STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 150134_409USPC_SEQUENCE_LISTING.txt. The text file is 33.5 KB, was created on Aug. 26, 2020, and is being submitted electronically via EFS-Web.

TECHNICAL FIELD

The present invention relates to a novel 8-lipoxygenase and a nucleic acid encoding the same, and a method of use thereof. The present invention also relates to a method of oxidizing a highly unsaturated fatty acid using a novel 8-lipoxygenase.

BACKGROUND ART

The physiological function of metabolites of highly unsaturated fatty acids has drawn attention. For example, an 8-hydroxyeicosapentaenoic acid (8-HEPE) is a derivative generated by oxidation (hydroxylation) of carbon at position 8 of an eicosapentaenoic acid (EPA). 8-HEPE is a compound that has a higher peroxisome proliferator-activated receptor (PPAR) activating action than EPA and is expected to have an effect of preventing/improving metabolic diseases such as obesity and diabetes (Non Patent Literature 1).

8-HEPE does not exist in nature at a large quantity. For example, Pacific krill (Euphausia pacifica) contains only 10 s of ppm of 8-HEPE (Non Patent Literature 2).

While there are currently efforts to prepare an 8-HEPE concentrated ingredient from Pacific krill for practical application thereof as an ingredient of functional food, the amount of 8-HEPE harvested from Pacific krill is miniscule, as described above. Besides 8-HEPE, Pacific krill contains several ppm of 8-hydroxyeicosatetraenoic acid (8-HETE) produced by introduction of a hydroxyl group to position 8 of an arachidonic acid and 10-hydroxydocosahexaenoic acid (10-HDoHE) produced by introduction of a hydroxyl group to position 10 of an docosahexaenoic acid (DHA). Such derivatives are also expected to be utilized as a novel functional component or pharmaceutical product (Non Patent Literatures 3 and 4).

However, utilization of such derivatives as a novel functional component or pharmaceutical product was challenging due to the low content of these derivatives in Pacific krill.

Information on prior art documents related to the invention of the present application includes the following.

CITATION LIST

Non Patent Literature

[NPL 1] Yamada H et al., Journal of Lipid Research, 55, 895-904, 2014[NPL 2] Yamada H et al., Scientific Reports, 7, 9944, 2017[NPL 3] Klawitter J et al., J Lipid Res. 2014 June; 55(6): 1139-49.[NPL 4] Hashimoto M et al., Biochim Biophys Acta. 2015 February; 1851 (2): 203-9

SUMMARY OF INVENTION

Technical Problem

In this regard, there is a demand for a technology for mass producing 8-HEPE, i.e., technology for enzymatically producing 8-HEPE, for utilization as a novel functional component or pharmaceutical product.

Solution to Problem

After noting that Pacific krill contains, besides 8-HEPE that is a derivative generated by oxidation (hydroxylation) of carbon at position 8 of an eicosapentaenoic acid (EPA), several ppm of 8-hydroxyeicosatetraenoic acid (8-HETE) produced by introduction of a hydroxyl group to position 8 of an arachidonic acid and 10-hydroxydocosahexaenoic acid (10-HDoHE) produced by introduction of a hydroxyl group to position 10 of a docosahexaenoic acid (DHA), and 8-HEPE is in fact produced by incubating a Pacific krill protein and EPA at 20° C., the inventors completed the present invention by isolating/identifying a lipoxygenase gene from Pacific krill and identifying an enzyme that converts an eicosapentaenoic acid (EPA) into an 8-hydroxyeicosapentaenoic acid (8-HEPE).

The inventors employed an approach combining next generation sequencer analysis and bioinformatic analysis for performing de novo RNA sequence analysis on Pacific krill (Euphausia pacifica) in order to identify the lipoxygenase gene sequence of Pacific krill, and narrowing down candidate lipoxygenase genetic sequences by Blast search to obtain candidate lipoxygenase genes of Pacific krill, i.e., Pacific krill Lipoxygenase clones 1 and 2. Since it was clone 1 between them that encoded a lipoxygenase domain, clone 1 was named PK LOX1. Since a PK LOX2 clone was considered a partial sequence, full length cDNA sequence analysis was performed by Rapid amplification of cDNA ends (RACE) to obtain a full length cDNA sequence. For PK LOX1 and PK LOX2, proteins were expressed in insect cells to test 70 to 80 kDA protein expression and lipoxygenase activity. In this manner, the technical problem described above was solved by finding an enzyme generating an 8-hydroxyeicosapentaenoic acid (8-HEPE) through oxidation of carbon at position 8 of an eicosapentaenoic acid (EPA), an 8-hydroxyeicosatetraenoic acid (8-HETE) through oxidation of carbon at position 8 of an arachidonic acid (ARA), and a 10-hydroxydocosahexaenoic acid (10-HDoHE) through oxidation of carbon at position 10 of a docosahexaenoic acid (DHA).

A lipoxygenase with the same effect as an 8-lipoxygenase of Pacific krill, which was first identified through the present invention, is present not only in Pacific krill, but also in other organisms belonging to Malacostraca, such as other organisms belonging to Euphausiacea and other organisms belonging to Decapoda. It is expected that such a lipoxygenase can be isolated/prepared as a membrane protein.

For example, the present invention provides the following.

(Item 1)

An 8-lipoxygenase derived from Malacostraca, having activity selected from the group consisting of:

(1) activity to oxidize carbon at position 8 of an arachidonic acid;

(2) activity to oxidize carbon at position 8 of an eicosapentaenoic acid; and

(3) activity to oxidize carbon at position 10 of a docosahexaenoic acid.

(Item 2)

The 8-lipoxygenase of item 1, wherein the 8-lipoxygenase is an 8-lipoxygenase derived from Euphausiacea or an 8-lipoxygenase derived from Decapoda.

(Item 3)

A polypeptide selected from the group consisting of:

(a) a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4;

(b) a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4;

(c) a polypeptide consisting of an amino acid sequence comprising one or several mutations, substitutions, insertions, or deletions in the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4;

(d) a polypeptide encoded by a nucleic acid comprising the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3;

(e) a polypeptide encoded by a nucleic acid that hybridizes with a complementary strand of a nucleic acid comprising the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3 under a high stringency condition;

(f) a polypeptide encoded by a nucleic acid comprising a sequence that is at least 90% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3; and

(g) a polypeptide encoded by a nucleic acid having a nucleic acid sequence comprising one or several mutations, substitutions, insertions, or deletions in the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3;

wherein the polypeptide has activity selected from the group consisting of:

(1) activity to oxidize carbon at position 8 of an arachidonic acid;

(2) activity to oxidize carbon at position 8 of an eicosapentaenoic acid; and

(3) activity to oxidize carbon at position 10 of a docosahexaenoic acid.

(Item 4)

The polypeptide of item 3, selected from the group consisting of:

(a) a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; and

(d) a polypeptide encoded by a nucleic acid comprising the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3.

(Item 5)

A method of generating an oxide by reacting the polypeptide of any one of items 1 to 4 with a highly unsaturated fatty acid or a derivative of a highly unsaturated fatty acid.

(Item 6)

The method of item 5, wherein:the highly unsaturated fatty acid is an arachidonic acid, and the oxide is an 8-hydroxyeicosatetraenoic acid;the highly unsaturated fatty acid is an eicosapentaenoic acid, and the oxide is an 8-hydroxyeicosapentaenoic acid;the highly unsaturated fatty acid is a docosahexaenoic acid, and the oxide is a 10-hydroxydocosahexaenoic acid; orthe highly unsaturated fatty acid is a docosapentaenoic acid, and the oxide is a 10-hydroxydocosapentaenoic acid.
(Item 7)

A composition for generating an oxide of a highly unsaturated fatty acid or a derivative of a highly unsaturated fatty acid from a highly unsaturated fatty acid or a derivative of a highly unsaturated fatty acid, comprising the polypeptide of any one of items 1 to 4.

(Item 8)

The composition of item 7, whereinthe highly unsaturated fatty acid is an arachidonic acid, and the oxide is an 8-hydroxyeicosatetraenoic acid;the highly unsaturated fatty acid is an eicosapentaenoic acid, and the oxide is an 8-hydroxyeicosapentaenoic acid; orthe highly unsaturated fatty acid is a docosahexaenoic acid, and the oxide is a 10-hydroxydocosahexaenoic acid.
(Item 9)

A nucleic acid encoding an 8-lipoxygenase derived from Malacostraca, having activity selected from the group consisting of:

(1) activity to oxidize carbon at position 8 of an arachidonic acid;

(2) activity to oxidize carbon at position 8 of an eicosapentaenoic acid; and

(3) activity to oxidize carbon at position 10 of a docosahexaenoic acid.

(Item 10)

The nucleic acid of item 9, wherein the 8-lipoxygenase is an 8-lipoxygenase derived from Euphausiacea or an 8-lipoxygenase derived from Decapoda.

(Item 11)

A nucleic acid selected from the group consisting of:

(h) a nucleic acid comprising the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3;

(i) a nucleic acid that hybridizes with a complementary strand of a nucleic acid comprising the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3 under a high stringency condition;

(j) a nucleic acid comprising a sequence that is at least 90% identical to the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3;

(k) a nucleic acid having a nucleic acid sequence comprising one or several mutations, substitutions, insertions, or deletions in the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3;

(l) a nucleic acid encoding a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4;

(m) a nucleic acid encoding a polypeptide consisting of an amino acid sequence that is at least 90% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4; and

(n) a nucleic acid encoding a polypeptide consisting of an amino acid sequence comprising one or several mutations, substitutions, insertions, or deletions in the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4;

wherein the nucleic acid encodes a polypeptide having activity selected from the group consisting of:

(1) activity to oxidize carbon at position 8 of an arachidonic acid;

(2) activity to oxidize carbon at position 8 of an eicosapentaenoic acid; and

(3) activity to oxidize carbon at position 10 of a docosahexaenoic acid.

(Item 12)

The nucleic acid of item 11, selected from the group consisting of:

(h) a nucleic acid comprising the nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 3; and

(l) a nucleic acid encoding a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 4.

(Item 13)

A recombinant vector comprising the nucleic acid of item 11 or 12.

(Item 14)

A transformant comprising the recombinant vector of item 13.

(Item 15)

A method of manufacturing an 8-lipoxygenase, comprising:

culturing the transformant of item 14;

generating and accumulating the 8-lipoxygenase of any one of items 1 to 4 in a culture; and

retrieving the generated and accumulated 8-lipoxygenase.

(Item 16)

A composition for performing a lipoxygenase reaction, comprising a membrane protein of an organism belonging to Malacostraca.

(Item 17)

The composition of item 16, wherein the membrane protein is a membrane protein of an organism belonging to Euphausiacea or a membrane protein of an organism belonging to Decapoda.

(Item 18)

The composition of item 16, wherein the membrane protein is a membrane protein of Pacific krill.

(Item 19)

The composition of any one of items 16 to 18 for generating an oxide of a highly unsaturated fatty acid or a derivative of a highly unsaturated fatty acid from a highly unsaturated fatty acid or a derivative of a highly unsaturated fatty acid.

(Item 20)

The composition of item 19, whereinthe highly unsaturated fatty acid is an arachidonic acid, and the oxide is an 8-hydroxyeicosatetraenoic acid;the highly unsaturated fatty acid is an eicosapentaenoic acid, and the oxide is an 8-hydroxyeicosapentaenoic acid; orthe highly unsaturated fatty acid is a docosahexaenoic acid, and the oxide is a 10-hydroxydocosahexaenoic acid.

A polynucleotide having a nucleic acid sequence homologous to SEQ ID NO: 1 or SEQ ID NO: 3, or a polypeptide having an amino acid sequence homologous to SEQ ID NO: 2 or SEQ ID NO: 4 can be used in the present invention. A polypeptide isolated from a cell belonging toEuphausia, having activity selected from the group consisting of: (1) activity to oxidize carbon at position 8 of an arachidonic acid; (2) activity to oxidize carbon at position 8 of an eicosapentaenoic acid; and (3) activity to oxidize carbon at position 10 of a docosahexaenoic acid, or a polynucleotide encoding such a polynucleotide can also be used in the present invention.

Advantageous Effects of Invention

A lipoxygenase (8-lipoxygenase) provided by the present invention enables highly efficient generation of an 8-hydroxyeicosatetraenoic acid from an arachidonic acid, highly efficient generation of an 8-hydroxyeicosapentaenoic acid from an eicosapentaenoic acid, and highly efficient generation of a 10-hydroxydocosahexaenoic acid from a docosahexaenoic acid.

