METHOD FOR PRODUCING RARE FATTY ACID USING NOVEL ENZYME, AND NOVEL RARE FATTY ACID

- KYOTO UNIVERSITY

The present invention provides production of hydroxylated fatty acid by a hydration reaction using a novel enzyme derived from lactobacillus and using fatty acid as a substrate, and further, a production method of oxo fatty acid by an enzyme reaction or chemical oxidation reaction using the hydroxylated fatty acid as a substrate. In addition, a valuable novel rare fatty acid obtained by such production method is also provided.

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Description
TECHNICAL FIELD

The present invention relates to a production method of a fatty acid. More particularly, the present invention relates to a production of a hydroxylated fatty acid by using an unsaturated fatty acid as a starting material, which is characterized by a hydration reaction by a novel enzyme, and further relates to a production of an oxo fatty acid from hydroxylated fatty acid by an enzyme reaction or chemical oxidation reaction. In addition, it relates to a novel rare fatty acid obtained by the production method.

BACKGROUND ART

Conjugated fatty acid represented by conjugated linoleic acid (CLA) has been reported to have various physiological activities such as a lipid metabolism improving effect, an anti-arteriosclerosis action, a body fats decreasing action and the like (non-patent documents 1-3), and is a functional lipid expected to be applicable to various fields of medicament, functional food and the like (patent documents 1, 2). While CLA is known to be contained in dairy products and meat products since it is produced by microorganisms present in the stomach of ruminant and to be present in a small amount in vegetable oil, the detailed mechanism of production thereof is not known.

The present inventors reported that enzymes present in the fungus of Lactobacillus plantarum (CLA-HY, CLA-DC, CLA-DH) are essential for the reaction to convert linoleic acid to conjugated linoleic acid (patent document 1). The mechanism of a series of specific reactions, the presence of an intermediate and the like in these enzyme reactions have been reported (patent document 4). While these enzyme reactions were effective for the production of a rare fatty acid having a hydroxyl group, a carbonyl group at the 10-position of an unsaturated fatty acid having 18 carbon atoms such as linoleic acid and the like, a production method of a rare fatty acid having a hydroxyl group, a carbonyl group at the 13-position has not been clarified.

In addition, it has been reported in recent years that oxo fatty acids such as 9-oxo-octadecadienoic acid, 13-oxo-octadecadienoic acid and the like contained in tomato have an activity to improve lifestyle-related diseases, such as lipid metabolism improvement and the like (patent document 3, non-patent documents 4, 5). Furthermore, hydroxylated fatty acid and oxo fatty acid having a hydroxyl group, an oxo group at the 10-position have been reported to have an activity to improve lifestyle-related diseases, such as metabolism improvement, lipid metabolism improvement and the like, and an activity to improve the intestine barrier function (patent documents 5, 6), hydroxylated fatty acid and oxo fatty acid are drawing an increasing attention. However, synthesis of functional hydroxylated fatty acid, functional oxo fatty acid from unsaturated fatty acid is difficult since, in hydroxylated fatty acid and oxo fatty acid, it is necessary to distinguish double bonds present in a plurality in a molecule of unsaturated fatty acid and introduce hydroxyl group, carbonyl group into particular positions.

DOCUMENT LIST Patent Documents

  • patent document 1: JP-A-2007-259712
  • patent document 2: JP-A-2007-252333
  • patent document 3: JP-A-2011-184411
  • patent document 4: WO 2013/168310
  • patent document 5: WO 2014/069227
  • patent document 6: WO 2014/129384

Non-Patent Documents

  • non-patent document 1: Ha Y L, (1987), Carcinogenesis, vol. 8, no. 12, p. 1881-1887
  • non-patent document 2: Clement Ip, (1991), Cancer Res., vol. 51, p. 6118-6124
  • non-patent document 3: Kisun N L, (1994), Atherosclerosis, vol. 108, p. 19-25
  • non-patent document 4: Kim Y-I, (2011), Mol. Nutr. Food Res., vol. 55, p. 585-593
  • non-patent document 5: Kim Y-I, (2012), PLoS ONE, vol. 7, no. 2, e31317

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

An object of the present invention is to provide a method of producing hydroxylated fatty acid, oxo fatty acid by using unsaturated fatty acid as a starting material and using a novel enzyme.

Means of Solving the Problems

The present inventors have clarified a reaction wherein Lactobacillus acidophilus hydroxylates the 13-position of an unsaturated fatty acid having 18 carbon atoms and identified a novel enzyme (FA-HY) involved in the production.

The present inventors have found a method of converting unsaturated fatty acid having 16, 18, 20 carbon atoms, cis-4, cis-7, cis-10, cis-13, cis-16, cis-19-docosahexaenoic acids (DHA) and cis-9-tetradecenoic acid (myristoleic acid) to hydroxylated fatty acid by using a novel enzyme (FA-HY), and further, a method of oxidizing a hydroxyl group of the produced substance by an enzyme reaction or chemical reaction.

The present inventor have produced found hydroxylated fatty acid and oxo fatty acid having a structure not found heretofore, by a production method of a new rare fatty acid by using a novel enzyme (FA-HY), and found that they have a nuclear receptor PPARα and PPARγ agonist activity.

To be specific, the present inventors have found that 13-hydroxy-cis-9-octadecenoic acid is produced from linoleic acid by using a novel enzyme (FA-HY), and further found that 13-oxo-cis-9-octadecenoic acid is produced from 13-hydroxy-cis-9-octadecenoic acid by introducing an enzyme reaction or a chemical oxidation method using chromic acid.

The present inventors have further studied and found that fatty acids hydroxylated at the 13-position, 10-position, the 15-position, the 12-position are produced, using a novel enzyme (FA-HY), respectively from an unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position (linoleic acid, γ-linolenic acid, α-linolenic acid, stearidonic acid etc.), unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position (pulmitoleic acid etc.), unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position (Dihomo-γ-linolenic acid or arachidonic acid etc.), unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position (cis-vaccenic acid, dihomo-γ-linolenic acid, mead acid etc.) as a substrate. Furthermore, they have found that 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid is produced by using DHA as a substrate and 10-hydroxy-tetradecanoic acid is produced by using myristoleic acid as a substrate. In addition, they have found that some of the hydroxylated fatty acids in the obtained resultant products have a structure not found before and they are novel substances. They have found that such hydroxylated fatty acid containing a novel fatty acid has an activity as an agonist of nuclear receptor PPARα and PPARγ, and is a substances utilizable as a metabolism improving agent and the like.

They have also found that hydroxylated tatty acid obtained by the present invention as a starting material can be converted to oxo fatty acid by introducing an enzyme reaction or a chemical oxidation method using chromic acid. They have found a novel substance having a structure not found before among oxo fatty acids of such resultant products. They have found that such oxo fatty acid including a novel fatty acid has an activity as an agonist of nuclear receptors PPARα and PPARγ, and they are substances utilizable as a metabolism improving agent and the like. The present invention has been completed based on the above findings.

Accordingly, the present invention provides the following:

[1] An enzyme protein of any of the following (a)-(c):
(a) the enzyme protein consisting of the amino acid sequence shown in SEQ ID NO: 2,
(b) a protein comprising an amino acid sequence wherein one or plural amino acids in the amino acid sequence shown in SEQ ID NO: 2 are deleted and/or substituted and/or inserted and/or added, and having an enzyme activity that catalyzes a hydration reaction,
(c) a protein encoded by a base sequence that hybridizes to a nucleic acid consisting of a chain sequence complementary to the base sequence shown in SEQ ID NO: 1 under stringent conditions, and having an enzyme activity that catalyzes a hydration reaction;
[2] Lactobacillus acidophilus fungus or fungal debris thereof, comprising the enzyme protein of [1];
[3] a nucleic acid encoding the enzyme protein of [1];
[4] a vector comprising the nucleic acid of [3];
[5] a host cell transformed with the vector of [4];
[6] a method of producing the enzyme protein of [1], comprising culturing the host cell of [5] and recovering the enzyme from the culture;
[7] a method of producing a hydroxylated fatty acid having 18 carbon atoms and a hydroxyl group at the 13-position, comprising subjecting an unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position to a hydration reaction using the enzyme protein of [1];
[8] a method of producing an oxo fatty acid having 18 carbon atoms and a carbonyl group at the 13-position, comprising subjecting an unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position to a hydration reaction using the enzyme protein of [1] to induce a hydroxylated fatty acid having 18 carbon atoms and a hydroxyl group at the 13-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation;
[9] a method of producing a hydroxylated fatty acid having 16 carbon atoms and a hydroxyl group at the 10-position, comprising subjecting an unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position to a hydration reaction using the enzyme protein of [1];
[10] a method of producing an oxo fatty acid having 16 carbon atoms and a carbonyl group at the 10-position, comprising subjecting an unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position to a hydration reaction using the enzyme protein of [1] to induce a hydroxylated fatty acid having 16 carbon atoms and a hydroxyl group at the 10-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation;
[11] a method of producing a hydroxylated fatty acid having 20 carbon atoms and a hydroxyl group at the 15-position, comprising subjecting an unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position to a hydration reaction using the enzyme protein of [1];
[12] a method of producing an oxo fatty acid having 20 carbon atoms and a carbonyl group at the 15-position, comprising subjecting an unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position to a hydration reaction using the enzyme protein of [1] to induce a hydroxylated fatty acid having 20 carbon atoms and a hydroxyl group at the 15-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation;
[13] a method of producing a hydroxylated fatty acid having 18 or 20 carbon atoms and a hydroxyl group at the 12-position, comprising subjecting an unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position to a hydration reaction using the enzyme protein of [1];
[14] a method of producing an oxo fatty acid having 18 or 20 carbon atoms and a carbonyl group at the 12-position, comprising subjecting an unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position to a hydration reaction using the enzyme protein of [1] to induce a hydroxylated fatty acid having 18 or 20 carbon atoms and a hydroxyl group at the 12-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation;
[15] the method of [7] or [8], wherein the unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position is linoleic acid, γ-linolenic acid, α-linolenic acid, stearidonic acid, 10-hydroxy-cis-12-octadecenoic acid, 10-hydroxy-cis-6, cis-12-octadecadienoic acid, 10-hydroxy-cis-12,cis-15-octadecadienoic acid, 10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid, 10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoic acid, 10-oxo-cis-12,cis-15-octadecadienoic acid, 10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, pinolenic acid or columbinic acid;
[16] the method of [9] or [10], wherein the unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position is pulmitoleic acid;
[17] the method of [11] or [12], wherein the unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position is cis-11,cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic acid, dihomo-γ-linolenic acid, cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, arachidonic acid, sciadonic acid or juniperonic acid;
[18] the method of [13] or [14], wherein the unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position is cis-vaccenic acid, cis-11-cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic, dihomo-γ-linolenic acid, mead acid, cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid or arachidonic acid;
[19] a method of producing 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, comprising subjecting cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid to a hydration reaction using the enzyme protein of [1];
[20] a method of producing 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, comprising subjecting cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid to a hydration reaction using the enzyme protein of [1] to induce 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation;
[21] a method of producing 10-hydroxy-tetradecanoic acid, comprising subjecting cis-9-tetradecenoic acid (myristoleic acid) to a hydration reaction using the enzyme protein of [1];
[22] a method of producing 10-oxo-tetradecanoic acid, comprising subjecting cis-9-tetradecenoic acid (myristoleic acid) to a hydration reaction using the enzyme protein of [1] to induce 10-hydroxy-tetradecanoic acid, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation;
[23] the method of [8], [10], [12], [14]-[18], [20] or [22], wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus;
[24] the method of [23], wherein the lactobacillus is Lactobacillus plantarum PERM BP-10549 strain;
[25] 13-hydroxy-cis-9,cis-15-octadecadienoic acid, 13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid, 10,13-dihydroxy-cis-6-octadecenoic acid, 10,13-dihydroxy-cis-15-octadecenoic acid, 10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid, 10-oxo-13-hydroxy-cis-6-octadecenoic acid, 10-oxo-13-hydroxy-cis-15-octadecenoic acid, 10-oxo-13-hydroxy-cis-6, cis-15-octadecadienoic acid, 13-hydroxy-cis-5, cis-9-octadecadienoic acid or 13-hydroxy-trans-5,cis-9-octadecadienoic acid, which is produced by the method of [7];
[26] 15-hydroxy-cis-11,cis-17-eicosadienoic acid, 15-hydroxy-cis-8,cis-11-eicosadienoic acid, 15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid, 15-hydroxy-cis-5,cis-11-eicosadienoic acid or 15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid, which is produced by the method of [11];
[27] 12-hydroxy-cis-14-eicosenoic acid, 12-hydroxy-cis-14,cis-17-eicosadienoic acid or 12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid, which is produced by the method of [13];
[28] 13-oxo-cis-6,cis-9-octadecadienoic acid, 13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid, 10,13-dioxo-cis-6-octadecenoic acid, 10,13-dioxo-cis-15-octadecenoic acid, 10,13-dioxo-cis-6,cis-15-octadecadienoic acid, 13-oxo-cis-5,cis-9-octadecadienoic acid or 13-oxo-trans-5,cis-9-octadecadienoic acid, which is produced by the method of [8];
[29] 15-oxo-cis-11,cis-17-eicosadienoic acid, 15-oxo-cis-8,cis-11-eicosadienoic acid, 15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid, 15-oxo-cis-5,cis-11-eicosadienoic acid or 15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid, which is produced by the method of [12];
[30] 12-oxo-cis-14-eicosenoic acid, 12-oxo-cis-14,cis-17-eicosadienoic acid, 12-oxo-cis-8,cis-14-eicosadienoic acid, 12-oxo-cis-5,cis-8-eicosadienoic acid or 12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid, which is produced by the method of [14];
[31] 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid produced by the method of [19];
[32] 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, which is produced by the method of [20];
[33] a fatty acid-containing substance comprising the hydroxylated fatty acid or oxo fatty acid of any one of [25]-[32];
[34] use of Lactobacillus acidophilus fungus or fungal debris thereof, comprising the enzyme protein of [1] in the production of the hydroxylated fatty acid or oxo fatty acid of any one of [25]-[32].

Effect of the Invention

In the present invention, a fatty acid hydration enzyme (FA-HY) conventionally not known was found, a method of converting unsaturated fatty acid having 16, 18 or 20 carbon atoms, DHA or myristoleic acid to a hydroxylated fatty acid was found, and further, a method of oxidizing a hydroxyl group of the resulting substance by an enzyme reaction or a chemical reaction was found. The rare fatty acid etc. to be produced are extremely useful since they are used in various fields such as medicament, food, cosmetic and the like. In addition, a novel rare fatty acid can be produced by the production using a novel enzyme, and the novel rare fatty acid also has an activity as an agonist of nuclear receptors PPARα and PPARγ. Therefore, it can be a useful substance in various fields such as medicament, food, cosmetic and the like.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of PPARα agonist activity of rare fatty acids derived from linoleic acid, α-linolenic acid, γ-linolenic acid, and arachidonic acid. EtOH shows a negative control (ethanol addition), and GW7647 shows a positive control (PPARα agonist addition). The vertical axis shows relative luciferase activity.

FIG. 2 shows the results of PPARγ agonist activity of rare fatty acids derived from linoleic acid, α-linolenic acid, γ-linolenic acid, and arachidonic acid. EtOH shows a negative control (ethanol addition), and Tro shows a positive control (PPARγ agonist addition). The vertical axis shows relative luciferase activity.

 DESCRIPTION OF EMBODIMENTS

The present invention is explained in detail below.

the present invention provides a novel fatty acid hydration enzyme “FA-HY”.

Specifically, the novel enzyme “FA-HY” of the present invention is

(a) the enzyme protein consisting of the amino acid sequence shown in SEQ ID NO: 2,
(b) a protein comprising an amino acid sequence wherein one or plural amino acids in the amino acid sequence shown in SEQ ID NO: 2 are deleted and/or substituted and/or inserted and/or added, and having an enzyme activity that the enzyme protein consisting of the amino acid sequence shown in SEQ ID NO: 2 has, or
(c) a protein encoded by a base sequence that hybridizes to a nucleic acid consisting of a chain sequence complementary to the base sequence shown in SEQ ID NO: 1 under stringent conditions, and having an enzyme activity that the enzyme protein consisting of the amino acid sequence shown in SEQ ID NO: 2 has.

More specific examples of the above-mentioned (b) include a protein containing (i) an amino acid sequence which is the amino acid sequence shown in SEQ ID NO: 2, wherein 1-20, preferably 1-10, more preferably 1-several (5, 4, 3 or 2) amino acids are deleted, (ii) an amino acid sequence which is the amino acid sequence shown in SEQ ID NO: 2, wherein 1-20, preferably 1-10, more preferably 1-several number (5, 4, 3 or 2) amino acids are added, (iii) an amino acid sequence which is the amino acid sequence shown in SEQ ID NO: 2, wherein 1-20, preferably 1-10, more preferably 1-several (5, 4, 3 or 2) amino acids are inserted, (iv) an amino acid sequence which is the amino acid sequence shown in SEQ ID NO: 2, wherein 1-20, preferably 1-10, more preferably 1-several (5, 4, 3 or 2) amino acids are substituted by other amino acids, or (v) an amino acid sequence obtained by combining them. When amino acids with similar properties (e.g., glycine and alanine, valine and leucine and isoleucine, serine and threonine, aspartic acid and glutamic acid, asparagine and glutamine, lysine and arginine, cysteine and methionine, phenylalanine and tyrosine etc.) are substituted with each other and the like, a greater number of substitutions and the like are possible.

When amino acids are deleted, substituted or inserted as mentioned above, the positions of deletion, substitution and insertion are not particularly limited as long as the above-mentioned enzyme activity is maintained.

In the above-mentioned (c), the “stringent conditions” are conditions under which nucleotide sequences having high identity, for example, identity of 70, 80, 90, 95 or 99% or above, hybridize to each other and nucleotide sequences having identity lower than that do not hybridize; specifically, conditions of washing once, more preferably 2-3 times, at the salt concentration and temperature corresponding to those in the washing conditions of general Southern hybridization (60° C., 1×SSC, 0.1% SDS, preferably, 0.1×SSC, 0.1% SDS, more preferably. 68° C., 0.1×SSC, 0.1% SDS) and the like.

Regarding the above-mentioned (b) or (c), the enzyme activity that the enzyme protein consisting of the amino acid sequence shown in SEQ ID NO: 2 has is not particularly limited as long as it has at least one, preferably all, of (1) an enzyme activity capable of converting an unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position (hereinafter sometimes to be abbreviated as “cis-12 unsaturated fatty acid”) utilized as a substrate to a hydroxylated fatty acid having 18 carbon atoms and a hydroxyl group at the 13-position (hereinafter sometimes to be abbreviated as “13-hydroxy fatty acid”) (reaction 1), (2) an enzyme activity capable of converting an unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position (hereinafter sometimes to be abbreviated as “cis-9 unsaturated fatty acid”)) utilized as a substrate to a hydroxylated fatty acid having 16 carbon atoms and a hydroxyl group at the 10-position (hereinafter sometimes to be abbreviated as “10-hydroxy fatty acid”) (reaction 2), (3) an enzyme activity capable of converting an unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position (hereinafter sometimes to be abbreviated as “cis-14 unsaturated fatty acid”)) utilized as a substrate to a hydroxylated fatty acid having 20 carbon atoms and a hydroxyl group at the 15-position (hereinafter sometimes to be abbreviated as “15-hydroxy fatty acid”) (reaction 3), (4) an enzyme activity capable of converting an unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position (hereinafter sometimes to be abbreviated as “cis-11 unsaturated fatty acid”)) utilized as a substrate to a hydroxylated fatty acid having 18 or 20 carbon atoms and a hydroxyl group at the 12-position (hereinafter sometimes to be abbreviated as “12-hydroxy fatty acid”) (reaction 4), an enzyme activity capable of converting cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid (DHA) to 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid (reaction A), and an enzyme activity capable of converting cis-9-tetradecenoic acid (myristoleic acid) to 10-hydroxy-tetradecanoic acid (reaction I)

The above-mentioned “cis-12 unsaturated fatty acid”, “cis-9 unsaturated fatty acid”, “cis-14 unsaturated fatty acid”, and “cis-11 unsaturated fatty acid” are not particularly limited as long as they are an unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position, an unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position, an unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position, an unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position, respectively and, for example, monovalent unsaturated fatty acid, divalent unsaturated fatty acid, trivalent unsaturated fatty acid, tetravalent unsaturated fatty acid, pentavalent unsaturated fatty acid and the like can be mentioned. In the present specification, the “fatty acid” encompasses not only free acids but also ester form, salt with basic compound and the like. The “DHA” and “myristoleic acid” also encompass not only free acids but also ester form, salt with basic compound and the like.

The FA-HY of the present invention can be isolated from, for example, the fungus, culture medium of Lactobacillus acidophilus by a protein separation and purification technique known per se. Alternatively, FA-HY may be used as the fungus of Lactobacillus acidophilus containing FA-HY or fungal debris thereof. The fungus of Lactobacillus acidophilus containing FA-HY is not particularly limited as long as it contains the FA-HY of the present invention and, for example, NITE BP-01788 and the like can be mentioned. Alternatively, FA-HY can also be produced as a recombinant protein by isolating a gene encoding FA-HY according to the method described in Example 2, subcloning same into a suitable vector, introducing same into a suitable host such as Escherichia coli and the like and culturing same. FA-HY may be a purified one or a crudely purified one. Alternatively, hydratase may be expressed in fungus such as Escherichia coli and the like and the fungus itself may be used or culture medium thereof may be used. Furthermore, the enzyme may be of a free form, or immobilized by various carriers.

As a vector containing a nucleic acid encoding FA-HY of the present invention, one suitable for a host cell to be introduced with the vector may be appropriately selected according to the object (e.g., protein expression) and can be used. In the case of an expression vector, it contains the nucleic acid of the present invention, which is operably linked to an appropriate promoter, and preferably contains a transcription termination signal, i.e., terminator region, at the downstream of the nucleic acid of the present invention. Furthermore, it can also contain a selection marker gene for selection of a transformant (drug resistance gene, gene that complements auxotrophic mutation etc.). Also, it may contain a sequence encoding a tag sequence useful for separation and purification of the expressed protein and the like. In addition, the vector may be incorporated into the genome of a target host cell. The vector of the present invention can be introduced into a target host cell by a transformation method known per se such as a competent cell method, a protoplast method, a calcium phosphate coprecipitation method and the like.

In the present invention, the “host cell” may be any cell as long as it can express a vector containing a nucleic acid encoding EA-HY of the present invention, and bacterium, yeast, fungi, higher eukaryotic cell and the like can be mentioned. Examples of the bacterium include gram-positive bacteria such as bacillus, Streptomyces and the like and gram negative bacteria such as Escherichia coli and the like. A recombinant cell introduced with a vector containing a nucleic acid encoding FA-HY can be cultivated by a method known per se which is suitable for the host cell.

“Purification” of the FA-HY of the present invention can be performed by a method known per se, for example, fungi collected by centrifugation and the like are ruptured by ultrasonication or glass beads and the like, solid such as cell debris is removed by centrifugation and the like, and the like to give a crude enzyme solution, which is subjected to a salting out method using ammonium sulfate, sodium sulfate and the like, chromatographys such as ion exchange chromatography, gel filtration chromatography, affinity chromatography and the like, gel electrophoresis and the like.

The FA-HY of the present invention has, as mentioned above, an enzyme activity capable of converting cis-12 unsaturated fatty acid, cis-9 unsaturated fatty acid, cis-14 unsaturated fatty acid, cis-11 unsaturated fatty acid, DHA, myristoleic acid utilized as substrates to 13-hydroxy fatty acid, 10-hydroxy fatty acid, 15-hydroxy fatty acid, 12-hydroxy fatty acid, 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, 10-hydroxy-tetradecanoic acid, respectively. Therefore, the present invention also provides [1] a method of producing 13-hydroxy fatty acid from cis-12 unsaturated fatty acid by a hydration reaction using the FA-HY of the present invention (production method 1), [2] a method of producing 10-hydroxy fatty acid from cis-9 unsaturated fatty acid by a hydration reaction using the FA-HY of the present invention (production method 2), [3] a method of producing 15-hydroxy fatty acid from cis-14 unsaturated fatty acid by a hydration reaction using the FA-HY of the present invention (production method 3), [4] a method of producing 12-hydroxy fatty acid from cis-11 unsaturated fatty acid by a hydration reaction using the FA-HY of the present invention (production method 4), [A] a method of producing 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid from DHA by a hydration reaction using the FA-HY of the present invention (production method A), and [I] a method of producing 10-hydroxy-tetradecanoic acid from myristoleic acid by a hydration reaction using the FA-HY of the present invention (production method I).

Examples of the “cis-12 unsaturated fatty acid” in the production method 1 of the present invention include cis-9,cis-12-octadecadienoic acid (linoleic acid), cis-6,cis-9,cis-12-octadecatrienoic acid (γ-linolenic acid), cis-9,cis-12,cis-15-octadecatrienoic acid (α-linolenic acid), cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (stearidonic acid), as well as 10-hydroxy-cis-12-octadecenoic acid, 10-hydroxy-cis-6,cis-12-octadecadienoic acid, 10-hydroxy-cis-12,cis-15-octadecadienoic acid, 10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid, 10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoic acid, 10-oxo-cis-12,cis-15-octadecadienoic acid, 10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, cis-5,cis-9,cis-12-octadecatrienoic acid(pinolenic acid), trans-5,cis-9,cis-12-octadecatrienoic acid (columbinic acid), which are now producible by WO 2013/168310, and the like. These substrates may be obtained by a method other than WO 2013/168310.

Examples of the “13-hydroxy fatty acid” produced by production method 1 of the present invention include 13-hydroxy-cis-9-octadecenoic acid induced from cis-9,cis-12-octadecadienoic acid (linoleic acid), 13-hydroxy-cis-6,cis-9-octadecadienoic acid induced from cis-6,cis-9,cis-12-octadecatrienoic acid (γ-linolenic acid), 13-hydroxy-cis-9,cis-15-octadecadienoic acid induced from cis-9,cis-12,cis-15-octadecatrienoic acid (α-linolenic acid), 13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid induced from cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (stearidonic acid), 10,13-dihydroxy-octadecanoic acid induced from 10-hydroxy-cis-12-octadecenoic acid, 10,13-dihydroxy-cis-6-octadecenoic acid induced from 10-hydroxy-cis-6,cis-12-octadecadienoic acid, 10,13-dihydroxy-cis-15-octadecenoic acid induced from 10-hydroxy-cis-12, cis-15-octadecadienoic acid, 10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid induced from 10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid, 10-oxo-13-hydroxy-octadecanoic acid induced from 10-oxo-cis-12-octadecenoic acid, 10-oxo-13-hydroxy-cis-6-octadecenoic acid induced from 10-oxo-cis-6, cis-12-octadecadienoic acid, 10-oxo-13-hydroxy-cis-15-octadecenoic acid induced from 10-oxo-cis-12,cis-15-octadecadienoic acid, 10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid induced from 10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, 13-hydroxy-cis-5, cis-9-octadecadienoic acid induced from cis-5,cis-9,cis-12-octadecatrienoic acid (pinolenic acid), 13-hydroxy-trans-5, cis-9-octadecadienoic acid induced from trans-5,cis-9,cis-12-octadecatrienoic acid (columbinic acid) and the like.

Examples of the “cis-9 unsaturated fatty acid” in production method 2 of the present invention include cis-9-hexadecenoic acid (pulmitoleic acid) and the like.

Examples of the “10-hydroxy fatty acid” produced by production method 2 of the present invention include 10-hydroxy-hexadecanoic acid induced from cis-9-hexadecenoic acid (pulmitoleic acid) and the like.

Examples of the “cis-14 unsaturated fatty acid” in production method 3 of the present invention include cis-11,cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic acid, cis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid), cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid), cis-5,cis-11,cis-14-eicosatrienoic acid (sciadonic acid), cis-5,cis-11,cis-14,cis-17-eicosatetraenoic acid (juniperonic acid) and the like.

Examples of the “15-hydroxy fatty acid” produced by production method 3 of the present invention include 15-hydroxy-cis-11-eicosenoic acid induced from cis-11,cis-14-eicosadienoic acid, 15-hydroxy-cis-11,cis-17-eicosadienoic acid induced from cis-11,cis-14,cis-17-eicosatrienoic acid, 15-hydroxy-cis-8,cis-11-eicosadienoic acid induced from cis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid), 15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid induced from cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, 15-hydroxy-cis-5,cis-8,cis-11-eicosatrienoic acid induced from cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid), 15-hydroxy-cis-5,cis-11-eicosadienoic acid induced from cis-5,cis-11,cis-14-eicosatrienoic acid (sciadonic acid), 15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid induced from cis-5,cis-11,cis-14,cis-17-eicosatetraenoic acid (juniperonic acid) and the like.

Examples of the “cis-11 unsaturated fatty acid” in production method 4 of the present invention include cis-11-octadecenoic acid (cis-vaccenic acid), cis-11,cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic acid, cis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid), cis-5,cis-8,cis-11-eicosatrienoic acid (mead acid), cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid) and the like.

Examples of the “12-hydroxy fatty acid” produced by production method 4 of the present invention include 12-hydroxy-octadecanoic acid induced from cis-11-octadecenoic acid(cis-vaccenic acid), 12-hydroxy-cis-14-eicosenoic acid induced from cis-11,cis-14-eicosadienoic acid, 12-hydroxy-cis-14,cis-17-eicosadienoic acid induced from cis-11,cis-14,cis-17-eicosatrienoic acid, 12-hydroxy-cis-8,cis-14-eicosadienoic acid induced from cis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid), 12-hydroxy-cis-5,cis-8-eicosadienoic acid induced from cis-5,cis-8,cis-11-eicosatrienoic acid (mead acid), 12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid induced from cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, 12-hydroxy-cis-5,cis-8,cis-14-eicosatrienoic acid induced from cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid) and the like.

The hydration reaction may be performed in a suitable buffer (e.g., phosphate buffer, tris buffer, borate buffer etc.) by mixing unsaturated fatty acid, which is a substrate, and FA-HY of the present invention at suitable concentrations and incubating the mixture. The substrate concentration is, for example, 1-1000 g/L, preferably 10-500 g/L, more preferably 20-250 g/L. The amount of the aforementioned FA-HY to be added is, for example, 0.001-10 mg/mL, preferably 0.1-5 mg/mL, more preferably 0.2-2 mg/mL.

A “cofactor” may be used for a hydration reaction (reaction 1-4, reaction A or reaction I) and, for example, FAD and the like can be used. The concentration of addition may be any as long as the hydration reaction proceeds efficiently. It is preferably 0.001-20 mM, more preferably 0.01-10 mM.

Furthermore, an “activator” may be used for the hydration reaction and, for example, 1 or 2 compounds selected from the group consisting of NADH and NADPH can be mentioned. The concentration of addition thereof may be any as long as the hydration reaction proceeds efficiently. It is preferably 0.1-20 mM, more preferably 1-10 mM.

The hydration reaction is desirably performed at a preferable temperature and in a preferable pH range for the FA-HY of the present invention. For example, the reaction temperature is 5-50° C., preferably 20-45° C. The pH of the reaction mixture is, for example, pH 4-10, preferably pH 5-9. The reaction time is not particularly limited and it is, for example, 10 min-72 hr, preferably 30 min-36 hr.

In one preferable embodiment of the present invention, the FA-HY of the present invention is provided to the reaction system in the form of recombinant cells (e.g., Escherichia coli, Bacillus subtilis, yeast, insect cell, animal cell etc.) introduced with an expression vector containing a nucleic acid encoding same. In this case, the hydration reaction can also be performed by cultivating the cells in a liquid medium suitable for the culture of the cells and added with cofactor and a substrate and, where necessary, an activator.

Furthermore, by a dehydrogenation reaction or chemical oxidation using chrome acid, an oxo fatty acid having 18 carbon atoms and a carbonyl group at the 13-position (hereinafter sometimes to be abbreviated as “13-oxo fatty acid”) is produced from 13-hydroxy fatty acid obtained in production methods 1-4, production method A, production method I of the present invention (reaction 5), an oxo fatty acid having 16 carbon atoms and a carbonyl group at the 10-position (hereinafter sometimes to be abbreviated as “10-oxo fatty acid”) is produced from 10-hydroxy fatty acid (reaction 6), an oxo fatty acid having 20 carbon atoms and a carbonyl group at the 15-position (hereinafter sometimes to be abbreviated as “15-oxo fatty acid”) is produced from 15-hydroxy fatty acid (reaction 7), an oxo fatty acid having 18 or 20 carbon atoms and a carbonyl group at the 12-position (hereinafter sometimes to be abbreviated as “12-oxo fatty acid”) is produced from 12-hydroxy fatty acid (reaction 8), 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid is produced from 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid (reaction B), and 10-oxo-tetradecanoic acid is produced from 10-hydroxy-tetradecanoic acid (reaction II).

Therefore, the present invention also provides [5] a method of producing 13-oxo fatty acid, comprising subjecting cis-12 unsaturated fatty acid to a hydration reaction using the FA-HY of the present invention to induce 13-hydroxy fatty acid, and subjecting the 13-hydroxy fatty acid to a dehydrogenation reaction or chemical oxidation (production method 5), [6] a method of producing 10-oxo fatty acid, comprising subjecting cis-9 unsaturated fatty acid to a hydration reaction using the FA-HY of the present invention to induce 10-hydroxy fatty acid, and subjecting the 10-hydroxy fatty acid to a dehydrogenation reaction or chemical oxidation (production method 6), [7] a method of producing 15-oxo fatty acid, comprising subjecting cis-14 unsaturated fatty acid to a hydration reaction using the FA-HY of the present invention to induce 15-hydroxy fatty acid, and subjecting the 15-hydroxy fatty acid to a dehydrogenation reaction or chemical oxidation (production method 7), [8] a method of producing 12-oxo fatty acid, comprising subjecting cis-11 unsaturated fatty acid to a hydration reaction using the FA-HY of the present invention to induce 12-hydroxy fatty acid, and subjecting the 12-hydroxy fatty acid to a dehydrogenation reaction or chemical oxidation (production method 8), [B] a method of producing 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, comprising subjecting DHA to a hydration reaction using the EA-HY of the present invention to induce 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, and subjecting the 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid to a dehydrogenation reaction or chemical oxidation (production method B), and [II] a method of producing 10-oxo-tetradecanoic acid, comprising subjecting myristoleic acid to a hydration reaction using the FA-HY of the present invention to induce 10-hydroxy-tetradecanoic acid, and subjecting the 10-hydroxy-tetradecanoic acid to a dehydrogenation reaction or chemical oxidation (production method II).

The “cis-12 unsaturated fatty acid”, “cis-9 unsaturated fatty acid”, “cis-14 unsaturated fatty acid”, “cis-11 unsaturated fatty acid” in the production methods 5-8 of the present invention are the same as the substrates in the above-mentioned production methods 1-4.

Examples of the “13-oxo fatty acid” produced by the production method 5 of the present invention include 13-oxo-cis-9-octadecenoic acid induced from cis-9,cis-12-octadecadienoic acid (linoleic acid), 13-oxo-cis-6,cis-9-octadecadienoic acid induced from cis-6,cis-9,cis-12-octadecatrienoic acid (γ-linolenic acid), 13-oxo-cis-9,cis-15-octadecadienoic acid induced from cis-9,cis-12,cis-15-octadecatrienoic acid (α-linolenic acid), 13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid induced from cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (stearidonic acid), 10,13-dioxo-octadecanoic acid induced from 10-hydroxy-cis-12-octadecenoic acid or 10-oxo-cis-12-octadecenoic acid, 10,13-dioxo-cis-6-octadecenoic acid induced from 10-hydroxy-cis-6,cis-12-octadecadienoic acid or 10-oxo-cis-6,cis-12-octadecadienoic acid, 10,13-dioxo-cis-15-octadecenoic acid induced from 10-hydroxy-cis-12,cis-15-octadecadienoic acid or 10-oxo-cis-12,cis-15-octadecadienoic acid, 10,13-dioxo-cis-6,cis-15-octadecadienoic acid induced from 10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid or 10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, 13-oxo-cis-5,cis-9-octadecadienoic acid induced from cis-5,cis-9,cis-12-octadecatrienoic acid (pinolenic acid), 13-oxo-trans-5,cis-9-octadecadienoic acid induced from trans-5,cis-9,cis-12-octadecatrienoic acid (columbinic acid) and the like.

Examples of the “10-oxo fatty acid” produced by the production method 6 of the present invention include 10-oxo-hexadecanoic acid induced from cis-9-hexadecenoic acid (pulmitoleic acid) and the like.

Examples of the “15-oxo fatty acid” produced by the production method 7 of the present invention include 15-oxo-cis-11-eicosenoic acid induced from cis-11,cis-14-eicosadienoic acid, 15-oxo-cis-11,cis-17-eicosadienoic acid induced from cis-11,cis-14,cis-17-eicosatrienoic acid, 15-oxo-cis-8,cis-11-eicosadienoic acid induced from cis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid), 15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid induced from cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, 15-oxo-cis-5,cis-8,cis-11-eicosatrienoic acid induced from cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid), 15-oxo-cis-5,cis-11-eicosadienoic acid induced from cis-5,cis-11,cis-14-eicosatrienoic acid (sciadonic acid), 15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid induced from cis-5,cis-11,cis-14,cis-17-eicosatetraenoic acid (juniperonic acid) and the like.

Examples of the “12-oxo fatty acid” produced by the production method 8 of the present invention include 12-oxo-octadecanoic acid induced from cis-11-octadecenoic acid (cis-vaccenic acid), 12-oxo-cis-14-eicosenoic acid induced from cis-11,cis-14-eicosadienoic acid, 12-oxo-cis-14,cis-17-eicosadienoic acid induced from cis-11,cis-14,cis-17-eicosatrienoic acid, 12-oxo-cis-8, cis-14-eicosadienoic acid induced from cis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid), 12-oxo-cis-5,cis-8-eicosadienoic acid induced from cis-5,cis-8,cis-11-eicosatrienoic acid (mead acid), 12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid induced from cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, 12-oxo-cis-5,cis-8,cis-14-eicosatrienoic acid induced from cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid) and the like.

The dehydrogenase to be used in the production methods 5-8, production method B or production method II of the present invention is not particularly limited as long as it is an enzyme capable of converting 13-hydroxy fatty acid, 10-hydroxy fatty acid, 15-hydroxy fatty acid, 12-hydroxy fatty acid, 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, 10-hydroxy-tetradecanoic acid utilized as substrates to 13-oxo fatty acid, 10-oxo fatty acid, 15-oxo fatty acid, 12-oxo fatty acid, 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, 10-oxo-tetradecanoic acid, respectively and, for example, lactobacillus-derived hydroxylated fatty acid-dehydrogenase (CLA-DH) is preferable. More preferred is Lactobacillus plantarum-derived CLA-DH, and particularly preferred is L. plantarum FERN BP-10549 strain-derived CLA-DH. CLA-DH can be obtained by the method described in JP-A-2007-259712, the method described in WO 2013/168310. Dehydrogenase may be a purified one or a crudely purified one. Alternatively, dehydrogenase may be expressed in fungus such as Escherichia coli and the like and the fungus itself may be used or culture medium thereof may be used. Furthermore, the enzyme may be of a free form, or immobilized by various carriers.

The dehydrogenation reaction is performed in a suitable buffer (e.g., phosphate buffer, tris buffer, borate buffer etc.) by mixing 13-hydroxy fatty acid, 10-hydroxy fatty acid, 15-hydroxy fatty acid, 12-hydroxy fatty acid, 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, 10-hydroxy-tetradecanoic acid as substrates and dehydrogenase at suitable concentrations and incubating the mixture. The substrate concentration is, for example, 0.01-100 g/L, preferably 0.05-50 g/L, more preferably 0.1-5 g/L. The amount of dehydrogenase to be added is, for example, 0.001-10 mg/mL, preferably 0.005-1 mg/mL, more preferably 0.05-0.2 mg/mL.

A “cofactor” may be used for the dehydrogenation reaction and, for example, NAD+, NADP+ and the like can be used. The concentration of addition thereof may be any as long as the hydration reaction proceeds efficiently. It is preferably 0.001-20 mM, more preferably 0.01-10 mM.

The dehydrogenation reaction is desirably performed within the ranges of preferable temperature and preferable pH of dehydrogenase. For example, the reaction temperature is 5-50° C., preferably 20-45° C. The pH of the reaction mixture is, for example, pH 4-10, preferably pH 5-9. The reaction time is not particularly limited and it is, for example, 10 min-72 hr, preferably 30 min-36 hr.

In one embodiment of the present invention, dehydrogenase is subjected to the reaction system in the form of recombinant cells (e.g., Escherichia coli, Bacillus subtilis, yeast, insect cell, animal cell etc.) introduced with an expression vector containing a nucleic acid encoding same. In this case, the oxidation reaction can also be performed by cultivating the cells in a liquid medium suitable for the culture of the cells and added with a substrate and, where necessary, a cofactor and an activator.

In addition, by replacing the dehydrogenation reaction with a chemical oxidation using chromic acid, an oxo fatty acid similar to that by enzyme reaction can be chemically obtained.

As the chemical oxidation, methods known per se, for example, chromic acid oxidation, preferably Jones oxidation and the like can be mentioned. As the chromic acid, salts or complexes of the compound such as anhydrous chromic acid CrO3, chromic acid H2CrO4 and dichromic acid H2Cr2O7 can be used.

To be specific, sulfuric acid (2.3 ml) and water (7.7 ml) are added to anhydrous chromic acid (2.67 g), and acetone (90 ml) is added to the mixture to give a chromic acid solution. 2 g of hydroxylated fatty acid and 40 ml of acetone are added in an Erlenmeyer flask, and the above-mentioned chromic acid solution is added by one drop while stirring in a stirrer on ice. When the solution turns from blue to tea green, dropwise addition of the chromic acid solution is stopped, and the reaction is discontinued with isopropyl alcohol. The precipitated sediment is filtered through filter paper, placed in a separating funnel, diethyl ether (150 ml) and Milli-Q water (300 ml) are added, the mixture is shaken well, and the diethyl ether layer is washed several times with Milli-Q water. To the diethyl ether layer after washing is added an appropriate amount of sodium sulfate (anhydrous), the mixture is stirred and the residual water is removed. The anhydrous sodium sulfate added is filtered off through filter paper, the obtained diethyl ether layer is concentrated by a rotary evaporator, and the reaction product (oxo fatty acid) and unreacted substrate are extracted.

An extract obtained by an oxidation reaction with anhydrous chromic acid (mixture containing substrate and resultant product (oxo fatty acid)) is subjected to moderate-pressure chromatography, a solution that comes out from the column is recovered in fractions. The recovered each fraction is analyzed by LC/MS and gas chromatography, fractions containing oxo fatty acid alone are collected and concentrated by a rotary evaporator. A part of the obtained final resultant product is methylesterified, the purity of oxo fatty acid is evaluated by gas chromatography, and oxo fatty acid having a purity of not less than 98% can be obtained.

The following hydroxylated fatty acid and oxo fatty acid obtained by the production methods 1-8 and production methods A, B of the present invention are novel fatty acids having structures conventionally not known.

<Hydroxylated Fatty Acid>

  • 13-hydroxy-cis-9, cis-15-octadecadienoic acid
  • 13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid
  • 13-hydroxy-cis-5, cis-9-octadecadienoic acid
  • 13-hydroxy-trans-5, cis-9-octadecadienoic acid
  • 12-hydroxy-cis-14-eicosenoic acid
  • 12-hydroxy-cis-14,cis-17-eicosadienoic acid
  • 15-hydroxy-cis-11,cis-17-eicosadienoic acid
  • 15-hydroxy-cis-8,cis-11-eicosadienoic acid
  • 12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid
  • 15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid
  • 15-hydroxy-cis-5,cis-11-eicosadienoic acid
  • 15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid
  • 10,13-dihydroxy-cis-6-octadecenoic acid
  • 10,13-dihydroxy-cis-15-octadecenoic acid
  • 10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid
  • 10-oxo-13-hydroxy-cis-6-octadecenoic acid
  • 10-oxo-13-hydroxy-cis-15-octadecenoic acid
  • 10-oxo-13-hydroxy-cis-6, cis-15-octadecadienoic acid
  • 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid

<Oxo Fatty Acid>

  • 13-oxo-cis-6, cis-9-octadecadienoic acid
  • 13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid
  • 13-oxo-cis-5, cis-9-octadecadienoic acid
  • 13-oxo-trans-5, cis-9-octadecadienoic acid
  • 12-oxo-cis-14-eicosenoic acid
  • 12-oxo-cis-14,cis-17-eicosadienoic acid
  • 15-oxo-cis-11,cis-17-eicosadienoic acid
  • 15-oxo-cis-8,cis-11-eicosadienoic acid
  • 12-oxo-cis-8,cis-14-eicosadienoic acid
  • 12-oxo-cis-5,cis-8-eicosadienoic acid
  • 12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid
  • 15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid
  • 15-oxo-cis-5,cis-11-eicosadienoic acid
  • 15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid
  • 10,13-dioxo-cis-6-octadecenoic acid
  • 10,13-dioxo-cis-15-octadecenoic acid
  • 10,13-dioxo-cis-6,cis-15-octadecadienoic acid
  • 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid

Since the hydroxylated fatty acids and oxo fatty acids obtained by the present invention have an activity as an agonist of nuclear receptors PPARα and PPARγ, they can be utilized as metabolism improving agents and the like. Therefore, novel hydroxylated fatty acids and oxo fatty acids are also novel substances that can be utilized as metabolism improving agents and the like.

The hydroxylated fatty acid and oxo fatty acid obtained in the present invention can be used by being blended with, for example, medicament, food or cosmetic agent based on the conventionally-known physiological activity.

The dosage form of the medicaments containing hydroxylated fatty acid, oxo fatty acid includes, for example, powder, granule, pill, soft capsule, hard capsules, tablet, chewable tablet, quick-integrating tablet, syrup, liquid, suspension, suppository, ointment, cream, gel, adhesive, inhalant, injection and the like. A preparation thereof is formulated according to a conventional method. Since oxo fatty acid and the like are poorly soluble in water, they are dissolved in a non-hydrophilic organic solvent such as plant-derived oil, animal-derived oil and the like or dispersed or emulsified in an aqueous solution together with an emulsifier, a dispersing agent, a surfactant and the like by a homogenizer (high-pressure homogenizer) and used.

Examples of the additives that can be used for formulating include animal and plant oils such as soybean oil, safflower oil, olive oil, germ oil, sunflower oil, beef fat, sardine oil and the like, polyalcohols such as polyethylene glycol, propylene glycol, glycerol, sorbitol and the like, surfactants such as sorbitan ester of fatty acid, sucrose ester of fatty acid, glycerin fatty acid ester, polyglycerol ester of fatty acid and the like, excipients such as purified water, lactose, starch, crystalline cellulose, D-mannitol, lecithin, gum arabic, sorbitol solution, carbohydrate solution and the like, sweetener, colorant, pH adjuster, flavor and the like. A liquid preparation may be dissolved or suspended in water or other suitable medium when in use. Also, tablet and granules may be coated by a well-known method.

For administration in the form of an injection, intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal, intraarticular, intrasynovial, intrathecal, intraperiosteum, sublingual, oral administrations and the like are preferable, and intravenous administration or intraperitoneal administration is particularly preferable. The intravenous administration may be any of drip administration and bolus administration.

Examples of the form of the “food” containing the hydroxylated fatty acid, oxo fatty acid and the like obtained by the present invention include supplement (powder, granule, soft capsule, hard capsules, tablet, chewable tablet, quick-integrating tablet, syrup, liquid etc.), drinks (tea drinks, carbonic acid drinks, lactic acid drinks, sport drinks etc.), confectionery (gummy candy, jelly, gum, chocolate, cookie, candy etc.), oil, fat and oil foods (mayonnaise, dressing, butter, cream, margarine etc.) and the like.

The above-mentioned foods can contain, where necessary, various nutrients, various vitamins (vitamin A, vitamin B1, vitamin B2, vitamin B6, vitamin C, vitamin D, vitamin E, vitamin K etc.), various minerals (magnesium, zinc, iron, sodium, potassium, selenium etc.), dietary fiber, dispersing agent, stabilizer such as emulsifier and the like, sweetener, flavor components (citric acid, malic acid etc.), flavor, royal jelly, propolis, Agaricus and the like.

Examples of the “cosmetics” containing the hydroxylated fatty acid, oxo fatty acid and the like obtained by the present invention include cream, skin milk, toner, microemulsion essence, bathing powder and the like, and flavor and the like may be added.

The present invention is explained in more detail in the following by referring to Examples. The Examples are mere exemplifications of the present invention and do not limit the scope of the present invention in any manner.

Example 1 Culture Method of Lactobacillus acidophilus

Lactobacillus acidophilus was inoculated in 15 ml of MRS liquid medium (manufactured by Difco; pH 6.5) from MRS high layer medium containing 2% agar and preserved at 4° C., and cultured at 37° C. for 20 hr. After the culture, the cells were collected by centrifugation at 3,000 rpm, 4° C. for 10 min to give the fungus of Lactobacillus acidophilus.

The aforementioned Lactobacillus acidophilus was deposited on Jan. 17, 2014 at the NITE Patent Microorganisms Depositary (NPMD) under deposit number NITE BP-01788.

Example 2 Cloning of Gene of Enzyme (FA-HY: Fatty Acid Hydration Enzyme) Having the Amino Acid Sequence Shown in SEQ ID NO: 2 (1) Obtainment of Genome DNA

The aforementioned Lactobacillus acidophilus was inoculated in 10 ml of MRS liquid medium (manufactured by Difco) and standing culture was performed at 37° C. overnight, and the cells were collected by centrifugation. The obtained fungus was washed twice with 1 ml of sterile water and added with 0.1 ml of sterile water, 0.125 ml of lysis buffer (80 mM EDTA containing 200 mM Tris-HCl buffer (pH 8.0)), 0.25 ml of TE saturated phenol, and the mixture was vortexed for 1 min and the supernatant was recovered by centrifugation. To the obtained supernatant (0.2 ml) was added 0.2 ml of PCI solution (manufactured by Nacalai Tesque), and the mixture was hand-shaken, and the supernatant (0.125 ml) was recovered by centrifugation at 15,000 rpm, 4° C., 5 min. 0.0125 ml of 5 M NaCl, 0.31 ml of ethanol were added, and the mixture was stood at room temperature for 10 min and centrifuged at 15,000 rpm, 4° C., for 5 min. The supernatant was discarded and the pellets were washed with 0.2 ml of 70% ethanol. 0.1 ml of sterile water was added to the pellets and the mixture was incubated at 65° C. for 10 min to give genomic DNA.

(2) Obtainment of FA-HY Gene by PCR

In the published total genomic gene sequence of Lactobacillus acidophilus strain, open-reading-frame (ORF) having homology (about 32%) with gene sequence of CLA-HY in WO/2013/168310 was targeted. A sense primer (SEQ ID NO: 3) was designed based on the sequence of the 5′ side of the initiation codon of ORF, and an antisense primer (SEQ ID NO: 4) was designed based on the sequence of the 3′ side of the stop codon. Using these primers, genome DNA of Lactobacillus acidophilus as a templates, PCR was performed. The base sequence of about 1.8 kbp gene segment amplified as a result of PCR was analyzed. It was clarified that this gene segment contained a 1,773 bp one open-reading-frame (ORF) (SEQ ID NO: 1) starting from the initiation codon ATG and ending at stop codon TAG, and this gene was taken as FA-HY gene. The FA-HY gene encodes a protein consisting of 590 residual amino acids shown in SEQ ID NO: 2.

Example 3 Expression of (FA-HY) in Escherichia coli

A host vector system consisting of Escherichia coli expression vector pET21b (Novagen) and Rosetta 2 (DE3) strain was used. FA-HY gene segment amplified by PCR using genomic DNA of Lactobacillus acidophilus as a template was inserted into pET21b to construct an expression vector (pFA-HY). Rosetta 2 (DE3) strain was transformed with pFA-HY to give a transformed Rosetta/pFA-HY strain. The obtained Rosetta/pFA-HY strain was aerobically cultured at 37° C., 300 rpm in a 10 ml LB medium containing 0.5 mg ampicillin, 0.3 mg chloramphenicol (medium containing 1% Bacto Tripton (Difco), 0.5% yeast extract, 1% sodium chloride (pH 7.0)), 1 μl of 1 M IPTG was added when OD600 nm was 0.5, and the mixture was further cultured at 16° C. for 18 hr. After culture, the mixture was centrifuged at 3,000 rpm for 10 min to give wet fungus of Rosetta/pFA-HY strain.

Example 4 Production of Hydroxylated Fatty Acid from Unsaturated Fatty Acid Using Transformed Escherichia coli Expressing Fatty Acid Hydration Enzyme

Hydroxylated fatty acid production test from various unsaturated fatty acids was performed using fatty acid hydration enzyme induction transformed Escherichia coli. The total amount of the reaction mixture was adjusted to 1 ml with 100 mM potassium phosphate buffer (pH 6.5) containing fatty acid hydration enzyme induction transformed Escherichia coli (wet body weight 0.3 g/ml), NADH (5 mM), FAD (0.1 mM), unsaturated fatty acid (100 mg), BSA (10 mg). The reaction was performed by anaerobically shaking at 37° C., 120 rpm for 16 to 60 hr in the presence of an oxygen adsorbent Anaeropack (Mitsubishi Chemical Corporation). After the reaction, 2 ml of chloroform, 2 ml of methanol, and 1 ml of 0.5% KCl were added to the reaction mixture (1 ml), and the mixture was stirred and the chloroform layer was recovered. The recovered chloroform layer was concentrated by a centrifugation evaporator, and the reaction product and unreacted substrate were extracted. A part of the extract was methylesterified and the reaction product was evaluated by gas chromatography.

Example 5 Purification of Resultant Product from Extract Obtained in Example 4 (Mixture Containing Substrate and Resultant Product (Hydroxylated Fatty Acid))

An extract obtained from Example 4 (mixture containing substrate and resultant product (hydroxylated fatty acid)) was subjected to moderate-pressure chromatography, a solution that came out from the column was recovered in fractions. The recovered each fraction was analyzed by LC/MS and gas chromatography, fractions containing resultant product (hydroxylated fatty acid) alone were collected and concentrated by a rotary evaporator. A part of the obtained final resultant product (hydroxylated fatty acid) was methylesterified, and the purity of the resultant product was evaluated by gas chromatography. As a result, a resultant product having a purity of not less than 98% was obtained from each substrate. The chemical structure of the resultant product was determined by NMR, two-dimensional NMR, GC-MS analysis and the like. As a result, 10-hydroxy-hexadecanoic acid having a purity of not less than 98% could be obtained from cis-9-hexadecenoic acid (pulmitoleic acid). 12-Hydroxy-octadecanoic acid having a purity of not less than 98% could be obtained from cis-11-octadecenoic acid(cis-vaccenic acid). 13-Hydroxy-cis-9-octadecenoic acid having a purity of not less than 98% could be obtained from cis-9,cis-12-octadecadienoic acid (linoleic acid). 13-Hydroxy-cis-6,cis-9-octadecadienoic acid having a purity of not less than 98% could be obtained from cis-6,cis-9,cis-12-octadecatrienoic acid (γ-linolenic acid). 13-hydroxy-cis-9,cis-15-octadecadienoic acid having a purity of not less than 98% could be obtained from cis-9,cis-12,cis-15-octadecatrienoic acid (α-linolenic acid). 13-Hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid having a purity of not less than 98% could be obtained from cis-6,cis-9,cis-12,cis-15-octadecatetraenoic acid (stearidonic acid). 12-Hydroxy-cis-14-eicosenoic acid and 15-hydroxy-cis-11-eicosenoic acid having a purity of not less than 98% could be obtained from cis-11,cis-14-eicosadienoic acid. 12-Hydroxy-cis-14,cis-17-eicosadienoic acid and 15-hydroxy-cis-11,cis-17-eicosadienoic acid having a purity of not less than 98% could be obtained from cis-11,cis-14,cis-17-eicosatrienoic acid. 15-Hydroxy-cis-8,cis-11-eicosadienoic acid and 12-hydroxy-cis-8,cis-14-eicosadienoic acid having a purity of not less than 98% could be obtained from cis-8,cis-11,cis-14-eicosatrienoic acid (dihomo-γ-linolenic acid). 12-Hydroxy-cis-5,cis-8-eicosadienoic acid having a purity of not less than 98% could be obtained from cis-5,cis-8,cis-11-eicosatrienoic acid (mead acid). 12-Hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid and 15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid having a purity of not less than 98% could be obtained from cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid. 15-Hydroxy-cis-5,cis-8,cis-11-eicosatrienoic acid and 12-hydroxy-cis-5,cis-8,cis-14-eicosatrienoic acid having a purity of not less than 98% could be obtained from cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid (arachidonic acid). 10,13-Dihydroxy-octadecanoic acid having a purity of not less than 98% could be obtained from 10-hydroxy-cis-12-octadecenoic acid. 10,13-Dihydroxy-cis-6-octadecenoic acid having a purity of not less than 98% could be obtained from 10-hydroxy-cis-6, cis-12-octadecadienoic acid. 10,13-Dihydroxy-cis-15-octadecenoic acid having a purity of not less than 98% could be obtained from 10-hydroxy-cis-12,cis-15-octadecadienoic acid. 10,13-Dihydroxy-cis-6,cis-15-octadecadienoic acid having a purity of not less than 98% could be obtained from 10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid. 10-Oxo-13-hydroxy-octadecanoic acid having a purity of not less than 98% could be obtained from 10-oxo-cis-12-octadecenoic acid. 10-Oxo-13-hydroxy-cis-6-octadecenoic acid having a purity of not less than 98% could be obtained from 10-oxo-cis-6,cis-12-octadecadienoic acid. 10-Oxo-13-hydroxy-cis-15-octadecenoic acid having a purity of not less than 98% could be obtained from 10-oxo-cis-12,cis-15-octadecadienoic acid. 10-Oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid having a purity of not less than 98% could be obtained from 10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid. 13-Hydroxy-cis-5,cis-9-octadecadienoic acid having a purity of not less than 98% could be obtained from cis-5,cis-9,cis-12-octadecatrienoic acid (pinolenic acid). 13-Hydroxy-trans-5,cis-9-octadecadienoic acid having a purity of not less than 98% could be obtained from trans-5,cis-9,cis-12-octadecatrienoic acid (columbinic acid). 15-Hydroxy-cis-5,cis-11-eicosadienoic acid having a purity of not less than 98% could be obtained from cis-5,cis-11,cis-14-eicosatrienoic (Sciadonic acid). 15-Hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid having a purity of not less than 98% could be obtained from cis-5,cis-11,cis-14,cis-17-eicosatetraenoic acid (juniperonic acid). 14-Hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid having a purity of not less than 98% could be obtained from cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid (DHA). 10-Hydroxy-tetradecanoic acid having a purity of not less than 98% could be obtained from cis-9-tetradecenoic acid(myristoleic acid).

Example 6 Production of Oxo Fatty Acid from Hydroxylated Fatty Acid by Using Dehydrogenase (CLA-DH) Expressed in Escherichia coli

An oxo fatty acid production test from hydroxylated fatty acid was performed using purified dehydrogenase (CLA-DH) derived from L. plantarum FERN BP-10549 strain obtained by the method described in JP-A-2007-259712 or the method described in WO/2013/168310. The reaction mixture was adjusted to the total amount of 1 ml with 50 mM potassium phosphate buffer (pH 8.0) containing purified dehydrogenase (enzyme amount 83 μg), 0.5 mM NAD+, 0.5 mg hydroxylated fatty acid. The reaction was performed by anaerobically shaking at 37° C., 120 rpm for 24 hr in the presence of an oxygen adsorbent Anaeropack (Mitsubishi Chemical Corporation). After the reaction, lipid was extracted by Bligh-Dyer method from the reaction mixture. The extract was methylesterified and production of oxo fatty acid was evaluated by gas chromatography. As a result, production of oxo fatty acid 13-oxo-cis-9-octadecenoic acid (0.01 mg) was confirmed when hydroxylated fatty acid 13-hydroxy-cis-9-octadecenoic acid was used as a substrate. Production of 13-oxo-cis-9,cis-15-octadecadienoic acid (0.02 mg) was confirmed when 13-hydroxy-cis-9,cis-15-octadecadienoic acid was used as a substrate, and production of 13-oxo-cis-6,cis-9-octadecadienoic acid (0.06 mg) was confirmed when 13-hydroxy-cis-6,cis-9-octadecadienoic acid was used as a substrate.

Using a similar method, the resultant product oxo fatty acid can be obtained by using each hydroxylated fatty acid as a substrate.

Example 7 Production of Oxo Fatty Acid from Hydroxylated Fatty Acid by Using Anhydrous Chromic Acid (CrO3)

To anhydrous chromic acid (2.67 g) were added sulfuric acid (2.3 ml) and water (7.7 ml), and acetone (90 ml) was added thereto to give a chromic acid solution. 2 g of hydroxylated fatty acid and 40 ml of acetone were added into an Erlenmeyer flask, and the above-mentioned chromic acid solution was added drop by drop on ice while stirring the mixture with a stirrer. When the solution turned from blue to the color of powdered green tea, the dropwise addition of the chromic acid solution was stopped and the reaction was quenched with isopropyl alcohol. The precipitated sediment was filtered with a filter paper and placed in a partitioning funnel. Diethyl ether (150 ml) and Milli Q water (300 ml) were further added and the mixture was shaken well. The diethyl ether layer was washed several times with Milli Q water. To the diethyl ether layer after washing was added an appropriate amount of sodium sulfate (anhydrous), the mixture was stirred, and the residual water was removed. The anhydrous sodium sulfate added was filtered off with a filter paper, the obtained diethyl ether layer was concentrated in a rotary evaporator, and the reaction product (oxo fatty acid) and an unreacted substrate were extracted.

Example 8 Purification of Resultant Product from Extracts (Mixture Containing Substrate and Resultant Product (Oxo Fatty Acid)) Obtained in Examples 6, 7

The extracts (mixture containing substrate and resultant product (oxo fatty acid)) obtained in Examples 6, 7 were subjected to moderate-pressure chromatography, a solution that came out from the column was recovered in fractions. The recovered each fraction was analyzed by LC/MS and gas chromatography, fractions containing hydroxylated fatty acid alone were collected and concentrated by a rotary evaporator. A part of the obtained final resultant product was methylesterified, and the purity of oxo fatty acid was evaluated by gas chromatography. As a result, oxo fatty acid having a purity of not less than 98% was obtained from each substrate. The chemical structure of the resultant product was determined by NMR, two-dimensional NMR, GC-MS analysis and the like. As a result, 13-oxo-cis-9-octadecenoic acid having a purity of not less than 98% could be obtained from 13-hydroxy-cis-9-octadecenoic acid. 13-Oxo-cis-6,cis-9-octadecadienoic acid having a purity of not less than 98% could be obtained from 13-hydroxy-cis-6, cis-9-octadecadienoic acid. 13-Oxo-cis-9,cis-15-octadecadienoic acid having a purity of not less than 98% could be obtained from 13-hydroxy-cis-9,cis-15-octadecadienoic acid.

Using a similar method, 10-oxo-hexadecanoic acid having a purity of not less than 98% can be obtained from 10-hydroxy-hexadecanoic acid. 12-Oxo-octadecanoic acid having a purity of not less than 98% can be obtained from 12-hydroxy-octadecanoic acid. 13-Oxo-cis-6,cis-9,cis-15-octadecatrienoic acid having a purity of not less than 98% can be obtained from 13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid. 15-Oxo-cis-11-eicosenoic acid having a purity of not less than 98% can be obtained from 15-hydroxy-cis-11-eicosenoic acid. 12-Oxo-cis-14-eicosenoic acid having a purity of not less than 98% can be obtained from 12-hydroxy-cis-14-eicosenoic acid. 12-Oxo-cis-14,cis-17-eicosadienoic acid having a purity of not less than 98% can be obtained from 12-hydroxy-cis-14,cis-17-eicosadienoic acid. 15-Oxo-cis-11,cis-17-eicosadienoic acid having a purity of not less than 98% can be obtained from 15-hydroxy-cis-11,cis-17-eicosadienoic acid. 15-Oxo-cis-8,cis-11-eicosadienoic acid having a purity of not less than 98% can be obtained from 15-hydroxy-cis-8,cis-11-eicosadienoic acid. 12-Oxo-cis-8,cis-14-eicosadienoic acid having a purity of not less than 98% can be obtained from 12-hydroxy-cis-8,cis-14-eicosadienoic acid. 12-Oxo-cis-5,cis-8-eicosadienoic acid having a purity of not less than 98% can be obtained from 12-hydroxy-cis-5,cis-8-eicosadienoic acid. 12-Oxo-cis-8,cis-14,cis-17-eicosatrienoic acid having a purity of not less than 98% can be obtained from 12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid. 15-Oxo-cis-8,cis-11,cis-17-eicosatrienoic acid having a purity of not less than 98% can be obtained from 15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid. 15-Oxo-cis-5,cis-8,cis-11-eicosatrienoic acid having a purity of not less than 98% can be obtained from 15-hydroxy-cis-5,cis-8,cis-11-eicosatrienoic acid. 12-Oxo-cis-5,cis-8,cis-14-eicosatrienoic acid having a purity of not less than 98% can be obtained from 12-hydroxy-cis-5,cis-8,cis-14-eicosatrienoic acid. 10,13-Dioxo-octadecanoic acid having a purity of not less than 98% can be obtained from 10,13-dihydroxy-octadecanoic acid or 10-oxo-13-hydroxy-octadecanoic acid. 10,13-Dioxo-cis-6-octadecenoic acid having a purity of not less than 98% can be obtained from 10,13-dihydroxy-cis-6-octadecenoic acid or 10-oxo-13-hydroxy-cis-6-octadecenoic acid. 10,13-Dioxo-cis-15-octadecenoic acid having a purity of not less than 98% can be obtained from 10,13-dihydroxy-cis-15-octadecenoic acid or 10-oxo-13-hydroxy-cis-15-octadecenoic acid. 10,13-Dioxo-cis-6,cis-15-octadecadienoic acid having a purity of not less than 98% can be obtained from 10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid or 10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid. 13-Oxo-cis-5, cis-9-octadecadienoic acid having a purity of not less than 98% can be obtained from 13-hydroxy-cis-5, cis-9-octadecadienoic acid. 13-Oxo-trans-5, cis-9-octadecadienoic acid having a purity of not less than 98% can be obtained from 13-hydroxy-trans-5,cis-9-octadecadienoic acid. 15-Oxo-cis-5,cis-11-eicosadienoic acid having a purity of not less than 98% can be obtained from 15-hydroxy-cis-5,cis-11-eicosadienoic acid. 15-Oxo-cis-5,cis-11,cis-17-eicosatrienoic acid having a purity of not less than 98% can be obtained from 15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid. 14-Oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid having a purity of not less than 98% can be obtained from 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid. 10-Oxo-tetradecanoic acid having a purity of not less than 98% can be obtained from 10-hydroxy-tetradecanoic acid.

Example 9 Measurement of Activity as Agonist of Nuclear Receptors PPARα and PPARγ

The PPARα,γ activation ability of the PPAR ligand of the present invention was measured in reference to Nobuyuki Takahashi et al., FEBS Letters 514 (2002) p. 315-322, “Dual action of isoprenols from herbal medicines on both PPARgamma and PPARalpha in 3T3-L1 adipocytes and HepG2 hepatocytes.”, the section of Material and Methods “Reporter plasmids and luciferase assays”. To be specific, PPARα,γ ligand activity was measured by a reporter assay that evaluates binding to a fusion protein of PPAR ligand binding region and GAL4 DNA binding region and target gene activation, based on the expression of luciferase. Specifically, a plasmid comprising a DNA encoding a fusion protein of PPARα,γ ligand binding region and GAL4 DNA binding region and a reporter plasmid wherein luciferase linked to GAL4 bound DNA sequence were introduced into CV-1 cell, a ligand described below was added to the cell, the cell was incubated and the luciferase activity was detected.

The concentration of the sample was adjusted with ethanol. Ethanol was used as a negative control, and PPARα,γ ligands GW7647 (10 nM) and troglitazone (5 μM) were used as a positive control.

The PPARα, γ ligand activity data of representative hydroxylated fatty acid, oxo fatty acid, (1) 13-hydroxy-cis-9-octadecenoic acid (indicated as “13-OH LA”), (2) 13-oxo-cis-9-octadecenoic acid (indicated as “13-OXO LA”), (3) 10,13-dihydroxy-octadecanoic acid (indicated as “10,13-OH LA”), (4) 13-hydroxy-cis-9,cis-15-octadecadienoic acid (indicated as “13-OH ALA”), (5) 13-oxo-cis-9,cis-15-octadecadienoic acid (indicated as “13-OXO ALA”), (6) 10,13-dihydroxy-cis-15-octadecenoic acid (indicated as “10,13-OH ALA”) (7) 13-hydroxy-cis-6,cis-9-octadecadienoic acid (indicated as “13-OH GLA”), (8) 13-oxo-cis-6,cis-9-octadecadienoic acid (indicated as “13-OXO GLA”), (9) 10,13-dihydroxy-cis-6-octadecenoic acid (indicated as “10,13-OH GLA”), (10) 15-hydroxy-cis-5,cis-8,cis-11-eicosatrienoic acid (indicated as “15-OH AA”) are shown in FIG. 1 and FIG. 2. Of these, rare fatty acids of (4), (6), (8) and (9) are novel fatty acids.

While the present invention has been described with emphasis on preferred embodiments, it is obvious to those skilled in the art that the preferred embodiments can be modified.

The contents disclosed in any publication cited herein, including patents and patent applications, are hereby incorporated in their entireties by reference, to the extent that they have been disclosed herein.

INDUSTRIAL APPLICABILITY

According to the method of the present invention, various hydroxylated fatty acids and oxo fatty acids can be produced, and therefore, the hydroxylated fatty acids and oxo fatty acids can be applied to various field of medicament, food and the like. In addition, according to the method of the present invention, novel rare fatty acid can be produced, which is extremely useful.

This application is based on a patent application No. 2014-011855 filed in Japan (filing date: Jan. 24, 2014), the contents of which are incorporated in full herein.

Claims

1. An enzyme protein of any of the following (a)-(c):

(a) the enzyme protein consisting of the amino acid sequence shown in SEQ ID NO: 2,
(b) a protein comprising an amino acid sequence wherein one or plural amino acids in the amino acid sequence shown in SEQ ID NO: 2 are deleted and/or substituted and/or inserted and/or added, and having an enzyme activity that catalyzes a hydration reaction,
(c) a protein encoded by a base sequence that hybridizes to a nucleic acid consisting of a chain sequence complementary to the base sequence shown in SEQ ID NO: 1 under stringent conditions, and having an enzyme activity that catalyzes a hydration reaction.

2. Lactobacillus acidophilus fungus or fungal debris thereof, comprising the enzyme protein according to claim 1.

3. A nucleic acid encoding the enzyme protein according to claim 1.

4. A vector comprising the nucleic acid according to claim 3.

5. A host cell transformed with the vector according to claim 4.

6. A method of producing the enzyme protein according to claim 1, comprising culturing the host cell according to claim 5 and recovering the enzyme from the culture.

7. A method of producing a hydroxylated fatty acid having 18 carbon atoms and a hydroxyl group at the 13-position, comprising subjecting an unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position to a hydration reaction using the enzyme protein according to claim 1, wherein the unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position is linoleic acid, γ-linolenic acid, α-linolenic acid, stearidonic acid, 10-hydroxy-cis-12-octadecenoic acid, 10-hydroxy-cis-6,cis-12-octadecadienoic acid, 10-hydroxy-cis-12,cis-15-octadecadienoic acid, 10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid, 10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoic acid, 10-oxo-cis-12,cis-15-octadecadienoic acid, 10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, pinolenic acid, or columbinic acid.

8. A method of producing an oxo fatty acid having 18 carbon atoms and a carbonyl group at the 13-position, comprising subjecting an unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position to a hydration reaction using the enzyme protein according to claim 1 to induce a hydroxylated fatty acid having 18 carbon atoms and a hydroxyl group at the 13-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the unsaturated fatty acid having 18 carbon atoms and a cis double bond at the 12-position is linoleic acid, γ-linolenic acid, α-linolenic acid, stearidonic acid, 10-hydroxy-cis-12-octadecenoic acid, 10-hydroxy-cis-6,cis-12-octadecadienoic acid, 10-hydroxy-cis-12,cis-15-octadecadienoic acid, 10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid, 10-oxo-cis-12-octadecenoic acid, 10-oxo-cis-6,cis-12-octadecadienoic acid, 10-oxo-cis-12,cis-15-octadecadienoic acid, 10-oxo-cis-6,cis-12,cis-15-octadecatrienoic acid, pinolenic acid, or columbinic acid, and wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.

9. A method of producing a hydroxylated fatty acid having 16 carbon atoms and a hydroxyl group at the 10-position, comprising subjecting an unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position to a hydration reaction using the enzyme protein according to claim 1, wherein the unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position is pulmitoleic acid.

10. A method of producing an oxo fatty acid having 16 carbon atoms and a carbonyl group at the 10-position, comprising subjecting an unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position to a hydration reaction using the enzyme protein according to claim 1 to induce a hydroxylated fatty acid having 16 carbon atoms and a hydroxyl group at the 10-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the unsaturated fatty acid having 16 carbon atoms and a cis double bond at the 9-position is pulmitoleic acid, and wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.

11. A method of producing a hydroxylated fatty acid having 20 carbon atoms and a hydroxyl group at the 15-position, comprising subjecting an unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position to a hydration reaction using the enzyme protein according to claim 1, wherein the unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position is cis-11,cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic acid, dihomo-γ-linolenic acid, cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, arachidonic acid, sciadonic acid, or juniperonic acid.

12. A method of producing an oxo fatty acid having 20 carbon atoms and a carbonyl group at the 15-position, comprising subjecting an unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position to a hydration reaction using the enzyme protein according to claim 1 to induce a hydroxylated fatty acid having 20 carbon atoms and a hydroxyl group at the 15-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the unsaturated fatty acid having 20 carbon atoms and a cis double bond at the 14-position is cis-11,cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic acid, dihomo-γ-linolenic acid, cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, arachidonic acid, sciadonic acid, or juniperonic acid, and wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.

13. A method of producing a hydroxylated fatty acid having 18 or 20 carbon atoms and a hydroxyl group at the 12-position, comprising subjecting an unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position to a hydration reaction using the enzyme protein according to claim 1, wherein the unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position is cis-vaccenic acid, cis-11-cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic, dihomo-γ-linolenic acid, mead acid, cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, or arachidonic acid.

14. A method of producing an oxo fatty acid having 18 or carbon atoms and a carbonyl group at the 12-position, comprising subjecting an unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position to a hydration reaction using the enzyme protein according to claim 1 to induce a hydroxylated fatty acid having 18 or 20 carbon atoms and a hydroxyl group at the 12-position, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the unsaturated fatty acid having 18 or 20 carbon atoms and a cis double bond at the 11-position is cis-vaccenic acid, cis-11-cis-14-eicosadienoic acid, cis-11,cis-14,cis-17-eicosatrienoic, dihomo-γ-linolenic acid, mead acid, cis-8,cis-11,cis-14,cis-17-eicosatetraenoic acid, or arachidonic acid, and wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.

15.-18. (canceled)

19. A method of producing 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, comprising subjecting cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid to a hydration reaction using the enzyme protein according to claim 1.

20. A method of producing 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, comprising subjecting cis-4,cis-7,cis-10,cis-13,cis-16,cis-19-docosahexaenoic acid to a hydration reaction using the enzyme protein according to claim 1 to induce 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.

21. A method of producing 10-hydroxy-tetradecanoic acid, comprising subjecting cis-9-tetradecenoic acid (myristoleic acid) to a hydration reaction using the enzyme protein according to claim 1.

22. A method of producing 10-oxo-tetradecanoic acid, comprising subjecting cis-9-tetradecenoic acid (myristoleic acid) to a hydration reaction using the enzyme protein according to claim 1 to induce 10-hydroxy-tetradecanoic acid, and subjecting the hydroxylated fatty acid to a dehydrogenation reaction or chemical oxidation, wherein the dehydrogenation reaction uses a dehydrogenase derived from lactobacillus.

23.-24. (canceled)

25. 13-Hydroxy-cis-9,cis-15-octadecadienoic acid, 13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid, 10,13-dihydroxy-cis-6-octadecenoic acid, 10,13-dihydroxy-cis-15-octadecenoic acid, 10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid, 10-oxo-13-hydroxy-cis-6-octadecenoic acid, 10-oxo-13-hydroxy-cis-15-octadecenoic acid, 10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid, 13-hydroxy-cis-5,cis-9-octadecadienoic acid, or 13-hydroxy-trans-5,cis-9-octadecadienoic acid, which is produced by the method according to claim 7.

26. 15-Hydroxy-cis-11,cis-17-eicosadienoic acid, 15-hydroxy-cis-8, cis-11-eicosadienoic acid, 15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid, 15-hydroxy-cis-5,cis-11-eicosadienoic acid, or 15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid, which is produced by the method according to claim 11.

27. 12-Hydroxy-cis-14-eicosenoic acid, 12-hydroxy-cis-14,cis-17-eicosadienoic acid or 12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid, which is produced by the method according to claim 13.

28. 13-Oxo-cis-6,cis-9-octadecadienoic acid, 13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid, 10,13-dioxo-cis-6-octadecenoic acid, 10,13-dioxo-cis-15-octadecenoic acid, 10,13-dioxo-cis-6,cis-15-octadecadienoic acid, 13-oxo-cis-5,cis-9-octadecadienoic acid or 13-oxo-trans-5,cis-9-octadecadienoic acid, which is produced by the method according to claim 8.

29. 15-Oxo-cis-11,cis-17-eicosadienoic acid, 15-oxo-cis-8,cis-11-eicosadienoic acid, 15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid, 15-oxo-cis-5,cis-11-eicosadienoic acid or 15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid, which is produced by the method according to claim 12.

30. 12-Oxo-cis-14-eicosenoic acid, 12-oxo-cis-14,cis-17-eicosadienoic acid, 12-oxo-cis-8,cis-14-eicosadienoic acid, 12-oxo-cis-5,cis-8-eicosadienoic acid or 12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid, which is produced by the method according to claim 14.

31. 14-Hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid produced by the method according to claim 19.

32. 14-Oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, which is produced by the method according to claim 20.

33. A fatty acid-containing substance comprising 13-hydroxy-cis-9,cis-15-octadecadienoic acid, 13-hydroxy-cis-6,cis-9,cis-15-octadecatrienoic acid, 10,13-dihydroxy-cis-6-octadecenoic acid, 10,13-dihydroxy-cis-15-octadecenoic acid, 10,13-dihydroxy-cis-6,cis-15-octadecadienoic acid, 10-oxo-13-hydroxy-cis-6-octadecenoic acid, 10-oxo-13-hydroxy-cis-15-octadecenoic acid, 10-oxo-13-hydroxy-cis-6,cis-15-octadecadienoic acid, 13-hydroxy-cis-5,cis-9-octadecadienoic acid, 13-hydroxy-trans-5,cis-9-octadecadienoic acid, 15-hydroxy-cis-11,cis-17-eicosadienoic acid, 15-hydroxy-cis-8,cis-11-eicosadienoic acid, 15-hydroxy-cis-8,cis-11,cis-17-eicosatrienoic acid, 15-hydroxy-cis-5,cis-11-eicosadienoic acid, 15-hydroxy-cis-5,cis-11,cis-17-eicosatrienoic acid, 12-hydroxy-cis-14-eicosenoic acid, 12-hydroxy-cis-14,cis-17-eicosadienoic acid, 12-hydroxy-cis-8,cis-14,cis-17-eicosatrienoic acid, 13-oxo-cis-6,cis-9-octadecadienoic acid, 13-oxo-cis-6,cis-9,cis-15-octadecatrienoic acid, 10,13-dioxo-cis-6-octadecenoic acid, 10,13-dioxo-cis-15-octadecenoic acid, 10,13-dioxo-cis-6,cis-15-octadecadienoic acid, 13-oxo-cis-5,cis-9-octadecadienoic acid, 13-oxo-trans-5,cis-9-octadecadienoic acid, 15-oxo-cis-11,cis-17-eicosadienoic acid, 15-oxo-cis-8,cis-11-eicosadienoic acid, 15-oxo-cis-8,cis-11,cis-17-eicosatrienoic acid, 15-oxo-cis-5,cis-11-eicosadienoic acid, 15-oxo-cis-5,cis-11,cis-17-eicosatrienoic acid, 12-oxo-cis-14-eicosenoic acid, 12-oxo-cis-14,cis-17-eicosadienoic acid, 12-oxo-cis-8,cis-14-eicosadienoic acid, 12-oxo-cis-5,cis-8-eicosadienoic acid, 12-oxo-cis-8,cis-14,cis-17-eicosatrienoic acid, 14-hydroxy-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid, or 14-oxo-cis-4,cis-7,cis-10,cis-16,cis-19-docosapentaenoic acid.

34. (canceled)

Patent History
Publication number: 20170022526
Type: Application
Filed: Jan 23, 2015
Publication Date: Jan 26, 2017
Applicants: KYOTO UNIVERSITY (Kyoto-shi), NITTO PHARMACEUTICAL INDUSTRIES, LTD. (Muko-shi)
Inventors: Jun OGAWA (Kyoto-shi), Shigenobu KISHINO (Kyoto-shi), Teruo KAWADA (Kyoto-shi), Nobuyuki TAKAHASHI (Kyoto-shi), Tsuyoshi GOTO (Kyoto-shi), Yasunori YONEJIMA (Muko-shi)
Application Number: 15/113,604
Classifications
International Classification: C12P 7/64 (20060101); C12N 9/04 (20060101);