Cyanobacteria that produce sulfated polysaccharides and method for producing sulfated polysaccharides derived from cyanobacteria

Abstract

To provide a method for producing sulfated polysaccharides derived from cyanobacteria, with the aim of mass-producing and modifying the composition of sulfated polysaccharides.SOLUTION: Provided are cyanobacteria capable of producing sulfated polysaccharides and having a sulfated polysaccharide synthesis gene and a sulfated polysaccharide synthesis control gene. The sulfated polysaccharide synthesis control gene is composed of a gene encoding a transcriptional regulator, a gene encoding a response regulator, and a gene encoding a sensor histidine kinase. In the cyanobacteria, the activation of transcription of any one of the gene encoding the transcriptional regulator, the gene encoding the response regulator, and the gene encoding the sensor histidine kinase is regulated.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to cyanobacteria that produce sulfated polysaccharides and a method for producing sulfated polysaccharides derived from cyanobacteria.

Background Art

[0002] Polysaccharides are very important biopolymers having a structure in which monosaccharides are linked in a chain, along with nucleic acids and proteins, and are rich in diversity due to differences in monosaccharide composition and modification. Sulfated polysaccharides are a type of acidic polysaccharide modified by sulfate groups and are present in animals, eukaryotic algae, archaea, and cyanobacteria. They are involved in various important functions of organisms and have attracted attention in recent years as thickeners, water retention agents, and pharmaceuticals (anticancer, antiviral, anticoagulants) (Hayashi, 2008). Currently, sulfated polysaccharides derived from microorganisms are obtained by a method of recovering and purifying those produced by wild strains of bacteria such as algae and cyanobacteria, or by a method of sulfating non-sulfated polysaccharides with a chemical catalyst.

[0003] For example, International Publication No. 2003 / 023045 discloses a method for producing sulfated polysaccharides, in which a Pseudomonas sp. WAK-1 strain isolated from the surface of wakame thalli living in the ocean is inoculated and cultured in a medium containing a nutrient source, and the culture is treated with an organic solvent such as alcohol to obtain a precipitate fraction (Patent Document 1).

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] Research on bacterial extracellular polysaccharides has advanced, and various synthetic systems have been elucidated (Becker et al., 1998, Schmid et al., 2015). However, the sulfated polysaccharide synthesis system in cyanobacteria remains unclear. In addition, typical cyanobacteria in which the presence of sulfated polysaccharides is known are difficult to transform due to the following circumstances: the extracellular polysaccharide is a major obstacle to transformation, the endogenous restriction-modification system is strong and degrades foreign DNA, and the genome is multicopy. These two points are obstacles to the establishment of biotechnological production methods.

[0006] Therefore, the present invention aims to establish a method for producing sulfated polysaccharides derived from cyanobacteria for the purpose of mass-producing sulfated polysaccharides and modifying their composition.

Means for Solving the Problems

[0007] In order to solve the above problems, the present inventors identified a group of genes involved in the sulfated polysaccharide synthesis system and examined means for controlling the genes related to transcriptional regulation therein, and obtained the finding that a significant amount of sulfated polysaccharide can be accumulated in the culture medium.

[0008] The present invention is based on such findings, and provides a cyanobacterium that produces a sulfated polysaccharide, comprising a sulfated polysaccharide synthesis system gene and a sulfated polysaccharide synthesis control system gene, wherein the sulfated polysaccharide synthesis control system gene is composed of a gene encoding a transcriptional regulator, a gene encoding a response regulator, and a gene encoding a sensor histidine kinase, and the transcription of any one of the gene encoding the transcriptional regulator, the gene encoding the response regulator, and the gene encoding the sensor histidine kinase is controlled to be activated.

[0009] The present invention also provides a method for producing a sulfated polysaccharide, comprising the step of culturing the cyanobacterium.

Effects of the Invention

[0010] According to the present invention, a sulfated polysaccharide derived from cyanobacteria can be efficiently produced.

Brief Description of the Drawings

[0011]

Figure 1

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Figure 7

Mode for Carrying Out the Invention

[0012] 1. Recombinant cyanobacteria (1) Preparation of recombinant cyanobacteria The first aspect of the present invention provides a recombinant cyanobacterium that efficiently produces a sulfated polysaccharide, wherein the gene encoding a sensor histidine kinase is disrupted.

[0013] Examples of the types of cyanobacteria that can be used in the present invention include cyanobacteria belonging to the genus Synechocystis, Trichodesmium, Acaryochloris, Crocosphaera, Aphanothece, Anabaena, Nostoc, Rivularia, Arthrospira, Cyanothece, Microcystis, Oscillatoria, or Leptolyngbya. Preferably, examples include Synechocystis, Aphanothece, Oscillatoria, Cyanothece, Crocosphaera, Anabaena, or Nostoc. Among them, Synechocystis sp. PCC6803 substr. PCC-P strain is preferable in that transformation operation is very easy.

[0014] Sensor histidine kinase is an enzyme involved in signal transduction via phosphorylation and is abundant in cyanobacteria. The recombinant cyanobacteria of the present invention knockout a specific enzyme that controls the synthesis of sulfated polysaccharides, enabling mass production of sulfated polysaccharides.

[0015] Methods for knocking out sensor histidine kinases can employ various genetic engineering techniques. For example, a knockout method by homologous recombination with foreign DNA containing a drug resistance gene cassette can be mentioned. Note that since cyanobacteria have a multi-copy genome, that is, there are multiple copies of the genome, the gene to be disrupted also exists in the same number as the genome. Therefore, although a small amount of wild-type genome may sometimes be detected in the disrupted strain, in this embodiment, in addition to the disrupted strain in which all sensor histidine kinases are knocked out, recombinant strains in which a small amount of wild-type genome is also detected if a prominent phenotype appears are also included in the disrupted strains in which sensor histidine kinases are knocked out.

[0016] The recombinant cyanobacteria of the present invention have a prominent ability to produce sulfated polysaccharides. In addition, since the recombinant cyanobacteria of the present invention significantly release sulfated polysaccharides into the medium, they are easy to recover. Therefore, if the recombinant cyanobacteria of the present invention are cultured under appropriate conditions and then the secreted sulfated polysaccharides are recovered, efficient production of sulfated polysaccharides can be realized.

[0017] (2) Culture method The culture of recombinant cyanobacteria can generally be carried out based on liquid culture using BG-11 medium (J Gen Microbiol., 1979, 111:1-61) or a modified method thereof. For sulfated polysaccharide production, it is preferable to culture until the metabolism of the cells is activated. For example, it is suitable to carry out aeration and agitation culture or shaking culture for 1 to 7 days. The knockout strain of the sensor histidine kinase prepared in the present invention forms a hard biofilm on an agar medium because it significantly accumulates sulfated polysaccharides. Therefore, when starting liquid culture based on this, it is preferable to suspend it well and then inoculate. In addition, if the agitation during liquid culture is weak, cells form aggregates at an early stage, which hinders growth. Therefore, it is preferable to agitate strongly in either aeration and agitation culture or shaking culture.

[0018] (3) Recovery method Through the above cultivation, cyanobacteria produce sulfated polysaccharides and release the sulfated polysaccharides into the culture medium. When the culture solution is allowed to stand in a state where sulfated polysaccharides have accumulated in the culture medium, the cells and sulfated polysaccharides aggregate to form a viscous cell mass like a bloom on the liquid surface. When recovering the secreted sulfated polysaccharides, for the former case, from the whole culture solution, and for the latter case, from the viscous cell mass, solid components such as cells are removed by filtration, centrifugation, etc., and after recovering the remaining liquid components, the sulfated polysaccharides are recovered or purified on a filter by suction filtration, etc. In the method for producing sulfated polysaccharides according to the present invention, since the sulfated polysaccharides are secreted outside the cells of cyanobacteria, it is not necessary to disrupt the cells for recovering the sulfated polysaccharides. The cells remaining after recovering the sulfated polysaccharides can be repeatedly used for producing sulfated polysaccharides.

[0019] As one embodiment, when sulfated polysaccharides were produced using the recombinant cyanobacteria of the present invention, in the culture supernatant cultured in BG11 liquid medium for 2 days, it was possible to produce about 19 times as much sulfated polysaccharides as the wild strain.

[0020] 2. Low-temperature induction (1) Cyanobacteria The second aspect of the present invention is a method for producing sulfated polysaccharides using cyanobacteria that efficiently produce sulfated polysaccharides and in which a sulfated polysaccharide synthesis gene group is induced by low temperature.

[0021] The types of cyanobacteria that can be used in the present invention are the same as those shown in the above 1(1). For example, cyanobacteria belonging to the genus Synechocystis, Trichodesmium, Acaryochloris, Crocosphaera, Aphanothece, Anabaena, Nostoc, Rivularia, Arthrospira, Cyanothece, Microcystis, Oscillatoria, or Leptolyngbya, preferably Synechocystis, Aphanothece, Oscillatoria, Cyanothece, Crocosphaera, Anabaena or Nostoc can be mentioned. Among them, Synechocystis sp. PCC6803 substr. PCC-P strain is preferable in that the transcriptional regulation of the sulfated polysaccharide synthesis system by low temperature has already been demonstrated by transcriptome analysis.

[0022] The environmental stimulus of low temperature is sensed by the sensor histidine kinase XssS and transmitted to downstream XssR and XssQ, and the transcription of the sulfated polysaccharide synthesis system Xss is controlled by the transcription regulator XssQ, enabling the mass production of sulfated polysaccharides.

[0023] The method of applying the environmental stimulus of low temperature to cyanobacteria is to set the culture temperature condition to a temperature about 10 degrees lower than the optimum growth temperature. However, it is necessary to examine the optimum low temperature conditions for each type of cyanobacteria to be handled.

[0024] By using the present invention, cyanobacteria exhibit a high ability to produce sulfated polysaccharides as compared to the optimal growth conditions. Further, since the cyanobacteria of the present invention significantly release sulfated polysaccharides into the culture medium, their recovery is easy. Therefore, by culturing the cyanobacteria of the present invention and then recovering the secreted sulfated polysaccharides, efficient production of sulfated polysaccharides can be achieved.

[0025] (2) Culture method Generally, it can be carried out based on liquid culture using BG-11 medium (J Gen Microbiol., 1979, 111:1-61) or its modified method. For sulfated polysaccharide production, it is preferable to culture until the metabolism of cells is activated. For example, it is suitable to carry out aeration and agitation culture or shaking culture for 1 to 7 days. In the present invention, the temperature during this culture period is set to a low temperature. Since the cyanobacteria using the present invention accumulate a large amount of sulfated polysaccharides, if the agitation in liquid culture is weak, cells will form aggregates at an early stage, which will hinder growth. Therefore, it is preferable to agitate strongly in either aeration and agitation culture or shaking culture.

[0026] (3) Recovery method The recovery of sulfated polysaccharides can be carried out in the same manner as described in 1(3) above. By the above culture, cyanobacteria produce sulfated polysaccharides and release the sulfated polysaccharides into the culture medium. Also, when the culture solution is allowed to stand in a state where sulfated polysaccharides have accumulated in the culture medium, cells and sulfated polysaccharides aggregate to form a viscous cell mass like a bloom on the liquid surface. When recovering the secreted sulfated polysaccharides, for the former, solid components such as cells are removed from the whole culture solution by filtration, centrifugation, etc., and after recovering the remaining liquid components, the sulfated polysaccharides are recovered or purified on a filter by suction filtration, etc. In the method for producing sulfated polysaccharides according to the present invention, since sulfated polysaccharides are secreted outside the cells of cyanobacteria, it is not necessary to disrupt the cells for sulfated polysaccharide recovery. The cells remaining after sulfated polysaccharide recovery can be repeatedly used for sulfated polysaccharide production.

[0027] As an embodiment, when the culture method of the present invention was used to produce sulfated polysaccharide, sulfated polysaccharide about three times that of normal culture conditions could be produced in the culture supernatant cultured in BG11 liquid medium for 2 days.

[0028] 3. Enhancement of the expression of response regulator gene or transcriptional regulator gene (1) Preparation method The third aspect of the present invention provides a recombinant cyanobacterium that efficiently produces sulfated polysaccharide, wherein the genes encoding a response regulator and a transcriptional regulator constituting a sulfated polysaccharide synthesis control system are enhanced in expression.

[0029] The types of cyanobacteria that can be used in the present invention are the same as those shown in the above 1(1). For example, cyanobacteria belonging to the genus Synechocystis, Trichodesmium, Acaryochloris, Crocosphaera, Aphanothece, Anabaena, Nostoc, Rivularia, Arthrospira, Cyanothece, Microcystis, Oscillatoria, or Leptolyngbya can be mentioned, and preferably Synechocystis, Aphanothece, Oscillatoria, Cyanothece, Crocosphaera, Anabaena, or Nostoc can be mentioned. Among them, Synechocystis sp. PCC6803 substr. PCC-P strain is preferable in that the transformation operation is very easy.

[0030] A response regulator is a protein involved in signal transduction via phosphorylation and is abundant in cyanobacteria. In most cases, it receives signals from sensor histidine kinases and plays a role in further downstream signal transduction. A transcription regulator is a group of proteins involved in the transcriptional regulation of organisms and is very diverse. However, those dealt with in the present invention are transcription regulators specific to cyanobacteria that receive signals from a two-component control system consisting of a specific sensor histidine kinase and a response regulator and perform transcriptional regulation of the sulfated polysaccharide synthesis system. The recombinant cyanobacteria of the present invention enable mass production of sulfated polysaccharides by enhancing the expression of the xssR gene, which is a specific response regulator that controls the synthesis of sulfated polysaccharides, and the xssQ gene, which is a transcription regulator.

[0031] As a method for enhancing the expression of a response regulator or a transcription regulator, various genetic engineering techniques can be employed. For example, a method of introducing into a neutral site on a target genome by homologous recombination with foreign DNA containing a drug resistance gene cassette, a promoter sequence having strong transcriptional activity, a gene to be enhanced in expression, and a terminator sequence can be mentioned.

[0032] The recombinant cyanobacteria of the present invention have a remarkable ability to produce sulfated polysaccharides. In addition, since the recombinant cyanobacteria of the present invention significantly release sulfated polysaccharides into the medium, they are easy to recover. Therefore, if the recombinant cyanobacteria of the present invention are cultured under appropriate conditions and then the secreted sulfated polysaccharides are recovered, efficient production of sulfated polysaccharides can be achieved.

[0033] (2) Culture method The cultivation of recombinant cyanobacteria can generally be carried out based on liquid culture using BG-11 medium (J Gen Microbiol., 1979, 111:1-61) or its modified methods. For the production of sulfated polysaccharides, it is preferable to culture until the metabolism of the cells is activated. For example, it is suitable to carry out aerated stirring culture or shaking culture for 1 to 7 days. The expression-enhanced strains of the response regulator XssR or the transcriptional regulator XssQ prepared in the present invention significantly accumulate sulfated polysaccharides and form hard biofilms on agar medium. Therefore, when starting liquid culture based on this, it is preferable to suspend it well and then inoculate. Also, if the stirring during liquid culture is weak, cells will form aggregates at an early stage, which will hinder growth. Therefore, it is preferable to stir strongly in either aerated stirring culture or shaking culture.

[0034] (3) Recovery method The recovery of sulfated polysaccharides can be carried out in the same manner as described in 1(3) above. By the above cultivation, cyanobacteria produce sulfated polysaccharides and release the sulfated polysaccharides into the medium. Also, when the culture solution is left standing in a state where sulfated polysaccharides have accumulated in the medium, the cells and sulfated polysaccharides aggregate to form viscous cell masses like blooms on the liquid surface. When recovering the secreted sulfated polysaccharides, for the former, solid components such as cells are removed from the whole culture solution by filtration, centrifugation, etc., and after recovering the remaining liquid components, the sulfated polysaccharides are recovered or purified on a filter by suction filtration, etc. In the method for producing sulfated polysaccharides according to the present invention, since the sulfated polysaccharides are secreted outside the cells of cyanobacteria, it is not necessary to break the cells for sulfated polysaccharide recovery. The cells remaining after sulfated polysaccharide recovery can be repeatedly used for sulfated polysaccharide production.

[0035] As one embodiment, when sulfated polysaccharides were produced using the recombinant cyanobacteria of the present invention, in the culture supernatant cultured in BG11 liquid medium for 2 days, it was possible to produce about 20 times as much sulfated polysaccharides as the wild strain.

[0036] Hereinafter, the present invention will be described in more detail using specific examples, but the present invention is not limited to the following embodiments.

Example

[0037] 1. Preparation of transformants In the preparation of transformants, Synechocystis sp. PCC6803 substr. PCC-P strain (hereinafter referred to as 6803P strain), a model cyanobacterium with a completely sequenced genome, was used as the host.

[0038] The transformation plasmid used for transformation was prepared by the same method as in the previous research paper (J. Biotechnol., 2018, 276:25-33). Briefly, the following procedure was carried out, and substitution of the target gene region by homologous recombination was performed.

[0039] In the preparation of gene disruption strains, a fragment amplified by PCR using the pPCRscript vector as a template and PrimeSTAR (registered trademark) Max DNA Polymerase for the vector region, a fragment amplified in the same manner for a region corresponding to approximately 1000 bp upstream of the target gene region using the 6803P strain genome as a template, a fragment amplified in the same manner for the drug resistance gene cassette region using an arbitrary plasmid as a template, and a fragment amplified in the same manner for a region corresponding to approximately 1000 bp downstream of the target gene region using the 6803P strain genome as a template were ligated using the In-Fusion (registered trademark) HD Cloning Kit and transformed into Escherichia coli.

[0040] In the preparation of gene overexpression strains, a fragment amplified for the vector region using as a template a plasmid in which 1000 bp upstream of a neutral site for 6803 (IS203 region or near the slr0846 region), a strong trc promoter, an rrnB terminator, and 1000 bp downstream of the neutral site were cloned in this order into the pPCRscript vector, and a fragment amplified for the target gene region were ligated using the In-Fusion (registered trademark) HD Cloning Kit and transformed into Escherichia coli. The target plasmid was extracted and purified by the general alkaline-SDS method.

[0041] The transformation of the obtained plasmid into the 6803P strain was carried out using the natural transformation method. For 300 μL of the cell culture solution with aeration and agitation culture and an OD 730 = 0.5 - 1.0, 2 μL of the transformation plasmid was mixed without mixing, and it was spread on a BG11 agar medium (without drugs) with a nitrocellulose membrane placed on it. After culturing for recovery under normal white light for 1 - 2 days, the membrane was transferred to a BG11 agar medium containing the drug for selection of transformants. Colonies of transformants were obtained in about 1 - 2 weeks. The drug concentrations of spectinomycin, kanamycin, and chloramphenicol used for selection were all 20 μg / mL. After subculturing for several generations, it was confirmed by PCR whether the desired homologous recombination was completed in all of the cyanobacterial multicopy genomes. A list of the primers used in the present invention is shown in Tables 1 - 3. The obtained transformants were used as samples for the following sulfated polysaccharide analysis.

[0042]

Table 1

[0043]

Table 2

[0044]

Table 3

[0045] 2. Production of sulfated polysaccharide Using the wild strain and the obtained transformants, sulfated polysaccharide was produced in the following manner. Each strain maintained on an agar medium was inoculated into a BG11 liquid medium containing drugs and cultured with aeration and agitation, which was used as a preculture. Next, using the preculture solution with an OD 730 of 1.0 - 2.0, in 50 mL of BG11 liquid medium with an OD 730The cells were inoculated so that [the value] would be 0.2, and the main culture was started. Both the agar culture and the liquid culture were carried out at a normal culture temperature of 31°C. Only under low-temperature conditions, the main culture was carried out at 20°C, which is 11°C lower than the optimal temperature of the wild strain. Sulfated polysaccharide was constantly released into the medium during the culture. In the following experiment, the sulfated polysaccharide was recovered two days after the start of the main culture.

[0046] When the aerated stirring culture of the 6803P strain culture solution was stopped, as shown in Fig. 1, the culture that was initially suspended throughout the culture solution gradually gathered on the liquid surface over time. The formation of this bloom-like viscous cell mass was due to the buoyancy of photosynthesis-derived air bubbles trapped in the extracellular viscous substance, causing the cells and the viscous substance to float.

[0047] This phenomenon was not observed in another strain of the same species, Synechocystis sp. PCC 6803 substr. GT (hereinafter referred to as the 6803GT strain).

[0048] 3. Analysis of the Viscous Substance To analyze the obtained viscous substance, a simple fractionation method shown in Fig. 2 was constructed. First, the cells were removed by centrifugation (10,000 × g, 10 minutes). Next, the centrifuged supernatant was suction-filtered using a PTFE membrane filter (manufactured by Merck, pore size 0.45 μm) to trap the viscous substance on the membrane. Then, the viscous substance was peeled off and recovered using forceps from the membrane to which about 1 mL of ultrapure water was dropped (the viscous substance fraction in Fig. 2). Samples in this state were used for quantitative analysis. The samples used for the following compositional analysis were further centrifuged (20,000 × g, 10 minutes) to remove fine dust, and then dialyzed three times against 2 L of ultrapure water using a cellulose dialysis tube for 12 hours, 3 hours, and 3 hours. Furthermore, freeze-drying was carried out using a freeze-drying apparatus FDU-810 (manufactured by EYELA), and this was used as the compositional analysis sample.

[0049] As a result of quantitatively analyzing the viscous substance obtained by this method, almost no proteins or nucleic acids were detected, but polysaccharides were detected. Furthermore, as a result of commissioning the above composition analysis sample to Toray Research Center for composition analysis, as shown in Table 4, it was revealed that this polysaccharide is a sulfated polysaccharide.

[0050]

Table 4

[0051] 4. Identification of the synthesis system of sulfated polysaccharide The synthesis system of the obtained sulfated polysaccharide was identified. The inventor prepared a large number of disrupted strains of factors important for polysaccharide synthesis in the same manner as the method described in 1 above. Then, they were cultured by the culture method described in 2 above and screened using the ability to form viscous cell masses as an index.

[0052] Part of the results is shown in FIGS. 3 and 4. As shown in FIG. 3, it was observed that the wild strain (denoted as "WT" in FIG. 3) formed viscous cell masses, but in the disrupted strain of the gene region sll5052 - slr5054 on the pSYSM plasmid of the 6803P strain (denoted as "Δsll5052 - slr5054" in FIG. 3), bloom-like cell masses were not formed. Also, as a result of quantitatively analyzing the viscous substance by the method described in 3 above, as shown in FIG. 4, it became clear that the total sugar content in the viscous substance fraction was significantly decreased. From these results, sll5052 - slr5054 was considered to be a gene involved in sulfated polysaccharide synthesis.

[0053] FIG. 5 is a diagram showing the sulfated polysaccharide synthesis gene cluster of the 6803P strain. White indicates synthesis-related genes, gray indicates control-related genes, and black indicates genes of unknown function. As shown in FIG. 5, the control-related genes were present on a large gene cluster on pSYSM. And on this gene cluster, genes presumed to be related to polysaccharide synthesis and control were accumulated.

[0054] Table 5 shows a list of genes within the sulfated polysaccharide synthesis gene cluster. As a result of creating disruption strains of individual genes presumed to be involved in synthesis and analyzing their phenotypes (data not shown), the contribution of almost all genes to sulfated polysaccharide synthesis was shown.

[0055]

Table 5

[0056] Based on these results, the inventors named this gene cluster xss (extracellular sulfated polysaccharide biosynthesis). Within the xss cluster, there were genes related to polysaccharide synthesis, genes for sulfotransferases, genes for transcriptional regulators, and genes for two-component regulatory systems. The bacterial polysaccharide synthesis system can be broadly divided into three types: the Wzx / Wzy type, the ABC transporter type, and the synthase type (Schmid et al., 2015), and the xss cluster had a gene group characteristic of the Wzx / Wzy type. On the other hand, the gene encoding OPX, a protein that forms an outer membrane pore for discharging sugar chains extracellularly in the Wzx / Wzy type synthesis system, was not present on the xss cluster. However, from experiments using disruption strains, it was shown that the OPX gene sll1581 present on the chromosome was necessary for the synthesis of sulfated polysaccharides (Δsll1581 in Fig. 6(A)), so this gene was named xssT. As a result of the above investigations, a novel sulfated polysaccharide and the gene group involved in its synthesis were identified.

[0057] 5. Production of Sulfated Polysaccharides by Various Gene Disruption Strains There are three regulatory factors in the xss cluster. XssQ was presumed to be a transcriptional regulator from its amino acid sequence. XssR and XssS are factors of the two-component regulatory system, which is a common regulatory system in bacteria, and it was presumed that XssS is a sensor histidine kinase and XssR is a response regulator.

[0058] Thus, we prepared strains in which each gene was disrupted by the method described in 1 above, and examined the phenotypes of these strains in which each gene was disrupted by the culture method described in 2 above. Figure 6 shows the results of sulfated polysaccharide accumulation (A) and viscous cell mass formation (B) in the strains in which the control-related genes were disrupted.

[0059] As shown in Fig. 6(A), the xssQ and xssR disruptants (ΔxssQ and ΔxssR) hardly accumulated sulfated polysaccharides. On the other hand, the xssS disruptant (ΔxssS) accumulated sulfated polysaccharides at a level approximately 19-fold higher than that of the wild-type strain (WT). The ΔxssS strain further transformed with xssK-M disruption (ΔxssS+ΔxssK-M) no longer accumulated sulfated polysaccharides. Furthermore, the xssR and xssQ double disruptant (ΔxssR-S) did not accumulate sulfated polysaccharides. In addition, both the xssR expression-enhanced strain (OX-xssR) and the xssQ expression-enhanced strain (OX-xssQ) showed high accumulation of sulfated polysaccharides, similar to that of the xssS disruptant. These results suggest that xssS suppresses sulfated polysaccharide synthesis by negatively regulating xssQ via xssR.

[0060] As shown in Table 4, the compositional analysis results of the sulfated polysaccharides that accumulated in the xssS-disrupted strain were different from those of the wild-type strain. Since it is common for one species of cyanobacteria to have multiple extracellular polysaccharides, it is considered that the composition of the sulfated polysaccharides synthesized by the xss gene cluster in the xssS-disrupted strain is significantly reflected by the dramatic increase in the amount of accumulated sulfated polysaccharides.

[0061] 6. Measurement by Real-time Quantitative PCR Since XssQ was assumed to be a transcription factor, we investigated its relationship with the transcription of major xss genes by real-time quantitative PCR. The procedure for real-time quantitative PCR is as follows.

[0062] At the same time as the recovery of the sulfated polysaccharide, the cells were recovered by centrifugation (5000 × g, 4°C, 10 minutes). RNA extraction was performed using the RNeasy Mini kit for bacteria (Qiagen) according to the protocol recommended by the kit. cDNA synthesis was carried out using the PrimeScript RT reagent kit with gDNA eraser (manufactured by Takara). The real-time quantitative PCR reaction was performed using the THUNDERBIRD SYBR qPCR Mix (manufactured by Toyobo) and the Thermal Cycler Dice Real Time System II (manufactured by Takara). The rnpB gene was used as the internal standard for comparing gene expression levels. The primers used are shown in Table 6.

[0063] In the xssS disruption strain, which is a sulfated polysaccharide high-accumulating strain, an increasing trend in the transcription levels of some genes was confirmed, while in the xssQ disruption strain, which is a sulfated polysaccharide non-accumulating strain, a significant decreasing trend in the transcription levels of some genes was confirmed (Figure 7).

[0064]

Table 6

[0065] From these results, it was considered that the synthesis of the sulfated polysaccharide was controlled by the transcriptional regulation of some xss genes by the transcriptional regulator XssQ. In addition, it was speculated that the sulfated polysaccharide high-accumulating strain of the present invention was due to the enhancement of the transcriptional level of the xss gene by the release of suppression by the knockout of XssS.

[0066] 7. Measurement of the accumulation amount of sulfated polysaccharide at low temperature In the previous transcriptome study of the 6803P strain, it was shown that a group of genes with significant transcriptional variation were transcriptionally induced at low temperature (20°C) (Kopf et al. 2014). Therefore, by performing the main culture of the operation described in 2 at 20°C and conducting quantitative analysis in the same way as other strains, the amount of sulfated polysaccharide accumulated at low temperature was measured. As a result, it increased by about three times compared to the amount accumulated at the normal culture temperature (31°C) of the wild strain (Figure 6, Table 7).

[0067] This result suggests that the environmental stimulus sensed by the sensor XssS may be low temperature. That is, even in species where it is difficult to disrupt the gene of the sensor histidine kinase, it was shown that the sulfated polysaccharide synthesis system can be activated by exposing to low temperature, improving the sulfated polysaccharide productivity.

[0068]

Table 7

Claims

Claim 1 A cyanobacterium that produces a sulfated polysaccharide, comprising a sulfated polysaccharide synthesis gene system and a sulfated polysaccharide synthesis control gene system, wherein the sulfated polysaccharide synthesis control gene system is composed of a gene encoding a transcriptional regulator, a gene encoding a response regulator, and a gene encoding a sensor histidine kinase, wherein the activation of the transcription of any one of the gene encoding the transcriptional regulator, the gene encoding the response regulator, and the gene encoding the sensor histidine kinase is controlled, wherein the cyanobacterium satisfies any one of the following (a) to (c): (a) The gene encoding the sensor histidine kinase is the xssS gene and the xssS gene is disrupted; (b) The gene encoding the response regulator is the xssR gene and the expression of the xssR gene is enhanced; (c) The gene encoding the transcriptional regulator is the xssQ gene and the expression of the xssQ gene is enhanced. Claim 2 The cyanobacterium according to claim 1, wherein the cyanobacterium is Synechocystis sp. PCC6803 substr. PCC-P strain. Claim 3 A method for producing a sulfated polysaccharide, comprising the step of culturing the cyanobacterium according to claim 1 or 2. Claim 4 The method for producing a sulfated polysaccharide according to claim 3, comprising, after the culturing step, a step of separating the cyanobacterium and the culture supernatant by centrifugation, and a step of collecting the sulfated polysaccharide from the supernatant by suction filtration through filtration. A method for producing a sulfated polysaccharide.

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