POLYUNSATURATED FATTY ACID PRODUCTION IN HETEROLOGOUS ORGANISMS USING PUFA POLYKETIDE SYNTHASE SYSTEMS
Disclosed are novel acyl-CoA synthetases and novel acyltransferases, nucleic acid molecules encoding the same, recombinant nucleic acid molecules and recombinant host cells comprising such nucleic acid molecules, genetically modified organisms (microorganisms and plants) comprising the same, and methods of making and using the same. Also disclosed are genetically modified organisms (e.g., plants, microorganisms) that have been genetically modified to express a PKS-like system for the production of PUFAs (a PUFA PKS system or PUFA synthase), wherein the organisms have been modified to express an acyl-CoA synthetase, to express an acyl transferase, to delete or inactivate a fatty acid synthase (FAS) expressed by the organism, to reduce competition for malonyl CoA with the PUFA synthase or to increase the level of malonyl CoA in the plant or plant cell, and in one aspect, to inhibit KASII or KASIII. Additional modifications, and methods to make and use such organisms, in addition to PUFAs and oils obtained from such organisms, are disclosed, alone with various products including such PUFAs and oils.
1 . A genetically modified plant, part of the plant, or plant cell, wherein the plant or plant cell has been genetically modified with a PUFA synthase that produces at least one polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl transferase (PPTase), and wherein the plant or plant cell contains a genetic modification to inhibit the expression or activity of a protein selected from the group consisting of KASII and KASIII.
2 . A genetically modified plant, part of the plant, or plant cell, wherein the plant or plant cell has been genetically modified with a PUFA synthase that produces at least one polyunsaturated fatty acid (PUFA) and a phosphopantetheinyl transferase (PPTase), and wherein at least one nucleic acid molecule encoding the PUFA synthase or the PPTase is operatively linked to a nucleic acid sequence encoding a plastid-targeting sequence represented by SEQ ID NO:81.
3 . The genetically modified plant, part of the plant, or plant cell of claim 2 , wherein the plant contains a genetic modification to inhibit the expression or activity of a protein selected from the group consisting of KASII and KASIII.
4 . The genetically modified plant, part of the plant, or plant cell of claim 1 or claim 3 , wherein the plant or plant cell produces an increased level of said at least one PUFA as compared to in the absence of said inhibition of KASII or KASIII.
5 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 3 , wherein the plant or plant cell has been genetically modified to inhibit the expression or activity of KASII.
6 . The genetically modified plant, part of the plant, or plant cell of claim 5 , wherein the genetic modification comprises the transformation of the plant or plant cell with an RNAi construct that inhibits the expression or activity of KASII.
7 . The genetically modified plant, part of the plant, or plant cell of claim 6 , wherein the RNAi construct comprises a nucleic acid sequence represented herein by SEQ ID NO:122.
8 . The genetically modified plant, part of the plant, or plant cell of claim 5 , wherein the genetic modification comprises the transformation of the plant or plant cell with an antisense nucleic acid molecule that inhibits the expression or activity of KASII.
9 . The genetically modified plant, part of the plant, or plant cell of claim 8 , wherein the antisense nucleic acid molecule comprises a nucleic acid sequence represented herein by SEQ ID NO:123.
10 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 3 , wherein the plant has been genetically modified to inhibit the expression or activity of KASIII.
11 . The genetically modified plant, part of the plant, or plant cell of claim 10 , wherein the genetic modification comprises the transformation of the plant or plant cell with an RNAi construct that inhibits the expression or activity of KASIII.
12 . The genetically modified plant, part of the plant, or plant cell of claim 11 , wherein the RNAi construct comprises a nucleic acid sequence represented herein by SEQ ID NO:124.
13 . The genetically modified plant, part of the plant, or plant cell of claim 10 , wherein the genetic modification comprises the transformation of the plant or plant cell with an antisense nucleic acid molecule that inhibits the expression or activity of KASIII.
14 . The genetically modified plant, part of the plant, or plant cell of claim 13 , wherein the antisense nucleic acid molecule comprises a nucleic acid sequence represented herein by SEQ ID NO:125.
15 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 14 , wherein the plant or plant cell contains an additional genetic modification to express one or more heterologous acyl-CoA synthetases (ACoAS) or a homologue thereof that catalyzes the conversion of long chain PUFA free fatty acids (FFA) to acyl-CoA.
16 . The genetically modified plant, part of the plant, or plant cell of claim 15 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) or a homologue thereof from Crypthecodinium cohnii , wherein the ACoAS or homologue thereof catalyzes the conversion of long chain PUFA free fatty acids (FFA) to acyl-CoA.
17 . The genetically modified plant, part of the plant, or plant cell of claim 15 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) from Schizochytrium or a homologue that is at least 60% identical to the amino acid sequence encoding the ACoAS from Schizochytrium , wherein the ACoAS or homologue thereof catalyzes the conversion of long chain PUFA free fatty acids (FFA) to acyl-CoA.
18 . The genetically modified plant, part of the plant, or plant cell of claim 15 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) that is at least 60% identical to an ACoAS having an amino acid sequence selected from the group consisting of: SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97 and SEQ ID NO:99.
19 . The genetically modified plant, part of the plant, or plant cell of claim 15 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) having an amino acid sequence selected from the group consisting of: SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97 and SEQ ID NO:99.
20 . The genetically modified plant, part of the plant, or plant cell of claim 15 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) having an amino acid sequence selected from the group consisting of: SEQ ID NO:83, SEQ ID NO:85 and SEQ ID NO:97.
21 . The genetically modified plant, part of the plant, or plant cell of claim 15 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) having an amino acid sequence of SEQ ID NO:83 or SEQ ID NO:85, and with a nucleic acid molecule comprising a nucleic acid sequence encoding an acyl-CoA synthetase (ACoAS) having an amino acid sequence of SEQ ID NO:97.
22 . The genetically modified plant, part of the plant, or plant cell of claim 15 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, and SEQ ID NO:98.
23 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 22 , wherein the plant or plant cell contains an additional genetic modification to express one or more heterologous proteins from an organism that endogenously produces PUFAs, wherein the protein utilizes PUFA-CoA as substrates in forming phospholipids (PL) or triacylglycerols (TAG).
24 . The genetically modified plant, part of the plant, or plant cell of claim 23 , wherein the organism endogenously expresses a PUFA synthase.
25 . The genetically modified plant, part of the plant, or plant cell of claim 23 , wherein the protein is a DAGAT or an LPAAT.
26 . The genetically modified plant, part of the plant, or plant cell of claim 23 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding a protein from a Thraustochytrid or a Labyrinthulid that utilizes PUFA-CoA as substrates in forming phospholipids (PL) or triacylglycerols (TAG).
27 . The genetically modified plant, part of the plant, or plant cell of claim 23 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding a protein from Schizochytrium that utilizes PUFA-CoA as substrates in forming phospholipids (PL) or triacylglycerols (TAG).
28 . The genetically modified plant, part of the plant, or plant cell of claim 27 , wherein the nucleic acid sequence encodes a protein comprising an amino acid sequence that is at least 60% identical to an amino acid sequence selected from the group consisting of: SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:100, and SEQ ID NO:113.
29 . The genetically modified plant, part of the plant, or plant cell of claim 27 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:107, SEQ ID NO:110, and SEQ ID NO:113.
30 . The genetically modified plant, part of the plant, or plant cell of claim 27 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding a protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:102 and SEQ ID NO:104.
31 . The genetically modified plant, part of the plant, or plant cell of claim 27 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding a protein comprising an amino acid sequence of SEQ ID NO:102 and with a nucleic acid sequence encoding a protein comprising an amino acid sequence of SEQ ID NO:104.
32 . The genetically modified plant, part of the plant, or plant cell of claim 27 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:111, and SEQ ID NO:112.
33 . The genetically modified plant, part of the plant, or plant cell of claim 23 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding a protein from Crypthecodinium cohnii that utilizes PUFA-CoA as substrates in forming phospholipids (PL) or triacylglycerols (TAG).
34 . The genetically modified plant, part of the plant, or plant cell of claim 33 , wherein the plant or plant cell is transformed with a nucleic acid molecule comprising a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of: SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120 and SEQ ID NO:121.
35 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 34 , wherein the plant or plant cell comprises an additional genetic modification to delete or inactivate an endogenous fatty acid synthase (FAS) or protein associated with an FAS expressed by the plant.
36 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 35 , wherein the plant or plant cell comprises an additional genetic modification to reduce competition for malonyl CoA with the PUFA synthase or to increase the level of malonyl CoA in the plant or plant cell.
37 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase comprises at least one functional domain from a PUFA synthase from a Thraustochytrid or a Labyrinthulid.
38 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase comprises at least one functional domain from a PUFA synthase from a Thraustochytriales microorganism.
39 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase comprises at least one functional domain from a PUFA synthase from an organism selected from the group consisting of: Schizochytrium, Thraustochytrium, Ulkenia , and Labyrinthula.
40 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase comprises at least one functional domain from a PUFA synthase from Schizochytrium.
41 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase comprises at least one functional domain from a PUFA synthase from an organism selected from the group consisting of Schizochytrium sp. American Type Culture Collection (ATCC) No. 20888 , Thraustochytrium 23B ATCC No. 20892, and a mutant of any of said microorganisms.
42 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase comprises at least one functional domain from a PUFA synthase from a marine bacterium.
43 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase comprises at least one functional domain from a PUFA synthase from an organism selected from the group consisting of Shewanella, Moritella and Photobacterium.
44 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase consists of one or more proteins comprising:
a) at least one enoyl-ACP reductase (ER) domain;
b) at least four acyl carrier protein (ACP) domains;
c) at least two β-ketoacyl-ACP synthase (KS) domains;
d) at least one acyltransferase (AT) domain;
e) at least one β-ketoacyl-ACP reductase (KR) domain;
f) at least two FabA-like β-hydroxyacyl-ACP dehydrase (DH) domains; and
g) at least one chain length factor (CLF) domain;
h) at least one malonyl-CoA:ACP acyltransferase (MAT) domain.
45 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase consists of one or more proteins comprising:
a) two enoyl ACP-reductase (ER) domains;
b) eight or nine acyl carrier protein (ACP) domains;
c) two β-keto acyl-ACP synthase (KS) domains;
d) one acyltransferase (AT) domain;
e) one ketoreductase (KR) domain;
f) two FabA-like β-hydroxy acyl-ACP dehydrase (DH) domains;
g) one chain length factor (CLF) domain; and
h) one malonyl-CoA:ACP acyltransferase (MAT) domain.
46 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase is a bacterial PUFA synthase that produces PUFAs at temperatures of at least about 25° C., and wherein the PUFA synthase consists of one or more proteins comprising:
a) at least one enoyl ACP-reductase (ER) domain;
b) at least six acyl carrier protein (ACP) domains;
c) at least two β-keto acyl-ACP synthase (KS) domains;
d) at least one acyltransferase (AT) domain;
e) at least one ketoreductase (KR) domain;
f) at least two FabA-like β-hydroxy acyl-ACP dehydrase (DH) domains;
g) at least one chain length factor (CLF) domain;
h) at least one malonyl-CoA:ACP acyltransferase (MAT) domain; and
i) at least one 4′-phosphopantetheinyl transferase (PPTase) domain.
47 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 36 , wherein the PUFA synthase comprises one or more sequences selected from the group consisting of: any one of SEQ ID NOs:1-32 and any one of SEQ ID NOs:35-80.
48 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 47 , wherein one or more nucleic acid sequences encoding the PUFA synthase has been optimized to improve the expression of the PUFA synthase in the plant or plant cell.
49 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 47 , wherein expression of the PUFA synthase and the PPTase is targeted to the plastid of the plant or plant cell.
50 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 49 , wherein the plant is an oil seed plant.
51 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 50 , wherein the plant is a dicotyledonous plant.
52 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 49 , wherein the plant is selected from the group consisting of: canola, soybean, rapeseed, linseed, corn, safflower, sunflower and tobacco.
53 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 52 , wherein the plant or plant cells produces at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of: EPA (C20:5, n-3), DHA (C22:6, n-3), DPA (C22:5, n-6 or n-3), ARA (C20:4, n-6), GLA (C18:3, n-6), and/or SDA (C18:4, n-3)).
54 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 52 , wherein the plant or plant cells produces at least one polyunsaturated fatty acid (PUFA) selected from the group consisting of: DHA, EPA and DPAn-6.
55 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 52 , wherein the plant or plant cells produces DHA and DPAn-6.
56 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 52 , wherein the plant or plant cells produces ARA.
57 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 56 , wherein the total fatty acid profile in the plant, part of the plant, or plant cell comprises at least 0.5% by weight of said at least one PUFA.
58 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 56 , wherein the total fatty acids produced by said PUFA synthase, other than said at least one PUFA, comprises less than about 10% by weight of the total fatty acids produced by said plant or plant cell.
59 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 56 , wherein the total fatty acids produced by said PUFA synthase, other than said at least one PUFA, comprises less than about 5% by weight of the total fatty acids produced by said plant or plant cell.
60 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 56 , wherein the total fatty acid profile in the plant, part of the plant, or plant cell comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) having at least twenty carbons and four or more carbon-carbon double bonds, 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.
61 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 56 , wherein the total fatty acid profile in the plant, part of the plant, or plant cell comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) having at least twenty carbons and four or more carbon-carbon double bonds, and wherein the total fatty acid profile in the plant or part of the plant contains less than 1% of each 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 genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 56 , wherein the total fatty acid profile in the plant, part of the plant, or plant cell comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) having at least twenty carbons and four or more carbon-carbon double bonds, 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).
63 . The genetically modified plant, part of the plant, or plant cell of claim 63 , wherein the total fatty acid profile in the plant, part of the plant, or plant cell contains less than 1% by weight of gamma-linolenic acid (GLA; 18:3, n-6) and dihomo-gamma-linolenic acid (DGLA or HGLA; 20:3, n-6).
64 . The genetically modified plant, part of the plant, or plant cell of any one of claims 1 to 56 , wherein the total fatty acid profile in the plant, part of the plant, or plant cell comprises at least about 0.5% by weight of at least one polyunsaturated fatty acid (PUFA) having at least twenty carbons and four or more carbon-carbon double bonds, 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).
65 . The genetically modified plant, part of the plant, or plant cell of claim 64 , wherein the total fatty acid profile in the plant, part of the plant, or plant cell contains less than 0.5% by weight of gamma-linolenic acid (GLA; 18:3, n-6).
66 . An oil obtained from the plant, part of the plant, or plant cell of any one of claims 1 to 65 .
67 . A plant oil comprising detectable amounts of DHA (docosahexaenoic acid (C22:6, n-3)), and DPA (docosapentaenoic acid (C22:5, n-6), wherein the ratio of DPAn-6 to DHA is 1:1 or greater than 1:1, wherein the plant oil is obtained from the plant, plant part, or plant cell of any one of claims 1 to 65 .
68 . Seeds obtained from the plant of any one of claims 1 to 65 .
69 . A food product that contains an oil of claim 66 or claim 67 or the seeds from claim 68 .
70 . A pharmaceutical product that contains an oil of claim 66 or claim 67 .
71 . A method to produce an oil comprising at least one PUFA, comprising recovering an oil from the seed of claim 68 .
72 . A method to produce an oil comprising at least one PUFA, comprising recovering an oil from the plant, part of plant, or plant cell of any one of claims 1 to 65 .
73 . A method to produce at least one polyunsaturated fatty acid (PUFA), comprising growing the plant of any one of claims 1 to 65 .
74 . A method to provide a supplement or therapeutic product containing at least one PUFA to an individual, comprising providing to the individual a plant, part of plant, or plant cell of any one of claims 1 to 65 , seeds of claim 68 , an oil of claim 66 or claim 67 , a food product of claim 69 , or a pharmaceutical product of claim 70 .
75 . A method to produce the genetically modified plant, part of plant, or plant cell of claim 1 or claim 2 , comprising transforming a plant or plant cell with one or more nucleic acid molecules encoding the PUFA synthase and the PPTase, wherein the plant or plant cell contains a genetic modification to inhibit the expression or activity of a protein selected from the group consisting of KASII and KASIII.
76 . A method to produce the genetically modified plant, part of plant, or plant cell of claim 1 or claim 2 , comprising transforming a plant or plant cell with one or more nucleic acid molecules encoding the PUFA synthase and the PPTase, and further genetically modifying the plant or plant cell to inhibit the expression or activity of a protein selected from the group consisting of KASII and KASIII.