Method for producing sulfated polysaccharides and method for producing PAPS

Abstract

To provide a method for easily producing sulfated polysaccharides by reacting a PAPS production / regeneration system utilizing the metabolic activity of a microorganism or a treated matter thereof with a microorganism expressing a sulfation enzyme or a treated matter or extract thereof upon mixing of inexpensive raw materials such as magnesium sulfate; and to provide a practical method for producing PAPS from inexpensive raw materials.SOLUTION: The present invention relates to a method for producing a sulfated polysaccharide and a method for producing PAPS. The methods comprise a step of preparing a transformant (a) of a bacterium of the genus Corynebacterium, which comprises at least a gene encoding an ATP sulfurylase and a gene encoding an APS kinase which are introduced thereinto in an expressible manner, and in which a cell plasma membrane of the transformant (a) is substance-permeable, or a treated matter of the transformant (a); and a step of conducting a reaction for producing PAPS by using a reaction solution containing ATP or an ATP source, a sulfate ion source, and the transformant (a) or the treated matter thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for producing 3'-phosphoadenosine 5'-phosphosulfate (hereinafter referred to as PAPS) from inexpensive raw materials such as glucose and adenine using coryneform bacteria expressing ATP sulfurylase and APS kinase (Adenylsulfate kinase), and a method for producing sulfated polysaccharides using microorganisms belonging to prokaryotes expressing the coryneform bacteria and various sulfating enzymes.

Background Art

[0002] PAPS widely exists from microorganisms to higher organisms and is a coenzyme that functions as a sulfate group donor in vivo. In higher organisms, it is required for the biosynthesis of glycosaminoglycans such as chondroitin sulfate and heparan sulfate. Sulfated in vivo metabolites are known to have various physiological functions, and PAPS itself is expected to be used in applications such as hair growth agents (Patent Document 1) and topical skin agents having a moisturizing effect (Patent Document 2).

[0003] As methods for producing PAPS, there are a method using yeast-derived ATP sulfurylase, Aspergillus niger-derived APS kinase, and Escherichia coli-derived pyrophosphatase purified using ATP as a raw material (Non-Patent Document 1), and also a method using thermostable ATP sulfurylase, APS kinase derived from thermophilic bacteria, and yeast-derived pyrophosphatase using ATP as a raw material (Patent Document 3), and a method using ATP sulfurylase, APS kinase derived from thermophilic bacteria, and Pseudomonas aeruginosa-derived polyphosphate kinase using adenosine 5'-monophosphate (hereinafter referred to as AMP) as a raw material (Patent Document 4), etc. are known.

[0004] On the other hand, a method for industrially producing ATP using inexpensive raw materials such as glucose and adenine by treating the cell membrane of a mutant strain of Corynebacterium ammoniagenes with xylene or a surfactant to impart permeability is known (Non-Patent Document 2).

[0005] PAPS is a very unstable compound. Even in the method using crude enzymes, crude enzymes of ATP sulfurylase and APS kinase derived from a thermotolerant bacterium recombinantly expressed with Escherichia coli as the host are prepared, heat-treated before the reaction, and PAPS is produced by suppressing the contaminating enzyme activity derived from Escherichia coli (Patent Document 4).

[0006] It is described in PTL8 that PAPS is decomposed in the crude enzyme solution of Escherichia coli. Escherichia coli has several enzymes involved in the decomposition of PAPS, and the decomposition of PAPS can be suppressed by deleting the genes of those enzymes.

[0007] Heparin, a sulfated polysaccharide, is a major anticoagulant and is used for the treatment of thromboembolic diseases, disseminated intravascular coagulation syndrome, and for preventing coagulation during hemodialysis and extracorporeal circulation. Industrially, heparin is mainly extracted and purified from porcine intestinal mucosa. Since a fatal accident occurred in 2008 due to the contamination of porcine-derived heparin with impurities, research and development of manufacturing management / quality control of non-animal-derived heparin production have been carried out.

[0008] As a specific example, N-acetylheparosan (hereinafter abbreviated as "heparosan"), which is a capsular polysaccharide of Gram-negative microorganisms, is fermentatively produced and purified, chemically N-deacetylated and N-sulfated, and then epimerized enzymatically to produce heparin having the same structure and anticoagulant activity as the porcine-derived product (Patent Documents 5 to 7, Non-Patent Documents 3 and 4).

[0009] Another useful sulfated polysaccharide is chondroitin sulfate, which is used as a pharmaceutical for joint pain and an eye drop for corneal surface protection. Although chondroitin sulfate purified from various animal tissues is used, a method of producing chondroitin sulfate using a sulfating enzyme with a capsular polysaccharide derived from Escherichia coli as a raw material has been reported in recent years (Non-Patent Documents 5 and 6).

[0010] In the method of sulfation using an enzyme purified from a polysaccharide derived from the capsule of a microorganism, 3'-phosphoadenosine 5'-phosphate (hereinafter referred to as PAP) generated by the transfer of a sulfate group of PAPS to the substrate polysaccharide requires PAPS as a coenzyme in the reaction of the sulfating enzyme. In the method for producing a sulfated polysaccharide using the polysaccharide derived from the capsule of the above microorganism as a raw material, the sulfate group of p-nitrophenyl sulfate is enzymatically transferred to PAP to regenerate PAPS, and the enzymatic sulfation reaction of the polysaccharide proceeds (Patent Document 6, Non-Patent Document 3).

Prior Art Documents

Patent Documents

[0011]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

Patent Document 6

Patent Document 7

Patent Document 8

Non-Patent Documents

[0012]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Summary of the Invention

Problems to be Solved by the Invention

[0013] In the conventional methods for producing PAPS described in Non-Patent Document 1, Patent Documents 1 and 2 above, all use relatively expensive nucleotides such as ATP and AMP as raw materials. After culturing purified enzymes or bacteria, the cells are separated, and crude enzymes prepared by disrupting the cells by ultrasonic treatment or the like and centrifugation are used. Industrial-scale implementation is not easy.

[0014] On the other hand, as described above, regarding the production of PAPS, methods using purified enzymes or crude enzymes with ATP or AMP as raw materials are known, but examples of producing PAPS using the microorganism itself have not been known so far. Also, as described above, PAPS is a very unstable compound, and in the prior art, when using crude enzymes, complicated steps such as suppressing contaminating enzyme activity by heat treatment have been carried out.

[0015] Considering the above, it is difficult to predict that PAPS can be produced by a method similar to the method for industrially producing ATP by simply expressing ATP sulfurylase and APS kinase using corynebacterium bacteria as a host. This is because, like Escherichia coli, corynebacterium bacteria also have several enzymes involved in the degradation of PAPS.

[0016] In addition, as described above, in the conventional enzymatic method for sulfating polysaccharides, microorganisms expressing sulfating enzymes are cultured, collected, and a plurality of enzymes purified from a disrupted cell solution prepared by ultrasonic disruption or the like are used, and it is not easy to carry out on an industrial scale. Also, expensive PAPS or p-nitrophenyl sulfate is required as a raw material.

[0017] Therefore, an object of the present invention is to provide a method for easily producing sulfated polysaccharides by mixing a PAPS production / regeneration system utilizing the metabolic activity of a microorganism or its treated product with a microorganism or its treated product or extract expressing a sulfating enzyme and inexpensive raw materials such as magnesium sulfate and reacting them. Another object of the present invention is to provide a method for producing practical PAPS from inexpensive raw materials.

Means for Solving the Problems

[0018] The present inventors completed the present invention based on the following findings (1) and (2). (1) After culturing a strain in which the activities of ATP sulfurylase and APS kinase have been enhanced by recombinant DNA techniques using a coryneform bacterium having ATP-producing ability as a host, imparting membrane permeability with a surfactant or the like, and adding raw materials such as glucose and adenine, PAPS can be produced inexpensively and simply as compared with the conventional method. (2) Using the strain in which the activities of ATP sulfurylase and APS kinase described in (1) have been enhanced as a microbial cell body responsible for the PAPS production / regeneration reaction, and performing a mixing reaction by imparting membrane permeability to a microorganism belonging to a prokaryote expressing an epimerase and / or a sulfating enzyme, sulfated polysaccharides can be produced using the cell body and inexpensive raw materials such as magnesium sulfate without purifying the enzyme or adding PAPS.

[0019] That is, the present invention is as follows. 1. A method for producing a sulfated polysaccharide, comprising the following steps (1-1) and (1-2). (1-1) A step of preparing a transformant (a) of a Corynebacterium bacterium or a processed product thereof, which contains at least a gene encoding ATP sulfurylase and a gene encoding APS kinase that are expressibly introduced into the transformant (1-2) A step of performing a production reaction of PAPS using a reaction solution containing ATP or an ATP source, a sulfate ion source, and the transformant (a) or a processed product thereof 2. The method for producing a sulfated polysaccharide according to 1 above, further comprising steps (2-1) and (2-2). (2-1) A step of preparing a transformant (b) of a microorganism belonging to prokaryotes or a processed product or extract thereof, which contains at least a gene encoding C5-epimerase that is expressibly introduced into the transformant (2-2) A step of performing C5-epimerization by adding the transformant (b) or a processed product or extract thereof to the reaction solution in the presence of N-sulfoheparosan 3. The method for producing a sulfated polysaccharide according to claim 1 or 2, comprising a step of sulfating with a transformant containing a gene encoding a sulfotransferase that is expressibly introduced or a processed product or extract thereof. 4. The method for producing a sulfated polysaccharide according to 3 above, further comprising steps (3-1) and (3-2). (3-1) A step of preparing a transformant (c) of a microorganism belonging to prokaryotes or a processed product or extract thereof, which contains at least a gene encoding 2-O-sulfotransferase that is expressibly introduced into the transformant (3-2) A step of performing 2-O-sulfation by adding the transformant (c) or a processed product or extract thereof to the reaction solution in the presence of N-sulfoheparosan 5. The method for producing a sulfated polysaccharide according to any one of 3 or 4 above, further comprising steps (3'-1) to (3'-3). (3'-1) A step of preparing a transformant (b) of a microorganism belonging to prokaryotes or a processed product or extract thereof, which contains at least a gene encoding C5-epimerase that is expressibly introduced into the transformant Step of preparing a transformant (c) that is a transformant of a microorganism belonging to prokaryotes and contains a gene encoding at least 2-O-sulfotransferase that has been expressibly introduced into the transformant, or a processed product or extract thereof (3´-3) Step of causing the transformant (b) or a processed product or extract thereof and the transformant (c) or a processed product or extract thereof to be contained in the reaction solution in the presence of N-sulfoheparosan to perform C5-epimerization and 2-O-sulfation 6. The method for producing a sulfated polysaccharide according to any one of 3 to 5 above, further including steps (4-1) and (4-2). (4-1) Step of preparing a transformant (d) that is a transformant of a microorganism belonging to prokaryotes and contains a gene encoding at least 6-O-sulfotransferase that has been expressibly introduced into the transformant, or a processed product or extract thereof (4-2) Step of causing the transformant (d) or a processed product or extract thereof to be contained in the reaction solution in the presence of N-sulfoheparosan to perform 6-O-sulfation 7. The method for producing a sulfated polysaccharide according to any one of 3 to 6 above, further including steps (5-1) and (5-2). (5-1) Step of preparing a transformant (e) that is a transformant of a microorganism belonging to prokaryotes and contains a gene encoding at least 3-O-sulfotransferase that has been expressibly introduced into the transformant, or a processed product or extract thereof (5-2) Step of causing the transformant (e) or a processed product or extract thereof to be contained in the reaction solution in the presence of N-sulfoheparosan to perform 3-O-sulfation 8. In the presence of ATP or an ATP source, a sulfate ion source, and N-sulfoheparosan a transformant (a) that is a transformant of a bacterium belonging to the genus Corynebacterium and contains a gene encoding at least ATP sulfurylase and a gene encoding APS kinase that have been expressibly introduced into the transformant, or a processed product thereof, and A transformant of a microorganism belonging to prokaryotes, which contains a gene encoding a C5-epimerase that is at least expressibly introduced into the transformant (b), or a processed product or extract thereof, A transformant of a microorganism belonging to prokaryotes, which contains a gene encoding a 2-O-sulfotransferase that is at least expressibly introduced into the transformant (c), or a processed product or extract thereof, A transformant of a microorganism belonging to prokaryotes, which contains a gene encoding a 6-O-sulfotransferase that is at least expressibly introduced into the transformant (d), or a processed product or extract thereof, and A method for producing a sulfated polysaccharide, which comprises incorporating at least one selected from a transformant of a microorganism belonging to prokaryotes, which contains a gene encoding a 3-O-sulfotransferase that is at least expressibly introduced into the transformant (e), or a processed product or extract thereof, into a reaction solution to produce a sulfated polysaccharide. 9. In the presence of ATP or an ATP source, a sulfate ion source, and N-sulfoheparosan, The method for producing a sulfated polysaccharide according to item 8 above, which comprises incorporating the transformant (a) or a processed product thereof and the transformants (b) to (e) or processed products or extracts thereof into a reaction solution to produce a sulfated polysaccharide. 10. The method for producing a sulfated polysaccharide according to any one of items 1 to 9 above, which is a method for producing heparin. 11. A method for producing PAPS, which comprises the following steps (i) and (ii). (i) A step of preparing a transformant of a bacterium belonging to the genus Corynebacterium, which contains a gene encoding an ATP sulfurylase and a gene encoding an APS kinase that are at least expressibly introduced into the transformant, or a processed product thereof (ii) A step of performing a production reaction of PAPS using a reaction solution containing ATP or an ATP source, a sulfate ion source, and the transformant or a processed product thereof prepared in step (i)

Advantages of the Invention

[0020] According to the method for producing a sulfated polysaccharide of the present invention, by using a PAPS production / regeneration system utilizing the metabolic activity of microorganisms and a microorganism expressing a sulfating enzyme, or a processed product or extract thereof, a sulfated polysaccharide can be produced simply and inexpensively. Further, according to the method for producing PAPS of the present invention, PAPS can be produced simply and inexpensively by utilizing the metabolic activity of a microorganism or a processed product thereof. Furthermore, by using a bacterium belonging to the genus Corynebacterium in the method of the present invention, complicated genetic modification for preventing the degradation of PAPS is unnecessary.

Brief Description of the Drawings

[0021]

Figure 1

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Figure 8

Figure 9

Mode for Carrying Out the Invention

[0022] <Transformant> The method for producing a sulfated polysaccharide of the present invention is characterized in that 1) the supply / regeneration of PAPS is carried out by a reaction using a bacterial cell. In the method of the present invention, it is preferable that at least one of 2) C5-epimerization, 3) 2-O-sulfation, 4) 6-O-sulfation, and 5) 3-O-sulfation is carried out by a reaction using a bacterial cell. The transformants (a) to (e) used in each of the following reactions will be described.

[0023] <<Transformant (a): Supply / Regeneration of PAPS>> In the method for producing a sulfated polysaccharide of the present invention, the supply / regeneration of PAPS is carried out by a transformant (a) which is a transformant of a microorganism belonging to the genus Corynebacterium and contains at least a gene encoding ATP sulfurylase and a gene encoding APS kinase that are expressibly introduced into the transformant, or a processed product thereof.

[0024] Examples of bacteria belonging to the genus Corynebacterium include Corynebacterium ammoniagenes, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Corynebacterium alkanolyticum, Corynebacterium callunae, Corynebacterium crenatum, Corynebacterium glutamicum, Corynebacterium lilium, Corynebacterium melassecola, Corynebacterium thermoaminogenes (Corynebacterium efficiens), and Corynebacterium herculis.

[0025] Examples of strains of bacteria belonging to the genus Corynebacterium include, for example, Corynebacterium ammoniagenes (Corynebacterium stationis) ATCC 6871, ATCC 6872, Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium acetoglutamicum ATCC 15806, Corynebacterium alkanolyticum ATCC 21511, Corynebacterium callunae ATCC 15991, Corynebacterium crenatum AS1.542, Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869, FERM BP-734, Corynebacterium lilium ATCC 15990, Corynebacterium melassecola ATCC 17965, Corynebacterium efficiens (Corynebacterium thermoaminogenes) AJ12340 (FERM BP-1539), Corynebacterium herculis ATCC 13868, Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020, Brevibacterium flavum (Corynebacterium glutamicum) ATCC 13826, ATCC 14067, AJ12418 (FERM BP-2205), Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869.

[0026] Note that Corynebacterium bacteria include those that were previously classified in the genus Brevibacterium but are now integrated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). In addition, Corynebacterium stationis includes bacteria that were previously classified as Corynebacterium ammoniagenes but were reclassified as Corynebacterium stationis by analysis of the nucleotide sequence of 16S rRNA, etc. [Int. J. Syst. Evol. Microbiol., 60, 874-879 (2010)].

[0027] These strains are available, for example, from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is assigned, and the strain can be obtained using this registration number (see https: / / www.atcc.org / ). The registration number corresponding to each strain is described in the catalog of the American Type Culture Collection. In addition, these strains can be obtained, for example, from the depository institution where each strain is deposited. Corynebacterium bacteria may be wild strains, mutant strains, or artificial gene recombinants thereof.

[0028] In the present invention, a gene encoding ATP sulfurylase and a gene encoding APS kinase are introduced into Corynebacterium bacteria so as to be expressible.

[0029] ATP sulfurylase generates APS (Adenosine 5’-phosphosulfate) from sulfate according to the following reaction formula.

[0030]

Chemical formula

[0031] One example of ATP sulfurylase is MET3. The origin of ATP sulfurylase is not particularly limited. For example, it may be derived from Saccharomyces.Cerevisiae, Candida albicans, Shizosaccharomyces pombe, Yarrowia lipolytica, Neurospora crassa, Penicillium chrysogenum, Kluyveromyces lactis, Fusarium fujikuroi, Aspergillus oryzae, or Ashbya gossypii. Among these, it is preferably derived from Saccharomyces.Cerevisiae.

[0032] Examples of the gene encoding ATP sulfurylase include the nucleotide sequence shown in SEQ ID NO: 22. Also, a DNA containing a nucleotide sequence having preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more identity with the nucleotide sequence shown in SEQ ID NO: 22, and encoding a polypeptide having ATP sulfurylase activity; a DNA containing a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 22 or a nucleotide sequence complementary to the nucleotide sequence, and encoding a polypeptide having ATP sulfurylase activity. "Stringent conditions" refer to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, conditions under which DNAs with high identity, such as 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more identity hybridize with each other, and DNAs with lower identity do not hybridize with each other, or conditions corresponding to 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, more preferably 68°C, 0.1×SSC, 0.1% SDS, which are the washing conditions for ordinary Southern hybridization, and washing once, preferably 2 - 3 times, can be mentioned.

[0033] The ATP sulfurylase activity in the transformant is confirmed by an increase in the ATP sulfurylase activity value in the cell extract of the transformant. The activity of ATP sulfurylase can be confirmed by the method described in the literature [Medina D. C. et al., Temperature effects on the allosteric transition of ATP sulfurylase from Penicillium chrysogenum. Arch. Biochem. Biophys. 1;393(1):51-60 (2001)].

[0034] The numerical value of identity in the present invention may be a numerical value calculated using a homology search program known to those skilled in the art, unless otherwise specified. For nucleotide sequences, it may be a numerical value calculated using the default parameters in BLAST [J. Mol. Biol., 215, 403 (1990)], and for amino acid sequences, it may be a numerical value calculated using the default parameters in BLAST2 [Nucleic Acids Res., 25, 3389 (1997), Genome Res., 7, 649 (1997), http: / / www.ncbi.nlm.nih.gov / Education / BLASTinfo / information3.htmL], etc.

[0035] APS kinase generates PAPS from APS according to the following reaction formula.

[0036]

Chemical formula

[0037] One aspect of APS kinase includes MET14. The origin of APS kinase is not particularly limited. For example, it may be derived from Saccharomyces cerevisiae, Candida albicans, Shizosaccharomyces pombe, Yarrowia lipolytica, Neurospora crassa, Penicillium chrysogenum, Kluyveromyces lactis, Fusarium fujikuroi, Aspergillus oryzae, or Ashbya gossypii, and among these, it is preferably derived from Saccharomyces cerevisiae.

[0038] Examples of the gene encoding APS kinase include the nucleotide sequence shown in SEQ ID NO: 23. Also, it is a DNA containing a nucleotide sequence having preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more identity with the nucleotide sequence shown in SEQ ID NO: 23, and encoding a polypeptide having APS kinase activity; a DNA containing a nucleotide sequence shown in SEQ ID NO: 23 or a nucleotide sequence complementary to the nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence, and encoding a polypeptide having APS kinase activity.

[0039] The APS kinase activity in the transformant is confirmed by an increase in the APS kinase activity value in the cell extract of the transformant. The activity of APS kinase can be confirmed by the method described in the literature [Renosto F. et al., Adenosine 5'-phosphosulfate kinase from Penicillium chrysogenum. Purification and kinetic characterization. J. Biol.Chem. 259(4):2113-2123 (1984)].

[0040] Introduction of the gene encoding ATP sulfurylase and the gene encoding APS kinase into Corynebacterium bacteria may be achieved by integrating the above-mentioned DNAs into the chromosome of the host respectively, or by cloning them into an appropriate plasmid vector that can be amplified in the host and introducing the resulting vector into the host.

[0041] The plasmid vector may contain a gene responsible for autonomous replication in bacteria of the genus Corynebacterium. Specific examples thereof include pAM330 derived from Brevibacterium lactofermentum 2256 [Japanese Patent Laid-Open No. 58-67699], [Miwa, K. et al., Cryptic plasmids in glutamic acid-producing bacteria. Agric. Biol. Chem. 48:2901-2903(1984)] and [Yamaguchi, R. et al., Determination of the complete nucleotide sequence of the Brevibacterium lactofermentum plasmid pAM330 and the analysis of its genetic information. Nucleic Acids Symp. Ser. 16:265-267(1985)], pHM1519 derived from Corynebacterium glutamicum ATCC3058 [Miwa, K. et al., Cryptic plasmids in glutamic acid-producing bacteria. Agric. Biol.Chem. 48:2901-2903(1984)] and pCRY30 [Kurusu, Y. et al., Identification of plasmid partition function in coryneform bacteria. Appl. Environ. Microbiol. 57:759-764 (1991)], pCG4 derived from Corynebacterium glutamicum T250 [Japanese Patent Laid-Open No. 57-183799], [Katsumata, R. et al., Protoplast transformation of glutamate-producing bacteria with plasmid DNA. J. Bacteriol., 159:306-311 (1984)], pAG1, pAG3, pAG14, pAG50 [Japanese Patent Laid-Open No. 62-166890], pEK0, pEC5, pEKEx1 [Eikmanns, B.J. et al., A family of Corynebacterium glutamicum / Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene, 102:93-98 (1991)], and the like can be mentioned.

[0042] As the promoter, it may be a promoter derived from the host or a promoter derived from a heterologous source. For example, the promoter PgapA of the glyceraldehyde 3-phosphate dehydrogenase A gene (gapA) derived from Corynebacterium glutamicum R, the promoter Pmdh of the malate dehydrogenase gene (mdh), the promoter PldhA of the lactate dehydrogenase A gene (ldhA), and the like can be mentioned.

[0043] As the terminator, for example, the rrnB T1T2 terminator of the E. coli rRNA operon, the trpA terminator of E. coli, the trp terminator of Brevibacterium lactofermentum, and the like can be mentioned.

[0044] In one aspect of the present invention, the expression of the gene of the enzyme involved in the degradation of PAPS is not attenuated. Examples of the gene of the enzyme involved in the degradation of PAPS include the cysQ gene and the CP01850 gene. As one aspect of the transformant (a), the expression of at least one selected from the cysQ gene and the CP01850 gene is not attenuated.

[0045] <<Transformant (b): C5-epimerization>> In the method for producing a sulfated polysaccharide of the present invention, the C5-epimerization of N-sulfoheparosan is carried out by a transformant of a microorganism belonging to prokaryotes, which contains a gene encoding C5-epimerase that is expressibly introduced into at least the transformant, or a processed product or extract thereof.

[0046] The C5-epimerase is not particularly limited as long as it can catalyze the isomerization of glucuronic acid (GlcUA) residues to iduronic acid (IdoA) residues. The C5-epimerase may be derived from any of animals, plants, microorganisms, etc. As the C5-epimerase, for example, human C5-epimerase can be used.

[0047] Examples of the gene encoding C5-epimerase include the nucleotide sequence shown in SEQ ID NO: 24. Also, a DNA containing a nucleotide sequence having preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more identity with the nucleotide sequence shown in SEQ ID NO: 24, and encoding a polypeptide having C5-epimerase activity; a DNA containing a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence shown in SEQ ID NO: 24 or a nucleotide sequence complementary to the nucleotide sequence, and encoding a polypeptide having C5-epimerase activity.

[0048] The C5-epimerase activity in the transformant is confirmed by an increase in the C5-epimerase activity value in the cell extract of the transformant. The C5-epimerase activity can be measured by the method described in the literature [Babu P. et al., A rapid, nonradioactive assay for measuring heparansulfate C-5 epimerase activity using hydrogen / deuterium exchange-mass spectrometry. Methods. Mol. Biol. 1229:209-219 (2015)].

[0049] The transformant expressing sulfotransferase will be described below. In the method for producing a sulfated polysaccharide of the present invention, sulfation is carried out using a transformant containing a gene encoding a sulfotransferase that has been introduced into the transformant so as to be expressible, or a processed product or extract thereof. In the present invention, the sulfotransferase is not particularly limited as long as it transfers a sulfate group to a polysaccharide to produce a sulfated polysaccharide. Examples thereof include 2-O-sulfotransferase (2-OST), 6-O-sulfotransferase (6-OST), and 3-O-sulfotransferase (3-OST).

[0050] <<Transformant (c): 2-O-sulfation>> In the method for producing a sulfated polysaccharide of the present invention, 2-O-sulfation is carried out using a transformant (c) that is a transformant of a microorganism belonging to prokaryotes and contains a gene encoding at least 2-O-sulfotransferase (2-OST) that has been introduced into the transformant so as to be expressible, or a processed product or extract thereof.

[0051] 2-OST is not particularly limited as long as it can catalyze the sulfation of the O-2 position of the IdoA residue. Further, 2-OST may be derived from any of animals, plants, microorganisms, etc. As 2-OST, for example, 2-OST derived from hamster can be used.

[0052] Examples of the gene encoding 2-OST include the nucleotide sequence represented by SEQ ID NO: 19. Further, it is a DNA containing a nucleotide sequence having preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more identity with the nucleotide sequence represented by SEQ ID NO: 19, and encoding a polypeptide having 2-OST activity; a DNA containing a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 19 or a nucleotide sequence complementary to the nucleotide sequence, and encoding a polypeptide having 2-OST activity.

[0053] The 2-OST activity in the transformant is confirmed by the increase in the 2-OST activity value in the cell extract of the transformant. The 2-OST activity can be confirmed by the method described in the literature [Zhang J. et al., High cell density cultivation of recombinant Escherichia coli strains expressing 2-O-sulfotransferase and C5-epimerase for the production of bioengineered heparin. Appl. Biochem. Biotechnol.175(6):2986-2995 (2015)].

[0054] <<Transformant (d): 6-O-sulfation>> In the method for producing the sulfated polysaccharide of the present invention, 6-O-sulfation is carried out by a transformant (d) belonging to a prokaryotic microorganism, which contains a gene encoding at least 6-O-sulfotransferase (6-OST) that is expressibly introduced into the transformant, or a processed product or extract thereof.

[0055] 6-OST is not particularly limited as long as it can catalyze the sulfation at the O-6 position of the N-sulfated glucosamine (GlcNS) residue. 6-OST may be derived from any of animals, plants, microorganisms, etc. Examples of 6-OST include 6-OST-1 derived from hamster and 6-OST-3 derived from mouse.

[0056] Examples of the gene encoding 6-OST include the nucleotide sequence represented by SEQ ID NO: 25. Further, it is DNA containing a nucleotide sequence having preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more identity with the nucleotide sequence represented by SEQ ID NO: 25, and encoding a polypeptide having 6-OST activity; DNA containing a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 25 or a nucleotide sequence complementary to the nucleotide sequence, and encoding a polypeptide having 6-OST activity.

[0057] The 6-OST activity in the transformant is confirmed by an increase in the 6-OST activity value in the cell extract of the transformant. The activity of 6-OST can be confirmed by the method described in the literature [Zhang J. et al., High cell density cultivation of a recombinant Escherichia coli strain expressing a 6-O-sulfotransferase for the production of bioengineered heparin. J. Appl. Microbiol. 118(1):92-98(2015)].

[0058] <<Transformant (e): 3-O-sulfation>> In the method for producing the sulfated polysaccharide of the present invention, 3-O-sulfation is carried out by a transformant (e) of a microorganism belonging to prokaryotes, which contains a gene encoding at least 3-O-sulfotransferase (3-OST) that is expressibly introduced into the transformant, or a processed product or extract thereof.

[0059] 3-OST is not particularly limited as long as it can catalyze the sulfation at the O-3 position of N-sulfated and 6-O-sulfated glucosamine residues. 3-OST may be derived from any of animals, plants, microorganisms, etc. As 3-OST, for example, 3-OST-1 derived from a mouse can be used.

[0060] Examples of the gene encoding 3-OST include the nucleotide sequence represented by SEQ ID NO: 26. Further, it is a DNA containing a nucleotide sequence having preferably 80% or more, more preferably 90% or more, still more preferably 95% or more, and particularly preferably 98% or more identity with the nucleotide sequence represented by SEQ ID NO: 26, and encoding a polypeptide having 3-OST activity; a DNA containing a nucleotide sequence that hybridizes under stringent conditions with the nucleotide sequence represented by SEQ ID NO: 26 or a nucleotide sequence complementary to the nucleotide sequence, and encoding a polypeptide having 3-OST activity.

[0061] The 3-OST activity in the transformant is confirmed by an increase in the 3-OST activity value in the cell extract of the transformant. The 3-OST activity can be confirmed by the method described in the literature [Jin W. et al., Increased soluble heterologous expression of a rat brain 3-O-sulfotransferase 1 - A key enzyme for heparin biosynthesis. Protein Expr. Purif. 151:23-29 (2018)].

[0062] <<Microorganisms belonging to prokaryotes>> In the present invention, the microorganisms belonging to prokaryotes used as the host of the transformant are not particularly limited as long as they can express the gene specified in the present invention. Examples include bacteria such as those belonging to the genus Escherichia, Serratia, Bacillus, Corynebacterium, Microbacterium, and Pseudomonas, and bacteria belonging to the genus Escherichia are preferably used.

[0063] The Escherichia bacteria used in the present invention are not particularly limited, and examples include bacteria classified into the genus Escherichia according to the classification known to experts in microbiology. Examples of Escherichia bacteria include those described in the literature [Backmann, B. J. 1996. Derivations and Genotypes of some mutant derivatives of Escherichia coli K-12, p. 2460-2488. Table 1. In F. D. Neidhardt(ed.), Escherichia coli and Salmonella Cellular and Molecular Biology / Second Edition, American Society for Microbiology Press, Washington, D.C.].

[0064] Examples of Escherichia bacteria include, for example, Escherichia coli. Examples of Escherichia coli include Escherichia coli K-12 strains such as W3110 strain (ATCC 27325) and MG1655 strain (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); Escherichia coli B strains such as BL21(DE3) strain; Escherichia coli Nissle 1917 strain (DSM 6601); and their derivative strains.

[0065] These strains are available, for example, from the American Type Culture Collection (12301 Parklawn Drive, Rockville, Maryland 20852 P.O. Box 1549, Manassas, VA 20108, United States of America). That is, a registration number corresponding to each strain is assigned, and the strain can be obtained using this registration number (see https: / / www.atcc.org / ). The registration number corresponding to each strain is described in the catalog of the American Type Culture Collection. Also, the BL21(DE3) strain is available, for example, from Life Technologies (product number C6000-03).

[0066] For the purpose of introducing a gene so that it can be expressed, as a vector used for the transformation of a microorganism belonging to prokaryotes, a vector that can replicate autonomously in the host cell can be used. The vector is preferably a multicopy vector. Also, for selecting a transformant, the vector preferably has a marker such as an antibiotic resistance gene or other genes described in the literature [Karl Friehs, Plasmid Copy Number and Plasmid Stability, Adv Biochem Engin / Biotechnol 86: 47-82 (2004)]. Further, the vector may be provided with a promoter and a terminator for expressing the inserted gene. Examples of the vector include vectors derived from bacterial plasmids, vectors derived from yeast plasmids, vectors derived from bacteriophages, cosmids, or phagemids, etc.

[0067] Examples of vectors capable of autonomous replication in Escherichia coli bacteria include, specifically, pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all from Takara Bio Inc.), pACYC184, pMW219 (from Nippon Gene Co., Ltd.), pTrc99A (from Pharmacia), pPROK vectors (from Clontech), pKK233-2 (from Clontech), pET vectors (from Novagen), pQE vectors (from Qiagen), and the broad host range vector RSF1010.

[0068] The promoter may be a promoter derived from the host or a promoter derived from a heterologous source. The promoter may be the specific promoter of the gene to be introduced or the promoter of another gene.

[0069] Examples of terminators include the T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0070] In the above-mentioned transformants (b) to (e), two or more of the genes introduced into the microorganisms belonging to prokaryotes may be introduced into one transformant. Specifically, for example, a gene encoding C5-epimerase and a gene encoding 2-O-sulfotransferase may be introduced into a microorganism belonging to one prokaryote to obtain one transformant.

[0071] <<Processed product of the transformant>> The processed product of the transformant in the present invention refers to a product in which the cell cytoplasmic membrane of the bacterial cells has substance permeability. In the present invention, the fact that the cell cytoplasmic membrane has substance permeability means that various molecules, including small molecules (such as ions) and large molecules (such as proteins), can pass through the cell membrane by diffusion and freely enter and exit. The processed product of the transformant in the present invention is preferably a resting cell body that has lost its growth ability by treatment for imparting membrane permeability.

[0072] Examples of processed products of the transformant include those containing viable cells that retain the same functions as the culture of the cells as an enzyme source, such as surfactant-treated products of the cells as the transformant, solvent-treated products of the cells, enzyme-treated products of the cells, immobilized products of the cells, ultrasonic-treated products of the cells, and mechanically ground products of the cells.

[0073] Examples of methods for making the cell plasma membrane of the transformant permeable to substances include chemical treatment and mechanical treatment. In the production method of the present invention, the timing for making the cell plasma membrane of the transformant permeable to substances is not particularly limited as long as the effects of the present invention can be achieved. The cell plasma membrane of each transformant may be made permeable to substances in advance, or when the transformants used in the reaction are brought into contact with each other for reaction.

[0074] Examples of chemical treatment include methods using surfactants, methods using organic solvents, and methods using enzymes. Nonionic surfactants are preferred because their action on proteins, etc. is milder (compared to ionic surfactants). Examples of such surfactants include digitonin, saponin, Triton X100, Triton X114, Tween 20, Tween 80, N,N-Bis(3-D-gluconamidopropyl)cholamide [BIGCHAP], N,N-Bis(3-D-gluconamidopropyl)deoxycholamide [Deoxy-BIGCHAP], NIKKOL BL-9EX [Polyoxyethylene(9)Lauryl Ether], Octanoyl-N-methylglucamide [MEGA-8], benzalkonium chloride, etc.

[0075] Examples of organic solvents include Benzene, Toluene, Xylene, and other alcohols. Examples of enzymes include lysozyme, achromopeptidase, etc.

[0076] The conditions such as the concentration, temperature, and time of the treatment with the above-mentioned substance vary depending on the cell type, and appropriate conditions for performing the desired analysis must be set. Generally, the treatment concentration is 10 - 1000 μg / ml, more commonly 20 - 200 μg / ml, the temperature is 2 - 37 °C, and the time is 1 - 30 minutes.

[0077] Examples of mechanical treatments include ultrasonic treatment and mechanical grinding treatment.

[0078] <<Extract of transformant>> Examples of the extract of the transformant in the present invention include a crude enzyme extract obtained from the cells that are the transformant, a purified enzyme obtained from the cells treated as described above (for example, chemical treatment or mechanical treatment), a concentrate of a culture obtained by culturing the above-described transformant or its treated product, and a dried product of the culture. In addition, the extract of the transformant also includes the cells obtained by centrifuging or filtering the culture, the dried product of the cells, and the freeze-dried product of the cells.

[0079] <<Transformation method and culture method of transformant>> Known methods can be used without limitation for the transformation method. Examples of such known methods include the calcium chloride - rubidium chloride method, calcium phosphate method, DEAE - dextran - mediated transfection, electropulse method, etc. Among them, the electropulse method is suitable for coryneform bacteria, and the electropulse method can be carried out by a known method [Kurusu, Y. et al., Electroporation - transformation system for Coryneform bacteria by auxotrophic complementation. Agric. Biol. Chem. 54:443 - 447 (1990)].

[0080] The transformant is preferably grown by culturing it using a medium commonly used for culturing microorganisms prior to each reaction. As this medium, a natural medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, and other nutrients, etc. can usually be used.

[0081] The carbon source can be an ATP source. Examples of the carbon source include carbohydrates or sugar alcohols such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, arabinose, galactose, starch, molasses, sorbitol, glycerin, etc.; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid or gluconic acid; and alcohols such as ethanol, propanol, etc. The carbon source can be used alone as one kind, or two or more kinds can be mixed. The concentration of these carbon sources in the medium is usually about 0.1 - 10 (w / v%).

[0082] Examples of the nitrogen source include inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium acetate, etc., urea, aqueous ammonia, sodium nitrate, potassium nitrate, etc. Also, nitrogen-containing organic compounds such as corn steep liquor, meat extract, peptone, NZ - amine, protein hydrolysate, amino acids, etc. can be used. The nitrogen source can be used alone as one kind, or two or more kinds can be mixed. The nitrogen source concentration in the medium varies depending on the nitrogen compound used, but is usually about 0.1 - 10 (w / v %).

[0083] Examples of the inorganic salts include, in addition to sources of sulfate ions such as magnesium sulfate, manganese sulfate, zinc sulfate, cobalt sulfate, etc., potassium dihydrogen phosphate, dipotassium hydrogen phosphate, ferrous nitrate, sodium chloride, or calcium carbonate, etc. These inorganic salts can be used alone as one kind, or two or more kinds can be mixed. The inorganic salt concentration in the medium varies depending on the inorganic salt used, but is usually about 0.01 - 1 (w / v%).

[0084] Examples of nutrients include meat extract, peptone, polypeptone, yeast extract, dried yeast, corn steep liquor, skim milk powder, hydrochloric acid hydrolyzate of defatted soybean, or extracts and degradation products of animal, plant, or microbial cells. Usually, it may be about 0.1 - 10 (w / v%). Furthermore, vitamins can be added as needed. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid, etc. The pH of the medium is preferably about 6 - 8.

[0085] Preferred microbial culture media include Medium A [Inui, M. et al., Metabolic analysis of Corynebacterium glutamicum during lactate and succinate productions under oxygen deprivation conditions. J. Mol. Microbiol. Biotechnol. 7:182 - 196 (2004)], Medium BT [Omumasaba, C.A. et al., Corynebacterium glutamicum glyceraldehyde - 3 - phosphate dehydrogenase isoforms with opposite, ATP - dependent regulation. J. Mol. Microbiol. Biotechnol. 8:91 - 103 (2004)], etc. Specific culture conditions include, for example, a culture temperature of about 15 - 45°C and a culture time of about 1 - 7 days.

[0086] <Method for producing sulfated polysaccharide> One aspect of the method for producing a sulfated polysaccharide of the present invention is a method for producing a sulfated polysaccharide, which comprises incorporating into a reaction solution at least one selected from the above - mentioned transformant (a) or its treated product and the above - mentioned transformants (b) - (e) or their treated products or extracts in the presence of ATP or an ATP source, a sulfate ion source, and N - sulfated heparosan to generate a sulfated polysaccharide.

[0087] As one aspect of the method for producing a sulfated polysaccharide of the present invention, for example, in the presence of ATP or an ATP source, a sulfate ion source, and N-sulfoheparosan, a reaction solution containing the above-mentioned transformant (a) or a processed product thereof and the above-mentioned transformants (b) to (e) or their processed products or extracts is used to produce a sulfated polysaccharide. A method for producing a sulfated polysaccharide can be mentioned.

[0088] In addition, one aspect of the method for producing a sulfated polysaccharide of the present invention is a method for producing a sulfated polysaccharide in which at least one selected from the above-mentioned transformants (b) to (e) or their processed products or extracts is contained in a reaction solution in the presence of PAPS and N-sulfoheparosan to produce a sulfated polysaccharide.

[0089] In the production method of the present invention, each transformant may be initially added to the reaction solution, sequentially added, or a combination thereof may be used. As an aspect of initially adding each transformant to the reaction solution, specifically, for example, in the presence of ATP or an ATP source, a sulfate ion source, and N-sulfoheparosan, the above-mentioned transformant (a) or a processed product thereof and the above-mentioned transformants (b) to (e) or their processed products or extracts are initially contained in the reaction solution to produce a sulfated polysaccharide.

[0090] In the method for producing a sulfated polysaccharide of the present invention, the above-mentioned transformant (a) or a processed product thereof and the above-mentioned transformants (b) to (e) or their processed products or extracts are used to perform 1) supply / regeneration of PAPS by the transformant (a) or a processed product thereof, 2) C5-epimerization by the transformant (b) or a processed product or extract thereof, 3) 2-O-sulfation by the transformant (c) or a processed product or extract thereof, 4) 6-O-sulfation by the transformant (d) or a processed product or extract thereof, and 5) 3-O-sulfation by the transformant (e) or a processed product or extract thereof.

[0091] Hereinafter, it will be described separately for each step. However, the order of 2) C5-epimerization, 3) 2-O-sulfation, 4) 6-O-sulfation, and 5) 3-O-sulfation is not particularly limited as long as the desired sulfated polysaccharide can be obtained.

[0092] In the following, the reactions of 1) PAPS supply / regeneration, 2) C5-epimerization, 3) 2-O-sulfation, 4) 6-O-sulfation, and 5) 3-O-sulfation will be described separately for each reaction, but these reactions may be carried out simultaneously for two or more of them. For example, these reactions may be carried out simultaneously using a reaction solution containing all of the transformant (a) or a processed product thereof and the transformants (b) to (e) or their processed products or extracts.

[0093] Examples of the sulfated polysaccharide produced by the method for producing a sulfated polysaccharide of the present invention include heparin.

[0094] <<PAPS supply / regeneration>> The method for producing a sulfated polysaccharide of the present invention is characterized by including the following steps (1-1) and (1-2). (1-1) Preparing a transformant (a) of a bacterium belonging to the genus Corynebacterium, which contains at least a gene encoding ATP sulfurylase and a gene encoding APS kinase, which are expressibly introduced into the transformant, or a processed product thereof (1-2) Performing a production reaction of PAPS using a reaction solution containing ATP or an ATP source, a sulfate ion source, and the transformant (a) or a processed product thereof

[0095] ATP sulfurylase and APS kinase expressed by the transformant (a) or a processed product thereof react with ATP or an ATP source and a sulfate ion source in the transformant (a) or a processed product thereof to produce PAPS. PAPS functions as a donor of sulfate groups in the production of sulfated polysaccharides. Further, when PAPS produced by the transformant (a) or a processed product thereof is used in the method for producing a sulfated polysaccharide, PAP is obtained, and PAPS can be produced from the PAP by the transformant (a) or a processed product thereof. Therefore, by including the transformant (a) or a processed product thereof in the reaction solution, supply / regeneration of PAPS becomes possible.

[0096] <<C5-epimerization>> The method for producing a sulfated polysaccharide of the present invention preferably includes the following steps (2-1) and (2-2). (2-1) Preparing a transformant (b) that is a transformant of a microorganism belonging to prokaryotes and contains a gene encoding at least C5-epimerase that has been expressibly introduced into the transformant, or a processed product or extract thereof (2-2) A step of performing C5-epimerization by including the transformant (b) or a processed product or extract thereof in a reaction solution in the presence of N-sulfoheparosan

[0097] In step (2-2), C5-epimerization of N-sulfoheparosan is performed by the transformant (b) expressing C5-epimerase or a processed product or extract thereof.

[0098] <<2-O-sulfation>> The method for producing a sulfated polysaccharide of the present invention preferably includes the following steps (3-1) and (3-2). (3-1) Preparing a transformant (c) that is a transformant of a microorganism belonging to prokaryotes and contains a gene encoding at least 2-O-sulfotransferase that has been expressibly introduced into the transformant, or a processed product or extract thereof (3-2) A step of performing 2-O-sulfation by including the transformant (c) or a processed product or extract thereof in a reaction solution in the presence of N-sulfoheparosan

[0099] In step (3-2), PAPS produced by the transformant (a) or a processed product thereof is used as a sulfate group donor, and 2-O-sulfation of N-sulfoheparosan is performed by the transformant (c) expressing 2-O-sulfotransferase or a processed product or extract thereof. When the sulfated polysaccharide produced in step (2-2) is present in the reaction solution, 2-O-sulfation of the sulfated polysaccharide produced in this step is performed. For example, when C5-epimerized N-sulfoheparosan is present, 2-O-sulfation of C5-epimerized N-sulfoheparosan is performed.

[0100] The above C5-epimerization and 2-O-sulfation may be carried out simultaneously. That is, as one aspect of the method for producing a sulfated polysaccharide of the present invention, it preferably includes the following steps (3'-1) to (3'-3). (3'-1) Preparing a transformant (b) of a microorganism belonging to prokaryotes, which contains a gene encoding at least C5-epimerase that has been introduced into the transformant so as to be expressible, or a processed product or extract thereof (3'-2) Preparing a transformant (c) of a microorganism belonging to prokaryotes, which contains a gene encoding at least 2-O-sulfotransferase that has been introduced into the transformant so as to be expressible, or a processed product or extract thereof (3'-3) In the presence of N-sulfoheparosan, adding the transformant (b) or a processed product or extract thereof and the transformant (c) or a processed product or extract thereof to the reaction solution to carry out C5-epimerization and 2-O-sulfation

[0101] <<6-O-sulfation>> The method for producing a sulfated polysaccharide of the present invention preferably includes the following steps (4-1) and (4-2). (4-1) Preparing a transformant (d) of a microorganism belonging to prokaryotes, which contains a gene encoding at least 6-O-sulfotransferase that has been introduced into the transformant so as to be expressible, or a processed product or extract thereof (4-2) In the presence of N-sulfoheparosan, adding the transformant (d) or a processed product or extract thereof to the reaction solution to carry out 6-O-sulfation

[0102] In step (4-2), PAPS produced by the transformant (a) or a processed product thereof is used as a sulfate group donor, and 6-O-sulfation of N-sulfoheparosan is carried out by the transformant (d) that expresses 6-O-sulfotransferase or a processed product or extract thereof.

[0103] In addition, when the sulfated polysaccharide produced in step (2-2), step (3-2) or step (3'-3) is present in the reaction solution, 6-O-sulfation of the sulfated polysaccharide produced in the step is performed.

[0104] For example, when C5-epimerized N-sulfoheparosan is present, 6-O-sulfation of C5-epimerized N-sulfoheparosan is performed. When 2-O-sulfated N-sulfoheparosan is present, 6-O-sulfation of 2-O-sulfated N-sulfoheparosan is performed.

[0105] In addition, when C5-epimerized and 2-O-sulfated N-sulfoheparosan is present, 6-O-sulfation of C5-epimerized and 2-O-sulfated N-sulfoheparosan is performed.

[0106] <<3-O-sulfation>> The method for producing a sulfated polysaccharide of the present invention preferably includes the following steps (5-1) and (5-2). (5-1) Preparing a transformant (e) that is a transformant of a microorganism belonging to prokaryotes and contains a gene encoding at least 3-O-sulfotransferase that is expressibly introduced into the transformant, or a processed product or extract thereof (5-2) A step of subjecting 3-O-sulfation by containing the transformant (e) or a processed product or extract thereof in a reaction solution in the presence of N-sulfoheparosan

[0107] In step (5-2), PAPS produced by the transformant (a) or a processed product thereof is used as a sulfate group donor, and 3-O-sulfation of N-sulfoheparosan is performed by the transformant (e) that expresses 3-O-sulfotransferase or a processed product or extract thereof.

[0108] In addition, when the sulfated polysaccharide produced in step (2-2), step (3-2), step (3'-3) or step (4-2) is present in the reaction solution, 3-O-sulfation of the sulfated polysaccharide produced in the step is performed.

[0109] For example, when C5-epimerized N-sulfoheparosan is present, 3-O-sulfation of C5-epimerized N-sulfoheparosan is carried out. When 2-O-sulfated N-sulfoheparosan is present, 3-O-sulfation of 2-O-sulfated N-sulfoheparosan is carried out. When 6-O-sulfated N-sulfoheparosan is present, 3-O-sulfation of 6-O-sulfated N-sulfoheparosan is carried out.

[0110] When C5-epimerized and 2-O-sulfated N-sulfoheparosan is present, 3-O-sulfation of C5-epimerized and 2-O-sulfated N-sulfoheparosan is carried out. When C5-epimerized and 6-O-sulfated N-sulfoheparosan is present, 3-O-sulfation of C5-epimerized and 6-O-sulfated N-sulfoheparosan is carried out. When 2-O-sulfated and 6-O-sulfated N-sulfoheparosan is present, 3-O-sulfation of 2-O-sulfated and 6-O-sulfated N-sulfoheparosan is carried out.

[0111] Also, when C5-epimerized, 2-O-sulfated, and 6-O-sulfated N-sulfoheparosan is present, 3-O-sulfation of C5-epimerized, 2-O-sulfated, and 6-O-sulfated N-sulfoheparosan is carried out.

[0112] <<Reaction conditions>> The reaction conditions for 1) supply / regeneration of PAPS, 2) C5-epimerization, 3) 2-O-sulfation, 4) 6-O-sulfation, and 5) 3-O-sulfation described above will be explained below.

[0113] The pH of the reaction solution is preferably about 6 to 8. During the reaction, it is preferable to react while controlling the pH of the reaction solution to be near neutral, particularly about 7, using an aqueous solution such as aqueous ammonia, sodium hydroxide, potassium hydroxide, etc. with a pH controller.

[0114] The reaction temperature, i.e., the survival temperature of the transformant during the reaction, is preferably 20 to 50°C, more preferably 25 to 47°C. The reaction time is preferably about 1 to 7 days, more preferably about 1 to 3 days. The culture may be any of batch, fed-batch, and continuous types. Among them, the batch type is preferred.

[0115] The aeration condition reaction may be carried out under reducing conditions or microaerobic conditions. The reducing conditions are defined by the redox potential of the reaction solution. The redox potential of the reaction solution is preferably about -200 mV to -500 mV, more preferably -250 mV to -500 mV.

[0116] The reducing state of the reaction solution can be simply estimated with a resazurin indicator, but can be accurately measured using a redox potentiometer. As a method for adjusting the reaction solution under reducing conditions, known methods can be used without limitation.

[0117] Specifically, by subjecting distilled water or the like to heat treatment or reduced pressure treatment to remove dissolved gas, an aqueous solution for the reaction solution under reducing conditions can be obtained. In addition, an appropriate reducing agent (for example, thioglycolic acid, ascorbic acid, cystine hydrochloride, mercaptoacetic acid, thioacetic acid, glutathione, sodium sulfide, etc.) can be added to adjust the aqueous solution for the reaction solution under reducing conditions. Appropriately combining these methods is also an effective method for adjusting the aqueous solution for the reaction solution under reducing conditions.

[0118] When maintaining the reducing conditions during the reaction, it is desirable to prevent the intrusion of oxygen from outside the reaction system as much as possible. Specifically, methods such as enclosing the reaction system with an inert gas such as nitrogen gas or carbon dioxide gas can be mentioned.

[0119] When maintaining microaerobic conditions during the reaction, the aeration rate can be set to a low value such as 0.5 vvm or less, and the reaction can be carried out under conditions where the stirring speed is a low value such as 500 rpm or less. In some cases, after the start of the reaction, aeration can be interrupted at an appropriate time, and the reaction can be carried out in combination with a state where the degree of anaerobiosis is improved under conditions where the stirring speed is 100 rpm or less.

[0120] <<Recovery of sulfated polysaccharide>> By culturing as described above, sulfated polysaccharide is produced in the reaction solution. The sulfated polysaccharide can be recovered by collecting the reaction solution, and furthermore, the sulfated polysaccharide can also be separated from the reaction solution by a known method. Such known methods include, for example, distillation method, membrane permeation method, organic solvent extraction method, etc.

[0121] <<Preparation of N-sulfoheparosan>> The N-sulfoheparosan used in the method for producing a sulfated polysaccharide of the present invention is obtained by deacetylating, depolymerizing and N-sulfating heparosan. The production of heparosan and the production of N-sulfoheparosan from heparosan can be carried out by known methods (for example, International Publication No. 2018 / 048973).

[0122] As a method for producing heparosan, for example, culturing a microorganism belonging to a prokaryote having the following gene modification (a1) and having the ability to produce heparosan in a medium to produce heparosan in the medium, and recovering heparosan from the medium, The manufacturing method including this is mentioned. (a1) Gene modification that increases the expression of the kpsS gene The microorganism belonging to the prokaryote may further have at least one of the following gene modifications (a2) and (a3) in addition to the above (a1). (a2) Gene modification that increases the expression of at least one gene selected from the kfiA, kfiB, kfiC and kfiD genes (a3) Gene modification that impairs the function of the yhbJ gene

[0123] The kpsS gene is a gene encoded in Region I among the gene groups of Region I, Region II, and Region III. kpsS is involved in the initiation of heparosan synthesis, and in heparosan production, kpsS, together with kpsC, plays a role of adding a plurality of Kdo linkers to phosphatidylglycerol in the inner membrane.

[0124] As the kpsS gene, a kpsS gene derived from the genus Escherichia is preferred. Specifically, for example, the kpsS gene of Escherichia coli K5 strain can be mentioned. The nucleotide sequence of the kpsS gene of Escherichia coli K5 strain and the amino acid sequence of the protein encoded by the gene can be obtained from public databases. The kpsS gene of Escherichia coli K5 strain is registered as GenBank accession CAA52659.1.

[0125] As the kpsS gene, DNA containing the nucleotide sequence shown in SEQ ID NO: 27, or DNA containing a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 27 and having the property of increasing the heparosan-producing ability of a microorganism belonging to prokaryotes when the expression level is increased can be mentioned.

[0126] kfiA, kfiB, kfiC, and kfiD are genes encoded in Region II among the gene groups of Region I, Region II, and Region III. As shown in Figure 2, kfiA, kfiB, kfiC, and kfiD are involved in the synthesis of heparosan and play a role in synthesizing heparosan by adding sugars.

[0127] As the kfiA, kfiB, kfiC, or kfiD gene, a kfiA, kfiB, kfiC, or kfiD gene derived from the genus Escherichia is preferred. Specifically, for example, the kfiA, kfiB, kfiC, or kfiD gene of Escherichia coli K5 strain can be mentioned. The nucleotide sequence of the kfiA, kfiB, kfiC, or kfiD gene of Escherichia coli K5 strain and the amino acid sequence of the protein encoded by the gene can be obtained from public databases. kfiA is registered as GenBank accession CAA54711.1; kfiB is registered as GenBank accession CAE55824.1; kfiC is registered as GenBank accession CAA54709.1; kfiD is registered as GenBank accession CAA54708.1.

[0128] As the kfiA gene, there is DNA containing the nucleotide sequence shown in SEQ ID NO: 28, or DNA containing a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 28 and having the property of increasing the heparosan-producing ability of a microorganism belonging to prokaryotes having heparosan-producing ability when the expression level is increased.

[0129] As the kfiB gene, there is DNA containing the nucleotide sequence shown in SEQ ID NO: 29, or DNA containing a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 29 and having the property of increasing the heparosan-producing ability of a microorganism belonging to prokaryotes having heparosan-producing ability when the expression level is increased.

[0130] As the kfiC gene, there is DNA containing the nucleotide sequence shown in SEQ ID NO: 30, or DNA containing a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 30 and having the property of increasing the heparosan-producing ability of a microorganism belonging to prokaryotes having heparosan-producing ability when the expression level is increased.

[0131] As the kfiD gene, there is DNA containing the nucleotide sequence shown in SEQ ID NO: 31, or DNA containing a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 31 and having the property of increasing the heparosan-producing ability of a microorganism belonging to prokaryotes having heparosan-producing ability when the expression level is increased.

[0132] Figure 3 shows a schematic diagram of the biosynthetic pathway of heparosan. GlmS is the first enzyme in the UDP-N-acetylglucosamine supply pathway, which is a precursor of heparosan, and is an enzyme that catalyzes the reaction from fructose-6-phosphate to glucosamine-6-phosphate. YhbJ is an enzyme that negatively regulates GlmS.

[0133] As the yhbJ gene, a yhbJ gene derived from the genus Escherichia is preferred. Specifically, for example, the yhbJ gene of Escherichia coli K-12 strain can be mentioned. The nucleotide sequence of the yhbJ gene of Escherichia coli K-12 strain and the amino acid sequence of the protein encoded by the gene can be obtained from public databases. The yhbJ gene of Escherichia coli K-12 strain is registered as GenBank accession BAE77249.1.

[0134] As the yhbJ gene, there can be mentioned DNA containing the nucleotide sequence shown in SEQ ID NO: 32, or DNA containing a nucleotide sequence having 90% or more identity with the nucleotide sequence shown in SEQ ID NO: 32 and having the property of increasing the heparosan-producing ability of a microorganism belonging to prokaryotes when the expression level is decreased in the microorganism.

[0135] Each of the genes in the above (a1) to (a3) can be easily obtained from public databases, for example, by BLAST search or FASTA search using the nucleotide sequences of the respective genes described above. Also, homologs of each gene can be obtained, for example, by PCR using oligonucleotides prepared based on these known gene sequences as primers with the chromosome of a microorganism such as bacteria as a template.

[0136] Each of the genes in the above (a1) to (a3) may be a variant of the gene as long as the original function (for example, activity or property) of the protein encoded by the gene is maintained. Whether the protein encoded by the variant of the gene maintains the original function can be specifically confirmed, for example, when the original function is a function of improving the heparosan-producing ability, by introducing the variant of the gene into a microorganism belonging to prokaryotes having the heparosan-producing ability and checking whether it has the original function.

[0137] The variants of each gene in the above (a1) to (a3) can be obtained, for example, by modifying the coding region of the gene by a site-directed mutagenesis method so that the amino acid residues at specific sites of the encoded protein include substitution, deletion, insertion or addition. Also, the variants of each gene in the above (a1) to (a3) can be obtained, for example, by a mutagenesis treatment.

[0138] As long as the original function is maintained, each gene in the above (a1) to (a3) may encode a protein having an amino acid sequence in which one or several amino acids are substituted, deleted, inserted or added at one or several positions in the above amino acid sequence. For example, the N-terminus and / or C-terminus of the encoded protein may be extended or shortened. The above "one or several" varies depending on the position and type of the amino acid residue in the three-dimensional structure of the protein, but specifically means, for example, 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, still more preferably 1 to 5, and particularly preferably 1 to 3.

[0139] The above-described substitution, deletion, insertion, or addition of one or several amino acids is a conservative mutation that maintains the normal function of the protein. A typical example of a conservative mutation is a conservative substitution. A conservative substitution means that when the substitution site is an aromatic amino acid, it is between Phe, Trp, and Tyr; when the substitution site is a hydrophobic amino acid, it is between Leu, Ile, and Val; when it is a polar amino acid, it is between Gln and Asn; when it is a basic amino acid, it is between Lys, Arg, and His; when it is an acidic amino acid, it is between Asp and Glu; and when it is an amino acid with a hydroxyl group, it is a mutation where they substitute each other between Ser and Thr. Specifically, substitutions considered to be conservative substitutions include substitution of Ala with Ser or Thr, substitution of Arg with Gln, His, or Lys, substitution of Asn with Glu, Gln, Lys, His, or Asp, substitution of Asp with Asn, Glu, or Gln, substitution of Cys with Ser or Ala, substitution of Gln with Asn, Glu, Lys, His, Asp, or Arg, substitution of Glu with Gly, Asn, Gln, Lys, or Asp, substitution of Gly with Pro, substitution of His with Asn, Lys, Gln, Arg, or Tyr, substitution of Ile with Leu, Met, Val, or Phe, substitution of Leu with Ile, Met, Val, or Phe, substitution of Lys with Asn, Glu, Gln, His, or Arg, substitution of Met with Ile, Leu, Val, or Phe, substitution of Phe with Trp, Tyr, Met, Ile, or Leu, substitution of Ser with Thr or Ala, substitution of Thr with Ser or Ala, substitution of Trp with Phe or Tyr, substitution of Tyr with His, Phe, or Trp, and substitution of Val with Met, Ile, or Leu. In addition, the above-described amino acid substitution, deletion, insertion, addition, or inversion, etc. also includes those caused by naturally occurring mutations (mutant or variant) such as those based on individual differences or species differences of the organism from which the gene is derived.

[0140] In addition, as long as the original function is maintained, each gene in the above (a1) to (a3) may be a gene encoding a protein having an identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more with respect to the entire amino acid sequence.

[0141] In addition, as long as the original function is maintained, each gene in the above (a1) to (a3) may be a DNA that hybridizes under stringent conditions with a probe that can be prepared from a known gene sequence, for example, a complementary sequence to the whole or a part of the above nucleotide sequence. "Stringent conditions" refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. For example, conditions under which DNAs with high identity, for example, DNAs with an identity of 80% or more, preferably 90% or more, more preferably 95% or more, still more preferably 97% or more, and particularly preferably 99% or more hybridize with each other, and DNAs with lower identity do not hybridize with each other, or conditions of washing in ordinary Southern hybridization, that is, washing once, preferably 2 to 3 times, at a salt concentration and temperature corresponding to 60°C, 1×SSC, 0.1% SDS, preferably 60°C, 0.1×SSC, 0.1% SDS, and more preferably 68°C, 0.1×SSC, 0.1% SDS can be mentioned.

[0142] The probe used for the above hybridization may be a part of the complementary sequence of each gene. Such a probe can be prepared by PCR using an oligonucleotide prepared based on a known gene sequence as a primer and a DNA fragment containing each gene in the above (a1) to (a3) as a template. For example, a DNA fragment with a length of about 300 bp can be used as the probe. When a DNA fragment with a length of about 300 bp is used as the probe, conditions for washing the hybridization include 50°C, 2×SSC, 0.1% SDS.

[0143] In addition, since the codon degeneracy varies depending on the host, each gene in the above (a1) to (a3) may be a substitution of any codon with an equivalent codon as long as its original function is maintained. For example, the genes in Tables 1 to 3 may be modified to have optimal codons according to the codon usage frequency of the host to be used.

[0144] Examples of the mutation treatment include a method of in vitro treating a DNA molecule having the nucleotide sequence of each gene in the above (a1) to (a3) with hydroxylamine or the like, and a method of treating a microorganism retaining each gene in the above (a1) to (a3) with a mutagen such as X-ray, ultraviolet ray, or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS).

[0145] <<<Gene modification for increasing gene expression>>> "Increase in gene expression" means that the expression of the gene is increased as compared with the unmodified strain. As one aspect of the increase in gene expression, for example, the expression of the gene is preferably increased by 1.5-fold or more, more preferably 2-fold or more, and even more preferably 3-fold or more as compared with the unmodified strain.

[0146] In addition, "increase in gene expression" includes not only increasing the expression level of the target gene in a strain in which the target gene is originally expressed, but also expressing the target gene in a strain in which the target gene is not originally expressed. That is, "increase in gene expression" includes, for example, introducing the target gene into a strain that does not carry the target gene and expressing the target gene. Note that "increase in gene expression" is also referred to as "enhancement of gene expression" or "increase in gene expression".

[0147] An increase in gene expression can be achieved, for example, by increasing the copy number of the gene. An increase in the copy number of the gene can be achieved by introducing the gene into the host chromosome. The introduction of the gene into the chromosome can be carried out, for example, using homologous recombination (Miller I, J. H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Only one copy of the gene may be introduced, or two or more copies may be introduced.

[0148] For example, by performing homologous recombination targeting a sequence with multiple copies on the chromosome, multiple copies of the gene can be introduced into the chromosome. Examples of sequences with multiple copies on the chromosome include repetitive DNA sequences and inverted repeats present at both ends of transposons.

[0149] Alternatively, homologous recombination may be performed targeting an appropriate sequence on the chromosome such as a gene unnecessary for the production of the target substance. Homologous recombination can be carried out, for example, by a method using linear DNA, a method using a plasmid containing a temperature-sensitive origin of replication, a method using a conjugative plasmid, a method using a suicide vector having no origin of replication functioning in the host, or a transduction method using a phage. Also, the gene can be randomly introduced onto the chromosome using a transposon or Mini-Mu (Japanese Patent Laid-Open No. 2-109985).

[0150] Confirmation that the target gene has been introduced onto the chromosome can be made by Southern hybridization using a probe having a sequence complementary to all or part of the gene, or by PCR using primers prepared based on the sequence of the gene.

[0151] In addition, an increase in the copy number of a gene can also be achieved by introducing a vector containing the gene into a host. For example, a DNA fragment containing a target gene is ligated to a vector that functions in the host to construct an expression vector for the gene, and the host is transformed with the expression vector to increase the copy number of the gene. The DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism having the target gene as a template. The transformation method is not particularly limited, and conventionally known methods can be used.

[0152] As the vector, a vector capable of autonomous replication in the host cell can be used. The vector is preferably a multicopy vector. In addition, in order to select a transformant, the vector preferably has a marker such as an antibiotic resistance gene or other genes described in the literature [Karl Friehs, Plasmid Copy Number and Plasmid Stability, Adv Biochem Engin / Biotechnol 86: 47-82 (2004)]. Further, the vector may be provided with a promoter and a terminator for expressing the inserted gene. Examples of the vector include vectors derived from bacterial plasmids, vectors derived from yeast plasmids, vectors derived from bacteriophages, cosmids, or phagemids.

[0153] Specific examples of vectors capable of autonomous replication in bacteria of the family Enterobacteriaceae such as Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, pSTV29 (all from Takara Bio Inc.), pACYC184, pMW219 (Nippon Gene Co., Ltd.), pTrc99A (Pharmacia), pPROK series vectors (Clontech), pKK233-2 (Clontech), pET series vectors (Novagen), pQE series vectors (Qiagen), and the broad host range vector RSF1010.

[0154] When introducing a gene, the gene only needs to be retained in a microorganism belonging to a prokaryote having the gene modification in the present invention. Specifically, the gene only needs to be introduced so as to be expressed under the control of a promoter sequence that functions in the bacterium of the present invention. The promoter may be a promoter derived from the host or a promoter derived from a heterologous source. The promoter may be the native promoter of the gene to be introduced or the promoter of another gene. As the promoter, for example, a more powerful promoter as described below may be used.

[0155] A terminator for transcription termination can be arranged downstream of the gene. The terminator is not particularly limited as long as it functions in the bacterium of the present invention. The terminator may be a terminator derived from the host or a terminator derived from a heterologous source. The terminator may be the native terminator of the gene to be introduced or the terminator of another gene. Specifically, examples of the terminator include T7 terminator, T4 terminator, fd phage terminator, tet terminator, and trpA terminator.

[0156] Regarding vectors, promoters, and terminators that can be used in various microorganisms, they are described in detail in, for example, "Basic Microbiology Course 8 Genetic Engineering, Kyoritsu Shuppan, 1987", and it is possible to use them.

[0157] Also, when introducing two or more genes, each gene only needs to be retained in the bacterium of the present invention so that it can be expressed. For example, all of the genes may be retained on a single expression vector, or all may be retained on the chromosome. Also, each gene may be separately retained on a plurality of expression vectors, or may be separately retained on a single or a plurality of expression vectors and on the chromosome. Further, an operon may be constituted by two or more genes and introduced. Examples of "when introducing two or more genes" include cases where genes encoding two or more enzymes are introduced respectively, cases where genes encoding two or more subunits constituting a single enzyme are introduced respectively, and combinations thereof.

[0158] The gene to be introduced is not particularly limited as long as it encodes a protein that functions in the host. The gene to be introduced may be a gene derived from the host or a gene derived from a heterologous source. The gene to be introduced can be obtained, for example, by PCR using primers designed based on the nucleotide sequence of the gene and using genomic DNA of an organism having the gene or a plasmid carrying the gene as a template. Also, the gene to be introduced may be, for example, fully synthesized based on the nucleotide sequence of the gene [Gene, 60(1), 115 - 127 (1987)].

[0159] Also, an increase in gene expression can be achieved by improving the transcription efficiency of the gene. Improvement of the transcription efficiency of the gene can be achieved, for example, by replacing the promoter of the gene on the chromosome with a stronger promoter. A "stronger promoter" means a promoter by which the transcription of the gene is improved compared to the wild - type promoter originally present.

[0160] Examples of "stronger promoters" include, for example, the uspA promoter, T7 promoter, trp promoter, lac promoter, thr promoter, tac promoter, trc promoter, tet promoter, araBAD promoter, rpoH promoter, PR promoter, and PL promoter, which are known high-expression promoters.

[0161] In addition, as a stronger promoter, a highly active form of an endogenous promoter may be obtained by using various reporter genes. For example, the activity of a promoter can be enhanced by making the -35 and -10 regions in the promoter region closer to the consensus sequence (International Publication No. 2000 / 18935).

[0162] Examples of highly active promoters include various tac-like promoters (Katashkina JI et al., Russian Federation Patent Application 2006134574) and the pnlp8 promoter (International Publication No. 2010 / 027045). Methods for evaluating the strength of promoters and examples of strong promoters are described in known literature [Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105 - 128 (1995), etc.].

[0163] In addition, an increase in gene expression can be achieved by improving the translation efficiency of the gene. Improvement of the translation efficiency of a gene can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence [also referred to as the ribosome binding site (RBS)] of the gene on the chromosome with a stronger SD sequence.

[0164] "More potent SD sequence" means an SD sequence in which mRNA translation is improved compared to the wild-type SD sequence originally present. Examples of more potent SD sequences include, for example, the RBS of gene 10 derived from phage T7 [Olins P. O. et al, Gene, 1988, 73, 227-235]. Furthermore, it is known that substitutions, insertions, or deletions of several nucleotides in the spacer region between the RBS and the start codon, particularly in the sequence (5'-UTR) immediately upstream of the start codon, have a very significant impact on mRNA stability and translation efficiency, and the translation efficiency of the gene can also be improved by modifying these.

[0165] In the present invention, sites that affect gene expression such as promoters, SD sequences, and the spacer region between the RBS and the start codon are collectively referred to as "expression regulatory regions". Expression regulatory regions can be determined using gene analysis software such as promoter search vectors and GENETYX. Modification of these expression regulatory regions can be carried out, for example, by methods using temperature-sensitive vectors or the Red-driven integration method (International Publication No. 2005 / 010175).

[0166] Improvement of gene translation efficiency can also be achieved, for example, by codon modification. Specifically, for example, when heterologous expression of a gene is carried out, the translation efficiency of the gene can be improved by replacing rare codons present in the gene with synonymous codons that are used more frequently.

[0167] Codon substitution can be carried out, for example, by site-directed mutagenesis methods that introduce the desired mutation into the target site of DNA. Examples of site-directed mutagenesis methods include methods using PCR [Higuchi, R., 61, in PCR technology, Erlich, H. A. Eds., Stockton press (1989); Carter, P., Meth. in Enzymol., 154, 382 (1987)] and methods using phage [Kramer, W. and Frits, H. J., Meth. in Enzymol., 154, 350 (1987); Kunkel, T. A. et al., Meth. in Enzymol., 154, 367 (1987)]. Alternatively, the gene fragment with codon substitution may be fully synthesized. The codon usage frequencies in various organisms are disclosed in the "Codon Usage Database" [http: / / www.kazusa.or.jp / codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000)].

[0168] In addition, an increase in gene expression can also be achieved by amplifying a regulator that increases gene expression or deleting or weakening a regulator that decreases gene expression. The methods for increasing gene expression as described above may be used alone or in any combination.

[0169] That the gene expression has increased can be confirmed, for example, by confirming that the transcription level of the gene has increased or that the amount of the protein expressed from the gene has increased. Also, that the gene expression has increased can be confirmed, for example, by confirming that the activity of the protein expressed from the gene has increased.

[0170] Confirmation of increased gene transcription levels can be achieved by comparing the amount of mRNA transcribed from the gene with that of an unmodified strain such as a wild-type strain or parental strain. Methods for evaluating the amount of mRNA include Northern hybridization, RT-PCR, etc. [Sambrook, J., et al., Molecular Cloning A Laboratory Manual / Third Edition, Cold spring Harbor Laboratory Press, Cold spring Harbor (USA), 2001]. An increase in the amount of mRNA is, for example, preferably at least 1.5-fold, more preferably at least 2-fold, and even more preferably at least 3-fold higher compared to the unmodified strain.

[0171] An increase in the amount of protein can be confirmed, for example, by Western blot using an antibody. An increase in the amount of protein is, for example, preferably at least 1.5-fold, more preferably at least 2-fold, and even more preferably at least 3-fold higher compared to the unmodified strain.

[0172] An increase in protein activity can be confirmed, for example, by measuring the activity of the protein. An increase in protein activity is, for example, preferably at least 1.5-fold, more preferably at least 2-fold, and even more preferably at least 3-fold higher compared to the unmodified strain.

[0173] The methods for increasing the expression of the above-mentioned genes can be used for enhancing the expression of each of the genes (a1) and (a2) described above.

[0174] As gene modifications for increasing the expression of the kpsS gene, it is preferable that they are at least one of modification of the expression regulatory region of the kpsS gene and gene modification for increasing the copy number. Among the heparosan production gene group, there are kpsFEDUCS genes. However, as described later in the examples, the inventors have found that by increasing the expression of only the kpsS gene among these, a remarkable effect of improving heparosan production can be obtained. Therefore, as the gene modification for increasing the expression of the kpsS gene, gene modification for increasing the copy number of the kpsS gene is particularly preferable.

[0175] As gene modifications for increasing the expression of at least one gene selected from the kfiA, kfiB, kfiC, and kfiD genes, it is preferable that they are at least one of modification of the expression regulatory region of at least one gene selected from the kfiA, kfiB, kfiC, and kfiD genes and increasing the copy number of the gene. As shown in FIG. 1, the kfiA, kfiB, kfiC, and kfiD genes form an operon. Gene modification for strengthening the entire operon composed of the kfiA, kfiB, kfiC, and kfiD genes is preferable, and modification of the expression regulatory region of the kfiA, kfiB, kfiC, and kfiD genes is more preferable.

[0176] <<<Gene modification for disabling gene function>>> As the gene modification for disabling the function of the yhbJ gene in (a3) described above, examples include modifying the DNA encoding the portion corresponding to yhbJ in the genomic DNA of a microorganism belonging to a prokaryote as the host, so as to reduce or completely stop the function of the protein encoded by the portion corresponding to yhbJ.

[0177] In the method of the present invention, the form of the modification added to the DNA encoding the portion corresponding to yhbJ is not particularly limited as long as it is a form that reduces or completely stops the function of the protein encoded by the gene corresponding to yhbJ, and known methods can be appropriately used.

[0178] Examples of forms that reduce or completely stop the function of the protein encoded by the portion corresponding to yhbJ include, for example, any one of the following modifications (I) to (III). (I) Remove all or part of the DNA encoding the portion corresponding to yhbJ. (II) Perform one or several substitutions, deletions or additions to the DNA encoding the portion corresponding to yhbJ. (III) Replace the DNA encoding the portion corresponding to yhbJ with a DNA sequence having less than 80% identity with the DNA sequence before modification.

[0179] Examples of the loss of function of the yhbJ gene include, for example, that the activity of yhbJ is preferably 20% or less, more preferably 10% or less, and still more preferably 5% or less compared to the unmodified strain. The activity of yhbJ can be confirmed by examining the expression level of glmS by methods such as Northern blotting and Western blotting [Kalamorz F. et al, (2007) “Feedback control of glucosamine-6-phosphate synthase GlmS expression depends on the small RNA GlmZ and involves the novel protein YhbJ in Escherichia coli.” Mol Microbiol.65(6):1518-33].[[]END]]

[0180] In the method for producing the sulfated polysaccharide of the present invention, N-sulfoheparosan obtained by chemically modifying heparosan obtained by the above-described method for producing heparosan by a known method can be used in the reaction solution.

[0181] <Method for Producing PAPS> The method for producing PAPS of the present invention is characterized in that a production reaction of PAPS is carried out by adding to a reaction solution containing an ATP source, a sulfate ion source, the above-described transformant (a) or a processed product thereof. That is, the method for producing PAPS of the present invention includes the following steps (i) and (ii). (i) A step of preparing a transformant of a Corynebacterium bacterium, which contains at least a gene encoding ATP sulfurylase and a gene encoding APS kinase that are expressibly introduced into the transformant, and a transformant or a processed product thereof in which the cell plasma membrane has substance permeability (ii) A step of performing a production reaction of PAPS using a reaction solution containing an ATP source, a sulfate ion source, and the transformant or a processed product thereof prepared in step (i)

[0182] According to the method for producing PAPS of the present invention, PAPS can be produced by a cell reaction using a transformant of a Corynebacterium bacterium that expresses ATP sulfurylase and APS kinase or a processed product thereof.

Example

[0183] Examples are shown below, but the present invention is not limited to the following examples.

[0184] [Example 1] Construction of a DE3 plasmid of Corynebacterium ammoniagenes A plasmid for inserting λDE3 containing the T7 RNA polymerase gene between gltD and purT on the chromosome of the Corynebacterium ammoniagenes wild strain ATCC 6872 was constructed as follows. Two primers [gltD-purT_1 (SEQ ID NO: 1) and gltD-purT_2BX (SEQ ID NO: 2)] for amplifying the gltD side, and two primers [gltD-purT_3BX (SEQ ID NO: 3) and gltD-purT_4 (SEQ ID NO: 4)] for amplifying the purT side were designed.

[0185] At that time, in order to perform the second PCR (fusion PCR) for ligating the gltD-side fragment and the purT-side fragment amplified in the first PCR, a complementary sequence of approximately 25 bases on the 3'-side of the 5'-primer (gltD-purT_4; SEQ ID NO: 4) for amplifying the purT-side fragment was added to the 5'-side of the 3'-primer (gltD-purT_2BX; SEQ ID NO: 2) for amplifying the gltD-side fragment. Furthermore, sequences were added to the 5'-sides of the 5'-primer (gltD-purT_1) for amplifying the gltD-side fragment and the 3'-primer (gltD-purT_4) for amplifying the purT-side fragment for In fusion cloning. In addition, Bgl II and Xho I recognition sequences were added to the ligation portions on the gltD side and the purT side for inserting λDE3.

[0186] Chromosomal DNA of Corynebacterium ammoniagenes ATCC 6872 strain (hereinafter referred to as ATCC 6872 strain), which is a wild-type strain of Corynebacterium ammoniagenes, was prepared according to the method of Saito et al. [Biochim. Biophys. Acta 72, 619 (1963)].

[0187] Using this chromosomal DNA as a template, the first PCR was performed to amplify the gltD side and the purT side, and a DNA fragment of approximately 0.8 kb on the gltD side and a DNA fragment of approximately 0.8 kb on the purT side were obtained. Subsequently, the second PCR was performed to ligate these gltD-side fragment and purT-side fragment, and a DNA fragment of approximately 1.6 kb (gltD-BX-purT) was obtained.

[0188] A 2.6 kb PstI DNA fragment [Mol. Microbiol., 6, 1195 (1992)] containing the levansucrase gene sacB of Bacillus subtilis was ligated to the PstI cleavage site of the vector pHSG299 of Escherichia coli having a kanamycin resistance gene [Gene, 61, 63, (1987)]. After plasmid pESB30 thus obtained was cleaved with BamHI, the gltD-BX-purT fragment obtained above was ligated using an In-Fusion cloning kit (Takara Bio Inc.). Using the reaction product, Escherichia coli DH5α [manufactured by Toyobo Co., Ltd.] was transformed according to a conventional method [Molecular cloning: a laboratory manual, 3 rd ed., 2001, Cold Spring Harbor Laboratory Press].

[0189] The resulting strain was cultured on an LB agar medium [medium containing 10 g of Bacto Tryptone (manufactured by Difco), 5 g of yeast extract (manufactured by Difco), 10 g of sodium chloride, 16 g of Bacto Agar (manufactured by Difco) in 1 L of water, adjusted to pH 7.0] containing 20 μg / ml of kanamycin to select transformants. After selecting the target clone by colony PCR, the transformant was inoculated into an LB medium (medium having the same composition as the LB agar medium except for not containing agar) containing 20 μg / ml of kanamycin and cultured overnight, and a plasmid was prepared from the resulting culture solution using a QIAprep Spin Miniprep Kit (Qiagen). By nucleotide sequence analysis, it was confirmed that the plasmid had a structure in which a gltD-BX-purT fragment of about 1.6 kb was inserted into pESB30.

[0190] Subsequently, a 4.5-kb λDE3 fragment was amplified by PCR using chromosomal DNA extracted from Escherichia coli BL21(DE3) as a template and primers DE3-for_Xho (SEQ ID NO: 5) and DE3-rev_Bgl (SEQ ID NO: 6). An Xho I recognition sequence was added to DE3-for_Xho, and a Bgl II recognition sequence was added to DE3-rev_Bgl. A plasmid having a structure in which a fragment of about 1.6 kb of gltD-BX-purT was inserted into this fragment and pESB30 was digested with Xho I and Bgl II, and then ligated using a DNA ligation kit (manufactured by Takara Bio Inc.).

[0191] In the same manner as described above, Escherichia coli DH5α was transformed, and the resulting strain was cultured on an LB agar medium containing 20 μg / ml of kanamycin to select transformants. The transformants were inoculated into an LB medium containing 20 μg / ml of kanamycin and cultured overnight, and a plasmid was prepared from the resulting culture solution using a QIAprep Spin Miniprep Kit (Qiagen). By nucleotide sequence analysis, it was confirmed that the plasmid had a structure in which a 4.5-kb λDE3 fragment was inserted between about 1.6 kb of gltD-purT on pESB30. This plasmid was named pC-DE3.

[0192] [Example 2] Construction of a λDE3 Insertion Strain of Corynebacterium ammoniagenes pC-DE3 was introduced into the ATCC 6872 strain by electroporation according to the method of Resta et al. [Appl. Microbiol. Biotech., 52, 541 (1999)], and kanamycin-resistant strains were selected. When the chromosomal structure obtained from one of the kanamycin-resistant strains was examined by Southern hybridization [Molecular cloning: a laboratory manual, 3 rd ed., 2001, Cold Spring Harbor Laboratory Press], it was confirmed that pC-DE3 was integrated into the chromosome by Campbell-type homologous recombination.

[0193] The transformed strain (single recombinant) was spread on Suc agar medium [a medium containing 100 g of sucrose, 7 g of meat extract, 10 g of peptone, 3 g of sodium chloride, 5 g of yeast extract (manufactured by Difco), 15 g of Bacto Agar (manufactured by Difco) in 1 L of water, adjusted to pH 7.2], and colonies that grew after culturing at 30°C for 1 day were selected. Strains in which the sacB gene is present cannot grow on this medium because they convert sucrose into a suicide substrate [J. Bacteriol., 174, 5462 (1991)]. In contrast, strains in which the sacB gene has been deleted by a second homologous recombination between the wild-type and λDE3 insertion types that are present adjacent to each other on the chromosome can grow on this medium because no suicide substrate is produced. During this homologous recombination, either the wild-type structure or the strain with the λDE3 fragment inserted between gltD and purT is lost together with sacB. When the wild-type structure is lost together with sacB, it means that gene replacement to the λDE3 insertion type has occurred.

[0194] The double recombinant thus obtained was used as a template for colony PCR with primers DE3-for_Xho and DE3-rev_Bgl to obtain a strain in which λDE3 was inserted between gltD and purT of ATCC 6872. This strain was named ATCC 6872(DE3).

[0195] [Example 3] Construction of a plasmid having a DNA fragment for MET3-MET14 expression The plasmid pCS299P [Appl. Microbiol. Biotech., 63, 592 (2004)] was digested with BamHI. Using pET21b as a template, PCR was performed with pCET_Fw2 (SEQ ID NO: 7) and pCET_Rv2 (SEQ ID NO: 8) to obtain a DNA fragment containing lacI-PT7. These were purified using the QIAquick PCR Purification Kit (Qiagen) and ligated using the In-Fusion cloning kit (manufactured by Takara Bio Inc.). Using the reaction product, Escherichia coli DH5α (manufactured by Toyobo Co., Ltd.) was transformed according to a conventional method and cultured on an LB agar medium containing 20 μg / ml kanamycin to select transformants. After selecting the target clone by colony PCR, the transformant was inoculated into an LB medium containing 20 μg / ml kanamycin and cultured overnight, and a plasmid was prepared from the obtained culture solution using the QIAprep Spin Miniprep Kit (Qiagen). By nucleotide sequence analysis, it was confirmed that the plasmid had a structure in which a pET21b-derived lacI-PT7 DNA fragment of approximately 1.9 kb was inserted into pCS299P. This plasmid was named pCET212.

[0196] Subsequently, a plasmid in which MET3 and MET14 derived from Saccharomyces cerevisiae were inserted downstream of the T7 promoter of pCET212 was constructed. Two kinds of primers (MET3_1 (SEQ ID NO: 9) and MET3_2 (SEQ ID NO: 10)) for amplifying the MET3 side and two kinds of primers (MET14_3 (SEQ ID NO: 11) and MET14_4 (SEQ ID NO: 12)) for amplifying MET14 were designed from the chromosomal DNA of the Saccharomyces cerevisiae S288C strain (hereinafter referred to as S288C).

[0197] At that time, in order to perform the second PCR (fusion PCR) for ligating the MET3 fragment and the MET14 fragment amplified in the first PCR, a complementary sequence of approximately 15 bases on the 5'-side of the 5'-primer for MET14 amplification (MET14_3; SEQ ID NO: 11) was added to the 5'-side of the 3'-primer for MET3 amplification (MET3_2; SEQ ID NO: 10), and a complementary sequence of approximately 15 bases on the 5'-side of the 5'-primer for MET3 amplification (MET3_3) was added to the 5'-side of MET14_3. Furthermore, sequences were added to the 5'-sides of the 5'-primer for MET3 amplification (MET3_1) and the 3'-primer for MET13 amplification (MET14_4) for In fusion cloning. Using the chromosomal DNA of the S288C strain as a template, the first PCR was performed to amplify MET3 and MET14, and a DNA fragment of approximately 1.5 kb of MET3 and a DNA fragment of approximately 0.6 kb of the downstream region were obtained. Subsequently, the second PCR was performed to ligate these MET3 fragments and MET14 fragments, and a DNA fragment of approximately 2.1 kb (MET3-MET14) was obtained. After purifying this DNA fragment with the QIAquick PCR Purification Kit (Qiagen), it was ligated to pCET212 digested with Nde I and Xho I using the In-Fusion cloning kit (manufactured by Takara Bio Inc.). Using the reaction product, Escherichia coli DH5α (manufactured by Toyobo Co., Ltd.) was transformed according to a conventional method. The obtained strain was cultured on an LB agar medium containing 20 μg / ml of kanamycin, and transformants were selected. After selecting the target clone by colony PCR, the transformant was inoculated into an LB medium containing 20 μg / ml of kanamycin and cultured overnight, and a plasmid was prepared from the obtained culture solution using the QIAprep Spin Miniprep Kit (Qiagen). By nucleotide sequence analysis, it was confirmed that the plasmid had a structure in which a MET3-MET14 fragment of approximately 2.1 kb was inserted into pCET212. This plasmid was named pSC-3-13. By transforming pSC-3-13 into ATCC 6872(DE3), the PAPS-producing strain Corynebacterium ammoniagenes ATCC 6872(DE3) / pSC-3-13 was obtained.

[0198] [Example 4] Construction of a plasmid having a DNA fragment for chaperone protein expression Using the chromosomal DNA of Escherichia coli BL21(DE3) strain as a template, PCR was performed with Gro_F (SEQ ID NO: 13) and Gro_R (SEQ ID NO: 14) to obtain a DNA fragment containing GroES-GroEL. Next, using pKD46 [Datsenko, K.A., Warner, B.L., Proceedings of the National Academy of Science of the United States of America, Vol. 97. 6640-6645(2000)] as a template, PCR was performed with AraC_ParaB_F (SEQ ID NO: 15) and AraC_ParaB_R (SEQ ID NO: 16) to obtain a DNA fragment containing AraC_ParaB. Using pCDF-Duet1 (Novagen) as a template, PCR was performed with pCDF-SmOri-F (SEQ ID NO: 17) and pCDF-SmOri-R (SEQ ID NO: 18) to obtain a DNA fragment containing the streptomycin resistance gene and the ColdDF replication origin. These were purified using the QIAquick PCR Purification Kit (Qiagen) and ligated using the In-Fusion cloning kit (manufactured by Takara Bio Inc.). Using the reaction product, Escherichia coli DH5α (manufactured by Toyobo Co., Ltd.) was transformed according to a conventional method and cultured on an LB agar medium containing 50 μg / ml of streptomycin to select transformants. After selecting the target clone by colony PCR, the transformant was inoculated into an LB medium containing 50 μg / ml of streptomycin and cultured overnight, and a plasmid was prepared from the obtained culture solution using the QIAprep Spin Miniprep Kit (Qiagen). By nucleotide sequence analysis, it was confirmed that the plasmid had a structure in which a pKD46-derived AraC-ParaB DNA fragment of about 1.2 kb and a BL21(DE3)-derived GroES-GroEL DNA fragment of about 2.0 kb were inserted into pCDF-Duet1. This plasmid was named pGro(Sm).

[0199] [Example 5] Construction of Escherichia coli Expressing C5-epimerase, 2OST, 6OST-3, and 3OST-1 The catalytic domain region (E53-N609) of human-derived C5-epimerase was cloned into the pMAL-C2X vector (New England Biolabs) by the method described in the literature [Biochemical and Biophysical Research Communications Volume 339, Issue 2, 13 January 2006, Pages 597-602] to construct MBP-C5. MBP-C5 was transformed into Origami-B(DE3) (Novagen) together with pGro7 (Takara Bio Inc.) to construct Escherichia coli Origami-B(DE3) / MBP-C5_pGro7 strain expressing C5-epimerase.

[0200] The nucleotide sequence of the catalytic domain (R51-N356) of Chinese hamster-derived 2-O-sulfotransferase isoform 1 optimized for codons for expression in Escherichia coli is shown in SEQ ID NO: 19. The above synthetic sequence was used as a template and PCR amplified using the primers described in SEQ ID NOs: 20 and 21. This PCR fragment was cloned into pET-His6-MBP-TEV-LIC (Addgene) by the Ligation independent cloning method [Methods Mol Biol. 2009; 498: 105-115] to construct H-MBP-2OST. H-MBP-2OST was transformed into Origami-B(DE3) (Novagen) together with pGro(Sm) to construct Escherichia coli Origami-B(DE3) / H-MBP-2OST_pGro(Sm) strain expressing 2OST.

[0201] The catalytic domain (P120-L424) of mouse-derived 6-O-sulfotransferase isoform 3 was cloned into the pMAL-C2X vector (New England Biolabs) by the method described in the literature [Chemistry & Biology Volume 14, Issue 9, 21 September 2007, Pages 986-993] to construct MBP-6OST3. MBP-6OST3 was transformed into Origami-B(DE3) (Novagen) together with pGro7 (Takara Bio) to construct an Escherichia coli, Origami-B(DE3) / MBP-6OST3_pGro7 strain expressing 6OST-3.

[0202] The catalytic domain (G48-H311) of mouse-derived 3-O sulfotransferase was cloned into the pET28a vector (Novagen) by the method described in the literature [J Biol Chem. 2004 Jun 11;279(24):25789-97] to construct HIS-3OST1. HIS-3OST1 was transformed into BL21-CodonPlus(DE3)-RIL (Agilent Technologies) together with pGro(Sm) to construct an Escherichia coli RIL / HIS-3OST1_pGro(Sm) strain expressing 3OST1.

[0203] [Example 6] Preparation of N-sulfoheparosan and 2-O-sulfated N-sulfoheparosan The fermentative production of heparosan was carried out using Escherichia coli K5 strain or Nissle strain by the method described in the patent document [WO2018 / 048973 A1]. The obtained heparosan was chemically deacetylated and depolymerized according to the same document, and then chemically N-sulfated. Thereafter, fractionation was performed by ethanol precipitation to obtain N-sulfoheparosan. The obtained N-sulfoheparosan was subjected to epimerization at the C5 position of the uronic acid residue and 2-O-sulfation by an enzymatic reaction according to the same document, and then fractionation was performed by ethanol precipitation to obtain 2-O-sulfated N-sulfoheparosan.

[0204] [Example 7] Cultivation of ATCC 6872(DE3) / pSC-3-13 The ATCC 6872(DE3) / pSC-3-13 strain obtained in Example 3 was inoculated into a BY-Glucose agar medium containing 50 μg / ml kanamycin [a medium containing 10 g of glucose, 20 g of normal broth medium (manufactured by Kyokuto Pharmaceutical Industry Co., Ltd.), 5 g of yeast extract (manufactured by Difco), and 20 g of bacto agar (manufactured by Difco) in 1 L of water], and cultured overnight at 30°C.

[0205] The cells from two plates were inoculated into 350 ml of a first seed medium [50 g / L of glucose, 10 g / L of polypeptone (manufactured by Nippon Pharmaceutical Co., Ltd.), 10 g / L of yeast extract (manufactured by Asahi), KH 2 PO 4 1 g / L, K 2 HPO 4 (NH 4 ) 2 SO 4 0.5 g / L, 0.5 g / L of urea, 0.03 g / L of L-cystine, MgSO 4 ·7H 2 O 1 g / L, CaCl 2 ·2H 2 O 0.1 g / L, ZnSO 4 ·7H 2 O 0.01 g / L, FeSO 4 ·7H 2 O 0.01 g / L, MnSO 4 ·5H 2 O 0.02 g / L, 0.01 g / L of calcium D-pantothenate, 40 μg / L of biotin, 0.005 g / L of thiamine hydrochloride, 0.005 g / L of nicotinic acid, adjusted to pH 7.2 with sodium hydroxide, sterilized at 122°C for 20 minutes using an autoclave, and then separately added with 0.1 g / L of L-cysteine, 1 g / L of sodium thiosulfate, and 0.1 g / L of kanamycin] in a 2 L Erlenmeyer flask, and cultured at 30°C with a stirring speed of 220 rpm for 24 hours.

[0206] 300 ml of the above culture solution was added to 1700 ml of the secondary seed medium [glucose 1000 g / L, fructose 4 g / L, yeast extract (manufactured by Asahi Breweries, Ltd.) 10 g / L, KH 2 PO 4 1.25 g / L, K 2 HPO 4 1 g / L, sodium glutamate monohydrate 2.1 g / L, L-cystine 0.02 g / L, MgSO 4 ·7H 2 O 1.25 g / L, CaCl 2 ·2H 2 O 0.1 g / L, CuSO 4 ·5H 2 O 0.002 g / L, ZnSO 4 ·7H 2 O 0.01 g / L, FeSO 4 ·7H 2 O 0.02 g / L, MnSO 4 ·5H 2 O 0.02 g / L, calcium D-pantothenate 0.015 g / L, biotin 40 μg / L, nicotinic acid 0.005 g / L, Adekanol LG-109 (manufactured by Adeka Corporation) 1 ml / L, adjusted to pH 7.2 with sodium hydroxide, and after separately adding urea to a concentration of 3.2 g / L, sterilized at 122°C for 20 minutes using an autoclave, and then separately added with thiamine hydrochloride 0.1 g / L, L-cysteine 0.3 g / L, sodium thiosulfate 2.5 g / L, and kanamycin 0.2 g / L] in a 6 L culture tank, and cultured at 30°C under the culture conditions of a stirring speed of 650 rpm and an aeration rate of 2 L / min while adjusting the pH to 6.8 with 18% aqueous ammonia for 24 hours.

[0207] 300 ml of the above culture solution was added to 1700 ml of the main tank medium [KH 2 PO 4 10 g / L, K 2 HPO 4 10 g / L, sodium glutamate monohydrate 1 g / L, L-cysteine 0.02 g / L, CaCl 2 ·2H 2 O 0.1 g / L, CuSO 4 ·5H 2 O 0.005 g / L, ZnSO4 ·7H 2 O 0.01 g / L, FeSO 4 ·7H 2 O 0.02 g / L, biotin 150 μg / L, nicotinic acid 0.005 g / L, urea 2 g / L, Adekanol LG - 109 (manufactured by Adeka) 1 ml / L. After sterilizing at 122 °C for 20 minutes using an autoclave, glucose 125 g / L, fructose 25 g / L, MgSO 4 ·7H 2 O 10 g / L, MnSO 4 ·5H 2 O 0.02 g / L, calcium D - pantothenate 0.015 g / L, thiamine hydrochloride 0.005 g / L, L - cysteine 0.15 g / L, sodium thiosulfate 2.5 g / L, kanamycin 0.2 g / L were separately added to obtain a medium. The cells were inoculated into this medium, and under the culture conditions of 30 °C and an aeration rate of 2 L / min, the stirring speed was adjusted between 650 rpm and 900 rpm so that the dissolved oxygen content did not fall below 1 ppm, and the culture was carried out for 25 hours while adjusting the pH to 6.8 with 18% aqueous ammonia.

[0208] During this period, IPTG was added at a final concentration of 1 mM 6 hours after the start of the culture, and biotin 100 μg / L, nicotinic acid 0.015 g / L, calcium D - pantothenate 0.015 g / L, and thiamine hydrochloride 0.005 g / L were additionally added 10 hours after the start of the culture. After the culture was completed, the culture solution was separated into cells and the culture supernatant using a centrifuge. The culture solution was centrifuged to obtain the pellet as wet cells and frozen at - 80 °C.

[0209] [Example 8] Cultivation of Origami - B(DE3) / MBP - C5_pGro7, Origami - B(DE3) / H - MBP - 2OST_pGro(Sm), Origami - B(DE3) / MBP - 6OST3_pGro7, RIL / HIS - 3OST1_pGro(Sm) The Origami-B(DE3) / MBP-C5_pGro7 strain obtained in Example 5 was inoculated into a large test tube containing 5 mL of TB medium containing 50 μg / mL of ampicillin, 20 μg / mL of chloramphenicol, 15 μg / mL of tetracycline, and 15 μg / mL of kanamycin, and cultured at 30 °C for 16 hours. The culture solution was inoculated at 1.2% into a baffled Erlenmeyer flask containing 500 mL of TB medium containing 50 μg / mL of ampicillin, 20 μg / mL of chloramphenicol, 15 μg / mL of tetracycline, and 15 μg / mL of kanamycin, and cultured with shaking at 37 °C for 6 hours. Then, IPTG with a final concentration of 1 mM and arabinose with a final concentration of 4 mM were added, and the culture was continued at 28 °C for 20 hours. Thereafter, the culture solution was centrifuged to obtain the pellet as wet cells and frozen at -80 °C.

[0210] The Origami-B(DE3) / H-MBP-2OST_pGro(Sm) strain obtained in Example 5 was inoculated into a large test tube containing 5 mL of TB medium containing 50 μg / mL of ampicillin, 50 μg / mL of streptomycin, 15 μg / mL of tetracycline, and 15 μg / mL of kanamycin, and cultured at 30 °C for 16 hours. The culture solution was inoculated at 1.2% into a baffled Erlenmeyer flask containing 500 mL of TB medium containing 50 μg / mL of ampicillin, 50 μg / mL of streptomycin, 15 μg / mL of tetracycline, and 15 μg / mL of kanamycin, and cultured with shaking at 30 °C for 12 hours. Then, IPTG with a final concentration of 1 mM and arabinose with a final concentration of 4 mM were added, and the culture was continued at 28 °C for 20 hours. Thereafter, the culture solution was centrifuged to obtain the pellet as wet cells and frozen at -80 °C.

[0211] The Origami-B(DE3) / MBP-6OST3_pGro7 strain obtained in Example 5 was inoculated into a large test tube containing 5 mL of TB medium containing 50 μg / mL of ampicillin, 20 μg / mL of chloramphenicol, 15 μg / mL of tetracycline, and 15 μg / mL of kanamycin, and cultured at 30 °C for 16 hours. The culture solution was inoculated at 1.2% into a baffled Erlenmeyer flask containing 500 mL of TB medium containing 50 μg / mL of ampicillin, 20 μg / mL of chloramphenicol, 15 μg / mL of tetracycline, and 15 μg / mL of kanamycin, and cultured with shaking at 37 °C for 4 hours. Then, IPTG with a final concentration of 1 mM and arabinose with a final concentration of 4 mM were added, and the culture was continued at 28 °C for 20 hours. Thereafter, the culture solution was centrifuged to obtain the pellet as wet cells, which were frozen at -80 °C.

[0212] The RIL / HIS-3OST1_pGro(Sm) strain obtained in Example 5 was inoculated into a large test tube containing 5 mL of TB medium containing 50 μg / mL of streptomycin and 15 μg / mL of kanamycin, and cultured at 30 °C for 16 hours. The culture solution was inoculated at 1.2% into a baffled Erlenmeyer flask containing 500 mL of TB medium containing 50 μg / mL of streptomycin and 15 μg / mL of kanamycin, and cultured with shaking at 37 °C for 4 hours. Then, IPTG with a final concentration of 1 mM and arabinose with a final concentration of 4 mM were added, and the culture was continued at 28 °C for 20 hours. Thereafter, the culture solution was centrifuged to obtain the pellet as wet cells, which were frozen at -80 °C.

[0213] [Example 9] 2-O Sulfation Reaction Test of N-Sulfoheparosan Using ATCC 6872(DE3) / pSC-3-13 and Origami-B(DE3) / MBP-C5_pGro7, Origami-B(DE3) / H-MBP-2OST_pGro(Sm) The frozen cells of ATCC 6872(DE3) / pSC-3-13, Origami-B(DE3) / MBP-C5_pGro7, and Origami-B(DE3) / H-MBP-2OST_pGro(Sm) obtained in Examples 7 and 8 were each suspended in distilled water so that the weight of the frozen cells was 333 g / L to prepare cell suspensions. 30 ml of the thus-obtained ATCC 6872(DE3) / pSC-3-13 cell suspension, 6 ml of the Origami-B(DE3) / MBP-C5_pGro7 cell suspension, and 6 ml of the Origami-B(DE3) / H-MBP-2OST_pGro(Sm) cell suspension were added to 18 ml of a reaction solution [an aqueous solution containing 60 g / L of glucose, 8.75 g / L of KH 2 PO 4 8.75 g / L, K 2 HPO 4 15 g / L, MgSO 4 ·7H 2 O 20 g / L, calcium D-pantothenate 0.3 g / L, nicotinic acid 0.2 g / L, adenine 4.05 g / L, benzalkonium chloride 1.25 g / L, Adekanol LG-109 (manufactured by Adeka) 1 ml / L, N-sulfoheparosan (prepared in Example 6) 1 g / L] in a 250-ml culture tank, and reacted for 22 hours under culture conditions of 37°C, a stirring speed of 500 rpm, and an aeration rate of 0.75 mL / min while adjusting the pH to 6.5 with a 2.8% ammonia aqueous solution. During this period, 60 g / L of glucose, 5.0 g / L of MgSO 4 ·7H 2 O, and 2.7 g / L of adenine were additionally added 6 hours after the start of the reaction.

[0214] Samples were appropriately taken during the reaction to obtain reaction solutions. The obtained reaction solutions were appropriately diluted and then centrifuged, and PAPS was detected and quantified by measuring the absorbance at 254 nm with a UV detector using HPLC manufactured by Shimadzu Corporation. The results are shown in Figure 4. Also, according to Patent Document [WO2018 / 048973 A1], unsaturated disaccharide production by enzymatic digestion and analysis by HPLC were performed.

[0215] That is, the obtained reaction solution was centrifuged, and the resulting supernatant was heated and held at 80°C for 10 minutes to denature the protein. After protein denaturation, the solution was centrifuged, and the resulting supernatant was desalted by ultrafiltration using a molecular weight 3k filter device (manufactured by Merck). The desalted solution was subjected to a heparinase reaction solution [composition consisting of 50 mM ammonium acetate and 2 mM calcium chloride] containing 0.5 U / ml of heparinase I, II, and III (manufactured by Sigma and others) respectively, and enzymatically digested at 35°C for 2 hours. The solution after enzymatic digestion was held at 95°C for 15 minutes to inactivate heparinase.

[0216] The solution after inactivation of heparinase was subjected to gradient elution mode analysis using mobile phase A [aqueous solution containing 1.8 mM sodium dihydrogen phosphate and adjusted to pH 3.0 with phosphoric acid] and mobile phase B [aqueous solution containing 1.8 mM sodium dihydrogen phosphate and 1 M sodium perchlorate and adjusted to pH 3.0 with phosphoric acid] with Shimadzu HPLC and a strong anion exchange column (spherisorb-SAX chromatography column, 4.0×250 mm, 5 μm, manufactured by Waters) to perform unsaturated disaccharide analysis.

[0217] The unsaturated disaccharide was detected by measuring the absorbance at 232 nm with a UV detector. The retention times of ΔUA-GlcNS and ΔUA,2S-GlcNS were confirmed by comparison with an unsaturated disaccharide standard (manufactured by Iduron). 2-O-sulfation was confirmed by determining the area ratio of ΔUA,2S-GlcNS to the total area of the detected ΔUA-GlcNS and ΔUA,2S-GlcNS. The results are shown in Figure 5.

[0218] [Example 10] 6-O-sulfation reaction test of 2-O-sulfated N-sulfoheparosan using ATCC 6872(DE3) / pSC-3-13 and Origami-B(DE3) / MBP-6OST3_pGro7 The frozen cells of ATCC 6872(DE3) / pSC-3-13 and Origami-B(DE3) / MBP-6OST3_pGro7 obtained in Examples 7 and 8 were each suspended in distilled water so that the weight of the frozen cells was 333 g / L to prepare cell suspensions. 30 ml of the thus-obtained ATCC 6872(DE3) / pSC-3-13 cell suspension, 6 ml of the Origami-B(DE3) / MBP-6OST3_pGro7 cell suspension, and 6 ml of distilled water were added to 18 ml of a reaction solution [glucose 60 g / L, KH 2 PO 4 8.75 g / L, K 2 HPO 4 15 g / L, MgSO 4 ·7H 2 O 20 g / L, calcium D-pantothenate 0.3 g / L, nicotinic acid 0.2 g / L, adenine 4.05 g / L, benzalkonium chloride 1.25 g / L, Adekanol LG-109 (manufactured by Adeka) 1 ml / L, 2-O-sulfated N-sulfoheparosan (prepared in Example 6) 0.5 g / L] in a 250 ml culture tank, and reacted for 22 hours under culture conditions of 32°C, a stirring speed of 500 rpm, and an aeration rate of 0.75 mL / min while adjusting the pH to 7.4 with an aqueous solution of 2 N potassium hydroxide.

[0219] During this period, 60 g / L of glucose, 5.0 g / L of MgSO 4 ·7H 2 O, and 2.7 g / L of adenine were additionally added 6 hours after the start of the reaction. Sampling was appropriately performed during the reaction to obtain a reaction solution. Using the same method as in Example 9, PAPS was detected and quantified. The results are shown in Figure 6.

[0220] Also, using the same method as in Example 9, the sulfation composition of the sugar chain after the reaction was analyzed. The retention times of ΔUA,2S-GlcNS and ΔUA,2S-GlcN,6S were confirmed by comparison with an unsaturated disaccharide standard (manufactured by Iduron). 6-O-sulfation was confirmed by determining the area ratio of ΔUA,2S-GlcN,6S to the total area of the detected ΔUA,2S-GlcNS and ΔUA,2S-GlcN,6S. The results are shown in Figure 7.

[0221] [Example 11] 6-O-position and 3-O-position Sulfation Reaction Test of 2-O-Sulfated N-Sulfoheparosan Using ATCC 6872(DE3) / pSC-3-13 and Origami-B(DE3) / MBP-6OST3_pGro7, RIL / HIS-3OST1_pGro(Sm) The reaction was carried out for 22 hours under the conditions shown in Example 10. During this period, 6 ml of a cell suspension prepared by suspending the frozen cells of IL / HIS-3OST1_pGro(Sm) obtained in Example 8 in distilled water so that the weight of the frozen cells was 333 g / L was added 3 hours after the start of the reaction. Sampling was carried out appropriately during the reaction to obtain the reaction solution. Detection and quantification of PAPS were carried out using the same method as in Example 9. The results are shown in Fig. 8.

[0222] After completion of the reaction, the reaction solution was centrifuged, and the obtained supernatant was appropriately diluted and then the anti-IIa activity was measured using BIOPHEN™ ANTI-IIa Measurement Kit (manufactured by Hyphen Biomed) to confirm 3-O-sulfation. Also, the anti-IIa activity of the solution after the reaction of Example 10 was measured as a negative control without addition of RIL / HIS-3OST1_pGro(Sm). BIOPHEN™ UFH Calibrator (manufactured by Hyphen Biomed) was used to prepare the calibration curve. The results are shown in Table 1.

[0223]

Table 1

[0224] [Example 12] The frozen cells of ATCC 6872(DE3) / pSC3-13 obtained in Example 7 were suspended in distilled water so that the weight of the frozen cells was 333 g / L to prepare a cell suspension. 30 ml of the thus obtained cell suspension was added to 30 ml of a reaction solution [glucose 60 g / L, KH 2 PO 4 8.75 g / L, K 2 HPO 4 15 g / L, MgSO 4 ·7H2 It was added to an aqueous solution containing 20 g / L of O, 0.3 g / L of calcium D-pantothenate, 0.2 g / L of nicotinic acid, 4.05 g / L of adenine, 0.625 g / L of benzalkonium chloride, and 1 mL / L of Adekanol LG-109 (manufactured by Adeka Corporation), and reacted for 22 hours while adjusting the pH to 7.4 with 2N potassium hydroxide aqueous solution under the culture conditions of 32 °C, a stirring speed of 500 rpm, and an aeration rate of 0.75 mL / min.

[0225] During this period, 60 g / L of glucose, KH 2 PO 4 2.19 g / L, K 2 HPO 4 3.75 g / L, MgSO 4 ·7H 2 O 5.0 g / L, and 2.7 g / L of adenine were additionally added so as to obtain these concentrations. Sampling was appropriately performed during the reaction to obtain a reaction solution.

[0226] The obtained reaction solution was appropriately diluted and then centrifuged, and PAPS was detected and quantified by measuring the absorbance at 254 nm with a UV detector using an HPLC manufactured by Shimadzu Corporation. The results are shown in Figure 9.

[0227] As described above, by adding a bacterium belonging to the genus Corynebacterium capable of producing and regenerating PAPS from raw materials such as glucose, adenine, and magnesium sulfate and a microorganism belonging to prokaryotes expressing a sulfating enzyme and causing a reaction, sulfated polysaccharides could be efficiently produced from polysaccharides.

[0228] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. All references cited herein are incorporated in their entirety. This application is based on an international patent application (PCT / JP2020 / 015388) filed on April 3, 2020, which is incorporated herein by reference in its entirety.

Claims

1. In the presence of ATP or an ATP source, a sulfate ion source, and N-sulfoheparasan, A transformant of a bacterium belonging to the genus Corynebacterium, the transformant (a) comprising at least a gene encoding ATP sulfurylase and a gene encoding APS kinase introduced into the transformant in an expressible manner, or a processed product thereof; A transformant of a prokaryotic microorganism, the transformant comprising at least a gene encoding a C5-epimerase introduced into the transformant in an expressible manner, or a processed product or extract thereof; A transformant of a prokaryotic microorganism, comprising at least a gene encoding 2-O-sulfotransferase introduced into the transformant in an expressible manner, or a processed product or extract thereof (c); A transformant of a prokaryotic microorganism, comprising at least a gene encoding 6-O-sulfotransferase introduced into the transformant in an expressible manner, (d) or a processed product or extract thereof; and A method for producing a sulfated polysaccharide, comprising adding to a reaction solution at least one selected from a transformant (e) of a prokaryotic microorganism, the transformant comprising at least a gene encoding 3-O-sulfotransferase introduced into the transformant in an expressible manner, or a processed product or extract thereof, to produce a sulfated polysaccharide.

2. In the presence of ATP or an ATP source, a sulfate ion source, and N-sulfoheparasan, The method for producing a sulfated polysaccharide according to claim 1, wherein the transformant (a) or a processed product thereof and the transformants (b) to (e) or a processed product or extract thereof are contained in a reaction solution to produce the sulfated polysaccharide.

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