Modified 2-o-sulfotransferase

Abstract

The purpose of the present invention is to provide a 2-O-sulfotransferase having higher 2-O-sulfation activity than the conventional art and exhibiting excellent selectivity for iduronic acid residue in heparosan. The protein comprises a catalytic domain of a protein comprising an amino acid sequence represented by SEQ ID NO: 1 with the 111th amino acid residue substituted with an L-glutamic acid residue or an L-asparagine residue, and has 2-O-sulfation activity. The selectivity of the 2-O-sulfation activity for iduronic acid residues is higher than that of a protein comprising a catalytic domain of a protein comprising an amino acid sequence represented by SEQ ID NO: 1.

Description

Modified 2-O-sulphotransferase

[0001] The present disclosure relates to a modified 2-O-sulphotransferase, DNA encoding the modified 2-O-sulphotransferase, a microorganism containing the DNA, and a method for producing a modified heparosan compound using the modified 2-O-sulphotransferase.

[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 disaccharides consisting of uronic acid residues and N-acetylglucosamine residues. Examples of modified heparosan compounds include naturally occurring polysaccharides such as heparin and heparan sulfate, which are mainly present in animal tissues and the extracellular matrix. Modified heparosan compounds have specific properties and functions conferred by changing the molecular structure of heparosan, for example, by chemical modification or modification by enzymatic reactions. 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 thromboembolism, disseminated intravascular coagulation syndrome, prevention of coagulation in hemodialysis and extracorporeal circulation, etc. Industrially, heparin extracted and purified mainly from porcine intestinal mucosa is utilized. Since a fatal accident occurred in 2008 due to the contamination of porcine-derived heparin with impurities, the need for the production of non-animal-derived heparin with controlled production and quality has been increasing (Non-Patent Document 1). To date, methods for producing heparin having a structure and anticoagulant activity equivalent to those of porcine-derived products have been reported by deacetylating, isomerizing, and sulfating N-acetylheparosan (hereinafter referred to as heparosan), which is a capsular polysaccharide of microorganisms, by chemical and enzymatic methods (Patent Documents 1 and 2 and Non-Patent Document 2).

[0004] Enzymes necessary for the heparin production described above include, for example, N-deacetylase / N-sulfotransferase (hereinafter referred to as NDST), C5-epimerase (hereinafter referred to as C5-epi), 2-O-sulfotransferase (hereinafter referred to as 2OST), 6-O-sulfotransferase (hereinafter referred to as 6OST), and 3-O-sulfotransferase (hereinafter referred to as 3OST) (Patent Documents 1 and 2 and Non-Patent Document 2).

[0005] There are two types of uronic acid residues that make up polysaccharides such as heparin: glucuronic acid and iduronic acid. Natural heparin contains a large amount of iduronic acid. On the other hand, the uronic acid residue usually contained in heparosan derived from microorganisms is glucuronic acid. Therefore, when attempting to produce heparin from heparosan derived from microorganisms, the process of epimerizing the glucuronic acid contained in heparosan into iduronic acid is essential.

[0006] Patent Document 1 discloses that in the production of non-animal-derived heparin, glucuronic acid in heparosan can be epimerized to iduronic acid by using C5-epi. Epimerization by C5-epi is a reversible equilibrium reaction, and the ratio of the products depends on the reaction conditions.

[0007] In vitro, it has been reported that the iduronic acid content in heparosan reaches 30% to 40% after the reaction with C5-epi, but does not reach a higher percentage (Non-Patent Literature 3). On the other hand, it has been disclosed that when the sulfate enzyme 2OST is reacted simultaneously with C5-epi, the iduronic acid content in the final heparin becomes higher than the equilibrium state, and heparin closer to the natural heparin structure can be obtained (Non-Patent Literature 4).

[0008] 2OST is a sulfate enzyme that transfers a sulfate group to the hydroxyl group at the 2 position of uronic acid residues in polysaccharides, and is known to have sulfate activity for both glucuronic acid and iduronic acid in heparosan (Non-Patent Literature 4). Natural heparin contains 2-O-sulfated forms of both iduronic acid and glucuronic acid, but it contains a particularly large amount of the 2-O-sulfated form of iduronic acid (Non-Patent Literature 5). In fact, 2OST usually preferentially performs 2-O-sulfation on iduronic acid residues.

[0009] However, it has been suggested that 2-O-sulfation of glucuronic acid residues is more likely to occur in in vitro reactions (Non-Patent Documents 4, 7). In addition, it is known that in in vitro reactions, if the glucuronic acid residues in heparosan are first sulfated by 2OST, epimerization from glucuronic acid to iduronic acid by C5-epi does not occur (Non-Patent Document 4).

[0010] On the other hand, Non-Patent Document 5 discloses that introducing the Y94A mutation into 2OST significantly reduces the selectivity for glucuronic acid residues in 2-O-sulfate transfer activity (hereinafter also abbreviated as "selectivity for glucuronic acid residues") compared to wild-type 2OST. Furthermore, Non-Patent Document 2 discloses that introducing the K111A mutation into 2OST maintains the selectivity for iduronic acid residues in 2-O-sulfate transfer activity (hereinafter also abbreviated as "selectivity for iduronic acid residues") while reducing only the selectivity for glucuronic acid residues.

[0011] U.S. Patent No. 8,771,995, International Publication No. 2018 / 048973, International Publication No. 2021 / 201282

[0012] Natural Product Reports, 2009, 26, 313-321Proc. Natl. Acad. Sci. USA. 2008 105, 18724-9. Biochem. J. 347, 69-75, 2000 Proc Natl Acad Sci USA. 2024 Apr 2;121(14):e2315586121. J. Biol. Chem. 2014, 289, 13407-13418Glycobiology 2014, 24, 681-692Biochemistry 2001, 40, 5548-5555

[0013] As mentioned above, it has been suggested that 2-O-sulfation of glucuronic acid residues by 2OST is more likely to occur in the in vitro reaction. Furthermore, in the in vitro reaction, if the glucuronic acid residues in heparosan are first sulfated by 2OST, epimerization from glucuronic acid to iduronic acid by C5-epi is inhibited.

[0014] Considering these factors, when attempting to produce modified heparosan compounds using heparosan as a substrate via enzymatic methods employing C5-epi and 2OST, the resulting modified heparosan compounds may have a higher proportion of 2-O-sulfated glucuronic acid residues compared to the natural product. Therefore, in order to produce modified heparosan compounds with a structure close to that of the natural product using non-animal-derived methods, it is necessary to reduce the selectivity of 2OST for glucuronic acid residues while improving the selectivity for iduronic acid residues.

[0015] Non-patent document 5 discloses a 2OST with the Y94A mutation introduced, but the selectivity for iduronic acid residues of the 2OST with this mutation is reduced, and the 2-O-sulfate transfer activity of the 2OST is insufficient, which are technical limitations. Non-patent document 2 discloses a 2OST with the K111A mutation introduced, but the selectivity for glucuronic acid residues of the 2OST with this mutation is still high at 62% of that of the wild type, and the desired selectivity control has not been achieved.

[0016] To produce modified heparosan compounds more efficiently using non-animal-derived methods, it is necessary to search for 2OSTs that exhibit higher 2-O-sulfate transfer activity and superior selectivity for iduronic acid residues compared to conventional methods.

[0017] Therefore, this disclosure aims to provide a 2OST that exhibits higher 2-O-sulfate transfer activity compared to conventional products and superior selectivity for iduronic acid residues in heparosan.

[0018] As a result of investigating the above-mentioned problems, the present inventors found that a modified 2OST in which the 111th amino acid residue of the amino acid sequence shown in Sequence ID No. 1 is replaced with an L-glutamic acid residue or an L-asparagine residue exhibits excellent selectivity for iduronic acid residues in heparosan, and thus the present invention was made.

[0019] In other words, the present disclosure is as follows: 1. A protein comprising a catalytic domain of a protein consisting of an amino acid sequence in which the 111th amino acid residue of the amino acid sequence shown in Sequence ID No. 1 is substituted with an L-glutamic acid residue or an L-asparagine residue, and having 2-O-sulfotransfer activity, wherein the selectivity for the iduronic acid residue in the 2-O-sulfotransfer activity is higher than that of a protein comprising a catalytic domain of a protein consisting of an amino acid sequence in which the 111th amino acid residue of the amino acid sequence shown in Sequence ID No. 1 is substituted with an L-glutamic acid residue or an L-asparagine residue, and having 2-O-sulfotransfer activity, wherein the selectivity for the iduronic acid residue in the 2-O-sulfotransfer activity is higher than that of a protein comprising a catalytic domain of a protein consisting of an amino acid sequence in which the 111th amino acid residue of the amino acid sequence shown in Sequence ID No. 1 is not an L-glutamic acid residue or an L-asparagine residue. [A1] A protein containing a catalytic domain of a protein consisting of an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted, inserted and / or added from the amino acid sequence shown in Sequence ID No. 1. [A2] A protein containing a catalytic domain of a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in Sequence ID No. 1. 3. A protein having 2-O-sulfate transfer activity, wherein the protein described in [B1] or [B2] below is the original protein, and the original protein contains a catalytic domain of a protein consisting of an amino acid sequence in which the amino acid residue corresponding to the 111th amino acid sequence shown in Sequence ID No. 1 in the amino acid sequence of the original protein is substituted with an L-glutamic acid residue or an L-asparagine residue, and the selectivity for the iduronic acid residue in the 2-O-sulfate transfer activity is higher than that of the original protein.[B1] A protein comprising a catalytic domain of a protein having 2-O-sulfate transfer activity, wherein 1 to 20 amino acid residues from the amino acid sequence shown in SEQ ID NO: 1 are deleted, substituted, inserted and / or added. [B2] A protein comprising a catalytic domain of a protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and having 2-O-sulfate transfer activity. 4. DNA encoding the protein described in any one of 1 to 3 above. 5. A microorganism containing DNA encoding the protein described in any one of 1 to 3 above. 6. A method for producing a modified heparosan compound, comprising reacting the protein described in any one of 1 to 3 above with the heparosan compound. 7. A method for producing a modified heparosan compound, comprising 2-O-sulfating the uronic acid residue of the heparosan compound in the presence of the protein described in any one of 1 to 3 above. 8. The method for producing the heparosan compound according to 7 above, wherein the uronic acid residue includes an iduronic acid residue and a glucuronic acid residue. 9. The method for producing the modified heparosan compound according to claim 7, wherein the production of the modified heparosan compound is carried out in the presence of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and a protein having C5-epimerase activity. 10. The method for producing the method according to claim 7, wherein the heparosan compound is N-sulfohepalosan. 11. The method for producing the method according to claim 7, wherein the modified heparosan compound is heparin.

[0020] The disclosure also includes the following embodiments: • A protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, comprising a catalytic domain of a protein consisting of an amino acid sequence including the following substitutions (C1) or (C2), and having 2-O-sulfotransfer activity, wherein the selectivity for iduronic acid residues in the 2-O-sulfotransfer activity is higher than that of a protein comprising a catalytic domain of a protein consisting of an amino acid sequence shown in SEQ ID NO: 1. (C1) Substitution of the amino acid residue corresponding to the 111th position of the amino acid sequence shown in SEQ ID NO: 1 with an L-asparagine residue. (C2) Substitution of the amino acid residue corresponding to the 111th position of the amino acid sequence shown in SEQ ID NO: 1 with an L-glutamic acid residue. • A protein comprising a catalytic domain of a protein consisting of any one of the following amino acid sequences (D1) to (D4), and having 2-O-sulfotransfer activity, wherein the selectivity for iduronic acid residues in the 2-O-sulfotransfer activity is higher than that of a protein comprising a catalytic domain of a protein consisting of an amino acid sequence shown in SEQ ID NO: 1. (D1) An amino acid sequence having 1 to 20 further substitutions, deletions, insertions, or additions of amino acid residues in a sequence other than the amino acid residue corresponding to position 111 of the amino acid sequence shown in SEQ ID NO: 55. (D2) An amino acid sequence having 1 to 20 further substitutions, deletions, insertions, or additions of amino acid residues in a sequence other than the amino acid residue corresponding to position 111 of the amino acid sequence shown in SEQ ID NO: 56. (D3) An amino acid sequence having 80% or more identity in a sequence other than the amino acid residue corresponding to position 111 of the amino acid sequence shown in SEQ ID NO: 55. (D4) An amino acid sequence having 80% or more identity in a sequence other than the amino acid residue corresponding to position 111 of the amino acid sequence shown in SEQ ID NO: 56.

[0021] The protein disclosed herein exhibits excellent 2-O-sulfate transfer activity and high selectivity for iduronic acid residues in heparosan. Therefore, the protein disclosed herein allows for the efficient production of modified heparosan compounds with structures close to those of nature using non-animal methods.

[0022] The present invention will be described below based on embodiments, but the present invention is not limited to these embodiments. This embodiment includes the first to third embodiments described below, and these embodiments together are referred to as this embodiment.

[0023] 1. Protein The protein of the first embodiment of this disclosure is a protein that includes a catalytic domain of a protein consisting of an amino acid sequence in which the 111th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is substituted with an L-glutamic acid residue or an L-asparagine residue, and has 2-O-sulfate transfer activity, characterized in that the selectivity for iduronic acid residues in the 2-O-sulfate transfer activity is higher than that of a protein containing a catalytic domain of a protein consisting of an amino acid sequence shown in SEQ ID NO: 1.

[0024] The protein of the second embodiment of this disclosure is the protein described in [A1] or [A2] below, which includes a catalytic domain of a protein consisting of an amino acid sequence in which the amino acid residue corresponding to the 111th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is an L-glutamic acid residue or an L-asparagine residue, and is a protein having 2-O-sulfate transfer activity, characterized in that the selectivity for the iduronic acid residue in the 2-O-sulfate transfer activity is higher than that of a protein in which the amino acid residue corresponding to the 111th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is other than an L-glutamic acid residue or an L-asparagine residue. [A1] A protein including a catalytic domain of a protein consisting of an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted, inserted and / or added from the amino acid sequence shown in SEQ ID NO: 1 [A2] A protein including a catalytic domain of a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1

[0025] The protein of the third embodiment of this disclosure is a protein having 2-O-sulfotransfer activity, comprising a catalytic domain of a protein consisting of an amino acid sequence in which the amino acid residue corresponding to the 111th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 in the amino acid sequence of the original protein is substituted with an L-glutamic acid residue or an L-asparagine residue, and characterized in that the selectivity for the iduronic acid residue in the 2-O-sulfotransfer activity is higher than that of the original protein. [B1] A protein having 2-O-sulfotransfer activity comprising a catalytic domain of a protein consisting of an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted, inserted and / or added from the amino acid sequence shown in SEQ ID NO: 1 [B2] A protein having 2-O-sulfotransfer activity comprising a catalytic domain of a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1

[0026] The protein of this embodiment includes a catalytic domain. In this embodiment, the catalytic domain is a region that includes a binding site between the substrate and the reactant, and amino acid residues involved in the catalytic reaction, and in which these elements interact to promote the 2-O-sulfate transfer reaction (THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 11, pp. 8356-8367, 2007). The protein containing the catalytic domain of this embodiment also includes proteins to which tags (e.g., histidine tags, Strep-tagII tags, etc.) have been attached. Furthermore, it is preferable that the protein of this embodiment is a protein without a transmembrane domain.

[0027] The amino acid sequence shown in Sequence ID No. 1 is the amino acid sequence of 2-O-sulfotransferase isoform 1 from the Chinese hamster (heparan sulfate 2-O-sulfotransferase 1 isoform X2 [Cricetrus griseus].ACCESSION XP_003514811), and the nucleotide sequence represented by Sequence ID No. 2 (ACCESSION XM_003514763.4:931-2001 PREDICTED: Cricetrus griseus heparan sulfate 2-O-sulfotransferase 1 (LOC100760748)). It is encoded in the transcription variant X2 (mRNA).

[0028] The 111th amino acid residue in the amino acid sequence shown in Sequence ID No. 1 interacts with the sulfate group donor 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and the substrate polysaccharide, together with the arginine residue at the 188th arginine residue in the catalytic site of 2OST (Glycobiology 2018, 28, 885-897).

[0029] In the first embodiment, the 111th amino acid residue in the amino acid sequence shown in SEQ ID NO: 1 is substituted with an L-glutamic acid residue or an L-asparagine residue, and is preferably substituted with an L-glutamic acid residue.

[0030] In the second embodiment, the amino acid residue corresponding to the 111th position in the amino acid sequence shown in SEQ ID NO: 1 is either an L-glutamic acid residue or an L-asparagine residue, and is preferably an L-glutamic acid residue.

[0031] In the third embodiment, the amino acid residue corresponding to the 111th position in the amino acid sequence shown in SEQ ID NO: 1 of the amino acid sequence of the protein before modification is substituted with an L-glutamic acid residue or an L-asparagine residue, and it is preferable that it is substituted with an L-glutamic acid residue.

[0032] In the second and third embodiments, the amino acid residue corresponding to the 111th position of the amino acid sequence shown in SEQ ID NO: 1 can be identified by amino acid sequence alignment. Amino acid sequence alignment can be created using the known alignment program CrystalW [Nucleic Acids Research 22, 4673, (1994)]. CrystalW is available from http: / / www.ebi.ac.uk / clustalw / (European Bioinformatics Institute). When creating alignment using CrystalW, default values ​​can be used for the parameters.

[0033] In [A1] of the second embodiment and [B1] of the third embodiment, the number of deleted, substituted, inserted and / or added amino acid residues in the amino acid sequence shown in 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 it may be introduced into multiple different regions.

[0034] 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.

[0035] 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.

[0036] 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

[0037] 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

[0038] In [A2] of the second embodiment and [B2] of the third embodiment, the identity with the amino acid sequence shown in Sequence ID No. 1 is 80% or more, preferably 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, in that order.

[0039] In this embodiment, 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 the residues they contain are most identical. For example, the percentage of sequence identity can be determined using a mathematical algorithm.

[0040] 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.

[0041] 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.

[0042] Alignment using these programs can be performed, for example, using initial parameters. For the CLUSTAL program, see 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.

[0043] For BLAST, see 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.

[0044] The protein of this embodiment may further have a desired mutation at a predetermined site as long as it can retain the target property. The positions of amino acid residues at which mutations may be introduced while retaining the target property will be apparent to those skilled in the art.

[0045] As used herein, "2-O-sulfotransferase activity" refers to the activity of transferring a sulfate group from a sulfate donor [e.g., PAPS] to the hydroxyl group at the 2-position of a uronic acid residue to generate a "-O-sulfate group" structure at the 2-position of the uronic acid residue. Examples of uronic acid residues include iduronic acid residues and glucuronic acid residues, with iduronic acid residues being preferred.

[0046] The selectivity of 2OST for iduronic acid residues in 2-O-sulfotransferase activity can be evaluated as appropriate, for example, by a method including the following steps (I) to (III), as described in the examples. (I) A 2-O-sulfotransferase reaction is carried out with a heparosan compound as a substrate in the presence of 2OST. (II) After the 2-O-sulfotransferase reaction in (I), the obtained sample is reacted with nitrite to carry out a nitrite decomposition reaction. (III) The disaccharide containing N-sulfated glucosamine (NS-containing disaccharide) produced by the nitrite decomposition reaction in (II) is analyzed for composition by HPLC. From the results of the compositional analysis, the proportion of disaccharides consisting of N-sulfated glucosamine and 2-O-sulfated uronic acid (iduronic acid or glucuronic acid) (NS2S), disaccharides consisting of 2-O-sulfated glucuronic acid and N-sulfated glucosamine (hereinafter also abbreviated as GlcA2SNS), or disaccharides consisting of 2-O-sulfated iduronic acid and N-sulfated glucosamine (hereinafter also abbreviated as IdoA2SNS) in the total NS-containing disaccharides in the modified heparosan compound produced by the 2-O-sulfotransferase reaction is determined. Here, GlcA2SNS and IdoA2SNS are obtained by identifying the residue with 2-position sulfated (2S) from NS2S.

[0047] More specifically, the selectivity of 2OST for iduronic acid residues can be evaluated by a method including the following steps (i) to (iii): (i) Add 30 μL of 2OST-containing solution to 0.3 mL of a reaction solution containing a reaction mixture (1 g / L heparosan compound (substrate), 4.4 mM PAPS (sulfate group donor), 50 mmol / L dipotassium hydrogen phosphate, and 10.1 mmol / L magnesium sulfate), and carry out the enzymatic reaction at 37°C and 660 rpm for 22 hours.

[0048] As the 2OST-containing solution, purified enzyme solution or cell-free extract can be used. As the heparosan compound (substrate), N-sulfated heparosan, epimerized heparosan, or N-sulfated epimerized heparosan is preferred. These heparosans may be low molecular weight. The heparosan compound (substrate) may also be N-sulfated epimerized low molecular weight heparosan.

[0049] After the enzymatic reaction of (ii)(i) is complete, add 400 μL of 389.2 g / L citric acid solution and 200 μL of 49.5 g / L sodium nitrite solution to 200 μL of the obtained sample, mix well, and react for 2 hours at 65°C and 600 rpm with shaking.

[0050] (iii) After the reaction in (iii) is complete, the NS-containing disaccharides produced by nitrite decomposition are analyzed by HPLC. The percentage (%) of total NS2S, GlcA2SNS, or IdoA2SNS in the total NS-containing disaccharides in the N-sulfated 2-O-sulfated heparosan produced by the enzymatic reaction in (i) is calculated. The "IdoA2SNS / total NS2S" (%) is calculated by dividing the percentage of IdoA2SNS relative to total NS-containing disaccharides by the percentage of total NS2S relative to total NS-containing disaccharides × 100 (%), and this value is used as an indicator to evaluate the 2-O-sulfate transfer activity to the iduronic acid residue.

[0051] In the first embodiment, "the selectivity for iduronic acid residues in 2-O-sulfate transfer activity is higher than that of a protein containing the catalytic domain of a protein consisting of the amino acid sequence shown in SEQ ID NO: 1" means, specifically, for example, that the "IdoA2SNS / Total NS2S" (%) calculated by the above procedure is preferably 100% or more, and more preferably 101% or more, 102% or more, and 103% or more, respectively, based on the value of a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0052] In this embodiment, the "IdoA2SNS / TotalNS2S" (%) calculated by the above procedure is preferably 90% or higher in absolute value, more preferably 91% or higher, 92% or higher, 93% or higher, and even more preferably 94% or higher.

[0053] In the second embodiment, "the selectivity for iduronic acid residues in 2-O-sulfate transfer activity is higher than that of a protein containing a catalytic domain of a protein whose amino acid sequence is such that the amino acid residue corresponding to position 111 of the amino acid sequence shown in SEQ ID NO: 1 is not an L-glutamic acid residue or an L-asparagine residue" means, for example, that the "IdoA2SNS / TotalNS2S" (%) calculated by the above procedure is preferably 100% or more, and more preferably 101% or more, 102% or more, and 103% or more, in that order.

[0054] In the third embodiment, "higher selectivity for iduronic acid residues compared to the original protein" specifically means, for example, that the "IdoA2SNS / totalNS2S" (%) calculated by the above procedure is preferably 100% or more, and more preferably 101% or more, 102% or more, and 103% or more, in that order.

[0055] The protein of this embodiment preferably has a "total NS2S / total NS" (%) calculated in step (iii) above that is 100% or more, more preferably 101% or more, based on the value of the protein before modification. The "total NS2S / total NS" (%) calculated in step (iii) above is preferably 80% or more, more preferably 81% or more, and even more preferably 82% or more.

[0056] The protein of this embodiment may also be a fusion protein linked to a heterologous portion via a peptide bond. Examples of such heterologous portions include peptide components that facilitate the purification of the target protein (mutant) (e.g., tag portions such as histidine tag and Strep-tag II; proteins used for the purification of the target protein, such as glutathione-S-transferase, maltose-binding protein, and their variants), 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 protein or a part thereof), and linkers.

[0057] Examples of amino acid sequences include those of natural proteins, naturally occurring homologs thereof, or artificially created mutant or homologous proteins.

[0058] Mutant proteins or homologous proteins can be obtained, for example, by introducing mutations into the DNA encoding the target protein and then 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).

[0059] 2. DNA The DNA in this embodiment is the DNA that encodes the protein of this embodiment as described above. The DNA in this embodiment can constitute an expression unit. The DNA that encodes the protein of this embodiment may be naturally derived DNA or artificially synthesized DNA. In this specification, "expression unit" means the smallest unit that enables the transcription of the DNA and, consequently, the production of the protein encoded by the DNA, including a predetermined DNA to be expressed as a protein and a promoter operably linked thereto. The expression unit may further include elements such as a terminator, a ribosome binding site, and a drug resistance gene.

[0060] 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 embodiment, one or both of the polynucleotide encoding the target protein or the promoter are derived from organisms other than the host cell (e.g., prokaryotes, eukaryotes, and animals such as microorganisms, insects, plants, and mammals) or viruses, or are artificially synthesized. 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.

[0061] 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 transcriptional output by deleting the terminator region upstream of the ilv promoter may be used. 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.

[0062] 3. Microorganisms The microorganisms of this embodiment include the DNA of this embodiment as described above. The microorganisms of this embodiment are obtained by introducing the DNA of this embodiment into a host cell and transforming it.

[0063] In this embodiment, 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.

[0064] Examples of Escherichia coli include, but are not limited to, Escherichia coli Origami B (DE3) (Novagen), Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli BL21 (DE3) pLysS (Merck Millipore), Escherichia coli BL21, Escherichia coli DH5α, Escherichia coli HST08 Premium, and Escherichia coli 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.

[0065] The host cells in this embodiment may inherently possess the ability to produce heparosan compounds, or they may be modified to possess the ability to produce heparosan compounds. Microorganisms possessing the ability to produce heparosan compounds can be obtained, for example, by conferring the ability to produce heparosan compounds to the aforementioned microorganisms.

[0066] 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.

[0067] Proteins involved in the production of heparosan compounds include glycosyltransferases and heparosan efflux carrier proteins. In the host cells of this embodiment, 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 gene, and the copy number of the 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.

[0068] The microorganisms of this embodiment can be produced by any method known in the art. For example, the expression units described above are contained in the host cell either 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 the host cells with an expression vector using any method known in the art (e.g., competent cell method, electroporation method).

[0069] 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.

[0070] 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)].

[0071] In this embodiment, the expression vector may further include elements such as a terminator that functions in the host cell, a ribosome binding site, and a drug resistance gene, in addition to the minimum unit described above as the expression unit. Examples of drug resistance genes include resistance genes to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0072] 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.

[0073] 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, or 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.

[0074] The expression vector may also be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector may also be a commonly used expression vector. Examples of such expression vectors include 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 are used.

[0075] One embodiment of the microorganism of this embodiment is a host cell containing an expression unit comprising DNA encoding the protein of this embodiment and a promoter operably linked thereto.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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: 57). 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.

[0081] 4. Method for Producing Modified Heparosan Compounds The method for producing modified heparosan compounds according to this embodiment (hereinafter also referred to as "this production method") is characterized by reacting the protein of this embodiment described above with the heparosan compound. This production method includes 2-O-sulfating the uronic acid residues of the heparosan compound in the presence of the protein of this embodiment described above. Examples of uronic acid residues include iduronic acid residues and glucuronic acid residues, but iduronic acid residues are preferred.

[0082] The protein of this embodiment exhibits excellent 2-O-sulfate transfer activity and high selectivity for iduronic acid residues in heparosan. Therefore, this manufacturing method allows for the production of modified heparosan compounds with structures close to those of nature, with higher efficiency than conventional methods.

[0083] Preferred performance indicators of the modified heparosan compounds obtained by this manufacturing method, based on NS-containing disaccharide analysis, include the following: • IdoA2SNS / Total NS2S (%): Preferably 90% or more, more preferably 92% or more, and even more preferably 94% or more. • Total NS2S / Total NS (%): Preferably 80% or more, more preferably 81% or more, and even more preferably 82% or more. These indicators are easily achieved by using K111E or modified K111N 2OST, supplying a sufficient amount of PAPS, and simultaneous reaction with C5-epimery.

[0084] The protein used in this manufacturing method may be from a microorganism containing the DNA encoding the protein of this embodiment, or it may be extracted and purified from a microorganism.

[0085] This manufacturing method is carried out in the presence of a sulfate group donor. Examples of sulfate group donors include 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and aryl sulfate compounds, with PAPS being preferred. The sulfate group donor may be a commercially available product or one that is manufactured as appropriate.

[0086] The method for producing the sulfate group donor is not particularly limited, and known methods can be used, for example. Instead of adding the sulfate group donor, a product of a microorganism capable of producing sulfate group donors may be added. The product of the microorganism will be described in detail in section (ii) Cellular Reaction Method below. Alternatively, instead of adding the sulfate group donor, a microorganism capable of producing sulfate group donors may be cultured together with the microorganism of this embodiment. The sulfate group donor may be supplied from the microorganism of this embodiment. This microorganism may be obtained using a host cell that inherently has the ability to produce sulfate group donors, or it may be modified to have that ability.

[0087] When PAPS is used as a sulfate group donor, it is preferable to supply PAPS by combining external addition with regeneration within the reaction system. Specifically, referring to Patent Document 3, a regeneration system is used that regenerates PAPS using ATP sulfate and APS kinase as substrates. This suppresses reaction termination due to PAPS degradation and depletion, and maintains both IdoA selectivity and total 2-O-sulfation degree.

[0088] In this specification, "acting the protein of this embodiment with a heparosan compound" means bringing the two into contact under conditions that allow the reaction to proceed, so that the catalytic action of the protein extends to the heparosan compound. Such "contact" includes, but is not limited to, the following: - Contact by mixing and stirring in solution (e.g., batch, fed-batch, or continuous flow) - Contact by flowing or immersing the heparosan compound solution over the protein immobilized on a solid support - Contact by coexisting the heparosan compound with a microorganism expressing and maintaining the protein (e.g., live cells, dormant cells, cell lysate, membrane fraction, or soluble fraction) or a product of the microorganism described later, in the same reaction system - Contact in simultaneous and sequential reactions (e.g., 2-O-sulfation in the simultaneous presence of C5-epimerase, or 2-O-sulfation after C5-epimerase treatment) Here, "action" includes the protein catalyzing the transfer reaction of a 2-O-sulfate group (sulfo group) to a uronic acid residue of the heparosan compound, which is the reaction substrate, and includes setting conditions under which the reaction can proceed (e.g., temperature, pH, ionic strength, cofactor PAPS concentration, reaction time, etc.).

[0089] In this specification, “in the presence of” means that the protein, and optionally a sulfate group donor (such as PAPS) and other enzymes (e.g., C5-epimerase), are present in a reaction system (e.g., a solution, suspension system, immobilized reactor, or culture system) in a form capable of exhibiting catalytic activity. “In the presence of” includes the following embodiments: - A mode in which the protein is included in the reaction system by adding a purified product, crude extract, cell-free extract, culture supernatant, immobilized enzyme, fusion protein, or tagged protein. - A mode in which a microorganism expressing the protein (e.g., live or resting cells) or a processed product of the microorganism described below is introduced into the reaction system to utilize intracellular or membrane-localized enzymatic activity. - A mode in which a heparosan compound (addition or biosynthesis) is simultaneously present in a culture system in which the protein is expressed in a host. - A mode in which PAPS is added externally or regenerated within the reaction system (ATP sulfaterase, APS kinase, etc.) and the 2-O-sulfate transfer activity of the protein can be expressed. - A mode in which a substrate solution flows in the vicinity of the protein immobilized on a solid support. Note that "in the presence of" includes free-diffusion, immobilized, and intracellular types, and also includes simultaneous and sequential addition.

[0090] In this specification, "effective amount" means the amount of each component, such as the protein, PAPS, and C5-epimerase, that allows the target 2-O-sulfation reaction to proceed detectably and reproducibly, and that achieves the desired composition ratio (e.g., IdoA2SNS / totalNS2S, totalNS2S / totalNS). The concentration, ratio, and timing of administration of each component can be appropriately optimized by those skilled in the art depending on the reaction scale, molecular weight of the substrate, degree of N-sulfation, and degree of epimerization.

[0091] The following are preferred embodiments of this manufacturing method. Preferably, a modified 2OST of SEQ ID NO: 55 (K111E) or SEQ ID NO: 56 (K111N) is used as the 2-O-sulfotransferase. Preferably, 2-O-sulfation is carried out simultaneously with C5-epimerization in the presence of PAPS and a protein having C5-epimerase activity (simultaneous reaction). The simultaneous reaction contributes to improving the IdoA2SNS ratio through the coordinated generation of IdoA residues and 2-O-sulfation. More preferably, a divalent metal ion (e.g., magnesium sulfate) is added to the reaction system and the pH is adjusted to preferably 6.5 to 7.5 with phosphate buffer (e.g., 50 mM dipotassium hydrogen phosphate). Preferred reaction indicators are conditions in which the IdoA2SNS / total NS2S ratio relative to total NS-containing disaccharides is preferably 94% or more, and the total NS2S / total NS ratio is preferably 82% or more. These indicators are calculated according to the measurement conditions described herein.

[0092] This manufacturing method is preferably carried out in the presence of a protein having C5-epimerase activity. The protein having C5-epimerase activity may be from a microorganism containing DNA encoding the protein having C5-epimerase activity, or it may be extracted and purified from a microorganism.

[0093] 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.

[0094] For example, when using immobilized enzymes, it is preferable to co-immobilize 2OST (K111E or K111N) and C5-epimerase on the same support. Examples of support materials include epoxidized polysaccharide resins, amination magnetic particles, and porous membranes. In a flow-through reaction, the residence time of the substrate solution is set to, for example, 5 to 60 minutes, and the process is carried out by recirculation until the desired degree of sulfation is reached.

[0095] 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.

[0096] (i) Enzymatic method: One embodiment of this manufacturing method includes 2-O-sulfation of uronic acid residues using the protein of this embodiment described above. For example, when recombinant protein is used as the protein of this embodiment, the recombinant protein can be obtained using a cell-free vector or from a microorganism that produces the protein of this embodiment. The protein of this embodiment can be used as unpurified, crude, or purified protein. These proteins may be used as immobilized proteins in the reaction.

[0097] The culture medium for culturing the microorganisms of this embodiment is well known and can be used, for example, by adding a carbon source, nitrogen source, vitamin source, etc. to a nutrient medium such as LB medium or a minimal medium such as M9 medium. Depending on the host, the microorganisms of this embodiment 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 protein 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 culturing.

[0098] The method for extracting the protein of this embodiment from the microorganism of this embodiment is not particularly limited, and known methods can be used, for example. Such methods include surfactant treatment, organic solvent treatment, sonication, mechanical grinding, and freeze-thaw treatment. These treatments can be used individually or in appropriate combinations.

[0099] 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 reverse-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.

[0100] An example of preferred reaction conditions in the enzymatic method is shown below. • Substrate (heparosan compound): N-sulfohepalosan and N-sulfated epimerized low molecular weight heparosan are preferred. The concentration is preferably 0.2 to 5.0 g / L, more preferably 0.5 to 2.0 g / L. • Sulfate group donor (PAPS): Preferably 1.0 to 10 mM, more preferably 3.0 to 5.0 mM. • Buffer solution / ion: Phosphate buffer, preferably 25 to 100 mM MgSO4. ４ , preferably 1 to 20 mM, more preferably 5 to 12 mM. • Enzyme amount: Preferably 0.05 to 1.0 mg / mL, more preferably 0.1 to 0.5 mg / mL as the total protein concentration of 2OST. C5-epimerase may be added in the same concentration range as needed. • Reaction temperature and time: Preferably 25 to 40°C, more preferably 37°C; preferably 4 to 48 hours, preferably 12 to 24 hours. Stirring preferably at 600 to 700 rpm. • Form: Can be solubilized enzyme, crude extract, or immobilized enzyme (resin, membrane, magnetic particles). For immobilization, a flow-through reaction (space velocity 0.1 to 2.0 h) －１ ) is preferable. Under the above conditions, it is easier to achieve both improvement in IdoA2SNS / allNS2S and maintenance (or improvement) of allNS2S / allNS.

[0101] (ii) Cellular Reaction Method One embodiment of the present manufacturing method includes a method in which the microorganism of this embodiment is treated with a drug or the like to make the cell plasma membrane permeable (hereinafter referred to as the treated microorganism), and an enzyme or substrate is incorporated into the treated product, and the uronic acid residue is 2-O-sulfated. 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 microorganism is preferably a dormant cell that has lost its ability to proliferate due to treatment to impart membrane permeability.

[0102] Examples of microbial treatment products include microbial surfactant-treated products, microbial solvent-treated products, microbial freeze-thaw products, microbial enzyme-treated products, immobilized microbial products containing live cells that maintain the same function as microbial cultures as enzyme sources, microbial ultrasonic treatment products, and microbial mechanical grinding treatment products, with surfactant-treated or solvent-treated products being preferred.

[0103] 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.

[0104] Chemical treatments include, for example, methods using surfactants, organic solvents, and enzymes. As for surfactants, nonionic surfactants are preferred because they have less impact on proteins and the like (compared to ionic surfactants). Examples of such surfactants include digitonin, saponin, Triton X100, Triton X114, Tween 20, Tween 80, N,N-Bis(3-D-gluconamidepropyl)cholamide [BIGCHAP], N,N-Bis(3-D-gluconamidepropyl)deoxycholamide [Deoxy-BIGCHAP], NIKKOLBL-9EX [Polyoxyethylene(9)LaurylEther], Octanoyl-N-methylglucamide [MEGA-8], and benzalkonium chloride.

[0105] Examples of organic solvents include benzene, toluene, xylene, and other alcohols. Examples of enzymes include lysozyme and achromopeptidase.

[0106] 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, more generally 20 to 200 μg / ml, with a temperature of 2 to 37°C and a time of 1 to 30 minutes.

[0107] Examples of mechanical treatments include ultrasonic treatment and mechanical grinding.

[0108] (iii) Culture Method One embodiment of the present manufacturing method includes 2-O-sulfation of uronic acid residues in the presence of the microorganism of this embodiment. The uronic acid residues include iduronic acid residues and glucuronic acid residues in a mixed state, and the microorganism of this embodiment 2-O-sulfates the iduronic acid residues with higher selectivity than the glucuronic acid residues, thereby efficiently increasing the proportion of IdoA2SNS in the resulting 2-O-sulfated product.

[0109] The culture medium used in this embodiment is not particularly limited, as long as the microorganisms of this embodiment 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). As the culture medium, for example, a culture medium containing a carbon source, a nitrogen source, and other components selected from various organic and inorganic components as needed can be used. The types and concentrations of culture medium components may be appropriately set by those skilled in the art.

[0110] In this embodiment, the carbon source is not particularly limited as long as it can be assimilated by the bacteria of the present invention to produce modified heparosan 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.

[0111] 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.

[0112] 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.

[0113] 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.

[0114] In this embodiment, the culture conditions are not particularly limited, as long as the bacteria of the present invention can grow and the modified heparosan compound is produced and accumulated. Culture can be carried out, for example, under the usual conditions used for bacterial culture. The culture conditions may be set as appropriate by those skilled in the art.

[0115] 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.

[0116] By culturing a microorganism according to one aspect of the present invention as described above, the modified heparosan compound accumulates in the microorganism and in at least one of the culture medium.

[0117] The following are preferred embodiments of the culture method: • Composition: Express 2OST (K111E or K111N), C5-epimerase, N-deacetylase, and N-sulfotransferase in a host cell, and continuously carry out N-deacetylated heparosan biosynthesis, N-sulfoheparosan biosynthesis, C5-epimery, and 2-O-sulfation in a culture system. Alternatively, express 2OST (K111E or K111N), C5-epimerase, and N-sulfotransferase in a host cell, and continuously carry out N-sulfoheparosan biosynthesis, C5-epimery, and 2-O-sulfation in a culture system. The host cells are preferably microorganisms capable of producing heparosan compounds. • Culture medium: Add a carbon source (e.g., glucose or glycerol), a nitrogen source, trace metals, and vitamins to the M9 minimal medium. Sulfates and ATP precursors are supplied as needed to promote the intrinsic production of PAPS. Heparosan compounds that serve as substrates may also be added as needed. Conditions: Preferably 25-37°C, batch or fed-add, preferably 8-72 hours. When using an inducible promoter, an appropriate inducer (e.g., IPTG, arabinose) is added. Dissolved oxygen is maintained and the balance between specific growth rate and target glycosylation modification is optimized. Target indicators: For the extract at the end of culture, conditions are preferred in which IdoA2SNS / totalNS2S relative to total NS-containing disaccharides is preferably 92-96%, and total NS2S / total NS is preferably 81-85%. This embodiment is excellent in process consolidation and scale-up suitability.

[0118] (Heparosan Compounds) In the manufacturing method of this embodiment, a predetermined target substance can be produced by using a heparosan compound as a starting material. In the present invention, "heparosan compound" means heparosan or a heparosan derivative.

[0119] Heparosan is a disaccharide with a repeating structure consisting of a β-D-glucuronic acid (GlcA) residue and an N-acetyl-α-D-glucosamine (GlcNAc) residue [→4)-β-D-GlcA-(1→4)-α-D-GlcNAc-(1→] ｎ It is a polysaccharide composed of the following. Heparosan can be prepared, for example, by fermentation using microorganisms capable of producing heparosan (e.g., International Publication No. 2015 / 050184).

[0120] Examples of heparosan derivatives include heparosan having one or more modifications selected from the group consisting of (1) to (5) below.

[0121] (1) N-deacetylation of glucosamine residues; (2) depolymerization; (3) N-sulfation of glucosamine residues; (4) C5-epimery of uronic acid residues; or (5) 2-O-sulfation of uronic acid residues

[0122] In this specification, "uronic acid" means glucuronic acid (GlcA) or iduronic acid (IdoA).

[0123] In this embodiment, glucuronic acid is preferred as the "uronic acid residue" in (4) "C5-epimery of uronic acid residue". Therefore, in the C5-epimery of (4), it is preferable that iduronic acid is produced by isomerization of glucuronic acid.

[0124] In this embodiment, iduronic acid is preferred as the "uronic acid residue" in (5) "2-O-sulfation of uronic acid residue". Therefore, in the 2-O-sulfation of (5), it is preferable that the hydroxyl group at position 2 of iduronic acid is sulfated.

[0125] In one embodiment of this product, the starting material, the 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 (1) and (3). The N-sulfated heparosan compound may further have one or more modifications selected from (2) or (4) above.

[0126] 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.

[0127] In one embodiment of this product, 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 providing heparosan to the treatment described in (4) above. The epimerized heparosan compound may further have one or more modifications selected from the group consisting of (1) to (3) above.

[0128] In one embodiment of this product, the starting material, a heparosan compound, may be a low-molecular-weight heparosan compound. In this specification, "low-molecular-weight" means that the material is 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 heparosan to the treatment described in (2) above. A low-molecular-weight heparosan compound may further have one or more modifications selected from the group consisting of (1), (3), and (4) above.

[0129] In one embodiment of this product, the heparosan compound used as the starting material may be an N-sulfated epimerized low-molecular-weight heparosan compound. The terms "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 heparosan to the treatments described in (1) to (4) above.

[0130] In one embodiment of this product, the heparosan compound used as the starting material 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 heparosan to the treatments described in (1) to (4) above.

[0131] In one embodiment of this product, the heparosan compound used as the starting material may be N-deacetylated heparosan. In this specification, "deacetylation" means that the acetyl group is removed from the amino group at the C2 position of the glucosamine residue. N-deacetylated heparosan can be obtained by subjecting heparosan to the treatment described in (1) above. Specifically, N-deacetylated heparosan can be obtained by chemical treatment using sodium hydroxide, for example, referring to Appl Microbiol Biotechnol., 2011, Jul; 91(1):91-9, etc.

[0132] (Modified Heparosan Compounds) As the modified heparosan compound produced by the production method of this embodiment, a modified heparosan compound containing a 2-O-sulfated uronic acid residue is preferred. Examples of modified heparosan compounds containing a 2-O-sulfated uronic acid residue 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.

[0133] One embodiment of the manufacturing method of this embodiment involves subjecting heparosan to a process including (s1) N-deacetylation of glucosamine residues, (s2) depolymerization, (s3) N-sulfation of glucosamine residues, (s4) C5-epimery of uronic acid residues, (s5) 2-O-sulfation of uronic acid residues, (s6) 6-O-sulfation of glucosamine residues, and (s7) 3-O-sulfation of glucosamine residues to produce heparan sulfate, wherein (s5) 2-O-sulfation of uronic acid residues is carried out in the presence of the protein of this embodiment described above.

[0134] In a preferred configuration of the process sequence, 2-O-sulfation (s5) is preferably carried out simultaneously with C5-epimerization (s4). Simultaneous carryout tends to rapidly 2-O-sulfate the iduronic acid residues generated by C5-epimerization, suppressing excessive 2-O-sulfation of glucuronic acid residues. As a result, the proportion of IdoA2SNS increases, making it easier to obtain a modified heparosan compound that closely resembles the structure of natural heparin.

[0135] When linking each step, continuous processing is preferred, where the reaction solution is passed directly to the next step. If necessary, enzyme inactivation, pH readjustment, salt intensity adjustment, and removal of low-molecular-weight by-products by membrane separation or ultrafiltration may be incorporated. When immobilized enzymes are used, process integration can be achieved by connecting reactors in series.

[0136] In one embodiment of the manufacturing method of this embodiment, it is preferable that the modified heparosan compound is produced in the presence of the protein of this embodiment, the heparosan compound, 3'-phosphoadenosine-5'-phosphosulfate (PAPS), and a protein having C5-epimerase activity.

[0137] The heparosan treatment 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) low molecular weight reduction 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. Two or more of the above treatments can be carried out simultaneously or separately.

[0138] 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.

[0139] As described above, the following configurations are disclosed in this specification: 1. A protein comprising a catalytic domain of a protein consisting of an amino acid sequence in which the 111th amino acid residue of the amino acid sequence shown in Sequence ID No. 1 is substituted with an L-glutamic acid residue or an L-asparagine residue, and having 2-O-sulfotransfer activity, wherein the selectivity for the iduronic acid residue in the 2-O-sulfotransfer activity is higher than that of a protein comprising a catalytic domain of a protein consisting of an amino acid sequence in which the 111th amino acid residue of the amino acid sequence shown in Sequence ID No. 1 is substituted with an L-glutamic acid residue or an L-asparagine residue, and having 2-O-sulfotransfer activity, wherein the selectivity for the iduronic acid residue in the 2-O-sulfotransfer activity is higher than that of a protein comprising a catalytic domain of a protein consisting of an amino acid sequence in which the 111th amino acid residue of the amino acid sequence shown in Sequence ID No. 1 is not an L-glutamic acid residue or an L-asparagine residue. [A1] A protein containing a catalytic domain of a protein consisting of an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted, inserted and / or added from the amino acid sequence shown in Sequence ID No. 1. [A2] A protein containing a catalytic domain of a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in Sequence ID No. 1. 3. A protein having 2-O-sulfate transfer activity, wherein the protein described in [B1] or [B2] below is the original protein, and the original protein contains a catalytic domain of a protein consisting of an amino acid sequence in which the amino acid residue corresponding to the 111th amino acid sequence shown in Sequence ID No. 1 in the amino acid sequence of the original protein is substituted with an L-glutamic acid residue or an L-asparagine residue, and the selectivity for the iduronic acid residue in the 2-O-sulfate transfer activity is higher than that of the original protein.[B1] A protein comprising a catalytic domain of a protein having 2-O-sulfate transfer activity, wherein 1 to 20 amino acid residues from the amino acid sequence shown in SEQ ID NO: 1 are deleted, substituted, inserted and / or added. [B2] A protein comprising a catalytic domain of a protein having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1, and having 2-O-sulfate transfer activity. 4. DNA encoding the protein described in any one of 1 to 3 above. 5. A microorganism containing DNA encoding the protein described in any one of 1 to 3 above. 6. A method for producing a modified heparosan compound, comprising reacting the protein described in any one of 1 to 3 above with the heparosan compound. 7. A method for producing a modified heparosan compound, comprising 2-O-sulfating the uronic acid residue of the heparosan compound in the presence of the protein described in any one of 1 to 3 above. 8. The method for producing the heparosan compound according to 7 above, wherein the uronic acid residue includes an iduronic acid residue and a glucuronic acid residue. 9. The method for producing the modified heparosan compound according to 7 or 8, wherein the production of the modified heparosan compound is carried out in the presence of a protein having 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and C5-epimerase activity. 10. The method for producing the modified heparosan compound according to any one of 7 to 9, wherein the heparosan compound is N-sulfohepalosan. 11. The method for producing the modified heparosan compound according to any one of 7 to 10, wherein the modified heparosan compound is heparin.

[0140] [Analysis Example] In the example, high-performance liquid chromatography Prominence (Shimadzu Corporation) was used to analyze the NS-containing disaccharides. The analytical conditions are as follows.

[0141] [Analysis Conditions] Column: YMC-Triart C18 (YMC) Column temperature: 25°C Mobile phase: (Mobile phase A) 50 mM ammonium formate buffer (pH 4.5) (Mobile phase B) 100% acetonitrile Mixing ratio of mobile phase A and mobile phase B: (0-1 min) 20:80 (1-27 min) Slope from 20:80 to 46:54 (27-28 min) Slope from 46:54 to 90:10 (28-29 min) 90:10 (29-30 min) Slope from 90:10 to 20:80 (30-50 min) 20:80 Flow rate: 0.5 mL / min Detector: SPD-20A (Shimadzu Corporation) (365 nm UV absorption)

[0142] 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.

[0143] [Example 1] Preparation of microorganisms expressing 2OST (1) Preparation of plasmids for chaperone protein expression Using DNA consisting of the base sequences shown in "Primer Set" in Table 1 as a primer set and the DNA described in "Template" in Table 1 as a template, PCR was performed to obtain each amplified DNA fragment.

[0144]

[0145] The chromosomal DNA of Escherichia coli BL21 (DE3) strain was prepared by standard method. The amplified DNA fragment GroES-GroEL contains the ORF sequences of the GroES gene and the GroEL gene on the chromosomal DNA of Escherichia coli BL21 (DE3) strain. The amplified DNA fragment AraC_ParaB is pKD46 [Datsenko, K. A., Warner, B. L., Proceedings of the National Academy of Science of the United States of America, Vol. 97.] Includes the ORF sequence of the AraC gene and the full-length region of the AraBAD promoter as shown in [6640-6645 (2000)].

[0146] Using DNA consisting of the nucleotide sequences represented by SEQ ID NOs: 7 and 8 as a primer set, PCR was performed on plasmid pCDFDuet-1 (Novagen) as a template to obtain a vector fragment of approximately 2.1 kb.

[0147] The GroES-GroEL fragment, AraC_ParaB fragment, and vector fragment obtained above were ligated using an In-Fusion cloning kit (manufactured by Takara Bio Inc.) to obtain the chaperone protein expression plasmid pCDFDuet1-AraC-ParaB-GroES-GroEL (hereinafter referred to as pGro-Sm).

[0148] (2) Preparation of a plasmid for wild-type 2OST expression A DNA sequence (SEQ ID NO: 10) with codon optimization for expression in E. coli was prepared by artificial synthesis of the base sequence (SEQ ID NO: 9) of the gene encoding the catalytic domain (R51-N356) of 2-O-sulfotransferase isoform 1 (2OST) derived from Chinese hamsters. In SEQ ID NO: 10, an MBP tag was added to the N-terminus of the catalytic domain of 2OST, and a His tag was added to the C-terminus.

[0149] Using the DNA represented by Sequence ID No. 10 as a template, PCR was performed using the DNA consisting of the nucleotide sequences represented by Sequence ID Nos. 12 and 13 as a primer set to obtain a 2OST fragment. Following the Ligation-independent cloning method [Methods Mol Biol. 2009; 498: 105-115], the obtained 2OST fragment was cloned using pET-His6-MBP-TEV-LIC (Addgene), thereby obtaining the wild-type 2OST expression plasmid p2OST.

[0150] (3) Preparation of modified 2OST expression plasmid A modified 2OST expression plasmid was prepared by replacing the 111th lysine residue in the amino acid sequence of the Chinese hamster-derived 2OST represented by Sequence ID No. 1 with another amino acid, using the following procedure.

[0151] Using the plasmid p2OST obtained in (2) above as a template, fragments 1 and 2 for various amino acid residue substitutions were amplified using a primer set consisting of DNA with the base sequence shown in Table 1 as either the "primer set for amplifying fragment 1" or the "primer set for amplifying fragment 2".

[0152]

[0153] By ligating the various amino acid residue substitution fragments 1 and 2 obtained above using an In-Fusion cloning kit (manufactured by Takara Bio Inc.), a total of 18 modified 2OST expression plasmids were created.

[0154] (4) Production of microorganisms expressing wild-type 2OST or modified 2OST Using the wild-type 2OST expression plasmid p2OST obtained in (2) above and a total of 18 modified 2OST expression plasmids having each mutation site obtained in (3) above, Origami-B (DE3) (Novagen) was transformed together with the pGr-Sm obtained in (1) above to obtain transformants having each plasmid.

[0155] [Comparative Example] Preparation of a microorganism expressing K111A modified 2OST A plasmid for expressing K111A modified 2OST was prepared by replacing the 111th lysine residue in the amino acid sequence of the Chinese hamster-derived 2OST represented by Sequence ID No. 1 with an alanine residue, using the following procedure.

[0156] Using the plasmid p2OST obtained in (2) above as a template, PCR was performed using DNA consisting of the base sequences represented by SEQ ID NOs. 14 and 52, and SEQ ID NOs. 15 and 53, as primer sets, to obtain K111A fragment 1 and K111A fragment 2.

[0157] The obtained K111A fragment 1 and K111A fragment 2 were ligated using an In-Fusion cloning kit (manufactured by Takara Bio Inc.) to create a modified K111A 2OST expression plasmid.

[0158] Using the obtained plasmid, Origami-B (DE3) (Novagen) was transformed together with pGr-Sm obtained in Example 1(1) to obtain transformants having K111A modified 2OST.

[0159] [Example 2] Evaluation of 2-O-sulfate transfer activity of modified 2OST A total of 18 transformants having the modified 2OST prepared in Example 1 (4) were used to evaluate the 2-O-sulfate transfer activity of each modified 2OST to N-sulfohepalosan. For control, a transformant having the wild-type 2OST prepared in Example 1 (4) and a transformant having the K111A modified 2OST prepared in the comparative example were used.

[0160] The 2-O-sulfotransfer activity was evaluated using the following procedure. First, each of the two OST enzymes was used in an enzymatic reaction with N-sulfohepalosan as a substrate in the presence of C5-epimerase to produce N-sulfated 2-O-sulfated heparosan. Next, referring to International Publication No. 2017 / 115675, the obtained N-sulfated 2-O-sulfated heparosan was decomposed with sodium nitrite to obtain NS-containing disaccharides. The obtained NS-containing disaccharides were analyzed, and the ratio of GlcA2SNS and IdoA2SNS in the NS-containing disaccharides was confirmed to evaluate the 2-O-sulfotransfer activity of each of the two OSTs to GlcA residues or IdoA residues.

[0161] (1) Preparation of enzyme samples for activity evaluation Various transformants were inoculated into large test tubes containing 5 mL of LB medium containing 100 mg / L ampicillin and 50 mg / L streptomycin, and incubated with shaking at 30°C for 16 hours. 500 μL of the resulting culture solution was inoculated into a flask containing 40 mL of LB medium containing 100 mg / L ampicillin and 50 mg / L streptomycin, and incubated at 30°C.

[0162] After the bacterial cells reached an OD660nm range of 0.4–0.8, isopropyl-β-D-thiogalactopyranoside (IPTG) and arabinose were added to final concentrations of 1 mL / L and 200 mg / L, respectively, and the culture was continued with shaking for 5 hours. After the culture was complete, the culture medium was centrifuged to obtain wet bacterial cells. The obtained wet bacterial cells were adjusted to 100 g / L, and then the cells were disrupted by sonication. The resulting lysate was used as the enzyme sample for activity evaluation.

[0163] (2) Preparation of C5-epimerase enzyme solution Escherichia coli expressing human-derived C5-epimerase was prepared in accordance with Patent Document 3. The obtained C5-epimerase-expressing Escherichia coli was cultured using the method described in Patent Document 3, and the culture solution was centrifuged to obtain wet bacterial cells.

[0164] The obtained wet bacterial cells were prepared to a concentration of 100 g / L, and then the cells were disrupted by sonication. The resulting lysate was used as the C5-epimerase enzyme solution.

[0165] (3) Evaluation of the 2-O-sulfotransferase activity of modified 2OST. N-sulfohepalosan, the substrate for the enzyme reaction, and PAPS, the sulfate group donor, were prepared according to previously reported information (Chem Biol, 2007, 14:986-993; J. Org. Chem., 2000, 65:5565-5574).

[0166] A reaction solution consisting of 30 μL of the enzyme sample obtained in (1) above, 30 μL of the C5-epimerase enzyme solution obtained in (2), 50 mmol / L of dipotassium hydrogen phosphate, 4.4 mmol / L of PAPS, 1 g / L of N-sulfohepalosan, and 10.1 mmol / L of magnesium sulfate was prepared in 0.3 mL, and the N-sulfated 2-O-sulfated heparosan synthesis reaction was carried out at 37°C and 660 rpm for 22 hours. At the end of the reaction, the mixture was heated at 80°C for 10 minutes to inactivate the enzyme.

[0167] After the enzymatic reaction was complete, 400 μL of 389.2 g / L citric acid solution and 200 μL of 49.5 g / L sodium nitrite solution were added to 200 μL of the obtained sample, mixed well, and reacted for 2 hours at 65°C and 600 rpm with shaking. Then, 400 μL of 20.4 g / L (acetonitrile) DNPH solution was added, and reacted for 2 hours at 45°C and 600 rpm with shaking.

[0168] After the reaction was complete, the NS-containing disaccharides produced by nitrite decomposition were analyzed by HPLC. Table 3 shows the proportion of NS2S, GlcA2SNS, or IdoA2SNS in the total NS-containing disaccharides in N-sulfated 2-O-sulfated heparosan produced by each 2OST enzyme reaction, and the 2-O-sulfate transfer activity of each 2OST enzyme to IdoA (IdoA2SNS / total NS2S). Table 4 shows the values ​​for each modified 2OST compared to the wild-type 2OST [WT(K111)] for each value shown in Table 3.

[0169] In Tables 3 and 4, "IdoA2SNS / Total NS2S" represents the percentage obtained by dividing the ratio of IdoA2SNS to total NS-containing disaccharides by the value of total NS2S to total NS-containing disaccharides.

[0170]

[0171]

[0172] As shown in Table 3, the K111A modified 2OST described in Non-Patent Literature 2 showed an improvement in IdoA2SNS / totalNS2S ratio from 89% to 91%, while the NS2S ratio decreased from 81.1% to 76.6%. From these results, it was confirmed that the K111A mutation improves selectivity for IdoA residues, while decreasing the 2-O-sulfate transfer activity itself.

[0173] On the other hand, as shown in Tables 3 and 4, the modified 2OSTs of K111E or K111N improved the IdoA2SNS ratio while maintaining 2-O-sulfate transfer activity.

[0174] From the above, it was found that by using a modified 2OST in which the 111th lysine residue in the amino acid sequence of 2OST is replaced with a glutamic acid residue or an asparagine residue, it is possible to preferentially 2-O-sulfate the IdoA residue while maintaining a high 2-O-sulfate transfer activity equivalent to or better than that of wild-type 2OST.

[0175] 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. 2024-208605, filed on 29 November 2024, which is incorporated by reference in its entirety. All references incorporated herein are incorporated as a whole.

[0176]

Claims

1. A protein comprising a catalytic domain of an amino acid sequence in which the 111th amino acid residue of the amino acid sequence shown in Sequence ID No. 1 is substituted with an L-glutamic acid residue or an L-asparagine residue, and which has 2-O-sulfate transfer activity, wherein the selectivity for the iduronic acid residue in the 2-O-sulfate transfer activity is higher than that of a protein comprising a catalytic domain of an amino acid sequence shown in Sequence ID No.

1.

2. A protein described in [A1] or [A2] below, which contains a catalytic domain of a protein consisting of an amino acid sequence in which the amino acid residue corresponding to the 111th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is an L-glutamic acid residue or an L-asparagine residue, and which has 2-O-sulfate transfer activity, and which has higher selectivity for the iduronic acid residue in the 2-O-sulfate transfer activity compared to a protein containing a catalytic domain of a protein consisting of an amino acid sequence in which the amino acid residue corresponding to the 111th amino acid residue of the amino acid sequence shown in SEQ ID NO: 1 is other than an L-glutamic acid residue or an L-asparagine residue. [A1] A protein containing a catalytic domain of a protein consisting of an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted, inserted and / or added from the amino acid sequence shown in SEQ ID NO:

1. [A2] A protein containing a catalytic domain of a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO:

1.

3. A protein having 2-O-sulfotransfer activity, wherein the protein described in [B1] or [B2] below is the original protein, and the original protein contains a catalytic domain of a protein consisting of an amino acid sequence in which the amino acid residue corresponding to the 111th amino acid sequence shown in SEQ ID NO: 1 in the amino acid sequence of the original protein is substituted with an L-glutamic acid residue or an L-asparagine residue, and the selectivity for the iduronic acid residue in the 2-O-sulfotransfer activity is higher than that of the original protein. [B1] A protein having 2-O-sulfotransfer activity, containing a catalytic domain of a protein consisting of an amino acid sequence in which 1 to 20 amino acid residues are deleted, substituted, inserted and / or added from the amino acid sequence shown in SEQ ID NO: 1 [B2] A protein having 2-O-sulfotransfer activity, containing a catalytic domain of a protein consisting of an amino acid sequence having 80% or more identity with the amino acid sequence shown in SEQ ID NO: 1 4. DNA encoding the protein according to any one of claims 1 to 3.

5. A microorganism comprising DNA encoding a protein according to any one of claims 1 to 3.

6. A method for producing a modified heparosan compound, comprising reacting a heparosan compound with the protein described in any one of claims 1 to 3.

7. A method for producing a modified heparosan compound, comprising 2-O-sulfating a uronic acid residue of a heparosan compound in the presence of the protein described in any one of claims 1 to 3.

8. The method for producing the product according to claim 7, wherein the uronic acid residue includes an iduronic acid residue and a glucuronic acid residue.

9. The method for producing the modified heparosan compound according to claim 7, wherein the production of the modified heparosan compound is carried out in the presence of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) and a protein having C5-epimerase activity.

10. The method for producing the product according to claim 7, wherein the heparosan compound is N-sulfohepalosan.

11. The method for producing the modified heparosan compound according to claim 7, wherein the modified heparosan compound is heparin.

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