Method for producing microorganism having N-deacetylation activity and N-sulfation activity, method for producing heparan derivative compound, and microorganism having N-deacetylation activity and N-sulfation activity

Abstract

The present invention relates to a method for producing a microorganism having N-deacetylation activity and N-sulfation activity, the method comprising a step (I) of modifying the nucleotide sequence of DNA encoding an N-deacetylase / N-sulfotransferase, and a step (II) of introducing DNA containing the nucleotide sequence modified in step (I) into the microorganism in an expressible manner, wherein the step (I) is a step of codon optimization according to the frequency of use of codons of a biological species different from the microorganism. Furthermore, the present invention relates to a method for producing a microorganism having N-deacetylation activity and N-sulfation activity, the method comprising the step (i) of expressing a specific protein in the microorganism.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for producing a microorganism having N-deacetylation activity and N-sulfation activity, a method for producing a heparosan derivative compound, and a microorganism having N-deacetylation activity and N-sulfation activity. BACKGROUND

[0002] Heparin is a sulfated polysaccharide, and is an anticoagulant used for treating thromboembolism and disseminated intravascular coagulation syndrome, preventing coagulation during artificial dialysis and extracorporeal circulation, and the like. In industry, heparin is extracted and purified from animal organs, mainly from the intestinal mucosa of pigs.

[0003] Since the heparin adulteration incident in 2008 in which a large number of deaths were caused by contamination of pig-derived heparin with impurities such as oversulfated chondroitin sulfate, production of non-animal-derived heparin under controlled production and quality-controlled production (NPL 1) has been required. As a specific example, a method for producing heparin having a structure and anticoagulant activity equivalent to that of pig-derived products by chemically and enzymatically deacetylating and sulfating heparosan (a microbial capsular polysaccharide) has been reported (PTL 1 and PTL 2).

[0004] In the natural process of heparin biosynthesis in vivo, the modification that occurs after the formation of the heparosan-like disaccharide repeat sequence main chain is the de-N-acetylation and N-sulfation of glucosamine, and these two reactions are carried out by the action of one polypeptide, N-deacetylase / N-sulfotransferase (hereinafter referred to as "NDST") (NPL 1).

[0005] NDST is a family of 4 isozymes that are 65% to 80% identical to each other and cloned from different mammals (NPL 2). NDST contains an N-deacetylase domain and an N-sulfatase domain, and its activity differs depending on the source and isozyme (NPL 2). Expression of NDST activity has been achieved in various eukaryotic systems such as COS cells, HEK 293 cells, CHO cells, and the like, as well as in insect cells and yeast (NPL 3 and NPL 4). On the other hand, although expression of the N-sulfatase domain in Escherichia coli has been reported, expression of active full-length NDST, especially the N-deacetylase domain, in bacteria has not been achieved (NPL 5). In recent years, a research group at Jiangnan University has published documents and patents on expression of NDST activity in Pichia pastoris, but it is also described therein that it is difficult to confirm the deacetylation activity of NDST in Escherichia coli (PTL 3 and NPL 6).

[0006] LIST OF CITATIONS

[0007] PATENT LITERATURE

[0008] PTL 1: US 8,771,995 B

[0009] PTL 2: WO 2018 / 048973

[0010] PTL 3: CN 107384990 B

[0011] Non-Patent Literature

[0012] NPL 1: Advanced Drug Delivery Reviews 97 (2016) 237-249

[0013] NPL 2: Journal of Biological Chemistry 276 (2001) 5876-5882

[0014] NPL 3: Proceedings of the National Academy of Sciences of the United States of America 90 (1993) 3885-3888

[0015] NPL 4: Glycobiology 12 (2004) 1217-1228

[0016] NPL 5: FEBS Letters 433 (1998) 211-214

[0017] NPL 6: Green Chemistry 24 (2022) 3180-3192 SUMMARY

[0018] TECHNICAL PROBLEM

[0019] The N-deacetylation reaction of heparin alkali can also be performed by a chemical method, but if it can be performed in a living organism, it can be performed together with a subsequent O-sulfation reaction, which is considered to be more desirable in the production of heparin of non-animal origin (NPL 6). Expression of N-deacetylation reaction activity has been achieved in Pichia pastoris. On the other hand, expression of N-deacetylation reaction activity has not been achieved in Escherichia coli, in which expression of sulfotransferase has been achieved many times, and expression of the activity in bacteria including E. coli is expected.

[0020] In view of this, an object of the present application is to provide a new method of producing a microorganism having N-deacetylation activity and N-sulfation activity, and the microorganism.

[0021] Heterologous expression of genes can sometimes be improved by optimizing the nucleotide sequence of the gene according to the codon usage frequency of the host, for example, in NPL 6, the expression of various sulfotransferases was codon-optimized. This takes advantage of the fact that even if the amino acid codons are the same, the amount of tRNA varies depending on the host, and in view of this property, codon optimization according to the codon usage frequency of the host is natural, and it is generally considered that optimization according to the codon usage frequency of living organisms of other species would be a suboptimal choice. For example, the codon usage frequency of the budding yeast and the codon usage frequency of Escherichia coli are different, and in the case of arginine codons, E. coli prefers CGU or CGC, but the budding yeast prefers the AGA codon, which is rare in E. coli. Therefore, when heterologous expression is performed using E. coli as the host, there are few attempts at codon optimization according to the budding yeast.

[0022] Technical Solution

[0023] To achieve the above object, the present inventors have conducted intensive studies, and as a result, it has been found that introduction of NDST into a microorganism using a sequence that has been codon-optimized according to different biological species can produce a microorganism having N-deacetylation activity and N-sulfation activity. In addition, it has been found that by expressing a specific protein in a microorganism, a microorganism having N-deacetylation activity and N-sulfation activity can be produced.

[0024] That is, the present application relates to the following.

[0025] ((1)) A method of producing a microorganism having N-deacetylation activity and N-sulfation activity, the method comprising:

[0026] Step (I): modifying the nucleotide sequence of DNA encoding an N-deacetylase / N-sulfotransferase; and

[0027] Step (II): introducing the DNA containing the nucleotide sequence modified in Step (I) into a microorganism in an expressible manner, wherein

[0028] The Step (I) is a step of codon optimization according to the codon usage frequency of a biological species different from the microorganism.

[0029] ((2)) The method of producing a microorganism having N-deacetylation activity and N-sulfation activity according to the above ((1)), wherein the microorganism is a bacterium, and the biological species is a budding yeast.

[0030] ((3)) The method for producing a microorganism having N-deacetylation activity and N-sulfation activity according to the above ((2)), wherein the DNA containing the modified nucleotide sequence is the DNA in (A-1) or (A-2):

[0031] (A-1) DNA containing a nucleotide sequence represented by SEQ ID NO: 6; or

[0032] (A-2) DNA containing a nucleotide sequence having 80% or more identity with the nucleotide sequence represented by SEQ ID NO: 6 and encoding a protein having N-deacetylation activity and N-sulfation activity.

[0033] ((4)) A method for producing a heparin-alkane derivative compound, the method comprising step (III): producing an N-deacetylated and N-sulfated heparin-alkane derivative compound from heparin-alkane in the presence of a microorganism having N-deacetylation activity and N-sulfation activity produced by the production method according to the above ((1)) or an extract thereof.

[0034] ((5)) The production method according to the above ((1)), wherein the microorganism is a bacterium of the genus Escherichia.

[0035] ((6)) The production method according to the above ((5)), wherein the bacterium of the genus Escherichia is Escherichia coli.

[0036] ((7)) The production method according to the above ((4)), wherein the heparin-alkane derivative compound is an N-sulfated heparin-alkane.

[0037] ((8)) A method for producing a microorganism having N-deacetylation activity and N-sulfation activity, the method comprising step (i): expressing in a microorganism any one protein selected from the group consisting of (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3), and (E-1) to (E-3):

[0038] (B-1) a protein containing an amino acid sequence represented by SEQ ID NO: 13;

[0039] (B-2) a protein containing an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 13 and having N-deacetylation activity and N-sulfation activity;

[0040] (B-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 13, and having N-deacetylating activity and N-sulfating activity;

[0041] (C-1) a protein containing the amino acid sequence represented by SEQ ID NO: 14;

[0042] (C-2) a protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 14, and having N-deacetylating activity and N-sulfating activity;

[0043] (C-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 14, and having N-deacetylating activity and N-sulfating activity;

[0044] (D-1) a protein containing the amino acid sequence represented by SEQ ID NO: 15;

[0045] (D-2) a protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 15, and having N-deacetylating activity and N-sulfating activity;

[0046] (D-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 15, and having N-deacetylating activity and N-sulfating activity;

[0047] (E-1) a protein containing the amino acid sequence represented by SEQ ID NO: 16;

[0048] (E-2) a protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 16, and having N-deacetylating activity and N-sulfating activity; and

[0049] (E-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 16, and having N-deacetylating activity and N-sulfating activity.

[0050] ((9)) The method for producing a microorganism having N-deacetylating activity and N-sulfating activity according to the above-described ((8)), wherein:

[0051] The step (i) is a step of introducing any one of DNA selected from the group consisting of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3), and (e-1) to (e-3) below into a microorganism in an expressible manner:

[0052] (b-1) DNA containing a nucleotide sequence represented by SEQ ID NO: 9;

[0053] (b-2) DNA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 13;

[0054] (b-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 9 and encoding a protein having N-deacetylation activity and N-sulfation activity;

[0055] (c-1) DNA containing a nucleotide sequence represented by SEQ ID NO: 10;

[0056] (c-2) DNA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 14;

[0057] (c-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 10 and encoding a protein having N-deacetylation activity and N-sulfation activity;

[0058] (d-1) DNA containing a nucleotide sequence represented by SEQ ID NO: 11;

[0059] (d-2) DNA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 15;

[0060] (d-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 11 and encoding a protein having N-deacetylation activity and N-sulfation activity;

[0061] (e-1) DNA containing a nucleotide sequence represented by SEQ ID NO: 12;

[0062] (e-2) DNA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 16; and

[0063] (e-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 12 and encoding a protein having N-deacetylation activity and N-sulfation activity.

[0064] ((10)) A method for producing a heparin alkane derivative compound, the method comprising step (ii): producing an N-deacetylated and N-sulfated heparin alkane derivative compound from heparin alkane in the presence of a microorganism having N-deacetylation activity and N-sulfation activity or an extract thereof produced by the production method according to the above-described ((8)).

[0065] ((11)) The production method according to the above-described ((8)), wherein the microorganism is a bacterium of the genus Escherichia.

[0066] ((12)) The production method according to the above-described ((11)), wherein the bacterium of the genus Escherichia is Escherichia coli.

[0067] ((13)) The production method according to the above-described ((10)), wherein the heparin alkane derivative compound is an N-sulfated heparin alkane.

[0068] ((14)) A microorganism having N-deacetylation activity and N-sulfation activity, which expresses any one protein selected from the group consisting of the following (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3), and (E-1) to (E-3):

[0069] (B-1) a protein containing the amino acid sequence represented by SEQ ID NO: 13;

[0070] (B-2) a protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 13 and having N-deacetylation activity and N-sulfation activity;

[0071] (B-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 13 and having N-deacetylation activity and N-sulfation activity;

[0072] (C-1) a protein containing the amino acid sequence represented by SEQ ID NO: 14;

[0073] (C-2) a protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 14 and having N-deacetylation activity and N-sulfation activity;

[0074] (C-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 14 and has N-deacetylation activity and N-sulfation activity;

[0075] (D-1) A protein containing the amino acid sequence represented by SEQ ID NO: 15;

[0076] (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 15 and has N-deacetylation and N-sulfation activities;

[0077] (D-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 15 and has N-deacetylation activity and N-sulfation activity;

[0078] (E-1) A protein containing the amino acid sequence represented by SEQ ID NO: 16;

[0079] (E-2) A protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylation and N-sulfation activities; and

[0080] (E-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylation activity and N-sulfation activity.

[0081] Beneficial effects

[0082] In the production method according to the first aspect of the invention, by introducing NDST, which has been codon-optimized according to different biological species, into microorganisms, microorganisms with superior N-deacetylation and N-sulfation activities compared with related technologies can be produced.

[0083] In the production method according to the second aspect of the invention, by expressing a protein having a specific amino acid sequence in microorganisms, microorganisms with superior N-deacetylation and N-sulfation activities compared to related technologies can be produced. Attached Figure Description

[0084] [ Figure 1 ] Figure 1The results show the analytical results of the amount of disaccharide composition in the NDST reaction experiment using heparinane as a substrate and without the addition of PAPS.

[0085] [ Figure 2 ] Figure 2 The results show the analytical results of the amount of disaccharide composition in the NDST reaction assay using deacetylated heparinane as a substrate and with the addition of PAPS.

[0086] [ Figure 3 ] Figure 3 The results show the analytical results of the amount of disaccharides in the NDST reaction experiment using heparane as a substrate and with the addition of PAPS. Detailed Implementation

[0087] The present invention will be described in detail below, but these descriptions show examples of preferred embodiments and the invention is not limited thereto.

[0088] The range of values ​​indicated by “to” is the range of values ​​before and after “to”. For example, “0 mass% to 100 mass%” means a range of 0 mass% or higher and 100 mass% or lower.

[0089] 1. A method for producing microorganisms with N-deacetylation and N-sulfation activities (1)

[0090] According to one embodiment of the present invention, a method for producing microorganisms having N-deacetylation and N-sulfation activities (hereinafter also referred to as "microorganisms expressing NDST activity") includes:

[0091] Step (I): Modify the nucleotide sequence of the DNA encoding NDST; and

[0092] Step (II): Introduce DNA containing the nucleotide sequence modified in step (I) into the microorganism in an expressible manner, wherein

[0093] Step (I) is a codon optimization step based on the codon usage frequency of biological species different from the microorganism.

[0094] In this specification, "N-deacetylation" refers to, for example, the formation of an amino group by N-deacetylation of the N-acetyl group of an α-D-glucosamine residue of heparinane. Examples of N-deacetylation include partial N-deacetylation.

[0095] Furthermore, in this specification, "N-sulfation" refers to the sulfation of the amino group of, for example, the N-acetyl-D-glucosamine residue of heparan.

[0096] Examples of nucleotide sequences encoding NDST used in step (I) include the nucleotide sequence represented by SEQ ID NO: 1.

[0097] In step (I), which modifies the nucleotide sequence of the DNA encoding NDST, codon optimization is performed based on the frequency of codon usage in a biological species different from the microorganism in which the DNA was introduced (hereinafter also referred to as the "parental strain"). Generally, "codon optimization" refers to changing the codons for each amino acid forming the peptide to codons that are more frequently used in the biological species in which the DNA was introduced. In this invention, it has been surprisingly found that microorganisms expressing NDST activity can be obtained by performing codon optimization based on the frequency of codon usage in a biological species different from the microorganism in which the DNA was introduced. This result implies that this species-mismatched codon optimization achieves active expression of NDST by slowing protein expression and avoiding accumulation in the cell.

[0098] Here, "codon optimization" does not necessarily mean changing the codons of 100% of the amino acids in the polypeptide's amino acid sequence, but rather changing the codons corresponding to at least one amino acid. However, it is preferable to change 50% or more of the codons.

[0099] In step (I) above, preferably, at least one codon among the codons corresponding to the nucleotide sequence of the DNA encoding NDST is optimized based on the codon usage frequency of biological species different from the parent strain.

[0100] The biological species that differ from the microorganism (parental strain) into which DNA is introduced in step (II) above are preferably yeast, insect cells, and filamentous fungi, with budding yeast being particularly preferred based on previous results of heterologous expression. The frequently used codons in these biological species can be identified, for example, through a codon usage database (https: / / www.kazusa.or.jp / codon / ). Those skilled in the art are familiar with codon optimization methods for each biological species.

[0101] In this specification, the parent strain refers to the original strain that will undergo gene modification, transformation, etc. In particular, in the "Method for producing microorganisms with N-deacetylation activity and N-sulfation activity (1)", the parent strain of the microorganisms with N-deacetylation activity and N-sulfation activity of the present invention refers to the strain in which DNA containing the modified nucleotide sequence has been introduced in an expressible manner in step (II) above.

[0102] From the viewpoint of ease of industrial cultivation, the microbial species (parental strain) into which DNA is introduced in an expressible manner in step (II) above is preferably a bacterium belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, Pseudomonas, etc., more preferably a bacterium of the genus Escherichia, and particularly preferably Escherichia coli.

[0103] Specific, non-limiting examples of *Escherichia coli* include *Escherichia coli* Origami B(DE3) (manufactured by Novagen), *Escherichia coli* BL21 codon plus, *Escherichia coli* XL1-Blue, *Escherichia coli* XL2-Blue (both manufactured by Agilent Technologies), *Escherichia coli* BL21(DE3) pLysS (manufactured by Merck Millipore), *Escherichia coli* BL21, *Escherichia coli* DH5α, *Escherichia coli* HST08 Premium, *Escherichia coli* HST02, *Escherichia coli* HST04 dam- / dcm-, *Escherichia coli* JM109, *Escherichia coli* HB101, *Escherichia coli* CJ236, *Escherichia coli* BMH 71-18 mutS, *Escherichia coli* MV1184, *Escherichia coli* TH2 (all manufactured by Takara Bio), and *Escherichia coli* W... (ATCC9637), Escherichia coli B (ATCC23226), Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG 1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli K5 and Escherichia coli Nissle1917.

[0104] The parental strain used in this invention can be a strain that inherently possesses the ability to produce heparin, or a strain that has been modified to possess the ability to produce heparin. As described above, by endowing microorganisms with the ability to produce heparin, microorganisms capable of producing heparin can be obtained.

[0105] Referring to Metabolic Engineering, 2012, 14, pp. 521-527, Carbohydrate Research, 2012, 360, pp. 19-24, US9,975,928B, etc., the ability to produce heparan can be conferred by introducing a gene encoding a protein involved in heparan production. Examples of proteins involved in heparan production include glycosyltransferases and heparan efflux carrier proteins. In the parental strain used in this invention, one type of gene may be introduced, or two or more types of genes may be introduced. The introduction of genes can also be achieved by introducing a vector containing the gene into the host. For example, a DNA fragment containing the target gene is ligated into a vector that functions in the host to construct an expression vector for the gene, and the host is transformed with this expression vector, thereby increasing the copy number of the gene. The DNA fragment containing the target gene can be obtained, for example, by PCR, using the genomic DNA of a microorganism with the target gene as a template. There are no particular limitations on the transformation method; conventionally known methods can be used.

[0106] In step (II), DNA containing the nucleotide sequence modified in step (I) is introduced into the microorganism in an expressible manner, preferably using recombinant DNA of the microorganism (parental strain) having DNA containing the nucleotide sequence modified in step (I).

[0107] The DNA containing the nucleotide sequence modified in step (I) above is preferably one of the following (A-1) or (A-2).

[0108] (A-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 6

[0109] (A-2) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 6 and encoding a protein having N-deacetylation and N-sulfation activities (hereinafter also referred to as "NDST activity").

[0110] When the DNA containing the nucleotide sequence modified in step (I) above is the DNA in (A-1) or (A-2) above, the biological species that is different from the parent strain in step (II) above is budding yeast.

[0111] Examples of DNA described in (A-2) above include DNA consisting of a nucleotide sequence having 80% or more, preferably 85% or more, 90% or more, 95% or more, more preferably 98% or more, most preferably 99% or more identity with the nucleotide sequence represented by SEQ ID NO: 6 and encoding a protein having NDST activity.

[0112] In this specification, the identity of nucleotide or amino acid sequences can be determined using the Lipman-Pearson method [Science, 227 (4693), 1435-41 (1985)], the BLAST algorithm of Karlin and Altschul [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)], or FASTA [Methods Enzymol., 183, 63 (1990)]. Based on the BLAST algorithm, a program called BLASTN or BLASTX has been developed [J. Mol. Biol., 215, 403 (1990)]. For example, when analyzing nucleotide sequences using BLASTN based on BLAST, the parameters are set as follows: score = 100, word length = 12. Furthermore, for example, when analyzing amino acid sequences using BLASTX based on BLAST, the parameters are set as follows: score = 50, word length = 3. When using the BLAST and empty BLAST procedures, the default parameters for each procedure are used. The specific methodology for this analysis is known.

[0113] The DNA described in (A-1) above can be prepared, for example, by chemical synthesis using an NTS M series DNA synthesizer manufactured by Nihon Techno Service Co., Ltd., based on the nucleotide sequence represented by SEQ ID NO: 6.

[0114] For example, the DNA described above (A-2) can be prepared by searching various gene sequence databases for nucleotide sequences that have 80% or higher, preferably 85% or higher, 90% or higher, 95% or higher, preferably 97% or higher, more preferably 98% or higher, and most preferably 99% or higher identity with the nucleotide sequence represented by SEQ ID NO: 6, and by chemically synthesizing based on the nucleotide sequences obtained through the search using an NTS M series DNA synthesizer manufactured by Nihon Techno Service Co., Ltd.

[0115] Recombinant DNA having DNA containing the nucleotide sequence modified in step (I) above (preferably the DNA described in (A-1) or (A-2) above), for example, refers to DNA that can autonomously replicate in the parental strain and contains DNA containing the nucleotide sequence modified in step (I) above (preferably the DNA described in (A-1) or (A-2) above), is integrated into an expression vector containing a promoter and is located at the position where the DNA described in (A-1) or (A-2) above can be transcribed.

[0116] The recombinant DNA is DNA capable of integrating into the chromosome of the parental strain and containing the nucleotide sequence modified in step (I) above (preferably the DNA described in (A-1) or (A-2) above). When the recombinant DNA is DNA capable of integrating into the chromosomal DNA of the parental strain, it does not need to contain a promoter.

[0117] When prokaryotes such as bacteria are used as parental strains, the recombinant DNA that can autonomously replicate in the parental strain is preferably a recombinant DNA consisting of a promoter, a ribosome-binding sequence, a nucleotide sequence modified in step (I) above (preferably the DNA described in (A-1) or (A-2) above), and a transcription termination sequence. It may contain a gene controlling the promoter. It is preferable to use recombinant DNA in which the distance between the Shine-Dalgarno sequence (as the ribosome-binding sequence) and the start codon is appropriately adjusted (e.g., 6 to 18 nucleotides).

[0118] In recombinant DNA that can replicate autonomously in the parental strain, the transcription termination sequence is not essential for DNA expression, but the transcription termination sequence is preferably located immediately downstream of the structural gene.

[0119] This specification does not impose any particular restrictions on the expression vector, as long as it is a nucleic acid molecule suitable for the introduction, proliferation and expression of target DNA in the host. Not only plasmids, but also vectors utilizing artificial chromosomes or transposons, or granules can be used.

[0120] In this specification, examples of expression vectors used when microorganisms belonging to the genus *Escherichia* as parental strains include pColdI, pSTV28, pSTV29, pUC118 (both manufactured by Takara Bio), pMW119 (manufactured by Nippon Gene Ltd.), pET21a, pCOLADuet-1, pCDFDuet-1, pCDF-1b, pRSF-1b (both manufactured by Merck Millipore), pMAL-c5x (manufactured by New England Biolabs), pGEX-4T-1, pTrc99A (both manufactured by GE Healthcare Bio-Sciences), pTrcHis, pSE280 (both manufactured by Thermo Fisher Scientific), pGEMEX-1 (manufactured by Promega), pQE-30, pQE80L (both manufactured by Qiagen NV), pET-3, pBluescriptII SK(+), and pBluescriptII. KS(-) (all manufactured by Agilent Technologies), pKYP10 (JPS58-110600A), pKYP200 [Agric. Biol. Chem., 48,669 (1984)], pLSA1 [Agric. Biol. Chem., 53, 277 (1989)], pGEL1 [Proc. Natl. Acad. Sci., USA, 82, 4306 (1985)], pTrS30 [prepared from Escherichia coli JM109 / pTrS30 (FERM BP-5407)], pTrS32 [prepared from Escherichia coli JM109 / pTrS32 (FERM BP-5408)], pTK31 [Applied and Environmental Microbiology, 2007, Vol. 73, No. 20] Pages 6378-6385], pPAC31 (WO98 / 12343), pUC19 [Gene, 33, 103 (1985)] and pPA1 (JPS63-233798A).

[0121] As promoters, any promoter can be used when using the aforementioned expression vectors, as long as it functions in the cells of microorganisms belonging to the genus *Escherichia*. Examples include promoters of genes involved in amino acid biosynthesis, such as the trp and ilv promoters, as well as promoters derived from *Escherichia coli*, bacteriophages, etc., such as the uspA, lac, PL, PR, and PSE promoters. In addition, artificially designed and modified promoters can also be used, such as promoters consisting of two trp promoters connected in tandem, tac, trc, lacT7, or letI promoters.

[0122] The recombinant DNA used in this invention as described herein can be prepared, for example, by treating a DNA fragment prepared by the above method with a restriction enzyme or the like and inserting it downstream of the promoter of the appropriate expression vector.

[0123] In this specification, examples of methods for introducing recombinant DNA containing DNA encoding proteins with N-deacetylation and N-sulfation activities into autonomously replicable plasmids in host cells include, for example, methods using calcium ions, protoplast methods, electroporation methods, globular methods [Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)], and lithium acetate methods [J. Bacteriol., 153, 163 (1983)].

[0124] Furthermore, in this specification, when recombinant DNA containing DNA encoding proteins with N-deacetylation and N-sulfation activities is inserted into the genome of a parental strain, for example, a method utilizing homologous recombination can be used. That is, DNA attached to a portion of a chromosomal region that leads to the introduction of the target DNA is incorporated into the microbial cell to induce homologous recombination in a portion of that chromosomal region, thereby integrating the DNA into the genome. For example, as a method frequently used in *Escherichia coli*, the method of introducing recombinant DNA using the homologous recombination system of λ phage is exemplified [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)]. Here, there is no particular limitation on the chromosomal region leading to the introduction, but a non-essential gene region or a non-gene region upstream of a non-essential gene region is preferred. As a method for incorporating DNA into a microbial cell, any method can be used, as long as it is a method of introducing DNA into a host cell; examples include the methods described above using calcium ions, protoplast methods, and electroporation methods.

[0125] In this specification, whether a microorganism is obtained by introducing recombinant DNA containing DNA encoding proteins with N-deacetylation and N-sulfation activities into a parental strain in an expressible manner can be confirmed, for example, by comparing the amount of DNA transcribed in a microorganism obtained using Northern blotting or the amount of protein in a microorganism obtained using Western blotting with the amount in the parental strain.

[0126] In this specification, whether the microorganisms produced by the above method are recombinant microorganisms with N-deacetylation and N-sulfation activities can be confirmed, for example, by the following method. First, the parental strain and the produced microorganisms are cultured separately in a culture medium, and a cell extract containing proteins with N-deacetylation and N-sulfation activities is prepared from the obtained culture. Subsequently, the cell extract is contacted with heparinane as a substrate and PAPS as a sulfate group donor to produce N-deacetylated and N-sulfation heparinane. Finally, by detecting the N-deacetylated and N-sulfation heparinane in the reaction solution using conventional analytical methods such as high-performance liquid chromatography and gas chromatography, it can be confirmed that the produced microorganisms are recombinant microorganisms with N-deacetylation and N-sulfation activities.

[0127] 2. Methods for producing microorganisms with N-deacetylation and N-sulfation activities (2)

[0128] A method for producing microorganisms expressing NDST activity according to one embodiment of the present invention comprises the step (i): expressing in the microorganism any one of the following proteins selected from the group consisting of (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3) and (E-1) to (E-3):

[0129] (B-1) A protein containing the amino acid sequence represented by SEQ ID NO: 13;

[0130] (B-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 13 and having NDST activity;

[0131] (B-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 13 and has NDST activity;

[0132] (C-1) A protein containing the amino acid sequence represented by SEQ ID NO: 14;

[0133] (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 14 and having NDST activity;

[0134] (C-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 14 and has NDST activity;

[0135] (D-1) A protein containing the amino acid sequence represented by SEQ ID NO: 15;

[0136] (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 15 and has N-deacetylation and N-sulfation activities;

[0137] (D-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 15 and has N-deacetylation activity and N-sulfation activity;

[0138] (E-1) A protein containing the amino acid sequence represented by SEQ ID NO: 16;

[0139] (E-2) A protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 16 and possessing NDST activity; and

[0140] (E-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 16 and has NDST activity.

[0141] Examples of proteins described in (B-2), (C-2), (D-2) or (E-2) above include proteins comprising amino acid sequences having 80% or higher, preferably 85% or higher, 90% or higher, 95% or higher, more preferably 98% or higher, and most preferably 99% or higher identity with the amino acid sequences represented by SEQ ID NO: 13, 14, 15 or 16, respectively, and having NDST activity.

[0142] Examples of proteins described in (B-3), (C-3), (D-3) or (E-3) above include proteins containing amino acid sequences in which one or more, preferably 1 to 20, more preferably 1 to 10, even more preferably 1 to 8, and most preferably 1 to 5 amino acids are deleted, substituted, added or inserted, respectively, in the amino acid sequences shown in SEQ ID NO: 13, 14, 15 or 16, and which have NDST activity.

[0143] In order to obtain microorganisms with high N-deacetylation and N-sulfation activities, in step (i) above, it is preferable to express any one of the proteins (B-1) to (B-3), (C-1) to (C-3) and (E-1) to (E-3) selected from the above proteins in the microorganisms, and more preferably to express any one of the proteins (C-1) to (C-3) selected from the above proteins in the microorganisms.

[0144] An amino acid sequence that has been 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 the amino acid sequence.

[0145] It is not important whether the missing, substituted, added, or inserted amino acid is natural or non-natural. Examples of natural 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.

[0146] Examples of amino acids that can be substituted for each other are shown below. Amino acids included in the same group can be substituted for each other.

[0147] Group A: Leucine, Isoleucine, Norleucine, Valine, Norvaline, Alanine, 2-Aminobutyric Acid, Methionine, O-Methylserine, Tert-Butylglycine, Tert-Butylalanine, and Cyclohexylalanine

[0148] Group B: Aspartic acid, glutamic acid, isaspartic acid, isoglutamic acid, 2-aminohexanoic acid, and 2-aminosuccinic acid

[0149] Group C: Asparagine and glutamine

[0150] Group D: Lysine, arginine, ornithine, 2,4-diaminobutyric acid and 2,3-diaminopropionic acid

[0151] Group E: Proline, 3-hydroxyproline, and 4-hydroxyproline

[0152] Group F: Serine, Threonine, and Homoserine

[0153] Group G: Phenylalanine, Tryptophan, and Tyrosine

[0154] Whether a protein possesses N-deacetylation and N-sulfation activities (NDST activities) can be confirmed by methods such as the following.

[0155] First, recombinant DNA containing DNA encoding the protein is prepared using the following method. Then, microorganisms whose NDST activity cannot be confirmed, such as *Escherichia coli* Origami B(DE3), are cultured using the recombinant DNA, and a cell extract containing the protein is prepared from the obtained culture. Subsequently, the cell extract is reacted using heparan as a substrate in the presence of 3'-adenosine-5'-phosphate sulfate (hereinafter also referred to as "PAPS"). Finally, conventional analytical methods such as high-performance liquid chromatography (HPLC) and gas chromatography (GC) are performed. The N-deacetylation and N-sulfation activities of the target protein can be confirmed by detecting the production of N-deacetylation and N-sulfation of heparan.

[0156] The preferred microbial species used in step (i) above (hereinafter also referred to as "parental strain") is the same as the preferred microbial species in step (II) of 1 above, which is the microorganism (parental strain) that introduces DNA in an expressible manner.

[0157] In this specification, the parent strain refers to the original strain that will undergo gene modification, transformation, etc. In particular, in the "Method for producing microorganisms with N-deacetylation activity and N-sulfation activity (2)", the parent strain of the microorganisms with N-deacetylation activity and N-sulfation activity of the present invention refers to the strain before expressing any one of the proteins selected from (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3), and (E-1) to (E-3) in step (i) above.

[0158] Step (i) above is preferably the step of introducing DNA selected from the group consisting of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3) and (e-1) to (e-3) into the microorganism in an expressible manner:

[0159] (b-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 9;

[0160] (b-2) DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 13;

[0161] (b-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 9 and encoding a protein having N-deacetylation and N-sulfation activities;

[0162] (c-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 10;

[0163] (c-2) DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 14;

[0164] (c-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 10 and encoding a protein having N-deacetylation and N-sulfation activities;

[0165] (d-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 11;

[0166] (d-2) DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 15;

[0167] (d-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 11 and encoding a protein having N-deacetylation and N-sulfation activities;

[0168] (e-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 12;

[0169] (e-2) DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 16; and

[0170] (e-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 12 and encoding a protein having N-deacetylation and N-sulfation activities.

[0171] In order to obtain microorganisms with high N-deacetylation and N-sulfation activities, in step (i) above, it is preferable to introduce any one of the DNAs selected from (b-1) to (b-3), (c-1) to (c-3) and (e-1) to (e-3) of the above DNAs into the microorganisms in an expressible manner, and more preferably to introduce any one of the DNAs selected from (c-1) to (c-3) of the above DNAs into the microorganisms in an expressible manner.

[0172] The DNA described in (b-1), (c-1), (d-1), (e-1), (b-2), (c-2), (d-2), and (e-2) above can be prepared, for example, by chemical synthesis using an NTS M series DNA synthesizer manufactured by Nihon Techno Service Co., Ltd., based on the nucleotide sequence represented by SEQ ID NO: 9, 10, 11, or 12 and the amino acid sequence represented by SEQ ID NO: 13, 14, 15, or 16, respectively.

[0173] The DNA described in (b-3), (c-3), (d-3), or (e-3) above can be prepared, for example, by searching various gene sequence databases for nucleotide sequences that have 80% or higher, preferably 87% or higher, 88% or higher, 90% or higher, 95% or higher, more preferably 98% or higher, and most preferably 99% or higher identity with the nucleotide sequences represented by SEQ ID NO: 9, 10, 11, or 12, respectively, and by chemically synthesizing based on the nucleotide sequences obtained through the search using an NTS M series DNA synthesizer manufactured by Nihon Techno Service Co., Ltd.

[0174] An example of introducing any one of the DNAs selected from (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3) and (e-1) to (e-3) into a microorganism in an expressible manner in step (i) above includes transforming the microorganism (parental strain) with recombinant DNA having any one of the DNAs selected from (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3) and (e-1) to (e-3).

[0175] The recombinant DNA having any of the DNA described in (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3) and (e-1) to (e-3) above refers to, for example, DNA that can autonomously replicate in the parental strain, and the DNA described in any one or more of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3) and (e-1) to (e-3) above is integrated into an expression vector containing a promoter and is located at a position where the DNA can be transcribed from any one or more of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3) and (e-1) to (e-3) above.

[0176] The recombinant DNA is capable of integrating into the chromosome of the parental strain and is the DNA described in any one or more of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3), and (e-1) to (e-3) above, and also has the DNA described in any one or more of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3), and (e-1) to (e) above. When the recombinant DNA is DNA capable of integrating into the chromosomal DNA of the parental strain, it does not need to contain a promoter.

[0177] When a prokaryote, such as a bacterium, is used as the parental strain, the recombinant DNA that can autonomously replicate in the parental strain is preferably a recombinant DNA consisting of a promoter, a ribosome-binding sequence, the DNA described in any one or more of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3), and (e-1) to (e-3) above, and a transcription termination sequence. It may contain a gene controlling the promoter. Preferably, recombinant DNA is used in which the distance between the Shine-Dalgarno sequence, which serves as the ribosome-binding sequence, and the start codon is appropriately adjusted (e.g., 6 to 18 nucleotides).

[0178] In recombinant DNA that can replicate autonomously in the parental strain, the transcription termination sequence is not essential for DNA expression, but the transcription termination sequence is preferably located immediately downstream of the structural gene.

[0179] 3. Microorganisms with N-deacetylation and N-sulfation activities

[0180] According to one embodiment of the present invention, the microorganisms having N-deacetylation and N-sulfation activities express any one of the proteins selected from the group consisting of (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3), and (E-1) to (E-3):

[0181] (B-1) A protein containing the amino acid sequence represented by SEQ ID NO: 13;

[0182] (B-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 13 and has N-deacetylation and N-sulfation activities;

[0183] (B-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 13 and has N-deacetylation activity and N-sulfation activity;

[0184] (C-1) A protein containing the amino acid sequence represented by SEQ ID NO: 14;

[0185] (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 14 and has N-deacetylation and N-sulfation activities;

[0186] (C-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 14 and has N-deacetylation activity and N-sulfation activity;

[0187] (D-1) A protein containing the amino acid sequence represented by SEQ ID NO: 15;

[0188] (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 15 and has N-deacetylation and N-sulfation activities;

[0189] (D-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 15 and has N-deacetylation activity and N-sulfation activity;

[0190] (E-1) A protein containing the amino acid sequence represented by SEQ ID NO: 16;

[0191] (E-2) A protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylation and N-sulfation activities; and

[0192] (E-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylation activity and N-sulfation activity.

[0193] The microorganisms possessing N-deacetylation and N-sulfation activities described above can be produced by the method described in step 2 above. The preferred microbial species used are the same as those used in step (i) of step 2 above.

[0194] 4. Methods for producing heparan derivatives

[0195] One embodiment of the method for producing heparan derivative compounds of the present invention includes step (III): producing N-deacetylated and N-sulfated heparan derivative compounds from heparan in the presence of microorganisms or extracts thereof that have N-deacetylation and N-sulfation activities produced by steps (I) and (II) of 1 above.

[0196] Furthermore, one embodiment of the method for producing heparan derivative compounds of the present invention includes step (ii): producing N-deacetylated and N-sulfated heparan derivative compounds from heparan in the presence of microorganisms or extracts thereof that have N-deacetylation and N-sulfation activities produced by step (i) of step 2 above.

[0197] The preferred heparin derivatives to be produced are N-sulfated heparinane, N-sulfated epimerized heparinane, N-sulfated depolyheparinane, N-sulfated epimerized depolyheparinane, N-sulfated-6-O-sulfated heparinane, N-sulfated-6-O-sulfated epimerized heparinane, N-sulfated-2-O-sulfated-6-O-sulfated heparinane, and N-sulfated-2-O-sulfated-6-O-sulfated heparinane. The polyheparinane is sulfated epimerized, N-sulfated-6-O-sulfated polyheparinane, N-sulfated-6-O-sulfated epimerized polyheparinane, N-sulfated-2-O-sulfated-6-O-sulfated polyheparinane, N-sulfated-2-O-sulfated-6-O-sulfated epimerized polyheparinane or heparin, more preferably N-sulfated heparinane or heparin, and most preferably N-sulfated heparinane.

[0198] "Epomerization" refers to the conversion of the β-D-glucuronic acid residue of heparinane into the α-L-iduronic acid residue.

[0199] “Depolymerization” refers to a process to reduce the molecular weight. For example, the number-average molecular weight (Mn) of the “depolymerized” heparinane compound is 1,000 to 150,000, preferably 8,000 to 60,000, and the weight-average molecular weight (Mw) is 2,000 to 300,000, preferably 10,000 to 100,000, which are values ​​measured by GPC using pullulan as a standard.

[0200] "6-O-sulfation" refers to the sulfation of the 6-hydroxyl group of the N-acetyl-D-glucosamine residue.

[0201] "2-O-sulfation" refers to the sulfation of the 2-hydroxyl group of a hexuronic acid residue (preferably an α-L-iduronic acid residue).

[0202] Examples of methods for producing the heparin alkyl derivatives of the present invention include (α) a method for producing N-deacetylated and N-sulfated heparin alkyl derivatives by fermentation, and (β) a method for producing N-deacetylated and N-sulfated heparin alkyl derivatives by adding a culture of microorganisms having N-deacetylation and N-sulfation activity or a treatment material of such culture to a heparin alkyl substrate to induce a reaction. The corresponding production methods will be described below.

[0203] (α) A method for producing N-deacetylated and N-sulfated heparan derivatives by fermentation

[0204] The production of N-deacetylated and N-sulfated heparin alkyl derivatives via fermentation can be carried out by culturing microorganisms with N-deacetylation and N-sulfation activities in a culture medium, allowing the production of N-deacetylated and N-sulfated heparin alkyl derivatives in the culture. The production method may include, for example, producing N-deacetylated and N-sulfated heparin alkyl derivatives in a culture, then accumulating and collecting the N-deacetylated and N-sulfated heparin alkyl derivatives from the culture.

[0205] The recombinant microorganisms used in the method for producing N-deacetylated and N-sulfated heparan derivatives by fermentation are microorganisms produced by steps (I) and (II) above or by step (i) above, and are preferably microorganisms capable of producing heparan as a substrate for proteins with NDST activity, and / or microorganisms capable of artificially enhanced production of the sulfate donor PAPS. The microorganisms may be microorganisms with one or more of the activities of epimerization, 2-O-sulfation, 6-O-sulfation, and 3-O-sulfation.

[0206] Referring to *Metabolic Engineering*, 2012, 14, pp. 521-527; *Carbohydrate Research*, 2012, 360, pp. 19-24; and US9,975,928B, the ability to produce heparan can be conferred by introducing genes encoding proteins involved in heparan production. Referring to *J. Org. Chem.*, 2000, 65, 18, 5565-5574; and WO2021 / 201282, the ability to produce PAPS can be conferred by introducing genes encoding proteins involved in PAPS production. Referring to WO2021 / 201282, the ability to produce epimerase, 2-O-sulfotransferase, 6-O-sulfotransferase, and 3-O-sulfotransferase, including related isotypes, can be conferred by introducing genes encoding C5 epimerase, 2-O-sulfotransferase, 6-O-sulfotransferase, or 3-O-sulfotransferase, including related isotypes.

[0207] Microorganisms can be cultured using conventional methods. As the culture medium for culturing microorganisms, either natural or synthetic media can be used, as long as they contain heparin and carbon, nitrogen, and inorganic salts that can be assimilated by microorganisms and can effectively promote microbial culture.

[0208] The carbon source can be any carbon source that can be assimilated by microorganisms. Examples include glucose, fructose, sucrose, molasses containing these substances, carbohydrates such as starch and starch hydrolysate, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol, propanol and glycerol.

[0209] Examples of nitrogen sources include ammonia, ammonium salts of inorganic or organic acids such as ammonium chloride, ammonium sulfate, ammonium acetate, and ammonium phosphate, other nitrogen-containing compounds, peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermenting bacterial cells and their digestion products.

[0210] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0211] In methods for producing heparan derivatives via fermentation, heparan can be added to the culture medium when the microorganisms used do not have the ability to produce heparan as a substrate for proteins with NDST activity.

[0212] Furthermore, in the method for producing heparane-derived compounds by fermentation, when the microorganisms used do not have the ability to produce heparane as a substrate for proteins with NDST activity, heparane can be provided to the microorganisms used in this invention by co-culturing microorganisms capable of producing heparane with the recombinant microorganisms of this invention, instead of adding heparane to the culture medium.

[0213] In methods for producing heparan derivatives via fermentation, PAPS can be added to the culture medium when the microorganisms used do not have the ability to provide PAPS necessary for the N-sulfation reaction.

[0214] Furthermore, in the method of producing heparan derivatives by fermentation, when the microorganisms used do not have the ability to provide PAPS necessary for the N-sulfation reaction, PAPS can be provided to the microorganisms used in this invention by co-culturing microorganisms capable of producing PAPS with the recombinant microorganisms of this invention instead of adding PAPS to the culture medium.

[0215] It is preferred to culture under aerobic conditions, such as shaking culture or deep aerated and stirred culture. The preferred culture temperature is 15°C to 40°C, and the culture time is usually 5 hours to 7 days. The pH during culture is preferably maintained between 3.0 and 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0216] In addition, antibiotics such as ampicillin or tetracycline can be added to the culture medium as needed during the cultivation process. When culturing microorganisms transformed using expression vectors with inducible promoters, inducers can be added to the culture medium as needed.

[0217] For example, when culturing microorganisms transformed with an expression vector using a lac promoter, isopropyl-β-D-thiogalactopyranoside can be added to the culture medium; when culturing microorganisms transformed with an expression vector using a trp promoter, indoleacrylic acid can be added to the culture medium.

[0218] Heparan derivatives can be produced by generating heparan derivatives in the culture through the above-described culturing process. The quantification of heparan derivatives can be performed using HPLC (e.g., an analyzer SPD-M20A manufactured by Shimadzu Corporation).

[0219] Heparin derivatives can typically be collected from cultures using a combination of ion exchange resins, precipitation, and other known methods. When heparin derivatives accumulate in bacterial cells, the cells can be disrupted, for example by sonication, followed by centrifugation to remove the cells, and the compounds can be collected from the resulting supernatant using methods such as ion exchange resins.

[0220] (β) A method for producing N-deacetylated and N-sulfated heparan derivatives by adding a culture of microorganisms with N-deacetylation and N-sulfation activities or a treatment material of such a culture to a heparan as a substrate to induce a reaction.

[0221] The production of the aforementioned heparinane-derived compounds can be carried out by presenting a microorganism or its extract with N-deacetylation and N-sulfation activity as an enzyme source, heparinane as a substrate, and PAPS as a sulfate group donor in an aqueous culture medium, and producing N-deacetylated and N-sulfated heparinane-derived compounds in the aqueous culture medium. The production method may include, for example, producing N-deacetylated and N-sulfated heparinane-derived compounds in an aqueous culture medium, then accumulating and collecting the N-deacetylated and N-sulfated heparinane-derived compounds from the aqueous culture medium.

[0222] The methods and culture media for culturing microorganisms are the same as those described in (α) above.

[0223] In this specification, examples of culture processing materials include concentrations of the above-mentioned cultures, dried culture materials, bacterial cells obtained by centrifuging, filtering, etc., of cultures, dried bacterial cell materials, lyophilized bacterial cells, surfactant-treated bacterial cell materials, solvent-treated bacterial cell materials, enzyme-treated bacterial cell materials, and materials containing live bacterial cells that retain the same function as the culture as the enzyme source, such as immobilized bacterial cell materials, ultrasonically treated bacterial cell materials, mechanically ground bacterial cell materials, crude enzyme extracts obtained from treated bacterial cells, and purified enzymes obtained from treated bacterial cells.

[0224] Among these, preferred examples include the above-mentioned culture concentrate, dried culture material, bacterial cells obtained by centrifuging, filtering, etc., bacterial cell dried material, lyophilized bacterial cell material, bacterial cell surfactant-treated material, bacterial cell solvent-treated material, bacterial cell enzyme-treated material, and materials containing live bacterial cells that retain the same function as the culture as the enzyme source, such as bacterial cell immobilization material, bacterial cell sonication material, and bacterial cell mechanical grinding material. Most preferred examples include the above-mentioned culture concentrate, dried culture material, bacterial cells obtained by centrifuging, filtering, etc., bacterial cell dried material, bacterial cell lyophilized material, bacterial cell surfactant-treated material, bacterial cell solvent-treated material, bacterial cell enzyme-treated material, and materials containing live bacterial cells that retain the same function as the culture as the enzyme source, such as bacterial cell immobilization material.

[0225] The concentration of the protein with NDST activity used as an enzyme source is preferably from 1 mg / L to 500 g / L, more preferably from 1 mg / L to 300 g / L, and most preferably from 1 mg / L to 5 g / L.

[0226] The concentration of heparinane as a substrate is preferably from 1 mg / L to 100 g / L, more preferably from 10 mg / L to 20 g / L.

[0227] The concentration of PAPS as a sulfate group donor is preferably from 0.1 to 500 mM, more preferably from 0.5 to 50 mM. Furthermore, when used in an N-sulfation reaction, PAPS is converted to 3'-adenosine-5'-phosphate (hereinafter also referred to as "PAP"), so an enzyme and / or substrate for regenerating PAPS from PAP can be added. Examples of enzymes and / or substrates for regenerating PAPS include ATP sulfate kinase, adenosine 5'-phosphate sulfate kinase, ATP or an ATP source, a sulfate ion source (WO2021 / 201282), 3'-adenosine-5'-phosphate sulfate-sulfotransferase, p-nitrobenzene sulfate (pNPS) (WO2020 / 013346), etc.

[0228] Examples of aqueous culture media include water, buffers such as phosphates, carbonates, acetates, borates, citrates, and Tris, alcohols such as methanol and ethanol, esters such as ethyl acetate, ketones such as acetone, and amides such as acetamide. Furthermore, the culture medium of microorganisms used as enzyme sources can be used as an aqueous culture medium.

[0229] Heparan derivatives produced in aqueous culture media can be quantified and collected by the method described in (α) above.

[0230] In addition to step (III) or step (ii), one embodiment of the method for producing heparinane-derived compounds of the present invention may also include at least one of the following steps. Examples of the steps in the production of heparin from heparinane are WO2017 / 115674 and WO2017 / 115675.

[0231] - A step to produce heparinane with a lower molecular weight by decomposing heparinane.

[0232] - The steps to isomerize the β-D-glucuronic acid residue in heparinane to the epimer α-L-iduronic acid (IdoA) residue.

[0233] - The step of sulfating the 2-hydroxyl group of the hexuronic acid residue (preferably α-L-iduronic acid residue) in heparinane.

[0234] - The step of sulfating the 6-hydroxyl group of the α-D-glucosamine residue in heparinane.

[0235] - A step of sulfating the 3-hydroxyl group of the α-D-glucosamine residue in heparane.

[0236] As stated above, the following are disclosed in this specification.

[0237] {1} A method for producing microorganisms with N-deacetylation and N-sulfation activities, the method comprising:

[0238] Step (I): Modify the nucleotide sequence of the DNA encoding N-deacetylase / N-sulfotransferase; and

[0239] Step (II): Introduce DNA containing the nucleotide sequence modified in step (I) into the microorganism in an expressible manner, wherein

[0240] Step (I) is a codon optimization step based on the codon usage frequency of biological species different from the microorganism.

[0241] {2} The method for producing microorganisms with N-deacetylation and N-sulfation activities according to {1} above, wherein the microorganisms are bacteria and the biological species are budding yeasts.

[0242] {3} The method for producing microorganisms with N-deacetylation and N-sulfation activities according to {2} above, wherein the DNA containing the modified nucleotide sequence is the DNA in (A-1) or (A-2):

[0243] (A-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 6; or

[0244] (A-2) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 6 and encoding a protein having N-deacetylation and N-sulfation activities.

[0245] {4} A method for producing heparan derivative compounds, the method comprising step (III): producing N-deacetylated and N-sulfated heparan derivative compounds from heparan in the presence of microorganisms having N-deacetylation and N-sulfation activities produced by the production method according to any one of {1} to {3} above.

[0246] {5} The production method according to any one of {1} to {4} above, wherein the microorganism is a bacterium of the genus Escherichia.

[0247] {6} According to the production method described in {5} above, the Escherichia coli is a bacterium of the genus Escherichia coli.

[0248] {7} According to the production method described in {4} above, the heparan derivative compound is N-sulfated heparan.

[0249] {8} A method for producing microorganisms having N-deacetylation and N-sulfation activities, the method comprising the step (i): expressing in the microorganisms a protein selected from the group consisting of (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3) and (E-1) to (E-3):

[0250] (B-1) A protein containing the amino acid sequence represented by SEQ ID NO: 13;

[0251] (B-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 13 and has N-deacetylation and N-sulfation activities;

[0252] (B-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 13 and has N-deacetylation activity and N-sulfation activity;

[0253] (C-1) A protein containing the amino acid sequence represented by SEQ ID NO: 14;

[0254] (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 14 and has N-deacetylation and N-sulfation activities;

[0255] (C-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 14 and has N-deacetylation activity and N-sulfation activity;

[0256] (D-1) A protein containing the amino acid sequence represented by SEQ ID NO: 15;

[0257] (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 15 and has N-deacetylation and N-sulfation activities;

[0258] (D-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 15 and has N-deacetylation activity and N-sulfation activity;

[0259] (E-1) A protein containing the amino acid sequence represented by SEQ ID NO: 16;

[0260] (E-2) A protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylation and N-sulfation activities; and

[0261] (E-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylation activity and N-sulfation activity.

[0262] {9} The method for producing microorganisms with N-deacetylation and N-sulfation activities according to {8} above, wherein:

[0263] Step (i) is the step of introducing DNA, selected from the group consisting of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3), and (e-1) to (e-3), into the microorganism in an expressible manner:

[0264] (b-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 9;

[0265] (b-2) DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 13;

[0266] (b-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 9 and encoding a protein having N-deacetylation and N-sulfation activities;

[0267] (c-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 10;

[0268] (c-2) DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 14;

[0269] (c-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 10 and encoding a protein having N-deacetylation and N-sulfation activities;

[0270] (d-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 11;

[0271] (d-2) DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 15;

[0272] (d-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 11 and encoding a protein having N-deacetylation and N-sulfation activities;

[0273] (e-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 12;

[0274] (e-2) DNA encoding a protein containing the amino acid sequence represented by SEQ ID NO: 16; and

[0275] (e-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 12 and encoding a protein having N-deacetylation and N-sulfation activities.

[0276] {10} A method for producing heparan derivative compounds, the method comprising step (ii): producing N-deacetylated and N-sulfated heparan derivative compounds from heparan in the presence of microorganisms having N-deacetylation and N-sulfation activities produced by the production method according to {8} or {9} above.

[0277] {11} The production method according to any one of {8} to {10} above, wherein the microorganism is a bacterium of the genus Escherichia.

[0278] {12} According to the production method described in {11} above, the Escherichia coli bacteria mentioned therein are Escherichia coli.

[0279] {13} According to the production method described in {10} above, the heparan derivative compound is N-sulfated heparan.

[0280] {14} A microorganism having N-deacetylation and N-sulfation activities, expressing a protein selected from the group consisting of (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3), and (E-1) to (E-3):

[0281] (B-1) A protein containing the amino acid sequence represented by SEQ ID NO: 13;

[0282] (B-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 13 and has N-deacetylation and N-sulfation activities;

[0283] (B-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 13 and has N-deacetylation activity and N-sulfation activity;

[0284] (C-1) A protein containing the amino acid sequence represented by SEQ ID NO: 14;

[0285] (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 14 and has N-deacetylation and N-sulfation activities;

[0286] (C-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 14 and has N-deacetylation activity and N-sulfation activity;

[0287] (D-1) A protein containing the amino acid sequence represented by SEQ ID NO: 15;

[0288] (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence represented by SEQ ID NO: 15 and has N-deacetylation and N-sulfation activities;

[0289] (D-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 15 and has N-deacetylation activity and N-sulfation activity;

[0290] (E-1) A protein containing the amino acid sequence represented by SEQ ID NO: 16;

[0291] (E-2) A protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylation and N-sulfation activities; and

[0292] (E-3) A protein containing an amino acid sequence in which one or more amino acid residues are missing, substituted, added or inserted in the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylation activity and N-sulfation activity.

[0293] Example

[0294] The present invention will be described in detail below with reference to the embodiments, but the present invention is not limited to the following embodiments, as long as it does not depart from its spirit.

[0295] Analysis of Examples

[0296] The activity of heparinane in N-deacetylation and N-sulfation reactions was analyzed by HPLC as follows.

[0297] According to WO2018 / 048973, unsaturated disaccharides were generated by enzymatic digestion and analyzed by HPLC. The resulting reaction solution was centrifuged, and the supernatant was heated at 80°C for 10 minutes to denature the protein. The denatured solution was centrifuged, and the supernatant was desalted by ultrafiltration using an Amicon 3K ultracentrifuge and filtration system (Merck). The desalted solution was placed in a heparinase reaction solution (a composition of 50 mM ammonium acetate and 2 mM calcium chloride) containing 0.5 U / ml each of heparinase I, heparinase II, and heparinase III (Sigma), and enzymatically digested at 35°C for 2 hours. The digested solution was then held at 95°C for 15 minutes to inactivate the heparinase.

[0298] Unsaturated disaccharides were analyzed by using a general-purpose HPLC Prominence (Shimadzu) and a strong anion exchange column (Spherisorb-SAX column, 4.0 × 250 mm, 5 μm, Waters) in gradient elution mode with mobile phase A [an aqueous solution containing 1.8 mM sodium dihydrogen phosphate, adjusted to pH 3.0 with phosphoric acid] and mobile phase B [an aqueous solution containing 1.8 mM sodium dihydrogen phosphate and 1 M sodium perchlorate, adjusted to pH 3.0 with phosphoric acid] to analyze the solution after heparinase inactivation.

[0299] Unsaturated disaccharides were detected by measuring absorbance at 232 nm using a UV detector SPD-20A (Shimadzu Corporation). The retention times of ΔUA-GlcNAc, ΔUA-GlcN, and ΔUA-GlcNS were confirmed by comparison with unsaturated disaccharide standards (Iduron Corporation). ΔUA refers to 4,5-unsaturated uronic acid, GlcNAc refers to N-acetylglucosamine, GlcN refers to glucosamine, and GlcNS refers to N-sulfoglucosamine. The activities of N-deacetylase and N-sulfatase were confirmed by determining the area ratio of ΔUA-GlcNS to the total area of ​​the detected ΔUA-GlcNAc, ΔUA-GlcN, and ΔUA-GlcNS. Furthermore, the activity of N-deacetylase was confirmed by determining the area ratio of ΔUA-GlcN to the total area of ​​the detected ΔUA-GlcNAc, ΔUA-GlcN, and ΔUA-GlcNS. In the following text, ΔUA-GlcNAc is abbreviated as GlcNAc, ΔUA-GlcN as GlcN, and ΔUA-GlcNS as GlcNS.

[0300] Example 1

[0301] Production of microorganisms expressing NDST (1)

[0302] (1) Construction of expression plasmid for wild-type rat NDST-1

[0303] The following procedure was used to produce a plasmid for expressing the wild-type NDST gene (SEQ ID NO: 1) from rats.

[0304] The wild-type NDST gene from rats was amplified by PCR using first-strand cDNA from mouse livers (purchased from Genostaff) as a template, with primers consisting of SEQ ID NO: 2 and 3. Subsequently, PCR was performed using the expression vector pGEX-4T3 (GE Healthcare Life Sciences) as a template, with primers consisting of SEQ ID NO: 4 and 5. The resulting fragment and DNA obtained by manipulating the previously prepared wild-type NDST gene fragment from rats were ligated using the In-Fusion HD Cloning Kit (Takara Bio) to obtain the expression plasmid pGEX-NDST (WT).

[0305] (2) Construction of expression plasmid for rat NDST-1 with codon optimization of budding yeast

[0306] The following procedure was used to produce a plasmid for expressing the codon-optimized NDST gene (SEQ ID NO: 6) of budding yeast.

[0307] The wild-type NDST gene from rats was amplified by PCR using Eurofins-synthesized DNA as a template and primers consisting of SEQ ID NO: 7 and 8. Subsequently, PCR was performed using the expression vector pGEX-4T3 (GE Healthcare Life Sciences) as a template and primers consisting of SEQ ID NO: 4 and 5. The obtained fragments and DNA obtained by manipulating the previously prepared wild-type NDST gene fragment from rats were treated with restriction enzymes BamHI and EcoRI, respectively, and the resulting fragments were ligated to obtain the expression plasmid pGEX-NDST (SC).

[0308] (3) Construction of Escherichia coli strain expressing NDST-1 transfected with plasmid

[0309] Using ampicillin screening, the constructed expression plasmid vector was used to transform Escherichia coli Origami B(DE3)(Novagen) to obtain Escherichia coli strains expressing NDST.

[0310] Example 2

[0311] NDST reaction test (1)

[0312] (1) Cultivation methods

[0313] Escherichia coli Origami B(DE3) transformed with the expression plasmid was cultured overnight at 30°C on LB agar supplemented with ampicillin. The grown bacterial cells were then inoculated into LB liquid medium supplemented with ampicillin and cultured overnight in test tubes with shaking at 30°C to obtain a seed culture. The seed culture was further inoculated into LB liquid medium supplemented with ampicillin at a volume of 5%. This culture was cultured with shaking at 30°C. When the absorbance at 660 nm (using a spectrophotometer) reached 0.4 to 0.6, IPTG was added to a final concentration of 0.1 mM, and induction culture was performed at 20°C for 24 hours. After induction, the bacterial cells were recovered and centrifuged at 9,000 rpm for 5 minutes at 4°C. The bacterial cells were stored at -80°C.

[0314] (2) Reaction method

[0315] The recovered *E. coli* cells were resuspended in extraction buffer [0.1 M MES / NaOH (6.5), 20 mM NCl2, 5% (v / v) glycerol, and 1 mM PMSF] to a wet weight of 140 g / L. The *E. coli* suspension was disrupted by sonication on ice and centrifuged at 12,000 rpm for 5 min at 4 °C. The supernatant was collected as the *E. coli* extract and used for enzymatic reactions.

[0316] (3) Enzyme-catalyzed reaction

[0317] The substrate and E. coli extract solution were mixed in a 1.5 mL tube to obtain [50 mM MES / NaOH (6.5), 10 mM MnCl2, 1 g / L heparinane, 4.4 mM PAPS, and 50% (v / v) crude E. coli extract solution], and allowed to react in an incubator at 30 °C for 24 h. The reaction was stopped by heat treatment at 80 °C for 10 min.

[0318] (4) Results of the reaction test

[0319] The results of the reaction experiments are summarized in Table 1. GlcNAc indicates the presence of unreacted heparinane, GlcN indicates the presence of N-deacetylated heparinane, and GlcNS indicates the presence of N-sulfated heparinane.

[0320]

[0321] pGEX-NDST(SC) of NDST, which incorporates codon optimization based on the codon usage frequency of budding yeast, exhibited unexpectedly high N-deacetylation and N-sulfation reactions.

[0322] Example 3

[0323] Production of microorganisms expressing NDST (2)

[0324] In each of the human-derived NDST-1 to NDST-4 genes, consecutive rare codons were changed to the optimal codons for *E. coli*, the nucleotide sequences at sites on the mRNA where loops might form were altered, the GC content was modified to more closely resemble that found in *E. coli*, and the amino acid arrangement on the tertiary surface was changed. The nucleotide sequences designed in this manner, namely the modified NDST-1 to NDST-4 represented by SEQ ID NOs: 9-12, were synthesized. Each DNA sequence was cut with NdeI and BamHI and inserted into the BamHI-NdeI site of PET15b, thereby obtaining NDST1_pET15b, NDST2_pET15b, NDST3_pET15b, and NDST4_pET15b, respectively. The amino acid sequences of the modified NDST-1 to NDST-4 are represented by SEQ ID NOs: 13 to 16, respectively.

[0325] Example 4

[0326] NDST reaction test (2)

[0327] Each plasmid was introduced into *E. coli* Origami B(DE3), and the *E. coli* were cultured in LB medium at 22°C. When the *E. coli* grew and the OD600 reached 0.8 to 1.0, IPTG was added to a concentration of 1 mM, and the *E. coli* were cultured for another 16 to 20 hours. Subsequently, bacterial cells were collected and sonicated, and the supernatant was used to prepare a cell extract. The following substrate was added to a solution of 50% of this cell extract (pH 6.8 to 7.0) consisting of 50 mM MES, 125 mM NaCl, and 10 mM MgCl2 to obtain a concentration of 2 mg / mL, and the reaction was carried out at 37°C for 18 hours. In cases (b) and (c) below, PAPS was added to the solution to obtain a final concentration of 5 mM.

[0328] The reaction is carried out under the following three conditions.

[0329] (a) Using heparinane as a substrate and without adding PAPS.

[0330] (b) Using N-deacetylated heparinane as a substrate and adding PAPS.

[0331] (c) Using heparinane as a substrate and adding PAPS.

[0332] The reaction was stopped by heat treatment at 95°C for 10 minutes, and the amount of disaccharides was analyzed by HPLC after the sugars produced were decomposed by heparinase I, heparinase II, and heparinase III. The results for case (a) above are shown in... Figure 1 In the above case (b), the results are shown in Figure 2 In the above case (c), the results are shown in Figure 3 In the figure, GlcNAc indicates the presence of unreacted heparin, GlcN indicates the presence of deacetylated heparin, and GlcNS indicates the presence of N-sulfated heparin.

[0333] like Figure 1 As shown in Table 2, in the case of (a) using heparinane as a substrate and in the absence of PAPS, approximately 50% of GlcNAc is deacetylated by NDST-1 and NDST-2. Figure 2 As shown in Table 2, in case (b) where deacetylated heparinane was used as a substrate and the reaction was carried out in the presence of PAPS, a large amount of GlcNS was observed to be produced when any enzyme was used, and the activities of NDST-1, NDST-2, and NDST-4 were high. Figure 3 As shown in Table 2, GlcNS were generated in (c) with heparinane as the substrate and in the presence of PAPS, in the cases of NDST-1, NDST-2, and NDST-4. Therefore, NDST-1, NDST-2, and NDST-4 were found to possess both N-deacetylation and N-sulfation activities. NDST-2 showed an 85% or higher proportion of GlcNS, thus demonstrating particularly high N-deacetylation and N-sulfation activities as an enzyme expressed by bacteria.

[0334]

[0335] The invention has been described in detail with reference to specific aspects, but it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the invention. This application is based on U.S. Patent Application No. 18 / 326,155, filed May 31, 2023, which is incorporated herein by reference in its entirety. Furthermore, all references cited herein are incorporated in their entirety.

[0336] Sequence List Free Text

[0337] SEQ ID NO: 1: Nucleotide sequence of wild-type NDST gene from rats

[0338] SEQ ID NO: 2: Nucleotide sequence of primer F for amplifying the wild-type NDST gene from rats.

[0339] SEQ ID NO: 3: Nucleotide sequence of primer R for amplifying the wild-type NDST gene from rats.

[0340] SEQ ID NO: 4: Nucleotide sequence of primer F used for amplifying pGEX-4T3

[0341] SEQ ID NO: 5: Nucleotide sequence of primer R used to amplify pGEX-4T3

[0342] SEQ ID NO: 6: Nucleotide sequence of the codon-optimized NDST gene in budding yeast

[0343] SEQ ID NO: 7: Nucleotide sequence of primer F for amplifying the NDST gene of budding yeast with codon optimization

[0344] SEQ ID NO: 8: Nucleotide sequence of primer R for amplifying the NDST gene of budding yeast with codon optimization

[0345] SEQ ID NO: 9: Nucleotide sequence of the modified NDST-1 gene

[0346] SEQ ID NO: 10: Nucleotide sequence of the modified NDST-2 gene

[0347] SEQ ID NO: 11: Nucleotide sequence of the modified NDST-3 gene

[0348] SEQ ID NO: 12: Nucleotide sequence of the modified NDST-4 gene

[0349] SEQ ID NO: 13: Amino acid sequence of modified NDST-1

[0350] SEQ ID NO: 14: Amino acid sequence of modified NDST-2

[0351] SEQ ID NO: 15: Amino acid sequence of modified NDST-3

[0352] SEQ ID NO: 16: Amino acid sequence of modified NDST-4

Claims

1. A method of producing a microorganism having N-deacetylation activity and N-sulfation activity, the method comprising: Step (I): modifying a nucleotide sequence of DNA encoding an N-deacetylase / N-sulfotransferase; and Step (II): introducing the DNA containing the nucleotide sequence modified in Step (I) into a microorganism in an expressible manner, wherein the Step (I) is a step of codon optimization according to codon usage frequency of a biological species different from the microorganism.

2. The method of producing a microorganism having N-deacetylation activity and N-sulfation activity according to claim 1, wherein the microorganism is a bacterium, and the biological species is a budding yeast.

3. The method of producing a microorganism having N-deacetylation activity and N-sulfation activity according to claim 2, wherein the DNA containing the modified nucleotide sequence is the DNA in (A-1) or (A-2): (A-1) DNA containing a nucleotide sequence represented by SEQ ID NO: 6; or (A-2) DNA containing a nucleotide sequence having 80% or more identity to the nucleotide sequence represented by SEQ ID NO: 6 and encoding a protein having N-deacetylation activity and N-sulfation activity.

4. A method of producing a heparin alkane derivative compound, the method comprising Step (III): producing an N-deacetylated and N-sulfated heparin alkane derivative compound from a heparin alkane in the presence of a microorganism having N-deacetylation activity and N-sulfation activity produced by the production method according to any one of claims 1 to 3 or an extract thereof.

5. The production method according to any one of claims 1 to 3, wherein the microorganism is a bacterium of the genus Escherichia.

6. The production method according to claim 5, wherein the bacterium of the genus Escherichia is Escherichia coli.

7. The production method according to claim 4, wherein the heparin alkane derivative compound is an N-sulfated heparin alkane.

8. A method of producing a microorganism having N-deacetylation activity and N-sulfation activity, the method comprising Step (i): expressing in a microorganism any one protein selected from the group consisting of (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3), and (E-1) to (E-3): (B-1) a protein containing an amino acid sequence represented by SEQ ID NO: 13; (B-2) a protein containing an amino acid sequence having 80% or more identity to the amino acid sequence represented by SEQ ID NO: 13 and having N-deacetylation activity and N-sulfation activity; (B-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 13 and having N-deacetylation activity and N-sulfation activity; (C-1) a protein comprising the amino acid sequence represented by SEQ ID NO: 14; (C-2) a protein comprising an amino acid sequence having 80% or higher identity to the amino acid sequence represented by SEQ ID NO: 14 and having N-deacetylating activity and N-sulfating activity; (C-3) a protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 14 and having N-deacetylating activity and N-sulfating activity; (D-1) a protein comprising the amino acid sequence represented by SEQ ID NO: 15; (D-2) a protein comprising an amino acid sequence having 80% or higher identity to the amino acid sequence represented by SEQ ID NO: 15 and having N-deacetylating activity and N-sulfating activity; (D-3) a protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 15 and having N-deacetylating activity and N-sulfating activity; (E-1) a protein comprising the amino acid sequence represented by SEQ ID NO: 16; (E-2) a protein comprising an amino acid sequence having 80% or higher identity to the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylating activity and N-sulfating activity; and (E-3) a protein comprising an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylating activity and N-sulfating activity.

9. The method for producing a microorganism having N-deacetylating activity and N-sulfating activity according to claim 8, wherein: the step (i) is a step of introducing into the microorganism any one DNA selected from the group consisting of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3), and (e-1) to (e-3) in an expressible manner: (b-1) DNA comprising the nucleotide sequence represented by SEQ ID NO: 9; (b-2) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 13; (b-3) DNA comprising a nucleotide sequence having 80% or higher identity to the nucleotide sequence represented by SEQ ID NO: 9 and encoding a protein having N-deacetylating activity and N-sulfating activity; (c-1) DNA comprising the nucleotide sequence represented by SEQ ID NO: 10; (c-2) DNA encoding a protein comprising the amino acid sequence represented by SEQ ID NO: 14; (c-3) DNA comprising a nucleotide sequence in which one or several nucleotide residues are deleted, substituted, added, or inserted in the nucleotide sequence represented by SEQ ID NO: 10 and encoding a protein having N-deacetylating activity and N-sulfating activity; (c-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 10 and encoding a protein having N-deacetylation activity and N-sulfation activity; (d-1) DNA containing a nucleotide sequence represented by SEQ ID NO: 11; (d-2) DNA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 15; (d-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 11 and encoding a protein having N-deacetylation activity and N-sulfation activity; (e-1) DNA containing a nucleotide sequence represented by SEQ ID NO: 12; (e-2) DNA encoding a protein containing an amino acid sequence represented by SEQ ID NO: 16; and (e-3) DNA containing a nucleotide sequence having 80% or higher identity with the nucleotide sequence represented by SEQ ID NO: 12 and encoding a protein having N-deacetylation activity and N-sulfation activity.

10. A method for producing a heparosan derivative compound, the method comprising step (ii): producing an N-deacetylated and N-sulfated heparosan derivative compound from heparosan in the presence of a microorganism having N-deacetylation activity and N-sulfation activity or an extract thereof produced by the production method according to claim 8 or 9.

11. The production method according to claim 8 or 9, wherein the microorganism is a bacterium of the genus Escherichia.

12. The production method according to claim 11, wherein the bacterium of the genus Escherichia is Escherichia coli.

13. The production method according to claim 10, wherein the heparosan derivative compound is an N-sulfated heparosan.

14. A microorganism having N-deacetylation activity and N-sulfation activity, which expresses any one protein selected from the group consisting of (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3), and (E-1) to (E-3): (B-1) a protein containing an amino acid sequence represented by SEQ ID NO: 13; (B-2) a protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 13 and having N-deacetylation activity and N-sulfation activity; (B-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 13 and having N-deacetylation activity and N-sulfation activity; (C-1) a protein containing an amino acid sequence represented by SEQ ID NO: 14; (C-2) a protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 14 and having N-deacetylation activity and N-sulfation activity; and (C-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 14 and having N-deacetylation activity and N-sulfation activity. (C-2) a protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 14 and having N-deacetylating activity and N-sulfating activity; (C-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 14 and having N-deacetylating activity and N-sulfating activity; (D-1) a protein containing the amino acid sequence represented by SEQ ID NO: 15; (D-2) a protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 15 and having N-deacetylating activity and N-sulfating activity; (D-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 15 and having N-deacetylating activity and N-sulfating activity; (E-1) a protein containing the amino acid sequence represented by SEQ ID NO: 16; (E-2) a protein containing an amino acid sequence having 80% or higher identity with the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylating activity and N-sulfating activity; and (E-3) a protein containing an amino acid sequence in which one or several amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence represented by SEQ ID NO: 16 and having N-deacetylating activity and N-sulfating activity.

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