JP7710705B2 - Cyanobacteria producing sulfated polysaccharides and method for producing sulfated polysaccharides derived from cyanobacteria - Google Patents

Description

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

Polysaccharides, which have a structure in which monosaccharides are linked in a chain, are extremely important biopolymers along with nucleic acids and proteins, and are highly diverse due to differences in monosaccharide composition and modifications. Sulfated polysaccharides are a type of acidic polysaccharide modified with sulfate groups, and are found in animals, eukaryotic algae, archaea, and cyanobacteria. They are not only involved in various important functions in living organisms, but have also attracted attention in recent years as thickeners, water-retaining agents, and pharmaceuticals (anticancer, antiviral, and anticoagulant agents) (Hayashi, 2008). Currently, sulfated polysaccharides derived from microorganisms are obtained by collecting and purifying polysaccharides produced by wild strains of bacteria such as algae and cyanobacteria, or by sulfating non-sulfated polysaccharides using chemical catalysts.

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

International Publication No. 2003/023045

Research on bacterial extracellular polysaccharides has progressed, and various synthesis systems have been elucidated (Becker et al., 1998, Schmid et al., 2015). However, the sulfate polysaccharide synthesis system in cyanobacteria is unknown. In addition, typical cyanobacteria that are known to have sulfate polysaccharides are difficult to transform because the extracellular polysaccharides are 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.

The present invention aims to establish a method for producing sulfate polysaccharides derived from cyanobacteria, and to mass-produce sulfate polysaccharides and modify their composition.

In order to solve the above problems, the present inventors identified a group of genes involved in the synthesis of sulfated polysaccharides and investigated means for controlling genes involved in transcriptional control among them, and discovered that it was possible to accumulate a significant amount of sulfated polysaccharides in a medium.

The present invention is based on this finding, and provides a cyanobacterium that produces sulfate polysaccharide, the cyanobacterium comprising sulfate polysaccharide synthesis system genes and sulfate polysaccharide synthesis control system genes, the sulfate polysaccharide synthesis control system genes being composed of a gene encoding a transcription control factor, a gene encoding a response regulator, and a gene encoding a sensor histidine kinase, and the transcriptional activation of any one of the gene encoding the transcription control factor, the gene encoding the response regulator, and the gene encoding the sensor histidine kinase is controlled.

The present invention also provides a method for producing sulfated polysaccharides, the method including a step of culturing the cyanobacteria.

According to the present invention, sulfate polysaccharides derived from cyanobacteria can be efficiently produced.

These are images showing the time course of the formation of bloom-like viscous cell masses produced in a culture medium in which a transformant (Δ6803P strain) was cultured. FIG. 1 is a diagram for explaining a fractionation method for analyzing viscous substances. 13 shows images depicting the results of observing the ability of viscous cell mass formation in the sll5052-slr5054 disruptant of the 6803P strain. FIG. 1 shows the results of analysis of viscous substances produced by the sll5052-slr5054 disruptant of the 6803P strain. FIG. 1 shows the sulfated polysaccharide synthesis gene cluster of 6803P. FIG. 1 shows the results of sulfated polysaccharide accumulation (A) and bloom formation (B) in the regulatory gene disruptant. FIG. 1 shows the results of examining the transcriptional changes of major xss genes due to xssQ and xssS disruption by real-time PCR.

1. Recombinant Cyanobacteria (1) Preparation of Recombinant Cyanobacteria The first aspect of the present invention provides a recombinant cyanobacterium that efficiently produces sulfated polysaccharides, in which a gene encoding a sensor histidine kinase has been disrupted.

Examples of species of cyanobacteria that can be used in the present invention include the genera Synechocystis, Trichodesmium, Acaryochloris, Crocosphaera, Aphanothece, Anabaena, Nostoc, Rivularia, Arthrospira, Cyanothece, It is a cyanobacterium belonging to the genus Microcystis, Oscillatoria, or Leptolyngbya, and preferably includes the genus Synechocystis, Aphanothece, Oscillatoria, Cyanothece, Crocosphaera, Anabaena, or Nostoc. Among them, Synechocystis sp. PCC6803 substr. PCC-P strain is preferred because it is very easy to transform.

Sensor histidine kinase is an enzyme involved in signal transduction via phosphorylation and is abundant in cyanobacteria. The recombinant cyanobacteria of the present invention knock out a specific enzyme that controls the synthesis of sulfate polysaccharides, making it possible to mass-produce sulfate polysaccharides.

Various genetic engineering techniques can be used to knock out the sensor histidine kinase, for example, a knockout method using homologous recombination with foreign DNA containing a drug resistance gene cassette can be mentioned. Note that cyanobacteria have a multicopy genome, i.e., multiple copies of the genome exist, so there are as many genes to be disrupted as there are genomes. Therefore, although a small amount of wild-type genome may be detected in a disrupted strain, in this embodiment, in addition to disrupted strains in which all sensor histidine kinases are knocked out, recombinant strains in which a small amount of wild-type genome is detected if a significant phenotype is observed are also included in the disrupted strains in which sensor histidine kinases are knocked out.

The recombinant cyanobacteria of the present invention have a remarkable ability to produce sulfate polysaccharides. In addition, the recombinant cyanobacteria of the present invention release sulfate polysaccharides into the medium in a remarkable manner, making them easy to recover. Therefore, efficient sulfate polysaccharide production can be achieved by culturing the recombinant cyanobacteria of the present invention under appropriate conditions and then recovering the secreted sulfate polysaccharides.

(2) Cultivation method The recombinant cyanobacteria can generally be cultured in liquid culture using BG-11 medium (J Gen Microbiol., 1979, 111:1-61) or a modified method thereof. For sulfate polysaccharide production, it is preferable to culture until the metabolism of the cells is activated, for example, it is preferable to perform 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 accumulates sulfate polysaccharide significantly and forms a hard biofilm on the agar medium. Therefore, when starting liquid culture based on it, it is preferable to suspend it well and then seed it. In addition, if the agitation during liquid culture is weak, the cells will form aggregates at an early stage and growth will be hindered, so it is preferable to agitate strongly in either aeration and agitation culture or shaking culture.

(3) Recovery method By the above-mentioned culture, the cyanobacteria produce sulfate polysaccharides and release the sulfate polysaccharides into the medium. When the culture solution is allowed to stand with sulfate polysaccharides accumulated in the medium, the cells and sulfate polysaccharides aggregate to form a viscous cell mass like a bloom on the liquid surface. When recovering the secreted sulfate polysaccharides, solids such as cells are removed by filtration, centrifugation, etc. from the entire culture solution in the former case, or from the viscous cell mass in the latter case, and the remaining liquid components are recovered, and then the sulfate polysaccharides are recovered or purified in a filter by suction filtration, etc. In the method for producing sulfate polysaccharides according to the present invention, since sulfate polysaccharides are secreted outside the cells of the cyanobacteria, there is no need to destroy the cells to recover the sulfate polysaccharides. The cells remaining after the recovery of sulfate polysaccharides can be repeatedly used for sulfate polysaccharide production.

As one embodiment, sulfate polysaccharides were produced using the recombinant cyanobacteria of the present invention. After culturing for two days in BG11 liquid medium, the culture supernatant produced approximately 19 times as much sulfate polysaccharide as the wild-type strain.

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

The types of cyanobacteria that can be used in the present invention are the same as those listed in 1(1) above. For example, the genera Synechocystis, Trichodesmium, Acaryochloris, Crocosphaera, Aphanothece, Anabaena, Nostoc, Rivularia, Arthrospira, Cyanothece, Microcystis, and the like. cystis, Oscillatoria, or Leptolyngbya, and preferably Synechocystis, Aphanothece, Oscillatoria, Cyanothece, Crocosphaera, Anabaena, or Nostoc. Among them, Synechocystis sp. PCC6803 substr. PCC-P strain is preferred because transcriptional regulation of sulfate polysaccharide synthesis by low temperature has already been demonstrated by transcriptome analysis.

The environmental stimulus of low temperature is sensed by the sensor histidine kinase XssS and transmitted to the downstream XssR and XssQ. The transcription of the Xss pathway in the sulfate polysaccharide synthesis pathway is controlled by the transcription factor XssQ, enabling mass production of sulfate polysaccharides.

The method for providing the cyanobacteria with the environmental stimulus of low temperature is to set the culture temperature at a temperature about 10 degrees lower than the optimal growth temperature. However, the optimal low temperature conditions must be considered for each cyanobacteria species being treated.

By using the present invention, cyanobacteria can exhibit a high sulfate polysaccharide production ability compared to optimal growth conditions. In addition, since the cyanobacteria of the present invention release a significant amount of sulfate polysaccharide into the medium, it is easy to recover it. Therefore, by culturing the cyanobacteria of the present invention and then recovering the secreted sulfate polysaccharide, efficient sulfate polysaccharide production can be achieved.

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

(3) Recovery Method Sulfate polysaccharide can be recovered by the same method as in 1(3) above. By the above-mentioned culture, cyanobacteria produce sulfate polysaccharide and release the sulfate polysaccharide into the medium. In addition, when the culture solution is left to stand with sulfate polysaccharide accumulated in the medium, the cells and sulfate polysaccharide aggregate to form a viscous cell mass like a bloom on the liquid surface. When the secreted sulfate polysaccharide is recovered, solids such as cells are removed by filtration, centrifugation, etc. from the entire culture solution in the former case, or from the viscous cell mass in the latter case, and the remaining liquid components are recovered, and then the sulfate polysaccharide is recovered or purified in a filter by suction filtration, etc. In the method for producing sulfate polysaccharide according to the present invention, sulfate polysaccharide is secreted outside the cells of cyanobacteria, so there is no need to destroy the cells to recover sulfate polysaccharide. The cells remaining after sulfate polysaccharide recovery can be repeatedly used for sulfate polysaccharide production.

As one embodiment, the culture method of the present invention was used to produce sulfated polysaccharides. After culturing for two days in BG11 liquid medium, the culture supernatant produced about three times as much sulfated polysaccharide as under normal culture conditions.

3. Enhancement of Expression of Response Regulator Gene or Transcriptional Control Factor Gene (1) Production Method The third aspect of the present invention provides a recombinant cyanobacterium that efficiently produces sulfate polysaccharides, in which expression of genes encoding a response regulator and a transcriptional control factor that constitute a sulfate polysaccharide synthesis control system is enhanced.

The types of cyanobacteria that can be used in the present invention are the same as those shown in 1(1) above. For example, the genera Synechocystis, Trichodesmium, Acaryochloris, Crocosphaera, Aphanothece, Anabaena, Nostoc, Rivularia, Arthrospira, Cyanothece, Microcystis, and the like. cystis, Oscillatoria, or Leptolyngbya, and preferably Synechocystis, Aphanothece, Oscillatoria, Cyanothece, Crocosphaera, Anabaena, or Nostoc. Among them, Synechocystis sp. PCC6803 substr. PCC-P strain is preferred because it is very easy to transform.

Response regulators are proteins involved in signal transduction via phosphorylation and are abundant in cyanobacteria. In most cases, they receive signals from sensor histidine kinases and transmit signals downstream. Transcription factors are a group of proteins involved in transcriptional control in organisms and are extremely diverse, but the one dealt with in the present invention is a transcription factor specific to cyanobacteria that receives signals from a two-component control system consisting of a specific sensor histidine kinase and a response regulator and controls the transcription of the sulfate polysaccharide synthesis system. The recombinant cyanobacteria of the present invention enable mass production of sulfate polysaccharides by enhancing the expression of the xssR gene, a specific response regulator that controls the synthesis of sulfate polysaccharides, and the xssQ gene, a transcription factor.

Various genetic engineering techniques can be used to enhance the expression of a response regulator or transcription factor, but examples include a method of introducing a drug resistance gene cassette, a promoter sequence with strong transcriptional activity, a gene whose expression is enhanced, or foreign DNA containing a terminator sequence into a neutral site on the target genome by homologous recombination.

The recombinant cyanobacteria of the present invention have a remarkable ability to produce sulfate polysaccharides. In addition, the recombinant cyanobacteria of the present invention release sulfate polysaccharides into the medium in a remarkable manner, making them easy to recover. Therefore, efficient sulfate polysaccharide production can be achieved by culturing the recombinant cyanobacteria of the present invention under appropriate conditions and then recovering the secreted sulfate polysaccharides.

(2) Cultivation method Recombinant cyanobacteria can generally be cultured using BG-11 medium (J Gen Microbiol., 1979, 111:1-61) or a modified method thereof. For sulfate polysaccharide production, it is preferable to culture until the metabolism of the cells is activated, for example, it is preferable to perform aeration and agitation culture or shaking culture for 1 to 7 days. The strains with enhanced expression of the response regulator XssR or transcriptional regulator XssQ prepared in the present invention accumulate sulfate polysaccharides significantly and form hard biofilms on agar media. Therefore, when starting liquid culture based on the strain, it is preferable to suspend the cells well before seeding. In addition, if the agitation during liquid culture is weak, the cells will form aggregates at an early stage, which will hinder growth, so it is preferable to agitate the cells strongly in either aeration and agitation culture or shaking culture.

(3) Recovery Method Sulfate polysaccharide can be recovered by the same method as in 1(3) above. By the above-mentioned culture, cyanobacteria produce sulfate polysaccharide and release the sulfate polysaccharide into the medium. In addition, when the culture solution is left to stand with sulfate polysaccharide accumulated in the medium, the cells and sulfate polysaccharide aggregate to form a viscous cell mass like a bloom on the liquid surface. When the secreted sulfate polysaccharide is recovered, solids such as cells are removed by filtration, centrifugation, etc. from the entire culture solution in the former case, or from the viscous cell mass in the latter case, and the remaining liquid components are recovered, and then the sulfate polysaccharide is recovered or purified in a filter by suction filtration, etc. In the method for producing sulfate polysaccharide according to the present invention, sulfate polysaccharide is secreted outside the cells of cyanobacteria, so there is no need to destroy the cells to recover sulfate polysaccharide. The cells remaining after sulfate polysaccharide recovery can be repeatedly used for sulfate polysaccharide production.

As one embodiment, sulfate polysaccharides were produced using the recombinant cyanobacteria of the present invention. After culturing for two days in BG11 liquid medium, the culture supernatant produced about 20 times as much sulfate polysaccharide as the wild-type strain.

The present invention will be described in more detail below using specific examples, but the present invention is not limited to the following embodiments.

1. Preparation of transformants To prepare transformants, Synechocystis sp. PCC6803 substr. PCC-P strain (hereinafter referred to as 6803P strain), a model cyanobacterium whose genome has been completely sequenced, was used as the host.

The transformation plasmid used for transformation was prepared using the same method as in a previous research paper (J. Biotechnol., 2018, 276:25-33). Briefly, the procedure was as follows, and the target gene region was replaced by homologous recombination.

To prepare the gene disruption strain, a total of four fragments were ligated using the In-Fusion (registered trademark) HD Cloning Kit and transformed into E. coli: a fragment obtained by amplifying the vector region by PCR using PrimeSTAR (registered trademark) Max DNA Polymerase with the pPCRscript vector as a template; a fragment obtained by similarly amplifying a region equivalent to approximately 1000 bp upstream of the target gene region with the 6803P strain genome as a template; a fragment obtained by similarly amplifying a drug resistance gene cassette region with a plasmid of your choice as a template; and a fragment obtained by similarly amplifying a region equivalent to approximately 1000 bp downstream of the target gene region with the 6803P strain genome as a template.

To create a strain with enhanced gene expression, a plasmid in which 1000 bp upstream of the neutral site for 6803 (IS203 region or region adjacent to slr0846), a strong trc promoter, an rrnB terminator, and 1000 bp downstream of the neutral site were cloned in this order into the pPCRscript vector was used as a template, and a fragment in which the vector region was amplified was combined with a fragment in which the target gene region was amplified using the In-Fusion (registered trademark) HD Cloning Kit, and transformed into E. coli. The target plasmid was extracted and purified using a standard alkaline-SDS method.

The resulting plasmid was transformed into the 6803P strain by natural transformation. 2 μL of the transformation plasmid was mixed with 300 μL of the bacterial culture solution, which had been cultured with aeration and agitation to an OD ₇₃₀ of 0.5 to 1.0, and the mixture was spread on a BG11 agar medium (without drugs) with a nitrocellulose membrane. After 1 to 2 days of recovery culture under normal white light, the membrane was transferred to a BG11 agar medium containing a drug for selecting transformants. Colonies of transformants were obtained in about 1 to 2 weeks. The drug concentrations of spectinomycin, kanamycin, and chloramphenicol used for selection were all 20 μg/mL. After several generations of subculture, it was confirmed by PCR whether the desired homologous recombination had been completed in all of the cyanobacterial multicopy genomes. The primers used in the present invention are listed in Tables 1 to 3. The resulting transformants were used as samples for the following sulfated polysaccharide analysis.

2. Production of sulfated polysaccharides Using the wild-type strain and the obtained transformants, sulfated polysaccharides were produced as follows. Each strain maintained on agar medium was inoculated into BG11 liquid medium containing a drug and cultured under aeration and agitation, which was used as a preculture. Next, using the preculture liquid with an OD ₇₃₀ of 1.0 to 2.0, the cells were inoculated into 50 mL of BG11 liquid medium to give an OD ₇₃₀ of 0.2, and main culture was started. The normal culture temperature for both agar culture and liquid culture was 31°C. Only under the low-temperature condition, main culture was performed at 20°C, which is 11°C lower than the optimal temperature for the wild-type strain. Sulfated polysaccharides were constantly released into the medium during culture. In the following experiments, sulfated polysaccharides were collected two days after the start of main culture.

When the aeration and agitation culture of the 6803P strain culture medium was stopped, the culture material that was initially suspended throughout the medium gradually gathered at the surface over time, as shown in Figure 1. The formation of this bloom-like viscous cell mass was caused by the cells and viscous material rising to the surface due to the buoyancy provided by photosynthetic air bubbles trapped in the extracellular viscous material.

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

3. Analysis of Viscous Substances To analyze the obtained viscous substances, a simple fractionation method was developed as shown in Figure 2. First, the bacterial cells were removed by centrifugation (10,000 × g, 10 min). Next, the centrifugal supernatant was suction filtered using a PTFE membrane filter (Merck, pore size 0.45 μm) to trap the viscous substances in the membrane. Then, the viscous substances were peeled off and collected using tweezers from the membrane on which about 1 mL of ultrapure water was dropped (viscous substance fraction in Figure 2). The sample in this state was used for quantitative analysis. The sample subjected to the composition analysis described below was further centrifuged (20,000 × g, 10 min) to remove fine debris, and then dialyzed three times against 2 L of ultrapure water using a cellulose dialysis tube, overnight, for 3 hours, and for 3 hours. Furthermore, the sample was freeze-dried using a freeze-drying device FDU-810 (EYELA), and this was used as the composition analysis sample.

Quantitative analysis of the viscous substance obtained by this method detected almost no protein or nucleic acid, but only polysaccharide. Furthermore, the composition analysis of the above composition analysis sample was entrusted to Toray Research Center, and the results showed that the polysaccharide was sulfate polysaccharide, as shown in Table 4.

4. Identification of the synthesis system of sulfated polysaccharides The synthesis system of the obtained sulfated polysaccharides was identified. The inventors prepared many strains in which factors important for polysaccharide synthesis were disrupted, using the same method as described in 1 above. Then, they were cultured using the culture method described in 2 above, and screened using the ability to form viscous cell masses as an index.

Part of the results are shown in Figures 3 and 4. As shown in Figure 3, the wild-type strain (indicated as "WT" in Figure 3) was observed to form viscous cell masses, but in the 6803P strain, a disruptant of the gene region sll5052 - slr5054 on the pSYSM plasmid (indicated as "Δsll5052 - slr5054" in Figure 3), no bloom-like cell masses were formed. Furthermore, quantitative analysis of the viscous substance using the method described in 3 above revealed a significant decrease in the total sugar content in the viscous substance fraction, as shown in Figure 4. From these results, it was believed that sll5052 - slr5054 are genes involved in sulfated polysaccharide synthesis.

Figure 5 shows the sulfated polysaccharide synthesis gene cluster of the 6803P strain. Synthesis-related genes are indicated in white, regulation-related genes in grey, and genes of unknown function in black. As shown in Figure 5, the regulation-related genes were present on a large gene cluster on pSYSM. Furthermore, genes presumed to be related to polysaccharide synthesis and regulation were concentrated on this gene cluster.

Table 5 shows a list of genes in the sulfate polysaccharide synthesis gene cluster. We created mutants of individual genes presumed to be involved in synthesis and analyzed the phenotypes (data not shown), which showed that almost all genes contribute to sulfate polysaccharide synthesis.

Based on these results, the inventors named this gene cluster xss (extracellular sulfated polysaccharide biosynthesis). The xss cluster contained genes involved in polysaccharide synthesis, genes for sulfotransferases, genes for transcriptional regulators, and genes for two-component control systems. Bacterial polysaccharide synthesis systems are broadly divided into three types: Wzx/Wzy type, ABC transporter type, and synthase type (Schmid et al., 2015), and the xss cluster contained a group of genes characteristic of the Wzx/Wzy type. On the other hand, the xss cluster did not contain a gene encoding OPX, a protein that forms an outer membrane pore to export glycans to the outside of the cell in the Wzx/Wzy synthesis system. However, experiments using a disruptant showed that the OPX gene sll1581 present on the chromosome is necessary for the synthesis of sulfated polysaccharides (Δsll1581 in Figure 6 (A)), and this gene was named xssT. As a result of the above investigations, a novel sulfate polysaccharide and the gene cluster involved in its synthesis were identified.

5. Production of sulfated polysaccharides by various gene disruptants
The xss cluster contains three regulatory factors. XssQ is predicted to be a transcription factor based on its amino acid sequence. XssR and XssS are factors of a two-component regulatory system, which is a common regulatory system in bacteria. XssS is predicted to be a sensor histidine kinase, and XssR is predicted to be a response regulator.

Therefore, we prepared knockout strains of each gene using the method described in 1 above, and investigated the phenotypes of these knockout strains using 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 knockout strains of the control-related genes.

As shown in Fig. 6 (A), the xssQ and xssR disruptants (ΔxssQ and ΔxssR) showed little accumulation of sulfated polysaccharides. On the other hand, the xssS disruptant (ΔxssS) showed an increase in sulfated polysaccharide accumulation of about 19-fold compared to the wild-type strain (WT). The ΔxssS strain further transformed with xssK-M disruption (ΔxssS+ΔxssK-M) showed no sulfated polysaccharide accumulation. Furthermore, the xssR and xssQ double disruptant (ΔxssR-S) showed no sulfated polysaccharide accumulation. In addition, both the xssR expression-enhanced strain (OX-xssR) and the xssQ expression-enhanced strain (OX-xssQ) showed high sulfated polysaccharide accumulation, similar to that of the xssS disruptant. These results suggest that xssS inhibits sulfate polysaccharide synthesis by negatively regulating xssQ via xssR.

As shown in Table 4, the compositional analysis results of the sulfated polysaccharides that accumulated in high amounts 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 thought that the composition of the sulfated polysaccharides synthesized by the xss gene group in the xssS-disrupted strain is significantly reflected by the dramatic increase in the amount of accumulated sulfated polysaccharides.

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.

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

The xssS disruptant, a strain that accumulates sulfated polysaccharides, showed a tendency for the transcription levels of some genes to increase, while the xssQ disruptant, a strain that does not accumulate sulfated polysaccharides, showed a tendency for the transcription levels of some genes to decrease significantly (Figure 7).

From these results, it was considered that the synthesis of the sulfated polysaccharides was controlled by the transcriptional regulation of some of the xss genes by the transcription factor XssQ. It was also speculated that the high sulfated polysaccharide accumulation strain of the present invention was caused by the enhanced transcription level of the xss genes due to the release of repression by knocking out XssS.

7. Measurement of sulfated polysaccharide accumulation at low temperatures Previous transcriptome studies of the 6803P strain showed that the genes whose transcription levels were found to vary significantly this time were transcriptionally induced at low temperatures (20°C) (Kopf et al. 2014). Thus, we performed the main culture at 20°C as described in 2 and quantitatively analyzed the amount of sulfated polysaccharide accumulated at low temperatures, which was approximately three times higher than the amount accumulated at the normal culture temperature (31°C) of the wild-type strain (Figure 6, Table 7).

This result suggests that the environmental stimulus sensed by the sensor XssS may be low temperature. In other words, even in species where gene disruption of the sensor histidine kinase is difficult, exposure to low temperature may be able to activate the sulfate polysaccharide synthesis pathway and improve sulfate polysaccharide productivity.

Claims (4)

A cyanobacterium that produces sulfated polysaccharides,
The present invention provides a sulfated polysaccharide synthesis system gene and a sulfated polysaccharide synthesis control system gene,
the sulfated polysaccharide synthesis control system genes are composed of a gene encoding a transcription control factor, a gene encoding a response regulator, and a gene encoding a sensor histidine kinase;
the transcriptional activation of any one of the gene encoding the transcription control factor, the gene encoding the response regulator, and the gene encoding the sensor histidine kinase is regulated;
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 has been disrupted; (b) the gene encoding the response regulator is the xssR gene, and the expression of the xssR gene has been enhanced; (c) the gene encoding the transcription control factor is the xssQ gene, and the expression of the xssQ gene has been enhanced. The cyanobacterium according to claim 1, wherein the cyanobacterium is Synechocystis sp. PCC6803 substr. PCC-P strain. 1. A method for producing sulfated polysaccharides, comprising the steps of:
A method for producing sulfated polysaccharides, comprising a step of culturing the cyanobacterium according to claim 1 or 2. A method for producing the sulfated polysaccharide according to claim 3, comprising the steps of:
After the culturing step, a step of separating the cyanobacteria and the culture supernatant by centrifugation;
filtering the supernatant to separate sulfated polysaccharides by suction filtration;
A method for producing a sulfated polysaccharide, comprising: