KR102086298B1 - Method for producing lipoxin analogues by combination reation using recombinant lipoxigenases and lipoxin analogues therefrom - Google Patents

Abstract

The present invention relates to a method for preparing a novel lipoxin analogue or a leukotriene analogue and composition thereof using two kinds of lipoxygenases. According to the present invention, 8-lipox from Mus musculus mice By using 15-lipoxygenase from sigenase and Burkholderia thailandensis strains, new lipoxin analogues and leukotriene analogues which have not been reported to date in an environmentally friendly manner can be produced with high productivity and high yield. Therefore, it can be usefully used in various industries such as medicine, food and cosmetics.

Description

[Method for producing lipoxin analogues by combination reation using recombinant lipoxigenases and lipoxin analogues therefrom]

The present invention relates to a method for preparing a leukotriene analog or a lipoxin analog using two lipoxygenases, a composition thereof, and a novel leukotriene analog or a lipoxin analog prepared therefrom.

Hydroxylated fatty acids generally refer to substances that have a hydroxyl group in fatty acids, and are substances of nature that exist in the lipids of various organisms such as animals, plants, insects, and microorganisms. In particular, hydroxylated fatty acids are used as raw materials industrially because of their high reactivity, and they are used as raw materials for cosmetics because of their high antibacterial and antifungal activity, as well as reducing surface tension due to the action of hydroxyl groups. Hydroxylated fatty acids found in animals among many organisms are used as precursors for signaling substances in the human body, and even a single substance is involved in various physiological activities. Among the lipid mediators, a type of human signaling material, lipoxin is mainly converted from polyunsaturated fatty acids by lipoxygenase and epoxide hydrolase. Lipoxin-containing lipid modulators are important substances involved in various physiological functions such as homeostasis regulation and immune response in mammals including humans. Specifically, lipoxin (LX) is a product of the interaction of lipoxygenase (LOX), and is a substance belonging to an eicosanoid in the form of trihydroxy fatty acid, which is mainly produced by the sequential reaction of lipoxygenase from arachidonic acid. The structure of these substances is formed in the process of cell-cell interaction and is an important substance involved in immune mechanisms and anti-inflammatory reactions in the human body. Lipoxin is a substance that acts on specific G-protein-coupled receptors and regulates cellular responses to inflammation and inflammation, and lipoxin A4 (lipoxin A4, LXA4) and lipoxin B4 (LXB4) act similarly to aspirin's mechanism of action. Among them, 15-epi-LX is synthesized in the acetylation process of cyclooxygenase (COX), which is involved in the inflammatory reaction, and has an effect on anti-inflammatory activity by inhibiting the production of TNF-α. These lipoxins have been proven to be eicosanoid compounds that promote anti-inflammatory and inflammation in vivo through animal models such as rabbits and mice, and inflammation, asthma, arthritis, cardiovascular disease, gastrointestinal disease, periodontal disease, kidney disease and grafts As a substance that plays an important role in many diseases including large-host disease, LXA4's representative immune function relieves inflammation by binding to the leukotriene receptor of T-cell acting in the inflammatory response, inhibits bronchial contraction, and asthma. It is known to have the effect of alleviating the contracted period of the patient. Accordingly, the lipoxin studied so far provides a new approach to the treatment of common diseases related to inflammation.If LX analogues having a similar material structure in addition to the previously known lipoxin are developed, the next-generation medical treatment is similar to the existing lipoxin. It is considered necessary to develop a new type of lipoxin analog in that it can present its functionality as a substance, and it can be used as a research material in various disciplines such as medicine, biology, and biotechnology.

Lipoxygenase (LOX) is a dioxygenase that converts unsaturated fatty acids into hydroperoxy fatty acids containing two oxygens, and converted peroxy fatty acids into hydroxyl fatty acids containing one oxygen in nature. It is gradually converted. It is mainly named as a site-specific oxidase according to its position to oxidize to arikidonic acid, an unsaturated fatty acid having 20 carbon atoms, and the lipoxygenases identified so far are 5-, 8-, 11-, 12-, and 15-lipoxygenases according to their site specificity. Aje exists. This oxidative enzyme catalyzes the reaction to synthesize oxidized fatty acid and is an enzyme containing iron, which is a multifunctional enzyme that catalyzes two reactions. Characteristically, only polyunsaturated fatty acids having one or more cis, cis-1,4-pentadiene are used as a substrate to catalyze stereospecificity and reaction specificity through dioxygenation. The lipoxin produced by these lipoxygenases has been reported as a substance produced by a combination reaction of human-derived 5-lipoxygenase, 12-lipoxygenase, and 15-lipoxygenase.

Until now, no new site-specific lipoxin analogs other than the previously reported substances have been synthesized by combining site-specific lipoxygenases, and in particular, arachidonic acid or eicosine by combining 8-lipoxygenase and 15-lipoxygenase. The production of lipoxin analogs and leukotriene analogs from sapentaenoic acid has not been reported.

The present inventors devised a synthetic route through a combination reaction of specific lipoxygenases to biosynthesize a new lipoxin analog, which has not been previously reported, and confirmed that a new type of lipoxin analog is synthesized through biotransformation. Finished.

Accordingly, an object of the present invention is to provide a composition capable of producing lipoxin analogs in a high yield through a bioconversion process, a method for producing a lipoxin analog, and a lipoxin analog produced thereby.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention in order to achieve the above object, Mus musculus (Mus musculus) mouse-derived 8-lipoxygenase; It provides a composition for preparing 8,15-diperoxygenated fatty acids or leukotriene analogues comprising, as an active ingredient; 15-lipoxygenase derived from Burkholderia thailandensis strain.

The 8,15-diperhydroxide fatty acid may be represented by any one of the following Chemical Formulas 1 to 4, and the leukotriene analogue may be represented by the following Chemical Formula 5 or 6.

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

[Formula 5]

[Formula 6]

The 8-lipoxygenase may be composed of the amino acid sequence of SEQ ID NO: 1, and the 15-lipoxygenase may be composed of the amino acid sequence of SEQ ID NO: 3.

The composition may further include arachidonic acid or eicosapentaenoic acid as a substrate.

In another aspect, the present invention provides a Mus musculus mouse-derived 8-lipoxygenase; 15-lipoxygenase from Burkholderia thailandensis strain; And Myxococcus xanthus strain-derived epoxide hydrolase as an active ingredient, wherein the lipoxin analog is a composition represented by any one of the following Formulas 7 to 10:

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

The 8-lipoxygenase is composed of the amino acid sequence of SEQ ID NO: 1, the 15-lipoxygenase is composed of the amino acid sequence of SEQ ID NO: 3, and the epoxide hydrolase is composed of the amino acid sequence of SEQ ID NO: 5 I can.

The composition may further include arachidonic acid or eicosapentaenoic acid as a substrate.

In another aspect, the present invention relates to a gene encoding 8-lipoxygenase derived from Mus musculus mice; And a gene encoding 15-lipoxygenase derived from Burkholderia thailandensis strain; as an active ingredient, a composition for preparing an 8,15-hyperhydroxide fatty acid or a leukotriene analogue, wherein the 8,15-hyperhydroxide Fatty acid is represented by any one of Formulas 1 to 4, and the leukotriene analog provides a composition represented by Formula 5 or 6.

In addition, the present invention is Mus musculus (Mus musculus) a gene encoding a mouse-derived 8-lipoxygenase; A gene encoding 15-lipoxygenase derived from Burkholderia thailandensis strain; And a gene encoding an epoxide hydrolase derived from Myxococcus xanthus strain; as an active ingredient, the lipoxin analogue is represented by any one of Formulas 7 to 10. The composition is provided.

In another aspect, the present invention provides a Mus musculus mouse-derived 8-lipoxygenase; 15-lipoxygenase from Burkholderia thailandensis strain; Epoxide hydrolase derived from Myxococcus xanthus strain; And it provides a method for producing a lipoxin analog represented by any one of Formulas 7 to 10 by bioconversion by reacting a substrate.

The method may use arachidonic acid or eicosapentaenoic acid in the range of 0.5 mM to 5 mM as a substrate.

In the above method, the reaction can be carried out in the range of pH 6.5 to 7.5.

In the above method, the reaction may be carried out at a temperature ranging from 10°C to 30°C.

In another aspect, the present invention is a gene encoding 8-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 2; A gene encoding a 15-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 4; Any one gene selected from; And a gene encoding an epoxide hydrolase consisting of the amino acid sequence of SEQ ID NO: 6, wherein the lipoxin analog provides a recombinant expression vector represented by any one of Formulas 7 to 10. do.

In another aspect, the present invention provides a transformant transformed with the recombinant expression vector according to claim 13 in a host cell.

In another aspect, the present invention provides a method for preparing a lipoxin analog by treating the transformant on a substrate.

The concentration of the transformant may be treated in the range of 10 g/L to 50 g/L.

In another aspect, the present invention provides an 8,15-diperhydroxide fatty acid represented by any one of Formulas 1 to 4.

In addition, the present invention provides a leukotriene analog represented by Chemical Formula 5 or 6.

In addition, the present invention provides a lipoxin analog represented by any one of Formulas 7 to 10.

In addition, the present invention provides a compound represented by any one of the following formulas 11 to 14.

[Formula 11]

[Formula 12]

[Formula 13]

[Formula 14]

According to the present invention, by using 8-lipoxygenase derived from Mus musculus mouse and 15-lipoxygenase derived from Burkholderia thailandensis strain, a novel lipoxin not reported to date Analogs and leukotriene analogs can be produced.

In addition, according to the present invention, since lipoxin analogues and leukotriene analogues can be manufactured with high productivity and high yield in an environmentally friendly manner, they can be usefully used in various industrial fields such as medicine, food, and cosmetics.

The lipoxin analog prepared according to the present invention is a signaling material and is expected to be involved in various physiologically active functions in animals including humans.

1 shows the synthesis pathway of lipoxin A4 and B4 analogs produced using arachidonic acid as a substrate using 8-lipoxygenase, 15-lipoxygenase, and epoxide hydrolase of the present invention.
Figure 2a is an HPLC chromatogram confirming the generation of the lipoxin A4 analog, Figure 2b is an HPLC chromatogram confirming the production of the lipoxin B4 analog, Figure 2c is a graph showing the synthesis degree of the lipoxin B4 analog over time.
FIG. 3A is a diagram showing substance identification using LC-MS/MS for substance identification of lipoxin A4 analogues, FIG. 3B is a substance identification result of lipoxin B4 analogue, and FIG. 3C is a substance identification result of leukotriene B4 analogue. to be.
Figure 4 shows the synthesis pathway of lipoxin A5 and B5 analogs produced using eicosapentaenoic acid as a substrate using 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase of the present invention. .
Figure 5a is an HPLC chromatogram confirming the generation of the lipoxin A5 analog, Figure 5b is an HPLC chromatogram confirming the production of the lipoxin B5 analog.
Figure 6a is a diagram showing the substance identification using LC-MS/MS for substance identification of lipoxin A5 analogue, Figure 6b is the substance identification result of lipoxin B5 analogue, and Figure 6c is the substance identification result of leukotriene B5 analogue to be.
Figure 7 is a Mus musculus of the present invention (Mus musculus) derived from 8-lipoxygenase, Burkholderia tylandensis (Burkholderia thailandensis ) derived 15-lipoxygenase and Myxococcus xanthus (Myxococcus xanthus) derived epoxide from It shows a recombinant vector in which each side hydrolase was synthesized.

Hereinafter, the present invention will be described in detail.

The present inventors have conducted continuous research to more effectively prepare 8-hydroxylated fatty acids, 15-hydroxylated fatty acids or lipoxin analogs through a bioconversion process. Specifically, the present inventors are Mus musculus (Mus musculus) mouse-derived 8-lipoxygenase, Burkholderia tylandensis (Burkholderia thailandensis ) strain derived 15-lipoxygenase and Myxococcus xanthus strains (Myxococcus xanthus) strain The derived epoxide hydrolase is cloned to produce a recombinant expression vector and a microorganism transformed therefrom, and then whole cells are produced using this, and then treated on a substrate to produce 8-hydroxylated fatty acids, 15-hydroxylated fatty acids, It was confirmed that a kind of leukotriene analogue or four kinds of lipoxin analogues can be prepared with high productivity and high yield, and the present invention was completed.

In the present specification, the term "8-hydroxylated fatty acid" means that one hydroxyl group or peroxy group is substituted with the fatty acid, and it is preferable to have a hydroxyl group or a peroxy group on the eighth carbon of an unsaturated fatty acid having 20 carbon atoms, but is not limited thereto. Does not. More specifically, the 8-hydroxylated fatty acid is 8-hydroxyarachidonic acid, 8-hydroperoxyarachidonic acid, 8-hydroxyeicosapentaenoic acid, 8 -hydroxy-5,8,11,14 ( Z , E , Z,Z )-eicosatetraenoic acid also called) and 8-peroxyeicosapentaenoic acid (8-hydroperoxyeicosapentaenoic acid) containing at least one selected from the group consisting of It is preferable to do, but is not limited thereto.

In the present specification, the term "15-hydroxylated fatty acid" preferably has a hydroxyl group or a peroxidation group on the 15th carbon of the unsaturated fatty acid having 20 carbon atoms, but is not limited thereto. More specifically, the 15-hydroxylated fatty acid is 15-hydroxyarachidonic acid, 15-hydroperoxyarachidonic acid, 15-hydroxyeicosapentaenoic acid, 15 -hydroxy-5,8,11,14 ( Z , Z , Z , E )-eicocatetraenoicacid also called) and 15-peroxyeicosapentaenoic acid (15-hydroperoxyeicosapentaenoic acid) containing at least one selected from the group consisting of It is preferred, but is not limited thereto.

In the specification of the present application, "8,15-diperhydroxylated fatty acid" refers to 8-hydroxylated, 15-perhydroxylated arachidonic acid (8,15(P)-DiHETE, 8-hydroxy&15-hydroperoxyarachidonic acid), 8-perhydroxylated, 15-hydroxylated arachidone Acids (8-hydroperoxy&15-hydroxyarachidonic acid), 8-hydroxylated, 15-perhydroxylated eicosapentaenoic acid (8,15(P)-DiHEPE, 8-hydroxy&15-hydroperoxyeicosapentaenoic acid), 8-perhydroxylated, 15-hydroxyarachidonic acid Sapentaenoic acid (8-hydroperoxy&15-hydroxyeicosapentaenoic acid), which means any one selected from the group consisting of, and is a novel compound, each represented by the following formulas 1 to 4 in order:

[Formula 1]

[Formula 2]

[Formula 3]

[Formula 4]

In the present specification, "leukotriene analog" refers to any one selected from the group consisting of leukotriene B4 analogues (LTA4 analogue, 8,15-dihydroxyeicosatetraenoic acid) and leukotriene B5 analogues (LTB5 analogue, 8,15-dihydroxyeicosapentaenoic acid), and as a novel compound , Each in order is represented by the following Chemical Formula 5 and Chemical Formula 6:

[Formula 5]

[Formula 6]

"Lipoxin analogue" in the present specification refers to a lipoxin A4 analogue (LXA4 analogue, 8,9,15-trihydroxyeicosatetraenoic acid), a lipoxin B4 analogue (LXB4 analogue, 8,14,15-trihydroxyeicosatetraenoic acid), a lipoxin A5 analogue ( It means any one selected from the group consisting of LXA5 analogue, 8,9,15-trihydroxyeicosapentaenoic acid), and lipoxin B5 analogue (LXB5 analogue, 8,14,15-trihydroxyeicosapentaenoic acid), and as a novel compound, each of the following formulas in order It is represented by the formula 7 to 10:

[Formula 7]

[Formula 8]

[Formula 9]

[Formula 10]

The present invention is Mus musculus (Mus musculus) 8-lipoxygenase derived from mice; It provides a composition for preparing 8,15-diperoxygenated fatty acids or leukotriene analogues comprising, as an active ingredient; 15-lipoxygenase derived from Burkholderia thailandensis strain.

In addition, the present invention Mus musculus (Mus musculus) mouse derived 8-lipoxygenase; 15-lipoxygenase from Burkholderia thailandensis strain; And Myxococcus xanthus (Myxococcus xanthus) provides a composition for producing a lipoxin analogue comprising an epoxide hydrolase derived from the strain as an active ingredient.

In addition, the present invention is Mus musculus (Mus musculus) a gene encoding a mouse-derived 8-lipoxygenase; And a gene encoding 15-lipoxygenase derived from a strain of Burkholderia thailandensis ; and an 8,15-diperoxygenated fatty acid or a composition for preparing a leukotriene analogue comprising as an active ingredient.

In addition, the present invention is Mus musculus (Mus musculus) a gene encoding a mouse-derived 8-lipoxygenase; A gene encoding 15-lipoxygenase derived from Burkholderia thailandensis strain; And Myxococcus xanthus (Myxococcus xanthus) provides a composition for producing a lipoxin analogue comprising a gene encoding the epoxide hydrolase derived from the strain; as an active ingredient.

In one embodiment of the present invention, the 8-lipoxygenase derived from the Mus musculus mouse is composed of the amino acid sequence of SEQ ID NO: 1; The 15-lipoxygenase derived from the Burkholderia thailandensis strain is preferably composed of the amino acid sequence of SEQ ID NO: 3, but one or more substitutions, deletions, additions, etc. to the sequence provide the desired effect of the present invention. All mutant enzymes that can be achieved are included in the scope of the present invention. The 8-lipoxygenase may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 2, and the 15-lipoxygenase may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 4.

In addition, the epoxide hydrolase derived from the Myxococcus xanthus strain is not only composed of the amino acid sequence of SEQ ID NO: 5, but also a functional equivalent thereof, that is, mutations such as one or more substitutions or deletions in the sequence. It means to include all mutants to achieve the object of the present invention by causing, the epoxide hydrolase may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 6.

In one embodiment of the present invention, the composition is an unsaturated fatty acid having 20 carbon atoms as a substrate, in particular, an unsaturated fatty acid having 20 to 22 carbon atoms with one or more cis, cis-1,4 pentadiene, preferably arachidonic acid. (arachidonic acid, AA) or eicosapentaenoic acid (eicosapentaenoic acid, EPA) may be for treatment on a substrate containing.

In another aspect, the present invention provides a Mus musculus mouse-derived 8-lipoxygenase; 15-lipoxygenase from Burkholderia thailandensis strain; Epoxide hydrolase derived from Myxococcus xanthus strain; And a method of producing lipoxin analogues (lipoxin A4, lipoxin B4, lipoxin A5, lipoxin B5) through biotransformation by reacting a substrate.

In one embodiment of the present invention, the 8-lipoxygenase derived from the Mus musculus mouse is composed of the amino acid sequence of SEQ ID NO: 1; The 15-lipoxygenase derived from the Burkholderia thailandensis strain is preferably composed of the amino acid sequence of SEQ ID NO: 3, but one or more substitutions, deletions, additions, etc. to the sequence provide the desired effect of the present invention. All mutant enzymes that can be achieved are included in the scope of the present invention. The 8-lipoxygenase may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 2, and the 15-lipoxygenase may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 4.

In addition, the epoxide hydrolase derived from the Myxococcus xanthus strain is not only composed of the amino acid sequence of SEQ ID NO: 5, but also a functional equivalent thereof, that is, mutations such as one or more substitutions or deletions in the sequence. It means to include all mutants to achieve the object of the present invention by causing, the epoxide hydrolase may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 6.

In another embodiment of the present invention, the substrate is preferably arachidonic acid (AA) and eicosapentaenoic acid (EPA), and the method comprises arachidonic acid (AA) as a substrate. ) And eicosapentaenoic acid (EPA) are more preferably used in the range of 0.1 mM to 3 mM, but are not limited thereto.

In an embodiment of the present invention, the method preferably performs the reaction in a pH range of 6.5 to 7.5, and the method preferably performs the reaction in a temperature range of 10° C. to 30° C., but is not limited thereto.

In another aspect, the present invention provides a recombinant expression vector comprising 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase, respectively; And an expression vector containing all of the enzymes.

According to one embodiment of the present invention, the present invention is a gene encoding 8-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 2; A gene encoding a 15-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 4; And a recombinant expression vector for producing a lipoxin analog (Lipoxin A4, B4, A5, and B5) comprising a gene encoding an epoxide hydrolase consisting of the amino acid sequence of SEQ ID NO: 6, and the recombinant expression vector is transformed into a host cell. Transformed transformants are provided.

The genes encoding the 8-lipoxygenase and 15-lipoxygenase are not only composed of the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4, respectively, but also functional equivalents thereof, that is, one or more substitutions in the sequence, It means to include all mutants that achieve the object of the present invention by causing mutations such as deletions.

In addition, the gene encoding the epoxide hydrolase is not only composed of the nucleotide sequence shown in SEQ ID NO: 6, but also a functional equivalent thereof, that is, by causing mutations such as one or more substitutions or deletions in the sequence, according to the present invention. It is meant to include all mutants that achieve the purpose.

Specifically, the entire cell containing the 8-lipoxygenase is a gene encoding the amino acid sequence described in SEQ ID NO: 1; Alternatively, it is preferable to cultivate a transformant transformed with a recombinant expression vector containing a gene consisting of the nucleotide sequence of SEQ ID NO: 2, and obtain and use the transformant. In addition, the whole cell containing the 15-lipoxygenase is a gene encoding the amino acid sequence shown in SEQ ID NO: 3; Alternatively, it is preferable to cultivate a transformant transformed with a recombinant expression vector containing a gene consisting of the nucleotide sequence of SEQ ID NO: 4, and obtain and use the transformant. In addition, the whole cell containing the epoxide hydrolase is a gene encoding the amino acid sequence shown in SEQ ID NO: 4; Alternatively, it is preferable to cultivate a transformant transformed with a recombinant expression vector containing a gene consisting of the nucleotide sequence of SEQ ID NO: 6, and obtain and use the transformant.

As the recombinant expression vector, any vector may be used as long as it is a plasmid vector used in the art for gene recombination, and it is more preferable to specifically use pET-28a(+) or pACYC duet plasmid, but is not limited thereto. .

As the transformed transformant, any microorganism may be used as long as it is transformed with a recombinant expression vector to overexpress the desired gene and produce an active enzyme protein, specifically E. coli ER 2566 strain can be used. However, it is not limited thereto.

In addition, the present invention is to (1) construct a recombinant expression vector comprising 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase genes; (2) culturing the microorganism transformed with the recombinant expression vector; (3) Inducing the expression of 8-lipoxygenase, 15-lipoxygenase, and epoxide hydrolase genes (4) The recombinant protein expressed and a method for producing a strain including the same are provided.

The whole cells containing the 8-, 15 lipoxygenase and/or epoxide hydrolase are i) centrifuged to recover the primary whole cells by centrifuging the culture medium of the microorganism; ii) washing the recovered whole cells with saline solution; iii) removing the supernatant by second centrifuging the washed whole cells to obtain whole cells; And iv) washing the secondly recovered whole cells with physiological saline. Specifically, the recovery of whole cells in step i) can be performed in a range of around 13,000 g using a device known in the art such as a centrifuge, and washing of the whole cells in step ii) is performed with 0.9% or less sodium chloride solution. It is appropriate to perform.

The present invention provides a production method for synthesizing 8-hydroxylated fatty acid, 15-hydroxylated fatty acid, and four lipoxin analogs using cells containing cultured 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase. .

E. coli transformed with a recombinant expression vector containing the 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase genes was cultured to induce expression of the recombinant enzyme gene, and then the strain containing the expressed protein was recovered. It is preferable to use it.

In addition, it is preferable to use a fatty acid as a substrate, more preferably arachidonic acid and eicosapentaenoic acid. In addition, the concentration of the substrate is preferably 0.5 mM to 5 mM, and more preferably around 1 mM.

In addition, the enzymatic reaction is preferably performed in the range of pH 6.5 to 7.5, more preferably in the range of about pH 7.0. In order to maintain this pH condition, a HEPES buffer solution can be used as a reaction solvent.

In addition, the enzymatic reaction is preferably performed at a temperature in the range of 10°C to 30°C, more preferably in a temperature range of around 25°C. By maintaining these conditions, it is possible to improve the production activity of 8- and 15-hydroxylated fatty acids and lipoxin analogs.

In addition, the time of the whole cell reaction can be appropriately adjusted according to a conventional method.

In another embodiment of the present invention, the method increases the concentration of cells containing lipoxygenase and epoxide hydrolase, which are enzymes that produce hydroxylated fatty acids and lipoxin analogs from unsaturated fatty acids, from 10 g/L to 50 g/L. It is preferable to process in the range, but is not limited thereto.

As described above, according to the present invention, Mus musculus 8-lipoxygenase derived from mice; And by using 15-lipoxygenase derived from Burkholderia thailandensis strain, 8,15-pohydrylated fatty acids, leukotriene analogs and new lipoxin analogs can be prepared with high productivity and high yield, It is expected to be useful in various industrial fields such as, food and cosmetics.

The lipoxin analog, prepared according to the present invention, is a signaling material and is expected to be involved in various physiologically active functions in animals including humans.

Hereinafter, a preferred embodiment is presented to aid the understanding of the present invention. However, the following examples are provided for easier understanding of the present invention, and the contents of the present invention are not limited by the following examples.

Example 1. Construction of a recombinant expression vector and a transformed microorganism for the expression of 8-lipoxygenase, 15-lipoxygenase or epoxide hydrolase

In the present invention, in order to prepare 8-lipoxygenase, 15-lipoxygenase or epoxide hydrolase, mice, Burkholderia thailandensis strains and Myxococcus xanthus strains Genes encoding 8-lipoxygenase, 15-lipoxygenase, and epoxide hydrolase were isolated from the genes, and a recombinant expression vector for overexpressing them was constructed.

Specifically, the gene sequence and amino acid sequence are already specified at the Korea Microbial Conservation Center (Seoul). Mus musculus , Burkholderia thailandensis and Myxococcus xanthus were purchased and selected, and 8-lipoxygenase, 15-lipoxygenase derived therefrom or genomic mouse cDNA, member kolde Ria tailran den sheath (Burkholderia thailandensis) or dynamic Socorro kusu glass tooth (Myxococcus xanthus) in order on the basis of the DNA nucleotide sequence of the epoxide hydrolase carrying out the polymerase chain reaction (PCR) DNA was extracted and used as a template for PCR, and primers based on the DNA sequence of 8-lipoxygenase, 15-lipoxygenase, or epoxide hydrolase were each devised and subjected to polymerase chain reaction. (PCR) was carried out. Nde I and XhoI were used as restriction enzymes for each gene. The cloning method was Gibson assembly method, and each primer is as follows. The primers of the mouse-derived 8-lipoxygenase sequence are F: AGC GGC CTG GTG CCG CGC GGC AGC CAT ATG GCG AAA TGC AGG GTG AGA GTA TCC ACG (SEQ ID NO: 7), R: ATC TCA GTG GTG GTG GTG GTG GTG CTC GAG TTA GAT GGA GAC ACT GTT CTC AAT GAG GGG (SEQ ID NO: 8), and the primer of the 15-lipoxygenase sequence derived from Burkholderia thailandensis is F: AGC GGC CTG GTG CCG CGC GGC AGC CAT ATG GTC AAT CAC AAA ACC GGG TCA AAT ATG (SEQ ID NO: 9), R: ATC TCA GTG GTG GTG GTG GTG GTG CTC GAG TCA AAT GTT CGT GCT TGC CGG AAT CCG GCT (SEQ ID NO: 10), Myxococcus The primers of the epoxide hydrolase sequence derived from Xanthus ( Myxococcus xanthus ) are F: AGC GGC CTG GTG CCG CGC GGC AGC CAT ATG GCT GAC ATC ACG CAT CGA ACC GTA AAG (SEQ ID NO: 11), R: ATC TCA GTG GTG GTG It consisted of GTG GTG GTG CTC GAG TCA GGC CGG CAG CTT CTT CAG GAA GGC CAG (SEQ ID NO: 12). In addition, in order to clone each gene into pET 28a, each vector was also prepared using PCR and then ligated using isothermal buffer to produce recombinant 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase. I did.

Then, the recombinant expression vector obtained as described above was transformed into E. coli ER 2566 strain purchased from New England Biolabs (Hertfordshire, UK) by a conventional transformation method, and the transformed microorganisms used a 20% glycerin solution. After addition, 8-hydroxylated fatty acid, 11-hydroxylated fatty acid and lipoxin analogs were incubated for trial production and stored frozen at -70°C before use.

Example 2. Expression of 8-lipoxygenase, 15-lipoxygenase or epoxide hydrolase

For protein expression of enzymes, the transformed microorganisms stored frozen in (1) were aerated at 200 rev/min in a flask containing 500 ml of LB (Difco, Sparks, MD, USA) medium and 20 μg/ml kanamycin. Incubated at 37°C under conditions. When the absorbance of bacteria reached 0.6 to 0.8 at 600 nm, a final concentration of 0.1 mM IPTG was added to induce protein expression of enzymes, and the culture was incubated with stirring at 16°C for 16 hours at 150 rev/min. .

E. coli cells containing cultured 8-lipoxygenase, 15-lipoxygenase, or epoxide hydrolase were collected and used. In addition, 8-lipoxygenase, 15-lipoxygenase, or epoxide hydrolase produced by overexpressing as described above is 0.85% sodium chloride by centrifuging the culture medium of the transformed strain at 6,000xg for 30 minutes at 4°C. After washing twice with (NaCl), it was used as a recombinant cell to produce 8-hydroxylated fatty acid, 15-hydroxylated fatty acid and four lipoxin analogs.

Example 3. Construction of a pathway for the synthesis of lipoxin A4 and B4 analogs from arachidonic acid using 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase

In order to construct a pathway for the synthesis of lipoxin A4 and B4 analogs using the 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase, arachidonic acid (AA) was used as a substrate, and the lipoxin A4 analog synthesis pathway In the case of, 15-hydroxylated arachidonic acid was prepared and used from 15-lipoxygenase, and 8-hydroxylated arachidonic acid was prepared and used from 8-lipoxygenase for the synthesis pathway of lipoxin B4 analog. The lipoxin A4 analog was 40 g/L of recombinant 8-lipoxygenase from 1 mM of 15-hydroxylated arachidonic acid, and the lipoxin B4 analog was 40 g/L of recombinant 15 from 1 mM of 8-hydroxylated arachidonic acid. -Lipoxygenase was used, and in both reactions, a whole cell reaction was carried out with epoxide hydrolase at pH 7.0 and 25°C for 1 hour.

15-HPETE and 8-HPETE formed from 15-lipoxygenase and 8-lipoxygenase from 1 mM arachidonic acid were reduced to 15-HETE and 8-HETE by treatment with 100 mM Cysteine. When each substance was once again used as a substrate, 8-lipoxygenase and 15-lipoxygenase were used to form each perhydroxylated fatty acid, and then 8,15-dihydroxylated when treated with 100 mM cysteine. It was confirmed that a leukotriene B4 analogue, which is a fatty acid (8,15-DiHETE, LTB4 analogue), was formed. In addition, in order to synthesize analogs of lipoxin A4 and B4, 8-lipoxygena using 15-HPETE and 8-HPETE formed from 15-lipoxygenase and 8-lipoxygenase from 1 mM arachidonic acid under the reaction conditions presented above as substrates. 15-OH-8,9-Ep-HETE and 8-OH-14,15-Ep-HETE obtained through the two-stage reaction of the enzyme and 15-lipoxygenase were produced, and the formed epoxide was produced through an epoxide hydrolase. The lipoxin analog was synthesized by hydrolysis. As a result, as a result, a synthesis route as shown in [Fig. 1] was constructed. The lipoxin A4 and B4 analogs converted under the above-described reaction conditions were confirmed using HPLC, and the amount of lipoxin B4 analogues that can be quantified was confirmed over time (FIG. 2). In the case of substance identification, it was confirmed through LC-MS/MS, and as a result of the confirmation, it was confirmed that they were lipoxin A4 and B4 analogues and leukotriene B4 analogues (Fig. 3).

Example 4. Construction of a pathway for the synthesis of lipoxin A5 and B5 analogs from eicosapentaenoic acid using 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase

Eicosapentaenoic acid (EPA) was used as a substrate in order to construct a pathway for the synthesis of lipoxin A5 and B5 analogs using the 8-lipoxygenase, 15-lipoxygenase, and epoxide hydrolase. In the case of the A5 analog synthesis route, 15-hydroxylated eicosapentaenoic acid was prepared and used from 15-lipoxygenase, and in the case of the lipoxin B5 analog synthesis route, 8-hydroxylated eicosapentaenoic acid from 8-lipoxygenase. Was prepared and used. The lipoxin A5 analog was 1 mM of 15-hydroxylated eicosapentaenoic acid and 40 g/L of recombinant 8-lipoxygenase was used, and the lipoxin B5 analog was 1 mM of 8-hydroxylated eicosapentaenoic acid. 40 g/L of recombinant 15-lipoxygenase was used, and both reactions were performed with epoxide hydrolase at pH 7.0 and 25° C. for 1 hour.

15-HPEPE and 8-HPEPE formed from 15-lipoxygenase and 8-lipoxygenase from 1 mM eicosapentaphosphate in the reaction conditions presented above were treated with 100 mM Cysteine to yield 15-HEPE and 8-HEPE. Reduction, and when each of the substances was once again used as a substrate, 8-lipoxygenase and 15-lipoxygenase were used to form each perhydroxylated fatty acid and then treated with 100 mM cysteine. 8,15- It was confirmed that a leukotriene B5 analog, which is a dihydroxy fatty acid (8,15-DiHEPE, LTB5 analogue), was formed. In addition, in order to synthesize analogs of lipoxin A5 and B5, 8-lipoxygena using 15-HPEPE and 8-HPEPE formed from 15-lipoxygenase and 8-lipoxygenase from 1 mM arachidonic acid under the reaction conditions presented above. 15-OH-8,9-Ep-HEPE and 8-OH-14,15-Ep-HEPE obtained through the two-stage reaction of the enzyme and 15-lipoxygenase were produced. The lipoxin analog was synthesized by hydrolysis. As a result, as a result, a synthetic route as shown in [Fig. 4] was constructed. In addition, the produced lipoxin A5 and B5 analogs were confirmed using HPLC (Fig. 5), and the substance identification was confirmed by LC-MS/MS, and as a result of the confirmation, it was confirmed that the lipoxin A4 and B4 analogues and the leukotriene B5 analogues ( Fig. 6).

As described above, the present invention is a mouse-derived 8-lipoxygenase, a 15-lipoxygenase derived from Burkholderia thailandensis , and an epoxide hydrolase derived from Myxococcus xanthus. Hydroxy fatty acids, leukotriene B4, B5 analogs, and lipoxin A4, B4, A5 and B5 analogs that have not been reported so far, which can act as lipid mediators in animals including humans, are produced using biotransformation methods. It's about the method. Specifically, arachidonic acid and eicosapentaenoic acid are used as substrates, and 8-lipoxygenase derived from mice, 15-lipoxygenase derived from Burkholderia thailandensis , and Myxococcus xanthus derived Since it is possible to produce 8,15-diperhydroxide fatty acids, leukotriene analogs and lipoxin A4, B4, A5 and B5 analogs by biotransformation method using transformed cells containing the epoxide hydrolase of , Compared to the conventional chemical method, not only is it overcome by environmentally friendly conditions, but also the production of lipid modulators that have not been reported before and the specific production by bioconversion method have great significance.

As described above, specific parts of the present invention have been described in detail. For those of ordinary skill in the fatty acid industry and medical field, these specific techniques are only preferred embodiments, and the scope of the present invention is not limited thereby. The point will be clear. Therefore, it will be said that the practical scope of the present invention is defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp <120> Method for producing leukotriene analogues or lipoxin analogues by combination reation using recombinant lipoxigenases and leukotriene analogues and lipoxin analogues therefrom <130> 1064119 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 677 <212> PRT <213> Mus musculus <400> 1 Met Ala Lys Cys Arg Val Arg Val Ser Thr Gly Glu Ala Cys Gly Ala 1 5 10 15 Gly Thr Trp Asp Lys Val Ser Val Ser Ile Val Gly Thr His Gly Glu 20 25 30 Ser Pro Leu Val Pro Leu Asp His Leu Gly Lys Glu Phe Ser Ala Gly 35 40 45 Ala Glu Glu Asp Phe Glu Val Thr Leu Pro Gln Asp Val Gly Thr Val 50 55 60 Leu Met Leu Arg Val His Lys Ala Pro Pro Glu Val Ser Leu Pro Leu 65 70 75 80 Met Ser Phe Arg Ser Asp Ala Trp Phe Cys Arg Trp Phe Glu Leu Glu 85 90 95 Trp Leu Pro Gly Ala Ala Leu His Phe Pro Cys Tyr Gln Trp Leu Glu 100 105 110 Gly Ala Gly Glu Leu Val Leu Arg Glu Gly Ala Ala Lys Val Ser Trp 115 120 125 Gln Asp His His Pro Thr Leu Gln Asp Gln Arg Gln Lys Glu Leu Glu 130 135 140 Ser Arg Gln Lys Met Tyr Ser Trp Lys Thr Tyr Ile Glu Gly Trp Pro 145 150 155 160 Arg Cys Leu Asp His Glu Thr Val Lys Asp Leu Asp Leu Asn Ile Lys 165 170 175 Tyr Ser Ala Met Lys Asn Ala Lys Leu Phe Phe Lys Ala His Ser Ala 180 185 190 Tyr Thr Glu Leu Lys Val Lys Gly Leu Leu Asp Arg Thr Gly Leu Trp 195 200 205 Arg Ser Leu Arg Glu Met Arg Arg Leu Phe Asn Phe Arg Lys Thr Pro 210 215 220 Ala Ala Glu Tyr Val Phe Ala His Trp Gln Glu Asp Ala Phe Phe Ala 225 230 235 240 Ser Gln Phe Leu Asn Gly Ile Asn Pro Val Leu Ile Arg Arg Cys His 245 250 255 Ser Leu Pro Asn Asn Phe Pro Val Thr Asp Glu Met Val Ala Pro Val 260 265 270 Leu Gly Pro Gly Thr Ser Leu Gln Ala Glu Leu Glu Lys Gly Ser Leu 275 280 285 Phe Leu Val Asp His Gly Ile Leu Ser Gly Val His Thr Asn Ile Leu 290 295 300 Asn Gly Lys Pro Gln Phe Ser Ala Ala Pro Met Thr Leu Leu His Gln 305 310 315 320 Ser Ser Gly Ser Gly Pro Leu Leu Pro Ile Ala Ile Gln Leu Lys Gln 325 330 335 Thr Pro Gly Pro Asp Asn Pro Ile Phe Leu Pro Ser Asp Asp Thr Trp 340 345 350 Asp Trp Leu Leu Ala Lys Thr Trp Val Arg Asn Ser Glu Phe Tyr Ile 355 360 365 His Glu Ala Val Thr His Leu Leu His Ala His Leu Ile Pro Glu Val 370 375 380 Phe Ala Leu Ala Thr Leu Arg Gln Leu Pro Arg Cys His Pro Leu Phe 385 390 395 400 Lys Leu Leu Ile Pro His Ile Arg Tyr Thr Leu His Ile Asn Thr Leu 405 410 415 Ala Arg Glu Leu Leu Val Ala Pro Gly Lys Leu Ile Asp Lys Ser Thr 420 425 430 Gly Leu Gly Thr Gly Gly Phe Ser Asp Leu Ile Lys Arg Asn Met Glu 435 440 445 Gln Leu Asn Tyr Ser Val Leu Cys Leu Pro Glu Asp Ile Arg Ala Arg 450 455 460 Gly Val Glu Asp Ile Pro Gly Tyr Tyr Tyr Arg Asp Asp Gly Met Gln 465 470 475 480 Ile Trp Gly Ala Ile Lys Ser Phe Val Ser Glu Ile Val Ser Ile Tyr 485 490 495 Tyr Pro Ser Asp Thr Ser Val Gln Asp Asp Gln Glu Leu Gln Ala Trp 500 505 510 Val Arg Glu Ile Phe Ser Glu Gly Phe Leu Gly Arg Glu Ser Ser Gly 515 520 525 Met Pro Ser Leu Leu Asp Thr Arg Glu Ala Leu Val Gln Tyr Ile Thr 530 535 540 Met Val Ile Phe Thr Cys Ser Ala Lys His Ala Ala Val Ser Ser Gly 545 550 555 560 Gln Phe Asp Ser Cys Val Trp Met Pro Asn Leu Pro Pro Thr Met Gln 565 570 575 Leu Pro Pro Pro Thr Ser Lys Gly Gln Ala Arg Pro Glu Ser Phe Ile 580 585 590 Ala Thr Leu Pro Ala Val Asn Ser Ser Ser Tyr His Ile Ile Ala Leu 595 600 605 Trp Leu Leu Ser Ala Glu Pro Gly Asp Gln Arg Pro Leu Gly His Tyr 610 615 620 Pro Asp Glu His Phe Thr Glu Asp Ala Pro Arg Arg Ser Val Ala Ala 625 630 635 640 Phe Gln Arg Lys Leu Ile Gln Ile Ser Lys Gly Ile Arg Glu Arg Asn 645 650 655 Arg Gly Leu Ala Leu Pro Tyr Thr Tyr Leu Asp Pro Pro Leu Ile Glu 660 665 670 Asn Ser Val Ser Ile 675 <210> 2 <211> 2034 <212> DNA <213> Mus musculus <400> 2 atggcgaaat gcagggtgag agtatccacg ggggaagcct gtggggctgg cacatgggac 60 aaagtgtctg tcagcatcgt gggaacccac ggagagagcc ccttagtacc tctggaccat 120 ctgggcaagg agttcagcgc cggtgctgaa gaagacttcg aggtgacgct tccccaggac 180 gtaggcactg tgctgatgct gcgagtccac aaagcacccc cggaagtgtc cctcccgctt 240 atgtctttcc gttctgatgc ctggttctgc cgctggttcg agctggagtg gctacctggg 300 gctgcactcc acttcccctg ttatcagtgg ctggaagggg cgggggagct ggtgctgaga 360 gagggagcag caaaggtgtc ctggcaagac catcacccta cactgcagga tcagcgccag 420 aaggagcttg agtccaggca gaagatgtac agctggaaga cttacattga aggttggcct 480 cgctgccttg accacgagac tgtgaaagac ttggacctca acatcaagta ctctgcgatg 540 aagaatgcca aactcttctt taaagcccac tccgcgtata cggagctgaa agtcaaaggg 600 ctcctggacc gcacaggact ctggaggagt ctgagggaga tgagaaggct gtttaacttc 660 cgcaagactc cagcagcaga gtatgtgttt gcacactggc aggaagatgc cttcttcgcc 720 tcccagttcc taaatggcat caacccggtc ctgattcgcc gctgtcacag tctcccaaac 780 aacttcccgg tcactgatga aatggtggcc ccagtgctgg gccctggaac cagtctgcag 840 gctgagttgg agaagggctc cctgttcttg gtggatcatg gcattctttc tggagtccac 900 accaacatcc tcaatggaaa gcctcagttc tctgcagccc cgatgaccct gttacaccag 960 agctcagggt ccggacccct gcttcccatt gccatccagc tcaaacagac tcccgggcca 1020 gacaacccca tcttcctgcc cagcgatgac acgtgggact ggttgctggc caagacctgg 1080 gttcgcaatt ctgagtttta catccatgag gctgtcacac atctgctgca tgcccatctg 1140 attccagaag tctttgcctt ggccacatta cgtcagctgc ctaggtgtca ccctctcttc 1200 aagctattga ttcctcacat tcggtacaca ctgcacatca acacgcttgc ccgggagctg 1260 ctcgttgccc ctgggaagtt gatagacaag tccacaggcc ttggcactgg gggattctct 1320 gacctgataa agagaaacat ggagcagctg aactactctg tcctgtgtct ccctgaagat 1380 atccgagccc gaggtgtgga agacatccca ggctactatt accgagatga tgggatgcag 1440 atctgggggg caataaagag ctttgtctct gaaatagtca gcatctacta tccaagtgac 1500 acatccgtcc aagatgacca agagctccag gcctgggtga gggagatctt ctctgagggc 1560 ttcctcggcc gagaaagctc aggtatgccc tccttgttgg atacccggga agccctggtc 1620 cagtatatca ccatggtgat attcacctgc tcagccaagc atgcagctgt cagttcaggc 1680 cagttcgact cttgtgtttg gatgcccaat ctgccaccta ccatgcagct accaccacct 1740 acttccaaag gccaggcccg gcctgagagt ttcatagcca cgctcccagc agttaattcg 1800 tcaagttatc acatcattgc tctctggctg ctaagcgcag aacctgggga ccaaaggccc 1860 ctgggccact atccagatga acacttcaca gaggatgccc cccggcgaag cgtggctgcc 1920 ttccagagaa agctgatcca gatctccaag ggcatcaggg agaggaaccg aggcctggca 1980 ctgccctaca cctacctgga tcctcccctc attgagaaca gtgtctccat ctaa 2034 <210> 3 <211> 695 <212> PRT <213> Burkholderia sp. <400> 3 Met Val Asn His Lys Thr Gly Ser Asn Met Asn Arg Arg Asp Leu Ile 1 5 10 15 Lys Phe Leu Ser Phe Ala Ala Ser Gly Thr Ala Phe Ala Gly Leu Val 20 25 30 Arg Ser Thr Leu Ser Ser Pro Ala Ala Ser Ser Ile Thr Ala Ser Pro 35 40 45 Arg Thr Leu Asp Ala Gly Ile Gly Ile Ser Ser Pro Gln Ala Val Arg 50 55 60 Ala Ala Ala Pro Val Leu Pro Gln Lys Asp Thr Ala Ala Gly Arg Ile 65 70 75 80 Ala Arg Ala Gly Phe Leu Ala Thr Gln Arg Leu Ser Tyr Ile Trp Thr 85 90 95 Glu His Val Pro Thr Ala Ser Gly Ile Pro Leu Ala Leu Val Thr Pro 100 105 110 Gln Asp Leu Pro Thr Ile Glu Trp Leu Ile Lys Phe Ile Ala Ile Val 115 120 125 Val Gly Val Ile Glu Asn Phe Leu Gly Ser Ala Pro Ala Thr Ala Val 130 135 140 Ala Leu Trp Arg Asp Gln Phe Ala Lys Ile Arg Val Asp Leu Leu Ser 145 150 155 160 Leu Glu Asn Leu Tyr Ser Asp Leu Thr His Asp Pro Asn Leu Gln Asp 165 170 175 Pro Val Ala Ile Ala Gln Ala Ala Ser Ile Gln Ala Ala Leu Ile Ala 180 185 190 Leu Leu Ala Asn Val Gly Val Leu Ser Lys Asp Ile Ile Ser Arg Leu 195 200 205 Gly Glu Ile Val Ser Asn His Asp Thr Arg Ser Glu Glu Asn Phe Lys 210 215 220 Ala Leu Phe Ser Thr Phe Pro Leu Pro Asp Ile Ser Ala Ala Tyr Gln 225 230 235 240 Arg Asp Asp His Phe Ala Ser Leu Arg Val Ala Gly Gln Asn Pro Val 245 250 255 Leu Ile Lys Arg Ile Ser Gly Leu Pro Ser Lys Phe Pro Leu Thr Asn 260 265 270 Ala Gln Phe Gln Gln Val Met Gly Pro Ala Asp Asn Leu Val Ser Ala 275 280 285 Ala Ala Glu Asn Arg Leu Tyr Leu Leu Asp Tyr Val Asp Asn Gly Leu 290 295 300 Leu Ala Thr Ser Arg Ala Val Ala Lys Pro Leu Thr Gly Ile Gly Tyr 305 310 315 320 Ser Tyr Ala Pro Ile Ala Leu Phe Ala Leu Pro Arg Gly Gly Ala Ser 325 330 335 Leu Val Pro Val Ala Ile Gln Cys Asp Gln Asp Pro Ala Thr Asn Pro 340 345 350 Leu Phe Leu Pro Ala Asp Pro Ser Gln Glu Ser Ala Tyr Trp Ala Trp 355 360 365 Gln Met Ala Lys Thr Val Val Gln Cys Ala Glu Glu Asn Tyr His Glu 370 375 380 Met Phe Val His Leu Ala Arg Thr His Leu Val Thr Gly Ala Ile Cys 385 390 395 400 Val Ala Thr His Arg Asn Leu Ala Ser Thr His Pro Leu Tyr Ala Leu 405 410 415 Leu Met Pro His Phe Glu Gly Thr Leu Tyr Ile Asn Glu Leu Ala Ala 420 425 430 Leu Thr Leu Leu Pro Pro Leu Met Phe Ile Asp Thr Leu Phe Ala Ala 435 440 445 Pro Ile Gln Gln Thr Gln Gln Met Val Ala Ser Asp Arg Leu Ala Phe 450 455 460 Asp Phe Tyr Asp His Met Leu Pro Asn Asp Ile Glu Met Arg Gly Val 465 470 475 480 Gly Ala Ala Asn Leu Pro Asp Tyr Pro Tyr Arg Asp Asp Gly Leu Leu 485 490 495 Ile Trp Asn Ala Ile Ala Glu Trp Ala Lys Ala Tyr Val Asp Val Tyr 500 505 510 Tyr Lys Ser Asp Gln Asp Val Val Asp Asp Tyr Glu Leu Arg Ser Trp 515 520 525 Ala Ala Asp Ile Ile Ala Asn Gly Lys Val Lys Gly Phe Arg Pro Val 530 535 540 Arg Ser Lys Ala Gln Leu Ile Asp Val Leu Thr Met Ile Ile Phe Thr 545 550 555 560 Ala Ser Ala Gln His Ala Ala Val Asn Phe Ser Gln Ser Asp Phe Ser 565 570 575 Thr Tyr Ala Pro Ala Leu Ser Ala Leu Leu Ser Ala Pro Ala Pro Thr 580 585 590 Ser Ala Val Gly Lys Ser Lys Ala Asp Trp Leu Lys Met Leu Pro Pro 595 600 605 Leu Val Ser Gly Ile Glu Arg Val Ala Ile Tyr Glu Ile Leu Ala Gly 610 615 620 Val Gln His Ser Ala Leu Gly Gln Tyr Arg Ser Asn Val Phe Pro Tyr 625 630 635 640 Arg Pro Leu Ile Thr Asp Pro Ala Ile Thr Gly Ser Asn Gly Pro Leu 645 650 655 Glu His Phe Arg Gln Ala Leu Gly Asp Val Glu Ser Gln Ile Asn Ala 660 665 670 Arg Asn Ser Ile Arg Lys Thr Pro Tyr Glu Tyr Leu Leu Pro Ser Arg 675 680 685 Ile Pro Ala Ser Thr Asn Ile 690 695 <210> 4 <211> 2088 <212> DNA <213> Burkholderia sp. <400> 4 atggtcaatc acaaaaccgg gtcaaatatg aaccgaaggg atttaattaa attcttgagc 60 ttcgccgcca gcggaaccgc gtttgcgggg ctcgtcaggt cgactctgtc gtcaccggcc 120 gcgtcgtcga tcaccgcaag ccctcgcacg ctcgacgccg gcatcggcat ttcgtcgccg 180 caggcggttc gcgcagccgc gccggtgctg ccgcaaaaag acacggccgc aggcaggatc 240 gcacgggcgg gttttctcgc cacgcaaaga ctgagttaca tctggacgga acatgtgccg 300 accgcgagcg gcattccact tgcgctggtc acgccgcaag atcttccaac cattgaatgg 360 ctgatcaaat tcatcgcgat cgttgtgggc gtcatcgaga attttctcgg ctccgcaccc 420 gcgacggcgg ttgccctctg gcgcgatcag ttcgccaaga tcagggtgga tctgctttcc 480 ctcgaaaacc tgtattcgga tctcacccac gatccgaact tgcaagaccc ggttgccatt 540 gctcaggcag cgagcattca ggcggccttg atcgcgctgt tggcgaacgt cggcgtcttg 600 tcgaaggaca tcatttcaag attgggcgaa atcgtctcca accacgacac tcgaagcgaa 660 gagaacttca aggcgctgtt ttccaccttc ccgcttcccg acatctccgc cgcgtaccag 720 cgggacgatc atttcgcgtc tctccgggtt gccggccaga atccggtgtt gatcaagcgc 780 atctccggct tgccgtcgaa gttcccactg acgaacgctc aattccagca agtcatgggg 840 cccgccgaca atctcgtcag cgccgccgcg gagaatcggc tgtatctcct cgattacgtc 900 gacaacggtc tgttggcgac gtcgcgggcc gtggccaaac cactcacggg catcggctac 960 tcctatgcgc ccatcgcact tttcgcgctg ccgaggggcg gcgcgtccct cgttcccgtg 1020 gcgattcaat gcgatcagga tcccgcgacg aatcccctgt tcctccctgc cgatcccagt 1080 caggaatccg cctattgggc gtggcaaatg gccaagacgg tcgttcaatg cgcggaggaa 1140 aactatcacg agatgtttgt tcatctcgcg cgaacgcacc tggtgaccgg cgcgatttgc 1200 gtcgccactc accggaatct cgcgtcgacg catccgctct acgcgcttct gatgccgcac 1260 ttcgagggca ctttgtacat caacgaactc gccgcgctca cgctgcttcc accgttgatg 1320 ttcatcgata cgctgtttgc cgcgcccatc cagcagacgc aacagatggt cgcaagcgat 1380 cggctcgcgt tcgatttcta cgatcacatg ttgcccaacg acatcgaaat gcgcggagtc 1440 ggcgcggcca acctgccgga ctatccgtat cgcgacgacg gcctcctgat ttggaacgcc 1500 atcgccgaat gggcgaaggc gtatgtcgat gtctattaca agtccgatca ggatgtcgtc 1560 gatgactacg agctcaggtc ctgggccgcc gacatcatcg ccaacggcaa ggtcaaggga 1620 ttccggccgg tgcgttcgaa ggcgcaattg atcgacgtgc tgaccatgat catctttacc 1680 gcaagcgccc agcacgccgc cgtcaacttc tcgcagtcgg atttttcgac ctacgcgccc 1740 gcgctttccg cactgctgtc cgcgccggcc ccgacaagcg ccgtgggaaa aagcaaggcc 1800 gattggctga agatgctccc tcccctcgtt tcagggatcg agcgggttgc gatctacgag 1860 atcttggcgg gcgtccagca cagcgcgttg ggccagtatc gcagcaacgt atttccatat 1920 cggccgctca tcacggaccc cgcgatcacc ggaagcaacg ggccgctcga gcatttccga 1980 caggcgctcg gcgacgtcga atcgcagatc aacgctcgca acagcatacg caagacgcct 2040 tatgaatatc tgctgccgag ccggattccg gcaagcacga acatttga 2088 <210> 5 <211> 319 <212> PRT <213> Myxococcus xanthus <400> 5 Met Ala Asp Ile Thr His Arg Thr Val Lys Thr Asn Gly Ile Asn Leu 1 5 10 15 His Leu Ala Glu Ala Gly Ser Gly Pro Leu Val Leu Leu Leu His Gly 20 25 30 Trp Pro Glu Ser Trp Tyr Ser Trp Arg His Gln Leu Pro Ala Leu Ala 35 40 45 Ala Ala Gly Tyr His Ala Val Ala Pro Asp Val Arg Gly Tyr Gly Gln 50 55 60 Ser Asp Lys Pro Glu Ala Ile Glu Ala Tyr Ser Met Lys Gln Leu Val 65 70 75 80 Gly Asp Ala Val Gly Leu Leu Asp Ala Leu Gly Glu Arg Thr Ala Ile 85 90 95 Val Ile Gly His Asp Trp Gly Ser Ala Ile Ala Trp Asn Cys Ala Ala 100 105 110 Leu His Pro Asp Arg Phe Arg Ala Val Val Gly Met Ser Val Pro His 115 120 125 Leu Gly Arg Ala Pro Met Pro Pro Met Gln Leu Phe Gln Arg Met Phe 130 135 140 Gly Glu Lys Trp Phe Tyr Ile Leu Tyr Phe Gln Glu Pro Gly Val Ala 145 150 155 160 Glu Ala Glu Phe Glu Ala Asp Val Pro Arg Thr Val Arg Ala Ile Leu 165 170 175 Thr Gly Thr Pro Gly Phe Asp Val Thr Asn Pro Ala Val Leu Ala Lys 180 185 190 Lys Lys Gly Glu Gly Phe Leu Ala Arg Leu Asp Val Pro Glu Thr Leu 195 200 205 Pro Gly Trp Leu Thr Glu Ala Asp Val Ala Tyr Phe Ala Lys Glu Leu 210 215 220 Ala Gly Ser Gly Phe Arg Gly Gly Leu Asn Arg Tyr Arg Asn Met Asp 225 230 235 240 Arg Asp Trp His Glu Leu Pro Glu Leu Ala Thr Ala Val Ile Ser Gln 245 250 255 Pro Ala Leu Tyr Ile Val Gly Glu Lys Asp Pro Val Arg Ala Phe Ser 260 265 270 Pro Val Asp Pro Met Lys Ala Leu Val Pro Asn Leu Ala Asp Ile His 275 280 285 Val Ile Pro Gly Ala Gly His Trp Val Gln Gln Glu His Ala Ala Glu 290 295 300 Val Asn Ala Ala Leu Leu Ala Phe Leu Lys Lys Leu Pro Ala Glx 305 310 315 <210> 6 <211> 957 <212> DNA <213> Myxococcus xanthus <400> 6 atggctgaca tcacgcatcg aaccgtaaag acgaatggca tcaacctgca cctcgcggag 60 gcgggttcag ggccgctcgt gttgctcctg catggttggc cggagtcctg gtactcgtgg 120 cgccatcagc ttccggcgtt ggcggcggcg gggtaccacg cggtggcccc agacgtccgt 180 ggctacggcc agagcgacaa gccggaggcc atcgaggcgt acagcatgaa gcagttggtg 240 ggtgatgcgg ttggcctgct ggatgcgctg ggggagagga cggccatcgt catcggccac 300 gactggggct cggcgattgc ctggaactgt gccgccctgc atcccgaccg cttccgcgcg 360 gtggtgggaa tgagcgttcc ccatctgggg cgcgcgccca tgccgcccat gcagttgttt 420 cagcgcatgt tcggtgagaa gtggttctac atcctctact tccaggagcc cggcgtggcc 480 gaggccgagt tcgaggcgga cgtcccgagg acggtccggg cgattctcac gggcaccccc 540 ggcttcgatg tgacgaatcc cgccgtgctg gcgaagaaga agggggaagg cttcctcgcg 600 aggctcgacg tacctgagac gttgcccggc tggctcaccg aggcggacgt cgcgtacttc 660 gcgaaggagc tcgccggcag tggattccgg ggcgggctca atcgctaccg caacatggac 720 cgggactggc acgaactgcc ggagctggcg acggcggtca tctcgcagcc ggccctctac 780 atcgtcggag agaaggaccc ggtgcgtgcc ttctctcccg ttgacccgat gaaggccctg 840 gtgccgaacc tggccgacat ccacgtcatt cccggcgcgg gtcattgggt tcagcaggag 900 cacgccgccg aggtgaatgc cgcgctgctg gccttcctga agaagctgcc ggcctga 957 <210> 7 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 agcggcctgg tgccgcgcgg cagccatatg gcgaaatgca gggtgagagt atccacg 57 <210> 8 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 atctcagtgg tggtggtggt ggtgctcgag ttagatggag acactgttct caatgagggg 60 60 <210> 9 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 agcggcctgg tgccgcgcgg cagccatatg gtcaatcaca aaaccgggtc aaatatg 57 <210> 10 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 atctcagtgg tggtggtggt ggtgctcgag tcaaatgttc gtgcttgccg gaatccggct 60 60 <210> 11 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 agcggcctgg tgccgcgcgg cagccatatg gctgacatca cgcatcgaac cgtaaag 57 <210> 12 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 atctcagtgg tggtggtggt ggtgctcgag tcaggccggc agcttcttca ggaaggccag 60 60

Claims (12)

8-lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 1 from Mus musculus mouse;
15-lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 3 from Burkholderia thailandensis strain; And
Arachidonic acid or eicosapentaenoic acid as an substrate comprising an epoxide hydrolase consisting of the amino acid sequence of SEQ ID NO: 5 derived from Myxococcus xanthus strain as an active ingredient A composition for preparing a lipoxin analogue represented by any one of the following Chemical Formulas 7 to 10 using
[Formula 7]

,
[Formula 8]

,
[Formula 9]

,
[Formula 10]

.
delete A gene encoding 8-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 2 derived from Mus musculus mouse;
A gene encoding 15-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 4 from a Burkholderia thailandensis strain; And
Gene encoding an epoxide hydrolase consisting of the nucleotide sequence of SEQ ID NO: 6 derived from Myxococcus xanthus strain; as an active ingredient, arachidonic acid or eicosapentaeno A composition for preparing a lipoxin analogue represented by any one of the following Chemical Formulas 7 to 10 using eicosapentaenoic acid:
[Formula 7]

,
[Formula 8]

,
[Formula 9]

,
[Formula 10]

. 8-lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 1 from Mus musculus mouse;
15-lipoxygenase consisting of the amino acid sequence of SEQ ID NO: 3 from Burkholderia thailandensis strain;
An epoxide hydrolase consisting of the amino acid sequence of SEQ ID NO: 5 from Myxococcus xanthus strains; And arachidonic acid or eicosapentaenoic acid as a substrate to produce a lipoxin analogue represented by any one of the following Chemical Formulas 7 to 10 by bioconversion:
[Formula 7]

,
[Formula 8]

,
[Formula 9]

,
[Formula 10]

.
The method of claim 4, wherein
The method is characterized by using arachidonic acid (arachidonic acid) or eicosapentaenoic acid (eicosapentaenoic acid) in the range of 0.5 mM to 5 mM as a substrate.
The method of claim 4, wherein
The method is characterized in that the reaction is carried out in the pH 6.5 to 7.5 range.
The method of claim 4, wherein
The method is characterized in that the reaction is carried out in the temperature range 10 ℃ to 30 ℃.
A gene encoding 8-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 2 derived from a mus musculus mouse;
A gene encoding 15-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 4 from a Burkholderia thailandensis strain; And
Arachidonic acid or eicosapentaenoic acid as a substrate containing a gene encoding an epoxide hydrolase consisting of the nucleotide sequence of SEQ ID NO: 6 derived from Myxococcus xanthus strain Recombinant expression vector for preparing a lipoxin analogue represented by any one of the following Formulas 7 to 10 using
[Formula 7]

,
[Formula 8]

,
[Formula 9]

,
[Formula 10]

.
A transformant transformed with a recombinant expression vector according to claim 8 in a host cell.
A method for preparing a lipoxin analogue by treating the transformant of claim 9 with arachidonic acid or eicosapentaenoic acid as a substrate.
The method of claim 10,
The concentration of the transformant is characterized in that the treatment in the range of 10 g / L to 50 g / L.
Lipoxin analogs represented by any one of the following Formulas 7-10:
[Formula 7]

,
[Formula 8]

,
[Formula 9]

,
[Formula 10]

.

Images