Delta 17 desaturase and its use in making polyunsaturated fatty acids

Abstract

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 fragment comprising a nucleic acid sequence encoding a delta-17 desaturase polypeptide comprising at least one amino acid sequence motif selected from the group consisting of SEQ ID NO:96 and SEQ ID NO:98,
 wherein the delta-17 desaturase polypeptide does not comprise SEQ ID NO:43 and SEQ ID NO:95, and   wherein the delta-17 desaturase polypeptide has delta-17 desaturase activity.   
     
     
         2 . A delta-17 desaturase polypeptide comprising at least one amino acid motif selected from the group consisting of SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98. 
     
     
         3 . (canceled) 
     
     
         4 . A method for the identification and isolation of a delta-17 desaturase polypeptide comprising:
 a) probing a genomic library with:
 i) an isolated nucleic acid fragment encoding an amino acid sequence selected from the group consisting of SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98; or, 
 ii) an isolated nucleic acid fragment that is complementary to (i); 
   b) identifying a DNA clone that hybridizes with the nucleic acid fragment of step (a); and,   c) sequencing the genomic fragment that comprises the clone identified in step (b);   wherein the sequenced genomic fragment encodes a delta-17 desaturase polypeptide.   
     
     
         5 . A method for the identification and isolation of a delta-17 desaturase polypeptide comprising:
 a) synthesizing at least one oligonucleotide primer corresponding to a portion of an isolated nucleic acid sequence encoding an amino acid motif selected from the group consisting of SEQ ID NO:96, SEQ ID NO:97 and SEQ ID NO:98; and,   b) amplifying an insert present in a cloning vector using the oligonucleotide primer of step (a);   wherein the amplified insert encodes a portion of an amino acid sequence encoding a delta-17 desaturase enzyme.   
     
     
         6 . The isolated nucleic acid fragment of  claim 1 , wherein the delta-17 desaturase polypeptide further comprises three His Box motifs set forth as H(X) 3 H (SEQ ID NO:99), H(X) 2 HH (SEQ ID NO:100) and H/Q(X) 2 HH (SEQ ID NO:101).