Multizymes and their use in making polyunsaturated fatty acids

Abstract

Isolated nucleic acid fragments and recombinant constructs comprising such fragments encoding multizymes (i.e., single polypeptides having at least two independent and separable enzymatic activities) along with a method of making long-chain polyunsaturated fatty acids (PUFAs) using these multizymes in plants and oleaginous yeast are disclosed.

1 . A multizyme comprising a single polypeptide having at least two independent and separable enzymatic activities. 
     
     
         2 . (canceled) 
     
     
         3 . The multizyme of  claim 1 , wherein the enzymatic activities comprises at least one fatty acid elongase linked to at least one fatty acid desaturase. 
     
     
         4 . The multizyme of  claim 3 , wherein the fatty acid desaturase is selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-9 desaturase, a delta-12 desaturase, a delta-15 desaturase, and a delta-17 desaturase. 
     
     
         5 . The multizyme of  claim 3 , wherein the fatty acid elongase is selected from the group consisting of a delta-9 elongase, a C 14/16  elongase, a C 16/18  elongase, a C 18/20  elongase, and a C 20/22  elongase. 
     
     
         6 . The multizyme of  claim 1 , wherein a first enzymatic activity is linked to a second enzymatic activity and said link is selected from the group consisting of a polypeptide bond, SEQ ID NO:198 (EgDHAsyn1 linker), SEQ ID NO:200 (EgDHAsyn2 linker), SEQ ID NO:235 (EaDHAsyn1 linker), SEQ ID NO:438, SEQ ID NO:472, SEQ ID NO:445, and SEQ ID NO:504. 
     
     
         7 . An isolated polynucleotide encoding a DHA synthase comprising:
 (a) a nucleotide sequence encoding a polypeptide having DHA synthase activity, wherein the polypeptide has at least 80% amino acid identity, based on the Clustal V method of alignment, when compared to an amino acid sequence as set forth in SEQ ID NO:12, SEQ ID NO:22, SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97;   (b) a nucleotide sequence encoding a polypeptide having DHA synthase activity wherein the nucleotide sequence has at least 80% sequence identity, based on the BLASTN method of alignment, when compared to a nucleotide sequence as set forth in SEQ ID NO:11, SEQ ID NO:205, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO:410;   (c) a nucleotide sequence encoding a polypeptide having DHA synthase activity, wherein the nucleotide sequence hybridizes under stringent conditions to a nucleotide sequence as set forth in SEQ ID NO:11, SEQ ID NO:205, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO:410; or   (d) a complement of the nucleotide sequence of (a), (b), or (c), wherein the complement and the nucleotide sequence consist of the same number of nucleotides and are 100% complementary.   
     
     
         8 . The polynucleotide of  claim 7 , wherein the nucleotide sequence comprises SEQ ID NO:11, SEQ ID NO:21, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, or SEQ ID NO: 410. 
     
     
         9 . The polynucleotide of  claim 7 , wherein the amino acid sequence of the polypeptide comprises SEQ ID NO:12, SEQ ID NO:22,
 SEQ ID NO:95, SEQ ID NO:96, or SEQ ID NO:97.   
     
     
         10 - 14 . (canceled) 
     
     
         15 . A recombinant construct comprising the isolated polynucleotide of  claim 7  operably linked to at least one regulatory sequence. 
     
     
         16 . A host cell comprising in its genome the recombinant construct of  claim 15 . 
     
     
         17 . The host cell of  claim 16 , wherein said cell is selected from the group consisting of plants and yeast. 
     
     
         18 . The host cell of  claim 17 , wherein the host cell is a transformed  Yarrowia  sp. 
     
     
         19 - 22 . (canceled) 
     
     
         23 . A plant comprising in its genome the recombinant construct of  claim 15 . 
     
     
         24 . The plant of  claim 23 , wherein the plant is an oilseed plant. 
     
     
         25 . The plant of  claim 23 , wherein the plant is soybean. 
     
     
         26 . Seed obtained from the plant of  claim 23 . 
     
     
         27 - 36 . (canceled) 
     
     
         37 . A method for making a multizyme which comprises:
 (a) linking a first polypeptide with at least a second polypeptide wherein each polypeptide has an independent and separable enzymatic activity; and   (b) evaluating the product of step (a) for the independent and separable enzymatic activities.   
     
     
         38 . (canceled) 
     
     
         39 . The method of  claim 37 , wherein the enzymatic activities comprises at least one fatty acid elongase linked to at least one fatty acid desaturase. 
     
     
         40 . The method of  claim 39 , wherein the fatty acid desaturase is selected from the group consisting of a delta-4 desaturase, a delta-5 desaturase, a delta-6 desaturase, a delta-8 desaturase, a delta-9 desaturase, a delta-12 desaturase, a delta-15 desaturase, and a delta-17 desaturase. 
     
     
         41 . The method of  claim 39 , wherein the fatty acid elongase is selected from the group consisting of a delta-9 elongase, a C 14/16  elongase, a C 16/18  elongase, a C 18/20  elongase, and a C 20/22  elongase. 
     
     
         42 . The method of  claim 37 , wherein the link is selected from the group consisting of a polypeptide bond, SEQ ID NO:198 (EgDHAsyn1 linker amino acid sequence), SEQ ID NO:200 (EgDHAsyn2 linker), SEQ ID NO:235 (EaDHAsyn1 linker), SEQ ID NO:435, SEQ ID NO:438, SEQ ID NO:472, and SEQ ID NO:504. 
     
     
         43 - 50 . (canceled) 
     
     
         51 . A method for the conversion of eicosapentaenoic acid to docosahexaenoic acid comprising:
 a) providing a recombinant microbial host cell comprising:
 i) a DHA synthase comprising:
 1) at least one polypeptide encoding a C20 elongase; 
 2) at least one polypeptide encoding a delta-4 desaturase; and 
 3) a polypeptide linker wherein the linker is interposed between the C20 elongase and the delta-4 desaturase; and 
 
 ii) a source of eicosapentaenoic acid; and 
   b) growing the host cell of (a) under conditions whereby docosahexaenoic acid is produced.   
     
     
         52 - 65 . (canceled)