KR102086235B1 - Method for producing resolvin D2 analogues by combination reaction using recombinant lipoxigenases and resolvin D2 analogues therefrom - Google Patents

Abstract

The present invention relates to a method and a composition for preparing a novel resolbin D2 analogue using two kinds of lipoxygenases. According to the present invention, 8-lipoxygenase derived from Mus musculus mice And by using 15-lipoxygenase from Burkholderia thailandensis strain, new resolbin analogs not reported to date in an environmentally friendly manner can be produced with high productivity and high yield, It can be usefully used in various industries such as food and cosmetics.

Description

Method for producing resolvin D2 analogues by combination reaction using recombinant lipoxigenases and resolvin D2 analogues therefrom}

The present invention relates to a method for the preparation of novel resolbin D2 analogs and compositions thereof using two lipoxygenases.

Hydroxylated fatty acids generally mean substances that have hydroxyl groups in fatty acids and are natural substances present in the lipids of various organisms such as animals, plants, insects and microorganisms. In particular, hydroxyamic acid has excellent reactivity and is used as a raw material industrially, and it is used as a raw material of cosmetics because of high activity such as reduction of surface tension, antibacterial and antifungal activity by the action of hydroxyl group. In particular, hydroxide fatty acids found in animals among various organisms are used as precursors of signal transduction substances in the human body, and a single substance is involved in various physiological activities. Among the lipid signaling mediators, resolbin is a substance produced mainly from polyunsaturated fatty acids and is converted by lipoxygenase and epoxide hydrolase. Liposomal regulators, including resolbin, are important substances involved in various physiological activities such as homeostasis control and immune response in mammals including humans.

Specifically, resolvin (RV) is a product of a combination reaction of lipoxygenases (lipoxygenase, LOX) and is mainly omega 3 polyunsaturated fatty acid eicosapentaenoic acid (EPA) or docosahexaenoic acid. (docosahexaenoic acid, DHA) is a substance belonging to the trihydroxy fatty acid form of eicosanoids produced by the sequential reaction of LOX. Depending on the starting substrate, it is classified as resolvin E using eicosaptananoic acid and resolbin D using docosahexaenoic acid. These substances are endogenous lipid mediators derived from the phase of acute inflammation, confirming potent anti-inflammatory and early inflammation in several animal models. Resolvin E1 and Resolbin D1 were able to prevent anti-inflammatory and postoperative pain, respectively. Specifically, Resolvin E1 was treated with nanograms of substances in animal models treated with inflammatory substances. Confirmed to have analgesic effects. Resolbin E1 and Resolbin D1 have been found to prevent incision-induced pain, especially Resolbin D1, which has been shown to heal or prevent hyperalgesia and allodynia following surgery in rats. There is a finding that the resolution effect is more effective. Resolbin E1, Resolbin D1, and Resolbin D2 have also been reported to stimulate the dermis of mouse skin to reduce neutrophil infiltration and promote skin regeneration, while D1 and D2 of the Resolbin D family are wound in a diabetic mouse model. It has been reported to promote healing. Therefore, if a resolvin analog (RV analogue) having a similar material structure is developed in addition to the previously studied resolbins, the strong bioactivity like the existing resolbins may suggest the possibility of replacing drugs related to other inflammatory remedies. It is thought that research and development is necessary in that it can be used, and suggests that it can be used as a research material in various fields such as medicine, biology, and biotechnology, as in other lipid mediators, as well as the possibility of being a medical substance related to the next generation inflammatory treatment.

Lipoxygenase (LOX) is a dioxygenase, and is named as a site-specific oxidase according to the position of oxidizing it to aridic acid, which is an unsaturated fatty acid having 20 carbon atoms. -, 11-, 12- and 15-LOX are present. These enzymes catalyze the reaction of synthesizing oxidized fatty acids and are enzymes containing iron and are multifunctional enzymes catalyzing two reactions. Characterized by using a polyunsaturated fatty acid having only one or more cis, cis-1,4-pentadiene as a substrate as a substrate to catalyze stereospecificity and reaction specificity through the dioxygenation reaction. Among them, in the case of arachidonic acid 8-lipoxygenase (hereinafter referred to as 8-lipoxygenase) which generates a hydroxyl group at position 8 of the animal polyunsaturated fatty acid, specifically, in an unsaturated fatty acid having 20 or more carbon atoms such as arachidonic acid The hydroxyl group is only formed at position 8 carbon. Further, in unsaturated fatty acids having 22 or more carbon atoms, a hydroxyl group is formed at the carbon number 10. In the case of 8-lipoxygenase, so far has not been found in animals other than mice, and recently, its existence has been reported in marine corals. In addition, arachidonic acid 15-lipoxygenase (hereinafter referred to as 15-lipoxygenase), which is a lipoxygenase to be used for biosynthesis of resolbin, has a hydroxyl group only at the carbon position of 15 at a carbon number of 20 or more unsaturated fatty acids such as arachidonic acid. To form. Further, in unsaturated fatty acids having 22 or more carbon atoms, a hydroxyl group is formed at the carbon number 17.

Resolbinates known to date have been reported with substances produced by acetylated cycloxygenase-2, P450 or human-derived 5-lipoxygenase and 15-lipoxygenase. However, a combination of site-specific oxidases and epoxide hydrolases has not been synthesized, and new site-specific resolbins and analogs thereof have not been previously reported, especially 8-lipoxygenase, 15-lipoxygenase and epoxy. The production of resolbin D2 analogs from docosahexaenoic acid in combination with side hydrolases has not been reported.

As a result of continuous efforts to biosynthesize new resolvin analogs that have not been reported previously, the present inventors have developed a synthetic route through a combination reaction of a specific lipoxygenase, and a new form of resolbin D2 analog is synthesized through bioconversion. By confirming that the present invention was completed.

Accordingly, the present invention is to provide a composition capable of producing a high yield of resolbin D2 analogues in a bioconversion process, a method for producing a resolbin D2 analogue and the resolbin D2 analogue produced thereby.

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

8-lipoxygenase from Mus musculus mice to achieve the above object; And a 15-lipoxygenase derived from a strain of Burkholderia thailandensis ( Burkholderia thailandensis ); provides a composition for preparing 10,17-dihydric fatty acid represented by Formula 1 or Formula 2 below.

[Formula 1]

[Formula 2]

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

The composition may further include docosahexaenoic acid (DHA) as a substrate.

In another aspect, the present invention provides a mus musculus mouse-derived 8-lipoxygenase; 15-lipoxygenase from Burkholderia thailandensis strains; And an epoxide hydrolase derived from Myxococcus xanthus strain as an active ingredient, and provides a composition for preparing resolbin D2 analogue represented by any one of the following Chemical Formulas 3 and 4.

[Formula 3]

[Formula 4]

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, the epoxide hydrolase is composed of the amino acid sequence of SEQ ID NO: 5 Can be.

The composition may further include docosahexaenoic acid (DHA) as a substrate.

In another aspect, the invention provides a gene encoding 8-lipoxygenase from Mus musculus mice; And a gene encoding 15-lipoxygenase derived from a Burkholderia thailandensis strain, as an active ingredient, the composition for preparing 10,17-dihydric fatty acid represented by Formula 1 or Formula 2 above is provided. .

In addition, the present invention provides a gene encoding 8-lipoxygenase derived from Mus musculus mice; Gene encoding 15-lipoxygenase from Burkholderia thailandensis strain; And a gene encoding an epoxide hydrolase derived from Myxococcus xanthus strain, as an active ingredient, a composition for preparing resolbin D2 analogue represented by Chemical Formula 3 or Chemical Formula 4 above.

In another aspect, the present invention provides a mus musculus mouse-derived 8-lipoxygenase; 15-lipoxygenase from Burkholderia thailandensis strains; Epoxide hydrolase from Myxococcus xanthus strains; And reacting the substrate to produce a resolbin D2 analogue represented by Formula 3 or Formula 4 by bioconversion.

The method may use docosahexaenoic acid (DHA) in the range of 0.5 mM to 5 mM as a substrate.

The method may be carried out the reaction in the pH 6.5 to 7.5 range.

The method may be carried out the reaction at a temperature of 10 ℃ to 30 ℃ range.

In another aspect, the present invention provides a gene encoding 8-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 2; A gene encoding 15-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 4; Any one gene selected from among; And it provides a recombinant expression vector for preparing resolbin D2 analogue comprising a gene encoding an epoxide hydrolase consisting of the amino acid sequence of SEQ ID NO: 6.

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

In another aspect, the present invention provides a method of preparing a resolbin D2 analog by treating the transformant with 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 a 10,17-dihydric fatty acid represented by the formula (1) or (2).

In addition, the present invention provides a resolbin D2 analogue represented by Formula 3 or Formula 4.

The present invention also provides an epoxide compound represented by the following formula (5) or (6).

[Formula 5]

[Formula 6]

According to the present invention, a novel resolbin has not been reported to date by using 8-lipoxygenase from Mus musculus mouse and 15-lipoxygenase from Burkholderia thailandensis strains. D2 analogs can be produced.

In addition, according to the present invention, since resolbin D2 analogues can be produced 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.

Resolbin D2 analogues prepared according to the present invention are signaling agents and are expected to be involved in various bioactive functions in animals including humans.

Figure 1 shows the synthetic pathway of the two types of resolbin D2 analogues produced using docosahexaenoic acid as a substrate using 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase of the present invention. .
Figure 2a is an HPLC chromatogram confirming the production of resolbin D2 analog-1, Figure 2b is an HPLC chromatogram confirming the production of resolbin D2 analog-2, Figure 2c shows the degree of synthesis of resolbin D2 analogs over time The graph shown.
FIG. 3A illustrates a material identification using LC-MS / MS for material identification of resolbin D2 analog-1. FIG. 3B shows a material identification result of resolbin D2 analog-2.
Figure 4 is an epoxide derived from 8-lipoxygenase from Mus musculus , 15-lipoxygenase from Burkholderia thailandensis and Myxococcus xanthus from the present invention . The recombinant vector synthesize | combined side hydrolase is shown respectively.

Hereinafter, the present invention will be described in detail.

The inventors have carried out ongoing studies to produce resolbin analogs more effectively through bioconversion processes. Specifically, the present inventors have found that non-mousse School Ruth (Mus musculus) mouse-derived 8-Lipoxygenase, member kolde Syrian den tailran sheath (Burkholderia thailandensis) derived from strain 15-Lipoxygenase and mix Socorro kusu glass tooth (Myxococcus xanthus) strains Cloning the derived epoxide hydrolase to produce a recombinant expression vector and a microorganism transformed therefrom, using the same to produce whole cells, and then treating the substrate with 10, 17-dihydric fatty acids and two kinds of It was found that resolvin D2 analogs can be produced with high productivity and high yield, and the present invention was completed.

As used herein, the term “10-hydroxylated fatty acid” means that one hydroxyl group or peroxide group is substituted for a fatty acid, and preferably has a hydroxyl group or a peroxide group on the 10th carbon of an unsaturated fatty acid having 22 carbon atoms. , 10-hydroxydoxahexaenoic acid (8-hydroxydocosahexaenoic acid) is preferred, but is not limited thereto.

In the present specification, "17-hydroxy fatty acid" preferably has a hydroxyl group or a peroxide group on the 157th carbon of an unsaturated fatty acid having 20 carbon atoms, and more specifically, 17-hydroxydocosahexaenoic acid is preferable. However, the present invention is not limited thereto.

As used herein, "10,17-dihydroperoxydocosahexaenoic acid" refers to 10-hydroxyl, 17-dihydroperoxide docosahexaenoic acid (10,17 (P) -DiHETE, 10-hydroxy & 17-hydroperoxydocosahexaenoic acid Or 10-peroxide, 17-docosahexaenoic acid, and as a novel compound, represented by the following Chemical Formulas 1 and 2, respectively, in order:

[Formula 1]

[Formula 2]

As used herein, “resolvin D analogues (RVs) are either resolvin D2 analogue-1 (10,16,17-trihydroxydocosahexaenoic acid) or resolvin D2 analogue-2 (resolvin D analogues-). 2, 10,11,17-trihydroxydocosahexaenoic acid), and as new compounds, are represented by the following general formulas (3) and (4), respectively, in order.

[Formula 3]

[Formula 4]

The present invention is a mus musculus mouse-derived 8-lipoxygenase; And 15-lipoxygenase derived from Burkholderia thailandensis strain, as an active ingredient, provides a composition for preparing 10,17-dihydric fatty acid.

In addition, the present invention is a mus musculus mouse-derived 8-lipoxygenase; 15-lipoxygenase from Burkholderia thailandensis strains; And it provides a composition for the preparation of resolbin D2 analogs comprising an epoxide hydrolase derived from Myxococcus xanthus strain as an active ingredient.

In addition, the present invention provides a gene encoding 8-lipoxygenase derived from Mus musculus mice; And a gene encoding a 15-lipoxygenase derived from a Burkholderia thailandensis strain, as an active ingredient.

In addition, the present invention provides a gene encoding 8-lipoxygenase derived from Mus musculus mice; Gene encoding 15-lipoxygenase from Burkholderia thailandensis strain; And a gene encoding an epoxide hydrolase derived from Myxococcus xanthus strain, as an active ingredient.

In one embodiment, the 8-lipoxygenase derived from Mus musculus mouse is composed of the amino acid sequence of SEQ ID NO: 1; The 15-lipoxygenase derived from the strain Burkholderia thailandensis ( Burkholderia thailandensis ) is preferably composed of the amino acid sequence of SEQ ID NO: 3, but one or more substitutions, deletions, additions, etc. to the sequence to achieve the desired effect All mutant enzymes that can be achieved are within the scope of this 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. It means to include all the mutants to achieve the object of the present invention, 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 invention, the composition is an unsaturated fatty acid having 20 to 22 carbon atoms as a substrate, in particular, at least one cis, cis-1,4 pentadiene and an unsaturated fatty acid having 22 carbon atoms, preferably docosa It may be for treatment with a substrate comprising hexanoic acid (docosahexaenoic acid, DHA).

In another aspect, the present invention provides a mus musculus mouse-derived 8-lipoxygenase; 15-lipoxygenase from Burkholderia thailandensis strains; Epoxide hydrolase from Myxococcus xanthus strains; And reacting the substrate to produce resolvin D2 analogues by bioconversion.

In one embodiment, the 8-lipoxygenase derived from Mus musculus mouse is composed of the amino acid sequence of SEQ ID NO: 1; The 15-lipoxygenase derived from the strain Burkholderia thailandensis ( Burkholderia thailandensis ) is preferably composed of the amino acid sequence of SEQ ID NO: 3, but one or more substitutions, deletions, additions, etc. to the sequence to achieve the desired effect All mutant enzymes that can be achieved are within the scope of this 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 functional equivalents thereof, that is, one or more substitutions, deletions, etc. mutations in the sequence. It means to include all the mutants to achieve the object of the present invention, 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 docosahexaenoic acid (docosahexaenoic acid, DHA), the method is more preferably using docosahexaenoic acid in the range of 0.1 mM to 3 mM as a substrate. Preferred but not limited to this.

In an embodiment of the present invention, the method is preferably carried out the reaction in the pH 6.5 to 7.5 range, the method is preferably carried out in the temperature range 10 ℃ to 30 ℃, but is not limited thereto.

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

According to an embodiment of the present invention, the present invention provides a gene encoding 8-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 2; A gene encoding 15-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 4; And a recombinant expression vector for preparing resolvin D2 analogue comprising a gene encoding an epoxide hydrolase consisting of the amino acid sequence of SEQ ID NO: 6, and a transformant transformed with the recombinant expression vector in a host cell. To provide.

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

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

Specifically, the whole cell containing the 8-lipoxygenase is a gene encoding the amino acid sequence represented by SEQ ID NO: 1; Or it is preferable to culture the transformant transformed with a recombinant expression vector comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 2, obtain and use it. In addition, whole cells including the 15-lipoxygenase may include a gene encoding an amino acid sequence set forth in SEQ ID NO: 3; Or it is preferable to culture the transformant transformed with a recombinant expression vector comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 4, obtain and use it. In addition, the whole cell containing the epoxide hydrolase is a gene encoding the amino acid sequence described in SEQ ID NO: 4; Or it is preferable to culture the transformant transformed with a recombinant expression vector comprising a gene consisting of the nucleotide sequence of SEQ ID NO: 6, obtain and use it.

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. Specifically, pET-28a (+) or pACYC duet plasmid is more preferably used, but 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 a desired gene and produce an active enzyme protein, and specifically, E. coli ER 2566 strain may be used. However, it is not limited thereto.

In addition, the present invention (1) to produce a recombinant expression vector comprising 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase gene; (2) culturing the microorganism transformed with the recombinant expression vector; (3) inducing the expression of 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase genes, and (4) providing a recombinant protein expressed and a method for producing a strain comprising the same.

Whole cells comprising the 8-, 15-lipoxygenase and / or epoxide hydrolase is i) recovering the primary whole cells by centrifuging the culture of the microorganism; ii) washing the recovered whole cells with saline solution; iii) centrifuging the washed whole cells to remove the supernatant and obtaining whole cells; And iv) washing the second-recovered whole cells with physiological saline once again. Specifically, the recovery of whole cells in step i) can be performed in the range of about 13,000 g using a device known in the art such as a centrifuge, and in step ii) washing of the whole cells with 0.9% or less sodium chloride solution. It is suitable to carry out.

The present invention provides a production method for synthesizing 10,17-dihydroxide fatty acid and resolbin D2 analogues using cultured cells comprising 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase.

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

In addition, it is preferable to use fatty acid as a substrate, and more preferably, docosahexaenoic acid (DHA). In addition, the concentration of the substrate is preferably in a concentration of 0.5 mM to 5 mM, more preferably around 1 mM.

In addition, the enzyme reaction is preferably made in the range of pH 6.5 to pH 7.5, more preferably in the range of pH 7.0. In order to maintain these pH conditions, HEPES buffer solution may be used as the reaction solvent.

In addition, the enzyme reaction is preferably made in the temperature range of 10 ° C to 30 ° C, more preferably in the temperature range of about 25 ° C. By maintaining these conditions, it is possible to enhance the 10,17-dihydroxylated fatty acid and resolbin D2 analogue production activity.

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 provides a concentration of 10 g / cell with lipoxygenase and epoxide hydrolase, enzymes that produce 10,17-dihydric fatty acid and resolbin D2 analogs from unsaturated fatty acids. It is preferred to treat in the range of 1 to 50 g / L, but is not limited thereto.

As described above, according to the present invention, mus musculus mice-derived 8-lipoxygenase; And 15-lipoxygenase derived from Burkholderia thailandensis strain, the new resolbin D2 analogues can be produced with high productivity and high yield, which is useful in various industries such as medicine, food and cosmetics. It is expected to be able to be used.

Resolbin D2 analogues prepared according to the present invention, as signaling agents, are expected to be involved in various bioactive functions in animals, including humans.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example 1 Construction of Recombinant Expression Vectors and Transgenic Microorganisms for Expression of 8-lipoxygenase, 15-lipoxygenase or Epoxide Hydrolase

In the present invention, to prepare 8-lipoxygenase, 15-lipoxygenase or epoxide hydrolase, mouse, Burkholderia thailandensis strain and Myxococcus xanthus strain Genes encoding 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase, respectively, were isolated from the genes, and recombinant expression vectors were constructed to overexpress them.

Specifically, the gene base sequence and amino acid sequence are already specified in the Korea Microorganism 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. Polymerase chain reaction was performed by designing primers based on DNA sequences of 8-lipoxygenase, 15-lipoxygenase or epoxide hydrolase, respectively. (PCR) was performed. Nde I and XhoI were used as restriction enzymes of each gene. The cloning method was performed using Gibson assembly method.

Primers of mouse-derived 8-lipoxygenase sequence were 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), the primer of 15-lipoxygenase sequence from Burkholderia thailandensis was 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) Primers of the epoxide hydrolase sequence derived from Myxococcus xanthus were 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 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 ligated using isothermal buffer to prepare recombinant 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase. It was.

Then, the recombinant expression vector obtained as described above is transformed into E. coli ER 2566 strain purchased from New England Biolabs (Hertfordshire, UK) by a conventional transformation method, the transformed microorganism is a 20% glycerin (glycerine) solution Addition was incubated for prototyping of 10-hydroxyfatty acid, 17-hydroxyfatty acid and resolbin D2 analogs and then stored frozen at -70 ° C prior to use.

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

For protein expression of enzymes, the transformed microorganisms frozen in (1) were aspirated 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 the conditions. When the absorbance of bacteria reached 0.6 to 0.8 at 600 nm, the final concentration of 0.1 mM IPTG was added to induce protein expression of the enzymes, and the culture was incubated with stirring at 16 rev for 150 hours at 16 ° C. .

E. coli cells comprising cultured 8-lipoxygenase, 15-lipoxygenase or epoxide hydrolase were collected and used. In addition, 8-lipoxygenase, 15-lipoxygenase, or epoxide hydrolase produced by overexpression as described above is 0.85% sodium chloride by centrifuging the culture of the transformed strain at 6,000xg for 30 minutes at 4 ℃ Washed twice with (NaCl) and then used as recombinant cells to produce 10,17-dihydrohydroxyfatty acid and two resolbin D2 analogs.

Example 3 Synthesis of Resolbin D2 Analogue from Docosahexaenoic Acid Using 8-lipoxygenase, 15-lipoxygenase and Epoxide Hydrolase

Docosahexaenoic acid (DHA) was used as a substrate to construct a synthetic pathway of resolbin D2 analogue using the 8-lipoxygenase, 15-lipoxygenase and epoxide hydrolase, and the resolbin D2 analogue- 1 Synthetic route was prepared by using 10-hydroxyl docosahexaenoic acid from 8-lipoxygenase, and 17-hydroxyl docosahexaenoic acid from 15-lipoxygenase for the synthetic route of resolbin D2 analog-2. Was prepared and used. Resolbin D2 analog-1 used 40 g / L recombinant 8-lipoxygenase from 1 mM 10-hydroxy docosahexaenoic acid, and resolbin D2 analog-2 was 1 mM 17-hydroxy docosahexa. 40 g / L of recombinant 8-lipoxygenase from enoic acid was used, and both reactions were performed with whole cell reaction for 1 hour at pH 7.0, 25 ° C with epoxide hydrolase.

As a result, a synthetic route was constructed as shown in FIG. Two kinds of resolbin D2 analogs converted at the reaction conditions presented above were identified using HPLC (FIG. 2). In the case of material identification, it was confirmed that it was a resolbin D2 analogue through LC-MS / MS (FIG. 3).

As described above, the present invention relates to 8-lipoxygenase derived from mouse, 15-lipoxygenase derived from Burkholderia thailandensis and epoxide hydrolase derived from Myxococcus xanthus . The present invention relates to a method for producing hydroxylated fatty acids that can act as lipid mediators in animals including humans and resolvin D2 analogs not reported so far by using bioconversion. Specifically, the substrate uses docosahexaenoic acid, and the epoxide derived from mouse-derived 8-lipoxygenase, 15-lipoxygenase from Burkholderia thailandensis , and Myxococcus xanthus. 10,17-dihydrohydroxyfatty acid and resolbin D2 analogs can be produced using biotransformation using transformed cells containing hydrolase, so that they are environmentally friendly compared to conventional chemical methods In addition, the production of lipid regulators, which have not been reported previously, and the specific production by bioconversion methods have great significance.

As mentioned above, specific portions of the contents of the present invention have been described in detail, and for those skilled in the art of fatty acids and the medical field, these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereto. The point will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp <120> Method for producing resolvin D2 analogues by combination          reaction using recombinant lipoxigenases and resolvin D2          analogues therefrom <130> 1064118 <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 (19)

8-lipoxygenase from Mus musculus mice; As a composition for preparing 10,17-dihydroperoxydocosahexaenoic acid comprising 15-lipoxygenase derived from Burkholderia thailandensis strain, as an active ingredient,
The 10,17-dihydric fatty acid is a composition represented by the following formula (1) or (2):
[Formula 1]

[Formula 2]

The method of claim 1,
The 8-lipoxygenase is composed of the amino acid sequence of SEQ ID NO: 1, wherein the 15-lipoxygenase is composed of the amino acid sequence of SEQ ID NO: 3.
The method of claim 1,
The composition further comprises a docosahexaenoic acid (dochahexaenoic acid, DHA) as a substrate.
8-lipoxygenase from Mus musculus mice;
15-lipoxygenase from Burkholderia thailandensis strains; And
As a composition for preparing resolvin D2 analogue comprising an epoxide hydrolase derived from Myxococcus xanthus strain as an active ingredient,
The resolbin D2 analogue is a composition represented by any one of the following formula (3) or (4):
[Formula 3]

[Formula 4]

The method of claim 4, wherein
Wherein 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 .
The method of claim 4, wherein
The composition further comprises a docosahexaenoic acid (dochahexaenoic acid, DHA) as a substrate.
A gene encoding 8-lipoxygenase from Mus musculus mouse; And a gene encoding 15-lipoxygenase derived from a strain of Burkholderia thailandensis , comprising 10,17-dihydric fatty acid as an active ingredient.
The 10,17-dihydric fatty acid is a composition represented by the following formula (1) or (2):
[Formula 1]

[Formula 2]

A gene encoding 8-lipoxygenase from Mus musculus mouse;
Gene encoding 15-lipoxygenase from Burkholderia thailandensis strain; And
As a composition for preparing a resolvin D2 analogue comprising a gene encoding an epoxide hydrolase derived from Myxococcus xanthus strain as an active ingredient,
The resolbin D2 analogue is a composition represented by the following formula (3) or formula (4):
[Formula 3]

[Formula 4]

8-lipoxygenase from Mus musculus mice;
15-lipoxygenase from Burkholderia thailandensis strains;
Epoxide hydrolase from Myxococcus xanthus strains; And reacting the substrate to produce a resolvin D2 analogue represented by the following formula (3) or (4) by bioconversion:
[Formula 3]

[Formula 4]

The method of claim 9,
The method is characterized in that the docosahexaenoic acid (dochahexaenoic acid, DHA) is used as a substrate in the range of 0.5 mM to 5 mM.
The method of claim 9,
The method is characterized in that the reaction is carried out in the pH 6.5 to 7.5 range.
The method of claim 9,
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; A gene encoding 15-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 4; And a recombinant expression vector for preparing a resolbin D2 analogue comprising a gene encoding an epoxide hydrolase consisting of the nucleotide sequence of SEQ ID NO: 6,
The resolbin D2 analogue is a recombinant expression vector represented by Formula 3 or Formula 4 below:
[Formula 3]

[Formula 4]

A transformant transformed with a recombinant expression vector according to claim 13 in a host cell.
A method of preparing a resolbin D2 analog by treating the substrate of claim 14 with a substrate.
The method of claim 15,
The concentration of the transformant is characterized in that the treatment in the range of 10 g / L to 50 g / L.
delete Rezobin D2 analogs represented by Formula 3 or Formula 4 below:
[Formula 3]

[Formula 4]

Compound represented by the following formula (5) or (6):
[Formula 5]

[Formula 6]

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