Δ8 desaturases and their use in making polyunsaturated fatty acids
View Patent ↗The present invention relates to Δ8 desaturase genes, which have the ability to convert eicosadienoic acid (EDA; 20:2 ω-6) to dihomo-γ-linolenic acid (DGLA; 20:3 ω-6) and/or eicosatrienoic acid (ETrA; 20:3 ω-3) to eicosatetraenoic acid (ETA; (20:3 ω-3). Isolated nucleic acid fragments and recombinant DNA constructs comprising such fragments encoding Δ8 desaturases along with a method of making long-chain polyunsaturated fatty acids (PUFAs) using these Δ8 desaturases in oleaginous yeast are disclosed.
1. A method for the production of at least one polyunsaturated fatty acid comprising:
a) providing an isolated microbial host cell comprising:
i) a recombinant nucleotide molecule encoding a polypeptide having delta-8 desaturase activity, wherein the polypeptide has at least 90% amino acid identity, based on the Clustal V method of alignment, when compared to amino acid sequence selected from the group consisting of: SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:24; and
ii) a source of a substrate polyunsaturated fatty acid selected from the group consisting of eicosadienoic acid and eicosatrienoic acid;
b) growing the isolated microbial host cell of step (a) under conditions wherein eicosadienoic acid is converted to dihomo-qamma-linolenic acid and eicosatrienoic acid is converted to eicosatetraenoic acid; and
c) optionally recovering the dihomo-gamma-linolenic acid or eicosatetraenoic acid or both of step (b).
2. The method of claim 1 , wherein the isolated microbial host cell is a Yarrowia , said polypeptide has the full-length amino acid sequence set forth in SEQ ID NO:40, and the recombinant nucleotide molecule comprises at least 208 codons which are optimized for expression in Yarrowia.
3. The method according to claim 1 , wherein:
a) the recombinant nucleotide molecule has the full-length nucleic acid sequence selected from the group consisting of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 and SEQ ID NO:39; and
b) the host cell is Yarrowia lipolytica.
4. The method according to claim 1 wherein the isolated microbial host cell is selected from the group consisting of yeast, algae, bacteria, euglenoids, stramenopiles and fungi.
5. The method according to claim 4 wherein the isolated microbial host cell is a fungus of the genus Mortierella.
6. The method according to claim 4 wherein the isolated microbial host cell is a stramenopile selected from the group consisting of: Thraustochytrium and Schizochytrium.
7. The method according to claim 4 wherein the isolated microbial host cell is an oleaginous yeast.
8. The method according to claim 7 wherein the isolated microbial host cell is an oleaginous yeast selected from the group consisting of: Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.
9. The method of claim 1 , wherein the isolated microbial host further comprises a recombinant nucleotide molecule encoding a polypeptide having delta-9 elongase activity.