KR20230004466A - Process for producing sulfated polysaccharide and process for producing PAPS - Google Patents

Abstract

An object of the present invention is to produce sulfated polysaccharides by reacting a PAPS production/regeneration system using the metabolic activity of microorganisms or their processed products when mixing inexpensive raw materials such as magnesium sulfate with microorganisms expressing sulfate enzymes or their processed products or extracts. It is to provide an easy manufacturing method. Another object of the present invention is to provide a practical method for producing PAPS from inexpensive raw materials. The present invention relates to a method for producing sulfated polysaccharide and a method for producing PAPS, said methods comprising at least a gene encoding an ATP sulfurylase and a gene encoding an APS kinase introduced in an expressible manner. As a bacterial transformant (a) of the genus Bacterium ( Corynebacterium ), the cell plasma membrane of the transformant (a) is material-permeable to prepare a transformant (a) or a treated product of the transformant (a) step; and performing a reaction for producing PAPS using a reaction solution containing ATP or an ATP source, a sulfate ion source, and the transformant (a) or a processed product thereof.

Description

Process for producing sulfated polysaccharide and process for producing PAPS

The present invention uses bacteria of the genus Corynebacterium expressing ATP sulfurylase and APS kinase (adenylyl sulfate kinase) to produce 3'-phosphoadenosine 5' from inexpensive raw materials such as glucose and adenine. -A method for producing phosphosulfate (hereinafter referred to as PAPS) and a method for producing sulfated polysaccharides using bacteria belonging to the genus Corynebacterium and microorganisms belonging to prokaryotes expressing various sulfate enzymes will be.

PAPS is a coenzyme that exists widely from microorganisms to higher organisms and functions as a donor of sulfate groups in vivo. In higher organisms, PAPS 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 as a hair growth agent (PTL 1) and an external skin preparation having a moisturizing effect (PTL 2).

A method for producing PAPS, using ATP as a raw material, using ATP sulfurylase derived from purified yeast, APS kinase derived from blue mold, and pyrophosphatase derived from Escherichia coli ( NPL 1); a method using thermostable ATP sulfurylase derived from thermophilic bacteria, APS kinase and pyrophosphatase derived from yeast using ATP as a raw material as in the above method (PTL 3); Using adenosine 5'-monophosphate (hereinafter referred to as AMP) as a raw material, ATP sulfurylase derived from thermostable bacteria, APS kinase, and polyphosphate kinase derived from Pseudomonas aeruginosa method (PTL 4); etc. are known.

On the other hand, when using inexpensive raw materials such as glucose and adenine, the bacterial cell membrane is treated with xylene or a surfactant to impart permeability to it. Using a mutant strain of Corynebacterium ammoniagenes , industrially producing ATP Production methods are known (NPL 2).

PAPS is a very unstable compound. Even in the method using crude enzymes, crude enzymes of ATP sulfurylase and APS kinase derived from a thermostable bacterium recombinantly expressed by Escherichia coli as a host are prepared, followed by heat treatment before reaction, and PAPS is produced by inhibiting the activity of a contaminating enzyme derived from Escherichia coli via (PTL 4)

PTL8 discloses that PAPS is degraded in a coenzyme solution of Escherichia coli. Escherichia coli has some enzymes involved in the degradation of PAPS, and the degradation of PAPS can be inhibited by deleting the genes for these enzymes.

Heparin, a sulfated polysaccharide, is the principal anticoagulant and is used for thromboembolism and disseminated intravascular coagulation syndrome and for anticoagulation during artificial dialysis and in extracorporeal circulation. Industrially, most heparin is extracted and purified from porcine intestinal mucosa. Since the fatal accident caused by contamination of porcine-derived heparin with impurities in 2008, research and development on non-animal-derived production control/quality control heparin has been conducted.

As a specific example, after chemically N-deacetylation and N-sulfation of fermentatively produced and purified N-acetylheparoic acid (hereinafter referred to as heparoic acid), which is a capsular polysaccharide of some Gram-negative microorganisms, There are methods by enzymatic epimerization and sulphation to yield heparin with the same structure and anticoagulant activity as that derived from porcine (PTLs 5-7 and NPLs 3 and 4).

Chondroitin sulfate is known as another useful sulfated polysaccharide and has been used as a drug for joint pain and as an eye drop to protect the superficial layer of the cornea. Chondroitin sulfate extracted and purified from various animal tissues has been used, but recently a method using a sulfation enzyme using capsular polysaccharide derived from Escherichia coli as a raw material in the same way as heparin has been reported (NPL 5 and 6).

In a method for carrying out sulfation using a purified enzyme, which uses polysaccharides derived from capsular membranes of microorganisms as a raw material, PAPS serves as a donor of sulfate groups in the reaction of sulfation enzymes and is required as a cofactor. In the above-mentioned method for producing sulfated polysaccharide using capsular polysaccharide derived from the capsular membrane of microorganisms as a raw material, 3'-phosphoadenosine 5'-phosphate (hereinafter, PAP) is generated and the sulfate group of p-nitrophenyl sulfate is enzymatically transferred to PAP, thereby regenerating PAPS and enzymatic sulfation of the polysaccharide proceeds (PTL 6 and NPL 3).

PTL 1; WO2012/057336 PTL 2: JP-A-2012-201665 PTL 3: JP-A-H5-137588 PTL 4: Japanese Patent No. 4505011 PTL 5: Japanese Patent No. 5830464 PTL 6: US Patent No. 8771995 PTL 7: WO2018/048973 PTL 8: WO2020/013346

NPL 1: Glycobiology vol. 21, no. 6, p. 771-780, 2011 NPL 2: Biosci. Biotechnol. Biochem. 65 (3), 644-650, 2001 NPL 3: Carbohydrate Polymers 122 (2015) 399-407 NPL 4: Advanced Drug Delivery Reviews 97 (2016) 237-249 NPL 5: Metabolic Engineering 27 (2015) 92-100 NPL 6: Biotechnology and Bioengineering 115 (2018) 1561-1570

Conventional PAPS production methods described in NPL 1 and PTL 1 and 2 each use relatively expensive nucleotides such as ATP and AMP as raw materials, culture bacteria, separate bacterial cells, destroy bacterial cells by ultrasonic treatment, etc. Enzymes or coenzymes produced by centrifugation are used. Therefore, it is not easy to carry out these methods on an industrial scale.

On the other hand, as described above, although methods using purified enzymes or coenzymes using ATP or AMP as raw materials for the production of PAPS are known, examples of producing PAPS using microorganisms themselves are not known. In addition, as described above, PAPS is a very unstable compound, and in the prior art, when a coenzyme is used, complicated steps such as inhibiting the activity contaminating the enzyme by heat treatment are performed.

Considering the above, it is difficult to predict that PAPS can be produced by a method similar to the method of industrially producing ATP by simply expressing ATP sulfurylase and APS kinase in a bacterium of the genus Corynebacterium as a host. This is because, as in the case of Escherichia coli, Corynebacterium rates have some enzymes involved in the degradation of PAPS.

In addition, as described above, in the conventional method of enzymatically sulfating polysaccharides, a plurality of sulfuric acids purified from cell lysates produced by culturing microorganisms expressing sulfating enzymes, collecting and ultrasonicating, etc. use enzymes These methods were not easy to implement on an industrial scale. In addition, expensive PAPS and p-nitrophenyl sulfate were required as raw materials.

Therefore, an object of the present invention is to react a PAPS production / regeneration system using the metabolic activity of microorganisms or their processed products when mixing inexpensive raw materials such as magnesium sulfate with microorganisms expressing sulfate enzymes or their processed products or extracts to produce sulfated It is to provide a method for easily producing polysaccharides. Another object of the present invention is to provide a practical method for producing PAPS from inexpensive raw materials.

The inventors of the present invention completed the present invention based on the following findings (1) and (2).

(1) PAPS cultivates strains with improved ATP sulfurylase and APS kinase activities by recombinant DNA technology using bacteria of the genus Corynebacterium having ATP-producing ability as a host, and membrane permeability thereof with a surfactant or the like It can be produced more easily and inexpensively than methods in the related art by adding raw materials such as glucose and adenine.

(2) For sulfated polysaccharides, the strains with improved ATP sulfurylase and APS kinase activities described in (1) are used as microbial bacterial cells responsible for PAPS production/regeneration reactions, and epimerase and/or sulfation enzymes are used. By imparting membrane permeability to microorganisms belonging to expressing prokaryotes and performing a mixing reaction, it can be produced using bacterial cells and inexpensive raw materials such as magnesium sulfate without enzymatic purification or addition of PAPS.

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 bacterial transformant (a) or transformant of the genus Corynebacterium, which contains at least a gene encoding ATP sulfurylase and a gene encoding APS kinase, introduced in an expressible manner ( preparing the treated product of a); and

(1-2) A step of carrying out a reaction for producing PAPS using a reaction solution containing ATP or an ATP source, a sulfate ion source, and a transformant (a) or a processed product thereof.

2. The method for producing a sulfated polysaccharide according to 1, further comprising the following steps (2-1) and (2-2):

(2-1) Transformants (b) or processed products or extracts of transformants (b) of microorganisms belonging to prokaryotes, which contain at least a gene encoding C5-epimerase introduced in an expressible manner manufacturing; and

(2-2) Incorporating the transformant (b) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform C5-epimerization.

3. Sulfuric acid according to 1 or 2, comprising the step of performing sulfation with a transformant containing a gene encoding a sulfotransferase introduced in an expressible manner or a treated product or extract of the transformant. Methods for preparing polysaccharides.

4. The method for producing a sulfated polysaccharide according to 3, further comprising the following steps (3-1) and (3-2):

(3-1) a transformant (c) of a microorganism belonging to a prokaryote or a processed product of a transformant (c), which contains at least a gene encoding 2-O-sulfotransferase introduced in an expressible manner; preparing an extract; and

(3-2) Incorporating the transformant (c) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform 2-O-sulfation.

5. The method for producing a sulfated polysaccharide according to 3 or 4, further comprising the following steps (3'-1) to (3'-3):

(3'-1) Transformants (b) or processed products or extracts of transformants (b) of microorganisms belonging to prokaryotes, which contain at least a gene encoding C5-epimerase introduced in an expressible manner Preparing;

(3'-2) Transformants (c) or treated products of transformants (c) of microorganisms belonging to prokaryotes, which contain at least a gene encoding 2-O-sulfotransferase introduced in an expressible manner or preparing an extract; and

(3'-3) Transformant (b) or its processed product or extract, and transformant (c) or its processed product or extract were incorporated into the reaction solution in the presence of N-sulfohepharoic acid to form C5-epi performing merization and 2-O-sulfation.

6. The method for producing a sulfated polysaccharide according to any one of 3 to 5, further comprising the following steps (4-1) and (4-2):

(4-1) Transformants (d) or treated products of transformants (d) of microorganisms belonging to prokaryotes, which contain at least a gene encoding 6-O-sulfotransferase introduced in an expressible manner, or preparing an extract; and

(4-2) A step of incorporating the transformant (d) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform 6-O-sulfation.

7. The method for producing a sulfated polysaccharide according to any one of 3 to 6, further comprising the following steps (5-1) and (5-2):

(5-1) Transformants (e) or treated products of transformants (e) of microorganisms belonging to prokaryotes, which contain at least a gene encoding 3-O-sulfotransferase introduced in an expressible manner, or preparing an extract; and

(5-2) Incorporating the transformant (e) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform 3-O-sulfation.

8. As a method for producing sulfated polysaccharides,

Transformant (a) or treatment of transformant (a) of a bacterium of the genus Corynebacterium comprising at least a gene encoding ATP sulfurylase and a gene encoding APS kinase, introduced in an expressible manner And, incorporating at least one selected from the following into a reaction solution in the presence of ATP or an ATP source, a sulfate ion source, and N-sulfoheparoic acid to produce a sulfated polysaccharide, wherein the production of a sulfated polysaccharide Way:

Transformants (b) or treated products or extracts of transformants (b) of microorganisms belonging to prokaryotes, which contain at least a gene encoding C5-epimerase introduced in an expressible manner;

Transformants (c) or processed products or extracts of transformants (c) of microorganisms belonging to prokaryotes, which contain at least a gene encoding 2-O-sulfotransferase introduced in an expressible manner;

A transformant (d) or a treated product or extract of a transformant (d) of a microorganism belonging to a prokaryote, which contains at least a gene encoding 6-O-sulfotransferase introduced in an expressible manner, and

A transformant (e) or a treated product or extract of a transformant (e) of a microorganism belonging to a prokaryote, which contains at least a gene encoding 3-O-sulfotransferase introduced in an expressible manner.

9. The transformant (a) or the treated product thereof, and the transformants (b) to (e) or the treated product or extract thereof according to 9. 7 are ATP or an ATP source, a sulfate ion source, and N-sulfoheparoic acid A method for producing a sulfated polysaccharide, further comprising the step of generating a sulfated polysaccharide by incorporating into the reaction solution in the presence of

10. The method for producing a sulfated polysaccharide according to any one of 1 to 9, which is for producing heparin.

11. A method for preparing PAPS, comprising the following steps (i) and (ii):

(i) Preparation of a bacterial transformant of the genus Corynebacterium or a treatment of the transformant, comprising at least a gene encoding ATP sulfurylase and a gene encoding APS kinase, introduced in an expressible manner doing; and

(ii) carrying out a reaction for producing PAPS using a reaction solution containing ATP or an ATP source, a sulfate ion source, and the transformant prepared in step (i) or a processed product thereof.

According to the method for producing sulfated polysaccharide of the present invention, sulfated polysaccharide is easily and inexpensively produced by using a PAPS production/regeneration system using the metabolic activity of microorganisms and microorganisms expressing sulfate enzymes or processed products or extracts thereof. It can be. In addition, according to the method for producing PAPS of the present invention, PAPS can be easily and inexpensively produced by using the metabolic activity of microorganisms or their processed products. Moreover, by using the genus Corynebacterium in the method of the present invention, it is not necessary to modify the gene in a complex way to avoid degradation of PAPS.

[Figure 1] Figure 1 shows a schematic diagram of the heparoic acid synthesis gene cluster on the chromosome of Escherichia coli K5 strain.
[Figure 2] Figure 2 shows a schematic diagram of enzymes involved in heparoic acid production.
[Figure 3] Figure 3 shows a schematic diagram of the heparoic acid biosynthetic pathway.
[Fig. 4] Fig. 4 is a graph showing the time-dependent change in PAPS concentration contained in the supernatant of the reaction solution in the 2-O-sulfation reaction test of N-sulfoheparoic acid.
[Fig. 5] Fig. 5 is a graph showing the time-dependent change in the area ratio of delta-UA and 2S-GlcNS in the HPLC analysis of unsaturated disaccharides in the 2-O-sulfation reaction test of N-sulfoheparoic acid.
[Figure 6] Figure 6 is a graph showing the time-dependent change in PAPS concentration contained in the supernatant of the reaction solution in the 6-O-sulfation reaction test of 2-O-sulfated N-sulfoheparoic acid.
[Figure 7] Figure 7 shows the time-of area ratios of delta-UA, 2S-GlcN, and 6S in HPLC analysis of unsaturated disaccharides in the 6-O-sulfation reaction test of 2-O-sulfated N-sulfoheparoic acid. It is a graph showing the dependent change.
[Figure 8] Figure 8 is a time-dependent analysis of the PAPS concentration contained in the supernatant of the reaction solution in the 6-O-position and 3-O-position sulfuration reaction test of 2-O-sulfated N-sulfoheparoic acid. It is a graph showing change.
[Figure 9] Figure 9 is a graph showing the time-dependent change of the PAPS concentration contained in the supernatant of the reaction solution in the PAPS production test.

<Transformant>

The method for producing sulfated polysaccharide of the present invention is characterized in that 1) the step of supplying/reproducing PAPS is performed by a reaction using bacterial cells. In the method of the present invention, preferably, at least one of the steps of 2) C5-epimerization, 3) 2-O-sulfation, 4) 6-O-sulfation and 5) 3-O-sulfation is carried out in bacterial cells. It is carried out by a reaction using Hereinafter, transformants (a) to (e) used in each reaction will be described.

<<Transformant (a): supply/regeneration of PAPS>>

In the production method of the sulfated polysaccharide of the present invention, the supply/regeneration of PAPS is introduced in an expressible manner into Corynebacterium genus, which contains at least a gene encoding ATP sulfurylase and a gene encoding APS kinase. It is performed using a transformant (a) or a treated product of the transformant (a) of a microorganism to which it belongs.

Examples of bacterial species of the genus Corynebacterium are Corynebacterium ammoniagenes, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum , Corynebacterium alkanoli Ticum ( Corynebacterium alkanolyticum ), Corynebacterium callunae ( Corynebacterium callunae ), Corynebacterium crenatum ( Corynebacterium crenatum ) Corynebacterium glutamicum ( Corynebacterium glutamicum ), Corynebacterium lilium ( Corynebacterium lilium ) , Corynebacterium melassecola , Corynebacterium thermoaminogenes ( Corynebacterium efficiens ) and Corynebacterium herculis ( Corynebacterium herculis ) include

Examples of strains of the genus Corynebacterium include Corynebacterium ammoniagenes ( Corynebacterium stationis ) ATCC 6871 and ATCC 6872, Corynebacterium acetoacidophilum ATCC 13870, Corynebacterium Acetoglutamicum ATCC 15806, Corynebacterium alkanoliticum ATCC 21511, Corynebacterium calunaae ATCC 15991, Corynebacterium crenatum AS1.542, Corynebacterium glutamicum ATCC 13020, ATCC 13032, ATCC 13060, ATCC 13869 and FERM BP-734, Corynebacterium Lilium ATCC 15990, Corynebacterium melasecol ATCC 17965, Corynebacterium efficiency (Corynebacterium thermoaminogenes) AJ12340 (FERM BP -1539), Corynebacterium herculis ATCC 13868, Brevibacterium divaricatum (Corynebacterium glutamicum) ATCC 14020, Brevibacterium flavum (Corynebacterium flavum) glutamicum) ATCC 13826, ATCC 14067, and AJ12418 (FERM BP-2205) and Brevibacterium lactofermentum (Corynebacterium glutamicum) ATCC 13869.

Bacteria belonging to the genus Corynebacterium also include bacteria previously classified in the genus Brevibacterium but now incorporated into the genus Corynebacterium (Int. J. Syst. Bacteriol., 41, 255 (1991)). In addition, Corynebacterium stethosis also includes bacteria conventionally classified as Corynebacterium ammoniagenes, but now reclassified as Corynebacterium stethosis by 16S rRNA nucleotide sequence analysis or the like [Int. J Syst. Evol. Microbiol., 60, 874-879 (2010)].

These strains are available, for example, from the American Type Culture Collection, 12301 Park Lawn Drive, Rockville, Md. 20852, P.O. Box 1549, Manassas, Va. 20108 USA. That is, a registration number is assigned to each strain, and the strain can be obtained using the 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. Bacteria of the genus Corynebacterium may be wild-type strains, mutant strains thereof, or artificial recombinants thereof.

In the present invention, the gene encoding ATP sulfurylase and the gene encoding APS kinase are introduced into a bacterium of the genus Corynebacterium in an expressible manner.

ATP sulfurylase generates adenosine 5'-phosphosulfate (APS) from sulfate by the following reaction scheme.

[Scheme 1]

(In the above formula, ATP represents adenosine 5'-triphosphate.)

One aspect of ATP sulfurylase is MET3. The origin of ATP sulfurylase is not particularly limited, examples thereof include Saccharomyces cerevisiae , Candida albicans , Shizosaccharomyces pombe , Yarrowia lipopoly Tika ( Yarrowia lipolytica ), Neurospora crassa , Penicillium chrysogenum , Kluyveromyces lactis , Fusarium fujikuroi , Aspergillus ATP sulfurylase derived from Aspergillus oryzae or Ashbya gossypii is included, and among these, ATP sulfurylase derived from Saccharomyces cerevisiae is preferred.

An example of a gene encoding ATP sulfurylase includes the nucleotide sequence shown in SEQ ID NO: 22. An example thereof is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more and particularly preferably 95% or more and particularly preferably 80% or more, more preferably 90% or more, and particularly preferably 90% or more of the nucleotide sequence shown in SEQ ID NO: 22 that encodes a polypeptide having ATP sulfurylase activity. DNA comprising nucleotide sequences that are at least 98% identical; and DNA comprising a nucleotide sequence encoding a polypeptide having ATP sulfurylase activity and hybridizing under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 22 or a nucleotide sequence complementary to the nucleotide sequence. The term “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Examples thereof are DNAs that are identical to each other at a higher level, for example, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more and particularly preferably 99% or more of each other. conditions in which identical DNAs hybridize to each other and, at a lower level, identical DNAs to each other do not hybridize to each other; or a salt concentration and temperature where the wash corresponds to 60°C, 1 x SSC and 0.1% SDS, preferably 60°C, 0.1 x SSC and 0.1% SDS, and more preferably 68°C, 0.1 x SSC and 0.1% SDS. It includes conditions that are conditions for washing in a standard Southern hybridization, which is performed once, preferably 2 to 3 times in .

ATP sulfurylase activity in the transformant is confirmed by an increase in the ATP sulfurylase activity value in the cell extract of the transformant. ATP sulfurylase activity is described by Medina DC et al., Temperature effects on the allosteric transition of ATP sulfurylase from Penicillium chrysogenum. Arch. Biochem. Biophys. One; 393 (1): 51-60 (2001).

In the present invention, unless otherwise specified, numerical values related to identity may be numerical values calculated using a homology search program known to those skilled in the art. Examples of numerical values for nucleotide sequences are BLAST [J. Mol. Biol., 215, 403 (1990)], examples of numerical values for amino acid sequences include BLAST2 [Nucleic Acids Res., 25, 3389 (1997), Genome Res., 7 , 649 (1997), http://www.ncbi.nlm.nih.gov/Education/BLASTinfo/information3.htmL].

APS kinase generates PAPS from APS according to the reaction scheme below.

[Scheme 2]

(In the above formula, ADP represents adenosine 5'-diphosphate.)

One aspect of APS kinase includes MET14. The origin of APS kinase is not particularly limited, and examples thereof are Saccharomyces cerevisiae, Candida albicans, Shizoscharomyces pombe, Yarrowia lipolytica, Neurospora crassa, Penicillium chrysogenum , including APS kinases derived from Kluyveromyces lactis, Fusarium fujicuroi, Aspergillus duckweed or Asiatic gossipii, among which APS kinases derived from Saccharomyces cerevisiae are preferred. .

An example of a gene encoding APS kinase includes the nucleotide sequence shown in SEQ ID NO:23. An example thereof further encodes a polypeptide having APS kinase activity and is preferably at least 80%, more preferably at least 90%, even more preferably at least 95% and particularly preferably at least 95% identical to the nucleotide sequence shown in SEQ ID NO: 23. DNA comprising nucleotide sequences that are at least 98% identical; and DNA comprising a nucleotide sequence encoding a polypeptide having APS kinase activity and hybridizing under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 23 or a nucleotide sequence complementary to the nucleotide sequence.

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

Introducing the gene encoding ATP sulfurylase and the gene encoding APS kinase into a bacterium of the genus Corynebacterium involves either incorporating the above-described DNA into the host's chromosome or using an appropriate plasmid vector capable of amplifying the above-described DNA in the host. cloning and introducing the vector into the host.

The plasmid vector may be any plasmid vector as long as it has a gene controlling autonomous replication function in bacteria of the genus Corynebacterium. Specific examples thereof are Brevibacterium lactofermentum 2256 [JP-A-S58-67699], [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 [Miwa, K. et al., Cryptic plasmids in glutamic acid-producing bacteria. Agric. Biol. Chem. 48: 2901-2903 (1984)] and pCRY30 derived from Corynebacterium glutamicum ATCC 3058 [Kurusu, Y. et al., Identification of plasmid partition function in coryneform bacteria. Appl. Environ. Microbiol. 57: 759-764 (1991)]; pCG4 [JP-A-S57-183799] and [Katsumata, R. et al., Protoplast transformation of glutamate-producing bacteria with plasmid DNA. J. Bacteriol., 159: 306-311 (1984)], pAG1, pAG3, pAG14 and pAG50 [JP-A-S62-166890], and pEK0, pEC5, and pEKEx1 derived from Corynebacterium glutamicum T250. [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)];

The promoter may be a promoter derived from a host or a heterologous promoter. Examples thereof include promoter PgapA of glyceraldehyde 3-phosphate dehydrogenase A gene (gapA) derived from Corynebacterium glutamicum R, promoter Pmdh of malate dehydrogenase gene (mdh), lactate dehydrogenase gene (mdh), and promoter PldhA of hydrogenase A gene (ldhA).

Examples of the terminator include the rrnB T1T2 terminator of the Escherichia coli rRNA operon, the trpA terminator of Escherichia coli, the trp terminator of Brevibacterium lactofermentum, and the like.

As one aspect of the present invention, gene expression of enzymes involved in the degradation of PAPS is not attenuated. Examples of genes of enzymes 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 gene selected from the cysQ gene and the CP01850 gene is not attenuated.

<<Transformant (b): C5-epimerization>>

In the method for preparing sulfated polysaccharide according to the present invention, the C5-epimerization of N-sulfoheparoic acid is carried out by a prokaryotic microorganism belonging to prokaryotes, which contains at least a gene encoding C5-epimerase introduced in an expressible manner. It is carried out using a transformant (b) of or a treated product or extract of transformant (b).

C5-epimerase is not particularly limited as long as it can catalyze the isomerization of glucuronic acid (GlcUA) residues to iduronic acid (IdoA) residues. C5-epimerase may be derived from animals, plants, microorganisms, and the like. For example, human C5-epimerase can be used as the C5-epimerase.

An example of a gene encoding C5-epimerase includes the nucleotide sequence shown in SEQ ID NO: 24. An example thereof is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more and particularly preferably 95% or more of the nucleotide sequence shown in SEQ ID NO: 24 encoding a polypeptide having C5-epimerase activity. DNA having a nucleotide sequence that is 98% or more identical; and DNA comprising a nucleotide sequence encoding a polypeptide having C5-epimerase activity and hybridizing under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 24 or a nucleotide sequence complementary to the nucleotide sequence.

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. C5-epimerase activity is described by Babu P. et al., A rapid, nonradioactive assay for measuring heparan sulfate C-5 epimerase activity using hydrogen/deuterium exchange-mass spectrometry. Methods. Mol. Biol. 1229: 209-219 (2015)].

Hereinafter, transformants expressing sulfotransferase will be described. In the production method of the sulfated polysaccharide of the present invention, the sulfated is carried out using a transformant containing a gene encoding a sulfotransferase introduced in an expressible manner, or a treated product or extract of the transformant. . In the present invention, the sulfotransferase is not particularly limited as long as it produces a sulfated polysaccharide by transferring a sulfate group to the polysaccharide. Examples of sulfotransferases include 2-O-sulfotransferase (2-OST), 6-O-sulfotransferase (6-OST) and 3-O-sulfotransferase (3-OST).

<<Transformant (c): 2-O-sulfation>>

In the production method of the sulfated polysaccharide of the present invention, 2-O-sulfation belongs to prokaryotes, which contain at least a gene encoding 2-O-sulfotransferase (2-OST) introduced in an expressible manner. It is carried out using the transformant (c) of the microorganism or a treated product or extract of the transformant (c).

2-OST is not particularly limited as long as it can catalyze the sulfation of the IdoA residue at the O-2 position. Additionally, 2-OSTs may be derived from animals, plants, microorganisms, and the like. For example, a 2-OST derived from a hamster can be used as the 2-OST.

An example of a gene encoding 2-OST includes the nucleotide sequence shown in SEQ ID NO: 19. An example of this is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more and particularly preferably 98% or more of the nucleotide sequence shown in SEQ ID NO: 19 encoding a polypeptide having 2-OST activity. DNA containing nucleotide sequences that are at least % identical; and DNA comprising a nucleotide sequence encoding a polypeptide having 2-OST activity and hybridizing under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 19 or a nucleotide sequence complementary to the nucleotide sequence.

2-OST activity in the transformant was confirmed by an increase in the 2-OST activity value in the cell extract of the transformant. 2-OST activity is described by 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).

<Transformant (d): 6-O-sulfated>>

In the production method of the sulfated polysaccharide of the present invention, 6-O-sulfation belongs to prokaryotes, which contain at least a gene encoding 6-O-sulfotransferase (6-OST) introduced in an expressible manner. It is carried out using a transformant (d) of a microorganism or a treated product or extract of a transformant (d).

6-OST is not particularly limited as long as it can catalyze the sulfation of an N-sulfated glucosamine (GlcNS) residue at the O-6 position. 6-OSTs can be derived from any of animals, plants, microorganisms, and the like. Examples of 6-OST include 6-OST-1 derived from hamsters and 6-OST-3 derived from mice.

An example of a gene encoding 6-OST includes the nucleotide sequence shown in SEQ ID NO: 25. An example of this is preferably 80% or more, more preferably 90% or more, still more preferably 95% or more and particularly preferably 98% or more of the nucleotide sequence shown in SEQ ID NO: 22 encoding a polypeptide having 6-OST activity. DNA containing nucleotide sequences that are at least % identical; and DNA comprising a nucleotide sequence encoding a polypeptide having 6-OST activity and hybridizing under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 25 or a nucleotide sequence complementary to the nucleotide sequence.

6-OST activity in the transformant was confirmed by an increase in the 6-OST activity value in the cell extract of the transformant. 6-OST activity is described by 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).

<<Transformant (e): 3-O-sulfation>>

In the production method of the sulfated polysaccharide of the present invention, 3-O-sulfation belongs to prokaryotes, which contain at least a gene encoding 3-O-sulfotransferase (3-OST) introduced in an expressible manner. It is carried out using a transformant (e) of a microorganism or a treated product or extract of the transformant (e).

3-OST is not particularly limited as long as it can catalyze the sulfation of the N-sulfated/6-O-sulfated glucosamine residue at the O-3 position. 3-OSTs can be derived from animals, plants, microorganisms, and the like. For example, 3-OST-1 derived from mice can be used as 3-OST.

An example of a gene encoding 3-OST includes the nucleotide sequence shown in SEQ ID NO: 26. An example of this is preferably 80% or more, more preferably 90% or more, even more preferably 95% or more and particularly preferably 98% or more of the nucleotide sequence shown in SEQ ID NO: 26 encoding a polypeptide having 3-OST activity. DNA containing nucleotide sequences that are at least % identical; and DNA comprising a nucleotide sequence encoding a polypeptide having 3-OST activity and hybridizing under stringent conditions to the nucleotide sequence shown in SEQ ID NO: 26 or a nucleotide sequence complementary to the nucleotide sequence.

3-OST activity in the transformant was confirmed by an increase in the 3-OST activity value in the cell extract of the transformant. 3-OST activity is described by 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)].

<<Microorganism belonging to prokaryotic organisms>>

Microorganisms belonging to prokaryotes to be used as hosts of transformants in the present invention are not particularly limited as long as they can express a predetermined gene of the present invention. Examples thereof include bacteria such as microorganisms belonging to the genus Escherichia, the genus Serratia , the genus Bacillus , the genus Corynebacterium, the genus Microbacterium and the genus Pseudomonas, Bacteria are preferably used.

The bacteria belonging to the genus Escherichia used in the present invention are not particularly limited, and examples thereof include bacteria classified into the genus Escherichia by a classification known to microbiology experts. Examples of bacteria belonging to the genus Escherichia are described in Backmann, BJ 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, DC.

Examples of bacteria belonging to the genus Escherichia include Escherichia coli. Examples of Escherichia coli include Escherichia coli strain K-12, such as strain W3110 (ATCC 27325) and strain MG1655 (ATCC 47076); Escherichia coli K5 strain (ATCC 23506); Escherichia coli B strains such as the BL21 (DE3) strain; Escherichia coli Nissle 1917 strain (DSM 6601); and their derivative strains.

These strains are available, for example, from the American Type Culture Collection (address: 12301 Park Lawn Drive, Rockville, MD 20852, P.O. Box 1549, Manassas, VA 20108). That is, a registration number is assigned to each strain, and the strain can be obtained using the 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, the BL21(DE3) strain is available, for example, from Life Technologies (product number C6000-03).

For the purpose of introducing a gene in an expressible manner, a vector capable of autonomously replicating in a host may be used as a vector used for transformation of microorganisms belonging to prokaryotes. The vector is preferably a multicopy vector. In addition, the vector is preferably combined with an antibiotic resistance gene or other gene as described in Karl Friehs, Plasmid Copy Number and Plasmid Stability, Adv Biochem Engin/Biotechnol 86: 47-82 (2004) to select transformants. have the same markers. Additionally, vectors may have promoters or terminators to express the inserted gene. Examples of vectors include vectors derived from bacterial plasmids, vectors derived from yeast plasmids, vectors derived from bacteriophages, cosmids, phagemids, and the like.

Specific examples of vectors capable of autonomous replication in bacteria of the genus Escherichia coli include pUC19, pUC18, pHSG299, pHSG399, pHSG398, pBR322, and pSTV29 (all from Takara Bio Inc.), pACYC184 and pMW219 (Nippon Gene), pTrc99A ( Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen) and wide host range vector RSF1010.

The promoter may be a promoter derived from a host or a heterologous promoter. The promoter may be an endogenous promoter of the gene to be introduced or a promoter of another gene.

Examples of terminators include T7 terminators, T4 terminators, fd phage terminators, tet terminators and trpA terminators.

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

<<Processed product of transformants>>

In the present invention, the treated material of a transformant refers to a substance in which the cell plasma membrane of a bacterial cell becomes permeable. In the present invention, when a cell plasma membrane is referred to as material-permeable, it means that various small (such as ions) and large (such as proteins) molecules can freely enter and exit the cell membrane by penetrating the cell membrane by diffusion. In the present invention, the treated product of the transformant is preferably a dormant bacterial cell that has lost growth ability due to treatment for imparting membrane permeability.

Examples of a transformant treatment include a surfactant treatment of bacterial cells as a transformant, a solvent treatment of bacterial cells, an enzyme treatment of bacterial cells, and live bacterial cells that maintain the same function as the culture of bacterial cells as an enzyme source. processing products containing, such as immobilized products of bacterial cells, sonicated products of bacterial cells and mechanically disrupted products of bacterial cells.

Examples of methods for making cell plasma membrane material-permeable include chemical treatment and mechanical treatment. In the production method of the present invention, the time to make the cell plasma membrane of the transformant permeable is not particularly limited as long as the effect of the present invention is exhibited. The cell plasma membrane of each transformant can be made material-permeable in advance, or it can be made when the reaction is carried out by bringing the transformants used in the reaction into contact with each other.

Examples of chemical treatment include a method using a surfactant, a method using an organic solvent, and a method using an enzyme. As surfactants, nonionic surfactants are preferred because their action on proteins and the like is weaker (compared to ionic surfactants). Examples of surfactants are digitonin, saponin, Triton X100, Triton X114, Tween 20, Tween 80, N,N-bis(3-D-gluconamidopropyl)colamide [BIGCHAP], N,N-bis( 3-D-gluconamidopropyl) deoxycholamide [deoxy-BIGCHAP], NIKKOLBL-9EX [polyoxyethylene (9) lauryl ether], octanoyl-N-methylglucamide [MEGA-8] , benzalkonium chloride, and the like.

Examples of organic solvents include benzene, toluene, xylene, other alcohols, and the like. Examples of enzymes include lysozyme, achromopeptidase, and the like.

Conditions such as concentration, temperature, and time of treatment with the above-mentioned substances vary depending on the type of cell, and appropriate conditions must be set in order to perform the desired assay. 10 to 1000 micrograms/ml and more usually 20 to 200 micrograms/ml in a time of minutes.

Examples of mechanical treatment include ultrasonic treatment and mechanical polishing treatment.

<<Extract of transformants>>

Examples of the extract of the transformant in the present invention include the coenzyme extract obtained from the transformant bacterial cells, the purified enzyme obtained from the bacterial cells treated as above (e.g., chemically or mechanically), the transformants described above. It includes a concentrate of a culture obtained by culturing a sieve or a processed product thereof, and a dried product of the culture. Examples of transformant extracts also include bacterial cells obtained by centrifuging or filtering cultures, dried bacterial cells, and lyophilized bacterial cells.

<<Transformation method and method for culturing transformants>>

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

The transformant is preferably grown by culturing using a medium generally used for culturing microorganisms before each reaction. As a medium, a natural or synthetic medium can be used, which generally contains a carbon source, a nitrogen source, inorganic salts and other nutrients.

The carbon source may be an ATP source. Examples of carbon sources include carbohydrates or sugar alcohols such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, arabinose, galactose, starch, molasses, sorbitol and glycerin; organic acids such as acetic acid, citric acid, lactic acid, fumaric acid, maleic acid and gluconic acid; and alcohols such as ethanol and propanol. As a carbon source, one type may be used alone, or two or more types thereof may be mixed. Generally, a concentration of about 0.1 to 10 (w/v%) of these carbon sources in the medium is sufficient.

Examples of the nitrogen source include inorganic or organic ammonium compounds such as ammonium chloride, ammonium sulfate, ammonium nitrate and ammonium acetate, urea, aqueous ammonia, sodium nitrate, potassium nitrate and the like. It is also possible to use corn steep liquor, meat extracts, peptones, NZ-amines, protein hydrolysates, nitrogen-containing organic compounds such as amino acids, and the like. As a nitrogen source, one type may be used alone, or two or more types thereof may be mixed and used. The concentration of the nitrogen source in the medium varies depending on the nitrogen compound used, but is generally 0.1 to 10 (w/v%).

Examples of inorganic salts include monobasic potassium phosphate, dibasic potassium phosphate, ferrous nitrate, sodium chloride, calcium carbonate, and the like, in addition to sulfate ion sources such as magnesium sulfate, manganese sulfate, zinc sulfate, and cobalt sulfate. As the inorganic salt, one type may be used alone, or two or more types thereof may be mixed and used. The concentration of the inorganic salt in the medium varies depending on the inorganic salt used, but is generally about 0.01 to 1 (w/v%).

Examples of nutritional substances include meat extract, peptone, polypeptone, yeast extract, dried yeast, corn steep liquor, skim milk powder, skim soybean hydrochloride hydrolysate, extracts of animal and plant or microbial bacterial cells and their degradation products, and the like; , its concentration is generally about 0.1 to 10 (w/v%). In addition, vitamins may be added if necessary. Examples of vitamins include biotin, thiamine (vitamin B1), pyridoxine (vitamin B6), pantothenic acid, inositol, nicotinic acid and the like. The pH of the medium is preferably about 6 to 8.

Preferred examples of culture media for microorganisms include A medium [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)], BT medium [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)]; As specific culture conditions, for example, the culture temperature is about 15° C. to 45° C., and the culture time is about 1 day to 7 days.

<Method for producing sulfated polysaccharide>

One aspect of the method for producing a sulfated polysaccharide according to the present invention is a method for producing a sulfated polysaccharide, which includes transformants (a) or treated products thereof, and transformants (b) to (e) or treated products thereof. A method comprising introducing at least one selected from water or an extract into a reaction solution in the presence of ATP or an ATP source, a sulfate ion source, and N-sulfoheparoic acid to produce a sulfated polysaccharide.

As one aspect of the method for producing a sulfated polysaccharide according to the present invention, an example thereof is a method for producing a sulfated polysaccharide, wherein a transformant ( a) or a treated product thereof, and a reaction solution containing transformants (b) to (e) or treated products or extracts thereof to produce a sulfated polysaccharide.

In addition, one aspect of the method for producing a sulfated polysaccharide according to the present invention is a method for producing a sulfated polysaccharide, wherein at least one selected from transformants (b) to (e) or a processed product or extract thereof is used as PAPS and A method comprising the step of introducing a sulfated polysaccharide into a reaction solution in the presence of N-sulfoheparoic acid.

In the production method of the present invention, each transformant may be initially added to the reaction solution, may be added sequentially, or may be added initially and sequentially. A specific example of the aspect of adding each transformant to the reaction solution at the beginning is the transformant (a) or a processed product thereof, and the transformants (b) to (e) or a processed product or extract thereof, ATP or ATP It includes an aspect in which sulfated polysaccharide is produced by initial addition to the reaction solution in the presence of a source, a source of sulfate ions, and N-sulfoheparoic acid.

In the method for producing a sulfated polysaccharide of the present invention using a transformant (a) or a processed product thereof, and a transformant (b) to (e) or a processed product or extract thereof, 1) a transformant (a) ) or supply / regeneration of PAPS using its processed material, 2) C5-epimerization using transformant (b) or its processed material or extract, 3) transformant (c) or its processed material or extract 2-O-sulfation, 4) 6-O-sulfation using a transformant (d) or a treatment or extract thereof, and 5) 3-O- using a transformant (e) or a treatment or extract thereof. Sulfation can be carried out.

Hereinafter, each step will be described individually, but as long as the desired sulfated polysaccharide can be obtained, 2) C5-epimerization, 3) 2-O-sulfation, 4) 6-O-sulfation and 5) The order of 3-O-sulfation is not particularly limited.

In addition, each reaction of 1) supply/regeneration of PAPS, 2) C5-epimerization, 3) 2-O-sulfation, 4) 6-O-sulfation, and 5) 3-O-sulfation is separately However, two or more of these reactions may be carried out simultaneously. For example, these reactions can be carried out simultaneously using a reaction solution containing both transformants (a) or treated products thereof, and transformants (b) to (e) or treated products or extracts thereof. .

An example of the sulfated polysaccharide produced by the method for producing a sulfated polysaccharide of the present invention includes heparin.

<<Supply/regeneration of PAPS>>

The method for producing a sulfated polysaccharide of the present invention is characterized by comprising the following steps (1-1) and (1-2):

(1-1) A bacterial transformant (a) or transformant of the genus Corynebacterium, which contains at least a gene encoding ATP sulfurylase and a gene encoding APS kinase, introduced in an expressible manner ( preparing the treated product of a); and

(1-2) A step of carrying out a reaction for producing PAPS using a reaction solution containing ATP or an ATP source, a sulfate ion source, and a transformant (a) or a processed product thereof.

ATP sulfurylase and APS kinase expressed by the transformant (a) or a treatment thereof react with ATP or an ATP source and a sulfate ion source in the transformant (a) or a treatment thereof, thereby producing PAPS . PAPS functions as a donor of sulfate groups in sulfated polysaccharide production. In addition, PAPS produced by the transformant (a) or its processed product is used in a method for producing sulfated polysaccharide, through which PAP is obtained. PAPS can be produced from this PAP by the transformant (a) or a processed product thereof. Therefore, PAPS can be supplied/regenerated by incorporating the transformant (a) or its treated product into the reaction solution.

<<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) of a microorganism belonging to a prokaryote, or a processed product or extract thereof, containing at least a gene encoding C5-epimerase introduced in an expressible manner; and

(2-2) Incorporating the transformant (b) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform C5-epimerization.

In step (2-2), C5-epimerization of N-sulfoheparoic acid is carried out by the transformant (b) expressing C5-epimerase or a treated product or extract thereof.

<<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) a transformant (c) of a microorganism belonging to a prokaryote or a processed product of a transformant (c), which contains at least a gene encoding 2-O-sulfotransferase introduced in an expressible manner; preparing an extract; and

(3-2) Incorporating the transformant (c) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform 2-O-sulfation.

In step (3-2), 2-O-sulfation of N-sulfoheparoic acid is carried out using PAPS produced by the transformant (a) or its treatment as a sulfate group donor to form 2-O-sulfo Transformants expressing transferase (c) or treated products or extracts thereof.

When the sulfated polysaccharide prepared in step (2-2) is present in the reaction solution, 2-O-sulfation of the sulfated polysaccharide prepared in this step is performed. For example, when N-sulfoheparoic acid with C5-epimerization is present, 2-O-sulfation of N-sulfoheparoic acid with C5-epimerization is performed.

The aforementioned C5-epimerization and 2-O-sulfation can be carried out simultaneously. That is, one aspect of the method for producing a sulfated polysaccharide of the present invention preferably includes the following steps (3'-1) to (3'-3):

(3'-1) Transformants (b) or processed products or extracts of transformants (b) of microorganisms belonging to prokaryotes, which contain at least a gene encoding C5-epimerase introduced in an expressible manner Preparing;

(3'-2) Transformants (c) or treated products of transformants (c) of microorganisms belonging to prokaryotes, which contain at least a gene encoding 2-O-sulfotransferase introduced in an expressible manner or preparing an extract; and

(3'-3) Transformant (b) or its processed product or extract, and transformant (c) or its processed product or extract were incorporated into the reaction solution in the presence of N-sulfohepharoic acid to form C5-epi performing merization and 2-O-sulfation.

<<6-O-Sulfated>>

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 a prokaryote, or a processed product or extract thereof, containing at least a gene encoding 6-O-sulfotransferase introduced in an expressible manner; and

(4-2) A step of incorporating the transformant (d) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform 6-O-sulfation.

In step (4-2), 6-O-sulfation of N-sulfoheparoic acid is carried out using PAPS produced by the transformant (a) or its treatment as a sulfate group donor to form 6-O-sulfo It is carried out by the transformant (d) expressing the transferase or a treatment or extract thereof or a treatment or extract thereof.

When the sulfated polysaccharide prepared in step (2-2), (3-2) or (3′-3) is present in the reaction solution, 6-O-sulfation of the sulfated polysaccharide prepared in this step is carried out

For example, when N-sulfoheparoic acid with C5-epimerization is present, 6-O-sulfation of N-sulfoheparoic acid with C5-epimerization is performed. When N-sulfoheparosone with 2-O-sulfation is present, 6-O-sulfation of N-sulfoheparosic acid with 2-O-sulfation is performed.

When N-sulfoheparoic acid with C5-epimerization and 2-O-sulfation is present, 6-O-sulfation of N-sulfoheparoic acid with C5-epimerization and 2-O-sulfation takes place do.

<<3-O-Sulfated>>

The method for producing a sulfated polysaccharide of the present invention preferably includes the following steps (5-1) and (5-2):

(5-1) Transformants (e) or treated products of transformants (e) of microorganisms belonging to prokaryotes, which contain at least a gene encoding 3-O-sulfotransferase introduced in an expressible manner, or preparing an extract; and

(5-2) Incorporating the transformant (e) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform 3-O-sulfation.

In step (5-2), 3-O-sulfation of N-sulfoheparoic acid is performed as a sulfate group donor using PAPS produced by transformant (a) or its treatment product to obtain 3-O-sulfo Transformants expressing transferase (e) or treated products or extracts thereof.

When the sulfated polysaccharide prepared in step (2-2), (3-2), (3′-3) or (4-2) is present in the reaction solution, the amount of the sulfated polysaccharide prepared in this step 3-O-sulfation is performed.

For example, when N-sulfoheparoic acid with C5-epimerization is present, 3-O-sulfation of N-sulfoheparoic acid with C5-epimerization is performed. When N-sulfoheparoic acid with 2-O-sulfation is present, 3-O-sulfation of N-sulfoheparoic acid with 2-O-sulfation is performed. When N-sulfoheparoic acid with 6-O-sulfation is present, 3-O-sulfation of N-sulfoheparoic acid with 6-O-sulfation is performed.

When N-sulfoheparoic acid with C5-epimerization and 2-O-sulfation is present, 3-O-sulfation of N-sulfoheparoic acid with C5-epimerization and 2-O-sulfation takes place do. In the presence of N-sulfoheparoic acid with C5-epimerization and 6-O-sulfation, 3-O-sulfation of N-sulfoheparoic acid with C5-epimerization and 6-O-sulfation takes place do. 3-O of N-sulfoheparoic acid with 2-O-sulfation and 6-O-sulfation, if present - sulfation is carried out;

When N-sulfoheparoic acid with C5-epimerization, 2-O-sulfation and 6-O-sulfation is present, it has C5-epimerization, 2-O-sulfation and 6-O-sulfation 3-O-sulfation of N-sulfoheparoic acid is performed.

<<reaction conditions>>

The reaction conditions for the above-mentioned 1) supply/regeneration of PAPS, 2) C5-epimerization, 3) 2-O-sulfation, 4) 6-O-sulfation, and 5) 3-O-sulfation are described below. I will explain.

The pH of the reaction solution is preferably about 6 to 8. During the reaction, it is preferable to carry out the reaction using a pH control agent for controlling the pH of the reaction solution close to neutral, particularly about 7 together with an aqueous ammonia solution, an aqueous sodium hydroxide solution, an aqueous potassium hydroxide solution or the like.

The reaction temperature during the reaction, that is, the survival temperature of the transformant is preferably 20°C to 50°C and more preferably 25°C to 47°C. The reaction time is preferably about 1 to 7 days and more preferably about 1 to 3 days. Cultivation can be any of batch, fed-batch and continuous. Among them, a batch type is preferable.

Aeration conditions The reaction may be conducted under reducing conditions or under aerobic conditions. Reduction conditions are defined by the oxidation-reduction potential of the reaction solution. The oxidation-reduction potential of the reaction solution is preferably about -200mV to -500mV and more preferably -250mV to -500mV.

The reduction state of the reaction solution can be easily estimated using a resazurin indicator and can be accurately measured using an oxidation-reduction potentiometer. As a method for preparing a reaction solution under reducing conditions, a known method can be used without limitation.

Specifically, an aqueous solution for the reaction solution can be obtained under reducing conditions by subjecting distilled water or the like to heat treatment or reduced pressure to remove dissolved gases. In addition, an appropriate reducing agent (eg, thioglycolic acid, ascorbic acid, cystine hydrochloride, mercaptoacetic acid, thiolacetic acid, glutathione, sodium sulfide, etc.) may be added to prepare an aqueous solution for the reaction solution under reducing conditions. An appropriate combination of these methods is also an effective method for preparing an aqueous solution for a reaction solution under reducing conditions.

When reducing conditions are maintained during the reaction, it is preferable to prevent mixing of oxygen from the outside of the reaction system as much as possible, and specific examples of such a method include a method of sealing the reaction system with an inert gas such as nitrogen gas or carbon dioxide gas. do.

When microaerobic conditions are maintained during the reaction, the reaction can be carried out under conditions in which the aeration rate is set to a value such as 0.5 vvm or lower, and the agitation rate is set to a lower value such as 500 rpm or lower. In some cases, the reaction may be carried out in combination with a state in which aeration is stopped at an appropriate time after initiation of the reaction and the degree of anaerobic state is increased under the condition of an agitation speed of 100 rpm or less.

<<Collection of sulfated polysaccharides>>

Sulfated polysaccharide is produced in the reaction solution by culturing as described above. The sulfated polysaccharide can be collected by recovering the reaction solution, and also the sulfated polysaccharide can be separated from the reaction solution by a known method. Examples of such known methods include distillation, membrane permeation, organic solvent extraction, and the like.

<<Manufacture of N-sulfoheparoic acid>>

The N-sulfoheparoic acid used in the method for preparing the sulfated polysaccharide of the present invention is obtained by deacetylation, depolymerization and N-sulfation of heparoic acid. The preparation of heparoic acid and the preparation of N-sulfoheparoic acid from heparoic acid can be carried out by known methods (eg WO2018/048973).

An example of a method for producing heparoic acid is to culture a microorganism belonging to a prokaryotic organism having the genetic modification of (a1) and having the ability to produce heparoic acid in a medium to produce heparoic acid in the medium, and producing heparosic acid from the medium. How to collect includes:

(a1) Genetic modification that increases the expression level of the kpsS gene.

Microorganisms belonging to prokaryotes may further have at least one of the following genetic modifications (a2) and (a3) in addition to (a1):

(a2) genetic modification to increase the expression level of at least one gene selected from kfiA, kfiB, kfiC and kfiD genes; and

(a3) Genetic alterations causing loss of function of the yhbJ gene.

The kpsS gene is a gene encoded by region I in a group of genes of region I, region II and region III. kpsS is involved in the initiation of heparoic acid synthesis. In heparoic acid production, kpsS, together with kpsC, is responsible for adding multiple Kdo linkers to the phosphatidylglycerol of the inner membrane.

As the kpsS gene, the kpsS gene derived from the genus Escherichia is preferred. A specific example thereof includes the kpsS gene of Escherichia coli K5 strain. The nucleotide sequence of the kpsS gene of Escherichia coli K5 strain and the amino acid sequence of the protein encoded by this gene can be obtained from public databases. The kpsS gene of Escherichia coli K5 strain has been registered with GenBank under accession number CAA52659.1.

An example of the kpsS gene is DNA having the nucleotide sequence shown in SEQ ID NO: 27, or a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 27, and the expression level is increased in microorganisms belonging to prokaryotes having the ability to produce heparoic acid It contains DNA that has the property of increasing the ability of microorganisms to produce heparoic acid when they are produced.

kfiA, kfiB, kfiC and kfiD are genes encoded by region II in the gene clusters of region I, region II and region III. As shown in FIG. 2, kfiA, kfiB, kfiC, and kfiD are involved in heparoic acid synthesis, and play a role in synthesizing heparoic acid by adding saccharides.

As the kfiA, kfiB, kfiC or kfiD gene, the kfiA, kfiB, kfiC or kfiD gene derived from the genus Escherichia is preferred. Specific examples thereof include the kfiA, kfiB, kfiC or kfiD genes of Escherichia coli K5 strain. The nucleotide sequences of the kfiA, kfiB, kfiC or kfiD genes of Escherichia coli K5 strain and the amino acid sequences of the proteins encoded by these genes can be obtained from public databases. kfiA is registered as GenBank Accession No. CAA54711.1; kfiB is registered with Genbank accession number CAE55824.1; kfiC is registered with GenBank accession number CAA54709.1; kfiD is registered with Genbank under the accession number CAA54708.1.

An example of the kfiA gene is DNA having the nucleotide sequence shown in SEQ ID NO: 28, or a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 28, and the expression level is increased in microorganisms belonging to prokaryotes having the ability to produce heparose acid. It contains DNA that has the property of increasing the ability of microorganisms to produce heparoic acid when they are produced.

An example of the kfiB gene is DNA having the nucleotide sequence shown in SEQ ID NO: 29, or a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 29, and the expression level is increased in microorganisms belonging to prokaryotes having the ability to produce heparoic acid. It contains DNA that has the property of increasing the ability of microorganisms to produce heparoic acid when they are produced.

An example of the kfiC gene is DNA having the nucleotide sequence shown in SEQ ID NO: 30, or a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 30, and the expression level is increased in microorganisms belonging to prokaryotes having the ability to produce heparoic acid. It contains DNA that has the property of increasing the ability of microorganisms to produce heparoic acid when they are produced.

An example of the kfiD gene is DNA having the nucleotide sequence shown in SEQ ID NO: 31, or a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 31, and the expression level is increased in microorganisms belonging to prokaryotes having the ability to produce heparoic acid It contains DNA that has the property of increasing the ability of microorganisms to produce heparoic acid when they are produced.

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

As the yhbJ gene, the yhbJ gene derived from the genus Escherichia is preferred. A specific example thereof includes the yhbJ gene of Escherichia coli K-12 strain. The nucleotide sequence of the yhbJ gene of Escherichia coli strain K-12 and the amino acid sequence of the protein encoded by this gene can be obtained from public databases. The yhbJ gene of Escherichia coli strain K-12 has been registered with GenBank accession number BAE77249.1.

An example of the yhbJ gene is DNA having the nucleotide sequence shown in SEQ ID NO: 32, or a nucleotide sequence that is 90% or more identical to the nucleotide sequence shown in SEQ ID NO: 32, and the expression level is increased in microorganisms belonging to prokaryotes having the ability to produce heparoic acid It contains DNA that has the property of increasing the ability of microorganisms to produce heparoic acid when they are produced.

Each of the genes (a1) to (a3) can be easily obtained from a public database by, for example, BLAST search or FASTA search using the nucleotide sequence of each gene described above. Further, for example, homologs of each gene can be obtained by PCR using a chromosome of a microorganism such as a bacterium as a template and oligonucleotides prepared based on these known gene sequences as primers.

Each of the genes (a1) to (a3) may be a variant of the gene as long as the original function (eg, activity or characteristic) of the protein encoded by the gene is maintained. It can be confirmed whether the protein encoded by the variant of the gene retains its original function; Specifically, for example, when the original function is to improve the heparoic acid-producing ability, it can be confirmed by introducing a variant of the gene into a microorganism belonging to prokaryotic organisms having the heparoic acid-producing ability.

Variants of each gene of (a1) to (a3) can be obtained by site-directed mutagenesis by modifying the coding region of a gene such that an amino acid residue at a specific position of the encoded protein is substituted, deleted, inserted, or added. there is. In addition, variants of each gene of (a1) to (a3) can also be obtained by, for example, mutagenesis.

Each of the genes (a1) to (a3) is a gene encoding a protein having an amino acid sequence in which one or several amino acids at one or several positions are substituted, deleted, inserted, or added as long as their original functions are maintained. can be For example, the N-terminus and/or C-terminus of an encoded protein may be extended or shortened. The phrase “one or several” differs depending on the position and type of amino acid residues in the three-dimensional structure of a protein. Specific examples thereof include 1 to 50, 1 to 40, 1 to 30, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 5, and especially Preferably it is 1-3 pieces.

Substitution, deletion, insertion or addition of one or several amino acids as described above is a conservative mutation that maintains normal protein function. Representative of conservative mutations are conservative substitutions. Conservative substitutions are mutations in which substitution occurs between Phe, Trp, and Tyr when the substitution site is an aromatic amino acid, and substitution occurs between Leu, Ile, and Val when the substitution site is a hydrophobic amino acid. When is a polar amino acid, substitution occurs between Gln and Asn, when the substitution site is a basic amino acid, substitution occurs between Lys, Arg, and His, and when the substitution site is an acidic amino acid, substitution occurs between Asp and Glu occurs, and substitution occurs between Ser and Thr when the substitution site is an amino acid having a hydroxyl group. Specific examples of substitutions that are considered conservative include Ala to Ser or Thr; substitution of Arg with Gln, His or Lys; substitution of Asn with Glu, Gln, Lys, His or Asp; replacement 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, Gin, 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, He or Leu; substitution of Ser for Thr or Ala; substitution of Thr with Ser or Ala; substitution of Trp for Phe or Tyr; substitution of Tyr with His, Phe or Trp; and substitution of Val with Met, He or Leu. In addition, substitution, deletion, insertion, addition, and reversal of amino acids as described above are caused by naturally occurring mutations such as mutations based on individual differences or species differences in organisms from which genes are derived, substitutions, deletions, insertions and Includes additions, inversions, etc. (mutants or variants).

In addition, each gene of (a1) to (a3) is 80% or more, preferably 90% or more, more preferably 95% or more of the entire amino acid sequence, even more preferably, as long as their original functions are maintained. may be a gene encoding a protein that is 97% or more and particularly preferably 99% or more identical.

In addition, each gene of (a1) to (a3) hybridizes under stringent conditions with a probe that can be prepared from a known gene sequence, such as a sequence complementary to all or part of the nucleotide sequence, as long as their original function is maintained It may be DNA that The term “stringent conditions” refers to conditions under which so-called specific hybrids are formed and non-specific hybrids are not formed. Examples thereof are DNAs that are identical to each other at a higher level, for example, 80% or more, preferably 90% or more, more preferably 95% or more, even more preferably 97% or more and particularly preferably 99% or more of each other. conditions in which identical DNAs hybridize to each other and, at a lower level, DNAs identical to each other do not hybridize to each other; or a salt concentration and temperature where the wash corresponds to 60°C, 1 x SSC and 0.1% SDS, preferably 60°C, 0.1 x SSC and 0.1% SDS, and more preferably 68°C, 0.1 x SSC and 0.1% SDS. It includes conditions that are conditions for washing in a standard Southern hybridization, which is performed once, preferably 2 to 3 times in .

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

In addition, since codon degeneracy differs depending on the host, each of the above genes (a1) to (a3) may be a gene obtained by replacing an arbitrary codon with an equivalent codon as long as their original functions are maintained. For example, the genes in Tables 1 to 3 can be modified to have optimal codons according to the codon usage frequency of the host used.

Examples of the mutation treatment include in vitro treatment of a DNA molecule having the nucleotide sequence of each gene of (a1) to (a3) with hydroxylamine or the like; Microorganisms having each gene of (a1) to (a3) are irradiated with X-rays, ultraviolet light, or N-methyl-N'-nitro-N-nitrosoguanidine (NTG), ethyl methanesulfonate (EMS) and methyl methane and treatment with mutagens such as sulfonate (MMS).

<<<Gene modification to increase gene expression>>>

The phrase “increase in gene expression” means that the expression of a gene is elevated compared to that of an unmodified strain. An example of one aspect of increasing the expression of a gene is that the expression of the gene is preferably increased by 1.5 times or more, more preferably by 2 times or more, and even more preferably by 3 times or more compared to that of the unmodified strain. include the aspect

In addition, the phrase "gene expression is increased" means that the target gene is expressed in a strain in which the target gene is not naturally expressed, as well as an increase in the expression of the target gene in a strain in which the target gene is naturally expressed. That is, the phrase "gene expression is increased" includes, for example, the case where the target gene is introduced into a strain having no target gene and the target gene is expressed therein. The phrase "gene expression is increased" is also referred to as the phrase "gene expression is enhanced" or "gene expression is elevated".

An increase in gene expression can be achieved, for example, by increasing the copy number of a gene. Increasing the copy number of a gene can be achieved by introducing the gene into the host's chromosome. Introduction of genes into chromosomes can be performed using, for example, homologous recombination (Miller I, J.H. Experiments in Molecular Genetics, 1972, Cold Spring Harbor Laboratory). Only one copy of a gene may be introduced, or two or more copies of it may be introduced.

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

Alternatively, homologous recombination can be performed while targeting appropriate sequences on the chromosome, such as genes unnecessary for the production of the target substance. Homologous recombination includes, for example, the use of linear DNA, the use of plasmids containing a temperature-sensitive origin of replication, the use of plasmids capable of conjugative delivery, and the use of suicide vectors without an origin of replication that function in the host. Alternatively, it may be performed by a transduction method using phage. Also, the gene may be randomly introduced into the chromosome using a transposon or Mini-Mu (JP-A-H2-109985).

Whether or not the target gene has been introduced into the chromosome can be confirmed by Southern hybridization using a probe having a sequence complementary to all or part of the gene, PCR using primers prepared based on the sequence of the gene, or the like.

Also, the copy number of a gene can be increased by introducing a vector containing the gene into a host. For example, copy number of the gene can be increased by constructing an expression vector for the gene by ligating a DNA fragment containing the target gene to a vector that functions in the host and transforming the host with the expression vector. . A DNA fragment containing the target gene can be obtained, for example, by PCR using 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.

As a vector, a vector capable of autonomous replication in a host cell can be used. The vector is preferably a multicopy vector. In addition, the vector is preferably combined with an antibiotic resistance gene or other gene as described in Karl Friehs, Plasmid Copy Number and Plasmid Stability, Adv Biochem Engin/Biotechnol 86: 47-82 (2004) to select transformants. have the same markers. In addition, the vector may have a promoter or terminator to express the inserted gene. Examples of vectors include vectors derived from bacterial plasmids, vectors derived from yeast plasmids, vectors derived from bacteriophages, cosmids, phagemids, and the like.

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 and pSTV29 (all from Takara Bio Inc.), pACYC184 and pMW219 (Nippon Gene), pTrc99A (Pharmacia), pPROK vector (Clontech), pKK233-2 (Clontech), pET vector (Novagen), pQE vector (Qiagen) and wide host range vector RSF1010.

In the case of introducing a gene, it is sufficient if the gene is retained in a microorganism belonging to a prokaryotic organism having genetic modification in the present invention. Specifically, it is sufficient if the gene is 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 host-derived promoter or a heterologous promoter. The promoter may be an endogenous promoter of the gene to be introduced or a promoter of another gene. As a promoter, a stronger promoter described below can be used, for example.

A terminator for terminating transcription can be placed downstream of the gene. The terminator is not particularly limited as long as it functions in the bacterium of the present invention. Terminators can be host-derived terminators or heterologous terminators. The terminator may be a terminator specific to the gene to be introduced or may be a terminator of another gene. Specific examples of the terminator include T7 terminator, T4 terminator, fd phage terminator, tet terminator and trpA terminator.

Vectors, promoters, and terminators that can be used in various microorganisms are described in detail in, for example, "Basic Lecture 8 on Microbiology, Gene Engineering, KYORITSU SHUPPAN, 1987", and these can be used.

In addition, when two or more genes are introduced, it is sufficient if each gene is maintained in a manner capable of being expressed in the bacterium of the present invention. For example, each gene may be all carried in a single expression vector or may be all carried on a chromosome. In addition, each gene may be individually carried on a plurality of expression vectors, or individually carried on a single or a plurality of expression vectors and chromosomes. Also, two or more genes can be introduced by forming an operon. Examples of "when two or more genes are introduced" are when genes encoding two or more enzymes are introduced, when genes encoding two or more subunits forming a single enzyme are introduced, and combinations thereof. includes

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 heterologous gene. 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 or the like as a template. Also, the gene to be introduced can be entirely synthesized based on, for example, the nucleotide sequence of the gene [Gene, 60(1), 115-127 (1987)].

In addition, an increase in gene expression can be achieved by improving the efficiency of transcription of a gene. Improving the transcription efficiency of a gene can be achieved, for example, by replacing the gene's promoter on the chromosome with a stronger promoter. "Strong promoter" refers to a promoter that enhances gene transcription relative to a naturally occurring wild-type promoter.

Examples of "strong promoters" include known high expression promoters such as 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. include

In addition, as a more powerful promoter, a conventional highly active type of promoter can be obtained using various reporter genes. For example, promoter activity can be increased by bringing the -35 and -10 regions of the promoter region closer to the consensus sequence (WO2000/18935).

Examples of highly active type promoters include various tac-like promoters (Katashkina JI et al. Russian Federation Patent Application 2006134574) and the pnlp8 promoter (International WO2010/027045). Methods for evaluating promoter strength and examples of strong promoters are described in Prokaryotic promoters in biotechnology. Biotechnol. Annu. Rev., 1, 105-128 (1995), etc.].

In addition, an increase in the level of gene expression can be achieved by improving the translation efficiency of a gene. An improvement in the efficiency of translation of a gene can be achieved, for example, by replacing the Shine-Dalgarno (SD) sequence (also called the ribosome binding site (RBS)) of the gene on the chromosome with a more robust SD sequence.

"More potent SD sequences" refers to SD sequences whose mRNA translation is improved over naturally occurring wild-type SD sequences. Examples of more robust SD sequences include the RBS of gene 10 of phage T7 [Olins P.O. et al, Gene, 1988, 73, 227-235]. In addition, it is known that substitution, insertion or deletion of several nucleotides in the spacer region between the RBS and the initiation codon, especially in the sequence immediately upstream of the initiation codon (5'-UTR), significantly affects the stability and translation efficiency of mRNA, Thus, the translational efficiency of genes can be improved by modifying them.

In the present invention, sites that affect gene expression, such as promoters, SD sequences, and spacer regions between RBS and initiation codons, are also collectively referred to as "expression control regions". Expression control regions can be determined using promoter search vectors or gene search software such as GENETYX. Modification of these expression control regions can be performed, for example, by a method using a thermosensitive vector or a Red driven integration method (WO2005/010175).

Improvements in gene translation efficiency can also be achieved, for example, by codon modification. Specifically, for example, when heterologous expression of a gene is performed, the translation efficiency of a gene can be improved by replacing rare codons present in the gene with more frequently used synonymous codons.

Codon substitution can be performed, for example, by site-directed mutagenesis methods that introduce target mutations into target sites of DNA. An example of a site-directed mutagenesis method is a method using PCR [Higuchi, R., 61, in PCR technology, Erlich, H.A. Eds., Stockton press (1989); Carter, P., Meth. In Enzymol., 154, 382 (1987)] and phage methods [Kramer, W. and Frits, H.J., Meth. In Enzymol., 154, 350 (1987); Kunkel, T.A. et al., Meth. In Enzymol., 154, 367 (1987)]. Alternatively, gene fragments with codon substitutions may be wholly synthesized. Codon usage frequencies in various organisms can be found in the "Codon Usage Database" [http://www.kazusa.or.jp/codon; Nakamura, Y. et al, Nucl. Acids Res., 28, 292 (2000).

Expression of a gene may also be increased by amplifying a regulator that increases expression of a gene or deleting or attenuating a regulator that decreases expression of a gene. The techniques for increasing the expression of genes as described above may be used alone or in any combination.

An increase in expression of a gene can be confirmed, for example, by confirming an increase in the amount of transcription of the gene or an increase in the amount of protein expressed from the gene. In addition, increased expression of a gene can be confirmed, for example, by confirming an increase in the activity of a protein expressed from the gene.

Confirmation of an increase in the amount of gene transcription can be performed by comparing the amount of mRNA transcribed from the gene with a wild strain or an unmodified strain such as a parental strain. Examples of 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 refers to, for example, a case in which the amount of mRNA is preferably increased by 1.5 times or more, more preferably by 2 times or more, and even more preferably by 3 times or more compared to that of the unmodified strain.

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 refers to, for example, a case in which the amount of protein is preferably increased by 1.5 times or more, more preferably by 2 times or more, and even more preferably by 3 times or more compared to that of the unmodified strain.

An increase in protein activity can be confirmed, for example, by measuring protein activity. An increase in protein activity refers to, for example, a case in which the protein activity is preferably increased 1.5 times or more, more preferably 2 times or more, and even more preferably 3 times or more compared to that of the unmodified strain.

The technique of increasing the expression of the gene described above can be used to enhance the expression of each of the genes (a1) and (a2).

The genetic modification to increase the expression of the kpsS gene is preferably at least one of modification of the expression control region of the kpsS gene and genetic modification to increase the copy number. The kpsFEDUCS gene exists as a group of heparoic acid production genes, but as described later in the Examples, the inventors of the present invention found that the effect of particularly improving heparoic acid production was obtained by increasing the expression of only the kpsS gene among them. . Therefore, as the genetic modification that increases the expression of the kpsS gene, genetic modification that increases the copy number of the kpsS gene is particularly preferred.

The genetic modification to increase the expression of at least one gene selected from the kfiA, kfiB, kfiC and kfiD genes is preferably a modification of the expression control region and copy number of the gene selected from the kfiA, kfiB, kfiC and kfiD genes. at least one of the increases. As shown in Figure 1, the kfiA, kfiB, kfiC and kfiD genes constitute an operon. Gene modification that enhances the entire operon composed of the kfiA, kfiB, kfiC and kfiD genes is preferred, and modification of the expression control region of the kfiA, kfiB, kfiC and kfiD genes is more preferred.

<<<Genetic alterations that cause gene loss of function>>>

An example of the genetic modification causing loss of function of the yhbJ gene of (a3) above is that the function of the protein encoded by the portion corresponding to yhbJ is a host that encodes the portion corresponding to yhbJ in the genomic DNA of a microorganism belonging to prokaryotes. Includes genetic alterations that are reduced or stopped completely by altering DNA.

In the method of the present invention, the form of modification added to the DNA encoding the portion corresponding to yhbJ is not particularly limited as long as the function of the protein encoded by the gene corresponding to yhbJ is reduced or completely stopped, and known methods can be used appropriately.

Examples of forms that reduce or completely stop the function of the protein encoded by the portion corresponding to yhbJ include any one of the following modifications (I) to (III):

(I) All or part of the DNA encoding the region corresponding to yhbJ is removed.

(II) One or several substitutions, deletions or additions are made to the DNA encoding the portion corresponding to yhbJ.

(III) DNA encoding the portion corresponding to yhbJ is replaced with a DNA sequence that is less than 80% identical to the DNA sequence prior to modification.

Examples of loss of function of the yhbJ gene include cases where the activity of the yhbJ gene is preferably 20% or less, more preferably 10% or less, and still more preferably 5% or less compared to that of the unmodified strain. The activity of yhbJ can be confirmed by examining the expression level of glmS by Northern blotting method, Western blotting method, etc. [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].

In the method for producing the sulfated polysaccharide of the present invention, in the case of the reaction solution, N-sulfoheparoic acid obtained by chemically transforming heparoic acid by a known method can be used, wherein heparoic acid is Obtained by the heparoic acid production method.

<Method for producing PAPS>

The method for producing PAPS of the present invention is characterized in that the reaction for producing PAPS is carried out by incorporating an ATP source, a sulfate ion source, and the above-described transformant (a) or a treated product thereof into a reaction solution. That is, the method for producing PAPS of the present invention includes the following steps (i) and (ii):

(i) a bacterial transformant of the genus Corynebacterium comprising at least a gene encoding ATP sulfurylase and a gene encoding APS kinase, introduced in an expressible manner, wherein the cell plasma membrane of the transformant is generating a substance-permeable transformant or a treatment product thereof; and

(ii) performing a reaction for producing PAPS using a reaction solution containing an ATP source, a sulfate ion source, and the transformant prepared in step (i) or a treated product thereof.

According to the method for producing PAPS of the present invention, PAPS can be produced by bacterial cell reaction using transformants of bacteria of the genus Corynebacterium expressing ATP sulfurylase and APS kinase or processed products thereof.

Example

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

Example 1

Construction of Corynebacterium ammoniagenes DE3 plasmid

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

At this time, in order to perform the second PCR (fusion PCR) for linking the gltD flanking fragment amplified in the first PCR and the purT flanking fragment, a 5' primer (gltD-purT_4; sequence) for amplifying the purT flanking fragment A sequence complementary to the sequence of about 25 bases on the 3' side of No. 4) was added to the 5' side of the 3' primer (gltD-purT_2BX; SEQ ID NO: 2) for amplifying the gltD flanking fragment. In addition, sequences for In-Fusion cloning were added to the 5' side of the 5' primer (gltD-purT_1) to amplify the gltD flanking fragment and the 3' primer (gltD-purT_4) to amplify the purT flanking fragment. . Additionally, lambda DE3 was inserted by adding BglII and XhoI recognition sequences to the preferred parts of the gltD and purT sides.

The chromosomal DNA of Corynebacterium ammoniagenes ATCC 6872 strain (hereinafter referred to as ATCC 6872), a wild type strain of Corynebacterium ammoniagenes, was prepared by Saito et al. [Biochim. Biophys. Acta 72, 619 (1963)].

Using chromosomal DNA as a template, primary PCR was performed to amplify the gltD flanking fragment and the purT flanking fragment, through which a DNA fragment of about 0.8 kb on the gltD flanking and a DNA of about 0.8 kb on the purT flanking was performed. A fragment was obtained. Next, secondary PCR was performed to link these gltD flanking fragments and purT flanking fragments, thereby obtaining a DNA fragment (gltD-BX-purT) of about 1.6 kb.

A 2.6 kb PstI DNA fragment containing the levansucrase gene sacB of Bacillus subtilis [Mol. Microbiol., 6, 1195 (1992)] plasmid pESB30 contained in the PstI cut site [Gene, 61, 63, (1987)] of Escherichia coli vector pHSG299 having kanamycin resistance gene was digested with BamHI. Then, the gltD-BX-purT fragment obtained above was ligated using an In-Fusion cloning kit (Takara Bio Inc.). Using the reaction product, Escherichia coli DH5 alpha [TOYOBO CO., LTD. production] was transformed according to a conventional method [Molecular cloning: a laboratory manual, 3 ^rd ed., 2001, Cold Spring Harbor Laboratory Press].

The obtained strain was prepared in LB agar medium containing 20 micrograms/ml of kanamycin [10 g of bactotryptone (manufactured by Difco), 5 g of yeast extract (manufactured by Difco), 10 g of sodium chloride and bacto agar (manufactured by Difco) in 1 L of water. ) in a medium containing 16 g and its pH adjusted to pH 7.0], and transformed strains were selected. After target clones were selected by colony PCR, the transformed strain was inoculated into LB medium containing 20 micrograms/ml of kanamycin (a medium having the same composition as LB agar medium except that it did not contain agar), An overnight culture was used to prepare plasmids from the resulting culture broth using the QIAprep Spin Miniprep kit (Qiagen). Nucleotide sequence analysis confirmed that this plasmid had a structure in which a gltD-BX-purT fragment of about 1.6 kb was inserted into pESB30.

Subsequently, chromosomal DNA extracted from Escherichia coli BL21 (DE3) was used as a template and amplified by PCR using primers of DE3-for_Xho (SEQ ID NO: 5) and DE3-rev_Bgl (SEQ ID NO: 6). The XhoI recognition sequence was added to DE3-for_Xho and the BglII recognition sequence was added to DE3-rev_Bgl. A plasmid having a structure in which this fragment and a gltD-BX-purT fragment of about 1.6 kb were inserted into pESB30 was digested with XhoI and BglII, and then ligated with a DNA ligation kit (Takara Bio Inc.).

Escherichia coli DH5 alpha was transformed in the same manner as described above, and the strain thus obtained was cultured on LB agar medium containing 20 micrograms/ml of kanamycin, and the transformed strain was selected. Transformed strains were inoculated into LB medium containing 20 micrograms/ml of kanamycin and cultured overnight, whereby plasmids were prepared from the obtained culture broth using the QIAprep Spin Miniprep kit (Qiagen). Nucleotide sequence analysis confirmed that this plasmid had a structure in which a 4.5 kb lambda DE3 fragment was inserted between about 1.6 kb of gltD-purT on pESB30. This plasmid was designated pC-DE3.

Example 2

Construction of lambda DE3-inserted strains of Corynebacterium ammoniagenes

PC-DE3 is based on the method of Rest et al. [Appl. Microbiol. Biotech., 52, 541 (1999)] into ATCC 6872 strain by electroporation, and kanamycin-resistant strains were selected. When the structure of the chromosome obtained from the kanamycin-resistant strain was investigated by Southern hybridization [Molecular cloning: a laboratory manual, 3 ^rd ed., 2001, Cold Spring Harbor Laboratory Press], pC-DE3 was found to be of the Campbell type. It was confirmed that it was integrated into the chromosome by homologous recombination.

The transformed strain (single recombinant) was mixed with Suc agar medium [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) and bacto agar (manufactured by Difco) in 1 L of water. medium containing 15 g and its pH adjusted to pH 7.2], incubated at 30° C. for 1 day, and growing colonies were selected. Strains with the sacB gene could not grow on this medium because it converts sucrose into a suicide substrate [J. Bacteriol., 174, 5462 (1991)]. On the other hand, the strain in which the sacB gene was deleted by secondary homologous recombination between the lambda DE3-inserted type and the wild type present near the chromosome could grow on this medium without generating a suicide substrate. During this homologous recombination, strains or wild-type constructs in which the lambda DE3 fragment was inserted between gltD-purT were eliminated along with acB. At this time, gene replacement with the lambda DE3-inserted type occurred in the strain in which the wild-type structure was eliminated together with sacB.

A strain in which lambda DE3 was inserted between gltD-purT of ATCC 6872 was obtained by colony PCR using a second recombinant obtained with primers of DE3-for_Xho and DE3-rev_Bgl. This strain was designated ATCC 6872 (DE3).

Example 3

Construction of a plasmid with a DNA fragment for expressing MET3-MET14A

Plasmid pCS299P [Appl. Microbiol. Biotech., 63, 592 (2004)] was digested with BamHI. PCR was performed using pCET_Fw2 (SEQ ID NO: 7) and pCET_Rv2 (SEQ ID NO: 8) and pET21b as a template to obtain a DNA fragment containing lacI-PT7. They were purified with the QIAquick PCR purification kit (Qiagen) and ligated using the In-Fusion cloning kit (Takara Bio Inc.). Using the reaction product, Escherichia coli DH5 alpha (manufactured by TOYOBO CO., LTD.) was transformed according to a conventional method, cultured in LB agar medium containing 20 micrograms/ml kanamycin, and transformed strains were selected. After target clones were selected by colony PCR, the transformed strain was inoculated into LB medium containing 20 micrograms/ml of kanamycin and cultured overnight, thereby culturing obtained using the QIAprep Spin Miniprep kit (Qiagen) Plasmids were prepared from the broth. Nucleotide sequence analysis confirmed that this plasmid had a structure in which a lacI-PT7 DNA fragment of about 1.9 kb derived from pET21b was inserted into pCS299P. This plasmid was designated pCET212.

Subsequently, plasmids in which MET3 and MET14 derived from Saccharomyces cerevisiae were inserted downstream of the T7 promoter of pCET212 were constructed. Two primers (MET3_1 (SEQ ID NO: 9) and MET3_2 (SEQ ID NO: 10)) for amplifying MET3 from chromosomal DNA of Saccharomyces cerevisiae S288C strain (hereinafter referred to as S288C) and 2 for amplifying MET14 Species primers (MET14_3 (SEQ ID NO: 11) and MET14_4 (SEQ ID NO: 12)) were designed.

At this time, in order to perform the second PCR (fusion PCR) linking the MET3 fragment and the MET14 fragment amplified in the first PCR, about 15 on the 5' side of the 5' primer (MET14_3; SEQ ID NO: 11) for amplifying MET14. A sequence complementary to a sequence of 15 bases was added to the 5' side of a 3' primer (MET3_2; SEQ ID NO: 10) for amplifying MET3, and a sequence complementary to a sequence of about 15 bases on the 5' side of MET3_3 was added to MET14_3. was added to the 5' side of In addition, sequences for In-Fusion cloning were added to the 5' side of the 5' primer (MET3_1) to amplify MET3 and the 3' primer (MET14_4) to amplify MET13. Using the chromosomal DNA of strain S288C as a template, primary PCR was performed to amplify MET3 and MET14, thereby obtaining a DNA fragment of about 1.5 kb of MET3 and a DNA fragment of about 0.6 kb of the downstream region. Next, secondary PCR was performed to link these MET3 and MET14 fragments, and through this, a DNA fragment (MET3-MET14) of about 2.1 kb was obtained. The DNA fragment was purified using a QIAquick PCR purification kit (Qiagen) and then ligated into pCET212 digested with NdeI and XhoI 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. The strains thus obtained were cultured on LB agar medium containing 20 micrograms/ml of kanamycin, and transformed strains were selected. After target clones were selected by PCR, the transformed strains were inoculated into LB medium containing 20 micrograms/ml of kanamycin and cultured overnight, thereby obtaining culture broth using the QIAprep Spin Miniprep kit (Qiagen). A plasmid was prepared from Nucleotide sequence analysis confirmed that the plasmid had a structure in which a MET3-MET14 fragment of about 2.1 kb was inserted into pCET212. This plasmid was designated pSC-3-13. pSC-3-13 was transformed into ATCC 6872(DE3) to obtain a PAPS-producing ATCC 6872(DE3)/pSC-3-13 strain of Corynebacterium ammoniagenes.

Example 4

Construction of Plasmids with DNA Fragments for Expressing Chaperone Proteins

PCR was performed using Gro_F (SEQ ID NO: 13) and Gro_R (SEQ ID NO: 14) and chromosomal DNA of Escherichia coli BL21 (DE3) strain as a template, thereby obtaining a DNA fragment containing GroES-GroEL did Next, 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)] and PCR was performed using AraC_ParaB_F (SEQ ID NO: 15) and AraC_ParaB_R (SEQ ID NO: 16), thereby obtaining a DNA fragment containing AraC_ParaB. PCR was performed using pCDF-SmOri-F (SEQ ID NO: 17) and pCDF-SmOri-R (SEQ ID NO: 18) and pCDF-Duet1 (Novagen) as a template, through which streptomycin resistance gene and ColdDF origin of replication A DNA fragment containing was obtained. They were purified by the QIAquick PCR purification kit (Qiagen) and ligated using the In-Fusion cloning kit (Takara Bio Inc.). Using the reaction product, Escherichia coli DH5 alpha (manufactured by TOYOBO CO., LTD.) was transformed according to a conventional method, cultured on LB agar medium containing 50 micrograms/ml streptomycin, and Transformed strains were selected. After target clones were selected by colony PCR, the transformed strain was inoculated into LB medium containing 50 micrograms/ml of streptomycin and cultured overnight, thereby culturing obtained using the QIAprep Spin Miniprep kit (Qiagen) Plasmids were prepared from the broth. Nucleotide sequence analysis confirmed that the plasmid had a structure in which an AraC-ParaB DNA fragment of about 1.2 kb derived from pKD46 and a GroES-GroEL DNA fragment of about 2.0 kb derived from BL21 (DE3) were inserted into pCDF-Duet1. This plasmid was designated pGro(Sm).

Example 5

Construction of Escherichia coli Expressing C5-Epimerase, 2OST, 6OST-3 and 3OST-1

The catalytic domain region of human C5-epimerase (E53-N609) was transferred to the pMAL-C2X vector (New England Biolabs) to construct MBP-C5. MBP-C5 was transformed with Origami-B(DE3) (Novagen) together with pGro7 (Takara Bio Inc.) and Origami-B(DE3)/MBP- A C5_pGro7 strain was constructed.

SEQ ID NO: 19 shows the nucleotide sequence of the catalytic domain (R51-N356) of 2-O-sulfotransferase isoform 1, which is derived from Chinese hamster and codon optimized for expression in Escherichia coli. PCR was performed using the sequence artificially synthesized as described above and the primers of SEQ ID NOs: 20 and 21 as templates. This PCR fragment was subjected to a ligation independent cloning method [Methods Mol Biol. 2009; 498: 105-115] to construct H-MBP-2OST by cloning into pET-His6-MBP-TEV-LIC (Addgene). H-MBP-2OST was transformed with Origami-B (DE3) (Novagen) together with pGro (Sm), whereby Origami-B (DE3) / H-MBP-2OST_pGro ( Sm) strains were constructed.

The catalytic domain (P120-L424) of mouse-derived 6-O-sulfotransferase isoform 3 was isolated from pMAL by the method described in Chemistry & Biology Volume 14, Issue 9, 21 September 2007, pages 986-993. MBP-6OST3 was constructed by cloning into the -C2X vector (New England Biolabs). Origami-B(DE3)/MBP-6OST3_pGro7 of Escherichia coli expressing 6OST-3 by transforming MBP-6OST3 with Origami-B(DE3) (Novagen) together with pGro7 (Takara Bio Inc.) Strains were constructed.

The catalytic domain (G48-H311) of 3-O sulfotransferase derived from mouse was prepared by J Biol Chem. 2004 Jun 11; 279 (24): 25789-97] to construct HIS-3OST1 by cloning into the pET28a vector (Novagen). HIS-3OST1 was transformed with BL21-CodonPlus(DE3)-RIL (Agilent Technologies) together with pGro(Sm), through which the RIL/HIS-3OST1_pGro(Sm) strain of Escherichia coli expressing 3OST1 was constructed .

Example 6

Preparation of N-sulfoheparoic acid and 2-O-sulfated N-sulfoheparoic acid

According to the method described in the patent document [WO2018/048973 A1], heparoic acid was prepared by fermentation using Escherichia coli K5 strain or Nissl strain. According to the above literature, the obtained heparoic acid was chemically deacetylated and depolymerized and then chemically N-sulfated. Thereafter, fractionation by ethanol precipitation gave N-sulfoheparoic acid. The obtained N-sulfoheparoic acid was subjected to epimerization at the C5 position of the uronic acid residue and sulfation at the 2-O position by an enzymatic reaction according to the same literature, followed by fractionation by ethanol precipitation and 2-O- Sulfated N-sulfoheparoic acid was obtained.

Example 7

Cultivation of ATCC 6872 (DE3)/pSC-3-13

The ATCC 6872 (DE3) / pSC-3-13 strain obtained in Example 3 was mixed with BY-glucose agar medium containing 50 micrograms / ml of kanamycin [10 g of glucose in 1 L of water, a conventional bouillon medium (Kyokuto Pharmaceutical Industrial Co., Ltd.) 20 g, yeast extract (manufactured by Difco) 5 g, and bacto agar (manufactured by Difco) 20 g] and incubated overnight at 30°C.

Two plates of bacterial cells were cultured in 350 ml of a primary seed medium [glucose 50 g/L, High Polypeptone (manufactured by Nihon Pharmaceutical Co., Ltd.) 10 g/L, yeast extract in a 2 L Erlenmeyer flask. (Asahi) 10 g/L, KH ₂ PO ₄ 1 g/L, K ₂ HPO ₄ 1 g/L, (NH ₄ ) ₂ SO ₄ 0.5 g/L, Urea 0.5 g/L, L-Cystine 0.03 g/L, MgSO ₄ -7H ₂ O 1g/L, CaCl ₂ -2H ₂ O 0.1g/L, ZnSO ₄ -7H ₂ O 0.01g/L, FeSO ₄ -7H ₂ O 0.01g/L, MnSO ₄ -5H ₂ O 0.02g /L, D-calcium pantothenate 0.01 g/L, biotin 40 micrograms/L, thiamine hydrochloride 0.005 g/L and nicotinic acid 0.005 g/L, the pH of which is adjusted to pH 7.2 with sodium hydroxide; After sterilization at 122°C for 20 minutes using an autoclave, it was inoculated into a medium to which 0.1 g/L of L-cysteine, 1 g/L of sodium thiosulfate, and 0.1 g/L of kanamycin were individually added] and 220 rpm at 30°C. Incubated for 24 hours at an agitation speed.

1700 ml of secondary seed medium in a 6 L culture tank [glucose 100 g/L, fructose 4 g/L, yeast extract (manufactured by Asahi) 10 g/L, KH ₂ PO ₄ 1.25 g/L, K ₂ HPO ₄ 1 g/L , monosodium 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, D-calcium pantothenate 0.015 g/L, biotin 40 micrograms/L L, 0.005 g/L of nicotinic acid and 1 ml/L of ADEKA NOL LG-109 (manufactured by ADEKA), the pH of which was adjusted to pH 7.2 with sodium hydroxide, and after individually adding urea at 3.2 g/L, 122 Sterilized in an autoclave at °C for 20 minutes, 0.1 g/L of thiamine hydrochloride, 0.3 g/L of L-cysteine, 2.5 g/L of sodium thiosulfate, and 0.2 g/L of kanamycin were individually added] to 300 ml of the above medium. The culture medium was inoculated and cultured for 24 hours under culture conditions of 30°C, agitation rate of 650 rpm and aeration rate of 2 L/min while adjusting its pH to 6.8 with 18% aqueous ammonia.

1700 ml of main culture medium [KH ₂ PO ₄ 10 g/L, K ₂ HPO ₄ 10 g/L, monosodium glutamate monohydrate 1 g/L, L-cystine 0.02 g/L, CaCl ₂ -2H ₂ O in 6 L culture tank 0.1 g/L, CuSO ₄ -5H ₂ O 0.005 g/L, ZnSO ₄ -7H ₂ O 0.01 g/L, FeSO ₄ -7H ₂ O 0.02 g/L, Biotin 150 micrograms/L, Nicotinic acid 0.005 g/L , containing 2 g/L of urea and 1 ml/L of ADEKA NOL LG-109 (manufactured by ADEKA), sterilized in an autoclave at 122° C. for 20 minutes, 125 g/L glucose, 25 g/L fructose, and MgSO ₄ -7H ₂ O 10 g/L, MnSO ₄ -5H ₂ O 0.02 g/L, D-calcium pantothenate 0.015 g/L, thiamine hydrochloride 0.005 g/L, L-cysteine 0.15 g/L, sodium thiosulfate 2.5 g /L and 0.2 g/L of kanamycin were added separately] with 300 ml of the culture medium, and the stirring speed was adjusted to 650 rpm to 900 rpm so that the amount of dissolved oxygen did not fall below 1 ppm, and 18% ammonia water was added. It was cultured for 25 hours under culture conditions of 30°C and an aeration rate of 2 L/min while adjusting its pH to 6.8.

During this period, IPTG was added to a final concentration of 1 mM 6 hours after the start of the culture, 100 micrograms/L of biotin, 0.015 g/L of nicotinic acid, 0.015 g/L of calcium D-pantothenate and thiamine hydrochloride. 0.005 g/L was further added 10 hours after the start of the culture. After the end of the culture, the culture medium was separated into bacterial cells and culture supernatant by centrifugation, whereby a pellet was obtained as wet bacterial cells and frozen at -80°C.

Example 8

Culture of Origami-B (DE3)/MBP-C5_pGro7, Origami-B (DE3)/H-MBP-2OST_pGro (Sm), Origami-B (DE3)/MBP-6OST3_pGro7 and RIL/HIS-3OST1_pGro (Sm)

The Origami-B(DE3)/MBP-C5_pGro7 strain obtained in Example 5 was treated with 5 micrograms/mL ampicillin, 20 micrograms/mL chloramphenicol, 15 micrograms/mL tetracycline and 15 micrograms/mL kanamycin. It was inoculated into a large test tube containing mL of TB medium and incubated for 16 hours at 30°C. Inoculate 1.2% culture into baffled Erlenmeyer flasks containing 500 mL of TB medium containing 50 micrograms/mL ampicillin, 20 micrograms/mL chloramphenicol, 15 micrograms/mL tetracycline and 15 micrograms/mL kanamycin and incubated with shaking at 37°C for 6 hours. Thereafter, IPTG at a final concentration of 1 mM and arabinose at a final concentration of 4 mM were added thereto, and culture was performed at 28° C. for 20 hours. The culture was then centrifuged to obtain a pellet as wet bacterial cells through which it was frozen at -80°C.

The Origami-B(DE3)/H-MBP-2OST_pGro(Sm) strain obtained in Example 5 was treated with 50 microgram/mL ampicillin, 50 microgram/mL streptomycin, 15 microgram/mL tetracycline and 15 microgram/mL ampicillin. It was inoculated into a large test tube containing 5 mL of TB medium containing mL kanamycin and incubated at 30°C for 16 hours. 1.2% culture was inoculated into baffled Erlenmeyer flasks containing 500 mL of TB medium containing 50 micrograms/mL ampicillin, 50 micrograms/mL streptomycin, 15 micrograms/mL tetracycline and 15 micrograms/mL kanamycin and incubated with shaking at 30°C for 12 hours. Thereafter, IPTG at a final concentration of 1 mM and arabinose at a final concentration of 4 mM were added thereto, and culture was performed at 28° C. for 20 hours. The culture was then centrifuged to obtain a pellet as wet bacterial cells through which it was frozen at -80°C.

The Origami-B(DE3)/MBP-6OST3_pGro7 strain obtained in Example 5 was treated with 5 micrograms/mL ampicillin, 20 micrograms/mL chloramphenicol, 15 micrograms/mL tetracycline and 15 micrograms/mL kanamycin. It was inoculated into a large test tube containing mL of TB medium and incubated for 16 hours at 30°C. Inoculate 1.2% culture into baffled Erlenmeyer flasks containing 500 mL of TB medium containing 50 micrograms/mL ampicillin, 20 micrograms/mL chloramphenicol, 15 micrograms/mL tetracycline and 15 micrograms/mL kanamycin and incubated at 37°C for 4 hours with shaking. Thereafter, IPTG at a final concentration of 1 mM and arabinose at a final concentration of 4 mM were added thereto, and culture was performed at 28° C. for 20 hours. The culture was then centrifuged to obtain a pellet as wet bacterial cells through which it was frozen at -80°C.

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 micrograms/ml of streptomycin and 15 micrograms/mL of kanamycin, and incubated at 30°C. incubated for 16 hours. The 1.2% culture was inoculated into a baffled Erlenmeyer flask containing 500 mL of TB medium containing 50 micrograms/mL streptomycin and 15 micrograms/mL kanamycin, and cultured at 37°C for 4 hours with shaking. Thereafter, IPTG at a final concentration of 1 mM and arabinose at a final concentration of 4 mM were added thereto, and culture was performed at 28° C. for 20 hours. The culture was then centrifuged to obtain a pellet as wet bacterial cells through which it was frozen at -80°C.

Example 9

2-O of N-sulfoheparoic acid using ATCC 6872 (DE3)/pSC-3-13, Origami-B (DE3)/MBP-C5_pGro7 and Origami-B (DE3)/H-MBP-2OST_pGro (Sm) Test for in situ sulfation reaction

Freeze-dried bacteria 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 A bacterial cell suspension was prepared by suspending each of the cells in distilled water so that the frozen bacterial cell weight was 333 g/L. 30 ml of thus obtained ATCC 6872 (DE3)/pSC-3-13 bacterial cell suspension, 6 ml of Origami-B (DE3)/MBP-C5_pGro7 bacterial cell suspension and 6 mL of Origami-B (DE3)/H- MBP-2OST_pGro (Sm) bacterial cell suspension was mixed with 18 ml of reaction solution in a 250 ml bioreactor [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 L, D-calcium pantothenate 0.3 g/L, nicotinic acid 0.2 g/L, adenine 4.05 g/L, benzalkonium chloride 1.25 g/L, ADEKA NOL LG-109 (manufactured by ADEKA) 1 ml/L and N-sulfo in an aqueous solution containing 1 g/L of heparoic acid (prepared in Example 6)] and adjusting its pH to 6.5 with 2.8% aqueous ammonia at 37°C, a stirring speed of 500 rpm and aeration of 0.75 ml/min. It was reacted for 22 hours under the culture condition of speed. 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 further added 6 hours after the start of the culture.

Sampling was appropriately performed during the reaction to obtain a reaction solution. The obtained reaction solution was appropriately diluted and then centrifuged, and the absorbance was measured at 254 nm with a UV detector using HPLC manufactured by Shimadzu Corporation to detect and quantify PAPS. The results are shown in FIG. 4 . In addition, the production of unsaturated disaccharide by enzymatic degradation and analysis by HPLC were performed according to the patent document [WO2018/048973 A1].

That is, the obtained reaction solution was centrifuged, and the resulting supernatant was heated and maintained at 80° C. for 10 minutes to denature the protein. The solution after protein denaturation was centrifuged, and the resulting supernatant was desalted by ultrafiltration using a 3k molecular weight filter device (manufactured by Merck). The solution after desalting was prepared as a heparanase reaction solution [consisting of 50 mM ammonium acetate and 2 mM calcium chloride] containing 0.5 U/ml of each of heparanase I, II, and III (manufactured by Sigma, but other heparinase may also be used). and enzymatically digested at 35° C. for 2 hours. The solution after enzymatic digestion was maintained at 95° C. for 15 minutes to inactivate heparinase.

The solution after heparanase inactivation was purified by Shimadzu HPLC and a strong anion exchange column (spherisorb-SAX chromatography column, 4.0 x 250 mm, 5 micrometers, manufactured by Waters), mobile phase A [containing 1.8 mM sodium dihydrogen phosphate and phosphoric acid Unsaturation by subjecting an aqueous solution whose pH was adjusted to pH 3.0 with 3.0] and mobile phase B [aqueous solution containing 1.8 mM sodium dihydrogenphosphate and 1 M sodium perchlorate, the pH of which was adjusted to pH 3.0 with phosphoric acid] to gradient elution mode analysis. Disaccharide analysis was performed.

Unsaturated disaccharides were detected by measuring absorbance at 232 nm with a UV detector. The retention times of delta-UA-GlcNS and delta-UA, 2S-GlcNS were compared with an unsaturated disaccharide standard reagent (manufactured by Iduron) and confirmed. 2-O-sulfation was confirmed by calculating the area ratio of delta-UA and 2S-GlcNS to the total area of delta-UA-GlcNS, delta-UA and 2S-GlcNS detected. The results are shown in FIG. 5 .

Example 10

Test for sulfation reaction at the 6-O position of 2-O-sulfated N-sulfoheparoic acid using ATCC 6872 (DE3)/pSC-3-13 and Origami-B (DE3)/MBP-6OST3_pGro7

Each of the frozen bacterial cells of ATCC 6872 (DE3) / pSC-3-13 and Origami-B (DE3) / MBP-6OST3_pGro7 obtained in Examples 7 and 8 was prepared so that the weight of the frozen bacterial cells was 333 g / L. A bacterial cell suspension was prepared by suspending in distilled water. 30 ml of ATCC 6872 (DE3)/pSC-3-13 bacterial cell suspension and 6 ml of Origami-B (DE3)/MBP-6OST3_pGro7 bacterial cell suspension thus obtained, as well as 6 ml of distilled water were added to 18 ml in a 250 ml bioreactor. ml of the 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, D-calcium pantonate 0.3 g/L, nicotinic acid 0.2 g /L, adenine 4.05 g/L, benzalkonium chloride 1.25 g/L, ADEKA NOL LG-109 (manufactured by ADEKA) 1 ml/L and 2-O-sulfated N-sulfoheparoic acid 0.5 g/L (Example prepared in Example 6)], and reacted for 22 hours under culturing conditions of 32° C., stirring rate of 500 rpm and aeration rate of 0.75 ml/min while adjusting its pH to 7.4 with 2N aqueous potassium hydroxide solution. made it

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 further added 6 hours after the start of the culture. Sampling was appropriately performed during the reaction to obtain a reaction solution. Detection and quantification of PAPS was performed using the same method as in Example 9. The results are shown in FIG. 6 .

In addition, the sulfated composition contained in the sugar chain after the reaction was analyzed by the same method as in Example 9. The retention times of delta-UA, 2S-GlcNS, and delta-UA, 2S-GlcN, and 6S were compared with unsaturated disaccharide standard reagents (manufactured by Iduron) and confirmed. 6-O-sulfation was confirmed by calculating the area ratio of delta-UA, 2S-GlcN, and 6S to the total area of delta-UA, 2S-GlcNS, and delta-UA, 2S-GlcN, and 6S detected. The results are shown in FIG. 7 .

Example 11

6-O of 2-O-sulfated N-sulfoheparoic acid using ATCC 6872 (DE3)/pSC-3-13, Origami-B (DE3)/MBP-6OST3_pGro7 and RIL/HIS-3OST1_pGro (Sm) Test for sulfation reaction at position and 3-O position

The reaction was carried out for 22 hours under the conditions described in Example 10. During this period, 3 hours after starting the reaction, the frozen bacterial cells of IL/HIS-3OST1_pGro (Sm) obtained in Example 8 were suspended in distilled water so that the weight of the frozen bacterial cells was 333 g/L and 6 ml of the prepared bacterial cell suspension was added. Sampling was appropriately performed during the reaction to obtain a reaction solution. Detection and quantification of PAPS was performed using the same method as in Example 9. The results are shown in FIG. 8 .

After completion of the reaction, the reaction solution was centrifuged, and the resulting supernatant was appropriately diluted, and then the anti-IIa activity was measured using a BIOPHEN (registered trademark) ANTI-IIa assay kit (manufactured by Hyphen Biomed), and 3-O-sulfuric acid Anger was confirmed. In addition, as a negative control in which RIL/HIS-3OST1_pGro(Sm) was not added, the anti-IIa activity of the solution after the reaction of Example 10 was also measured. A calibration curve was prepared using a BIOPHEN (registered trademark) UFH calibrator (manufactured by Hyphen Biomed). The results are shown in Table 1.

Table 1. Measurement results of anti-IIa activity Addition of RIL/HIS-30ST1_pGro(Sm) Anti-IIa activity (IU/ml - supernatant) adding 375 not added -36

Example 12

A bacterial cell suspension was prepared by suspending the frozen bacterial cells of ATCC 6872(DE3)/pSC-3-13 obtained in Example 7 in distilled water so that the weight of the frozen bacterial cells was 333 g/L. 30 ml of the bacterial cell suspension thus obtained was mixed with 30 ml of the 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 in a 250 ml bioreactor. /L, D-calcium pantothenate 0.3 g/L, nicotinic acid 0.2 g/L, adenine 4.05 g/L, benzalkonium chloride 0.625 g/L, ADEKA NOL LG-109 (manufactured by ADEKA) 1 ml/L aqueous solution], and reacted for 22 hours under culture conditions of 32°C, a stirring rate of 500 rpm, and an aeration rate of 0.75 mL/min while adjusting its pH to 7.4 with a 2N aqueous solution of potassium hydroxide.

During this period, 60 g/L of glucose, 2.19 g/L of KH ₂ PO ₄ , 3.75 g/L of K ₂ HPO ₄ , 5.0 g/L of MgSO ₄ -7H ₂ O and 2.7 g/L of adenine were added 6 hours after starting the reaction. Additional L was added. Sampling was appropriately performed during the reaction to obtain a reaction solution.

The obtained reaction solution was appropriately diluted and then centrifuged, and the absorbance was measured at 254 nm with a UV detector using HPLC manufactured by Shimadzu Corporation to detect and quantify PAPS. The results are shown in FIG. 9 .

As described above, bacteria belonging to the genus Corynebacterium capable of producing and regenerating PAPS and microorganisms belonging to prokaryotes expressing sulfate enzymes were added to raw materials such as glucose, adenine, and magnesium sulfate, and reacted, thereby reducing polysaccharides from polysaccharides. Sulfated polysaccharides were produced efficiently.

Although the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention. All references cited herein are incorporated in their entirety. This application is based on International Application No. PCT/JP2020/015388 filed on April 3, 2020, the entire contents of which are incorporated herein by reference.

SEQUENCE LISTING <110> RENSSELAER POLYTECHNIC INSTITUTE OTSUKA PHARMACEUTICAL FACTORY, INC KIRIN BIOMATERIALS CO., LTD. <120> Method for producing sulfated polysaccharide and method for producing PAPS <130> W531376 <150> PCT/JP2020/015388 <151> 2020-04-03 <160> 32 <170> PatentIn version 3.5 <210> 1 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 1 cggtacccgg ggatccgact ggccagtaca ggctg 35 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> < 223> Synthesized Oligonucleotide for primer <400> 2 agcgactcga gtttagatct tcaatgctca acccgacctg 40 <210> 3 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 3 attgaagatc taaactcgacag tcgctttgacg atgtttgacg 40 <210> 4 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 4 cgactctaga ggatcgtttg tcgcgggcga cgata 35 <210> 5 <211> 30 <212> DNA < 213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 5 tttctcgaga actgcgcaac tcgtgaaagg 30 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence < 220> <223> Synthesized Oligonucleotide for primer <400> 6 tttagatctg ttacgcgaac gcgaagtc 28 <210> 7 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 7 cggtacccgg ggatcgctta atgcgccgct acaggg 36 <210> 8 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 8 atgcctgcag gtcgaggagc tgactgggtt gaagg 35 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 9 gaaggata tacatatgcc tgctcctcac ggtgg 35 <210> 10 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 10 gttgtgtctc ctctattaaa atacaaaaaa gccat 35 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 11 tagaggagac acaacatggc tactaatattacttg 35 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 12 ggtggtggtg ctcgatta ca aatgcttacg gatga 35 <210> 13 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 13 ttgggaattc gagctctaag gaggttataa aaaatgaata ttcgtccatt gcatgatcgc 60 g 61 <210> 14 > 24 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 14 ttacatcatg ccgcccatgc cacc 24 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> < 223> Synthesized Oligonucleotide for primer <400> 15 ttatgacaac ttgacggcta catcattcac 30 <210> 16 <211> 61 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 16 cgcgatcatg caatggacga atattcattt tttagtaacct ccttagtaacct cgaattccca 60 a 61 <210> 17 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 17 ggcggcatga tgtaattatt tgccgactac cttggtgatc tc 42 <210> 18 <211> 45 < 212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 18 gtcaagttgt cataatctag agcggtt cag tagaaaagat caaag 45 <210> 19 <211> 918 <212> DNA <213> Chinese hamster <400> 19 cgcgaaatcg aacagcgtca cacgatggat ggcccgcgcc aagatgcaac cctggacgaa 60 gaagaagata tggttatcat ctacaaccgc gtcccgaaaa ccgcttcaac gtcgtttacc 120 aatattgcgt acgacctgtg cgccaaaaac aaatatcatg tgctgcacat caataccacg 180 aaaaacaatc cggttatgag cctgcaggat caagtccgtt ttgtgaaaaa catcacctct 240 tggaaagaaa tgaaaccggg cttctaccat ggtcacgtta gttatctgga ctttgccaaa 300 ttcggcgtga agaaaaaacc gatctacatc aacgttatcc gtgatccgat cgaacgcctg 360 gtctcttatt actattttct gcgtttcggc gatgactatc gcccgggtct gcgtcgccgt 420 aaacagggtg acaagaaaac ctttgatgaa tgcgtcgcag aaggcggttc agattgtgct 480 ccggaaaaac tgtggctgca gatcccgttt ttctgcggcc atagctctga atgttggaac 540 gtgggttcgc gctgggcaat ggaccaagct aaatacaacc tgatcaacga atattttctg 600 gtgggtgtta ccgaagaact ggaagatttc atcatgctgc tggaagccgc actgccgcgc 660 tttttccgtg gcgcgacgga actgtaccgt accggcaaaa aatctcatct gcgcaaaacc 720 acggagaaaa aactgccgac gaaacagacc attgcgaaac tgcagcaatc cgacatctgg 780 aaaatgg aaa acgaattcta cgaattcgcc ctggaacagt ttcaattcat tcgtgcgcac 840 gccgttcgcg aaaaagatgg tgacctgtat atcctggcac aaaacttctt ctatgaaaaa 900 atctatccga aatcaaat 918 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 20 tacttccaat ccaatcgcga aatcgaacag cgtca 35 <210> 21 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Synthesized Oligonucleotide for primer <400> 21 ttatccactt ccaatatttg atttcggata gattt 35 <210> 22 <211> 1536 <212> DNA <213> Saccharomyces cerevisiae <400> 22 atgcctgctc ctcacggtgg tattctacaa gacttgattg ctagagatgc gttaaagaag 60 aatgaattgt tatctgaagc gcaatcttcg gacattttag tatggaactt gactcctaga 120 caactatgtg atattgaatt gattctaaat ggtgggtttt ctcctctgac tgggtttttg 180 aacgaaaacg attactcctc tgttgttaca gattcgagat tagcagacgg cacattgtgg 240 accatcccta ttacattaga tgttgatgaa gcatttgcta accaaattaa accagacaca 300 agaattgccc ttttccaaga tgatgaaatt cctattgcta tacttactgt ccaggatgtt 360 tacaagccaa acaaaactat cgaagccgaa aaagtcttc a gaggtgaccc agaacatcca 420 gccattagct atttatttaa cgttgccggt gattattacg tcggcggttc tttagaagcg 480 attcaattac ctcaacatta tgactatcca ggtttgcgta agacacctgc ccaactaaga 540 cttgaattcc aatcaagaca atgggaccgt gtcgtagctt tccaaactcg taatccaatg 600 catagagccc acagggagtt gactgtgaga gccgccagag aagctaatgc taaggtgctg 660 atccatccag ttgttggact aaccaaacca ggtgatatag accatcacac tcgtgttcgt 720 gtctaccagg aaattattaa gcgttatcct aatggtattg ctttcttatc cctgttgcca 780 ttagcaatga gaatgagtgg tgatagagaa gccgtatggc atgctattat tagaaagaat 840 tatggtgcct cccacttcat tgttggtaga gaccatgcgg gcccaggtaa gaactccaag 900 ggtgttgatt tctacggtcc atacgatgct caagaattgg tcgaatccta caagcatgaa 960 ctggacattg aagttgttcc attcagaatg gtcacttatt tgccagacga agaccgttat 1020 gctccaattg atcaaattga caccacaaag acgagaacct tgaacatttc aggtacagag 1080 ttgagacgcc gtttaagagt tggtggtgag attcctgaat ggttctcata tcctgaagtg 1140 gttaaaatcc taagagaatc caacccacca agaccaaaac aaggtttttc aattgtttta 1200 ggtaattcat taaccgtttc tcgtgagcaa ttatccattg ctttgttgtc aac attcttg 1260 caattcggtg gtggcaggta ttacaagatc tttgaacaca ataataagac agagttacta 1320 tctttgattc aagatttcat tggttctggt agtggactaa ttattccaaa tcaatgggaa 1380 gatgacaagg actctgttgt tggcaagcaa aacgtttact tattagatac ctcaagctca 1440 gccgatattc agctagagtc agcggatgaa cctatttcac atattgtaca aaaagttgtc 1500 ctattcttgg aagacaatgg cttttttgta ttttaa 1536 <210> 23 <211> 609 <212> DNA <213> Saccharomyces cerevisiae <400> 23 atggctacta atattacttg gcatccaaat cttacttacg acgaacgcaa ggcattgaga 60 aaacaggacg gttgtactat ttggttaaca ggtctaagtg cgtcaggtaa aagtacaatc 120 gcctgtgcgc tagaacagtt actgctccaa aaaaacttgt ctgcatatag attggatggt 180 gacaacattc gttttggatt gaacaaggat ttgggtttct cagaaaagga cagaaatgaa 240 aacattcgta gaattagcga agtttctaag ctatttgctg attcatgtgc tatttcaatc 300 acctcattta tctctccata cagagttgac agagatagag ctcgtgaact acataaggag 360 gctggtttga agttcattga aatatttgtt gatgttccat tagaagtcgc tgagcaaagg 420 gaccctaagg gtttatacaa gaaagctagg gagggtgtaa tcaaggagtt tacaggtatt 480 tctgccccat atgaagcgcc aaaagctc ca gagctacatt tgagaaccga ccagaagacg 540 gttgaagaat gtgctaccat tatttatgag tacttaatca gtgaaaaaat catccgtaag 600 catttgtaa 609 <210> 24 <211> 1695 <212> DNA <213> Homo sapiens <400> 24 gaaaaaagag cagcagcatc tgagagtaac aactatatga accacgtggc caaacaacag 60 tctgaggaag cattccctca ggaacagcag aaagcacccc ctgttgttgg gggcttcaat 120 agcaatgtgg gaagtaaggt gttagggctc aaatatgaag aaattgactg tctcataaat 180 gatgaacaca caattaaagg gagacgagag gggaacgaag tctttcttcc attcacttgg 240 gttgagaaat attttgatgt ttatggaaag gtggttcagt atgatggcta tgatcggttt 300 gaattctctc atagctattc caaagtctat gcacagagag ccccctatca ccccgatggt 360 gtgtttatgt cttttgaagg ctacaatgtg gaagtccgag acagagtcaa gtgcataagt 420 ggggttgaag gtgtgccatt atctacacaa tggggacctc aaggctattt ctatccaatc 480 cagattgcac agtatggatt aagtcattac agcaagaatc taactgagaa acctcctcac 540 atagaggtat atgaaacagc agaagacaga gacaaaaaca agcctaatga ctggactgtg 600 ccaaagggct gctttatggc gaatgtggct gataagtcta gattcaccaa tgtcaaacag 660 tttatgcac cagaaaccag tgaaggtgta tccttgcaac tggga aacac aaaagatttt 720 attatttcat ttgacctcaa gttcttgaca aatggaagtg tgtccgtggt tctagagacc 780 acagaaaaga atcagctctt cactatacat tatgtctcaa atgctcagct aattgctttt 840 aaagaaagag atatatacta tggcattggg cccagaactt catggagcac agttaccagg 900 gacctggtca ctgacctcag gaaaggagtg ggtctttcaa acacaaaagc tgtcaagcca 960 accaaaataa tgcccaagaa ggtggttagg ttgattgcaa aaggtaaggg attcctcgac 1020 aacattacca tctctaccac agcccacatg gctgcatttt ttgctgctag tgattggcta 1080 gtaaggaacc aggatgagaa aggtggctgg ccaattatgg tgacccgtaa gttaggggaa 1140 gggttcaagt ctttagagcc aggatggtat tctgccatgg cccaagggca agccatttct 1200 acattagtca gggcctatct gttaacaaaa gaccatatat tcctcaattc agctttaagg 1260 gcaacagccc cttataagtt tctatctgag cagcatggag ttaaagctgt gtttatgaat 1320 aaacatgact ggtatgaaga atatccaacc acacctagct cttttgtttt aaatggcttt 1380 atgtattctt taattgggct gtatgactta aaagaaactg caggggaaaa actcggaaaa 1440 gaagcaaggt ccttgtatga gcgtggcatg gaatctctta aagccatgct gcccttgtat 1500 gacactggct caggaaccat ctatgacctc cgtcacttca tgcttggcat cgctc ctaac 1560 ctggctcgct gggactatca taccacccac atcaatcagt tgcagctact cagtaccatt 1620 gatgagtccc caatcttcaa agaatttgtc aagaggtgga aaagctacct taaaggcagc 1680 agggcaaagc acaac 1695 <210> 25 <211> 1695 <212> DNA <213> Mus musculus <400> 25 gaaaaaagag cagcagcatc tgagagtaac aactatatga accacgtggc caaacaacag 60 tctgaggaag cattccctca ggaacagcag aaagcacccc ctgttgttgg gggcttcaat 120 agcaatgtgg gaagtaaggt gttagggctc aaatatgaag aaattgactg tctcataaat 180 gatgaacaca caattaaagg gagacgagag gggaacgaag tctttcttcc attcacttgg 240 gttgagaaat attttgatgt ttatggaaag gtggttcagt atgatggcta tgatcggttt 300 gaattctctc atagctattc caaagtctat gcacagagag ccccctatca ccccgatggt 360 gtgtttatgt cttttgaagg ctacaatgtg gaagtccgag acagagtcaa gtgcataagt 420 ggggttgaag gtgtgccatt atctacacaa tggggacctc aaggctattt ctatccaatc 480 cagattgcac agtatggatt aagtcattac agcaagaatc taactgagaa acctcctcac 540 atagaggtat atgaaacagc agaagacaga gacaaaaaca agcctaatga ctggactggg 600 ccaaagggct gctttatggc gaatgtggct gataagtcta gattcaccaa tgtcaaacag 66 0 tttattgcac cagaaaccag tgaaggtgta tccttgcaac tgggaaacac aaaagatttt 720 attatttcat ttgacctcaa gttcttgaca aatggaagtg tgtccgtggt tctagagacc 780 acagaaaaga atcagctctt cactatacat tatgtctcaa atgctcagct aattgctttt 840 aaagaaagag atatatacta tggcattggg cccagaactt catggagcac agttaccagg 900 gacctggtca ctgacctcag gaaaggagtg ggtctttcaa acacaaaagc tgtcaagcca 960 accaaaataa tgcccaagaa ggtggttagg ttgattgcaa aaggtaaggg attcctcgac 1020 aacattacca tctctaccac agcccacatg gctgcatttt ttgctgctag tgattggcta 1080 gtaaggaacc aggatgagaa aggtggctgg ccaattatgg tgacccgtaa gttaggggaa 1140 gggttcaagt ctttagagcc aggatggtat tctgccatgg cccaagggca agccatttct 1200 acattagtca gggcctatct gttaacaaaa gaccatatat tcctcaattc agctttaagg 1260 gcaacagccc cttataagtt tctatctgag cagcatggag ttaaagctgt gtttatgaat 1320 aaacatgact ggtatgaaga atatccaacc acacctagct cttttgtttt aaatggcttt 1380 atgtattctt taattgggct gtatgactta aaagaaactg caggggaaaa actcggaaaa 1440 gaagcaaggt ccttgtatga gcgtggcatg gaatctctta aagccatgct gcccttgtat 1500 gacactggc t caggaaccat ctatgacctc cgtcacttca tgcttggcat cgctcctaac 1560 ctggctcgct gggactatca taccacccac atcaatcagt tgcagctact cagtaccatt 1620 gatgagtccc caatcttcaa agaatttgtc aagaggtgga aaagctacct taaaggcagc 1680 agggcaaagc acaac 1695 <210> 26 <211> 792 <212> DNA <213> Mus musculus <400> 26 ggcacagcat ccaatggttc cacacagcag ctgccacaga ccatcatcat tggggtgcgc 60 aagggtggta cccgagccct gctagagatg ctcagcctgc atcctgatgt tgctgcagct 120 gaaaacgagg tccatttctt tgactgggag gagcattaca gccaaggcct gggctggtac 180 ctcacccaga tgcccttctc ctcccctcac cagctcaccg tggagaagac acccgcctat 240 ttcacttcgc ccaaagtgcc tgagagaatc cacagcatga accccaccat ccgcctgctg 300 cttatcctga gggacccatc agagcgcgtg ctgtccgact acacccaggt gttgtacaac 360 caccttcaga agcacaagcc ctatccaccc attgaggacc tcctaatgcg ggacggtcgg 420 ctgaacctgg actacaaggc tctcaaccgc agcctgtacc atgcacacat gctgaactgg 480 ctgcgttttt tcccgttggg ccacatccac attgtggatg gcgaccgcct catcagagac 540 cctttccctg agatccagaa ggtcgaaaga ttcctgaagc tttctccaca gatcaacgcc 600 tcgaacttct actttaa caa aaccaagggc ttctactgcc tgcgggacag tggcaaggac 660 cgctgcttac acgagtccaa aggccgggcg cacccccagg tggatcccaa actacttgat 720 aaactgcacg aatactttca tgagccaaat aagaaatttt tcaagctcgt gggcagaaca 780 ttcgactggc ac 792 <210> 27 <211> 1170 <212> DNA <213> Escherichia coli <400> 27 atgcaaggta atgcactaac cgttttatta tccggtaaaa aatatctgct attgcagggg 60 ccaatgggac cctttttcag tgatgttgcc gagtggctag agtcattagg tcgtaacgct 120 gtgaatgttg tattcaacgg tggggatcgt ttttactgcc gccatcgaca atacctagct 180 tactaccaga caccgaaaga gtttcccgga tggttacggg atctccaccg gcaatatgac 240 tttgacacaa tcctctgctt tggcgactgc cgcccattgc ataaagaagc aaaacgctgg 300 gcaaagtcga aagggatccg cttcctggca tttgaagaag gatatttacg cccgcaattt 360 attaccgttg aagaaggcgg agtgaacgca tattcatcgc taccgcgcga tccggatttt 420 tatcgtaagt taccagatat gcctacgccg cacgttgaga acttaaaacc ttcaacgatg 480 aaacgtatag gccatgctat gtggtattac ctgatgggct ggcattaccg tcatgagttt 540 cctcgctacc gccaccacaa atcattttcc ccctggtatg aagcacgttg ctgggttcgt 600 gcatactggc gcaagcaact ttacaag gta acacagcgta aggtattacc gaggttaatg 660 aacgaactgg accagcgtta ttatcttgct gttttgcagg tgtataacga tagccagatt 720 cgtaaccaca gcagttataa cgatgtgcgt gactatatta atgaagtcat gtactcattt 780 tcgcgtaaag cgccgaaaga aagttatttg gtgatcaaac atcatccgat ggatcgtggt 840 cacagactct atcgaccatt aattaaacgg ttgagtaagg aatatggctt aggtgagcga 900 atcctttatg tgcacgatct cccgatgccg gaattattac gccatgcaaa agcggtggtg 960 acgattaaca gtacggcggg gatctctgcg ctgattcata acaaaccact caaagtgatg 1020 ggcaatgccc tgtacgacat caagggcttg acgtatcaag ggcatttgca ccagttctgg 1080 caggctgatt ttaaaccaga tatgaaactg tttaagaagt ttcgtgggta tttattggtg 1140 aagacgcagg ttaatgcggt ttattattaa 1170 <210> 28 <211> 717 <212> DNA <213> Escherichia coli <400> 28 atgattgttg caaatatgtc atcataccca cctcgaaaaa aagagttggt gcattctata 60 caaagtttac atgctcaagt agataaaatt aatctttgcc tgaatgagtt tgaagaaatt 120 cctgaggaat tagatggttt ttcaaaatta aatccagtta ttccagataa agattataag 180 gatgtgggca aatttatatt tccttgcgct aaaaatgata tgatcgtact tacagatgat 240 gatattattt accctcc cga ttatgtagaa aaaatgctca atttttataa ttcctttgca 300 atattcaatt gcattgttgg gattcatggc tgtatataca tagatgcatt tgatggagat 360 cagtctaaaa gaaaagtatt ttcatttact caagggctat tgcgaccgag agttgtaaat 420 caattaggta cagggactgt ttttcttaag gcagatcaat taccatcttt aaaatatatg 480 gatggttctc aacgattcgt cgatgttaga ttttctcgct atatgttaga gaatgaaatt 540 ggtatgatat gtgttcccag agaaaaaaac tggctaagag aggtctcatc aggttcaatg 600 gaaggacttt ggaacacatt tacaaaaaaa tggcctttag acatcataaa agaaacacaa 660 gcaatcgcag gatattcaaa acttaacctc gaattagtgt ataatgtgga agggtaa 717 <210> 29 <211> 1689 <212> DNA <213> Escherichia coli <400> 29 atgaataaat tagtgctagt cggacatcct ggctcaaagt atcagatagt tgaacatttt 60 ttgaaagaaa ttggcatgaa ctcaccaaat tattctacaa gtaataaaat ttccccagaa 120 tatatcaccg cttcattatg tcaattttat caaacaccag aagttaatga tgtagtagat 180 gagagagaat tctcagctgt tcaagtctca accatgtggg atagcatggt tcttgaacta 240 atgatgaaca atctaaataa caaactttgg gggtgggcag atccatctat aatatttttt 300 cttgattttt ggaaaaatat agataaaagc ataaaattca tcatga tata tgatcaccct 360 aaatataatt taatgcgttc agtaaataat gcccctctct ctttaaatat aaataatagt 420 gtagataact ggattgcata taataaaaga ttgcttgatt tttttttgga gaataaagaa 480 cgatgtgtgt tgattaattt tgaggcgttt caaagcaata agaaaaatat tataaagcca 540 ttgagtaata ttataaaaat agataatcta atgtctgcgc attacaaaaa ttcaatattg 600 tttgatgtgg ttgagaataa tgattataca aaatcaaatg aaattgccct gcttgaaaaa 660 tatacaactt tattttcttt aagtgcaaat gagactgaaa ttacatttaa tgatacaaag 720 gttagtgagt acttagtatc tgaattaata aaagaaagaa ccgaggttct gaagctttat 780 aatgagttac aagcctatgc aaacctacct tatatagaaa catcgaaaga taacgtttcg 840 gctgaggctg cattatggga ggtagtcgaa gagagaaatt ctatcttcaa tattgtatct 900 catttggtgc aagagtcaaa aaagaaggat gcagatattg aattgactaa atctatattt 960 aagaaaagac aatttttatt attgaacagg attaatgagc taaaaaaaga aaaggaagag 1020 gtaattaaac tttcaaaaat aaatcacaac gatgttgtga gacaagaaaa atatccagat 1080 gatattgaaa aaaaaataaa tgacatacag aaatatgaag aagagataag cgaaaaagaa 1140 tcaaaactca ctcaggcaat atcagaaaaa gaacagattt taaaacaatt gcataaatat One 200 gaagaagaga taagcgaaaa agaatcaaaa ctcactcagg caatatcaga aaaagaacag 1260 attttaaaac aattgcatat agtgcaagag cagttggaac actattttat agaaaatcag 1320 gaaattaaaa agaaacttcc acctgtgcta tatggagcag ctgagcagat aaaacaagag 1380 ttaggttatc gacttggtta tattatagtc tcgtattcta aatccctcaa ggggattatt 1440 accatgccat ttgcacttat ccgtgagtgt gtttttgaaa aaaaacgtaa gaagagttat 1500 ggcgttgatg tgccactcta tttatatgct gatgctgata aggctgaaag agttaagaaa 1560 catttatctt atcaattagg gcaggctatt atctccagtg ctaattcgat atttggattc 1620 attacccttc catttaagtt aattgttgtt gtttataaat ataggagagc taaaatcaag 1680 ggctgttaa 1689 <210> 30 <211 > 1563 <212> DNA <213> Escherichia coli <400> 30 atgaacgcag aatatataaa tttagttgaa cgtaaaaaga aattagggac aaatattggt 60 gctcttgatt ttttattatc aattcataag gagaaagttg atcttcaaca taaaaactcg 120 cctttaaaag gtaacgataa ccttattcac aaaagaataa acgaatacga caatgtactt 180 gaactatcta agaatgtatc agctcagaat tctggcaatg agttttctta tttattggga 240 tatgcagatt ctcttagaaa agttggtatg ttggatactt atattaaaat tgtttgttat 300 ctaacaattc aatctcgtta ttttaaaaat ggcgaacgag ttaagctttt tgaacatata 360 agtaacgctc tacggtattc aaggagtgat tttctcatta atcttatttt tgaacgatat 420 atcgaata ta taaaccatct aaaattgtcg cccaaacaaa aagattttta tttttgtacg 480 aagttttcaa aatttcatga ttatactaaa aatggatata aatatttagc atttgataat 540 caagccgatg cagggtatgg cctgacttta ttattaaatg caaacgatga tatgcaagat 600 agttataatc tactccctga gcaagaactt tttatttgta atgctgtaat agataatatg 660 aatatttata ggagtcaatt taacaaatgt ctacgaaaat acgatttatc agaaataact 720 gatatatacc caaataaaat tatattgcaa ggaattaagt tcgataagaa aaaaaatgtt 780 tatggaaaag atcttgttag tataataatg tcagtattca attcagaaga tactattgca 840 tactcattac attcattgtt gaatcaaaca tatgaaaata ttgaaattct cgtgtgcgat 900 gattgttcat cggacaaaag ccttgaaata attaagagca tagcttattc tgattcaaga 960 gtgaaagtat atagctcacg aaaaaaccaa ggcccttata atataagaaa tgagctaata 1020 aaaaaagcac acggtaattt catcaccttt caagatgcag atgatctttc tcatccggag 1080 agaatacaaa gacaagttga ggttcttcgc aataataagg ctgtaatctg tatggctaac 1140 tggatccgtg ttgcgtcaaa tggaaaaatt caattcttct atgatgataa agccacaaga 1200 atgtctgttg tatcgtcaat gataaaaaaa gatatttttg cgacagttgg tggctataga 1260 caatctttaa ttggtgcaga t acggagttt tatgaaacag taataatgcg ttatgggcga 1320 gaaagtattg taagattact gcagccattg atattggggt tatggggaga ctccggactt 1380 accaggaata aaggaacaga agctctacct gatggatata tatcacaatc tcgaagagaa 1440 tatagtgata tcgcggcaag acaacgagtg ttagggaaaa gtatcgtaag tgataaagat 1500 gtacgtggtt tattatctcg ctatggtttg tttaaagatg tatcaggaat aattgaacaa 1560 tag 1563 <210> 31 <211> 1179 <212> DNA <213> Escherichia coli <400> 31 atgttcggaa cactaaaaat aactgtttca ggcgctggtt acgttgggct ttcaaatgga 60 attctaatgg ctcaaaatca tgaagtggtt gcatttgata cccatcaaaa aaaagttgac 120 ttacttaatg ataaactctc tcctatagag gataaggaaa ttgaaaatta tctttcaact 180 aaaatactta attttcgcgc aactactaac aaatatgaag cctataaaaa tgccaattac 240 gttattattg ctacaccaac gaattatgac ccaggttcaa attactttga tacatcaagc 300 gttgaagctg tcattcgtga cgtaacggaa atcaacccaa acgcaattat ggtggttaaa 360 tctacggtcc cagtaggttt cacaaaaaca attaaagaac atttaggtat taataatatt 420 atcttctctc cagaattttt acgagaagga agagccctat acgataatct ccatccatct 480 cgcattatta tcggtgaatg ttctgaacgg gcaga acgtt tggcagtgtt atttcaggaa 540 ggagcgatta aacaaaatat acccgtttta tttacagatt ctacggaagc ggaagcgatt 600 aagttatttt caaatactta tttggctatg cgagttgcat tttttaatga attggatagt 660 tacgcagaaa gttttggtct gaatacgcgt cagattattg acggtgtttg tttggatccg 720 cgcattggta attactacaa taatccttct tttggttatg gtggctactg tttgccaaaa 780 gataccaagc aattattagc caactatcag tctgttccga ataaacttat atctgcaatt 840 gttgatgcta accgtacacg taaggacttt atcactaatg ttattttgaa acatagacca 900 caagttgtgg gggtttatcg tttgattatg aaaagtggtt cagataattt tagagattct 960 tctattcttg gtattataaa gcgtatcaag aaaaaaggcg tgaaagtaat tatttatgag 1020 ccgcttattt ctggagatac attctttaac tcacctttgg aacgggagct ggcgatcttt 1080 aaagggaaag ctgatattat tatcactaac cgaatgtcag aggagttgaa cgatgtggtc 1140 gacaaagtct atagtcgcga tttgtttaaa tgtgactaa 1179 <210> 32 <211> 855 <212> DNA <213> Escherichia coli <400> 32 atggtactga tgatcgtcag cggacgttca ggttcaggta aatctgtcgc cctgcgtgcg 60 ctggaagata tgggttttta ctgcgtggat aaccttcccg tagtgttgtt acccgatctg 120 gctcgaactc tggccg atcg agagatttct gccgccgtca gcattgatgt tcgtaatatg 180 ccggagtcac cagaaatatt cgaacaggcg atgagtaacc tgcctgacgc tttctcaccg 240 caactactgt tcctggatgc cgaccgtaat accttaattc gtcgttacag tgacacgcgc 300 cgactgcatc cgctttccag caaaaacctg tcgctggaaa gtgctatcga caaagaaagc 360 gatttgctgg agcctctgcg ttcgcgagcg gatctgattg tcgacacctc agaaatgtcc 420 gttcacgagc tggcagaaat gctgcgtacc cgtctgctgg gtaaacgtga acgtgaactg 480 accatggtct ttgagtcttt cggcttcaaa cacggtatcc ctatcgatgc agattacgtc 540 tttgacgtgc gcttcttgcc gaacccgcac tgggatccga aactgcgtcc aatgacaggt 600 cttgataaac ctgtcgccgc gttcctcgac cgccacacag aagtacacaa ttttatctac 660 cagacgcgaa gctatcttga gctatggtta cctatgctgg aaaccaacaa ccgtagctac 720 ctgacggtcg ccattggttg taccggcggg aagcaccgtt cggtgtatat tgcagagcaa 780 ctggcagact acttccgctc gcgcggtaaa aacgtccagt cacgccatcg gacgctggaa 840aaacgtaaac catga 855

Claims (11)

A method for producing a sulfated polysaccharide comprising the following steps (1-1) and (1-2):
(1-1) Transformant (a) or transformation of a bacterium of the genus Corynebacterium , which contains at least a gene encoding ATP sulfurylase and a gene encoding APS kinase, introduced in an expressible manner Preparing a treated product of the conversion product (a); and
(1-2) A step of carrying out a reaction for producing PAPS using a reaction solution containing ATP or an ATP source, a sulfate ion source, and a transformant (a) or a processed product thereof. The method for producing a sulfated polysaccharide according to claim 1, further comprising the following steps (2-1) and (2-2):
(2-1) Transformants (b) or processed products or extracts of transformants (b) of microorganisms belonging to prokaryotes, which contain at least a gene encoding C5-epimerase introduced in an expressible manner manufacturing; and
(2-2) Incorporating the transformant (b) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform C5-epimerization. The method of claim 1 or 2, comprising the step of performing sulfation with a transformant containing a gene encoding a sulfotransferase introduced in an expressible manner or a treated product or extract of the transformant , A method for producing sulfated polysaccharides. The method for producing a sulfated polysaccharide according to claim 3, further comprising the following steps (3-1) and (3-2):
(3-1) a transformant (c) of a microorganism belonging to a prokaryote or a processed product of a transformant (c), which contains at least a gene encoding 2-O-sulfotransferase introduced in an expressible manner; preparing an extract; and
(3-2) Incorporating the transformant (c) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform 2-O-sulfation. The method for producing a sulfated polysaccharide according to claim 3 or 4, further comprising the following steps (3'-1) to (3'-3):
(3'-1) Transformants (b) or processed products or extracts of transformants (b) of microorganisms belonging to prokaryotes, which contain at least a gene encoding C5-epimerase introduced in an expressible manner Preparing;
(3'-2) Transformants (c) or treated products of transformants (c) of microorganisms belonging to prokaryotes, which contain at least a gene encoding 2-O-sulfotransferase introduced in an expressible manner or preparing an extract; and
(3'-3) Transformant (b) or its processed product or extract, and transformant (c) or its processed product or extract were incorporated into the reaction solution in the presence of N-sulfohepharoic acid to form C5-epi performing merization and 2-O-sulfation. The method for producing a sulfated polysaccharide according to any one of claims 3 to 5, further comprising the following steps (4-1) and (4-2):
(4-1) Transformants (d) or treated products of transformants (d) of microorganisms belonging to prokaryotes, which contain at least a gene encoding 6-O-sulfotransferase introduced in an expressible manner, or preparing an extract; and
(4-2) A step of incorporating the transformant (d) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform 6-O-sulfation. The method for producing a sulfated polysaccharide according to any one of claims 3 to 6, further comprising the following steps (5-1) and (5-2):
(5-1) Transformants (e) or treated products of transformants (e) of microorganisms belonging to prokaryotes, which contain at least a gene encoding 3-O-sulfotransferase introduced in an expressible manner, or preparing an extract; and
(5-2) Incorporating the transformant (e) or a treated product or extract thereof into a reaction solution in the presence of N-sulfohepharoic acid to perform 3-O-sulfation. As a method for producing a sulfated polysaccharide,
Transformant (a) or treatment of transformant (a) of a bacterium of the genus Corynebacterium comprising at least a gene encoding ATP sulfurylase and a gene encoding APS kinase, introduced in an expressible manner And, incorporating at least one selected from the following into a reaction solution in the presence of ATP or an ATP source, a sulfate ion source, and N-sulfoheparoic acid to produce a sulfated polysaccharide, wherein the production of a sulfated polysaccharide Way:
Transformants (b) or treated products or extracts of transformants (b) of microorganisms belonging to prokaryotes, which contain at least a gene encoding C5-epimerase introduced in an expressible manner;
Transformants (c) or processed products or extracts of transformants (c) of microorganisms belonging to prokaryotes, which contain at least a gene encoding 2-O-sulfotransferase introduced in an expressible manner;
A transformant (d) or a treated product or extract of a transformant (d) of a microorganism belonging to a prokaryote, which contains at least a gene encoding 6-O-sulfotransferase introduced in an expressible manner, and
A transformant (e) or a treated product or extract of a transformant (e) of a microorganism belonging to a prokaryote, which contains at least a gene encoding 3-O-sulfotransferase introduced in an expressible manner. The method of claim 8, wherein the transformant (a) or a treated product thereof and the transformants (b) to (e) or a treated product or extract thereof are prepared from ATP or an ATP source, a sulfate ion source and N-sulfoheparoic acid A method for producing a sulfated polysaccharide, further comprising the step of generating a sulfated polysaccharide by incorporating into the reaction solution in the presence of 10. The method for producing a sulfated polysaccharide according to any one of claims 1 to 9 for producing heparin. A process for preparing PAPS, comprising the following steps (i) and (ii):
(i) Preparation of a bacterial transformant of the genus Corynebacterium or a treatment of the transformant, comprising at least a gene encoding ATP sulfurylase and a gene encoding APS kinase, introduced in an expressible manner doing; and
(ii) carrying out a reaction for producing PAPS using a reaction solution containing ATP or an ATP source, a sulfate ion source, and the transformant prepared in step (i) or a processed product thereof.

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