Δ 5 desaturase and its use in making polyunsaturated fatty acids
View Patent ↗The present invention relates to a Δ5 desaturase, which has the ability to convert dihomo-γ-linolenic acid (DGLA; 20:3 ω-6) to arachidonic acid (ARA; 20:4 ω-6) and/or eicosatetraenoic acid (ETA; 20:4 ω-3) to eicosapentaenoic acid (EPA; 20:5 ω-3). Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding Δ5 desaturase along with a method of making long chain polyunsaturated fatty acids (PUFAs) using this Δ5 desaturase in oleaginous yeast are disclosed.
1. An isolated nucleic acid molecule selected from the group consisting of:
(a) an isolated nucleotide sequence encoding a Δ5 desaturase enzyme as set forth in SEQ ID NO:2;
(b) an isolated nucleotide sequence that hybridizes with (a) under the following hybridization conditions: 0.1×SSC, 0.1% SDS, 65° C. and washed with 2×SSC, 0.1% SDS, wherein said nucleotide sequence encodes a delta-5 desaturase followed by 0.1×SSC, 0.1% SDS; and,
(c) an isolated nucleotide sequence that is completely complementary to (a) or (b).
2. The isolated nucleic acid molecule of claim 1 , wherein at least 189 codons are codon-optimized for expression in Yarrowia.
3. The isolated nucleic acid molecule of claim 1 selected from the group of SEQ ID NOs:1 and 3.
4. An isolated nucleic acid fragment of claim 1 isolated from Euglena gracilis.
5. An isolated nucleic acid molecule comprising a first nucleotide sequence encoding a Δ5 desaturase enzyme of at least 449 amino acids that has at least 95% identity based on BLASTP algorithms when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2;
or a second nucleotide sequence comprising the complement of the first nucleotide sequence.
6. A chimeric gene comprising the isolated nucleic acid molecule of any one of claims 1 - 5 operably linked to at least one regulatory sequence.
7. A transformed Yarrowia sp. comprising the isolated nucleic acid molecule of claim 1 .
8. The transformed Yarrowia sp. of claim 7 selected from the group consisting of Yarrowia lipolytica ATCC #20362 , Yarrowia lipolytica ATCC #8862 , Yarrowia lipolytica ATCC #18944 , Yarrowia lipolytica ATCC #76982 and Yarrowia lipolytica LGAM S(7)1.
9. A method for the production of arachidonic acid comprising:
a) providing a host cell comprising:
(i) an isolated nucleotide molecule encoding a Δ5 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 BLASTP algorithms; and,
(ii) a source of dihomo-γ-linolenic acid;
b) growing the host cell of step (a) under conditions wherein the nucleic acid fragment encoding the Δ5 desaturase polypeptide is expressed and the dihomo-γ-linolenic acid is converted to arachidonic acid; and,
c) optionally recovering the arachidonic acid of step (b).
10. A method for the production of eicosapentaenoic acid comprising:
a) providing a host cell comprising:
(i) an isolated nucleotide molecule encoding a Δ5 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 BLASTP algorithms; and,
(ii) a source of eicosatetraenoic acid;
b) growing the host cell of step (a) under conditions wherein the nucleic acid fragment encoding the Δ5 desaturase polypeptide is expressed and the eicosatetraenoic acid is converted to eicosapentaenoic acid; and,
c) optionally recovering the eicosapentaenoic acid of step (b).
11. A method according to either of claims 9 or 10 wherein the isolated nucleic acid molecule encoding the Δ5 desaturase polypeptide is set forth in SEQ ID NO:2, wherein at least 189 codons are codon-optimized for expression in Yarrowia.
12. A method according to either of claims 9 or 10 wherein the isolated nucleic acid molecule encodes a Δ5 desaturase polypeptide having the amino acid sequence as set forth in SEQ ID NO:2.
13. A method according to either of claims 9 or 10 wherein:
a) the isolated nucleic acid molecule has the nucleic acid sequence selected from the group consisting of SEQ ID NOs:1 and SEQ ID NO:3; and,
b) the host cell is Yarrowia lipolytica.
14. A method according to either of claims 9 or 10 , wherein the host cell is selected from the group consisting of: algae, bacteria, yeast, euglenoids and fungi.
15. A method according to claim 14 wherein the host cell is a fungus selected from the group consisting of: Thraustochytrium, Schizochytrium and Mortierella.
16. A method according to claim 14 wherein the yeast is an oleaginous yeast.
17. A method according to claim 16 wherein the oleaginous yeast is selected from the group consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.