Fad4, fad5, fad5-2, and fad6, novel fatty acid desaturase family members and uses thereof

Abstract

The invention provides isolated nucleic acid molecules which encode novel fatty acid desaturase family members. The invention also provides recombinant expression vectors containing desaturase nucleic acid molecules, host cells into which the expression vectors have been introduced, and methods for large-scale production of long chain polyunsaturated fatty acids (LCPUFAs), e.g., DHA.

We claim: 
     
         1 . A method for producing n−3 long chain polyunsaturated fatty acids comprising at least 20 carbons in a transgenic plant, comprising:
 a) selecting a first plant line having high content of n−6 fatty acid linoleic acid (LA) relative to a second plant line of the same plant species; 
 b) providing at least one nucleic acid molecule encoding a Δ6 desaturase in said first plant line to produce a transgenic plant line, wherein the level of n−6 gamma-linolenic acid (GLA) produced in said first transgenic plant line is higher than the level of GLA produced in a reference transgenic plant line of the second plant line expressing a nucleic acid molecule encoding the same Δ6 desaturase, 
 c) further manipulating the first transgenic plant to comprise the desaturase and elongase activities for biosynthetic production of n−3 long chain polyunsaturated fatty acids comprising at least 20 from GLA; 
 d) cultivating the transgenic plant line under conditions to produce the n−3 long chain polyunsaturated fatty acids comprising at least 20 carbon; and 
 e) optionally recovering the n−3 long chain polyunsaturated fatty acids comprising at least 20 carbons. 
 
     
     
         2 . The method of  claim 1 , wherein the n−3 long chain polyunsaturated fatty acids comprises at least one of eicosapentaenioc acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6). 
     
     
         3 . The method of  claim 1 , wherein the n−3 long chain polyunsaturated fatty acids comprises docosahexaenoic acid (DHA, 22:6). 
     
     
         4 . The method of  claim 1 , wherein said at least one Δ6 desaturase comprises a cytochrome b5-like domain. 
     
     
         5 . The method of  claim 4 , wherein said at least one Δ6 desaturase further comprises at least two histidine motifs. 
     
     
         5 . The method of  claim 4 , wherein said at least one Δ6 desaturase comprises three histidine motifs. 
     
     
         6 . The method of  claim 1 , wherein said at least one Δ6 desaturase has at least 70% sequence identity to the amino acid sequence of SEQ ID NO: 8. 
     
     
         7 . The method of  claim 1 , wherein said at least one Δ6 desaturase comprises:
 a) the amino acid sequence of SEQ ID NO: 8; or 
 b) an amino acid sequence encoded by the nucleotide sequence of SEQ ID NO: 7. 
 
     
     
         8 . The method of  claim 1 , wherein the plant species is a  Brassica  species. 
     
     
         9 . The method of  claim 3 , wherein recovering the n−3 long chain polyunsaturated fatty acids comprises isolating unsaturated fatty acids from said transgenic plant line. 
     
     
         10 . The method of  claim 3 , wherein d) cultivating the transgenic plant line comprises producing a transgenic plant to produce the n−3 long chain polyunsaturated fatty acids comprising at least 20 carbons. 
     
     
         11 . The method of  claim 10 , wherein recovering the n−3 long chain polyunsaturated fatty acids comprises isolating unsaturated fatty acids from said transgenic plant. 
     
     
         12 . The method of  claim 11 , wherein said unsaturated fatty acids are isolated from seeds of said transgenic plant. 
     
     
         13 . The method of  claim 11 , wherein said n−3 long chain polyunsaturated fatty acids comprises DHA. 
     
     
         14 . The method of  claim 9 , wherein said unsaturated fatty acids are isolated in the form of lipids. 
     
     
         15 . The method of  claim 9 , wherein said n−3 long chain polyunsaturated fatty acids comprises DHA. 
     
     
         16 . A composition comprising the unsaturated fatty acids produced in  claim 11 . 
     
     
         17 . A dietary supplement, animal feed, a neutraceutical, or a pharmaceutical composition comprising the unsaturated fatty acids produced in  claim 11 .