Method for determining detachment of outer membrane of cyanobacterium

Abstract

The present disclosure provides a method for determining the detachment of the outer membrane of a cyanobacterium, which is advantageous in terms of simplicity and accuracy. This method for determining the detachment of the outer membrane of a cyanobacterium includes determining whether or not the outer membrane of a cyanobacterium has been detached by measuring the concentration of a target substance T in a measurement sample L3. The amount of contaminants I contained in a culture medium L1 of the cyanobacteria is reduced. The contaminants I contain at least one component selected from the group consisting of proteins, polysaccharides, metal ions, salts, and solid substances. The concentration of the target substance T is increased, and thereby the measurement sample L3 is obtained. The target substance T is at least one substance selected from the group consisting of amino acids and adenosine.

Description

Method for determining outer membrane detachment of cyanobacteria

[0001] The present disclosure relates to a method for determining outer membrane shedding of cyanobacteria.

[0002] Photosynthetic microorganisms such as cyanobacteria have attracted attention as a next-generation tool for material production with low environmental impact. Material production using photosynthetic microorganisms is a carbon-neutral technology, and has the advantages of proceeding under ambient temperature and pressure, using light as an energy source, and being able to utilize water and carbon dioxide (CO2) in the air.

[0003] Recent advances in genetic engineering technology have made it possible to produce a wide variety of compounds using photosynthetic microorganisms, including sucrose (see Non-Patent Document 1), isobutanol (see Non-Patent Document 2), fatty acids (see Non-Patent Document 3), amino acids (see Non-Patent Document 4), and proteins (see Non-Patent Document 5).

[0004] Furthermore, Patent Document 1 describes a modified cyanobacterium in which the function of a protein involved in binding between the outer membrane and the cell wall is suppressed or eliminated, and the modified cyanobacterium improves protein secretion productivity, enabling efficient protein production.

[0005] International Publication No. 2021 / 100640

[0006] Daniel C Ducat et al., “Rerouting Carbon Flux To Enhance Photosynthetic Productivity”, Applied and Environmental Microbiology, American Society for Microbiology, April 2012, Vol.78, No.8, pp.2660-2668Shota Atsumi et al., “Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde”, Nature Biotechnology, Nature Publishing Group, November 2009, Vol.27, No.12, pp.1177-1180Xinyao Liu et al., "Fatty acid production in genetically modified cyanobacteria", The Proceedings of the Natural Academy of Sciences(PNAS), National Academy of Sciences, April 2011, Vol.108, No.17, pp.6899-6904Arnav Deshpande et al., "Combining random mutagenesis and metabolic engineering for enhanced tryptophan production in Synechocystis sp. strain PCC 6803", Applied Environmental Microbiology, May 2020, Vol.86, No.9, e02816-19James A. Gregory et al., "Alga-Produced Cholera Toxin-Pfs25 Fusion Proteins as Oral Vaccines", Applied and Environmental Microbiology, American Society for Microbiology, June 2013, Vol.79, No. 13, pp. 3917-3925.

[0007] The techniques described in the above documents need to be reconsidered from the viewpoint of easily and accurately determining the detachment of the outer membrane of cyanobacteria. Therefore, the present disclosure provides a method for determining the detachment of the outer membrane of cyanobacteria that is advantageous in terms of ease and accuracy.

[0008] The present disclosure provides a method for determining whether the outer membrane of a cyanobacterium has been detached, the method comprising: determining whether the outer membrane of the cyanobacterium has been detached by measuring the concentration of a target substance in a measurement sample in which the amount of impurities, including at least one selected from the group consisting of proteins, polysaccharides, metal ions, salts, and solid substances, contained in a cyanobacterial culture solution has been reduced and the concentration of the target substance has been increased by concentration, wherein the target substance is at least one selected from the group consisting of amino acids and adenosine.

[0009] The method of the present disclosure for determining outer membrane detachment of cyanobacteria is advantageous in terms of simplicity and accuracy.

[0010] FIG. 1 is a flowchart showing an example of a method for determining outer membrane detachment of cyanobacteria according to this embodiment. FIG. 2 is a diagram schematically showing a method for preparing a measurement sample. FIG. 3 is a diagram schematically showing the cell surface of a cyanobacterium. FIG. 4 is a diagram schematically showing a plasmid used for genetic modification of cyanobacteria. FIG. 5 is a diagram showing the results of electrophoresis of DNA fragments amplified from modified cyanobacteria according to an example. FIG. 6 is a graph showing the results of measuring the activity of lipopolysaccharide (LPS) in a culture supernatant.

[0011] (Findings that Form the Basis of the Present Disclosure) In substance production using cyanobacteria, it is important to efficiently and simply evaluate or manage the state of cyanobacterial cells and the state of cyanobacterial culture. As described above, the detachment of the outer membrane of cyanobacteria can improve the production capacity of substances such as proteins. Therefore, from the perspective of improving the production capacity of substances using cyanobacteria, it is expected that there will be an increasing need to determine whether the outer membrane of cyanobacteria has detached. Meanwhile, according to the above literature, it is believed that electron microscope observation or complicated biochemical analysis methods are necessary to determine whether the outer membrane of cyanobacteria has detached. Therefore, if a method for easily and accurately determining whether the outer membrane of cyanobacteria has detached can be provided, it is believed that the spread of substance production using cyanobacteria can be promoted.

[0012] In light of these circumstances, the present inventors have conducted extensive research into a method for easily and accurately determining whether outer membrane detachment has occurred in cyanobacteria. As a result, they have newly discovered that outer membrane detachment in cyanobacteria can be easily and accurately determined by measuring the concentration of a predetermined target substance in a measurement sample obtained by subjecting a cyanobacterial culture solution to a predetermined treatment. Based on this new finding, the present inventors have completed the disclosed method for determining outer membrane detachment in cyanobacteria.

[0013] (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 arrangement positions, component connection configurations, process conditions, steps, and step order shown in the following embodiments are merely examples and are not intended to limit the present disclosure. Furthermore, among the components in the following embodiments, components that are not recited in the independent claims that represent the highest concepts 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 redundant explanations may be omitted or simplified. Furthermore, below, numerical ranges do not only represent strict meanings but also include substantially equivalent ranges. Furthermore, in this specification, each of the bacterial body and cell represents a single cyanobacterial individual.

[0014] (Embodiment) In this specification, the homology between nucleotide 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 on cyanobacterial genes and the proteins they encode is publicly available, for example, in the above-mentioned NCBI database and Cyanobase (http: / / genome.microbedb.jp / cyanobase / ). The amino acid sequences of target proteins and the nucleotide sequences of the genes encoding those proteins can be obtained from these databases.

[0015] 1 is a flowchart showing an example of a method for determining whether the outer membrane of a cyanobacterium has been detached according to the present embodiment. As shown in FIG. 1, the method for determining whether the outer membrane of a cyanobacterium has been detached includes determining whether the outer membrane of a cyanobacterium has been detached by measuring the concentration of a target substance T in a measurement sample L3.

[0016] FIG. 2 is a schematic diagram showing a method for preparing a measurement sample L3. As shown in FIG. 2, the amount of impurities I contained in a cyanobacterial culture solution L1 is reduced. The impurities I include at least one selected from the group consisting of proteins, polysaccharides, metal ions, salts, and solid substances. The cyanobacterial culture solution L1 may contain a target substance T. By concentration, the concentration of the target substance T is increased, and a measurement sample L3 is obtained. The measurement sample L3 thus obtained is subjected to concentration measurement of the target substance T, and based on the concentration measurement result, it is determined whether the outer membrane of the cyanobacterium has peeled off. The target substance T is at least one selected from the group consisting of amino acids and adenosine.

[0017] Cyanobacteria, also known as blue-green algae or cyanobacteria, are a group of prokaryotes that capture light energy using chlorophyll and use the energy to electrolyze water and generate oxygen through photosynthesis. Cyanobacteria are highly diverse, ranging from unicellular species such as Synechocystis sp. PCC6803 to multicellular, filamentous species such as Anabaena sp. PCC7120. Cyanobacteria also vary in their habitats, from thermophilic species such as Thermosynechococcus elongatus to marine species such as Synechococcus elongatus and freshwater species such as Synechocystis. Many species also have 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.

[0018] Figure 3 is a schematic diagram of the cell surface of a cyanobacterium. As shown in Figure 3, the cell surface of a cyanobacterium is composed of, from the inside out, an inner membrane 1, which is a plasma membrane; peptidoglycan 2; and an outer membrane 5, which is a lipid membrane forming the outermost layer of the cell. Sugar chains 3 composed of glucosamine, mannosamine, and the like are covalently bound to peptidoglycan 2, and pyruvate is bound to these covalently bound sugar chains 3 (see Jurgens and Weckesser, 1986, J. Bacteriol., 168:568-573). In this specification, the peptidoglycan 2 and the covalently bound sugar chains 3 are collectively referred to as the cell wall 4. The space between the inner membrane 1, which is a plasma membrane, and the outer membrane 5 is called the periplasm, where various enzymes involved in protein degradation or formation of three-dimensional structures, lipid or nucleic acid degradation, and uptake of extracellular nutrients are present.

[0019] As described in Patent Document 1, modified cyanobacteria modified so that the function of a protein involved in binding between the outer membrane and the cell wall is suppressed or eliminated improve protein secretion productivity, enabling efficient protein production. Therefore, cyanobacteria with detached outer membranes are thought to have a high substance production capacity. For example, the modified cyanobacteria described in Patent Document 1 can be used as cyanobacteria with detached outer membranes. The modified cyanobacteria and substance production using the modified cyanobacteria can be performed, for example, according to the method described in Patent Document 1. The cyanobacteria with detached outer membranes may be cyanobacteria genetically modified using the pNRSmazF-petE0688 plasmid described in the Examples.

[0020] According to the inventors' studies, the concentration of target substance T in measurement sample L3 obtained from culture solution L1 of cyanobacteria in which outer membrane detachment has occurred is significantly higher than the concentration of target substance T in measurement sample L3 obtained from culture solution L1 of cyanobacteria in which outer membrane detachment has not occurred. This is thought to be because outer membrane detachment of cyanobacteria makes it easier to secrete target substance T, which is normally difficult to secrete outside the cyanobacterial cell. Further studies by the inventors have shown that cyanobacterial culture solution may contain impurities I that inhibit the measurement of the concentration of target substance T. Therefore, in order to accurately measure the concentration of target substance T, it is important to reduce the amount of impurities I. Furthermore, it is also important to prepare measurement sample L3 by concentrating it so that the concentration of target substance T is increased. According to the above-mentioned determination method, outer membrane detachment of cyanobacteria can be easily and accurately determined by measuring the concentration of the target substance in measurement sample L3.

[0021] The amino acid that can be the target substance T is not limited to a specific amino acid, and is, for example, at least one amino acid selected from the group consisting of isoleucine, leucine, tyrosine, phenylalanine, L-amino acids, and branched-chain amino acids.

[0022] 1, in step S11, a cyanobacterial culture solution L1 is collected. The culture solution L1 may be, for example, a culture supernatant.

[0023] Next, in step S12, the amount of impurities I contained in the culture solution L1 is reduced. The method for reducing the amount of impurities I is not limited to a specific method. For example, the amount of impurities I, which is at least one selected from the group consisting of proteins and polysaccharides, is reduced by ultrafiltration of a solution containing the impurities I. The solution subjected to ultrafiltration may be the culture solution L1, or a solution obtained by subjecting the culture solution L1 to some kind of treatment. For example, a solution obtained by subjecting the culture solution L1 to a concentration treatment may be subjected to ultrafiltration.

[0024] In step S12, the solution containing the impurity I may be brought into contact with a chelating resin. In this case, for example, the amount of metal ions that are the impurity I may be reduced. The metal ions may be, for example, heavy metal ions. For example, the solution containing the impurity I may be brought into contact with the chelating resin by passing the solution through a column on which the chelating resin is immobilized. The chelating resin is not limited to a specific resin. Examples of chelating resins include iminodiacetic acid, isothiouronium, and methylglucamine.

[0025] In step S12, the solution containing the impurity I may be brought into contact with a porous material. In this case, for example, the amount of salts that are the impurity I may be reduced. The impurity I may be adsorbed onto the porous material. Examples of the porous material include activated carbon, zeolite, and porous silica.

[0026] When a solution containing impurities I is brought into contact with a porous substance, the target substance T may be adsorbed onto the porous substance. Therefore, the target substance T may be eluted from the porous substance into an organic solvent. The organic solvent is not limited to a specific organic solvent. Examples of the organic solvent include polar solvents such as ethanol and acetone.

[0027] In step S12, at least one selected from the group consisting of filtration of the solution containing impurity I and centrifugation of the solution containing impurity I may be performed. The filtration may include filtration other than ultrafiltration. In this case, the solid substance that is impurity I may be reduced. The solid substance may be a precipitate in the solution containing impurity I.

[0028] In step S12, a plurality of operations for reducing the amount of impurities I may be performed in combination.

[0029] In step S13, the concentration of the target substance T is increased by concentration. As shown in Fig. 2, concentration may be performed on a solution in which the amount of the impurity I has been reduced, or on the culture solution L1 before the amount of the impurity I has been reduced.

[0030] The concentration method is not limited to a specific method. Concentration includes, for example, at least one method selected from the group consisting of freeze-drying, removal of solvent by heating, and dissolving the residue obtained by removing the solvent by heating in a solvent. Removal of the solvent by heating is performed using, for example, an evaporator or a block thermostatic bath. The solvent for dissolving the residue is not limited to a specific solvent. For example, the solvent is phosphate-buffered saline (PBS Buffer). In this way, the measurement sample L3 is obtained.

[0031] Next, in step S14, the concentration of the target substance T in the measurement sample L3 is measured. The method for measuring the concentration of the target substance T in the measurement sample L3 is not limited to a specific method. The concentration of the target substance T in the measurement sample L3 can be measured, for example, by high-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LC / MS), or by using an enzyme assay kit. In particular, when the concentration of the target substance T in the measurement sample L3 is measured using an enzyme assay kit, no large-scale device is required for measuring the concentration of the target substance T, which is particularly simple.

[0032] The measurement of the concentration of the target substance T in the measurement sample L3 may include optical measurement of the sample obtained by using the enzyme assay kit described above. This allows for easy measurement of the concentration of the target substance T in the measurement sample L3 and easy determination of outer membrane detachment of the cyanobacterium. Examples of optical measurement include fluorescence measurement and absorbance measurement.

[0033] The enzyme assay kit is not limited to a specific kit. For example, the enzyme assay kit may be an adenosine assay kit. In this case, the concentration of the target substance T, adenosine, can be easily measured.

[0034] The enzyme assay kit may be an L-amino acid assay kit, in which case the concentration of the target substance T, an L-amino acid, can be easily measured.

[0035] The enzyme assay kit may be a branched-chain amino acid assay kit, in which case the concentration of the target substance T, that is, the branched-chain amino acid, can be easily measured.

[0036] Next, in step S15, it is determined whether the outer membrane of the cyanobacteria has detached. For example, it is determined that the cyanobacteria have detached when the concentration of the target substance T measured by measuring the concentration of the target substance T in the measurement sample L3 is greater than a predetermined threshold. The determination of whether the outer membrane of the cyanobacteria has detached may be made based on the concentration of a single type of amino acid, or may be made based on the concentrations of multiple types of amino acids.

[0037] The threshold concentration of the target substance T for determining whether the outer membrane of the cyanobacterium has been detached is adjusted as appropriate depending on the type of target substance T. For example, the threshold concentration of the target substance T can be determined based on the results of measuring the concentration of the target substance T using a sample whose outer membrane is known to have been detached or not.

[0038] When the target substance T is adenosine, it is determined that the outer membrane of the cyanobacterium has detached when the concentration of adenosine measured by measuring the concentration of the target substance T is greater than a first threshold value.

[0039] When the target substance T is an L-amino acid, it is determined that the outer membrane of the cyanobacterium has detached when the concentration of the L-amino acid measured by measuring the concentration of the target substance T is greater than a second threshold value.

[0040] When the target substance T is a branched-chain amino acid, it is determined that the outer membrane of the cyanobacterium has detached when the concentration of the branched-chain amino acid measured by measuring the concentration of the target substance T is greater than a third threshold value.

[0041] The determination of whether the outer membrane of a cyanobacterium has detached may be made using a computer that includes a storage device storing a program for this determination and a processor that reads and executes the program from the storage device and performs calculations for the determination, or the determination may be made by a human.

[0042] (Additional Note) From the above description, the following techniques are disclosed.

[0043] (Technology 1) A method for determining whether the outer membrane of a cyanobacterium has been detached, comprising: determining whether the outer membrane of the cyanobacterium has been detached by measuring the concentration of a target substance in a measurement sample in which the amount of impurities, including at least one selected from the group consisting of proteins, polysaccharides, metal ions, salts, and solid substances, contained in a cyanobacterial culture solution has been reduced and the concentration of the target substance has been increased by concentration, wherein the target substance is at least one selected from the group consisting of amino acids and adenosine.

[0044] (Technology 2) The determination method according to Technology 1, wherein the amino acid is at least one selected from the group consisting of isoleucine, leucine, tyrosine, phenylalanine, L-amino acids, and branched-chain amino acids.

[0045] (Technology 3) The method according to Technology 1 or 2, wherein the amount of at least one selected from the group consisting of the protein and the polysaccharide is reduced by ultrafiltration of the solution containing the impurities.

[0046] (Technology 4) The determination method according to any one of Technologies 1 to 3, wherein the amount of the metal ions is reduced by contacting the solution containing the impurities with a chelating resin.

[0047] (Technology 5) The method according to any one of Techniques 1 to 4, wherein the amount of the salt is reduced by contacting the solution containing the impurities with a porous substance.

[0048] (Technology 6) The determination method according to Technology 5, wherein the target substance is eluted from the porous substance into an organic solvent.

[0049] (Technology 7) The method according to any one of Techniques 1 to 6, wherein the amount of the solid material is reduced by at least one selected from the group consisting of filtration of the solution containing the impurities and centrifugation of the solution.

[0050] (Technology 8) The method according to any one of technologies 1 to 7, wherein the concentration includes at least one selected from the group consisting of freeze-drying, removing the solvent by heating, and dissolving the residue obtained by removing the solvent by heating in a solvent.

[0051] (Technology 9) The method according to any one of Techniques 1 to 8, wherein the concentration of the target substance is measured by high performance liquid chromatography, liquid chromatography mass spectrometry, or an enzyme assay kit.

[0052] (Technology 10) The determination method according to Technology 9, wherein the measurement of the concentration of the target substance includes optical measurement of a sample obtained by using the enzyme assay kit.

[0053] (Technology 11) The determination method according to Technology 9 or 10, wherein the enzyme assay kit is an adenosine assay kit.

[0054] (Technology 12) The determination method according to Technology 9 or 10, wherein the enzyme assay kit is an L-amino acid assay kit.

[0055] (Technology 13) The determination method according to Technology 9 or 10, wherein the enzyme assay kit is a branched-chain amino acid assay kit.

[0056] (Technology 14) The method according to any one of Techniques 1 to 11, wherein the outer membrane of the cyanobacterium is determined to be detached when the concentration of adenosine measured by measuring the concentration of the target substance is greater than a first threshold value.

[0057] (Technology 15) The method according to any one of Techniques 1 to 12, wherein the outer membrane of the cyanobacterium is determined to be detached when the concentration of the L-amino acid measured by measuring the concentration of the target substance is greater than a second threshold value.

[0058] (Technology 16) The method according to any one of Techniques 1 to 10 and 13, wherein the outer membrane of the cyanobacterium is determined to be detached when the concentration of the branched-chain amino acid measured by measuring the concentration of the target substance is greater than a third threshold value.

[0059] 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.

[0060] (Preparation of cyanobacterial culture supernatant solution) Escherichia coli HST08 strain and wild-type cyanobacterium Synechocystis sp. PCC 6803 strain (parent cyanobacteria) were each cultured in 5 mL of LB medium for 16 hours. In the LB medium, the Tripton concentration was 1.0% by mass, the yeast extract concentration was 0.5% by mass, and the NaCl concentration was 1.0% by mass. Additionally, these were cultured in 10 mL of BG-11 medium for 120 hours. BG-11 medium was prepared as a 1 L aqueous solution containing the components shown in Table 1. Cells derived from 1 mL of fully grown culture medium were subjected to Promega's Wizard® Genomic DNA Purification Kit to extract cellular genomic DNA.

[0061]

[0062] Next, a plasmid for genetic modification was constructed. Figure 4 is a schematic diagram of the plasmid used for genetic modification of cyanobacteria. As shown in Figure 4, the plasmid contains homologous region 1 (see SEQ ID NO: 1), which is the sequence immediately upstream of the transcription start point of the slr0688 gene, and homologous region 2 (see SEQ ID NO: 2), which is the sequence immediately downstream of the transcription start point of the slr0688 gene. In the plasmid, the sequence for suppressing expression of the slr0688 gene is the promoter sequence of the cyanobacterial petE gene (see SEQ ID NO: 3). This sequence enables suppression of expression of the slr0688 gene. The plasmid contains a resistance gene for the antibiotic kanamycin (see SEQ ID NO: 4) as a genetic modification marker. This gene serves as an indicator for selecting modified cyanobacteria into which new sequences have been introduced by homologous recombination. The plasmid also contains the E. coli mazF gene (see SEQ ID NO: 5) as a genetic modification marker. This 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 this gene is the promoter sequence of the cyanobacterial nrsB gene (see SEQ ID NO: 6). This sequence allows for the expression of the lethal gene to be selectively controlled.

[0063] To construct the above plasmids, a total of seven DNA fragments (fragments 1 to 7) were amplified by PCR. Toyobo's KOD One® PCR Master Mix-Blue was used for PCR. For fragment 1, the E. coli replication origin and the ampicillin antibiotic resistance gene were amplified using primer 1 (see SEQ ID NO:7) and primer 2 (see SEQ ID NO:8) with the pUC19 plasmid as a template. Next, for fragments 2 to 5, the promoter sequence of the petE gene was amplified using primer 3 (see SEQ ID NO:9) and primer 4 (see SEQ ID NO:10) with the cyanobacterium-derived genomic DNA as a template. For fragment 2, the promoter sequence of the nrsB gene was amplified using primer 5 (see SEQ ID NO:11) and primer 6 (see SEQ ID NO:12). For fragment 4, the homologous region 1 of the slr0688 gene was amplified using primer 7 (see SEQ ID NO:13) and primer 8 (see SEQ ID NO:14). For fragment 5, primer 9 (see SEQ ID NO: 15) and primer 10 (see SEQ ID NO: 16) were used to amplify homologous region 2 of the slr0688 gene. For fragment 6, primer 11 (see SEQ ID NO: 17) and primer 12 (see SEQ ID NO: 18) were used to amplify the mazF gene using the above-mentioned genomic DNA derived from E. coli as a template. For fragment 7, primer 13 (see SEQ ID NO: 19) and primer 14 (see SEQ ID NO: 20) were used to amplify the resistance gene for the antibiotic kanamycin using the pSL2680 plasmid as a template.

[0064] The seven fragments were ligated using Clontech's In-Fusion® Snap Assembly Master Mix. The resulting plasmid 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 had been introduced were selected. The replicated plasmid in these cells was extracted using Promega's Wizard® Plus SV Minipreps DNA Purification System to obtain the pNRSmazF-petE0688 plasmid (see Figure 4) used for gene modification.

[0065] The parent cyanobacterial cells were cultured in 10 mL of BG-11 medium inside a 125 mL Erlenmeyer flask. The culture was incubated at 30°C for 120 hours with 100 μmol photons m -2 s -1 The transformation was performed in an artificial climate chamber under illumination with 1000 W of white LED light and shaking at 160 rotations per minute (rpm). A 0.5 mL culture of the fully grown parent cyanobacterium was mixed with 3 μg of the pNRSmazF-petE0688 plasmid, and homologous recombination was carried out by natural transformation to introduce the petE promoter sequence immediately upstream of the slr0688 gene. The transformed cells were grown on BG-11 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).

[0066] The modified cyanobacterial colonies thus obtained were transferred twice to new agar plates and grown there, allowing homologous recombination to propagate 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 5 shows the results of electrophoresis of DNA fragments amplified by colony PCR. The left side of Figure 5 shows the results of colony PCR using primer 15 (see SEQ ID NO: 21) and primer 16 (see SEQ ID NO: 22). This colony PCR involved amplification of the genomic sequence located upstream of the site of homologous recombination and the genomic sequence located downstream of that site. The right side of Figure 5 shows the results of colony PCR using primer 16 and primer 17 (see SEQ ID NO: 23). This colony PCR involved amplification of the genomic sequence located downstream of the site of homologous recombination and the sequence in the kanamycin resistance gene where homologous recombination occurred. The center lane in Figure 5 shows the results of electrophoresis using Nippon Gene's Gene Ladder Wide 2 as a DNA size marker. Regarding the electrophoresis results for each DNA fragment in the left and right lanes, the numbers 1 to 5 indicate the colony numbers used as templates. In addition, "P" indicates the results of electrophoresis for DNA fragments amplified using the pNRSmazF-petE0688 plasmid as a control, and "N" indicates the results of electrophoresis for DNA fragments amplified using the genomic DNA derived from the parent cyanobacterium as a control. The results shown in Figure 5 confirm that homologous recombination was widespread throughout the genome, and it is understood that a modified cyanobacterium into which the promoter sequence of the petE gene was introduced was obtained.

[0067] The modified cyanobacterial cells were cultured in 1 L of BG-11 medium for 120 hours, similar to the parent cyanobacterium. As a control, parent cyanobacterial cells were also cultured simultaneously. The resulting culture solutions were then passed through a Merck Millipore Millex®-GS Syringe Filter Unit (0.22 μm) to remove the cells. This resulted in a culture supernatant solution containing the secretions of the modified cyanobacterium according to the Example, and a culture supernatant solution of the parent cyanobacterial cells according to the Reference Example.

[0068] (LPS Detection) The culture supernatant solutions of the Examples and the Reference Example were each subjected to measurement of lipopolysaccharide (LPS), a characteristic component indicative of outer membrane detachment in cyanobacteria. Specifically, 0.2 mL of each culture supernatant solution was measured for LPS activity using a Seikagaku Corporation Endospecy ES-24S set and Pyrocolor diazo reagent. Figure 6 is a graph showing the results of measuring the LPS activity of the culture supernatants. As shown in Figure 6, the LPS activity of the culture supernatant solutions of the Examples was higher than that of the Reference Example, suggesting that the outer membrane of the modified cyanobacteria had partially detached. In contrast, the LPS activity of the culture supernatant solution of the Reference Example was lower, suggesting that the outer membrane of the cyanobacteria had not detached.

[0069] (Adenosine Concentration Measurement) A solution containing 100 mL of the culture supernatant solution of the Example, to which adenosine had been added at a concentration of 400 nM, was ultrafiltered. The molecular weight cutoff (MWCO) in this ultrafiltration was 3K. This ultrafiltration is believed to have reduced the amount of protein and sugar contained in the solution. The solvent from the filtrate obtained by ultrafiltration was removed using an aluminum block thermostatic bath equipped with a nitrogen gas blowing unit, a concentrating device manufactured by Taitec Corporation. The resulting residue was dissolved in 1.0 mL of PBS buffer to obtain a 100-fold concentrated solution. This solution was filtered through a filter with a pore size of 0.22 μm to obtain a measurement sample of Example 1. A measurement sample of Comparative Example 1 was obtained in the same manner as the measurement sample of Example 1, except that ultrafiltration was not performed.

[0070] The adenosine concentrations of the measurement samples of Example 1 and Comparative Example 1 were measured using an adenosine assay kit manufactured by Cell Biolabs. Adenosine in the measurement sample was converted to inosine by adenosine deaminase included in the assay kit. Inosine was converted to hypoxanthine by purine nucleoside phosphorylase included in the assay kit. Hypoxanthine was converted to xanthine and hydrogen peroxide by xanthine oxidase included in the assay kit. The generated hydrogen peroxide was detected with a specific fluorescent probe, and the adenosine-induced fluorescence intensity was calculated from the difference in fluorescence intensity between the sample with and without adenosine deaminase. The adenosine concentration was calculated based on a previously prepared calibration curve. For fluorescence intensity measurements, the excitation wavelength was adjusted to 570 nm and the fluorescence wavelength to 610 nm. The results are shown in Table 2.

[0071] As shown in Table 2, in the measurement using the measurement sample according to Comparative Example 1, the fluorescence intensity remained almost unchanged regardless of whether adenosine deaminase was added or not, making it difficult to measure the adenosine concentration. This is thought to be because the measurement sample according to Comparative Example 1 did not undergo ultrafiltration, and therefore the amount of impurities such as proteins and polysaccharides was not reduced. On the other hand, the adenosine concentration of the measurement sample according to Example 1 was determined to be 20.0 μM. This suggests that the adenosine concentration can be measured simply and accurately by obtaining a measurement sample through a process including ultrafiltration.

[0072]

[0073] (Measurement of branched-chain amino acid concentration) A solution in which isoleucine was added at a concentration of 400 nM to 100 mL of the culture supernatant solution according to the example was ultrafiltered. The molecular weight cutoff (MWCO) in this ultrafiltration was 3K. This ultrafiltration is believed to have reduced the amount of protein and sugar contained in the solution. The solvent from the filtrate obtained by ultrafiltration was removed using an aluminum block thermostatic chamber equipped with a nitrogen gas blowing unit, a concentrating device manufactured by Taitec Corporation. The resulting residue was dissolved in 1.0 mL of PBS buffer to obtain a 100-fold concentrated solution. This solution was filtered through a filter with a pore size of 0.22 μm to obtain a measurement sample according to Example 2. A measurement sample according to Comparative Example 2 was obtained in the same manner as the measurement sample according to Example 2, except that ultrafiltration was not performed.

[0074] The concentrations of the measurement samples in Example 2 and Comparative Example 2 were measured using a branched-chain amino acid assay kit manufactured by Cell Biolabs. L-leucine, L-valine, and L-isoleucine were measured using an excess of nicotinamide adenine dinucleotide (NAD). +In the presence of leucine dehydrogenase, branched-chain amino acids are converted to α-keto acids, ammonia, and NADH by leucine dehydrogenase. NADH is detected colorimetrically using WST-1, which is converted to formazan by an electron mediator. The absorbance attributable to branched-chain amino acids was calculated from the difference in absorbance between the sample with leucine dehydrogenase and the sample without leucine dehydrogenase, and the concentration of branched-chain amino acids was calculated based on a previously prepared calibration curve. Light with a wavelength of 450 nm was used to measure the absorbance. The results are shown in Table 3.

[0075] As shown in Table 3, in the measurement using the measurement sample of Comparative Example 2, when leucine dehydrogenase was not added, the absorbance was high and the concentration could not be calculated. This is thought to be because the measurement sample of Comparative Example 2 was not subjected to ultrafiltration, and therefore remaining impurities such as proteins and polysaccharides reacted with the colorimetric probe. On the other hand, the concentration of branched-chain amino acids in the measurement sample of Example 2 was determined to be 46.3 μM. This suggests that the concentration of branched-chain amino acids can be measured simply and accurately by obtaining a measurement sample through a process including ultrafiltration.

[0076]

[0077] (Measurement of L-amino acid concentration) 100 mL of the culture supernatant solution according to the example was ultrafiltered. The molecular weight cutoff (MWCO) in this ultrafiltration was 3K. This ultrafiltration is believed to have reduced the amount of protein and sugar contained in the solution. The solvent from the filtrate obtained by ultrafiltration was removed using an aluminum block thermostatic bath equipped with a nitrogen gas blowing unit, a concentrating device manufactured by Taitec Corporation. The resulting residue was dissolved in 1.0 mL of PBS buffer to obtain a 100-fold concentrated solution. This solution was filtered through a 0.22 μm pore filter to obtain a measurement sample according to Example 3. A measurement sample according to Comparative Example 3 was obtained in the same manner as the measurement sample according to Example 3, except that the culture supernatant solution according to the above Reference Example was used instead of the culture supernatant solution according to the example. A measurement sample according to Comparative Example 4 was obtained in the same manner as the measurement sample according to Example 3, except that ultrafiltration was not performed.

[0078] The concentrations of L-amino acids in the measurement samples of Example 3, Comparative Example 3, and Comparative Example 4 were measured using an L-amino acid assay kit manufactured by Cell Biolabs. The L-amino acids in the measurement samples were oxidatively deaminated by L-amino acid oxidase to produce α-keto acids, ammonia, and hydrogen peroxide. The produced hydrogen peroxide was detected using a colorimetric probe. The absorbance intensity attributable to L-amino acids was calculated from the difference in absorbance between the sample with and without L-amino acid oxidase, and the concentration was calculated based on a previously prepared calibration curve. Light with a wavelength of 570 nm was used to measure absorbance. The results are shown in Table 4.

[0079] When the measurement sample of Comparative Example 4 was used, even without the addition of L-amino acid oxidase, the absorbance exceeded 1.0, making it impossible to detect the concentration of L-amino acids. This is thought to be because the measurement sample of Comparative Example 4 did not undergo ultrafiltration, resulting in the reaction of remaining impurities such as proteins and polysaccharides with the colorimetric probe. On the other hand, the concentration of L-amino acids in the measurement sample of Example 3 was determined to be 404 μM, while the concentration of L-amino acids in the measurement sample of Comparative Example 3, which was measured using a supernatant solution obtained from a control strain in which the outer membrane was difficult to detach, was determined to be 0 μM. These results suggest that obtaining a measurement sample through a process including ultrafiltration allows for the easy and accurate measurement of L-amino acid concentrations, and that the measurement results can be used to determine whether the outer membrane of cyanobacteria has detached.

[0080]

[0081] (Amino acid analysis by LC / MS / MS) A modified cyanobacterium in which expression of the slr0688 gene was suppressed was obtained in the same manner as in Example 2 of Patent Document 1. According to Patent Document 1, detachment of the outer membrane was confirmed in this modified cyanobacterium using an electron microscope. In addition, a control strain was obtained in the same manner as in Comparative Example 1 of Patent Document 1. According to Patent Document 1, detachment of the outer membrane was not confirmed in the control cyanobacterium strain.

[0082] The modified cyanobacteria in which the expression of the slr0688 gene was suppressed and the control strain were cultured at the initial bacterial density OD 730 The bacteria were inoculated into 12 mL of BG-11 medium (see Table 1) containing 1 μg / mL aTc in a 125 mL Erlenmeyer flask so that the β-glucan concentration was 0.1, and the light intensity was 100 μmol / m 2 The mixture was cultured with shaking at 100 / s and 30°C for 3 days.

[0083] After 3 days of incubation, the culture medium was centrifuged at 2500 g for 10 minutes at room temperature, and the culture supernatant was filtered through a Millex-GV Syringe Filter Unit, 0.22 μm (Millipore). The filtered supernatant was subjected to amino acid analysis by LC / MS / MS.

[0084] The concentrations of isoleucine, leucine, tyrosine, and phenylalanine contained in the culture supernatants of the above two types of cyanobacteria were measured by LC / MS / MS.

[0085] One mL of the filtered culture supernatant from each cyanobacterial strain was lyophilized and then dissolved in 195 μL of APDS Tag® Wako borate buffer. 5 μL of a solution of 100 mg of 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS) tag reagent dissolved in 5 mL of acetonitrile was added, mixed well, and heated at 55 to 60°C for 5 to 15 minutes. In this way, samples for LC / MS / MS analysis were obtained.

[0086] The obtained samples were analyzed using an LC / MS / MS device, UltiMate 3000 RSLCnano LC System, under the following conditions.

[0087] Sample injection volume: 10 μL Column: Tosoh TSKgel ODS-100V 2.0 mm ID x 15 cm Mobile phase: Solution A is APDS Tag (registered trademark) Wako eluent, Solution B is 60% acetonitrile Elution time table (solution B %): 0 min, 6%; 0.05-1.70 min, 8%; 1.71 min, 12%; 4.95 min, 30%; 5.95 min, 60%; 6.70 min, 95%; 6.71 min, 6%; 12.0 min, 6% Flow rate: 0.3 mL / min Column temperature: 40°C Ionization mode: ESI positive The concentrations of isoleucine, leucine, tyrosine, and phenylalanine in each sample are shown in Table 5. As shown in Table 5, samples derived from the culture supernatant of modified cyanobacteria in which expression of the slr0688 gene was suppressed were confirmed to contain isoleucine, leucine, tyrosine, and phenylalanine at concentrations of 130 nM to 320 nM. In contrast, samples derived from the culture supernatant of the control strain were confirmed to contain almost no of these four amino acids. Therefore, outer membrane detachment of cyanobacteria can be determined based on the results of measuring the concentrations of amino acids such as isoleucine, leucine, tyrosine, and phenylalanine in the above-mentioned measurement samples obtained by reducing and concentrating the amount of contaminants.

[0088]

[0089] The method of determining outer membrane detachment of cyanobacteria disclosed herein can be used for substance production using cyanobacteria.

[0090] Contaminants I Culture solution L1 Measurement sample L3 Target substance T

Claims

1. A method for determining whether the outer membrane of a cyanobacterium has been detached, comprising: determining whether the outer membrane of the cyanobacterium has been detached by measuring the concentration of a target substance in a measurement sample in which the amount of impurities contained in a cyanobacterial culture solution, the impurities including at least one selected from the group consisting of proteins, polysaccharides, metal ions, salts, and solid substances, has been reduced and the concentration of the target substance has been increased by concentration, wherein the target substance is at least one selected from the group consisting of amino acids and adenosine.

2. The method of claim 1, wherein the amino acid is at least one selected from the group consisting of isoleucine, leucine, tyrosine, phenylalanine, L-amino acids, and branched-chain amino acids.

3. The method according to claim 1, wherein the amount of at least one selected from the group consisting of proteins and polysaccharides is reduced by ultrafiltration of the solution containing the impurities.

4. The method according to claim 1, wherein the amount of the metal ions is reduced by contacting the solution containing the impurities with a chelating resin.

5. The method according to claim 1, wherein the amount of the salt is reduced by contacting the solution containing the impurities with a porous substance.

6. The method according to claim 5, further comprising the step of eluting the target substance from the porous substance into an organic solvent.

7. The method according to claim 1, wherein the amount of the solid material is reduced by at least one selected from the group consisting of filtering the solution containing the impurities and centrifuging the solution.

8. The method according to claim 1, wherein the concentration includes at least one method selected from the group consisting of freeze-drying, removal of the solvent by heating, and dissolving the residue obtained by removing the solvent by heating in a solvent.

9. The method according to claim 1, wherein the concentration of the target substance is measured by using high performance liquid chromatography, liquid chromatography mass spectrometry, or an enzyme assay kit.

10. The method of claim 9, wherein said measuring the concentration of said target substance comprises optical measurement of a sample obtained by use of said enzyme assay kit.

11. The method according to claim 9, wherein the enzyme assay kit is an adenosine assay kit.

12. The method according to claim 9, wherein the enzyme assay kit is an L-amino acid assay kit.

13. The method according to claim 9, wherein the enzyme assay kit is a branched-chain amino acid assay kit.

14. The method according to claim 1, wherein the outer membrane of the cyanobacterium is determined to be detached when the concentration of adenosine measured by measuring the concentration of the target substance is greater than a first threshold value.

15. The method according to claim 1, wherein the outer membrane of the cyanobacterium is determined to be detached when the concentration of the L-amino acid measured by measuring the concentration of the target substance is greater than a second threshold value.

16. The method according to claim 1, wherein the outer membrane of the cyanobacterium is determined to be detached when the concentration of branched-chain amino acids measured by measuring the concentration of the target substance is greater than a third threshold value.

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