Method for producing microorganisms having N-deacetylation activity and N-sulfation activity, method for producing heparosan-derived compounds, and microorganisms 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, comprising the steps of (I) modifying the nucleotide sequence of DNA encoding N-deacetylase / N-sulfotransferase and (II) introducing the DNA containing the modified nucleotide sequence in step (I) into a microorganism so that it can express, wherein step (I) is a step of codon optimization to match the codon usage frequency of a different species of organism than the microorganism. The present invention also relates to a method for producing a microorganism having N-deacetylation activity and N-sulfation activity, comprising the step of (i) expressing a specific protein in the microorganism.

Description

Technical Field

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

Background Art

[0002] Heparin, a sulfated polysaccharide, is one of the anticoagulants and is used for the treatment of thromboembolism, disseminated intravascular coagulation syndrome, and prevention of coagulation in hemodialysis and extracorporeal circulation. Industrially, heparin is extracted and purified from animal organs, mainly porcine intestinal mucosa.

[0003] In 2008, the contamination of heparin with impurities caused many deaths due to the contamination of porcine-derived heparin with impurities such as oversulfated chondroitin sulfate, and thus the production of non-animal-derived heparin with controlled production and quality has been demanded (Non-Patent Document 1). As a specific example, a method has been reported for producing heparin having the same structure and anticoagulant activity as porcine-derived products by deacetylating and sulfating heparosan, which is a capsular polysaccharide of microorganisms, by chemical and enzymatic methods (Patent Documents 1 and 2).

[0004] In the natural process of heparin biosynthesis in vivo, the modifications that occur after the formation of the backbone of the repeating sequence of heparosan-like disaccharides are N-deacetylation and N-sulfation of glucosamine, and these two reactions are carried out by the action of a single polypeptide, N-deacetylase / N-sulfotransferase (hereinafter referred to as "NDST") (Non-Patent Document 1).

[0005] NDST is a family of four isozymes, each with 65% to 80% identity, and has been cloned from various mammals (Non-Patent Literature 2). NDST consists of an N-deacetylation domain and an N-sulfation domain, and their activity differs depending on the origin and isozyme (Non-Patent Literature 2). NDST activity has been achieved in various eukaryotic systems such as COS cells, HEK293 cells, and CHO cells, and has also been successfully demonstrated in insect cells and yeast (Non-Patent Literature 3, 4). On the other hand, while there are reports of N-sulfation domain expression in E. coli, the full-length expression of NDST, particularly the N-deacetylation domain, has not been achieved in bacteria (Non-Patent Literature 5). Recently, a group from Jiangnan University has published literature and patents on NDST activity expression in Pichia yeast, but it is noted that confirming the deacetylation activity of NDST in E. coli is difficult (Patent Literature 3, Non-Patent Literature 6). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent No. 8,771,995 [Patent Document 2] International Publication No. 2018 / 048973 [Patent Document 3] Chinese Patent No. 107384990 Specification [Non-patent literature]

[0007] [Non-Patent Document 1] Advanced Drug Delivery Reviews 97 (2016) 237-249 [Non-Patent Document 2] Journal of Biological Chemistry 276 (2001) 5876-5882 [Non-Patent Document 3] Proceedings of the National Academy of Sciences of the United States of America 90 (1993) 3885-3888 [Non-Patent Document 4] Glycobiology 12 (2004) 1217-1228 [Non-Patent Document 5] FEBS Letters 433 (1998) 211-214 [Non-Patent Document 6] Green Chemistry 24 (2022) 3180-3192 [Non-Patent Document 7] Biochemical Society Transactions 21 (1993) 835-841 [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] While the N-deacetylation reaction of heparosan can be carried out chemically, if it can be performed biologically, it can be carried out in conjunction with the subsequent O-sulfation reaction, which is considered more desirable for the production of non-animal-derived heparin (Non-Patent Literature 6). Activity of the N-deacetylation reaction has already been demonstrated in Pichia yeast. On the other hand, activity of the N-deacetylation reaction has not been demonstrated in Escherichia coli, which has a proven track record of sulfase expression, and activity expression in bacteria, including Escherichia coli, is desired.

[0009] Therefore, the present invention aims to provide a new method for producing microorganisms having N-deacetylation activity and N-sulfation activity, as well as to provide such microorganisms.

[0010] In heterologous gene expression, the gene's base sequence can be optimized to match the host's codon usage frequency, and for example, Non-Patent Literature 6 describes codon optimization for the expression of various sulfate enzymes. This utilizes the fact that the amount of tRNA differs depending on the host, even for the same amino acid codon. Given this property, it was generally believed that codon optimization should naturally be optimized to match the host's codon usage frequency, and that optimizing to match the codon usage frequency of a different organism would only reach the lower limit of the optimal range. For example, the codon usage frequency of budding yeast differs from that of E. coli. In the case of arginine codons, E. coli prefers CGU and CGC, while budding yeast prefers the AGA codon, which is not very common in E. coli. Therefore, when heterologous expression using E. coli as the host, it is not common practice to attempt codon optimization to match budding yeast. [Means for solving the problem]

[0011] The inventors conducted intensive research to solve the above problems and discovered that by introducing NDST into microorganisms using codon-optimized sequences tailored to different biological species, it is possible to produce microorganisms possessing N-deacetylation activity and N-sulfation activity. Furthermore, they found that by expressing specific proteins in microorganisms, it is possible to produce microorganisms possessing N-deacetylation activity and N-sulfation activity.

[0012] In other words, the present invention relates to the following. ((1)) Step (I) involves modifying the nucleotide sequence of the DNA encoding N-deacetylase / N-sulfotransferase, The process includes (I) a step of introducing DNA containing the modified nucleotide sequence into a microorganism so that it can express the DNA, The above step (I) is a step of codon optimization that matches the codon usage frequency of a different biological species from the microorganism in question. A method for producing microorganisms having N-deacetylation activity and N-sulfation activity. ((2)) The method for 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 Saccharomyces cerevisiae. ((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 of (A-1) or (A-2). (A-1) DNA containing the nucleotide sequence represented by SEQ ID NO: 6 (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 ((4)) A method for producing a heparosan-derived compound, comprising step (III) of producing an N-deacetylated and N-sulfated heparosan-derived 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 the above ((1)). ((5)) The production method according to the above ((1)), wherein the microorganism is a bacterium belonging to the genus Escherichia. ((6)) The production method according to the above ((5)), wherein the bacterium belonging to the genus Escherichia is Escherichia coli. ((7)) The production method according to the above ((4)), wherein the heparosan-derived compound is N-sulfated heparosan. ((8)) A method for producing a microorganism having N-deacetylation activity and N-sulfation activity, comprising step (i) of expressing 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) in a microorganism. (B-1) A protein containing the amino acid sequence represented by SEQ ID NO: 13 (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 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: 13, and having N-deacetylation activity and N-sulfation activity A protein comprising the amino acid sequence represented by SEQ ID NO: 14 A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 14, and having N-deacetylation activity and N-sulfation activity 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-deacetylation activity and N-sulfation activity A protein comprising the amino acid sequence represented by SEQ ID NO: 15 A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 15, and having N-deacetylation activity and N-sulfation activity 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-deacetylation activity and N-sulfation activity A protein comprising the amino acid sequence represented by SEQ ID NO: 16 A protein comprising an amino acid sequence having 80% or more identity with the amino acid sequence represented by SEQ ID NO: 16, and having N-deacetylation activity and N-sulfation activity 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-deacetylation activity and N-sulfation activity ((9)) A method for producing a microorganism having N-deacetylation activity and N-sulfation activity as described in (8) above, wherein step (i) is a step of introducing into the microorganism in a manner that enables expression of 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). (b-1) DNA containing the nucleotide sequence shown in Sequence ID No. 9 (b-2) DNA encoding a protein containing the amino acid sequence shown in Sequence ID No. 13 (b-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 9, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (c-1) DNA containing the nucleotide sequence shown in Sequence ID No. 10 (c-2) DNA encoding a protein containing the amino acid sequence shown in Sequence ID No. 14 (c-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 10, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (d-1) DNA containing the nucleotide sequence shown in SEQ ID NO: 11 (d-2) DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO: 15 (d-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 11, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (e-1) DNA containing the nucleotide sequence shown in Sequence ID No. 12 (e-2) DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO: 16 (e-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 12, and which encodes a protein having N-deacetylation activity and N-sulfation activity. ((10)) A method for producing a heparosan-derived compound, comprising the step (ii) of producing an N-deacetylated and N-sulfated heparosan-derived compound from heparosan in the presence of a microorganism having N-deacetylation activity and N-sulfation activity or an extract thereof, produced by the manufacturing method described in (8) above. ((11)) The method for producing the product described in (8) above, wherein the microorganism is a bacterium of the genus Escherichia. ((12)) The method for producing the product according to (11) above, wherein the Escherichia bacterium is Escherichia coli. ((13)) The method for producing the product according to (10) above, wherein the heparosan-derived compound is N-sulfated heparosan. ((14)) A microorganism expressing one of the proteins selected from the groups (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3), and (E-1) to (E-3) below, and possessing N-deacetylation activity and N-sulfation activity. (B-1) Protein containing the amino acid sequence shown in Sequence ID No. 13 (B-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 13, and which also possesses N-deacetylation activity and N-sulfation activity. (B-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 13, and that have N-deacetylation activity and N-sulfation activity. (C-1) Protein containing the amino acid sequence shown in Sequence ID No. 14 (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 14, and possessing N-deacetylation activity and N-sulfation activity. (C-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 14, and that have N-deacetylation activity and N-sulfation activity. (D-1) Protein containing the amino acid sequence shown in Sequence ID No. 15 (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 15, and possessing N-deacetylation activity and N-sulfation activity. (D-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 15, and that have N-deacetylation activity and N-sulfation activity. (E-1) Protein containing the amino acid sequence shown in Sequence ID No. 16 (E-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 16, and possessing N-deacetylation activity and N-sulfation activity. (E-3) A protein having N-deacetylation activity and N-sulfation activity, which includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 16. [Effects of the Invention]

[0013] The manufacturing method according to the first invention makes it possible to produce microorganisms having superior N-deacetylation activity and N-sulfation activity compared to conventional methods by introducing codon-optimized NDST to microorganisms according to different biological species.

[0014] The manufacturing method according to the second invention makes it possible to produce microorganisms having superior N-deacetylation activity and N-sulfation activity compared to conventional methods by expressing a protein having a specific amino acid sequence in a microorganism. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 shows the results of the disaccharide analysis of the NDST reaction test using heparosan as the substrate and without the addition of PAPS. [Figure 2]Figure 2 shows the results of the disaccharide analysis of the NDST reaction test under conditions where deacetylated heparosan was used as a substrate and PAPS was added. [Figure 3] Figure 3 shows the results of the disaccharide analysis of the NDST reaction test using heparosan as a substrate and PAPS added. [Modes for carrying out the invention]

[0016] The present invention will be described in detail below, but these are merely examples of preferred embodiments and are not limiting to these. The "~" in a numerical range indicates a range that includes the numbers before and after it. For example, "0 mass%~100 mass%" means a range that is greater than or equal to 0 mass% and less than or equal to 100 mass%.

[0017] 1. Method for producing microorganisms having N-deacetylation activity and N-sulfation activity - 1 A method for producing a microorganism having N-deacetylation activity and N-sulfation activity (hereinafter also referred to as "NDST activity-expressing microorganism") according to one aspect of the present invention is: Step (I) involves modifying the nucleotide sequence of the DNA encoding NDST, The process includes (I) a step of introducing DNA containing the modified nucleotide sequence into a microorganism in a way that enables expression, Step (I) described above is a step of codon optimization that matches the codon usage frequency of a different biological species from the microorganism in question.

[0018] In this specification, "N-deacetylation" means, for example, N-deacetylating the N-acetyl group of the α-D-glucosamine residue of heparosan to produce an amino group. Examples of N-deacetylation include partial N-deacetylation. Furthermore, in this specification, "N-sulfation" means, for example, the sulfated amino group of the N-acetyl-D-glucosamine residue of heparosan.

[0019] An example of the nucleotide sequence of the DNA encoding NDST used in process (I) is the nucleotide sequence shown in SEQ ID NO: 1.

[0020] In step (I), which involves modifying the nucleotide sequence of the DNA encoding NDST as described above, codon optimization is performed to match the codon usage frequency of a different species from the microorganism into which the DNA described above is introduced in step (II) (hereinafter also referred to as the "parent strain"). Normally, "codon optimization" refers to changing the codons for each amino acid constituting the peptide to codons that are frequently used in the species into which it is introduced. Surprisingly, in this invention, it was found that NDST-expressing microorganisms could be obtained by performing codon optimization to match the codon usage frequency of a different species from the microorganism into which the DNA described above is introduced. This result suggests that codon optimization that is mismatched for this species leads to the active expression of NDST by slowing down protein expression and avoiding aggregation in cells. 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 that the codon corresponding to at least one amino acid is changed. However, it is preferable that 50% or more of the codons are changed.

[0021] In step (I) described above, it is preferable that at least one codon corresponding to the nucleotide sequence of the DNA encoding NDST is codon-optimized to match the codon usage frequency of a different species than the parent strain.

[0022] From the viewpoint of heterologous expression experience, yeast, insect cells, and filamentous fungi are preferred as the different species of organism from the microorganism (parent strain) into which DNA is introduced in step (II) above, and budding yeast is particularly preferred. Frequently used codons in these species can be confirmed, for example, by the Codon Usage Database (https: / / www.kazusa.or.jp / codon / ). Methods for codon optimization in each species are well known to those skilled in the art.

[0023] In this specification, "parent strain" refers to the original strain that is the subject of genetic modification and transformation. In particular, in "Method for producing microorganisms having N-deacetylation activity and N-sulfation activity - 1", the parent strain in the microorganism having N-deacetylation activity and N-sulfation activity of the present invention refers to the strain into which DNA containing the modified nucleotide sequence is introduced in the above step (II) in an expressible manner.

[0024] As for the type of microorganism (parent strain) into which DNA can be expressed in the above step (II), from the viewpoint of ease of industrial cultivation, bacteria belonging to the genera Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, Microbacterium, or Pseudomonas are preferred, Escherichia is more preferred, and Escherichia coli is particularly preferred.

[0025] Examples of specific, non-limiting Escherichia coli species include Escherichia coli Origami B (DE3) (Novagen), Escherichia coli BL21 codon plus, Escherichia coli XL1-Blue, Escherichia coli XL2-Blue (all manufactured by Agilent Technologies), Escherichia coli BL21(DE3)pLysS (Merck Millipore), Escherichia coli BL21, Escherichia coli DH5α, Escherichia coli HST08 Premium, Escherichia coli HST02, Escherichia coli HST04 dam- / dcm-, Escherichia coli JM109, Escherichia coli HB101, Escherichia coli CJ236, Escherichia coli BMH71-18 mutS, and Escherichia coli. Examples include MV1184, Escherichia coli TH2 (both manufactured by Takara Bio), Escherichia coli W (ATCC9637), Escherichia coli B (ATCC23226), Escherichia coli JM101, Escherichia coli W3110, Escherichia coli MG1655, Escherichia coli DH1, Escherichia coli MC1000, Escherichia coli W1485, Escherichia coli MP347, Escherichia coli NM522, Escherichia coli K5, and Escherichia coli Nissle1917.

[0026] The parent strain used in this invention may be one that inherently possesses heparosan production ability, or it may be one that has been modified to possess heparosan production ability. A microorganism possessing heparosan production ability can be obtained, for example, by conferring heparosan production ability to such a microorganism.

[0027] Heparosan production ability can be conferred by introducing a gene encoding a protein involved in heparosan production, referring to Metabolic Engineering, 2012, 14, pp. 521-527, Carbohydrate Research, 2012, 360, pp. 19-24, and U.S. Patent No. 9,975,928, etc. Examples of proteins involved in heparosan production include glycosyltransferase and heparosan efflux carrier proteins. In the parental strain used in this invention, one gene may be introduced, or two or more genes may be introduced. Gene introduction can also be achieved by introducing a vector containing the gene into the host. For example, the copy number of the gene can be increased by constructing an expression vector for the gene by linking a DNA fragment containing the target gene with a vector that functions in the host, and then transforming the host with this expression vector. A DNA fragment containing the target gene can be obtained, for example, by PCR using the genomic DNA of a microorganism possessing the target gene as a template. The transformation method is not particularly limited, and conventionally known methods can be used.

[0028] In step (II) described above, it is preferable to transform the microorganism (parent strain) with recombinant DNA having DNA containing the nucleotide sequence modified in step (I) so that the DNA containing the nucleotide sequence modified in step (I) can be expressed.

[0029] The DNA containing the modified nucleotide sequence in step (I) above is preferably the DNA of (A-1) or (A-2) below. (A-1) DNA containing the nucleotide sequence shown in Sequence ID No. 6 (A-2) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 6, and which encodes a protein having N-deacetylation activity and N-sulfation activity (hereinafter also referred to as "NDST activity").

[0030] When the DNA containing the modified nucleotide sequence in step (I) is the DNA of (A-1) or (A-2) above, the biological species different from the parent strain in step (II) is budding yeast.

[0031] The DNA described in (A-2) above includes DNA that encodes a protein having NDST activity, comprising a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 6, preferably in the following order: 85% or more, 90% or more, 95% or more, more preferably 98% or more, and most preferably 99% or more.

[0032] In this specification, the identity of nucleotide and amino acid sequences can be determined using the Lipman-Pearson method [Science, 227(4693), 1435-41 (1985)], the BLAST algorithm by Karlin and Altschul [Pro. Natl. Acad. Sci. USA, 90, 5873 (1993)], or FASTA [Methods Enzymol., 183, 63 (1990)]. Based on the BLAST algorithm, programs called BLASTN and BLASTX have been developed [J. Mol. Biol., 215, 403 (1990)]. When analyzing a nucleotide sequence using BLASTN based on BLAST, the parameters are, for example, Score=100 and wordlength=12. When analyzing an amino acid sequence using BLASTX based on BLAST, the parameters are, for example, score=50 and wordlength=3. When using the BLAST and Gapped BLAST programs, use the default parameters for each program. The specific methods for these analysis techniques are publicly known.

[0033] The DNA described in (A-1) above can be prepared, for example, by chemical synthesis using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd., based on the nucleotide sequence represented by Sequence ID No. 6.

[0034] The DNA described in (A-2) above can be prepared, for example, by searching various gene sequence databases for nucleotide sequences that have 80% or more identity with the nucleotide sequence represented by Sequence ID No. 6, preferably in the following order: 85% or more, 90% or more, 95% or more, preferably 97% or more, more preferably 98% or more, and most preferably 99% or more identity, and then chemically synthesizing the nucleotide sequence obtained by the search using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd.

[0035] Recombinant DNA having DNA containing the nucleotide sequence modified in step (I), preferably the DNA described in (A-1) or (A-2) above, means, for example, DNA in which DNA is capable of autonomous replication in the parent strain, and the DNA containing the nucleotide sequence modified in step (I), preferably the DNA described in (A-1) or (A-2) above, is incorporated into an expression vector containing a promoter at a position where the DNA containing the nucleotide sequence modified in step (I), preferably the DNA described in (A-1) or (A-2) above, can be transcribed.

[0036] The recombinant DNA is DNA that can be incorporated into the chromosomes of the parent plant and contains the nucleotide sequence modified in step (I) above, preferably the DNA described in (A-1) or (A-2) above. The recombinant DNA is DNA that contains the nucleotide sequence modified in step (I) above, preferably the DNA described in (A-1) or (A-2) above. If the recombinant DNA is DNA that can be incorporated into the chromosomal DNA of the parent plant, it does not need to contain a promoter.

[0037] When using prokaryotes such as bacteria as the parent strain, the recombinant DNA capable of autonomous replication in the parent strain is preferably recombinant DNA composed of a promoter, a ribosome binding sequence, DNA containing the 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 also include genes that control the promoter. It is preferable to use recombinant DNA in which the distance between the Shine-Dalgarno sequence, which is the ribosome binding sequence, and the start codon is adjusted to an appropriate distance (e.g., 6 to 18 bases).

[0038] In recombinant DNA that can autonomously replicate in the parental plant, a transcription termination sequence is not necessarily required for the expression of the DNA, but it is preferable to place the transcription termination sequence directly below the structural gene.

[0039] In this specification, the expression vector is not particularly limited as long as it is a suitable nucleic acid molecule for introducing, amplifying, and expressing the target DNA in a host. This includes not only plasmids, but also, for example, artificial chromosomes, vectors using transposons, and cosmids.

[0040] In this specification, when using a microorganism belonging to the genus Escherichia as the parent strain, the expression vectors include, for example, pColdI, pSTV28, pSTV29, pUC118 (all manufactured by Takara Bio Inc.), pMW119 (manufactured by Nippon Gene Inc.), pET21a, pCOLADuet-1, pCDFDuet-1, pCDF-1b, pRSF-1b (all manufactured by Merck Millipore Corporation), pMAL-c5x (manufactured by New England Biolabs Inc.), pGEX-4T-1, pTrc99A (all manufactured by GE Healthcare Biosciences Inc.), pTrcHis, pSE280 (all manufactured by Thermo Fisher Scientific Inc.), pGEMEX-1 (manufactured by Promega Inc.), pQE-30, pQE80L (all manufactured by Qiagen Inc.), pET-3, pBluescriptII SK(+), and pBluescriptII KS(-) (both manufactured by Agilent Technologies), pKYP10 (Japanese Patent Publication No. 58-110600), 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 Examples include Microbiology, 2007, Vol.73, No.20, pp. 6378-6385, pPAC31 (International Publication No. 98 / 12343), pUC19 [Gene, 33, 103 (1985)], and pPA1 (Japanese Patent Publication No. 63-233798).

[0041] When using the expression vector described above, any promoter that functions in the cells of microorganisms belonging to the genus Escherichia may be used. Examples include promoters of genes involved in amino acid biosynthesis, such as the trp promoter and the ilv promoter, and promoters derived from Escherichia coli or phages, such as the uspA promoter, lac promoter, PL promoter, PR promoter, and PSE promoter. In addition, artificially designed and modified promoters such as two trp promoters in series, the tac promoter, the trc promoter, the lacT7 promoter, and the letI promoter can also be used.

[0042] Recombinant DNA used in the invention described herein can be prepared, for example, by restricting the DNA fragment prepared by the method described above and inserting it downstream of the promoter of a suitable expression vector.

[0043] In this specification, methods for introducing recombinant DNA having DNA encoding a protein with N-deacetylation activity and N-sulfation activity as an autonomously replicating plasmid in a host cell include, for example, the calcium ion method, the protoplast method, the electroporation method, and the spheroplast method [Proc. Natl. Acad. Sci., USA, 81, 4889 (1984)] and the lithium acetate method [J. Bacteriol., 153, 163 (1983)].

[0044] Furthermore, in this specification, when inserting recombinant DNA containing DNA encoding a protein having N-deacetylation activity and N-sulfation activity into the genome of a parent strain, a method such as homologous recombination may be used. That is, DNA to which a portion of the chromosomal region to which the target DNA will be introduced is attached can be taken up into a microbial cell, and homologous recombination can be caused in that portion of the chromosomal region to be incorporated into the genome. For example, a method using homologous recombination frequently used in Escherichia coli is the introduction of recombinant DNA using a lambda phage homologous recombination system [Proc. Natl. Acad. Sci. USA, 97, 6641-6645 (2000)]. Here, the chromosomal region to which the introduction will occur is not particularly limited, but a non-essential gene region or a non-gene region upstream of a non-essential gene region is preferred. As for the method of taking up the DNA into the microbial cell, any method of introducing DNA into a host cell can be used, for example, the calcium ion method, the protoplast method, the electroporation method, etc.

[0045] In this specification, a microorganism obtained by introducing recombinant DNA having DNA encoding a protein having N-deacetylation activity and N-sulfation activity into a parent strain in an expressible manner can be confirmed, for example, by comparing the transcription amount of the DNA of the microorganism with that of the parent strain by Northern blotting, or by comparing the production amount of the protein of the microorganism with that of the parent strain by Western blotting.

[0046] In this specification, it can be confirmed, for example, that the microorganisms created by the above method are recombinant microorganisms possessing N-deacetylation activity and N-sulfation activity by the following method. First, the parent strain and the created microorganisms are cultured in culture media, and a cell extract containing proteins possessing N-deacetylation activity and N-sulfation activity is prepared from the resulting cultures. Next, the cell extract is brought into contact with heparosan, which is the substrate, and PAPS, which is the sulfate group donor, to produce N-deacetylated and N-sulfated heparosan. Finally, it can be confirmed that the created microorganisms are recombinant microorganisms possessing N-deacetylation activity and N-sulfation activity by detecting the N-deacetylated and N-sulfated heparosan in the reaction solution using a general analytical method such as high-performance liquid chromatography or gas chromatography.

[0047] 2. Method for producing microorganisms having N-deacetylation activity and N-sulfation activity - 2 A method for producing NDST-active microorganisms according to one aspect of the present invention includes step (i) of expressing 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) in a microorganism. (B-1) Protein containing the amino acid sequence shown in Sequence ID No. 13 (B-2) A protein that contains an amino acid sequence having 80% or more identity with the amino acid sequence shown in Sequence ID No. 13, and that has NDST activity. (B-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 13, and that have NDST activity. (C-1) Protein containing the amino acid sequence shown in Sequence ID No. 14 (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 14, and which has NDST activity. (C-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 14, and that have NDST activity. (D-1) Protein containing the amino acid sequence shown in Sequence ID No. 15 (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 15, and possessing N-deacetylation activity and N-sulfation activity. (D-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 15, and that have N-deacetylation activity and N-sulfation activity. (E-1) Protein containing the amino acid sequence shown in Sequence ID No. 16 (E-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 16, and which has NDST activity. (E-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 16, and that have NDST activity.

[0048] The proteins described in (B-2), (C-2), (D-2), or (E-2) above include proteins that have NDST activity and consist of an amino acid sequence that has 80% or more identity with the amino acid sequence shown in SEQ ID NOs. 13, 14, 15, or 16, and more preferably 85% or more, 90% or more, 95% or more, more preferably 98% or more, in that order.

[0049] The proteins described in (B-3), (C-3), (D-3), or (E-3) above include proteins that contain an amino acid sequence in which one or several amino acids, 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 in the amino acid sequence represented by SEQ ID NOs. 13, 14, 15, or 16, respectively, and that have NDST activity.

[0050] In order to obtain microorganisms having high N-deacetylation activity and N-sulfation activity, in step (i) above, it is preferable to express in the microorganism one protein selected from the group consisting of (B-1) to (B-3), (C-1) to (C-3), and (E-1) to (E-3), and it is more preferable to express in the microorganism one protein selected from the group consisting of (C-1) to (C-3).

[0051] An amino acid sequence in which amino acids are deleted, substituted, added, or inserted refers to an amino acid sequence obtained by artificially deleting or substituting amino acid residues in the original amino acid sequence, or by artificially adding or inserting amino acid residues into said amino acid sequence.

[0052] The amino acids deleted, substituted, added, or inserted may be native or unnatural forms. Examples of native amino acids include L-alanine, L-asparagine, L-aspartic acid, L-glutamine, L-glutamic acid, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-arginine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and L-cysteine.

[0053] Below are examples of amino acids that are mutually substituted. Amino acids belonging to the same group are mutually substituted. Group A: Leucine, Isoleucine, Norleucine, Valine, Norvaline, Alanine, 2-Aminobutanoic Acid, Methionine, O-Methylserine, t-Butylglycine, t-Butylalanine, Cyclohexylalanine Group B: Aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid Group C: Asparagine, Glutamine Group D: Lysine, Arginine, Ornithine, 2,4-Diaminobutanoic acid, 2,3-Diaminopropionic acid Group E: Proline, 3-hydroxyproline, 4-hydroxyproline Group F: Serine, Threonine, Homoserine Group G: Phenylalanine, Tryptophan, Tyrosine

[0054] The fact that the above protein possesses N-deacetylation activity and N-sulfation activity (NDST activity) can be confirmed, for example, by the following method. First, recombinant DNA containing the DNA encoding the protein is prepared using the method described later. Next, microorganisms that do not exhibit NDST activity, such as Escherichia coli OrigamiB(DE3), are transformed with the recombinant DNA, and the resulting microorganisms are cultured. A cell extract containing the protein is then prepared from the resulting culture. Subsequently, the cell extract is reacted with heparosan as a substrate in the presence of 3'-phosphoadenosine-5'-phosphosulfate (hereinafter also referred to as "PAPS"), and finally, a general analytical method such as high-performance liquid chromatography or gas chromatography is performed. By detecting the formation of N-deacetylated and N-sulfated heparosan, it can be confirmed that the target protein possesses N-deacetylation activity and N-sulfation activity.

[0055] The preferred types of microorganisms used in step (i) above (hereinafter also referred to as "parent strain") are the same as the preferred types of microorganisms listed as the microorganisms (parent strain) into which DNA can be expressed in step (II) above.

[0056] In this specification, "parent strain" refers to the original strain that is the subject of genetic modification and transformation. In particular, in "Method for producing microorganisms having N-deacetylating activity and N-sulfating activity - 2", the parent strain in the microorganism having N-deacetylating activity and N-sulfating activity of the present invention refers to the strain before the expression of 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) in step (i) above.

[0057] The above step (i) is preferably a step of introducing into the microorganism in an expressible state 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). (b-1) DNA containing the nucleotide sequence shown in Sequence ID No. 9 (b-2) DNA encoding a protein containing the amino acid sequence shown in Sequence ID No. 13 (b-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 9, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (c-1) DNA containing the nucleotide sequence shown in Sequence ID No. 10 (c-2) DNA encoding a protein containing the amino acid sequence shown in Sequence ID No. 14 (c-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 10, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (d-1) DNA containing the nucleotide sequence shown in SEQ ID NO: 11 (d-2) DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO: 15 (d-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 11, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (e-1) DNA containing the nucleotide sequence shown in Sequence ID No. 12 (e-2) DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO: 16 (e-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 12, and which encodes a protein having N-deacetylation activity and N-sulfation activity.

[0058] In order to obtain microorganisms having high N-deacetylation activity and N-sulfation activity, in step (i) above, it is preferable to introduce into the microorganism any one DNA selected from the group consisting of (b-1) to (b-3), (c-1) to (c-3), and (e-1) to (e-3) so as to be expressible, and it is more preferable to introduce into the microorganism any one DNA selected from the group consisting of (c-1) to (c-3) so as to be expressible.

[0059] 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 by chemical synthesis using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd., based on, for example, the nucleotide sequences represented by SEQ ID NOs: 9, 10, 11, and 12 and the amino acid sequences represented by SEQ ID NOs: 13, 14, 15, and 16.

[0060] The DNA described in (b-3), (c-3), (d-3), and (e-3) above can be prepared, for example, by searching various gene sequence databases for nucleotide sequences that have 80% or more identity with the nucleotide sequences represented by SEQ ID NOs: 9, 10, 11, and 12, preferably in the following order: 87% or more, 88% or more, 90% or more, 95% or more, more preferably 98% or more, and most preferably 99% or more, nucleotide sequences that have 80% or more identity with the nucleotide sequences represented by SEQ ID NOs: 9, 10, 11, and 12, respectively, and then chemically synthesizing them using an NTS M series DNA synthesizer manufactured by Nippon Techno Service Co., Ltd. or the like based on the nucleotide sequences obtained by the search.

[0061] In step (i) above, 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) is introduced into the microorganism in an expressible manner. For example, this involves transforming the microorganism (parent strain) with recombinant DNA having 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).

[0062] Recombinant DNA having the DNA described in any one of (b-1)~(b-3), (c-1)~(c-3), (d-1)~(d-3), and (e-1)~(e-3) above refers, for example, to DNA in which the DNA is capable of autonomous replication in the parent strain, and an expression vector containing a promoter at a position where one or more of the DNA described in any one of (b-1)~(b-3), (c-1)~(c-3), (d-1)~(d-3), and (e-1)~(e-3) above can be transcribed, into which one or more of the DNA described in (b-1)~(b-3), (c-1)~(c-3), (d-1)~(d-3), and (e-1)~(e-3) above is incorporated.

[0063] DNA that can be incorporated into the chromosomes of the parental plant, and which is described in 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 also recombinant DNA having one or more of the DNA described in 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. If the recombinant DNA is DNA that can be incorporated into the chromosomal DNA of the parental plant, it does not need to contain a promoter.

[0064] When using prokaryotes such as bacteria as the parent strain, it is preferable that the recombinant DNA capable of autonomous replication in the parent strain is composed of a promoter, a ribosome binding sequence, one or more 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), and a transcription termination sequence. A gene that controls the promoter may also be included. It is preferable to use recombinant DNA in which the distance between the Shine-Dalgarno sequence, which is the ribosome binding sequence, and the start codon is adjusted to an appropriate distance (e.g., 6 to 18 bases).

[0065] In recombinant DNA that can autonomously replicate in the parental plant, a transcription termination sequence is not necessarily required for the expression of the DNA, but it is preferable to place the transcription termination sequence directly below the structural gene.

[0066] 3. Microorganisms possessing N-deacetylation activity and N-sulfation activity A microorganism having N-deacetylation activity and N-sulfation activity according to one aspect of the present invention expresses 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) Protein containing the amino acid sequence shown in Sequence ID No. 13 (B-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 13, and which also possesses N-deacetylation activity and N-sulfation activity. (B-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 13, and that have N-deacetylation activity and N-sulfation activity. (C-1) Protein containing the amino acid sequence shown in Sequence ID No. 14 (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 14, and possessing N-deacetylation activity and N-sulfation activity. (C-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 14, and that have N-deacetylation activity and N-sulfation activity. (D-1) Protein containing the amino acid sequence shown in Sequence ID No. 15 (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 15, and possessing N-deacetylation activity and N-sulfation activity. (D-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 15, and that have N-deacetylation activity and N-sulfation activity. (E-1) Protein containing the amino acid sequence shown in Sequence ID No. 16 (E-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 16, and possessing N-deacetylation activity and N-sulfation activity. (E-3) A protein having N-deacetylation activity and N-sulfation activity, which includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 16.

[0067] Microorganisms having the above-mentioned N-deacetylation activity and N-sulfation activity can be produced by the method described in 2. The preferred type of microorganism is the same as the preferred type of microorganism used in step (i) of 2.

[0068] 4. Method for producing heparosan-derived compounds One embodiment of the present invention for producing a heparosan-derived compound includes a step (III) of producing an N-deacetylated and N-sulfated heparosan-derived compound from heparosan in the presence of a microorganism having N-deacetylation activity and N-sulfation activity or an extract thereof, produced by the above steps (I) and (II) of 1. above.

[0069] Furthermore, one embodiment of the method for producing a heparosan-derived compound of the present invention includes a step (ii) of producing an N-deacetylated and N-sulfated heparosan-derived compound from heparosan in the presence of a microorganism having N-deacetylation activity and N-sulfation activity or an extract thereof, produced by step (i) of 2. above.

[0070] The heparosan-derived compounds produced include N-sulfated heparosan, N-sulfated epimerized heparosan, N-sulfated low molecular weight heparosan, N-sulfated low molecular weight epimerized heparosan, N-sulfated 6-O-sulfated heparosan, N-sulfated 6-O-sulfated epimerized heparosan, N-sulfated 2-O-sulfated 6-O-sulfated heparosan, and N-sulfated 2-O-sulfated 6-O-sulfated low molecular weight heparosan. Molecular-weight heparosan, N-sulfated 6-O-sulfated low molecular weight heparosan, N-sulfated 6-O-sulfated epimerized low molecular weight heparosan, N-sulfated 2-O-sulfated 6-O-sulfated low molecular weight heparosan, N-sulfated 2-O-sulfated 6-O-sulfated low molecular weight heparosan, or heparin are preferred, N-sulfated heparosan or heparin are more preferred, and N-sulfated heparosan is most preferred.

[0071] "Epimeriation" means that the β-D-glucuronic acid residue of heparosan is converted to an α-L-iduronic acid residue. "Depolymerization" means that a substance is treated to reduce its molecular weight. For example, a "depolymerized" heparosan compound has a number-average molecular weight (Mn) of 1,000 to 150,000, preferably 8,000 to 60,000, and a weight-average molecular weight (Mw) of 2,000 to 300,000, preferably 10,000 to 100,000, as measured by GPC with pullulan as the standard. "6-O-sulfation" means that the hydroxyl group at position 6 of the N-acetyl-D-glucosamine residue is sulfated. "2-O-sulfation" means that the hydroxyl group at position 2 of a hexuronic acid residue (preferably an α-L-iduronic acid residue) is sulfated.

[0072] The present invention provides methods for producing heparosan-derived compounds, including (α) a method for producing N-deacetylated and N-sulfated heparosan-derived compounds by fermentation, and (β) a method for producing N-deacetylated and N-sulfated heparosan-derived compounds by adding a culture of a microorganism having N-deacetylation activity and N-sulfation activity, or a processed product of said culture, to heparosan as a substrate and reacting them. Each production method will be described below.

[0073] (α) Method for producing N-deacetylated and N-sulfated heparosan-derived compounds by fermentation. The production of N-deacetylated and N-sulfated heparosan-derived compounds by fermentation can be carried out by culturing microorganisms having N-deacetylation activity and N-sulfation activity in a culture medium to generate N-deacetylated and N-sulfated heparosan-derived compounds in the culture. This production method may include, for example, generating N-deacetylated and N-sulfated heparosan-derived compounds in a culture, accumulating the culture, and then collecting the N-deacetylated and N-sulfated heparosan-derived compounds from the culture.

[0074] The recombinant microorganism used in the method for producing N-deacetylated and N-sulfated heparosan-derived compounds by fermentation is preferably the microorganism produced in step (I) and step (II) above, or the microorganism produced in step (i) above, and is also preferably a microorganism capable of producing heparosan, which is a substrate for proteins having NDST activity, and / or a microorganism in which the ability to produce PAPS, which is a sulfate group donor, has been artificially enhanced. The microorganism may further be a microorganism having one or more of the activities of epimerization, 2-O-sulfation, 6-O-sulfation, and 3-O-sulfation.

[0075] Heparosan production ability can be conferred by introducing a gene encoding a protein involved in heparosan production, referring to Metabolic Engineering, 2012, 14, pp. 521-527, Carbohydrate Research, 2012, 360, pp. 19-24, U.S. Patent No. 9,975,928, etc. The ability to produce PAPS can be conferred by introducing a gene encoding a protein involved in PAPS production, referring to J. Org. Chem. 2000, 65, 18, 5565-5574, International Publication No. 2021 / 201282, etc. The activities of epimerization, 2-O-sulfation, 6-O-sulfation, and 3-O-sulfation can be conferred by introducing genes encoding C5-epimerase, 2-O-sulfotransferase, 6-O-sulfotransferase, 3-O-sulfotransferase, and related isoforms, as referenced in International Publication No. 2021 / 201282, etc.

[0076] The culture of microorganisms can be carried out according to conventional methods. The culture medium for the microorganisms may be either a natural or synthetic medium, as long as it contains heparosan, a carbon source, a nitrogen source, and inorganic salts that the microorganisms can utilize, and is capable of efficiently culturing the microorganisms.

[0077] Any carbon source that the microorganism can utilize is acceptable, and examples include carbohydrates such as glucose, fructose, sucrose, molasses containing these, starch and starch hydrolysates, organic acids such as acetic acid and propionic acid, and alcohols such as ethanol, propanol, and glycerol.

[0078] 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, as well as peptone, meat extract, yeast extract, corn steep liquor, casein hydrolysate, soybean meal and soybean meal hydrolysate, various fermentation microorganisms, and their digests.

[0079] Examples of inorganic salts include monopotassium phosphate, dipotassium phosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, copper sulfate, and calcium carbonate.

[0080] In a method for producing heparosan-derived compounds by fermentation, if the microorganisms used do not have the ability to produce heparosan, which is a substrate for proteins having NDST activity, heparosan may be added to the culture medium.

[0081] Furthermore, in a method for producing heparosan-derived compounds by fermentation, if the microorganism used does not have the ability to produce heparosan, which is a substrate for proteins having NDST activity, instead of adding heparosan to the culture medium, heparosan may be supplied to the microorganism used in the present invention by co-culturing a microorganism capable of producing heparosan with the recombinant microorganism of the present invention.

[0082] In a method for producing heparosan-derived compounds by fermentation, if the microorganisms used do not have the ability to supply PAPS necessary for the N-sulfation reaction, PAPS may be added to the culture medium.

[0083] Furthermore, in the method for producing heparosan-derived compounds by fermentation, if the microorganism used does not have the ability to supply PAPS necessary for the N-sulfation reaction, instead of adding PAPS to the culture medium, PAPS may be supplied to the microorganism used in the present invention by co-culturing a microorganism capable of producing PAPS with the recombinant microorganism of the present invention.

[0084] Culturing is usually carried out under aerobic conditions such as shaking culture or deep aeration stirring culture. The culture temperature is preferably 15 to 40°C, and the culture time is usually 5 hours to 7 days. The pH during cultivation is preferably maintained between 3.0 and 9.0. pH adjustment is performed using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.

[0085] Furthermore, antibiotics such as ampicillin or tetracycline may be added to the culture medium as needed during cultivation. When culturing microorganisms transformed with an expression vector using an inducible promoter, an inducer may be added to the culture medium as needed.

[0086] For example, when culturing microorganisms transformed with an expression vector using the lac promoter, isopropyl-β-D-thiogalactopyranoside or the like may be added to the culture medium, and when culturing microorganisms transformed with an expression vector using the trp promoter, indoleacrylic acid or the like may be added to the culture medium.

[0087] The above cultivation method allows for the production of heparosan-derived compounds in the culture medium. Quantitative analysis of the heparosan-derived compounds can be performed using HPLC (for example, the SPD-M20A analyzer manufactured by Shimadzu Corporation).

[0088] The collection of heparosan-derived compounds from the culture can usually be carried out by combining the ion exchange resin method, the precipitation method, and other known methods. Furthermore, if heparosan-derived compounds accumulate within the bacterial cells, the compounds can be collected from the supernatant obtained by, for example, disintegrating the bacterial cells using ultrasound and removing them by centrifugation, using the ion exchange resin method.

[0089] A method for producing N-deacetylated and N-sulfated heparosan-derived compounds by adding a culture of a microorganism having (β)N-deacetylation activity and N-sulfation activity, or a processed product of said culture, to heparosan as a substrate and reacting the two. The above-mentioned heparosan-derived compound can be produced by a method in which a microorganism having N-deacetylation activity and N-sulfation activity, or an extract thereof, as an enzyme source, heparosan as a substrate, and PAPS as a sulfate group donor are placed in an aqueous medium, and an N-deacetylated and N-sulfated heparosan-derived compound is produced in the aqueous medium. This production method may include, for example, producing an N-deacetylated and N-sulfated heparosan-derived compound in an aqueous medium, accumulating it, and then collecting the N-deacetylated and N-sulfated heparosan-derived compound from the aqueous medium.

[0090] The method and culture medium for culturing microorganisms are the same as those described above in (α).

[0091] In this specification, examples of processed products of a culture include concentrates of the culture, dried products of the culture, bacterial cells obtained by centrifuging or filtering the culture, dried products of the bacterial cells, freeze-dried products of the bacterial cells, surfactant-treated products of the bacterial cells, solvent-treated products of the bacterial cells, enzyme-treated products of the bacterial cells, and immobilized products of the bacterial cells, which contain live bacterial cells that have the same function as the culture as an enzyme source, as well as ultrasonically treated products of the bacterial cells, mechanically ground products of the bacterial cells, crude enzyme extracts obtained from the treated bacterial cells, and purified enzymes obtained from the treated bacterial cells.

[0092] Among these, preferred are concentrated cultures, dried cultures, bacterial cells obtained by centrifuging or filtering the culture, dried bacterial cells, freeze-dried bacterial cells, surfactant-treated bacterial cells, solvent-treated bacterial cells, enzyme-treated bacterial cells, and immobilized bacterial cells, which contain live bacterial cells that have the same function as the culture as an enzyme source, as well as ultrasonically treated bacterial cells and mechanically ground bacterial cells. Most preferred are concentrated cultures, dried cultures, bacterial cells obtained by centrifuging or filtering the culture, dried bacterial cells, freeze-dried bacterial cells, surfactant-treated bacterial cells, solvent-treated bacterial cells, enzyme-treated bacterial cells, and immobilized bacterial cells, which contain live bacterial cells that have the same function as the culture as an enzyme source.

[0093] The concentration of the protein having NDST activity as an enzyme source is preferably 1 mg / L to 500 g / L, more preferably 1 mg / L to 300 g / L, and most preferably 1 mg / L to 5 g / L.

[0094] The concentration of the substrate heparosan is preferably 1 mg / L to 100 g / L, and more preferably 10 mg / L to 20 g / L.

[0095] The concentration of PAPS, which is a sulfate group donor, is preferably 0.1 to 500 mM, and more preferably 0.5 to 50 mM. Furthermore, since PAPS is converted to 3'-phosphoadenosine-5'-phosphate (hereinafter also referred to as "PAP") when used in the N-sulfation reaction, an enzyme and / or substrate for regenerating PAP to PAPS may be added. Examples of enzymes and / or substrates for regenerating PAPS include ATP sulfurylase, adenosine 5'-phosphosulfate kinase, ATP or an ATP source and a sulfate ion source (International Publication No. 2021 / 201282), 3'-phosphoadenosine 5'-phosphosulfate-sulfotransferase and p-nitrophenyl sulfate (pNPS) (International Publication No. 2020 / 013346).

[0096] Examples of aqueous 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. In addition, the culture medium of microorganisms used as an enzyme source can be used as an aqueous medium.

[0097] The heparosan-derived compound generated in the aqueous medium can be quantified and collected in (α) by the method described above.

[0098] One embodiment of the method for producing a heparosan-derived compound of the present invention may include at least one of the following steps in addition to step (III) or step (ii). The following steps are examples of steps for producing heparin from heparosan (International Publication No. 2017 / 115674, International Publication No. 2017 / 115675). • A process of breaking down heparosan to produce heparosan with a lower molecular weight. • The process of isomerizing the β-D-glucuronic acid residue in heparosan to the epimer, α-L-iduronic acid (IdoA) residue. • A step of sulfating the hydroxyl group at position 2 of the hexuronic acid residue (preferably an α-L-iduronic acid residue) in heparosan. • A process of sulfating the hydroxyl group at position 6 of the α-D-glucosamine residue in heparosan. • A process of sulfating the hydroxyl group at position 3 of the α-D-glucosamine residue in heparosan.

[0099] As explained above, the following matters are disclosed in this specification. {1} Step (I) involves modifying the nucleotide sequence of the DNA encoding N-deacetylase / N-sulfotransferase, The process includes (I) a step of introducing DNA containing the modified nucleotide sequence into a microorganism so that it can express the DNA, The above step (I) is a step of codon optimization that matches the codon usage frequency of a different biological species from the microorganism in question. A method for producing microorganisms having N-deacetylation activity and N-sulfation activity. {2} A method for producing a microorganism having N-deacetylation activity and N-sulfation activity as described in {1} above, wherein the microorganism is a bacterium and the species is budding yeast. {3} A method for producing a microorganism having N-deacetylation activity and N-sulfation activity as described in {2} above, wherein the DNA containing the modified nucleotide sequence is DNA of (A-1) or (A-2). (A-1) DNA containing the nucleotide sequence shown in Sequence ID No. 6 (A-2) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 6, and which encodes a protein having N-deacetylation activity and N-sulfation activity. {4} A method for producing a heparosan-derived compound, comprising the step (III) of producing an N-deacetylated and N-sulfated heparosan-derived compound from heparosan in the presence of a microorganism or extract thereof having N-deacetylation activity and N-sulfation activity as described in any one of {1} to {3} above. {5} The method for producing a microorganism according to any one of {1} to {4} above, wherein the microorganism is a bacterium of the genus Escherichia. {6} The method for producing a bacterial strain of the genus Escherichia as described in {5} above, wherein the Escherichia bacterium is Escherichia coli. {7} The method for producing the product according to {4} above, wherein the heparosan-derived compound is N-sulfated heparosan. {8} A method for producing a microorganism having N-deacetylation activity and N-sulfation activity, comprising step (i) expressing in a microorganism 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). (B-1) Protein containing the amino acid sequence shown in Sequence ID No. 13 (B-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 13, and which also possesses N-deacetylation activity and N-sulfation activity. (B-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 13, and that have N-deacetylation activity and N-sulfation activity. (C-1) Protein containing the amino acid sequence shown in Sequence ID No. 14 (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 14, and possessing N-deacetylation activity and N-sulfation activity. (C-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 14, and that have N-deacetylation activity and N-sulfation activity. (D-1) Protein containing the amino acid sequence shown in Sequence ID No. 15 (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 15, and possessing N-deacetylation activity and N-sulfation activity. (D-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 15, and that have N-deacetylation activity and N-sulfation activity. (E-1) Protein containing the amino acid sequence shown in Sequence ID No. 16 (E-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 16, and possessing N-deacetylation activity and N-sulfation activity. (E-3) A protein having N-deacetylation activity and N-sulfation activity, which includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 16. {9} A method for producing a microorganism having N-deacetylation activity and N-sulfation activity as described in {8} above, wherein step (i) is a step of introducing into the microorganism in a manner that enables expression of any one DNA selected from the group consisting of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3) below. (b-1) DNA containing the nucleotide sequence shown in Sequence ID No. 9 (b-2) DNA encoding a protein containing the amino acid sequence shown in Sequence ID No. 13 (b-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 9, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (c-1) DNA containing the nucleotide sequence shown in Sequence ID No. 10 (c-2) DNA encoding a protein containing the amino acid sequence shown in Sequence ID No. 14 (c-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 10, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (d-1) DNA containing the nucleotide sequence shown in SEQ ID NO: 11 (d-2) DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO: 15 (d-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 11, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (e-1) DNA containing the nucleotide sequence shown in Sequence ID No. 12 (e-2) DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO: 16 (e-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 12, and which encodes a protein having N-deacetylation activity and N-sulfation activity. {10} A method for producing a heparosan-derived compound, comprising the step (ii) of producing an N-deacetylated and N-sulfated heparosan-derived compound from heparosan in the presence of a microorganism or extract thereof having N-deacetylation activity and N-sulfation activity as described in {8} or {9} above. {11} The method for producing a microorganism according to any one of {8} to {10} above, wherein the microorganism is a bacterium of the genus Escherichia. {12} The method for producing a bacterial strain of the genus Escherichia as described in {11} above, wherein the Escherichia bacterium is Escherichia coli. {13} The method for producing the product according to {10} above, wherein the heparosan-derived compound is N-sulfated heparosan. {14} A microorganism expressing one of the proteins selected from the groups (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3), and (E-1) to (E-3) below, and possessing N-deacetylation activity and N-sulfation activity. (B-1) Protein containing the amino acid sequence shown in Sequence ID No. 13 (B-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 13, and which also possesses N-deacetylation activity and N-sulfation activity. (B-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 13, and that have N-deacetylation activity and N-sulfation activity. (C-1) Protein containing the amino acid sequence shown in Sequence ID No. 14 (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 14, and possessing N-deacetylation activity and N-sulfation activity. (C-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in SEQ ID NO: 14, and that have N-deacetylation activity and N-sulfation activity. (D-1) Protein containing the amino acid sequence shown in Sequence ID No. 15 (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 15, and possessing N-deacetylation activity and N-sulfation activity. (D-3) Proteins that contain an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 15, and that have N-deacetylation activity and N-sulfation activity. (E-1) Protein containing the amino acid sequence shown in Sequence ID No. 16 (E-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 16, and possessing N-deacetylation activity and N-sulfation activity. (E-3) A protein having N-deacetylation activity and N-sulfation activity, which includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 16. [Examples]

[0100] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples unless it exceeds the gist of the invention.

[0101] [Example of analysis] The N-deacetylation and N-sulfation reaction activity of heparosan was analyzed by HPLC as follows.

[0102] In accordance with Patent Document 2 (WO2018 / 048973), unsaturated disaccharides were produced by enzymatic digestion and analyzed by HPLC. The obtained reaction solution was centrifuged, and the resulting supernatant was heated and held at 80°C for 10 minutes to denature the protein. The solution after protein denaturation was centrifuged, and the resulting supernatant was desalted by ultrafiltration using an Amicon Ultra centrifugal filter 3K device (Merck). The desalted solution was subjected to a heparinase reaction solution [composed of 50 mM ammonium acetate and 2 mM calcium chloride] containing 0.5 U / ml each of heparinase I, II, and III (Sigma-A) and enzymatically digested at 35°C for 2 hours. The heparinase was inactivated by holding the solution after enzymatic digestion at 95°C for 15 minutes.

[0103] Unsaturated disaccharides were analyzed by subjecting the solution after heparinase inactivation to gradient elution mode analysis using a general-purpose HPLC Prominence (Shimadzu Corporation) and a strong anion exchange column (spherisorb-SAX chromatography column, 4.0 × 250 mm, 5 μm, Waters). This analysis involved mobile phase A [aqueous solution containing 1.8 mM sodium dihydrogen phosphate, adjusted to pH 3.0 with phosphoric acid] and mobile phase B [aqueous solution containing 1.8 mM sodium dihydrogen phosphate and 1 M sodium perchlorate, adjusted to pH 3.0 with phosphoric acid].

[0104] Unsaturated disaccharides were detected by measuring the 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). Here, ΔUA represents 4,5-unsaturated uronic acid, GlcNAc represents N-acetylglucosamine, GlcN represents glucosamine, and GlcNS represents N-sulfoglucosamine. N-deacetylation and N-sulfation activity can be confirmed by determining the area ratio of ΔUA-GlcNS to the total area of ​​detected ΔUA-GlcNAc, ΔUA-GlcN, and ΔUA-GlcNS, and N-deacetylation activity can be confirmed by determining the area ratio of ΔUA-GlcN to the total area of ​​ΔUA-GlcNAc, ΔUA-GlcN, and ΔUA-GlcNS. Hereafter, ΔUA-GlcNAc will be abbreviated as GlcNAc, ΔUA-GlcN as GlcN, and ΔUA-GlcNS as GlcNS. [Example 1]

[0105] Generation of microorganisms expressing NDST - 1 (1) Creation of a plasmid for expressing wild-type rat NDST-1 A plasmid expressing the rat-derived wild-type NDST gene (SEQ ID NO: 1) was constructed using the following procedure. First strand cDNA derived from mouse liver, purchased from Genostaff, was used as a template, and PCR was performed using primers consisting of SEQ ID NOs. 2 and 3 to amplify the rat-derived wild-type NDST gene. Next, expression vector pGEX-4T3 (GE Healthcare Life Sciences) was used as a template, and PCR was performed using primers consisting of SEQ ID NOs. 4 and 5. The resulting fragment and the previously prepared rat-derived wild-type NDST gene fragment were ligated using the In-Fusion HD Cloning Kit (Takara Bio) to obtain the expression plasmid pGEX-NDST (WT).

[0106] (2) Creation of a plasmid for expressing NDST-1 in budding yeast codon-optimized rats A plasmid expressing the budding yeast codon-optimized NDST gene (SEQ ID NO: 6) was constructed using the following procedure. Using DNA synthesized with Eurofins as a template, PCR was performed with primers consisting of SEQ ID NOs. 7 and 8 to amplify the rat-derived wild-type NDST gene. Next, using the expression vector pGEX-4T3 (GE Healthcare Life Sciences) as a template, PCR was performed with primers consisting of SEQ ID NOs. 4 and 5. The resulting fragments and the rat-derived wild-type NDST gene fragment prepared earlier were used to obtain the expression plasmid pGEX-NDST(SC) by restriction enzyme treatment with BamHI and EcoRI, respectively, and then ligated.

[0107] (3) Creation of NDST-1 expressing E. coli strains into which plasmids have been introduced. E. coli Origami B (DE3) (Novagen), transformed using the constructed expression plasmid vector, was selected with ampicillin to obtain an E. coli strain expressing NDST. [Example 2]

[0108] NDST Reaction Test-1 (1)Culture method E. coli Origami B (DE3) transformed with an expression plasmid was cultured overnight at 30°C on LB agar medium supplemented with ampicillin. The grown cells were inoculated into LB liquid medium supplemented with ampicillin and cultured overnight at 30°C in a test tube with shaking to prepare a seed culture. The seed culture was then inoculated into LB liquid medium supplemented with ampicillin at a volume of 5%. This culture was cultured at 30°C with shaking, and when the absorbance at 660 nm was 0.4-0.6 as measured by spectrophotometer, IPTG was added to a final concentration of 0.1 mM and the culture was inducible at 20°C for 24 hours. After the culture was completed, the cells were collected and centrifuged at 9,000 rpm, 4°C, for 5 minutes. The cells were stored at -80°C.

[0109] (2) Reaction method The recovered E. coli cells were suspended in extraction buffer [0.1M MES / NaOH (6.5%), 20mM MnCl2, 5% (v / v) glycerol, 1mM PMSF] to a wet weight of 140 g / L. The E. coli suspension was sonicated on ice and centrifuged at 12,000 rpm, 4°C, for 5 minutes. The supernatant was collected as the E. coli extract and used in the enzymatic reaction.

[0110] (3) Enzyme reaction The substrate and E. coli extract were mixed in a 1.5 mL tube to the following composition: [50 mM MES / NaOH (6.5%), 10 mM MnCl2, 1 g / L heparosan, 4.4 mM PAPS, 50% (v / v) crude E. coli extract]. The mixture was incubated at 30°C for 24 hours. The reaction was stopped by heat treatment at 80°C for 10 minutes.

[0111] (4) Reaction test results The results of the reaction test are summarized in Table 1. GlcNAc indicates the presence of unreacted heparosan, GlcN indicates the presence of N-deacetylated heparosan, and GlcNS indicates the presence of N-sulfated heparosan.

[0112] [Table 1]

[0113] pGEX-NDST(SC), which incorporates NDST with codon optimization to match the codon usage frequency of budding yeast, showed unexpectedly high N-deacetylation and N-sulfation reactions. [Example 3]

[0114] Generation of microorganisms expressing NDST - 2 For each of the human-derived NDST-1 to NDST-4 genes, we modified the sequence by changing consecutive rare codons to optimal codons for E. coli, altering the base sequence of regions that may form loops on the mRNA, modifying the GC content to be closer to that of E. coli, and changing the amino acids positioned on the surface in terms of three-dimensional structure. Modified NDST-1 to NDST-4, represented by SEQ ID NOs. 9 to 12, were synthesized using the base sequences designed in this way. Each DNA sequence was cleaved at NdeI and BamHI and inserted into the BamHI-NdeI site of PET15b to obtain NDST1_pET15b, NDST2_pET15b, NDST3_pET15b, and NDST4_pET15b, respectively. The amino acid sequences of the modified NDST-1 to NDST-4 are shown in SEQ ID NOs. 13 to 16. [Example 4]

[0115] NDST Reaction Test-2 Each plasmid was introduced into Escherichia coli OrigamiB(DE3), and each was cultured in LB medium at 22°C. When the OD600 increased from 0.8 to 1.0, IPTG was added to a final concentration of 1 mM, and the cells were cultured for a further 16-20 hours. After that, the cells were collected, lysed by sonication, and the supernatant was prepared as a cell extract. A pH 6.8-7.0 solution consisting of 50 mM MES, 125 mM NaCl, and 10 mM MgCl2, containing 50% of this cell extract, was mixed with the following substrates at a concentration of 2 mg / mL, and the mixture was reacted at 37°C for 18 hours. In (b) and (c) below, PAPS was added to the solution to a final concentration of 5 mM.

[0116] The reaction was carried out under the following three conditions. (a) Heparosan was used as the substrate, and PAPS was not added. (b) N-deacetylated heparosan was used as a substrate, and PAPS was added. (c) Heparosan was used as a substrate, and PAPS was added.

[0117] The reaction was stopped by heat treatment at 95°C for 10 minutes. The resulting sugars were decomposed with heparinases I, II, and III, and the amount of the two constituent sugars was analyzed by HPLC. The results of (a) are shown in Figure 1, the results of (b) are shown in Figure 2, and the results of (c) are shown in Figure 3. GlcNAc indicates the presence of unreacted heparosan, GlcN indicates the presence of deacetylated heparosan, and GlcNS indicates the presence of N-sulfated heparosan.

[0118] As shown in Figure 1, when heparosan was used as a substrate in the absence of PAPS (a), approximately 50% of GlcNAc was deacetylated in NDST-1 and NDST-2. As shown in Figure 2, when deacetylated heparosan was reacted as a substrate in the presence of PAPS (b), significant GlcNS production was observed in all cases, but the activity of NDST-1, 2, and 4 was higher. As shown in Figure 3, when heparosan was reacted as a substrate in the presence of PAPS (c), GlcNS was produced in NDST-1, 2, and 4, indicating that NDST-1, 2, and 4 possess both deacetylation and N-sulfation activity. NDST-2 was found to have particularly high N-deacetylation and N-sulfation activity as an enzyme expressed in bacteria, with a GlcNS ratio of over 85%.

[0119] [Table 2]

[0120] While the present invention is described in detail with reference to certain embodiments, it will be obvious 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 on 31 March 2023, the contents of which are incorporated herein by reference. [Sequence Listing Free Text]

[0121] Sequence ID 1: Nucleotide sequence of the rat-derived wild-type NDST gene Sequence ID 2: Nucleotide sequence of primer F for amplification of rat-derived wild-type NDST gene Sequence ID 3: Nucleotide sequence of primer R for amplification of rat-derived wild-type NDST gene Sequence ID 4: Base sequence of primer F for pGEX-4T3 amplification Sequence ID 5: Base sequence of primer R for pGEX-4T3 amplification Sequence ID 6: Base sequence of the budding yeast codon-optimized NDST gene Sequence ID 7: Base sequence of primer F for codon-optimized NDST gene amplification in budding yeast Sequence ID 8: Base sequence of primer R for codon-optimized NDST gene amplification in budding yeast Sequence ID 9: DNA sequence of the modified NDST-1 gene Sequence ID No. 10: Base sequence of the modified NDST-2 gene Sequence ID 11: Base sequence of the modified NDST-3 gene Sequence ID 12: Base sequence of the modified NDST-4 gene Sequence ID 13: Amino acid sequence of modified NDST-1 Sequence ID 14: Amino acid sequence of modified NDST-2 Sequence ID 15: Amino acid sequence of modified NDST-3 Sequence ID 16: Amino acid sequence of modified NDST-4

Claims

1. Step (I) involves modifying the nucleotide sequence of the DNA encoding N-deacetylase / N-sulfotransferase, The process includes (I) a step of introducing DNA containing the modified nucleotide sequence into a microorganism so that it can express the DNA, The above step (I) is a step of codon optimization that matches the codon usage frequency of a different biological species from the microorganism in question. A method for producing microorganisms having N-deacetylation activity and N-sulfation activity.

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

3. A method for producing a microorganism having N-deacetylation activity and N-sulfation activity according to claim 2, wherein the DNA containing the modified nucleotide sequence is DNA of (A-1) or (A-2). (A-1) DNA containing the nucleotide sequence shown in Sequence ID No. 6 (A-2) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 6, and which encodes a protein having N-deacetylation activity and N-sulfation activity.

4. A method for producing a heparosan-derived compound, comprising the step (III) of producing an N-deacetylated and N-sulfated heparosan-derived compound from heparosan in the presence of a microorganism having N-deacetylation activity and N-sulfation activity or an extract thereof, produced by the manufacturing method described in any one of claims 1 to 3.

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

6. The method for producing a bacterial organism of the genus Escherichia according to claim 5, wherein the Escherichia bacterium is Escherichia coli.

7. The method for producing the product according to claim 4, wherein the heparosan-derived compound is N-sulfated heparosan.

8. A method for producing a microorganism having N-deacetylation activity and N-sulfation activity, comprising step (i) expressing in a microorganism 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) below. (B-1) Protein containing the amino acid sequence shown in Sequence ID No. 13 (B-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 13, and which has N-deacetylation activity and N-sulfation activity. (B-3) A protein having N-deacetylation activity and N-sulfation activity, which includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No.

13. (C-1) Protein containing the amino acid sequence shown in Sequence ID No. 14 (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 14, and which has N-deacetylation activity and N-sulfation activity. (C-3) A protein having N-deacetylation activity and N-sulfation activity, which includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No.

14. (D-1) Protein containing the amino acid sequence shown in Sequence ID No. 15 (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 15, and which has N-deacetylation activity and N-sulfation activity. (D-3) A protein having N-deacetylation activity and N-sulfation activity, which includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No.

15. (E-1) Protein containing the amino acid sequence shown in Sequence ID No. 16 (E-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 16, and which has N-deacetylation activity and N-sulfation activity. (E-3) A protein having an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 16, and which also has N-deacetylation activity and N-sulfation activity.

9. The method for producing a microorganism having N-deacetylation activity and N-sulfation activity according to claim 8, wherein step (i) is a step of introducing into the microorganism in a manner that enables expression of any one DNA selected from the group consisting of (b-1) to (b-3), (c-1) to (c-3), (d-1) to (d-3) below. (b-1) DNA containing the nucleotide sequence shown in Sequence ID No. 9 (b-2) DNA encoding a protein containing the amino acid sequence shown in Sequence ID No. 13 (b-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 9, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (c-1) DNA containing the nucleotide sequence shown in Sequence ID No. 10 (c-2) DNA encoding a protein containing the amino acid sequence shown in Sequence ID No. 14 (c-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 10, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (d-1) DNA containing the nucleotide sequence shown in Sequence ID No. 11 (d-2) DNA encoding a protein containing the amino acid sequence shown in Sequence ID No. 15 (d-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 11, and which encodes a protein having N-deacetylation activity and N-sulfation activity. (e-1) DNA containing the nucleotide sequence shown in Sequence ID No. 12 (e-2) DNA encoding a protein containing the amino acid sequence shown in SEQ ID NO: 16 (e-3) DNA containing a nucleotide sequence that has 80% or more identity with the nucleotide sequence shown in Sequence ID No. 12, and which encodes a protein having N-deacetylation activity and N-sulfation activity.

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

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

12. The method for producing the product according to claim 11, wherein the Escherichia bacterium is Escherichia coli.

13. The method for producing the product according to claim 10, wherein the heparosan-derived compound is N-sulfated heparosan.

14. A microorganism expressing one of the proteins selected from the groups (B-1) to (B-3), (C-1) to (C-3), (D-1) to (D-3), and (E-1) to (E-3) below, and possessing N-deacetylation activity and N-sulfation activity. (B-1) Protein containing the amino acid sequence shown in Sequence ID No. 13 (B-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 13, and which has N-deacetylation activity and N-sulfation activity. (B-3) A protein having N-deacetylation activity and N-sulfation activity, which includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No.

13. (C-1) Protein containing the amino acid sequence shown in Sequence ID No. 14 (C-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 14, and which has N-deacetylation activity and N-sulfation activity. (C-3) A protein having N-deacetylation activity and N-sulfation activity, which includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No.

14. (D-1) Protein containing the amino acid sequence shown in Sequence ID No. 15 (D-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 15, and which has N-deacetylation activity and N-sulfation activity. (D-3) A protein having N-deacetylation activity and N-sulfation activity, which includes an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No.

15. (E-1) Protein containing the amino acid sequence shown in Sequence ID No. 16 (E-2) A protein containing an amino acid sequence that has 80% or more identity with the amino acid sequence shown in Sequence ID No. 16, and which has N-deacetylation activity and N-sulfation activity. (E-3) A protein having an amino acid sequence in which one or more amino acid residues are deleted, substituted, added, or inserted in the amino acid sequence shown in Sequence ID No. 16, and which also has N-deacetylation activity and N-sulfation activity.

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