KR102190311B1 - Method for the production of the protectin DX by combination reaction of arachidonate mouse 8-lipoxygenase and microbial arachidonate 15-lipoxygenase - Google Patents

Abstract

In the present invention, protectin DX (Protectin DX) from docosahexaenoic acid (DHA), an unsaturated fatty acid It relates to a method of producing and a composition for producing Protectin DX. According to the present invention, since it is possible to manufacture Protectin DX with high productivity and high yield by an eco-friendly method that has not been reported to date, medicine, food And it can be usefully used in various industrial fields such as cosmetics, and protectin DX prepared according to the present invention is expected to be involved in various physiologically active functions in animals, including humans, as a signal transducer.

Description

Method for the production of the protectin DX by combination reaction of arachidonate mouse 8-lipoxygenase and microbial arachidonate 15-lipoxygenase}

The present invention relates to a method for preparing dihydroxy fatty acid by combining two lipoxygenases having different regiospecificities and a composition for preparing the same, and more particularly, to a composition for preparing the same, and more specifically, docosahexa, an animal unsaturated fatty acid. It relates to a method for preparing Protectin DX (Protectin DX) from docosahexaenoic acid (DHA) and a composition for preparing the same.

Hydroxylated fatty acids are substances that exist in nature, and exist in the lipids of various organisms such as animals, plants, insects, and microorganisms, including triglycerol, cerebroside, and wax, and are generally substances of nature that have a hydroxyl group. Since it has a hydroxyl group, it is used as a raw material industrially because of its excellent reactivity, and it is also used as a raw material for cosmetics due to the reduction of surface tension and an increase in anti-fungal activity due to the action of this hydroxyl group. In particular, in animals, hydroxylated fatty acids are used as precursors for signaling substances, and even a single substance is involved in various physiological activities. Lipid mediator, a type of signaling substance, is an important substance involved in various physiologically active functions such as immune response and homeostasis regulation in animals including humans. Among these substances, protectin is mainly lipoxygenase. It is converted by acting on 20 and 22 polyunsaturated fatty acids (C20 and C22).

Protectin D1 (PD1), which is generally known among the protectins, is an endogenous stereoselective lipid mediator, a substance derived from docosahexaenoic acid, an omega fatty acid, and has been mainly found in tissues such as the retina, lungs and nervous system. This substance plays the role of an anti-inflammatory, anticancer and neuroprotective agent. As a result of a study in stroke patients and Alzheimer's disease animal models, it was confirmed that treatment with this substance could potentially reduce inflammation caused by oxidative stress and prevent cell degeneration by inhibiting the induction of apoptosis. I did. In addition, it is known that it has the ability to inhibit the infectivity of avian influenza viruses such as H5N1 and relieve inflammation by weakening the replication ability of the influenza virus. Considering the efficacy confirmed that it does not induce harmful points to host RNA, which may cause harmful side effects in host cells, through the treatment of protectin, this material with strong anti-inflammatory ability in viral or bacterial inflammation is used as a biotechnology against viral infection. It suggests that it may play a role as a new antiviral agent as well as a marker. On the other hand, depending on the chirality of the hydroxyl group, protectin D1 (protectin D1, PD1) in the case of 10R, 17S-dihydroxydocosahexaenoic acid, 10S, 17S-protectin in the case of docosahexaenoic acid dihydroxylated by Protec. It is referred to as protectin DX (PDX). PDX, like PD1, is also an anti-inflammatory substance and inhibits the influx of circulating white blood cells into the peritoneum in an inflammatory mouse model. It also inhibits the formation of prostaglandins by inhibiting cyclooxygenase and blocks the accumulation of platelets. Protectin has the potential as a next-generation medical substance in place of chemically synthesized drugs with side effects, and can be used as a research substance in various disciplines such as medicine, biology, and biotechnology, but biological production methods have not yet been developed. .

Lipoxygenase (LOX) is a dioxygenase and catalyzes the reaction to synthesize oxidized fatty acids. Although it is an oxidative enzyme, it is characterized by not having heme, and instead is an enzyme that contains iron. Characteristically, a polyunsaturated fatty acid having one or more cis, cis-1,4-pentadiene is used as a substrate to catalyze stereospecificity and reaction specificity through dioxygenation. The site specificity is different depending on the type of polyunsaturated fatty acid used as a substrate. Among them, arachidonic acid 8-lipoxygenase (hereinafter referred to as 8-lipoxygenase) that generates a hydroxyl group at the 8th position of arikidonic acid of animal polyunsaturated fatty acids. In the case of zero), specifically, a hydroxyl group is formed only at the 8th carbon position in an unsaturated fatty acid having 20 or more carbon atoms such as arachidonic acid. In addition, this enzyme forms a hydroxyl group at the 10th carbon position in an unsaturated fatty acid having 22 or more carbon atoms. In the case of 8-lipoxygenase, it has not been found in animals other than mice until now, and it has recently been reported in marine organisms, corals. Mouse-derived 8-lipoxygenase specifically undergoes a double dioxide process.After forming 8-hydroxylated arachidonic acid from arachidonic acid, it forms a hydroxyl group at carbon position 15 to form 8,15-dihydroxylated arachidonic acid. . As an additional enzyme to be used in the biosynthesis of protectin, arachidonic acid 15-lipoxygenase (hereinafter referred to as 15-lipoxygenase) forms a hydroxyl group only at the 15th carbon position in an unsaturated fatty acid having 20 or more carbon atoms such as arachidonic acid. . In addition, in an unsaturated fatty acid having 22 or more carbon atoms, a hydroxyl group is formed at the 17th carbon position.

As a synthesis method of protectin reported so far, an 8-step convergent stereoselective chemical synthesis method has been reported and is known to be synthesized with a yield of 15%. This process is a method of synthesis using organic solvents, heavy metals such as palladium, and hydrogenation reactions, and contaminants and by-products produced in the chemical synthesis process are not biodegradable in nature and thus cause environmental pollution.

In the present invention, a corresponding pathway was constructed to biosynthesize protectin by using a site-specific lipoxygenase alone or in combination, and its conversion was confirmed. Until now, a method of preparing a protectin from docosahexaenoic acid by using 8-lipoxygenase (8-LOX) alone or in combination with 8-lipoxygenase and 15-lipoxygenase has been reported. There is no bar.

The present invention was conceived by the necessity of the above, and an object of the present invention is a method capable of producing Protectin DX in high yield through a combination reaction of mouse-derived and microorganism-derived lipoxygenase, and for the production of protectin DX It is intended to provide a composition.

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

The present invention for achieving the above object is Mus musculus ( Mus musculus ) 8-lipoxygenase derived from mouse; It provides a composition for producing a protectin DX (protectin DX, PDX) comprising a 15-lipoxygenase derived from a strain of Burkholderia thailandensis and Burkholderia thailandensis as an active ingredient.

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 docosahexaenoic acid (DHA) 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, it provides a composition for producing Protectin DX.

In another aspect, the present invention provides a Mus musculus mouse-derived 8-lipoxygenase; And Burkholderia tylandensis ( Burkholderia thailandensis ) 15-lipoxygenase derived from strain; by treating a substrate to provide a method for producing a protectin DX by biotransformation.

The substrate may be docosahexaenoic acid (DHA).

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

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 can be carried out at a temperature ranging from 10°C to 30°C.

The method can treat the concentration of cells containing lipoxygenase in the range of 10 g/L to 50 g/L.

According to the present invention, a recombinant expression vector including 8-lipoxygenase and 15-lipoxygenase genes, a method for producing protectin DX using the expression vector and strain, and a composition for producing protectin DX can be provided. have.

According to the present invention, since protectin DX can be manufactured with high productivity and high yield by an eco-friendly method not reported so far, it can be usefully used in various industrial fields such as medicine, food and cosmetics.

Protectin DX prepared according to the present invention is a signal transducer and is expected to be involved in various physiologically active functions in animals including humans.

Figure 1 shows the synthesis pathway of Protectin DX produced by using docosahexaenoic acid as a substrate using 8-lipoxygenase and 15-lipoxygenase of the present invention.
2 is an HPLC chromatogram confirming the production of Protectin DX.
Figure 3 is a diagram showing the progress of substance identification of Protectin DX using LC-MS/MS.
Figure 4 is a single reaction of 8-lipoxygenase-expressed recombinant E. coli of the present invention, the addition of 15-lipoxygenase-expressed recombinant E. This is a diagram confirming the amount of protectin DX produced when 8-lipoxygenase-expressing recombinant E. coli is added after reaction by lipoxygenase-expressing recombinant E. coli.
Figure 5 is a diagram showing the amount of time-dependent protectin DX production of 15-lipoxygenase-expressed recombinant E. coli after reaction with 8-lipoxygenase-expressed recombinant E. coli.
FIG. 6 shows the conversion of protectin DX by time after purifying 10-hydroxy docosahexaenoic acid produced by 8-lipoxygenase from docosahexaenoic acid and then treating the purified substrate with 15-lipoxygenase. This is the degree confirmed by.
Figure 7 shows a recombinant vector in which the mouse-derived 8-lipoxygenase and the 15-lipoxygenase derived from Bercholderia tylandensis of the present invention were synthesized, respectively.

Hereinafter, the present invention will be described in detail.

The present inventors conducted continuous research to more effectively produce 8-hydroxylated fatty acids, 15-hydroxylated fatty acids or lipoxin analogs through a bioconversion process. As a result, 8-lipoxygenase and burr derived from Mus musculus mice By cloning a 15-lipoxygenase derived from Burkholderia thailandensis strain, a recombinant expression vector and a transformed microorganism therefrom are produced, and whole cells are produced using this, and then the whole cell is produced by treating it on a substrate, thereby making it eco-friendly. As a new production route as a bioconversion method, 10-hydroxydocosahexaenoic acid, 10-hydroxy-4,7,11,13,16,19 ( Z , Z , E , Z,Z , Z ) -Docosahexaenoic acid), 17-hydroxydocosahexaenoic acid, 17-hydroxy-4,7,10,13,15,19 ( Z , Z , Z , Z , E,Z )- Docosahexaenoic acid) and Protectin DX (PDX) were confirmed to be able to be prepared with high productivity and high yield, and the present invention was completed.

Thus, the present invention Mus musculus ( Mus musculus ) mouse derived 8-lipoxygenase; It provides a composition for the production of Protectin DX (Protectin DX) comprising a; and Burkolderia tyrandensesis ( Burkholderia thailandensis ) strain derived 15-lipoxygenase; as an active ingredient.

In addition, the present invention is Mus musculus ( Mus musculus ) gene encoding 8-lipoxygenase derived from mouse; It provides a composition for producing a protectin DX (Protectin DX) comprising a; and a gene encoding the 15-lipoxygenase derived from Burkholderia thailandensis strains.

In the present specification, protectin DX (PDX) is a compound represented by the following Formula 1, 10S,17S-dihydroxy-4Z,7Z,11E,13Z,15E,19Z-docosahexaenoic acid (docosahexaenoic acid), also referred to as 10S,17S-dihydroxydocosahexaenoic acid (10,17-dihydroxydocosahexaenoic acid). As described above, Protectin DX can be used as a research material for various disciplines such as medicine, biology, and biotechnology, but biological production methods have not been developed yet.

[Formula 1]

In one embodiment of the present invention, the 8-lipoxygenase derived from the Mus musculus mouse consists of the amino acid sequence of SEQ ID NO: 1, the Burkholderia tylandensis ( Burkholderia thailandensis ) strain derived 15 -Lipoxygenase is preferably composed of the amino acid sequence of SEQ ID NO: 3, but all mutant enzymes capable of achieving the desired effect of the present invention by one or more substitutions, deletions, additions, etc. to the sequence are included in the scope of the present invention. do. 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 one embodiment of the present invention, the composition is an unsaturated fatty acid having 22 carbon atoms as a substrate, particularly, one or more cis, cis-1,4 pentadiene, and an unsaturated fatty acid having 20 to 22 carbon atoms, preferably docosa. It may be for treatment on a substrate containing hexaenoic acid (DHA).

In another aspect, the present invention provides a Mus musculus mouse derived 8-lipoxygenase; And Burkholderia tyrandensis ( Burkholderia thailandensis ) strain derived 15-lipoxygenase; It provides a method of producing a protectin DX (Protectin DX) by treating a substrate.

In one embodiment of the present invention, the 8-lipoxygenase derived from the Mus musculus mouse consists of the amino acid sequence of SEQ ID NO: 1, the Burkholderia tylandensis ( Burkholderia thailandensis ) strain derived 15 -Lipoxygenase is preferably composed of the amino acid sequence of SEQ ID NO: 3, but all mutant enzymes capable of achieving the desired effect of the present invention by one or more substitutions, deletions, additions, etc. to the sequence are included in the scope of the present invention. do. 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 another embodiment of the present invention, the substrate is preferably docosahexaenoic acid, and the method uses docosahexaenoic acid as a substrate in the range of 0.5 mM to 5 mM. It is more preferable, but is not limited thereto.

In a preferred 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 and 15-lipoxygenase, respectively, and an expression vector comprising all of the above enzymes.

According to one embodiment of the present invention, the present invention is a protec comprising a gene encoding an 8-lipoxygenase consisting of a nucleotide sequence of SEQ ID NO: 2 and a gene encoding a 15-lipoxygenase consisting of a nucleotide sequence of SEQ ID NO: 4 A recombinant expression vector for producing Protectin DX, and a transformant transformed with the recombinant expression vector are provided to host cells.

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.

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.

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 use pET-28a(+) or pACYC duet plasmid specifically, 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, and 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 and 15-lipoxygenase and genes; (2) culturing the microorganism transformed with the recombinant expression vector; (3) Inducing the expression of 8-lipoxygenase and 15-fatty acid enzyme genes (4) The recombinant protein expressed and a method for producing a strain including the same are provided.

The whole cells containing the 8-lipoxygenase and/or 15-lipoxygenase are i) centrifuging the culture medium of the microorganism to recover the primary whole cells; 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 a sodium chloride solution of 0.9% or less. It is appropriate to perform.

The present invention provides a method for producing Protectin DX using cells containing cultured 8-lipoxygenase and 15-lipoxygenase.

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

Further, it is preferable to use a fatty acid as a substrate, more preferably docosahexaenoic 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 carried out in the range of pH 6.5 to 7.5, more preferably in the range of 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 8-hydroxylated fatty acid, 15-hydroxylated fatty acid, and protectin DX 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 is preferable to treat the concentration of cells with lipoxygenase, an enzyme that produces hydroxylated fatty acids and protectin DX from unsaturated fatty acids, in the range of 10 g/L to 50 g/L. However, it 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, Protectin DX is more productive than those obtained by chemical synthesis using conventional heavy metals and organic solvents. Since it can be manufactured in high yield without producing, it is expected to be useful in various industrial fields such as medicine, food and cosmetics.

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 expression of 8-lipoxygenase or 15-lipoxygenase

In order to prepare the 8-lipoxygenase or 15-lipoxygenase of the present invention, From the genes of Mus musculus mouse and Burkholderia thailandensis strain, respectively, the genes encoding 8-lipoxygenase and 15-lipoxygenase were first isolated, and recombination to overexpress them An expression vector was constructed.

Specifically, the gene sequence and amino acid sequence are already specified at the Korea Microbial Conservation Center (Seoul) Polymerase based on the DNA sequence of 8-lipoxygenase or 15-lipoxygenase derived from purchased and selected Mus musculus and Burkholderia thailandensis In order to perform the chain reaction (PCR), mouse cDNA or genomic DNA of Burkholderia thailandensis was extracted, and this was used as a template for PCR, and DNA of 8-lipoxygenase or 15-lipoxygenase Polymerase chain reaction (PCR) was performed by devising each primer based on the nucleotide sequence. Nde I and XhoI were used as restriction enzymes for each gene, and 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: 5), R: ATC TCA GTG GTG GTG GTG GTG GTG CTC GAG TTA GAT GGA GAC ACT GTT CTC AAT GAG GGG (SEQ ID NO: 6), 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: 7), R: ATC TCA GTG GTG GTG GTG GTG GTG CTC GAG TCA AAT GTT CGT GCT TGC CGG AAT CCG GCT (SEQ ID NO: 8). 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 and 15-lipoxygenase.

Thereafter, 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 culturing for the production of 10-hydroxylated fatty acid, 17-hydroxylated fatty acid and protectin by addition, it was stored frozen at -70°C before use.

Example 2. Preparation of 8-lipoxygenase or 15-lipoxygenase

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 or 15-lipoxygenase were collected and used. In addition, 8-lipoxygenase or 15-lipoxygenase produced by overexpressing as described above is centrifuged at 6,000xg for 30 minutes at 4°C for 30 minutes with 0.85% sodium chloride (NaCl). After washing, it was used as a recombinant cell to produce 10-hydroxylated fatty acid, 17-hydroxylated fatty acid and protectin.

Example 3. Construction of a synthetic pathway of protectin from docosahexaenoic acid using 8-lipoxygenase and 15-lipoxygenase

Docosahexaenoic acid was used as a substrate in order to establish a pathway for synthesizing protectin using the 8-lipoxygenase and 15-lipoxygenase. As for the protectin, 30 g/L of recombinant 8-lipoxygenase and 15-lipoxygenase were used from 1 mM docosahexaenoic acid, respectively, and whole cell reaction at pH 7.0 and 25°C for 1 hour Was carried out.

As a result, a synthetic route as shown in Fig. 1 was constructed. In addition, it was confirmed by HPLC whether or not protectin was generated (FIG. 2), and it was confirmed that it was protectin through LS-MS (FIG. 3), and 10S, 17S-dihydroxy-4Z, through NMR analysis, 7Z, 11E, 13Z, 15E, 19Z-docosahexaenoic acid (docosahexaenoic acid) was identified and named as Protectin DX (PDX) (Chemical Formula 1, Table 1).

[Formula 1]

C No. ^One H (δ) multiplet J (Hz) Protons ¹³ C (δ) One 2 2.39 t 2H 33.9 3 2.31 m 2H 23.1 4 3.38-4.41 m 1H 128.3 5 3.38-4.41 m 1H 129.0 6 5.64 t 6.85 2H 24.7 7 6.52-6.81 m 1H 130.2 8 7.18 m 1H 124.9 9 2.49 d 15.01, 6.85 2H 35.3 10 3.04 dd 14.01, 5.88 1H 72.1 11 8.19 dd 15.01, 5.82 1H 137.3 12 3.29-3.39 m 1H 124.1 13 5.61-5.72 m 1H 128.9 14 5.62 t 10.23 1H 128.9 15 4.40 dd 15.29, 10.23 1H 125.1 16 1.20 dd 15.29, 5.94 1H 135.1 17 1.29 dt 5.94, 6.72 1H 73.1 18 2.11-2.30 m 2H 34.9 19 3.29-3.44 m 1H 125.4 20 3.29-3.44 m 1H 133.7 21 1.98 dt 7.49, 7.11 2H 22.5 22 0.84 t 3.97 3H 11.1

Example 4. Synthesis of Protectin DX using a single whole-cell reaction of 8-lipoxygenase and a combination reaction of two kinds of recombinant enzymes of 8-lipoxygenase and 15-lipoxygenase

Addition of 15-lipoxygenase-expressing recombinant E. coli after reaction with 8-lipoxygenase-expressed recombinant E. coli and 8-lipoxygenase-expressed recombinant E. coli, 15 -After the reaction by the lipoxygenase-expressing recombinant E. coli, the amount of protectin DX produced when the 8-lipoxygenase-expressing recombinant E. coli was added was confirmed. A whole cell reaction was carried out for 1 hour at a pH of 7.0 and a temperature of 25° C. for 1 mM docosahexaenoic acid as a substrate, and the degree of production of protectin for each reaction catalyst was confirmed.

As a result, as shown in [Fig. 4], it was confirmed that about 300 μM of Protectin DX was produced by a single reaction in which 8-lipoxygenase was expressed, and the reaction by the recombinant E. coli 8-lipoxygenase was expressed. Then, by the combination reaction with the addition of 15-lipoxygenase-expressing recombinant E. coli, about 400 μM of Protectin DX was produced, confirming the production amount increased by about 1.3 times than that of a single reaction. When combined, it was found that the reactivity was improved. The results showing the production of protectin DX over time under the same conditions are shown in FIG. 5.

Next, referring to the fact that the reactivity was better when the two enzymes were combined, 10-hydroxy docosahexaenoic acid produced by 8-lipoxygenase from docosahexaenoic acid was purified, and then the purified substrate was used. Conversion of Protectin DX was confirmed by treatment with 15-lipoxygenase. The whole cell reaction was carried out with 2.5 mM 10-hydroxy docosahexaenoic acid at pH 7.5 and at 30° C. for 30 minutes. As a result, as shown in [Fig. 6], it was confirmed that about 1.9 mM of Protectin DX was produced.

As described above, the present invention uses 8-lipoxygenase derived from Mus musculus mouse and 15-lipoxygenase derived from Burkholderia thailandensis strain in animals including humans. It relates to a method of producing a hydroxylated fatty acid and protectin DX, which can act as a lipid mediator, by bioconversion. Specifically, the substrate used docosahexaenoic acid and transformed cells containing 8-lipoxygenase derived from Mus musculus and 15-lipoxygenase derived from Burkholderia thailandensis . Using a bioconversion method, specifically 10-hydroxylated fatty acids, 17-hydroxylated fatty acids, and protectin DX can be produced using bioconversion methods, thus overcoming environmentally friendly conditions compared to conventional chemical methods, and not previously reported. The production of lipid modulators and specific production by biotransformation have great significance.

As described above, specific parts of the present invention have been described in detail, and for those of ordinary skill in the fatty acid industry and pharmacy, 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 the production of the protectin DX by combination reaction of arachidonate mouse 8-lipoxygenase and microbial arachidonate 15-lipoxygenase <130> 1065359 <150> KR 2018/0090890 <151> 2018-08-03 <160> 8 <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> 57 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 agcggcctgg tgccgcgcgg cagccatatg gcgaaatgca gggtgagagt atccacg 57 <210> 6 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 atctcagtgg tggtggtggt ggtgctcgag ttagatggag acactgttct caatgagggg 60 60 <210> 7 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 agcggcctgg tgccgcgcgg cagccatatg gtcaatcaca aaaccgggtc aaatatg 57 <210> 8 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 atctcagtgg tggtggtggt ggtgctcgag tcaaatgttc gtgcttgccg gaatccggct 60 60

Claims (10)

8-lipoxygenase from Mus musculus mice; And
Including 15-lipoxygenase derived from Burkholderia thailandensis strain as an active ingredient,
Docosahexaenoic acid (docosahexaenoic acid) further comprising as a substrate, Protectin DX (Protectin DX, PDX) composition for production.
The composition of claim 1, wherein the 8-lipoxygenase consists of the amino acid sequence of SEQ ID NO: 1, and the 15-lipoxygenase consists of the amino acid sequence of SEQ ID NO: 3.
delete A gene encoding an 8-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 2 derived from Mus musculus mouse; And
Including, as an active ingredient, a gene encoding 15-lipoxygenase consisting of the nucleotide sequence of SEQ ID NO: 4 derived from Burkholderia thailandensis strain,
Docosahexaenoic acid (docosahexaenoic acid) further comprising as a substrate, Protectin DX (Protectin DX, PDX) composition for production.
8-lipoxygenase from Mus musculus mice; And
Burkholderia tylandensis ( Burkholderia thailandensis ) strain derived 15-lipoxygenase; a method for producing a protectin DX (Protectin DX, PDX) by bioconversion by treating with docosahexaenoic acid as a substrate.
delete The method of claim 5,
The method, characterized in that the use of docosahexaenoic acid in the range of 0.5 mM to 5 mM as a substrate.
The method of claim 5,
The method is characterized in that the reaction is carried out in the range of pH 6.5 to 7.5.
The method of claim 5,
The method is characterized in that the reaction is carried out at a temperature in the range of 10 ℃ to 30 ℃.
The method of claim 5,
The method, characterized in that the treatment of the concentration of cells containing lipoxygenase in the range of 10 g / L to 50 g / L.

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