Protein having n-sulfotransferase activity and DNA encoding said protein

Abstract

The purpose of the present invention is to provide a means for producing a non-animal-derived modified heparosan compound with higher efficiency than conventional means. Provided is any one of the following proteins [1] to [3] fused to a TRX tag. [1] A protein which comprises the amino acid sequence represented by SEQ ID NO: 1. [2] A protein which comprises an amino acid sequence having a structure wherein 1 to 20 amino acid residues are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1 and which has N-sulfotransferase activity. [3] A protein which comprises an amino acid sequence having 70% or more identity to the amino acid sequence represented by SEQ ID NO: 1 and has N-sulfotransferase activity.

Description

Protein having N-sulfotransferase activity and DNA encoding the protein

[0001] The present invention relates to a protein having N-sulfotransferase activity, DNA encoding the protein, a microorganism containing the DNA, and a method for producing a modified heparosan compound using the protein.

[0002] A modified heparosan compound is a compound obtained by chemically or enzymatically modifying heparosan. Heparosan is a polysaccharide composed of a repeating structure of a disaccharide consisting of an uronic acid residue and an N-acetylglucosamine residue. As modified heparosan compounds, for example, naturally occurring polysaccharides such as heparin and heparan sulfate are known, and these are mainly present in animal tissues and extracellular matrices. A modified heparosan compound is imparted with specific properties and functions by changing the molecular structure of heparosan, for example, by chemical modification or modification by an enzymatic reaction. Modified heparosan compounds play an important role as anticoagulants, etc. particularly in the pharmaceutical field, and are also applied to cosmetics, food additives, and agricultural uses.

[0003] Heparin is a kind of sulfated modified heparosan compound and is used as an anticoagulant for the treatment of thromboembolic diseases, disseminated intravascular coagulation syndrome, and prevention of coagulation in hemodialysis and extracorporeal circulation. Industrially, heparin extracted and purified mainly from porcine intestinal mucosa is used. Since a fatal accident occurred in 2008 due to contamination of porcine-derived heparin with impurities, the need for non-animal-derived heparin with controlled production and quality has been increasing (Non-Patent Document 1).

[0004] So far, a method for producing heparin having the same structure and anticoagulant activity as porcine-derived products by deacetylating and sulfating N-acetyl heparosan (hereinafter referred to as heparosan), which is a capsular polysaccharide of a microorganism, by chemical and enzymatic methods has been reported (Patent Documents 1 and 2).

[0005] Specifically, a method for producing non-animal heparin is known in which the substrate heparosan is subjected to (1) N-deacetylation and N-sulfation by chemical or enzymatic methods, (2) epimerization of glucuronic acid residues to iduronic acid residues by C5 epimerase, (3) 2-O-sulfation of uronic acid residues at the 2-position by 2-O-sulfotransferase (hereinafter referred to as 2OST), (4) further 6-O-sulfation by 6-O-sulfotransferase (hereinafter referred to as 6OST), and (5) further 3-O-sulfation by 3-O-sulfotransferase (hereinafter referred to as 3OST) (Patent Documents 1 and 2 and Non-Patent Document 2).

[0006] Regarding the reaction described in (1) above, Patent Document 2 discloses a method that includes chemically N-deacetylating and N-sulfating heparosan using reagents.

[0007] U.S. Patent No. 8,771,995, International Publication No. 2018 / 048973

[0008] Natural Product Reports, 2009, 26(3), 313-321 Proc Natl Acad Sci USA. , 2024; 121(14): e2315586121. J. Biol. Chem. , 274(16), 1999, 10673-10676

[0009] As mentioned above, there is a need for non-animal-derived production methods for modified heparosan compounds, including heparin, with controlled manufacturing and quality. Patent Document 2 discloses a method that includes chemically N-deacetylating and N-sulfating heparosan, followed by conversion to heparin using isomerase and sulfatease enzymes, but it has not yet been put into practical use. The reagents used for the N-sulfation reaction of heparosan in this process are expensive, so if the N-sulfation reaction can be carried out more cheaply and efficiently, modified heparosan compounds can be produced with higher efficiency than conventional methods.

[0010] Non-patent document 3 discloses the N-sulfotransferase (NST) domain of N-deacetylase N-sulfotransferase (NDST) as an enzyme responsible for N-sulfation. The document discloses a method for expressing only the NST domain of NDST in E. coli, but this is for the purpose of crystal structure analysis and does not disclose a method for producing heparin using NST.

[0011] Non-patent document 3 describes the use of a GST (glutathione-S-transferase) tag to efficiently express NST. While tag fusion to proteins is a commonly used technique to improve protein solubility and efficient expression, selecting the optimal tag is not easy due to the need to choose the right tag for the target protein and the existence of numerous tag types. The optimal tag for efficiently expressing NST in microorganisms such as E. coli has not yet been identified.

[0012] Based on the above, this disclosure aims to provide a means for producing non-animal-derived modified hepalosan compounds with higher efficiency than conventional methods.

[0013] As a result of investigating the aforementioned problems, the inventors of the present invention discovered that by expressing a protein with a specific tag attached to the NST domain in microorganisms, a protein capable of exhibiting higher N-sulfotransferase activity than conventional proteins can be obtained, and thus the present invention was conceived.

[0014] In other words, the present disclosure is as follows: 1. A protein fused with a TRX tag, one of the following [1] to [3]: [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 1. [2] A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1, and which has N-sulfotransferase activity. [3] A protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and which has N-sulfotransferase activity. 2. DNA encoding the protein described in 1 above. 3. DNA consisting of the base sequence represented by SEQ ID NO: 12 or its complementary sequence. 4. A microorganism containing the DNA described in 2 or 3 above. 5. A method for producing a modified heparosan compound, comprising the step of reacting a heparosan compound with at least one protein selected from the following [1] to [3], fused with a TRX tag: [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 1. [2] A protein having N-sulfotransferase activity, comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 1. [3] A protein having N-sulfotransferase activity, comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by Sequence ID No. 1. 6. The method for producing the product according to 5, wherein at least one protein selected from [1] to [3], to which a TRX tag has been fused, is produced by a microorganism containing the following DNA: (i) DNA encoding at least one protein selected from [1] to [3], to which a TRX tag has been fused; (ii) DNA consisting of the base sequence represented by Sequence ID No. 12 or its complementary sequence. 7. The method for producing the product according to 5, wherein the heparosan compound is deacetylated heparosan. 8. The method for producing the product according to 5, wherein the modified heparosan compound is heparin.

[0015] The protein disclosed herein has a TRX tag attached to its NST domain, resulting in superior N-sulfotransferase activity compared to conventional proteins. By using the protein disclosed herein, the N-sulfation reaction of N-deacetylated heparosan can be efficiently carried out, enabling the highly efficient production of modified heparosan compounds using non-animal-derived methods.

[0016] The present invention will be described below based on embodiments, but the present invention is not limited to these embodiments.

[0017] 1. Protein The protein of this disclosure is a protein fused with a TRX tag and is one of the following [1] to [3]: [1] A protein consisting of the amino acid sequence represented by Sequence ID No. 1. [2] A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 1, and which has N-sulfotransferase activity. [3] A protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence represented by Sequence ID No. 1, and which has N-sulfotransferase activity.

[0018] A TRX (thioredotoxin) tag is a tag consisting of a thioredotoxin protein. While known proteins can be used as the TRX tag, for example, a protein consisting of the amino acid sequence represented by SEQ ID NO: 14, encoded by the nucleotide sequence represented by SEQ ID NO: 11, can be used. In this disclosure, "TRX tag fused to a protein" means that the TRX tag is attached to the protein via a peptide bond. In the protein of this disclosure, the TRX tag is preferably located at the N-terminus or C-terminus, more preferably at the N-terminus. Furthermore, an arbitrary amino acid sequence consisting of 1 to 10 amino acid residues may be included as a linker between the TRX tag and the protein. The amino acid sequence of the linker is not particularly limited, but an example is glycine-serine. The protein of this disclosure, being a protein in which a TRX tag is fused to any one of the proteins [1] to [3] above, can exhibit excellent N-sulfotransferase activity.

[0019] Sequence ID 1 is the amino acid sequence of the N-sulfotransferase region (L558-R882) of human (Homo sapiens) N-deacetylase and N-sulfotransferase (NDST1) (UniProtKB Accession No. P52848.1), and is encoded by the nucleotide sequence represented by Sequence ID 2.

[0020] In [2] above, the number of deleted, substituted, inserted and / or added amino acid residues in the amino acid sequence represented by Sequence ID No. 1 is 1 to 20, preferably 1 to 15, more preferably 1 to 10, and even more preferably 1 to 5, in that order. The amino acid residue mutation may be introduced into one region of the amino acid sequence, or into multiple different regions.

[0021] An amino acid sequence in which amino acids are deleted, substituted, added, or inserted refers to an amino acid sequence obtained by artificially deleting or substituting amino acid residues in the original amino acid sequence, or by artificially adding or inserting amino acid residues into said amino acid sequence.

[0022] The amino acids deleted, substituted, added, or inserted may be either native or unnatural forms. Examples of native amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0023] The following are examples of mutually substitutable amino acids. Amino acids within the same group are mutually substitutable. • Group A: Hydrophobic amino acids • Group B: Acidic amino acids • Group C: Polar amino acids • Group D: Basic amino acids • Group E: Secondary amino acids • Group F: Amino acids with a hydroxyl group • Group G: Aromatic amino acids • Group H: Sulfur-containing amino acids

[0024] More specifically, they are as follows: Group A: Leucine, Isoleucine, Norleucine, Valine, Norvaline, Alanine, 2-Aminobutanoic acid, Methionine, O-Methylserine, t-Butylglycine, t-Butylalanine, Cyclohexylalanine Group B: Aspartic acid, Glutamic acid, Isoaspartic acid, Isoglutamic acid, 2-Aminoadipic acid, 2-Aminosveric acid Group C: Asparagine, Glutamine Group D: Lysine, Arginine, Ornithine, 2,4-Diaminobutanoic acid, 2,3-Diaminopropionic acid Group E: Proline, 3-Hydroxyproline, 4-Hydroxyproline Group F: Serine, Threonine, Homoserine Group G: Phenylalanine, Tryptophan, Tyrosine

[0025] In the above [3], the identity with the amino acid sequence represented by Sequence ID No. 1 is 70% or more, preferably 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0026] In this disclosure, the percentage of sequence identity between two amino acid sequences or two nucleotide sequences is calculated as the ratio of matching residues when the two sequences are aligned to the extent that they are most identical. For example, the percentage of sequence identity can be determined using a mathematical algorithm.

[0027] Examples of such mathematical algorithms include the local homology algorithm of Smith et al (1981) Adv. Appl. Math. 2:482, the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453, the similarity search method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448, and Karlin and Altschul (1993) Proc. Natl. Acad. Sci. Examples include the improved Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264 algorithm, as described in USA 90:5873-5877. However, mathematical algorithms are not limited to the above examples.

[0028] These mathematical algorithms can be used to perform alignment to determine the percentage of sequence identity. This program can be executed by a computer as needed. Such programs are not limited to, but include the PC / Gene programs CLUSTAL (available from Intelligenetics, Mountain View, Calif.), MAFFT (Katoh, K., Misawa, K., Kuma, K., & Miyata, T. (2002), 30(14), 3059-3066., http: / / mafft.cbrc.jp / alignment / server / ), MUSCLE (Edgar R. C. (2004). Nucleic Acids Research, 32(5)), 1792–1797. (http: / / www.ebii.ac.uk / Tools / msa / muscle / ), BLAST, FASTA, and TFASTA are examples.

[0029] Alignment using these programs can be performed, for example, using initial parameters. CLUSTAL programs are described in Higgins et al. (1988) Gene 73:237-244, Higgins et al. (1989) CABIOS 5:151-153, Corpet et al. (1988) Nucleic Acids Res. 16:10881-90, Huang et al. (1992) CABIOS 8:155-65, and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.

[0030] BLAST is described in Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). 215(3), 403-410., Mount D. W. (2007). CSH protocols, 2007, pdb. top17., etc. Based on BLAST, programs called BLASTP and BLASTN have been developed, and the percentage of sequence identity can be calculated using these programs with default settings.

[0031] In this disclosure, "N-sulfotransferase activity" refers to the sulfate group (SO ３ This is a reaction that transfers a specific molecule to the amino group of a particular molecule. An example of such a particular molecule is the glucosamine residue in N-deacetylated heparosan.

[0032] The N-sulfotransferase activity can be evaluated by a method comprising the following steps (i) to (iii) in order, as described later in the examples. (i) Using the target protein, an enzymatic reaction is carried out with N-deacetylated heparosan as a substrate in the presence of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to produce N-sulfated heparosan. (ii) Next, referring to Proc Natl Acad Sci U S A. 2024 Apr 2;121(14):e2315586121. (Non-patent Literature 2), the N-sulfated heparosan obtained in step (i) is decomposed using heparinase to obtain an unsaturated disaccharide. (iii) The N-sulfotransferase activity of the target protein with respect to glucosamine residues in heparosan is evaluated by analyzing the unsaturated disaccharide obtained in step (iii) and confirming the proportion of N-sulfated glucosamine residues (ΔUA-GlcNS).

[0033] In [2] and [3] above, "having N-sulfotransferase activity" means that ΔUA-GlcNS is detected in the analysis in procedure (iii) above.

[0034] The proteins of this disclosure may be fusion proteins linked to other heterologous regions via peptide bonds in addition to the TRX tag. Examples of such heterologous regions include peptide components that improve the solubility of the target protein (e.g., Nus-tag), peptide components that act as chaperones (e.g., trigger factors), peptide components with other functions (e.g., full-length proteins or parts thereof), and linkers.

[0035] Examples of the amino acid sequences in [2] and [3] above include amino acid sequences of natural proteins, naturally occurring homologs thereof, or artificially produced mutant or homologous proteins. Mutant or homologous proteins can be obtained, for example, by introducing a mutation into the DNA encoding the target protein and producing the protein using the resulting mutant DNA. Examples of mutagenesis methods include site-directed mutagenesis and random mutagenesis (e.g., treatment with mutagens and ultraviolet irradiation).

[0036] 2. DNA The DNA of the Disclosure is DNA that encodes the protein of the Disclosure as described above. The DNA of the Disclosure may constitute an expression unit. One embodiment of the DNA that encodes the protein of the Disclosure is DNA that encodes a protein having N-sulfotransferase activity, comprising the nucleotide sequence represented by Sequence ID No. 12, its complementary sequence, or homologous sequences thereof.

[0037] Examples of DNA encoding a protein having N-sulfotransferase activity, consisting of homologous sequences of the base sequence represented by Sequence ID No. 12 or its complementary sequence, include (d1) and (d2) below: (d1) DNA encoding a protein having N-sulfotransferase activity, consisting of a base sequence that preferably has 60% or more identity with the base sequence represented by Sequence ID No. 12 or its complementary sequence, more preferably 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, even more preferably 97% or more, 98% or more, and most preferably 99% or more identity with the base sequence represented by Sequence ID No. 12 or its complementary sequence, and (d2) DNA encoding a protein having N-sulfotransferase activity, hybridizing under stringent conditions with the base sequence represented by Sequence ID No. 12 or its complementary sequence.

[0038] "Hybridizing" refers to the hybridization of complementary DNA with DNA having a specific base sequence, or a portion of such DNA. Therefore, DNA having this specific base sequence, or a portion of such DNA, can be used as a probe for Northern or Southern blot analysis, and can also be used as oligonucleotide primers for PCR analysis.

[0039] Examples of DNA used as a probe include DNA with at least 100 bases, preferably 200 bases, and more preferably 500 bases. Examples of DNA used as a primer include DNA with at least 10 bases, preferably 15 bases.

[0040] Methods for DNA hybridization experiments are well known. For example, the conditions for hybridization can be determined and experiments can be conducted according to Molecular Cloning, 4th Edition (Cold Spring Harbor Laboratory Press (2012)), Methods for General and Molecular Bacteriology (ASM Press (1994)), Immunology methods manual (Academic press (1997)), and many other standard textbooks.

[0041] Also, DNA that hybridizes under stringent conditions can be obtained by following the instructions attached to commercially available hybridization kits. Commercially available hybridization kits include, for example, the Random Prime DNA Labeling Kit (manufactured by Roche Diagnostics) that prepares a probe by the random prime method and performs hybridization under stringent conditions.

[0042] Examples of the stringent conditions include incubating a filter immobilized with DNA and probe DNA overnight at 42°C in a solution containing 50% formamide, 5×SSC (750 mM sodium chloride, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg / l of denatured salmon sperm DNA, and then washing the filter in a 0.2×SSC solution at about 65°C.

[0043] The various conditions described above can also be set by adding or changing blocking reagents used to suppress the background of hybridization experiments. The addition of the blocking reagents described above may be accompanied by changes in hybridization conditions to adapt the conditions.

[0044] As the DNA capable of hybridizing under the stringent conditions, for example, when calculated based on the above parameters using BLAST, FASTA, etc., there is DNA having at least 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity with the DNA consisting of the base sequence represented by SEQ ID NO: 12.

[0045] The DNA described in the above (d1) or (d2) can be obtained, for example, by subjecting DNA consisting of the base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 or its complementary sequence, or DNA consisting of the base sequence represented by SEQ ID NO: 12 or its complementary sequence to error-prone PCR or the like using it as a template.

[0046] Also, the DNA described in the above (d1) or (d2) can also be obtained by PCR [Gene, 77, 51 (1989)] using a set of PCR primers each having a base sequence designed so that the target mutation (deletion, substitution, insertion or addition) is inserted at the 5'-end.

[0047] That is, a sense primer corresponding to the 5'-end of DNA consisting of the base sequence encoding the amino acid sequence represented by SEQ ID NO: 1 or its complementary sequence, or DNA consisting of the base sequence represented by SEQ ID NO: 12 or its complementary sequence, and an antisense primer corresponding to the sequence immediately before (5'-side) the mutation-introducing site, having a sequence complementary to the mutated sequence at the 5'-end, are used to perform PCR using this DNA as a template to amplify fragment A (with the mutation introduced on the 3'-side) from the 5'-end to the mutation-introducing site of this DNA.

[0048] Next, a sense primer corresponding to the sequence immediately after (3'-side) the mutation-introducing site, having the mutated sequence at the 5'-end, and an antisense primer corresponding to the 3'-end of this DNA are used to perform PCR using this DNA as a template to amplify fragment B from the mutation-introducing site to the 3'-end of this DNA with the mutation introduced at the 5'-end.

[0049] After purifying these amplified fragments, if they are mixed and PCR is performed without adding templates or primers, the sense strand of amplified fragment A and the antisense strand of amplified fragment B hybridize because they share a common mutation site. This hybridizes the DNA, which then acts as both a primer and a template, allowing the PCR reaction to proceed and amplifying the mutated DNA.

[0050] In this specification, “expression unit” means the smallest unit that enables the transcription of a given DNA to be expressed as a protein and, consequently, the production of the protein encoded by that DNA, including a promoter operably linked to it. An expression unit may further include elements such as a terminator, a ribosome binding site, and a drug resistance gene.

[0051] The expression units may be homogeneous or heterogeneous with respect to the host cell. The term "heterogeneous expression units" means that the expression units are heterogeneous with respect to the host cell. In this disclosure, one or both of the polynucleotide encoding the target protein or the promoter are derived from or artificially synthesized from organisms other than host cells (e.g., prokaryotes, eukaryotes, and animals such as microorganisms, insects, plants, and mammals) or viruses. Alternatively, the polynucleotide encoding the target protein may be heterogeneous with respect to the host cell. Preferably, the target protein is heterogeneous with respect to the host cell.

[0052] The promoter constituting the heterologous expression unit is not particularly limited as long as it can express the protein encoded by the polynucleotide linked downstream in the host cell. For example, the promoter may be homogeneous or heterologous to the host cell. For example, a promoter commonly used for recombinant protein production or an inducible promoter can be used. Examples of such promoters include the PhoA promoter, PhoC promoter, T7 promoter, T5 promoter, T3 promoter, lac promoter, trp promoter, ilv promoter, trc promoter, tac promoter, PR promoter, PL promoter, SP6 promoter, arabinose-inducible promoter, cold shock promoter, and tetracycline-inducible promoter. Alternatively, a derepressive ilv promoter (Pilv*) with improved transcription levels may be used by deleting the terminator region upstream of the ilv promoter. Preferably, a promoter with potent transcriptional activity in the host cell can be used. Examples of promoters with potent transcriptional activity in the host cell include promoters of genes highly expressed in the host cell and virus-derived promoters.

[0053] 3. Microorganisms The microorganisms of this disclosure include the DNA of this disclosure as described above. The microorganisms of this disclosure can be obtained, for example, by introducing an expression unit containing the DNA of this disclosure into a host cell and transforming it.

[0054] In this disclosure, the host cell is preferably a bacterium belonging to the genera Escherichia (e.g., Escherichia coli), Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas, more preferably Escherichia, and particularly preferably Escherichia coli.

[0055] Examples of Escherichia choli include, but are not limited to, Escherichia choli Origami B (DE3) (Novagen), Escherichia choli BL21 codon plus, Escherichia choli XL1-Blue, Escherichia choli XL2-Blue (all manufactured by Agilent Technologies), Escherichia choli BL21 (DE3) pLysS (Merck Millipore), Escherichia choli BL21, Escherichia choli DH5α, Escherichia choli HST08 Premium, and Escherichia choli HST02, Escherichia coli HST04 dam － / dcm － Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, Escherichia coli MV1184, Escherichia coli TH2 (all manufactured by Takara Bio), Escherichia coli W (ATCC9637), Escherichia coli B (ATCC23226), Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG1655, Escherichia Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli coli K5, Escherichia coli Nissle 1917, and the like.

[0056] The host cells in this disclosure may be microorganisms that inherently possess the ability to produce heparosan compounds, or they may be modified to possess the ability to produce heparosan compounds. Microorganisms that possess the ability to produce heparosan compounds can be obtained, for example, by conferring the ability to produce heparosan compounds to the aforementioned microorganisms.

[0057] The ability to produce heparosan compounds can be conferred by introducing a gene encoding a protein involved in heparosan production, referring to Metabolic Engineering, 2012, 14, pp. 521-527, Carbohydrate Research, 2012, 360, pp. 19-24, and U.S. Patent No. 9,975,928, etc. Furthermore, a gene encoding N-deacetylase / N-sulfotransferase (NDST) can also be introduced, referring to PCT / JP2024 / 019404, etc.

[0058] Proteins involved in the production of heparosan compounds include glycosyltransferases and heparosan efflux carrier proteins. In the host cells described herein, one gene may be introduced, or two or more genes may be introduced. Gene introduction can also be achieved by introducing a vector containing the gene into the host. For example, a DNA fragment containing the target gene can be linked to a vector that functions in the host to construct an expression vector for the target gene, and the copy number of the target gene can be increased by transforming the host with this expression vector. The DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism having the target gene as a template. The method of transformation is not particularly limited, and conventionally known methods can be used.

[0059] The microorganisms of this disclosure can be produced by any method known in the art. For example, the expression units described above are contained in host cells in a form integrated into the host cell's genomic DNA or in a form not integrated into the host cell's genomic DNA (e.g., in the form of an expression vector). Host cells containing the expression units can be obtained by transforming host cells with an expression vector by any method known in the art (e.g., competent cell method, electroporation).

[0060] If the expression vector is an integrative vector that undergoes homologous recombination with the host cell's genomic DNA, the expression units can be incorporated into the host cell's genomic DNA through transformation.

[0061] On the other hand, if the expression vector is a non-integrating vector that does not undergo homologous recombination with the host cell's genomic DNA, the expression units are not incorporated into the host cell's genomic DNA by transformation and can exist independently of the genomic DNA within the host cell in the state of the expression vector. Alternatively, the expression units can be incorporated into the host cell's genomic DNA using genome editing technology [e.g., the CRISPR / Cas system, or Translation Activator-Like Effector Nucleases (TALEN)].

[0062] The expression vectors in this disclosure may further include, in addition to the minimum units described above, elements such as terminators that function in host cells, ribosome binding sites, and drug resistance genes. Examples of drug resistance genes include resistance genes to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0063] The microorganisms of this embodiment may have an expression unit that includes a configuration in which a TRX tag (thioredoxin tag) is fused to the N-terminus of NST (N-sulfotransferase) in order to efficiently express NST. The TRX tag is known to contribute to improved solubility and increased expression levels, and examples of host cells include Escherichia coli Origami B (DE3) (Novagen), which has an oxidative cytoplasmic environment. For NST expression, for example, an expression system using a pMALcx vector can be used, and by inserting the TRX tag-fused NST gene into the vector, highly efficient expression becomes possible. Furthermore, co-expression of the GroES-GroEL chaperone promotes accurate folding and maintenance of activity. Transformants obtained with these configurations can produce soluble and active NST protein by culturing under IPTG induction.

[0064] The expression vector may also further include a region that enables homologous recombination with the host cell's genome for homologous recombination with the host cell's genomic DNA. For example, the expression vector may be designed so that the expression units contained therein are located between a pair of homologous regions (e.g., homology arms, loxP, FRT) homologous to a specific sequence in the host cell's genome. The host cell genomic region to which the expression units should be introduced (the target of the homologous region) is not particularly limited, but may be a locus of a gene that is highly expressed in the host cell.

[0065] The expression vector may be a plasmid, viral vector, phage, or artificial chromosome. The expression vector may also be an integrative or non-integrative vector. An integrative vector may be a vector that is entirely integrated into the host cell's genome. Alternatively, an integrative vector may be a vector in which only a portion (e.g., an expression unit) is integrated into the host cell's genome.

[0066] The expression vector may also be a DNA vector or an RNA vector (e.g., a retrovirus). A commonly used expression vector may also be used. Examples of such expression vectors include pMAL (e.g., pMALcx, pMALcx2), pET (e.g., pET-His6-MBP-TEV-LIC), pCDFDuet-1, pTrc99A, pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), and pQE (e.g., pQE30), as well as their derivatives. Furthermore, when selecting Corynebacterium glutamicum as host cells, high-copy vectors such as pPK4, pCS299P, and pRI109 can be used.

[0067] One embodiment of the microorganisms of this disclosure is a host cell comprising an expression unit including DNA encoding the protein of this disclosure and a promoter operably linked thereto.

[0068] The microorganisms of this embodiment may have reduced or deleted expression of thioredoxin reductase, glutathione reductase, etc., for the purpose of enhancing the expression of the proteins of this disclosure. Such microorganisms can be produced by any method known in the art.

[0069] In bacteria of the genus Escherichia, the trxB gene is an example of a gene encoding thioredoxin reductase, and the gor gene is an example of a gene encoding glutathione reductase.

[0070] The nucleotide sequences of the trxB and gor genes, and the amino acid sequences of the proteins encoded by these genes, can be obtained from public databases. For example, the nucleotide sequence of the trxB gene of Escherichia koli BL21 strain is registered as nucleotides 906603 to 907568 in Genbank Accession No. CP053601.1, and the amino acid sequence is registered as Genbank Accession No. QJZ03384.1. Similarly, the nucleotide sequence of the gor gene of Escherichia koli BL21 strain is registered as nucleotides 3477850 to 3479202 in Genbank Accession No. CP060121.1, and the amino acid sequence is registered as Genbank Accession No. It is registered as QNG34301.1.

[0071] Furthermore, in microorganisms in which the expression of thioredoxin reductase, glutathione reductase, or both is reduced or deleted, one or more amino acid mutations may be introduced into the amino acid sequence of the endogenous alkylhydroperoxide reductase C (AhpC) for the purpose of improving the deterioration of growth caused by such modification.

[0072] When using Escherichia coli BL21 strain as the host, the amino acid sequence of AhpC is the amino acid sequence of NCBI Reference Sequence WP_160515916.1 (SEQ ID NO: 15). In this amino acid sequence, amino acid mutations that can improve growth include, for example, the deletion of the 37th amino acid residue and the substitution of the 39th amino acid residue with an L-aspartic acid residue. Such amino acid mutations can be introduced by any method known in this field.

[0073] 4. Method for Producing Modified Heparosan Compounds The method for producing modified heparosan compounds according to this disclosure (hereinafter also referred to as "this production method") is characterized by including the action of the protein described above on the heparosan compound. That is, this production method includes the step of including the action of at least one protein selected from the following [1] to [3], to which a TRX tag has been fused, on the heparosan compound. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO: 1. [2] A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1, and which has N-sulfotransferase activity. [3] A protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and which has N-sulfotransferase activity.

[0074] A preferred embodiment of this manufacturing method includes the following conditions: • It is preferable to use N-deacetylated heparosan as the substrate. • It is preferable to perform N-sulfation in the presence of TRX-tagged NST. • For the sulfate group donor (PAPS), in addition to external addition, it is preferable to supply it using a processed product or culture supernatant of a microorganism capable of producing PAPS. • The N-sulfation step (s3) can be combined with C5 epimerization (s4), 2-O-sulfation (s5), 6-O-sulfation (s6), and 3-O-sulfation (s7) simultaneously or sequentially. For example, when producing heparin, a one-pot reaction is preferred in which all the enzymes required for steps (s3) to (s7) are reacted simultaneously in the same reaction solution.

[0075] This manufacturing method includes N-sulfating a heparosan compound with the protein disclosed herein. Since the protein disclosed herein has excellent N-sulfotransferase activity, this manufacturing method allows for the efficient N-sulfation of N-deacetylated heparosan, even when using heparosan derived from microorganisms, thereby enabling the production of modified heparosan compounds more efficiently by a non-animal method.

[0076] The proteins used in this manufacturing method may be from the microorganisms of this disclosure, or may be extracted and purified from such microorganisms. One embodiment of this manufacturing method preferably includes a step of reacting a heparosan compound with at least one protein selected from [1] to [3], which is produced by a microorganism containing DNA encoding any one of the proteins in [1] to [3] above, or DNA consisting of the base sequence represented by SEQ ID NO: 12 or its complementary sequence.

[0077] This manufacturing method is carried out in the presence of a sulfate group donor. 3'-phosphoadenosine-5'-phosphosulfate (PAPS) is preferred as the sulfate group donor. The sulfate group donor may be a commercially available product or one obtained through appropriate manufacturing. The method for producing the sulfate group donor is not particularly limited; for example, known methods can be used.

[0078] In this specification, "to act" means bringing one of the proteins [1] to [3] fused with a TRX tag (a fusion protein containing an NST domain having N-sulfotransferase activity) into contact with a heparosan compound (heparosan or its derivative) in the same reaction system so that it can catalyze the compound. The manner of contact is not limited and includes the following: - Contact by mixing and stirring in a solution system (batch, fed-add, or continuous) - Contact by flow or immersion between the protein immobilized on a solid support (solid-phase protein) and the substrate solution - Contact by coexistence with extracts, crude products, culture supernatants derived from microorganisms containing the protein, or microorganisms expressing the protein (live cells, resting cells, membrane fractions, solubilized fractions) or processed products of the microorganism described later - Simultaneous and sequential reactions (when present simultaneously with other enzymes, or when the N-sulfation step by the protein is carried out sequentially with preceding and succeeding steps) The term "action" here includes setting conditions such as temperature, pH, ionic strength, cofactor concentration, and reaction time that enable the catalytic reaction (N-sulfation) to proceed, which transfers sulfate groups to the amino groups of glucosamine residues in heparosan compounds under the influence of a sulfate group donor such as PAPS.

[0079] In this specification, "in the presence of" means a state in which the protein (TRX-tagged fusion NST) and the components necessary for the reaction to proceed (e.g., sulfate group donor PAPS, buffer, divalent metal ions, etc.) are present in the reaction system (e.g., solution, suspension system, immobilized reactor, culture system) in a form and concentration that allows catalytic activity to be expressed. "In the presence of" includes the following embodiments: - Embodiments by adding purified protein, crude extract, culture supernatant, immobilized protein, or tagged fusion protein - Embodiments by introducing microorganisms expressing the protein (live or resting cells) or processed products of such microorganisms described later into the reaction system (utilizing intracellular or membrane-localized enzymatic activity) - Embodiments by adding PAPS from an external source, or by supplying it in combination with processed products or culture systems of microorganisms capable of producing PAPS - Embodiments by simultaneously adding or sequentially adding the reaction components

[0080] In this specification, "effective amount" means the amount of reaction components such as the protein, PAPS, and divalent metal ions that allows for the reproducible detection of the progress of the N-sulfation reaction and that allows for the confirmation of the N-sulfotransferase activity by the unsaturated disaccharide analysis (detection of ΔUA-GlcNS) described in the analytical example. The concentration, ratio, and timing of addition of each component can be appropriately optimized by those skilled in the art according to the reaction scale, the molecular weight of the substrate (heparosan compound), the degree of N-deacetylation, etc.

[0081] Instead of adding a sulfate donor, a product of a microorganism capable of producing sulfate donors may be added. The product of such microorganism will be described in detail in section (ii) Cellular Reaction Method. Alternatively, instead of adding a sulfate donor, a microorganism capable of producing sulfate donors may be cultured together with the microorganism of this embodiment. The sulfate donor may be supplied from the microorganism of this embodiment. The microorganism of this embodiment may be obtained using host cells that inherently have the ability to produce sulfate donors, or it may be modified to have the ability to produce sulfate donors.

[0082] In this manufacturing method, a heparosan compound that serves as a substrate may be added. Alternatively, instead of adding a heparosan compound, a product of microorganisms capable of producing heparosan compounds may be added. Alternatively, instead of adding a heparosan compound, microorganisms capable of producing heparosan compounds may be cultured together with the microorganisms of this embodiment.

[0083] Embodiments of this manufacturing method include (i) an enzymatic method using extracted enzymes (hereinafter referred to as the "enzymatic method"), (ii) a microbial reaction method, and (iii) a culture method.

[0084] (i) Enzymatic method: One embodiment of the present manufacturing method includes reacting the protein of the present disclosure itself with a heparosan compound. Specifically, for example, when recombinant protein is used as the protein of the present disclosure, the recombinant protein can be obtained using a cell-free vector or from the microorganism of the present disclosure. The protein of the present disclosure can be used as unpurified, crude, or purified protein. These proteins may be used in a state immobilized on a solid phase during the reaction (immobilized protein).

[0085] Culture media for culturing the microorganisms of this disclosure are known and can be used, for example, by adding a carbon source, nitrogen source, vitamin source, etc. to nutrient media such as LB medium or minimal media such as M9 medium. Depending on the host, the microorganisms of this disclosure are usually cultured at 16 to 42°C, preferably 25 to 37°C, for 5 to 168 hours, preferably 8 to 72 hours. Depending on the host, either shaking culture or static culture is possible, but stirring or aeration may be performed as needed. When actinomycetes are selected as the expression host, conditions that can be used to produce proteins can be used as appropriate. In addition, when an inducible promoter is used for protein expression, a promoter inducer can be added to the culture medium before cultivation.

[0086] The method for extracting the proteins of this disclosure from the microorganisms of this disclosure is not particularly limited, and known methods can be used, for example. Such methods include, for example, surfactant treatment, organic solvent treatment, sonication, mechanical grinding, and freeze-thaw treatment. These treatments can be used individually or in appropriate combinations.

[0087] The produced protein can be extracted from the microorganism by the method described above, and then purified and isolated by known precipitation methods such as salting out, isoelectric focusing, or solvent precipitation; methods utilizing molecular weight differences such as dialysis, ultrafiltration, or gel filtration; methods utilizing specific affinity such as ion exchange chromatography; methods utilizing differences in hydrophobicity such as hydrophobic chromatography or reversed-phase chromatography; and other methods such as affinity chromatography, SDS polyacrylamide electrophoresis, isoelectric focusing, or a combination thereof. If the target protein is secreted and expressed, the culture supernatant containing the protein can be obtained by removing the bacterial cells from the culture medium obtained by culturing the microorganisms using centrifugation or the like. The protein can also be purified and isolated from this culture supernatant.

[0088] One preferred configuration for the N-sulfation reaction using TRX-tagged NST in the enzymatic method is as follows: • Substrate: N-deacetylated heparosan or a precursor of an N-sulfated heparosan derivative. The concentration is preferably 0.2 to 2.0 g / L, more preferably 0.5 to 1.0 g / L. • PAPS: Preferably 3.0 to 6.0 mM, more preferably 4.0 to 5.0 mM. • Buffer solution: MOPS buffer, preferably 25 to 100 mM (preferably pH 6.8 to 7.2), MgCl ２　 Preferably 5 to 20 mM, more preferably 7 to 15 mM. Protein amount: The total protein concentration of the TRX tag-fused NST is preferably 0.05 to 1.0 mg / mL, more preferably 0.1 to 0.5 mg / mL. Crude extract, purified product, culture supernatant, or immobilized protein can all be used. Reaction temperature and time: Preferably 25 to 37°C, more preferably 30 to 37°C; preferably 0.5 to 24 hours, more preferably 1 to 6 hours. Stirring is preferably 300 to 700 rpm. Solidification: A flow-through method using TRX tag-fused NST immobilized on a carrier (e.g., resin, membrane, magnetic particles, etc.) is also possible, with a residence time of preferably several minutes to several tens of minutes, and recirculation as needed to reach the desired degree of N-sulfation.

[0089] (ii) Microbial Reaction Method One embodiment of the present manufacturing method includes a method in which an enzyme or substrate is incorporated into a treated product (hereinafter also abbreviated as "treated product") in which the cell plasma membrane of the microorganism of the present disclosure has been made permeable by treatment with a drug or the like, and palosan is N-sulfated to N-deacetylation. In this embodiment, the permeability of the cell plasma membrane means that various molecules, small (ions, etc.) and large (proteins, etc.), can freely enter and exit the cell membrane by diffusion. In this embodiment, the treated product of the microorganism is preferably a dormant cell that has lost its ability to proliferate due to treatment to impart membrane permeability.

[0090] Examples of microbial treatment products include surfactant-treated products of microbial cells, solvent-treated products of the microbial cells, freeze-thawed products of the microbial cells, enzyme-treated products of the microbial cells, immobilized products of the microbial cells that contain live cells that maintain the same function as the culture of the microbial cells as an enzyme source, ultrasonically treated products of the microbial cells, and mechanically ground products of the microbial cells.

[0091] Methods for making the cell plasma membrane permeable to substances include, for example, chemical treatment, mechanical treatment, and freeze-thaw treatment. In the production method of the present invention, the timing for making the cell plasma membrane of microorganisms permeable to substances is not particularly limited, as long as the effects of the present invention are achieved, the cell plasma membrane of each microorganism may be made permeable to substances in advance, or it may be done when the microorganisms used in the reaction are brought into contact with each other and reacted.

[0092] Chemical treatments include, for example, methods using surfactants, organic solvents, and enzymes. Among surfactants, nonionic surfactants are preferred because they have a milder effect on proteins (compared to ionic surfactants). Examples of such surfactants include digitonin, saponin, Triton X100 (hereinafter, Triton is a registered trademark), Triton X114, Tween 20 (hereinafter, Tween is a registered trademark), Tween 80, and N,N-Bis(3-D-gluconamidepropyl)cholamide [BIGCHAP]. Examples include N,N-Bis(3-D-gluconamidepropyl)deoxycholamide [Deoxy-BIGCHAP], NIKKOLBL-9EX [Polyoxyethylene(9)LaurylEther], Octanoyl-N-methylglucamide [MEGA-8], and benzalkonium chloride.

[0093] Examples of organic solvents include benzene, toluene, xylene, and other alcohols. Examples of enzymes include lysozyme and achromopeptidase.

[0094] The conditions for treatment with the above-mentioned substance, such as concentration, temperature, and time, vary depending on the type of cell, and appropriate conditions must be set to perform the desired analysis. However, a typical treatment concentration is 10 to 1000 μg / ml, preferably 20 to 200 μg / ml, with a temperature of 2 to 37°C and a duration of 1 to 30 minutes.

[0095] Examples of mechanical treatments include ultrasonic treatment and mechanical grinding.

[0096] The preferred embodiments of the bacterial cell reaction method are as follows: • Enzyme source: Microorganisms expressing TRX tag-fused NST (e.g., Escherichia coli Origami B (DE3), etc.) can be used. If necessary, it is preferable to use dormant cells that have lost their growth ability by membrane permeabilization treatment. • Permeabilization treatment: A nonionic surfactant (e.g., saponin, Triton X-100, Tween 20) is used, preferably at a concentration of 20 to 200 μg / mL, at a temperature of preferably 25 to 30°C, and for a time of preferably 5 to 20 minutes. • Reaction conditions: The substrate concentration is preferably 0.001 to 100 g / L, the PAPS concentration is preferably 4 to 500 mM, and the buffer is preferably 0.05 M phosphate buffer (pH 6.5) and 0.25% magnesium sulfate. The reaction temperature is preferably 27 to 37°C, and the reaction time is preferably 24 hours. • PAPS supply: In addition to external addition, PAPS can be supplied using processed products of microorganisms capable of producing PAPS. • Cell morphology: Whole cell suspension or immobilized resting cells (e.g., immobilized with alginate, polyacrylamide, etc.) can be used. Immobilization improves reusability. This configuration makes it possible to efficiently carry out the N-sulfation reaction while omitting the enzyme extraction step.

[0097] (iii) Culture Method One embodiment of the present manufacturing method includes a step of reacting the microorganism of the present disclosure with a heparosan compound. The protein produced by the microorganism of the present disclosure has N-sulfotransferase activity and can N-sulfate N-deacetylated heparosan.

[0098] The culture medium used in this embodiment is not particularly limited, as long as the microorganisms of this disclosure can grow and modified heparosan compounds are produced and accumulated. In this embodiment, for example, a conventional culture medium used for bacterial culture can be used as the culture medium. Examples of culture media include, but are not limited to, LB medium (Luria-Bertani medium) and mineral medium (Carbohydrate Research, 2012, 360, 19-24). For example, a culture medium can be used that contains, as necessary, a carbon source, a nitrogen source, and other components selected from various organic and inorganic components. The types and concentrations of culture medium components may be appropriately set by those skilled in the art.

[0099] In this embodiment, the carbon source is not particularly limited as long as it can be assimilated by the microorganisms of this disclosure to produce modified hepalosan compounds. Examples of carbon sources include sugars such as glucose, fructose, sucrose, lactose, galactose, xylose, arabinose, molasses, starch hydrolysates, and biomass hydrolysates; organic acids such as acetic acid, fumaric acid, citric acid, succinic acid, and malic acid; alcohols such as glycerol, crude glycerol, and ethanol; and fatty acids. One carbon source may be used, or two or more carbon sources may be combined.

[0100] In this embodiment, examples of nitrogen sources include ammonium salts such as ammonium sulfate, ammonium chloride, and ammonium phosphate, peptone, organic nitrogen sources such as yeast extract, meat extract, and soy protein hydrolysate, ammonia, and urea. One nitrogen source may be used, or two or more nitrogen sources may be combined.

[0101] In this embodiment, other various organic and inorganic components specifically include, for example, inorganic salts such as sodium chloride and potassium chloride; trace metals such as iron, manganese, magnesium, and calcium; vitamins such as vitamin B1, vitamin B2, vitamin B6, nicotinic acid, nicotinamide, and vitamin B12; amino acids; nucleic acids; and organic components containing these, such as peptone, casamino acid, yeast extract, and soy protein hydrolysate. These other various organic and inorganic components may be used individually, or in combination of two or more components.

[0102] Furthermore, in this embodiment, when using a nutrient-requiring mutant strain that requires amino acids or other nutrients for growth, it is preferable to supplement the culture medium with the required nutrients. Also, when introducing a gene using a vector carrying an antibiotic resistance gene, it is preferable to add the antibiotic corresponding to the culture medium.

[0103] In this embodiment, the culture conditions are not particularly limited, as long as the microorganisms of this disclosure can grow and the modified heparosan compound is produced and accumulated. The culture can be carried out, for example, under the usual conditions used for culturing microorganisms. The culture conditions may be set as appropriate by those skilled in the art.

[0104] In this embodiment, the culture is typically performed at 16 to 42°C, preferably 25 to 37°C, for 5 to 168 hours, preferably 8 to 72 hours, depending on the host. Depending on the host, either shaking culture or static culture is possible, but stirring or aeration may be performed as needed. The culture can be carried out by batch culture, fed-batch culture, continuous culture, or a combination thereof. Furthermore, the culture may be divided into pre-culture and main culture. Pre-culture may be performed using, for example, plate medium or liquid medium.

[0105] By culturing the microorganism of this disclosure in the manner described above, the modified heparosan compound accumulates in the microorganism and in at least one of the culture medium.

[0106] The preferred embodiment of the culture method is as follows: • Composition: TRX tag-fused NST is expressed in the host, and heparosan compounds (added externally or produced by the host) and PAPS (added externally or produced by the host) are present in the same culture system. • Culture medium: A carbon source (e.g., glucose, glycerol, etc.), nitrogen source, trace metals, vitamins, etc. are added to LB medium or M9 minimal medium. Antibiotics are added as needed. • Culture conditions: Preferably cultured at 25-37°C for 8-72 hours. Batch culture, fed-batch culture, or continuous culture are all possible. When using an inducible promoter, an inducer (e.g., IPTG) is added at an appropriate time. • Goal: Preferably, at the end of the culture period, ΔUA-GlcNS is detected from the obtained product under the conditions of the analysis example.

[0107] (Heparosan Compounds) In this manufacturing method, a predetermined target substance can be produced by using heparosan compounds as a starting material. In this invention, "heparosan compound" means heparosan or heparosan derivatives.

[0108] Heparosan is a polysaccharide composed of a repeating disaccharide structure consisting of β-D-glucuronic acid (GlcA) residues and N-acetyl-α-D-glucosamine (GlcNAc) residues [→4)-β-D-GlcA-(1→4)-α-D-GlcNAc-(1→]. Heparosan can be prepared, for example, by fermentation using microorganisms capable of producing heparosan (e.g., International Publication No. 2015 / 050184).

[0109] Examples of heparosan derivatives include heparosan having one or more modifications selected from the group consisting of (x1) to (x5) below: (x1) N-deacetylation of glucosamine residue (x2) Depolymerization (x3) N-sulfation of glucosamine residue (x4) C5-epimery of uronic acid residue (x5) 2-O-sulfation of uronic acid residue

[0110] The heparosan compound used as a substrate can be prepared by conventionally known methods (for example, Japanese Patent Publication No. 2024-060562). In this specification, "uronic acid" means glucuronic acid (GlcA) or iduronic acid (IdoA).

[0111] In this disclosure, glucuronic acid is preferred as the "uronic acid residue" in the "C5-epimery of the uronic acid residue" of (x4). Therefore, in the C5-epimery of (x4), it is preferable that iduronic acid is produced by isomerization of glucuronic acid.

[0112] In this disclosure, iduronic acid is preferred as the "uronic acid residue" in the "2-O-sulfation of the uronic acid residue" of (x5). Therefore, in the 2-O-sulfation of (x5), it is preferable that the hydroxyl group at position 2 of iduronic acid is sulfated.

[0113] In one embodiment of this disclosure, the starting material, a heparosan compound, is preferably a deacetylated heparosan. In this specification, "deacetylated" means that the acetyl group has been removed from the amino group at the C2 position of the glucosamine residue.

[0114] Deacetylated heparosan can be obtained by providing a heparosan compound to the treatment described in (x1) above. Specifically, N-deacetylated heparosan can be obtained by chemical treatment with sodium hydroxide, for example, referring to Appl Microbiol Biotechnol., 2011, Jul; 91(1):91-9, etc.

[0115] In one embodiment of this disclosure, the starting material, heparosan compound, may be an N-sulfated heparosan compound. In this specification, "N-sulfated" means that the amino group of the N-acetyl-D-glucosamine residue is sulfated. An N-sulfated heparosan compound can be obtained by subjecting heparosan to both of the above treatments (x1) and (x3). The N-sulfated heparosan compound may further have one or more modifications selected from (x2) or (x4) above.

[0116] An example of an N-sulfated heparosan compound is N-sulfohepalosan (Genes Dev, 1998, 12, 1894-1906). The degree of N-sulfation of N-sulfohepalosan is arbitrary, but 50% or more is preferred, more preferably 60% or more, 70% or more, and even more preferably 80% or more.

[0117] In one embodiment of this disclosure, the starting material, a heparosan compound, may be an epimerized heparosan compound. In this specification, "epimerization" means that a glucuronic acid residue has been converted to an iduronic acid residue in relation to a uronic acid residue. An epimerized heparosan compound can be obtained by subjecting the heparosan compound to the treatment described in (x4) above. The epimerized heparosan compound may further have one or more modifications selected from the group consisting of (x1) to (x3) above.

[0118] In one embodiment of this disclosure, the starting material, a heparosan compound, may be a low-molecular-weight heparosan compound. In this specification, "low-molecular-weight" means being processed to reduce its molecular weight. For example, a "low-molecular-weight" heparosan compound has a number-average molecular weight (Mn) of 1,000 to 150,000, preferably 8,000 to 60,000, and a weight-average molecular weight (Mw) of 2,000 to 300,000, preferably 10,000 to 100,000, as measured by gel permeation chromatography (GPC) with pullulan as the standard. A low-molecular-weight heparosan compound can be obtained by subjecting a heparosan compound to the treatment described in (x2) above. A low-molecular-weight heparosan compound may further have one or more modifications selected from the group consisting of (x1), (x3), and (x4) above.

[0119] In one embodiment of this disclosure, the starting material, the heparosan compound, may be an N-sulfated epimerized low-molecular-weight heparosan compound. The "N-sulfation," "epimerization," and "low-molecular-weight" in the N-sulfated epimerized low-molecular-weight heparosan compound are as described above. The N-sulfated epimerized low-molecular-weight heparosan compound can be obtained by providing the heparosan compound to the treatments (x1) to (x4) described above.

[0120] In one embodiment of this disclosure, the starting material, the heparosan compound, may be N-sulfated epimerized low molecular weight heparosan. The terms "N-sulfation," "epimerization," and "low molecular weight" in N-sulfated epimerized low molecular weight heparosan are as described above. N-sulfated epimerized low molecular weight heparosan can be obtained by providing the heparosan compound to the treatments (x1) to (x4) described above.

[0121] (Modified Heparosan Compounds) Modified heparosan compounds containing N-sulfated glucosamine residues are preferred as modified heparosan compounds produced by this manufacturing method. Examples of modified heparosan compounds containing N-sulfated glucosamine residues include heparan sulfate, heparin, dermatan sulfate, acetylated heparosan, methylated heparosan, and silylated heparosan, with heparan sulfate and heparin being preferred, and heparin being more preferred.

[0122] In one embodiment of this manufacturing method, it is preferable that the modified heparosan compound is produced in the presence of the protein, the heparosan compound, and PAPS of this disclosure.

[0123] One embodiment of the present manufacturing method includes subjecting a heparosan compound to the following treatments (s1) to (s7) to produce a modified heparosan compound, wherein (s3) N-sulfation of the glucosamine residue is preferably carried out in the presence of the protein of the present disclosure described above, and more preferably in the presence of the microorganism of the present disclosure or its processed product. (s1) N-deacetylation of the glucosamine residue (s2) Depolymerization (s3) N-sulfation of the glucosamine residue (s4) C5-epimery of the uronic acid residue by C5 epimerase (s5) 2-O-sulfation of the uronic acid residue by 2OST (s6) 6-O-sulfation of the glucosamine residue by 6OST (s7) 3-O-sulfation of the glucosamine residue by 3OST

[0124] In one embodiment of the present disclosure, it is preferable that the production of the modified heparosan compound is carried out in the presence of the protein of the present disclosure, the heparosan compound, PAPS, a protein having C5-epimerase activity, a protein having 2-OST activity, a protein having 6-OST activity, and a protein having 3-OST activity.

[0125] C5 epimerase, 2OST, 6OST, and 3OST can be prepared according to conventionally known methods (for example, the method described in International Publication No. 2021 / 201282). In one embodiment of this production method, for example, C5 epimerase, 2OST, 6OST, or 3OST may be from microorganisms expressing each protein, or may be extracted and purified from microorganisms. In this embodiment, each protein may be added to the reaction mixture initially, sequentially, or in combination.

[0126] The treatment of the heparosan compound according to (s1) to (s7) above can be carried out by methods well known in the art. The above treatments can be carried out in any order. For example, (s2) depolymerization can be carried out before, during, or after the treatments according to (s1) and (s3) to (s7), but it may also be carried out after (s1) and before (s3). Furthermore, the treatments according to (s5) to (s7) can be carried out in any order, but typically they can be carried out in the order of 2-O-sulfation, 3-O-sulfation and 6-O-sulfation, or in the order of 2-O-sulfation, 6-O-sulfation and 3-O-sulfation. The above treatments can also be carried out in numerical order, and two or more treatments can be carried out simultaneously or separately. For example, when producing highly modified heparin, it is preferable to add enzymes (s3) to (s7) simultaneously to the reaction solution and carry out the reaction.

[0127] The products from each step may be used in the next step while still contained in the reaction solution of the previous step, or they may be recovered from the reaction solution and used in the next step. The means for recovering each product from the reaction solution are not particularly limited. Means for recovering each product include known methods used for the separation and purification of compounds, such as membrane treatment and precipitation. The products from each step may be subjected to treatments such as purification, dilution, concentration, drying, dissolution, and enzyme inactivation as appropriate before being used in the next step. Purification may be carried out to the desired degree. These treatments may be carried out individually or in combination as appropriate.

[0128] As described above, the following configurations are disclosed in this specification: 1. A protein fused with a TRX tag, one of the following [1] to [3]: [1] A protein consisting of the amino acid sequence represented by Sequence ID No. 1. [2] A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 1, and having N-sulfotransferase activity. [3] A protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence represented by Sequence ID No. 1, and having N-sulfotransferase activity. 2. DNA encoding the protein described in 1 above. 3. DNA consisting of the base sequence represented by Sequence ID No. 12 or its complementary sequence. 4. A microorganism containing the DNA described in 2 or 3 above. 5. A method for producing a modified heparosan compound, comprising the step of reacting a heparosan compound with at least one protein selected from the following [1] to [3], fused with a TRX tag: [1] A protein consisting of the amino acid sequence represented by Sequence ID No. 1. [2] A protein having N-sulfotransferase activity, comprising an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by Sequence ID No. 1. [3] A protein having N-sulfotransferase activity, comprising an amino acid sequence having 70% or more identity with the amino acid sequence represented by Sequence ID No. 1. 6. The method for producing a protein according to 5, wherein at least one protein selected from [1] to [3], to which a TRX tag has been fused, is produced by a microorganism containing the following DNA: (i) DNA encoding at least one protein selected from [1] to [3], to which a TRX tag has been fused; (ii) DNA consisting of the base sequence represented by Sequence ID No. 12 or its complementary sequence. 7. The method for producing a protein according to 5 or 6, wherein the heparosan compound is deacetylated heparosan. 8. The method for producing a protein according to any one of 5 to 7, wherein the modified heparosan compound is heparin.

[0129] [Analysis Example] In the example, high-performance liquid chromatography Prominence (manufactured by Shimadzu Corporation) was used for the analysis of unsaturated disaccharides. The analysis conditions are shown below. [Analysis Conditions] Column: spherisorb-SAX chromatography column (Waters) Column temperature: 25°C Mobile phase: (Mobile phase A) 1.8 mM sodium dihydrogen phosphate (pH 3.0) (Mobile phase B) 1.8 mM sodium dihydrogen phosphate, 1 M sodium perchlorate (pH 3.0) Mixing ratio of mobile phase A and mobile phase B: (0-10 min) 90:10 (10-50 min) 70:30 (50-60 min) 35:65 (60-65 min) 0:100 (65-67 min) 0:100 (67-68 min) 90:10 (68-90 min) 90:10 Flow rate: 0.45 mL / min Detector: UV detector SPD-20A (Shimadzu Corporation) (232 nm)

[0130] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist of the invention.

[0131] [Example 1] Preparation of microorganisms expressing TRX tag-fused NST Referring to J. Biol. Chem., 274(16), 1999, 10673-10676, DNA consisting of the base sequence (SEQ ID NO: 2) of the gene encoding the N-sulfotransferase region (L558-R882) of human (Homo sapiens) N-deacetylase and N-sulfotransferase (NDST1) (UniProtKB Accession No. P52848.1) was prepared by artificial synthesis.

[0132] Using the DNA represented by Sequence ID No. 2 as a template, PCR was performed using DNA consisting of the base sequences represented by Sequence ID No. 3 and Sequence ID No. 4 as a primer set to obtain the NST gene fragment.

[0133] Next, using the genomic DNA of Escherichia coli BL21 (DE3) strain prepared by a conventional method as a template, PCR was performed using a primer set consisting of DNA with the nucleotide sequences represented by SEQ ID NOs. 5 and 6 to obtain a TRX tag fragment. Furthermore, using pMALcx2 (New England Biolabs) as a template, PCR was performed using a primer set consisting of DNA with the nucleotide sequences represented by SEQ ID NOs. 7 and 8 to obtain a pMALcx vector fragment.

[0134] Four TRX-tagged NST expression plasmids, pTRX-NST, were constructed by ligating the NST gene fragment, TRX tag fragment, and pMALcx vector fragment obtained above using an In-Fusion cloning kit (Takara Bio Inc.).

[0135] The plasmids pTRX-NST obtained above were transformed into OrigamiB (DE3) (Novagen), and the resulting transformants were named TRX-1, TRX-2, TRX-3, and TRX-4, respectively.

[0136] [Comparative Example 1] Production of microorganisms expressing GST-tagged NST Using pGEX4T-3 (manufactured by GE Healthcare Life Sciences) as a template, PCR was performed using DNA consisting of the base sequences represented by SEQ ID NO: 9 and SEQ ID NO: 10 as a primer set to obtain GST-tagged fragments.

[0137] Four GST-tagged NST expression plasmids, pGST-NST, were prepared by ligating the obtained GST tag fragment, along with the NST gene fragment and pMALcx vector fragment obtained in Example 1, using an In-Fusion cloning kit (Takara Bio Inc.). The nucleotide sequences of the GST-tagged NSTs are shown in Sequence ID No. 13.

[0138] The four plasmid pGST-NSTs obtained above were each transformed into OrigamiB (DE3) (Novagen), and the resulting transformants were named GST-1, GST-2, GST-3, and GST-4, respectively.

[0139] [Example 2] Evaluation of N-sulfotransferase activity The N-sulfotransferase activity of TRX-tagged NSTs was evaluated. GST-tagged NSTs were used as a comparison. The N-sulfation activity of each NST was evaluated by the following procedure. First, using the NST enzyme, an enzymatic reaction was carried out with N-deacetylated heparosan as a substrate in the presence of PAPS to produce N-sulfated heparosan.

[0140] Next, referring to International Publication No. 2018 / 048973 (Patent Document 2), the obtained N-sulfated heparosan was decomposed using heparinase to obtain unsaturated disaccharides. The obtained unsaturated disaccharides were analyzed, and the proportion of N-sulfated glucosamine residues (ΔUA-GlcNS) was confirmed to evaluate the N-sulfotransferase activity of each NST enzyme toward glucosamine residues in heparosan.

[0141] (1) Preparation of enzyme samples for activity evaluation TRX-1, TRX-2, TRX-3 and TRX-4 prepared in Example 1, and GST-1, GST-2, GST-3 and GST-4 prepared in Comparative Example 1 were inoculated into 5 mL of LB medium containing 100 mg / L ampicillin and cultured with shaking at 30°C and 290 rpm.

[0142] Three hours after the start of cultivation, isopropyl-β-D-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM, and the culture was continued for another 22 hours. After the culturing was complete, the culture medium was centrifuged to obtain wet cells. The obtained wet cells were prepared to have a bacterial weight of 20 g / L, and then the cells were lysed by sonication. The resulting suspension after lysation was used as a sample for activity evaluation.

[0143] (2) Evaluation of N-sulfotransferase activity A reaction solution of 100 μL was prepared consisting of 60 μL of the activity evaluation sample obtained in (1) above, 50 mM MOPS buffer (pH 7.0), 10 mM magnesium chloride, 0.5 g / L N-deacetylated heparosan, and 4.4 mM PAPS. Using the reaction solution, an enzymatic reaction was carried out at 30°C and 350 rpm for 30 minutes to produce N-sulfated heparosan. Thereafter, the reaction was stopped by heating at 95°C for 10 minutes.

[0144] The solution after the enzymatic reaction was centrifuged, and the resulting supernatant was heat-treated at 80°C for 10 minutes to denature proteins derived from the bacterial cells. The solution after heat treatment was centrifuged again, and the resulting supernatant was ultrafiltered using Amicon Ultra Centrifugal Filters 0.5 mL-3K (Merck) to remove salt.

[0145] 50 μL of the desalted solution was mixed with 50 μL of heparinase reaction solution (50 mM ammonium acetate, 2 mM calcium chloride) containing 0.5 U / ml each of heparinase I, II, and III (all manufactured by Sigma-Ace). The mixture was reacted at 35°C for 2 hours to decompose N-sulfated heparosan. Subsequently, the reaction was stopped by heat treatment at 95°C for 15 minutes.

[0146] After the reaction was complete, the unsaturated disaccharides (ΔUA-GlcN, ΔUA-GlcNAc, and ΔUA-GlcNS) produced by the heparinase reaction were analyzed by HPLC. Table 1 shows the percentage (%) of ΔUA-GlcN, ΔUA-GlcNAc, or ΔUA-GlcNS in the N-sulfated heparosan produced by each NST enzyme reaction.

[0147] Furthermore, ΔUA-GlcN indicates the presence of deacetylated glucosamine residues in the sugar chain, ΔUA-GlcNAc indicates the presence of N-acetylated glucosamine residues in the sugar chain, and ΔUA-GlcNS indicates the presence of N-sulfated glucosamine residues in the sugar chain.

[0148]

[0149] As shown in Table 1, the proportion of ΔUA-GlcNS in N-sulfated heparosan obtained using GST-tagged NST as previously reported [J. Biol. Chem., 274(16), 1999, 10673-10676] was approximately 30%.

[0150] On the other hand, when using TRX-tagged NST, the proportion of ΔUA-GlcNS in the resulting N-sulfated heparosan was close to 70%, confirming high N-sulfotransferase activity. From these results, it was found that fusing the TRX tag to NST significantly improves N-sulfotransferase activity compared to GST tags.

[0151] [Production Example 1] Production of heparin using TRX tag-fused NST (1) Preparation of NST-containing solution Escherichia coli expressing TRX tag-fused NST is prepared according to Example 1. The prepared strain is cultured according to Example 1. The bacterial cells are collected from the obtained culture solution and frozen at -80°C. The frozen bacterial cells are suspended in distilled water, and the resulting suspension is used as the NST-containing solution.

[0152] (2) Preparation of C5-epimerase-containing solution Escherichia coli expressing C5-epimerase is prepared according to previously reported information (International Publication No. 2021 / 201282). The prepared strain is cultured according to the same literature. The cells are collected from the culture medium and frozen at -80°C. The frozen cells are suspended in distilled water, and the resulting suspension is used as the C5-epimerase-containing solution.

[0153] (3) Preparation of 2OST-containing solution Escherichia coli expressing 2OST is prepared according to previously reported information (International Publication No. 2021 / 201282). The prepared strain is cultured according to the same literature. The cells are collected from the culture medium and frozen at -80°C. The frozen cells are suspended in distilled water, and the resulting suspension is used as the 2OST-containing solution.

[0154] (4) Preparation of 6OST-containing solution Escherichia coli expressing 6OST-3 is prepared according to previously reported information (International Publication No. 2021 / 201282). The prepared strain is cultured according to the same literature. The cells are collected from the culture medium and frozen at -80°C. The frozen cells are suspended in distilled water, and the resulting suspension is used as the 6OST-containing solution.

[0155] (5) Preparation of 3OST-containing solution Escherichia coli expressing 3OST-1 is prepared according to previously reported information (International Publication No. 2021 / 201282). The prepared strain is cultured according to the same literature. The cells are collected from the culture medium and frozen at -80°C. The frozen cells are suspended in distilled water, and the resulting suspension is used as the 3OST-containing solution.

[0156] (6) Preparation of PAPS-containing solution Corynebacterium that produces PAPS is prepared according to a previously published report (International Publication No. 2021 / 201282). The prepared strain is cultured according to the same literature to produce PAPS. The bacterial cells are collected from the obtained culture solution and frozen at -80°C. The frozen bacterial cells are suspended in distilled water, and the resulting suspension is used as the PAPS-containing solution.

[0157] (7) Preparation of N-deacetylated heparosan Escherichia coli that produces heparosan is prepared in accordance with a previously published document (Japanese Patent Publication No. 2024-060562), and heparosan is produced by the method described in the same document. Using the obtained heparosan, chemical N-deacetylation treatment is performed in accordance with a previously published document [Appl Microbiol Biotechnol., 2011, Jul; 91(1): 91-9] to obtain N-deacetylated heparosan.

[0158] (8) Method for producing heparin The NST-containing solution, C5-epimerase-containing solution, 2OST-containing solution, 6OST-containing solution, and 3OST-containing solution obtained in (1) to (5) above, and the PAPS-containing solution obtained in (6) above are added to the reaction mixture [0.05 M phosphate buffer (pH 6.5), 1 mg / L deacetylated heparosan, 0.25% magnesium sulfate] and reacted at 27 to 37°C for 24 hours to produce heparin. At this time, each enzyme-containing solution is added to the total volume to about 2%, and the PAPS-containing solution to about 5%. After that, the reaction is stopped by heating at 80°C for 10 minutes. The solution after the reaction is centrifuged and the supernatant is collected. Heparin is obtained by purifying the supernatant as appropriate.

[0159] Although the present invention has been described in detail with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications are possible without departing from the spirit and scope of the invention. This application is based on Japanese Patent Application No. 2024-208814, filed on 29 November 2024, which is incorporated by reference in its entirety. All references incorporated herein are incorporated as a whole.

[0160] Sequence ID 1: Amino acid sequence of the N-sulfotransferase region (L558-R882) of human (Homo sapiens) N-deacetylase and N-sulfotransferase (NDST1) Sequence ID 2: Human (Homo sapiens) N-deacetylase and Sequence ID 3: Base sequence of the gene encoding the N-sulfotransferase region (L558-R882) of N-sulfotransferase (NDST1) Sequence ID 4: Base sequence of primer F for NST gene fragment amplification Sequence ID 5: Base sequence of primer F for TRX tag fragment amplification Sequence ID 6: Base sequence of primer R for TRX tag fragment amplification Sequence ID 7: Base sequence of primer F for pMALcx vector fragment amplification Sequence ID 8: Base sequence of primer R for pMALcx vector fragment amplification Sequence ID 9: Base sequence of primer F for GST tag fragment amplification Sequence ID 10: Base sequence of primer R for GST tag fragment amplification Sequence ID 11: Base sequence of TRX tag Sequence ID 12: Base sequence of TRX tag fused NST Sequence ID 13: Base sequence of GST tag fused NST Sequence ID 14: Amino acid sequence of TRX tag Sequence ID 15: Amino acid sequence of AhpC

Claims

1. A protein fused with a TRX tag, which is one of the following [1] to [3]: [1] A protein consisting of the amino acid sequence represented by Sequence ID No.

1. [2] A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added to the amino acid sequence represented by Sequence ID No. 1, and which has N-sulfotransferase activity. [3] A protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence represented by Sequence ID No. 1, and which has N-sulfotransferase activity.

2. DNA encoding the protein described in claim 1.

3. DNA consisting of the base sequence represented by Sequence ID No. 12 or its complementary sequence.

4. A microorganism containing DNA as described in claim 2 or 3.

5. A method for producing a modified heparosan compound, comprising the step of reacting a heparosan compound with at least one protein selected from [1] to [3] below, which has a TRX tag fused to it. [1] A protein consisting of the amino acid sequence represented by SEQ ID NO:

1. [2] A protein consisting of an amino acid sequence in which 1 to 20 amino acids are deleted, substituted, inserted, or added in the amino acid sequence represented by SEQ ID NO: 1, and which has N-sulfotransferase activity. [3] A protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence represented by SEQ ID NO: 1, and which has N-sulfotransferase activity.

6. The method for producing the product according to claim 5, wherein at least one protein selected from [1] to [3] fused with a TRX tag is produced by a microorganism containing the following DNA: (i) DNA encoding at least one protein selected from [1] to [3] fused with a TRX tag; (ii) DNA consisting of the base sequence represented by Sequence ID No. 12 or its complementary sequence.

7. The method for producing the product according to claim 5, wherein the heparosan compound is deacetylated heparosan.

8. The method for producing the modified heparosan compound according to claim 5, wherein the modified heparosan compound is heparin.

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