Production of DHA and Other LC-PUFAs in Plants

Abstract

The invention provides recombinant host organisms (e.g., plants) genetically modified with a polyunsaturated fatty acid (PUFA) synthase system and one or more accessory proteins (e.g., PPTase and/or ACoAS) that allow for and/or improve the production of PUFAs in the host organism. The present invention also relates to methods of making and using such organisms (e.g., to obtain PUFAs) as well as products obtained from such organisms (e.g., oil and/or seed).

1 - 68 . (canceled) 
     
     
         69 . A genetically modified  Brassica  plant, descendant, cell, tissue, seed or part thereof, comprising:
 (i) a nucleic acid sequence encoding a polyunsaturated fatty acid (PUFA) synthase system that produces at least one PUFA; and   (ii) a nucleic acid sequence encoding a phosphopantetheinyl transferase (PPTase) that transfers a phosphopantetheinyl cofactor to a PUFA synthase system ACP domain.   
     
     
         70 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , wherein the plant, descendant, cell, tissue, seed, or part thereof is  Brassica napus  or  Brassica juncea.    
     
     
         71 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , wherein the PUFA synthase system comprises an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO:1, 2 or 3. 
     
     
         72 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , wherein the nucleic acid sequence encoding the PUFA synthase system comprises a nucleic acid sequence at least 80% identical to the nucleic acid sequence of SEQ ID NO:6, 7 or 8. 
     
     
         73 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , wherein the PUFA synthase system comprises the amino acid sequences of SEQ ID NOs: 1, 2 and 3. 
     
     
         74 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , wherein the PPTase comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:5. 
     
     
         75 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , wherein the nucleic acid sequence encoding the PPTase is at least 80% identical to the nucleic acid sequence of SEQ ID NO:10. 
     
     
         76 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , wherein the nucleic acid sequences of (i) and (ii) are contained in a single recombinant expression vector. 
     
     
         77 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , wherein the nucleic acid sequences of (i) and (ii) are operably linked to a promoter selected from the group consisting of: PvDlec2, LfKCS3 and FAE 1. 
     
     
         78 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , further comprising:
 (iii) a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) that catalyzes the conversion of long chain PUFA free fatty acids (FFA) to acyl-CoA.   
     
     
         79 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 78 , wherein the ACoAS comprises an amino acid sequence that is at least 80% identical to SEQ ID NO:4. 
     
     
         80 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 78 , wherein the nucleic acid sequence encoding, the ACoAS comprises a nucleic acid sequence that is at least 80% identical to the nucleic acid sequence of SEQ ID NO:9 or SEQ ID NO:34. 
     
     
         81 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , further comprising a nucleic acid sequence encoding an acetyl CoA carboxylase (ACCase) or a nucleic acid sequence encoding a type 2 diacylglycerol acyltransferase (DGAT2). 
     
     
         82 . An isolated nucleic acid molecule comprising a nucleic acid sequence selected from SEQ ID NOs:6-10. 
     
     
         83 . A recombinant expression vector pDAB7361, pDAB7362 or pDAB7363. 
     
     
         84 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 , wherein the plant, descendant, cell, tissue, seed, or part thereof comprises 0.01% to 15% by weight of docosahexaenoic acid (DHA). 
     
     
         85 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 84 , wherein the plant, descendant, cell, tissue, seed, or part thereof comprises 0.01% to 5% by weight of eicosapentaenoic acid (EPA). 
     
     
         86 . The genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 84 , wherein the plant, descendant, cell, tissue, seed, or part thereof comprises 0.01% to 5% by weight docosapentaenoic acid (DPA). 
     
     
         87 . An oil obtained from the genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 . 
     
     
         88 . A seed obtained from a genetically modified  Brassica  plant, descendant, cell, tissue, or part thereof of  claim 69 . 
     
     
         89 . A functional food comprising; the oil obtained from the genetically modified  Brassica  plant, descendant cell, tissue, seed, or part thereof of  claim 69 . 
     
     
         90 . A pharmaceutical product comprising the oil obtained from the genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 . 
     
     
         91 . A method to produce an oil comprising at least one PUFA, comprising recovering oil from the genetically modified  Brassica  plant, descendant, cell, tissue, seed, or part thereof of  claim 69 . 
     
     
         92 . An oil composition from seeds of a  Brassica  plant comprising about 0.05% to 5% by weight of DHA. 
     
     
         93 . The oil composition of  claim 92 , farther comprising a fatty acid profile of >70% by weight of oleic acid. 
     
     
         94 . The oil composition of  claim 92 , further comprising a fatty acid profile of <3.5% by weight of alpha-linolenic acid. 
     
     
         95 . A method to produce the genetically modified  Brassica  plant, comprising transforming a  Brassica  plant or plant cell with an expression cassette comprising: (i) a nucleic acid sequence encoding a polyunsaturated fatty acid (PUFA) synthase that produces at least one PUFA; (ii) a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) that catalyzes the conversion of long chain PUFA free fatty acids (FFA) to acyl-CoA; and (iii) a nucleic acid sequence encoding a phosphopantetheinyl transferase (PPTase) that activates a PUFA synthase system ACP domain.