KR102402601B1 - Composition and method for producing hydroxy fatty acid or dihydroxy fatty acid using 9-lipoxygenase or variant thereof - Google Patents

Abstract

The present invention is to prepare a novel hydroxylated fatty acid or dihydroxylated fatty acid through a bioconversion process using a novel microorganism-derived 9-lipoxygenase, Sphingomonas macrogoltabidus ( Sphingomonas macrogoltabidus ) derived 9-lipoxygenase; Or from the imanoic acid sequence of the 9-lipoxygenase, a 9-lipoxygenase variant in which valine (V), which is the 569th amino acid, is substituted with phenylalanine (F) as an active ingredient, a composition for preparing hydroxylated fatty acid or dihydroxylated fatty acid; It relates to a manufacturing method.

Description

Composition for preparing hydroxylated fatty acid or dihydroxylated fatty acid using 9-lipoxygenase or a variant thereof, and a production method using the same

The present invention is Sphingomonas macrogoltabidus ( Sphingomonas macrogoltabidus ) derived from 9-lipoxygenase; Or the 9-lipoxygenase is directed to a composition and method for preparing a hydroxylated fatty acid or dihydroxylated fatty acid using a modified 9-lipoxygenase variant (V569F) by site-directed mutation.

Hydroxated fatty acids are substances present in the lipids of various living organisms in the natural world, such as animals, plants, insects, and microorganisms, and generally have hydroxyl groups in fatty acids. Hydroxated fatty acids are used as raw materials industrially because of their excellent reactivity by hydroxyl groups, and are used as raw materials for cosmetics because of their high antibacterial and antifungal activity and reduction of surface tension due to the action of hydroxyl groups. Among various living things, especially the hydroxylated fatty acids found in humans, are used as precursors of signal transduction substances in the human body, and are involved in various physiological activities with only a single substance. Lipid mediator, a kind of human signal transduction material, is an important substance involved in various physiological functions such as homeostasis regulation and immune response in mammals including humans.

Lipoxygenase (LOX) is a dioxygenase, and although it is an oxidase enzyme, it does not contain heme and contains iron. Converted and converted peroxidized fatty acid is a multifunctional enzyme that naturally catalyzes one oxygenated hydroxylated fatty acid. Characteristically, it catalyzes stereospecificity and reaction specificity through dioxygenation by using only polyunsaturated fatty acids having a large number of cis, cis-1,4-pentadiene as substrates. The site specificity is different depending on the type of polyunsaturated fatty acid used as a substrate, and it is named as a site-specific oxidase depending on the position where it is oxidized to arichidonic acid, an unsaturated fatty acid having 20 carbon atoms. 5-, 8-, 11-, 12-, 15-lipoxygenases exist. Among them, 9-lipoxygenase, which produces a hydroxyl group at the 9th position of animal polyunsaturated fatty acids, specifically forms a hydroxyl group at the 9th position in unsaturated fatty acids having 20 or more carbon atoms, such as arachidonic acid. . In addition, this enzyme forms a hydroxyl group at the 11th carbon position in unsaturated fatty acids having 22 or more carbon atoms. 9-Lipoxygenase has not been found in animals, including humans, and 9-hydroxyarachidonic acid has been reported in marine organisms such as red algae, but the identification and biochemical properties of 9-lipoxygenase have not been reported. none.

The present invention is to prepare a novel hydroxylated fatty acid or dihydroxylated fatty acid through a bioconversion process using a novel microorganism-derived 9-lipoxygenase, Sphingomonas macrogoltabidus ( Sphingomonas macrogoltabidus ) derived 9-lipoxygenase; Or from the imanoic acid sequence of 9-lipoxygenase, a 9-lipoxygenase variant in which valine (V), the 569th amino acid, is substituted with phenylalanine (F) as an active ingredient, a hydroxylated fatty acid or a composition for preparing dihydroxylated fatty acid, etc. is to provide

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

The present invention is Sphingomonas macrogoltabidus ( Sphingomonas macrogoltabidus ) derived from 9-lipoxygenase; Or, from the imanoic acid sequence of the 9-lipoxygenase, a 9-lipoxygenase variant in which valine (V), which is the 569th amino acid, is substituted with phenylalanine (F) as an active ingredient. , The hydroxylated fatty acid or dihydroxylated fatty acid is 9-hydroxyarachidonic acid, 9,15-dihydroxyarachidonic acid, 9-hydroxyeicosapentaenoic acid acid), 9,15-dihydroxyeicopentaenoic acid, 11-hydroxydocosapentaenoic acid, 11,17-dihydroxyeicosapentaenoic acid (11 , 17-dihydroxydocosapentaenoic acid), 11-hydroxydocosahexaenoic acid (11-hydroxydocosahexaenoic acid) and 11,17-dihydroxydocosahexaenoic acid (11,17-dihydroxydocosahexaenoic acid) at least one selected from the group consisting of, hydroxylated fatty acids Or it provides a composition for preparing dihydroxy fatty acid.

The 9-lipoxygenase may consist of the amino acid sequence of SEQ ID NO: 1, and the 9-lipoxygenase variant may consist of the amino acid sequence of SEQ ID NO: 2.

The composition has at least one carbon number selected from the group consisting of arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid and docosahexaenoic acid, 20- 22 may be for processing on a substrate comprising unsaturated fatty acids.

In one embodiment of the present invention, 9-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 3; Or it provides a composition for preparing the hydroxylated fatty acid or dihydroxylated fatty acid comprising the 9-lipoxygenase mutant gene consisting of the nucleotide sequence of SEQ ID NO: 4 as an active ingredient.

In another embodiment of the present invention, there is provided a method for preparing the hydroxylated fatty acid or dihydroxylated fatty acid comprising the step of treating the composition to a substrate.

The substrate has at least one carbon number selected from the group consisting of arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid, and docosahexaenoic acid 20- 22 unsaturated fatty acids.

The treatment may be carried out at a pH of 6.5 to 9.0 and a temperature of 20 to 40 °C.

In another embodiment of the present invention, 9-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 3; Alternatively, it provides a recombinant expression vector for preparing the hydroxylated fatty acid or dihydroxylated fatty acid comprising a 9-lipoxygenase mutant gene consisting of the nucleotide sequence of SEQ ID NO: 4.

In another embodiment of the present invention, there is provided a transformant transformed with the recombinant expression vector in a host cell.

In another embodiment of the present invention, there is provided a novel hydroxylated fatty acid represented by any one of the following Chemical Formulas 1 to 4:

[Formula 1]

,

,

[Formula 2]

,

,

[Formula 3]

,

,

[Formula 4]

.

.

In another embodiment of the present invention, there is provided a novel dihydroxylated fatty acid represented by any one of the following Chemical Formulas 5 to 8:

[Formula 5]

,

,

[Formula 6]

,

,

[Formula 7]

,

,

[Formula 8]

.

.

According to the present invention, a novel hydroxy fatty acid or dihydroxy fatty acid can be produced with high productivity and high yield through a bioconversion process using 9-lipoxygenase derived from a novel microorganism, so it is useful in various industrial fields such as medicine, food and cosmetics expected to be used.

The novel hydroxylated fatty acid or dihydroxylated fatty acid prepared according to the present invention is expected to be involved in various physiologically active functions in animals, including humans, as signal transmitters.

1 shows the synthesis route of 9-hydroxydonic acid and 9,15-dihydroxydonic acid produced using arachidonic acid as a substrate using 9-lipoxygenase of the present invention.
2 is an HPLC chromatogram confirming the production of 9-hydroxydonic acid and 9,15-dihydroxydonic acid.
3 is a result of 1D NMR for the identification of the substance of 9,15- arachidonic acid dihydroxy.
Figure 4 shows the synthesis route of 9-hydroxyl eicosapentaenoic acid and 9,15-eicosapentaenoic acid produced using eicosapentaenoic acid as a substrate using the 9-lipoxygenase of the present invention. will be.
5 is an HPLC chromatogram confirming the production of 9-hydroxylated eicosapentaenoic acid and 9,15-dihydroxylated eicosapentaenoic acid.
6 is a result of 1D NMR for material identification of 9,15- dihydrate eicosapentaenoic acid.
7 shows the synthesis route of 11-hydroxylated docosapentaenoic acid and 11,17-dihydroxylated docosapentaenoic acid produced using docosapentaenoic acid as a substrate using 9-lipoxygenase of the present invention. .
8 is an HPLC chromatogram confirming the production of 11-hydroxylated docosapentaenoic acid and 11,17-dihydroxylated docosapentaenoic acid.
9 is a result of 1D NMR for material identification of 11,17-docosapentaenoic acid dihydrate.
10 shows the synthesis route of 11-hydroxylated docosahexaenoic acid and 11,17-dihydroxylated docosahexaenoic acid produced using docosahexaenoic acid as a substrate using 9-lipoxygenase of the present invention. .
11 is an HPLC chromatogram confirming the production of 11-hydroxylated docosahexaenoic acid and 11,17-dihydroxylated docosahexaenoic acid.
12 is a result of 1D NMR for material identification of 11,17-dihydroxylated docosahexaenoic acid.
13a shows the effect of the pH of 9-lipoxygenase of the present invention on the 9-hydroxydonic acid, 9,15-dihydroxydonic acid production activity (●: HEPES buffer, ▼: HEPPS buffer, ■: CHES buffer), Figure 13b shows the effect of the temperature of the 9-lipoxygenase of the present invention on the production activity of 9-hydroxydonic acid and 9,15-dihydroxydonic acid.
14 shows the hourly production of 9-hydroxydonic acid from arachidonic acid using the 9-lipoxygenase variant (V569F) of the present invention (■: arachidonic acid, ▼: 9-hydroxydonic acid, ●: 9,15 -Arachidonic acid dihydrate).
15 shows the hourly production of 9,15-dihydroxydonic acid from arachidonic acid using 9-lipoxygenase of the present invention (■: arachidonic acid, ▼: 9-hydroxydonic acid, ●: 9,15- arachidonic acid dihydroxy).
Figure 16 shows the hourly production of 9-hydroxyl eicosapentaenoic acid from eicosapentaenoic acid using the 9-lipoxygenase variant (V569F) of the present invention (■: arachidonic acid, ▼: 9-hydroxyl eicosa pentaenoic acid, ●: 9,15-eicosapentaenoic acid dihydrate).
Figure 17 shows the hourly production of 9,15-dihydroxyeicosapentaenoic acid from eicosapentaenoic acid using 9-lipoxygenase of the present invention (■: eicosapentaenoic acid, ▼: 9-hydroxyl eicosapentaenoic acid, ●: 9,15-eicosapentaenoic acid).
Figure 18 shows the hourly production of 11-hydroxylated docosapentaenoic acid from docosapentaenoic acid using the 9-lipoxygenase variant (V569F) of the present invention (■: arachidonic acid, ▼: 11-hydroxylated docosa) pentaenoic acid, ●: 11,17-docosapentaenoic acid dihydrate).
19 shows the hourly production of 11,17-dihydroxylated docosapentaenoic acid from docosapentaenoic acid using 9-lipoxygenase of the present invention (■: docosapentaenoic acid, ▼: 11-hydroxylation degree cosapentaenoic acid, ●: 11,17-docosapentaenoic acid dihydrate).
Figure 20 shows the hourly production of 11-hydroxylated docosahexaenoic acid from docosahexaenoic acid using the 9-lipoxygenase variant (V569F) of the present invention (■: arachidonic acid, ▼: 11-hydroxylated docosa hexaenoic acid, ●: 11,17-dihydroxydocosahexaenoic acid).
21 shows the hourly production of 11,17-dihydroxylated docosahexaenoic acid from docosahexaenoic acid using 9-lipoxygenase of the present invention (■: docosahexaenoic acid, ▼: 11-hydroxylation degree cosahexaenoic acid, ●: 11,17-docosahexaenoic acid dihydrate).
22 shows a recombinant vector synthesizing 9-lipoxygenase of the present invention.

The present inventors first identified the gene and enzyme of 9-lipoxygenase from Sphingomonas macrogoltabidus corresponding to Gram-negative bacteria. Thereafter, the present inventors conducted continuous research to more effectively produce hydroxylated fatty acids and dihydroxylated fatty acids through a bioconversion process using them.

Specifically, the present inventors cloned 9-lipoxygenase derived from Sphingomonas macrogoltabidus DSM 8826 to prepare a recombinant expression vector and a transformed microorganism therefrom, and to use this to produce whole cells. Next, it was confirmed that hydroxylated fatty acids and dihydroxylated fatty acids could be produced with high productivity and high yield through such an eco-friendly process by treating them on a substrate, and the present invention was completed.

Hereinafter, the present invention will be described in detail.

As used herein, the term "hydroxylated fatty acid" refers to a fatty acid, preferably an unsaturated fatty acid having 20 to 22 carbon atoms, in which one hydroxyl group is substituted, specifically, 9-hydroxyarachidonic acid (9-hydroxyarachidonic acid; 9 -HETE), 9-hydroxyeicosapentaenoic acid (9-HEPE), 11-hydroxydocosapentaenoic acid (11-hydroxydocosapentaenoic acid; 11-HDOPE), and 11-hydroxydocosahexaenoic acid (11-hydroxydocosahexaenoic acid; 11-HDOHE) may be any one or more selected from the group consisting of, all of which are novel compounds, and may be sequentially represented by the following Chemical Formulas 1 to 4:

[Formula 1]

,

,

[Formula 2]

,

,

[Formula 3]

,

,

[Formula 4]

.

.

As used herein, the term "dihydroxylated fatty acid" refers to a fatty acid, preferably an unsaturated fatty acid having 20 to 22 carbon atoms, in which two hydroxyl groups are substituted, specifically, 9,15-dihydroxylated arachidonic acid (9, 15-dihydroxyarachidonic acid; 9,15-diHETE), 9,15-dihydroxyeicosapentaenoic acid (9,15-dihydroxyeicosapentaenoic acid; 9,15-diHEPE), 11,17-dihydroxyeicosapentaenoic acid (11) , 17-dihydroxydocosapentaenoic acid; 11,17-diHDOPE) and 11,17-dihydroxydocosahexaenoic acid (11,17-dihydroxydocosahexaenoic acid; 11,17-diHDOHE) may be at least one selected from the group consisting of, all of which are novel. As a compound, it may be represented by the following formulas 5 to 8 in order:

[Formula 5]

,

,

[Formula 6]

,

,

[Formula 7]

,

,

[Formula 8]

.

.

Composition for preparing hydroxylated fatty acid or dihydroxylated fatty acid

The present invention is Sphingomonas macrogoltabidus ( Sphingomonas macrogoltabidus ) derived from 9-lipoxygenase; Or, from the imanoic acid sequence of the 9-lipoxygenase, the 9-lipoxygenase variant in which valine (V), which is the 569th amino acid, is substituted with phenylalanine (F) as an active ingredient. provides

The composition for preparing a hydroxylated fatty acid or dihydroxylated fatty acid according to the present invention comprises: 9-lipoxygenase derived from Sphingomonas macrogoltabidus ; Or, from the imanoic acid sequence of the 9-lipoxygenase, the 9-lipoxygenase variant in which valine (V), which is the 569th amino acid, is substituted with phenylalanine (F) as an active ingredient, as an active ingredient, the 9-lipoxygenase or Whole cells containing the 9-lipoxygenase variant may be included.

First, the 9-lipoxygenase is a wild-type, and can be used to produce hydroxylated fatty acid or dihydroxylated fatty acid from fatty acids. Specifically, hydroxylated fatty acid is produced from fatty acid through oxidation reaction, and then added Through the oxidation reaction, the two-step oxidation reaction to produce dihydroxy fatty acid can be performed well. Therefore, dihydroxy fatty acid may be the main product, and hydroxylated fatty acid may be a by-product. In this case, the dihydroxylated fatty acid may be one in which a hydroxyl group is formed at the 9th carbon position of an unsaturated fatty acid having 20 carbon atoms, and then a hydroxyl group is formed at the 15th carbon position, or a hydroxyl group is formed at the 11th carbon position of an unsaturated fatty acid having 22 or more carbon atoms. Then, a hydroxyl group may be formed at the 17th carbon position.

In addition, the 9-lipoxygenase is not only composed of the amino acid sequence of SEQ ID NO: 1, but also functional equivalents thereof, that is, any one or more substitutions, deletions, etc. that achieve the object of the present invention by inducing mutations in the sequence It is meant to include a mutant, and the 9-lipoxygenase may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 3.

Next, the 9-lipoxygenase variant is the 9-lipoxygenase modified by site-directed mutation, and can be used to produce hydroxylated fatty acids or dihydroxylated fatty acids from fatty acids. Specifically, oxidation reaction from fatty acids Through this, the first-step oxidation reaction to produce hydroxylated fatty acids can be performed well. Therefore, hydroxylated fatty acid may be the main product, and dihydroxylated fatty acid may be a by-product. In this case, the hydroxylated fatty acid may be one in which a hydroxyl group is formed at the 9th carbon position of an unsaturated fatty acid having 20 carbon atoms, or a hydroxyl group formed at the 11th carbon position in an unsaturated fatty acid having 22 or more carbon atoms.

In addition, the 9-lipoxygenase variant not only consists of the amino acid sequence of SEQ ID NO: 2, but also its functional equivalent, that is, one or more substitutions, deletions, etc. in the sequence to achieve the object of the present invention by inducing mutations It is meant to include all mutants, and the 9-lipoxygenase mutant may be a product expressed from a gene consisting of the nucleotide sequence of SEQ ID NO: 3.

On the other hand, the optimal active pH of the 9-lipoxygenase or 9-lipoxygenase variant is 6.5 to 9.0, preferably 8.0 to 9.0, and the optimal activity temperature is 20 ° C. to 40 ° C., preferably 25 ° C. to 35 ° C. .

Specifically, the whole cell comprising the 9-lipoxygenase or the 9-lipoxygenase variant comprises a gene encoding the amino acid sequence of SEQ ID NO: 1 or 2 or a gene consisting of the nucleotide sequence of SEQ ID NO: 3 or 4 It is preferable to culture the transformed microorganism transformed with the recombinant expression vector, and to obtain and use the same.

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, it is more preferable to use a pET-28a-c(+) vector, but is not limited thereto.

As the transformation microorganism, any microorganism may be used as long as it is a microorganism used in the art as a system for overexpressing a desired protein by transformation with a recombinant vector, and specifically, it is more preferable to use Escherichia coli ER 2566 strain, not limited

The whole cells are obtained by: i) centrifuging the culture solution of the microorganism to recover primary whole cells; ii) washing the recovered whole cells with saline solution; iii) second centrifuging the washed whole cells to remove the supernatant to obtain whole cells; and iv) washing the secondarily recovered whole cells with physiological saline once again. Specifically, the recovery of whole cells in step i) can be performed in a range of around 6,000xg using a device known in the art such as a centrifuge, and washing of whole cells in step ii) is performed with 0.85% or less sodium chloride solution. It is appropriate to perform

In this case, the concentration of whole cells containing the lipoxygenase variant in the composition is preferably 0.01 g/L to 1 g/L, more preferably 0.05 g/L to 0.5 g/L, but is not limited thereto. does not

The composition has at least one carbon number selected from the group consisting of arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid and docosahexaenoic acid, 20- for treatment on a substrate comprising 22 unsaturated fatty acids (preferably with one or more cis, cis-1,4 pentadiene).

The concentration of the unsaturated fatty acid having 20 to 22 carbon atoms in the substrate is preferably 0.1 mM to 10 mM, but is not limited thereto. The unsaturated fatty acid having 20 to 22 carbon atoms may improve the production concentration of hydroxylated fatty acid or dihydroxylated fatty acid by maintaining this concentration.

In addition, the present invention is a 9-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 3; Or it provides a composition for preparing the hydroxylated fatty acid or dihydroxylated fatty acid comprising the 9-lipoxygenase mutant gene consisting of the nucleotide sequence of SEQ ID NO: 4 as an active ingredient.

In addition, the present invention is a 9-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 3; Alternatively, the recombinant expression vector for preparing the hydroxylated fatty acid or dihydroxylated fatty acid comprising a 9-lipoxygenase mutant gene consisting of the nucleotide sequence of SEQ ID NO: 4, and a transformant transformed with the recombinant expression vector into a host cell is provided.

First, the lipoxygenase gene not only consists of the nucleotide sequence of SEQ ID NO: 3, but also its functional equivalent, that is, all mutations that achieve the object of the present invention by inducing mutations such as one or more substitutions and deletions in the sequence means to include a body.

Next, the lipoxygenase mutant gene not only consists of the nucleotide sequence of SEQ ID NO: 4, but also its functional equivalent, that is, one or more substitutions, deletions, etc. in the sequence to achieve the object of the present invention by inducing mutations It is meant to include all mutants.

Method for producing hydroxylated fatty acid or dihydroxylated fatty acid

The present invention provides a method for producing the hydroxylated fatty acid or dihydroxylated fatty acid comprising the step of treating the composition on a substrate.

The method for producing a hydroxylated fatty acid or a dihydroxylated fatty acid according to the present invention includes treating the composition to a substrate.

The composition comprises: 9-lipoxygenase from Sphingomonas macrogoltabidus ; Or, from the imanoic acid sequence of the 9-lipoxygenase, a 9-lipoxygenase variant in which valine (V), the 569th amino acid, is substituted with phenylalanine (F) as an active ingredient, or consisting of the nucleotide sequence of SEQ ID NO: 3 9-lipoxygenase gene; Or to include the 9-lipoxygenase mutant gene consisting of the nucleotide sequence of SEQ ID NO: 4 as an active ingredient, the specific details are as described above.

The substrate has at least one carbon number selected from the group consisting of arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid, and docosahexaenoic acid 20- 22 unsaturated fatty acids.

The concentration of the unsaturated fatty acid having 20 to 22 carbon atoms in the substrate is preferably 0.1 mM to 10 mM, but is not limited thereto. The unsaturated fatty acid having 20 to 22 carbon atoms may improve the production concentration of hydroxylated fatty acid or dihydroxylated fatty acid by maintaining this concentration.

The treatment is preferably carried out at a temperature of pH 6.5 ~ 9.0 and 20 ~ 40 ℃, more preferably carried out at a temperature of pH 8.0 ~ 9.0 and 25 ~ 35 ℃, but is not limited thereto. In order to maintain these pH conditions, HEPES, EPPS, or CHES buffer may be used as a reaction solvent. By maintaining such a pH condition, the enzyme used can be optimally activated, and finally, hydroxylated fatty acid or dihydroxylated fatty acid can be produced with high productivity and high yield. In addition, the treatment is preferably performed for 10 minutes or more, but is not limited thereto.

Meanwhile, the treatment may be performed through reaction with cysteine at a concentration of 100 mM to 400 mM, and preferably through reaction with cysteine at a concentration of 100 mM to 300 mM, but is not limited thereto. Thereby, the enzyme used can be optimally activated, and finally, hydroxylated fatty acid or dihydroxylated fatty acid can be produced with high productivity and high yield.

As described above, new hydroxy fatty acid or dihydroxy fatty acid can be produced with high productivity and high yield through the bioconversion process using the novel microorganism-derived 9-lipoxygenase, so it is useful in various industries such as medicine, food and cosmetics. expected to be used.

The novel hydroxylated fatty acid or dihydroxylated fatty acid prepared according to the present invention is expected to be involved in various physiologically active functions in animals, including humans, as signal transmitters.

Hereinafter, preferred examples are presented to help 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 microorganism containing a gene for the expression of 9-lipoxygenase

In order to prepare the 9-lipoxygenase gene of the present invention, the 9-lipoxygenase gene derived from Sphingomonas macrogoltabidus DSM 8826 was first isolated.

Specifically, Sphingomonas macrogoltabidus ( Sphingomonas macrogoltabidus ) DSM 8826 Based on the DNA nucleotide sequence in which the gene nucleotide sequence and the amino acid sequence are already specified, to carry out the polymerase chain reaction (PCR), Sphingomonas macrogol The genomic DNA of Sphingomonas macrogoltabidus DSM 8826 was extracted (Intron) and used as a template for PCR, and each primer was designed based on the DNA sequence of 9-lipoxygenase and polymerase chain reaction (PCR) was performed. Restriction enzymes Nde I and Hind III were used as primers for the sequence of 9-lipoxygenase, and the primer sequence was F: GCT CGA CCA TAT GTC CTT TGT GTC GCC TTC GCT G and R: GCG CCC AAG CTT TCA GAT ATT GAT It consisted of GCT CGT CGG G. In addition, a plasmid vector pET 28a(+) (manufactured by Novagen) was prepared by treatment with a restriction enzyme, and ligated to prepare pET 28a(+)/9-lipoxygenase, respectively.

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 microorganism was added with 20% glycerine solution. Therefore, it was stored frozen at -70°C before culturing for the production of 9-hydroxylated fatty acid and 9,15-dihydroxylated fatty acid.

Example 2. Preparation of 9-lipoxygenase

For the protein expression of the enzyme, the transformed microorganisms stored frozen in Example 1 were aerated at 200 rev/min in a flask with 500 ml of LB (Difco, Sparks, MD, USA) medium and 20 μg/ml of kanamycin. Incubated at 37°C under conditions. When the absorbance of the bacteria reached 0.6 to 0.8 at 600 nm, after adding the final concentration of 0.1 mM IPTG to induce protein expression of the enzymes, the culture medium was stirred at 16°C for 16-18 hours at 150 rev/min. cultured.

E. coli cells containing cultured 9-lipoxygenase were collected and used. In addition, 9-lipoxygenase produced by overexpression as described above was centrifuged at 6,000xg at 4°C for 30 minutes, washed twice with 0.85% sodium chloride (NaCl), and then washed with hydroxylated fatty acid or It was used as a recombinant cell to produce dihydroxy fatty acid. Since 9-lipoxygenase produces dihydroxy fatty acid by performing a two-step oxidation reaction, a 9-lipoxygenase mutant (V569F) that performs well in the first-step oxidation reaction through site-directed mutagenesis was prepared Thus, it was used for the production of hydroxylated fatty acids.

Example 3. Construction of a synthetic route of 9-hydroxydonic acid and 9,15-dihydroxydonic acid from arachidonic acid using 9-lipoxygenase

Arachidonic acid (ARA) was used as a substrate to construct a synthesis pathway for 9-hydroxydonic acid and 9,15-dihydroxydonic acid using the 9-lipoxygenase. For 9-hydroxydonic acid and 9,15-dihydroxydonic acid, with respect to 1 mM arachidonic acid, 0.05 g/L of 9-lipoxygenase was used, 200 mM cysteine was added, and pH 8.5 and 30° C. for 15 minutes carried out.

As a result, a synthetic route as shown in FIG. 1 was constructed. In addition, the produced 9-hydroxylated arachidonic acid and 9,15-dihydroxylated arachidonic acid were confirmed using HPLC (FIG. 2), and 9,15-dihydroxylated arachidonic acid was confirmed through material identification (FIG. 3).

Example 4. Construction of a synthetic route of 9-hydroxylated eicosapentaenoic acid and 9,15-dihydroxylated eicosapentaenoic acid from eicosapentaenoic acid using 9-lipoxygenase

Eicosapentaenoic acid (EPA) was used as a substrate to construct a synthesis route of 9-hydroxylated eicosapentaenoic acid and 9,15-dihydroxylated eicosapentaenoic acid using the 9-lipoxygenase. 9-eicosapentaenoic acid and 9,15-dihydrate eicosapentaenoic acid, 0.5 g/L of 9-lipoxygenase was used with respect to 1 mM eicosapentaenoic acid, 200 mM cysteine was added, pH 8.5 and 30° C. for 60 min.

As a result, a synthetic route as shown in FIG. 4 was constructed. In addition, the produced 9-hydroxyl eicosapentaenoic acid and 9,15-eicosapentaenoic acid were confirmed using HPLC (FIG. 5), and 9,15-eicosapentaenoic acid dihydrate was identified through material identification. was confirmed (FIG. 6).

Example 5. Construction of a synthetic route of 11-hydroxylated docosapentaenoic acid and 11,17-dihydroxylated docosapentaenoic acid from docosapentaenoic acid using 9-lipoxygenase

Docosapentaenoic acid (DPA) was used as a substrate to construct a synthesis route of 11-hydroxylated docosapentaenoic acid and 11,17-dihydroxylated docosapentaenoic acid using the 9-lipoxygenase. For 11-hydroxylated docosapentaenoic acid and 11,17-dihydroxylated docosapentaenoic acid with respect to 1 mM docosapentaenoic acid, 0.1 g/L of 9-lipoxygenase was used, 200 mM cysteine was added, and pH 8.5 and 30° C. for 20 min.

As a result, a synthetic route as shown in FIG. 7 was constructed. Also, the produced 11-hydroxylated docosapentaenoic acid and 11,17-dihydroxylated docosapentaenoic acid were confirmed using HPLC (FIG. 8), and 11,17-dihydroxylated docosapentaenoic acid was confirmed through material identification. (FIG. 9).

Example 6. Construction of a synthesis route of 11-hydroxylated docosahexaenoic acid and 11,17-dihydroxylated docosahexaenoic acid from docosahexaenoic acid using 9-lipoxygenase

Docosahexaenoic acid (DHA) was used as a substrate to construct a synthetic route of 11-hydroxylated docosahexaenoic acid and 11,17-dihydroxylated docosahexaenoic acid using the 9-lipoxygenase. For 11-hydroxylated docosahexaenoic acid and 11,17-dihydroxylated docosahexaenoic acid, 0.1 g/L of 9-lipoxygenase was used with respect to 1 mM docosahexaenoic acid, 200 mM cysteine was added, and pH 8.5 and 30° C. for 20 min.

As a result, a synthetic route as shown in FIG. 10 was constructed. In addition, the produced 11-hydroxylated docosahexaenoic acid and 11,17-dihydroxylated docosahexaenoic acid were confirmed using HPLC (FIG. 11), and 11,17-dihydroxylated docosahexaenoic acid was identified through material identification. (Fig. 12).

Example 7. Effect of pH and temperature of 9-lipoxygenase on production activity of 9-hydroxydonic acid and 9,15-dihydroxydonic acid

To investigate the pH effect of 9-lipoxygenase on the production of 9-hydroxydonic acid and 9,15-dihydroxydonic acid, HEPES buffer (HEPES buffer, pH) with respect to 1 mM arachidonic acid as a substrate. 6.5-7.5), EPPS buffer (EPPS buffer, pH 7.5-8.5) and chess buffer (CHES buffer, pH 8.5-9.0) for 10 minutes. As a result, as shown in FIG. 13a , it was found that the optimum pH was 8.5.

In addition, in order to investigate the effect of temperature on the production of 9-hydroxydonic acid and 9,15-dihydroxydonic acid of 9-lipoxygenase, 50 mM EPPS buffer pH with respect to 1 mM arachidonic acid as a substrate At 8.5, the temperature was ranged from 20 °C to 40 °C in 5 °C increments, and then the enzymatic reaction was performed for 10 minutes. As a result, as shown in FIG. 13b , it was found that the optimum temperature was 30°C.

Example 8. Conversion rate of 9-hydroxydonic acid using a 9-lipoxygenase variant (V569F)

Sphingomonas macrogoltabidus of the present invention In order to confirm the production of 9-hydroxyl arachidonic acid using 9-lipoxygenase variant (V569F) derived from Sphingomonas macrogoltabidus , 9-lipoxygenase variant (V569F) 0.05 g/ The conversion rate of 9-hydroxydonic acid over time was measured by using 1 mM arachidonic acid as a substrate in a 50 mM EPPS buffer containing L at a pH of 8.5 and a temperature of 30°C. The results are shown in FIG. 14, and the 9-lipoxygenase mutant (V569F) produced 0.95 mM 9-hydroxylated arachidonic acid. The final conversion yield of 9-hydroxylated arachidonic acid was 95%.

Example 9. Conversion rate of 9,15-dihydroxydonic acid using 9-lipoxygenase

To confirm the production of 9,15-dihydroxydonic acid using 9-lipoxygenase derived from Sphingomonas macrogoltabidus of the present invention, a 50 containing 9-lipoxygenase 0.05 g/L The conversion rate of 9,15-dihydroxydonic acid over time was measured using 1 mM arachidonic acid as a substrate in an mM EPPS buffer at pH 8.5 and a temperature of 30°C. The results are shown in FIG. 15, and 9-lipoxygenase produced 1 mM 9,15-dihydroxydonic acid. The final conversion yield of 9,15-dihydroxydonic acid was 100%.

Example 10. Conversion rate of 9-hydroxyeicosapentaenoic acid using 9-lipoxygenase variant (V569F)

In order to confirm the production of 9-hydroxylated eicosapentaenoic acid using a 9-lipoxygenase variant (V569F) derived from Sphingomonas macrogoltabidus of the present invention, a 9-lipoxygenase variant (V569F) The time-wise conversion of 9-hydroxyeicosapentaenoic acid was measured using 1 mM eicosapentaenoic acid as a substrate in a 50 mM EPPS buffer containing 0.1 g/L at a pH of 8.5 and a temperature of 30°C. The results are shown in FIG. 16, and the 9-lipoxygenase mutant (V569F) produced 0.72 mM 9-hydroxyl eicosapentaenoic acid. The final conversion yield of 9-hydroxyl eicosapentaenoic acid was 72%. .

Example 11. Conversion rate of 9,15-dihydroxyeicosapentaenoic acid using 9-lipoxygenase

To confirm the production of 9,15-dihydroxyeicosapentaenoic acid using 9-lipoxygenase derived from Sphingomonas macrogoltabidus of the present invention, 9-lipoxygenase 0.5 g/L The conversion rate of 9,15-eicosapentaenoic acid dihydroxylated over time was measured using the 50 mM EPPS buffer containing 1 mM eicosapentaenoic acid as a substrate at a pH of 8.5 and a temperature of 30°C. The results are shown in FIG. 17, and 9-lipoxygenase produced 0.83 mM of 9,15-dihydroxyeicosapentaenoic acid. The final conversion yield of 9,15-dihydroxyeicosapentaenoic acid was 83%.

Example 12. Conversion rate of 11-hydroxydocosapentaenoic acid using 9-lipoxygenase variant (V569F)

In order to confirm the production of 11-hydroxylated docosapentaenoic acid using a 9-lipoxygenase variant (V569F) derived from Sphingomonas macrogoltabidus of the present invention, a 9-lipoxygenase variant (V569F) The time-wise conversion of 11-hydroxydocosapentaenoic acid was measured using 1 mM docosapentaenoic acid as a substrate in a 50 mM EPPS buffer containing 0.1 g/L at a pH of 8.5 and a temperature of 30°C. The results are shown in FIG. 18, and the 9-lipoxygenase mutant (V569F) produced 0.76 mM 11-hydroxylated docosapentaenoic acid. The final conversion yield of 11-hydroxylated docosapentaenoic acid was 76%. .

Example 13. Conversion rate of 11,17-dihydroxylated docosapentaenoic acid using 9-lipoxygenase

In order to confirm the production of 11,17-dihydroxylated docosapentaenoic acid using 9-lipoxygenase derived from Sphingomonas macrogoltabidus of the present invention, it contains 0.1 g/L of 9-lipoxygenase In a 50 mM EPPS buffer, the conversion rate of 11,17-dihydroxylated docosapentaenoic acid over time was measured using 1 mM docosapentaenoic acid as a substrate at a pH of 8.5 and a temperature of 30°C. The results are shown in FIG. 19, and 9-lipoxygenase produced 0.94 mM of 11,17-dihydroxylated docosapentaenoic acid. The final conversion yield of 11,17-dihydroxylated docosapentaenoic acid was 94%.

Example 14. Conversion rate of 11-hydroxylated docosahexaenoic acid using 9-lipoxygenase variant (V569F)

In order to confirm the production of 11-hydroxylated docosahexaenoic acid using a 9-lipoxygenase variant (V569F) derived from Sphingomonas macrogoltabidus of the present invention, a 9-lipoxygenase variant (V569F) The conversion rate of 11-hydroxydocosahexaenoic acid over time was measured by using 1 mM docosahexaenoic acid as a substrate in a 50 mM EPPS buffer containing 0.1 g/L at a pH of 8.5 and a temperature of 30°C. The results are shown in FIG. 20, and the 9-lipoxygenase mutant (V569F) produced 0.78 mM of 11-hydroxylated docosahexaenoic acid. The final conversion yield of 11-hydroxylated docosahexaenoic acid was 78%. .

Example 15. Conversion rate of 11,17-dihydroxylated docosahexaenoic acid using 9-lipoxygenase

In order to confirm the production of 11,17-dihydroxylated docosahexaenoic acid using 9-lipoxygenase derived from Sphingomonas macrogoltabidus of the present invention, it contains 0.1 g/L of 9-lipoxygenase The conversion rate of 11,17-dihydroxylated docosahexaenoic acid over time was measured using 1 mM docosahexaenoic acid as a substrate in a 50 mM EPPS buffer at a pH of 8.5 and a temperature of 30°C. The results are shown in FIG. 21, and 9-lipoxygenase produced 0.95 mM 11,17-dihydroxylated docosahexaenoic acid. The final conversion yield of 11,17-dihydroxylated docosahexaenoic acid was 95%.

Above, a specific part of the content of the present invention has been described in detail, for those of ordinary skill in the fatty acid industry and the medical community, these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereto. The point will be clear. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.

<110> Konkuk University Industrial Cooperation Corp <120> COMPOSITION AND METHOD FOR PRODUCING HYDROXY FATTY ACID OR DIHYDROXY FATTY ACID USING 9-LIPOXYGENASE OR VARIANT THEREOF <130> 2020-I141 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 646 <212> PRT <213> Sphingomonas macrogoltabidus <400> 1 Met Pro Ser Ala Asn Pro Ser Leu Pro Gln Asn Asp Thr Pro Ala Glu 1 5 10 15 Gln Ala Ala Arg Ala Ala Gln Leu Ala Ala Ser Gln Ala Val Tyr Val 20 25 30 Trp Thr Thr Asp Val Pro Thr Leu Pro Gly Val Pro Leu Ala Thr Asp 35 40 45 Val Pro Lys Asn Asp Glu Pro Thr Ile Ala Trp Phe Gly Ile Leu Ile 50 55 60 Gly Val Gly Leu Ala Ile Val Arg Asn Ala Leu Thr Val Lys Leu Gly 65 70 75 80 Gly Val Asp Arg Gly Glu Leu Asp Thr Pro Arg Ala Glu Tyr Glu Thr 85 90 95 Ala Leu Ala Glu Cys Asp Ala Ile Glu Leu Ser Thr Ala Lys Ile Val 100 105 110 Ala Glu His Gly Val His Ser Gly Gly Asn Ile Phe Glu Arg Ile Val 115 120 125 Gly Asp Val Glu Asn Ala Val Ala Ala Ala Glu Arg Asp Val His Leu 130 135 140 Ala Leu Leu Gln Gly Tyr Lys Glu Arg Leu Glu Asp Leu Met Lys Val 145 150 155 160 Asp Glu Ala Glu Val Ala Gly Leu Gly Ser Lys Thr Pro Arg Ser Leu 165 170 175 Asp Ala Tyr Arg Ala Leu Phe Ala Thr Leu Pro Val Pro Gly Ile Ser 180 185 190 Tyr Met Phe Gln Asp Asp Lys Glu Phe Ala Arg Leu Arg Val Gln Gly 195 200 205 Pro Asn Cys Met Leu Ile Ala Ala Val Glu Gly Ala Leu Pro Ala Asn 210 215 220 Phe Pro Leu Ser Glu Lys Ala Tyr Ala Ala Val Val Asn Gly Asp Thr 225 230 235 240 Leu Ala Ala Ala Leu Ala Asp Gly Arg Leu Phe Met Leu Asp Tyr Lys 245 250 255 Pro Leu Ala Ile Leu Asp Pro Gly Thr Tyr Gly Gly Glu Ala Lys Tyr 260 265 270 Val Ser Gln Pro Met Ala Leu Phe Ala Val Pro Pro Gly Gly Ala Ser 275 280 285 Leu Ile Pro Val Ala Ile Gln Cys Gly Gln Asp Pro Ala Asp Cys Pro 290 295 300 Ile Phe Thr Pro Ser Pro Ala Ala Asp Arg Gln Trp Gly Trp Glu Met 305 310 315 320 Ala Lys Phe Val Val Gln Val Ala Asp Gly Asn Tyr His Glu Leu Phe 325 330 335 Ala His Leu Ala Arg Thr His Leu Val Ile Glu Ala Phe Ala Val Ala 340 345 350 Thr His Arg His Leu Ala Glu Ala His Pro Val Trp Ala Leu Leu Val 355 360 365 Pro His Phe Glu Gly Thr Leu Phe Ile Asn Glu Gln Ala Ala Thr Ser 370 375 380 Leu Ile Ala Ala Asn Gly Pro Ile Asp His Ile Phe Ala Gly Thr Ile 385 390 395 400 Ala Ser Ser Gln Leu Ala Ala Val Asp Ala Arg Leu Ala Phe Asp Phe 405 410 415 Arg Gly Lys Met Pro His Ala Asp Phe Ala Ala Arg Gly Val Gly Val 420 425 430 Asp Ser Ala Leu Ala Asp Tyr Pro Tyr Arg Asp Asp Ala Leu Leu Val 435 440 445 Trp Asp Ala Ile His Glu Trp Ala Arg Gln Tyr Val Asp Leu Tyr Tyr 450 455 460 Thr Gly Asp Thr Asp Ile Val Ala Asp Thr Glu Leu Ala Ala Trp Ala 465 470 475 480 Ala Cys Leu Ala Gly Glu Ala Lys Val Gly Gly Leu Gly Pro Val Thr 485 490 495 Thr Arg Asn Gln Leu Ala Glu Ile Cys Ala Met Val Met Phe Thr Ala 500 505 510 Ser Ala Gln His Ala Ala Val Asn Leu Pro Gln Lys Asp Ile Met Ala 515 520 525 Phe Ala Pro Ala Val Thr Gly Ala Ala Trp Gln Pro Ala Pro Asn Gly 530 535 540 Gln Arg Gly His Asp Lys Thr Gly Trp Leu Ala Met Met Pro Pro Met 545 550 555 560 Ala Leu Ala Leu Glu Gln Leu Asn Val Leu Glu Leu Leu Gly Ser Val 565 570 575 His Tyr Arg Pro Leu Gly Asp Tyr Arg Ser Asn Ala Phe Pro Tyr Pro 580 585 590 Gln Trp Phe Gln Asp Pro Arg Val Thr Ala Ala Glu Gly Pro Leu Ala 595 600 605 Trp Phe Gln Ala Ala Leu Ala Asp Val Glu Ala Glu Ile Val Thr Arg 610 615 620 Asn Ala Glu Arg Met Gln Pro Tyr Pro Tyr Leu Gln Pro Ser Leu Ile 625 630 635 640 Pro Thr Ser Ile Asn Ile 645 <210> 2 <211> 646 <212> PRT <213> Artificial Sequence <220> <223> V569F mutant of 9-lipoxygenase <400> 2 Met Pro Ser Ala Asn Pro Ser Leu Pro Gln Asn Asp Thr Pro Ala Glu 1 5 10 15 Gln Ala Ala Arg Ala Ala Gln Leu Ala Ala Ser Gln Ala Val Tyr Val 20 25 30 Trp Thr Thr Asp Val Pro Thr Leu Pro Gly Val Pro Leu Ala Thr Asp 35 40 45 Val Pro Lys Asn Asp Glu Pro Thr Ile Ala Trp Phe Gly Ile Leu Ile 50 55 60 Gly Val Gly Leu Ala Ile Val Arg Asn Ala Leu Thr Val Lys Leu Gly 65 70 75 80 Gly Val Asp Arg Gly Glu Leu Asp Thr Pro Arg Ala Glu Tyr Glu Thr 85 90 95 Ala Leu Ala Glu Cys Asp Ala Ile Glu Leu Ser Thr Ala Lys Ile Val 100 105 110 Ala Glu His Gly Val His Ser Gly Gly Asn Ile Phe Glu Arg Ile Val 115 120 125 Gly Asp Val Glu Asn Ala Val Ala Ala Ala Glu Arg Asp Val His Leu 130 135 140 Ala Leu Leu Gln Gly Tyr Lys Glu Arg Leu Glu Asp Leu Met Lys Val 145 150 155 160 Asp Glu Ala Glu Val Ala Gly Leu Gly Ser Lys Thr Pro Arg Ser Leu 165 170 175 Asp Ala Tyr Arg Ala Leu Phe Ala Thr Leu Pro Val Pro Gly Ile Ser 180 185 190 Tyr Met Phe Gln Asp Asp Lys Glu Phe Ala Arg Leu Arg Val Gln Gly 195 200 205 Pro Asn Cys Met Leu Ile Ala Ala Val Glu Gly Ala Leu Pro Ala Asn 210 215 220 Phe Pro Leu Ser Glu Lys Ala Tyr Ala Ala Val Val Asn Gly Asp Thr 225 230 235 240 Leu Ala Ala Ala Leu Ala Asp Gly Arg Leu Phe Met Leu Asp Tyr Lys 245 250 255 Pro Leu Ala Ile Leu Asp Pro Gly Thr Tyr Gly Gly Glu Ala Lys Tyr 260 265 270 Val Ser Gln Pro Met Ala Leu Phe Ala Val Pro Pro Gly Gly Ala Ser 275 280 285 Leu Ile Pro Val Ala Ile Gln Cys Gly Gln Asp Pro Ala Asp Cys Pro 290 295 300 Ile Phe Thr Pro Ser Pro Ala Ala Asp Arg Gln Trp Gly Trp Glu Met 305 310 315 320 Ala Lys Phe Val Val Gln Val Ala Asp Gly Asn Tyr His Glu Leu Phe 325 330 335 Ala His Leu Ala Arg Thr His Leu Val Ile Glu Ala Phe Ala Val Ala 340 345 350 Thr His Arg His Leu Ala Glu Ala His Pro Val Trp Ala Leu Leu Val 355 360 365 Pro His Phe Glu Gly Thr Leu Phe Ile Asn Glu Gln Ala Ala Thr Ser 370 375 380 Leu Ile Ala Ala Asn Gly Pro Ile Asp His Ile Phe Ala Gly Thr Ile 385 390 395 400 Ala Ser Ser Gln Leu Ala Ala Val Asp Ala Arg Leu Ala Phe Asp Phe 405 410 415 Arg Gly Lys Met Pro His Ala Asp Phe Ala Ala Arg Gly Val Gly Val 420 425 430 Asp Ser Ala Leu Ala Asp Tyr Pro Tyr Arg Asp Asp Ala Leu Leu Val 435 440 445 Trp Asp Ala Ile His Glu Trp Ala Arg Gln Tyr Val Asp Leu Tyr Tyr 450 455 460 Thr Gly Asp Thr Asp Ile Val Ala Asp Thr Glu Leu Ala Ala Trp Ala 465 470 475 480 Ala Cys Leu Ala Gly Glu Ala Lys Val Gly Gly Leu Gly Pro Val Thr 485 490 495 Thr Arg Asn Gln Leu Ala Glu Ile Cys Ala Met Val Met Phe Thr Ala 500 505 510 Ser Ala Gln His Ala Ala Val Asn Leu Pro Gln Lys Asp Ile Met Ala 515 520 525 Phe Ala Pro Ala Val Thr Gly Ala Ala Trp Gln Pro Ala Pro Asn Gly 530 535 540 Gln Arg Gly His Asp Lys Thr Gly Trp Leu Ala Met Met Pro Pro Met 545 550 555 560 Ala Leu Ala Leu Glu Gln Leu Asn Phe Leu Glu Leu Leu Gly Ser Val 565 570 575 His Tyr Arg Pro Leu Gly Asp Tyr Arg Ser Asn Ala Phe Pro Tyr Pro 580 585 590 Gln Trp Phe Gln Asp Pro Arg Val Thr Ala Ala Glu Gly Pro Leu Ala 595 600 605 Trp Phe Gln Ala Ala Leu Ala Asp Val Glu Ala Glu Ile Val Thr Arg 610 615 620 Asn Ala Glu Arg Met Gln Pro Tyr Pro Tyr Leu Gln Pro Ser Leu Ile 625 630 635 640 Pro Thr Ser Ile Asn Ile 645 <210> 3 <211> 1941 <212> DNA <213> Sphingomonas macrogoltabidus <400> 3 ttgccatcgg ccaatcccag cctgccgcaa aacgatacgc ccgccgagca agcggcgcgc 60 gcggcacagc tcgccgcttc gcaggccgtc tatgtctgga ccaccgacgt cccgacgctg 120 cccggcgtgc cgctcgccac cgacgtgccg aagaacgacg aaccgacgat cgcctggttc 180 ggcatcttga tcggcgtcgg cctcgcgatc gtgcgcaatg cgctgacggt gaagctcggc 240 ggcgtcgacc ggggcgagct cgacacgccg cgcgccgaat atgaaaccgc gctcgccgag 300 tgcgacgcga tcgagctatc gaccgcgaag atcgtcgccg aacatggcgt tcacagcggc 360 ggcaatatct tcgaacgcat cgtcggcgac gtggaaaatg ccgtcgccgc ggctgagcgc 420 gacgtgcatc tcgctttgct tcagggatat aaggagcggc tcgaggatct catgaaggtc 480 gacgaggccg aggtggcggg gctgggcagc aagacgccgc gcagcctcga cgcatatcgc 540 gcgctgttcg ctaccctgcc ggtgccgggc atcagctata tgttccagga cgacaaggag 600 ttcgcccgcc tgcgcgtgca ggggccgaac tgcatgctga tcgcggcggt cgagggagcg 660 ctgcccgcca atttcccgct gagcgaaaag gcttacgccg cggtggtgaa tggcgacacg 720 ctggccgccg cgctcgccga cggccgcctc ttcatgctcg attacaagcc gctcgcgatc 780 ctcgaccccg gcacctatgg cggcgaagcc aaatatgtct cgcagccgat ggcgctgttc 840 gcggtaccgc cgggcggcgc atcgctcatc cccgtcgcga tccagtgcgg ccaggacccc 900 gccgactgtc cgattttcac gccctcaccg gcggccgaca ggcaatgggg ctgggaaatg 960 gcgaaattcg tcgtgcaggt cgccgacggt aattatcatg agctcttcgc ccatctggca 1020 cgcacccatc tggtgatcga ggcgttcgcg gtcgcgacgc accgccatct tgccgaggcg 1080 catccggtgt gggcgctgct cgtcccgcat ttcgagggga cattgttcat caacgaacag 1140 gcggcgacct cgctgatcgc ggcgaacggc ccgatcgacc atattttcgc ggggacgatc 1200 gcgtcgagcc agcttgccgc agtcgatgcg cggctggcgt tcgatttccg gggcaagatg 1260 ccgcacgccg attttgccgc gcggggcgtc ggggtcgatt cggcgctcgc cgactatccg 1320 tatcgcgacg acgcgctgct ggtgtgggac gcgatccacg aatgggcgcg gcaatatgtc 1380 gatctttatt atacgggcga caccgatatc gtcgccgata ccgagctggc ggcatgggcc 1440 gcgtgcctcg cgggcgaagc caaggtcggc gggctcggcc cggtgacgac gcgcaaccag 1500 ctcgccgaaa tctgcgcgat ggtgatgttc accgccagcg cgcagcatgc ggcggtcaat 1560 ttaccgcaaa aggacatcat ggccttcgcc cccgcggtga ccggcgcagc gtggcaaccg 1620 gcgccgaacg gccagcgcgg acacgacaag acgggctggc tcgcgatgat gccgccgatg 1680 gccctcgcgc tcgaacaatt gaacgtgctc gaactgctgg ggtcggtgca ttatcgcccg 1740 ctcggcgact atcgcagcaa cgcctttccc tatccgcagt ggttccagga tccgcgcgtc 1800 accgcggcgg agggcccgct cgcctggttt caggcggcgc ttgccgacgt cgaggcggaa 1860 atcgtcacgc gcaatgccga gcggatgcag ccctatccct atctgcagcc gagcctgatc 1920 ccgacgagca tcaatatctg a 1941 <210> 4 <211> 1941 <212> DNA <213> Artificial Sequence <220> <223> V569F mutant of 9-lipoxygenase <400> 4 ttgccatcgg ccaatcccag cctgccgcaa aacgatacgc ccgccgagca agcggcgcgc 60 gcggcacagc tcgccgcttc gcaggccgtc tatgtctgga ccaccgacgt cccgacgctg 120 cccggcgtgc cgctcgccac cgacgtgccg aagaacgacg aaccgacgat cgcctggttc 180 ggcatcttga tcggcgtcgg cctcgcgatc gtgcgcaatg cgctgacggt gaagctcggc 240 ggcgtcgacc ggggcgagct cgacacgccg cgcgccgaat atgaaaccgc gctcgccgag 300 tgcgacgcga tcgagctatc gaccgcgaag atcgtcgccg aacatggcgt tcacagcggc 360 ggcaatatct tcgaacgcat cgtcggcgac gtggaaaatg ccgtcgccgc ggctgagcgc 420 gacgtgcatc tcgctttgct tcagggatat aaggagcggc tcgaggatct catgaaggtc 480 gacgaggccg aggtggcggg gctgggcagc aagacgccgc gcagcctcga cgcatatcgc 540 gcgctgttcg ctaccctgcc ggtgccgggc atcagctata tgttccagga cgacaaggag 600 ttcgcccgcc tgcgcgtgca ggggccgaac tgcatgctga tcgcggcggt cgagggagcg 660 ctgcccgcca atttcccgct gagcgaaaag gcttacgccg cggtggtgaa tggcgacacg 720 ctggccgccg cgctcgccga cggccgcctc ttcatgctcg attacaagcc gctcgcgatc 780 ctcgaccccg gcacctatgg cggcgaagcc aaatatgtct cgcagccgat ggcgctgttc 840 gcggtaccgc cgggcggcgc atcgctcatc cccgtcgcga tccagtgcgg ccaggacccc 900 gccgactgtc cgattttcac gccctcaccg gcggccgaca ggcaatgggg ctgggaaatg 960 gcgaaattcg tcgtgcaggt cgccgacggt aattatcatg agctcttcgc ccatctggca 1020 cgcacccatc tggtgatcga ggcgttcgcg gtcgcgacgc accgccatct tgccgaggcg 1080 catccggtgt gggcgctgct cgtcccgcat ttcgagggga cattgttcat caacgaacag 1140 gcggcgacct cgctgatcgc ggcgaacggc ccgatcgacc atattttcgc ggggacgatc 1200 gcgtcgagcc agcttgccgc agtcgatgcg cggctggcgt tcgatttccg gggcaagatg 1260 ccgcacgccg attttgccgc gcggggcgtc ggggtcgatt cggcgctcgc cgactatccg 1320 tatcgcgacg acgcgctgct ggtgtgggac gcgatccacg aatgggcgcg gcaatatgtc 1380 gatctttatt atacgggcga caccgatatc gtcgccgata ccgagctggc ggcatgggcc 1440 gcgtgcctcg cgggcgaagc caaggtcggc gggctcggcc cggtgacgac gcgcaaccag 1500 ctcgccgaaa tctgcgcgat ggtgatgttc accgccagcg cgcagcatgc ggcggtcaat 1560 ttaccgcaaa aggacatcat ggccttcgcc cccgcggtga ccggcgcagc gtggcaaccg 1620 gcgccgaacg gccagcgcgg acacgacaag acgggctggc tcgcgatgat gccgccgatg 1680 gccctcgcgc tcgaacaatt gaacttcctc gaactgctgg ggtcggtgca ttatcgcccg 1740 ctcggcgact atcgcagcaa cgcctttccc tatccgcagt ggttccagga tccgcgcgtc 1800 accgcggcgg agggcccgct cgcctggttt caggcggcgc ttgccgacgt cgaggcggaa 1860 atcgtcacgc gcaatgccga gcggatgcag ccctatccct atctgcagcc gagcctgatc 1920 ccgacgagca tcaatatctg a 1941

Claims (11)

9-lipoxygenase from Sphingomonas macrogoltabidus ; Or, from the imanoic acid sequence of the 9-lipoxygenase, a 9-lipoxygenase variant in which valine (V), which is the 569th amino acid, is substituted with phenylalanine (F) as an active ingredient. ,
The hydroxylated fatty acid or dihydroxylated fatty acid is 9-hydroxyarachidonic acid, 9,15-dihydroxyarachidonic acid, 9-hydroxyeicopentaenoic acid ), 9,15-dihydroxyeicopentaenoic acid, 11-hydroxydocosapentaenoic acid, 11,17-dihydroxyeicosapentaenoic acid (11, At least one selected from the group consisting of 17-dihydroxydocosapentaenoic acid, 11-hydroxydocosahexaenoic acid, and 11,17-dihydroxydocosahexaenoic acid, a hydroxylated fatty acid or A composition for preparing a dihydroxylated fatty acid.
According to claim 1,
The 9-lipoxygenase is composed of the amino acid sequence of SEQ ID NO: 1, and the 9-lipoxygenase variant is composed of the amino acid sequence of SEQ ID NO: 2, a composition for preparing hydroxylated fatty acid or dihydroxylated fatty acid.
According to claim 1,
The composition has at least one carbon number selected from the group consisting of arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid and docosahexaenoic acid, 20- A composition for preparing a hydroxylated fatty acid or a dihydroxylated fatty acid for treating a substrate containing 22 unsaturated fatty acids.
9-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 3; Or a composition for preparing hydroxylated fatty acid or dihydroxylated fatty acid comprising the 9-lipoxygenase mutant gene consisting of the nucleotide sequence of SEQ ID NO: 4 as an active ingredient,
The hydroxylated fatty acid or dihydroxylated fatty acid is 9-hydroxyarachidonic acid, 9,15-dihydroxyarachidonic acid, 9-hydroxyeicopentaenoic acid ), 9,15-dihydroxyeicopentaenoic acid, 11-hydroxydocosapentaenoic acid, 11,17-dihydroxyeicosapentaenoic acid (11, At least one selected from the group consisting of 17-dihydroxydocosapentaenoic acid, 11-hydroxydocosahexaenoic acid, and 11,17-dihydroxydocosahexaenoic acid, a hydroxylated fatty acid or A composition for preparing a dihydroxylated fatty acid.
A method for producing a hydroxylated fatty acid or a dihydroxylated fatty acid comprising the step of treating a substrate with the composition according to any one of claims 1 to 4,
The hydroxylated fatty acid or dihydroxylated fatty acid is 9-hydroxyarachidonic acid, 9,15-dihydroxyarachidonic acid, 9-hydroxyeicopentaenoic acid ), 9,15-dihydroxyeicopentaenoic acid, 11-hydroxydocosapentaenoic acid, 11,17-dihydroxyeicosapentaenoic acid (11, At least one selected from the group consisting of 17-dihydroxydocosapentaenoic acid, 11-hydroxydocosahexaenoic acid, and 11,17-dihydroxydocosahexaenoic acid, a hydroxylated fatty acid or Method for producing dihydroxy fatty acid.
6. The method of claim 5,
The substrate has at least one carbon number selected from the group consisting of arachidonic acid, eicosapentaenoic acid, eicosapentaenoic acid, and docosahexaenoic acid 20- A method for producing a hydroxylated fatty acid or a dihydroxylated fatty acid, comprising 22 unsaturated fatty acids.
6. The method of claim 5,
Wherein the treatment is carried out at a pH of 6.5 to 9.0 and a temperature of 20 to 40 ° C.
9-lipoxygenase gene consisting of the nucleotide sequence of SEQ ID NO: 3; Or as a recombinant expression vector for producing hydroxylated fatty acid or dihydroxylated fatty acid comprising a 9-lipoxygenase mutant gene consisting of the nucleotide sequence of SEQ ID NO: 4,
The hydroxylated fatty acid or dihydroxylated fatty acid is 9-hydroxyarachidonic acid, 9,15-dihydroxyarachidonic acid, 9-hydroxyeicopentaenoic acid ), 9,15-dihydroxyeicopentaenoic acid, 11-hydroxydocosapentaenoic acid, 11,17-dihydroxyeicosapentaenoic acid (11, At least one selected from the group consisting of 17-dihydroxydocosapentaenoic acid, 11-hydroxydocosahexaenoic acid, and 11,17-dihydroxydocosahexaenoic acid, a hydroxylated fatty acid or Recombinant expression vector for production of dihydroxylated fatty acid.
A transformant obtained by transforming a host cell with the recombinant expression vector according to claim 8 .
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