ENZYMES AND METHODS FOR PRODUCING OMEGA-3 FATTY ACIDS
The present invention relates to methods of synthesizing long-chain polyunsaturated fatty acids, especially eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid, in recombinant cells such as yeast or plant cells. Also provided are recombinant cells or plants which produce long-chain polyunsaturated fatty acids. Furthermore, the present invention relates to a group of new enzymes which possess desaturase or elongase activity that can be used in methods of synthesizing long-chain polyunsaturated fatty acids. In particular, the present invention provides ω3 destaurases, Δ5 elongases and Δ6 desaturases with novel activities. Also provided are methods and DNA constructs for transiently and/or stably transforming cells, particularly plant cells, with multiple genes.
1 - 175 . (canceled)
176 . A process for producing lipid comprising docosahexaenoic acid (DHA) in an esterified form, the process comprising a step of extracting lipid from a plant cell which comprises exogenous polynucleotides encoding
a Δ6 desaturase,
a Δ6 elongase,
a Δ5 desaturase,
a Δ5 elongase, and
a Δ4 desaturase,
wherein each exogenous polynucleotide is operably linked to a promoter that directs expression of said polynucleotide in the plant cell,
wherein more than one of the Δ6 desaturase, Δ5 desaturase and Δ4 desaturase is a microalgal desaturase,
wherein one or more of the Δ6 desaturase, Δ5 desaturase and Δ4 desaturase has greater activity on an acyl-CoA substrate than on a corresponding acyl-phosphatidylcholine (acyl-PC) substrate, and wherein DHA is present in the extracted lipid at a level of between 3% and 5% of the extracted lipid's total fatty acid content,
thereby producing the lipid comprising DHA.
177 . The process of claim 176 , wherein the Δ6 desaturase is a microalgal Δ6 desaturase which has greater activity on ALA-CoA as fatty acid substrate than on a-linolenic acid (ALA) joined to the sn-2 position of PC as fatty acid substrate.
178 . The process of claim 176 , wherein the microalgal Δ6 desaturase is an Ostreococcus tauri Δ6 desaturase.
179 . The process of claim 177 , wherein the microalgal Δ6 desaturase has the amino acid sequence set forth as SEQ ID NO:30.
180 . The process of claim 176 , wherein the Δ5 elongase is a microalgal Δ5 elongase.
181 . The process of claim 176 , wherein the Δ5 elongase has the amino acid sequence provided by Accession No. AAV67798.
182 . The process of claim 176 , wherein the Δ4 desaturase has the amino acid sequence provided by Accession No. AY332747.
183 . The process of claim 176 , wherein each exogenous polynucleotide is operably linked to a seed-specific promoter.
184 . The process of claim 176 , wherein 4% of the total fatty acid content is DPA.
185 . The process of claim 176 , which comprises a step of purifying the extracted lipid.
186 . A process for producing lipid comprising docosahexaenoic acid (DHA) in an esterified form, the process comprising a step of extracting lipid from a plant cell which comprises exogenous polynucleotides encoding
a Δ6 desaturase,
a Δ6 elongase,
a Δ5 desaturase,
a Δ5 elongase, and
a Δ4 desaturase,
wherein each exogenous polynucleotide is operably linked to a promoter that directs expression of said polynucleotide in the plant cell,
wherein one or more of the Δ5 desaturase and Δ4 desaturase is a microalgal desaturase,
wherein the Δ6 desaturase is a microalgal Δ6 desaturase which has greater activity on ALA-CoA as fatty acid substrate than on α-linolenic acid (ALA) joined to the sn-2 position of PC as fatty acid substrate, and has greater Δ6 desaturase activity on ALA than linoleic acid (LA) as fatty acid substrate,
and wherein DHA is present in the extracted lipid at a level of between 3% and 5% of the extracted lipid's total fatty acid content,
thereby producing the lipid comprising DHA.
187 . A process for producing lipid comprising docosahexaenoic acid (DHA) in an esterified form, the process comprising a step of extracting lipid from a plant cell which comprises exogenous polynucleotides encoding
a Δ6 desaturase,
a Δ6 elongase,
a Δ5 desaturase,
a Δ5 elongase, and
a Δ4 desaturase,
wherein each exogenous polynucleotide is operably linked to a promoter that directs expression of said polynucleotide in the plant cell,
wherein more than one of the Δ6 desaturase, Δ5 desaturase and Δ4 desaturase is a microalgal desaturase,
wherein one or more of the Δ6 desaturase, Δ5 desaturase and Δ4 desaturase has greater activity on an acyl-CoA substrate than on a corresponding acyl-phosphatidylcholine (acyl-PC) substrate, wherein the Δ4 desaturase is a Δ4 desaturase from Pavlova lutheri or Pavlova salina,
and wherein DHA is present in the extracted lipid at a level of between 3% and 5% of the extracted lipid's total fatty acid content,
thereby producing the lipid comprising DHA.