substance according to the present embodiment. As shown in FIG. 1, the method for controlling the production of a plant growth-promoting substance and the method for producing a plant growth-promoting substance are carried out by measuring the activity value V of lipopolysaccharide contained in the culture supernatant of a cyanobacterium. A Based on the progress of plant growth promoter production V P This allows the progress of the production of plant growth-promoting substances to be determined easily. According to the studies of the present inventors, the activity value V of lipopolysaccharides contained in the culture supernatant of cyanobacteria is A is the progress of the production of plant growth promoters V PIt was found that there is a high correlation between

[0018] As shown in FIG. 1, first, in step S101, the cultivation of cyanobacteria is started. As a result, plant growth-promoting substances are produced and accumulated in the cyanobacterial culture solution. Next, after a predetermined time has elapsed since the start of the cultivation of the cyanobacteria, the cyanobacterial culture supernatant is collected, and in step S102, the activity value V of the lipopolysaccharide contained in the cyanobacterial culture supernatant is measured. A The predetermined time is, for example, one day. The activity value V of lipopolysaccharide is A The method for measuring the activity value V of lipopolysaccharide is not limited to a specific method. For example, the activity value V of lipopolysaccharide can be measured by a colorimetric method. A is measured.

[0019] Next, in step S103, the activity value V of the lipopolysaccharide is A Based on the progress of plant growth promoter production V P is determined. A Progression V based on P The method for determining the activity value V is not limited to a specific method. A If is less than the predetermined value, the progress V P is determined to be "less than 50." The predetermined value is, for example, 100 to 200 endotoxin units (EU) / mL.

[0020] Progress V P is the lipopolysaccharide activity value V from the start of cyanobacterial culture. A In this case, the progress V P The reliability of the

[0021] Progress V P is, for example, the activity value V of the lipopolysaccharide. A The activation value V calculated from the time series data A In this case, the progress degree V P is likely to be more reliable.

[0022] For example, the activity value V Ais equal to or greater than a predetermined value, the lipopolysaccharide activity value V in the nth measurement (n is an integer of 2 or more) from the start of the cyanobacterial culture A_n The corresponding progress V P is the activity value V A rate of change (V A_n -V A_n-1 ) / V A_n-1 The smaller the value of V, the higher the probability of A_n-1 is the lipopolysaccharide activity value in the n-1th measurement from the start of cyanobacterial culture. P is the activity value V A is equal to or greater than a predetermined value, for example, (V A_n -V A_n-1 ) / V A_n-1 The values ​​are determined as shown in Table 1 below.

[0023]

[0024] Next, in step S104, the progress V P It is determined whether the progress level V is equal to or greater than the threshold value T0. For example, according to Table 1, P If the determination result in step S104 is negative, the process proceeds to step S108, where the progress degree V P is output, and the process returns to step S102. In detail, after a predetermined time has elapsed since the n-1th measurement, the activity value V of the lipopolysaccharide contained in the culture supernatant of the cyanobacteria is A The nth measurement is performed. The predetermined time is, for example, one day. P The progress V P The determination of the lipopolysaccharide activity value V A The measurements are taken at regular time intervals, for example, at daily intervals.

[0025] If the determination result in step S104 is affirmative, the process proceeds to step S105, and the determined progress degree V P Next, in step S106, the culture of the cyanobacteria is stopped. Next, in step S107, the culture supernatant of the cyanobacteria is collected, and the series of processes is completed. Progress V Pis equal to or greater than the threshold value T0, a sufficient amount of the plant growth-promoting substance has been produced, and the cyanobacterial culture supernatant can contain the desired amount of the plant growth-promoting substance. In this way, the plant growth-promoting substance can be produced.

[0026] The above-mentioned method for managing the production of a plant growth-promoting substance can be executed, for example, using a computer. Therefore, a program for causing a computer to execute the above-mentioned production management method can be provided. This program can be provided by a recording medium or via a telecommunications line.

[0027] 2 is a diagram showing a schematic diagram of an example of a production management device according to the present embodiment. As shown in FIG. 2, the production management device 1a includes, for example, an input device 10 and a calculation device 20. The input device 10 receives the activity value V of lipopolysaccharide contained in the culture supernatant of cyanobacteria. A The calculation device 20 receives the input of the measurement data of the lipopolysaccharide activity value V A Based on the progress of plant growth promoter production V P The production management device 1a stores, for example, the above program. The calculation device 20 determines the activity value V of lipopolysaccharide input by the input device 10. A Based on the measurement data, this program will P Determine.

[0028] The production management device 1a further includes, for example, a storage device 30 in which the program is stored. In the production management device 1a, the arithmetic device 20 is included in, for example, a central processing unit (CPU). The storage device 30 includes, for example, a main storage device and an auxiliary storage device.

[0029] 2, the production management device 1a further includes an output device 40. The output device 40 outputs the determined progress V P Examples of the output device 40 are a display and an indicator light.

[0030] According to the above production control method and production method, the progress of the production of plant growth-promoting substances in the culture of cyanobacteria is V. PBased on this, it can be determined whether or not to continue culturing the cyanobacteria, which tends to reduce the time and energy required to produce the plant growth-promoting substance.

[0031] Cyanobacteria, also known as blue-green algae or cyanobacteria, are a group of prokaryotic organisms that capture light energy using chlorophyll and use the energy to split water and generate oxygen through photosynthesis. Cyanobacteria are highly diverse, ranging from unicellular species such as Synechocystis sp. PCC6803 to multicellular, filamentous species such as Anabaena sp. PCC7120. Their habitats vary widely, from thermophilic species such as Thermosynechococcus elongatus to marine species such as Synechococcus elongatus and freshwater species such as Synechocystis. Many species also possess unique characteristics, such as species that possess gas vesicles and produce toxins, such as Microcystis aeruginosa, and Gloeobacter violaceus, which lacks thylakoids but instead possesses light-harvesting proteins called phycobilisomes in the plasma membrane.

[0032] The cyanobacteria may be, for example, an outer membrane-detached cyanobacterium. An outer membrane-detached cyanobacterium is a cyanobacterium obtained by artificially modifying the intracellular genomic DNA of a wild-type cyanobacterium (parent cyanobacterium) to mutate the phenotype of the parent cyanobacterium. The method for modifying the genomic DNA of the parent cyanobacterium is not limited to a specific method. Examples of such methods include genetic recombination, genome editing, and mutagenesis. For example, natural transformation-based genetic recombination (see Non-Patent Document 6) or genome editing using the CRISPR-Cpf1 system (see Non-Patent Document 7) may be used to modify the genomic DNA of a cyanobacterium.

[0033] An outer membrane-detached cyanobacterium is, for example, a cyanobacterium in which the function of a protein involved in binding between the outer membrane and the cell wall is suppressed or lost. In this case, the amount and strength of the bond between the cell wall and the outer membrane are partially reduced in the outer membrane-detached cyanobacterium, making it easier for the outer membrane to partially detach from the cell wall. As a result, in the outer membrane-detached cyanobacterium, intracellularly produced substances such as proteins and metabolites are more likely to leak out of the outer membrane, i.e., outside the cell. This makes it easier for proteins and metabolites produced inside the cell of the outer membrane-detached cyanobacterium to be secreted outside the cell. This eliminates the need for extraction processes for intracellularly produced substances, such as disrupting the cell. This reduces the physiological activity and yield of intracellularly produced substances, and also reduces the physiological activity and yield of substances that can contribute to the exertion of plant growth-promoting effects among intracellularly produced substances of outer membrane-detached cyanobacteria. As a result, the plant growth-promoting substances contained in the cyanobacterial culture supernatant can exert the desired plant growth-promoting effect.

[0034] The target site for genetic modification in the genomic DNA of outer membrane-detached cyanobacteria may be, for example, a gene encoding at least one of a Surface Layer Homology (SLH) domain-containing outer membrane protein or a cell wall-pyruvate-modifying enzyme, both of which are involved in binding between the outer membrane and the cell wall. Modifications that suppress the expression of such genes reduce the amount and strength of the bond between the SLH domain of the SLH domain-containing outer membrane protein in the outer membrane and the covalently bound glycans on the surface of the cell wall, facilitating partial detachment of the outer membrane from the cell wall, as described above. Examples of such genes include slr1841, slr0688, and slr1908 found in Synechocystis sp. PCC 6803, as well as corresponding genes found in cyanobacteria of the genera Synechococcus, Anabaena, and Nostoc. The SLH domain-containing outer membrane proteins encoded by the above-mentioned slr1841 and slr1908 consist of a C-terminal region embedded in the lipid membrane (also known as the outer membrane) and an N-terminal SLH domain protruding from the lipid membrane, and are widely distributed in cyanobacteria (see Non-Patent Document 8). The region embedded in the lipid membrane forms a channel that allows hydrophilic substances to pass through the outer membrane. Meanwhile, the SLH domain functions to bind to the cell wall (see Non-Patent Document 9). For the SLH domain to bind to the cell wall, covalently bound glycans on peptidoglycan must be modified with pyruvate (see Non-Patent Document 10). The cell wall-pyruvate-modifying enzyme, which catalyzes the pyruvate modification of covalently bound glycans on peptidoglycan, was identified in the Gram-positive bacterium Bacillus anthracis and named CsaB (see Non-Patent Document 11), and corresponds to the above-mentioned slr0688.

[0035] Vector DNA for genetic modification of outer membrane-detached cyanobacteria is, for example, a plasmid or virus designed for genetic modification of the target site of genetic modification. For example, the vector DNA may contain the sequence of a homologous region of the target site of genetic modification, an optional sequence for suppressing expression of the gene, and the sequence of a gene used as a marker for genetic modification.

[0036] Figure 3 is a schematic diagram showing an example of vector DNA for genetic modification. The target site for genetic modification by the vector DNA shown in Figure 3 is the slr0688 gene of cyanobacteria. Homologous region 1 (SEQ ID NO: 1), which is a sequence immediately upstream of the transcription start point of the slr0688 gene, and homologous region 2 (SEQ ID NO: 2), which is a sequence immediately downstream of the transcription start point, have been introduced. In Figure 3, the sequence for suppressing expression of the slr0688 gene is the promoter sequence of the cyanobacterial petE gene (SEQ ID NO: 3). This sequence enables suppression of expression of the slr0688 gene. In Figure 3, the gene used as a marker for genetic modification is the antibiotic kanamycin resistance gene (SEQ ID NO: 4). This gene serves as an indicator for selecting modified cyanobacteria into which new sequences have been introduced by homologous recombination. Furthermore, in Figure 3, the gene used as a marker for genetic modification is the mazF gene of Escherichia coli (SEQ ID NO: 5). The mazF gene acts as a lethal gene and serves as a marker for selecting modified cyanobacteria in which the existing sequence has been deleted by homologous recombination. The sequence for regulating the mazF gene is the promoter sequence of the cyanobacterial nrsB gene (SEQ ID NO: 6). This sequence allows for the selective control of the expression of the lethal gene.

[0037] The method for constructing the vector DNA is not particularly limited. The vector DNA can be constructed, for example, by artificial gene synthesis. For example, genomic DNA is extracted from cells of a parent cyanobacterium, and a sequence corresponding to the homologous region of the target site for gene modification is amplified from this genomic DNA by the polymerase chain reaction (PCR) method. The vector DNA can be constructed by introducing the gene fragment obtained in this manner into a plasmid. The plasmid DNA from which the gene fragment is introduced is not particularly limited. For example, pUC19, which is easily introduced into and replicated within E. coli, can be preferably used. The method for introducing the gene fragment into the plasmid is not particularly limited. The gene fragment can be easily introduced into the plasmid by linearizing the plasmid DNA using any restriction enzyme and then ligating it using, for example, Takara Bio's DNA Ligation Kit or Clontech's In-Fusion® HD Cloning Kit.

[0038] The method for suppressing the expression of a target gene by genetic modification is not limited to a specific method. For example, the expression of a target gene can be suppressed by introducing, immediately upstream of the target gene, a promoter sequence of a gene whose expression is weaker than that of the target gene, or a promoter sequence whose expression can be arbitrarily regulated. A promoter is a regulatory sequence involved in the efficiency of the transcription initiation reaction of a gene, and includes both regulatory sequences that exist relatively close to the transcription start point and those that exist far from the transcription start point and operate. Examples of promoters whose expression can be regulated in cyanobacteria are the promoters of the cyanobacterial petE gene, the nirA gene, and the psbA2 gene (see Non-Patent Document 12). For example, the petE gene promoter is a promoter that can be regulated when cells containing the promoter are Cu 2+ This is a promoter that is expressed only in a growth environment in the presence of ions (see Non-Patent Document 13).

[0039] The gene used as a marker for genetic modification in cyanobacteria is not limited to a specific gene. As described above, the gene is preferably an antibiotic resistance gene or a lethal gene. When introducing a gene sequence into a parent cyanobacterium, adding an antibiotic resistance gene to the introduced gene sequence allows only outer membrane-detached cyanobacteria into which the gene sequence has been introduced to grow even in the presence of an antibiotic. This facilitates the selection of outer membrane-detached cyanobacteria. The antibiotic resistance gene is not limited to a specific resistance gene. Examples of antibiotic resistance genes are kanamycin, spectinomycin, and chloramphenicol. When deleting a gene sequence from outer membrane-detached cyanobacteria, if the gene sequence contains a lethal gene, only outer membrane-detached cyanobacteria lacking the gene sequence can grow even under conditions in which the lethal gene is induced. This facilitates the selection of outer membrane-detached cyanobacteria. The gene used as a lethal gene is not limited to a specific gene. Examples of lethal genes are preferably the mazF gene of Escherichia coli and the sacB gene of Bacillus subtilis (see Non-Patent Document 14). Preferably, a promoter sequence capable of regulating the expression of the lethal gene in cyanobacteria, as described above, is added upstream of the lethal gene. By regulating the expression of the lethal gene using such a sequence, the lethal gene is not expressed before genetic modification, and is less likely to impair the growth of the cyanobacteria.

[0040] Bacteria are classified as Gram-positive and Gram-negative bacteria based on their Gram staining ability. This difference in staining ability is due to differences in the thickness of their cell walls (peptidoglycan layer). Gram-positive bacteria have thick cell walls (peptidoglycan layer), while Gram-negative bacteria have thin cell walls. Therefore, in Gram-positive bacteria, the thick cell wall prevents the dye from being decolorized during the Gram staining process, leaving the dye intact. On the other hand, Gram-negative bacteria do not show Gram staining because their thin peptidoglycan layer is decolorized. Cyanobacteria are a typical Gram-negative bacteria. Gram-negative bacteria have an outer membrane that Gram-positive bacteria do not have, where lipopolysaccharide (LPS) is present. Lipopolysaccharide has a structure in which a glycan chain consisting of multiple sugars is bound to a lipid called lipid A. The glycan portion consists of a portion called a core polysaccharide and a portion called an O antigen. The lipid A portion of lipopolysaccharide is embedded in the lipid layer of the outer membrane. Lipopolysaccharide activates immune cells via Toll-like receptor 4 (TLR4), MD-2, and CD14 on their cell surfaces (see Patent Document 2). Thus, lipopolysaccharide is considered to be useful as an innate immune activator. Therefore, the progress of plant growth-promoting substance production can be determined based on the activity of lipopolysaccharide contained in the cyanobacterial culture supernatant, as in the above-mentioned production control method.

[0041] Second Embodiment Hereinafter, a second embodiment will be specifically described with reference to FIGS.

[0042] 4 is a flowchart showing an example of a method for determining the state of a cyanobacterial culture supernatant according to the second embodiment. As shown in FIG. 4, in this determination method, the activity value V of lipopolysaccharide contained in the cyanobacterial culture supernatant is A When the activity value V of lipopolysaccharide is equal to or greater than the threshold value T0, the culture supernatant is judged to be capable of exhibiting the desired plant growth-promoting effect. A When the activity value V of lipopolysaccharide contained in the cyanobacterial culture supernatant is less than the threshold value T0, the culture supernatant is judged to be incapable of exhibiting the desired plant growth-promoting effect. AThis can be easily determined based on the above.

[0043] As shown in FIG. 4, first, in step S301, the activity value V of lipopolysaccharide contained in the culture supernatant of cyanobacteria, for example, is calculated. A The activity value V of lipopolysaccharide is measured. A The method for measuring the activity value V of lipopolysaccharide is not limited to a specific method. For example, the activity value V of lipopolysaccharide can be measured by a colorimetric method. A is measured.

[0044] Next, in step S302, the activity value V of the lipopolysaccharide is calculated. A is greater than or equal to the threshold value T0. If the determination result in step S302 is positive, the process proceeds to step S303, where it is determined that the cyanobacterial culture supernatant is capable of exhibiting the desired plant growth-promoting effect. If the determination result in step S302 is negative, the process proceeds to step S304, where it is determined that the cyanobacterial culture supernatant is incapable of exhibiting the desired plant growth-promoting effect. Next, the process proceeds to step S305, where the determination result in step S303 or step S304 is output, and the series of processes ends.

[0045] The threshold value T in step S302 is not limited to a specific value. For example, the threshold value T is 10 endotoxin units (EU) / mL or more and 500 EU / mL or less. This tends to increase the reliability of the determination result. The threshold value T may be 15 EU / mL or more, 20 EU / mL or more, or 30 EU / mL or more, or 400 EU / mL or less, 300 EU / mL or less, 200 EU / mL or less, or 100 EU / mL or less.

[0046] The above-described determination method can be executed, for example, by using a computer. Therefore, a program for causing a computer to execute the above-described determination method can be provided. This program can be provided by a recording medium or via a telecommunications line.

[0047] 5 is a diagram schematically illustrating an example of a determination device according to this embodiment. As shown in FIG. 5, the determination device 1b includes, for example, an input device 12 and a calculation device 22. The input device 12 receives the activity value V of lipopolysaccharide contained in the culture supernatant of cyanobacteria. A The calculation device 22 receives the input of the measurement data of the lipopolysaccharide activity value V A is equal to or greater than the threshold value T0, the culture supernatant is determined to be capable of exhibiting the desired plant growth-promoting effect, and the lipopolysaccharide activity value V A is less than the threshold value T0, the culture supernatant is determined to be incapable of exhibiting the desired plant growth-promoting effect. The determination device 1b stores, for example, the above program. The calculation device 22 calculates the lipopolysaccharide activity value V A Based on the measurement data, this program is used to determine whether the cyanobacterial culture supernatant exhibits the desired plant growth-promoting effect.

[0048] The determination device 1b further includes, for example, a storage device 32 in which the program is stored. In the determination device 1b, the arithmetic device 22 is included in, for example, a central processing unit (CPU). The storage device 32 includes, for example, a main storage device and an auxiliary storage device.

[0049] 5, the determination device 1b further includes an output device 42. The output device outputs the determination result as to whether or not the cyanobacterial culture supernatant exhibits the desired plant growth-promoting effect. Examples of the output device 42 include a display and an indicator light.

[0050] According to the above-described determination method, only the culture supernatant of a cyanobacterium determined to be capable of exerting the desired plant growth-promoting effect can be shipped. Thus, the above-described determination method can be used, for example, for quality control of the culture supernatant of a cyanobacterium expected to have a plant growth-promoting effect.

[0051] For example, a culture supernatant obtained by collecting a portion of the cyanobacterial culture solution in the above-mentioned determination method may be used. In this case, the lipopolysaccharide activity value V of the culture supernatant thus obtained may be Ais equal to or greater than the threshold value T0, the culture supernatant is further recovered from the culture broth to produce a plant growth promoter. The cyanobacterial culture supernatant may be provided as a plant growth promoter as is, or the method for producing a plant growth promoter may include at least one step selected from the group consisting of diluting the cyanobacterial culture supernatant and adding an additive to the cyanobacterial culture supernatant. On the other hand, the lipopolysaccharide activity value V of the culture supernatant thus obtained is A When T is less than the threshold value T0, the culture medium may be discarded without recovering the culture supernatant.

[0052] The explanation regarding cyanobacteria in the first embodiment also applies to the second embodiment, unless technically inconsistent. As described above, a cyanobacterial culture supernatant can exhibit the desired plant growth-promoting effect. On the other hand, an unintended event during cyanobacterial cultivation may result in a cyanobacterial culture supernatant that does not exhibit the desired plant growth-promoting effect. According to the above-described determination method, if a cyanobacterial culture supernatant that does not exhibit the desired plant growth-promoting effect is obtained, the culture supernatant can be discarded without being shipped, thereby ensuring the quality of the shipped product.

[0053] As mentioned above, lipopolysaccharides are thought to be useful as innate immune activators. Therefore, the condition of cyanobacterial culture supernatants can be determined based on the activity of lipopolysaccharides, as in the above-mentioned determination method.

[0054] (Additional Notes) The above disclosure discloses the following technologies. (Technology 1) A method for managing the production of a plant growth-promoting substance, comprising determining the degree of progress of production of the plant growth-promoting substance based on the activity value of lipopolysaccharide contained in a cyanobacterial culture supernatant. (Technology 2) A method for managing the production of a plant growth-promoting substance according to Technology 1, in which the degree of progress is determined based on time-series data of the activity value of the lipopolysaccharide from the start of cyanobacterial cultivation. (Technology 3) A method for managing the production of a plant growth-promoting substance according to Technology 2, in which the degree of progress is determined according to a rate of change of the activity value calculated from the time-series data. (Technology 4) A method for managing the production of a plant growth-promoting substance according to any one of Technology 1 to 3, in which determination of the degree of progress is repeated until the degree of progress reaches or exceeds a threshold. (Technology 5) A method for producing a plant growth-promoting substance, comprising culturing cyanobacteria to produce the plant growth-promoting substance, and determining the degree of progress of production of the plant growth-promoting substance based on the activity value of lipopolysaccharide contained in the cyanobacterial culture supernatant. (Technology 6) The method for producing a plant growth-promoting substance according to Technology 5, wherein the degree of progress is determined based on time-series data of the activity value of the lipopolysaccharide from the start of cyanobacterial cultivation. (Technology 7) The method for producing a plant growth-promoting substance according to Technology 6, wherein the degree of progress is determined according to a rate of change of the activity value calculated from the time-series data. (Technology 8) The method for producing a plant growth-promoting substance according to any one of Technology 5 to 7, further comprising stopping the cultivation of the cyanobacteria and recovering the cyanobacterial culture supernatant when the degree of progress is equal to or greater than a threshold. (Technology 9) A production management device for a plant growth-promoting substance, comprising: an input device that accepts input of measurement data of the activity value of lipopolysaccharide contained in the cyanobacterial culture supernatant; and a calculation device that determines the degree of progress of production of the plant growth-promoting substance based on the activity value. (Technology 10) A program for causing a computer to execute the production management method according to any one of Technology 1 to 4.(Technology 11) A method for determining the state of a cyanobacterial culture supernatant, comprising determining that the culture supernatant is capable of exerting a desired plant growth-promoting effect when the activity value of lipopolysaccharide contained in the cyanobacterial culture supernatant is equal to or greater than a threshold, and determining that the culture supernatant is incapable of exerting the desired plant growth-promoting effect when the activity value is less than the threshold. (Technology 12) The determination method according to Technology 11, wherein the threshold is equal to or greater than 10 endotoxin units (EU) / mL and equal to or less than 500 EU / mL. (Technology 13) A determination device comprising: an input device that accepts input of measurement data of the activity value of lipopolysaccharide contained in the cyanobacterial culture supernatant; and a computing device that determines that the culture supernatant is capable of exerting a desired plant growth-promoting effect when the activity value is equal to or greater than the threshold, and determines that the culture supernatant is incapable of exerting the desired plant growth-promoting effect when the activity value is less than the threshold. (Technology 14) A program for causing a computer to execute the determination method according to Technology 11. (Technology 15) A method for producing a plant growth promoter, comprising collecting a portion of a cyanobacterial culture solution and recovering the culture supernatant from the culture solution when the lipopolysaccharide activity in the culture supernatant is equal to or greater than a threshold value.

[0055] The present disclosure will be described in more detail below with reference to examples, but the present disclosure is not limited to the following examples.

[0056] (Production Example 1) Escherichia coli HST08 strain and wild-type cyanobacterium Synechocystis sp. PCC 6803 strain (parent cyanobacteria) were each cultured in 5 mL of LB medium (1.0% by mass of Tripton, 0.5% by mass of Yeast Extract, 1.0% by mass of NaCl) for 16 hours. These were then cultured in 10 mL of BG11 medium for 120 hours. Cells derived from 1 mL of fully grown culture were subjected to Promega's Wizard® Genomic DNA Purification Kit to extract cellular genomic DNA. BG11 medium is an aqueous solution containing the following per 1 L: 2mL Solution I (0.5 g / L Na2ethylenediaminetetraacetic acid (EDTA), 2H2O, 3 g / L ammonium iron (III) citrate,3 g / L citric acid), 25mL Solution II-a (60 g / L NaNO3, 3 g / L MgSO4), 25mL Solution II-b (1.56 g / L K2HPO4), 2mL of Solution III(14.3 g / L CaCl2), 1mL of A6 Solution(2.86 g / L H3BO3, 1.81 g / L MnCl2・4H2O, 0.22 g / L ZnSO4・7H2O, 0.08 g / L CuSO4・5H2O, 0.021 g / L Na2MoO4・H2O, 0.0494 g / L Co(NO3)2・6H2O, 1 droplet of H2SO4), 20 mL of 1 M TES-KOH (pH 7.5)

[0057] Next, to construct the plasmid DNA used for gene modification, a total of seven DNA fragments were amplified by PCR. These fragments are referred to as fragments 1 to 7. For PCR, Toyobo's KOD One® PCR Master Mix -Blue- was used. Fragment 1, which contains the E. coli replication origin and the resistance gene for the antibiotic ampicillin, was amplified using primer 1 (SEQ ID NO: 7) and primer 2 (SEQ ID NO: 8) with the pUC19 plasmid as a template. Next, fragments 2 to 5 were amplified using the genomic DNA derived from the cyanobacterium as a template. Fragment 2, which contains the promoter sequence of the petE gene, was amplified using primer 3 (SEQ ID NO: 9) and primer 4 (SEQ ID NO: 10). Fragment 3, which contains the promoter sequence of the nrsB gene, was amplified using primer 5 (SEQ ID NO: 11) and primer 6 (SEQ ID NO: 12). Fragments 4 and 5, which correspond to homologous regions 1 and 2 of the slr0688 gene, were amplified using primer 7 (SEQ ID NO: 13) and primer 8 (SEQ ID NO: 14), as well as primer 9 (SEQ ID NO: 15) and primer 10 (SEQ ID NO: 16), respectively. Fragment 6, which corresponds to the mazF gene, was amplified using the above-mentioned genomic DNA derived from E. coli as a template and primer 11 (SEQ ID NO: 17) and primer 12 (SEQ ID NO: 18). Finally, fragment 7, which corresponds to the resistance gene for the antibiotic kanamycin, was amplified using primer 13 (SEQ ID NO: 19) and primer 14 (SEQ ID NO: 20) and the pSL2680 plasmid as a template.

[0058] The seven fragments were ligated using Clontech's In-Fusion® Snap Assembly Master Mix. The resulting plasmid DNA was introduced into Takara Bio's E. coli HST08 Premium Competent Cells using the heat shock method. The resulting E. coli was cultured in the presence of 50 μg / mL of the antibiotic ampicillin, and cells into which the plasmid DNA had been introduced were selected. The replicated plasmid DNA in these cells was extracted using Promega's Wizard® Plus SV Minipreps DNA Purification System to obtain the pNRSmazF-petE0688 plasmid (see Figure 3) used for gene modification.

[0059] The parent cyanobacterial cells were cultured in 10 mL of BG11 medium in a 125 mL Erlenmeyer flask. The parent cyanobacterial cell culture was incubated in a 30°C controlled atmosphere at 100 μmol photons m -2 s -1 The culture was irradiated with white LED light for 120 hours under the conditions of [0.01]. In addition, the Erlenmeyer flask was shaken at 160 rotations per minute (rpm). 0.5 mL of the fully grown culture was mixed with 3 μg of pNRSmazF-petE0688 plasmid to carry out natural transformation, resulting in homologous recombination, which introduced the petE gene promoter sequence immediately upstream of the slr0688 gene. The transformed cells were grown on BG11 agar medium for 2 days and then transferred to agar medium containing 20 μg / mL of the antibiotic kanamycin and grown for 7 days. Cells were transferred to another agar medium using an Immobilon-NC Triton-free MCE 0.45 μm 82 mm disc membrane filter (Merck Millipore).

[0060] The outer membrane-detached cyanobacterial colonies obtained by the above procedure were transferred to new agar medium and grown twice more to allow homologous recombination to spread throughout the entire genome of the cells. To confirm this, colony PCR was performed using the obtained colonies as templates. Promega's GoTaq® Green Master Mix was used for colony PCR. Figure 6 shows the results of electrophoresis of DNA fragments amplified by colony PCR. The electrophoresis results on the left side of Figure 6 correspond to colony PCR using primer 15 (SEQ ID NO: 21) and primer 16 (SEQ ID NO: 22), which amplified the genomic sequence located upstream of the site of homologous recombination and the sequence located downstream of that site. The electrophoresis results on the right side of Figure 6 correspond to colony PCR using primer 16 and primer 17 (SEQ ID NO: 23), which amplified the genomic sequence located downstream of the site of homologous recombination and the sequence within the kanamycin resistance gene where homologous recombination occurred. The center lane in Figure 6 shows the results of electrophoresis using Nippon Gene's Gene Ladder Wide 2 as a DNA size marker. For each DNA fragment in the left and right lanes, the numbers 1 to 5 indicate the colony numbers used as templates. "P" indicates the results of electrophoresis of a DNA fragment amplified using the pNRSmazF-petE0688 plasmid as a template as a control. "N" indicates the results of electrophoresis of a DNA fragment amplified using the genomic DNA derived from the parent cyanobacterium as a template as a control. These results confirmed that homologous recombination had spread throughout the genome. In this way, an outer membrane-detached cyanobacterium into which the promoter sequence of the petE gene had been introduced was obtained.

[0061] The above outer membrane-detached cyanobacteria were cultured in 50 mL of BG11 medium in a 500 mL Erlenmeyer flask. This culture was carried out in an environment of 30°C. In the culture of the outer membrane-detached cyanobacteria, 100 μmol photons m -2 s -1The culture solution was irradiated with white LED light for 7 days under the conditions of 100 μmol photons m , and the Erlenmeyer flask was shaken at 10 rpm. The culture solution that had grown sufficiently in this way was placed inside a colorless, transparent plastic bag, and 3 L of BG11 medium was added and further cultured. This culture was carried out in an environment of 30°C, and 300 μmol photons m . -2 s -1 The culture solution was irradiated with white LED light for 7 days under the conditions. In addition, air was supplied to the culture solution at an aeration rate of 1.67 L / min through a silicone tube. Three sets of 3 L culture solution of the outer membrane-detached cyanobacteria obtained in this way (total of 9 L) were prepared, and 0.5 ml 3 The culture solution was placed in a stainless steel SUS tank with an internal volume of 1000 ml. 400 L of BG11 medium containing all ingredients except CuSO4·5H2O was then added to the tank. Eight LED long white underwater fish-attracting lights manufactured by Ks Garage were evenly arranged inside the tank. While maintaining the temperature of the culture solution at 30°C, the underwater fish-attracting lights were energized at 3.5 A to irradiate the culture solution with light, and an aeration volume of 0.3 m was maintained. 3Mass-production culture was performed at 1000 kJ / min. During mass-production culture, 50 mL of culture medium was withdrawn every day from the start of culture, and the turbidity (OD730) of the culture medium at a wavelength of 730 nm was measured using a spectrophotometer with a 1-cm pathlength cell. The results are shown in Figure 7. As shown in Figure 7, it was suggested that outer membrane-detached cyanobacteria were growing inside the tank and undergoing logarithmic growth. Each culture medium obtained every day from the start of culture was passed through a Merck Millipore Millex®-GS Syringe Filter Unit (0.22 μm) to remove cells, yielding a culture supernatant containing components secreted by the outer membrane-detached cyanobacteria. The lipopolysaccharide content of the culture supernatant was quantified. The culture supernatant was diluted 5000-fold, and 0.1 mL of the diluted solution was measured for lipopolysaccharide activity using a Seikagaku Corporation Endospecy ES-50M set and Pyrocolor diazo reagent. The lipopolysaccharide activity was converted to the value for the culture supernatant solution before dilution. The results are shown in Figure 8. As shown in Figure 8, the lipopolysaccharide activity monotonically increased over time and accumulated in the culture solution until day 6 of culture. On the other hand, there was almost no increase in the lipopolysaccharide activity after day 7 of culture, suggesting that a sufficient amount of plant growth-promoting substance was produced by day 6 of culture at the latest.

[0062] The culture supernatant solution obtained on the 5th, 6th, 7th, and 8th days after the start of the mass production culture of the cyanobacteria was diluted 50-fold and the diluted solution was sprayed on the leaves of spinach. Commercially available culture soil was placed in a 9 cm diameter round cultivation pot, and Sakata Seed's Mirage spinach was used as the spinach, with three spinach seeds sown per pot. A total of 40 cultivation pots sown with spinach seeds were prepared. Spinach was cultivated in an artificial climate chamber adjusted to 20°C, with 100 μmol photons m -2 s -1The spinach plants were irradiated with white LED light under these conditions for 40 days, alternating between 12 hours of light and 12 hours of darkness. During this time, each pot was subirrigated with 50 mL of pure water every three days. Seven days after the start of cultivation, when the cotyledons had developed, the plants were thinned out to a uniform size. Then, 14 and 28 days after the start of cultivation, each of the eight cultivation pots was sprayed with 5 mL of the diluted solution using a spray bottle to wet the entire leaf surface. As a control, the remaining eight cultivation pots were sprayed with pure water instead of the diluted solution. Forty days after the start of cultivation, all spinach plants were harvested, and the fresh weight of the aboveground parts was measured. The results are shown in Figure 9. As shown in Figure 9, the growth of plants treated with the culture supernatant solution obtained from the fifth day onward after the start of cyanobacterial mass production cultivation was enhanced compared to the control plants that were not treated with the culture supernatant solution.

[0063] These results suggest that the progress of plant growth-promoting substance production can be determined based on the activity of lipopolysaccharides contained in the culture supernatant of outer membrane-detached cyanobacteria. Based on the determined progress, the production of plant growth-promoting substances accompanying cyanobacterial cultivation can be managed, and the timing to terminate the production of plant growth-promoting substances can be appropriately determined.

[0064] (Production Example 2) Mass-production culture of outer membrane-detached cyanobacteria was carried out in the same manner as in Production Example 1. During mass-production culture, 50 mL of culture medium was withdrawn every day from the start of culture, and the turbidity (OD730) of the culture medium was measured at a wavelength of 730 nm using a spectrophotometer with a 1 cm light path length cell. The results are shown in Figure 10. As shown in Figure 10, it was suggested that outer membrane-detached cyanobacteria were growing inside the tank and undergoing logarithmic growth. The culture medium obtained every day from the start of culture was passed through a Merck Millipore Millex®-GS Syringe Filter Unit (0.22 μm) to remove cells, and a culture supernatant solution containing components secreted by the outer membrane-detached cyanobacteria was obtained. The lipopolysaccharide content of the culture supernatant solution was quantified. The culture supernatant solution was diluted 5000-fold, and 0.1 mL of the diluted solution was measured for lipopolysaccharide activity using a Seikagaku Corporation Endospecy ES-50M set and Pyrocolor diazo reagent. The lipopolysaccharide activity was converted to the value relative to the culture supernatant solution before dilution. The results are shown in Figure 11. As shown in Figure 11, the lipopolysaccharide activity monotonically increased over time, indicating that lipopolysaccharide accumulated in the culture supernatant solution.

[0065] The culture supernatant solution was diluted 50-fold one day, two days, three days, four days, and five days after the start of the mass production culture of the cyanobacteria, and the diluted solution was sprayed on the leaves of spinach. Commercially available culture soil was placed in a 9 cm diameter round cultivation pot, and Sakata Seed's Mirage spinach was used as the spinach, with three spinach seeds sown per pot. A total of 48 cultivation pots sown with spinach seeds were prepared. Spinach was cultivated in an artificial climate chamber adjusted to 20°C, with 100 μmol photons m -2 s -1The spinach plants were irradiated with white LED light under these conditions for 40 days, alternating between 12 hours of light and 12 hours of darkness. During this time, each pot was subirrigated with 50 mL of pure water every three days. Seven days after the start of cultivation, when the cotyledons had developed, the plants were thinned out to a uniform size in each pot. Then, 14 and 28 days after the start of cultivation, each of the above-mentioned diluted solutions was sprayed onto eight cultivation pots with a spray bottle to wet the entire leaf surface. The amount of diluted solution was 5 mL. As a control, the remaining eight cultivation pots were sprayed with pure water instead of the diluted solution. Forty days after the start of cultivation, all spinach plants were harvested, and the fresh weight of the aboveground parts was measured. The results are shown in Figure 12. In Figure 12, the vertical axis represents the relative fresh weight of the spinach plants to the fresh weight of the spinach plants sprayed with pure water, expressed as a percentage. It can be seen from Figure 12 that the growth of spinach can be promoted by foliar spraying of the culture supernatant on spinach. For example, from Figures 11 and 12, if the lipopolysaccharide activity of the culture supernatant is 100 EU / mL or more, it is expected that a plant growth-promoting effect capable of increasing the fresh weight of spinach by 10% or more compared to when the culture supernatant is not used is exerted.

[0066] A polysaccharide fraction solution was separated from the culture supernatant 5 days after the start of culture. 2 mL of ethanol was added to 1 mL of this solution, and the mixture was mixed. The resulting liquid was allowed to stand at -20°C for 16 hours to precipitate the polysaccharide fraction. The resulting solution was centrifuged at 13,000 rpm for 30 minutes, and the supernatant was removed to obtain a precipitate of the polysaccharide fraction. This precipitate was suspended in 1 mL of pure water to obtain a polysaccharide fraction solution.

[0067] The polysaccharide fraction solution thus obtained was used to verify its plant growth promoting effect. The polysaccharide fraction solution was diluted 500 times and sprayed onto the leaves of spinach. Sakata Seed's Mirage was used as the spinach, and commercially available culture soil was placed in round cultivation pots with a diameter of 9 cm, with three spinach seeds sown per pot. Sixteen cultivation pots were prepared. The spinach was cultivated in an artificial climate chamber adjusted to 20°C, with 100 μmol photons m -2 s -1The spinach plants were irradiated with white LED light under these conditions for 40 days, alternating between 12 hours of light and 12 hours of darkness. During this time, each pot was subirrigated with 50 mL of pure water every three days. Seven days after the start of cultivation, when the cotyledons had developed, the plants were thinned out to a uniform size. Then, half of the eight cultivation pots were sprayed with 5 mL of the diluted solution using a spray bottle, ensuring that the entire leaf surface was wet, 14 and 28 days after the start of cultivation. As a control, the other half of the eight cultivation pots were sprayed with pure water instead of the diluted solution. After 40 days of cultivation, all spinach plants were harvested and the fresh weight of the aboveground parts was measured. The results are shown in Figure 13. As shown in Figure 13, it can be seen that the application of a polysaccharide fraction solution isolated from the culture supernatant of outer membrane-detached cyanobacteria promoted plant growth compared to the control plants that were not applied with the polysaccharide fraction solution.

[0068] These results suggest that lipopolysaccharides contained in the culture supernatant of outer membrane-detached cyanobacteria function as an active ingredient in promoting plant growth, and that the activity of lipopolysaccharides contained in the culture supernatant of outer membrane-detached cyanobacteria can be used to easily determine whether the culture supernatant can exert the desired plant growth-promoting effect.

[0069] The method for controlling the production of plant growth-promoting substances disclosed herein can be used for the production of plant growth-promoting substances involving the cultivation of cyanobacteria.

Claims

1. A method for controlling the production of a plant growth-promoting substance, comprising determining the progress of the production of the plant growth-promoting substance based on the activity value of lipopolysaccharide contained in the culture supernatant of a cyanobacterium.

2. The method for managing production of a plant growth-promoting substance according to claim 1, wherein the degree of progress is determined based on time-series data of the activity value of the lipopolysaccharide from the start of the culture of the cyanobacteria.

3. The method for managing production of a plant growth-promoting substance according to claim 2, wherein the degree of progress is determined according to the rate of change of the activity value calculated from the time-series data.

4. The method for managing production of a plant growth-promoting substance according to claim 1, wherein the determination of the degree of progress is repeated until the degree of progress becomes equal to or greater than a threshold value.

5. A method for producing a plant growth-promoting substance, comprising: culturing cyanobacteria to produce a plant growth-promoting substance; and determining the progress of production of the plant growth-promoting substance based on the activity value of lipopolysaccharide contained in the culture supernatant of the cyanobacteria.

6. The method for producing a plant growth-promoting substance according to claim 5, wherein the degree of progress is determined based on time-series data of the activity value of the lipopolysaccharide from the start of the culture of the cyanobacteria.

7. The method for producing a plant growth-promoting substance according to claim 6, wherein the degree of progress is determined according to a rate of change in the activity value calculated from the time-series data.

8. The method for producing a plant growth-promoting substance according to claim 5, further comprising stopping the cultivation of the cyanobacteria when the degree of progress is equal to or greater than a threshold value and recovering the culture supernatant of the cyanobacteria.

9. A production management device for a plant growth-promoting substance, comprising: an input device that receives input of measurement data on the activity value of lipopolysaccharide contained in a cyanobacterial culture supernatant; and a calculation device that determines the progress of production of the plant growth-promoting substance based on the activity value.

10. A program for causing a computer to execute the production management method according to claim 1.

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