Δ17 desaturase and its use in making polyunsaturated fatty acids
View Patent ↗The present invention relates to Δ17 desaturases, which have the ability to convert ω-6 fatty acids into their ω-3 counterparts (i.e., conversion of arachidonic acid [20:4, ARA] to eicosapentaenoic acid [20:5, EPA]). Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding Δ17 desaturases along with a method of making long-chain polyunsaturated fatty acids (PUFAs) using these Δ17 desaturases in oleaginous yeast are disclosed.
1. An isolated nucleic acid molecule selected from the group consisting of:
a) an isolated nucleotide molecule encoding a Δ17 desaturase enzyme, selected from the group consisting of SEQ ID NO:2 and SEQ ID NO:3;
b) an isolated nucleotide molecule encoding Δ17 desaturase enzyme that hybridizes with (a) under the following hybridization conditions: 0.1X SSC, 0.1% SDS, 65° C. and washed with 2X SSC, 0.1% SDS followed by 0.1X SSC, 0.1% SDS; or
c) an isolated nucleotide molecule that is completely complementary to (a) or (b).
2. The isolated nucleic acid molecule which encodes a Δ17 desaturase enzyme selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 4.
3. The isolated nucleic acid molecule which encodes a Δ17 desaturase enzyme as set forth in SEQ ID NO: 2, wherein at least 175 codons are codon-optimized for expression in Yarrowia.
4. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a Δ17 desaturase enzyme of at least 359 amino acids that has at least 95% identity based on Clustal W algorithms when compared to a polypeptide having the sequence as set forth in SEQ ID NO: 2; or a second nucleotide sequence comprising the full-complement of the first nucleotide sequence.
5. A chimeric gene comprising the isolated nucleic acid molecule of claim 1 operably linked to suitable regulatory sequences.
6. An isolated transformed host cell comprising the isolated nucleic acid molecule of claim 1 .
7. The transformed host cell of claim 6 selected from the group consisting of algae, bacteria, yeast, oomycetes and fungi.
8. The transformed host cell of claim 7 wherein the yeast is an oleaginous yeast.
9. The transformed host cell of claim 8 wherein the oleaginous yeast is selected from the group consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
10. A method for the production of eicosapentaenoic acid comprising:
a) providing a host cell comprising:
(i) an isolated nucleotide molecule encoding a bifunctional Δ17/ Δ15 desaturase polypeptide having at least 95% identity when compared to a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2, based on the Clustal W method of alignment; and
(ii) a source of arachidonic acid;
b) growing the host cell of step (a) under conditions wherein the nucleic acid molecule encoding the bifunctional Δ17/ Δ15 desaturase polypeptide is expressed and the arachidonic acid is converted to eicosapentaenoic acid; and
c) optionally recovering the eicosapentaenoic acid of step (b).
11. A method for the production of eicosatetraenoic acid comprising:
a) providing a host cell comprising:
(i) an isolated nucleotide molecule encoding a bifunctional Δ17/ Δ15 desaturase polypeptide having at least 95% identity when compared to a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2, based on the Clustal W method of alignment; and
(ii) a source of dihomo-γ-linolenic acid;
b) growing the host cell of step (a) under conditions wherein the nucleic acid molecule encoding the bifunctional Δ17/ Δ15 desaturase polypeptide is expressed and the dihomo-γ-linolenic acid is converted to eicosatetraenoic acid; and
c) optionally recovering the eicosapentaenoic acid of step (b).
12. A method for the production of polyunsaturated fatty acids comprising:
a) providing a host cell comprising:
i) an isolated nucleotide molecule encoding a bifunctional Δ17/ Δ15 desaturase polypeptide having at least 95% identity when compared to a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2, based on the Clustal W method of alignment; and
ii) a source of fatty acid selected from the group consisting of: linoleic acid and eicosadienoic acid;
b) growing the host cell of step (a) under conditions wherein the nucleic acid molecule encoding the bifunctional Δ17/ Δ15 desaturase polypeptide is expressed and the linoleic acid is converted to α-linolenic acid and the eicosadienoic acid is converted to eicosatrienoic acid; and
c) optionally recovering the fatty acid of step (b).
13. A method according to any of claims 10 , 11 or 12 wherein the isolated nucleic acid molecule encodes a bifunctional Δ17/ Δ15 desaturase polypeptide having the amino acid sequence as set forth in SEQ ID NO:2, wherein at least 175 codons are codon-optimized for expression in Yarrowia.
14. A method according to any of claims 10 , 11 or 12 wherein the isolated nucleic acid molecule encodes a bifunctional Δ17/ Δ15 desaturase polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:2 and SEQ ID NO: 3.
15. A method according to any of claims 10 , 11 or 12 wherein:
a.) the isolated nucleic acid molecule has a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 4; and,
b.) the host cell is Yarrowia lipolytica.
16. A method according to any of claims 10 , 11 or 12 , wherein the host cell is selected from the group consisting of: algae, bacteria, yeast, oomycetes and fungi.
17. A method according to claim 16 wherein the host cell is a fungus selected from the group consisting of: Thraustochytrium sp., Schizochytrium sp. and Mortierella sp.
18. A method according to claim 16 wherein the yeast is an oleaginous yeast.
19. A method according to claim 18 wherein the oleaginous yeast is selected from the group consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.