PUFA Polyketide Synthase Systems and Uses Thereof

Abstract

Disclosed are the complete polyunsaturated fatty acid (PUFA) polyketide synthase (PKS) systems from Schizochytrium, and biologically active fragments and homologues thereof. More particularly, this invention relates to nucleic acids encoding such PUFA PKS systems, to proteins and domains thereof that comprise such PUFA PKS systems, to genetically modified organisms (plants and microorganisms) comprising such PUFA PKS systems, and to methods of making and using the PUFA PKS systems disclosed herein. This invention also relates to genetically modified plants and microorganisms and methods to efficiently produce lipids enriched in various polyunsaturated fatty acids (PUFAs) as well as other bioactive molecules by manipulation of a PUFA polyketide synthase (PKS) system.

1 . An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
 a) a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 1 SEQ ID NO:3, and SEQ ID NO:5;   b) a nucleic acid sequence encoding an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6;   c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:2 or that is a fragment of SEQ ID NO:2, wherein said amino acid sequence has β-keto acyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, and wherein said amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO:2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO:2;   d) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:4 or that is a fragment of SEQ ID NO:4, wherein said amino acid sequence has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, and wherein said amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO:4; and   e) a nucleic acid sequence encoding an amino aid sequence that is at least 90% identical to SEQ ID NO:6 or that is a fragment of SEQ ID NO:6, wherein said amino acid sequence has FabA-like β-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, and wherein said amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 876-890 of SEQ ID NO:6.   
     
     
         2 - 7 . (canceled) 
     
     
         8 . An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
 a) a first nucleic acid sequence encoding a first amino acid sequence that has β-keto acyl-ACP synthase (KS) activity, malonyl-CoA:ACP acyltransferase (MAT) activity, acyl carrier protein (ACP) activity and ketoreductase (KR) activity, wherein the first nucleic acid sequence hybridizes under very high stringency conditions to the complement of a second nucleic acid sequence encoding a second amino acid sequence of SEQ ID NO:2, and wherein said first amino acid sequence comprises an aspartate at a position corresponding to amino acid 667 of SEQ ID NO:2 and a histidine at a position corresponding to amino acid 668 of SEQ ID NO:2;   b) a first nucleic acid sequence encoding a first amino acid sequence that has KS activity, chain length factor (CLF) activity, acyl transferase (AT) activity, and enoyl ACP-reductase (ER) activity, wherein the first nucleic acid sequence hybridizes under very high stringency conditions to the complement of a second nucleic acid sequence encoding a second amino acid sequence of SEQ ID NO:4, and wherein said first amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:4 and a glutamate at a position corresponding to amino acid 1415 of SEQ ID NO:4; and   c) a first nucleic acid sequence encoding a first amino acid sequence that has FabA-like β-hydroxy acyl-ACP dehydrase (DH) activity and ER activity, wherein the first nucleic acid sequence hybridizes under very high stringency conditions to the complement of a second nucleic acid sequence encoding a second amino acid sequence of SEQ ID NO:6, and wherein said first amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 876-890 of SEQ ID NO:6.   
     
     
         9 - 10 . (canceled) 
     
     
         11 . An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
 a) a nucleic acid sequence of SEQ ID NO:9;   b) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:10; and   c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:10 or that is a fragment of SEQ ID NO:10, wherein the amino acid sequence has malonyl-CoA:ACP acyltransferase (MAT) activity, and wherein said amino acid sequence comprises an aspartate at a position corresponding to amino acid 93 of SEQ ID NO:10 and a histidine at a position corresponding to amino acid 94 of SEQ ID NO:10.   
     
     
         12 - 13 . (canceled) 
     
     
         14 . An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
 a) a nucleic acid sequence of SEQ ID NO:19;   b) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:20; and   c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:20 or that is a fragment of SEQ ID NO:20, wherein the amino acid sequence has β-keto acyl-ACP synthase (KS) activity, and wherein said amino acid sequence comprises a valine at a position corresponding to amino acid 371 of SEQ ID NO:20.   
     
     
         15 - 16 . (canceled) 
     
     
         17 . An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of:
 a) a nucleic acid sequence of SEQ ID NO:29;   b) a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO:30; and   c) a nucleic acid sequence encoding an amino acid sequence that is at least 90% identical to SEQ ID NO:30 or that is a fragment of SEQ ID NO:30, wherein the amino acid sequence has FabA-like β-hydroxy acyl-ACP dehydrase (DH) activity, and wherein said amino acid sequence comprises the sequence of H-G-I-A-N-P-T-F-V-H-A-P-G-K-I (positions 876-890 of SEQ ID NO:6) at positions corresponding to amino acids 426-440 of SEQ ID NO:30.   
     
     
         18 - 19 . (canceled) 
     
     
         20 . A recombinant nucleic acid molecule comprising the nucleic acid molecule of  claim 1 , operatively linked to at least one transcription control sequence. 
     
     
         21 . A recombinant cell transfected with the nucleic acid molecule of  claim 1 . 
     
     
         22 . The recombinant cell of  claim 21 , wherein said recombinant cell is a microorganism. 
     
     
         23 . The recombinant cell of  claim 21 , wherein said recombinant cell is a plant cell. 
     
     
         24 . An isolated nucleic acid molecule consisting essentially of a nucleic acid sequence that is fully complementary to the nucleic acid molecule of  claim 1 . 
     
     
         25 . A genetically modified microorganism that has been transformed with the nucleic acid molecule of  claim 1 . 
     
     
         26 . A genetically modified plant or part of a plant that has been transformed with the nucleic acid molecule of  claim 1 . 
     
     
         27 - 28 . (canceled) 
     
     
         29 . The genetically modified microorganism of  claim 25 , wherein said microorganism endogenously expresses a PUFA PKS system. 
     
     
         30 . The genetically modified microorganism of  claim 25 , wherein said microorganism has been further transformed with a recombinant nucleic acid molecule encoding a phosphopantetheine transferase. 
     
     
         31 . The genetically modified microorganism of  claim 25 , wherein said microorganism is a Thraustochytriales microorganism. 
     
     
         32 . The genetically modified microorganism of  claim 25 , wherein said microorganism is a bacterium. 
     
     
         33 . The genetically modified microorganism of  claim 25 , wherein said microorganism is a yeast. 
     
     
         34 . A method to produce a bioactive molecule, comprising culturing under conditions effective to produce said bioactive molecule a genetically modified organism according to  claim 25 . 
     
     
         35 - 36 . (canceled) 
     
     
         37 . The genetically modified plant or part of a plant of  claim 26 , wherein said plant has been further genetically modified to express a recombinant nucleic acid molecule encoding a phosphopantetheine transferase. 
     
     
         38 - 39 . (canceled) 
     
     
         40 . The genetically modified plant or part of a plant of  claim 26 , wherein said plant is selected from the group consisting of: canola, soybean, rapeseed, linseed, corn, safflower, sunflower and tobacco. 
     
     
         41 . The genetically modified plant or part of a plant of  claim 26 , wherein the plant is an oilseed plant and wherein the part of the plant is a mature oilseed. 
     
     
         42 . The genetically modified plant or part of a plant of  claim 26 , wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one PUFA selected from the group consisting of DHA (docosahexaenoic acid (C22:6, n-3)) and DPA (docosapentaenoic acid (C22:5, n-6), and wherein the total fatty acids produced as a result of transformation with said nucleic acid molecules, other than said at least one PUFA, comprise less than about 10% of the total fatty acids produced by said plant. 
     
     
         43 . The genetically modified plant or part of a plant of  claim 42 , wherein the total fatty acids produced as a result of transformation with said nucleic acid molecules, other than said at least one PUFA, comprise less than 5% by weight of the total fatty acids produced by said plant. 
     
     
         44 . The genetically modified plant or part of a plant of  claim 42 , wherein the fatty acids consisting of gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds, comprise less than 5% by weight of the total fatty acids produced by said plant. 
     
     
         45 . The genetically modified plant or part of a plant of  claim 42 , wherein gamma-linolenic acid (GLA; 18:3, n-6) comprises less than 1% by weight of the total fatty acids produced by said plant. 
     
     
         46 . A plant or a part of the plant, wherein the total fatty acid profile in the plant or part of the plant comprises detectable amounts of DHA (docosahexaenoic acid (C22:6, n-3)) and DPA (docosapentaenoic acid (C22:5, n-6). 
     
     
         47 . The plant or a part of the plant of  claim 46 , wherein the ratio of DPAn-6 to DHA is less than 1:1. 
     
     
         48 . The plant or a part of the plant of  claim 46 , wherein the total fatty acid profile in the plant or part of the plant contains less than 5% by weight in total of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds. 
     
     
         49 . The plant or a part of the plant of  claim 46 , wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 5% in total of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds. 
     
     
         50 . The plant or a part of the plant of  claim 46 , wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds. 
     
     
         51 . The plant or a part of the plant of  claim 46 , wherein the total fatty acid profile in the plant or part of the plant comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 2% of gamma-linolenic acid (GLA; 18:3, n-6) and dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6). 
     
     
         52 . Seeds obtained from the plant or part of the plant of  claim 46 . 
     
     
         53 . A food product comprising the seeds of  claim 52 . 
     
     
         54 . An oil obtained from seeds of the plant of  claim 46 . 
     
     
         55 . A food product comprising the oil of  claim 54 . 
     
     
         56 . An oil blend comprising the oil of  claim 54  and another oil. 
     
     
         57 - 59 . (canceled) 
     
     
         60 . The oil of  claim 54 , comprising the following fatty acids: DHA (C22:6n-3), DPAn-6 (C22:5n-6), oleic acid (C18:1), linolenic acid (C18:3), linoleic acid (C18:2), C16:0, C18.0, C20:0, C20:1n-9, C20:2n-6, C22:1n-9; wherein the oil comprises less than 0.5% of any of the following fatty acids: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds. 
     
     
         61 . The plant of  claim 26 , wherein said plant produces mature seeds in which the total seed fatty acid profile comprises at least 1.0% by weight of at least one polyunsaturated fatty acid selected from the group consisting of DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 5% in total of all of the following PUFAs: gamma-linolenic acid (GLA; 18:3, n-6), PUFAs having 18 carbons and four carbon-carbon double bonds, PUFAs having 20 carbons and three carbon-carbon double bonds, and PUFAs having 22 carbons and two or three carbon-carbon double bonds. 
     
     
         62 . The plant of  claim 61 , where said plant produces mature seeds in which the total seed fatty acid profile comprises at least 1.0% by weight of at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of DHA (C22:6n-3) and DPAn-6 (C22:5n-6), and wherein the total fatty acid profile in the plant or part of the plant contains less than 1% of gamma-linolenic acid (GLA; 18:3, n-6). 
     
     
         63 . A method to produce a bioactive molecule, comprising growing under conditions effective to produce said bioactive molecule a genetically modified plant according to  claim 26 . 
     
     
         64 - 66 . (canceled) 
     
     
         67 . The plant or a part of the plant of  claim 46 , wherein the ratio of DPAn-6 to DHA is 1:1 or greater than 1:1.