Delta-15 desaturases genes suitable for increasing levels of omega-3 fatty acids

Abstract

The present invention relates to fungal Δ-15 fatty acid desaturases that are able to catalyze the conversion of linoleic acid (18:2, LA) to alpha-linolenic acid (18:3, ALA). Nucleic acid sequences encoding the desaturases, nucleic acid sequences which hybridize thereto, DNA constructs comprising the desaturase genes, and recombinant host plants and microorganisms expressing increased levels of the desaturases are described. Methods of increasing production of specific omega-3 and omega-6 fatty acids by over-expression of the Δ-15 fatty acid desaturases are also described herein.

1 . An isolated nucleic acid fragment encoding a fungal Δ5 desaturase enzyme, selected from the group consisting of:
 (a) an isolated nucleic acid fragment encoding the amino acid sequence as set forth in SEQ ID NO:2;   (b) an isolated nucleic acid fragment 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 followed by 0.1×SSC, 0.1% SDS; or,   an isolated nucleic acid fragment that is complementary to (a) or (b).   
     
     
         2 . The isolated nucleic acid fragment of  claim 1  as set forth in SEQ ID NO:1. 
     
     
         3 . The isolated nucleic acid fragment of  claim 1  isolated from  Fusarium moniliforme.    
     
     
         4 . An isolated nucleic acid fragment comprising a first nucleotide sequence encoding a Δ5 desaturase enzyme of at least 402 amino acids that has at least 86% identity based on the Clustal method of alignment 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.   
     
     
         5 . A polypeptide encoded by the isolated nucleic acid fragment of any one of  claims 1 - 4  which binds both oleic and linoleic acid as enzymatic substrates. 
     
     
         6 . A chimeric gene comprising the isolated nucleic acid fragment of any of  claims 1 - 4  operably linked to suitable regulatory sequences. 
     
     
         7 . A transformed host cell comprising the isolated nucleic acid fragment of any of  claims 1 - 5 . 
     
     
         8 . A transformed host cell according to  claim 7  selected from the group consisting of: plants, algae, bacteria, yeast and fungi. 
     
     
         9 . A transformed host cell according to  claim 8  wherein the host cell is a plant selected from the group consisting of: soybean, corn, rapeseed, primrose, flax, canola, maize, safflower and sunflower. 
     
     
         10 . A transformed host cell according to  claim 8  wherein the host cell is a fungus selected from the group consisting of:  Thraustochytrium  sp.,  Schizochytrium  sp. and  Mortierella  sp. 
     
     
         11 . A transformed host cell according to  claim 8  wherein the yeast is an oleaginous yeast. 
     
     
         12 . The transformed yeast of  claim 11  wherein the oleaginous yeast is selected from the group consisting of:  Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon  and  Lipomyces.    
     
     
         13 . The transformed yeast of  claim 12  wherein the oleaginous yeast is  Yarrowia  sp. 
     
     
         14 . The transformed  Yarrowia  sp. of  claim 13  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. 
     
     
         15 . A method for the production of a-linolenic acid comprising:
 a) providing a host cell comprising:
 (i) an isolated nucleic acid fragment encoding a protein having Δ5 desaturase activity that has at least 46.2% identity based on the Clustal method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2; and 
 (ii) a source of linoleic acid; 
   b) growing the host cell of step (a) under conditions wherein the nucleic acid fragment encoding a protein having Δ5 desaturase activity is expressed and the linoleic acid is converted to Δ-linolenic acid; and   c) optionally recovering the α-linolenic acid of step (b).   
     
     
         16 . A method for the production of α-linolenic acid comprising:
 a) providing a host cell comprising:
 (i) an isolated nucleic acid fragment encoding a protein having Δ5 desaturase activity that has at least 46.2% identity based on the Clustal method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2; and 
 (ii) a source of oleic acid; 
   b) growing the host cell of step (a) under conditions wherein the nucleic acid fragment encoding a protein having Δ5 desaturase activity is expressed and the oleic acid is converted to a-linolenic acid; and   c) optionally recovering the α-linolenic acid of step (b).   
     
     
         17 . A method according to  claim 15  or  16  wherein the isolated nucleic acid fragment encoding a protein having Δ15 desaturase activity is isolated from a fungus selected from the group consisting of:  Fusarium moniliforme, Magnaporthe grisea, Neurospora crassa, Fusarium graminearium  and  Aspergillus nidulans.    
     
     
         18 . A method according to  claim 15  or  16  wherein the isolated nucleic acid fragment encoding a protein having Δ15 desaturase activity encodes an amino acid sequence selected from the group consisting of SEQ ID NO:2, 6, 10, 14 and 18. 
     
     
         19 . A method according to  claim 15  wherein the protein having Δ5 desaturase activity has a percent substrate conversion of linoleic acid to α-linolenic acid of at least about 50%. 
     
     
         20 . A method according to  claim 15  wherein the protein having Δ5 desaturase activity has a percent substrate conversion of linoleic acid to α-linolenic acid of at least about 80%. 
     
     
         21 . A method according to  claim 15  wherein the protein having Δ5 desaturase activity has a percent substrate conversion of linoleic acid to α-linolenic acid of at least about 90%. 
     
     
         22 . A method according to  claim 15  wherein the protein having Δ5 desaturase activity has a percent substrate conversion of linoleic acid to α-linolenic acid that is greater than at least about 95%. 
     
     
         23 . A method according to  claim 15  or  16  wherein the protein having Δ5 desaturase activity binds both oleic acid and linoleic acid as enzymatic substrates. 
     
     
         24 . A method for the production of ω-3 fatty acids in a host cell comprising:
 a) providing a host cell comprising:
 (ii) an isolated nucleic acid fragment encoding a protein having Δ5 desaturase activity that has at least 46.2% identity based on the Clustal method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2; and 
 (ii) genes encoding a functional ω-3/ω-6 fatty acid biosynthetic pathway; 
   b) providing a source of desaturase substrate consisting of oleic acid;   c) growing the host cell of step (a) with the desaturase substrate of step (b) under conditions wherein ω-3 fatty acids are produced; and   d) optionally recovering the ω-3 fatty acids of step (c).   
     
     
         25 . A method of increasing the ratio of ω-3 fatty acids to ω-6 fatty acids in a host cell producing ω-3 fatty acids to ω-6 fatty acids comprising:
 a) providing a host cell producing ω-3 fatty acids and ω-6 fatty acids;   b) introducing into the host cell of (a) an isolated nucleic acid fragment encoding a protein having at least 46.2% identity based on the Clustal method of alignment when compared to a polypeptide having the sequence as set forth in SEQ ID NO:2, wherein the polypeptide binds both oleic acid and linoleic acid as an enzyme substrate, wherein the ratio of ω-3 fatty acids to ω-6 fatty acids are increased.   
     
     
         26 . A method according to  claim 25  wherein the host cell producing ω-3 fatty acids and ω-6 fatty acids lacks a polypeptide having delta-12 desaturase activity. 
     
     
         27 . A method according to  claim 24  of  25  wherein the isolated nucleic acid fragment encoding a protein having Δ5 desaturase activity encodes an amino acid sequence selected from the group consisting of SEQ ID NOs:2, 6, 10, 14 and 18. 
     
     
         28 . A method according to  claim 24  wherein the ω-3 fatty acids are selected from the group consisting of: a-linolenic acid, stearidonic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, docosapentaenoic acid and docosahexaenoic acid. 
     
     
         29 . A method according to  claim 24  wherein the genes encoding enzymes of the ω-3/ω-6 fatty acid biosynthetic pathway are selected from the group consisting of: a Δ6 desaturase, an elongase, a Δ5 desaturase, a Δ4 desaturase, a Δ8 desaturase, a Δ9 desaturase, a Δ9 elongase and a Δ17 desaturase. 
     
     
         30 . A method according to any one of  claims 15 ,  16 ,  24  and  25 , wherein the host cell is selected from the group consisting of: plants, algae, bacteria, yeast and fungi. 
     
     
         31 . A method according to  claim 30  wherein the host cell is a plant selected from the group consisting of: soybean, corn, flax, rapeseed, primrose, canola, maize, safflower and sunflower. 
     
     
         32 . A method according to  claim 30  wherein the host cell is a fungus selected from the group consisting of:  Thraustochytrium  sp.,  Schizochytrium  sp. and  Mortierella  sp. 
     
     
         33 . A method according to  claim 30  wherein the yeast is an oleaginous yeast. 
     
     
         34 . A method according to  claim 33  wherein the oleaginous yeast is selected from the group consisting of:  Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon  and  Lipomyces.    
     
     
         35 . A method according to  claim 34  wherein the  Yarrowia  is 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. 
     
     
         36 . A microbial oil produced by the method of any one of  claims 15 ,  16 ,  24  and  25 . 
     
     
         37 . A method of obtaining a nucleic acid fragment encoding a Δ5 desaturase enzyme comprising:
 (a) probing a genomic library with the nucleic acid fragment of  claim 1 ;   (b) identifying a DNA clone that hybridizes with the nucleic acid fragment of  claim 1 ; and   (c) sequencing the genomic fragment that comprises the clone identified in step (b),   
       wherein the sequenced genomic fragment encodes a Δ15 desaturase enzyme. 
     
     
         38 . A method of obtaining a nucleic acid fragment encoding a Δ15 desaturase enzyme comprising:
 (a) synthesizing at least one oligonucleotide primer corresponding to a portion of the sequence as set forth in SEQ ID NO:1; 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 Δ15 desaturase enzyme.