8R-lipoxygenase-based enzyme synthesis technology for inflammation resolution mediators

Abstract

To provide a method for synthesizing a lipid mediator having an inflammation resolution effect from a polyunsaturated fatty acid.SOLUTION: According to a method, the R configuration of a polyunsaturated fatty acid is oxidized to synthesize a substance selected from the group consisting of an oxide of a hydroxy polyunsaturated fatty acid and an oxide of a hydroperoxy polyunsaturated fatty acid. The method includes the step of performing an enzymatic reaction using 8-lipoxygenase derived from Malacostraca.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for synthesizing a lipid mediator using 8R-lipoxygenase derived from crustaceans. [Background technology]

[0002] Lipid mediators that are polyunsaturated fatty acid metabolites such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and have an inflammation-resolving effect are collectively called specialized pro-resolving mediators (SPMs). Inflammation is a major risk factor for cancer, heart disease, brain disease, metabolic disease, rheumatism, and other conditions, and SPMs that have an anti-inflammatory effect are expected to have an effect of preventing and improving these diseases. Most SPMs are unsaturated fatty acids with two or three hydroxyl groups introduced, and the stereochemical configuration of the hydroxyl groups is important for the activity of SPMs.

[0003] In vivo, SPM is produced at the site of inflammation. Specifically, SPM is synthesized in one or multiple steps from omega-3 fatty acids by the action of lipoxygenase, cyclooxygenase, and epoxide hydrolase. In order to put this metabolite into practical use as a medicine, it is necessary to synthesize it efficiently and in large quantities at low cost. For example, the reaction shown in Figure 1 can be considered to produce protectin D1 from DHA, but a method for synthesizing a high-purity product that can be used as an active ingredient in medicine at low cost and in large quantities has not yet been established. Protectin D1 is biosynthesized at the site of inflammation, but it is difficult to use the biosynthetic enzymes at the site of inflammation for the synthesis, and no attempts have been made to do so.

[0004] Various lipoxygenases exist universally in the living world, each with a different biological function. In the first step of biosynthesis of protectin D1, an SPM at an inflammatory site, 17S-HpDHA is produced from DHA. For example, soybean lipoxygenase also has the activity of introducing a hydroxyl group to the 17th position of DHA to produce 17S-HpDHA. In addition, a method using plant lipoxygenase for the enzymatic synthesis of SPMs such as protectin D1 has been reported (Non-Patent Document 1). However, since LOX in plants such as soybeans is an enzyme that introduces a hydroxyl group to unsaturated fatty acids so that they have an S configuration, it has been difficult to enzymatically synthesize metabolites that bind a hydroxyl group in the R configuration (e.g., resolvin D1: 7S,8R,17S-trihydroxy-DHA and protectin D1: 10R,17S-dihydroxy-DHA).

[0005] It was discovered that by varying the reaction conditions of soybean lipoxygenase, metabolites were produced in which a hydroxyl group was introduced at the 10th position in addition to the 17th position. However, the SPM produced here is 10S,17S-DiH-DHA, an isomer with a different three-dimensional structure from protectin D1, which is 10R,17S-DiH-DHA, and it has been revealed that it has weaker anti-inflammatory activity than protectin D1 (Non-Patent Document 2).

[0006] Yamada et al. published Non-Patent Document 3, which shows that 8R-H-EPE, 8R-H-ETE, and 10R-H-DHA are contained in Euphausia pacifica extract, and that 8R-H-EPE is produced from EPA through an enzymatic reaction. However, just because 8R-H-EPE is contained in Euphausia pacifica, it is not easy to assume that Euphausia pacifica has an R-type lipoxygenase that specifically oxidizes the 10th carbon of DHA. In the discussion of the paper in Non-Patent Document 3, Yamada et al. state that 8R-H-ETE, 8R-H-EPE, and 10R-H-DHA are rare compounds, but do not mention the R-type lipoxygenase of Euphausia pacifica. In studies preceding Non-Patent Document 3, the general interpretation was that the R-configuration oxidation of highly unsaturated fatty acids occurs not in lipoxygenase but in P450 or cyclooxygenase treated with aspirin. In the biosynthetic pathway of protectin D1, a paper preceding Non-Patent Document 3 showed that the hydroxyl group of 10R is generated by hydrolysis by a hydrolase after the peroxide at the 17th position is epoxidized. Thus, for those skilled in the art, finding a compound having a hydroxyl group in the R configuration was not synonymous with demonstrating the presence of an R-type lipoxygenase. In addition, the present invention shows that PK-LOX1 produces 10R,17S-DiH-DHA using 17S-H-DHA as a substrate, but it is not possible to predict whether an enzyme that synthesizes 10R-H-DHA can specifically oxidize the R configuration of the 10th carbon by reacting with 17S-H-DHA, even if such an enzyme exists. Therefore, even if a person skilled in the art comes across the paper by Yamada et al., he or she will not easily come up with a method for easily and economically producing an SPM having a preferred three-dimensional structure.

[0007] Therefore, a method for easily and economically producing SPMs with preferred three-dimensional structures has been desired. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Dobson et al., Journal of Lipid Research, Volume 54, 2013, 1439-1447 [Non-Patent Document 2] Balas et al., Biochimie, 99(2014), 1-7 [Non-Patent Document 3] Yamada et al., Bioscience, Biotechnology, and Biochemistry Volume 84, 2020 - Issue 3 Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a method for easily and economically producing SPMs having a preferred three-dimensional structure. [Means for solving the problem]

[0010] The inventors demonstrated that Euphausia pacifica lipoxygenase (PK-LOX1) is an enzyme that can oxidize arachidonic acid, EPA, and DHA to produce hydroperoxy (-OOH) products, and that by utilizing PK-LOX1, it is possible to enzymatically synthesize SPMs that bind hydroxyl groups in the R-configuration (i.e., SPMs with a preferred three-dimensional structure), thereby solving the above-mentioned problem.

[0011] For example, the present invention provides the following: (Item 1) A method for synthesizing a substance selected from the group consisting of oxides of highly unsaturated fatty acids in the hydroxyl type and oxides of highly unsaturated fatty acids in the hydroperoxy type by oxidizing the R configuration of a highly unsaturated fatty acid, the method comprising a step of performing an enzymatic reaction using 8-lipoxygenase derived from Malacostraca. (Item 2) 2. The method according to item 1, wherein the oxidized polyunsaturated fatty acid is SPM. (Item 3) The 8-lipoxygenase derived from the class Malacostraca is (1) The activity of oxidizing the 8-carbon atom of arachidonic acid; (2) the activity of oxidizing the 8-carbon atom of eicosapentaenoic acid; and (3) Activity of oxidizing the 10-carbon atom of docosahexaenoic acid The method according to item 1 or 2, wherein the compound has an activity selected from the group consisting of: (Item 4) 3. The method according to item 1 or 2, wherein the 8-lipoxygenase derived from Malacostraca is an 8-lipoxygenase derived from Euphausiacea or an 8-lipoxygenase derived from Decapoda. (Item 5) The 8-lipoxygenase derived from the class Malacostraca is (a) a polypeptide comprising an amino acid sequence comprising SEQ ID NO:2; (b) a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2; (c) a polypeptide consisting of an amino acid sequence of SEQ ID NO:2 containing one or several mutations, substitutions, insertions or deletions; (d) a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence consisting of SEQ ID NO:1; (e) a polypeptide encoded by a nucleic acid that hybridizes under highly stringent conditions to the complement of a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:1; (f) a polypeptide encoded by a nucleic acid comprising a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:1; (g) a polypeptide encoded by a nucleic acid sequence having a nucleic acid sequence containing one or more mutations, substitutions, insertions or deletions in the nucleic acid sequence of SEQ ID NO:1; A polypeptide selected from the group consisting of: (1) The activity of oxidizing the 8-carbon atom of arachidonic acid; (2) the activity of oxidizing the 8-carbon atom of eicosapentaenoic acid; and (3) Activity of oxidizing the 10-carbon atom of docosahexaenoic acid The method according to item 1 or 2, wherein the polypeptide has an activity selected from the group consisting of: (Item 6) The 8-lipoxygenase derived from the class Malacostraca is (a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2, and (d) a polypeptide encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:1. The method according to item 1 or 2, selected from the group consisting of: (Item 7) An SPM-containing material made by combining (i) a crustacean-derived food product having 8R-LOX activity, (ii) a food product having 15S-LOX activity, and (iii) a substance selected from the group consisting of highly unsaturated fatty acids and their derivatives. (Item 8) By oxidizing the R configuration of highly unsaturated fatty acids, (a) Oxidation of polyunsaturated fatty acids in the hydroxy form; (b) an oxide of a highly unsaturated fatty acid in the hydroperoxy form; and (c) Oxidized polyunsaturated fatty acids containing hydroxyl and hydroperoxy groups; A method for synthesizing an oxide selected from the group consisting of: (1) 8-lipoxygenase from Malacostraca and (2) 15-LOX lipoxygenase The method includes carrying out an enzymatic reaction using (Item 9) 9. The method according to item 8, wherein the oxide of the highly unsaturated fatty acid is SPM. (Item 10) From DHA 10R,17S-dihydroxy-DHA (10R,17S-DiH-DHA), 10R-hydroperoxy-,17S-hydroxy-DHA (10R-Hp-,17S-H-DHA), 10R-hydroxy-,17S-hydroperoxy-DHA (10R-H-,17S-Hp-DHA), and 1. A method for producing a substance selected from the group consisting of 10R,17S-dihydroperoxy-DHA (10R,17S-DiHp-DHA), comprising: (1) 8-lipoxygenase derived from Malacostraca, and (2) 15-LOX lipoxygenase The method includes carrying out an enzymatic reaction using (Item 11) The 8-lipoxygenase derived from the class Malacostraca is (1) The activity of oxidizing the 8-carbon atom of arachidonic acid; (2) the activity of oxidizing the 8-carbon atom of eicosapentaenoic acid; and (3) Activity of oxidizing the 10-carbon atom of docosahexaenoic acid 11. The method according to any one of items 8 to 10, wherein the compound has an activity selected from the group consisting of: (Item 12) 11. The method according to any one of items 8 to 10, wherein the 8-lipoxygenase derived from Malacostraca is 8-lipoxygenase derived from Euphausiacea or 8-lipoxygenase derived from Decapoda. (Item 13) The 8-lipoxygenase derived from the class Malacostraca is (a) a polypeptide comprising an amino acid sequence comprising SEQ ID NO:2; (b) a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2; (c) a polypeptide consisting of an amino acid sequence of SEQ ID NO:2 containing one or several mutations, substitutions, insertions or deletions; (d) a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence consisting of SEQ ID NO:1; (e) a polypeptide encoded by a nucleic acid that hybridizes under highly stringent conditions to the complement of a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:1; (f) a polypeptide encoded by a nucleic acid comprising a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:1; (g) a polypeptide encoded by a nucleic acid sequence having a nucleic acid sequence containing one or more mutations, substitutions, insertions or deletions in the nucleic acid sequence of SEQ ID NO:1; A polypeptide selected from the group consisting of: (1) The activity of oxidizing the 8-carbon atom of arachidonic acid; (2) the activity of oxidizing the 8-carbon atom of eicosapentaenoic acid; and (3) Activity of oxidizing the 10-carbon atom of docosahexaenoic acid 11. The method according to any one of items 8 to 10, wherein the polypeptide has an activity selected from the group consisting of: (Item 14) The 8-lipoxygenase derived from the class Malacostraca is selected from the group consisting of: (a) a polypeptide comprising an amino acid sequence consisting of SEQ ID NO:2; and (d) a polypeptide encoded by a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:1. 10. The method according to item 8 or 9, selected from the group consisting of: (Item 15) The 15-LOX lipoxygenase is (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, and 8; (b) a polypeptide consisting of an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, and 8; (c) a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, and 8, which contains one or several mutations, substitutions, insertions, or deletions; (d) a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 7; (e) a polypeptide encoded by a nucleic acid that hybridizes under highly stringent conditions to the complement of a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 7; (f) a polypeptide encoded by a nucleic acid comprising a sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 7; (g) a polypeptide encoded by a nucleic acid sequence having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 7, which comprises one or several mutations, substitutions, insertions, or deletions; 11. The method according to any one of items 8 to 10, wherein the polypeptide is selected from the group consisting of: Effect of the Invention

[0012] PK-LOX1 and enzymes produced by modifying the PK-LOX1 gene enable the enzymatic synthesis of metabolites having a hydroxyl group in the R configuration (e.g., resolvin D1: 7S,8R,17S-trihydroxy-DHA and protectin D1: 10R,17S-dihydroxy-DHA). Furthermore, in the present invention, 10R,17S-dihydroxy-DHA (10R,17S-DiH-DHA), 10R-hydroperoxy-, 17S-hydroxy-DHA (10R-Hp-, 17S-H-DHA), 10R-hydroxy-,17S-hydroperoxy-DHA (10R-H-,17S-Hp-DHA), and It is possible to produce a substance selected from the group consisting of 10R,17S-dihydroperoxy-DHA (10R,17S-DiHp-DHA). [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows a conventional method for producing protectin D1 from DHA. [Diagram 2] FIG. 2 shows the nucleic acid sequence (SEQ ID NO:1) of the lipoxygenase gene (PK LOX1) isolated from Euphausia pacifica. [Diagram 3] FIG. 3 shows the amino acid sequence (SEQ ID NO:2) of lipoxygenase (PK LOX1) isolated from Euphausia pacifica. [Figure 4] FIG. 4 shows the results showing the activity of recombinantly expressed PK-LOX1. [Diagram 5] FIG. 5 shows the results demonstrating that the PK-LOX1 reaction product is 10,17S-DiH-DHA. [Figure 6] Figure 6 shows the results showing that 10,17-DiH-DHA is produced by combining soybeans and Euphausia pacifica. [Figure 7] FIG. 7 shows (SEQ ID NO:3) of a lipoxygenase gene isolated from soybean. [Figure 8] FIG. 8 shows the amino acid sequence of a lipoxygenase isolated from soybean (SEQ ID NO:4). [Figure 9] FIG. 9 shows the nucleic acid sequence of the mouse 15-LOX gene (SEQ ID NO:5). [Figure 10] FIG. 10 shows the nucleic acid sequence of the human 15-LOX gene (SEQ ID NO:7). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described below. Throughout this specification, the singular expression should be understood to include the concept of the plural form, unless otherwise specified. In addition, it should be understood that the terms used in this specification are used in the sense that they are commonly used in the field, unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In case of conflict, this specification (including definitions) shall prevail.

[0015] (Definition of terms) Listed below are definitions of terms specifically used in this specification.

[0016] As used herein, "enzyme" includes, but is not limited to, enzymes that produce fatty acid hydroperoxides or hydroxy fatty acids, or both, from fatty acids having double bonds.

[0017] As used herein, the term "SPM" is used interchangeably with "specialized pro-resolving mediator", "inflammatory-resolving lipid metabolite" or "anti-inflammatory mediator" and refers to lipids that have an inflammation-resolving effect. Examples of SPMs include, but are not limited to, resolvin E (e.g., resolvin E1), resolvin D (e.g., resolvin D1), and protectin (e.g., protectin D1).

[0018] As used herein, the term "unsaturated fatty acid" refers to a fatty acid that has one or more unsaturated carbon bonds. An unsaturated carbon bond is an unsaturated bond between carbon atoms in a carbon molecular chain, i.e., a carbon double bond or a triple bond. Unsaturated fatty acids found in nature have one or more double bonds, which replace saturated fatty acids in fats, thereby changing the properties of the fat, such as melting point and fluidity.

[0019] The term "highly unsaturated fatty acid" as used herein means an unsaturated fatty acid having 16 or more carbon atoms and two or more double bonds in the molecule, and examples thereof include docosahexaenoic acid (C22:6, DHA), eicosapentaenoic acid (C20:5, EPA), arachidonic acid (C20:4, AA), docosapentaenoic acid (C22:5, DPA), stearidonic acid (C18:4), linolenic acid (C18:3), linoleic acid (C18:2), tetracosahexaenoic acid (C24:6), eicosatetraenoic acid (C24:7), tetracosahexaenoic acid (C24:8), tetracosahexaenoic acid (C24:9), tetracosahexaenoic acid (C24:10), tetracosahexaenoic acid (C24:11), tetracosahexaenoic acid (C24:12), tetracosahexaenoic acid (C24:13), tetracosahexaenoic acid (C24:14), tetracosahexaenoic acid (C24:15), tetracosahexaenoic acid (C24:16), tetracosahexaenoic acid (C24:17), tetracosahexaenoic acid (C24:18), tetracosahexaenoic acid (C24:19 ... 0:4, ETA), eicosatrienoic acid (C20:3), hexadecadienoic acid (C16:2), hexadecatrienoic acid (C16:3), hexadecatetraenoic acid (C16:4), eicosadienoic acid (C20:2), docosadienoic acid (C22:2), pinolenic acid (C18:3), eleostearic acid (C18:3), mead acid (C20:3), adrenic acid (C22:4), bosseopentaenoic acid (C18:5), and tetracosapentaenoic acid (C24:5), etc., but are not limited thereto. The highly unsaturated fatty acid may be a free fatty acid.

[0020] The "polyunsaturated fatty acid" used in the method of the present invention refers to fatty acids that are not free and those that are free, and includes, but is not limited to, polyunsaturated fatty acids, phospholipids that bind polyunsaturated fatty acids, triglycerides that bind polyunsaturated fatty acids, and ester derivatives of polyunsaturated fatty acids such as methyl esters and ethyl esters, amide derivatives such as ethylamides and methylamides, fatty alcohol derivatives, triglycerides, diglycerides, and monoglycerides. Unsaturated fatty acid derivatives, such as polyunsaturated fatty acid derivatives, can also be used as substrates. Examples of derivatives of highly unsaturated fatty acids include, but are not limited to, ethyl esters of highly unsaturated fatty acids, triglycerides containing highly unsaturated fatty acids, diglycerides containing highly unsaturated fatty acids, monoglycerides containing highly unsaturated fatty acids, phospholipids containing highly unsaturated fatty acids, lysophospholipids containing highly unsaturated fatty acids, sphingolipids containing highly unsaturated fatty acids, and glycolipids containing highly unsaturated fatty acids, as well as amide derivatives of highly unsaturated fatty acids (e.g., fatty acid amides such as ethanolamides of highly unsaturated fatty acids). Derivatives of highly unsaturated fatty acids also include esters of highly unsaturated fatty acids. Unsaturated fatty acid esters (e.g., unsaturated fatty acid ethyl esters), particularly highly unsaturated fatty acid esters (e.g., highly unsaturated fatty acid ethyl esters), can also be used as substrates for enzyme reactions. The above-mentioned highly unsaturated fatty acids can be used as substrates for the enzymes of the present invention.

[0021] As used herein, "oxidation" of an unsaturated fatty acid refers to a reaction in which a functional group (e.g., carbon) of an unsaturated fatty acid is oxidized, in particular, a reaction in which a hydrogen atom is converted to a hydroxyl group, a reaction in which a hydrogen atom is converted to a hydroperoxy group, and a reaction in which a hydrogen atom is converted to a hydroperoxy group and the hydroperoxy group is converted to a hydroxyl group, a carbonyl group, or an epoxy group.

[0022] The term "oxide of polyunsaturated fatty acid" as used herein refers to a substance obtained by the oxidation reaction of polyunsaturated fatty acid, and examples thereof include substances in which one or more hydrogen atoms bonded to the main chain of polyunsaturated fatty acid are replaced with hydroxyl groups and / or hydroperoxy groups, as well as hydroxides, carbonyl compounds, and epoxy compounds derived from such substances.

[0023] As used herein, a "kit" refers to an article of manufacture comprising multiple containers, and manufacturer's instructions, each container containing a nucleic acid and / or polypeptide of the invention.

[0024] A "polynucleotide", "nucleic acid" or "nucleic acid molecule" can refer to ribonucleotides (adenosine, guanosine, uridine, or cytidine; "RNA molecules") or deoxyribonucleotides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine "DNA molecules") in the phosphate polymeric form, in single-stranded, double-stranded, or other form, or any phosphoester analogs thereof (e.g., phosphorothioates and thioesters). A "polynucleotide", "nucleic acid" or "nucleic acid molecule" carries "sequence" information.

[0025] A "polynucleotide sequence," "base sequence," "nucleic acid sequence," or "nucleotide sequence" is a sequence of a series of nucleotide bases (also called "nucleosides") in a nucleic acid (e.g., DNA or RNA) and refers to a sequence of two or more nucleotides in any strand or its complementary strand. Preferred nucleic acids of the invention include the nucleic acids set forth in SEQ ID NO:1 and / or SEQ ID NO:3, as well as complementary strands, variants, and fragments thereof.

[0026] "Complementary strand" refers to a strand of nucleotides that can form base pairs with a nucleic acid sequence. For example, the strands of a double-stranded DNA have complementary base sequences, and one strand is complementary to the other strand.

[0027] A "coding sequence" or a sequence that "encodes" an expression product (e.g., RNA, polypeptide, protein, or enzyme), is a nucleotide sequence that, when expressed, results in the production of that product.

[0028] A "protein," "peptide" or "polypeptide" comprises a consecutive string of two or more amino acids. Preferred peptides of the invention include the peptides set forth in SEQ ID NO:2 and / or SEQ ID NO:4, as well as variants and fragments thereof.

[0029] "Protein sequence," "peptide sequence," or "polypeptide sequence" or "amino acid sequence" refers to the sequence of a series of two or more amino acids in a protein, peptide, or polypeptide.

[0030] As used herein, the identity of a sequence (such as a nucleic acid sequence or an amino acid sequence) refers to the degree of identical sequences (individual nucleic acids, amino acids, etc.) between two or more comparable sequences. Thus, the higher the homology between two genes, the higher the identity or similarity between those sequences. Whether two types of genes have a high identity can be examined by direct comparison of the sequences, or, in the case of nucleic acids, by hybridization methods under stringent conditions. When two gene sequences are directly compared, the genes have homology if the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical. Alternatively, the amino acid sequences compared have homology if the amino acid sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical. As used herein, "similarity" of genes (e.g., nucleic acid sequences, amino acid sequences, etc.) refers to the degree of identity between two or more gene sequences when conservative substitutions are considered positive (identical) in the above-mentioned homology. Therefore, when conservative substitutions are present, homology and similarity differ depending on the presence of the conservative substitutions. Also, when there are no conservative substitutions, homology and similarity show the same numerical value.

[0031] In the present specification, the comparison of similarity, identity and homology of amino acid sequences and base sequences is calculated using the sequence analysis tool BLAST with default parameters. Alternatively, the identity of amino acid sequences and base sequences can be calculated by aligning two sequences to be compared and calculating the ratio of identical residues to the entire sequence.

[0032] As used herein, the term "fragment" refers to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length is n). The length of the fragment can be changed appropriately depending on the purpose. For example, the lower limit of the length of a polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit. In addition, in the case of a polynucleotide, the lower limit can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be suitable as the lower limit. In the present specification, the length of a polypeptide and a polynucleotide can be expressed by the number of amino acids or nucleic acids, respectively, as described above, but the above-mentioned number is not absolute, and it is intended that the above-mentioned number as an upper or lower limit includes several numbers above or below that number (or, for example, 10% above or below) as long as it has the same function. In order to express such an intention, the number may be expressed in the present specification by adding "about" before it. However, it should be understood that the presence or absence of "about" does not affect the interpretation of the numerical value in the present specification. The length of a fragment useful in the present specification can be determined by whether at least one function of the full-length protein that is the reference for the fragment is retained.

[0033] As used herein, an "isolated" biological factor (e.g., a nucleic acid or a protein) refers to a biological factor that is substantially separated or purified from other biological factors (e.g., in the case of a nucleic acid, factors other than the nucleic acid and a nucleic acid containing a nucleic acid sequence other than the nucleic acid of interest; in the case of a protein, factors other than the protein and a protein containing an amino acid sequence other than the protein of interest, etc.) in the cells of an organism in which the biological factor naturally occurs. "Isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.

[0034] As used herein, a "purified" biological agent (e.g., a nucleic acid or a protein) refers to a biological agent from which at least a portion of the factors naturally associated with the biological agent has been removed. Thus, the purity of the biological agent in a purified biological agent is usually higher (i.e., more concentrated) than the state in which the biological agent normally exists.

[0035] As used herein, the terms "purified" and "isolated" mean that at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological factor is present.

[0036] As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to well-known conditions commonly used in the art. Such polynucleotides can be obtained by colony hybridization, plaque hybridization, Southern blot hybridization, or the like, using a polynucleotide selected from the polynucleotides of the present invention as a probe. Specifically, the term refers to a polynucleotide that can be identified by hybridizing a filter on which DNA derived from colonies or plaques is immobilized in the presence of 0.7 to 1.0 M NaCl at 65°C, and then washing the filter at 65°C using a 0.1 to 2-fold concentration of SSC (saline-sodium citrate) solution (the composition of 1-fold concentration SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). Hybridization can be carried out according to the method described in experimental textbooks such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplements 1-38, and DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). Preferably, sequences that hybridize under stringent conditions exclude sequences that contain only A or T sequences. The term "hybridizable polynucleotide" refers to a polynucleotide that can hybridize to another polynucleotide under the above-mentioned hybridization conditions. Specific examples of hybridizable polynucleotides include polynucleotides that have at least 60% homology to the base sequence of a DNA encoding a polypeptide having an amino acid sequence specifically shown in the present invention, preferably polynucleotides that have 80% or more homology, and more preferably polynucleotides that have 95% or more homology.

[0037] As used herein, "highly stringent conditions" refer to conditions designed to allow hybridization of DNA strands with a high degree of complementarity in nucleic acid sequences and to exclude hybridization of DNA with a significant number of mismatches. Hybridization stringency is determined primarily by temperature, ionic strength, and denaturing agent conditions such as formamide. Examples of "highly stringent conditions" for such hybridization and washing include, but are not limited to, typically: (1) 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65 to 68°C; (2) 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide, 42°C; (3) 0.3 M sodium chloride, 0.03 M sodium citrate, 0.1% SDS, 65 to 68°C; (4) 0.15 M sodium chloride, 0.015 M sodium citrate, 0.1% SDS, 65 to 68°C; or (5) 0.03 M sodium chloride, 0.003 M sodium citrate, 0.1% SDS, 65 to 68°C. For such highly stringent conditions, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory (Cold Spring Harbor, N,Y. 1989); and Anderson et al., Nucleic Acid Hybridization: a Practical approach, IV, IRL Press Limited (Oxford, England). Limited, Oxford, England. If necessary, more stringent conditions (e.g., higher temperature, lower ionic strength, higher formamide, or other denaturing agents) may be used. Other agents may be included in the hybridization and washing buffers to reduce non-specific and / or background hybridization.Examples of such other agents are 0.1% bovine serum albumin, 0.1% polyvinylpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO4 or SDS), Ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non-complementary DNA) and dextran sulfate, although other suitable agents may also be used. The concentrations and types of these additives may be varied without substantially affecting the stringency of the hybridization conditions. Hybridization experiments are usually performed at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is nearly pH independent. See Anderson et al., Nucleic Acid Hybridization: a Practical Approach, Chapter 4, IRL Press Limited (Oxford, England).

[0038] Factors that affect the stability of DNA duplexes include base composition, length and the degree of base pair mismatch. Hybridization conditions can be adjusted by those skilled in the art to accommodate these variables and allow DNA of different sequence relatedness to form hybrids. The melting temperature of a perfectly matched DNA duplex can be estimated by the following formula: Tm(℃)=81.5+16.6(log[Na + ]) + 0.41 (% G + C) - 600 / N - 0.72 (% formamide) where N is the length of the duplex formed and [Na + ] is the molar concentration of sodium ions in the hybridization or washing solution, and %G+C is the percentage of (guanine + cytosine) bases in the hybrid. For imperfectly matched hybrids, the melting temperature decreases by approximately 1°C for each 1% mismatch.

[0039] As used herein, "moderately stringent conditions" refer to conditions under which a DNA duplex having a higher degree of base pair mismatch than can occur under "highly stringent conditions" can be formed. Representative examples of "moderately stringent conditions" are 0.015 M sodium chloride, 0.0015 M sodium citrate, 50-65°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20% formamide, 37-50°C. As an example, "moderately stringent" conditions in 0.015 M sodium ion at 50°C allow about 21% mismatch.

[0040] For oligonucleotide probes up to about 20 nucleotides, a good estimate of the melting temperature in 1M NaCl is given by Tm=(2° C. per AT base pair)+(4° C. per GC base pair), where the sodium ion concentration in 6× sodium citrate (SSC) is 1M (see Suggs et al., Developmental Biology Using Purified Genes, p. 683, Brown and Fox (eds.) (1981)).

[0041] Naturally occurring nucleic acids encoding proteins such as polypeptides having the amino acid sequence of SEQ ID NO:2 and / or SEQ ID NO:4 or variants or fragments thereof are readily isolated, for example, from cDNA libraries with PCR primers and hybridization probes that include portions of the nucleic acid sequence of SEQ ID NO:1 and / or SEQ ID NO:3 or variants thereof. Nucleic acids encoding ... 3 PO 41 mM EDTA; 7% SDS at a temperature of 42° C., and a wash buffer essentially containing 2×SSC (600 mM NaCl; 60 mM sodium citrate); 0.1% SDS at 50° C., more preferably 1% bovine serum albumin (BSA); 500 mM sodium phosphate (Na phosphate); 1% EDTA; 1% SDS at a temperature of 42° C. 3 PO 4 15% formamide; 1 mM EDTA; 7% SDS, and a wash buffer containing 1×SSC (300 mM NaCl; 30 mM sodium citrate); 1% SDS at essentially 50° C., most preferably 1% bovine serum albumin (BSA); 200 mM sodium phosphate (Na phosphate); 200 mM EDTA; 200 mM sodium phosphate (Na phosphate); 200 mM sodium phosphate; 200 mM sodium citrate; 200 mM sodium phosphate ... 3 PO 4 ); 15% formamide; 1 mM EDTA; 7% SDS, and a wash buffer essentially containing 0.5xSSC (150 mM NaCl; 15 mM sodium citrate); 0.1% SDS at 65°C, or a portion thereof.

[0042] As used herein, the percentage of "identity", "homology" and "similarity" of sequences (such as amino acids or nucleic acids) is determined by comparing two sequences aligned optimally in a comparison window. Here, the portion of the comparison window of a polynucleotide sequence or polypeptide sequence may contain additions or deletions (i.e., gaps) when compared with a reference sequence for optimal alignment of two sequences (where gaps may occur if the other sequence contains additions, but the reference sequence here is assumed to have no additions or deletions). The number of matched positions is determined by determining the number of positions where the same nucleic acid base or amino acid residue is found in both sequences, and the number of matched positions is divided by the total number of positions in the comparison window, and the result is multiplied by 100 to calculate the percentage of identity. When used in a search, homology is evaluated using an appropriate one of various sequence comparison algorithms and programs known in the art. Such algorithms and programs include TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Thompson et al., 1994, Nucleic Acids Res. 22(2):4673-4680; Higgins et al., 1996, Methods Enzymol. 266:383-402; Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Altschul et al., 1993, Nature Genetics 3:266-272), but is in no way limited to these.In a particularly preferred embodiment, the homology of protein and nucleic acid sequences is evaluated using the Basic Local Alignment Search Tool (BLAST), which is well known in the art (see, for example, Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268; Altschul et al., 1990, J. Mol. Biol. 215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; Altschul et al., 1997, Nuc. Acids Res. 25:3389-3402). In particular, comparison or search can be accomplished by carrying out the following operations using five dedicated BLAST programs:

[0043] (1) Compare amino acid query sequences against protein sequence databases using BLASTP and BLAST3; (2) BLASTN compares a nucleotide query sequence against a nucleotide sequence database; (3) BLASTX comparison of conceptual translation products of nucleotide query sequences (both strands) in six reading frames against protein sequence databases; (4) comparing the protein query sequence to a nucleotide sequence database converted in all six reading frames (both strands) with TBLASTN; (5) The nucleotide query sequence was converted into six reading frames using TBLASTX, and compared to a nucleotide sequence database converted into six reading frames.

[0044] The BLAST program identifies homologous sequences by identifying similar segments, called "high-score segment pairs", between an amino acid or nucleic acid query sequence and a subject sequence, preferably obtained from a protein or nucleic acid sequence database. The high-score segment pairs are preferably identified (i.e., aligned) by a scoring matrix, many of which are well known in the art. Preferably, the BLOSUM62 matrix (Gonnet et al., 1992, Science 256:1443-1445; Henikoff and Henikoff, 1993, Proteins 17:49-61) is used as the scoring matrix. Although less preferred, the PAM or PAM250 matrix can also be used (see, for example, Schwartz and Dayhoff, eds., 1978, Matrices for Detecting Distance Relationships: Atlas of Protein Sequence and Structure, Washington: National Biomedical Research Foundation). The BLAST program evaluates the statistical significance of all identified high-scoring segment pairs and preferably selects segments that meet a user-defined threshold level of significance, such as a user-specified percentage of homology. The statistical significance of high-scoring segment pairs is preferably evaluated using the Karlin formula for statistical significance (see Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87:2267-2268).

[0045] As used herein, "substitution, addition, or deletion" of a polypeptide or polynucleotide refers to the replacement, addition, or removal of an amino acid or its substitute, or a nucleotide or its substitute, from the original polypeptide or polynucleotide, respectively. Such techniques for substitution, addition, or deletion are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. The number of substitutions, additions, or deletions may be any number greater than or equal to one, and such number may be as large as long as the intended function is retained in the variant having the substitution, addition, or deletion. For example, such a number may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or several, and may preferably be within 20% or 10% of the total length, or 100 or less, 50 or less, 25 or less, etc.

[0046] The molecular biological, biochemical and microbiological techniques used herein are well known and commonly used in the art, and are described in, for example, Sambrook J. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, FM (1987). Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience; Ausubel, FM (1989). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associat ES and Wiley-Interscience; Innis, MA (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; Ausubel, FM (1992). Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub.Associates;Ausubel,FM(1995).Short Protocols in Molecular Biology:A Compendium of Methods from Current Protocols in Molecular Biology,Greene Pub.Associates;Innis,MAet al.(1995).PCR Strategies,Academic Press;Ausubel,FM(1999).These methods are described in Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Wiley, and annual updates; Sninsky, JJ et al. (1999); PCR Applications: Protocols for Functional Genomics, Academic Press, Special Edition of Experimental Medicine: "Gene Introduction & Expression Analysis Experimental Methods" Yodosha, 1997, etc., and the relevant parts (possibly the entirety) of these are incorporated herein by reference.

[0047] DNA synthesis techniques and nucleic acid chemistry for producing artificially synthesized genes are described, for example, in Gait, MJ (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, MJ (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F. (1991). Oligonucleotides and Analogues: A Practical Approach, IRL Press; Adams, RL et al. (1992). The Biochemistry of the Nucleic Acids, Chapman & Hall; Shabarova, Z. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, GM et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, GT (1996). Bioconjugate Techniques, Academic Press, and the like, the relevant portions of which are incorporated herein by reference.

[0048] In the present specification, the presence of a nucleic acid can be confirmed by any appropriate method, including a molecular biological measurement method such as a radioactivity method, a fluorescence method, a Northern blot method, a dot blot method, or a PCR method.

[0049] The terms "express" and "expression" mean to enable or cause the information in a gene, RNA sequence, or DNA sequence to become apparent (e.g., to produce a protein by activating the cellular functions involved in transcription and translation of the corresponding gene). A DNA sequence is expressed in or by a cell to form an "expression product" (e.g., an RNA (e.g., mRNA) or a protein). The expression product itself may also be said to be "expressed" by the cell.

[0050] Expression of the nucleic acid encoding the polypeptide according to SEQ ID NO:2 and / or SEQ ID NO:4 of the present invention can be carried out by conventional methods, preferably in eukaryotic cells. Suitable host cells for expressing the nucleic acid include higher eukaryotic organisms, including established tissue culture cell lines from animal cells, both of non-mammalian origin (e.g., insect cells) and mammalian origin (e.g., human, primate, and rodent).

[0051] Higher eukaryotic tissue culture cells may also be used for recombinant production of the polypeptides (or variants thereof) set forth in SEQ ID NO:2 and / or SEQ ID NO:4 of the present invention. Any higher eukaryotic tissue culture cell line may be used, including insect baculovirus expression systems, but mammalian cells are preferred. Transformation, transfection and growth of such cells have become routine procedures. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, J774 cells, Caco2 cells, baby rat kidney (BRK) cell lines, insect cell lines, avian cell lines, and monkey (COS) cell lines. Expression vectors for such cell lines usually contain an origin of replication, a promoter, a translation initiation site, an RNA splice site (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection or amplification gene. Suitable expression vectors can be plasmids, viruses or retroviruses, carrying promoters derived from sources such as adenovirus, SV40, parvovirus, vaccinia virus or cytomegalovirus.Exemplary expression vectors include pCR®3.1, pCDNA1, pCD (Okayama et al., (1985) Mol.Cell Biol.5:1136), pMC1neo Poly-A (Thomas et al., (1987) Cell 51:503), pREP8, pSVSPORT and their derivatives, and baculovirus vectors (e.g., pAC373 or pAC610).

[0052] As used herein, "operably linked" refers to the expression (operation) of a desired sequence being placed under the control of a certain transcription / translation regulatory sequence (e.g., promoter, enhancer, etc.) or translation regulatory sequence. In order for a promoter to be operably linked to a gene, the promoter is usually placed immediately upstream of the gene, but is not necessarily required to be placed adjacent to the gene.

[0053] (Production of SPM using 8-lipoxygenase) In the present invention, SPM can be produced from polyunsaturated fatty acids by combining multiple enzymes (e.g., PK-LOX1 and soybean lipoxygenase). Enzymes that can be used in the present invention include, but are not limited to, the following: 8-lipoxygenase from Malacostraca, which is (1) The activity of oxidizing the 8-carbon atom of arachidonic acid; (2) the activity of oxidizing the 8-carbon atom of eicosapentaenoic acid; and (3) Activity of oxidizing the 10-carbon atom of docosahexaenoic acid 8-Lipoxygenase having an activity selected from the group consisting of: 8-lipoxygenase from Euphausiacea or 8-lipoxygenase from Decapoda. ·below: (a) a polypeptide comprising an amino acid sequence comprising SEQ ID NO:2; (b) a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO:2; (c) a polypeptide consisting of an amino acid sequence of SEQ ID NO:2 containing one or several mutations, substitutions, insertions or deletions; (d) a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence consisting of SEQ ID NO:1; (e) a polypeptide encoded by a nucleic acid that hybridizes under highly stringent conditions to the complement of a nucleic acid comprising the nucleic acid sequence of SEQ ID NO:1; (f) a polypeptide encoded by a nucleic acid comprising a sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO:1; (g) a polypeptide encoded by a nucleic acid sequence having a nucleic acid sequence containing one or more mutations, substitutions, insertions or deletions in the nucleic acid sequence of SEQ ID NO:1; A polypeptide selected from the group consisting of: (1) The activity of oxidizing the 8-carbon atom of arachidonic acid; (2) the activity of oxidizing the 8-carbon atom of eicosapentaenoic acid; and (3) Activity of oxidizing the 10-carbon atom of docosahexaenoic acid A polypeptide having an activity selected from the group consisting of: ·below: (a) a polypeptide comprising the amino acid sequence of SEQ ID NO:2; and (d) a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence consisting of SEQ ID NO:1; A polypeptide selected from the group consisting of:

[0054] (Enzyme reaction by 8-lipoxygenase) By using the lipoxygenase having the above-mentioned 8R-LOX activity, it is possible to oxidize the R configuration of highly unsaturated fatty acids and synthesize a substance selected from the group consisting of oxides of highly unsaturated fatty acids in the hydroxyl type and oxides of highly unsaturated fatty acids in the hydroperoxy type. For example, by using the lipoxygenase having the above-mentioned 8R-LOX activity, it is possible to oxidize the R configuration of highly unsaturated fatty acids and synthesize a substance selected from the group consisting of oxides of highly unsaturated fatty acids in the hydroperoxy type. (a) Oxidation of polyunsaturated fatty acids in the hydroxy form; (b) an oxide of a highly unsaturated fatty acid in the hydroperoxy form; or (c) a mixture of oxides of highly unsaturated fatty acids in the hydroxyl form and oxides of highly unsaturated fatty acids in the hydroperoxy form; can be synthesized. (8-lipoxygenase and other enzyme combinations) The above-mentioned lipoxygenase having 8R-LOX activity can be used in combination with other lipoxygenases. Examples of other lipoxygenases include, but are not limited to, enzymes selected from the group consisting of soybean lipoxygenase, human and mouse 15-lipoxygenase, 5-lipoxygenase, and 12-lipoxygenase. The lipoxygenase having 8R-LOX activity can also be used in combination with lipoxygenase having 15S-LOX activity. Examples of lipoxygenase having 15S-LOX activity include, but are not limited to, soybean lipoxygenase (SEQ ID NOs: 3 and 4, and Figures 7 and 8), mouse 15-lipoxygenase (SEQ ID NOs: 5 and 6, and Figure 9), and human 15-lipoxygenase (SEQ ID NOs: 7 and 8, and Figure 10). The 15-LOX lipoxygenase can also be modified in the same manner as the lipoxygenase having 8R-LOX activity and used in the present invention. In the present invention, by using a combination of an enzyme that oxidizes the R-configuration of highly unsaturated fatty acids and an enzyme that oxidizes the S-configuration of highly unsaturated fatty acids, it is possible to prepare an oxide product in which both the R-configuration and the S-configuration of highly unsaturated fatty acids are oxidized. For example, when the 17-carbon of DHA is oxidized with soybean lipoxygenase to obtain 17S-H-DHA, and the 10-carbon is further oxidized with Isada 8R-lipoxygenase, a product in which the 10-carbon is R-configuration while the 17-carbon remains S-configuration can be obtained. As a result, for example, 10R,17S-dihydroxy-DHA can be obtained. (Manufacturing of SPM-containing materials) For example, in the present invention, it is possible to produce an SPM-containing material by combining (i) a crustacean-derived food product having 8R-LOX activity, (ii) a food product having 15S-LOX activity, and (iii) a substance selected from the group consisting of highly unsaturated fatty acids and derivatives thereof.

[0055] For example, the following enzymes having lipoxygenase activity can be used in combination with 8-lipoxygenase: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, and 8; (b) a polypeptide consisting of an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, and 8; (c) a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 6, and 8, which contains one or several mutations, substitutions, insertions, or deletions; (d) a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 7; (e) a polypeptide encoded by a nucleic acid that hybridizes under highly stringent conditions to the complement of a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 7; (f) a polypeptide encoded by a nucleic acid comprising a sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 7; (g) a polypeptide encoded by a nucleic acid sequence having a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 5, and 7, which contains one or several mutations, substitutions, insertions, or deletions; A polypeptide selected from the group consisting of:

[0056] When a lipoxygenase having 8R-LOX activity and a lipoxygenase having 15S-LOX activity are combined, stereoselective oxidation reactions occur successively, and for example, 10R,17S-DiH-DHA can be produced from DHA. 10R,17S-dihydroxy-DHA (10R,17S-DiH-DHA), 10R-hydroperoxy-, 17S-hydroxy-DHA (10R-Hp-, 17S-H-DHA), 10R-hydroxy-,17S-hydroperoxy-DHA (10R-H-,17S-Hp-DHA), and It is possible to produce a substance selected from the group consisting of 10R,17S-dihydroperoxy-DHA (10R,17S-DiHp-DHA).

[0057] An example of a crustacean-derived food having 8R-LOX activity is, but is not limited to, frozen krill.

[0058] An example of a food having 15S-LOX activity is, but is not limited to, dried soybeans.

[0059] (Enzyme reaction conditions) When the enzymes of the present invention are used in the production of the above SPM, the enzyme reaction conditions are determined taking into consideration the characteristics of each enzyme. For example, lipoxygenase isolated from soybean (e.g., lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 4) preferably reacts at an alkaline pH. On the other hand, lipoxygenase isolated from Euphausia pacifica (e.g., lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 2) preferably reacts in the range of acidic to neutral pH. In addition, each of the above enzyme reactions is preferably carried out at 20°C. EXAMPLES

[0060] The SPM production method of the present invention will be specifically described below based on examples, but the present invention is not limited to these.

[0061] Example 1: 8R-LOX activity of PK-LOX1 A baculovirus containing a His-tagged PK-LOX1 gene (a gene encoding the amino acid sequence of SEQ ID NO:2 with a His tag) was cloned and incubated at 5 × 10 5Sf9 cells were infected at MOI=1. Two days after infection, the PK-LOX1-expressing Sf9 cells were washed with PBS, dispensed into 12 tubes, centrifuged at 5000g for 3 minutes at 4°C, and stored at -80°C after removing the PBS. 25 μL of Tris-HCl buffer containing 50 μM fatty acid (arachidonic acid, EPA, or DHA) was added to the PK-LOX1-expressing Sf9 cell mass dissolved on ice, and after stirring by pipetting, the mixture was allowed to react at 27°C for 1 hour. 75 μL of acetonitrile containing 1% formic acid was added to the reaction solution, which was then vortexed and centrifuged at 20000g for 10 minutes. After centrifugation, the supernatant was collected and 5 μL was subjected to a high-performance liquid chromatograph-quadrupole time-of-flight hybrid mass spectrometer (LC / QTOFMS) using CHIRALPAC ID to analyze the conformations of 8-H-ETE, 8-H-EPE, and 10-H-DHA. For the analysis, (±)8-H-ETE, (±)8-H-EPE, (±)10-H-DHA, 8S-H-ETE, 8R-H-ETE, and 8S-H-EPE purchased from Cayman Chemical, and 8S-H-ETE, 8S-H-EPE, and 10S-H-DHA obtained by mouse Alox8 were used for comparison. The results are shown in Figure 4.

[0062] Example 2 25 μL of Tris-HCl buffer containing 10 μM 17S-H-DHA (17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid, purchased from Cayman Chemical) was added to the PK-LOX1-expressing Sf9 cell mass thawed on ice, and after stirring by pipetting, the mixture was reacted at 27°C for 1 hour. 75 μL of acetonitrile containing 1% formic acid was added to the reaction solution, vortexed, and centrifuged at 20000 g for 10 minutes. The supernatant was collected from the centrifuged sample, and 5 μL was subjected to LC / QTOF MS using a Luna C18-2 column (150×2 mm, 5 μm, Shimadzu GLC) to analyze 10R,17S-DiH-DHA. When 17S-hydroxy-4Z,7Z,10Z,13Z,15E,19Z-docosahexaenoic acid is used as a substrate and the 10R configuration is oxidized by PK-LOX1, the expected reaction product is 10R,17S-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid. In a paper reported in the Journal of Immunology in 2006 by Charles N. Serhan et al., it was shown that 10S,17S-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid and 10R,17S-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid can be separated on a Luna C18-2 column, and that 10R,17S-DiH-DHA has a longer column retention time. The MS / MS fragment patterns of the reaction product of 17S-H-DHA and PK-LOX1 are completely consistent with those of the 10S,17S-DiH-DHA standard, indicating that the PK-LOX1 reaction product is 10,17S-DiH-DHA (Figure 5A). Furthermore, the retention times of the 10S,17S-DiH-DHA standard and the PK-LOX1 reaction product are different in Luna C18-2, with the PK-LOX1 reaction product having a longer retention time, indicating that the PK-LOX1 reaction product is 10R,17S-DiH-DHA (Figure 5B).

[0063] Example 3 In the above, we have shown that PK LOX1 derived from Euphausia pacifica metabolizes DHA to 10R-H-DHA and 17S-H-DHA to 10R,17S-DiH-DHA. PK LOX1 is an enzyme contained in Euphausia pacifica, and 10R-H-DHA increases when Euphausia pacifica is incubated at 20°C. On the other hand, soybeans contain LOX that metabolizes DHA to 17S-H-DHA. We thought that by combining soybeans and Euphausia pacifica, we could easily produce 10,17-DiH-DHA.

[0064] The materials used were Euphausia pacifica caught in Iwate Prefecture in 2019 and stored at -50°C or below, and soybeans harvested in 2018 and dried and stored. The soybeans were soaked overnight in 10 times the amount of water, pretreated at 70°C for 10 minutes, washed with water, and crushed using a mortar and pestle. The isada was thawed under running water and crushed using a mortar and pestle. 20 grams of soybeans, 20 grams of Euphausia pacifica, and 80 mL of water were placed in an Erlenmeyer flask and incubated at 160 rpm and 20°C for 24 hours using a medium-sized shaker. Lipids were extracted from 5 mL of the incubated solution using the Bligh-Dyer method and analyzed by LC / QTOFMS. Although 10,17-DiH-DHA was not detected in the sample incubated with Euphausia pacifica alone, the production of 10,17-DiH-DHA was confirmed in the sample incubated with soybeans and Euphausia pacifica mixed together (Figure 6). It was shown that 10,17-DiH-DHA, which has an R-type hydroxyl group at the 10-position of DHA, can be easily produced by mixing and incubating soybeans and Euphausia pacifica. Unexpectedly, the reaction by lipoxygenase was able to oxidize the R-configuration of highly unsaturated fatty acids such as DHA and generate hydroxyl (-OH) products rather than hydroperoxy (-OOH) products.

[0065] In addition, the structure of the product using DHA as the starting material has a trans double bond between positions 15 and 16, which is different from the cis type in protectin D1. However, this product, like protectin D1, has also been found at sites of inflammation and has been reported to have inflammation-resolving properties (Colas et al., Am. J. Physiol. Cell Physiol., 2014, 307, C39-C54).

[0066] As described above, the present invention has been illustrated using the preferred embodiment of the present invention, but the present invention should not be interpreted as being limited to this embodiment. It is understood that the scope of the present invention should be interpreted only by the claims. It is understood that a person skilled in the art can implement an equivalent scope based on the description of the present invention and technical common sense from the description of the specific preferred embodiment of the present invention. It is understood that the patents, patent applications and literature cited in this specification should be incorporated by reference to this specification in the same manner as if the contents themselves were specifically described in this specification. [Sequence List Free Text]

[0067] SEQ ID NO:1 is the nucleic acid sequence of a lipoxygenase gene (PK LOX1) isolated from Euphausia pacifica. SEQ ID NO:2 is the amino acid sequence of a lipoxygenase (PK LOX1) isolated from Euphausia pacifica. SEQ ID NO:3 is the nucleic acid sequence of a lipoxygenase gene isolated from soybean. SEQ ID NO:4 is the amino acid sequence of a lipoxygenase isolated from soybean. SEQ ID NO:5 is the nucleic acid sequence of the mouse 15-LOX gene. SEQ ID NO:6 is the amino acid sequence of mouse 15-LOX. SEQ ID NO:7 is the nucleic acid sequence of the human 15-LOX gene. SEQ ID NO:8 is the amino acid sequence of human 15-LOX.

Claims

1. A method for synthesizing a substance selected from the group consisting of oxides of highly unsaturated fatty acids that are hydroxyl type and oxides of highly unsaturated fatty acids that are hydroperoxy type, by oxidizing an R configuration of a highly unsaturated fatty acid or an oxide of said highly unsaturated fatty acid, comprising the steps of: The method includes the step of performing an enzymatic reaction using 8-lipoxygenase; The 8-lipoxygenase may be any of the following: (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2; (b) a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO:2; (c) a polypeptide consisting of an amino acid sequence containing one or several mutations, substitutions, insertions or deletions in the amino acid sequence shown in SEQ ID NO:2; (d) a polypeptide encoded by a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO:1; (e) a polypeptide encoded by a nucleic acid that hybridizes under highly stringent conditions with the complementary strand of a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO:1; (f) a polypeptide encoded by a nucleic acid comprising a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence set forth in SEQ ID NO:1; (g) a polypeptide encoded by a nucleic acid sequence containing one or more mutations, substitutions, insertions or deletions in the nucleic acid sequence shown in SEQ ID NO:1; A polypeptide selected from the group consisting of: (1) the activity of oxidizing the 8-carbon atom of arachidonic acid; (2) the activity of oxidizing the 8-carbon atom of eicosapentaenoic acid; and (3) Activity of oxidizing the 10-carbon atom of docosahexaenoic acid A polypeptide having an activity selected from the group consisting of: wherein the highly unsaturated fatty acid is selected from the group consisting of docosahexaenoic acid (C22:6, DHA), eicosapentaenoic acid (C20:5, EPA), and arachidonic acid (C20:4, AA); The oxide of the polyunsaturated fatty acid is a substance in which one or more hydrogen atoms bonded to the main chain of the highly unsaturated fatty acid are substituted with a hydroxyl group; A substance in which one or more hydrogen atoms bonded to the main chain of the highly unsaturated fatty acid are substituted with a hydroperoxy group, and a substance in which one or more hydrogen atoms bonded to the main chain of the highly unsaturated fatty acid are substituted with a hydroxyl group, and one or more hydrogen atoms bonded to the main chain of the highly unsaturated fatty acid are substituted with a hydroperoxy group; The method of claim 1, wherein the

2. The method according to claim 1, wherein the oxidized polyunsaturated fatty acid is a lipid mediator (SPM) having an inflammation-resolving effect.

3. The method according to claim 1, wherein the method is a method for oxidizing a highly unsaturated fatty acid selected from the group consisting of docosahexaenoic acid (C22:6, DHA), eicosapentaenoic acid (C20:5, EPA), arachidonic acid (C20:4, AA), and 17S-H-DHA.

4. The 8-lipoxygenase is (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:2, and (d) a polypeptide encoded by a nucleic acid comprising the nucleic acid sequence set forth in SEQ ID NO:

1. The method of claim 1 or 2, selected from the group consisting of:

5. The method of claim 1, comprising: 10R,17S-dihydroxy-DHA (10R,17S-DiH-DHA), 10R-hydroperoxy-, 17S-hydroxy-DHA (10R-Hp-, 17S-H-DHA), 10R-hydroxy-,17S-hydroperoxy-DHA (10R-H-,17S-Hp-DHA), and 10R,17S-dihydroperoxy-DHA (10R,17S-DiHp-DHA), wherein the enzymatic reaction further employs 15-LOX lipoxygenase.

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